U.S. patent application number 17/082955 was filed with the patent office on 2021-04-22 for separation moieties and methods of use thereof.
The applicant listed for this patent is Werewolf Therapeutics, Inc.. Invention is credited to Vinay BHASKAR, Heather BRODKIN, Luke EVNIN, Daniel HICKLIN, Giselle KNUDSEN, Jose Andres SALMERON GARCIA, Cynthia SEIDEL-DUGAN, William WINSTON.
Application Number | 20210115102 17/082955 |
Document ID | / |
Family ID | 1000005326084 |
Filed Date | 2021-04-22 |
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United States Patent
Application |
20210115102 |
Kind Code |
A1 |
WINSTON; William ; et
al. |
April 22, 2021 |
SEPARATION MOIETIES AND METHODS OF USE THEREOF
Abstract
Provided herein are separation moieties that are suitable for
use in conjunction with a variety of therapeutic payloads. The
separation moieties serve to generate conditionally active
macromolecules whereby the macromolecules have reduced or minimal
biological activity until the separation moieties are modified
under specific conditions.
Inventors: |
WINSTON; William; (West
Newton, MA) ; EVNIN; Luke; (San Francisco, CA)
; BHASKAR; Vinay; (San Francisco, CA) ; KNUDSEN;
Giselle; (San Anselmo, CA) ; HICKLIN; Daniel;
(Montclair, NJ) ; SEIDEL-DUGAN; Cynthia; (Belmont,
MA) ; SALMERON GARCIA; Jose Andres; (Westminster,
MA) ; BRODKIN; Heather; (West Newton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Werewolf Therapeutics, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
1000005326084 |
Appl. No.: |
17/082955 |
Filed: |
October 28, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US2020/032988 |
May 14, 2020 |
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17082955 |
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62938786 |
Nov 21, 2019 |
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62847914 |
May 14, 2019 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/02 20130101;
C07K 2319/30 20130101; C07K 2317/92 20130101; C07K 2317/73
20130101; C07K 2317/622 20130101; C07K 2319/03 20130101; C07K 14/57
20130101; C07K 14/7155 20130101; C07K 2319/50 20130101; C07K
2319/31 20130101; C07K 16/2887 20130101; C07K 14/55 20130101; A61K
2039/505 20130101 |
International
Class: |
C07K 14/55 20060101
C07K014/55; C07K 14/715 20060101 C07K014/715; C07K 14/57 20060101
C07K014/57; C07K 16/28 20060101 C07K016/28 |
Claims
1-74. (canceled)
75. A polypeptide comprising Formula I: [D1]-[L1]-[D2], wherein D1
is a first domain of interest; L1 is a separation moiety that
connects or links D1 to D2, wherein the separation moiety comprises
an amino acid sequence selected from SEQ ID NOs: 195-220 or an
amino acid sequence that has at least about 75% identity to SEQ ID
NOs:195-220; and D2 is a second domain of interest.
76. The polypeptide of claim 1, wherein the separation moiety
comprises an amino acid sequence that is a substrate for at least
one protease present in a tumor microenvironment of a human
tumor.
77. The polypeptide of claim 1, comprising two or more separation
moieties, wherein the two or more separation moieties are each a
substrate for a protease and, wherein each separation moiety
independently comprises an amino acid sequence selected from SEQ ID
NOs: 195-220 or an amino acid sequence that has at least about 75%
identity to SEQ ID NOs:195-220.
78. The polypeptide of claim 1, further comprising a non-cleavable
linker sequence.
79. The polypeptide of claim 1, wherein [D1] or [D2] comprises a
cytokine, chemokine, growth factor, a soluble receptor, or a
fragment thereof, or any combination thereof.
80. The polypeptide of claim 1, wherein the polypeptide comprises
at least one of an extracellular domain, a transmembrane domain,
and an intracellular domain.
81. The polypeptide of claim 1, wherein [D1] or [D2] comprises a
cell surface receptor, a chimeric antigen receptor (CAR), or a T
Cell Receptor (TCR) subunit, or a fragment thereof.
82. The polypeptide of claim 1, wherein [D1] or [D2] comprises an
antigen-binding polypeptide, an antibody or an antigen-binding
portion thereof.
83. The polypeptide of claim 1, wherein [D1] or [D2] comprises a
half-life extension domain.
84. The polypeptide of claim 1, wherein the cleavable moiety is
cleaved with either (a) greater catalytic efficiency, (b) greater
specificity, or (c) both (a) and (b), by one or more proteases than
a reference polypeptide sequence.
85. The polypeptide of claim 1, wherein the cleavable moiety is
cleaved with reduced catalytic efficiency by one or more proteases
than a reference polypeptide sequence.
86. The polypeptide of claim 1, wherein the cleavable moiety is
cleaved with reduced catalytic efficiency by one or more serum
proteases, one or more hepatic proteases, or a protease present in
a normal healthy tissue.
87. A recombinant pro-protein comprising: a. a recombinant
polypeptide comprising a cleavable moiety that is a substrate for a
protease, wherein the cleavable moiety comprises an amino acid
sequence selected from the group consisting of SEQ ID NOs: 195-220
or an amino acid sequence at least at least 75% identical to an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 195-220; and b. a polypeptide with biological activity,
wherein the pro-protein has attenuated biological activity, and
wherein cleavage of the cleavable moiety by the protease produces a
polypeptide with biological activity that is not attenuated.
88. The recombinant pro-protein of claim 87, wherein the
polypeptide with biological activity comprises a cytokine,
chemokine, growth factor, a soluble receptor or a combination
thereof.
89. The recombinant pro-protein of claim 87, wherein the
polypeptide with biological activity comprises at least one of an
extracellular domain, a transmembrane domain, and an intracellular
domain.
90. The recombinant pro-protein of claim 87, wherein the
polypeptide with biological activity comprises a cell surface
receptor, a chimeric antigen receptor (CAR), or a T Cell Receptor
(TCR) subunit.
91. The recombinant pro-protein of claim 87, wherein the
polypeptide with biological activity comprises an antigen-binding
polypeptide, an antibody or an antigen-binding portion thereof.
92. A fusion protein comprising: a. a signaling protein or
molecule; b. a blocking moiety selected from a steric blocking
moiety, a specific blocking moiety, and the combination thereof;
and c. a peptide linker that comprises a cleavable moiety that is a
substrate for a protease, wherein the cleavable moiety comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 195-220 or an amino acid sequence at least at least 75%
identical to an amino acid sequence selected from the group
consisting of SEQ ID NOs: 195-220, wherein (a) and (b) are operably
linked by (c).
93. The fusion protein of claim 92, wherein the steric blocking
moiety comprises human serum albumin (HSA), an anti-HSA antibody,
an immunoglobulin Fc, or a fragment thereof.
Description
[0001] The present application claims the benefit of U.S.
Provisional Application No. 62/847,914 filed on May 14, 2019 and
U.S. Provisional Application No. 62/938,786 filed on Nov. 21, 2019,
each of which are incorporated herein by reference in their
entireties.
1. SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 14, 2020, is named 761146_000140_SL.txt and is 896,815 bytes
in size.
2. BACKGROUND
[0003] Recombinant fusion proteins containing two or more
functional polypeptides have utility in many fields including, for
use in protein purification, imaging, as therapeutics, and drug
delivery. For example, protein drugs can be fused to Fc domains of
antibodies or to carrier proteins (i.e., human serum albumin) for
targeting, to extend their plasma half-lives and/or to achieve
therapeutic effects. Chen et al., (2013), Adv Drug Deliv Rev.
65(10):1357-1369.
[0004] Direct fusion of functional polypeptide or domains without a
linker may lead to many undesirable outcomes, including misfolding
of the fusion proteins (Zhao et al., (2008), Protein Expr. Purif.,
61:73-77), low yield in protein production (Amet et al., (2009),
Pharm. Res. 26:523-528), or impaired bioactivity (Bai et al.,
(2006) Proc. Nat Acad. Sci. USA, 102:7292-7296). One approach to
overcome these difficulties is to use linker sequences between the
component polypeptides or domains in a fusion protein. However, the
selection of a suitable linker to join protein domains together can
be complicated and is often neglected in the design of fusion
proteins. Chen et al., (2013), Adv Drug Deliv Rev.
65(10):1357-1369. The properties of linker sequences, such as
length, hydrophobicity, amino acid composition, secondary
structures, and the overall folding can affect linker suitability
and need to be taken into consideration in designing and selecting
an appropriate linker. In addition, linkers that can be cleaved
under selected conditions or in selected biological locations
(e.g., in tumor microenvironment) to deliver active therapeutic
agents (e.g., therapeutic polypeptides) can provide targeted
pharmacological activity of the therapeutic agents and reduce
unwanted systemic effects. This introduces further complexity into
designing suitable linkers.
[0005] There is a need for improved liner sequences that can be
used to prepare stable fusion proteins, including linkers that can
be cleaved under selected conditions. Accordingly, novel separation
moieties or linkers are disclosed herein. The separation moieties
or linkers disclosed herein can be utilized, for example to
specifically deliver prodrugs such as conditionally active and/or
targeted cytokines to target sites where the linkers are processed
to activate bioactivity.
3. SUMMARY
[0006] Provided herein are compositions and methods to generate and
use high efficiency separation moieties and/or linkers. The linkers
can confer site-selectivity with regards to biological activity of
the attached payload or payloads. In some embodiments, the
separation moieties and/or linkers are used in conjugation with
therapeutic proteins to treat a disease or disorder, such as
proliferative disease, a tumorous disease, an inflammatory disease,
an immunological disorder, an autoimmune disease, an infectious
disease, a viral disease, an allergic reaction, a parasitic
reaction, graft-versus-host disease and the like.
[0007] Disclosed herein are recombinant polypeptides comprising a
separation moiety, wherein the separation moiety comprises an amino
acid sequence is a substrate for an enzyme, specifically a
protease. The protease can be selected from the group consisting of
Fibroblast activation protein alpha (FAP.alpha., also known as
prolyl endopeptidase FAP), Cathepsin L (CTSL1), an ADAM selected
from ADAM 8, ADAM 9, ADAM 10, ADAM12 ADAM17, and ADAMTS1, and an
MMP selected from MMP1, MMP2, MMP9 or MMP14. The cleavable moiety
can also be a substrate for a cathepsin, such as cathepsin B,
cathepsin C, cathepsin D, cathepsin E, cathepsin G, cathepsin K
and/or cathepsin L. Preferably, the cathepsin is Cathepsin L.
Preferably, the protease is MMP14 or cathepsin L. The cleavable
moiety can comprise an amino acid sequence that is a substrate for
at least two proteases. In another embodiment, the separation
moiety comprises two or more cleavable moieties, each of which is a
substrate for a protease. The separation moiety can comprise a
first cleavable moiety comprising a first amino acid sequence that
is a substrate for a first protease and a second cleavable moiety
comprising a second amino acid sequence that is a substrate for a
second protease. In embodiments, the disclosure related to
recombinant polypeptides that comprise a separation moiety that
contains a protease cleavage motif as disclosed herein. The
recombinant polypeptide can comprise a separation moiety that
comprises an amino acid sequence selected from the group consisting
of SEQ ID NOs: 195-220, or an amino acid sequence that has at least
90% identity to SEQ ID NOs:195-220. Preferred separation moieties
comprise the sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID
NO: 198). The disclosure also relates to functional variants of
separation moieties comprising SEQ ID NOs: 195-220. The functional
variants of SEQ ID NO: 195 can comprise any of SEQ ID NOS: 258-331.
The functional variants of SEQ ID NO: 198 can comprise of SEQ ID
NO:198 comprises SEQ ID NOS: 199 or any of SEQ ID NOS: 332-408. The
separation moieties disclosed herein can comprise Formula I:
[D1]-[L1]-[D2]. D1 is a first domain of interest. L1 is a
separation moiety that connects or links D1 to D2, wherein the
separation moiety comprises an amino acid sequence selected from
SEQ ID NOs: 195-220 or an amino acid sequence that has at least
about 90% identity to SEQ ID NOs:195-220. D2 is a second domain of
interest.
[0008] In one embodiment, the recombinant polypeptide can further
comprise a non-cleavable linker sequence. The recombinant
polypeptide can include a therapeutic protein, such as a cytokine,
chemokine, growth factor, soluble receptor, antigen-binding portion
of an antibody (e.g., scFV, dAb) and the like. In another
embodiment, the recombinant polypeptide comprises a cytokine,
chemokine, growth factor, a soluble receptor, or any combination
thereof. In another embodiment, the recombinant polypeptide
comprises at least one of an extracellular domain, a transmembrane
domain, and an intracellular domain. In one embodiment, the
recombinant polypeptide comprises a cell surface receptor, a
chimeric antigen receptor (CAR), or a T Cell Receptor (TCR)
subunit. In one embodiment, the recombinant polypeptide comprises
an antigen-binding polypeptide, an antibody or an antigen-binding
portion thereof.
[0009] In one embodiment, the cleavable moiety is cleaved with
either (a) greater catalytic efficiency or (b) greater specificity
or (c) both (a) and (b), by one or more proteases than a reference
polypeptide sequence. In another embodiment, the one or more
proteases are selected from the group consisting of FAP.alpha.,
CTSL1, an ADAM selected from ADAM 8, ADAM 9, ADAM 10, ADAM12
ADAM17, and ADAMTS1, and an MMP selected from MMP1, MMP2, MMP9 and
MMP14. The one or more proteases can also include a protease
selected from cathepsins, such as cathepsin B, cathepsin C,
cathepsin D, cathepsin E, cathepsin G, cathepsin K and/or cathepsin
L. In another embodiment, the reference polypeptide sequence is
present in a naturally occurring polypeptide substrate for
FAP.alpha., CTSL1, an ADAM selected from ADAM 8, ADAM 9, ADAM 10,
ADAM12 ADAM17, and ADAMTS1, and an MMP selected from MMP1, MMP2,
MMP9 and MMP14, a cathepsin, such as cathepsin B, cathepsin C,
cathepsin D, cathepsin E, cathepsin G, cathepsin K and/or cathepsin
L or a combination thereof. In another embodiment, the cleavable
moiety is cleaved with reduced catalytic efficiency by one or more
proteases than a reference polypeptide sequence. In another
embodiment the cleavable moiety is cleaved with reduced catalytic
efficiency by one or more serum proteases. In another embodiment,
the cleavable moiety is cleaved with reduced catalytic efficiency
by one or more hepatic proteases. In another embodiment, the
cleavable moiety is cleaved with reduced catalytic efficiency by
one or more Factor Xa, hepsin, or thrombin.
[0010] In one embodiment, the recombinant polypeptide comprises two
or more separation moieties. In one embodiment, the recombinant
polypeptide is operably linked to a moiety selected from the group
consisting of a polypeptide moiety, a lipid moiety, a nucleic acid
moiety, a detectable moiety, and a small molecule.
[0011] Provided herein is a recombinant pro-protein comprising: a
recombinant polypeptide comprising a cleavable moiety that is a
substrate for a protease, wherein the cleavable moiety comprises an
amino acid sequence selected from the group consisting of SEQ ID
NOs: 195-220; and a polypeptide with biological activity, wherein
the pro-protein has attenuated biological activity and wherein
cleavage of the cleavable moiety by the protease produces a
polypeptide with biological activity that is not attenuated. In one
embodiment, the polypeptide with biological activity comprises a
cytokine, chemokine, growth factor, a soluble receptor or a
combination thereof. In some preferred aspects, the linkers are
components of fusion proteins with therapeutic utility, and the
linkers are not cleaved or are cleaved with low efficiency in the
peripheral circulation but are cleaved with higher efficiency at a
desired location in the body, such as a tumor microenvironment or
site of inflammation. In another embodiment, the polypeptide with
biological activity comprises at least one of an extracellular
domain, a transmembrane domain, and an intracellular domain. In
another embodiment, the polypeptide with biological activity
comprises a cell surface receptor, a chimeric antigen receptor
(CAR), or a T Cell Receptor (TCR) subunit. In another embodiment,
the polypeptide with biological activity comprises an
antigen-binding polypeptide, an antibody or an antigen-binding
portion thereof.
[0012] The disclosure also relates to a fusion protein comprising:
a. a signaling protein or molecule; and b. a blocking moiety
selected from a steric blocking moiety, a specific blocking moiety,
and the combination thereof; and c. a peptide linker that comprises
a cleavable moiety, e.g., a cleavable moiety disclosed herein,
having at least one protease-cleavable sequence. In one embodiment,
the fusion protein, wherein (a) and (b) are operably linked by (c).
In another embodiment, the fusion protein, wherein the peptide
linker comprises two or more copies of the same cleavable moiety.
In another embodiment, the fusion protein wherein the steric
blocking moiety comprises human serum albumin (HSA) or an anti-HSA
antibody. In another embodiment, the fusion protein wherein the
signaling protein is an interleukin-2 amino acid sequence
comprising: (i) a non-native N terminus and/or (ii) a non-native C
terminus. In another embodiment, the fusion protein wherein the
signaling protein is an interleukin-2 amino acid sequence. In
another embodiment, the fusion protein further comprising one or
more half-life extension domains that are not also a specific
blocker. In another embodiment, the fusion protein wherein the
non-native N and/or C termini are generated by circular
permutation.
[0013] The fusion polypeptide provided herein can a first
polypeptide fusion partner linked to a ligand by a protease
cleavable linker, wherein the cleavable linker has been optimized
for catalytic efficiency, and wherein the ligand has been
optionally modified, wherein the first polypeptide fusion partner
is a blocking moiety which prevents binding of the modified ligand
to a target receptor or a subunit of a target receptor until
cleavage of the protease cleavable linker.
[0014] The fusion polypeptide provided herein can also a fusion
polypeptide comprising a first polypeptide fusion partner linked to
a ligand by a protease cleavable linker, wherein the cleavable
linker has been optimized for catalytic efficiency, and wherein the
ligand has been optionally modified, including by circularly
permutation to create a non-native N-terminus and a new C-terminus
as compared to a native ligand, and wherein at least one of the new
N-terminus or the new C-terminus of the modified ligand is operably
linked to a first polypeptide fusion partner to form a fusion
polypeptide wherein the first polypeptide fusion partner is a
blocking moiety which prevents binding of the modified ligand to a
target receptor or a subunit of a target receptor until cleavage of
the protease cleavable linker.
[0015] In one embodiment, the first polypeptide fusion partner is
selected from the group consisting of an antibody, an antibody
fragment, and an albumin molecule. In another embodiment, the first
polypeptide fusion partner further comprising a second polypeptide
fusion partner comprising a second blocking moiety. In another
embodiment, the second polypeptide fusion partner is a different
kind of blocking moiety than the first polypeptide fusion partner.
In another embodiment, the first polypeptide fusion partner is
albumin and the second polypeptide fusion partner is a domain
comprising a complementary amino acid sequence that blocks activity
of the cytokine. In another embodiment, the first polypeptide
fusion partner is a steric blocker, such as albumin, and the second
polypeptide is a specific blocker, such as a cytokine receptor,
portion of a cytokine receptor, a de novo affinity peptide specific
for the cytokine, or an antibody or antibody fragment that
specifically binds the cytokine of the fusion polypeptide. In
another embodiment, the second polypeptide fusion partner is the
same kind of blocking moiety as the first polypeptide fusion
partner.
[0016] In one embodiment, the fusion protein further comprises a
tumor antigen binding component. In another embodiment, the fusion
protein further comprises a serum half-life extension domain. In
another embodiment, the ligand is selected from the group
consisting of helix bundle proteins and cytokines (including, but
not limited to, growth hormone, IL-2, IL-4, IL-5, IL-6, IL-10,
IL-22, IL-23p19, IL-11, IL-13, IL-15, IL-12p35, IL-21, IL-30
(IL27p28), IL-34, IL-35, IL-35p35, IFN-.alpha., IFN-.beta.,
IFN.gamma., LIF, CNTF, oncostatin M, CLCF-1, GCSF, GM-CSF, EPO,
ferritin, leptin, placental lactogen, prolactin, apolipoprotein e),
b-trefoil proteins (including, but not limited to, IL-1.alpha.,
IL-1.beta., IL-1Ra, IL18, IL-33, IL-36Ra, IL-36a, IL-36b, IL-36g,
IL-37, IL-38, IL1Hy2, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6,
FGF-7, FGF-8a, FGF-8b, FGF-8e, FGF-8f, FGF-9, FGF-10, FGF-11,
FGF-12, FGF-13, FGF-14, FGF-16, FGF-17, FGF-18, FGF-19, FGF-20,
FGF-21, FGF-22, FGF-23), .alpha./.beta. (TIM) barrel proteins
(including, but not limited to, triosephosphate isomerase), beta
sandwich proteins (including, but not limited to, galectin-1,
galectin-3, TNF-beta, seven .beta.-propeller proteins, class 1 MHC
.alpha.1.alpha.2 domain, integrin I domain, GYF domain, C1 domain,
C2 domain (for example, from cPLA2, PKC, synaptotagmin), PDZ
domains, C3d, C5a. In one embodiment, wherein the ligand comprises
IL-2 polypeptide or a fragment or fragments thereof. In another
embodiment, the protease-cleavable linker polypeptide comprises a
sequence that is capable of being cleaved by at least one protease
selected from the group consisting of a kallikrein, thrombin,
chymase, carboxypeptidase A, cathepsin G, an elastase, a FAP, an
ADAM selected from ADAM 8, ADAM 9, ADAM 10, ADAM12 ADAM17, and
ADAMTS1, PR-3, granzyme M, a calpain, a matrix metalloproteinase
(MMP), a plasminogen activator, a cathepsin, a caspase, a tryptase,
and a tumor cell surface protease. In another embodiment, the
cytokine or fragment or mutein thereof is substantially dissociated
from the cytokine blocking moiety after the protease-cleavable
polypeptide linker is cleaved by a protease.
[0017] Disclosed herein are fusion polypeptide comprising at least
one of each of: a cytokine polypeptide or functional fragment or
mutein thereof [A]; a cytokine blocking moiety [B]; and an
optimized protease-cleavable polypeptide linker [L]; wherein the
blocking moiety is selected from the group consisting of an
antibody, an antibody fragment, and an albumin, and wherein the
cytokine comprises a circularly permuted cytokine. In some
embodiments, the fusion protein further comprises a tumor antigen
binding component and/or a serum half-life extension domain. In
some embodiment, the fusion polypeptide wherein the cytokine
peptide or functional fragment or mutein thereof is selected from
the group consisting of helix bundle proteins and cytokines
(including, but not limited to, growth hormone, IL-2, IL-4, IL-5,
IL-6, IL-10, IL-22, IL-23p19, IL-11, IL-13, IL-15, IL-12p35, IL-21,
IL-30 (IL27p28), IL-34, IL-35, IL-35p35, IFN-.beta., IFN.gamma.,
LIF, CNTF, oncostatin M, CLCF-1, GCSF, GM-CSF, EPO, ferritin,
leptin, placental lactogen, prolactin, apolipoprotein e), a FAP
(e.g., Fap.alpha.), an ADAM selected from ADAM 8, ADAM 9, ADAM 10,
ADAM12 ADAM17, and ADAMTS1, b-trefoil proteins (including, but not
limited to, IL-1.alpha., IL-1.beta., IL-1Ra, IL18, IL-33, IL-36Ra,
IL-36a, IL-36b, IL-36g, IL-37, IL-38, IL1Hy2, FGF-1, FGF-2, FGF-3,
FGF-4, FGF-5, FGF-6, FGF-7, FGF-8a, FGF-8b, FGF-8e, FGF-8f, FGF-9,
FGF-10, FGF-11, FGF-12, FGF-13, FGF-14, FGF-16, FGF-17, FGF-18,
FGF-19, FGF-20, FGF-21, FGF-22, FGF-23), .alpha./.beta. (TIM)
barrel proteins (including, but not limited to, triosephosphate
isomerase), beta sandwich proteins (including, but not limited to,
galectin-1, galectin-3, TNF-.beta., seven .beta.-propeller
proteins, class 1 MHC .alpha.1.alpha.2 domain, integrin I domain,
GYF domain, C1 domain, C2 domain (for example, from cPLA2, PKC,
synaptotagmin), PDZ domains, C3d, C5a. In one embodiment, the
cytokine peptide or functional fragment or mutein thereof comprises
IL-2. In another embodiment, the cytokine blocking moiety comprises
a ligand binding domain or fragment or mutein of a cognate receptor
for the cytokine, a single domain antibody or scFv that binds the
cytokine polypeptide or functional fragment or mutein thereof, or
an antibody or antibody fragment that binds a receptor of the
cytokine. In another embodiment, antibody is a single domain
antibody or scFv. In another embodiment, the blocking moiety
extends the serum half-life of the cytokine or fragment
thereof.
[0018] Disclosed herein are fusion polypeptides comprising a
protease cleavable moiety, wherein the sequence is catalytically
optimized for cleavage by certain proteases and wherein protease
cleavage renders the composition inducible in a tumor
microenvironment. In one embodiment, the fusion protein further
comprises a biologically inactive polypeptide, wherein cleavage of
the cleavable moiety by the protease converts the biologically
inactive polypeptide to a biologically active polypeptide. In one
embodiment, the biologically inactive polypeptide comprises a
cytokine, chemokine, growth factor, or soluble receptor. In another
embodiment, the biologically inactive polypeptide comprises at
least one of an extracellular domain, a transmembrane domain, and
an intracellular domain. In another embodiment, the biologically
inactive polypeptide comprises a cell surface receptor, a chimeric
antigen receptor (CAR), or a T Cell Receptor (TCR) subunit. In
another embodiment, the biologically inactive polypeptide comprises
an antigen-binding polypeptide, an antibody or an antigen-binding
portion thereof.
[0019] The disclosure further relates to a nucleic acid encoding
any of the polypeptides disclosed herein, a vector comprising any
of the nucleic acids encoding any of the polypeptides disclosed
herein, and a host cell comprising said vector.
[0020] Methods of making a pharmaceutical composition, comprising
culturing the host cell comprising a vector comprising a nucleic
acid encoding any of the polypeptides disclosed herein, under
suitable conditions for expression and collection of desired
polypeptides are provided herein. Method of using any of the
polypeptides disclosed herein comprising administering an effective
amount of a pharmaceutical composition comprising such polypeptides
to a subject in need thereof are described. For example, use for
treating a subject with a disease or disorder disclosed herein.
[0021] Provided herein are pharmaceutical compositions comprising
an effective amount of any of the recombinant polypeptides, any of
the pro-proteins, any of the fusion proteins, any of the fusion
polypeptides, any of the nucleic acids, any of the vectors, or any
of the host cells comprising such vectors disclosed herein. For
example, a pharmaceutical composition for treating a subject with a
disease or disorder disclosed herein.
[0022] Also disclosed is the use of the recombinant polypeptides,
pro-proteins, fusion proteins, fusion polypeptides, nucleic acids,
vectors, or host cells comprising such vectors disclosed herein for
the manufacture of a medicament for treating a disease or disorder
disclosed herein.
4. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1A is a schematic illustrating a protease-activated
cytokine or chemokine that includes a blocking moiety. The blocking
moiety may optionally function as a serum half-life extending
domain. To the left of the arrow the drawing shows that a cytokine
is connected to a blocking moiety via a protease-cleavable linker,
thus blocking its ability to bind to its receptor. To the right of
the arrow the drawing shows that in an inflammatory or tumor
environment a protease cleaves at a protease-cleavage site on the
linker, releasing the blocking moiety and allowing the cytokine to
bind to its receptor.
[0024] FIG. 1B is a schematic illustrating a protease-activated
cytokine or chemokine wherein HSA (blocking moiety) is directly
bound to the cytokine or chemokine of interest, with a protease
cleavage site between the HSA and a cytokine or chemokine of
interest. To the left of the arrow the drawing shows that a
cytokine is connected to a blocking moiety via a protease-cleavable
linker, thus blocking its ability to bind to its receptor. To the
right of the arrow the drawing shows that in an inflammatory or
tumor environment, the protease cleaves at a protease-cleavage site
on linker, releasing the blocking moiety and allowing the cytokine
to bind to its receptor.
[0025] FIG. 1C is a schematic illustrating a protease-activated
cytokine or chemokine wherein more than one HSA (blocking moiety)
is bound directly to the molecule of interest. If desired, one or
more of the HSA can be bonded to the cytokine or chemokine through
a linker, such as a linker that contains a protease cleavage site.
To the left of the arrow the drawing shows that a cytokine is
connected to a blocking moiety via a protease-cleavable linker,
thus blocking its ability to bind to its receptor. To the right of
the arrow the drawing shows that in an inflammatory or tumor
environment, protease cleaves at protease-cleavage site on linker,
releasing the blocking moiety and allowing cytokine to bind
receptor. The cytokine now has similar pK properties as compared to
the native cytokine (e.g., has a short half-life).
[0026] FIG. 1D is a schematic illustrating a protease-activated
cytokine or chemokine comprising more than one cytokine, of the
same type or different type, each of which is bonded to a binding
domain through a protease-cleavable linker. To the left of the
arrow the drawing shows that a cytokine is connected to a blocking
moiety via a protease-cleavable linker, thus blocking its ability
to bind to its receptor. To the right of the arrow the drawing
shows that in an inflammatory or tumor environment a protease
cleaves at a protease cleavage site on linker, releasing the
blocking moiety and allowing the cytokine to bind to its
receptor.
[0027] FIG. 2 is a schematic illustrating a protease-activated
cytokine or chemokine comprising a cytokine or chemokine
polypeptide, a blocking moiety, and a serum half-life extending
domain connected by at least one protease-cleavable linker. To the
left of the arrow the drawing shows that a cytokine is connected to
a blocking moiety via protease-cleavable linkers, thus blocking its
ability to bind to its receptor. It is also bound to a separate
half-life extension element, which extends half-life in serum. To
the right of the arrow the drawing shows that in an inflammatory or
tumor environment a protease cleaves at a protease-cleavage site on
linker, thus releasing the serum half-life extension element and
the blocking moiety and allowing the cytokine to bind to its
receptor. The cytokine now has similar pK properties as compared to
the native cytokine (e.g., a short half-life).
[0028] FIG. 3 is a schematic illustrating a protease-activated
cytokine or chemokine comprising a cytokine or chemokine
polypeptide, a blocking moiety, and a targeting domain connected by
at least one protease-cleavable linker. To the left of the arrow
the drawing shows that a cytokine is connected to a blocking moiety
and a targeting domain via a protease-cleavable linker, thus
blocking its ability to bind to its receptor. To the right of the
arrow the drawing shows that in an inflammatory or tumor
microenvironment a protease cleaves at the protease cleavage site
in the linker, releasing the targeting domain and the blocking
moiety and allowing the cytokine to bind to its receptor.
[0029] FIG. 4A is a schematic illustrating a protease-activated
cytokine or chemokine comprising a cytokine or chemokine
polypeptide, a blocking moiety, a targeting domain, and a serum
half-life extending domain connected by at least one
protease-cleavable linker, wherein the cytokine polypeptide and the
targeting domain are connected by a protease-cleavable linker. To
the left of the arrow, the drawing shows that a cytokine or
chemokine is connected to targeting domain, blocking moiety, and
half-life extension element via protease-cleavable linker(s), thus
blocking its ability to bind to its receptor. To the right of the
arrow the drawing shows that in an inflammatory or tumor
environment, the protease cleaves at a protease-cleavage site on
linker(s), releasing the half-life extension element, the targeting
domain, and the blocking moiety, and allowing the cytokine to bind
to its receptor. The cytokine now has similar pK properties as
compared to the native cytokine (e.g., short half-life).
[0030] FIG. 4B is a schematic illustrating a protease-activated
cytokine or chemokine comprising a cytokine or chemokine
polypeptide, a blocking moiety, a targeting domain, and a serum
half-life extending domain connected by at least one
protease-cleavable linker. To the left of the arrow, the drawing
shows that a cytokine is connected to targeting domain, a blocking
moiety, and a half-life extension element via protease-cleavable
linker(s), thus blocking its ability to bind to its receptor. To
the right of the arrow the drawing shows that in an inflammatory or
tumor environment, the protease cleaves at a protease-cleavage site
on linker(s), releasing the half-life extension element and the
blocking moiety and allowing the cytokine to bind to the receptor.
The targeting moiety remains bound, keeping the cytokine in the
tumor microenvironment. The cytokine now has similar pK properties
as compared to the native cytokine (e.g., a short half-life).
[0031] FIG. 5 depicts a graph showing that Linkers-2 (GPAGLYAQ, SEQ
ID NO: 195) and Linkers-3 (ALFKSSFP, SEQ ID NO: 198) are minimally
cleaved in lung, kidney, and livery cells.
[0032] FIGS. 6A-6B show graphs that polypeptides containing
recombinant human IL-2 and the sequence for Linker-1 (GPAGMKGL, SEQ
ID NO: 196), Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or Linker-3
(ALFKSSFP, SEQ ID NO: 198) are not processed by healthy lung
fibroblasts.
[0033] FIGS. 7A-7H is a series of graphs showing activity of
exemplary IL-2 fusion proteins in IL-2 dependent cytotoxic T
lymphocyte cell line CTLL-2. Each graph shows results of the IL-2
proliferation assay as quantified by CellTiter-Glo.RTM. (Promega)
luminescence-based cell viability assay. Each proliferation assay
was performed with HSA (FIGS. 7B, 7D, 7F, 7H) or without (FIGS. 7A,
7C, 7E, 7G). Each fusion protein comprises an anti-HSA binder, and
both uncleaved and MMP9 protease cleaved versions of the fusion
protein were used in each assay.
[0034] FIGS. 8A-8F is a series of graphs showing activity of
exemplary IL-2 fusion proteins in IL-2 dependent cytotoxic T
lymphocyte cell line CTLL-2. Each graph shows results of the IL-2
proliferation assay as quantified by CellTiter-Glo (Promega)
luminescence-based cell viability assay. Both uncleaved and MMP9
protease cleaved versions of the fusion protein were used in each
assay.
[0035] FIGS. 9A-9Z is a series of graphs showing activity of
exemplary IL-2 fusion proteins in IL-2 dependent cytotoxic T
lymphocyte cell line CTLL-2. Each graph shows results of the IL-2
proliferation assay as quantified by CellTiter-Glo (Promega)
luminescence-based cell viability assay. Both uncleaved and MMP9
protease cleaved versions of the fusion protein were used in each
assay.
[0036] FIG. 10 shows results of protein cleavage assay. Fusion
protein ACP16 was run on an SDS-PAGE gel in both cleaved and
uncleaved form. As can be seen in the gel, cleavage was
complete.
[0037] FIGS. 11A-11B are a series of graphs depicting results from
a HEK-Blue IL-12 reporter assay performed on human p40/murine p35
IL12 fusion proteins and recombinant human IL12 (Rec hIL-12).
Analysis was performed based on quantification of Secreted Alkaline
Phosphatase (SEAP) activity using the reagent QUANTI-Blue.RTM.
(InvivoGen). Results confirm that IL12 protein fusion proteins are
active.
[0038] FIGS. 12A-12F show a series of graphs depicting the results
of HEK-blue assay of four IL-12 fusion proteins, before and after
cleavage by MMP9. Analysis was performed based on quantification of
Secreted Alkaline Phosphatase (SEAP) activity using the reagent
QUANTI-Blue (InvivoGen). The data show greater activity in the
cleaved IL12 than in the full fusion protein. Constructs tested
were ACP06 (FIG. 12A), ACP07 (FIG. 12C), ACP08 (FIG. 12B), ACP09
(FIG. 12D), ACP10 (FIG. 12E), ACP11 (FIG. 12F)
[0039] FIG. 13 shows results of protein cleavage assay. Fusion
protein ACP11 was run on an SDS-PAGE gel in both cleaved and
uncleaved form. As can be seen in the gel, cleavage was
complete.
[0040] FIG. 14 is a schematic which depicts a non-limiting example
of an inducible cytokine protein, wherein the construct is
activated upon protease cleavage of a linker attached between two
subunits of the cytokine.
[0041] FIGS. 15A-15D are graphs depicting results from a HEK-Blue
assay performed on human p40/murine p35 IL12 fusion proteins and
recombinant human IL12 (Rec hIL-12). Results confirm that IL12
protein fusion proteins are active. Each proliferation assay was
performed with HSA or without HSA.
[0042] FIGS. 16A-16F are a series of graphs showing activity of
exemplary IFN.gamma. fusion proteins compared to activity of mouse
IFN.gamma. control using WEHI 279 cell survival assay. Each assay
was performed with medium containing HSA (+HSA) or not containing
HSA (-HSA). Each fusion protein comprises an anti-HSA binder, and
both uncleaved and MMP9 protease cleaved versions of the fusion
protein were used in each assay.
[0043] FIGS. 17A-17F are a series of graphs showing activity of
exemplary IFN.gamma. fusion proteins compared to activity of mouse
IFN.gamma. control using B16 reporter assay. Each assay was
performed with medium containing HSA (+HSA) or not containing HSA
(-HSA). Each fusion protein comprises an anti-HSA binder, and both
uncleaved and MMP9 protease cleaved versions of the fusion protein
were used in each assay.
[0044] FIGS. 18A-18B shows results of protein cleavage assay. Two
constructs, ACP31 (IFN-a fusion protein; FIG. 18A) and ACP55
(IFN-.gamma. fusion protein; 18B), were run on an SDS-PAGE gel in
both cleaved and uncleaved form. As can be seen in the gel,
cleavage was complete.
[0045] FIGS. 19A-19B are a series of graphs showing activity of
exemplary IFN fusion proteins compared to activity of mouse
IFN.gamma. control using B16 reporter assay. Each assay was
performed with culture medium containing HSA, and each fusion
protein comprises an anti-HSA binder. Both uncleaved and MMP9
protease cleaved versions of the fusion protein were used in each
assay.
[0046] FIGS. 20A-20B are a series of graphs showing activity of
exemplary IFN.alpha. fusion proteins compared to activity of mouse
IFNalphaA control using a B16 reporter assay. Each assay was
performed with medium containing HSA, and each fusion protein
comprises an anti-HSA binder. Both uncleaved and MMP9 protease
cleaved versions of the fusion protein were used in each assay.
[0047] FIGS. 21A-21D are a series of graphs depicting the results
of tumor growth studies using the MC38 cell line. FIG. 21A-21C show
the effect of IFN.gamma. and IFN.gamma. fusion proteins on tumor
growth when injected intraperitoneally (IP) using different dosing
levels and schedules (.mu.g=micrograms, BID=twice daily, BIW=twice
weekly, QW=weekly). FIG. 21D shows the effect of intratumoral (IT)
injection of IFN.gamma. and IL-2 on tumor growth.
[0048] FIGS. 22A-22B are a series of graphs showing activity of
exemplary IFN.gamma. fusion proteins (ACP51 and ACP52) cleaved by
MMP9 protease compared to activity of uncleaved fusion proteins
using B16 reporter assay. Each fusion protein comprises an anti-HSA
binder and a tumor targeting domain.
[0049] FIGS. 23A-23B are a series of graphs showing activity of
exemplary IFN.gamma. fusion proteins (ACP53 and ACP54) cleaved by
MMP9 protease compared to activity of uncleaved fusion proteins
using B16 reporter assay. Each fusion protein comprises IFN.gamma.
directly fused to albumin.
[0050] FIGS. 24A-24B are two graphs showing the stability of IL-2
fusion proteins containing Linker-1 (GPAGMKGL, SEQ ID NO: 196),
Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID
NO: 198) in human serum in normal patient and a cancer patient.
FIG. 24A depicts the stability of the IL-2 fusion proteins
containing Linker-1 (GPAGMKGL, SEQ ID NO: 196), Linker-2 (GPAGLYAQ,
SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID NO: 198) at 24
hours. FIG. 24B depicts the stability of the IL-2 fusion proteins
containing Linker-1 (GPAGMKGL, SEQ ID NO: 196), Linker-2 (GPAGLYAQ,
SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID NO: 198) at 72
hours.
[0051] FIGS. 25A and 25B are two graphs showing analysis of ACP16
(FIG. 25A) and ACP124 (FIG. 25B) in a HEKBlue IL-2 reporter assay
in the presence of HSA. Circles depict the activity of the uncut
polypeptide, squares depict activity of the cut polypeptide, and
triangles depict IL-2 alone as a control. FIG. 25C is a graph
showing results of a CTLL-2 proliferation assay. CTLL2 cells (ATCC)
were plated in suspension at a concentration of 500,000 cells/well
in culture media with or without 40 mg/ml human serum albumin (HSA)
and stimulated with a dilution series of recombinant hIL2 or
activatable hIL2 for 72 hours at 37.degree. C. and 5% CO.sub.2.
Activity of uncleaved and cleaved activatable ACP16 was tested.
Cleaved activatable hIL2 was generated by incubation with active
MMP9 Cell activity was assessed using a CellTiter-Glo (Promega)
luminescence-based cell viability assay. Triangles show wile-type
cytokine, circles depict intact fusion protein, and squares depict
protease-cleaved fusion protein.
[0052] FIGS. 26A-26C are a series of graphs showing activity of
fusion proteins in an HEKBlue IL-12 reporter assay. FIG. 26A is a
graph showing activity of cut and uncut ACP11 (a human p40/murine
p35 IL12 fusion protein). FIG. 26B is a graph showing analysis of
ACP91 (a chimeric IL-12 fusion protein). Squares depict activity of
the uncut ACP91 polypeptide, and triangles depict the activity of
the cut polypeptide (ACP91+MMP9). EC50 values for each are shown in
the table. FIG. 26C is a graph showing analysis of ACP136 (a
chimeric IL-12 fusion protein). Squares depict activity of the
uncut ACP136 polypeptide, and triangles depict the activity of the
cut polypeptide (ACP136+MMP9). EC50 values for each are shown in
the table insert.
[0053] FIGS. 27A-27F are a series of graphs showing that cleaved
IL-12 polypeptides are active in a HEKBlue IL2 reporter assay.
Fusion proteins are evaluated both uncut (circles) and cut
(squares) form, and wild type IL2 is used as a control +HSA for
FIGS. 27A-C; ACP131 is used as a control (triangles) for FIGS.
27D-27F. Shown are data for APC31+HSA (FIG. 27A), ACP125+HSA (FIG.
27B), ACP126+HSA (FIG. 27C), ACP127 (FIG. 27D), ACP128 (FIG. 27E),
and ACP129 (FIG. 27F). The EC50 values for each are shown in the
table below each graph.
[0054] FIGS. 28A-28N are a series of graphs depicting the activity
of APC56 (FIG. 28A), APC57 (FIG. 28B) APC58 (FIG. 28C), APC59 (FIG.
28D), APC60 (FIG. 28E), APC61+HSA (FIG. 28F), ACP30+HSA (FIG. 28G),
ACP73 (FIG. 28H), ACP70+HSA (FIG. 28I), ACP71 (FIG. 28J), ACP72
(FIG. 28K), ACP 73 (FIG. 28L), ACP74 (FIG. 28M), and ACP75 (FIG.
28N) in a HEKBlue IFN.alpha. reporter assay. Each fusion was tested
for its activity when cut (squares) and uncut (circles). Analysis
of murine IFN.gamma. is included in each graph as a comparator.
[0055] FIGS. 29A-29B is two graphs showing results of analyzing
ACP31 (mouse IFN.alpha.1 fusion protein) and ACP11 (a human
p40/murine p35 IL12 fusion protein) in a tumor xenograft model.
FIG. 29A shows tumor volume over time in mice treated with 33 .mu.g
ACP31 (circles), 110 .mu.g ACP31 (triangles), 330 .mu.g ACP31
(diamonds), and as controls 1 .mu.g murine wild type IFN.alpha.1
(dashed line, squares) and 10 .mu.g mIFN.alpha.1 (dashed line,
small circles). Vehicle alone is indicated by large open circles.
The data show tumor volume decreasing over time in a dose-dependent
manner in mice treated with ACP31. FIG. 29B shows tumor volume over
time in mice treated with 17.5 .mu.g ACP11 (squares), 175 .mu.g
ACP31 (triangles), 525 .mu.g ACP31 (circles), and as controls 2
.mu.g ACP04 (dashed line, triangles) and 10 .mu.g ACP04 (dashed
line, diamonds). Vehicle alone is indicated by large open circles.
The data show tumor volume decreasing over time in a dose-dependent
manner in mice treated with both ACP11 and ACP04 (a human
p40/murine p35 IL12 fusion protein).
[0056] FIGS. 30A-30F are a series of spaghetti plots showing tumor
volume over time in a mouse xenograft tumor model in mice each
treated with vehicle alone (FIG. 30A), 2 .mu.g ACP04 (FIG. 30B), 10
.mu.g ACP04 (FIG. 30C), 17.5 .mu.g ACP11 (FIG. 30D), 175 .mu.g
ACP11 (FIG. 30E), and 525 .mu.g ACP11 (FIG. 30F). Each line
represents a single mouse.
[0057] FIGS. 31A-31C depicts three graphs showing results of
analyzing ACP16 and ACP124 in a tumor xenograft model. FIG. 31A
shows tumor volume over time in mice treated with 4.4 .mu.g ACP16
(squares), 17 .mu.g ACP16 (triangles), 70 .mu.g ACP16 (downward
triangles), 232 .mu.g ACP16 (dark circles), and as a comparator 12
.mu.g wild type IL-2 (dashed line, triangles) and 36 .mu.g wild
type IL-2 (dashed line, diamonds. Vehicle alone is indicated by
large open circles. The data show tumor volume decreasing over time
in a dose-dependent manner in mice treated with ACP16 at higher
concentrations. FIG. 31B shows tumor volume over time in mice
treated with 17 .mu.g ACP124 (squares), 70 .mu.g ACP124
(triangles), 230 .mu.g ACP124 (downward triangles), and 700 .mu.g
ACP124. Vehicle alone is indicated by large open circles. FIG. 31C
shows tumor volume over time in mice treated with 17 .mu.g ACP16
(triangles), 70 .mu.g ACP16 (circles), 232 .mu.g ACP16 (dark
circles), and as a comparator 17 .mu.g ACP124 (dashed line,
triangles) 70 .mu.g ACP124 (dashed line, diamonds), 230 .mu.g
ACP124 (dashed line, diamonds). Vehicle alone is indicated by dark
downward triangles. The data show tumor volume decreasing over time
in a dose-dependent manner in mice treated with ACP16, but not
ACP124.
[0058] FIGS. 32A-32C are a series of spaghetti plots showing
activity of fusion proteins in an MC38 mouse xenograft model. Each
line in the plots is a single mouse.
[0059] FIG. 33 is a graph showing tumor volume over time in a mouse
xenograft model showing tumor growth in control mice (open circles)
and AP16-treated mice (squares).
[0060] FIGS. 34A-34D are a series of survival plots showing
survival of mice over time after treatment with cleavable fusion
proteins. FIG. 34A shows data for mice treated with vehicle alone
(gray line), 17 .mu.g ACP16 (dark line), and 11 .mu.g ACP124
(dashed line). FIG. 34B shows data for mice treated with vehicle
alone (gray line), 70 .mu.g ACP16 (dark line), and 70 .mu.g ACP124
(dashed line). FIG. 34C shows data for mice treated with vehicle
alone (gray line), 232 .mu.g ACP16 (dark line), and 230 .mu.g
ACP124 (dashed line). FIG. 34D shows data for mice treated with
vehicle alone (gray line), 232 .mu.g ACP16 (dark line), and 700
.mu.g ACP124 (dashed line).
[0061] FIG. 35 a series of spaghetti plots showing activity of
fusion proteins in an MC38 mouse xenograft model. All mouse groups
were given four doses total except for the highest three doses of
APC132, wherein fatal toxicity was detected after 1 week/2 doses.
Shown are vehicle alone (top), 17, 55, 70, and 230 .mu.g ACP16 (top
full row), 9, 28, 36, and 119 .mu.g ACP132 (middle full row), and
13, 42, 54, and 177 .mu.g ACP21 (bottom full row). Each line in the
plots represents an individual animal.
[0062] FIG. 36 is a schematic illustrating a substrate cleavage
activity in conditioned complete (+FBS) media by FRET endpoint
assay across four cell lines. The ratio of tumor vs control
activity was approximated by averaging the three tumor cell lines
and comparing to the control myofibroblast cell line where signal
was detectable. FIG. 36 discloses SEQ ID NOs: 201, 198, 197, 196
and 195, respectively, in order of appearance.
[0063] FIG. 37 is a schematic illustrating ADAM17_2 substrate
kinetics in cell culture conditioned media. FIG. 37 discloses SEQ
ID NO: 235.
[0064] FIG. 38 is a schematic illustrating FAP.alpha._1 substrate
kinetics in conditioned media. FIG. 38 discloses SEQ ID NO:
197.
[0065] FIG. 39 is a schematic illustrating FAP.alpha._1 substrate
kinetics in cell lysates. FIG. 39 discloses SEQ ID NO: 197.
[0066] FIG. 40 is a schematic illustrating MMP9_1 substrate
kinetics in cell lysates. FIG. 40 discloses SEQ ID NO: 196.
[0067] FIG. 41 is a schematic illustrating a substrate cleavage
activity in cell lysates by FRET endpoint assay. FIG. 41 discloses
SEQ ID NOS 198 and 197, respectively, in order of appearance.
[0068] FIG. 42 is a schematic illustrating CTSL1_1 substrate
kinetics in cell lysates. FIG. 42 discloses SEQ ID NO: 198.
[0069] FIG. 43 is a schematic illustrating MMP14_1 substrate
kinetics in cell lysates. FIG. 43 discloses SEQ ID NO: 195.
[0070] FIG. 44 is a schematic illustrating calculated concentration
of enzyme equivalents per cell culture-derived sample. FIG. 44
discloses SEQ ID NOS 201, 198, 197, 197, 196 and 195, respectively,
in order of appearance.
[0071] FIG. 45 is a schematic illustrating an enzyme progress curve
for CTSL1 cleavage of CSTL1_2 vs CTSL1_1. FIG. 45 discloses SEQ ID
NOS 198, 199 and 236, respectively, in order of appearance.
[0072] FIG. 46 is a schematic illustrating 30-mer cleavage of
CTSL1_1 (ALFKSSFP, SEQ ID NO: 198) vs CTSL1_2 (ALFFSSPP, SEQ ID NO:
199).
[0073] FIG. 47 is a schematic illustrating susceptibility of the
CTSL1 FRET substrates to CTSK cleavage. Rates of product formation
were measured as a specific activity in units pmol min.sup.-1
.mu.g.sup.-1. The threshold value for the reference substrate,
Z-LR-AMC is shown as a dashed line. FIG. 47 discloses SEQ ID NOS
198 and 199, respectively, in order of appearance.
[0074] FIG. 48 is a schematic illustrating 30-mer Substrate
Degradation by MMP9. Ranking of the substrates by relative rates of
degradation are shown with "+"; uncleaved substrates are indicated
as "-". FIG. 48 discloses SEQ ID NOS 204, 205, 214, 216, 202, 217,
203, 211, 219, 207, 215, 212, 213, 206, 208, 209, 210, 218 and 220,
respectively, in order of appearance.
[0075] FIG. 49 is a schematic illustrating tandem MMP14_1 Motif
degradation by MMP9. Top: substrate degradation traces, modeled
with first-order kinetics. Bottom: product formation traces showing
complex kinetics. FIG. 49 discloses SEQ ID NOS 202-205,
respectively, in order of appearance.
[0076] FIG. 50 is a schematic illustrating 30-mer Substrate
Degradation by FAP.alpha.. Ranking of the substrates by relative
rates of degradation are shown with "+"; uncleaved substrates are
indicated as "-". FIG. 50 discloses SEQ ID NOS 205, 204, 206, 217,
203, 218, 219, 213, 216, 207, 214, 210, 202, 211, 208, 209, 212,
215 and 220, respectively, in order of appearance.
[0077] FIG. 51 is a schematic illustrating 30-mer Substrate
Degradation by CTSL1. Ranking of the substrates by relative rates
of degradation are shown with "+"; uncleaved substrates are
indicated as "-". FIG. 51 discloses SEQ ID NOS 207, 208, 202, 218,
219, 212, 215, 217, 211, 209, 214, 206, 213, 210, 216, 203, 204,
205 and 220, respectively, in order of appearance.
[0078] FIG. 52 is a schematic illustrating 30-mer Substrate
Degradation by ADAM17. Ranking of the substrates by relative rates
of degradation are shown with "+"; uncleaved substrates are
indicated as "-". FIG. 52 discloses SEQ ID NOS 208, 209, 211, 214,
217, 219, 213, 218, 215, 210, 212, 216, 207, 206, 202, 203, 204,
205 and 220, respectively, in order of appearance.
[0079] FIG. 53 is a schematic illustrating 30-mer Substrate
Degradation by Factor Xa. Ranking of the substrates by relative
rates of degradation are shown with "+"; uncleaved substrates are
indicated as "-". FIG. 53 discloses SEQ ID NOS 220, 206, 202, 214,
208, 209, 215, 210, 218, 217, 207, 213, 216, 211, 212, 219, 203,
204 and 205, respectively, in order of appearance.
[0080] FIG. 54 is a schematic illustrating 30-mer Substrate
Degradation by Thrombin. Ranking of the substrates by relative
rates of degradation are shown with "+"; uncleaved substrates are
indicated as "-". FIG. 54 discloses SEQ ID NOS 220, 204, 202, 207,
205, 211, 212, 215, 209, 218, 219, 217, 210, 213, 216, 214, 208,
206 and 203, respectively, in order of appearance.
[0081] FIG. 55 is a schematic illustrating 30-mer Substrate
Degradation by hepsin. Ranking of the substrates by relative rates
of degradation are shown with "+"; uncleaved substrates are
indicated as "-". FIG. 55 discloses SEQ ID NOS 220, 209, 216, 215,
210, 213, 206, 214, 212, 207, 217, 208, 211, 218, 219, 202, 203,
204 and 205, respectively, in order of appearance.
[0082] FIGS. 56A-56C show western blots probed with an IL-2
antibody demonstrating the stability of ACP16 (FIG. 56A), ACP153
(FIG. 56B), and ACP157 (FIG. 56C) in 90% serum. Serum was pooled
from three human donors. Constructs of interest were incubated with
PBS, Serum, or MMP9 protease and cleavage was assessed at T=0 hours
and at T=24 hours.
[0083] FIG. 57A-57B show western blots probed with an IL-2 antibody
demonstrating the stability of ACP153, ACP155, ACP156, ACP16, and
ACP372 in 90% serum. Serum was pooled from three human donors.
Constructs of interest were incubated with PBS, Serum, or MMP9
protease and cleavage was assessed at T=24 hours and at T=72 hours.
FIG. 57A shows the result using serum from a human donor and FIG.
57B shows the result using serum from a mouse donor.
[0084] FIGS. 58A-58D show a series of spaghetti plots showing
activity of fusion proteins in an MC38 mouse xenograft model. Shown
are vehicle alone (FIG. 58A, top), 17, 55, and 230 .mu.g ACP16
(FIG. 58A), 55 and 230 .mu.g ACP153 (FIG. 58B), 55 and 230 .mu.g
ACP155 (FIG. 58C), and 55 and 230 .mu.g ACP156 (FIG. 58D). Each
line in the plots represents an individual animal.
[0085] FIG. 59 shows a graph depicting results from a STAT
activation reporter assay performed on IL-2 fusion proteins and
recombinant human IL2 (Rec hIL-2). Analysis was performed based on
quantification of Secreted Alkaline Phosphatase (SEAP) activity
using the reagent QUANTI-Blue (InvivoGen).
[0086] FIG. 60 shows a graph depicting results from a STAT
activation reporter assay performed on IL-2 fusion proteins and
recombinant human IL2 (Rec hIL-2). Analysis was performed based on
quantification of Secreted Alkaline Phosphatase (SEAP) activity
using the reagent QUANTI-Blue (InvivoGen).
[0087] FIG. 61 shows a graph depicting results from a STAT
activation reporter assay performed on IL-2 fusion proteins and
recombinant human IL2 (Rec hIL-2). Analysis was performed based on
quantification of Secreted Alkaline Phosphatase (SEAP) activity
using the reagent QUANTI-Blue (InvivoGen).
[0088] FIG. 62 shows a graph depicting results from a STAT
activation reporter assay performed on IL-2 fusion proteins and
recombinant human IL2 (Rec hIL-2). Analysis was performed based on
quantification of Secreted Alkaline Phosphatase (SEAP) activity
using the reagent QUANTI-Blue (InvivoGen).
[0089] FIG. 63 shows a table reporting engineered cleavage
substrates described herein and the extent of cleavage observed
using relevant proteases. Flanking sequences are lower case, the
first cleavable sequence is underlined, the second is in bold font,
and the third cleavable sequence is in italics. In some cases,
there is overlap between cleavable sequences, which are indicated
accordingly. FIG. 63 discloses SEQ ID NOS 202-220, respectively, in
order of appearance.
[0090] FIG. 64 is a schematic of an inducible tetravalent antibody
format.
[0091] FIG. 65A-65B show that multivalent 4-1BB antibodies are able
to inducibly agonize 4-1BB.
5. DETAILED DESCRIPTION
[0092] This disclosure relates to novel separation moieties or
linkers and to polypeptides, such as fusion proteins, that contain
the linkers. The linkers are preferably protease cleavable and link
a first amino acid sequence of interest (e.g. a first domain of
interest) to a second amino acid sequence of interest (e.g. a
second domain of interest).
[0093] The disclosed separation moieties confer site-selectivity
with regard to the action of the attached payload or payloads. The
payload can be a therapeutic agent, a half-life extender, a
blocking agent and the like, or any combination thereof. The
separation moieties may be used to attach any payload of interest,
including e.g. cytokines, antibodies, cell-based therapies, etc.
The separation moieties may be used individually or be used in
tandem, triplicate, quadruplicate, and so forth, as long as the
separation moiety is smaller than about 100 amino acids. Individual
separation moieties may be directly joined to each other, or may be
interspersed with non-cleavable linkers, whichever promotes high
efficiency and site-specificity.
[0094] The various embodiments of the present disclosure are
further described in detail in the paragraphs below.
[0095] Unless otherwise defined, all terms of art, notations and
other scientific terminology used herein are intended to have the
meanings commonly understood by those of skill in the art to which
this invention pertains. In some cases, terms with commonly
understood meanings are defined herein for clarity and/or for ready
reference, and the inclusion of such definitions herein should not
necessarily be construed to represent a difference over what is
generally understood in the art. The techniques and procedures
described or referenced herein are generally well understood and
commonly employed using conventional methodologies by those skilled
in the art, such as, for example, the widely utilized molecular
cloning methodologies described in Sambrook et al., Molecular
Cloning: A Laboratory Manual 4th ed. (2012) Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,
procedures involving the use of commercially available kits and
reagents are generally carried out in accordance with
manufacturer-defined protocols and conditions unless otherwise
noted.
[0096] "Cytokine" is a well-known term of art that refers to any of
a class of immunoregulatory proteins (such as interleukin or
interferon) that are secreted by cells especially of the immune
system and that are modulators of the immune system. Cytokine
polypeptides that can be used in the fusion proteins disclosed
herein include, but are not limited to transforming growth factors,
such as TGF-.alpha. and TGF-.beta. (e.g., TGFbeta1, TGFbeta2,
TGFbeta3); interferons, such as interferon-.alpha.,
interferon-.beta., interferon-.gamma., interferon-kappa and
interferon-omega; interleukins, such as IL-1, IL-1.alpha., 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-21 and IL-25; tumor
necrosis factors, such as tumor necrosis factor alpha and
lymphotoxin; transforming growth factor beta (TGFbeta) family
proteins, chemokines (e.g., C-X-C motif chemokine 10 (CXCL10),
CCL19, CCL20, CCL21), and granulocyte macrophage-colony stimulating
factor (GM-CS), as well as fragments of such polypeptides that
active the cognate receptors for the cytokine (i.e., functional
fragments of the foregoing). "Chemokine" is a term of art that
refers to any of a family of small cytokines with the ability to
induce directed chemotaxis in nearby responsive cells.
[0097] Cytokines are well-known to have short serum half-lives that
frequently are only a few minutes. Even forms of cytokines that
have altered amino acid sequences intended to extend the serum
half-life yet retain receptor agonist activity typically also have
short serum half-lives. As used herein, a "short-half-life
cytokine" refers to a cytokine that has a substantially brief
half-life circulating in the serum of a subject, such as a serum
half-life that is less than 10, less than 15, less than 30, less
than 60, less than 90, less than 120, less than 240, or less than
480 minutes. As used herein, a short half-life cytokine includes
cytokines which have not been modified in their sequence to achieve
a longer than usual half-life in the body of a subject and
polypeptides that have altered amino acid sequences intended to
extend the serum half-life yet retain receptor agonist activity.
This latter case is not meant to include the addition of
heterologous protein domains, such as a bona fide half-life
extension element, such as serum albumin.
[0098] A "conservative" amino acid substitution, as used herein,
generally refers to substitution of one amino acid residue with
another amino acid residue from within a recognized group which can
change the structure of the peptide but biological activity of the
peptide is substantially retained. Conservative substitutions of
amino acids are known to those skilled in the art. Conservative
substitutions of amino acids can include, but not limited to,
substitutions made amongst amino acids within the following groups:
(a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f)
Q, N; and (g) E, D. For instance, a person of ordinary skill in the
art reasonably expect that an isolated replacement of a leucine
with an isoleucine or valine, an aspartate with a glutamate, a
threonine with a serine, or a similar replacement of an amino acid
with a structurally related amino acid will not have a major effect
on the biological activity of the resulting molecule.
[0099] "Sortases" are transpeptidases that modify proteins by
recognizing and cleaving a carboxyl-terminal sorting signal
embedded in or terminally attached to a target protein or peptide.
Sortase A catalyzes the cleavage of the LPXTG motif (where X is any
standard amino acid) (SEQ ID NO: 237) between the Thr and Gly
residue on the target protein, with transient attachment of the Thr
residue to the active site Cys residue on the enzyme, forming an
enzyme-thioacyl intermediate. To complete transpeptidation and
create the peptide-monomer conjugate, a biomolecule with an
N-terminal nucleophilic group, typically an oligoglycine motif,
attacks the intermediate, displacing Sortase A and joining the two
molecules.
[0100] As used herein, the term "steric blocker" refers to a
polypeptide or polypeptide moiety that can be covalently bonded to
a cytokine polypeptide directly or indirectly through other
moieties such as linkers, for example in the form of a chimeric
polypeptide (fusion protein), but otherwise does not covalently
bond to the cytokine polypeptide. A steric blocker can
non-covalently bond to the cytokine polypeptide, for example though
electrostatic, hydrophobic, ionic or hydrogen bonding. A steric
blocker typically inhibits or blocks the activity of the cytokine
moiety due to its proximity to the cytokine moiety and comparative
size.
[0101] As used and described herein, a "half-life extension
element" is apart of the chimeric polypeptide that increases the
serum half-life and improve pK, for example, by altering its size
(e.g., to be above the kidney filtration cutoff), shape,
hydrodynamic radius, charge, or parameters of absorption,
biodistribution, metabolism, and elimination.
