U.S. patent application number 12/617018 was filed with the patent office on 2010-12-09 for methods of using il-21 for adoptive immunotherapy and identification of tumor antigens.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Cassian Yee.
Application Number | 20100310533 12/617018 |
Document ID | / |
Family ID | 36588349 |
Filed Date | 2010-12-09 |
United States Patent
Application |
20100310533 |
Kind Code |
A1 |
Yee; Cassian |
December 9, 2010 |
METHODS OF USING IL-21 FOR ADOPTIVE IMMUNOTHERAPY AND
IDENTIFICATION OF TUMOR ANTIGENS
Abstract
Methods for preparing ex vivo T cell cultures using IL-21
compositions for use in adoptive immunotherapy are described.
Addition of IL-21 to cultures of non-terminally differentiated T
cells population, either isolated or present in peripheral blood
mononuclear cells are exposed to one or more tumor antigens, and in
the presence of IL-21 compositions and antigen presenting cells
(APCs), the resulting T cell population has an enhanced
antigen-specificity, and can be reintroduced into the patient.
Methods are also disclosed for identifying tumor antigens by
culturing T cell populations exposed to IL-21 compositions and APCs
in the presence of tumor material.
Inventors: |
Yee; Cassian; (Seattle,
WA) |
Correspondence
Address: |
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
1201 EASTLAKE AVENUE EAST
SEATTLE
WA
98102-3702
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
36588349 |
Appl. No.: |
12/617018 |
Filed: |
November 12, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11285970 |
Nov 23, 2005 |
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12617018 |
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60630727 |
Nov 24, 2004 |
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Current U.S.
Class: |
424/93.71 ;
435/29; 435/372.3; 435/6.16 |
Current CPC
Class: |
C12N 2502/1121 20130101;
G01N 33/57484 20130101; C12N 2501/23 20130101; G01N 33/5047
20130101; G01N 33/57492 20130101; C12N 5/0636 20130101; A61K 35/17
20130101; C12N 5/0638 20130101; C12N 2501/2321 20130101; G01N
33/574 20130101; G01N 33/6863 20130101; G01N 33/505 20130101; A61P
37/00 20180101; A61P 35/00 20180101 |
Class at
Publication: |
424/93.71 ;
435/29; 435/6; 435/372.3 |
International
Class: |
A61K 35/12 20060101
A61K035/12; C12Q 1/02 20060101 C12Q001/02; C12Q 1/68 20060101
C12Q001/68; C12N 5/0783 20100101 C12N005/0783; A61P 37/00 20060101
A61P037/00 |
Claims
1. A method for identifying a tumor antigen comprising:
co-culturing tumor material isolated from a subject with peripheral
blood mononuclear cells (PBMCs) in the presence of an IL-21
composition and antigen presenting cells (APCs); isolating a T cell
population from the culture; cloning individual T cells from the T
cell population; and characterizing T cell clones for
antigen-specificity.
2. The method of claim 1, wherein the T cell population is a T cell
population that is non-terminally differentiated.
3. The method of claims 1, wherein the PBMCs or T cell population
is an autologous cell population.
4. The method of claim 1, wherein the tumor material comprises
total RNA, lysed tumor cells or apoptotic bodies.
5. The method of claim 2, wherein the isolated T cell population
does not include CD4+ cells.
6. The method of claim 1, wherein co-culturing is in the presence
of the IL-21 composition and one or more additional cytokines
7. A method of preparing a naive CD8+ human T cell population for
in use adoptive immunotherapy comprising: isolating peripheral
blood mononuclear cells (PBMCs) from a biological sample;
identifying PBMCs having a histocompatible phenotype to a subject
having a tumor; isolating a naive CD8+ human T cell population;
co-culturing tumor material isolated from the subject with the
naive CD8+ human T cell population in the presence of an IL-21
composition and antigen presenting cells (APCs); and expanding the
cells in culture.
8. The method of claim 7, wherein the PBMCs are autologous.
9. The method of claim 7, wherein after the step of co-culturing
the tumor material and with the T cell population, the T cells are
enriched.
10. A method of preparing a naive CD8+ human T cell population for
use in adoptive immunotherapy comprising: isolating a biological
sample containing T cells; identifying a T cell population having a
histocompatible phenotype to a subject having a tumor; isolating a
naive CD8+ human T cell population; co-culturing tumor material
from the subject with the naive CD8+ human T cell population in the
presence of an IL-21 composition and APCs; and expanding these
cells in culture.
11. The method of claim 10, wherein the T cell population is
autologous.
12. The method of claim 7, 10, or 16 wherein the tumor material
comprises total RNA, lysed tumor cells or apoptotic bodies.
13. The method of claim 10, wherein after the step of co-culturing
the tumor material and with the T cell population, the T cells are
enriched.
14. A method for ex vivo expansion of antigen-specific cytotoxic T
cells comprising: isolating a biological sample containing T cells;
identifying a T cell population having a histocompatible phenotype
to a subject; co-culturing antigenic material from the subject with
the T cell population in the presence of an IL-21 composition;
expanding the cells in culture; and and reintroducing the cells
back into the subject.
15. The method of claim 14, wherein after the step of co-culturing
the tumor material and with the T cell population, the T cells are
enriched.
16. A method of preparing a non-terminally differentiated CD8+
human T cell population for use in adoptive immunotherapy
comprising: isolating a biological sample containing T cells;
identifying a T cell population having a histocompatible phenotype
to a subject having a tumor; isolating a non-terminally
differentiated CD8+ human T cell population; co-culturing tumor
material from the subject with the non-terminally differentiated
CD8+ human T cell population in the presence of an IL-21
composition and APCs; and expanding these cells in culture.
17. The method of claim 16, wherein the T cell population is
autologous.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/285,970, filed Nov. 23, 2005; which claims
the benefit of U.S. Provisional Application Ser. No. 60/630,727,
filed Nov. 24, 2004, both of which are herein incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] Cytokines generally stimulate proliferation or
differentiation of cells of the hematopoietic lineage or
participate in the immune and inflammatory response mechanisms of
the body. The interleukins are a family of cytokines that mediate
immunological responses. Central to an immune response is the T
cell, which produces many cytokines and plays a role in adaptive
immunity to antigens. Cytokines produced by the T cell have been
classified as type 1 and type 2 (Kelso, A. Immun. Cell Biol.
76:300-317, 1998). Type 1 cytokines include IL-2, IFN-.gamma.,
LT-.alpha., and are involved in inflammatory responses, viral
immunity, intracellular parasite immunity and allograft rejection.
Type 2 cytokines include IL-4, IL-5, IL-6, IL-10 and IL-13, and are
involved in humoral responses, helminth immunity and allergic
response. Shared cytokines between Type 1 and 2 include IL-3,
GM-CSF and TNF-.alpha.. There is some evidence to suggest that Type
1 and Type 2 producing T cell populations preferentially migrate
into different types of inflamed tissue.
[0003] IL-21 has been shown to be a potent modulator of cytotoxic T
cells and NK cells. (Parrish-Novak, et al. Nature 408:57-63, 2000;
Parrish-Novak, et al., J. Leuk. Bio. 72:856-863, 2002; Collins et
al., Immunol Res. 28:131-140, 2003; Brady, et al. J. Immunol
172:2048-58, 2004.) T cell responses include enhancement of primary
antigen response as modulation of memory T cell functions.
[0004] In murine studies, IL-21 potentiates the maturation and
effector function of NK cells and promotes T cell activation in
response to alloantigen (Kasaian et al., Immunity 16:559-569,
2002). As a cytokine which limits NK cell expansion and promotes
activation of murine CD8 T cells, IL-21 is believed to play a role
in the transition from innate to adaptive immunity (Kasaian, supra,
2002). Among CD4 T cells, IL-21 has been described as both a Th1 (T
helper 1) cytokine which upregulates the expression of genes
associated with innate immunity (Strengell et al., J. Immunol.
170:5464, 2003) as well as a Th2 cytokine that inhibits the
differentiation of naive Th cells into IFN-gamma-producing Thl
cells (Wurster et al., J. Exp. Med. 196:969, 2002). The effects of
IL-21 in the development of innate immunity and CD4 Th responses
are well-characterized (Strengell supra, 2003; Strengell et al., J.
Leukoc. Biol. 76:416, 2004), but its role in the antigen-specific
CD8+ T cell response, particularly in humans, had not been fully
explored. The present invention provides methods for inducing a
high affinity antigen-specific cytotoxic T cell response by
administering IL-21. Induction of a high affinity CD8 response
against self antigens, which are represented increasingly as
potential immune targets in cancer immunotherapy demonstrate a
significant role for IL-21 in antigen-specific anti-tumor
strategies. Thus, the present invention provides methods for
administering IL-21 as an adjuvant in tumor vaccines and ex vivo
expansion of tumor antigen-specific cytotoxic T cells for use in
adoptive immunotherapy. The present invention also provides methods
for administering IL-21 as an adjuvant for vaccines and ex vivo
expansion of antigen-specific cytotoxic T cells in general against
viruses, and other target antigens. These and other uses should be
apparent to those skilled in the art from the teachings herein.
SUMMARY OF THE INVENTION
[0005] In one aspect, the present invention provides a method for
identifying a tumor antigen comprising co-culturing tumor material
isolated from a subject with peripheral blood mononuclear cells
(PBMCs) in the presence of an IL-21 composition and antigen
presenting cells (APCs), isolating a Tcell population from the
culture, enriching for individual T cells from the T cell
populating, and characterizing the T cell clones for antigen
specificity. In certain embodiments, enrichment is cloning of
individual T cells.
[0006] In another aspect of the present invention a method of
identifying a tumor antigen is provided which comprises
co-culturing tumor material from a subject with an isolated T cell
population that is non-terminally differentiated in the presence of
an IL-21 composition; cloning individual T cells from the T cell
population; and characterizing T cell clones for
antigen-specificity. In one embodiment, the isolated T cell
population does not include CD4+ cells.
[0007] In certain embodiments of these methods, the PBMCs or T cell
population is an autologous cell population. In other embodiments,
the tumor material comprises total RNA, lysed tumor cells, necrotic
tumor cells, tumor proteins or apoptotic bodies. In another
embodiment, co-culturing is in the presence of the IL-21
composition and one or more additional cytokines.
