U.S. patent application number 14/268147 was filed with the patent office on 2014-08-21 for methods of obtaining antigen-specific t cell populations.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Department of Health and Human Serv. The applicant listed for this patent is The United States of America, as represented by the Secretary, Department of Health and Human Serv, The United States of America, as represented by the Secretary, Department of Health and Human Serv. Invention is credited to Udai S. Kammula.
Application Number | 20140234353 14/268147 |
Document ID | / |
Family ID | 40637782 |
Filed Date | 2014-08-21 |
United States Patent
Application |
20140234353 |
Kind Code |
A1 |
Kammula; Udai S. |
August 21, 2014 |
METHODS OF OBTAINING ANTIGEN-SPECIFIC T CELL POPULATIONS
Abstract
The invention provides a method of obtaining a population of
antigen-specific T cells from peripheral blood of a host. An
embodiment of the method of the invention comprises (i) dividing
PBMCs from peripheral blood of a host into more than one
sub-population; (ii) contacting the PBMCs with an antigen and IL-2;
(iii) obtaining a sample of PBMCs from each sub-population; (iv)
identifying an antigen-reactive sub-population by determining by
high throughput quantitative PCR the expression of a factor
produced by the PBMCs of each sample; (v) dividing the
antigen-reactive sub-population into microcultures; (vi)
identifying the antigen-reactive microculture; and (vii) expanding
the microculture, thereby obtaining a population of T cells
specific for the antigen. The invention also provides a population
of T cells obtained by the inventive method, a pharmaceutical
composition comprising the same, and a method of treating a disease
in a host using the pharmaceutical composition. Related isolating
and screening methods are further provided.
Inventors: |
Kammula; Udai S.; (Bethesda,
MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Department of Health and Human Serv |
Bethesda |
MD |
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary, Department of Health and Human
Serv
Bethesda
MD
|
Family ID: |
40637782 |
Appl. No.: |
14/268147 |
Filed: |
May 2, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12866919 |
Aug 17, 2010 |
8759014 |
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PCT/US09/33649 |
Feb 10, 2009 |
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14268147 |
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61027623 |
Feb 11, 2008 |
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Current U.S.
Class: |
424/185.1 ;
424/196.11; 435/377; 506/9 |
Current CPC
Class: |
A61K 39/001168 20180801;
A61P 35/00 20180101; A61K 39/001186 20180801; A61K 39/0011
20130101; C12Q 1/6881 20130101; C12N 5/0636 20130101; A61K
39/001192 20180801; G01N 33/56972 20130101; C12N 5/0638 20130101;
A61P 31/12 20180101; A61K 39/001188 20180801; A61K 2035/124
20130101 |
Class at
Publication: |
424/185.1 ;
435/377; 424/196.11; 506/9 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C12Q 1/68 20060101 C12Q001/68; C12N 5/0783 20060101
C12N005/0783 |
Claims
1. A clinical grade population of antigen-specific T cells obtained
by a method comprising: (i) dividing peripheral blood mononuclear
cells (PBMCs) from peripheral blood of a host into more than one
sub-population; (ii) contacting the PBMCs of each sub-population
with an antigen and Interleukin-2 (IL-2); (iii) obtaining a sample
of the contacted PBMCs from each sub-population; (iv) identifying
an antigen-reactive sub-population by determining by high
throughput quantitative PCR (HT-qPCR) the expression of a factor
produced by the PBMCs of each sample; (v) dividing the
antigen-reactive sub-population into microcultures; (vi)
identifying an antigen-reactive microculture; and (vii) expanding
the microculture, thereby obtaining a clinical grade population of
T cells specific for the antigen.
2. The population of claim 1, wherein the method is carried out in
less than about 7 weeks.
3. The population of claim 2, wherein the method is carried out in
about 5 to about 6 weeks.
4. The population of any of claim 1, wherein (i) to (iv) is carried
out within about 1 week.
5. The population of any of claim 1, wherein (i) to (vi) is carried
out in about 30 days or less.
6. The population of claim 1, wherein the number of PBMCs of the
antigen-reactive sub-population identified in (iv) is less than
about 10% of the number of PBMCs of (i).
7. The population of claim 6, wherein the number of PBMCs of the
antigen-reactive sub-population identified in (iv) is less than
about 1% of the number of PBMCs of (i).
8. The population of claim 1, wherein the PBMCs are divided into
about 96 sub-populations.
9. The population of claim 1, wherein about 3.times.10.sup.5 PBMCs
are contacted in (ii).
10. The population of claim 9, wherein each sample of (iii)
comprises about 1.times.10.sup.5 PBMCs.
11. The population of claim 1, comprising contacting each sample of
(iii) with an antigenic peptide presented by a carrier cell prior
to (iv).
12. The method population of claim 1, wherein the factor is
Interferon-.gamma. (IFN-.gamma.).
13. The population of claim 1, wherein the PMBCs are contacted in
(ii) with a viral antigen or a cancer antigen.
14. The population of claim 13, wherein the cancer antigen is
selected from the group consisting of gp100, NY-ESO-1, MART-1,
MAGE-A1, and mesothelin.
15. The population of claim 14, wherein the epitope is
gp100.sub.154-162 (SEQ ID NO: 2), NY-ESO-1.sub.157-165 (SEQ ID NO:
6), MAGE-A1.sub.278-286 (SEQ ID NO: 10), mesothelin.sub.18-26 (SEQ
ID NO: 11), or mesothelin.sub.21-29 (SEQ ID NO: 12).
16. The population of claim 13, wherein the antigen is an influenza
viral antigen.
17. (canceled)
18. The population of claim 1, wherein the population of
antigen-specific T cells is greater than about 90% clonal.
19. The population of claim 18, wherein the population of
antigen-specific T cells is about 99% clonal.
20. The population of claim 1, wherein the antigen-specific T cells
have high functional avidity for the antigen, recognize tumor cells
expressing the antigen, and/or are CD27.sup.+.
21. The population of claim 20, wherein the antigen-specific T
cells recognize target cells pulsed with about 10.sup.-10 to about
10.sup.-11 M antigen.
22. The population of claim 20, wherein at least 80% of the
antigen-specific T cells are CD27.sup.+ T cells.
23. The population of claim 1, wherein the antigen-specific T cells
are CD8.sup.+ T cells or CD4.sup.+ T cells.
24. A pharmaceutical composition comprising the population of claim
1 and a pharmaceutically acceptably carrier.
25. A method of treating a disease in a host, comprising
administering to the host the pharmaceutical composition of claim
24 in an amount effective to treat the disease in the host.
26. The method of claim 25, wherein the antigen-specific T cells of
the population are autologous to the host.
27. The method of claim 25, wherein the disease is a viral disease
or a cancer.
28. The method of claim 27, wherein the cancer is selected from a
group consisting of melanoma, breast cancer, colorectal cancer,
esophageal cancer, gastric cancer, non-small cell lung cancer, a
sarcoma, pancreatic cancer, mesothelioma, and ovarian cancer.
29. A method of isolating antigen-specific T cells from peripheral
blood of a host, comprising: (i) dividing peripheral blood
mononuclear cells (PBMCs) from peripheral blood of a host into more
than one sub-population; (ii) contacting the PBMCs with an antigen
and Interleukin-2 (IL-2); (iii) obtaining a sample of the contacted
PBMCs from each sub-population; (iv) identifying an
antigen-reactive sub-population by determining by high throughput
quantitative PCR (HT-qPCR) the expression of a factor produced by
the PBMCs of each sample; (v) dividing the antigen-reactive
sub-population into microcultures; and (vi) identifying an
antigen-reactive microculture; whereupon T cells specific for the
antigen are isolated from the peripheral blood.
30. A method of screening candidate cancer antigen epitopes,
comprising: (i) dividing PBMCs from peripheral blood of a host into
more than one sub-population; (ii) contacting the PBMCs with one or
more candidate cancer antigen epitopes and IL-2; (iii) obtaining a
sample of the contacted PBMCs from each sub-population; and (iv)
identifying an antigen-reactive sub-population by determining by
high throughput quantitative PCR (HT-qPCR) the expression of a
factor produced by the PBMCs of each sample.
31. The method of claim 30, wherein, when a cancer antigen epitope
is identified, the method further comprises: (v) dividing the
antigen-reactive subpopulation into microcultures; (vi) identifying
the antigen-reactive microculture; and (vii) expanding the
microculture; thereby obtaining a population of T cells specific
for the cancer antigen epitope.
32. The method of claim 31, further comprising assaying the
population for tumor reactivity against a tumor cell line which
expresses the cancer antigen epitope.
33. The method of claim 31, further comprising determining the
cancer antigen of which the cancer antigen epitope is a part,
thereby identifying a cancer antigen.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application claims the benefit of U.S.
Provisional Patent Application No. 61/027,623, filed Feb. 11, 2008,
which is incorporated by reference.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0002] Incorporated by reference in its entirety herein is a
computer-readable nucleotide/amino acid sequence listing submitted
concurrently herewith and identified as follows: One 4,216 Byte
ASCII (Text) file named "704352ST25.TXT," created on Jan. 30,
2009.
BACKGROUND OF THE INVENTION
[0003] Adoptive immunotherapy with autologous tumor infiltrating
lymphocytes (TIL) has been shown to mediate significant tumor
regression in .about.50% of patients with refractory metastatic
melanoma (Dudley et al., Science 298: 850-854 (2002) and Dudley et
al., J. Clin. Oncol. 23: 2346-2357 (2005)). However, the isolation
of TIL requires invasive surgery, which can lead to post-operative
complications and delays in initiating adoptive immunotherapy with
TIL.
[0004] The use of lymphocytes from peripheral blood (i.e.,
peripheral blood lymphocytes (PBL) or peripheral blood mononuclear
cells (PBMCs)) in adoptive immunotherapy, in place of TIL, has been
postulated as having several advantages. For example, procuring
tumor reactive PBLs from a blood draw or leukapheresis avoids the
need for invasive surgery. Also, the broad repertoire of PBL might
allow for the isolation of unique populations of tumor-reactive
lymphocytes that are not commonly found in TIL. Finally, the use of
PBL might allow for the use of a generalized strategy to obtain
tumor-reactive lymphocyte populations from patients, regardless of
the diversity of the histology, thereby, expanding the therapeutic
relevance of this approach.
[0005] A significant obstacle to the use of PBL in adoptive
immunotherapy has been the lack of the availability of efficient in
vitro methods to rapidly isolate and expand tumor reactive T cell
clones from the peripheral repertoire. Many attractive tumor
antigens are derived from normal self proteins, and conventional
views of immunologic tolerance suggest that T cells reactive
against these self antigens are rare in the natural peripheral
repertoire and are predominantly of low functional avidity,
incapable of recognizing tumor cells.
[0006] In view of the foregoing, there is a need for a rapid and
efficient method of obtaining a population of antigen-specific T
lymphocytes, especially rare antigen-specific T lymphocytes, from
the peripheral blood of a host.
BRIEF SUMMARY OF THE INVENTION
[0007] The invention provides a rapid and efficient method of
obtaining a population of antigen-specific T cells, e.g., rare
antigen-specific T cells, from the peripheral blood of a host. The
method allows for high throughput screening of bulk PBMCs, and yet
is highly sensitive. For example, the method can detect low
frequency or rare antigen-specific T cells (e.g., T cells that
exist in the peripheral blood at a frequency of about 1 of about
1.times.10.sup.5 bulk PBMCs or lower).
[0008] An embodiment of the method of the invention comprises (i)
dividing peripheral blood mononuclear cells (PBMCs) from peripheral
blood of a host into more than one sub-population; (ii) contacting
the PBMCs with an antigen and Interleukin-2 (IL-2); (iii) obtaining
a sample of the contacted PBMCs from each sub-population; (iv)
identifying an antigen-reactive sub-population by determining by
high throughput quantitative PCR (HT-qPCR) the expression of a
factor produced by the PBMCs of each sample; (v) dividing the
antigen-reactive sub-population into microcultures; (vi)
identifying an antigen-reactive microculture; and (vii) expanding
the microculture, thereby obtaining a population of T cells
specific for the antigen.
