U.S. patent application number 14/776661 was filed with the patent office on 2016-05-26 for compositions and methods to modify cells for therapeutic objectives.
The applicant listed for this patent is FRED HUTCHINSON CANCER RESEARCH CENTER. Invention is credited to Matthias Stephan.
Application Number | 20160145348 14/776661 |
Document ID | / |
Family ID | 51581420 |
Filed Date | 2016-05-26 |
United States Patent
Application |
20160145348 |
Kind Code |
A1 |
Stephan; Matthias |
May 26, 2016 |
COMPOSITIONS AND METHODS TO MODIFY CELLS FOR THERAPEUTIC
OBJECTIVES
Abstract
The present disclosure provides compositions and methods that
rapidly and selectively modify cells of the immune system to
achieve therapeutic objectives. The methods can be practiced in
vivo and any cell type that expresses a known marker can be
targeted for a therapeutic objective. The present disclosure
provides compositions and methods that rapidly and selectively
modify cells of the immune system to achieve therapeutic
objectives. The methods can be practiced in vivo and any cell type
that expresses or is associated with a known marker can be targeted
for a therapeutic objective by the modified cell.
Inventors: |
Stephan; Matthias; (Seattle,
WA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
FRED HUTCHINSON CANCER RESEARCH CENTER |
Seattle |
WA |
US |
|
|
Family ID: |
51581420 |
Appl. No.: |
14/776661 |
Filed: |
March 14, 2014 |
PCT Filed: |
March 14, 2014 |
PCT NO: |
PCT/US14/29137 |
371 Date: |
September 14, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61785907 |
Mar 14, 2013 |
|
|
|
Current U.S.
Class: |
424/450 ;
424/498; 514/44R |
Current CPC
Class: |
C07K 14/70521 20130101;
A61K 47/645 20170801; C07K 16/3069 20130101; C07K 16/40 20130101;
A61K 38/177 20130101; A61K 9/1271 20130101; A61K 38/1774 20130101;
A61K 39/3955 20130101; C07K 2319/03 20130101; C12N 15/88 20130101;
C07K 14/7051 20130101; A61K 48/00 20130101; C07K 2317/622 20130101;
C07K 2319/74 20130101; A61K 9/5123 20130101; A61K 47/6913 20170801;
A61K 9/0019 20130101; A61K 47/6455 20170801; A61K 47/6849 20170801;
C07K 16/2815 20130101; C12N 2810/859 20130101 |
International
Class: |
C07K 16/40 20060101
C07K016/40; C07K 14/725 20060101 C07K014/725; A61K 39/395 20060101
A61K039/395; C07K 16/28 20060101 C07K016/28; A61K 38/17 20060101
A61K038/17; C07K 14/705 20060101 C07K014/705; A61K 47/48 20060101
A61K047/48 |
Claims
1-42. (canceled)
43. A synthetic nanocarrier comprising (i) a lipid-coated porous
nanoparticle (ii) a lymphocyte-directing agent extending from the
surface of the nanoparticle; and (iii) a polynucleotide encoding a
chimeric antigen receptor (CAR) targeting agent within the pores of
the nanoparticle.
44. A synthetic nanocarrier of claim 43 further comprising an
endosomal release agent extending from the surface of the
nanoparticle and (ii) a nuclear localization signal within the
pores of the nanoparticle.
45. A synthetic nanocarrier of claim 43 wherein the synthetic
nanocarrier comprises a liposome, polymeric particle, metallic
particle, polymeric micelle, polyethyleneimine (PEI)/DNA complex,
or a combination thereof.
46. A synthetic nanocarrier of claim 43 wherein the
lymphocyte-directing agent selectively binds to lymphocytes in
vivo.
47. A synthetic nanocarrier of claim 43 wherein the
lymphocyte-directing agent selectively binds T cells, NK cells,
monocytes, macrophages, B cells, hematopoietic stem cells or a
combination thereof.
48. A synthetic nanocarrier of claim 43 wherein the
lymphocyte-directing agent selectively binds CCR7; CD3; CD4; CD5;
CD8; CD16; CD19; CD20; CD21; CD22; CD25; CD28; CD35; CD40; CD45RA;
CD45RO; CD52; CD62L; CD80; CD95; CD127; or CD137.
49. A synthetic nanocarrier of claim 43 wherein the
lymphocyte-directing agent selectively binds CD8.
50. A synthetic nanocarrier of claim 43 wherein the
lymphocyte-directing agent comprises a binding domain selected from
a lymphocyte receptor ligand, lymphocyte receptor antibody,
lymphocyte receptor peptide aptamer, lymphocyte receptor nucleic
acid aptamer, lymphocyte receptor spiegelmer or a combination
thereof.
51. A synthetic nanocarrier of claim 49 wherein the binding domain
consists of an ScFv fragment of a T-cell a chain antibody; T-cell
.beta. chain antibody; T-cell .gamma. chain antibody; T-cell
.DELTA. chain antibody; CCR7 antibody; CD3 antibody; CD4 antibody;
CD5 antibody; CD7 antibody; CD8 antibody; CD11b antibody; CD11c
antibody; CD16 antibody; CD19 antibody; CD20 antibody; CD21
antibody; CD22 antibody; CD25 antibody; CD28 antibody; CD34
antibody; CD35 antibody; CD40 antibody; CD45RA antibody; CD45RO
antibody; CD52 antibody; CD56 antibody; CD62L antibody; CD68
antibody; CD80 antibody; CD95 antibody; CD117 antibody; CD127
antibody; CD133 antibody; CD137 (4-1BB) antibody; CD163 antibody;
F4/80 antibody; IL-4R.alpha. antibody; Sca-1 antibody; CTLA-4
antibody; GITR antibody GARP antibody; LAP antibody; granzyme B
antibody; LFA-1 antibody; transferrin antibody; or perforin
antibody.
52. A synthetic nanocarrier of claim 43 wherein the polynucleotide
is a plasmid, a minicircle plasmid, or an mRNA molecule.
53. A synthetic nanocarrier of claim 52 wherein the polynucleotide
is a minicircle plasmid encoding a hyperactive transposase.
54. A synthetic nanocarrier of claim 43 comprising a S/MAR element,
a PiggyBac transposase-containing plasmid, a Sleeping Beauty
transposase-containing plasmid; a homo sapiens transposon-derived
Buster1 transposase-like protein gene; a human endogenous
retrovirus H protease/integrase-derived ORF1; a homo sapiens
Cas-Br-M (murine) ecotropic retroviral transforming sequence; a
homo sapiens endogenous retroviral sequence K; a homo sapiens
endogenous retroviral family W sequence; a homo sapiens LINE-1 type
transposase domain; or a homo sapiens pogo transposable
element.
55. A synthetic nanocarrier of claim 43 wherein the CAR targeting
agent comprises a binding domain for a marker associated with an
unwanted cell type.
56. A synthetic nanocarrier of claim 55 wherein the unwanted cell
type is a cancer cell.
57. A synthetic nanocarrier of claim 43 wherein the CAR targeting
agent is a surface antigen receptor or a receptor for an
intracellular antigen presented by a Major Histocompatibility
Complex antigen-presenting pathway.
58. A synthetic nanocarrier of claim 44 wherein the endosomal
release agent is selected from any one of SEQ ID NOs.29-50.
59. A synthetic nanocarrier of claim 44 wherein the NLS is selected
from any of SEQ ID NOs. 51-93.
60. A method for treating a disease associated with an antigen, the
method comprising: administering to a subject in need thereof, a
synthetic nanocarrier comprising (i) a lipid-coated porous
nanoparticle with an endosomal release agent extending from the
surface of the nanoparticle (ii) a lymphocyte-directing agent
extending from the surface of the nanoparticle; (iii) a
polynucleotide encoding a chimeric antigen receptor (CAR) targeting
agent within the pores of the nanoparticle; and (iv) a nuclear
localization signal within the pores of the nanoparticle thereby
treating the disease in the subject.
61. A method of selectively transfecting lymphocytes in a subject
in vivo, the method comprising: contacting lymphocytes within the
subjection with a synthetic nanocarrier comprising (i) a
lipid-coated porous nanoparticle with an endosomal release agent
extending from the surface of the nanoparticle (ii) a
lymphocyte-directing agent extending from the surface of the
nanoparticle; (iii) a polynucleotide encoding a chimeric antigen
receptor (CAR) targeting agent within the pores of the
nanoparticle; and (iv) a nuclear localization signal within the
pores of the nanoparticle thereby transfecting lymphocytes in the
subject in vivo.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Provisional Patent
Application No. 61/785,907, filed Mar. 14, 2013, which is
incorporated herein by reference in its entirety.
FIELD OF THE DISCLOSURE
[0002] The present disclosure provides compositions and methods
that rapidly and selectively modify cells of the immune system to
achieve therapeutic objectives. The methods can be practiced in
vivo and any cell type that expresses or is associated with a known
marker can be targeted for a therapeutic objective by the modified
cell.
BACKGROUND OF THE DISCLOSURE
[0003] One of the primary goals of clinical health research is to
develop compositions and methods that rapidly and selectively
direct cells of the immune system to achieve therapeutic
objectives. For example, vaccines are used to prime the immune
system to target antigens associated with unwanted cells. The
biological processes underlying conventional vaccines, however, can
render them ineffective against many unwanted cells based on, among
other factors, the time it takes to prime the immune system, the
amount or degree to which the natural immune system can be primed
against certain unwanted cell types and over time, the depletion of
immune system resources. As examples, conventional vaccine
approaches can be ineffective against cancer cells and cells
affected by certain infectious diseases.
[0004] Using cancer cells as an example of an unwanted cell type,
vaccines can be capable of targeting the immune system to destroy
cancer cells in some patients. The immune response using this
approach, however, requires months to mature and during this time,
cancers can significantly progress and become fatal. Thus,
conventional vaccines do not provide an adequate method to target
and destroy unwanted cancer cells.
[0005] To achieve more rapid and potent cancer cell destruction,
infusions of autologous T cells genetically targeted to tumor
antigen are currently being tested in the clinic and represent a
promising treatment option. However, T-cell transfer therapies are
also time and labor-intense and must be personalized for each
patient in cell production facilities, which are available only at
a few highly specialized cancer centers worldwide. Similar issues
are encountered with a number of other unwanted cell types. Thus,
additional solutions are needed that allow rapid and selective
direction of cells of the immune system to achieve therapeutic
objectives
SUMMARY OF THE DISCLOSURE
[0006] The present disclosure provides compositions and methods
that can rapidly and selectively direct cells of the immune system
to achieve therapeutic objectives. In particular embodiments, the
compositions and methods modify cells of the immune system, such as
T cells or natural killer (NK) cells, to target and destroy
unwanted cell types. In other embodiments, the compositions and
methods modify cells of the immune system, such as
monocytes/macrophages, to target and destroy viruses before they
infect cells and/or to target bacteria or fungus. In further
embodiments, the compositions and methods modify cells of the
immune system, such as B cells, to produce and release antibodies,
such as broadly-neutralizing antibodies. In additional embodiments,
the compositions and methods modify cells of the immune system,
such as immunosuppressive regulatory T cells (T.sub.REG) to target
and protect, rather than destroy, cell types. Compositions and
methods disclosed herein can also be used to modify stem cells to
achieve therapeutic objectives.
[0007] The described methods can be practiced in vivo rather than
requiring patient-specific isolation and culturing, as is currently
required by many cancer treatments. The methods can be practiced in
vivo because following administration to a subject, the
compositions selectively modify cells of the immune system to
achieve selected therapeutic objectives.
[0008] The compositions and methods can be used to target any cell
type for which a marker is now or later becomes known. The
compositions and methods achieve this benefit by modifying cells of
the immune system to express targeting agents for the marker of
interest.
[0009] In particular examples, the cells of the immune system are
modified to express targeting agents that bind markers, such as
antigens, on unwanted cells. Once bound to an unwanted cell, the
immune cells mediate its destruction. Alternatively, the cells of
the immune system can be modified to express targeting agents that
bind markers expressed by wanted cells or cells in the vicinity of
wanted cells. Once bound to a wanted cell or in the vicinity of a
wanted cell, the immune cells can mediate protection of the wanted
cell.
[0010] The compositions and methods disclosed herein also provide
further advantages over the current state of the art. For example,
the compositions and methods can selectively destroy unwanted cells
leaving healthy tissue undamaged. The compositions can be
manufactured on a large scale in a stable form with a long shelf
life rendering them compatible with wide distribution and
inexpensive administration to large patient populations in
outpatient settings (i.e., they provide "off-the-shelf" directed
treatments). Further, the compositions can be administered in
booster doses to reinforce immune cell targeting. Alternatively,
the administered composition can be altered over time as a
population of unwanted or wanted cell types (collectively "targets"
herein) evolves.
[0011] The compositions and methods achieve the described benefits
by providing nanocarriers. In their simplest form, the nanocarriers
include a polynucleotide encoding a targeting agent. The
nanocarrier is taken up by a cell of the immune system, which then
expresses the encoded targeting agent. The targeting agent
selectively binds a marker on a target, directing the cells of the
immune system to the site of the therapeutic objective. If the
expressed targeting agent is an unwanted cell-targeting agent (such
as an antibody or a receptor for a cancer antigen), once bound, the
modified immune cell will mediate the destruction of the unwanted
cell. If the expressed targeting agent is a wanted cell-targeting
agent (such as a receptor for a marker expressed by a cell
undergoing autoimmune attack), once bound, the modified immune cell
will mediate the protection of the wanted cell.
[0012] In some embodiments, nanocarriers further include
lymphocyte-directing agents. Lymphocyte-directing agents can
achieve selective uptake of the nanocarriers by cells of interest
for a particular therapeutic objective. For example, the
lymphocyte-directing agents can include binding domains extending
from the surface of the nanocarriers that facilitate uptake by
lymphocytes or particular classes of lymphocytes. Nanocarriers can
also include lymphocyte-directing agents that achieve selective
uptake by more than one cell type.
[0013] Nanocarriers can also further include one or more of: an
endosomal release agent to facilitate release of the polynucleotide
from endosomal compartments of the lymphocytes and/or a nuclear
localization signal (NLS) to direct the polynucleotide into the
nucleus of the lymphocyte for expression, particularly when, for
example, the polynucleotide comprises plasmid DNA.
[0014] In particular embodiments, the nanocarriers comprise a
porous nanoparticle surrounded by a coating. In these embodiments,
the polynucleotide (and optionally the NLS) can be within the pores
of the nanoparticle and the optional lymphocyte-directing agent and
endosomal release agent can extend from the surface of the
coating.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1: Schematic of described strategy to rapidly and
selectively modify immune cells for therapeutic objectives using
synthetic nanocarriers. Nanocarriers are loaded with
polynucleotides that encode a targeting agent (e.g. tumor- or
virus-specific T-cell receptor). Surface-anchored lymphocyte
directing agents (e.g. anti-CD3 antibody) enable these nanocarriers
to bind lymphocytes selectively. Upon infusion into a patient's
bloodstream, the nanocarriers transfer the polynucleotide molecules
into lymphocytes, which subsequently express the targeting agent on
their surface. Lymphocytes then recognize and lyse cells of
interest (e.g. cancerous or virus-infected cells).
[0016] FIG. 2: (A) Schematic representation of the protocell
nanoparticle used in studies described herein. (B) A representative
TEM image of a protocell nanoparticle.
[0017] FIG. 3: Schematic representation of minicircle DNA construct
used in studies described herein. Structure of the P28z minicircle.
The prostate-specific membrane antigen (PSMA)-targeting chimeric
antigen receptor P28z is expressed under the control of the
T-cell-specific CD3-delta promoter.
[0018] FIG. 4: Redirecting T-cell specificity toward prostate tumor
via nanoparticle-mediated gene transfer. (A) Prostate-Specific
Membrane Antigen (PSMA)-specific chimeric antigen receptor P28z (B)
Flow cytometric measurement of surface P28z expression on mouse
effector T cells 30 hours after incubation with "empty" (left
panel) or P28z minicircle-loaded (right panel) protocell
nanoparticles. (C) 51Cr release assay of T cells 30 h after
nanoparticle transfection targeting PSMA-positive TRAMP prostate
tumor cells. (D) Light microscope images of
nanoparticle-transfected T cells co-cultured on a TRAMP prostate
tumor cell monolayer. (E) Flow cytometric measurement of protocell
binding to circulating host T cells 6 hours after intravenous
injection of 1.times.10.sup.11 fluorescently tagged
nanoparticles.
[0019] FIG. 5: Repeated injections of nanocarriers loaded DNA
encoding the P28z chimeric antigen receptor brings about T-cell
mediated regression of prostate tumor in mice. Luciferase tagged
TRAMP-PSMA prostate tumor cells were transplanted into the dorsal
lobe of the prostate gland of C57BL/6 mice. Two weeks later (Day
0), mice were treated with five high-dose bolus injections of
1.times.10.sup.12 CD3-targeting nanoparticles carrying
P28z-encoding transgenes (Day 0, Day 2, Day 4, Day 6, and Day 8).
Control mice received no nanoparticles. (A) Sequential
bioluminescence imaging of Firefly luciferase-expressing TRAMP-PSMA
tumors. (B) Quantified bioluminescent tumor signal. Pairwise
differences in bioluminescent photon counts between treatment
groups were statistically analyzed with the Wilcoxon rank-sum test.
*, **=Significant P<0.0001.
[0020] FIG. 6: Schematic representation of minicircle DNA
constructs. (A) Structure of the P28z minicircle. The
prostate-specific membrane antigen (PSMA)-targeting chimeric
antigen receptor P28z is expressed under the control of the T-cell
specific CD3-delta promoter. (B) A scaffold/matrix attachment
region (S/MAR) is shown upstream of the poly-A signal to allow
sustained episomal replication. (C) Alternatively, the gene
expression cassette can be flanked by the piggyBac inverted
terminal repeats. The piggyBac transposon is a mobile genetic
element that efficiently transposes between vector and chromosome
via a "cut and paste" mechanism. This integration event is mediated
by piggyBac transposase. Therefore, in piggyBac transposon studies,
a plasmid encoding the hyperactive form of piggyBac transposase
iPB7 will be co-encapsulated into protocell nanoparticles.
DETAILED DESCRIPTION
[0021] The present disclosure provides compositions and methods
that can rapidly and selectively direct cells within the body to
achieve therapeutic objectives. In particular embodiments, the
compositions and methods modify cells of the immune system, such as
T-cells or NK cells, to target and destroy unwanted cell types. In
other embodiments, the compositions and methods modify cells of the
immune system, such as monocytes/macrophages to target and destroy
viruses before they infect cells and/or bacterial or fungal cells.
In further embodiments, the compositions and methods modify cells
of the immune system, such as B cells, to produce and release
antibodies, such as broadly-neutralizing antibodies. In additional
embodiments, the compositions and methods modify cells of the
immune system, such as immunosuppressive T.sub.REG cells to target
and protect cell types from, for example, autoimmune attack.
Compositions and methods disclosed herein can also be used to
modify stem cells to achieve therapeutic objectives.
[0022] The described methods can be practiced in vivo rather than
requiring patient-specific isolation and culturing, as is currently
required by many treatments. The methods can be practiced in vivo
because following administration to a subject, the compositions
selectively modify cells of interest to achieve the therapeutic
objective.
[0023] As an example, one of the primary goals of clinical health
research is to develop compositions and methods to rapidly and
selectively direct the immune system to destroy unwanted cells. For
example, vaccines are used to prime the immune system to target
antigens associated with unwanted cells. The biological processes
underlying conventional vaccines, however, can render them
ineffective against many unwanted cells based on, among other
factors, the time it takes to prime the immune system, the amount
or degree to which the natural immune system can be primed against
certain unwanted cell types and over time, the depletion of immune
system resources.
[0024] The present disclosure provides compositions and methods
that can rapidly modify cells of the immune system to target and
destroy unwanted cell types. The methods can be practiced in vivo
rather than requiring patient-specific isolation and culturing, as
is currently required by many cancer treatments. The methods can be
practiced in vivo because following administration to a subject,
the compositions selectively modify cells of the immune system to
target unwanted cell types.
[0025] The compositions and methods can be used to target any cell
type for which a marker is now or later becomes known. The
compositions and methods achieve this benefit by modifying cells of
the immune system to express targeting agents for the marker
expressed by the target or in the vicinity of a target. In
particular examples, the cells of the immune system are modified to
express targeting agents that bind markers, such as antigens, on
unwanted cells. Once bound to an unwanted cell, the immune cells
mediate its destruction. Alternatively, cells of the immune system
can be modified to express targeting agents that bind markers on or
in the vicinity of wanted cells. Once bound to a wanted cell or in
the wanted cell's vicinity, the immune cell can mediate its
protection.
[0026] The compositions and methods achieve the described benefits
by providing nanocarriers that include a polynucleotide encoding a
targeting agent. Cells that uptake the nanocarrier will begin to
express the polynucleotide, thereby expressing the targeting agent.
The targeting agent directs the modified immune cell to the site of
the therapeutic objective. In one example, a lymphocyte uptakes the
nanocarrier and begins to express an unwanted cell targeting agent.
In this embodiment, the lymphocyte then binds and mediates the
destruction of the unwanted cell type.
[0027] Additional embodiments of the nanocarriers include
lymphocyte-directing agents that selectively deliver the
nanocarriers to cells of interest. The compositions can further
include one or more of: an endosomal release agent to facilitate
release of the polynucleotide from endosomal compartments of cells
of the immune system and/or a nuclear localization signal (NLS) to
direct the polynucleotide into the nucleus of the cell for
expression if, for example, the polynucleotide includes plasmid
DNA.
[0028] In particular embodiments, the nanocarriers comprise a
porous nanoparticle surrounded by a coating. In these embodiments,
the polynucleotide (and optionally the NLS) can be within the pores
of the nanoparticle and the lymphocyte-directing agent (and
optionally the endosomal release agent) can extend from the surface
of the coating. Each of these components is now described in
further detail.
[0029] Lymphocyte-Directing Agents. The lymphocyte-directing agents
of the disclosed compositions selectively bind immune cells of
interest. In particular embodiments, the cells are lymphocytes. In
these embodiments, lymphocyte-directing agents can direct the
compositions to any lymphocyte capable of, without limitation, (i)
targeting and killing unwanted cells, (ii) targeting unwanted cells
for killing by other cell types, (iii) mediating unwanted cell
killing; (iv) targeting viruses for destruction before viral entry
into cells, (v) antibody production and/or (vi) targeting and
protecting beneficial cells. As described herein, lymphocytes
include T-cells, B cells, natural killer (NK) cells,
monocytes/macrophages and hematopoietic stem cells.
[0030] Several different subsets of T-cells have been discovered,
each with a distinct function. In particular embodiments,
lymphocyte-directing agents achieve selective direction to
particular lymphocyte populations through receptor-mediated
endocytosis. For example, a majority of T-cells have a T-cell
receptor (TCR) existing as a complex of several proteins. The
actual T-cell receptor is composed of two separate peptide chains,
which are produced from the independent T-cell receptor alpha and
beta (TCR.alpha. and TCR.beta.) genes and are called .alpha.- and
.beta.-TCR chains. Lymphocyte directing agents disclosed herein can
bind .alpha.- and/or .beta.-TCR chains to achieve selective
delivery of a polynucleotide to these T cells.
[0031] .gamma..delta. T-cells represent a small subset of T-cells
that possess a distinct T-cell receptor (TCR) on their surface. In
.gamma..delta. T-cells, the TCR is made up of one .gamma.-chain and
one .delta.-chain. This group of T-cells is much less common (2% of
total T-cells) than the .alpha..beta.-cells. Nonetheless,
lymphocyte-directing agents disclosed herein can bind .gamma.-
and/or .delta. TCR chains to achieve selective delivery of a
polynucleotide to these T cells.
[0032] CD3 is expressed on all mature T cells. Accordingly,
lymphocyte-directing agents disclosed herein can bind CD3 to
achieve selective delivery of a polynucleotide to all mature
T-cells. Activated T-cells express 4-1BB (CD137). Accordingly,
lymphocyte-directing agents disclosed herein can bind 4-1 BB to
achieve selective delivery of a polynucleotide to activated
T-cells. CD5 and transferrin receptor are also expressed on T-cells
and can be used to achieve selective delivery of a polynucleotide
to T-cells.
[0033] T-cells can further be classified into helper cells (CD4+
T-cells) and cytotoxic T-cells (CTLs, CD8+ T-cells), which comprise
cytolytic T-cells. T helper cells assist other white blood cells in
immunologic processes, including maturation of B cells into plasma
cells and activation of cytotoxic T-cells and macrophages, among
other functions. These cells are also known as CD4+ T-cells because
they express the CD4 protein on their surface. Helper T-cells
become activated when they are presented with peptide antigens by
MHC class II molecules that are expressed on the surface of antigen
presenting cells (APCs). Once activated, they divide rapidly and
secrete small proteins called cytokines that regulate or assist in
the active immune response. Lymphocyte-directing agents disclosed
herein can bind CD4 to achieve selective delivery of a
polynucleotide to T helper cells.
[0034] Cytotoxic T-cells destroy virally infected cells and tumor
cells, and are also implicated in transplant rejection. These cells
are also known as CD8+ T-cells because they express the CD8
glycoprotein at their surface. These cells recognize their targets
by binding to antigen associated with MHC class I, which is present
on the surface of nearly every cell of the body.
Lymphocyte-directing agents disclosed herein can bind CD8 to
achieve selective delivery of a polynucleotide to CTL.
[0035] "Central memory" T-cells (or "T.sub.CM") as used herein
refers to an antigen experienced CTL that expresses CD62L or CCR7
and CD45RO on the surface thereof, and does not express or has
decreased expression of CD45RA as compared to naive cells. In
particular embodiments, central memory cells are positive for
expression of CD62L, CCR7, CD25, CD127, CD45RO, and CD95, and have
decreased expression of CD45RA as compared to naive cells.
Lymphocyte-directing agents disclosed herein can bind CD62L, CCR7,
CD25, CD127, CD45RO and/or CD95 to achieve selective delivery of a
polynucleotide to T.sub.CM.
[0036] "Effector memory" T-cell (or "T.sub.EM") as used herein
refers to an antigen experienced T-cell that does not express or
has decreased expression of CD62L on the surface thereof as
compared to central memory cells, and does not express or has
decreased expression of CD45RA as compared to a naive cell. In
particular embodiments, effector memory cells are negative for
expression of CD62L and CCR7, compared to naive cells or central
memory cells, and have variable expression of CD28 and CD45RA.
Effector T-cells are positive for granzyme B and perforin as
compared to memory or naive T-cells. Lymphocyte-directing agents
disclosed herein can bind granzyme B and/or perform to achieve
selective delivery of a polynucleotide to T.sub.EM.
[0037] Regulatory T cells ("T.sub.REG") are a subpopulation of T
cells, which modulate the immune system, maintain tolerance to
self-antigens, and abrogate autoimmune disease. T.sub.REG express
CD25, CTLA-4, GITR, GARP and LAP. Lymphocyte-directing agents
disclosed herein can bind CD25, CTLA-4, GITR, GARP and/or LAP to
achieve selective delivery of a polynucleotide to naive
T.sub.REG.
[0038] "Naive" T-cells as used herein refers to a non-antigen
experienced T cell that expresses CD62L and CD45RA, and does not
express CD45RO as compared to central or effector memory cells. In
some embodiments, naive CD8+ T lymphocytes are characterized by the
expression of phenotypic markers of naive T-cells including CD62L,
CCR7, CD28, CD127, and CD45RA. Lymphocyte-directing agents
disclosed herein can bind CD62L, CCR7, CD28, CD127 and/or CD45RA to
achieve selective delivery of a polynucleotide to naive
T-cells.
[0039] Natural killer cells (also known as NK cells, K cells, and
killer cells) are activated in response to interferons or
macrophage-derived cytokines. They serve to contain viral
infections while the adaptive immune response is generating
antigen-specific cytotoxic T cells that can clear the infection. NK
cells express CD8, CD16 and CD56 but do not express CD3.
Lymphocyte-directing agents disclosed herein can bind CD8, CD16
and/or CD56 to achieve selective delivery of a polynucleotide to NK
cells.
[0040] Macrophages (and their precursors, monocytes) reside in
every tissue of the body (in certain instances as microglia,
Kupffer cells and osteoclasts) where they engulf apoptotic cells,
pathogens and other non-self components. Because
monocytes/macrophages engulf non-self components, a particular
macrophage- or monocyte-directing agent is not required on the
nanocarriers described herein for selective uptake by these cells.
Alternatively, lymphocyte-directing agents disclosed herein can
bind CD11b, F4/80; CD68; CD11c; IL-4R.alpha.; and/or CD163 to
achieve selective delivery of a polynucleotide to
monocytes/macrophages.
[0041] B cells can be distinguished from other lymphocytes by the
presence of the B cell receptor (BCR). The principal function of B
cells is to make antibodies. B cells express CD5, CD19, CD20, CD21,
CD22, CD35, CD40, CD52, and CD80. Lymphocyte-directing agents
disclosed herein can bind CD5, CD19, CD20, CD21, CD22, CD35, CD40,
CD52, and/or CD80 to achieve selective delivery of a polynucleotide
to B-cells.
[0042] Lymphocyte function-associated antigen 1 (LFA-1) is
expressed by all T-cells, B-cells and monocytes/macrophages.
