U.S. patent application number 17/709510 was filed with the patent office on 2022-07-14 for interferon alpha 2b variants.
The applicant listed for this patent is Teva Pharmaceuticals Australia PTY LTD.. Invention is credited to Collette Behrens, Adam Clarke, Teresa Domagala, Anthony Doyle, Matthew Pollard.
Application Number | 20220220184 17/709510 |
Document ID | / |
Family ID | 1000006242177 |
Filed Date | 2022-07-14 |
United States Patent
Application |
20220220184 |
Kind Code |
A1 |
Behrens; Collette ; et
al. |
July 14, 2022 |
INTERFERON ALPHA 2B VARIANTS
Abstract
The present invention provides a fusion polypeptide comprising a
first domain and a second domain, wherein the first domain
comprises a polypeptide ligand which binds to a cell
surface-associated antigen and the second domain comprises
aglycosylated interferon .alpha. 2b (IFN.alpha.2b) having a
sequence of SEQ ID NO: 1 or SEQ ID NO: 2. The aglycosylated
IFN.alpha.2b further comprises one or more amino acid substitutions
or deletions which attenuate the activity of the aglycosylated
IFN.alpha.2b.
Inventors: |
Behrens; Collette;
(Macquarie Park, AU) ; Doyle; Anthony; (Macquarie
Park, AU) ; Clarke; Adam; (Macquarie Park, AU)
; Pollard; Matthew; (Macquarie Park, AU) ;
Domagala; Teresa; (Macquarie Park, AU) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Teva Pharmaceuticals Australia PTY LTD. |
Macquarie Park |
|
AU |
|
|
Family ID: |
1000006242177 |
Appl. No.: |
17/709510 |
Filed: |
March 31, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16710574 |
Dec 11, 2019 |
11319356 |
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17709510 |
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14921420 |
Oct 23, 2015 |
10544199 |
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16710574 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/2896 20130101;
C07K 2319/70 20130101; C07K 16/2833 20130101; C07K 14/56 20130101;
C07K 2319/33 20130101 |
International
Class: |
C07K 14/56 20060101
C07K014/56; C07K 16/28 20060101 C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Oct 29, 2014 |
AU |
2014904326 |
Claims
1. A method of treating a tumour in a subject, the method
comprising: administering to the subject a fusion polypeptide
comprising a first domain and a second domain, wherein the first
domain comprises a polypeptide ligand which binds to a cell
surface-associated antigen on cells of the tumour and the second
domain comprises aglycosylated interferon .alpha. 2b (IFN.alpha.2b)
having a sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and wherein the
aglycosylated IFN.alpha.2b further comprises one or more amino acid
substitutions or deletions which attenuate the activity of the
aglycosylated IFN.alpha.2b.
2. The method of claim 1, wherein the tumour is selected from
multiple myeloma or non-Hodgkin's lymphoma.
3. A method of generating a polypeptide ligand-attenuated
IFN.alpha.2b fusion polypeptide in mammalian cells, wherein the
polypeptide ligand-attenuated IFN.alpha.2b fusion polypeptide has
improved heterogeneity and/or enhanced FcRn binding, the method
comprising: culturing a recombinant mammalian cell comprising a
polynucleotide encoding the polypeptide ligand-attenuated
IFN.alpha.2b fusion polypeptide wherein T106 of the IFN.alpha.2b
sequence is replaced with another amino acid or is deleted such
that on expression the IFN.alpha.2b component of the fusion protein
is aglycosylated.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S.
application Ser. No. 16/710,574, filed Dec. 11, 2019, which is a
divisional application of U.S. application Ser. No. 14/921,420,
filed Oct. 23, 2015 (U.S. Pat. No. 10,544,199), which claims
priority to Australian Patent Application No. 2014904326 filed on
Oct. 29, 2014, the contents of each of which are incorporated by
reference herein, in their entirety and for all purposes.
REFERENCE TO A SEQUENCE LISTING
[0002] This application includes a Sequence Listing submitted
electronically as a text file named 101017.000014.txt, created on
Mar. 28, 2022, with a size of 298 KB. The Sequence Listing is
incorporated by reference herein.
FIELD OF INVENTION
[0003] The present invention relates to polypeptides comprising
ligands targeted against cell surface antigens and aglycosylated
interferon .alpha. 2b (IFN.alpha.2b) and the use of these
polypeptides in the treatment of cancer.
BACKGROUND OF INVENTION
[0004] Numerous peptide and polypeptide molecules have been
described to function by interacting with a receptor on a cell
surface, and thereby stimulating, inhibiting, or otherwise
modulating a biological response, usually involving signal
transduction pathways inside the cell that bears the said receptor.
Examples of such molecules include peptide and polypeptide
hormones, cytokines, chemokines, growth factors, apoptosis-inducing
factors and the like. These molecules can be either soluble or can
be attached to the surface of another cell.
[0005] Due to the biological activity of such molecules, some have
potential use as therapeutics. Several peptide or polypeptide
molecules have been approved by regulatory agencies as therapeutic
products, including, for example, human growth hormone, insulin,
interferon IFN.alpha.2b, IFN.alpha.2a, IFN.beta., erythropoietin,
G-CSF and GM-CSF. Many of these and other peptides have
demonstrated potential in therapeutic applications, but have also
exhibited toxicity when administered to human patients. One reason
for toxicity is that most of these molecules trigger receptors on a
variety of cells, including cells other than those that mediate the
therapeutic effect. For example, when IFN.alpha.2b is used to treat
multiple myeloma its utility resides, at least in part, in its
binding to type I interferon receptors on the myeloma cells, which
in turn triggers reduced proliferation and hence limits disease
progression. Unfortunately, however, this IFN also binds to
numerous other, normal cells within the body, triggering a variety
of other cellular responses, some of which are harmful (e.g.
flu-like symptoms, neutropenia, depression). A consequence of such
"off target" activity of peptides is that many peptides are not
suitable as drug candidates. In this context, "off target activity"
refers to activity on the peptide's natural receptor, but on the
surface of cells other than those that mediate therapeutically
beneficial effects.
[0006] Even though some peptides, such as IFN.alpha.2b, are
approved for the treatment of medical conditions, they are poorly
tolerated due to their "off target" biological activity. The
off-target activity and associated poor tolerability also mean that
some of these peptide based drugs cannot be administered at
sufficiently high dosages to produce optimal therapeutic effects on
the target cells which mediate the therapeutic effect.
[0007] Similarly, it has been known since the mid-1980's that
interferons, in particular IFN.alpha., are able to increase
apoptosis and decrease proliferation of certain cancer cells. These
biological activities are mediated by type I interferon receptors
on the surface of the cancer cells which, when stimulated, initiate
various signal transduction pathways leading to reduced
proliferation and/or the induction of terminal differentiation or
apoptosis. IFN.alpha. has been approved by the FDA for the
treatment of several cancers including melanoma, renal cell
carcinoma, B cell lymphoma, multiple myeloma, chronic myelogenous
leukemia (CML) and hairy cell leukemia. A "direct" effect of
IFN.alpha. on the tumour cells is mediated by the IFN.alpha.
binding directly to the type I IFN receptor on those cells and
stimulating apoptosis, terminal differentiation or reduced
proliferation. One "indirect" effect of IFN.alpha. on non-cancer
cells is to stimulate the immune system, which may produce an
additional anti-cancer effect by causing the immune system to
reject the tumour.
[0008] Unfortunately, the type I interferon receptor is also
present on most non-cancerous cells. Activation of this receptor on
such cells by IFN.alpha. causes the expression of numerous
pro-inflammatory cytokines and chemokines, leading to toxicity.
Such toxicity prevents the dosing of IFN.alpha. to a subject at
levels that exert the maximum anti-proliferative and pro-apoptotic
activity on the cancer cells.
[0009] Ozzello et al. (Breast Cancer Research and Treatment
25:265-76, 1993) described covalently attaching human IFN.alpha. to
a tumour-targeting antibody, thereby localizing the direct
inhibitory activity of IFN.alpha. to the tumour as a way of
reducing tumour growth rates, and demonstrated that such conjugates
have anti-tumour activity in a xenograft model of a human cancer.
The mechanism of the observed anti-cancer activity was attributed
to a direct effect of IFN.alpha. on the cancer cells, since the
human IFN.alpha. used in the experiments did not interact
appreciably with the murine type I IFN receptor, which could have
lead to an indirect anti-cancer effect. Because of this lack of
binding of the human IFN.alpha. to the murine cells, however, the
authors could not evaluate the toxicity of the antibody-IFN.alpha.
conjugate relative to free INF.alpha.. These authors used a
chemical method to attach the IFN.alpha. to the antibody.
[0010] Alkan et al., (Journal of Interferon Research, volume 4,
number 3, p. 355-63, 1984) demonstrated that attaching human
IFN.alpha. to an antibody that binds to the Epstein-Barr virus
(EBV) membrane antigen (MA) increased its antiproliferative
activities towards cells that express the EBV-MA antigen. This
increased potency was dependent on both antigen expression by the
target cells and the binding specificity of the antibody. The cell
line tested was the cancer cell line QIMR-WIL, a myeloblastic
leukemia. The authors suggested that the attachment of IFN.alpha.
to an antibody could be used as a treatment for cancer since it
would reduce tumour growth. Alkan et al did not address the
potential toxicity of these antibody-IFN.alpha. conjugates arising
from their interactions with normal, antigen-negative cells.
[0011] It is also known that the linkage between an antibody and
IFN.alpha. may be accomplished by making a fusion protein
construct. For example, IDEC (WO01/97844) disclose a direct fusion
of human IFN.alpha. to the C terminus of the heavy chain of an IgG
targeting the tumour antigen CD20. Other groups have disclosed the
use of various linkers between the C-terminus of an IgG heavy chain
and the IFN.alpha.. For example, U.S. Pat. No. 7,456,257 discloses
that the C-terminus of an antibody heavy chain constant region may
be connected to IFN.alpha. via an intervening serine-glycine rich
(S/G) linker of the sequence (GGGGS).sub.n(SEQ ID NO: 113), where n
may be 1, 2 or 3, and that there are no significant differences in
the IFN.alpha. activity of the fusion protein construct regardless
of linker length.
[0012] Morrison et al. (US2011/0104112 A1; and Xuan C, Steward K K,
Timmerman J M, Morrison S L. Targeted delivery of
interferon-.alpha. via fusion to anti-CD20 results in potent
antitumor activity against B-cell lymphoma. Blood 2010;
115:2864-71) also disclose IFN.alpha. linked to the C-terminus of
the heavy chain of a cancer-targeting IgG antibody, with an
intervening S/G linker, and observed that the fusion of the IgG and
linker to the IFN.alpha. reduced the activity of IFN.alpha. on
cells that did not express the corresponding antigen on the cell
surface. The decreased IFN activity of these fusion protein
constructs was modest when compared to human non-fusion protein
IFN.alpha. (free IFN.alpha.) acting on human cells, but appeared to
be more significant for murine IFN.alpha. on murine cells. The
decrease in the activity of human IFN.alpha. that results from
fusing it to the C-terminus of an antibody, as observed by Morrison
et al, and in U.S. Pat. No. 7,456,257 is modest and is generally
considered to be a disadvantage since it reduces potency of the
IFN. This disadvantage was pointed out, for example, by Rossi et al
(Blood vol. 114, No. 18, pp 3864-71), who used an alternative
strategy of attaching the IFN.alpha. to a tumor targeting antibody
in such a way that no loss in IFN.alpha. activity was observed.
[0013] In general the prior art teaches to use a potent IFN and to
target this IFN to cancer cells. While this approach results in an
increase in activity of the IFN against cancer cells, it does not
address the issue of activity of the IFN on normal "off-target"
cells. In prior art examples referred to above, the human
IFN.alpha. portion of the antibody-IFN.alpha. fusion protein
maintained a high proportion of native IFN.alpha. activity when
exposed to human cells that do not express the corresponding
antigen on their cell surfaces. This activity may lead to toxicity
arising from the activation of non-cancerous, normal ("off target")
cells by the IFN.alpha. portion of the fusion protein. Accordingly,
there exists a need to decrease the "off-target" activity of
IFN-based drugs, while retaining the "on-target", therapeutic
effect of such drugs. The maintenance of target-specific activity
and at the same time a reduction in non-target toxicity of these
types of therapeutic agents would create a greater therapeutic
concentration window for therapeutically useful peptides. It would
for example be desirable to use human IFN.alpha. in a form such
that its activity can be directed to the cancer cells while
minimizing its effects on normal human cells. Ideally the type I
interferon receptor on the cancer cells would be maximally
stimulated, while the same receptor on non-cancerous cells would
experience minimal stimulation. There is a need to target human
IFN.alpha. to the cancer cells in such a way that it has
dramatically more activity on the cancer cells, which display the
antigen, than on the normal cells, which do not display the
antigen. The same logic applies to other potentially therapeutic
molecules, e.g. other cytokines, peptide and polypeptide hormones,
chemokines, growth factors, apoptosis-inducing factors and the
like.
[0014] The logic of this approach has been demonstrated in WO
2013/059885, and WO 2014/178820, the disclosure of each of which is
incorporated herein by cross reference.
SUMMARY OF INVENTION
[0015] In a first aspect the present invention provides a fusion
polypeptide comprising a first domain and a second domain, wherein
the first domain comprises a polypeptide ligand which binds to a
cell surface-associated antigen and the second domain comprises
human aglycosylated interferon .alpha. 2b (IFN.alpha.2b) having a
sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and wherein the
aglycosylated IFN.alpha.2b further comprises one or more amino acid
substitutions or deletions which attenuate the activity of the
aglycosylated IFN.alpha.2b.
[0016] In another aspect the present invention provides a fusion
polypeptide comprising a sequence selected from the group
consisting of SEQ ID NOs: 31, 61 to 77, 83 and 87, and a sequence
selected from the group consisting of SEQ ID NOs: 81, 82 and
84.
[0017] In another aspect the present invention provides a fusion
polypeptide comprising SEQ ID NO: 87 and SEQ ID NO: 81.
[0018] In another aspect the present invention provides a fusion
polypeptide comprising SEQ ID NO: 79 and SEQ ID NO: 85.
[0019] In another aspect the present invention provides a fusion
polypeptide comprising SEQ ID NO: 80 and SEQ ID NO: 86.
[0020] In another aspect the present invention provides a fusion
polypeptide comprising SEQ ID NO: 78.
[0021] In another aspect the present invention provides a
composition comprising the fusion polypeptide of the present
invention and a pharmaceutically acceptable carrier or diluent.
[0022] In another aspect the present invention provides a method of
treating a tumour in a subject comprising administering to the
subject the fusion polypeptide of the present invention or the
composition of the present invention wherein the first domain of
the fusion polypeptide binds to cells of the tumour.
[0023] In another aspect the present invention provides the use of
the fusion polypeptide of the present invention in the treatment of
a tumour wherein first domain of the fusion polypeptide binds to
the tumour.
[0024] In another aspect the present invention provides an isolated
polynucleotide(s) encoding the fusion polypeptide(s) of the present
invention.
[0025] In another aspect the present invention provides a vector
comprising the one or more polynucleotides of the present
invention.
[0026] In another aspect the present invention provides a
transformed cell comprising the vector of the present
invention.
[0027] In another aspect the present invention provides a method of
generating a polypeptide ligand-attenuated IFN.alpha.2b fusion
polypeptide in mammalian cells, wherein the polypeptide
ligand-attenuated IFN.alpha.2b fusion polypeptide has reduced
heterogeneity and/or enhanced FcRn binding and/or improved target
selectivity, the method comprising culturing a recombinant
mammalian cell comprising a polynucleotide encoding the polypeptide
ligand-attenuated IFN.alpha.2b fusion polypeptide wherein T106 of
the IFN.alpha.2b sequence is replaced with another amino acid or is
deleted such that on expression in mammalian cells the IFN.alpha.2b
component of the fusion protein is aglycosylated.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1, parts A and B: Anti-proliferative activity upon
treatment of (A) ARP1 and (B) NCI-H929 cells with
anti-CD38-attenuated IFN.alpha.2b fusion proteins in IgG1 or IgG4
format with and without O-linked glycosylation of the
IFN.alpha.2b.
[0029] FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D and FIG. 2E.
Anti-proliferative activity of anti-CD38-attenuated IFN.alpha.2b
fusion proteins with different amino acid substitutions removing
the O-linked glycosylation site from the attenuated
IFN.alpha.2b.
[0030] FIG. 3, parts A and B: On-target activities of (A) A10.21
and (B) A10.43 anti-CD38-attenuated IFN.alpha.2b fusion proteins
with (T106T) and without (T106A) O-linked glycosylation of the
IFN.alpha.2b.
[0031] FIG. 4, parts A and B: Off-target activities of (A) A10.21
and (B) A10.43 anti-CD38-attenuated IFN.alpha.2b fusion proteins
with (T106T) and without (T106A) O-linked glycosylation of the
IFN.alpha.2b.
[0032] FIG. 5: Off-target activity by anti-CD38-attenuated
IFN.alpha.2b fusion proteins with (T106T) and without (T106A or
.DELTA.T106)O-linked glycosylation of the IFN.alpha.2b.
[0033] FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E and FIG. 6F:
Off-target activity of anti-CD38-attenuated IFN.alpha.2b fusion
proteins with different amino acid substitutions removing the
O-linked glycosylation site from the attenuated IFN.alpha.2b.
[0034] FIG. 7: Off-target activity of A10.43 anti-CD38-attenuated
IFN.alpha.2b fusion proteins with (T106T) or without (T106A)
O-linked glycosylation of the IFN.alpha.2b.
[0035] FIG. 8: Potency of sub-optimal dosages of
anti-CD38-attenuated IFN.alpha.2b fusion proteins with (T106T) or
without (T106A) O-linked glycosylation in treating tumours in a
murine model of multiple myeloma.
[0036] FIG. 9: Number of charged species of A10.21
anti-CD38-attenuated IFN.alpha.2b fusion proteins with (T106T) or
without (T106A, .DELTA.T106, T106S, T106V, T106G, T106E) 0-linked
glycosylation of the IFN.alpha.2b, as assessed by the number of
bands on IEF gel.
[0037] FIG. 10: Number of charged species of A10.21
anti-CD38-attenuated IFN.alpha.2b fusion proteins with (T106T) or
without (T106A) O-linked glycosylation of the IFN.alpha.2b and with
varying Fc isotypes, as assessed by the number of bands on IEF
gel.
[0038] FIG. 11: Number of charged species of A10.21 (IgG4 with
S228P) anti-CD38-attenuated IFN.alpha.2b fusion proteins with
(T106T) or without (T106A) O-linked glycosylation of the
IFN.alpha.2b in the presence of YTE substitutions in the antibody
constant region, as assessed by the number of bands on IEF gel.
[0039] FIG. 12: Number of charged species of A10.21 (IgG4 with
S228P) anti-CD38-attenuated IFN.alpha.2b fusion proteins with
(T106T) or without (T106A) O-linked glycosylation of the
IFN.alpha.2b in the presence of a variety of IFN attenuating
substitutions, as assessed by the number of bands on IEF gel.
[0040] FIG. 13: Number of charged species of antibodies with
differing target specificities; anti-CD138 antibody, anti-HLA
antibody and anti-CD38 antibody (A02.12) (all IgG4 with S228P)
fused to attenuated IFN.alpha.2b with (T106T) or without (T106A)
O-linked glycosylation of the IFN.alpha.2b, as assessed by the
number of bands on IEF gel.
[0041] FIG. 14: "On-target" activities of anti-CD38-attenuated
IFN.alpha.2b fusion proteins (A10.21 IgG4 (S228P) IFN (A145D)) with
(T106T) and without (T106A, .DELTA.T106, T106S, T106V, T106G,
T106E)O-linked glycosylation of the IFN.alpha.2b.
[0042] FIG. 15: "On-target" activities of two different anti-CD38
antibody-attenuated IFN.alpha.2b fusion proteins (A02.12 and
A10.21, both IgG4 with S228P) which bind different epitopes on CD38
with (T106T) or without (T106A) O-linked glycosylation of the
IFN.alpha.2b.
[0043] FIG. 16: "On-target" activities of A10.21
anti-CD38-attenuated IFN.alpha.2b fusion proteins (A10.21 IgG4
(S228P) IFN) with (T106T) and without (T106A) O-linked
glycosylation of the IFN.alpha.2b, with a variety of IFN
attenuating substitutions (R33A, R144I, R145Q, A145K or A145G).
[0044] FIG. 17: "On-target" activities of antibodies with differing
target specificities; anti-CD138 antibody and anti-HLA antibody
(both IgG4 with S228P) fused to attenuated IFN.alpha.2b with
(T106T) or without (T106A) O-linked glycosylation of the
IFN.alpha.2b.
[0045] FIG. 18: "On-target" activities of A10.21
anti-CD38-attenuated IFN.alpha.2b fusion proteins (A10.21 IgG4
(S228P) IFN (A145D)) with (T106T) and without (T106A) O-linked
glycosylation of the IFN.alpha.2b, in the presence of YTE
substitution in the antibody heavy chain.
[0046] FIG. 19: "On-target" activities of A10.21
anti-CD38-attenuated IFN.alpha.2b (A145D) fusion proteins with
(T106T) and without (T106A) O-linked glycosylation of the
IFN.alpha.2b, with with a variety of immunoglobulin Fc
isotypes.
[0047] FIG. 20: Selectivity index of A10.21 anti-CD38-attenuated
IFN.alpha.2b fusion proteins with and without O-linked
glycosylation of the IFN.alpha.2b in the presence of a variety of
amino acid substitutions to remove glycosylation of the IFN, YTE
substitutions in the imunoglobulin constant region for extended
half-life, IFN attenuation and Fc isotypes.
DETAILED DESCRIPTION OF THE INVENTION
[0048] Throughout this specification, unless the context requires
otherwise, the word "comprise", or variations such as "comprises"
or "comprising", will be understood to imply the inclusion of a
stated element or integer or group of elements or integers but not
the exclusion of any other element or integer or group of elements
or integers.
[0049] The reference in this specification to any prior publication
(or information derived from it), or to any matter which is known,
is not, and should not be taken as an acknowledgment or admission
or any form of suggestion that prior publication (or information
derived from it) or known matter forms part of the common general
knowledge in the field of endeavour to which this specification
relates.
[0050] All publications mentioned in this specification are herein
incorporated by reference in their entirety.
[0051] It must be noted that, as used in the subject specification,
the singular forms "a", "an" and "the" include plural aspects
unless the context clearly dictates otherwise. Thus, for example,
reference to "an agent" includes a single agent, as well as two or
more agents; reference to "a molecule" includes a single molecule,
as well as two or more molecules; and so forth.
[0052] The constructs of the present invention are polypeptide
ligand-attenuated aglycosylated IFN.alpha.2b fusion constructs,
which show an elevated antigen-selectivity index with respect to
activating signaling pathways due to the action of both the ligand
targeting to a cell surface receptor on a cell of interest and the
attenuated IFN.alpha.2b having reduced affinity to a cell surface
IFN receptor. These constructs are based on the discovery outlined
in WO 2013/059885 that, in the context of an antibody-IFN fusion
construct, the IFN portion can be mutated in such a way that the
IFN activity on antigen-negative cells is dramatically attenuated,
while the IFN activity on antigen-positive cells is only modestly,
if at all, attenuated. Such constructs display one, two, three,
four or five orders of magnitude greater potency on
antigen-positive cells compared to antigen negative cells than does
the free IFN. In one embodiment, the antibody-attenuated IFN
construct retains at least 1%, at least 10%, at least 20%, at least
30%, at least 40% or at least 50% of the potency on
antigen-positive cells as the non-attenuated free (i.e. not
attached to an antibody) IFN. In addition, in one embodiment the
antibody-attenuated IFN construct retains at least 30%, at least
50%, at least 75% or at least 90% of the maximal activity of the
non-attenuated free (i.e. not attached to an antibody) IFN; in this
context, "maximal activity" should be understood as meaning the
amount of signaling activity (or downstream effect thereof) at the
high, plateau portion of a dose-response curve, where further
increases in the agent does not further increase the amount of
response).
[0053] The present inventors have now found that an unexpected
advantage is obtained by using constructs comprising aglycosylated
IFN.alpha.2b as compared to the constructs comprising
O-glycosylated IFN.alpha.2b. In some embodiments these advantages
include one or more of an increase in ON-target activity, increased
target selectivity and enhanced affinity to FcRn, whilst providing
a less heterogeneous product than the O-glycosylated IFN.alpha.2b
when produced in a mammalian cell expression system. Enhanced FcRn
binding is desirable to improve the pK of a biological therapeutic
agent which comprises an Fc region. Increased target selectivity is
desirable as it potentially reduces OFF-target toxicity whilst
substantially maintaining ON-target activity. A reduction in
heterogeneity allows increases in yields of purified product from a
mammalian cell culture system.
[0054] Accordingly, in a first aspect, the present invention
provides a fusion polypeptide comprising a first and a second
domain, wherein the first domain comprises a polypeptide ligand
which binds to a cell surface-associated antigen and the second
domain comprises aglycosylated interferon .alpha. 2b (IFN.alpha.2b)
having a sequence of SEQ ID NO: 1 or SEQ ID NO: 2, and wherein the
aglycosylated IFN.alpha.2b further comprises one or more amino acid
substitutions or deletions which attenuate the activity of the
aglycosylated IFN.alpha.2b.
[0055] In an embodiment of the present invention the sequence of
the aglycosylated IFN.alpha.2b is SEQ ID NO: 1 in which the residue
at position 106 is A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S,
V, W or Y. This represents a substitution of the T106 normally
present in human IFN.alpha.2b with a naturally occurring amino acid
which does not permit O-linked glycosylation at this position when
produced in mammalian cell culture. In another embodiment of the
present invention the sequence of the aglycosylated IFN.alpha.2b is
SEQ ID NO: 2. This represents a deletion of residue T106 found in
normal human IFN.alpha.2b, which also removes the O-linked
glycosylation site found in this molecule. As demonstrated herein,
each of the substitutions or deletion removes the O-glycosylation
site from human attenuated IFN.alpha.2b and reduces the
heterogeneity of the molecule as measured in IEF gels, when
expressed by CHO cells, and whilst at least substantially
maintaining the activity of the attenuated IFN.alpha.2b to bind
cell surface IFN receptors and to initiate downstream
signaling.
[0056] In additional embodiments the sequence of the aglycosylated
IFN.alpha.2b is SEQ ID NO: 1 modified by an attenuating mutation
selected from the group consisting of LISA, R22A, R23A, S25A, L26A,
F27A, L30A, L30V, K31A, D32A, R33A, R33K, R33Q, H34A, Q40A, D114R,
L117A, R120A, R120E, R125A, R125E, K131A, E132A, K133A, K134A,
M148A, R149A, S152A, L153A, N156A, (L30A, H57Y, E58N and Q61S),
(M148A, H57Y, E58N and Q61S), (L153A, H57Y, E58N and Q61S), (R144A,
H57Y, E58N and Q61S), (N65A, L80A, Y85A and Y89A,) (N65A, L80A,
Y85A, Y89A and D114A), (N65A, L80A, Y85A, Y89A and L117A), (N65A,
L80A, Y85A, Y89A and R120A), (Y85A, Y89A and D114A), (D114A and
R120A), (L117A and R120A), (L117A, R120A and K121A), (R120A and
K121A), (R120E and K121E), replacement of R at position 144 with A,
D, E, G, H, I, K, L, N, Q, S, T, V or Y, replacement of A at
position 145 with D, E, G, H, I, K, L, M, N, Q, S, T, V or Y, and
deletion of residues L161 to E165.
[0057] In additional embodiments the sequence of the aglycosylated
IFN.alpha.2b is SEQ ID NO: 2 modified by an attenuating mutation
selected from the group consisting of LISA, R22A, R23A, S25A, L26A,
F27A, L30A, L30V, K31A, D32A, R33A, R33K, R33Q, H34A, Q40A, D113R,
L116A, R119A, R119E, R124A, R124E, K130A, E131A, K132A, K133A,
M147A, R148A, S149A, L152A, N155A, (L30A, H57Y, E58N and Q61S),
(M147A, H57Y, E58N and Q61S), (L152A, H57Y, E58N and Q61S), (R143A,
H57Y, E58N and Q61S), (N65A, L80A, Y85A and Y89A,) (N65A, L80A,
Y85A, Y89A and D113A), (N65A, L80A, Y85A, Y89A and L116A), (N65A,
L80A, Y85A, Y89A and R1190A), (Y85A, Y89A and D113A), (D113A and
R119A), (L116A and R119A), (L116A, R119A and K120A), (R119A and
K120A), (R119E and K120E), replacement of R at position 143 with A,
D, E, G, H, I, K, L, N, Q, S, T, V or Y, replacement of A at
position 144 with D, E, G, H, I, K, L, M, N, Q, S, T, V or Y, and
deletion of residues L160 to E164.
[0058] In another embodiment the sequence of the aglycosylated
IFN.alpha.2b modified by an attenuating mutation is selected from
the group consisting of SEQ ID NOs: 3 to 30 and SEQ ID NOs: 32 to
47.
[0059] In another embodiment the cell surface-associated antigen is
selected from the group consisting of CD38, CD138, RANK-Ligand,
HM1.24, CD56, CS1, CD20, CD74, IL-6R, Blys (BAFF), BCMA, HLA-SR,
HLA-DR, Kininogen, beta2 microglobulin, FGFR3, ICAM-1, matriptase,
CD52, EGFR, GM2, alpha4-integrin, IFG-1R, KIR, CD3, CD4, CD8, CD24,
CD44, CD69, CD71, CD79, CD83, CD86, CD96, HLA, PD-1, ICOS, CD33,
CD115, CD11c, CD19, CD52, CD14, FSP1, FAP, PDGFR alpha, PDGFR beta,
ASGR1, ASGR2, FSP1, RTI140/Ti-alpha, HTI56, VEGF receptor, CD241
the product of the RCHE gene, CD117 (c-kit), CD71 (transferrin
receptor), CD36 (thrombospondin receptor), CD34, CD45RO, CD45RA,
CD115, CD168, CD235, CD236, CD237, CD238, CD239 and CD240.
[0060] In certain embodiments the polypeptide ligand is an antibody
or antigen binding portion thereof.
[0061] In another embodiment the polypeptide ligand is an antibody
which binds CD38. It is preferred that the V.sub.H sequence of the
antibody is selected from the group consisting of SEQ ID Nos: 48 to
56 and 58 and that the V.sub.L sequence of the antibody is selected
from the group consisting of SEQ ID Nos: 81, 82 and 84.
[0062] In another embodiment the polypeptide ligand is an antibody
which binds CD138. It is preferred that the V.sub.H sequence of the
antibody is SEQ ID NO: 59 and that the V.sub.L sequence of the
antibody is SEQ ID NO: 85.
[0063] In another embodiment the polypeptide ligand binds
RANK-Ligand. It is preferred that the sequence of the polypeptide
ligand is SEQ ID NO: 57.
[0064] In another embodiment the first domain is linked to the
second domain via a peptide bond. The first domain may be linked to
the second domain directly by a peptide bond (a "zero-length
linker") or via a peptide linker of from 1 to 20 amino acids in
length. The linker may be (SGGGGS).sub.n (SEQ ID NO: 114) where n
is 1 to 3. Examples of linkers include SGGGGS (SEQ ID NO: 114) and
SGGGGSGGGGSGGGGS (SEQ ID NO: 115).
[0065] In another embodiment the C-terminus of the first domain is
linked to N-terminus of the second domain.
[0066] In another embodiment the amino acid sequence of the first
domain is glycosylated.
[0067] In another aspect the present invention provides a fusion
polypeptide comprising a sequence selected from the group
consisting of SEQ ID NOs: 31, 61 to 77, 83 and 87, and a sequence
selected from the group consisting of SEQ ID NOs: 81, 82 and
84.
[0068] In another aspect the present invention provides a fusion
polypeptide comprising SEQ ID NO: 87 and SEQ ID NO: 81.
[0069] In another aspect the present invention provides a fusion
polypeptide comprising SEQ ID NO: 79 and SEQ ID NO: 85.
[0070] In another aspect the present invention provides a fusion
polypeptide comprising SEQ ID NO: 80 and SEQ ID NO: 86.
[0071] In another aspect the present invention provides a fusion
polypeptide comprising SEQ ID NO: 78.
[0072] As will be understood from the discussion above particular
forms of the fusion polypeptide of the current invention are as
follows: [0073] a. The fusion polypeptide in which the sequence of
the aglycosylated IFN.alpha.2b is SEQ ID NO: 1 in which the residue
at position 106 is A. [0074] b. The fusion polypeptide in which the
sequence of the aglycosylated IFN.alpha.2b is SEQ ID NO: 1 in which
the residue at position 106 is C. [0075] c. The fusion polypeptide
in which the sequence of the aglycosylated IFN.alpha.2b is SEQ ID
NO: 1 in which the residue at position 106 is D. [0076] d. The
fusion polypeptide in which the sequence of the aglycosylated
IFN.alpha.2b is SEQ ID NO: 1 in which the residue at position 106
is E. [0077] e. The fusion polypeptide in which the sequence of the
aglycosylated IFN.alpha.2b is SEQ ID NO: 1 in which the residue at
position 106 is F. [0078] f. The fusion polypeptide in which the
sequence of the aglycosylated IFN.alpha.2b is SEQ ID NO: 1 in which
the residue at position 106 is G. [0079] g. The fusion polypeptide
in which the sequence of the aglycosylated IFN.alpha.2b is SEQ ID
NO: 1 in which the residue at position 106 is H. [0080] h. The
fusion polypeptide in which the sequence of the aglycosylated
IFN.alpha.2b is SEQ ID NO: 1 in which the residue at position 106
is I. [0081] i. The fusion polypeptide in which the sequence of the
aglycosylated IFN.alpha.2b is SEQ ID NO: 1 in which the residue at
position 106 is K. [0082] j. The fusion polypeptide in which the
sequence of the aglycosylated IFN.alpha.2b is SEQ ID NO: 1 in which
the residue at position 106 is L. [0083] k. The fusion polypeptide
in which the sequence of the aglycosylated IFN.alpha.2b is SEQ ID
NO: 1 in which the residue at position 106 is M. [0084] l. The
fusion polypeptide in which the sequence of the aglycosylated
IFN.alpha.2b is SEQ ID NO: 1 in which the residue at position 106
is N. [0085] m. The fusion polypeptide in which the sequence of the
aglycosylated IFN.alpha.2b is SEQ ID NO: 1 in which the residue at
position 106 is P. [0086] n. The fusion polypeptide in which the
sequence of the aglycosylated IFN.alpha.2b is SEQ ID NO: 1 in which
the residue at position 106 is Q. [0087] o. The fusion polypeptide
in which the sequence of the aglycosylated IFN.alpha.2b is SEQ ID
NO: 1 in which the residue at position 106 is R. [0088] p. The
fusion polypeptide in which the sequence of the aglycosylated
IFN.alpha.2b is SEQ ID NO: 1 in which the residue at position 106
is V. [0089] q. The fusion polypeptide in which the sequence of the
aglycosylated IFN.alpha.2b is SEQ ID NO: 1 in which the residue at
position 106 is W. [0090] r. The fusion polypeptide in which the
sequence of the aglycosylated IFN.alpha.2b is SEQ ID NO: 1 in which
the residue at position 106 is Y. [0091] s. The fusion polypeptide
in which the sequence of the aglycosylated IFN.alpha.2b is SEQ ID
NO: 1 or SEQ ID NO: 2 modified by the attenuating mutation L15A.
