Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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INTERFERON VARIANTS WITH IMPROVED PROPERTIES
[001] This application claims benefit of priority under 35 USC 119(e)(1) to
USSN: 60/415,541, filed
October 1, 2002; USSN: 60/477,246, filed June 10, 2003, and 60/489,725, filed
July 24, 2003,
and 101676,705, filed September 30, 2003, all hereby incorporated by reference
in their entirety.
[002] FIELD OF THE INVENTION
[003] The invention relates to variants of type I interferons with improved
properties, and to
methods of making and to methods and compositions utilizing these variants.
the use of a variety
of computational methods, including Protein Design Automation~ (PDA~)
technology, to identify
interferon variants with improved properties, generate computationally
prescreened secondary
libraries of proteins, and to methods of making and methods and compositions
utilizing soluble
variants and the libraries.
[004] BACKGROUND OF THE INVENTION
[005] Interferons (IFNs) are a well-known family of cytokines secreted by a
large variety of
eukaryotic cells. A possessing a range of biological activities is associated
with interferons
including antiviral, anti-proliferative, neoplastic and immunoregulatory
immunomodulatory
activities. Interferons have demonstrated utility in the treatment of a
variety of diseases, and are
in widespread use for the treatment of multiple sclerosis and viral hepatitis;
the most common
therapeutic applications are currently treatment of hepatitis C and multiple
sclerosis.
[006] lnterferons include a number of related proteins, such as interferon-
alpha (IFN-a), interferon-
beta (IFN-[3), interferon-gamma (IFN-y) interferon-kappa (IFN-K, also known as
interferon-epsilon
or IFN-~), interferon-tau (IFN-r), and interferon-omega (IFN-w). These
interferon proteins are
produced in a variety of cell types: IFN-a (leukocytes), IFN-(3 (fibroblasts),
IFN-g (lymphocytes),
IFN-s or K (keratinocytes), IFN-c~ (leukocytes) and IFN-r (trophoblasts). IFN-
a, IFN-(3, IFN-~ or ~e,
IFN-w, and 1FN-t are classified as type I interferons, while IFN-y is
classified as a type fl
interferon. Interferon alpha is encoded by a multi-gene family, while the
other interferons appear
to each be coded by a single gene in the human genome. Furthermore, there is
some allelic
variation in interferon sequences among different members of the human
population.
[007] Type-I interferons all appear to bind a common receptor, type I IFN-R,
composed of IFNAR1
and IFNAR2 subunits. The exact binding mode and downstream signal transduction
cascades
differ somewhat among the type I interferons. However, in general, the
JAK/STAT signal
transduction pathway is activated following binding of interferon to the
interferon receptor. STAT
transcription factors then translocate to the nucleus, leading to the
expression of a number of
proteins with antiviral, antineoplastic, and immunomodulatory activities.
[008] The properties of naturally occurring type f interferon proteins are not
optimal for therapeutic
use. Type I interferons induce injection site reactions and a number of other
side effects. They
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are highly immunogenic, eliciting neutralizing and non-neutralizing antibodies
in a significant
fraction of patients. Ilnterferons are poorly absorbed from the subcutaneous
injection site and
have short serum half- lives. Finally, type I interferons do not express
solubly in prokaryotic
hosts, thus necessitating more costly and difficult refolding or mammalian
expression protocols.
[009] The present invention is directed to identification of interferon
proteins with improved
properties. A number of groups have generated modified interFerons with
improved properties;
the references below are all expressly incorporated by reference in their
entirety.
[010] Cysteine-depleted variants have been generated to minimize formation of
unwanted inter- or
intra-molecular disulfide bonds (US 4,518,584;, US 4,588,585;, US 4,959,314).
Methionine-
depleted variants have been generated to minimize susceptibility to oxidation
(EPO 260350).
[011] Interferons.with modified activity have been generated (US 6,514,729; US
4,738,844; US
4,738,845; US 4,753,795; US 4,766,106; WO 00/78266). US Patent Nos. 5,545,723
and
6,127,332 disclose substitution mutants of interferon beta at position 101.
Chimeric interferons
comprising sequences from one or more interferons have been made (Chang et.
al. Nature
Biotech. 17: 793-797 (1999), US 4,758,428; US 4,885,166; US 5,382,657; US
5,738,846).
Substitution mutations to interferon beta at positions 49 and 51 have also
been described (US
6,531,122).
[012] Interferon beta variants with enhanced stability have been claimed, in
which the hydrophobic
core was optimized using rational design methods (WO 00/68387). Alternate
formulations that
promote interferon stability or solubility have also been disclosed (US
4,675,483; US 5,730,969;
US 5,766,582; WO 02/38170).
[013] Interferon beta muteins with enhanced solubility have been claimed, in
which several leucine
and phenylalanine residues are replaced with serine, threonine, or tyrosine
residues (WO
98148018). However, it is not clear whether any of the variants claimed are
sufficiently soluble,
stable, and active to constitute improved variants.
[014] Interferon alpha and interferon beta variants with reduced
immunogenicity have been claimed
(See WO 02/085941 and WO 02/074783). However, due to the large number of
variants
disclosed and the apparent lack of consideration of the structural and
functional effects of the
introduced mutations, one skilled in the art faces a problem in identifying a
variant that would be a
functional, less immunogenic interferon variant suitable for administration to
patients may be
difficult.
[015] Immunogenicity is a major limitation of current interferon (including
interferon beta)
therapeutics. Although immune responses are typically most severe for non-
human proteins,
even therapeutics based on human proteins, such as interferon beta, are often
observed to be
immunogenic. Immunogenicity is a complex series of responses to a substance
that is perceived
as foreign and may include production of neutralizing and non-neutralizing
antibodies, formation
of immune complexes, complement activation, mast cell activation,
inflammation, and
anaphylaxis.
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[016] Several factors can contribute to protein immunogenicity, including but
not limited to the
protein sequence, the route and frequency of administration, and the patient
population.
Aggregation has been linked to the immunogenicity of a related protein
therapeutic, interferon
alpha [Braun et. at. Pharm. Res. 1997 14: 1472-1478]. Another study suggests
that the presence
of DR15 MHC alleles increases susceptibility to neutralizing antibody
formation; interestingly, the
same alleles also confer susceptibility to multiple sclerosis [Stickler et.
al. Genes Immun. 2004 5:
1-7].
[017] As aggregation may contribute to the immunogenicity of interferons
(particularly interferon
beta), variants engineered for improved solubility may also possess reduced
immunogenicity.
Cysteine-depleted variants have been generated to minimize formation of
unwanted inter- or
intra-molecular disulfide bonds (US 4,518,584; US 4,588,585; US 4,959,314);
such variants show
a reduced propensity for aggregation. Interferon beta variants with enhanced
stability have been
claimed, in which the hydrophobic core was optimized using rational design
methods (WO
00/68387); in some cases solubility may be enhanced by improvements in
stability. Alternate
formulations that promote interferon stability and solubility have also been
disclosed (US
4,675,483; US 5,730,969; US 5,766,582; WO 02/38170). Interferon beta muteins
with enhanced
solubility have been claimed, in which several leucine and phenylalanine
residues are replaced
with serine, threonine, or tyrosine residues (WO 98/48018). However, it is not
clear whether any
of the variants claimed are sufficiently soluble, stable, and active to
constitute improved variants.
[018] Interferons have been modified by the addition of polyethylene glycol
("PEG") (see US
4,917,888; US 5,382,657; WO 99/55377; WO 02/09766; WO 02/3114). PEG addition
can
improve serum half-life and solubility. In some cases, PEGylation has been
observed to reduce
the fraction of patients who raise neutralizing antibodies by sterically
blocking access to antibody
agretopes (see for example, Hershfield et. al. PNAS 1991 88:7185-7189 (1991);
Baifon. et ai.
Bioconjug. Chem. 12: 195-202(2001); He et al. Life Sci. 65: 355-368 (1999)).
[019] Interferon beta variants have also been generated that are predicted to
bind class II MHC
alleles with decreased affinity relative to the wild type protein; in both
examples primarily alanine
mutations were used to confer decreased binding [WO 02/074783; Stickler
supra].
[020] Several methods have been developed to modulate the immunogenicity of
proteins; a
preferred approach is to disrupt T-cell activation by removing MHC-binding
agretopes. This
approach is more tractable than evading T-cell receptor or antibody binding,
as the diversity of
MHC molecules comprises only 103 alleles, while the antibody repertoire is
estimated to be
approximately 108 and the T-cell receptor repertoire is larger still. By
identifying and removing or
modifying class II MHC-binding peptides within a protein sequence, the
molecuVar basis of
immunogenicity can be evaded. The elimination of such agretopes for the
purpose of generating
less immunogenic proteins has been disclosed previously; see for example WO
98/52976, WO
02/079232, and WO 00/3317.
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[021] While a large number of mutations in MHC-binding agretopes may be
identified that are
predicted to confer reduced immunogenicity, most of these amino acid
substitutions will be
energetically unfavorable. As a result, the vast majority of the reduced
immunogenicity
sequences identified using the methods described above will be incompatible
with the structure
and/or function of the protein. In order for MHC agretope removal to be a
viable approach for
reducing immunogenicity, it is crucial that simultaneous efforts are made to
maintain a protein's
structure, stability, and biological activity.
[022] Immunogenicity may limit the efficacy and safety of interferon
therapeutics in multiple ways.
Therapeutic efficacy may be reduced directly by the formation of neutralizing
antibodies. Efficacy
may also be reduced indirectly, as binding to either neutralizing or non-
neutralizing antibodies
may alter serum half-life. Unwanted immune responses may take the form of
injection site
reactions, including but not limited to delayed-type hypersensitivity
reactions. It is also possible
that anti-interferon beta neutralizing antibodies may cross-react with
endogenous interferon beta
and block its function.
[023] There remains a need for novel interferon proteins having reduced
immunogenicity. Variants
of interferon with reduced immunogenicity could find use in the treatment of a
number of
interferon responsive conditions.
[024] As a result, there exists a need for the development and discovery of
interferon proteins with
improved properties, including but not limited to increased efficacy,
decreased side effects,
decreased immunogenicity, increased solubility, and enhanced soluble
prokaryotic expression.
Improved interferon therapeutics could may be useful for the treatment of a
variety of diseases
and conditions, including autoimmune diseases, viral infections, and ,
inflammatory diseases, cell
proliferation diseases, bacterial infections, enhancing fertility, and cancer,
among others.and
transplant rejection. In addition, interferons may be used to promote the
establishment of
pregnancy in certain mammals.
[025] SUMMARY OF THE INVENTION
[026] The present invention is related to variants of type I human interferons
with at least one
improved property, including but not limited to increased solubility (for
example by an inability to
multimerize, particularly upon administration), increased specific activity,
and modified
immunogenicity.
[027] In one aspect, the invention provides variant type 1 interferon beta
(IFN-[3) proteins exhibiting
modified immunogenicity as compared to a wild type (IFN-[3). A number of wild-
type interferons of
use in the present invention are shown in SEQ ID NOS: 1-18. Modified
immunogenicity includes
reduced immunogenicity, for example where the variant protein demonstrates
reduced binding to
at least one human class II MHC allele, or when the variant exhibits improved
solubility.
Increased solubility can be obtained by substituting at least one solvent-
exposed hydrophobic
residue. Modified immunogenicity also includes increased immunogenicity.
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[028] The invention also provides variant interferons that exhibit modified
immunogenicity while
substantially maintaining interferon biological activity, including, but not
limited to,
immunomodulatory activity, antiviral activity and antineoplastic activities.
[029] In an additional aspect, the invention provides IFN-[i variants
exhibiting modified
immunogenicity comprising at least one modification at a position selected
from the group
consisting of 1,2, 3, 4, 5, 6, 8, 9, 12, 15, 16, 22, 28, 30, 32, 36, 42, 43,
46, 47, 48, 49, 51, 92, 93,
96, 100, 101, 104, 111, 113, 116, 117, 120, 121, 124,130, 148, and 155. In
some cases, the
modifications to residues 5, 8, 15, 47, 111, 116, and 120 are substitution
mutations preferably
selected from the group consisting of alanine, arginine, aspartic acid,
asparagine, glutamic acid,
glutamine, glycine, histidine, and lysine. Similarly, the modifications to
residues 22, 28, 30, 32,
36, 92, 130, 148, and 155 are preferably selected from the group including
alanine, arginine,
aspartic acid, asparagine, glutamic acid, glutamine, glycine, histidine,
serine, threonine and
lysine. These variants are particularly preferred for increased solubility
leading to reduced
immunogenicity.
[030] Partioularly preferred variant IFN-~ proteins comprise at least one
modification selected from
the group consisting of:LSA, LSD, LSE, LSK, LSN, LSQ, LSR, LSS, LST, F8A, FBD,
F8E, F8K,
FBN, F80, FBR, F8S, S12E, S12K, S12Q, S12R, W22E, L280, Y30H, L32A, E43K,
E43R, L47K,
Y92Q, E104R, E104K, E104H, E104Q, E104A, F111 N, R113D, R113E, R113Q, R113A,
L116D,
L116E, L116N, L116Q, L116R, M117R, L120D, L120R, L130R, V148A, and Y155S.
Particularly
preferred variants have the sequences outlined in SEQ ID N0;19 (variant 2),
SEQ ID N0:20
(variant 7), SEQ ID N0:21 (variant 15), SEQ ID N0:22 (variant 23), SEQ ID
N0:23 (variant 36),
SEQ ID N0:24 (variant 39) and SEO ID N0:25 (variant 64).
[031] In a further aspect, variant IFN-a proteins with reduced immunogenicity
exhibit reduced
binding at least one human class Il MHC allele. In this embodiment (as well as
for other modified
immunogenicity variants) at least one amino acid modification is made in at
least one of the
following positions: agretope 1: residues 3-11; agretope 2: residues 5-13;
agretope 3: residues 8-
16; agretope 4: residues 9-17; agretope 5: residues 15-23; agretope 6:
residues 22-30; agretope
7: residues 30-38; agretope 8: residues 36-44; agretope 9: residues 47-55;
agretope 10: residues
57-65; agretope 11: residues 60-68; agretope 12: residues 63-71; agretope 13:
residues 70-78;
agretope 14: residues 79-87; agretope 15: residues 95-103; agretope 16:
residues 122-130;
agretope 17: residues 125-133; agretope 18: residues 129-137; agretope 19:
residues 130-138;
agretope 20: residues 143-151; agretope 21: residues 145-153; agretope 22:
residues 146-154;
agretope 23: residues 148-156; agretope 24: residues 151-159; agretope 25:
residues 154-162;
agretope 26: residues 156-164; agretope 27: residues 157-165. Preferred
variants also include
amino acid modifications in 2 or more of these agretopes.
[032] Preferred variants in agretope 6 include SEQ ID NOS:**1-14. Preferred
variants in agretope 8
include SEQ lD NOS:**15-45. Preferred variants in agretope 11 include SEQ ID
NOS:**46-54.
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Preferred variants in agretope 20 include SEQ ID NOS:**55-65. Preferred
variants in agretope 24
include SEQ ID NOS:**66-100. A preferred variants in agretope 25 includes SEQ
ID NO: **101.
[033] In a further aspect, the invention provides variant type 1 interferon
alpha (IFN-a) proteins
exhibiting modified immunogenicity as compared to a wild type IFN-a comprising
at least one
modification at a position selected from the group consisting of 16, 27, 30,
89, 100, 110, 111, 117,
128, and 161. In a preferred embodiment, the modifications are substitution
mutations selected
from the group consisting of alanine, arginine, aspartic acid, asparagine,
glutamic acid, glutamine,
glycine, histidine, serine, threonine, and lysine. In a preferred embodiment,
the variants exhibit
enhanced solubility.
[034] In an additional aspect, the invention provides variant type 1
interferon kappa (IFN-x) proteins
exhibiting modified immunogenicity as compared to a wild type IFN-x comprising
at least one
modification at a position selected from the group consisting of 1, 5, 8, 15,
18, 28, 30, 33, 37, 46,
48, 52, 65, 68, 76, 79, 89, 97, 112, 115, 120, 127, 133, 151, 161, 168, and
171. Preferred
substitutions are selected from the group consisting of alanine, arginine,
aspartic acid,
asparagine, glutamic acid, glutamine, glycine, histidine, serine, threonine,
and lysine. In a
preferred embodiment, the variants exhibit enhanced solubility.
[035] Particularly preferred IFN-x variants comprise at least one modification
selected from the
group consisting of LSQ, V8N, W15R, F28Q, F28S, V30R, 137N, Y48Q, M52N, M52Q,
F76S,
Y78A, 189T, Y97D, M112T, M115G, L133Q, V161A, C166A, Y168S, and Y171T.
[036] In a further aspect, the invention provides recombinant nucleic acids
encoding the variant
proteins, expression vectors containing the variant nucleic acids, host cells
comprising the variant
nucleic acids and/or expression vectors, and methods for producing the variant
proteins.
[037] In an additional aspect, the invention provides treating an interferon
responsive disorder by
administering to a patient a variant protein, usually with a pharmaceutical
carrier, in a
therapeutically effective amount.
[038] In a further aspect, the invention provides methods for modulating
immunogenicity
(particularly reducing immunogenicity) of interferons (particularly IFN-(3) by
altering MHC Class II
epitopes.
[039] BRIEF DESCRIPTION OF THE DRAWINGS
[040] Figure 1 shows amino acid sequences for human type I interferons and
some preferred
variants SEO ID NOS:1-30.
[041] Figure 2 shows a sequence alignment of human interferon-alpha subtypes,
SEQ ID NOs:31-
43.
[042] Figure 3 shows the sequence alignment of IFN-a2a (11TF), IFN-b (1AU1),
IFN-k (IFNI<), and
IFN-t (1 B5L) (SEQ ID NOs:44-47) that was used to construct the homology model
of interferon-
kappa.
[043] Figure 4 shows ISRE assay dose-response curves for interferon beta
variants.
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[044] Figure 5 shows a dot blot assay used to test for soluble expression of
interferon-kappa
variants. G12 and H12 are positive controls, whereas E12 and F12 are soluble
extracts from cells
expressing WT interferon-kappa (negative control). Wells C5, C8, D4, E5 and F2
represent
clones expressing soluble interferon-kappa variants.
[045] Figure 6 shows a dot blot assay used to test for soluble expression of
interferon-kappa
variants. G12 and H12 are positive controls, whereas E12 and F12 are soluble
extracts from cells
expressing WT interferon-kappa (negative control). Most of the putative
soluble clones test
positive (soluble expression) upon reexpression.
[046] Figure 7 shows a western blot of solubly expressed interferon kappa
variants. The arrow
indicates the expected position of interferon-kappa protein. Lanes 2 and 3 are
total soluble
fraction from WT interferon-kappa expressing cells, respectively. Lanes 4-15
are soluble fractions
from the lysates of different variants. exposed hydrophobic residues in
interferon-kappa and
suitable polar replacements as determined by PDA~ technology calculations.
[047] Figure 8 shows exposed hydrophobic residues in interferon-kappa and
suitable polar
replacements according to sequence alignment data.
[048) Figure 9 depicts a method for engineering less immunogenic interferon
derivatives,
particularly IFN-[i.
[049] Figure 10 depicts a schematic representation of a method for in vitro
testing of the
immunogenicity of interferon peptides or proteins with IW technology,
particularly IFN-[3.
[050] Figure 11 graphically shows decreased aggregation of SEQ ID NO: 20
variant as compared
to BetaSeron~ (Schering AG/Berlex) at pH 3.0 over time (9h at 37°C).
The top graph shows
XENP806, the middle graph shows BetaSeron and the bottom graph shows wild type
IFNB.
[051] Figure 12 graphically shows decreased aggregation of SEQ ID NO: 20
variant as compared
to BetaSeron~ (Schering AG/Berlex) at 6 pH 6.0 ~ 1.0 over time (9h at
37°C). The top graph
shows XENP806, the middle graph shows BetaSeron and the bottom graph shows
wild type
IFNB.
[052] DETAILED DESCRIPTION OF THE INVENTION
[053] Definitions: By "control sequences" and grammatical equivalents herein
is meant nucleic
acid sequences necessary for the expression of an operably linked coding
sequence in a
particular host organism. The control sequences that are suitable for
prokaryotes, for example,
include a promoter, optionally an operator sequence, and a ribosome binding
site. Eukaryotic
cells are known to utilize promoters, polyadenylation signals, and enhancers.
[054) Nucleic acids are "operably linked" when it is placed into a functional
relationship with another
nucleic acid sequence. For example, DNA for a presequence or secretory leader
is operably
linked to DNA for a polypeptide if it is expressed as a preprotein that
participates in the secretion
of the polypeptide; a promoter or enhancer is operably linked to a coding
sequence if it affects the
transcription of the sequence; or a ribosome binding site is operably linked
to a coding sequence
if it is positioned so as to facilitate translation. Generally, "operably
linked" means that the DNA
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sequences being linked are contiguous, and, in the case of a secretory leader,
contiguous and in
reading frame. However, elements such as enhancers do not have to be
contiguous.
[055] By "immunogenicity" and grammatical equivalents herein is meant the
ability of a protein to
elicit an immune response, including but not limited to production of
neutralizing and non-
neutralizing antibodies, formation of immune complexes, complement activation,
mast cell
activation, inflammation, and anaphylaxis.
[056] By "reduced immunogenicity" and grammatical equivalents herein is meant
a decreased
ability to activate the immune system, when compared to the wild type protein.
For example, an
IFN variant protein can be said to have "reduced immunogenicity" if it elicits
neutralizing or non-
neutralizing antibodies in lower titer or in fewer patients than wild type
IFN. In a preferred
embodiment, the amount of neutralizing antibodies is decreased by at least 5
%, with at least 50
or 90 % decreases being especially preferred. Therefore, if a wild type
produces an immune
response in 10 % of patients, a variant with reduced immunogenicity would
produce an immune
response in not more than 9.5 % of patients, with less than 5
°I° or less than 1 % being especially
preferred. An IFN variant protein is also said to have "reduced
immunogenicity" if it shows
decreased binding to one or more MHC alleles or if it induces T-cell
activation in a decreased
fraction of patients relative to wild type IFN. In a preferred embodiment, the
probability of T-cell
activation is decreased by at least 5 %, with at least 50 % or 90 % decreases
being especially
preferred.
[057] By "interferon aggregates" and grammatical equivalents herein is meant
protein-protein
complexes comprising at least one interferon molecule (and often multiple IFN
molecules) and
possessing less bioactivity as compared to the wild-type or parent molecule,
including
immunomodulatory, antiviral, or antineoplastic activity than the corresponding
monomeric
interferon molecule. Interferon aggregates include interferon dimers,
interferon-albumin dimers,
higher order species, etc.
[058] By "interferon-responsive disorders" and grammatical equivalents herein
is meant diseases,
disorders, and conditions that can benefit from treatment with a type I
interferon. Examples of
interferon-responsive disorders include, but are not limited to, autoimmune
diseases (e.g. multiple
sclerosis, diabetes mellitus, Vupus erythematosus, Crohn's disease, rheumatoid
arthritis,
stomatitis, asthma, allergies and psoriasis), infectious diseases including
viral infections (hepatitis
C, papilloma viruses, hepatitis B, herpes viruses, viral encephalitis,
cytomegalovirus, and
rhinovirus), and cell proliferation diseases including cancer (e.g.
osteosarcoma, basal cell
carcinoma, cervical dysplasia, glioma, acute myeloid leukemia, multiple
myeloma, chronic
lymphocytic leukemia, Kaposi's sarcoma, chronic myelogenous leukemia, renal-
cell carcinoma,
ovarian cancers, hairy-cell leukemia, and Hodgkin's disease). Interferons may
also be used to
promote the establishment of pregnancy in certain mammals, and to reduce
transplant rejection.
[059] By "library" as used herein is meant a collection of protein sequences
that are likely to take on
a particular fold or have particular protein properties. The library
preferably comprises a set of
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sequences resulting from computation, which may include energy calculations or
statistical or
knowledge based approaches. Libraries that range in size from about 50 to
about 10'3
sequences are preferred. Libraries are generally generated experimentally and
analyzed for the
presence of members possessing desired protein properties.
[060] By "modification" and grammatical equivalents is meant insertions,
deletions, or substitutions
to a protein or nucleic acid sequence, with substitutions being preferred.
[061] By "naturally occurring" or "wild -type" or "wt" and grammatical
equivalents thereof herein is
meant an amino acid sequence or a nucleotide sequence that is found in nature
and includes
allelic variations. In a preferred embodiment, the wild-type sequence is the
most prevalent human
sequence. In addition, in most cases the modifications of amino acids of the
invention are in
relation to a wild-type "starting" molecule. However, as will be appreciated
by those in the art,
other starting molecules, generally referred to herein as "parents", are
already non-naturally
occurring IFN derivatives that are further modified using the methods of the
invention. While
human sequences are generally preferred, in some embodiments the wild type fFN
proteins may
be from any number of organisms, include, but are not limited to, rodents
(rats, mice, hamsters,
guinea pigs, etc.), primates, and farm animals (including sheep, goats, pigs,
cows, horses, etc).
[062] By "nucleic acid" and grammatical equivalents herein is meant DNA, RNA,
or molecules,
which contain both deoxy- and ribonucleotides. Nucleic acids include genomic
DNA, cDNA and
oligonucleotides including sense and anti-sense nucleic acids. Nucleic acids
may also contain
modifications, such as modifications in the ribose-phosphate backbone that
confer increased
stability and half-life.
[063] A "patient" for the purposes of the present invention includes both
humans and other animals,
particularly mammals, and organisms. Thus the methods are applicable to both
human therapy
and veterinary applications. In the preferred embodiment the patient is a
mammal, and in the
most preferred embodiment the patient is human.
[064] "Pharmaceutically acceptable carrier" or grammatical equivalents
includes pharmaceutically
acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical
Sciences 16th edition,
Osol, A. Ed.,1980), in the form of lyophilized formulations, aqueous
solutions, etc. Acceptable
carriers, excipients, or stabilizers are nontoxic to recipients at the dosages
and concentrations
employed, and include buffers such as phosphate, citrate, acetate, and other
organic acids;
antioxidants including ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride,
benzethonium chloride; phenol, butyl orbenzyl alcohol; alkyl parabens such as
methyl or propyl
paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight
(less than about 10 residues) polypeptides; proteins, such as serum albumin,
gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino
acids such as glycine,
glutamine, asparagine, histidine, arginine, or lysine; monosaccharides,
disaccharides, and other
carbohydrates including glucose, mannose, or dextrins; chelating agents such
as EDTA; sugars
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such as sucrose, mannitol, trehalose or sorbitol; sweeteners and other
flavoring agents; fillers
such as microcrystalline cellulose, lactose, corn and other starches; binding
agents; additives;
coloring agents; salt-forming counter-ions such as sodium; metal complexes
(e.g. Zn-protein
complexes); and/or non-ionic surfactants such as TWEENT"", PLURON1CS1"' or
polyethylene
glycol (PEG). In a preferred embodiment, the pharmaceutical composition that
comprises the
compositions of the present invention is in a water-soluble form, such as
being present as
pharmaceutically acceptable salts, which is meant to include both acid and
base addition salts.
Acceptable carriers include, but are not limited to pharmaceutically
acceptable acid and base
salts. "Pharmaceutically acceptable acid addition salt" refers to those salts
that retain the
biological effectiveness of the free bases and that are not biologically or
otherwise undesirable,
formed with inorganic acids such as hydrochloric acid, hydrobromic acid,
sulfuric acid, nitric acid,
phosphoric acid and the like, and organic acids such as acetic acid, propionic
acid, glycolic acid,
pyruvic acid, oxalic acid, malefic acid, malonic acid, succinic acid, fumaric
acid, tartaric acid, citric
acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid,
ethanesulfonic acid, p-
toluenesulfonic acid, salicylic acid and the like. "Pharmaceutically
acceptable base addition salts"
include those derived from inorganic bases such as sodium, potassium, lithium,
ammonium,
calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the
like. Particularly
preferred are the ammonium, potassium, sodium, calcium, and magnesium salts.
Salts derived
from pharmaceutically acceptable organic non-toxic bases include salts of
primary, secondary,
and tertiary amines, substituted amines including naturally occurring
substituted amines, cyclic
amines and basic ion exchange resins, such as isopropylamine, trimethylamine,
diethylamine,
triethylamine, tripropylamine, and ethanolamine.
[065] The following residues are defined herein to be "polar" residues:
aspartic acid, asparagine,
glutamic acid, gfutamine, lysine, arginine, histidine, serine, and threonine.
fn some embodiments,
as is further outlined below, these residues are particularly useful to
replace hydrophobic residues
on the surface of proteins to avoid aggregation.
[066] By "protein" herein is meant a molecule comprising at least two
covalently attached amino
acids, which includes proteins, polypeptides, oligopeptides and peptides. The
protein may be
made up of naturally occurring amino acids and peptide bonds, or synthetic
peptidomimetic
structures, i.e., "analogs " such as peptoids ([see Simon et al., Proc. Natl.
Acad. Sci. U.S.A.
89(20:9367-71 (1992))]. For example, homo-phenylalanine, citrulline, and
noreleucine are
considered amino acids for the purposes of the invention. "Amino acid" also
includes amino acid
residues such as proline and hydroxyproline. B, and both D- and L- amino acids
may be utilized.
j067] By "protein properties" herein is meant biological, chemical, and
physical properties including
but not limited to enzymatic activity, specificity (including substrate
specificity, kinetic association
and dissociation rates, reaction mechanism, and pH profile), stability
(including thermal stability,
stability as a function of pH or solution conditions, resistance or
susceptibility to ubiquitination or
proteolytic degradation), solubility, aggregation, structural integrity,
crystallizability, binding affinity
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and specificity (to one or more molecules including proteins, nucleic acids,
polysaccharides,
lipids, and small molecules), oligomerization state, dynamic properties
(including conformational
changes, allostery, correlated motions, flexibility, rigidity, folding rate),
subcellular localization,
ability to be secreted, ability to be displayed on the surface of a cell,
posttranslational modification
(including N- or C-linked glycosylation, lipidation, and phosphorylation),
ammenability to synthetic
modification (including PEGylation, attachment to other molecules or
surfaces), and ability to
induce altered phenotype or changed physiology (including cytotoxic activity,
immunogenicity,
toxicity, ability to signal, ability to stimulate or inhibit cell
proliferation, ability to induce apoptosis,
and ability to treat disease). When a biological activity is the property,
modulation in this context
includes both an increase or a decrease in activity.
[068] By "solubility" and grammatical equivalents herein is meant the maximum
possible
concentration of monomeric protein in a solution of specified condition. By
"soluble expression"
and grammatical equivalents herein is meant that the protein is able to be
produced at least
partially in soluble form rather than in inclusion bodies when expressed in a
prokaryotic host. It is
preferred that at least 1 mg soluble protein is produced per 100 mL culture,
with at least 10 mg or
100 mg being especially preferred. By "improved solubility" and grammatical
equivalents herein is
meant an increase in the maximum possible concentration of monomeric protein
in solution.
