U.S. patent application number 17/528422 was filed with the patent office on 2022-03-10 for compositions and methods related to multimodal therapeutic cell systems for autoimmune indications.
This patent application is currently assigned to Rubius Therapeutics, Inc.. The applicant listed for this patent is Rubius Therapeutics, Inc.. Invention is credited to Noubar B. Afeyan, Tiffany F. Chen, Robert J. Deans, Nathan Dowden, Avak Kahvejian, Jordi Mata-Fink, John Round, Torben Straight Nissen, Tom Wickham.
Application Number | 20220072048 17/528422 |
Document ID | / |
Family ID | 57907000 |
Filed Date | 2022-03-10 |
United States Patent
Application |
20220072048 |
Kind Code |
A1 |
Kahvejian; Avak ; et
al. |
March 10, 2022 |
Compositions and Methods Related to Multimodal Therapeutic Cell
Systems for Autoimmune Indications
Abstract
The invention includes compositions and methods related to
multimodal therapies, e.g., for treating immune conditions. A
multimodal therapy described herein provides and/or administers a
plurality of agents that function in a coordinated manner to
provide a therapeutic benefit to a subject in need thereof, e.g., a
subject having an autoimmune disease or inflammatory disease.
Inventors: |
Kahvejian; Avak; (Arlington,
MA) ; Mata-Fink; Jordi; (Somerville, MA) ;
Deans; Robert J.; (Riverside, CA) ; Chen; Tiffany
F.; (Cambridge, MA) ; Round; John; (Cambridge,
MA) ; Afeyan; Noubar B.; (Lexington, MA) ;
Straight Nissen; Torben; (Chestnut Hill, MA) ;
Dowden; Nathan; (Winchester, MA) ; Wickham; Tom;
(Groton, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Rubius Therapeutics, Inc. |
Cambridge |
MA |
US |
|
|
Assignee: |
Rubius Therapeutics, Inc.
Cambridge
MA
|
Family ID: |
57907000 |
Appl. No.: |
17/528422 |
Filed: |
November 17, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15905445 |
Feb 26, 2018 |
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17528422 |
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PCT/US2017/013033 |
Jan 11, 2017 |
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15905445 |
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62420973 |
Nov 11, 2016 |
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62370915 |
Aug 4, 2016 |
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62359448 |
Jul 7, 2016 |
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62277130 |
Jan 11, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/00 20130101;
A61P 17/00 20180101; A61K 35/18 20130101; A61K 2039/505 20130101;
C12N 5/0641 20130101; A61K 39/0011 20130101; A61K 38/191 20130101;
C12N 2510/00 20130101; C07K 14/705 20130101; A61K 39/3955 20130101;
A61K 2039/515 20130101; A61K 35/12 20130101 |
International
Class: |
A61K 35/18 20060101
A61K035/18; A61K 35/12 20060101 A61K035/12; A61K 38/19 20060101
A61K038/19; A61K 39/00 20060101 A61K039/00; A61K 39/395 20060101
A61K039/395; C12N 5/078 20060101 C12N005/078; A61P 17/00 20060101
A61P017/00; C07K 14/705 20060101 C07K014/705 |
Claims
1. A method of treating sepsis in a subject, the method comprising
administering a population of enucleated erythroid cells to the
subject, wherein the enucleated erythroid cells of the population
comprise two or more exogenous polypeptides present on the surface
of the enucleated erythroid cells, wherein at least two of the two
or more exogenous polypeptides comprise a binding domain that binds
specifically to an inflammatory cytokine.
2. The method of claim 1, wherein the binding domain is an antibody
or an antibody fragment.
3. The method of claim 2, wherein the antibody fragment is an
scFv.
4. The method of claim 1, wherein the binding domain is a receptor
of an inflammatory cytokine.
5. The method of claim 1, wherein a first exogenous polypeptide of
the at least two exogenous polypeptides comprises a binding domain
that binds to a first inflammatory cytokine, and a second exogenous
polypeptide of the at least two exogenous polypeptides comprises a
binding domain that binds to a second inflammatory cytokine,
wherein the first and the second inflammatory cytokines are
different.
6. The method of claim 5, wherein the first inflammatory cytokine
and the second inflammatory cytokine are independently selected
from the group consisting of: tumor necrosis factor-alpha, IL-6,
interferon-gamma, IL-12, IL-1, G-CSF, IL-8, IL-11, IL-17, IL-18,
interferon-alpha, interferon-beta, and tumor necrosis factor
beta.
7. The method of claim 5, wherein the enucleated erythroid cells of
the population comprise a third exogenous polypeptide present on
the surface of the enucleated erythroid cells, wherein the third
exogenous polypeptide comprises a binding domain that binds
specifically to a third inflammatory cytokine.
8. The method of claim 7, wherein the first, the second, and the
third inflammatory cytokines are different.
9. The method of claim 8, wherein the first inflammatory cytokine,
the second inflammatory cytokine, and the third inflammatory
cytokine are independently selected from the group consisting of:
tumor necrosis factor-alpha, IL-6, interferon-gamma, IL-12, IL-1,
G-CSF, IL-8, IL-11, IL-17, IL-18, interferon-alpha,
interferon-beta, and tumor necrosis factor beta.
10. The method of claim 7, wherein the enucleated erythroid cells
of the population comprise a fourth exogenous polypeptide present
on the surface of the enucleated erythroid cells, wherein the
fourth exogenous polypeptide comprises a binding domain that binds
specifically to a fourth inflammatory cytokine.
11. The method of claim 10, wherein the first, the second, the
third, and the fourth inflammatory cytokines are different.
12. The method of claim 11, wherein the first inflammatory
cytokine, the second inflammatory cytokine, the third inflammatory
cytokine, and the fourth inflammatory cytokine are independently
selected from the group consisting of: tumor necrosis factor-alpha,
IL-6, interferon-gamma, IL-12, IL-1, G-CSF, IL-8, IL-11, IL-17,
IL-18, interferon-alpha, interferon-beta, and tumor necrosis factor
beta.
13. The method of claim 1, wherein the population of enucleated
erythroid cells are reticulocytes.
14. The method of claim 1, wherein the population of enucleated
erythroid cells are erythrocytes.
15. The method of claim 1, wherein the enucleated erythroid cells
are not hypotonically loaded cells.
16. The method of claim 1, wherein the enucleated erythroid cells
exhibit substantially the same osmotic membrane fragility as an
isolated, unmodified, uncultured erythroid cell that does not
comprise the at least two exogenous polypeptides.
17. The method of claim 1, wherein the enucleated erythroid cells
are human enucleated erythroid cells.
18. The method of claim 1, wherein the enucleated erythroid cells
were produced by a process comprising: providing a nucleated
erythroid cell precursor comprising exogenous nucleic acid encoding
the two or more exogenous polypeptides; and culturing the nucleated
erythroid cell precursor under conditions suitable for enucleation
of the nucleated erythroid cell precursor and for the production of
the two or more exogenous polypeptides.
19. The method of claim 18, wherein the process further comprises
introducing the exogenous nucleic acid into the nucleated erythroid
cell precursor.
20. The method of claim 18, wherein the nucleated erythroid cell
precursor is a CD34.sup.+ hematopoietic stem cell.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of PCT Application No.
PCT/US2017/013033, filed Jan. 11, 2017, which claims priority to
U.S. Ser. No. 62/277,130, filed Jan. 11 2016, U.S. Ser. No.
62/359,448, filed Jul. 7, 2016, U.S. Ser. No. 62/370,915, filed
Aug. 4, 2016, and U.S. Ser. No. 62/420,973, filed Nov. 11, 2016,
the contents of which are incorporated herein by reference in their
entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jan. 5, 2017, is named R2081-7013WO SL.txt and is 24,800 bytes
in size.
BACKGROUND
[0003] Red blood cells have been considered for use as drug
delivery systems, e.g., to degrade toxic metabolites or inactivate
xenobiotics, and in other biomedical applications.
SUMMARY OF THE INVENTION
[0004] The invention includes compositions and methods related to
multimodal therapies. The therapies are useful, e.g., for treating
immune conditions, e.g., autoimmune or inflammatory diseases. A
multimodal therapy described herein provides and/or administers a
plurality of agents that function in a coordinated manner to
provide a therapeutic benefit to a subject in need thereof, e.g., a
subject having an autoimmune or inflammatory disease. In general, a
multimodal therapy described herein includes the administration to
a subject of a preparation of engineered red blood cells, e.g.,
enucleated red blood cells, comprising (e.g., expressing or
containing) a plurality of agents (e.g., polypeptides) that
function in a coordinated manner (e.g., agent-additive,
agent-synergistic, multiplicative, independent function,
localization-based, proximity-dependent, scaffold-based,
multimer-based, or compensatory).
[0005] In some aspects, the present disclosure provides an
enucleated red blood cell, e.g., a reticulocyte, comprising a
plurality of agents, e.g., a plurality of polypeptides (e.g.,
exogenous polypeptides), e.g., a first exogenous polypeptide, a
second exogenous polypeptide, and a third exogenous
polypeptide.
[0006] In some aspects, the present disclosure provides an
enucleated red blood cell, e.g., a reticulocyte, comprising a
plurality of exogenous polypeptides, wherein a first and a second
exogenous polypeptide of the plurality have agent-additive,
agent-synergistic, multiplicative, independent function,
localization-based, proximity-dependent, scaffold-based,
multimer-based, or compensatory activity.
[0007] In some aspects, the present disclosure provides an
enucleated red blood cell, e.g., a reticulocyte, comprising a first
exogenous polypeptide and a second exogenous polypeptide, wherein:
[0008] a) the first and second exogenous polypeptides act on the
same target, wherein optionally the target is a cell surface
receptor and/or an endogenous human protein; [0009] b) the first
exogenous polypeptide binds to a first endogenous human protein and
the second exogenous polypeptide binds to a second endogenous human
target protein, e.g., with a Kd of less than 500, 200, 100, 50, 20,
10, 5, 2, or 1 nM; [0010] c) the first exogenous polypeptide acts
on (e.g., binds) a first target, and the second exogenous
polypeptide act on (e.g., binds) a second target, wherein the first
and second targets are members of the same biological pathway,
wherein optionally the targets are cell surface receptors,
endogenous human proteins, or both; [0011] d) the first exogenous
polypeptide comprises a first pro-apoptotic polypeptide and the
second exogenous polypeptide comprises a second pro-apoptotic
polypeptide, e.g., a TRAIL receptor ligand, e.g., a TRAIL
polypeptide; [0012] e) the first and second exogenous polypeptides
are in close proximity to each other, e.g., are less than 10, 7, 5,
4, 3, 2, 1, 0.5, 0.2, or 0.1 nm apart for a duration of at least 1,
2, 5, 10, 30, or 60 seconds; 1, 2, 5, 10, 30, or 60 minutes, or 1,
2, 3, 6, 12, or 14 hours; [0013] f) the first and second exogenous
polypeptides have a Kd of less than 500, 200, 100, 50, 20, 10, 5,
2, or 1 nM for each other; [0014] g) the first exogenous
polypeptide comprises an antigen-presenting polypeptide, e.g., an
MHC molecule, e.g., an MHC class II molecule, and the second
exogenous polypeptide comprises an antigen; [0015] h) the first and
second exogenous polypeptides act on different targets, wherein
optionally at least one of the targets is a cell surface receptor
and/or an endogenous human protein, e.g., the first exogenous
polypeptide binds a first cell type, e.g., an immune effector cell,
and the second exogenous polypeptide binds a second cell type,
e.g., an immune effector cell, e.g., a T cell; [0016] i) the first
exogenous polypeptide and the second exogenous polypeptide have an
abundance ratio of about 1:1, from about 2:1 to 1:2, from about 5:1
to 1:5, from about 10:1 to 1:10, from about 20:1 to 1:20, from
about 50:1 to 1:50, from about 100:1 to 1:100 by weight or by copy
number; [0017] j) the first exogenous polypeptide and the second
exogenous polypeptide have a Kd for a first target and a second
target, respectively, with a ratio of about 1:1, from about 2:1 to
1:2, from about 5:1 to 1:5, from about 10:1 to 1:10, from about
20:1 to 1:20, from about 50:1 to 1:50, from about 100:1 to 1:100;
[0018] k) the first exogenous polypeptide has a first activity
(e.g., binding) towards a first target, and the second exogenous
polypeptide has a second activity (e.g., binding) towards the first
target, e.g., the first and second exogenous polypeptides bind a
single target; [0019] l) the first exogenous polypeptide acts on
(e.g., binds) a first target and the second exogenous polypeptide
acts on (e.g., binds) a second target, and the first and second
targets are part of the same pathway, wherein optionally the first
exogenous polypeptide acts on the first target and the second
exogenous polypeptide acts on the second target simultaneously;
[0020] m) the first exogenous polypeptide acts on (e.g., binds) a
first target and the second exogenous polypeptide acts on (e.g.,
binds) a second target, and the first and second targets are part
of different pathways, wherein optionally the first and second
pathways both act to promote a given cellular response; [0021] n)
the first exogenous polypeptide localizes the enucleated red blood
cell to a desired site, e.g., a human cell, and the second
exogenous polypeptide has a therapeutic activity, e.g., an
immunomodulation activity such as a T cell inhibition activity or
antigen presenting activity; [0022] o) the first exogenous
polypeptide binds a first target, e.g., a first cell, e.g., a first
cell type, e.g., an immune effector cell, and the second exogenous
polypeptide binds a second target, e.g., a second cell, e.g., a
second cell type, e.g., an immune effector cell, e.g., a T cell;
[0023] p) the first exogenous polypeptide and the second exogenous
polypeptide are non-human proteins; [0024] q) the first exogenous
polypeptide and the second exogenous polypeptide are both enzymes,
e.g., biosynthetic enzymes; [0025] r) the first exogenous
polypeptide promotes formation of an intermediate molecule and the
second exogenous polypeptide acts on the intermediate molecule; or
[0026] s) the first exogenous polypeptide and the second exogenous
polypeptide act on successive steps of a pathway.
[0027] Any of the aspects herein, e.g., the aspects above, can be
characterized by one or more of the embodiments herein, e.g., the
embodiments below.
[0028] In some embodiments, the exogenous polypeptides have
synergistic activity. In some embodiments, the exogenous
polypeptides have additive activity.
[0029] In some embodiments, the exogenous polypeptides have
proximity-dependent activity. The proximity between the plurality
of polypeptides, before, during, or after, interaction with a
target moiety or moieties, may confer a property or result which is
not seen in the absence of such proximity in vivo or in vitro.
[0030] In some embodiments, the first exogenous polypeptide
interacts with, e.g., binds, a first target moiety, e.g., a first
target cell polypeptide on a target cell (e.g., an immune effector
cell, e.g., a T cell), and the second exogenous polypeptide
interacts with, e.g., binds, a second target moiety, e.g., a second
target cell polypeptide on the target cell (e.g., wherein binding
of the first and second target cell polypeptide alters a biological
property of the target cell). In an embodiment the first and second
targets are subunits of a multimeric complex on the target
cell.
[0031] In some embodiments, the first exogenous polypeptide
promotes fusion of the red blood cell with a target cell and the
second exogenous polypeptide is a polypeptide of any of Table 1,
Table 2, Table 3, or Table 4 (e.g., a human polypeptide of any of
Table 1, Table 2, Table 3, or Table 4, e.g., a polypeptide having
the amino acid sequence of the human wild type polypeptide).
[0032] In some embodiments the first and second exogenous
polypeptides interact with one another, e.g., the first modifies,
e.g., by cleavage or phosphorylation, the second, or the first and
second form a dimeric or multimeric protein.
[0033] In some embodiments, the enucleated red blood cell comprises
3, 4, 5, 6, 7, 8, 9, or 10 different exogenous polypeptides. In an
embodiment a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10), or
all, of the different exogenous polypeptides, have a preselected
level of homology to each other, e.g., at least 40, 50, 60, 70, 75,
80, 85, 90, 95, 96, 97, 98, 99, or 99.5% sequence identity to each
other. In an embodiment a plurality (e.g., 2, 3, 4, 5, 6, 7, 8, 9,
or 10), or all, of the different exogenous polypeptides, have a
preselected level of homology to a reference sequence, e.g., at
least 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.5%, or
100% sequence identity with a reference sequence (which reference
sequence, includes an entire polypeptide sequence, or a portion
thereof, e.g., a preselected domain), e.g., a plurality or all of
the different exogenous polypeptides are antibodies or antibody
molecules. In some embodiments, the reference sequence is an
antibody sequence or fragment thereof. In some embodiments, the
reference sequence comprises a heavy chain constant region or
portion thereof, light chain constant region or fragment thereof,
heavy chain variable region or portion thereof, light chain
variable region or fragment thereof, or any combination of the
foregoing. In embodiments, the enucleated red blood cells are used
to treat an immunodeficient subject, e.g., as a substitute for (or
combination with) an IVIG therapy.
[0034] In some embodiments, the enucleated red blood cell comprises
at least 2 but no more than 5, 6, 7, 8, 9, or 10 different
exogenous polypeptides, e.g., exogenous polypeptides that are
encoded by one or more exogenous nucleic acids that are not
retained by the enucleated red blood cell.
[0035] In some embodiments, the exogenous polypeptides are encoded
by one or more exogenous nucleic acids that are not retained by the
enucleated red blood cell.
[0036] In some embodiments, one or more (e.g., two or three) of the
first, second, and optionally third exogenous polypeptides are
transmembrane polypeptides or surface-anchored polypeptides.
[0037] In some embodiments, the first exogenous polypeptide
interacts with, e.g., binds, a moiety on a target cell, and the
second exogenous polypeptide alters a property of the target cell,
e.g., kills, activates, inactivates, or induces tolerance or anergy
in the target cell.
[0038] In some embodiments, the first exogenous polypeptide and the
second exogenous polypeptide have an abundance ratio of about 1:1,
from about 2:1 to 1:2, from about 5:1 to 1:5, from about 10:1 to
1:10, from about 20:1 to 1:20, from about 50:1 to 1:50, or from
about 100:1 to 1:100 by weight or by copy number. In some
embodiments, both the first and second polypeptides have a
stoichiometric mode of action, or both have a catalytic mode of
action, and both are present at a similar abundance, e.g., about
1:1 or from about 2:1 to 1:2. In some embodiments, the first
exogenous polypeptide is more abundant than the second exogenous
polypeptide by at least about 10%, 20%, 30%, 50%, or a factor of 2,
3, 4, 5, 10, 20, 50, or 100 (and optionally up to 10 or 100 fold)
by weight or copy number. In some embodiments, the second exogenous
polypeptide is more abundant than the first exogenous polypeptide
by at least about 10%, 20%, 30%, 50%, or a factor of 2, 3, 4, 5,
10, 20, 50, or 100 (and optionally up to 10 or 100 fold) by weight
or copy number. In some embodiments, the first polypeptide has a
stoichiometric mode of action and the second polypeptide has a
catalytic mode of action, and the first polypeptide is more
abundant than the second polypeptide. In some embodiments, the
second polypeptide has a stoichiometric mode of action and the
first polypeptide has a catalytic mode of action, and the second
polypeptide is more abundant than the first polypeptide.
[0039] In some embodiments, the first exogenous polypeptide
comprises a targeting moiety.
[0040] In some embodiments, the enucleated red blood cell has one
or more of the following characteristics: [0041] a) an osmotic
fragility of less than 50% cell lysis at 0.3%, 0.35%, 0.4%, 0.45%,
or 0.5% NaCl; [0042] b) a cell volume of about 10-200 fL or a cell
diameter of between about 1 micron and about 20 microns, between
about 2 microns and about 20 microns, between about 3 microns and
about 20 microns, between about 4 microns and about 20 microns,
between about 5 microns and about 20 microns, between about 6
microns and about 20 microns, between about 5 microns and about 15
microns, or between about 10 microns and about 30 microns; [0043]
c) greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% fetal
hemoglobin; or at least about 20, 25, or 30 pg/cell of hemoglobin;
or [0044] d) phosphatidylserine content of the outer leaflet is
less than 30%, 25%, 20%, 15%, 10%, or 5% as measured by Annexin V
staining.
[0045] In some embodiments, at least one, e.g., all, of the
plurality of exogenous polypeptides are glycosylated. In some
embodiments, at least one, e.g., all, of the plurality of exogenous
polypeptides are phosphorylated.
[0046] In some embodiments, the enucleated red blood cell is a
reticulocyte.
[0047] In some embodiments, the exogenous polypeptide or
polypeptides lack a sortase transfer signature (i.e., a sequence
that can be created by a sortase reaction) such as LPXTG (SEQ ID
NO: 17).
[0048] In some aspects, the present disclosure provides a method of
treating a disease or condition described herein, comprising
administering to a subject in need thereof an enucleated red blood
cell, e.g., a reticulocyte, described herein. In some embodiments,
the disease or condition is an inflammatory disease, or an
autoimmune disease.
[0049] In some aspects, the present disclosure provides a method of
bringing into proximity a first and a second cell surface moiety,
e.g., transmembrane receptors, comprising administering to a
subject in need thereof an enucleated red blood cell, e.g., a
reticulocyte, described herein.
[0050] In some aspects, the present disclosure provides a method of
delivering, presenting, or expressing a plurality of
proximity-dependent molecules comprising providing an enucleated
red blood cell, e.g., a reticulocyte, described herein.
[0051] In some aspects, the present disclosure provides a method of
producing an enucleated red blood cell, e.g., a reticulocyte,
described herein, providing contacting a red blood cell precursor
with one or more nucleic acids encoding the exogenous polypeptides
and placing the cell in conditions that allow enucleation to
occur.
[0052] In some aspects, the present disclosure provides a
preparation, e.g., pharmaceutical preparation, comprising a
plurality of enucleated red blood cells, e.g., reticulocytes,
described herein, e.g., at least 10.sup.8, 10.sup.9, 10.sup.10,
10.sup.11, or 10.sup.12 cells.
[0053] In some aspects, the present disclosure provides a cell
complex, e.g., an in vitro or in vivo complex, of an engineered red
blood cell (RBC), e.g., an enucleated red blood cell, e.g., a
reticulocyte, and a target cell, the complex mediated by one of the
exogenous polypeptides. In some embodiments, the cell complex
comprises at least 2, 3, 4, 5, 10, 20, 50, or 100 cells.
[0054] In some aspects, the present disclosure proves a reaction
mixture comprising an engineered RBC, e.g., an enucleated red blood
cell, e.g., a reticulocyte, and nucleic acid, e.g., one or more
nucleic acid molecules, encoding a multimodal pair described
herein. In some embodiments, the nucleic acid comprises at least
one promoter that is active in a red blood cell. In some
embodiments, nucleic acid encodes at least two proteins described
herein (e.g., in Table 1, Table 2, Table 3, and Table 4). In some
embodiments, the nucleic acid encodes a third exogenous
polypeptide.
[0055] In some aspects, the present disclosure comprises a method
of making an engineered RBC (e.g., an enucleated red blood cell,
e.g., a reticulocyte) described herein, comprising: providing,
e.g., receiving, information about a target cell or subject,
responsive to that information selecting a plurality of exogenous
polypeptides, and introducing nucleic acids encoding the exogenous
polypeptides into a RBC or RBC precursor.
[0056] In some aspects, the present invention comprises a method of
evaluating an engineered RBC (e.g., enucleated RBC, e.g., a
reticulocyte), comprising providing a candidate RBC, and
determining if nucleic acid encoding a plurality of exogenous
polypeptides, e.g., a multimodal pair of the exogenous
polypeptides, are present.