[0092] t. The fusion polypeptide in which the sequence of the
aglycosylated IFN.alpha.2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified
by the attenuating mutation A19W. [0093] u. The fusion polypeptide
in which the sequence of the aglycosylated IFN.alpha.2b is SEQ ID
NO: 1 or SEQ ID NO: 2 modified by the attenuating mutation R22A.
[0094] v. The fusion polypeptide in which the sequence of the
aglycosylated IFN.alpha.2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified
by the attenuating mutation R23A. [0095] w. The fusion polypeptide
in which the sequence of the aglycosylated IFN.alpha.2b is SEQ ID
NO: 1 or SEQ ID NO: 2 modified by the attenuating mutation S25A.
[0096] x. The fusion polypeptide in which the sequence of the
aglycosylated IFN.alpha.2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified
by the attenuating mutation L26A. [0097] y. The fusion polypeptide
in which the sequence of the aglycosylated IFN.alpha.2b is SEQ ID
NO: 1 or SEQ ID NO: 2 modified by the attenuating mutation F27A.
[0098] z. The fusion polypeptide in which the sequence of the
aglycosylated IFN.alpha.2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified
by the attenuating mutation L30A or L30V. [0099] aa. The fusion
polypeptide in which the sequence of the aglycosylated IFN.alpha.2b
is SEQ ID NO: 1 or SEQ ID NO: 2 modified by the attenuating
mutation K31A. [0100] bb. The fusion polypeptide in which the
sequence of the aglycosylated IFN.alpha.2b is SEQ ID NO: 1 or SEQ
ID NO: 2 modified by the attenuating mutation D32A. [0101] cc. The
fusion polypeptide in which the sequence of the aglycosylated
IFN.alpha.2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified by the
attenuating mutation R33A, R33K or R33Q. [0102] dd. The fusion
polypeptide in which the sequence of the aglycosylated IFN.alpha.2b
is SEQ ID NO: 1 or SEQ ID NO: 2 modified by the attenuating
mutation H34A. [0103] ee. The fusion polypeptide in which the
sequence of the aglycosylated IFN.alpha.2b is SEQ ID NO: 1 or SEQ
ID NO: 2 modified by the attenuating mutation Q40A. [0104] ff. The
fusion polypeptide in which the sequence of the aglycosylated
IFN.alpha.2b is SEQ ID NO: 1 modified by the attenuating mutation
D114R or SEQ ID NO: 2 modified by the attenuating mutation D113R.
[0105] gg. The fusion polypeptide in which the sequence of the
aglycosylated IFN.alpha.2b is SEQ ID NO: 1 modified by the
attenuating mutation L117A or SEQ ID NO: 2 modified by the
attenuating mutation L116A. [0106] hh. The fusion polypeptide in
which the sequence of the aglycosylated IFN.alpha.2b is SEQ ID NO:
1 modified by the attenuating mutation R120A or R120E or SEQ ID NO:
2 modified by the attenuating mutation R119A or R119E. [0107] ii.
The fusion polypeptide in which the sequence of the aglycosylated
IFN.alpha.2b is SEQ ID NO: 1 modified by the attenuating mutation
R125A or R125E or SEQ ID NO: 2 modified by the attenuating mutation
R124A or R124E. [0108] jj. The fusion polypeptide in which the
sequence of the aglycosylated IFN.alpha.2b is SEQ ID NO: 1 modified
by the attenuating mutation K131A or SEQ ID NO: 2 modified by the
attenuating mutation K130A. [0109] kk. The fusion polypeptide in
which the sequence of the aglycosylated IFN.alpha.2b is SEQ ID NO:
1 modified by the attenuating mutation E132A or SEQ ID NO: 2
modified by the attenuating mutation E131A. [0110] ll. The fusion
polypeptide in which the sequence of the aglycosylated IFN.alpha.2b
is SEQ ID NO: 1 modified by the attenuating mutation K133A or SEQ
ID NO: 2 modified by the attenuating mutation K132A. [0111] mm. The
fusion polypeptide in which the sequence of the aglycosylated
IFN.alpha.2b is SEQ ID NO: 1 modified by the attenuating mutation
K134A or SEQ ID NO: 2 modified by the attenuating mutation K133A.
[0112] nn. The fusion polypeptide in which the sequence of the
aglycosylated IFN.alpha.2b is SEQ ID NO: 1 modified by the
attenuating mutation M148A or SEQ ID NO: 2 modified by the
attenuating mutation M147A. [0113] oo. The fusion polypeptide in
which the sequence of the aglycosylated IFN.alpha.2b is SEQ ID NO:
1 modified by the attenuating mutation R149A or SEQ ID NO: 2
modified by the attenuating mutation R148A. [0114] pp. The fusion
polypeptide in which the sequence of the aglycosylated IFN.alpha.2b
is SEQ ID NO: 1 modified by the attenuating mutation S152A or SEQ
ID NO: 2 modified by the attenuating mutation S151A. [0115] qq. The
fusion polypeptide in which the sequence of the aglycosylated
IFN.alpha.2b is SEQ ID NO: 1 modified by the attenuating mutation
L153A or SEQ ID NO: 2 modified by the attenuating mutation L152A.
[0116] rr. The fusion polypeptide in which the sequence of the
aglycosylated IFN.alpha.2b is SEQ ID NO: 1 modified by the
attenuating mutation N156A or SEQ ID NO: 2 modified by the
attenuating mutation N155A. [0117] ss. The fusion polypeptide in
which the sequence of the aglycosylated IFN.alpha.2b is SEQ ID NO:
1 or SEQ ID NO: 2 modified by the attenuating mutations L30A, H57Y,
E58N and Q61S. [0118] tt. The fusion polypeptide in which the
sequence of the aglycosylated IFN.alpha.2b is SEQ ID NO: 1 modified
by the attenuating mutations M148A, H57Y, E58N and Q61S or SEQ ID
NO: 2 modified by the attenuating mutations M147A, H57Y, E58N and
Q61S. [0119] uu. The fusion polypeptide in which the sequence of
the aglycosylated IFN.alpha.2b is SEQ ID NO: 1 modified by the
attenuating mutations L153A, H57Y, E58N and Q61S or SEQ ID NO: 2
modified by the attenuating mutations L152A, H57Y, E58N and Q61S.
[0120] vv. The fusion polypeptide in which the sequence of the
aglycosylated IFN.alpha.2b is SEQ ID NO: 1 modified by the
attenuating mutations R144A, H57Y, E58N and Q61S or SEQ ID NO: 2
modified by the attenuating mutations R143A, H57Y, E58N and Q61S.
[0121] ww. The fusion polypeptide in which the sequence of the
aglycosylated IFN.alpha.2b is SEQ ID NO: 1 or SEQ ID NO: 2 modified
by the attenuating mutations N65A, L80A, Y85A and Y89A. [0122] xx.
The fusion polypeptide in which the sequence of the aglycosylated
IFN.alpha.2b is SEQ ID NO: 1 modified by the attenuating mutations
N65A, L80A, Y85A, Y89A and D114A or SEQ ID NO: 2 modified by the
attenuating mutations N65A, L80A, Y85A, Y89A and D113A. [0123] yy.
The fusion polypeptide in which the sequence of the aglycosylated
IFN.alpha.2b is SEQ ID NO: 1 modified by the attenuating mutations
N65A, L80A, Y85A, Y89A and L117A or SEQ ID NO: 2 modified by the
attenuating mutations N65A, L80A, Y85A, Y89A and L116A. [0124] zz.
The fusion polypeptide in which the sequence of the aglycosylated
IFN.alpha.2b is SEQ ID NO: 1 modified by the attenuating mutations
N65A, L80A, Y85A, Y89A and R120A or SEQ ID NO: 2 modified by the
attenuating mutations N65A, L80A, Y85A, Y89A and R119A. [0125] aaa.
The fusion polypeptide in which the sequence of the aglycosylated
IFN.alpha.2b is SEQ ID NO: 1 modified by the attenuating mutations
Y85A, Y89A and D114A or SEQ ID NO: 2 modified by the attenuating
mutations Y85A, Y89A and D113A. [0126] bbb. The fusion polypeptide
in which the sequence of the aglycosylated IFN.alpha.2b is SEQ ID
NO: 1 modified by the attenuating mutations D114A and R120A or SEQ
ID NO: 2 modified by the attenuating mutations D113A and R119A.
[0127] ccc. The fusion polypeptide in which the sequence of the
aglycosylated IFN.alpha.2b is SEQ ID NO: 1 modified by the
attenuating mutations L117A and R120A or SEQ ID NO: 2 modified by
the attenuating mutations L116A and R119A. [0128] ddd. The fusion
polypeptide in which the sequence of the aglycosylated IFN.alpha.2b
is SEQ ID NO: 1 modified by the mutations L117A, R120A and K121A or
SEQ ID NO: 2 modified by the mutations L116A, R119A and K120A.
[0129] eee. The fusion polypeptide in which the sequence of the
aglycosylated IFN.alpha.2b is SEQ ID NO: 1 modified by the
mutations R120A and K121A or SEQ ID NO: 2 modified by the mutations
R119A and K120A. [0130] fff. The fusion polypeptide in which the
sequence of the aglycosylated IFN.alpha.2b is SEQ ID NO: 1 modified
by the mutations R120E and K121E or SEQ ID NO: 2 modified by the
mutations R119E and K120E. [0131] ggg. The fusion polypeptide in
which the sequence of the aglycosylated IFN.alpha.2b SEQ ID NO: 1
is modified by replacement of R at position 144 with A, D, E, G, H,
I, K, L, N, Q, S, T, V or Y or SEQ ID NO: 2 is modified by
replacement of R at position 143 with A, D, E, G, H, I, K, L, N, Q,
S, T, V or Y. [0132] hhh. The fusion polypeptide in which the
sequence of the aglycosylated IFN.alpha.2b is SEQ ID NO: 1 modified
by replacement of A at position 145 with D, E, G, H, I, K, L, M, N,
Q, S, T, V or Y or SEQ ID NO: 2 modified by replacement of A at
position 144 with D, E, G, H, I, K, L, M, N, Q, S, T, V or Y.
[0133] iii. The fusion polypeptide in which the sequence of the
aglycosylated IFN.alpha.2b is SEQ ID NO: 1 modified by deletion of
residues L161 to E165 or SEQ ID NO: 2 modified by deletion of
residues L161 to E165. [0134] jjj. The fusion polypeptide as
claimed in claim 1 in which the sequence of the aglycosylated
IFN.alpha.2b is selected from the group consisting of SEQ ID NOs: 3
to 30 and SEQ ID NOs: 32 to 47.
[0135] The term "antibody", as used herein, broadly refers to any
immunoglobulin (Ig) molecule comprised of four polypeptide chains,
two heavy (H) chains and two light (L) chains, or any functional
fragment, mutant, variant, or derivation thereof, which retains the
essential epitope binding features of an Ig molecule. Such mutant,
variant, or derivative antibody formats are known in the art,
non-limiting embodiments of which are discussed below.
[0136] In a full-length antibody, each heavy chain is comprised of
a heavy chain variable region (abbreviated herein as HCVR or
V.sub.H) and a heavy chain constant region. The heavy chain
constant region is comprised of three domains, CH1, CH2 and CH3.
Each light chain is comprised of a light chain variable region
(abbreviated herein as LCVR or V.sub.L) and a light chain constant
region. The light chain constant region is comprised of one domain,
CL, which in humans may be of either the .kappa. or .lamda. class.
The V.sub.H and V.sub.L regions can be further subdivided into
regions of hypervariability, termed complementarity determining
regions (CDR), interspersed with regions that are more conserved,
termed framework regions (FR). Each V.sub.H and V.sub.L is composed
of three CDRs and four FRs, arranged from amino-terminus to
carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,
CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG,
IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4,
IgA1 and IgA2) or subclass.
[0137] The term "antigen binding domain" or "antigen binding
portion" of an antibody, as used herein, refers to one or more
fragments of an antibody or protein that retain the ability to
specifically bind to an antigen (e.g., CD38). It has been shown
that the antigen-binding function of an antibody can be performed
by fragments of a full-length antibody. Such antibody embodiments
may also be bispecific, dual specific, or multi-specific formats,
specifically binding to two or more different antigens. Examples of
binding fragments encompassed within the term "antigen-binding
portion" of an antibody include (i) a Fab fragment, a monovalent
fragment consisting of the V.sub.L, V.sub.H, CL and CH1 domains;
(ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab
fragments in addition to a portion of the hinge region, linked by a
disulfide bridge at the hinge region; (iii) an Fd fragment
consisting of the V.sub.H and CH1 domains; (iv) an Fv fragment
consisting of the V.sub.L and V.sub.H domains of a single arm of an
antibody, (v) a domain antibody (dAb) (Ward et al. 1989 Nature 341
544-6, Winter et al., PCT publication WO 90/05144 Al herein
incorporated by reference), which comprises a single variable
domain; and (vi) an isolated complementarity determining region
(CDR). Furthermore, although the two domains of the Fv fragment,
V.sub.L and V.sub.H, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the
V.sub.L and V.sub.H regions pair to form monovalent molecules
(known as single chain Fv (scFv); see e.g., Bird et al. 1988
Science 242 423-6; Huston et al. 1988 Proc Natl Acad Sci USA 85
5879-83). Such single chain antibodies are also intended to be
encompassed within the term "antigen-binding portion" of an
antibody. Other forms of single chain antibodies, such as
diabodies, are also encompassed. Diabodies are bivalent, bispecific
antibodies in which V.sub.H and V.sub.L domains are expressed on a
single polypeptide chain, but using a linker that is too short to
allow for pairing between the two domains on the same chain,
thereby forcing the domains to pair with complementary domains of
another chain and creating two antigen binding sites (see e.g.,
Holliger, P., et al., 1993, Proc. Natl. Acad. Sci. USA
90:6444-6448; Poljak, R. J., et al., 1994, Structure 2:1121-1123).
Such antibody binding portions are known in the art (Kontermann and
Dubel eds., Antibody Engineering 2001 Springer-Verlag. New York.
790 pp., ISBN 3-540-41354-5). In an embodiment the antibody binding
portion is a Fab fragment.
[0138] The antibody described herein may be a humanized antibody.
The term "humanized antibody" shall be understood to refer to a
protein comprising a human-like variable region, which includes
CDRs from an antibody from a non-human species (e.g., mouse or rat
or non-human primate) grafted onto or inserted into FRs from a
human antibody (this type of antibody is also referred to a
"CDR-grafted antibody"). Humanized antibodies also include proteins
in which one or more residues of the human protein are modified by
one or more amino acid substitutions and/or one or more FR residues
of the human protein are replaced by corresponding non-human
residues. Humanized antibodies may also comprise residues which are
found in neither the human antibody or in the non-human antibody.
Any additional regions of the protein (e.g., Fc region) are
generally human. Humanization can be performed using a method known
in the art, e.g., U.S. Pat. Nos. 5,225,539, 6,054,297, 7,566,771 or
U.S. Pat. No. 5,585,089. The term "humanized antibody" also
encompasses a super-humanized antibody, e.g., as described in U.S.
Pat. No. 7,732,578.
[0139] The antibody described herein may be human. The term "human
antibody" as used herein refers to proteins having variable and,
optionally, constant antibody regions found in humans, e.g. in the
human germline or somatic cells or from libraries produced using
such regions. The "human" antibodies can include amino acid
residues not encoded by human sequences, e.g. mutations introduced
by random or site directed mutations in vitro (in particular
mutations which involve conservative substitutions or mutations in
a small number of residues of the protein, e.g. in 1, 2, 3, 4 or 5
of the residues of the protein). These "human antibodies" do not
necessarily need to be generated as a result of an immune response
of a human, rather, they can be generated using recombinant means
(e.g., screening a phage display library) and/or by a transgenic
animal (e.g., a mouse) comprising nucleic acid encoding human
antibody constant and/or variable regions and/or using guided
selection (e.g., as described in or U.S. Pat. No. 5,565,332). This
term also encompasses affinity matured forms of such antibodies.
For the purposes of the present disclosure, a human protein will
also be considered to include a protein comprising FRs from a human
antibody or FRs comprising sequences from a consensus sequence of
human FRs and in which one or more of the CDRs are random or
semi-random, e.g., as described in U.S. Pat. No. 6,300,064 and/or
U.S. Pat. No. 6,248,516.
[0140] The antibody portions of polypeptides of the present
invention may be full length antibodies of any class, preferably
IgG1, IgG2 or IgG4. The constant domains of such antibodies are
preferably human. The variable regions of such antibodies may be of
non-human origin or, preferably, be of human origin or be
humanized. Antibody fragments may also be used in place of the full
length antibodies.
[0141] The term "antibody" also includes engineered antibodies. As
will be appreciated there are many variations of engineered
antibodies (e.g. mouse monoclonal, chimeric, humanized and human
monoclonal antibodies, single chain variable antibody fragments
(scFv's), minibodies, aptamers, as well as bispecific antibodies
and diabodies as described above).
[0142] Single variable region domains (termed dAbs) are, for
example, disclosed in (Ward et al., 1989, Nature 341: 544-546;
Hamers-Casterman et al., 1993, Nature 363: 446-448; Davies &
Riechmann, 1994, FEBS Lett. 339: 285-290).
[0143] Minibodies are small versions of whole antibodies, which
encode in a single chain the essential elements of a whole
antibody. Suitably, the minibody is comprised of the V.sub.H and
V.sub.L domains of a native antibody fused to the hinge region and
CH3 domain of the immunoglobulin molecule as, for example,
disclosed in U.S. Pat. No. 5,837,821.
[0144] In an alternate embodiment, the engineered antibody may
comprise non-immunoglobulin derived, protein frameworks. For
example, reference may be made to (Ku & Schutz, 1995, Proc.
Natl. Acad. Sci. USA 92: 6552-6556) which discloses a four-helix
bundle protein cytochrome b562 having two loops randomized to
create CDRs, which have been selected for antigen binding.
[0145] There is a plethora of non-antibody recognition protein or
protein domain scaffolds that may be utilised as the antigen
binding domains in the constructs of this invention. These include
scaffolds based on cytotoxic T lymphocyte-associated antigen 4
(CTLA-4) (Evibody; U.S. Pat. No. 7,166,697); human transferrin
(Trans-body); a three-helix bundle from the Z-domain of Protein A
(Affibody); a monomeric or trimeric human C-type lectin domain
(Tetranectin); the tenth human fibronectin type III domain
(AdNectin); the Kunitz-type domain of human or bovine trypsin
inhibitor; insect Defensin A (IICA29), APPI (Kuntiz domains);
lipocalins, FABP, Bilin-binding protein, Apoloproptein D
(Anticalins); human .alpha.-crystallin or ubiquitin molecule
(Affilin); trypsin inhibitor II (Microbody); .alpha.2p8 or Ankyrin
repeat (repeat-motif proteins), Charybdotoxin (Scorpion toxins),
Min-23, Cellulose binding domain (Knottins); Neocarzinostatin,
CBM4-2 and Tendamistat.
[0146] Further, in addition to scaffolds provided for by
antibody-derived domains or non-antibody folds as described above,
there are naturally occurring ligand binding proteins or protein
domains that may be utilised as the ligand binding domains in this
invention. For example, protein domains that possess ligand binding
properties include extracellular domains of receptors, PDZ modules
of signaling proteins, such as Ras-binding protein AF-6, adhesion
molecules, and enzymes.
[0147] Using methods well known in the art to increase binding, by
for example, affinity maturation, or to decrease immunogenicity by
removing predicted MHC class II-binding motifs. The therapeutic
utility of the antibodies described herein can be further enhanced
by modulating their functional characteristics, such as
antibody-dependent cell-mediated cytotoxicity (ADCC),
complement-dependent cytotoxicity (CDC), serum half-life,
biodistribution and binding to Fc receptors or the combination of
any of these. This modulation can be achieved by
protein-engineering, glyco-engineering or chemical methods.
Depending on the therapeutic application required, it could be
advantageous to either increase or decrease any of these
activities.
[0148] An example of glyco-engineering used the Potelligent.RTM.
method as described in Shinkawa T. et al., 2003 (J Biol Chem 278:
3466-73).
[0149] Numerous methods for affinity maturation of antibodies are
known in the art. Many of these are based on the general strategy
of generating panels or libraries of variant proteins by
mutagenesis followed by selection and/or screening for improved
affinity. Mutagenesis is often performed at the DNA level, for
example by error prone PCR (Thie, Voedisch et al. 2009, Methods Mol
Biol 525: 309-322), by gene shuffling (Kolkman and Stemmer 2001,
Nat Biotechnol. May; 19(5):423-8), by use of mutagenic chemicals or
irradiation, by use of `mutator` strains with error prone
replication machinery (Greener 1996, In Vitro Mutagenesis
Protocols. Humana press, NJ) or by somatic hypermutation approaches
that harness natural affinity maturation machinery (Peled, Kuang et
al. 2008, Annu Rev Immunol. 26:481-511). Mutagenesis can also be
performed at the RNA level, for example by use of Q.beta. replicase
(Kopsidas, Roberts et al. 2006, Immunol Lett. 2006 Nov. 15;
107(2):163-8). Library-based methods allowing screening for
improved variant proteins can be based on various display
technologies such as phage, yeast, ribosome, bacterial or mammalian
cells, and are well known in the art (Benhar 2007, Expert Opin Biol
Ther. May; 7(5): 763-79). Affinity maturation can be achieved by
more directed/predictive methods for example by site-directed
mutagenesis or gene synthesis guided by findings from 3D protein
modeling (see for example Queen, Schneider et al. 1989, PNAS,
86(24): 10029-33 or U.S. Pat. Nos. 6,180,370 or 5,225,539).
[0150] Methods of increasing ADCC have been described by Ferrara,
Brunker et al. 2006, Biotechnol Bioeng; 93:851-61; Li, Sethuraman
et al. 2006, Nat Biotechnol; 24:210-5; Stavenhagen, Gorlatov et al.
2007, Cancer Res; 67:8882-90; Shields, Namenuk et al. 2001, J Biol
Chem; 276:6591-604; Shinkawa, Nakamura et al. 2003, J Biol Chem;
278:3466-73; and WO 2008/006554.
[0151] Methods of increasing CDC have been described by Idusogie,
Wong et al. 2001, J Immunol; 176:346-56; Dall'Acqua, Cook et al.
2006, J Biol Chem; 281:23514-24; Michaelsen, Aase et al. 1990,
Scand J Immunol; 32:517-28; Brekke, Bremnes et al. 1993, Mol
Immunol; 30:1419-25; Tan, Shopes et al. 1990, PNAS; 87:162-6; and
Norderhaug, Brekke et al. 1991, Eur J Immunol; 21:2379-84.
[0152] References describing methods of increasing ADCC and CDC
include Natsume, In et al. 2008, Cancer Res; 68:3863-72. The
disclosure of each of these references is included herein by cross
reference. In certain embodiments it may be advantageous to reduce
or eliminate ADCC and CDC activities of the antibody component of
the polypeptide of the invention, so that the IFN.alpha.2b activity
is the principal activity of the polypeptide which modulates target
cell survival.
[0153] A number of methods for modulating antibody serum half-life
and biodistribution are based on modifying the interaction between
antibody and the neonatal Fc receptor (FcRn), a receptor with a key
role in protecting IgG from catabolism, and maintaining high serum
antibody concentration. Dall'Acqua et al describe substitutions in
the Fc region of IgG1 that enhance binding affinity to FcRn,
thereby increasing serum half-life (Dall'Acqua, Woods et al. 2002,
J Immunol; 169:5171-80) and further demonstrate enhanced
bioavailability and modulation of ADCC activity with triple
substitution of M252Y/S254T/T256E (with residue numbering according
to the EU Index) or M265Y/S267T/T269 (with residue numbering
according to the Kabat numbering system) (Dall'Acqua, Kiener et al.
2006, J Biol Chem; 279:6213-6). See also U.S. Pat. Nos. 6,277,375;
6,821,505; and U.S. Pat. No. 7,083,784. Hinton et al have described
constant domain amino acid substitutions at positions 250 and 428
that confer increased in vivo half-life (Hinton, Johlfs et al.
2004, J Biol Chem; 279:6213-6; Hinton, Xiong et al. 2006, J
Immunol; 176:346-56). See also U.S. Pat. No. 7,217,797. Petkova et
al have described constant domain amino acid substitutions at
positions 307, 380 and 434 that confer increased in vivo half-life
(Petkova, Akilesh et al. 2006, Int Immunol; 18:1759-69). See also
Shields et al 2001, J Biol Chem; 276:6591-604 and WO 2000/42072.
Other examples of constant domain amino acid substitutions which
modulate binding to Fc receptors and subsequent function mediated
by these receptors, including FcRn binding and serum half-life, are
described in U.S Pat. Application Nos 20090142340; 20090068175 and
20090092599. The substitution referred to herein as "S228P" which
is numbered according to the EU index as in Kabat has also been
referred to as "S241P" according to Kabat et al. (1987 Sequences of
proteins of immunological interest. United States Department of
Health and Human Services, Washington D.C.). This substitution
stabilizes the hinge region of IgG4 molecules, having the effect of
making the sequence of the core of the hinge region the same as
that of an IgG1 or IgG2 isotype antibody. This results in a
reduction in the spontaneous dissociation and reassociation of the
heavy chains which often leads to the production of heterodimeric
IgG4 antibodies.
[0154] The glycans linked to antibody molecules are known to
influence interactions of antibody with Fc receptors and glycan
receptors and thereby influence antibody activity, including serum
half-life (Kaneko, Nimmerjahn et al. 2006, Science; 313:670-3;
Jones, Papac et al. 2007, Glcobiology; 17:529-40; and Kanda, Yamada
et al. 2007, Glycobiology; 17:104-18). Hence, certain glycoforms
that modulate desired antibody activities can confer therapeutic
advantage. Methods for generating engineered glycoforms are known
in the art and include but are not limited to those described in
U.S. Pat. Nos U.S. Pat. Nos. 6,602,684; 7,326,681; 7,388,081 and in
WO 2008/006554.
[0155] Extension of half-life by addition of polyethylene glycol
(PEG) has been widely used to extend the serum half-life of
proteins, as reviewed, for example, by Fishburn 2008, J Pharm Sci;
97:4167-83.
[0156] As will be recognised it is possible to make conservative
amino acid substitutions within the sequences of the current
invention. By "conservative substitution" is meant amino acids
having similar properties. As used in this specification the
following groups of amino acids are to be seen as conservative
substitutions: H, R and K; D, E, N and Q; V, I and L; C and M; S,
T, P, A and G; and F, Y and W. It is not intended, however, that
substitutions other than those specifically recited are made at the
sites of attenuation and/or glycosylation.
[0157] The term "cell surface-associated antigen", as used herein,
broadly refers to any antigen expressed on surfaces of cells,
including without limitation malignant cells or infectious or
foreign cells.
[0158] In certain aspects of the present invention, the fusion
polypeptide constructs or compositions of the present invention may
be used to treat patients with cancer. Cancers contemplated herein
include: a group of diseases and disorders that are characterized
by uncontrolled cellular growth (e.g. formation of tumor) without
any differentiation of those cells into specialized and different
cells. Such diseases and disorders include ABL1 protooncogene, AIDS
related cancers, acoustic neuroma, acute lymphocytic leukaemia,
acute myeloid leukaemia, adenocystic carcinoma, adrenocortical
cancer, agnogenic myeloid metaplasia, alopecia, alveolar soft-part
sarcoma, anal cancer, angiosarcoma, aplastic anaemia, astrocytoma,
ataxia-telangiectasia, basal cell carcinoma (skin), bladder cancer,
bone cancers, bowel cancer, brain stem glioma, brain and CNS
tumors, breast cancer, carcinoid tumors, cervical cancer, childhood
brain tumors, childhood cancer, childhood leukaemia, childhood soft
tissue sarcoma, chondrosarcoma, choriocarcinoma, chronic
lymphocytic leukaemia, chronic myeloid leukaemia, colorectal
cancers, cutaneous T-Cell lymphoma,
dermatofibrosarcoma-protuberans,
desmoplastic-small-round-cell-tumor, ductal carcinoma, endocrine
cancers, endometrial cancer, ependymoma, esophageal cancer, Ewing's
sarcoma, extra-hepatic bile duct cancer, eye cancer, eye: melanoma,
retinoblastoma, fallopian tube cancer, fanconi anemia,
fibrosarcoma, gall bladder cancer, gastric cancer, gastrointestinal
cancers, gastrointestinal-carcinoid-tumor, genitourinary cancers,
germ cell tumors, gestational-trophoblastic-disease, glioma,
gynaecological cancers, hematological malignancies, hairy cell
leukaemia, head and neck cancer, hepatocellular cancer, hereditary
breast cancer, histiocytosis, Hodgkin's disease, human
papillomavirus, hydatidiform mole, hypercalcemia, hypopharynx
cancer, intraocular melanoma, islet cell cancer, Kaposi's sarcoma,
kidney cancer, Langerhan's-cell-histiocytosis, laryngeal cancer,
leiomyosarcoma, leukemia, Li-Fraumeni syndrome, lip cancer,
liposarcoma, liver cancer, lung cancer, lymphedema, lymphoma,
Hodgkin's lymphoma, non-Hodgkin's lymphoma, male breast cancer,
malignant-rhabdoid-tumor-of-kidney, medulloblastoma, melanoma,
merkel cell cancer, mesothelioma, metastatic cancer, mouth cancer,
multiple endocrine neoplasia, mycosis fungoides, myelodysplastic
syndromes, multiple myeloma, myeloproliferative disorders, nasal
cancer, nasopharyngeal cancer, nephroblastoma, neuroblastoma,
neurofibromatosis, nijmegen breakage syndrome, non-melanoma skin
cancer, non-small-cell-lung-cancer-(NSCLC), ocular cancers,
oesophageal cancer, oral cavity cancer, oropharynx cancer,
osteosarcoma, ostomy ovarian cancer, pancreas cancer, paranasal
cancer, parathyroid cancer, parotid gland cancer, penile cancer,
peripheral-neuroectodermal-tumors, pituitary cancer, polycythemia
vera, prostate cancer, rare-cancers-and-associated-disorders, renal
cell carcinoma, retinoblastoma, rhabdomyosarcoma, Rothmund-Thomson
syndrome, salivary gland cancer, sarcoma, schwannoma, Sezary
syndrome, skin cancer, small cell lung cancer (SCLC), small
intestine cancer, soft tissue sarcoma, spinal cord tumors,
squamous-cell-carcinoma-(skin), stomach cancer, synovial sarcoma,
testicular cancer, thymus cancer, thyroid cancer,
transitional-cell-cancer-(bladder),
transitional-cell-cancer-(renal-pelvis-/-ureter), trophoblastic
cancer, urethral cancer, urinary system cancer, uroplakins, uterine
sarcoma, uterus cancer, vaginal cancer, vulva cancer,
Waldenstrom's-macroglobulinemia and Wilms' tumor. In an embodiment
the tumor is selected from a group of multiple myeloma or
non-hodgkin's lymphoma.
[0159] As contemplated for the treatment of cancer, the antibody
portions of the fusion constructs of the present invention may bind
to tumour-associated antigens, i.e., cell surface antigens that are
selectively expressed by cancer cells or over-expressed in cancer
cells relative to most normal cells. There are many
tumour-associated antigens (TAAs) known in the art. Non-limiting
examples of TAAs include enzyme tyrosinase; melanoma antigen GM2;
alphafetoprotein (AFP); carcinoembryonic antigen (CEA); Mucin 1
(MUC1); Human epidermal growth factor receptor (Her2/Neu); T-cell
leukemia/lymphoma 1 (TCL1) oncoprotein. Exemplary TAAs associated
with a number of different cancers are telomerase (hTERT);
prostate-specific membrane antigen (PSMA); urokinase plasminogen
activator and its receptor (uPA/uPAR); vascular endothelial growth
factor and its receptor (VEGF/VEGFR); extracellular matrix
metalloproteinase inducer (EMMPRIN/CD147); epidermal growth factor
(EGFR); platelet-derived growth factor and its receptor
(PDGF/PDGFR) and c-kit (CD117).
[0160] A list of other TAAs is provided in US 2010/0297076, the
disclosure of which is included herein by reference. Of particular
interest are cell surface antigens associated with multiple myeloma
leukemia or lymphoma cells, including but not limited to CD38,
CD138, CD79, CS1, and HM1.24. In one embodiment an antigen for
ligand-attenuated IFN constructs, for example, an
antibody-attenuated interferon construct, is CD38.
[0161] CD38 is a 46 kDa type II transmembrane glycoprotein. It has
a short N-terminal cytoplasmic tail of 20 amino acids, a single
transmembrane helix and a long extracellular domain of 256 amino
acids (Bergsagel, P., Blood; 85:436, 1995 and Liu, Q., Structure,
13:1331, 2005). It is expressed on the surface of many immune cells
including CD4 and CD8 positive T cells, B cells, NK cells,
monocytes, plasma cells and on a significant proportion of normal
bone marrow precursor cells (Malavasi, F., Hum. Immunol. 9:9,
1984). In lymphocytes, however, the expression appears to be
dependent on the differentiation and activation state of the cell.
Resting T and B cells are negative while immature and activated
lymphocytes are predominantly positive for CD38 expression (Funaro,
A., J. Immunol. 145:2390, 1990). Additional studies indicate mRNA
expression in non-hemopoeitic organs such as pancreas, brain,
spleen and liver (Koguma, T., Biochim. Biophys. Acta 1223:160,
1994.)