[0102] The term "separation moiety" or "linker" as used herein
refers to an amino acid sequence typically less than about 100
amino acids that connects or links a first amino acid sequence of
interest (e.g., an amino acid sequence that folds to form a first
protein domain) to a second amino acid sequence of interest (e.g.,
an amino acid sequence that folds to form a second protein domain)
in a contiguous polypeptide chain. The separation moiety or linker
typically include one or more protease cleavage sites and thus is
protease cleavable. A "tandem linker" refers to a linker that
comprises two or more protease cleavages sites which can be cleaved
by the same or different proteases, and which can be arranged in
any desired orientation, such as one cleavage site adjacent to
another cleavage site, one cleavage site overlapping another
cleavage site, one cleavage site following by another cleavage site
with intervening amino acids between the two cleavage sites.
[0103] As used herein, the terms "activatable," "activate,"
"induce," and "inducible" refer to the ability of a protein, i.e. a
cytokine, that is part of a conjugate, to bind its receptor and
effectuate activity upon cleavage of additional elements from the
conjugate.
[0104] As used herein, "plasmids" or "viral vectors" are agents
that transport the disclosed nucleic acids into the cell without
degradation and include a promoter yielding expression of the
nucleic acid molecule and/or polypeptide in the cells into which it
is delivered.
[0105] As used herein, the terms "peptide", "polypeptide", or
"protein" are used broadly to mean two or more amino acids linked
by a peptide bond. Protein, peptide, and polypeptide are also used
herein interchangeably to refer to amino acid sequences. It should
be recognized that the term polypeptide is not used herein to
suggest a particular size or number of amino acids comprising the
molecule and that a peptide of the invention can contain up to
several amino acid residues or more.
[0106] As used throughout, "subject" can be a vertebrate, more
specifically a mammal (e.g. a human, horse, cat, dog, cow, pig,
sheep, goat, mouse, rabbit, rat, and guinea pig), birds, reptiles,
amphibians, fish, and any other animal. The term does not denote a
particular age or sex. Tus, adult and newborn subjects, whether
male or female, are intended to be covered.
[0107] As used herein, "patient" or "subject" may be used
interchangeably and can refer to a subject with a disease or
disorder (e.g. cancer). The term patient or subject includes human
and veterinary subjects.
[0108] As used herein the terms "treatment", "treat", "treating,"
or grammatically related terms refer to a method of reducing the
effects of a disease or condition or symptom of the disease or
condition. Thus, in the disclosed method, treatment can refer to at
least about 10%, at least about 20%, at least about 30%, at least
about 40%, at least about 50%, at least about 60%, at least about
70%, at least about 80%, at least about 90%, or substantially
complete reduction in the severity of an established disease or
condition or symptom of the disease or condition. For example, a
method for treating a disease is considered to be a treatment if
there is a 10% reduction in one or more symptoms of the disease in
a subject as compared to a control. Thus, the reduction can be a
10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent
reduction in between 10% and 100% as compared to native or control
levels. It is well understood in the art that treatment does not
necessarily refer to a cure or complete ablation of the disease,
condition, or symptoms of the disease or condition. Desirable
effects of treatment include, but are not limited to, preventing
occurrence or recurrence of disease, alleviation of symptoms,
diminishment of any direct or indirect pathological consequences of
the disease, preventing metastasis, decreasing the rate of disease
progression, amelioration or palliation of the disease state, and
remission or improved prognosis.
[0109] As used herein, the terms "prevent", "preventing", and
"prevention" of a disease or disorder refers to an action, for
example, administration of the chimeric polypeptide or nucleic acid
sequence encoding the chimeric polypeptide, that occurs before or
at about the same time a subject begins to show one or more
symptoms of the disease or disorder, which inhibits or delays onset
or exacerbation of one or more symptoms of the disease or
disorder.
[0110] As used herein, references to "decreasing", "reducing", or
"inhibiting" include a change of at least about 10%, of at least
about 20%, of at least about 30%, of at least about 40%, of at
least about 50%, of at least about 60%, of at least about 70%, of
at least about 80%, of at least about 90% or greater as compared to
a suitable control level. Such terms can include but do not
necessarily include complete elimination of a function or property,
such as agonist activity.
[0111] An "attenuated cytokine receptor agonist" is a cytokine
receptor agonist that has decreased receptor agonist activity as
compared to the cytokine receptor's naturally occurring agonist. An
attenuated cytokine agonist may have at least about 10.times., at
least about 50.times., at least about 100.times., at least about
250.times., at least about 500.times., at least about 1000.times.
or less agonist activity as compared to the receptor's naturally
occurring agonist. When a fusion protein that contains a cytokine
polypeptide as described herein is described as "attenuated" or
having "attenuated activity", it is meant that the fusion protein
is an attenuated cytokine receptor agonist.
[0112] An "intact fusion protein" is a fusion protein in which no
domain has been removed from the fusion protein, for example by
protease cleavage. A domain may be removable by protease cleavage
or other enzymatic activity, but when the fusion protein is
"intact", this has not occurred.
[0113] As used herein "moiety" refers to a portion of a molecule
that has a distinct function within that molecule, and that
function may be performed by that moiety in the context of another
molecule. A moiety may be a chemical entity with a particular
function, or a portion of a biological molecule with a particular
function. For example, a "blocking moiety" within a fusion protein
is a portion of the fusion protein which is capable of blocking the
activity of some or all of the fusion polypeptide. his may be a
protein domain, such as serum albumin.
[0114] A. Separation Moiety or Linker
[0115] The disclosure relates to novel protease cleavable
separation moieties. As described herein, the protease cleavable
separation moieties were designed so that the separation moieties
are cleaved with high efficiency by proteases at a desired location
(e.g., proteases that are selectively expressed or expressed at
high levels in the tumor microenvironment) but are stable and not
cleaved or cleaved with low efficiency in other locations (e.g., in
the periphery, for example healthy tissue or serum).
[0116] The protease cleavable separation moieties were designed
using a process that included prioritizing proteases that would be
suitable for cleaving the separation moieties based on expression
in target indications, such as, expression in particular types of
tumors (e.g., colon, lung, breast, melanoma). Multiple data sources
for increased expression or specific expression of proteases in the
target indications were used, including mRNA, proteomics and tissue
staining data. The proteases were also prioritized based on their
specific activity as well as intrinsic specificity, with high
specific activity and high intrinsic activity preferred. Stability
in the serum is an important design consideration, and to avoid
potential off-target cleavage of the separation moieties by serum
proteases, proteases that are not dependent on arginine in their
substrate were selected, since many off-target enzymes are active
towards arginine residues.
[0117] Starting sequences for the design process were selected
using a diverse peptide library as substrates for proteases with
mass spectrometric detection of protease cleaved products to
identify preferred sequence motifs for each candidate protease. For
selected initial motifs, a new peptide library that was tailored to
the preferred sequence motif for the candidate protease was
designed, created and analyzed. The peptide motifs were also
counter-screened for cleavage by the serum proteases thrombin and
Factor Xa as well as the liver/kidney protease hepsin. his process
yielded peptides containing sequence motifs that are cleaved by
certain tumor-associated proteases (e.g., Matrix Metaloprotease 9
(MMP9), MMP14 and/or Cathepsin L) with high efficiency, but are
stable (not cleaved or cleaved with low efficiency) in the serum or
normal healthy tissue (e.g., by thrombin, Factor Xa, hepsin and the
like). The separation moieties disclosed herein are efficient
cleavage by human tumors and minimal cleavage by normal tissues or
serum.
[0118] This disclosure relates to separation moieties or linkers
that connect a first amino acid sequence of interest (e.g. a first
domain of interest) to a second amino acid sequence of interest
(e.g. a second domain of interest). Typically, the first amino acid
sequence of interest and the second amino acid sequence of interest
are not found together in a naturally occurring protein. For
example, the separation moiety can connect or link a first domain
of interest to a second domain of interest in a fusion protein. The
separation moiety is an amino acid sequence that can be of any
suitable length, and preferably can be cleaved by a protease.
[0119] The separation moieties disclosed herein can confer
functionality, including flexibility as well as the ability to be
cleaved. Flexible linkers are usually applied when joined domains
requires a certain degree of movement or interaction. Cleavable
linkers are introduced to release free and functional domains in
vivo at a target site. The separation moieties disclosed herein
serve to connect at least two domains of interest. The separation
moieties can maintain cooperative inter-domain interactions or
preserving biological activity. The separation moieties can join
functional domains (e.g., a payload and half-life extension
element) that are released from the separation moiety at a target
site (e.g. a tumor microenvironment).
[0120] In a preferred embodiment, the separation moiety is
cleavable by a cleaving agent, e.g., an enzyme. Preferably, the
separation moiety comprises a protease cleavage site. In some
cases, the separation moiety comprises one or more cleavage sites.
The separation moiety can comprise a single protease cleavage site.
The separation moiety can also comprise 2 or more protease cleavage
sites. For example, 2 cleavage sites, 3 cleavage sites, 4, cleavage
sites, 5 cleavage sites, or more. In cases the separation moiety
comprises 2 or more protease cleavage sites, the cleavage sites can
be cleaved by the same protease or different proteases. A
separation moiety comprising two or more cleavage sites is referred
to as a "tandem linker." The two or more cleavage sites can be
arranged in any desired orientation, including, but not limited tom
one cleavage site adjacent to another cleavage site, one cleavage
site overlapping another cleavage site, or one cleavage site
following by another cleavage site with intervening amino acids
between the two cleavage sites.
[0121] Of particular interest in the present invention are disease
specific protease-cleavable linkers. Also preferred are
protease-cleavable linkers that are preferentially cleaved at a
desired location in the body, such as the tumor microenvironment,
relative to the peripheral circulation. For example, the rate at
which the protease-cleavable linker is cleaved in the tumor
microenvironment can be at least about 10 times, at least about 100
times, at least about 1000 times or at least about 10,000 times
faster in the desired location in the body, e.g., the tumor
microenvironment, in comparison to in the peripheral circulation
(e.g., in plasma).
[0122] Proteases known to be associated with diseased cells or
tissues include but are not limited to serine proteases, cysteine
proteases, aspartate proteases, threonine proteases, glutamic acid
proteases, metalloproteases, asparagine peptide lyases, serum
proteases, cathepsins, Cathepsin B, Cathepsin C, Cathepsin D,
Cathepsin E, Cathepsin G, Cathepsin K, Cathepsin L, kallikreins,
hK1, hK10, hK15, plasmin, collagenase, Type IV collagenase,
stromelysin, Factor Xa, chymotrypsin-like protease, trypsin-like
protease, elastase-like protease, subtilisin-like protease,
actinidain, bromelain, calpain, caspases, caspase-3, Mirl-CP,
papain, HIV-1 protease, HSV protease, CMV protease, chymosin,
renin, pepsin, matriptase, legumain, plasmepsin, nepenthesin,
metalloexopeptidases, metalloendopeptidases, matrix
metalloproteases (MMP), MMP1, MMP2, MMP3, MMP8, MMP9, MMP13, MMP11,
MMP14, urokinase plasminogen activator (uPA), enterokinase,
prostate-specific antigen (PSA, hK3), interleukin-1.beta.
converting enzyme, thrombin, FAP (FAP.alpha.), dipeptidyl
peptidase, meprins, granzymes and dipeptidyl peptidase IV
(DPPIV/CD26). Proteases capable of cleaving linker amino acid
sequences (which can be encoded by the chimeric nucleic acid
sequences provided herein) can, for example, be selected from the
group consisting of a prostate specific antigen (PSA), a matrix
metalloproteinase (MMP), an A Disintigrin and a Metalloproteinase
(ADAM), a plasminogen activator, a cathepsin, a caspase, a tumor
cell surface protease, and an elastase. The MMP can, for example,
be matrix metalloproteinase 2 (MMP2), matrix metalloproteinase 9
(MMP9), matrix metalloproteinase 14 (MMP14). In addition, or
alternatively, the linker can be cleaved by a cathepsin, such as,
Cathepsin B, Cathepsin C, Cathepsin D, Cathepsin E, Cathepsin G,
Cathepsin K and/or Cathepsin L. Preferably, the linker can be
cleaved by MMP14 or Cathepsin L.
[0123] Proteases useful for cleavage of linkers and for use in the
methods disclosed herein are presented in Table 1, and exemplary
proteases and their cleavage site are presented in Table 1a:
TABLE-US-00001 TABLE 1 Proteases relevant to inflammation and
cancer Protease Specificity Other aspects Secreted by killer T
cells: Granzyme B (grB) Cleaves after Asp Type of serine protease;
strongly residues (asp-ase) implicated in inducing
perforin-dependent target cell apoptosis Granzyme A (grA)
trypsin-like, cleaves after Type of serine protease; basic residues
Granzyme H (grH) Unknown substrate Type of serine protease;
specificity Other granzymes are also secreted by killer T cells,
but not all are present in humans Caspase-8 Cleaves after Asp Type
of cysteine protease; plays essential residues role in TCR-induced
cellular expansion- exact molecular role unclear Mucosa-associated
Cleaves after arginine Type of cysteine protease; likely acts both
lymphoid tissue residues as a scaffold and proteolytically active
(MALT1) enzyme in the CBM-dependent signaling pathway Tryptase
Targets: angiotensin I, Type of mast cell-specific serine protease;
fibrinogen, prourokinase, trypsin-like; resistant to inhibition by
TGF.beta.; preferentially macromolecular protease inhibitors
cleaves proteins after expressed in mammals due to their lysine or
arginine tetrameric structure, with all sites facing residues
narrow central pore; also associated with inflammation Associated
with inflammation: Thrombin Targets: FGF-2, Type of serine
protease; modulates HB-EGF, Osteo-pontin, activity of vascular
growth factors, PDGF, VEGF chemokines and extracellular proteins;
strengthens VEGF-induced proliferation; induces cell migration;
angiogenic factor; regulates hemostasis Chymase Exhibit
chymotrypsin- Type of mast cell-specific serine protease like
specificity, cleaving proteins after aromatic amino acid residues
Carboxypeptidase A Cleaves amino acid Type of zinc-dependent
metalloproteinase (MC-CPA) residues from C-terminal end of peptides
and proteins Kallikreins Targets: high molecular Type of serine
protease; modulate weight relaxation response; contribute to
kininogen, pro-urokinase inflammatory response; fibrin degradation
Elastase Targets: E-cadherin, GM- Type of neutrophil serine
protease; CSF, IL-1, IL-2, IL-6, degrades ECM components; regulates
IL8, p38.sup.MAPK, TNF.alpha., VE- inflammatory response; activates
pro- cadherin apoptotic signaling Cathepsin G Targets: EGF, ENA-78,
Type of serine protease; degrades ECM IL-8, MCP-1, MMP-2,
components; chemo-attractant of MT1-MMP, leukocytes; regulates
inflammatory PAI-1, RANTES, TGF.beta., response; promotes apoptosis
TNF.alpha. PR-3 Targets: ENA-78, IL-8, Type of serine protease;
promotes IL-18, JNK, p38.sup.MAPK, inflammatory response; activates
pro- TNF.alpha. apoptotic signaling Granzyme M (grM) Cleaves after
Met and Type of serine protease; only expressed in other long,
unbranched NK cells hydrophobic residues Calpains Cleave between
Arg and Family of cysteine proteases; calcium- Gly dependent;
activation is involved in the process of numerous inflammation-
associated diseases
TABLE-US-00002 TABLE 1a Exemplary Proteases and Protease
Recognition Sequences SEQ ID Protease Cleavage Domain Sequence NO:
MMP7 KRALGLPG 3 MMP7 (DE).sub.8RPLALWRS(DR).sub.8 4 MMP9
PR(S/T)(L/I)(S/T) 5 MMP9 LEATA 6 MMP11 GGAANLVRGG 7 MMP14
SGRIGFLRTA 8 MMP PLGLAG 9 MMP PLGLAX 10 MMP PLGC(me)AG 11 MMP
ESPAYYTA 12 MMP RLQLKL 13 MMP RLQLKAC 14 MMP2, MMP9, MMP14
EP(Cit)G(Hof)YL 15 Urokinase plasminogen SGRSA 16 activator (uPA)
Urokinase plasminogen DAFK 17 activator (uPA) Urokinase plasminogen
GGGRR 18 activator (uPA) Lysosomal Enzyme GFLG 19 Lysosomal Enzyme
ALAL 20 Lysosomal Enzyme FK 21 Cathepsin B NLL 22 Cathepsin D
PIC(Et)FF 23 Cathepsin K GGPRGLPG 24 Prostate Specific Antigen
HSSKLQ 25 Prostate Specific Antigen HSSKLQL 26 Prostate Specific
Antigen HSSKLQEDA 27 Herpes Simplex Virus Protease LVLASSSFGY 28
HIV Protease GVSQNYPIVG 29 CMV Protease GVVQASCRLA 30 Thrombin
F(Pip)RS 31 Thrombin DPRSFL 32 Thrombin PPRSFL 33 Caspase-3 DEVD 34
Caspase-3 DEVDP 35 Caspase-3 KGSGDVEG 36 Interleukin 1.beta.
converting enzyme GWEHDG 37 Enterokinase EDDDDKA 38 FAP KQEQNPGST
39 Kallikrein 2 GKAFRR 40 Plasmin DAFK 41 Plasmin DVLK 42 Plasmin
DAFK 43 TOP ALLLALL 44 GPLGVRG 221 IPVSLRSG 222 VPLSLYSG 223
SGESPAYYTA 224
[0124] Exemplary protease linkers include, but are not limited to
kallikrein cleavable linkers, thrombin cleavable linkers, chymase
cleavable linkers, carboxypeptidase A cleavable linkers, cathepsin
cleavable linkers, elastase cleavable linkers, FAP cleavable
linkers, ADAM cleavable linkers, PR-3 cleavable linkers, granzyme M
cleavable linkers, a calpain cleavable linkers, a matrix
metalloproteinase (MMP) cleavable linkers, a plasminogen activator
cleavable linkers, a caspase cleavable linkers, a tryptase
cleavable linkers, or a tumor cell surface protease. Specifically,
MMP9 cleavable linkers, ADAM cleavable linkers, CTSL1 cleavable
linkers, FAP.alpha. cleavable linkers, and cathepsin cleavable
linkers. Some preferred protease-cleavable linkers are cleaved by a
MMP and/or a cathepsin.
[0125] The separation moieties disclosed herein are typically less
than 100 amino acids. Such separation moieties can be of different
lengths, such as from 1 amino acid (e.g., Gly) to 30 amino acids,
from 1 amino acid to 40 amino acids, from 1 amino acid to 50 amino
acids, from 1 amino acid to 60 amino acids, from 1 to 70 amino
acids, from 1 to 80 amino acids, from 1 to 90 amino acids, and from
1 to 100 amino acids. In some embodiments, the linker is at least
about 1, about 2, about 3, about 4, about 5, about 10, about 15,
about 20, about 25, about 30, about 35, about 40, about 45, about
50, about 55, about 60, about 65, about 70, about 75, about 80,
about 85, about 90, about 95, or about 100 amino acids in length.
Preferred linkers are typically from about 5 amino acids to about
30 amino acids.
[0126] Preferably the lengths of linkers vary from 2 to 30 amino
acids, optimized for each condition so that the linker does not
impose any constraints on the conformation or interactions of the
linked domains.
TABLE-US-00003 In some embodiments, the separation moiety comprises
the sequence (SEQ ID NO: 195) GPAGLYAQ; (SEQ ID NO: 196) GPAGMKGL;
(SEQ ID NO: 197) PGGPAGIG; (SEQ ID NO: 198) ALFKSSFP; (SEQ ID NO:
199) ALFFSSPP; (SEQ ID NO: 200) LAQRLRSS; (SEQ ID NO; 201)
LAQKLKSS; (SEQ ID NO: 202) GALFKSSFPSGGGPAGLYAQGGSGKGGSGK; (SEQ ID
NO: 203) RGSGGGPAGLYAQGSGGGPAGLYAQGGSGK; (SEQ ID NO: 204)
KGGGPAGLYAQGPAGLYAQGPAGLYAQGSR; (SEQ ID NO: 205)
RGGPAGLYAQGGPAGLYAQGGGPAGLYAQK; (SEQ ID NO: 206)
KGGALFKSSFPGGPAGIGPLAQKLKSSGGS; (SEQ ID NO: 207)
SGGPGGPAGIGALFKSSFPLAQKLKSSGGG; (SEQ ID NO: 208)
RGPLAQKLKSSALFKSSFPGGPAGIGGGGK; (SEQ ID NO: 209)
GGGALFKSSFPLAQKLKSSPGGPAGIGGGR; (SEQ ID NO: 210)
RGPGGPAGIGPLAQKLKSSALFKSSFPGGG; (SEQ ID NO: 211)
RGGPLAQKLKSSPGGPAGIGALFKSSFPGK; (SEQ ID NO: 212)
RSGGPAGLYAQALFKSSFPLAQKLKSSGGG; (SEQ ID NO: 213)
GGPLAQKLKSSALFKSSFPGPAGLYAQGGR; (SEQ ID NO: 214)
GGALFKSSFPGPAGLYAQPLAQKLKSSGGK; (SEQ ID NO: 215)
RGGALFKSSFPLAQKLKSSGPAGLYAQGGK; (SEQ ID NO: 216)
RGGGPAGLYAQPLAQKLKSSALFKSSFPGG; (SEQ ID NO: 217)
SGPLAQKLKSSGPAGLYAQALFKSSFPGSK; (SEQ ID NO: 218)
KGGPGGPAGIGPLAQRLRSSALFKSSFPGR; (SEQ ID NO: 219)
KSGPGGPAGIGALFFSSPPLAQKLKSSGGR; or (SEQ ID NO: 220)
SGGFPRSGGSFNPRTFGSKRKRRGSRGGGG
[0127] Certain preferred separation moieties comprises the sequence
GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198). The
separation moieties disclosed herein can comprise one or more
cleavage motif or functional variants that are the same or
different. The separation moieties can comprise 1, 2, 3, 4, 5, or
more cleavage motifs or functional variants. Separation moieties
comprising 30 amino acids can contain 2 cleavage motifs or
functional variants, 3 cleavage motifs or functional variants or
more. A "functional variant" of a separation moiety retains the
ability to be cleaved with high efficiency at a target site (e.g.,
a tumor microenvironment that expresses high levels of the
protease) and are not cleaved or cleaved with low efficiency in the
periphery (e.g., serum). For example, the functional variants
retain at least about 50%, about 55%, about 60%, about 70%, about
80%, about 85%, about 95% or more of the cleavage efficiency of a
separation moiety comprising any one of SEQ ID NOs. 195-220.
[0128] The separation moieties comprising more than one cleavage
motif can be selected from SEQ ID NOs: 195-201 and combinations
thereof. Preferred separation moieties comprising more than one
cleavage motif comprise the amino acids selected from SEQ ID NO:
202-220.
[0129] The separation moiety can comprise both ALFKSSFP (SEQ ID NO:
198) and GPAGLYAQ (SEQ ID NO: 195). The separation moiety can
comprise two cleavage motifs that each have the sequence GPAGLYAQ
(SEQ ID NO: 195). Alternatively or additionally, the separation
moiety can comprise two cleavage motifs that each have the sequence
ALFKSSFP (SEQ ID NO: 198). The separation moiety can comprise a
third cleavage motif that is the same or different.
[0130] In some embodiments, the separation moiety comprises an
amino acid sequence that is at least about 90%, at least about 95%,
at least about 96%, at least about 97%, at least about 98%, or at
least 99% identical to SEQ ID NOs: 195 to SEQ ID NO: 220 over the
full length of SEQ ID NO: 195-220.
[0131] The disclosure also relates to functional variants of
separation moieties comprising SEQ ID NOs. 195-220. The functional
variants of separation moieties comprising SEQ ID NOs: 195-220
generally differ from SEQ ID NOs. 195-220 by one or a few amino
acids (including substitutions, deletions, insertions, or any
combination thereof), and substantially retain their ability to be
cleaved by a protease.
[0132] The functional variants can contain at least one or more
amino acid substitutions, deletions, or insertions relative to the
separation moieties comprising SEQ ID NOs. 195-220. The functional
variant can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
alterations comparted to the separation moieties comprising SEQ ID
NOs. 195-220. In some preferred embodiments, the functional variant
differs from the separation moiety comprising SEQ ID NOs. 195-220
by less than 10, less, than 8, less than 5, less than 4, less than
3, less than 2, or one amino acid alterations, e.g., amino acid
substitutions or deletions. In other embodiments, the functional
variant may comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid
substitutions compared to SEQ ID NOs. 195-220. The amino acid
substitution can be a conservative substitution or a
non-conservative substitution, but preferably is a conservative
substitution.
[0133] In other embodiments, the functional variants of the
separation moieties may comprise 1, 2, 3, 4, or 5 or more
non-conservative amino acid substitutions compared the separation
moieties comprising SEQ ID NOs: 195-220. Non-conservative amino
acid substitutions could be recognized by one of skill in the art.
The functional variant of the separation moiety preferably contains
no more than 1, 2, 3, 4, or 5 amino acid deletions.
[0134] The amino acid sequences disclosed in the separation
moieties can be described by the relative linear position in the
separation moiety with respect to the sissile bond. As will be
well-understood by persons skilled in the art, separation moieties
comprising 8 amino acid protease substrates (e.g., SEQ ID Nos:
195-201) contain amino acid at positions P4, P3, P2, P1, P1', P2',
P3', P4', wherein the sissile bond is between P1 and P1'. For
example, amino acid positions for the separation moiety comprising
the sequence GPAGLYAQ (SEQ ID NO: 195) can be described as
follows:
TABLE-US-00004 G P A G L Y A Q P4 P3 P2 P1 P1` P2` P3` P4`
[0135] Amino acids positions for the separation moiety comprising
the sequence ALFKSSFP (SEQ ID NO: 198) can be described as
follows:
TABLE-US-00005 A L F K S S F P P4 P3 P2 P1 P1` P2` P3` P4`
[0136] Preferably, the amino acids surrounding the cleavage site
(e.g., positions P1 and P1' for SEQ ID NOs: 195-201) are not
substituted.
[0137] In embodiments, the separation moiety comprises the sequence
GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) or a
functional variant of SEQ ID NO: 195 or a function variant of SEQ
ID NO: 198. As described herein, a functional variant of PAGLYAQ
(SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198) can comprise one or
more amino acid substitutions, and substantially retain their
ability to be cleaved by a protease. Specifically, the functional
variants of GPAGLYAQ (SEQ ID NO: 195) is cleaved by MMP14, and the
functional variant of ALFKSSFP (SEQ ID NO: 198) is cleaved by
Capthepsin L (CTSL1). The functional variants also retain their
ability to be cleaved with high efficiency at a target site (e.g.,
a tumor microenvironment that expresses high levels of the
protease). For example, the functional variants of GPAGLYAQ (SEQ ID
NO: 195) or ALFKSSFP (SEQ ID NO: 198) retain at least about 50%,
about 55%, about 60%, about 70%, about 80%, about 85%, about 95% or
more of the cleavage efficiency of a separation moiety comprising
amino acid sequence GPAGLYAQ (SEQ ID NO: 195) or ALFKSSFP (SEQ ID
NO: 198), respectively.
[0138] Preferably, the functional variant of GPAGLYAQ (SEQ ID NO:
195) or ALFKSSFP (SEQ ID NO: 198) comprise no more than 1, 2, 3, 4,
or 5 conservative amino acid substitutions compared to GPAGLYAQ
(SEQ ID NO: 195) or ALFKSSFP (SEQ ID NO: 198). Preferably, the
amino acids at position P1 and P1' are not substituted. The amino
acids at positions P1 and P1' in SEQ ID NO: 195 are G and L, and
the amino acids at positions P1 and P1' in SEQ ID NO: 198 are K and
S.
[0139] The functional variant of GPAGLYAQ (SEQ ID NO: 195) can
preferably comprise one or more of the following: a) an arginine
amino acid substitution at position P4, b) a leucine, valine,
asparagine, or proline amino acid substitution at position P3, c) a
asparagine amino acid substitution at position P2, d) a histidine,
asparagine, or glycine amino acid substitution at position P1, e) a
asparagine, isoleucine, or leucine amino acid substitution at
position P1', f) a tyrosine or arginine amino acid substitution at
position P2', g) a glycine, arginine, or alanine amino acid
substitution at position P3', h) or a serine, glutamine, or lysine
amino acid substitution at position P4'. The following amino acid
substitutions are disfavored in functional variants of GPAGLYAQ
(SEQ ID NO: 195): a) arginine or isoleucine at position P3, b)
alanine at position P2, c) valine at position P1, d) arginine,
glycine, asparagine, or threonine at position P1', e) aspartic acid
or glutamic acid at position P2', f) isoleucine at position P3', g)
valine at position P4'. In some embodiments, the functional variant
of GPAGLYAQ (SEQ ID NO: 195) does not comprise an amino acid
substitution at position P1 and/or P1'.
[0140] The amino acid substitution of the functional variant of
GPAGLYAQ (SEQ ID NO: 195) preferably comprises an amino acid
substitution at position P4 and/or P4'. For example, the functional
variant of GPAGLYAQ (SEQ ID NO: 195) can comprises a leucine at
position P4, or serine, glutamine, lysine, or phenylalanine at
position P4. Alternatively or additionally, the functional variant
of GPAGLYAQ (SEQ ID NO: 195) can comprises a glycine,
phenylalanine, or a proline at position P4'.
[0141] In some embodiments, the amino acid substitutions at
position P2 or P2' of GPAGLYAQ (SEQ ID NO: 195) are not
preferred.
[0142] In some embodiments, the functional variant of GPAGLYAQ (SEQ
ID NO: 195) comprises the amino acid sequence selected from SEQ ID
NOs: 258-331. Specific functional variants of GPAGLYAQ (SEQ ID NO:
195) include GPLGLYAQ (SEQ ID NO: 295), and GPAGLKGA (SEQ ID NO:
285).
[0143] The functional variants of LFKSSFP (SEQ ID NO: 198)
preferably comprises hydrophobic amino acid substitutions. The
functional variant of LFKSSFP (SEQ ID NO: 198) can preferably
comprise one or more of the following: (a) lysine, histidine,
serine, glutamine, leucine, proline, or phenylalanine at position
P4; (b) lysine, histidine, glycine, proline, asparagine,
phenylalanine at position P3; (c) arginine, leucine, alanine,
glutamine, or histatine at position P2; (d) phenylalanine,
histidine, threonine, alanine, or glutamine at position P1; (e)
histidine, leucine, lysine, alanine, isoleucine, arginine,
phenylalanine, asparagine, glutamic acid, or glycine at position
P1', (f) phenylalanine, leucine, isoleucine, lysine, alanine,
glutamine, or proline at position P2'; (g) phenylalanine, leucine,
glycine, serine, valine, histidine, alanine, or asparagine at
position P3'; and phenylalanine, histidine, glycine, alanine,
serine, valine, glutamine, lysine, or leucine.
[0144] The inclusion of aspartic acid and/or glutamic acid in
functional variants of SEQ ID NO:198 are generally disfavored and
avoided. The following amino acid substitutions are also disfavored
in functional variants of LFKSSFP (SEQ ID NO: 198): (a) alanine,
serine, or glutamic acid at position P3; (b) proline, threonine,
glycine, or aspartic acid at position P2; (c) proline at position
P1; (d) proline at position P1'; (e) glycine at position P2'; (f)
lysine or glutamic acid at position P3'; (g) aspartic acid at
position P4'.
[0145] The amino acid substitution of the functional variant of
LFKSSFP (SEQ ID NO: 198) preferably comprises an amino acid
substitution at position P4 and/or P1. In some embodiments, an
amino acid substitution of the functional variant of LFKSSFP (SEQ
ID NO: 198) at position P4' is not preferred.
[0146] In some embodiments, the functional variant of LFKSSFP (SEQ
ID NO: 198) comprises the amino acid sequence selected from SEQ ID
NOs: 332-408. Specific functional variants of LFKSSFP (SEQ ID NO:
198) include ALFFSSPP (SEQ ID NO: 199), ALFKSFPP (SEQ ID NO: 381),
ALFKSLPP (SEQ ID NO: 382); ALFKHSPP (SEQ ID NO: 370); ALFKSIPP (SEQ
ID NO: 383); ALFKSSLP (SEQ ID NO: 390); or SPFRSSRQ (SEQ ID NO:
333).
[0147] The separation moieties disclosed herein can form a stable
complex under physiological conditions with the amino acid
sequences (e.g. domains) that they link, while being capable of
being cleaved by a protease. For example, the separation moiety is
stable (e.g., not cleaved or cleaved with low efficiency) in the
circulation and cleaved with higher efficiency at a target site
(i.e. a tumor microenvironment). Accordingly, fusion polypeptides
that include the linkers disclosed herein can, if desired, have a
prolonged circulation half-life and/or lower biological activity in
the circulation in comparison to the components of the fusion
polypeptide as separate molecular entities. Yet, when in the
desired location (e.g., tumor microenvironment) the linkers can be
efficiently cleaved to release the components that are joined
together by the linker and restoring or nearly restoring the
half-life and biological activity of the components as separate
molecular entities.
[0148] The separation moiety desirably remains stable in the
circulation for at least 2 hours, at least 5, hours, at least 10
hours, at least 15 hours, at least 20 hours, at least 24 hours, at
least 30 hours, at least 35 hours, at least 40 hours, at least 45
hours, at least 50 hours, at least 60 hours, at least 65 hours, at
least 70 hours, at least 80 hours, at least 90 hours, or
longer.
[0149] In some embodiments, the separation moiety is cleaved by
less than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 20%, 5%, or 1% in
the circulation as compared to the target location. The separation
moiety is also stable in the absence of an enzyme capable of
cleaving the linker. However, upon expose to a suitable enzyme
(i.e., a protease), the separation moiety is cleaved resulting in
separation of the linked domain.
[0150] B. Polypeptides and Compositions Comprising a Separation
Moiety
[0151] The separation moieties disclosed herein can be used in a
wide range of applications. Without limitation, they are suitable
for us in fusion proteins. As further described herein, the
separation moieties are particularly useful for preparing
therapeutic fusion proteins in which the therapeutic of biological
activity of the fusion protein is attenuated and the attenuation is
removed upon cleavage of the separation moiety. The separation
moieties can also be used to conjugate a variety of payloads such
as therapeutic and/or diagnostic agents to carriers or targeting
agents (e.g., antibodies and fragments of antibodies,
nanoparticles). Suitable methods for prepare such conjugates are
well-known in the art, see for example, Bioconjugate Techniques,
Third Ed., G. T. Hermanson (Ed.) Academic Press 2013. Exemplary
payloads include, but are not limited to cytokines, antibodies,
cell based-therapies, antibiotics, cytotoxic drugs, or other
recombinant polypeptide complexes. Of specific interest are
separation moieties that are suitable for use in conjugation with
payloads that target or are targeted to tumor
microenvironments.
[0152] This disclosure relates recombinant polypeptides in which a
separation moiety as disclosed herein links a first amino acid
sequence of interest (e.g. a first domain of interest) to a second
amino acid sequence of interest (e.g. a second domain of interest).
Typically, the first amino acid sequence of interest and the second
amino acid sequence of interest are not found together in a
naturally occurring protein. Preferred linkers are SEQ ID
NOS:195-220. In embodiments, at least one of the first amino acid
sequence of interest and the second amino acid sequence of interest
is the amino acid sequence of a therapeutic polypeptide. In some
embodiments in which at least one of the first amino acid sequence
of interest and the second amino acid sequence of interest is the
amino acid sequence of a therapeutic polypeptide, the other amino
acid sequence of interest can be the amino acid sequence of a
targeting polypeptide, a half-life extending polypeptide and/or a
blocking polypeptide.
[0153] The polypeptides that contain a separation moiety can be
represented by Formula I: [D1]-[L1]-[D2], wherein D1 is a first
amino acid sequence of interest (e.g., a domain of interest), L1 is
a separation moiety that connects or links D1 to D2; and D2 is a
second amino acid sequence of interest (e.g., a second domain of
interest). Preferably, L1 is a protease-cleavable separation
moiety, and more preferably L1 comprises or consists of any of SEQ
ID NOS: 195-220.
[0154] The polypeptides can also be represented by Formula II:
[D1]-[L1]-[D2]-[L2]-[D3], wherein D1 is a first amino acid of
interest (e.g., a domain of interest), L1 and L2 are each,
independently, a linker; D2 is a second amino acid sequence of
interest (e.g., a domain of interest), and D3 is a third amino acid
of interest (e.g., a domain of interest), wherein at least one of
L1 and L2 is a protease-cleavable separation moiety, and preferably
at least one of L1 and L2 comprises or consists of any of SEQ ID
NOS: 195-220.
[0155] The polypeptide can also be represented by Formula III:
[D1]-[L1]-[D2]-[L2]-[D3]-[L3]-[D4], wherein D1 is a first amino
acid of interest (e.g., a domain of interest), L1, L2 and L3 are
each, independently, a linker; D2 is a second amino acid sequence
of interest (e.g., a domain of interest), D3 is a third amino acid
of interest (e.g., a domain of interest); and wherein D4 is a
fourth amino acid of interest (e.g., a domain of interest), wherein
at least one of L1, L2 and L3 is a protease-cleavable separation
moiety, and preferably at least one of L1, L2 and L3 comprises or
consists of any of SEQ ID NOS: 195-220.
[0156] Additional specific applications of the separation moieties
are described in more detail herein.
[0157] i. Delivery of Payloads
[0158] The separation moieties as described herein can be used to
attach a therapeutic drug-moiety. In this approach, a therapeutic
drug moiety is attached to the separation moiety to make a
therapeutic drug moiety complex. The separate drug moiety complex
can be a prodrug that is inactive until the target protease cleaves
the prodrug, releasing the free drug.
[0159] ii. Antibody-Drug Conjugates
[0160] Another example of use of the separation moiety is in the
field of antibody-drug conjugates (ADCs) that are mainly directed
toward the treatment of cancer. ADCs typically are an antibody that
is linked to a cytotoxic moiety, such as a cytotoxic drug. The ADCs
discriminate between the healthy and diseased cells and provide
targeted delivery of drug (e.g. cytotoxic drug) to diseased cells.
ADCs typically comprise an antibody that targets a tumor marker
that is specific to tumor cells, whereupon the antibody attaches
itself to the tumor cell, causing the ADC to be absorbed into the
cell, which enables the cytotoxic component to be released to kill
the tumor cell. A key aspect of ADCs is the provision of a stable
linker between the antibody component and the cytotoxic agent. In
such applications, linkers may be cleavable or non-cleavable. For
non-cleavable linkers, the antibody, linker and cytotoxic unit is
incorporated into the tumor cell. The nature of the linker
typically determines the release profile of the cytotoxic agent.
For example, cleavable linkers between the antibody and the
cytotoxic agent are typically catalyzed by enzymes in the tumor
cell or in the tumor microenvironment, wherein the antibody and the
cytotoxic agents are cleaved to release the cytotoxic agent.
[0161] In certain embodiments, the separation moiety disclosed
herein links or connects a drug moiety to an antibody moiety.
[0162] iii. Peptide-Drug Conjugates
[0163] The separation moieties disclosed herein are suitable for
use in peptide-drug conjugates. These compounds typically comprise
a cytotoxic payload and linker; however, instead of antibodies,
peptide-drug conjugates are equipped with peptides that have the
ability to penetrate tumors, thus allowing the cargo to be
delivered inside the tumor. In some embodiments, the separation
moiety links or connects a cytotoxic payload with a peptide. The
peptide-drug conjugate remains stable and has no biological
activity until the target protease cleaves the separation
moiety.
[0164] iv. Inducible Adoptive Cell Therapy
[0165] The separation moieties disclosed herein are suitable for
use in constructs engineered for use in adoptive cell transfer
(ACT) therapies. The field of adoptive cell transfer (ACT) is
currently comprised of chimeric antigen receptor (CAR) engineered T
cells (and next generation therapies) that target T cells to cell
surface expressed targets (e.g., tumor cells expressing surface
targets), and T cell receptor (TCR) engineered T cells that can
target intracellular antigens.
[0166] In one embodiment, the separation moiety is used to tether a
targeting moiety to a CAR construct. A CAR replaces the endogenous
TCR complex with a new receptor that uses a fragment of a human or
mouse antibody to bind to targets outside of a cancer cell. The
antibody fragment is linked to various signaling proteins inside
T-cell which mediate receptor activation when the CAR binds to its
target. Porter et al., (2011) NEJM, 365:725-733; Grupp et al.,
(2013) NEJM, 368:1509-1518; U.S. Ser. No.
10/221,245/WO/2014/153270, Treatment of cancer using humanized
anti-CD19 chimeric antigen receptor.
[0167] In another embodiment, the separation moiety is used to
tether a targeting moiety to a TCR construct. A TCR is based on the
gene for the protein receptor that is already naturally present in
T-cells. The gene for a desired TCR can be discovered in a single
patient, e.g., a patient that is able to mount an effective immune
response against a type of cancer. This gene can then be introduced
into other patients by incorporating it into TCR T cell constructs
or reengineered to improve the binding interaction with its MHC
target. Guy et al., (2013) Nat Immunol., 14(3):262-70; Kuhns et
al., (2012) Front Immunol., 25; 3:159; Fesnak et al., (2016) Nat
Rev Cancer, 16(9): 566-581.
[0168] These types of engineered T cells, whether autologous or
allogenic, comprise engineered T cell receptor components that
comprise a targeting agent such as an isolated human or humanized
antibody. In CAR-T and TCR-T cells, the binding affinity of the
targeting moiety may be affected by the steric, chemical, or
flexibility properties of the separation moiety tethering the
targeting moiety to the rest of the construct and the T cell. The
separation moieties disclosed herein are suitable for use with
engineered constructs for making CAR T and TCR T cells.
[0169] v. Antigen-Binding Proteins
[0170] The separation moieties disclosed herein are suitable for
use in antigen-binding proteins. An "antigen-binding protein" (ABP)
is a protein comprising one or more antigen-binding domains that
specifically bind to an antigen or epitope. In some embodiments,
the antigen-binding domain binds the antigen or epitope with
specificity and affinity similar to that of naturally occurring
antibodies. Typically, the separation moieties link a polypeptide
that blocks the antigen-binding site of the ABP from binding to its
cognate antigen. But when the separation moiety is cleaved the
blocking polypeptide can diffuse away from the ABP antigen binding
site and the ABP can bind to its cognate antigen. Exemplary binding
polypeptides that can block the antigen binding site of an ABP
include steric blocker such as human serum albumin, and peptides
that interact with one or more of the complementarity determining
regions (CDRs) in the antigen binding site of the ABP. Such
blocking peptides can be obtained by screening libraries or by
screening peptide fragments of the cognate antigen of an ABP of
interest. Typically, when the ABP contains and an antigen binding
site of an antibody, the separation moiety and blocker will be
bonded to the amino terminus of the antibody light chain, or the
amino terminus of the antibody heavy chain, so that the blocker is
tethered close to and will readily block the antigen binding site.
Suitable variations of this approach are used when the ABP contains
an alternative scaffold for the binding site. Similarly, when
single chain antibody binding sites are use, such as scFV of dAb,
the blocker-separation moiety is typically bonded to the amino
terminus near the antigen binding site. In certain embodiments, the
ABP comprises an antibody binding site that comprises a VH and a
VL, and the blocker-separation moiety is bonded to the amino
terminus of the VL.
[0171] The ABP can be an antibody (e.g., the first and second
antigen binding domains are in the form of an antibody).
Preferably, at least one antigen binding domain of the ABP is in
the form of an antibody. In another preferred embodiment, a first
or second antigen binding domain is in the form of an antibody, and
a first or second antigen binding domain is in the form of an
antigen-binding fragment (e.g., the first antigen binding domain is
an antibody and the second antigen binding domain is an
antigen-binding fragment. Alternatively, the first antigen binding
domain is an antigen binding fragment and the second antigen
binding domain is an antibody).
[0172] In some embodiments, the ABP consists of an antibody. In
some embodiments, the ABP consists essentially of an antibody. In
some embodiments, the ABP comprises an alternative scaffold. In
some embodiments, the ABP consists of an alternative scaffold. In
some embodiments, the ABP consists essentially of an alternative
scaffold. In some embodiments, the ABP comprises an antibody
fragment. In some embodiments, the ABP consists of an antibody
fragment. In some embodiments, the ABP consists essentially of an
antibody fragment.
[0173] In some embodiments, the separation moieties disclosed
herein are suitable for use in conjunction with an antibody. The
term "antibody" is used herein in its broadest sense and includes
certain types of immunoglobulin molecules comprising one or more
antigen-binding domains that specifically bind to an antigen or
epitope. An antibody specifically includes intact antibodies (e.g.,
intact immunoglobulins), antibody fragments, and multi-specific
antibodies. An antibody is one type of ABP.
[0174] In some embodiments, the separation moieties disclosed
herein are suitable for use in conjunction with an antigen binding
protein comprising an alternative scaffold. The term "alternative
scaffold" refers to a molecule in which one or more regions may be
diversified to produce one or more antigen-binding domains that
specifically bind to an antigen or epitope.
[0175] In some embodiments, the antigen-binding domain binds the
antigen or epitope with specificity and affinity similar to that of
an antibody. Exemplary alternative scaffolds include those derived
from fibronectin (e.g., Adnectins.TM.), the .beta.-sandwich (e.g.,
iMab), lipocalin (e.g., Anticalins.RTM.), EETI-II/AGRP,
BPTI/LACI-D1/ITI-D2 (e.g., Kunitz domains), thioredoxin peptide
aptamers, protein A (e.g., Affibody.RTM.), ankyrin repeats (e.g.,
DARPins), gamma-B-crystallin/ubiquitin (e.g., Affilins), CTLD3
(e.g., Tetranectins), Fynomers, and (LDLR-A module) (e.g.,
Avimers). Additional information on alternative scaffolds is
provided in Binz et al., Nat. Biotechnol., 2005 23:1257-1268;
Skerra, Current Opin. in Biotech., 2007 18:295-304; and Silacci et
al., J. Biol. Chem., 2014, 289:14392-14398; each of which is
incorporated by reference in its entirety. An alternative scaffold
is one type of ABP.
[0176] In some embodiments, the separation moieties disclosed
herein are suitable for use in conjunction with an antibody
fragment. An "antibody fragment" comprises a portion of an intact
antibody, such as the antigen-binding or variable region of an
intact antibody. Antibody fragments include, for example, Fv
fragments, Fab fragments, F(ab')2 fragments, Fab' fragments, scFv
(sFv) fragments, and scFv-Fc fragments.
[0177] In some embodiments, the separation moieties disclosed
herein are suitable for use in conjunction with one or more Fv,
Fab, or F(ab')2 fragments. "Fv" fragments comprise a
non-covalently-linked dimer of one heavy chain variable domain and
one light chain variable domain. "Fab" fragments comprise, in
addition to the heavy and light chain variable domains, the
constant domain of the light chain and the first constant domain
(CH1) of the heavy chain. Fab fragments may be generated, for
example, by recombinant methods or by papain digestion of a
full-length antibody. "F(ab')2" fragments contain two Fab'
fragments joined, near the hinge region, by disulfide bonds.
F(ab')2 fragments may be generated, for example, by recombinant
methods or by pepsin digestion of an intact antibody. The F(ab')
fragments can be dissociated, for example, by treatment with
1-mercaptoethanol.
[0178] In some embodiments, the separation moieties disclosed
herein are suitable for use in conjunction with an scFv or scFv-Fc.
"Single-chain Fv" or "sFv" or "scFv" antibody fragments comprise a
VH domain and a VL domain in a single polypeptide chain. The VH and
VL are generally linked by a peptide linker. See Pluckthun A.
(1994). Any suitable linker may be used.
[0179] In some embodiments, the linker is a (GGGGS)n (SEQ ID NO:
231). In some embodiments, n=1, 2, 3, 4, 5, or 6. See Antibodies
from Escherichia coli. In Rosenberg M. & Moore G. P. (Eds.),
The Pharmacology of Monoclonal Antibodies vol. 113 (pp. 269-315).
Springer-Verlag, New York, incorporated by reference in its
entirety. "scFv-Fc" fragments comprise an scFv attached to an Fc
domain. For example, an Fc domain may be attached to the C-terminal
of the scFv. The Fc domain may follow the VH or VL, depending on
the orientation of the variable domains in the scFv (i.e., VH-VL or
VL-VH). Any suitable Fc domain known in the art or described herein
may be used. In some cases, the Fc domain comprises an IgG4 Fc
domain.
[0180] In some embodiments, the separation moieties disclosed
herein are suitable for use in conjunction with a single domain
antibody. The term "single domain antibody" refers to a molecule in
which one variable domain of an antibody specifically binds to an
antigen without the presence of another variable domain. Single
domain antibodies, and fragments thereof, are described in Arabi
Ghahroudi et al., FEBS Letters, 1998, 414:521-526 and Muyldermans
et al., Trends in Biochem. Sci., 2001, 26:230-245, each of which is
incorporated by reference in its entirety. Single domain antibodies
are also known as sdAbs or nanobodies.
[0181] In some embodiments, the separation moieties disclosed
herein are suitable for use in conjunction with a monospecific ABP.
A "monospecific ABP" is an ABP that comprises one or more binding
sites that specifically bind to the same epitope. An example of a
monospecific ABP is a naturally occurring IgG molecule which, while
divalent (i.e., having two antigen-binding domains), recognizes the
same epitope at each of the two antigen-binding domains. The
binding specificity may be present in any suitable valency.
[0182] In some embodiments, the separation moieties disclosed
herein are suitable for use in conjunction with a monoclonal
antibody. The term "monoclonal antibody" refers to an antibody from
a population of substantially homogeneous antibodies. A population
of substantially homogeneous antibodies comprises antibodies that
are substantially similar and that bind the same epitope(s), except
for variants that may normally arise during production of the
monoclonal antibody. Such variants are generally present in only
minor amounts. A monoclonal antibody is typically obtained by a
process that includes the selection of a single antibody from a
plurality of antibodies. For example, the selection process can be
the selection of a unique clone from a plurality of clones, such as
a pool of hybridoma clones, phage clones, yeast clones, bacterial
clones, or other recombinant DNA clones. The selected antibody can
be further altered, for example, to improve affinity for the TNFR
superfamily member proteins ("affinity maturation"), to humanize
the antibody, to improve its production in cell culture, and/or to
reduce its immunogenicity in a subject.
[0183] In some embodiments, the separation moieties disclosed
herein are suitable for use in conjunction with a chimeric
antibody. The term "chimeric antibody" refers to an antibody in
which a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0184] In some embodiments, the separation moieties disclosed
herein are suitable for use in conjunction with a humanized
antibody. "Humanized" forms of non-human antibodies are chimeric
antibodies that contain minimal sequence derived from the non-human
antibody. A humanized antibody is generally a human antibody
(recipient antibody) in which residues from one or more CDRs are
replaced by residues from one or more CDRs of a non-human antibody
(donor antibody). The donor antibody can be any suitable non-human
antibody, such as a mouse, rat, rabbit, chicken, or non-human
primate antibody having a desired specificity, affinity, or
biological effect. In some instances, selected framework region
residues of the recipient antibody are replaced by the
corresponding framework region residues from the donor antibody.
Humanized antibodies may also comprise residues that are not found
in either the recipient antibody or the donor antibody. Such
modifications may be made to further refine antibody function. For
further details, see Jones et al., Nature, 1986, 321:522-525;
Riechmann et al., Nature, 1988, 332:323-329; and Presta, Curr. Op.
Struct. Biol., 1992, 2:593-596, each of which is incorporated by
reference in its entirety.
[0185] In some embodiments, the separation moieties disclosed
herein are suitable for use in conjunction with a human antibody. A
"human antibody" is one which possesses an amino acid sequence
corresponding to that of an antibody produced by a human or a human
cell, or derived from a non-human source that utilizes a human
antibody repertoire or human antibody-encoding sequences (e.g.,
obtained from human sources or designed de novo). Human antibodies
specifically exclude humanized antibodies.
[0186] In some embodiments, the ABPs provided herein specifically
bind to the extracellular domain of a TNFR Superfamily Protein. In
some embodiments, the TNFR superfamily protein is CD27, CD137,
CD40, GITR, LT-betaR, CD30, HVEM, TNFR1, TNFR2, or OX-40. The TNFR
superfamily protein may be expressed on the surface of any suitable
target cell. In some embodiments, the target cell is a T cell. In
some embodiments, the target cell is an effector T cell. In some
embodiments, the target cell is a regulatory T cell. In some
embodiments, the target cell is a natural killer (NK) cell. In some
embodiments, the target cell is a natural killer T (NKT) cell. In
some embodiments, the target cell is a B cell. In some embodiments,
the target cell is a myeloid derived cell. In some embodiments, the
target cell is a myeloid derived suppressor cell. In some
embodiments, the target cell is a dendritic cell.
[0187] In some embodiments, an ABP provided herein is an antibody.
In some embodiments, an ABP provided herein is an antibody
fragment. In some embodiments, an ABP provided herein is an
alternative scaffold.
[0188] In some embodiments, the ABPs provided herein comprise an
immunoglobulin molecule. In some embodiments, the ABPs provided
herein consist of an immunoglobulin molecule. In some embodiments,
the ABPs provided herein consist essentially of an immunoglobulin
molecule. In some aspects, the immunoglobulin molecule comprises an
antibody. In some aspects, the immunoglobulin molecule consists of
an antibody. In some aspects, the immunoglobulin molecule consists
essentially of an antibody.
[0189] In some embodiments, the ABPs provided herein comprise
alight chain. In some aspects, the light chain is a kappa light
chain. In some aspects, the light chain is a lambda light
chain.
[0190] In some embodiments, the ABPs provided herein comprise a
heavy chain. In some aspects, the heavy chain is an IgA. In some
aspects, the heavy chain is an IgD. In some aspects, the heavy
chain is an IgE. In some aspects, the heavy chain is an IgG. In
some aspects, the heavy chain is an IgM. In some aspects, the heavy
chain is an IgG1. In some aspects, the heavy chain is an IgG2. In
some aspects, the heavy chain is an IgG3. In some aspects, the
heavy chain is an IgG4. In some aspects, the heavy chain is an
IgA1. In some aspects, the heavy chain is an IgA2.
[0191] In some embodiments, the separation moieties connect to a
further blocking moiety.
[0192] In some embodiments, the separation moieties are positioned
such that movement of the antigen-binding protein subunits is
restricted.
[0193] a. Multispecific and Monospecific Multivalent Antigen
Binding Proteins
[0194] In some embodiments, the separation moieties disclosed
herein are suitable for use in conjunction with a multi-specific
antigen binding protein (ABP). Multispecific ABP's provided herein
bind more than one antigen. For instance, a multispecific antibody
can bind 2 antigens, 3 antigens, binds 4 antigens, 5 antigens, or
more. Alternatively, the multispecific ABP can bind 2 or more
different epitopes. When the ABP binds two or more epitopes, the
two or more different epitopes may be epitopes on the same antigen
(e.g., a single TNFR superfamily protein molecule expressed by a
cell) or on different antigens (e.g., different TNFR superfamily
member protein molecules expressed by the same cell, or a TNFR
superfamily member protein molecule and a non-TNFR Superfamily
member protein molecule). In some aspects, a multi-specific ABP
binds two different epitopes (i.e., a "bispecific ABP"). In some
aspects, a multi-specific ABP binds three different epitopes (i.e.,
a "trispecific ABP"). In some aspects, a multi-specific ABP binds
four different epitopes (i.e., a "quadspecific ABP"). In some
aspects, a multi-specific ABP binds five different epitopes (i.e.,
a "quintspecific ABP"). In some aspects, a multi-specific ABP binds
6, 7, 8, or more different epitopes. Each binding specificity may
be present in any suitable valency.
[0195] In various embodiments, the antigen binding protein
comprises a blocking domain. The separation moiety disclosed herein
links the blocking domain to a first antigen binding domain and
consequently the blocking domain inhibits (a) binding affinity or
avidity of the antigen binding protein on the epitope and/or (b)
agonist or antagonist activity of the antigen binding protein on
the epitope. Preferably the separation moiety comprises a cleavage
site. Upon cleavage of the separation moiety by a disease-specific
enzyme (i.e., a protease) (a) the binding affinity or avidity of
the antigen binding protein on the epitope and/or (b) agonist or
antagonist activity of the antigen binding protein on the epitope
is increased. In some embodiments, the antigen binding protein
comprises an additional separation moiety that links or connects an
additional blocking domain to an antigen binding domain. The
additional separation moiety comprises a cleavage site.
[0196] In various embodiments, the antigen binding protein
comprises an additional blocking domain (e.g., a second, third, or
fourth blocking domain) that is/are operably linked to an antigen
binding domain (e.g., the second, third, or fourth antigen binding
domain) by a cleavable separation moiety as disclosed herein. The
blocking domain can be a steric blocker or a specific blocker. In
some embodiments, the steric blocker is independently selected from
the group consisting of a fragment of the extracellular portion of
the antibody binding protein, serum albumin, a fragment of serum
albumin, and an antibody or antigen binding fragment thereof that
binds serum albumin. In some embodiments, the specific blocker is
independently selected from a fragment of an antibody or
antigen-binding fragment thereof that binds to the first, second,
third or fourth antigen binding domain of the antigen binding
protein described herein. Preferably, the blocking domain is serum
albumin or a fragment thereof, or an antibody or antigen binding
fragment thereof that binds serum albumin.
[0197] The antigen binding domain may further comprise a third
antigen binding domain and a fourth antigen binding domain each
with binding specificity for a target antigen. TNFR superfamily
target antigens are well known in the art and are encompassed by
this disclosure. Exemplary TNFR superfamily members include, but
are not limited to CD27, CD30, CD137 (4-1BB), TNFR1 (CD120a), TNFR2
(CD120b), CD40, CD95 (Fas/Apo-1), HVEM, LT-betaR, GITR, nerve
growth factor receptor or OX-40 (CD34). Preferred TNFR superfamily
target antigens are CD27, CD137, and OX40.
[0198] The antigen binding domains with specificity for an antigen
can have binding specificity for the same epitope or for a
different epitope on the same antigen. For example, the first,
second and third antigen binding domains can have binding
specificity for the same epitope, for a different epitope, two of
the antigen binding domains may binding specificity for the same
epitope, or three of the antigen binding domains may have binding
specificity for the same epitope. In some embodiments, the antigen
binding protein may further comprise a fifth antigen binding domain
with specificity for a tumor antigen.
[0199] In some embodiments, the antigen binding polypeptide
comprises an Fc region. In one format, two antigen binding domains
can be located on each end of the Fc region. Another format
comprises two antibody arms attached to the N terminus of the Fc
region, each arm comprising 2 antigen binding domains. The antigen
binding domain can also comprise a half-life extension domain. The
half-life extension domain can be albumin, an antigen binding
domain that recruits albumin, or an immunoglobulin Fc, or a
fragment thereof. In some embodiments, the half-life extension
domain is operably linked to the antigen binding polypeptide by a
cleavable linker.
[0200] The disclosure also relates to an antigen binding protein
comprising at least a first polypeptide and a second polypeptide.
The first polypeptide comprises at least a portion of an antibody
heavy chain constant region and an antibody heavy chain variable
region (VH). The second polypeptide comprises at least a portion of
an antibody light chain constant region and an antibody light chain
variable region (VL). At least one of the first and second
polypeptide further comprises a blocking domain that is operably
linked to the VH or VL through a protease cleavable linker. The
first polypeptide associates with the second polypeptide, and VH
and VL form an antigen binding site with binding specificity for a
target antigen (e.g., 4-1BB or CS27). The blocking domain inhibits
binding of the antigen binding site to the target antigen (e.g.,
4-1BB or CD27). In some embodiments, the first polypeptide further
comprises a second VH, and the first polypeptide associates with
two of said second polypeptides to form two VH/VL antigen binding
sites that each have specificity for human 4-1BB. In some
embodiments, the antigen binding protein is a dimer of the first
polypeptide with the associated second polypeptides. The antigen
binding protein can further comprise a third, fourth, fifth, or a
sixth polypeptide.
[0201] In some embodiments the ABP is monospecific multivalent ABP.
Such formats can have a variety of structures and be prepared using
suitable antibody engineering techniques. For example, a dual Fab
antibody that contains a heavy chain with the structure
VH-CH1-noncleavable liner-VH-CH1-CH2-CH3 can be prepare. Such a
heavy chain can be expressed and can pair with two light chains.
The heavy chain can dimerize through conventional interchain
disulfide bonds to form an antibody format that contains 4 Fab
antigen binding sites. In such formats, the separation moieties
described herein can be bonded to the amino terminus of the light
chain polypeptides to link a blocker to each of the Fab antigen
binding sites. Other suitable monospecific, multivalent ABP formats
can be readily envisioned by those of skill in the art. In one such
example a heavy chain is prepared that has the structure
VH-CH1-CH2-CH3-VH-CH1. Two such heavy chains and dimerize and
associate with four light chains to form a monospecific tetravalent
ABP format. In such formats, the separation moieties described
herein can be bonded to the amino terminus of the light chain
polypeptides to link a blocker to each of the Fab antigen binding
sites. The binding activity of such monospecific tetravalent ABPs
is masked by the blocking domain, and the mask is removed upon
cleavage of the separation domain (e.g., in a tumor
microenvironment) and the ABP is able to bind its cognate
antigen.
[0202] The monospecific multivalent ABP formats can have binding
specificity for any desired antigen. In some embodiments, the
monospecific multivalent ABP formats specifically bind to the
extracellular domain of a TNFR Superfamily Protein, such as CD27,
CD30, CD137 (4-1BB), TNFR1 (CD120a), TNFR2 (CD120b), CD40, CD95
(Fas/Apo-1), HVEM, LT-betaR, GITR, nerve growth factor receptor or
OX-40 (CD34).
[0203] In embodiments, the multivalent antigen binding protein can
comprise a first antigen binding site with specificity for a target
antigen (e.g., CD27 or TNFR1), second antigen binding site with
specificity for the target antigen, a blocking polypeptide, at
least one protease cleavable linker, and an optional half-life
extension element. In such embodiments, a first blocking
polypeptide is operably linked to the first antigen binding site by
a protease cleavable linker and, optionally, a second blocking
polypeptide is operably linked to the second antigen binding site
by a protease cleavable linker. Preferably, the blocking
polypeptide is operably linked to the second antigen binding site
by a protease cleavable linker. The blocking polypeptide inhibits
(a) binding affinity or avidity of the antigen binding protein on
the target antigen and/or (b) agonist or antagonist activity of the
antigen binding protein on the target antigen and upon cleavage of
the protease cleavable linker (a) the binding affinity or avidity
of the antigen binding protein on the target antigen and/or (b)
agonist activity of the antigen binding protein on the target
antigen is increased. The optional half-life extension element can
be operably linked to the first antigen binding site and/or second
antigen binding site through a optionally protease cleavable
linker. In such embodiments, the first and second binding sites can
have specificity for the same epitope or different epitopes.