Preferred solutions in this context include the pharmaceutically acceptable
carrier, as well as the
site of administration. For example, if the naturally occurring protein can be
concentrated to 1 mM
and the variant can be concentrated to 5 mM under the same solution
conditions, the variant can
be said to have improved solubility. In a preferred embodiment, solubility is
increased by at least
a factor of 2, with increases of at least 5x or 10x being especially
preferred. As will be
appreciated by those skilled in the art, solubility is a function of solution
conditions. For the
purposes of this invention, solubility should be assessed under solution
conditions that are
pharmaceutically acceptable. Specifically, pH should be between 6.0 and 8.0,
salt concentration
should be between 50 and 250 mM. Additional buffer components such as
excipients may also
be included, although it is preferred that albumin is not required.
[069] By "therapeutically effective dose" herein is meant a dose that produces
the effects for which
it is administered. The exact dose will depend on the purpose of the
treatment, and will be
ascertainable by one skilled in the art using known techniques. In a preferred
embodiment,
dosages of about 5 Ng/kg are used, administered either intravenously or
subcutaneously. As is
known in the art, adjustments for variant IFN protein degradation, systemic
versus localized
delivery, and rate of new protease synthesis, as well as the age, body weight,
general health, sex,
diet, time of administration, drug interaction and the severity of the
condition may be necessary,
and will be ascertainable with routine experimentation by those skilled in the
art.
[070] By "treatment" herein is meant to include therapeutic treatment, as well
as prophylactic, or
suppressive measures for the disease or disorder. Thus, for example,
successful administration
of a variant IFN protein prior to onset of the disease may result in treatment
of the disease. As
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another example, successful administration of a variant IFN protein after
clinical manifestation of
the disease to combat the symptoms of the disease comprises "treatment" of the
disease.
"Treatment" also encompasses administration of a variant IFN protein after the
appearance of the
disease in order to ameliorate or eradicate the disease. Successful
administration of an agent
after onset and after clinical symptoms have developed, with possible
abatement of clinical
symptoms and perhaps amelioration of the disease, further comprises
"treatment" of the disease.
[071] By "variant IFN interferon nucleic acids" and grammatical equivalents
herein is meant nucleic
acids that encode variant IFN interferon proteins. Due to the degeneracy of
the genetic code, an
extremely large number of nucleic acids may be made, all of which encode the
variant IFN
proteins of the present invention, by simply modifying the sequence of one or
more codons in a
way which that does not change the amino acid sequence of the variant IFN.
[072] By "variant IFN proteins" or "non-naturally occurring IFN interferon
proteins" and grammatical
equivalents thereof herein is meant non-naturally occurring IFN proteins which
differ from the wild
type IFN protein by at least one (1) amino acid insertion, deletion, or
substitution. 1t should be
noted that unless otherwise stated, all positional numbering of variant IFN
proteins and variant
IFN nucleic acids is based on the wild-type sequences. IFN variants are
characterized by the
predetermined nature of the variation, a feature that sets them apart from
naturally occurring
allelic or interspecies variation of the IFN protein sequence. The IFN
variants must typically either
exhibit the same qualitative biological activity as the naturally occurring
IFN or have been
specifically engineered to have alternate biological properties. Preferably
the IFN variants of the
present invention retain at least 50 % of at least one wild type interferon
activity, as determined
using the ISRE assay described below. Variants that retain at least 75 % or 90
% of wild type
activity are more preferred, and variants that are more active than wild type
are especially
preferred. The variant interferon IFN proteins may contain insertions,
deletions, and/or
substitutions at the N-terminus, C-terminus, or internally. In a preferred
embodiment, variant IFN
proteins have at least 1 residue that differs from the most similar human IFN
sequence, with at
least 2, 3, 4, or 5 different residues being more preferred. Variant IFN
proteins may contain
further modifications, for instance mutations that alter solubility or
additional protein properties
such as stability or immunogenicity or which enable or prevent
posttranslational modifications
such as PEGylation or glycosylation. Variant IFN interferon proteins may be
subjected to co- or
post-translational modifications, including but not limited to synthetic
derivatization of one or more
side chains or termini, glycosylation, PEGylation, circular permutation,
cyclization, fusion to
proteins or protein domains, and addition of peptide tags or labels.
[073] By "9-mer peptide frame" and grammatical equivalents herein is meant a
linear sequence of
nine amino acids that is located in a protein of interest. 9-mer frames may be
analyzed for their
propensity to bind one or more class II MHC alleles as is further described
below.
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[074] By "allele" and grammatical equivalents herein is meant an alternative
form of a gene.
Specifically, in the context of class II MHC molecules, alleles comprise all
naturally occurring
sequence variants of DRA, DRB1, DRB3/4/5, DQA1, DQB1, DPA1, and DPB1
molecules.
[075] By "hit" and grammatical equivalents herein is meant, in the context of
the matrix method, that
a given peptide is predicted to bind to a given class II MHC allele. In a
preferred embodiment, a
hit is defined to be a peptide with binding affinity among the top 5%, or 3%,
or 1 % of binding
scores of random peptide sequences. In an alternate embodiment, a hit is
defined to be a peptide
with a binding affinity that exceeds some threshold, for instance a peptide
that is predicted to bind
an MHC allele with at least 100 ~M or 10 ~M or 1 pM affinity.
[076] By "matrix method" and grammatical equivalents thereof herein is meant a
method for
calculating peptide - MHC affinity in which a matrix is used that contains a
score for each
possible residue at each position in the peptide, interacting with a given MHC
allele. The binding
score for a given peptide - MHC interaction is obtained by summing the matrix
values for the
amino acids observed at each position in the peptide.
[077] By "MHC-binding agretopes" and grammatical equivalents herein is meant
peptides that are
capable of binding to one or more class II MHC alleles with appropriate
affinity to enable the
formation of MHC - peptide - T-cell receptor complexes and subsequent T-cell
activation. MHC-
binding agretopes are linear peptide sequences that comprise at least
approximately 9 residues.
[078] Naturally occurring interferons possess antiviral, antiproliferative,
and immunomodulatory
activities, making interferons valuable therapeutics. However, drugs based on
naturally occurring
interferons suffer from a number of liabilities, including a high incidence of
side effects and
immunogenicity.
[079] Accordingly, the present invention provides novel variants of type I
interferon proteins. These
interferon variants comprise one or more modifications that were selected to
improve biophysical
properties and clinical performance. In general, two main properties are
altered as described
herein. In a preferred embodiment, solubility is altered. Poor solubility
contributes to many of the
liabilities of current interferon therapeutics. Accordingly, a primary focus
of this invention is
interferon variants with improved solubility. Secondly, as further described
below, IFNs can be
engineered to alter the binding of the IFN to human class II MHC alleles,
particularly exhibiting
reduced binding.
[080] Although type I interferons are biologically active as monomers, they
are known to form
dimers and higher order species. These species can consist primarily of
interferon proteins, or
may also contain additional proteins such as human serum albumin. Non-
monomeric interferon
species exhibit significantly decreased activity, as even dimer formation
interferes with receptor
binding (Utsumi et. al. Biochim. Biophys. Acta 998: 167 (1989) and Runkel et.
al. Pharm. Res. 15:
641 (1998)). Interferon therapeutics are known to elicit neutralizing
antibodies in a substantial
fraction of patients (Antonelli et. al. Eur. Cytokine Netw. 10: 413 (1999)).
Poor solubility may be a
significant contributing factor to the immunogenicity of interferon
therapeutics, as aggregates are
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typically more immunogenic than soluble proteins (Speidel et. al. Eur. J.
Immunol. 27: 2391
(1997)), and aggregation has been demonstrated to increase the immunogenicity
of interferon-
alpha (Braun et. al. Pharm. Res. 14: 1472 (1997)). Furthermore, poor
solubility results in reduced
absorption following subcutaneous injection (Clodfelter et. al. Pharm. Res.
15: 254 (1998)).
[081] Protein solubility is essential for the discovery, manufacturing, and
clinical utilization of protein
therapeutics. Protein solubility comprises two features: soluble expression
and resistance to
aggregation. Poor solubility hinders structural and functional
characterization and drives up
production costs by necessitating refolding or mammalian expression. In
protein therapeutics,
aggregates exhibit decreased efficacy due to intrinsic inactivity of
aggregated species (see
Utsumi et. al. Biochim. Biophys. Acta 998: 167 (1989) and Runkel et. al.
Pharm. Res. 15: 641
(1998)) and reduced absorption following subcutaneous injection (Clodfelter
et. al. Pharm. Res.
15: 254 (1998)). To compensate, larger doses can be used, although this often
causes side
effects. Aggregates are also far more likely to raise an immune response than
soluble proteins
(Braun et. al. Pharm. Res. 14: 1472 (1997), Speidel et. al. Eur. J. Immunol.
27: 2391 (1997)).
[082] Poor solubility contributes to many of the liabilities of current
interferon therapeutics.
Although type I interferons are biologically active as monomers, they are
known to form dimers
and higher order species. Non-monomeric interferon species exhibit
significantly decreased
activity, as even dimer formation interferes with receptor binding (Utsumi,
Runkel supra).
Interferon therapeutics are known to elicit neutraiizing antibodies in a
substantial fraction of
patients (Antonelli et. al. Eur. Cytokine Netw. 10: 413 (1999)). Aggregates
are typically more
immunogenic than soluble proteins, and aggregation has been demonstrated to
increase the
immunogenicity of interferon-alpha (Braun, supra).
[083] A variety of strategies may be utilized to design IFN variants with
improved solubility. In a
preferred embodiment, one or more of the following strategies are used: 1)
reduce hydrophobicity
by substituting one or more solvent-exposed hydrophobic residues with suitable
polar residues,
2) increase polar character by substituting one or more neutral polar residues
with charged polar
residues, 3) decrease formation of intermolecular disulfide bonds by modifying
one or more non-
disulfide bonded cysteine residues (unpaired cysteines) with suitable non-
cysteine residues, and
4) reduce the occurrence of known unwanted protein-protein interactions by
modifying one or
more residues located at protein-protein interaction sites such as dimer
interfaces with alternate
residues with a decreased propensity to form protein-protein interactions. As
will be appreciated
by those in the art, several alternative strategies in combination are also
contemplated. For
example, modifications that, 5) increase protein stability, for example by one
or more
modifications that improve packing in the hydrophobic core, improve helix
capping and dipole
interactions, or remove unfavorable electrostatic interactions, and 6) modify
one or more residues
that can affect the isoelectric point of the protein (that is, aspartic acid,
glutamic acid, histidine,
lysine, arginine, tyrosine, and cysteine residues) to decrease the isoelectric
point of the protein
below physiological pH. Increasing the stability of a protein can improve
solubility by decreasing
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the population of partially folded or misfolded states. Additionally, protein
solubility is typically at a
minimum when the isoelectric point of the protein is equal to the pH of the
surrounding solution.
Modifications that perturb the isoelectric point of the protein away from the
pH of a relevant
environment, such as serum, may therefore serve to improve solubility.
Furthermore,
modifications that decrease the isoelectric point of a protein can improve
injection site absorption
(Holash et. al. PNAS 99: 11393-11398 (2002)).
[084] Type I interferons typically have one free cysteine residue and several
exposed hydrophobic
residues. These positions can be targeted for mutagenesis in order to improve
solubility.
Replacing exposed hydrophobic residues with appropriate polar residues can
also decrease the
number of MHC-binding epitopes. (See USSN: 10/039,170, filed January 8, 2003,
hereby
incorporated by reference). Since MHC binding is a key step in the initiation
of an immune
response, such mutations may decrease immunogenicity by multiple mechanisms.
[085] In two cases, type I interferons have been observed to crystallize as
dimers or higher order
species. While the dimeric structure is not biologically significantly less
active than the monomer,
it may represent an inactive species that is present in interferon
therapeutics. Accordingly,
residues located at or close to the protein-protein interfaces can be targeted
for modification.
[086] The present invention is directed to identification of interferon
proteins with improved
properties, see US 6, 188,965; US 6,269,312; US 6,403,312; US 6,433,145; USSN:
60/368,014;
PCTl00/13216; W09848018A1; WO00/05371 and W001/07608, all expressly
incorporated by
reference in their entirety.
[087] A number of methods can be used to identify modifications (that is,
insertion, deletion, or
substitution mutations) that will yield interferon variants with improved
solubility and while
retaining or improving other bioactivities, including but not limited to
immunomodulatory, antiviral,
or antineoplastic activity. These include, but are not limited to, sequence
profiling (Bowie and
f;
Eisenberg, Science 253(5016): 164-70, (1991 )), rotamer library selections
(Dahiyat and Mayo,
Protein Sci 5(5): 895-903 (1996); Dahiyat and Mayo, Science 278(5335): 82-7
(1997); Desjarlais
and Handel, Protein Science 4: 2006-2018 (1995); Harbury et al, PNAS USA
92(18): 8408-8412
(1995); Kono et al., Proteins: Structure, Function and Genetics 19: 244-255
(1994); Hellinga and
Richards, PNAS USA 91: 5803-5807 (1994)); and residue pair potentials (Jones,
Protein Science
3: 567-574, (1994)).
[088] In an especially preferred embodiment, rational design of improved IFN
variants is achieved
by using Protein Design Automation~ (PDA~) technology. (See U.S. Patent Nos.
6,188,965;
6,269,312; 6,403,312; W098/47089 and USSNs 09/058,459, 09/127,926, 60/104,612,
60/158,700, 09/419,351, 60/181,630, 60/186,904, 09/419,351, 09/782,004 and
091927,790,
60/347,772, and 10/218,102; and PCT/US01/218,102 and U.S.S.N. 10/218,102,
U.S.S.N.
60/345,805; U.S.S.N. 60/373,453 and U.S.S.N. 60/374,035, all references
expressly incorporated
herein in their entirety.)
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[089] PDA~ technology couples computational design algorithms that generate
quality sequence
diversity with experimental high-throughput screening to discover proteins
with improved
properties. The computational component uses atomic level scoring functions,
side chain rotamer
sampling, and advanced optimization methods to accurately capture the
relationships between
protein sequence, structure, and function. Calculations begin with the three-
dimensional structure
of the protein and a strategy to optimize one or more properties of the
protein. PDA~ technology
then explores the sequence space comprising all pertinent amino acids
(including unnatural
amino acids, if desired) at the positions targeted for design. This is
accomplished by sampling
conformational states of allowed amino acids and scoring them using a
parameterized and
experimentally validated function that describes the physical and chemical
forces governing
protein structure. Powerful combinatorial search algorithms are then used to
search through the
initial sequence space, which may constitute 105° sequences or more,
and quickly return a
tractable number of sequences that are predicted to satisfy the design
criteria. Useful modes of
the technology span from combinatorial sequence design to prioritized
selection of optimal single
site substitutions. PDA~ technology has been applied to numerous systems
including important
pharmaceutical and industrial proteins and has a demonstrated record of
success in protein
optimization.
[090] In a preferred embodiment, each polar residue is represented using a set
of discrete low-
energy side-chain conformations (see for example Dunbrack Curr. Opin. Struct.
Biol. 12:431-440
(2002)). A preferred force field may include terms describing van der Waals
interactions,
hydrogen bonds, electrostatic interactions, and solvation, among others.
[091] In a preferred embodiment, preferred suitable polar residues are defined
as those polar
residues: 1) Whose energy in the optimal rotameric configuration is more
favorable than the
energy of the exposed hydrophobic residue at that position and 2) Whose energy
in the optimal
rotameric configuration is among the most favorable of the set of energies of
all polar residues at
that position
[092] In a preferred embodiment, Dead-End Elimination (DEE) is used to
identify the rotamer for
each polar residue that has the most favorable energy (see Gordon et. al. J.
Comput Chem. 24:
232-243 (2003), Goidstein Biophys. J. 66: 1335-1340 (1994) and Lasters and
Desmet, Prot. Eng.
6: 717-722 (1993)).
[093] In an alternate embodiment, Monte Carlo can be used in conjunction with
DEE to identify
groups of polar residues that have favorable energies.
[094] In an alternate preferred embodiment, a sequence prediction algorithm
(SPA) is used to
design proteins that are compatible with a known protein backbone structure as
is described in
Raha, IC., et al. (2000) Protein Sci., 9: 1106-1119; USSN 09/877,695, filed
June 8, 2001 and
10/071,859, filed February 6, 2002.
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[095] In a preferred embodiment, after performing one or more computational
calculations,
including PDA~ technology calculations, a library of variant proteins is
designed, experimentally
constructed, and screened for desired properties.
[096] In one embodiment, the library is a combinatorial library, meaning that
the library comprises
all possible combinations of allowed residues at each of the variable
positions. For example, if
positions 3 and 9 are allowed to vary, allowed choices at position 3 are A, V,
and I, and allowed
choices at position 9 are E and Q, the library includes the following
sequences: 3A/9E, 3A/9Q,
3V/9E, 3V/9Q, 31/9E, and 31/9Q. In a preferred embodiment, PDA~ technology
calculations may
be used to modify wild type interteron sequences to generate novel, non-
naturally occurring,
soluble proteins from known interferon sequences (see for example, Figure 1 ).
See US
6,188,965; US 6,269,312; US 6,403,312, expressly incorporated by reference
herein.
[097] In a preferred embodiment, the polar residues that are included in the
library at each variable
position are deemed suitable by both PDA~ technology calculations and by
sequence alignment
data. Alternatively, one or more of the polar residues that are included in
the library are deemed
suitable by either PDA~ technology calculations or sequence alignment data.
[098] In a preferred embodiment, residues that are close in sequence are
"coupled" in the library,
meaning that all combinatorial possibilities are not sampled. For instance, if
the library includes
residues L and Q at position 5 and residues F and E at position 8, a "coupled"
library could
include L5iF8 and Q51E8 but not include L51E8 or Q5iF8. Coupling residues
decreases the
overall combinatorial complexity of the library, thereby simplifying
screening. Furthermore,
coupling can be used to avoid the introduction of two or more modifications
that are incompatible
with each other. For interferon kappa, preferred suitable residues based on
both PDA~
calculations and sequence alignment data are described herein. In an alternate
embodiment, the
polar residues that are included in the library at each variable position are
deemed suitable by
either PDA~ technology calculations or by sequence alignment data or by both
methods.
Preferred suitable residues based on both PDA~ calculations and sequence
alignment data are
shown in Figure 2.
[099] Preferred, suitable residues based on both PDA~ technology calculations
and sequence
alignment data are shown in Figure 5.
[0100] Obtaining structures of type 1 interferons PDA~ technology
calculations, described above,
require a template protein structure. In the a most preferred embodiment, the
structure of a type I
interferon is obtained by solving its crystal structure or NMR structure by
techniques well known in
the art. High- resolution structures are available for type I interferons
including interferon-a2a
(interferon-alpha2a), .interferon -a2b (interferon-alpha2b), interferon-b
(interferon-beta), and
interferon-t (interferon-tau) (see Radhakrishnan et. al. J. Mol. Biol. 286:151-
162 (1999), Karpusas
et. al. Proc. Nat, Acad. Sci. USA 94:22 (1997), Klaus et. al. J. Mol. Biol.
274:661-675 (1997),
Radhakrishnan et. al. Structure 4:1453-1463 (1996)).
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[0101] In an alternate embodiment, a homology model is built, using methods
known to those in the
art. Homology models of interferons have been constructed earlier: See for
example Seto et. al.
Protein Sci. 4:655-670 (1995). The homology model may be derived from one or
more of the high
resolution structures listed above.
[0102] In a preferred embodiment, the BLAST alignment algorithm is used to
generate alignments
proteins that are homologs of an interferon of interest. Examples of
homologous proteins include
other classes of type I interferons, allelic variants of interferon, and
interferons from other species.
[0103] Identifying solvent exposed hydrophobic residue positions . Hydrophobic
residues as used
herein generally are identified as valine, leucine, isoleucine, methionine,
phenylalanine, tyrosine,
and tryptophan. Exposed residues as used herein as those residues whose side
chains have at
least 30 A~ (square Angstroms) of solvent accessible surface area. As will be
appreciated by
those skilled in the art, other values such as 50 A~ or fractional values such
as 50% could be used
instead. Furthermore, alternative methods such as contact models, among
others, may be used
to identify exposed residues.
[0104] Identifying suitable polar residues for each exposed hydrophobic
position. In a preferred
embodiment, solvent exposed hydrophobic residues are replaced with
structurally and functionally
compatible polar residues. As used herein, polar residues generally include
serine, threonine,
histidine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, and
lysine. Alanine and
giycine may also serve as suitable replacements, constituting a reduction in
hydrophobicity.
Solvent exposed hydrophobic residues can be defined according to absolute or
fractional solvent
accessibility, as defined above. It is also possible to use other methods,
such as contact models,
to identify exposed residues.
[0105] In a preferred embodiment, suitable polar residues include alanine,
serine, threonine,
histidine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, and
lysine.
[0106] In a preferred embodiment, suitable polar residues include only the
subset of polar residues
that are observed in analogous positions in homologous proteins, especially
other interferons.
[0107] In an especially preferred embodiment, suitable polar residues include
only the subset of
polar residues with low or favorable energies as determined using PDA~
technology or SPATM
calculations.
[0108] As used herein, for example, solvent exposed hydrophobic residues in
interferon-alpha 2a
include, but are not limited to, Met 16, Phe 27, Leu 30, Tyr 89, Ile 100, Leu
110, Met 111, Leu
117, Leu 128, and Leu 161.
[0109] Especially preferred solvent exposed hydrophobic residues are those
that have not been
implicated in interferon alpha function or receptor binding (see for example
Piehler et. al. J. Biol.
Chem. 275: 40425-40433 (2000), Hu et. al. J. Immunol. 163: 854-860 (1999), Hu
et. al. J.
Immunol. 167: 1482-1489 (2001)), including Met 16, Phe 27, Ile 100, Leu 110,
Met 111, Leu 117,
and Leu 161.
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[0110] Especially preferred modifications to interferon-alpha include, but are
not limited to, M16D,
F270, 1100Q, L110N, M111 Q, L117R, and L161 E.
[0111] As used herein, for example, solvent exposed hydrophobic residues in
interteron-beta
include, but are not limited to, Leu 5, Phe 8, Phe 15, Trp 22, Leu 28, Tyr 30,
Leu 32, Met 36, Leu
47, Tyr 92, Phe 111, Leu 116, Leu 120, Leu 130, Val 148, and Tyr 155.
[0112] Especially preferred modifications to interferon-beta include, but are
not limited to, LSQ, FBE,
F111 N, L116E, and L120R.
[0113] Especially preferred solvent exposed hydrophobic residues are those
residues that have not
been implicated in interferon beta function or receptor binding (see for
example Runkel et. al.
Biochem. 39: 2538-2551 (2000), Runkel et. al. J. Int. Cytokine Res. 21: 931-
941 (2001)), include
Leu 5, Phe 8, Leu 47, Phe 111, Leu 116, and Leu 120.
[0114] As used herein, for example, solvent exposed hydrophobic residues in
interferon-kappa
include, but are not limited to, Leu 1, Leu 5, Val 8, Trp 15, Leu 18, Phe 28,
Val 30, Leu 33, Ile 37,
Leu 46, Tyr 48, Met 52, Leu 65, Phe 68, Phe 76, Tyr 78, Trp 79, Ile 89, Tyr
97, Met 112, Met 115,
Met 120, Val 127, Leu 133, Tyr 151, Val 161, Tyr 168, and Tyr 171.
[0115] Especially preferred solvent exposed hydrophobic residues are located
at positions that are
polar in other interferon sequences, and include Leu 5, Val 8, Trp 15, Phe 28,
Val 30, Ile 37, Tyr
48, Met 52, Phe 76, Tyr 78, Ile 89, Tyr 97, Val 161, Tyr 168, and Tyr 171.
[0116] Especially preferred modifications to interferon-kappa include, but are
not limited to, L5Q,
VBN, W15R, F28Q, V30R, 137N, Y480, M52N, F76S, Y78A, 189T, Y97D, M112T, M115G,
L133Q,
V161A, Y168S, and Y171T.
[0117] Identifying unpaired cysteine positions. Unpaired cysteines are defined
to be cysteines that
do not form a disulfide bond in the folded protein. Unpaired cysteines can be
identified, for
example, by visual analysis of the structure or by analysis of the disulfide
bond patterns of related
proteins.
[0118] Interferon alpha-1 and interferon alpha-13 contain one unpaired
cysteine at position 86 (Cys
86).
[0119] Interferon-beta contains one unpaired cysteine at position 17 (Cys 17).
[0120] Interferon-kappa contains one unpaired cysteine at position 166 (Cys
166).
[0121] Ovine interferon-tau contains one unpaired cysteine at position 86 (Cys
86).
[0122] Identifying suitable non-cysteine residues for each unpaired cysteine
position. Suitable non-
cysteine residues as used herein are meant all amino acid residues other than
cysteine.
[0123] In a preferred embodiment, if the cysteine position is substantially
buried in the protein core,
suitable non-cysteine residues include alanine and the hydrophobic residues
valine, leucine,
isoleucine, methionine, phenylalanine, tyrosine, and tryptophan.
[0124] In a preferred embodiment, if the cysteine position is substantially
exposed to solvent,
suitable non-cysteine residues include alanine and the polar residues serine,
threonine, histidine,
aspartic acid, asparagine, glutamic acid, glutamine, arginine, and lysine.
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[0125] In a preferred embodiment, suitable residues are defined as those with
low (favorable)
energies as calculated using PDA~ technology.
[0126] In a preferred embodiment, suitable residues defined as those that are
observed at
homologous positions in proteins that are interferon-kappa homologs. For
example, position 86 is
an unpaired cysteine in some interferon-alpha1 and interferon-alpha13, but is
replaced with
tyrosine or serine in other interferon alpha subtypes. Also, position 166 is
an unpaired cysteine in
interferon-kappa, but is frequently alanine in other interferon sequences.
[0127] In a more preferred embodiment, suitable residues are those, which that
have both low
(favorable) energies as calculated using PDA~ technology and are observed at
homologous
positions in proteins that are interferon-kappa homologsin the analogous
position in other
interferon proteins.
[0128] In a most preferred embodiment, Cys 86 in interferon-alpha 1 or
interferon alpha-13 replaced
by glutamic acid, lysine, or glutamine.
[0129] In a most preferred embodiment, Cys 17 in interferon-beta is replaced
by alanine, aspartic
acid, asparagine, serine or threonine.
[0130] In a most preferred embodiment, Cys 166 in interferon-kappa is replaced
by Alanine, glutamic
acid, or histidine.
[0131] In some embodiments, the variant INF proteins of the invention do not
include substitutions at
unpaired cysteine postions.
[0132] Identifying dimer interface residues. In a preferred embodiment,
residues that mediate
intermolecular interactions between interferon monomers or between interferon
and human serum
albumin are replaced with structurally and functionally compatible residues
that confer decreased
propensity for unwanted intermolecular interactions.
[0133] In a preferred embodiment, interface residues are defined as those
residues located within 8
A of a protein-protein contact. Distances of less than 5 A are especially
preferred. Distances
may be measured using any structure, with high- resolution crystal structures
being especially
preferred.
[0134] Preferred interface residues in interferon alpha include, but are not
limited to, residues 16, 19,
20, 25, 27, 28, 30, 33, 35-37, 39-41, 44-46, 54, 58, 61, 65, 68, 85, 91, 99,
112-115, 117, 118, 121,
122, 125, and 149.
[0135] Preferred interface residues in interferon beta include, but are not
limited to, residues 1-6, 8,
9, 12, 16, 42, 43, 46, 47,49, 51, 93, 96, 97, 100, 101, 104, 113, 116, 117,
120, 121, and 124.
[0136] Identifying suifable residues for each interface position. Suitable
residues for interface
residues as used herein are meant all amino acid residues that are compatible
with the structure
and function of a type I interferon, but which are substantially incapable of
forming unwanted
intermolecular interactions, including but not limited to interactions with
other interferon molecules
and interactions with human serum albumin.
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[0137] Typically, the interface positions will be substantially exposed to
solvent. In such cases,
preferred substitutions include alanine and the polar residues serine,
threonine, histidine, aspartic
acid, asparagine, glutamic acid, glutamine, arginine, and lysine. However, for
interface positions
that are substantially buried in the monomer structure, hydrophobic
replacements are preferred.
[0138] In a preferred embodiment, suitable polar residues include only the
subset of polar residues
that are observed in analogous positions in homologous proteins, especially
other interterons, that
do not form a given unwanted intermolecular interaction.
[0139] In an especially preferred embodiment, suitable polar residues include
only the subset of
polar residues with low or favorable energies as determined using PDAO
technology calculations
or SPA calculations (described above).
[0140] In a most especially preferred embodiment, suitable polar residues
include only the subset of
polar residues that are determined to be compatible with the monomer structure
and incompatible
with a given unwanted intermolecular interaction, as determined using PDA~
technology
calculations or SPA calculations.
[0141] Especially preferred modifications to interferon-beta include LSA, LSD,
LSE, LSK, LSN, LSQ,
LSR, L5S, L5T, FBA, FBD, FBE, FBK, F8N, FBQ, FBR, FBS, S12E, S12K, S12Q, S12R,
E43K,
E43R, R113D, L11fiD, L116E, L116N, L116Q, L116R, and M117R.
[0142] In a preferred embodiment, each polar residue is represented using a
set of discrete low-
energy side-chain conformations obtained from the 1996 Dunbrak and Karplus
rotamer library. A
preferred force field may include terms describing van der Waals interactions,
hydrogen bonds,
electrostatic interactions, and solvation, among others.
[0143] In a preferred embodiment, DEE is used to identify the rotamer for each
polar residue that
has the most favorable energy.
[0144] In an alternate embodiment, Monte Carlo can be used in conjunction with
DEE to identify
groups of polar residues that have favorable energies.
[0145] In a preferred embodiment, the frequency of occurrence of each polar
residue at each
position in interferon-kappa homologs is normalized using the method of
Henikoff & Henikoff (J.
Mol. Biol. 243: 547-578 (1994)). In an alternate embodiment, a simple count of
the number of
occurrences of each polar residue at each position is made. For interferon
kappa, preferred
suitable residues based on sequence alignment data include those shown in
Figure x.Preferred
suitable residues based on sequence alignment data include those shown in
Figure 3.
[0146] Especially preferred modifications to interferon-alpha include, but are
not limited to, M16D,
F27Q, 1100Q, L110N, M111Q, L117R, and L161E.
[0147] Additional preferred, suitable residues are shown in Figure 6. These
residues are located at
positions that are close in primary sequence to the residues in Figure 5 and
are suitable based on
either PDAT"' technology calculations or sequence alignment data. Diversity at
such positions
can be incorporated into a library of protein variants without increasing the
total combinatorial
complexity of the library.
21
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[0148] Identification of MHC-binding agretopes in interferons The following
discussion is centered
on INF-[i. Although as will be appreciated by those in the art, these
techniques apply to the other
IFNs as well. MHC-binding peptides are obtained from proteins by a process
called antigen
processing. First, the protein is transported into an antigen presenting cell
(APC) by endocytosis
or phagocytosis. A variety of proteolytic enzymes then cleave the protein into
a number of
peptides. These peptides can then be loaded onto class II MHC molecules, and
the resulting
peptide-MHC complexes are transported to the cell surface. Relatively stable
peptide-MHC
complexes can be recognized by T-cell receptors that are present on the
surface of naive T cells.
