U.S. patent application number 12/165601 was filed with the patent office on 2009-09-24 for mutant forms of fas ligand and uses thereof.
Invention is credited to Keting Chu.
Application Number | 20090239240 12/165601 |
Document ID | / |
Family ID | 27363738 |
Filed Date | 2009-09-24 |
United States Patent
Application |
20090239240 |
Kind Code |
A1 |
Chu; Keting |
September 24, 2009 |
Mutant Forms of Fas Ligand and Uses Thereof
Abstract
The invention provides for DNA encoding Fas ligand muteins and
chimeras and the proteins encoded thereby. The invention further
includes the use of DNA and vectors to produce transformed cells
expressing the mutant or chimeric Fas ligand. When the Fas ligand
of the invention is a non cleavable form, the cells expressing the
Fas ligand are useful in vitro for identifying Fas expressing cells
or in vivo for reducing populations of Fas expressing cells. Thus,
in other embodiments, the present invention is also directed to a
method for treating a patient, for example a mammal, for autoimmune
disease or transplant rejection by administering a Fas ligand
therapeutic agent. The therapeutic agent is a polypeptide, a
polynucleotide encoding the polypeptide or a small molecule. The
polypeptides include full-length Fas ligand polypeptide, or a
biologically active variant, derivative, portion, fusion or peptide
thereof.
Inventors: |
Chu; Keting; (Emeryville,
CA) |
Correspondence
Address: |
NOVARTIS VACCINES AND DIAGNOSTICS INC.
INTELLECTUAL PROPERTY- X100B, P.O. BOX 8097
Emeryville
CA
94662-8097
US
|
Family ID: |
27363738 |
Appl. No.: |
12/165601 |
Filed: |
June 30, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10404466 |
Apr 1, 2003 |
7393942 |
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12165601 |
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08968686 |
Nov 12, 1997 |
6544523 |
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10404466 |
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60039972 |
Feb 10, 1997 |
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60030871 |
Nov 13, 1996 |
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Current U.S.
Class: |
435/7.2 |
Current CPC
Class: |
C12N 2799/021 20130101;
A61P 37/06 20180101; A61P 25/00 20180101; A61K 38/00 20130101; A61P
3/10 20180101; A61P 21/04 20180101; A61P 13/12 20180101; A61P 21/00
20180101; A61P 7/06 20180101; A61P 7/04 20180101; A61P 29/00
20180101; A61P 1/16 20180101; A61P 15/08 20180101; A61P 17/00
20180101; A61P 19/02 20180101; A61P 11/06 20180101; A61P 43/00
20180101; A61P 9/10 20180101; A61P 37/08 20180101; C07K 2319/00
20130101; C12N 2799/022 20130101; A61P 17/04 20180101; C07K
14/70575 20130101; C12N 2799/027 20130101; A61P 37/02 20180101 |
Class at
Publication: |
435/7.2 |
International
Class: |
G01N 33/53 20060101
G01N033/53 |
Claims
1. A method for determining the presence in a sample of cells
expressing Fas, the method comprising the steps of: a) providing a
transformed cell having a recombinant Fas ligand surface bound
thereto, said surface bound, recombinant Fas ligand being resistant
to cleavage; b) contacting said transformed cell with a sample
containing cells suspected of having surface bound Fas thereon to
obtain a cell mixture; c) allowing said cell bound Fas to bind to
said surface bound recombinant Fas ligand; and d) observing said
cell mixture for rosette formation or clumping, whereby the binding
of cells from said sample to said transformed cell indicates the
presence of Fas on the surface of cells in said sample.
2. The method of claim 1, wherein said recombinant Fas ligand is
selected from the group consisting of a Fas ligand deletion mutein
and a Fas ligand chimera, said Fas ligand deletion mutein being a
deletion mutein of pro-Fas ligand lacking a continuous segment of
10 to 17 amino acid residues beginning at residue position 130 of
pro-Fas ligand (SEQ ID NO: 12), said Fas ligand chimera being a
fusion protein having an amino acid sequence that corresponds to
the fusion product of the carboxy terminus of a transmembrane
domain of a cell surface protein other than Fas ligand, said
transmembrane domain being substantially non-cleavable from a cell
membrane by proteinases or convertases and being fused to the amino
terminus of the extracellular domain of Fas ligand, said
extracellular domain lacking a continuous segment of 10 to 17 amino
acid residues beginning at about residue position 130 of pro-Fas
ligand (SEQ ID NO: 12).
3. The method of claim 2, wherein said Fas ligand mutein has an
amino acid sequence that is the same as the amino acid sequence in
SEQ ID NO: 6 or SEQ ID NO: 10.
4. The method of claim 3, wherein said Fas ligand mutein has an
amino acid sequence that is the same as the amino acid sequence in
SEQ ID NO: 6.
5. The method of claim 3, wherein said Fas ligand mutein has an
amino acid sequence that is the same as the amino acid sequence in
SEQ ID NO: 10.
6. The method of claim 2, wherein said Fas ligand chimera has an
amino acid sequence that corresponds to the fusion product of the
carboxy terminus of the transmembrane domain of CD30 ligand fused
to the amino terminus of the extracellular domain of Fas ligand,
said extracellular domain lacking a continuous segment of 10 to 17
amino acid residues beginning at about residue position 130 of
pro-Fas ligand.
7. The method of claim 6, wherein said Fas ligand chimera has an
amino acid sequence corresponding to the fusion product of the
carboxy terminus of residues 1-86 of CD30 ligand fused to the amino
terminus of the extracellular domain of Fas ligand, said
extracellular domain beginning 10 to 17 amino acid residues beyond
about residue position 130 and extending to residue position 281 of
pro-Fas.
8. The method of claim 2, wherein said Fas ligand chimera has an
amino acid sequence corresponding to the fusion product of the
carboxy terminus of the transmembrane domain of CD40 ligand fused
to the amino terminus of the extracellular domain of Fas ligand,
said extracellular domain lacking a continuous segment of 10 to 17
amino acid residues beginning at about residue position 130 of
pro-Fas ligand.
9. The method of claim 8, wherein said Fas ligand chimera has an
amino acid sequence corresponding to the fusion product of the
carboxy terminus of residues 1-107 of CD40 ligand fused to the
amino terminus of the extracellular domain of Fas ligand, said
extracellular domain beginning 10 to 17 amino acid residues beyond
about residue position 130 and extending to residue position 281 of
pro-Fas ligand.
10. The method of claim 1, wherein said recombinant Fas ligand is a
polypeptide comprising: a transmembrane domain of a cell surface
protein; and a Fas ligand portion, wherein said Fas ligand portion
comprises a sequence at least 90% homologous to SEQ ID NO:7, and
wherein the Fas ligand portion binds Fas; wherein said polypeptide
does not contain 17 contiguous amino acid residues starting at
position 130 of SEQ ID NO: 12.
11. The method of claim 10, wherein said transmembrane domain is an
uncleavable transmembrane portion of a transmembrane protein other
than pro-Fas ligand.
12. The method of claim 10, wherein said Fas ligand portion
comprises SEQ ID NO:7.
13. The method of claim 10, wherein the transmembrane domain is a
transmembrane domain of a polypeptide selected from the group
consisting of a membrane-bound antibody, a membrane bound viral
antigen, and a member of the tumor necrosis factor receptor
superfamily of polypeptides.
14. The method of claim 13, wherein the transmembrane domain
comprises a transmembrane domain of a tumor necrosis factor
receptor superfamily polypeptide.
15. The method of claim 14, wherein the tumor necrosis factor
receptor superfamily polypeptide is selected from the group
consisting of tumor necrosis factor receptor, CD30, nerve growth
factor receptor, CD27, CD40, CD120a, CD120b, lymphotoxin beta
receptor, and TRAIL receptor.
16. The method of claim 10, wherein the transmembrane domain
comprises a transmembrane domain of CD30 ligand.
17. The method of claim 10, wherein the transmembrane domain
comprises a transmembrane domain of CD40 ligand.
Description
[0001] This application is a divisional of application Ser. No.
10/404,466, filed Apr. 1, 2003, which is a divisional of
application Ser. No. 08/968,686 filed Nov. 12, 1997, now U.S. Pat.
No. 6,544,523, which claims the benefit of provisional application
Ser. No. 60/039,972, filed Feb. 10, 1997 and provisional
application Ser. No. 60/030,871 filed Jan. 13, 1996, which are
hereby incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] The invention relates to Fas ligand muteins and chimeric
proteins that act as Fas ligand agonists, and the DNA encoding the
same. More particularly, the present invention relates to novel
forms of Fas ligand, which when expressed in a transformed host
cell, are surface bound in a conventional type II prohormone form
but non-cleavable therefrom. These non-cleavable forms of Fas
ligand and the transformed host cells expressing them are useful in
diagnostic assays and in reducing populations of Fas expressing
cells (e.g., activated T cells and activated B cells) both in vitro
and in vivo. The Fas ligand and transformed host cells of the
present invention are particularly useful as pharmaceutical agents
in the treatment of transplant rejection and the various autoimmune
diseases that are well known in the art.
BACKGROUND OF THE INVENTION
[0003] The tumor necrosis factor superfamily includes ligands that
bind the corresponding members of the tumor necrosis factor
receptor superfamily of receptors. Such members of the receptor
superfamily include, for example, tumor necrosis factor receptors
(TNFR) (type I or 55K or TNFR60 and type II or 75K or TNFR80),
CD30, nerve growth factor receptor, CD27, CD40, CD95/APO-1 or Fas,
CD120a, CD120b, lymphtoxin beta receptor (LT beta R), and a TRAIL
receptor. The receptors of this family are membrane bound and
recognize soluble or membrane bound ligands that mediate diverse
cellular responses. The corresponding ligands for these receptors
include in some cases membrane or soluble forms and include, for
example, tumor necrosis factor alpha (TNF.alpha.), CD30 ligand,
nerve growth factor, CD70/CD27 ligand, CD40 ligand, Fas ligand, and
TNF-related apoptosis-inducing ligand TRAIL (as described in Wiley
et al, Immunity 3:673-82 (1995)). The ligands of the superfamily,
including Fas ligand, are type II transmembrane glycoproteins with
beta strands that form a jelly-roll beta-sandwich as described in
Lotz et al, J. of Leukocyte Biol 60:1-7 (1996), which is hereby
incorporated by reference. Treatment of autoimmune diseases present
a unique challenge to molecular biology and medical research.
Autoimmune diseases affect between 5 and 7% of the human
population, often causing chronic debilitating illnesses, as
described in Kuby, IMMUNOLOGY, (W.H. Freeman, NY 1992).
[0004] Although precise details of an autoimmune response are
incompletely understood, the outcome of antigenic stimulation,
whether antibody formation or activated T-cells, or tolerance,
seems to depend on the same factors whether a reaction to
auto-antigen or exogenous antigen, as described in THE MERCK
MANUAL, 16th edition (Merck & Co. Inc. 1992). Also described in
THE MERCK MANUAL are four classes of auto-antigens. Class 1 is
antigens from intracellular regions of the cells of the body that,
by virtue of their sequestration from the immune system, are not
recognized as "self" in the body once secreted. For example,
sympathetic ophthalmia causes the release of eye antigens, and a
subsequent self reaction to the antigen. Class 2 is represented by
self antigens that may become immunogenic by chemical, physical, or
biological alteration, for example, when a chemical couples to a
self antigen and produces a "foreign" reaction, for example in
contact dermatitis and hypersensitivity to drugs. Class 3 is
represented by foreign antigen that cross reacts with self antigen,
and induces a self reaction to the self antigen, for example, as
shown with the development of encephalitis after rabies
vaccination. Class 4 is represented by a mutation in
immunoincompetent cells, such as the autoimmune phenomena seen with
mammals having lymphoma. Finally, an autoimmune reaction may be
epiphenomena, developing secondarily after an immune response to an
obscure antigen, for example, a virus. All autoimmune diseases have
in common an involvement of the immune system, and many involve
either activated B-cells, activated T-cells, or both.
[0005] Although various ameliorative and palliative therapies exist
for some autoimmune diseases, and while the autoimmune diseases can
spontaneously regress in a remission, effective treatment has yet
to be developed for treating autoimmune diseases. It would be
desirable to advance the capacity of medical and clinical research
to develop effective treatments of autoimmune diseases by discovery
of new methods and new therapeutic agents targeting the molecular
biology of autoimmune diseases.
SUMMARY OF THE INVENTION
[0006] The present invention has multiple aspects. In a first
aspect, the present invention is directed to non-cleavable forms of
Fas ligand, including Fas-ligand muteins and Fas ligand chimeras,
and to the polynucleotides encoding them. Preferably, the Fas
ligand deletion mutein comprises an effective length of the
transmembrane region of Fas ligand up to residue 129 of SEQ ID NO:
12 linked by a substantially non-cleavable peptide linkage to the
Fas binding domain of human Fas ligand beginning from about residue
139 to residue 146 of SEQ ID NO: 12, and the Fas ligand chimera
comprises the transmembrane region of a cell surface protein linked
by a substantially non-cleavable peptide linkage to the Fas binding
domain of human Fas ligand beginning from about residue 139 to
residue 146 of SEQ ID NO: 12.
[0007] A second aspect is directed to a vector (DNA or RNA)
comprising a promoter operably linked to a gene encoding a Fas
ligand mutein or chimera that when membrane bound is substantially
non-cleavable. Preferably, the vector comprises a promoter operably
linked to a recombinant gene encoding a Fas ligand deletion mutein
or a Fas ligand chimera, the Fas ligand deletion mutein comprising
an effective length of the transmembrane region of Fas ligand up to
residue 129 of SEQ ID NO: 12 linked by a substantially
non-cleavable peptide linkage to the Fas binding domain of human
Fas ligand beginning from about residue 139 to residue 146 of SEQ
ID NO: 12, said Fas ligand chimera comprising the transmembrane
region of a cell surface protein linked by a substantially
non-cleavable peptide linkage to the Fas binding domain of human
Fas ligand beginning from about residue 139 to residue 146 of SEQ
ID NO: 12.
[0008] A third aspect is directed to a transformed host cell,
wherein the host cell is transformed with vector (DNA or RNA)
encoding a Fas ligand mutein or chimeric protein that when
expressed in the cell remains membrane bound.
[0009] A fourth aspect of the present invention is directed to
compositions comprising the above described host cell and a
carrier. In a preferred embodiment, the composition is a
pharmaceutical composition and the carrier is a pharmaceutically
acceptable carrier.
[0010] Another aspect of the present invention is directed to a
method for determining the presence in a sample of cells expressing
Fas, the method comprising the steps of: [0011] a) providing a
transformed cell having a recombinant Fas ligand surface bound
thereto, said surface bound, recombinant Fas ligand being resistant
to cleavage; [0012] b) contacting said transformed cell with a
sample containing cells suspected of having surface bound Fas
thereon to obtain a cell mixture; [0013] c) allowing said cell
bound Fas to bind to said surface bound recombinant Fas ligand; and
[0014] d) observing said cell mixture for rosette formation or
clumping, whereby the binding of cells from said sample to said
transformed cell indicates the presence of Fas on the surface of
cells in said sample.
[0015] In another aspect, the present invention is directed to a
method for reducing a population of Fas expressing cells, the
method comprising the steps of: [0016] a) providing a transformed
cell having a recombinant Fas ligand surface bound thereto, said
surface bound, recombinant Fas ligand being resistant to cleavage;
and [0017] b) contacting a population of Fas expressing cells with
said transformed cell, whereby said Fas ligand binds to Fas on said
Fas expressing cells, causing said Fas expressing cells that are so
bound to undergo apoptosis such that said population is reduced. In
a preferred embodiment of the above method, the Fas expressing
cells are activated T cells and/or B cells and said transformed
cell expresses a second ligand that is recognized by said activated
T cell and/or B cell.
[0018] In another aspect, the present invention is directed to a
method of treating a patient having an autoimmune diseases
manifesting activated T-cells or activated B-cells or both by
providing a Fas ligand therapeutic agent, which can include a
polypeptide, polynucleotide encoding a polypeptide, a peptide, a
peptoid, or an organic small molecule agonist, and by administering
an effective amount of the Fas ligand therapeutic agent to a mammal
having an autoimmune disease.
[0019] Another aspect of the invention is a composition comprising
a gene delivery vehicle capable of expressing a polynucleotide
sequence encoding a Fas ligand polypeptide.
[0020] Additionally, another aspect of the invention is a
therapeutic agent for treating an autoimmune disease where the
therapeutic agent is derived from a Fas ligand and has also a
pharmaceutically acceptable carrier for administering the agent to
a mammal having the autoimmune disease.
[0021] Another aspect of the invention is a Fas ligand polypeptide
capable of remaining on a cell membrane longer than native Fas
ligand.
[0022] Another aspect of the invention is a polynucleotide encoding
said Fas ligand polypeptide capable of remaining on a cell membrane
longer than native Fas ligand.
[0023] Another aspect of the invention is a host cell transformed
with a polynucleotide sequence encoding a Fas ligand polypeptide
that is either a full length Fas ligand polypeptide, a biologically
active portion of Fas ligand polypeptide, a membrane-bound Fas
ligand polypeptide, or a sequence encoding a soluble Fas ligand
polypeptide.
[0024] Another aspect of the invention is a method for reducing a
population of activated T-cells or activated B-cells that comprises
transforming a cell to express a non-cleavable Fas ligand
polypeptide that remains membrane-bound thereto, wherein the
transformed cell also bears a second ligand recognized by the
activated T-cells or activated B-cells, and re-introducing this
transformed cell into the population of activated T-cells or
activated B-cells.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The invention described herein draws on previously published
work and pending patent applications. By way of example, such work
consists of scientific papers, patents or pending patent
applications. All such published work and pending patent
applications cited herein are hereby expressly incorporated by
reference in full.
DEFINITIONS
[0026] "Fas ligand" refers to a substance provided with an activity
to induce apoptosis of a Fas-antigen presenting cell by binding to
the Fas antigen expressed on the surface of the cell. Apoptosis in
such a cell is believed to result when Fas ligand binds Fas
expressed on a cell surface Fas ligand can be membrane bound or
soluble. Both the DNA (SEQ ID NO: 11) and the amino acid (SEQ ID
NO: 12) sequences for human Fas ligand are well known in the art
and are disclosed in EP 675 200, Suda et al, Cell, 75: 1169-1178
(1993), Takahashi et al, Int'l Immunol. 6(10):1567-74 (1994), and
Nagata, Adv. in Immun. 57: 129-144 (1994), all incorporated by
reference in full. Fas ligand is a member of the membrane-type
tumor necrosis factor (TNF) cytokine family. Many of the members of
this family are associated with apoptosis activity. The activity of
Fas ligand includes the ability to bind Fas and induce apoptosis in
cells that express Fas. The membrane-bound human Fas ligand is
converted to a soluble form by the action of a matrix
metalloproteinase-like enzyme. Sera from healthy humans do not
contain a detectable level of soluble Fas ligand, as described in
Tanaka et al, Nature Medicine 2(3): 317-22 (1996). Mammalian
expression of Fas ligand in healthy individuals indicates low
levels of expression in lymphoid organs (thymus, lymph node,
spleen), lung and small intestine, demonstrating Fas ligand
involvement in the general immune system and mucosal immunity. The
testis expresses a splice variant of Fas ligand that is shorter
than the T-cell Fas ligand. Activation of the splenocytes with the
T-cell activators such as PMA and ionomycin, Con A, anti-CD3, or
IL-2 alone, induce Fas ligand expression. CD8 splenocytes express
Fas ligand more abundantly than CD4 splenocytes upon activation, as
described in Suda et al, J. of Immunol. 154(8):3806-13 (1995).