[0009] The invention also provides a population of T cells obtained
by the above inventive method and a pharmaceutical composition
comprising the same. Further provided by the invention is a method
of treating a disease in a host. The method comprises administering
to the host a population of antigen-specific T cells.
[0010] A method of isolating antigen-specific T cells is
furthermore provided by the invention. An embodiment of the method
of the invention comprises (i) dividing PBMCs from peripheral blood
of a host into more than one sub-population; (ii) contacting the
PBMCs with an antigen and IL-2; (iii) obtaining a sample of the
contacted PBMCs from each sub-population; (iv) identifying an
antigen-reactive sub-population by determining by HT-qPCR the
expression of a factor produced by the PBMCs of each sample; (v)
dividing the antigen-reactive sub-population into microcultures;
and (vi) identifying an antigen-reactive microculture. T cells
specific for the antigen are isolated from the peripheral blood
upon the inventive method.
[0011] The invention moreover provides a method of screening
candidate cancer antigen epitopes. An embodiment of the method of
the invention comprises (i) dividing PBMCs from peripheral blood of
a host into more than one sub-population; (ii) contacting the PBMCs
with one or more candidate cancer antigen epitopes and IL-2; (iii)
obtaining a sample of the contacted PBMCs from each sub-population;
and (iv) identifying an antigen-reactive sub-population by
determining by high throughput quantitative PCR (HT-qPCR) the
expression of a factor produced by the PBMCs of each sample.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
[0012] FIG. 1 is a schematic of a method of obtaining a population
of antigen-specific T cells for adoptive immunotherapy in
accordance with an embodiment of the invention.
[0013] FIG. 2A is a graph of the stimulation index (SI;
SI=IFN-.gamma. mRNA (gp100.sub.154-162)/IFN-.gamma. mRNA (HIVpol))
of samples comprising the indicated number of C6E4 gp100.sub.154 T
cell clones spiked into 150,000 PBMC as determined by qPCR.
[0014] FIG. 2B is a graph of the stimulation index (SI;
SI=IFN-.gamma. mRNA (gp100.sub.154-162)/IFN-.gamma. mRNA (HIVpol))
of samples comprising the indicated number of C6E4 gp100.sub.154 T
cell clones spiked into 150,000 PBMC as determined by ELISA.
[0015] FIGS. 3A-3E is a set of graphs of the stimulation index
((SI)=IFN-.gamma. mRNA (peptide x)/IFN-.gamma. mRNA (HIVpol)) of
PBMC from 17 HLA-A2+ melanoma patients which were individually
sensitized for 6 days with either 1 .mu.M of FLU M1 peptide (FIG.
3A), MART.sub.27-35 (FIG. 3B), gp100.sub.209-217 (FIG. 3C),
gp100.sub.154-162 (FIG. 3D) or no peptide (DMSO; FIG. 3E) in the
presence of IL-2 (90 IU/ml) and then assayed for T cell recognition
of the sensitizing peptide versus the HIV.sub.pol peptide pulsed
onto T2 cells as determined by qPCR. (O) represents the SI for each
microwell. Bar is median SI value. Shaded area represents range of
non-specific reactivity (SI=0.5-2.0).
[0016] FIG. 4A represents the SI of multiple samples of PBMCs from
Patient 1 which were stimulated with gp100.sub.209-217 peptide for
6 days as determined by ELISA or qPCR (left most panel). (O)
represents the SI for each microwell. Shaded area represents range
of non-specific reactivity (SI=0.5-2.0). Sub-populations with high
or low SI (as determined by qPCR) were selected for rapid
expansion. After about 8 days of expansion, the cells were assayed
by FACs for percent stained positive for CD8 and gp100.sub.209
tetramer (middle column). Functional reactivity of the expanded
cultures was then assayed by stimulating cells with peptide pulsed
T2 cells followed by ELISA measurement of IFN-.gamma. production 24
hours after stimulation. ELISA data represents the average of
replicate co-culture wells. (*), not detectable.
[0017] FIG. 4B represents the SI of multiple samples of PBMCs from
Patient 3 which were stimulated with gp100.sub.209-217 peptide for
6 days as determined by ELISA or qPCR (left most panel). (O)
represents the SI for each microwell. Shaded area represents range
of non-specific reactivity (SI=0.5-2.0). Sub-populations with high
or low SI (as determined by qPCR) were selected for rapid
expansion. After about 8 days of expansion, the cells were assayed
by FACs for percent stained positive for CD8 and gp100.sub.209
tetramer (middle column). Functional reactivity of the expanded
cultures was then assayed by stimulating cells with peptide pulsed
T2 cells followed by ELISA measurement of IFN-.gamma. production 24
hours after stimulation. ELISA data represents the average of
replicate co-culture wells. (*), not detectable.
DETAILED DESCRIPTION OF THE INVENTION
[0018] The invention provides a method of obtaining a population of
antigen-specific T cells from the peripheral blood of a host. An
embodiment of the method of the invention comprises (i) dividing
peripheral blood mononuclear cells (PBMCs) from peripheral blood of
a host into more than one sub-population; (ii) contacting the PBMCs
with an antigen and Interleukin-2 (IL-2); (iii) obtaining a sample
of the contacted PBMCs from each sub-population; (iv) identifying
an antigen-reactive sub-population by determining by high
throughput quantitative PCR (HT-qPCR) the expression of a factor
produced by the PBMCs of each sample; (v) dividing the
antigen-reactive sub-population into microcultures; (vi)
identifying an antigen-reactive microculture; and (vii) expanding
the microculture, thereby obtaining a population of T cells
specific for antigen.
[0019] Contacting PBMCs from Peripheral Blood
[0020] An embodiment of the method of the invention comprises
contacting PBMCs from the peripheral blood of a host. The PBMCs of
the peripheral blood can be obtained from the host by any suitable
means known in the art. For example, the PBMCs can be obtained from
the host by a blood draw or a leukapheresis.
[0021] The host referred to herein can be any host. Preferably, the
host is a mammal. As used herein, the term "mammal" refers to any
mammal, including, but not limited to, mammals of the order
Rodentia, such as mice and hamsters, and mammals of the order
Logomorpha, such as rabbits. It is preferred that the mammals are
from the order Carnivora, including Felines (cats) and Canines
(dogs). It is more preferred that the mammals are from the order
Artiodactyla, including Bovines (cows) and Swines (pigs) or of the
order Perssodactyla, including Equines (horses). It is most
preferred that the mammals are of the order Primates, Ceboids, or
Simoids (monkeys) or of the order Anthropoids (humans and apes). An
especially preferred mammal is the human.
[0022] The PBMCs of the peripheral blood of the host are contacted
with an antigen and IL-2 in the method of the invention. By
"contact" as used herein refers to providing conditions which
promote the antigen and IL-2 to physically contact the PBMCs.
Depending on the contacting antigen and the PBMCs contacted with
the antigen, one or more PBMCs may be stimulated by the contacting
antigen. By "stimulate" as used herein refers to the elicitation of
the signal transduction pathways characteristic of an immune
response, which signal transduction pathways are initiated by the
binding of the T cell receptor (TCR) with the appropriate
antigen-MHC complex. The term "stimulate" as used herein is
synonymous with "sensitize." Methods of determining whether a T
cell is stimulated by an antigen, e.g., the contacting antigen, are
known in the art and include, for example, cytokine release assays,
e.g., ELISA assays and qPCR assays (such as those described herein
in Examples 2 and 3), cytotoxicity assays, and proliferation
assays, and the like.
[0023] Any antigen can be used to contact the PBMCs. As used
herein, the term "antigen" refers to any molecule that can bind
specifically to an antibody. For example, the antigen can be any
molecule that can be recognized by a T cell in the context of the
MHC molecule by which the T cell is restricted. The antigen can be,
for example, an antigen which is characteristic of a disease. The
disease can be any disease involving an antigen, as discussed
herein, e.g., an infectious disease, an autoimmune disease, or a
cancer. The antigen could be, for example, a viral antigen, a
bacterial antigen, a cancer antigen, etc.
[0024] Preferably, the antigen is a cancer antigen or a viral
antigen. By "cancer antigen" is meant any molecule (e.g., protein,
peptide, lipid, carbohydrate, etc.) solely or predominantly
expressed or over-expressed by a tumor cell or cancer cell, such
that the antigen is associated with the tumor or cancer. The cancer
antigen additionally can be expressed by normal, non-tumor, or
non-cancerous cells. However, in such a situation, the expression
of the cancer antigen by normal, non-tumor, or non-cancerous cells
is not as robust as the expression by tumor or cancer cells. In
this regard, the tumor or cancer cells can over-express the antigen
or express the antigen at a significantly higher level, as compared
to the expression of the antigen by normal, non-tumor, or
non-cancerous cells. Also, the cancer antigen additionally can be
expressed by cells of a different state of development or
maturation. For instance, the cancer antigen can be additionally
expressed by cells of the embryonic or fetal stage, which cells are
not normally found in an adult host. Alternatively, the cancer
antigen additionally can be expressed by stem cells or precursor
cells, which cells are not normally found in an adult host. Another
group of cancer antigens are represented by the differentiation
antigens that are expressed in only a limited set of tissues in the
adult, such as the melanocytes differentiation antigens, whose
expression is limited to normal melanocytes. Although it is not
known why these molecules elicit immune responses, the limited
expression pattern of these proteins may allow these molecules to
be recognized by the immune system.
[0025] The cancer antigen can be an antigen expressed by any cell
of any cancer or tumor, including the cancers and tumors described
herein. The cancer antigen may be a cancer antigen of only one type
of cancer or tumor, such that the cancer antigen is associated with
or characteristic of only one type of cancer or tumor.
Alternatively, the cancer antigen may be a cancer antigen (e.g.,
may be characteristic) of more than one type of cancer or tumor.
For example, the cancer antigen may be expressed by both breast and
prostate cancer cells and not expressed at all by normal,
non-tumor, or non-cancer cells. In a preferred embodiment of the
invention, the cancer antigen is a melanoma cancer antigen or a
breast cancer antigen. In a more preferred embodiment, the cancer
antigen is selected from the group consisting of gp100, MART-1,
NY-ESO-1, a member of the MAGE family of proteins, e.g., MAGE-A1,
mesothelin, Tyrosinase, TRP-1, TRP-2, PMSA, Her-2, and p53. In a
most preferred embodiment, the cancer antigen is selected from the
group consisting of gp100, NY-ESO-1, and MAGE-1.
[0026] Alternatively, the antigen can be a viral antigen. By "viral
antigen" is meant those antigens encoded by a part of a viral
genome which can be detected by a specific immunological response.
Viral antigens include, for example, a viral coat protein, an
influenza viral antigen, an HIV antigen, a Hepatitis B antigen, or
a Hepatitis C antigen.
[0027] With regard to the invention, the antigen can be the whole,
full-length, or intact antigen or an immunogenic portion thereof.
By "immunogenic portion" as used herein is meant any part of the
antigen to which a T cell receptor (TCR) specifically binds, such
that an immune response is elicited as a result of the TCR binding
to the part of the antigen. As used herein, the term "antigen"
encompasses the whole, full-length, or intact antigenic protein and
any immunogenic portion thereof.
[0028] The antigen can be naturally, artificially, synthetically,
or recombinantly produced. In this respect, the antigen can be a
synthetic, recombinant, isolated, and/or purified protein,
polypeptide, or peptide. Methods of making or obtaining such
antigens are known in the art. For example, suitable methods of de
novo synthesizing polypeptides and proteins (e.g., antigenic
polypeptides and proteins) are described in Chan et al., Fmoc Solid
Phase Peptide Synthesis, Oxford University Press, Oxford, United
Kingdom, 2005; Peptide and Protein Drug Analysis, ed. Reid, R.,
Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al.,
Oxford University Press, Oxford, United Kingdom, 2000; and U.S.