[0057] The present disclosure provides, in some aspects, an
enucleated erythroid cell comprising:
[0058] a first exogenous polypeptide that interacts with a target,
and
[0059] a second exogenous polypeptide that modifies the target;
[0060] wherein one or more of:
[0061] (a) the second exogenous polypeptide comprises a moiety that
cleaves an antibody, e.g., that cleaves at a hinge region, a CH2
region, or between a hinge and CH2 region, e.g., an IdeS
polypeptide;
[0062] (b) the second exogenous polypeptide comprises an enzyme
(e.g., a protease) that modifies, e.g., is specific, e.g., binds to
a site on target, binds (e.g., specifically) and modifies, e.g.,
covalently modifies, e.g., cleaves, or removes or attaches a moiety
to, the target, wherein the target is optionally an antibody or a
complement factor;
[0063] (c) the second exogenous polypeptide comprises a
polypeptide, e.g., an enzyme, e.g., a protease, that modifies the
secondary, tertiary, or quaternary structure of the target, and, in
embodiments, alters, e.g., decreases or increases, the ability of
the target to interact with another molecule, e.g., the first
exogenous polypeptide or a molecule other than the first exogenous
polypeptide, wherein optionally the target comprises an antibody,
or complement factor;
[0064] (d) the second exogenous polypeptide comprises a
polypeptide, e.g., an enzyme (e.g., a protease) that cleaves the
target, e.g., a polypeptide, between a first target domain and a
second target domain, e.g., a first target domain that binds a
first substrate and a second target domain that binds a second
substrate;
[0065] (e) the target is a polypeptide (e.g., an autoantibody or a
complement factor); a carbohydrate (e.g., a glycan), a lipid (e.g.,
a phospholipid), or a nucleic acid (e.g., DNA, or RNA);
[0066] (f) the first exogenous polypeptide binds a target, e.g., an
antibody, but does not cleave, and the second exogenous polypeptide
cleaves a bond e.g., a covalent bond, e.g., a covalent bond in the
antibody;
[0067] (g) the target comprises an antibody and the first exogenous
polypeptide binds the variable region of the antibody target;
[0068] (h) the target comprises an antibody and first exogenous
polypeptide binds the constant region of the antibody target;
[0069] (i) the first exogenous polypeptide has an affinity for the
target that is about 1-2 pM, 2-5 pM, 5-10 pM, 10-20 pM, 20-50 pM,
50-100 pM, 100-200 pM, 200-500 pM, 500-1000 pM, 1-2 nM, 2-5 nM,
5-10 nM, 10-20 nM, 20-50 nM, 50-100 nM, 100-200 nM, 200-500 nM,
500-1000 nM, 1-2 .mu.M, 2-5 .mu.M, 5-10 .mu.M, 10-20 .mu.M, 20-50
.mu.M, or 50-100 .mu.M;
[0070] (j) the second exogenous polypeptide has a K.sub.M for the
target of about 10.sup.-1-10.sup.-7M, 10.sup.-1-10.sup.-2M,
10.sup.-2-10.sup.-3M, 10.sup.-3-10.sup.4M, 10.sup.-4-10.sup.-5M,
10.sup.-5-10.sup.-6M, or 10.sup.-6-10.sup.-7M;
[0071] (k) a ratio of the K.sub.d of the first exogenous
polypeptide for the target (measured in M) divided by the KM of the
second exogenous polypeptide for the target (measured in M) is
about 1.times.10.sup.-9-2.times.10.sup.-9,
2.times.10.sup.-9-5.times.10.sup.-9,
5.times.10.sup.-9-1.times.10.sup.-8,
1.times.10.sup.-8-2.times.10.sup.-8,
2.times.10.sup.-8-5.times.10.sup.-8,
5.times.10.sup.-8-1.times.10.sup.-7,
1.times.10.sup.-7-2.times.10.sup.-7,
2.times.10.sup.-7-5.times.10.sup.-7,
5.times.10.sup.-7-1.times.10.sup.-6,
1.times.10.sup.-6-2.times.10.sup.-6,
2.times.10.sup.-6-5.times.10.sup.-6,
5.times.10.sup.-6-1.times.10.sup.-5,
1.times.10.sup.-5-2.times.10.sup.-5,
2.times.10.sup.-5-5.times.10.sup.-5,
5.times.10.sup.-5-1.times.10.sup.4
1.times.10.sup.-4-2.times.10.sup.-4,
2.times.10.sup.-4-5.times.10''.sup.4
5.times.10.sup.-4-1.times.10.sup.-3,
1.times.10.sup.-3-2.times.10.sup.-3,
2.times.10.sup.-3-5.times.10.sup.-3,
5.times.10.sup.-3-1.times.10.sup.-2,
1.times.10.sup.-2-2.times.10.sup.-2,
2.times.10.sup.-2-5.times.10.sup.-2,
5.times.10.sup.-2-1.times.10.sup.-1,
1.times.10.sup.-1-2.times.10.sup.-1,
2.times.10.sup.-1-5.times.10.sup.-1, or 5.times.10.sup.-1-1;
[0072] (l) the observed reaction rate of the second exogenous
polypeptide modifying the target is greater than the reaction rate
of an enucleated cell which is similar but which lacks the first
exogenous polypeptide under otherwise similar reaction
conditions;
[0073] (m) a ratio of the average number of the first exogenous
polypeptide on the erythroid cell to the average number of the
second exogenous polypeptide on the erythroid cell is about 50:1,
20:1, 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:6, 1:8, 1:10,
1:20, or 1:50;
[0074] (n) affinity of the first exogenous polypeptide for the
target is greater than the affinity of the first exogenous
polypeptide for the modified (e.g., cleaved) target;
[0075] (o) a therapeutically effective dose of the enucleated
erythroid cell is less than stoichiometry (e.g., less by 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or
99.99%) to the amount of target in a subject's peripheral blood at
the time of administration;
[0076] (p) the number of enucleated erythroid cells in an effective
dose, is less than (e.g., less by 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 99.99%) the number
of targets, e.g., target molecules, in the subject's peripheral
blood at the time of administration;
[0077] (q) the number of second exogenous polypeptides comprised by
a preselected amount of enucleated erythroid cells, e.g., an
effective dose, or in vitro effective amount of enucleated
erythroid cells, is less than (e.g., less by 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 99.99%) a
reference value for targets, e.g., less than the number of targets
in the peripheral blood of the subject at the time of
administration;
[0078] (r) the number of first exogenous polypeptides comprised by
a preselected amount of enucleated erythroid cells, e.g., an
effective dose, or in vitro effective amount of enucleated
erythroid cells, is less than (e.g., less by 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 99.99%) a
reference value for targets, e.g., less than the number of targets
in the peripheral blood of the subject at the time of
administration;
[0079] (s) the number of first exogenous polypeptides and the
number of second exogenous polypeptides comprised by a preselected
amount of enucleated erythroid cells, e.g., an effective dose,
enucleated erythroid cells, is each less than a reference value for
targets, e.g., less than the number of targets in the peripheral
blood of the subject at the time of administration;
[0080] (t) the second exogenous polypeptide modifies (e.g. cleaves)
the target with a KM of at least 10.sup.-1 M, 10.sup.-2 M,
10.sup.-3M, 10.sup.-4 M, 10.sup.-5M, 10.sup.-6 M, or 10.sup.-7
M;
[0081] (u) the second exogenous polypeptide comprises a
chaperone;
[0082] (v) the first exogenous polypeptide comprises a
surface-exposed portion and the second exogenous polypeptide
comprises a surface exposed portion; or
[0083] (w) an effective amount of the enucleated erythroid cells is
less than (e.g., less by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 95%, 98%, 99%, 99.5%, 99.9%, or 99.99%) an effective amount of
otherwise similar enucleated erythroid cells that lack the second
exogenous polypeptide.
[0084] In embodiments, (b) the second exogenous polypeptide
comprises an enzyme (e.g., a protease) that modifies, e.g., is
specific, e.g., binds to a site on target, binds (e.g.,
specifically) and modifies, e.g., covalently modifies, e.g.,
cleaves, or removes or attaches a moiety to, the target, wherein
the target is optionally an antibody or a complement factor. In
embodiments the modification alters, e.g., increases or decreases,
the ability of the target to interact with another molecule, e.g.,
the first exogenous polypeptide or a molecule other than the first
exogenous polypeptide.
[0085] In embodiments, (d) the second exogenous polypeptide
comprises a polypeptide, e.g., an enzyme (e.g., a protease) that
cleaves the target, e.g., a polypeptide, between a first target
domain and a second target domain, e.g., a first target domain that
binds a first substrate and a second target domain that binds a
second substrate. In embodiments the first target domain is
released from the second target domain. In embodiments cleavage
alters the affinity one or both of the first target domain for a
first substrate and the affinity of the second target domain for a
second substrate. In an embodiment the target comprises an antibody
and the first target domain comprises one or more CDRs and the
second target domain comprises a portion of the constant region,
e.g., a Fc region.
[0086] In embodiments, at least two (e.g., at least 3, 4, 5, 6, 7,
8, 9, or 10) of (a)-(w) are present. In embodiments, at least (a)
and (e) are present. In embodiments, at least (a) and (i) are
present.
[0087] In embodiments, at least (a) and (j) are present. In
embodiments, at least (a) and (m) are present. In embodiments, at
least (a) and (q) are present. In embodiments, at least (a) and (r)
are present. In embodiments, at least (a) and (s) are present. In
embodiments, at least (e) and (i) are present. In embodiments, at
least (e) and (j) are present. In embodiments, at least (e) and (m)
are present. In embodiments, at least (e) and (q) are present. In
embodiments, at least (e) and (r) are present. In embodiments, at
least (e) and (s) are present. In embodiments, at least (i) and (j)
are present. In embodiments, at least (i) and (m) are present. In
embodiments, at least (i) and (q) are present. In embodiments, at
least (i) and (r) are present. In embodiments, at least (i) and (s)
are present. In embodiments, at least (j) and (m) are present. In
embodiments, at least (j) and (q) are present. In embodiments, at
least (j) and (r) are present. In embodiments, at least (j) and (s)
are present. In embodiments, at least (m) and (q) are present. In
embodiments, at least (m) and (r) are present. In embodiments, at
least (m) and (s) are present. In embodiments, at least (q) and (r)
are present. In embodiments, at least (q) and (s) are present. In
embodiments, at least (r) and (s) are present.
[0088] In embodiments, at least:
[0089] (a) and (b), (a) and (c), (a) and (d), (a) and (e), (a) and
(f), (a) and (g), (a) and (h), (a) and (i), (a) and (j), (a) and
(k), (a) and (l), (a) and (m), (a) and (n), (a) and (o), (a) and
(p), (a) and (q), (a) and (r), (a) and (s), (a) and (t), (a) and
(u), (a) and (v), (a) and (w),
[0090] (b) and (c), (b) and (d), (b) and (e), (b) and (f), (b) and
(g), (b) and (h), (b) and (i), (b) and (j), (b) and (k), (b) and
(l), (b) and (m), (b) and (n), (b) and (o), (b) and (p), (b) and
(q), (b) and (r), (b) and (s), (b) and (t), (b) and (u), (b) and
(v), (b) and (w),
[0091] (c) and (d), (c) and (e), (c) and (f), (c) and (g), (c) and
(h), (c) and (i), (c) and (j), (c) and (k), (c) and (l), (c) and
(m), (c) and (n), (c) and (o), (c) and (p), (c) and (q), (c) and
(r), (c) and (s), (c) and (t), (c) and (u), (c) and (v), (c) and
(w),
[0092] (d) and (e), (d) and (f), (d) and (g), (d) and (h), (d) and
(i), (d) and (j), (d) and (k), (d) and (l), (d) and (m), (d) and
(n), (d) and (o), (d) and (p), (d) and (q), (d) and (r), (d) and
(s), (d) and (t), (d) and (u), (d) and (v), (d) and (w),
[0093] (e) and (f), (e) and (g), (e) and (h), (e) and (i), (e) and
(j), (e) and (k), (e) and (l), (e) and (m), (e) and (n), (e) and
(o), (e) and (p), (e) and (q), (e) and (r), (e) and (s), (e) and
(t), (e) and (u), (e) and (v), (e) and (w),
[0094] (f) and (g), (f) and (h), (f) and (i), (f) and (j), (f) and
(k), (f) and (l), (f) and (m), (f) and (n), (f) and (o), (f) and
(p), (f) and (q), (f) and (r), (f) and (s), (f) and (t), (f) and
(u), (f) and (v), (f) and (w),
[0095] (g) and (h), (g) and (i), (g) and (j), (g) and (k), (g) and
(l), (g) and (m), (g) and (n), (g) and (o), (g) and (p), (g) and
(q), (g) and (r), (g) and (s), (g) and (t), (g) and (u), (g) and
(v), (g) and (w),
[0096] (h) and (i), (h) and (j), (h) and (k), (h) and (l), (h) and
(m), (h) and (n), (h) and (o), (h) and (p), (h) and (q), (h) and
(r), (h) and (s), (h) and (t), (h) and (u), (h) and (v), (h) and
(w),
[0097] (i) and (j), (i) and (k), (i) and (l), (i) and (m), (i) and
(n), (i) and (o), (i) and (p), (i) and (q), (i) and (r), (i) and
(s), (i) and (t), (i) and (u), (i) and (v), (i) and (w),
[0098] (j) and (k), (j) and (l), (j) and (m), (j) and (n), (j) and
(o), (j) and (p), (j) and (q), (j) and (r), (j) and (s), (j) and
(t), (j) and (u), (j) and (v), (j) and (w),
[0099] (k) and (l), (k) and (m), (k) and (n), (k) and (o), (k) and
(p), (k) and (q), (k) and (r), (k) and (s), (k) and (t), (k) and
(u), (k) and (v), (k) and (w),
[0100] (l) and (m), (l) and (n), (l) and (o), (l) and (p), (l) and
(q), (l) and (r), (l) and (s), (l) and (t), (l) and (u), (l) and
(v), (l) and (w),
[0101] (m) and (n), (m) and (o), (m) and (p), (m) and (q), (m) and
(r), (m) and (s), (m) and (t), (m) and (u), (m) and (v), (m) and
(w),
[0102] (n) and (o), (n) and (p), (n) and (q), (n) and (r), (n) and
(s), (n) and (t), (n) and (u), (n) and (v), (n) and (w),
[0103] (o) and (p), (o) and (q), (o) and (r), (o) and (s), (o) and
(t), (o) and (u), (o) and (v), (o) and (w),
[0104] (p) and (q), (p) and (r), (p) and (s), (p) and (t), (p) and
(u), (p) and (v), (p) and (w),
[0105] (q) and (r), (q) and (s), (q) and (t), (q) and (u), (q) and
(v), (q) and (w),
[0106] (r) and (s), (r) and (t), (r) and (u), (r) and (v), (r) and
(w),
[0107] (s) and (t), (s) and (u), (s) and (v), (s) and (w),
[0108] (t and (u), (t) and (v), (t) and (w),
[0109] (u) and (v), (u) and (w), or
[0110] (v) and (w), are present.
[0111] In embodiments, the target is other than an infectious
component, e.g., other than a bacterial component, a viral
component, a fungal component, or a parasitic component. In
embodiments, the first exogenous polypeptide comprises an
autoantigen. In embodiments, the surface-exposed portion of the
first exogenous polypeptide binds the target. In embodiments, the
surface-exposed portion of the second exogenous polypeptide
comprises enzymatic activity, e.g., protease activity. In
embodiments, the surface-exposed portion of the second exogenous
polypeptide enzymatically modifies, e.g., cleaves, the target. In
embodiments, the target comprises an autoantibody, the first
exogenous polypeptide comprises an autoantigen, and the second
exogenous polypeptide comprises a protease that cleaves the
autoantibody to produce a Fab portion and an Fc portion. In
embodiments, the enucleated red blood cell is capable of clearing
the target from a subject's body at a faster rate than an otherwise
similar enucleated red blood cell that lacks the second exogenous
polypeptide. In embodiments, the enucleated red blood cell is
complexed with the target or a reaction product of the second
exogenous protein acting on the target, e.g., during cleavage.
[0112] The present disclosure also provides, in certain aspects, an
enucleated erythroid cell comprising:
[0113] a first exogenous polypeptide comprising a transmembrane
domain and a surface-exposed autoantigen capable of binding an
autoantibody, and
[0114] a second exogenous polypeptide comprising a transmembrane
domain and a surface-exposed IdeS polypeptide.
[0115] The present disclosure also provides, in some aspects, a
polypeptide comprising a protease that can cleave an antibody,
e.g., an IdeS polypeptide, and a membrane anchor domain, e.g., a
transmembrane domain, e.g., type I or type II red blood cell
transmembrane domain. The disclosure also provides a nucleic acid
encoding said polypeptide.
[0116] The present disclosure also provides, in some aspects, a
nucleic acid comprising:
[0117] a first sequence encoding a protease that can cleave an
antibody, e.g., an IdeS polypeptide,
[0118] a second sequence encoding a membrane anchor domain, e.g., a
transmembrane domain, wherein the first and second sequences are
operatively linked to form a fusion protein; and
[0119] optionally, a promoter sequence that is active in an
erythroid cell.
[0120] The present disclosure also provides, in some aspects, a
nucleic acid composition comprising:
[0121] a first nucleic acid sequence encoding a first exogenous
polypeptide that interacts with a target, e.g., a first exogenous
polypeptide described herein,
[0122] a second nucleic acid sequence encoding a second exogenous
polypeptide that modifies the target, e.g., a second nucleic acid
sequence described herein and
[0123] optionally, a promoter sequence that is active in an
erythroid cell.
[0124] In embodiments, the first nucleic acid sequence and second
nucleic acid sequence are contiguous or are separate molecules
(e.g., admixed molecules or in separate containers). In
embodiments, the first nucleic acid sequence and second nucleic
acid sequence are part of the same open reading frame and have a
protease cleavage site situated therebetween. In embodiments, the
first nucleic acid is operatively linked to a first promoter and
the second nucleic acid is operatively linked to a second
promoter.
[0125] The disclosure provides, in some aspects, a kit
comprising:
[0126] (A) nucleic acids encoding: (A-i) a plurality of binding
moieties (e.g., antibody molecules, e.g., scFv domains), fused to
(A-ii) a membrane anchor domain, e.g., a transmembrane domain,
wherein (A-i) and (A-ii) are operatively linked to a nucleic acid
that directs expression in an erythroid cell; and
[0127] (B) nucleic acids encoding (B-i) a plurality of enzymes
(e.g., proteases), optionally fused to (B-ii) a membrane anchor
domain, e.g., a transmembrane domain, wherein (B-i) and (B-ii) are
operatively linked to nucleic acid that directs expression in an
erythroid cell.
[0128] The present disclosure provides, in some aspects, a method
of making a fragment of a target, e.g., a target polypeptide, e.g.,
a method of making a fragment of a target comprising a first target
domain, e.g., a method of making a variable region fragment, or a
method of making a constant region containing fragment, comprising
contacting the target polypeptide with an erythroid cell described
herein. In embodiments, the second exogenous polypeptide cleaves
the target to provide the fragment. In embodiments, the target
polypeptide is an antibody, e.g., an autoantibody. In embodiments,
the fragment of the target polypeptide binds an autoantigen without
activating an immune response and/or inflammation. In embodiments,
the contacting comprises administering the erythroid cell to a
subject that comprises the target polypeptide.
[0129] The present disclosure also provides, in certain aspects, a
method of making an inhibitor, e.g., a competitive inhibitor,
comprising, e.g., contacting a precursor of the inhibitor (a
target) with an erythroid cell described herein. In embodiments,
the second exogenous polypeptide interacts with the target, e.g.,
cleaves the target. In embodiments, the inhibitor is an antibody
fragment (e.g., a Fab fragment). In embodiments, the target is an
antibody which is cleaved to produce an inhibitor which is an
antibody fragment, e.g., Fab fragment. In embodiments, the
inhibitor binds an autoantigen without activating an immune
response and/or inflammation. In embodiments, the precursor of the
inhibitor is an antibody, e.g., an autoantibody. In embodiments,
the contacting comprises administering the erythroid cell to a
subject that comprises the precursor of the inhibitor.
[0130] The present disclosure also provides, in some aspects, a
method of converting or activating a target, e.g., a polypeptide,
e.g., converting a prodrug to a drug, comprising contacting the
polypeptide with an erythroid cell described herein. In
embodiments, the second exogenous polypeptide interacts with the
target (e.g., prodrug), e.g., cleaves the target. In embodiments,
the prodrug is an antibody, e.g., an autoantibody. In embodiments,
the drug is an antibody fragment, e.g., a Fab fragment. In
embodiments, the drug binds an autoantigen without activating an
immune response and/or inflammation. In embodiments, the contacting
comprises administering the erythroid cell to a subject that
comprises the polypeptide, e.g., prodrug.
[0131] The present disclosure also provides, in some aspects, a
method of converting an endogenous polypeptide from a first
activity state to a second activity state (e.g., from an inactive
state to an active state or an active state to an inactive state),
comprising contacting the endogenous polypeptide with an erythroid
cell described herein. In embodiments, the second exogenous
polypeptide interacts with the target, e.g., covalently modifies,
e.g., cleaves the target, or alters its ability to interact with,
e.g., bind, another molecule. In embodiments, the endogenous
polypeptide is an antibody, e.g., an autoantibody. In embodiments,
the contacting comprises administering the erythroid cell to a
subject that comprises the endogenous polypeptide.
[0132] The disclosure provides, in some aspects, a method of
reducing a level of a target (e.g., an antibody, e.g., an
autoantibody) in a subject, comprising administering to the subject
an erythroid cell described herein. In embodiments, the second
exogenous polypeptide interacts with the target, e.g., covalently
modifies, e.g., cleaves the target, or alters its ability to
interact with, e.g., bind, another molecule. The disclosure also
provides, in certain aspects, a method of generating an inhibitory
fragment of an antibody (e.g., a Fab fragment) in a subject,
comprising administering to the subject an erythrocyte cell
described herein. The disclosure provides, in addition, a method of
treating an autoimmune disease in a subject, comprising
administering to the subject an erythroid cell described
herein.
[0133] In embodiments, e.g., embodiments of any of the methods
described above, the erythroid cell comprises:
[0134] a first exogenous polypeptide that interacts with a target,
and
[0135] a second exogenous polypeptide that modifies the target;
[0136] wherein one or more of:
[0137] (a) the second exogenous polypeptide comprises a moiety that
cleaves an antibody, e.g., that cleaves at a hinge region, a CH2
region, or between a hinge and CH2 region, e.g., an IdeS
polypeptide;
[0138] (b) the second exogenous polypeptide comprises an enzyme
(e.g., a protease) that modifies, e.g., is specific, e.g., binds to
a site on target, binds (e.g., specifically) and modifies, e.g.,
covalently modifies, e.g., cleaves, or removes or attaches a moiety
to, the target, wherein the target is optionally an antibody or a
complement factor;
[0139] (c) the second exogenous polypeptide comprises a
polypeptide, e.g., an enzyme, e.g., a protease, that modifies the
secondary, tertiary, or quaternary structure of the target, and, in
embodiments, alters, e.g., decreases or increases, the ability of
the target to interact with another molecule, e.g., the first
exogenous polypeptide or a molecule other than the first exogenous
polypeptide, wherein optionally the target comprises an antibody,
or complement factor;
[0140] (d) the second exogenous polypeptide comprises a
polypeptide, e.g., an enzyme (e.g., a protease) that cleaves the
target, e.g., a polypeptide, between a first target domain and a
second target domain, e.g., a first target domain that binds a
first substrate and a second target domain that binds a second
substrate;
[0141] (e) the target is a polypeptide (e.g., an autoantibody or a
complement factor); a carbohydrate (e.g., a glycan), a lipid (e.g.,
a phospholipid), or a nucleic acid (e.g., DNA, or RNA);
[0142] (f) the first exogenous polypeptide binds a target, e.g., an
antibody, but does not cleave, and the second exogenous polypeptide
cleaves a bond e.g., a covalent bond, e.g., a covalent bond in the
antibody;
[0143] (g) the target comprises an antibody and the first exogenous
polypeptide binds the variable region of the antibody target;
[0144] (h) the target comprises an antibody and first exogenous
polypeptide binds the constant region of the antibody target;
[0145] (i) the first exogenous polypeptide has an affinity for the
target that is about 1-2 pM, 2-5 pM, 5-10 pM, 10-20 pM, 20-50 pM,
50-100 pM, 100-200 pM, 200-500 pM, 500-1000 pM, 1-2 nM, 2-5 nM,
5-10 nM, 10-20 nM, 20-50 nM, 50-100 nM, 100-200 nM, 200-500 nM,
500-1000 nM, 1-2 .mu.M, 2-5 .mu.M, 5-10 .mu.M, 10-20 .mu.M, 20-50
.mu.M, or 50-100 .mu.M;
[0146] (j) the second exogenous polypeptide has a K.sub.M for the
target of about 10.sup.-1-10.sup.-7M, 10.sup.-1-10.sup.-2M,
10.sup.-2-10.sup.-3M, 10.sup.-3-10.sup.4M, 10.sup.-4-10.sup.-5M,
10.sup.-5-10.sup.-6M, or 10.sup.-6-10.sup.-7M;
[0147] (k) a ratio of the K.sub.d of the first exogenous
polypeptide for the target (measured in M) divided by the KM of the
second exogenous polypeptide for the target (measured in M) is
about 1.times.10.sup.-9-2.times.10.sup.-9,
2.times.10.sup.-9-5.times.10.sup.-9,
5.times.10.sup.-9-1.times.10.sup.-8,
1.times.10.sup.-8-2.times.10.sup.-8,
2.times.10.sup.-8-5.times.10.sup.-8,
5.times.10.sup.-8-1.times.10.sup.-7,
1.times.10.sup.-7-2.times.10.sup.-7,
2.times.10.sup.-7-5.times.10.sup.-7,
5.times.10.sup.-7-1.times.10.sup.-6,
1.times.10.sup.-6-2.times.10.sup.-6,
2.times.10.sup.-6-5.times.10.sup.-6,
5.times.10.sup.-6-1.times.10.sup.-5,
1.times.10.sup.-5-2.times.10.sup.-5,
2.times.10.sup.-5-5.times.10.sup.-5,
5.times.10.sup.-5-1.times.10.sup.-4,
1.times.10.sup.-4-2.times.10.sup.-4,
2.times.10.sup.-4-5.times.10.sup.-4
5.times.10.sup.-4-1.times.10.sup.-3,
1.times.10.sup.-3-2.times.10.sup.-3,
2.times.10.sup.-3-5.times.10.sup.-3,
5.times.10.sup.-3-1.times.10.sup.-2,
1.times.10.sup.-2-2.times.10.sup.-2,
2.times.10.sup.-2-5.times.10.sup.-2,
5.times.10.sup.-2-1.times.10.sup.-1,
1.times.10.sup.-1-2.times.10.sup.-1,
2.times.10.sup.-1-5.times.10.sup.-1, or 5.times.10.sup.-1-1;
[0148] (l) the observed reaction rate of the second exogenous
polypeptide modifying the target is greater than the reaction rate
of an enucleated cell which is similar but which lacks the first
exogenous polypeptide under otherwise similar reaction
conditions;
[0149] (m) a ratio of the average number of the first exogenous
polypeptide on the erythroid cell to the average number of the
second exogenous polypeptide on the erythroid cell is about 50:1,
20:1, 10:1, 8:1, 6:1, 4:1, 2:1, 1:1, 1:2, 1:4, 1:6, 1:8, 1:10,
1:20, or 1:50;
[0150] (n) affinity of the first exogenous polypeptide for the
target is greater than the affinity of the first exogenous
polypeptide for the modified (e.g., cleaved) target;
[0151] (o) a therapeutically effective dose of the enucleated
erythroid cell is less than (e.g., less by 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 99.99%)
stoichiometry to the amount of target in a subject's peripheral
blood at the time of administration;
[0152] (p) the number of enucleated erythroid cells in an effective
dose, is less than (e.g., less by 10%, 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or 99.99%) the number
of targets, e.g., target molecules, in the subject's peripheral
blood at the time of administration;
[0153] (q) the number of second exogenous polypeptides comprised by
a preselected amount of enucleated erythroid cells, e.g., an
effective dose of enucleated erythroid cells, is less than (e.g.,
less by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%,
99.5%, 99.9%, or 99.99%) a reference value for targets, e.g., less
than the number of targets in the peripheral blood of the subject
at the time of administration;
[0154] (r) the number of first exogenous polypeptides comprised by
a preselected amount of enucleated erythroid cells, e.g., an
effective dose of enucleated erythroid cells, is less than (e.g.,
less by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%,
99.5%, 99.9%, or 99.99%) a reference value for targets, e.g., less
than the number of targets in the peripheral blood of the subject
at the time of administration;
[0155] (s) the number of first exogenous polypeptides and the
number of second exogenous polypeptides comprised by a preselected
amount of enucleated erythroid cells, e.g., an effective dose,
enucleated erythroid cells, is each less than (e.g., less by 10%,
20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%,
99.9%, or 99.99%) a reference value for targets, e.g., less than
the number of targets in the peripheral blood of the subject at the
time of administration;
[0156] (t) the second exogenous polypeptide modifies (e.g. cleaves)
the target with a KM of at least 10.sup.-1 M, 10.sup.-2 M,
10.sup.-3 M, 10.sup.-4 M, 10.sup.-5 M, 10.sup.-6 M, or
10.sup.-7M;
[0157] (u) the second exogenous polypeptide comprises a
chaperone;
[0158] (v) the first exogenous polypeptide comprises a
surface-exposed portion and the second exogenous polypeptide
comprises a surface exposed portion; or
[0159] (w) an effective amount of the enucleated erythroid cells is
less than (e.g., less by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%,
90%, 95%, 98%, 99%, 99.5%, 99.9%, or 99.99%) an effective amount of
otherwise similar enucleated erythroid cells that lack the second
exogenous polypeptide.