Further, functional soluble Fas ligand is expressed in activated
lymphocytes as a 40 kDa protein, and is believed to exist in humans
as a trimer in the soluble form, as described in Tanaka et al, EMBO
J. 14(6):1129-35 (1995).
[0027] A "Fas ligand polypeptide" embodies variants, derivatives,
analogues, fragments, chimeras and mutants of the native sequence
of Fas ligand. The polypeptides are encoded by recombinantly
produced polynucleotides sequences designed to encode the specific
Fas ligand polypeptide intended for expression in a host cell.
[0028] A "Fas ligand therapeutic agent" includes molecules derived
from Fas ligand native polynucleotide or polypeptide sequence, and
variants, mutants, analogues, chimeras, and fragments of such Fas
ligand polynucleotide or polypeptide. Polynucleotide Fas ligand
therapeutic agents are generally sequences encoding a Fas ligand
polypeptide that can be recombinantly expressed in a host cell.
Additionally, a Fas ligand therapeutic agent can be a small
molecule agonist of Fas ligand activity. Other Fas ligand
therapeutic agents may include modulators of Fas ligand activity
that effect Fas ligand and cause a therapeutic effect associated
with Fas ligand activity or its absence. A modulator of Fas ligand
can be, for example, a polynucleotide, a polypeptide, or a small
molecule.
[0029] "Fas antigen" or "Fas" refers to a membrane-associated
polypeptide expressed on an antigen presenting cell, capable of
binding Fas ligand and inducing apoptosis, or cell death, of the
cell bearing Fas. Fas antigen is described in Itoh et al, Cell
66:233-243 (1991), and Nagata, Adv. in Immun. 57: 129-144 (1994),
both incorporated by reference in full. Activated human T and B
cells, for example, APCs, abundantly express Fas as described in
Trauth et al, Science 245: 301-305 (1989). Fas is a cell surface
receptor that belong to the tumor necrosis factor (TNF)/nerve
growth factor receptor family. Fas can transduce an apoptotic
signal through the death domain in the cytoplasmic region.
[0030] The term "diagnostic agent," as used herein, refers to any
agent that contributes to one or more of the diagnostic uses of the
invention. These diagnostic uses include methods for determining
the presence of Fas presenting cells. The diagnostic agents include
the following: DNA encoding a Fas ligand mutein (preferably a
mutein which is substantially non-cleavable from the surface of the
cell in which it is expressed), the non-cleavable Fas ligand mutein
(which is capable of specifically binding to Fas), and cells or the
membranes of cells having the non-cleavable Fas ligand mutein
surface bound thereto.
[0031] A "therapeutic agent" as used herein can be any agent that
accomplishes or contributes to the accomplishment of one or more of
the therapeutic elements of the invention. For example, where the
therapeutic agent is a polynucleotide designed to express a Fas
ligand polypeptide, that agent is a polynucleotide that can be
administered to and expressed in a cell in the mammal. Thus, the
active form of the agent will initially be the expressed
polypeptide. A Fas ligand therapeutic agent is a therapeutic agent
with the bioactivity of Fas ligand or a therapeutic agent derived
from native Fas ligand, such as a polynucleotide encoding a Fas
ligand polypeptide capable of remaining membrane-bound longer than
native Fas ligand. A therapeutic agent achieves a therapeutic goal,
alone or in combination with other agents, for example, the use of
other known treatments for a particular autoimmunity used in
conjunction with administration of Fas ligand, or a gene delivery
vehicle capable of facilitating expression of Fas ligand in the
mammal. The therapeutic agents including for example agonists of
Fas ligand or drugs developed for other purposes can be, for
example, a small organic molecule, a peptide, a peptoid (defined
below), a polynucleotide encoding a Fas ligand polypeptide, a
polypeptide Fas ligand therapeutic agent, or a transformed cell
expressing a Fas ligand mutant that is surface bound thereto and
substantially non-cleavable therefrom.
[0032] A "combination therapeutic agent" is a therapeutic
composition having several components or agents that produce their
separate effects when administered together, and may produce a
synergistic effect when administered together to treat a disease.
Preferably, the separate effects of the combination therapeutic
agent combine to result in a larger therapeutic effect, for example
recovery from an autoimmune disease and long term survival. An
example of separate effects resulting from administration of a
combination therapeutic agent is the combination of such effects as
short-term, or long-term remission, or decrease of an auto-immune
response to a particular type of cell in the patient. An example of
a combination therapeutic agent of this invention would be
administration of a gene delivery vehicle including a
polynucleotide encoding a mutant or native Fas ligand and HSV
thymidine kinase. Alternatively, two gene delivery vectors can be
used, one expressing Fas ligand and one encoding HSV thymidine
kinase. Also by example, IFN.gamma., or a gene delivery vehicle
expressing IFN.gamma., can be administered to upregulate Fas
expression in anticipation of an administration of Fas ligand for
inducing apoptosis in the Fas expressing cells. The various
therapeutic agents can be administered in the same pharmaceutically
acceptable carrier at the same time, followed, for example, by
repeated administration of one or all of the individual agents as
needed to make the therapy efficacious.
[0033] A "gene delivery vehicle" refers to a component that
facilitates delivery to a cell of a coding sequence for expression
of a polypeptide in the cell. The cell can be inside the mammal, as
in in vivo gene therapy, or can be removed from the mammal for
transfection and returned to the mammal for expression of the
polypeptide as in ex vivo gene therapy. The gene delivery vehicle
can be any component or vehicle capable of accomplishing the
delivery of a gene to a cell, for example, a liposome, a particle,
or a vector. A gene delivery vehicle is a recombinant vehicle, such
as a recombinant viral vector, a nucleic acid vector (such as
plasmid), a naked nucleic acid molecule such as genes, a nucleic
acid molecule complexed to a polycationic molecule capable of
neutralizing the negative charge on the nucleic acid molecule and
condensing the nucleic acid molecule into a compact molecule, a
nucleic acid associated with a liposome (U.S. Pat. No. 5,166,320,
issued Nov. 24, 1992; and Wang, et al., PNAS 84:7851, 1987), and a
bacterium. Gene delivery vehicles include certain eukaryotic cells
such as a producer cell, that are capable of delivering a nucleic
acid molecule having one or more desirable properties to host cells
in an organism. As discussed further below, the desirable
properties include the ability to express a desired substance, such
as, for example, a protein, enzyme, or antibody, and/or the ability
to provide a biological activity, which is where the nucleic acid
molecule carried by the gene delivery vehicle is itself the active
agent without requiring the expression of a desired substance. One
example of such biological activity is gene therapy where the
delivered nucleic acid molecule incorporates into a specified gene
so as to inactivate the gene and "turn off" the product the gene
was making. Gene delivery vehicle refers to an assembly which is
capable of directing the expression of the sequence(s) or gene(s)
of interest. The gene delivery vehicle generally includes promoter
elements and may include a signal that directs polyadenylation. In
addition, the gene delivery vehicle includes a sequence which, when
transcribed, is operably linked to the sequence(s) or gene(s) of
interest and acts as a translation initiation sequence. The gene
delivery vehicle may also include a selectable marker such as Neo,
SV.sup.2 Neo, TK, hygromycin, phleomycin, histidinol, or DHFR, as
well as one or more restriction sites and a translation termination
sequence. Gene delivery vehicles as used within the present
invention refers to recombinant vehicles, such as viral vectors
(Jolly, Cancer Gen. Therapy 1:51-64, 1994), nucleic acid vectors,
naked DNA, liposomal DNA, cosmids, bacteria, and certain eukaryotic
cells (including producer cells; see U.S. Ser. No. 08/240,030 and
U.S. Ser. No. 07/800,921), that are capable of eliciting an immune
response within an animal.
[0034] "Biologically active" refers to a molecule that retains a
specific activity. A biologically active Fas ligand polypeptide,
for example, retains the ability to bind Fas antigen on a cell
surface, and activate the apoptotic pathway leading to apoptosis of
the Fas antigen expressing cell.
[0035] "B-cells" or "B lymphocytes" refer to lymphocytes that
mature in the bone marrow and are precursors of antibody-secreting
plasma cells, as described in Kuby, IMMUNOLOGY, (W.H. Freeman, NY
1992). B-cells can be activated by antigen to become antigen
presenting cells (APCs) and expresses Fas upon such activation. An
activated B-cell undergoes apoptosis in the presence of either
soluble or cell membrane associated Fas ligand.
[0036] "T-cells" or "T lymphocytes" refer to lymphocytes that
mature in the thymus, where they undergo differentiation. T-cell
possess a rearranged T-cell receptor. A "CD4 T-cell" refers to a
T-cell that possesses a cell membrane molecule identifies the T
lymphocyte or T-cell as a subset of lymphocytes. CD4 is a cell
surface glycoprotein found on a subset of the T-cells that
recognize antigenic peptides complexed to class II MHC, as
described in Kuby, IMMUNOLOGY, (W.H. Freeman & Co., NY 1992).
CD4 T-cells can express cytotoxic activity. While activation of CD4
CTLs (cytotoxic T-lymphocytes) is MHC-class II-restricted, the
target cell killing activity is unrestricted and non-specific for
antigen. CD4 CTLs preferentially lyse their targets via Fas-Fas
ligand interaction, which is a different mechanism than that used
by CD8 T-cells which used granule exocytosis. Although CD8 T-cells
can also express the Fas ligand, their lytic activity through
interaction with Fas is of less importance.
[0037] A "nucleic acid molecule" or a "polynucleotide," as used
herein, refers to either RNA or DNA molecule that encodes a
specific amino acid sequence or its complementary strand. A "coding
sequence" as used herein, refers to either RNA or DNA that encodes
a specific amino acid sequence or its complementary strand. A
polynucleotide may include, for example, an antisense
oligonucleotide, or a ribozyme, and may also include such items as
a 3' or 5' untranslated region of a gene, or an intron of a gene,
or other region of a gene that does not make up the coding region
of the gene. The DNA or RNA may be single stranded or double
stranded. Synthetic nucleic acids or synthetic polynucleotides can
be chemically synthesized nucleic acid sequences, and may also be
modified with chemical moieties to render the molecule resistant to
degradation. A polynucleotide, and can be generated, for example,
by polymerase chain reaction (PCR) amplification, or recombinant
expression of complementary DNA or RNA, or by chemical
synthesis.
[0038] The term "an expression control sequence" or a "regulatory
sequence" refers to a sequence that is conventionally used to
effect expression of a gene that encodes a polypeptide and include
one or more components that affect expression, including
transcription and translation signals. The expression control
sequence that is appropriate for expression of the present
polypeptides differs depending upon the host system in which the
polypeptide is to be expressed.
[0039] Any "polypeptide" of the invention, including a Fas ligand
polypeptide, includes any part of the Fas ligand protein including
the mature protein, and further include truncations, variants,
alleles, analogs and derivatives thereof. Variants can be spliced
variants expressed from the same gene as the related protein.
Unless specifically mentioned otherwise, such a polypeptide
possesses one or more of the bioactivities of the protein,
including for example binding activity to a specific partner. This
term is not limited to a specific length of the product of the
gene. Thus, polypeptides that are identical or contain at least
60%, preferably 70%, more preferably 80%, and most preferably 90%
homology to the target protein or the mature protein, wherever
derived, from human or nonhuman sources are included within this
definition of a polypeptide. Also included, therefore, are alleles
and variants of the product of the gene that contain amino acid
substitutions, deletions, or insertions. The amino acid
substitutions can be conservative amino acid substitutions or
substitutions to eliminate non-essential amino acid residues such
as to alter a glycosylation site, a phosphorylation site, an
acetylation site, or to alter the folding pattern by altering the
position of the cysteine residue that is not necessary for
function. Conservative amino acid substitutions are those that
preserve the general charge, hydrophobicity/hydrophilicity and/or
steric bulk of the amino acid substituted, for example,
substitutions between the members of the following groups are
conservative substitutions: Gly/Ala, Val/Ile/Leu, Asp/Glu, Lys/Arg,
Asn/Gln, Ser/Cys/Thr and Phe/Trp/Tyr. Analogs include peptides
having one or more peptide mimics, also known as peptoids, that
possess the target protein-like activity. Included within the
definition are, for example, polypeptides containing one or more
analogs of an amino acid (including, for example, unnatural amino
acids, etc.), polypeptides with substituted linkages, as well as
other modifications known in the art, both naturally occurring and
non-naturally occurring. The term "polypeptide" also does not
exclude post-expression modifications of the polypeptide, for
example, glycosylations, acetylations, phosphorylations,
myristoylations and the like.
[0040] The term "naked DNA" refers to polynucleotide DNA for
administration to a mammal for expression in the mammal. The
polynucleotide can be, for example, a coding sequence, and the
polynucleotide DNA can be directly or indirectly connected to an
expression control sequence that can facilitate the expression of
the coding sequence once the DNA is inside a cell. The direct or
indirect connection is equivalent from the perspective of
facilitating the expression of the DNA in the mammal's cells, and
merely allows the possibility of the inclusion of other sequences
between the regulatory region and the coding sequence that may
facilitate the expression further, or may merely act a linker or
spacer to facilitate connecting the two polynucleotide regions
together to form a nonviral vector.
[0041] "Vector" refers to an assembly which is capable of directing
the expression of the sequencers) or gene(s) of interest. The
vector must include transcriptional promoter/enhancer or locus
defining element(s), or other elements which control gene
expression by other means such as alternate splicing, nuclear RNA
export, post-translational modification of messenger, or
post-transcriptional modification of protein. In addition, the
vector must include a sequence which, when transcribed, is operably
linked to the sequence(s) or gene(s) of interest and acts as a
translation initiation sequence. Optionally, the vector may also
include a signal which directs polyadenylation, a selectable marker
such as Neo, TK, hygromycin, phleomycin, histidinol, or DHFR, as
well as one or more restriction sites and a translation termination
sequence. In addition, if the vector is placed into a retrovirus,
the vector must include a packaging signal, long terminal repeats
(LTRs), and positive and negative strand primer binding sites
appropriate to the retrovirus used (if these are not already
present).
[0042] "Tissue-specific promoter" refers to transcriptional
promoter/enhancer or locus defining elements, or other elements
which control gene expression as discussed above, which are
preferentially active in a limited number of tissue types.
Representative examples of such tissue-specific promoters include
the PEPCK promoter, HER2/neu promoter, casein promoter, IgG
promoter, Chorionic Embryonic Antigen promoter, elastase promoter,
porphobilinogen deaminase promoter, insulin promoter, growth
hormone factor promoter, tyrosine hydroxylase promoter, albumin
promoter, alphafetoprotein promoter, acetyl-choline receptor
promoter, alcohol dehydrogenase promoter, a or h globin promoters,
T-cell receptor promoter, or the osteocalcin promoter.
[0043] "Event-specific promoter" refers to transcriptional
promoter/enhancer or locus defining elements, or other elements
which control gene expression as discussed above, whose
transcriptional activity is altered upon response to cellular
stimuli. Representative examples of such event-specific promoters
include thymidine kinase or thymidilate synthase promoters, .alpha.
or .beta. interferon promoters and promoters that respond to the
presence of hormones (either natural, synthetic or from other
non-host organisms, e.g., insect hormones).
[0044] The term "fusion protein" or "fusion polypeptide" refers to
the recombinant expression of more than one heterologous coding
sequence in a vector or contiguous connection such that expression
of the polypeptide in the vector results in expression of one
polypeptide that includes more than one protein or portion of more
than one protein. Most optimally, the fusion protein retains the
biological activity of at least one of the polypeptide units from
which it is built, and preferably, the fusion protein generates a
synergistic improved biological activity by combining the portion
of the separate proteins to form a single polypeptide. A fusion
protein can also be created with a polypeptide that has function
and a peptide or polypeptide that has no function when expressed,
but which serves a purpose for the expression of the polypeptide
with activity. Examples of fusion proteins useful for the invention
include any Fas ligand fusion polypeptide genetically engineered to
some advantage for the therapy. The term "chimera" or "chimeric
protein" is equivalent to fusion protein or fusion polypeptide. A
"chimeric molecule" can be a fusion polypeptide, or a
polynucleotide fusion molecule encoding a fusion polypeptide. Thus,
for example, a fusion protein can have an extracellular receptor
binding domain of Fas ligand, and a transmembrane proximal and
transmembrane domain of another protein, for example, a protein in
the same family as Fas ligand, for example, TNF or CD30 ligand. The
chimera can be constructed from ligated DNA coding sequences and
expressed in a cell system, or administered in a vector for
expression in vivo in an animal. For example, a chimera or fusion
protein including a membrane bound Fas ligand can be administered
in a gene therapy protocol in vivo or ex vivo. The chimeric
polypeptide can be, for example, a fusion polypeptide comprising a
Fas binding portion of Fas ligand fused to another transmembrane
portion incapable of being cleaved from the membrane. The chimeric
polypeptide can also be a fusion polypeptide of a Fas binding
portion of Fas ligand fused to a synthetically derived sequence of
amino acids that provide a fusion polypeptide incapable of being
cleaved from the cell membrane.
[0045] The phrase "sequence encoding a membrane-bound Fas ligand"
is meant to include a polynucleotide sequence encoding a Fas ligand
mutein or a Fas ligand chimera molecule, the polynucleotide
comprising a first DNA sequence encoding the Fas binding domain of
Fas ligand linked to a second DNA sequence encoding the
intracellular (and transmembrane) region of Fas ligand or a
membrane bound receptor. For example, a membrane-bound Fas ligand
can include a fusion protein having a Fas-binding domain and a
polypeptide sequence of another transmembrane protein including
replacement of the Fas ligand cleavage site, or a sequence that is
not cleaved from the cell membrane. A fusion protein that makes up
a membrane-bound Fas ligand can also be, for example, a Fas binding
domain of Fas ligand polypeptide, and a synthetic sequence of amino
acids such that the proximal extracellular portion of the fusion
cannot be cleaved from the cell membrane, and such that the
sequence of amino acids includes sufficient hydrophobicity for a
transmembrane sequence, and such that the sequence of amino acids
proximal intracellular is also appropriately configured with charge
for constituting a fusion polypeptide capable of expression on but
not cleavage from the cell membrane. For example a membrane-bound
Fas ligand can include a chimera that has an extracellular and
Fas-binding portion of the Fas ligand, an uncleavage domain and
transmembrane portion of another protein of the same family as Fas
ligand, and a cytoplasmic portion of the same or yet another
protein in the same family as Fas ligand. Thus, for example, a
sequence encoding a membrane-bound Fas ligand includes a
polynucleotide sequence encoding an extracellular and Fas-binding
portion of the Fas ligand ligated to a polynucleotide sequence
encoding the intracellular and transmembrane domains of a member of
the TNF receptor family. In addition to the Fas ligand chimeras
being made with Fas ligand and portions of other members of the TNF
superfamily, Fas ligand chimeras that enable Fas ligand to remain
membrane-bound when expressed in cells can also be made of Fas
ligand and portions of any other transmembrane protein such that
cleavage of the Fas ligand from the membrane is prevented and the
Fas ligand polypeptide remains membrane bound.