This recognition event is required for the initiation of an immune response.
Accordingly, blocking
the formation of stable peptide-MHC complexes is an effective approach for
preventing unwanted
immune responses.
[0149] The factors that determine the affinity of peptide-MHC interactions
have been characterized
using biochemical and structural methods. Peptides bind in an extended
conformation bind along
a groove in the class Il MHC molecule. While peptides that bind class II MHC
molecules are
typically approximately 13-18 residues long, a nine-residue region is
responsible for most of the
binding affinity and specificity. The peptide binding groove can be subdivided
into "pockets",
commonly named P1 through P9, where each pocket is comprises the set of MHC
residues that
interacts with a specific residue in the peptide. A number of polymorphic
residues face into the
peptide-binding groove of the MHC molecule. The identity of the residues
lining each of the
peptide-binding pockets of each MHC molecule determines its peptide binding
specificity.
Conversely, the sequence of a peptide determines its affinity for each MHC
allele.
[0150] Several methods of identifying MHC-binding agretopes in protein
sequences are known in the
art and may be used to identify agretopes in type 1 interferons, including IFN-
(3.
[0151] Sequence-based information can be used to determine a binding score for
a given peptide-
MHC interaction (see for example Mallios, Bioinformatics 15: 432-439 (1999);
Mallios,
Bioinformatics 17: p942-948 (2001 ); Sturniolo et. al. Nature Biotech. 17: 555-
561 (1999)). It is
possible to use structure-based methods in which a given peptide is
computationally placed in the
peptide-binding groove of a given MHC molecule and the interaction energy is
determined (for
example, see WO 98/59244 and WO 021069232). Expressly incorporated herein by
reference.
Such methods may be referred to as "threading" methods. Alternatively, purely
experimental
methods can be used; for example a set of overlapping peptides derived from
the protein of
interest can be experimentally tested for the ability to induce T-cell
activation and/or other aspects
of an immune response. (see for example WO 02/77187).
[0152] In a preferred embodiment, MHC-binding propensity scores are calculated
for each 9-residue
frame along the interferon beta sequence using a matrix method (see Sturniolo
et. al., supra;
Marshall et. al., J. Immunol. 154: 5927-5933 (1995), and Hammer et. al., J.
Exp. Med. 180: 2353-
2358 (1994)). Expressly incorporated herein by reference. It is also possible
to consider scores
for only a subset of these residues, or to consider also the identities of the
peptide residues
22
CA 02528964 2005-12-09
WO 2005/003157 PCT/US2004/009824
before and after the 9-residue frame of interest. The matrix comprises binding
scores for specific
amino acids interacting with the peptide binding pockets in different human
class II MHC
molecule. In the most preferred embodiment, the scores in the matrix are
obtained from
experimental peptide binding studies. In an alternate preferred embodiment,
scores for a given
amino acid binding to a given pocket are extrapolated from experimentally
characterized alleles to
additional alleles with identical or similar residues lining that pocket.
Matrices that are produced
by extrapolation are referred to as "virtual matrices".
[0153] In a preferred embodiment, the matrix method is used to calculate
scores for each peptide of
interest binding to each allele of interest. Several methods can then be used
to determine
whether a given peptide will bind with significant affinity to a given MHC
allele. In one
embodiment, the binding score for the peptide of interest is compared with the
binding propensity
scores of a large set of reference peptides. Peptides whose binding propensity
scores are large
compared to the reference peptides are likely to bind MHC and may be
classified as "hits". For
example, if the binding propensity score is among the highest 1 % of possible
binding scores for
that allele, it may be scored as a "hit" at the 1 % threshold. The total
number of hits at one or
more threshold values is calculated for each peptide. In some cases, the
binding score may
directly correspond with a predicted binding affinity. Then, a hit may be
defined as a peptide
predicted to bind with at least 100 NM or 10 pM or 1 NM affinity.
[0154] In a preferred embodiment, the number of hits for each 9-mer frame in
the protein is
calculated using one or more threshold values ranging from 0.5% to 10%. In an
especially
preferred embodiment, the number of hits is calculated using 1 %, 3%, and 5%
thresholds.
[0155] In a preferred embodiment, MHC-binding agretopes are identified as the
9-mer frames that
bind to several class II MHC alleles. In an especially preferred embodiment,
MHC-binding
agretopes are predicted to bind at least 10 alleles at 5% threshold and/or at
least 5 alleles at 1
threshold. Such 9-mer frames may be especially likely to elicit an immune
response in many
members of the human population.
[0156] In a preferred embodiment, MHC-binding agretopes are predicted to bind
MHC alleles that
are present in at least 0.01 -10 % of the human population. Alternatively, to
treat conditions that
are linked to specific class II MHC alleles, MHC-binding agretopes are
predicted to bind MHC
alleles that are present in at least 0.01 -10 % of the relevant patient
population.
[0157] Data about the prevalence of different MHC alleles in different ethnic
and racial groups has
been acquired by groups such as the National Marrow Donor Program (NMDP); for
example see
Mignot et al. Am. J. Hum. Genet. 68: 686-699 (2001 ), Southwood et al. J.
"Immunol. 160: 3363-
3373 (1998), Hurley et al. Bone Marrow Transplantation 25: 136-137 (2000),
Sintasath Hum.
Immunol. 60: 1001 (1999), Collins et al. Tissue Antigens 55: 48 (2000), Tang
et al. Hum.
Immunol. 63: 221 (2002), Chen et al. Hum. Immunol. 63: 665 (2002), Tang et al.
Hum. Immunol.
61: 820 (2000), Gans et al. Tissue Antigens 59: 364-369, and Baldassarre et
al. Tissue Antigens
61: 249-252 (2003).
23
CA 02528964 2005-12-09
WO 2005/003157 PCT/US2004/009824
[0158] In a preferred embodiment, MHC binding agretopes are predicted for MHC
heterodimers
comprising highly prevalent MHC alleles. Class II MHC alleles that are present
in at least 10 % of
the US population include but are not limited to: DPA1*0103, DPA1*0201,
DPB1*0201,
DPB1 *0401, DPB1 *0402, DQA1 *0101, DQA1 *0102, DQA1 *0201, DQA1 *0501, DQB1
*0201,
DQB1*0202, DQB1*0301, DQB1*0302, DQB1*0501, DQB1*0602, DRA*0101, DRB1*0701,
DRB1*1501, DRB1*0301, DRB1*0101, DRB1*1101, DRB1*1301, DRB3*0101, DRB3*0202,
DRB4*0101, DRB4*0103, and DRB5*0101.
[0159] In a preferred embodiment, MHC binding agretopes are also predicted for
MHC heterodimers
comprising moderately prevalent MHC alleles. Class I1 MHC alleles that are
present in 1% to
10% of the US population include but are not limited to: DPA1*0104, DPA1*0302,
DPA1*0301,
DPB1*0101, DPB1*0202, DPB1*0301, DPB1* 0501, DPB1*0601, DPB1*0901, DPB1*1001,
DPB1*1101, DPB1*1301, DPB1*1401, DPB1*1501, DPB1*1701, DPB1*1901, DPB1*2001,
DQA1 *0103, DQA1 *0104, DQA1 *0301, DQA1 *0302, DQA1 *0401, DQB1 *0303, DQB1
*0402,
DQB1*0502, DQB1*0503, DQB1*0601, DQB1*0603, DRB1*1302, DRB1*0404, DRB1*0801,
DRB1*0102, DRB1*1401, DRB1*1104, DRB1*1201, DRB1*1503, DRB1*0901, DRB1*1601,
DRB1*0407, DRB1*1001, DRB1*1303, DRB1*0103, DRB1*1502, DRB1*0302, DRB1*0405,
DRB1*0402, DRB1*1102, DRB1*0803, DRB1*0408, DRB1*1602, DRB1*0403, DRB3*0301,
DRB5*0102, and DRB5*0202.
[0160] MHC binding agretopes may also be predicted for MHC heterodimers
comprising less
prevalent alleles. Information about MHC alleles in humans and other species
can be obtained,
for example, from the IMGT/HLA sequence database (www.ebi.ac.uk/imat/hla/).
[0161] In an additional preferred embodiment, MHC-binding agretopes are
identified as the 9-mer
frames that are located among "nested" agretopes, or overlapping 9-residue
frames that are each
predicted to bind a significant number of alleles. Such sequences may be
especially likely to elicit
an immune response.
[0162] Preferred MHC-binding agretopes are those agretopes that are predicted
to bind, at a 3
threshold, to MHC alleles that are present in at least 5 % of the population.
Preferred MHC-
binding agretopes in interferon beta include, but are not limited to, agretope
2: residues 5-13;
agretope 3: residues 8-16; agretope 5: residues 15-23; agretope 6: residues 22-
30; agretope 7:
residues 30-38; agretope 8: residues 36-44; agretope 10: residues 57-65;
agretope 11: residues
60-68; agretope 12: residues 63-71; agretope 13: residues 70-78; agretope 16:
residues 122-130;
agretope 18: residues 129-137; agretope 20: residues 143-151; agretope 21:
residues 145-153;
agretope 22: residues 146-154; agretope 23: residues 148-156; agretope 24:
residues 151-159;
agretope 25: residues 154-162; and agretope 26: residues 156-164.
[0163] Especially preferred MHC-binding agretopes are those agretopes that are
predicted to bind,
at a 1 % threshold, to MHC alleles that are present in at least 10 % of the
population. Especially
preferred MHC-binding agretopes in interferon beta include, but are not
limited to, agretope 6:
24
CA 02528964 2005-12-09
WO 2005/003157 PCT/US2004/009824
residues 22-30; agretope 8: residues 36-44; agretope 11: residues 60-68;
agretope 20: residues
143-151; agretope 24: residues 151-159; and agretope 25: residues 154-162.
[0164] Additional especially preferred MHC-binding agretopes are those
agretopes whose
sequences partially overlap with additional MHC-binding agretopes. Sets of
overlapping MHC-
binding agretopes in interferon beta include, but are not limited to, residues
5-44; residues 57-78;
residues 122--137; and residues 143-164.
[0165] Interferon beta is commonly used to treat multiple sclerosis. As
multiple sclerosis has been
linked to the presence of certain MHC alleles, especially preferred MHC-
binding agretopes are
those agretopes that are predicted to bind, at a 3% threshold, to MHC alleles
that are commonly
present in multiple sclerosis patients. The HLA DRB1*1501-DQB1*0602 haplotype
has been
repeatedly demonstrated to confer susceptibility to multiple sclerosis.
Additional alleles that are
associated with increased risk of multiple sclerosis include, but are not
limited to, DRB1*1503
(Quelvennec et. al. Tissue Antigens 2003 61: 166-171), DPB1*0501 (Fukuzawa et.
al. J. Neurol.
2000 247: 175-178), DRB1 *1303 (Kohn et. al. Muft. Scler. 1999 5: 410-415),
DQA1 *0101 and
DQA1*0102 (Arcos-Burgos et. al. Exp. Clin. Immunogenet. 1999 16: 131-138).
Agretopes in
interferon beta that are predicted to bind one or more MHC alleles which
confer susceptibility to
multiple sclerosis include, but are not limited to, agretope 10: residues 57-
65; agretope 16
residues 122-130; and agretope 24: residues 151-159.
[0166] Confirmation of MHC-bindine~ aclrefopes
[0167] In a preferred embodiment, the immunogenicity of the above-predicted
MHC-binding
agretopes is experimentally confirmed by measuring the extent to which
peptides comprising
each predicted agretope can elicit an immune response. However, it is possible
to proceed from
agretope prediction to agretope removal without the intermediate step of
agretope confirmation.
[0168] Several methods, discussed in more detail below, can be used for
experimental confirmation
of agretopes. For example, sets of naive T cells and antigen presenting cells
from matched
donors can be stimulated with a peptide containing an agretope of interest,
and T-cell activation
can be monitored. It is also possible to first stimulate T cells with the
whole protein of interest,
and then re-stimulate with peptides derived from the whole protein. If sera
are available from
patients who have raised an immune response to interferon beta, it is possible
to detect mature T
cells that respond to specific epitopes. In a preferred embodiment, interferon
gamma or IL-5
production by activated T-cells is monitored using Elispot assays, although it
is also possible to
use other indicators of T-cell activation or proliferation such as tritiated
thymidine incorporation or
production of other cytokines.
[0169] Patient aenotyt~e analysis and screening
[0170] HLA genotype is a major determinant of susceptibility to specific
autoimmune diseases (see
for example Nepom Clin. Immunol. Immunopathol. 67: S50-S55 (1993)) and
infections (see for
example Singh et. al. Emerg. Infect. Dis. 3: 41-49 (1997)). Furthermore, the
set of MHC alleles
present in an individual can affect the efficacy of some vaccines (see for
example Cailat-Zucman
CA 02528964 2005-12-09
WO 2005/003157 PCT/US2004/009824
et. al. Kidney Int. 53: 1626-1630 (1998) and Poland et. al. Vaccine 20: 430-
438 (2001)). HLA
genotype may also confer susceptibility for an individual to elicit an
unwanted immune response
to a interferon beta therapeutic.
[0171] In a preferred embodiment, class II MHC alleles that are associated
with increased or
decreased susceptibility to elicit an immune response to interferon beta
proteins are identified.
For example, patients treated with interferon beta therapeutics may be tested
for the presence of
anti-interferon beta antibodies and genotyped for class II MHC. Alternatively,
T-cell activation
assays such as those described above may be conducted using cells derived from
a number of
genotyped donors. Alleles that confer susceptibility to interferon beta
immunogenicity may be
defined as those alleles that are significantly more common in those who
elicit an immune
response versus those who do not. Similarly, alleles that confer resistance to
interferon beta
immunogenicity may be defined as those that are significantly less common in
those who do not
elicit an immune response versus those that do. It is also possible to use
purely computational
techniques to identify which alleles are likely to recognize interferon beta
therapeutics.
[0172] In one embodiment, the genotype association data is used to identify
patients who are
especially likely or especially unlikely to raise an immune response to a
interferon beta
therapeutic.
[0173] Design of active, less-immunogenic variants. In a preferred embodiment,
the above-
determined MHC-binding agretopes are replaced with alternate amino acid
sequences to
generate active variant interferon beta proteins with reduced or eliminated
immunogenicity.
Alternatively, the MHC-binding agretopes are modified to introduce one or more
sites that are
susceptible to cleavage during protein processing. If the agretope is cleaved
before it binds to a
MHC molecule, it will be unable to promote an immune response. There are
several possible
strategies for integrating methods for identifying less immunogenic sequences
with methods for
identifying structured and active sequences, including but not limited to
those presented below.
[0174] In one embodiment, for one or more 9-mer agretope identified above, one
or more possible
alternate 9-mer sequences are analyzed for immunogenicity as well as
structural and functional
compatibility. The preferred alternate 9-mer sequences are then defined as
those sequences that
have low predicted immunogenicity and a high probability of being structured
and active. It is
possible to consider only the subset of 9-mer sequences that are most likely
to comprise
structured, active, less immunogenic variants. For example, it may be
unnecessary to consider
sequences that comprise highly non-conservative mutations or mutations that
increase predicted
immunogenicity.
[0175] In a preferred embodiment, less immunogenic variants of each agretope
are predicted to bind
MHC alleles in a smaller fraction of the population than the wild type
agretope. tn an especially
preferred embodiment, the less immunogenic variant of each agretope is
predicted to bind to
MHC alleles that are present in not more than 5 % of the population, with not
more than 1 % or
0.1 % being most preferred.
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CA 02528964 2005-12-09
WO 2005/003157 PCT/US2004/009824
[0176] Substitution matrices In another especially preferred embodiment,
substitution matrices or
other knowledge-based scoring methods are used to identify alternate sequences
that are likely to
retain the structure and function of the wild type protein. Such scoring
methods can be used to
quantify how conservative a given substitution or set of substitutions is. In
most cases,
conservative mutations do not significantly disrupt the structure and function
of proteins (see for
example, Bowie et. al. Science 247: 1306-1310 (1990), Bowie and Sauer Proc.
Nat. Acad. Sci.
USA 86: 2152-2156 (1989), and Reidhaar-Oison and Sauer Proteins 7: 306-316
(1990)).
However, non-conservative mutations can destabilize protein structure and
reduce activity (see
for example, Lim et. al. Biochem. 31: 4324-4333 (1992)). Substitution matrices
including but not
limited to BLOSUM62 provide a quantitative measure of the compatibility
between a sequence
and a target structure, which can be used to predict non-disruptive
substitution mutations (see
Topham et al. Prot. Eng. 10: 7-21 (1997)). The use of substitution matrices to
design peptides
with improved properties has been disclosed; see Adenot et al. J. Mol. Graph.
Model. 17: 292-309
(1999).Substitution matrices include, but are not limited to, the BLOSUM
matrices (Henikoff and
Henikoff, Proc. Nat. Acad. Sci. USA 89. 10917 (1992), the PAM matrices, the
Dayhoff matrix, and
the like. For a review of substitution matrices, see for example Henikoff
Curr. Opin. Struct. Biol. 6:
353-360 (1996). It is also possible to construct a substitution matrix based
on an alignment of a
given protein of interest and its homologs; see for example Henikoff and
Henikoff Comput. Appl.
Biosci. 12: 135-143 (1996).1n a preferred embodiment, each of the substitution
mutations that are
considered has a BLOSUM 62 score of zero or higher. According to this metric,
preferred
substitutions include, but are not limited to:
[0177]
Table 1. Conservative
mutations
Wild type residuepreferred substitutions
A CSTAGV
C CA
D SNDEQ
E SNDEQHRK
F MILFYW
G SAGN
H NEQHRY
I MILVF
K SNEQRK
L MILVF
M G2MILVF
N STGNDEQHRK
P P
Q SNDEQHRKM
R NEQHRK
S STAGNDEQK
T TAMILV
V STANV
W FYW
Y HFYW
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WO 2005/003157 PCT/US2004/009824
[001] In addition, it is preferred that the total BLOSUM 62 score of an
alternate sequence for a nine
residue MHC-binding agretope is decreased only modestly when compared to the
BLOSUM 62
score of the wild type nine residue agretope. In a preferred embodiment, the
score of the variant
9mer is at least 50 % of the wild type score, with at least 67%, 75% or 90%
being especially
preferred.
[002] Alternatively, alternate sequences can be selected that minimize the
absolute reduction in
BLOSUM score; for example it is preferred that the score decrease for each 9-
mer is less than 20,
with score decreases of less than about 10 or about 5 being especially
preferred. The exact
value may be chosen to produce a library of alternate sequences that is
experimentally tractable
and also sufficiently diverse to encompass a number of active, stable, less
immunogenic variants.
[003] In a preferred embodiment, substitution mutations are preferentially
introduced at positions
that are substantially solvent exposed. As is known in the art, solvent
exposed positions are
typically more tolerant of mutation than positions that are located in the
core of the protein.
[004] In a preferred embodiment, substitution mutations are preferentially
introduced at positions
that are not highly conserved. As is known in the art, positions that are
highly conserved among
members of a protein family are often important for protein function,
stability, or structure, while
positions that are not highly conserved often may be modified without
significantly impacting the
structural or functional properties of the protein.
[005] Aianine substitutions. In an alternate embodiment, one or more alanine
substitutions may be
made, regardless of whether an alanine substitution is conservative or non-
conservative. As is
known in the art, incorporation of sufficient alanine substitutions may be
used to disrupt
intermolecular interactions.
[006] In a preferred embodiment, variant 9-mers are selected such that
residues that have been or
can be identified as especially critical for maintaining the structure or
function of interferon beta
retain their wild type identity. In alternate embodiments, it may be desirable
to produce variant
interferon beta proteins that do not retain wild type activity. In such cases,
residues that have
been identified as critical for function may be specifically targeted for
modification.
[007] Mutagenesis studies have identified three residues, R27, R35, and K123,
that are important
for interferon beta activity [Runkel et. al. J. Biol. Chem. 1998 273: 8003-
8008]. A number of
additional residues in interferon beta have been implicated in receptor
binding, based on the
results of cassette alanine scanning experiments, including F15, Q16, Q18,
K19, W22, Q23, L28,
E29, Y30, L32, K33, M36, N37, D39, E42, Q64, F67, A68, R71, Q72, D73, Y92,
N96, K99, T100,
8128, L130, H131, K134, V148, 8152, Y155, and N158 [Runkel et. al. Biochem
2000 39: 2538-
28
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2551]. However, since these residues were mutated in groups, it is likely that
only a subset are
actually required for interferon beta function.
[0178] Protein design methods and MHC agretope identification methods may be
used together to
identify stable, active, and minimally immunogenic protein sequences (see
W003/006154). The
combination of approaches provides significant advantages over the prior art
for immunogenicity
reduction, as most of the reduced immunogenicity sequences identified using
other techniques
fail to retain sufficient activity and stability to serve as therapeutics.
[0179] Protein design methods may identify non-conservative or unexpected
mutations that
nonetheless confer desired functional properties and reduced immunogenicity,
as well as
identifying conservative mutations. Nonconservative mutations are defined
herein to be all
substitutions not included in Table 1 above; nonconservative mutations also
include mutations
that are unexpected in a given structural context, such as mutations to
hydrophobic residues at
the protein surface and mutations to polar residues in the protein core.
[0180] Furthermore, protein design methods may identify compensatory
mutations. For example, if a
given first mutation that is introduced to reduce immunogenicity also
decreases stability or
activity, protein design methods may be used to find one or more additional
mutations that serve
to recover stability and activity while retaining reduced immunogenicity.
Similarly, protein design
methods may identify sets of two or more mutations that together confer
reduced immunogenicity
and retained activity and stability, even in cases where one or more of the
mutations, in isolation,
fails to confer desired properties.
[0181] In a preferred embodiment, the results of matrix method calculations
are used to identify
which of the 9 amino acid positions within the agretope(s) contribute most to
the overall binding
propensities for each particular allele "hit". This analysis considers which
positions (P1-P9) are
occupied by amino acids which consistently make a significant contribution to
MHC binding
affinity for the alleles scoring above the threshold values. Matrix method
calculations are then
used to identify amino acid substitutions at said positions that would
decrease or eliminate
predicted immunogenicity and PDA~ technology is used to determine which of the
alternate
sequences with reduced or eliminated immunogenicity are compatible with
maintaining the
structure and function of the protein,
[0182] In an alternate preferred embodiment, the residues in each agretope are
first analyzed by one
skilled in the art to identify alternate residues that are potentially
compatible with maintaining the
structure and function of the protein. Then, the set of resulting sequences
are computationally
screened to identify the least immunogenic variants. Finally, each of the less
immunogenic
sequences are analyzed more thoroughly in PDA~ technology protein design
calculations to
identify protein sequences that maintain the protein structure and function
and decrease
immunogenicity.
29
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WO 2005/003157 PCT/US2004/009824
[0183] In an alternate preferred embodiment, each residue that contributes
significantly to the MHC
binding affinity of an agretope is analyzed to identify a subset of amino acid
substitutions that are
potentially compatible with maintaining the structure and function of the
protein. This step may be
performed in several ways, including PDA~ calculations or visual inspection by
one skilled in the
art. Sequences may be generated that contain all possible combinations of
amino acids that were
selected for consideration at each position. Matrix method calculations can be
used to determine
the immunogenicity of each sequence. The results can be analyzed to identify
sequences that
have significantly decreased immunogenicity. Additional PDA~ calculations may
be performed to
determine which of the minimally immunogenic sequences are compatible with
maintaining the
structure and function of the protein.
[0184] In an alternate preferred embodiment, pseudo-energy terms derived from
the peptide binding
propensity matrices are incorporated directly into the PDA~ technology
calculations. In this way,
it is possible to select sequences that are active and less immunogenic in a
single computational
step.
[0185] Combining immunogenicity reduction strategies In a preferred
embodiment, more than one
method is used to generate variant proteins with desired functional and
immunological properties.
For example, substitution matrices may be used in combination with PDA~
technology
calculations. Strategies for immunogenicity reduction include, but are not
limited to, those
described in US.S.N. , Optimized Fc Variants and Methods for Generation, filed
March 26, 2004, incorporated by reference.
[0186] In a preferred embodiment, a variant protein with reduced binding
affinity for one or more
class II MHC alleles is further engineered to confer improved solubility. As
protein aggregation
may contribute to unwanted immune responses, increasing protein solubility may
reduce
immunogenicity (see for example SIFN).
[0187] In an additional preferred embodiment, a variant protein with reduced
binding affinity for one
or more class II MHC alleles is further modified by derivitization with PEG or
another molecule.
As is known in the art, PEG may sterically interfere with antibody binding or
improve protein
solubility, thereby reducing immunogenicity. In an especially preferred
embodiment, rational
PEGyiation methods are used (see U.S.S.N. 60/459,094 and U.S.S.N. ,
"Generating
Protein ProDrugs using Reversible PPG Linkages, filed 3!19/04, hereby
incorporated by
reference).
[0188] In a preferred embodiment, PDA~ technology and matrix method
calculations are used to
remove more than one MHC-binding agretope from a protein of interest.
[0189] Additional modifications. Additional insertions, deletions, and
substitutions may be
incorporated into the variant interferon proteins of the invention in order to
confer other desired
properties.
[0190] In a preferred embodiment, the immunogenicity of interferons may be
modulated. See for
example USSNs: 09/903,378; 10/039,170; 10/339,788 (filed January 8, 2003,
titled Novel Protein
CA 02528964 2005-12-09
WO 2005/003157 PCT/US2004/009824
with Altered Immunogenicity); and PCT/US01/21823; and PCT/US02/00165. All
references
expressly incorporated by reference in their entirety. See for example USSNs:
091903,378;
10/039,170; / (filed January 8, 2003, titled Novel Protein with Altered
Immunogenicity);
and PCT/US01/21823; and PCT/US02/00165. All references cited herein are
expressly
incorporated by reference in their entirety.
[0191] In an alternate preferred embodiment, the interferon variant is further
modified to increase
stability. As discussed above, modifications that improve stability can also
improve solubility, for
example by decreasing the concentration of partially unfolded, aggregation-
prone species. For
example, modifications can be introduced to the protein core that improve
packing or remove
polar or charged groups that are not forming favorable hydrogen bond or
electrostatic
interactions. It is also possible to introduce modifications that introduce
stabilizing electrostatic
interactions or remove destabilizing interactions. Additional stabilizing
modifications also may be
used.
[0192] In one embodiment, the sequence of the variant interferon protein is
modified in order to add
or remove one or more N-linked or O-linked glycosylation sites. Addition of
glycosylation sites to
variant interferon polypeptides may be accomplished, for example, by the
incorporation of one or
more serine or threonine residues to the native sequence or variant interferon
polypeptide (for O-
linked glycosylation sites) or by the incorporation of a canonical N-linked
glycosylation site,
including but not limited to, N-X-Y, where X is any amino acid except for
proline and Y is
preferably threonine, serine or cysteine. Glycosylation sites may be removed
by replacing one or
more serine or threonine residues or by replacing one or more canonical N-
linked glycosylation
sites.
[0193] In another preferred embodiment, one or more cysteine, lysine,
histidine, or other reactive
amino acids are designed into variant interferon proteins in order to
incorporate labeling sites or
PEGylation sites. It is also possible to remove one or more cysteine, lysine,
histidine, or other
reactive amino acids in order to prevent the incorporation of labeling sites
or PEGylations sites at
specific locations. For example, in a preferred embodiment, non-labile
PEGylation sites are
selected to be well removed from any required receptor binding sites in order
to minimize loss of
activity.
[0194] Variant interferon polypeptides of the present invention may also be
modified to form chimeric
molecules comprising a variant interferon polypeptide fused to another,
heterologous polypeptide
or amino acid sequence. In one embodiment, such a chimeric molecule comprises
a fusion of a
variant interferon polypeptide with a tag polypeptide which provides an
epitope to which an anti-
tag antibody can selectively bind. The epitope tag is generally placed at the
amino-or carboxyl-
terminus of the variant interferon polypeptide. The presence of such epitope-
tagged forms of a
variant interferon polypeptide can be detected using an antibody against the
tag polypeptide.
Also, provision of the epitope tag enables the variant interferon polypeptide
to be readily purified
by affinity purification using an anti-tag antibody or another type of
affinity matrix that binds to the
31
CA 02528964 2005-12-09
WO 2005/003157 PCT/US2004/009824
epitope tag. Various tag polypeptides and their respective antibodies are well
known in the art.
Examples include poly-histidine (poly-His) or poly-histidine-glycine (poly-His-
Gly) tags; the flu HA
tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol. 8:2159-
2165 (1988)]; the c-
myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et
al., Molecular and
Cellular Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus
glycoprotein D (gD) tag and
its antibody [Paborsky et al., Protein Engineering, 3(6): 547-553 (1990)].
Other tag polypeptides
include the Flag-peptide [Hope et al., BioTechnology 6:1204-1210 (1988)]; the
KT3 epitope
peptide [Martin et al., Science 255:192-194 (1992)]; tubulin epitope peptide
[Skinner et al., J. Biol.
Chem. 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-
Freyermuth et al.,
Proc. Natl. Acad. Sci. U.S.A. 87:6393-6397 (1990)].
[0195] In an alternative embodiment, the chimeric molecule may comprise a
fusion of a variant
interferon polypeptide with another protein. Various fusion partners are well
known in the art, and
include but are not limited to the following examples. The variant interferon
proteins of the
invention may be fused to an immunogiobulin or the Fc region of an
immunoglobulin, such as an
IgG molecule. The interferon variants can also be fused to albumin, other
interferon proteins,
other cytokine proteins, the extracellular domains of the interferon receptor
protein,
etc.immunoglobulin or a particular region of an immunoglobulin. For a bivalent
form of the
chimeric molecule, such a fusion could be to the Fc region of an IgG molecule.
[0196] In another embodiment, the N- and C-termini of a variant IFN protein
are joined to create a
cyclized or circularly permutated IFN protein. Various techniques may be used
to permutate
proteins. See US 5,981,200; Maki K, Iwakura M., Seikagaku. 2001 Jan; 73(1): 42-
6; Pan T.,
Methods Enzymol. 2000; 317:313-30; Heinemann U, Hahn M., Prog Biophys Mol
Biol. 1995;
64(2-3): 121-43; Harris ME, Pace NR, Mol Biol Rep. 1995-96; 22(2-3):115-23;
Pan T, Uhlenbeck
OC., 1993 Mar 30; 125(2): 111-4; Nardulli AM, Shapiro DJ. 1993 Winter;
3(4):247-55, EP
1098257 A2; WO 02/22149; WO 01/51629; WO 99/51632; Hennecke, et al., 1999, J.
Mol. Biol.,
286, 1197-1215; Goldenberg et al J. Mol. Biol 165, 407-413 (1983); Luger et
al, Science, 243,
206-210 (1989); and Zhang et al., Protein Sci 5, 1290-1300 (1996); all hereby
incorporated by
reference.