[0162] CD38 is a multifunctional ectoenzyme that is involved in
transmembrane signaling and cell adhesion. It is also known as
cyclic ADP ribose hydrolase because it can transform NAD.sup.+ and
NADP.sup.+ into cADPR, ADPR and NAADP, depending on extracellular
pH. These products induce Ca.sup.2+-mobilization inside the cell
which can lead to tyrosine phosphorylation and activation of the
cell. CD38 is also a receptor that can interact with a ligand,
CD31. Activation of receptor via CD31 leads to intracellular events
including Ca.sup.2+ mobilization, cell activation, proliferation,
differentiation and migration (reviewed in Deaglio, S., Trends in
Mol. Med. 14:210, 2008.)
[0163] CD38 is expressed at high levels on multiple myeloma cells,
in most cases of T- and B-lineage acute lymphoblastic leukemias,
some acute myelocytic leukemias, follicular center cell lymphomas
and T lymphoblastic lymphomas. (Malavasi, F., J. Clin Lab Res.
22:73, 1992). More recently, CD38 expression has become a reliable
prognostic marker in B-lineage chronic lymphoblastic leukemia
(B-CLL) (Ibrahim, S., Blood. 98:181, 2001 and Dung, J., Leuk. Res.
25:927, 2002). Independent groups have demonstrated that B-CLL
patients presenting with a CD38.sup.+ clone are characterized by an
unfavorable clinical course with a more advance stage of disease,
poor responsiveness to chemotherapy and shorter survival time
(Morabito, F., Haematologica. 87:217, 2002). The consistent and
enhanced expression of CD38 on lymphoid tumors makes this an
attractive target for therapeutic antibody technologies.
[0164] Preferred antigens for the development of
antibody-attenuated aglycosylated IFN.alpha.2b fusion protein
constructs which target cancer are antigens which show selective or
greater expression on the cancer cells than on most other,
non-transformed cells within the body. Non-protein examples of such
antigens include, sphingolipids, ganglioside GD2 (Saleh et al.,
1993, J. Immunol., 151, 3390-3398), ganglioside GD3 (shitara et
al., 1993, Cancer Immunol. Immunother. 36:373-380), ganglioside GM2
(Livingston et al., 1994, J. Clin. Oncol. 12:1036-1044),
ganglioside GM3 (Hoon et al., 1993, Cancer Res. 53:5244-5250) and
Lewis.sup.x, lewis.sup.y and lewis.sup.xy carbohydrate antigens
that can be displayed on proteins or glycolipids. Examples of
protein antigens are HER-2/neu, human papillomavirus-E6 or -E7,
MUC-1; KS 1/4 pan-carcinoma antigen (Perez and Walker, 1990, J.
Immunol. 142:3662-3667; Bumal, 1988, Hybridoma 7(4):407-415);
ovarian carcinoma antigen CA125 (Yu et al., 1991, Cancer Res.
51(2):468-475); prostatic acid phosphate (Tailor et al., 1990,
Nucl. Acids Res. 18(16):4928); prostate specific antigen (Henttu
and Vihko, 1989, Biochem. Biophys. Res. Comm. 160(2):903-910;
Israeli et al., 1993, Cancer Res. 53:227-230); melanoma-associated
antigen p97 (Estin et al., 1989, J. Natl. Cancer Instit.
81(6):445-446); melanoma antigen gp75 (Vijayasardahl et al., 1990,
J. Exp. Med. 171(4):1375-1380); prostate specific membrane antigen;
carcinoembryonic antigen (CEA) (Foon et al., 1994, Proc. Am. Soc.
Clin. Oncol. 13:294), MUC16 (antibodies include MJ-170, MJ-171,
MJ-172 and MJ-173 [U.S. Pat. No. 7,202,346], 3A5 [U.S. Pat. No.
7,723,4851]).NMB (U.S. Pat. No. 8,039,593), malignant human
lymphocyte antigen-APO-1 (Bernhard et al., 1989, Science
245:301-304); high molecular weight melanoma antigen (HMW-MAA)
(Natali et al., 1987, Cancer 59:55-63; Mittelman et al., 1990, J.
Clin. Invest. 86:2136-2144); Burkitt's lymphoma antigen-38.13; CD19
(Ghetie et al., 1994, Blood 83:1329-1336); human B-lymphoma
antigen-CD20 (Reff et al., 1994, Blood 83:435-445); GICA 19-9
(Herlyn et al., 1982, J. Clin. Immunol. 2:135), CTA-1 and LEA; CD33
(Sgouros et al., 1993, J. Nucl. Med. 34:422-430); oncofetal
antigens such as alpha-fetoprotein for liver cancer or bladder
tumor oncofetal antigen (Hellstrom et al., 1985, Cancer. Res.
45:2210-2188); differentiation antigens such as human lung
carcinoma antigen L6 or L20 (Hellstrom et al., 1986, Cancer Res.
46:3917-3923); antigens of fibrosarcoma; human leukemia T cell
antigen-Gp37 (Bhattacharya-Chatterjee et al., 1988, J. Immunol.
141:1398-1403); tumor-specific transplantation type of cell-surface
antigen (TSTA) such as virally-induced tumor antigens including
T-antigen, DNA tumor virus and envelope antigens of RNA tumor
viruses; neoglycoproteins, breast cancer antigens such as EGFR
(Epidermal growth factor receptor), polymorphic epithelial mucin
(PEM) (Hilkens et al., 1992, Trends in Bio. Chem. Sci. 17:359);
polymorphic epithelial mucin antigen; human milk fat globule
antigen; colorectal tumor-associated antigens such as TAG-72
(Yokata et al., 1992, Cancer Res. 52:3402-3408), CO 17-1A
(Ragnhammar et al., 1993, Int. J. Cancer 53:751-758);
differentiation antigens (Feizi, 1985, Nature 314:53-57) such as
I(Ma) found in gastric adenocarcinomas, SSEA-1 found in myeloid
cells, VEP8, VEP9, Myl, VIM-D5, M18 and M39 found in breast
epithelial cancers, D156-22 found in colorectal cancer, TRA-1-85
(blood group H), C14 found in colonic adenocarcinoma, F3 found in
lung adenocarcinoma, AH6 found in gastric cancer, Y hapten found in
embryonal carcinoma cells, TL5 (blood group A), E1 series (blood
group B) antigens found in pancreatic cancer, FC10.2 found in
embryonal carcinoma cells, gastric adenocarcinoma antigen, CO-514
(blood group Le.sup.a) found in adenocarcinoma, NS-10 found in
adenocarcinomas, CO-43 (blood group Le.sup.b), G49 found in A431
cells, 19.9 found in colon cancer; gastric cancer mucins; R24 found
in melanoma, MH2 (blood group ALe.sup.b/Le.sup.y) found in colonic
adenocarcinoma, 4.2, D1.1, OFA-1, G.sub.M2, OFA-2 and M1:22:25:8
found in embryonal carcinoma cells and SSEA-3 and SSEA-4. HMW-MAA
(SEQ ID NO:433), also known as melanoma chondroitin sulfate
proteoglycan, is a membrane-bound protein of 2322 residues which is
overexpressed on over 90% of the surgically removed benign nevi and
melanoma lesions (Camploi, et. al, Crit Rev Immunol.; 24:267,
2004). Accordingly it may be a potential target cell surface
associated antigen.
[0165] Other example cancer antigens for targeting with fusion
protein constructs of the present invention include (exemplary
cancers are shown in parentheses): CD5 (T-cell leukemia/lymphoma),
CA15-3 (carcinomas), CA19-9 (carcinomas), L6 (carcinomas), CA 242
(colorectal), placental alkaline phosphatase (carcinomas),
prostatic acid phosphatase (prostate), MAGE-1 (carcinomas), MAGE-2
(carcinomas), MAGE-3 (carcinomas), MAGE-4 (carcinomas), transferrin
receptor (carcinomas), p97 (melanoma), MUC1 (breast cancer), MART1
(melanoma), CD20 (non Hodgkin's lymphoma), CD52 (leukemia), CD33
(leukemia), human chorionic gonadotropin (carcinoma), CD38
(multiple myeloma), CD21 (B-cell lymphoma), CD22 (lymphoma), CD25
(B-cell Lymphoma), CD37 (B-cell lymphoma), CD45 (acute myeloblastic
leukemia), HLA-DR (B-cell lymphoma), IL-2 receptor (T-cell leukemia
and lymphomas), CD40 (lymphoma), CD79 (B cell leukemia or lymphoma,
Hodgkin lymphoma), various mucins (carcinomas), P21 (carcinomas),
MPG (melanoma), Ep-CAM (Epithelial Tumors), Folate-receptor alpha
(Ovarian), A33 (Colorectal), G250 (renal), Ferritin (Hodgkin
lymphoma), de2-7 EGFR (glioblastoma, breast, and lung), Fibroblast
activation protein (epithelial) and tenascin metalloproteinases
(glioblastoma). Some specific, useful antibodies include, but are
not limited to, BR64 (Trail et al., 1997, Cancer Research 57:100
105), BR96 mAb (Trail et al., 1993, Science 261:212-215), mAbs
against the CD40 antigen, such as S2C6 mAb (Francisco et al., 2000,
Cancer Res. 60:3225-3231) or other anti-CD40 antibodies, such as
those disclosed in U.S Patent Publication Nos. 2003-0211100 and
2002-0142358; mAbs against the CD30 antigen, such as AC10 (Bowen et
al., 1993, J. Immunol. 151:5896-5906; Wahl et al., 2002 Cancer Res.
62(13):3736-42) or MDX-0060 (U.S. Patent Publication No.
2004-0006215) and mAbs against the CD70 antigen, such as 1F6 mAb
and 2F2 mAb (see, e.g., U.S. Patent Publication No. 2006-0083736)
or antibodies 2H5, 10B4, 8B5, 18E7, 69A7 (U.S. Pat. No. 8,124,738).
Other antibodies have been reviewed elsewhere (Franke et al., 2000,
Cancer Biother. Radiopharm. 15:459 76; Murray, 2000, Semin. Oncol.
27:64 70; Breitling, F., and Dubel, S., Recombinant Antibodies,
John Wiley, and Sons, New York, 1998).
[0166] In certain embodiments, useful antibodies can bind to a
receptor or a complex of receptors expressed on a target cell. The
receptor or receptor complex can comprise an immunoglobulin gene
superfamily member, a major histocompatibility protein, a cytokine
receptor, a TNF receptor superfamily member, a chemokine receptor,
an integrin, a lectin, a complement control protein, a growth
factor receptor, a hormone receptor or a neuro-transmitter
receptor. Non-limiting examples of appropriate immunoglobulin
superfamily members are CD2, CD3, CD4, CD8, CD19, CD22, CD79, CD90,
CD152/CTLA-4, PD-1, B7-H4, B7-H3, and ICOS. Non-limiting examples
of suitable TNF receptor superfamily members are TACI, BCMA, CD27,
CD40, CD95/Fas, CD134/0X40, CD137/4-1BB, TNFR1, TNFR2, RANK,
osteoprotegerin, APO 3, Apo2/TRAIL R1, TRAIL R2, TRAIL R3, and
TRAIL R4. Non-limiting examples of suitable integrins are CD11a,
CD11 b, CD11c, CD18, CD29, CD41, CD49a, CD49b, CD49c, CD49d, CD49e,
CD49f, CD103 and CD104. Non-limiting examples of suitable lectins
are S type, C type, and I type lectin. Examples of antibodies to
CEA are shown in Table 1.
TABLE-US-00001 TABLE 1 CEA Antibodies Ab Clones patent Assignee
Comments COL-1 U.S. Pat. No. The Dow Chemical Humanized 6,417,337
Company 806.077 U.S. Pat. No. AstraZeneca UK Ltd. Humanized
6,903,203 T84.66 U.S. Pat. No. City of Hope Humanized 7,776,330
[0167] Antibodies that bind the CD22 antigen expressed on human B
cells include, for example, HD6, RFB4, UV22-2, To15, 4KB128 and a
humanized anti-CD22 antibody (hLL2) (see, e.g., Li et al. (1989)
Cell. Immunol. 111: 85-99; Mason et al. (1987) Blood 69: 836-40;
Behr et al. (1999) Clin. Cancer Res. 5: 3304s-3314s; Bonardi et al.
(1993) Cancer Res. 53: 3015-3021).
[0168] Antibodies to CD33 include, for example, HuM195 (see, e.g.,
Kossman et al. (1999) Clin. Cancer Res. 5: 2748-2755; U.S. Pat. No.
5,693,761) and CMA-676 (see, e.g., Sievers et al., (1999) Blood 93:
3678-3684).
[0169] Illustrative anti-MUC-1 antibodies include, but are not
limited to Mc5 (see, e.g., Peterson et al. (1997) Cancer Res. 57:
1103-1108; Ozzello et al. (1993) Breast Cancer Res. Treat. 25:
265-276), and hCTMO1 (see, e.g., Van Hof et al. (1996) Cancer Res.
56: 5179-5185).
[0170] Illustrative anti-TAG-72 antibodies include, but are not
limited to CC49 (see, e.g., Pavlinkova et al. (1999) Clin. Cancer
Res. 5: 2613-2619), B72.3 (see, e.g., Divgi et al. (1994) Nucl.
Med. Biol. 21: 9-15), and those disclosed in U.S. Pat. No.
5,976,531.
[0171] Illustrative anti-HM1.24 antibodies include, but are not
limited to a mouse monoclonal anti-HM1.24 and a humanized
anti-HM1.24 IgGlkappa antibody (see, e.g., Ono et al. (1999) Mol.
Immuno. 36: 387-395).
[0172] In certain embodiments the targeting moiety comprises an
anti-Her2 antibody. The erBB 2 gene, more commonly known as
(Her-2/neu), is an oncogene encoding a transmembrane receptor.
Several antibodies have been developed against Her-2/neu, and some
of these are in clinical use. These include trastuzumab (e.g.,
HERCEPTIN.TM.; Fornir et al. (1999) Oncology (Huntingt) 13:
647-58), TAB-250 (Rosenblum et al. (1999) Clin. Cancer Res. 5:
865-874), BACH-250 (Id.), TA1 (Maier et al. (1991) Cancer Res. 51:
5361-5369), and the mAbs described in U.S. Pat. Nos. 5,772,997;
5,770,195 (mAb 4D5; ATCC CRL 10463); and U.S. Pat. No.
5,677,171.
[0173] Other fully human anti-Her2/neu antibodies are well known to
those of skill in the art. Such antibodies include, but are not
limited to the C6 antibodies such as C6.5, DPLS, G98A, C6MH3-B1,
B1D2, C6VLB, C6VLD, C6VLE, C6VLF, C6MH3-D7, C6MH3-D6, C6MH3-D5,
C6MH3-D3, C6MH3-D2, C6MH3-D1, C6MH3-C4, C6MH3-C3, C6MH3-B9,
C6MH3-B5, C6MH3-B48, C6MH3-B47, C6MH3-B46, C6MH3-B43, C6MH3-B41,
C6MH3-B39, C6MH3-B34, C6MH3-B33, C6MH3-B31, C6MH3-B27, C6MH3-B25,
C6MH3-B21, C6MH3-B20, C6MH3-B2, C6MH3-B16, C6MH3-B15, C6MH3-B11,
C6MH3-B1, C6MH3-A3, C6MH3-A2, and C6ML3-9. These and other
anti-HER2/neu antibodies are described in U.S. Pat. Nos. 6,512,097
and 5,977,322, in PCT Publication WO 97/00271, in Schier et al.
(1996) J Mol Biol 255: 28-43, Schier et al. (1996) J Mol Biol 263:
551-567, and the like.
[0174] More generally, antibodies directed to various members of
the epidermal growth factor receptor family are well suited for use
as targeting antibodies or antigen binding portions thereof in the
constructs of the present invention. Such antibodies include, but
are not limited to anti-EGFR antibodies as described in U.S. Pat.
Nos. 5,844,093 and 5,558,864, and in European Patent No. 706,799A.
Other illustrative anti-EGFR family antibodies include, but are not
limited to antibodies such as C6.5, C6ML3-9, C6MH3-B1, C6-B1D2, F5,
HER3.A5, HER3.F4, HER3.H1, HER3.H3, HER3.E12, HER3.B12, EGFR.E12,
EGFR.C10, EGFR.B11, EGFR.E8, HER4.B4, HER4.G4, HER4.F4, HER4.A8,
HER4.B6, HER4.D4, HER4.D7, HER4.D11, HER4.D12, HER4.E3, HER4.E7,
HER4.F8 and HER4.C7 and the like (see, e.g., U.S. Patent
publications US 2006/0099205 A1 and US 2004/0071696 A1 which are
incorporated herein by reference).
[0175] CD38 is of particular interest as an antibody target for
fusion protein constructs of the present invention. Antibodies to
CD38 include for example, .DELTA.T13/5 (see, e.g., Ellis et al.
(1995) J. Immunol. 155: 925-937), HB7, and the like.
[0176] The present invention also provides compositions comprising
the fusion polypeptides of the present invention. These
compositions can further comprise at least one of any suitable
auxiliary, such as, but not limited to, diluent, binder,
stabiliser, buffers, salts, lipophilic solvents, preservative,
adjuvant or the like. Pharmaceutically acceptable auxiliaries are
preferred. Non-limiting examples of, and methods of preparing such
sterile solutions are well known in the art, such as, but not
limited to, Gennaro, Ed., Remington's Pharmaceutical Sciences, 18th
Edition, Mack Publishing Co. (Easton, Pa.) 1990. Pharmaceutically
acceptable carriers can be routinely selected that are suitable for
the mode of administration, solubility and/or stability of the
antibody composition as well known in the art or as described
herein.
[0177] Pharmaceutical excipients and additives useful in the
present composition include but are not limited to proteins,
peptides, amino acids, lipids, and carbohydrates (e.g., sugars,
including monosaccharides, di-, tri-, tetra-, and oligosaccharides;
derivatised sugars such as alditols, aldonic acids, esterified
sugars and the like; and polysaccharides or sugar polymers), which
can be present singly or in combination, comprising alone or in
combination 1-99.99% by weight or volume. Exemplary protein
excipients include serum albumin, such as human serum albumin
(HSA), recombinant human albumin (rHA), gelatin, casein, and the
like. Representative amino acids which can also function in a
buffering capacity include alanine, glycine, arginine, betaine,
histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine,
isoleucine, valine, methionine, phenylalanine, aspartame, and the
like. One preferred amino acid is histidine. A second preferred
amino acid is arginine.
[0178] Carbohydrate excipients suitable for use in the invention
include, for example, monosaccharides, such as fructose, maltose,
galactose, glucose, D-mannose, sorbose, and the like;
disaccharides, such as lactose, sucrose, trehalose, cellobiose, and
the like; polysaccharides, such as raffinose, melezitose,
maltodextrins, dextrans, starches, and the like; and alditols, such
as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol
(glucitol), myoinositol and the like. Preferred carbohydrate
excipients for use in the present invention are mannitol,
trehalose, and raffinose.
[0179] Antibody compositions can also include a buffer or a pH
adjusting agent; typically, the buffer is a salt prepared from an
organic acid or base. Representative buffers include organic acid
salts, such as salts of citric acid, ascorbic acid, gluconic acid,
carbonic acid, tartaric acid, succinic acid, acetic acid, or
phthalic acid; Tris, tromethamine hydrochloride, phosphate buffers
or amino acid buffers. Preferred buffers for use in the present
compositions are organic acid salts, such as citrate or amino
acids.
[0180] Additionally, the compositions of the invention can include
polymeric excipients/additives, such as polyvinylpyrrolidones,
ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such
as 2-hydroxypropyl-.beta.-cyclodextrin), polyethylene glycols,
flavoring agents, antimicrobial agents, sweeteners, antioxidants,
antistatic agents, surfactants (e.g., polysorbates such as
"TWEEN.RTM. 20" and "TWEEN.RTM. 80"), lipids (e.g., phospholipids,
fatty acids), steroids (e.g., cholesterol), and chelating agents
(e.g., EDTA).
[0181] These and additional known pharmaceutical excipients and/or
additives suitable for use in the antibody compositions according
to the invention are known in the art, e.g., as listed in
"Remington: The Science & Practice of Pharmacy", 19 th ed.,
Williams & Williams, (1995), and in the "Physician's Desk
Reference", 52 nd ed., Medical Economics, Montvale, N.J. (1998),
the disclosures of which are entirely incorporated herein by
reference. Preferred carrier or excipient materials are
carbohydrates (e.g., saccharides and alditols) and buffers (e.g.,
citrate) or polymeric agents.
[0182] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications
which fall within the spirit and scope. The invention also includes
all of the steps, features, compositions and compounds referred to
or indicated in this specification, individually or collectively,
and any and all combinations of any two or more of said steps or
features.
[0183] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any materials and methods similar or equivalent to those described
herein can be used to practice or test the present invention, the
preferred materials and methods are now described.
EXAMPLES
General Methods
Production of Antibody Fusion Constructs in HEK-293E Cells.
[0184] The DNA sequences of a number of the domains in the fusion
polypeptides of the present are provided in the attached Sequence
listing incorporated herein. DNA plasmids encoding protein
constructs (antibody-attenuated IFN.alpha.2b fusion constructs)
were prepared using HiSpeed Plasmid Maxi Kit (Qiagen, Valencia,
Calif.) and then transfected into HEK293E cells (CNRC, Montreal,
Canada), grown in F17 synthetic medium supplemented with 0.45%
(w/v) D-(+)-Glucose (Sigma, Castle Hill, NSW), 25 .mu.g/mL
Geneticin (Invitrogen, Carlsbad, Calif.), and 1.times.GlutaMAX
(Invitrogen, Carlsbad, Calif.) using a commercially available
transfection reagent and OptiMEM medium (Invitrogen, Carlsbad,
Calif.). After allowing for expression for 6 days in an incubator
supplied with 5% CO.sub.2 and 120 rpm shaking, the culture media
was isolated and subjected to affinity purification using Protein A
MABSELECT SURE.RTM. agarose beads (GE Healthcare, Piscataway,
N.J.). Purified protein constructs were buffer-exchanged into 0.2M
arginine HCl, 25 mM citric acid, 71.5 mM sodium hydroxide at pH 6.0
using a PD Midi-Trap G-25 column (GE Healthcare, Piscataway, N.J.)
or a HiPrep 26/10 Desalting column (HiTrap Desalting HiPrep 26/10
Desalting). Purified protein constructs were then concentrated
using 50 kDa Amicon Ultra centrifugal filter devices (Millipore,
Billerica, Mass.), followed by protein concentration determination
by reading absorbance at 280 nm.
Production of Antibody-Fusion Constructs in EXPI293 Cells.
[0185] DNA plasmids encoding protein constructs
(antibody-IFN.alpha.2b related constructs) were prepared using
HiSpeed Plasmid Maxi Kit (Qiagen, Valencia, Calif.) and then
transfected into EXPI293 cells (Life Technologies, Carlsbad,
Calif.), grown in EXPI Expression medium (Life Technologies,
Carlsbad, Calif.) using transfection reagent provided in the
EXPI293 transfection kit and OptiMEM medium (Invitrogen, Carlsbad,
Calif.). After allowing for expression for 3 days in an incubator
supplied with 5% CO.sub.2 and 125 rpm shaking, the culture media
was isolated and subjected to affinity purification using Protein A
MABSELECT SURE.RTM. agarose beads (GE Healthcare, Piscataway,
N.J.). Purified protein constructs were buffer-exchanged into 0.2M
arginine HCl, at pH 6.0 using a PD Midi-Trap G-25 column (GE
Healthcare, Piscataway, N.J.) or a HiPrep 26/10 Desalting column
(HiTrap Desalting HiPrep 26/10 Desalting). Purified protein
constructs were then concentrated using 50 kDa Amicon Ultra
centrifugal filter devices (Millipore, Billerica, Mass.), followed
by protein concentration determination by reading absorbance at 280
nm.
Production of Antibody-Fusion Constructs in CHO Cells.
[0186] DNA plasmids encoding protein constructs
(antibody-IFN.alpha.2b related constructs) were prepared using
HiSpeed Plasmid Maxi Kit (Qiagen, Valencia, Calif.) and then
transfected into CHO cells (Lonza) grown in FREESTYLE.TM. CHO
Expression Medium (Invitrogen, Carlsbad, Calif.) using a
commercially available transfection reagent and OptiPro SFM.TM.
medium (Invitrogen, Carlsbad, Calif.). After allowing for
expression for 6 days in an incubator supplied with 10% CO.sub.2
and 120 rpm shaking, the culture media was isolated and subjected
to affinity purification using Protein A MABSELECT SURE.RTM.
agarose beads (GE Healthcare, Piscataway, N.J.). Purified protein
constructs were buffer-exchanged into 0.2M arginine.HCl, 25 mM
citric acid, 71.5 mM sodium hydroxide at pH 6.0 using a PD
Midi-Trap G-25 column (GE Healthcare, Piscataway, N.J.) or a HiPrep
26/10 Desalting column (HiTrap Desalting HiPrep 26/10 Desalting).
Purified protein constructs were then concentrated using 50 kDa
Amicon Ultra centrifugal filter devices (Millipore, Billerica,
Mass.), followed by protein concentration determination by reading
absorbance at 280 nm.
Method for Measuring Antigen-Targeted Activity of
Antibody-IFN.alpha.2b Fusion Protein Constructs
[0187] "On target (Daudi) assay": This assay was used to quantify
the anti-proliferative activity of IFN.alpha.2b and
antibody-IFN.alpha.2b fusion protein constructs on cells that
display both IFN receptor and the antigen targeted by the antibody
to which the IFN.alpha.2b is fused. Daudi cells express both CD20
and CD38 as cell surface associated antigens, as well as cell
surface IFN receptors. The viability of the Daudi cells was
measured using the reagent CELLTITER-GLO.RTM., Cat #G7570, from
Promega (Madison, Wis.). This is a luminescence-based assay that
determines the viability of cells in culture based on quantitation
of ATP. The signal strength is proportional to the number of viable
cells in a microtiter plate well. The details of the assay are as
follows:
[0188] Daudi cells (obtained from ATCC, Manassas, Va.) were
cultured in a T75 flask (TPP, Trasadingen, Switzerland, cat #90076)
to a preferred density of between 0.5.times.10.sup.5 and
0.8.times.10.sup.5 viable cells/ml in RPMI 1640 (Mediatech, Inc.,
Manassas, Va., cat #10-040-CV) with 10% Fetal Bovine Serum (FBS;
Hyclone, Logan, Utah cat # SH30070.03). Cells were harvested by
centrifuging at 400 g for five minutes, decanting the supernatant,
and resuspending the cell pellet in RPMI 1640+10% FBS. Cells were
then counted and the density was adjusted to 3.0.times.10.sup.5
cells/ml in RPMI 1640+10% FBS. Then, 50 .mu.l of the cell
suspension was aliquoted into each well of a 96 well round bottom
tissue culture plate (hereafter, "experimental plate") (TPP, cat
#92067). On a separate, sterile 96 well plate (hereafter, "dilution
plate"; Costar, Corning, N.Y. cat #3879), test articles were
serially diluted in duplicate in RPMI 1640+10% FBS. Then, 50
.mu.l/well was transferred from the dilution plate to the
experimental plate. The experimental plate was then incubated for
four days at 37.degree. C. with 5% CO.sub.2.
[0189] A mixture of the manufacturer-supplied assay buffer and
assay substrate (hereafter, "CELLTITER-GLO.RTM. reagent", mixed
according to the manufacturer's instructions) was added to the
experimental plate at 100 .mu.l/well. The plate was shaken for two
minutes. Then, 100 .mu.l/well was transferred from the experimental
plate to a 96 well flat bottom white opaque plate (hereafter,
"assay plate"; BD Biosciences, Franklin Lakes, N.J. cat #35 3296).
The content of the assay plate was then allowed to stabilize in the
dark for 15 minutes at room temperature. The plate was read on a
Victor 3V Multilabel Counter (Perkin Elmer, Waltham, Mass., model #
1420-041) on the luminometry channel and the luminescence was
measured. Results are presented as "relative luminescence units
(RLU)".
[0190] Data was analyzed using Prism 5 (Graphpad, San Diego,
Calif.) using non-linear regression and three parameter curve fit
to determine the midpoint of the curve (EC50). For each test
article, potency relative to free IFN.alpha.2b (or some other form
of IFN with a known potency relative to IFN.alpha.2b) was
calculated as a ratio of EC50s.
[0191] One of ordinary skill in the art will appreciate that there
are many other commonly used assays for measuring cell viability
that could also be used.
[0192] "On target (ARP) assay" (also sometimes referred to herein
as a "targeted assay"): The multiple myeloma cell line ARP-1 was a
gift from Bart Barlogie Md., PhD, Director of the Myeloma Institute
at the University of Arkansas Medical Center (Little Rock, Ak.). It
is described in Hardin J. et al. (Interleukin-6 prevents
dexamethasone-induced myeloma cell death. Blood; 84:3063, 1994).
ARP-1 cells (CD38.sup.+) were used to test CD38 targeting
antibody-IFN fusion protein constructs. Culture and assay
conditions were the same as for Daudi-based assay outlined above,
with the following exceptions: ARP-1 was cultured to a density of
4.0.times.10.sup.5 to 6.0.times.10.sup.5 cells/ml. ARP-1
concentration was adjusted to 1.0.times.10.sup.4 cells/ml prior to
assay.
Example 1
Isoelectric Points of Anti-CD38 Antibody Attenuated IFN.alpha.2b
Fusion Proteins
[0193] Various transiently transfected cells expressing an
anti-CD38 antibody-attenuated IFN.alpha.2b fusion constructs (Table
2) were harvested and purified using a MABSELECT SURE.RTM. Protein
A column. Samples were desalted into 200 mM Arginine, 25 mM
Histidine pH 6.5 using a HiLoad Superdex 200 column.
TABLE-US-00002 TABLE 2 Table of Constructs SEQ ID No. Heavy Light
VH + Antibody Constructs Chain Chain IFN IFN A10.21 IgG4 (S228P)
IFN (A145D, T106T) 48 81 107 A10.21 IgG4 (S228P) IFN (A145D, T106A)
48 81 25 87, 31 A10.21 IgG4 (S228P) IFN (A145D, .DELTA.T106) 48 81
110 61 A10.21 IgG1 IFN (A145D, T106T) 50 81 107 A10.21 IgG1 IFN
(A145D, T106A) 50 81 25 68 A10.21 IgG1 IFN (A145D, .DELTA.T106) 50
81 83 A10.43 IgG4 (S228P) IFN (A145D, T106T) 55 81 107 A10.43 IgG4
(S228P) IFN (A145D, T106A) 55 81 25 74 A10.21 IgG4 (S228P) IFN
(A145D, T106S) 48 81 25 31 A10.21 IgG4 (S228P) IFN (A145D, T106V)
48 81 25 31 A10.21 IgG4 (S228P) IFN (A145D, T106G) 48 81 25 31
A10.21 IgG4 (S228P) IFN (A145D, T106E) 48 81 25 31 A10.21 IgG4
(S228P, M252Y, S254T, 49 81 107 T256E) IFN (A145D, T106T) A10.21
IgG4 (S228P, M252Y, S254T, 49 81 25 62 T256E) IFN (A145D, T106A)
A10.21 IgG4 (S228P) IFN (R144I T106T) 48 81 108 A10.21 IgG4 (S228P)
IFN (R144I, T106A) 48 81 24 63 A10.21 IgG4 (S228P) IFN (A145K,
T106T) 48 81 107 A10.21 IgG4 (S228P) IFN (A145K, T106A) 48 81 25 64
A10.21 IgG4 (S228P) IFN (A145G, T106T) 48 81 107 A10.21 IgG4
(S228P) IFN (A145G, T106A) 48 81 25 64 A10.21 IgG4 (S228P) IFN
(R33A, T106T) 48 81 109 A10.21 IgG4 (S228P) IFN (R33A, T106A) 48 81
13 65 A10.21 IgG4 (S228P) IFN (A145Q, T106T) 48 81 107 A10.21 IgG4
(S228P) IFN (A145Q, T106A) 48 81 25 66 A10.21 IgG1 (L235A, G237A)
IFN (A145D, T106T) 51 81 107 A10.21 IgG1 (L235A, G237A) IFN (A145D,
T106A) 51 81 25 69 A10.21 IgG2 IFN (A145D, T106T) 53 81 107 A10.21
IgG2 IFN (A145D, T106A) 53 81 25 72 A10.21 IgG2 (A330S, P331S) IFN
(A145D, T106T) 81 107 A10.21 IgG2 (A330S, P331S) IFN (A145D, T106A)
81 25 A02.12 IgG4 (S228P) IFN (A145D, T106T) 58 84 107 A02.12 IgG4
(S228P) IFN (A145D, T106A) 58 84 25 77 Anti-CD138 IgG4 (S228P) IFN
(A145D, T106T) 59 85 107 Anti-CD138 IgG4 (S228P) IFN (A145D, T106A)
59 85 25 Anti-HLA IgG4 (S228P) IFN (A145D, T106T) 60 86 107
Anti-HLA IgG4 (S228P) IFN (A145D, T106A) 60 86 25
[0194] Isoelectric focusing gels were used to determine the
isoelectric point (pI) of the fusion polypeptide and to detect
minor changes in the protein due to post-translational
modifications such as phosphorylation and glycosylation.
[0195] Pre-cast IEF gel was setup in gel tanks ensuring a tight
seal between gel and buffer. Then 200 mL of 1.times. Cathode buffer
was poured into inner chamber ensuring no buffer enters the outer
chamber. 500 mL of 1.times. Anode buffer was then poured into the
outer chamber and filled 3/4 of the tank. After the samples and
ladder were loaded onto the gel, it was then run for 1 hour at 100
volt, 1 hour at 200 volt and 1/2 hour at 500 volt. As soon as gel
run was finished, the gel was taken out and fixed in TCA solution
in a glass container for 30 min. The gel was then immediately
washed with deionised water 3 times. The gel was stained in
SimplyBlue SafeStain (Invitrogen Life Technologies) for a full
hour, and left overnight in water to destain. The final image
scanned using a scanner.
[0196] The O-linked glycosylation site of the IFN.alpha.2b portion
of the antibody-attenuated interferon fusion constructs was removed
by either substituting the interferon's threonine 106 (T106) to
alanine (shown as T106A), serine (T106S), valine (T106V), glycine
(T106G) or glutamic acid (T106E) or by deleting T106 (shown as
.DELTA.T106). The effect of these changes on pI and the
heterogeneity of the fusion constructs was investigated by
comparing constructs with and without O-linked glycosylation by
separation on IEF gels.
[0197] In each case, deleting T106 or substituting T106 with
alanine, serine, valine, glycine or glutamic acid decreased the
number of observed charged species on an IEF gel, as evidenced by a
reduced number of bands when directly compared to unmodified T106,
and hence the heterogeneity of the fusion construct was reduced
(FIG. 9). The reduced number of charged species on the IEF gel and
therefore the reduced heterogeneity of the molecule incorporating
T106S is consistent with removal of the O-linked glycosylation at
residue 106 of IFN.alpha.2b.