[0046] A "patient" can be any treatable living organism, including
but not limited to a eukaryote or a prokaryote. The patient
eukaryote can be, for example, a vertebrate or an invertebrate.
Thus, for example, the patient can be a fish, a bird, a worm, an
insect, a mammal, a reptile, an amphibian, a fungi, or a plant,
preferably a mammal. The mammal can be, for example a human.
General Methods of Making and Using a Fas Ligand
[0047] Therapeutic Agent and/or Diagnostic Agent
[0048] In one aspect, the present invention includes a method for
treating an autoimmune disease manifesting activated T-cells or
activated B-cells or both by providing a Fas ligand therapeutic
agent, and administering an effective amount of the Fas ligand
therapeutic agent to a mammal having an autoimmune disease. An
"autoimmune disease", "autoimmune disease" and "autoimmunity" all
refer to a disorder characterized by autoimmunity in the mammal
which is the response of an immune system against self components.
An autoimmune response can develop into a condition manifesting
clinical symptoms. Although strictly speaking transplantation
rejection is not an autoimmune reaction, where patient condition
prescribes surgery to replace or graft cells, tissue or an organ,
the body receiving the allograft can react immunologically against
the foreign graft. "Transplantation rejection" occurs when during
an allograft of cells, tissue, or an organ, from one member of a
species to another, an immune response in the recipient results,
sufficient to reject the transplanted cells, tissue or organ.
[0049] Examples of "autoimmune diseases" that can be treated by the
method and therapeutic agent of the invention include multiple
sclerosis, Hashimoto's thyroiditis, systemic lupus erythematosus
(SLE), Goodpasture's syndrome, Pemphigus, receptor autoimmunity,
autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura,
osteoarthritis, rheumatoid arthritis, schleroderma with
anticollagen antibodies, mixed connective tissue disease,
polymyositis, pernicious anemia, idiopathic Addison's disease,
spontaneous infertility, glomerulonephritis, bullous pemphigoid,
adrenergic drug resistance, chronic active hepatitis, primary
biliary cirrhosis, autoimmune-based endocrine gland failure,
vitiligo, vasculitis, post-myocardial infarction, cardiotomy
syndrome, urticaria, atopic dermatitis, autoimmune-based asthma,
autoimmune-based inflammatory reactions, granulomatous disorders,
alkylizing spondylitis, poststreptococcal glomerulonephritis,
autoimmune hemolytic anemia, encephalitis, autoimmune reaction
secondary to lymphoma, degenerative disorders, and atrophic
disorders. Autoimmune diseases manifesting receptor autoimmunity
include, for example, Grave's disease, myasthenia gravis, and
insulin resistance. Autoimmune diseases of adrenergic drug
resistance include, for example, asthma and cystic fibrosis.
[0050] Other autoimmune diseases appropriate for the invention
include, for example those for which an animal model exists,
including for example, Sjogren's syndrome (autoimmune dacryodentis
or immune-mediated sialadenitis), autoimmune myocarditis, primary
biliary cirrhosis (PBC), inflammatory heart disease,
mercury-induced renal autoimmunity, insulin dependent diabetes
mellitus (type I diabetes or IDD), post-thymectomy autoimmunity, a
central nervous system (CNS) demyelination disorder, CNS lupus,
narcolepsy, a immune-mediated PNS disorder, osteoarthritis,
rheumatoid arthritis, uveitis, medullary cystic fibrosis,
autoimmune hemolytic disease, autoimmune vasculitis, ovarian
autoimmune disease, and human schleroderma. An autoimmune disease
characterized by a central nervous system (CNS) demyelination
disorder can be, for example, multiple sclerosis (MS). A peripheral
nervous system (PNS) autoimmune disease can be, for example,
Guillain-Barre syndrome (GBS).
[0051] The Fas ligand therapeutic agent can be a polypeptide, a
polynucleotide, a small organic molecule, a peptide or a peptoid.
The Fas ligand therapeutic agent can be a Fas ligand polypeptide, a
polynucleotide encoding a Fas ligand polypeptide, a fusion
polypeptide comprising a portion of native Fas ligand polypeptide,
a polynucleotide encoding a fusion polypeptide comprising a portion
of native Fas ligand polypeptide, a biologically active peptide
derivative of Fas ligand polypeptide, a biologically active peptoid
derived from Fas ligand polypeptide, or a small organic molecule
agonist of Fas ligand activity. The Fas ligand polypeptide can be a
biologically active Fas ligand polypeptide such as a Fas ligand
polypeptide variant, a Fas ligand polypeptide derivative, a
modified Fas ligand polypeptide, or a truncated Fas ligand
polypeptide. The polynucleotide encoding a Fas ligand polypeptide
can be a polynucleotide sequence encoding full length Fas ligand
polypeptide, a sequence encoding a biologically active portion of
Fas ligand polypeptide, a sequence encoding a biologically active
peptide derived from Fas ligand polypeptide, a sequence encoding
membrane-bound Fas ligand polypeptide, or a sequence encoding a
soluble Fas ligand polypeptide. Another embodiment of the invention
is a composition having a gene delivery vehicle capable of
expressing a polynucleotide sequence encoding a Fas ligand
polypeptide.
[0052] The Fas ligand polypeptide of the compositions of the
invention include a membrane-bound Fas ligand polypeptide. In a
preferred embodiment, the Fas ligand polypeptide is a recombinant
Fas ligand that is capable of remaining on a cell membrane longer
than native Fas ligand, more preferably, the membrane bound
recombinant Fas ligand is substantially non-cleavable from the
surface of the expressing cell. The invention also includes,
therefore, a host cell, preferably a human host cell, transformed
with a polynucleotide sequence encoding a Fas ligand polypeptide
selected from the group consisting of a full length Fas ligand
polypeptide, a biologically active portion of Fas ligand
polypeptide, a membrane-bound Fas ligand polypeptide, and a
sequence encoding a soluble Fas ligand polypeptide. Preferably, the
host cell is a T-cell. Thus, in its therapeutic embodiment, the
invention is also directed to a method of reducing a population of
activated T-cells or activated B-cells by transforming a host cell
with a Fas ligand polypeptide that remains membrane-bound, wherein
the host cell also bears another ligand recognized by the activated
T-cells or activated B-cells, and re-introducing the transformed
cell into the population of activated T-cells or activated B-cells.
This method is particularly suited to treatment of an autoimmune
disease (which is mediated by activated T cells or B cells) or
transplantation rejection. In this method, the other ligand is
natively expressed by the cell, or a polynucleotide encoding the
other ligand is used to transform the host cell for expression
therein. Examples of some of the autoimmune diseases treatable by
the method of the present invention include rheumatoid arthritis
(RA), primary biliary cirrhosis (PBS), systemic lupus erythmatosis
(SLE), and idiopathic thrombocytopenic purpura (ITP).
[0053] When used in the therapeutic embodiment, the host cell is
more preferably an allogeneic host cell, and most preferably a
syngeneic host cell. Transduction or transformation of the host
cell is performed in vivo or ex vivo, more preferably ex vivo using
techniques and vectors that are well known in the art. See U.S.
Pat. No. 5,399,346 (Anderson) and U.S. Ser. No. 08/463,122, filed
Jun. 5, 1995, now allowed, both of which are hereby expressly
incorporated herein by reference for their disclosures of said
techniques and vectors.
[0054] The same host cell having the non-cleavable (or
substantially non-cleavable) Fas-ligand on its surface is capable
of being used in a diagnostic embodiment to detect activated T cell
or B cells having Fas (antigen) expressed on their surface. In
particular, the diagnostic method of this embodiment comprises:
[0055] a) providing a transformed host cell having a recombinant
Fas ligand surface bound thereto, said surface bound Fas ligand
mutein being resistant to cleavage; [0056] b) contacting said
transformed cell with a sample containing an excess of cells
suspected of having surface bound Fas thereon to obtain a cell
mixture; [0057] c) allowing said cell bound Fas to bind to said
cell bound recombinant Fas ligand; and [0058] d) observing said
cell mixture for rosette formation or clumping, whereby rosette
formation or clumping indicates the presence of cells having Fas
thereon in said sample.
[0059] The cells to be tested in the present inventory are
typically centrifuged to separate them from any soluble interfering
substances. The centrifuged cells are resuspended in any of a
variety of balanced salt solutions, such including Hank's, Earle's
saline, or Dulbecco's buffered saline. To obtain rosette formation,
the cells suspected of having Fas expressed on their surface are
provided in excess, relative to the RFL cell, typically 25:1,
preferably 40:1, more preferably 100:1. See Smith et al. "A
Modified Assay For The Detection Of Human Adult Active T Rosette
Forming Lymphocytes," J. Immunol. Methods, 8:175-184 (1975);
Fandenberg et al., "T-rosette-Forming Cells: Cellular Immunity and
Cancer," N. Eng. J. Of Med., 292:465-475 (1975); and Kerman et al.,
"Active T Rosette Forming Cells in the Peripheral Blood of Cancer
Patients," Cancer Res., 36:3274-3278 (1976).
[0060] As the above cited references reflect, various methods for
observing rosette formation are well known to the art.
[0061] When the above described method of detecting activated T
cells and B cells is performed with human T cell and B cells, the
transformed host cell is preferably a human host cell.
[0062] Ultimately, when cell bound Fas binds to a Fas ligand,
including the membrane bound Fas ligand of the present invention,
the cell with the Fas surface bound thereon goes into apoptosis and
dies. Thus, the present invention is also directed to a method for
reducing the population of activated T cells and/or B cells, the
method comprising the steps of: [0063] a) contacting a population
of activated T cells and/or B cells with a transformed cell
expressing a recombinant Fas ligand that is surface bound thereon,
said activated T cells and/or B cells having a cell membrane with
Fas expressed thereon, said Fas ligand on said cell binding to said
Fas on said activated T cells and/or B cells, and inducing
apoptosis in said activated T cells and or B cells, whereby said
population is reduced. The term "membrane bound Fas ligand" as used
in the present invention, not only includes muteins of Fas ligand
that are non-cleavable (and thus remain membrane bound), but also a
chimera or a fusion protein of Fas ligand.
[0064] The present invention is also directed to a chimera or
fusion protein for providing a membrane bound Fas ligand that
induces apoptosis in activated T-cells and B-cells, for the purpose
of a treatment of certain autoimmune diseases is that Fas ligand
binds to Fas and induces apoptosis of Fas expressing cells. It has
been shown that Fas ligand is cleaved from the cell surface to
produce a soluble form of Fas ligand. The goal of construction of a
chimera or fusion protein including a portion of the Fas ligand
sequence is to restrict Fas ligand expression to the cell surface
to protecting the expressing cell, tissue or organ from CTL
(cytotoxic T-cell lymphocyte) mediated killing. Because such non
cleavable receptor proteins TNF and CD30 ligand and other ligands
of the same superfamily are also type II transmembrane proteins
they are suited to forming a chimera with Fas ligand that allows
Fas ligand to retain a similar transmembrane and cytoplasmic
component as native Fas ligand. However, the advantage of using,
for example, TNF for the transmembrane and cytoplasmic portion is
that the site of cleavage on TNF is well defined, and the TNF
portion of the fusion protein can be mutated to be substantially
noncleavable. Fusions can be made of the extracellular domain of
Fas ligand, retaining at least the portion of Fas ligand that binds
to Fas, and uncleavable transmembrane and intracellular domain of,
for example a modified TNF sequence, or other polypeptide, for
example, uncleavable transmembrane and intracellular domains of
other members of that ligand superfamily. Thus, the chimera as
described binds Fas and induces apoptosis in Fas-expressing cells,
but is not cleaved from the surface of the cell in which the
chimera is expressed. The Fas ligand chimera can also be
constructed using portions of any other transmembrane protein for
the purpose of making the Fas ligand extracellular portion of the
chimera that is capable of binding Fas and inducing apoptosis in
Fas expressing cells but that remains membrane-bound. Examples of
such transmembrane proteins from which can be derived a polypeptide
sequence appropriate for constructing a Fas ligand fusion protein
having the ability to bind Fas but also the ability to remain
membrane-bound, include such membrane-bound transmembrane proteins
as membrane-bound antibodies, viral antigens that remain
membrane-bound, or proximally extracellular/transmembrane/and
intracellular portions of other known transmembrane proteins.
Additionally, any operable combination of protein sequence and
synthetically designed sequence, for example a sequence of
hydrophobic amino acids to anchor the fusion protein in the
membrane can be figured into the design of a Fas ligand fusion
polypeptide.
[0065] Chimeric molecules or fusion proteins can be created that
express a membrane-bound Fas ligand chimera. For example, the
region of pro-Fas ligand (SEQ ID NO: 12) extending 129 residues
from M1 to Q130 of the pro-protein (i.e., SEQ ID NO: 2) can be
replaced by another polypeptide sequence that provides a fusion
protein capable of expression on the cell membrane and capable of
binding to Fas, but also capable of remaining on the cell membrane
longer than native Fas ligand. The polypeptide sequence that is
used in the fusion with the Fas binding portion of Fas ligand can
be another transmembrane protein, preferably one that is not
normally cleaved from the cell membrane, or a polypeptide sequence
that includes a synthetically assimilated sequence that provides
for the proper amino acid content for the proximal extracellular
domain, the transmembrane domain, and the proximal intracellular
domain.
[0066] For example, the extracellular sequence of Fas ligand from
I131 to L281, and which binds Fas, can be retained as the Fas
binding portion of the fusion protein. The proximal extracellular
domain (of the fusion polypeptide) corresponding to about 14 amino
acids (in the native Fas ligand protein) can be a multiple proline
sequence, or a sequence having mostly proline amino acids or amino
acids forming a sequence that is not normally cleaved from the cell
membrane by a protease or convertase. The sequence of the
transmembrane portion of the fusion polypeptide can be of
hydrophobic amino acids having nonpolar side chains, including for
example the amino acids alanine, valine, leucine, isoleucine,
proline, phenylalanine, tryptophan, and methionine. The sequence of
the fusion polypeptide of amino acids proximally intracellular can
be amino acids with charged side chains including, for example,
aspartic acid and glutamic acid which are negatively charged at pH
6.0, and lysine, arginine, and histidine which are positively
charged at pH 6.0. The remainder of the fusion polypeptide can be a
sequence appropriate for an intracellular portion of a
transmembrane polypeptide, and might preferably be a sequence of
Fas ligand or of a member of the TNF family of ligands so that any
effects that the intracellular portion of the molecule may have on
the biological activity desired from the Fas ligand derived
polypeptide fusion might be retained.
[0067] The Fas ligand fusion polypeptide can also be made up of a
Fas ligand extracellular portion including the portion of Fas
ligand that binds Fas and promotes signaling, and the transmembrane
and intracellular portion of another member of the same family (the
tumor necrosis factor family). The fusion protein construct is
especially effective where the intracellular region of Fas ligand
and the Fas ligand cleavage site is replaced with an uncleavable
domain, such as, for example, the intracellular and transmembrane
domains one of the members of the TNF receptor family of proteins,
such as TNFR, CD30 or CD40. The result is a membrane-bound
polypeptide having Fas ligand capable of binding Fas for activating
apoptosis in Fas expressing cells. Such a chimera is well suited to
a gene therapy administration by using a gene delivery vehicle, and
can thus facilitate activating apoptosis in Fas expressing cells,
such as, for example activated T-cells or activated B-cells
involved to protect target cells/tissues/organs in the context of
an autoimmune disease or transplant rejection. The DNA sequences
for the various members of the TNF receptor family are well known
in the art. In particular, the cloning of TNFR and CD40 is
disclosed in the following references, each of which is hereby
incorporated by reference: Schall, et al., "Molecular Cloning and
Expression of a Receptor for Human Tomor Necrosis Factor," Cell
61:361-370 (1990); and Stamenkovic et al., "A B-lymphocyte
Activation Molecule Related to the Nerve Growth Factor Receptor and
Induced by Cytokines in Carcinomas," EMBO J 8(5):1403-1410
(1989).
[0068] Fusion proteins are constructed, for example, by replacing a
fragment of Fas ligand with an uncleavable fragment from a cell
surface receptor, for example CD30, CD40, or a TNFR, or even an
uncleavable portion of another transmembrane protein. For example,
substitution of the fragment of Fas ligand from L127 to R144 with a
fragment of CD30 ligand from S83 to A100 or a fragment of CD40
ligand from E104 to Q121 results in a mutant or fusion protein Fas
ligand with the replacement in the cleavage site that provides that
the mutant remains membrane-bound.
[0069] Yet another variation is construction of a chimeric molecule
of C-terminal from Fas ligand from I131 to L281 fused to the
N-terminal CD30 ligand from M1 to L86 or the N-terminal of CD40
ligand from M1 to K107. The resulting chimera is membrane bound and
uncleavable.
[0070] Using standard gene splicing techniques that are well known
in the art, gene constructs are made that encode a chimeric Fas
ligand polypeptide that is expressed in membrane bound form. In
such constructs, approximately the first fourteen amino acid region
of the extracellular region of the Fas ligand may be deleted or
replaced. Deletion of the DNA coding for this region provides a Fas
ligand molecule that is not be cleaved, as cleavage of
native-sequence Fas ligand from the cell membrane is believed to
occur within this region. Replacement of this coding region with
the corresponding coding region of any transmembrane receptor
protein that is not cleaved in that region, creates a DNA encoding
a fusion protein that is also not susceptible to cleavage, and thus
remains membrane-bound.