[0197] To produce a circularly permuted IFN protein, a novel set of N- and C-
termini are created at
amino acid positions normally internal to the protein's primary structure, and
the original N- and C-
termini are joined via a peptide linker consisting of from 0 to 30 amino acids
in length (in some
cases, some of the amino acids located near the original termini are removed
to accommodate
the linker design). In a preferred embodiment, the novel N- and C-termini are
located in a non-
regular secondary structural element, such as a loop or turn, such that the
stability and activity of
the novel protein are similar to those of the original protein. The circularly
permuted IFN protein
may be further PEGylated, glycosylated, or otherwise modified. In a further
preferred
embodiment PDA~ technology may be used to further optimize the IFN variant,
particularly in the
32
CA 02528964 2005-12-09
WO 2005/003157 PCT/US2004/009824
regions affected by circular permutation. These include the novel N- and C-
termini, as well as the
original termini and linker peptide.
[0198] In addition, a completely cyclic IFNTPO may be generated, wherein the
protein contains no
termini. This is accomplished utilizing intein technology. Thus, peptides can
be cyclized and in
particular inteins may be utilized to accomplish the cyclization.
[0199] Generating the variants. Variant interferon nucleic acids and proteins
of the invention may be
produced using a number of methods known in the art and described herein.
[0200] Preparing nucleic acids encoding the IFN variants. In a preferred
embodiment, nucleic acids
encoding IFN variants are prepared by total gene synthesis, or by site-
directed mutagenesis of a
nucleic acid encoding wild type or variant IFN protein. Methods including
template-directed
ligation, recursive PCR, cassette mutagenesis, site-directed mutagenesis or
other techniques that
are well known in the art may be utilized (see for example Strizhov et. al.
PNAS 93:15012-15017
(1996), Prodromou and Perl, Prot. Eng. 5: 827-829 (1992), Jayaraman and
Puccini,
Biotechniques 12: 392-398 (1992), and Chafmers et. at. Biotechniques 30: 249-
252 (2001)).
[0201] Expression vectors. In a preferred embodiment, an expression vector
that comprises the
components described below and a gene encoding a variant IFN protein is
prepared. Numerous
types of appropriate expression vectors and suitable regulatory sequences for
a variety of host
cells are known in the art for a variety of host cells. The expression vectors
may contain
transcriptional and transfational regulatory sequences including but not
limited to promoter
sequences, ribosomal binding sites, transcriptional start and stop sequences,
translational start
and stop sequences, transcription terminator signals, polyadenylation signals,
and enhancer or
activator sequences. In a preferred embodiment, the regulatory sequences
include a promoter
and transcriptional start and stop sequences. In addition, the expression
vector may comprise
additional elements. For example, the expression vector may have two
replication systems, thus
allowing it to be maintained in two organisms, for example in mammalian or
insect cells for
expression and in a prokaryotic host for cloning and amplification.
Furthermore, for integrating
expression vectors, the expression vector contains at least one sequence
homologous to the host
cell genome, and preferably two homologous sequences, which flank the
expression construct.
The integrating vector may be directed to a specific locus in the host cell by
selecting the
appropriate homologous sequence for inclusion in the vector. Constructs for
integrating vectors
are well known in the art. In addition, in a preferred embodiment, the
expression vector contains
a selectable marker gene to allow the selection of transformed host cells.
Selection genes are
well known in the art and will vary with the host cell used. The expression
vectors may be either
self-replicating extrachromosomal vectors or vectors which integrate into a
host genome.
[0202] The expression vector may include a secretory leader sequence or signal
peptide sequence
that provides for secretion of the variant IFN protein from the host cell.
Suitable secretory leader
sequences that lead to the secretion of a protein are known in the art. The
signal sequence
typically encodes a signal peptide comprised of hydrophobic amino acids, which
direct the
33
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WO 2005/003157 PCT/US2004/009824
secretion of the protein from the cell, as is well known in the art. The
protein is either secreted
into the growth media or , for prokaryotes, into the periplasmic space,
located between the inner
and outer membrane of the cell. For expression in bacteria, usually bacterial
secretory leader
sequences, operabfy linked to a variant fFN encoding nucleic acid, are usually
preferred.
[0203] TransfectionlTransformation. The variant IFN nucleic acids are
introduced into the cells either
alone or in combination with an expression vector in a manner suitable for
subsequent expression
of the nucleic acid. The method of introduction is largely dictated by the
targeted cell type, as
discussed below. Exemplary methods include CaP04 precipitation, liposome
fusion, Llipofectin~,
electroporation, viral infection, dextran-mediated transfection, polybrene
mediated transfection,
protoplast fusion, direct microinjection, etc. The variant IFN nucleic acids
may stably integrate
into the genome of the host cell or may exist either transiently or stably in
the cytoplasm. As
outlined herein, a particularly preferred method utilizes retroviral
infection, as outlined in PCT/
US97/01019, incorporated by reference.
[0204] ,4ppropriate host cells for the expression of IFN varianfs. Appropriate
host cells for the
expression of IFN variants include yeast, bacteria, archaebacteria, fungi, and
insect and animal
cells, including mammalian cells. Of particular interest are bacteria such as
E. coli and Bacillus
subtilis, fungi such as Saccharomyces cerevisiae, Pichia pastoris, and
Neurospora, insects such
as Drosophila melangaster and insect cell lines such as SF9, mammalian cell
lines including 293,
CHO, COS, Jurkat, NIH3T3, etc (see the ATCC cell line catalog; hereby
expressly incorporated
by reference), as well as primary cell lines.
[0205] Interferon variants can also be produced in more complex organisms,
including but not limited
to plants (such as corn, tobacco, and algae) and animals (such as chickens,
goats, cows); see
for example Dove, Nature Biotechnol. 20: 777-779 (2002).
[0206] In one embodiment, the cells may be additionally genetically
engineered, that is, contain
exogenous nucleic acid other than the expression vector comprising the variant
IFN nucleic acid.
[0207] Expression methods. The variant IFN proteins of the present invention
are produced by
culturing a host cell transformed with an expression vector containing nucleic
acid encoding a
variant IFN protein, under the appropriate conditions to induce or cause
expression of the variant
IFN protein. The conditions appropriate for variant IFN protein expression
will vary with the
choice of the expression vector and the host cell, and will be easily
ascertained by one skilled in
the art through routine experimentation. For example, the use of constitutive
promoters in the
expression vector will require optimizing the growth and proliferation of the
host cell, while the use
of an inducible promoter requires the appropriate growth conditions for
induction. In addition, in
some embodiments, the timing of the harvest is important. For example, the
baculoviral systems
used in insect cell expression are lytic viruses, and thus harvest time
selection can be crucial for
product yield.
[0208] Purification. In a preferred embodiment, the IFN variants are purified
or isolated after
expression. Standard purification methods include electrophoretic, molecular,
immunological and
34
CA 02528964 2005-12-09
WO 2005/003157 PCT/US2004/009824
chromatographic techniques, including ion exchange, hydrophobic, affinity, and
reverse-phase
HPLC chromatography, and chromatofocusing. For example, a IFN variant may be
purified using
a standard anti-recombinant protein antibody column. Ultrafiltration and
diafiltration techniques, in
conjunction with protein concentration, are also useful. For general guidance
in suitable
purification techniques, see Scopes, R., Protein Purification, Springer-
Verlag, NY, 3dr ed. (1994).
The degree of purification necessary will vary depending on the desired use,
and in some
instances no purification will be necessary. For further references on
purification of type I
interferons, see for example Moschera et. al. Meth. Enzym. 119: 177-192
(1986). For further
references on purification of type I interferons, see for example Moschera et.
al. Meth. Enzym.
119: 177-183 (1986) ; Tarnowski et. al. Meth. Enzym. 119:153-165(1986);
Thatcher et. al. Meth.
Enzym. 119:166-177 (1986); Lin et. al. Meth. Enzym. 119:183-192 (1986).
Methods for
purification of interferon beta are disclosed in US 4,462,940 and US 4,894,
330.
[0209] Posttranslational modification and derivitization. Once made, the
variant IFN proteins may be
covalentfy modified. Covalent and non-covalent modifications of the protein
are thus included
within the scope of the present invention. Such modifications may be
introduced into a variant
IFN polypeptide by reacting targeted amino acid residues of the polypeptide
with an organic
derivatizing agent that is capable of reacting with selected side chains or
terminal residues.
Optimal sites for modification can be chosen using a variety of criteria,
including but not limited to,
visual inspection, structural analysis, sequence analysis and molecular
simulation.
[0210] In one embodiment, the variant IFN proteins of the invention are
labeled with at least one
element, isotope or chemical compound. In general, labels fall into three
classes: a) isotopic
labels, which may be radioactive or heavy isotopes; b) immune labels, which
may be antibodies
or antigens; and c) colored or fluorescent dyes. The labels may be
incorporated into the
compound at any position. Labels include but are not limited to biotin, tag
(e.g. FLAG, Myc) and
fluorescent labels (e.g. fluorescein).
[0211] Derivatization with bifunctional agents is useful, for instance, for
cross linking a variant IFN
protein to a water-insoluble support matrix or surface for use in the method
for purifying anti-
variant IFN antibodies or screening assays, as is more fully described below.
Commonly used
cross linking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,
glutaraldehyde, N-
hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid,
homobifunctional
imidoesters, including disuccinimidyl esters such as 3,3'-
dithiobis(succinimidylpropionate),
bifunctional maleimides such as bis-N-maleimido-1,8-octane and agents such as
methyl-3-[(p-
azidophenyl)dithio] propioimidate.
j0212] Other modifications include deamidation of gfutaminyi and asparaginyi
residues to the
corresponding glutamyl and aspartyl residues, respectively, hydroxylation of
proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation
of the "-amino
groups of lysine, arginine, and histidine side chains ([T.E. Creighton,
Proteins: Structure and
CA 02528964 2005-12-09
WO 2005/003157 PCT/US2004/009824
Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983))],
acetylation of the
N-terminal amine, and amidation of any C-terminal carboxyl group.
[0213] Such derivitization may improve the solubility, absorption,
permeability across the blood brain
barrier, serum half life, and the like. Modifications of variant IFN
polypeptides may alternatively
eliminate or attenuate any possible undesirable side effect of the protein.
Moieties capable of
mediating such effects are disclosed, for example, in Remington's
Pharmaceutical Sciences, 16th
ed., Mack Publishing Co., Easton, Pa. (1980).
[0214] Another type of covalent modification of variant IFN comprises linking
the variant IFN
polypeptide to one of a variety of nonproteinaceous polymers, e.g.,
polyethylene glycol ("PEG"),
polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S.
Patent Nos. 4,640,835;
4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337. A variety of coupling
chemistries may
be used to achieve PEG attachment, as is well known in the art. Examples,
include but are not
limited to, the technologies of Shearwater and Enzon, which allow modification
at primary amines,
including but not limited to, cysteine groups, histidine groups, lysine groups
and the N- terminus.
S (see, Kinstler et al, Advanced Drug Deliveries Reviews, 54, 477-485 (2002)
and MJ Roberts et
al, Advanced Drug Delivery Reviews, 54, 459-476 (2002), both hereby
incorporated by
reference). Both labile and non-labile PEG linkages may be used.
[0215] Addition of Nobex polymers and the like. An additional form of covalent
modification includes
coupling of the variant 1FN polypeptide with one or more molecules of a
polymer comprised of a
lipophililic and a hydrophilic moiety. Such composition may enhance resistance
to hydrolytic or
enzymatic degradation of the IFN protein. Polymers utilized may incorporate,
for example, fatty
acids for the lipophilic moiety and linear polyalkylene glycols for the
hydrophilic moiety. The
polymers may additionally incorporate acceptable sugar moieties as well as
spacers used for IFN
protein attachment. Polymer compositions and methods for covalent conjugation
are described,
for example, in U.S. Patent Nos. 5,681,811; 5,359,030.
[0216] Another type of modification is chemical or enzymatic coupling of
glycosides to the variant
IFN protein. Such methods are described in the art, e.g., in WO 87!05330
published 11
September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306
(1981).
[0217] Alternatively, removal of carbohydrate moieties present on the variant
IFN polypeptide may
be accomplished chemically or enzymatically. Chemical deglycosylation
techniques are known in
the art and described, for instance, by Hakimuddin, et al., Arch. Biochem.
Biophys., 259:52 (1987)
and by Edge et al., Anal. Biochem., 118:131 (1981 ). Enzymatic cleavage of
carbohydrate
moieties on polypeptides can be achieved by the use of a variety of endo-and
exo-glycosidases
as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).
[0218] A primary object of the invention is the identification of variant
interferon proteins with
improved solubility. Accordingly, in a preferred embodiment, the variant
interferon proteins are
assayed for solubility using methods including but not limited to those
described below.
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[0219] In all preferred embodiments, the variant and wild-type proteins are
usually compared directly
in the same assay system and under the same conditions in order to evaluate
the solubility of
each variant.
[0220] The solubility of the interferon variant proteins may be determined
under a number of solution
conditions. A variety of excipients, including solubilizing and stabilizing
agents, may be tested for
their ability to promote the highest stable IFN concentration. In addition,
different salt
concentrations and varying pH may be tested. In a preferred embodiment,
solubility is assayed
under pharmaceutically acceptable conditions.
[0221] In order to evaluate the effectiveness of the modifications introduced
to the protein, suitable
biophysical measurements must be taken. In this instance, the protein
concentration over time,
relative mobility on native gel electrophoresis, and oligomeric state are the
primary criteria of
interest. In the case of poor solubility, aggregates will form over time in
the protein solution, and
eventually precipitate entirely. In the latter case, a drop in solution
protein concentration over time
will be observed, following centrifugation and sampling of the solution phase.
A description of
techniques to evaluate oligomeric state, including detection of aggregates,
follows below (DLS,
AUC, SEC, etc).
[0222] To determine the impact of mutations introduced to the molecule,
variant and wild-type
proteins should be compared directly in the same assay system. When comparable
data are
available, the evaluation of the influence of a given mutation (the variant)
on solubility may be
rendered.
[0223] Soluble expression. The variables to be tested in order to achieve
expression of IFN as a
soluble protein rather than in inclusion bodies are to lower the fermentation
temperature, to lower
the concentration of the inducing agent, and to attempt the application of a
commercially-available
enzyme system engineered to to optimize soluble protein expression (Athena
Expression
Systems, Baltimore, MD).
[0224] Maximum concentration under different solution conditions. A variety of
excipients, including
solubilizing and stabilizing agents will be tested for their ability to
promote the highest stable IFNb
concentration. In addition, different salt concentrations and varying pH will
be tested. The tests
utilized will be the same as in the assay of protein solubility, because
essentially the solubility of
the protein at varying high concentrations will be tested in this methodology.
[0225] Besides native gel electrophoresis, these are the methods of choice for
evaluation of protein
oligomeric state.
[0226] Dynamic light scattering (DLS) -- DLS is a method useful for
determination of diffusion
coefficients based on signal correlation from fluctuation of laser light
scattered from Brownian
motion of particles in solution (Light scattering by Polymer Solutions, Paul
C. Heimenz, Chapter
in Polymer Chemistry, Marcel Dekker, Inc., NY, 1984, pp. 659-701 ).
Commercially- available
instruments provide graphical or table readouts of particle populations) by
sizes) after
transforming the diffusion coefficients) measured by
deconvolution/autocorrelation of laser light
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scattering data using the Stokes-Einstein equation. The size is therefore the
hydrodynamic radius.
Particle size standards aremay be used to check the accuracy of the instrument
settings
(nanoparticles obtained from Duke Scientific Corporation, Palo Alto, CA). The
distribution of
particle sizes within a populations) is the dispersity, and this factor
provides data on the
uniformity of the particle population(s). Both dispersity and the appearance
of aggregates over
time may be monitored to test for solubility. - this should be monitored as
well as the appearance
of aggregates over time for an indication of stability.
[0227] Aggregated protein will may be easily resolved by DLS, and readily
detected at low levels due
to the physical property of aggregates: they scatter more laser light per unit
due to the greater
target surface area. Concentration requirements can be on the order of mg/ml
to obtain
reasonable signal, however as little as 10 microliter in some model
instruments can be detected,
for a total sample amount requirement on the order of 10 microgram per sample
condition tested
(allowing additional material for sample handling losses). The sample is may
be directly
introduced into the cuvette (i.e. it is not necessary to perform a
chromatographic step first) and
not separated on a separate matrix requiring time and removal from the test
solution conditions
(in the event of a differently formulated mobile phase) as in the case of
static light scattering. A
relative ratio of monodisperse to aggregate particle population is generally
available from this line
of instruments, sometimes weighted by mass sometimes by light-scattering
intensity, and
sometimes a choice of either, depending on software provided by the
manufacturermay be
determined. Optionally, this ratio may be weighted by mass or by light
scattering intensity. Thus,
DLS is an idea preferred technique to monitor formation of aggregates, and
holds the advantage
in that it is a non-intrusive technique.
[0228] Analytical ultracentrifugation (AUC). In another preferred embodiment
(AUC) is used to
determine the oligomerization state of the variant interferon proteins. AUC
can be performed in
two different 'modes', either velocity or equilibrium. Equilibrium AUC can be
considered the 'gold
standard' for determining protein molecular weight and oligomeric state
measurement.
[0229] Size exclusion chromatography (SEC) -- A further preferred embodiment
is to use size-
exclusion chromatography (SEC) to determine the oligomerization state of the
variant interferon
proteins. Utilizing high performance liquid chromatography, sample may be
introduced to an.
isocratic mobile phase and separated on a gel permeation matrix designed to
exclude protein on
the basis of size. Thus, the samples will be "sieved" such that the aggregated
protein will elute
first with the shortest retention time, and will be easily separated from the
remainder. This will
unequivocallycan identify aggregates and allow a relative quantification by
peak integration using
the peak analysis software provided with the instrument.
[0230] In an alternate embodiment, protein concentration is monitored as a
function of time. In the
case of poor solubility, aggregates will form over time in the protein
solution, and eventually
precipitate entirely. This may be performed following centrifugation and
sampling of the solution
38
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phase, in which case insolubility can be measured as a drop in solution
protein concentration over
time will be observed following centrifugation.
[0231] In an alternate embodiment, the oligomerization state is determined by
monitoring relative
mobility on native gel electrophoresis.
[0232] In another embodiment, the amount of protein that is expressed solubly
in a prokaryotic host
is determined. While factors other than the solubility of the native protein
can impact levels of
soluble expression, improvements in soluble expression may correlate with
improvements in
solubility. Any of a number of methods may be used; for example, following
expression, SDS-
pofyacryfamide gel electrophoresis and/or western blots can be done on the
soluble fraction of
crude cell lysates or the expression media. There are also high throughput
screens for soluble
expression. In one embodiment, the protein of interest is fused to a
fluorescent protein such as
GFP, and the cells monitored for fluorescence (Waldo et. al. Nat. Biotechnol.
17: 691 (1999)). In
an alternate embodiment, the protein of interest is fused to the antibiotic
resistance enzyme
chloramphenicol transferase. If the protein expresses sofubly, the enzyme will
be functional,
thereby allowing growth on media with increased concentration of the
antibiotic chloramphenicol
(Maxwell et. al. Protein Sci. 8: 1908 (1999)). In another embodiment, the
protein of interest is
expressed as a fusion with the alpha domain of the enzyme beta-galactosidase.
If the protein
expresses in soluble form, the alpha domain will complement the omega domain
to yield a
functional enzyme. This may be detected as blue rather than white colony
formation when the
cells are plated on media containing the indicator X-gal (Wigley et. al. Nat.
Biotechnol. 19: 131
(2001 )).
[0233] Assaying the activity of the variants. In a preferred embodiment, the
wild-type and variant
proteins are analyzed for biological activities by suitable methods known in
the art. Such assays
include but are not limited to activation of interferon-responsive genes,
receptor binding assays,
antiviral activity assays, cytopathic effect inhibition assays, (Familletti
et. al., Meth. Enzymol.
78:387-394), antiproliferative assays, (Aebersold and Sample, Meth. Enzymol.
119:579-582),
immunomodulatory assays (US 4,914,033;4,753,795), and assays that monitor the
induction of
MHC molecules (for example, Hokland et al, Meth. Enzymol. 119:688-693)., as
described in
Meager, J. Immunol. Meth., 261:21-36 (2002).
[0234] In a preferred embodiment, wild type and variant proteins will be
analyzed for their ability to
activate interferon-sensitive signal transduction pathways. One example is the
interferon-
stimulated response element (ISRE) assay, described below and in the Examples.
Cells which
constitutively express the type I interferon receptor (for example Hela cells,
293T cells) are
transiently transfected with an ISRE-luciferase vector. After transfection,
the cells are treated with
an interferon variant. In a preferred embodiment, a number of protein
concentrations, for example
from 0.0001 -10 ng/mL, are tested to generate a dose-response curve. In an
alternate
embodiment, two or more concentrations are tested. If the variant binds and
activates its
receptor, the resulting signal transduction cascade induces luciferase
expression.
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Luminseescence can be measured in a number of ways, for example by using a
TopCountTM or
FusionTM microplate reader.
[0235] In a preferred embodiment, wild type and variant proteins will be
analyzed for their ability to
bind to the type 1 interferon receptor (IFNAR). Suitable binding assays
include, but are not limited
to, BIAcore assays (Pearce et al., Biochemistry 38:81-89 (1999)) and
AIphaScreenTM assays
(commercially available from PerkinElmer) (Bosse R., Illy C., and Chelsky D
(2002). Principles of
AIphaScreenTM PerkinElmer Literature Application Note Ref# s4069.
AIphaScreenTM is a bead-
based non-radioactive luminescent proximity assay where the donor beads are
excited by a laser
at 680 nm to release singlet oxygen. The singlet oxygen diffuses and reacts
with the thioxene
derivative on the surface of acceptor beads leading to fluorescence emission
at 600 nm. The
fluorescence emission occurs only when the donor and acceptor beads are
brought into close
proximity by molecular interactions occurring when each is linked to ligand
and receptor
respectively. This ligand-receptor interaction can be competed away using
receptor-binding
variants while non-binding variants will not compete.
[0236] In an alternate preferred embodiment, wild type and variant proteins
will be analyzed for their
efficacy in treating an animal model of disease, such as the mouse or rat EAE
model for multiple
sclerosis.
[0237] In an alternate preferred embodiment, wild type and variant proteins
will be analyzed for their
antiviral activity.
[0238] Antiproliferative activity: In an alternate preferred embodiment, wild
type and variant proteins
will be analyzed for their efficacy in treating an animal model of disease,
such as the mouse or rat
EAE model for multiple sclerosis.
[0239] Determining the immunogenicity of the variants In the case of
engineered MHC
immunogenicity, the variants are tested. Also, in some cases, increased
protein solubility may
decrease immunogenicity by reducing uptake by antigen presenting cells.
Accordingly, in a
preferred embodiment, uptake of wild type and variant interferon proteins by
professional antigen
presenting cells is monitored.
[0240] In a preferred embodiment, the immunogenicity of the interferon
variants is determined
experimentally to confirm that the variants do have reduced or eliminated
immunogenicity relative
to the parent protein. Again the discussion below is centered around IFN-[3,
but the methods can
be utilized for any IFN of the invention.
[0241] In a preferred embodiment, ex vivo T-cell activation assays are used to
experimentally
quantitate immunogenicity. In this method, antigen presenting cells and naive
T cells from
matched donors are challenged with a peptide or whole protein of interest one
or more times.
Then, T cell activation can be detected using a number of methods, for example
by monitoring
production of cytokines or measuring uptake of tritiated thymidine. In the
most preferred
embodiment, interferon gamma production is monitored using Elispot assays (see
Schmittel et. al.
J. Immunol. Meth., 24: 17-24 (2000)).
CA 02528964 2005-12-09
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[0242] Other suitable T-cell assays include those disclosed in Meidenbauer, et
al. Prostate 43, 88
100 (2000); Schultes, B.C and Whiteside, T.L., J. Immunol. Methods 279, 1-15
(2003); and
Stickler, et al., J. Immunotherapy, 23, 654-660 (2000).
[0243] fn a preferred embodiment, the PBMC donors used for the above-described
T-cell activation
assays will comprise class II MHC alleles that are common in patients
requiring treatment for
interferon beta responsive disorders. For example, for most diseases and
disorders, it is
desirable to test donors comprising all of the alleles that are prevalent in
the population.
However, for diseases or disorders that are linked with specific MHC alleles,
it may be more
appropriate to focus screening on alleles that confer susceptibility to
interferon beta responsive
disorders.
[0244] In a preferred embodiment, the MHC haplotype of PBMC donors or patients
that raise an
immune response to the wild type or variant interferon beta are compared with
the MHC
haplotype of patients who do not raise a response. This data may be used to
guide preclinical
and clinical studies as well as aiding in identification of patients who will
be especially likely to
respond favorably or unfavorably to the interferon beta therapeutic.
[0245] In an alternate preferred embodiment, immunogenicity is measured in
transgenic mouse
systems. For example, mice expressing fully or partially human class II MHC
molecules may be .,-
used.
[0246] In an alternate embodiment, immunogenicity is tested by administering
the interferon beta
variants to one or more animals, including rodents and primates, and
monitoring for antibody
formation. Non-human primates with defined MHC haplotypes may be especially
useful, as the
sequences and hence peptide binding specificities of the MHC molecules in non-
human primates
may be very similar to the sequences and peptide binding specificities of
humans. Similarly,
genetically engineered mouse models expressing human MHC peptide-binding
domains may be
used (see for example Sonderstrup et. al. Immunol. Rev. 172: 335-343 (1999)
and Forsthuber et.
al. J. Immunol. 167: 119-125 (2001)).
[0247] Administration and Treatment using IFN variants. Once made, the variant
IFN proteins and
nucleic acids of the invention find use in a number of applications. In a
preferred embodiment, a
variant IFN protein or nucleic acid is administered to a patient to treat an
IFN related disorder.
[0248] The administration of the variant IFN proteins of the present
invention, preferably in the form
of a sterile aqueous solution, may be done in a variety of ways, including,
but not limited to, orally,
parenterally, subcutaneously, intravenously, intranasally, transdermally,
intraperitoneally,
intramuscularly, intrapulmonary, vaginally, rectally, intranasally or
intraocularly. In some
instances, the variant IFN protein may be directly applied as a solution or
spray. Depending upon
the manner of introduction, the pharmaceutical composition may be formulated
in a variety of
ways.
[0249] The pharmaceutical compositions of the present invention comprise a
variant IFN protein in a
form suitable for administration to a patient. In the preferred embodiment,
the pharmaceutical
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compositions are in a water-soluble form, such as being present as
pharmaceutically acceptable
salts, which is meant to include both acid and base addition salts.
[0250] The pharmaceutical compositions may also include one or more of the
following: carrier
proteins such as serum albumin; buffers such as NaOAc; fillers such as
microcrystalline cellulose,
lactose, corn and other starches; binding agents; sweeteners and other
flavoring agents; coloring
agents; and polyethylene glycol. Additives are well known in the art, and are
used in a variety of
formulations, and discussed above.
[0251] In a further embodiment, the variant IFN proteins are added in a
micellular formulation; see
U.S. Patent No. 5,833,948, hereby expressly incorporated by reference in its
entirety.
[0252] Combinations of pharmaceutical compositions may be administered.
Moreover, the
compositions may be administered in combination with other therapeutics.
[0253] In a preferred embodiment, the nucleic acid encoding the variant IFN
proteins may also be
used in gene therapy. In gene therapy applications, genes are introduced into
cells in order to
achieve in vivo synthesis of a therapeutically effective genetic product, for
example for
replacement of a defective gene. "Gene therapy" includes both conventional
gene therapy where
a lasting effect is achieved by a single treatment, and the administration of
gene therapeutic
agents, which involves the one time or repeated administration of a
therapeutically effective DNA
or mRNA. The oligonucleotides canmay be modified to enhance their uptake, e.g.
by substituting
their negatively charged phosphodiester groups by uncharged groups.
[0254] There are a variety of techniques available for introducing nucleic
acids into viable cells. The
techniques vary depending upon whether the nucleic acid is transferred into
cultured cells in vitro,
or in vivo in the cells of the intended host. Techniques suitable for the
transfer of nucleic acid into
mammalian cells in vitro include the use of liposomes, electroporation,
microinjection, cell fusion,
DEAE-dextran, the calcium phosphate precipitation method, etc. The currently
preferred in vivo
gene transfer techniques include transfection with viral (typically
retroviral) vectors and viral coat
protein-liposome mediated transfection [(Dzau et al., Trends in Biotechnology
11:205-210
(1993))]. In some situations it is desirable to provide the nucleic acid
source with an agent that
targets the target cells, such as an antibody specific for a cell surface
membrane protein or the
target cell, a ligand for a receptor on the target cell, etc. Where Gposomes
are employed, proteins
which bind to a cell surface membrane protein associated with endocytosis may
be used for
targeting and/or to facilitate uptake, e.g. capsid proteins or fragments
thereof tropic for a particular
cell type, antibodies for proteins which undergo internalization in cycling,
proteins that target
intracellular localization and enhance intracellular half-life. The technique
of receptor-mediated
endocytosis is described, for example, by Wu et ai., J. Biol. Chem. 262:4429-
4432 (1987); and
Wagner et al., Proc. Natl. Acad. Sci. U.S.A. 87:3410-3414 (1990). For review
of gene marking
and gene therapy protocols see Anderson et al., Science 256:808-813 (1992).
[0255] While the foregoing invention has been described above, it will be
clear to one skilled in the
art that various changes and additional embodiments made be made without
departing from the
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scope of the invention. All publications, patents, patent applications
(provisional, utility and PCT)
or other documents cited herein are incorporated by references in their
entirety.
[0256] EXAMPLES
[0257] Example 1: Construction of a homology model of interferon kappa
[0258] A homology model of interferon kappa was constructed based on the
sequence of human
interferon kappa (GenBank code 14488028), the crystal structures for
interferon tau (PDB code
1BL5) and interferon beta (PDB code 1AU1), as well as the NMR structure for
interferon alpha-2a
(PDB code 11TF). The sequences for interferons alpha-2a, beta, kappa, and tau
were aligned
using the multiple sequence alignment tool in the Homology model of the
Insightll software
package (Accelrys), as shown in Figure 2. As the sequences share only
approximately 35%
identity, slightly different sequence alignments could have been used instead
(see for example
LaFleur et. al. J. Biol. Chem. 276: 39765-39771 (2001)). Based on similarity
to the other
interferon sequences, disulfide bonds are expected to be formed between
residues C3 and C102
and between residues C32 and C155 (LaFleur supra); these disufides were used
as constraints in
the generation of the homology models. A total of nine homology models were
generated using
the Modeler tool in the Insightll software package (Accelrys). The structures
were analyzed for
quality and the top four models were used in the analysis and design
calculations described
below.
[0259] Example 2: Identification of exposed hydrophobic residues in type I
interferons
[0260] A number of type I interferon structures were analyzed to identify
solvent-exposed
hydrophobic residues. The absolute and fractional solvent-exposed hydrophobic
surface area of
each residue was calculated using the method of Lee and Richards (J. Mol.
Biol. 55: 379-400
(1971)) using an add-on radius of 1.4 A (Angstroms). Each residue was also
classified as core,
boundary, or surface (see Dahiyat and Mayo Science 278: 82-87 (1997)).