[0008] In another aspect, the present invention provides method of
preparing a T cell population for use in adoptive immunotherapy
comprising identifying PBMCs having a histocompatible phenotype to
a subject having a tumor; co-culturing tumor material or tumor
associated peptides from the patient with the peripheral blood
mononuclear cells (PBMCs) in the presence of an IL-21 composition
and APCs; expanding these cells in culture; and and reintroducing
these cells back into the patient. In one embodiment, the PBMCs are
autologous. In one embodiment, the T cell population is autologous.
In another embodiment, the tumor material comprises total RNA,
lysed tumor cells or apoptotic bodies. In another embodiment, the
PBMCs or T cells are allogenic.
[0009] In another aspect, the present invention provides a method
of preparing a T cell population for use in adoptive immunotherapy
comprising identifying T cell population having a histocompatible
phenotype to a subject having a tumor; co-culturing tumor material
from the subject with the T cell population in the presence of an
IL-21 composition and APCs; expanding these cells in culture; and
and reintroducing these cells back into the subject. In one
embodiment, T cell population is naive or non-terminally
differentiated. In another embodiment, the T cell population is
autologous. In another embodiment, the tumor material comprises
total RNA, lysed tumor cells or apoptotic bodies.
BRIEF DESCRIPTION OF THE FIGURES
[0010] FIGS. 1 and 2 illustrate that IL-21 enhances the generation
of MART-1 specific CTL. CD8+ T cells from healthy HLA-A2+ donors
CG, NE and LD were stimulated in vitro with autologous mature
dendritic cells pulsed with the MART-1, M26 peptide as described in
the Examples.
[0011] FIG. 1. On day 7 after stimulation, 10.sup.6 Cells from each
experiment group were harvested and stained with 20 .mu.g/ml of
peptide/MHC tetramer (PE, vertical axis) and a vital dye (PI or
DAPI, horizontal axis). Data are expressed as percentage of
tetramer positive cells among gated lymphocytes (purified CD8+
cells).
[0012] FIG. 2. The absolute number in millions of tetramer+ cells
corresponding to untreated and IL-21 treated cultures from donors
CG, NE and LD depicted in A., and the fold increase in absolute
numbers of IL-21 treated to untreated cultures. C. Cultures from a
normal healthy donor, CG, and a patient with metastatic melanoma,
ST, were analyzed on Day 7 after the first (Stim 1) and second
(Stim 2) stimulation in the presence or absence of IL-21 during
Stim 1. IL-2 and IL-7 were added following Stim 2. Data are
expressed as percentage of tetramer positive cells among gated
lymphocytes (purified CD8+ cells). Results above are representative
of three separate experiments for each donor.
[0013] FIG. 3 illustrates that exposure to other gamma-chain
cytokines, IL-2, IL-7 or IL-15 does not enhance generation of
antigen-specific CTL. CD8+ T cells from a healthy HLA-A2+ donor
were stimulated in vitro with autologous mature dendritic cells
pulsed with the MART-1, M26 peptide as described the Examples. On
day 7 after stimulation, 10.sup.6 Cells from each experiment group
were harvested and stained with 20 .mu.g/ml of peptide/MHC tetramer
(PE, vertical axis) and a vital dye (PI or DAPI, horizontal axis).
Data are expressed as percentage of tetramer positive cells among
gated lymphocytes (purified CD8+ cells). Data are representative of
cultures from 3 HLA-A2+ donors.
[0014] FIG. 4 illustrates IL-21 treated cells undergo increased
proliferation and decreased apoptosis during primary in vitro
stimulation. Purified populations of naive (CD45RA+, CD62L+)
lymphocytes were pre-incubated with CFSE and stimulatied with
MART-1 peptide in the absence (No Cytokine) or presence of IL-21
(IL-21-treated). On Day 7, cells were stained with MART-1
peptide-MHC Tetramer-PE and analyzed for the fraction of dividing
cells (CFSE) or cells undergoing apoptosis (Annexin V).
CFSE-stained cells lose fluorescence intensity with successive
divisions. Results are expressed as percentage of non-dividing
(rightmost box), dividing (center) and rapidly dividing (leftmost)
tetramer+cells. For apoptotic cells, Annexin V staining
tetramer+cells are represented as a percentage in the right upper
quadrant.
[0015] FIG. 5 illustrates IL-21 influences primarily naive vs.
memory CD8+ T cells. Highly purified populations of naive (CD45RA+,
CD62L+) or memory (CD45RO+) T cells from a healthy normal donor
(CG) and a donor with melanoma (ST) were evaluated for induction of
antigen-specific responses to MART-1 peptide in the absence or
presence of IL-21. The percentage of MART-1-specific CTL are
expressed as percentage tetramer+ cells among total gated
lymphocytes (CD8+ purified cells) after two cycles of in vitro
stimulation. Results are representative of experiments performed on
2 other normal healthy donors (NE, LD) and 2 other patient donors
(AM, RE).
[0016] FIG. 6 illustrates IL-21 preferentially induces the
generation of high avidity antigen specific CTL. Individual MART-1
specific CD8+ T cell clones were isolated from cultures stimulated
in the absence (Control) or presence (IL-21) of IL-21. 8-12
representative CTL clones from each experimental condition were
evaluated for target affinity in a chromium release assay using T2
cells pulsed with decreasing concentrations of M26 peptide (peptide
dose titration analysis). Data are expressed as the concentration
(nM) required to reach 50% maximal lysis of target cells. On
average, CTL clones generated by IL-21, demonstrated a decreased
peptide dose requirement for specific lysis compared to CTL clones
generated in no cytokine control (* p<0.01). A: healthy donor,
CG ( ); B: melanoma patient, ST (.DELTA.). These same clones were
evaluated for specific reactivity to a MART-1+tumor cell line (526)
at E/T ratio of 10 to 1 in a standard 4 hr .sup.51Chromium release
assay (CRA). Significantly greater lysis of antigen-positive tumor
(o , .tangle-solidup.) with background lysis of antigen-negative
tumor (375 0, A) was observed in CTL clones isolated from IL-21
treated culture than those isolated from no cytokine control.
(.dagger. p<0.01). C: healthy donor, CG ( , .largecircle.) ; D:
melanoma patient, ST (.DELTA., .tangle-solidup.). Results are
representative of 3 normal healthy donors and 3 patient donors.
[0017] FIG. 7 illustrates IL-21 treated cultures enrich for CTL
expressing higher affinity TCR:tetramer dissociation assay. The
tetramer dissociation assay, which depicts the fraction of bound
tetramer remaining over time in the presence of an excess of
unlabeled tetramer, was used as a surrogate measure of the TCR
dissociation rate or TCR affinity of individual clones. The
fraction of PE-tetramer labelled MART-1-specific CTL clones from
IL-21 treated cultures ( ) was compared with clones from untreated
cultures (.largecircle.) over time (2 to 60 minutes). The rate of
decrease in the percent fluorescence intensity of tetramer staining
from Time 0 is inversely correlated with TCR affinity. Results are
representative of 4 donors.
[0018] FIG. 8 illustrates IL-21 treated cultures yield a population
of CD28.sup.hi antigen-specific CTL. Cells were collected from
pre-stimulation PBMC, and then 7 days following stimulation with
MART-1 peptide-pulsed autologous dendritic cells in the absence or
presence of IL-21. Cells were stained for MART-1-Tetramer and
simultaneously with either CD45RA or CD45 RO, CD28 and CCR7 (FIG.
7). Histogram analysis for individual phenotypic markers was
performed on gated MART-1-tetramer staining cells. CD28 expression
on gated tetramer-staining cells is also shown for culture from Day
27 after stimulation.These results are representative of cultures
from 3 donors.
[0019] FIG. 9 illustrates IL-2 production following
antigen-specific stimulation of CD28.sup.hi and CD28-low expressing
CTL. MART-1 tetramer+ CD8+ T cells from IL-21 treated (CD28.sup.hi)
or untreated (CD28.sup.1ow) cultures were sorted and co-cultivated
with either unpulsed T2 cells, MART-1 (M26) peptide pulsed T2 cells
alone, or with CTLA4-Ig (0.5 .mu.g/m1) to block B7-CD28 engagement.
Supernants were collected 48 hr later and analyzed for IL-2 by
ELISA. Specific induction of IL-2 production among CD28-hi
expressing MART-1-specific CTL following antigen stimulation, and
inhibited by CTLA4-Ig, is observed suggesting IL-2 induction among
IL-21 treated cells is CD28-dependent. Results are the mean of
triplicate assays, error bars as shown.
[0020] FIG. 10 illustrates IL-2 production following
antigen-specific stimulation of CD28.sup.hi and CD28.sup.low
expressing CTLs. MART-1 tetramer-positive CD8 cells from IL-21
treated or untreated cultures were sorted and co-cultivated with
either unpulsed T2 cells, MART-1 peptide-pulsed T2 cells alone, or
with CTLA4-Ig to block B7-CD28 engagement. Supernatatnts were
collected 48 f later and analyzed for IL-2 by ELISA. Specific
induction of IL-2 production among CD28.sup.hi expressing MART-1
specific CTLs following antigen stimulation and inhibition by
CTLA4-Ig is observed. Results are the mean of triplicate
assays.
[0021] FIG. 11 illustrates that IL-21 influences the CD8 T cells
response to gp100 and NY-ESO-1 antigen. CD8 T cells were stimulated
both in vitro with autologous DCs pulsed with NY-ESO-1 or gp100
peptide. IL-21 was added to IL-21 treated cultures. Six days after
primary in vitro stimulation cultures were analyzed for antigen
specificity and surface phenotype by tetramer staining and
multiparametric analysis on flow cytometry. In panel A, NY-ESO-1
and G154-specific CTL frequency are shown as percentage of all CD8
T cells next to the boxed gates. The fold increase in absolute
numbers of of NY-ESO-1 specific CTLs was calculated based on
numbers of cells in respective cultures and for NY-ESO-1 was found
to be almost 20-fold greater among IL-21 treated cells. Panel B.
Gated tetramer-positive cells from control or IL-21 treated
cultures were analyzed for CD8 expression. All cells were CD45RO+,
CCR7-. Histogram analysis for CD28 expression among NY-OEST-1 or
gp1000-specific CTLSs was found to be significantly upregulated
among IL-21 treated cultures compared to controls. There results
are representative of six separate experiments from threee HLA-2+
individuals.