[0071] Alternatively, as described above, the Fas ligand can be the
basis of a chimeric but non-cleavable starting with replacement of
membrane bound Fas ligand, the portion of the extracellular region
of Fas ligand believed to be the cleavage site for a protease that
releases soluble Fas ligand, (preferably the first 14 amino acids
of the extracellular region), and the remainder of the N-terminal
region of the Fas ligand can be replaced with the corresponding
sequence of CD30, or CD40, or other member of the TNF receptor
family provided that the TNF receptor is not cleaved at the
replaced sequence, or that the replaced sequence is altered to
prevent cleavage. Finally, the Fas ligand sequence can be
mutagenized at the region where the clipping of the membrane-bound
molecule is believed to occur, to prevent or reduce the likelihood
of cleavage, using standard mutagenesis techniques, such as taught
by Gilman et al., Gene, 8:81 (1979) and Roberts et al., Nature,
328:731 (1987). The resulting Fas ligand mutant is also a cleavage
resistant variant of the native Fas ligand, and thus, stays
membrane bound longer when expressed in a host cell. The resulting
membrane bound ligand when expressed in a transduced host cell
protects cells/tissues/organ expressing Fas and eliminates side
effects of soluble Fas ligand when cleaved off the membrane.
[0072] Also within the scope of this invention are Fas ligand
deletion muteins which when expressed in a host cell become
membrane bound Fas ligand agonists that are substantially
non-cleavable. In one embodiment of the present invention, the Fas
ligand deletion mutein is a deletion mutein of pro-Fas ligand that
lacks a segment of 4 to 17 amino acid residues beginning at about
residue position 127 of pro-Fas ligand (SEQ ID NO: 12). Preferably,
the deletion mutein of pro-Fas ligand lacks a continuous segment of
10 to 17 amino acids beginning at about residue 130 of pro-Fas
ligand (SEQ ID NO: 12). More preferably, the Fas ligand deletion
mutein has a deletion selected from the group consisting of
130.fwdarw.139, 130.fwdarw.140, 130.fwdarw.141, 130.fwdarw.142,
130.fwdarw.143, 130.fwdarw.144, 130.fwdarw.145, 130.fwdarw.146,
131.fwdarw.140, 131.fwdarw.141, 131.fwdarw.142, 131.fwdarw.143,
131.fwdarw.144, 131.fwdarw.145 and 131.fwdarw.146.
[0073] To make a membrane-bound uncleavable Fas ligand, DNA
encoding four to seventeen residues from residue positions L127 to
R144 of Fas ligand native sequence is deleted from a genomic or
cDNA (SEQ ID NO: 11). Expression of this deletion mutein of Fas
ligand produces a Fas ligand agonist that is substantially
non-cleavable. The preparation of specific deletion muteins of Fas
ligand are disclosed in the specification at Examples 5 and 6. In
Example 5, a polynucleotide (SEQ ID NO: 1) encoding the first 129
amino acid residues (SEQ ID NO: 2) of Fas ligand was ligated to a
second polynucleotide (SEQ ID NO: 3) encoding residues 143-282 of
Fas ligand. The resulting polynucleotide (SEQ ID NO: 5) encoded the
non-cleavable Fas ligand.sub.130.fwdarw.142 deletion mutein (SEQ ID
NO: 6). In example 6, a polynucleotide (SEQ ID NO: 1) encoding the
first 129 amino acid residues (SEQ ID NO: 2) of Fas ligand was
ligated to a second polynucleotide (SEQ ID NO: 7) encoding residues
146-282 of Fas ligand. The resulting polynucleotide (SEQ ID NO: 9)
encoded the non-cleavable Fas ligand.sub.130.fwdarw.146 deletion
mutein (SEQ ID NO: 10).
[0074] A Fas ligand fusion protein having a Fas binding portion of
amino acid residues R144 to L281, and a cell surface molecule
binding portion can be constructed. For example, the cell surface
molecule binding portion can be heparin, and the cell surface
molecule to which it binds can be a glycosaminoglycan molecule.
Such a polypeptide fusion protein, for example using heparin and
Fas ligand, can be constructed as described in U.S. Ser. No.
07/608,539, filed Nov. 1, 1990, and U.S. Ser. No. 07/608,569, filed
Nov. 2, 1990. This Fas ligand fusion polypeptide can be expressed
and purified in any standard expression system suitable for
producing sufficient quantities of the recombinant protein, and for
any necessary post translational modifications. Once purified the
polypeptide can be used, for example, to bathe the organ or tissue
to be transplanted, before entry into the transplant recipient. The
Fas ligand polypeptide binds at its heparin portion, cell surfaces
on the external cells of the organ or tissue. When the organ, so
treated, is placed in the patient, any activated T-cells attempting
to generate an immune rejection to the foreign organ or tissue
encounters Fas ligand coated cells. Upon binding to Fas of the Fas
ligand portion coating the cells, the activated T-cells expressing
Fas undergoes apoptosis, thus reducing the immune rejection of the
organ or tissue.
[0075] In all cases of the above described fusion polypeptides, and
all obvious variations and permutations of these fusion
polypeptides, construction can be facilitated by constructing a
polynucleotide chimera capable of expressing the desired
polypeptide fusion. Any mutations and deletions in such
polynucleotide constructs can be conducted using standard
mutagenesis (such as taught by Gilman et al., Gene, 8:81 (1979) and
Roberts et al., Nature, 328:731 (1987)) and restriction digest
cleavage and ligation in standard plasmids or vectors. In many
cases, the fusion polypeptide can be made by starting with DNA
encoding native Fas ligand and DNA encoding another transmembrane
protein. Addition of synthetic sequences can also be accomplished
by standard techniques. Any member of the TNF family of ligands,
and in some cases any transmembrane protein, may be suitable for
arranging a chimera or mutant along the lines described in order
that a Fas ligand signaling polypeptide is created that remains
membrane bound for the purpose of binding and signaling through Fas
on another cell, for example an activated T-cell or B-cell.
[0076] In the case of polynucleotides encoding Fas ligand
polypeptides for expression in cells to target activated T-cells or
B-cells or other Fas expressing cells harmful to an organism, the
polynucleotide constructs, once designed, for example, as described
herein, can be constructed by standard recombinant DNA technology
and manipulation. For example, polynucleotide constructs having
deletions, mutations, substitutions, fusions, and which otherwise
encode polypeptide variants, derivatives, mutants, analogues, or
chimeras of Fas ligand can be constructed by conventional
techniques of molecular biology, microbiology, and recombinant DNA
technology that are within the skill of the art. The Fas ligand
polynucleotide can be placed into a vector which directs its
expression.
[0077] Such techniques for polynucleotide and polypeptide
construction and expression are explained fully in the literature,
for example in Sambrook, et al. MOLECULAR CLONING; A LABORATORY
MANUAL, SECOND EDITION (1989); DNA CLONING, VOLUMES I AND II (D. N
Glover ed. 1985); OLIGONUCLEOTIDE SYNTHESIS (M. J. Gait ed, 1984);
NUCLEIC ACID HYBRIDIZATION (B. D. Hames & S. J. Higgins eds.
1984); TRANSCRIPTION AND TRANSLATION (B. D. Hames & S. J.
Higgins eds. 1984); B. Perbal, A PRACTICAL GUIDE TO MOLECULAR
CLONING (1984); the series, METHODS IN ENZYMOLOGY (Academic Press,
Inc.); GENE TRANSFER VECTORS FOR MAMMALIAN CELLS (J. H. Miller and
M. P. Calos eds. 1987, Cold Spring Harbor Laboratory), Methods in
Enzymology Vol. 154 and Vol. 155 (Wu and Grossman, and Wu, eds.,
respectively), Mayer and Walker, eds. (1987), IMMUNOCHEMICAL
METHODS IN CELL AND MOLECULAR BIOLOGY (Academic Press, London),
Scopes, (1987), PROTEIN PURIFICATION: PRINCIPLES AND PRACTICE,
Second Edition (Springer-Verlag, N.Y.), HANDBOOK OF EXPERIMENTAL
IMMUNOLOGY, VOLUMES I-IV (D. M. Weir and C. C. Blackwell eds 1986);
and VACCINES (R. W. Ellis, ed., 1992, Butterworth-Heinemann,
London). Standard abbreviations for nucleotides and amino acids are
used in this specification. All publications, patents, and patent
applications cited herein are incorporated by reference. Further,
sequences that encode the above-described genes may also be
synthesized, for example, on an Applied Biosystems Inc. DNA
synthesizer (e.g., ABI DNA synthesizer model 392 (Foster City,
Calif.)). Additionally, the polynucleotides can be constructed and
cloned as described in PCR PROTOCOLS, Cold Spring Harbor, N.Y.
1991. The desired gene can also be isolated from cells and tissues
containing the gene, using phenol extraction, PCR of cDNA, or
genomic DNA. The gene of interest can also be produced
synthetically, rather than cloned, as described in Edge, Nature
292: 756 (1981), Nambair et al, Science 223: 1299 (1984), and Jay
et al, J. Biol. Chem. 259: 6311 (1984). Additionally, variations of
any polynucleotide or polypeptide can be made by conventional
techniques, including PCR or site-directed mutagenesis (such as
taught by Gilman et al., Gene, 8:81 (1979) and Roberts et al.,
Nature, 328:731 (1987)). The DNA construct so synthesized can be
ligated to an expression plasmid containing an appropriate promoter
for expression in a desired host expression system. The host system
can be in vitro, in vivo or ex vivo. In all cases, assay for
whether the Fas ligand polypeptide (expressed in vivo, ex vivo or
in vitro) is functional in terms of binding Fas when expressed on a
cell, and whether, more specifically, any of the Fas ligand fusion
polypeptides are capable of remaining membrane bound longer than
native Fas ligand, can be determined by standard assays, for
example, as described herein.
[0078] A Fas ligand therapeutic agent including a variant,
derivative, mutant, chimera, or analogue of a Fas ligand
polypeptide, or a biologically active Fas ligand derived peptide
therapeutic agent, can be made using the following exemplary
expression systems. Below are some exemplary expression systems in
bacteria, yeast, insects, and mammals. In the particular case of
any of the Fas ligand polypeptides of the invention, the
polypeptides can be expressed recombinantly by methods standard in
the art, including using expression systems described herein, for
example.
Expression Systems
[0079] Although the methodology described below is believed to
contain sufficient details to enable one skilled in the art to
practice the present invention, other constructs can be constructed
and purified using standard recombinant DNA techniques as described
in, for example, Sambrook et al. (1989), MOLECULAR CLONING: A
LABORATORY MANUAL, 2nd ed. (Cold Spring Harbor Press, Cold Spring
Harbor, N.Y.); and under current regulations described in United
States Dept. of HHS, NATIONAL INSTITUTE OF HEALTH (NLH) GUIDELINES
FOR RECOMBINANT DNA RESEARCH. The polypeptides of the invention can
be expressed in any expression system, including, for example,
bacterial, yeast, insect, amphibian and mammalian systems.
Expression systems in bacteria include those described in Chang et
al., Nature (1978) 275: 615, Goeddel et al., Nature (1979) 281:
544, Goeddel et al., Nucleic Acids Res. (1980) 8: 4057, EP 36,776,
U.S. Pat. No. 4,551,433, deBoer et al., Proc. Natl. Acad. Sci. USA
(1983) 80: 21-25, and Siebenlist et al., Cell (1980) 20: 269.
Expression systems in yeast include those described in Hinnen et
al., Proc. Natl. Acad. Sci. USA (1978) 75: 1929; Ito et al., J.
Bacteriol. (1983) 153: 163; Kurtz et al., Mol. Cell. Biol. (1986)
6: 142; Kunze et al., J. Basic Microbiol. (1985) 25: 141; Gleeson
et al., J. Gen. Microbiol. (1986) 132: 3459, Roggenkamp et al.,
Mol. Gen. Genet. (1986) 202:302) Das et al., J. Bacteriol. (1984)
158: 1165; De Louvencourt et al., J. Bacteriol. (1983) 154: 737,
Van den Berg et al., Bio/Technology (1990) 8: 135; Kunze et al., J.
Basic Microbiol. (1985) 25: 141; Cregg et al., Mol. Cell. Biol.
(1985) 5: 3376, U.S. Pat. No. 4,837,148, U.S. Pat. No. 4,929,555;
Beach and Nurse, Nature (1981) 300: 706; Davidow et al., Curr.
Genet. (1985) 10: 380, Gaillardin et al., Curr. Genet. (1985) 10:
49, Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112:
284-289; Tilburn et al., Gene (1983) 26: 205-221, Yelton et al.,
Proc. Natl. Acad. Sci. USA (1984).sub.--81: 1470-1474, Kelly and
Hynes, EMBO J. (1985) 4: 475-479; EP 244,234, and WO 91/00357.
Expression of heterologous genes in insects can be accomplished as
described in U.S. Pat. No. 4,745,051, Friesen et al. (1986) "The
Regulation of Baculovirus Gene Expression" in: THE MOLECULAR
BIOLOGY OF BACULOVIRUSES (W. Doerfler, ed.), EP 127,839, EP
155,476, and Vlak et al., J. Gen. Virol. (1988) 69: 765-776, Miller
et al., Ann. Rev. Microbiol. (1988) 42: 177, Carbonell et al., Gene
(1988) 73: 409, Maeda et al., Nature (1985) 315: 592-594,
Lebacq-Verheyden et al., Mol. Cell. Biol. (1988) 8: 3129; Smith et
al., Proc. Natl. Acad. Sci. USA (1985) 82: 8404, Miyajima et al.,
Gene (1987) 58: 273; and Martin et al., DNA (1988) 7:99. Numerous
baculoviral strains and variants and corresponding permissive
insect host cells from hosts are described in Luckow et al.,
Bio/Technology (1988) 6: 47-55, Miller et al., in GENERIC
ENGINEERING (Setlow, J. K. et al. eds.), Vol. 8 (Plenum Publishing,
1986), pp. 277-279, and Maeda et al., Nature, (1985) 315:
592-594._Mammalian expression can be accomplished as described in
Dijkema et al., EMBO J. (1985) 4: 761, Gorman et al., Proc. Natl.
Acad. Sci. USA (1982b) 79: 6777, Boshart et al., Cell (1985) 41:
521 and U.S. Pat. No. 4,399,216. Other features of mammalian
expression can be facilitated as described in Ham and Wallace,
Meth. Enz. (1979) 58: 44, Barnes and Sato, Anal. Biochem. (1980)
102: 255, U.S. Pat. No. 4,767,704, U.S. Pat. No. 4,657,866, U.S.
Pat. No. 4,927,762, U.S. Pat. No. 4,560,655, WO 90/103430, WO
87/00195, and U.S. RE 30,985.
Small Molecule Library Synthesis
[0080] The Fas ligand derived therapeutic agents can include small
molecule mimics or agonists of Fas ligand activity. They can be
screened for the ability to bind Fas, or bind and activate Fas
expressed on a cell. Activation can be noted by the death of the
cell contacted by the small molecule Fas ligand agonist. Small
molecule agonists of Fas ligand activity, for example, a peptide,
peptoid, or organic small molecule can be screened from a library
for ability to bind Fas antigen, or additionally for the ability to
bind and activate Fas antigen expressed on a cell for the purpose
of inducing apoptosis in the Fas expressing cell.
[0081] Therapeutic agents of the invention can include peptides and
peptoids derived from Fas ligand polypeptide sequence, and can be
designed or modified to accomplish improved biological activity
over Fas ligand polypeptide, such as, for example higher affinity
for Fas ligand receptor, or resistance to degradation in the
mammal. Peptides and peptoids can be prepared synthetically in
libraries for screening for a favored biological activity, for
example, increased binding to Fas ligand receptor. Exemplary
synthesis of some of these small molecules are described below. The
small molecules libraries designed based on variations in Fas
ligand receptor binding sites of a Fas ligand polypeptide can be
screened for ability to bind to Fas ligand receptor and induce
apoptosis, for example, in a cell-base assay in microwell
plates.
[0082] Small molecule libraries that are peptide and peptoid
derivatives of Fas ligand are made as follows. A "library" of
peptides may be synthesized and used following the methods
disclosed in U.S. Pat. No. 5,010,175, (the '175 patent) and in PCT
WO91/17823. In method of the '175 patent, a suitable peptide
synthesis support, for example, a resin, is coupled to a mixture of
appropriately protected, activated amino acids. The method
described in WO91/17823 is similar but simplifies the process of
determining which peptides are responsible for any observed
alteration of gene expression in a responsive cell. The methods
described in WO91/17823 and U.S. Pat. No. 5,194,392 enable the
preparation of such pools and subpools by automated techniques in
parallel, such that all synthesis and resynthesis may be performed
in a matter of days.
[0083] Further alternative agents include small molecules,
including peptide analogs and derivatives, that can act as
stimulators or inhibitors of gene expression, or as ligands or
antagonists. Some general means contemplated for the production of
peptides, analogs or derivatives are outlined in CHEMISTRY AND
BIOCHEMISTRY OF AMINO ACIDS, PEPTIDES, AND PROTEINS--A SURVEY OF
RECENT DEVELOPMENTS, Weinstein, B. ed., Marcell Dekker, Inc., publ.
New York (1983). Moreover, substitution of D-amino acids for the
normal L-stereoisomer can be carried out to increase the half-life
of the molecule.
[0084] Peptoids, polymers comprised of monomer units of at least
some substituted amino acids, can act as small molecule stimulators
or inhibitors herein and can be synthesized as described in PCT
91/19735. Presently preferred amino acid substitutes are
N-alkylated derivatives of glycine, which are easily synthesized
and incorporated into polypeptide chains. However, any monomer
units which allow for the sequence specific synthesis of pools of
diverse molecules are appropriate for use in producing peptoid
molecules. The benefits of these molecules for the purpose of the
invention is that they occupy different conformational space than a
peptide and as such are more resistant to the action of
proteases.
[0085] Peptoids are easily synthesized by standard chemical
methods. The preferred method of synthesis is the "submonomer"
technique described by R. Zuckermann et al., J. Am. Chem. Soc.
(1992) 114:10646-7. Synthesis by solid phase techniques of
heterocyclic organic compounds in which N-substituted glycine
monomer units forms a backbone is described in copending
application entitled "Synthesis of N-Substituted Oligomers" filed
on Jun. 7, 1995 and is herein incorporated by reference in full.
Combinatorial libraries of mixtures of such heterocyclic organic
compounds can then be assayed for the ability to alter gene
expression.
[0086] Synthesis by solid phase of other heterocyclic organic
compounds in combinatorial libraries is also described in copending
application U.S. Ser. No. 08/485,006 entitled "Combinatorial
Libraries of Substrate-Bound Cyclic Organic Compounds" filed on
Jun. 7, 1995, herein incorporated by reference in full. Highly
substituted cyclic structures can be synthesized on a solid support
by combining the submonomer method with powerful solution phase
chemistry. Cyclic compounds containing one, two, three or more
fused rings are formed by the submonomer method by first
synthesizing a linear backbone followed by subsequent
intramolecular or intermolecular cyclization as described in the
same application.