[0261] Solvent exposed hydrophobic residues in interferon-alpha 2a were
defined to be hydrophobic
residues with at least 75 A2 (square Angstroms) exposed hydrophobic surface
area in the
interferon alpha-2a NMR structure (PDB code 11TF, first molecule).
[0262] Table 1. Exposed hydrophobic residues in interferon-alpha 2a.
core / exposed
boundary l hydrophobic percent hydrophobic
residue# surface surface areaarea exposed
MET 16 surface 93.90 44.50
PHE 27 surface 172.10 69.10
LEU 30 surface 84.20 39.40
TYR 89 surface 80.00 41.10
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ILE 100 surface 103.60 50.00
LEU 110 surface 151.30 70.20
MET 111 surface 76.40 35.60
LEU 117 surface 78.60 37.80
LEU 128 surface 104.30 50.40
LEU 161 surface 90.10 45.30
[0263] Solvent exposed hydrophobic residues in interferon beta were defined to
be hydrophobic
residues with at least 75 A~ (square Angstroms) exposed hydrophobic surface
area in the
interferon-beta crystal structure (PDB code 1AU1, chain A)
[0264] Table 2. Exposed hydrophobic residues in interferon-beta.
exposed
core / percent hydrophobic
surface
hydrophobic
residue# / boundarysurface area buried
area
LEU 5 boundary 100.30 48.30
PHE 8 surface 131.00 54.90
PHE 15 surface 151.90 63.30
TRP 22 surface 147.90 58.30
LEU 28 boundary 61.90 31.00
TYR 30 surface 129.00 66.80
LEU 32 surface 50.40 23.70
MET 36 boundary 82.60 40.00
LEU 47 boundary 72.20 35.50
TYR 92 surface 84.60 44.40
PHE 111 surface 196.30 80.10
LEU 116 surface 94.60 45.70
LEU 120 surface 67.20 32.50
LEU 130 surface 57.10 27.40
VAL 148 boundary 77.40 42.80
TYR 155 surface 88.60 46.30
[0265] Solvent exposed hydrophobic residues in interferon-kappa were defined
to be hydrophobic
residues with at least 30 A2 (square Angstroms) exposed hydrophobic surface
area in at least one
of the top four homology models (see above) and which were classified as
boundary (B) or
surface (S) in at least 3 of the 4 top structures. Solvent exposed hydrophobic
residues in
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interferon kappa, along with their exposed hydrophobic surface area and ClS/B
classification, are
shown below.
[0266] Table 3. Exposed hydrophobic residues in interferon kappa.
Solvent exposed hydrophobic surface areas in square Angstroms are given for
the top four
homology models. Core / surface / boundary classification is indicated as "C",
"S", or "B" below.
model1 model2 model3 model4
LEU1 134.57S 135.88 B 91.03 B 134.11
S
LEU5 102.62S 89.78 B 70.67 S 103.39
S
VAL8 70.36 S 76.97 S 70.19 S 72.51 S
TRP15 155.63S 161.08 S 149.83 S 153.22
S
LEU18 33.86 B 42.72 B 64.82 B 34.39 B
PHE28 39.03 S 32.47 B 16.19 B 34.43 S
VAL30 118.49S 112.38 S 43.12 S 118.23
S
LEU33 92.00 S 73.35 S 72.73 S 93.60 S
ILE37 106.52B 127.16 B 99.30 B 106.28
B
LEU46 84.43 S 86.04 S 84.47 S 83.90 S
TYR48 79.98 B 60.73 B 93.88 B 81.91 B
MET52 101.62B 149.86 S 149.37 S 104.68
S
LEU65 109.14B 98.21 S 111.58 B 91.38 S
PHE68 55.88 B 107.51 B 104.30 B 57.45 B
PHE76 61.69 B 66.90 B 53.90 B 59.28 B
TYR78 104.70B 112.65 S 135.51 B 111.51
B
TRP79 57.96 S 138.78 B 133.03 C 58.32 S
ILE88 104.67S 77.94 S 77.75 S 111.79
S
TYR96 98.61 B 118.35 B 63.52 B 97.46 B
MET111 118.98B 152.74 S 115.40 B 109.32
B
MET114 141.73S 188.48 S 174.59 S 134.99
B
MET119 147.52S 173.09 S 159.56 S 134.72
S
VAL126 23.49 C 77.29 S 70.45 B 54.01 S
LEU132 86.27 S 95.70 S 81.83 S 84.16 S
TYR150 41.55 B 62.57 B 86.01 B 45.22 B
~
VAL160 49.02 B 69.23 S 70.61 B 49.02 B
TYR167 99.52 S 84.23 S 149.46 S 100.52
S
TYR170 63.85 S 77.37 S 110.88 S 61.83 S
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[0267) The results in Table 3 were combined with the sequence analysis
described in Example 4 to
identify exposed hydrophobic residues in interferon kappa that could be
replaced with polar
residues without compromising the structure or function of the resulting
variant protein.
[0268] Solvent exposed hydrophobic residues in ovine interferon tau were
defined to be hydrophobic
residues that were at least 25 % exposed to solvent in the crystal structure
of interferon tau (PDB
code 1 B5L).
[0269] Table 4. Exposed hydrophobic residues in interferon-tau. The exposed
hydrophobic surface
areas
Percent
C/S/B Exposed hydrophobic
area
Residue# classificationhydrophobic burial
area
TYR 2 surface 153.9 22.9
LEU 9 surface 85.8 59.1
LEU 24 boundary 121.1 42.5
LEU 30 surface 152.2 25.8
TYR 69 surface 71.6 62.5
TRP 77 surface 233.3 6.3
MET 114 surface 137.6 36.9
VAL 118 surface 103.9 42.9
TYR 136 boundary 53.3 72.6
VAL 146 boundary 64.5 63.9
[0270) Example 3: Identification of dimer interface residues in type I
interferons
[0271) Potential sites of interactions between interferon monomers were
identified by examining
contacts between monomers in the crystal structures of interferon molecules.
[0272) Interferon alpha-2b crystallized as a trimer of dimers (PDB code 1
RH2), in which the dimer
interface is zinc-mediated (see Radhakrishnan et. al. Structure 4: 1453-1463
(1996)). The zinc-
mediated dimer is referred to herein as the "AB dimer", while the interface
between AB dimers is
referred to as the "BC" dimer interface. The zinc-binding site comprises the
residues Glu 41 and
Glu 42. Additional residues that have been implicated in stabilizing the AB
dimer interface include
Lys 121, Asp 114, Gly 44, and Arg 33 (Radhakrishnan, supra).
[0273) Next, distance measurements were used to identify additional residues
that may participate in
intermolecular interactions. Residues that are within 8 A (Angstroms) of the
AB dimer interface
(as measured by CA-CA distances) include: 35-37, 39-41, 44-46, 114-115, 117-
118, 121-122,
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and 125. Residues that are within 8 A of the BC dimer-dimer interface (as
measured by CA-CA
distances) include: 16, 19, 20, 25, 27, 28, 30, 33, 54, 58, 61, 65, 68, 85,
93, 99, 112, 113, and
149.
[0274] Interferon beta crystallized as an asymmetric dimer (PDB code 1AU1).
Residues that are
within 5 ,4 of the dimer interface (minimum heavy atom-heavy atom distance)
include 42, 43, 46-
49, 51, 113, 116, 117, 120, 121, and 124 (on chain A), as well as 1-6, 8, 9,
12, 16, 93, 96, 97,
100, 101, and 104 (on chain B).
[0275] Example 4: Identification of residues observed at each position in the
interferon family
[0276] A large number of type f interferon sequences are known to exist,
comprising interferons of
different subtypes (e.g. alpha-2, alpha-4, beta, kappa), allelic variants
(e.g. alpha-2a vs. alpha-
2b), and interferons from different species. Analysis of these different
interferon sequences can
suggest substitutions that will be compatible with maintaining the structure
and function of type I
interferons.
[0277] The BLAST sequence alignment program was used to identify the 100
protein sequences in
the nonredundant protein sequence database that are most closely related to
interferon kappa.
The annotations for these sequences were analyzed to confirm that all of the
sequences are type
one interferons. Next, the number of occurrences of each residue (and of
deletions, denoted "-")
at each position in interferon kappa was determined. For example, the
frequency of each residue
at the exposed hydrophobic positions in interferon kappa is shown below.
[0278] Table 5. Frequency of each residue at exposed hydrophobic positions in
interferon kappa.
# wt - A C D E F G H I K L M N P Q R S T V W
Y
1 L 0 0 0 0 0 0 0 0 0 0 39 0 0 0 0 0 0 0 0 0
0
L 0 0 0 0 0 0 0 0 0 0 39 0 0 0 0 0 0 0 0 0
0
8 V 0 0 0 5 0 0 0 1 1 0 0 0 15 0 0 0 2 12 3 0
0
W 17 0 0 0 0 0 0 0 0 0 5 2 0 0 0 8 1 2 0 4
0
18 L 0 0 0 0 0 1 0 0 0 0 73 0 0 0 0 0 0 0 0 0
0
28 F 10 0 0 0 0 3 0 0 0 0 0 0 0 1 0 0 70 0 0 0
0
30 V 0 1 0 0 0 16 0 44 0 0 0 0 0 2 0 4 9 0 8 0
0
33 L 0 0 0 0 0 0 0 0 0 0 88 0 0 0 2 0 0 0 0 0
0
37 I 0 0 0 0 0 0 0 4 3 51 0 5 12 1 0 12 0 1 0 0
1
46 L 0 1 0 0 0 12 0 0 0 6 13 0 0 0 0 0 0 0 580
0
48 Y 0 0 0 1 0 0 780 0 0 5 0 0 1 1 0 0 0 1 0
3
52 M 1 0 0 0 0 0 0 0 0 0 0 3 0 2 80 4 0 0 0 0
0
65 L 0 0 0 0 0 0 0 0 0 0 3 0 0 0 87 0 0 0 0 0
0
68 F 0 0 0 0 0 85 0 0 0 0 2 0 0 0 0 0 1 0 1 0
1
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76 F 0 111 0 0 4 0 0 0 0 0 0 0 1 0 0 73 0 0 0 0
78 Y 0 680 0 0 0 0 0 0 0 0 1 0 0 0 0 5 9 4 0 3
79 W 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 900
89 I 0 120 2 0 0 0 0 4 0 0 0 1 0 0 0 3 68 0 0 0
97 Y 0 0 0 46 6 0 5 26 0 0 0 0 4 0 0 0 0 0 0 0 3
112M 43 130 0 0 6 0 0 1 0 1 5 0 3 0 0 1 10 7 0 0
115M 19 4 0 0 3 0 6 0 5 1 8 16 0 0 0 0 27 1 0 0 0
120M 38 0 0 0 0 0 0 0 1 0 5 44 0 0 0 0 0 0 2 0 0
127V 77 0 0 0 0 0 4 0 0 0 2 3 0 0 0 0 0 0 4 0 0
133L 4 0 0 0 0 0 0 0 0 0 64 0 0 0 0 0 0 0 22 0 0
151Y 0 0 0 1 0 0 0 13 0 0 0 0 0 0 0 0 0 0 0 0 76
161V 0 210 0 0 0 0 0 0 0 3 17 0 0 0 0 0 0 49 0 0
168Y 12
171Y 0 0 0 0 0 0 0 0 0 0 0 0 6 0 0 0 0 12 0 0 3
[0279] The raw frequencies above were normalized using the method of Henikoff
& Henikoff (J. Mol.
Biol. 243: 547-578 (1994)). Numerical values are only included for cells in
which the number of
occurrences in the table above is greater than 0.
[0280] Tabie 6. Normalized frequency of each residue at exposed hydrophobic
positions in
interferon kappa.
# wt A C D E F G H I K L M P Q R S T V Y
- N W
1 L _ _ _ _ _ _ _ _ _ 0.6- _ _ _ _ _ _ _
- _ _
L _ _ _ _ _ _ _ _ _ 0.6- _ _ _ _ _ _ _
- _ _
8 V - - 0 - - - 0 0 - - - - - - 0.10.20.2 -
- 0.1 -
15W - - - - - - - - - 0.10.1 - - 0 0 0.1- -
- - 0.2
18L _ _ _ - p _ _ _ _ 0.7- _ _ _ _ _ _ _
- _ _
28F - - - - 0.2 - - - - - - 0 - - 0.4- - -
- - -
30V 0 - - - 0.2 - 0.1- - - - 0 - 0 0.3- 0.2 -
- - -
33L _ _ _ _ _ _ _ _ _ 1 _ _ 0 _ _ _ _ _
- _ _
37I - - - - - - 0.10.2 - 0.1 0 - 0.1- 0 - 0
- 0.1 0.3 -
46L 0 - - - 0.2 - - - 0.2 0.5- - - - - - 0.2 -
- - -
48Y - - 0 - - 0.6 - - - 0.1- 0 0 - - - 0 0.2
- - -
52M _ _ _ _ _ _ _ _ _ _ 0.2 0 0.8 0 - _ - _
- - _
65L _ _ _ _ _ _ _ _ _ 0.2- _ 0.8 - _ _ _ _
- _ _
68F - - - - 0.9 - - - - 0 - - - - 0 - 0 0
- - -
48 '
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76 F 0.2 - - 0.2 - - - - - - 0 - - 0.5 - - - -
- 0 -
78 Y 0.4 - - - - - - - - 0 - - - - 0 0.3 0.1 0.2
- - -
79 W _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 1 _
-
89 I 0.4 0.1 - - - 0.2 - - 0 - - - 0 0.4 - -
- - - - -
97 Y - - 0.4 0 - 0.3 - - - 0.1 - - - - - - 0.2
- 0 - -
112M 0.2 - - 0.2 - 0 - 0 0.2 - 0 - 0 0.1 0 -
- - - - -
115M 0 - - 0 - 0.2 - 0.1 0.20.3 - - - 0.1 0 - -
- 0 -
120M - - - - - - - 0 - 0 0.3 - - - - - 0 -
- -
127V _ _ _ _ _ 0 _ _ _ 0.10 - _ _ _ _ _ 0.2 _
- -
133L _ _ _ _ _ _ _ _ _ 0.9- _ _ _ _ _ _ p _ _
-
151Y _ _ 0 _ _ _ 0.3 - _ _ _ _ _ _ _ _ _ _ 0.7
- _
161V 0.4 _ _ _ _ _ _ _ 0 0.1 - _ _ _ _ 0.5 -
- - _ -
168Y 0.4
171Y _ _ _ _ _ _ _ _ _ _ _ 0.2 _ _ _ 0.4 - 0.2
- _ -
[0281] This sequence alignment data was used in conjunction with the PDA~
technology
calculations described above to identify suitable residues for different
variable positions. If
hydrophobicity at a given position was found to be conserved among interferons
(i.e. the
frequency of polar residues at that position was zero or very low), the
position was not considered
further. At the remaining positions, PDA~ technology calculations were
performed to aid in the
identification of suitable polar replacements.
[0282] Exposed hydrophobic positions at which polar residues are observed with
a normalized
frequency of 0.1 or greater include:
[0283) Table 7. Exposed hydrophobic positions in interferon-kappa at which
polar residues are
observed with a normalized frequency of at least 0.1 in other interferon
proteins.
# wt A D E G H K N Q R S T
-
8 V - 0 - - 0 - 0.1 - - 0.10.2
-
15W _ _ _ _ _ _ _ _ 0 0 0.1
-
28F _ _ _ _ _ _ _ _ _ 0.4-
-
30V 0 - - - 0.1 - - - 0 0.3-
-
371 - - - - 0.1 0.1 0.3 - - 0
- 0.1
46L 0 - - - - 0.2 - - - - -
-
48Y - 0 - 0.6 - - - 0 - - -
-
52M _ _ _ _ _ _ _ 0.8 0 - _
-
65L _ _ _ _ _ _ _ 0.8 - _ _
-
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76 F 0.2 - - - - - - - - 0.5
- -
78 Y 0.4 - - - - - - - - 0 0.3
-
89 I 0.4 0.1 - - - - 0 - - 0 0.4
-
97 Y - 0.4 0 0 0.3 - 0.1 - - -
- -
112M 0.2 - - - - - - - - 0
- 0.1
115M 0 - 0 0.2 - 0 - - - 0.1
- 0
151Y - 0 _ _ 0.3 - _ - _ _
-
161V 0.4 - - - - - - - - -
- -
168Y - _ _ _ _ _ _ _ _ 0.4
- -
171Y - - - - - - 0.2 - - -
- 0.4
[0284] Example 5: Identification of suitable replacements for exposed
hydrophobic residues
[0285] PDA~ technology calculations were performed to identify polar residues
that are compatible
with the structure and function of type I interferons. Energies were
calculated for alanine and
each of the polar residues at each exposed hydrophobic position, using a force
field describing
van der Waals interactions (VDW), electrostatics (Elec), hydrogen bonds
(Hbond), and solvation
(Sole). The energy of the wild type hydrophobic residue was also calculated.
Polar residues with
total energies that were similar to or more favorable than the wild type
hydrophobic residue (the
first line below for each position) were considered to be compatible with the
target interferon (*
below), and the polar residues with the most favorable energies were
especially preferred (**
below). Histidine was modeled in two possible states: "HSP~ is the doubly-
protonated state of
histidine, while "HIS" is neutral histidine.
[0286] Table 8. Interferon-alpha calculation results, exposed hydrophobic
residues
# AA Total VDW Elec HBond Solv
16 MET 9.68 -4.05 0.00 0.00 13.729
* 16 ALA 3.87 -1.65 0.00 0.00 5.522
** 16 ASP -1.33 -2.85 -0.40 0.00 1.9233
* 16 GLU 1.55 -3.19 -0.40 0.00 5.1371
* 16 HIS 3.90 -3.60 0.00 0.00 7.4983
* 16 HSP 3.91 -3.62 0.27 0.00 7.2511
* 16 LYS 5.22 -3.31 0.31 0.00 8.2164
* 16 ASN 0.86 -2.88 0.01 0.00 3.7346
* 16 GLN 0.70 -3.20 -0.04 0.00 3.9397
* 16 ARG 0.73 -3.36 0.22 0.00 3.8702
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* 16 SER 0.00 -1.94 0.00 0.00 1.9394
* 16 THR 3.55 -2.89 0.04 0.00 6.4007
27 PHE 20.55 -2.52 0.00 0.00 23.0764
* 27 ALA 6.99 -0.82 0.00 0.00 7.8098
* 27 ASP 1.27 -1.51 -0.38 0.00 3.1569
* 27 GLU 1.76 -1.53 -0.22 0.00 3.5092
* 27 HIS 11.57 -1.76 -0.01 0.00 13.3424
* 27 HSP 11.16 -1.76 0.16 0.00 12.7635
* 27 LYS 7.36 -2.10 0.25 0.00 9.2138
** 27 ASN 0.52 -1.52 -0.06 0.00 2.091
** 27 GLN 0.89 -1.54 0.00 0.00 2.4286
* 27 ARG 5.35 -1.59 0.21 0.00 6.7299
* 27 SER 1.63 -1.00 -0.03 0.00 2.6514
* 27 THR 6.62 -1.40 -0.03 0.00 8.0523
100 ILE 6.17 -4.09 0.00 0.00 10.2668
* 100 ALA 3.44 -1.47 0.00 0.00 4.9013
* 100 ASP -0.59 -2.28 0.24 0.00 1.4537
** 100 GLU -1.26 -3.19 0.50 0.00 1.4374
100 HIS 15.87 0.86 -0.01 0.00 15.0219
100 HSP 15.16 0.98 -0.20 0.00 14.3823
* 100 LYS 1.23 -3.37 -0.38 0.00 4.9902
* 100 ASN 0.38 -3.14 0.00 0.00 3.5252
** 100 GLN -2.56 -3.28 0.02 0.00 0.7041
** 100 ARG -1.57 -3.39 -0.27 0.00 2.0909
* 100 SER -0.30 -1.72 -0.01 0.00 1.4346
* 100 THR 4.32 -2.62 0.00 0.00 6.9432
110 LEU 18.52 -1.89 0.00 0.00 20.4107
* 110 ALA 8.94 -0.77 0.00 0.00 9.7089
* 110 ASP 3.92 -1.36 0.17 0.00 5.1126
* 110 GLU 4.44 -2.34 0.61 0.00 6.1639
* 110 HlS 13.80 -1.79 0.00 0.00 15.5913
* 110 HSP 13.11 -1.79 -0.10 0,00 15.0058
* 110 LYS 11.14 -1.96 -0.23 0.00 13.3274
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** 110 ASN 2.75 -1.37 -0.04 0.00 4.1649
** 110 GLN 2.83 -2.34 0.06 0.00 5.1235
* 110 ARG 6.17 -0.09 -0.23 0.00 6.4996
** 110 SER 3.03 -0.94 -0.02 0.00 3.9872
* 110 THR 4.82 -1.84 -0.03 0.00 6.7023
111 MET 1.37 -4.94 0.00 0.00 6.308
111 ALA 5.58 -1.21 0.00 0.00 6.7846
* 111 ASP 0.88 -2.06 0.41 0.00 2.534
* 111 GLU 0.33 -2.52 0.42 0.00 2.4273
111 HIS 2.55 -3.90 -0.01 0.00 6.4709
111 HSP 3.57 -3.92 -1.10 0.00 8.5877
111 LYS 2.18 -2.62 -0.28 0.00 5.0789
* 111 ASN 0.14 -2.09 0.05 0.00 2.1808
** 111 GLN -0.92 -2.54 -0.05 0.00 1.6617
* 111 ARG 1.21 -2.71 -0.44 0.00 4.3527
* 111 SER 1.29 -1.46 0.02 0.00 2.7337
** 111 THR -0.16 -3.15 0.05 0.00 2.9415
117 LEU 3.03 -4.07 0.00 0.00 7.0989
* 117 ALA -1.03 -1.74 0.00 0.00 0.7126
** 117 ASP -3.58 -3.54 0.63 0.00 -0.6613
** 117 GLU -3.35 -3.35 0.26 0.00 -0.2511
117 HIS 3.54 -3.46 -0.08 0.00 7.0827
117 HSP 3.69 -3.26 0.46 0.00 6.5019
* 117 LYS -1.42 -4.06 -0.48 0.00 3.1122
* 117 ASN -0.83 -3.24 -0.11 0.00 2.5211
** 117 GLN -4.34 -3.37 0.06 0.00 -1.0372
** 117 ARG -3.91 -1.54 -0.49 -2.87 0.9774
** 117 SER -3.47 -2.09 -0.03 0.00 -1.3545
* 117 THR -1.87 -3.00 -0.02 0.00 1.1538
161 LEU 10.25 -3.57 0.00 0.00 13.8222
* 161 ALA 2.72 -1.25 0.00 0.00 3.9705
* 161 ASP -0.17 -2.59 -0.04 -0.11 2.5728
** 161 GLU -2.33 -3.04 0.15 0.00 0.5566
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* 161 H I 2.94 -4.91 -0.03 0.00 7.8882
S
* 161 HSP 4.64 -4.93 -0.19 0.00 9.7575
** 161 LYS -1.13 -3.55 -0.20 0.00 2.6196
* 161 ASN -0.29 -2.17 -0.07 0.00 1.943
161 GLN -0.66 -3.07 -0.03 0.00 2.4459
* 161 ARG -0.43 -4.56 -1.02 -4.78 9.9354
* 161 SER 0.34 -1.58 -0.04 0.00 1.9577
* 161 THR 0.71 -2.75 -0.04 0.00 3.4958
[0287] Table 9. Interferon beta calculation results, exposed hydrophobic
residues
# AA Total VDW Elec HBond Solv
LEU 6.86 -4.43 0.00 0.00 11.28
* 5 ALA 1.42 -1.74 0.00 0,00 3.16
**5 ASP -2.63 -2.74 -0.37 0.00 0.47
**5 GLU -3.43 -3.98 -0.31 0.00 0.87
5 HIS 13.88 -0.11 -0.09 0.00 14.07
5 HSP 13.62 -0.01 0.08 0.00 13.55
* 5 LYS -0.35 -4.39 0.18 0.00 3.86
* 5 ASN -0.15 -2.77 0.02 0.00 2.61
**5 GLN -3.95 -4.00 -0.03 0.00 0.08
* 5 ARG 0.17 -3.17 0.21 0.00 3.12
**5 SER -3.45 -2.03 -0.02 0.00 -1.40
**5 THR -2.86 -3.43 -0.02 0.00 0.59
8 PHE 11.34 -4.41 0.00 0.00 15.75
* 8 ALA -0.23 -1.77 0.00 0.00 1.54
**8 ASP -3.43 -2.73 -0.34 0.00 -0.37
**8 GLU -2.58 -4.05 -0.30 0.00 1.77
* 8 HIS 6.12 -3.53 0.08 0.00 9.57
* 8 HSP 6.14 -3.54 0.47 0.00 9.20
* 8 LYS 2.74 -3.94 0.24 0.00 6.44
* 8 ASN -1.13 -2.74 -0.02 0.00 1.63
**8 GLN -2.86 -2.46 -0.08 -2.76 2.44
* 8 ARG -1.50 -4.00 0.33 0.00 2.17
**8 SER -4.37 -2.02 -0.02 0.00 -2.33
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* 8 THR 3.32 -3.02 -0.08 0.00 6.42
15 PHE 16.43 -3.32 0.00 0.00 19.75
* 15 ALA 4.13 -1.43 0.00 0.00 5.55
**15 ASP -2.05 -2.23 -0.22 0.00 0.40
* 15 GLU -0.61 -2.42 -0.19 0.00 2.01
* 15 HIS 8.24 -2.87 -0.01 0.00 11.11
* 15 HSP 7.89 -2.87 0.22 0.00 10.54
* 15 LYS 4.45 -2.65 0.18 0.00 6.92
* 15 ASN -0.40 -2.86 0.01 0.00 2.45
**15 GLN -1.29 -2.45 0.01 0.00 1.15
* 15 ARG 0.02 -2.55 0.20 0.00 2.36
**15 SER -1.36 -1.64 0.00 0.00 0.27
* 15 THR 4.55 -2.43 0.02 0.00 6.96
22 TRP 18.45 -5.92 0.00 0.00 24.37
* 22 ALA 4.20 -1.41 0.00 0.00 5.61
* 22 ASP 0.36 -2.04 -0.31 0.00 2.71
**22 GLU -1.48 -3.44 -0.22 0.00 2.18
* 22 HIS 11.29 0.90 -0.15 0.00 10.54
* 22 HSP 10.51 0.24 -0.05 0.00 10.32
* 22 LYS 1.76 -3.78 0.24 0.00 5.31
* 22 ASN 0.23 -2.05 -0.05 0.00 2.33
**22 GLN -2.43 -3.44 0.01 0.00 1.00
* 22 ARG 0.66 -3.42 0.23 0.00 3.84
**22 SER -1.24 -1.58 -0.01 0.00 0.35
* 22 THR 3.43 -2.85 0.05 0.00 6.22
28 LEU 2.83 -5.56 0.00 0.00 8.40
* 28 ALA 2.61 -1.61 0.00 0.00 4.21
* 28 ASP 1.55 -3.49 0.01 0.00 5.03
* 28 GLU -1.66 -3.82 -0.04 0.00 2.20
28 HIS 4.28 -5.06 0.06 0.00 9.28
28 HSP 5.23 -4.96 0.04 -0.73 10.88
* 28 LYS -0.87 -4.43 -0.01 0.00 3.57
* 28 ASN 0.72 -3.46 0.04 0.00 4.14
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**28GLN -6.92 -3.78 -0.11 -5.30 2.27
28ARG 3.10 -6.28 0.21 0.00 9.17
* 28SER 0.59 -2.01 -0.01 0.00 2.62
28THR 7.09 -2.50 0.01 0.00 9.57
30TYR 13.74 -3.59 -0.05 0.00 17.38
* 30ALA 10.72 -0.88 0.00 0.00 11.60
**30ASP 3.32 -1.36 -0.24 0.00 4.92
* 30GLU 5.32 -1.88 -0.29 0.00 7.49
* 30HIS 9.66 -2.99 -0.08 0.00 12.73
* 30HSP 12.47 -3.00 0.74 0.00 14.73
* 30LYS 8.65 -2.26 0.19 0.00 10.72
**30ASN 2.78 -1.37 0.01 0.00 4.15
* 30GLN 4.45 -1.89 -0.01 0.00 6.35
* 30ARG 7.17 -1.90 0.15 0.00 8.93
* 30SER 4.49 -1.03 -0.02 0.00 5.54
* 30THR 7.17 -1.69 -0.02 0.00 8.88
32LEU 0.79 -4.68 0.00 0.00 5.47
**32ALA -0.14 -1.52 0.00 0.00 1.38
32ASP 1.58 -3.02 -0.21 0.00 4.81
* 32GLU 0.18 -4.32 -0.47 0.00 4.97
* 32H -0.42 -4.84 -0.17 0.00 4.58
I
S
**32HSP -0.93 -4.84 -0.22 0.00 4.13
32LYS 2.85 -4.41 0.39 0.00 6.87
32ASN 3.94 -3.09 -0.04 0.00 7.06
* 32GLN 0.22 -4.00 0.01 0.00 4.21
* 32ARG 0.95 -4.74 0.36 0.00 5.33
* 32SER 0.83 -1.93 0.06 0.00 2.70
32TH 1.72 -3.10 0.06 0.00 4.76
R
36MET 0.14 -5.60 0.00 0.00 5.74
36' 0.38 -1.86 0.00 0.00 2.24
ALA
**36AS -3.06 -3.47 0.02 -0.03 0.43
P
**36GLU -3.53 -3.34 -0.05 0.00 -0.14
* 36HIS -0.84 -5.33 0.03 0.00 4.46
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36 HSP0.32 -5.04 -0.08 0.00 5.44
**36 LYS-3.76 -4.99 0.00 0.00 1.22
* 36 ASN-1.09 -3.53 0.00 -0.05 2.48
**36 GLN-5.26 -2.66 -0.10 -2.32 -0.18
* 36 ARG-2.19 -2.92 0.05 0.00 0.69
* 36 SER-2.41 -2.27 0.02 0.00 -0.17
2**36 THR-3.93 -1.20 0.02 0.00 -2.76
47 LEU1.86 -6.08 0.00 0.00 7.94
* 47 ALA0.52 -2.11 0.00 0.00 2.62
**47 ASP-7.26 -4.20 -0.37 -2.90 0.22
* 47 GLU-2.33 -4.94 0.02 0.00 2.59
47 HIS217.36 213.11 0.09 0.00 4.16
47 HSP4313.02 4309.27 -2.51 0.00 6.27
**47 LYS-5.22 -5.97 0.01 0.00 0.74
**47 ASN-4.27 -4.31 -0.18 -2.14 2.37
* 47 GLN-1,65 -5.40 -0.07 -2.13 5.95
* 47 ARG-3.84 -4.76 -0.27 -6.29 7.49
* 47 SER-1.23 -2.64 0.03 0.00 1.37
* 47 THR-0.02 -2.58 0.01 0.00 2.56
92 TYR3.84 -5.11 0.01 0.00 8.95
* 92 ALA-1.94 -1.95 0.00 0.00 0.01
**92 ASP-5,45 -3.06 -0.33 -0.01 -2.04
**92 GLU-5.14 -3.67 -0.08 0.00 -1.40
* 92 HlS3.04 -4.25 -0.04 0.00 7.33
* 92 HSP2.94 -4.25 0.28 0.00 6.91
* 92 LYS-1,75 -3.96 0.00 0.00 2.21
* 92 ASN-3.30 -3.13 -0.12 -0.03 -0.02
**92 GLN-5.55 -3.69 0.02 0.00 -1.89
* 92 ARG-0.49 -3.72 0.14 0.00 3.10
**92 SER-4.90 -2.25 -0.03 0.00 -2.62
92 THR4.46 0.21 0.00 0.00 4.25
111PHE29.59 -2.42 0.00 0.00 32.01
* 111ALA15.98 -0.76 0.00 0.00 16.74
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**111 8.56 -1.11 0.03 0,00 9.64
ASP
* 111 13.15 -1.18 -0.07 0,00 14.39
GLU
* 111 19.66 -1.33 0.00 0.00 20.99
HIS
* 111 19.06 -1.33 -0.02 0.00 20.41
HSP
* 111 20.27 -1.30 0.08 0.00 21.49
LYS
**111 7.32 -1.10 0.00 0.00 8.41
ASN
* 111 11.91 -1.18 -0.03 0.00 13.12
GLN
* 111 15.55 -1.25 0.02 0.00 16.78
ARG
**111 9.49 -0.86 0.01 0.00 10.34
SER
* 111 14.87 -0.10 -0.10 -0.71 15.78
THR
116 4.71 -3.66 0.00 0.00 8.37
LEU
* 116 1.74 -1.32 0.00 0.00 3.06
ALA
**116 -2,58 -2.25 -0.19 0.00 -0.13
ASP
* 116 -1.53 -3.11 -0.11 0.00 1.69
GLU
116 7.67 -3.22 0.11 0.00 10.78
HIS
116 7.44 , -3.22 0.50 0.00 10.16
HSP
* 116 1.45 -3.27 0.03 0.00 4.68
LYS
**116 -2.54 -2.29 -0,05 0.00 -0.20
ASN
* 116 -1.95 -3.13 -0.01 0.00 1.18
GLN
* 116 -1.05 -3.53 0.29 0.00 2.18
ARG .