Pat. No. 5,449,752. Also, polypeptides and proteins (e.g.,
antigenic polypeptides and proteins) can be recombinantly produced
using nucleic acids which encode the polypeptide or protein using
standard recombinant methods. See, for instance, Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 3.sup.rd ed., Cold Spring
Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al.,
Current Protocols in Molecular Biology, Greene Publishing
Associates and John Wiley & Sons, NY, 1994. The nucleotide
sequences of many antigens are known in the art and are available
from the GenBank database of the National Center for Biotechnology
Information (NCBI) website. Further, the antigen can be isolated
and/or purified from a source, such as a plant, a bacterium, an
insect, a mammal, e.g., a rat, a human, etc. Methods of isolation
and purification are well-known in the art.
[0029] Also, the antigen can be a free antigen, e.g., unbound
antigenic peptide (e.g., a free peptide), or can be a bound
antigen, e.g., an MHC-peptide tetramer or an antigenic peptide
presented by a carrier cell which was pulsed with the peptide. For
example, the antigen can be a peptide portion of the antigen gp100,
e.g., amino acids 154-162 of gp100 (gp100.sub.154-162; SEQ ID NO:
2), or a peptide portion of the antigen NY-ESO-1, e.g., amino acids
157-165 of NY-ESO-1 (NY-ESO-1.sub.157-165; SEQ ID NO: 6). Also, for
example, the antigen can be a carrier cell, e.g., T2 cell, which
was pulsed with the peptide of SEQ ID NO: 2 or 6.
[0030] The PBMCs of the peripheral blood obtained from the host are
additionally contacted with IL-2. The IL-2 can be, for example, a
recombinantly produced IL-2, such as those that are commercially
available from BD Pharmingen, Franklin Lakes, N.J., and BioLegend,
San Diego, Calif. The PBMCs can be contacted with any non-toxic
dose of IL-2, e.g., a dose which is less than 1000 CU/ml.
Preferably, the PBMCs are contacted with an amount of IL-2 ranging
from about 10 CU/ml to about 20 CU/ml. More preferably, the PBMCs
are stimulated with about 10 CU/ml IL-2.
[0031] The PBMCs can be contacted with antigen and IL-2 by any
number of suitable means, which means are well-known to those
skilled in the art. Strictly by way of example, the PBMCs can be
plated into a culture dish containing culture medium comprising the
antigen and IL-2. Alternatively, the antigen and IL-2 can be
simultaneously or sequentially added to culture medium comprising
the PBMCs.
[0032] The culture dish containing the PBMCs during contact with
the antigen and IL-2 can be any tissue culture plate. As the PBMCs
are divided into more than one sub-population before being
contacted, the culture dish preferably is a multi-well plate, such
as, for example, a 6-, 24-, or 96-well U-bottom plate. In a
preferred embodiment, PBMCs from peripheral blood are plated into a
96-well plate comprising culture medium and the antigen and IL-2
are subsequently added to the culture medium comprising the
PBMCs.
[0033] Any number of PBMCs from peripheral blood can be contacted
with the antigen and IL-2. Preferably, a total of about
3.times.10.sup.5 PBMCs are contacted among the 96
sub-populations.
[0034] Obtaining a Sample
[0035] The method of the invention comprises obtaining a sample
(e.g., a fraction) of the contacted PBMCs from each sub-population.
Preferably, a sample from each sub-population is transferred to a
culture dish which is of similar type to the culture dish
comprising the contacted PBMCs. For instance, if the contacted
PBMCs were contacted in a 96-well plate, then the sample of each
sub-population is transferred to a corresponding well of another
96-well plate.
[0036] The amount of PBMCs of the sample can be any amount,
provided that the sample is only a fraction of the contacted
sub-population. Preferably, the sample is about 1/3 of the
sub-population of the contacted PBMCs. Advantageously, each sample
can comprise as little as about 1.times.10.sup.5 PBMCs of the
sub-population.
[0037] Identifying an Antigen-Reactive Sub Population
[0038] The method of the invention comprises identifying an
antigen-reactive sub-population, e.g., a sub-population which
comprises one or more PBMCs that react to the contacting antigen or
are stimulated by the contacting antigen. The antigen-reactive
sub-population is identified by determining the expression of a
factor produced by the PBMCs of each sample. The expression of a
factor produced by the PBMCs of each sample is determined by high
throughput quantitative PCR (HT-qPCR). "High throughput
quantitative PCR" as used herein, refers to any of the high
throughput quantitative PCR methods known in the art, including,
for example, any of those described herein in Example 2, Morrison
et al., Nucleic Acids Research, e-publication on Sep. 25, 2006;
Ryncarz et al., J. Clin. Microbiol. 37: 1941-1947 (1999); and Loeb
et al., Hepatology 32: 626-629 (published on line Dec. 20, 2003).
The HT-qPCR may be carried out on any suitable machine
appropriately equipped for such assaying. The HT-qPCR machine can
be, for example, the ABI Prism.RTM. 7900HT Sequence Detection
System, which is commercially available from Applied Biosystems,
Foster City, Calif.
[0039] The high throughput qPCR can comprise the simultaneous
analysis of multiple samples of sub-populations. Preferably, the
HT-qPCR comprises the simultaneous analysis of at least 20 samples.
More preferably, the HT-qPCR comprises the simultaneous analysis of
at least 40 samples. Most preferably, the HT-qPCR comprises the
simultaneous analysis of at least 75 samples, if not more, e.g.,
90, 96, more than 100.
[0040] The PCR primers used in the HT-qPCR can be any PCR primers
provided that they allow for the amplification of a portion of a
nucleic acid encoding the factor. In a preferred embodiment, each
of the forward and reverse PCR primers comprises the nucleotide
sequence of SEQ ID NOs: 7 and 8, respectively. Also, while the
probe used in the HT-qPCR can comprise any suitable nucleotide
sequence, the probe preferably comprises the nucleotide sequence of
SEQ ID NO: 9.
[0041] The factor for which the level of expression is determined
through HT-qPCR can be any T cell factor which is produced in
response to antigen binding. The factor can be, for example,
Interferon-.gamma. (IFN-.gamma.), Granulocyte Macrophage Colony
Stimulating Factor (GM-CSF), Tumor Necrosis Factor-.alpha.
(TNF-.alpha.), or Interleukin-2 (IL-2). Preferably, the factor is
IFN-.gamma..
[0042] Desirably, immediately before determining the expression of
the factor produced by the PBMCs of each sample, the method further
comprises an additional contacting of each sample of PBMCs with
antigen and optionally IL-2. Methods of contacting PBMCs with
antigen and optionally IL-2 are well-known in the art and include
any of the methods described herein. Preferably, the contacting
antigen is in the form of a peptide antigen presented by a carrier
cell, e.g., T2 cell.
[0043] As HT-qPCR determines the copy numbers of expressed mRNAs of
the factor of the contacted PBMCs, one or more antigen-reactive
sub-populations are identified as those with increased copy numbers
of the expressed mRNAs of the factor as compared to a negative
control, e.g., a sub-population not contacted with an antigen with
or without IL-2, a sub-population contacted with DMSO, a
sub-population contacted with an irrelevant peptide, e.g., a
peptide which is known to go unrecognized by any of the PBMCs.
[0044] One or more antigen-reactive sub-populations as identified
via HT-qPCR are then divided into microcultures for purposes of
limiting dilution cloning. For example, the single sub-population
with the highest copy number of the expressed mRNA of the factor
can be selected for limiting dilution cloning. Also, for example,
the sub-populations exhibiting the top ten highest copy numbers are
selected for limiting dilution cloning. Limiting dilution cloning
procedures are well-known in the art, and include, methods such as
the one described herein in Example 4. Briefly, the number of PBMCs
of an identified sub-population is determined and a calculated
amount of the sub-population is placed into a calculated volume of
medium in a single well of a multi-well tissue culture plate, such
that the calculated cell density of the well is about 1 cell per
well.
[0045] After culturing the microcultures for a sufficient amount of
time, e.g., preferably about 2 weeks, each well containing the
microcultures are inspected for growth. The inspection can be a
visual inspection in which the bottom of the tissue culture plate
containing the micro-cultures are visually inspected (with or
without a microscope) for cell clusters, which are representative
of cell growth.
[0046] Growth positive wells are subsequently assayed for
antigen-reactivity to identify the wells containing
antigen-reactive clones. The antigen-reactivity can be assayed by
any suitable means known in the art, including, for instance, the
qPCR methodology described herein in Example 2, the ELISA assay
described herein in Example 3, or the visual microcytotoxicity
assay described herein in Example 4.
[0047] Identification of the antigen-reactive microculture allows
for the expansion thereof. Any suitable microculture expansion
protocol known in the art can be used. Preferably, the
microcultures are expanded in accordance with the rapid expansion
protocols described herein in Examples 4 and 14.
[0048] Nature of the Antigen-Specific T Cells Obtained by the
Inventive Method
[0049] The method of the invention obtains a population of
antigen-specific T cells, e.g., T cells specific for the contacting
antigen. As used herein, the term "antigen-specific" refers to a T
cell comprising T cell receptors (TCRs) which specifically bind to
and immunologically recognize the contacting antigen, such that
binding of the TCRs to the contacting antigen elicits an immune
response. The TCRs of the antigen-specific T cell, in contrast, do
not bind to a control peptide or irrelevant peptide, which are
different from the contacting antigen, and thereby do not elicit an
immune response.
[0050] In a preferred embodiment of the invention, the
antigen-specific T cells of the population obtained by the method
of the invention are highly avid for the contacting antigen, in
that the TCRs expressed on the surface of the T cells strongly and
specifically bind to the antigen for which the TCRs are specific,
e.g., the contacting antigen. High avidity can be demonstrated by
assaying the minimum amount of antigenic peptide pulsed into target
cells required for the target cells to be recognized and killed by
the T cells. Highly avid T cells can recognize, for example, target
cells pulsed with as little as about 10.sup.-10 to about 10.sup.-11
M antigenic peptide.
[0051] In one embodiment of the invention, the antigen-specific T
cells are specific for a cancer antigen. In this instance, it is
preferable for the antigen-specific T cells to recognize tumor
cells which express the cancer antigen for which the T cells are
specific, e.g., express the contacting antigen. Tumor cell
recognition refers to the ability of the T cells to immunologically
recognize the antigen and cause killing of the tumor cell. Methods
of testing whether T cells recognize tumor cells are well-known in
the art and include, for example, the method set forth herein in
Example 10.
[0052] The antigen-specific T cells of the population obtained by
the inventive method can be of any phenotype. Preferably, the T
cells of the obtained population are CD27.sup.+ (e.g., express the
CD27 protein). Additionally, the T cells can have a phenotype which
is similar to those described in Examples 6 and 12. In one
embodiment of the invention, at least 80% of the antigen-specific T
cells of the population obtained by the inventive method are
CD27.sup.+ T cells.
[0053] The antigen-specific T cells can be any T cells, including,
but not limited to CD8.sup.+ T cells, CD4.sup.+ T cells,
CD8.sup.+/CD4.sup.+ T cells, and the like. As the antigen-specific
T cells are obtained from bulk PBMCs from peripheral blood, it is
understood that the antigen-specific T cells of the population are
not tumor infiltrating lymphocytes (TILs), since TILs are not
considered to be in the peripheral blood.
[0054] Sensitivity
[0055] Advantageously, the method is highly sensitive in that low
frequency or rare antigen-specific T cells are detected. For
example, the inventive method can detect T cells which naturally
exist in the peripheral blood at a frequency of about 1 of about
5.times.104 PBMCs from peripheral blood (bulk PBMCs) or at an even
lower frequency, e.g., about 1 in 10.sup.5 bulk PBMCs. In contrast,
ELISA assays are unable to detect such low frequency T cells.