Assays for Activity of Fas Ligand Therapeutic Agents
[0087] The Fas ligand therapeutic agents of the invention can be
tested for binding of Fas in an in vitro assay or in vivo assay by
contacting Fas with the Fas ligand therapeutic agent. The Fas
ligand therapeutic agents can be tested for activation of Fas in a
in vivo cell-based assay. In a cell-based assay, cells expressing
Fas are cultured in the presence of the candidate Fas ligand
therapeutic, and apoptosis of the cells represents a functional Fas
ligand therapeutic agent. Such an assay for cytotoxic activity can
be performed as described, for example, in Tanaka et al, Nature
Medicine 2(3): 317-322 (1996). Alternatively, transcription of a
gene known to be upregulated by activation of the Fas pathway can
be measured, as an indicator of activation of Fas by binding to a
ligand.
[0088] Where the Fas ligand therapeutic agent is a polynucleotide
encoding a Fas ligand polypeptide, the polynucleotide can be
expressed in the cells expressing Fas. Activation of Fas can be
detected as just described. Alternatively, the polynucleotide can
be expressed in a different population of cells, and either
co-cultured with the Fas expressing cells, or the supernatants of
the Fas ligand polypeptide expressing cells can be added to the Fas
expressing cells.
[0089] Standard binding assays can be used to detect molecules
capable of binding Fas, preferably the extracellular portion of
Fas. Such binding can be detected by standard techniques, including
labeled antibodies, or labeled Fas polypeptides, or labeled
candidate molecules. Standard in vivo cell assays for detecting
molecules capable of binding and activating Fas can be constructed
using Fas expressing cell lines, and identifying apoptosis in cells
that have contacted a molecule positive for the ability to bind and
activate Fas. It is possible that the screening assays can be
conducted by first screening for molecules that bind Fas, and from
those molecules screening for molecules that also then activate Fas
expressed on a cell.
[0090] Assays for determining whether a Fas ligand polypeptide has
been constructed that is capable of remaining on the cell membrane
longer than native Fas ligand can be conducted as described in
Tanaka et al, Nature Medicine 2(3): 317-322 (1996) using anti-Fas
ligand antibodies and making a comparison of staining intensity of
mutant Fas ligand polypeptide with, for example, native Fas ligand
expressed in the same cell type.
Diagnostic and Therametric Procedures
[0091] Practice of the invention may begin by diagnosing the mammal
as is appropriate for the particular autoimmunity they may be
exhibiting. The diagnosis may also continue during treatment, as a
therametric procedure, to monitor the progress of treatment, and to
direct modification of such parameters as the dosage or frequency
in continued treatments, for example. Additional diagnosis that
might aid in determining appropriateness for administration of a
Fas ligand therapeutic agent include an analysis of expression
levels of Fas in the mammals lymphocytes, and a comparison of these
levels between lymphocytes distal from the site of autoimmunity,
and those proximal to the site or autoimmunity. The autoimmune
disease in the mammal being treated can be monitored by detecting
Fas antigen on a cell surface. This monitoring can include
contacting a sample of the mammal's lymphocytes with a Fas-specific
antibody, and detecting binding of the antibody to the sample.
Gene Delivery Vehicles
[0092] Gene therapy vehicles for delivery of constructs including a
coding sequence of a therapeutic of the invention, to be delivered
to the mammal for expression in the mammal, for example, a Fas
ligand coding sequence, or also including a nucleic acid sequence
of all or a portion of Fas ligand for delivery can be administered
either locally or systemically. These constructs can utilize viral
or non-viral vector approaches in in vivo or ex vivo modality.
Expression of such coding sequence can be induced using endogenous
mammalian or heterologous promoters. Expression of the coding
sequence in vivo can be either constitutive or regulated. Where the
Fas ligand is expressed in the mammal, it can be expressed as
soluble Fas ligand, or as a membrane-bound Fas ligand, both or
either including, for example, all of the Fas ligand, or a
biologically active portion, variant, derivative or fusion of Fas
ligand.
[0093] The invention includes gene delivery vehicles capable of
expressing the contemplated Fas ligand nucleic acid sequences. The
gene delivery vehicle is preferably a viral vector and, more
preferably, a retroviral, adenoviral, adeno-associated viral (AAV),
herpes viral, or alphavirus vectors. The viral vector can also be
an astrovirus, coronavirus, orthomyxovirus, papovavirus,
paramyxovirus, parvovirus, picornavirus, poxvirus, togavirus viral
vector. See generally, Jolly, Cancer Gene Therapy 1 (1994) 51-64,
Kimura, Human Gene Therapy 5 (1994) 845-852, Connelly, Human Gene
Therapy 6 (1995) 185-193 and Kaplitt, Nature Genetics 6 (1994)
148-153.
[0094] Retroviral vectors are well known in the art and we
contemplate that any retroviral gene therapy vector is employable
in the invention, including B, C and D type retroviruses,
xenotropic retroviruses (for example, NZB-X1, NZB-X2 and NZB9-1
(see O'Neill, J. Vir. 53 (1985) 160) polytropic retroviruses (for
example, MCF and MCF-MLV (see Kelly, J. Vir. 45 (1983) 291),
spumaviruses and lentiviruses. See RNA Tumor Viruses, Second
Edition, Cold Spring Harbor Laboratory, 1985.
[0095] Portions of the retroviral gene therapy vector may be
derived from different retroviruses. For example, retrovector LTRs
may be derived from a Murine Sarcoma Virus, a tRNA binding site
from a Rous Sarcoma Virus, a packaging signal from a Murine
Leukemia Virus, and an origin of second strand synthesis from an
Avian Leukosis Virus.
[0096] These recombinant retroviral vectors may be used to generate
transduction competent retroviral vector particles by introducing
them into appropriate packaging cell lines (see U.S. Ser. No.
07/800,921, filed Nov. 29, 1991). Retrovirus vectors can be
constructed for site-specific integration into host cell DNA by
incorporation of a chimeric integrase enzyme into the retroviral
particle. See, U.S. Ser. No. 08/445,466 filed May 22, 1995. It is
preferable that the recombinant viral vector is a replication
defective recombinant virus.
[0097] Packaging cell lines suitable for use with the
above-described retrovirus vectors are well known in the art, are
readily prepared (see U.S. Ser. No. 08/240,030, filed May 9, 1994;
see also WO 92/05266), and can be used to create producer cell
lines (also termed vector cell lines or "VCLs") for the production
of recombinant vector particles. Preferably, the packaging cell
lines are made from human parent cells (e.g., HT1080 cells) or mink
parent cell lines, which eliminates inactivation in human
serum.
[0098] Preferred retroviruses for the construction of retroviral
gene therapy vectors include Avian Leukosis Virus, Bovine Leukemia,
Virus, Murine Leukemia Virus, Mink-Cell Focus-Inducing Virus,
Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma
Virus. Particularly preferred Murine Leukemia Viruses include 4070A
and 1504A (Hartley and Rowe, J. Virol 19 (1976) 19-25), Abelson
(ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC
No. VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No.
VR-998) and Moloney Murine Leukemia Virus (ATCC No. VR-190). Such
retroviruses may be obtained from depositories or collections such
as the American Type Culture Collection ("ATCC") in Rockville, Md.
or isolated from known sources using commonly available
techniques.
[0099] Exemplary known retroviral gene therapy vectors employable
in this invention include those described in GB 2200651, EP
0415731, EP 0345242, WO 89/02468; WO 89/05349, WO 89/09271, WO
90/02806, WO 90/07936, WO 90/07936, WO 94/03622, WO 93/25698, WO
93/25234, WO 93/11230, WO 93/10218, WO 91/02805, in U.S. Pat. No.
5,219,740, U.S. Pat. No. 4,405,712, U.S. Pat. No. 4,861,719, U.S.
Pat. No. 4,980,289 and U.S. Pat. No. 4,777,127, in U.S. Ser. No.
07/800,921 and in Vile, Cancer Res 53 (1993) 3860-3864, Vile,
Cancer Res 53 (1993) 962-967, Ram, Cancer Res 53 (1993) 83-88,
Takamiya, J Neurosci Res 33 (1992) 493-503, Baba, J Neurosurg 79
(1993) 729-735, Mann, Cell 33 (1983) 153, Cane, Proc Natl Acad Sci
81 (1984) 6349 and Miller, Human Gene Therapy 1 (1990).
[0100] Human adenoviral gene therapy vectors are also known in the
art and employable in this invention. See, for example, Berkner,
Biotechniques 6 (1988) 616, and Rosenfeld, Science 252 (1991) 431,
and WO 93/07283, WO 93/06223, and WO 93/07282. Exemplary known
adenoviral gene therapy vectors employable in this invention
include those described in the above referenced documents and in WO
94/12649, WO 93/03769, WO 93/19191, WO 94/28938, WO 95/11984, WO
95/00655, WO 95/27071, WO 95/29993, WO 95/34671, WO 96/05320, WO
94/08026, WO 94/11506, WO 93/06223, WO 94/24299, WO 95/14102, WO
95/24297, WO 95/02697, WO 94/28152, WO 94/24299, WO 95/09241, WO
95/25807, WO 95/05835, WO 94/18922 and WO 95/09654. Alternatively,
administration of DNA linked to killed adenovirus as described in
Curiel, Hum. Gene Ther. 3 (1992) 147-154 may be employed.
[0101] The gene delivery vehicles of the invention also include
adenovirus associated virus (AAV) vectors. Leading and preferred
examples of such vectors for use in this invention are the AAV-2
basal vectors disclosed in Srivastava, WO 93/09239. Most preferred
AAV vectors comprise the two AAV inverted terminal repeats in which
the native D-sequences are modified by substitution of nucleotides,
such that at least 5 native nucleotides and up to 18 native
nucleotides, preferably at least 10 native nucleotides up to 18
native nucleotides, most preferably 10 native nucleotides are
retained and the remaining nucleotides of the D-sequence are
deleted or replaced with non-native nucleotides. The native
D-sequences of the AAV inverted terminal repeats are sequences of
20 consecutive nucleotides in each AAV inverted terminal repeat
(i.e., there is one sequence at each end) which are not involved in
HP formation. The non-native replacement nucleotide may be any
nucleotide other than the nucleotide found in the native D-sequence
in the same position. Other employable exemplary AAV vectors are
pWP-19, pWN-1, both of which are disclosed in Nahreini, Gene 124
(1993) 257-262. Another example of such an AAV vector is psub201.
See Samulski, J. Virol. 61 (1987) 3096. Another exemplary AAV
vector is the Double-D ITR vector. How to make the Double D ITR
vector is disclosed in U.S. Pat. No. 5,478,745. Still other vectors
are those disclosed in Carter, U.S. Pat. No. 4,797,368 and
Muzyczka, U.S. Pat. No. 5,139,941, Chartejee, U.S. Pat. No.
5,474,935, and Kotin, PCT Patent Publication WO 94/288157. Yet a
further example of an AAV vector employable in this invention is
SSV9AFABTKneo, which contains the AFP enhancer and albumin promoter
and directs expression predominantly in the liver. Its structure
and how to make it are disclosed in Su, Human Gene Therapy 7 (1996)
463-470. Additional AAV gene therapy vectors are described in U.S.
Pat. No. 5,354,678, U.S. Pat. No. 5,173,414, U.S. Pat. No.
5,139,941, and U.S. Pat. No. 5,252,479.
[0102] The gene therapy vectors of the invention also include
herpes vectors. Leading and preferred examples are herpes simplex
virus vectors containing a sequence encoding a thymidine kinase
polypeptide such as those disclosed in U.S. Pat. No. 5,288,641 and
EP 0176170 (Roizman). Additional exemplary herpes simplex virus
vectors include HFEM/ICP6-LacZ disclosed in WO 95/04139 (Wistar
Institute), pHSVlac described in Geller, Science 241 (1988)
1667-1669 and in WO 90/09441 and WO 92/07945, HSV Us3::pgC-lacZ
described in Fink, Human Gene Therapy 3 (1992) 11-19 and HSV 7134,
2 RH 105 and GAL4 described in EP 0453242 (Breakefield), and those
deposited with the ATCC as accession numbers ATCC VR-977 and ATCC
VR-260.
[0103] We also contemplate that alpha virus gene therapy vectors
may be employed in this invention. Preferred alpha virus vectors
are Sindbis viruses vectors. Togaviruses, Semliki Forest virus
(ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR-370), Ross
River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine
encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC
VR-532), and those described in U.S. Pat. Nos. 5,091,309,
5,217,879, and WO 92/10578. More particularly, those alpha virus
vectors described in U.S. Ser. No. 08/405,627, filed Mar. 15, 1995
and U.S. Ser. No. 08/198,450 and in PCT Patent Publications WO
94/21792, WO 92/10578, WO 95/07994, U.S. Pat. No. 5,091,309 and
U.S. Pat. No. 5,217,879 are employable. Such alpha viruses may be
obtained from depositories or collections such as the ATCC in
Rockville, Md. or isolated from known sources using commonly
available techniques. Preferably, alphavirus vectors with reduced
cytotoxicity are used (see co-owned U.S. Ser. No. 08/679,640).
[0104] DNA vector systems such as eukaryotic layered expression
systems are also useful for expressing the Fas ligand nucleic acids
of the invention. See WO 95/07994 for a detailed description of
eukaryotic layered expression systems. Preferably, the eukaryotic
layered expression systems of the invention are derived from
alphavirus vectors and most preferably from Sindbis viral
vectors.
[0105] Other viral vectors suitable for use in the present
invention include those derived from poliovirus, for example ATCC
VR-58 and those described in Evans, Nature 339 (1989) 385 and
Sabin, J. Biol. Standardization 1 (1973) 115; rhinovirus, for
example ATCC VR-1110 and those described in Arnold, J Cell Biochem
(1990) L401; pox viruses such as canary pox virus or vaccinia
virus, for example ATCC VR-111 and ATCC VR-2010 and those described
in Fisher-Hoch, Proc Natl Acad Sci 86 (1989) 317, Flexner, Ann NY
Acad Sci 569 (1989) 86, Flexner, Vaccine 8 (1990) 17; in U.S. Pat.
No. 4,603,112 and U.S. Pat. No. 4,769,330 and in WO 89/01973; SV40
virus, for example ATCC VR-305 and those described in Mulligan,
Nature 277 (1979) 108 and Madzak, J Gen Vir 73 (1992) 1533;
influenza virus, for example ATCC VR-797 and recombinant influenza
viruses made employing reverse genetics techniques as described in
U.S. Pat. No. 5,166,057 and in Enami, Proc Natl Acad Sci 87 (1990)
3802-3805, Enami and Palese, J Virol 65 (1991) 2711-2713 and
Luytjes, Cell 59 (1989) 110, (see also McMicheal., NE J Med 309
(1983) 13, and Yap, Nature 273 (1978) 238 and Nature 277 (1979)
108); human immunodeficiency virus as described in EP 0386882 and
in Buchschacher, J. Vir. 66 (1992) 2731; measles virus, for example
ATCC VR-67 and VR-1247 and those described in EP 0440219; Aura
virus, for example ATCC VR-368; Bebaru virus, for example ATCC
VR-600 and ATCC VR-1240; Cabassou virus, for example ATCC VR-922;
Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; Fort
Morgan Virus, for example ATCC VR-924; Getah virus, for example
ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example ATCC
VR-927; Mayaro virus, for example ATCC VR-66; Mucambo virus, for
example ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example ATCC
VR-371; Pixuna virus, for example ATCC VR-372 and ATCC VR-1245;
Tonate virus, for example ATCC VR-925; Triniti virus, for example
ATCC VR-469; Una virus, for example ATCC VR-374; Whataroa virus,
for example ATCC VR-926; Y-62-33 virus, for example ATCC VR-375;
O'Nyong virus, Eastern encephalitis virus, for example ATCC VR-65
and ATCC VR-1242; Western encephalitis virus, for example ATCC
VR-70, ATCC VR-1251, ATCC VR-622 and ATCC VR-1252; and coronavirus,
for example ATCC VR-740 and those described in Hamre, Proc Soc Exp
Biol Med 121 (1966) 190.
[0106] Delivery of the compositions of this invention into cells is
not limited to the above mentioned viral vectors. Other delivery
methods and media may be employed such as, for example, nucleic
acid expression vectors, polycationic condensed DNA linked or
unlinked to killed adenovirus alone, for example see U.S. Ser. No.
08/366,787, filed Dec. 30, 1994 and Curiel, Hum Gene Ther 3 (1992)
147-154 ligand linked DNA, for example see Wu, J Biol Chem 264
(1989) 16985-16987, eucaryotic cell delivery vehicles cells, for
example see U.S. Ser. No. 08/240,030, filed May 9, 1994, and U.S.
Ser. No. 08/404,796, deposition of photopolymerized hydrogel
materials, hand-held gene transfer particle gun, as described in
U.S. Pat. No. 5,149,655, ionizing radiation as described in U.S.
Pat. No. 5,206,152 and in WO 92/11033, nucleic charge
neutralization or fusion with cell membranes. Additional approaches
are described in Philip, Mol Cell Biol 14 (1994) 2411-2418 and in
Woffendin, Proc Natl Acad Sci 91 (1994) 1581-585.
[0107] Particle mediated gene transfer may be employed, for example
see U.S. Ser. No. 60/023,867. Briefly, the sequence can be inserted
into conventional vectors that contain conventional control
sequences for high level expression, and then be incubated with
synthetic gene transfer molecules such as polymeric DNA-binding
cations like polylysine, protamine, and albumin, linked to cell
targeting ligands such as asialoorosomucoid, as described in Wu and
Wu, J. Biol. Chem. 262 (1987) 4429-4432, insulin as described in
Hucked, Biochem Pharmacol 40 (1990) 253-263, galactose as described
in Plank, Bioconjugate Chem 3 (1992) 533-539, lactose or
transferrin.
[0108] Naked DNA may also be employed. Exemplary naked DNA
introduction methods are described in WO 90/11092 and U.S. Pat. No.
5,580,859. Uptake efficiency may be improved using biodegradable
latex beads. DNA coated latex beads are efficiently transported
into cells after endocytosis initiation by the beads. The method
may be improved further by treatment of the beads to increase
hydrophobicity and thereby facilitate disruption of the endosome
and release of the DNA into the cytoplasm.
[0109] Liposomes that can act as gene delivery vehicles are
described in U.S. Pat. No. 5,422,120, WO 95/13796, WO 94/23697, WO
91/144445 and EP 524,968. As described in co-owned U.S. Ser. No.
60/023,867, on non-viral delivery, the nucleic acid sequences
encoding a Fas ligand polypeptide can be inserted into conventional
vectors that contain conventional control sequences for high level
expression, and then be incubated with synthetic gene transfer
molecules such as polymeric DNA-binding cations like polylysine,
protamine, and albumin, linked to cell targeting ligands such as
asialoorosomucoid, insulin, galactose, lactose, or transferrin.
Other delivery systems include the use of liposomes to encapsulate
DNA comprising the gene under the control of a variety of
tissue-specific or ubiquitously-active promoters. Further non-viral
delivery suitable for use includes mechanical delivery systems such
as the approach described in Woffendin et al., Proc. Natl. Acad.