[0160] In some embodiments of any of the compositions and methods
described herein involving an exogenous polypeptide, e.g., a fusion
protein: [0161] i) at least 50, 60, 70, 80, 90, 95, or 99% of the
fusion proteins on the surface of the erythroid cell have an
identical sequence, [0162] ii) at least 50, 60, 70, 80, 90, 95, or
99% of the fusion protein have the same transmembrane region,
[0163] iii) the fusion protein does not include a full length
endogenous membrane protein, e.g., comprises a segment of a full
length endogenous membrane protein, which segment lacks at least 1,
2, 3, 4, 5, 10, 20, 50, 100, 200, or 500 amino acids of the full
length endogenous membrane protein; [0164] iv) at least 50, 60, 70,
80, 90, 95, or 99% of the fusion proteins do not differ from one
another by more than 1, 2, 3, 4, 5, 10, 20, or 50 amino acids,
[0165] v) the exogenous polypeptide lacks a sortase transfer
signature, [0166] vi) the exogenous polypeptide comprises a moiety
that is present on less than 1, 2, 3, 4, or 5 sequence distinct
fusion polypeptides; [0167] vii) the exogenous polypeptide is
present as a single fusion polypeptide; [0168] viii) the fusion
protein does not contain Gly-Gly at the junction of an endogenous
transmembrane protein and the moiety; [0169] ix) the fusion protein
does not contain Gly-Gly, or the fusion protein does not contain
Gly-Gly, or does not contain Gly-Gly in an extracellular region,
does not contain Gly-Gly in an extracellular region that is within
1, 2, 3, 4, 5, 10, 20, 50, or 100 amino acids of a transmembrane
segment; or a combination thereof.
[0170] The cell systems described herein may be used in combination
with another (one or more) autoimmune therapies or
anti-inflammatory agents. Such therapies and agents include a
steroid, e.g., a corticosteroid (e.g., methylprednisolone,
prednisolone, hydrocortisone, cortisone, dexamethasone,
betamethasone, triamcinolone), or an interferon, e.g., interferon
beta-1.
[0171] The disclosure contemplates all combinations of any one or
more of the foregoing aspects and/or embodiments, as well as
combinations with any one or more of the embodiments set forth in
the detailed description and examples.
[0172] Although methods and materials similar or equivalent to
those described herein can be used in the practice or testing of
the present invention, suitable methods and materials are described
below. All publications, patent applications, patents, and other
references (e.g., sequence database reference numbers) mentioned
herein are incorporated by reference in their entirety. For
example, all GenBank, Unigene, and Entrez sequences referred to
herein, e.g., in any Table herein, are incorporated by reference.
Unless otherwise specified, the sequence accession numbers
specified herein, including in any Table herein, refer to the
database entries current as of Jan. 11, 2016. When one gene or
protein references a plurality of sequence accession numbers, all
of the sequence variants are encompassed.
BRIEF DESCRIPTION OF THE DRAWINGS
[0173] FIG. 1 is a set of graphs showing results of a Raji
apoptosis assay measured through flow cytometry. Raji cells are
CFSE labeled and co-cultured with erythroid differentiated cells
that are untransduced (control) and transduced with single or
multiple TRAIL variants or co-cultured with two different singly
transduced cells. Percent apoptosis determined by percent of cells
that are Raji (CFSE+) and annexin V+. (Top) Flow cytometry plots of
CFSE and annexin V staining of various conditions. (Bottom) Graph
of percent apoptosis of the various conditions.
[0174] FIG. 2 is a bar graph showing the mean fluorescent intensity
from control erythroid cells (UNT) or IdeS-expressing erythroid
cells (IDES) labelled with an anti-Rabbit Fc fluorophore labeled
antibody, before or after a 5 hour incubation.
[0175] FIG. 3 is a Western blot showing intact heavy chain of
target antibodies or fragments of the heavy chain in supernatant
from control cells (UNT) or Ide-S expressing cells (IdeS-RCT).
Arrows indicate the heavy chain (Hc), heavy chain fragment
(Hc-fragment), and light chain (Lc).
[0176] FIG. 4 is a diagram of an erythroid cell comprising a first
exogenous polypeptide (white), a second exogenous polypeptide
(hatching), and a third exogenous polypeptide (close hatching)
wherein each exogenous polypeptide comprises a capture agent
capable of trapping a target, e.g., an unwanted target. The
erythroid cell can engage in dual trapping, where it uses more than
one exogenous polypeptide to bind a single or multiple soluble
factors.
[0177] FIG. 5 is a diagram of an erythroid cell comprising a first
exogenous polypeptide and a second exogenous polypeptide wherein
each exogenous polypeptide is capable of trapping an antibody,
e.g., unwanted antibody.
[0178] FIG. 6 is a diagram of an erythroid cell comprising a first
exogenous polypeptide that binds a target, e.g., an antibody, e.g.,
an unwanted antibody, and a second exogenous polypeptide that
modifies the target, e.g., cleaves the target. The second exogenous
polypeptide may comprise a protease such as IdeS.
[0179] FIG. 7 is a diagram of an erythroid cell comprising a first
exogenous polypeptide that binds a target, e.g., an unwanted
anti-drug antibody produced by a subject in reaction to treatment
with a drug, a second exogenous polypeptide that cleaves the
target, and an optional third exogenous polypeptide comprising a
therapeutic protein, e.g., an alternative to the drug against which
the subject produced anti-drug antibodies.
[0180] FIG. 8 is a diagram of an erythroid cell comprising a first
exogenous polypeptide with therapeutic activity, a second exogenous
polypeptide that inhibits the first exogenous polypeptide, and
optionally a third exogenous polypeptide that comprises a targeting
agent, e.g., an anti-CD20 antibody molecule.
[0181] FIG. 9 is a diagram of an erythroid cell comprising a first
exogenous polypeptide with a first targeting agent (e.g., an
anti-VCAM antibody molecule) and a second exogenous polypeptide
with a second targeting agent (e.g., an anti-E-selectin antibody
molecule). One or both of the first or second exogenous
polypeptides can be used to target the erythroid cell to an
inflamed tissue.
[0182] FIG. 10 is a diagram of an erythroid cell comprising an
antagonist and/or agonist.
[0183] FIG. 11 is a diagram or an erythroid cell comprising a
targeting agent (e.g., an anti-CD4 antibody molecule) and an
internal payload (e.g., IDO).
[0184] FIG. 12 is a diagram of an erythroid cell comprising a first
exogenous polypeptide comprising a targeting agent (e.g., an
anti-MAdCAM-1 antibody molecule) and a second exogenous polypeptide
comprising an agonist of a target (e.g., wherein the agonist
comprises IL10 and the target comprises IL10 Receptor).
[0185] FIG. 13 is a diagram of an erythroid cell comprising a first
exogenous polypeptide comprising a targeting agent (e.g., an
anti-BCMA antibody molecule) and a second exogenous polypeptide
comprising a capture agent.
[0186] FIG. 14 is a diagram of an erythroid cell comprising a first
exogenous polypeptide comprising a targeting agent (e.g., an AQP4
epitope) and a second exogenous polypeptide (e.g., TRAIL) that
promotes a given activity, e.g., apoptosis.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0187] As used herein, the term "antibody molecule" refers to a
protein, e.g., an immunoglobulin chain or fragment thereof,
comprising at least one immunoglobulin variable domain sequence.
The term "antibody molecule" encompasses antibodies and antibody
fragments. In an embodiment, an antibody molecule is a
multispecific antibody molecule, e.g., a bispecific antibody
molecule. Examples of antibody molecules include, but are not
limited to, Fab, Fab', F(ab')2, Fv fragments, scFv antibody
fragments, disulfide-linked Fvs (sdFv), a Fd fragment consisting of
the VH and CH1 domains, linear antibodies, single domain antibodies
such as sdAb (either VL or VH), camelid VHH domains, multi-specific
antibodies formed from antibody fragments such as a bivalent
fragment comprising two Fab fragments linked by a disulfide bridge
at the hinge region, an isolated epitope binding fragment of an
antibody, maxibodies, minibodies, nanobodies, intrabodies,
diabodies, triabodies, tetrabodies, v-NAR and bis-scFv.
[0188] As used herein, a "combination therapy" or "administered in
combination" means that two (or more) different agents or
treatments are administered to a subject as part of a treatment
regimen for a particular disease or condition. The treatment
regimen includes the doses and periodicity of administration of
each agent such that the effects of the separate agents on the
subject overlap. In some embodiments, the delivery of the two or
more agents is simultaneous or concurrent and the agents may be
co-formulated. In other embodiments, the two or more agents are not
co-formulated and are administered in a sequential manner as part
of a prescribed regimen. In some embodiments, administration of two
or more agents or treatments in combination is such that the
reduction in a symptom, or other parameter related to the disorder
is greater than what would be observed with one agent or treatment
delivered alone or in the absence of the other. The effect of the
two treatments can be partially additive, wholly additive, or
greater than additive (e.g., synergistic). Sequential or
substantially simultaneous administration of each therapeutic agent
can be effected by any appropriate route including, but not limited
to, oral routes, intravenous routes, intramuscular routes, and
direct absorption through mucous membrane tissues. The therapeutic
agents can be administered by the same route or by different
routes. For example, a first therapeutic agent of the combination
may be administered by intravenous injection while a second
therapeutic agent of the combination may be administered
orally.
[0189] The term "coordinated" or "coordinated manner" means that a
plurality of agents work together to provide a therapeutic benefit.
Types of coordinated activity include agent-additive,
agent-synergistic, multiplicative, independent function,
localization-based, proximity-dependent, scaffold-based,
multimer-based, and compensatory activity. In an embodiment the
level of therapeutic benefit conferred by a plurality of exogenous
polypeptides delivered in the same enucleated RBC is greater than
would be seen if each of the plurality of polypeptides were
delivered from different enucleated RBCs.
[0190] As used herein, "enucleated" refers to a cell that lacks a
nucleus, e.g., a cell that lost its nucleus through differentiation
into a mature red blood cell.
[0191] As used herein, the term "exogenous polypeptide" refers to a
polypeptide that is not produced by a wild-type cell of that type
or is present at a lower level in a wild-type cell than in a cell
containing the exogenous polypeptide. In some embodiments, an
exogenous polypeptide is a polypeptide encoded by a nucleic acid
that was introduced into the cell, which nucleic acid is optionally
not retained by the cell.
[0192] As used herein, the term "multimodal therapy" refers to a
therapy, e.g., an enucleated red blood cell therapy, that provides
a plurality (e.g., 2, 3, 4, or 5 or more) of exogenous agents
(e.g., polypeptides) that have a coordinated function (e.g.,
agent-additive, agent-synergistic, multiplicative, independent
function, localization-based, proximity-dependent, scaffold-based,
multimer-based, or compensatory activity).
[0193] As used herein, the term "pathway" or "biological pathway"
refers to a plurality of biological molecules, e.g., polypeptides,
that act together in a sequential manner. Examples of pathways
include signal transduction cascades and complement cascades. In
some embodiments, a pathway begins with detection of an
extracellular signal and ends with a change in transcription of a
target gene. In some embodiments, a pathway begins with detection
of a cytoplasmic signal and ends with a change in transcription of
a target gene. A pathway can be linear or branched. If branched, it
can have a plurality of inputs (converging), or a plurality of
outputs (diverging).
[0194] As used herein, a "proximity-dependent" molecule refers to a
first molecule that has a different, e.g., greater, activity when
in proximity with a second molecule than when alone. In some
embodiments, a pair of proximity-dependent ligands activates a
downstream factor more strongly when the ligands are in proximity
than when they are distant from each other. As used herein,
"receptor component" refers to a polypeptide that functions as a
receptor, by itself or as part of a complex. Thus a receptor
component encompasses a polypeptide receptor and a polypeptide that
functions as part of a receptor complex.
[0195] The term "synergy" or "synergistic" means a more than
additive effect of a combination of two or more agents (e.g.,
polypeptides that are part of an enucleated red blood cell)
compared to their individual effects. In certain embodiments,
synergistic activity is a more-than-additive effect of an
enucleated red blood cell comprising a first polypeptide and a
second polypeptide, compared to the effect of an enucleated red
blood cell comprising the first polypeptide and an enucleated red
blood cell comprising the second polypeptide. In some embodiments,
synergistic activity is present when a first agent produces a
detectable level of an output X, a second agent produces a
detectable level of the output X, and the first and second agents
together produce a more-than-additive level of the output X.
[0196] As used herein, the term "variant" of a polypeptide refers
to a polypeptide having at least one sequence difference compared
to that polypeptide, e.g., one or more substitutions, insertions,
or deletions. In some embodiments, the variant has at least 70%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to that
polypeptide. A variant includes a fragment. In some embodiments, a
fragment lacks up to 1, 2, 3, 4, 5, 10, 20, or 100 amino acids on
the N-terminus, C-terminus, or both (each independently), compared
to the full-length polypeptide.
[0197] Exemplary exogenous polypeptides and uses thereof.
[0198] In embodiments, the red blood cell therapeutics described
herein comprise one or more (e.g., 2, 3, 4, 5, 6, 10 or more)
different exogenous agents, e.g., exogenous polypeptides, lipids,
or small molecules. In some embodiments, a red blood cell
therapeutic comprises an exogenous fusion polypeptide comprising
two or more different proteins described herein. In some
embodiments, an enucleated red blood cell, e.g., a reticulocyte,
comprises two or more different exogenous polypeptides described
herein. In some embodiments, one or more (e.g., all) of the
exogenous polypeptides are human polypeptides or fragments or
variants thereof.
[0199] In some embodiments, the two or more polypeptides act on the
same target, and in other embodiments, they act on two or more
different targets. In some embodiments, the single target or
plurality of targets is chosen from an endogenous human protein or
a soluble factor (e.g., a polypeptide, small molecule, or cell-free
nucleic acid).
[0200] One or more of the exogenous proteins may have
post-translational modifications characteristic of eukaryotic
cells, e.g., mammalian cells, e.g., human cells. In some
embodiments, one or more (e.g., 2, 3, 4, 5, or more) of the
exogenous proteins are glycosylated, phosphorylated, or both. In
vitro detection of glycoproteins is routinely accomplished on
SDS-PAGE gels and Western Blots using a modification of Periodic
acid-Schiff (PAS) methods. Cellular localization of glycoproteins
may be accomplished utilizing lectin fluorescent conjugates known
in the art. Phosphorylation may be assessed by Western blot using
phospho-specific antibodies.
[0201] Post-translation modifications also include conjugation to a
hydrophobic group (e.g., myristoylation, palmitoylation,
isoprenylation, prenylation, or glypiation), conjugation to a
cofactor (e.g., lipoylation, flavin moiety (e.g., FMN or FAD), heme
C attachment, phosphopantetheinylation, or retinylidene Schiff base
formation), diphthamide formation, ethanolamine phosphoglycerol
attachment, hypusine formation, acylation (e.g. O-acylation,
N-acylation, or S-acylation), formylation, acetylation, alkylation
(e.g., methylation or ethylation), amidation, butyrylation,
gamma-carboxylation, malonylation, hydroxylation, iodination,
nucleotide addition such as ADP-ribosylation, oxidation, phosphate
ester (O-linked) or phosphoramidate (N-linked) formation, (e.g.,
phosphorylation or adenylylation), propionylation, pyroglutamate
formation, S-glutathionylation, S-nitrosylation, succinylation,
sulfation, ISGylation, SUMOylation, ubiquitination, Neddylation, or
a chemical modification of an amino acid (e.g., citrullination,
deamidation, eliminylation, or carbamylation), formation of a
disulfide bridge, racemization (e.g., of proline, serine, alanine,
or methionine). In embodiments, glycosylation includes the addition
of a glycosyl group to arginine, asparagine, cysteine,
hydroxylysine, serine, threonine, tyrosine, or tryptophan,
resulting in a glycoprotein. In embodiments, the glycosylation
comprises, e.g., O-linked glycosylation or N-linked
glycosylation.
[0202] In some embodiments, one or more of the exogenous
polypeptides is a fusion protein, e.g., is a fusion with an
endogenous red blood cell protein or fragment thereof, e.g., a
transmembrane protein, e.g., GPA or a transmembrane fragment
thereof. In some embodiments, one or more of the exogenous
polypeptides is fused with a domain that promotes dimerization or
multimerization, e.g., with a second fusion exogenous polypeptide,
which optionally comprises a dimerization domain. In some
embodiments, the dimerization domain comprises a portion of an
antibody molecule, e.g., an Fc domain or CH3 domain. In some
embodiments, the first and second dimerization domains comprise
knob-in-hole mutations (e.g., a T366Y knob and a Y407T hole) to
promote heterodimerization.
[0203] An exemplary human polypeptide, e.g., a human polypeptide
selected from any of Tables 1-6, includes:
[0204] a) a naturally occurring form of the human polypeptide,
e.g., a naturally occurring form of the human polypeptide that is
not associated with a disease state;
[0205] b) the human polypeptide having a sequence appearing in a
database, e.g., GenBank database, on Jan. 11, 2017, for example a
naturally occurring form of the human polypeptide that is not
associated with a disease state having a sequence appearing in a
database, e.g., GenBank database, on Jan. 11, 2017;
[0206] c) a human polypeptide having a sequence that differs by no
more than 1, 2, 3, 4, 5 or 10 amino acid residues from a sequence
of a) or b);
[0207] d) a human polypeptide having a sequence that differs at no
more than 1, 2, 3, 4, 5 or 10% its amino acids residues from a
sequence of a) or b);
[0208] e) a human polypeptide having a sequence that does not
differ substantially from a sequence of a) or b); or
[0209] f) a human polypeptide having a sequence of c), d), or e)
that does not differ substantially in a biological activity, e.g.,
an enzymatic activity (e.g., specificity or turnover) or binding
activity (e.g., binding specificity or affinity) from a human
polypeptide having the sequence of a) or b). Candidate peptides
under f) can be made and screened for similar activity as described
herein and would be equivalent hereunder if expressed in enucleated
RBCs as described herein).
[0210] In embodiments, an exogenous polypeptide comprises a human
polypeptide or fragment thereof, e.g., all or a fragment of a human
polypeptide of a), b), c), d), e), or f) of the preceding
paragraph. In an embodiment, the exogenous polypeptide comprises a
fusion polypeptide comprising all or a fragment of a human
polypeptide of a), b), c), d), e), or f) of the preceding paragraph
and additional amino acid sequence. In an embodiment the additional
amino acid sequence comprises all or a fragment of human
polypeptide of a), b), c), d), e), or f) of the preceding paragraph
for a different human polypeptide.
[0211] The invention contemplates that functional fragments or
variants thereof (e.g., a ligand-binding fragment or variant
thereof of the receptors listed in Table 1, 2 or 3 or the antigens
listed in Table 4) can be made and screened for similar activity as
described herein and would be equivalent hereunder if expressed in
enucleated RBCs as described herein).
[0212] In embodiments, the two or more exogenous agents (e.g.,
polypeptides) have related functions that are agent-additive,
agent-synergistic, multiplicative, independent function,
localization-based, proximity-dependent, scaffold-based,
multimer-based, or compensatory, as described herein. In some
embodiments, more than one of these descriptors applies to a given
RBC.
[0213] Agent-Additive Configurations
[0214] When two or more agents (e.g., polypeptides) are
agent-additive, the effect of the agents acting together is greater
than the effect of either agent acting alone. In an embodiment, two
agents have different (e.g., complementary) functions in the RBC
(e.g., on the RBC surface) and act together to have a stronger
effect (compared to either of the agents acting alone), e.g., a
higher binding affinity for the target, or a greater degree of
modulation of signal transduction by the target, e.g., a single
target. In some embodiments, two or more agents each bind to the
same target, e.g., to different epitopes within the same target
protein.
[0215] In an embodiment the agents associate with one another,
e.g., are members of a heterodimeric complex. In an embodiment, the
agents have greater avidity for a target when acting together than
when acting alone.
[0216] In some embodiments, the two or more agents enable tighter
binding to a target than either agent alone. In some embodiments, a
heterodimer of receptor components, e.g., cytokine receptor
components, e.g., interleukin receptor components, e.g., IL-1
receptor components, bind to a target, e.g., IL-1, with higher
affinity than either receptor component alone. Many signaling
molecules form heterodimers or heteromultimers on the cell surface
to bind to their ligand. Cytokine receptors, for example, can be
heterodimers or heteromultimers. For instance, IL-2 receptor
comprises three different molecules: IL2Ra, IL2Rb, and IL2Rg. The
IL-13 receptor is a heterodimer of IL13Ra and IL4R. The IL-23
receptor is a heterodimer of IL23R and IL12Rb1. The TNFa receptor
is, in embodiments, a heterodimer of TNFR1 and TNFR2. Without
wishing to be bound by theory, in some instances of disease, for
example in inflammatory diseases or sepsis, it may be desirous to
bind and clear a cytokine from circulation. In embodiments, this is
achieved by administering a red blood cell (e.g., a reticulocyte)
that expresses one or more (e.g., 2 or 3) of the receptors for the
target molecule simultaneously. For example, for the treatment of
psoriasis, a red blood cell (e.g., a reticulocyte) engineered to
express IL23R and IL12Rb1 binds and sequesters IL-23, an
inflammatory mediator of the disease. A table of cytokines and
their receptors is provided herein as Table 1. In some embodiments
the agents are antibody molecules that bind cytokines, e.g., one or
more cytokines of Table 1. In some embodiments, an enucleated RBC
comprises one or more (e.g., 2, 3, 4, 5, or more) cytokine receptor
subunits from Table 1 or cytokine-binding variants or fragments
thereof. In some embodiments, an enucleated RBC comprises two or
three (e.g., all) cytokine receptor subunits from a single row of
Table 1 or cytokine-binding variants or functional fragments
thereof. The cytokine receptors can be present on the surface of
the RBC. The expressed receptors typically have the wild type human
receptor sequence or a variant or fragment thereof that is able to
bind and sequester its target ligand. In embodiments, two or more
cytokine receptor subunits are linked to each other, e.g., as a
fusion protein.
[0217] In some embodiments the first polypeptide comprises a
cytokine or fragment or variant thereof, e.g., a cytokine of Table
1 or a fragment or variant thereof. In embodiments, the second
polypeptide comprises a second cytokine or fragment or variant
thereof, e.g., a cytokine of Table 1 or a fragment or variant
thereof. In embodiments, one or more (e.g., 2 or all) of the
cytokines are fused to transmembrane domains (e.g., a GPA
transmembrane domain or other transmembrane domain described
herein), e.g., such that the cytokine is on the surface of the
erythroid cell. In embodiments, the erythroid cell further
comprises a targeting moiety, e.g., a targeting moiety described in
FIG. 12 herein or the section herein entitled "Localization
Configurations." In some embodiments, an erythroid cell comprising
a plurality of cytokines is used to treat an autoimmune disease,
e.g., SLE.
TABLE-US-00001 TABLE 1 Cytokines and Receptors Name Cytokine
Receptor(s)(Da) and Form Interleukins IL-1-like IL-1.alpha. CD121a,
CDw121b IL-1.beta. CD121a, CDw121b IL-1RA CD121a IL-18
IL-18R.alpha., .beta. Common g chain (CD132) IL-2 CD25, 122, 132
IL-4 CD124, 213a13, 132 IL-7 CD127, 132 IL-9 IL-9R, CD132 IL-13
CD213a1, 213a2, IL-15 IL-15Ra, CD122, 132 Common b chain (CD131)
IL-3 CD123, CDw131 IL-5 CDw125, 131 Also related GM-CSF CD116,
CDw131 IL-6-like IL-6 CD126, 130 IL-11 IL-11Ra, CD130 Also related
G-CSF CD114 IL-12 CD212 LIF LIFR, CD130 OSM OSMR, CD130 IL-10-like
IL-10 CDw210 IL-20 IL-20R.alpha., .beta. Others IL-14 IL-14R IL-16
CD4 IL-17 CDw217 Interferons IFN-.alpha. CD118 IFN-.beta. CD118
IFN-.gamma. CDw119 TNF CD154 CD40 LT-.beta. LT.beta.R TNF-.alpha.