[0022] FIG. 12 illustrates that the addition of IL-21 to IL-6 and
IL-12, IL-2, IL-7 or IL-15 alone significantly enhances the CD8 T
cell response, compared to IL-6 and IL-12, IL-2, IL-7 or IL-15
alone.
[0023] FIG. 13 illustrates that IL-21 can increase T cell frequency
to levels that are high enough for expansion and adoptive transfer
without further antigen-specific T cell enrichment
DESCRIPTION OF THE INVENTION
[0024] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms:
[0025] The term "allelic variant" is used herein to denote any of
two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in phenotypic polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered amino acid
sequence. The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
[0026] The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides. Where the context
allows, these terms are used with reference to a particular
sequence or portion of a polypeptide to denote proximity or
relative position. For example, a certain sequence positioned
carboxyl-terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus of the
complete polypeptide.
[0027] The term "cancer" or "cancer cell" is used herein to denote
a tissue or cell found in a neoplasm which possesses
characteristics which differentiate it from normal tissue or tissue
cells. Among such characteristics include but are not limited to:
degree of anaplasia, irregularity in shape, indistinctness of cell
outline, nuclear size, changes in structure of nucleus or
cytoplasm, other phenotypic changes, presence of cellular proteins
indicative of a cancerous or pre-cancerous state, increased number
of mitoses, and ability to metastasize. Words pertaining to
"cancer" include carcinoma, sarcoma, tumor, epithelioma, leukemia,
lymphoma, polyp, and scirrus, transformation, neoplasm, and the
like.
[0028] The term "co-administration" is used herein to denote that
an IL-21 polypeptide or protein and a second therapeutic molecule
may be given concurrently or at a different times. The
co-adminstration may be a single co-administration of both IL-21
and the second therapeutic molecule or multiple cycles of
co-administration. Co-administration need not be the only times
either IL-21 or the second therapeutic molecule is administered to
a patient.
[0029] The term "combination therapy" is used herein to denote that
a subject is administered at least one therapeutically effective
dose of IL-21 and a second therapeutic molecule. The IL-21 may be a
mature polypeptide, fragment thereof, fusion or conjugate that
demonstrates IL-21 biological activity.
[0030] The term "enhance" when in reference to an immune response
is used herein to mean increasing the scale and/or efficiency of an
immune response or extending the duration of the immune response.
The term is used interchangeably with "augument." An immune
response includes, but is not limited to, enhanced cytolytic
activity, apoptotic activity or increases in CD8+ T cell numbers or
survival.
[0031] The term "isolated", when applied to a polynucleotide,
denotes that the polynucleotide has been removed from its natural
genetic milieu and is thus free of other extraneous or unwanted
coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions such as promoters and
terminators. The identification of associated regions will be
evident to one of ordinary skill in the art (see for example, Dynan
and Tijan, Nature 316:774-78, 1985).
[0032] An "isolated" polypeptide or protein is a polypeptide or
protein that is found in a condition other than its native
environment, such as apart from blood and animal tissue. In a
preferred form, the isolated polypeptide is substantially free of
other polypeptides, particularly other polypeptides of animal
origin. It is preferred to provide the polypeptides in a highly
purified form, i.e. greater than 95% pure, more preferably greater
than 99% pure. When used in this context, the term "isolated" does
not exclude the presence of the same polypeptide in alternative
physical forms, such as dimers or alternatively glycosylated or
derivatized forms.
[0033] The term "level" when referring to immune cells, such as NK
cells, T cells, in particular cytotoxic T cells, B cells and the
like, refers to the number of cells or the activity of cells. An
increased level is either increased number of cells or enhanced
activity of cell function.
[0034] The term "level" when referring to viral infections refers
to a change in the level of viral infection and includes, but is
not limited to, a change in the level of CTLs or NK cells (as
described above), a decrease in viral load, an increase antiviral
antibody titer, decrease in serological levels of alanine
aminotransferase, or improvement as determined by histological
examination of a target tissue or organ. Determination of whether
these changes in level are significant differences or changes is
well within the skill of one in the art.
[0035] The term "neoplastic", when referring to cells, indicates
cells undergoing new and abnormal proliferation, particularly in a
tissue where in the proliferation is uncontrolled and progressive,
resulting in a neoplasm. The neoplastic cells can be either
malignant, i.e. invasive and metastatic, or benign.
[0036] A "polynucleotide" is a single- or double-stranded polymer
of deoxyribonucleotide or ribonucleotide bases read from the 5' to
the 3' end. Polynucleotides include RNA and DNA, and may be
isolated from natural sources, synthesized in vitro, or prepared
from a combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides ("nt"), or kilobases ("kb"). Where the context allows,
the latter two terms may describe polynucleotides that are
single-stranded or double-stranded. When the term is applied to
double-stranded molecules it is used to denote overall length and
will be understood to be equivalent to the term "base pairs". It
will be recognized by those skilled in the art that the two strands
of a double-stranded polynucleotide may differ slightly in length
and that the ends thereof may be staggered as a result of enzymatic
cleavage; thus all nucleotides within a double-stranded
polynucleotide molecule may not be paired.
[0037] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides".
[0038] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0039] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule (i.e., a ligand) and mediates the
effect of the ligand on the cell. Membrane-bound receptors are
characterized by a multi-peptide structure comprising an
extracellular ligand-binding domain and an intracellular effector
domain that is typically involved in signal transduction. Binding
of ligand to receptor results in a conformational change in the
receptor that causes an interaction between the effector domain and
other molecule(s) in the cell. This interaction in turn leads to an
alteration in the metabolism of the cell. Metabolic events that are
linked to receptor-ligand interactions include gene transcription,
phosphorylation, dephosphorylation, increases in cyclic AMP
production, mobilization of cellular calcium, mobilization of
membrane lipids, cell adhesion, hydrolysis of inositol lipids and
hydrolysis of phospholipids. In general, receptors can be membrane
bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating
hormone receptor, beta-adrenergic receptor) or multimeric (e.g.,
PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF
receptor, G-CSF receptor, erythropoietin receptor and IL-6
receptor).
[0040] Molecular weights and lengths of polymers determined by
imprecise analytical methods (e.g., gel electrophoresis) will be
understood to be approximate values. When such a value is expressed
as "about" X or "approximately" X, the stated value of X will be
understood to be accurate to .+-.10%.
[0041] All references used are herein incorporated by
reference.
[0042] The present inventions are directed to use of IL-21 to
increase proliferation and survival of antigen-specific T cells.
IL-21 enhancement of antigen-specific T cells will be useful for
increasing the frequency of antigen-specific T cells, enriching for
populations of antigen-specific T cells with enhanced affinity, and
generating a population of antigen-specific T cells with increased
CD28 expression and "helper-independent" phenotype (Widmer et al.,
Nature 294:750, 1981; Topp et al., J. Exp Med 198(6):947, 2003;
Cheng et al., J Immunol 169:4990, 2002)
[0043] In certain aspects the present invention provides methods
that comprise culturing naive T cells in the presence of an IL-21
composition and an antigen resulting in a cytotoxic T cell
population that has a higher affinity for an antigen than T cells
cultured in the absence of IL-21. Of particular interest are tumor
antigens. High affinity antigen-specific T cells have the capacity
to recognize and kill tumor, whereas a low affinity T cell may have
tumor antigen-specificity, yet still not recognize and kill a tumor
cell. Affinity of a T cell for a tumor is in part dependent on the
antigen density that is presented by the tumor cell. If antigen
presentation on the tumor cell is low, then is more likely that
only high affinity T cells will be able recognize the tumor cell
and initiate cytolysis. Moreover, the methods of the present
invention can be used to increase T cell frequency to levels that
are high enough for expansion and adoptive cell transfer without
further antigen-specific T cell enrichment and thereby greatly
decrease the time to therapy and obviate the requirement for
further selection or cloning.
[0044] In another aspect of the present invention, the methods of
the present invention comprise generating an antigen-specific CTL
population that has a high affinity for self antigens by culturing
T cells that have non-terminally differentiated to an IL-21
composition. In one embodiment, the T cells are isolated naive T
cells. Once the antigen-specific T cell population has been
expanded ex vivo, the cells are reintroduced into the patient. In
certain embodiments, IL-21 administration to the patient will
continue and may be in combination with other therapies.
[0045] In another aspect, the methods of the present invention
provide methods for enhancing the repetoire of antigens recognized
by a T cell population. The methods comprise co-culturing tumor
material such as a tumor cell line or derivative thereof (e.g.
total RNA, lysed tumor cells, apoptotic bodies) with autologous T
cells isolated from a subject. The tumor material and the T cells
are cultured in the presence of an IL-21 composition, and after
allowing T cells to proliferate, the T cells are cloned.
Antigen-specific T cells are identified and further analyzed to
characterize the antigen-specificity.
A. Description of IL-21.
[0046] Human IL-21 (SEQ ID NO:1 and SEQ ID NO:2) was originally
designated zalpha11 Ligand, and is described in commonly-owned U.S.
Pat. Nos. 6,307,024, and 6,686,178, which are incorporated herein
by reference. The IL-21 receptor is described in U.S. Pat. No.
6,057,128. The IL-21 receptor, previously designated zalphal 1 (SEQ
ID NO:5 and SEQ ID NO:6), and heterodimeric receptor IL-21R/IL-2Ry
are also described in commonly-owned U.S Pat. Nos. 6,576,744,
6,803,451, 6,692,924 and WO 00/17235, which are incorporated herein
by reference. As described in these publications, IL-21 was
isolated from a cDNA library generated from activated human
peripheral blood cells (hPBCs), which were selected for CD3. CD3 is
a cell surface marker unique to cells of lymphoid origin,
particularly T cells.
[0047] The amino acid sequence for the IL-21R indicated that the
encoded receptor belonged to the Class I cytokine receptor
subfamily that includes, but is not limited to, the receptors for
IL-2, IL-4, IL-7, IL-15, EPO, TPO, GM-CSF and G-CSF (for a review
see, Cosman, "The Hematopoietin Receptor Superfamily" in Cytokine
5(2): 95-106, 1993). The IL-21 receptor has been identified on NK
cells, T cells and B cell indicating -21 acts on hematopoietic
lineage cells, in particular lymphoid progenitor cells and lymphoid
cells. Other known four-helical-bundle cytokines that act on
lymphoid cells include IL-2, IL-4, IL-7, and IL-15. For a review of
four-helical-bundle cytokines, see, Nicola et al., Advances in
Protein Chemistry 52:1-65, 1999 and Kelso, A., Immunol Cell Biol.