Sci. USA (1994) 91(24): 11581-11585. Moreover, the coding sequence
and the product of expression of such can be delivered through
deposition of photopolymerized hydrogel materials. Other
conventional methods for gene delivery that can be used for
delivery of the coding sequence include, for example, use of
hand-held gene transfer particle gun, as described in U.S. Pat. No.
5,149,655; use of ionizing radiation for activating transferred
gene, as described in U.S. Pat. No. 5,206,152 and WO 92/11033
[0110] Exemplary liposome and polycationic gene delivery vehicles
are those described in U.S. Pat. Nos. 5,422,120 and 4,762,915, in
WO 95/13796, WO 94/23697, and WO 91/14445, in EP 0524968 and in
Stryer, Biochemistry, pages 236-240 (1975) W.H. Freeman, San
Francisco, Szoka, Biochem Biophys Acta 600 (1980) 1, Bayer, Biochem
Biophys Acta 550 (1979) 464, Rivnay, Meth Enzymol 149 (1987) 119,
Wang, Proc Natl Acad Sci 84 (1987) 7851, Plant, Anal Biochem 176
(1989) 420.
Pharmaceutical Compositions and Therapeutic Methods
[0111] The invention discloses a method of treating mammals
afflicted with an autoimmune disease that includes activated
lymphocytes, including activated T-cells or activated B-cells, by
administration of a Fas ligand or Fas ligand derived therapeutic
agent, for example, either Fas ligand polypeptide as a therapeutic
agent or a polynucleotide encoding a Fas ligand polypeptide for
expression in the mammal, or a small molecule Fas ligand agonist
therapeutic agent Autoimmune diseases that can be treated by the
method and compositions of the invention include any autoimmune
disease, or transplantation rejection, including, but not limited
to, for example, those autoimmune diseases listed herein.
[0112] Fas ligand can be administered, for example, as a
recombinantly expressed polypeptide, or as a variant, derivative,
or fusion protein of Fas ligand polypeptide, delivered either
locally or systemically to the mammal. DNA or RNA encoding Fas
ligand, or a derivative or variant of Fas ligand, or a Fas ligand
fusion, can be administered in a gene therapy protocol, as naked
plasmid DNA including regulatory regions for expression in the
mammal, or in a viral vector for expression in the mammal. Delivery
of Fas ligand polypeptide for expression can be accomplished with a
pharmaceutically acceptable carrier capable of facilitating the
delivery. Treatment of a mammal having an autoimmune disease with a
Fas ligand derived therapeutic agent can result in amelioration or
remission or the autoimmune disease, or in absence of clinical
symptoms attributable to the autoimmunity.
[0113] Although the invention is not limited to theories of how the
invention works, it is posited by the inventor that activated
T-cells and B-cells that cause the self-recognition and subsequent
harm in autoimmunity express Fas. By expressing Fas ligand or
causing Fas ligand to be expressed, or by administering a Fas
ligand derived therapeutic agent, the activated lymphocytes
expressing Fas are preferentially targeted for apoptosis by binding
the Fas ligand moiety made available. The Fas ligand polypeptide or
a Fas ligand derived therapeutic agent can be administered in the
region exhibiting the autoimmunity, for example, in the localized
region that characterized the particular autoimmune disease being
treated. This optimizes the contact between the administered Fas
ligand or other therapeutic agent and the Fas expressing activated
T-cells and B-cells which are specific for the self antigens
expressed on the cells of that region. The cells of the region are
thus also good candidates for expressing, by aid of a gene delivery
vehicle, a polynucleotide encoding a Fas ligand polypeptide
administered to the region. Thus, in various permutations and
applications of the invention, the expression of the Fas ligand
polypeptide can be recombinantly engineered to facilitate
expression in cells that are under attack by the activated T-cells
and B-cells. In the case of transplantation rejection, the inventor
proposes a Fas ligand polypeptide fusion with a binding portion of
a molecule capable of binding a protein ubiquitously expressed on
the cell surfaces of many cell types. This binding portion can be,
for example, heparin, and the molecule on the cell surface to which
it binds can be a glycosaminoglycan. Alternatively, the binding
portion may be a single chain antibody binding domain, specific for
any selected cell surface antigen. The expressed and purified
fusion protein of a Fas binding portion of Fas ligand and
glycosaminoglycan binding portion of heparin can be administered to
an organ, tissue or cell in preparation for a transplant, and upon
transplantation, the organ, tissue or cell has Fas ligand
polypeptide portions available as a first line of defense against
activated T-cells or B-cells which would otherwise attack and
immunologically reject the allograft.
[0114] "Administration" or "administering" as used herein refers to
the process of delivering to a mammal a therapeutic agent, or a
combination of therapeutic agents. The process of administration
can be varied, depending on the therapeutic agent, or agents, and
the desired effect. Administration can be accomplished by any means
appropriate for the therapeutic agent, for example, by parenteral
or oral delivery. The parenteral delivery can be, for example,
subcutaneous, intravenous, intramuscular, intra-arterial, injection
into the tissue of an organ, mucosal, pulmonary, topical, or
catheter-based. Oral means is by mouth, including pills or other
gastroenteric delivery means, including a drinkable liquid. Mucosal
delivery can include, for example, intranasal delivery. Pulmonary
delivery can include inhalation of the agent. Catheter-based
delivery can include delivery by iontophoretic catheter-based
delivery. Administration generally also includes delivery with a
pharmaceutically acceptable carrier, such as, for example, a
buffer, a polypeptide, a peptide, a polysaccharide conjugate, a
liposome, and a lipid. A gene therapy protocol is considered an
administration in which the therapeutic agent is a polynucleotide
capable of accomplishing a therapeutic goal when expressed as a
transcript or a polypeptide in the mammal, and can be applied to
both parenteral and oral delivery means. Such administration means
are selected as appropriate for the disease being treated. For
example, where the disease is organ-based, delivery may be local,
and for example, where the disease is systemic, the delivery may be
systemic.
[0115] "Co-administration" refers to administration of one or more
therapeutic agents in course of a given treatment of a patient. The
agents may be administered with the same pharmaceutical carrier, or
different carriers. They may be administered by the same or
different administration means. The agents may be the same type of
agent or different types of agents, for example, different types
can include polynucleotides, polypeptide, or small molecules. The
time of administration may be exactly the same time, or one
therapeutic agent may be administered before or after another
agent. Thus a co-administration can be simultaneous, or
consecutive. The exact protocol for a given combination of
therapeutic agents is determined considering the agents and the
condition being treated, among other considerations.
[0116] The term "in vivo administration" refers to administration
to a patient, for example a mammal, of a polynucleotide encoding a
polypeptide for expression in the mammal. In particular, direct in
viva administration involves transfecting a mammal's cell with a
coding sequence without removing the cell from the mammal. Thus,
direct in vivo administration may include direct injection of the
DNA encoding the polypeptide of interest in the region afflicted by
the autoimmune disease, resulting in expression in the patient's
cells.
[0117] The term "ex vivo administration" refers to transfecting a
cell, for example, a cell from a population of cells that are under
auto immune attack, after the cell is removed from the patient, for
example a mammal. After transfection the cell is then replaced in
the mammal. Ex vivo administration can be accomplished by removing
cells from a mammal, optionally selecting for cells to transform,
(i.e. cells under attack by an autoimmune mechanism) rendering the
selected cells incapable of replication, transforming the selected
cells with a polynucleotide encoding a gene for expression, (i.e.
Fas ligand), including also a regulatory region for facilitating
the expression, and placing the transformed cells back into the
patient for expression of the Fas ligand.
[0118] A "therapeutically effective amount" is that amount that
generates the desired therapeutic outcome. For example, if the
therapeutic effect desired is a remission from autoimmunity, the
therapeutically effective amount is that amount that facilitates
the remission. A therapeutically effective amount can be an amount
administered in a dosage protocol that includes days or weeks of
administration, for example. Where the therapeutic effect is a
reduction of the effects of an autoimmune response in the mammal,
for example, during the manifestations of symptoms of an autoimmune
disease, the effective amount of an agent to accomplish this in the
mammal is that amount that results in reduction of the symptoms of
autoimmunity.
[0119] The term "pharmaceutically acceptable carrier" refers to a
carrier for administration of a therapeutic agent, such as, for
example, a polypeptide, polynucleotide, small molecule, peptoid, or
peptide, refers to any pharmaceutically acceptable carrier that
does not itself induce the production of antibodies harmful to the
individual receiving the composition, and which may be administered
without undue toxicity. Within another aspect of the invention,
pharmaceutical compositions are provided, comprising a recombinant
viral vector as described above, in combination with a
pharmaceutically acceptable carrier or diluent. Such pharmaceutical
compositions may be prepared either as a liquid solution, or as a
solid form (e.g., lyophilized) which is suspended in a solution
prior to administration. In addition, the composition may be
prepared with suitable carriers or diluents for either surface
administration, injection, oral, or rectal administration.
Pharmaceutically acceptable carriers or diluents are nontoxic to
recipients at the dosages and concentrations employed.
Representative examples of carriers or diluents for injectable
solutions include water, isotonic saline solutions which are
preferably buffered at a physiological pH (such as
phosphate-buffered saline or Tris-buffered saline), mannitol,
dextrose, glycerol, and ethanol, as well as polypeptides or
proteins such as human serum albumin. A particularly preferred
composition comprises a vector or recombinant virus in 10 mg/ml
mannitol, 1 mg/ml HSA, 20 mM Tris, pH 7.2, and 150 mM NaCl. In this
case, since the recombinant vector represents approximately 1 mg of
material, it may be less than 1% of high molecular weight material,
and less than 1/100,000 of the total material (including water).
This composition is stable at -70.degree. C. for at least six
months.
[0120] Pharmaceutical compositions of the present invention may
also additionally include factors which stimulate cell division,
and hence, uptake and incorporation of a recombinant retroviral
vector. Preserving recombinant viruses is described in U.S.
applications entitled "Methods for Preserving Recombinant Viruses"
(U.S. Ser. No. 08/135,938, filed Oct. 12, 1993) which is
incorporated herein by reference in full.
[0121] All of the therapeutic agents that make up the proposed
therapy of the invention can be incorporated into an appropriate
pharmaceutical composition that includes a pharmaceutically
acceptable carrier for the agent. The pharmaceutical carrier for
the agents may be the same or different for each agent. Suitable
carriers may be large, slowly metabolized macromolecules such as
proteins, polysaccharides, polylactic acids, polyglycolic acids,
polymeric amino acids, amino acid copolymers, and inactive viruses
in particles. Such carriers are well known to those of ordinary
skill in the art. Pharmaceutically acceptable salts can be used
therein, for example, mineral acid salts such as hydrochlorides,
hydrobromides, phosphates, sulfates, and the like; and the salts of
organic acids such as acetates, propionates, malonates, benzoates,
and the like. A thorough discussion of pharmaceutically acceptable
excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES
(Mack Pub. Co., N.J. 1991). Pharmaceutically acceptable carriers in
therapeutic compositions may contain liquids such as water, saline,
glycerol and ethanol. Additionally, auxiliary substances, such as
wetting or emulsifying agents, pH buffering substances, and the
like, may be present in such vehicles. Typically, the therapeutic
compositions are prepared as injectables, either as liquid
solutions or suspensions; solid forms suitable for solution in, or
suspension in, liquid vehicles prior to injection may also be
prepared. Liposomes are included within the definition of a
pharmaceutically acceptable carrier.
[0122] Pharmaceutical compositions are provided comprising a
recombinant retrovirus or virus carrying one of the above-described
vector constructs, in combination with a pharmaceutically
acceptable carrier or diluent. The composition may be prepared
either as a liquid solution, or as a solid form (e.g., lyophilized)
which is suspended in a solution prior to administration. In
addition, the composition may be prepared with suitable carriers or
diluents for either surface administration, injection, oral, or
rectal administration.
[0123] Pharmaceutically acceptable carriers or diluents are
nontoxic to recipients at the dosages and concentrations employed.
Representative examples of carriers or diluents for injectable
solutions include water, isotonic saline solutions which are
preferably buffered at a physiological Ph (such as
phosphate-buffered saline or Tris-buffered saline), mannitol,
dextrose, glycerol, and ethanol, as well as polypeptides or
proteins such as human serum albumin. A vector or recombinant virus
can be delivered in a pharmaceutical composition in 10 mg/ml
mannitol, 1 mg/ml HSA, 20 Mm Tris, Ph 7.2, and 150 Mm NaCl. In this
case, since the recombinant vector represents approximately 1 g of
material, it may be less than 1% of high molecular weight material,
and less than 1/100,000 of the total material (including water).
This composition is stable at -70.degree. C. for at least six
months.
[0124] The pharmaceutically acceptable carrier or diluent may be
combined with the gene delivery vehicles to provide a composition
either as a liquid solution, or as a solid form (e.g., lyophilized)
which can be resuspended in a solution prior to administration. The
two or more gene delivery vehicles are typically administered via
traditional direct routes, such as buccal/sublingual, rectal, oral,
nasal, topical, (such as transdermal and ophthalmic), vaginal,
pulmonary, intraarterial, intramuscular, intraperitoneal,
subcutaneous, intraocular, intranasal or intravenous, or
indirectly.
[0125] Any therapeutic of the invention, including, for example,
polynucleotides for expression in the mammal, can be formulated
into an enteric coated tablet or gel capsule according to known
methods in the art. These are described in the following patents:
U.S. Pat. No. 4,853,230, EP 225,189, AU 9,224,296, AU 9,230,801,
and WO 92144,52. Such a capsule is administered orally to be
targeted to the jejunum. At 1 to 4 days following oral
administration expression of the polypeptide, or inhibition of
expression by, for example a ribozyme or an antisense
oligonucleotide, is measured in the plasma and blood, for example
by antibodies to the expressed or non-expressed proteins.
[0126] The gene delivery vehicle can be introduced into a mammal,
for example, by injection, particle gun, topical administration,
parental administration, inhalation, or iontophoretic delivery, as
described in U.S. Pat. No. 4,411,648 and U.S. Pat. No. 5,222,936,
U.S. Pat. No. 5,286,254; and WO 94/05369.
[0127] A therapeutic composition or therapeutic agent can be
administered with other therapeutic agents capable of ameliorating
the autoimmune disease, or capable of enhancing the therapeutic
benefits of administration of a Fas ligand therapeutic agent. For
example, administration for treatment of an allergic reaction can
be by aerosol administration of Fas ligand polynucleotide for
expression in the cells present in mucosal, nasal, bronchial or
lung tissue, and may be most favorably administered in repeat
administrations, for example by nasal or aerosol spray several
times daily for a period of time until the allergic reaction
subsides.
[0128] The gene delivery vehicle may be administered at single or
multiple sites to a mammal directly, for example by direct
injection, or alternatively, through the use of target cells
transduced ex vivo. The present invention also provides
pharmaceutical compositions (including, for example, various
excipients) suitable for administering the gene delivery
vehicles.
[0129] A vector construct which directs the expression of a Fas
ligand polypeptide, variant, derivative, analogue, mutant, or
chimera can be directly administered to a site exhibiting
autoimmunity, for example the pancreas, kidney, liver, joints,
brain, the spinal fluid, skin, or other region or organ of the
body. Various methods may be used within the context of the present
invention in order to directly administer the vector construct. For
example, arteries which serve the region may be identified, and the
vector injected into such an artery, in order to deliver the vector
directly into the site. Similarly, the vector construct may be
directly administered to the skin surface, for example, by
application of a topical pharmaceutical composition containing the
vector construct.
[0130] In a direct administration, combination therapeutic agents
including a Fas ligand therapeutic agent and other anti-autoimmune
agents can be administered together. The co-administration can be
simultaneous, achieved for example by placing polynucleotides
encoding the agents in the same vector, or by putting the agents,
whether polynucleotide, polypeptide, or other drug, in the same
pharmaceutical composition, or by administering the agents in
different pharmaceutical compositions injected at about the same
time, and perhaps in the same location. If the co-administration is
not simultaneous, for example, in the case of administration of the
prodrug after administration of the prodrug activator, the second
agent can be administered by direct injection as appropriate for
the goals of the therapy. Thus, for example, in the case of an
administration of a prodrug, the prodrug is administered at the
same location as the prodrug activator. Thus, a co-administration
protocol can include a combination of administrations to achieve
the goal of the therapy. Further, the co-administration can include
subsequent administrations as is necessary, for example, repeat in
vivo direct injection administrations of a Fas ligand.
[0131] Within the context of the present invention, it should be
understood that the removed cells may be returned to the same
animal, or to another allogenic animal or mammal. In such a case it
is generally preferable to have histocompatibility matched animals
(although not always, see, e.g., Yamamoto et al., "Efficacy of
Experimental FIV Vaccines," 1st International Conference of FIV
Researchers, University of California at Davis, September 1991.
[0132] Cells may be removed from a variety of locations in the
patient. In addition, within other embodiments of the invention, a
vector construct may be inserted into, for example, cells from the
skin (dermal fibroblasts), or from the blood (e.g., peripheral
blood leukocytes). If desired, particular fractions of cells such
as a T cell subset or stem cells may also be specifically removed
from the blood (see, for example, PCT WO 91/16116, an application
entitled "Immunoselection Device and Method") Vector constructs may
then be contacted with the removed cells utilizing any of the
above-described techniques, followed by the return of the cells to
the warm-blooded animal, preferably to or within the vicinity of
the region exhibiting autoimmunity.
[0133] Once the patient, for example a mammal, has been diagnosed,
practice of the invention includes providing a Fas ligand
therapeutic agent, and administering it to the mammal in a manner
and dose appropriate for the particular autoimmune disease being
treated, and monitoring the mammal for determining the need for
continued or modified administrations of the therapeutic agent.
Practice of the invention is accomplished by identifying the
disease to be treated, and determining the probable cell-type or
region of the body to which a targeted gene therapy can be applied.
The Fas ligand polynucleotide is constructed, including either a
plasmid with regulatory regions for expression in the mammal, or a
viral vector for the expression. Some of the mammal's cells can be
removed, transfected with the polynucleotide encoding Fas ligand,
and replaced into the mammal for expression of Fas ligand.
Alternatively the polynucleotide can be administered to the mammal,
for example in the region where the disease is manifest, for
expression in the mammal's cells in that region.
[0134] Thus, for example, in the case of rheumatoid arthritis, the
sinovoid cells can be transfected ex vivo with Fas ligand, or Fas
ligand in a gene delivery vehicle can be administered to the joints
which includes large populations of sinovoid cells, for expression
of the Fas ligand polypeptide in these cells.
[0135] For example, in treatment of multiple sclerosis, Fas ligand
can be injected into the region of the brain being effected, or in
the spinal fluid, to facilitate expression of Fas ligand in the
cells that are under attack by the activated T-cells or B-cells in
an autoimmune type of reaction. Also, by example, in the case of
multiple sclerosis, Fas ligand DNA can be locally injected into the
mammal's brain, or oligodendrites from the spinal fluid can be
removed, transfected with Fas ligand DNA, and returned to the
region of the spinal cord.