CD120a, b TNF-.beta. (LT-.alpha.) CD120a, b 4-1BBL CD137 (4-1BB)
APRIL BCMA, TACI CD70 CD27 CD153 CD30 CD178 CD95 (Fas) GITRL GITR
LIGHT LTbR, HVEM OX40L OX40 TALL-1 BCMA, TACI TRAIL TRAILR1-4 TWEAK
Apo3 TRANCE RANK, OPG TGF-.beta. TGF-.beta.1 TGF-.beta.R1
TGF-.beta.2 TGF-.beta.R2 TGF-.beta.3 TGF-.beta.R3 Miscellaneous
hematopoietins Epo EpoR Tpo TpoR Flt-3L Flt-3 SCF CD117 M-CSF CD115
MSP CDw136
[0218] In some embodiments the agents are different
antibody-binding molecules, e.g., Fc-binding molecules, for the
capture of antibodies in circulation. In embodiments, the agents
are non-competitive with one another to enable higher affinity
binding of individual antibodies or opsonized particles. For
example, in embodiments, one or more agent is chosen from protein
A, Fc receptor 1 (FcR1), FcR2a, FcR2b, FcR3, FcR4, FcRn (neonatal
Fc receptor) or an antibody-binding fragment or variant thereof. In
embodiments, one or more agent is chosen from an autoantigen or
autoantibody-binding fragments or variants thereof, or an
anti-idiotypic antibody molecule that binds autoantibodies. In
embodiments, the enucleated RBC is used to treat an autoimmune
disease.
[0219] An enucleated erythroid cell can comprise a first exogenous
polypeptide (e.g., an autoantigen) that interacts with a target
(e.g., an autoantibody) and a second exogenous polypeptide (e.g., a
protease, e.g., IdeS) that modifies the target. In embodiments, the
erythroid cell is administered to a subject, e.g., a subject having
an autoimmune disorder of Table 4.
[0220] In embodiments, an effective amount of the enucleated
erythroid cells comprising a first exogenous polypeptide and a
second exogenous polypeptide is less than (e.g., less by 10%, 20%,
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.9%, or
99.99%) an effective amount of otherwise similar enucleated
erythroid cells that lack the first exogenous polypeptide or lack
the second exogenous polypeptide. In embodiments, the preselected
amount is an effective dose or an in vitro effective amount of
enucleated erythroid cells. In embodiments, the preselected amount
(e.g., in vitro effective amount) is an amount that is effective in
an assay, e.g., to convert at least 10%, 20%, 30%, 405, 50%, 60%,
70%, 80%, or 90% of substrate into produce in a preselected amount
of time, e.g., 1, 2, 3, 4, 5, or 6 hours. In embodiments, the
preselected amount (e.g., in vitro effective amount) is effective
to cleave at least 50% of a target antibody in 5 hours. The assay
may measure, e.g., reduction in levels of soluble, unmodified
(e.g., non-cleaved) target in a solution.
[0221] In embodiments, the reference value for targets is the
number of targets in the peripheral blood of the subject at the
time of administration. In embodiments (e.g., embodiments involving
an in vitro effective amount of cells) the reference value for
targets is the number of targets in a reaction mixture for an
assay.
[0222] First Exogenous Polypeptide (e.g., Autoantigen)
[0223] The first exogenous polypeptide can bind a target. In
embodiments, the first exogenous polypeptide comprises a binding
domain that recognizes an antibody, e.g., an autoantibody. In
embodiments, the first domain comprises an autoantigen, e.g.,
comprises a full length protein or a fragment thereof that binds an
autoantibody. In embodiments, the first exogenous polypeptide
comprises an autoantigen from a protein of Table 4, e.g., comprises
a full length protein or a fragment thereof that binds an
autoantibody.
[0224] In embodiments, the first exogenous polypeptide comprises a
binding domain (e.g., an autoantigen capable of binding an
autoantibody) and a membrane anchor domain (e.g., a transmembrane
domain, e.g., type I or type II red blood cell transmembrane
domain). In embodiments, the membrane anchor domain is C-terminal
or N-terminal of the modifier (e.g., protease) domain. In
embodiments, the transmembrane domain comprises GPA or a
transmembrane portion thereof, e.g., as set out in SEQ ID NO: 9
herein or a transmembrane portion thereof, or a polypeptide having
at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity
to any of the foregoing. In embodiments, the GPA polypeptide is
C-terminal of the binding domain.
[0225] In embodiments, the first exogenous polypeptide comprises an
address moiety or targeting moiety described in WO2007030708, e.g.,
in pages 34-45 therein, which application is herein incorporated by
reference in its entirety.
[0226] Other examples of proteins that can be suitably adapted for
use as the first exogenous polypeptide include ligand binding
domains of receptors, such as where the target is the receptor
ligand. Conversely, the first exogenous polypeptide can comprise a
receptor ligand where the target is the receptor. A target ligand
can be a polypeptide or a small molecule ligand.
[0227] In a further embodiment, a first exogenous polypeptide may
comprise a domain derived from a polypeptide that has an
immunoglobulin-like fold, such as the 10th type III domain of human
fibronectin ("Fn3"). See U.S. Pat. Nos. 6,673,901; 6,462,189. Fn3
is small (about 95 residues), monomeric, soluble and stable. It
does not have disulfide bonds which permit improved stability in
reducing environments. The structure may be described as a
beta-sandwich similar to that of Ab VH domain except that Fn3 has
seven beta-strands instead of nine. There are three loops on each
end of Fn3; and the positions of three of these loops correspond to
those of CDR1, 2 and 3 of the VH domain. The 94 amino acid Fn3
sequence is:
TABLE-US-00002 (SEQ ID NO: 18)
VSDVPRDLEWAATPTSLLISWDAPAVTVRYYRITYGETGGNSPVQEFT
VPGSKSTATISGLKPGVDYTITGYAVTGRGDSPASSKPISINYRT
[0228] The amino acid positions of the CDR-like loops will be
defined as residues 23-30 (BC Loop), 52-56 (DE Loop) and 77-87 (FG
Loop). Accordingly, one or more of the CDR-like loops may be
modified or randomized, to generate a library of Fn3 binding
domains which may then be screened for binding to a desired address
binding site. See also PCT Publication WO0232925. Fn3 is an example
of a large subfamily of the immunoglobulin superfamily (IgSF). The
Fn3 family includes cell adhesion molecules, cell surface hormone
and cytokine receptors, chaperonin, and carbohydrate-binding
domains, all of which may also be adapted for use as binding
agents. Additionally, the structure of the DNA binding domains of
the transcription factor NF-kB is also closely related to the Fn3
fold and may also be adapted for use as a binding agent. Similarly,
serum albumin, such as human serum albumin contains an
immunoglobulin-like fold that can be adapted for use as a targeting
moiety.
[0229] In still other embodiments, the first exogenous polypeptide
can comprise an engineered polypeptide sequence that was selected,
e.g., synthetically evolved, based on its kinetics and selectivity
for binding to the address site. In embodiments, the sequence of
the first exogenous polypeptide is designed using a screen or
selection method, e.g., by phage display or yeast two-hybrid
screen.
[0230] In some embodiments, the first exogenous polypeptide
comprises a peptide ligand for a soluble receptor (and optionally
the target comprises a soluble receptor), a synthetic peptide that
binds a target, a complement regulatory domain (and optionally the
target comprises a complement factor), or a ligand for a cell
surface receptor (and optionally the target comprises the cell
surface receptor).
[0231] Second Exogenous Polypeptide (e.g., Protease)
[0232] In embodiments, the second exogenous polypeptide (which
modifies the target) is a factor set out in Table 5. In some
embodiments, the protease is a protease set out in Table 5. In
embodiments, the protease is a bacterial protease, a human
protease, or a plant protease, or a fragment or variant
thereof.
[0233] In embodiments, the second exogenous polypeptide (which
modifies the target) is a protease. Exemplary proteases include
those classified as Aminopeptidases; Dipeptidases;
Dipeptidyl-peptidases and tripeptidyl peptidases;
Peptidyl-dipeptidases; Serine-type carboxypeptidases;
Metallocarboxypeptidases; Cysteine-type carboxypeptidases;
Omegapeptidases; Serine proteinases; Cysteine proteinases; Aspartic
proteinases; Metalloproteinases; or Proteinases of unknown
mechanism.
[0234] Aminopeptidases include cytosol aminopeptidase (leucyl
aminopeptidase), membrane alanyl aminopeptidase, cystinyl
aminopeptidase, tripeptide aminopeptidase, prolyl aminopeptidase,
arginyl aminopeptidase, glutamyl aminopeptidase, x-pro
aminopeptidase, bacterial leucyl aminopeptidase, thermophilic
aminopeptidase, clostridial aminopeptidase, cytosol alanyl
aminopeptidase, lysyl aminopeptidase, x-trp aminopeptidase,
tryptophanyl aminopeptidase, methionyl aminopeptidase,
d-stereospecific aminopeptidase, and aminopeptidase. Dipeptidases
include x-his dipeptidase, x-arg dipeptidase, x-methyl-his
dipeptidase, cys-gly dipeptidase, glu-glu dipeptidase, pro-x
dipeptidase, x-pro dipeptidase, met-x dipeptidase,
non-stereospecific dipeptidase, cytosol non-specific dipeptidase,
membrane dipeptidase, and beta-ala-his dipeptidase.
Dipeptidyl-peptidases and tripeptidyl peptidases include
dipeptidyl-peptidase I, dipeptidyl-peptidase II, dipeptidyl
peptidase III, dipeptidyl-peptidase IV, dipeptidyl-dipeptidase,
tripeptidyl-peptidase I, and tripeptidyl-peptidase II.
Peptidyl-dipeptidases include peptidyl-dipeptidase A and
peptidyl-dipeptidase B. Serine-type carboxypeptidases include
lysosomal pro-x carboxypeptidase, serine-type D-ala-D-ala
carboxypeptidase, carboxypeptidase C, and carboxypeptidase D.
Metallocarboxypeptidases include carboxypeptidase A,
carboxypeptidase B, lysine(arginine) carboxypeptidase, gly-X
carboxypeptidase, alanine carboxypeptidase, muramoylpentapeptide
carboxypeptidase, carboxypeptidase H, glutamate carboxypeptidase,
carboxypeptidase M, muramoyltetrapeptide carboxypeptidase, zinc
D-ala-D-ala carboxypeptidase, carboxypeptidase A2, membrane pro-x
carboxypeptidase, tubulinyl-tyr carboxypeptidase, and
carboxypeptidase T. Omegapeptidases include
acylaminoacyl-peptidase, peptidyl-glycinamidase,
pyroglutamyl-peptidase I, beta-aspartyl-peptidase,
pyroglutamyl-peptidase II, n-formylmethionyl-peptidase,
pteroylpoly-[gamma]-glutamate carboxypeptidase, gamma-glu-X
carboxypeptidase, and acylmuramoyl-ala peptidase. Serine
proteinases include chymotrypsin, chymotrypsin C, metridin,
trypsin, thrombin, coagulation factor Xa, plasmin, enteropeptidase,
acrosin, alpha-lytic protease, glutamyl, endopeptidase, cathepsin
G, coagulation factor VIIa, coagulation factor IXa, cucumisi,
prolyl oligopeptidase, coagulation factor XIa, brachyurin, plasma
kallikrein, tissue kallikrein, pancreatic elastase, leukocyte
elastase, coagulation factor XIIa, chymase, complement component
clr55, complement component cls55, classical-complement pathway
c3/c5 convertase, complement factor I, complement factor D,
alternative-complement pathway c3/c5 convertase, cerevisin,
hypodermin C, lysyl endopeptidase, endopeptidase 1a, gamma-reni,
venombin AB, leucyl endopeptidase, tryptase, scutelarin, kexin,
subtilisin, oryzin, endopeptidase K, thermomycolin, thermitase,
endopeptidase SO, T-plasminogen activator, protein C, pancreatic
endopeptidase E, pancreatic elastase II, IGA-specific serine
endopeptidase, U-plasminogen, activator, venombin A, furin,
myeloblastin, semenogelase, granzyme A or cytotoxic T-lymphocyte
proteinase 1, granzyme B or cytotoxic T-lymphocyte proteinase 2,
streptogrisin A, treptogrisin B, glutamyl endopeptidase II,
oligopeptidase B, limulus clotting factor C, limulus clotting
factor, limulus clotting enzyme, omptin, repressor lexa, bacterial
leader peptidase I, and togavirin, flavirin. Cysteine proteinases
include cathepsin B, papain, ficin, chymopapain, asclepain,
clostripain, streptopain, actinide, cathepsin 1, cathepsin H,
calpain, cathepsin T, glycyl, endopeptidase, cancer procoagulant,
cathepsin S, picornain 3C, picornain 2A, caricain, ananain, stem
bromelain, fruit bromelain, legumain, histolysain, and interleukin
1-beta converting enzyme. Aspartic proteinases include pepsin A,
pepsin B, gastricsin, chymosin, cathepsin D, neopenthesin, renin,
retropepsin, pro-opiomelanocortin converting enzyme,
aspergillopepsin I, aspergillopepsin II, penicillopepsin,
rhizopuspepsin, endothiapepsin, mucoropepsin, candidapepsin,
saccharopepsin, rhodotorulapepsin, physaropepsin,
acrocylindropepsin, polyporopepsin, pycnoporopepsin,
scytalidopepsin A, scytalidopepsin B, xanthomonapepsin, cathepsin
E, barrierpepsin, bacterial leader peptidase I, pseudomonapepsin,
and plasmepsin. Metalloproteinases include atrolysin A, microbial
collagenase, leucolysin, interstitial collagenase, neprilysin,
envelysin, IgA-specific metalloendopeptidase, procollagen
N-endopeptidase, thimet oligopeptidase, neurolysin, stromelysin 1,
meprin A, procollagen C-endopeptidase, peptidyl-lys
metalloendopeptidase, astacin, stromelysin 2, matrilysin
gelatinase, aeromonolysin, pseudolysin, thermolysin, bacillolysin,
aureolysin, coccolysin, mycolysin, beta-lytic metalloendopeptidase,
peptidyl-asp metalloendopeptidase, neutrophil collagenase,
gelatinase B, leishmanolysin, saccharolysin, autolysin,
deuterolysin, serralysin, atrolysin B, atrolysin C, atroxase,
atrolysin E, atrolysin F, adamalysin, horrilysin, ruberlysin,
bothropasin, bothrolysin, ophiolysin, trimerelysin I, trimerelysin
II, mucrolysin, pitrilysin, insulysin, 0-syaloglycoprotein
endopeptidase, russellysin, mitochondrial, intermediate, peptidase,
dactylysin, nardilysin, magnolysin, meprin B, mitochondrial
processing peptidase, macrophage elastase, choriolysin, and
toxilysin. Proteinases of unknown mechanism include thermopsin and
multicatalytic endopeptidase complex. In embodiments, the second
exogenous polypeptide comprises a fragment or variant of any of the
foregoing.
[0235] In embodiments, the second exogenous polypeptide comprises
an IdeS polypeptide. In some embodiments, the IdeS polypeptide
comprises the sequence set out below as SEQ ID NO: 8 or a
proteolytically active fragment of the sequence of SEQ ID NO: 8
(e.g., a fragment of at least 100, 150, 200, 250, or 300 amino
acids) or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%,
96%, 97%, 98%, 99% identity to any of the foregoing. In some
embodiments involving nucleic acids, the nucleic acid encodes an
IdeS polypeptide having the sequence set out below as SEQ ID NO: 8,
or a proteolytically active fragment thereof, or a sequence having
at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity
to any of the foregoing.
[0236] Ides Polypeptide:
TABLE-US-00003 (SEQ ID NO: 8)
DSFSANQEIRYSEVTPYHVTSVWTKGVTPPAKFTQGEDVFHAPYVANQ
GWYDITKTFNGKDDLLCGAATAGNMLHWWFDQNKEKIEAYLKKHPDKQ
KIMFGDQELLDVRKVINTKGDQTNSELFNYFRDKAFPGLSARRIGVMP
DLVLDMFINGYYLNVYKTQTTDVNRTYQEKDRRGGIFDAVFTRGDQSK
LLTSRHDFKEKNLKEISDLIKKELTEGKALGLSHTYANVRINHVINLW
GADFDSNGNLKAIYVTDSDSNASIGMKKYFVGVNSAGKVAISAKEIKE
DNIGAQVLGLFTLSTGQDSWNQTN
[0237] In embodiments, the second exogenous polypeptide comprises a
modifier domain (e.g., a protease domain, e.g., an IdeS
polypeptide) and a membrane anchor domain (e.g., a transmembrane
domain, e.g., type I or type II red blood cell transmembrane
domain). In embodiments, the membrane anchor domain is C-terminal
or N-terminal of the modifier (e.g., protease) domain. In
embodiments, the transmembrane domain comprises GPA or a
transmembrane portion thereof. In embodiments, the GPA polypeptide
has a sequence of:
TABLE-US-00004 (SEQ ID NO: 9)
LSTTEVAMHTSTSSSVTKSYISSQTNDTHKRDTYAATPRAHEVSEISV
RTVYPPEEETGERVQLAHHFSEPEITLIIFGVMAGVIGTILLISYGIR
RLIKKSPSDVKPLPSPDTDVPLSSVEIENPETSDQ
[0238] or a transmembrane portion thereof, or a polypeptide having
at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identity
to any of the foregoing. In embodiments, the GPA polypeptide is
C-terminal of the modifier (e.g., protease) domain.
[0239] In some embodiments, a linker is disposed between the IdeS
polypeptide and the transmembrane polypeptide, e.g., a
glycine-serine linker, e.g., a linker comprising a sequence of
GGSGGSGG (SEQ ID NO: 10) and/or GGGSGGGS (SEQ ID NO: 11).
[0240] In some embodiments, the exogenous polypeptide, e.g., the
second exogenous polypeptide, e.g., a protease, e.g., IdeS
polypeptide, comprises a leader sequence, e.g., a GPA leader
sequence, e.g., MYGKIIFVLLLSEIVSISA (SEQ ID NO: 12).
[0241] In some embodiments, the exogenous polypeptide, e.g., the
second exogenous polypeptide further comprises a tag, e.g., an HA
tag or a FLAG tag.
[0242] In some embodiments, the protease (e.g., immunoglobulin
degrading enzyme, e.g., immunoglobulin-G degrading enzyme, e.g.,
IdeS) cleaves an immunoglobulin at a hinge region, a CH2 region, or
between a hinge and CH2 region. In embodiments, the protease
cleaves an immunoglobulin at one of the sequences below, e.g.,
between the two italicized glycines or the italicized alanine and
glycine in the sequences below.
[0243] Human IgG1 Hinge/CH2 Sequence CPPCPAPELLGGPSVF (SEQ ID NO:
13)
[0244] Human IgG2 Hinge/CH2 Sequence CPPCPAPPVAGPSVF (SEQ ID NO:
14)
[0245] Human IgG3 Hinge/CH2 Sequence CPRCPAPELLGGPSVF (SEQ ID NO:
15)
[0246] Human IgG4 Hinge/CH2 Sequence AHHAQAPEFLGGPSVF (SEQ ID NO:
16)
[0247] In embodiments, the protease (e.g., a bacterial protease)
cleaves IgG, e.g., IdeS or IgA protease.
[0248] In embodiments, the protease (e.g., a papain family
protease, e.g., papain) cleaves an immunoglobulin between the Fc
and Fab regions, e.g., a histidine-threonine bond between positions
224 and 225 of the heavy chain and/or a glutamic acid-leucine bond
between positions 233 and 234 of the heavy chain.
[0249] In embodiments, the protease or other modifier acts on a
target listed in Table 5 or Table 6.
[0250] In embodiments, the protease or other modifier acts on
(e.g., inactivates or inhibits) a TNF molecule (such as TNF-alpha),
e.g., in a subject having sepsis, e.g. bacterial sepsis. For
instance, the first exogenous polypeptide can comprise a TNF-alpha
binding moiety such as an anti-TNF-alpha antibody, and the second
exogenous polypeptide can comprise a protease that cleaves
TNF-alpha, e.g., MT1-MMP, MMP12, tryptase, MT2-MMP, elastase, MMPI,
chymotrypsin, or trypsin, or active variants or fragments
thereof.
[0251] In embodiments, the second exogenous polypeptide comprises a
catalytic moiety described in WO2007030708, e.g., in pages 45-46
therein, which application is herein incorporated by reference in
its entirety.
[0252] The second exogenous polypeptide can comprise a moiety
capable of acting on a target to induce a chemical change, thereby
modulate its activity, e.g., a moiety capable of catalyzing a
reaction within a target. The second exogenous polypeptide can
comprise a naturally occurring enzyme, an active (e.g.,
catalytically active) fragment thereof, or an engineered enzyme,
e.g., a protein engineered to have an enzymatic activity, such as a
protein designed to contain a serine protease active motif. A
catalytic domain of a second exogenous polypeptide may comprise the
arrangement of amino acids that are effective to induce the desired
chemical change in the target. They may be N-terminal or C-terminal
truncated versions of natural enzymes, mutated versions, zymogens,
or complete globular domains.
[0253] The second exogenous polypeptide can comprise an
enzymatically active site that alone is promiscuous, binding with a
cleavage site it recognizes on many different biomolecules, and may
have relatively poor reaction kinetics. In embodiments, the first
exogenous polypeptide supplies or improves specificity by
increasing the local concentration of target near the second
exogenous polypeptide.
[0254] The second exogenous polypeptide can, in embodiments, modify
the target so that it is recognized and acted upon by another
enzyme (e.g., an enzyme that is already present in a subject). In
an embodiment, the second exogenous polypeptide comprises a moiety
that alters the structure of the target so that its activity is
inhibited or upregulated. Many naturally occurring enzymes activate
other enzymes, and these can be exploited in accordance with the
compositions and methods described herein.
[0255] The second exogenous polypeptide can comprise a protease, a
glycosidase, a lipase, or other hydrolases, an amidase (e.g.,
N-acetylmuramoyl-L-alanine amidase, PGRP-L amidase), or other
enzymatic activity, including isomerases, transferases (including
kinases), lyases, oxidoreductases, oxidases, aldolases, ketolases,
glycosidases, transferases and the like. In embodiments, the second
exogenous polypeptide comprises human lysozyme, a functional
portion of a human lysozyme, a human PGRP-L, a functional portion
of a human PGRP-L, a phospholipase A2, a functional portion of a
phospholipase A2, or a matrix metalloproteinase (MMP) extracellular
enzyme such as MMP-2 (gelatinase A) or MMP-9 (gelatinase B).
[0256] In embodiments, the second exogenous polypeptide is a serine
proteinase, e.g., of the chymotrypsin family which includes the
mammalian enzymes such as chymotrypsin, trypsin or elastase or
kallikrein, or the substilisin family which includes the bacterial
enzymes such as subtilisin. The general three-dimensional structure
is different in the two families but they have the same active site
geometry and catalysis proceeds via the same mechanism. The serine
proteinases exhibit different substrate specificities which are
related to amino acid substitutions in the various enzyme subsites
interacting with the substrate residues. Three residues which form
the catalytic triad are important in the catalytic process: His-57,
Asp-102 and Ser-195 (chymotrypsinogen numbering).
[0257] In embodiments, the second exogenous polypeptide is a
cysteine proteinase which includes the plant proteases such as
papain, actinidin or bromelain, several mammalian lysosomal
cathepsins, the cytosolic calpains (calcium-activated), and several
parasitic proteases (e.g., Trypanosoma, Schistosoma). Papain is the
archetype and the best studied member of the family. Like the
serine proteinases, catalysis proceeds through the formation of a
covalent intermediate and involves a cysteine and a histidine
residue. The essential Cys-25 and His-159 (papain numbering) play
the same role as Ser-195 and His-57 respectively. The nucleophile
is a thiolate ion rather than a hydroxyl group. The thiolate ion is
stabilized through the formation of an ion pair with neighboring
imidazolium group of His-159. The attacking nucleophile is the
thiolate-imidazolium ion pair in both steps and then a water
molecule is not required.
[0258] In embodiments, the second exogenous polypeptide is an
aspartic proteinase, most of which belong to the pepsin family. The
pepsin family includes digestive enzymes such as pepsin and
chymosin as well as lysosomal cathepsins D, processing enzymes such
as renin, and certain fungal proteases (penicillopepsin,
rhizopuspepsin, endothiapepsin). A second family comprises viral
proteinases such as the protease from the AIDS vims (HIV) also
called retropepsin. In contrast to serine and cysteine proteinases,
catalysis by aspartic proteinases does not involve a covalent
intermediate, though a tetrahedral intermediate exists. The
nucleophilic attack is achieved by two simultaneous proton
transfers: one from a water molecule to the dyad of the two
carboxyl groups and a second one from the dyad to the carbonyl
oxygen of the substrate with the concurrent CO--NH bond cleavage.
This general acid-base catalysis, which may be called a "push-pull"
mechanism leads to the formation of a non-covalent neutral
tetrahedral intermediate.
[0259] In embodiments, the second exogenous polypeptide is a
metalloproteinase, which can be found in bacteria, fungi as well as
in higher organisms. They differ widely in their sequences and
their structures but the great majority of enzymes contain a zinc
(Zn) atom which is catalytically active. In some cases, zinc may be
replaced by another metal such as cobalt or nickel without loss of
the activity. Bacterial thermolysin has been well characterized and
its crystallographic structure indicates that zinc is bound by two
histidines and one glutamic acid. Many enzymes contain the sequence
HEXXH, which provides two histidine ligands for the zinc whereas
the third ligand is either a glutamic acid (thermolysin,
neprilysin, alanyl aminopeptidase) or a histidine (astacin). Other
families exhibit a distinct mode of binding of the Zn atom. The
catalytic mechanism leads to the formation of a non-covalent
tetrahedral intermediate after the attack of a zinc-bound water
molecule on the carbonyl group of the scissile bond. This
intermediate is further decomposed by transfer of the glutamic acid
proton to the leaving group.
[0260] In embodiments, the second exogenous polypeptide comprises
an isomerase (e.g., an isomerase that breaks and forms chemical
bonds or catalyzes a conformational change). In embodiments, the
isomerase is a racemase (e.g., amino acid racemase), epimerase,
cis-trans isomerase, intramolecular oxidoreductase, intramolecular
transferase, or intramolecular lyase.