[0198] Removal of the O-linked glycosylation site in the attenuated
IFN portion of the antibody fusion constructs resulted in an
increased pI relative to 0-link glycosylated proteins. Using the
same antibody front-end of A10.21, this trend was consistent
regardless of whether the isotype of the antibody was IgG4, IgG1,
IgG1 AA (IgG1 L235A, G237A, an effector function reduced form of
IgG1), IgG2 or IgG2 SS (IgG2 (A330S, P331S)) in the constructs
(FIG. 10).
[0199] YTE substitutions (M252Y, S254T, T256E) have been shown to
confer increased affinity to FcRn, presumably increasing the
half-life of antibodies. Further experiments examined whether
substitutions in other parts of the antibody IFN fused constructs
will affect the reduced heterogeneity which resulted from a T106
deletion or substitution. The heterogeneity of glycosylated A10.21
anti-CD38-attenuated IFN.alpha.2b fusion construct (YTE, T106T) and
the non-glycosylated IFN fusion construct with the YTE
substitutions (YTE, T106A) was assessed on IEF gels. Removal of
glycosylation of the IFN.alpha.2b component of fusion constructs
bearing the YTE mutations decreased heterogeneity (FIG. 11).
[0200] Attenuation of IFN.alpha.2b is attained by substitutions of
key amino acid residues which are responsible for binding to IFN
receptors. The number of charged species of A10.21 IgG4 (S228P) IFN
constructs with various attenuating amino acid substitutions in the
IFN.alpha.2b, together with (T106T) or without O-linked
glycosylation (T106A) of the IFN.alpha.2b component was evaluated.
Individual amino acid residues Arginine-33, Arginine-144 and
Alanine-145 of IFN.alpha.2b were substituted one at a time with
Alanine for residue 33 (R33A), Isoleucine for residue 144 (R144I)
or Lysine, Glycine or Glutamine for residue 145 (A145K, A145G,
A145Q). When directly compared the aglycosylated IFN fusion
constructs were consistently less heterogeneous than their
glycosylated counterparts (FIG. 12).
[0201] The reduction in heterogeneity exhibited was independent of
the antibody portion of the construct. Removal of the O-linked
glycosylation site in the attenuated IFN.alpha.2b portion of the
antibody (IgG4 (S228P))-attenuated IFN fusion constructs with
specificity against HLA, CD138 and CD38 (a different epitope on
CD38 to antibody A10.21-antibody A02.12) also resulted in a
decrease in heterogeneity (FIG. 13) as detected by IEF.
Example 2
Anti-Proliferative Activity of Antibody Attenuated
Interferon.alpha.2b Fusion Proteins
[0202] The anti-proliferative effects of IFN.alpha.2b consist of
direct and indirect activities. Direct activity occurs through
cancer cell growth inhibition by cell cycle arrest (Matsui et al.
2003), apoptosis by death receptor-dependent (Crowder et al. 2005)
and -independent (Otsuki et al. 1998) pathways, or differentiation
(Matsui et al. 2003). Target-specific direct cytotoxicity of
antibody attenuated interferon fusion proteins were measured
against target positive cell lines using a luminescent cell
viability assay.
Anti CD38-IFN Lead Sequence Samples
[0203] The constructs (Table 3) were either stably cloned or
transiently transfected and were harvested and purified using a
MABSELECT SURE.RTM. Protein A column. Samples were desalted into
200 mM Arginine, 25 mM Histidine pH 6.5 using a HiLoad Superdex 200
column.
TABLE-US-00003 TABLE 3 Table of Constructs Seq Id No. Heavy Light
VH + Antibody Constructs Chain Chain IFN IFN A10.21 IgG4 (S228P)
IFN (A145D, 48 81 107 T106T) A10.21 IgG4 (S228P) IFN (A145D, 48 81
25 87, 31 T106A) A10.21 IgG4 (S228P) IFN (A145D, 48 81 110 61
.DELTA.T106) A10.21 IgG4 (S228P) IFN (A145D, 48 81 25 31 T106S)
A10.21 IgG4 (S228P) IFN (A145D, 48 81 25 31 T106V) A10.21 IgG4
(S228P) IFN (A145D, 48 81 25 31 T106G) A10.21 IgG4 (S228P) IFN
(A145D, 48 81 25 31 T106E) A10.21 IgG4 (S228P, M252Y, S254T, 49 81
107 T256E) IFN (A145D, T106T) A10.21 IgG4 (S228P, M252Y, S254T, 49
81 25 62 T256E) IFN (A145D, T106A) A10.21 IgG4 (S228P) IFN (R144I
T106T) 48 81 108 A10.21 IgG4 (S228P) IFN (R144I, 48 81 24 63 T106A)
A10.21 IgG4 (S228P) IFN (A145K, 48 81 107 T106T) A10.21 IgG4
(S228P) IFN (A145K, 48 81 25 64 T106A) A10.21 IgG4 (S228P) IFN
(A145G, 48 81 107 T106T) A10.21 IgG4 (S228P) IFN (A145G, 48 81 25
64 T106A) A10.21 IgG4 (S228P) IFN (R33A, T106T) 48 81 109 A10.21
IgG4 (S228P) IFN (R33A, T106A) 48 81 13 65 A10.21 IgG4 (S228P) IFN
(A145Q, 48 81 107 T106T) A10.21 IgG4 (S228P) IFN (A145Q, 48 81 25
66 T106A) A10.21 IgG4 (S228P) IFN (A145N, 48 81 107 T106T) A10.21
IgG4 (S228P) IFN (A145N, 48 81 25 66 T106A) A10.21 IgG4 (S228P) IFN
(R144N, 48 81 108 T106T) A10.21 IgG4 (S228P) IFN (R144N, 48 81 24
67 T106A) A10.21 IgG4 (S228P) IFN (R144H, 48 81 108 T106T) A10.21
IgG4 (S228P) IFN (R144H, 48 81 24 67 T106A) A10.21 IgG1 IFN (A145D,
T106T) 50 81 107 A10.21 IgG1 IFN (A145D, T106A) 50 81 25 68 A10.21
IgG1 IFN (A145D, .DELTA.T106) 50 81 110 83 A10.21 IgG1 (L235A,
G237A) IFN 51 81 107 (A145D, T106T) A10.21 IgG1 (L235A, G237A) IFN
51 81 25 69 (A145D, T106A) A10.21 IgG1 (L235A, G237A, M252Y, 52 81
107 S254T, T256E) IFN (A145D, T106T) A10.21 IgG1 (L235A, G237A,
M252Y, 52 81 25 S254T, T256E) IFN (A145D, T106A) A10.21 IgG1
(M252Y, S254T, T256E) 52 81 107 IFN (A145D, T106T) A10.21 IgG1
(M252Y, S254T, T256E) 81 25 70 IFN (A145D, T106A) A10.21 IgG2 IFN
(A145D, T106T) 53 81 107 A10.21 IgG2 IFN (A145D, T106A) 53 81 25 72
A10.21 IgG2 (A330S, P331S) IFN (A145D, 81 107 T106T) A10.21 IgG2
(A330S, P331S) IFN (A145D, 81 25 T106A) A10.21 IgG2 (M252Y, S254T,
T256E) 54 81 107 IFN (A145D, T106T) A10.21 IgG2 (M252Y, S254T,
T256E) 54 81 25 73 IFN (A145D, T106A) A10.43 IgG4 (S228P) IFN
(A145D, 55 81 107 T106T) A10.43 IgG4 (S228P) IFN (A145D, 55 81 25
74 T106A) R10A2 IgG4 (S228P) IFN (A145D, 75 82 107 T106T) R10A2
IgG4 (S228P) IFN (A145D, 75 82 25 T106A) R10A2 IgG4 (S228P) IFN
(A145D, 75 82 25 T106R) R10A2 IgG4 (S228P) IFN (A145D, 75 82 25
T106N) R10A2 IgG4 (S228P) IFN (A145D, 75 82 25 T106D) R10A2 IgG4
(S228P) IFN (A145D, 75 82 25 T106C) R10A2 IgG4 (S228P) IFN (A145D,
75 82 25 T106E) R10A2 IgG4 (S228P) IFN (A145D, 75 82 25 T106Q)
R10A2 IgG4 (S228P) IFN (A145D, 75 82 25 T106G) R10A2 IgG4 (S228P)
IFN (A145D, 75 82 25 T106H) R10A2 IgG4 (S228P) IFN (A145D, 75 82 25
T106I) R10A2 IgG4 (S228P) IFN (A145D, 75 82 25 T106L) R10A2 IgG4
(S228P) IFN (A145D, 75 82 25 T106K) R10A2 IgG4 (S228P) IFN (A145D,
75 82 25 T106M) R10A2 IgG4 (S228P) IFN (A145D, 75 82 25 T106F)
R10A2 IgG4 (S228P) IFN (A145D, 75 82 25 T106P) R10A2 IgG4 (S228P)
IFN (A145D, 75 82 25 T106S) R10A2 IgG4 (S228P) IFN (A145D, 75 82 25
T106W) R10A2 IgG4 (S228P) IFN (A145D, 75 82 25 T106Y) R10A2 IgG4
(S228P) IFN (A145D, 75 82 25 T106V) A02.12 IgG4 (S228P) IFN (A145D,
58 84 107 T106T) A02.12 IgG4 (S228P) IFN (A145D, 58 84 25 77 T106A)
OPG-Fc (IgG2) IFN (A145D, T106T) 57 107 OPG-Fc (IgG2) IFN (A145D,
T106A) 57 25 78 Anti-CD138 IgG4 (S228P) IFN (A145D, 59 85 107
T106T) Anti-CD138 IgG4 (S228P) IFN (A145D, 59 85 25 T106A) Anti-HLA
IgG4 (S228P) IFN (A145D, 60 86 107 T106T) Anti-HLA IgG4 (S228P) IFN
(A145D, 60 86 25 T106A)
Commercial IFN.alpha.2b
[0204] INTRON.RTM. A, a commercial bacterially produced
IFN.alpha.2b from Schering-Plough, was used as a positive
control.
Anti-Proliferative Activity Measurement
[0205] Anti-proliferative activity was measured using the methods
described above "Daudi cell proliferation assay" and "ARP-1 Cell
proliferation assay". The ARP-1 cell proliferation assay method was
used with an additional cell line NCI-H929 for the measurement of
the anti-proliferative activity of antibody-attenuated IFN.alpha.2b
fusion protein. In some experiments the plates were read on a
dedicated GLOMAX.RTM. 96 Microplate Luminometer and
CELLTITER-GLO.RTM. 2.0 reagents were used instead of the original
CELLTITER-GLO.RTM., neither of which impacted the results. The
ARP-1/NCI-H929 or Daudi cell proliferation assay was used to
quantify the anti-proliferative activity of IFNs and
antibody-attentuated IFN.alpha.2b fusion protein constructs on
cells that display CD38. Daudi, ARP1 and NCI-H929 cells express
CD38 as a cell surface associated antigen. The details of the
assays are as follows.
[0206] In the ARP-1/NCI-H929 cell proliferation assay viability of
cells was measured using the reagent CELLTITER-GLO.RTM. 2.0, Cat #
G9242, from Promega (Madison, Wis.). This is a luminescence-based
assay that determines the viability of cells in culture based on
quantitation of ATP. The signal strength is proportional to the
number of viable cells in a microtiter plate well. Cells (NCI-H929
from ATCC, Manassas, Va. and ARP-1, a gift from Bart Barlogie Md.,
PhD, Director of the Myeloma Institute at the University of
Arkansas Medical Center; Little Rock, Ak.) were cultured in a T75
flask (TPP, Trasadingen, Switzerland, cat #90076) to a preferred
density of between 0.5.times.10.sup.5 and 0.8.times.10.sup.5 viable
cells/mL in RPMI 1640 (Mediatech, Inc., Manassas, Va., cat
#10-040-CV) with 10% Fetal Bovine Serum (FBS; Hyclone, Logan, Utah
cat # SH30070.03). Cells were harvested by centrifuging at
400.times.g for five minutes, decanting the supernatant, and
resuspending the cell pellet in RPMI 1640+10% FBS. Cells were then
counted and the density was adjusted to 3.0.times.10.sup.5 cells/mL
in RPMI 1640+10% FBS. 50 .mu.L of cell suspension was seeded into
each well of a 96 well round bottom tissue culture plate
(hereafter, "experimental plate") (TPP, cat #92067). Cells were
incubated at 4.degree. C. for 1 hour prior to the addition of test
compounds. On a separate, sterile 96 well plate (hereafter,
"dilution plate"; Costar, Corning, N.Y. cat #3879), test articles
were serially diluted in duplicate in RPMI 1640+10% FBS. 50
.mu.L/well was transferred from the dilution plate to the
experimental plate. The experimental plate was then incubated for
four days at 37.degree. C. with 5% CO.sub.2. A "CELLTITER-GLO.RTM.
reagent 2.0", was added to the experimental plate at 100
.mu.L/well. The plate was shaken for two minutes. 100 .mu.L/well
was transferred from the experimental plate to a 96 well flat
bottom white opaque plate (hereafter, "assay plate"; BD
Biosciences, Franklin 5 Lakes, N.J. cat #35 3296). The content of
the assay plate was then allowed to stabilize in the dark for 15
minutes at room temperature. The plate was read on GLOMAX.RTM. 96
Microplate Luminometer. Results are presented as "relative
luminescence units" (RLU).
[0207] Data was analyzed using Prism 5 (Graphpad, San Diego,
Calif.) using non-linear regression and four parameter curve fit to
determine the IC50.
[0208] The O-linked glycosylation site of anti-CD38 antibody
attenuated interferon fusion constructs were removed by either
substituting threonine 106 (T106) to alanine (T106A), serine
(T106S), valine (T106V), glycine (T106G) or glutamic acid (T106E)
or deleting T106 (shown as .DELTA.T106). The effect on cell
proliferation was investigated by comparing A10.21 anti-CD38
antibody fused to attenuated IFN.alpha.2b with and without O-linked
glycosylation. Removal of the O-linked glycosylation site in the
attenuated IFN portion of the antibody fusion constructs resulted
in increased anti-proliferative activity as shown by lower IC50
(nM) relative to the corresponding O-linked glycosylated fusion
proteins i.e. A10.21 IgG4 (S228P) IFN (A145D, T106T) and A10.21
IgG1 IFN (A145D, T106T) in both ARP1 (FIG. 1A) and NCI-H929 cells
(FIG. 1B). This trend was consistent regardless of whether the
antibody isotype was IgG4 and IgG1.
[0209] Substitution of threonine 106 (T106) to alanine (T106A),
serine (T106S), valine (T106V), glycine (T106G) or glutamic acid
(T106E) resulted in increased anti-proliferative activity as shown
by lower IC50 (nM) relative to O-linked glycosylated fusion
proteins. All non-glycosylated constructs showed higher "on-target"
potency relative to their glycosylated counterpart in NCI-H929
cells (FIG. 14).
[0210] The impact on anti-proliferative activity of the removal of
the O-linked glycosylation site from the attenuated IFN.alpha.2b
component was examined in a range of anti-CD38 antibody-attenuated
IFN.alpha.2b fusion proteins. Variants of the anti-CD38
antibody-attenuated IFN.alpha.2b fusion protein R10A2 IgG4 (S228P)
IFN (A145D) possessing different amino acid substitutions at T106
to remove the O-linked glycosylation site on the IFN component
(T106A, T106G, T106N, T106F, T106R, T106D, T106E, T106Q, T106H,
T1061, T106L, T106K, T106M, T106F, T106P, T106S, T106V, T106Y and
T106W, (Table 4) were examined and the results shown in FIG. 2.
[0211] While the level of potency varied between different
substitutions at the T106 site, all substitutions that are
predicted to result in the removal of O-linked glycosylation
increased potency relative to the corresponding O-linked
glycosylated protein (FIG. 2).
TABLE-US-00004 TABLE 4 R10A2 Anti-CD38 antibody- attenuated IFN
fusion construct variants with various amino acid substitutions at
T106 for removal of O-linked glycosylation. Seq Id No. Heavy Light
Construct Name Chain Chain IFN R10A2 IgG4 (S228P) IFN (A145D,
T106T) 75 82 107 R10A2 IgG4 (S228P) IFN (A145D, T106A) 75 82 25
R10A2 IgG4 (S228P) IFN (A145D, T106R) 75 82 25 R10A2 IgG4 (S228P)
IFN (A145D, T106N) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106D)
75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106C) 75 82 25 R10A2 IgG4
(S228P) IFN (A145D, T106E) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D,
T106Q) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106G) 75 82 25
R10A2 IgG4 (S228P) IFN (A145D, T106H) 75 82 25 R10A2 IgG4 (S228P)
IFN (A145D, T106I) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106L)
75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106K) 75 82 25 R10A2 IgG4
(S228P) IFN (A145D, T106M) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D,
T106F) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106P) 75 82 25
R10A2 IgG4 (S228P) IFN (A145D, T106S) 75 82 25 R10A2 IgG4 (S228P)
IFN (A145D, T106W) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106Y)
75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106V) 75 82 25
[0212] The relative changes in the potency between two different
anti-CD38 antibody interferon fusion proteins (A02.12 and A10.21)
which bind different epitopes on CD38, with or without the O-linked
glycosylation (A02.12 IgG4 (S228P) IFN (A145D, T106T), A02.12 IgG4
(S228P) IFN (A145D, T106A), A10.21 IgG4 (S228P) IFN (A145D, T106T)
and A10.21 IgG4 (S228P) IFN (A145D, T106A) was assessed in a
similar manner. Removal of the O-linked glycosylation site in the
attenuated IFN portion of the antibody (IgG4 (S228P)) fusion
constructs with differing specificity against CD38 resulted in an
increase in anti-proliferative activity regardless of the target of
the antibody portion of the construct (FIG. 15).
[0213] Attenuation of IFN.alpha.2b is achieved by various
attenuating substitutions. The anti-proliferative activity of
A10.21 IgG4 (S228P) IFN constructs with various attenuating
substitutions in the IFN.alpha.2b with (T106T) and without O-linked
glycosylation (T106A) (Table 5) was also evaluated. The removal of
the O-linked glycosylation site in the IFN.alpha.2b component of
constructs with various attenuating substitutions all had higher
potency than their glycosylated counterpart (FIG. 16).
TABLE-US-00005 TABLE 5 A10.21 IgG4 (S228P) IFN constructs with
various attenuating substitutions in the IFN.alpha.2b with (T106T)
and without O-linked glycosylation (T106A). Seq Id No. Heavy Light
VH + Construct Name Chain Chain IFN IFN A10.21 IgG4 (S228P) IFN
(R144I T106T) 48 81 108 A10.21 IgG4 (S228P) IFN (R144I, T106A) 48
81 24 63 A10.21 IgG4 (S228P) IFN (A145K, T106T) 48 81 107 A10.21
IgG4 (S228P) IFN (A145K, T106A) 48 81 25 64 A10.21 IgG4 (S228P) IFN
(A145G, T106T) 48 81 107 A10.21 IgG4 (S228P) IFN (A145G, T106A) 48
81 25 64 A10.21 IgG4 (S228P) IFN (R33A, T106T) 48 81 109 A10.21
IgG4 (S228P) IFN (R33A, T106A) 48 81 13 65 A10.21 IgG4 (S228P) IFN
(A145Q, T106T) 48 81 107 A10.21 IgG4 (S228P) IFN (A145Q, T106A) 48
81 25 66 A10.21 IgG4 (S228P) IFN (A145N, T106T) 48 81 107 A10.21
IgG4 (S228P) IFN (A145N, T106A) 48 81 25 66 A10.21 IgG4 (S228P) IFN
(R144N, T106T) 48 81 108 A10.21 IgG4 (S228P) IFN (R144N, T106A) 48
81 24 67 A10.21 IgG4 (S228P) IFN (R144H, T106T) 48 81 108 A10.21
IgG4 (S228P) IFN (R144H, T106A) 48 81 24 67
[0214] To investigate whether other attenuated IFN.alpha.2b fused
antibodies/proteins which have different binding targets have
modulated anti-proliferative activity as a result of removal of
O-linked glycosylation, 2 constructs were created (an anti-CD138
antibody and an anti-HLA antibody and fused to IFN (A145D) with or
without O-linked glycosylation (Table 6) and were tested for
anti-proliferative activity.
[0215] Removal of the O-linked glycosylation site in the attenuated
IFN portion of the antibody (IgG4 (S228P)) fusion constructs with
specificity against HLA and CD138 resulted in an increase in
anti-proliferative activity regardless of the target of the
antibody portion of the construct. This was demonstrated in
antibody fusion constructs against HLA and CD138 (FIG. 17).
TABLE-US-00006 TABLE 6 Constructs of various IFN fusion proteins
-anti- CD138 antibody and anti-HLA antibody fused to IFN (A145D)
with or without O-linked glycosylation Seq Id No. Heavy Light VH +
Antibody Constructs Chain Chain IFN IFN Anti-CD138 IgG4 (S228P) IFN
(A145D, 59 85 107 59 T106T) Anti-CD138 IgG4 (S228P) IFN (A145D, 59
85 25 59 T106A) Anti-HLA IgG4 (S228P) IFN (A145D, 60 86 107 T106T)
Anti-HLA IgG4 (S228P) IFN (A145D, 60 86 25 T106A)
Effect on Anti Proliferative Activity of the Removal of O-Linked
Glycosylation from Fusion Proteins Comprising Substitutions in the
Fc Regions for Half-Life Extension or Reduced Effector Function
[0216] YTE substitutions have been shown to confer increased
affinity to FcRn, presumably increasing the half-life of
antibodies. IFN fused antibodies containing the YTE substitutions
were tested for anti-proliferative activity in the presence and
absence of O-linked glycosylation. The following variants were made
of the A10.21 anti-CD38 antibody attenuated IFN fusion protein
(Table 7).
[0217] The introduction of the YTE substitution to A10.21 IgG4
(S228P) (A145D, T106A) did not impact the increase in potency
caused by removal of the O-linked glycosylation from the attenuated
IFN (FIG. 18).
[0218] L235A and G237A substitutions in the Fc portion of IgG1
variants and A330S and P331S substitutions in the Fc portion of
IgG2 variants results in reduced effector function. Attenuated IFN
fused IgG1 antibodies containing L235A and G237A substitutions and
attenuated IFN fused IgG2 antibodies containing A330S and P331S
substitutions were tested for anti-proliferative activity in the
presence and absence of O-linked glycosylation. These variants were
made of the A10.21 anti-CD38 antibody attenuated IFN fusion protein
(Table 7). Removal of the O-linked glycosylation site in the
attenuated IFN portion of the antibody fusion constructs resulted
in an increase in potency regardless of whether the isotype of the
antibody in the antibody constructs was IgG1 or IgG1 AA (IgG1
(L235A, G237A)), IgG2 or IgG2 SS (IgG2 (A330S, P331S)) (FIG.
19).
TABLE-US-00007 TABLE 7 A10.21 fusion proteins composed of
substitutions in the Fc regions for half-life extension or reduced
effector function Seq Id No. Heavy Light VH + Antibody Constructs
Chain Chain IFN IFN A10.21 IgG4 (S228P, M252Y, S254T, T256E) 49 81
107 IFN (A145D, T106T) A10.21 IgG4 (S228P, M252Y, S254T, T256E) 49
81 25 62 IFN (A145D, T106A) A10.21 IgG1 (L235A, G237A) IFN (A145D,
51 81 107 T106T) A10.21 IgG1 (L235A, G237A) IFN (A145D, 51 81 25 69
T106A) A10.21 IgG1 (L235A, G237A, M252Y, S254T, 52 81 107 T256E)
IFN (A145D, T106T) A10.21 IgG1 (L235A, G237A, M252Y, S254T, 52 81
25 T256E) IFN (A145D, T106A) A10.21 IgG1 (M252Y, S254T, T256E) IFN
52 81 107 (A145D, T106T) A10.21 IgG1 (M252Y, S254T, T256E) IFN 111
81 25 70 (A145D, T106A) A10.21 IgG2 IFN (A145D, T106T) 53 81 107
A10.21 IgG2 IFN (A145D, T106A) 53 81 25 72 A10.21 IgG2 (A330S,
P331S) IFN (A145D, T106T) 112 81 107 A10.21 IgG2 (A330S, P331S) IFN
(A145D, T106A) 112 81 25 A10.21 IgG2 (M252Y, S254T, T256E) IFN 54
81 107 (A145D, T106T) A10.21 IgG2 (M252Y, S254T, T256E) IFN 54 81
25 73 (A145D, T106A)
[0219] Removal of the O-linked glycosylation site from the
attenuated interferon portion of the anti-CD38 antibody interferon
fusion proteins by amino acid substitution or deletion of T106
resulted in an increase in anti-proliferative activity on target
CD38, CD138 or HLA positive cells (1.3-12 fold).
Example 3
On/Off Target Activity of Antibody-Attenuated IFN.alpha.2b Fusion
Proteins
[0220] The iLite.TM. reporter gene assay was performed for the
quantitative determination of Human Interferon Alpha (IFN.alpha.2b)
bioactivity (IU/ml) using luciferase generated-bioluminescence. The
cells used in this assay express CD38 and were used to measure the
`ON-target` activity of anti-CD38-attenuated IFN.alpha.2b fusion
proteins. These cells can also be used to measure `OFF-target`
activity when CD38 is blocked with an anti-CD38 antibody
recognizing the same epitope. These assays can be used to determine
the Selectivity Index (SI) which is a measure of how selectively
active anti-CD38 IFN fusion proteins are against CD38+ target cells
and non-active on cells where CD38 is blocked (mimicking CD38.sup.-
cells). The larger the SI the more selective the agents are against
the target, while a number close to 1 indicates that there is no
selectivity against the target or non-target. Intron A was used as
a positive control as it is active against cells that express
interferon alpha receptors, IFNAR1/2 but not selective against
other cell surface expressed antigens (i.e. CD38) having an SI of
approximately 1.
Antibody-Attenuated IFN Constructs
[0221] The sequences for the constructs used are set out in
Sequence Listing and are listed in the following Table 8.
TABLE-US-00008 TABLE 8 Seq Id No. Heavy Light VH + Antibody
Constructs Chain Chain IFN IFN A10.21 IgG4 (S228P) IFN (A145D,
T106T) 48 81 107 A10.21 IgG4 (S228P) IFN (A145D, T106A) 48 81 25
87, 31 A10.21 IgG4 (S228P) IFN (A145D, .DELTA.T106) 48 81 110 61
A10.21 IgG4 (S228P) IFN (A145D, T106S) 48 81 25 31 A10.21 IgG4
(S228P) IFN (A145D, T106V) 48 81 25 31 A10.21 IgG4 (S228P) IFN
(A145D, T106G) 48 81 25 31 A10.21 IgG4 (S228P) IFN (A145D, T106E)
48 81 25 31 A10.21 IgG4 (S228P, M252Y, S254T, T256E) 49 81 107 IFN
(A145D, T106T) A10.21 IgG4 (S228P, M252Y, S254T, T256E) 49 81 25 62
IFN (A145D, T106A) A10.21 IgG4 (S228P) IFN (R144I T106T) 48 81 108
A10.21 IgG4 (S228P) IFN (R144I, T106A) 48 81 24 63 A10.21 IgG4
(S228P) IFN (A145K, T106T) 48 81 107 A10.21 IgG4 (S228P) IFN
(A145K, T106A) 48 81 25 64 A10.21 IgG4 (S228P) IFN (A145G, T106T)
48 81 107 A10.21 IgG4 (S228P) IFN (A145G, T106A) 48 81 25 64 A10.21
IgG4 (S228P) IFN (R33A, T106T) 48 81 109 A10.21 IgG4 (S228P) IFN
(R33A, T106A) 48 81 13 65 A10.21 IgG4 (S228P) IFN (A145Q, T106T) 48
81 107 A10.21 IgG4 (S228P) IFN (A145Q, T106A) 48 81 25 66 A10.21
IgG4 (S228P) IFN (A145N, T106T) 48 81 107 A10.21 IgG4 (S228P) IFN
(A145N, T106A) 48 81 25 66 A10.21 IgG4 (S228P) IFN (R144N, T106T)
48 81 108 A10.21 IgG4 (S228P) IFN (R144N, T106A) 48 81 24 67 A10.21
IgG4 (S228P) IFN (R144H, T106T) 48 81 108 A10.21 IgG4 (S228P) IFN
(R144H, T106A) 48 81 24 67 A10.21 IgG1 IFN (A145D, T106T) 50 81 107
A10.21 IgG1 IFN (A145D, T106A) 50 81 25 68 A10.21 IgG1 IFN (A145D,
.DELTA.T106) 50 81 110 83 A10.21 IgG1 (L235A, G237A) IFN (A145D, 51
81 107 T106T) A10.21 IgG1 (L235A, G237A) IFN (A145D, 51 81 25 69
T106A) A10.21 IgG1 (L235A, G237A, M252Y, 52 81 107 S254T, T256E)
IFN (A145D, T106T) A10.21 IgG1 (L235A, G237A, M252Y, 52 81 25
S254T, T256E) IFN (A145D, T106A) A10.21 IgG1 (M252Y, S254T, T256E)
IFN 52 81 107 (A145D, T106T) A10.21 IgG1 (M252Y, S254T, T256E) IFN
111 81 25 70 (A145D, T106A) A10.21 IgG2 IFN (A145D, T106T) 53 81
107 A10.21 IgG2 IFN (A145D, T106A) 53 81 25 72 A10.21 IgG2 (M252Y,
S254T, T256E) IFN 54 81 107 (A145D, T106T) A10.21 IgG2 (M252Y,
S254T, T256E) IFN 54 81 25 73 (A145D, T106A) A10.43 IgG4 (S228P)
IFN (A145D, T106T) 55 81 107 A10.43 IgG4 (S228P) IFN (A145D, T106A)
55 81 25 74 R10A2 IgG4 (S228P) IFN (A145D, T106T) 75 82 107 R10A2
IgG4 (S228P) IFN (A145D, T106A) 75 82 25 R10A2 IgG4 (S228P) IFN
(A145D, T106R) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106N) 75 82
25 R10A2 IgG4 (S228P) IFN (A145D, T106D) 75 82 25 R10A2 IgG4
(S228P) IFN (A145D, T106C) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D,
T106E) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106Q) 75 82 25
R10A2 IgG4 (S228P) IFN (A145D, T106G) 75 82 25 R10A2 IgG4 (S228P)
IFN (A145D, T106H) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106I)
75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106L) 75 82 25 R10A2 IgG4
(S228P) IFN (A145D, T106K) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D,
T106M) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106F) 75 82 25
R10A2 IgG4 (S228P) IFN (A145D, T106P) 75 82 25 R10A2 IgG4 (S228P)
IFN (A145D, T106S) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106W)
75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106Y) 75 82 25 R10A2 IgG4
(S228P) IFN (A145D, T106V) 75 82 25 A02.12 IgG4 (S228P) IFN (A145D,
T106T) 58 84 107 A02.12 IgG4 (S228P) IFN (A145D, T106A) 58 84 25 77
OPG-Fc (IgG2) IFN (A145D, T106T) 57 107 OPG-Fc (IgG2) IFN (A145D,
T106A) 57 25 78 Anti-CD138 IgG4 (S228P) IFN (A145D, 59 85 107
T106T) Anti-CD138 IgG4 (S228P) IFN (A145D, 59 85 25 T106A) Anti-HLA
IgG4 (S228P) IFN (A145D, T106T) 60 86 107 Anti-HLA IgG4 (S228P) IFN
(A145D, 60 86 25 T106A)
[0222] INTRON.RTM. A was used as a positive control.
ON/OFF-Target Activity Measurement
[0223] The ON/OFF-target activity was measured using the same
method described above as "iLite gene reporter assay"
[0224] iLite reporter gene assay (PBL Interferon Source,
Piscataway, N.J., Cat #51100) was performed largely as described by
the manufacturer, with the addition of a human IgG blocking step.
The iLite cell line is described by the manufacturer as "a stable
transfected cell line derived from a commercially available
pro-monocytic human cell line characterized by the expression of
MHC Class II antigens, in particular the human lymphocyte antigen
(HLADR), on the cell surface." The cell line expresses CD38 and
contains a stably transfected luciferase gene, the expression of
which is driven by an interferon-response element (IRE), which
allows for interferon activity to be quantified based on
luminescence output.
[0225] The manufacturer supplied iLite plate (hereafter, assay
plate) and diluent were removed from the -80.degree. C. freezer and
allowed to equilibrate to room temperature. 50 .mu.L of the diluent
was added per well to the assay plate. The vial of
manufacturer-supplied reporter cells was removed from the
-80.degree. C. freezer and thawed in a 37.degree. C. water bath. 25
.mu.L aliquots of cells were dispensed into each well of the assay
plate. Next, 12.5 .mu.L of 8 mg/mL human IgG diluted in RPMI
1640+10% FBS (Sigma Chemicals, St. Louis, Mo.; cat #14506) was
added per well. The contents were mixed and incubated at 37.degree.
C. for 15 minutes. On a separate "dilution plate," test articles
were serially diluted in duplicate in RPMI 1640+10% FBS. Then, 12.5
.mu.L of the test articles were transferred from the dilution plate
to the assay plate. The assay plate was then incubated at
37.degree. C. with 5% CO.sub.2 for 17 hours. The
manufacturer-supplied assay buffer and substrate were removed from
the -80.degree. C. freezer and allowed to equilibrate to room
temperature for two hours. The manufacturer-supplied assay buffer
was added to the manufacturer-supplied substrate vial and mixed
well according to the manufacturer's instructions to create the
"luminescence solution." Then, 100 .mu.L of the luminescence
solution was added to each well of the assay plate. The plate was
shaken for 2 minutes. The plate was then incubated at room
temperature for 5 minutes in the dark and read on a Victor 3V
Multilabel Counter on a luminometry channel and the luminescence
measured and presented as RLU.
[0226] To test the off-target activity of anti-CD38 antibody-IFN
fusion protein constructs in the iLite assay, manufacturer-supplied
diluent was supplemented with 0.25 mg/mL anti-CD38 antibody (an
antibody recognizing the same epitope on CD38 as the antibody-IFN
fusion protein construct being tested, to block any binding of the
anti-CD38 antibody-IFN fusion protein constructs to the CD38
expressed on the iLite cells). This blocking stage was followed by
treatment with anti-CD38 antibody-IFN fusion protein or
IFN.alpha.2b.