[0136] Further by example, for treating a mammal having Sojgren's
syndrome, the organ targeted by the disease is selected for
administration Fas ligand polypeptide by injection. Also, by
example, for mammal's suffering from Sojgren's syndrome, the
affected organ can be identified, for example the kidney, and Fas
ligand DNA administered to the organ directly, or cells from the
organ removed, transfected, and replaced in the body for expression
of Fas ligand in those cells in the mammal.
[0137] For example, in the case of preventing transplantation
rejection, the animal to receive the transplant can receive
localized or systemic administration of a Fas ligand therapeutic
agent in order to kill any activated patient cells that express Fas
which attacks the foreign cells, tissue or organ, or a Fas ligand
polypeptide can be expressed in cells on the external surface of
the organ just prior to the transplant, in order the protect the
organ once inside the patient's body. Continued administration of
the Fas ligand therapeutic agent may be necessary while the
recipient's immune system adjusts to the foreign cells, tissue or
organ.
[0138] The Fas ligand therapeutic agent is expected to act
analogously to native Fas ligand. Accordingly, it will trimerize
Fas to cause an apoptotic reaction in the cells expressing Fas.
Thus, stoichiometrically, the clinician would be able to be aware
of the amount of Fas ligand that needs to be expressed or otherwise
administered to the mammal for achieving the desired activation of
Fas and subsequent death of the cell that expressed Fas. Various
proposed mechanisms of action of Fas ligand are explained and the
production of Fas ligand is described in EP 675 200, incorporated
by reference in full. Within other aspects of the present
invention, the vector constructs described herein may also direct
the expression of additional non-vector derived genes.
[0139] For example, a prodrug system applied in conjunction with
administration of Fas ligand can act as a safety mechanism for the
gene therapy, or can act as a combination therapeutic agent. As a
safety mechanism, the prodrug activator, for example HSV TK, is
expressed in a vector along with the Fas ligand. When it is
determined that the system should be arrested, the prodrug, for
example gancyclovir, is administered and HSV TK activates
gancyclovir, which kills the cells expressing Fas. This allows the
clinician a measure of control over the gene therapy. The HSV
TK/gancyclovir system may be useful for inactivating the
transfected cells in the mammal, where, for example, the
autoimmunity is exacerbated by the Fas ligand expression. The HSV
TK/gancyclovir system can also be administered as combination
therapeutic agent, in a combination therapy protocol, for achieving
cell killing using the prodrug activation provided by the HSV
TK/gancyclovir system as just described.
[0140] The therapy including administration of a polynucleotide
encoding a Fas ligand polypeptide, in conjunction with a prodrug
activator and prodrug, can also be immunomodulatory.
"Immunomodulatory" refers to use of factors which, when
manufactured by one or more of the cells involved in an immune
response, or, which when added exogenously to the cells, causes the
immune response to be different in quality or potency from that
which would have occurred in the absence of the factor. The quality
or potency of a response may be measured by a variety of assays
known to one of skill in the art including, for example, in vitro
assays which measure cellular proliferation (e.g., .sup.3H
thymidine uptake), and in vitro cytotoxic assays (e.g., which
measure .sup.51Cr release) (see, Warner et al., AIDS Res. and Human
Retroviruses 7:645-655, 1991). Immunomodulatory factors may be
active both in vivo and ex vivo. Representative examples of such
factors include cytokines, such as interleukins 2, 4, 6, 12 and 15
(among others), alpha interferons, beta interferons, gamma
interferons, GM-CSF, G-CSF, and tumor necrosis factors (TNFs).
Other immunomodulatory factors include, for example, CD3, ICAM-1,
ICAM-2, LFA-1, LFA-3, MHC class I molecules, MHC class II
molecules, B7.1-0.3, b.sub.2-microglobulin, chaperones, or analogs
thereof. If the gene delivery vehicle, however, does not express an
immunomodulatory cofactor which is a cytokine, this cytokine may be
included in the above-described compositions, or may be
administered separately (concurrently or subsequently) with the
above-described compositions. Briefly, within such an embodiment,
the immunomodulatory cofactor is preferably administered according
to standard protocols and dosages as prescribed in The Physician's
Desk Reference. For example, alpha interferon may be administered
at a dosage of 1-5 million units/day for 2-4 months, and IL-2 at a
dosage of 10,000-100,000 units/kg of body weight, 1-3 times/day,
for 2-12 weeks. Gamma interferon may be administered at dosages of
150,000-1,500,000 units 2-3 times/week for 2-12 weeks for example,
for upregulating Fas expression in activated T-cells for achieving
more effective therapy with the administration of Fas ligand.
[0141] As a combination therapeutic agent, the prodrug activator
can be expressed from its own vector, or from the same vector as
the Fas ligand polypeptide. Either vector system (a single vector,
or two vectors) can be administered by in vivo or ex vivo means. In
an autoimmune therapy, for example, the addition of HSV TK or other
prodrug activator facilitates further immunomodulatory effect
supporting the effect achieved by Fas ligand and in addition,
addition of the prodrug can activate the killing of transfected
cells.
[0142] A chaperon molecule can be administered before,
contemporaneously with or after administration of the
polynucleotide therapeutic, and the chaperon molecule can be, for
example, a heat shock protein, such as, for example hsp70. Further,
the polynucleotide being expressed in the mammal can be linked to
an inducible promoter, for example a tissue specific promoter, for
the purpose of, for example, ensuring expression of the
polynucleotide only in the desired target cells. Additionally, for
the purpose of effectively delivering the polynucleotide to a
tissue, the polynucleotide can be flanked by nucleotide sequences
suitable for integration into genome of the cells of that
tissue.
[0143] For this and many other aspects of the invention,
effectiveness of treating humans may first be tested in animal
models for a given autoimmune disease. Such existing animal models
include those for the following autoimmune disease: Sjogren's
syndrome (autoimmune dacryodentis or immune-mediated sialadenitis),
autoimmune myocarditis, primary biliary cirrhosis (PBC),
inflammatory heart disease, mercury-induced renal autoimmunity,
insulin dependent diabetes mellitus (type I diabetes or IDD),
post-thymectomy autoimmunity, a central nervous system (CNS)
demyelination disorder, CNS lupus, narcolepsy, myasthenia gravis
(MG), Grave's disease, a immune-mediated PNS disorder,
osteoarthritis, rheumatoid arthritis, uveitis, medullary cystic
fibrosis, autoimmune hemolytic disease, autoimmune vasculitis,
ovarian autoimmune disease, and human schleroderma.
[0144] The multiple gene delivery vehicles may be administered to
animals or plants. In preferred embodiments, the animal is a
warm-blooded animal, further preferably selected from the group
consisting of mice, chickens, cattle, pigs, pets such as cats and
dogs, horses, and humans.
[0145] For polypeptide therapeutics, for example, Fas ligand or
other cytokine, the dosage can be in the range of about 5 .mu.g to
about 50 .mu.g/kg of mammal body weight, also about 50 .mu.g to
about 5 mg/kg, also about 100 .mu.g to about 500 .mu.g/kg of mammal
body weight, and about 200 to about 250 ug/kg.
[0146] For polynucleotide therapeutics, for example a
polynucleotide encoding a native or mutant Fas ligand polypeptide,
depending on the expression of the polynucleotide in the patient,
for example a mammal, for tissue targeted administration, vectors
containing expressible constructs of coding sequences, or
non-coding sequences can be administered in a range of about 100 ng
to about 200 mg of DNA for local administration in a gene therapy
protocol, also about 500 ng to about 50 mg, also about 1 ug to
about 2 mg of DNA, about 5 ug of DNA to about 500 ug of DNA, and
about 20 ug to about 100 ug during a local administration in a gene
therapy protocol, and for example, a dosage of about 500 ug, per
injection or administration. Where greater expression is desired,
over a larger area of tissue, larger amounts of DNA or the same
amounts readministered in a successive protocol of administrations,
or several administrations to different adjacent or close tissue
portions of for example, a tumor site, may be required to effect a
positive therapeutic outcome.
[0147] For administration of small molecule therapeutics, depending
on the potency of the small molecule, the dosage may vary. For a
very potent inhibitor, microgram (.mu.) amounts per kilogram of
mammal may be sufficient, for example, in the range of about 1
.mu.g/kg to about 500 mg/kg of mammal weight, and about 100
.mu.g/kg to about 5 mg/kg, and about 1 .mu.g/kg to about 50
.mu.g/kg, and, for example, about 10 ug/kg. For administration of
peptides and peptoids the potency also affects the dosage, and may
be in the range of about 1 .mu.g/kg to about 500 mg/kg of mammal
weight, and about 100 .mu.g/kg to about 5 mg/kg, and about 1
.mu.g/kg to about 50 .mu.g/kg, and a usual dose might be about 10
ug/kg.
[0148] In all cases, routine experimentation in clinical trials
would narrow the therapeutic range for optimal therapeutic effect,
for each therapeutic, each administrative protocol, and
administration to specific mammals will also be adjusted to within
effective and safe ranges depending on the mammal condition and
responsiveness to initial administrations.
[0149] Further objects, features, and advantages of the present
invention will become apparent from the detailed description. It
should be understood, however, that the detailed description, while
indicating preferred embodiments of the invention, is given by way
of illustration only, since various changes and modifications
within the spirit and scope of the invention will become apparent
to those skilled in the art from this detailed description. Also,
the invention is not limited by any theories of mechanism of the
method of the invention.
[0150] The present invention is illustrated by reference to the
following examples which set forth particularly advantageous
embodiments. However, it should be noted that these embodiments are
illustrative and are not to be construed as restricting the
invention in any way.
Example 1
[0151] A human patient with rheumatoid arthritis is diagnosed. The
sinovoid cells of the patient are targeted for therapy. A
population of the sinovoid cells of the patient are removed and
transfected with a polynucleotide (SEQ ID NO: 5 or 9) encoding a
non-cleavable Fas ligand (e.g., SEQ ID NO: 6 or 10) under control
of a temperature sensitive promoter. The transfected ex vivo cells
are replaced into the region of the patient's joints from which
they were removed. Heating pads are applied to the patient's joints
to induce expression of non-cleavable membrane bound Fas ligand.
The patient is monitored for reduction in symptoms.
Example 2
[0152] A patient is diagnosed with multiple sclerosis. An
adeno-associated viral (AAV) vector and its necessary components is
prepared having a polynucleotide sequence encoding a Fas ligand
polypeptide fusion protein capable of remaining on the cell
membrane longer than native Fas ligand. This AAV is injected into
the spinal fluid of the patient, and also into regions of the
brain. Repeat administrations are directed as the patient is
monitored for improvement.
Example 3
[0153] A human patient is diagnosed as having Sojgren's syndrome
manifested in the kidney. A Fas ligand polynucleotide is prepared
in a plasmid under regulatory control of a kidney specific
promoter. The plasmid DNA is encapsulated in liposomes, and the
composition is administered directly to several regions of the
kidney. The patient is monitored for improvement, and for
readministration of the Fas ligand as directed by the progress.
Example 4
[0154] A Fas ligand chimera is constructed by replacing the
putative cleavage portion of Fas ligand with an uncleavable
fragment from a similar region of a polypeptide of the same family.
A 17 residue portion of Fas ligand between residues L127 to R144 is
replaced with a 17 residue Fas ligand molecule corresponds to the
wild type fragment of CD30 ligand from S83 to A100. The remainder
of the Fas ligand molecule corresponds to the wild type Fas ligand
sequence. The resulting fusion polypeptide remains membrane bound
longer than native Fas.
Example 5
Fas Ligand.sub.130.fwdarw.142 Deletion Mutein
[0155] A cDNA fragment (SEQ ID NO: 1) encoding the first 129
residues of Fas ligand (from M1 to Q130), designated as the "MQ"
fragment was isolated and amplified using standard PCR techniques.
A second cDNA fragment (SEQ ID NO: 3) encoding residues 143 to 281
of Fas ligand (from L143 to the stop codon), designated as the "LT"
fragment, was isolated and amplified using standard PCR
techniques.
[0156] The 3' end of the MQ fragment was ligated to the 5' end of
the LT fragment MQ/LT using a standard ligase reaction to produce
polynucleotide (SEQ ID NO: 5) encoding the Fas ligand
130.fwdarw.142 deletion mutein.
[0157] The MQ/LT polynucleotide is cloned into an appropriate high
titer retroviral vector, such as pLNL6 [Bender, et al., (1987) J.
Virol., 61(5): 1639-1646 or U.S. Pat. No. 5,324,655, which are
hereby incorporated herein by reference] or a commercially
available vector that is capable of infecting a mammalian host
cell, preferably a human host cell. The transfected or transduced
host cell is cultured whereupon the non-cleavable Fas
ligand.sub.130.fwdarw.142 deletion mutein (i.e., a mutein lacking
residues +1 to +12 from the P1 site) is expressed in said host cell
and remains surface bound thereto.
Example 6
Fas Ligand.sub.130.fwdarw.145 Deletion Mutein
[0158] The cDNA fragment (SEQ ID NO: 1) encoding the first 129
residues of Fas ligand (from M1 to Q130), designated as the "MQ"
fragment was isolated and then amplified using standard PCR
techniques. A second DNA fragment (SEQ ID NO: 7) encoding residues
146 to 281 of Fas ligand (from V146 to the stop codon), designated
as the "VT" fragment.
[0159] The 3' end of the MQ fragment was ligated to the 5' end of
the VT fragment using a standard ligase reaction to produce the
MQ/VT polynucleotide (SEQ ID NO: 9) encoding the Fas ligand
130.fwdarw.145 deletion mutein.
[0160] The MQ/VT polynucleotide is cloned into an appropriate high
titer retroviral vector, such as pLNL6 [Bender et al., (1987), J.
Virol., 61(5): 1639-1646 or U.S. Pat. No. 5,324,655, both of which
are hereby incorporated herein by reference] or a commercially
available expression vector that is capable of infecting a
mammalian host cell. The transfected or transduced host cell is
cultured, whereupon the non-cleavable Fas ligand.sub.130.fwdarw.145
deletion mutein (i.e., a mutein lacking residues +1 to +15 from the
P1 site) is expressed in the host cell and remains surface bound
thereto.