[0056] As used herein, the term "naturally exist" refers to the
number of T cells which are present in the peripheral blood of an
untreated host, e.g., a host which has not been administered an
agent which affects (increases or decreases) the number of T cells
in the peripheral blood. An untreated host refers to, for example,
a host which has not undergone an adoptive cell transfer procedure
and/or has not received a heteroclitic peptide immunization or
vaccine within, e.g., 2 weeks, 1 month, 2 months, 3 months, 6
months, 1 year, 5 years, or 10 years, such that the number of T
cells in the peripheral blood might increase or decrease. An
untreated host can be, for example, a host who has never undergone
adoptive cell transfer and/or received a heteroclitic peptide
immunization or vaccine. Methods of determining the frequency of a
given antigen-specific T cell are known in the art and include, for
example, the method set forth herein in Example 5.
[0057] While the inventive method can be highly sensitive with
regard to the detection of rare or low frequency T cells, as
exemplified above, the invention is not limited to just this
aspect. Rather, the inventive method can be used to detect and
obtain a population of antigen-specific T cells which naturally
exist in the peripheral blood at a relatively higher frequency
which one of ordinary skill in the art recognizes as having a
potential benefit. For example, the method can be used to detect
and obtain a population of antigen-specific T cells which naturally
exist in the peripheral blood at a frequency which is greater than
about 1 of about 1.times.10.sup.5 PBMCs.
[0058] Rapidity
[0059] Also, the method is advantageously rapid, in that a
population of antigen-specific T cells, e.g., clinical grade
antigen-specific T cells, can be obtained from the peripheral blood
of a host in a relatively short period of time. For example,
embodiments of the inventive method (comprising (i) to (vii)) can
be carried out in less than about 7 weeks, e.g., about 5 to about 6
weeks, such that a population of clinical grade antigen-specific T
cells, e.g., clinical grade antigen-specific T cells, is obtained
from the peripheral blood of a host in this time frame. Also, for
instance, embodiments of the method can be tailored such that (i)
to (iv) is carried out within about 1 week. Alternatively or
additionally, embodiments of the method can be tailored such that
(i) to (vi) is carried out in about 30 days or less.
[0060] While the inventive method can be rapid, as exemplified
above, the invention is not limited to just this aspect. Rather,
the inventive method can occur in a relatively longer period of
time of which one of ordinary skill in the art recognizes as having
a potential benefit. For example, the method can be carried out in
a time frame which is greater than 7 weeks, e.g., 8, 9, 10 or more
weeks.
[0061] Efficiency
[0062] Furthermore, the method is advantageously efficient in that
the method is highly sensitive for low frequency, antigen-specific
T cells and detects and isolates a clinical grade population of low
frequency, antigen-specific T cells in a relatively short period of
time. For instance, the number of PBMCs of the antigen-reactive
sub-population identified in (iv) can be less than about 10% of the
number of the PBMCs of (i) (the starting amount of PBMCs in (i)).
That is to say that (i) to (iv) of the inventive method can
effectively eliminate greater than about 90% of the PBMCs of (i)
(e.g., the starting number of PBMCs). The number of PBMCs of the
antigen-reactive sub-population identified in (iv) also can be, for
example, less than about 1% of the number of the PBMCs of (i),
which is to say that (i) to (iv) of the inventive method can
effectively eliminate greater than about 99% of the PBMCs of
(i).
[0063] The efficiency of the inventive method also can be
exemplified by the degree of homogeneity, e.g., the % clonality, of
the obtained population of antigen-specific T cells. For example,
the method can obtain a population of antigen-specific T cells
which is greater than about 90% clonal, e.g., about 93%, about 95%,
about 98%, about 99%, or about 100% clonal.
[0064] As the inventive method can be efficient, as exemplified
above, the invention is not limited to just this aspect. Rather,
the inventive method can be tailored to detect less rare
antigen-specific T cells, to detect and isolate a population of
antigen-specific T cells in a relatively longer period of time,
and/or to obtain a less clonal population of antigen-specific T
cells of which one of ordinary skill in the art recognizes as
having a potential benefit.
[0065] Population of Antigen-Specific T Cells and Pharmaceutical
Compositions Comprising Same
[0066] The invention provides a population of antigen-specific T
cells which is obtained by the inventive method. By virtue of being
obtained by the inventive method, the population of the
antigen-specific T cells and the antigen-specific T cells are as
described herein. Namely, the population can be oligoclonal or
clonal as described above. Also, at least 80% of the population can
be CD27.sup.+. The T cells can be CD8+ and/or CD4+. The T cells can
be specific to any antigen including any of those described
herein.
[0067] The inventive population of antigen specific T cells is a
clinical grade population of antigen specific T cells. The term
"clinical grade" is synonymous with "good manufacturing practice
grade" and is meant appropriate for human administration per the
guidelines set forth by the Food and Drug Administration (FDA).
See, for example, 21 C.F.R. Section 606.
[0068] Accordingly, the inventive populations of antigen-specific T
cells can be formulated into a composition, such as a
pharmaceutical composition. In this regard, the invention provides
a pharmaceutical composition comprising any of the populations of
antigen-specific T cells described herein and a pharmaceutically
acceptable carrier. The inventive pharmaceutical compositions
containing any of the inventive populations of antigen-specific T
cells can comprise more than one type of population of
antigen-specific T cells, e.g., a population of gp100-specific T
cells along with a population of NY-ESO-1-specific T cells.
Alternatively, the pharmaceutical composition can comprise an
inventive population of T cells in combination with another
pharmaceutically active agent or drug, such as a T cell growth
supporting factor, e.g., IL-2.
[0069] With respect to pharmaceutical compositions, the
pharmaceutically acceptable carrier can be any of those
conventionally used and is limited only by chemico-physical
considerations, such as solubility and lack of reactivity with the
active compound(s), and by the route of administration. The
pharmaceutically acceptable carriers described herein, for example,
vehicles, adjuvants, excipients, and diluents, are well-known to
those skilled in the art and are readily available to the public.
It is preferred that the pharmaceutically acceptable carrier be one
which is chemically inert to the active agent(s) and one which has
no detrimental side effects or toxicity under the conditions of
use.
[0070] The choice of carrier will be determined in part by the
particular inventive populations of antigen-specific T cells, as
well as by the particular method used to administer the inventive
populations of antigen-specific T cells. Accordingly, there are a
variety of suitable formulations of the pharmaceutical composition
of the invention. In a preferred embodiment of the invention, the
pharmaceutical composition is a parenteral formulation or an
intravenous formulation.
[0071] Formulations suitable for parenteral administration include
aqueous and non-aqueous, isotonic sterile injection solutions,
which can contain anti-oxidants, buffers, bacteriostats, and
solutes that render the formulation isotonic with the blood of the
intended recipient, and aqueous and non-aqueous sterile suspensions
that can include suspending agents, solubilizers, thickening
agents, stabilizers, and preservatives. The inventive material can
be administered in a physiologically acceptable diluent in a
pharmaceutical carrier, such as a sterile liquid or mixture of
liquids, including water, saline, aqueous dextrose and related
sugar solutions, an alcohol, such as ethanol or hexadecyl alcohol,
a glycol, such as propylene glycol or polyethylene glycol,
dimethylsulfoxide, glycerol, ketals such as
2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol)
400, oils, fatty acids, fatty acid esters or glycerides, or
acetylated fatty acid glycerides with or without the addition of a
pharmaceutically acceptable surfactant, such as a soap or a
detergent, suspending agent, such as pectin, carbomers,
methylcellulose, hydroxypropylmethylcellulose, or
carboxymethylcellulose, or emulsifying agents and other
pharmaceutical adjuvants.
[0072] Oils, which can be used in parenteral formulations include
petroleum, animal, vegetable, or synthetic oils. Specific examples
of oils include peanut, soybean, sesame, cottonseed, corn, olive,
petrolatum, and mineral. Suitable fatty acids for use in parenteral
formulations include oleic acid, stearic acid, and isostearic acid.
Ethyl oleate and isopropyl myristate are examples of suitable fatty
acid esters.
[0073] The parenteral formulations will typically contain from
about 0.5% to about 25% by weight of the inventive material in
solution. Preservatives and buffers may be used. In order to
minimize or eliminate irritation at the site of injection, such
compositions may contain one or more nonionic surfactants having a
hydrophile-lipophile balance (HLB) of from about 12 to about 17.
The quantity of surfactant in such formulations will typically
range from about 5% to about 15% by weight. Suitable surfactants
include polyethylene glycol sorbitan fatty acid esters, such as
sorbitan monooleate and the high molecular weight adducts of
ethylene oxide with a hydrophobic base, formed by the condensation
of propylene oxide with propylene glycol. The parenteral
formulations can be presented in unit-dose or multi-dose sealed
containers, such as ampoules and vials, and can be stored in a
freeze-dried (lyophilized) condition requiring only the addition of
the sterile liquid excipient, for example, water, for injections,
immediately prior to use. Extemporaneous injection solutions and
suspensions can be prepared from sterile powders, granules, and
tablets of the kind previously described.
[0074] Injectable formulations are in accordance with the
invention. The requirements for effective pharmaceutical carriers
for injectable compositions are well-known to those of ordinary
skill in the art (see, e.g., Pharmaceutics and Pharmacy Practice,
J.B. Lippincott Company, Philadelphia, Pa., Banker and Chalmers,
eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs,
Toissel, 4th ed., pages 622-630 (1986)). Preferably, when
administering cells, e.g., dendritic cells, the cells are
administered via injection.
[0075] It will be appreciated by one of skill in the art that, in
addition to the above-described pharmaceutical compositions, the
populations of the invention can be formulated as inclusion
complexes, such as cyclodextrin inclusion complexes, or
liposomes.
[0076] For purposes of the invention, the amount or dose of the
inventive pharmaceutical composition administered should be
sufficient to effect, e.g., a therapeutic or prophylactic response,
in the subject or animal over a reasonable time frame. For example,
the dose of the inventive pharmaceutical composition should be
sufficient to cause tumor regression, or treat or prevent a disease
(e.g., cancer or viral disease in a period of from about 2 hours or
longer, e.g., 12 to 24 or more hours, from the time of
administration. In certain embodiments, the time period could be
even longer. The dose will be determined by the efficacy of the
particular inventive pharmaceutical composition and the condition
of the animal (e.g., human), as well as the body weight of the
animal (e.g., human) to be treated.
[0077] Many assays for determining an administered dose are known
in the art. For purposes of the invention, an assay, which
comprises comparing the extent to which tumors regress, upon
administration of a given dose of an inventive pharmaceutical
composition to a mammal among a set of mammals of which is each
given a different dose of the inventive pharmaceutical composition,
could be used to determine a starting dose to be administered to a
mammal. The extent to which tumors regress upon administration of a
certain dose can be assayed by methods known in the art, including,
for instance, the methods described in Therasse et al., J. Natl.
Cancer Inst. 92: 205-216 (2000).
[0078] The dose of the inventive pharmaceutical composition also
will be determined by the existence, nature and extent of any
adverse side effects that might accompany the administration of a
particular inventive pharmaceutical composition. Typically, the
attending physician will decide the dosage of the inventive
pharmaceutical composition with which to treat each individual
patient, taking into consideration a variety of factors, such as
age, body weight, general health, diet, sex, inventive material to
be administered, route of administration, and the severity of the
condition being treated. By way of example and not intending to
limit the invention, the dose of the inventive pharmaceutical
composition can be about 1.times.10.sup.9 cells to about
3.times.10.sup.11 T cells.
[0079] One of ordinary skill in the art will readily appreciate
that the inventive pharmaceutical composition of the invention can
be modified in any number of ways, such that the therapeutic or
prophylactic efficacy of the inventive pharmaceutical compositions
is increased through the modification. For instance, the inventive
pharmaceutical compositions can be modified to express T cell
growth supporting molecules, e.g., IL-2. Such methods of modifying
T cells to express IL-2 genes are known in the art.