Accordingly, lymphocyte-directing agents disclosed herein can bind
LFA-1to achieve selective delivery of a polynucleotide to T-cells,
B-cells and monocytes/macrophages.
[0043] Hematopoietic stem cells can also be targeted for selective
delivery of nanocarriers disclosed herein. Hematopoietic stem cells
express CD34, CD133, Sca-1 and CD117. Lymphocyte-directing agents
disclosed herein can bind CD34, CD133, Sca-1 and/or CD117 to
achieve selective delivery of a polynucleotide to hematopoietic
stem cells.
[0044] "Selective delivery" means that polynucleotides are
delivered and expressed by one or more selected lymphocyte
populations. In particular embodiments, selective delivery is
exclusive to a selected lymphocyte population. In further
embodiments, at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% of
administered polynucleotides are delivered and/or expressed by a
selected lymphocyte population. In further embodiments, selective
delivery ensures that non-lymphocyte cells do not express delivered
polynucleotides. For example, when the targeting agent is a T-cell
receptor (TCR) gene, selectivity is ensured because only T cells
have the zeta chains required for TCR expression. Selective
delivery can also be based on lack of polynucleotide uptake into
unselected cells or based on the presence of a specific promoter
within the polynucleotide sequence when the polynucleotide includes
plasmid DNA. For example, plasmid DNA can include a T-cell-specific
CD3-delta promoter. Additional promoters that can achieve selective
delivery include: the murine stem cell virus promoter or the distal
Ick promoter for T cells or hematopoietic stem cells; the CD45
promoter, WASP promoter or IFN-beta promoter for hematopoietic stem
cells; the B29 promoter for B cells; or the CD14 promoter or the
CD11b promoter for monocytes/macrophages.
[0045] As indicated, lymphocyte-directing agents can include
binding domains for motifs found on lymphocyte cells.
Lymphocyte-directing agents can also include any selective binding
mechanism allowing selective uptake into lymphocytes. In particular
embodiments, lymphocyte-directing agents include binding domains
for T-cell receptor motifs; T-cell .alpha. chains; T-cell .beta.
chains; T-cell .gamma. chains; T-cell .delta. chains; CCR7; CD3;
CD4; CD5; CD7; CD8; CD11b; CD11c; CD16; CD19; CD20; CD21; CD22;
CD25; CD28; CD34; CD35; CD40; CD45RA; CD45RO; CD52; CD56; CD62L;
CD68;CD80; CD95; CD117; CD127; CD133; CD137 (4-1BB); CD163; F4/80;
IL-4R.alpha.; Sca-1; CTLA-4; GITR; GARP; LAP; granzyme B; LFA-1;
transferrin receptor; and combinations thereof.
[0046] In particular embodiments, binding domains include cell
marker ligands, receptor ligands, antibodies, peptides, peptide
aptamers, nucleic acids, nucleic acid aptamers, spiegelmers or
combinations thereof. Within the context of lymphocyte-directing
agents, binding domains include any substance that binds to another
substance to form a complex capable of mediating endocytosis.
[0047] "Antibodies" are one example of binding domains and include
whole antibodies or binding fragments of an antibody, e.g., Fv,
Fab, Fab', F(ab').sub.2, Fc, and single chain Fv fragments (scFvs)
or any biologically effective fragments of an immunoglobulin that
bind specifically to a motif expressed by a lymphocyte. Antibodies
or antigen binding fragments include all or a portion of polyclonal
antibodies, monoclonal antibodies, human antibodies, humanized
antibodies, synthetic antibodies, chimeric antibodies, bispecific
antibodies, mini bodies, and linear antibodies.
[0048] Antibodies from human origin or humanized antibodies have
lowered or no immunogenicity in humans and have a lower number of
non-immunogenic epitopes compared to non-human antibodies.
Antibodies and their fragments will generally be selected to have a
reduced level or no antigenicity in human subjects.
[0049] Antibodies that specifically bind a motif expressed by a
lymphocyte can be prepared using methods of obtaining monoclonal
antibodies, methods of phage display, methods to generate human or
humanized antibodies, or methods using a transgenic animal or plant
engineered to produce antibodies as is known to those of ordinary
skill in the art (see, for example, U.S. Pat. Nos. 6,291,161 and
6,291,158). Phage display libraries of partially or fully synthetic
antibodies are available and can be screened for an antibody or
fragment thereof that can bind to a lymphocyte motif. For example,
binding domains may be identified by screening a Fab phage library
for Fab fragments that specifically bind to a target of interest
(see Hoet et al., Nat. Biotechnol. 23:344, 2005). Phage display
libraries of human antibodies are also available. Additionally,
traditional strategies for hybridoma development using a target of
interest as an immunogen in convenient systems (e.g., mice, HuMAb
mouse.RTM., TC mouse.TM., KM-mouse.RTM., llamas, chicken, rats,
hamsters, rabbits, etc.) can be used to develop binding domains. In
particular embodiments, antibodies specifically bind to motifs
expressed by a selected lymphocyte and do not cross react with
nonspecific components or unrelated targets. Once identified, the
amino acid sequence or polynucleotide sequence coding for the
antibody can be isolated and/or determined.
[0050] In particular embodiments, binding domains of
lymphocyte-directing agents include T-cell receptor motif
antibodies; T-cell .alpha. chain antibodies; T-cell .beta. chain
antibodies; T-cell .gamma. chain antibodies; T-cell .delta. chain
antibodies; CCR7 antibodies; CD3 antibodies; CD4 antibodies; CD5
antibodies; CD7 antibodies; CD8 antibodies; CD11b antibodies; CD11c
antibodies; CD16 antibodies; CD19 antibodies; CD20 antibodies; CD21
antibodies; CD22 antibodies; CD25 antibodies; CD28 antibodies; CD34
antibodies; CD35 antibodies; CD40 antibodies; CD45RA antibodies;
CD45R0 antibodies; CD52 antibodies; CD56 antibodies; CD62L
antibodies; CD68 antibodies; CD80 antibodies; CD95 antibodies;
CD117 antibodies; CD127 antibodies; CD133 antibodies; CD137 (4-1BB)
antibodies; CD163 antibodies; F4/80 antibodies; IL-4R.alpha.
antibodies; Sca-1 antibodies; CTLA-4 antibodies; GITR antibodies
GARP antibodies; LAP antibodies; granzyme B antibodies; LFA-1
antibodies; or transferrin receptor antibodies. These binding
domains also can consist of scFv fragments of the foregoing
antibodies. In one particular embodiment, the lymphocyte-directing
agent binding domain includes the scFv fragment (SEQ ID NO. 1) of
the PSMA-specific chimeric antigen receptor (CAR), P28z.
[0051] Peptide aptamers include a peptide loop (which is specific
for a target protein) attached at both ends to a protein scaffold.
This double structural constraint greatly increases the binding
affinity of the peptide aptamer to levels comparable to an
antibody. The variable loop length is typically 8 to 20 amino acids
(e.g., 8 to 12 amino acids), and the scaffold may be any protein
which is stable, soluble, small, and non-toxic (e.g.,
thioredoxin-A, stefin A triple mutant, green fluorescent protein,
eglin C, and cellular transcription factor Spl). Peptide aptamer
selection can be made using different systems, such as the yeast
two-hybrid system (e.g., Gal4 yeast-two-hybrid system) or the LexA
interaction trap system.
[0052] Nucleic acid aptamers are single-stranded nucleic acid (DNA
or RNA) ligands that function by folding into a specific globular
structure that dictates binding to target proteins or other
molecules with high affinity and specificity, as described by
Osborne et al., Curr. Opin. Chem. Biol. 1:5-9, 1997; and Cerchia et
al., FEBS Letters 528:12-16, 2002. In particular embodiments,
aptamers are small (.about.15 KD; or between 15-80 nucleotides or
between 20-50 nucleotides). Aptamers are generally isolated from
libraries consisting of 10.sup.14-10.sup.15 random oligonucleotide
sequences by a procedure termed SELEX (systematic evolution of
ligands by exponential enrichment; see, for example, Tuerk et al.,
Science, 249:505-510, 1990; Green et al., Methods Enzymology.
75-86, 1991; and Gold et al., Annu. Rev. Biochem., 64: 763-797,
1995). Further methods of generating aptamers are described in, for
example, U.S. Pat. Nos. 6,344,318; 6,331,398; 6,110,900; 5,817,785;
5,756,291; 5,696,249; 5,670,637; 5,637,461; 5,595,877; 5,527,894;
5,496,938; 5,475,096; and 5,270,16. Spiegelmers are similar to
nucleic acid aptamers except that at least one .beta.-ribose unit
is replaced by .beta.-D-deoxyribose or a modified sugar unit
selected from, for example, .beta.-D-ribose, .alpha.-D-ribose,
.beta.-L-ribose.
[0053] Other agents that can facilitate internalization by and/or
transfection of lymphocytes, such as poly(ethyleneimine)/DNA
(PEI/DNA) complexes can also be used.
[0054] Polynucleotides Encoding Targeting Agents. As used herein,
the term "polynucleotide" includes a nucleic acid molecule that
contains a nucleic acid sequence such that upon introduction into a
targeted lymphocyte, the nucleic acid molecule can cause
transcription and resulting translation of targeting agents encoded
by the nucleic acid sequence of the nucleic acid molecule. In
particular embodiments, the targeting agent is an unwanted cell
targeting agent. In further embodiments, the targeting agent is a
wanted cell targeting agent.
[0055] As used herein, the term "gene" refers to a nucleic acid
sequence that encodes a targeting agent. This definition includes
various sequence polymorphisms, mutations, and/or sequence variants
wherein such alterations do not affect the function of the encoded
targeting agent. The term "gene" may include not only coding
sequences but also regulatory regions such as promoters, enhancers,
and termination regions. The term further can include all introns
and other DNA sequences spliced from the mRNA transcript, along
with variants resulting from alternative splice sites. Nucleic acid
sequences encoding the targeting agent can be DNA or RNA that
directs the expression of the targeting agent. These nucleic acid
sequences may be a DNA strand sequence that is transcribed into RNA
or an RNA sequence that is translated into protein. The nucleic
acid sequences include both the full-length nucleic acid sequences
as well as non-full-length sequences derived from the full-length
protein. The sequences can also include degenerate codons of the
native sequence or sequences that may be introduced to provide
codon preference in a specific lymphocyte. Gene sequences to encode
targeting agents disclosed herein are available in publicly
available databases and publications, incorporated by reference
herein.
[0056] As used herein, the term "encoding" refers to a property of
sequences of nucleotides in a polynucleotide, such as a plasmid, a
gene, cDNA, mRNA, to serve as templates for synthesis of targeting
agents. A polynucleotide can, e.g., encode a protein if
transcription and translation of mRNA produced by that gene
produces the protein in a cell or other biological system. Unless
otherwise specified, polynucleotides having a sequence encoding a
targeting agent include all nucleotide sequences that are
degenerate versions of each other and that encode the same amino
acid sequence. The polynucleotides that encode proteins and RNA can
also include introns.
[0057] In some embodiments, the polynucleotide includes a plasmid,
a cDNA, or an mRNA that can include, e.g., a sequence (e.g., a
gene) for expressing a targeting agent. Suitable plasmids include
standard plasmid vectors and minicircle plasmids that can be used
to transfer a gene to a lymphocyte. The polynucleotides (e.g.,
minicircle plasmids) can further include any additional sequence
information to facilitate transfer of the genetic material (e.g., a
sequence encoding a receptor to an antigen) to lymphocytes. For
example, the polynucleotides can include promoters, such as general
promoters, tissue-specific promoters, cell-specific promoters,
and/or promoters specific for the nucleus or cytoplasm. Promoters
and plasmids (e.g., minicircle plasmids) are generally well known
in the art and can be prepared using conventional techniques. As
described further herein, the polynucleotides can be used to
transfect lymphocytes. Unless otherwise specified, the terms
transfect, transfected, or transfecting can be used to indicate the
presence of exogenous polynucleotides or the expressed polypeptide
therefrom in a lymphocyte. A number of vectors are known to be
capable of mediating transfer of genes to lymphocytes, as is known
in the art.
[0058] In particular embodiments, the transfected polynucleotides
can edit the antigen-specificity of lymphocytes without affecting
off-target bystander cells (i.e., provide for selective delivery as
defined herein). For example, delivered genes can be expressed
under the control of a lymphocyte-specific promoter. In particular
embodiments, the promoters can be included in minicircle plasmids
that are a form of supercoiled DNA molecule for nonviral gene
transfer, which have neither bacterial origin of replication nor
antibiotic resistance marker. They are thus smaller and potentially
safer than the standard plasmids currently used in gene
therapy.
[0059] To sustain the expression of transferred targeting agent
genes, for example, in rapidly dividing lymphocytes, a
scaffold/matrix attachment region can also be inserted into the
polynucleotides. Polynucleotides containing an expression cassette
linked to a S/MAR element, can autonomously replicate
extra-chromosomally in dividing cells. In some embodiments,
PiggyBac or Sleeping Beauty transposase-containing plasmids can
also be used to stably integrate nanocarrier-delivered targeting
agent genes into the genome of transfected lymphocytes. Other
options to sustain expression include homo sapiens
transposon-derived Buster1 transposase-like protein gene; human
endogenous retrovirus H protease/integrase-derived ORF1; homo
sapiens Cas-Br-M (murine) ecotropic retroviral transforming
sequence; homo sapiens endogenous retroviral sequence K; homo
sapiens endogenous retroviral family W; homo sapiens LINE-1 type
transposase domain; or homo sapiens pogo transposable element.
[0060] When a delivered polynucleotide is mRNA, backbone
modifications can increase the mRNA's stability making resistant to
premature cleavage.
[0061] Targeted Cells & Associated Markers. Targeted cells can
be unwanted cells or wanted cells. Unwanted cells include any cell
type that is (i) capable of recognition and destruction by the
immune system; and (ii) deemed undesirable by a subject, physician,
veterinarian or researcher. Unwanted cells include (i) eukaryotic
cells that are either cancerous or infected with a pathogen such as
a virus and (ii) prokaryotic cells, such as certain bacteria, fungi
or yeast. Wanted cells include any cell type that is (i) capable of
recognition and protection by the immune system; and (ii) deemed
desirable by a subject, physician, veterinarian or researcher.
Wanted cells can include cells undergoing auto-immune attack or
bacteria that are beneficial to the health of a microbiome.
[0062] For targeting according to the compositions and methods
disclosed herein, unwanted or wanted cells must be associated with
a marker that is currently known or later discovered. In particular
embodiments, the markers are antigens. Antigens refer to substances
capable of either binding to an antigen binding region of an
immunoglobulin molecule or of eliciting an immune response, e.g., a
T cell-mediated immune response by the presentation of the antigen
on Major Histocompatibility Antigen (MHC) cellular proteins.
"Antigens" include antigenic determinants, haptens, and immunogens,
which may be peptides, small molecules, carbohydrates, lipids,
nucleic acids or combinations thereof. When referencing antigens
that are processed for presentation to T cells, the term "antigen"
refers to those portions of the antigen (e.g., a peptide fragment)
that is a T cell epitope presented by MHC to the T cell receptor.
When used in the context of a B cell mediated immune response in
the form of an antibody that is specific for an "antigen", the
portion of the antigen that binds to the complementarity
determining regions of the variable domains of the antibody (light
and heavy) is referenced. The bound portion may be a linear or
three-dimensional epitope.
[0063] Cancer Markers. In particular embodiments, markers are
expressed by unwanted cells from cancers. Exemplary cancers include
adrenal cancers, bladder cancers, blood cancers, bone cancers,
brain cancers, breast cancers, carcinoma, cervical cancers, colon
cancers, colorectal cancers, corpus uterine cancers, ear, nose and
throat (ENT) cancers, endometrial cancers, esophageal cancers,
gastrointestinal cancers, head and neck cancers, Hodgkin's disease,
intestinal cancers, kidney cancers, larynx cancers, leukemias,
liver cancers, lymph node cancers, lymphomas, lung cancers,
melanomas, mesothelioma, myelomas, nasopharynx cancers,
neuroblastomas, non-Hodgkin's lymphoma, oral cancers, ovarian
cancers, pancreatic cancers, penile cancers, pharynx cancers,
prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers,
stomach cancers, teratomas, testicular cancers, thyroid cancers,
uterine cancers, vaginal cancers, vascular tumors, and metastases
thereof.
[0064] Particular antigen markers associated with cancers cells
include A33; BAGE; Bcl-2; .beta.-catenin; CAl25; CA19-9; CD5; CD19;
CD20; CD21; CD22; CD33; CD37; CD45; CD123; CEA; c-Met; CS-1; cyclin
B1; DAGE; EBNA; EGFR; ephrinB2; estrogen receptor; FAP; ferritin;
folate-binding protein; GAGE; G250; GD-2; GM2; gp75, gp100 (Pmel
17); HER-2/neu; HPV E6; HPV E7; Ki-67; LRP; mesothelin, p53, PRAME;
progesterone receptor; PSA; PSMA; MAGE; MART; mesothelin; MUC;
MUM-1-B; myc; NYESO-1; ras; RORI; survivin; tenascin; TSTA
tyrosinase; VEGF; and WT1.
[0065] Without limiting the foregoing, the particular following
cancers can be treated by targeting the associated provided
antigens: leukemia/lymphoma (CD19, CD20, CD22, ROR1, CD33);
multiple myeloma (B-cell maturation antigen (BCMA)); prostate
cancer (PSMA, WT1, Prostate Stem Cell antigen (PSCA), SV40 T);
breast cancer (HER2, ERBB2); stem cell cancer (CD133); ovarian
cancer (L1-CAM, extracellular domain of MUC16 (MUC-CD), folate
binding protein (folate receptor), Lewis Y); renal cell carcinoma
(carboxy-anhydrase-IX (CAIX); melanoma (GD2); and pancreatic cancer
(mesothelin, CEA, CD24).
[0066] In further particular examples, cancer cell antigens
include:
TABLE-US-00001 Cancer Antigen Sequence SEQ ID NO. PSMA
MWNLLHETDSAVATARRPRWLCAGALVLAGGFFLL 2
GFLFGWFIKSSNEATNITPKHNMKAFLDELKAENIKK
FLYNFTQIPHLAGTEQNFQLAKQIQSQWKEFGLDSV
ELAHYDVLLSYPNKTHPNYISIINEDGNEIFNTSLFEP
PPPGYENVSDIVPPFSAFSPQGMPEGDLVYVNYAR
TEDFFKLERDMKINCSGKIVIARYGKVFRGNKVKNA
QLAGAKGVILYSDPADYFAPGVKSYPDGWNLPGG
GVQRGNILNLNGAGDPLTPGYPANEYAYRRGIAEA
VGLPSIPVHPIGYYDAQKLLEKMGGSAPPDSSWRG
SLKVPYNVGPGFTGNFSTQKVKMHIHSTNEVTRIYN
VIGTLRGAVEPDRYVILGGHRDSWVFGGIDPQSGA
AWHEIVRSFGTLKKEGWRPRRTILFASWDAEEFGL
LGSTEWAEENSRLLQERGVAYINADSSIEGNYTLRV
DCTPLMYSLVHNLTKELKSPDEGFEGKSLYESWTK
KSPSPEFSGMPRISKLGSGNDFEVFFQRLGIASGRA
RYTKNWETNKFSGYPLYHSVYETYELVEKFYDPMF
KYHLTVAQVRGGMVFELANSIVLPFDCRDYAWLR
KYADKIYSISMKHPQEMKTYSVSFDSLFSAVKNFTEI
ASKFSERLQDFDKSNPIVLRMMNDQLMFLERAFIDP
LGLPDRPFYRHVIYAPSSHNKYAGESFPGIYDALFDI
ESKVDPSKAWGEVKRQIYVAAFTVQAAAETLSEVA PSCA
MKAVLLALLMAGLALQPGTALLCYSCKAQVSNEDC 3
LQVENCTQLGEQCWTARIRAVGLLTVISKGCSLNCV
DDSQDYYVGKKNITCCDTDLCNASGAHALQPAAAIL ALLPALGLLLWGPGQL Mesothelin
MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRT 4
LAGETGQEAAPLDGVLANPPNISSLSPRQLLGFPCA
EVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRL
SEPPEDLDALPLDLLLFLNPDAFSGPQACTHFFSRIT
KANVDLLPRGAPERQRLLPAALACWGVRGSLLSEA
DVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPL
DQDQQEAARAALQGGGPPYGPPSTWSVSTMDAL
RGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQP
ERTILRPRFRREVEKTACPSGKKAREIDESLIFYKKW
ELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLD
ELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLE
TLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKD
TLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQD
LDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFL
GGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLT
VAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDD
LDTLGLGLQGGIPNGYLVLDLSVQEALSGTPCLLGP GPVLTVLALLLASTLA CD19
MPPPRLLFFLLFLTPMEVRPEEPLWKVEEGDNAVL 5
QCLKGTSDGPTQQLTWSRESPLKPFLKLSLGLPGL
GIHMRPLASWLFIFNVSQQMGGFYLCQPGPPSEKA
WQPGWTVNVEGSGELFRWNVSDLGGLGCGLKNR
SSEGPSSPSGKLMSPKLYVWAKDRPEIWEGEPPC
VPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVS
RGPLSWTHVHPKGPKSLLSLELKDDRPARDMWVM
ETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITARP
VLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQ
RALVLRRKRKRMTDPTRRFFKVTPPPGSGPQNQY
GNVLSLPTPTSGLGRAQRWAAGLGGTAPSYGNPS
SDVQADGALGSRSPPGVGPEEEEGEGYEEPDSEE
DSEFYENDSNLGQDQLSQDGSGYENPEDEPLGPE
DEDSFSNAESYENEDEELTQPVARTMDFLSPHGSA
WDPSREATSLGSQSYEDMRGILYAAPQLRSIRGQP
GPNHEEDADSYENMDNPDGPDPAWGGGGRMGT WSTR CD20
MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRM 6
SSLVGPTQSFFMRESKTLGAVQIMNGLFHIALGGLL
MIPAGIYAPICVTVVVYPLWGGIMYIISGSLLAATEKN
SRKCLVKGKMIMNSLSLFAAISGMILSIMDILNIKISH
FLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQYC
YSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCS
RPKSNIVLLSAEEKKEQTIEIKEEWGLTETSSQPKN
EEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSS P ROR1
MHRPRRRGTRPPLLALLAALLLAARGAAAQETELSV 7
SAELVPTSSWNISSELNKDSYLTLDEPMNNITTSLG
QTAELHCKVSGNPPPTIRWFKNDAPVVQEPRRLSF
RSTIYGSRLRIRNLDTTDTGYFQCVATNGKEVVSST
GVLFVKFGPPPTASPGYSDEYEEDGFCQPYRGIAC
ARFIGNRTVYMESLHMQGEIENQITAAFTMIGTSSH
LSDKCSQFAIPSLCHYAFPYCDETSSVPKPRDLCRD
ECEILENVLCQTEYIFARSNPMILMRLKLPNCEDLPQ
PESPEAANCIRIGIPMADPINKNHKCYNSTGVDYRG
TVSVTKSGRQCQPWNSQYPHTHTFTALRFPELNG
GHSYCRNPGNQKEAPWCFTLDENFKSDLCDIPACD
SKDSKEKNKMEILYILVPSVAIPLAIALLFFFICVCR
NNQKSSSAPVQRQPKHVRGQNVEMSMLNAYKPKSKA
KELPLSAVRFMEELGECAFGKIYKGHLYLPGMDHA
QLVAIKTLKDYNNPQQWTEFQQEASLMAELHHPNI
VCLLGAVTQEQPVCMLFEYINQGDLHEFLIMRSPHS
DVGCSSDEDGTVKSSLDHGDFLHIAIQIAAGMEYLS
SHFFVHKDLAARNILIGEQLHVKISDLGLSREIYSAD
YYRVQSKSLLPIRWMPPEAIMYGKFSSDSDIWSFG
WLWEIFSFGLQPYYGFSNQEVIEMVRKRQLLPCSE
DCPPRMYSLMTECWNEIPSRRPRFKDIHVRLRSWE
GLSSHTSSTTPSGGNATTQTTSLSASPVSNLSNPR
YPNYMFPSQGITPQGQIAGFIGPPIPQNQRFIPINGY
PIPPGYAAFPAAHYQPTGPPRVIQHCPPPKSRSPSS
ASGSTSTGHVTSLPSSGSNQEANIPLLPHMSIPNHP GGMGITVFGNKSQKPY
KIDSKQASLLGDANIHGHTESMISAEL WT1 MGHHHHHHHHHHSSGHIEGRHMRRVPGVAPTLVR
8 SASETSEKRPFMCAYPGCNKRYFKLSHLQMHSRK
HTGEKPYQCDFKDCERRFFRSDQLKRHQRRHTGV
KPFQCKTCQRKFSRSDHLKTHTRTHTGEKPFSCR
WPSCQKKFARSDELVRHHNMHQRNMTKLQLAL
[0067] In particular embodiments disclosed herein, modified T
cells, NK cells and/or monocytes/macrophages target and destroy
cancer cells. B cells can also be modified to secrete
tumor-specific antibodies.
[0068] Viral Markers. In particular embodiments, markers are
expressed by unwanted virally-infected cells. Exemplary viruses
include adenoviruses, arenaviruses, bunyaviruses, coronavirusess,
flavirviruses, hantaviruses, hepadnaviruses, herpesviruses,
papilomaviruses, paramyxoviruses, parvoviruses, picornaviruses,
poxviruses, orthomyxoviruses, retroviruses, reoviruses,
rhabdoviruses, rotaviruses, spongiform viruses or togaviruses. In
additional embodiments, viral antigen markers include peptides
expressed by CMV, cold viruses, Epstein-Barr, flu viruses,
hepatitis A, B, and C viruses, herpes simplex, HIV, influenza,
Japanese encephalitis, measles, polio, rabies, respiratory
syncytial, rubella, smallpox, varicella zoster or West Nile
virus.
[0069] As further particular examples, cytomegaloviral antigens
include envelope glycoprotein B and CMV pp65; Epstein-Barr antigens
include EBV EBNAI, EBV P18, and EBV P23; hepatitis antigens include
the S, M, and L proteins of hepatitis B virus, the pre-S antigen of
hepatitis B virus, HBCAG DELTA, HBV HBE, hepatitis C viral RNA, HCV
NS3 and HCV NS4; herpes simplex viral antigens include immediate
early proteins and glycoprotein D; HIV antigens include gene
products of the gag, pol, and env genes such as HIV gp32, HIV gp41,
HIV gp120, HIV gp160, HIV P17/24, HIV P24, HIV P55 GAG, HIV P66
POL, HIV TAT, HIV GP36, the Nef protein and reverse transcriptase;
influenza antigens include hemagglutinin and neuraminidase;
Japanese encephalitis viral antigens include proteins E, M-E,
M-E-NS1, NS1, NS1-NS2A and 80% E; measles antigens include the
measles virus fusion protein; rabies antigens include rabies
glycoprotein and rabies nucleoprotein; respiratory syncytial viral
antigens include the RSV fusion protein and the M2 protein;
rotaviral antigens include VP7sc; rubella antigens include proteins
E1 and E2; and varicella zoster viral antigens include gpl and
gpll.
[0070] Additional particular exemplary viral antigen sequences
include:
TABLE-US-00002 SEQ ID Source Sequence NO. Nef (66-97):
VGFPVTPQVPLRPMTYKAAVDLSH 9 FLKEKGGL Nef (116-145)
HTQGYFPDWQNYTPGPGVRYPLTF 10 GWLYKL Gag p17 (17-35)
EKIRLRPGGKKKYKLKHIV 11 Gag p17-p24 NPPIPVGEIYKRWIILGLNKIVRM 12
(253-284) YSPTSILD Pol 325-355 AIFQSSMTKILEPFRKQNPDIVI 13 (RT
158-188) YQYMDDLY
See Fundamental Virology, Second Edition, eds. Fields, B. N. and
Knipe, D. M. (Raven Press, New York, 1991) for additional examples
of viral antigens.
[0071] In particular embodiments disclosed herein, modified T cells
recognize and destroy virally-infected cells. Alternatively, or in
addition, modified monocytes/macrophages can remove viruses from
peripheral tissue or the blood stream (extracellular) before
cellular infection by a viral particle. B cells can also be
modified to express broadly neutralizing antibodies. In one
example, B cells can be modified to express broadly neutralizing
anti-HIV antibodies.
[0072] In particular embodiments, the targeting agent targets HIV
gag protein, gp120 or the Hepatitis B envelope protein (S
domain).
[0073] Bacterial Markers. In particular embodiments, markers are
expressed by cells associated with unwanted bacterial infections.
Exemplary bacteria include anthrax; gram-negative bacilli,
chlamydia, diptheria, haemophilus influenza, Helicobacter pylori,
malaria, Mycobacterium tuberculosis, pertussis toxin, pneumococcus,
rickettsiae, staphylococcus, streptococcus and tetanus.