Sequence CWU 1
1
121387DNAHomo sapiensCDS(1)...(387) 1atg cag cag ccc ttc aat tac
cca tat ccc cag atc tac tgg gtg gac 48Met Gln Gln Pro Phe Asn Tyr
Pro Tyr Pro Gln Ile Tyr Trp Val Asp1 5 10 15agc agt gcc agc tct ccc
tgg gcc cct cca ggc aca gtt ctt ccc tgt 96Ser Ser Ala Ser Ser Pro
Trp Ala Pro Pro Gly Thr Val Leu Pro Cys 20 25 30cca acc tct gtg ccc
aga agg cct ggt caa agg agg cca cca cca cca 144Pro Thr Ser Val Pro
Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro 35 40 45ccg cca ccg cca
cca cta cca cct ccg ccg ccg ccg cca cca ctg cct 192Pro Pro Pro Pro
Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro 50 55 60cca cta ccg
ctg cca ccc ctg aag aag aga ggg aac cac agc aca ggc 240Pro Leu Pro
Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr Gly65 70 75 80ctg
tgt ctc ctt gtg atg ttt ttc atg gtt ctg gtt gcc ttg gta gga 288Leu
Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val Gly 85 90
95ttg ggc ctg ggg atg ttt cag ctc ttc cac cta cag aag gag ctg gca
336Leu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala
100 105 110gaa ctc cga gag tct acc agc cag atg cac aca gca tca tct
ttg gag 384Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser
Leu Glu 115 120 125aag 387Lys2129PRTHomo sapiens 2Met Gln Gln Pro
Phe Asn Tyr Pro Tyr Pro Gln Ile Tyr Trp Val Asp1 5 10 15Ser Ser Ala
Ser Ser Pro Trp Ala Pro Pro Gly Thr Val Leu Pro Cys 20 25 30Pro Thr
Ser Val Pro Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro 35 40 45Pro
Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro 50 55
60Pro Leu Pro Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr Gly65
70 75 80Leu Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val
Gly 85 90 95Leu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu
Leu Ala 100 105 110Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala
Ser Ser Leu Glu 115 120 125Lys3420DNAHomo sapiensCDS(1)...(420)
3ctg agg aaa gtg gcc cat tta aca ggc aag tcc aac tca agg tcc atg
48Leu Arg Lys Val Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser Met1
5 10 15cct ctg gaa tgg gaa gac acc tat gga att gtc ctg ctt tct gga
gtg 96Pro Leu Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu Ser Gly
Val 20 25 30aag tat aag aag ggt ggc ctt gtg atc aat gaa act ggg ctg
tac ttt 144Lys Tyr Lys Lys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu
Tyr Phe 35 40 45gta tat tcc aaa gta tac ttc cgg ggt caa tct tgc aac
aac ctg ccc 192Val Tyr Ser Lys Val Tyr Phe Arg Gly Gln Ser Cys Asn
Asn Leu Pro 50 55 60ctg agc cac aag gtc tac atg agg aac tct aag tat
ccc cag gat ctg 240Leu Ser His Lys Val Tyr Met Arg Asn Ser Lys Tyr
Pro Gln Asp Leu65 70 75 80gtg atg atg gag ggg aag atg atg agc tac
tgc act act ggg cag atg 288Val Met Met Glu Gly Lys Met Met Ser Tyr
Cys Thr Thr Gly Gln Met 85 90 95tgg gcc cgc agc agc tac ctg ggg gca
gtg ttc aat ctt acc agt gct 336Trp Ala Arg Ser Ser Tyr Leu Gly Ala
Val Phe Asn Leu Thr Ser Ala 100 105 110gat cat tta tat gtc aac gta
tct gag ctc tct ctg gtc aat ttt gag 384Asp His Leu Tyr Val Asn Val
Ser Glu Leu Ser Leu Val Asn Phe Glu 115 120 125gaa tct cag acg ttt
ttc ggc tta tat aag ctc taa 420Glu Ser Gln Thr Phe Phe Gly Leu Tyr
Lys Leu 130 1354139PRTHomo sapiens 4Leu Arg Lys Val Ala His Leu Thr
Gly Lys Ser Asn Ser Arg Ser Met1 5 10 15Pro Leu Glu Trp Glu Asp Thr
Tyr Gly Ile Val Leu Leu Ser Gly Val 20 25 30Lys Tyr Lys Lys Gly Gly
Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe 35 40 45Val Tyr Ser Lys Val
Tyr Phe Arg Gly Gln Ser Cys Asn Asn Leu Pro 50 55 60Leu Ser His Lys
Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu65 70 75 80Val Met
Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met 85 90 95Trp
Ala Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala 100 105
110Asp His Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu
115 120 125Glu Ser Gln Thr Phe Phe Gly Leu Tyr Lys Leu 130
1355807DNAHomo sapiensCDS(1)...(807) 5atg cag cag ccc ttc aat tac
cca tat ccc cag atc tac tgg gtg gac 48Met Gln Gln Pro Phe Asn Tyr
Pro Tyr Pro Gln Ile Tyr Trp Val Asp1 5 10 15agc agt gcc agc tct ccc
tgg gcc cct cca ggc aca gtt ctt ccc tgt 96Ser Ser Ala Ser Ser Pro
Trp Ala Pro Pro Gly Thr Val Leu Pro Cys 20 25 30cca acc tct gtg ccc
aga agg cct ggt caa agg agg cca cca cca cca 144Pro Thr Ser Val Pro
Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro 35 40 45ccg cca ccg cca
cca cta cca cct ccg ccg ccg ccg cca cca ctg cct 192Pro Pro Pro Pro
Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro 50 55 60cca cta ccg
ctg cca ccc ctg aag aag aga ggg aac cac agc aca ggc 240Pro Leu Pro
Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr Gly65 70 75 80ctg
tgt ctc ctt gtg atg ttt ttc atg gtt ctg gtt gcc ttg gta gga 288Leu
Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val Gly 85 90
95ttg ggc ctg ggg atg ttt cag ctc ttc cac cta cag aag gag ctg gca
336Leu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala
100 105 110gaa ctc cga gag tct acc agc cag atg cac aca gca tca tct
ttg gag 384Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser
Leu Glu 115 120 125aag ctg agg aaa gtg gcc cat tta aca ggc aag tcc
aac tca agg tcc 432Lys Leu Arg Lys Val Ala His Leu Thr Gly Lys Ser
Asn Ser Arg Ser 130 135 140atg cct ctg gaa tgg gaa gac acc tat gga
att gtc ctg ctt tct gga 480Met Pro Leu Glu Trp Glu Asp Thr Tyr Gly
Ile Val Leu Leu Ser Gly145 150 155 160gtg aag tat aag aag ggt ggc
ctt gtg atc aat gaa act ggg ctg tac 528Val Lys Tyr Lys Lys Gly Gly
Leu Val Ile Asn Glu Thr Gly Leu Tyr 165 170 175ttt gta tat tcc aaa
gta tac ttc cgg ggt caa tct tgc aac aac ctg 576Phe Val Tyr Ser Lys
Val Tyr Phe Arg Gly Gln Ser Cys Asn Asn Leu 180 185 190ccc ctg agc
cac aag gtc tac atg agg aac tct aag tat ccc cag gat 624Pro Leu Ser
His Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp 195 200 205ctg
gtg atg atg gag ggg aag atg atg agc tac tgc act act ggg cag 672Leu
Val Met Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln 210 215
220atg tgg gcc cgc agc agc tac ctg ggg gca gtg ttc aat ctt acc agt
720Met Trp Ala Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr
Ser225 230 235 240gct gat cat tta tat gtc aac gta tct gag ctc tct
ctg gtc aat ttt 768Ala Asp His Leu Tyr Val Asn Val Ser Glu Leu Ser
Leu Val Asn Phe 245 250 255gag gaa tct cag acg ttt ttc ggc tta tat
aag ctc taa 807Glu Glu Ser Gln Thr Phe Phe Gly Leu Tyr Lys Leu 260
2656268PRTHomo sapiens 6Met Gln Gln Pro Phe Asn Tyr Pro Tyr Pro Gln
Ile Tyr Trp Val Asp1 5 10 15Ser Ser Ala Ser Ser Pro Trp Ala Pro Pro
Gly Thr Val Leu Pro Cys 20 25 30Pro Thr Ser Val Pro Arg Arg Pro Gly
Gln Arg Arg Pro Pro Pro Pro 35 40 45Pro Pro Pro Pro Pro Leu Pro Pro
Pro Pro Pro Pro Pro Pro Leu Pro 50 55 60Pro Leu Pro Leu Pro Pro Leu
Lys Lys Arg Gly Asn His Ser Thr Gly65 70 75 80Leu Cys Leu Leu Val
Met Phe Phe Met Val Leu Val Ala Leu Val Gly 85 90 95Leu Gly Leu Gly
Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala 100 105 110Glu Leu
Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser Leu Glu 115 120
125Lys Leu Arg Lys Val Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser
130 135 140Met Pro Leu Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu
Ser Gly145 150 155 160Val Lys Tyr Lys Lys Gly Gly Leu Val Ile Asn
Glu Thr Gly Leu Tyr 165 170 175Phe Val Tyr Ser Lys Val Tyr Phe Arg
Gly Gln Ser Cys Asn Asn Leu 180 185 190Pro Leu Ser His Lys Val Tyr
Met Arg Asn Ser Lys Tyr Pro Gln Asp 195 200 205Leu Val Met Met Glu
Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln 210 215 220Met Trp Ala
Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser225 230 235
240Ala Asp His Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe
245 250 255Glu Glu Ser Gln Thr Phe Phe Gly Leu Tyr Lys Leu 260
2657411DNAHomo sapiensCDS(1)...(411) 7gtg gcc cat tta aca ggc aag
tcc aac tca agg tcc atg cct ctg gaa 48Val Ala His Leu Thr Gly Lys
Ser Asn Ser Arg Ser Met Pro Leu Glu1 5 10 15tgg gaa gac acc tat gga
att gtc ctg ctt tct gga gtg aag tat aag 96Trp Glu Asp Thr Tyr Gly
Ile Val Leu Leu Ser Gly Val Lys Tyr Lys 20 25 30aag ggt ggc ctt gtg
atc aat gaa act ggg ctg tac ttt gta tat tcc 144Lys Gly Gly Leu Val
Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr Ser 35 40 45aaa gta tac ttc
cgg ggt caa tct tgc aac aac ctg ccc ctg agc cac 192Lys Val Tyr Phe
Arg Gly Gln Ser Cys Asn Asn Leu Pro Leu Ser His 50 55 60aag gtc tac
atg agg aac tct aag tat ccc cag gat ctg gtg atg atg 240Lys Val Tyr
Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val Met Met65 70 75 80gag
ggg aag atg atg agc tac tgc act act ggg cag atg tgg gcc cgc 288Glu
Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp Ala Arg 85 90
95agc agc tac ctg ggg gca gtg ttc aat ctt acc agt gct gat cat tta
336Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp His Leu
100 105 110tat gtc aac gta tct gag ctc tct ctg gtc aat ttt gag gaa
tct cag 384Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu
Ser Gln 115 120 125acg ttt ttc ggc tta tat aag ctc taa 411Thr Phe
Phe Gly Leu Tyr Lys Leu 130 1358136PRTHomo sapiens 8Val Ala His Leu
Thr Gly Lys Ser Asn Ser Arg Ser Met Pro Leu Glu1 5 10 15Trp Glu Asp
Thr Tyr Gly Ile Val Leu Leu Ser Gly Val Lys Tyr Lys 20 25 30Lys Gly
Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr Ser 35 40 45Lys
Val Tyr Phe Arg Gly Gln Ser Cys Asn Asn Leu Pro Leu Ser His 50 55
60Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val Met Met65
70 75 80Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp Ala
Arg 85 90 95Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp
His Leu 100 105 110Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe
Glu Glu Ser Gln 115 120 125Thr Phe Phe Gly Leu Tyr Lys Leu 130
1359798DNAHomo sapiensCDS(1)...(798) 9atg cag cag ccc ttc aat tac
cca tat ccc cag atc tac tgg gtg gac 48Met Gln Gln Pro Phe Asn Tyr
Pro Tyr Pro Gln Ile Tyr Trp Val Asp1 5 10 15agc agt gcc agc tct ccc
tgg gcc cct cca ggc aca gtt ctt ccc tgt 96Ser Ser Ala Ser Ser Pro
Trp Ala Pro Pro Gly Thr Val Leu Pro Cys 20 25 30cca acc tct gtg ccc
aga agg cct ggt caa agg agg cca cca cca cca 144Pro Thr Ser Val Pro
Arg Arg Pro Gly Gln Arg Arg Pro Pro Pro Pro 35 40 45ccg cca ccg cca
cca cta cca cct ccg ccg ccg ccg cca cca ctg cct 192Pro Pro Pro Pro
Pro Leu Pro Pro Pro Pro Pro Pro Pro Pro Leu Pro 50 55 60cca cta ccg
ctg cca ccc ctg aag aag aga ggg aac cac agc aca ggc 240Pro Leu Pro
Leu Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr Gly65 70 75 80ctg
tgt ctc ctt gtg atg ttt ttc atg gtt ctg gtt gcc ttg gta gga 288Leu
Cys Leu Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val Gly 85 90
95ttg ggc ctg ggg atg ttt cag ctc ttc cac cta cag aag gag ctg gca
336Leu Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala
100 105 110gaa ctc cga gag tct acc agc cag atg cac aca gca tca tct
ttg gag 384Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser
Leu Glu 115 120 125aag gtg gcc cat tta aca ggc aag tcc aac tca agg
tcc atg cct ctg 432Lys Val Ala His Leu Thr Gly Lys Ser Asn Ser Arg
Ser Met Pro Leu 130 135 140gaa tgg gaa gac acc tat gga att gtc ctg
ctt tct gga gtg aag tat 480Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu
Leu Ser Gly Val Lys Tyr145 150 155 160aag aag ggt ggc ctt gtg atc
aat gaa act ggg ctg tac ttt gta tat 528Lys Lys Gly Gly Leu Val Ile
Asn Glu Thr Gly Leu Tyr Phe Val Tyr 165 170 175tcc aaa gta tac ttc
cgg ggt caa tct tgc aac aac ctg ccc ctg agc 576Ser Lys Val Tyr Phe
Arg Gly Gln Ser Cys Asn Asn Leu Pro Leu Ser 180 185 190cac aag gtc
tac atg agg aac tct aag tat ccc cag gat ctg gtg atg 624His Lys Val
Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val Met 195 200 205atg
gag ggg aag atg atg agc tac tgc act act ggg cag atg tgg gcc 672Met
Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp Ala 210 215
220cgc agc agc tac ctg ggg gca gtg ttc aat ctt acc agt gct gat cat
720Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp
His225 230 235 240tta tat gtc aac gta tct gag ctc tct ctg gtc aat
ttt gag gaa tct 768Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn
Phe Glu Glu Ser 245 250 255cag acg ttt ttc ggc tta tat aag ctc taa
798Gln Thr Phe Phe Gly Leu Tyr Lys Leu 260 26510265PRTHomo sapiens
10Met Gln Gln Pro Phe Asn Tyr Pro Tyr Pro Gln Ile Tyr Trp Val Asp1
5 10 15Ser Ser Ala Ser Ser Pro Trp Ala Pro Pro Gly Thr Val Leu Pro
Cys 20 25 30Pro Thr Ser Val Pro Arg Arg Pro Gly Gln Arg Arg Pro Pro
Pro Pro 35 40 45Pro Pro Pro Pro Pro Leu Pro Pro Pro Pro Pro Pro Pro
Pro Leu Pro 50 55 60Pro Leu Pro Leu Pro Pro Leu Lys Lys Arg Gly Asn
His Ser Thr Gly65 70 75 80Leu Cys Leu Leu Val Met Phe Phe Met Val
Leu Val Ala Leu Val Gly 85 90 95Leu Gly Leu Gly Met Phe Gln Leu Phe
His Leu Gln Lys Glu Leu Ala 100 105 110Glu Leu Arg Glu Ser Thr Ser
Gln Met His Thr Ala Ser Ser Leu Glu 115 120 125Lys Val Ala His Leu
Thr Gly Lys Ser Asn Ser Arg Ser Met Pro Leu 130 135 140Glu Trp Glu
Asp Thr Tyr Gly Ile Val Leu Leu Ser Gly Val Lys Tyr145 150 155
160Lys Lys Gly Gly Leu Val Ile Asn Glu Thr Gly Leu Tyr Phe Val Tyr
165 170 175Ser Lys Val Tyr Phe Arg Gly Gln Ser Cys Asn Asn Leu Pro
Leu Ser 180 185 190His Lys Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln
Asp Leu Val Met 195 200 205Met Glu Gly Lys Met Met Ser Tyr Cys Thr
Thr Gly Gln Met Trp Ala 210 215 220Arg Ser Ser Tyr Leu Gly Ala Val
Phe Asn Leu Thr Ser Ala Asp His225 230 235
240Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu Ser
245 250 255Gln Thr Phe Phe Gly Leu Tyr Lys Leu 260 26511972DNAHomo
sapiensCDS(65)...(910) 11tctagactca ggactgagaa gaagtaaaac
cgtttgctgg ggctggcctg actcaccagc 60tgcc atg cag cag ccc ttc aat tac
cca tat ccc cag atc tac tgg gtg 109Met Gln Gln Pro Phe Asn Tyr Pro
Tyr Pro Gln Ile Tyr Trp Val1 5 10 15gac agc agt gcc agc tct ccc tgg
gcc cct cca ggc aca gtt ctt ccc 157Asp Ser Ser Ala Ser Ser Pro Trp
Ala Pro Pro Gly Thr Val Leu Pro 20 25 30tgt cca acc tct gtg ccc aga
agg cct ggt caa agg agg cca cca cca 205Cys Pro Thr Ser Val Pro Arg
Arg Pro Gly Gln Arg Arg Pro Pro Pro 35 40 45cca ccg cca ccg cca cca
cta cca cct ccg ccg ccg ccg cca cca ctg 253Pro Pro Pro Pro Pro Pro
Leu Pro Pro Pro Pro Pro Pro Pro Pro Leu 50 55 60cct cca cta ccg ctg
cca ccc ctg aag aag aga ggg aac cac agc aca 301Pro Pro Leu Pro Leu
Pro Pro Leu Lys Lys Arg Gly Asn His Ser Thr 65 70 75ggc ctg tgt ctc
ctt gtg atg ttt ttc atg gtt ctg gtt gcc ttg gta 349Gly Leu Cys Leu
Leu Val Met Phe Phe Met Val Leu Val Ala Leu Val80 85 90 95gga ttg
ggc ctg ggg atg ttt cag ctc ttc cac cta cag aag gag ctg 397Gly Leu
Gly Leu Gly Met Phe Gln Leu Phe His Leu Gln Lys Glu Leu 100 105 110
gca gaa ctc cga gag tct acc agc cag atg cac aca gca tca tct ttg
445Ala Glu Leu Arg Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser Leu
115 120 125gag aag caa ata ggc cac ccc agt cca ccc cct gaa aaa aag
gag ctg 493Glu Lys Gln Ile Gly His Pro Ser Pro Pro Pro Glu Lys Lys
Glu Leu 130 135 140agg aaa gtg gcc cat tta aca ggc aag tcc aac tca
agg tcc atg cct 541Arg Lys Val Ala His Leu Thr Gly Lys Ser Asn Ser
Arg Ser Met Pro 145 150 155ctg gaa tgg gaa gac acc tat gga att gtc
ctg ctt tct gga gtg aag 589Leu Glu Trp Glu Asp Thr Tyr Gly Ile Val
Leu Leu Ser Gly Val Lys160 165 170 175tat aag aag ggt ggc ctt gtg
atc aat gaa act ggg ctg tac ttt gta 637Tyr Lys Lys Gly Gly Leu Val
Ile Asn Glu Thr Gly Leu Tyr Phe Val 180 185 190tat tcc aaa gta tac
ttc cgg ggt caa tct tgc aac aac ctg ccc ctg 685Tyr Ser Lys Val Tyr
Phe Arg Gly Gln Ser Cys Asn Asn Leu Pro Leu 195 200 205agc cac aag
gtc tac atg agg aac tct aag tat ccc cag gat ctg gtg 733Ser His Lys
Val Tyr Met Arg Asn Ser Lys Tyr Pro Gln Asp Leu Val 210 215 220atg
atg gag ggg aag atg atg agc tac tgc act act ggg cag atg tgg 781Met
Met Glu Gly Lys Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp 225 230
235gcc cgc agc agc tac ctg ggg gca gtg ttc aat ctt acc agt gct gat
829Ala Arg Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala
Asp240 245 250 255cat tta tat gtc aac gta tct gag ctc tct ctg gtc
aat ttt gag gaa 877His Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val
Asn Phe Glu Glu 260 265 270tct cag acg ttt ttc ggc tta tat aag ctc
taa gagaagcact ttgggattct 930Ser Gln Thr Phe Phe Gly Leu Tyr Lys
Leu 275 280ttccattatg attctttgtt acaggcaccg agatgttcta ga 97212281
PRTHomo sapiens 12Met Gln Gln Pro Phe Asn Tyr Pro Tyr Pro Gln Ile
Tyr Trp Val Asp1 5 10 15Ser Ser Ala Ser Ser Pro Trp Ala Pro Pro Gly
Thr Val Leu Pro Cys 20 25 30Pro Thr Ser Val Pro Arg Arg Pro Gly Gln
Arg Arg Pro Pro Pro Pro 35 40 45Pro Pro Pro Pro Pro Leu Pro Pro Pro
Pro Pro Pro Pro Pro Leu Pro 50 55 60Pro Leu Pro Leu Pro Pro Leu Lys
Lys Arg Gly Asn His Ser Thr Gly65 70 75 80Leu Cys Leu Leu Val Met
Phe Phe Met Val Leu Val Ala Leu Val Gly 85 90 95Leu Gly Leu Gly Met
Phe Gln Leu Phe His Leu Gln Lys Glu Leu Ala 100 105 110Glu Leu Arg
Glu Ser Thr Ser Gln Met His Thr Ala Ser Ser Leu Glu 115 120 125Lys
Gln Ile Gly His Pro Ser Pro Pro Pro Glu Lys Lys Glu Leu Arg 130 135
140Lys Val Ala His Leu Thr Gly Lys Ser Asn Ser Arg Ser Met Pro
Leu145 150 155 160Glu Trp Glu Asp Thr Tyr Gly Ile Val Leu Leu Ser
Gly Val Lys Tyr 165 170 175Lys Lys Gly Gly Leu Val Ile Asn Glu Thr
Gly Leu Tyr Phe Val Tyr 180 185 190Ser Lys Val Tyr Phe Arg Gly Gln
Ser Cys Asn Asn Leu Pro Leu Ser 195 200 205His Lys Val Tyr Met Arg
Asn Ser Lys Tyr Pro Gln Asp Leu Val Met 210 215 220Met Glu Gly Lys
Met Met Ser Tyr Cys Thr Thr Gly Gln Met Trp Ala225 230 235 240Arg
Ser Ser Tyr Leu Gly Ala Val Phe Asn Leu Thr Ser Ala Asp His 245 250
255Leu Tyr Val Asn Val Ser Glu Leu Ser Leu Val Asn Phe Glu Glu Ser
260 265 270Gln Thr Phe Phe Gly Leu Tyr Lys Leu 275 280
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