76:300-317, 1998.
[0048] For IL-21, a secretory signal sequence is comprised of amino
acid residues 1 (Met) to 29 (Ser), and a mature polypeptide is
comprised of amino acid residues 30 (Gln) to 162 (Ser) (as shown in
SEQ ID NO: 2). The corresponding polynucleotide sequence is shown
in SEQ ID NO:1. Those skilled in the art will recognize that the
sequence disclosed in SEQ ID NO:1 represents a single allele of
human IL-21 and that allelic variation and alternative splicing are
expected to occur.
B. Use of IL-21 Vaccine Therapy, Adoptive Immunotherapy and
Identification of Tumor Specific Antigens.
[0049] The present invention is based in part on a study of both
human healthy donors and melanoma patients where a positive
regulatory role for IL-21 in the induction of a primary
antigen-specific human CD8+ T cell response was demonstrated. Using
peptide-MHC tetramers to track a rare but measurable naive T cell
population recognizing a normal self antigen, in the presence of
IL-21, the frequency and absolute numbers of antigen-specific CD8 T
cells that could be elicited increased by more than 20-fold
compared to cultures grown in the absence of IL-21. The enhanced
generation of an antigen-specific T cell response is specific to
this gamma-chain receptor cytokine since the addition of IL-2, IL-7
or IL-15 during initial priming had no added effect over cultures
that received no cytokine. IL-21-exposed and antigen-primed T cells
retained the capacity to respond to growth-promoting cytokines,
such as IL-2 and IL-7, and could be readily isolated and expanded.
The present invention provides IL-21 enhanced generation of human
antigen-specific CD8+ T cells characterized by CD28 upregulation
and expression of high affinity TCR resulting in antigen-driven
helper-independent IL-2 production, increased target avidity, and
augmented antigen-specific tumor killing. The present invention
provides methods of using IL-21 for induction of a human
antigen-specific CD8+ T cell responses and immunotherapy,
particularly adoptive cell therapy.
[0050] In the studies described herein, the IL-21 augmented
antigen-specific response was limited to the naive and not memory T
cell population using pre-selected responder T cells. Naive T cells
have not previously seen antigen and have the potential to
recognize and bind a single, unique antigen. A tumor antigen is a
peptide or polypeptide or peptide complex that has a different
expression profile from antigen found on a non-tumor cells. For
example, a non-tumor antigen may be expressed in higher frequency
or density by tumor cells than by non-tumor cells. A tumor antigen
may differ from a non-tumor antigen structurally, for example, the
antigen could be expressed as a truncated polypeptide, have some
mutation in the amino acid sequence or polynucleotide sequence
encoding the antigen, be misfolded, or improperly modified
post-translationally. Similarity to antigens that are present on
normal, non-tumor cells in the host organism allow the tumor cells
to escape the host's immunological surveillance mechanisms.
[0051] Observation that tumor-associated antigens generated
specific immunological responses which attacked tumors provided
researchers a basis to develop tumor specific antigen cancer
therapies. However, tumors express a multitude of antigens, many of
which have not been isolated or characterized. Moreover, not all
tumor antigens are expressed at levels high enough to stimulate a
sufficient immune response.
[0052] In recent years, many genes encoding tumor antigens that can
be recognized by cytotoxic T lymphocytes have been identified from
cDNA of human tumor cells (Gomi et al., J. Immunol. 163:4994-5004,
1999.) Examples include the genes HER/neu (Peoples et al., Proc.
Natl Acad. Sci. USA, 92:432-436, 1995) and mutant CASP-8
(Mandruzzato et al., J. Exp. Med., 186:785-793, 1997). Tumor
antigen-specific T cells can be isolated from patients, however
maintaining these T cell cultures has been difficult. Tumor
antigen-specific T cells can be localized in blood, lymphoid tissue
such as the spleen, or can be from the tumor itself. Generally,
tumor tissue is biopsied and a cell suspension is cultured in
vitro. It has been shown that antigen-specific tumor cells in the
presence of cytokine, such as IL-2, IL-7, IL-4, and IL-15 survive
longer (Vella et al., PNAS 95:3810-3815, 1998). In the instant
invention, addition of IL-21 to cultures of tumor antigen-specific
T cells in the presence of primary antigen presentation by
dendritic cells resulted in a significant increase in the absolute
numbers of antigen-specific T cells beyond that seen with IL-15,
IL-6, IL-12, 2 or IL-7 alone. Thus, the present invention provides
methods for identifying new tumor specific antigens and enhancing
tumor antigen-specific T cell populations to target those tumors by
exposing T cells to IL-21. The methods for enhancing the repetoire
of antigens recognized by a T cell population by generating
tumor-specific cell lines arose from having demonstrated that IL-21
compositions enhance proliferation of antigen-specific T cell
populations when antigen is presented to non-terminally
differentiated T cells. The methods comprise co-culturing tumor
material such as a tumor cell line or derivative thereof (e.g.
total RNA, lysed tumor cells, apoptotic bodies) with autologous T
cells isolated from a subject. The tumor material and the T cells
are cultured in the presence of an IL-21 composition, and after
allowing T cells to proliferate, the T cells are enriched, for
example the T cells can be cloned. In some embodiments, the need
for IL-2 or other growth factors is minimized by administration of
IL-21. In other embodiments, it is not necessary to have CD4+ T
cells present in the culture. Antigen-specific T cells are
identified are further analyzed to characterize the
antigen-specificity. (See, van der Bruggen Science 254:1643, 1991
and Engelhard et al., Mol. Immunol 39:127, 2002).
[0053] It is known that a larger number of MART-1 specific T cells
reside among the naive population (Pittet et al, J. Exp. Med.
190:705, 1999) however, measurable frequencies of MART-1-specific T
cells can also be detected among the memory population (D'Souza et
al., Int. J. Cancer 78:699, 2004), and yet these failed to expand
when IL-21 was added. In the case of patients with melanoma, prior
encounter with antigen-bearing tumor cells may lead to defective
signalling among memory T cells rendering them unresponsive to
IL-21 mediated proliferation in vitro (Zippelius, et al, Cancer
Res. 64:2865, 2004; Lee et al., Nature Medicine 5:677, 1999).
[0054] Antigen-primed T cells undergo increased proliferation and
decreased apoptosis when exposed to IL-21 compared to their
untreated counterparts, hence providing methods for enhancing T
cell-mediated vaccines and providing an adjuvant for
immunotherapeutic cancer treatments. IL-21 treatment led to
upregulated CD28 expression and enriched for a population of T
cells expressing a stable unique phenotype, CD45RO+, CD28hi , CCR7-
CD8+. This phenotype may be characterized as intermediate between a
naive (CD45RO-, CD28+, CCR7+) and memory (CD45RO+, CD28-, CCR7-/+)
T cell (Tomiyama, et al., J. Exp. Med. 198:947, 2003). CD28+CD8+ T
cells represent potentially more effective CTL for adoptive
immunotherapy since they can provide an antigen-driven autocrine
signal for proliferation. Such helper-independent CD8 T cells would
not require exogenous help in the form of IL-2 or CD4+ T cells to
survive and expand (Ho et al., Cancer Cell 3:431. 2003; and Topp et
al., J. Exp. Med. 198:947, 2003). Thus, the present invention
provides methods for treating an immune-mediated disease by
providing a subject with a CD8+ T cell population that has enhanced
cytotoxic activity in the absence or reduced presence of additional
cytokines, such as IL-2, or CD4+ T cells. The methods are
particularly useful for ex vivo expansion of cytolytic,
antigen-specific CD8+ T cells, but may also be used in vivo when
additional cytokines result in unwanted side effects or CD4+ cell
populations are compromised.
[0055] The examples disclosed herein demonstrate that exposure to
IL-21 during primary in vitro stimulation also led to the
generation of antigen-specific T cell clones of uniformly higher
affinity and target cell avidity. These clones were represented by
diverse TCR Vbetas suggesting that this was not likely the result
of an expanded population of a few high affinity clones, but a more
global effect on the T cell repertoire. Previous studies have shown
an increased probability of isolating higher affinity T cell clones
when cytokines such as IL-10, that downregulate the stimulatory
capacity of APCs, are used in culture (Tsai et al., Critical Rev.
Immunol 18:65, 1998). IL-21 (Brandt et al., Blood 102:4090, 2003)
has been shown to lead to maturational arrest among murine DC
resulting in reduced MHC expression and decreased stimulatory
capacity for T cell activation. However, in that case, IL-21 was
added to human DCs that had already undergone full maturation. In
preliminary studies, the addition of IL-21 to mature DC did not
affect surface expression of MHC-Class I, HLA-DR, CD80, CD83 or
CD86 compared to untreated DC. While not intending to be bound by
theory, the results suggest dampened expression of surface
stimulatory molecules is not likely an explanation for the enhanced
generation of high affinity T cells in vitro. Pre-incubation of
mature human DC with IL-21 also had no effect on the frequency or
affinity of CD8+ tetramer+T cells that could be generated.
[0056] The use of IL-21 in augmenting an antigen-specific CD8 T
cell response has been explored in mouse models and found to be
highly effective in eradicating aggressive tumors (Ma et al., J.
Immunol. 171:608, 2002; Kishida et al., Mol. Ther. 8:552, 2003;
Moroz et al., J. Immunol 173:900, 2003). The selective effect of
IL-21 on naive vs memory T cells suggests a greater influence
during priming, and in fact, murine studies demonstrate a strong
priming effect characterized by a slow rejection response and
induction of prolonged antitumor memory. IL-21 promotes longterm
survival of previously activated antigen-specific CD8 T cells in
vivo as a result of reduced apoptosis through an indeterminate
mechanism possibly involving STAT3 phsophorylation or induction of
a central memory phenotype (Brenne et al., Blood 99:3756, 2002).
Some of these effects may be attributable to CD28 upregulation
among IL-21-treated CD8 T cells.
[0057] Methods of using T cell populations for adoptive cell
therapy in treatment of human subjects are known to clinicians
skilled in the art. T cell populations prepared according to the
methods described herein and known in the art can be used in such
methods. For example, adoptive cell therapy using
tumor-infiltrating lymphocytes, with MART-1 antigen specific T
cells have been tested in the clinic (Powell et al., Blood
105:241-250, 2005). Patients with renal cell carcinoma have been
vaccinated with irradiated autologous tumor cells. Harvested cells
were secondarily activated with anti-CD3 monoclonal antibody and
IL-2, then readministered to the patients (Chang et al., J.