[0074] As particular examples of bacterial antigen markers, anthrax
antigens include anthrax protective antigen; gram-negative bacilli
antigens include lipopolysaccharides; haemophilus influenza
antigens include capsular polysaccharides; diptheria antigens
include diptheria toxin; Mycobacterium tuberculosis antigens
include mycolic acid, heat shock protein 65 (HSP65), the 30 kDa
major secreted protein and antigen 85A; pertussis toxin antigens
include hemagglutinin, pertactin, FIM2, FIM3 and adenylate cyclase;
pneumococcal antigens include pneumolysin and pneumococcal capsular
polysaccharides; rickettsiae antigens include rompA; streptococcal
antigens include M proteins; and tetanus antigens include tetanus
toxin.
[0075] In certain embodiments where the presence of bacteria is
beneficial to the health of a microbiome, bacterial cells can also
be wanted cell types.
[0076] Monocytes/macrophages are particularly useful to modify when
the therapeutic objective is treatment of a bacterial infection. In
one particular embodiment, monocytes/macrophages can be modified
with a ligand recognizing the surface component lipoteichoic acid
of Staphyloccus aureus or the Staphylococcus aureus clumping factor
A (CIfA). Immunosuppressive T.sub.REG can be useful to modify when
a bacteria is a wanted cell type.
[0077] Superbugs. In particular embodiments, lymphocytes are
modified to target multi-drug resistant "superbugs". Examples of
superbugs include Enterococcus faecium, Clostridium difficile,
Acinetobacter baumannii, Pseudomonas aeruginosa, and
Enterobacteriaceae (including Escherichia coli, Klebsiella
pneumoniae, Enterobacter spp.).
[0078] Fungal Markers. In particular embodiments, markers are
expressed by cells associated with unwanted fungal infections.
Exemplary fungi include candida, coccidiodes, cryptococcus,
histoplasma, leishmania, plasmodium, protozoa, parasites,
schistosomae, tinea, toxoplasma, and trypanosoma cruzi.
[0079] As further particular examples of fungal antigens,
coccidiodes antigens include spherule antigens; cryptococcal
antigens include capsular polysaccharides; histoplasma antigens
include heat shock protein 60 (HSP60); leishmania antigens include
gp63 and lipophosphoglycan; plasmodium falciparum antigens include
merozoite surface antigens, sporozoite surface antigens,
circumsporozoite antigens, gametocyte/gamete surface antigens,
protozoal and other parasitic antigens including the blood-stage
antigen pf 155/RESA; schistosomae antigens include
glutathione-S-transferase and paramyosin; tinea fungal antigens
include trichophytin; toxoplasma antigens include SAG-1 and p30;
and trypanosoma cruzi antigens include the 75-77 kDa antigen and
the 56 kDa antigen.
[0080] Monocytes/macrophages are particularly useful to modify when
the therapeutic objective is treatment of a fungal infection.
[0081] Autoimmune or Allergy Markers. In particular embodiments,
markers are expressed by cells associated with unwanted autoimmune
or allergic conditions. Exemplary autoimmune conditions include
acute necrotizing hemorrhagic encephalopathy, allergic asthma,
alopecia areata, anemia, aphthous ulcer, arthritis (including
rheumatoid arthritis, juvenile rheumatoid arthritis,
osteoarthritis, psoriatic arthritis), asthma, autoimmune
thyroiditis, conjunctivitis, Crohn's disease, cutaneous lupus
erythematosus, dermatitis (including atopic dermatitis and
eczematous dermatitis), diabetes, diabetes mellitus, erythema
nodosum leprosum, keratoconjunctivitis, multiple sclerosis,
myasthenia gravis, psoriasis, scleroderma, Sjogren's syndrome,
including keratoconjunctivitis sicca secondary to Sjogren's
syndrome, Stevens-Johnson syndrome, systemic lupus erythematosis,
ulcerative colitis, vaginitis and Wegener's granulomatosis.
[0082] Examples of autoimmune antigens include glutamic acid
decarboxylase 65 (GAD 65), native DNA, myelin basic protein, myelin
proteolipid protein, acetylcholine receptor components,
thyroglobulin, and the thyroid stimulating hormone (TSH) receptor.
Examples of allergic antigens include pollen antigens such as
Japanese cedar pollen antigens, ragweed pollen antigens, rye grass
pollen antigens, animal derived antigens (such as dust mite
antigens and feline antigens), histocompatibility antigens, and
penicillin and other therapeutic drugs.
[0083] Immunosuppressive T.sub.REG can be useful to modify to
protect wanted cells from autoimmune attack or to reduce immune
system activity in an area. Exemplary wanted cells to protect from
autoimmune attack include neurons in multiple sclerosis or
amylotrophic lateral sclerosis; connective tissue in rheumatoid
arthritis; colon epithelium in Chrohn's disease; and the pancreas
in Diabetes mellitus type 1. In one particular embodiment,
T.sub.REG are modified to express a chimeric antigen receptor (CAR)
against KIR4.1 (a potassium channel) that has been identified as an
immune target in multiple sclerosis.
[0084] Without limiting any of the foregoing examples, markers can
also include B-cell targets, TNF receptor superfamily members,
Hedgehog family members, receptor tyrosine kinases,
proteoglycan-related molecules, TGF-.beta. superfamily members,
Wnt-related molecules, T-cell targets, dendritic cell targets, NK
cell targets, a monocyte/macrophage cell targets, and angiogenesis
targets.
[0085] Without limiting any of the foregoing examples, markers can
also include CEACAM6, c-Met, EGFR, ErbB2, ErbB3, ErbB4, EphA2,
IGF1R, GHRHR, GHR, FLT1, KDR, FLT4, CD44v6, CAl25, CEA, BTLA,
TGFBR2, TGFBR1, IL6R, gp130, TNFR1, TNFR2, PD1, PD-L1, PD-L2, HVEM,
mesothelin, PSMA, RANK, ROR1, TNFRSF4, TWEAK-R, HLA, tumor or
pathogen derived peptides bound to HLA (such as from hTERT,
tyrosinase, or WT-1), LT.beta.R, LIFR.beta., LRP5, MUC1,
OSMR.beta., TCR.alpha., TCR.beta., B7H4, TLR7, TLR9, PTCH1, PTCH1,
Robo1, .alpha.-fetoprotein (AFP) or Frizzled.
[0086] Targeting Agents. Targeting agents include any binding
domain capable of (i) expression by a lymphocyte; and (ii) binding
to a marker associated with a target. Binding of the targeting
agent to the marker then mediates destruction or protection of the
target.
[0087] Binding domains include any substance that binds to another
substance to form a complex. Examples of binding domains include
cell marker ligands, receptor ligands, antibodies, peptides,
peptide aptamers, receptors and chimeric antigen receptors (CAR) or
combinations thereof. As will be understood by one of ordinary
skill in the art, targeting agent binding domains can include the
same components, options and identification methods as described
above in relation to lymphocyte-directing agent binding domains
with altered specificity, as appropriate.
[0088] Targeting agent binding domains can particularly include any
peptide that specifically binds a marker on a targeted cell.
Sources of targeting agent binding domains include antibody
variable regions from various species (which can be in the form of
antibodies, sFvs, scFvs, Fabs, scFv-based grababody, or soluble VH
domain or domain antibodies). These antibodies can form
antigen-binding regions using only a heavy chain variable region,
i.e., these functional antibodies are homodimers of heavy chains
only (referred to as "heavy chain antibodies") (Jespers et al.,
Nat. Biotechnol. 22:1161, 2004; Cortez-Retamozo et al., Cancer Res.
64:2853, 2004; Baral et al., Nature Med. 12:580, 2006; and
Barthelemy et al., J. Biol. Chem. 283:3639, 2008).
[0089] An alternative source of targeting agent binding domains
includes sequences that encode random peptide libraries or
sequences that encode an engineered diversity of amino acids in
loop regions of alternative non-antibody scaffolds, such as scTCR
(see, e.g., Lake et al., Int. Immunol. 11:745, 1999; Maynard et
al., J. Immunol. Methods 306:51, 2005; U.S. Pat. No. 8,361,794),
fibrinogen domains (see, e.g., Weisel et al., Science 230:1388,
1985), Kunitz domains (see, e.g., U.S. Pat. No. 6,423,498),
designed ankyrin repeat proteins (DARPins) (Binz et al., J. Mol.
Biol. 332:489, 2003 and Binz et al., Nat. Biotechnol. 22:575,
2004), fibronectin binding domains (adnectins or monobodies)
(Richards et al., J. Mol. Biol. 326:1475, 2003; Parker et al.,
Protein Eng. Des. Selec. 18:435, 2005 and Hackel et al. (2008) J.
Mol. Biol. 381:1238-1252), cysteine-knot miniproteins (Vita et al.
(1995) Proc. Natl. Acad. Sci. (USA) 92:6404-6408; Martin et al.
(2002) Nat. Biotechnol. 21:71, 2002 and Huang et al. (2005)
Structure 13:755, 2005), tetratricopeptide repeat domains (Main et
al., Structure 11:497, 2003 and Cortajarena et al., ACS Chem. Biol.
3:161, 2008), leucine-rich repeat domains (Stumpp et al., J. Mol.
Biol. 332:471, 2003), lipocalin domains (see, e.g., WO 2006/095164,
Beste et al., Proc. Natl. Acad. Sci. (USA) 96:1898, 1999 and
Schonfeld et al., Proc. Natl. Acad. Sci. (USA) 106:8198, 2009),
V-like domains (see, e.g., U.S. Patent Application Publication No.
2007/0065431), C-type lectin domains (Zelensky and Gready, FEBS J.
272:6179, 2005; Beavil et al., Proc. Natl. Acad. Sci. (USA) 89:753,
1992 and Sato et al., Proc. Natl. Acad. Sci. (USA) 100:7779, 2003),
mAb.sup.2 or Fcab.TM. (see, e.g., PCT Patent Application
Publication Nos. WO 2007/098934; WO 2006/072620), armadillo repeat
proteins (see, e.g., Madhurantakam et al., Protein Sci. 21: 1015,
2012; PCT Patent Application Publication No. WO 2009/040338),
affilin (Ebersbach et al., J. Mol. Biol. 372: 172, 2007), affibody,
avimers, knottins, fynomers, atrimers, cytotoxic T-lymphocyte
associated protein-4 (Weidle et al., Cancer Gen. Proteo. 10:155,
2013) or the like (Nord et al., Protein Eng. 8:601, 1995; Nord et
al., Nat. Biotechnol. 15:772, 1997; Nord et al., Euro. J. Biochem.
268:4269, 2001; Binz et al., Nat. Biotechnol. 23:1257, 2005;
Boersma and Pluckthun, Curr. Opin. Biotechnol. 22:849, 2011).
[0090] In some embodiments, a binding domain is a single chain T
cell receptor (scTCR) comprising V.sub..alpha./.beta. and
C.sub..alpha./.beta. chains (e.g., V.sub..alpha.-C.sub..alpha.,
V.sub..beta.-C.sub..beta., V.sub..alpha.-V.sub..beta.) or
comprising V.sub..alpha.-C.sub..alpha., V.sub..beta.-C.sub..beta.,
V.sub..alpha.-V.sub..beta. pair specific for a target of interest
(e.g., peptide-MHC complex).
[0091] In another embodiment, the targeting agent is an unwanted
cell targeting agent and the binding domain can be an antibody
targeting PSMA. A number of antibodies specific for PSMA are known
to those of skill in the art and can be readily characterized for
sequence, epitope binding, and affinity. Unwanted cell targeting
agent binding domains can also include anti-Mesothelin ligands
(associated with treating ovarian cancer, pancreatic cancer, and
mesothelioma); anti-WT-1 (associated with treating leukemia and
ovarian cancer); anti-HIV-gag (associated with treating HIV
infections); or anti-cytomegalovirus (associated with treating CMV
diseases such as herpes virus). As will be understood by one of
ordinary skill in the art, the unwanted cell targeting agent
binding domain can be any ligand that binds to any marker
associated with an unwanted cell type as described herein.
[0092] In one embodiment, the targeting agent is an unwanted cell
targeting agent and the binding domain can be an antibody targeting
CD19. In some embodiments, a binding domain is a single chain Fv
fragment (scFv) that comprises VH and VL regions specific for CD19.
In certain embodiments, the V.sub.H and V.sub.L regions are human.
Exemplary V.sub.H and V.sub.L regions include the segments of
anti-CD19 specific monoclonal antibody FMC63. In particular
embodiments, the scFV is a human or humanized ss comprising a
variable light chain comprising a CDRL1 sequence of RASQDISKYLN
(SEQ ID NO. 14), CDRL2 sequence of SRLHSGV (SEQ ID NO. 15), and a
CDRL3 sequence of GNTLPYTFG (SEQ ID NO. 16). In other embodiments,
the scFV is a human or humanized ScFv comprising a variable heavy
chain comprising CDRHI sequence of DYGVS (SEQ ID NO. 17), CDRH2
sequence of VTWGSETTYYNSALKS (SEQ ID NO. 18), and a CDRH3 sequence
of YAMDYWG (SEQ ID NO. 19). Other CD19-targeting antibodies such as
SJ25C1 and HD37 are known. (SJ25C1: Bejcek et al. Cancer Res 2005,
PMID 7538901; HD37: Pezutto et al. JI 1987, PMID 2437199). SEQ ID
NO. 20 provides the anti-CD19 scFv (VH-VL) FMC63 DNA sequence and
SEQ ID NO. 21 provides the anti-CD19 scFv (VH-VL) FMC63 amino acid
sequence.
[0093] In another embodiment, the targeting agent is an unwanted
cell targeting agent and the binding domain can be an antibody
targeting RORI. In a particular embodiment, the scFV is a human or
humanized scFv comprising a variable light chain comprising a CDRL1
sequence of ASGFDFSAYYM (SEQ ID NO. 22), CDRL2 sequence of TIYPSSG
(SEQ ID NO. 23), and a CDRL3 sequence of ADRATYFCA (SEQ ID NO. 24).
In other embodiments, the scFV is a human or humanized scFv
comprising a variable heavy chain comprising CDRH1 sequence of
DTIDWY (SEQ ID NO. 25), CDRH2 sequence of VQSDGSYTKRPGVPDR (SEQ ID
NO. 26), and a CDRH3 sequence of YIGGYVFG (SEQ ID NO. 27). A number
of antibodies specific for RORI are known to those of skill in the
art and can be readily characterized for sequence, epitope binding,
and affinity.
[0094] In certain embodiments, targeting agent binding domains
comprise a sequence that is at least 90%, at least 91%, at least
92%, at least 93%, at least 94%, at least 95%, at least 96%, at
least 97%, at least 98%, at least 99%, at least 99.5%, or 100%
identical to an amino acid sequence of a TCR V.sub..alpha.,
V.sub..beta., C.sub..alpha., or C.sub..beta., wherein each CDR
comprises zero changes or at most one, two, or three changes, from
a TCR or fragment or derivative thereof that specifically binds to
target of interest.
[0095] In certain embodiments, targeting agent binding domain
V.sub..alpha., V.sub..beta., C.sub..alpha., or C.sub..beta. region
of the present disclosure can be derived from or based on a
V.sub..alpha., V.sub..beta., C.sub..alpha., or C.sub..beta. of a
known TCR (e.g., a high-affinity TCR) and contains one or more
(e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g.,
2, 3, 4, 5, 6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4,
5, 6, 7, 8, 9, 10) amino acid substitutions (e.g., conservative
amino acid substitutions or non-conservative amino acid
substitutions), or a combination of the above-noted changes, when
compared with the V.sub..alpha., V.sub..beta., C.sub..alpha., or
C.sub..beta.of a known TCR. An insertion, deletion or substitution
may be anywhere in a V.sub..alpha., V.sub..beta., C.sub..alpha., or
C.sub..beta. region, including at the amino- or carboxy-terminus or
both ends of these regions, provided that each CDR comprises zero
changes or at most one, two, or three changes and provided a
binding domain containing a modified V.sub..alpha., V.sub..beta.,
C.sub..alpha., or C.sub..beta. region can still specifically bind
its target with an affinity similar to wild type.
[0096] In certain embodiments, a binding domain V.sub.H region of
the present disclosure can be derived from or based on a V.sub.H of
a known monoclonal antibody and can contain one or more (e.g., 2,
3, 4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5,
6, 7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8,
9, 10) amino acid substitutions (e.g., conservative amino acid
substitutions or non-conservative amino acid substitutions), or a
combination of the above-noted changes, when compared with the
V.sub.H of a known monoclonal antibody. An insertion, deletion or
substitution may be anywhere in the V.sub.H region, including at
the amino- or carboxy-terminus or both ends of this region,
provided that each CDR comprises zero changes or at most one, two,
or three changes and provided a binding domain containing the
modified V.sub.H region can still specifically bind its target with
an affinity similar to the wild type binding domain.
[0097] In further embodiments, a V.sub.L region in a binding domain
of the present disclosure is derived from or based on a V.sub.L of
a known monoclonal antibody and contains one or more (e.g., 2, 3,
4, 5, 6, 7, 8, 9, 10) insertions, one or more (e.g., 2, 3, 4, 5, 6,
7, 8, 9, 10) deletions, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9,
10) amino acid substitutions (e.g., conservative amino acid
substitutions), or a combination of the above-noted changes, when
compared with the V.sub.L of the known monoclonal antibody. An
insertion, deletion or substitution may be anywhere in the V.sub.L
region, including at the amino- or carboxy-terminus or both ends of
this region, provided that each CDR comprises zero changes or at
most one, two, or three changes and provided a binding domain
containing the modified V.sub.L region can still specifically bind
its target with an affinity similar to the wild type binding
domain.
[0098] In certain embodiments, a binding domain comprises or is a
sequence that is at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, at least 99%, at least 99.5%, or 100% identical to an
amino acid sequence of a light chain variable region (V.sub.L) or
to a heavy chain variable region (V.sub.H), or both, wherein each
CDR comprises zero changes or at most one, two, or three changes,
from a monoclonal antibody or fragment or derivative thereof that
specifically binds to target of interest.
[0099] As stated, cell-targeting agents disclosed herein include
chimeric antigen receptors. "Chimeric antigen receptors" or "CARs"
refer to synthetically designed receptors comprising at least a
binding domain and an effector domain and optionally a spacer
domain and/or a transmembrane domain. Binding domains are described
elsewhere herein.
[0100] Effector domains are capable of transmitting functional
signals to a cell. In certain embodiments, an effector domain will
directly or indirectly promote a cellular response by associating
with one or more other proteins that directly promote a cellular
response. Effector domains can provide for activation of at least
one function of a transduced lymphocyte expressing the CAR upon
binding to the marker expressed on a targeted cell. Activation of
the lymphocyte can include one or more of proliferation,
differentiation, activation or other effector functions. In
particular embodiments, the delivered polynucleotide encodes for
the effector domain.
[0101] An effector domain may include one, two, three or more
receptor signaling domains, intracellular signaling domains,
costimulatory domains, or combinations thereof. Any intracellular
effector domain, costimulatory domain or both from any of a variety
of signaling molecules (e.g., signal transduction receptors) may be
used in the CARs of this disclosure.
[0102] Exemplary effector domains include those from 4-1BB,
CD3.epsilon., CD3.delta., CD3.zeta., CD27, CD28 (e.g., SEQ ID
NO.:28), CD79A, CD79B, CARD11, DAP10, FcR.alpha., FcR.beta.,
FcR.gamma., Fyn, HVEM, ICOS, Lck, LAG3, LAT, LRP, NOTCH1, Wnt,
NKG2D, OX40, ROR2, Ryk, SLAMF1, Slp76, pTa, TCR.alpha., TCR.beta.,
TRIM, Zap70, PTCH2, or any combination thereof.
[0103] T cell activation can be said to be mediated by two distinct
classes of cytoplasmic signaling sequence: those that initiate
antigen-dependent primary activation and provide a T cell receptor
like signal (primary cytoplasmic signaling sequences) and those
that act in an antigen-independent manner to provide a secondary or
co-stimulatory signal (secondary cytoplasmic signaling sequences).
Primary cytoplasmic signaling sequences that act in a stimulatory
manner may contain signaling motifs which are known as receptor
tyrosine-based activation motifs or iTAMs. Examples of iTAM
containing primary cytoplasmic signaling sequences include those
derived from CD3 zeta, FeR gamma, CD3 gamma, CD3 delta, CD3
epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
[0104] In particular embodiments, an effector domain comprises a
cytoplasmic portion that associates with a cytoplasmic signaling
protein, wherein the cytoplasmic signaling protein is a lymphocyte
receptor or signaling domain thereof, a protein comprising a
plurality of ITAMs, a costimulatory factor, or any combination
thereof.
[0105] Examples of intracellular signaling domains include the
cytoplasmic sequences of the CD3 zeta chain, and/or co-receptors
that act in concert to initiate signal transduction following CAR
engagement, as well as any derivative or variant of these sequences
and any synthetic sequence that has the same functional capability.
In particular embodiments, an intracellular signaling domain of a
CAR can be designed to comprise an intracellular signaling domain
combined with any other desired cytoplasmic domain(s). For example,
the intracellular signaling domain of a CAR can comprise an
intracellular signaling domain and a costimulatory signaling
region. The costimulatory signaling region refers to a portion of
the CAR comprising the intracellular domain of a costimulatory
molecule. A costimulatory molecule is a cell surface molecule other
than the expressed marker ligand that is required for a response of
lymphocytes to a marker. Examples of such molecules include CD27,
CD28, 4-1BB (CD 137), OX40, CD30, CD40, lymphocyte
function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C,
B7-H3, and a ligand that specifically binds with CD83.
[0106] In certain embodiments, CAR polynucleotides can comprise a
sequence encoding for a spacer region. The length of the spacer
region can be customized for individual markers on targets to
optimize target recognition and destruction or protection. In
particular embodiments, a spacer length can be selected based upon
the location of a marker epitope, affinity of an antibody for the
epitope, and/or the ability of the lymphocytes expressing the CAR
to proliferate in vitro and/or in vivo in response to marker
recognition.
[0107] Typically a spacer region is found between the binding
domain and a transmembrane domain of the CAR. Spacer regions can
provide for flexibility of the binding domain and allows for high
expression levels in the modified cells. In particular embodiments,
a spacer region can have at least 10 to 250 amino acids, at least
10 to 200 amino acids, at least 10 to 150 amino acids, at least 10
to 100 amino acids, at least 10 to 50 amino acids or at least 10 to
25 amino acids and including any integer between the endpoints of
any of the listed ranges. In further embodiments, a spacer region
has 250 amino acids or less; 200 amino acids or less, 150 amino
acids or less; 100 amino acids or less; 50 amino acids or less; 40
amino acids or less; 30 amino acids or less; 20 amino acids or
less; or 10 amino acids or less.
[0108] In particular embodiments, spacer regions can be derived
from a hinge region of an immunoglobulin like molecule, for example
all or a portion of the hinge region from a human IgG1, human IgG2,
a human IgG3, or a human IgG4. Hinge regions can be modified to
avoid undesirable structural interactions such as dimerization. In
some embodiments, all or a portion of a hinge region can be
combined with one or more domains of a constant region of an
immunoglobulin. For example, a portion of a hinge region can be
combined with all or a portion of a CH2 or CH3 domain or variant
thereof.
[0109] CARs disclosed herein can also include transmembrane
domains. In particular embodiments, the CAR polynucleotide encodes
the transmembrane domain. The transmembrane domain provides for
anchoring of the CAR in the lymphocyte membrane. The transmembrane
domain may be derived either from a natural or a synthetic source.
When the source is natural, the domain may be derived from any
membrane-bound or transmembrane protein. Transmembrane regions
comprise at least the transmembrane region(s) of) the alpha, beta
or zeta chain of the T-cell receptor, CD28, CD3, CD45, CD4, CDS,
CD9, CDI6, CD22; CD33, CD37, CD64, CD80, CD86, CDl34, CDl37 and
CD154. In further particular embodiments, synthetic or variant
transmembrane domains comprise predominantly hydrophobic residues
such as leucine and valine.
[0110] In a particular embodiment, the CAR comprises a P28z fusion
receptor composed of a single-chain antibody (scFv) specific for
the extracellular domain of PSMA (J591) combined with CD28 and
CD3.zeta. cytoplasmic signaling domains. In another embodiment, the
CAR comprises a P28z CAR of SEQ ID NO. 94. SEQ ID NO. 94 includes
murine components and was utilized in studies described herein.
Amino acid positions 1-797 include the anti-PSMA scFv (J592)
whereas positions 797-1477 include the murine CD8 transmembrane
domain, murine CD28 signaling domain and the murine CD3zeta
signaling domain. Any P28z domain can be individually replaced with
optimized domains. In particularized embodiments, the transmembrane
domain and signaling domains within positions 797-1477 of SEQ ID
NO. 94 can be particularly replaced with domains optimized for use
in humans or other animals. In additional embodiments, any whole or
portion of a binding domain, any whole or portion of an effector
domain, any whole or portion of a spacer domain and/or any whole or
portion of a transmembrane domain can be optimized for use in
humans or other animals. In additional embodiments, the P28z CAR is
optimized for use in humans. When optimized for humans, the P28z
CAR can have lowered or no immunogenicity in humans and have a
lower number of non-immunogenic epitopes compared to non-human
antibodies.
[0111] Endosomal Release Agents. As used herein, "endosomal release
agents" include any compound or peptide sequence that facilitates
cargo exit from the endosome of a lymphocyte. Exemplary endosomal
release agents include imidazoles, poly or oligoimidazoles, PEIs,
peptides, fusogenic peptides, polycarboxylates, polycations, masked
oligo or poly cations or anions, acetals, polyacetals,
ketals/polyketyals, orthoesters, polymers with masked or unmasked
cationic or anionic charges, amphiphilic block copolymers and
dendrimers with masked or unmasked cationic or anionic charges.
[0112] Many endosomal release agents are adapted from viral
elements that promote escape from the endosome and deliver
polynucleotides intact into the nucleus. As one particular example,
the H5WYG peptide can be used to induce the lysis of membranes at
low pH. The histidine-rich peptide H5WYG is a derivative of the
N-terminal sequence of the HA-2 subunit of the influenza virus
hemagglutinin in which 5 of the amino acids have been replaced with
histidine residues. H5WYG is able to selectively destabilize
membranes at a slightly acidic pH as the histidine residues are
protonated. The E1 protein from Semliki Forrest virus is also a
useful endosomal release agent.
[0113] In particular embodiments, endosomal release agents include
a hydrophobic membrane translocation sequence (MTS). An exemplary
hydrophobic MTS-containing peptide is RFGF having the amino acid
sequence AAVALLPAVLLALLAP (SEQ ID NO. 29). An RFGF analogue (e.g.,
amino acid sequence AALLPVLLAAP (SEQ ID NO. 30)) containing a
hydrophobic MTS can also be used.
[0114] Additional exemplary endosomal release agents include:
TABLE-US-00003 Source Sequence SEQ ID NO. Influenza virus
GLFEAIAGFIENGWEG 31 hemagglutinin HA-2 TAT of HIV YGRKKRRQRRR 32
N-terminal region MSGTFGGILAGLIGLL 33 of the S protein of duck
hepatitis B S protein of woodchuck MSPSSLLGLLAGLQW 34 hepatitis B
Synthetic, Duguid GLFEALLELLESLWELL 35 et al. 1998 Synthetic, Gupta
LKKLLKKLLKKLLKKL 36 & Kothekar, 1997 Derossi et al., J.
RQIKIWFQNRRMKWKK 37 Biol. Chem. 269: 10444, 1994 Tat fragment
(48-60) GRKKRRQRRRPPQC 38 Chaloin et al., Biochem.
GALFLGWLGAAGSTMGAWSQ 39 peptide Biophys. Res. PKKKRKV Commun., 243:
601, 1998 PVEC LLIILRRRIRKQAHAHSK 40 Transportan
GWTLNSAGYLLKINLKALAALAK 41 KIL Amphiphilic model KLALKLALKALKAALKLA
42 peptide; Oehlke et al., Mol. Ther., 2: 339, 2000 Arg.sub.9
RRRRRRRRR 43 LL-37 LLGDFFRKSKEKIGKEFKRIVQR 44 IKDFLRNLVPRTES
Cecropin P1 SWLSKTAKKLENSAKKRISEGIA 45 IAIQGGPR .alpha.-defensin
ACYCRIPACIAGERRYGTCIYQ 46 GRLWAFCC .beta.-defensin
DHYNCVSSGGQCLYSACPIFTK 47 IQGTCYRGKAKCCK Bactenecin RKCRIWIRVCR 48
PR-3 RRRPRPPYLPRPRPPPFFPPRL 49 PPRIPPGFPPRFPPRFPGKR- NH.sub.2
Indolicidin ILPWKWPWWPWRR-NH2 50
[0115] Nuclear Localization Signals. "Nuclear localization signals"
(NLS) refer to sequences that direct associated sequences into the
nucleus of a cell. Generally, NLS are a class of short amino acid
sequences from 3 to 100 amino acids in length, from 3 to 50, 4 to
30, or 4 to 20 amino acids in length.
[0116] Exemplary NLS sequences include (i) monopartite NLS
exemplified by the SV40 large T antigen NLS (PKKKRKV) (SEQ ID NO:
51); (ii) bipartite NLS consisting of two basic domains separated
by a variable number of spacer amino acids and exemplified by the
Xenopus nucleoplasmin NLS (KRXXXXXXXXXXKKKL) (SEQ ID NO: 52); and
(iii) noncanonical sequences such as M9 of the hnRNP A1 protein,
the influenza virus nucleoprotein NLS, and the yeast Ga14 protein
NLS (Dingwall and Laskey, Trends Biochem Sci 16:478-481, 1991). In
particular embodiments, the NLS can be a highly cationic or basic
peptide. In other embodiments, the NLS comprises two or more Arg or
Lys amino acid residues. In further embodiments, the NLS can bind
cytosolic proteins, such as importins and karyopherins, which
recognize and transport NLS-containing sequences to the nuclear
pore complex.