* 116 -1.66 -1.55 -0.01 0.00 -0.10
SER
* 116 1.59 -1.87 -0.01 0.00 3.47
THR
120 0.81 -6.47 0.00 0.00 7.28
LEU
120 2.03 -1.44 0.00 0.00 3.46
ALA
**120 -2.85 -2.28 -0.33 0.00 -0.24
ASP
120 1.19 -2.64 -0.16 0.00 3.99
GLU
120 10.00 -3.07 0.08 0.00 12.99
HIS
120 9.96 -2.91 0.20 0.00 12.68
HSP
120 6.44 -2.73 0.30 0.00 8.87
LYS
* 120 -1.33 -2.21 -0.05 0.00 0.94
ASN
* 120 0.39 -2.66 0.04 0.00 3.01
GLN
120 4.28 -2.64 0.23 0.00 6.69
ARG '
**120 -2.59 -1.64 -0.05 0.00 -0.90
SER
120 3.04 -3.74 -0.01 0.00 6.80
THR
57
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130 -4.92 -5.89 0.00 0.00 0.98
LEU
130 0.46 -1.57 0.00 0.00 2.03
ALA
* 130 -4.43 -2.75 -0.13 0.00 -1.55
ASP
**130 -6.43 -3.00 -0.16 0.00 -3.28
GLU
130 0.41 -4.27 -0.03 0.00 4.71
HIS
130 2.99 -4.38 0.03 0.00 7.34
HSP
* 130 -4.72 -5.08 0.18 0.00 0.19
LYS
* 130 -4.59 -2.79 0.00 0.00 -1.80
ASN
**130 -6.62 -4.38 0.01 0.00 -2.25
GLN
**130 -5.87 -5.87 -0.01 -2.32 2.33
ARG
130 -3.50 -1.84 0.00 0.00 -1.66
SER
130 -3.29 -3.41 0.02 0.00 0.09
THR
148 6.65 -3.33 0.00 0.00 9.98
VAL
148 7.09 -1.45 0.00 0.00 8.54
ALA
**148 0.64 -2.35 -0.29 0.00 3.28
ASP
**148 1.02 -3.73 -0.30 0.00 5.06
GLU
148 7.65 -3.09 -0.04 0.00 10.79
HIS
148 7.26 -3.10 0.16 0.00 10.20
HSP
* 148 2.96 -4.18 0.36 0.00 6.77
LYS
* 148 2.53 -2.37 -0.02 0.00 4.92
ASN
* 148 2.96 -2.72 0.03 0.00 5.64
GLN
**148 1.86 -3.88 0.34 0.00 5.40
ARG
**148 1.08 -1.68 0.00 0.00 2.77
SER
* 148 5.24 -2.58 0.03 0.00 7.79
THR
155 6.95 -4.80 -0.01 0.00 11.76
TYR
* 155 4.11 -1.52 0.00 0.00 5.63
ALA
**155 -1.98 -2.45 -0.29 0.00 0.76
ASP
* 155 -0.57 -3.62 -0.27 0.00 3.31
GLU 1
155 8.86 -3.52 0.01 0.00 12.37
HIS
155 9.02 -3.52 0.31 0.00 12.23
HSP
* 155 5.53 -2.99 0.25 0.00 8.27
LYS
* 155 0.17 -2.47 -0.01 0.00 2.65
ASN
**155 -1.50 -3.63 0.00 0.00 2.13
GLN
58
CA 02528964 2005-12-09
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** 155 -3.63 0.28 0.00 4.65
ARG
1.29
* 155 -1.77 0.01 0.00 0.94
SER
-0.82
* 155 -2.70 0.00 0.00 7.75
THR
5.05
[0288] nterferon n results,
Table kappa exposed
10. calculatio hydrophobic
I residues
# AA Total vdW Elec HbondSolv
1 LEU 16.16 -1.74 0.00 0.00 17.90
* 1 ALA 8.55 -0.56 0.00 0.00 9.12
* 1 ARG 5.07 -1.90 -0.32 0.00 7.29
* 1 ASN 2.47 -1.03 0.12 0.00 3.38
** 1 ASP 0.82 -1.11 -0.05 -3.985.96
* 1 GLN 2.37 -1.39 0.03 0.00 3.73
* 1 GLU 3.52 -1.14 0.22 0.00 4.45
* 1 GLY 2.79 -0.09 0.00 0.00 2.88
* 1 HIS 10.39 -1.90 -0.15 -2.5414.97
* 1 HSP 9.14 -1.90 -1.03 -2.5314.61
* 1 LYS 7.37 -0.82 -0.27 0.00 8.46
* 1 SER 3.41 -0.54 0.03 0.00 3.92
* 1 THR 6.26 -1.13 0.03 0.00 7.37
5 LEU 9.28 -3.12 0.00 0.00 12,40
* 5 ALA 6.92 -1.11 0.00 0.00 8.03
* 5 ARG 2.30 -2.28 0.16 0.00 4.42
** 5 ASN -1.00 -1.73 0.02 0.00 0.71
** 5 ASP -0.31 -1.73 -0.28 0.00 1.69
* 5 GLN 0.46 -2.44 0.00 0.00 2.91
* 5 GLU ~ 1.43 -2.42 -0.17 0.00 4.02
* 5 GLY 6.79 -0.17 0.00 0.00 6.96
* 5 HIS 6.18 -2.38 -0.01 0.00 8.57
* 5 HSP 6.04 -2.38 0.23 0.00 8.19
* 5 LYS 2.82 -3.46 0.42 -3.199.05
* 5 SER 1.03 -1.26 -0.01 0.00 2.29
* 5 TH R 1.09 -2.29 -0.01 0.00 3.39
8 VAL 5.07 -3.35 0.00 0.00 8.42
59
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* 8 ALA 5.02 -1.40 0.00 0.00 6.43
* 8 ARG -0.04 -3.23 0.36 0.00 2.83
** 8 ASN -3.01 -2.45 -0.09 -2.84 2.37
* 8 ASP -0.54 -2.52 -0.30 0.00 2.29
** 8 GLN -2.05 -2.96 0.04 0.00 0.88
** 8 GLU -1.27 -2.68 -0.26 0.00 1.66
* 8 GLY 2.09 -0.22 0.00 0.00 2.30
* 8 HIS 2.94 -3.79 0.03 0.00 6.70
* 8 HSP 3.07 -3.79 0.37 0.00 6.49
* 8 LYS 0.38 -3.42 0.33 0.00 3.47
* 8 SER 0.32 -1.69 0.00 0.00 2.01
* 8 THR 2.44 -2.69 0.00 0.00 5.13
15 TRP 2.66 -6.08 0.00 0.00 8.74
* 15 ALA 2.27 -1.39 0.00 0.00 3.66
* 15 ARG -0.49 -3.53 0.41 0.00 2.63
** 15 ASN -4.15 -2.97 0.05 -2.71 1.48
** 15 ASP -3.09 -2.99 -0.43 0.00 0.32
** 15 GLN -4.26 -3.24 -0.01 0.00 -1.01
** 15 GLU -3.94 -3.19 -0.36 0.00 -0.37
* 15 GLY 1.98 -0.30 0.00 0.00 2.28
15 HIS 3.07 -3.90 0.01 0.00 6.96
15 HSP 3.13 -3.88 0.42 0.00 6.59
* 15 LYS -0.64 -2.80 0.43 0.00 1.73
* 15 SER -1.70 -1.75 -0.01 0,00 0.07
15 THR 5.05 -0.75 0.03 0.00 5.77
18 LEU -7.96 -6.28 0.00 0.00 -1.69
18 ALA -3.37 -2.20 0.00 0.00 -1.16
18 ARG -3.90 -5.75 0.36 0.00 1.48
18 ASN -3.50 -4.51 0.00 0.00 1.02
18 ASP -5.98 -4.64 -0.35 0.00 -0.99
* 18 GLN -7.59 -4.63 -0.01 0.00 -2.95
* 18 GLU -8.87 -5.82 -0.43 0.00 -2.61
18 GLY 0.11 -0.37 0.00 0,00 0.48
18 HIS -0.92 -4.87 -0.02 0.00 3.96
CA 02528964 2005-12-09
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18 HSP 3.12 -3.46 0.42 0.00 6.16
* 18 LYS -6.70 -6.21 0.30 0.00 -0.79
18 SER -3.95 -2.68 0.00 0.00 -1.27
18 THR -1.25 -3.94 0.07 0.00 2.61
28 PHE 18.32 -4.71 0.00 0.00 23.02
* 28 ALA 5.85 -1.85 0.00 0.00 7.69
* 28 ARG 3.35 -3.31 -0.03 0.00 6.69
** 28 ASN -2.32 -3.19 -0.19 -3.03 4.09
* 28 ASP 1.28 -2.94 0.28 0.00 3.93
* 28 GLN 0.95 -3.74 -0.14 -3.37 8.21
* 28 GLU 3.31 -3.39 0.15 0.00 6.55
* 28 GLY 6.33 -0.28 0.00 0.00 6.62
* 28 HIS 7.67 -4.12 0.03 0.00 11.76
* 28 HSP 6.77 -4.11 -0.24 0.00 11.12
* 28 LYS 4.45 -3.59 -0.52 -5.05 13.61
* 28 SER 1.76 -2.16 0.01 0.00 3.91
* 28 THR 9.75 2.16 0.00 0.00 7.60
30 VAL 10.27 -2.35 0.00 0,00 12.62
* 30 ALA 6.08 -0.92 0.00 0.00 7.00
* 30 ARG 2.49 -2.42 0.06 0.00 4.85
* 30 ASN 0.13 -1.83 0.00 0.00 1.97
* 30 ASP 1.13 -1.82 0.04 0.00 2.91
** 30 GLN -0.65 -1.87 -0.02 0.00 1.24
* 30 GLU 0.68 -1.87 0.01 0.00 2.54
* 30 GLY 2.71 -0.16 0.00 0.00 2.87
* 30 HIS 7.83 -3.68 -0.01 0.00 11.52
* 30 HSP 7.87 -3.56 -0.13 0.00 11.56
* 30 LYS 5.43 -3.08 0.01 0.00 8.51
* 30 SER 1.64 -1.15 0.00 0.00 2.78
* 30 THR 5.28 -1.93 0.01 0.00 7.20
33 LEU 8.89 -3.10 0.00 0.00 12.00
* 33 ALA 5.67 -0.99 0.00 0.00 6.67
* 33 ARG -0.88 -2.82 -0.07 0.00 2.01
61 '
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** 33 ASN -1.09 -1.86 0.00 0.00 0.78
* 33 AS P 0.12 -1.86 0.12 0.00 1.86
** 33 GLN -3.13 -2.90 -0.09 -2.65 2.51
* 33 GLU -0.44 -2.85 0.16 0.00 2.24
* 33 GLY 2.91 -0.15 0.00 0.00 3.07
* 33 HIS 6.16 -2.83 0.01 0.00 8.98
* 33 HSP 5.57 -2.83 -0.12 0.00 8.51
* 33 LYS 1.75 -2.89 -0.09 0.00 4.73
* 33 SER 0.39 -1.19 0.01 0.00 1.58
* 33 THR 1.15 -2.27 -0.01 0.00 3.42
37 ILE 0.71 -5.77 0.00 0.00 6.48
37 ALA 3.26 -1.68 0.00 0.00 4.94
* 37 ARG -1.63 -2.56 -0.39 -5.88 7.21
* 37 ASN -1.24 -3.19 0.03 0.00 1.92
* 37 ASP -3.15 -2.98 0.23 -0.10 -0.30
** 37 GLN -6.08 -3.22 -0.06 -4.23 1.44
* 37 GLU -2.78 -3.25 0.27 0,00 0.19
37 GLY 2.71 -0.21 0.00 0,00 2.92
37 HIS 2.18 -5.14 0.01 0.00 7.30
37 HSP 2.77 -4.28 -0.34 -1.12 8.51
* 37 LYS -1.72 -4.15 -0.21 0, 00 2.64
* 37 SER -0.42 -1.99 0.01 0.00 1.55
** 37 THR -4.92 -4.32 0.01 0.00 -0.62
46 LEU 0.03 -4.37 0.00 0.00 4.40
* 46 ALA -2.83 -1.86 0.00 0.00 -0.97
** 46 ARG -5.84 -4.27 -0.18 -2.39 1.00
* 46 ASN -4.07 -3.26 0.00 0.00 -0.81
** 46 ASP -6.38 -3.22 -0.25 0.00 -2.92
** 46 GLN -7.53 -3.68 0.01 0.00 -3.86
** 46 GLU -7.16 -3.55 -0.12 0.00 -3.48
* 46 GLY -0.53 -0.26 0.00 0.00 -0.27
46 HIS 0.17 -4.16 -0.02 0.00 4.35
* 46 HSP -0.20 -4.15 0.17 0.00 3.78
* 46 LYS -3.15 -3.48 0.15 0.00 0.19
62
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** 46 SER -5.21 -2.19 0.01 0.00 -3.03
* 46 THR -0.91 1.44 0.01 0.00 -2.37
48 TYR -3.30 -5.42 0.01 0.00 2.10
48 ALA -1.88 -1.89 0.00 0.00 0.01
* 48 ARG -5.36 -5.53 -0.11 0.00 0.28
48 ASN -2.23 -3.76 -0.03 0.00 1.55
** 48 ASP -9.47 -3.96 0.00 -2.99 -2.52
* 48 GLN -7.50 -4.51 -0.11 -2.67 -0.22
** 48 GLU -9.11 -4.52 -0.05 -2.71 -1.83
48 GLY 1.29 -0.24 0.00 0.00 1.52
48 HIS -1.45 -5.38 -0.03 0.00 3.96
48 HSP -2.14 -5.37 -0.15 0.00 3.37
* 48 LYS -5.37 -4.29 -0.11 0.00 -0.96
48 SER -3.16 -2.27 -0.01 0.00 -0.88
* 48 THR -4.68 -1.54 -0.01 0.00 -3.13
52 MET 12.92 -3.56 0.00 0.00 16.48
* 52 ALA 5.97 -1.54 0.00 0.00 7.51
* 52 ARG 3.75 -2.96 0.15 0.00 6.56
** 52 ASN -1.71 -1.11 -0.27 -5.77 5.43
** 52 ASP -1.46 -1.59 -1.25 -3.93 5.32
* 52 GLN 1.34 -3.03 -0.07 0.00 4.44
* 52 GLU 2.17 -2.98 -0.28 0.00 5.43
52 GLY 4.74 -0.23 0.00 0.00 4.97
* 52 H1S 7.79 -2.91 -0.28 -3.46 14.44
* 52 HSP 6.75 -2.89 -0.70 -3.48 13.82
* 52 LYS 6.71 -3.15 0.16 0.00 9.70
* 52 SER 0.84 -1.76 0.04 0.00 2.56
* 52 THR 5.25 -1.27 0.04 0.00 6.48
65 LEU -2.31 -4.75 0.00 0.00 2.44
65 ALA -1.88 -1.76 0.00 0.00 -0.12
* 65 ARG -3.62 -4.35 -0.05 0.00 0.79
* 65 ASN -2.88 -3.75 0.01 0.00 0.86
* 65 ASP -4.97 -3.88 0.30 0.00 -1.39
63 '
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** 65 GLN -6.92 -4.78 0.03 0.00 -2.18
** 65 GLU -6.66 -4.91 0.23 0.00 -1.98
65 GLY 0.31 -0.25 0.00 0.00 0.56
65 HIS 11.96 10.19 0.01 0.00 1.75
65 HSP 13.91 8.82 0.17 0.00 4.91
* 65 LYS -3.12 -4.48 -0.18 0.00 1.54
* 65 SER -3.53 -2.15 0.01 0.00 -1.39
* 65 THR -4.25 -3.45 -0.02 0.00 -0.78
68 PHE -5.87 -7.03 0.00 0.00 1.16
68 ALA -3.75 -2.01 0.00 0.00 -1.74
* 68 ARG -6.84 -5.85 -0.53 0.00 -0.46
68 ASN -4.99 -4.40 -0.04 0.00 -0.55
* 68 ASP -6.55 -3.87 0.34 0.00 -3.02
* 68 GLN -8.01 -5.42 -0.02 0.00 -2.56
** 68 GLU -9.36 -5.40 0.34 0.00 -4.30
68 GLY -0.85 -0.30 0.00 0.00 -0.54
* 68 HlS -6.00 -6.05 0.04 0.00 0.02
* 68 HSP -6.74 -5.97 -0.34 0.00 -0.42
** 68 LYS -9.96 -5.89 -0.41 0.00 -3.66
68 SER -3.46 -2.41 -0.03 0.00 -1.02
68 THR -2.31 -3.42 -0.14 0.00 1.25
76 PHE 17.46 -4.29 0.00 0.00 21.75
* 76 ALA 6.77 -1.11 0.00 0.00 7.88
* 76 ARG 3.07 -2.50 -0.10 0.00 5.67
** 76 ASN -1.69 -1.48 -0.15 -2.30 2.24
** 76 ASP -0.22 -1.71 0.06 0.00 1.43
* 76 GLN 1.69 -2.19 -0.04 0.00 3.93
* 76 GLU 2.66 -2.09 0.09 0.00 4.65
* 76 GLY 6.19 -0.15 0.00 0.00 6.35
* 76 HIS 9.14 -3.17 0.06 0.00 12.25
* 76 HSP 8.48 -3.17 -0.34 0.00 11.99
* 76 LYS 8.39 -2.70 -0.15 0.00 11.24
* 76 SER 0.59 -1.28 -0.02 0.00 1.89
* 76 THR 2.57 -2.46 -0.02 0.00 5.05
64
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78 TYR 6.54 -5.49 -0.04 0.00 12.07
78 ALA 7.63 -1.15 0.00 0.00 8.79
* 78 ARG 4.88 -2.52 -0.07 0.00 7.47
* 78 ASN 3.23 -2.44 -0.02 0.00 5.69
* 78 ASP 3.05 -2.26 0.07 -0.94 6.18
** 78 GLN 1.98 -2.21 -0.04 0.00 4.23
** 78 GLU 1.67 -2.22 -0.02 0.00 3.91
78 GLY 6.81 -0.14 0.00 0.00 6.96
* 78 HIS 5.82 -6.20 -0.02 0.00 12.03
* 78 HSP 3.01 -6.07 -0.46 -2.67 12,22
* 78 ~ LYS 4.97 -3.96 -0.48 0.00 9.41
* 78 SER 3.33 -1.23 -0.12 -5.35 10.03
* 78 THR 2.95 -1.98 -0.12 -5.18 10.22
79 TRP 10.75 -4.92 0.01 0.00 15.65
* 79 ALA 3.38 -1.21 0.00 0.00 4.59
* 79 ARG 0.30 -2.70 -0.07 0.00 3.06
** 79 ASN -1.20 -2.37 0.13 0.00 1.04
* 79 ASP -0.65 -2.21 0.26 0.00 1.31
** 79 GLN -2.65 -2.77 -0.10 -7.46 7.69
* 79 GLU 0.31 -2.79 0.14 0.00 2.96
* 79 GLY 1.45 -0.20 0.00 0.00 1.66
* 79 HIS 6.19 -2.99 0.04 0.00 9.15
* 79 HSP 5.75 -2.99 -0.17 0.00 8.90
* 79 LYS 1.55 -3.33 -0.19 0.00 5.07
* 79 SER -0.73 -1.40 0.00 0.00 0.67
* 79 THR 3.74 -2.24 -0.05 -0.02 6.05
89 ILE 5.42 -4.08 0.00 0.00 9.50
* 89 ALA 3.77 -1.15 0.00 0.00 4.92
* 89 ARG -1.59 -4.17 0.11 0.00 2.48
** 89 ASN -3.80 -1.93 0.02 0.00 -1.89
** 89 ASP -3.01 -1.82 0.08 0.00 -1.26
* 89 GLN -1.06 -2.39 0.10 0.00 1.23
* 89 GLU -0.26 -2.18 -0.25 0.00 2.17
CA 02528964 2005-12-09
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* 89 GLY 3.72 -0.17 0.00 0.00 3.89
* 89 HIS 4.04 -2.39 -0.03 0.00 6.46
* 89 HSP 3.42 -2.39 -0.14 0.00 5.96
* 89 LYS 3.92 -2.39 0.08 0.00 6.22
* 89 SER -1.60 -1.33 0.04 0.00 -0.31
* 89 THR -1.68 -2.51 0.04 0.00 0.79
97 TYR -1.92 -5.22 -0.02 0.00 3.32
97 ALA 0.39 -1.49 0.00 0.00 1.87
** 97 ARG -3.91 -4.23 -0.68 -3.13 4.13
97 ASN -1.28 -2.95 0.10 0.00 1.56
97 ASP -1.03 -2.50 0.18 0.00 1.29
* 97 GLN -2.98 -3.34 0.02 0.00 0.35
* 97 GLU -2.53 -3.45 0.21 0.00 0.71
97 GLY 2.13 -0.21 0.00 0.00 2.33
97 HIS 1.22 -4.20 0.01 0.00 5.41
97 HSP 0.98 -4.21 0.18 0.00 5.04
97 LYS -0.50 -4.16 -0.11 0.00 3.77
97 SER 0.18 -1.76 -0.06 0.00 2.01
** 97 THR -3.47 -3.33 -0.03 0.00 -0.12
112 MET 0.07 -5.90 0.00 0.00 5.97
112 ALA 3.69 -1.52 0.00 0.00 5.21
** 112 ARG -3.11 -4.06 -0.40 -2.39 3.74
** 112 ASN -2.04 -2.63 0.01 0,00 0.58
* 112 ASP -1.23 -2.33 0.50 0.00 0.61
* 112 GLN -1.40 -2.90 0.09 0.00 1.42
* 112 GLU -1.83 -2.95 0.47 0.00 0.65
112 GLY 2.47 -0.19 0.00 0.00 2.66
112 HIS 1.58 -4.34 0.02 0.00 5.90
112 HSP 1.55 -4.36 -0.56 0.00 6.48
** 112 LYS -2.09 -3.70 -0.37 0.00 1.99
* 112 SER -0.70 -1.75 -0.01 0.00 1.07
* 112 THR -0.57 -2.95 -0.01 0.00 2.39
115 MET 20.53 -1.89 0.00 0.00 22.43
66
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* 115 ALA 11.10 -0.75 0.00 0.00 11.85
* 115 ARG 8.78 -1.98 -0.22 0.00 10.97
** 115 ASN 3.56 -1.30 0.01 0.00 4.87
** 115 ASP 4.09 -0.30 -0.30 -2.86 7.55
* 115 GLN 6.25 -1.40 -0.02 0.00 7.67
* 115 GLU 7.28 -1.41 0.17 0.00 8.52
** 115 GLY 4.47 -0.15 0.00 0.00 4.63
* 115 HIS 14.96 -1.92 0.02 0.00 16.86
* 115 HSP 14.25 -1.92 -0.20 0.00 16.37
* 115 LYS 11.59 -2.01 -0.21 0.00 13.81
** 115 SER 4.62 -0.91 0.00 0.00 5.53
* 115 THR 11.38 0.32 0.00 0.00 11.06
120 MET 14.72 -3.42 0.00 0.00 18.15
* 120 ALA 10.26 -0.70 0.00 0.00 10.96
* 120 ARG 4.52 -2.66 -0.24 0.00 7.42
** 120 ASN 2.06 -1.28 -0.02 0.00 3.36
** 120 ASP 3.57 -1.28 0.24 0.00 4.61
** 120 GLN 3.28 -1.52 0.01 0.00 4.79
* 120 GLU 4.92 -1.64 0.32 0.00 6.23
* 120 GLY 6.29 -0.11 0.00 0.00 6.41
* 120 HIS 10.39 -2.74 -0.03 0.00 13.16
* 120 HSP 9.47 -2.75 -0.48 0.00 12.70
* 120 LYS 7.88 -2.63 -0.26 0.00 10.77
* 120 SER 4.15 -0.85 0.02 0.00 4.98
* 120 THR 8.44 -1.54 0.00 0.00 9.99
127 VAL 7.26 8.43 0.00 0.00 -1.17
** 127 ALA -3.43 -1.35 0.00 0.00 -2.09
* 127 ARG 0.00 -7.82 -0.88 0.00 8.70
** 127 ASN -4.70 -3.66 -0.13 -4.04 3.13
** 127 ASP -6.95 -3.82 0.68 -3.10 -0.71
* 127 GLN -0.81 -5.91 -0.07 -0.29 5.46
** 127 GLU -3.83 -5.90 0.78 0.00 1.29
** 127 GLY -2.85 -0.30 0.00 0.00 -2.55
127 HIS 16.59 12.31 -0.12 0.00 4.41
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127 HSP 19.54 14.09 -1.04 0.00 6.50
* 127 LYS -1.30 -4.78 -0.07 0.00 3.56
* 127 SER -0.99 -2.21 -0.04 0.00 1.26
** 127 THR -3.15 -4.29 -0.04 0.00 1.17
133 LEU 9.92 -3.97 0.00 0.00 13.89
* 133 ALA 8.39 -0.97 0.00 0.00 9.35
* 133 ARG 3.29 -3.25 -0.18 0.00 6.72
* 133 ASN 2.32 -1.71 -0.19 0.00 4.22
* 133 ASP 3.00 -1.70 -027 0.00 4.97
** 133 GLN -2.05 -2.51 -0.14 -5.10 5.69
* 133 GLU 2.24 -3.06 0.42 0.00 4.88
* 133 GLY 2.12 -0.15 0.00 0.00 2.27
* 133 HIS 9.18 -2.46 0.01 0.00 11,64
* 133 HSP 9.02 -2.47 0.30 0.00 11.19
* 133 LYS 3.76 -3.26 -0.26 0.00 7.28
* 133 SER 3.26 -1.17 -0.02 0.00 4.45
* 133 THR 4.07 -2.42 -0.04 0.00 6.53
151 TYR -2.01 -5.96 -0.20 -2.23 6.37
151 ALA 2.45 -1.62 0.00 0.00 4.07
* 151 ARG -2.32 -3.34 0.09 0.00 0.94
151 ASN 0.06 -3.31 0.03 0.00 3.34
151 ASP -1.42 -2.87 0.05 0.00 1.40
** 151 GLN -3.98 -4.25 0.03 0.00 0.24
** 151 GLU -4.41 -4.75 -0.09 0.00 0.43
151 GLY 0.89 -0.23 0.00 0.00 1.12
** 151 HIS -3.72 -5.32 0.02 0.00 1.58
151 HSP -1.50 -5.39 0.06 0.00 3.83
151 LYS -1.43 -4.88 0.21 0.00 3.24
151 SER 0.50 -2.12 -0.03 -2.79 5.44
151 THR -0.98 -3.30 0.02 0.00 2.30
161 VAL -2.90 -4.54 0.00 0.00 1.64
161 ALA -1.30 -1.78 0.00 0.00 0.48
* 161 ARG -5.02 -4.50 0.12 0.00 -0,63
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* 161 ASN -3.65 -3.44 -0.21 -1.47 1.46
** 161 ASP -6.06 -3.46 -0.40 0.00 -2.21
* 161 GLN -4.93 -4.30 -0.01 0.00 -0.62
** 161 GLU -7.22 -4.29 -0.28 0.00 -2.66
161 GLY -1.08 -0.25 0.00 0.00 -0.83
161 HIS -1.44 -4.70 0.22 0.00 3.04
161 HSP -1.34 -4.71 0.85 0.00 2.51
* 161 LYS -4.79 -4.47 0.14 0.00 -0.45
* 161 SER -2.99 -2.12 -0.03 0.00 -0.84
161 THR -0.47 -3.87 -0.03 0.00 3.42
168 TYR 1.50 -7.16 -0.05 0.00 8.71
168 ALA 1.77 -1.79 0.00 0.00 3.56
* 168 ARG -0.38 -4.14 0.40 0.00 3.37
** 168 ASN -1.76 -3.23 -0.07 -2.62 4.16
** 168 ASP -2.08 -3.56 -0.38 0.00 1.85
** 168 GLN -1.72 -3.90 -0.01 0.00 2.19
** 168 GLU -1.52 -3.79 -0.36 0.00 2.62
168 GLY 1.91 -0.28 0.00 0.00 2.18
168 HIS 2.66 -5.84 0.00 0.00 8.51
168 HSP 5.46 -5.83 0.59 0.00 10.70
168 LYS 2.36 -4.49 0.38 0.00 6.48
* 168 SER -0.98 -2.17 -0.01 0.00 1.20
* 168 THR 1.15 -3.18 -0.01 0.00 4.34
171 TYR 1.43 -4.26 -0.04 0.00 5.73
* 171 ALA -0.78 -1.66 0.00 0.00 0.87
* 171 ARG -4.70 -3.96 0.36 0.00 -1.10
* 171 ASN -3.30 -2.81 -0.01 0.00 -0.47
** 171 ASP -5.70 -2.80 -0.41 0.00 -2.49
** 171 GLN -6.16 -3.14 0.01 0.00 -3.03
** 171 GLU -6.10 -4.42 -0.32 0.00 -1.35
* 171 GLY 0.09 -0.22 0.00 0.00 0.31
* 171 HIS -0.40 -5.05 -0.06 -0.38 5.09
* 171 HSP 1.13 -4.02 0.46 0.00 4.69
* 171 LYS -3.45 -5.26 0.43 0.00 1.38
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** 171 SER -4.54 -1.92 0.00 0.00 -2.62
* 171 THR -2.12 -2.78 0.00 0.00 0.66
[0289] Next, we simultaneously designed sets ofi exposed hydrophobic residues
that are located
close to each other in space. These calculations were performed to account for
coupling between
interacting positions. As before, sets of residues were considered to be
compatible with interferon
structure if their energy was similar to or more favorable than the energy of
the wild type residues
at that set of positions. The most preferred sets of residues are those with
the most favorable
energies.