Clinical Oncology 21:884-890, 2003.)
[0058] The present invention provides methods for enhancing
adoptive immunotherapy by providing a patient with a level of
enhanced immunity by stimulating cells ex vivo, and then
readministering them to the patient. The cells are histocompatible
with the subject, and may be allogenic or autologous. The method of
preparing comprises isolating peripheral blood mononuclear cells
(PBMCs) from a patient, expanding these cells in culture to very
high numbers in a culture media comprising an IL-21 composition,
and then to reintroducing these cells back into patients. The
growth of these effector cells, which include NK cells, LAK cells,
and tumor-specific T-cells, may require additional cytokines such
as IL-2 (Dudley et al., J. Immunother. 24:363-73, 2001) or IL-15
(Marks-Konczalik et al., Proc Natl Acad Sci USA, 97:11445-50 2000;
Waldmann T A. Nat Med., 9:269-77, 2003; Fehniger et al., Cytokine
Growth Factor Rev., 13:169-83, 2002.) Following the transfer of
cells back into patients, methods are employed to maintain their
viability by treating patients with cytokines that could include
IL-21 and IL-2 (Bear et al., Cancer Immunol Immunother. 50:269-74,
2001; and Schultze et al., Br. J. Haematol. 113:455-60, 2001). In
another embodiment, once PBMCs are isolated, the cells can be
further isolated to provide a more homogeneous culture of CD8+
cells, and these cells are cultured in the presence of an IL-21
composition and then readministered to the patient. Because IL-21
can increase T cell frequency to levels that are high enough for
expansion and adoptive transfer without further antigen-specific T
cell enrichment, the present invention provides methods that can
greatly decrease the time to therapy and obviate the requirement
for further selection and/or cloning.
[0059] The present invention provides a method of preparing a T
cell population for use in adoptive immunotherapy comprising
identifying PBMCs having a histocompatible phenotype to a tumor
patient; co-culturing tumor material from the patient with the
peripheral blood mononuclear cells (PBMCs) in the presence of an
IL-21 composition and antigen presenting cells (APCs), such as
autologous dendritic cells, monocytes, B cells, EBV-transformed B
cell lines, allogeneic EBV transformed B cell lines expressing the
shared restricting allele, artificial antigen presenting cells (Yee
et al., Proc Natl Acad Sci. 99(25):16168, 2002; Oelke et al., Nat
Med. 9(5):619-24, 2003; Maus et al., Clin Immunol. 106(1):16-22,
2003; Cai et al., Immunol Rev. 165:249-65, 1998); expanding these
cells in culture; and reintroducing these cells back into the
patient. In one embodiment, the PBMCs are autologous. In another
embodiment, the tumor material comprises peptide, total RNA, lysed
tumor cells or apoptotic bodies.
[0060] The present invention also provides a method of preparing a
T cell population for use in adoptive immunotherapy comprising
identifying a T cell population having a histocompatible phenotype
to a tumor patient; co-culturing tumor material from the patient
with the T cell population in the presence of an IL-21 composition
and APCs; expanding these cells in culture; and and reintroducing
these cells back into the patient. In one embodiment, the T cell
population is autologous (Dudley et al., Science 290:850,
2002).
[0061] The present invention provides a method of preparing a T
cell population for use in adoptive immunotherapy comprising T
cells, bone marrow cells or PBMCs (including NK cells) engineered
(by viral transduction, transfection, electroporation or other
methods of introducing genetic material) to express a T cell
receptor or a chimeric T cell receptor fused with signaling
molecules, that recognize the target antigen; culturing in the
presence of an IL-21 composition; expanding these cells in culture;
and and reintroducing these cells back into the patient. (Hughes et
al., Hum Gene Ther 16(4):457, 2005; Roszkowski et al., Cancer Res
65(4):1570, 2005; Cooper et al., Blood 101:1637, 2003; Alajez et
al., Blood 105:4583, 2005). In one embodiment, the T cell
population is autologous.
[0062] The present invention also provides methods for improving
cancer vaccine therapy. Many tumors express foreign antigens that
can potentially serve as targets for destruction by the immune
system (Boon, T., Adv. Cancer Res. 58:177-211, 1992). Cancer
vaccines generate a systemic tumor-specific immune response in a
subject that comprises both humoral and cellular components. The
response is elicited from the subject's own immune system by
administering a vaccine composition at a site distant from the
tumor or at the site of a localized tumor. The antibodies or immune
cells bind the tumor antigen and lyse the tumor cells. However,
there remains a need for increased proliferation of T cell
populations capable of produced enhanced immune responses in
vivo.
[0063] Numerous methods for immunizing patients with cancer
antigens have been employed, and a variety of techniques are being
used to amplify the strength of the immune response following
antigen delivery (reviewed in Rosenberg, S A. (Ed.), Principles and
practice of the Biologic Therapy of Cancer., 3rd edition,
Lippincott Williams & Wilkins, Philadelphia, Pa., 2000).
Methods in which IL-21 can be used in combination with a tumor
vaccine include, but are not limited to, the delivery of autologous
and allogeneic tumor cells that either express the IL-21 gene or in
which IL-21 is delivered in the context of a adjuvant protein.
Similarly, IL-21 can be delivered in combination with injection of
purified tumor antigen protein, tumor antigen expressed from
injected DNA, or tumor antigen peptides that are presented to
effector cells using dendritic cell-based therapies. Examples of
these types of therapies include the use of cytokines like IL-2 in
the context of vaccination with modified tumor cells (Antonia et
al., J. Urol. 167:1995-2000, 2002; and Schrayer et al., Clin. Exp.
Metastasis 19:43-53, 2002), DNA (Niethammer et al., Cancer Res.
61:6178-84, 2001), and dendritic cells (Shimizu et al., Proc. Nat.
Acad. Sci USA 96:2268-73, 1999). IL-21 can be used as an
anti-cancer vaccine adjuvant.
[0064] The determination of vaccine efficacy is difficult to
evaluate. The ultimate demonstration of efficacy is the rate of
tumor regression, duration of disease-free survival, or, at least,
time-to progression (TTP), these end-points require following
patient progress for years. To provide a more immediate means for
evaluating efficacy there is an ongoing search for so called
"surrogate markers" that would permit early measurements and be
predictive of clinical outcome. As of today, in vitro measurements
of tumor- and/or vaccine-specific immune responses have not proven
successful as surrogate markers (see, Srivastava P., Nat Immunol
1:363-366, 2000).
[0065] For any cancer therapy each protocol may define tumor
response accessments differently, but exemplary guidelines can be
found in Clinical Research Associates Manual, Southwest Oncology
Group, CRAB, Seattle, Wash., Oct. 6, 1998, updated August 1999.
According to the CRA Manual (see, chapter 7 "Response Accessment"),
tumor response means a reduction or elimination of all measurable
lesions or metastases. Disease is generally considered measurable
if it comprises bidimensionally measurable lesions with clearly
defined margins by medical photograph or X-ray, computerized axial
tomography (CT), magnetic resonance imaging (MRI), or palpation.
Evaluable disease means the disease comprises unidimensionally
measurable lesions, masses with margins not clearly defined, lesion
with both diameters less than 0.5 cm, lesions on scan with either
diameter smaller than the distance between cuts, palpable lesions
with diameter less than 2 cm, or bone disease. Non-evaluable
disease includes pleural effusions, ascites, and disease documented
by indirect evidence. Previously radiated lesions which have not
progressed are also generally considered non-evaluable.
[0066] Positive therapeutic outcome can be measured using objective
status protocols to assess solid tumor response. Representative
criteria include the following: (1) Complete Response (CR) defined
as complete disappearance of all measurable and evaluable disease
with no new lesions, and no disease related symptoms. No evidence
of non-evaluable disease; (2) Partial Response (PR) defined as
greater than or equal to 30% decrease from baseline in the sum of
products of perpendicular diameters of all measurable lesions, with
no progression of evaluable disease, no new lesions. According the
RESIST criteria, patients with at least one measurable lesion; (3)
Progression defined as 20% or an increase of 10 cm.sup.2 in the sum
of products of measurable lesions over the smallest sum observed
using same techniques as baseline, or clear worsening of any
evaluable disease, or reappearance of any lesion which had
disappeared, or appearance of any new lesion, or failure to return
for evaluation due to death or deteriorating condition (unless
unrelated to this cancer); (4) Stable or No Response defined as not
qualifying for CR, PR, or Progression. (See, Clinical Research
Associates Manual, supra.)
[0067] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
A. Cell Lines and Reagents
[0068] Melanoma cell lines A375 (CRL 1619; American Type Culture
Collection (ATCC), Manassas, Va.), and Mel 526 (Arrighi et al.,
Cancer Res. 60(16):4446-52, 2000; Marcinola et al. J Immunother
Emphasis Tumor Immunol. 19(3):192-205, 1996.) were maintained in
RPMI with HEPES (25 mM), L-glutamine (4 mM), penicillin (50 U/ml),
streptomycin (50 mg/ml), sodium pyruvate (10 mM), non-essential
amino acids (1 mM), and 10% fetal bovine serum (Hyclone, Utah).
Both lines express the HLA-A2 allele, but only Mel 526 expresses
the MART-1 antigen. The T2 cell line is a TAP-deficient T-B-cell
hybrid expressing the HLA-A2 allele. EBV-LCL cell lines are
Epstein-Ban virus transformed lymphoblastoid cell lines (Yee,
FHCRC, Seattle, Wash.).