[0227] Data was analyzed using Prism 5 (Graphpad, San Diego,
Calif.) using non-linear regression and three parameter curve fit
to determine the midpoint of the curve (EC50). Selectivity Index
(SI) was calculated by EC50 (OFF-target activity)/EC50 (ON-target
activity). Selectivity Index (SI) is a measure of how selectively
active anti-CD38 IFN constructs are against CD38-expressing cells
and non-active in cells with no CD38. The larger the number the
more selective it is against the target, while a number close to 1
indicates that there is no selectivity against the target. Intron A
was used as a positive control, has an SI of approximately 1.
[0228] The O-linked glycosylation site of anti-CD38 antibody
attenuated interferon fusion constructs were removed by
substituting threonine 106 (T106) to alanine (shown as T106A). The
activity was investigated by comparing A10.21 and A10.43 anti-CD38
antibodies fused to IFN.alpha.2b with and without O-linked
glycosylation.
[0229] Removal of the O-linked glycosylation site in the attenuated
IFN portion of the antibody fusion constructs (shown as T106A)
resulted in slight increase in ON-target activity (FIG. 3 and Table
9) as well as slight to no increase in the OFF-target activity
relative to 0-linked glycosylated proteins. Both O-linked
glycosylated and non-glycosylated proteins showed high selectivity
towards CD38+ cells, while Intron A did not show selectivity.
TABLE-US-00009 TABLE 9 EC50 and SI of anti-CD38 antibody attenuated
IFN.alpha.2b fusion proteins A10.21 and A10.43 with and without
O-linked glycosylation EC50 (nM) ON OFF Test Article Target Target
SI Intron A 0.00017 0.00016 0.90 A10.21 IgG4 (S228P) IFN(A145D,
T106T) 0.00240 2.33 972 A10.21 IgG4 (S228P) IFN (A145D, T106A)
0.00182 2.23 1223 A10.43 IgG4 (S228P) IFN (A145D, T106T) 0.00560
2.31 412 A10.43 IgG4 (S228P) IFN (A145D, T106A) 0.00124 2.25
1822
[0230] The impact on ON/OFF target activity of the removal of the
O-linked glycosylation site from the attenuated IFN of fusion
proteins may also be examined in a similar manner.
[0231] The iLite.TM. reporter gene ON-OFF target activity assays
were conducted to demonstrate the selective activity of anti-CD38
attenuated IFN fusion proteins on target CD38+ cells and the
limited activity on cells when CD38 is blocked with an anti-CD38
antibody (recognizing the same epitope) mimicking the activity
anticipated on target negative cells.
[0232] Removal of the O-linked glycosylation site in the attenuated
IFN portion of the antibody fusion constructs (by T106A
substitution) resulted in a slight increase in ON-target activity
as well as slight to no increase in the OFF-target activity
relative to O-linked glycosylated proteins. Both O-linked
glycosylated and non-glycosylated anti-CD38 antibody attenuated
interferon fusion proteins showed high selectivity towards CD38+
cells, while Intron A did not show selectivity.
[0233] To further examine the modulation in selectivity index as a
result of the removal of the O-linked glycosylation site in the
attenuated IFN portion of the antibody fusion constructs, ON-OFF
target activity was examined in a range of constructs. The changes
examined were based on the A10.21 IgG4 (S228P) IFN (A145D, T106T)
construct. These included deletion of threonine 106 (.DELTA.T106),
substitution of T106 to serine (T106S), YTE substitution (YTE T106T
and YTE T106A), IFN attenuation by substitution from alanine 145 to
glutamine (A145Q) and varying antibody isotype to IgG1 (IgG1 T106T
and IgG1 T106A).
[0234] The majority of constructs demonstrated a selectivity
index>1, the magnitude of selectivity towards targeted CD38+
cells varied depending on the anti-CD38 IFN fused construct (FIG.
20). The highest selectivity was observed in A10.21 IgG4 (S228P)
IFN (A145D, T106A).
Example 4
Off-Target Activity of Anti-CD38 Antibody-Attenuated IFN.alpha.2b
Fusion Proteins
[0235] HEK-Blue.TM. IFN-.alpha./.beta. cells allow the detection of
bioactive human type I IFNs by monitoring the activation of the
ISGF3 pathway. Stimulation of HEK-Blue.TM. IFN-.alpha./.beta. cells
with human IFN-.alpha. activates the JAK/STAT/ISGF3 pathway and
subsequently induces the production of SEAP (a reporter gene under
the control of the IFN-.alpha./.beta. inducible ISG54 promoter).
Levels of SEAP in the supernatant can be easily determined with
QUANTI-Blue.TM.. The effects of removal of O-linked glycosylation
from the attenuated interferon portion of the antibody interferon
fusion proteins were evaluated using this reporter gene assay.
Antibody/Fc Fusion Protein Construct Production
[0236] Various transiently transfected cells expressing interferon
fusion proteins were harvested and purified using a MABSELECT
SURE.RTM. Protein A column. Samples were desalted into 200 mM
Arginine, 25 mM Histidine pH 6.5 using a HiLoad Superdex 200
column.
TABLE-US-00010 TABLE 10 Seq Id No. Heavy Light VH + Antibody
Constructs Chain Chain IFN IFN A10.21 IgG4 (S228P) IFN (A145D,
T106T) 48 81 107 A10.21 IgG4 (S228P) IFN (A145D, T106A) 48 81 25
87, 31 A10.21 IgG4 (S228P) IFN (A145D, .DELTA.T106) 48 81 110 61
A10.21 IgG4 (S228P) IFN (A145D, T106S) 48 81 25 31 A10.21 IgG4
(S228P) IFN (A145D, T106V) 48 81 25 31 A10.21 IgG4 (S228P) IFN
(A145D, T106G) 48 81 25 31 A10.21 IgG4 (S228P) IFN (A145D, T106E)
48 81 25 31 A10.21 IgG4 (S228P, M252Y, S254T, T256E) 49 81 107 IFN
(A145D, T106T) A10.21 IgG4 (S228P, M252Y, S254T, T256E) 49 81 25 62
IFN (A145D, T106A) A10.21 IgG4 (S228P) IFN (R144I T106T) 48 81 108
A10.21 IgG4 (S228P) IFN (R144I, T106A) 48 81 24 63 A10.21 IgG4
(S228P) IFN (A145K, T106T) 48 81 107 A10.21 IgG4 (S228P) IFN
(A145K, T106A) 48 81 25 64 A10.21 IgG4 (S228P) IFN (A145G, T106T)
48 81 107 A10.21 IgG4 (S228P) IFN (A145G, T106A) 48 81 25 64 A10.21
IgG4 (S228P) IFN (R33A, T106T) 48 81 109 A10.21 IgG4 (S228P) IFN
(R33A, T106A) 48 81 13 65 A10.21 IgG4 (S228P) IFN (A145Q, T106T) 48
81 107 A10.21 IgG4 (S228P) IFN (A145Q, T106A) 48 81 25 66 A10.21
IgG4 (S228P) IFN (A145N, T106T) 48 81 107 A10.21 IgG4 (S228P) IFN
(A145N, T106A) 48 81 25 66 A10.21 IgG4 (S228P) IFN (R144N, T106T)
48 81 108 A10.21 IgG4 (S228P) IFN (R144N, T106A) 48 81 24 67 A10.21
IgG4 (S228P) IFN (R144H, T106T) 48 81 108 A10.21 IgG4 (S228P) IFN
(R144H, T106A) 48 81 24 67 A10.21 IgG1 IFN (A145D, T106T) 50 81 107
A10.21 IgG1 IFN (A145D, T106A) 50 81 25 68 A10.21 IgG1 IFN (A145D,
.DELTA.T106) 50 81 110 83 A10.21 IgG1 (L235A, G237A) IFN (A145D, 51
81 107 T106T) A10.21 IgG1 (L235A, G237A) IFN (A145D, 51 81 25 69
T106A) A10.21 IgG1 (L235A, G237A, M252Y, S254T, 52 81 107 T256E)
IFN (A145D, T106T) A10.21 IgG1 (L235A, G237A, M252Y, S254T, 52 81
25 T256E) IFN (A145D, T106A) A10.21 IgG1 (M252Y, S254T, T256E) IFN
52 81 107 (A145D, T106T) A10.21 IgG1 (M252Y, S254T, T256E) IFN 81
25 70 (A145D, T106A) A10.21 IgG2 IFN (A145D, T106T) 53 81 107
A10.21 IgG2 IFN (A145D, T106A) 53 81 25 72 A10.21 IgG2 (M252Y,
S254T, T256E) IFN 54 81 107 (A145D, T106T) A10.21 IgG2 (M252Y,
S254T, T256E) IFN 54 81 25 73 (A145D, T106A) A10.43 IgG4 (S228P)
IFN (A145D, T106T) 55 81 107 A10.43 IgG4 (S228P) IFN (A145D, T106A)
55 81 25 74 R10A2 IgG4 (S228P) IFN (A145D, T106T) 75 82 107 R10A2
IgG4 (S228P) IFN (A145D, T106A) 75 82 25 R10A2 IgG4 (S228P) IFN
(A145D, T106R) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106N) 75 82
25 R10A2 IgG4 (S228P) IFN (A145D, T106D) 75 82 25 R10A2 IgG4
(S228P) IFN (A145D, T106C) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D,
T106E) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106Q) 75 82 25
R10A2 IgG4 (S228P) IFN (A145D, T106G) 75 82 25 R10A2 IgG4 (S228P)
IFN (A145D, T106H) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106I)
75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106L) 75 82 25 R10A2 IgG4
(S228P) IFN (A145D, T106K) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D,
T106M) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106F) 75 82 25
R10A2 IgG4 (S228P) IFN (A145D, T106P) 75 82 25 R10A2 IgG4 (S228P)
IFN (A145D, T106S) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106W)
75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106Y) 75 82 25 R10A2 IgG4
(S228P) IFN (A145D, T106V) 75 82 25 A02.12 IgG4 (S228P) IFN (A145D,
T106T) 58 84 107 A02.12 IgG4 (S228P) IFN (A145D, T106A) 58 84 25 77
OPG-Fc (IgG2) IFN (A145D, T106T) 57 107 OPG-Fc (IgG2) IFN (A145D,
T106A) 57 25 78 Anti-CD138 IgG4 (S228P) IFN (A145D, T106T) 59 85
107 Anti-CD138 IgG4 (S228P) IFN (A145D, T106A) 59 85 25 Anti-HLA
IgG4 (S228P) IFN (A145D, T106T) 60 86 107 Anti-HLA IgG4 (S228P) IFN
(A145D, T106A) 60 86 25
[0237] INTRON.RTM. A was used as a positive control.
Off-Target Activity Measurement by HEK-Blue Assay
[0238] Off-target activity of antibody-IFN fusion was measured
using the same method as described in "HEK-Blue Off-target assay"
except for the volumes of cells seeded, diluted antibodies and
supernatant used. The details of the assay are as follows.
[0239] HEK-Blue Off-target assay was used to quantify the ability
of antibody-IFN fusion constructs to bind interferon-alpha/0
receptor (IFNAR) using the HEK-Blue.TM. IFN-alpha/0 cell line
(InvivoGen, San Diego, Calif.). The "off-target (HB-IFN) assay" was
performed largely as described by the manufacturer of the HEK-Blue
IFN-alpha/.beta. cell line. HEK-Blue.TM. IFN-alpha/.beta. Cells are
specifically designed to monitor the activation of the JAK-STAT
pathway, which is induced by type I IFNs. The cells were generated
by introducing the human STAT2 and IRF9 genes into HEK293 cells to
obtain a fully active type I IFN signaling pathway. The
HEK-Blue.TM. IFN-alpha/.beta. cells stably express a reporter gene,
secreted embryonic alkaline phosphatase (SEAP), under the control
of the ISG54 promoter. ISG54 is a well-known ISG activated through
an ISRE-dependent mechanism by type I IFNs. Upon IFN-alpha or
IFN.beta. stimulation, HEK-Blue.TM. IFN-alpha/.beta. cells activate
the JAK-STAT pathway and then the expression of the SEAP reporter
gene. SEAP is secreted into the media and can be quantitated using
the colorimetric reagent QUANTI-Blue.TM..
[0240] Briefly, HEK-Blue IFN-alpha/.beta. cells (Invivogen, San
Diego Calif. cat # hkb-ifnab) were thawed and cultured in DMEM
media (Mediatech, Manassas Va., cat #10-013-CV)+10% FBS (Hyclone,
Logan Utah, cat # SH30070.03) that had been heat inactivated (HI
FBS). When the cells reached 60-80% confluence, they were lifted
with Cell Stripper (Mediatech, cat #25-056-Cl). Cells were washed
twice in DMEM+HI FBS and counted. Cells were adjusted to
2.77.times.10.sup.5 viable cells/mL in DMEM+HI FBS and was seeded
at 180 .mu.L per well into a flat bottom 96 well tissue culture
plate (hereafter, the "experimental plate"). Then, 20 .mu.L of
IFN-alpha2b or fusion protein construct, diluted into DMEM+HI FBS,
was added per well. The plate was incubated at 37.degree. C. 5%
CO.sub.2 for 16-24 hours. QUANTI-Blue (Invivogen, cat # rep-qb1)
was prepared according to the manufacturer's directions.
QUANTI-Blue (180 .mu.L) was seeded into each well of a flat bottom
plate (hereafter, the "assay plate"). Then, 20 .mu.L supernatant
per well from the experimental plate was transferred to assay
plate. Assay plate was then incubated at 37.degree. C. for 1-3
hours. Assay plate absorbance at 620 nm was read on a model
SpectraMaxPlus 384 Microplate Reader from Molecular Devices. The
data was analyzed using Graph Pad Prism.
[0241] The impact the presence or the absence of O-linked
glycosylation in IFN.alpha.2b has on the off-target activity of the
attenuated IFN fused anti-CD38 antibodies was evaluated.
[0242] The O-linked glycosylation site of an anti-CD38 antibody
with attenuated interferon fusion constructs was removed by either
substituting threonine 106 (T106) to alanine (shown as T106A) or
deleting T106 (shown as .DELTA.T106). The effect on off-target
activity was investigated by comparing X10.21 anti-CD38 antibody
fused to attenuated IFN.alpha.2b with and without O-linked
glycosylation.
[0243] Removal of the O-linked glycosylation site in the attenuated
IFN portion of the antibody fusion constructs resulted in a slight
increase in off-target activity relative to O-linked glycosylated
proteins i.e. A10.21 IgG4 IFN (145D, T106T) and A10.21 IgG1 IFN
(145D, T106T) (FIG. 5). This trend was consistent regardless of
isotype (FIG. 5). However IFN.alpha.2b (A145D) is so attenuated
that the level of induction of SEAP in this assay is limited even
at the highest doses used. Therefore the EC50 is an approximation
only.
[0244] The off-target activity of antibodies fused with attenuated
IFN (R10A2 IgG4 (S228P) IFN (A145D)) possessing different amino
acid substitutions for the removal of O-linked glycosylation was
examined. All possible amino acid substitutions were tested and the
construct are listed in Table 11.
TABLE-US-00011 TABLE 11 Seq Id No. Heavy Light Construct Name Chain
Chain IFN R10A2 IgG4 (S228P) IFN (A145D, T106T) 75 82 107 R10A2
IgG4 (S228P) IFN (A145D, T106A) 75 82 25 R10A2 IgG4 (S228P) IFN
(A145D, T106R) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106N) 75 82
25 R10A2 IgG4 (S228P) IFN (A145D, T106D) 75 82 25 R10A2 IgG4
(S228P) IFN (A145D, T106C) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D,
T106E) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106Q) 75 82 25
R10A2 IgG4 (S228P) IFN (A145D, T106G) 75 82 25 R10A2 IgG4 (S228P)
IFN (A145D, T106H) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106I)
75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106L) 75 82 25 R10A2 IgG4
(S228P) IFN (A145D, T106K) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D,
T106M) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106F) 75 82 25
R10A2 IgG4 (S228P) IFN (A145D, T106P) 75 82 25 R10A2 IgG4 (S228P)
IFN (A145D, T106S) 75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106W)
75 82 25 R10A2 IgG4 (S228P) IFN (A145D, T106Y) 75 82 25 R10A2 IgG4
(S228P) IFN (A145D, T106V) 75 82 25
[0245] It was shown that each of these substitutions demonstrated
similar or slightly less off-target activity (increased EC.sub.50)
relative to the O-glycosylated IFN fused antibody R10A2 IgG4
(S228P) IFN (A145D, T106T). However, IFN.alpha.2b (A145D) is so
attenuated that the level of induction of SEAP in this assay is
limited even at the highest doses used. Therefore the EC.sub.50 is
an approximation only.
[0246] The off-target activity of another anti-CD38 antibody
attenuated IFN.alpha.2b construct and the impact of removal of
O-linked glycosylation by the T106A substitution was investigated.
A10.43 has 7 amino acid changes in the heavy chain compared to
A10.21 while the two constructs share the same light chain sequence
(see Sequence Listing). The results demonstrated that removal of
the O-linked glycosylation site by T106A substitution slightly
increased the off-target activity of A10.43. However IFN.alpha.2b
(A145D) is so attenuated that the level of induction of SEAP in
this assay is limited even at the highest doses used. Therefore the
EC.sub.50 is an approximation only.
[0247] Removal of the O-linked glycosylation site in the attenuated
IFN portion of the antibody interferon fusion constructs resulted
in slight to no increase in the OFF-target activity as well as a
slight increase in ON-target activity relative to O-linked
glycosylated proteins. Both O-linked glycosylated and
non-glycosylated anti-CD38 antibody attenuated interferon fusion
proteins showed high selectivity towards CD38+ cells, while Intron
A did not show selectivity.
Example 5
Evaluation of Human Neonatal FC Receptor (FCRN) Binding to
Anti-CD38-Attenuated IFN
[0248] Poly-his-tagged FcRn was immobilized onto the designated
active flow cells of a CMS sensor chip in a Biacore T200 enhanced
instrument using an amine coupling protocol, while a blank
immobilization was performed on the reference flow cells. Pulses of
poly-his-tagged FcRn were injected over the active surfaces to
ensure that the solution would pre-concentrate onto the flow cell.
The surfaces were then washed with 50 mM NaOH. Both the reference
and active surfaces were activated for seven minutes with a mix of
50:50 EDC/NHS. This was followed by a series of pulses of
poly-his-tagged FcRn at 2 .mu.g/mL in 10 mM Sodium Acetate pH 5.0
over the active surfaces only. Once the target of 150 RU was
reached, both surfaces were deactivated with 1 M ethanolamine pH
8.5 for seven minutes. This protocol resulted in the immobilization
of approximately 150 RU of poly-his-tagged FcRn onto the active
surfaces. Each active surface was used for one run.
[0249] The test antibodies were desalted in PBS-P running buffer
(DPBS, pH 7.4 and 0.005% Tween-20 adjusted to pH 6.0 with HCl at
25.degree. C.) using Zeba Spin Desalting Column, 7K MWCO, 0.5 mL
(Pierce Product #89882). Following desalting, the concentration of
each sample was adjusted to 1 mg/mL.
[0250] On the day of the assay the 1 mg/mL solutions were further
diluted in running buffer to prepare the highest concentration, and
then diluted 1:2 to create the concentration series. The test
samples were passed over the surface using a flow rate of 50
.mu.L/minute. The association phase was 100 seconds while the
dissociation phase was 300 seconds for all concentrations tested.
The active and reference surfaces were regenerated using a 60
second injection of 100 mM Tris, 50 mM NaCl at pH 8.0 to remove the
test antibody. Binding constants were determined at 25.degree.
C.
[0251] The antibody poly-his-FcRn binding interaction was evaluated
using the two state reaction model where the R.sub.max was set to
local and RI (refractive index) parameter was set to local. All
data was double reference subtracted: first, the signal in the
reference cell (blank immobilization) resulting from antibody
binding to the dextran matrix was subtracted, and second, the
signal of 0 nM antibody on the active surface was subtracted.
[0252] Binding constants were determined on at least two separate
runs for each antibody. Prolia Lot 1035726 was tested alongside the
anti-CD38/IFN samples as a positive control for each run. Average
values for k.sub.a1, k.sub.d1, k.sub.a2, k.sub.d2 and K.sub.D were
determined.
[0253] The average kinetic and affinity values for FcRn binding for
the 4 anti-CD38/IFN (A10.21 IgG4 (S228P) IFN (A145D, T106A), A10.21
IgG4 (S228P) IFN (A145D, T106T) A10.43 IgG4 (S228P) IFN (A145D,
T106A) A10.43 IgG4 (S228P) IFN (A145D, T106T)) were measured. In
this sample set the clone A10.21 IgG4 (S228P) IFN (A145D, T106A)
displayed the highest affinity for FcRn.
Example 6
[0254] Efficacy of Anti-CD38 Antibodies Fused to Attenuated
Interferon Alpha 2B with and without O-Linked Glycosylation in the
Mouse NCI-H929 Multiple Myeloma Model
Drugs and Treatment:
TABLE-US-00012 [0255] TABLE 12 Regimen 1 Gr. N Agent .mu.g/animal
Route Schedule .sup. 1.sup.# 8 vehicle 20 ip biwk x 5 2 8 A10.21
IgG4 (S228P) 20 ip biwk x 5 IFN (A145D, T106T) 3 8 A10.21 IgG4
(S228P) 20 ip biwk x 5 IFN (A145D, T106A) 4 8 A10.43 IgG4 (S228P)
20 ip biwk x 5 IFN (A145D, T106T) 5 8 A10.43 IgG4 (S228P) 20 ip
biwk x 5 IFN (A145D, T106A) .sup.#Control Group
Procedures:
[0256] Set up CR female CB.17 SCID mice with 1.times.10.sup.7 H929
tumor cells in 50% Matrigel sc in flank. [0257] Cell Injection
Volume is 0.2 mL/mouse. [0258] Age at Start Date: 8 to 12 weeks.
[0259] Perform a pair match when tumors reach an average size of
170-350 mm.sup.3, and begin treatment. [0260] Dosing volume=0.2
mL/mouse. Do not adjust for body weight. [0261] Body Weight:
qd.times.5 then biwk to end [0262] Caliper Measurement: biwk to end
[0263] Endpoint TGD. Animals are to be monitored individually. The
endpoint of the experiment is a tumor volume of 2000 mm.sup.3 or 60
days, whichever comes first. Responders can be followed longer.
When the endpoint is reached, the animals are to be euthanized per
SOP #687.
[0264] The results are shown in FIG. 8. Roughly equivalent efficacy
was seen with and without O-linked glycosylation, with the
aglycosylated form slightly more potent in impeding tumour
growth.
Example 7
[0265] In the evaluation of human neonatal Fc receptor (FcRn)
binding to anti-CD38 antibodies fused to attenuated IFN with and
without O-linked glycosylation the proteins without O-linked
glycosylation displayed the highest affinity for FcRn. The effect
of this may be evaluated in cynomolgus monkeys and humanised FcRn
mice.
Cynomolgus Monkey Study
[0266] Monkey study design- to compare +/-O-glyc [0267] Single 3
mg/kg intravenous infusion (1 hour) through an indwelling catheter
[0268] A10.21 IgG4 (S228P) IFN (145D, T106A) (n=4) [0269] A10.21
IgG4 (S228P) IFN (145D, T106T) (n=4) [0270] A10.21 IgG4 (S228P) IFN
(145D, T106A) (n=4) [0271] A10.21 IgG4 (S228P) IFN (145D, T106T)
(n=4) [0272] Compare PK and PD (biological effect i.e. serum
neopterin levels) [0273] PK: all monkeys, .ltoreq.1 ml, 11
timepoints (Pre dose, 0 minutes (immediately post end of infusion).
2, 6, 12, 24, 48, 96, 120, 168 and at 240 hours post infusion.
Samples (80) are analysed by ELISA. [0274] TK modelling: WinNonlin
Table Assembly {non-compartmental analysis) [0275] Clinical
pathology: Hematology and blood chemistry--all monkeys, 3 occasions
(pre-treatment, 24 hours post dose and Day 8) [0276] Serum
neopterin all monkeys, .about.0.5 ml 3 occasions at 5 timepoints
(Predose, 12, 24, 96, 168 hours post dose)
Pharmacokinetic Study in Humanised FcRN Mice
[0277] 1. Thirty two (32) B6.Cg-Fcgr.sup.tm1Dcr
Tg(CAG-FCGRT)276Dcr/DcrJ (JAX stock #004919) female mice are dosed
on day 1 by IP injection of 1 mg/kg of; [0278] A10.21 IgG4 (S228P)
IFN (145D, T106A) (n=8) [0279] A10.21 IgG4 (S228P) IFN (145D,
T106T) (n=8) [0280] A10.43 IgG4 (S228P) IFN (145D, T106A) (n=8)
[0281] A10.43 IgG4 (S228P) IFN (145D, T106T) (n=8) 2. Body weight
is measured 3 days prior to dosing, the day of dosing and then
weekly. 3. Cage side observations are made daily and clinical
observations made weekly. 4. Pharmacokinetic blood collection: Mice
are bled (25 .mu.I) at pre-dose 3 days and postdose 1, 12, 24, 48
and 72 hours and 5, 7, 10, and 14 days. Mice are bled in 2 cohorts
(4 mice/group/cohort). 5. All mice are sacrificed on day 14.
Terminal cardiocentesis is performed to collect blood. 6. Blood is
collected in lithium heparin tubes and centrifuged at 10,000 rpm
for 2 min at 4.degree. C. 7. Plasma samples are diluted 1:10 in PBS
and are frozen prior to analysis by binding ELISA for A10.21 IgG4
IFN(145D) A106, A10.21 IgG4 IFN(145D) T106, A10.43 IgG4 IFN(145D)
A106 or A10.43 IgG4 IFN(145D) T106.