[0117] In particular embodiments, to direct import of delivered
polynucleotides, particularly plasmid DNA, into the nucleus,
polynucleotides (in one embodiment nanoparticle-encapsulated
plasmids) can be conjugated to the SV40 T-Ag-derived NLS peptides.
Exemplary SV40 T-Ag-derived NLS peptides include: PKKKRKV (SEQ ID
NO. 51); PKKKRMV (SEQ ID NO. 53); PKKKRKVEDP (SEQ ID NO. 54);
PKKGSKKA (SEQ ID NO. 55); PKTKRKV (SEQ ID NO. 56); CGGPKKKRKVG (SEQ
ID NO. 57); PKKKIKV (SEQ ID NO. 58);
CYDDEATADSQHSTPPKKKRKVEDPKDFESELLS (SEQ ID NO. 59); and
CGYGPKKKRKVGG (SEQ ID NO. 60).
[0118] Additional exemplary NLS sequences include:
TABLE-US-00004 SEQ ID Source Sequence NO. Polyoma large T protein
PKKARED 61 Polyoma large T protein CGYGVSRKRPRPG 62 SV40 VP1 capsid
polypeptide APTKRKGS 63 Polyoma virus major capsid APKRKSGVSKC 64
protein VP1 SV40 VP2 capsid protein PNKKKRK 65 Polyoma virus capsid
protein VP2 EEDGPQKKKRRL 66 Yeast histone H2B GKKRSKA 67 Adenovirus
E1a KRPRP 68 Adenovirus type 2/5 E1a CGGLSSKRPRP 69 Xenopus NLS2
LKDKDAKKSKQE 70 v-Rel or p59.sup.v-rel GNKAKRQRST 71 Influenza A
NS1 protein PFLDRLRRDQK 72 Human lamin A SVTKKRKLE 73 Xenopus lamin
A SASKRRRLE 74 Adenovirus 5 DBP PPKKRMRRRIE 75 Rat glucocorticoid
receptor YRKCLQAGMNLEARKTKK 76 KIKGIQQATA Human estrogen receptor
RKDRRGGRMLKHKRQRD 77 DGEGRGEVGSAGDMRAM INACIDNLWPSPLMIKRSK K Rabbit
progesterone receptor RKFKKFNK 78 c-myb gene product PLLKKIKQ 79
N-myc gene product PPQKKIKS 80 p53 PQPKKKP 81 c-erb-A gene product
SKRVAKRKL 82 Yeast ribosomal protein L29 MTGSKTRKHRGSGA 83 Yeast
ribosomal protein L29 RHRKHP 84 Yeast ribosomal protein L29 KRRKHP
85 Yeast ribosomal protein L29 KYRKHP 86 Yeast ribosomal protein
L29 KHRRHP 87 Yeast ribosomal protein L29 KHKKHP 88 Yeast ribosomal
protein L29 RHLKHP 89 Hepatitis B core antigen PETTWRRRGRSPRRRTP 90
SPRRRRSPRRRRSQS Viral jun ASKSRKRKL 91 Human T-cell leukemia virus
Tax GGLCSARLHRHALLAT 92 trans-activator protein Mouse nuclear Mx1
protein DTREKKKFLKRRLLRLDE 93
[0119] Exemplary NLS are also described in Cokol et al., 2000, EMBO
Reports, 1(5):411-415; Boulikas, 1993, Crit. Rev. Eukaryot. Gene
Expr., 3:193-227; Collas et al., 1996, Transgenic Research, 5:
451-458; Collas and Alestrom, 1997, Biochem. Cell Biol. 75:
633-640; Collas and Alestrom, 1998, Transgenic Research, 7:
303-309; Collas and Alestrom, 1996, Mol. Reprod. Devel.,
45:431-438, and U.S. Pat. Nos. 7,531,624; 7,498,177; 7,332,586; and
7,550,650.
[0120] Nanocarriers. Compositions disclosed herein include
nanocarriers. Nanocarriers can include a porous nanoparticle at
least substantially covered by a coating. In particular
embodiments, polynucleotides and optionally NLSs can be found
within the porous nanoparticle whereas optional
lymphocyte-directing agents and endosomal release agents can be
anchored to the coating.
[0121] Porous Nanoparticles. Porous nanoparticles of particular
compositions can be constructed from any material capable of
forming a porous network. Exemplary materials include a variety of
material including, without limitation, biocompatible polymers,
metals, transition metals and metalloids. Exemplary biocompatible
polymers include, but not limited to, agar, agarose, alginate,
alginate/calcium phosphate cement (CPC), beta-galactosidase
(.beta.-GAL), (1,2,3,4,6-pentaacetyl a-D-galactose), cellulose,
chitin, chitosan, collagen, elastin, gelatin, hyaluronic acid
collagen, hydroxyapatite,
poly(3-hydroxybutyrate-co-3-hydroxy-hexanoate) (PHBHHx),
poly(lactide), poly(caprolactone) (PCL), poly(lactide-co-glycolide)
(PLG), poly(Iactic-co-glycolic acid) (PLGA),poly(vinyl alcohol)
(PVA), silk, soy protein, and soy protein isolate, alone or in
combination with any other polymer composition, in any
concentration and in any ratio. Blending different polymer types in
different ratios using various grades can result in characteristics
that borrow from each of the contributing polymers. Various
terminal group chemistries can also be adopted. Exemplary metals,
transition metals and metalloids include lithium, magnesium, zinc,
aluminum and silica. In one embodiment, the porous nanoparticles
comprise silica. The exceptionally high surface area of mesoporous
silica (exceeding 1,000 m.sup.2/g) enables polynucleotide loading
at levels exceeding conventional DNA carriers such as liposomes or
polymer conjugates. In additional embodiments, pores range in size
from 10-20 nm.
[0122] Useful nanocarriers of particular embodiments also include
those based on (i) lipid-based delivery systems, including cationic
lipids, ionizable cationic lipids, lipid-like molecules and
pH-sensitive amphiphiles; and/or (ii) polymeric RNA/DNA delivery
systems such as polyethyleniminie (PEI)-based polymeric vectors,
chitosan-based vectors, dendrimers (highly branched, spherical
macromolecules synthesized from poly-amidoamine (PAMAM) and
poly-propylene iminie (PPI), and block copolymers such as
PAA/BMA/DMAEMA and PDMAEMA.
[0123] The porous nanoparticles can be a variety of different
shapes, including spheroidal, cuboidal, pyramidal, oblong,
cylindrical, toroidal, and the like. The polynucleotides can be
included in the porous nanoparticles in a variety of ways. For
example, the polynucleotides can be encapsulated in the porous
nanoparticles. In other aspects, the polynucleotides can be
associated (e.g., covalently and/or non-covalently) with the
surface or close underlying vicinity of the surface of the porous
nanoparticles. In some embodiments, the polynucleotides can be
incorporated in the porous nanoparticles e.g., integrated in the
material of the porous nanoparticles. For example, the
polynucleotides can be incorporated into a polymer matrix of
polymer nanoparticles. One of ordinary skill in the art will
appreciate the various ways to carry the polynucleotides so as to
allow delivery of the polynucleotide molecules to the
lymphocytes.
[0124] In particular embodiments, porous nanoparticles include
liposomes. Liposomes are microscopic vesicles consisting of at
least one concentric lipid bilayer. Vesicle-forming lipids are
selected to achieve a specified degree of fluidity or rigidity of
the final complex. In some embodiments, liposomes provide a lipid
composition that is an outer layer surrounding a porous
nanoparticle.
[0125] Liposomes can be neutral (cholesterol) or bipolar and
include phospholipids, such as phosphatidylcholine (PC),
phosphatidylethanolamine (PE), phosphatidylinositol (PI), and
sphingomyelin (SM) and other type of bipolar lipids including but
not limited to dioleoylphosphatidylethanolamine (DOPE), with a
hydrocarbon chain length in the range of 14-22, and saturated or
with one or more double C.dbd.C bonds. Examples of lipids capable
of producing a stable liposome, alone, or in combination with other
lipid components are phospholipids, such as hydrogenated soy
phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine,
lysolecithin, lysophosphatidylethanolamine, phosphatidylserine,
phosphatidylinositol, sphingomyelin, cephalin, cardiolipin,
phosphatidic acid, cerebro sides,
distearoylphosphatidylethanolamine (DSPE),
dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine
(DPPC), palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoylphosphatidylethanolamine (POPE) and
dioleoylphosphatidylethanolamine
4-(N-maleimido-methyl)cyclohexane-1-carboxylate (DOPE-mal).
Additional non-phosphorous containing lipids that can become
incorporated into liposomes include stearylamine, dodecylamine,
hexadecylamine, isopropyl myristate, triethanolamine-lauryl
sulfate, alkyl-aryl sulfate, acetyl palmitate, glycerol
ricinoleate, hexadecyl stereate, amphoteric acrylic polymers,
polyethyloxylated fatty acid amides, and the cationic lipids
mentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA),
DOSPA, DPTAP, DSTAP, DC-Chol). Negatively charged lipids include
phosphatidic acid (PA), dipalmitoylphosphatidylglycerol (DPPG),
dioleoylphosphatidylglycerol and (DOPG), dicetylphosphate that are
able to form vesicles. In particular embodiments, lipids used to
create liposomes disclosed herein include cholesterol, hydrogenated
soy phosphatidylcholine (HSPC) and, the derivatized vesicle-forming
lipid PEG-DSPE.
[0126] Methods of forming liposomes are described in, for example,
U.S. Pat. Nos. 4,229,360; 4,224,179; 4,241,046; 4,737,323;
4,078,052; 4,235,871; 4,501,728; and 4,837,028, as well as in Szoka
et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980) and Hope et al.,
Chem. Phys. Lip. 40:89 (1986).
[0127] The size of the nanocarriers can vary over a wide range and
can be measured in different ways. For example, the nanocarriers of
the present disclosure can have a minimum dimension of 100 nm. The
nanocarriers of the present disclosure can also have a minimum
dimension of equal to or less than 500 nm, less than 150 nm, less
than 100 nm, less than 90 nm, less than 80 nm, less than 70 nm,
less than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm,
less than 20 nm, or less than 10 nm. In certain embodiments, the
nanocarriers can have a minimum dimension ranging between 5 nm and
500 nm, between 10 nm and 100 nm, between 20 nm and 90 nm, between
30 nm and 80 nm, between 40 nm and 70 nm, and between 40 nm and 60
nm. In some embodiments, the dimension is the diameter of
nanoparticles or coated nanoparticles. In some embodiments, a
population of nanocarriers of the present disclosure can have a
mean minimum dimension of equal to or less than 500 nm, less than
100 nm, less than 90 nm, less than 80 nm, less than 70 nm, less
than 60 nm, less than 50 nm, less than 40 nm, less than 30 nm, less
than 20 nm, or less than 10 nm. In certain embodiments, a
population of nanocarriers in a composition of the present
disclosure can have a mean diameter ranging between 5 nm and 500
nm, between 10 nm and 100 nm, between 20 nm and 90 nm, between 30
nm and 80 nm, between 40 nm and 70 nm, and between 40 nm and 60 nm.
Dimensions of the nanocarriers can be determined using, e.g.,
conventional techniques, such as dynamic lightscattering and/or
electron microscopy.
[0128] In particular embodiments, the compositions include
protocells as nanocarriers. Protocells can be formed via fusion of
liposomes to porous silica nanoparticles. The high pore volume and
surface area of the spherical mesoporous silica core allow
high-capacity encapsulation of a spectrum of cargos, including
plasmid DNA. The supported lipid bilayer, whose composition can be
modified for specific biological applications, can serve as a
modular, reconfigurable scaffold, allowing the attachment of a
variety of molecules, such as lymphocyte-directing agents, to
provide cell-specific targeting and controlled intracellular
trafficking. As provided further herein, protocells can efficiently
introduce polynucleotides into lymphocytes.
[0129] In one particular embodiment intended to illustrate the
foregoing, anti-CD3 antibodies can be coupled onto protocell
nanocarriers to selectively target the nanocarriers to T cells for
rapid receptor-induced endocytosis. Protocells can be formed via
fusion of liposomes with porous silica nanoparticles (FIGS. 2A, B).
The high pore volume and surface area of the spherical mesoporous
silica core allow high-capacity encapsulation of a spectrum of
cargos, including plasmid DNA. The membrane serves as a modular
scaffold for the attachment of a variety of targeting moieties. In
the embodiment depicted in FIG. 2, the pH-sensitive fusogenic
peptide H5WYG is tethered to the nanocarrier surface to facilitate
endosomal escape. The plasmid DNA was also modified before
encapsulation into nanoparticles with the SV40 large T antigen
nuclear localization signal peptide (FIG. 2A).
[0130] Particular nanocarrier embodiments include:
TABLE-US-00005 Selected Lymphocyte Lymphocyte- Endosomal Population
Directing Agent Target Targeting Agent Release Agent NLS T cells
Anti-CD3 Leukemia Anti-CD19 CAR Fusogenic SV40 antibody cells
(1928zeta or peptide 194-1BBzeta) H5WYG CD8 T cells Anti-CD8
Ovarian Anti-mesothelin "Proton Sponge" NLS antibody cancer CAR
(with or effect of Ku70 cells without integrated polymeric
costimulatory domains) nanoparticles T cells Anti-LFA Pancreatic
Affinity-enhanced TAT None antibody cancer T cell receptor peptide
cells (TCR) specific for mesothelin T cells 4-1BB HIV- HIV-gag
protein- Cationic- hnRNP (CD137) infected specific T-cell polymer-
(M9) targeting cells receptor based aptamers nanocarrier Monocytes/
Anti-CD14 Staphylococcus Clumping factor Pas nona- SV40 macrophages
antibodies aureus A (ClfA) arginine (PR9) NK cells Anti-CD56
Prostate Anti-PSMA CAR Cationic None antibodies cancer (P28zeta or
lipid-based cells P4-1BB zeta) nanocarrier T.sub.REG Anti-CTLA-4
Neurons CAR specific for Cationic SV40 or anti-GARP KIR4.1 for the
polymer-based antibodies treatment of nanocarrier multiple
sclerosis Hematopoietic Anti-CD34 Leukemia Affinity-enhanced
"Proton Sponge" NLS stem cells antibodies cells T cell receptor
effect of Ku70 (TCR) specific for polymeric Wilms` tumor
nanoparticles antigen (WT1)
[0131] Compositions. The nanoparticles, porous nanoparticles and
nanocarriers (all collectively referred to herein as "active
ingredients") disclosed herein can be provided as part of
compositions that comprise, consist of or consist essentially of
the nanoparticles, porous nanoparticles and/or nanocarriers. The
compositions can be formulated for administration to subjects.
[0132] In some embodiments, the active ingredients are provided as
part of a composition that can comprise, for example, at least 0.1%
w/v of active ingredient(s); at least 1% w/v of active
ingredient(s); at least 10% w/v of active ingredient(s); at least
20% w/v of active ingredient(s); at least 30% w/v of active
ingredient(s); at least 40% w/v of active ingredient(s); at least
50% w/v of active ingredient(s); at least 60% w/v of active
ingredient(s); at least 70% w/v of active ingredient(s); at least
80% w/v of active ingredient(s); at least 90% w/v of active
ingredient(s); at least 95% w/v of active ingredient(s); or at
least 99% w/v of active ingredient(s).
[0133] In other embodiments, the active ingredients are provided as
part of a composition that can comprise, for example, at least 0.1%
w/w of active ingredient(s); at least 1% w/w of active
ingredient(s); at least 10% w/w of active ingredient(s); at least
20% w/w of active ingredient(s); at least 30% w/w of active
ingredient(s); at least 40% w/w of active ingredient(s); at least
50% w/w of active ingredient(s); at least 60% w/w of active
ingredient(s); at least 70% w/w of active ingredient(s); at least
80% w/w of active ingredient(s); at least 90% w/w of active
ingredient(s); at least 95% w/w of active ingredient(s); or at
least 99% w/w of active ingredient(s).
[0134] The compositions disclosed herein can be formulated for
administration by, without limitation, injection, inhalation,
infusion, perfusion, lavage or ingestion. The compositions
disclosed herein can further be formulated for, without limitation,
intravenous, intradermal, intraarterial, intranodal,
intralymphatic, intraperitoneal, intralesional, intraprostatic,
intravaginal, intrarectal, topical, intrathecal, intratumoral,
intramuscular, intravesicular, oral and/or subcutaneous
administration and more particularly by intravenous, intradermal,
intraarterial, intranodal, intralymphatic, intraperitoneal,
intralesional, intraprostatic, intravaginal, intrarectal, topical,
intrathecal, intratumoral, intramuscular, intravesicular, oral
and/or subcutaneous injection.
[0135] For injection, compositions can be formulated as aqueous
solutions, such as in buffers including Hanks' solution, Ringer's
solution, or physiological saline. The aqueous solutions can
contain formulatory agents such as suspending, stabilizing and/or
dispersing agents. Alternatively, the formulation can be in
lyophilized and/or powder form for constitution with a suitable
vehicle, e.g., sterile pyrogen-free water, before use.
[0136] For oral administration, the compositions can be formulated
as tablets, pills, dragees, capsules, liquids, gels, syrups,
slurries, suspensions and the like. For oral solid formulations
such as, for example, powders, capsules and tablets, suitable
excipients include binders (gum tragacanth, acacia, cornstarch,
gelatin), fillers such as sugars, e.g. lactose, sucrose, mannitol
and sorbitol; dicalcium phosphate, starch, magnesium stearate,
sodium saccharine, cellulose, magnesium carbonate; cellulose
preparations such as maize starch, wheat starch, rice starch,
potato starch, gelatin, gum tragacanth, methyl cellulose,
hydroxypropylmethyl-cellulose, sodium carboxy-methylcellulose,
and/or polyvinylpyrrolidone (PVP); granulating agents; and binding
agents. If desired, disintegrating agents can be added, such as
corn starch, potato starch, alginic acid, cross-linked
polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such
as sodium alginate. If desired, solid dosage forms can be
sugar-coated or enteric-coated using standard techniques. Flavoring
agents, such as peppermint, oil of wintergreen, cherry flavoring,
orange flavoring, etc. can also be used.
[0137] For administration by inhalation, compositions can be
formulated as aerosol sprays from pressurized packs or a nebulizer,
with the use of a suitable propellant, e.g.,
dichlorodifluoromethane, trichlorofluoromethane,
dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In
the case of a pressurized aerosol the dosage unit may be determined
by providing a valve to deliver a metered amount. Capsules and
cartridges of gelatin for use in an inhaler or insufflator may be
formulated containing a powder mix of the therapeutic and a
suitable powder base such as lactose or starch.
[0138] Any composition formulation disclosed herein can
advantageously include any other pharmaceutically acceptable
carriers which include those that do not produce significantly
adverse, allergic or other untoward reactions that outweigh the
benefit of administration, whether for research, prophylactic
and/or therapeutic treatments. Exemplary pharmaceutically
acceptable carriers and formulations are disclosed in Remington's
Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990.
Moreover, formulations can be prepared to meet sterility,
pyrogenicity, general safety and purity standards as required by
United States FDA Office of Biological Standards and/or other
relevant foreign regulatory agencies.
[0139] Exemplary generally used pharmaceutically acceptable
carriers include any and all bulking agents or fillers, solvents or
co-solvents, dispersion media, coatings, surfactants, antioxidants
(e.g., ascorbic acid, methionine, vitamin E), preservatives,
isotonic agents, absorption delaying agents, salts, stabilizers,
buffering agents, chelating agents (e.g., EDTA), gels, binders,
disintegration agents, and/or lubricants.
[0140] Exemplary buffering agents include citrate buffers,
succinate buffers, tartrate buffers, fumarate buffers, gluconate
buffers, oxalate buffers, lactate buffers, acetate buffers,
phosphate buffers, histidine buffers and/or trimethylamine
salts.
[0141] Exemplary preservatives include phenol, benzyl alcohol,
meta-cresol, methyl paraben, propyl paraben,
octadecyldimethylbenzyl ammonium chloride, benzalkonium halides,
hexamethonium chloride, alkyl parabens such as methyl or propyl
paraben, catechol, resorcinol, cyclohexanol and 3-pentanol.
[0142] Exemplary isotonic agents include polyhydric sugar alcohols
including trihydric or higher sugar alcohols, such as glycerin,
erythritol, arabitol, xylitol, sorbitol or mannitol.
[0143] Exemplary stabilizers include organic sugars, polyhydric
sugar alcohols, polyethylene glycol; sulfur-containing reducing
agents, amino acids, low molecular weight polypeptides, proteins,
immunoglobulins, hydrophilic polymers or polysaccharides.
[0144] Compositions can also be formulated as depot preparations.
Depot preparations can be formulated with suitable polymeric or
hydrophobic materials (for example as an emulsion in an acceptable
oil) or ion exchange resins, or as sparingly soluble derivatives,
for example, as a sparingly soluble salts.
[0145] Additionally, compositions can be formulated as
sustained-release systems utilizing semipermeable matrices of solid
polymers containing at least one active ingredient. Various
sustained-release materials have been established and are well
known by those of ordinary skill in the art. Sustained-release
systems may, depending on their chemical nature, release active
ingredients following administration for a few weeks up to over 100
days.
[0146] When formulated to treat cancer, the disclosed compositions
can also include plasmid DNA carrying one or more anticancer genes
selected from p53, RB, BRCA1, E1A, bcl-2, MDR-1, p21, p16, bax,
bcl-xs, E2F, IGF-I VEGF, angiostatin, oncostatin, endostatin,
GM-CSF, IL-12, IL-2, IL-4, IL-7, IFN-.gamma., TNF-.alpha. and/or
HSV-tk. Compositions can also include or be administered in
combination with one or more antineoplastic drugs including
adriamycin, angiostatin, azathioprine, bleomycin, busulfane,
camptothecin, carboplatin, carmustine, chlorambucile,
chlormethamine, chloroquinoxaline sulfonamide, cisplatin,
cyclophosphamide, cycloplatam, cytarabine, dacarbazine,
dactinomycin, daunorubicin, didox, doxorubicin, endostatin,
enloplatin, estramustine, etoposide, extramustinephosphat,
flucytosine, fluorodeoxyuridine, fluorouracil, gallium nitrate,
hydroxyurea, idoxuridine, interferons, interleukins, leuprolide,
lobaplatin, lomustine, mannomustine, mechlorethamine,
mechlorethaminoxide, melphalan, mercaptopurine, methotrexate,
mithramycin, mitobronitole, mitomycin, mycophenolic acid,
nocodazole, oncostatin, oxaliplatin, paclitaxel, pentamustine,
platinum-triamine complex, plicamycin, prednisolone, prednisone,
procarbazine, protein kinase C inhibitors, puromycine, semustine,
signal transduction inhibitors, spiroplatin, streptozotocine,
stromelysin inhibitors, taxol, tegafur, telomerase inhibitors,
teniposide, thalidomide, thiamiprine, thioguanine, thiotepa,
tiamiprine, tretamine, triaziquone, trifosfamide, tyrosine kinase
inhibitors, uramustine, vidarabine, vinblastine, vinca alcaloids,
vincristine, vindesine, vorozole, zeniplatin, zeniplatin or
zinostatin.
[0147] Methods. Methods disclosed herein include treating subjects
(humans, veterinary animals, livestock and research animals) with
compositions, active ingredients, nanoparticles, porous
nanoparticles and/or nanocarriers disclosed herein. Treating
subjects includes delivering a therapeutically effective amount. An
"effective amount" is the amount of a compound necessary to result
in a desired physiological change in the subject. Effective amounts
are often administered for research purposes. Effective amounts
disclosed herein reduce the number of unwanted cell types in a
subject.
[0148] A "prophylactic treatment" includes a treatment administered
to a subject who does not display signs or symptoms of a disease or
condition associated with or caused by a target or displays only
early signs or symptoms of the disease or condition such that
treatment is administered for the purpose of diminishing,
preventing, or decreasing the risk of developing the disease or
condition further. Thus, a prophylactic treatment functions as a
preventative treatment against a disease or disorder associated
with or caused by a target.
[0149] A "therapeutic treatment" includes a treatment administered
to a subject who displays symptoms or signs of a disease or
condition associated with or caused by a target and is administered
to the subject for the purpose of diminishing or eliminating those
signs or symptoms of the disease or condition.
[0150] "Therapeutically effective amounts" include those that
provide effective amounts, prophylactic treatment and/or
therapeutic treatment. Therapeutically effective amounts need not
fully prevent or cure the disease or condition but can also provide
a partial benefit, such as reduction in the number of unwanted
targets; reduction of destruction of wanted targets; and/or a delay
of onset or alleviation or improvement of at least one symptom of
the disease or condition.
[0151] For administration, effective amounts and therapeutically
effective amounts (also referred to herein as doses) can be
initially estimated based on results from in vitro assays and/or
animal model studies. For example, a dose can be formulated in
animal models to achieve a circulating concentration range that
includes an IC.sub.50 as determined in cell culture against a
particular target. Such information can be used to more accurately
determine useful doses in subjects of interest.
[0152] The actual dose amount administered to a particular subject
can be determined by a physician, veterinarian or researcher taking
into account parameters such as physical and physiological factors
including target, body weight, severity of condition, type of
disease, previous or concurrent therapeutic interventions,
idiopathy of the subject and route of administration.
[0153] Useful doses often range from 0.1 to 5 .mu.g/kg or from 0.5
to 1 .mu.g/kg. In other non-limiting examples, a dose can comprise
1 .mu.g/kg, 5 .mu.g/kg, 10 .mu.g/kg, 15 .mu.g/kg, 20 .mu.g/kg, 25
.mu.g/kg, 30 .mu.g/kg, 35 .mu.g/kg, 40 .mu.g/kg, 45 .mu.g/kg, 50
.mu.g/kg, 55 .mu.g/kg, 60 .mu.g/kg, 65 .mu.g/kg, 70 .mu.g/kg, 75
.mu.g/kg, 80 .mu.g/kg, 85 .mu.g/kg, 90 .mu.g/kg, 95 .mu.g/kg, 100
.mu.g/kg, 150 .mu.g/kg, 200 .mu.g/kg, 250 .mu.g/kg, 350 .mu.g/kg,
400 .mu.g/kg, 450 .mu.g/kg, 500 .mu.g/kg, 550 .mu.g/kg, 600
.mu.g/kg, 650 .mu.g/kg, 700 .mu.g/kg, 750 .mu.g/kg, 800 .mu.g/kg,
850 .mu.g/kg, 900 .mu.g/kg, 950 .mu.g/kg, 1000 .mu.g/kg, 0.1 to 5
mg/kg or from 0.5 to 1 mg/kg. In other non-limiting examples, a
dose can comprise 1 mg/kg, 5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg,
25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 55
mg/kg, 60 mg/kg, 65 mg/kg, 70 mg/kg, 75 mg/kg, 80 mg/kg, 85 mg/kg,
90 mg/kg, 95 mg/kg, 100 mg/kg, 150 mg/kg, 200 mg/kg, 250 mg/kg, 350
mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 650
mg/kg, 700 mg/kg, 750 mg/kg, 800 mg/kg, 850 mg/kg, 900 mg/kg, 950
mg/kg, 1000 mg/kg or more.
[0154] Therapeutically effective amounts can be achieved by
administering single or multiple doses during the course of a
treatment regimen (e.g., daily, every other day, every 3 days,
every 4 days, every 5 days, every 6 days, weekly, every 2 weeks,
every 3 weeks, monthly, every 2 months, every 3 months, every 4
months, every 5 months, every 6 months, every 7 months, every 8
months, every 9 months, every 10 months, every 11 months or
yearly.
[0155] Exemplary methods disclosed herein include administering
nanocarriers to a subject in need thereof. The nanocarriers are
directed to chosen lymphocytes in the subject and are designed to
be internalized by the lymphocytes. Once internalized, the
nanocarriers further deliver a polynucleotide having a sequence
that encodes a targeting agent. The polynucleotide modifies the
lymphocytes to express the targeting agent, which subsequently
binds a marker associated with the target. Upon binding, the
lymphocytes can kill or otherwise trigger the destruction of
unwanted targets such as unwanted cells, thereby treating a disease
or condition associated with the unwanted cell type. Alternatively,
upon binding, the lymphocytes can protect wanted targets such as
wanted cells, thereby treating a disease or condition associated
with unwanted destruction of the wanted cell type.
[0156] In another particular embodiment, nanocarriers can be loaded
with polynucleotides (e.g., Transgenes) that encode for a defined
tumor- or virus-specific TCR. Surface-anchored lymphocyte-directing
agents that recognize T-cell-specific proteins enable the
nanocarriers to selectively bind T-cells. Upon infusion into a
subject's bloodstream, the nanocarriers can deliver TCR genes into
T-cells, which can subsequently express this TCR on their surface.
Equipped with a therapeutically relevant TCR, the T-cells can
recognize and lyse malignant cells or virus-infected cells or other
targeted unwanted cell types.