[0290] Calculations were performed on the following clusters of exposed
hydrophobic residues in
interferon beta: 5 and 8; 15 and 155; 22 and 148; 22, 30, 32, and 36; and 116
and 120. Results
of the cluster calculations for interferon beta are given in the table below:
[0291] Table 11. Interferon beta calculation results, exposed hydrophobic
clusters
# Most preferred preferred
T S, N,K,E
8 E D, N, Q, S, R
D
22 E K, D, S, Q, R, N
28 Q K
30 D T, S, N, E
32 S E
36 T K, E
116 T K, S, N, D,H,E
120 R D, K, E, T, S
148 E
155 D E, N, S, Q
[0292] Finally, we reconciled the results of the PDA~ technology calculations
and the sequence
alignment data for interferon kappa. The most preferred polar substitution for
each exposed
hydrophobic residue was defined to be the residue with the highest normalized
frequency of
occurrence, among the set of polar residues with favorable energies in the
PDA~ technology
calculations. The most preferred substitutions are: VBN, W15R, V30R, 137N,
Y48Q, F76S, 189T,
Y97D, M112T, M115G, V161A, Y168S, and Y171T. In the case of Y97D and V161A,
the
replacements have slightly less favorable energies than the wild type
hydrophobic residue.
However, since the energy difference is only slight and the alternate residues
are frequently
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observed in other interferons, it is likely that these substitutions are
structurally and functionally
suitable.
[0293] A few of these substitutions are close in sequence to other exposed
hydrophobic residues.
As a result, it was possible to test the effect of altering a small number of
additional residues
without increasing the overall library complexity. Preferred polar residues
for these additional
exposed hydrophobic residues were selected for favorable PDA~ technology
energies or high
normalized frequency in other interferons; the most preferred substitutions
are: LSQ, F28Q,
M52N, Y78A, and L133Q.
[0294] Example 6: Identification of suitable replacements for dimer interface
residues
[0295] PDA~ technology calculations were performed to identify residues that
form favorable
intermolecular interactions in the interferon-beta dimer. Each of the residues
identified as dimer
interface residues was considered. The interaction energy between each dimer
interface residue
in chain A and each dimer interface residue in chain B was calculated using a
force field
describing van der Waals interactions, electrostatics, hydrogen bonds, and
solvation. The
residues were all held fixed in the crystallographically observed
conformations. Half- interaction
energies are as shown below; the energies are symmetric and the total
interaction energy is
twice the value shown.
[0296] Table 12. Interactions across the interferon-beta dimer interface.
Glu 42 Glu 43 Gln 46 Leu Gln 48 Gln 49 Gln 51 Arg Leu Met Leu His Arg
A A A 47A A A A 113A 116A 117A 120A 121 A 124A
ME
T 1 B 0.0 0.0 0.0 0.0 0.0 0.0 -1.0-1.4-0.1 0.0 0.0 0.0 0.0
SER 2 B 0.0 0.0 0.0 0.0 0.0 0.0 -1.8-2.4 0.0 0.0 0.0 0.0
0.0
TYR 3 B 0.0 0.0 0.0 0.0 0.0 0.0 0.6 0.0 0.0 0.0 0.0 0.0
0.0
ASN 4 B 0.0 0.0 -0.20.0 -1.4 1.9 0.0 0.0 0.0 0.0 0.0 0.0
0.0
LEU 5 B -2.20.0 -2.00.0 0.0 0.0 0.0 -1.5 -2.5-1.0-1.00.0
0.0
LEU 6 B 0.0 0.0 0.0 0.0 0.0 0.0 -0.2-0.7 0.0 0.0 0.0 0.0
0.0
PHE 8 B -1.5-1.7 -1.2-0.20.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
-2.0
LEU 9 B -1.80.0 -0.10.0 0.0 0.0 0.0 -1.0 -0.3-2.4-3.30.0
-0.7
SER 12 0.0 1.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
B 0.2
GLN 16 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
B 0.9
HIS 93 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -0.8-2.10.9
B 0.0
ASN 96 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -0.40.0 1.0
B 0.0
HIS 97 0.0 0.0 0.0 0.0 0.0 0.0 0.0 -0.8 0.0 -2.4-2.01.9
B 0.0
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100
TH R B 0.0 0.0 0.0 0.0 0,0 0.0 0.0 0.3 -1.7 0.0 -0.7 0.0 0.0
101
VAL B 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.6 -1.6 0.0 0.0 0.0 0.0
104
G LU B 0.0 0.0 0.0 0.0 0,0 0.0 0.0 -2.6 -0.5 0.0 0.0 0.0 0.0
[0297] Residues that participate in at least one intermolecular interaction
that is at least 1 kcal/mol in
magnitude may play a role in dimer formation; those residues that form several
favorable
interactions are especially likely to be critical for dimerization.
[0298] Next, SPA calculations were used to identify suitable replacements for
the dimer interface
residues. Two sets of calculations were performed for each interface residue.
First, the energy of
the most favorable rotamer for each possible residue was determined in the
context of the
monomer structure (chain A or chain B, PDB code 1AU1). Next, the energy of the
most
favorable rotamer for each possible residue was determined in the context of
the dimer structure
(chains A and B, PDB code 1AU1). These energies were analyzed to identify
residues that are
compatible with the monomer structure but not the dimer structure. Residues
were deemed
compatible with the monomer structure if their energy score in the monomer
structure was better
than 2, and residues were deemed incompatible with the dimer structure if
their energy score in
the dimer structure was worse than 2.
[0299] Tabte 13. SPA energies in the context of the monomer structure.
The residue number and chain identifier are shown in the left, along with the
residue observed in wild type interferon beta. Energy scores were truncated at
50Ø
A C D E F G H I K L M N P Q R S T V W Y
42 A E 0.5 2.0 0.3 0.9 3.0 3.8 3.1 1.5 1.5 1.4 2.3 0.1 0.0 0.4 1.3 0.1 0.5 2.1
5.4 2.7
43 A E 1.4 1.9 2.9 1.3 1.1 6.6 3.0 1.8 0.9 0.0 1.8 2.5 2.0 1.2 0.7 2.2 1.1 0.6
3.7 1.5
46 A Q 0.9 1.9 1.7 0.6 1.8 4.1 2.2 11.2 0.4 1.1 2.7 0.0 50.0 0,0 0.4 0.6 2.1
8.5 5.7 1.4
47 A L 3.6 4.0 4.2 1.7 20.0 6.8 5.7 20.01.4 3.9 2.4 2.6 50.0 0, 0 2.5 3.7 7.5
20.0 50.0 50.0
48 A Q 1.7 2.8 1.1 1.6 4.3 4.6 3.3 2.1 2.1 2.9 2.9 0.0 3.9 0.9 2.3 1.2 1.2 2.9
7.0 3.7
49 A Q 1.0 2.1 0.5 0.8 3.4 3.3 2.8 3.7 1.9 2.3 3.4 0.0 4.9 0.5 1.4 0.2 1.6 2.9
5.8 3.3
51 A Q 1.0 2.8 3.5 1.3 3.2 4.9 2.5 4.0 1.0 1.9 3.3 1.0 0.0 0.9 1.3 0.5 3.2 3.2
5.6 3.2
113 A R 0.9 1.8 1.5 0.5 1.5 3.4 1.7 2.6 1.1 1.5 2.0 0.0 50.0 0.3 0.3 0.2 1.8
2.2 5.0 1.4
1 16 A L 0.3 2.0 1.4 0.0 2.7 4.1 3.4 1.7 1.2 1.0 2.8 0.5 50.0 0.1 1.5 0.2 0.7
1.8 5.4 3.0
117 A M 2.2 4.0 5.1 8.0 19.77.7 12.91.1 4.7 7.3 3.3 3.7 5.0 6.9 1.8 2.9 1.7
0.0 20.013.8
1 20 A L 1.9 2.9 1.5 2.2 2.1 4.5 3.4 9.4 1.4 1.8 2.8 0.0 1 7.7 2,6 2.6 2.1 3.9
8.2 5.9 1.7
121 A H 1.5 3.1 1.9 1.6 1.5 5.6 2.9 20.00.1 1.6 2.6 0.0 20.00.9 0.8 1.9 1.1
10.24.2 1.8
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124AR 0.31.61.30.04.04.21.70.71.00.92.11.050.0 1.30.40.90.56.54.0
0.5
1 BM 0.52.00.40.53.92.82.93.41.52.43.40.13.6 0.90.01.72.66.53.7
0.2
2 BS 4.14.64.33.95.50.04.03.92.44.74.42.550.0 3.43.32.16.37.86.3
3.3
3 BY 5.75.87.35.82.19.25.511.94.2 4.23.85.450.06.08.26.014.712.90.0
2.5
4 BL 1.92.40.50.64.55.45.21.51.92.83.70.05.8 2.31.21.51.46.54.8
1.1
BL 0.51.80.30.02.44.42.70.71.00.61.60.45.5 0.60.40.60.34.12.3
0.2
6 BL 5.47.06.45.520.010.1 12.320.05.5 0.04.56.050.06.316.67.4
10.850.020.020.0
7 BG 50.0 50.0
50.0 50.0
50.0 50.0
50.0 50.0
50.0 50.0
0.0 50.0
50.0
50.0
50.0
50.0
50.0
50.0
50.0
50.0
8 BF 0.81.91.20.02.44.53.05.91.50.93.20.650.0 1.30.82.69.54.32.9
0.2
9 BL 2.33.54.02.57.07.63.71.40.30.02.13.150.0 3.21.12.72.58.87.0
2.2
12 BS 0.31.20.30.31.84.43.40.50.80.31.40.050.0 1.00.60.70.92.92.3
0.1
16 BQ 0.01.50.00.34.74.51.80.30.41.10.90.750.0 1.90.11.30.47.54.5
0.6
93 BH 0.11.71.70.55.34.31.60.70.40.11.90.950.0 1.00.40.81.38.14.7
0.0
96 BN 1.32.01.60.03.05.22.00.60.60.02.01.750.0 1.31.21.71.65.93.4
0.3
97 BH 1.63.13.42.36.57.12.70.01.53.82.80.150.0 2.61.82.00.08.11
2.6 0.4
1 BT 0.92.22.41.12.85.02.80.70.80.02.41.550.0 0.81.31.61.86.53.1
0 0.6
0
101BV 2.43.64.59.220.0 8.91.43.913.0 4.050.0 6.43.52.00.020.0
8.3 7.9 9.9 20.0
1 BE 1.73.64.51.34.65.43.63.20.40.82.12.750.0 1.40.01.04.17.84.9
04 0.0
[0300] Table 14. SPA energies in the context of the dimer structure.
The residue number and chain identifier are shown in the left, along with the
residue observed in wild type interferon beta. Energy scores were truncated at
50Ø
A C D E F G H 1 K L M N P Q R S T V W Y
42 A E 0.9 2.6 1.0 1.3 2.8 4.9 3.4 0.6 1.2 0.9 2.6 0.8 0.0 0,2 2.2 1.0 1.0 1.8
5.5 2.8
43 A E 0.5 1.7 6.2 2.5 20.0 7.0 8.0 0.9 3.0 7.7 2.6 5.7 0.2 2.7 11.8 2.1 0.9
0.0 20.0 20.0
46 A Q 0.7 1.9 1.9 0.4 1.0 4.5 2.0 20.0 0.0 0.5 2.4 0.3 50.0 0.1 0.3 0.5 4.8
20.0 5.0 0.8
47 A L 4.0 4.3 4.1 1.7 14.0 8.3 3.8 20.01.4 1.9 1.3 2.6 50.0 0.0 3.7 4.8 8.0
20.0 50.0 50.0
48 A Q 1.7 2.6 0.9 1.6 3.8 4.6 3.2 1.9 2.2 2.7 2.8 0.0 4.0 0.9 2.0 1.0 1.0 2.9
6.0 3.4
49 A Q 1.4 2.9 0.8 2.3 2.5 4.8 2.9 3.0 2.5 3.9 3.6 0.0 4.3 2.4 1.9 1.6 2.3 2.3
4.3 2.6
51 A Q 1.2 2.7 3.6 1.9 2.1 5.5 3.2 3.9 1.2 1.5 2.8 2.1 0.0 1.6 1.7 0.7 3.6 3.4
2.0 1.7
113 A R 1.7 3.4 4.1 2.2 0.0 5.1 1.0 2.0 0.0 0.3 2.6 0.8 50.0 1.7 0.3 1.7 2.3
2.0 2.7 0.3
116 A L 1.9 3.3 4.4 2.3 0.0 6.9 2.7 1.3 1.7 3.0 2.0 3.7 50.0 2.9 5.1 1.3 0.9
1.6 20.0 1.8
117 A M 2.3 4.3 5.1 7.2 20.0 8.1 15.5 3.0 6.6 7.1 3.3 4.0 4.9 6.9 4.8 3.1 1.5
0.0 20.0 20.0
120 A L 1.6 2.7 1.9 2.3 0.7 4.7 2.6 8.0 0.9 0.6 1.7 0.0 19.0 2.9 3.4 2.0 2.1
7.0 3.4 0.3
121 A H 2.5 3.9 3.0 2.3 3.0 6.7 3.4 20.00.3 1.9 2.4 0.0 20.02.3 2.1 2.5 1.1
10.612.38.9
1 24 A R 0.4 1.6 1.4 0.0 3.8 4.3 1.9 0.9 1.2 0.9 2.1 1.2 5 0.0 0.7 1.4 0.3 0.9
0.5 6.3 4.3
1 B M 0.4 1.9 0.7 1.2 2.1 3.3 3.1 3.1 0.5 1.7 3.0 0.1 2.9 1.0 0.5 0.0 1.4 1.7
5.8 4.2
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2 B S 2.93.05,99.312.80.0 5.76.05.820.06.4 4.250.017.711.02.3
1.54.220.08.9
3 B Y 5.96.06.45.52.39.45.612.25.2 4.44.07.250.06.39.36.515.312.60.0
2.2
4 B N 2.42.90.21.68.66.96.92.02.12.03.00.06.1 3.72.22.42.750.0
1.9 9.3
B L 4.05.75.26.73.49.83.80.05.36.94.04.68.4 1 3.51.120.0
8.4 0.1 4.4
5.0
6 B L 5.47.06.54.920.010.1 14.020.05.9 0.04.46.150.06.317.97.3
11.050.020.020.0
7 B G 50.0 50.0
50.0 50.0
50.0 50.0
50.0 50.0
50.0 50.0
0.0 50.0
50.0
50.0
50.0
50.0
50.0
50.0
50.0
50.0
8 B F 4.96.07.34.40.09.84.717.54.1 5.24.95.950.03,78.06.15.713.810.25.6
9 B L 2.94.75.94.22.88.52.61.90.00.12.14.95 0.0 4.31.63.83.220.0
3,6 3.1
12 B S 0.11.50.77.39.14.91 5.96.04.80.450.0 7.60.91.20.09.8
6.5 7,4 8.4
2.0
16 B Q 0.11.60.30.74.74.62.00.30.01.11.20.95 0.0 0.50.11.20.36.0
0,6 4.7
93 B H 0.01.71.10.05.44.31.60.60.70.01.51.050.0 1.60.70.91.18.9
0.1 4.6
96 B N 1.42.01.60.13.15.31.80.81.00.02.12.050.0 2.11.21.81.65.7
0.5 3.5
97 B H 1.93.43.42.75.37.62.80.01.53.42.10.850.0 3.82.52.30.520.0
2.9 20.0
100B T 1.12.62.31.31.85.52.60.71.30.02.52.150.0 1.61.91.91.96.1
1.1 2.5
101B V 2.02.63.19.020.0 15.018.3 20.012.3 50.010.510.3
1.50.020.0
7.9 6.5 3.3 3.4 20.0
1 B E 2.03.44.32.62.86.45.63.20.07.92.54.35 0.0 3.00.10.63.63.6
04 1.6 4.2
[0301] Table 15. Suitable replacements for dimer interface positions,
as determined by the above SPA calculations.
A C D E F G H I K L M N P Q R S T V W Y
42 A E
43 A E F I< L R Y
46 A Q
47 A L
48 A Q
49 A Q
51 A Q
113A R D
116A L D E L N Q R
117A M R
120A L
121A H Y
124A R
1 B M
2 B S
3 B Y
4 B L
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B L A C D E K L M N Q R S T
6 B L
7 B G
8 B F A C D E K L N Q R S
9 B L
12 B S E F K L M Q R
16 B Q
93 B H
96 B N
97 B H
100 B T
101 B V I
104 B E L
[0302] As can be observed in the tables above, positions 5, 8, 12, 43, and 116
are all involved in
stabilizing the dimer structure of interferon-beta, and a number of
modifications at these positions
are predicted to significantly prevent dimerization.
[0303] Further analysis was performed to determine which of the above
modifications is most likely
to significantly prevent dimerization. Hydrophobic interactions and
electrostatic interactions
(including salt bridges and hydrogen bonds) can stabilize protein-protein
interfaces. These
interactions may be effectively disrupted by hydrophobic to polar and charge
reversal mutations.
[0304] Hydrophobic residues that are significantly less solvent exposed in the
dimer structure versus
the monomer structure were defined to be those residues that are classified as
surface in the
monomer and core or boundary in the dimer, and residues that are classified as
boundary in the
monomer and core in the dimer, as shown below:
[0305] Table 16. Hydrophobic residues that are more buried in the dimer than
in the monomer.
Residue Monomer Dimer
Leu 5 Boundary Core
Phe 8 Surface Core
Leu 9 Boundary Core
Leu 47 Boundary Core
Leu 116 Surface Boundary
[0306] Debye-Huckel scaled Coulomb's law calculations were performed on the
1AU1 dimer and
monomers, using an ionic strength of 0.15 M, to determine the electrostatic
potential at each
75 '
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position in the context of the monomer versus the dimer. The following
positions were found to
have a change in potential of at least 0.20 kcal/mol:
[0307] Table 17. Positions that experience a significant difference
in electrostatic potential in the dimer versus monomer structure.
Dimer Monomer Difference
SER 2 B 0.36 -0.30 0.66
LEU 5 B -0.10 0.11 -0.21
PHE 8 B 0.14 0.42 -0.28
LEU 9 B -0.11 0.16 -0.27
SER 12 B -0.42 0.29 -0.71
LEU 47 A 0.25 0.04 0.21
GLN 49 A 0.32 0.08 0.24
HIS 93 B 0.29 0.04 0.25
ASN 96 B 0.24 0.04 0.20
THR 100 B -0.22 -0.45 0.23
VAL 101 B 0.15 -0.39 0.54
GLU 104 B 0.58 -0.02 0.60
ARG 113 A -1.37 -0.36 -1.01
[0308] Modifications of the electrostatic properties of the residues at these
positions can be selected
to favor the monomer structure and disfavor the dimer structure. For example,
Glu 104 and Arg
113 form a salt bridge in the dimer structure, which can be observed in the
crystal structure. In
the table above, Glu 104 is in a region of positive potential in the dimer and
neutral potential in the
monomer, while Arg 113 is in a region of negative potential in the dimer
structure and slightly
negative potential in the monomer structure. Modifications that could disrupt
this interaction
include, but are not limited to, E104R, E104K, E104H, E104Q, E104A, R113D,
R113E, R113Q,
and R113A.
[0309] Example 7: Identification of suitable replacements for free cysteine
residues
[0310] PDA~ technology calculations were also performed to identify suitable
replacements for free
cysteine residues. These calculations were performed using the methods
described above for the
hydrophobic to polar point mutations, except that both polar and nonpolar
replacements were
considered. Alternate residues with favorable energies are marked with a star
(*) below.
[0311] Table 18. Free cysteine calculation results
76
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IFNa
AA Total VDW Elec HBond Solv
TYR-C -10.45 -0.11 -2.32 -0.59
-13.47
ILE 15.37 13.90 0.00 0.00 1.47
* LEU -5.58 -5.38 0.00 0.00 -0.20
* MET -6.17 -5.42 0.00 0.00 -0.75
PHE 887.53 893.12 0.00 0.00 -5.59
TRP 0.98 -6.86 -0.01 0.00 7.86
TYR 803.08 804.33 -0.02 0.00 -1.23
VAL 27.93 29.08 0.00 0.00 -1.15
ALA -2.53 -1.89 0.00 0.00 -0.63
* ASP -4.45 -4.05 0.33 0.00 -0.73
* GLU -7.53 -4.66 0.39 0.00 -3.26
* HIS -5.94 -6.12 -0.12 0.00 0.30
* HSP -4.19 -5.94 -0.76 0.00 2.51
* LYS -8.48 -5.48 -0.38 0.00 -2.63
ASN -3.00 -4.12 -0.03 0.00 1.15
* GLN -8.21 -4.70 -0.01 0.00 -3.50
* ARG -4.73 -5.42 -0.24 0.00 0.93
SER -4.04 -2.17 -0.02 0.00 -1.85
* THR -5.10 -3.08 -0.02 0.00 -2.01
IFNb
CYS-C -7.06 0.00 0.00 -6.91
-13.97
ILE 324.91 334.90 0.00 0.00 -9.99
LEU 840.30 846.29 0.00 0.00 -5.99
MET 2082.912089.080.00 0.00 -6.17
PHE 5529.905539.670.00 0.00 -9.77
TYR 6341.296346.98-0.26 0.00 -5.43
VAL 82.62 89.33 0.00 0.00 -6.70
* ALA -8.69 -3.42 0.00 0.00 -5.27
* ASP -10.20 -7.37 0.12 0.00 -2.96
GLU 357.99 358.18 0.42 0.00 -0.62
NIS 501.55 504.61 -0.05 0.00 -3.01
HSP 506.45 506.93 0.35 0.00 -0.83
LYS 2087.792085.18-0.04 0.00 2.64
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* ASN -5.08 -6.54 0.11 0.00 1.36
GLN 483.14 479.27 0.10 0.00 3.77
ARG 15093.5915085.560.04 0.00 7.99
* SER -5.96 -4.41 -0.08 0.00 -1.47
* THR -9.17 -5.20 0.06 0.00 -4.03
IFNk
LEU-C 5514.27-0.41 0.00 -6.01
5507.86
ILE 44.93 50.89 0.00 0.00 -5.96
LEU -13.20 -7.12 0.00 0.00 -6.08
* MET -3.21 3.30 0.00 0.00 -6.51
PHE 36.05 43.81 0.00 0.00 -7.76
TRP 292.31 298.19 -0.01 0.00 -5.87
TYR 196.77 200.15 -0.01 0.00 -3.37
VAL 37.53 42.27 0.00 0.00 -4.74
* ALA -7.83 -2.63 0.00 0.00 -5.20
ASP -4.81 -5.70 -0.12 0.00 1.01
* GLU -9.02 -8.02 -0.17 0.00 -0.83
* HIS -10.31 -9.00 -0.11 0.00 -1.21
* HSP -7.47 -8.25 -0.23 0.00 1.00
LYS 2.43 0.20 0.02 0.00 2.22
ASN -0.48 -5.83 0.00 0.00 5.35
* GLN -4.21 -7.92 -0.03 0.00 3.74
ARG 52.67 44.39 0.01 0.00 8.27
* SER -4.86 -3.32 0.00 0.00 -1.54
* THR -3.56 -3.63 -0.10 0.00 0.18
j0312] Example 8: Generation of interferon beta variants
[0313] Construction of the interferon beta gene as a template formutagenesis.
The DNA sequence,
GenBank accession number NM 002176, encompassing the full-length human
interferon beta
cDNA gene containing the native signal sequence was modified to remove the
signal sequence
and facilitate high level expression in bacterial cells. Primers were designed
to synthesize the
region between positions 65-561 by recursive PCR. The primer sequences also
biased the codon
usage towards highly expressed E. coli bacterial genes. In addition, the codon
for cysteine 17
(amino acid numbering with the signal sequence removed) was changed to serine.
An internal
Sacl DNA restriction enzyme site was designed for ease of later mutagenesis as
well as Ndel and
Xhol restriction sites flanking the ends of the gene for cassette cloning into
various expression
J
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vectors. The bacterial expression vectors pET28a and pET24a (Novagen) were
used to sub-
clone the interferon beta gene containing C17S between the Ndel and Xhol
multiple cloning
restriction sites. Cloning into pET24a expression in E coli produces a C17S
interferon beta
variant while cloning into pET28a introduces the additional amino acid
sequence
MGSSHHHHHHSSGLVPRGSH to the N-terminus of C17S. This amino acid sequence
includes a
6-His purification tag and a thrombin cleavage site for later removal of the
added amino acid
sequences.
[0314] Construction of interferon beta variants containing exposed hydrophobic
to polar mutations
[0315] Sixteen solvent exposed hydrophobic residues were identified in the
interferon beta structure.
Polar amino acid residues to substitute at these positions were designed by
computational
analysis as described above. The list of substitutions are listed in the table
below:
[0316] Table 19. List of substitutions used in library of interferon beta
variants
positionwt LIB
L Q
8 F E
F D
22 W E
28 L Q
30 Y N
32 L E
36 M K
47 L K
92 Y D
111 F N
116 L E
120 L R
130 L T
148 V E
155 Y S
[0317] Mutagenesis experiments were done to construct variants containing
these amino acid
substitutions in the interferon beta-C17S gene background (referred to as
"wild type" throughout
the following examples).
[0318] For a library containing combinations of the wild-type or substitution
listed in the table above,
a template directed ligation-PCR method was used as described in Strizhov et.
al. PNAS
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93:15012-15017 (1996). Variants constructed contain single or multiple
combinations of the
substitutions.
[0319] For a 64-member library containing all possible combinations of wild-
type or above-listed
substitution at positions 5,8,47,111,116, andlor 120, multiple rounds of site-
directed mutagenesis
reactions were done using the Quikchange kit (commercially available from
Stratagene) following
the manufacturer's protocol. Positive clones were identified by sequencing.
[0320] Production of interferon beta variants in E. coli. Sequence verified
clones in pET28a were
transformed into BL21 (DE3) star cells (commercially available from
invitrogen) and cultures were
grown in auto-inducing media, a rich medium for growth with little or no
induction during log phase
and auto-induction of expression as the culture approaches saturation. Media
components
include 25 mM (NH4)~S04, 50 mM KH2PO4, 50 mM Na2HP04, 1 mM MgS04, 0.5%
glycerol,
0.05% glucose, 0.2% alpha-lactose, 0.1% tryptone, and 0.05% yeast extract. The
cultures were
grown for 7 hours to an OD between 4 and 5 and cells harvested by
centrifugation. Cells were
lysed by sonication, inclusion pellets denatured in 8M guanidine HCI and bound
to a column
containing Ni-NTA resin. A dilution series of guanidine HCI with decreasing pH
was used to purify
and refold the protein.
[0321] An alternative method for purification of clones with and without the N-
terminal 6-His tag was
followed as disclosed in US 4,462,940, Lin et al, Meth. Enzymoi. 119:183-192.
[0322] Example 9: Soluble expression of interferon beta variants. Each of the
64 members of the
library described above were tested for soluble expression. Western blot
analysis utilizing an
anti-His antibody was done for the soluble fractions of cell lysates. A band
running at the
expected size of approximately 20 kilodaltons was present for at least 33 of
the variants but was
not detectable for the C17S variant, suggesting that many of the designed
variants exhibit
improved soluble expression.
[0323] Example 10: Activity analysis of constructed variants
[0324] A standard ISRE (interferon-stimulated response element) reporter assay
was used to
determine the activity of interferon beta variants. In this assay, 293T cells
which constitutively
express the type I interferon receptor were transiently transfected with an
ISRE-luciferase vector
(pISRE-luc, commercially available from Clontech). Twelve hours after
transfection, the cells
were treated with a dilution series of concentrations for an interferon beta
variant. Variants which
bind the interferon receptor and trigger the JAK/STAT signal transduction
cascade activate
transcription of the luciferase gene operably linked to the ISRE. Luciferase
activity was detected
using the Steady-Glo~ Luciferase Assay System (commercially available from
Promega) with the
TopCount NXTTM microplate reader used to measure luminescence.
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[0325] Initial activity determination utilizing the ISRE reporter assay was
done for the 64 member
library described in example 8. Cultures were grown, cells harvested and
lysed. The inclusion
pellet was resuspended in a 0.025% SDS solution and tested in the ISRE
activity assay. Activity
was demonstrated for the 37 variants listed in the table below. However, since
the amount of
protein tested in this assay was not quantitated first, it is possible that
additional variants are
active but were present in insufficient quantity to be detected in the assay.
[0326] Table 20: Amino acid sequences at exposed hydrophobic positions for
active interferon beta
variants
Amino acid position
Variant 5 8 47 111 116 120
IFB1 2 Q F L F L L
IFB1 3 Q F K F L L
IFB1 4 L E L F L L
IFB1 5 L E K F L L
IFB1 6 L F K F L L
IFB1 7 Q E L F L L
IFB1 8 Q E K F L L
IFB1 9 L F L N L L
IFB1 10 Q F L N L L
IFB1 11 Q F K N L L
IFB1 15 Q E L N L L
IFB1 16 Q E K N L L
IFB1 23 Q E L F E L
IFB1 26 Q F L F L R
IFB1 27 Q F K F L R
IFB1 28 L E L F L R
IFB1 29 L E K F L R
IFB1 31 Q E L F L R
IFB1 32 Q E K F L R
IFB1 33 L F L N E L
IFB1 34 Q F L N E L
IFB1 35 Q F K N E L
IFB1 36 L E L N E L
IFB1 37 L E K N E L
IFB1 39 Q E L N E L
IFB1 40 Q E K N E L
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IFB141 L F L N L R
IFB142 Q F L N L R
IFB144 L E L N L R
IFB147 Q E L N L R
IFB1_48 Q E K N L R
IFB150 Q F ' L F E R
IFB151 Q F K F E R
IFB152 L E L F E R
IFB155 Q E L F E R
IFB156 Q E K F E R
IFB163 Q E L N E R
IFB164 Q E K N E R
[0327] Those variants exhibiting increased activity relative to the wild type
(interferon beta C17S)
were tested for more quantitative activity measurements. Selected variants
were purified and
refolded as described in example 8 above. Each variant was then assayed using
a ten point half
log dilution series in the ISRE reporter assay. GraphPad Prism, version 4
(GraphPad Software,
Inc.) was used to plot the data and calculate EC50 values. The dose response
curves for the
retested variants are shown in figure 4. Ail of the variants exhibited
improved activity, with EC50
values ranging from 12-30 fold better activity than C17S interferon beta, as
shown in the table
below.