Sequence CWU 1
1
1151165PRTArtificialHuman interferon alpha-2-b with mutation at
amino acid position 106VARIANT(106)..(106)Xaa can be any amino acid
selected from A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, V, W, Y
1Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5
10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys
Asp 20 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln
Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln
Gln Ile Phe 50 55 60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp
Asp Glu Thr Leu65 70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln
Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val
Xaa Glu Thr Pro Leu Met Lys 100 105 110Glu Asp Ser Ile Leu Ala Val
Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys
Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile
Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser145 150 155
160Leu Arg Ser Lys Glu 1652164PRTArtificialHuman Interferon
alpha-2-b with deletion of amino acid at position 106 2Cys Asp Leu
Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu
Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg
His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40
45Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe
50 55 60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr
Leu65 70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn
Asp Leu Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Glu Thr Pro
Leu Met Lys Glu 100 105 110Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe
Gln Arg Ile Thr Leu Tyr 115 120 125Leu Lys Glu Lys Lys Tyr Ser Pro
Cys Ala Trp Glu Val Val Arg Ala 130 135 140Glu Ile Met Arg Ser Phe
Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu145 150 155 160Arg Ser Lys
Glu3165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation L15A 3Cys Asp Leu Pro Gln
Thr His Ser Leu Gly Ser Arg Arg Thr Ala Met1 5 10 15Leu Leu Ala Gln
Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp
Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala
Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75
80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met
Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg
Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
1654165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation A19W 4Cys Asp Leu Pro Gln
Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Trp Gln
Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp
Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala
Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75
80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met
Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg
Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
1655165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation R22A 5Cys Asp Leu Pro Gln
Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln
Met Ala Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp
Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala
Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75
80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met
Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg
Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
1656165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation R23A 6Cys Asp Leu Pro Gln
Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln
Met Arg Ala Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp
Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala
Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75
80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met
Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg
Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
1657165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation S25A 7Cys Asp Leu Pro Gln
Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln
Met Arg Arg Ile Ala Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp
Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala
Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75
80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met
Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg
Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
1658165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation L26A 8Cys Asp Leu Pro Gln
Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln
Met Arg Arg Ile Ser Ala Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp
Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala
Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75
80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met
Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg
Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
1659165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation F27A 9Cys Asp Leu Pro Gln
Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln
Met Arg Arg Ile Ser Leu Ala Ser Cys Leu Lys Asp 20 25 30Arg His Asp
Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala
Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn
Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75
80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met
Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg
Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16510165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation L30X, where X can be any
amino acid selected from A, VVARIANT(30)..(30)Xaa can be any amino
acid selected from A, V 10Cys Asp Leu Pro Gln Thr His Ser Leu Gly
Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser
Leu Phe Ser Cys Xaa Lys Asp 20 25 30Arg His Asp Phe Gly Phe Pro Gln
Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro Val
Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn Leu Phe Ser Thr Lys
Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75 80Leu Asp Lys Phe
Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys Val
Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys 100 105 110Glu
Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120
125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg
130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln
Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16511165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation K31A 11Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Ala Asp 20 25 30Arg His
Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55
60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65
70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu
Met Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16512165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation D32A 12Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Ala 20 25 30Arg His
Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55
60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65
70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu
Met Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16513165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation R33X, where X can be any
amino acid selected from K, A, QVARIANT(33)..(33)Xaa can be any
amino acid selected from K, A, Q 13Cys Asp Leu Pro Gln Thr His Ser
Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln Met Arg Arg
Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Xaa His Asp Phe Gly Phe
Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile
Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn Leu Phe Ser
Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75 80Leu Asp
Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala
Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys 100 105
110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn
Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16514165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation H34A 14Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg Ala
Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55
60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr
Leu65
70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu
Met Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16515165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation Q40A 15Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His
Asp Phe Gly Phe Pro Ala Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55
60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65
70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu
Met Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16516165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation D114R 16Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His
Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55
60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65
70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu
Met Lys 100 105 110Glu Arg Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16517165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation L117A 17Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His
Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55
60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65
70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu
Met Lys 100 105 110Glu Asp Ser Ile Ala Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16518165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation R120X where X can be any
amino acid selected from A, EVARIANT(120)..(120)Xaa can be any
amino acid selected from A, E 18Cys Asp Leu Pro Gln Thr His Ser Leu
Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln Met Arg Arg Ile
Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp Phe Gly Phe Pro
Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro
Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn Leu Phe Ser Thr
Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75 80Leu Asp Lys
Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys
Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys 100 105
110Glu Asp Ser Ile Leu Ala Val Xaa Lys Tyr Phe Gln Arg Ile Thr Leu
115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn
Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16519165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation R125X where X can be any
amino acid selected from A, EVARIANT(125)..(125)Xaa can be any
amino acid selected from A, E 19Cys Asp Leu Pro Gln Thr His Ser Leu
Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln Met Arg Arg Ile
Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp Phe Gly Phe Pro
Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro
Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn Leu Phe Ser Thr
Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75 80Leu Asp Lys
Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys
Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys 100 105
110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Xaa Ile Thr Leu
115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn
Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16520165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation K131A 20Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His
Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55
60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65
70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu
Met Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125Tyr Leu Ala Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16521165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation E132A 21Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His
Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55
60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65
70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu
Met Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125Tyr Leu Lys Ala Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16522165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation K133A 22Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His
Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55
60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65
70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu
Met Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu Ala Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16523165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation K134A 23Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His
Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55
60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65
70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu
Met Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Ala Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16524165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation R144X where X can be any
amino acid selected from A, D, E, G, H, I, K, L, N, Q, S, T, V,
YVARIANT(144)..(144)Xaa can be any amino acid selected from A, D,
E, G, H, I, K, L, N, Q, S, T, V, Y 24Cys Asp Leu Pro Gln Thr His
Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln Met Arg
Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp Phe Gly
Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr
Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn Leu Phe
Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75 80Leu
Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90
95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys
100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile
Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp
Glu Val Val Xaa 130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu Ser
Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16525165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation A145X where X can be any
amino acid selected from D, E, G, H, I, K, L, M, N, Q, R, S, T, V,
YVARIANT(145)..(145)Xaa can be any amino acid selected from D, E,
G, H, I, K, L, M, N, Q, R, S, T, V, Y 25Cys Asp Leu Pro Gln Thr His
Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln Met Arg
Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp Phe Gly
Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr
Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn Leu Phe
Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75 80Leu
Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90
95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys
100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile
Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp
Glu Val Val Arg 130 135 140Xaa Glu Ile Met Arg Ser Phe Ser Leu Ser
Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16526165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation M148A 26Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His
Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55
60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65
70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu
Met Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile Ala Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16527165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation R149A 27Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His
Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55
60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65
70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
Glu 85 90 95Ala Cys Val Ile Gln Gly
Val Gly Val Ala Glu Thr Pro Leu Met Lys 100 105 110Glu Asp Ser Ile
Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125Tyr Leu
Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135
140Ala Glu Ile Met Ala Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu
Ser145 150 155 160Leu Arg Ser Lys Glu
16528165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation S152A 28Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His
Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55
60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65
70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu
Met Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ala
Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16529165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation L153A 29Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His
Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55
60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65
70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu
Met Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser
Ala Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16530165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutation N156A 30Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala
Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His
Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55
60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65
70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu
Met Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Ala Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16531612PRTArtificialA10.21 IgG4 (S228P) IFN (A592D, T553X) where X
can be any amino acid selected from A, G, E, S,
VVARIANT(553)..(553)Xaa can be any amino acid selected from A, G,
E, S, V 31Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe
Thr Asp Ser 20 25 30Val Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly
Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro Glu Tyr Gly Arg Thr Asp
Val Ala Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr
Ser Thr Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Lys Tyr Asn Ser
Gly Tyr Gly Phe Pro Tyr Trp Gly Gln 100 105 110Gly Thr Thr Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150
155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys
Asn Val Asp His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg
Val Glu Ser Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro Cys Pro Ala
Pro Glu Phe Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu
Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265
270Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys
275 280 285Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly Leu Pro
Ser Ser Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390
395 400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
Arg 405 410 415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Leu Gly Lys Cys 435 440 445Asp Leu Pro Gln Thr His Ser Leu Gly
Ser Arg Arg Thr Leu Met Leu 450 455 460Leu Ala Gln Met Arg Arg Ile
Ser Leu Phe Ser Cys Leu Lys Asp Arg465 470 475 480His Asp Phe Gly
Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys 485 490 495Ala Glu
Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn 500 505
510Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu
515 520 525Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
Glu Ala 530 535 540Cys Val Ile Gln Gly Val Gly Val Xaa Glu Thr Pro
Leu Met Lys Glu545 550 555 560Asp Ser Ile Leu Ala Val Arg Lys Tyr
Phe Gln Arg Ile Thr Leu Tyr 565 570 575Leu Lys Glu Lys Lys Tyr Ser
Pro Cys Ala Trp Glu Val Val Arg Asp 580 585 590Glu Ile Met Arg Ser
Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu 595 600 605Arg Ser Lys
Glu 61032165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutations L30A, H57Y, E58N and
Q61S 32Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu
Met1 5 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Ala
Lys Asp 20 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn
Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro Val Leu Tyr Asn Met Ile
Ser Gln Ile Phe 50 55 60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Thr Leu65 70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr
Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly
Val Ala Glu Thr Pro Leu Met Lys 100 105 110Glu Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu
Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140Ala Glu
Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser145 150 155
160Leu Arg Ser Lys Glu 16533165PRTArtificialAglycosylated (T106A)
human interferon alpha-2-b comprising attenuating mutations R33A,
H57Y, E58N and Q61S 33Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser
Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Leu
Phe Ser Cys Leu Lys Asp 20 25 30Ala His Asp Phe Gly Phe Pro Gln Glu
Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro Val Leu
Tyr Asn Met Ile Ser Gln Ile Phe 50 55 60Asn Leu Phe Ser Thr Lys Asp
Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75 80Leu Asp Lys Phe Tyr
Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys Val Ile
Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys 100 105 110Glu Asp
Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120
125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg
130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln
Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16534165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutations M148A, H57Y, E58N and
Q61S 34Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu
Met1 5 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu
Lys Asp 20 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn
Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro Val Leu Tyr Asn Met Ile
Ser Gln Ile Phe 50 55 60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Thr Leu65 70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr
Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly
Val Ala Glu Thr Pro Leu Met Lys 100 105 110Glu Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu
Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140Ala Glu
Ile Ala Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser145 150 155
160Leu Arg Ser Lys Glu 16535165PRTArtificialAglycosylated (T106A)
human interferon alpha-2-b comprising attenuating mutations L153A,
H57Y, E58N and Q61S 35Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser
Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Leu
Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp Phe Gly Phe Pro Gln Glu
Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro Val Leu
Tyr Asn Met Ile Ser Gln Ile Phe 50 55 60Asn Leu Phe Ser Thr Lys Asp
Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75 80Leu Asp Lys Phe Tyr
Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys Val Ile
Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys 100 105 110Glu Asp
Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120
125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg
130 135 140Ala Glu Ile Met Arg Ser Phe Ser Ala Ser Thr Asn Leu Gln
Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16536165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutations R144A, H57Y, E58N and
Q61S 36Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu
Met1 5 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu
Lys Asp 20 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn
Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro Val Leu Tyr Asn Met Ile
Ser Gln Ile Phe 50 55 60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Thr Leu65 70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr
Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly
Val Ala Glu Thr Pro Leu Met Lys 100 105 110Glu Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu
Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Ala 130 135 140Ala Glu
Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser145 150 155
160Leu Arg Ser Lys Glu 16537165PRTArtificialAglycosylated (T106A)
human interferon alpha-2-b comprising attenuating mutations
N65A,L80A, Y85A, Y89A 37Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser
Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Leu
Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp Phe Gly Phe Pro Gln Glu
Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro Val Leu
His Glu Met Ile Gln Gln Ile Phe 50 55 60Ala Leu Phe Ser Thr Lys Asp
Ser Ser Ala Ala Trp Asp Glu Thr Ala65 70 75 80Leu Asp Lys Phe Ala
Thr Glu Leu Ala Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys Val Ile
Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys 100 105 110Glu Asp
Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120
125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg
130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln
Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16538165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutations N65A,L80A, Y85A, Y89A
and D114A 38Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr
Leu Met1 5 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys
Leu Lys Asp 20 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly
Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro Val Leu His Glu Met
Ile Gln Gln Ile Phe 50 55 60Ala Leu Phe Ser Thr Lys Asp Ser Ser Ala
Ala Trp Asp Glu Thr Ala65 70 75
80Leu Asp Lys Phe Ala Thr Glu Leu Ala Gln Gln Leu Asn Asp Leu Glu
85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met
Lys 100 105 110Glu Ala Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg
Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu
Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16539165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutations N65A,L80A, Y85A, Y89A
and L117A 39Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr
Leu Met1 5 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys
Leu Lys Asp 20 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly
Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro Val Leu His Glu Met
Ile Gln Gln Ile Phe 50 55 60Ala Leu Phe Ser Thr Lys Asp Ser Ser Ala
Ala Trp Asp Glu Thr Ala65 70 75 80Leu Asp Lys Phe Ala Thr Glu Leu
Ala Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys Val Ile Gln Gly Val
Gly Val Ala Glu Thr Pro Leu Met Lys 100 105 110Glu Asp Ser Ile Ala
Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125Tyr Leu Lys
Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140Ala
Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser145 150
155 160Leu Arg Ser Lys Glu 16540165PRTArtificialAglycosylated
(T106A) human interferon alpha-2-b comprising attenuating mutations
N65A,L80A, Y85A, Y89A and R120A 40Cys Asp Leu Pro Gln Thr His Ser
Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln Met Arg Arg
Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp Phe Gly Phe
Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile
Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Ala Leu Phe Ser
Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Ala65 70 75 80Leu Asp
Lys Phe Ala Thr Glu Leu Ala Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala
Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys 100 105
110Glu Asp Ser Ile Leu Ala Val Ala Lys Tyr Phe Gln Arg Ile Thr Leu
115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn
Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
16541165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutations Y85A, Y89A and R120A
41Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1
5 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys
Asp 20 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln
Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln
Gln Ile Phe 50 55 60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp
Asp Glu Thr Leu65 70 75 80Leu Asp Lys Phe Ala Thr Glu Leu Ala Gln
Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val
Ala Glu Thr Pro Leu Met Lys 100 105 110Glu Asp Ser Ile Leu Ala Val
Ala Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys
Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile
Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser145 150 155
160Leu Arg Ser Lys Glu 16542165PRTArtificialAglycosylated (T106A)
human interferon alpha-2-b comprising attenuating mutations
D114A,R120A 42Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg
Thr Leu Met1 5 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser
Cys Leu Lys Asp 20 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe
Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro Val Leu His Glu
Met Ile Gln Gln Ile Phe 50 55 60Asn Leu Phe Ser Thr Lys Asp Ser Ser
Ala Ala Trp Asp Glu Thr Leu65 70 75 80Leu Asp Lys Phe Tyr Thr Glu
Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys Val Ile Gln Gly
Val Gly Val Ala Glu Thr Pro Leu Met Lys 100 105 110Glu Ala Ser Ile
Leu Ala Val Ala Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125Tyr Leu
Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135
140Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu
Ser145 150 155 160Leu Arg Ser Lys Glu
16543165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutations L117A,R120A 43Cys Asp
Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu
Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25
30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln
35 40 45Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile
Phe 50 55 60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu
Thr Leu65 70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu
Asn Asp Leu Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu
Thr Pro Leu Met Lys 100 105 110Glu Asp Ser Ile Ala Ala Val Ala Lys
Tyr Phe Gln Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr
Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg
Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg
Ser Lys Glu 16544165PRTArtificialAglycosylated (T106A) human
interferon alpha-2-b comprising attenuating mutations
L117A,R120A,K121A 44Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg
Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
Ser Cys Leu Lys Asp 20 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu
Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro Val Leu His
Glu Met Ile Gln Gln Ile Phe 50 55 60Asn Leu Phe Ser Thr Lys Asp Ser
Ser Ala Ala Trp Asp Glu Thr Leu65 70 75 80Leu Asp Lys Phe Tyr Thr
Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys Val Ile Gln
Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys 100 105 110Glu Asp Ser
Ile Ala Ala Val Ala Ala Tyr Phe Gln Arg Ile Thr Leu 115 120 125Tyr
Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135
140Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu
Ser145 150 155 160Leu Arg Ser Lys Glu
16545165PRTArtificialAglycosylated (T106A) human interferon
alpha-2-b comprising attenuating mutations R120A,K121A 45Cys Asp
Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu
Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25
30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln
35 40 45Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile
Phe 50 55 60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu
Thr Leu65 70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu
Asn Asp Leu Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu
Thr Pro Leu Met Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Ala Ala
Tyr Phe Gln Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr
Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile Met Arg
Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg
Ser Lys Glu 16546165PRTArtificialAglycosylated (T106A) human
interferon alpha-2-b comprising attenuating mutations R120E,K121E
46Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met1
5 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys
Asp 20 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln
Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln
Gln Ile Phe 50 55 60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp
Asp Glu Thr Leu65 70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln
Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly Val
Ala Glu Thr Pro Leu Met Lys 100 105 110Glu Asp Ser Ile Leu Ala Val
Glu Glu Tyr Phe Gln Arg Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys
Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140Ala Glu Ile
Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser145 150 155
160Leu Arg Ser Lys Glu 16547160PRTArtificialAglycosylated (T106A)
human interferon alpha-2-b comprising attenuating deletion of
residues L161-E165 47Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser
Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Leu
Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp Phe Gly Phe Pro Gln Glu
Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro Val Leu
His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn Leu Phe Ser Thr Lys Asp
Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75 80Leu Asp Lys Phe Tyr
Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys Val Ile
Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys 100 105 110Glu Asp
Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120
125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg
130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln
Glu Ser145 150 155 16048447PRTArtificialVH sequence of 10.21
formatted as IgG4 incorporating hinge stabilisation substitution
S228P 48Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ala1 5 10 15Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr
Asp Ser 20 25 30Val Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu
Glu Trp Met 35 40 45Gly Trp Ile Asp Pro Glu Tyr Gly Arg Thr Asp Val
Ala Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser
Thr Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Lys Tyr Asn Ser Gly
Tyr Gly Phe Pro Tyr Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155
160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
Val Asp His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro Cys Pro Ala Pro
Glu Phe Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270Val
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280
285Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395
400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
405 410 415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly Lys 435 440 44549447PRTArtificialVH sequence of 10.21
formatted as an IgG4 incorporating hinge stabilisation substitution
S228P and YTE residues substituted in positions 252, 254, 256
respectively 49Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys
Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr
Phe Thr Asp Ser 20 25 30Val Met Asn Trp Val Gln Gln Ala Pro Gly Lys
Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro Glu Tyr Gly Arg Thr
Asp Val Ala Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp
Thr Ser Thr Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg
Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Lys Tyr Asn
Ser Gly Tyr Gly Phe Pro Tyr Trp Gly Gln 100 105 110Gly Thr Thr Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135
140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Lys Thr
Tyr Thr Cys Asn Val Asp His Lys 195 200 205Pro Ser Asn Thr Lys Val
Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro
Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val225 230 235 240Phe
Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Tyr Ile Thr Arg Glu 245 250 255Pro Glu Val Thr Cys
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270Val Gln Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295
300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410
415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys 435 440 44550450PRTArtificialVH sequence of 10.21 formatted as
an IgG1 50Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro
Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe
Thr Asp Ser 20 25 30Val Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly
Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro Glu Tyr Gly Arg Thr Asp
Val Ala Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr
Ser Thr Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Lys Tyr Asn Ser
Gly Tyr Gly Phe Pro Tyr Trp Gly Gln 100 105 110Gly Thr Thr Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu
Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150
155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
Asn Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys
Val Glu Pro Lys Ser Cys Asp 210 215 220Lys Thr His Thr Cys Pro Pro
Cys Pro Ala Pro Glu Leu Leu Gly Gly225 230 235 240Pro Ser Val Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265
270Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr
Tyr Arg 290 295 300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp
Leu Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365Thr Cys Leu
Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390
395 400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp 405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His 420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro 435 440 445Gly Lys 45051450PRTArtificialVH
sequence of 10.21 formatted as an IgG1 incorporating substitutions
L238A and G240A to reduce effector function 51Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile
Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30Val Met Asn
Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp
Ile Asp Pro Glu Tyr Gly Arg Thr Asp Val Ala Glu Lys Phe 50 55 60Gln
Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr Gly Phe Pro Tyr Trp Gly
Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Ala
Gly Ala225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly Lys 45052450PRTArtificialVH sequence of 10.21 formatted as
an IgG1 incorporating substitutions L235A and G237A (reduce
effector function) and M255Y, S257T, T259E 52Glu Val Gln Leu Val
Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile
Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30Val Met Asn
Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp
Ile Asp Pro Glu Tyr Gly Arg Thr Asp Val Ala Glu Lys Phe 50 55 60Gln
Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr Gly Phe Pro Tyr Trp Gly
Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Ala
Gly Ala225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Tyr Ile 245 250 255Thr Arg Glu Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly Lys 45053446PRTArtificialVH sequence of 10.21 formatted onto
an IgG2 backbone 53Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr
Thr Phe Thr Asp Ser 20 25 30Val Met Asn Trp Val Gln Gln Ala Pro Gly
Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro Glu Tyr Gly Arg
Thr Asp Val Ala Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala
Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Lys Tyr
Asn Ser Gly Tyr Gly Phe Pro Tyr Trp Gly Gln 100 105 110Gly Thr Thr
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135
140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro 180 185 190Ser Ser Asn Phe Gly Thr Gln Thr
Tyr Thr Cys Asn Val Asp His Lys 195 200 205Pro Ser Asn Thr Lys Val
Asp Lys Thr Val Glu Arg Lys Cys Cys Val 210 215 220Glu Cys Pro Pro
Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe225 230 235 240Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250
255Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg
Val Val Ser Val 290 295 300Leu Thr Val Val His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys Gly Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335Lys Thr Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375
380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp 405 410 415Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 435 440 44554446PRTArtificialVH sequence of
10.21 formatted onto an IgG2 backbone incorporating substitutions
M251Y, S253T and T255E 54Glu Val Gln Leu Val Gln Ser Gly Ala Glu
Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys Lys Val Ser
Gly Tyr Thr Phe Thr Asp Ser 20 25 30Val Met Asn Trp Val Gln Gln Ala
Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro Glu Tyr
Gly Arg Thr Asp Val Ala Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Ile
Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser
Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr
Lys Tyr Asn Ser Gly Tyr Gly Phe Pro Tyr Trp Gly Gln 100 105 110Gly
Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120
125Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala
130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Asn Phe Gly Thr Gln
Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205Pro Ser Asn Thr Lys
Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val 210 215 220Glu Cys Pro
Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe225 230 235
240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile Thr Arg Glu Pro
245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro
Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr
Phe Arg Val Val Ser Val 290 295 300Leu Thr Val Val His Gln Asp Trp
Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys
Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330 335Lys Thr Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser
Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360
365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly
370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp
Ser
Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp 405 410 415Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 435 440 44555447PRTArtificialVH sequence of
10.43 formatted onto an IgG4 backbone incorporating the hinge
stabilisation substitution S228P 55Gln Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Val Ser Gly Tyr Thr Leu Thr Asp Ser 20 25 30Val Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro
Glu Tyr Gly Arg Thr Asp Val Ala Gln Lys Phe 50 55 60Gln Gly Arg Val
Thr Met Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80Met Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Thr Lys Tyr Asn Ser Gly Tyr Gly Phe Pro Tyr Trp Gly Gln 100 105
110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr
Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly
Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr
Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly
Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205Pro Ser Asn
Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220Pro
Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val225 230
235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
Asp Pro Glu 260 265 270Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser
Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335Ser Lys
Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345
350Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Arg
Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Glu Gly Asn Val Phe
Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr
Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440
44556447PRTArtificialVH sequence of 10.152 formatted as an IgG4
incorporating the hinge stabilisation substitution S228P 56Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr
Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Ser 20 25
30Val Met Asn Trp Val Gln Gln His Pro Gly Lys Gly Leu Glu Trp Met
35 40 45Gly Trp Ile Asp Pro Glu Tyr Gly Arg Thr Asp Val Ala Glu Lys
Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr Gly Phe
Pro Tyr Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp
His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270Val Gln Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295
300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410
415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys 435 440 44557401PRTArtificialOPG-Fc construct 57Glu Thr Phe Pro
Pro Lys Tyr Leu His Tyr Asp Glu Glu Thr Ser His1 5 10 15Gln Leu Leu
Cys Asp Lys Cys Pro Pro Gly Thr Tyr Leu Lys Gln His 20 25 30Cys Thr
Ala Lys Trp Lys Thr Val Cys Ala Pro Cys Pro Asp His Tyr 35 40 45Tyr
Thr Asp Ser Trp His Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro 50 55
60Val Cys Lys Glu Leu Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His65
70 75 80Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu Gly Glu
Phe 85 90 95Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe Gly Val Val
Gln Ala 100 105 110Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg Cys
Pro Asp Gly Phe 115 120 125Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro
Cys Arg Lys His Thr Asn 130 135 140Cys Ser Val Phe Gly Leu Leu Leu
Thr Gln Lys Gly Asn Ala Thr His145 150 155 160Asp Asn Ile Cys Ser
Gly Asn Ser Glu Ser Thr Gln Lys Glu Arg Lys 165 170 175Cys Cys Val
Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro 180 185 190Ser
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 195 200
205Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
210 215 220Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn225 230 235 240Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr Phe Arg Val 245 250 255Val Ser Val Leu Thr Val Val His Gln
Asp Trp Leu Asn Gly Lys Glu 260 265 270Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ala Pro Ile Glu Lys 275 280 285Thr Ile Ser Lys Thr
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 290 295 300Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr305 310 315
320Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
325 330 335Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Met Leu 340 345 350Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 355 360 365Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu 370 375 380Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly385 390 395
400Lys58448PRTArtificialX2.12 VH sequence formatted as an IgG4