[0204] In embodiments, the antigen binding protein further
comprises a third antigen binding site with specificity for the
same target antigen. The antigen binding protein can further
comprise a fourth antigen binding site with specificity for the
same target antigen as the first and second and third antigen
binding sites. The third and fourth antigen binding sites can each
further comprise a blocking polypeptide that is operable linked to
the antigen-binding sight through a protease cleavable linker.
[0205] This disclosure also relates to a tetravalent antigen
binding protein that can comprise a first polypeptide that
comprises at least a portion of an antibody heavy chain constant
region and an antibody heavy chain variable region (VH) and a
second polypeptide that comprises at least a portion of an antibody
light chain constant region, an antibody light chain variable
region (VL). The first polypeptide associates with the second
polypeptide and the VH and VL form an antigen binding site with
binding specificity for a target antigen. The tetravalent antigen
binding protein further comprises a blocking polypeptide that is
operably linked to the VH or VL through a protease cleavable
linker. The blocking polypeptide inhibits binding of the antigen
binding site to the target antigen. In some embodiments, the first
polypeptide can further comprise a second VH. The first polypeptide
associates with two of the second polypeptides to form two VH/VL
antigen binding site that each have specificity for the target
antigen.
[0206] The tetravalent antigen binding protein can further comprise
a third polypeptide, a fourth polypeptide, a fifth polypeptide, a
sixth polypeptide, a seventh polypeptide, and an eight polypeptide.
The third polypeptide can comprise at least a portion of an
antibody heavy chain constant region and an antibody heavy chain
variable region (VH). The fourth polypeptide can comprise at least
a portion of an antibody light chain constant region, an antibody
light chain variable region (VL). The firth polypeptide can
comprise at least portion of an antibody heavy chain constant
region and an antibody heavy chain variable region (VH). The sixth
polypeptide can comprise at least a portion of an antibody light
chain constant region, an antibody light chain variable region
(VL). The seventh polypeptide can comprise at least portion of an
antibody heavy chain constant region and an antibody heavy chain
variable region (VH). The eighth polypeptide can comprise at least
a portion of an antibody light chain constant region, an antibody
light chain variable region (VL).
[0207] In some embodiments at least one of the third and fourth
polypeptide further comprises a blocking polypeptide that is
operably linked to the VH or VL through a protease cleavable
linker. The third polypeptide associates with the fourth
polypeptide and VH and VL form an antigen binding site with binding
specificity for a target antigen, and the blocking domain inhibits
binding of the antigen binding site to the target antigen.
[0208] In some embodiments, the fifth and sixth polypeptide further
comprises a blocking polypeptide that is operably linked to the VH
or VL through a protease cleavable linker. The fifth polypeptide
associates with the sixth polypeptide and VH and VL form an antigen
binding site with binding specificity for a target antigen, and the
blocking domain inhibits binding of the antigen binding site to the
target antigen.
[0209] In some embodiments, the seventh and eighth polypeptide
further comprises a blocking polypeptide that is operably linked to
the VH or VL through a protease cleavable linker. The seventh
polypeptide associates with the eighth polypeptide and VH and VL
form an antigen binding site with binding specificity for a target
antigen, and the blocking domain inhibits binding of the antigen
binding site to the target antigen.
[0210] vi. Inducible Cytokines
[0211] Disclosed herein are methods and compositions to engineer
and use constructs comprising inducible cytokines. Cytokines are
potent immune agonists, which lead to them being considered
promising therapeutic agents for oncology. However, cytokines have
a very narrow therapeutic window. Cytokines have short serum
half-lives and are also considered to be highly potent.
Consequently, therapeutic administration of cytokines produces
undesirable systemic effects and toxicities. These were exacerbated
by the need to administer large quantities of cytokine in order to
achieve the desired levels of cytokine at the intended site of
cytokine action (e.g., a tumor). Unfortunately, due to the biology
of cytokines and inability to effectively target and control their
activity, cytokines have not achieved the hoped-for clinical
advantages in the treatment of tumors.
[0212] Disclosed herein are fusion proteins that overcome the
toxicity and short half-life problems that have severely limited
the clinical use of cytokines in oncology. The fusion proteins
contain cytokine polypeptides that have receptor agonist activity.
But in the context of the fusion protein, the cytokine receptor
agonist activity is attenuated, and the circulating half-life is
extended. The fusion proteins include protease cleave sites, which
are cleaved by proteases that are associated with a desired site of
cytokine activity (e.g., a tumor), and are typically enriched or
selectively present at the site of desired activity. Thus, the
fusion proteins are preferentially (or selectively) and efficiently
cleaved at the desired site of activity to limit cytokine activity
substantially to the desired site of activity, such as the tumor
microenvironment. Protease cleavage at the desired site of
activity, such as in a tumor microenvironment, releases a form of
the cytokine from the fusion protein that is much more active as a
cytokine receptor agonist than the fusion protein (typically at
least about 100.times. more active than the fusion protein). The
form of the cytokine that is released upon cleavage of the fusion
protein typically has a short half-life, which is often
substantially similar to the half-life of the naturally occurring
cytokine, further restricting cytokine activity to the tumor
microenvironment. Even though the half-life of the fusion protein
is extended, toxicity is dramatically reduced or eliminated because
the circulating fusion protein is attenuated, and active cytokine
is targeted to the tumor microenvironment. The fusion proteins
described herein, for the first time, enable the administration of
an effective therapeutic dose of a cytokine to treat tumors with
the activity of the cytokine substantially limited to the tumor
microenvironment, and dramatically reduces or eliminates unwanted
systemic effects and toxicity of the cytokine.
[0213] In general, the therapeutic use of cytokines is strongly
limited by their systemic toxicity. TNF, for example, was
originally discovered for its capacity of inducing the hemorrhagic
necrosis of some tumors, and for its in vitro cytotoxic effect on
different tumoral lines, but it subsequently proved to have strong
pro-inflammatory activity, which can, in case of overproduction
conditions, dangerously affect the human body. As the systemic
toxicity is a fundamental problem with the use of pharmacologically
active amounts of cytokines in humans, novel derivatives and
therapeutic strategies are now under evaluation, aimed at reducing
the toxic effects of this class of biological effectors while
keeping their therapeutic efficacy.
[0214] IL-2 exerts both stimulatory and regulatory functions in the
immune system and is, along with other members of the common
.gamma. chain (.gamma.c) cytokine family, central to immune
homeostasis. IL-2 mediates its action by binding to IL-2 receptors
(IL-2R), consisting of either trimeric receptors made of
IL-2R.alpha. (CD25), IL-2R.beta. (CD122), and IL-2R.gamma.
(.gamma.c, CD132) chains or dimeric .beta..gamma. IL-2Rs (1, 3).
Both IL-2R variants are able to transmit signal upon IL-2 binding.
However, trimeric .alpha..beta..gamma. IL-2Rs have a roughly 10-100
times higher affinity for IL-2 than dimeric .beta..gamma. IL-2Rs
(3), implicating that CD25 confers high-affinity binding of IL-2 to
its receptor but is not crucial for signal transduction. Trimeric
IL-2Rs are found on activated T cells and CD4+ forkhead box P3
(FoxP3)+ T regulatory cells (Treg), which are sensitive to IL-2 in
vitro and in vivo. Conversely, antigen-experienced (memory) CD8+,
CD44 high memory-phenotype (MP) CD8+, and natural killer (NK) cells
are endowed with high levels of dimeric .beta..gamma. IL-2Rs, and
these cells also respond vigorously to IL-2 in vitro and in
vivo.
[0215] Expression of the high-affinity IL-2R is critical for
endowing T cells to respond to low concentrations of IL-2 that is
transiently available in vivo. IL-2R.alpha. expression is absent on
naive and memory T cells but is induced after antigen activation.
IL-2R.beta. is constitutively expressed by NK, NKT, and memory CD8+
T cells but is also induced on naive T cells after antigen
activation. .gamma.c is much less stringently regulated and is
constitutively expressed by all lymphoid cells. Once the
high-affinity IL-2R is induced by antigen, IL-2R signaling
upregulates the expression of IL-2Ra in part through
Stat5-dependent regulation of Il2ra transcription (Kim et al.,
2001). This process represents a mechanism to maintain expression
of the high-affinity IL-2R and sustain IL-2 signaling while there
remains a source of IL-2.
[0216] IL-2 is captured by IL-2R.alpha. through a large hydrophobic
binding surface surrounded by a polar periphery that results in a
relatively weak interaction (Kd 10-8 M) with rapid on-off binding
kinetics. However, the IL-2R.alpha.-IL-2 binary complex leads to a
very small conformational change in IL-2 that promotes association
with IL-2R.beta. through a distinct polar interaction between IL-2
and IL-2R.beta.. The pseudo-high affinity of the IL2/.alpha./.beta.
trimeric complex (i.e. Kd.about.300 pM) clearly indicates that the
trimeric complex is more stable than either IL2 bound to the
.alpha. chain alone (Kd=10 nM) or to the .beta. chain alone (Kd=450
nM). In any event, the IL2/.alpha./.beta. trimer then recruits the
.gamma. chain into the quaternary complex capable of signaling,
which is facilitated by the large composite binding site on the
IL2-bound .beta. chain for the .gamma. chain.
[0217] In other words, the ternary IL-2R.alpha.-IL-2R.beta.-IL-2
complex then recruits .gamma.c through a weak interaction with IL-2
and a stronger interaction with IL-2R.beta. to produce a stable
quaternary high-affinity IL-2R (Kd 10-11 M which is 10 pM). The
formation of the high-affinity quaternary IL-2-IL-2R complex leads
to signal transduction through the tyrosine kinases Jak1 and Jak3,
which are associated with IL-2R.beta. and .gamma.c, respectively
(Nelson and Willerford, 1998). The quaternary IL-2-IL-2R complex is
rapidly internalized, where IL-2, IL-2R.beta., and .gamma.c are
rapidly degraded, but IL-2R.alpha. is recycled to the cell surface
(Hemar et al., 1995; Yu and Malek, 2001). Thus, those functional
activities that require sustained IL-2R signaling require a
continued source of IL-2 to engage IL-2R.alpha. and form additional
IL-2-IL-2R signaling complexes.
[0218] Interleukin-15 (IL-15), another member of the 4-alpha-helix
bundle family of cytokines, has also emerged as an immunomodulator
for the treatment of cancer. IL-15 is initially captured via
IL-15R.alpha., which is expressed on antigen-presenting dendritic
cells, monocytes and macrophages. IL-15 exhibits broad activity and
induces the differentiation and proliferation of T, B and natural
killer (NK) cells via signaling through the IL-15/IL-2-R-.beta.
(CD122) and the common .gamma. chain (CD132). It also enhances
cytolytic activity of CD8.sup.+ T cells and induces long-lasting
antigen-experienced CD8.sup.+CD44 memory T cells. IL-15 stimulates
differentiation and immunoglobulin synthesis by B cells and induces
maturation of dendritic cells. It does not stimulate
immunosuppressive T regulatory cells (Tregs). Thus, boosting IL-15
activity selectively in the tumor micro-environment could enhance
innate and specific immunity and fight tumors (Waldmann et al.,
2012). IL-15 was initially identified for its ability to stimulate
T cell proliferation in an IL-2-like manner through common receptor
components (IL-2R/15R.beta.-.gamma.c) and signaling through
JAK1/JAK3 and STAT3/STAT5. Like IL-2, IL-15 has been shown to
stimulate proliferation of activated CD4-CD8-, CD4+CD8+, CD4+ and
CD8+ T cells as well as facilitate the induction of cytotoxic
T-lymphocytes, and the generation, proliferation and activation of
NK cells (Waldmann et al., 1999). However, unlike IL-2 which is
required to maintain forkhead box P3 (FOXP3)-expressing CD4+CD25+
Treg cells and for the retention of these cells in the periphery,
IL-15 has little effect on Tregs (Berger et al., 2009). This is
important as FOXP3-expressing CD4+CD25+ Tregs inhibit effector T
cells, thereby inhibiting immune responses including those directed
against the tumor. IL-2 also has a crucial role in initiating
activation induced cell death (AICD), a process that leads to the
elimination of self-reactive T cells, whereas IL-15 is an
anti-apoptotic factor for T cells (Marks-Konczalik et al., 2000).
IL-15 co-delivered with HIV peptide vaccines has been shown to
overcome CD4+ T cell deficiency by promoting longevity of
antigen-specific CD8+ T cells and blocking TRAIL-mediated apoptosis
(Oh et al., 2008). Furthermore, IL-15 promotes the long-term
maintenance of CD8+CD44hi memory T cells (Kanegane et al.,
1996).
[0219] The importance of IL-15 and IL-15R.alpha. to T and NK cell
development is further highlighted by the phenotype of
IL-15R.alpha..sup.-/- and IL-15.sup.-/- mice. Knockout mice
demonstrate decreased numbers of total CD8+ T cells, and are
deficient in memory-phenotype CD8+ T cells, NK cells, NK/T cells
and some subsets of intestinal intraepithelial lymphocytes,
indicating that IL-15 provides essential positive homeostatic
functions for these subsets of cells (Lodolce et al., 1996; Kennedy
et al., 1998). The similarities in the phenotypes of these two
strains of knockout mice suggest the importance of IL-15R.alpha. in
maintaining physiologically relevant IL-15 signals.
[0220] IL-15 is presented in trans by the IL-15 receptor
alpha-chain to the IL-15R.beta..gamma.c complex displayed on the
surface of T cells and natural killer (NK) cells (Han et al.,
2011). The IL-15Ra-chain plays a role of chaperone protein,
stabilizes, and increases IL-15 activity (Desbois et al., 2016). It
has been shown that exogenous IL-15 may have a limited impact on
patients with cancer due to its dependency on IL-15Ra frequently
downregulated in cancer patients. Therefore, the fusion protein
RLI, composed of the sushi+ domain of IL15Ra coupled via a linker
to IL-15, has been suggested as an alternative approach to IL15
therapy (Bessard et al., 2009). It was found that administration of
soluble IL-15/IL-15R.alpha. complexes greatly enhanced IL-15 serum
half-life and bioavailability in vivo (Stoklasek et al., 2010).
[0221] In addition to the effects on T and NK cells, IL-15 also has
several effects on other components of the immune system. IL-15
protects neutrophils from apoptosis, modulates phagocytosis and
stimulates the secretion of IL-8 and IL-1R antagonist. It functions
through the activation of JAK2, p38 and ERK1/2 MAPK, Syk kinase and
the NF-kB transcriptional factor (Pelletier et al., 2002). In mast
cells, IL-15 can act as a growth factor and an inhibitor of
apoptosis. In these cells IL-15 activates the JAK2/STAT5 pathway
without the requirement of .gamma.c binding (Tagaya et al., 1996).
IL-15 also induces B lymphocyte proliferation and differentiation,
and increases immunoglobulin secretion (Armitage et al., 1995). It
also prevents Fas-mediated apoptosis and allows induction of
antibody responses partially independent of CD4-help (Demerci et
al., 2004; Steel et al., 2010). Monocytes, macrophages and
dendritic cells effectively transcribe and translate IL-15. They
also respond to IL-15 stimulation. Macrophages respond by
increasing phagocytosis, inducing IL-8, IL-12 and MCP-1 expression,
and secreting IL-6, IL-8 and TNF.alpha. (Budagian et al., 2006).
Dendritic cells incubated with IL-15 demonstrate maturation with
increased CD83, CD86, CD40, and MHC class II expression, are also
resistant to apoptosis, and show enhanced interferon-.gamma.
secretion (Anguille et al., 2009).
[0222] IL-15 has also been shown to have effects on
non-hematological cells including myocytes, adipocytes, endothelial
and neural cells. IL-15 has an anabolic effect on muscle and may
support muscle cell differentiation (Quinn et al., 1995). It
stimulates myocytes and muscle fibers to accumulate contractile
protein and is able to slow muscle wasting in rats with
cancer-related cachexia (Figueras et al., 2004). IL-15 has also
been shown to stimulate angiogenesis (Angiolillo et al., 1997) and
induce microglial growth and survival (Hanisch et al., 1997).
[0223] Interleukin-7 (IL-7), also of the IL-2/IL-15 family, is a
well-characterized pleiotropic cytokine, and is expressed by
stromal cells, epithelial cells, endothelial cells, fibroblasts,
smooth muscle cells and keratinocytes, and following activation, by
dendritic cells (Alpdogan et al., 2005). Although it was originally
described as a growth and differentiation factor for precursor B
lymphocytes, subsequent studies have shown that IL-7 is critically
involved in T-lymphocyte development and differentiation.
Interleukin-7 signaling is essential for optimal CD8 T-cell
function, homeostasis and establishment of memory (Schluns et al.,
2000); it is required for the survival of most T-cell subsets, and
its expression has been proposed to be important for regulating
T-cell numbers.
[0224] IL-7 binds to a dimeric receptor, including IL-7R.alpha. and
.gamma..sub.c to form a ternary complex that plays fundamental
roles in extracellular matrix remodeling, development, and
homeostasis of T and B cells (Mazzucchelli and Durum, 2007).
IL-7R.alpha. also cross-reacts to form a ternary complex with
thymic stromal lymphopoietin (TSLP) and its receptor (TSLPR), and
activates the TSLP pathway, resulting in T and dendritic cell
proliferation in humans and further B cell development in mice
(Leonard, 2002). Tight regulation of the signaling cascades
activated by the complexes are therefore crucial to normal cellular
function. Under-stimulation of the IL-7 pathway caused by mutations
in the IL-7Ra ectodomain inhibits T and B cell development,
resulting in patients with a form of severe combined
immunodeficiency (SCID) (Giliani et al., 2005; Puel et al.,
1998).
[0225] IL-7 has a potential role in enhancing immune reconstitution
in cancer patients following cytotoxic chemotherapy. IL-7 therapy
enhances immune reconstitution and can augment even limited thymic
function by facilitating peripheral expansion of even small numbers
of recent thymic emigrants. Therefore, IL-7 therapy could
potentially repair the immune system of patients who have been
depleted by cytotoxic chemotherapy (Capitini et al., 2010).
[0226] Interleukin-12 (IL-12) is a disulfide-linked heterodimer of
two separately encoded subunits (p35 and p40), which are linked
covalently to give rise to the so-called bioactive heterodimeric
(p70) molecule (Lieschke et al., 1997; Jana et al., 2014). Apart
from forming heterodimers (IL-12 and IL-23), the p40 subunit is
also secreted as a monomer (p40) and a homodimer (p40.sub.2). It is
known in the art that synthesis of the heterodimer as a single
chain with a linker connecting the p35 to the p40 subunit preserves
the full biological activity of the heterodimer. IL-12 plays a
critical role in the early inflammatory response to infection and
in the generation of Th1 cells, which favor cell-mediated immunity.
It has been found that overproduction of IL-12 can be dangerous to
the host because it is involved in the pathogenesis of a number of
autoimmune inflammatory diseases (e.g. MS, arthritis, type 1
diabetes).
[0227] The IL-12 receptor (IL-12R) is a heterodimeric complex
consisting of IL-12R.beta.1 and IL-12R.beta.2 chains expressed on
the surface of activated T-cells and natural killer cells
(Trinchieri et al., 2003). The IL-12R.beta.1 chain binds to the
IL-12p40 subunit, whereas IL-12p35 in association with
IL-12R.beta.2 confers an intracellular signaling ability (Benson et
al., 2011). Signal transduction through IL-12R induces
phosphorylation of Janus kinase (Jak2) and tyrosine kinase (Tyk2),
that phosphorylate and activate signal transducer and activator of
transcription (STAT)1, STAT3, STAT4, and STAT5. The specific
cellular effects of IL-12 are due mainly to activation of STAT4.
IL-12 induces natural killer and T-cells to produce cytokines, in
particular interferon (IFN).gamma., that mediate many of the
proinflammatory activities of IL-12, including CD4+ T-cell
differentiation toward the Th1 phenotype (Montepaone et al.,
2014).
[0228] Treg cells actively suppress activation of the immune system
and prevent pathological self-reactivity and consequent autoimmune
disease. Developing drugs and methods to selectively activate
regulatory T cells for the treatment of autoimmune disease is the
subject of intense research and, until the development of the
present invention, which can selectively deliver active
interleukins at the site of inflammation, has been largely
unsuccessful. Treg are a class of CD4+CD25+ T cells that suppress
the activity of other immune cells. Treg are central to immune
system homeostasis and play a major role in maintaining tolerance
to self-antigens and in modulating the immune response to foreign
antigens. Multiple autoimmune and inflammatory diseases, including
Type 1 Diabetes (TD), Systemic Lupus Erythematosus (SLE), and
Graft-versus-Host Disease (GVHD) have been shown to have a
deficiency of Treg cell numbers or Treg function.
[0229] Consequently, there is great interest in the development of
therapies that boost the numbers and/or function of Treg cells. One
treatment approach for autoimmune diseases being investigated is
the transplantation of autologous, ex vivo-expanded Treg cells
(Tang, Q., et al, 2013, Cold Spring Harb. Perspect. Med., 3:1-15).
While this approach has shown promise in treating animal models of
disease and in several early stage human clinical trials, it
requires personalized treatment with the patient's own T cells, is
invasive, and is technically complex. Another approach is treatment
with low dose Interleukin-2 (IL-2). Treg cells characteristically
express high constitutive levels of the high affinity IL-2
receptor, IL2R.alpha..beta..gamma., which is composed of the
subunits IL2R.alpha. (CD25), IL2R.beta. (CD122), and IL2R.gamma.
(CD132), and Treg cell growth has been shown to be dependent on
IL-2 (Malek, T. R., et al., 2010, Immunity, 33:153-65).
[0230] Conversely, immune activation has also been achieved using
IL-2, and recombinant IL-2 (Proleukin.RTM.) has been approved to
treat certain cancers. High-dose IL-2 is used for the treatment of
patients with metastatic melanoma and metastatic renal cell
carcinoma with a long-term impact on overall survival.
[0231] Clinical trials of low-dose IL-2 treatment of chronic GVHD
(Koreth, J., et al., 2011, N Engl J Med., 365:2055-66) and
HCV-associated autoimmune vasculitis patients (Saadoun, D., et al.,
2011, N Engl J Med., 365:2067-77) have demonstrated increased Treg
levels and signs of clinical efficacy. New clinical trials
investigating the efficacy of IL-2 in multiple other autoimmune and
inflammatory diseases have been initiated. The rationale for using
so-called low dose IL-2 was to exploit the high IL-2 affinity of
the trimeric IL-2 receptor which is constitutively expressed on
Tregs while leaving other T cells which do not express the high
affinity receptor in the inactivated state. Aldesleukin (marketed
as Proleukin.RTM. by Prometheus Laboratories, San Diego, Calif.),
the recombinant form of IL-2 used in these trials, is associated
with high toxicity. Aldesleukin is approved for the treatment of
metastatic melanoma and metastatic renal cancer, but its side
effects are so severe that its use is only recommended in a
hospital setting with access to intensive care (Web address:
www.proleukin.com/assets/pdf/proleukin.pdf).
[0232] The clinical trials of IL-2 in autoimmune diseases have
employed lower doses of IL-2 in order to target Treg cells, because
Treg cells respond to lower concentrations of IL-2 than many other
immune cell types due to their expression of IL2R alpha (Klatzmann
D, 2015 Nat Rev Immunol. 15:283-94). However, even these lower
doses resulted in safety and tolerability issues, and the
treatments used have employed daily subcutaneous injections, either
chronically or in intermittent 5-day treatment courses. Therefore,
there is a need for an autoimmune disease therapy that potentiates
Treg cell numbers and function, that targets Treg cells more
specifically than IL-2, that is safer and more tolerable, and that
is administered less frequently.
[0233] One approach that has been suggested for improving the
therapeutic index of IL-2-based therapy is to use variants of IL-2
that are selective for Treg cells relative to other immune cells.
IL-2 receptors are expressed on a variety of different immune cell
types, including T cells, NK cells, eosinophils, and monocytes, and
this broad expression pattern likely contributes to its pleiotropic
effect on the immune system and high systemic toxicity. In
particular, activated T effector cells express
IL2R.alpha..beta..gamma., as do pulmonary epithelial cells. But,
activating T effector cells runs directly counter to the goal of
down-modulating and controlling an immune response, and activating
pulmonary epithelial cells leads to known dose-limiting side
effects of IL-2 including pulmonary edema. In fact, the major side
effect of high-dose IL-2 immunotherapy is vascular leak syndrome
(VLS), which leads to accumulation of intravascular fluid in organs
such as lungs and liver with subsequent pulmonary edema and liver
cell damage. There is no treatment of VLS other than withdrawal of
IL-2. Low-dose IL-2 regimens have been tested in patients to avoid
VLS, however, at the expense of suboptimal therapeutic results.
[0234] According to the literature, VLS is believed to be caused by
the release of proinflammatory cytokines from IL-2-activated NK
cells. However, there is strong evidence that pulmonary edema
results from direct binding of IL-2 to lung endothelial cells,
which expressed low to intermediate levels of functional
.alpha..beta..gamma. IL-2Rs. And, the pulmonary edema associated
with interaction of IL-2 with lung endothelial cells was abrogated
by blocking binding to CD25 with an anti-CD25 monoclonal antibody
(mAb), in CD25-deficient host mice, or by the use of CD122-specific
IL-2/anti-IL-2 mAb (IL-2/mAb) complexes, thus preventing VLS.
[0235] Treatment with interleukin cytokines other than IL-2 has
been more limited. IL-15 displays immune cell stimulatory activity
similar to that of IL-2 but without the same inhibitory effects,
thus making it a promising immunotherapeutic candidate. Clinical
trials of recombinant human IL-15 for the treatment of metastatic
malignant melanoma or renal cell cancer demonstrated appreciable
changes in immune cell distribution, proliferation, and activation
and suggested potential antitumor activity (Conlon et. al., 2014).
IL-15 is currently in clinical trials to treat various forms of
cancer. However, IL-15 therapy is known to be associated with
undesired and toxic effects, such as exacerbating certain
leukemias, graft-versus-host disease, hypotension,
thrombocytopenia, and liver injury. (Mishra A., et al., Cancer
Cell, 2012, 22(5):645-55; Alpdogan O. et al., Blood, 2005,
105(2):866-73; Conlon K C et al., J Clin Oncol, 2015,
33(1):74-82.)
[0236] IL-7 promotes lymphocyte development in the thymus and
maintains survival of naive and memory T cell homeostasis in the
periphery. Moreover, it is important for the organogenesis of lymph
nodes (LN) and for the maintenance of activated T cells recruited
into the secondary lymphoid organs (SLOs) (Gao et. al., 2015). In
clinical trials of IL-7, patients receiving IL-7 showed increases
in both CD4+ and CD8+ T cells, with no significant increase in
regulatory T cell numbers as monitored by FoxP3 expression (Sportes
et al., 2008). In clinical trials reported in 2006, 2008 and 2010,
patients with different kinds of cancers such as metastatic
melanoma or sarcoma were injected subcutaneously with different
doses of IL-7. Little toxicity was seen except for transient fevers
and mild erythema. Circulating levels of both CD4+ and CD8+ T cells
increased significantly and the number of Treg reduced. TCR
repertoire diversity increased after IL-7 therapy. However, the
anti-tumor activity of IL-7 was not well evaluated (Gao et. al.,
2015). Results suggest that IL-7 therapy could enhance and broaden
immune responses.
[0237] IL-12 is a pleiotropic cytokine, the actions of which create
an interconnection between the innate and adaptive immunity. IL-12
was first described as a factor secreted from PMA-induced
EBV-transformed B-cell lines. Based on its actions, IL-12 has been
designated as cytotoxic lymphocyte maturation factor and natural
killer cell stimulatory factor. Due to bridging the innate and
adaptive immunity and potently stimulating the production of
IFN.gamma., a cytokine coordinating natural mechanisms of
anticancer defense, IL-12 seemed ideal candidate for tumor
immunotherapy in humans. However, severe side effects associated
with systemic administration of IL-12 in clinical investigations
and the very narrow therapeutic index of this cytokine markedly
tempered enthusiasm for the use of this cytokine in cancer patients
(Lasek et. al., 2014). Approaches to IL-12 therapy in which
delivery of the cytokine is tumor-targeted, which may diminish some
of the previous issues with IL-12 therapy, are currently in
clinical trials for cancers.
[0238] The direct use of IL-2 as an agonist to bind the IL-2R and
modulate immune responses therapeutically has been problematic due
its well-documented therapeutic risks, e.g., its short serum
half-life and high toxicity. These risks have also limited the
therapeutic development and use of other cytokines. New forms of
cytokines that reduce these risks are needed. Disclosed herein are
compositions and methods comprising IL-2 and IL-15 and other
cytokines, functional fragments and muteins of cytokines as well as
conditionally active cytokines designed to address these risks and
provide needed immunomodulatory therapeutics.
[0239] The present invention is designed to address the
shortcomings of direct IL-2 therapy and therapy using other
cytokines, for example using cytokine blocking moieties, e.g.
steric blocking polypeptides, serum half-life extending
polypeptides, targeting polypeptides, linking polypeptides,
including protease cleavable linkers, and combinations thereof.
Cytokines, including interleukins (e.g., IL-2, IL-7, IL-12, IL-15,
IL-18, IL-21 IL-23), interferons (IFNs, including IFN.alpha.,
IFN.beta. and IFN.gamma.), tumor necrosis factors (e.g.,
TNF.alpha., lymphotoxin), transforming growth factors (e.g.,
TGF.beta.1, TGF.beta.2, TGF.beta.3), chemokines (C-X-C motif
chemokine 10 (CXCL10), CCL19, CCL20, CCL21), and granulocyte
macrophage-colony stimulating factor (GM-CS) are highly potent when
administered to patients. As used herein, "chemokine" means a
family of small cytokines with the ability to induce directed
chemotaxis in nearby responsive cells Cytokines can provide
powerful therapy but are accompanied by undesired effects that are
difficult to control clinically and which have limited the clinical
use of cytokines. This disclosure relates to new forms of cytokines
that can be used in patients with reduced or eliminated undesired
effects. In particular, this disclosure relates to pharmaceutical
compositions including chimeric polypeptides (fusion proteins),
nucleic acids encoding fusion proteins and pharmaceutical
formulations of the foregoing that contain cytokines or active
fragments or muteins of cytokines that have decreased cytokine
receptor activating activity in comparison to the corresponding
cytokine. However, under selected conditions or in a selected
biological environment the chimeric polypeptides activate their
cognate receptors, often with the same or higher potency as the
corresponding naturally occurring cytokine. As described herein,
this is typically achieved using a cytokine blocking moiety that
blocks or inhibits the receptor activating function of the
cytokine, active fragment or mutein thereof under general
conditions but not under selected conditions, such as those present
at the desired site of cytokine activity (e.g., an inflammatory
site or a tumor).
[0240] The chimeric polypeptides and nucleic acids encoding the
chimeric polypeptides can be made using any suitable method. For
example, nucleic acids encoding a chimeric polypeptide can be made
using recombinant DNA techniques, synthetic chemistry or
combinations of these techniques, and expressed in a suitable
expression system, such as in CHO cells. Chimeric polypeptides can
similarly be made, for example by expression of a suitable nucleic
acid, using synthetic or semi-synthetic chemical techniques, and
the like. In some embodiments, the blocking moiety can be attached
to the cytokine polypeptide via sortase-mediated conjugation.
"Sortases" are transpeptidases that modify proteins by recognizing
and cleaving a carboxyl-terminal sorting signal embedded in or
terminally attached to a target protein or peptide. Sortase A
catalyzes the cleavage of the LPXTG motif (where X is any standard
amino acid) (SEQ ID NO: 237) between the Thr and Gly residue on the
target protein, with transient attachment of the Thr residue to the
active site Cys residue on the enzyme, forming an enzyme-thioacyl
intermediate. To complete transpeptidation and create the
peptide-monomer conjugate, a biomolecule with an N-terminal
nucleophilic group, typically an oligoglycine motif, attacks the
intermediate, displacing Sortase A and joining the two
molecules.
[0241] To form the cytokine-blocking moiety conjugate, the cytokine
polypeptide is first tagged at the N-terminus with a polyglycine
sequence, or alternatively, with at the C-terminus with a LPXTG
(SEQ ID NO: 237) motif. The blocking moiety or other element has
respective peptides attached that serve as acceptor sites for the
tagged polypeptides. For conjugation to domains carrying a LPXTG
(SEQ ID NO: 237) acceptor peptide attached via its N-terminus, the
polypeptide will be tagged with an N-terminal poly-glycine stretch.
For conjugation to domain carrying a poly-glycine peptide attached
via its C-terminus, the polypeptide will be tagged at its
C-terminus with a LPXTG (SEQ ID NO: 237) sortase recognition
sequence. Recognizing poly-glycine and LPXTG (SEQ ID NO: 237)
sequences, sortase will form a peptide bond between polymer-peptide
and tagged polypeptides. The sortase reaction cleaves off glycine
residues as intermediates and occurs at room temperature.
[0242] A variety of mechanisms can be exploited to remove or reduce
the inhibition caused by the blocking moiety. For example, the
pharmaceutical compositions can include a cytokine moiety and a
blocking moiety, e.g. a steric blocking moiety, with a protease
cleavable linker comprising a protease cleavage site located
between the cytokine and cytokine blocking moiety or within the
cytokine blocking moiety. When the protease cleavage site is
cleaved, the blocking moiety can dissociate from cytokine, and the
cytokine can then activate cytokine receptor.
[0243] Any suitable linker can be used. For example, the linker can
comprise glycine-glycine, a sortase-recognition motif, or a
sortase-recognition motif and a peptide sequence
(Gly.sub.4Ser).sub.n (SEQ ID NO: 238) or (Gly.sub.3Ser).sub.n (SEQ
ID NO: 239), wherein n is 1, 2, 3, 4 or 5. Typically, the
sortase-recognition motif comprises a peptide sequence LPXTG (SEQ
ID NO: 237), where X is any amino acid. In some embodiments, the
covalent linkage is between a reactive lysine residue attached to
the C-terminal of the cytokine polypeptide and a reactive aspartic
acid attached to the N-terminal of the blocker or other domain. In
other embodiments, the covalent linkage is between a reactive
aspartic acid residue attached to the N-terminal of the cytokine
polypeptide and a reactive lysine residue attached to the
C-terminal of the blocker or another domain.
[0244] Accordingly, as described in detail herein, the cytokine
blocking moieties used can be steric blockers. As used herein, a
"steric blocker" refers to a polypeptide or polypeptide moiety that
can be covalently bonded to a cytokine polypeptide directly or
indirectly through other moieties such as linkers, for example in
the form of a chimeric polypeptide (fusion protein), but otherwise
does not covalently bond to the cytokine polypeptide. A steric
blocker can non-covalently bond to the cytokine polypeptide, for
example though electrostatic, hydrophobic, ionic or hydrogen
bonding. A steric blocker typically inhibits or blocks the activity
of the cytokine moiety due to its proximity to the cytokine moiety
and comparative size. The steric inhibition of the cytokine moiety
can be removed by spatially separating the cytokine moiety from the
steric blocker, such as by enzymatically cleaving a fusion protein
that contains a steric blocker and a cytokine polypeptide at a site
between the steric blocker and the cytokine polypeptide.
[0245] As described in greater detail herein, the blocking function
can be combined with or due to the presence of additional
functional components in the pharmaceutical composition, such as a
targeting domain, a serum half-life extension element, and
protease-cleavable linking polypeptides. For example, a serum
half-life extending polypeptide can also be a steric blocker.
[0246] In the interest of presenting a concise disclosure of the
full scope of the invention, aspects of the invention are described
in detail using the cytokine IL-2 as an exemplary cytokine.
However, the invention and this disclosure are not limited to IL-2.
It will be clear to a person of skill in the art that this
disclosure, including the disclosed methods, polypeptides and
nucleic acids, adequately describes and enables the use of other
cytokines, fragments and muteins, such as IL-2, IL-7, IL-12, IL-15,
IL-18, IL-21 IL-23, IFN.alpha., IFN.beta., IFN.gamma., TNF.alpha.,
lymphotoxin, TGF-.beta.1, TGF.beta.2, TGF.beta.3, GM-CSF, CXCL10,
CCL19, CCL20, CCL21 and functional fragments or muteins of any of
the foregoing.
[0247] Various elements ensure the delivery and activity of IL-2
preferentially at the site of desired IL-2 activity and to severely
limit systemic exposure to the interleukin via a blocking and/or a
targeting strategy preferentially linked to a serum half-life
extension strategy. In this serum half-life extension strategy, the
blocked version of interleukin circulates for extended times
(preferentially 1-2 or more weeks) but the activated version has
the typical serum half-life of the interleukin.
[0248] By comparison to a serum half-life extended version, the
serum half-life of IL-2 administered intravenously is only
.about.10 minutes due to distribution into the total body
extracellular space, which is large, .about.15 L in an average
sized adult. Subsequently, IL-2 is metabolized by the kidneys with
a half-life of .about.2.5 hours. (Smith, K. "Interleukin 2
immunotherapy." Therapeutic Immunology 240 (2001)). By other
measurements, IL-2 has a very short plasma half-life of 85 minutes
for intravenous administration and 3.3 hours subcutaneous
administration (Kirchner, G. I., et al., 1998, Br J Clin Pharmacol.
46:5-10). In some embodiments of this invention, the half-life
extension element is linked to the interleukin via a linker which
is cleaved at the site of action (e.g. by inflammation-specific
proteases) releasing the interleukin's full activity at the desired
site and also separating it from the half-life extension of the
uncleaved version. In such embodiments, the fully active and free
interleukin would have very different pharmacokinetic (pK)
properties--a half-life of hours instead of weeks. In addition,
exposure to active cytokine is limited to the site of desired
cytokine activity (e.g., an inflammatory site or tumor) and
systemic exposure to active cytokine, and associated toxicity and
side effects, are reduced.
[0249] Other cytokines envisioned in this invention have similar
pharmacology (e.g. IL-15 as reported by Blood 2011 117:4787-4795;
doi: doi.org/10.1182/blood-2010-10-311456) as IL-2 and accordingly,
the designs of this invention address the shortcomings of using
these agents directly, and provide chimeric polypeptides that can
have extended half-life and/or be targeted to a site of desired
activity (e.g., a site of inflammation or a tumor).
[0250] If desired, IL-2 can be engineered to bind the IL-2R complex
generally or one of the three IL-2R subunits specifically with an
affinity that differs from that of the corresponding wild-type
IL-2, for example to selectively activate Tregs or Teff (Effector T
Cell). For example, IL-2 polypeptides that are said to have higher
affinity for the trimeric form of the IL-2 receptor relative to the
dimeric beta/gamma form of the I1-2 receptor in comparison to wild
type IL-2 can have an amino acid sequence that includes one of the
following sets of mutations with respect to SEQ ID NO: 1 (a mature
IL-2 protein comprising amino acids 21-153 of human IL-2 having the
UniProt Accession No. P60568-1): (a) K64R, V69A, and Q74P; (b)
V69A, Q74P, and T101A; (c) V69A, Q74P, and I128T; (d) N30D, V69A,
Q74P, and F103S; (e) K49E, V69A, A73V, and K76E; (f) V69A, Q74P,
T101A, and T133N; (g) N30S, V69A, Q74P, and I128A; (h) V69A, Q74P,
N88D, and S99P; (i) N30S, V69A, Q74P, and I128T; (j) K9T, Q11R,
K35R, V69A, and Q74P; (k) A1T, M46L, K49R, E61D, V69A, and H79R;
(l) K48E, E68D, N71T, N90H, F103S, and I114V; (m) S4P, T10A, Q11R,
V69A, Q74P, N88D, and T133A; (n) E15K, N30S Y31H, K35R, K48E, V69A,
Q74P, and I92T; (o) N30S, E68D, V69A, N71A, Q74P, S75P, K76R, and
N90H; (p) N30S, Y31C, T37A, V69A, A73V, Q74P, H79R, and I128T; (q)
N26D, N29S, N30S, K54R, E67G, V69A, Q74P, and I92T; (r) K8R, Q13R,
N26D, N30T, K35R, T37R, V69A, Q74P, and 192T; and (s) N29S, Y31H,
K35R, T37A, K48E, V69A, N71R, Q74P, N88D, and I89V. This approach
can also be applied to prepare muteins of other cytokines including
interleukins (e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-23),
interferons (IFNs, including IFNalpha, IFNbeta and IFNgamma), tumor
necrosis factors (e.g., TNFalpha, lymphotoxin), transforming growth
factors (e.g., TGFbeta1, TGFbeta2, TGFbeta3) and granulocyte
macrophage-colony stimulating factor (GM-CS). For example, muteins
can be prepared that have desired binding affinity for a cognate
receptor.
[0251] As noted above, any of the mutant IL-2 polypeptides
disclosed herein can include the sequences described; they can also
be limited to the sequences described and otherwise identical to
SEQ ID NO:1. Moreover, any of the mutant IL-2 polypeptides
disclosed herein can optionally include a substitution of the
cysteine residue at position 125 with another residue (e.g.,
serine) and/or can optionally include a deletion of the alanine
residue at position 1 of SEQ ID NO:1.
[0252] Another approach to improving the therapeutic index of an
IL-2 based therapy is to optimize the pharmacokinetics of the
molecule to maximally activate Treg cells. Early studies of IL-2
action demonstrated that IL-2 stimulation of human T cell
proliferation in vitro required a minimum of 5-6 hours exposure to
effective concentrations of IL-2 (Cantrell, D. A., et. al., 1984,
Science, 224: 1312-1316). When administered to human patients, IL-2
has a very short plasma half-life of 85 minutes for intravenous
administration and 3.3 hours subcutaneous administration (Kirchner,
G. I., et al., 1998, Br J Clin Pharmacol. 46:5-10). Because of its
short half-life, maintaining circulating IL-2 at or above the level
necessary to stimulate T cell proliferation for the necessary
duration necessitates high doses that result in peak IL-2 levels
significantly above the EC50 for Treg cells or will require
frequent administration. These high IL-2 peak levels can activate
IL2R.beta..gamma. receptors and have other unintended or adverse
effects, for example VLS as noted above. An IL-2 analog, or a
multifunctional protein with IL-2 attached to a domain that enables
binding to the FcRn receptor, with a longer circulating half-life
than IL-2 can achieve a target drug concentration for a specified
period of time at a lower dose than IL-2, and with lower peak
levels. Such an IL-2 analog will therefore require either lower
doses or less frequent administration than IL-2 to effectively
stimulate Treg cells. Less frequent subcutaneous administration of
an IL-2 drug will also be more tolerable for patients. A
therapeutic with these characteristics will translate clinically
into improved pharmacological efficacy, reduced toxicity, and
improved patient compliance with therapy. Alternatively, IL-2 or
muteins of IL-2 (herein, "IL-2*") can be selectively targeted to
the intended site of action (e.g. sites of inflammation). This
targeting can be achieved by one of several strategies, including
the addition of domains to the administered agent that comprise
blockers of the IL-2 (or muteins) that are cleaved away or by
targeting domains or a combination of the two.
[0253] In some embodiments, IL-2* partial agonists can be tailored
to bind with higher or lower affinity depending on the desired
target; for example, an IL-2* can be engineered to bind with
enhanced affinity to one of the receptor subunits and not the
others. These types of partial agonists, unlike full agonists or
complete antagonists, offer the ability to tune the signaling
properties to an amplitude that elicits desired functional
properties while not meeting thresholds for undesired properties.
Given the differential activities of the partial agonists, a
repertoire of IL-2 variants could be engineered to exhibit an even
finer degree of distinctive signaling activities, ranging from
almost full to partial agonism to complete antagonism.
[0254] In some embodiments, the IL-2* has altered affinity for
IL-2R.alpha.. In some embodiments, the IL-2* has a higher affinity
for IL-2R.alpha. than wild-type IL-2. In other embodiments, the
IL-2* has altered affinity for IL-2R.beta.. In one embodiment,
IL-2* has enhanced binding affinity for IL-2R.beta., e.g., the
N-terminus of IL-2R.beta., that eliminates the functional
requirement for IL-2R.alpha.. In another embodiment, an IL-2* is
generated that has increased binding affinity for IL-2R.beta. but
that exhibited decreased binding to IL-2R.gamma., and thereby is
defective IL-2R.gamma..beta. heterodimerization and signaling.
[0255] Blocking moieties, described in further detail below, can
also be used to favor binding to or activation of one or more
receptors. In one embodiment, blocking moieties are added such that
IL-2R.beta..gamma. binding or activation is blocked but
IL-2R.alpha. binding or activation is not changed. In another
embodiment, blocking moieties are added such that IL-2R.alpha.
binding or activation is diminished. In another embodiment,
blocking moieties are added such that binding to and or activation
of all three receptors is inhibited. This blocking may be
relievable by removal of the blocking moieties in a particular
environment, for example by proteolytic cleavage of a linker
linking one or more blocking moieties to the cytokine.
[0256] A similar approach can be applied to improve other
cytokines, particularly for use as immunostimulatory agents, for
example for treating cancer. For example, in this aspect, the
pharmacokinetics and/or pharmacodynamics of the cytokine (e.g.,
IL-2, IL-7, IL-12, IL-15, IL-18, IL-21 IL-23, IFN.alpha., IFN.beta.
and IFN.gamma., TNF.alpha., lymphotoxin, TGF-.beta.1, TGF.beta.2,
TGF.beta.3, GM-CSF, CXCL10, CCL19, CCL20, and CCL21 can be tailored
to maximally activate effector cells (e.g., effect T cells, NK
cells) and/or cytotoxic immune response promoting cells (e.g.,
induce dendritic cell maturation) at a site of desired activity,
such as in a tumor, but preferably not systemically.
[0257] Thus, provided herein are pharmaceutical compositions
comprising at least one cytokine polypeptide, such as interleukins
(e.g., IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, IL-23), interferons
(IFNs, including IFN.alpha., IFN.beta. and IFN.gamma.), tumor
necrosis factors (e.g., TNF.alpha., lymphotoxin), transforming
growth factors (e.g., TGF-.beta.1, TGF.beta.2, TGF.beta.3),
chemokines (e.g. CXCL10, CCL19, CCL20, CCL21) and granulocyte
macrophage-colony stimulating factor (GM-CS) or a functional
fragment or mutein of any of the foregoing. The polypeptide
typically also includes at least one linker amino acid sequence,
wherein the amino acid sequence is in certain embodiments capable
of being cleaved by an endogenous protease. In one embodiment, the
linker comprises an amino acid sequence comprising HSSKLQ (SEQ ID
NO: 25), GPLGVRG (SEQ ID NO: 221), IPVSLRSG (SEQ ID NO: 222),
VPLSLYSG (SEQ ID NO: 223), or SGESPAYYTA (SEQ ID NO: 224). In other
embodiments, the chimeric polypeptide further contains a blocking
moiety, e.g. a steric blocking polypeptide moiety, capable of
blocking the activity of the interleukin polypeptide. The blocking
moiety, for example, can comprise a human serum albumin (HSA)
binding domain or an optionally branched or multi-armed
polyethylene glycol (PEG). Alternatively, the pharmaceutical
composition comprises a first cytokine polypeptide or a fragment
thereof, and blocking moiety, e.g. a steric blocking polypeptide
moiety, wherein the blocking moiety blocks the activity of the
cytokine polypeptide on the cytokine receptor, and wherein the
blocking moiety in certain embodiments comprises a protease
cleavable domain. In some embodiments, blockade and reduction of
cytokine activity is achieved simply by attaching additional
domains with very short linkers to the N or C terminus of the
interleukin domain. In such embodiments, it is anticipated the
blockade is relieved by protease digestion of the blocking moiety
or of the short linker that tethers the blocker to the interleukin.
Once the domain is clipped or is released, it will no longer be
able to achieve blockade of cytokine activity.
[0258] The pharmaceutical composition e.g., chimeric polypeptide
can comprise two or more cytokines, which can be the same cytokine
polypeptide or different cytokine polypeptides. For example, the
two or more different types of cytokines have complementary
functions. In some examples, a first cytokine is IL-2 and a second
cytokine is IL-12. In some embodiments, each of the two or more
different types of cytokine polypeptides have activities that
modulate the activity of the other cytokine polypeptides. In some
examples of chimeric polypeptides that contain two cytokine
polypeptides, a first cytokine polypeptide is T-cell activating,
and a second cytokine polypeptide is non-T-cell-activating. In some
examples of chimeric polypeptides that contain two cytokine
polypeptides, a first cytokine is a chemoattractant, e.g., CXCL10,
and a second cytokine is an immune cell activator.
[0259] Preferably, the cytokine polypeptides (including functional
fragments) that are included in the fusion proteins disclosed
herein are not mutated or engineered to alter the properties of the
naturally occurring cytokine, including receptor binding affinity
and specificity or serum half-life. However, changes in amino acid
sequence from naturally occurring (including wild type) cytokine
are acceptable to facilitate cloning and to achieve desired
expression levels, for example.
[0260] a. Blocking Moiety
[0261] The blocking moiety can be any moiety that inhibits the
ability of the cytokine to bind and/or activate its receptor. The
blocking moiety can inhibit the ability of the cytokine to bind
and/or activate its receptor sterically blocking and/or by
noncovalently binding to the cytokine. Examples of suitable
blocking moieties include the full length or a cytokine-binding
fragment or mutein of the cognate receptor of the cytokine.
Antibodies and fragments thereof including, a polyclonal antibody,
a recombinant antibody, a human antibody, a humanized antibody a
single chain variable fragment (scFv), single-domain antibody such
as a heavy chain variable domain (VH), a light chain variable
domain (VL) and a variable domain of camelid-type nanobody (VHH), a
sdAb and the like that bind the cytokine can also be used. Other
suitable antigen-binding domain that bind the cytokine can also be
used, include non-immunoglobulin proteins that mimic antibody
binding and/or structure such as, anticalins, affilins, affibody
molecules, affimers, affitins, alphabodies, avimers, DARPins,
fynomers, kunitz domain peptides, monobodies, and binding domains
based on other engineered scaffolds such as SpA, GroEL,
fibronectin, lipocalin and CTLA4 scaffolds. Further examples of
suitable blocking polypeptides include polypeptides that sterically
inhibit or block binding of the cytokine to its cognate receptor.
Advantageously, such moieties can also function as half-life
extending elements. For example, a peptide that is modified by
conjugation to a water-soluble polymer, such as PEG, can sterically
inhibit or prevent binding of the cytokine to its receptor.
Polypeptides, or fragments thereof, that have long serum half-lives
can also be used, such as serum albumin (human serum albumin),
immunoglobulin Fc, transferring and the like, as well as fragments
and muteins of such polypeptides.
[0262] Antibodies and antigen-binding domains that bind to, for
example, a protein with a long serum half-life such as HSA,
immunoglobulin or transferrin, or to a receptor that is recycled to
the plasma membrane, such as FcRn or transferrin receptor, can also
inhibit the cytokine, particularly when bound to their antigen.
Examples of such antigen-binding polypeptides include a single
chain variable fragment (scFv), single-domain antibody such as a
heavy chain variable domain (VH), a light chain variable domain
(VL) and a variable domain of camelid-type nanobody (VHH), a sdAb
and the like. Other suitable antigen-binding domain that bind the
cytokine can also be used, include non-immunoglobulin proteins that
mimic antibody binding and/or structure such as, anticalins,
affilins, affibody molecules, affimers, affitins, alphabodies,
avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and
binding domains based on other engineered scaffolds such as SpA,
GroEL, fibronectin, lipocalin and CTLA4 scaffolds.
[0263] In illustrative examples, when IL-2 is the cytokine in the
chimeric polypeptide, the blocking moiety can be the full length or
fragment or mutein of the alpha chain of IL-2 receptor
(IL-2R.alpha.) or beta (IL-2R.beta.) or gamma chain of IL-2
receptor (IL-2R.gamma.), an anti-IL-2 single-domain antibody (sdAb)
or scFv, an anti-CD25 antibody or fragment thereof, and anti-HSA
sdAb or scFv, and the like.
[0264] b. In Vivo Half-Life Extension Elements
[0265] Preferably, the chimeric polypeptides comprise an in vivo
half-life extension element. Increasing the in vivo half-life of
therapeutic molecules with naturally short half-lives allows for a
more acceptable and manageable dosing regimen without sacrificing
effectiveness. As used herein, a "half-life extension element" is a
part of the chimeric polypeptide that increases the in vivo
half-life and improve pK, for example, by altering its size (e.g.,
to be above the kidney filtration cutoff), shape, hydrodynamic
radius, charge, or parameters of absorption, biodistribution,
metabolism, and elimination. An exemplary way to improve the pK of
a polypeptide is by expression of an element in the polypeptide
chain that binds to receptors that are recycled to the plasma
membrane of cells rather than degraded in the lysosomes, such as
the FcRn receptor on endothelial cells and transferrin receptor.
Three types of proteins, e.g., human IgGs, HSA (or fragments), and
transferrin, persist for much longer in human serum than would be
predicted just by their size, which is a function of their ability
to bind to receptors that are recycled rather than degraded in the
lysosome. These proteins, or fragments of them that retain the FcRn
binding are routinely linked to other polypeptides to extend their
serum half-life. In one embodiment, the half-life extension element
is a human serum albumin (HSA) binding domain. HSA (SEQ ID NO:2)
may also be directly bound to the pharmaceutical compositions or
bound via a short linker. Fragments of HSA may also be used. HSA
and fragments thereof can function as both a blocking moiety and a
half-life extension element. Human IgGs can also carry out a
similar function.
[0266] The serum half-life extension element can also be
antigen-binding polypeptide that binds to a protein with a long
serum half-life such as serum albumin, transferrin and the like.
Examples of such polypeptides include antibodies and fragments
thereof including, a polyclonal antibody, a recombinant antibody, a
human antibody, a humanized antibody a single chain variable
fragment (scFv), single-domain antibody such as a heavy chain
variable domain (VH), a light chain variable domain (VL) and a
variable domain of camelid-type nanobody (VHH), a sdAb and the
like. Other suitable antigen-binding domain include
non-immunoglobulin proteins that mimic antibody binding and/or
structure such as, anticalins, affilins, affibody molecules,
affimers, affitins, alphabodies, avimers, DARPins, fynomers, kunitz
domain peptides, monobodies, and binding domains based on other
engineered scaffolds such as SpA, GroEL, fibronectin, lipocalin and
CTLA4 scaffolds. Further examples of antigen-binding polypeptides
include a ligand for a desired receptor, a ligand-binding portion
of a receptor, a lectin, and peptides that binds to or associates
with one or more target antigens.
[0267] Some preferred serum half-life extension elements are
polypeptides that comprise complementarity determining regions
(CDRs), and optionally non-CDR loops. Advantageously, such serum
half-life extension elements can extend the serum half-life of the
cytokine, and also function as inhibitors of the cytokine (e.g.,
via steric blocking, non-covalent interaction or combination
thereof) and/or as targeting domains. In some instances, the serum
half-life extension elements are domains derived from an
immunoglobulin molecule (Ig molecule) or engineered protein
scaffolds that mimic antibody structure and/or binding activity.
The Ig may be of any class or subclass (IgG1, IgG2, IgG3, IgG4,
IgA, IgE, IgM etc.). A polypeptide chain of an Ig molecule folds
into a series of parallel beta strands linked by loops. In the
variable region, three of the loops constitute the "complementarity
determining regions" (CDRs) which determine the antigen binding
specificity of the molecule. An IgG molecule comprises at least two
heavy (H) chains and two light (L) chains inter-connected by
disulfide bonds, or an antigen binding fragment thereof. Each heavy
chain is comprised of a heavy chain variable region (abbreviated
herein as 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 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 (CDRs)
with are hypervariable in sequence and/or involved in antigen
recognition and/or usually form structurally defined loops,
interspersed with regions that are more conserved, termed framework
regions (FR). Each VH and VL is composed of three CDRs and four
FRs, arranged from amino-terminus to carboxy-terminus in the
following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some
embodiments of this disclosure, at least some or all of the amino
acid sequences of FR1, FR2, FR3, and FR4 are part of the "non-CDR
loop" of the binding moieties described herein. A variable domain
of an immunoglobulin molecule has several beta strands that are
arranged in two sheets. The variable domains of both light and
heavy immunoglobulin chains contain three hypervariable loops, or
complementarity-determining regions (CDRs). e three CDRs of a V
domain (CDR1, CDR2, CDR3) cluster at one end of the beta barrel.
The CDRs are the loops that connect beta strands B-C, C'-C'', and
F-G of the immunoglobulin fold, whereas the bottom loops that
connect beta strands AB, CC', C''-D and E-F of the immunoglobulin
fold, and the top loop that connects the D-E strands of the
immunoglobulin fold are the non-CDR loops. In some embodiments of
this disclosure, at least some amino acid residues of a constant
domain, CH1, CH2, or CH3, are part of the "non-CDR loop" of the
binding moieties described herein. Non-CDR loops comprise, in some
embodiments, one or more of AB, CD, EF, and DE loops of a C1-set
domain of an Ig or an Ig-like molecule; AB, CC', EF, FG, BC, and
EC' loops of a C2-set domain of an Ig or an Ig-like molecule; DE,
BD, GF, A(A1A2)B, and EF loops of I(Intermediate)-set domain of an
Ig or Ig-like molecule.
[0268] Within the variable domain, the CDRs are believed to be
responsible for antigen recognition and binding, while the FR
residues are considered a scaffold for the CDRs. However, in
certain cases, some of the FR residues play an important role in
antigen recognition and binding. Framework region residues that
affect Ag binding are divided into two categories. The first are FR
residues that contact the antigen, thus are part of the
binding-site, and some of these residues are close in sequence to
the CDRs. Other residues are those that are far from the CDRs in
sequence but are in close proximity to it in the 3-D structure of
the molecule, e.g., a loop in heavy chain.
[0269] The binding moieties are any kinds of polypeptides. For
example, in certain instances the binding moieties are natural
peptides, synthetic peptides, or fibronectin scaffolds, or
engineered bulk serum proteins. The bulk serum protein comprises,
for example, albumin, fibrinogen, or a globulin. In some
embodiments, the binding moieties are engineered scaffolds.
Engineered scaffolds comprise, for example, a sdAb, a scFv, a Fab,
a VHH, a fibronectin type III domain, immunoglobulin-like scaffold
(as suggested in Halaby et al., 1999. Prot Eng 12(7):563-571),
DARPin, cysteine knot peptide, lipocalin, three-helix bundle
scaffold, protein G-related albumin-binding module, or a DNA or RNA
aptamer scaffold.
[0270] In some cases, the serum half-life extending element
comprises a binding site for a bulk serum protein. In some
embodiments, the CDRs provide the binding site for the bulk serum
protein. The bulk serum protein is, in some examples, a globulin,
albumin, transferrin, IgG1, IgG2, IgG4, IgG3, IgA monomer, Factor
XIII, Fibrinogen, IgE, or pentameric IgM. In some embodiments, the
CDR form a binding site for an immunoglobulin light chain, such as
an Ig.kappa. free light chain or an Ig.lamda. free light chain.
[0271] The serum half-life extension element can be any type of
binding domain, including but not limited to, domains from a
monoclonal antibody, a polyclonal antibody, a recombinant antibody,
a human antibody, a humanized antibody. In some embodiments, the
binding moiety is a single chain variable fragment (scFv),
single-domain antibody such as a heavy chain variable domain (VH),
a light chain variable domain (VL) and a variable domain (VHH) of
camelid derived nanobody. In other embodiments, the binding
moieties are non-Ig binding domains, i.e., antibody mimetic, such
as anticalins, affilins, affibody molecules, affimers, affitins,
alphabodies, avimers, DARPins, fynomers, kunitz domain peptides,
and monobodies.
[0272] In other embodiments, the serum half-life extension element
can be a water-soluble polymer or a peptide that is conjugated to a
water-soluble polymer, such as PEG. "PEG," "polyethylene glycol"
and "poly(ethylene glycol)" as used herein, are interchangeable and
encompass any nonpeptidic water-soluble poly(ethylene oxide). The
term "PEG" also means a polymer that contains a majority, that is
to say, greater than 50%, of --OCH.sub.2CH.sub.2-- repeating
subunits. With respect to specific forms, the PEG can take any
number of a variety of molecular weights, as well as structures or
geometries such as "branched," "linear," "forked,"
"multifunctional," and the like, to be described in greater detail
below. The PEG is not limited to a particular structure and can be
linear (e.g., an end capped, e.g., alkoxy PEG or a bifunctional
PEG), branched or multi-armed (e.g., forked PEG or PEG attached to
a polyol core), a dendritic (or star) architecture, each with or
without one or more degradable linkages. Moreover, the internal
structure of the PEG can be organized in any number of different
repeat patterns and can be selected from the group consisting of
homopolymer, alternating copolymer, random copolymer, block
copolymer, alternating tripolymer, random tripolymer, and block
tripolymer. PEGs can be conjugated to polypeptide and peptides
through any suitable method. Typically a reactive PEG derivative,
such as N-hydroxysuccinimidyl ester PEG, is reacted with a peptide
or polypeptide that includes amino acids with a side chain that
contains an amine, sulfhydryl, carboxylic acid or hydroxyl
functional group, such as cysteine, lysine, asparagine, glutamine,
threonine, tyrosine, serine, aspartic acid, and glutamic acid.
[0273] c. Targeting and Retention Domains
[0274] For certain applications, it may be desirable to maximize
the amount of time the construct is present in its desired location
in the body. This can be achieved by including one further domain
in the chimeric polypeptide (fusion protein) to influence its
movements within the body. For example, the chimeric nucleic acids
can encode a domain that directs the polypeptide to a location in
the body, e.g., tumor cells or a site of inflammation; this domain
is termed a "targeting domain" and/or encode a domain that retains
the polypeptide in a location in the body, e.g., tumor cells or a
site of inflammation; this domain is termed a "retention domain".
In some embodiments a domain can function as both a targeting and a
retention domain. In some embodiments, the targeting domain and/or
retention domain are specific to a protease-rich environment. In
some embodiments, the encoded targeting domain and/or retention
domain are specific for regulatory T cells (Tregs), for example
targeting the CCR4 or CD39 receptors. Other suitable targeting
and/or retention domains comprise those that have a cognate ligand
that is overexpressed in inflamed tissues, e.g., the IL-1 receptor,
or the IL-6 receptor. In other embodiments, the suitable targeting
and/or retention domains comprise those who have a cognate ligand
that is overexpressed in tumor tissue, e.g., Epcam, CEA or
mesothelin. In some embodiments, the targeting domain is linked to
the interleukin via a linker which is cleaved at the site of action
(e.g. by inflammation or cancer specific proteases) releasing the
interleukin full activity at the desired site. In some embodiments,
the targeting and/or retention domain is linked to the interleukin
via a linker which is not cleaved at the site of action (e.g. by
inflammation or cancer specific proteases), causing the cytokine to
remain at the desired site.