[0261] In embodiments, the second exogenous protease comprises a
chaperone, or an active variant or fragment thereof. For instance,
the chaperone can be a general chaperone (e.g., GRP78/BiP, GRP94,
GRP170), a lectin chaperone (e.g., calnexin or calreticulin), a
non-classical molecular chaperone (e.g., HSP47 or ERp29), a folding
chaperone (e.g., PDI, PPI, or ERp57), a bacterial or archaeal
chaperone (e.g., Hsp60, GroEL/GroES complex, Hsp70, DnaK, Hsp90,
HtpG, Hsp100, Clp family (e.g., ClpA and ClpX), Hsp104). In
embodiments, the enucleated erythrocyte comprises a co-chaperone,
or an active variant or fragment thereof, e.g., immunophilin, Stil,
p50 (Cdc37), or Ahal. In embodiments, the molecular chaperone is a
chaperonin.
[0262] Candidates for the second exogenous protein (which modifies
a target) can be screened based on their activity. Depending on the
specific activity of each molecule being tested, an assay
appropriate for that molecule can be used. For example, if the
second exogenous protein is a protease, the assay used to screen
the protease can be an assay to detect cleavage products generated
by the protease, e.g., a chromatography or gel electrophoresis
based assay.
[0263] In an example, the second exogenous polypeptide may have
kinase activity. An assay for kinase activity could measure the
amount of phosphate that is covalently incorporated into the target
of interest. For example, the phosphate incorporated into the
target of interest could be a radioisotope of phosphate that can be
quantitated by measuring the emission of radiation using a
scintillation counter.
[0264] Targets (e.g., Antibodies or Complement Pathway Factors) and
Indications
[0265] In embodiments, the target is a target listed in Table 5 or
Table 6.
[0266] In embodiments, the target is an immune checkpoint molecule
selected from PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1,
CEACAM-3 and/or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160,
2B4 or TGF beta. In embodiments, the target is an inhibitory ligand
listed in Table 3, and the first exogenous polypeptide optionally
comprises a binding domain from a corresponding target receptor of
Table 3. In some embodiments, the target is a target receptor of
Table 3, and the first exogenous polypeptide optionally comprises a
binding domain from a corresponding inhibitory ligand of Table 3.
In some embodiments, the second exogenous polypeptide comprises a
protease that cleaves an immune checkpoint molecule, e.g., trypsin.
In embodiments, e.g., for treating an autoimmune disease, a T cell
is inhibited, e.g., by contacting its receptor (e.g., a receptor of
Table 3) with an inhibitory ligand of Table 3.
[0267] In embodiments, the target is an antibody e.g., a human
antibody. The antibody can be, e.g., an autoantibody. The antibody
can be, e.g., IgA, IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3, or IgG4),
or IgM. In embodiments, the target is an autoantibody that binds an
autoantigen of Table 4. In embodiments, the target is an anti-AQP4
autoantibody, wherein the first exogenous polypeptide binds an
anti-AQP4 autoantibody and the second polypeptide cleaves the
autoantibody (e.g., comprises an IdeS polypeptide), e.g., for
treating Neuromyelitis optica. In embodiments, the target is an
anti-PLA2R autoantibody, wherein the first exogenous polypeptide
binds an anti-PLA2R autoantibody and the second polypeptide cleaves
the autoantibody (e.g., comprises an IdeS polypeptide), e.g., for
treating membranous nephropathy.
[0268] In embodiments, the target is a complement factor, e.g., a
factor that acts in the classical complement pathway or the
alternative complement pathway. In embodiments, the complement
factor is a pro-protein or an activated (e.g., cleaved) protein. In
embodiments, the complement factor comprises C1, C2a, C4b, C3, C3a,
C3b, C5, C5a, C5b, C6, C7, C8, or C9. In embodiments, the second
exogenous polypeptide cleaves the complement factor. In
embodiments, the second exogenous polypeptide activates the
complement factor, e.g., to promote an immune response. For
instance, the second exogenous polypeptide may comprise a
complement control protein and/or complement activation family
protein such as Factor H or Factor I (which promote C3b cleavage),
or a fragment or variant thereof. In embodiments, the second
exogenous polypeptide inactivates the complement factor, e.g., by
cleaving it to yield one or more inactive fragments, e.g., to
reduce an unwanted immune response, e.g., to treat an autoimmune or
inflammatory disease. In embodiments, the first exogenous
polypeptide binds a complement factor (e.g., binds complement
factor C3b) and the second exogenous polypeptide cleaves the
complement factor, e.g., to iC3b. For instance, the first exogenous
polypeptide could comprise Factor H or CR1 or a fragment or variant
thereof and the second exogenous polypeptide could comprise Factor
I or a fragment or variant thereof.
[0269] Engineered erythroid cells described herein can also be used
to treat a subject that has antibodies against a drug (e.g., see
FIG. 7). The erythroid cell can reduce levels of anti-drug
antibodies in a subject, and can optionally further comprise a
therapeutic protein that treats the disease. For instance, the
erythroid cell comprises a first exogenous polypeptide that binds a
target, e.g., wherein the target is an anti-drug antibody. The
erythroid cell can further comprise a second exogenous polypeptide
(e.g., IdeS, or a fragment or variant thereof) that inactivates,
e.g., cleaves the target. The erythroid cell may optionally further
comprise a third exogenous polypeptide, e.g., a therapeutic protein
that treats the same disease as the prior therapeutic to which the
subject developed anti-drug antibodies, e.g., a therapeutic protein
which is the same as or different from the prior therapeutic to
which the subject developed anti-drug antibodies. In embodiments,
the subject comprises anti-drug antibodies against erythropoietin,
an anti-TNF antibody molecule (adalimumab or infliximab), an
anti-EGFR antibody (e.g., cetuximab), an anti-CD20 antibody
molecule, insulin, an anti-alpha4 integrin antibody molecule (e.g.,
natalizumab), or an interferon e.g. IFN.beta.1a or IFN.beta.1b. In
embodiments, the first polypeptide comprises an anti-MAdCAM-1
antibody molecule and the second polypeptide comprises LysC or LysN
(which can cleave MAdCAM-1 at 1 or more (e.g., 2, 3, or 4 sites),
or a fragment or variant thereof having protease activity, e.g.,
wherein the target tissue is inflamed gut or liver tissue. In such
methods of treatment, the patient may be tested for the presence of
anti-drug antibodies, e.g., for the presence of neutralizing
anti-drug antibodies, before, during and/or after administration of
the engineered erythroid cells described herein.
[0270] Agent-Synergistic Configurations
[0271] When two or more agents (e.g., polypeptides) are
agent-synergistic, the agents act on two or more different targets
within a single pathway. In an embodiment, the action of the two or
more agents together is greater than the action of any of the
individual agents. For example, the first and second polypeptides
are ligands for cellular receptors that signal to the same
downstream target. For example, the first exogenous polypeptide
comprises a ligand for a first target cellular receptor, and the
second exogenous polypeptide comprises a ligand for a second target
cellular receptor, e.g., which first and second target cellular
receptors signal to the same downstream target. In embodiments, the
first exogenous polypeptide acts on the first target and the second
exogenous polypeptide acts on the second target simultaneously,
e.g., there is some temporal overlap in binding of the first
exogenous polypeptide to the first target and binding of the second
exogenous polypeptide to the second target. In some embodiments the
simultaneous action generates a synergistic response of greater
magnitude than would be expected when either target is acted on
alone or in isolation.
[0272] In an embodiment, the first and second polypeptides are
ligands for a first cellular receptor and a second cellular
receptor that mediates apoptosis. In an embodiment the agents
comprise two or more TRAIL receptor ligands, e.g., wild-type or
mutant TRAIL polypeptides, or antibody molecules that bind TRAIL
receptors, and induce apoptosis in a target cell, e.g., an
autoreactive T cell. In some embodiments, a RBC comprising TRAIL
receptor ligands further comprises a targeting moiety, e.g., a
targeting moiety described herein. In an embodiment the first
target and the second target interacts with the same substrate,
e.g., a substrate protein. In an embodiment the first target and
the second target interact with different substrates.
[0273] TRAIL (TNF-related apoptosis inducing ligand) is a member of
the TNF family that induces apoptosis. TRAIL has at least two
receptors, TRAIL R1 and TRAIL R2. TRAIL receptor agonists, e.g.,
mutants of TRAIL that bind one or more of the receptors, or
antibody molecules that bind one or both of TRAIL R1 or TRAIL R2
(see, e.g. Gasparian et al., Apoptosis 2009 Jun 14(6), Buchsbaum et
al. Future Oncol 2007 Aug. 3(4)), have been developed as a clinical
therapy for a wide range of cancers. Clinical trials of TRAIL
receptor agonists have failed for, among other reasons, the fact
that many primary cancers are not sensitive to signaling through a
single receptor but rather require engagement of both receptors to
induce cytotoxicity (Marconi et al., Cell Death and Disease (2013)
4, e863). In one embodiment the agents expressed on the engineered
blood cell are single receptor-specific TRAIL agonists that, in
combination, enable the cell to engage and agonize both TRAIL
receptors simultaneously, thus leading to a synergistic induction
of apoptosis of a target cell. Thus, in some embodiments, the
enucleated red blood cell (e.g., reticulocyte) comprises on its
surface a first polypeptide that binds TRAIL R1 and a second
polypeptide that binds TRAIL R2. In embodiments, each polypeptide
has a Kd for TRAIL R1 or TRAIL R2 that is 2, 3, 4, 5, 10, 20, 50,
100, 200, or 500-fold stronger than the Kd for the other receptor.
While not wishing to be bound by theory, in some embodiments an
enucleated red blood cell comprising a TRAIL R1-specific ligand and
a TRAIL R2-specific ligand promote better heterodimerization of
TRAIL R1 and TRAIL R2 than an enucleated red blood cell comprising
a ligand that binds to TRAIL R1 and TRAIL R2 with about the same
affinity.
[0274] In some embodiments, one, two, or more of the exogenous
polypeptides are members of the TNF superfamily. In some
embodiments, the exogenous polypeptides bind to one or both of
death receptors DR4 (TRAIL-R1) and DR5 (TRAIL-R2). In some
embodiments, the exogenous polypeptides bind to one or more of
TNFRSF10A/TRAILR1, TNFRSF10B/TRAILR2, TNFRSF10C/TRAILR3,
TNFRSF10D/TRAILR4, or TNFRSF11B/OPG. In some embodiments, the
exogenous polypeptides activate one or more of MAPK8/JNK, caspase
8, and caspase 3.
[0275] In some embodiments, a TRAIL polypeptide is a TRAIL agonist
having a sequence of any of SEQ ID NOS: 1-5 herein, or a sequence
with at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%
identity thereto. Sequence identity is measured, e.g., by BLAST
(Basic Local Alignment Search Tool). SEQ ID Nos. 1-5 are further
described in Mohr et al. BMC Cancer (2015) 15:494), which is herein
incorporated by reference in its entirety.
TABLE-US-00005 Soluble TRAIL variant DR4-1 SEQ ID NO: 1
MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDK
YSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTS
EETISTVQEKQQNISPLVRERGPQRVAAHITGTRRRSNTLSSPNSKNE
KALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRF
QEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLY
SIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG Soluble TRAIL variant
DR4-2 SEQ ID NO: 2 MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDK
YSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTS
EETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNE
KALGRKINSWESSRRGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRF
QEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLY
SIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG Soluble TRAIL variant
DR4-3 SEQ ID NO: 3 MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDK
YSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTS
EETISTVQEKQQNISPLVRERGPQRVAAHITGTRRRSNTLSSPNSKNE
KALGIKINSWESSRRGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRF
QEEIKENTKNDKQMVQYIYKYTDYPDPILLMKSARNSCWSKDAEYGLY
SIYQGGIFELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG Soluble TRAIL variant
DR5-1 SEQ ID NO: 4 MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDK
YSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTS
EETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNE
KALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRF
QEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLY
SIYQGGIFELKENDRIFVSVTNEHLIDMHHEASFFGAFLVG Soluble TRAIL variant
DR5-2 SEQ ID NO: 5 MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELKQMQDK
YSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQLRQLVRKMILRTS
EETISTVQEKQQNISPLVRERGPQRVAAHITGTRGRSNTLSSPNSKNE
KALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYSQTYFRF
QERIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLY
SIYQGGIFELKENDRIFVSVTNEHLIDMHHEASFFGAFLVG
[0276] All combinations of the TRAIL receptor ligands are
envisioned. In some embodiments, the first and second agents
comprise SEQ ID NO: 1 and SEQ ID NO: 2; SEQ ID NO: 1 and SEQ ID NO:
3; SEQ ID NO: 1 and SEQ ID NO: 4; SEQ ID NO: 1 and SEQ ID NO: 5;
SEQ ID NO: 2 and SEQ ID NO: 3; SEQ ID NO: 2 and SEQ ID NO: 4; SEQ
ID NO: 2 and SEQ ID NO: 5; SEQ ID NO: 3 and SEQ ID NO: 4; SEQ ID
NO: 3 and SEQ ID NO: 5; or SEQ ID NO: 4 and SEQ ID NO: 5, or a
fragment or variant of any of the foregoing.
[0277] In some embodiments, the TRAIL receptor ligand comprises an
antibody molecule. In embodiments, the antibody molecule recognizes
one or both of TRAIL R1 and TRAIL R2. The antibody molecule may be,
e.g., Mapatumumab (human anti-DR4 mAb), Tigatuzumab (humanized
anti-DR5 mAb), Lexatumumab (human anti-DR5 mAb), Conatumumab (human
anti-DR5 mAb), or Apomab (human anti-DR5 mAb), or a fragment or
variant thereof, e.g., a variant having the same CDRs as any of the
aforementioned antibodies, e.g., by the Chothia or Kabat
definitions. In some embodiments, the enucleated red blood cell
(e.g., reticulocyte) comprises two or more (e.g., three, four,
five, or more) different antibody molecules that bind a TRAIL
receptor. In some embodiments, the enucleated red blood cell (e.g.,
reticulocyte) comprises at least one antibody molecule that binds a
TRAIL receptor and at least one TRAIL polypeptide.
[0278] In some embodiments, the agents are modulators of a
multi-step pathway that act agent-synergistically by targeting
upstream and downstream steps of the pathway, e.g., simultaneously.
In one embodiment, the target pathway is the complement cascade,
which has several parallel activation paths (classical,
alternative, lectin pathways) and multiple auto-catalytic enzymes
to enhance its potency in responding to infection and leading to
membrane-attack complex formation (see, e.g. Bu et al., Clin Dev
Immunol. 2012; 2012: 370426). Inhibitors of complement cascade
exist, both as synthetic antibodies and peptides, that act on
different levels of the cascade, e.g. anti-05 (eculizumab) and
anti-C3 (compstatin), and as endogenous proteins and polypeptides,
e.g. CFH, CFI, CD46/MCP, CD55/DAF, CD59, and CR1. Non-enzymatic
complement inhibitors include include Efb (extracellular
fibrinogen-binding protein, e.g., from S. aureus, which binds C3b),
Ehp (binds C3d and inhibits C3 conversion), SCIN (staphylococcal
complement inhibitor, which stabilize C3 convertase into a
non-functional state), CHIPS (chemotaxis inhibitory protein of S.
aureus, which antagonizes C5a receptor), and SSL-7 (Staphyloccal
superantigen-like protein-7, which binds C5). Enzymatic complement
inhibitors include LysC, LysN, PaE, PaAP, 56 kDa protease from
Serratia marcescens, C5a peptidases, Plasmin, SpeB, PrtH,
Staphylokinase, and MMPs (see Table 1). The exogenous polypeptide
can also comprise a fragment or variant of any of the complement
inhibitors described herein. To treat diseases of complement
over-activation it can be beneficial to inhibit the complement
cascade, and it can be especially beneficial to intervene at two or
more stages of the cascade to obtain a more potent inhibition. In
some embodiments the agents expressed on the engineered red blood
cell (e.g., reticulocyte) are inhibitors of the complement cascade
that act on different levels of the cascade, e.g. CFI and MCP, CD55
and CD59, or anti-C3 and anti-CS.
[0279] In some embodiments, the enucleated RBC comprises two or
more agents that are anti-inflammatory. For instance, the agents
can comprise an anti-TNFa antibody molecule (e.g., humira), an
anti-IgE antibody molecule (e.g., Xolair), or a molecule that
inhibits T cells (e.g., IL-10 or PD-L1; also see Table 1 and Table
3), or any combination thereof. In embodiments, one or more agents
capture a factor such as a cytokine. In embodiments, one or more
agents modulate immune cells.
[0280] Multiplicative Configurations
[0281] When two or more agents (e.g., polypeptides) are
multiplicative, a first agent acts on a first molecule that is part
of a first pathway and a second agent acts on a second molecule
that is part of a second pathway, which pathways act in concert
toward a desired response.
[0282] In some embodiments, the desired response is inactivation
(e.g., anergy) of an inappropriately activated immune cell, e.g., T
cell. In some embodiments, the agents inhibit multiple T cell
activation pathways. In embodiments, one or more (e.g., 2, 3, 4, or
5 or more) T cell inhibition ligands comprise an inhibiting variant
(e.g., fragment) of a ligand of Table 2. In embodiments, one or
more (e.g., 2, 3, 4, or 5 or more) T cell inhibition ligands
comprise an inhibitory antibody molecule that binds a target
receptor of Table 2 or a T-cell inhibiting variant (e.g., fragment)
thereof. In embodiments, these proteins signal through
complementary activation pathways. In some embodiments the ligands
are inhibitory cytokines, interferons, or TNF family members. In
some embodiments the agents are combinations of the above classes
of molecules. The agents can be derived from endogenous ligands or
antibody molecules to the target receptors.
TABLE-US-00006 TABLE 2 T cell activation Activating Ligand Target
Receptor on T cell B7-H2 (e.g., Accession Number ICOS, CD28 (e.g.,
Accession NP_056074.1) Number NP_006130.1) B7-1 (e.g., Accession
Number CD28 (e.g., Accession Number NP_005182.1) NP_006130.1) B7-2
(e.g., Accession Number CD28 (e.g., Accession Number AAA86473)
NP_006130.1) CD70 (e.g., Accession Number CD27 (e.g., Accession
Number NP_001243.1) NP_001233.1) LIGHT (e.g., Accession Number HVEM
(e.g., Accession Number NP_003798.2) AAQ89238.1) HVEM (e.g.,
Accession Number LIGHT (e.g., Accession Number AAQ89238.1)
NP_003798.2) CD40L (e.g., Accession Number CD40 (e.g., Accession
Number BAA06599.1) NP_001241.1) 4-1BBL (e.g., Accession Number
4-1BB (e.g., Accession NP_003802.1) NP_001552.2) OX40L (e.g.,
Accession Number OX40 (e.g., Accession Number NP_003317.1)
NP_003318.1) TL1A (e.g., Accession Number DR3 (e.g., Accession
Number NP_005109.2) NP_683866.1) GITRL (e.g., Accession Number GITR
(e.g., Accession Number NP_005083.2) NP_004186.1) CD30L (e.g.,
Accession Number CD30 (e.g., Accession Number NP_001235.1),
NP_001234.3) TIM4 (e.g., Accession Number TIM1 (e.g., Accession
Number NP_612388.2) NP_036338.2) SLAM (e.g., Accession Number SLAM
(e.g., Accession Number AAK77968.1) AAK77968.1) CD48 (e.g.,
Accession Number CD2 (e.g., Accession Number CAG33293.1)
NP_001315538.1) CD58 (e.g., Accession Number CD2 (e.g., Accession
Number CAG33220.1) NP_001315538.1) CD155 (e.g., Accession Number
CD226 (e.g., Accession Number NP_001129240.1) NP_006557.2) CD112
(e.g., Accession Number CD226 (e.g., Accession Number
NP_001036189.1) NP_006557.2) CD137L (e.g., Accession Number CD137
(e.g., Accession NP_003802.1) NP_001552.2)
[0283] In some embodiments, an anti-IL6 or TNFa antibody molecule
comprises a sequence of either of SEQ ID NO: 6 or 7 herein, or a
sequence with at least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or
99% identity thereto.
TABLE-US-00007 Anti-IL6 scFv SEQ ID NO: 6
EVQLVESGGGLVQPGGSLRLSCAASGFNFNDYFMNWVRQAPGKGLEWV
AQMRNKNYQYGTYYAESLEGRFTISRDDSKNSLYLQMNSLKTEDTAVY
YCARESYYGFTSYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSPS
SLSASVGDRVTITCQASQDIGISLSWYQQKPGKAPKLLIYNANNLADG
VPSRFSGSGSGTDFTLTISSLQPEDFATYYCLQHNSAPYTFGQGTKLE IKR
Anti-TNF.alpha. scFv SEQ ID NO: 7
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWV
SAITWNSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC
AKVSYLSTASSLDYWGQGTLVTVSSGGGGSGGGGSGGGGSDIQMTQSP
SSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQS
GVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKV EIK
[0284] In some embodiments, a T cell (e.g., autoreactive T cell) is
inhibited using a ligand or receptor of Table 3, or a fragment or
variant thereof. For instance, the first and second exogenous
polypeptide can comprise a T cell inhibiting ligand (e.g., an
inhibitory ligand of Table 3 or a fragment or variant thereof) and
an agent that inhibits activation of a T cell (e.g., through a
receptor of Table 2). In some embodiments, the agent that inhibits
a T cell is an inhibitory ligand of Table 3, or a fragment or
variant thereof. In some embodiments, the agent is an antibody
molecule that binds a target receptor of Table 3, or a fragment or
variant thereof.
TABLE-US-00008 TABLE 3 T cell inhibition Inhibitory Ligand Target
Receptor on T cell B7-1 CTLA4, B7H1 B7-2 CTLA4 B7DC PD1 B7H1 PD1,
B7-1 HVEM CD160, BTLA COLLAGEN LAIR1 GALECTIN9 TIM3 CD48, TIM4
TIM4R CD48 2B4 CD155, CD112, CD113 TIGIT PDL1 PD1
[0285] Without wishing to be bound by theory, in some embodiments
the objective is to dampen an immune response by inhibiting T cell
activation. Thus, in embodiments, an engineered red blood cell
(e.g., reticulocyte) targets multiple T cell inhibitory pathways in
combination (e.g., as described in Table 3), e.g., using ligands or
antibody molecules, or both, co-expressed on an engineered red
blood cell. In some embodiments, the agents comprise a receptor or
antibody molecule that captures inflammatory cytokines, e.g., to
prevent additional activation of the target T cell. For example,
the agents may comprise an agonist antibody molecule to the
receptor CTLA-4 and an antibody molecule against TNFalpha.
[0286] In some embodiments the objective is to activate or to
inhibit T cells. To ensure that T cells are preferentially targeted
over other immune cells that may also express either activating or
inhibitory receptors as described herein, one of the agents on the
red blood cell (e.g., reticulocyte) may comprise a targeting
moiety, e.g., an antibody molecule that binds the T cell receptor
(TCR) or another T cell marker. Targeting moieties are described in
more detail in the section entitled "Localization configurations"
herein. In some embodiments, a specific T cell subtype or clone may
be enhanced (a T cell with anti-tumor specificity) or inhibited (a
T cell with autoimmune reactivity). In some embodiments, one or
more of the agents on the red blood cell (e.g., reticulocyte) is a
peptide-MHC molecule that will selectively bind to a T cell
receptor in an antigen-specific manner.
[0287] In the context of inducing a tolerogenic response, the first
and second exogenous polypeptides comprise, in some embodiments, an
antigen and an inhibitory ligand so that antigen presentation by
APCs induces tolerance instead of immunity. In embodiments, the
antigen is an autoimmune antigen, e.g., an autoantigen of Table 4,
or an allergen, e.g., an allergen from plants such as pollen, an
allergen from food, or an allergen from an animal. In embodiments,
the inhibitory ligand comprises FasL, B7, PD1, PD-L1, CTLA4, TIM3,
CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), LAG3, VISTA,
BTLA, TIGIT, LAIR1, CD160, 2B4 and TGF beta, or a functional
variant (e.g., fragment) thereof. In some embodiments, the agent
that inhibits a T cell is an inhibitory ligand of Table 3, or an
inhibitory fragment or variant thereof. In some embodiments, the
agent that inhibits a T cell is an antibody molecule that binds a
target receptor of Table 3, or an inhibitory fragment or variant
thereof. In some embodiments, the red blood cell (e.g.,
reticulocyte) further comprises an antibody molecule that binds a
dendritic cell receptor, e.g. CD205 or CD206. In embodiments this
agent promotes uptake by antigen presenting cells. In some
embodiments, CD205 induces tolerogenic antigen presentation in the
absence of additional co-stimulatory signals.
[0288] In some embodiments, the first and second exogenous
polypeptides comprise, in some embodiments, an antigen and a
costimulatory molecule, e.g., wherein the erythroid cell can act as
an APC.