[0080] Method of Treating a Disease
[0081] The inventive pharmaceutical compositions comprising the
antigen-specific T cell populations can be used in methods of
treating a disease. In this regard, the invention provides a method
of treating a disease in a host. The method comprises administering
to the host any of the pharmaceutical compositions described
herein.
[0082] The disease can be any disease involving an antigen, e.g.,
an infectious disease, an autoimmune disease, or a cancer. For
purposes herein, "infectious disease" means a disease that can be
transmitted from person to person or from organism to organism, and
is caused by a microbial agent (e.g., common cold). Infectious
diseases are known in the art and include, for example, a viral
disease, a bacterial disease, or a parasitic disease, which
diseases are caused by a virus, a bacterium, and a parasite,
respectively. In this regard, the infectious disease can be, for
example, a hepatitis, sexually transmitted diseases (e.g.,
Chlamydia, gonorrhea), tuberculosis, HIV/AIDS, diphtheria,
hepatitis B, hepatitis C, cholera, SARS, the bird flu, and
influenza.
[0083] For purposes herein, "autoimmune disease" refers to a
disease in which the body produces an immunogenic (i.e., immune
system) response to some constituent of its own tissue. In other
words the immune system loses its ability to recognize some tissue
or system within the body as "self" and targets and attacks it as
if it were foreign. Autoimmune diseases can be classified into
those in which predominantly one organ is affected (e.g., hemolytic
anemia and anti-immune thyroiditis), and those in which the
autoimmune disease process is diffused through many tissues (e.g.,
systemic lupus erythematosus). For example, multiple sclerosis is
thought to be caused by T cells attacking the sheaths that surround
the nerve fibers of the brain and spinal cord. This results in loss
of coordination, weakness, and blurred vision. Autoimmune diseases
are known in the art and include, for instance, Hashimoto's
thyroiditis, Grave's disease, lupus, multiple sclerosis, rheumatic
arthritis, hemolytic anemia, anti-immune thyroiditis, systemic
lupus erythematosus, celiac disease, Crohn's disease, colitis,
diabetes, scleroderma, psoriasis, and the like.
[0084] The disease can be a cancer. The cancer can be any cancer,
including any of acute lymphocytic cancer, acute myeloid leukemia,
alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast
cancer, cancer of the anus, anal canal, or anorectum, cancer of the
eye, cancer of the intrahepatic bile duct, cancer of the joints,
cancer of the neck, gallbladder, or pleura, cancer of the nose,
nasal cavity, or middle ear, cancer of the oral cavity, cancer of
the vulva, chronic lymphocytic leukemia, chronic myeloid cancer,
colon cancer, esophageal cancer, cervical cancer, gastrointestinal
carcinoid tumor, Hodgkin lymphoma, hypopharynx cancer, kidney
cancer, larynx cancer, liver cancer, lung cancer, malignant
mesothelioma, melanoma, multiple myeloma, nasopharynx cancer,
non-Hodgkin lymphoma, ovarian cancer, pancreatic cancer,
peritoneum, omentum, and mesentery cancer, pharynx cancer, prostate
cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma
(RCC)), small intestine cancer, soft tissue cancer, stomach cancer,
testicular cancer, thyroid cancer, ureter cancer, and urinary
bladder cancer. Preferably, the cancer is breast cancer, prostate
cancer, ovarian cancer, stomach cancer (e.g., gastric
adenocarcinoma), colon cancer, liver cancer, melanoma, basal cell
carcinoma, rhabdomyosarcoma, or medulloblastoma. Preferably, the
cancer is a melanoma, breast cancer, colorectal cancer, esophageal
cancer, gastric cancer, non-small cell lung cancer, a sarcoma,
pancreatic cancer, mesothelioma, or ovarian cancer.
[0085] The term "treat" as used herein does not necessarily imply
100% or complete treatment. Rather, there are varying degrees of
treatment of which one of ordinary skill in the art recognizes as
having a potential benefit or therapeutic effect. In this respect,
the inventive methods can provide any amount of any level of
treatment of cancer in a mammal. Furthermore, the treatment
provided by the inventive method can include treatment of one or
more conditions or symptoms of the disease, e.g., cancer, being
treated.
[0086] The pharmaceutical composition administered to the host can
be any of those described herein. The T cells of the population of
the pharmaceutical composition can be allogeneic or autologous to
the host. Preferably, the T cells of the pharmaceutical composition
are autologous to the host.
[0087] Also, the pharmaceutical composition can be administered to
the host through any route. Preferably, the pharmaceutical
composition is administered to the host via injection or
intravenously.
[0088] Method of Isolating Antigen Specific T Cells
[0089] The invention also provides a method of isolating
antigen-specific T cells from peripheral blood of a host. An
embodiment of the method of the invention comprises (i) dividing
peripheral blood mononuclear cells (PBMCs) from peripheral blood of
a host into more than one sub-population; (ii) contacting the PBMCs
with an antigen and Interleukin-2 (IL-2); (iii) obtaining a sample
of the contacted PBMCs from each sub-population; (iv) identifying
an antigen-reactive sub-population by determining by high
throughput quantitative PCR (HT-qPCR) the expression of a factor
produced by the PBMCs of each sample; (v) dividing the
antigen-reactive sub-population into microcultures; and (vi)
identifying an antigen-reactive microculture; whereupon T cells
specific for the contacting antigen are isolated from the
peripheral blood.
[0090] The method of isolating antigen-specific T cells from
peripheral blood of the invention can be carried out in accordance
with any of the embodiments of (i) to (vi) as described herein with
regard to the inventive method of obtaining a clinical population
of antigen-specific T cells.
[0091] Screening Candidate Cancer Antigen Epitopes
[0092] The invention further provides a method of screening
candidate cancer antigen epitopes. An embodiment of the method of
the invention comprises (i) dividing PBMCs from peripheral blood of
a host into more than one sub-population; (ii) contacting the PBMCs
with one or more candidate cancer antigen epitopes and IL-2; (iii)
obtaining a sample of the contacted PBMCs from each sub-population;
and (iv) identifying an antigen-reactive sub-population by
determining by high throughput quantitative PCR (HT-qPCR) the
expression of a factor produced by the PBMCs of each sample.
[0093] A cancer antigen epitope is identified when the
sub-population which was contacted by the cancer antigen epitope is
identified as antigen reactive, e.g., reactive to the contacting
antigen. The identification of an antigen-reactive sub-population
can comprise a comparison of the expression level of the factor by
the sub-population(s) with a positive control sub-population and a
negative control sub-population. The positive control
sub-population can be, for example, a sub-population stimulated
with IL-2 and a known cancer epitope, e.g., gp100.sub.154-162,
whereas the negative control sub-population can be, for example, a
sub-population stimulated with IL-2 and a peptide which is known to
not be a cancer epitope.
[0094] When a cancer antigen epitope is identified, the method can
further comprise (v) dividing the antigen-reactive subpopulation
into microcultures; (vi) identifying the antigen-reactive
microculture; and (vii) expanding the microculture to thereby
obtain a population of T cells specific for the cancer antigen
epitope.
[0095] In yet another embodiment of the inventive method, the
method can further comprise assaying the population for tumor
reactivity against a tumor cell line, e.g., a tumor cell line
expressing the cancer antigen epitope.
[0096] In yet another embodiment of the invention, the method can
further comprise determining the cancer antigen of which the cancer
antigen epitope is a part, thereby identifying a cancer antigen.
Methods of determining the cancer antigen of which the cancer
antigen epitope is a part are known in the art, and include, for
example, performing a BLAST search for the sequence of the epitope
and identifying candidate cancer antigens. Cells expressing the
candidate cancer antigens can be produced by known methods of
engineering and the cells can be assayed for recognition by the T
cell clones which recognize the cancer antigen epitope.
[0097] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
EXAMPLE 1
[0098] This example demonstrates a method of stimulating PBMCs from
peripheral blood of a host, which PBMCs are divided into more than
one sub-population, with an antigen, or an epitope thereof, and
IL-2.
[0099] Synthetic peptides are made for in vitro stimulation of
PBMCs using a solid phase method on a peptide synthesizer at the
Surgery Branch (NCI). The purity of each peptide is confirmed by
mass spectrometry and each is resuspended to 1 mg/ml for in vitro
use. Peptides of the following sequences are made:
gp100.sub.209-217 (ITDQVPFSV; SEQ ID NO: 1), gp100.sub.154-162
(KTWGQYWQV; SEQ ID NO: 2), MART-1.sub.27-35 (AAGIGILTV; SEQ ID NO:
3), HIVpol.sub.476-484 (ILKEPVHGV SEQ ID NO: 4), and FLU
M1.sub.58-66 (GILGFVFTL SEQ ID NO: 5).
[0100] PBMCs obtained by leukapheresis from HLA-A2.sup.+ metastatic
melanoma patients are in vitro stimulated in accordance with a 6-
or 10-day procedure. The 6-day procedure comprises the following:
On day 0, cryopreserved PBMCs are thawed, washed twice with CM, and
plated in a 96-well plate (3.times.10.sup.5 cells/well; 0.2
mL/well). Plates are incubated at 37.degree. C. in 5% CO.sub.2
overnight. On day 1, the sensitizing or stimulating peptide is
added to the PBMC culture plate at a final concentration of 1
.mu.g/ml. On day 2, 90 IU/ml recombinant interleukin 2 (IL-2;
Chiron Co., Emeryville, Calif.) is added to the cultures. On day 6,
the sensitized cultures are assayed for peptide reactivity by
either a qPCR assay or ELISA-based cytokine release assay.
[0101] The 10-day in vitro stimulation procedure is the same as the
6-day stimulation procedure, except that an additional peptide
exposure is performed on day 6, IL-2 (90 IU/ml) is added on day 7,
and the cultures are assayed for reactivity on day 10.
EXAMPLE 2
[0102] This example demonstrates a method of identifying an
antigen-reactive sub-population by determining by high throughput
qPCR the expression of a factor produced by PBMCs.
[0103] PBMCs undergo the 6- or 10-day in vitro stimulation as
described in Example 1. On the last day of stimulation (Day 6 or
Day 10), T2 cells (HLA-A2.sup.+ peptide transporter-associated
protein deficient T-B hybrid) are pulsed with either a relevant
sensitizing (stimulating) peptide or an irrelevant (control)
peptide at 1 .mu.g/ml in medium for .about.2 hrs at 37.degree. C.
T2 cells are washed three times to remove unbound peptide. From
each bulk PBMC culture to be assayed, two equal aliquots of cells
(each .about.50 .mu.l) are removed and incubated in parallel with
3.times.10.sup.4 T2 cells (pulsed with either relevant or
irrelevant peptides) in a 0.2-ml volume in individual wells of a 96
well U-bottom tissue culture plate. After 3 hours of incubation,
the 96-well plate is spun (900 RPM, 5 minutes), the supernatant is
completely discarded, and the cell pellet placed in RLT lysis
buffer (Qiagen, Valencia, Calif.).
[0104] RNA isolation is performed in a 96-well format using the
RNeasy 96 BioRobot 8000 kit (Qiagen). Total RNA for each sample is
transcribed into complementary DNA (cDNA) using TaqMan Reverse
Transcription Reagants (Applied Biosystems, Foster City, Calif.).
Quantitative real-time PCR is performed to determine the copy
number for interferon-.gamma. (IFN-.gamma.) mRNA in each sample, as
described previously (Kammula et al., J. Immuol. 163: 6867-6875
(1999) and Kammula et al., J. Natl. Cancer Inst. 92: 1336-1344
(2000)) using the ABI 7500 Fast Real-Time PCR System (Applied
Biosystems, Foster City, Calif.). The IFN-.gamma. mRNA levels in
response to the relevant peptide is divided by the IFN-.gamma. mRNA
levels induced by the irrelevant HIV.sub.pol peptide to define a
stimulation index (SI) for each parental PBMC culture:
SI=IFN-.gamma. (relevant peptide)/IFN-.gamma. (irrelevant peptide
(HIV.sub.pol)). A PBMC sample with a SI>2 is considered as
having specific peptide reactivity. All samples analyzed have
C.sub.T values less than 35 cycles to ensure the quality of the
PBMC samples in the assay.