[0275] Antigens of choice, in some cases, are expressed on the
surface of a diseased cell or tissue, for example a tumor or a
cancer cell. Antigens useful for tumor targeting and retention
include but are not limited to EpCAM, EGFR, HER-2, HER-3, c-Met,
FoIR, and CEA. Pharmaceutical compositions disclosed herein, also
include proteins comprising two targeting and/or retention domains
that bind to two different target antigens known to be expressed on
a diseased cell or tissue. Exemplary pairs of antigen binding
domains include but are not limited to EGFR/CEA, EpCAM/CEA, and
HER-2/HER-3.
[0276] Suitable targeting and/or retention domains include
antigen-binding domains, such as antibodies and fragments thereof
including, a polyclonal antibody, a recombinant antibody, a human
antibody, a humanized antibody a single chain variable fragment
(scFv), single-domain antibody such as a heavy chain variable
domain (VH), a light chain variable domain (VL) and a variable
domain of camelid-type nanobody (VHH), a sdAb and the like. Other
suitable antigen-binding domain include non-immunoglobulin proteins
that mimic antibody binding and/or structure such as, anticalins,
affilins, affibody molecules, affimers, affitins, alphabodies,
avimers, DARPins, fynomers, kunitz domain peptides, monobodies, and
binding domains based on other engineered scaffolds such as SpA,
GroEL, fibronectin, lipocalin and CTLA4 scaffolds. Further examples
of antigen-binding polypeptides include a ligand for a desired
receptor, a ligand-binding portion of a receptor, a lectin, and
peptides that binds to or associates with one or more target
antigens.
[0277] In some embodiments, the targeting and/or retention domains
specifically bind to a cell surface molecule. In some embodiments,
the targeting and/or retention domains specifically bind to a tumor
antigen. In some embodiments, the targeting polypeptides
specifically and independently bind to a tumor antigen selected
from at least one of EpCAM, EGFR, HER-2, HER-3, cMet, CEA, and
FoIR. In some embodiments, the targeting polypeptides specifically
and independently bind to two different antigens, wherein at least
one of the antigens is a tumor antigen selected from EpCAM, EGFR,
HER-2, HER-3, cMet, CEA, and FoIR.
[0278] The targeting and/or retention antigen can be a tumor
antigen expressed on a tumor cell. Tumor antigens are well known in
the art and include, for example, EpCAM, EGFR, HER-2, HER-3, c-Met,
FolR, PSMA, CD38, BCMA, and CEA. 5T4, AFP, B7-H3, Cadherin-6, CAIX,
CD117, CD123, CD138, CD166, CD19, CD20, CD205, CD22, CD30, CD33,
CD352, CD37, CD44, CD52, CD56, CD70, CD71, CD74, CD79b, DLL3,
EphA2, FAP, FGFR2, FGFR3, GPC3, gpA33, FLT-3, gpNMB, HPV-16 E6,
HPV-16 E7, ITGA2, ITGA3, SLC39A6, MAGE, mesothelin, Muc1, Muc16,
NaPi2b, Nectin-4, P-cadherin, NY-ESO-1, PRLR, PSCA, PTK7, ROR1,
SLC44A4, SLTRK5, SLTRK6, STEAP1, TIM1, Trop2, WT1.
[0279] The targeting and/or retention antigen can be an immune
checkpoint protein. Examples of immune checkpoint proteins include
but are not limited to CD27, CD137, 2B4, TIGIT, CD155, ICOS, HVEM,
CD40L, LIGHT, TIM-1, OX40, DNAM-1, PD-L1, PD1, PD-L2, CTLA-4, CD8,
CD40, CEACAM1, CD48, CD70, A2AR, CD39, CD73, B7-H3, B7-H4, BTLA,
IDO1, IDO2, TDO, KIR, LAG-3, TIM-3, or VISTA.
[0280] The targeting and/or retention antigen can be a cell surface
molecule such as a protein, lipid or polysaccharide. In some
embodiments, a targeting and/or retention antigen is a on a tumor
cell, virally infected cell, bacterially infected cell, damaged red
blood cell, arterial plaque cell, inflamed or fibrotic tissue cell.
The targeting and/or retention antigen can comprise an immune
response modulator. Examples of immune response modulator include
but are not limited to granulocyte-macrophage colony stimulating
factor (GM-CSF), macrophage colony stimulating factor (M-CSF),
granulocyte colony stimulating factor (G-CSF), interleukin 2
(IL-2), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin
15 (IL-15), B7-1 (CD80), B7-2 (CD86), GITRL, CD3, or GITR.
[0281] The targeting and/or retention antigen can be a cytokine
receptor. Examples, of cytokine receptors include but are not
limited to Type I cytokine receptors, such as GM-CSF receptor,
G-CSF receptor, Type I IL receptors, Epo receptor, LIF receptor,
CNTF receptor, TPO receptor; Type II Cytokine receptors, such as
IFN-alpha receptor (IFNAR1, IFNAR2), IFB-beta receptor, IFN-gamma
receptor (IFNGR1, IFNGR2), Type II IL receptors; chemokine
receptors, such as CC chemokine receptors, CXC chemokine receptors,
CX3C chemokine receptors, XC chemokine receptors; tumor necrosis
receptor superfamily receptors, such as TNFRSF5/CD40, TNFRSF8/CD30,
TNFRSF7/CD27, TNFRSF1A/TNFR1/CD120a, TNFRSF1B/TNFR2/CD120b;
TGF-beta receptors, such as TGF-beta receptor 1, TGF-beta receptor
2; Ig super family receptors, such as IL-1 receptors, CSF-R, PDGFR
(PDGFRA, PDGFRB), SCFR.
[0282] d. Linkers
[0283] As stated above, the pharmaceutical compositions comprise
one or more linker sequences. A linker sequence serves to provide
flexibility between polypeptides, such that, for example, the
blocking moiety is capable of inhibiting the activity of the
cytokine polypeptide. The linker sequence can be located between
any or all of the cytokine polypeptide, the serum half-life
extension element, and/or the blocking moiety. As described herein,
at least one of the linkers is protease cleavable, and contains a
(one or more) cleavage site for a (one or more) desired protease.
Preferably, the desired protease is enriched or selectively
expressed at the desired site of cytokine activity (e.g., the tumor
microenvironment). Thus, the fusion protein is preferentially or
selectively cleaved at the site of desired cytokine activity.
[0284] The orientation of the components of the pharmaceutical
composition, are largely a matter of design choice and it is
recognized that multiple orientations are possible, and all are
intended to be encompassed by this disclosure. For example, a
blocking moiety can be located C-terminally or N-terminally to a
cytokine polypeptide.
[0285] Provided herein are pharmaceutical compositions comprising
polypeptide sequences. As with all peptides, polypeptides, and
proteins, including fragments thereof, it is understood that
additional modifications in the amino acid sequence of the chimeric
polypeptides (amino acid sequence variants) can occur that do not
alter the nature or function of the peptides, polypeptides, or
proteins. Such modifications include conservative amino acid
substitutions and are discussed in greater detail below.
[0286] The compositions provided herein have a desired function.
The compositions are comprised of at least a cytokine polypeptide,
such as IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, or IFN.gamma., or a
chemokine, such as CXCL10, CCL19, CCL20, CCL21, a blocking moiety,
e.g. a steric blocking polypeptide, and an optional serum half-life
extension element, and an optional targeting polypeptide, with one
or more linkers connecting each polypeptide in the composition. The
first polypeptide, e.g., an IL-2 mutein, is provided to be an
active agent. The blocking moiety is provided to block the activity
of the interleukin. The linker polypeptide, e.g., a protease
cleavable polypeptide, is provided to be cleaved by a protease that
is specifically expressed at the intended target of the active
agent. Optionally, the blocking moiety blocks the activity of the
first polypeptide by binding the interleukin polypeptide. In some
embodiments, the blocking moiety, e.g. a steric blocking peptide,
is linked to the interleukin via a protease-cleavable linker which
is cleaved at the site of action (e.g. by inflammation specific
proteases) releasing the cytokine full activity at the desired
site.
[0287] In some embodiments, the linker comprises glycine-glycine, a
sortase-recognition motif, or a sortase-recognition motif and a
peptide sequence (Gly.sub.4Ser).sub.n (SEQ ID NO: 238) or
(Gly.sub.3Ser).sub.n (SEQ ID NO: 239), wherein n is 1, 2, 3, 4 or
5. In one embodiment, the sortase-recognition motif comprises a
peptide sequence LPXTG, where X is any amino acid (SEQ ID NO: 237).
In one embodiment, the covalent linkage is between a reactive
lysine residue attached to the C-terminal of the cytokine
polypeptide and a reactive aspartic acid attached to the N-terminal
of the blocking or other moiety. In one embodiment, the covalent
linkage is between a reactive aspartic acid residue attached to the
N-terminal of the cytokine polypeptide and a reactive lysine
residue attached to the C-terminal of the blocking or other
moiety.
[0288] e. Cleavage and Inducibility
[0289] As described herein, the activity of the cytokine
polypeptide the context of the fusion protein is attenuated, and
protease cleavage at the desired site of activity, such as in a
tumor microenvironment, releases a form of the cytokine from the
fusion protein that is much more active as a cytokine receptor
agonist than the fusion protein. For example, the cytokine-receptor
activating (agonist) activity of the fusion polypeptide can be at
least about 10.times., at least about 50.times., at least about
100.times., at least about 250.times., at least about 500.times.,
or at least about 1000.times. less than the cytokine receptor
activating activity of the cytokine polypeptide as a separate
molecular entity. The cytokine polypeptide that is part of the
fusion protein exists as a separate molecular entity when it
contains an amino acid that is substantially identical to the
cytokine polypeptide and does not substantially include additional
amino acids and is not associated (by covalent or non-covalent
bonds) with other molecules. If necessary, a cytokine polypeptide
as a separate molecular entity may include some additional amino
acid sequences, such as a tag or short sequence to aid in
expression and/or purification.
[0290] In other examples, the cytokine-receptor activating
(agonist) activity of the fusion polypeptide is at least about
10.times., at least about 50.times., at least about 100.times., at
least about 250.times., at least about 500.times., or about
1000.times. less than the cytokine receptor activating activity of
the polypeptide that contains the cytokine polypeptide that is
produced by cleavage of the protease cleavable linker in the fusion
protein. In other words, the cytokine receptor activating (agonist)
activity of the polypeptide that contains the cytokine polypeptide
that is produced by cleavage of the protease cleavable linker in
the fusion protein is at least about 10.times., at least about
50.times., at least about 100.times., at least about 250.times., at
least about 500.times., or at least about 1000.times. greater than
the cytokine receptor activating activity of the fusion protein. In
other examples, a recombinant polypeptide is in conjugation with a
cleavable moiety wherein the cleavable moiety is cleaved with
reduced catalytic efficiency by one or more proteases than a
reference polypeptide sequence.
[0291] In some embodiments, the cleavable moiety is resistant to
proteolytic cleavage by one or more proteases. A cleavable moiety
is resistant to a protease if the sequence comprises a binding site
that is altered from the canonical cleavable motif sequence for the
specific protease. In some embodiments, a binding site is altered
compared to a reference sequence by making one or more
substitutions in the protease cleavage motif sequence. For example,
the protease cathepsin S cleaves the sequence GAVVRGA (SEQ ID NO:
240); a sequence is made to substitution can be made to substitute
the arginine (R) with a glutamine (Q), thus changing a charged
residue to a shorter, polar residue, reducing the ability of
cathepsin S to bind and cleave the sequence. Such semi-conservative
amino acid substitutions in protease target sequence motifs can
lead to reduced binding ability, and such altered sequence motifs
are therefore protease resistant. An uncleavable moiety can be made
by inserting a disruptive amino acid into the protease target
sequence motif, such as a proline (causes curves in the secondary
structure of the peptide) or histidine (causes steric interference
with other amino acid side chains). As used herein, a
"protease-resistant" peptide linker is one with reduced or
undetectable cleavage by one or more specified proteases. Exemplary
protease-resistant peptide linkers can be tested, e.g., in vitro by
incubation with a specific protease followed by analysis of the
digestion products by western blot.
[0292] f. Polypeptide Substitutions
[0293] The polypeptides described herein can include components
(e.g., the cytokine, the blocking moiety) that have the same amino
acid sequence of the corresponding naturally occurring protein
(e.g., IL-2, IL-15, HSA) or can have an amino acid sequence that
differs from the naturally occurring protein so long as the desired
function is maintained. It is understood that one way to define any
known modifications and derivatives or those that might arise, of
the disclosed proteins and nucleic acids that encode them is
through defining the sequence variants in terms of identity to
specific known reference sequences. Specifically disclosed are
polypeptides and nucleic acids which have at least, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity to the chimeric
polypeptides provided herein. For example, provided are
polypeptides or nucleic acids that have at least, 70, 71, 72, 73,
74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity to the sequence
of any of the nucleic acids or polypeptides described herein. This
includes polypeptides or nucleic acids that have at least, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent identity to the
sequence of the cleavable linkers provided herein. This also
includes variants of the linker or inducible polypeptides that
comprise 1, 2, 3, 4, 5, or 6 variants from the cleavage domain
sequences. Those of skill in the art readily understand how to
determine the identity of two polypeptides or two nucleic acids.
For example, the identity can be calculated after aligning the two
sequences so that the identity is at its highest level.
[0294] Another way of calculating identity can be performed by
published algorithms. Optimal alignment of sequences for comparison
may be conducted by the local identity algorithm of Smith and
Waterman Adv. Appl. Math. 2:482 (1981), by the identity alignment
algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by
the search for similarity method of Pearson and Lipman, Proc. Natl.
Acad. Sci. USA 85:2444 (1988), by computerized implementations of
these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin
Genetics Software Package, Genetics Computer Group, 575 Science
Dr., Madison, Wis.), or by inspection.
[0295] The same types of identity can be obtained for nucleic acids
by, for example, the algorithms disclosed in Zuker, Science
244:48-52 (1989); Jaeger et al., Proc. Natl. Acad. Sci. USA
86:7706-7710 (1989); Jaeger et al., Methods Enzymol. 183:281-306
(1989), which are herein incorporated by reference for at least
material related to nucleic acid alignment. It is understood that
any of the methods typically can be used and that in certain
instances the results of these various methods may differ, but the
skilled artisan understands if identity is found with at least one
of these methods, the sequences would be said to have the stated
identity, and be disclosed herein.
[0296] Protein modifications include amino acid sequence
modifications. Modifications in amino acid sequence may arise
naturally as allelic variations (e.g., due to genetic
polymorphism), may arise due to environmental influence (e.g., by
exposure to ultraviolet light), or may be produced by human
intervention (e.g., by mutagenesis of cloned DNA sequences), such
as induced point, deletion, insertion and substitution mutants.
These modifications can result in changes in the amino acid
sequence, provide silent mutations, modify a restriction site, or
provide other specific mutations. Amino acid sequence modifications
typically fall into one or more of three classes: substitutional,
insertional or deletional modifications. Insertions include amino
and/or carboxyl terminal fusions as well as intrasequence
insertions of single or multiple amino acid residues. Insertions
ordinarily will be smaller insertions than those of amino or
carboxyl terminal fusions, for example, on the order of one to four
residues. Deletions are characterized by the removal of one or more
amino acid residues from the protein sequence. Typically, no more
than about from 2 to 6 residues are deleted at any one site within
the protein molecule. Amino acid substitutions are typically of
single residues but can occur at a number of different locations at
once; insertions usually will be on the order of about from 1 to 10
amino acid residues; and deletions will range about from 1 to 30
residues. Deletions or insertions preferably are made in adjacent
pairs, i.e. a deletion of 2 residues or insertion of 2 residues.
Substitutions, deletions, insertions or any combination thereof may
be combined to arrive at a final construct. The mutations must not
place the sequence out of reading frame and preferably will not
create complementary regions that could produce secondary mRNA
structure. Substitutional modifications are those in which at least
one residue has been removed and a different residue inserted in
its place. Such substitutions generally are made in accordance with
the following Table 2 and are referred to as conservative
substitutions.
TABLE-US-00006 TABLE 2 Exemplary amino acid substitutions Amino
Acid Exemplary Substitutions Ala Ser, Gly, Cys Arg Lys, Gln, Met,
Ile Asn Gln, His, Glu, Asp Asp Glu, Asn, Gln Cys Ser, Met, Thr Gln
Asn, Lys, Glu, Asp Glu Asp, Asn, Gln Gly Pro, Ala His Asn, Gln Ile
Leu, Val, Met Leu Ile, Val, Met Lys Arg, Gln, Met, Ile Met Leu,
Ile, Val Phe Met, Leu, Tyr, Trp, His Ser Thr, Met, Cys Thr Ser,
Met, Val Trp Tyr, Phe Tyr Trp, Phe, His Val Ile, Leu, Met
[0297] Modifications, including the specific amino acid
substitutions, are made by known methods. For example,
modifications are made by site specific mutagenesis of nucleotides
in the DNA encoding the polypeptide, thereby producing DNA encoding
the modification, and thereafter expressing the DNA in recombinant
cell culture. Techniques for making substitution mutations at
predetermined sites in DNA having a known sequence are well known,
for example M13 primer mutagenesis and PCR mutagenesis.
[0298] Modifications can be selected to optimize binding. For
example, affinity maturation techniques can be used to alter
binding of the scFv by introducing random mutations inside the
complementarity determining regions (CDRs). Such random mutations
can be introduced using a variety of techniques, including
radiation, chemical mutagens or error-prone PCR. Multiple rounds of
mutation and selection can be performed using, for example, phage
display.
[0299] The disclosure also relates to nucleic acids that encode the
chimeric polypeptides described herein, and to the use of such
nucleic acids to produce the chimeric polypeptides and for
therapeutic purposes. For example, the invention includes DNA and
RNA molecules (e.g., mRNA, self-replicating RNA) that encode a
chimeric polypeptide and to the therapeutic use of such DNA and RNA
molecules.
[0300] g. Exemplary Compositions
[0301] Exemplary fusion proteins of the invention combine the above
described elements in a variety of orientations. The orientations
described in this section are meant as examples and are not to be
considered limiting.
[0302] In some embodiments, the fusion protein comprises a
cytokine, a blocking moiety and a half-life extension element. In
some embodiments, the cytokine is positioned between the half-life
extension element and the blocking moiety. In some embodiments, the
cytokine is N-terminal to the blocking moiety and the half-life
extension element. In some such embodiments, the cytokine is
proximal to the blocking moiety; in some such embodiments, the
cytokine is proximal to the half-life extension element. At least
one protease-cleavable linker must be included in all embodiments,
such that the cytokine may be active upon cleavage. In some
embodiments, the cytokine is C-terminal to the blocking moiety and
the half-life extension element. Additional elements may be
attached to one another by a cleavable linker, a non-cleavable
linker, or by direct fusion.
[0303] In some embodiments, the blocking domains used are capable
of extending half-life, and the cytokine is positioned between two
such blocking domains. In some embodiments, the cytokine is
positioned between two blocking domains, one of which is capable of
extending half-life.
[0304] In some embodiments, two cytokines are included in the same
construct. In some embodiments, the cytokines are connected to two
blocking domains each (three in total in one molecule), with a
blocking domain between the two cytokine domains. In some
embodiments, one or more additional half-life extension domains may
be included to optimize pharmacokinetic properties.
[0305] In some embodiments, three cytokines are included in the
same construct. In some embodiments, the third cytokine may
function to block the other two in place of a blocking domain
between the two cytokines.
[0306] Preferred half-life extension elements for use in the fusion
proteins are human serum albumin (HSA), an antibody or antibody
fragment (e.g., scFV, dAb) which binds serum albumin, a human or
humanized IgG, or a fragment of any of the foregoing. In some
preferred embodiments, the blocking moiety is human serum albumin
(HSA), or an antibody or antibody fragment which binds serum
albumin, an antibody which binds the cytokine and prevents
activation of binding or activation of the cytokine receptor,
another cytokine, or a fragment of any of the foregoing. In
preferred embodiments comprising an additional targeting domain,
the targeting domain is an antibody which binds a cell surface
protein which is enriched on the surface of cancer cells, such as
EpCAM, FOLR1, and Fibronectin.
[0307] vii. Other Uses
[0308] The separation moieties disclosed herein can be used for
antibody-antibiotic conjugates. The separation moiety disclosed
herein connects or links an antimicrobial antibiotic to an antibody
specific for a bacterial strain (e.g., Staphylococcus aureus Ab).
The antibody-antibiotic conjugate does not display antibacterial
activity when the antibody is linked to the antibiotic. However,
upon internalization into host cells, the separation moiety is
cleaved by proteases releasing the free antibiotic. The free
antibiotic kills the intracellular bacteria.
[0309] The separation moieties disclosed herein can be used for
applications in chemical probes use for detection and isolation of
proteins. Chemical probes are designed based on small molecule
interaction with proteins. The probes typically comprise a covalent
binding motif in order for the probe to interact with the target
protein, a detection/purification tag for
visualization/purification of the protein target and a linker
group. The separation moieties described herein can be incorporated
to enable the target protein to be detected and isolated.
[0310] C. Methods of Treatment and Pharmaceutical Compositions
[0311] Further provided are methods of treating a subject with or
at risk of developing an of a disease or disorder, such as
proliferative disease, a tumorous disease, an inflammatory disease,
an immunological disorder, an autoimmune disease, an infectious
disease, a viral disease, an allergic reaction, a parasitic
reaction, or graft-versus-host disease. The methods administering
to a subject in need thereof an effective amount of a fusion
protein as disclosed herein that is typically administered as a
pharmaceutical composition. In some embodiments, the method further
comprises selecting a subject with or at risk of developing such a
disease or disorder. The pharmaceutical composition preferably
comprises a blocked cytokine, fragment or mutein thereof that is
activated at a site of inflammation. In one embodiment, the
chimeric polypeptide comprises a cytokine polypeptide, fragment or
mutein thereof and a serum half-life extension element. In another
embodiment, the chimeric polypeptide comprises a cytokine
polypeptide, fragment or mutein thereof and a blocking moiety, e.g.
a steric blocking polypeptide, wherein the steric blocking
polypeptide is capable of sterically blocking the activity of the
cytokine polypeptide, fragment or mutein thereof. In another
embodiment, the chimeric polypeptide comprises a cytokine
polypeptide, fragment or mutein thereof, a blocking moiety, and a
serum half-life extension element.
[0312] Inflammation is part of the complex biological response of
body tissues to harmful stimuli, such as pathogens, damaged cells,
or irritants, and is a protective response involving immune cells,
blood vessels, and molecular mediators. The function of
inflammation is to eliminate the initial cause of cell injury,
clear out necrotic cells and tissues damaged from the original
insult and the inflammatory process, and to initiate tissue repair.
Inflammation can occur from infection, as a symptom or a disease,
e.g., cancer, atherosclerosis, allergies, myopathies, HIV, obesity,
or an autoimmune disease. An autoimmune disease is a chronic
condition arising from an abnormal immune response to a
self-antigen. Autoimmune diseases that may be treated with the
polypeptides disclosed herein include but are not limited to lupus,
celiac disease, diabetes mellitus type 1, Graves' disease,
inflammatory bowel disease, multiple sclerosis, psoriasis,
rheumatoid arthritis, and systemic lupus erythematosus.
[0313] The pharmaceutical composition can comprise one or more
protease-cleavable linker sequences. The linker sequence serves to
provide flexibility between polypeptides, such that each
polypeptide is capable of inhibiting the activity of the first
polypeptide. The linker sequence can be located between any or all
of the cytokine polypeptide, fragment or mutein thereof, the
blocking moiety, and serum half-life extension element. Optionally,
the composition comprises, two, three, four, or five linker
sequences. The linker sequence, two, three, or four linker
sequences can be the same or different linker sequences. In one
embodiment, the linker sequence comprises GGGGS (SEQ ID NO: 232),
GSGSGS (SEQ ID NO: 233), or G(SGGG).sub.2SGGT (SEQ ID NO: 234). In
another embodiment, the linker comprises a protease-cleavable
sequence selected from group consisting of HSSKLQ (SEQ ID NO: 25),
GPLGVRG (SEQ ID NO:221), IPVSLRSG (SEQ ID NO: 222), VPLSLYSG (SEQ
ID NO: 223), or SGESPAYYTA (SEQ ID NO: 224). In some embodiments,
the linker is cleaved by a protease selected from the group
consisting of a kallikrein, thrombin, chymase, carboxypeptidase A,
cathepsin G, an elastase, PR-3, granzyme M, a calpain, a matrix
metalloproteinase (MMP), a plasminogen activator, a cathepsin, a
caspase, a tryptase, or a tumor cell surface protease.
[0314] Further provided are methods of treating a subject with or
at risk of developing cancer. The methods comprise administering to
the subject in need thereof an effective amount of a chimeric
polypeptide (a fusion protein) as disclosed herein that is
typically administered as a pharmaceutical composition. In some
embodiments, the method further comprises selecting a subject with
or at risk of developing cancer. The pharmaceutical composition
preferably comprises a blocked cytokine, fragment or mutein thereof
that is activated at a tumor site. Preferably, the tumor is a solid
tumor. The cancer may be a colon cancer, a lung cancer, a melanoma,
a sarcoma, a renal cell carcinoma, a breast cancer,
[0315] The method can further involve the administration of one or
more additional agents to treat cancer, such as chemotherapeutic
agents (e.g., Adriamycin, Cerubidine, Bleomycin, Alkeran, Velban,
Oncovin, Fluorouracil, Thiotepa, Methotrexate, Bisantrene,
Noantrone, Thiguanine, Cytaribine, Procarabizine), immuno-oncology
agents (e.g., anti-PD-L1, anti-CTLA4, anti-PD-1, anti-CD47,
anti-GD2), cellular therapies (e.g, CAR-T, T-cell therapy),
oncolytic viruses and the like.
[0316] Provided herein are pharmaceutical formulations or
compositions containing the chimeric polypeptides and a
pharmaceutically acceptable carrier. The herein provided
compositions are suitable for administration in vitro or in vivo.
By pharmaceutically acceptable carrier is meant a material that is
not biologically or otherwise undesirable, i.e., the material is
administered to a subject without causing undesirable biological
effects or interacting in a deleterious manner with the other
components of the pharmaceutical formulation or composition in
which it is contained. The carrier is selected to minimize
degradation of the active ingredient and to minimize adverse side
effects in the subject.
[0317] Suitable carriers and their formulations are described in
Remington: The Science and Practice ofPharmacy 21.sup.st Edition,
David B. Troy, ed., Lippicott Williams & Wilkins (2005).
Typically, an appropriate amount of a pharmaceutically acceptable
salt is used in the formulation to render the formulation isotonic,
although the formulate can be hypertonic or hypotonic if desired.
Examples of the pharmaceutically acceptable carriers include, but
are not limited to, sterile water, saline, buffered solutions like
Ringer's solution, and dextrose solution. The pH of the solution is
generally about 5 to about 8 or from about 7 to 7.5. Other carriers
include sustained release preparations such as semipermeable
matrices of solid hydrophobic polymers containing the immunogenic
polypeptides. Matrices are in the form of shaped articles, e.g.,
films, liposomes, or microparticles. Certain carriers may be more
preferable depending upon, for instance, the route of
administration and concentration of composition being administered.
Carriers are those suitable for administration of the chimeric
polypeptides or nucleic acid sequences encoding the chimeric
polypeptides to humans or other subjects.
[0318] The pharmaceutical formulations or compositions are
administered in a number of ways depending on whether local or
systemic treatment is desired and, on the area to be treated. The
compositions are administered via any of several routes of
administration, including topically, orally, parenterally,
intravenously, intra-articularly, intraperitoneally,
intramuscularly, subcutaneously, intracavity, transdermally,
intrahepatically, intracranially, nebulization/inhalation, or by
installation via bronchoscopy. In some embodiments, the
compositions are administered locally (non-systemically), including
intratumorally, intra-articularly, intrathecally, etc.
[0319] Preparations for parenteral administration include sterile
aqueous or non-aqueous solutions, suspensions, and emulsions.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oils such as olive oil, and injectable organic
esters such as ethyl oleate. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions or suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringer's dextrose, dextrose and sodium chloride,
lactated Ringer's, or fixed oils. Intravenous vehicles include
fluid and nutrient replenishers, electrolyte replenishers (such as
those based on Ringer's dextrose), and the like. Preservatives and
other additives are optionally present such as, for example,
antimicrobials, antioxidants, chelating agents, and inert gases and
the like.
[0320] Formulations for topical administration include ointments,
lotions, creams, gels, drops, suppositories, sprays, liquids, and
powders. Conventional pharmaceutical carriers, aqueous, powder, or
oily bases, thickeners and the like are optionally necessary or
desirable.
[0321] Compositions for oral administration include powders or
granules, suspension or solutions in water or non-aqueous media,
capsules, sachets, or tables. Thickeners, flavorings, diluents,
emulsifiers, dispersing aids or binders are optionally
desirable.
[0322] Optionally, the chimeric polypeptides or nucleic acid
sequences encoding the chimeric polypeptides are administered by a
vector. There are a number of compositions and methods which can be
used to deliver the nucleic acid molecules and/or polypeptides to
cells, either in vitro or in vivo via, for example, expression
vectors. These methods and compositions can largely be broken down
into two classes: viral based delivery systems and non-viral based
delivery systems. Such methods are well known in the art and
readily adaptable for use with the compositions and methods
described herein. Such compositions and methods can be used to
transfect or transduce cells in vitro or in vivo, for example, to
produce cell lines that express and preferably secrete the encoded
chimeric polypeptide or to therapeutically deliver nucleic acids to
a subject. The components of the chimeric nucleic acids disclosed
herein typically are operably linked in frame to encode a fusion
protein.
[0323] As used herein, plasmid or viral vectors are agents that
transport the disclosed nucleic acids into the cell without
degradation and include a promoter yielding expression of the
nucleic acid molecule and/or polypeptide in the cells into which it
is delivered. Viral vectors are, for example, Adenovirus,
Adeno-associated virus, herpes virus, Vaccinia virus, Polio virus,
Sindbis, and other RNA viruses, including these viruses with the
HIV backbone. Also preferred are any viral families which share the
properties of these viruses which make them suitable for use as
vectors. Retroviral vectors, in general are described by Coffin et
al., Retroviruses, Cold Spring Harbor Laboratory Press (1997),
which is incorporated by reference herein for the vectors and
methods of making them. The construction of replication-defective
adenoviruses has been described (Berkner et al., J. Virol.
61:1213-20 (1987); Massie et al., Mol. Cell. Biol. 6:2872-83
(1986); Haj-Ahmad et al., J. Virol. 57:267-74 (1986); Davidson et
al., J. Virol. 61:1226-39 (1987); Zhang et al., BioTechniques
15:868-72 (1993)). The benefit and the use of these viruses as
vectors is that they are limited in the extent to which they can
spread to other cell types, since they can replicate within an
initial infected cell, but are unable to form new infectious viral
particles. Recombinant adenoviruses have been shown to achieve high
efficiency after direct, in vivo delivery to airway epithelium,
hepatocytes, vascular endothelium, CNS parenchyma, and a number of
other tissue sites. Other useful systems include, for example,
replicating and host-restricted non-replicating vaccinia virus
vectors.
[0324] The provided polypeptides and/or nucleic acid molecules can
be delivered via virus like particles. Virus like particles (VLPs)
consist of viral protein(s) derived from the structural proteins of
a virus. Methods for making and using virus like particles are
described in, for example, Garcea and Gissmann, Current Opinion in
Biotechnology 15:513-7 (2004).
[0325] The provided polypeptides can be delivered by subviral dense
bodies (DBs). DBs transport proteins into target cells by membrane
fusion. Methods for making and using DBs are described in, for
example, Pepperl-Klindworth et al., Gene Therapy 10:278-84
(2003).
[0326] The provided polypeptides can be delivered by tegument
aggregates. Methods for making and using tegument aggregates are
described in International Publication No. WO 2006/110728.
[0327] Non-viral based delivery methods can include expression
vectors comprising nucleic acid molecules and nucleic acid
sequences encoding polypeptides, wherein the nucleic acids are
operably linked to an expression control sequence. Suitable vector
backbones include, for example, those routinely used in the art
such as plasmids, artificial chromosomes, BACs, YACs, or PACs.
Numerous vectors and expression systems are commercially available
from such corporations as Novagen (Madison, Wis.), Clonetech (Pal
Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen/Life
Technologies (Carlsbad, Calif.). Vectors typically contain one or
more regulatory regions. Regulatory regions include, without
limitation, promoter sequences, enhancer sequences, response
elements, protein recognition sites, inducible elements, protein
binding sequences, 5' and 3' untranslated regions (UTRs),
transcriptional start sites, termination sequences, polyadenylation
sequences, and introns. Such vectors can also be used to make the
chimeric polypeptides by expression is a suitable host cell, such
as CHO cells.
[0328] Preferred promoters controlling transcription from vectors
in mammalian host cells may be obtained from various sources, for
example, the genomes of viruses such as polyoma, Simian Virus 40
(SV40), adenovirus, retroviruses, hepatitis B virus, and most
preferably cytomegalovirus (CMV), or from heterologous mammalian
promoters, e.g. .beta.-actin promoter or EF1.alpha. promoter, or
from hybrid or chimeric promoters (e.g., CMV promoter fused to the
.beta.-actin promoter). Of course, promoters from the host cell or
related species are also useful herein.
[0329] Enhancer generally refers to a sequence of DNA that
functions at no fixed distance from the transcription start site
and can be either 5' or 3' to the transcription unit. Furthermore,
enhancers can be within an intron as well as within the coding
sequence itself. hey are usually between 10 and 300 base pairs (bp)
in length, and they function in cis. Enhancers usually function to
increase transcription from nearby promoters. Enhancers can also
contain response elements that mediate the regulation of
transcription. While many enhancer sequences are known from
mammalian genes (globin, elastase, albumin, fetoprotein, and
insulin), typically one will use an enhancer from a eukaryotic cell
virus for general expression. Preferred examples are the SV40
enhancer on the late side of the replication origin, the
cytomegalovirus early promoter enhancer, the polyoma enhancer on
the late side of the replication origin, and adenovirus
enhancers.
[0330] The promoter and/or the enhancer can be inducible (e.g.
chemically or physically regulated). A chemically regulated
promoter and/or enhancer can, for example, be regulated by the
presence of alcohol, tetracycline, a steroid, or a metal. A
physically regulated promoter and/or enhancer can, for example, be
regulated by environmental factors, such as temperature and light.
Optionally, the promoter and/or enhancer region can act as a
constitutive promoter and/or enhancer to maximize the expression of
the region of the transcription unit to be transcribed. In certain
vectors, the promoter and/or enhancer region can be active in a
cell type specific manner. Optionally, in certain vectors, the
promoter and/or enhancer region can be active in all eukaryotic
cells, independent of cell type. Preferred promoters of this type
are the CMV promoter, the SV40 promoter, the .beta.-actin promoter,
the EF1.alpha. promoter, and the retroviral long terminal repeat
(LTR).
[0331] The vectors also can include, for example, origins of
replication and/or markers. A marker gene can confer a selectable
phenotype, e.g., antibiotic resistance, on a cell. The marker
product is used to determine if the vector has been delivered to
the cell and once delivered is being expressed. Examples of
selectable markers for mammalian cells are dihydrofolate reductase
(DHFR), thymidine kinase, neomycin, neomycin analog G418,
hygromycin, puromycin, and blasticidin. When such selectable
markers are successfully transferred into a mammalian host cell,
the transformed mammalian host cell can survive if placed under
selective pressure. Examples of other markers include, for example,
the E. coli lacZ gene, green fluorescent protein (GFP), and
luciferase. In addition, an expression vector can include a tag
sequence designed to facilitate manipulation or detection (e.g.,
purification or localization) of the expressed polypeptide. Tag
sequences, such as GFP, glutathione S-transferase (GST),
polyhistidine, c-myc, hemagglutinin, or FLAG.TM. tag (Kodak; New
Haven, Conn.) sequences typically are expressed as a fusion with
the encoded polypeptide. Such tags can be inserted anywhere within
the polypeptide including at either the carboxyl or amino
terminus.
[0332] As used herein, the terms peptide, polypeptide, or protein
are used broadly to mean two or more amino acids linked by a
peptide bond. Protein, peptide, and polypeptide are also used
herein interchangeably to refer to amino acid sequences. It should
be recognized that the term polypeptide is not used herein to
suggest a particular size or number of amino acids comprising the
molecule and that a peptide of the invention can contain up to
several amino acid residues or more. As used throughout, subject
can be a vertebrate, more specifically a mammal (e.g. a human,
horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and
guinea pig), birds, reptiles, amphibians, fish, and any other
animal. The term does not denote a particular age or sex. Thus,
adult and newborn subjects, whether male or female, are intended to
be covered. As used herein, patient or subject may be used
interchangeably and can refer to a subject with a disease or
disorder (e.g. cancer). The term patient or subject includes human
and veterinary subjects.
[0333] A subject at risk of developing a disease or disorder can be
genetically predisposed to the disease or disorder, e.g., have a
family history or have a mutation in a gene that causes the disease
or disorder, or show early signs or symptoms of the disease or
disorder. A subject currently with a disease or disorder has one or
more than one symptom of the disease or disorder and may have been
diagnosed with the disease or disorder.
[0334] The methods and agents as described herein are useful for
both prophylactic and therapeutic treatment. For prophylactic use,
a therapeutically effective amount of the chimeric polypeptides or
chimeric nucleic acid sequences encoding the chimeric polypeptides
described herein are administered to a subject prior to onset
(e.g., before obvious signs of cancer or inflammation) or during
early onset (e.g., upon initial signs and symptoms of cancer or
inflammation). Prophylactic administration can occur for several
days to years prior to the manifestation of symptoms of cancer or
inflammation. Prophylactic administration can be used, for example,
in the preventative treatment of subjects diagnosed with a genetic
predisposition to cancer. Therapeutic treatment involves
administering to a subject a therapeutically effective amount of
the chimeric polypeptides or nucleic acid sequences encoding the
chimeric polypeptides described herein after diagnosis or
development of cancer or inflammation (e.g., an autoimmune
disease). Prophylactic use may also apply when a patient is
undergoing a treatment, e.g., a chemotherapy, in which inflammation
is expected.
[0335] According to the methods taught herein, the subject is
administered an effective amount of the agent (e.g., a chimeric
polypeptide). The terms effective amount and effective dosage are
used interchangeably. The term effective amount is defined as any
amount necessary to produce a desired physiologic response.
Effective amounts and schedules for administering the agent may be
determined empirically, and making such determinations is within
the skill in the art. The dosage ranges for administration are
those large enough to produce the desired effect in which one or
more symptoms of the disease or disorder are affected (e.g.,
reduced or delayed). The dosage should not be so large as to cause
substantial adverse side effects, such as unwanted cross-reactions,
anaphylactic reactions, and the like. Generally, the dosage will
vary with the age, condition, sex, type of disease, the extent of
the disease or disorder, route of administration, or whether other
drugs are included in the regimen, and can be determined by one of
skill in the art. The dosage can be adjusted by the individual
physician in the event of any contraindications. Dosages can vary
and can be administered in one or more dose administrations daily,
for one or several days. Guidance can be found in the literature
for appropriate dosages for given classes of pharmaceutical
products.
[0336] As used herein the terms treatment, treat, or treating
refers to a method of reducing the effects of a disease or
condition or symptom of the disease or condition. Thus, in the
disclosed method, treatment can refer to a 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, or 100% reduction in the severity of an
established disease or condition or symptom of the disease or
condition. For example, a method for treating a disease is
considered to be a treatment if there is a 10% reduction in one or
more symptoms of the disease in a subject as compared to a control.
Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 100%, or any percent reduction in between 10% and 100% as
compared to native or control levels. It is understood that
treatment does not necessarily refer to a cure or complete ablation
of the disease, condition, or symptoms of the disease or
condition.
[0337] As used herein, the terms prevent, preventing, and
prevention of a disease or disorder refers to an action, for
example, administration of the chimeric polypeptide or nucleic acid
sequence encoding the chimeric polypeptide, that occurs before or
at about the same time a subject begins to show one or more
symptoms of the disease or disorder, which inhibits or delays onset
or exacerbation of one or more symptoms of the disease or disorder.
As used herein, references to decreasing, reducing, or inhibiting
include a change of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or
greater as compared to a control level. Such terms can include but
do not necessarily include complete elimination.
[0338] IL-2 variants have been developed that are selective for
IL2R.alpha..beta..gamma. relative to IL2R.beta..gamma. (Shanafelt,
A. B., et al., 2000, Nat Biotechnol. 18:1197-202; Cassell, D. J.,
et. al., 2002, Curr Pharm Des., 8:2171-83). These variants have
amino acid substitutions which reduce their affinity for IL2RB.
Because IL-2 has undetectable affinity for IL2RG, these variants
consequently have reduced affinity for the IL2R.beta..gamma.
receptor complex and reduced ability to activate
IL2R.beta..gamma.-expressing cells but retain the ability to bind
IL2RA and the ability to bind and activate the
IL2R.alpha..beta..gamma. receptor complex.
[0339] One of these variants, IL2/N88R (Bay 50-4798), was
clinically tested as a low-toxicity version of IL-2 as an immune
system stimulator, based on the hypothesis that
IL2R.beta..gamma.-expressing NK cells are a major contributor to
toxicity. Bay 50-4798 was shown to selectively stimulate the
proliferation of activated T cells relative to NK cells, and was
evaluated in phase I/I clinical trials in cancer patients
(Margolin, K., et. al., 2007, Clin Cancer Res., 13:3312-9) and HIV
patients (Davey, R. T., et. al., 2008, J Interferon Cytokine Res.,
28:89-100). These clinical trials showed that Bay 50-4798 was
considerably safer and more tolerable than aldesleukin, and also
showed that it increased the levels of CD4+CD25+ T cells, a cell
population enriched in Treg cells. Subsequent to these trials,
research in the field more fully established the identity of Treg
cells and demonstrated that Treg cells selectively express
IL2R.alpha..beta..gamma. (reviewed in Malek, T. R., et al., 2010,
Immunity, 33:153-65). Based on this new research, it can now be
understood that IL2R.alpha..beta..gamma. selective agonists should
be selective for Treg cells.
[0340] In addition, mutants can be made that selectively alter the
affinity for the CD25 chain relative to native I1-2.
[0341] IL-2 can be engineered to produce mutants that bind the
IL-2R complex generally or the IL-2Ra subunit specifically with an
affinity that differs from that of the corresponding wild-type IL-2
or of a presently available mutant (referred to as C125S, as the
cysteine residue at position 125 is replaced with a serine
residue).
[0342] Accordingly, the present invention features mutant
interleukin-2 (IL-2*) polypeptides that include an amino acid
sequence that is at least 80% identical to wild-type IL-2 (e.g.,
85, 87, 90, 95, 97, 98, or 99% identical) and that bind, as
compared to WT IL-2, with higher to the IL-2 trimeric receptor
relative to the dimeric IL-2 receptor. Typically, the muteins will
also bind an IL-2 receptor .alpha. subunit (IL-2R.alpha.) with an
affinity that is greater than the affinity with which wild type
IL-2 binds the IL-2R.alpha.. The amino acid sequence within mutant
IL-2 polypeptides can vary from SEQ ID NO:1 (UniProtKB accession
number P60568) by virtue of containing (or only containing) one or
more amino acid substitutions, which may be considered conservative
or non-conservative substitutions. Non-naturally occurring amino
acids can also be incorporated. Alternatively, or in addition, the
amino acid sequence can vary from SEQ ID NO:1 (which may be
considered the "reference" sequence) by virtue of containing and
addition and/or deletion of one or more amino acid residues. More
specifically, the amino acid sequence can differ from that of SEQ
ID NO:1 by virtue of a mutation at least one of the following
positions of SEQ ID NO:1: 1, 4, 8, 9, 10, 11, 13, 15, 26, 29, 30,
31, 35, 37, 46, 48, 49, 54, 61, 64, 67, 68, 69, 71, 73, 74, 75, 76,
79, 88, 89, 90, 92, 99, 101, 103, 114, 125, 128, or 133 (or
combinations thereof). As noted, as few as one of these positions
may be altered, as may two, three, four, five, six, seven, eight,
nine, ten, or 11 or more (including up to all) of the positions.
For example, the amino acid sequence can differ from SEQ ID NO:1 at
positions 69 and 74 and further at one or more of positions 30, 35,
and 128. The amino acid sequence can also differ from SEQ ID NO:2
(as disclosed in U.S. Pat. No. 7,569,215, incorporated herein by
reference) at one of the following sets of positions: (a) positions
64, 69, and 74; (b) positions 69, 74, and 101; (c) positions 69,
74, and 128; (d) positions 30, 69, 74, and 103; (e) positions 49,
69, 73, and 76; (f) positions 69, 74, 101, and 133; (g) positions
30, 69, 74, and 128; (h) positions 69, 74, 88, and 99; (i)
positions 30, 69, 74, and 128; (j) positions 9, 11, 35, 69, and 74;
(k) positions 1, 46, 49, 61, 69, and 79; (l) positions 48, 68, 71,
90, 103, and 114; (m) positions 4, 10, 11, 69, 74, 88, and 133; (n)
positions 15, 30 31, 35, 48, 69, 74, and 92; (O) positions 30, 68,
69, 71, 74, 75, 76, and 90; (p) positions 30, 31, 37, 69, 73, 74,
79, and 128; (q) positions 26, 29, 30, 54, 67, 69, 74, and 92; (r)
positions 8, 13, 26, 30, 35, 37, 69, 74, and 92; and (s) positions
29, 31, 35, 37, 48, 69, 71, 74, 88, and 89. Aside from mutations at
these positions, the amino acid sequence of the mutant IL-2
polypeptide can otherwise be identical to SEQ ID NO:1. With respect
to specific substitutions, the amino acid sequence can differ from
SEQ ID NO:1 by virtue of having one or more of the following
mutations: A1T, S4P, K8R, K9T, T10A, Q11R, Q13R, E15K, N26D, N29S,
N30S, N30D, N30T, Y31H, Y31C, K35R, T37A, T37R, M46L, K48E, K49R,
K49E, K54R, E61D, K64R, E67G, E68D, V69A, N71T, N71A, N71R, A73V,
Q74P, S75P, K76E, K76R, H79R, N88D, I89V, N90H, I92T, S99P, T101A,
F103S, I114V, I128T, I128A, T133A, or T133N. Our nomenclature is
consistent with that of the scientific literature, where the single
letter code of the amino acid in the wild-type or reference
sequence is followed by its position within the sequence and then
by the single letter code of the amino acid with which it is
replaced. Thus, A1T designates a substitution of the alanine
residue a position 1 with threonine. Other mutant polypeptides
within the scope of the invention include those that include a
mutant of SEQ ID NO:2 having substitutions at V69 (e.g. A) and Q74
(e.g., P). For example, the amino acid sequence can include one of
the following sets of mutations with respect to SEQ ID NO:2: (a)
K64R, V69A, and Q74P; (b) V69A, Q74P, and T101A; (c) V69A, Q74P,
and I128T; (d) N30D, V69A, Q74P, and F103S; (e) K49E, V69A, A73V,
and K76E; (f) V69A, Q74P, T101A, and T133N; (g) N30S, V69A, Q74P,
and I128A; (h) V69A, Q74P, N88D, and S99P; (i) N30S, V69A, Q74P,
and I128T; (j) K9T, Q11R, K35R, V69A, and Q74P; (k) A1T, M46L,
K49R, E61D, V69A, and H79R; (l) K48E, E68D, N71T, N90H, F103S, and
I114V; (m) S4P, T10A, Q1R, V69A, Q74P, N88D, and T133A; (n) E15K,
N30S Y31H, K35R, K48E, V69A, Q74P, and I92T; (o) N30S, E68D, V69A,
N71A, Q74P, S75P, K76R, and N90H; (p) N30S, Y31C, T37A, V69A, A73V,
Q74P, H79R, and I128T; (q) N26D, N29S, N30S, K54R, E67G, V69A,
Q74P, and I92T; (r) K8R, Q13R, N26D, N30T, K35R, T37R, V69A, Q74P,
and I92T; and (s) N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P,
N88D, and I89V. SEQ ID NO:2 is disclosed in U.S. Pat. No.
7,569,215, which is incorporated herein by reference as an
exemplary IL-2 polypeptide sequence that can be used in the
invention.
[0343] As noted above, any of the mutant IL-2 polypeptides
disclosed herein can include the sequences described; they can also
be limited to the sequences described and otherwise identical to
SEQ ID NO:1. Moreover, any of the mutant IL-2 polypeptides
described herein can optionally include a substitution of the
cysteine residue at position 125 with another residue (e.g.,
serine) and/or can optionally include a deletion of the alanine
residue at position 1 of SEQ ID NO:1.
[0344] The mutant IL-2 polypeptides disclosed herein can bind to
the IL-2R.alpha. subunit with a K.sub.d of less than about 28 nM
(e.g., less than about 25 nM; less than about 5 nM; about 1 nM;
less than about 500 pM; or less than about 100 pM). More
specifically, a mutant IL-2 polypeptide can have an affinity
equilibrium constant less than 1.0 nM (e.g., about 0.8, 0.6, 0.4,
or 0.2 nM). Affinity can also be expressed as a relative rate of
dissociation from an IL-2R.alpha. subunit or from an IL-2 receptor
complex (e.g., a complex expressed on the surface of a cell or
otherwise membrane bound). For example, the mutant IL-2
polypeptides can dissociate from, e.g., IL-2R.alpha., at a
decreased rate relative to a wild-type polypeptide or to an IL-2
based therapeutic, e.g., IL-2*. Alternatively, affinity can be
characterized as the time, or average time, an IL-2* polypeptide
persists on, for example, the surface of a cell expressing an
IL-2R. For example, an IL-2*polypeptide can persist on the receptor
for at least about 2, 5, 10, 50, 100, or 250 times (or more).
[0345] Disclosed are materials, compositions, and components that
can be used for, can be used in conjunction with, can be used in
preparation for, or are products of the disclosed methods and
compositions. These and other materials are disclosed herein, and
it is understood that when combinations, subsets, interactions,
groups, etc. of these materials are disclosed that while specific
reference of each various individual and collective combinations
and permutations of these compounds may not be explicitly
disclosed, each is specifically contemplated and described herein.
For example, if a method is disclosed and discussed and a number of
modifications that can be made to a number of molecules including
the method are discussed, each and every combination and
permutation of the method, and the modifications that are possible
are specifically contemplated unless specifically indicated to the
contrary. Likewise, any subset or combination of these is also
specifically contemplated and disclosed. his concept applies to all
aspects of this disclosure including, but not limited to, steps in
methods using the disclosed compositions. Thus, if there are a
variety of additional steps that can be performed, it is understood
that each of these additional steps can be performed with any
specific method steps or combination of method steps of the
disclosed methods, and that each such combination or subset of
combinations is specifically contemplated and should be considered
disclosed.
[0346] Publications cited herein and the material for which they
are cited are hereby specifically incorporated by reference in
their entireties.
6. INCORPORATION BY REFERENCE
[0347] All publications, patents, and patent applications mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication, patent, or patent
application was specifically and individually indicated to be
incorporated by reference. However, the citation of a reference
herein should not be construed as an acknowledgement that such
reference is prior art to the present invention. To the extent that
any of the definitions or terms provided in the references
incorporated by reference differ from the terms and discussion
provided herein, the present terms and definitions control.
7. EXAMPLES
[0348] The following are examples of methods and compositions of
the invention. It is understood that various other embodiments may
be practiced, given the general description provided herein.
Example 1. Detection of IL-2, IL-2 Mutein, IL-2R.alpha. and
IL-2R.gamma. in Fusion Proteins by ELISA
[0349] IL-2 mutein is detected with a commercially available
antibody, e.g., the anti-IL-2 monoclonal (JES6-1A12) (BD
Pharmingen; San Jose, Calif.). A positive control is used to show
whether the monoclonal antibody recognizes the cytokine or mutein.
Antibodies against IL-2R.alpha. and IL-2R.gamma. chain are also
used. Wells of a 96-well plate are coated with an antibody (2.5
.mu.g/ml) in PBS. Wells are blocked with 5% non-fat milk in PBS
with 0.2% Tween.RTM. 20 (PBS-M-Tw) and fusion proteins are added
for 1-2 hours at 37.degree. C. After washing, an anti-IL-2
biotin-labeled antibody, e.g., JES5H4 (BD Pharmingen) is added and
binding is detected using Streptavidin HRP (Southern Biotechnology
Associates; Birmingham, Ala.). The ELISA plate is developed by
adding 50 .mu.l O-phenylenediamine (OPD) (Sigma-Aldrich) in 0.1M
Citrate pH 4.5 and 0.04% H.sub.2O.sub.2, stopped by adding 50
.mu.l/well 2N H.sub.2SO.sub.4 and the absorbance was read at 490
nm.
Example 2: Protease Cleavage of Fusion Protein by MMP9 Protease
[0350] One of skill in the art would be familiar with methods of
setting up protein cleavage assay. 100 .mu.g of protein in
1.times.PBS pH 7.4 were cleaved with 1 .mu.g active MMP9 (Sigma
catalog #SAE0078-50 or Enzo catalog BML-SE360) and incubated at
room temperature for up to 16 hours. Digested protein is
subsequently used in functional assays or stored at -80.degree. C.
prior to testing. Extent of cleavage was monitored by SDS PAGE
using methods well known in the art. As shown in FIGS. 10, 13, 18A,
18b, and 27A full cleavage of the fusion proteins by MMP9 protease
is seen.
Example 3: CTLL-2 Assay
[0351] CTLL2 cells (ATCC) were plated in suspension at a
concentration of 500,000 cells/well in culture media with or
without 40 mg/ml human serum albumin (HSA) and stimulated with a
dilution series of recombinant hIL2 or activatable hIL2 for 72
hours at 37.degree. C. and 5% CO.sub.2. Activity of uncleaved and
cleaved activatable hIL2 was tested. Cleaved activatable hIL2 was
generated by incubation with active MMP9. Cell activity was
assessed using a CellTiter-Glo.RTM. (Promega) luminescence-based
cell viability assay. Results are shown in FIGS. 8A-8F, FIGS.
9A-9Z, FIG. 25C.
Example 4: Protease Cleavage of the IL-2/IL-2R.alpha./IL-2R.gamma.
Chimeric Polypeptide Results in Increased Accessibility to
Antibodies and Biologically Active IL-2 Mutein
[0352] The IL-2 mutein fusion proteins are biochemically
characterized before and after cleavage with a protease, e.g., PSA.
Immunoblot analyses will show that the fusion proteins can be
cleaved by PSA and that there is an increase in intensity of the
predicted low molecular weight cleavage product of approximately 20
kDa reactive with an anti-IL-2 antibody after treatment of the
samples with PSA. The degree of cleavage is dependent upon the
amount of PSA as well as the time of incubation. Interestingly,
when the fusion protein is analyzed before and after PSA treatment
by ELISA, it was found that the apparent amount of IL-2 is
increased after PSA cleavage. In this experiment, there is an
approximately 2 or 4-fold increase in the apparent amount of IL-2
detected using this sandwich ELISA depending on the construct,
suggesting that the antibody binding is partially hindered in the
intact fusion protein. Aliquots of the same samples are also
analyzed after PSA treatment using the CTLL-2 cell line that
requires IL-2 for growth and survival and the viability of cells
can be ascertained using the colorimetric MTT assay. In this assay,
the more a supernatant can be diluted, the more biologically active
IL-2 it contains, and there is an increase in the amount of
biologically active IL-2 after PSA cleavage. The amount of IL-2
mutein increase will suggest that after PSA cleavage there is an
increase in the predicted low molecular weight cleavage fragment of
approximately 20 kDa reactive with an anti-IL-2 antibody, an
increase in antibody accessibility, and most importantly, an
increase in the amount of biologically active IL-2 mutein.
Example 5. In Vivo Delivery of a Protease Activated Fusion Protein
Results in Decreased Tumor Growth
[0353] The chimeric polypeptide is examined to determine if it
could have biological effects in vivo. For these experiments a
system is used in which tumor cells injected intraperitoneally
rapidly and preferentially attach and grow initially on the milky
spots, a series of organized immune aggregates found on the omentum
(Gerber et al., Am. J. Pathol. 169:1739-52 (2006)). This system
offers a convenient way to examine the effects of fusion protein
treatment on tumor growth since fusion proteins can be delivered
intraperitoneally multiple times and tumor growth can be analyzed
by examining the dissociated omental cells. For these experiments,
the Colon 38 cell line, a rapidly growing tumor cell line that
expresses both MMP2 and MMP9 in vitro, may be used. The omental
tissue normally expresses a relatively small amount of MMP2 and
MMP9, but, when Colon 38 tumor is present on the omentum, MMP
levels increase. Using this tumor model, the ability of IL-2 mutein
fusion proteins to affect tumor growth is examined. Colon 38 cells
are injected intraperitoneally, allowed to attach and grow for 1
day, and then treated daily with fusion protein intraperitoneally.
At day 7, the animals are sacrificed and the omenta examined for
tumor growth using flow cytometry and by a colony-forming
assay.
Example 6: Determination of Antigen Affinity by Flow Cytometry
[0354] Activatable interleukin proteins are tested for their
binding affinities to human CD20.sup.+ cells and cynomolgus
CD20.sup.+ cells.
[0355] CD20.sup.+ cells are incubated with 100 .mu.L of serial
dilutions of the activatable interleukin proteins and at least one
protease. After washing three times with FACS buffer the cells are
incubated with 0.1 mL of 10 .mu.g/mL mouse monoclonal anti-idiotype
antibody in the same buffer for 45 min on ice. After a second
washing cycle, the cells are incubated with 0.1 mL of 15 g/mL
FITC-conjugated goat anti-mouse IgG antibodies under the same
conditions as before. As a control, cells are incubated with the
anti-His IgG followed by the FITC-conjugated goat anti-mouse IgG
antibodies without the activatable IL2 proteins. The cells were
then washed again and resuspended in 0.2 mL of FACS buffer
containing 2 .mu.g/mL propidium iodide (PI) in order to exclude
dead cells. The fluorescence of 1.times.10.sup.4 living cells is
measured using a Beckman-Coulter FC500 MPL flow cytometer using the
MXP software (Beckman-Coulter, Krefeld, Germany) or a Millipore
Guava EasyCyte flow cytometer using the Incyte software (Merck
Millipore, Schwalbach, Germany). Mean fluorescence intensities of
the cell samples are calculated using CXP software
(Beckman-Coulter, Krefeld, Germany) or Incyte software (Merck
Millipore, Schwalbach, Germany). After subtracting the fluorescence
intensity values of the cells stained with the secondary and
tertiary reagents alone the values are then used for calculation of
the K.sub.D values with the equation for one-site binding
(hyperbola) of the GraphPad Prism (version 6.00 for Windows,
GraphPad Software, La Jolla Calif. USA).
[0356] CD20 binding and crossreactivity are assessed on the human
CD20.sup.+ tumor cell lines. The K.sub.D ratio of crossreactivity
is calculated using the K.sub.D values determined on the CHO cell
lines expressing either recombinant human or recombinant cynomolgus
antigens.
Example 7: Cytotoxicity Assay
[0357] Activatable interleukin protein is evaluated in vitro on its
mediation of immune response to CD20.sup.+ target cells.
[0358] Fluorescence labeled CD20.sup.+ REC-1 cells (a Mantle cell
lymphoma cell line, ATCC CRL-3004) are incubated with isolated PBMC
of random donors or CB15 T-cells (standardized T-cell line) as
effector cells in the presence of the activatable IL2 protein and
at least one protease. After incubation for 4 h at 37.degree. C. in
a humidified incubator, the release of the fluorescent dye from the
target cells into the supernatant is determined in a
spectro-fluorimeter. Target cells incubated without the activatable
IL2 protein and target cells totally lysed by the addition of
saponin at the end of the incubation serve as negative and positive
controls, respectively.
[0359] Based on the measured remaining living target cells, the
percentage of specific cell lysis is calculated according to the
following formula: [1-(number of living targets.sub.(sample)/number
of living targets.sub.(spontaneous)].times.100%. Sigmoidal dose
response curves and EC.sub.50 values are calculated by non-linear
regression/4-parameter logistic fit using the GraphPad Software.
The lysis values obtained for a given antibody concentration are
used to calculate sigmoidal dose-response curves by 4 parameter
logistic fit analysis using the Prism.RTM. GraphPad.RTM.
software.
Example 8: Pharmacokinetics of Activatable Interleukin Proteins
[0360] Activatable interleukin protein is evaluated for half-time
elimination in animal studies.
[0361] The activatable IL2 protein is administered to cynomolgus
monkeys as a 0.5 mg/kg bolus injection into the saphenous vein.
Another cynomolgus monkey group receives a comparable IL2 construct
in size but lacking a serum half-life extension element. A third
and fourth group receive an IL2 construct with serum half-life
extension element and a cytokine with CD20 and serum half-life
extension elements respectively, and both comparable in size to the
activatable interleukin protein. Each test group consists of 5
monkeys. Serum samples are taken at indicated time points, serially
diluted, and the concentration of the proteins is determined using
a binding ELISA to CD20.
[0362] Pharmacokinetic analysis is performed using the test article
plasma concentrations. Group mean plasma data for each test article
conforms to a multi-exponential profile when plotted against the
time post-dosing. The data are fit by a standard two-compartment
model with bolus input and first-order rate constants for
distribution and elimination phases. The general equation for the
best fit of the data for i.v. administration is:
c(t)=Ae.sup.-.alpha.t+Be.sup.-.beta.t, where c(t) is the plasma
concentration at time t, A and B are intercepts on the Y-axis, and
.alpha. and .beta. are the apparent first-order rate constants for
the distribution and elimination phases, respectively. The
.alpha.-phase is the initial phase of the clearance and reflects
distribution of the protein into all extracellular fluid of the
animal, whereas the second or .beta.-phase portion of the decay
curve represents true plasma clearance. Methods for fitting such
equations are well known in the art. For example,
A=D/V(.alpha.-k21)/(.alpha.-.beta.),
B=D/V(.beta.-k21)/(.alpha.-.beta.), and .alpha. and .beta. (for
.alpha.>.beta.) are roots of the quadratic equation:
r.sup.2+(k12+k21+k10)r+k21k10=0 using estimated parameters of
V=volume of distribution, k10=elimination rate, k12=transfer rate
from compartment 1 to compartment 2 and k21=transfer rate from
compartment 2 to compartment 1, and D=the administered dose.
[0363] Data analysis: Graphs of concentration versus time profiles
are made using KaleidaGraph (KaleidaGraph.TM. V. 3.09 Copyright
1986-1997. Synergy Software. Reading, Pa.). Values reported as less
than reportable (LTR) are not included in the PK analysis and are
not represented graphically. Pharmacokinetic parameters are
determined by compartmental analysis using WinNonlin software
(WinNonlin.RTM. Professional V. 3.1 WinNonlin.RTM. Copyright
1998-1999. Pharsight Corporation. Mountain View, Calif.).
Pharmacokinetic parameters are computed as described in Ritschel W
A and Kearns G L, 1999, IN: Handbook of Basic Pharmacokinetics
Including Clinical Applications. 5th edition, American
Pharmaceutical Assoc., Washington, D.C.