[0157] In additional embodiments, NK cells are selectively modified
to express CARs or high-affinity TCRs. In additional embodiments,
hematopoietic stem cells (HSGs) are selectively modified to express
CARs or high-affinity TCRs. In additional embodiments,
monocytes/macrophages cells are selectively modified to express
CARs or high-affinity ligands specific for viruses, bacteria,
fungus or yeast antigens. In additional embodiments, B cells are
selectively modified to express tumor- or virus-specific
antibodies. In additional embodiments, T.sub.REG cells are
selectively modified to express CARs or high-affinity ligands
specific for autoimmune markers, allergic reaction markers or
beneficial bacteria.
[0158] Additional embodiments include methods of delivering
pre-designed synthetic nanocarriers to lymphocytes (e.g., T-cells),
in which the nanocarriers can be loaded with polynucleotides (e.g.,
plasmids) that encode a receptor for an antigen (e.g., a prostate
tumor-targeting receptor P28z). Internalization of the nanocarriers
can render transfected lymphocytes (e.g., T-cells) capable of
lysing cells associated with the antigen (e.g., a prostate tumor).
In some embodiments, delivery of the nanocarriers including the
receptor genes into lymphocytes (e.g., T-cells) can include, e.g.,
(1) specific binding to the lymphocytes (e.g., T-cells), (2)
internalization of the nanocarriers by the lymphocytes, (3) escape
from endocytic vesicles into the cytoplasm after internalization,
(4) release of the polynucleotide, which (5) can be transported
into the nucleus of the lymphocytes and (6) transcribed to deliver
genes for expressing a receptor for the antigen.
[0159] In particular embodiments, the methods are used to target
unwanted cancer cells. Thus, the disclosed methods provide a new
paradigm for the treatment of cancer that can involve programming
circulating lymphocytes with tumor-recognizing capabilities in
vivo. This paradigm contrasts with those currently used to generate
T cells with defined anti-cancer specificities, which involve
isolation of the lymphocytes from the patient and genetically
modifying them in the laboratory with tumor antigen-specific
receptors using retroviral or lentiviral vectors; the programmed
cells are then expanded and infused back into the patient where
they can recognize and destroy cancer cells. This ex vivo
production of modified cells requires the production of a new
lymphocyte cohort for each patient, a laborious process that can
only be accomplished at elaborate cell-production facilities
available at just a few cancer centers worldwide.
[0160] The disclosed methods provide a more practical and
widespread approach, allowing use of an "off-the-shelf" solution
that can quickly modify lymphocytes to recognize and destroy tumors
while they are circulating in a subject, thus avoiding the
complications of laboratory modification of extracted cells. In
comparison to in vitro methods that modify and expand T cells for
each patient, the compositions and methods disclosed herein can
produce targeting effects within a subject's circulatory system in
only days.
[0161] The disclosed methods provide the first implementation of
nanocarriers for the genetic engineering of immune cells to
selectively target cells associated with markers for various
therapeutic objectives. For example, and in relation to cancer
cells as an unwanted cell type, previous nanotechnology-based
clinical research has focused on particles that selectively
accumulate chemotherapeutics, siRNA, or imaging agents at tumor
sites while minimizing off-target toxicities. The methods described
herein are different: instead of introducing therapeutics into
tumor tissue, the disclosed methods introduce genes encoding
tumor-recognizing receptors into circulating lymphocytes, which in
turn bind and destroy tumor cells. This strategy has the advantage
that, unlike agent-loaded nanoparticles (which are quickly cleared
by phagocytes), the modified lymphocytes can persist and
proliferate in the subject for a long-term effect. Thus, in
relation to cancer treatments specifically, the current disclosure
provides a new, more effective therapy. The disclosure shifts the
focus from broad-impact chemotherapy or radiotherapy (which have
many negative side-effects) to tumor-specific immunotherapeutics
(which do not harm healthy tissue). Nanoparticle gene therapy will
provide clinicians with the ability to instantly treat diagnosed
patients with an off-the shelf composition that can be widely
distributed at low cost, and is amenable to changes in dose and
specificity as the treatment evolves.
[0162] In the context of cancers, therapeutically effective amounts
can decrease the number of tumor cells, decrease the number of
metastases, decrease tumor volume, increase life expectancy, induce
apoptosis of cancer cells, induce cancer cell death, induce chemo-
or radiosensitivity in cancer cells, inhibit angiogenesis near
cancer cells, inhibit cancer cell proliferation, inhibit tumor
growth, prevent metastasis, prolong a subject's life, reduce
cancer-associated pain, reduce the number of metastases, and/or
reduce relapse or re-occurrence of the cancer following
treatment.
[0163] While the methods disclosed herein are advantageously
practiced in vivo, additional embodiments may also be practiced ex
vivo. For example, the methods can include obtaining lymphocytes
from a subject. Lymphocytes can, e.g., be obtained from a subject
using any procedure generally known in the art. For example, blood
can be obtained from a subject and lymphocytes can be isolated. The
isolated lymphocytes can then be combined with nanocarriers (or a
composition comprising nanocarriers) including a polynucleotide
having a sequence that encodes a targeting agent. The nanocarriers
can be internalized by the lymphocytes such that the lymphocytes
then incorporate the polynucleotide and express the targeting
agent. The modified lymphocytes expressing the targeting agent can
be administered to the subject such that, after the administering,
the lymphocytes bind to the targeted markers on cells associated
with the disease, thereby treating the disease. It will be
appreciated, for example, that the modifying of the lymphocytes can
be fully accomplished ex vivo prior to administration, and/or
nanocarriers can be internalized and the lymphocytes can be
administered to the subject while modifying is being carried out
leading to expression of the targeting agents.
EXEMPLARY EMBODIMENTS--Set 1.
[0164] 1. A synthetic nanocarrier comprising (i) a lipid-coated
porous nanoparticle (ii) a lymphocyte-directing agent extending
from the surface of the lipid-coated porous nanoparticle; and (iii)
a polynucleotide encoding a chimeric antigen receptor (CAR)
targeting agent within the pores of the lipid-coated porous
nanoparticle nanoparticle. [0165] 2. A synthetic nanocarrier of
embodiment 1 further comprising an endosomal release agent
extending from the surface of the lipid-coated porous nanoparticle
and (ii) a nuclear localization signal (NLS) within the pores of
the lipid-coated porous nanoparticle. [0166] 3. A synthetic
nanocarrier of embodiments 1 or 2 wherein the CAR is P28z. [0167]
4. A synthetic nanocarrier of any one of embodiments 1, 2 or 3
wherein the lipid coating is a liposome, a lipid bilayer or a
polymeric micelle. [0168] 5. A synthetic nanocarrier of any one of
embodiments 1-4 wherein the synthetic nanocarriers comprise
liposomes, polymeric particles, metallic particles, polymeric
micelles, polyethyleneimine (PEI)/DNA complexes, or a combination
thereof. [0169] 6. A synthetic nanocarrier of any one of
embodiments 1-5 wherein the lipid coating encapsulates the
lipid-coated porous nanoparticle. [0170] 7. A synthetic nanocarrier
of any one of embodiments 1-6 wherein the lymphocyte-directing
agent selectively binds to lymphocytes in vivo. [0171] 8. A
synthetic nanocarrier of any one of embodiments 1-7 wherein the
lymphocyte-directing agent comprises a binding domain selected from
a lymphocyte receptor ligand, lymphocyte receptor antibody,
lymphocyte receptor peptide aptamer, lymphocyte receptor nucleic
acid aptamer, lymphocyte receptor spiegelmer, or a combination
thereof. [0172] 9. A synthetic nanocarrier of any one of
embodiments 1-8 wherein the lymphocyte-directing agent selectively
binds T cells, NK cells, monocytes, macrophages, B cells,
hematopoietic stem cells, or a combination thereof. [0173] 10.A
synthetic nanocarrier of any one of embodiments 1-9 wherein the
lymphocyte-directing agent selectively binds T-cell receptor
motifs; T-cell .alpha. chains; T-cell .beta. chains; T-cell .gamma.
chains; T-cell .delta. chains; CCR7; CD3; CD4; CD5; CD7; CD8;
CD11b; CD11c; CD16; CD19; CD20; CD21; CD22; CD25; CD28; CD34; CD35;
CD40; CD45RA; CD45RO; CD52; CD56; CD62L; CD68; CD80; CD95; CD117;
CD127; CD133; CD137 (4-1BB); CD163; F4/80; IL-4R.alpha.; Sca-1;
CTLA-4; GITR; GARP; LAP; granzyme B; LFA-1; or transferrin
receptor. [0174] 11. A synthetic nanocarrier of any one of
embodiments 1-9 wherein the lymphocyte-directing agent selectively
binds CCR7; CD3; CD4; CD5; CD8; CD16; CD19; CD20; CD21; CD22; CD25;
CD28; CD35; CD40; CD45RA; CD45RO; CD52; CD62L; CD80; CD95; CD127;
or CD137. [0175] 12. A synthetic nanocarrier of any one of
embodiments 1-9 wherein the lymphocyte-directing agent comprises a
binding domain selected from a T-cell .alpha. chain antibody;
T-cell .beta. chain antibody; T-cell .gamma. chain antibody; T-cell
.delta. chain antibody; CCR7 antibody; CD3 antibody; CD4 antibody;
CD5 antibody; CD7 antibody; CD8 antibody; CD11 b antibody; CD11c
antibody; CD16 antibody; CD19 antibody; CD20 antibody; CD21
antibody; CD22 antibody; CD25 antibody; CD28 antibody; CD34
antibody; CD35 antibody; CD40 antibody; CD45RA antibody; CD45RO
antibody; CD52 antibody; CD56 antibody; CD62L antibody; CD68
antibody; CD80 antibody; CD95 antibody; CD117 antibody; CD127
antibody; CD133 antibody; CD137 (4-1BB) antibody; CD163 antibody;
F4/80 antibody; IL-4R.alpha. antibody; Sca-1 antibody; CTLA-4
antibody; GITR antibody GARP antibody; LAP antibody; granzyme B
antibody; LFA-1 antibody; or transferrin receptor antibody. [0176]
13. A synthetic nanocarrier of embodiment 12 wherein the binding
domain consists of or consists essentially of an scFv fragment of a
T-cell .alpha. chain antibody; T-cell .beta. chain antibody; T-cell
.gamma. chain antibody; T-cell .delta. chain antibody; CCR7
antibody; CD3 antibody; CD4 antibody; CD5 antibody; CD7 antibody;
CD8 antibody; CD11 b antibody; CD11c antibody; CD16 antibody; CD19
antibody; CD20 antibody; CD21 antibody; CD22 antibody; CD25
antibody; CD28 antibody; CD34 antibody; CD35 antibody; CD40
antibody; CD45RA antibody; CD45RO antibody; CD52 antibody; CD56
antibody; CD62L antibody; CD68 antibody; CD80 antibody; CD95
antibody; CD117 antibody; CD127 antibody; CD133 antibody; CD137
(4-1BB) antibody; CD163 antibody; F4/80 antibody; IL-4R.alpha.
antibody; Sca-1 antibody; CTLA-4 antibody; GITR antibody GARP
antibody; LAP antibody; granzyme B antibody; LFA-1 antibody; or
transferrin receptor antibody. [0177] 14. A synthetic nanocarrier
of embodiment 12 wherein the binding domain consists of consists of
or consists essentially of the scFv fragment (SEQ ID NO. 1) of the
PSMA-specific chimeric antigen receptor (CAR), P28z. [0178] 15. A
synthetic nanocarrier of any of embodiments 1-14 wherein the
polynucleotide is a plasmid, a minicircle plasmid, or an mRNA
molecule. [0179] 16. A synthetic nanocarrier of any of embodiments
1-15 wherein the CAR targeting agent comprises a binding domain for
a marker associated with an unwanted cell type. [0180] 17. A
synthetic nanocarrier of embodiment 16 wherein the unwanted cell
type is a cancer cell. [0181] 18. A synthetic nanocarrier of
embodiment 16 wherein the marker is a cancer antigen. [0182] 19. A
synthetic nanocarrier of embodiment 16 wherein the marker is a
cancer antigen selected from A33; BAGE; Bcl-2; .beta.-catenin;
CAl25; CA19-9; CD5; CD19; CD20; CD21; CD22; CD33; CD37; CD45;
CD123; CEA; c-Met; CS-1; cyclin B1; DAGE; EBNA; EGFR; ephrinB2;
estrogen receptor; FAP; ferritin; folate-binding protein; GAGE;
G250; GD-2; GM2; gp75, gp100 (Pmel 17); HER-2/neu; HPV E6; HPV E7;
Ki-67; LRP; mesothelin, p53, PRAME; progesterone receptor; PSA;
PSMA; MAGE; MART; mesothelin; MUC; MUM-1-B; myc; NYESO-1; ras;
RORI; survivin; tenascin; TSTA tyrosinase; VEGF; or WT1. [0183] 20.
A synthetic nanocarrier of embodiment 16 wherein the marker is
PSMA. [0184] 21. A synthetic nanocarrier of any of embodiments 1-20
wherein the CAR targeting agent is a surface antigen receptor or a
receptor for an intracellular antigen presented by a Major
Histocompatibility Complex antigen-presenting pathway. [0185] 22. A
synthetic nanocarrier of any one of embodiments 2-21 wherein the
endosomal release agent is selected from any one of SEQ ID NOs.
29-50 or combinations thereof. [0186] 23. A synthetic nanocarrier
of any one of embodiments 2-22 wherein the NLS is selected from any
one of SEQ ID NOs. 51-93 or combinations thereof. [0187] 24. A
synthetic nanocarrier of any one of embodiments 1-23 comprising a
S/MAR element, a PiggyBac transposase-containing plasmid, a
Sleeping Beauty transposase-containing plasmid; a homo sapiens
transposon-derived Buster1 transposase-like protein gene; a human
endogenous retrovirus H protease/integrase-derived ORF1; a homo
sapiens Cas-Br-M (murine) ecotropic retroviral transforming
sequence; a homo sapiens endogenous retroviral sequence K; a homo
sapiens endogenous retroviral family W sequence; a homo sapiens
LINE-1 type transposase domain; or a homo sapiens pogo transposable
element. [0188] 25. A composition comprising a synthetic
nanocarrier of any one of embodiments 1-24. [0189] 26. A method of
treating a subject having a condition associated with a cell type
comprising: administering a therapeutically effective amount of a
synthetic nanocarrier of any one of embodiments 1-24 to the subject
thereby treating the subject. [0190] 27. A method of treating a
subject having a condition associated with a cell type comprising:
administering a therapeutically effective amount of a composition
of embodiment 25 to the subject thereby treating the subject.
[0191] 28. A method of embodiments 26 or 27 wherein the unwanted
cell type is an unwanted cancer cell. [0192] 29. A method of
embodiment 28 wherein the unwanted cancer cell is selected from an
adrenal cancer cell, a bladder cancer cell, a blood cancer cell, a
bone cancer cell, a brain cancer cell, a breast cancer cell, a
carcinoma cell, a cervical cancer cell, a colon cancer cell, a
colorectal cancer cell, a corpus uterine cancer cell, an ear, nose
and throat (ENT) cancer cell, an endometrial cancer cell, an
esophageal cancer cell, a gastrointestinal cancer cell, a head and
neck cancer cell, a Hodgkin's disease cell, an intestinal cancer
cell, a kidney cancer cell, a larynx cancer cell, a leukemia cell,
a liver cancer cell, a lymph node cancer cell, a lymphoma cell, a
lung cancer cell, a melanoma cell, a mesothelioma cell, a myeloma
cell, a nasopharynx cancer cell, a neuroblastoma cell, a
non-Hodgkin's lymphoma cell, an oral cancer cell, an ovarian cancer
cell, a pancreatic cancer cell, a penile cancer cell, a pharynx
cancer cell, a prostate cancer cell, a rectal cancer cell, a
sarcoma cell, a seminoma cell, a skin cancer cell, a stomach cancer
cell, a teratoma cell, a testicular cancer cell, a thyroid cancer
cell, a uterine cancer cell, a vaginal cancer cell, or a vascular
tumor cell. [0193] 30. A method of any one of embodiments 26-29
wherein the administering results in expression of the
polynucleotide selectively by lymphocytes within 10 days; within 9
days; within 8 days; within 7 days; within 6 days; within 5 days;
within 4 days; or within 3 days of administration. [0194] 31. A
method for treating a disease associated with an antigen, the
method comprising: administering to a subject in need thereof, a
composition comprising a therapeutically effective amount of
nanocarriers including a polynucleotide having a sequence that
encodes a receptor for the antigen, thereby treating the disease.
[0195] 32. A method of embodiment 31 wherein after the
administering the nanocarriers are selectively incorporated into
lymphocytes in the subject such that the lymphocytes express the
receptor and subsequently bind to the antigen on cells associated
with the disease thereby killing the cells. [0196] 33. A method for
treating a disease associated with an antigen, the method
comprising: [0197] obtaining lymphocytes from a subject in need
thereof; [0198] combining the lymphocytes with a composition
comprising nanocarriers including a polynucleotide having a
sequence that encodes a receptor for the antigen, [0199] wherein
the nanocarriers are selectively incorporated into the lymphocytes
such that the lymphocytes express the receptor; and [0200]
administering the lymphocytes expressing the receptor to the
subject, thereby treating the disease. [0201] 34. A method of
embodiment 33 wherein after the administering, the lymphocytes bind
to the antigen on cells associated with the disease thereby killing
the cells. [0202] 35. A method of selectively transfecting
lymphocytes in vivo, the method comprising: [0203] contacting
lymphocytes with nanocarriers comprising a polynucleotide having a
sequence that encodes a receptor for an antigen associated with a
disease, [0204] wherein the nanocarriers are selectively
incorporated into the lymphocyte to release the polynucleotide such
that the lymphocyte expresses the receptor, thereby transfecting
the lymphocyte. [0205] 36. A method of embodiment 35 wherein the
antigen comprises a tumor antigen. [0206] 37. A method of
embodiment 35 wherein the antigen comprises a viral antigen. [0207]
38. A method of any one of embodiments 35-37 wherein the
lymphocytes comprise T-cells, NK cells, macrophages, monocytes, B
cells, hematopoietic stem cells, or a combination thereof. [0208]
39. A method of embodiment 38 wherein the lymphocytes comprise
T-cells. [0209] 40. A method of any one of embodiments 35-39
wherein the disease is a cancer. [0210] 41. A method of embodiment
40 wherein the cancer comprises a leukemia, a lymphoma, a
carcinoma, a sarcoma, or a melanoma. [0211] 42. A method of
embodiment 40 wherein the disease is prostate cancer.
EXEMPLARY EMBODIMENTS--Set 2.
[0211] [0212] 1. A synthetic nanocarrier comprising (i) a
lymphocyte-directing agent; and (ii) a polynucleotide encoding a
targeting agent. [0213] 2. A synthetic nanocarrier of embodiment 1
further comprising a nanoparticle. [0214] 3. A synthetic
nanocarrier of embodiments 1 or 2 further comprising a coating.
[0215] 4. A synthetic nanocarrier of embodiment 2 or 3 wherein the
nanoparticle is a porous nanoparticle. [0216] 5. A synthetic
nanocarrier of embodiment 3 or 4 wherein the coating is a liposome,
a lipid bilayer, or a polymeric micelle. [0217] 6. A synthetic
nanocarrier of any one of embodiments 1-5 wherein the synthetic
nanocarrier comprises liposomes, polymeric particles, metallic
particles, polymeric micelles, polyethyleneimine (PEI)/DNA
complexes, or a combination thereof. [0218] 7. A synthetic
nanocarrier of embodiments 3 or 5 wherein the coating encapsulates
the nanoparticle. [0219] 8. A synthetic nanocarrier of any one of
embodiments 1-7 wherein the polynucleotide is on the surface of the
nanocarrier, incorporated in the nanocarrier, encapsulated in the
nanocarrier, or a combination thereof. [0220] 9. A synthetic
nanocarrier of any one of embodiments 3-8 wherein the
lymphocyte-directing agent extends from the outer surface of the
coating. [0221] 10. A synthetic nanocarrier of any one of
embodiments 4-9 wherein the polynucleotide is within the pores of
the porous nanoparticle. [0222] 11. A synthetic nanocarrier of any
one of embodiments 1-10 wherein the lymphocyte-directing agent
selectively binds to lymphocytes in vivo. [0223] 12. A synthetic
nanocarrier of any one of embodiments 1-11 wherein the
lymphocyte-directing agent comprises a binding domain selected from
a lymphocyte receptor ligand, lymphocyte receptor antibody,
lymphocyte receptor peptide aptamer, lymphocyte receptor nucleic
acid aptamer, lymphocyte receptor spiegelmer, or a combination
thereof. [0224] 13. A synthetic nanocarrier of any one of
embodiments 1-12 wherein the lymphocyte-directing agent selectively
binds T cells, NK cells, monocytes, macrophages, B cells,
hematopoietic stem cells, or a combination thereof. [0225] 14. A
synthetic nanocarrier of any one of embodiments 1-13 wherein the
lymphocyte-directing agent selectively binds T-cell receptor
motifs; T-cell .alpha. chains; T-cell .beta. chains; T-cell .gamma.
chains; T-cell .DELTA. chains; CCR7; CD3; CD4; CD5; CD7; CD8; CD11
b; CD11c; CD16; CD19; CD20; CD21; CD22; CD25; CD28; CD34; CD35;
CD40; CD45RA; CD45RO; CD52; CD56; CD62L; CD68;CD80; CD95; CD117;
CD127; CD133; CD137 (4-1BB); CD163; F4/80; IL-4R.alpha.; Sca-1;
CTLA-4; GITR; GARP; LAP; granzyme B; LFA-1; or transferrin
receptor. [0226] 15.A synthetic nanocarrier of any one of
embodiments 1-13 wherein the lymphocyte-directing agent selectively
binds CCR7; CD3; CD4; CD5; CD8; CD16; CD19; CD20; CD21; CD22; CD25;
CD28; CD35; CD40; CD45RA; CD45RO; CD52; CD62L; CD80; CD95; CD127;
or CD137. [0227] 16.A synthetic nanocarrier of any one of
embodiments 1-13 wherein the lymphocyte-directing agent comprises a
binding domain selected from a T-cell .alpha. chain antibody;
T-cell .beta. chain antibody; T-cell .gamma. chain antibody; T-cell
.DELTA. chain antibody; CCR7 antibody; CD3 antibody; CD4 antibody;
CD5 antibody; CD7 antibody; CD8 antibody; CD11 b antibody; CD11c
antibody; CD16 antibody; CD19 antibody; CD20 antibody; CD21
antibody; CD22 antibody; CD25 antibody; CD28 antibody; CD34
antibody; CD35 antibody; CD40 antibody; CD45RA antibody; CD45RO
antibody; CD52 antibody; CD56 antibody; CD62L antibody; CD68
antibody; CD80 antibody; CD95 antibody; CD117 antibody; CD127
antibody; CD133 antibody; CD137 (4-1BB) antibody; CD163 antibody;
F4/80 antibody; IL-4R.alpha. antibody; Sca-1 antibody; CTLA-4
antibody; GITR antibody GARP antibody; LAP antibody; granzyme B
antibody; LFA-1 antibody; or transferrin receptor antibody. [0228]
17. A synthetic nanocarrier of embodiment 16 wherein the binding
domain consists of or consists essentially of an scFv fragment of a
T-cell .alpha. chain antibody; T-cell .beta. chain antibody; T-cell
.gamma. chain antibody; T-cell .delta. chain antibody; CCR7
antibody; CD3 antibody; CD4 antibody; CD5 antibody; CD7 antibody;
CD8 antibody; CD11b antibody; CD11c antibody; CD16 antibody; CD19
antibody; CD20 antibody; CD21 antibody; CD22 antibody; CD25
antibody; CD28 antibody; CD34 antibody; CD35 antibody; CD40
antibody; CD45RA antibody; CD45RO antibody; CD52 antibody; CD56
antibody; CD62L antibody; CD68 antibody; CD80 antibody; CD95
antibody; CD117 antibody; CD127 antibody; CD133 antibody; CD137
(4-1BB) antibody; CD163 antibody; F4/80 antibody; IL-4R.alpha.
antibody; Sca-1 antibody; CTLA-4 antibody; GITR antibody GARP
antibody; LAP antibody; granzyme B antibody; LFA-1 antibody; or
transferrin receptor antibody. [0229] 18. A synthetic nanocarrier
of embodiment 17 wherein the binding domain consists of consists of
or consists essentially of the scFv fragment (SEQ ID NO. 1) of the
PSMA-specific chimeric antigen receptor (CAR), P28z. [0230] 19. A
synthetic nanocarrier of any of embodiments 1-18 wherein the
polynucleotide is a plasmid, a minicircle plasmid, or an mRNA
molecule. [0231] 20. A synthetic nanocarrier of any of embodiments
1-19 wherein the targeting agent comprises a binding domain for a
marker associated with an unwanted cell type. [0232] 21. A
synthetic nanocarrier of embodiment 20 wherein the unwanted cell
type is a cancer cell, a virally infected cell, a bacterial cell,
or a fungal cell. [0233] 22. A synthetic nanocarrier of embodiment
20 wherein the marker is a cancer antigen, a viral antigen, a
bacterial antigen, or a fungal antigen. [0234] 23. A synthetic
nanocarrier of embodiment 20 wherein the marker is a cancer antigen
selected from A33; BAGE; Bcl-2; .beta.-catenin; CA125; CA19-9; CD5;
CD19; CD20; CD21; CD22; CD33; CD37; CD45; CD123; CEA; c-Met; CS-1;
cyclin B1; DAGE; EBNA; EGFR; ephrinB2; estrogen receptor; FAP;
ferritin; folate-binding protein; GAGE; G250; GD-2; GM2; gp75,
gp100 (Pmel 17); HER-2/neu; HPV E6; HPV E7; Ki-67; LRP; mesothelin,
p53, PRAME; progesterone receptor; PSA; PSMA; MAGE; MART;
mesothelin; MUC; MUM-1-B; myc; NYESO-1; ras; RORI; survivin;
tenascin; TSTA tyrosinase; VEGF; or WT1. [0235] 24. A synthetic
nanocarrier of embodiment 23 wherein the marker is PSMA. [0236] 25.
A synthetic nanocarrier of embodiment 20 wherein the marker is a
viral antigen selected from envelope glycoprotein B; CMV pp65; EBV;
EBNAI; EBV; P18; EBV P23; S protein of hepatitis B; of M protein of
hepatitis B; L proteins of hepatitis B; pre-S antigen of hepatitis
B virus; HBCAG DELTA; HBV; HBE; hepatitis C viral RNA; HCV NS3; HCV
NS4; herpes simplex immediate early proteins; glycoprotein D; HIV
gp32; HIV gp41; HIV gp120; HIV gp160; HIV P17/24; HIV P24; HIV P55
GAG; HIV P66 POL; HIV TAT; HIV GP36; Nef protein; hemagglutinin;
neuraminidase; Japanese encephalitis protein E; Japanese
encephalitis protein M-E; Japanese encephalitis protein M-E-NS1;
Japanese encephalitis protein NS1; Japanese encephalitis protein
NS1-NS2A; Japanese encephalitis protein 80% E; measles virus fusion
protein; rabies glycoprotein; rabies nucleoprotein; RSV fusion
protein; M2 protein; VP7sc; rubella protein El; rubella protein E2;
gpl; gpll; Nef (66-97); Nef (116-145); Gag p17 (17-35); Gag p17-p24
(253-284); and Pol 325-355 (RT 158-188). [0237] 26. A synthetic
nanocarrier of embodiment 20 wherein the marker is a bacterial
antigen selected from anthrax protective antigen;
lipopolysaccharide; capsular polysaccharide; diptheria toxin;
mycolic acid; heat shock protein 65 (HSP65); the 30 kDa major
secreted protein; antigen 85A; hemagglutinin; pertactin; FIM2;
FIM3; adenylate cyclase; pneumolysin; pneumococcal capsular
polysaccharide; rompA; M proteins; tetanus toxin; lipoteichoic
acid; and clumping factor A (CIfA). [0238] 27. A synthetic
nanocarrier of embodiment 20 wherein the marker is a fungal antigen
selected from spherule antigens; capsular polysaccharides; heat
shock protein 60; gp63; lipophosphoglycan; merozoite surface
antigens; sporozoite surface antigens; circumsporozoite antigens;
gametocyte/gamete surface antigens; blood-stage antigen pf
155/RESA; glutathione-S-transferase; paramyosin; trichophytin;
SAG-1; p30; trypanosoma cruzi 75-77 kDa antigen; and trypanosoma
cruzi 56 kDa antigen. [0239] 28. A synthetic nanocarrier of any of
embodiments 1-19 wherein the targeting agent comprises a binding
domain for a marker associated with a wanted cell type. [0240] 29.
A synthetic nanocarrier of embodiment 28 wherein the wanted cell
type is a cell associated with an autoimmune disorder, a cell
associated with an allergy, or a bacterial cell. [0241] 30. A
synthetic nanocarrier of embodiment 28 wherein the marker is an
autoimmune antigen, an allergic antigen, or a bacterial antigen.
[0242] 31. A synthetic nanocarrier of embodiment 30 wherein the
marker is an autoimmune antigen selected from glutamic acid
decarboxylase 65 (GAD 65); native DNA; myelin basic protein; myelin
proteolipid protein; acetylcholine receptor components;
thyroglobulin; and thyroid stimulating hormone (TSH) receptor.