EXAMPLE 3
[0105] This example demonstrates a method of identifying an
antigen-reactive sub-population by determining by a conventional
ELISA assay the expression of a factor produced by PBMCs.
[0106] PBMC and derived lymphocyte cultures are tested for
antigen-specific reactivity in a cytokine release assay using
commercially available IFN-.gamma. ELISA kits (Endogen, Pierce,
Rockford, Ill.). T2 cells are pulsed with relevant or irrelevant
peptide (1 .mu.g/ml) in medium for .about.2 hrs at 37.degree. C.,
followed by washing (three times) before initiation of co-cultures.
For these assays, 10.sup.5 responder cells (PBL or cloned T cells)
and 10.sup.5 stimulator cells (T2 cells or tumor lines) are
co-incubated in a 0.2-ml volume in individual wells of a 96-well
plate. Supernatants are harvested from duplicate wells after 20-24
hours and IFN-.gamma. secretion is measured in culture
supernatants, diluted to be within the linear range of the assay.
All data from the ELISA-based assays is presented herein as a mean
of duplicate samples. Cultures with IFN-.gamma. production greater
than 100 pg/ml and twice background are considered as having
specific antigen reactivity.
EXAMPLE 4
[0107] This example demonstrates a method of obtaining a population
of antigen-specific T cells in accordance with an embodiment of the
invention.
[0108] PBMCs obtained by leukapheresis from HLA-A2+ metastatic
melanoma patients are used to establish 96 independent
subpopulations cultured in complete medium (CM) consisting of RPMI
1640 supplemented with 10% heat-inactivated fetal bovine serum, 2
mM L-glutamine (Invitrogen, Carlsbad, Calif.), 50 units/mL
penicillin (Invitrogen), 50 .mu.g/mL streptomycin (Invitrogen), 50
.mu.g/mL gentamicin (Invitrogen), 10 mM Hepes (Invitrogen), and 250
ng/mL Amphotericin B (Invitrogen), along with 10% heat-inactivated
human AB serum (Gemini Bio-Products, Woodland, Calif.). Each
subpopulation is in vitro stimulated with 1 .mu.M of
gp100.sub.154-162 (KTWGQYWQV; SEQ ID NO: 2) for 10 days in the
presence of IL-2 (90 IU/ml) as essentially described in Example 1.
On day 10, a sample from each subpopulation is screened using a
qPCR assay for specific recognition of the gp100.sub.154-162
peptide versus the HIV.sub.pol peptide, as essentially described in
Example 2. The SI reactivities for the 96 wells are stratified by
their magnitude and the most reactive subpopulations are selected
for limiting dilution cloning.
[0109] Limiting dilution cloning is carried out by plating between
1 and 5 PBMCs from a reactive subpopulation/well in 96-well
U-bottom plates in 0.2 ml complete medium (CM) additionally
containing 30 ng/ml ortho-anti-CD3 (Ortho-Biotech, Raritan, N.J.)
and 300 IU/ml IL-2 with 5.times.10.sup.4 allogeneic irradiated
(4000 rad) PBMCs/well derived from at least 3 different donors. On
day 5 and every 3-4 days thereafter, half of the media in each well
is replaced with fresh media containing IL-2.
[0110] Approximately 2 weeks after initiating limiting dilution
cloning of PBMCs from reactive subpopulations, wells are inspected
for cell growth. Cell growth positive wells are screened in a
cytotoxicity assay to identify clones with cytolytic activity
against peptide pulsed T2 cells. Wells are further characterized by
assaying IFN-.gamma. secretion in response to limiting
concentrations of peptide pulsed onto T2 cells or to antigen
positive tumor lines via ELISA.
[0111] Selected clones are rapidly expanded with 30 ng/ml
ortho-anti-CD3 and 5.times.10.sup.6 irradiated allogeneic PBMCs in
upright 25-cm.sup.2 flasks as described previously (Dudley et al.,
J. Immunother. 24: 363-373 (1999)). Additional rapid expansions are
performed to determine proliferative capacity of clones. Expanded
clones are re-evaluated for peptide and tumor recognition and cell
surface phenotype by FACS.
[0112] The above method of obtaining a population of
antigen-specific T cells in accordance with an embodiment of the
invention is outlined in FIG. 1.
EXAMPLE 5
[0113] This example demonstrates the biological features of the
antigen-specific T cell populations obtained through a method of
the invention.
[0114] The strategy of Example 4 is applied to PBMC from four
melanoma patients (Patients 2, 5, 6, and 7). A sample of the bulk
PBMCs from each patient, prior to any in vitro manipulation,
undergoes staining with the gp100.sub.154-162 tetramer to determine
natural precursor frequency. The stained cells undergo FACS
analysis and it is determined that none of the patients demonstrate
a significant population of tetramer positive CD8.sup.+ T cells by
FACS on day 0, since less than 1% of cells of all four patients
were positive for CD8 and gp100.sub.154 specific T cell receptor
(Table 1).
TABLE-US-00001 TABLE 1 % cells positive for Highest % cells
positive for CD8 & gp100.sub.154-162 SI CD8 &
gp100.sub.154-162 Patient expression (Day 0) (Day 10) expression
(Day ~25-34) 2 <0.5 45 99 5 0 635 99 6 0 23 99 7 0 78 99
[0115] After 10 days of sensitization (stimulation) according to
the method of Example 1, the 96 independent subpopulations for each
patient are screened for peptide reactivity using the qPCR assay.
The stratified results for Patients 2, 5, and 6 demonstrate that
only 7%, 12%, and 8% of the wells had a SI.gtoreq.2, respectively;
1%, 3%, and 1% of the wells had a SI.gtoreq.10, respectively; and
the remaining majority of the wells has no detectable peptide
reactivity (SI<2). In contrast, for Patient 7, 92% of the wells
has a SI.gtoreq.2 and 60% of the wells has a SI.gtoreq.10.
[0116] The highest reactive subpopulations from patients 2, 5, 6,
and 7 (qPCR SI=45, 635, 23, and 78, respectively) are selected for
limiting dilution cloning. The frequencies of growth positive
clones with lytic ability against peptide pulsed targets are 0.2%,
28%, 0.1%, and 2.3% for Patients 2, 5, 6 and 7 which directly
correlate with the qPCR SI (r.sup.2=0.99, p<0.0001). These
selected clones are expanded with ortho-anti-CD3 and irradiated
allogeneic PBMCs as described in Example 4 and undergo FACS
analysis between days 25 and 34. FACS analysis with CD8 antibody
and gp100.sub.154 tetramer reveal highly enriched populations (99%)
of gp100.sub.154-162 tetramer positive CD8.sup.+ T cells (Table 2).
Further, the derived populations are confirmed to be clonal by the
sequencing of a single T cell receptor V13 chain for each patient.
The functional avidity of these isolated T cell clones are high, as
measured by their ability to recognize 10.sup.-10 to 10.sup.-11 M
of gp100.sub.154-162 peptide pulsed onto T2 cells (Table 1) and HLA
A2.sup.+/gp100.sup.+ melanoma tumor lines in vitro (Table 3).
TABLE-US-00002 TABLE 2 gp100.sub.154 (M) HIV (M) 10.sup.-6
10.sup.-7 10.sup.-8 10.sup.-9 10.sup.-10 10.sup.-11 10.sup.-12
10.sup.-6 Patient 2 clone 7105 6606 5102 2555 187 <10 <10
<10 Patient 5 clone 12067 11274 5323 1210 244 <10 <10
<10 Patient 7 clone 33349 29191 26858 18575 687 220 <10
<10
TABLE-US-00003 TABLE 3 Tumor cell Mel 526 Mel 624 Mel 888 Hep 3B
Media Tumor cell A2+/ A2+/ A2-/ A2-/ na Phenotype gp100+ gp100+
gp100+ gp100- Patient 2 clone 2724 5743 <10 <10 <10
Patient 5 clone 5114 7425 <10 <10 <10 Patient 7 clone 3975
8434 <10 <10 <10
[0117] gp100.sub.154-162CD8.sup.+ T cells in 6 of 8 patients (75%)
are successfully cloned in this manner. In pilot clinical scale
expansions, these clones demonstrated between 850-1000 fold
expansions in cell numbers over the initial 14 days after a single
rapid expansion in flasks. A second serial expansion of these
clones resulted in an additional 400-600 fold expansion over the
ensuing week. Thus, two consecutive rapid expansions is sufficient
to generate .about.10.sup.10 cells for potential clinical adoptive
transfer from each starting isolated clone.
[0118] This example demonstrated that the antigen-specific T cell
populations obtained through a method of the invention are clonal
populations of highly avid T lymphocytes.
EXAMPLE 6
[0119] This example demonstrates the phenotype of the T cell clones
obtained in Example 5.
[0120] The phenotype of the cells obtained in Example 5 is assessed
by cell surface FACS for CD27, CD28, CD45RO, CD45RA, CD62L, and
CD25. Specifically, approximately 1.times.10.sup.5 cells are
stained in a FACS buffer comprising PBS (BioWhittaker,
Walkersville, Md.) and 0.5% BSA with FITC-conjugated monoclonal
antibodies specific for CD8, CD25, CD27, CD28, CD45RO, CD45RA, or
CD62L (L-selectin) (BD Biosciences, San Jose, Calif.).
Immunofluorescence (which is analyzed as the relative log
fluorescence of live cells) is then determined using a FACScan flow
cytometer (BD Biosciences). A combination of forward angle light
scatter and propidium iodide staining is used to gate out dead
cells.
[0121] As shown in Table 4, the gp100.sub.154-162 tetramer positive
cells from patients 2, 5, and 7 all are uniformly CD45RO.sup.+ and
CD62L.sup.-, consistent with an effector memory phenotype. However,
unlike typical antigen experienced T cells, there is persistent
variable expression of CD45RA (19-96%). In addition, all of the
isolated clones continue to have significant expression of the
costimulatory molecule CD27 (90-99%). This phenotype differs from
the TIL derived MART.sub.27-35 specific clone, JKF6, which has no
significant expression of CD27.
TABLE-US-00004 TABLE 4 % Cells positive for staining Patient
Patient Patient JKF6 clone with antibody specific for: 2 5 7
(control) CD27 99 90 96 5 CD28 2 6 98 2 CD45RO 100 100 100 100
CD45RA 62 19 96 21 CD62L 1 5 13 2 CD25 1 27 12 ND
EXAMPLE 7
[0122] This example demonstrates the sensitivity of the qPCR assay
in comparison to a conventional ELISA assay.
[0123] Varying absolute numbers (between 1.5 and 3000) of the C6E4
gp100.sub.154-162 reactive CD8.sup.+ T cell clone are spiked into
individual microwells of a 96 well plate containing 150,000
nonreactive autologous bulk PBMC populations (FIG. 2). Exogenous
cytokines are not added to the PMBC populations and the cells of
the populations are not cultured. Rather, the spiked PBMCs are
immediately co-incubated with T2 cells pulsed with relevant peptide
(gp100.sub.154-162; 1 .mu.M) or an irrelevant peptide (HIV.sub.pol;
1 .mu.M). Cellular IFN-.gamma. mRNA production is measured by qPCR
at 3 hours after co-incubation, as essentially described in Example
2. Alternatively, supernatant IFN-.gamma. protein production is
measured at 24 hours by ELISA, as essentially described in Example
3. Stimulation indexes (SI) for each of the assays are determined
by dividing the reactivity against the relevant peptide by the
reactivity against the irrelevant peptide
(SI=gp100.sub.154-162/HIV.sub.pol).