[0364] It is expected that the activatable interleukin protein has
improved pharmacokinetic parameters such as an increase in
elimination half-time as compared to proteins lacking a serum
half-life extension element.
Example 9: Xenograft Tumor Model
[0365] Activatable IL2 protein is evaluated in a xenograft
model.
[0366] Female immune-deficient NOD/scid mice are sub-lethally
irradiated (2 Gy) and subcutaneously inoculated with
4.times.10.sup.6 Ramos RA1 cells into the right dorsal flank. When
tumors reach 100 to 200 mm.sup.3, animals are allocated into 3
treatment groups. Groups 2 and 3 (8 animals each) are
intraperitoneally injected with 1.5.times.10.sup.7 activated human
T-cells. Three days later, animals from Group 3 are subsequently
treated with a total of 9 intravenous doses of 50 .mu.g activatable
interleukin protein. Groups 1 and 2 are only treated with vehicle.
Body weight and tumor volume are determined for 30 days.
[0367] It is expected that animals treated with the activatable
interleukin protein have a statistically significant delay in tumor
growth in comparison to the respective vehicle-treated control
group.
Example 10: Mouse IFN.gamma. WEHI Cell Survival Assay
[0368] WEHI279 cells (ATCC) were plated in suspension at a
concentration of 25,000 cells/well in culture media with or without
1.5% human serum albumin (HSA) and stimulated with a dilution
series of recombinant mIFN.gamma. or inducible mIFN.gamma. for 72
hours at 37.degree. C. and 5% CO.sub.2. Activity of uncleaved and
cleaved inducible mIFN.gamma. was tested. Cleaved inducible
mIFN.gamma. was generated by incubation with active MMP9. Cell
survival was assessed using a CellTiter-Glo (Promega)
luminescence-based cell viability assay. The EC50 values for
cleaved inducible mIFN.gamma. molecules were at least 100.times.
more potent than un-cleaved inducible mIFN.gamma. molecules. As
shown in FIGS. 16A-16, greater inducibility was seen in assays
wherein the culture medium contained human serum albumin.
Example 11: Reserved
Example 12: Mouse IFN.gamma. B16 Reporter Cell Assay
[0369] B16-Blue IFN.gamma. cells (InvivoGen) were plated at a
concentration of 75,000 cells/well in culture media with or without
1.5% human serum albumin (HSA) and stimulated with a dilution
series of recombinant mIFN.gamma. or inducible mIFN.gamma. for 24
hours at 37.degree. C. and 5% CO.sub.2. Activity of uncleaved and
cleaved inducible mIFN.gamma. was tested. Cleaved inducible
mIFN.gamma. was generated by incubation with active MMP9.
Supernatants were harvested, and SEAP activation was assessed by
adding QUANTI-Blue Reagent (InvivoGen), incubating at 37.degree. C.
for 2 hours, and measuring absorbance at 620 nm. The EC50 values
for cleaved inducible mIFN.gamma. molecules were at least
100.times. more potent than un-cleaved inducible mIFN.gamma.
molecules. Results are shown in e.g., FIG. 19A-19B, FIGS. 22A-22B,
FIGS. 23A-23B. This experiment was repeated with for IFN.alpha.
conjugates using B16-Blue IFN.alpha./.beta. cells. The EC50 values
for cleaved inducible mIFN.alpha. molecules were at least
100.times. more potent than un-cleaved inducible mIFN.alpha.
molecules. See FIGS. 20A-20B.
Example 13. In Vivo Delivery of a Protease Activated Fusion Protein
Results in Decreased Tumor Growth
[0370] The chimeric polypeptide is examined to determine if it
could have biological effects in vivo. For these experiments a
system is used in which tumor cells injected intraperitoneally
rapidly and preferentially attach and grow initially on the milky
spots, a series of organized immune aggregates found on the omentum
(Gerber et al., Am. J. Pathol. 169:1739-52 (2006)). This system
offers a convenient way to examine the effects of fusion protein
treatment on tumor growth since fusion proteins can be delivered
intraperitoneally multiple times and tumor growth can be analyzed
by examining the dissociated omental cells. For these experiments,
the Colon 38 cell line, a rapidly growing tumor cell line that
expresses both MMP2 and MMP9 in vitro, may be used. The omental
tissue normally expresses a relatively small amount of MMP2 and
MMP9, but, when Colon 38 tumor is present on the omentum, MMP
levels increase. Using this tumor model, the ability of IFN fusion
proteins to affect tumor growth is examined. Colon 38 cells are
injected intraperitoneally, allowed to attach and grow for 1 day,
and then treated daily with fusion protein intraperitoneally. At
day 7, the animals are sacrificed and the omenta examined for tumor
growth using flow cytometry and by a colony-forming assay.
Example 14: The Chimeric Polypeptide was Examined to Determine its
Biological Effects in Vivo
[0371] The MC38 cell line, a rapidly growing colon adenocarcinoma
cell line that expresses MMP9 in vitro, was used. Using this tumor
model, the ability of IFN.gamma. fusion proteins to affect tumor
growth was examined. MC38 cells were injected subcutaneously,
allowed to grow for 10-14 days, and then treated with fusion
protein twice weekly intraperitoneally for a total of four doses.
As a comparator, wild type mIFN.gamma. was administered at the dose
levels indicated, twice daily for 2 weeks on a 5 day on/2 day off
schedule (10 total doses). Tumor growth and body weight were
monitored approximately twice per week for two weeks.
Example 15: Construction of an Exemplary IFN.gamma. Protein
Targeting CD20
[0372] 15.1 Generation of an Activatable Cytokine Domain
[0373] An IFN.gamma. polypeptide capable of binding to CD20
polypeptide present in a tumor or on a tumor cell is produced as
follows. A nucleic acid is produced that contains nucleic acid
sequences: (1) encoding an IFN.gamma. polypeptide sequence and (2)
one or more polypeptide linkers. Activatable IFN.gamma. plasmid
constructs can have optional Flag, His or other affinity tags, and
are electroporated into HEK293 or other suitable human or mammalian
cell lines and purified. Validation assays include T cell
activation assays using T cells responsive to IFN.gamma.
stimulation in the presence of a protease.
[0374] 15.2 Generation of a scFv CD20 Binding Domain
[0375] CD20 is one of the cell surface proteins present on
B-lymphocytes. CD20 antigen is found in normal and malignant pre-B
and mature B lymphocytes, including those in over 90% of B-cell
non-Hodgkin's lymphomas (NHL). The antigen is absent in
hematopoietic stem cells, activated B lymphocytes (plasma cells)
and normal tissue. As such, several antibodies mostly of murine
origin have been described: 1F5, 2B8/C2B8, 2H7, and 1H4.
[0376] Human or humanized anti-CD20 antibodies are therefore used
to generate scFv sequences for CD20 binding domains of an
activatable IFN.gamma. protein. DNA sequences coding for human or
humanized VL and VH domains are obtained, and the codons for the
constructs are, optionally, optimized for expression in cells from
Homo sapiens. The order in which the VL and VH domains appear in
the scFv is varied (i.e., VL-VH, or VH-VL orientation), and three
copies of the "G4S" (SEQ ID NO: 241) or "G.sub.4S" (SEQ ID NO: 241)
subunit (G.sub.4S).sub.3 (SEQ ID NO: 242) connect the variable
domains to create the scFv domain. Anti-CD20 scFv plasmid
constructs can have optional Flag, His or other affinity tags, and
are electroporated into HEK293 or other suitable human or mammalian
cell lines and purified. Validation assays include binding analysis
by FACS, kinetic analysis using Proteon, and staining of
CD20-expressing cells.
[0377] 15.3 Cloning of DNA Expression Constructs Encoding the
Activatable IFN.gamma. Protein
[0378] The activatable IFN.gamma. construct with protease cleavage
site domains is used to construct an activatable IFN.gamma. protein
in combination with an anti-CD20 scFv domain and a serum half-life
extension element (e.g., a HSA binding peptide or VH domain). For
expression of an activatable IFN.gamma. protein in CHO cells,
coding sequences of all protein domains are cloned into a mammalian
expression vector system. In brief, gene sequences encoding the
activatable IFN.gamma. domain, serum half-life extension element,
and CD20 binding domain along with peptide linkers L1 and L2 are
separately synthesized and subcloned. The resulting constructs are
then ligated together in the order of CD20 binding
domain-L1-IFN.gamma. subunit 1-L2-protease cleavage
domain-L3-IFN.gamma. subunit2-L4-anti-CD20 scFv-L5-serum half-life
extension element to yield a final construct. All expression
constructs are designed to contain coding sequences for an
N-terminal signal peptide and a C-terminal hexahistidine
(6.times.His)-tag (SEQ ID NO: 243) to facilitate protein secretion
and purification, respectively.
[0379] 15.4 Expression of Activatable IFN.gamma. Proteins in Stably
Transfected CHO Cells
[0380] A CHO cell expression system (Flp-In.RTM., Life
Technologies), a derivative of CHO-K1 Chinese Hamster ovary cells
(ATCC, CCL-61) (Kao and Puck, Proc. Natl. Acad. Sci. USA 1968;
60(4):1275-81), is used. Adherent cells are subcultured according
to standard cell culture protocols provided by Life
Technologies.
[0381] For adaption to growth in suspension, cells are detached
from tissue culture flasks and placed in serum-free medium.
Suspension-adapted cells are cryopreserved in medium with 10%
DMSO.
[0382] Recombinant CHO cell lines stably expressing secreted
activatable IFN.gamma. proteins are generated by transfection of
suspension-adapted cells. During selection with the antibiotic
Hygromycin B viable cell densities are measured twice a week, and
cells are centrifuged and resuspended in fresh selection medium at
a maximal density of 0.1.times.10.sup.6 viable cells/mL. Cell pools
stably expressing activatable IFN.gamma. proteins are recovered
after 2-3 weeks of selection at which point cells are transferred
to standard culture medium in shake flasks. Expression of
recombinant secreted proteins is confirmed by performing protein
gel electrophoresis or flow cytometry. Stable cell pools are
cryopreserved in DMSO containing medium.
[0383] Activatable IFN.gamma. proteins are produced in 10-day
fed-batch cultures of stably transfected CHO cell lines by
secretion into the cell culture supernatant. Cell culture
supernatants are harvested after 10 days at culture viabilities of
typically >75%. Samples are collected from the production
cultures every other day and cell density and viability are
assessed. On day of harvest, cell culture supernatants are cleared
by centrifugation and vacuum filtration before further use.
[0384] Protein expression titers and product integrity in cell
culture supernatants are analyzed by SDS-PAGE.
[0385] 15.5 Purification of Activatable IFN.gamma. Proteins
[0386] Activatable IFN.gamma. proteins are purified from CHO cell
culture supernatants in a two-step procedure. The constructs are
subjected to affinity chromatography in a first step followed by
preparative size exclusion chromatography (SEC) on Superdex 200 in
a second step. Samples are buffer-exchanged and concentrated by
ultrafiltration to a typical concentration of >1 mg/mL. Purity
and homogeneity (typically >90%) of final samples are assessed
by SDS PAGE under reducing and non-reducing conditions, followed by
immunoblotting using an anti-HSA or anti idiotype antibody as well
as by analytical SEC, respectively. Purified proteins are stored at
aliquots at -80.degree. C. until use.
Example 16: Determination of Antigen Affinity by Flow Cytometry
[0387] The activatable IFN.gamma. proteins are tested for their
binding affinities to human CD20.sup.+ cells and cynomolgus
CD20.sup.+ cells.
[0388] CD20.sup.+ cells are incubated with 100 .mu.L of serial
dilutions of the activatable IFN.gamma. proteins and at least one
protease. After washing three times with FACS buffer the cells are
incubated with 0.1 mL of 10 .mu.g/mL mouse monoclonal anti-idiotype
antibody in the same buffer for 45 min on ice. After a second
washing cycle, the cells are incubated with 0.1 mL of 15 .mu.g/mL
FITC-conjugated goat anti-mouse IgG antibodies under the same
conditions as before. As a control, cells are incubated with the
anti-His IgG followed by the FITC-conjugated goat anti-mouse IgG
antibodies without the activatable IFN.gamma. proteins. The cells
were then washed again and resuspended in 0.2 mL of FACS buffer
containing 2 .mu.g/mL propidium iodide (PI) in order to exclude
dead cells. The fluorescence of 1.times.10.sup.4 living cells is
measured using a Beckman-Coulter FC500 MPL flow cytometer using the
MXP software (Beckman-Coulter, Krefeld, Germany) or a Millipore
Guava EasyCyte flow cytometer using the Incyte software (Merck
Millipore, Schwalbach, Germany). Mean fluorescence intensities of
the cell samples are calculated using CXP software
(Beckman-Coulter, Krefeld, Germany) or Incyte software (Merck
Millipore, Schwalbach, Germany). After subtracting the fluorescence
intensity values of the cells stained with the secondary and
tertiary reagents alone the values are then used for calculation of
the K.sub.D values with the equation for one-site binding
(hyperbola) of the GraphPad Prism (version 6.00 for Windows,
GraphPad Software, La Jolla Calif. USA).
[0389] CD20 binding and cross-reactivity are assessed on the human
CD20.sup.+ tumor cell lines. The K.sub.D ratio of cross-reactivity
is calculated using the K.sub.D values determined on the CHO cell
lines expressing either recombinant human or recombinant cynomolgus
antigens.
Example 17: Cytotoxicity Assay
[0390] The activatable IFN.gamma. protein is evaluated in vitro on
its mediation of immune response to CD20.sup.+ target cells.
[0391] Fluorescence labeled CD20.sup.+ REC-1 cells (a Mantle cell
lymphoma cell line, ATCC CRL-3004) are incubated with isolated PBMC
of random donors or CB15 T-cells (standardized T-cell line) as
effector cells in the presence of the activatable IFN.gamma.
protein and at least one protease. After incubation for 4 h at
37.degree. C. in a humidified incubator, the release of the
fluorescent dye from the target cells into the supernatant is
determined in a spectrofluorometer. Target cells incubated without
the activatable IFN.gamma. protein and target cells totally lysed
by the addition of saponin at the end of the incubation serve as
negative and positive controls, respectively.
[0392] Based on the measured remaining living target cells, the
percentage of specific cell lysis is calculated according to the
following formula: [1-(number of living targets.sub.(sample)/number
of living targets.sub.(spontaneous)].times.100%. Sigmoidal dose
response curves and EC.sub.50 values are calculated by non-linear
regression/4-parameter logistic fit using the GraphPad Software.
The lysis values obtained for a given antibody concentration are
used to calculate sigmoidal dose-response curves by 4 parameter
logistic fit analysis using the Prism software.
Example 18: Pharmacokinetics of Activatable IFN.gamma. Proteins
[0393] The activatable IFN.gamma. protein is evaluated for
half-time elimination in animal studies.
[0394] The activatable IFN.gamma. protein is administered to
cynomolgus monkeys as a 0.5 mg/kg bolus injection into the
saphenous vein. Another cynomolgus monkey group receives a
comparable cytokine in size but lacking a serum half-life extension
element. A third and fourth group receive a cytokine with serum
half-life extension elements and a cytokine with CD20 and serum
half-life extension elements respectively, and both comparable in
size to the activatable IFN.gamma. protein. Each test group
consists of 5 monkeys. Serum samples are taken at indicated time
points, serially diluted, and the concentration of the proteins is
determined using a binding ELISA to CD20.
[0395] Pharmacokinetic analysis is performed using the test article
plasma concentrations. Group mean plasma data for each test article
conforms to a multi-exponential profile when plotted against the
time post-dosing. The data are fit by a standard two-compartment
model with bolus input and first-order rate constants for
distribution and elimination phases. The general equation for the
best fit of the data for i.v. administration is:
c(t)=Ae.sup.-.alpha.t+Be.sup.-.beta.t, where c(t) is the plasma
concentration at time t, A and B are intercepts on the Y-axis, and
.alpha. and .beta. are the apparent first-order rate constants for
the distribution and elimination phases, respectively. The
.alpha.-phase is the initial phase of the clearance and reflects
distribution of the protein into all extracellular fluid of the
animal, whereas the second or .beta.-phase portion of the decay
curve represents true plasma clearance. Methods for fitting such
equations are well known in the art. For example,
A=D/V(.alpha.-k21)/(.alpha.-.beta.),
B=D/V(.beta.-k21)/(.alpha.-.beta.), and .alpha. and .beta. (for
.alpha.>.beta.) are roots of the quadratic equation:
r.sup.2+(k12+k21+k10)r+k21k10=0 using estimated parameters of
V=volume of distribution, k10=elimination rate, k12=transfer rate
from compartment 1 to compartment 2 and k21=transfer rate from
compartment 2 to compartment 1, and D=the administered dose.
[0396] Data analysis: Graphs of concentration versus time profiles
are made using KaleidaGraph (KaleidaGraph.TM. V. 3.09 Copyright
1986-1997. Synergy Software. Reading, Pa.). Values reported as less
than reportable (LTR) are not included in the PK analysis and are
not represented graphically. Pharmacokinetic parameters are
determined by compartmental analysis using WinNonlin software
(WinNonlin.RTM. Professional V. 3.1 WinNonlin.TM. Copyright
1998-1999. Pharsight Corporation. Mountain View, Calif.).
Pharmacokinetic parameters are computed as described in Ritschel W
A and Kearns G L, 1999, IN: Handbook of Basic Pharmacokinetics
Including Clinical Applications. 5th edition, American
Pharmaceutical Assoc., Washington, D.C.
[0397] It is expected that the activatable IFN protein has improved
pharmacokinetic parameters such as an increase in elimination
half-time as compared to proteins lacking a serum half-life
extension element.
Example 19: Xenograft Tumor Model
[0398] The activatable IFN.gamma. protein is evaluated in a
xenograft model.
[0399] Female immune-deficient NOD/scid mice are sub-lethally
irradiated (2 Gy) and subcutaneously inoculated with
4.times.10.sup.6 Ramos RA1 cells into the right dorsal flank. When
tumors reach 100 to 200 mm.sup.3, animals are allocated into 3
treatment groups. Groups 2 and 3 (8 animals each) are
intraperitoneally injected with 1.5.times.10.sup.7 activated human
T-cells. Three days later, animals from Group 3 are subsequently
treated with a total of 9 intravenous doses of 50 .mu.g activatable
IFN.gamma. protein. Groups 1 and 2 are only treated with vehicle.
Body weight and tumor volume are determined for 30 days.
[0400] It is expected that animals treated with the activatable
IFN.gamma. protein have a statistically significant delay in tumor
growth in comparison to the respective vehicle-treated control
group.
[0401] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
Example 20: HEK Blue Assay
[0402] HEK-Blue IL12 cells (InvivoGen) were plated in suspension at
a concentration of 250,000 cells/well in culture media with or
without 40 mg/ml human serum albumin (HSA) and stimulated with a
dilution series of recombinant hIL12, chimeric IL12 (mouse
p35/human p40) or activatable hIL12 for 24 hours at 37.degree. C.
and 5% CO.sub.2. Activity of uncleaved and cleaved activatable
hIL12 was tested. Cleaved inducible hIL12 was generated by
incubation with active MMP9. IL12 activity was assessed by
quantification of Secreted Alkaline Phosphatase (SEAP) activity
using the reagent QUANTI-Blue (InvivoGen), a colorimetric based
assay.
[0403] HEK-Blue IL2 cells (InvivoGen) were plated in suspension at
a concentration of 50,000 cells/well in culture media with or
without 15-40 mg/ml human serum albumin (HSA) and stimulated with a
dilution series of recombinant hIL2 or activatable hIL2 for 24
hours at 37.degree. C. and 5% CO.sub.2. Activity of uncleaved and
cleaved activatable hIL2 was tested. Cleaved inducible hIL2 was
generated by incubation with active MMP9 or another protease. IL2
activity was assessed by quantification of Secreted Alkaline
Phosphatase (SEAP) activity using the reagent QUANTI-Blue
(InvivoGen), a colorimetric based assay. Results are shown in FIGS.
59-62.
Example 21: Splenocyte T-Blast Assay
[0404] T-Blasts were induced from murine splenocytes with a 6-day
incubation with PHA and a 24 hr incubation with recombinant hIL12.
T-blasts were then plated in suspension at a concentration of
200,000 cells/well in culture media with or without 40 mg/ml human
serum albumin (HSA) and stimulated with a dilution series of
recombinant hIL12 or chimeric IL12 (mouse p35/human p40) or mouse
IL12 for 72 hours at 37.degree. C. and 5% CO2. Activity of
uncleaved and cleaved IL12 was tested. Cleaved inducible hIL12 was
generated by incubation with active MMP9. IL12 activity was
assessed by downstream quantification of IFN.gamma. production
using a mIFN.gamma. alphaLISA.
Example 22: In Vivo Delivery of a Protease Activated Fusion Protein
Results in Decreased Tumor Growth
[0405] The chimeric polypeptide is examined to determine if it
could have biological effects in vivo. For these experiments a
system is used in which tumor cells injected intraperitoneally
rapidly and preferentially attach and grow initially on the milky
spots, a series of organized immune aggregates found on the omentum
(Gerber et al., Am. J. Pathol. 169:1739-52 (2006)). This system
offers a convenient way to examine the effects of fusion protein
treatment on tumor growth since fusion proteins can be delivered
intraperitoneally multiple times and tumor growth can be analyzed
by examining the dissociated omental cells. For these experiments,
the Colon 38 cell line, a rapidly growing tumor cell line that
expresses both MMP2 and MMP9 in vitro, may be used. The omental
tissue normally expresses a relatively small amount of MMP2 and
MMP9, but, when Colon 38 tumor is present on the omentum, MMP
levels increase. Using this tumor model, the ability of IL-2 mutein
fusion proteins to affect tumor growth is examined. Colon 38 cells
are injected intraperitoneally, allowed to attach and grow for 1
day, and then treated daily with fusion protein intraperitoneally.
At day 7, the animals are sacrificed and the omenta examined for
tumor growth using flow cytometry and by a colony-forming
assay.
Example 23A: Construction of an Exemplary Activatable Interleukin
Protein Targeting CD20
[0406] 23.1 Generation of an Activatable Interleukin Domain
[0407] The human IL-12p35 chain canonical sequence is UniProt
Accession No. P29459. The human IL-12p40 chain canonical sequence
is UniProt Accession No. P29460. IL-12p35 and IL-12p40 are cloned
into an expression construct. A protease cleavage site is included
between the IL-12p35 and IL-12p40 domains. An IL-12 polypeptide
capable of binding to CD20 polypeptide present in a tumor or on a
tumor cell is produced as follows. A nucleic acid is produced that
contains nucleic acid sequences: (1) encoding an IFN.gamma.
polypeptide sequence and (2) one or more polypeptide linkers.
Activatable interleukin plasmid constructs can have optional Flag,
His or other affinity tags, and are electroporated into HEK293 or
other suitable human or mammalian cell lines and purified.
Validation assays include T cell activation assays using T cells
responsive to IL-12 stimulation in the presence of a protease.
[0408] 23.2 Generation of a scFv CD20 Binding Domain
[0409] CD20 is one of the cell surface proteins present on
B-lymphocytes. CD20 antigen is found in normal and malignant pre-B
and mature B lymphocytes, including those in over 90/of B-cell
non-Hodgkin's lymphomas (NHL). The antigen is absent in
hematopoietic stem cells, activated B lymphocytes (plasma cells)
and normal tissue. As such, several antibodies mostly of murine
origin have been described: 1F5, 2B8/C2B8, 2H7, and 1H4.
[0410] Human or humanized anti-CD20 antibodies are therefore used
to generate scFv sequences for CD20 binding domains of an
activatable interleukin protein. DNA sequences coding for human or
humanized VL and VH domains are obtained, and the codons for the
constructs are, optionally, optimized for expression in cells from
Homo sapiens. The order in which the VL and VH domains appear in
the scFv is varied (i.e., VL-VH, or VH-VL orientation), and three
copies of the "G4S" (SEQ ID NO: 241) or "G.sub.4S" (SEQ ID NO: 241)
subunit (G.sub.4S).sub.3 (SEQ ID NO: 242) connect the variable
domains to create the scFv domain. Anti-CD20 scFv plasmid
constructs can have optional Flag, His or other affinity tags, and
are electroporated into HEK293 or other suitable human or mammalian
cell lines and purified. Validation assays include binding analysis
by FACS, kinetic analysis using Proteon, and staining of
CD20-expressing cells.
[0411] 23.3 Cloning of DNA Expression Constructs Encoding the
Activatable Interleukin Protein
[0412] The activatable interleukin construct with protease cleavage
site domains are used to construct an activatable interleukin
protein in combination with an anti-CD20 scFv domain and a serum
half-life extension element (e.g., a HSA binding peptide or VH
domain). For expression of an activatable interleukin protein in
CHO cells, coding sequences of all protein domains are cloned into
a mammalian expression vector system. In brief, gene sequences
encoding the activatable interleukin domain, serum half-life
extension element, and CD20 binding domain along with peptide
linkers L1 and L2 are separately synthesized and subcloned. The
resulting constructs are then ligated together in the order of CD20
binding domain-L1-IL-12p35-L2-protease cleavage
domain-L3-IL-12p40-L4-anti-CD20 scFv-L5-serum half-life extension
element to yield a final construct. All expression constructs are
designed to contain coding sequences for an N-terminal signal
peptide and a C-terminal hexahistidine (6.times.His)-tag (SEQ ID
NO: 243) to facilitate protein secretion and purification,
respectively.
[0413] 23.4 Expression of Activatable Interleukin Proteins in
Stably Transfected CHO Cells
[0414] A CHO cell expression system (Flp-In.RTM., Life
Technologies), a derivative of CHO-K1 Chinese Hamster ovary cells
(ATCC, CCL-61) (Kao and Puck, Proc. Nat. Acad Sci USA 1968;
60(4):1275-81), is used. Adherent cells are subcultured according
to standard cell culture protocols provided by Life
Technologies.
[0415] For adaption to growth in suspension, cells are detached
from tissue culture flasks and placed in serum-free medium.
Suspension-adapted cells are cryopreserved in medium with 10%
DMSO.
[0416] Recombinant CHO cell lines stably expressing secreted
activatable interleukin proteins are generated by transfection of
suspension-adapted cells. During selection with the antibiotic
Hygromycin B viable cell densities are measured twice a week, and
cells are centrifuged and resuspended in fresh selection medium at
a maximal density of 0.1.times.10.sup.6 viable cells/mL. Cell pools
stably expressing activatable interleukin proteins are recovered
after 2-3 weeks of selection at which point cells are transferred
to standard culture medium in shake flasks. Expression of
recombinant secreted proteins is confirmed by performing protein
gel electrophoresis or flow cytometry. Stable cell pools are
cryopreserved in DMSO containing medium.
[0417] Activatable interleukin proteins are produced in 10-day
fed-batch cultures of stably transfected CHO cell lines by
secretion into the cell culture supernatant. Cell culture
supernatants are harvested after 10 days at culture viabilities of
typically >75%. Samples are collected from the production
cultures every other day and cell density and viability are
assessed. On day of harvest, cell culture supernatants are cleared
by centrifugation and vacuum filtration before further use.
[0418] Protein expression titers and product integrity in cell
culture supernatants are analyzed by SDS-PAGE.
[0419] 23.5 Purification of Activatable Interleukin Proteins
[0420] Activatable interleukin proteins are purified from CHO cell
culture supernatants in a two-step procedure. The constructs are
subjected to affinity chromatography in a first step followed by
preparative size exclusion chromatography (SEC) on Superdex 200 in
a second step. Samples are buffer-exchanged and concentrated by
ultrafiltration to a typical concentration of >1 mg/mL. Purity
and homogeneity (typically >90%) of final samples are assessed
by SDS PAGE under reducing and non-reducing conditions, followed by
immunoblotting using an anti-HSA or anti idiotype antibody as well
as by analytical SEC, respectively. Purified proteins are stored at
aliquots at -80.degree. C. until use.
Example 23B: Determination of Antigen Affinity by Flow
Cytometry
[0421] Activatable interleukin proteins are tested for their
binding affinities to human CD20.sup.+ cells and cynomolgus
CD20.sup.+ cells.
[0422] CD20.sup.+ cells are incubated with 100 .mu.L of serial
dilutions of the activatable interleukin proteins and at least one
protease. After washing three times with FACS buffer the cells are
incubated with 0.1 mL of 10 .mu.g/mL mouse monoclonal anti-idiotype
antibody in the same buffer for 45 min on ice. After a second
washing cycle, the cells are incubated with 0.1 mL of 15 .mu.g/mL
FITC-conjugated goat anti-mouse IgG antibodies under the same
conditions as before. As a control, cells are incubated with the
anti-His IgG followed by the FITC-conjugated goat anti-mouse IgG
antibodies without the activatable interleukin proteins. The cells
were then washed again and resuspended in 0.2 mL of FACS buffer
containing 2 .mu.g/mL propidium iodide (PI) in order to exclude
dead cells. The fluorescence of 1.times.10.sup.4 living cells is
measured using a Beckman-Coulter FC500 MPL flow cytometer using the
MXP software (Beckman-Coulter, Krefeld, Germany) or a Millipore
Guava EasyCyte flow cytometer using the Incyte software (Merck
Millipore, Schwalbach, Germany). Mean fluorescence intensities of
the cell samples are calculated using CXP software
(Beckman-Coulter, Krefeld, Germany) or Incyte software (Merck
Millipore, Schwalbach, Germany). After subtracting the fluorescence
intensity values of the cells stained with the secondary and
tertiary reagents alone the values are then used for calculation of
the K.sub.D values with the equation for one-site binding
(hyperbola) of the GraphPad Prism (version 6.00 for Windows,
GraphPad Software, La Jolla Calif. USA).
[0423] CD20 binding and cross-reactivity are assessed on the human
CD20.sup.+ tumor cell lines. The K.sub.D ratio of cross-reactivity
is calculated using the K.sub.D values determined on the CHO cell
lines expressing either recombinant human or recombinant cynomolgus
antigens.
Example 24: Cytotoxicity Assay
[0424] The activatable interleukin protein is evaluated in vitro on
its mediation of immune response to CD20.sup.+ target cells.
[0425] Fluorescence labeled CD20.sup.+ REC-1 cells (a Mantle cell
lymphoma cell line, ATCC CRL-3004) are incubated with isolated PBMC
of random donors or CB15 T-cells (standardized T-cell line) as
effector cells in the presence of the activatable interleukin
protein and at least one protease. After incubation for 4 h at
37.degree. C. in a humidified incubator, the release of the
fluorescent dye from the target cells into the supernatant is
determined in a spectrofluorometer. Target cells incubated without
the activatable interleukin protein and target cells totally lysed
by the addition of saponin at the end of the incubation serve as
negative and positive controls, respectively.
[0426] Based on the measured remaining living target cells, the
percentage of specific cell lysis is calculated according to the
following formula: [1-(number of living targets.sub.(sample)/number
of living targets.sub.(spontaneous))].times.100%. Sigmoidal dose
response curves and EC.sub.50 values are calculated by non-linear
regression/4-parameter logistic fit using the GraphPad Software.
The lysis values obtained for a given antibody concentration are
used to calculate sigmoidal dose-response curves by 4 parameter
logistic fit analysis using the Prism software.
Example 25: Pharmacokinetics of Activatable Interleukin
Proteins
[0427] The activatable interleukin protein is evaluated for
half-time elimination in animal studies.
[0428] The activatable interleukin protein is administered to
cynomolgus monkeys as a 0.5 mg/kg bolus injection into the
saphenous vein. Another cynomolgus monkey group receives a
comparable cytokine in size but lacking a serum half-life extension
element. A third and fourth group receive a cytokine with serum
half-life extension elements and a cytokine with CD20 and serum
half-life extension elements respectively, and both comparable in
size to the activatable interleukin protein. Each test group
consists of 5 monkeys. Serum samples are taken at indicated time
points, serially diluted, and the concentration of the proteins is
determined using a binding ELISA to CD20.
[0429] Pharmacokinetic analysis is performed using the test article
plasma concentrations. Group mean plasma data for each test article
conforms to a multi-exponential profile when plotted against the
time post-dosing. The data are fit by a standard two-compartment
model with bolus input and first-order rate constants for
distribution and elimination phases. The general equation for the
best fit of the data for i.v. administration is:
c(t)=Ae.sup.-.alpha.t+Be.sup.-.beta.t, where c(t) is the plasma
concentration at time t, A and B are intercepts on the Y-axis, and
.alpha. and .beta. are the apparent first-order rate constants for
the distribution and elimination phases, respectively. The
.alpha.-phase is the initial phase of the clearance and reflects
distribution of the protein into all extracellular fluid of the
animal, whereas the second or .beta.-phase portion of the decay
curve represents true plasma clearance. Methods for fitting such
equations are well known in the art. For example,
A=D/V(.alpha.-k21)/(.alpha.-.beta.),
B=D/V(.beta.-k21)/(.alpha.-.beta.), and .alpha. and .beta. (for
.alpha.>.beta.) are roots of the quadratic equation:
r.sup.2+(k12+k21+k10)r+k21k10=0 using estimated parameters of
V=volume of distribution, k10=elimination rate, k12=transfer rate
from compartment 1 to compartment 2 and k21=transfer rate from
compartment 2 to compartment 1, and D=the administered dose.
[0430] Data analysis: Graphs of concentration versus time profiles
are made using KaleidaGraph (KaleidaGraph.TM. V. 3.09 Copyright
1986-1997. Synergy Software. Reading, Pa.). Values reported as less
than reportable (LTR) are not included in the PK analysis and are
not represented graphically. Pharmacokinetic parameters are
determined by compartmental analysis using WinNonlin software
(WinNonlin.RTM. Professional V. 3.1 WinNonlin.TM. Copyright
1998-1999. Pharsight Corporation. Mountain View, Calif.).
Pharmacokinetic parameters are computed as described in Ritschel W
A and Kearns G L, 1999, IN: Handbook of Basic Pharmacokinetics
Including Clinical Applications. 5th edition, American
Pharmaceutical Assoc., Washington, D.C.
[0431] It is expected that the activatable interleukin protein has
improved pharmacokinetic parameters such as an increase in
elimination half-time as compared to proteins lacking a serum
half-life extension element.
Example 26: Xenograft Tumor Model
[0432] Activatable interleukin protein is evaluated in a xenograft
model.
[0433] Female immune-deficient NOD/scid mice are sub-lethally
irradiated (2 Gy) and subcutaneously inoculated with
4.times.10.sup.6 Ramos RA1 cells into the right dorsal flank. When
tumors reach 100 to 200 mm.sup.3, animals are allocated into 3
treatment groups. Groups 2 and 3 (8 animals each) are
intraperitoneally injected with 1.5.times.10.sup.7 activated human
T-cells. Three days later, animals from Group 3 are subsequently
treated with a total of 9 intravenous doses of 50 .mu.g activatable
interleukin protein. Groups 1 and 2 are only treated with vehicle.
Body weight and tumor volume are determined for 30 days.
TABLE-US-00007 TABLE 3 Summary of the treatment modes Gr. N Agent
Formulation dose Route Schedule .sup. 1.sup.# 10 Vehicle -- ip biwk
x 3 2 7 ACP16 700 .mu.g/animal ip biwk x 3 3 7 ACP16 230
.mu.g/animal ip biwk x 3 4 7 ACP16 70 .mu.g/animal ip biwk x 3 5 7
ACP16 55 ug/animal ip biwk x 3 6 7 ACP16 17 .mu.g/animal ip biwk x
3 7 7 ACP132 361 .mu.g/animal ip biwk x 3 8 7 ACP132 119
.mu.g/animal ip biwk x 3 9 7 ACP132 36 .mu.g/animal ip biwk x 3 10
7 ACP132 28 .mu.g/animal ip biwk x 3 11 7 ACP132 9 .mu.g/animal ip
biwk x 3 12 7 ACP21 540 .mu.g/animal ip biwk x 3 13 7 ACP21 177
.mu.g/animal ip biwk x 3 14 7 ACP21 54 .mu.g/animal ip biwk x 3 15
7 ACP21 42 .mu.g/animal ip biwk x 3 16 7 ACP21 13 .mu.g/animal ip
biwk x 3 17 7 ACP133 210 .mu.g/animal ip bid x 5 then 2-day pause
then bid x 5 then 2-day pause 18 7 ACP133 105 .mu.g/animal ip bid x
5 then 2-day pause then bid x 5 then 2-day pause 19 7 ACP133 40
.mu.g/animal ip bid x 5 then 2-day pause then bid x 5 then 2-day
pause 20 7 ACP133 3 .mu.g/animal ip bid x 5 then 2-day pause then
bid x 5 then 2-day pause .sup.#Control Group
[0434] It is expected that animals treated with the activatable
interleukin protein have a statistically significant delay in tumor
growth in comparison to the respective vehicle-treated control
group.
[0435] While preferred embodiments of the present invention have
been shown and described herein, it will be obvious to those
skilled in the art that such embodiments are provided by way of
example only. Numerous variations, changes, and substitutions will
now occur to those skilled in the art without departing from the
invention. It should be understood that various alternatives to the
embodiments of the invention described herein may be employed in
practicing the invention. It is intended that the following claims
define the scope of the invention and that methods and structures
within the scope of these claims and their equivalents be covered
thereby.
[0436] The MC38 cell line, a rapidly growing colon adenocarcinoma
cell line that expresses MMP9 in vitro, was used. Using this tumor
model, the ability of fusion proteins to affect tumor growth was
examined.
Example 27A: MC38 IL-2POC
[0437] 27A.1 Agents and Treatment
[0438] Additional studies were carried out in non-tumor bearing
animals as described below.
TABLE-US-00008 TABLE 4 Summarizes the treatment regime. Gr. N Agent
Formulation dose Route Schedule 1 8 Vehicle -- ip biwk x 4 2 8
ACP16 700 .mu.g/animal ip biwk x 4 3 8 ACP16 230 .mu.g/animal ip
biwk x 4 4 8 ACP16 70 .mu.g/animal ip biwk x 4 8 8 ACP153 700
.mu.g/animal ip biwk x 4 9 8 ACP153 230 .mu.g/animal ip biwk x 4 10
8 ACP153 70 .mu.g/animal ip biwk x 4 11 8 ACP154 700 .mu.g/animal
ip biwk x 4 12 8 ACP154 230 .mu.g/animal ip biwk x 4 13 8 ACP154 70
.mu.g/animal ip biwk x 4 14 8 ACP155 700 .mu.g/animal ip biwk x 4
15 8 ACP155 230 .mu.g/animal ip biwk x 4 16 8 ACP155 70
.mu.g/animal ip biwk x 4 17 8 ACP156 700 .mu.g/animal ip biwk x 4
18 8 ACP156 230 .mu.g/animal ip biwk x 4 19 8 ACP156 70
.mu.g/animal ip biwk x 4 20 8 ACP157 700 .mu.g/animal ip biwk x 4
21 8 ACP157 230 .mu.g/animal ip biwk x 4 22 8 ACP157 70
.mu.g/animal ip biwk x 4
TABLE-US-00009 TABLE 5 Describes the constructs used in the MC38
IL-2POC animal study. Construct Name Description MW ACP16
IL2-X-HSA-LX-blocker Fusion protein- 58256 6xHis ACP133 IL-2 with C
term 6x His 16462 ACP132 IL2-L-HSA 29996 ACP21 IL2-XL-blocker
Fusion protein-6xHis 44843
Example 27: MC3IL-2
TABLE-US-00010 [0439] TABLE 6 Summarizes the treatment regime. Gr.
N Agent Formulation dose Route Schedule .sup. 1.sup.# 12 Vehicle --
ip biwk x 2 2 8 ACP16 4.4 .mu.g/animal ip biwk x 2 3 8 ACP16 17
.mu.g/animal ip biwk x 2 4 8 ACP16 70 .mu.g/animal ip biwk x 2 5 8
ACP16 232 .mu.g/animal ip biwk x 2 6 8 ACP130 19 .mu.g/animal ip
biwk x 2 7 8 ACP130 45 .mu.g/animal ip biwk x 2 8 8 ACP130 180
.mu.g/animal ip biwk x 2 9 8 ACP130 600 .mu.g/animal ip biwk x 1 12
8 ACP124 17 .mu.g/animal ip biwk x 2 13 8 ACP124 70 .mu.g/animal ip
biwk x 2 14 8 ACP124 230 .mu.g/animal ip biwk x 2 15 8 ACP124 700
.mu.g/animal ip biwk x 2 16 8 IL-2- 12 .mu.g/animal ip bid x 5 then
2-day WTI pause then bid x 5 then 2-day pause 17 8 IL-2- 36
.mu.g/animal ip bid x 5 then 2-day WTI pause then bid x 5 then
2-day pause .sup.#Control Group
[0440] 27B.1 Procedure
[0441] Mice were anaesthetized with isoflurane for implant of cells
to reduce the ulcerations. 308 CR female C57BL/6 mice were set up
with 5.times.105 MC38 tumor cells in 0% Matrigel sc in flank. Cell
Injection Volume was 0.1 mL/mouse. Mouse age at start date was 8 to
12 weeks. Pair matches were performed when tumors reach an average
size of 100-150 mm.sup.3 and begin treatment. Body weights were
taken at initiation and then biweekly to the end. Caliper
measurements were taken biweekly to the end. Any adverse reactions
were to be reported immediately. Any individual animal with a
single observation of >than 30% body weight loss or three
consecutive measurements of >25% body weight loss was
euthanized. Any group with a mean body weight loss of >20% or
>10% mortality stopped dosing; the group was not euthanized and
recovery is allowed. Within a group with >20% weight loss,
individuals hitting the individual body weight loss endpoint were
euthanized. If the group treatment related body weight loss is
recovered to within 10% of the original weights, dosing resumed at
a lower dose or less frequent dosing schedule. Exceptions to
non-treatment body weight % recovery were allowed on a case-by-case
basis. Endpoint was tumor growth delay (TGD). Animals were
monitored individually. The endpoint of the experiment was a tumor
volume of 1500 mm3 or 45 days, whichever comes first. Responders
were followed longer. When the endpoint was reached, the animals
are to be euthanized.
[0442] 27B.2 Dosing Instructions
[0443] No compounds in salt form were used. The amount needed per
week was calculated, aliquoted accordingly, and stored at -20 C.
For each week of dosing, one aliquot was thawed, stored at 4 C, and
diluted in the required amount with PBS right before each
injection. IL-2-WTI required protection from light; pre-formulation
stored at -4.degree. C., post-formulation--stored at -20.degree. C.
Lyophilized material was reconstituted as directed by instructions,
similar to above. ACP16, ACP130, ACP124, and IL-2 WTI were prepared
for dosing in PBS. IL-2-WTI indicates Proleukin (aldesleukin) in
PBS; the vehicle was PBS.
[0444] Dosing volume was 0.2 mL/mouse for IL-2-WTI; 0.3 mL for
ACP16, ACP130; 0.5 mL for ACP124. Do not adjust for body
weight.
[0445] 27B.3 Special Instructions
[0446] ACP16: current amount of required compound--13.45 mg
[0447] ACP130: current amount of required compound--25.83 mg
[0448] 2 ACP124: current amount of required compound--42.31 mg
[0449] 3 IL-2-WTI: current amount of required compound--9.98 mg
[0450] Necropsy was to be performed in case of unexpected
toxicity
Example 27C: MC38 IFN.alpha. and IL-12
[0451] 27C.1 Agents and Treatment:
TABLE-US-00011 TABLE 7 Summarizes the treatment regime. Gr. N Agent
Formulation dose Route Schedule .sup. 1.sup.# 12 Vehicle -- ip biwk
x 3 2 8 ACP11 17.5 .mu.g/animal ip biwk x 3 3 8 ACP11 175
.mu.g/animal ip biwk x 3 4 8 ACP11 525 .mu.g/animal ip biwk x 3 5 8
ACP31 33 .mu.g/animal ip biwk x 3 6 8 ACP31 110 .mu.g/animal ip
biwk x 3 7 8 ACP31 330 .mu.g/animal ip biwk x 3 8 8 ACP131 1
.mu.g/animal ip bid x 5 then 2-day pause then bid x 5 then 2-day
pause 9 8 ACP131 10 .mu.g/animal ip bid x 5 then 2-day pause then
bid x 5 then 2-day pause 10 8 ACP131 30 .mu.g/animal ip bid x 5
then 2-day pause then bid x 5 then 2-day pause 11 8 mIFNa1- 1
.mu.g/animal ip bid x 5 then 2-day WTI pause then bid x 5 then
2-day pause 12 8 mIFNa1- 10 .mu.g/animal ip bid x 5 then 2-day WTI
pause then bid x 5 then 2-day pause 13 8 IL-12-HM- 2 .mu.g/animal
ip bid x 5 then 2-day WTI pause then bid x 5 then 2-day pause 14 8
IL-12-HM- 10 .mu.g/animal ip bid x 5 then 2-day WTI pause then bid
x 5 then 2-day pause 15 8 ACP131 5 .mu.g/animal itu bid x 5 then
2-day pause then bid x 5 then 2-day pause .sup.#Control Group
[0452] 27C.2 Procedures
[0453] Mice were anaesthetized with isoflurane for implant of cells
to reduce the ulcerations. 308 CR female C57BL/6 mice were set up
with 5.times.10.sup.5 MC38 tumor cells in 0% Matrigel sc in flank.
Cell Injection Volume was 0.1 mL/mouse. Mouse age at start date was
8 to 12 weeks. Pair matches were performed when tumors reach an
average size of 100-150 mm.sup.3 and begin treatment. Body weights
were taken at initiation and then biweekly to the end. Caliper
measurements were taken biweekly to the end. Any adverse reactions
were to be reported immediately. Any individual animal with a
single observation of >than 30% body weight loss or three
consecutive measurements of >25% body weight loss was
euthanized. Any group with a mean body weight loss of >20% or
>10% mortality stopped dosing; the group was not euthanized and
recovery is allowed. Within a group with >20% weight loss,
individuals hitting the individual body weight loss endpoint were
euthanized. If the group treatment related body weight loss is
recovered to within 10% of the original weights, dosing resumed at
a lower dose or less frequent dosing schedule. Exceptions to
non-treatment body weight % recovery were allowed on a case-by-case
basis. Endpoint was tumor growth delay (TGD). Animals were
monitored individually. The endpoint of the experiment was a tumor
volume of 1500 mm.sup.3 or 45 days, whichever comes first.
Responders were followed longer. When the endpoint was reached, the
animals are to be euthanized.
[0454] 27C.3 Dosing Instructions
[0455] No compounds in salt form were used. Prepared dosing
solutions were as follows: IL-12-HM-WTI was stored to provide
protection from light; pre-formulation at -4.degree. C.,
post-formulation at -20.degree. C. mIFNa1-WTI was stored at
-20.degree. C., protected from light; pre-formulation stored at
-20.degree. C., post-formulation stored at -4.degree. C.
Lyophilized material was reconstituted as directed in instructions.
The amount needed per week was calculated, aliquoted accordingly,
and stored at -20.degree. C. For each week of dosing, one aliquot
was thawed and stored at 4 C, the required amount was diluted with
PBS right before each injection.
[0456] For ACP11, ACP31, ACP131, the amount needed per week was
calculated, aliquoted accordingly, and stored at -20 C. For each
week of dosing, one aliquot was thawed and stored at 4 C, the
required amount was diluted with PBS right before each injection.
PBS was used as the vehicle for all tests.
[0457] Intraperitoneal (ip) dosing volume ACP131, mIFNa1-WTI,
IL-12-HM-WTI=0.2 mL/mouse. Dosing volume for ACP11=0.4 mL (before
Day 18, 2/26/19) and 0.55 mL (starting on Day 18, 2/26/19).
[0458] Dosing volume for ACP31=0.3 mL. Dosage was not adjusted for
body weight. Intratumoral (itu) dosing volume for Gr.16 mIFNa1-WTI
and Gr.17 ACP131=0.05 mL/mouse. Dosage was not adjusted for body
weight.
Example 27D: MC38 Rechallenge
[0459] 27D.1 Agents and Treatment:
TABLE-US-00012 TABLE 8 Summarizes the treatment regime. Gr. N Agent
Formulation dose Route Schedule .sup. 1.sup.# 33 No -- -- --
Treatment 2 7 ACP16 70 .mu.g/animal ip MC38-e415 Gr 4 An 1-4, 6-8
(ACP16 biwkx2) 3 8 ACP16 232 .mu.g/animal ip MC38-e415 Gr 5 An 1-8
(ACP16 biwkx2) 5 5 IL-2- 12 .mu.g/animal ip MC38-e415 WTI Gr 16 An
1, 3-6 (IL-2-WTI bid x 5 then 2-day pause then bid x 5 then 2-day
pause) 6 7 IL-2- 36 .mu.g/animal ip MC38-e415 WTI Gr 17 An 1-7
(IL-2-WTI bid x 5 then 2-day pause then bid x 5 then 2-day pause)
.sup.#Control Group
[0460] 27D.2 Procedures
[0461] Mice were anaesthetized with isoflurane for implant of cells
to reduce the ulcerations. This portion of the study began on the
day of implant (Day 1). Group 1 consisted of 33 CR female C57BL/6
mice set up with 5.times.105 MC38 tumor cells in 0% Matrigel
subcutaneously in the flank. Groups 2-6 consisted of 33 CR female
C57BL/6 mice set up with 5.times.105 MC38 tumor cells in
0%/Matrigel sc in the left flank. The tumors from the previous MC38
experiment (example 25.times.) were implanted in the right flank of
each animal. Cell Injection Volume was 0.1 mL/mouse. Age of control
mice at initiation was 14 to 17 weeks. These mice were age matched
to mice from the previous MC38 experiment (example 25.times.). No
dosing of active agent occurred during rechallenge. Body Weights
were taken biweekly until end, as were caliper measurements. Any
adverse reactions or death were reported immediately. Any
individual animal with a single observation of >than 30% body
weight loss or three consecutive measurements of >25% body
weight loss was euthanized. Endpoint was tumor growth delay (TGD).
Animals were monitored individually. The endpoint of the experiment
was a tumor volume of 1000 mm3 or 45 days, whichever comes first.
Responders were followed longer when possible. When the endpoint is
reached, the animals were euthanized.
Example 27E: Treatment with ACP16, APC153, ACP155, and ACP156 (See
FIG. 58)
[0462] 27E.1 Agents and Treatment:
TABLE-US-00013 TABLE 9 Summarizes the treatment regime. Gr. N Agent
Formulation dose Route Schedule .sup. 1.sup.# 12 Vehicle -- ip biwk
x 2 2 8 ACP16 17 .mu.g/animal ip biwk x 2 3 8 ACP16 55 .mu.g/animal
ip biwk x 2 4 8 ACP16 230 .mu.g/animal ip biwk x 2 5 8 ACP155 55
.mu.g/animal ip biwk x 2 6 8 ACP155 230 .mu.g/animal ip biwk x 2 7
8 ACP153 55 .mu.g/animal ip biwk x 2 8 8 ACP153 230 .mu.g/animal ip
biwk x 2 9 8 ACP156 55 .mu.g/animal ip biwk x 2 10 8 ACP156 230
.mu.g/animal ip biwk x 2
[0463] 27E.2 Procedures:
[0464] Mice were anaesthetized with isoflurane for implant of cells
to reduce the ulcerations. CR female C57BL/6 mice were set up with
5.times.10.sup.5 MC38 tumor cells in 0% Matrigel sc in flank. Cell
Injection Volume was 0.1 mL/mouse. Mouse age at start date was 8 to
12 weeks. Pair matches were performed when tumors reach an average
size of 100-150 mm.sup.3 and begin treatment. ACP16 was dosed at
17, 55 or 230 g/animal; ACP153, ACP155 and ACP156 were dosed at 55
or 230 .mu.g/animal. Body weights were taken at initiation and then
biweekly to the end. Caliper measurements were taken biweekly to
the end. Any adverse reactions were to be reported immediately. Any
individual animal with a single observation of >than 30% body
weight loss or three consecutive measurements of >25% body
weight loss was euthanized. Any group with a mean body weight loss
of >20% or >10% mortality stopped dosing; the group was not
euthanized and recovery is allowed. Within a group with >20%
weight loss, individuals hitting the individual body weight loss
endpoint were euthanized. If the group treatment related body
weight loss is recovered to within 10% of the original weights,
dosing resumed at a lower dose or less frequent dosing schedule.
Exceptions to non-treatment body weight % recovery were allowed on
a case-by-case basis. Endpoint was tumor growth delay (TGD).
Animals were monitored individually. The endpoint of the experiment
was a tumor volume of 1500 mm.sup.3 or 45 days, whichever comes
first. Responders were followed longer. When the endpoint was
reached, the animals are to be euthanized. Results are shown in
FIG. 58A-58D.
Example 28: FRET Screens of Conditioned Media
[0465] Proteolytic activity was screened in the conditioned media
samples by fluorescent resonance energy transfer (FRET) assays with
the substrates listed in Table 10.
TABLE-US-00014 TABLE 10 Substrate motif sequences. Experimentally
Tested Substrate SEQ ID in vitro Conditioned 30-mer Name P4-P4'
Sequence NO: kinetics media MSP-MS MMP14_1 GPAGLYAQ 195 MMP9_1
GPAGMKGL 196 FAP.alpha._1 PGGPAGIG 197 CTSL1_1 ALFKSSFP 198 CTSL1_2
ALFFSSPP 199 ADAM17_1 LAQRLRSS 200 ADAM17_2 LAQKLKSS 201
[0466] 28.1 Reaction Conditions
[0467] Protease specificity screening was performed using
Multiplexed Substrate Profiling by Mass Spectrometry (MSP-MS), the
method described in, e.g. O'Donoghue A. J. et al., Nat Methods.
2012; 9(11): 1095-1100. This method employs a physico-chemically
diverse peptide library as substrates for proteases, and reactions
are monitored over time with mass spectrometric detection of
cleaved products. The resulting cleavages are assessed for specific
cleavage in the enzyme-treated sample by comparison with results
from a no-enzyme control incubation. Sequence logos for motif
analysis are generated with iceLogo software (v.1.2)
(iomics.ugent.be/icelogoserver/).
[0468] Recombinant human enzymes were sourced from R&D Systems
(Bio-techne): CTSL1 (#952-CY), ADAM17 (a.k.a. TACE, TNF-alpha
Converting Enzyme) (#930-ADB), FAP-alpha (#930-ADB-010), MMP-14
(#918-MP-010), furin (#1503-SE-010), MMP-9 (#911-MP-010), thrombin
(#2196-SE-200), thermolysin (#3097-ZN), Factor Xa (#1063-SE-010),
hepsin (#4776-SE) and ADAM-TS1 (#2197-AD). Enzymes were activated
and assayed following the manufacturer's recommendations.
[0469] For CTSL1, enzyme was activated by pre-incubation in assay
buffer (50 mM MES, 5 mM DTT, 1 mM EDTA, pH=6) for 15 min. MSP-MS
reactions were then started with the mixing of enzyme and
substrate, at final CTSL1 concentration of 0.04 ng/.mu.l (0.77 nM),
and peptide substrate concentration at 500 nM.
[0470] ADAM17 activity was monitored following manufacturer's
recommended conditions using the FRET substrate
Mca-PLAQAV-Dpa-RSSSR-NH.sub.2(SEQ ID NO: 244), in the recommended
assay buffer: 25 mM TRIS, 2.5 .mu.M ZnCl.sub.2 at the optimal pH
9.0, and also at physiological pH 7.4. ADAM17 enzyme specificity
was profiled in the MSP-MS experiment at pH 9.0 using 10 nM enzyme,
and at pH 7.4 using 50 nM enzyme.
[0471] MMP-14 was activated following manufacturer's recommended
conditions by pre-incubation with the enzyme furin, at a molar
ratio of 1:100 furin to MMP-14, at 37.degree. C. and pH 9.0 for 1.5
hours. FRET assays were performed using 20 nM MMP-14 at 37.degree.
C. in activation buffer: 50 mM TRIS HCl, 3 mM CaCl.sub.2, 1 .mu.M
ZnCl.sub.2 at pH 8.5.
[0472] MMP9 was activated by incubation with 1 mM p-amino phenyl
mercuric acetate (prepared from a stock at 100 mM in DMSO) for 24h
at 37.degree. C. in activation buffer: 50 mM TRIS HCl (pH 7.5), 10
mM CaCl.sub.2, 150 mM NaCl, 0.05% Brij35. The final enzyme
concentration used for fluorescence assays was 2.5 nM.
[0473] FAP.alpha. activity was tested with fluorescence detection
using a generic Z-GP-AMC substrate. For FRET assays, the final
enzyme concentration was 5 nM and the buffer was 50 mM TRIS HCl, 1
M NaCl, 1 mg/mL BSA, pH 7.5.
[0474] Thrombin was activated following the manufacturer's
recommended conditions by pre-incubation with the enzyme with
thermolysin for 15 min, and then the thermolysin was quenched with
1,10 phenanthroline treatment. Activated thrombin was assayed at
1.2 nM in activation buffer: 50 mM TRIS HCl, 10 mM CaCl.sub.2, 150
mM NaCl, 0.05% (w/v) Brij-35, pH 7.5.
[0475] Factor Xa was assayed at 4.7 nM enzyme, at 37.degree. C. in
the manufacturer recommended buffer: 50 mM TRIS, 10 mM CaCl.sub.2,
150 mM NaCl, pH 7.5.
[0476] Hepsin was pre-activated overnight at 37.degree. C. in the
manufacturer recommended buffer: 100 mM TRIS, 10 mM CaCl.sub.2, 150
mM NaCl, 0.05% Brij-35, pH 8.0. Activity was measured with 2.4 or
24 nM enzyme in the buffer: 50 mM TRIS HCl at pH 7.4.
[0477] ADAM-TS1 was assayed at 5 .mu.M enzyme concentration in the
manufacturer recommended buffer: 50 mM TRIS, 10 mM CaCl.sub.2), 150
mM NaCl at pH 7.5.
[0478] 28.2 Samples
[0479] Conditioned media and cell lysate samples were received from
Charles River Laboratories for the murine colon cancer cell lines:
MC38 (epithelial), CT26 (fibroblast), and CT26 transduced with a
lentivirus for doxycyclin-inducible expression of MMP9 (CT26 pLVX
MMP9-5T4+dox). An immortalized mouse intestinal myofibroblast cell
line (ABM Good #T0565) was used as a control stromal cell line,
grown under manufacturer recommended conditions. Conditioned media
and cell lysate samples were also received for screening purposes
from ABM Good for the immortalized mouse colonic epithelial cell
line YAMC (ABM Good #T0567).
[0480] Conditioned media samples were prepared according to the
standard protocol using logarithmic phase cells. Briefly, cells
were grown for 16h in either serum-free media or in complete media
containing 10% fetal bovine serum (FBS). After conditioned media
collection, the adherent cells were washed then lysed on plate with
non-denaturing lysis buffer to produce a lysed "cell pellet" to
capture any activity that remained associated with the cells. All
cell culture-derived conditioned media samples, cell lysates from
Charles River Laboratories, and cell lysates derived internally
were processed with identical methods, to allow comparison between
samples.
[0481] The resulting conditioned media was buffer exchanged with
PBS and concentrated to 10.times. the original titer as a stock
solution.
[0482] 28.3 Results
[0483] Method development experiments with these samples showed
that conditioned media containing fetal bovine serum (FBS) were
amenable to FRET screening, although a small amount of background
fluorescence for each substrate was obtained in the presence of
FBS. FBS background controls were therefore used for baseline
subtraction at each concentration of substrate throughout the FRET
experiments.
[0484] In the end-point screening assays, a fixed titer of
10.times. concentrated sample and a substrate concentration of 10
.mu.M was used. Values reported in the end-point experiments are
initial velocity measurements, in relative fluorescence units (RFU)
per second. End-point measurements are a starting point for a
screen, but the rate of the reaction is non-linearly related to
substrate concentration. Therefore, a more accurate representation
of activity would be given by steady-state kinetic measurements
covering a range of substrate concentrations. To compare activity
across cell lines, the data were treated with a Michaelis-Menten
model, and the concentration of equivalent enzyme units [E.sub.0]
was solved.
[0485] Accordingly, initial velocity was measured as a function of
substrate concentration, using a 2-fold dilution series from 250 to
1 .mu.M. Data were fitted using non-linear least squares fitting
with GraphPad Prism software v 8.0 to the Michaelis-Menten
equation:
velocity = k cat [ E 0 ] [ S ] ( K M + [ S ] ) ##EQU00001##
where k.sub.cat is the rate of product formation under steady-state
conditions in units (s.sup.-1), and K.sub.M is the Michaelis
constant that gives the substrate concentration in molar units (M)
at which half maximum velocity of the reaction is produced for a
given enzyme, [E.sub.0] is the enzyme concentration, and [S] the
substrate concentration in (M).
[0486] In the assays where purified recombinant enzyme is used, the
Michaelis-Menten parameters are as classically defined above. For
conditioned media samples, the effective titer of conditioned media
per assay was adjusted to approximately a 2.times. concentrate of
the original unprocessed media samples to assure that the detection
range matched the signal produced. The enzyme component in the
calculation is redefined as a mixture of possible proteases that
contribute to the cumulative observed activity, each with intrinsic
reactivity toward a substrate. In this case, it is more appropriate
to consider k.sub.cat and K.sub.M as macroscopic constants that
represent the overall efficiency of catalysis, and E.sub.0 is an
enzyme equivalent, expressed in units of concentration. To solve
the data fit, k.sub.cat/K.sub.M can be fixed at that of the
recombinant enzyme and enzyme equivalents per cell culture volume
can be output. The effective titer of enzyme equivalents in this
case can be used to compare the activity produced by the different
cell lines.
[0487] The ratios of enzyme equivalents between tumor and control
cell lines may be a contributing factor to therapeutic index in a
pro-drug employing protease-cleavable linkers.
[0488] For all six motif substrates, greater activity was detected
in the conditioned media from the tumor cell lines than from the
control myofibroblast cell line (FIG. 36).
[0489] The ADAM17 substrate with the sequence LAQKLKSS (ADAM17_2)
(SEQ ID NO: 201), based on the results in the end-point screen
experiments, had approximately 3-fold greater activity in the
conditioned media produced from the three tumor cell lines,
compared to the myofibroblast cell line (FIG. 36, black bars). For
reference, the activity of recombinant ADAM17 at 12 nM is shown in
the same assay format. The steady state kinetic curves for ADAM17
substrate are shown in FIG. 37. There was essentially undetectable
ADAM17_2 background activity in the myofibroblast sample.
[0490] The CTSL1 substrate, ALFKSSFP (SEQ ID NO: 198) (CTSL1_1),
based on the results in the end-point screen experiments, had
undetectable activity in the control myofibroblast cell line, as
well had low activity in the MC38 and CT26 parental cell lines, and
had undetectable activity in the CT26-MMP9+ cell line (FIG. 36).
This assay was benchmarked with CTSL1 at 1.5 nM, showing that the
extracellular concentration of secreted CTSL1 activity was below
this effective concentration across the four cell lines.