[0243] 32. A synthetic nanocarrier of embodiment 30 wherein the
marker is an allergic antigen selected from Japanese cedar pollen
antigens; ragweed pollen antigens; rye grass pollen antigens; dust
mite antigens; feline antigens; and canine antigens. [0244] 33. A
synthetic nanocarrier of any of embodiments 1-32 wherein the
targeting agent is a surface antigen receptor or a receptor for an
intracellular antigen presented by a Major Histocompatibility
Complex antigen-presenting pathway. [0245] 34. A synthetic
nanocarrier of any of embodiments 1-33 wherein the targeting agent
is an antigen receptor or a chimeric antigen receptor. [0246] 35. A
synthetic nanocarrier of embodiment 34 wherein the targeting agent
is a P28z chimeric antigen receptor. [0247] 36. A synthetic
nanocarrier of embodiment 34 wherein the marker is CD19. [0248] 37.
A synthetic nanocarrier of embodiment 36 wherein the targeting
agent is monoclonal antibody FMC63. [0249] 38. A synthetic
nanocarrier of embodiment 36 wherein the targeting agent is a human
or humanized scFv comprising a variable light chain comprising a
CDRL1 sequence of RASQDISKYLN (SEQ ID NO. 14), CDRL2 sequence of
SRLHSGV (SEQ ID NO. 15), and a CDRL3 sequence of GNTLPYTFG (SEQ ID
NO. 16). [0250] 39. A synthetic nanocarrier of embodiment 36
wherein the targeting agent is a human or humanized scFv comprising
a variable heavy chain comprising CDRHI sequence of DYGVS (SEQ ID
NO. 17), CDRH2 sequence of VTWGSETTYYNSALKS (SEQ ID NO. 18), and a
CDRH3 sequence of YAMDYWG (SEQ ID NO. 19). [0251] 40. A synthetic
nanocarrier of embodiment 34 wherein the marker is RORI. [0252] 41.
A synthetic nanocarrier of embodiment 40 wherein the targeting
agent is a human or humanized scFv comprising a variable light
chain comprising a CDRL1 sequence of ASGFDFSAYYM (SEQ ID NO. 22),
CDRL2 sequence of TIYPSSG (SEQ ID NO. 23), and a CDRL3 sequence of
ADRATYFCA (SEQ ID NO. 24). [0253] 42. A synthetic nanocarrier of
embodiment 40 wherein the targeting agent is a human or humanized
scFv comprising a variable heavy chain comprising CDRH1 sequence of
DTIDWY (SEQ ID NO. 25), CDRH2 sequence of VQSDGSYTKRPGVPDR (SEQ ID
NO. 26), and a CDRH3 sequence of YIGGYVFG (SEQ ID NO. 27). [0254]
43. A synthetic nanocarrier of any one of embodiments 1-42 wherein
the synthetic nanocarrier further comprises an endosomal release
agent. [0255] 44. A synthetic nanocarrier of any one of embodiments
43 wherein the endosomal release agent extends from the outer
surface of the coating. [0256] 45. A synthetic nanocarrier of
embodiments 43 or 44 wherein the endosomal release agent is
selected from any one of SEQ ID NOs. 29-50 or combinations thereof.
[0257] 46. A synthetic nanocarrier of any of embodiments 1-45
wherein the polynucleotide is associated with a nuclear
localization signal (NLS). [0258] 47. A synthetic nanocarrier of
any one of embodiments 46 wherein the NLS is within a pore of the
porous nanoparticle. [0259] 48. A synthetic nanocarrier of
embodiments 46 or 47 wherein the NLS is selected from any one of
SEQ ID NOs. 51-93 or combinations thereof. [0260] 49. A synthetic
nanocarrier of any one of embodiments 1-48 comprising a S/MAR
element, a PiggyBac transposase-containing plasmid, a Sleeping
Beauty transposase-containing plasmid; a homo sapiens
transposon-derived Busterl transposase-like protein gene; a human
endogenous retrovirus H protease/integrase-derived ORF1; a homo
sapiens Cas-Br-M (murine) ecotropic retroviral transforming
sequence; a homo sapiens endogenous retroviral sequence K; a homo
sapiens endogenous retroviral family W sequence; a homo sapiens
LINE-1 type transposase domain; or a homo sapiens pogo transposable
element. [0261] 50. A composition comprising a synthetic
nanocarrier of any one of embodiments 1-49. [0262] 51. A method of
treating a subject having a condition associated with a cell type
comprising: administering a therapeutically effective amount of a
synthetic nanocarrier of any one of embodiments 1-49 to the subject
thereby treating the subject. [0263] 52. A method of treating a
subject having a condition associated with a cell type comprising:
administering a therapeutically effective amount of a composition
of embodiment 50 to the subject thereby treating the subject.
[0264] 53. A method of embodiments 51 or 52 wherein the cell type
is an unwanted cell type selected from a cancer cell, a virally
infected cell, a bacterial cell, or a fungal cell. [0265] 54. A
method of embodiment 53 wherein the unwanted cell type is a cancer
cell selected from an adrenal cancer cell, a bladder cancer cell, a
blood cancer cell, a bone cancer cell, a brain cancer cell, a
breast cancer cell, a carcinoma cell, a cervical cancer cell, a
colon cancer cell, a colorectal cancer cell, a corpus uterine
cancer cell, an ear, nose and throat (ENT) cancer cell, an
endometrial cancer cell, an esophageal cancer cell, a
gastrointestinal cancer cell, a head and neck cancer cell, a
Hodgkin's disease cell, an intestinal cancer cell, a kidney cancer
cell, a larynx cancer cell, a leukemia cell, a liver cancer cell, a
lymph node cancer cell, a lymphoma cell, a lung cancer cell, a
melanoma cell, a mesothelioma cell, a myeloma cell, a nasopharynx
cancer cell, a neuroblastoma cell, a non-Hodgkin's lymphoma cell,
an oral cancer cell, an ovarian cancer cell, a pancreatic cancer
cell, a penile cancer cell, a pharynx cancer cell, a prostate
cancer cell, a rectal cancer cell, a sarcoma cell, a seminoma cell,
a skin cancer cell, a stomach cancer cell, a teratoma cell, a
testicular cancer cell, a thyroid cancer cell, a uterine cancer
cell, a vaginal cancer cell, or a vascular tumor cell. [0266] 55. A
method of any one of embodiments 51-54 wherein the administering
results in expression of the polynucleotide selectively by
lymphocytes within 10 days; within 9 days; within 8 days; within 7
days; within 6 days; within 5 days; within 4 days; or within 3 days
of administration.
[0267] 56. A method for treating a disease associated with an
antigen, the method comprising: administering to a subject in need
thereof, a composition comprising a therapeutically effective
amount of nanocarriers including a polynucleotide having a sequence
that encodes a receptor for the antigen, thereby treating the
disease. [0268] 57. A method of embodiment 56 wherein after the
administering the nanocarriers are selectively incorporated into
lymphocytes in the subject such that the lymphocytes express the
receptor and subsequently bind to the antigen on cells associated
with the disease thereby killing the cells. [0269] 58. A method for
treating a disease associated with an antigen, the method
comprising: [0270] obtaining lymphocytes from a subject in need
thereof; [0271] combining the lymphocytes with a composition
comprising nanocarriers including a polynucleotide having a
sequence that encodes a receptor for the antigen, [0272] wherein
the nanocarriers are selectively incorporated into the lymphocytes
such that the lymphocytes express the receptor; and [0273]
administering the lymphocytes expressing the receptor to the
subject, thereby treating the disease. [0274] 59. A method of
embodiment 58 wherein after the administering, the lymphocytes bind
to the antigen on cells associated with the disease thereby killing
the cells. [0275] 60. A method of selectively transfecting
lymphocytes in vivo, the method comprising: [0276] contacting
lymphocytes with nanocarriers comprising a polynucleotide having a
sequence that encodes a receptor for an antigen, wherein the
nanocarriers are selectively incorporated into the lymphocyte to
release the polynucleotide such that the lymphocyte expresses the
receptor, thereby transfecting the lymphocyte. [0277] 61. A method
of any one of embodiments 51-60 wherein the antigen comprises a
tumor antigen. [0278] 62. A method of any one of embodiments 51-60
wherein the antigen comprises a viral antigen. [0279] 63. A method
of any one of embodiments 51-62 wherein the lymphocytes comprise
T-cells, NK cells, macrophages, monocytes, B cells, hematopoietic
stem cells, or a combination thereof. [0280] 64. A method of
embodiment 63 wherein the lymphocytes comprise T-cells. [0281] 65.
A method of any one of embodiment 56 wherein the disease is a
cancer. [0282] 66. A method of embodiment 65 wherein the cancer
comprises a leukemia, a lymphoma, a carcinoma, a sarcoma, or a
melanoma. [0283] 67. A method of embodiment 65 wherein the disease
is prostate cancer. [0284] 68. A method of embodiment 62 wherein
the antigen is expressed by virus-infected cells associated with
the disease.
[0285] Each of the exemplary embodiments in Set 1 and Set 2 also
includes an embodiment wherein the lymphocyte-directing agent can
be removed. These embodiments are especially useful when the
selected cell types are monocytes/macrophages and broad
non-specific uptake of the nanocarriers can be expected.
EXAMPLES
[0286] The Examples below are included to demonstrate particular
embodiments of the disclosure. Those of ordinary skill in the art
should recognize in light of the present disclosure that many
changes can be made to the specific embodiments disclosed herein
and still obtain a like or similar result without departing from
the spirit and scope of the disclosure.
Example 1
[0287] This example demonstrates that synthetic nanoparticles
containing TCR genes can be used to generate functional tumor- or
virus-specific T-cells. Lipid nanoparticles (FIG. 2A) were loaded
with a minicircle gene (FIG. 3) encoding the chimeric antigen
receptor P28z. P28z is a fusion receptor composed of a single-chain
antibody (scFv) specific for the extracellular domain of PSMA
(J591) combined with CD28 and CD3 cytoplasmic signaling domains
(FIG. 4A; SEQ ID NO. 94). In this Example, chimeric antigen
receptors (CARs) are fusion receptors including an antigen-binding
domain, a transmembrane domain and an intracellular signaling
domain resulting in T-cell activation after antigen binding. The
P28z CAR directs T-cells toward the prostate-specific membrane
antigen (PSMA), which is highly expressed on prostate cancer cells.
Therefore, the introduction of the P28z gene into T-cells renders
them capable of recognizing and lysing prostate tumor. The P28z
gene was cloned under the control of the T-cell specific promoter
CD3 delta into a minicircle plasmid. Minicircles can include
episomal DNA vectors that are produced as circular expression
cassettes devoid of any bacterial plasmid DNA backbone. Their
smaller molecular size can enable more efficient transfections and
offers sustained expression over a period of weeks as compared to
standard plasmid vectors that only work for a few days.
[0288] The minicircle plasmid DNA was entrapped into nanocarriers.
DOPC, DOPE, cholesterol, and 18:1 PEG 2000 PE lipids were first
mixed in a 55:5:30:10 mass ratio, dried under a stream of nitrogen,
and placed in a vacuum oven overnight to remove residual
chloroform. The lipid film was then dissolved in tert-butanol and
mixed 1:1 (v/v) with a P28z minicircle plasmid solution (diluted in
10 mM Tris-HCl (pH 7.4) with 0.85% (w/v) NaCl and 0.25 M sucrose)
such that the final DOPC:DNA ratio was 10:1 (w/w). The mixture was
vortexed and passed through a 100 nm filter at least 10 times using
a Mini-Extruder set (Avanti Polar Lipids, Inc.; Alabaster, Al.,
USA).
[0289] To target nanocarriers to T-cells, anti-mouse CD8 antibodies
were coupled to the surface of the lipid envelope. Anti-CD8
antibodies (10 mg/ml) were mildly reduced with a 25.times. molar
excess of DTT for 20 min at 25.degree. C. in the presence of 10 mM
EDTA in PBS to expose free hinge region thiols. To remove DTT,
antibodies were passed through a desalting column. The
heterobifunctional cross-linker SM(PEG).sub.24 was used to anchor
antibodies to the surface of DNA-loaded liposomes (Amine groups are
present in the head groups of PE lipids, free thiol groups on
antibodies were created by DTT, SM(PEG) .sub.24 cross-links between
amines and thiol groups). Liposomes were first incubated with a
tenfold molar excess of SM(PEG).sub.24 for 2 h at room temperature
and centrifuged to remove unreacted cross-linker. Activated
liposomes were then incubated with a fivefold molar excess of
reduced anti-CD8 antibody for 2 h at room temperature. Unbound
antibody was removed using a centrifugal filtration device (10 kDa
MWCO). The final liposome used for subsequent experiments were
.about.100 nm in diameter.
[0290] P28z gene transfer into T-cells using targeted DNA
nanocarriers renders them capable of lysing prostate tumor. The
transfection efficiency of liposome-mediated gene transfer into
primary T-cells was assessed. 60.times.10.sup.6 mouse effector
CD8.sup.+ T-cells mL.sup.-1 were resuspended in RPMI medium and an
equal volume of lipid nanoparticles (loaded with P28z minicircle
DNA) were added with a 100 particles/T-cell ratio. Cells were
incubated at 37.degree. C. for 30 min with gentle agitation every
10 min and unbound particles were removed by a PBS wash. Two days
later, the percentage of T-cells expressing the P28z CAR was
determined by flow cytometry. Thirty hours after transfection
.about.23% of the cells expressed the P28z receptor on their
surface (FIG. 4B). High P28z expression persisted for three days in
vitro before declining toward undetectable expression by eight days
(data not shown). Nanoparticle-transfected T cells were functional,
selectively lysing PSMA-expressing TRAMP prostate tumor cells
(FIGS. 4C,D).
Example 2
[0291] CD3-targeted protocell nanoparticles selectively bind
circulating T cells in mice. A goal of the current disclosure is to
selectively and quickly edit lymphocyte specificity in vivo to
target unwanted cells. To examine how selectively protocells bind
circulating host T cells, mice were systemically injected with
1.times.10.sup.11 fluorescently tagged nanoparticles. After 6 hours
peripheral blood was collected by retro-orbital puncture and the
percentage of fluorescent T cells was quantified by flow cytometry.
CD3-targeted protocells labeled the majority of T cells in the
blood, with relatively low binding to off-target cells (FIG. 4E,
left panel). Confocal imaging of sorted T cells showed that
nanoparticles are rapidly internalized from the cell surface into
the cytoplasm as a result of receptor-induced endocytosis (FIG. 4E,
right panel).
Example 3
[0292] Generating an orthotopic bioluminescent mouse model for
analyzing treatment of metastatic prostate cancer. Male TRAMP
transgenic mice spontaneously develop orthotopic prostate tumors
following puberty. However, unlike human prostate adenocarcinoma,
TRAMP tumors do not express significant amounts of PSMA, a target
in experiments using the P28z CAR. Furthermore, longitudinal
studies to measure the prostate cancer volume in TRAMP animals rely
on expensive and time-consuming magnetic resonance imaging (MRI)
techniques, which preclude analysis of large cohorts of mice. To
overcome these issues, a cell line from a primary TRAMP tumor was
established and the PSMA gene was introduced through retroviral
transduction. To serially monitor tumor burden by bioluminescence
imaging, tumor cells were also genetically tagged with Firefly
luciferase (FLuc). Following orthotopic transplantation into the
dorsal lobe of the prostate gland of C57BL/6 mice, TRAMP-PSMA-FLuc
tumor cells reproducibly developed into lesions within three weeks,
with all animals displaying progressive metastatic tumor spread to
regional (pelvic, paraaortic) lymph nodes (FIG. 5).
Example 4
[0293] The data shown in FIGS. 2 and 4 establish the ability to
generate nanoparticles that efficiently program T cells with genes
encoding receptors specific for prostate tumor. While this strategy
rapidly generates tumor-reactive T cells, expression of transgenes
is transient because transferred plasmids are diluted out every
time the lymphocyte divides. The current example evaluates
persistent receptor gene expression in actively dividing T cells
caused by inserting into the plasmid either: 1) a scaffold/matrix
attachment region (S/MAR) sequence (which can undergo episomal
self-replication), or 2) a transposable piggyBac element (which
integrates the transgene into the genome). Stable and dependable
transgene expression in dividing T cells will allow
nanoparticle-transfected lymphocytes to serially kill unwanted cell
types providing long-lived immunity against such cells.
[0294] In this Example, a S/MAR sequence (provided by Dr. Lipps,
University Witten/Herdecke) or piggyBac inverted terminal repeats
(provided by Dr. Craig, Johns Hopkins University) will be cloned
into minicircle plasmids that encode the P28z receptor, as
illustrated in FIG. 6. Protocell nanoparticles loaded with
equivalent amounts of P28z, P28z-S/MAR, or P28z-piggyBac minicircle
DNA will be incubated with mouse CD8.sup.+ T lymphocytes at a
cell:particle ratio of 1:10. Following nanoparticle transfection, T
cells will be expanded with plate-bound anti-CD3/anti-CD28
antibodies. Flow cytometry will be used to assess P28z receptor
expression levels and persistence in proliferating T cells every 24
hours during a two week culture period.
[0295] To investigate the extent to which S/MAR sequences or
piggyBac transposable elements prevent nanoparticle-transferred
plasmids from being lost by dilution in dividing T cells, the
actual number of P28z gene copies per T cell over time will be
quantified. To this end, genomic and low-molecular weight
(episomal) DNA will be isolated from transfected T cells at each
time point during the two week period. Vector copy numbers will be
measured by multiplex quantitative PCR (qPCR) with a set of primers
and probes specific to the P28z minicircle plasmid. A set of
primers specific to the gene encoding mouse albumin will be
included as an internal two-copy control.
[0296] To discriminate between episomal (extrachromosomal) versus
genome-integrated P28z transgenes, Southern blot analysis will be
performed by digesting isolated DNA with Not1. This restriction
site is present only once in the P28z minicircle episome; it yields
a 2.8-kb band for the extrachromosomal episome but yields fragments
of various lengths for plasmids integrated into the genome.
[0297] The described studies will show that S/MAR-based episomes
and piggyBac transposons are two highly efficient tools to modify
cells to achieve stable gene expression. Incorporating S/MAR
sequences or piggyBac transposable elements into
nanocarrier-delivered plasmids will also maintain high-level P28z
gene expression in T cells over weeks as a result of episomal
self-replication or somatic integration, respectively. Because
plasmids containing S/MAR elements do not integrate into the host
genome, P28z gene expression is independent of chromosomal position
effects and therefore not subject to epigenetic silencing and
cis-acting sequences.
Example 6
[0298] This Example determines that systemic injections of DNA
nanocarriers can program sufficient quantities of T cells to target
and eliminate disseminated prostate cancer. The tests will be
conducted using nanoparticles loaded with minicircle DNA encoding
the P28z CAR (described above), to generate PSMA-specific
lymphocytes. The results of the studies will be positive following
testing of the following questions: (1) how many peripheral T cells
are genetically modified to express P28z following a single
intravenous dose of CD3-targeting nanoparticles loaded with genes
encoding the receptor?; (2) do the injected nanoparticles
selectively edit the antigen-specificity of peripheral T cells
without affecting off-target cells? And (3) what nanoparticle
dosage is required to bring about T cell-mediated regression of
metastatic prostate tumors in mice?
Example 6(1)
[0299] What percentage of peripheral T cells are modified by
nanoparticle gene therapy? The goal of this study is to edit the
antigen specificity of at least 10% of peripheral T cells within
five days following a single bolus injection of nanocarriers. For
comparison, some of the strongest vaccine vectors reported in the
literature induce frequencies of self/tumor antigen-specific T
cells of 1-4% following repeated immunizations over weeks. Mice
will be systemically injected with 1.times.10.sup.9,
1.times.10.sup.10, or 1.times.10.sup.11 nanocarriers loaded with
minicircle DNA encoding P28z, or with GFP as a control. After
collecting peripheral blood by retro-orbital puncture every four
days over a 12-day period, the percentage of P28z.sup.+ T cells
will be quantified by flow cytometry using fluorescent recombinant
PSMA protein as the reporter, as performed in previous gene
transfer studies (see, e.g., FIG. 4B).
Example 6(2)
[0300] Does nanoparticle gene therapy edit the antigen specificity
of peripheral T cells without affecting off-target cells? To
confirm in vivo studies, showing that CD3-targeting protocells
efficiently bind to host T cells after intravenous injection (FIG.
4E), how selectively nanoparticles introduce tumor-targeting
receptor genes into circulating T cells will also be determined. To
this end, P28z expression by other leukocyte subsets will be
evaluated, using the samples obtained in Example 6(1). The other
cell types will be identified using the following reporters:
anti-CD8 and anti-CD4 (T-cell markers), anti-B220 (B-cell marker),
anti-NK1.1 (NK-cell marker), anti-CD115, anti-F4/80 and anti-CD11b
(monocyte markers), anti-Ly6G and anti-CD11 b (neutrophil markers),
and anti-Gr-1 antibody (granulocyte marker).
Example 6(3)
[0301] What nanoparticle dosage is required to bring about T
cell-mediated regression of metastatic prostate tumors in mice? To
develop a reproducibly effective treatment for metastatic prostate
cancer, the therapeutically optimal frequency and dosage of
nanocarrier injections must be determined. A test system will be
created by injecting luciferase-expressing TRAMP-PSMA tumor cells
into the prostate of C57BL/6 mice and allowing them to develop for
three weeks before performing the tests (see, e.g., FIG. 5).
[0302] The mice will be systemically injected with CD3-targeting
nanocarriers carrying P28z-encoding transgenes, according to four
administration protocols: single high-dose bolus injection
(1.times.10.sup.10 nanoparticles, i.v.); high-frequency high-dose
injections (1.times.10.sup.10 nanoparticles, i.v. every 3 days for
30 days); single low-dose injection (1.times.10.sup.9
nanoparticles, i.v.); or high-frequency low-dose injections
(1.times.10.sup.9 nanoparticles, i.v. every 3 days for 30 days). To
compare the therapeutic efficacy of nanoparticle infusions with
conventional adoptive T-cell therapy, one additional group of mice
will be treated with a single dose of 10 million T cells transduced
ex vivo with P28z-encoding retroviral vectors. Differences in
TRAMP-PSMA tumor progression will be measured between treatment and
control groups using bioluminescence imaging. To correlate tumor
regression with the concentration of nanoparticle-programmed T
cells in the peripheral circulation, the percentage of P28z.sup.+ T
cells in whole blood will be quantified by flow cytometry every 6
days.
[0303] The results will show that circulating T cells can be
selectively programmed to target prostate tumors without
genetically modifying other cells. This specificity can be achieved
by coating the nanoparticles with CD3-recognizing antibodies, and
by expressing the P28z transgene under the control of the T
cell-specific CD3 delta promoter. If flow cytometry shows that more
than 20% of P28z-expressing cells in the peripheral blood are not
the targeted T cells, the density of anti-CD3 antibodies on the
surface of nanocarriers will be increased to improve T cell
targeting. If the CD3 delta promoter is too weak to mediate
sufficient levels of receptor gene expression in vivo, the murine
stem cell leukemia virus (MSCV) promoter can be used to express the
P28z CAR in T cells. The MSCV promoter exhibits strong activity in
hematopoietic cells and stem cells.
Example 7
[0304] Example 7 determines that nanocarriers can alternatively
modify host T cells with prostate tumor-specific T-cell receptor
(TCR) genes that target different antigens.
[0305] Gene transfer of DNA encoding CARs can only target T cells
to antigens located on the surface of tumor cells, so the many
tumor antigens that are intracellular are inaccessible to these
receptors. However, after degradation in the proteasome these
intracellular proteins are presented by major histocompatibility
complex (MHC) molecules where they can be recognized by specific T
cell receptors (TCRs).
[0306] A murine receptor (3D TCR) that has a high affinity for the
intracellular oncoprotein Wilms tumor 1 (WT1) has been successfully
engineered by a team of immunologists led by P. Greenberg at the
Fred Hutchinson Cancer Research Center. WT1 was ranked first in a
list of 75 cancer antigens in a recent National Cancer Institute
prioritization project. It is strongly expressed in high-grade
prostate tumor where it promotes the formation of metastases, but
is absent in non-neoplastic or benign prostatic hyperplasia
tissues. In line with these studies, high WT1 gene expression was
detected in the TRAMP prostate tumor cells used herein. WT1 is
detected at only very low levels in other normal tissues,
particularly hematopoietic stem cells and kidney podocytes. T cells
have been shown to be capable of selectively recognizing
transformed cells expressing high levels without toxicity to normal
tissues. In Example 7 it will be shown that systemic injections of
protocells loaded with genes encoding affinity-matured WT1-specific
TCRs can impart specificity for WT1 to host T cells and lead to
elimination of prostate cancer.
[0307] To determine how efficiently nanocarriers transfect T cells
with WT1-TCR genes in vivo, mice will be injected with
1.times.10.sup.10 nanoparticles carrying 3D TCR genes. Control
nanoparticles will be loaded with GFP-expressing plasmids.
Peripheral blood collected by retro-orbital puncture every four
days over a 12-day period will be used to quantify WT1-TCR.sup.+ T
cells and other leukocyte subsets by flow cytometry using a
fluorescent conjugate of the WT1-derived RMFPNAPYL epitope tetramer
as the reporter.
[0308] To investigate whether nanoparticle injections can cause
regression of metastatic prostate cancer in mice,
luciferase-expressing TRAMP tumors will be implanted into the
prostate of C57BL/6 mice. Three weeks later, animals will be
treated with: a single high-dose bolus injection (1.times.10.sup.10
nanoparticles i.v.); high-frequency high-dose injections
(1.times.10.sup.10 nanoparticles i.v. every 3 days for 30 days); a
single low-dose injection (1.times.10.sup.9 nanoparticles, i.v.);
or high-frequency low-dose injections (1.times.10.sup.9
nanoparticles, i.v. every 3 days for 30 days). To determine the
therapeutic advantage of nanoparticle infusions over conventional
adoptive T-cell therapy, one additional group of mice will be
injected with 10 million T cells, which were ex vivo transduced
with 3D TCR genes using retroviral vectors. Differences in TRAMP
prostate tumor regression between treatment and control groups will
be measured using bioluminescence imaging.
[0309] The strength of T cell responses in antitumor immunity can
be decisively dependent on the quality of the TCRs involved. Due to
thymic selection, the affinities of natural TCRs that target
oncogenic self-proteins like WT1 are generally much lower than
those of typical virus-targeting TCRs. However, the ability of a
naturally occurring TCR to recognize antigens like WT1 can be
markedly enhanced by in vitro affinity maturation. Based on these
data, if genes for an affinity-optimized, WT1-specific TCR are
introduced into circulating T cells using the disclosed
nanoparticle gene therapy approach, T cells will effectively
recognize and kill prostate cancer cells. 3D TCRs are fully
functional in CD4.sup.+ and CD8.sup.+ T cells, and CD4.sup.+ T
cells can directly mediate tumor destruction and/or provide
cytokine help for CD8.sup.+ T cells; however, tumor-specific
CD4.sup.+ regulatory T cells abrogate CD8 T cell-mediated tumor
rejection. If CD3-targeted nanoparticles generate undesirable
WT1-specific CD4.sup.+ regulatory T cells, nanoparticles can be
targeted to CD8.sup.+ T cells only. These studies will demonstrate
that nanoparticles can deliver rationally engineered TCR genes into
host T-cells and enable them to recognize intracellular
tumor-associated antigen.
Example 8
[0310] Modifying host lymphocytes with HIV-specific TCR genes to
control HIV infection. HIV-infected humanized NOD/shi-scid/.gamma.c
null (NOG) mice with nanoparticles carrying HIV-gag
protein-specific TCR transgenes, or with control plasmids
expressing green fluorescent protein will be studied. Differences
in HIV viral titers between treatment groups will be determined and
administration of the nanoparticles will show a beneficial
result.
[0311] Unless otherwise indicated, the practice of the present
disclosure can employ conventional techniques of immunology,
molecular biology, microbiology, cell biology and recombinant DNA.
These methods are described in the following publications. See,
e.g., Sambrook, et al. Molecular Cloning: A Laboratory Manual,
2.sup.nd Edition (1989); F. M. Ausubel, et al. eds., Current
Protocols in Molecular Biology, (1987); the series Methods IN
Enzymology (Academic Press, Inc.); M. MacPherson, et al., PCR: A
Practical Approach, IRL Press at Oxford University Press (1991);
MacPherson et al., eds. PCR 2: Practical Approach, (1995); Harlow
and Lane, eds. Antibodies, A Laboratory Manual, (1988); and R. I.
Freshney, ed. Animal Cell Culture (1987).
[0312] Sequence information provided by public database can be used
to identify nucleic acid sequences encoding peptides disclosed
herein and vice versa. Variants of the sequences disclosed and
referenced herein are also included.
[0313] Variants of peptides can include those having one or more
conservative amino acid substitutions. As used herein, a
"conservative substitution" involves a substitution found in one of
the following conservative substitutions groups: Group 1: Alanine
(Ala), Glycine (Gly), Serine (Ser), Threonine (Thr); Group 2:
Aspartic acid (Asp), Glutamic acid (Glu); Group 3: Asparagine
(Asn), Glutamine (Gin); Group 4: Arginine (Arg), Lysine (Lys),
Histidine (His); Group 5: Isoleucine (Ile), Leucine (Leu),
Methionine (Met), Valine (Val); and Group 6: Phenylalanine (Phe),
Tyrosine (Tyr), Tryptophan (Trp).