B. Induction of Human Antigen-Specific CD8+ T Cells
[0069] Melanoma M26.about.35 peptide specific T cells were
generated (Yee et al., PNAS 99:16168, 2002; Yee et al., J. Immunol
162:2227, 1999; Tsai et al., J. Immunol 158:1796, 1997). Donor
blood was typed by the HLA Typing Lab at the Puget Sound Blood
Center (Seattle, Wash.). CD8+ T cells were first isolated by a CD8
positive isolation kit (Dynabeads, Dynal, Oslo, Norway) from
leukapheresis PBMCs, suspended in CTL medium consisting of RPMI
1640, 25 mM HEPES, 2 mM L-glutamine, penicillin (50 U/ml),
streptomycin (50 mg/ml) (Life Technologies, Gaithersburg, Md.), and
10% human serum from normal donors, and then placed in 6 well
tissue culture dishes (Costar, Corning Incorporated, Coring, N.Y.)
at 6.times.10.sup.6 cell/well. Mature DCs were harvested and pulsed
with 40 .mu.g/ml of synthesised peptides at 2.times.10.sup.6
cell/ml in the presence of 3 .mu.g/ml of .beta.2 microglobulin
(Scripps Lab, San Diego, Calif.) in PBS with 1% human serum albumin
(Life Technologies, Gaithersburg, Md.) for 4 hrs at room
temperature. After washing three times with sterile PBS (Life
Technologies), DCs were mixed with purified CD8 T cells at
3.times.10.sup.5 cells/well in 6 well plate. Cytokines, IL-15 (10
ng/ml, R&D Systems, Minneapolis, Minn.), IL-2 (10 U/ml, Chiron,
Emeryville, Calif.), IL-7 (10 ng/ml, R&D Systems), or IL-21 (30
ng/ml, ZymoGenetics, Seattle, Wash.) were added individually to
each well immediately after the culture initiated. IL-2 (50 IU/ml)
and IL-7 (10 ng/ml) were added one day after 2nd stimulation to
further facilitated expansion of activated antigen-specific T
cells.
[0070] DC were generated (Bender et al., J. Immunol. Methods
196:121, 1996) by exposing adherent PBMC to IL-4 (500 U/ml,
R&D) and GM-CSF (800 U/ml, Amgen, Thousand Oaks, Calif.) in
AIM-V.RTM. medium (Life Technologies) followed by maturation using
IL-1.beta. at 2 ng/ml, IL-6 at 1000 U/ml, TNF-.alpha. at 10 ng/ml
(R&D Systems) and PGE-2 at 1 .mu.g/ml (Sigma-Aldrich, St.
Louis, Mo.) for an additional 2 days. The mature DC population
contained more than 90% CD83+ DCs on day 8 as determined by FACS
analysis.
C. Antibody Plus Peptide-MHC Tetramer Staining of T Cells
[0071] PE or APC labeled M26-MHC-Tetramer and G154-MHC-tetramers
were produced in the immune monitoring lab at Fred Hutchinson
Cancer Center based on previously described protocols (Altman et
al., Science 274:94, 1996). For sample analysis, 0.5.times.10.sup.6
cells in 25 .mu.l of 2% FCS/PBS were first stained with peptide
tetramer-PE or APC (final concentration of 20 .mu.g MHC/ml) for one
hour at room temperature, followed by anti-CD28-APC (BD,
PharMingen, San Diego, Calif.) or anti-CD28-FITC (Caltac Lab,
Burlingame, Calif.), anti-CCR7-PE and anti-CD45RO or
anti-CD45RA-FITC (BD, PharMingen, San Diego, Calif.) staining for
20 min at 4.degree. C. After washing with PBS, cells were
resuspended in PBS containing 2% FBS and DAPI was added. Data were
acquired using a FACScalibur flow cytometer and CellQuest (BD) and
analyzed using FlowJo software (Tree Star, San Carlos, Calif.).
D. Enrichment for Naive and Memory Subsets
[0072] T cells were purified from human peripheral blood
mononuclear cells by the sequential application of a combination of
magnetic beads and an Automacs magnetic sorter (Miltenyi Biotech,
Auburn Calif.). CD8+ cells were isolated using negative selection
with the CD8 isolation kit II. Subsequent naive
(CD8+CD45RO-CD45RA+CD62L+) cell selection involved depletion of
memory CD8 cells using a CD45RO bead, followed by positive
selection of CD62L positive cells by staining with PE-conjugated
CD62L antibody (BD Phamingen, San Diego, Calif.) and incubation
with an antiPE bead. Memory cell isolation (CD8+CD45RA-CD45RO+)
involved depletion of the naive population with a CD45RA+bead.
Typical purities assessed by FACs were in excess of 95%.
E. Cloning and Expansion of Ag-Specific CTL
[0073] The cloning and expansion procedures as described Yee,
supra, 2002; Riddell et al., J. Immunolog. Methods 128:189, 1990,
were used to isolate T cells. Tetramer+ sorted T cells were plated
at limiting dilution in 96-well round-bottomed plates (Nalge Nunc
International, Denmark) in the presence of irradiated feeder cells
(PBL and LCL) at a responder to stimulator ratio of 1:50,000
together with anti-CD3 mAb (OKT3, Ortho Tech, Raritan, N.J.) and 50
U IL-2/ml in 0.2 ml of CTL medium. Wells positive for clonal growth
were identified 10-14 days after plating and screened in a
microcytotoxicity assay. Peptide-specific clones were transferred
to 25-cm2 flasks (Costar, Corning Incorporated, Coring, N.Y.),
restimulated with anti-CD3 mAb, and irradiated allogeneic PBL and
LCL were added as feeder cells for rapid expansion. The cultures
were fed with IL-2 at 50 U/ml 24 hrs after restimulation and then
every 3 days. After 14 days, cells were used for further analyses
or cryopreserved.
F. In Vitro Cytotoxicity Assay
[0074] Target cells (375, 526 melanoma cell lines or T2 cells) were
labeled with 100 .mu.Ci .sup.51Cr and co-cultured with effector
cells for 4 hrs at 37.degree. C. plus 5% CO.sub.2. For peptide dose
titration studies, T2 were pulsed with a peptides at concentrations
ranging from 10.sup.1 to 10.sup.7 pg/ml for 1 hour and then washed
prior to .sup.51Cr labeling. Released .sup.51Cr was measured with a
gamma scintillation counter and percent specific lysis was
determined by using the formula: percent specific
release=Experimental release-Spontaneous release/Total release.
Spontaneous release was <10% of the total release in all
assays.
G. MHC/Peptide Dissociation Assay to Identify High and Low Affinity
CTL Clones
[0075] CTL clones were stained with APC-Tetramer (20 .mu.g/ml) for
1 h at room temperature and washed once with cold PBS to eliminate
unbound tetramer. Cells were incubated in the presence of an excess
(100 .mu.g/ml) of PE-labeled tetramer to prevent rebinding of
APC-Tetramer after their dissociation from TCR. During this period,
aliquots of cells were collected at different time points and fixed
in 1% paraformaldehyde for flow cytometry analysis. The rate of APC
tetramer dissociation is inversely correlated with TCR affinity
(Dutoit et al., J. Immunol 168:1167, 2002).
Example 2
IL-21 Augments the Frequency of Antigen-Specific CD8+ T Cells
Generated Following Primary In Vitro Stimulation
[0076] A model system for primary in vitro stimulation of
antigen-specific T cells was established by isolating CD8+ T cells
from PBMC of HLA A2+ healthy donors and co-culturing with
autologous mature dendritic cells pulsed with immunogenic epitopes
of the tumor-associated self antigen, MART-1 (M26-35 peptide).
Cultures were grown with no added cytokine or with IL-21 (FIG. 1).
The frequency of MART-1-specific CD8 T cell responses in cultures
was evaluated 7 days after stimulation by tetramer staining In
representative healthy donors (CG, NE and LD), a 16 to 20-fold
increase in MART-1 specific CD8+ T cell frequency was observed in
IL-21 exposed cultures compared with no cytokine control cultures
(0.12 vs 2.26% ; 0.12 vs 1.95 and 0.11 vs 2.2% respectively)
following one cycle of in vitro stimulation (FIG. 1). The absolute
numbers of antigen-specific T cells generated in IL-21-treated
cultures exceeded control cultures by more than 20 to 30-fold
(Table 1).
TABLE-US-00001 TABLE 1 CG NE LD Control 0.22 .times. 10.sup.6 0.30
.times. 10.sup.6 0.43 .times. 10.sup.6 IL-21 7.80 .times. 10.sup.6
8.15 .times. 10.sup.6 14.1 .times. 10.sup.6 Fold increase 35 27
33
[0077] The use of other cytokines belonging to the common
gamma-chain cytokine receptor family, IL-2, IL-7 and IL-15 during
primary in vitro stimulation produced no added effect on the
frequency of MART-1-specific CD8+ T cells compared to no cytokine
control cultures (FIG. 3).
[0078] The addition of IL-2 and IL-7, however, does promote the ex
vivo expansion of previously primed, antigen-experienced T cells as
demonstrated by our group and others (Gervois, et al., Clin Cancer
Res 6:1459-1467, 2000; Liao et al., Mol Ther 9:757-764, 2004). When
added to cultures following a second in vitro stimulation, IL-2 (10
U/ml) and IL-7 (10 ng/ml) produced a further increase in the
magnitude of the MART-1 specific CD8 T cell population among IL-21
treated (11.8%) over untreated cultures (2.43%) (FIG. 2, donor
CG).
[0079] These studies demonstrate that IL-21 has the capacity to
augment tumor-associated antigen-specific CTL responses in patients
with melanoma, a tumor which shares expression of MART-1. In a
representative patient, the frequency of MART-1-specific CTL
generated in IL-21 treated compared with untreated cultures after
two cycles of in vitro stimulation demonstrate a 40-fold increase
when IL-21 was added compared to untreated controls (19.1 vs 0.46%)
(FIG. 2, patient ST).
[0080] To evaluate if the increase in frequency and absolute
numbers of antigen-specific CD8 T cells generated among IL-21
treated cultures was due to enhanced proliferation and/or enhanced
survival, naive CD8 T cells were labelled with CFSE, stimulated in
vitro with MART-1 peptide pulsed autologous DC and at Day 7,
evaluated for fraction of dividing cells (as determined by quantum
decreases in CFSE staining accompanying each cell division) and
apoptosis (Annexin V staining) For CFSE staining, analyses
performed on the tetramer-positive (MART-1-specific) T cell
population demonstrate a substantially greater fraction of
non-dividing cells (rightmost compartment) among untreated cultures
(44%) than IL-21 treated cultures (18%) (FIG. 4). In fact, the
ratio of rapidly dividing (leftmost compartment) to nondividing
antigen-specific T cells is more than 3 fold greater among the
IL-21 treated compared to the untreated cultures (63:18% vs. 36:
44%). That the effect of IL-21 on T cell proliferation is
antigen-specific is demonstrated by the large fraction of
tetramer-negative (non-antigen-specific) T cells remaining in the
non-dividing phase (95.6 and 87.9%).