(S228P) 58Gln Leu Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro
Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile
Ser Ser Ser 20 25 30Ser Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly
Lys Gly Leu Glu 35 40 45Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr
Asn Tyr Asn Pro Ser 50 55 60Leu Lys Ser Arg Val Thr Ile Ser Val Asp
Thr Ser Lys Asn Gln Phe65 70 75 80Ser Leu Lys Leu Ser Ser Val Thr
Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95Cys Ala Arg Val Gly Gly Ala
Gly Gly Trp Pro Leu Asp Val Trp Gly 100 105 110Gln Gly Thr Thr Val
Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser 115 120 125Val Phe Pro
Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala 130 135 140Ala
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val145 150
155 160Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
Ala 165 170 175Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr Val 180 185 190Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr
Cys Asn Val Asp His 195 200 205Lys Pro Ser Asn Thr Lys Val Asp Lys
Arg Val Glu Ser Lys Tyr Gly 210 215 220Pro Pro Cys Pro Pro Cys Pro
Ala Pro Glu Phe Leu Gly Gly Pro Ser225 230 235 240Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro 260 265
270Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
275 280 285Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
Val Val 290 295 300Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr305 310 315 320Lys Cys Lys Val Ser Asn Lys Gly Leu
Pro Ser Ser Ile Glu Lys Thr 325 330 335Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350Pro Pro Ser Gln Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp385 390
395 400Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
Ser 405 410 415Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
His Glu Ala 420 425 430Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Leu Gly Lys 435 440 44559449PRTArtificialAnti-CD138 VH
sequence formatted onto an IgG4 (S228P) backbone 59Gln Val Gln Leu
Gln Gln Ser Gly Ser Glu Leu Met Met Pro Gly Ala1 5 10 15Ser Val Lys
Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Asn Tyr 20 25 30Trp Ile
Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45Gly
Glu Ile Leu Pro Gly Thr Gly Arg Thr Ile Tyr Asn Glu Lys Phe 50 55
60Lys Gly Lys Ala Thr Phe Thr Ala Asp Ile Ser Ser Asn Thr Val Gln65
70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Arg Asp Tyr Tyr Gly Asn Phe Tyr Tyr Ala Met Asp
Tyr Trp 100 105 110Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr145 150 155 160Val Ser Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro 165 170 175Ala Val Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr 180 185 190Val
Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp 195 200
205His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr
210 215 220Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly
Gly Pro225 230 235 240Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser 245 250 255Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser Gln Glu Asp 260 265 270Pro Glu Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn 275 280 285Ala Lys Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val 290 295 300Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu305 310 315
320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
Tyr Thr 340 345 350Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln
Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr Thr Pro Pro Val Leu385 390 395 400Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys 405 410 415Ser Arg Trp
Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly 435 440
445Lys60447PRTArtificialAnti-HLA VH sequence formatted onto IgG4
incorporating the hinge stabilising substitution S228P 60Gln Val
Gln Leu Lys Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln1 5 10 15Ser
Leu Ser Leu Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25
30Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu
35 40 45Gly Val Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe
Ile 50 55 60Ser Arg Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln Val
Phe Phe65 70 75 80Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile
Tyr Tyr Cys Ala 85 90 95Arg Thr Phe Thr Thr Ser Thr Ser Ala Trp Phe
Ala Tyr Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ala Ala
Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Lys Thr
Tyr Thr Cys Asn Val Asp His Lys 195 200 205Pro Ser Asn Thr Lys Val
Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro
Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val225 230 235 240Phe
Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250
255Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu
260 265 270Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Leu Gly Lys 435 440 44561611PRTArtificialA10.21VH
sequence formatted onto IgG4 backbone incorporating hinge
stabilisation subsitution S228P and the deletion of Threonine
residue at position 553 (ieT106 in interferon sequence) 61Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr
Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Ser 20 25
30Val Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met
35 40 45Gly Trp Ile Asp Pro Glu Tyr Gly Arg Thr Asp Val Ala Glu Lys
Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr Gly Phe
Pro Tyr Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp
His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270Val Gln Phe
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr
Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295
300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile
Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr Lys
Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp
Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410
415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu
420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly
Lys Cys 435 440 445Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg
Thr Leu Met Leu 450 455 460Leu Ala Gln Met Arg Arg Ile Ser Leu Phe
Ser Cys Leu Lys Asp Arg465 470 475 480His Asp Phe Gly Phe Pro Gln
Glu Glu Phe Gly Asn Gln Phe Gln Lys 485 490 495Ala Glu Thr Ile Pro
Val Leu His Glu Met Ile Gln Gln Ile Phe Asn 500 505 510Leu Phe Ser
Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu 515 520 525Asp
Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala 530 535
540Cys Val Ile Gln Gly Val Gly Val Glu Thr Pro Leu Met Lys Glu
Asp545 550 555 560Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile
Thr Leu Tyr Leu 565 570 575Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp
Glu Val Val Arg Asp Glu 580 585 590Ile Met Arg Ser Phe Ser Leu Ser
Thr Asn Leu Gln Glu Ser Leu Arg 595 600 605Ser Lys Glu
61062612PRTArtificialA10.21 IgG4 IFN 62Glu Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys
Lys Val Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30Val Met Asn Trp Val
Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp
Pro Glu Tyr Gly Arg Thr Asp Val Ala Glu Lys Phe 50 55 60Gln Gly Arg
Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr Gly Phe Pro Tyr Trp Gly Gln
100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val 115 120 125Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser
Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205Pro
Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215
220Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr
Ile Thr Arg Glu 245 250 255Pro Glu Val Thr Cys Val Val Val Asp Val
Ser Gln Glu Asp Pro Glu 260 265 270Val Gln Phe Asn Trp Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu
Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr Val
Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315 320Cys
Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330
335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
340 345 350Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr
Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Glu Gly Asn
Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His Asn His
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Cys 435 440 445Asp
Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu 450 455
460Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp
Arg465 470 475 480His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn
Gln Phe Gln Lys 485 490 495Ala Glu Thr Ile Pro Val Leu His Glu Met
Ile Gln Gln Ile Phe Asn 500 505 510Leu Phe Ser Thr Lys Asp Ser Ser
Ala Ala Trp Asp Glu Thr Leu Leu 515 520 525Asp Lys Phe Tyr Thr Glu
Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala 530 535 540Cys Val Ile Gln
Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys Glu545 550 555 560Asp
Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr 565 570
575Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Asp
580 585 590Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu
Ser Leu 595 600 605Arg Ser Lys Glu 61063612PRTArtificialA10.21 IgG4
IFN 63Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly
Ala1 5 10 15Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr
Asp Ser 20 25 30Val Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu
Glu Trp Met 35 40 45Gly Trp Ile Asp Pro Glu Tyr Gly Arg Thr Asp Val
Ala Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser
Thr Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu
Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Lys Tyr Asn Ser Gly
Tyr Gly Phe Pro Tyr Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val
Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala
Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140Leu Gly
Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155
160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
Val Asp His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro Cys Pro Ala Pro
Glu Phe Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270Val
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280
285Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395
400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
405 410 415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly Lys Cys 435 440 445Asp Leu Pro Gln Thr His Ser Leu Gly Ser
Arg Arg Thr Leu Met Leu 450 455 460Leu Ala Gln Met Arg Arg Ile Ser
Leu Phe Ser Cys Leu Lys Asp Arg465 470 475 480His Asp Phe Gly Phe
Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys 485 490 495Ala Glu Thr
Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn 500 505 510Leu
Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu 515 520
525Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala
530 535 540Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met
Lys Glu545 550 555 560Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu Tyr 565 570 575Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Ile Ala 580 585 590Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser Leu 595 600 605Arg Ser Lys Glu
61064612PRTArtificialA10.21 IgG4 IFNVARIANT(592)..(592)Where Xaa
can be any amino acid selected from G, K 64Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser
Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30Val Met Asn Trp
Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile
Asp Pro Glu Tyr Gly Arg Thr Asp Val Ala Glu Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr Gly Phe Pro Tyr Trp Gly
Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro
210 215 220Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser Gln Glu Asp Pro Glu 260 265 270Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Tyr
Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Leu Gly Lys Cys 435 440 445Asp Leu Pro Gln Thr His
Ser Leu Gly Ser Arg Arg Thr Leu Met Leu 450 455 460Leu Ala Gln Met
Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg465 470 475 480His
Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys 485 490
495Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn
500 505 510Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr
Leu Leu 515 520 525Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn
Asp Leu Glu Ala 530 535 540Cys Val Ile Gln Gly Val Gly Val Ala Glu
Thr Pro Leu Met Lys Glu545 550 555 560Asp Ser Ile Leu Ala Val Arg
Lys Tyr Phe Gln Arg Ile Thr Leu Tyr 565 570 575Leu Lys Glu Lys Lys
Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Xaa 580 585 590Glu Ile Met
Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu 595 600 605Arg
Ser Lys Glu 61065612PRTArtificialA10.21 IgG4 IFN 65Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys
Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30Val Met
Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly
Trp Ile Asp Pro Glu Tyr Gly Arg Thr Asp Val Ala Glu Lys Phe 50 55
60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr Gly Phe Pro Tyr Trp
Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr
Ser Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser
Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro
210 215 220Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser Gln Glu Asp Pro Glu 260 265 270Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Glu
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Cys 435 440
445Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu
450 455 460Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys
Asp Ala465 470 475 480His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly
Asn Gln Phe Gln Lys 485 490 495Ala Glu Thr Ile Pro Val Leu His Glu
Met Ile Gln Gln Ile Phe Asn 500 505 510Leu Phe Ser Thr Lys Asp Ser
Ser Ala Ala Trp Asp Glu Thr Leu Leu 515 520 525Asp Lys Phe Tyr Thr
Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala 530 535 540Cys Val Ile
Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys Glu545 550 555
560Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr
565 570 575Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val
Arg Ala 580 585 590Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu
Gln Glu Ser Leu 595 600 605Arg Ser Lys Glu
61066612PRTArtificialA10.21 IgG4 IFNVARIANT(592)..(592)Where Xaa
can be any amino acid selected from the group Q, N 66Glu Val Gln
Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val
Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30Val
Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40
45Gly Trp Ile Asp Pro Glu Tyr Gly Arg Thr Asp Val Ala Glu Lys Phe
50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala
Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr Gly Phe Pro
Tyr Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser
Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Cys Ser Arg
Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185
190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys
195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr
Gly Pro 210 215 220Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly
Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val
Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270Val Gln Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro
Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310
315 320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425
430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Cys
435 440 445Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu
Met Leu 450 455 460Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys
Leu Lys Asp Arg465 470 475 480His Asp Phe Gly Phe Pro Gln Glu Glu
Phe Gly Asn Gln Phe Gln Lys 485 490 495Ala Glu Thr Ile Pro Val Leu
His Glu Met Ile Gln Gln Ile Phe Asn 500 505 510Leu Phe Ser Thr Lys
Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu 515 520 525Asp Lys Phe
Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala 530 535 540Cys
Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys Glu545 550
555 560Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
Tyr 565 570 575Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
Val Arg Xaa 580 585 590Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn
Leu Gln Glu Ser Leu 595 600 605Arg Ser Lys Glu
61067612PRTArtificialA10.21 IgG4 IFNVARIANT(591)..(591)Where Xaa
can be any amino acid selected from H, N 67Glu Val Gln Leu Val Gln
Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser
Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30Val Met Asn Trp
Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile
Asp Pro Glu Tyr Gly Arg Thr Asp Val Ala Glu Lys Phe 50 55 60Gln Gly
Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75
80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr Gly Phe Pro Tyr Trp Gly
Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly
Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro
210 215 220Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser Gln Glu Asp Pro Glu 260 265 270Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile
325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Val Leu Asp Ser385 390 395 400Asp Gly Ser Phe Phe
Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415Trp Gln Glu
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430His
Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys Cys 435 440
445Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu
450 455 460Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys
Asp Arg465 470 475 480His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly
Asn Gln Phe Gln Lys 485 490 495Ala Glu Thr Ile Pro Val Leu His Glu
Met Ile Gln Gln Ile Phe Asn 500 505 510Leu Phe Ser Thr Lys Asp Ser
Ser Ala Ala Trp Asp Glu Thr Leu Leu 515 520 525Asp Lys Phe Tyr Thr
Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala 530 535 540Cys Val Ile
Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys Glu545 550 555
560Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr
565 570 575Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val
Xaa Ala 580 585 590Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu
Gln Glu Ser Leu 595 600 605Arg Ser Lys Glu
61068615PRTArtificialA10.21 IgG1 IFN 68Glu Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys
Lys Val Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30Val Met Asn Trp Val
Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp
Pro Glu Tyr Gly Arg Thr Asp Val Ala Glu Lys Phe 50 55 60Gln Gly Arg
Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr Gly Phe Pro Tyr Trp Gly Gln
100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215
220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His 275
280 285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
Arg 290 295 300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
Asn Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala
Leu Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395
400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His 420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 435 440 445Gly Lys Cys Asp Leu Pro Gln Thr His Ser
Leu Gly Ser Arg Arg Thr 450 455 460Leu Met Leu Leu Ala Gln Met Arg
Arg Ile Ser Leu Phe Ser Cys Leu465 470 475 480Lys Asp Arg His Asp
Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln 485 490 495Phe Gln Lys
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln 500 505 510Ile
Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu 515 520
525Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp
530 535 540Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr
Pro Leu545 550 555 560Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys
Tyr Phe Gln Arg Ile 565 570 575Thr Leu Tyr Leu Lys Glu Lys Lys Tyr
Ser Pro Cys Ala Trp Glu Val 580 585 590Val Arg Asp Glu Ile Met Arg
Ser Phe Ser Leu Ser Thr Asn Leu Gln 595 600 605Glu Ser Leu Arg Ser
Lys Glu 610 61569615PRTArtificialA10.21 IgG1 IFN 69Glu Val Gln Leu
Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys
Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30Val Met
Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly
Trp Ile Asp Pro Glu Tyr Gly Arg Thr Asp Val Ala Glu Lys Phe 50 55
60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65
70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr
Cys 85 90 95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr Gly Phe Pro Tyr Trp
Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys
Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr
Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr
Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala
Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser
Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser
Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Ala
Gly Ala225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
Asp Thr Leu Met Ile 245 250 255Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315
320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln
Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn
Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440
445Gly Lys Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr
450 455 460Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser
Cys Leu465 470 475 480Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu
Glu Phe Gly Asn Gln 485 490 495Phe Gln Lys Ala Glu Thr Ile Pro Val
Leu His Glu Met Ile Gln Gln 500 505 510Ile Phe Asn Leu Phe Ser Thr
Lys Asp Ser Ser Ala Ala Trp Asp Glu 515 520 525Thr Leu Leu Asp Lys
Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp 530 535 540Leu Glu Ala
Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu545 550 555
560Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile
565 570 575Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp
Glu Val 580 585 590Val Arg Asp Glu Ile Met Arg Ser Phe Ser Leu Ser
Thr Asn Leu Gln 595 600 605Glu Ser Leu Arg Ser Lys Glu 610
61570615PRTArtificialA10.21 IgG1 IFN 70Glu Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys
Lys Val Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30Val Met Asn Trp Val
Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp
Pro Glu Tyr Gly Arg Thr Asp Val Ala Glu Lys Phe 50 55 60Gln Gly Arg
Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr Gly Phe Pro Tyr Trp Gly Gln
100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215
220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Tyr Ile 245 250 255Thr Arg Glu Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330
335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly
Lys Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr 450 455
460Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys
Leu465 470 475 480Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu
Phe Gly Asn Gln 485 490 495Phe Gln Lys Ala Glu Thr Ile Pro Val Leu
His Glu Met Ile Gln Gln 500 505 510Ile Phe Asn Leu Phe Ser Thr Lys
Asp Ser Ser Ala Ala Trp Asp Glu 515 520 525Thr Leu Leu Asp Lys Phe
Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp 530 535 540Leu Glu Ala Cys
Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu545 550 555 560Met
Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile 565 570
575Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
580 585 590Val Arg Asp Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn
Leu Gln 595 600 605Glu Ser Leu Arg Ser Lys Glu 610
61571615PRTArtificialA10.21 IgG1 IFN 71Glu Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys
Lys Val Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30Val Met Asn Trp Val
Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp
Pro Glu Tyr Gly Arg Thr Asp Val Ala Glu Lys Phe 50 55 60Gln Gly Arg
Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr Gly Phe Pro Tyr Trp Gly Gln
100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val 115 120 125Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser
Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys 195 200 205Pro
Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp 210 215
220Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Ala Gly
Ala225 230 235 240Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Tyr Ile 245 250 255Thr Arg Glu Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His Glu 260 265 270Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val His 275 280 285Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys305 310 315 320Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 325 330
335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser Leu 355 360 365Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp 370 375 380Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val385 390 395 400Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435 440 445Gly
Lys Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr 450 455
460Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys
Leu465 470 475 480Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu
Phe Gly Asn Gln 485 490 495Phe Gln Lys Ala Glu Thr Ile Pro Val Leu
His Glu Met Ile Gln Gln 500 505 510Ile Phe Asn Leu Phe Ser Thr Lys
Asp Ser Ser Ala Ala Trp Asp Glu 515 520 525Thr Leu Leu Asp Lys Phe
Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp 530 535 540Leu Glu Ala Cys
Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu545 550 555 560Met
Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile 565 570
575Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
580 585 590Val Arg Asp Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn
Leu Gln 595 600 605Glu Ser Leu Arg Ser Lys Glu 610
61572611PRTArtificialA10.21 IgG2 IFN 72Glu Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys
Lys Val Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30Val Met Asn Trp Val
Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp
Pro Glu Tyr Gly Arg Thr Asp Val Ala Glu Lys Phe 50 55 60Gln Gly Arg
Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr Gly Phe Pro Tyr Trp Gly Gln
100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val 115 120 125Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205Pro
Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val 210 215
220Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val
Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val 260 265 270Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Phe Arg Val Val Ser Val 290 295 300Leu
Thr Val Val His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310
315 320Lys Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile
Ser 325 330 335Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
Leu Pro Pro 340 345 350Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser
Leu Thr Cys Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala
Val Glu Trp Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys
Thr Thr Pro Pro Met Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Gln
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425
430Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Cys Asp
435 440 445Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met
Leu Leu 450 455 460Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu
Lys Asp Arg His465 470 475 480Asp Phe Gly Phe Pro Gln Glu Glu Phe
Gly Asn Gln Phe Gln Lys Ala 485 490 495Glu Thr Ile Pro Val Leu His
Glu Met Ile Gln Gln Ile Phe Asn Leu 500 505 510Phe Ser Thr Lys Asp
Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu Asp 515 520 525Lys Phe Tyr
Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys 530 535 540Val
Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys Glu Asp545 550
555 560Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr
Leu 565 570 575Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val
Arg Asp Glu 580 585 590Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu
Gln Glu Ser Leu Arg 595 600 605Ser Lys Glu
61073611PRTArtificialA10.21 IgG2 IFN 73Glu Val Gln Leu Val Gln Ser
Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys
Lys Val Ser Gly Tyr Thr Phe Thr Asp Ser 20 25 30Val Met Asn Trp Val
Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp
Pro Glu Tyr Gly Arg Thr Asp Val Ala Glu Lys Phe 50 55 60Gln Gly Arg
Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80Met
Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr Gly Phe Pro Tyr Trp Gly Gln
100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val 115 120 125Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu
Ser Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro
Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr
Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly
Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Asn
Phe Gly Thr Gln Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205Pro
Ser Asn Thr Lys Val Asp Lys Thr Val Glu Arg Lys Cys Cys Val 210 215
220Glu Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val
Phe225 230 235 240Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Tyr Ile
Thr Arg Glu Pro 245 250 255Glu Val Thr Cys Val Val Val Asp Val Ser
His Glu Asp Pro Glu Val 260 265 270Gln Phe Asn Trp Tyr Val Asp Gly
Val Glu Val His Asn Ala Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln
Phe Asn Ser Thr Phe Arg Val Val Ser Val 290 295 300Leu Thr Val Val
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys305 310 315 320Lys
Val Ser Asn Lys Gly Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 325 330
335Lys Thr Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
340 345 350Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
Leu Val 355 360 365Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
Glu Ser Asn Gly 370 375 380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro
Pro Met Leu Asp Ser Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp 405 410 415Gln Gln Gly Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430Asn His Tyr
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Cys Asp 435 440 445Leu
Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu Leu 450 455
460Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg
His465 470 475 480Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln
Phe Gln Lys Ala 485 490 495Glu Thr Ile Pro Val Leu His Glu Met Ile
Gln Gln Ile Phe Asn Leu 500 505 510Phe Ser Thr Lys Asp Ser Ser Ala
Ala Trp Asp Glu Thr Leu Leu Asp 515 520 525Lys Phe Tyr Thr Glu Leu
Tyr Gln Gln Leu Asn Asp Leu Glu Ala Cys 530 535 540Val Ile Gln Gly
Val Gly Val Ala Glu Thr Pro Leu Met Lys Glu Asp545 550 555 560Ser
Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr Leu 565 570
575Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Asp Glu
580 585 590Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser
Leu Arg 595 600 605Ser Lys Glu 61074612PRTArtificialA10.43 IgG4 IFN
74Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Ser Val Lys Val Ser Cys Lys Val Ser Gly Tyr Thr Leu Thr Asp
Ser 20 25 30Val Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Asp Pro Glu Tyr Gly Arg Thr Asp Val Ala
Gln Lys Phe 50 55 60Gln Gly Arg Val Thr Met Thr Ala Asp Thr Ser Thr
Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr
Gly Phe Pro Tyr Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155
160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
Val Asp His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro Cys Pro Ala Pro
Glu Phe Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270Val
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280
285Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395
400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
405 410 415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly Lys Cys 435 440 445Asp Leu Pro Gln Thr His Ser Leu Gly Ser
Arg Arg Thr Leu Met Leu 450 455 460Leu Ala Gln Met Arg Arg Ile Ser
Leu Phe Ser Cys Leu Lys Asp Arg465 470 475 480His Asp Phe Gly Phe
Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys 485 490 495Ala Glu Thr
Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn 500 505 510Leu
Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu Leu 515 520
525Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala
530 535 540Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met
Lys Glu545 550 555 560Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln
Arg Ile Thr Leu Tyr 565 570 575Leu Lys Glu Lys Lys Tyr Ser Pro Cys
Ala Trp Glu Val Val Arg Asp 580 585 590Glu Ile Met Arg Ser Phe Ser
Leu Ser Thr Asn Leu Gln Glu Ser Leu 595 600 605Arg Ser Lys Glu
61075612PRTArtificialR10A2 IgG4 IFNVARIANT(553)..(553)Where Xaa can
be any amino acid selected from A, C, D, E, F, G, H, I, L, K, M, N,
P, Q, R, S, V, W, Y 75Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Val
Gly Arg Pro Gly Ser1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly
Tyr Thr Phe Thr Asp Ser 20 25 30Val Met Asn Trp Val Lys Gln Ser Pro
Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Trp Ile Asp Pro Glu Tyr Gly
Arg Thr Asp Val Ala Glu Lys Phe 50 55 60Lys Lys Lys Ala Thr Leu Thr
Ala Asp Ser Ser Ser Ser Thr Ala Tyr65 70 75 80Ile Tyr Leu Ser Gly
Leu Thr Ser Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95Ala Arg Thr Lys
Tyr Asn Ser Gly Tyr Gly Phe Pro Tyr Trp Gly Gln 100 105 110Gly Ser
Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120
125Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala
130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr
Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu
Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Lys
Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205Pro Ser Asn Thr Lys
Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220Pro Cys Pro
Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val225 230 235
240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
245 250 255Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
Pro Glu 260 265 270Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser
Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn
Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro
Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360
365Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn
370 375 380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
Asp Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr
Val Asp Lys Ser Arg 405 410 415Trp Gln Glu Gly Asn Val Phe Ser Cys
Ser Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys
Ser Leu Ser Leu Ser Leu Gly Lys Cys 435 440 445Asp Leu Pro Gln Thr
His Ser Leu Gly Ser Arg Arg Thr Leu Met Leu 450 455 460Leu Ala Gln
Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg465 470 475
480His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys
485 490 495Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile
Phe Asn 500 505 510Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp
Glu Thr Leu Leu 515 520 525Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln
Leu Asn Asp Leu Glu Ala 530 535 540Cys Val Ile Gln Gly Val Gly Val
Xaa Glu Thr Pro Leu Met Lys Glu545 550 555 560Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr 565 570 575Leu Lys Glu
Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Asp 580 585 590Glu
Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu 595 600
605Arg Ser Lys Glu 61076612PRTArtificialA10.152 IgG4 IFN 76Glu Val
Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Thr
Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Ser 20 25
30Val Met Asn Trp Val Gln Gln His Pro Gly Lys Gly Leu Glu Trp Met
35 40 45Gly Trp Ile Asp Pro Glu Tyr Gly Arg Thr Asp Val Ala Glu Lys
Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp Thr
Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr Gly Phe
Pro Tyr Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser Ala
Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Cys Ser
Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys Leu Val
Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn
Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp
His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser
Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe
Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys
Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260
265 270Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys 275 280 285Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
Val Val Ser 290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly Leu
Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Leu Gly Lys Cys 435 440 445Asp Leu Pro Gln Thr His
Ser Leu Gly Ser Arg Arg Thr Leu Met Leu 450 455 460Leu Ala Gln Met
Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg465 470 475 480His
Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys 485 490
495Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn
500 505 510Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr
Leu Leu 515 520 525Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn
Asp Leu Glu Ala 530 535 540Cys Val Ile Gln Gly Val Gly Val Ala Glu
Thr Pro Leu Met Lys Glu545 550 555 560Asp Ser Ile Leu Ala Val Arg
Lys Tyr Phe Gln Arg Ile Thr Leu Tyr 565 570 575Leu Lys Glu Lys Lys
Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Asp 580 585 590Glu Ile Met
Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu 595 600 605Arg
Ser Lys Glu 61077613PRTArtificialA02.12 IgG4 IFN 77Gln Leu Gln Leu
Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu Ser
Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Ser 20 25 30Ser Tyr
Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45Trp
Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Asn Tyr Asn Pro Ser 50 55
60Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe65
70 75 80Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr
Tyr 85 90 95Cys Ala Arg Val Gly Gly Ala Gly Gly Trp Pro Leu Asp Val
Trp Gly 100 105 110Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
Lys Gly Pro Ser 115 120 125Val Phe Pro Leu Ala Pro Cys Ser Arg Ser
Thr Ser Glu Ser Thr Ala 130 135 140Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val Thr Val145 150 155 160Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190Pro
Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His 195 200
205Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly
210 215 220Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
Pro Ser225 230 235 240Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
Leu Met Ile Ser Arg 245 250 255Thr Pro Glu Val Thr Cys Val Val Val
Asp Val Ser Gln Glu Asp Pro 260 265 270Glu Val Gln Phe Asn Trp Tyr
Val Asp Gly Val Glu Val His Asn Ala 275 280 285Lys Thr Lys Pro Arg
Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val 290 295 300Ser Val Leu
Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr305 310 315
320Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
325 330 335Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
Thr Leu 340 345 350Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val
Ser Leu Thr Cys 355 360 365Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu Trp Glu Ser 370 375 380Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro Val Leu Asp385 390 395 400Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser 405 410 415Arg Trp Gln
Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440
445Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met
450 455 460Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu
Lys Asp465 470 475 480Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe
Gly Asn Gln Phe Gln 485 490 495Lys Ala Glu Thr Ile Pro Val Leu His
Glu Met Ile Gln Gln Ile Phe 500 505 510Asn Leu Phe Ser Thr Lys Asp
Ser Ser Ala Ala Trp Asp Glu Thr Leu 515 520 525Leu Asp Lys Phe Tyr
Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 530 535 540Ala Cys Val
Ile Gln Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys545 550 555
560Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
565 570 575Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
Val Arg 580 585 590Asp Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn
Leu Gln Glu Ser 595 600 605Leu Arg Ser Lys Glu
61078566PRTArtificialOPG-Fc (IgG2) IFN 78Glu Thr Phe Pro Pro Lys
Tyr Leu His Tyr Asp Glu Glu Thr Ser His1 5 10 15Gln Leu Leu Cys Asp
Lys Cys Pro Pro Gly Thr Tyr Leu Lys Gln His 20 25 30Cys Thr Ala Lys
Trp Lys Thr Val Cys Ala Pro Cys Pro Asp His Tyr 35 40 45Tyr Thr Asp
Ser Trp His Thr Ser Asp Glu Cys Leu Tyr Cys Ser Pro 50 55 60Val Cys
Lys Glu Leu Gln Tyr Val Lys Gln Glu Cys Asn Arg Thr His65 70 75
80Asn Arg Val Cys Glu Cys Lys Glu Gly Arg Tyr Leu Glu Gly Glu Phe
85 90 95Cys Leu Lys His Arg Ser Cys Pro Pro Gly Phe Gly Val Val Gln
Ala 100 105 110Gly Thr Pro Glu Arg Asn Thr Val Cys Lys Arg Cys Pro
Asp Gly Phe 115 120 125Phe Ser Asn Glu Thr Ser Ser Lys Ala Pro Cys
Arg Lys His Thr Asn 130 135 140Cys Ser Val Phe Gly Leu Leu Leu Thr
Gln Lys Gly Asn Ala Thr His145 150 155 160Asp Asn Ile Cys Ser Gly
Asn Ser Glu Ser Thr Gln Lys Glu Arg Lys 165 170 175Cys Cys Val Glu
Cys Pro Pro Cys Pro Ala Pro Pro Val Ala Gly Pro 180 185 190Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 195 200
205Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp
210 215 220Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val
His Asn225 230 235 240Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn
Ser Thr Phe Arg Val 245 250 255Val Ser Val Leu Thr Val Val His Gln
Asp Trp Leu Asn Gly Lys Glu 260 265 270Tyr Lys Cys Lys Val Ser Asn
Lys Gly Leu Pro Ala Pro Ile Glu Lys 275 280 285Thr Ile Ser Lys Thr
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 290 295 300Leu Pro Pro
Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr305 310 315
320Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
325 330 335Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
Met Leu 340 345 350Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
Thr Val Asp Lys 355 360 365Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys Ser Val Met His Glu 370 375 380Ala Leu His Asn His Tyr Thr Gln
Lys Ser Leu Ser Leu Ser Pro Gly385 390 395 400Lys Cys Asp Leu Pro
Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu 405 410 415Met Leu Leu
Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys 420 425 430Asp
Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe 435 440
445Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile
450 455 460Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp
Glu Thr465 470 475 480Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln
Gln Leu Asn Asp Leu 485 490 495Glu Ala Cys Val Ile Gln Gly Val Gly
Val Ala Glu Thr Pro Leu Met 500 505 510Lys Glu Asp Ser Ile Leu Ala
Val Arg Lys Tyr Phe Gln Arg Ile Thr 515 520 525Leu Tyr Leu Lys Glu
Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val 530 535 540Arg Asp Glu
Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu545 550 555
560Ser Leu Arg Ser Lys Glu 56579614PRTArtificialAnti-CD138 IgG4 IFN
79Gln Val Gln Leu Gln Gln Ser Gly Ser Glu Leu Met Met Pro Gly Ala1
5 10 15Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Asn
Tyr 20 25 30Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu
Trp Ile 35 40 45Gly Glu Ile Leu Pro Gly Thr Gly Arg Thr Ile Tyr Asn
Glu Lys Phe 50 55 60Lys Gly Lys Ala Thr Phe Thr Ala Asp Ile Ser Ser
Asn Thr Val Gln65 70 75 80Met Gln Leu Ser Ser Leu Thr Ser Glu Asp
Ser Ala Val Tyr Tyr Cys 85 90 95Ala Arg Arg Asp Tyr Tyr Gly Asn Phe
Tyr Tyr Ala Met Asp Tyr Trp 100 105 110Gly Gln Gly Thr Ser Val Thr
Val Ser Ser Ala Ser Thr Lys Gly Pro 115 120 125Ser Val Phe Pro Leu
Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr 130 135 140Ala Ala Leu
Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr145 150 155
160Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro
165 170 175Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
Val Thr 180 185 190Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr
Cys Asn Val Asp 195 200 205His Lys Pro Ser Asn Thr Lys Val Asp Lys
Arg Val Glu Ser Lys Tyr 210 215 220Gly Pro Pro Cys Pro Pro Cys Pro
Ala Pro Glu Phe Leu Gly Gly Pro225 230 235 240Ser Val Phe Leu Phe
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255Arg Thr Pro
Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp 260 265 270Pro
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280
285Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
290 295 300Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys Glu305 310 315 320Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro
Ser Ser Ile Glu Lys 325 330 335Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr Thr 340 345 350Leu Pro Pro Ser Gln Glu Glu
Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu385 390 395
400Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
405 410 415Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met
His Glu 420 425 430Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
Leu Ser Leu Gly 435 440 445Lys Cys Asp Leu Pro Gln Thr His Ser Leu
Gly Ser Arg Arg Thr Leu 450 455 460Met Leu Leu Ala Gln Met Arg Arg
Ile Ser Leu Phe Ser Cys Leu Lys465 470 475 480Asp Arg His Asp Phe
Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe 485 490 495Gln Lys Ala
Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile 500 505 510Phe
Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr 515 520
525Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu
530 535 540Glu Ala Cys Val Ile Gln Gly Val Gly Val Ala Glu Thr Pro
Leu Met545 550 555 560Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr
Phe Gln Arg Ile Thr 565 570 575Leu Tyr Leu Lys Glu Lys Lys Tyr Ser
Pro Cys Ala Trp Glu Val Val 580 585 590Arg Asp Glu Ile Met Arg Ser
Phe Ser Leu Ser Thr Asn Leu Gln Glu 595 600 605Ser Leu Arg Ser Lys
Glu 61080612PRTArtificialAnti-HLA IgG4 IFN 80Gln Val Gln Leu Lys
Gln Ser Gly Pro Gly Leu Val Gln Pro Ser Gln1 5 10 15Ser Leu Ser Leu
Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Ser Tyr 20 25 30Gly Val His
Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Leu 35 40 45Gly Val
Ile Trp Ser Gly Gly Ser Thr Asp Tyr Asn Ala Ala Phe Ile 50 55 60Ser
Arg Leu Ser Ile Arg Lys Asp Asn Ser Lys Ser Gln Val Phe Phe65 70 75
80Lys Met Asn Ser Leu Gln Ala Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95Arg Thr Phe Thr Thr Ser Thr Ser Ala Trp Phe Ala Tyr Trp Gly
Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly
Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser
Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val Ser145 150 155 160Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190Ser Ser
Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200
205Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro
210 215 220Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro
Ser Val225 230 235 240Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu
Met Ile Ser Arg Thr 245 250 255Pro Glu Val Thr Cys Val Val Val Asp
Val Ser Gln Glu Asp Pro Glu 260 265 270Val Gln Phe Asn Trp Tyr Val
Asp Gly Val Glu Val His Asn Ala Lys 275 280 285Thr Lys Pro Arg Glu
Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300Val Leu Thr
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys305 310 315
320Cys Lys Val Ser Asn Lys Gly
Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln
Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val
Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375
380Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
Ser385 390 395 400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val
Asp Lys Ser Arg 405 410 415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser
Val Met His Glu Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Leu Gly Lys Cys 435 440 445Asp Leu Pro Gln Thr His
Ser Leu Gly Ser Arg Arg Thr Leu Met Leu 450 455 460Leu Ala Gln Met
Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp Arg465 470 475 480His
Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln Lys 485 490
495Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe Asn
500 505 510Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr
Leu Leu 515 520 525Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn
Asp Leu Glu Ala 530 535 540Cys Val Ile Gln Gly Val Gly Val Ala Glu
Thr Pro Leu Met Lys Glu545 550 555 560Asp Ser Ile Leu Ala Val Arg
Lys Tyr Phe Gln Arg Ile Thr Leu Tyr 565 570 575Leu Lys Glu Lys Lys
Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Asp 580 585 590Glu Ile Met
Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu 595 600 605Arg
Ser Lys Glu 61081214PRTArtificialA10.21 and A10.43 light chain
81Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1
5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asn Val Asp Ser
Asp 20 25 30Val Asp Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu
Leu Ile 35 40 45Tyr Lys Ala Ser Asn Asp Tyr Thr Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser
Ser Leu Gln Pro65 70 75 80Glu Asp Ile Ala Thr Tyr Tyr Cys Met Gln
Ser Asn Thr His Pro Arg 85 90 95Thr Phe Gly Gly Gly Thr Lys Val Glu
Ile Lys Arg Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro
Pro Ser Asp Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val
Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln
Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155
160Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
Val Tyr 180 185 190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro
Val Thr Lys Ser 195 200 205Phe Asn Arg Gly Glu Cys
21082214PRTArtificialR10A2 light chain 82Asp Ile Val Met Thr Gln
Ser Pro Thr Ser Ile Ser Ile Ser Val Gly1 5 10 15Glu Arg Val Thr Met
Asn Cys Lys Ala Ser Gln Asn Val Asp Ser Asp 20 25 30Val Asp Trp Tyr
Gln Gln Lys Thr Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala
Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly
Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Asn Met Gln Ala65 70 75
80Glu Asp Leu Ala Val Tyr Tyr Cys Met Gln Ser Asn Thr His Pro Arg
85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg Thr Val Ala
Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe
Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala
Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr
Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys
Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200
205Phe Asn Arg Gly Glu Cys 21083614PRTArtificialA10.21 IgG1 IFN
83Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp
Ser 20 25 30Val Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Asp Pro Glu Tyr Gly Arg Thr Asp Val Ala
Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr
Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr
Gly Phe Pro Tyr Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro
Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155
160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
Val Asn His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val
Glu Pro Lys Ser Cys Asp 210 215 220Lys Thr His Thr Cys Pro Pro Cys
Pro Ala Pro Glu Leu Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 245 250 255Ser Arg Thr
Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270Asp
Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280
285Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn
Gly Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
Pro Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Asp
Glu Leu Thr Lys Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn
Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395
400Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His 420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro 435 440 445Gly Lys Cys Asp Leu Pro Gln Thr His Ser
Leu Gly Ser Arg Arg Thr 450 455 460Leu Met Leu Leu Ala Gln Met Arg
Arg Ile Ser Leu Phe Ser Cys Leu465 470 475 480Lys Asp Arg His Asp
Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln 485 490 495Phe Gln Lys
Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln 500 505 510Ile
Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu 515 520
525Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp
530 535 540Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Glu Thr Pro
Leu Met545 550 555 560Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr
Phe Gln Arg Ile Thr 565 570 575Leu Tyr Leu Lys Glu Lys Lys Tyr Ser
Pro Cys Ala Trp Glu Val Val 580 585 590Arg Asp Glu Ile Met Arg Ser
Phe Ser Leu Ser Thr Asn Leu Gln Glu 595 600 605Ser Leu Arg Ser Lys
Glu 61084222PRTArtificialA02.12 lambda light chain 84Gln Ala Val
Leu Thr Gln Pro Ala Ser Leu Ser Ala Ser Pro Gly Glu1 5 10 15Ser Ala
Arg Leu Thr Cys Thr Leu Pro Ser Asp Ile Asn Val Arg Tyr 20 25 30Tyr
Asn Ile Tyr Trp Tyr Gln Gln Lys Pro Gly Ser Pro Pro Arg Tyr 35 40
45Leu Leu Tyr Tyr Tyr Ser Asp Ser His Lys Gly Gln Gly Ser Gly Val
50 55 60Pro Ser Arg Phe Ser Gly Ser Lys Asp Val Ser Thr Asn Ser Gly
Ile65 70 75 80Leu Leu Ile Ser Gly Leu Gln Ser Glu Asp Ile Ala Thr
Tyr Tyr Cys 85 90 95Met Thr Trp Ser Ser Asn Gly Ser Gly Val Phe Gly
Gly Gly Thr Gln 100 105 110Leu Thr Val Leu Gly Gln Pro Lys Ala Ala
Pro Ser Val Thr Leu Phe 115 120 125Pro Pro Ser Ser Glu Glu Leu Gln
Ala Asn Lys Ala Thr Leu Val Cys 130 135 140Leu Ile Ser Asp Phe Tyr
Pro Gly Ala Val Thr Val Ala Trp Lys Ala145 150 155 160Asp Ser Ser
Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys 165 170 175Gln
Ser Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro 180 185
190Glu Gln Trp Lys Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu
195 200 205Gly Ser Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser
210 215 22085214PRTArtificialAnti-CD138 light chain 85Asp Ile Gln
Met Thr Gln Ser Thr Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Asp Arg
Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Asn Asn Tyr 20 25 30Leu
Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Glu Leu Leu Ile 35 40
45Tyr Tyr Thr Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu
Pro65 70 75 80Glu Asp Ile Gly Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys
Leu Pro Arg 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
Thr Val Ala Ala 100 105 110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly 115 120 125Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln145 150 155 160Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205Phe Asn Arg Gly Glu Cys 21086215PRTArtificialAnti-HLA
light chain 86Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Leu Val
Ser Ala Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser
Val Ser Asn Asp 20 25 30Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser
Pro Lys Leu Leu Ile 35 40 45Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val
Pro Asp Arg Phe Thr Gly 50 55 60Ser Gly Tyr Gly Thr Asp Phe Thr Phe
Thr Ile Ser Thr Val Gln Ala65 70 75 80Glu Asp Leu Ala Val Tyr Phe
Cys Gln Gln Asp Tyr Ser Ser Pro Pro 85 90 95Trp Thr Phe Gly Gly Gly
Thr Lys Leu Glu Ile Arg Arg Thr Val Ala 100 105 110Ala Pro Ser Val
Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125Gly Thr
Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135
140Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
Ser145 150 155 160Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser
Thr Tyr Ser Leu 165 170 175Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp
Tyr Glu Lys His Lys Val 180 185 190Tyr Ala Cys Glu Val Thr His Gln
Gly Leu Ser Ser Pro Val Thr Lys 195 200 205Ser Phe Asn Arg Gly Glu
Cys 210 21587612PRTArtificialA10.21 IgG4 (S228P) IFN (A145D, T106A)
87Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1
5 10 15Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp
Ser 20 25 30Val Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu
Trp Met 35 40 45Gly Trp Ile Asp Pro Glu Tyr Gly Arg Thr Asp Val Ala
Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr
Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr
Gly Phe Pro Tyr Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser
Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro
Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140Leu Gly Cys
Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155
160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr
Val Pro 180 185 190Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn
Val Asp His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Arg Val
Glu Ser Lys Tyr Gly Pro 210 215 220Pro Cys Pro Pro Cys Pro Ala Pro
Glu Phe Leu Gly Gly Pro Ser Val225 230 235 240Phe Leu Phe Pro Pro
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255Pro Glu Val
Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270Val
Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280
285Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser
290 295 300Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr Lys305 310 315 320Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser
Ile Glu Lys Thr Ile 325 330 335Ser Lys Ala Lys Gly Gln Pro Arg Glu
Pro Gln Val Tyr Thr Leu Pro 340 345 350Pro Ser Gln Glu Glu Met Thr
Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser385 390 395
400Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg
405 410 415Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
Ala Leu 420 425 430His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
Leu Gly Lys Cys 435 440 445Asp Leu Pro Gln Thr His Ser Leu Gly Ser
Arg Arg Thr Leu Met Leu 450
455 460Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp
Arg465 470 475 480His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn
Gln Phe Gln Lys 485 490 495Ala Glu Thr Ile Pro Val Leu His Glu Met
Ile Gln Gln Ile Phe Asn 500 505 510Leu Phe Ser Thr Lys Asp Ser Ser
Ala Ala Trp Asp Glu Thr Leu Leu 515 520 525Asp Lys Phe Tyr Thr Glu
Leu Tyr Gln Gln Leu Asn Asp Leu Glu Ala 530 535 540Cys Val Ile Gln
Gly Val Gly Val Ala Glu Thr Pro Leu Met Lys Glu545 550 555 560Asp
Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr 565 570
575Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Asp
580 585 590Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu
Ser Leu 595 600 605Arg Ser Lys Glu 61088495DNAArtificialIFN
alpha-2-beta 88tgtgatctgc ctcaaaccca cagcctgggt agcaggagga
ccttgatgct cctggcacag 60atgaggagaa tctctctttt ctcctgcttg aaggacagac
atgactttgg atttccccag 120gaggagtttg gcaaccagtt ccaaaaggct
gaaaccatcc ctgtcctcca tgagatgatc 180cagcagatct tcaatctctt
cagcacaaag gactcatctg ctgcttggga tgagaccctc 240ctagacaaat
tctacactga actctaccag cagctgaatg acctggaagc ctgtgtgata
300cagggggtgg gggtgacaga gactcccctg atgaaggagg actccattct
ggctgtgagg 360aaatacttcc aaagaatcac tctctatctg aaagagaaga
aatacagccc ttgtgcctgg 420gaggttgtca gagcagaaat catgagatct
ttttctttgt caacaaactt gcaagaaagt 480ttaagaagta aggaa
495891341DNAArtificialPolynucleotide of 10.21 as an IgG2
incorporating S228P 89gaagtgcagc tggtgcagtc tggcgccgaa gtgaagaaac
ccggcgctac cgtgaagatc 60tcctgcaagg tgtccggcta caccttcacc gactccgtga
tgaactgggt gcagcaggcc 120cctggcaagg gcctggaatg gatgggctgg
atcgaccccg agtacggcag aaccgacgtg 180gccgagaagt tccagggcag
agtgaccatc accgccgaca cctccaccga caccgcctac 240atggaactgt
cctccctgcg gagcgaggac accgccgtgt actactgcgc ccggaccaag
300tacaactccg gctacggctt cccctactgg ggccagggca ccaccgtgac
cgtgtcctcc 360gcctccacca agggcccctc cgtgttccct ctggcccctt
gctcccggtc cacctccgag 420tctaccgccg ctctgggctg cctggtcaag
gactacttcc ccgagcctgt gacagtgtcc 480tggaactctg gcgccctgac
ctccggcgtg cacaccttcc ctgccgtgct gcagtcctcc 540ggcctgtact
ccctgtcctc cgtcgtgacc gtgccttcca gctccctggg caccaagacc
600tacacctgta acgtggacca caagccctcc aacaccaagg tggacaagcg
ggtggaatct 660aagtacggcc ctccctgccc cccctgccct gcccctgaat
ttctgggcgg accttccgtg 720ttcctgttcc ccccaaagcc caaggacacc
ctgatgatct cccggacccc cgaagtgacc 780tgcgtggtgg tggacgtgtc
ccaagaggac cccgaggtgc agttcaattg gtacgtggac 840ggcgtggaag
tgcacaacgc caagaccaag cccagagagg aacagttcaa ctccacctac
900cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa
agaatacaag 960tgcaaagtct ccaacaaggg cctgccctcc agcatcgaaa
agaccatctc caaggccaag 1020ggacagcccc gcgagcccca ggtgtacacc
ctgcccccta gccaagagga aatgaccaag 1080aaccaggtgt ccctgacctg
cctcgtgaag ggcttctacc cctccgatat cgccgtggaa 1140tgggagtcca
acggccagcc cgagaacaac tacaagacca ccccccctgt gctggactcc
1200gacggctcct tcttcctgta ctctcggctg accgtggaca agtcccggtg
gcaagagggc 1260aacgtgttct cctgctccgt gatgcacgag gccctgcaca
accactacac ccagaagtcc 1320ctgtccctga gcctgggcaa g
1341901341DNAArtificialPolynucleotide sequence of 10.43 as an IgG4
incorporating S228P 90caggtgcagc tggtgcagtc tggcgccgaa gtgaagaaac
ctggcgcctc cgtgaaggtg 60tcctgcaagg tgtccggcta caccctgacc gactccgtga
tgaactgggt ccgacaggcc 120cctggcaagg gcctggaatg gatgggctgg
atcgaccccg agtacggcag aaccgacgtg 180gcccagaaat tccagggcag
agtgaccatg accgccgaca cctccaccga caccgcctac 240atggaactgt
cctccctgcg gagcgaggac accgccgtgt actactgcgc ccggaccaag
300tacaactccg gctacggctt cccctactgg ggccagggca ccaccgtgac
cgtgtcctct 360gcttccacca agggcccctc cgtgttccct ctggcccctt
gctcccggtc cacctccgag 420tctaccgccg ctctgggctg cctggtcaag
gactacttcc ccgagcctgt gacagtgtcc 480tggaactctg gcgccctgac
ctccggcgtg cacaccttcc ctgccgtgct gcagtcctcc 540ggcctgtact
ccctgtcctc cgtcgtgacc gtgccttcca gctccctggg caccaagacc
600tacacctgta acgtggacca caagccctcc aacaccaagg tggacaagcg
ggtggaatct 660aagtacggcc ctccctgccc cccctgccct gcccctgaat
ttctgggcgg accttccgtg 720ttcctgttcc ccccaaagcc caaggacacc
ctgatgatct cccggacccc cgaagtgacc 780tgcgtggtgg tggacgtgtc
ccaagaggac cccgaggtgc agttcaattg gtacgtggac 840ggcgtggaag
tgcacaacgc caagaccaag cccagagagg aacagttcaa ctccacctac
900cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa
agaatacaag 960tgcaaagtct ccaacaaggg cctgccctcc agcatcgaaa
agaccatctc caaggccaag 1020ggacagcccc gcgagcccca ggtgtacacc
ctgcccccta gccaagagga aatgaccaag 1080aaccaggtgt ccctgacctg
cctcgtgaag ggcttctacc cctccgatat cgccgtggaa 1140tgggagtcca
acggccagcc cgagaacaac tacaagacca ccccccctgt gctggactcc
1200gacggctcct tcttcctgta ctctcggctg accgtggaca agtcccggtg
gcaagagggc 1260aacgtgttct cctgctccgt gatgcacgag gccctgcaca
accactacac ccagaagtcc 1320ctgtccctga gcctgggcaa g
1341911344DNAArtificialPolynucleotide of 2.12 formatted an an IgG4
incorporating S228P 91cagctgcagc tgcaggaatc tggccctggc ctcgtgaagc
ccagcgagac actgagcctg 60acctgtaccg tgtccggcgg cagcatcagc agcagctcct
actactggtc ctggatcaga 120cagcaccccg gcaagggcct ggaatggatc
ggctacatct actacagcgg cagcaccaac 180tacaacccca gcctgaagtc
cagagtgacc atcagcgtgg acaccagcaa gaaccagttc 240tccctgaagc
tgagcagcgt gacagccgcc gataccgccg tgtactactg tgccagagtg
300ggcggagctg gcggctggcc tctggatgtg tggggacagg gcaccaccgt
gacagtgtcc 360tcagctagca ccaagggccc cagcgtgttc cctctggccc
cttgtagcag aagcaccagc 420gagtctacag ccgccctggg ctgcctcgtg
aaggactact ttcccgagcc cgtcaccgtg 480tcctggaact ctggcgctct
gacaagcggc gtgcacacct ttccagccgt gctgcagagc 540agcggcctgt
actctctgag cagcgtcgtg accgtgccca gctctagcct gggcaccaag
600acctacacct gtaacgtgga ccacaagccc agcaacacca aggtggacaa
gcgggtggaa 660tctaagtacg gccctccctg ccctccttgc ccagcccctg
aatttctggg cggaccctcc 720gtgttcctgt tccccccaaa gcccaaggac
accctgatga tcagccggac ccccgaagtg 780acctgcgtgg tggtggatgt
gtcccaggaa gatcccgagg tgcagttcaa ttggtacgtg 840gacggcgtgg
aagtgcacaa cgccaagacc aagcccagag aggaacagtt caacagcacc
900taccgggtgg tgtccgtgct gacagtgctg caccaggact ggctgaacgg
caaagagtac 960aagtgcaagg tgtccaacaa gggcctgcct agcagcatcg
agaaaaccat cagcaaggcc 1020aagggccagc cccgcgaacc tcaggtgtac
acactgcccc ctagccagga agagatgacc 1080aagaaccagg tgtccctgac
ctgtctcgtg aaaggcttct acccctccga tatcgccgtg 1140gaatgggaga
gcaacggcca gcccgagaac aactacaaga ccaccccccc tgtgctggac
1200agcgacggct cattcttcct gtacagcaga ctgaccgtgg acaagagccg
gtggcaggaa 1260ggcaacgtgt tcagctgcag cgtgatgcac gaggccctgc
acaaccacta cacccagaag 1320tccctgtccc tgtctctggg caag
1344921341DNAArtificialPolynucleotide of R10A2 formatted as an IgG4
incorporating S228P 92gaagtccagc tgcagcagtc tggccccgaa gtgggcagac
ctggctcctc cgtgaagatc 60tcctgcaagg cctccggcta caccttcacc gactccgtga
tgaactgggt caagcagtct 120cccggccagg gcctggaatg gatcggatgg
atcgaccccg agtacggcag aaccgacgtg 180gccgagaagt tcaagaagaa
ggccaccctg accgccgact cctccagctc caccgcctac 240atctacctgt
ccggcctgac ctccgaggac accgccacct acttttgcgc ccggaccaag
300tacaacagcg gctacggctt cccctactgg ggacagggct ctctcgtgac
agtgtcctca 360gcctccacca agggcccctc cgtgttccct ctggcccctt
gctcccggtc cacctccgag 420tctaccgccg ctctgggctg cctggtgaaa
gactacttcc ccgagcctgt gaccgtgagc 480tggaactctg gcgccctgac
ctccggcgtg cacaccttcc ctgccgtgct gcagtcctcc 540ggcctgtact
ccctgtcctc cgtggtgaca gtgccctcct ccagcctggg caccaagacc
600tacacctgta acgtggacca caagccctcc aacaccaagg tggacaagcg
ggtggaatct 660aagtacggcc ctccctgccc cccctgccct gcccctgaat
ttctgggcgg accttccgtg 720tttctgttcc ccccaaagcc caaggacacc
ctgatgatct cccggacccc cgaagtgacc 780tgcgtggtgg tggacgtgtc
ccaggaagat ccagaggtgc agttcaattg gtacgtggac 840ggcgtggaag
tgcacaacgc caagaccaag cccagagagg aacagttcaa ctccacctac
900cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa
agagtacaag 960tgcaaggtgt ccaacaaggg cctgccctcc agcatcgaaa
agaccatctc caaggccaag 1020ggccagcccc gcgagcccca ggtgtacacc
ctgcccccta gccaggaaga gatgaccaag 1080aaccaggtgt ccctgacctg
tctggtgaaa ggcttctacc cctccgacat tgccgtggaa 1140tgggagtcca
acggccagcc cgagaacaac tacaagacca ccccccctgt gctggactcc
1200gacggctcct tcttcctgta ctctcggctg acagtggata agtcccggtg
gcaggaaggc 1260aacgtgttct cctgcagcgt gatgcacgag gccctgcaca
accactatac ccagaagtcc 1320ctgtccctga gcctgggcaa g
134193642DNAArtificialPolynucleotide sequence of R10A2 VK
93gacatcgtga tgacccagag ccccacctcc atctccatca gcgtgggcga gcgcgtgacc
60atgaactgca aggcctccca gaacgtggac agcgacgtgg actggtatca gcagaaaacc
120ggccagtccc ccaagctgct gatctacaag gccagcaaca gatacaccgg
cgtgcccgac 180aggttcaccg gctctggctc tggaaccgac ttcaccttca
ccatcagcaa catgcaggcc 240gaggatctgg ccgtgtacta ctgtatgcag
tccaacaccc acccccggac cttcggcgga 300ggcaccaaac tggaactgaa
gcggaccgtg gccgctccca gcgtgttcat cttccccccc 360agcgacgagc
agctgaagag cggcaccgcc agcgtggtct gcctgctgaa caacttctac
420ccccgggagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtctgg
caacagccag 480gaaagcgtca ccgagcagga cagcaaggat agcacctaca
gcctgagcag caccctgacc 540ctgagcaagg ccgactacga gaagcacaag
gtgtacgcct gcgaggtcac ccaccagggc 600ctgtctagcc ccgtcaccaa
gagcttcaac cggggcgagt gc 64294642DNAArtificialPolynucleotide
sequence of VK used to generate antibody 10.21 and antibody 10.43
94gacatccaga tgacccagtc cccctccagc ctgtccgctt ccgtgggcga cagagtgacc
60atcacatgca aggcctccca gaacgtggac tccgacgtgg actggtatca gcagaagccc
120ggcaaggccc ccaagctgct gatctacaag gcctccaacg actacaccgg
cgtgccctcc 180agattctccg gctccggctc tggcaccgac ttcaccttca
ccatctccag cctgcagccc 240gaggatatcg ccacctacta ctgcatgcag
agcaacaccc acccccggac cttcggcgga 300ggcaccaagg tggaaatcaa
gcggaccgtg gccgctccct ccgtgttcat cttcccaccc 360tccgacgagc
agctgaagtc cggcaccgcc tccgtcgtgt gcctgctgaa caacttctac
420ccccgcgagg ccaaggtgca gtggaaggtg gacaacgccc tgcagtccgg
caactcccaa 480gagtccgtga ccgagcagga ctccaaggac agcacctact
ccctgtcctc caccctgacc 540ctgtccaagg ccgactacga gaagcacaag
gtgtacgcct gcgaagtgac ccaccagggc 600ctgtccagcc ccgtgaccaa
gtccttcaac cggggcgagt gc 642951341DNAArtificialPolynucleotide
sequence of 10.152 formatted as an IgG4 incorporating S228P
95gaagtgcagc tggtgcagtc tggcgccgaa gtgaagaaac ccggcgctac cgtgaagatc
60tcctgcaagg tgtccggcta caccttcacc gactccgtga tgaactgggt gcagcagcat
120cccggcaagg gcctggaatg gatgggctgg atcgaccccg agtacggcag
aaccgacgtg 180gccgagaagt tccagggcag agtgaccatc accgccgaca
cctccaccga caccgcctac 240atggaactgt cctccctgcg gagcgaggac
accgccgtgt actactgcgc ccggaccaag 300tacaactccg gctacggctt
cccctactgg ggccagggca ccaccgtgac cgtgtcctct 360gcctccacca
agggcccctc cgtgttccct ctggcccctt gctcccggtc cacctccgag
420tctaccgccg ctctgggctg cctggtcaag gactacttcc ccgagcctgt
gacagtgtcc 480tggaactctg gcgccctgac ctccggcgtg cacaccttcc
ctgccgtgct gcagtcctcc 540ggcctgtact ccctgtcctc cgtcgtgacc
gtgccttcca gctccctggg caccaagacc 600tacacctgta acgtggacca
caagccctcc aacaccaagg tggacaagcg ggtggaatct 660aagtacggcc
ctccctgccc cccctgccct gcccctgaat ttctgggcgg accttccgtg
720ttcctgttcc ccccaaagcc caaggacacc ctgatgatct cccggacccc
cgaagtgacc 780tgcgtggtgg tggacgtgtc ccaagaggac cccgaggtgc
agttcaattg gtacgtggac 840ggcgtggaag tgcacaacgc caagaccaag
cccagagagg aacagttcaa ctccacctac 900cgggtggtgt ccgtgctgac
cgtgctgcac caggactggc tgaacggcaa agaatacaag 960tgcaaagtct
ccaacaaggg cctgccctcc agcatcgaaa agaccatctc caaggccaag
1020ggacagcccc gcgagcccca ggtgtacacc ctgcccccta gccaagagga
aatgaccaag 1080aaccaggtgt ccctgacctg cctcgtgaag ggcttctacc
cctccgatat cgccgtggaa 1140tgggagtcca acggccagcc cgagaacaac
tacaagacca ccccccctgt gctggactcc 1200gacggctcct tcttcctgta
ctctcggctg accgtggaca agtcccggtg gcaagagggc 1260aacgtgttct
cctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc
1320ctgtccctga gcctgggcaa g 134196214PRTArtificialAmino acid
sequence of 10.152 light chain 96Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys
Gln Ala Ser Gln Asn Val Asp Ser Asp 20 25 30Val Asp Trp Tyr Gln Gln
Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45Tyr Lys Ala Ser Asn
Asp Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly
Thr Asp Phe Thr Phe Thr Ile Ser Ser Leu Gln Pro65 70 75 80Glu Asp
Ile Ala Thr Tyr Tyr Cys Ala Gln Ser Asn Thr His Pro Arg 85 90 95Thr
Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 100 105
110Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
Glu Ala 130 135 140Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser
Gly Asn Ser Gln145 150 155 160Glu Ser Val Thr Glu Gln Asp Ser Lys
Asp Ser Thr Tyr Ser Leu Ser 165 170 175Ser Thr Leu Thr Leu Ser Lys
Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185 190Ala Cys Glu Val Thr
His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 195 200 205Phe Asn Arg
Gly Glu Cys 21097642DNAArtificialPolynucleotide sequence of 10.152
light chain 97gacatccaga tgactcagag cccgtcctcg ctttcggctt
ccgtcggcga ccgcgtgacc 60atcacttgtc aggcgtcgca gaacgtcgat tccgacgtgg
actggtacca acagaagccg 120gggaaagcgc ccaagctgct gatctacaag
gcctccaatg attacactgg agtgcctagc 180cggttcagcg gatcagggtc
gggaacggac ttcactttta ccatctcaag cctccaacca 240gaagatattg
ccacctatta ctgcgcacaa tcaaacaccc acccgagaac cttcggcgga
300ggaaccaagg tggagatcaa acgtacggtg gcggcgccca gcgtgttcat
cttcccaccc 360agcgacgagc agctgaagtc cggcacagcc agcgtggtgt
gcctgctgaa caacttctac 420ccccgcgagg ccaaggtgca gtggaaggtg
gacaacgccc tgcagagcgg caacagccag 480gaaagcgtga ccgagcagga
cagcaaggac tccacctaca gcctgagcag caccctgacc 540ctgagcaagg
ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc
600ctgtccagcc ccgtgaccaa gagcttcaac cggggcgagt gc
64298666DNAArtificialPolynucleotide sequence of 2.12 light chain
98caggccgtgc tgacccagcc tgcctccctg tctgcctctc ctggcgagtc cgccagactg
60acctgcaccc tgccctccga catcaacgtg cggtactaca acatctactg gtatcagcag
120aagcccggca gcccccccag atacctgctg tactactact ccgactccca
caagggccag 180ggctccggcg tgccctccag attctccggc tccaaggacg
tgtccaccaa ctccggcatc 240ctgctgatct ccggcctgca gtccgaggac
attgccacct actactgcat gacttggagc 300agcaacggca gcggcgtgtt
cggcggaggc acccagctga ccgtcctagg tcagcccaag 360gccgctccca
gcgtgaccct gttcccccca agcagcgagg aactgcaggc caacaaggcc
420accctggtgt gcctgatcag cgacttctac cctggggccg tgaccgtggc
ctggaaggcc 480gatagcagcc ctgtgaaggc cggcgtggaa accaccaccc
cctccaagca gagcaacaac 540aaatacgccg ccagcagcta cctgtccctg
acccccgagc agtggaagtc ccaccggtcc 600tacagctgcc aggtgacaca
cgagggcagc accgtggaaa agaccgtggc ccccaccgag 660tgcagc
66699642DNAArtificialPolynucleotide sequence of Anti-CD138 kappa
light chain 99gacatccaga tgacccagag cacatcctct ctgagcgcct
ccctgggcga tcgcgtgact 60atcagttgca gcgcttccca agggattaac aattacctca
actggtacca gcagaagccc 120gacggaaccg tcgagctgct catctattac
acatctacgc tgcaaagcgg cgtgccttcc 180aggttctcag ggagcggttc
cggaactgat tactctctga ccattagcaa tctcgaacca 240gaagacatcg
gcacatatta ctgtcagcag tactccaagc tgccccggac ttttggggga
300ggcaccaaac tggagatcaa gcgtacggtg gcggcgccca gcgtgttcat
cttcccaccc 360agcgacgagc agctgaagtc cggcacagcc agcgtggtgt
gcctgctgaa caacttctac 420ccccgcgagg ccaaggtgca gtggaaggtg
gacaacgccc tgcagagcgg caacagccag 480gaaagcgtga ccgagcagga
cagcaaggac tccacctaca gcctgagcag caccctgacc 540ctgagcaagg
ccgactacga gaagcacaag gtgtacgcct gcgaagtgac ccaccagggc
600ctgtccagcc ccgtgaccaa gagcttcaac cggggcgagt gc
6421001347DNAArtificialPolynucleotide sequence of Anti-CD138 IgG4
(S228P) 100caggtgcagc tccaacagag cggctccgaa ctgatgatgc ctggggcctc
tgtcaagatt 60tcctgcaaag ctaccggcta cacattcagc aactattgga ttgagtgggt
gaagcagcgc 120ccagggcacg gtctggagtg gatcggagag atcctgccag
gcaccgggag gactatttac 180aatgaaaagt ttaaaggaaa ggccacattc
accgcagaca tctctagcaa cactgttcaa 240atgcagctct cctctctgac
ctccgaggat agcgccgtgt attactgtgc tcggagagac 300tactatggca
atttttacta tgctatggat tactggggac agggcacatc tgtgaccgtc
360agctccgcta gcaccaaggg ccccagcgtg ttccccctgg ccccttgtag
cagaagcacc 420agcgagagca cagccgccct gggctgcctg gtgaaagact
acttccccga gcccgtcacc 480gtgtcctgga acagcggagc cctgaccagc
ggcgtgcaca cctttccagc cgtgctgcag 540agcagcggcc tgtacagcct
gagcagcgtg gtgacagtgc cctccagcag cctgggcacc 600aagacctaca
cctgtaacgt ggaccacaag cccagcaaca ccaaggtgga caagcgggtg
660gaatctaagt acggcccacc ctgccccccc tgccctgccc ctgaatttct
gggcggaccc 720tccgtgttcc tgttcccccc aaagcccaag gacaccctga
tgatcagccg gacccccgaa 780gtgacctgcg tggtggtgga cgtgtcccag
gaagatcccg aggtccagtt caattggtac 840gtggacggcg tggaagtgca
caacgccaag accaagccca gagaggaaca gttcaacagc 900acctaccggg
tggtgtccgt gctgaccgtg ctgcaccagg actggctgaa cggcaaagag
960tacaagtgca aagtctccaa caagggcctg cccagctcca tcgagaaaac
catcagcaag 1020gccaagggcc agccccgcga gcctcaggtg tacacactgc
cccccagcca ggaagagatg 1080accaagaacc aggtgtccct gacctgtctg
gtgaaaggct
tctaccccag cgatatcgcc 1140gtggaatggg agagcaacgg ccagcccgag
aacaactaca agaccacccc ccctgtgctg 1200gacagcgacg gcagcttctt
cctgtactcc cggctgaccg tggacaagag ccggtggcag 1260gaaggcaacg
tcttcagctg cagcgtgatg cacgaggccc tgcacaacca ctacacccag
1320aagtccctga gcctgagcct gggcaag
13471011341DNAArtificialPolynucleotide sequence of Anti-HLA IgG4
(S228P) 101caggtccaac tcaagcagtc cggtccgggg ctggtccagc catctcaatc
actgtctctt 60acttgcaccg tgtccggatt cagcctgacc tcatacggag tgcattgggt
gcggcagcct 120cccgggaaag gactggagtg gctcggagtc atttggtccg
gtggatcaac tgactacaat 180gccgctttca tcagcaggct gagcattcgg
aaggacaact ctaagagcca agtgttcttc 240aagatgaact cactccaggc
cgatgacacc gccatctact attgtgccag aaccttcacc 300accagcacct
ctgcatggtt tgcatactgg ggccagggca ctcttgtgac cgtgtcagct
360gctagcacca agggccccag cgtgttcccc ctggcccctt gtagcagaag
caccagcgag 420agcacagccg ccctgggctg cctggtgaaa gactacttcc
ccgagcccgt caccgtgtcc 480tggaacagcg gagccctgac cagcggcgtg
cacacctttc cagccgtgct gcagagcagc 540ggcctgtaca gcctgagcag
cgtggtgaca gtgccctcca gcagcctggg caccaagacc 600tacacctgta
acgtggacca caagcccagc aacaccaagg tggacaagcg ggtggaatct
660aagtacggcc caccctgccc cccctgccct gcccctgaat ttctgggcgg
accctccgtg 720ttcctgttcc ccccaaagcc caaggacacc ctgatgatca
gccggacccc cgaagtgacc 780tgcgtggtgg tggacgtgtc ccaggaagat
cccgaggtcc agttcaattg gtacgtggac 840ggcgtggaag tgcacaacgc
caagaccaag cccagagagg aacagttcaa cagcacctac 900cgggtggtgt
ccgtgctgac cgtgctgcac caggactggc tgaacggcaa agagtacaag
960tgcaaagtct ccaacaaggg cctgcccagc tccatcgaga aaaccatcag
caaggccaag 1020ggccagcccc gcgagcctca ggtgtacaca ctgcccccca
gccaggaaga gatgaccaag 1080aaccaggtgt ccctgacctg tctggtgaaa
ggcttctacc ccagcgatat cgccgtggaa 1140tgggagagca acggccagcc
cgagaacaac tacaagacca ccccccctgt gctggacagc 1200gacggcagct
tcttcctgta ctcccggctg accgtggaca agagccggtg gcaggaaggc
1260aacgtcttca gctgcagcgt gatgcacgag gccctgcaca accactacac
ccagaagtcc 1320ctgagcctga gcctgggcaa g
1341102645DNAArtificialPolynucleotide sequence of Anti-HLA VK
102tctatcgtca tgacccagac cccgaagttc ctcctggtgt cagctggtga
tcgggtgacc 60atcacttgta aggcctctca gtctgtctca aacgacgtcg catggtacca
acaaaagcct 120gggcagtcac ctaagcttct gatctactat gcttccaatc
gctacaccgg cgtgcccgac 180aggttcaccg gatcagggta cggaaccgac
ttcaccttta ctatttccac cgtgcaggcc 240gaggacctcg ccgtgtattt
ctgccagcaa gattacagca gcccaccctg gactttcggt 300ggaggaacta
aactggaaat tagacgtacg gtggcggcgc ccagcgtgtt catcttccca
360cccagcgacg agcagctgaa gtccggcaca gccagcgtgg tgtgcctgct
gaacaacttc 420tacccccgcg aggccaaggt gcagtggaag gtggacaacg
ccctgcagag cggcaacagc 480caggaaagcg tgaccgagca ggacagcaag
gactccacct acagcctgag cagcaccctg 540accctgagca aggccgacta
cgagaagcac aaggtgtacg cctgcgaagt gacccaccag 600ggcctgtcca
gccccgtgac caagagcttc aaccggggcg agtgc
6451031203DNAArtificialPolynucleotide sequence of OPG-Fc
103gaaacctttc cgccgaaata tctgcattat gatgaagaaa ccagccatca
gctgctgtgc 60gataaatgcc cgccgggcac ctatctgaaa cagcattgca ccgcgaaatg
gaaaaccgtg 120tgcgcgccgt gcccggatca ttattatacc gatagctggc
ataccagcga tgaatgcctg 180tattgcagcc cggtgtgcaa agaactgcag
tatgtgaaac aggaatgcaa ccgcacccat 240aaccgcgtgt gcgaatgcaa
agaaggccgc tatctggaag gcgaattttg cctgaaacat 300cgcagctgcc
cgccgggctt tggcgtggtg caggcgggca ccccggaacg caacaccgtg
360tgcaaacgct gcccggatgg cttttttagc aacgaaacca gcagcaaagc
gccgtgccgc 420aaacatacca actgcagcgt gtttggcctg ctgctgaccc
agaaaggcaa cgcgacccat 480gataacattt gcagcggcaa cagcgaaagc
acccagaaag aacgcaaatg ctgcgtggaa 540tgcccgccgt gcccggcgcc
gccggtggcg ggcccgagcg tgtttctgtt tccgccgaaa 600ccgaaagata
ccctgatgat tagccgcacc ccggaagtga cctgcgtggt ggtggatgtg
660agccatgaag atccggaagt gcagtttaac tggtatgtgg atggcgtgga
agtgcataac 720gcgaaaacca aaccgcgcga agaacagttt aacagcacct
ttcgcgtggt gagcgtgctg 780accgtggtgc atcaggattg gctgaacggc
aaagaatata aatgcaaagt gagcaacaaa 840ggcctgccgg cgccgattga
aaaaaccatt agcaaaacca aaggccagcc gcgcgaaccg 900caggtgtata
ccctgccgcc gagccgcgaa gaaatgacca aaaaccaggt gagcctgacc
960tgcctggtga aaggctttta tccgagcgat attgcggtgg aatgggaaag
caacggccag 1020ccggaaaaca actataaaac caccccgccg atgctggata
gcgatggcag cttttttctg 1080tatagcaaac tgaccgtgga taaaagccgc
tggcagcagg gcaacgtgtt tagctgcagc 1140gtgatgcatg aagcgctgca
taaccattat acccagaaaa gcctgagcct gagcccgggc 1200aaa
12031041350DNAArtificialPolynucleotide sequence of 10.21 formatted
as an IgG1 104gaagtgcagc tggtgcagtc tggcgccgaa gtgaagaaac
ctggcgccac cgtgaagatc 60agctgcaagg tgtccggcta caccttcacc gacagcgtga
tgaactgggt gcagcaggcc 120cctggcaagg gcctggaatg gatgggatgg
atcgaccccg agtacggcag aaccgacgtg 180gccgagaagt tccagggcag
agtgaccatc accgccgaca ccagcaccga caccgcctac 240atggaactga
gcagcctgcg gagcgaggac accgccgtgt actactgtgc ccggaccaag
300tacaacagcg gctacggctt cccctactgg ggccagggca caaccgtgac
agtgtcctca 360gctagcacca agggacccag cgtgttccct ctggccccta
gcagcaagag cacatctggc 420ggaacagccg ccctgggctg cctcgtgaag
gactactttc ccgagcctgt caccgtgtct 480tggaactctg gcgccctgac
aagcggcgtg cacacctttc cagccgtgct gcagagcagc 540ggcctgtact
ctctgagcag cgtcgtgaca gtgcccagct ctagcctggg cacccagacc
600tacatctgca acgtgaacca caagcccagc aacaccaagg tggacaagaa
ggtggaaccc 660aagagctgcg acaagaccca cacctgtccc ccttgtcctg
cccccgaact gctgggaggc 720ccttccgtgt tcctgttccc cccaaagccc
aaggacaccc tgatgatcag ccggaccccc 780gaagtgacct gcgtggtggt
ggatgtgtcc cacgaggacc ctgaagtgaa gttcaattgg 840tacgtggacg
gcgtggaagt gcacaacgcc aagaccaagc ccagagagga acagtacaac
900agcacctacc gggtggtgtc cgtgctgaca gtgctgcacc aggactggct
gaacggcaaa 960gagtacaagt gcaaggtgtc caacaaggcc ctgcctgccc
ccatcgagaa aaccatcagc 1020aaggccaagg gccagccccg cgaaccccag
gtgtacacac tgcccccaag cagggacgag 1080ctgaccaaga accaggtgtc
cctgacctgt ctcgtgaaag gcttctaccc ctccgatatc 1140gccgtggaat
gggagagcaa cggccagccc gagaacaact acaagaccac cccccctgtg
1200ctggacagcg acggctcatt cttcctgtac agcaagctga ccgtggacaa
gtcccggtgg 1260cagcagggca acgtgttcag ctgcagcgtg atgcacgagg
ccctgcacaa ccactacacc 1320cagaagtccc tgagcctgag ccccggcaag
13501051338DNAArtificialPolynucleotide sequence of 10.21 IgG2
105gaagtgcagc tggtgcagtc tggcgccgaa gtgaagaaac ctggcgccac
cgtgaagatc 60agctgcaagg tgtccggcta caccttcacc gacagcgtga tgaactgggt
gcagcaggcc 120cctggcaagg gcctggaatg gatgggatgg atcgaccccg
agtacggcag aaccgacgtg 180gccgagaagt tccagggcag agtgaccatc
accgccgaca ccagcaccga caccgcctac 240atggaactga gcagcctgcg
gagcgaggac accgccgtgt actactgtgc ccggaccaag 300tacaacagcg
gctacggctt cccctactgg ggccagggca caaccgtgac agtgtcctca
360gccagcacca agggccccag cgtgttcccc ctggccccct gcagcagaag
caccagcgag 420agcacagccg ccctgggctg cctggtgaag gactacttcc
ccgagcccgt gacagtgagc 480tggaacagcg gagccctgac ctccggtgta
cacaccttcc ccgccgtgct gcagagcagc 540ggcctgtact ccctgagcag
cgtggtgacc gtgcccagca gcaacttcgg cacccagacc 600tacacctgca
acgtggacca caagcccagc aacaccaagg tggacaagac cgtggagagg
660aagtgctgcg tggagtgccc cccctgccca gcccccccag tggccggacc
ctccgtgttt 720ctgttccccc ccaagcccaa ggacaccctg atgatcagca
ggacccccga ggtgacctgc 780gtggtggtgg acgtgagcca cgaggatccg
gaggtgcagt tcaactggta cgtggacggc 840gtggaggtgc acaacgccaa
gaccaagccc agagaggagc agtttaacag caccttcagg 900gtggtgtccg
tgctgaccgt ggtgcaccag gactggctga acggcaagga atacaagtgc
960aaggtctcca acaagggcct gccagccccc atcgagaaaa ccatcagcaa
gaccaagggc 1020cagccacggg agccccaagt gtataccctg ccccccagcc
gggaggagat gaccaagaac 1080caggtgtccc tgacctgtct ggtgaagggc
ttctacccca gcgacatcgc cgtggagtgg 1140gagagcaacg gccagcccga
gaacaactac aagaccaccc cccccatgct ggacagcgac 1200ggcagcttct
tcctgtactc caagctgaca gtggacaagt ccaggtggca gcagggcaac
1260gtgttcagct gcagcgtgat gcacgaggcc ctgcacaacc actacaccca
gaagagcctg 1320agcctgtccc ccggcaag
13381061491DNAArtificialPolynucleotide sequence of 10.21 IgG3
106gaagtgcagc tggtgcagtc tggcgccgaa gtgaagaaac ctggcgccac
cgtgaagatc 60agctgcaagg tgtccggcta caccttcacc gacagcgtga tgaactgggt
gcagcaggcc 120cctggcaagg gcctggaatg gatgggatgg atcgaccccg
agtacggcag aaccgacgtg 180gccgagaagt tccagggcag agtgaccatc
accgccgaca ccagcaccga caccgcctac 240atggaactga gcagcctgcg
gagcgaggac accgccgtgt actactgtgc ccggaccaag 300tacaacagcg
gctacggctt cccctactgg ggccagggca caaccgtgac agtgtcctca
360gcgagcacca aaggcccgag cgtgtttccg ctggcgccgt gcagccgcag
caccagcggc 420ggcaccgcgg cgctgggctg cctggtgaaa gattattttc
cggaaccggt gaccgtgagc 480tggaacagcg gcgcgctgac cagcggcgtg
catacctttc cggcggtgct gcagagcagc 540ggcctgtata gcctgagcag
cgtggtgacc gtgccgagca gcagcctggg cacccagacc 600tatacctgca
acgtgaacca taaaccgagc aacaccaaag tggataaacg cgtggaactg
660aaaaccccgc tgggcgatac cacccatacc tgcccgcgct gcccggaacc
gaaaagctgc 720gataccccgc cgccgtgccc gcgctgcccg gaaccgaaaa
gctgcgatac cccgccgccg 780tgcccgcgct gcccggaacc gaaaagctgc
gataccccgc cgccgtgccc gcgctgcccg 840gcgccggaac tgctgggcgg
cccgagcgtg tttctgtttc cgccgaaacc gaaagatacc 900ctgatgatta
gccgcacccc ggaagtgacc tgcgtggtgg tggatgtgag ccatgaagat
960ccggaagtgc agtttaaatg gtatgtggat ggcgtggaag tgcataacgc
gaaaaccaaa 1020ccgcgcgaag aacagtttaa cagcaccttt cgcgtggtga
gcgtgctgac cgtgctgcat 1080caggattggc tgaacggcaa agaatataaa
tgcaaagtga gcaacaaagc gctgccggcg 1140ccgattgaaa aaaccattag
caaaaccaaa ggccagccgc gcgaaccgca ggtgtatacc 1200ctgccgccga
gccgcgaaga aatgaccaaa aaccaggtga gcctgacctg cctggtgaaa
1260ggcttttatc cgagcgatat tgcggtggaa tgggaaagca gcggccagcc
ggaaaacaac 1320tataacacca ccccgccgat gctggatagc gatggcagct
tttttctgta tagcaaactg 1380accgtggata aaagccgctg gcagcagggc
aacattttta gctgcagcgt gatgcatgaa 1440gcgctgcata accgctttac
ccagaaaagc ctgagcctga gcccgggcaa a
1491107165PRTArtificialGlycosylated (T106T) human interferon
alpha-2-b comprising attenuating mutation A145X where X can be any
amino acid selected from D, E, G, H, I, K, L, M, N, Q, R, S, T, V,
YVARIANT(145)..(145)Xaa can be any amino acid selected from D, E,
G, H, I, K, L, M, N, Q, R, S, T, V, Y 107Cys Asp Leu Pro Gln Thr
His Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln Met
Arg Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp Phe
Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu
Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn Leu
Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75
80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu
85 90 95Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met
Lys 100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg
Ile Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala
Trp Glu Val Val Arg 130 135 140Xaa Glu Ile Met Arg Ser Phe Ser Leu
Ser Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
165108165PRTArtificialGlycosylated (T106T) human interferon
alpha-2-b comprising attenuating mutation R144X where X can be any
amino acid selected from A, D, E, G, H, I, K, L, N, Q, S, T, V,
YVARIANT(144)..(144)Xaa can be any amino acid selected from A, D,
E, G, H, I, K, L, N, Q, S, T, V, Y 108Cys Asp Leu Pro Gln Thr His
Ser Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln Met Arg
Arg Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Arg His Asp Phe Gly
Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr
Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn Leu Phe
Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75 80Leu
Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90
95Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys
100 105 110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile
Thr Leu 115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp
Glu Val Val Xaa 130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu Ser
Thr Asn Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
165109165PRTArtificialGlycosylated (T106T) human interferon
alpha-2-b comprising attenuating mutation R33X, where X can be any
amino acid selected from K, A, QVARIANT(33)..(33)Xaa can be any
amino acid selected from K, A, Q 109Cys Asp Leu Pro Gln Thr His Ser
Leu Gly Ser Arg Arg Thr Leu Met1 5 10 15Leu Leu Ala Gln Met Arg Arg
Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30Xaa His Asp Phe Gly Phe
Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile
Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60Asn Leu Phe Ser
Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu65 70 75 80Leu Asp
Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala
Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105
110Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu
115 120 125Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
Val Arg 130 135 140Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn
Leu Gln Glu Ser145 150 155 160Leu Arg Ser Lys Glu
165110164PRTArtificialHuman interferon alpha-2-b comprising with
deletion of amino acid at position 106 and comprising attenuating
mutation A144X where X, can be any amino acid selected from D, E,
G, H, I, K, L, M, N, Q, R, S, T, V, YVARIANT(144)..(144)Xaa can be
any amino acid selected from D, E, G, H, I, K, L, M, N, Q, R, S, T,
V, Y 110Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu
Met1 5 10 15Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu
Lys Asp 20 25 30Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn
Gln Phe Gln 35 40 45Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile
Gln Gln Ile Phe 50 55 60Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala
Trp Asp Glu Thr Leu65 70 75 80Leu Asp Lys Phe Tyr Thr Glu Leu Tyr
Gln Gln Leu Asn Asp Leu Glu 85 90 95Ala Cys Val Ile Gln Gly Val Gly
Val Glu Thr Pro Leu Met Lys Glu 100 105 110Asp Ser Ile Leu Ala Val
Arg Lys Tyr Phe Gln Arg Ile Thr Leu Tyr 115 120 125Leu Lys Glu Lys
Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg Xaa 130 135 140Glu Ile
Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser Leu145 150 155
160Arg Ser Lys Glu111450PRTArtificialVH sequence of 10.21 formatted
as an IgG1 incorporating substitutions M255Y, S257T, T259E 111Glu
Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala1 5 10
15Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr Thr Phe Thr Asp Ser
20 25 30Val Met Asn Trp Val Gln Gln Ala Pro Gly Lys Gly Leu Glu Trp
Met 35 40 45Gly Trp Ile Asp Pro Glu Tyr Gly Arg Thr Asp Val Ala Glu
Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala Asp Thr Ser Thr Asp
Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr
Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Lys Tyr Asn Ser Gly Tyr Gly
Phe Pro Tyr Trp Gly Gln 100 105 110Gly Thr Thr Val Thr Val Ser Ser
Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe Pro Leu Ala Pro Ser
Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala 130 135 140Leu Gly Cys Leu
Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser145 150 155 160Trp
Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170
175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn
His Lys 195 200 205Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
Lys Ser Cys Asp 210 215 220Lys Thr His Thr Cys Pro Pro Cys Pro Ala
Pro Glu Leu Leu Gly Gly225 230 235 240Pro Ser Val Phe Leu Phe Pro
Pro Lys Pro Lys Asp Thr Leu Tyr Ile 245 250 255Thr Arg Glu Pro Glu
Val Thr Cys Val Val Val Asp Val Ser His Glu 260 265 270Asp Pro Glu
Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285Asn
Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290
295 300Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
Lys305 310 315 320Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro
Ala Pro Ile Glu 325 330 335Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro
Arg Glu Pro Gln Val Tyr 340 345 350Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn Gln Val Ser Leu 355 360 365Thr Cys Leu Val Lys Gly
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 370 375 380Glu Ser Asn Gly
Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val385 390 395 400Leu
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410
415Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu
Ser Pro 435 440 445Gly Lys 450112446PRTArtificialVH sequence of
10.21 formatted onto an IgG2 backbone incorporating substitutions
A330S and P331S 112Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10 15Thr Val Lys Ile Ser Cys Lys Val Ser Gly Tyr
Thr Phe Thr Asp Ser 20 25 30Val Met Asn Trp Val Gln Gln Ala Pro Gly
Lys Gly Leu Glu Trp Met 35 40 45Gly Trp Ile Asp Pro Glu Tyr Gly Arg
Thr Asp Val Ala Glu Lys Phe 50 55 60Gln Gly Arg Val Thr Ile Thr Ala
Asp Thr Ser Thr Asp Thr Ala Tyr65 70 75 80Met Glu Leu Ser Ser Leu
Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Thr Lys Tyr
Asn Ser Gly Tyr Gly Phe Pro Tyr Trp Gly Gln 100 105 110Gly Thr Thr
Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125Phe
Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135
140Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
Ser145 150 155 160Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
Phe Pro Ala Val 165 170 175Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser
Ser Val Val Thr Val Pro 180 185 190Ser Ser Asn Phe Gly Thr Gln Thr
Tyr Thr Cys Asn Val Asp His Lys 195 200 205Pro Ser Asn Thr Lys Val
Asp Lys Thr Val Glu Arg Lys Cys Cys Val 210 215 220Glu Cys Pro Pro
Cys Pro Ala Pro Pro Val Ala Gly Pro Ser Val Phe225 230 235 240Leu
Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250
255Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
260 265 270Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
Lys Thr 275 280 285Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg
Val Val Ser Val 290 295 300Leu Thr Val Val His Gln Asp Trp Leu Asn
Gly Lys Glu Tyr Lys Cys305 310 315 320Lys Val Ser Asn Lys Gly Leu
Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335Lys Thr Lys Gly Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350Ser Arg Glu
Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365Lys
Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375
380Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Met Leu Asp Ser
Asp385 390 395 400Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
Lys Ser Arg Trp 405 410 415Gln Gln Gly Asn Val Phe Ser Cys Ser Val
Met His Glu Ala Leu His 420 425 430Asn His Tyr Thr Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 435 440 4451135PRTArtificialIntervening
serine-glycine rich (S/G) linker 113Gly Gly Gly Gly Ser1
51146PRTArtificialPeptide linker 114Ser Gly Gly Gly Gly Ser1
511516PRTArtificialPeptide linker 115Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser1 5 10 15
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