[0314] Additionally, amino acids can be grouped into conservative
substitution groups by similar function or chemical structure or
composition (e.g., acidic, basic, aliphatic, aromatic,
sulfur-containing). For example, an aliphatic grouping may include,
for purposes of substitution, Gly, Ala, Val, Leu, and Ile. Other
groups containing amino acids that are considered conservative
substitutions for one another include: sulfur-containing: Met and
Cysteine (Cys); acidic: Asp, Glu, Asn, and Gin; small aliphatic,
nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly;
polar, negatively charged residues and their amides: Asp, Asn, Glu,
and Gin; polar, positively charged residues: His, Arg, and Lys;
large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys;
and large aromatic residues: Phe, Tyr, and Trp. Additional
information is found in Creighton (1984) Proteins, W.H. Freeman and
Company.
[0315] Variants of the protein and nucleic acid sequences disclosed
or referenced herein also include sequences with at least 70%
sequence identity, 80% sequence identity, 85% sequence, 90%
sequence identity, 95% sequence identity, 96% sequence identity,
97% sequence identity, 98% sequence identity, or 99% sequence
identity to he protein and nucleic acid sequences disclosed or
referenced herein.
[0316] "% sequence identity" refers to a relationship between two
or more sequences, as determined by comparing the sequences. In the
art, "identity" also means the degree of sequence relatedness
between proteins or nucleic acid sequences as determined by the
match between strings of such sequences. "Identity" (often referred
to as "similarity") can be readily calculated by known methods,
including (but not limited to) those described in: Computational
Molecular Biology (Lesk, A. M., ed.) Oxford University Press, NY
(1988); Biocomputing: Informatics and Genome Projects (Smith, D.
W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence
Data, Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana
Press, N.J. (1994); Sequence Analysis in Molecular Biology (Von
Heijne, G., ed.) Academic Press (1987); and Sequence Analysis
Primer (Gribskov, M. and Devereux, J., eds.) Oxford University
Press, N.Y. (1992). Preferred methods to determine identity are
designed to give the best match between the sequences tested.
Methods to determine identity and similarity are codified in
publicly available computer programs. Sequence alignments and
percent identity calculations may be performed using the Megalign
program of the LASERGENE bioinformatics computing suite (DNASTAR,
Inc., Madison, Wis.). Multiple alignment of the sequences can also
be performed using the Clustal method of alignment (Higgins and
Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP
PENALTY=10, GAP LENGTH PENALTY=10). Relevant programs also include
the GCG suite of programs (Wisconsin Package Version 9.0, Genetics
Computer Group (GCG), Madison, Wis.); BLASTP, BLASTN, BLASTX
(Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR
(DNASTAR, Inc., Madison, Wis.); and the FASTA program incorporating
the Smith-Waterman algorithm (Pearson, Comput. Methods Genome Res.,
[Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s):
Suhai, Sandor. Publisher: Plenum, New York, N.Y. Within the context
of this disclosure it will be understood that where sequence
analysis software is used for analysis, the results of the analysis
are based on the "default values" of the program referenced. As
used herein "default values" will mean any set of values or
parameters, which originally load with the software when first
initialized.
[0317] As will be understood by one of ordinary skill in the art,
each embodiment disclosed herein can comprise, consist essentially
of or consist of its particular stated element, step, ingredient or
component. As used herein, the transition term "comprise" or
"comprises" means includes, but is not limited to, and allows for
the inclusion of unspecified elements, steps, ingredients, or
components, even in major amounts. The transitional phrase
"consisting of" excludes any element, step, ingredient or component
not specified. The transition phrase "consisting essentially of"
limits the scope of the embodiment to the specified elements,
steps, ingredients or components and to those that do not
materially affect the embodiment. As used herein, a material effect
would cause a statistically-significant reduction in the ability of
a nanocarrier to reduce the number of an unwanted cell type and/or
to protect a wanted cell type in vivo.
[0318] Unless otherwise indicated, all numbers expressing
quantities of ingredients, properties such as molecular weight,
reaction conditions, and so forth used in the specification and
claims are to be understood as being modified in all instances by
the term "about." Accordingly, unless indicated to the contrary,
the numerical parameters set forth in the specification and
attached claims are approximations that may vary depending upon the
desired properties sought to be obtained by the present invention.
At the very least, and not as an attempt to limit the application
of the doctrine of equivalents to the scope of the claims, each
numerical parameter should at least be construed in light of the
number of reported significant digits and by applying ordinary
rounding techniques. When further clarity is required, the term
"about" has the meaning reasonably ascribed to it by a person
skilled in the art when used in conjunction with a stated numerical
value or range, i.e. denoting somewhat more or somewhat less than
the stated value or range, to within a range of .+-.20% of the
stated value; .+-.19% of the stated value; .+-.18% of the stated
value; .+-.17% of the stated value; .+-.16% of the stated value;
.+-.15% of the stated value; .+-.14% of the stated value; .+-.13%
of the stated value; .+-.12% of the stated value; .+-.11% of the
stated value; .+-.10% of the stated value; .+-.9% of the stated
value; .+-.8% of the stated value; .+-.7% of the stated value;
.+-.6% of the stated value; .+-.5% of the stated value; .+-.4% of
the stated value; .+-.3% of the stated value; .+-.2% of the stated
value; or .+-.1% of the stated value.
[0319] Notwithstanding that the numerical ranges and parameters
setting forth the broad scope of the invention are approximations,
the numerical values set forth in the specific examples are
reported as precisely as possible. Any numerical value, however,
inherently contains certain errors necessarily resulting from the
standard deviation found in their respective testing
measurements.
[0320] The terms "a," "an," "the" and similar referents used in the
context of describing the invention (especially in the context of
the following claims) are to be construed to cover both the
singular and the plural, unless otherwise indicated herein or
clearly contradicted by context. Recitation of ranges of values
herein is merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range. Unless otherwise indicated herein, each individual value is
incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein is intended
merely to better illuminate the invention and does not pose a
limitation on the scope of the invention otherwise claimed. No
language in the specification should be construed as indicating any
non-claimed element essential to the practice of the invention.
[0321] Groupings of alternative elements or embodiments of the
invention disclosed herein are not to be construed as limitations.
Each group member may be referred to and claimed individually or in
any combination with other members of the group or other elements
found herein. It is anticipated that one or more members of a group
may be included in, or deleted from, a group for reasons of
convenience and/or patentability. When any such inclusion or
deletion occurs, the specification is deemed to contain the group
as modified thus fulfilling the written description of all Markush
groups used in the appended claims.
[0322] Certain embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Of course, variations on these described embodiments
will become apparent to those of ordinary skill in the art upon
reading the foregoing description. The inventor expects skilled
artisans to employ such variations as appropriate, and the
inventors intend for the invention to be practiced otherwise than
specifically described herein. Accordingly, this invention includes
all modifications and equivalents of the subject matter recited in
the claims appended hereto as permitted by applicable law.
Moreover, any combination of the above-described elements in all
possible variations thereof is encompassed by the invention unless
otherwise indicated herein or otherwise clearly contradicted by
context.
[0323] Furthermore, numerous references have been made to patents
and printed publications throughout this specification. Each of the
above-cited references and printed publications are individually
incorporated herein by reference for their particular cited
teachings.
[0324] In closing, it is to be understood that the embodiments of
the invention disclosed herein are illustrative of the principles
of the present invention. Other modifications that may be employed
are within the scope of the invention. Thus, by way of example, but
not of limitation, alternative configurations of the present
invention may be utilized in accordance with the teachings herein.
Accordingly, the present invention is not limited to that precisely
as shown and described.
[0325] The particulars shown herein are by way of example and for
purposes of illustrative discussion of the preferred embodiments of
the present invention only and are presented in the cause of
providing what is believed to be the most useful and readily
understood description of the principles and conceptual aspects of
various embodiments of the invention. In this regard, no attempt is
made to show structural details of the invention in more detail
than is necessary for the fundamental understanding of the
invention, the description taken with the drawings and/or examples
making apparent to those skilled in the art how the several forms
of the invention may be embodied in practice.
[0326] Definitions and explanations used in the present disclosure
are meant and intended to be controlling in any future construction
unless clearly and unambiguously modified in the following examples
or when application of the meaning renders any construction
meaningless or essentially meaningless. In cases where the
construction of the term would render it meaningless or essentially
meaningless, the definition should be taken from Webster's
Dictionary, 3.sup.rd Edition or a dictionary known to those of
ordinary skill in the art, such as the Oxford Dictionary of
Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford
University Press, Oxford, 2004).
Sequence CWU 1
1
941265PRTArtificial SequencescFv of the PSMA-specific chimeric
antigen receptor P28z 1Met Ala Ser Pro Leu Thr Arg Phe Leu Ser Leu
Asn Leu Leu Leu Leu 1 5 10 15 Gly Glu Ser Ile Ile Leu Gly Ser Gly
Glu Ala Glu Val Gln Leu Gln 20 25 30 Gln Ser Gly Pro Glu Leu Val
Lys Pro Gly Thr Ser Val Arg Ile Ser 35 40 45 Cys Lys Thr Ser Gly
Tyr Thr Phe Thr Glu Tyr Thr Ile His Trp Val 50 55 60 Lys Gln Ser
His Gly Lys Ser Leu Glu Trp Ile Gly Asn Ile Asn Pro 65 70 75 80 Asn
Asn Gly Gly Thr Thr Tyr Asn Gln Lys Phe Glu Asp Lys Ala Thr 85 90
95 Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Arg Ser
100 105 110 Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Ala Gly
Trp Asn 115 120 125 Phe Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val
Ser Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
Gly Gly Ser Asp Ile Val 145 150 155 160 Met Thr Gln Ser His Lys Phe
Met Ser Thr Ser Val Gly Asp Arg Val 165 170 175 Ser Ile Ile Cys Lys
Ala Ser Gln Asp Val Gly Thr Ala Val Asp Trp 180 185 190 Tyr Gln Gln
Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala 195 200 205 Ser
Thr Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser 210 215
220 Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser Glu Asp Leu
225 230 235 240 Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Ser Tyr Pro Leu
Thr Phe Gly 245 250 255 Ala Gly Thr Met Leu Asp Leu Lys Arg 260 265
2750PRTHomo sapiens 2Met Trp Asn Leu Leu His Glu Thr Asp Ser Ala
Val Ala Thr Ala Arg 1 5 10 15 Arg Pro Arg Trp Leu Cys Ala Gly Ala
Leu Val Leu Ala Gly Gly Phe 20 25 30 Phe Leu Leu Gly Phe Leu Phe
Gly Trp Phe Ile Lys Ser Ser Asn Glu 35 40 45 Ala Thr Asn Ile Thr
Pro Lys His Asn Met Lys Ala Phe Leu Asp Glu 50 55 60 Leu Lys Ala
Glu Asn Ile Lys Lys Phe Leu Tyr Asn Phe Thr Gln Ile 65 70 75 80 Pro
His Leu Ala Gly Thr Glu Gln Asn Phe Gln Leu Ala Lys Gln Ile 85 90
95 Gln Ser Gln Trp Lys Glu Phe Gly Leu Asp Ser Val Glu Leu Ala His
100 105 110 Tyr Asp Val Leu Leu Ser Tyr Pro Asn Lys Thr His Pro Asn
Tyr Ile 115 120 125 Ser Ile Ile Asn Glu Asp Gly Asn Glu Ile Phe Asn
Thr Ser Leu Phe 130 135 140 Glu Pro Pro Pro Pro Gly Tyr Glu Asn Val
Ser Asp Ile Val Pro Pro 145 150 155 160 Phe Ser Ala Phe Ser Pro Gln
Gly Met Pro Glu Gly Asp Leu Val Tyr 165 170 175 Val Asn Tyr Ala Arg
Thr Glu Asp Phe Phe Lys Leu Glu Arg Asp Met 180 185 190 Lys Ile Asn
Cys Ser Gly Lys Ile Val Ile Ala Arg Tyr Gly Lys Val 195 200 205 Phe
Arg Gly Asn Lys Val Lys Asn Ala Gln Leu Ala Gly Ala Lys Gly 210 215
220 Val Ile Leu Tyr Ser Asp Pro Ala Asp Tyr Phe Ala Pro Gly Val Lys
225 230 235 240 Ser Tyr Pro Asp Gly Trp Asn Leu Pro Gly Gly Gly Val
Gln Arg Gly 245 250 255 Asn Ile Leu Asn Leu Asn Gly Ala Gly Asp Pro
Leu Thr Pro Gly Tyr 260 265 270 Pro Ala Asn Glu Tyr Ala Tyr Arg Arg
Gly Ile Ala Glu Ala Val Gly 275 280 285 Leu Pro Ser Ile Pro Val His
Pro Ile Gly Tyr Tyr Asp Ala Gln Lys 290 295 300 Leu Leu Glu Lys Met
Gly Gly Ser Ala Pro Pro Asp Ser Ser Trp Arg 305 310 315 320 Gly Ser
Leu Lys Val Pro Tyr Asn Val Gly Pro Gly Phe Thr Gly Asn 325 330 335
Phe Ser Thr Gln Lys Val Lys Met His Ile His Ser Thr Asn Glu Val 340
345 350 Thr Arg Ile Tyr Asn Val Ile Gly Thr Leu Arg Gly Ala Val Glu
Pro 355 360 365 Asp Arg Tyr Val Ile Leu Gly Gly His Arg Asp Ser Trp
Val Phe Gly 370 375 380 Gly Ile Asp Pro Gln Ser Gly Ala Ala Val Val
His Glu Ile Val Arg 385 390 395 400 Ser Phe Gly Thr Leu Lys Lys Glu
Gly Trp Arg Pro Arg Arg Thr Ile 405 410 415 Leu Phe Ala Ser Trp Asp
Ala Glu Glu Phe Gly Leu Leu Gly Ser Thr 420 425 430 Glu Trp Ala Glu
Glu Asn Ser Arg Leu Leu Gln Glu Arg Gly Val Ala 435 440 445 Tyr Ile
Asn Ala Asp Ser Ser Ile Glu Gly Asn Tyr Thr Leu Arg Val 450 455 460
Asp Cys Thr Pro Leu Met Tyr Ser Leu Val His Asn Leu Thr Lys Glu 465
470 475 480 Leu Lys Ser Pro Asp Glu Gly Phe Glu Gly Lys Ser Leu Tyr
Glu Ser 485 490 495 Trp Thr Lys Lys Ser Pro Ser Pro Glu Phe Ser Gly
Met Pro Arg Ile 500 505 510 Ser Lys Leu Gly Ser Gly Asn Asp Phe Glu
Val Phe Phe Gln Arg Leu 515 520 525 Gly Ile Ala Ser Gly Arg Ala Arg
Tyr Thr Lys Asn Trp Glu Thr Asn 530 535 540 Lys Phe Ser Gly Tyr Pro
Leu Tyr His Ser Val Tyr Glu Thr Tyr Glu 545 550 555 560 Leu Val Glu
Lys Phe Tyr Asp Pro Met Phe Lys Tyr His Leu Thr Val 565 570 575 Ala
Gln Val Arg Gly Gly Met Val Phe Glu Leu Ala Asn Ser Ile Val 580 585
590 Leu Pro Phe Asp Cys Arg Asp Tyr Ala Val Val Leu Arg Lys Tyr Ala
595 600 605 Asp Lys Ile Tyr Ser Ile Ser Met Lys His Pro Gln Glu Met
Lys Thr 610 615 620 Tyr Ser Val Ser Phe Asp Ser Leu Phe Ser Ala Val
Lys Asn Phe Thr 625 630 635 640 Glu Ile Ala Ser Lys Phe Ser Glu Arg
Leu Gln Asp Phe Asp Lys Ser 645 650 655 Asn Pro Ile Val Leu Arg Met
Met Asn Asp Gln Leu Met Phe Leu Glu 660 665 670 Arg Ala Phe Ile Asp
Pro Leu Gly Leu Pro Asp Arg Pro Phe Tyr Arg 675 680 685 His Val Ile
Tyr Ala Pro Ser Ser His Asn Lys Tyr Ala Gly Glu Ser 690 695 700 Phe
Pro Gly Ile Tyr Asp Ala Leu Phe Asp Ile Glu Ser Lys Val Asp 705 710
715 720 Pro Ser Lys Ala Trp Gly Glu Val Lys Arg Gln Ile Tyr Val Ala
Ala 725 730 735 Phe Thr Val Gln Ala Ala Ala Glu Thr Leu Ser Glu Val
Ala 740 745 750 3123PRTHomo sapiens 3Met Lys Ala Val Leu Leu Ala
Leu Leu Met Ala Gly Leu Ala Leu Gln 1 5 10 15 Pro Gly Thr Ala Leu
Leu Cys Tyr Ser Cys Lys Ala Gln Val Ser Asn 20 25 30 Glu Asp Cys
Leu Gln Val Glu Asn Cys Thr Gln Leu Gly Glu Gln Cys 35 40 45 Trp
Thr Ala Arg Ile Arg Ala Val Gly Leu Leu Thr Val Ile Ser Lys 50 55
60 Gly Cys Ser Leu Asn Cys Val Asp Asp Ser Gln Asp Tyr Tyr Val Gly
65 70 75 80 Lys Lys Asn Ile Thr Cys Cys Asp Thr Asp Leu Cys Asn Ala
Ser Gly 85 90 95 Ala His Ala Leu Gln Pro Ala Ala Ala Ile Leu Ala
Leu Leu Pro Ala 100 105 110 Leu Gly Leu Leu Leu Trp Gly Pro Gly Gln
Leu 115 120 4622PRTHomo sapiens 4Met Ala Leu Pro Thr Ala Arg Pro
Leu Leu Gly Ser Cys Gly Thr Pro 1 5 10 15 Ala Leu Gly Ser Leu Leu
Phe Leu Leu Phe Ser Leu Gly Trp Val Gln 20 25 30 Pro Ser Arg Thr
Leu Ala Gly Glu Thr Gly Gln Glu Ala Ala Pro Leu 35 40 45 Asp Gly
Val Leu Ala Asn Pro Pro Asn Ile Ser Ser Leu Ser Pro Arg 50 55 60
Gln Leu Leu Gly Phe Pro Cys Ala Glu Val Ser Gly Leu Ser Thr Glu 65
70 75 80 Arg Val Arg Glu Leu Ala Val Ala Leu Ala Gln Lys Asn Val
Lys Leu 85 90 95 Ser Thr Glu Gln Leu Arg Cys Leu Ala His Arg Leu
Ser Glu Pro Pro 100 105 110 Glu Asp Leu Asp Ala Leu Pro Leu Asp Leu
Leu Leu Phe Leu Asn Pro 115 120 125 Asp Ala Phe Ser Gly Pro Gln Ala
Cys Thr His Phe Phe Ser Arg Ile 130 135 140 Thr Lys Ala Asn Val Asp
Leu Leu Pro Arg Gly Ala Pro Glu Arg Gln 145 150 155 160 Arg Leu Leu
Pro Ala Ala Leu Ala Cys Trp Gly Val Arg Gly Ser Leu 165 170 175 Leu
Ser Glu Ala Asp Val Arg Ala Leu Gly Gly Leu Ala Cys Asp Leu 180 185
190 Pro Gly Arg Phe Val Ala Glu Ser Ala Glu Val Leu Leu Pro Arg Leu
195 200 205 Val Ser Cys Pro Gly Pro Leu Asp Gln Asp Gln Gln Glu Ala
Ala Arg 210 215 220 Ala Ala Leu Gln Gly Gly Gly Pro Pro Tyr Gly Pro
Pro Ser Thr Trp 225 230 235 240 Ser Val Ser Thr Met Asp Ala Leu Arg
Gly Leu Leu Pro Val Leu Gly 245 250 255 Gln Pro Ile Ile Arg Ser Ile
Pro Gln Gly Ile Val Ala Ala Trp Arg 260 265 270 Gln Arg Ser Ser Arg
Asp Pro Ser Trp Arg Gln Pro Glu Arg Thr Ile 275 280 285 Leu Arg Pro
Arg Phe Arg Arg Glu Val Glu Lys Thr Ala Cys Pro Ser 290 295 300 Gly
Lys Lys Ala Arg Glu Ile Asp Glu Ser Leu Ile Phe Tyr Lys Lys 305 310
315 320 Trp Glu Leu Glu Ala Cys Val Asp Ala Ala Leu Leu Ala Thr Gln
Met 325 330 335 Asp Arg Val Asn Ala Ile Pro Phe Thr Tyr Glu Gln Leu
Asp Val Leu 340 345 350 Lys His Lys Leu Asp Glu Leu Tyr Pro Gln Gly
Tyr Pro Glu Ser Val 355 360 365 Ile Gln His Leu Gly Tyr Leu Phe Leu
Lys Met Ser Pro Glu Asp Ile 370 375 380 Arg Lys Trp Asn Val Thr Ser
Leu Glu Thr Leu Lys Ala Leu Leu Glu 385 390 395 400 Val Asn Lys Gly
His Glu Met Ser Pro Gln Val Ala Thr Leu Ile Asp 405 410 415 Arg Phe
Val Lys Gly Arg Gly Gln Leu Asp Lys Asp Thr Leu Asp Thr 420 425 430
Leu Thr Ala Phe Tyr Pro Gly Tyr Leu Cys Ser Leu Ser Pro Glu Glu 435
440 445 Leu Ser Ser Val Pro Pro Ser Ser Ile Trp Ala Val Arg Pro Gln
Asp 450 455 460 Leu Asp Thr Cys Asp Pro Arg Gln Leu Asp Val Leu Tyr
Pro Lys Ala 465 470 475 480 Arg Leu Ala Phe Gln Asn Met Asn Gly Ser
Glu Tyr Phe Val Lys Ile 485 490 495 Gln Ser Phe Leu Gly Gly Ala Pro
Thr Glu Asp Leu Lys Ala Leu Ser 500 505 510 Gln Gln Asn Val Ser Met
Asp Leu Ala Thr Phe Met Lys Leu Arg Thr 515 520 525 Asp Ala Val Leu
Pro Leu Thr Val Ala Glu Val Gln Lys Leu Leu Gly 530 535 540 Pro His
Val Glu Gly Leu Lys Ala Glu Glu Arg His Arg Pro Val Arg 545 550 555
560 Asp Trp Ile Leu Arg Gln Arg Gln Asp Asp Leu Asp Thr Leu Gly Leu
565 570 575 Gly Leu Gln Gly Gly Ile Pro Asn Gly Tyr Leu Val Leu Asp
Leu Ser 580 585 590 Val Gln Glu Ala Leu Ser Gly Thr Pro Cys Leu Leu
Gly Pro Gly Pro 595 600 605 Val Leu Thr Val Leu Ala Leu Leu Leu Ala
Ser Thr Leu Ala 610 615 620 5556PRTHomo sapiens 5Met Pro Pro Pro
Arg Leu Leu Phe Phe Leu Leu Phe Leu Thr Pro Met 1 5 10 15 Glu Val
Arg Pro Glu Glu Pro Leu Val Val Lys Val Glu Glu Gly Asp 20 25 30
Asn Ala Val Leu Gln Cys Leu Lys Gly Thr Ser Asp Gly Pro Thr Gln 35
40 45 Gln Leu Thr Trp Ser Arg Glu Ser Pro Leu Lys Pro Phe Leu Lys
Leu 50 55 60 Ser Leu Gly Leu Pro Gly Leu Gly Ile His Met Arg Pro
Leu Ala Ser 65 70 75 80 Trp Leu Phe Ile Phe Asn Val Ser Gln Gln Met
Gly Gly Phe Tyr Leu 85 90 95 Cys Gln Pro Gly Pro Pro Ser Glu Lys
Ala Trp Gln Pro Gly Trp Thr 100 105 110 Val Asn Val Glu Gly Ser Gly
Glu Leu Phe Arg Trp Asn Val Ser Asp 115 120 125 Leu Gly Gly Leu Gly
Cys Gly Leu Lys Asn Arg Ser Ser Glu Gly Pro 130 135 140 Ser Ser Pro
Ser Gly Lys Leu Met Ser Pro Lys Leu Tyr Val Trp Ala 145 150 155 160
Lys Asp Arg Pro Glu Ile Trp Glu Gly Glu Pro Pro Cys Val Pro Pro 165
170 175 Arg Asp Ser Leu Asn Gln Ser Leu Ser Gln Asp Leu Thr Met Ala
Pro 180 185 190 Gly Ser Thr Leu Trp Leu Ser Cys Gly Val Pro Pro Asp
Ser Val Ser 195 200 205 Arg Gly Pro Leu Ser Trp Thr His Val His Pro
Lys Gly Pro Lys Ser 210 215 220 Leu Leu Ser Leu Glu Leu Lys Asp Asp
Arg Pro Ala Arg Asp Met Trp 225 230 235 240 Val Met Glu Thr Gly Leu
Leu Leu Pro Arg Ala Thr Ala Gln Asp Ala 245 250 255 Gly Lys Tyr Tyr
Cys His Arg Gly Asn Leu Thr Met Ser Phe His Leu 260 265 270 Glu Ile
Thr Ala Arg Pro Val Leu Trp His Trp Leu Leu Arg Thr Gly 275 280 285
Gly Trp Lys Val Ser Ala Val Thr Leu Ala Tyr Leu Ile Phe Cys Leu 290
295 300 Cys Ser Leu Val Gly Ile Leu His Leu Gln Arg Ala Leu Val Leu