[0124] Neither assay demonstrates significant reactivity
(gp100.sub.154-162/HIV.sub.pol SI<2) for each of the eight
replicate wells without spiked C6E4 clone (PBMC alone). As shown in
FIG. 2A, the qPCR assay identifies T cell reactivity in all
replicate wells containing between 3000 and 150 spiked clones. For
at least 2 of the 8 replicate wells, the qPCR assay can detect
reactivity at every dilution down to 1.5 cells spiked into 150,000
PBMC. In contrast, the detection limit for IFN-.gamma. protein
ELISA is reached in samples with 300 cells spiked into 150,000 PBMC
(FIG. 2B). The qPCR functional assay thus demonstrates a
significantly higher sensitivity than the standard ELISA assay,
detecting the antigen-induced cytokine response of approximately a
single CD8.sup.+ T cell at precursor frequency of .about.1:100,000
in a 96 microwell format.
[0125] This example demonstrated the high sensitivity of the qPCR
assay.
EXAMPLE 8
[0126] This example demonstrates that qPCR functional screening
rapidly identifies melanoma antigen-specific T cells in short term
sensitized (stimulated) peripheral blood cultures.
[0127] The qPCR assay described in Example 2 is applied to the
screening of PBMC for natural CD8.sup.+ T cell reactivity against
known epitopes from the melanocytic differentiation antigens, gp100
and MART. Peripheral blood leukapheresis samples are obtained from
17 HLA-A2.sup.+ metastatic melanoma patients who had not previously
undergone antigen specific immunotherapy (i.e., vaccine or cell
based transfer therapy). Bulk PBMC from each patient are plated in
replicate microwells (n=24) containing .about.300,000 cells and
individually sensitized (stimulated) for 6 days with 1 .mu.M of FLU
M1, MART.sub.27-35, gp100.sub.209-217, gp100.sub.154-162, or no
peptide (DMSO) in the presence of IL-2 (90 IU/ml). On day 6, a
sample from every microculture (.about.100,000 cells) is screened
using the qPCR assay for recognition of the respective sensitizing
peptide versus the irrelevant HIV.sub.pol peptide pulsed onto T2
cells (FIG. 3).
[0128] The IFN-.gamma. gene expression is normalized as a SI
(peptide x/HIV.sub.pol). The bulk cells cultured in IL-2 with no
sensitizing peptide (DMSO alone) are used to define the level of
nonspecific background reactivity for each patient (FIG. 3E). The
median DMSO/HIV.sub.pol SI for all patients is 1.0 (S.D..+-.0.3)
with individual wells ranging from 0.5 to 2.0. By using a cutoff SI
value of 2.0, significant microculture reactivity against the FLUM1
peptide in all 17 patients is identified (FIG. 3A), which served as
an internal positive control for the sensitization procedure.
Variability in the median FLUM1/HIV.sub.pol SI of the replicate
wells is observed across patients (median range: 3.0 to 376),
consistent with varying degrees of natural peripheral blood
CD8.sup.+ T cell reactivity against the FLU epitope. Further,
despite uniform culture conditions, marked well to well variability
within the culture replicates is noted for several patients.
[0129] Among the cultures sensitized for 6 days with the melanoma
antigen epitopes, heterogenic immune reactivity is similarly
observed. qPCR analysis of the cultures sensitized with
MART.sub.27-35 (FIG. 3B) revealed three patients (Patients 1, 4,
and 6) with median MART/HIV SI well reactivity above 2. However, in
12 patients (70.5%), the qPCR assay identifies at least one
individual microculture replicate which met criteria for
significant MART peptide reactivity. Similarly, among the
gp100.sub.209-217 sensitized cultures, only 4 patients (Patients 1,
2, 6, and 11) have median culture reactivity >2, but 16 of 17
(94%) patients are found to have individual wells with peptide
reactivity above background (FIG. 3C). Among the 8 patients
sensitized with the gp100.sub.154-162 peptide, one patient (patient
7) has median culture reactivity >2, but 6 patients (75%) have
individual wells with peptide reactivity (FIG. 3D). In summary,
CD8.sup.+ T cell reactivity against at least one of the melanoma
epitopes is identified in 16 of the 17 patients (94%). It is
concluded that the qPCR assay can be used as a highly efficient and
rapid screen to detect the reactivity of a variety of melanoma
specific T cells in short term sensitized PBMC microcultures.
[0130] To determine whether the immune reactivity identified at day
6 by the qPCR assay could also be detected by ELISA,
gp100.sub.209-217 sensitized microcultures from Patients 1 and 3
are evaluated using both assays with an equivalent number of
sampled PBMC (.about.100,000 cells) from each of the replicate
wells (FIGS. 4A and B).
[0131] ELISA evaluation does not identify any wells from either
patient with reactivity above background. In contrast the qPCR
assay performed on the same wells demonstrates multiple cultures
with detectable peptide reactivity. To confirm that the qPCR
reactivity in these early cultures independently correlate with the
presence of gp100.sub.209-217 specific T cells, the microcultures
with the highest and lowest SIs are rapidly expanded with anti-CD3,
allogenic feeder cells, and IL-2 over 1 week and evaluated for the
presence and activity of gp100.sub.209-217 reactive CD8.sup.+ T
cells (FIGS. 4A and B). By day 14, the expanded cultures from the
wells with the high SI (Patient 1 SI=11.1 and 12.4; Patient 3
SI=3.3) demonstrate a distinct population of antigen specific
CD8.sup.+ T cells when stained with the gp100.sub.209-217 tetramer
(3-5% of CD8+ cells). When samples of these expanded cultures are
tested for functional recognition of T2 cells pulsed with the
gp100.sub.209-217 peptide, they release significant amounts of
interferon-.gamma. protein that is easily detected by ELISA. In
contrast, the expanded cultures from the low SI wells (Patient 1
SI=1.1; Patient 3 SI=0.8) have neither discernable tetramer
positive cells nor functional activity against peptide pulsed
targets.
[0132] This example demonstrated that the qPCR assay can be used at
an early time point to stratify the epitope reactivity of short
term sensitized PBMC microcultures to prospectively identify
selected wells enriched for functionally active antigen specific T
cells and to eliminate wells with no evidence of reactivity.
EXAMPLE 9
[0133] This example demonstrates a method of detecting
NY-ESO.sub.157-165 specific T cells in the peripheral blood of
cancer patients.
[0134] PBMC from 9 HLA A2+ melanoma patients and 1 HLA A2+ breast
cancer patient are plated in replicate microwells (n=96) containing
.about.300,000 cells and sensitized (stimulated) for 14 days in the
presence of 1 mM of NY-ESO.sub.157-165 peptide (SLLMWITQC; SEQ ID
NO: 6) in the presence of IL-2 (90 IU/ml). On day 14, a sample from
every microculture (.about.100,000 cells) is screened for T cell
recognition of T2 cells pulsed with NY-ESO.sub.157-165 peptide
versus a DMSO control using the quantitative RT-PCR assay of
Example 2. Cellular IFN-.gamma. mRNA production is measured by qPCR
at 3 hours and reported as a stimulation index (SI). SI=IFN-g mRNA
(NY-ESO.sub.157-165)/IFN-g mRNA (DMSO).
[0135] NY-ESO.sub.157-165 specific CD8+ T cell reactivity (SI>2)
is detected in individual subpopulations from the peripheral blood
of all 9 melanoma patients and in the one patient with breast
cancer. Specifically, all 10 patients demonstrate subpopulations
with SI>2, 6 of 10 patients demonstrate subpopulations with
SI>10, 4 out of 10 patients demonstrated subpopulations with
SI>100.
[0136] This example demonstrated that the qPCR method could detect
NY-ESO.sub.157-165 specific T cells in the peripheral blood of
cancer patients.
EXAMPLE 10
[0137] This example demonstrates another method of obtaining a
population of antigen reactive T cells in accordance with an
embodiment of the invention.
[0138] On Day 0, PBMC from HLA-A2+ cancer patients are stained with
NY-ESO.sub.157-165 tetramers and anti-CD8 to determine natural
precursor frequency. PBMC from each patient are plated in replicate
microwells (n=96) containing .about.300,000 cells and sensitized
(stimulated) for 14 days with 1 mM of NY-ESO.sub.157-165 peptide in
the presence of IL-2 (90 IU/ml). On day 14, a sample from every
microwell (subpopulation) is screened using the qPCR assay for
specific recognition of the NY-ESO.sub.157-165 peptide versus a
DMSO control. The wells with the highest SI reactivity (shown in
Table 5) are selected for limiting dilution cloning, which is
carried out as essentially described in Example 4. After
approximately 2 weeks, growth positive wells are screened for the
ability to lyse peptide pulsed T2 cells using a cytotoxicity assay.
T cell clones selected on their ability to lyse peptide pulsed T2
cells are rapidly expanded with ortho-anti-CD3 and irradiated
allogeneic PBMCs in accordance with Example 4. The clones are
stained with NY-ESO.sub.157-165 tetramer and analyzed via FACS to
reveal highly enriched (99%) populations of NY-ESO.sub.157-165
tetramer-positive CD8+ T cells (Table 5).
TABLE-US-00005 TABLE 5 % cells % cells positive for positive for
CD8 & NY- CD8 & NY- ESO.sub.157-165 Highest ESO.sub.157-165
Pa- Cancer expression SI expression tient Type (Day 0) (Day 14)
(Day ~30-40) D melanoma 0.1 7 99 F melanoma 0.7 20 99 B melanoma
0.4 43 TBD C melanoma 0 70 TBD H melanoma 0.06 79 TBD J breast 1.2
288 TBD TBD = to be determined
[0139] This example demonstrated that the method of the invention
can be successfully obtain clonal populations of NY-ESO-specific,
CD8+ T cells.
EXAMPLE 11
[0140] This example demonstrates the biological features of the
clonal populations of Example 10.
[0141] The functional avidity of the NY-ESO-1-reactive clones of
Patient D obtained through Example 10 are assayed for avidity by
tumor and peptide-specific stimulation. 1.times.10.sup.5 cloned T
cells are co-cultured overnight with an equal number tumor cell
lines or T2 cells pulsed with peptide (as specified in Tables 6 and
7), and assessed for IFN-.gamma. (pg/ml) by standard ELISA assay.
The results are shown in Tables 6 and 7. Values of 200 pg/ml and
twice background are bolded and underlined.
TABLE-US-00006 TABLE 6 NY-ESO.sub.157(M) gp100.sub.154 M 10.sup.-6
10.sup.-7 10.sup.-8 10.sup.-9 10.sup.-10 10.sup.-11 10.sup.-12
10.sup.-6 Frese 9A2 NY-ESO.sub.157-165 CD8+ Clone 16,055 14,270
6,172 642 11 2 4 2 Frese Cl07 gp 100.sub.154-162 CD8+ Clone 0 4 2
24 0 4 20 13,770 Media 0 2 3 1 20 14 1 1
TABLE-US-00007 TABLE 7 A2+/NY-ESO+ A2+/NY-ESO- A2-/NY-ESO- Mel 1363
Mel 1300 Mel 624.38 H1299-A2 COSA2:ESO COSA2:Vector Panc-1 Mel 888
Hep 3B Media Frese 9A2 NY-ESO.sub.157-165 5,140 1,398 830 1,191
3,911 4 83 2 5 11 CD8+Clone Frese Clo7 gp100154-162 3,494 20,900
7,161 31 4 5 19 2 3 1 CD8+Clone Media 1 5 4 2 20 25 5 17 2 2
[0142] This example demonstrated that the clones obtained by the
inventive method have sufficient avidity to recognize peptide
pulsed targets and naturally expressed epitope on tumor cells
lines.
EXAMPLE 12
[0143] This example demonstrates the phenotype of the T cell clones
obtained in Example 10.
[0144] Two clones of Patient D are stained with antibodies specific
for CD27, CD28, CD45RO, CD45RA, CD62L, and CD25 and subsequently
analyzed via FACS. The results are shown in Table 8.