TABLE-US-00009 TABLE 4 Autoimmune diseases and antigens Disease
Antigen Acute rheumatic fever cross reactive antibodies to cardiac
muscle alopecia areata Trychohyalin, keratin 16 ANCA-associated
vasculitis Neutrophil cytoplasmic antigen, proteinase 3,
myeloperodixase, bacterial permiability increasing factor
autoimmune gastritis H, K adenosine triphosphatase autoimmune
hemolytic Rh blood group antigens, I antigen anemia autoimmune
hepatitis nuclear protein, liver-kidney microsome type 1, liver
cytosol type 1 autoimmune myocarditis cardiac myosin Autoimmune
thyroiditis Thyroid peroxidase, thyroglobulin, thyroid-stimulating
hormone receptor Autoimmune uveitis Retinal arrestin (S-antigen)
dermatomyositis Mi2 ATPase diabetes (type 1) Pancreatic beta cell
antigen goodpasture's syndrome Noncollagenous domain of basement
membrane collagen type IV Graves' disease Thyroid stimulating
hormone receptor Guillain-Barre syndrome Neurofascin-186,
gliomedin, nodal adhesion molecueles Hypoglycemia Insulin receptor
idiopathic Platelet integrin GpIIb, GpIIIa thrombocytopenic purpura
Insulin resistant diabetes Insulin receptor Membranous nephritis
Phospholipase A2 mixed essential rheumatoid factor IgG complexes
cryoglobulinemia multiple sclerosis Myelin basic protein,
proteolipid protein, myelin oligodendrocyte glycoprotein myasthenia
gravis Acetylcholine receptor Myasthenia gravis - MUSC Muscarinic
receptor pemphigus/pemphigoid Epidermal cadherin pernicious anemia
intrinsic factor (Gastric) polymyositis nuclear and nucleolar
antigen primary biliary cirrhosis neutrophil nuclear antigen,
mitochondrial multienzyme complex psoriasis PSO p27 rheumatoid
arthritis rheumatoid factor IgG complexes, synovial joint antigen,
citrullinated protein, carbamylated protein scleroderma/systemic
Scl-86, nucleolar scleroderma antigen sclerosis Sjogren's syndrome
SS-B, Lupus La protein systemic lupus DNA, histones, ribosomes,
snRNP, scRNP erythematosus vitiligo VIT-90, VIT-75, VIT-40
Wegener's granulomatosis neutrophil nuclear antigen
Antiphospholipid Beta-2 glycoprotein 1 syndrome (APS) &
catastrophic APS Chemotherapy induced Neuronal antigens peripheral
neuropathy Thrombotic ADAMTS13 thrombocytopenic purpura Atypical
hemolytic uremic Complement factor H syndrome
[0289] In some embodiments, an enucleated red blood cell (e.g.,
reticulocyte) comprising a first exogenous polypeptide and a second
exogenous polypeptide is administered to a subject having a first
target and a second target. In embodiments, the first exogenous
polypeptide acts on (e.g., binds) the first target and the second
exogenous polypeptide acts on the second target. Optionally, the
enucleated red blood cell comprises a third exogenous polypeptide
and the patient comprises a third target. In embodiments, the third
exogenous polypeptide acts on the third target.
[0290] In some embodiments an erythroid cell comprises a first
exogenous polypeptide which is an agonist or antagonist of a first
target in a first pathway, and further comprises a second exogenous
polypeptide which is an agonist or antagonist of a second target in
a second pathway, wherein the first and second pathways act in
concert toward a desired response. The first and second exogenous
polypeptides can both be agonists; can both be antagonists; or one
can be an agonist and the other can be an antagonist. In some
embodiments (e.g., see FIG. 10), the first exogenous polypeptide
comprises a surface-exposed anti-CD28 antibody molecule and the
second exogenous polypeptide comprises a surface-exposed anti-CTLA4
antibody molecule, e.g., for treating systemic lupus erythematosus
or rheumatoid arthritis. In some embodiments, the first exogenous
polypeptide comprises a surface-exposed anti-ICOSL antagonist and
the second exogenous polypeptide comprises a surface-exposed
anti-CD40 antagonist, e.g., wherein the target is an antigen
presenting cell, e.g., for treating systemic lupus erythematosus.
In some embodiments, the target cell or tissue comprises immune
cells or diseased tissue. In some embodiments, one or more of the
exogenous polypeptides are immune checkpoint agonists or
antagonists. In some embodiments, the erythroid cell further
comprises a targeting agent.
[0291] Independent Function Configurations
[0292] When two or more agents (e.g., polypeptides) have an
independent function relationship, the agents have two distinct
(e.g., complementary) functions. For example, a first agent binds a
first target and the second agent binds a second target. The
patient may lack the first or second target. Optionally, the first
and second agents are in different pathways.
[0293] In sepsis, tumor lysis syndrome, and other conditions marked
by a cytokine storm, the damage is driven by a diverse mix of
inflammatory cytokines. Existing monotherapies against one cytokine
are often insufficient to treat these acute conditions. Furthermore
it can sometimes be impossible to measure the driver of the
cytokine storm in time to prevent clinical damage. In an
embodiment, the first and second peptides are molecules (e.g.,
antibody molecules) that bind two different cytokines. In some
embodiments the agents bind and neutralize different cytokines and
thus the engineered red cell product provides multifaceted
protection from cytokine storm.
[0294] In embodiments the cytokines comprise interleukins, e.g.,
IL-1, IL02, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11,
IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21,
IL-22, IL-23, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31,
IL-32, IL-33, IL-35, or IL-36. In some embodiments, the cytokine is
a cytokine of Table 1 or a fragment or variant thereof. In some
embodiments, the first cytokine is TNFa and the second is an
interleukin, e.g., IL-6, or a fragment or variant of any of the
foregoing. In some embodiments, the agents comprise anti-TNFa,
anti-IL-6, or anti-IFNg antibody molecules, or any combination
thereof, or a fragment or variant of any of the foregoing.
[0295] In some embodiments the agents comprise a plurality of
antibody molecules, which plurality of antibody molecules binds a
plurality of common bacterial, fungal, or viral pathogens. In
embodiments, the antibodies are generated from human B cells, e.g.,
a combinatorial library of pooled human B cells. In embodiments,
the poly-antibody engineered red blood cells serve as an
alternative, replacement, or supplement to intravenous
immunoglobulin (IVIG). IVIG is in embodiments administered to
immunodeficient patients, e.g., patients who lack the capacity to
produce endogenous antibodies. The red blood cell (e.g.,
reticulocyte) would provide long-lasting protection and be free
from the risks associated with plasma donation.
[0296] In some embodiments, an enucleated red blood cell (e.g.,
reticulocyte) comprising a first exogenous polypeptide and a second
exogenous polypeptide is administered to a subject having a first
target but not a second target, or wherein the patient is not known
to have a first target or second target. In embodiments, the first
exogenous polypeptide acts on (e.g., binds) the first target and
the second exogenous polypeptide remains substantially unbound.
Optionally, the enucleated red blood cell comprises a third
exogenous polypeptide and the patient lacks a third target, or is
not known to have the third target. In some embodiments, the
enucleated red blood cell comprises a plurality of exogenous
polypeptides, and the patient does not have, or is not known to
have, targets for one or a subset of the plurality of exogenous
polypeptides.
[0297] An example of an independent function configuration is shown
in FIG. 4. The erythroid cell of FIG. 4 comprises a first exogenous
polypeptide (white), a second exogenous polypeptide (hatching), and
an optional third exogenous polypeptide (close hatching). The first
exogenous polypeptide can bind a first target, e.g., cytokine A,
and the second exogenous polypeptide can independently bind a
second target, e.g., cytokine B. This engineered erythroid cell
trap and clear both cytokines if both are present in the subject.
If only one of the cytokines is present in the subject, the
engineered erythroid cell can clear that cytokine. In embodiments,
one or more (e.g., two or all) of the exogenous polypeptides
comprise antibody molecules, e.g., scFvs, and optionally further
comprise a transmembrane domain. In embodiments, the targets
comprise a plurality of cytokines, chemokines, or a combination
thereof. As examples, the first exogenous polypeptide could bind
TNF-alpha and the second exogenous polypeptide could bind IL6,
e.g., to treat rheumatoid arthritis; the first exogenous
polypeptide could bind IL4 and the second exogenous polypeptide
could bind IL13, e.g., to treat asthma and/or ectopic dermatitis;
the first exogenous polypeptide could bind IL33 and the second
exogenous polypeptide could bind IL15, e.g., to treat celiac
disease or inflammatory bowel disease; or the first exogenous
polypeptide could bind IL35 and the second exogenous polypeptide
could bind IL25, e.g., to treat allergy. In embodiments, the first
target and second target are unwanted antibodies, e.g., endogenous
antibodies, e.g., autoimmune antibodies or anti-drug antibodies
(e.g., as illustrated in FIG. 5). For instance, the first exogenous
polypeptide could bind MOG and the second exogenous polypeptide
could bind MBP, e.g., to treat multiple sclerosis.
[0298] Localization Configurations
[0299] When two or more agents (e.g., polypeptides) have a
localization relationship, a first agent localizes the RBC to a
site of action that enhances the activity of the second or other
agent or agents compared to their activity when not localized to
the site of action (e.g., by binding of the first agent to its
target, there is an increase in the local concentration of the
second agent in the area of its target). In some embodiments one
agent serves to target the red blood cell (e.g., reticulocyte) to a
site of action and one or more agents have a therapeutic effect. In
an embodiment, binding of the first agent increases the activity of
an entity, e.g., polypeptide, bound by the second agent. In an
embodiment, the first agent binds to a substrate or product of the
entity, e.g., polypeptide, bound by the second agent. The agent
that localizes the RBC may be, e.g., a ligand for a receptor on a
target cell, or an antibody that binds a cell surface molecule on a
target cell.
[0300] As another example, a RBC comprises a targeting agent that
binds to inflamed vasculature and also comprises an
anti-inflammatory molecule. In some embodiments, the targeting
agent binds an inflammatory integrin, e.g., a avB3 integrin or an
addressin such as MADCAM1. In some embodiments, the targeting agent
comprises a lymphocyte homing receptor (e.g., CD34 or GLYCAM-1) or
integrin-binding portion or variant thereof. In some embodiments,
the anti-inflammatory molecule comprises an anti-inflammatory
cytokine (e.g., IL-1 receptor antagonist, IL-4, IL-6, IL-10, IL-11,
and IL-13), an inhibitor of TNF (e.g., an antibody molecule such as
Enbrel), an inhibitor of a pro-inflammatory cytokine, an inhibitor
against alpha4beta7 integrin (e.g., an antibody molecule), a
colony-stimulating factor, a peptide growth factor, Monocyte
Locomotion Inhibitory Factor (MLIF), Cortistatin, or an inhibitor
of immune cell activation (e.g. PDL1 or another molecule described
in Table 3 or antibody molecule thereto), or an anti-inflammatory
variant (e.g., fragment) thereof.
[0301] In some embodiments, the erythroid cell targets an
endothelial cell, e.g., in an inflamed tissue. As shown in FIG. 9,
the cell can comprise one or more targeting agents, e.g., exogenous
polypeptides that bind surface markers of inflamed tissue. The
targeting agent can be an exogenous polypeptide comprising, e.g.,
an anti-VCAM antibody molecule or an anti-E-selectin antibody
molecule. In embodiments, an erythroid cell comprises two targeting
agents, which may increase the specificity and/or affinity and/or
avidity of the erythroid cell binding to its target, compared to an
otherwise similar erythroid cell comprising only one of the
targeting agents. In embodiments, the targeting moieties comprise:
a surface exposed anti-VCAM antibody molecule and a surface exposed
anti-E-selectin antibody molecule; a surface exposed alpha4Beta1
integrin or fragment or variant thereof and a surface exposed
anti-E-selectin antibody molecule; or a surface exposed alphavbeta2
integrin or fragment or variant thereof and a surface exposed
anti-E-selectin antibody molecule. The erythroid cell optionally
further comprises an exogenous polypeptide with therapeutic
activity, e.g., anti-inflammatory activity. The exogenous
polypeptide with therapeutic activity can comprise an enzyme,
capture reagent, agonist, or antagonist.
[0302] The erythroid cell can also target, in embodiments, immune
cells or diseased tissue. In embodiments, the targeting moiety
comprises a receptor or a fragment or variant thereof. In
embodiments, the targeting moiety comprises an antibody molecule
such as an scFv.
[0303] In some embodiments, e.g., for killing autoreactive cells,
the first exogenous polypeptide comprises a targeting moiety, e.g.,
a surface-exposed anti-CD20 antibody molecule that can target the
erythroid cell to a B cell or a targeting moiety specific to
autoreactive cells, and the second exogenous polypeptide comprises
a moiety that can kill or anergize the T cell, e.g., a TRAIL
ligand. The erythroid cell can further comprise an inhibitor of the
second exogenous polypeptide, e.g., as illustrated in FIG. 8.
[0304] The first exogenous polypeptide can comprise a targeting
agent and the second exogenous polypeptide can comprise an enzyme
(e.g., FIG. 11). For example, in some embodiments, e.g., for
treating an autoimmune disease, the erythroid cell comprises a
first polypeptide comprising a targeting agent that binds an immune
cell and a second polypeptide that inhibits (e.g., kills, induces
anergy in, inhibits growth of) the immune cell. For instance, the
targeting agent can comprise an anti-CD4 antibody which binds CD4
on the surface of a T cell, e.g., an autoreactive T cell. The
second polypeptide can comprise an enzyme which can be
surface-exposed or intracellular, e.g., intracellular and not
membrane associated. The enzyme may be IDO or a fragment or variant
thereof, which depletes tryptophan and can induce anergy in the
autoreactive T cell, or ADA or a fragment or variant thereof. The
enzyme may be a protease. In embodiments, the first polypeptide
comprises an anti-MAdCAM-1 antibody molecule and the second
polypeptide comprises LysC or LysN (which can cleave MAdCAM-1 at 1
or more (e.g., 2, 3, or 4 sites), e.g., wherein the target tissue
is inflamed gut or liver tissue. In some embodiments, the targeting
agent comprises an anti-IL13R antibody molecule or IL13 or a
fragment or variant thereof, and the payload comprises IDO or ADA,
e.g., for treating an allergy or asthma. In some embodiments, the
targeting agent comprises an anti-IL23R antibody molecule or IL23
or a fragment or variant thereof, and the payload comprises IDO or
ADA, e.g., for treating psoriasis.
[0305] In embodiments, the target cell is an immune cell, e.g., a T
cell, e.g., a helper T cell, and/or a disease cell. The targeting
agent may comprise an antibody molecule, e.g., an scFv.
[0306] The first exogenous polypeptide can comprise a targeting
agent and the second exogenous polypeptide can comprise an agonist
of a target (see, e.g., FIG. 12). For instance, in some
embodiments, the first exogenous polypeptide comprises an
anti-MAdCAM-1 antibody molecule, e.g., which can bind MAdCAM-1,
e.g., on inflamed tissue. The second exogenous polypeptide may
comprise an anti-inflammatory molecule, e.g., IL 10 or a fragment
or variant thereof. In some embodiments, the target cell is a
pathogenic immune cell. In embodiments, the targeting agent
comprises a receptor or fragment or variant thereof, an antibody
molecule, a ligand or fragment or variant thereof, a cytokine or
fragment or variant thereof. In embodiments, the second exogenous
polypeptide comprises an attenuator, an activator, a cell-killing
agent, or a cytotoxic molecule (e.g., a small molecule, protein,
RNA e.g., antisense RNA, or TLR ligand). In embodiments, the second
exogenous polypeptide is intracellular, e.g., not membrane
associated, and in some embodiments, the second exogenous
polypeptide is surface-exposed. In embodiments, the targeting
moiety binds a hepatocyte and the second exogenous polypeptide
comprises TGF beta or a fragment or variant thereof, e.g., for
treating non-alcoholic fatty liver disease (NAFLD) or non-alcoholic
steatohepatitis (NASH).
[0307] The erythroid cell can comprise a targeting agent and a
capture agent (e.g., FIG. 13). For example, the first exogenous
polypeptide can comprise a targeting agent that binds a plasma
cell, e.g., an anti-BCMA antibody molecule. The second exogenous
polypeptide may capture cytokines, e.g., may comprise TACI or a
fragment or variant thereof which can capture BLyS (also called
BAFF) and/or APRIL. In embodiments, the second exogenous
polypeptide binds its target in a way that prevents the target from
interacting with an endogenous receptor, e.g., binds the target at
a moiety that overlaps with the receptor binding site. In
embodiments, the targeting moiety binds a receptor at the site of
disease, e.g., binds an integrin. In embodiments, the target cell
is a pathogenic immune cell or diseased tissue. In embodiments, the
targeting agent comprises a ligand or a cytokine or fragment or
variant thereof, or an antibody molecule, e.g., an scFv. In
embodiments, the capture agent comprises a receptor or fragment or
variant thereof, or an antibody molecule, e.g., an scFv. In
embodiments, the ligand is an unwanted cytokine or chemokine.
[0308] A targeting agent can direct an erythroid cell to a
particular sub-type of cell, e.g., an immune cell with a particular
antigen specificity, e.g., an autoreactive immune cell. The cell
can further comprise a second exogenous polypeptide that promotes a
given activity or pathway in the target cell, e.g., can attenuate,
activate, or induce cell death. For instance, FIG. 14 depicts an
erythroid cell comprising a first exogenous polypeptide comprising
an AQP4 epitope that can bind an anti-AQP4 B-cell receptor (BCR) on
an autoreactive plasma cell; other exemplary first exogenous
polypeptides include ACHR or insulin or fragments or variants
thereof. The erythroid cell can further comprise a second exogenous
polypeptide that inhibits (e.g., kills, induces anergy in, inhibits
growth of) the immune cell. For instance, the second exogenous
polypeptide can comprise a cell-killing agent, e.g., a
pro-apoptotic agent, e.g., a TRAIL polypeptide that induces
apoptosis in an autoreactive immune cell. As another example, the
erythroid cell can have antigen presentation activity, e.g., can
comprise MOG and/or MBP peptide on MHCII, and further comprise one
or both or IL10 and TGFbeta or a fragment or variant thereof, e.g.,
for treating multiple sclerosis. The erythroid cell may also
comprise an antigen and an attenuator or activator that is surface
exposed or intracellular. For example, the cell can comprise an
antigen and PDL1 or a fragment or variant thereof. As another
example the cell can comprise an antigen and an enzyme such as IDO
or ADA. The erythroid cell may also comprise an antigen and a cell
targeting agent, e.g., an antigen specific for a type of B cell
(e.g., an autoantigen specific for autoreactive B cells) and an
anti-BCMA antibody molecule that targets plasma cells.
[0309] Proximity-Based Configurations
[0310] When two or more agents (e.g., polypeptides) have a
proximity-based relationship, the two agents function more
strongly, e.g., exert a more pronounced effect, when they are in
proximity to each other than when they are physically separate. In
embodiments, the two agents are in proximity when they are directly
binding to each other, when they are part of a complex (e.g.,
linked by a third agent), when they are present on the same cell
membrane, or when they are present on the same subsection of a cell
membrane (e.g., within a lipid raft, outside a lipid raft, or bound
directly or indirectly to an intracellular structure such as a
cytoskeleton component). In some embodiments, first polypeptide
binds a first target molecule and the second polypeptide binds a
second target molecule, and this binding causes the first target
molecule and the second target molecule to move into closer
proximity with each other, e.g., to bind each other. In some
embodiments, the first and second target molecules are cell surface
receptors on a target cells.
[0311] An example of a proximity-based configuration is shown in
FIG. 4. The erythroid cell of FIG. 4 comprises an optional first
exogenous polypeptide (white), a second exogenous polypeptide
(hatching), and a third exogenous polypeptide (close hatching). The
second and third exogenous polypeptides bind to different epitopes
within the same polypeptide chain of a target, e.g., cytokine B.
The second and third exogenous polypeptides, which are mounted on
the erythrocyte, bind to the target with higher avidity than if the
second and third exogenous polypeptides were free polypeptides. As
examples, two or more exogenous polypeptides could bind different
sites on the same target, wherein the target is a cytokine, a
complement factor (e.g., C5), an enzyme, an antibody, or an immune
complex. As an example, the first exogenous polypeptide comprises
an antigen (e.g., an autoantigen, e.g., an autoantigen of Table 6)
that binds a specific antibody (e.g., an autoantibody) and the
second exogenous polypeptide binds antibody Fc (e.g., could
comprise CD16A which binds Fc) to increase avidity for the antibody
that binds the first exogenous polypeptide.
[0312] Scaffold Configurations
[0313] When two or more agents (e.g., polypeptides) have a scaffold
relationship, the agents bring two or more targets together, to
increase the likelihood of the targets interacting with each other.
In an embodiment the first and second agent are associated with
each other (forming a scaffold) at the surface of the RBC, e.g.,
two complexed polypeptides. In an embodiment, the red blood cell
(e.g., reticulocyte) comprises a bispecific antibody molecule,
e.g., an antibody molecule that recognizes one or more (e.g., 2)
proteins described herein, e.g., in any of Table 1, Table 2, Table
3, and Table 4.
[0314] The targets may comprise, e.g., proteins, cells, small
molecules, or any combination thereof. In an embodiment, the first
and second targets are proteins. In an embodiment, the first and
second targets are cells.
[0315] In some embodiments, the RBC brings an immune effector cell
into proximity with another immune cell, e.g., to promote antigen
presentation (e.g., when one cell is an antigen presenting cell and
the other cell is a T cell) or anergy (e.g., when one cell is a
Treg and the other cell is an immune effector cell, e.g., an
autoreactive immune effector cell).
[0316] In some embodiments, a RBC expresses an exogenous fusion
polypeptide comprising a first antibody molecule domain and a
second antibody molecule domain, wherein the exogenous polypeptide
functions as a bispecific antibody, e.g., wherein the first
antibody molecule domain binds a first target on a first cell and
the second antibody molecule domain binds a second target on a
second cell, e.g., a different cell type.
[0317] Multimer Configurations
[0318] When two or more agents (e.g., polypeptides) have a multimer
configuration, the agents combine with each other, e.g., bind each
other, to form a complex that has a function or activity on a
target. In an embodiment, the agents are subunits of a cell surface
complex, e.g., WWI, and a function is to bind a peptide. In an
embodiment, the agents are subunits of WICK and a function is to
bind a peptide. In an embodiment, the agents are subunits of a cell
surface molecule, e.g., MHCI and a peptide, e.g., a peptide loaded
on the WWI molecule, and a function is to present the peptide. In
an embodiment, the agents are subunits of a MHCII and a peptide,
e.g., a peptide loaded on the WWII molecule, and a function is to
present the peptide. In one embodiment, the complex is a functional
MHC I, the agents are WIC I (alpha chain 1-3) and beta-2
microglobulin. In one embodiment the complex is WIC II and the
agents are WIC II alpha chain and MHC II beta chain. In some
embodiments, the MHC molecule comprises human MHC class I or II,
e.g., MHC II alpha subunit and MHC II beta subunit or a fusion
molecule comprising both subunits or antigen-presenting fragments
thereof. A RBC with these two polypeptides is used, in some
embodiments, for immune induction or antigen presentation. In some
embodiments, the RBC comprises a single protein that is a fusion
between an MHC molecule and an antigen, e.g., a single-chain
peptide-WIC construct. In some embodiments, a non-membrane tethered
component of the complex, e.g. the peptide, or the beta-2
microglobulin, is assembled with another agent within the cell
prior to trafficking to the surface, is secreted by the cell then
captured on the surface by the membrane-tethered component of the
multimer, or is added in a purified form to an engineered red blood
cell.
[0319] The antigen is, in some embodiments, an autoimmune antigen,
e.g., a protein or fragment or variant thereof of Table 4. In some
embodiments, the antigen is about 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 25, 30, or 35 amino acids in length.
[0320] In some embodiments, a red blood cell (e.g., reticulocyte)
acts on a complement cascade. Some complement regulatory proteins
act in concert as co-factors for one another, e.g. CFH and CD55 are
co-factors for the enzymatic activity of CFI. In some embodiments,
the agents comprise an enzymatic protein or domain, e.g., CFI, and
a co-factor, e.g., CFH or CD55, that accelerates and enhances the
activity of CFI on the target complement molecule.
[0321] In some embodiments the complex comprises multiple
subdomains derived from different polypeptide chains, all of which
must be expressed in order for the complex to be active.
[0322] Compensatory Configurations
[0323] When two or more agents (e.g., polypeptides) have a
compensatory relationship, a first agent reduces an undesirable
characteristic of a second agent. For example, in some embodiments,
the second agent has a given level of immunogenicity, and the first
agent reduces the immunogenicity, e.g., by negatively signaling
immune cells (see Table 3), or by shielding an antigenic epitope of
the second agent. In some embodiments, the second agent has a given
half-life, and the first agent increases the half-life of the
second agent. For example, the first agent can comprise a chaperone
or fragment or variant thereof.
[0324] An enucleated erythroid cell can co-express a therapeutic
protein and its inhibitor (e.g., FIG. 8). The inhibitor can be
released (e.g., cease binding the therapeutic but remain on the
surface of the cell) in at the desired location in the body, to
activate the therapeutic protein.
[0325] For instance, in some embodiments, the erythroid cell
comprises a first exogenous polypeptide with therapeutic activity
(e.g., an anti-TNFalpha antibody molecule), a second exogenous
polypeptide (e.g., TNFalpha or a fragment or variant thereof) that
inhibits the first exogenous polypeptide. The second polypeptide
(e.g., TNFalpha) may inhibit activity of the first exogenous
polypeptide (e.g., anti-TNFalpha) until the erythroid cell is at a
desired location, e.g., at inflamed tissue, e.g., limiting
off-target effects. The second exogenous polypeptide (e.g.,
TNFalpha) may comprise a variant of the target (e.g., endogenous
TNFalpha) that the first exogenous polypeptide (e.g.,
anti-TNFalpha) binds. For instance, the variant can be a
weakly-binding variant that is competed away in the presence of the
target. In embodiments, the Kd of the first exogenous polypeptide
for the second exogenous polypeptide is at least 2, 3, 5, 10, 20,
50, or 100-fold greater than the Kd of the first exogenous
polypeptide for its target. The erythroid cell optionally comprises
a third exogenous polypeptide that comprises a targeting agent.
[0326] In some embodiments, the enucleated erythroid cell comprises
a prodrug (e.g., pro-insulin) that becomes a drug (e.g., insulin)
at a desired site in a subject.
[0327] Enucleated Red Blood Cells Comprising Three or More Agents
(e.g., Polypeptides)
[0328] In embodiments, a red blood cell (e.g., reticulocyte)
described herein comprises three or more, e.g., at least 4, 5, 10,
20, 50, 100, 200, 500, or 1000 agents. In embodiments, a population
of red blood cells described herein comprises three or more, e.g.,
at least 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, or 5000
agents, e.g., wherein different RBCs in the population comprise
different agents or wherein different RBCs in the population
comprise different pluralities of agents. In embodiments, two or
more (e.g., all) of the agents in the RBC or population or RBCs
have agent-additive, agent-synergistic, multiplicative, independent
function, localization-based, proximity-dependent, scaffold-based,
multimer-based, or compensatory activity.