[0081] Annexin V staining of tetramer-positive T cells on Day 7
reveals a modest decrease in the fraction of apoptotic (Annexin V+)
antigen-specific T cells among IL-21 treated cultures compared to
untreated cultures (10.4 vs 5.4% of tetramer+T cells, respectively,
FIG. 2). Taken together, these results suggest that the increase in
frequency and absolute numbers of antigen-specific CD8 T cells
generated among IL-21 treated cultures was due predominantly to
enhanced antigen-specific cellular proliferation and in minor part
to increased survival or decreased apoptosis.
Example 3
IL-21 Enhances Antigen-Specific T Cell Response Among a
Predominantly Naive CTL Population
[0082] The capacity of IL-21 to enhance the generation of
antigen-specific CD8+ T cells was evaluated separately among naive
and memory T cells. Purified populations of naive (>98% CD45RA+,
CD62L+) CD8+ T cells were compared with memory (100% CD45R0+) CD8+
T cells from both a healthy normal donor (CG) (FIG. 5) and an
individual with metastastic melanoma (ST). Whereas IL-21 exerts
minimal effect on the frequency of MART-1 specific cells generated
from memory CD8+T cells (0.10 to 0.15% and 0.05 to 0.037%), a 12 to
90-fold increase is observed among naive CD8 T cells following
IL-21 exposure (0.94 to 12.5% and 0.08 to 7.08%) providing evidence
that IL-21 influences primarily naive T cells.
Example 4
CTL Generated From IL-21-Treated Cultures Represent a Population of
High Affinity Antigen-Specific T Cells with Enhanced Tumor
Reactivity
[0083] To further characterize the function of antigen-specific T
cell populations generated under the influence of IL-21 at the
clonal level, tetramer+CD8+ T cells from both a healthy donor (CG)
and melanoma patient (ST) were sorted on day 7 and cloned at
limiting dilution into 96-well plates. MART-1 specific clones
identified by microcytotoxicity assays were expanded and tested for
1) the peptide concentration required for 50% maximal lysis
(P.sub.50) of peptide pulsed T2 cells and 2), the ability to lyse
antigen-positive melanoma targets. For evaluating P.sub.50, the
HLA-A2-transfected EBV B cell line, T2, was titrated with peptide
concentrations ranging from 10.sup.7 to 10.sup.2 pM. Results are
presented as the peptide dose requirement (nM) for 50% lysis
(P.sub.50). CTL clones generated from IL-21 treated cultures
required a >one log lower peptide dose requirement than their
untreated counterparts-mean 3 nM (range 0.6-30 nM) vs. mean 80 nM
(range 16-500 nM), respectively (FIG. 6A). A similar effect of
IL-21 was seen for CTL clones generated from melanoma patient (ST)
(FIG. 6B).
[0084] At an effector to target (E:T) ratio of 10:1, T cell clones
isolated following stimulation in the presence of IL-21 displayed
much higher specific lytic activity against the MART-1 positive 526
melanoma cell line (35.about.45%) than those isolated in the
absence of IL-21 (FIGS. 6C and 6D). For each individual clone,
increased tumor reactivity was coincident with decreased peptide
dose requirement suggesting that CTL generated in the presence of
IL-21 exhibited a higher avidity interaction with its cognate
target.
[0085] That the increased tumor avidity is attributable to a higher
affinity TCR and not other accessory factors can be demonstrated
using tetramer-based TCR staining assays. Although the intensity of
tetramer staining can generally be correlated with TCR affinity
(Yee et al., J. Immunol 162:2227, 1999; Crawford et al., Immunity
8:675, 1998), a more precise definition of TCR affinity can be
obtained based on the rate of tetramer dissociation, Kd, from its
specific TCR ligand (Dutoit et al., J. Immunol 168:675, 1990). In
this assay, the Kd of the TCR-peptide-MHC interaction or TCR
affinity is inversely correlated with the fraction of bound
tetramer remaining over time in the presence of an excess of
unlabeled tetramer. CTL clones elicited from IL-21 treated or
untreated cultures were stained with M27 peptide-tetramer-PE and
incubated with excess unlabelled M27-tetramer. The fraction of
tetramer-bound CTL was determined by flow cytometry at specified
timepoints (2 to 60 minutes). TCR/Tetramer-peptide off-rates were
found to be significantly faster for clones isolated from untreated
cultures compared to clones generated in IL-21 treated cultures
(FIG. 7). Taken together, these results demonstrate that IL-21
treatment leads to the generation of T cells clones expressing high
affinity TCR.
[0086] To demonstrate whether IL-21 mediated enrichment for high
affinity T cells was due to oligoclonal expansion of a limited
number of antigen-specific T cells or represented a broader effect
on the T cell repertoire, TCR Vbeta expression among the cohort of
high and low affinity T cell clones using a panel of anti-Vbeta
antibodies was examined. For example, for patient CG, among nine
high affinity T cell clones, seven expressed unique Vbeta chains
(only two shared Vbeta expression) and a similarly diverse TCR
repertoire was observed among the group of low affinity T cell
clones in this patient (among ten different low affinity clones,
only two shared the same Vbeta) suggesting that the effect of IL-21
was not due merely to the expansion of an oligoclonal population of
high affinity T cell clones in vitro (FIG. 3).
Example 5
Culture of Antigen-Specific CD8 T Cells with IL-21 Sustains CD28
Expression, IFN.gamma. and IL-2 Production
[0087] CD28 is an important co-stimulatory molecule for generation
of both CD4 and CD8 T cell responses. Signaling via the CD28
receptor results in increased stability of IL-2 mRNA and increased
IL-2 production in both CD4 and CD8 T cells (Boise et al, Immunity
3:87-98, 1995; Ragheb et al, J. Immunol 163:120-129, 1999). CD28
expression is lost ina subset of human CD8+ T cells after
activation and this subset exhibits reduced proliferation after
anti-CD3 stimulation (Azuma et al, J. Immunol 150:1147-1159, 1993).
CD28-CD8+ T cells are increased in the elderly and in the CD8
memory T cell pool in people with persistent viral infections, EBV
and CMV (Posnett et al, Int. Immunol. 11:229-241, 1999). The loss
of CD28 expression is most pronounced in HIV patients (Appay et al,
Nat. Med. 8:379-385, 2002) and increases this population has been
reported in patients with melanoma. Recently, it has been shown
that restoring CD28 expression in CMV-specific CD8 T cells sustains
IL-2 production and increased survival of antigen-specific CD8 T
cells in vitro (Topp et al, J. Exp. Med. 198:947-955, 2005). This
pathway is therefore recognized as an important pathway for
prolonged CD8 survival and function.
[0088] CTLs recognizing self-antigen MART-1 are present at low
numbers in the peripheral blood of healthy donors and are usually
characterized by a naive phenotype (CD45RA+, CCR7+ and
CD28.sup.int. Differentiation of this rare population in the
presence of IL-21 was examined. Antigen-stimulated, untreated cells
showed a CD45RO+ phenotype accompanied by loss of CCR7 and CD28
expression after a week in culture. In contrast, IL-21 treated
cultures showed sustained levels of CD28 expression, even 4 weeks
after primary stimulation with antigen (FIG. 10). This CD28
up-regulation was observed in both naive healthy donors and
melanoma patients for both MART-1 and gp-100 specific CTLs.
[0089] To evaluate if upregulated CD28 expression led to a
functionally competent signal, antigen-driven IL-2 and IFNy
production was analyzed in these cultures. As shown in FIG. 11,
IL-2 production was significantly elevated in IL-21 treated
CD28.sup.hi cells compared to untreated CD28.sup.lo expressing
cells. Furthermore, IL-2 production was inhibited by addition of
CTLA-4Ig, suggesting that CD28 expression was essential for IL-2
production in these cells.
[0090] These data suggest, that in vitro, IL-21 is capable of
inducing a CD28 expressing memory CD8 T cell population capable of
IL-2 production. This may translate to increased survival and
activation of these CD8 T cells in vitro and in vivo and suggests
an important role for IL-21 treatment as a monotherapy and in
adoptive cell therapy for cancer and viral infections.
Example 6
IL-21 Influences the CD8 T Cell Response to gp100 and NY-ESO-1
Antigens
[0091] To demonstrate that T cells recognize other self antigens,
the influence of IL-21 on CD8+ T cells was evaluated in similar
fashion using two other tumor-associated self antigens, the
melanosomal antigen, gp100 (G154 peptide) and the cancer-testis
antigen, NY-ESO-1 (NY157). See, Li et al., J. Immunol
175:2261-2269, 2005.
[0092] CD8+ T cells were stimulated in vitro with autologous
dendritic cells (DC) pulsed with NY-ESO-1 (NY157) or gp100 (G154)
peptide. IL-21 (30 ng/ml) was added to IL-21-treated cultures. Six
days following primary in vitro stimulation, cultures were analyzed
for antigen-specificity and surface phenotype by tetramer staining
and multiparametric analysis on flow cytometry.
[0093] In FIG. 13 panel A., the NY157- and G154-specific CTL
frequency are shown as % of all CD8+ T cells next to boxed gates.
For example, the fold increase in NY-ESO-1-specific CTL was 9.8
fold greater among IL-21 treated cells over control (5.3%:0.54%).
The fold increase in absolute numbers of NY-ESO-1-specific CTL was
calculated based on numbers of cells in respective cultures and for
NY-ESO-1 was found to be almost 20-fold greater among IL-21-treated
cells.
[0094] In FIG. 13 panel B., gated tetramer-positive cells from
control or IL-21 treated cultures were analyzed for CD28
expression. (All cells were CD45RO+, CCR7-negative). Histogram
analysis for CD28 expression among NY-ESO-1 or gp100-specific CTL
was found to be significantly upregulated among IL-21 treated
cultures compared to control controls. These results were
representative of 6 separate experiments from 3
HLA-A2+individuals.
Example 7
Enhancement of Anti-Tumor Immunity in Melanoma Patients Receiving
IL-21 Treatment After Myeloablative Therapy and Adoptive Cell
Transfer
[0095] Adoptive cell therapy (ACT) is based on the ex vivo
selection of tumor-reactive lymphocytes, and their activation to
autologus tumor-bearing host. Tumor-specific T cells (TILs) are
activated and expanded in vitro in the presence of the patient's
own tumor antigens in the presence of cytokines and then
transferred into the same patient followed by maintenance treatment
with cytokines. In a significant number of patients, this leads to
increased numbers of antigen-specific T cells in the periphery
resulting in anti-tumor effects as seen by objective anti-tumor
responses. IL-21 is used both as an in vitro activator/expander of
antigen-specific T cells and also for maintenance therapy of the T
cells once transferred into the cancer patients.