[0328] Table 21. Specific activity data for interferon-beta variants.
[0329] The sequence for residues 5, 8, 47, 111, 116, and 120 is given for each
variant, along with
the total number of mutations, the EC50, and the ratio of the wild type to
variant EC50. Variant
IFN1 1 is the interferon beta wild type with C17S.
Variant 5 8 47 111 116120 EC50 (log EC50 wt l EC50
# ng/ml) var
mut
IFN11 L F L F L L 0 5.306 1.0
IFB12 Q F L F L L 1 0.428 12.4
IFB17 Q E L F L L 2 0.179 29.6
IFB115 Q E L N L L 3 0.319 16.6
IFB123 Q E L F E L 3 0.277 19.2
IFB136 L E L N E L 3 0.294 18.0
1FB139 Q E L N E L 4 0.193 27.5
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IFB1 64 Q E K N E R 6 0.240 22.1
[0330] Activity Comparison with claimed solubility mutant from US Patent No.
6,572,853.
[0331] Several variants with enhanced solubility were claimed in US 6,572,853.
Activity comparison
of one of these claimed variants with the C17S wild type and the most active
variant tested above
was done. Purification of all the variants and activity evaluation was done
under the same
conditions with the results shown in the table below. The claimed solubility
variant (IFB_GM2)
exhibited 67 fold less activity than the wild type C17S interferon beta. In
comparison, variant
IFB1 7 still exhibited better than 25 fold better activity than the wild type.
[0332] Table 22. Specific activity data for interferon-beta variants.
[0333] The sequence for residues 5, 8, 47, 50, 106, 111, 116, and 120 is given
for each variant,
along with the total number of mutations, the EC50, and the ratio of the wild
type to variant EC50.
All variants are in the C17S background.
Variant5 8 47 50 106111 116120# EC50 (ng/ml)EC50 wt / EC50
mut var
IFN1 L F L F L F L L 0 1.90 1.00
1
IFB1 Q E L F L F L L 2 0.074 25.7
7
IFB L F S S S S S S 6 130 0.015
GM2
[0334] Example 11: Mutagenesis, expression, and soluble expression screening
of interferon
kappa
[0335] Construction of interferon kappa variants
[0336] Interferon kappa variants (total library size = 1024) with the
mutations listed in the table below
(single and all possible multiple combinations) were constructed essentially
as described above
for the Interferon beta variants.
[0337] Table 23. List of substitutions used in library of interferon-kappa
variants.
[0338] Each position or set of positions could have either the wild type
hydrophobic residues) or the
alternate polar residues) listed in the "LIB" column.
positions)wt LIB
5-8 L-V Q-N
15 W R
28-30 F-V Q-R
37 I N
48-52 Y-M Q-N
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76-78 F-Y S-A
89 I T
97 Y D
161 V A
166-168-171C-Y-Y A-S-T
[0339] Expression and screening for soluble expression via dot-blot using anti-
His antibodies for
detection.
[0340] The soluble fraction of E coli lysates expressing individual interferon-
kappa variants were dot
-blotted on nitrocellulose membranes, and the presence of soluble His-tagged
protein was
detected using anti-His antibodies conjugated to HRP. Figure 5 shows the
results of a dot-blot
analysis. The positive clones expressing soluble interferon-kappa were
regrown, and expressed
protein was retested to confirm soluble expression. Figure 6 shows a retest
plate.
[0341] The soluble extract from interferon-kappa variants testing positive
during the secondary
screen were then analyzed by SDS-PAGE/Western blotting to confirm the presence
of the
correctly sized protein band. Figure 7 is an example of these SDS-PAGE/Western
blot
experiments, identifying several interferon-kappa variants expressing the
correctly sized protein
with solubility characteristics better than WT interferon-kappa. The arrow
indicates the expected
position of interferon-kappa protein. Lanes 2 and 3 are total soluble fraction
from WT interferon-
kappa expressing cells, respectively. Lanes 4-15 are soluble fractions from
the lysates of
different variants.
[0342) Table 24. Sequence analysis of selected interferon kappa variants with
improved soluble
expression.
WT Seq L-V W F-V I Y-M F-Y I Y V C-Y-Y
MutationQ-N R Q-R N Q-N S-A T D A A-S-T
166,
Mutant 5, 15 28, 37 48, 76, 89 97 161 168,
8 30 52 78 171
IK 4-G7L-N R F-V I Q-N S-A T Y V C-Y-Y
IK 12-E4L-N R F-V I Q-N S-A T Y V C-Y-Y
IK 2-C11L-N R Q-R N Y-M S-A T D A A-S-T
IK 10-D8L-N W F-V I Q-N F-Y T D V A-S-T
I K L-N W F-V I Q-N S-A T D A A-S-T
10-H
7
IK 20-B12L-N W Q-R I Q-N S-A T Y V A-S-T
IK 3-A11L-N W Q-R I Y-M S-A T D A A-S-T
IK_3-H7L-N W Q-R I Y-M S-A T D A IA-S-T
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IK 12-F11L-N W Q-R N Q-N S-A T Y V A-S-T
I K L-V R F-V Q-N S-A D V A-S-T
3-D I T
IK 3-C10L-V R F-V Q-N S-A T D V C-Y-Y
I
IK 3-H11L-V R F-V Q-N S-A T D V C-Y-Y
I
IK 21-E1L-V R F-V Y-M S-A D V A-S-T
I I
IK 4-H11L-V R F-V Y-M S-A T D A C-Y-Y
I
I K L-V R F-V Y-M S-A T D V A-S-T
3-A2 I
IK 10-D2L-V R F-V N Y-M S-A T D V C-Y-Y
IK 12-H4L-V W F-V Q-N S-A Y V C-Y-Y
I I
IK 27-A6L-V W F-V Q-N S-A T D A C-Y-Y
I
IK 2-B4L-V W F-V Q-N S-A T D V C-Y-Y
I
IK 3-F11L-V W F-V I Q-N S-A T D V C-Y-Y
IK 14-A9L-V W F-V I Y-M F-Y T Y V C-Y-Y
IK 19-A5L-V W F-V I Y-M S-A I D A C-Y-Y
I K L-V W F-V I Y-M S-A I D V C-Y-Y
3-G
10
IK 4-A2L-V W F-V I Y-M S-A I D V C-Y-Y
IK 4-A10L-V W F-V I Y-M S-A I D V C-Y-Y
IK 16-G2L-V W F-V I Y-M S-A T D A C-Y-Y
IK 22-A4L-V W F-V I Y-M S-A T D V A-S-T
IK 1-C L-V W F-V N Q-N S-A I D V C-Y-Y
8
I K L-V W F-V N Q-N S-A I D V C-Y-Y
23-C
10
IK 12-H11L-V W F-V N Q-N S-A T Y V C-Y-Y
IK_9-H4L-V W Q-R N Y-M IS-A I D V IA-S-T
[0343] Variants with improved soluble expression were tested for activity
using the ISRE assay,
essentially as in the initial activity assay described above. A number of
variants that retain
interferon activity were identified, including those listed below.
[0344] Table 25. Sequence analysis of some of the interferon-kappa variant,
which still retain activity, as tested in an ISRE assay as described above for
interferon beta.
WT seq L-V W F-V I Y-M F-Y I Y V C-Y-Y
MutationsQ-N R Q-R N Q-N S-A T D A A-S-T
166,
168,
Variant 5, 15 28, 30 48, 76, 78 97 161 171
8 37 52 89
IK1 4 L-N R F-V I Q-N S-A T Y V C-Y-Y
G7
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IK146 L-V R F-V N Q-N S-A T D A A-S-T
E2
!K147 L-V R F-V I Y-M S-A I Y V C-Y-Y
C4
IK123 L-V W F-V N Q-N S-A I D V C-Y-Y
C10
IK140 L-V R F-V N Y-M S-A I Y V C-Y-Y
A10
[0345] Example 12: Evaluation of oligomerization and aggregation at
physiological pHs
[0346] This experiment evaluated the efficacy of the IFN of the present
invention with respect to
stability at certain pHs. More specifically, with the purpose of decreasing
dimerization and higher
order oligomerization of the molecule at pH ranges close to physiological
ranges in order to
determine aggregation levels. Thus, the relative rates of aggregation of
XENP342 (C17S
background, SEQ ID NO: 31) and XENP806 (L5Q/F8E/C17S variant, SEQ ID NO: 20)
were
measured.
[0347] The proteins were purified by Ni-capture of His-tagged Gu-solubilized
inclusion bodies
released from bacteria. Ni-elution fractions were refolded and cleaved with
thrombin, followed by
application to a semi-prep scale C4 protein reversed phase column. Elution
fractions identified to
have the target were repurified on an analytical scale column with the same
matrix. Fractions
were 'speed-vac'-treated to remove acetonitrile prior to use.
[0348] Interferon aggregation was examined using dynamic light scattering
measurements on a
Malvern Zetasizer DLS (Malvern Instruments). Samples were analyzed by DLS
initially, and then
again after incubation under conditions designed to promote aggregation (9
hours at 37°C). Neat
samples measuring pH 1.6 due to residual TFA were diluted into either
phosphate-citrate buffer
resulting in pH 3, or glycine-NaOH (both 200 mM in buffer components, 300 mM
NaCI) buffer
resulting in pH 6 ~ 1.
[0349] Data shown in Figures 9 and 10 are from two sequential measurements
(green and red trace)
on each sample tested, with no disturbance of the sample between measurements.
In order to
minimize perturbation, samples were not filtered or centrifuged.
[0350] Data presented in Figure 9 are from pH 3 samples, and Figure 10 shows
samples at around
pH 6. The data. The commercial interferon (BetaSeron~, Schering AG/Berlex)
(middle graph in
Figs. 9 and 10) aggregates much more rapidly than XENP806 and qualitatively
shows more
aggregation than the interferon of the present invention.
[0351] ***examples from provisional
[0352] Example 13. Identification of MHC-binding agretopes in interferon beta
[0353] Matrix method calculations (Sturniolo, supra) were conducted using the
parent interferon beta
sequence shown in SEQ ID N0:1.
[0354] Agretopes were predicted for the following alleles, each of which is
present in at least 1 % of
the US population: DRB1*0101, DRB1*0102, DRB1*0301, DRB1*0401, DRB1*0402,
DRB1*0404,
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DRB1*0405, DRB1*0408, DRB1*0701, DRB1*0801, DRB1*1101, DRB1*1102, DRB1*1301,
DRB1*1302, DRB1*1501, and DRB1*1502.
[0355] For each 9-mer that is predicted to bind to at least one allele at a 5
% threshold, the number
of alleles that are hit at 1 %, 3% and 5°lo thresholds were given, as
well as the percent of the US
population that are predicted to react to the 9-mer. The worst 9-mers are
shown in bold. They are
predicted to be immunogenic in at least 10% of the US population, using a 1 %
threshold.
[0356] Table 26. Predicted MHC-binding agretopes in interferon beta. The
number of alleles and
percent of population hit at 1 %, 3%, and 5% thresholds are shown. Especially
preferred
agretopes are predicted to affect at least 10% of the population, using a 1 %
threshold.
Table 26.
Predicted
MHC-bindin
a retopes
in interferon
beta.
Agretope
number Residues Se uence 1 % 3% 5% hits1 % 3% 0 5%
hits hits 0 o
1 3 - 11 YNLLGFLQR0 0 1 0.0% 0.0% 11.4%
2 5 - 13 LLGFLQRSS0 3 4 0.0% 19.9% 21.2%
3 8 - 16 FLQRSSNFQ0 2 2 0.0% 6.7% 6.7%
4 9 -17 LQRSSNFQC0 0 2 0.0% 0.0% 7.5%
15 - 23 FQCQKLLWQ0 1 1 0.0% 11.4% 11.4%
6 22 - 30 WQLNGRLEY2 3 5 19.3% 20.9% 28.3%
7 30 - 38 YCLKDRMNF0 2 2 0.0% 13.5l0 13.5%
8 36-44 MNFDIPEEI1 1 1 21.3% 21.3% 21.3%
9 47 - 55 LQQFQKEDA0 0 1 0.0% 0.0% 1.7%
57 - 65 LTIYEMLQN0 2 2 0.0% 24.1 24.1
%
11 60 - 68 YEMLQNIFA2 7 7 15.0% 40.2% 40.2%
12 63 - 71 LQNIFAIFR0 1 1 0.0% 5.0% 5.0%
13 70 - 78 FRQDSSSTG0 1 3 0.0% 14.0% 33.5%
14 79 - 87 WNETIVENL0 0 1 0.0% 0.0% 24.7%
95 -103 INHLKTVLE1 1 1 1.8% 1.8% 1.8%
16 122 -130 LKRYYGRIL0 2 2 0.0% 24.1 24.1
%
17 125 -133 YYGRILHYL0 0 1 0.0% 0.0% 5.1
18 129 - I LHYLKAKE1 1 1 5.1 5.1 5.1
137 % %
19 130 -138 LHYLKAKEY0 0 1 0.0% 0.0% 5.0%
143 - WTIVRVEIL1 1 1 24.7% 24.7% 24.7%
151
21 145 -153 IVRVEILRN1 3 5 4.5% 19.7% 39.0%
22 146 -154 VRVEILRNF0 1 2 0.0% 10.5% 18.5%
23 148 -156 VEILRNFYF0 1 4 0.0% 5.9% 29.3%
24 151 - LRNFYFINR1 2 2 22.6% 24.1% 24.1%
159
154 - FYFINRLTG1 3 5 11.4% 17.1% 28.3%
162
26 156-164 FINRLTGYL1 1 1 5.1% 5.1% 5.1%
27 157 -165 INRLTGYLR0 0 1 0.0% 0.0% 5.0%
[0357] Alleles that are predicted as "hits" for each of the agretopes above
are snown in the tame
below. "1" indicates a hit using a 1 % threshold, "3" indicates a hit using a
3% threshold, and "5"
indicates a hit using a 5% threshold.
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[0358] Table 27. Predicted MHC-binding agretopes in interferon beta. DRB1
alleles that are
predicted to bind to each allele at 1 %, 3%, and 5% cutoffs are marked with "1
", "3", or "5",
respectively.
Table
27.
DRB1
alleles
predicted
to
bind
MHC
a reto
es
in
interferon
beta.
Agretope
number 01010102030104010402040404050408070108011101110211041301130215011502
g _ _ _ - - - - - - 5 1 - 5 - 1 - 3
g _ _ 1 _ _ _ _ _ _ _ _ _ _ _ _ _ _
11 3 - - 1 - 3 3 1 - - 3 - - - - - 3
20 _ _ _ _ _ _ _ _ 1 _ _ _ _ _ _ _ _
24 - _ _ _ _ _ _ _ _ _ _ _ _ _ _ 1 3
25 - - - - - - - - - 5 1 - 3 - 5 - 3
[0367] Example 14. identification of suitable less immunogenic sequences for
MHC-binding
agretopes in interferon beta: BLOSUM method.
[0368] MHC-binding agretopes that were predicted to bind alleles present in at
least 10% of the US
population, using a 1 % threshold, were analyzed to identify suitable less
immunogenic variants.
[0369] At each agretope, all possible combinations of amino acid substitutions
were considered, with
the following requirements: (1) each substitution has a score of 0 or greater
in the BLOSUM62
substitution matrix, (2) each substitution is capable of conferring reduced
binding to at least one of
the MHC alleles considered, and (3) once sufficient substitutions are
incorporated to prevent any
allele hits at a 1% threshold, no additional substitutions are added to that
sequence.
[0370] Alternate sequences were scored for immunogenicity and structural
compatibility. Preferred
alternate sequences were defined to be those sequences that are not predicted
to bind to any of
the 17 MHC alleles tested above using a 1 % threshold, and that have a total
BLOSUM62 score
that is at least 85% of the wild type score.
[0371 ] Table 28. Suitable less immunogenic variants of agretope 6 (residues
22-30). B(wt) is the
BLOSUM62 score of the wild type 9-mer, I(alt) is the percent of the US
population containing one
or more MHC alleles that are predicted to bind the alternate 9-mer at a 1%
threshold, and B(alt) is
the BLOSUM62 score of the alternate 9-mer.
Table 28.
Suitable
less immuno
enic variants
of a reto
a 6 (residues
22-30 .
Variant
Se uence se uence I alt B alt WT se B wt
ID uence
SEQ ID;1 WSLNGRLEY0 48 WQLNGRLEY53
SEQ 1D;2 WNLNGRLEY0 48 WQLNGRLEY53
SEQ ID;3 WDLNGRLEY0 48 WQLNGRLEY53
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SEQ ID:4 WELNGRLEY0 50 WQLNGRLEY53
SEQ ID:5 WHLNGRLEY0 48 WQLNGRLEY53
SEQ ID:6 WKVNGRLEY0 46 WQLNGRLEY53
SEQ ID:7 WQVNGRLEY0 50 WQLNGRLEY53
SEQ ID:8 WQFSGRLEY0 44 WQLNGRLEY53
SEQ ID:9 WQFTGRLEY0 43 WQLNGRLEY53
SEQ ID:10 WQFGGRLEY0 43 WQLNGRLEY53
SEQ ID:11 WQLSGRLEY0 48 WQLNGRLEY53
SEQ ID:12 WQLTGRLEY0 47 WQLNGRLEY53
SEQ ID:13 WQLGGRLEY0 47 WQLNGRLEY53
SEQ ID:14 WQLNSQLEY0 43 WQLNGRLEY53
[0372] Table 29. Suitable less immunogenic variants of agretope 8 (residues 36-
44). B(wt) is the
BLOSUM62 score of the wild type 9-mer, I(alt) is the percent of the US
population containing one
or more MHC alleles that are predicted to bind the alternate 9-mer at a 1 %
threshold, and B(alt) is
the BLOSUM62 score of the alternate 9-mer.
Table 29.
Suitable
less immuno
enic variants
of a reto
a 8 residues
36-44).
Variant WT
Se uence se uence I alt B alt se uence B wt
ID
SEQ ID:15 QSFDIPEEI0 39 MNFDIPEEI48
SEQ ID:16 QDFDIPEEI0 39 MNFDIPEEI48
SEQ ID:17 MSFDIPEEI0 43 MNFDIPEEI48
SEQ ID:18 MTFDIPEEI0 42 MNFDIPEEI48
SEQ ID:19 MGFDIPEEI0 42 MNFDIPEEI48
SEQ ID:20 MDFDIPEEI0 43 MNFDIPEEI48
SEQ ID:21 MEFDIPEEI0 42 MNFDIPEEI48
SEQ ID;22 MNYSIPEEI0 39 MNFDIPEEI48
SEQ ID:23 MNYNIPEEI0 40 MNFDIPEEI48
SEQ ID:24 MNYEIPEEI0 41 MNFDIPEEI48
SEQ ID;25 MNYQIPEEI0 39 MNFDIPEEI48
SEQ ID;26 MNFSIPEEI0 42 MNFDIPEEI48
SEQ ID;27 MNFNIPEEI0 43 MNFDIPEEI48
SEQ ID:28 MNFEIPEEI0 44 MNFDIPEEI48
SEQ ID:29 MNFQIPEEI0 42 MNFDIPEEI48
SEQ ID:30 MNFDIPESL0 41 MNFDIPEEI48
SEQ ID:31 MNFDIPESV0 42 MNFDIPEEI48
SEQ ID:32 MNFDIPENL0 41 MNFDIPEEI48
SEQ ID:33 MNFDIPENV0 42 MNFDIPEEI48
SEQ ID:34 MNFDIPEDL0 43 MNFDIPEEI48
SEQ ID:35 MNFDIPEDV0 44 MNFDIPEEI48
SEQ ID:36 MNFDIPEQL0 43 MNFDIPEEI48
SEQ ID:37 MNFDIPEQV0 44 MNFDIPEEI48
SEQ ID:38 MNFDIPEHL0 41 MNFDIPEEI48
SEQ ID:39 MNFDIPEHV0 42 MNFDIPEEI48
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SEQ ID:40 MNFDIPERL0 41 MNFDIPEEI48
SEQ ID:41 MNFDIPERV0 42 MNFDIPEEI48
SEQ ID:42 MNFDIPEKL0 42 MNFDIPEEI48
SEQ ID:43 MNFDIPEKV0 43 MNFDIPEEI48
SEO ID:44 MNFDIPEEL0 46 MNFDIPEEI48
SEQ ID:45 MNFDIPEEV0 47 ~ MNFDIPEEI48
I
[0373] Table 30 Suitable less immunogenic variants of agretope 11 (residues 60-
68). B(wt) is the
BLOSUM62 score of the wild type 9-mer, I(alt) is the percent of the US
population containing one
or more MHC alleles that are predicted to bind the alternate 9-mer at a 1 %
threshold, and B(alt) is
the BLOSUM62 score of the alternate 9-mer.
Table 30.
Suitable
less immuno
enic variants
of a reto
a 11 (residues
60-68).
Variant WT
Se uence se uence I alt B alt se uence B wt
ID
SEQ ID:46 HDMLQNIFA0 38 YEMLQNIFA46
SEQ ID:47 YSQLQNIFA0 37 YEMLQNIFA46
SEQ ID:48 YSLLQNIFA0 38 YEMLQN1FA46
SEQ ID:49 YSVLQNIFA0 37 YEMLQNIFA46
SEQ ID:50 YSFLQNIFA0 37 YEMLQNIFA46
SEQ ID:51 YEQLQNIFA0 42 YEMLQNIFA46
SEQ ID:52 YEMLQNIYT0 39 YEMLQN1FA46
SEQ ID:53 YEMLQNIWT0 37 YEMLQNIFA46
SEQ 1D:54 YEMLQN1FT0 42 ~ EMLQNIFA ' 46
[0374] Table 31. Suitable less immunogenic variants of agretope 20 (residues
143-151 ). B(wt) is
the BLOSUM62 score of the wild type 9-mer, I(alt) is the percent of the US
population containing
one or more MHC alleles that are predicted to bind the alternate 9-mer at a 1
% threshold, and
B(alt) is the BLOSUM62 score of the alternate 9-mer.
Table 31.
Suitable
less immuno
enic variants
of a reto
a 20 (residues
143-151
.
Variant WT
Se uence se uence I alt B alt se uence B wt
ID
SEQ ID:55 WSIVRVEIL0 42 WTIVRVEIL46
SEQ ID:56 WT1VRVSIL0 41 WTIVRVEIL46
SEQ ID:57 WTIVRVEMM0 41 WTIVRVEIL46
SEQ ID:58 WTIVRVEMV0 40 WTIVRVEIL46
SEQ ID:59 WTIVRVEMF0 39 WTIVRVEIL46
SEQ ID:60 WTIVRVELF0 40 WTIVRVEIL46
SEQ ID:61 WTIVRVEVF0 41 WTIVRVEIL46
SEQ ID:62 WTIVRVEFF0 38 WTIVRVEIL46
SEQ ID:63 WTIVRVEIM0 44 WTIVRVEIL46
SEQ ID:64 WTIVRVEIV0 43 WTIVRVEIL46
SEQ ID:65 WTIVRVEIF0 42 WTIVRVEIL46
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[0375] Table 32. Suitable less immunogenic variants of agretope 24 (residues
151-159). B(wt) is
the BLOSUM62 score of the wild type 9-mer, I(alt) is the percent of the US
population containing
one or more MHC alleles that are predicted to bind the alternate 9-mer at a 1
% threshold, and
B(alt) is the BLOSUM62 score of the alternate 9-mer.
Table 33.
Suitable
less immuno
epic variants
of a retope
24 residues
151-159
.
Variant WT
Se uence se uenceI alt B alt se uence B wt
ID
SEQ ID:66 MNNFYFINR0 42 LRNFYFINR49
SEQ ID:67 MENFYFINR0 42 LRNFYFINR49
SEQ ID:68 MQNFYFINR0 43 LRNFYFINR49
SEQ ID:69 MHNFYFINR0 42 LRNFYFINR49
SEQ ID:70 MKNFYFINR0 44 LRNFYFINR49
SEQ ID:71 LNNFYFINR0 44 LRNFYFINR49
SEQ ID:72 LENFYFINR0 44 LRNFYFINR49
SEQ ID:73 LQNFYFINR0 45 LRNFYFINR49
SEQ ID:74 LHNFYFINR0 44 LRNFYFINR49
SEQ ID:75 LKNFYFINR0 46 LRNFYFINR49
SEQ ID:76 LRSFYFINR0 44 LRNFYFINR49
SEQ ID:77 LRTFYFINR0 43 LRNFYFINR49
SEQ ID:78 LRGFYFINR0 43 LRNFYFINR49
SEQ ID:79 LRDFYFINR0 44 LRNFYFINR49
SEQ ID:80 LREFYFINR0 43 LRNFYFINR49
SEQ ID:81 LRQFYFINR0 43 LRNFYFINR49
SEQ ID:82 LRHFYFINR0 44 LRNFYF1NR49
SEQ ID:83 LRKFYFINR0 43 LRNFYFINR49
SEQ ID:84 LRNMYFINR0 43 LRNFYFINR49
SEQ ID:85 LRNIYFINR0 43 LRNFYFINR49
SEQ ID:86 LRNLYFINR0 43 LRNFYFINR49
SEQ ID:87 LRNFHYVNR0 40 LRNFYFINR49
SEQ 1D:88 LRNFYFISQ0 40 LRNFYFINR49
SEQ ID:89 LRNFYFISK0 41 LRNFYFINR49
SEQ 1D:90 LRNFYFITK0 40 LRNFYFINR49
SEQ ID:91 LRNFYFIGK0 40 LRNFYFINR49
SEQ ID:92 LRNFYFIDK0 41 LRNFYFINR49
SEQ ID:93 LRNFYFIEK0 40 LRNFYFINR49
SEQ ID:94 LRNFYFIQK0 40 LRNFYFINR49
SEQ ID:95 LRNFYFIHK0 41 LRNFYFINR49
SEQ ID:96 LRNFYFIRK0 40 LRNFYFINR49
SEQ ID:97 LRNFYFIKK0 40 LRNFYFINR49
SEQ fD:98 LRNFYFINE0 44 LRNFYFINR49
SEQ ID:99 LRNFYFINQ0 45 LRNFYFINR49
SEQ ID:100 LRNFYFINK0 46 ~NFYFINR ' 49
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[0376] Table 34. Suitable less immunogenic variants of agretope 25 (residues
154-162). B(wt) is
the BLOSUM62 score of the wild type 9-mer, I(alt) is the percent of the US
population containing
one or more MHC alleles that are predicted to bind the alternate 9-mer at a 1
% threshold, and
B(alt) is the BLOSUM62 score of the alternate 9-mer.
Table 34. retope
Suitable 25 residues
less immuno 154-162
enic variants .
of a
Variant WT
Se uence se uence I alt B alt se uence B wt
ID
SEQ 1D:101 FYFISQLTG0 ~ 40 I FYFiNRLTG49 I
I
[0377] Example 15. Identification of suitable less immunogenic sequences for
MHC-binding
agretopes in interferon beta: PDA~ technology
[0378] MHC-binding agretopes that were predicted to bind alleles present in at
least 10% of the US
population, using a 1 % threshold, were analyzed using PDA~ technology to
identify suitable less
immunogenic variants.
[0379] Each position in the agretopes of interest were analyzed to identify a
subset of amino acid
substitutions that are potentially compatible with maintaining the structure
and function of the
protein. This step may be performed in several ways, including PDA~
calculations or visual
inspection by one skilled in the art. Sequences may be generated that contain
all possible
combinations of amino acids that were selected for consideration at each
position. Matrix method
calculations were then used to determine the immunogenicity of each sequence.
The results
were analyzed to identify sequences that are predicted to have significantly
decreased
immunogenicity. PDA~ calculations may be performed to determine which of the
minimally
immunogenic sequences are compatible with maintaining the structure and
function of the protein.
[0380] Alternate sequences were scored for immunogenicity and structural
compatibility. Preferred
alternate sequences were defined to be those sequences in which (1) the
central 9-mer agretope
is not predicted to bind to any of the 17 MHC alleles tested above using a 5%
threshold, (2)
overlapping 9-mer agretopes are not predicted to bind any of the 17 MHC
alleles tested more
tightly than the analogous 9-mer in wild type interferon beta, and (3) the
total energy, determined
using PDA~ technology calculations, that is at most 1 kcal mol-~ worse than
the energy of the wild
type interferon beta.
[0381] Example 16. Analysis of immunogenic sequences in interferon beta
solubility variants
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[0382] A set of interferon beta variants were engineered for enhanced
solubility. These variants
comprise hydrophobic to polar substitutions at solvent exposed positions that
are not functionally
required.
[0383] Table 35. Specific activity data for interferon-beta variants. The
sequence for residues 5, 8,
47, 111, 116, and 120 is given for each variant, along with the total number
of mutations, the
EC50, and the ratio of the wild type to variant EC50. Variant IFN1 1 is the
interferon beta wild
type with the C17S substitution.
Table 35. Sequence and activity of interferon beta solubility variants.
Variant 5 8 47 111116 120# EC50 (log EC50 wt I EC50
mut nglml) var
IFN1 1 L F L F L L 0 5.306 1.0
IFB1 2 Q F L F L L 1 0.428 12.4
IFB1 7 Q E L F L L 2 0.179 29.6
IFB1 15Q E L N L L 3 0.319 16.6
IFB1 23Q E L F E L 3 0.277 19.2
IFB1 36L E L N E L 3 0.294 18.0
IFB1 39Q E L N E L 4 0.193 27.5
IFB1 64Q E K N E R 6 0.240 22.1
[0384] Tabfe 36. Comparison of MHC agretopes in interferon beta solubility
variants. Potential
agretopes that include residues that were altered in one or more of the
solubility variants are
shown, along with the fraction of the population for which each agretope is a
hit using a 3%
threshold.
Table 36.
Comparison
of MHC
agretopes
in interferon
beta solubility
variants
mutations residuessequence wt v2 v7 v15 v23 v36 v39 v64
L5Q,F8E 1 - 9 MSYNLLGFL0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
L5Q,F8E 3 - 11 YNLLGFLQR0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
L5Q,F8E 5 - 13 LLGFLQRSS0.20 0.00 0.00 0.00 0.00 0.00 0.00 0.00
F8E 6 - 14 LGFLQRSSN0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
F8E 8 - 16 FLQRSSNFQ0.07 0.07 0.00 0.00 0.00 0.00 0.00 0.00
L47K 40-48 IPEEIKQLQ0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
L47K 44 - IKQLQQFQK0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
52
L47K 47 - LQQFQKEDA0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
55
F111 N 106 - LEKEDFTRG0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
114
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F111N,L116E111 -119FTRGKLMSS0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
L116E,L120R116 - LMSSLHLKR0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
124
L120R 117 -125MSSLHLKRY0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
L120R 120 - LHLKRYYGR0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00
128
[0385] As can be seen, many of the solubility variants are predicted to have
reduced immunogenicity
relative to wild type interferon beta.
94