Arg 305 310 315 320 Arg Lys Arg Lys Arg Met Thr Asp Pro Thr Arg Arg
Phe Phe Lys Val 325 330 335 Thr Pro Pro Pro Gly Ser Gly Pro Gln Asn
Gln Tyr Gly Asn Val Leu 340 345 350 Ser Leu Pro Thr Pro Thr Ser Gly
Leu Gly Arg Ala Gln Arg Trp Ala 355 360 365 Ala Gly Leu Gly Gly Thr
Ala Pro Ser Tyr Gly Asn Pro Ser Ser Asp 370 375 380 Val Gln Ala Asp
Gly Ala Leu Gly Ser Arg Ser Pro Pro Gly Val Gly 385 390 395 400 Pro
Glu Glu Glu Glu Gly Glu Gly Tyr Glu Glu Pro Asp Ser Glu Glu 405 410
415 Asp Ser Glu Phe Tyr Glu Asn Asp Ser Asn Leu Gly Gln Asp Gln Leu
420 425 430 Ser Gln Asp Gly Ser Gly Tyr Glu Asn Pro Glu Asp Glu Pro
Leu Gly 435 440 445 Pro Glu Asp Glu Asp Ser Phe Ser Asn Ala Glu Ser
Tyr Glu Asn Glu 450 455 460 Asp Glu Glu Leu Thr Gln Pro Val Ala Arg
Thr Met Asp Phe Leu Ser 465 470 475 480 Pro His Gly Ser Ala Trp Asp
Pro Ser Arg Glu Ala Thr Ser Leu Gly 485 490 495 Ser Gln Ser Tyr Glu
Asp Met Arg Gly Ile Leu Tyr Ala Ala Pro Gln 500 505 510 Leu Arg Ser
Ile Arg Gly Gln Pro Gly Pro Asn His Glu Glu Asp Ala 515 520
525 Asp Ser Tyr Glu Asn Met Asp Asn Pro Asp Gly Pro Asp Pro Ala Trp
530 535 540 Gly Gly Gly Gly Arg Met Gly Thr Trp Ser Thr Arg 545 550
555 6297PRTHomo sapiens 6Met Thr Thr Pro Arg Asn Ser Val Asn Gly
Thr Phe Pro Ala Glu Pro 1 5 10 15 Met Lys Gly Pro Ile Ala Met Gln
Ser Gly Pro Lys Pro Leu Phe Arg 20 25 30 Arg Met Ser Ser Leu Val
Gly Pro Thr Gln Ser Phe Phe Met Arg Glu 35 40 45 Ser Lys Thr Leu
Gly Ala Val Gln Ile Met Asn Gly Leu Phe His Ile 50 55 60 Ala Leu
Gly Gly Leu Leu Met Ile Pro Ala Gly Ile Tyr Ala Pro Ile 65 70 75 80
Cys Val Thr Val Trp Tyr Pro Leu Trp Gly Gly Ile Met Tyr Ile Ile 85
90 95 Ser Gly Ser Leu Leu Ala Ala Thr Glu Lys Asn Ser Arg Lys Cys
Leu 100 105 110 Val Lys Gly Lys Met Ile Met Asn Ser Leu Ser Leu Phe
Ala Ala Ile 115 120 125 Ser Gly Met Ile Leu Ser Ile Met Asp Ile Leu
Asn Ile Lys Ile Ser 130 135 140 His Phe Leu Lys Met Glu Ser Leu Asn
Phe Ile Arg Ala His Thr Pro 145 150 155 160 Tyr Ile Asn Ile Tyr Asn
Cys Glu Pro Ala Asn Pro Ser Glu Lys Asn 165 170 175 Ser Pro Ser Thr
Gln Tyr Cys Tyr Ser Ile Gln Ser Leu Phe Leu Gly 180 185 190 Ile Leu
Ser Val Met Leu Ile Phe Ala Phe Phe Gln Glu Leu Val Ile 195 200 205
Ala Gly Ile Val Glu Asn Glu Trp Lys Arg Thr Cys Ser Arg Pro Lys 210
215 220 Ser Asn Ile Val Leu Leu Ser Ala Glu Glu Lys Lys Glu Gln Thr
Ile 225 230 235 240 Glu Ile Lys Glu Glu Val Val Gly Leu Thr Glu Thr
Ser Ser Gln Pro 245 250 255 Lys Asn Glu Glu Asp Ile Glu Ile Ile Pro
Ile Gln Glu Glu Glu Glu 260 265 270 Glu Glu Thr Glu Thr Asn Phe Pro
Glu Pro Pro Gln Asp Gln Glu Ser 275 280 285 Ser Pro Ile Glu Asn Asp
Ser Ser Pro 290 295 7937PRTHomo sapiens 7Met His Arg Pro Arg Arg
Arg Gly Thr Arg Pro Pro Leu Leu Ala Leu 1 5 10 15 Leu Ala Ala Leu
Leu Leu Ala Ala Arg Gly Ala Ala Ala Gln Glu Thr 20 25 30 Glu Leu
Ser Val Ser Ala Glu Leu Val Pro Thr Ser Ser Trp Asn Ile 35 40 45
Ser Ser Glu Leu Asn Lys Asp Ser Tyr Leu Thr Leu Asp Glu Pro Met 50
55 60 Asn Asn Ile Thr Thr Ser Leu Gly Gln Thr Ala Glu Leu His Cys
Lys 65 70 75 80 Val Ser Gly Asn Pro Pro Pro Thr Ile Arg Trp Phe Lys
Asn Asp Ala 85 90 95 Pro Val Val Gln Glu Pro Arg Arg Leu Ser Phe
Arg Ser Thr Ile Tyr 100 105 110 Gly Ser Arg Leu Arg Ile Arg Asn Leu
Asp Thr Thr Asp Thr Gly Tyr 115 120 125 Phe Gln Cys Val Ala Thr Asn
Gly Lys Glu Val Val Ser Ser Thr Gly 130 135 140 Val Leu Phe Val Lys
Phe Gly Pro Pro Pro Thr Ala Ser Pro Gly Tyr 145 150 155 160 Ser Asp
Glu Tyr Glu Glu Asp Gly Phe Cys Gln Pro Tyr Arg Gly Ile 165 170 175
Ala Cys Ala Arg Phe Ile Gly Asn Arg Thr Val Tyr Met Glu Ser Leu 180
185 190 His Met Gln Gly Glu Ile Glu Asn Gln Ile Thr Ala Ala Phe Thr
Met 195 200 205 Ile Gly Thr Ser Ser His Leu Ser Asp Lys Cys Ser Gln
Phe Ala Ile 210 215 220 Pro Ser Leu Cys His Tyr Ala Phe Pro Tyr Cys
Asp Glu Thr Ser Ser 225 230 235 240 Val Pro Lys Pro Arg Asp Leu Cys
Arg Asp Glu Cys Glu Ile Leu Glu 245 250 255 Asn Val Leu Cys Gln Thr
Glu Tyr Ile Phe Ala Arg Ser Asn Pro Met 260 265 270 Ile Leu Met Arg
Leu Lys Leu Pro Asn Cys Glu Asp Leu Pro Gln Pro 275 280 285 Glu Ser
Pro Glu Ala Ala Asn Cys Ile Arg Ile Gly Ile Pro Met Ala 290 295 300
Asp Pro Ile Asn Lys Asn His Lys Cys Tyr Asn Ser Thr Gly Val Asp 305
310 315 320 Tyr Arg Gly Thr Val Ser Val Thr Lys Ser Gly Arg Gln Cys
Gln Pro 325 330 335 Trp Asn Ser Gln Tyr Pro His Thr His Thr Phe Thr
Ala Leu Arg Phe 340 345 350 Pro Glu Leu Asn Gly Gly His Ser Tyr Cys
Arg Asn Pro Gly Asn Gln 355 360 365 Lys Glu Ala Pro Trp Cys Phe Thr
Leu Asp Glu Asn Phe Lys Ser Asp 370 375 380 Leu Cys Asp Ile Pro Ala
Cys Asp Ser Lys Asp Ser Lys Glu Lys Asn 385 390 395 400 Lys Met Glu
Ile Leu Tyr Ile Leu Val Pro Ser Val Ala Ile Pro Leu 405 410 415 Ala
Ile Ala Leu Leu Phe Phe Phe Ile Cys Val Cys Arg Asn Asn Gln 420 425
430 Lys Ser Ser Ser Ala Pro Val Gln Arg Gln Pro Lys His Val Arg Gly
435 440 445 Gln Asn Val Glu Met Ser Met Leu Asn Ala Tyr Lys Pro Lys
Ser Lys 450 455 460 Ala Lys Glu Leu Pro Leu Ser Ala Val Arg Phe Met
Glu Glu Leu Gly 465 470 475 480 Glu Cys Ala Phe Gly Lys Ile Tyr Lys
Gly His Leu Tyr Leu Pro Gly 485 490 495 Met Asp His Ala Gln Leu Val
Ala Ile Lys Thr Leu Lys Asp Tyr Asn 500 505 510 Asn Pro Gln Gln Trp
Thr Glu Phe Gln Gln Glu Ala Ser Leu Met Ala 515 520 525 Glu Leu His
His Pro Asn Ile Val Cys Leu Leu Gly Ala Val Thr Gln 530 535 540 Glu
Gln Pro Val Cys Met Leu Phe Glu Tyr Ile Asn Gln Gly Asp Leu 545 550
555 560 His Glu Phe Leu Ile Met Arg Ser Pro His Ser Asp Val Gly Cys
Ser 565 570 575 Ser Asp Glu Asp Gly Thr Val Lys Ser Ser Leu Asp His
Gly Asp Phe 580 585 590 Leu His Ile Ala Ile Gln Ile Ala Ala Gly Met
Glu Tyr Leu Ser Ser 595 600 605 His Phe Phe Val His Lys Asp Leu Ala
Ala Arg Asn Ile Leu Ile Gly 610 615 620 Glu Gln Leu His Val Lys Ile
Ser Asp Leu Gly Leu Ser Arg Glu Ile 625 630 635 640 Tyr Ser Ala Asp
Tyr Tyr Arg Val Gln Ser Lys Ser Leu Leu Pro Ile 645 650 655 Arg Trp
Met Pro Pro Glu Ala Ile Met Tyr Gly Lys Phe Ser Ser Asp 660 665 670
Ser Asp Ile Trp Ser Phe Gly Val Val Leu Trp Glu Ile Phe Ser Phe 675
680 685 Gly Leu Gln Pro Tyr Tyr Gly Phe Ser Asn Gln Glu Val Ile Glu
Met 690 695 700 Val Arg Lys Arg Gln Leu Leu Pro Cys Ser Glu Asp Cys
Pro Pro Arg 705 710 715 720 Met Tyr Ser Leu Met Thr Glu Cys Trp Asn
Glu Ile Pro Ser Arg Arg 725 730 735 Pro Arg Phe Lys Asp Ile His Val
Arg Leu Arg Ser Trp Glu Gly Leu 740 745 750 Ser Ser His Thr Ser Ser
Thr Thr Pro Ser Gly Gly Asn Ala Thr Thr 755 760 765 Gln Thr Thr Ser
Leu Ser Ala Ser Pro Val Ser Asn Leu Ser Asn Pro 770 775 780 Arg Tyr
Pro Asn Tyr Met Phe Pro Ser Gln Gly Ile Thr Pro Gln Gly 785 790 795
800 Gln Ile Ala Gly Phe Ile Gly Pro Pro Ile Pro Gln Asn Gln Arg Phe
805 810 815 Ile Pro Ile Asn Gly Tyr Pro Ile Pro Pro Gly Tyr Ala Ala
Phe Pro 820 825 830 Ala Ala His Tyr Gln Pro Thr Gly Pro Pro Arg Val
Ile Gln His Cys 835 840 845 Pro Pro Pro Lys Ser Arg Ser Pro Ser Ser
Ala Ser Gly Ser Thr Ser 850 855 860 Thr Gly His Val Thr Ser Leu Pro
Ser Ser Gly Ser Asn Gln Glu Ala 865 870 875 880 Asn Ile Pro Leu Leu
Pro His Met Ser Ile Pro Asn His Pro Gly Gly 885 890 895 Met Gly Ile
Thr Val Phe Gly Asn Lys Ser Gln Lys Pro Tyr Lys Ile 900 905 910 Asp
Ser Lys Gln Ala Ser Leu Leu Gly Asp Ala Asn Ile His Gly His 915 920
925 Thr Glu Ser Met Ile Ser Ala Glu Leu 930 935 8168PRTHomo sapiens
8Met Gly His His His His His His His His His His Ser Ser Gly His 1
5 10 15 Ile Glu Gly Arg His Met Arg Arg Val Pro Gly Val Ala Pro Thr
Leu 20 25 30 Val Arg Ser Ala Ser Glu Thr Ser Glu Lys Arg Pro Phe
Met Cys Ala 35 40 45 Tyr Pro Gly Cys Asn Lys Arg Tyr Phe Lys Leu
Ser His Leu Gln Met 50 55 60 His Ser Arg Lys His Thr Gly Glu Lys
Pro Tyr Gln Cys Asp Phe Lys 65 70 75 80 Asp Cys Glu Arg Arg Phe Phe
Arg Ser Asp Gln Leu Lys Arg His Gln 85 90 95 Arg Arg His Thr Gly
Val Lys Pro Phe Gln Cys Lys Thr Cys Gln Arg 100 105 110 Lys Phe Ser
Arg Ser Asp His Leu Lys Thr His Thr Arg Thr His Thr 115 120 125 Gly
Glu Lys Pro Phe Ser Cys Arg Trp Pro Ser Cys Gln Lys Lys Phe 130 135
140 Ala Arg Ser Asp Glu Leu Val Arg His His Asn Met His Gln Arg Asn
145 150 155 160 Met Thr Lys Leu Gln Leu Ala Leu 165 932PRTHuman
immunodeficiency virus type 1 9Val Gly Phe Pro Val Thr Pro Gln Val
Pro Leu Arg Pro Met Thr Tyr 1 5 10 15 Lys Ala Ala Val Asp Leu Ser
His Phe Leu Lys Glu Lys Gly Gly Leu 20 25 30 1030PRTHuman
immunodeficiency virus type 1 10His Thr Gln Gly Tyr Phe Pro Asp Trp
Gln Asn Tyr Thr Pro Gly Pro 1 5 10 15 Gly Val Arg Tyr Pro Leu Thr
Phe Gly Trp Leu Tyr Lys Leu 20 25 30 1119PRTHuman immunodeficiency
virus type 1 11Glu Lys Ile Arg Leu Arg Pro Gly Gly Lys Lys Lys Tyr
Lys Leu Lys 1 5 10 15 His Ile Val 1232PRTHuman immunodeficiency
virus type 1 12Asn Pro Pro Ile Pro Val Gly Glu Ile Tyr Lys Arg Trp
Ile Ile Leu 1 5 10 15 Gly Leu Asn Lys Ile Val Arg Met Tyr Ser Pro
Thr Ser Ile Leu Asp 20 25 30 1331PRTHuman immunodeficiency virus
type 1 13Ala Ile Phe Gln Ser Ser Met Thr Lys Ile Leu Glu Pro Phe
Arg Lys 1 5 10 15 Gln Asn Pro Asp Ile Val Ile Tyr Gln Tyr Met Asp
Asp Leu Tyr 20 25 30 1411PRTArtificial SequenceCDRL1 sequence 14Arg
Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn 1 5 10 157PRTArtificial
SequenceCDRL2 sequence 15Ser Arg Leu His Ser Gly Val 1 5
169PRTArtificial SequenceCDRL3 sequence 16Gly Asn Thr Leu Pro Tyr
Thr Phe Gly 1 5 175PRTArtificial SequenceCDRHI sequence 17Asp Tyr
Gly Val Ser 1 5 1816PRTArtificial SequenceCDRH2 sequence 18Val Thr
Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser 1 5 10 15
197PRTArtificial SequenceCDRH3 sequence 19Tyr Ala Met Asp Tyr Trp
Gly 1 5 20734DNAArtificial SequenceAnti-CD19 scFv (VH-VL) FMC63
20gacatccaga tgacccagac cacctccagc ctgagcgcca gcctgggcga ccgggtgacc
60atcagctgcc gggccagcca ggacatcagc aagtacctga actggtatca gcagaagccc
120gacggcaccg tcaagctgct gatctaccac accagccggc tgcacagcgg
cgtgcccagc 180cggtttagcg gcagcggctc cggcaccgac tacagcctga
ccatctccaa cctggaacag 240gaagatatcg ccacctactt ttgccagcag
ggcaacacac tgccctacac ctttggcggc 300ggaacaaagc tggaaatcac
cggcagcacc tccggcagcg gcaagcctgg cagcggcgag 360ggcagcacca
agggcgaggt gaagctgcag gaaagcggcc ctggcctggt ggcccccagc
420cagagcctga gcgtgacctg caccgtgagc ggcgtgagcc tgcccgacta
cggcgtgagc 480tggatccggc agccccccag gaagggcctg gaatggctgg
gcgtgatctg gggcagcgag 540accacctact acaacagcgc cctgaagagc
cggctgacca tcatcaagga caacagcaag 600agccaggtgt tcctgaagat
gaacagcctg cagaccgacg acaccgccat ctactactgc 660gccaagcact
actactacgg cggcagctac gccatggact actggggcca gggcaccagc
720gtgaccgtga gcag 73421245PRTArtificial SequenceAnti-CD19 scFv
(VH-VL) FMC63 21Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala
Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln
Asp Ile Ser Lys Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp
Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr His Thr Ser Arg Leu His
Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr
Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 65 70 75 80 Glu Asp Ile
Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95 Thr
Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly 100 105
110 Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys
115 120 125 Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser
Leu Ser 130 135 140 Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp
Tyr Gly Val Ser 145 150 155 160 Trp Ile Arg Gln Pro Pro Arg Lys Gly
Leu Glu Trp Leu Gly Val Ile 165 170 175 Trp Gly Ser Glu Thr Thr Tyr
Tyr Asn Ser Ala Leu Lys Ser Arg Leu 180 185 190 Thr Ile Ile Lys Asp
Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn 195 200 205 Ser Leu Gln
Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr 210 215 220 Tyr
Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser 225 230
235 240 Val Thr Val Ser Ser 245 2211PRTArtificial SequenceCDRL1
sequence 22Ala Ser Gly Phe Asp Phe Ser Ala Tyr Tyr Met 1 5 10
237PRTArtificial SequenceCDRL2 sequence 23Thr Ile Tyr Pro Ser Ser
Gly 1 5 249PRTArtificial SequenceCDRL3 sequence 24Ala Asp Arg Ala
Thr Tyr Phe Cys Ala 1 5 256PRTArtificial SequenceCDRH1 sequence
25Asp Thr Ile Asp Trp Tyr 1 5 2616PRTArtificial SequenceCDRH2
sequence 26Val Gln Ser Asp Gly Ser Tyr Thr Lys Arg Pro Gly Val Pro
Asp Arg 1 5 10 15 278PRTArtificial SequenceCDRH3 sequence 27Tyr Ile
Gly Gly Tyr Val Phe Gly 1 5 28123DNAArtificial SequenceCD28
effector domain 28cggagcaagc ggagcagagg cggccacagc gactacatga
acatgacccc cagacggcct 60ggccccaccc ggaagcacta ccagccctac gccccaccca
gggactttgc cgcctacaga 120agc 1232916PRTHomo sapiens 29Ala Ala Val
Ala Leu Leu Pro Ala Val Leu Leu Ala Leu Leu Ala Pro 1 5 10 15
3011PRTArtificial SequenceRFGF analogue 30Ala Ala Leu Leu Pro Val
Leu Leu Ala Ala Pro 1 5 10 3116PRTInfluenza virus 31Gly Leu Phe Glu
Ala Ile Ala Gly Phe Ile Glu Asn Gly Trp Glu Gly 1 5 10 15
3211PRTHuman immunodeficiency virus type 1 32Tyr Gly Arg Lys Lys
Arg Arg Gln Arg Arg Arg 1 5 10 3316PRTDuck hepatitis B virus 33Met
Ser Gly Thr Phe Gly Gly Ile Leu Ala Gly Leu Ile Gly Leu Leu 1
5 10 15 3416PRTWoodchuck hepatitis B virus 34Met Ser Pro Ser Ser
Leu Leu Gly Leu Leu Ala Gly Leu Gln Val Val 1 5 10 15
3517PRTArtificial SequenceSynthetic endosomal release agent 35Gly
Leu Phe Glu Ala Leu Leu Glu Leu Leu Glu Ser Leu Trp Glu Leu 1 5 10
15 Leu 3616PRTArtificial SequenceSynthetic endosomal release agent
36Leu Lys Lys Leu Leu Lys Lys Leu Leu Lys Lys Leu Leu Lys Lys Leu 1
5 10 15 3716PRTArtificial SequenceEndosomal release agent 37Arg Gln
Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10 15
3814PRTHuman immunodeficiency virus type 1 38Gly Arg Lys Lys Arg
Arg Gln Arg Arg Arg Pro Pro Gln Cys 1 5 10 3927PRTHuman
immunodeficiency virus type 1 39Gly Ala Leu Phe Leu Gly Trp Leu Gly
Ala Ala Gly Ser Thr Met Gly 1 5 10 15 Ala Trp Ser Gln Pro Lys Lys
Lys Arg Lys Val 20 25 4018PRTMus musculus 40Leu Leu Ile Ile Leu Arg
Arg Arg Ile Arg Lys Gln Ala His Ala His 1 5 10 15 Ser Lys
4126PRTArtificial SequenceTransportan 41Gly Trp Thr Leu Asn Ser Ala
Gly Tyr Leu Leu Lys Ile Asn Leu Lys 1 5 10 15 Ala Leu Ala Ala Leu
Ala Lys Lys Ile Leu 20 25 4218PRTArtificial SequenceAmphiphilic
model peptide 42Lys Leu Ala Leu Lys Leu Ala Leu Lys Ala Leu Lys Ala
Ala Leu Lys 1 5 10 15 Leu Ala 439PRTArtificial SequenceArg9 43Arg
Arg Arg Arg Arg Arg Arg Arg Arg 1 5 4437PRTArtificial SequenceLL-37
44Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu 1
5 10 15 Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu
Val 20 25 30 Pro Arg Thr Glu Ser 35 4531PRTAscaris suum 45Ser Trp
Leu Ser Lys Thr Ala Lys Lys Leu Glu Asn Ser Ala Lys Lys 1 5 10 15
Arg Ile Ser Glu Gly Ile Ala Ile Ala Ile Gln Gly Gly Pro Arg 20 25
30 4630PRTHomo sapiens 46Ala Cys Tyr Cys Arg Ile Pro Ala Cys Ile
Ala Gly Glu Arg Arg Tyr 1 5 10 15 Gly Thr Cys Ile Tyr Gln Gly Arg
Leu Trp Ala Phe Cys Cys 20 25 30 4736PRTHomo sapiens 47Asp His Tyr
Asn Cys Val Ser Ser Gly Gly Gln Cys Leu Tyr Ser Ala 1 5 10 15 Cys
Pro Ile Phe Thr Lys Ile Gln Gly Thr Cys Tyr Arg Gly Lys Ala 20 25
30 Lys Cys Cys Lys 35 4812PRTArtificial SequenceBactenecin 48Arg
Lys Cys Arg Ile Val Val Ile Arg Val Cys Arg 1 5 10 4942PRTSus
scrofaMOD_RES(42)..(42)AMIDATION 49Arg Arg Arg Pro Arg Pro Pro Tyr
Leu Pro Arg Pro Arg Pro Pro Pro 1 5 10 15 Phe Phe Pro Pro Arg Leu
Pro Pro Arg Ile Pro Pro Gly Phe Pro Pro 20 25 30 Arg Phe Pro Pro
Arg Phe Pro Gly Lys Arg 35 40 5013PRTBos
taurusMOD_RES(13)..(13)AMIDATION 50Ile Leu Pro Trp Lys Trp Pro Trp
Trp Pro Trp Arg Arg 1 5 10 517PRTSimian virus 40 51Pro Lys Lys Lys
Arg Lys Val 1 5 5216PRTArtificial Sequencebipartite NLS consisting
of two basic domains separated by a variable number of spacer amino
acids and exemplified by the Xenopus nucleoplasmin NLS 52Lys Arg
Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys Lys Lys Leu 1 5 10 15
537PRTSimian virus 40 53Pro Lys Lys Lys Arg Met Val 1 5
5410PRTSimian virus 40 54Pro Lys Lys Lys Arg Lys Val Glu Asp Pro 1
5 10 558PRTSimian virus 40 55Pro Lys Lys Gly Ser Lys Lys Ala 1 5
567PRTSimian virus 40 56Pro Lys Thr Lys Arg Lys Val 1 5
5711PRTSimian virus 40 57Cys Gly Gly Pro Lys Lys Lys Arg Lys Val
Gly 1 5 10 587PRTSimian virus 40 58Pro Lys Lys Lys Ile Lys Val 1 5
5934PRTSimian virus 40 59Cys Tyr Asp Asp Glu Ala Thr Ala Asp Ser
Gln His Ser Thr Pro Pro 1 5 10 15 Lys Lys Lys Arg Lys Val Glu Asp
Pro Lys Asp Phe Glu Ser Glu Leu 20 25 30 Leu Ser 6013PRTSimian
virus 40 60Cys Gly Tyr Gly Pro Lys Lys Lys Arg Lys Val Gly Gly 1 5
10 617PRTArtificial SequencePolyoma large T protein 61Pro Lys Lys
Ala Arg Glu Asp 1 5 6213PRTArtificial SequencePolyoma large T
protein 62Cys Gly Tyr Gly Val Ser Arg Lys Arg Pro Arg Pro Gly 1 5
10 638PRTSimian virus 40 63Ala Pro Thr Lys Arg Lys Gly Ser 1 5
6411PRTArtificial SequencePolyoma virus major capsid protein VP1
64Ala Pro Lys Arg Lys Ser Gly Val Ser Lys Cys 1 5 10 657PRTSimian
virus 40 65Pro Asn Lys Lys Lys Arg Lys 1 5 6612PRTArtificial
SequencePolyoma virus capsid protein VP2 66Glu Glu Asp Gly Pro Gln
Lys Lys Lys Arg Arg Leu 1 5 10 677PRTArtificial SequenceYeast
histone H2B 67Gly Lys Lys Arg Ser Lys Ala 1 5 685PRTArtificial
SequenceAdenovirus E1a 68Lys Arg Pro Arg Pro 1 5 6911PRTArtificial
SequenceAdenovirus type 2/5 E1a 69Cys Gly Gly Leu Ser Ser Lys Arg
Pro Arg Pro 1 5 10 7012PRTXenopus laevis 70Leu Lys Asp Lys Asp Ala
Lys Lys Ser Lys Gln Glu 1 5 10 7110PRTArtificial Sequencev-Rel or
p59v-rel 71Gly Asn Lys Ala Lys Arg Gln Arg Ser Thr 1 5 10
7211PRTInfluenza A virus 72Pro Phe Leu Asp Arg Leu Arg Arg Asp Gln
Lys 1 5 10 739PRTHomo sapiens 73Ser Val Thr Lys Lys Arg Lys Leu Glu
1 5 749PRTXenopus laevis 74Ser Ala Ser Lys Arg Arg Arg Leu Glu 1 5
7511PRTHuman adenovirus type 5 75Pro Pro Lys Lys Arg Met Arg Arg
Arg Ile Glu 1 5 10 7628PRTRattus norvegicus 76Tyr Arg Lys Cys Leu
Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr 1 5 10 15 Lys Lys Lys
Ile Lys Gly Ile Gln Gln Ala Thr Ala 20 25 7754PRTHomo sapiens 77Arg
Lys Asp Arg Arg Gly Gly Arg Met Leu Lys His Lys Arg Gln Arg 1 5 10
15 Asp Asp Gly Glu Gly Arg Gly Glu Val Gly Ser Ala Gly Asp Met Arg
20 25 30 Ala Met Ile Asn Ala Cys Ile Asp Asn Leu Trp Pro Ser Pro
Leu Met 35 40 45 Ile Lys Arg Ser Lys Lys 50 788PRTArtificial
SequenceRabbit progesterone receptor 78Arg Lys Phe Lys Lys Phe Asn
Lys 1 5 798PRTArtificial Sequencec-myb gene product 79Pro Leu Leu
Lys Lys Ile Lys Gln 1 5 808PRTArtificial SequenceN-myc gene produc
80Pro Pro Gln Lys Lys Ile Lys Ser 1 5 817PRTHomo sapiens 81Pro Gln
Pro Lys Lys Lys Pro 1 5 829PRTArtificial Sequencec-erb-A gene
product 82Ser Lys Arg Val Ala Lys Arg Lys Leu 1 5 8314PRTArtificial
SequenceYeast ribosomal protein L29 83Met Thr Gly Ser Lys Thr Arg
Lys His Arg Gly Ser Gly Ala 1 5 10 846PRTArtificial SequenceYeast
ribosomal protein L29 84Arg His Arg Lys His Pro 1 5
856PRTArtificial SequenceYeast ribosomal protein L29 85Lys Arg Arg
Lys His Pro 1 5 866PRTArtificial SequenceYeast ribosomal protein
L29 86Lys Tyr Arg Lys His Pro 1 5 876PRTArtificial SequenceYeast
ribosomal protein L29 87Lys His Arg Arg His Pro 1 5
886PRTArtificial SequenceYeast ribosomal protein L29 88Lys His Lys
Lys His Pro 1 5 896PRTArtificial SequenceYeast ribosomal protein
L29 89Arg His Leu Lys His Pro 1 5 9033PRTHepatitis B virus 90Pro
Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg 1 5 10
15 Thr Pro Ser Pro Arg Arg Arg Arg Ser Pro Arg Arg Arg Arg Ser Gln
20 25 30 Ser 919PRTArtificial SequenceViral jun 91Ala Ser Lys Ser
Arg Lys Arg Lys Leu 1 5 9216PRTHuman T-cell lymphotropic virus type
1 92Gly Gly Leu Cys Ser Ala Arg Leu His Arg His Ala Leu Leu Ala Thr
1 5 10 15 9318PRTMus musculus 93Asp Thr Arg Glu Lys Lys Lys Phe Leu
Lys Arg Arg Leu Leu Arg Leu 1 5 10 15 Asp Glu 941476DNAArtificial
SequenceP28z CAR 94atggcctcac cgttgacccg ctttctgtcg ctgaacctgc
tgctgctggg tgagtcgatt 60atcctgggga gtggagaagc tgaggtgcag ctgcagcagt
caggacctga actggtgaag 120cctgggactt cagtgaggat atcctgcaag
acttctggat acacattcac tgaatatacc 180atacactggg tgaagcagag
ccatggaaag agccttgagt ggattggaaa catcaatcct 240aacaatggtg
gtaccaccta caatcagaag ttcgaggaca aggccacatt gactgtagac
300aagtcctcca gtacagccta catggagctc cgcagcctaa catctgagga
ttctgcagtc 360tattattgtg cagctggttg gaactttgac tactggggcc
aagggaccac ggtcaccgtc 420tcctcaggtg gaggtggatc aggtggaggt
ggatctggtg gaggtggatc tgacattgtg 480atgacccagt ctcacaaatt
catgtccaca tcagtaggag acagggtcag catcatctgt 540aaggccagtc
aagatgtggg tactgctgta gactggtatc aacagaaacc aggacaatct
600cctaaactac tgatttattg ggcatccact cggcacactg gagtccctga
tcgcttcaca 660ggcagtggat ctgggacaga cttcactctc accattacta
atgttcagtc tgaagacttg 720gcagattatt tctgtcagca atataacagc
tatcccctca cgttcggtgc tgggaccatg 780ctggacctga aacgggcggc
cgcatctact actaccaagc cagtgctgcg aactccctca 840cctgtgcacc
ctaccgggac atctcagccc cagagaccag aagattgtcg gccccgtggc
900tcagtgaagg ggaccggatt ggacttcgcc tgtgatattt acatctgggc
acccttggcc 960ggaatctgcg tggcccttct gctgtccttg atcatcactc
tcatctgcta caatagtaga 1020aggaacagac tccttcaaag tgactacatg
aacatgactc cccggaggcc tgggctcact 1080cgaaagcctt accagcccta
cgcccctgcc agagactttg cagcgtaccg ccccagagca 1140aaattcagca
ggagtgcaga gactgctgcc aacctgcagg accccaacca gctctacaat
1200gagctcaatc tagggcgaag agaggaatat gacgtcttgg agaagaagcg
ggctcgggat 1260ccagagatgg gaggcaaaca gcagaggagg aggaaccccc
aggaaggcgt atacaatgca 1320ctgcagaaag acaagatggc agaagcctac
agtgagatcg gcacaaaagg cgagaggcgg 1380agaggcaagg ggcacgatgg
cctttaccag ggtctcagca ctgccaccaa ggacacctat 1440gatgccctgc
atatgcagac cctggcccct cgctaa 1476
* * * * *