[0329] In embodiments, the RBC is produced by contacting a RBC
progenitor cell with a plurality of mRNAs encoding the agents.
[0330] Eukaryotic display screening. In an embodiment, a
combinatorial, high-diversity pool of cells is produced, e.g., for
use in an in vitro or in vivo binding assay. A combinatorial,
high-diversity nucleic acid library encoding cell-surface proteins
can be created. Such a library could, e.g., consist of entirely
variable sequences, or comprise a fixed sequence fused to a highly
variable, combinatorial sequence. These can be introduced into red
blood cell progenitors as a mixture or individually, using methods
such as electroporation, transfection or viral transduction. In one
embodiment, the cells are subsequently grown in differentiation
media until the desired level of maturity. In one embodiment, the
cells are used for a highly multiplexed in-vitro assay. Cells are
incubated with a biological sample in a microtiter plate. Wells are
washed using a cell-compatible buffer, with a desired level of
stringency. The remaining cells are isolated and analyzed for the
enrichment of specific sequences. In one embodiment, the analysis
is performed at the protein level, e.g., using mass spectrometry,
to identify the amino acid motifs that are enriched in the bound
population. In an embodiment, the analysis is performed at the
nucleic acid level (RNA or DNA) to identify the nucleic acid
sequences identifying the corresponding amino-acid motif enriched
in the bound population. In an embodiment, the high-diversity cell
population is injected into an animal model (either healthy or
diseased). In one embodiment the cells are fluorescently labeled to
visualize their in vivo distribution or localization. Various
tissues of the animal could then be collected and analyzed for the
relative enrichment of specific amino-acid motifs or nucleic acid
sequences identifying the corresponding amino-acid motif.
[0331] Expression optimization. A large number of variants can be
simultaneously transfected into individual cells to assess their
relative transcription or translation ability. In embodiments, a
library of protein coding sequences are designed and synthesized
with a diversity of 5' untranslated regions, 3' untranslated
regions, codon representations, amino acid changes, and other
sequence differences. This library would be introduced into red
blood cell progenitors as a mixture or individually, using methods
such as electroporation, transfection or viral transduction. In one
embodiment, the cells are subsequently grown in differentiation
media until the desired level of maturity.
[0332] Physical Characteristics of Enucleated Red Blood Cells
[0333] In some embodiments, the RBCs (e.g., reticulocytes)
described herein have one or more (e.g., 2, 3, 4, or more) physical
characteristics described herein, e.g., osmotic fragility, cell
size, hemoglobin concentration, or phosphatidylserine content.
While not wishing to be bound by theory, in some embodiments an
enucleated RBC that expresses an exogenous protein has physical
characteristics that resemble a wild-type, untreated RBC. In
contrast, a hypotonically loaded RBC sometimes displays aberrant
physical characteristics such as increased osmotic fragility,
altered cell size, reduced hemoglobin concentration, or increased
phosphatidylserine levels on the outer leaflet of the cell
membrane.
[0334] In some embodiments, the enucleated RBC comprises an
exogenous protein that was encoded by an exogenous nucleic acid
that was not retained by the cell, has not been purified, or has
not existed fully outside an RBC. In some embodiments, the RBC is
in a composition that lacks a stabilizer.
[0335] Osmotic Fragility
[0336] In some embodiments, the enucleated red blood cell exhibits
substantially the same osmotic membrane fragility as an isolated,
uncultured erythroid cell that does not comprise an exogenous
polypeptide. In some embodiments, the population of enucleated red
blood cells has an osmotic fragility of less than 50% cell lysis at
0.3%, 0.35%, 0.4%, 0.45%, or 0.5% NaCl. Osmotic fragility can be
assayed using the method of Example 59 of WO2015/073587.
[0337] Cell Size
[0338] In some embodiments, the enucleated RBC has approximately
the diameter or volume as a wild-type, untreated RBC.
[0339] In some embodiments, the population of RBC has an average
diameter of about 4, 5, 6, 7, or 8 microns, and optionally the
standard deviation of the population is less than 1, 2, or 3
microns. In some embodiments, the one or more RBC has a diameter of
about 4-8, 5-7, or about 6 microns. In some embodiments, the
diameter of the RBC is less than about 1 micron, larger than about
20 microns, between about 1 micron and about 20 microns, between
about 2 microns and about 20 microns, between about 3 microns and
about 20 microns, between about 4 microns and about 20 microns,
between about 5 microns and about 20 microns, between about 6
microns and about 20 microns, between about 5 microns and about 15
microns or between about 10 microns and about 30 microns. Cell
diameter is measured, in some embodiments, using an Advia 120
hematology system.
[0340] In some embodiment the volume of the mean corpuscular volume
of the RBCs is greater than 10 fL, 20 fL, 30 fL, 40 fL, 50 fL, 60
fL, 70 fL, 80 fL, 90 fL, 100 fL, 110 fL, 120 fL, 130 fL, 140 fL,
150 fL, or greater than 150 fL. In one embodiment the mean
corpuscular volume of the RBCs is less than 30 fL, 40 fL, 50 fL, 60
fL, 70 fL, 80 fL, 90 fL, 100 fL, 110 fL, 120 fL, 130 fL, 140 fL,
150 fL, 160 fL, 170 fL, 180 fL, 190 fL, 200 fL, or less than 200
fL. In one embodiment the mean corpuscular volume of the RBCs is
between 80-100, 100-200, 200-300, 300-400, or 400-500 femtoliters
(fL). In some embodiments, a population of RBCs has a mean
corpuscular volume set out in this paragraph and the standard
deviation of the population is less than 50, 40, 30, 20, 10, 5, or
2 fL. The mean corpuscular volume is measured, in some embodiments,
using a hematological analysis instrument, e.g., a Coulter
counter.
[0341] Hemoglobin Concentration
[0342] In some embodiments, the enucleated RBC has a hemoglobin
content similar to a wild-type, untreated RBC. In some embodiments,
the RBCs comprise greater than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%
or greater than 10% fetal hemoglobin. In some embodiments, the RBCs
comprise at least about 20, 22, 24, 26, 28, or 30 pg, and
optionally up to about 30 pg, of total hemoglobin. Hemoglobin
levels are determined, in some embodiments, using the Drabkin's
reagent method of Example 33 of WO2015/073587.
[0343] Phosphatidylserine Content
[0344] In some embodiments, the enucleated RBC has approximately
the same phosphatidylserine content on the outer leaflet of its
cell membrane as a wild-type, untreated RBC. Phosphatidylserine is
predominantly on the inner leaflet of the cell membrane of
wild-type, untreated RBCs, and hypotonic loading can cause the
phosphatidylserine to distribute to the outer leaflet where it can
trigger an immune response. In some embodiments, the population of
RBC comprises less than about 30, 25, 20, 15, 10, 9, 8, 6, 5, 4, 3,
2, or 1% of cells that are positive for Annexin V staining.
Phosphatidylserine exposure is assessed, in some embodiments, by
staining for Annexin-V-FITC, which binds preferentially to PS, and
measuring FITC fluorescence by flow cytometry, e.g., using the
method of Example 54 of WO2015/073587.
[0345] Other Characteristics
[0346] In some embodiments, the population of RBC comprises at
least about 50%, 60%, 70%, 80%, 90%, or 95% (and optionally up to
90 or 100%) of cells that are positive for GPA. The presence of GPA
is detected, in some embodiments, using FACS.
[0347] In some embodiments, the RBCs have a half-life of at least
30, 45, or 90 days in a subject.
[0348] In some embodiments, a population of cells comprising RBCs
comprises less than about 10, 5, 4, 3, 2, or 1% echinocytes.
[0349] In some embodiments, an RBC is enucleated, e.g., a
population of cells comprising RBCs used as a therapeutic
preparation described herein is greater than 50%, 60%, 70%, 80%,
90% enucleated. In some embodiments, a cell, e.g., an RBC, contains
a nucleus that is non-functional, e.g., has been inactivated.
[0350] Methods of Manufacturing Enucleated Red Blood Cells
[0351] Methods of manufacturing enucleated red blood cells (e.g.,
reticulocytes) comprising (e.g., expressing) exogenous agent (e.g.,
polypeptides) are described, e.g., in WO2015/073587 and
WO2015/153102, each of which is incorporated by reference in its
entirety.
[0352] In some embodiments, hematopoietic progenitor cells, e.g.,
CD34+ hematopoietic progenitor cells, are contacted with a nucleic
acid or nucleic acids encoding one or more exogenous polypeptides,
and the cells are allowed to expand and differentiate in
culture.
[0353] In some embodiments, the two or more polypeptides are
encoded in a single nucleic acid, e.g. a single vector. In
embodiments, the single vector has a separate promoter for each
gene, has two proteins that are initially transcribed into a single
polypeptide having a protease cleavage site in the middle, so that
subsequent proteolytic processing yields two proteins, or any other
suitable configuration. In some embodiments, the two or more
polypeptides are encoded in two or more nucleic acids, e.g., each
vector encodes one of the polypeptides.
[0354] The nucleic acid may be, e.g., DNA or RNA. A number of
viruses may be used as gene transfer vehicles including
retroviruses, Moloney murine leukemia virus (MMLV), adenovirus,
adeno-associated virus (AAV), herpes simplex virus (HSV),
lentiviruses such as human immunodeficiency virus 1 (HIV 1), and
spumaviruses such as foamy viruses, for example.
[0355] In some embodiments, the cells are produced using
sortagging, e.g., as described in WO2014/183071 or WO2014/183066,
each of which is incorporated by reference in its entirety.
[0356] In some embodiments, the RBCs are expanded at least 1000,
2000, 5000, 10,000, 20,000, 50,000, or 100,000 fold (and optionally
up to 100,000, 200,000, or 500,000 fold). Number of cells is
measured, in some embodiments, using an automated cell counter.
[0357] In some embodiments, the population of RBC comprises at
least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or
98% (and optionally up to about 80, 90, or 100%) enucleated RBC. In
some embodiments, the population of RBC contains less than 1% live
enucleated cells, e.g., contains no detectable live enucleated
cells. Enucleation is measured, in some embodiments, by FACS using
a nuclear stain. In some embodiments, at least 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, or 80% (and optionally up to about 70, 80, 90,
or 100%) of RBC in the population comprise one or more (e.g., 2, 3,
4 or more) of the exogenous polypeptides. Expression of the
polypeptides is measured, in some embodiments, by FACS using
labeled antibodies against the polypeptides. In some embodiments,
at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% (and
optionally up to about 70, 80, 90, or 100%) of RBC in the
population are enucleated and comprise one or more (e.g., 2, 3, 4,
or more) of the exogenous polypeptides. In some embodiments, the
population of RBC comprises about
1.times.10.sup.9-2.times.10.sup.9,
2.times.10.sup.9-5.times.10.sup.9,
5.times.10.sup.9-1.times.10.sup.10,
1.times.10.sup.10-2.times.10.sup.10, 2.times.10.sup.10,
5.times.10.sup.10, 5.times.10.sup.10-1.times.10.sup.11,
1.times.10.sup.11-2.times.10.sup.11,
2.times.10.sup.11-5.times.10.sup.11,
5.times.10.sup.11-1.times.10.sup.12,
1.times.10.sup.12-2.times.10.sup.12,
2.times.10.sup.12-5.times.10.sup.12, or
5.times.10.sup.12-1.times.10.sup.13 cells.
[0358] Physically Proximal, Synergistic Agents
[0359] In some aspects, the present disclosure provides a
composition comprising a first agent and a second agent in physical
proximity to each other. In some embodiments, agents act
synergistically when they are in physical proximity to each other
but not when they are separate. In some embodiments, the first and
second agent are covalently linked, e.g., are part of a fusion
protein or are chemically conjugated together. In some embodiments,
the first and second agent are non-covalently linked, e.g., are
bound directly to each other or to a scaffold. In some embodiments,
the first and second agents are part of (e.g., linked to) a
nanoparticle (e.g., 1-100, 100-2,500, or 2,500-10,000 nm in
diameter) liposome, vesicle, bead, polymer, implant, or polypeptide
complex.
[0360] In some embodiments, the composition comprises at least 3,
4, 5, 6, 7, 8, 9, or 10 different agents that are in physical
proximity to each other (e.g., covalently or noncovalently
linked).
[0361] In some embodiments, the composition comprises one or more
(e.g., 2, 3, 4, 5, or more) agents described herein, e.g.,
exogenous polypeptides described herein, e.g., polypeptides of any
of Table 1, Table 2, Table 3, or Table 4, or a fragment or variant
thereof, or an antibody molecule thereto. In some embodiments, one
or more (e.g., 2, 3, or more) of the exogenous polypeptides
comprise cytokines, interleukins, cytokine receptors, Fc-binding
molecules, T-cell activating ligands, T cell receptors, immune
inhibitory molecules, costimulatory molecule, MEW molecules,
APC-binding molecule, autoantigen, allergen, toxin, targeting
agent, or TRAIL receptor ligands.
[0362] In some embodiments, one or more (e.g., 2, 3, or more) of
the exogenous polypeptides comprise TRAIL receptor ligands, e.g., a
sequence of any of SEQ ID NOS: 1-5 herein, or a sequence with at
least 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity
thereto, or an antibody molecule that binds a TRAIL receptor. In
some embodiments, the first agent binds to a first TRAIL receptor,
e.g., TRAIL-RI, and the second agent binds to a second TRAIL
receptor, e.g., TRAIL-MI. In embodiments, the two TRAIL receptor
ligands in proximity provide a synergistic degree of apoptosis in a
target cell, compared to either TRAIL receptor ligand alone.
Example 1 herein demonstrates a synergistic activity when target
cells are treated with a composition comprising two TRAIL receptor
ligands in close proximity (e.g., on the surface of an enucleated
red blood cell).
[0363] Engineered Red Blood Cells Comprising One or More Agents
[0364] In some aspects, the present disclosure provides an
engineered red blood cell (e.g., reticulocyte) comprising an
exogenous agent. More specifically, in some aspects, the present
disclosure provides an enucleated red blood cell (e.g.,
reticulocyte) comprising an exogenous polypeptide. The red blood
cell optionally further comprises a second, different, exogenous
polypeptide.
[0365] In some embodiments, the exogenous polypeptide (e.g., an
exogenous polypeptide comprised by a red blood cell that optionally
further comprises a second exogenous polypeptide) is an exogenous
polypeptide described herein. In embodiments, the polypeptide is
selected from any of Table 1, Table 2, Table 3, or Table 4, or a
fragment or variant thereof, or an antibody molecule thereto.
[0366] In some embodiments, the exogenous polypeptide (e.g., an
exogenous polypeptide comprised by a red blood cell that optionally
further comprises a second exogenous polypeptide) comprises a
stimulatory ligand, e.g., CD80, CD86, 41BBL, or any combination
thereof. In some embodiments, the exogenous polypeptide comprises a
T cell inhibitor such as IL-10 and PD-L1 or a combination thereof,
e.g., for the treatment of an inflammatory disease.
[0367] Vehicles for Polypeptides Described Herein
[0368] While in many embodiments herein, the one or more (e.g., two
or more) exogenous polypeptides are situated on or in a red blood
cell, it is understood that any exogenous polypeptide or
combination of exogenous polypeptides described herein can also be
situated on or in another vehicle. The vehicle can comprise, e.g.,
a cell, an erythroid cell, a corpuscle, a nanoparticle, a micelle,
a liposome, or an exosome. For instance, in some aspects, the
present disclosure provides a vehicle (e.g., a cell, an erythroid
cell, a corpuscle, a nanoparticle, a micelle, a liposome, or an
exosome) comprising, e.g., on its surface, one or more agents
described herein. In some embodiments, the one or more agent
comprises a polypeptide that binds PD-1 (e.g., an antibody molecule
that binds PD-1 or an agonist of PD-1 such as PD-L1), a polypeptide
that binds PD-L1 (e.g., an antibody molecule that binds PD-L1), a
polypeptide that binds CD20 (e.g., an antibody molecule that binds
CD20), or a polypeptide that binds a TRAIL receptor (e.g., an
agonist of a TRAIL receptor). In some embodiments, the one or more
agents comprise an agent selected a polypeptide of any of Table 1,
Table 2, Table 3, or Table 4, or a fragment or variant thereof, or
an agonist or antagonist thereof, or an antibody molecule thereto.
In some embodiments, the vehicle comprises two or more agents
described herein, e.g., any pair of agents described herein.
[0369] In some embodiments, the vehicle comprises an erythroid
cell. In embodiments, the erythroid cell is a nucleated red blood
cell, red blood cell precursor, or enucleated red blood cell. In
embodiments, the erythroid cell is a cord blood stem cell, a CD34+
cell, a hematopoietic stem cell (HSC), a spleen colony forming
(CFU-S) cell, a common myeloid progenitor (CMP) cell, a blastocyte
colony-forming cell, a burst forming unit-erythroid (BFU-E), a
megakaryocyte-erythroid progenitor (MEP) cell, an erythroid
colony-forming unit (CFU-E), a reticulocyte, an erythrocyte, an
induced pluripotent stem cell (iPSC), a mesenchymal stem cell
(MSC), a polychromatic normoblast, an orthochromatic normoblast, or
a combination thereof. In some embodiments, the erythroid cells are
immortal or immortalized cells.
[0370] Cells Encapsulated in a Membrane
[0371] In some embodiments, enucleated erythroid cells or other
vehicles described herein are encapsulated in a membrane, e.g.,
semi-permeable membrane. In embodiments, the membrane comprises a
polysaccharide, e.g., an anionic polysaccharide alginate. In
embodiments, the semipermeable membrane does not allow cells to
pass through, but allows passage of small molecules or
macromolecules, e.g., metabolites, proteins, or DNA. In
embodiments, the membrane is one described in Lienert et al.,
"Synthetic biology in mammalian cells: next generation research
tools and therapeutics" Nature Reviews Molecular Cell Biology 15,
95-107 (2014), incorporated herein by reference in its entirety.
While not wishing to be bound by theory, in some embodiments, the
membrane shields the cells from the immune system and/or keeps a
plurality of cells in proximity, facilitating interaction with each
other or each other's products.
[0372] Methods of treatment with compositions herein, e.g.,
enucleated red blood cells Methods of administering enucleated red
blood cells (e.g., reticulocytes) comprising (e.g., expressing)
exogenous agent (e.g., polypeptides) are described, e.g., in
WO2015/073587 and WO2015/153102, each of which is incorporated by
reference in its entirety.
[0373] In embodiments, the enucleated red blood cells described
herein are administered to a subject, e.g., a mammal, e.g., a
human. Exemplary mammals that can be treated include without
limitation, humans, domestic animals (e.g., dogs, cats and the
like), farm animals (e.g., cows, sheep, pigs, horses and the like)
and laboratory animals (e.g., monkey, rats, mice, rabbits, guinea
pigs and the like). The methods described herein are applicable to
both human therapy and veterinary applications.
[0374] In some embodiments, the RBCs are administered to a patient
every 1, 2, 3, 4, 5, or 6 months.
[0375] In some embodiments, a dose of RBC comprises about
1.times.10.sup.9-2.times.10.sup.9,
2.times.10.sup.9-5.times.10.sup.9,
5.times.10.sup.9-1.times.10.sup.10,
1.times.10.sup.10-2.times.10.sup.10,
2.times.10.sup.10-5.times.10.sup.10,
5.times.10.sup.10-1.times.10.sup.11,
1.times.10.sup.11-2.times.10.sup.11,
2.times.10.sup.11-5.times.10.sup.11,
5.times.10.sup.11-1.times.10.sup.12,
1.times.10.sup.12-2.times.10.sup.12,
2.times.10.sup.12-5.times.10.sup.12, or
5.times.10.sup.12-1.times.10.sup.13 cells.
[0376] In some embodiments, the RBCs are administered to a patient
in a dosing regimen (dose and periodicity of administration)
sufficient to maintain function of the administered RBCs in the
bloodstream of the patient over a period of 2 weeks to a year,
e.g., one month to one year or longer, e.g., at least 2 weeks, 4
weeks, 6 weeks, 8 weeks, 3 months, 6 months, a year, 2 years.
[0377] In some aspects, the present disclosure provides a method of
treating a disease or condition described herein, comprising
administering to a subject in need thereof a composition described
herein, e.g., an enucleated red blood cell (e.g., reticulocyte)
described herein. In some embodiments, the disease or condition is
an inflammatory disease or an autoimmune disease. In some aspects,
the disclosure provides a use of a RBC described herein for
treating a disease or condition described herein, e.g., an immune
condition, e.g., an inflammatory disease or an autoimmune disease,
e.g., an autoimmune disease of Table 4. In some aspects, the
disclosure provides a use of a RBC described herein for manufacture
of a medicament for treating a disease or condition described
herein, e.g., an immune condition, e.g., an inflammatory disease or
an autoimmune disease, e.g., an autoimmune disease of Table 4.
[0378] Inflammatory disease include sepsis e.g., bacterial sepsis,
rheumatoid arthritis, age related macular degeneration (AMD),
systemic lupus erythematosus (an inflammatory disorder of
connective tissue), glomerulonephritis (inflammation of the
capillaries of the kidney), Crohn's disease, ulcerative colitis,
celiac disease, or other idiopathic inflammatory bowel diseases,
and allergic asthma.
[0379] Autoimmune diseases include systemic lupus erythematosus,
glomerulonephritis, rheumatoid arthritis, multiple sclerosis, type
1 diabetes, or a disease of Table 4. Immunodeficiencies include
primary immunodeficiencies, e.g., Adenosine Deaminase
Deficiency-Severe Combined Immunodeficiency (ADA-SCID), and e.g.,
secondary immunodeficiencies, e.g., secondary to an
immunosuppressive therapy.
[0380] Additional Tables
TABLE-US-00010 TABLE 5 Exemplary modifiers, e.g., proteases
Modifier Exemplary target Proteases IdeS IgG IdeZ (an
immunoglobulin-degrading enzyme from IgG Streptococcus equi
subspecies zooepidemicus) IgA protease IgG Papain IgG ADAM17/TACE
TNF-alpha mesotrypsin Peptides comprising linkages involving the
carboxyl group of lysine or arginine Lysozyme peptidoglycan
Endolysin peptidoglycan Endoproteinase, e.g., LysC (can cleave
proteins on Protein having a Lys-Xaa motif C-terminal side of
lysine residues) Metalloendopeptidase, e.g., LysN (can cleave
Protein having an Xaa-Lys motif proteins on amino side of lysine
residues) Elastase, e.g., Pseudomonas elastase (PaE) C3 alkaline
protease (PaAP) C3 56 kDa protease from Serratia marcescens C5a,
C1-INH, alpha 2-antiplasmin, antithrombin III C5a peptidase, e.g.,
Streptocoocal C5a peptidase, C5a ScpA, ScpB, SCPA Plasmin IgG, C3b,
iC3b cysteine protease, e.g., Streptococcal pyrogenic IgG,
cytokines, extracellular matrix exotoxin B (SpeB) proteins PrtH
(e.g., from Porphyromonas) IgG or C3 Staphylokinase plasminogen,
IgG, C3b Matrix metalloproteinases (e.g., MMP1, MMP2, ECM proteins,
e.g., collagen, gelatin, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11,
fibronectin, laminin, aggrecan, elastin, MMP12, MMP13, MMP14,
MMP15, MMP16, fibrin MMP17, MMP19, MMP20, MMP21, MMP23A, MMP23B,
MMP24, MMP25, MMP26, MMP27, MMP28) Other modifiers Protein
disulfide isomerases Proteins comprising two cysteine residues
Glycosyltransferases, e.g., .alpha.-glucan-branching Protein
comprising tyrosine, serine, glycosyltransferase, enzymatic
branching factor, threonine, or asparagine glycosylation site
branching glycosyltransferase, enzyme Q, glucosan transglycosylase,
glycogen branching enzyme, amylose isomerase, plant branching
enzyme, .alpha.-1,4- glucan: .alpha.-1,4-glucan-6-,
glycosyltransferase, starch branching enzyme,
UDP-N-acetyl-D-galactosamine, polypeptide,
N-acetylgalactosaminyltransferase, GDP-fucose protein
O-fucosyltransferase 2, O- GlcNAc transferase Acetyltransferases or
deacetylases, e.g., histone nucleosome-histone acetyltransferase,
histone acetokinase, histone acetylase, histone transacetylase,
histone deacetylase Acyltransferases Protein comprising an acyl
group Phosphatases, e.g., protein-tyrosine-phosphatase,
phosphoprotein phosphotyrosine phosphatase, phosphoprotein
phosphatase (phosphotyrosine), phosphotyrosine histone phosphatase,
protein phosphotyrosine phosphatase, tyrosylprotein phosphatase,
phosphotyrosine protein phosphatase, phosphotyrosylprotein
phosphatase, tyrosine O- phosphate phosphatase, PPT-phosphatase,
PTPase, [phosphotyrosine]protein phosphatase, PTP- phosphatase
Kinases, e.g., non-specific serine/threonine protein Protein
comprising a serine or threonine kinase, Fas-activated
serine/threonine kinase, phosphorylation site Goodpasture
antigen-binding protein kinase, I.kappa.B kinase, cAMP-dependent
protein kinase, cGMP- dependent protein kinase, protein kinase C,
polo kinase, cyclin-dependent kinase, mitogen-activated protein
kinase, mitogen-activated protein kinase kinase kinase, receptor
protein serine/threonine kinase, dual-specificity kinase
Gamma-carboxylases Protein comprising glutamic acid
Methyltransferases Protein comprising a lysine methylation site;
DNA; RNA Complement-factor inactivating moiety, e.g., Complement
factor, e.g., C1, C2a, C4b, complement control protein, Factor H or
Factor I C3, C3a, C3b, C5, C5a, C5b, C6, C7, C8, or C9
TABLE-US-00011 TABLE 6 Exemplary first exogenous polypeptides that
interact with a target Polypeptide Exemplary target(s) CD16A IgG Fc
CD14 LPS TLR4 LPS scFv Complement (e.g., C3 or C5) or cytokine
(e.g., TNF-alpha or IL-6 or another cytokine of Table 1) Aquaporin
4 (AQP4) and variants Anti-AQP4 autoantibodies Phospholipase A2
receptor (PLA2R) and Anti-PLA2R autoantibodies variants (shortened
domains & peptides) Acetylcholine receptor (AChR) and variants
Anti-AChR autoantibodies (shortened domains & peptides) Insulin
and variants (proinsulin, preproinsulin, Anti-insulin
autoantibodies etc.) B2-glycoprotein 1 (b2GP1) and variants
Anti-b2GP1 autoantibodies (shortened domains & peptides)
ADAMTS13 and variants (shortened domains Anti-ADAMTS13
autoantibodies & peptides) GAD65 and variants (shortened
domains & Anti-GAD65 autoantibodies peptides) Desmogleins,
e.g., Desmoglein-3 or Anti-Desmoglein autoantibodies Desmoglein-1
and variants (shortened domains & peptides) Complement-factor
binding moiety, e.g., CD55 Complement factor, e.g., C1, C2a, C4b,
C3, or CD46 C3a, C3b, C5, C5a, C5b, C6, C7, C8, or C9
EXAMPLES
Example 1. Agent-Synergistic Activity of eRBC Expressing Two
Different TRAIL Receptor Ligands on the Surface
[0381] The genes for TRAIL receptor agonists DR4.2 (SEQ ID 2) and
DR5.2 (SEQ ID 5) were synthesized. The genes were cloned into a
lentivirus vector (SBI) upstream of the gene for human glycophorin
A and separated by a sequence encoding a 12-amino acid Gly-Ser
(GGGSGGGSGGGS (SEQ ID NO: 19)) flexible linker and an HA epitope
tag (YPYDVPDY (SEQ ID NO: 20)).