Re-analysis of conditioned media with the CTSL1_1 substrate using
the steady-state kinetics analysis yielded no measurable activity
above background.
[0491] The secreted activity for FAP.alpha., based on the results
in the end-point screen experiments, was very low, but was still
detected in all three tumor cell lines (FIG. 36, blue bars). The
FAP.alpha._1 activity was benchmarked with 5 nM FAP.alpha. enzyme.
This enzyme is also found in a plasma membrane-associated form,
therefore FAP.alpha. activity was also tested in the cell pellet
fraction that was collected after the media conditioning procedure.
These results will be discussed below.
[0492] As observed in the end-point screen, re-analysis of the
conditioned media for activity toward the FAP.alpha._1 substrate
showed lower activity than that associated with the cell pellet,
measured in cell lysates (FIG. 38 and FIG. 39). Myofibroblasts had
no measurable secreted activity but did have cell-associated
activity (FIG. 38 and FIG. 39).
[0493] The MMP substrates MMP9_1 and MMP14_1 all had high activity
in the end point screen, and the activity was significantly higher
in the tumor cell lines than in the myofibroblast control cells.
MMP9_1 (second column from right, FIG. 38) had approximately 4-fold
greater activity in the tumor lines than the myofibroblast control;
and MMP14_1 had approximately 57-fold greater activity in the tumor
lines. The MMP14_1 substrate had the lowest activity in the
myofibroblast cells, thus contributing to the higher ratio of tumor
vs control in this assay. Secreted activity was measured for the
MMP9_1 substrate in the conditioned media for each of the tumor
cell lines using the steady-state kinetics analysis (FIG. 36). The
myofibroblast cell line had an artefact in analysis of MMP9_1
secreted activity (FIG. 36); this will be addressed with a repeat
analysis at higher enzyme titer. As with the MMP9 substrates, the
MMP14_1 substrate had significant measurable activity in the
conditioned media from the tumor cells but not in the myofibroblast
cell line.
[0494] Taken together, the results of the FRET screens of
conditioned media demonstrated tumor-specific activity for the
enzymes ADAM17, CTSL1, MMP9 and MMP14. Soluble FAP-alpha activity
was low.
Example 29: Tumor-Specific Activity Toward the FAP.alpha. Substrate
in the Cell Lysates
[0495] To test for cell pellet-associated FAP.alpha. activity, the
cell lysates were clarified by ultracentrifugation and then assayed
neat in the FRET assay. As shown in FIG. 41, FAP.alpha._1 cleavage
activity was detected in all four cell lines (blue bars, right hand
bar of each pair). A modest 2-fold greater activity was detected in
the three tumor cell lines over the myofibroblast cell line. For
comparison, CTSL1 activity was also tested in the cell lysates.
CTSL1 is a lysosomal enzyme; therefore, activity toward CTSL1_1 was
expected to be similarly abundant in all four cell lines. The ratio
of CTSL1_1 cleavage in tumor vs control cell lines in this
screening format was .about.1.times. (grey bars, left-hand bar of
each pair, FIG. 41). FAP.alpha. (fibroblast activating
protein-alpha) is a marker for fibroblast (CT26) and myofibroblast
cell lines. Therefore, an additional non-fibroblast, epithelial
cell line was obtained as an alternate negative control cell line,
comparable to the MC38 epithelial cell line.
Example 30: Cell-Associated Activity Toward CTSL1 Substrate
[0496] Cell-associated activity toward CTSL1 substrate was detected
in all of the cell lysates, indicating cell-associated activity for
this enzyme. The activity was significantly greater in MC38 cells
over the myofibroblast, CT26 parental, and CT26-MMP9+ cells (FIG.
42).
Example 31: Secreted Activity in Conditioned Media from
Immortalized Myofibroblast Cell Line
[0497] Low background-corrected fluorescence was observed in all
the myofibroblast reactions. Thus, it can be concluded that the
immortalized myofibroblast cell line had low conditioned media
activity.
Example 32: Screening of an Additional Control "Normal" Epithelial
Cell Line
[0498] An immortalized mouse colonic epithelial cell line (YAMC)
was used to test activity in the presence of additional control
"normal" epithelial cells. YAMC's are given the calculated enzyme
equivalents (nM) resulting from all of these steady state
experiments, to allow for comparison of the amount of equivalent
activity per sample. For example, the YAMC control epithelial cell
line produced activity with the ADAM17_2 substrate equivalent to
that of 0.52.+-.0.03 nM of ADAM17 recombinant enzyme. This cell
line compares directly to MC38, also of epithelial origin, which
had activity equivalent to that of 0.32.+-.0.03 nM of ADAM17
recombinant enzyme. The results of the screening show that the YAMC
cell line generally had more background activity than did the
myofibroblast cell line with all of the substrates. This cell line
required non-standard cell culture conditions, including growth
with murine IFN.gamma., and at low temperature to maintain, and may
not be fully representative of epithelial cell lines.
Example 33: Cleavage of CTSL1_2 Motif in the Context of an 8-Mer
FRET Peptide and in the Context of an Extended Tandem Linker
[0499] Additional in vitro kinetic experiments were performed to
characterize a new candidate motif for CTSL1 enzyme; the CTSL1_2
motif has the sequence ALFFSSPP (SEQ ID NO: 199). CTSL1 cleaves the
FRET substrate bearing the CTSL1_1 motif highly efficiently (FIG.
46). Yet, initial experiments with the CTSL1_2 FRET substrate were
inconclusive, possibly due to artifacts in the FRET assay;
therefore, a new FRET peptide construct was designed with the 9-mer
sequence ALFFSSPPS (SEQ ID NO: 236). This 9-mer FRET substrate
bearing the CTSL1_2 motif produced comparable catalytic efficiency
to the CTSL1_1 motif.
[0500] All of the FRET substrates used in these studies have a
methoxycoumarin (Mca) fluorophore and dinitrophenyl-lysyl (Dnp)
quencher pair at the N- and C-termini of the sequences,
respectively. The motif ALFFSSPP (SEQ ID NO: 199) was efficiently
cleaved in previous experiments at ALFF|SSPP (SEQ ID NO: 199)
(where | indicates the scissile bond) within 14-mer peptide
constructs that were used in the MSP-MS tailored library assay. It
was cleaved also at the ALF|FSSPP (SEQ ID NO: 199) and ALFFS|SPP
(SEQ ID NO: 199) sites within 14-mer peptides, indicating that the
enzyme recognition in the CTSL1_2 motif may be shifted up- or
down-sequence by +/-1 site in the 14-mer peptide experiments. This
also suggests that the 8-mer design using Mca-ALFFSSPPK-Dnp (SEQ ID
NO: 245) may have unfavorable interactions (steric or otherwise)
with the fluor/quencher pair in the shifted P4 or P4' positions
that were alleviated by the redesign as a 9-mer substrate using
Mca-ALFFSSPPSK-Dnp (SEQ ID NO: 246). This FRET experiment indicated
that cleavage of this sequence most likely requires binding
recognition at the +/-1 positionally-shifted site and that the
fluor or quencher may interfere with this binding.
[0501] This hypothesis was tested in the context of the hybrid
linker design experiment. Shown in FIG. 46 are the results of
cleavage of two matched 30-mer peptides with the following
sequences:
TABLE-US-00015 (CTSL1_1) (SEQ ID NO: 207)
SGGPGGPAGIGALFKSSFPLAQKLKSSGGG (CTSL1_2) (SEQ ID NO: 219)
KSGPGGPAGIGALFFSSPPLAQKLKSSGGR
[0502] Both peptides showed rapid disappearance of substrate
(squares) and product formation from cleavage at the target
scissile bond (site 0, blue triangles, FIG. 46). Where the peptides
differed is in the permissive cleavage at the -1 and +1 sites.
CTSL1_1 had a -1 site product at ALF|KSSFP (SEQ ID NO: 198)
(downward triangles) but CTSL1_2 did not, and the +1 site product
was more rapidly formed from the CTSL1_2 substrate (ALFFS|SPP (SEQ
ID NO: 199)) than with the CTSL1_1 substrate (ALFKS|SFP) (SEQ ID
NO: 198).
[0503] Counter-screening of CTSL1_1 and CTSL1_2 was performed with
the enzyme Cathepsin K (CTSK). The substrate CTSL1_2, although
lacking a basic P1 residue (ALFFSSPP (SEQ ID NO: 199)) was
measurably cleaved by CTSK but at a 15.times. lower level than the
CTSL1_1 substrate (ALFKSSFP (SEQ ID NO: 198)) FIG. 47. For
comparison, the specific activity for reference substrates such as
Z-LR-AMC and Z-FR-AMC is 15,000-25,000 pmol/min/.mu.g, with the
average value indicated by a dashed line in FIG. 47.
[0504] Thus, these results show that the new CTSL1_2 motif was not
cleaved in the context of an 8-mer FRET peptide, but it was cleaved
as a 9-mer FRET peptide and it was rapidly cleaved in the context
of an extended tandem linker.
Example 34: Tandem Linker Analysis with Mass Spectrometric
Detection
[0505] 34.1 Substrate Profiling by Mass Spectrometry
[0506] To analyze the catalytic efficiency of candidate tandem
linker designs, a tailored library approach using MSP-MS was
applied. For the tailored library, a set of nineteen synthetic
peptides, 30 amino acids in length (30-mers), were designed to test
whether sequence context affects the efficiency of cleavage.
Peptides were assayed in a multiplex format together as a substrate
library with individual recombinant enzymes, using MSP-MS. For
quantitative comparisons, kinetic analysis was performed over a
time course, and results are reported either as a catalytic
efficiency k.sub.cat/K.sub.M for well-behaved first-order kinetics,
or as observed rates as k.sub.obs for more complex kinetic
behavior.
[0507] 34.2 the Library of Tandem Linker Sequences
[0508] The library of tandem linker sequences was designed to
incorporate three individual protease motifs within the context of
a longer, 30-mer peptide sequence. Table 11 lists the sequences and
motif arrangement of the 30-mer peptides.
TABLE-US-00016 TABLE 11 30-mer peptide sequences. Substrate SEQ ID
Name Sequence NO: ALU30-1 GALFKSSFPSGGGPAGLYAQGGSGKGGSGK 202
ALU30-2 RGSGGGPAGLYAQGSGGGPAGLYAQGGSGK 203 ALU30-3
KGGGPAGLYAQGPAGLYAQGPAGLYAQGSR 204 ALU30-4
RGGPAGLYAQGGPAGLYAQGGGPAGLYAQK 205 ALU30-5
KGGALFKSSFPGGPAGIGPLAQKLKSSGGS 206 ALU30-6
SGGPGGPAGIGALFKSSFPLAQKLKSSGGG 207 ALU30-7
RGPLAQKLKSSALFKSSFPGGPAGIGGGGK 208 ALU30-8
GGGALFKSSFPLAQKLKSSPGGPAGIGGGR 209 ALU30-9
RGPGGPAGIGPLAQKLKSSALFKSSFPGGG 210 ALU30-10
RGGPLAQKLKSSPGGPAGIGALFKSSFPGK 211 ALU30-11
RSGGPAGLYAQALFKSSFPLAQKLKSSGGG 212 ALU30-12
GGPLAQKLKSSALFKSSFPGPAGLYAQGGR 213 ALU30-13
GGALFKSSFPGPAGLYAQPLAQKLKSSGGK 214 ALU30-14
RGGALFKSSFPLAQKLKSSGPAGLYAQGGK 215 ALU30-15
RGGGPAGLYAQPLAQKLKSSALFKSSFPGG 216 ALU30-16
SGPLAQKLKSSGPAGLYAQALFKSSFPGSK 217 ALU30-17
KGGPGGPAGIGPLAQRLRSSALFKSSFPGR 218 ALU30-18
KSGPGGPAGIGALFFSSPPLAQKLKSSGGR 219 ALU30-19
SGGFPRSGGSFNPRTFGSKRKRRGSRGGGG 220
[0509] To test for additive effects of having one, two, or three
repeat MMP14_1 motifs with small differences in spacing between the
motifs, a set of four tandem linkers were designed (ALU30-1 to
-4).
[0510] To test whether neighboring effects from adjacent sites
could alter the catalytic efficiency of a given protease toward its
target motif, the three motifs in a set were arranged in all
permutations. For example, the combination of motifs A, B, and C
could be arranged as: A-B-C, C-A-B, B-C-A, B-A-C, A-C-B, and C-B-A.
These permutations were tested for the tandem combinations of
MMP14_1, ADAM17_2, and CTSL1_1 as a broad set of motifs susceptible
to matrix metalloprotease (MMP) activity (ALU30-11 to -16), and for
FAP.alpha._1 with ADAM17_2, and CTSL1_1 as a set with lower MMP
sensitivity (ALU30-5 to -10).
[0511] Also tested within the library were two alternate substrates
for ADAM17 and CTSL1. Two additional peptides were designed,
swapping the ADAM17_2 motif with ADAM17_1 (in ALU30-17), or CTSL1_1
with CTSL1_2 (in ALU30-18), demonstrating a strategy for testing
further tandem linker designs with new individual protease
motifs.
[0512] 34.3 Counter-Screening
[0513] Counter-screening was performed against the enzymes
Thrombin, Factor Xa and Hepsin using this library as well, and for
calibration of activity, a positive-control peptide bearing
favorable motifs for these enzymes was also designed
(ALU30-19).
[0514] Outside of these motifs, minor variations were made to the
bracketing sequences at the N- or C-terminus of each tandem linker
peptide to generate unique sequences that allow for differentiation
of peptides using mass spectrometric detection.
[0515] Catalytic efficiency (k.sub.cat/K.sub.M) estimations were
used to rank the top cleaved substrates in the MSP-MS reaction.
Peptides were quantified by mass spectrometric label-free
quantitation from the MS1 precursor ion peak areas for each
peptide. Enzyme progress curves were modeled from six-time point
measurements in GraphPad Prism. Data were fitted using non-linear
least squares fitting to the first order kinetics equation:
Y = e ( - k cat K M [ E 0 ] t ) ##EQU00002##
where Y=percent product formation or substrate consumption, and
t=time. The observed rate is a function of the enzyme concentration
[E.sub.0], and an observed catalytic efficiency (k.sub.cat/K.sub.M)
in units M.sup.-1s.sup.-1
[0516] 34.4 Results
[0517] Analyses with the 30-mer tandem linker library showed
specific cleavages with each of the recombinant enzymes. In this
experiment, the rates of substrate degradation and of product
formation were both useful comparisons for understanding the
efficiency of linker cleavage.
[0518] For example, the substrate degradation traces for the 30-mer
library peptides are shown from MMP9-treatment in FIG. 55. The most
efficient MMP9 substrates were those that bore three-repeats of the
MMP14_1 motif. The next most efficient substrates contained the
pair of motifs where MMP14_1 was followed directly by ADAM17_2. The
single or double MMP14_1 motif-bearing peptides were next in the
order. An unanticipated result was that the FAP.alpha._1 motif was
also cleaved within the peptides bearing FAP.alpha._1 directly
followed by the CTSL1_1 motif at PGGPAG|GALF (SEQ ID NO: 247);
these were lower efficiency cleavages. The FAP.alpha._1 motif was
not cleaved by MMP9, however, when followed either by the ADAM17_2
motif or the spacing residues GG. The slowest peptides bear the
MMP14_1 motif near the C-terminus of the 30-mer peptide. From this
experiment, a trend emerged that MMP9 has additional sequence
preferences in the downstream prime-side positions, and that the
combination of MMP14_1 upstream of ADAM17_2 is most efficient.
[0519] To better understand the intrinsic rates of product
formation with MMP9 treatment, it is also possible to monitor the
cleavage products at a specific peptide bond in these experiments.
A comparison of peptides bearing one, two or three MMP14_1 tandem
motifs is shown in FIG. 50. The most rapidly degraded peptides were
those bearing three repeat motifs of MMP14_1 (top panel). Overall,
the peptides bearing one or two MMP14_1 motifs were degraded at
approximately the same rate (top panel). However, considering
intrinsic rates of bond cleavage at individual sites, the cleavage
product of Alu30-2 at bond 9 appeared more rapidly than the
cleavage product of Alu30-1 at bond 16 or of Alu30-2 at bond 21
(bottom panel). Thus, the specific cleavage of the Alu30-2 peptide
was more efficient than the Alu30-1 peptide. These individual bond
cleavage events are monitored by tracking unique peptide fragments,
and although too complex for data fitting, the ranking of products
is possible.
[0520] FAP.alpha. was able to cleave both FAP.alpha._1 and MMP14_1
motifs, at PGGP|AGIG (SEQ ID NO: 197) and GP|AGLYAQ (SEQ ID NO:
195) respectively. Degradation of the 30-mer peptides by FAP.alpha.
showed highest cleavage activity toward the peptides bearing tandem
MMP14_1 motifs (FIG. 50, Alu30-4 or Alu30-3). The peptide bearing
two MMP14_1 motifs (Alu30-2) was also efficiently cleaved.
FAP.alpha._1 motifs were cleaved more efficiently when followed by
the ADAM17_1 or ADAM17_2 motifs than when followed by CTSL1_1 or
CTSL1_2. Among these peptides, the Lys-bearing ADAM17_2 or CTSL1_1
motifs were lower efficiency than the new motifs tested in this
experiment, ADAM17_1 and CTSL1_2.
[0521] Another trend was a preference for cleavage of the
FAP.alpha._1 motif in the first or second position relative to the
N-terminus of these peptides. When the FAP.alpha._1 motif was in
the third position closest to the C-terminus, these peptides were
not cleaved (Alu30-7 or Alu30-8). The MMP14_1 motif was readily
cleaved in the third position closest to the C-terminus, but this
was most evident in the tandem MMP14_1 motif-bearing peptides,
where N-terminal cleavages assist in shortening the 30-mer. Thus,
it may be that FAP.alpha. could serve as a secondary-cleaving
enzyme after activation with a first enzyme elsewhere in a hybrid
linker design.
[0522] CTSL1 treatment of the 30-mer peptide library produced
cleavages in all peptides bearing a CTSL1_1 or CTSL1_2 motif (FIG.
51). The most efficiently cleaved peptides were those containing
the FAPa_1, CTSL1_1 and ADAM17_2 motifs, in multiple orders.
Peptides bearing the MMP14_1 motif with CTSL1_1 and ADAM17_2 were
slightly lower efficiency. CTSL1 motifs in the middle of the 30-mer
peptides were also favorable, thus CTSL1 may be able to serve as a
first cleavage in the hybrid motifs.
[0523] ADAM17 produced cleavages in all peptides bearing an
ADAM17_1 or ADAM17_2 motif as well (FIG. 52). The most efficient
peptide cleavages occurred in the peptides containing ADAM17_2,
CTSL1_1 and FAP.alpha._1 motifs. Slightly lower efficiency
cleavages were obtained in peptides containing the MMP14_1 with
ADAM17_2 and CTSL1_1. Peptides bearing only the MMP14_1 motif were
not cleaved. Peptides Alu30-9 and Alu30-17, containing the ADAM17_2
and ADAM17_1 motifs respectively, had similar cleavage
efficiencies.
[0524] Counter-screening with Thrombin, Factor Xa and Hepsin was
also performed following the same analysis. The library peptide
Alu30-19 was designed to include authentic sites for each enzyme,
for comparison of overall cleavage efficiency as well as cleavage
site specificity within each motif. The substrate designed for
Factor Xa has the motif FNPR|TFGS (SEQ ID NO: 248), derived from
the Factor Xa cleavage site in Thrombin. The substrate motif for
Thrombin was FPR|, a common tool substrate motif. The substrate
motif for hepsin was RKRR|GSRG (SEQ ID NO: 249) from filaggrin.
[0525] Shown in FIG. 53 are the results of Factor Xa cleavage. The
authentic cleavage site motif was Peptide Alu30-19 with this motif
was completely degraded before the 5 min time point. Products of
the Alu30-19 cleavage were formed from all three sites in this
peptide. In comparison, the only library peptide with significant
cleavage was Alu30-5, cleaved at the CTSL1_1 motif ALFKS|SFP (SEQ
ID NO: 198), upstream of a FAP.alpha._1 motif.
[0526] Otherwise, all other peptides bearing a CTSL1_1 motif were
cleaved by Factor Xa at either ALF|KSSFP (SEQ ID NO: 198) or
ALFKS|SFP (SEQ ID NO: 198). The ADAM17 motifs were also cleaved at
KLK|SSGP (SEQ ID NO: 250), KLKSS|ALF (SEQ ID NO: 251), or RLR|SSALF
SEQ ID NO: 252), but these cleavages were all lower efficiency. The
peptide with the motif CTSL1_2 was cleaved at the lowest
efficiency.
[0527] Thrombin showed higher activity than Factor Xa, and it
cleaved each of the 30-mer peptides. The MMP14_1 motifs were
cleaved at GPAG|LYAQ (SEQ ID NO: 195), the CTSL1_1 motifs at
ALFK|SSFP (SEQ ID NO: 198), and the ADAM17_2 motifs at LAQK|LKSS
(SEQ ID NO: 201) or LAQKLK|SS (SEQ ID NO: 201) (or LAQR|LRSS (SEQ
ID NO: 200) and LAQRL|SS (SEQ ID NO: 253) in ADAM17_1). The two
alternate ADAM17 motifs were cleaved with similar efficiency
(peptides Alu30-17 vs Alu30-9). However, the CTSL1_2 motif had
lower cleavage activity than did the CTSL1_1 motif (peptides
Alu30-18 vs Alu30-6). The peptides Alu30-1 and Alu30-3 were the
most rapidly cleaved in the library, and Alu30-4 was also an
efficient substrate, showing the susceptibility of MMP14_1 motifs
in this experiment. The peptide Alu30-2, bearing two MMP14_1
motifs, was less susceptible to thrombin, however, potentially due
to altered spacing with additional glycine residues between motifs.
Also, the peptides Alu30-12, Alu30-13 and Alu30-15, with MMP14_1
motifs in combination with ADAM17_2 and CTSL1_1, were cleaved at
lower efficiency by thrombin. Thus, increased spacing between the
motifs or specific arrangements may rescue a thrombin-susceptible
linker design (FIG. 54).
[0528] The favored arrangements for reducing thrombin
susceptibility include using ADAM17_2 upstream of CTSL1_1, and
CTSL1_1 upstream of FAP.alpha._1.
[0529] Finally, hepsin treatment of the 30-mer library produced
lower efficiency cleavages overall (FIG. 55). The kinetics of these
peptide degradation reactions were complex due to the formation of
multiple cleavage produces; for example, Alu30-19 was cleaved at
all three motifs within the first 15 min of the reaction at RKRR|
(SEQ ID NO: 254), FPR| and FNPR| (SEQ ID NO: 255). The literature
value for the catalytic efficiency of a FRET peptide bearing this
motif was 3.5.times.10.sup.5 M.sup.-1s.sup.-1. The authentic
peptide Alu30-19, as well as the top cleaved peptides Alu30-8,-9,
-12, and -14 all had apparent catalytic efficiencies on the same
order of 10.sup.5 M.sup.-1s.sup.-1. The features that made peptides
more susceptible to hepsin appear to be placement of an ADAM17_2
motif in the second motif position of the peptide, or in the
combination of ADAM17_2 followed by CTSL1_1. In general, the
library peptides were all cleaved at P1 Lys sites, QKLK| (SEQ ID
NO: 256) or ALFK| (SEQ ID NO: 257). The ADAM17_1 motif was less
efficiently cleaved than ADAM17_2, and the alternate CTSL1_2 motif
was also cleaved at a much slower rate than CTSL1_1.
[0530] To conclude, the tandem linker analysis with mass
spectrometric detection revealed motif order preferences and
unexpected side-reactions.
Example 35: 30-Mer Tandem Linker Design
[0531] To generate the most efficient tandem linker, the cleavage
efficiencies for each of the targeted enzymes toward the 30-mer
library peptides can be compared (FIG. 64).
[0532] The tandem linker with the arrangement of
ADAM17_2-MMP14_1-CTSL1_1 in the peptide Alu30-16 had generally high
activity toward the full set of five targeted enzymes, as well as
the lowest susceptibility to thrombin, Factor Xa, and hepsin. The
next best configuration was MMP14_1-ADAM17_2-CTSL1_1 in Alu30-15
which, although it had slightly higher hepsin susceptibility and
lower CTSL1 activity may be rescued by the replacement with the
CTSL1_2 motif. Replacement of the ADAM17_2 motif with ADAM17_1 may
also enhance activity toward FAP-alpha and CTSL1, even if ADAM17
activity enhancement is minor, and hepsin susceptibility would be
predicted to be reduced.
Example 36. Stability in Serum and Plasma
[0533] The stability of constructs of interest was measured in
human or mouse serum. Each construct (approximately 30 mg/mL) was
combined with serum (1:9 ratio of construct to serum), MMP9, or
PBS. The mixture was incubated at 37.degree. C. for 24 or 72 hours.
Samples were taken at T=0 hours for comparison. After incubation,
samples were diluted 1:500 (in human serum, FIG. 56) or 1:200 (in
mouse serum, FIG. 57) in PBS and run on an SDS PAGE gel for western
blot analysis. A polyclonal anti-IL-2 antibody (R&D Systems)
was used to probe the blots. Results are shown in FIG. 56 and FIG.
57.
Example 37. IL-2 Serum Stability
[0534] The stability of IL2 fusion proteins in human serum (normal
and cancer patient) was measured using capillary
electrophoresis-based immunoassays (Jess instrument, Protein
Simple). Fusion proteins were concentrated to 10 mg/ml and
incubated with serum (90%). A time zero sample was immediately
stored on ice while 24 hour and 72 hour samples were placed at
37.degree. C. Post incubation, samples were diluted 1:1000 with
0.1.times. sample buffer (Protein Simple) and loaded on the Jess
cartridge per the manufacturer's protocol. The primary antibody was
a monoclonal human IL2 antibody (R&D Systems, cat #AF-202-NA,
stock concentration 0.2 mg/ml, working concentration 1:100), and
the secondary antibody was a peroxidase-conjugated AffiniPure
Bovine Anti Goat IgG (H+L) (Jackson Immuno Research, cat
#805-035-18, reconstituted at 0.8 mg/ml, working concentration
1:5000 dilution). All antibodies were diluted in milk free diluent
(Protein Simple). Quantitation of IL2 containing species was
quantitated using Compass software (Protein Simple) to determine
the extent of cleavage (i.e. amount of IL2 containing species
smaller than the input fusion protein). Results are shown in FIGS.
24A-24B.
Example 38: Tissue Stability
[0535] Primary human lung epithelial cells and renal epithelial
cells were obtained from ATCC. Primary hepatocytes were obtained
from Lonza and Sigma. Cells were thawed, counted, and plated at 1e4
cells per well in a 96 well round bottom plate in their respective
growth medias. Polypeptides containing recombinant human IL-2 and
the sequence for Linker-2 and Linker-3 were incubated with cells or
media alone for 24 and 72 hours at a concentration 5 .mu.g/mL. Cell
culture supernatants were collected, and cells were discarded.
Protein cleavage was measured by western blot for IL-2 using the
protein simple JESS system and Compass software. Results are shown
in FIG. 5.
[0536] Primary human lung fibroblasts were obtained from ATCC.
Prior to the processing assay, human PBMCs were stimulated with 5
ug/mL PHA for 72 hours to generate T blasts, which were then frozen
and used to measure polypeptide processing. Primary human
fibroblasts were thawed, counted, and plated at 1e5 cells per well
in a 96 well round bottom plate in X-Vivo 15 media. Polypeptides
containing recombinant human IL-2 and the sequence for Linker-1
(GPAGMKGL, SEQ ID NO: 196), Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or
Linker-3 (ALFKSSFP, SEQ ID NO: 198), or a non-cleavable sequence
were incubated with or without fibroblasts cells for 48 hours at a
concentration of either A) 3.3 nM or B) 0.33 nM. As a positive
control, some wells were also incubated with a polypeptide that was
pre-cut in vitro overnight with 1 .mu.g pre-activated MMP9 enzyme
at 37.degree. C. After 48 hours, cell culture supernatants were
collected and healthy fibroblasts were discarded. Polypeptide
processing was measured either by A) western blot for IL-2 using
the protein simple JESS system, or by B) measuring the capacity of
the cell culture supernatants to stimulate T blast proliferation.
Briefly, T blasts were incubated for 72 hours with cell culture
supernatants containing polypeptides previously exposed to healthy
human fibroblasts, before T blast proliferation was measured by
Cell Titer Glow analysis. Results are shown in FIG. 6A-6B.
Example 39: Linker-2 is Efficiently Cleaved in Human Tumor
Cells
[0537] Cleavage efficiency of Linker-2 (GPAGLYAQ, SEQ ID NO: 195)
was evaluated in tumor tissue derived from human samples. A total
of 66 samples were analyzed from seven solid tumor types. The tumor
types are shown in Table 5 below.
[0538] Briefly, frozen dissociated tumor cells (DTC) were thawed,
counted and plated in X-Vivo media. Cells or "No Cell Control"
wells were incubated with an inducible IL-2 cytokine containing
Linker 1 (a cleavable linker that was not designed using the
processes described herein) or Linker-2 (GPAGLYAQ, SEQ ID NO: 195).
These inducible IL-2 proteins have no or minimal IL-2 biological
activity when the linker is intact, and IL-2 biological activity is
induced when the linker is cleaved. Cell culture supernatants were
collected and frozen before analysis for IL-2 biological activity
using T-Blast proliferation and Jess protein assays.
[0539] The minimum fold change in IL-2 activity is 1.00, and the
maximum is 2.50. A 1.3 fold change is indicative of a significant
increase in biological activity relative to the uncleaved control.
Table 5 shows the average fold change for Linker-2 (GPAGLYAQ, SEQ
ID NO: 195) in comparison to an uncleaved control, a cleaved
control, and Linker-1. In Table 12 the symbol "-" indicate
essentially no increase in activity; "+/-" indicates some increase
in activity but not significant; "+," "++," and "+++" indicate the
relative significant increase in activity compared to the
uncleavable control. Linker-2 is sufficiently cleaved in human
tumor types to induce IL-2 biological activity.
TABLE-US-00017 TABLE 12 Cleavage efficiency of Linker-2 in human
tumor types Average Fold Change Over Non-Cleavable Diagnosis
Uncleavable Linker 1 Linker 2 Pre-cut Melanoma - - +/- +++ (n = 8)
Kidney Cancer, - - + +++ Renal Cell Carcinoma (n = 11) Head and
Neck Cancer, - +/- +++ +++ Squamous Cell (n = 6) Colorectal Cancer,
- +/- + +++ Adenocarcinoma (n = 10) Lung Cancer, - - ++ +++
Squamous Cell Carcinoma (n = 13) Lung Cancer, - - + +++
Adenocarcinoma (n = 10) Breast Cancer - - +/- +++ (n = 5)
Example 40: Linker-3 is Efficiently Cleaved in Human Tumor
Cells
[0540] Cleavage efficiency of Linker-3 (ALFKSSFP, SEQ ID NO: 198)
was evaluated in tumor tissue derived from human samples. A total
of 66 samples were analyzed from seven solid tumor types. The tumor
types are shown in Table 6 below.
[0541] Briefly, frozen DTC cells were thawed, counted and plated in
X-Vivo media. Cells or "No Cell Control" wells were incubated with
an inducible IL-2 cytokine containing Linker 1 (a cleavable linker
that was not designed using the processes described herein) or
Linker-2 (GPAGLYAQ, SEQ ID NO: 195). These inducible IL-2 proteins
have no or minimal IL-2 biological activity when the linker is
intact, and IL-2 biological activity is induced when the linker is
cleaved. Cell culture supernatants were collected and frozen before
analysis for IL-2 biological activity using T-Blast proliferation
and Jess protein assays.
[0542] The minimum fold change in IL-2 activity is 1.00, and the
maximum is 2.50. A 1.3 fold change is indicative of a significant
increase in biological activity relative to the uncleaved control.
Table 6 shows the average fold change for Linker-3 (ALFKSSFP, SEQ
ID NO: 198) in comparison to an uncleaved control, a cleaved
control, and Linker 1. In Table 13 the symbol "-" indicate
essentially no increase in activity; "+/-" indicates some increase
in activity but not significant; "+," "++," and "+++" indicate the
relative significant increase in activity compared to the
uncleavable control. Linker-3 (ALFKSSFP, SEQ ID NO: 198) is
sufficiently cleaved in human tumor types to induce IL-2 biological
activity.
TABLE-US-00018 TABLE 13 Cleavage efficiency of Linker-3 in human
tumor types Average Fold Change Over Non-Cleavable Diagnosis
Uncleavable Linker 1 Linker 3 Pre-cut Melanoma - - + +++ (n = 8)
Kidney Cancer, - - + +++ Renal Cell Carcinoma (n = 11) Head and
Neck Cancer, - +/- +++ +++ Squamous Cell (n = 6) Colorectal Cancer,
- +/- +++ +++ Adenocarcinoma (n = 10) Lung Cancer, - - +++ +++
Squamous Cell Carcinoma (n = 13) Lung Cancer, - - +++ +++
Adenocarcinoma (n = 10) Breast Cancer - - + +++ (n = 5)
Example 41: Measuring Inducibility of Agonist Anti-4-1BB Antibodies
PGP-41
[0543] A stable HT-1080 cell line expressing human 4-1BB was
established. Agonism of human-4-1BB in these cells resulted in
increased secretion of IL-8. Tetravalent monospecific antibodies,
inducible format tetravalent monospecific antibodies (protease
cleaved or uncleaved), or trimeric ligands were tested for ability
to agonize 4-1BB.
[0544] Before addition to the cultured cells, some samples of the
antibody to be tested were incubated with an appropriate protease
under suitable conditions for proteolysis. Some samples were
maintained in an uncleaved state. The extent of inducibility was
determined by comparison of uncleaved (uninduced) inducible format
tetravalent monospecific antibody with the corresponding protease
cleaved inducible format tetravalent monospecific antibody.
[0545] After incubation at 37.degree. C. and 5% CO2 for 6 hours,
the agonistic activities of the cleaved and uncleaved antibodies
were evaluated by the quantification of IL-8 production using an
IL-8 AlphaLISA or ELISA. The EC50s and maximum IL-8 levels were
compared. Results are shown in FIG. 65A-65B.
Example 42: Protease Cleavage of Anti-4-1BB Antibodies by MMP9
[0546] One of skill in the art would be familiar with methods of
setting up protein cleavage assay. 100 .mu.g of protein in
1.times.PBS pH 7.4 were cleaved with 1 .mu.g active MMP9 (Sigma
catalog #SAE0078-50 or Enzo catalog BML-SE360) and incubated at
room temperature for up to 16 hours. Digested protein is
subsequently used in functional assays or stored at -80.degree. C.
prior to testing. The extent of cleavage was monitored by SDS PAGE
using methods well known in the art.
Example 43: Protease Cleavage of Anti-4-1BB Antibodies by MMP14 or
CTSL1
[0547] One of skill in an be replaced with Linker-3. the art would
be familiar with methods of setting up protein cleavage assay. 100
.mu.g of protein in 1.times.PBS pH 7.4 are cleaved with 1 .mu.g
active MMP14 (R&D Systems Catalog #9518-MP-010) or CTSL1
(R&D Systems Catalog #952-CY) and incubated at room temperature
for up to 16 hours. Digested protein are subsequently used in
functional assays or stored at -80.degree. C. prior to testing. The
extent of cleavage is monitored by SDS PAGE using methods well
known in the art.
8. OTHER EMBODIMENTS
[0548] The disclosure set forth above may encompass multiple
distinct inventions with independent utility. Although each of
these inventions has been disclosed in its preferred form(s), the
specific embodiments thereof as disclosed and illustrated herein
are not to be considered in a limiting sense, because numerous
variations are possible. The subject matter of the inventions
includes all novel and nonobvious combinations and subcombinations
of the various elements, features, functions, and/or properties
disclosed herein. The following claims particularly point out
certain combinations and subcombinations regarded as novel and
nonobvious. Inventions embodied in other combinations and
subcombinations of features, functions, elements, and/or properties
may be claimed in this application, in applications claiming
priority from this application, or in related applications. Such
claims, whether directed to a different invention or to the same
invention, and whether broader, narrower, equal, or different in
scope in comparison to the original claims, also are regarded as
included within the subject matter of the inventions of the present
disclosure.
[0549] Exemplary polypeptide constructs are detailed herein in
Appendix A. While the exemplary polypeptides that contain Linker-2
(GPAGLYAQ, SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID NO: 198)
or other cleavable linkers are disclosed in Appendix A, for each
construct, the disclosed linker can be replaced with either
Linker-2 (GPAGLYAQ, SEQ ID NO: 195), or Linker-3 (ALFKSSFP, SEQ ID
NO: 198) or other cleavable linkers disclosed herein. For example
construct
ACP355 (IgG4_Fc(S228P)-X-CD25ecd_C213S-LX-IL2-LX
Blocker_(Blocker=VHVL.F2.high.A02_Vh\Vl_A46S;X=MMP14-1) can contain
Linker-2, however it can be replaced with Linker-3. Alternatively,
construct ACPT464
(IgG4_Fc(S228P)-X-IL2-LX-blocker_(Blocker=VHVL.F2.high.A02_Vh/Vl_VH105-VL-
43_disulfidel;X=CTSL1-1) can contain Linker-3, however, it can be
replaced with Linker-2.
[0550] The elements of the polypeptide constructs provided in
Appendix A contain the abbreviations as follows: "L," "X," "LX,"
and "XL" each refer to a linker. "X" refers to a cleavable linker.
"L" refers a linker that is optionally cleavable. When L is the
only linker in a polypeptide, L is cleavable. "LX" or "XL" each
refer to a cleavable linker with an extended non-cleavable sequence
adjacent to it. Cleav. Lin. Also refers to a cleavable linker.
Other abbreviations used include: "mIFNg" for mouse interferon
gamma (IFNg); (5) "hAlbumin" for human serum albumin (HSA); and (6)
"mAlbumin" indicates mouse serum albumin.
TABLE-US-00019 APPENDIX A CONSTRUCT PERMUTATION TABLE Construct
Name Construct Description ACP01 (anti-HSA)-(cleav. link.)-mouse
IFN.gamma.-(cleav. link.)-(anti-HSA)-6xHis ACP02 (anti-HSA)-(cleav.
link.)-mouse IFN.gamma.-(cleav. link.)-mouse IFN.gamma.-(cleav.
link.)-(anti- HSA)-6xHis ACP03 (anti-HSA)-(cleav. link.)-mouse
IFN.gamma.-mouse IFN.gamma.-(cleav. link.)-(anti-HSA)-6xHis ACP50
(anti-EpCAM)-(anti-HSA)-(cleav. link.)-mouse IFN.gamma.-mouse
IFN.gamma.-(cleav. link.)-(anti- HSA)-6xHis ACP51
(anti-EpCAM)-Linker-(anti-HSA)-(cleav. link.)-mIFN.gamma.-(cleav.
link.)-(anti-HSA)- 6xHis ACP52 (anti-HSA)-(cleav.
link.)-mIFN.gamma.-(cleav. link.)-(anti-HSA)-Linker-(anti-EpCAM)-
6xHis ACP53 mAlbumin-(cleav. link.)-mIFN.gamma.-(cleav.
link.)-mAlbumin-6xHis ACP54 mAlbumin-(cleav.
link.)-mIFN.gamma.-Linker-mIFN.gamma.-(cleav. link.)-mAlbumin-6xHis
ACP30 (anti-HSA)-(cleav. link.)-mouse IFN.gamma.-(cleav.
link.)-(anti-HSA)-(cleav. link.)-mouse IFN.gamma.-(cleav.
link.)-(anti-HSA)-6xHis ACP55 (anti-HSA)-(cleav. link.)-mouse
IFN.gamma.-(cleav. link.)-(anti-HSA)-(cleav. link.)-mouse
IFN.gamma.-(cleav. link.)-(anti-HSA)-6xHis-C-tag ACP56
(anti-FOLR1)-Linker-(anti-HSA)-(cleav. link.)-mIFN.gamma.-(cleav.
link.)-(anti-HSA)-6xHis ACP57 (anti-HSA)-(cleav.
link.)-mIFN.gamma.-(cleav.
link.)-(anti-HSA)-Linker-(anti-FOLR1)-6xHis ACP58
(anti-HSA)-(cleav. link.)-mIFN.gamma.-(cleav.
link.)-mIFN.gamma.-(cleav. link.)-(anti-HSA)-
Linker-(anti-EpCAM)-6xHis ACP59
(anti-FOLR1)-Linker-(anti-HSA)-(cleav. link.)-mIFN.gamma.-(cleav.
link.)-mIFN.gamma.-(cleav. link.)-(anti-HSA)-6xHis ACP60
(anti-HSA)-(cleav. link.)-mIFN.gamma.-(cleav.
link.)-mIFN.gamma.-(cleav. link.)-(anti-HSA)-
Linker-(anti-FOLR1)-6xHis ACP61 (anti-HSA)-(cleav.
link.)-mIFN.gamma.-(cleav. link.)-mIFN.gamma.-(cleav.
link.)-(anti-HSA)- Linker-FN(CGS-2)-6xHis ACP63 anti-FN CGS-2 scFv
(Vh/Vl)-6xHis ACP69 (anti-HSA)-(cleav. link.)-mouse
IFN.gamma.-(cleav. link.)-(anti-HSA)-(cleav. link.)-mouse
IFN.gamma. ACP70 mouse IFN.gamma.-(cleav. link.)-(anti-HSA)-(cleav.
link.)-mouse IFN.gamma.-(cleav. link.)-(anti- HSA) ACP71 mouse
IFN.gamma.-(cleav. link.)-mAlbumin-(cleav. link.)-mouse
IFN.gamma.-(cleav. link.)- mAlbumin ACP72 mAlbumin-(cleav.
link.)-mouse IFN.gamma.-(cleav. link.)-mAlbumin-(cleav.
link.)-mouse IFN.gamma. ACP73 mAlbumin-(cleav. link.)-mouse
IFN.gamma.-(cleav. link.)-mAlbumin-(cleav. link.)-mouse
IFN.gamma.-(cleav. link.)-mAlbumin ACP74 mAlbumin-(cleav.
link.)-mouse IFN.gamma.-(cleav. link.)-5mer linker-mAlbumin-5mer
linker-(cleav. link.)-mouse IFN.gamma.-(cleav. link.)-mAlbumin
ACP75 mAlbumin-(cleav. link.)-mouse IFN.gamma.-(cleav. link.)-10mer
linker-mAlbumin-10mer linker-(cleav. link.)-mouse
IFN.gamma.-(cleav. link.)-mAlbumin ACP78
(anti-HSA)-Linker-mouse_IFN.gamma.-Linker-(anti-HSA)-Linker-mouse_IF-
N.gamma.-Linker-(anti- HSA)_(non-cleavable_control) ACP134
Anti-HSA-X-mouse_IFN.gamma.-X-anti-HSA-X-mouse_IFN.gamma.-X-anti-HS-
A-L-anti-FOLR1 ACP135
Anti-FOLR1-L-HSA-X-mouse_IFN.gamma.-X-HSA-X-mouse_IFN.gamma.-X-HSA
ACP04 human p40-murine p35-6xHis ACP05 human p40-human p35-6xHis
ACP34 mouse p35-(Cleavable Linker)-mouse p40-6xHis ACP35 mouse
p35-GS-(Cleavable Linker)-GS-mouse p40-6xHis ACP36
(anti-HSA)-(Cleav. Linker)-mouse p40-mouse p35-(Cleav.
Linker)-(anti-HSA)-6xHis ACP37 (anti-EpCAM)-(anti-HSA)-(Cleav.
Linker)-mouse p40-mouse p35-(Cleav. Linker)- (anti-HSA)-6xHis ACP79
(anti-EpCAM)-Linker-(anti-HSA)-(Cleavable Linker)-mIL12-(Cleavable
Linker)- (Anti-HSA)-6xHis ACP80 (anti-HSA)-(Cleavable
Linker)-mIL12-(Cleavable Linker)-(anti-HSA)-Linker-(anti-
EpCAM)-6xHis ACP06 Blocker12-Linker-(Cleavable Linker)-human
p40-Linker-mouse p35-(Cleavable Linker)-(anti-HSA)-6xHis ACP07
Blocker12-Linker-(Cleavable Linker)-human p40-Linker-mouse
p35-(Cleavable Linker)-(anti-HSA)-Linker-(anti-FOLR1)-6xHis ACP08
(anti-FOLR1)-Linker-Blocker12-Linker-(Cleavable Linker)-human
p40-Linker-mouse p35-(Cleavable Linker)-(anti-HSA)-6xHis ACP09
(anti-HSA)-Linker-Blocker12-Linker-(Cleavable Linker)-human
p40-Linker-mouse p35-6xHis ACP10 (anti-HSA)-(Cleavable
Linker)-human p40-L-mouse p35-(Cleavable Linker)-Linker-
Blocker12-6xHis ACP11 hp40-Linker-mp35-(Cleavable
Linker)-Linker-Blocker12-Linker-(anti-HSA)-6xHis ACP91
human_p40-Linker-mouse_p35-Linker-Linker-Blocker-Linker-(anti-HSA)
(non- cleavable_control) ACP136 human p40-L-mouse p35-XL-Blocker
ACP138 human_p40-L-mouse_p35-XL-Blocker-L-HSA-L-FOLR1 ACP139
FOLR1-L-human_p40-L-mouse_p35-XL-Blocker-L-HSA ACP140
FOLR1-X-human_p40-L-mouse_p35-XL-Blocker-L-HSA ACP12
(anti-EpCAM)-IL2-(cleav. link.)-(anti-HSA)-blocker-6xHis ACP13
(anti-EpCAM)-Blocker2-(anti-HSA)-(cleav. link.)-IL2-6xHis ACP14
Blocker2-Linker-(cleav. link.)-IL2- (cleav. link.)-(anti-HSA)-6xHis
ACP15 Blocker2-Linker-(anti-HSA)-Linker-(cleav. link.)- IL2 -6xHis
ACP16 IL2-(cleav. link.)-(anti-HSA)-Linker-(cleav.
link.)-Blocker2-6xHis ACP17 (anti-EpCAM)-Linker-IL2-(cleav.
link.)-(anti-HSA)-Linker-(cleav. link.)-Blocker2- 6xHis ACP18
(anti-EpCAM)-Linker-IL2-(cleav. link.)-(anti-HSA)-Linker-vh(cleav.
link.)vl-6xHis ACP19 IL2-(cleav.
link.)-Linker-Blocker2-Linker-(anti-HSA)-Linker-(anti-EpCAM) -6xHis
ACP20 IL2-(cleav. link.)-Blocker2-6xHis ACP21 IL2-(cleav.
link.)-Linker-Blocker2-6xHis ACP22 IL2-(cleav.
link.)-Linker-blocker-(cleav.
link.)-(anti-HSA)-Linker-(anti-EpCAM)- 6xHis ACP23
(anti-FOLR1)-(cleav. link.)-Blocker2-Linker-(cleav.
link.)-(anti-HSA)-(cleav. link.)- IL2-6xHis ACP24
(Blocker2)-(cleav. link.)-(IL2)-6xHis ACP25 Blocker2-Linker-(cleav.
link.)-IL2-6xHis ACP26 (anti-EpCAM)-Linker-IL2-(cleav.
link.)-(anti-HSA)-Linker-blocker(NARA1 Vh/Vl) ACP27
(anti-EpCAM)-Linker-IL2-(cleav.
link.)-(anti-HSA)-Linker-blocker(NARA1 Vl/Vh) ACP28 IL2-(cleav.
link.)-Linker-Blocker2-(NARA1
Vh/Vl)-Linker-(anti-HSA)-Linker-(anti- EpCAM) ACP29 IL2-(cleav.
link.)-Linker-Blocker2-(NARA1
Vl/Vh)-Linker-(anti-HSA)-Linker-(anti- EpCAM) ACP38 IL2-(cleav.
link.)-blocker-(anti-HSA)-(anti-EpCAM)-6xHis ACP39
(anti-EpCAM)-(cleav. link.)-(anti-HSA)-(cleav.
link.)-Blocker2-(cleav. link.)-IL-2- 6xHis ACP40
CD25ecd-Linker-(cleav. link.)-IL2-6xHis ACP41 IL2-(cleav.
link.)-Linker-CD25ecd-6xHis ACP42
(anti-HSA)-Linker-CD25ecd-Linker-(cleav. link.)-IL2-6xHis ACP43
IL2-(cleav. link.)-Linker-CD25ecd-Linker-(anti-HSA)-6xHis ACP44
IL2-(cleav. link.)-Linker-CD25ecd-(cleav. link.)-(anti-HSA)-6xHis
ACP45 (anti-HSA)-(cleav. link.)-Blocker2-Linker-(cleav.
link.)-IL2-6xHis ACP46 IL2-(cleav. link.)-linkerL-vh(cleav.
link.)vl-Linker-(anti-HSA)-L-(anti-EpCAM)- 6xHis ACP47
(anti-EpCAM)-Linker-IL2-(Cleavable
Linker)-(anti-HSA)-Linker-Blocker2-6xHis ACP48 IL2-(cleav.
link.)-Blocker2-Linker-(anti-HSA)-6xHis ACP49 IL2-(cleav.
link.)-Linker-Blocker2-Linker-(anti-HSA)-6xHis ACP92
(anti-HSA)-(16mer Cleav. Link.)-IL2-(16mer Cleav.
Link.)-(anti-HSA)-6XHis ACP93
(anti-EpCAM)-(anti-HSA)-(anti-EpCAM)-Blocker2-(cleav.
link.)-IL2-6xHis ACP94 (anti-EpCAM)-(anti-HSA)-Blocker2-(cleav.
link.)-IL2-6xHis ACP95 (anti-EpCAM)-(anti-HSA)-(cleav.
link.)-IL2-6xHis ACP96 (anti-EpCAM)-(16mer cleav. link.)-IL2-(16mer
cleav. link.)-(anti-HSA) ACP38 IL2-(cleav.
link.)-blocker-(anti-HSA)-(anti-EpCAM)-6xHis ACP97
(anti-EpCAM)-(anti-HSA)-(cleav. link.)-IL2-(cleav.
link.)-(anti-HSA)-6xHis ACP99 (anti-EpCAM)-Linker-IL2-(cleav.
link.)-(anti-HSA)-6xHis ACP100 (anti-EpCAM)-Linker-IL2-6xHis ACP101
IL2-(cleav. link.)-(anti-HSA)-6xHis ACP102 (anti-EpCAM)-(cleav.
link.)-IL2-(cleav. link.)-(anti-HSA)-Linker-blocker-6xHis ACP103
IL2-(cleav.
link.)-Linker-Blocker2-Linker-(anti-HSA)-Linker-(antiI-FOLR1)-6xHis
ACP104 (anti-FOLR1)-IL2-(cleav.
link.)-(anti-HSA)-Linker-Blocker2-6xHis ACP105
Blocker2-Linker-(cleav. link.)-IL2-(cleav.
link.)-(anti-HSA)-Linker-(anti-FOLR1)- 6xHis ACP106
(anti-FOLR1)-Linker-(anti-HSA)-(cleav.
link.)-blocker-Linker-(cleav. link.)-IL2 - 6xHis ACP107
Blocker2-Linker-(anti-HSA)-(cleav.
link.)-IL2-Linker-(anti-FOLR1)-6xHis ACP108
(anti-EpCAM)-IL2-(Dually cleav.
link.)-(anti-HSA)-Linker-blocker-6xHis ACP117 anti-FN CGS-2 scFv
(Vh/Vl)-6xHis ACP118 NARA1 Vh/Vl non-cleavable ACP119 NARA1 Vh/Vl
cleavable ACP120 NARA1 Vl/Vh non-cleavable ACP121 NARA1 Vl/Vh
cleavable ACP124
IL2-Linker-(anti-HSA)-Linker-Linker-blocker_(non-cleavable_control)
ACP132 IL2-L-HSA ACP141 IL2-L-hAlb ACP142 IL2-X-hAlb ACP144
IL2-X-HSA-LX-blocker-L-FOLR1 ACP145 FOLR1-L-IL2-X-HSA-LX-blocker
ACP146 FOLR1-X-IL2-X-HSA-LX-blocker ACP133 IL-2-6x His ACP147
IL2-X-HSA-LX-blocker-L-TAA ACP148 TAA-L-IL2-X-HSA-LX-blocker ACP149
TAA-X-IL2-X-HSA-LX-blocker ACP31 (anti-HSA)-(cleav.
link.)-mIFNa1-(cleav. link.)-(anti-HSA) ACP32 (anti-HSA)-(cleav.