[0096] All studies using human subjects receive prior approval by
an Institutional Review Board at the hospital conducting clinical
trials. After informed consent, peripheral blood mononuclear cells
(PBMCs) are obtained and antigen-specific cytotoxic T lymphocytes
(CTLs) are generated by using autologous dendritic cells pulsed
with the A2-restricted peptide epitope of MART-1 (M27) or gp100
(G154) or by using a tumor cell lysate derived from a biopsy from
the patients own tumor. T cells are expanded in a GMP approved
reactor with appropriate cytokines (25-50 ng/ml of IL-2 or 10-50
ng/ml IrIL-21). In some cases, after three cycles of stimulation at
weekly intervals, T cells are cloned by and expanded for in vitro
testing. CTL clones demonstrating specific lysis of
antigen-positive tumor targets in a chromium release assay are
selected. Clones are expanded in 14-day cycles by using anti-CD3
antibody (OKT3, Orthoclone; Ortho Biotech, Raritan, N.J.) at 30
ng/ml, irradiated allogeneic PBMCs, at 10.sup.6 cells/ml,
irradiated allogeneic lymphoblastoid cell lines (2.times.10.sup.5
cells/ml), and serial IL-2 (aldesleukin; Chiron) at 25-50 units/ml
every 2-3 days. All clones are characterized as CD3.sup.+,
CD4.sup.-, CD8.sup.+ and expressed the high-affinity IL-2 receptor
(CD25) after antigen stimulation.
[0097] Patients with Stage III-IV metastatic melanoma receive
nonmyeloablative chemoptherapy consisting of 2 days
cyclophosphamide (60 mg/kg) followed by 5 days fludarabine (25
mg/m.sup.2). On the day following the final dose of of fludarabine,
patients receive cell infusion of tumor-reactive lymphocytes
(10.sup.6-10.sup.10 cells/infusion) and cytokine therapy (high dose
IL-2 720,000 IU/kg iv every 8 hours or 10-30 ug/kg rIL-21 in
various treatment regimens). Some patients receive a vaccination
with 1 mg MART-1:26-35 (27L) or gp100:209-217 (210M) peptide in
incomplete Freund's adjuvant (IFA) injected subcutaneously.
Patients hematologic parameters are monitored daily. Positive
therapeutic outcome can be measured using objective status
protocols to assess solid tumor response. Patient response is
assessed using standard radiographic studies and physical
examination. See, Clinical Research Associates Manual, Southwest
Oncology Group, CRAB, Seattle, Wash., Oct. 6, 1998, updated August
1999.
[0098] Presence of objective CR and/or PR responses in this trial
suggest a potent role for IL-21 in anti-tumor responses in the
context of adoptive cell therapy either by culturing cells in vitro
with IL-21 or by maintaining patients with IL-21 after ACT.
Example 8
In Vitro Culture with IL-21 Enhances Anti-Tumor Effects in a Mouse
Model of ACT
[0099] Pmel-1 transgenic mice are mice engineered to express a T
cell receptor (TCR) specific for the human melanoma-specific
peptide antigen gp100.sub.25-33 (Overwijk et al., J. Exp. Med.
198:569-580, 2003). Splenocytes from pmel-1 transgenic mice are
isolated and cultured in the presence of 1 uM human gp100.sub.25-33
peptide and culture media containing 30 IU/ml of recombinant human
IL-2 or 10-100 ng/ml murine IL-21 for 6-7 days.
[0100] Female C57B1/6 mice (6-12 weeks old, Charles River
Laboratories) are injected s.c. with 2-5.times.10.sup.5 B16-F10
melanoma cells and treated 10-14 days later with i.v. adoptive
transfer of in vitro cultured pmel-1 splenocytes (as specified
above). Lymphopenia is induced by sublethal total body irradiation
(5Gy) of tumor bearing mice on the day of transfer. Mice are then
either vaccinated with 2.times.10.sup.5 pfu recombinant fowlpox
virus expressing human gp100 followed by treatment with cytokines
(rhIL-2, 100 ug/dose for 6-15 doses) or treated with cytokines
alone (without vaccination). Tumors are measured using calipers and
volume measured using the formula tumor volume=1/2
(B.sup.2.times.L) where B is the shortest diameter of the tumor and
L is the longest diameter of the tumor.
[0101] Reduction in tumor growth when transferring IL-21 cultured
pmel cells provide evidence of a potent role for IL-21 in in vitro
activation and expansion of antigen-specific T cells for ACT.
Example 9
IL-21 Treatment In Vivo Enhances Anti-Tumor Effects in a Mouse
Model of ACT
[0102] Pmel-1 transgenic mice are mice engineered to express a T
cell receptor (TCR) specific for the human melanoma-specific
peptide antigen gp100.sub.25-33 (Klebanoff et al., PNAS
101:1969-1974, 2004) Splenocytes from pmel-1 transgenic mice are
isolated and cultured in the presence of 1 uM human gp100.sub.25-33
peptide and culture media containing 30 IU/ml of recombinant human
IL-2 or 10-100 ng/ml murine IL-21 for 6-7 days.
[0103] Female C57B1/6 mice (6-12 weeks old, Charles River
Laboratories) are injected s.c. with 2-5.times.10.sup.5 B16-F10
melanoma cells and treated 10-14 days later with i.v. adoptive
transfer of in vitro cultured pmel-1 splenocytes (as specified
above). Lymphopenia is induced by sublethal total body irradiation
(5 Gy) of tumor bearing mice on the day of transfer. Mice are then
either vaccinated with 2.times.10.sup.5 pfu recombinant fowlpox
virus expressing human gp100 followed by treatment with cytokines
(rhIL-2, 100 ug/dose or mIL-21, 20-100 ug/dose for 6-15 doses) or
treated with cytokines alone (without vaccination). Tumors are
measured using calipers and volume measured using the formula tumor
volume=1/2 (B.sup.2.times.L) where B is the shortest diameter of
the tumor and L is the longest diameter of the tumor.
[0104] Reduction in tumor growth after IL-21 treatment in vivo
demonstrates a role for IL-21 in maintenance and activation of
tumor-specific T cells after ACT.
[0105] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 1
1
21642DNAHomo sapiensCDS(47)...(532) 1gctgaagtga aaacgagacc
aaggtctagc tctactgttg gtactt atg aga tcc 55 Met Arg Ser 1agt cct
ggc aac atg gag agg att gtc atc tgt ctg atg gtc atc ttc 103Ser Pro
Gly Asn Met Glu Arg Ile Val Ile Cys Leu Met Val Ile Phe 5 10 15ttg
ggg aca ctg gtc cac aaa tca agc tcc caa ggt caa gat cgc cac 151Leu
Gly Thr Leu Val His Lys Ser Ser Ser Gln Gly Gln Asp Arg His20 25 30
35atg att aga atg cgt caa ctt ata gat att gtt gat cag ctg aaa aat
199Met Ile Arg Met Arg Gln Leu Ile Asp Ile Val Asp Gln Leu Lys Asn
40 45 50tat gtg aat gac ttg gtc cct gaa ttt ctg cca gct cca gaa gat
gta 247Tyr Val Asn Asp Leu Val Pro Glu Phe Leu Pro Ala Pro Glu Asp
Val 55 60 65gag aca aac tgt gag tgg tca gct ttt tcc tgt ttt cag aag
gcc caa 295Glu Thr Asn Cys Glu Trp Ser Ala Phe Ser Cys Phe Gln Lys
Ala Gln 70 75 80cta aag tca gca aat aca gga aac aat gaa agg ata atc
aat gta tca 343Leu Lys Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile
Asn Val Ser 85 90 95att aaa aag ctg aag agg aaa cca cct tcc aca aat
gca ggg aga aga 391Ile Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn
Ala Gly Arg Arg100 105 110 115cag aaa cac aga cta aca tgc cct tca
tgt gat tct tat gag aaa aaa 439Gln Lys His Arg Leu Thr Cys Pro Ser
Cys Asp Ser Tyr Glu Lys Lys 120 125 130cca ccc aaa gaa ttc cta gaa
aga ttc aaa tca ctt ctc caa aag atg 487Pro Pro Lys Glu Phe Leu Glu
Arg Phe Lys Ser Leu Leu Gln Lys Met 135 140 145att cat cag cat ctg
tcc tct aga aca cac gga agt gaa gat tcc 532Ile His Gln His Leu Ser
Ser Arg Thr His Gly Ser Glu Asp Ser 150 155 160tgaggatcta
acttgcagtt ggacactatg ttacatactc taatatagta gtgaaagtca
592tttctttgta ttccaagtgg aggagcccta ttaaattata taaagaaata
6422162PRTHomo sapiens 2Met Arg Ser Ser Pro Gly Asn Met Glu Arg Ile
Val Ile Cys Leu Met1 5 10 15Val Ile Phe Leu Gly Thr Leu Val His Lys
Ser Ser Ser Gln Gly Gln 20 25 30Asp Arg His Met Ile Arg Met Arg Gln
Leu Ile Asp Ile Val Asp Gln 35 40 45Leu Lys Asn Tyr Val Asn Asp Leu
Val Pro Glu Phe Leu Pro Ala Pro 50 55 60Glu Asp Val Glu Thr Asn Cys
Glu Trp Ser Ala Phe Ser Cys Phe Gln65 70 75 80Lys Ala Gln Leu Lys
Ser Ala Asn Thr Gly Asn Asn Glu Arg Ile Ile 85 90 95Asn Val Ser Ile
Lys Lys Leu Lys Arg Lys Pro Pro Ser Thr Asn Ala 100 105 110Gly Arg
Arg Gln Lys His Arg Leu Thr Cys Pro Ser Cys Asp Ser Tyr 115 120
125Glu Lys Lys Pro Pro Lys Glu Phe Leu Glu Arg Phe Lys Ser Leu Leu
130 135 140Gln Lys Met Ile His Gln His Leu Ser Ser Arg Thr His Gly
Ser Glu145 150 155 160Asp Ser
* * * * *