[0382] Human CD34+ cells derived from mobilized peripheral blood
cells from normal human donors were purchased frozen from AllCells
Inc. Cells were thawed in PBS with 1% FBS. Cells were then cultured
in StemSpan SFEM media with StemSpan CC100 Cytokine Mix at a
density of 1E5 cells/mL. Media was swapped to differentiation media
on day 5.
[0383] Virus production protocol was conducted as follows. Briefly,
HEK293T cells were seeded 24 hours before transfection. Cells were
transfected with lentivector containing the construct along with
packaging plasmids. A media swap was performed 24 hours after
transfection and viruses were harvested 72 hours after
transfection. On day 6 after thaw, cells were transduced with equal
volumes of each virus in a 1:1 cell volume to virus volume ratio,
and spinoculated at 845.times.g for 1.5 hours with 5-10 .mu.g/ml of
polybrene.
[0384] Transduced cells were differentiated in defined media to
erythroid lineage cells and to mature enucleated reticulocytes
following the method of Hu et al., Blood 18 Apr. 2013 Vol 121, 16.
In brief, the cell culture procedure was comprised of 3 phases.
Composition of the base culture medium was Iscove's Modified
Dulbecco's Medium, 2% human peripheral blood plasma, 3% human AB
serum, 200 mg/mL Holohuman transferrin, 3 IU/mL heparin, and 10
mg/mL insulin. In the first phase (day 0 to day 6), CD341 cells at
a concentration of 10-5/mL were cultured in the presence of 10
ng/mL stem cell factor, 1 ng/mL IL-3, and 3 IU/mL erythropoietin.
In the second phase (day 7 to day 11), IL-3 was omitted from the
culture medium. In the third phase that lasted until day 21, the
cell concentration was adjusted to 10{circumflex over ( )}6/mL on
day 11 and to 5.times.10{circumflex over ( )}6/mL on day 15,
respectively. The medium for this phase was the base medium plus 3
IU/mL erythropoietin, and the concentration of transferrin was
adjusted to 1 mg/mL.
[0385] Expression of the transgenes was monitored by labeling with
soluble TRAIL R1 and TRAIL R2 (purchased from Sigma-Aldrich Inc.)
that had been chemically conjugated to complementary fluorescent
dyes Fluorescein and DyLight 650 and staining by flow cytometry.
Expression levels of both ligands DR4.2 and DR5.2 were verified
through flow cytometry.
[0386] An apoptosis assay was conducted according to a modified
version of Marconi et al., Cell Death and Disease 2013. In short,
fully mature enucleated reticulocytes expressing DR4.2 and DR5.2
were incubated with CFSE-labeled Raji Cells for 24 hours at a 1:1
ratio. Afterwards cells were stained with annexin V and analyzed by
flow cytometry. Apoptosis percentages were determined from CFSE
positive Raji cells that also stained positive for annexin V.
[0387] As shown in FIG. 1, when CFSE-labeled Raji cells were
incubated with untransduced, DR4.2 transduced, DR5.2 transduced, or
a mixture of DR4.2 transduced and DR5.2 transduced cultured
reticulocytes, minimal cell death was observed over background.
However, when CFSE-labeled Raji cells were incubated with cultured
reticulocytes that had been simultaneously transduced with both
DR4.2 and DR5.2 and thus express both proteins simultaneously, a
significant amount of cell death was observed (equivalent to the
maximal amount of TRAIL-induced apoptosis achievable in this assay
with Raji cells --see, e.g. Marconi et al., Cell Death and Disease
2013). This data indicates that the coordinated action of TRAIL
receptor agonists on the surface of a single engineered red blood
cell is able to induce cell killing in a synergistic manner,
relative to cells expressing single TRAIL receptor agonists and
even a mixture of cells that each express a different TRAIL
receptor agonist.
[0388] A cell population comprising TRAIL receptor agonists, and
optionally further comprising a moiety that targets autoreactive
immune cells, may be formulated in AS-3 additive solution and
administered intravenously to a patient, e.g., a patient suffering
from an autoimmune disease.
Example 2. Generation of Capture eRBC Comprising 5 Cytokines for
Use in Treating Sepsis
[0389] The genes for anti-TNFa (SEQ ID 7), anti-IL6 (SEQ ID 6),
CD14 (Uniprot # P08571), IFNGR1 (Uniprot # P15260), and IL12R1
(Uniprot # P42701) are synthesized by a commercial vendor. The
genes are cloned into a lentivirus vector (SBI) upstream of the
gene for human glycophorin A and separated by a sequence encoding a
12-amino acid Gly-Ser (GGGSGGGSGGGS (SEQ ID NO: 19)) flexible
linker and an HA epitope tag (YPYDVPDY (SEQ ID NO: 20)).
[0390] Human CD34+ cells can be cultured, and virus can be
produced, as described in Example 1. Transduced cells are
differentiated as described herein.
[0391] To assess the expression of the transgenes, cells are
labeled simultaneously with the ligands TNFa, IL-6, IFNg, and IL-12
(purchased from Life Technologies), as well as lipopolysaccharide
(ThermoFisher), that are chemically conjugated to complementary
fluorescent dyes. The cells are analyzed by flow cytometry to
verify that (a) the agents are all expressed on the surface of the
cell and (b) the agents are capable of binding to their target
ligands.
[0392] The cell population is formulated in AS-3 additive solution
and administered intravenously to a patient who is developing
sepsis. It is anticipated that the patient then exhibits an
improvement in his symptoms as measured by a reduction in
circulating cytokine levels, a reduction or prevention of vascular
leak syndrome, and improved survival.
Example 3: Capture and Modification of a Target Protein
[0393] In this Example, transgenic enucleated erythroid cells were
used to capture and modify a target protein. The control cells and
the experimental cells each comprise endogenous glycophorin A (GPA)
in their membranes, which was used to bind the target protein. The
experimental cells expressed an exogenous protein comprising
surface-exposed IdeS fused to GPA as a membrane anchor. IdeS is
capable of cleaving antibodies to produce a F(ab')2 fragment and a
Fc fragment. The target protein is an anti-GPA antibody that is
fluorescently labelled with FITC. Both the constant and variable
regions of the target antibody were FITC-labelled, so that if the
antibody was cleaved, both fragments could be detected.
[0394] First, the control cells and IdeS-expressing cells were
tested by FACS for the ability to bind the anti-GPA antibody. Both
control and IdeS-expressing cells bound the antibody as measured by
association of FITC with the cells (data not shown). In addition,
both control and IdeS-expressing cells bound the antibody as
measured by or using a second detection method with a fluorescently
labeled anti-rabbit Fc antibody (data not shown). These
measurements were taken at an early timepoint, before cells were
incubated to allow IdeS-mediated cleavage of the target
antibody.
[0395] In contrast, only the IdeS-expressing cells were able to
cleave the target antibody. This was shown by incubating the
control or IdeS-expressing cells with the target antibody to allow
antibody cleavage to occur. Fluorescently labeled anti-rabbit Fc
antibody was added to the reaction in order to detect intact
antibodies on the surface of the erythroid cells. The
IdeS-expressing cells showed a decrease in anti-rabbit Fc
association with the cells (FIG. 2), indicating lower levels of Fc
on the surface of the IdeS-expressing cells compared to the control
cells. There was no decrease in the amount of the directly
FITC-labeled target antibody associated with control cells or
IdeS-expressing cells, indicating that at least the FITC-labeled
variable region of the target antibody still bound the
IdeS-expressing and control cells. This result was confirmed by
Western blot, where anti rabbit heavy chain and anti rabbit light
chain antibodies were used to detect intact and cleaved antibody in
the supernatant of control or IdeS-expressing cells. The experiment
showed that IdeS-expressing erythroid cells but not control
erythroid cells cleaved the anti-GPA-antibody, resulting in
appearance of the heavy chain fragment (FIG. 3).
[0396] Thus, the control cells were able to bind the target
antibody, but only the IdeS-expressing cells were able to bind and
cleave the target antibody.
Example 4: Red Cell Therapeutics Co-Expressing Anti-CD20 and TRAIL
Ligand
[0397] When erythroid cells were engineered to simultaneously
express anti-CD20 as well as Trail ligand (an apoptosis inducing
agent), co-culture of Ramos cells with RCT-antiCD20, RCT-Trail, and
RCT-antiCD20+Trail (co-expressed) exert 32%, 47% and 76% apoptosis
respectively after 48 hours, suggesting a synergistic cell-killing
effect of the co-expressing RCTs.
[0398] A cell population comprising TRAIL ligand, anti-CD20 moeity,
and optionally further comprising a moiety that specifically
targets autoreactive immune cells, may be formulated in AS-3
additive solution and administered intravenously to a patient,
e.g., a patient suffering from an autoimmune disease.
Sequence CWU 1
1
201281PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 1Met Ala Met Met Glu Val Gln Gly
Gly Pro Ser Leu Gly Gln Thr Cys1 5 10 15Val Leu Ile Val Ile Phe Thr
Val Leu Leu Gln Ser Leu Cys Val Ala 20 25 30Val Thr Tyr Val Tyr Phe
Thr Asn Glu Leu Lys Gln Met Gln Asp Lys 35 40 45Tyr Ser Lys Ser Gly
Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr 50 55 60Trp Asp Pro Asn
Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln Val65 70 75 80Lys Trp
Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu Arg Thr Ser 85 90 95Glu
Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro 100 105
110Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly
115 120 125Thr Arg Arg Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys
Asn Glu 130 135 140Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser
Ser Arg Ser Gly145 150 155 160His Ser Phe Leu Ser Asn Leu His Leu
Arg Asn Gly Glu Leu Val Ile 165 170 175His Glu Lys Gly Phe Tyr Tyr
Ile Tyr Ser Gln Thr Tyr Phe Arg Phe 180 185 190Gln Glu Glu Ile Lys
Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln 195 200 205Tyr Ile Tyr
Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys 210 215 220Ser
Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr225 230
235 240Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg
Ile 245 250 255Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp
His Glu Ala 260 265 270Ser Phe Phe Gly Ala Phe Leu Val Gly 275
2802281PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 2Met Ala Met Met Glu Val Gln Gly
Gly Pro Ser Leu Gly Gln Thr Cys1 5 10 15Val Leu Ile Val Ile Phe Thr
Val Leu Leu Gln Ser Leu Cys Val Ala 20 25 30Val Thr Tyr Val Tyr Phe
Thr Asn Glu Leu Lys Gln Met Gln Asp Lys 35 40 45Tyr Ser Lys Ser Gly
Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr 50 55 60Trp Asp Pro Asn
Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln Val65 70 75 80Lys Trp
Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu Arg Thr Ser 85 90 95Glu
Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro 100 105
110Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly
115 120 125Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys
Asn Glu 130 135 140Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser
Ser Arg Arg Gly145 150 155 160His Ser Phe Leu Ser Asn Leu His Leu
Arg Asn Gly Glu Leu Val Ile 165 170 175His Glu Lys Gly Phe Tyr Tyr
Ile Tyr Ser Gln Thr Tyr Phe Arg Phe 180 185 190Gln Glu Glu Ile Lys
Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln 195 200 205Tyr Ile Tyr
Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys 210 215 220Ser
Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr225 230
235 240Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg
Ile 245 250 255Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp
His Glu Ala 260 265 270Ser Phe Phe Gly Ala Phe Leu Val Gly 275
2803281PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 3Met Ala Met Met Glu Val Gln Gly
Gly Pro Ser Leu Gly Gln Thr Cys1 5 10 15Val Leu Ile Val Ile Phe Thr
Val Leu Leu Gln Ser Leu Cys Val Ala 20 25 30Val Thr Tyr Val Tyr Phe
Thr Asn Glu Leu Lys Gln Met Gln Asp Lys 35 40 45Tyr Ser Lys Ser Gly
Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr 50 55 60Trp Asp Pro Asn
Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln Val65 70 75 80Lys Trp
Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu Arg Thr Ser 85 90 95Glu
Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro 100 105
110Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly
115 120 125Thr Arg Arg Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys
Asn Glu 130 135 140Lys Ala Leu Gly Ile Lys Ile Asn Ser Trp Glu Ser
Ser Arg Arg Gly145 150 155 160His Ser Phe Leu Ser Asn Leu His Leu
Arg Asn Gly Glu Leu Val Ile 165 170 175His Glu Lys Gly Phe Tyr Tyr
Ile Tyr Ser Gln Thr Tyr Phe Arg Phe 180 185 190Gln Glu Glu Ile Lys
Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln 195 200 205Tyr Ile Tyr
Lys Tyr Thr Asp Tyr Pro Asp Pro Ile Leu Leu Met Lys 210 215 220Ser
Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr225 230
235 240Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg
Ile 245 250 255Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met Asp
His Glu Ala 260 265 270Ser Phe Phe Gly Ala Phe Leu Val Gly 275
2804281PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 4Met Ala Met Met Glu Val Gln Gly
Gly Pro Ser Leu Gly Gln Thr Cys1 5 10 15Val Leu Ile Val Ile Phe Thr
Val Leu Leu Gln Ser Leu Cys Val Ala 20 25 30Val Thr Tyr Val Tyr Phe
Thr Asn Glu Leu Lys Gln Met Gln Asp Lys 35 40 45Tyr Ser Lys Ser Gly
Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr 50 55 60Trp Asp Pro Asn
Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln Val65 70 75 80Lys Trp
Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu Arg Thr Ser 85 90 95Glu
Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro 100 105
110Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly
115 120 125Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys
Asn Glu 130 135 140Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser
Ser Arg Ser Gly145 150 155 160His Ser Phe Leu Ser Asn Leu His Leu
Arg Asn Gly Glu Leu Val Ile 165 170 175His Glu Lys Gly Phe Tyr Tyr
Ile Tyr Ser Gln Thr Tyr Phe Arg Phe 180 185 190Gln Glu Glu Ile Lys
Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln 195 200 205Tyr Ile Tyr
Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys 210 215 220Ser
Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr225 230
235 240Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg
Ile 245 250 255Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met His
His Glu Ala 260 265 270Ser Phe Phe Gly Ala Phe Leu Val Gly 275
2805281PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 5Met Ala Met Met Glu Val Gln Gly
Gly Pro Ser Leu Gly Gln Thr Cys1 5 10 15Val Leu Ile Val Ile Phe Thr
Val Leu Leu Gln Ser Leu Cys Val Ala 20 25 30Val Thr Tyr Val Tyr Phe
Thr Asn Glu Leu Lys Gln Met Gln Asp Lys 35 40 45Tyr Ser Lys Ser Gly
Ile Ala Cys Phe Leu Lys Glu Asp Asp Ser Tyr 50 55 60Trp Asp Pro Asn
Asp Glu Glu Ser Met Asn Ser Pro Cys Trp Gln Val65 70 75 80Lys Trp
Gln Leu Arg Gln Leu Val Arg Lys Met Ile Leu Arg Thr Ser 85 90 95Glu
Glu Thr Ile Ser Thr Val Gln Glu Lys Gln Gln Asn Ile Ser Pro 100 105
110Leu Val Arg Glu Arg Gly Pro Gln Arg Val Ala Ala His Ile Thr Gly
115 120 125Thr Arg Gly Arg Ser Asn Thr Leu Ser Ser Pro Asn Ser Lys
Asn Glu 130 135 140Lys Ala Leu Gly Arg Lys Ile Asn Ser Trp Glu Ser
Ser Arg Ser Gly145 150 155 160His Ser Phe Leu Ser Asn Leu His Leu
Arg Asn Gly Glu Leu Val Ile 165 170 175His Glu Lys Gly Phe Tyr Tyr
Ile Tyr Ser Gln Thr Tyr Phe Arg Phe 180 185 190Gln Glu Arg Ile Lys
Glu Asn Thr Lys Asn Asp Lys Gln Met Val Gln 195 200 205Tyr Ile Tyr
Lys Tyr Thr Ser Tyr Pro Asp Pro Ile Leu Leu Met Lys 210 215 220Ser
Ala Arg Asn Ser Cys Trp Ser Lys Asp Ala Glu Tyr Gly Leu Tyr225 230
235 240Ser Ile Tyr Gln Gly Gly Ile Phe Glu Leu Lys Glu Asn Asp Arg
Ile 245 250 255Phe Val Ser Val Thr Asn Glu His Leu Ile Asp Met His
His Glu Ala 260 265 270Ser Phe Phe Gly Ala Phe Leu Val Gly 275
2806243PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic polypeptide" 6Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Asn Phe Asn Asp Tyr 20 25 30Phe Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Gln Met Arg Asn
Lys Asn Tyr Gln Tyr Gly Thr Tyr Tyr Ala Glu 50 55 60Ser Leu Glu Gly
Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn Ser65 70 75 80Leu Tyr
Leu Gln Met Asn Ser Leu Lys Thr Glu Asp Thr Ala Val Tyr 85 90 95Tyr
Cys Ala Arg Glu Ser Tyr Tyr Gly Phe Thr Ser Tyr Trp Gly Gln 100 105
110Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser
Pro Ser 130 135 140Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile
Thr Cys Gln Ala145 150 155 160Ser Gln Asp Ile Gly Ile Ser Leu Ser
Trp Tyr Gln Gln Lys Pro Gly 165 170 175Lys Ala Pro Lys Leu Leu Ile
Tyr Asn Ala Asn Asn Leu Ala Asp Gly 180 185 190Val Pro Ser Arg Phe
Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 195 200 205Thr Ile Ser
Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu 210 215 220Gln
His Asn Ser Ala Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu225 230
235 240Ile Lys Arg7243PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
polypeptide" 7Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Asp Asp Tyr 20 25 30Ala Met His Trp Val Arg Gln Ala Pro Gly Lys
Gly Leu Glu Trp Val 35 40 45Ser Ala Ile Thr Trp Asn Ser Gly His Ile
Asp Tyr Ala Asp Ser Val 50 55 60Glu Gly Arg Phe Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr65 70 75 80Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Lys Val Ser Tyr Leu
Ser Thr Ala Ser Ser Leu Asp Tyr Trp Gly 100 105 110Gln Gly Thr Leu
Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125Gly Gly
Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 130 135
140Ser Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys
Arg145 150 155 160Ala Ser Gln Gly Ile Arg Asn Tyr Leu Ala Trp Tyr
Gln Gln Lys Pro 165 170 175Gly Lys Ala Pro Lys Leu Leu Ile Tyr Ala
Ala Ser Thr Leu Gln Ser 180 185 190Gly Val Pro Ser Arg Phe Ser Gly
Ser Gly Ser Gly Thr Asp Phe Thr 195 200 205Leu Thr Ile Ser Ser Leu
Gln Pro Glu Asp Val Ala Thr Tyr Tyr Cys 210 215 220Gln Arg Tyr Asn
Arg Ala Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val225 230 235 240Glu
Ile Lys8312PRTStreptococcus pyogenes 8Asp Ser Phe Ser Ala Asn Gln
Glu Ile Arg Tyr Ser Glu Val Thr Pro1 5 10 15Tyr His Val Thr Ser Val
Trp Thr Lys Gly Val Thr Pro Pro Ala Lys 20 25 30Phe Thr Gln Gly Glu
Asp Val Phe His Ala Pro Tyr Val Ala Asn Gln 35 40 45Gly Trp Tyr Asp
Ile Thr Lys Thr Phe Asn Gly Lys Asp Asp Leu Leu 50 55 60Cys Gly Ala
Ala Thr Ala Gly Asn Met Leu His Trp Trp Phe Asp Gln65 70 75 80Asn
Lys Glu Lys Ile Glu Ala Tyr Leu Lys Lys His Pro Asp Lys Gln 85 90
95Lys Ile Met Phe Gly Asp Gln Glu Leu Leu Asp Val Arg Lys Val Ile
100 105 110Asn Thr Lys Gly Asp Gln Thr Asn Ser Glu Leu Phe Asn Tyr
Phe Arg 115 120 125Asp Lys Ala Phe Pro Gly Leu Ser Ala Arg Arg Ile
Gly Val Met Pro 130 135 140Asp Leu Val Leu Asp Met Phe Ile Asn Gly
Tyr Tyr Leu Asn Val Tyr145 150 155 160Lys Thr Gln Thr Thr Asp Val
Asn Arg Thr Tyr Gln Glu Lys Asp Arg 165 170 175Arg Gly Gly Ile Phe
Asp Ala Val Phe Thr Arg Gly Asp Gln Ser Lys 180 185 190Leu Leu Thr
Ser Arg His Asp Phe Lys Glu Lys Asn Leu Lys Glu Ile 195 200 205Ser
Asp Leu Ile Lys Lys Glu Leu Thr Glu Gly Lys Ala Leu Gly Leu 210 215
220Ser His Thr Tyr Ala Asn Val Arg Ile Asn His Val Ile Asn Leu
Trp225 230 235 240Gly Ala Asp Phe Asp Ser Asn Gly Asn Leu Lys Ala
Ile Tyr Val Thr 245 250 255Asp Ser Asp Ser Asn Ala Ser Ile Gly Met
Lys Lys Tyr Phe Val Gly 260 265 270Val Asn Ser Ala Gly Lys Val Ala
Ile Ser Ala Lys Glu Ile Lys Glu 275 280 285Asp Asn Ile Gly Ala Gln
Val Leu Gly Leu Phe Thr Leu Ser Thr Gly 290 295 300Gln Asp Ser Trp
Asn Gln Thr Asn305 3109131PRTHomo sapiens 9Leu Ser Thr Thr Glu Val
Ala Met His Thr Ser Thr Ser Ser Ser Val1 5 10 15Thr Lys Ser Tyr Ile
Ser Ser Gln Thr Asn Asp Thr His Lys Arg Asp 20 25 30Thr Tyr Ala Ala
Thr Pro Arg Ala His Glu Val Ser Glu Ile Ser Val 35 40 45Arg Thr Val
Tyr Pro Pro Glu Glu Glu Thr Gly Glu Arg Val Gln Leu 50 55 60Ala His
His Phe Ser Glu Pro Glu Ile Thr Leu Ile Ile Phe Gly Val65 70 75
80Met Ala Gly Val Ile Gly Thr Ile Leu Leu Ile Ser Tyr Gly Ile Arg
85 90 95Arg Leu Ile Lys Lys Ser Pro Ser Asp Val Lys Pro Leu Pro Ser
Pro 100 105 110Asp Thr Asp Val Pro Leu Ser Ser Val Glu Ile Glu Asn
Pro Glu Thr 115 120 125Ser Asp Gln 130108PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 10Gly Gly Ser Gly Gly Ser Gly Gly1 5118PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 11Gly Gly Gly Ser Gly Gly Gly
Ser1 51219PRTHomo sapiens 12Met Tyr Gly Lys Ile Ile Phe Val Leu Leu
Leu Ser Glu Ile Val Ser1 5 10 15Ile Ser Ala1316PRTHomo sapiens
13Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe1
5 10 151415PRTHomo sapiens 14Cys Pro Pro Cys Pro Ala Pro Pro Val
Ala Gly Pro Ser Val Phe1 5 10 151516PRTHomo sapiens 15Cys Pro Arg
Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe1 5 10
151616PRTHomo sapiens 16Ala His His Ala Gln Ala Pro Glu Phe Leu Gly
Gly Pro Ser Val Phe1 5 10 15175PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide"MOD_RES(3)..(3)Any amino acid 17Leu Pro Xaa Thr Gly1
51893PRTHomo sapiens 18Val Ser Asp Val Pro Arg Asp Leu Glu Trp Ala
Ala Thr Pro Thr Ser1 5 10 15Leu Leu Ile Ser Trp Asp Ala Pro Ala Val
Thr Val Arg Tyr Tyr Arg 20 25 30Ile Thr Tyr Gly Glu Thr Gly Gly Asn
Ser Pro Val Gln Glu Phe Thr 35 40 45Val Pro Gly Ser Lys Ser Thr Ala
Thr Ile Ser Gly Leu Lys Pro Gly 50 55 60Val Asp Tyr Thr Ile Thr Gly
Tyr Ala Val Thr Gly Arg Gly Asp Ser65 70 75 80Pro Ala Ser Ser Lys
Pro Ile Ser Ile Asn Tyr Arg Thr 85 901912PRTArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
peptide" 19Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser1 5
10208PRTArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic peptide" 20Tyr Pro Tyr Asp Val Pro Asp Tyr1
5
* * * * *