link.)-mIFNa1(N + C trunc)-(cleav. link.)-(anti-HSA) ACP33
(anti-HSA)-(cleav. link.)-mIFNa1(C trunc)-(cleav. link.)-(anti-HSA)
ACP131 mIFNa1 ACP125 HSA-X-mIFNa1 ACP126 mIFNa1-X-HSA ACP127
mAlb-X-mIFNa1-X-mAlb ACP128 mAlb-X-mIFNa1 ACP129 mIFNa1-X-mAlb
ACP150 FOLR1-L-HSA-X-mIFNa1-X-HSA ACP151
FOLR1-L-HSA-X-mIFNa1-X-HSA-L-FOLR1 ACP152
HSA-L-mIFNa1-L-HSA_(non-cleavable_control) ACP203
HSA-X-mIFNa1-X-HSA_(X = MMP14-1) ACP204 HSA-X-mIFNa1-X-HSA_(X =
CTSL1-1) ACP205 HSA-X-mIFNa1-X-HSA_(X = ADAM17-2) ACP206
HSA-X-Human_IFNA2b-X-HSA_(X = MMP14-1) ACP207
HSA-X-Human_IFNA2b-X-HSA _(X = CTSL1-1) ACP208
HSA-X-Human_IFNA2b-X-HSA _(X = ADAM17-2) ACP336
IL2-X-anti-HSA-LX-blocker_(Blocker = VHVL.F2.high.A02_Vh-X-
Vl_A46S; X = MMP14-1) ACP337 IL2-X-anti-HSA-LX- blocker_(Blocker =
VHVL.F2.high.A02_Vh/Vl_A46S; X = MMP14-1) ACP338
IL2-X-anti-HSA-LX-blocker_(Blocker = VHVL.F2.high.F03_Vh-X-Vl; X =
MMP14-1) ACP339 IL2-X-anti-HSA-LX-blocker_(Blocker =
VHVL.F2.high.F03_Vh/Vl;X = MMP14-1) ACP340
IL2-X-anti-HSA-LX-blocker_(Blocker = Hu2TOW91_B; X = MMP14-1)
ACP341 IL2-X-anti-HSA-LX-blocker_(Blocker = Hu3TOW85_A; X =
MMP14-1) ACP342 CD25ecd_C213S-LX-IL2-X-anti-HSA-LX-
blocker_(Blocker = VHVL.F2.high.A02_Vh-X-Vl_A46S; X = MMP14-1)
ACP343 CD25ecd_C213 S-LX-IL2-X-anti-HS A-LX- blocker_(Blocker =
VHVL.F2.high.A02_Vh/Vl_A46S; X = MMP14-1) ACP344
CD25ecd_C213S-LX-IL2-X-anti-HSA-LX- blocker_(Blocker =
VHVL.F2.high.F03_Vh-X-Vl; X = MMP14-1) ACP345
CD25ecd_C213S-LX-IL2-X-anti-HSA-LX- blocker_(Blocker =
VHVL.F2.high.F03_Vh/Vl; X = MMP14-1) ACP346
CD25ecd_C213S-LX-IL2-X-anti-HSA-LX- blocker_(Blocker = Hu2TOW91_B;
X = MMP14-1) ACP347 CD25ecd_C213S-LX-IL2-X-anti-HSA-LX-
blocker_(Blocker = Hu3TOW85_A; X = MMP14-1) ACP348
IgG4_Fc(S228P)-X-IL2-LX-Blocker_(Blocker =
VHVL.F2.high.A02_Vh-X-
Vl_A46S; X = MMP14-1) ACP349 IgG4_Fc(S228P)-X-IL2-LX-
Blocker_(Blocker = VHVL.F2.high.A02_Vh\Vl_A46S; X = MMP14-1) ACP350
IgG4_Fc(S228P)-X-IL2-LX-Blocker_(Blocker = VHVL.F2.high.F03_Vh-X-
V1; X = MMP14-1) ACP351 IgG4_Fc(S228P)-X-IL2-LX- Blocker_(Blocker =
VHVL.F2.high.F03_Vh\Vl; X = MMP14-1) ACP352
IgG4_Fc(S228P)-X-IL2-LX-Blocker_(Blocker = Hu2TOW91_B; X = MMP14-1)
ACP353 IgG4_Fc(S228P)-X-IL2-LX-Blocker_(Blocker = Hu3TOW85_A; X =
MMP14-1) ACP354 IgG4_Fc(S228P)-X-CD25ecd_C213S-LX-IL2-LX-
Blocker_(Blocker = VHVL.F2.high.A02_Vh-X-Vl_A46S; X = MMP14-1)
ACP355 IgG4_Fc(S228P)-X-CD25ecd_C213S-LX-IL2-LX- Blocker_(Blocker =
VHVL.F2.high.A02_Vh\Vl_A46S; X = MMP14-1) ACP356
IgG4_Fc(S228P)-X-CD25ecd_C213S-LX-IL2-LX- Blocker_(Blocker =
VHVL.F2.high.F03_Vh-X-Vl; X = MMP14-1) ACP357
IgG4_Fc(S228P)-X-CD25ecd_C213S-LX-IL2-LX- Blocker_(Blocker =
VHVL.F2.high.F03_Vh\Vl; X = MMP14-1) ACP358
IgG4_Fc(S228P)-X-CD25ecd_C213S-LX-IL2-LX- Blocker_(Blocker =
Hu2TOW91_B; X = MMP14-1) ACP359
IgG4_Fc(S228P)-X-CD25ecd_C213S-LX-IL2-LX- Blocker_(Blocker =
Hu3TOW85_A; X = MMP14-1) ACP360 MT204_Vh/Vl_3xG4S_A46S ACP361
MT204_Vh-X-Vl_X = MMP14-1 ACP362 MT204_Vh-X-Vl_X = MMP14-1,_A46S
ACP365 VHVL.F2.high.A02_Vh-X-Vl_X = MMP14-1 ACP366
VHVL.F2.high.A02_Vh-X-Vl_X = MMP14-1,_A46S ACP368
VHVL.F2.high.F03_Vh-X-Vl_X = MMP14-1 ACP371
IL2-X-anti-HSA-LX-blocker_(Blocker = MT204_Vh/Vl_VH44-
VL100_disulfide; X = MMP14-1) ACP372
IL2-X-anti-HSA-LX-blocker_(Blocker = MT204_Vh/Vl_VH105-
VL43_disulfide; X = MMP14-1) ACP373
IL2-X-anti-HSA-LX-blocker_(Blocker = VHVL.F2.high.A02_Vh/Vl_VH44-
VL100_disulfide; X = MMP14-1) ACP374
IL2-X-anti-HSA-LX-blocker_(Blocker = VHVL.F2.high.A02_Vh/Vl_VH105-
VL43_disulfide; X = MMP14-1) ACP375
IL2-X-anti-HSA-LX-blocker_(Blocker = VHVL.F2.high.F03_Vh/Vl_VH44-
VL100_disulfide; X = MMP14-1) ACP376
IL2-X-anti-HSA-LX-blocker_(Blocker = VHVL.F2.high.F03_Vh/Vl_VH105-
VL43_disulfideX = MMP14-1) ACP377
IL2-X-anti-HSA-LX-blocker_(Blocker = Hu2TOW91_A; X = MMP14-1)
ACP378 IL2-X-anti-HSA-LX-Heavy_blocker_Fab_(Blocker = MT204_VH-CH1;
X = MMP14-1) ACP379
IgG4_Fc(S228P)-X-IL2-LX-Heavy_blocker_Fab_(Blocker = MT204_VH- CH1;
X = MMP14-1) ACP383 IgG4_Fc(S228P)-X-IL2-LX-blocker_(Blocker =
MT204_Vh/Vl_VH44- VL100_disulfide; X = MMP14-1) ACP384
IgG4_Fc(S228P)-X-IL2-LX-blocker_(Blocker = MT204_Vh/Vl_VH105-
VL43_disulfide; X = MMP14-1) ACP385
IgG4_Fc(S228P)-X-IL2-LX-blocker_(Blocker =
VHVL.F2.high.A02_Vh/Vl_VH44- VL100_disulfide; X = MMP14-1) ACP386
IgG4_Fc(S228P)-X-IL2-LX-blocker_(Blocker =
VHVL.F2.high.A02_Vh/Vl_VH105- VL43_disulfide; X = MMP14-1) ACP387
IgG4_Fc(S228P)-X-IL2-LX-blocker_(Blocker =
VHVL.F2.high.F03_Vh/Vl_VH44- VL100_disulfide; X = MMP14-1) ACP388
IgG4_Fc(S228P)-X-IL2-LX-blocker_(Blocker =
VHVL.F2.high.F03_Vh/Vl_VH105- VL43_disulfide; X = MMP14-1) ACP389
IgG4_Fc(S228P)-X-IL2-LX-blocker_(Blocker = Hu2TOW91_A; X = MMP14-1)
ACP390 IL2-X-anti-HSA-LX-blocker_(Blocker =
VHVL.F2.high.A02_Vh/Vl_A46S_VH44- VL100_disulfide; X = MMP14-1)
ACP391 IgG4_Fc(S228P)-X-IL2-LX- blocker_(Blocker =
VHVL.F2.high.A02_Vh/Vl_A46S_VH44- VL100_disulfide; X = MMP14-1)
ACP392 IL2-XL-CD25ecd_C213S-X-HSA-LX- blocker_(Blocker =
VHVL.F2.high.A02_Vh_G44C_Vl_A46S_G100C; X = MMP14-1) ACP393
IL2-XL-CD25ecd_C213S-X-HSA-LX- blocker_(Blocker =
VHVL.F2.high.A02_Vh_Q105CV1A43C; X = MMP14-1) ACP394
IL2-XL-CD25ecd_C213S-X-HSA-LX- blocker_(Blocker =
VHVL.F2.high.F03_Vh_G44C_Vl_G100C; X = MMP14-1) ACP395
IL2-XL-CD25ecd_C213S-X-HSA-LX- blocker_(Blocker =
VHVL.F2.high.F03_Vh_Q105C_Vl_A43C; X = MMP14-1) ACP396
IL2-XL-CD25ecd_C213S-X-HSA-LX-blocker_(Blocker = Hu2TOW91_A; X =
MMP14- 1) ACP397 IL2-XL-CD25ecd_C213S-X-HSA-LX-blocker_(Blocker =
Hu2TOW91_B; X = MMP14- 1) ACP398
IL2-XL-CD25ecd_C213S-X-HSA-LX-Heavy_blocker_Fab_(Blocker =
MT204_VH- CH1; X = MMP14-1) ACP399
Blocker-XL-HSA-X-IL2(Nterm-41)-X- HSA_(Blocker =
VHVL.F2.high.A02_Vh_G44C_Vl_A46S_G100C; X = MMP14-1) ACP400
Blocker-XL-HSA-X-IL2(Nterm-41)-X- HSA_(Blocker =
VHVL.F2.high.A02_Vh_Q105CV1A43C; X = MMP14-1) ACP401
Blocker-XL-HSA-X-IL2(Nterm-41)-X- HSA_(Blocker =
VHVL.F2.high.F03_Vh_G44C_Vl_G100C; X = MMP14-1) ACP402
Blocker-XL-HSA-X-IL2(Nterm-41)-X- HSA_(Blocker =
VHVL.F2.high.F03_Vh_Q105C_Vl_A43C; X = MMP14-1) ACP403
Blocker-XL-HSA-X-IL2(Nterm-41)-X-HSA_(Blocker = Hu2TOW91_A; X =
MMP14-1) ACP404 Blocker-XL-HSA-X-IL2(Nterm-41)-X-HSA_(Blocker =
Hu2TOW91_B; X = MMP14-1) ACP405
Heavy_Blocker_Fab-XL-HSA-X-IL2(Nterm-41)-X-HSA_(Blocker = MT204_VH-
CH1; X = MMP14-1) ACP406
mIgG1_Fc(S228P)-X-IL2-LX-Heavy_blocker_Fab_(Blocker = MT204_VH-
CH1; X = MMP14-1) ACP407 mIgG1_Fc(S228P)-X-IL2-LX-blocker_(Blocker
= VHVL.F2.high.A02_Vh/Vl_VH44- VL100_disulfide; X = MMP14-1) ACP408
mIgG1_Fc(S228P)-X-IL2-LX- blocker_(Blocker =
VHVL.F2.high.A02_Vh/Vl_A46S_VH44- VL100_disulfide; X = MMP14-1)
ACP409 mIgG1_Fc(S228P)-X-IL2-LX-blocker_(Blocker =
VHVL.F2.high.A02_Vh/Vl_VH105- VL43_disulfidel; X = MMP14-1) ACP410
mIgG1_Fc(S228P)-X-IL2-LX-blocker_(Blocker =
VHVL.F2.high.F03_Vh/Vl_VH44- VL100_disulfidel; X = MMP14-1) ACP411
mIgG1_Fc(S228P)-X-IL2-LX-blocker_(Blocker =
VHVL.F2.high.F03_Vh/Vl_VH105- VL43_disulfidel; X = MMP14-1) ACP412
mIgG1_Fc(S228P)-X-IL2-LX-blocker_(Blocker = Hu2TOW91_A; X =
MMP14-1) ACP415 IL2-XL-blocker-L-CD25_213S-X- HSA_Blocker =
VHVL.F2.high.A02_Vh_G44C_Vl_A46S_G100C; X = MMP14-1) ACP416
IL2-XL-blocker-L-CD25_213S-X- HSA_(Blocker =
VHVL.F2.high.A02_Vh_Q105C_Vl_A43C; X = MMP14-1) ACP417
IL2-XL-blocker-L-CD25_213 S-X- HSA_(Blocker =
VHVL.F2.high.F03_Vh_G44C_Vl_G100C; X = MMP14-1) ACP418
IL2-XL-blocker-L-CD25_213S-X- HSA_(Blocker =
VHVL.F2.high.F03_Vh_Q105C_Vl_A43C; X = MMP14-1) ACP419
IL2-XL-blocker-L-CD25_213 S-X-HSA_(Blocker = Hu2TOW91_A; X =
MMP14-1) ACP420 IL2-XL-blocker-L-CD25_213S-X-HSA_(Blocker =
Hu2TOW91_B; X = MMP14-1) ACP421 HSA-X-blocker-L-CD25_213S-LX-
IL2_(Blocker = VHVL.F2.high.A02_Vh_G44C_Vl_A46S_G100C; X = MMP14-1)
ACP422 HSA-X-blocker-L-CD25_213S-LX- IL2_(Blocker =
VHVL.F2.high.A02_Vh_Q105C_Vl_A43C; X = MMP14-1) ACP423
HSA-X-blocker-L-CD25_213S-LX- IL2_(Blocker =
VHVL.F2.high.F03_Vh_G44C_Vl_G100C; X = MMP14-1) ACP424
HSA-X-blocker-L-CD25_213S-LX- IL2_(Blocker =
VHVL.F2.high.F03_Vh_Q105CV1A43C; X = MMP14-1) ACP425
HSA-X-blocker-L-CD25_213S-LX-IL2_(Blocker = Hu2TOW91_A; X =
MMP14-1) ACP426 HSA-X-blocker-L-CD25_213S-LX-IL2_(Blocker =
Hu2TOW91B; X = MMP14-1) ACP427 IL2-X-anti-HSA-LX-Blocker1-L-
Blocker2_(Blocker1 = VHVL.F2.high.A02_Vh_G44C_Vl_A46S_G100C,
Blocker2 = Hu2TOW91_A; X = MMP14-1) ACP428
IL2-X-anti-HSA-LX-Blocker1-L- Blocker2_(Blocker1 =
VHVL.F2.high.A02_Vh_Q105C_Vl_A43C, Blocker2 = Hu2TOW91_A; X =
MMP14-1) ACP429 IL2-X-anti-HSA-LX-Blocker1-L- Blocker2_(Blocker1 =
VHVL.F2.high.F03_Vh_G44C_Vl_G100C, Blocker2 = Hu2TOW91_A; X =
MMP14-1) ACP430 IL2-X-anti-HSA-LX-Blocker1-L- Blocker2_(Blocker1 =
VHVL.F2.high.F03_Vh_Q105C_Vl_A43C, Blocker2 = Hu2TOW91_A; X =
MMP14-1) ACP431 IL2-X-anti-HSA-LX-Blocker1-L- Blocker2_(Blocker1 =
VHVL.F2.high.A02_Vh_G44C_Vl_A46S_G100C, Blocker2 = Hu2TOW91_B; X =
MMP14-1) ACP432 IL2-X-anti-HSA-LX-Blocker1-L- Blocker2_(Blocker1 =
VHVL.F2.high.A02_Vh_Q105C_Vl_A43C, Blocker2 = Hu2TOW91_B; X =
MMP14-1) ACP433 IL2-X-anti-HSA-LX-Blocker1-L- Blocker2_(Blocker1 =
VHVL.F2.high.F03_Vh_G44C_Vl_G100C, Blocker2 = Hu2TOW91_B; X =
MMP14-1) ACP434 IL2-X-anti-HSA-LX-Blocker1-L- Blocker2_(Blocker1 =
VHVL.F2.high.F03_Vh_Q105C_Vl_A43C, Blocker2 = Hu2TOW91_B; X =
MMP14-1) ACP439 IL2-X-anti-HSA-LX-blocker_(Blocker =
VHVL.F2.high.C07_Vh/Vl; X = MMP14-1) ACP440 IL2-X-anti-HSA-LX-
blocker_(Blocker = VHVL.F2.high.C07_Vh/Vl_A46S; X = MMP14-1) ACP441
IL2-X-anti-HSA-LX- blocker_(Blocker = VHVL.F2.high.C07_Vh/Vl_A46L;
X = MMP14-1) ACP442 IL2-X-anti-HSA-LX-blocker_(Blocker =
VHVL.F2.high.C07_Vh/Vl_A46S_VH44- VL100_disulfide; X = MMP14-1)
ACP443 IL2-X-anti-HSA-LX-blocker_(Blocker =
VHVL.F2.high.C07_Vh/Vl_A46L_VH44- VL100_disulfide; X = MMP14-1)
ACP444 IL2-X-anti-HSA-LX-blocker_(Blocker =
VHVL.F2.high.C07_Vh/Vl_VH105- VL43_disulfide; X = MMP14-1) ACP445
IL2-X-anti-HSA-LX-blocker_(Blocker = VHVL.F2.high.A02_Vh-X-
V1_A46L; X = MMP14-1) ACP446 IL2-X-anti-HSA-LX- blocker_(Blocker =
VHVL.F2.high.A02_Vh/Vl_A46L; X = MMP14-1) ACP447
IL2-X-anti-HSA-LX-blocker_(Blocker =
VHVL.F2.high.A02_Vh/Vl_A46L_VH44- VL100_disulfide; X = MMP14-1)
ACP451 IL2-X-anti-HSA-LX-blocker_(Blocker =
VHVL.F2.high.A02_Vh/Vl_A46S; X = CTSL1- 1) ACP452
IL2-X-anti-HSA-LX-blocker_(Blocker = VHVL.F2.high.F03_Vh/Vl; X =
CTSL1-1) ACP453 IL2-X-anti-HSA-LX-blocker_(Blocker =
VHVL.F2.high.A02_Vh/Vl_A46S_VH44- VL100_disulfide; X = CTSL1-1)
ACP454 IL2-X-anti-HSA-LX-blocker_(Blocker =
VHVL.F2.high.A02_Vh/Vl_VH105- VL43_disulfidel; X = CTSL1-1) ACP455
IL2-X-anti-HSA-LX-blocker_(Blocker = VHVL.F2.high.F03_Vh/Vl_VH44-
VL100_disulfide; X = CTSL1-1) ACP456
IL2-X-anti-HSA-LX-blocker_(Blocker = VHVL.F2.high.F03_Vh/Vl_VH105-
VL43_disulfideX = CTSL1-1) ACP457
IL2-X-anti-HSA-LX-Heavy_blocker_Fab_(Blocker = MT204_VH-CH1; X =
CTSL1-1) ACP458 IgG4_Fc(S228P)-X-IL2-LX-Heavy_blocker_Fab_(Blocker
= MT204_VH- CH1; X = CTSL1-1) ACP459 IgG4_Fc(S228P)-X-IL2-LX-
Blocker_(Blocker = VHVL.F2.high.A02_Vh\Vl_A46S; X = CTSL1-1) ACP460
IgG4_Fc(S228P)-X-IL2-LX- Blocker_(Blocker = VHVL.F2.high.F03_Vh\Vl;
X = CTSL1-1) ACP461 IgG4_Fc(S228P)-X-IL2-LX- blocker_(Blocker =
VHVL.F2.high.A02_Vh/Vl_A46S_VH44- VL100_disulfide; X = CTSL1-1)
ACP462 IgG4_Fc(S228P)-X-IL2-LX-blocker_(Blocker =
VHVL.F2.high.A02_Vh/Vl_VH105- VL43_disulfidel; X = CTSL1-1) ACP463
IgG4_Fc(S228P)-X-IL2-LX-blocker_(Blocker =
VHVL.F2.high.F03_Vh/Vl_VH44- VL100_disulfidel; X = CTSL1-1) ACP464
IgG4_Fc(S228P)-X-IL2-LX-blocker_(Blocker=VHVL.F2.high.F03_Vh/Vl_VH1-
05- VL43_disulfidel; X = CTSL1-1) ACP465 mIgG1_Fc-X-IL2-LX-
Blocker_(Blocker = VHVL.F2.high.A02_Vh\Vl_A46S; X = CTSL1-1) ACP466
mIgG1_Fc-X-IL2-LX-Blocker_(Blocker = VHVL.F2.high.F03_Vh\Vl; X =
CTSL1-1) ACP467 mIgGl_Fc-X-IL2-LX-blocker_(Blocker =
VHVL.F2.high.A02_Vh/Vl_A46S_VH44- VL100_disulfide; X = CTSL1-1)
ACP468 mIgG1_Fc-X-IL2-LX-blocker_(Blocker =
VHVL.F2.high.A02_Vh/Vl_VH105- VL43_disulfidel; X = CTSL1-1) ACP469
mIgG1_Fc-X-IL2-LX-blocker_(Blocker = VHVL.F2.high.F03_Vh/Vl_VH44-
VL100_disulfidel; X = CTSL1-1) ACP470
mIgG1_Fc-X-IL2-LX-blocker_(Blocker = VHVL.F2.high.F03_Vh/Vl_VH105-
VL43_disulfidel; X = CTSL1-1) ACP471
mIgG1_Fc-X-IL2-LX-Heavy_blocker_Fab_(Blocker = MT204_VH-CH1; X =
CTSL1-1)
TABLE-US-00020 APPENDIX B Sequences SEQ ID NO. Name Sequence 1
Human IL-2 MYRMQLLSCI ALSLALVTNS APTSSSTKKT QLQLEHLLLD LQMILNGINN
YKNPKLTRML TFKFYMPKKA TELKHLQCLE EELKPLEEVL NLAQSKNFHL RPRDLISNIN
VIVLELKGSE TTFMCEYADE TATIVEFLNR WITFCQSIISTLT 2 Human MKWVTFISLL
FLFSSAYSRG VFRRDAHKSE VAHRFKDLGE serum ENFKALVLIA FAQYLQQCPF
EDHVKLVNEV TEFAKTCVAD albumin ESAENCDKSL HTLFGDKLCT VATLRETYGE
MADCCAKQEP ERNECFLQHK DDNPNLPRLV RPEVDVMCTA FHDNEETFLK KYLYEIARRH
PYFYAPELLF FAKRYKAAFT ECCQAADKAA CLLPKLDELR DEGKASSAKQ GLKCASLQKF
GERAFKAWAV ARLSQRFPKA EFAEVSKLVT DLTKVHTECC HGDLLECADD RADLAKYICE
NQDSISSILK ECCEKPLLEK SHCIAEVEND EMPADLPSLA ADFVGSKDVC KNYAEAKDVF
LGMFLYEYAR RHPDYSVVLL LRLAKTYETT LEKCCAAADP HECYAKVFDE FKPLVEEPQN
LIKQNCELFE QLGEYKFQNA LLVRYTKKVP QVSTPILVEV SRNLGKVGSK CCKHPEAKRM
PCAEDCLSVF LNQLCVLHEK TPVSDRYTKC
CTESLVNGRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIK KQTALV ELVKHK
PKATKEQLKAVMDDFAAFVEKCCKADDKET CFAEEGKKLVAASQAALGL 45 ACP12 (IL2
QVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQREL conjugate)
VARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYY
CNALYGTDYWGKGTQVTVSSggggsggggsggggsaptssstkktqlqlehllldlqmil
nginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelk-
g settfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGG
LVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGR
DTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLS
VSSQGTLVTVSSggggsggggsggggsEVQLVESGGGLVQPGGSLRLSCAA
SGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISR
DNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTV
SSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVG
TNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHH 46 ACP13 (IL2
QVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQREL conjugate)
VARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYY
CNALYGTDYWGKGTQVTVSSggggsggggsggggsEVQLVESGGGLVQP
GGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPD
TVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDY
WGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVT
ITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGS
GTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggs
ggggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPG
KGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPE
DTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGSaptssstkkt
qlqlehlllqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrpr
dlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH 47 ACP14
(IL2 EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLE conjugate)
WVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAV
YYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQM
TQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYS
ASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTF
GGGTKVEIKggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSa
ptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnla-
q
sknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMK
GLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAP
GKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRP
EDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHH 48 ACP15 (IL2
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLE conjugate)
WVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAV
YYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQM
TQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYS
ASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTF
GGGTKVEIKggggsggggsggggsggggsggggsggggsEVQLVESGGGLVQPG
NSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAE
SVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGT
LVTVSSggggsggggsggggsSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmil
nginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelk-
g settfmceyadetativeflnrwitfcqsiistltHHHHHH 49 ACP16 (IL2
aptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnla
conjugate)
qsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMK
GLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAP
GKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRP
EDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggg
gsSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFS
SYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKN
SLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGG
GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVG
WYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPED
FATYYCQQYYTYPYTFGGGTKVEIKHHHHHH 50 ACP17 (IL2
QVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQREL conjugate)
VARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYY
CNALYGTDYWGKGTQVTVSSggggsggggsggggsaptssstkktqlqlehllldlqmil
nginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelk-
g settfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGG
LVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGR
DTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLS
VSSQGTLVTVSSggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLP
GSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKG
LEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTA
VYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQ
MTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIY
SASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYT FGGGTKVEIKHHHHHH
51 ACP18 (IL2 QVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQREL
conjugate) VARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYY
CNALYGTDYWGKGTQVTVSSggggsggggsggggsaptssstkktqlqlehllldlqmil
nginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelk-
g settmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGG
LVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGR
DTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLS
VSSQGTLVTVSSggggsggggsggggsggggsggggsggggsEVQLVESGGGLVQ
PGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSP
DTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALD
YWGQGTTVTVSSsggpgpagmkglpgsDIQMTQSPSSLSASVGDRVTITCK
ASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTD
FTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHH 52 ACP19 (IL2
aptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnla
conjugate)
qsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMK
GLPGSggggsggggsggggsggggsggggsggggsEVQLVESGGGLVQPGGSLRL
SCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRF
TISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTT
VTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQ
NVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLT
ISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsEVQ
LVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVS
SISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYC
TIGGSLSVSSQGTLVTVSSggggsggggsggggsQVQLQESGGGLVQAGGS
LRLSCAASGRIFSIDIMSWYRQAPGKQRELVARITRGGTISYDDSVKG
RFTISRDNAKNTVYLQMNSLKPEDTGVYYCNALYGTDYWGKGTQV TVSSHHHHHH** 53 ACP20
(IL2
aptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnla
conjugate)
qsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMK
GLPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPG
KGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAED
TAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDI
QMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALI
YSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPY TFGGGTKVEIKHHHHHH
54 ACP21 (IL2
aptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnla
conjugate)
qsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMK
GLPGSggggsggggsggggsggggsggggsggggsEVQLVESGGGLVQPGGSLRL
SCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRF
TISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTT
VTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQ
NVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLT
ISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHH 55 ACP22 (IL2
aptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnla
conjugate)
qsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMK
GLPGSggggsggggsggggsggggsggggsggggsEVQLVESGGGLVQPGGSLRL
SCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRF
TISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTT
VTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQ
NVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLT
ISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKSGGPGPAGMKGLPG
SEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGL
EWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA
VYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsQVQLQESGGGLVQ
AGGSLRLSCAASGRIFSIDIMSWYRQAPGKQRELVARITRGGTISYDD
SVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCNALYGTDYWGK GTQVTVSSHHHHHH 56
ACP23 (IL2 QVQLQESGGGLAQAGGSLSLSCAASGFTVSNSVMAWYRQTPGKQR conjugate)
EFVAIINSVGSTNYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAV
YVCNRNFDRIYWGQGTQVTVSSSGGPGPAGMKGLPGSEVQLVESGG
GLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSS
YTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNW
DALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASV
GDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSR
FSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggg
gsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSEVQLVESGGGL
VQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRD
TLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSV
SSQGTLVTVSSSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnykn
pkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmc-
e yadetativeflnrwitfcqsiistltHHEIHRH 57 ACP24 (IL2
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLE conjugate)
WVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAV
YYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQM
TQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYS
ASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTF
GGGTKVEIKSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkl
trmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceya-
d etativeflnrwitfcqsiistltHHHHHH 58 ACP25
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLE (IL2
WVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAV conjugate)
YYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQM
TQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYS
ASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTF
GGGTKVEIKggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSa
ptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnla-
q sknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH
59 ACP26 (IL2 QVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQREL
conjugate) VARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYY
CNALYGTDYWGKGTQVTVSSggggsggggsggggsaptssstkktqlqlehllldlqmil
nginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelk-
g settfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGG
LVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGR
DTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLS
VSSQGTLVTVSSggggsggggsggggsggggsQVQLQQSGAELVRPGTSVKV
SCKASGYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFKG
KATLTADKSSSTAYMQLSSLTSDDSAVYFCARWRGDGYYAYFDVW
GAGTTVTVSSggggsggggsggggsDIVLTQSPASLAVSLGQRATISCKASQ
SVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGT
DFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIKHHHHHHEPE A 60 ACP27 (IL2
QVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQREL conjugate)
VARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYY
CNALYGTDYWGKGTQVTVSSggggsggggsggggsaptssstkktqlqlehllldlqmil
nginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelk-
g settfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQLVESGGG
LVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGR
DTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLS
VSSQGTLVTVSSggggsggggsggggsggggsDIVLTQSPASLAVSLGQRATIS
CKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSG
SGSGTDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIKggggsg
gggsggggsQVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQ
RPGQGLEWIGVINPGSGGTNYNEKFKGKATLTADKSSSTAYMQLSSL
TSDDSAVYFCARWRGDGYYAYFDVWGAGTTVTVSSHEIREIHHEPE A 61 ACP28 (IL2
aptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnla
conjugate)
qsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMK
GLPGSggggsggggsggggsggggsggggsQVQLQQSGAELVRPGTSVKVSCK
ASGYAFTNYLIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFKGKAT
LTADKSSSTAYMQLSSLTSDDSAVYFCARWRGDGYYAYFDVWGAG
TTVTVSSggggsggggsggggsDIVLTQSPASLAVSLGQRATISCKASQSVD
YDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTL
NIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIKggggsggggsggggsEV
QLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEW
VSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVY
YCTIGGSLSVSSQGTLVTVSSggggsggggsggggsQVQLQESGGGLVQAG
GSLRLSCAASGRIFSIDIMSWYRQAPGKQRELVARITRGGTISYDDSV
KGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCNALYGTDYWGKGT QVTVSSHHEIHRHEPEA 62
ACP29 (IL2
aptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnla
conjugate)
qsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMK
GLPGSggggsggggsggggsggggsggggsDIVLTQSPASLAVSLGQRATISCKA
SQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSG0
TDFTLNIHPVEEEDAATYYCQQSNEDPYTFGGGTKLEIKggggsggggsg
gggsQVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQ
GLEWIGVINPGSGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDD
SAVYFCARWRGDGYYAYFDVWGAGTTVTVSSggggsggggsggggsEVQ
LVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVS
SISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYC
TIGGSLSVSSQGTLVTVSSggggsggggsggggsQVQLQESGGGLVQAGGS
LRLSCAASGRIFSIDIMSWYRQAPGKQRELVARITRGGTISYDDSVKG
RFTISRDNAKNTVYLQMNSLKPEDTGVYYCNALYGTDYWGKGTQV TVSSHHHHHHEPEA 63
IL2Ra 10 20 30 40 50 MDSYLLMWGL LTFIMVPGCQ AELCDDDPPE IPHATFKAMA
YKEGTMLNCE 60 70 80 90 100 CKRGFRRIKS GSLYMLCTGN SSHSSWDNQC
QCTSSATRNT TKQVTPQPEE 110 120 130 140 150 QKERKTTEMQ SPMQPVDQAS
LPGHCREPPP WENEATERIY HFVVGQMVYY 160 170 180 190 200 QCVQGYRALH
RGPAESVCKM THGKTRWTQP QLICTGEMET SQFPGEEKPQ 210 220 230 240 250
ASPEGRPESE TSCLVTTTDF QIQTEMAATM ETSIFTTEYQ VAVAGCVFLL 260 270
ISVLLLSGLT WQRRQRKSRR TI 64 IL2Rb 10 20 30 40 50 MAAPALSWRL
PLLILLLPLA TSWASAAVNG TSQFTCFYNS RANISCVWSQ 60 70 80 90 100
DGALQDTSCQ VHAWPDRRRW NQTCELLPVS QASWACNLIL GAPDSQKLTT 110 120 130
140 150 VDIVTLRVLC REGVRWRVMA IQDFKPFENL RLMAPISLQV VHVETHRCNI 160
170 180 190 200 SWEISQASHY FERHLEFEAR TLSPGHTWEE APLLTLTQKQ
EWICLETLTP 210 220 230 240 250 DTQYEFQVRV KPLQGEFTTW SPWSQPLAFR
TKPAALGKDT IPWLGHLLVG 260 270 280 290 300 LSGAFGFIIL VYLLINCRNT
GPWLKKVLKC NTPDPSKFFS QLSSEHGGDV 310 320 330 340 350 QKWLSSPFPS
SSFSPGGLAP EISPLEVLER DKVTQLLLQQ DKVPEPASLS 360 370 380 390 400
SNHSLTSCFT NQGYFFFHLP DALEIEACQV YFTYDPYSEE DPDEGVAGAP 410 420 430
440 450 TGSSPQPLQP LSGEDDAYCT FPSRDDLLLF SPSLLGGPSP PSTAPGGSGA 460
470 480 490 500 GEERMPPSLQ ERVPRDWDPQ PLGPPTPGVP DLVDFQPPPE
LVLREAGEEV 510 520 530 540 550 PDAGPREGVS FPWSRPPGQG EFRALNARLP
LNTDAYLSLQ ELQGQDPTHL V 65 IL2Rg 10 20 30 40 50 MLKPSLPFTS
LLFLQLPLLG VGLNTTILTP NGNEDTTADF FLTTMPTDSL 60 70 80 90 100
SVSTLPLPEV QCFVFNVEYM NCTWNSSSEP QPTNLTLHYW YKNSDNDKVQ 110 120 130
140 150 KCSHYLFSEE ITSGCQLQKK EIHLYQTFVV QLQDPRFPRR QATQMLKLQN 160
170 180 190 200 LVIPWAPENL TLHKLSESQL ELNWNNRFLN HCLEHLVQYR
TDWDHSWTEQ 210 220 230 240 250 SVDYRHKFSL PSVDGQKRYT FRVRSRFNPL
CGSAQHWSEW SHPIHWGSNT 260 270 280 290 300 SKENPFLFAL EAVVISVGSM
GLIISLLCVY FWLERTMPRI PTLKNLEDLV 310 320 330 340 350 TEYHGNFSAW
SGVSKGLAES LQPDYSERLC LVSEIPPKGG ALGEGPGASP 360 CNQHSPYWAP
PCYTLKPET 66 ACP04
iwelkkdvyvveldwypdapgemvvltcdtpeedgitwtldqssevlgsgktltiqvkefgdagq-
ytch (human
kggevlshsllllhkkedgiwstdilkdqkepknktflrceaknysgrftcwwlttistdltfsvk-
ssrgssd p40/murine
pqgvtcgaatlsaervrgdnkeyeysvecqedsacpaaeeslpievmvdavhklkyenytssffirdiik
p35 IL12
pdppknlqlkplknsrqvevsweypdtwstphsyfsltfcvqvqgkskrekkdrvftdktsatv-
icrkna conjugate)
sisvraqdryyssswsewasvpcsggggsggggsggggsrvipvsgparclsqsrnllkaddmvktar
eklkhysctaedidheditrdqtstlktclplelhknesclatretssttrgsclppqktslmmtlclgsiy-
edlk
myqtefqainaalqnhnhqqiildkgmlvaidelmqslnhngetlrqkppvgeadpyrvkmklcillha
fstrvvtinrvmgylssaHHHHHH 67 ACP05
iwelkkdvyvveldwypdapgemvvltcdtpeedgitwtldqssevlgsgktltiqvkefgdagq-
ytch (human
kggevlshsllllhkkedgiwstdilkdqkepknktflrceaknysgrftcwwlttistdltfsvk-
ssrgssd p40/murine
pqgvtcgaatlsaervrgdnkeyeysvecqedsacpaaeeslpievmvdavhklkyenytssffirdiik
p35 IL12
pdppknlqlkplknsrqvevsweypdtwstphsyfsltfcvqvqgkskrekkdrvftdktsatv-
icrkna conjugate)
sisvraqdryyssswsewasvpcsggggsggggsggggsrnlpvatpdpgmfpclhhsqnllravsn
mlqkarqtlefypctseeidheditkdktstveaclpleltknesclnsretsfitngsclasrktsfmmal-
cls
siyedlkmyqvefktmnakllmdpkrqifldqnmlavidelmqalnfnsetvpqkssleepdfyktkik
lcillhafriravtidrvmsylnasHHHHHH 68 ACP06
QSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTVKWYQQLPGTAPKLL (human
IYYNDQRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDRY p40/murine
THPALLFGTGTKVTVLggggsggggsggggsQVQLVESGGGVVQPGRSLR p35 IL12
LSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSV conjugate)
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCKTHGSHDNWGQGT
MVTVSSggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSiwelk
kdvyvveldwypdapgemvvltcdtpeedgitwtldqssevlgsgktltiqvkefgdagqytchkgge
vlshsllllhkkedgiwstdilkdqkepknktflrceaknysgrftcwwlttistdltfsvkssrgssdpqg-
vt
cgaatlsaervrgdnkeyeysvecqedsacpaaeeslpievmvdavhklkyenytssffirdiikpdppk
nlqlkplknsrqvevsweypdtwstphsyfsltfcvqvqgkskrekkdrvftdktsatvicrknasisvra
qdryyssswsewasvpcsggggsggggsggggsrvipvsgparclsqsrnllkttddmvktareklkhy
sctaedidheditrdqtstlktclplelhknesclatretssttrgsclppqktslmmtlclgsiyedlkmy-
qte
fqainaalqnhnhqqiildkgmlvaidelmqslnhngetlrqkppvgeadpyrvkmklcillhafstrvv
tinrvmgylssaSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCA
ASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTI
SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHH FIRHHEPEA 69 ACP07
QSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTVKWYQQLPGTAPKLL (human
IYYNDQRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDRY p40/murine
THPALLFGTGTKVTVLggggsggggsggggsQVQLVESGGGVVQPGRSLR p35 IL12
LSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSV conjugate)
KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCKTHGSHDNWGQGT
MVTVSSggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSiwelk
kdvyvveldwypdapgemvvltcdtpeedgitwtldqssevlgsgktltiqvkefgdagqytchkgge
vlshsllllhkkedgiwstdilkdqkepknktflrceaknysgrftcwwlttistdltfsvkssrgssdpqg-
vt
cgaatlsaervrgdnkeyeysvecqedsacpaaeeslpievmvdavhklkyenytssffirdiikpdppk
nlqlkplknsrqvevsweypdtwstphsyfsltfcvqvqgkskrekkdrvftdktsatvicrknasisvra
qdryyssswsewasvpcsggggsggggsggggsrvipvsgparclsqsrnllkttddmvktareklkhy
sctaedidheditrdqtstlktclplelhknesclatretssttrgsclppqktslmmtlclgsiyedlkmy-
qte
fqainaalqnhnhqqiildkgmlvaidelmqslnhngetlrqkppvgeadpyrvkmklcillhafstrvv
tinrvmgylssaSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCA
ASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTI
SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggg
gsggggsggggsQVQLQESGGGLAQAGGSLSLSCAASGFTVSNSVMAWY
RQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTVYLQMNN
LKPEDTAVYVCNRNFDRIYWGQGTQVTVSSHHHHHHEPEA 70 ACP08
QVQLQESGGGLAQAGGSLSLSCAASGFTVSNSVMAWYRQTPGKQR (human
EFVAIINSVGSTNYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAV p40/murine
YVCNRNFDRIYWGQGTQVTVSSggggsggggsggggsQSVLTQPPSVSGAP p35 IL12
GQRVTISCSGSRSNIGSNTVKWYQQLPGTAPKLLIYYNDQRPSGVPD conjugate)
RFSGSKSGTSASLAITGLQAEDEADYYCQSYDRYTHPALLFGTGTKV
TVLggggsggggsggggsQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYG
MHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNT
LYLQMNSLRAEDTAVYYCKTHGSHDNWGQGTMVTVSSggggsggggsg
gggsggggsggggsggggsSGGPGPAGMKGLPGSiwelkkdvyvveldwypdapgem
vvltcdtpeedgitwtldqssevlgsgktltiqvkefgdagqytchkggevlshsllllhkkedgiwstdil-
k
dqkepknktflrceaknysgrftcwwlttistdltfsvkssrgssdpqgvtcgaatlsaervrgdnkeyeys
vecqedsacpaaeeslpievmvdavhklkyenytssffirdiikpdppknlqlkplknsrqvevsweyp
dtwstphsyfsltfcvqvqgkskrekkdrvftdktsatvicrknasisvraqdryyssswsewasvpcsgg
ggsggggsggggsrvipvsgparclsqsrnllkttddmvktareklkhysctaedidheditrdqtstlktc-
l
plelhknesclatretssttrgsclppqktslmmtlclgsiyedlkmyqtefqainaalqnhnhqqiildkg
mlvaidelmqslnhngetlrqkppvgeadpyrvkmklcillhafstrvvtinrvmgylssaSGGPGP
AGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWV
RQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMN
SLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHHEPEA 71 ACP09
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLE (human
WVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAV p40/murine
YYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsQSVLTQPPSVSGAPG p35 IL12
QRVTISCSGSRSNIGSNTVKWYQQLPGTAPKLLIYYNDQRPSGVPDR conjugate)
FSGSKSGTSASLAITGLQAEDEADYYCQSYDRYTHPALLFGTGTKVT
VLggggsggggsggggsQVQLVESGGGVVQPGRSLRLSCAASGFTFSSYG
MHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRDNSKNT
LYLQMNSLRAEDTAVYYCKTHGSHDNWGQGTMVTVSSggggsggggsg
gggsggggsggggsggggsSGGPGPAGMKGLPGSiwelkkdvyvveldwypdapgem
vvltcdtpeedgitwtldqssevlgsgktltiqvkefgdagqytchkggevlshsllllhkkedgiwstdil-
k
dqkepknktflrceaknysgrftcwwlttistdltfsvkssrgssdpqgvtcgaatlsaervrgdnkeyeys
vecqedsacpaaeeslpievmvdavhklkyenytssffirdiikpdppknlqlkplknsrqvevsweyp
dtwstphsyfsltfcvqvqgkskrekkdrvftdktsatvicrknasisvraqdryyssswsewasvpcsgg
ggsggggsggggsrvipvsgparclsqsrnllkaddmvktareklkhysctaedidheditrdqtstlktcl
plelhknesclatretssttrgsclppqktslmmtlclgsiyedlkmyqtefqainaalqnhnhqqiildkg
mlvaidelmqslnhngeftrqkppvgeadpyrvkmklcillhafstrvvtinrvmgylssaHHHHH
HEPEA 72 ACP10 EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLE
(human WVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAV p40/murine
YYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGSiwelkkdvyvveld p35 IL12
wypdapgemvvltcdtpeedgitwtldqssevlgsgktltiqvkefgdagqytchkggevlshs-
llllhk conjugate)
kedgiwstdilkdqkepknktflrceaknysgrftcwwlttistdltfsvkssrgssdpqgvtcgaatlsaer
vrgdnkeyeysvecqedsacpaaeeslpievmvdavhklkyenytssffirdiikpdppknlqlkplkn
srqvevsweypdtwstphsyfsltfcvqvqgkskrekkdrvftdktsatvicrknasisvraqdryysssw
sewasvpcsggggsggggsggggsrvipvsgparclsqsrnllkttddmvktareklkhysctaedidhe
ditrdqtstlktclplelhknesclatretssttrgsclppqktslmmtlclgsiyedlkmyqtefqainaa-
lqn
hnhqqiildkgmlvaidelmqslnhngetlrqkppvgeadpyrvkmklcillhafstrvvtinrvmgyls
saSGGPGPAGMKGLPGSggggsggggsggggsggggsggggsggggsQSVLTQPPS
VSGAPGQRVTISCSGSRSNIGSNTVKWYQQLPGTAPKLLIYYNDQRP
SGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDRYTHPALLFG
TGTKVTVLggggsggggsggggsQVQLVESGGGVVQPGRSLRLSCAASGF
TFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYADSVKGRFTISRD
NSKNTLYLQMNSLRAEDTAVYYCKTHGSHDNWGQGTMVTVSSHH HHHHEPEA 73 ACP11
iwelkkdvyvveldwypdapgemvvltcdtpeedgitwtldqssevlgsgktltiqvkefgdagq-
ytch (human
kggevlshsllllhkkedgiwstdilkdqkepknktflrceaknysgrftcwwlttistdltfsvk-
ssrgssd p40/murine
pqgvtcgaatlsaervrgdnkeyeysvecqedsacpaaeeslpievmvdavhklkyenytssffirdiik
p35 IL12
pdppknlqlkplknsrqvevsweypdtwstphsyfsltfcvqvqgkskrekkdrvftdktsatv-
icrkna conjugate)
sisvraqdryyssswsewasvpcsggggsggggsggggsrvipvsgparclsqsrnllkttddmvktar
eklkhysctaedidheditrdqtstlktclplelhknesclatretssttrgsclppqktslmmtlclgsiy-
edlk
myqtefqainaalqnhnhqqiildkgmlvaidelmqslnhngetlrqkppvgeadpyrvkinklcillha
fstrvvtinrvmgylssaSGGPGPAGMKGLPGSggggsggggsggggsggggsggggsgg
ggsQSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTVKWYQQLPGTAP
KLLIYYNDQRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSY
DRYTHPALLFGTGTKVTVLggggsggggsggggsQVQLVESGGGVVQPGR
SLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYA
DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCKTHGSHDNWG
QGTMVTVSSggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASG
FTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRD
NAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHH HHEPEA 74 IL12 p40
10 20 30 40 50 human MCHQQLVISW FSLVFLASPL VAIWELKKDV YVVELDWYPD
APGEMVVLTC (Uniprot 60 70 80 90 100 Accession DTPEEDGITW TLDQSSEVLG
SGKTLTIQVK EFGDAGQYTC HKGGEVLSHS No. P29460) 110 120 130 140 150
LLLLHKKEDG IWSTDILKDQ KEPKNKTFLR CEAKNYSGRF TCWWLTTSIT 160 170 180
190 200 DLTFSVKSSR GSSDPQGVTC GAATLSAERV RGDNKEYEYS VECQEDSACP 210
220 230 240 250 AAEESLPIEV MVDAVHKLKY ENYTSSFFIR DIIKPDPPKN
LQLKPLKNSR 260 270 280 290 300 QVEVSWEYPD TWSTPHSYFS LTFCVQVQGK
SKREKKDRVF TDKTSATVIC 310 320 RKNASISVRA QDRYYSSSWS EWASVPCS 75 IL1
p35 10 20 30 40 50 mouse MCQSRYLLFL ATLALLNHLS LARVIPVSGP
ARCLSQSRNL LKTTDDMVKT (Uniprot 60 70 80 90 100 Accession AREKLKHYSC
TAEDIDHEDI TRDQTSTLKT CLPLELHKNE SCLATRETSS No. P43431) 110 120 130
140 150 TTRGSCLPPQ KTSLMMTLCL GSIYEDLKMY QTEFQAINAA LQNHNHQQII 160
170 180 190 200 LDKGMLVAID ELMQSLNHNG ETLRQKPPVG EAEPYRVKMK
LCILLHAFST 210 RVVTINRVMG YLSSA 76 IL12Rb-2 10 20 30 40 50 60 70 80
90 100 110 120 130 140 150 160 170 180 190 200 210 220 230 240 250
260 270 280 290 300 310 320 330 340 350 360 370 380 390 400 410 420
430 440 450 460 470 480 490 500 510 520 530 540 550 560 570 580 590
600 610 620 630 640 650 660 670 680 690 700 710 720 730 740 750 760
770 780 790 800 810 820 830 840 850 860 ML 77 IL12Rb-1 10 20 30 40
50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210 220
230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390
400 410 420 430 440 450 460 470 480 490 500 510 520 530 540 550 560
570 580 590 600 610 620 630 640 650 660 KM 79 IL-12 p35 10 20 30 40
50 human MCHQQLVISW FSLVFLASPL VAIWELKKDV YVVELDWYPD APGEMVVLTC
(Uniprot 60 70 80 90 100 accession DTPEEDGITW TLDQSSEVLG SGKTLTIQVK
EFGDAGQYTC HKGGEVLSHS no. P29459) 110 120 130 140 150 LLLLHKKEDG
IWSTDILKDQ KEPKNKTFLR CEAKNYSGRF TCWWLTTIST 160 170 180 190 200
DLTFSVKSSR GSSDPQGVTC GAATLSAERV RGDNKEYEYS VECQEDSACP 210 220 230
240 250 AAEESLPIEV MVDAVHKLKY ENYTSSFFIR KIIKPDPPKN LQLKPLKNSR 260
270 280 290 300 QVEVSWEYPD TWSTPHSYFS LTFCVQVQGK SKREKKDRVF
TDKTSATVIC 310 320 RKNASISVRA QDRYYSSSWS EWASVPCS 80 IL-12 p40 10
20 30 40 50 mouse MCPQKLTISW FAIVLLVSPL MAMWELEKDV YVVEVDWTPD
APGETVNLTC (Uniprot 60 70 80 90 100 accession DTPEEDDITW TSDQRHGVIG
SGKTLTITVK EFLDAGQYTC HKGGETLSHS no. P43432) 110 120 130 140 150
HLLLHKKENG IWSTEILKNF KNKTFLKCEA PNYSGRFTCS WLVQRNMDLK 160 170 180
190 200 FNIKSSSSSP DSRAVTCGMA SLSAEKVTLD QRDYEKYSVS CQEDVTCPTA 210
220 230 240 250 EETLPIELAL EARQQNKYEN YSTSFFIRDI IKPDPPKNLQ
MKPLKNSQVE 260 270 280 290 300 VSWEYPDSES TPHSYFSLKF FVRIQRKKEK
MKETEECGNQ KGAFLVEKTS 310 320 330 TEVQCKGGNV CVQAQDRYYN SSCSKWACVP
CRVRS 81 ACP01 EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLE
(mouse IFN.gamma. WVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAV
conjugate) YYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGShgtviesleslnnyfn
ssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnisvieshlittffsnskakk-
d afmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcSGGPGPAGMKGLPGSEVQ
LVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVS
SISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYC
TIGGSLSVSSQGTLVTVSSHHHHHH 82 ACP02
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLE (mouse IFN.gamma.
WVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAV conjugate)
YYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGShgtviesleslnnyfn
ssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnisvieshlittffsnskakk-
d afmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcSGGPGPAGMKGLPGShgtvi
esleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnisvieshli-
tt
ffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcSGGPGPAGMKG
LPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPG
KGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPE
DTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHH 83 ACP03
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLE (mouse IFN.gamma.
WVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAV conjugate)
YYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGShgtviesleslnnyfn
ssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnisvieshlittffsnskakk-
d
afmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcggggsggggsggggshgtvieslesln
nyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnisvieshlittffsns-
k akkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcSGGPGPAGMKGLPGS
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLE
WVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAV
YYCTIGGSLSVSSQGTLVTVSSHHHHHH 84 Human IFN- 10 20 30 40 50 g
(Uniprot MKYTSYILAF QLCIVLGSLG CYCQDPVVKE AENLKKYFNA GHSDVADNGT
Accession 60 70 80 90 100 No. P01579) LFLGILKNWK EESDRKIMQA
QIVSFYFKLF KNFKDDQSIG KSVETIKEHM 110 120 130 140 150 NVKFFNSNKK
KRDDFEKLTN YSVTDLNVQR KAIHELIQVM AELSPAAKTG 160 KRKSQMLFR GRRASQ 85
Mouse IFN- 10 20 30 40 50 g (Uniprot MNATHCILAL QLFLMAVSGC
YCHGTVIESL ESLNNYFNSS GIDVEEKSLF Accession 60 70 80 90 100 No.
P01580) LDIWRNWQKD GDMKILQSQI ISFYLRLFEV LKDNQAISNN ISVIESHLIT 110
120 130 140 150 TFFSNSKAKK DAFMSIAKFE VNNPQVQRQA FNELIRVVHQ
LLPESSLRKR KRSRC 86 ACP30
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF (mouse
IFN.gamma. GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT
conjugate) LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMK
GLPGShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqai
snnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcSGGP
GPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMS
WVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYL
QMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLP
GShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnis
vieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcSGGPGPA
GMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVR
QAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNS
LRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHH 87 ACP31
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLE (mouse
WVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAV INFa1
YYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGScdlpqthnlrnkraltl conjugate)
lvqmrrlsplsclkdrkdfgfpqekvdaqqikkaqaipvlseltqqilniftskdssaawnttlldsfcndlh
qqlndlqgclmqqvgvqefpltqedallavrkyfhritvylrekkhspcawevvraevwralsssanvlg
rlreekSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFT
FSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNA
KTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHHE PEA 88 ACP32
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLE (mouse
WVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAV INFa1
YYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGScdlpqthnlrnkraltl conjugate)
lvqmrrlsplsclkdrkdfgfpqekvdaqqikkaqaipvlseltqqilniftskdssaawnttlldsfcndlh
qqlndlqgclmqqvgvqefpltqedallavrkyfhritvylrekkhspcawevvraevwralsssanS
GGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHHEPEA 89 IFN.gamma.R1 10
20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200
210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370
380 390 400 410 420 430 440 450 460 470 480 90 IFN.gamma.R2 10 20
30 40 50 60 70 80 90 100 110 120 130 140 150
160 170 180 190 200 210 220 230 240 250 260 270 280 290 300 310 320
330 91 ACP51 QVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQREL Mouse
IFG VARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYY conjugate
CNALYGTDYWGKGTQVTVSSggggsggggsggggsEVQLVESGGGLVQP
GNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLY
AESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQ
GTLVTVSSSGGPGPAGMKGLPGShgtviesleslnnyfnssgidveekslfldiwrnwq
kdgdmkilqsqiisfylrlfevlkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqvqrqa-
f nelirvvhqllpesslrkrkrsrcSGGPGPAGMKGLPGSEVQLVESGGGLVQPGN
SLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAES
VKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTL VTVSSHHHHHH 92
ACP52 EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLE Mouse IFG
WVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAV conjugate
YYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGShgtviesleslnnyfn
ssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnisvieshlittffsnskakk-
d afmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcSGGPGPAGMKGLPGSEVQ
LVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVS
SISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYC
TIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGSEVQLVESGGGLVQ
PGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLY
AESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQ
GTLVTVSSggggsggggsggggsQVQLQESGGGLVQAGGSLRLSCAASGRI
FSIDIMSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKN
TVYLQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSHHHHHH 93 ACP53
eahkseiahryndlgeqhfkglvliafsqylqkcsydehaklvqevtdfaktcvadesaancdks-
lhtlfg Mouse IFG
dklcaipnlrenygeladcctkqepernecflqhkddnpslppferpeaeamctsfkenpttf-
mghylhe conjugate
varrhpyfyapellyyaeqyneiltqccaeadkescltpkidgykekalvssvrqrmkcssmq-
kfgeraf
kawavarlsqtfpnadfaeitklatdltkvnkecchgdllecaddraelakymcenqatissklqtccdkpl
lkkahclsevehdtmpadlpaiaadfvedqeycknyaeakdvflgtflyeysahpdysyslllrlakkye
atlekccaeanppacygtvlaefqplveepknlvktncdlyeklgeygfqnailvrytqkapqvstptlve
aarnlgrvgtkcctlpedqrlpcvedylsailnrvcllhektpysehvtkccsgslverrpcfsaltvdety-
vp
kefkaetftfhsdictlpekekqikkqtalaelvkhkpkataeqlktvmddfaqfldtcckaadkdtcfste
gpnlvtrckdalaSGGPGPAGMKGLPGShgtviesleslnnyfnssgidveekslfldiwrnw
qkdgdmkilqsqiisfylrlfevlkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqvqrq
afnelirvvhqllpesslrkrkrsrcSGGPGPAGMKGLPGSeahkseiahryndlgeqhfkglvl
iafsqylqkcsydehaklvqevtdfaktcvadesaancdkslhtlfgdklcaipnlrenygeladcctkqep
ernecflqhkddnpslppferpeaeamctsfkenpttfmghylhevarrhpyfyapellyyaeqyneilt
qccaeadkescltpkldgvkekalvssvrqrmkcssmqkfgerafkawavarlsqtfpnadfaeitklat
dltkvnkecchgdllecaddraelakymcenqatissklqtccdkpllkkahclsevehdtmpadlpaia
adfvedqevcknyaeakdvflgtflyeysrrhpdysvslllrlakkyeatlekccaeanppacygtvlaefq
plveepknlvktncdlyeklgeygfqnailvrytqkapqvstptlyeaarnlgrvgtkcctlpedqrlpcve
dylsailnrvcllhektpvsehvtkccsgslverrpcfsaltvdetyvpkefkaetftfhsdictlpekekq-
ik
kqtalaelvkhkpkataeqlktvmddfaqfldtcckaadkdtcfstegpnlvtrckdalaHHHHHH
94 ACP54
eahkseiahryndlgeqhfkglvliafsqylqkcsydehaklvqevtdfaktcvadesaancdks-
lhtlfg Mouse IFG
dklcaipnlrenygeladcctkqepernecflqhkddnpslppferpeaeamctsfkenpttf-
mghylhe conjugate
varrhpyfyapellyyaeqyneiltqccaeadkescltpkldgvkekalvssvrqrmkcssmq-
kfgeraf
kawavarlsqtfpnadfaeitklatdltkvnkecchgdllecaddraelakymcenqatissklqtccdkpl
lkkahclsevehdtmpadlpaiaadfvedqevcknyaeakdvflgtflyeysrrhpdysvslllrlakkye
atlekccaeanppacygtvlaefqplveepknlvktncdlyeklgeygfqnailvrytqkapqvstptlve
aarnlgrvgtkcctlpedqrlpcvedylsailnrvcllhektpvsehvtkccsgslverrpcfsaltvdety-
vp
kefkaetftfhsdictlpekekqikkqtalaelvkhkpkataeqlktvmddfaqfldtcckaadkdtcfste
gpnlvtrckdalaSGGPGPAGMKGLPGShgtviesleslnnyfnssgidveekslfldiwrnw
qkdgdmkilqsqiisfylrlfevlkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqvqrq
afnelirvvhqllpesslrkrkrsrcggggsggggsggggshgtviesleslnnyfnssgidveekslfldi-
w
rnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqv
qrqafnelirvvhqllpesslrkrkrsrcSGGPGPAGMKGLPGSeahkseiahryndlgeqhfk
glyliafsqylqkcsydehaklvqevtdfaktcvadesaancdkslhtlfgdklcaipnlrenygeladcct
kqepernecflqhkddnpslppferpeaeamctsfkenpttfmghylhevarrhpyfyapellyyaeqy
neiltqccaeadkescltpkldgykekalvssvrqrmkcssmqkfgerafkawavarlsqtfpnadfaeit
klatdltkvnkecchgdllecaddraelakymcenqatissklqtccdkpllkkahclsevehdtmpadlp
aiaadfvedqevcknyaeakdvflgtflyeysrrhpdysyslllrlakkyeatlekccaeanppacygtvla
efqplveepknlvktncdlyeklgeygfqnailvrytqkapqvstptlveaarnlgrvgtkcctlpedqrlp
cvedylsailnrvcllhektpvsehvtkccsgslverrpcfsaltvdetyvpkefkaetftfhsdictlpek-
ek
qikkqtalaelvkhkpkataeqlktvmddfaqfldtcckaadkdtcfstegpnlvtrckdalaHHHHH
H 95 ACP50 mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI
Mouse IFG MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVY conjugate
LQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSggggsggggsgggg
sEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGL
EWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA
VYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGShgtviesleslnnyf
nssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnisvieshlittffsnskak-
k
dafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcggggsggggsggggshgtvieslesl
nnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnisvieshlittffsn-
s kakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcSGGPGPAGMKGLPG
SEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGL
EWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA
VYYCTIGGSLSVSSQGTLVTVSSHHHHHH 96 ACP55
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF Mouse IFG
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT conjugate
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMK
GLPGShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqai
snnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcSGGP
GPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMS
WVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYL
QMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLP
GShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnis
vieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcSGGPGPA
GMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVR
QAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNS
LRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHH 97 ACP56
mdmrvpaqllgllllwlrgarcQVQLQESGGGLAQAGGSLSLSCAASGFTVSNS Mouse IFG
VMAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTV conjugate
YLQMNNLKPEDTAVYVCNRNFDRIYWGQGTQVTVSSggggsggggsggg
gsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGL
EWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA
VYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGShgtviesleslnnyf
nssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnisvieshlittffsnskak-
k dafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcSGGPGPAGMKGLPGSEV
QLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEW
VSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVY
YCTIGGSLSVSSQGTLVTVSSHEIREIHHEPEA 98 ACP57
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF Mouse IFG
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT conjugate
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMK
GLPGShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqai
snnisvieshlittffsnskakkdainsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcSGGP
GPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMS
WVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYL
QMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsQV
QLQESGGGLAQAGGSLSLSCAASGFTVSNSVMAWYRQTPGKQREFV
AIINSVGSTNYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYV
CNRNFDRIYWGQGTQVTVSSHHHHHHEPEA 99 ACP58
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF Mouse IFG
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT conjugate
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMK
GLPGShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqai
snnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcSGGP
GPAGMKGLPGShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrl
fevlkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrk-
r krsrcSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFT
FSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNA
KTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggs
ggggsQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGK
QRELVARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDT
GVYYCNALYGTDYWGKGTQVTVSSHHHHHHEPEA 100 ACP59
mdmrvpaqllgllllwlrgarcQVQLQESGGGLAQAGGSLSLSCAASGFTVSNS Mouse IFG
VMAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTV conjugate
YLQMNNLKPEDTAVYVCNRNFDRIYWGQGTQVTVSSggggsggggsggg
gsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGL
EWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA
VYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGShgtviesleslnnyf
nssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnisvieshlittffsnskak-
k dafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcSGGPGPAGMKGLPGShgt
viesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnisviesh-
l
ittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcSGGPGPAGMK
GLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAP
GKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRP
EDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHHEPEA 101 ACP60
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF Mouse IFG
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT conjugate
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMK
GLPGShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqai
snnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcSGGP
GPAGMKGLPGShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrl
fevlkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrk-
r krsrcSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFT
FSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNA
KTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggs
ggggsQVQLQESGGGLAQAGGSLSLSCAASGFTVSNSVMAWYRQTPG
KQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTVYLQMNNLKPED
TAVYVCNRNFDRIYWGQGTQVTVSSHHHHHHEPEA 102 ACP61
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF Mouse IFG
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT conjugate
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMK
GLPGShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqai
snnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcSGGP
GPAGMKGLPGShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrl
fevlkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrk-
r krsrcSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFT
FSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNA
KTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggs
ggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPG
KGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAE
DTAVYYCARGVGAFRPYRKHEWGQGTLVTVSRggggsggggsggggsSSE
LTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQKPGQAPVLVIYG
KNNRPSGIPDRFSGSSSGNTASLTTTGAQAEDEADYYCNSSPFEHNL
VVFGGGTKLTVLHHHHHHEPEA 103 ACP63
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGGSLRLSCAASGFTFSSY Anti-FN
AMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNT CGS-2 scFv
LYLQMNSLRAEDTAVYYCARGVGAFRPYRKHEWGQGTLVTVSRgg
ggsggggsggggsSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQ
KPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTTTGAQAEDEAD
YYCNSSPFEHNLVVFGGGTKLTVLHHHHHHEPEA 104 ACP69
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF Mouse IFG
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT conjugate
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMK
GLPGShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqai
snnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcSGGP
GPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMS
WVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYL
QMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLP
GShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnis
vieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcHHHHHH
EPEA
105 ACP 70
mdmrvpaqllgllllwlrgarchgtviesleslnnyfnssgidveekslfldiwrnwqkdgdm-
kilqsqii Mouse IFG
sfylrlfevlkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnel-
irvvhqllpes conjugate
slrkrkrsrcSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAAS
GFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISR
DNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGP
GPAGMKGLPGShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrl
fevlkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrk-
r krsrcSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFT
FSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNA
KTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHHE PEA 106 ACP 71
mdmrvpaqllgllllwlrgarchgtviesleslnnyfnssgidveekslfldiwrnwqkdgdm-
kilqsqii Mouse IFG
sfylrlfevlkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnel-
irvvhqllpes conjugate
slrkrkrsrcSGGPGPAGMKGLPGSEAHKSEIAHRYNDLGEQHFKGLVLIA
FSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGD
KLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPE
AEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEIL
TQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAF
KAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDR
AELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLP
AIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRL
AKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLY
EKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPE
DQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFS
ALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKP
KATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDA
LASGGPGPAGMKGLPGShgtviesleslnnyfnssgidveeksifidiwrnwqkdgdmkil
qsqiisfylrlfevlkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvv-
hq llpesslrkrkrsrcSGGPGPAGMKGLPGSEAHKSEIAHRYNDLGEQHFKGL
VLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHT
LFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPF
ERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQY
NEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFG
ERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLEC
ADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMP
ADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSL
LLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTN
CDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKC
CTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVER
RPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAEL
VKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVT RCKDALAHHHHHHEPEA 107
ACP72 mdmrvpaqllgllllwlrgarcEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQ Mouse
IFG KCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIP conjugate
NLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCT
SFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEA
DKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVA
RLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKY
MCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFV
EDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEAT
LEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYG
FQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCV
EDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETY
VPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQL
KTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALASGGPGP
AGMKGLPGShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfev
lkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkr-
sr cSGGPGPAGMKGLPGSEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYL
QKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAI
PNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMC
TSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAE
ADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAV
ARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAK
YMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADF
VEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEA
TLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEY
GFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPC
VEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDET
YVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQ
LKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALASGGPG
PAGMKGLPGShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfe
vlkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrk-
r srcHHHHHHEPEA 108 ACP 73
mdmrvpaqllgllllwlrgarcEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQ Mouse IFG
KCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIP conjugate
NLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCT
SFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEA
DKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVA
RLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKY
MCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFV
EDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEAT
LEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYG
FQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCV
EDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETY
VPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQL
KTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALASGGPGP
AGMKGLPGShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfev
lkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkr-
sr cSGGPGPAGMKGLPGSEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYL
QKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAI
PNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMC
TSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAE
ADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAV
ARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAK
YMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADF
VEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEA
TLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEY
GFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPC
VEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDET
YVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQ
LKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALASGGPG
PAGMKGLPGShgtviesleslnnyfnssgidveeksifidiwrnwqkdgdmkilqsqiisfylrlfe
vlkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrk-
r srcSGGPGPAGMKGLPGSEAHKSEIAHRYNDLGEQHFKGLVLIAFSQY
LQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLC
AIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEA
MCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQC
CAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKA
WAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAE
LAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAI
AADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAK
KYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEK
LGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPED
QRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSAL
TVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPK
ATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDAL AHHHHHHEPEA 109 ACP74
mdmrvpaqllgllllwlrgarcEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQ Mouse IFG
KCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIP conjugate
NLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCT
SFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEA
DKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVA
RLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKY
MCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFV
EDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEAT
LEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYG
FQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCV
EDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETY
VPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQL
KTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALASGGPGP
AGMKGLPGShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfev
lkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkr-
sr cSGGPGPAGMKGLPGSggggsEAHKSEIAHRYNDLGEQHFKGLVLIAF
SQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGD
KLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPE
AEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEIL
TQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAF
KAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDR
AELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLP
AIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRL
AKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLY
EKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPE
DQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFS
ALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKP
KATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDA
LAggggsSGGPGPAGMKGLPGShgtviesleslnnyfnssgidveekslfldiwrnwqkdg
dmkilqsqiisfylrlfevlkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafne-
li rvvhqllpesslrkrkrsrcSGGPGPAGMKGLPGSEAHKSEIAHRYNDLGEQHF
KGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKS
LHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSL
PPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAE
QYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQK
FGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLL
ECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDT
MPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSV
SLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKT
NCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTK
CCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVE
RRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAE
LVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVT RCKDALAHHHHHHEPEA
110 ACP75 mdmrvpaqllgllllwlrgarcEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQ
Mouse IFG KCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIP conjugate
NLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCT
SFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEA
DKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVA
RLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKY
MCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFV
EDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEAT
LEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYG
FQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCV
EDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETY
VPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQL
KTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALASGGPGP
AGMKGLPGShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfev
lkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkr-
sr cSGGPGPAGMKGLPGSggggsggggsEAHKSEIAHRYNDLGEQHFKGLV
LIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTL
FGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFE
RPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYN
EILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGE
RAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECA
DDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPA
DLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLL
RLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCD
LYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCT
LPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPC
FSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKH
KPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKD
ALAggggsggggsSGGPGPAGMKGLPGShgtviesleslnnyfnssgidveekslfldiwr
nwqkdgdmkilqsqiisfylrlfevlkdnqaisnnisvieshlittffsnskakkdafmsiakfevnnpqv
qrqafnelirvvhqllpesslrkrkrsrcSGGPGPAGMKGLPGSEAHKSEIAHRYNDL
GEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVADESAA
NCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECFLQHKD
DNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYFYAPEL
LYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVRQRMKC
SSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKVNKECC
HGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKAHCLSE
VEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYEYSRRH
PDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPLVEEPK
NLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEAARNLG
RVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHVTKCCS
GSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEKQIKKQ
TALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDTCFSTE
GPNLVTRCKDALAHHHHHHEPEA 111 ACP78
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF Mouse IFG
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT conjugate
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsgggg
shgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnisv-
i
eshlittffsnskakkdafmsiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcggggsggggsg
gggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGK
GLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPED
TAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggshgtviesleslnnyfnss
gidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnisvieshlittffsnskakkda-
f msiakfevnnpqvqrqafnelirvvhqllpesslrkrkrsrcggggsggggsggggsEVQLVESG
GGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGS
GRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGS
LSVSSQGTLVTVSSHHHHHHEPEA 112 ACP134
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF Mouse IFG
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT conjugate
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMK
GLPGShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqai
snnisvieshlittffsnskakkdafmsiakfevnnpqvqrqafnelftyvhqllpesslrkrkrsrcSGGP
GPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMS
WVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYL
QMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLP
GShgtviesleslnnyfnssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnis
vieshlittffsnskakkdafmsiakfevnnpqvqrqafneliryvhqllpesslrkrkrsrcSGGPGPA
GMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVR
QAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNS
LRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsQVQLQE
SGGGLAQAGGSLSLSCAASGFTVSNSVMAWYRQTPGKQREFVAIINS
VGSTNYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYVCNRN
FDRIYWGQGTQVTVSSHHHHHHEPEA 113 ACP135
mdmrypaqllgllllwlrgarcQVQLQESGGGLAQAGGSLSLSCAASGFTVSNS Mouse IFG
VMAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTV conjugate
YLQMNNLKPEDTAVYVCNRNFDRIYWGQGTQVTVSSggggsggggsggg
gsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGL
EWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA
VYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGShgtviesleslnnyf
nssgidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnisvieshlittffsnskak-
k dafmsiakfevnnpqvqrqafneliryvhqllpesslrkrkrsrcSGGPGPAGMKGLPGSEV
QLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEW
VSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVY
YCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGShgtviesleslnnyfnss
gidveekslfldiwrnwqkdgdmkilqsqiisfylrlfevlkdnqaisnnisvieshlittffsnskakkda-
f msiakfevnnpqvqrqafneliryvhqllpesslrkrkrsrcSGGPGPAGMKGLPGSEVQL
VESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSS
ISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI
GGSLSVSSQGTLVTVSSHHEIHRHEPEA 114 ACP34
mdmrvpaqllgllllwlrgarcrvipvsgparclsqsrnllkttddmvktareklkhysctaed-
idheditr Mouse IL-12
dqtstlktclplelhknesclatretssttrgsclppqktslmmtlclgsiyedlkmyqtefqainaalqnhn-
h conjugate
qqnldkgmlvaidelmqslnhngetlrqkppygeadpyrykmklcillhafstrvvtinrymg-
ylssaS GGPGPAGMKGLPGSmwelekdvyvveydwtpdapgetvnltcdtpeedditwtsdqrhgv
igsgktltitvkefldagqytchkggetlshshlllhkkengiwsteilknfknktflkceapnysgrftcs-
wl
vqrnmdlkfnikssssspdsravtcgmaslsaekvtldqrdyekysvscqedvtcptaeetlpielalearq
qnkyenystsffirdiikpdppknlqmkplknsqveysweypdswstphsyfslkffyriqrkkekmk
eteegcnqkgaflvektstevqckggnvcvqaqdryynsscskwacvpcrvrsHHHHHH 115
ACP35
mdmrvpaqllgllllwlrgarcrvipvsgparclsqsrnllkttddmvktareklkhysctaed-
idheditr Mouse IL-12
dqtstlktclplelhknesclatretssttrgsclppqktslmmtlclgsiyedlkmyqtefqainaalqnhn-
h conjugate
qqnldkgmlvaidelmqslnhngetlrqkppygeadpyrvkmklcillhafstrvvtinrvmg-
ylssag gggsggggsggggsSGGPGPAGMKGLPGSggggsggggsggggsmwelekdvyvvey
dwtpdapgetvnltcdtpeedditwtsdqrhgvigsgktltitvkefldagqytchkggetlshshlllhkk
engiwsteilknfknktflkceapnysgrftcswlvqrnmdlkfnikssssspdsravtcgmaslsaekvtl
dqrdyekysvscqedvtcptaeetlpielalearqqnkyenystsffirdiikpdppknlqmkplknsqve
vsweypdswstphsyfslkffvriqrkkekmketeegcnqkgaflvektstevqckggnvcvqaqdry
ynsscskwacvpcrvrsHHHHHH 116 ACP36
mdmrypaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF Mouse IL-12
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT conjugate
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMK
GLPGSmwelekdvyvvevdwtpdapgetvnltcdtpeedditwtsdqrhgvigsgktltitvkefld
agqytchkggetlshshlllhkkengiwsteilknfknktflkceapnysgrftcswlvqrnmdlkfnikss
ssspdsravtcgmaslsaekvtldqrdyekysyscqedvtcptaeetlpielalearqqnkyenystsffir-
d
iikpdppknlqmkplknsqvevsweypdswstphsyfslkffvriqrkkekmketeegcnqkgaflve
ktstevqckggnvcvqaqdryynsscskwacvpcrvrsggggsggggsggggsrvipvsgparclsqs
rnllkttddmvktareklkhysctaedidheditrdqtstlktclplelhknesclatretssttrgsclpp-
qktsl
mmtlclgsiyedlkmyqtefqainaalqnhnhqqiildkgmlvaidelmqslnhngefirqkppvgea
dpyrvkmklcillhafstrvvtinrvmgylssaSGGPGPAGMKGLPGSEVQLVESGGG
LVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGR
DTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLS VSSQGTLVTVSSHHHHHH
117 ACP37 mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI
Mouse IL-12 MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVY
conjugate LQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSggggsggggsgggg
sEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGL
EWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA
VYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGSmwelekdvyvve
vdwtpdapgetvnltcdtpeedditwtsdqrhgvigsgktltitvkefldagqytchkggetlshshlllhk
kengiwsteilknfknktflkceapnysgrftcswlvqrnmdlkfnikssssspdsravtcgmaslsaekv
tldqrdyekysvscqedvtcptaeetlpielalearqqnkyenystsffirdiikpdppknlqmkplknsqv
evsweypdswstphsyfslkffvriqrkkekmketeegcnqkgaflvektstevqckggnvcvqaqdr
yynsscskwacvpcrvrsggggsggggsggggsrvipvsgparclsqsrnllkttddmvktareklkhy
sctaedidheditrdqtstlktclplelhknesclatretssttrgsclppqktslmmtlclgsiyedlkmy-
qte
fqainaalqnhnhqqiildkgmlvaidelmqslnhngetlrqkppvgeadpyrvkmklcillhafstrvv
tinrvmgylssaSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCA
ASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTI
SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHH HHHH 118 ACP79
mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI Mouse IL-12
MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVY conjugate
LQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSggggsggggsgggg
sEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGL
EWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA
VYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGSmwelekdvyvve
vdwtpdapgetvnltcdtpeedditwtsdqrhgvigsgktltitvkefldagqytchkggetlshshlllhk
kengiwsteilknfknktflkceapnysgrftcswlvqrnmdlkfnikssssspdsravtcgmaslsaekv
tldqrdyekysvscqedvtcptaeetlpielalearqqnkyenystsffirdiikpdppknlqmkplknsqv
evsweypdswstphsyfslkffvriqrkkekmketeegcnqkgaflvektstevqckggnvcvqaqdr
yynsscskwacvpcrvrsggggsggggsggggsrvipvsgparclsqsrnllkttddmvktareklkhy
sctaedidheditrdqtstlktclplelhknesclatretssttrgsclppqktslmmtlclgsiyedlkmy-
qte
fqainaalqnhnhqqiildkgmlvaidelmqslnhngetlrqkppvgeadpyrvkmklcillhafstrvv
tinrvmgylssaSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCA
ASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTI
SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSShh HHHH 119 ACP 80
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF Mouse IL-12
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT conjugate
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMK
GLPGSmwelekdvyvvevdwtpdapgetvnltcdtpeedditwtsdqrhgvigsgktltitvkefld
agqytchkggetlshshlllhkkengiwsteilknfknktflkceapnysgrftcswlvqrnmdlkfnikss
ssspdsravtcgmaslsaekvtldqrdyekysvscqedvtcptaeetlpielalearqqnkyenystsffir-
d
iikpdppknlqmkplknsqvevsweypdswstphsyfslkffvriqrkkekmketeegcnqkgaflve
ktstevqckggnvcvqaqdryynsscskwacvpcrvrsggggsggggsggggsrvipvsgparclsqs
rnllkttddmvktareklkhysctaedidheditrdqtstlktclplelhknesclatretssttrgsclpp-
qktsl
mmtlclgsiyedlkmyqtefqainaalqnhnhqqiildkgmlvaidelmqslnhngefirqkppvgea
dpyrvkmklcillhafstrvvtinrvmgylssaSGGPGPAGMKGLPGSEVQLVESGGG
LVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGR
DTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLS
VSSQGTLVTVSSggggsggggsggggsQVQLQESGGGLVQAGGSLRLSCA
ASGRIFSIDIMSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISR
DNAKNTVYLQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSH HHHHH 120 ACP91
mdmrvpaqllgllllwlrgarciwelkkdvyvveldwypdapgemvvltcdtpeedgitwtldq-
ssevl Chimeric IL-
gsgktltiqvkefgdagqytchkggevlshsllllhkkedgiwstdilkdqkepknktflrceaknysgrft
12 conjugate
cwwlttistdltfsvkssrgssdpqgvtcgaatlsaervrgdnkeyeysvecqedsacpaaeeslpievmv
davhklkyenytssffirdiikpdppknlqlkplknsrqvevsweypdtwstphsyfsltfcvqvqgksk
rekkdrvftdktsatvicrknasisvraqdryyssswsewasvpcsggggsggggsggggsrvipvsgp
arclsqsrnllkttddmvktareklkhysctaedidheditrdqtstlktclplelhknesclatretsstt-
rgscl
ppqktslmmtlclgsiyedlkmyqtefqainaalqnhnhqqiildkgmlvaidelmqslnhngefirqk
ppvgeadpyrvkmklcillhafstrvvtinrvmgylssaggggsggggsggggsggggsggggsggg
gsggggsggggsggggsQSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTVK
WYQQLPGTAPKLLIYYNDQRPSGVPDRFSGSKSGTSASLAITGLQAE
DEADYYCQSYDRYTHPALLFGTGTKVTVLggggsggggsggggsQVQLVE
SGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIR
YDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCKT
HGSHDNWGQGTMVTVSSggggsggggsggggsEVQLVESGGGLVQPGNS
LRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESV
KGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLV TVSSHHHHHHEPEA 121
ACP136
mdmrvpaqllgllllwlrgarciwelkkdvyvveldwypdapgemvvltcdtpeedgitwtld-
qssevl Chimeric IL-
gsgktltiqvkefgdagqytchkggevlshsllllhkkedgiwstdilkdqkepknktflrceaknysgrft
12 conjugate
cwwlttistdltfsvkssrgssdpqgvtcgaatlsaervrgdnkeyeysvecqedsacpaaeeslpievmv
davhklkyenytssffirdiikpdppknlqlkplknsrqvevsweypdtwstphsyfsltfcvqvqgksk
rekkdrvftdktsatvicrknasisvraqdryyssswsewasvpcsggggsggggsggggsrvipvsgp
arclsqsrnllkttddmvktareklkhysctaedidheditrdqtstlktclplelhknesclatretsstt-
rgscl
ppqktslmmtlclgsiyedlkmyqtefqainaalqnhnhqqiildkgmlvaidelmqslnhngefirqk
ppvgeadpyrvkmklcillhafstrvvtinrvmgylssaSGGPGPAGMKGLPGSggggsgg
ggsggggsggggsggggsggggsQSVLTQPPSVSGAPGQRVTISCSGSRSNIGS
NTVKWYQQLPGTAPKLLIYYNDQRPSGVPDRFSGSKSGTSASLAITG
LQAEDEADYYCQSYDRYTHPALLFGTGTKVTVLggggsggggsggggsQV
QLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEW
VAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCKTHGSHDNWGQGTMVTVSSHHHHHHEPEA 122 ACP138
mdmrvpaqllgllllwlrgarciwelkkdvyvveldwypdapgemvvltcdtpeedgitwtld-
qssevl Chimeric IL-
gsgktltiqvkefgdagqytchkggevlshsllllhkkedgiwstdilkdqkepknktflrceaknysgrft
12 conjugate
cwwlttistdltfsvkssrgssdpqgvtcgaatlsaervrgdnkeyeysvecqedsacpaaeeslpievmv
davhklkyenytssffirdiikpdppknlqlkplknsrqvevsweypdtwstphsyfsltfcvqvqgksk
rekkdrvftdktsatvicrknasisvraqdryyssswsewasvpcsggggsggggsggggsrvipvsgp
arclsqsrnllkttddmvktareklkhysctaedidheditrdqtstlktclplelhknesclatretsstt-
rgscl
ppqktslmmticlgsiyedlkmyqtefqainaalqnhnhqqiildkgmlvaidelmqslnhngefirqk
ppvgeadpyrvkmklcillhafstrvvtinrvmgylssaSGGPGPAGMKGLPGSggggsgg
ggsggggsggggsggggsggggsQSVLTQPPSVSGAPGQRVTISCSGSRSNIGS
NTVKWYQQLPGTAPKLLIYYNDQRPSGVPDRFSGSKSGTSASLAITG
LQAEDEADYYCQSYDRYTHPALLFGTGTKVTVLggggsggggsggggsQV
QLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEW
VAFIRYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV
YYCKTHGSHDNWGQGTMVTVSSggggsggggsggggsEVQLVESGGGLV
QPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTL
YAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSS
QGTLVTVSSggggsggggsggggsQVQLQESGGGLAQAGGSLSLSCAASGF
TVSNSVMAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNA
KNTVYLQMNNLKPEDTAVYVCNRNFDRIYWGQGTQVTVSSHHHHH HEPEA 123 ACP139
mdmrvpaqllgllllwlrgarcQVQLQESGGGLAQAGGSLSLSCAASGFTVSNS Chimeric IL-
VMAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTV 12 conjugate
YLQMNNLKPEDTAVYVCNRNFDRIYWGQGTQVTVSSggggsggggsggg
gsiwelkkdvyvveldwypdapgemvvltcdtpeedgitwtldqssevlgsgktltiqvkefgdagqyt
chkggevlshsllllhkkedgiwstdilkdqkepknktfIrceaknysgrftcwwlttistdltfsvkssrg-
ss
dpqgvtcgaatlsaervrgdnkeyeysvecqedsacpaaeeslpievmvdavhklkyenytssffirdii
kpdppknlqlkplknsrqvevsweypdtwstphsyfsltfcvqvqgkskrekkdrvftdktsatvicrkn
asisvraqdryyssswsewasvpcsggggsggggsggggsrvipvsgparclsqsrnllkttddmvkta
reklkhysctaedidheditrdqtstlktclplelhknesclatretssttrgsclppqktslmmticlgsi-
yedl
kmyqtefqainaalqnhnhqqiildkgmlvaidelmqslnhngetlrqkppvgeadpyrvkmklcillh
afstrvvtinrvmgylssaSGGPGPAGMKGLPGSggggsggggsggggsggggsggggsg
gggsQSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTVKWYQQLPGTAP
KLLIYYNDQRPSGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSY
DRYTHPALLFGTGTKVTVLggggsggggsggggsQVQLVESGGGVVQPGR
SLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSNKYYA
DSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCKTHGSHDNWG
QGTMVTVSSggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASG
FTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRD
NAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHH HHEPEA 124 ACP140
mdmrvpaqllgllllwlrgarcQVQLQESGGGLAQAGGSLSLSCAASGFTVSNS Chimeric IL-
VMAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTV 12 conjugate
YLQMNNLKPEDTAVYVCNRNFDRIYWGQGTQVTVSSSGGPGPAGM
KGLPGSiwelkkdvyvveldwypdapgemvvltcdtpeedgitwtldqssevlgsgktltiqvkef
gdagqytchkggevlshsllllhkkedgiwstdilkdqkepknktfirceaknysgrftcwwlttistdltf-
s
vkssrgssdpqgvtcgaatlsaervrgdnkeyeysvecqedsacpaaeeslpievmvdavhklkyenyt
ssffirdiikpdppknlqlkplknsrqvevsweypdtwstphsyfsltfcvqvqgkskrekkdrvftdkts
atvicrknasisvraqdryyssswsewasvpcsggggsggggsggggsrvipvsgparclsqsrnllkttd
dmvktareklkhysctaedidheditrdqtstlktclplelhknesclatretssttrgsclppqktslmmt-
icl
gsiyedlkmyqtefqainaalqnhnhqqiildkgmlvaidelmqslnhngetlrqkppvgeadpyrvk
mklcillhafstrvvtinrvmgylssaSGGPGPAGMKGLPGSggggsggggsggggsggggs
ggggsggggsQSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTVKWYQQL
PGTAPKLLIYYNDQRPSGVPDRFSGSKSGTSASLAITGLQAEDEADY
YCQSYDRYTHPALLFGTGTKVTVLggggsggggsggggsQVQLVESGGGV
VQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFIRYDGSN
KYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCKTHGSHD
NWGQGTMVTVSSggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCA
ASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTI
SRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHH HHHHEPEA
125 ACP38
mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltf-
kfympkka IL-2
telkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativefl-
nrwitfc conjugate
qsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAASGF
TFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNA
KNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSG
GGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTN
VGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQP
EDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsEVQLVESG
GGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGS
GRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGS
LSVSSQGTLVTVSSggggsggggsggggsQVQLQESGGGLVQAGGSLRLSC
AASGRIFSIDIMSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTIS
RDNAKNTVYLQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSS HHHHHH 126 ACP39
mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI IL-2
MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVY conjugate
LQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSSGGPGPAGM
KGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQA
PGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLR
PEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGSEVQL
VESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAA
IDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
DSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSS
LSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYS
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKV
EIKSGGPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfy
mpkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativef-
ln rwitfcqsiistltHHHHHH** 127 ACP40
mdmrvpaqllgllllwlrgarcelcdddppeiphatfkamaykegtmlnceckrgfrriksgsl-
ymlctg IL-2
nsshsswdnqcqctssatrnttkqvtpqpeeqkerkttemqspmqpvdqaslpghcrepppweneate
conjugate
riyhfvvgqmvyyqcvqgyralhrgpaesyckmthgktrwtqpqlictgemetsqfpgeekpq-
aspe
grpesetsclvtadfqiqtemaatmetsiftteyqggggsggggsggggsggggsggggsggggsSG
GPGPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkate
khlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfc-
qsi istltHHHHHH 128 ACP41
mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltf-
kfympkka IL-2
telkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativefl-
nrwitfc conjugate
qsiistltSGGPGPAGMKGLPGSggggsggggsggggsggggsggggsggggselcdddpp
eiphatfkamaykegtmlnceckrgfrriksgslymlctgnsshsswdnqcqctssatrnttkqvtpqpe
eqkerkttemqspmqpvdqaslpghcrepppweneateriyhfvvgqmvyyqcvqgyralhrgpae
svckmthgktrwtqpqlictgemetsqfpgeekpqaspegrpesetsclvtadfqiqtemaatmetsiftt
eyqHHHHHH 129 ACP42
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF IL-2
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT conjugate
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsgggg
selcdddppeiphatfkamaykegtmlnceckrgfrriksgslymlctgnsshsswdnqcqctssatrntt
kqvtpqpeeqkerkttemqspmqpvdqaslpghcrepppweneateriyhfvvgqmvyyqcvqgy
ralhrgpaesyckmthgktrwtqpqlictgemetsqfpgeekpqaspegrpesetsclvtttdfqiqtema
atmetsiftteyqggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSapt
ssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleeylnlaqs-
k nfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH
130 ACP43
mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltf-
kfympkka IL-2
telkhlqcleeelkpleeylnlaqsknfhlrprdlisninvivlelkgsettfmceyadetatiyefl-
nrwitfc conjugate
qsiistltSGGPGPAGMKGLPGSggggsggggsggggsggggsggggsggggselcdddpp
eiphatfkamaykegtmlnceckrgfrriksgslymlctgnsshsswdnqcqctssatrnttkqvtpqpe
eqkerkttemqspmqpydqaslpghcrepppweneateriyhfvygqmyyyqcyqgyralhrgpae
svckmthgktrwtqpqlictgemetsqfpgeekpqaspegrpesetsclvtttdfqiqtemaatmetsiftt
eyqggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG
MSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTL
YLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHH 131 ACP44
mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltf-
kfympkka IL-2
telkhlqcleeelkpleeylnlaqsknfhlrprdlisninvivlelkgsettfmceyadetatiyefl-
nrwitfc conjugate
qsiistltSGGPGPAGMKGLPGSggggsggggsggggsggggsggggsggggselcdddpp
eiphatfkamaykegtmlnceckrgfrriksgslymlctgnsshsswdnqcqctssatrnttkqvtpqpe
eqkerkttemqspmqpydqaslpghcrepppweneateriyhfvygqmyyyqcyqgyralhrgpae
svckmthgktrwtqpqlictgemetsqfpgeekpqaspegrpesetsclytadfqiqtemaatmetsiftt
eyqSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTF
SKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNA
KTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHH 132 ACP45
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF IL-2
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT conjugate
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMK
GLPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPG
KGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAED
TAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDI
QMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALI
YSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPY
TFGGGTKVEIKggggsggggsggggsggggsggggsggggsSGGPGPAGMKGLPG
Saptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleeyln-
l
aqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH
133 ACP46
mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltf-
kfympkka IL-2
telkhlqcleeelkpleeylnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativefl-
nrwitfc conjugate
qsiistltSGGPGPAGMKGLPGSggggsggggsggggsggggsggggsggggsEVQLV
ESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAI
DSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
DSNWDALDYWGQGTTVTVSSsggpgpagmkglpgsDIQMTQSPSSLSASV
GDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSR
FSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggg
gsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVR
QAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNS
LRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsQVQLQE
SGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQRELVARITR
GGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCNALY
GTDYWGKGTQVTVSSHHHHHH 134 ACP47
mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI IL-2
MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVY conujgate
LQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSggggsggggsgggg
saptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleeyln-
l
aqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGM
KGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQA
PGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLR
PEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsgg
ggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKG
LEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTA
VYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQ
MTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIY
SASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYT FGGGTKVEIKHHHHHH
135 ACP48
mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltf-
ldympkka IL-2
telkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativefl-
nrwitfc conjugate
qsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAASGF
TFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNA
KNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSG
GGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTN
VGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQP
EDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsggggsEVQLVESG
GGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGS
GRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGS
LSVSSQGTLVTVSSHHHHHH 136 ACP49
mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltf-
kfympkka IL-2
telkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativefl-
nrwitfc conjugate
qsiistltSGGPGPAGMKGLPGSggggsggggsggggsggggsggggsggggsEVQLV
ESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAI
DSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
DSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSS
LSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYS
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKV
EIKggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG
MSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTL
YLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHEIREIREI 137 ACP92
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF IL-2
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT conjugate
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMK
GLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpl
eevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPG-
P AGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWV
RQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMN
SLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHH 138 ACP93
mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI IL-2
MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVY conjugate
LQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSgsgsgsgsgsgsgsg
sEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGL
EWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA
VYYCTIGGSLSVSSQGTLVTVSSgsgsgsgsgsgsgsgsQVQLQESGGGLVQ
AGGSLRLSCAASGRIFSIDIMSWYRQAPGKQRELVARITRGGTISYDD
SVKGRFTISRDNAKNTVYLQMNSLKPEDTGVYYCNALYGTDYWGK
GTQVTVSSgsgsgsgsgsgsgsgsEVQLVESGGGLVQPGGSLRLSCAASGFT
FSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAK
NSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGG
GGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNV
GWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPE
DFATYYCQQYYTYPYTFGGGTKVEIKSGGPGPAGMKGLPGSaptssstk
ktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhl-
r prdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH 139
ACP94 mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI IL-2
MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVY conjugate
LQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSgsgsgsgsgsgsgsg
sEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGL
EWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA
VYYCTIGGSLSVSSQGTLVTVSSgsgsgsgsgsgsgsgsEVQLVESGGGLVQ
PGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSP
DTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALD
YWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRV
TITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSG
SGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKSGGPGPA
GMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcle
eelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistl-
tHH HHHH 140 ACP95
mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI IL-2
MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVY conjugate
LQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSgsgsgsgsgsgsgsg
sEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGL
EWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA
VYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGSaptssstkktqlqle
hllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlis-
n invivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH 141 ACP96
mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI IL-2
MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVY conjugate
LQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSSGGPGPAGM
KGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelk
pleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGG-
PG PAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSW
VRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQM
NSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHH 142 ACP97
mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI IL-2
MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVY conjugate
LQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSggggsggggsgggg
sEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGL
EWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA
VYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGSaptssstkktqlqle
hllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlis-
n invivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQL
VESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSS
ISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI
GGSLSVSSQGTLVTVSSHHHHHH 143 ACP99
mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI IL-2
MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVY conjugate
LQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSggggsggggsgggg
saptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevln-
l
aqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGM
KGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQA
PGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLR
PEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHH 144 ACP100
mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI IL-2
MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVY conjugate
LQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSggggsggggsgggg
saptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevln-
l
aqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH
145 A CP101
mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkka
IL-2
telkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativefl-
nrwitfc conjugate
qsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGF
TFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDN
AKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHH H 146 ACP102
mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI IL-2
MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVY conjugate
LQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSSGGPGPAGM
KGLPGSaptssstkktqlqlehllldlqmilnginnyknpkhrmltfkfympkkatelkhlqcleeelk
pleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGG-
PG PAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSW
VRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQM
NSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsg
gggsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQA
PGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRA
EDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGG
SDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPK
ALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYT
YPYTFGGGTKVEIKHHHHHH 147 A CP103
mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkka
IL-2
telkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativefl-
nrwitfc conjugate
qsiistltSGGPGPAGMKGLPGSggggsggggsggggsggggsggggsggggsEVQLV
ESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAI
DSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAR
DSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSS
LSASVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYS
GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKV
EIKggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG
MSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTL
YLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggs
QVQLQESGGGLAQAGGSLSLSCAASGFTVSNSVMAWYRQTPGKQR
EFVAIINSVGSTNYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAV
YVCNRNFDRIYWGQGTQVTVSSHHHHHH 148 ACP104
mdmrvpaqllgllllwlrgarcQVQLQESGGGLAQAGGSLSLSCAASGFTVSNS IL-2
VMAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTV conjugate
YLQMNNLKPEDTAVYVCNRNFDRIYWGQGTQVTVSSaptssstkktqlqle
hllldlqmilnginnyknpkhrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisn
invivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGMKGLPGSEVQL
VESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSS
ISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTI
GGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsggggsEVQLVESG
GGLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSS
SYTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSN
WDALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSA
SVGDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVP
SRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIK HHHHHH 149 ACP105
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYT IL-2
LAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLY conjugate
LQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSG
GGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQ
QKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATY
YCQQYYTYPYTFGGGTKVEIKggggsggggsggggsggggsggggsggggsSGGP
GPAGMKGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkh
lqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqs-
iistl tSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSK
FGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKT
TLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggg
gsQVQLQESGGGLAQAGGSLSLSCAASGFTVSNSVMAWYRQTPGKQ
REFVAIINSVGSTNYADSVKGRFTISRDNAKNTVYLQMNNLKPEDTA
VYVCNRNFDRIYWGQGTQVTVSSHHHHHH 150 ACP106
mdmrvpaqllgllllwlrgarcQVQLQESGGGLAQAGGSLSLSCAASGFTVSNS IL-2
VMAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTV conjugate
YLQMNNLKPEDTAVYVCNRNFDRIYWGQGTQVTVSSggggsggggsggg
gsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGL
EWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA
VYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGSEVQLVESGG
GLVQPGGSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSS
YTYSPDTVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNW
DALDYWGQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASV
GDRVTITCKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSR
FSGSGSGTDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggg
gsggggsggggsggggsggggsggggsSGGPGPAGMKGLPGSaptssstkktqlqlehllld
lqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvi
vlelkgsettfmceyadetativeflnrwitfcqsiistltHHHHHH 151 ACP107
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYT IL-2
LAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLY conjugate
LQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSG
GGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQ
QKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATY
YCQQYYTYPYTFGGGTKVEIKggggsggggsggggsggggsggggsggggsEVQ
LVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVS
SISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYC
TIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGSaptssstkktqlqlehllldlq
milnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivl-
e lkgsettfmceyadetativeflnrwitfcqsiistltggggsggggsggggsQVQLQESGGGLA
QAGGSLSLSCAASGFTVSNSVMAWYRQTPGKQREFVAIINSVGSTNY
ADSVKGRFTISRDNAKNTVYLQMNNLKPEDTAVYVCNRNFDRIYW GQGTQVTVSSHHHHHH 152
ACP108 mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI IL-2
MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVY conjugate
LQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSggggsggggsgggg
saptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevln-
l
aqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGM
KGLPGSrgetgpaaPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFG
MSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTL
YLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggs
ggggsggggsggggsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAW
VRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQM
NSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGG
SGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKP
GKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQ
QYYTYPYTFGGGTKVEIKHEIHHHH 153 ACP117
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGGSLRLSCAASGFTFSSY Anti-FN
AMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNT CGS-2 scFv
LYLQMNSLRAEDTAVYYCARGVGAFRPYRKHEWGQGTLVTVSRgg
ggsggggsggggsSSELTQDPAVSVALGQTVRITCQGDSLRSYYASWYQQ
KPGQAPVLVIYGKNNRPSGIPDRFSGSSSGNTASLTTTGAQAEDEAD
YYCNSSPFEHNLVVFGGGTKLTVLHHHHHHEPEA 154 ACP118
mdmrvpaqllgllllwlrgarcQVQLQQSGAELVRPGTSVKVSCKASGYAFTN NARA1
YLIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATLTADKSSST Vh/Vl non-
AYMQLSSLTSDDSAVYFCARWRGDGYYAYFDVWGAGTTVTVSSgg cleavable
ggsggggsggggsDIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYM
NWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEEE
DAATYYCQQSNEDPYTFGGGTKLEIKHHHHHHEPEA 155 ACP119
mdmrvpaqllgllllwlrgarcQVQLQQSGAELVRPGTSVKVSCKASGYAFTN NARA1
YLIEWVKQRPGQGLEWIGVINPGSGGTNYNEKFKGKATLTADKSSST Vh/Vl
AYMQLSSLTSDDSAVYFCARWRGDGYYAYFDVWGAGTTVTVSSSG cleavable
GPGPAGMKGLPGSDIVLTQSPASLAVSLGQRATISCKASQSVDYDGD
SYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPV
EEEDAATYYCQQSNEDPYTFGGGTKLEIKHHHHHHEPEA 156 ACP120
mdmrvpaqllgllllwlrgarcDIVLTQSPASLAVSLGQRATISCKASQSVDYDG NARA1
DSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHP Vl/Vh non-
VEEEDAATYYCQQSNEDPYTFGGGTKLEIKggggsggggsggggsQVQLQ cleavable
QSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVI
NPGSGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCA
RWRGDGYYAYFDVWGAGTTVTVSSHHHHHHEPEA 157 ACP121
mdmrvpaqllgllllwlrgarcDIVLTQSPASLAVSLGQRATISCKASQSVDYDG NARA1
DSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHP Vl/Vh
VEEEDAATYYCQQSNEDPYTFGGGTKLEIKSGGPGPAGMKGLPGSQ cleavable
VQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLE
WIGVINPGSGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAV
YFCARWRGDGYYAYFDVWGAGTTVTVSSHHHHHHEPEA 158 ACP124
mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmlt-
fkfympkka IL-2
telkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativefl-
nrwitfc conjugate
qsiistltggggsggggsggggsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHHHEPEA 159 ACP132
mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkttrmlt-
fkfympkka IL-2
telkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativefl-
nrwitfc conjugate
qsiistltggggsggggsggggsdahksevahrfkdlgeenfkalvliafaqylqqcpfedhv-
klvnevte
faktcvadesaencdkslhtlfgdklctvatlretygemadccakqepernecflqhkddnpnlprlvrpe
vdvmctafhdneetflkkylyeiarrhpyfyapellffakrykaafteccqaadkaacllpkldelrdegka
ssakqrlkcaslqkfgerafkawavarlsqrfpkaefaevsklvtdltkvhtecchgdllecaddradlaky-
i
cenqdsissklkeccekpllekshciaevendempadlpslaadfveskdvcknyaeakdvflgmflye
yarrhpdysvvlllrlaktyettlekccaaadphecyakvfdefkplveepqnlikqncelfeqlgeykfqn
allvrytkkvpqvstptivevsrnlgkvgskcckhpeakrmpcaedylsvvinqlcvlhektpvsdrytk
ccteslvnapcfsalevdetyvpkefnaetftfhadictlsekerqikkqtalvelvkhkpkatkeqlkavm
ddfaafvekcckaddketcfaeegkklvaasqaalglHHHHHHEPEA 160 ACP141
mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmlt-
fkfympkka IL-2
telkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativefl-
nrwitfc conjugate
qsiistltggggsggggsggggsdahksevahrfkdlgeenfkalvliafaqylqqcpfedhv-
klvnevte
faktcvadesaencdkslhtlfgdklctvatlretygemadccakqepernecflqhkddnpnlprlvrpe
vdvmctafhdneetflkkylyeiarrhpyfyapellffakrykaafteccqaadkaacllpkldelrdegka
ssakqrlkcaslqkfgerafkawavarlsqrfpkaefaevsklvtdltkvhtecchgdllecaddradlaky-
i
cenqdsissklkeccekpllekshciaevendempadlpslaadfveskdvcknyaeakdvflgmflye
yarrhpdysvvlllrlaktyettlekccaaadphecyakvfdefkplveepqnlikqncelfeqlgeykfqn
allvrytkkvpqvstptivevsrnlgkvgskcckhpeakrmpcaedylsvvinqlcvlhektpvsdrytk
ccteslvnapcfsalevdetyvpkefnaetftfhadictlsekerqikkqtalvelvkhkpkatkeqlkavm
ddfaafvekcckaddketcfaeegkklvaasqaalglHHHHHHEPEA 161 ACP142
mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmlt-
fkfympkka IL-2
telkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativefl-
nrwitfc conjugate
qsiistltSGGPGPAGMKGLPGSdahksevahrfkdlgeenfkalvliafaqylqqcpfedhv- kl
vnevtefaktcvadesaencdkslhtlfgdklctvaftretygemadccakqepernecflqhkddnpnlp
rlvrpevdvmctafhdneetflkkylyeiarrhpyfyapellffakrykaafteccqaadkaacllpkldel-
r
degkassakqrlkcaslqkfgerafkawavarlsqrfpkaefaevsklvtdltkvhtecchgdllecaddra
dlakyicenqdsissklkeccekpllekshciaevendempadlpslaadfveskdvcknyaeakdvflg
mflyeyarrhpdysvvlllrlaktyettlekccaaadphecyakvfdefkplveepqnlikqncelfeqlge
ykfqnallvrytkkvpqvstptivevsrnlgkvgskcckhpeakrmpcaedylsvvinqlcvlhektpvs
drvtkccteslvnapcfsalevdetyvpkefnaetftfhadictlsekerqikkqtalvelvkhkpkatkeq-
l kavmddfaafvekcckaddketcfaeegkklvaasqaalglHHHHHHEPEA 162 ACP144
mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmlt-
fkfympkka IL-2
telkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativefl-
nrwitfc conjugate
qsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGF
TFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDN
AKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggg
gsggggsggggsggggsggggsSGGPGPAGMKGLPGSEVQLVESGGGLVQPG
GSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDT
VRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYW
GQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTIT
CKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSG
TDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsgg
ggsQVQLQESGGGLAQAGGSLSLSCAASGFTVSNSVMAWYRQTPGK
QREFVAIINSVGSTNYADSVKGRFTISRDNAKNTVYLQMNNLKPEDT
AVYVCNRNFDRIYWGQGTQVTVSSHHEIREIHEPEA 163 ACP145
mdmrvpaqllgllllwlrgarcQVQLQESGGGLAQAGGSLSLSCAASGFTVSNS IL-2
VMAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTV conjugate
YLQMNNLKPEDTAVYVCNRNFDRIYWGQGTQVTVSSggggsggggsggg
gsaptssstkktqlqlehllldlqmilnginnyknpkhrmltfkfympkkatelkhlqcleeelkpleevln
laqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGM
KGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQA
PGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLR
PEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsgg
ggsSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFS
SYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKN
SLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGG
GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVG
WYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPED
FATYYCQQYYTYPYTFGGGTKVEIKHHHHHHEPEA 164 ACP146
mdmrvpaqllgllllwlrgarcQVQLQESGGGLAQAGGSLSLSCAASGFTVSNS IL-2
VMAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTV conjugate
YLQMNNLKPEDTAVYVCNRNFDRIYWGQGTQVTVSSSGGPGPAGM
KGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelk
pleevinlaqsknfhlrprdlisninvivlelkgsettfinceyadetativeflnrwitfcqsiistltSG-
GPG PAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSW
VRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQM
NSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsg
gggsggggsSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAAS
GFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRD
NAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVS
SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGT
NVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQ
PEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHHEPEA 165 ACP133
mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmlt-
fkfympkka IL-2-6xHis
telkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwit-
fc qsnstltHHHHHH 166 ACP147
mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmlt-
fkfympkka IL-2
telkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativefl-
nrwitfc conjugate
qsiistltSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGF
TFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDN
AKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggg
gsggggsggggsggggsggggsSGGPGPAGMKGLPGSEVQLVESGGGLVQPG
GSLRLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDT
VRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYW
GQGTTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTIT
CKASQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSG
TDFTLTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKggggsggggsgg
ggsQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDIMSWYRQAPGKQ
RELVARITRGGTISYDDSVKGRFTISRDNAKNTVYLQMNSLKPEDTG
VYYCNALYGTDYWGKGTQVTVSSHHHHHHEPEA 167 ACP148
mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI IL-2
MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVY conjugate
LQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSggggsggggsgggg
saptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelkpleevln-
l
aqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGGPGPAGM
KGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQA
PGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLR
PEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsggggsgg
ggsSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAASGFTFS
SYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKN
SLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGG
GSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVG
WYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPED
FATYYCQQYYTYPYTFGGGTKVEIKHHHHHHEPEA 168 ACP149
mdmrvpaqllgllllwlrgarcQVQLQESGGGLVQAGGSLRLSCAASGRIFSIDI IL-2
MSWYRQAPGKQRELVARITRGGTISYDDSVKGRFTISRDNAKNTVY conjugate
LQMNSLKPEDTGVYYCNALYGTDYWGKGTQVTVSSSGGPGPAGM
KGLPGSaptssstkktqlqlehllldlqmilnginnyknpkltrmltfkfympkkatelkhlqcleeelk
pleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativeflnrwitfcqsiistltSGG-
PG PAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSW
VRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQM
NSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsggggsggggsg
gggsggggsSGGPGPAGMKGLPGSEVQLVESGGGLVQPGGSLRLSCAAS
GFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRD
NAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVS
SGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQNVGT
NVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQ
PEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHHEPEA 169 ACP33
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF Mouse IFNa-
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT conjugate
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMK
GLPGScdlpqthnlrnkraltllvqmalsplsclkdrkdfgfpqekvdaqqikkagaipvlseltqqiln
iftskdssaawnttldsfcndlhqqlndlqgclmqqvgvqefpltqedallavrkyfhritvylrekkhsp
cawevvraevwralsssanvSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNS
LRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESV
KGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLV TVSSHHHHHHEPEA 170
A CP131
mdmrvpaqllgllllwlrgarccdlpqthnlrnkraltllvqmalsplsclkdrkdfgfpqekvdaqqikk
Mouse IFNa
aqaipvlseltqqflniftskdssaawnttlldsfcndlhqqlndlqgclmqqvgvqefpltqedallavrky
fhritvylrekkhspcawevvraevwralsssanvlgrlreekHHHHHHEPEA 171 ACP125
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF Mouse IFNa-
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT conjugate
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMK
GLPGScdlpqthnlrnkraltllvqmrrlsplsclkdrkdfgfpqekvdaqqikkagaipvlseltqqiln
iftskdssaawnttlldsfcndlhqqlndlqgclmqqvgvqefpltqedallavrkyfhritvylrekkhsp
cawevvraevwralsssanvlgrlreekHHHHHHEPEA 172 ACP126
mdmrvpaqllgllllwlrgarccdlpqthnlrnkraltllvqmrrlsplsclkdrkdfgfpqe-
kvdaqqikk Mouse IFNa-
aqaipvlseltqqflniftskdssaawnttlldsfcndlhqqlndlqgclmqqvgvqefpltqedallavrky
conjugate
fhritvylrekkhspcawevvraevwralsssanvlgrlreekSGGPGPAGMKGLPGSEVQ
LVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVS
SISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYC
TIGGSLSVSSQGTLVTVSSHHHHHHEPEA 173 ACP127
mdmrvpaqllgllllwlrgarcEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQ Mouse IFNa-
KCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIP conjugate
NLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCT
SFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEA
DKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVA
RLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKY
MCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFV
EDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEAT
LEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYG
FQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCV
EDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETY
VPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQL
KTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALASGGPGP
AGMKGLPGScdlpqthnlrnkraltllvqmalsplsclkdrkdfgfpqekvdaqqikkaqaipvls
eltqqilniftskdssaawnttlldsfcndlhqqlndlqgclmqqvgvqefpltqedallavrkyfhrityy-
lr ekkhspcawevvraevwralsssanvlgrlreekSGGPGPAGMKGLPGSEAHKSEIAH
RYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDFAKTCVA
DESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQEPERNECF
LQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEVARRHPYF
YAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEKALVSSVR
QRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKLATDLTKV
NKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCDKPLLKKA
HCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVFLGTFLYE
YSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTVLAEFQPL
VEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVSTPTLVEA
ARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKTPVSEHV
TKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICTLPEKEK
QIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKAADKDT
CFSTEGPNLVTRCKDALAHHHHHHEPEA 174 ACP128
mdmrvpaqllgllllwlrgarcEAHKSEIAHRYNDLGEQHFKGLVLIAFSQYLQ Mouse IFNa-
KCSYDEHAKLVQEVTDFAKTCVADESAANCDKSLHTLFGDKLCAIP conjugate
NLRENYGELADCCTKQEPERNECFLQHKDDNPSLPPFERPEAEAMCT
SFKENPTTFMGHYLHEVARRHPYFYAPELLYYAEQYNEILTQCCAEA
DKESCLTPKLDGVKEKALVSSVRQRMKCSSMQKFGERAFKAWAVA
RLSQTFPNADFAEITKLATDLTKVNKECCHGDLLECADDRAELAKY
MCENQATISSKLQTCCDKPLLKKAHCLSEVEHDTMPADLPAIAADFV
EDQEVCKNYAEAKDVFLGTFLYEYSRRHPDYSVSLLLRLAKKYEAT
LEKCCAEANPPACYGTVLAEFQPLVEEPKNLVKTNCDLYEKLGEYG
FQNAILVRYTQKAPQVSTPTLVEAARNLGRVGTKCCTLPEDQRLPCV
EDYLSAILNRVCLLHEKTPVSEHVTKCCSGSLVERRPCFSALTVDETY
VPKEFKAETFTFHSDICTLPEKEKQIKKQTALAELVKHKPKATAEQL
KTVMDDFAQFLDTCCKAADKDTCFSTEGPNLVTRCKDALASGGPGP
AGMKGLPGScdlpqthnlrnkraltllvqmalsplsclkdrkdfgfpqekvdaqqikkaqaipvls
eltqqilniftskdssaawnttlldsfcndlhqqlndlqgclmqqvgvqefpltqedallavrkyfhrityy-
lr ekkhspcawevvraevwralsssanvlgrlreekHHHHHHEPEA 175 ACP129
mdmrvpaqllgllllwlrgarccdlpqthnlrnkraltllvqmalsplsclkdrkdfgfpqek-
vdaqqikk Mouse IFNa-
aqaipvlseltqqilniftskdssaawnttlldsfendlhqqlndlqgclmqqvgvqefpltqedallavrky
conjugate
fhritvylrekkhspcawevvraevwralsssanvlgrlreekSGGPGPAGMKGLPGSEAH
KSEIAHRYNDLGEQHFKGLVLIAFSQYLQKCSYDEHAKLVQEVTDF
AKTCVADESAANCDKSLHTLFGDKLCAIPNLRENYGELADCCTKQE
PERNECFLQHKDDNPSLPPFERPEAEAMCTSFKENPTTFMGHYLHEV
ARRHPYFYAPELLYYAEQYNEILTQCCAEADKESCLTPKLDGVKEK
ALVSSVRQRMKCSSMQKFGERAFKAWAVARLSQTFPNADFAEITKL
ATDLTKVNKECCHGDLLECADDRAELAKYMCENQATISSKLQTCCD
KPLLKKAHCLSEVEHDTMPADLPAIAADFVEDQEVCKNYAEAKDVF
LGTFLYEYSRRHPDYSVSLLLRLAKKYEATLEKCCAEANPPACYGTV
LAEFQPLVEEPKNLVKTNCDLYEKLGEYGFQNAILVRYTQKAPQVST
PTLVEAARNLGRVGTKCCTLPEDQRLPCVEDYLSAILNRVCLLHEKT
PVSEHVTKCCSGSLVERRPCFSALTVDETYVPKEFKAETFTFHSDICT
LPEKEKQIKKQTALAELVKHKPKATAEQLKTVMDDFAQFLDTCCKA
ADKDTCFSTEGPNLVTRCKDALAHHHHHHEPEA 176 ACP150
mdmrvpaqllgllllwlrgarcQVQLQESGGGLAQAGGSLSLSCAASGFTVSNS Mouse IFNa-
VMAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNAKNTV conjugate
YLQMNNLKPEDTAVYVCNRNFDRIYWGQGTQVTVSSggggsggggsggg
gsEVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGL
EWVSSISGSGRDTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTA
VYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMKGLPGScdlpqthnlrnkral
tllvqmalsplsclkdrkdfgfpqekvdaqqikkaqaipvlseltqqilniftskdssaawnttlldsfcnd-
l
hqqlndlqgclmqqvgvqefpltqedallavrkyfhritvylrekkhspcawevvraevwralsssanvl
grlreekSGGPGPAGMKGLPGSEVQLVESGGGLVQPGNSLRLSCAASGF
TFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDN
AKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSHHHHH HEPEA 177 ACP151
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF Mouse IFNa-
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT conjugate
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSSGGPGPAGMK
GLPGScdlpqthnlrnkraltllvqmalsplsclkdrkdfgfpqekvdaqqikkaqaipvlseltqqiln
iftskdssaawnttlldsfcndlhqqlndlqgclmqqvgvqefpltqedallavrkyfhritvylrekkhsp
cawevvraevwralsssanvlgrlreekSGGPGPAGMKGLPGSEVQLVESGGGLV
QPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTL
YAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSS
QGTLVTVSSggggsggggsggggsQVQLQESGGGLAQAGGSLSLSCAASGF
TVSNSVMAWYRQTPGKQREFVAIINSVGSTNYADSVKGRFTISRDNA
KNTVYLQMNNLKPEDTAVYVCNRNFDRIYWGQGTQVTVSSHHHHH HEPEA 178 ACP152
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGNSLRLSCAASGFTFSKF Mouse IFNa-
GMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAKTT conjugate
LYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsgggg
scdlpqthnlrnkraltllvqmalsplsclkdrkdfgfpqekvdaqqikkaqaipvlseltqqilniftskd-
s
saawnttlldsfcndlhqqlndlqgclmqqvgvqefpltqedallavrkyfhritvylrekkhspcawevv
raevwralsssanvlgrlreekggggsggggsggggsEVQLVESGGGLVQPGNSLRLSC
AASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRF
TISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSS HHHHHHEPEA 179
ACP153
mdmrvpaqllgllllwlrgarcaptssstkktqlqlehllldlqmilnginnyknpkltrmlt-
fkfympkka (IL-2
telkhlqcleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmceyadetativef-
lnrwitfc Conjugate)
qsiistltsggpGPAGLYAQpgsEVQLVESGGGLVQPGNSLRLSCAASGFTFS
KFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAK
TTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsg
gggsggggsggggsggggssggpGPAGLYAQpgsEVQLVESGGGLVQPGGSLR
LSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGR
FTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGT
TVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKAS
QNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFT
LTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHHEPEA 180 ACP154
mdmrvpaqllgllllwlrgareaptssstkktqlqlehllldlqmilnginnyknpkltrmlt-
fkfympkka (IL-2
telkhlqeleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmeeyadetativef-
lnrwitfe Conjugate)
qsiistltsggpPGGPAGIGpgsEVQLVESGGGLVQPGNSLRLSCAASGFTFS
KFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAK
TTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsg
gggsggggsggggsggggssggpPGGPAGIGpgsEVQLVESGGGLVQPGGSLRL
SCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRF
TISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTT
VTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQ
NVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLT
ISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHHEPEA 181 ACP155
mdmrvpaqllgllllwlrgareaptssstkktqlqlehllldlqmilnginnyknpkltrmlt-
fkfympkka (IL-2
telkhlqeleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmeeyadetativef-
lnrwitfe Conjugate)
qsiistltsggpALFKSSFPpgsEVQLVESGGGLVQPGNSLRLSCAASGFTFS
KFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNAK
TTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggsg
gggsggggsggggsggggssggpALFKSSFPpgsEVQLVESGGGLVQPGGSLRL
SCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRF
TISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQGTT
VTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKASQ
NVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFTLT
ISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHHEPEA 182 ACP156
mdmrvpaqllgllllwlrgareaptssstkktqlqlehllldlqmilnginnyknpkltrmlt-
fkfympkka (IL-2
telkhlqeleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmeeyadetativef-
lnrwitfe Conjugate)
qsiistltsggpPLAQKLKSSpgsEVQLVESGGGLVQPGNSLRLSCAASGFTF
SKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRDNA
KTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSSggggsggggs
ggggsggggsggggsggggssggpPLAQKLKSSpgsEVQLVESGGGLVQPGGSL
RLSCAASGFTFSSYTLAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRG
RFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDSNWDALDYWGQG
TTVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCKA
SQNVGTNVGWYQQKPGKAPKALIYSASFRYSGVPSRFSGSGSGTDFT
LTISSLQPEDFATYYCQQYYTYPYTFGGGTKVEIKHHHHHHEPEA 183 ACP157
mdmrvpaqllgllllwlrgareaptssstkktqlqlehllldlqmilnginnyknpkltrmlt-
fkfympkka (IL-2
telkhlqeleeelkpleevlnlaqsknfhlrprdlisninvivlelkgsettfmeeyadetativef-
lnrwitfe Conjugate)
qsiistltsggpPGGPAGIGalfkssfpPLAQKLKSSpgsEVQLVESGGGLVQPGN
SLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAES
VKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTL
VTVSSggggsggggsggggsggggsggggsggggssggpPGGPAGIGalfkssfpPLAQ
KLKSSpgsEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYTLAWVRQ
APGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLYLQMNSLR
AEDTAVYYCARDSNWDALDYWGQGTTVTVSSGGGGSGGGGSGGG
GSDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGKAP
KALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYY
TYPYTFGGGTKVEIKHHHHHHEPEA 184 Place hold 185 Place hold 186 Place
hold 187 Place hold 188 Place hold 189 Place hold 190 Place hold
191 Place hold 192 Blocker 2
mdmrvpaqllgllllwlrgarcEVQLVESGGGLVQPGGSLRLSCAASGFTFSSYT (IL2
blocker) LAWVRQAPGKGLEWVAAIDSSSYTYSPDTVRGRFTISRDNAKNSLY
LQMNSLRAEDTAVYYCARDSNWDALDYWGQGTTVTVSSggggsgggg
sggggsDIQMTQSPSSLSASVGDRVTITCKASQNVGTNVGWYQQKPGK
APKALIYSASFRYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQY
YTYPYTFGGGTKVEIKHHHHHH 193 Blocker 12
mdmrvpaqllgllllwlrgarcQSVLTQPPSVSGAPGQRVTISCSGSRSNIGSNTV (IL-12
KWYQQLPGTAPKLLIYYNDQRPSGVPDRFSGSKSGTSASLAITGLQA blocker)
EDEADYYCQSYDRYTHPALLFGTGTKVTVLggggsggggsggggsQVQLV
ESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAFI
RYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYC
KTHGSHDNWGQGTMVTVSSFIRREIHH 194
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQGGGGGLD
GNEEPGGLEWVSSISGSGRDTLYADSVKGRFTISRDNAKTTLYLQMN
SLRPEDTAVYYCTIGGSLSVSSQGTLVTVSS 225 ACP203
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGR
DTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSS
sggpGPAGLYAQpgscdlpqthnlrnkraltllvqmrrlsplsclkdrkdfgfpqekvdaqqikkaqai
pvlseltqqilniftskdssaawnttlldsfcndlhqqlndlqgclmqqvgvqefpltqedallavrkyfhr-
it
vylrekkhspcawevvraeywralsssanylgrlreeksggpGPAGLYAQpgsEVQLVESGGGLV
QPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFT
ISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSS 226 ACP204
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGR
DTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSS
sggpALFKSSFPpgscdlpqthnlrnkraltllvqmrrlsplsclkdrkdfgfpqekvdaqqikkaqaipv
lseltqqilniftskdssaawnttlldsfcndlhqqlndlqgclmqqvgvqefpltqedallavrkyfhrit-
yyl
rekkhspcawevvraeywralsssanylgrlreeksggpALFKSSFPpgsEVQLVESGGGLVQP
GNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTIS
RDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSS 227 ACP205
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGR
DTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSS
sggpPLAQKLKSSpgscdlpqthnlrnkraltllvqmrrlsplsclkdrkdfgfpqekvdaqqikkaqai
pvlseltqqilniftskdssaawnttlldsfcndlhqqlndlqgclmqqvgvqefpltqedallavrkyfhr-
it
vylrekkhspcawevvraeywralsssanylgrlreeksggpPLAQKLKSSpgsEVQLVESGGGL
VQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGR
FTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSS 228 ACP206
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGR
DTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSS
sggpGPAGLYAQpgscdlpqthslgsrrtlmllaqmrrislfsclkdrhdfgfpqeefgnqfqkaetipvl
hemiqqifnlfstkdssaawdetlldkfytelyqqlndleacviqgvgytetplmkedsilayrkyfqritl-
yl kekkyspcawevvraeimrsfslstnlqeslrskesggpGPAGLYAQpgsEVQLVESGGGLVQP
GNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTIS
RDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSS 229 ACP207
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGR
DTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSS
sggpALFKSSFPpgscdlpqthslgsrrtlmllaqmrrislfsclkdrhdfgfpqeefgnqfqkaetipylh
emiqqifnlfstkdssaawdetlldkfytelyqqlndleacviqgvgytetplmkedsilayrkyfqritly-
lk
ekkyspcawevvraeimrsfslstnlqeslrskesggpALFKSSFPpgsEVQLVESGGGLVQPGN
SLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTISRD
NAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSS 230 ACP208
EVQLVESGGGLVQPGNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGR
DTLYAESVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSS
sggpPLAQKLKSSpgscdlpqthslgsrrtlmllaqmrrislfsclkdrhdfgfpqeefgnqfqkaetipv
lhemiqqifnlfstkdssaawdetlldkfytelyqqlndleacviqgvgytetplmkedsilayrkyfqrit-
ly
lkekkyspcawevvraeimrsfslstnlqeslrskesggpPLAQKLKSSpgsEVQLVESGGGLVQP
GNSLRLSCAASGFTFSKFGMSWVRQAPGKGLEWVSSISGSGRDTLYAESVKGRFTIS
RDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSVSSQGTLVTVSS 258 MMP14 GPLGLKAQ
substrate motif sequence 259 MMP14 LPLGLKAQ substrate motif
sequence 260 MMP14 SPLGLKAQ substrate motif sequence 261 MMP14
QPLGLKAQ substrate motif sequence 262 MMP14 KPLGLKAQ substrate
motif sequence 263 MMP14 FPLGLKAQ substrate motif sequence 264
MMP14 HPLGLKAQ substrate motif sequence 265 MMP14 PPLGLKAQ
substrate motif sequence 266 MMP14 APLGLKAQ substrate motif
sequence 267 MMP14 DPLGLKAQ substrate motif sequence 268 MMP14
GPHGLKAQ substrate motif sequence 269 MMP14 GPSGLKAQ substrate
motif sequence 270 MMP14 GpQGLKAQ substrate motif sequence 271
MMP14 GPPGLKAQ substrate motif sequence 272 MMP14 GPEGLKAQ
substrate motif sequence 273 MMP14 GPFGLKAQ substrate motif
sequence 274 MMP14 GPRGLKAQ substrate motif sequence 275 MMP14
GPGGLKAQ substrate motif sequence 276 MMP14 GPAGLKAQ substrate
motif sequence 277 MMP14 LPAGLKGA substrate motif sequence 195
MMP14 GPAGLYAQ substrate motif sequence 278 MMP14 GPANLVAQ
substrate motif sequence 279 MMP14 GPAALVGA substrate motif
sequence 280 MMP14 GPANLRAQ substrate motif sequence 281 MMP14
GPAGLRAQ substrate motif sequence 282 MMP14 GPAGLVAQ substrate
motif sequence 283 MMP14 GPAGLRGA substrate motif sequence 284
MMP14 LPAGLVGA substrate motif sequence 285 MMP14 GPAGLKGA
substrate motif sequence 286 MMP14 GPLALKAQ substrate motif
sequence 287 MMP14 GPLNLKAQ substrate motif sequence 288 MMP14
GPLHLKAQ substrate motif sequence 289 MMP14 GPLYLKAQ substrate
motif sequence 290 MMP14 GPLPLKAQ substrate motif sequence 291
MMP14 GPLELKAQ substrate motif sequence 292 MMP14 GPLRLKAQ
substrate motif sequence 293 MMP14 GPLLLKAQ substrate motif
sequence 294 MMP14 GPLSLKAQ substrate motif sequence 295 MMP14
GPLGLYAQ substrate motif sequence 296 MMP14 GPLGLFAQ substrate
motif sequence 297 MMP14 GPLGLLAQ substrate motif sequence 298
MMP14 GPLGLHAQ substrate motif sequence 299 MMP14 GPLGLRAQ
substrate motif sequence 300 MMP14 GPLGLAAQ substrate motif
sequence 301 MMP14 GPLGLEAQ substrate motif sequence 302 MMP14
GPLGLGAQ substrate motif sequence 303 MMP14 GPLGLPAQ substrate
motif sequence 304 MMP14 GPLGLQAQ substrate motif sequence 305
MMP14 GPLGLSAQ substrate motif sequence 306 MMP14 GPLGLVAQ
substrate motif sequence 307 MMP14 GPLGLKLQ substrate motif
sequence 308 MMP14 GPLGLKFQ substrate motif sequence 309 MMP14
GPLGLKEQ substrate motif sequence 310 MMP14 GPLGLKKQ substrate
motif sequence 311 MMP14 GPLGLKQQ substrate motif sequence 312
MMP14 GPLGLKSQ substrate motif sequence 313 MMP14 GPLGLKGQ
substrate motif sequence 314 MMP14 GPLGLKHQ substrate motif
sequence 315 MMP14 GPLGLKPQ substrate motif sequence 316 MMP14
GPLGLKAG substrate motif sequence 317 MMP14 GPLGLKAF substrate
motif sequence 318 MMP14 GPLGLKAP substrate motif sequence 319
MMP14 GPLGLKAL substrate motif sequence 320 MMP14 GPLGLKAE
substrate motif sequence 321 MMP14 GPLGLKAA substrate motif
sequence 322 MMP14 GPLGLKAH substrate motif sequence 323 MMP14
GPLGLKAK substrate motif sequence 324 MMP14 GPLGLKAS substrate
motif sequence 325 MMP14 GPLGLFGA substrate motif sequence 326
MMP14 GPLGLQGA substrate motif sequence 327 MMP14 GPLGLVGA
substrate motif sequence 328 MMP14 GPLGLAGA substrate motif
sequence 329 MMP14 GPLGLLGA substrate motif sequence
330 MMP14 GPLGLRGA substrate motif sequence 331 MMP14 GPLGLYGA
substrate motif sequence 332 CTSL1 ALFKSSPP substrate motif
sequence 333 CTSL1 SPFRSSRQ substrate motif sequence 334 CTSL1
KLFKSSPP substrate motif sequence 335 CTSL1 HLFKSSPP substrate
motif sequence 336 CTSL1 SLFKSSPP substrate motif sequence 337
CTSL1 QLFKSSPP substrate motif sequence 338 CTSL1 LLFKSSPP
substrate motif sequence 339 CTSL1 PLFKSSPP substrate motif
sequence 340 CTSL1 FLFKSSPP substrate motif sequence 341 CTSL1
GLFKSSPP substrate motif sequence 342 CTSL1 VLFKSSPP substrate
motif sequence 343 CTSL1 ELFKSSPP substrate motif sequence 344
CTSL1 AKFKSSPP substrate motif sequence 345 CTSL1 AHFKSSPP
substrate motif sequence 346 CTSL1 AGFKSSPP substrate motif
sequence 347 CTSL1 APFKSSPP substrate motif sequence 348 CTSL1
ANFKSSPP substrate motif sequence 349 CTSL1 AFFKSSPP substrate
motif sequence 350 CTSL1 AAFKSSPP substrate motif sequence 351
CTSL1 ASFKSSPP substrate motif sequence 352 CTSL1 AEFKSSPP
substrate motif sequence 353 CTSL1 ALRKSSPP substrate motif
sequence 354 CTSL1 ALLKSSPP substrate motif sequence 355 CTSL1
ALAKSSPP substrate motif sequence 356 CTSL1 ALQKSSPP substrate
motif sequence 357 CTSL1 ALHKSSPP substrate motif sequence 358
CTSL1 ALPKSSPP substrate motif sequence 359 CTSL1 ALTKSSPP
substrate motif sequence 360 CTSL1 ALGKSSPP substrate motif
sequence 361 CTSL1 ALDKSSPP substrate motif sequence 199 CTSL1
ALFFSSPP substrate motif sequence 362 CTSL1 ALFHSSPP substrate
motif sequence 363 CTSL1 ALFTSSPP substrate motif sequence 364
CTSL1 ALFASSPP substrate motif sequence 365 CTSL1 ALFQSSPP
substrate motif sequence 366 CTSL1 ALFLSSPP substrate motif
sequence 367 CTSL1 ALFGSSPP substrate motif sequence 368 CTSL1
ALFESSPP substrate motif sequence 369 CTSL1 ALFPSSPP substrate
motif sequence 370 CTSL1 ALFKHSPP substrate motif sequence 371
CTSL1 ALFKLSPP substrate motif sequence 372 CTSL1 ALFKKSPP
substrate motif sequence 373 CTSL1 ALFKASPP substrate motif
sequence 374 CTSL1 substrate ALFKISPP motif sequence 375 CTSL1
ALFKGSPP substrate motif sequence 376 CTSL1 ALFKNSPP substrate
motif sequence 377 CTSL1 ALFKRSPP substrate motif sequence 378
CTSL1 ALFKESPP substrate motif sequence
379 CTSL1 ALFKFSPP substrate motif sequence 380 CTSL1 ALFKPSPP
substrate motif sequence 381 CTSL1 ALFKSFPP substrate motif
sequence 382 CTSL1 ALFKSLPP substrate motif sequence 383 CTSL1
ALFKSIPP substrate motif sequence 384 CTSL1 ALFKSKPP substrate
motif sequence 385 CTSL1 ALFKSAPP substrate motif sequence 386
CTSL1 ALFKSQPP substrate motif sequence 387 CTSL1 ALFKSPPP
substrate motif sequence 388 CTSL1 ALFKSEPP substrate motif
sequence 389 CTSL1 ALFKSGPP substrate motif sequence 198 CTSL1
ALFKSSFP substrate motif sequence 390 CTSL1 ALFKSSLP substrate
motif sequence 391 CTSL1 substrate ALFKSSGP motif sequence 392
CTSL1 ALFKSSSP substrate motif sequence 393 CTSL1 ALFKSSVP
substrate motif sequence 394 CTSL1 ALFKSSHP substrate motif
sequence 395 CTSL1 ALFKSSAP substrate motif sequence 396 CTSL1
ALFKSSNP substrate motif sequence 397 CTSL1 ALFKSSKP substrate
motif sequence 398 CTSL1 ALFKSSEP substrate motif sequence 399
CTSL1 ALFKSSPF substrate motif sequence 400 CTSL1 ALFKSSPH
substrate motif sequence 401 CTSL1 ALFKSSPG substrate motif
sequence 402 CTSL1 ALFKSSPA substrate motif sequence 403 CTSL1
ALFKSSPS substrate motif sequence 404 CTSL1 ALFKSSPV substrate
motif sequence 405 CTSL1 ALFKSSPQ substrate motif sequence 406
CTSL1 ALFKSSPK substrate motif sequence 407 CTSL1 ALFKSSPL
substrate motif sequence 408 CTSL1 ALFKSSPD substrate motif
sequence indicates data missing or illegible when filed
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210115102A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20210115102A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References