TABLE-US-00008 TABLE 8 % clones positive for expression of cell
surface marker Patient D CD27 CD28 CD45RO CD45RA CD62L CD25 Clone
9A2 66 5 99.8 33 3 17 Clone 2A11 87 7 99.8 34 5 14
[0145] This example demonstrated that the clones of Patient D have
a phenotype of effector memory, but also are CD27+.
EXAMPLE 13
[0146] This example demonstrates a method of screening candidate
cancer antigen epitopes in accordance with an embodiment of the
invention.
[0147] Mesothelin peptides of the following amino acid sequence are
synthesized as essentially described in Example 1: FLLFSLGWV (SEQ
ID NO: 12), SLLFLLFSL (SEQ ID NO: 11), NMNGSEYFV (SEQ ID NO: 13),
VLPLTVAEV (SEQ ID NO: 14), LIFYKKWEL (SEQ ID NO: 15), LLATQMDRV
(SEQ ID NO: 16), LLGFPCAEV (SEQ ID NO: 17), VLLPRLVSC (SEQ ID NO:
18), LPLDLLLFL (SEQ ID NO: 19), and RLSEPPEDL (SEQ ID NO: 20). The
peptides are used to contact PBMC sub-populations along with IL-2
in accordance with the procedures described in Example 4. HT-qPCR
is performed on a sample of each sub-population to determine the
expression of IFN-.gamma. by the contacted PBMC
sub-populations.
[0148] The highest SI of each PBMC sub-population contacted with
the indicated mesothelin peptide is shown in Table 9.
TABLE-US-00009 TABLE 9 Peptide SEQ ID Highest SI Sequence NO: (Day
14) SLLFLLFSL 11 119 FLLFSLGWV 12 29 NMNGSEYFV 13 *<2 VLPLTVAEV
14 <2 LIFYKKWEL 15 <2 LLATQMDRV 16 <2 LLGFPCAEV 17 <2
VLLPRLVSC 18 <2 LPLDLLLFL 19 <2 RLSEPPEDL 20 <2 * = SI
<2, not reactive
[0149] The highest mesothelin peptide-reactive sub-populations
which recognize SEQ ID NO: 11 and 12 are selected for limiting
dilution cloning as essentially described in Example 4.
[0150] Cell growth positive wells are screened by assaying
IFN-.gamma. secretion in response to varying concentrations of the
appropriate antigenic peptide pulsed onto T2 cells. As shown in
Table 10, the SLLFLLFSL-reactive clones react to as little as 100
.mu.M of SLLFLLFSL. As shown in Table 11, 6 of the 14
FLLFSLGWV-reactive clones demonstrate reactivity to T2 cells pulsed
with as little as 10 pM FLLFSLGWV, whereas all 14 clones exhibit
reactivity to T2 cells pulsed with as little as 100 pM
FLLFSLGWV.
TABLE-US-00010 TABLE 10 T2 + T2 + T2 + T2 + T2 + T2 + T2 + T2 +
Meso Meso Meso Meso Meso Meso Meso HIV Clone 1 uM 100 nM 10 nM 1 nM
100 pM 10 pM 1 pM 1 uM Clo 2 13482 13839 13108 10560 3061 37 <10
<10 Clo 13 11511 11732 11444 9371 3987 71 <10 <10 Clo 27
9490 9363 8921 3919 521 12 <10 <10 Clo 34 3723 3885 3307 2424
504 <10 <10 <10 Clo 25 12021 11877 12199 10603 3664 12
<10 <10 Clo 28 4029 4522 4598 3273 632 <10 <10 <10
Negative <10 <10 <10 <10 <10 <10 <10 <10
Control Media <10 <10 <10 <10 <10 <10 <10
<10
TABLE-US-00011 TABLE 11 T2 + T2 + T2 + T2 + T2 + T2 + T2 + T2 + T2
+ Meso Meso Meso Meso Meso Meso Meso gp154 HIV Clone 1 uM 100 nM 10
nM 1 nM 100 pM 10 pM 1 pM 1 uM 1 uM 2 10194 10213 9640 5128 1829
154 0 36 0 2p 18946 19294 15136 9149 2775 453 11 11 5 3 3877 4219
3895 1493 304 49 0 0 5 5 8825 9466 8551 4586 1381 123 0 0 0 6 7904
8034 7281 4007 1194 86 0 0 0 6p 11084 10767 8377 3945 1256 142 5 5
11 7 10655 10225 8850 4561 1063 17 5 17 17 7p 9173 9485 9217 4953
1461 217 11 5 5 11p 10095 10854 9136 4686 1567 154 5 24 5 17 11003
10674 6142 2526 515 17 10 5 0 20 2812 3024 2694 901 210 42 11 5 0
21 8358 8346 6790 3055 571 17 5 5 5 24 15585 14956 10804 5109 858
42 10 5 11 26 8875 8906 7997 4032 1107 55 5 0 0 Negative 36 17 11 5
55 5 5 14321 0 control Media <10 <10 <10 <10 <10
<10 <10 <10 <10
[0151] The phenotype of Clone 2, which is specific for the
mesothelin peptide of SEQ ID NO: 12 is determined by FACS analysis
as essentially described in Example 6. As shown in Table 12, the
phenotype of the cells is shown to have a moderately differentiated
phenotype (CD27.sup.+, CD28.sup.-, and CD45RA.sup.+). Also, 99% of
the clones were positive for expression of CD8.
TABLE-US-00012 TABLE 12 % clones positive for expression of cell
surface marker Patient D CD27 CD28 CD45RO CD45RA CD62L CD25 Clone
81 1 87 99 1 1
[0152] This example demonstrates a method of screening candidate
cancer antigen epitopes and a method of obtaining a population of
mesothelin-reactive T cells.
EXAMPLE 14
[0153] This example demonstrates a method of preparing cells for
administration to humans.
[0154] Peripheral blood mononuclear cells (PBMC) from patients are
obtained by leukopheresis. PBMC are enriched by centrifugation on
Lymphocyte separation medium (LSM), (ICN Biomed, Inc; Avrora,
Ohio), washed 2 times with Ca.sup.++-, Mg.sup.++-, Phenol red-free
Hanks' balanced salt solution (HBSS) (BioWhittaker), and
cryopreserved at 1.times.10.sup.8 cells/vial in one ml of human
serum (Biowhittaker) with 10% DMSO.
[0155] One vial of PBMC is thawed by warming rapidly to 37.degree.
C. Cells are transferred directly into complete medium (CM), which
consists of RPMI-1640 with 10% human serum (Approved source,
heat-inactivated 56.degree. C. for 30 minutes) with final
concentrations of penicillin G (100 units/ml), streptomycin (100
.mu.g/ml), gentamicin (50 .mu.g/ml), L-glutamine (146 .mu.g/ml, 1
mM). PBMC are washed twice with CM and an aliquot is counted.
1-3.times.105 PBMC are plated in each well of a 96 well flat bottom
tissue culture plate in 0.1 ml of CM. Plates are incubated at
37.degree. C. in 5% CO2 overnight to recover from the thaw.
[0156] On the following day, hgp100.sub.154-162 peptide (NeoMPS,
Inc.) is added to the culture plate at a final concentration of 1.0
microgram/ml (approximately 1.0 micromolar). IL-2 is added to each
well to 10 CU/ml final concentration on the next day. Four and 5
days later, peptide and IL-2 are added respectively as above.
[0157] Between days 10-14 from the date that PMBCs are thawed, an
aliquot of cells is removed from each bulk culture well and assayed
for activity. Briefly, 50 .mu.l of parental culture is plated per
well of a 96 well U-bottom tissue culture plate with
3.times.10.sup.4 T2 cells pulsed with 1.0 micromolar
hgp100.sub.154-462 or T2 pulsed with DMSO. After 3 hours, the
co-cultured cells are lysed, RNA isolated and cDNA is synthesized.
Quantitative RT-PCR is performed to measure levels of
interferon-.gamma. mRNA. The wells that exhibit the highest peptide
specificity are selected for subcloning.
[0158] Active bulk cultures are cloned by limiting dilution in 96
well U-bottom plates. Briefly, allogeneic PBMC are prepared. PBMC
are obtained by thawing frozen leukopheresis vials from normal
donors as described above. PBMC are thawed directly into CM, washed
twice, resuspended in CM, and then irradiated (340 Gy, Nordion
gammacell 1000 Cs137 irradiator. Enough cloning reagents for 40
plates are mixed together: 800 ml CM, 4.times.108 irradiated PBMC
(either allogenic or autologous), 30 ng/ml OKT3, and 50 CU/ml IL-2.
Responder CTL for subcloning are harvested by removing the entire
contents of the most active bulk culture well and adding this to
the cloning reagent mixture. These are mixed well and plated in 40
U-bottom plates using a repeating multichannel pipette. Each well
is roughly estimated to contain 1 to 4 cells per well. The final
components of each well are set forth in Table 13.
TABLE-US-00013 TABLE 13 Component per well viable cells 1 to 4
allogeneic or autologous PBMC 1 .times. 10.sup.5 OKT3 30 ng/ml IL-2
50 CU/ml CM 200 microliters
[0159] Wells are screened visually for clonal growth 10-14 days
after plating.
[0160] Aliquots of all growth positive wells are tested by
co-culture assay for specificity and activity. 50 .mu.l aliquots of
cells from each well are re-plated in duplicate wells of a 96 well
flat bottom plate. 5.times.10.sup.4 T2 target cells are added to
each well. Typically, one well receives T2 pulsed with
hgp100.sub.154-162 and the other well receives T2 pulsed with a
control peptide. After a 24 hr co-incubation period, the co-culture
wells are visually screened for specific lysis of the T2 pulsed
targets. Wells demonstrating lysis of the hgp100.sub.154-162 pulsed
T2 targets are selected for further expansion.
[0161] Each active subclone is expanded using a Rapid Expansion
Protocol (REP).
[0162] On day 0, autologous or allogeneic PBMC are thawed, washed
twice, resuspended in CM and irradiated (340 Gy) as described
above. PBMC (2.5.times.107) and OKT3 (30 ng/ml) are added to CM (25
ml), mixed well, and aliquots are transferred to tissue culture
flasks. Viable CTLs from the well from the limiting dilution
cloning procedure (approximately 1.times.10.sup.5 cells) are added
last. Flasks (25 mm.sup.2) are incubated upright at 37.degree. C.
in 5% CO.sub.2. On day 2, IL-2 is added to 50 CU/ml. On day 5, 20
ml (130 ml for a 175 cm.sup.2 flask) of culture supernatant is
removed by aspiration (cells are retained on the bottom of the
flask). Media is replaced with CM containing 50 CU/ml IL-2. On day
8, an aliquot of cells is removed for counting and re-assay. Cells
are assayed for peptide specificity and tumor recognition by
co-incubation assay and ELISA. If cell density is greater than
1.times.10.sup.6/ml, cells are split into additional flasks or
transferred to Baxter 3 liter culture bags. IL-2 is added to 50
CU/ml. Fungizone is added to 1.25 mcg/ml and 1 ml/l Cipro is added.
On day 11, IL-2 is added to 50 CU/ml. Cells are split if density
exceeds 1.5.times.10.sup.6 cells/ml. On day 14, cells are harvested
and either prepared for additional REP cycles or cryopreserved.
[0163] Cells are tested for activity and specificity by
co-culturing with target cells (either tumor cells or T2 cells
pulsed with antigenic peptide) followed by measurement of cytokine
release via ELISA as described above. The most active clones are
expanded further to therapeutic numbers with additional REP cycles.
These additional REP cycles are the same as the first REP cycle
(described above), except that 1.times.10.sup.6 CTLs are added to
75 ml of CM additionally containing AIM V, 2.times.10.sup.8
allogeneic or autologous PBMCs, and 30 ng/ml OKT3 in 150 cm.sup.2
flasks.
[0164] In the REP cycle immediately preceding infusion, Fungizone
and Cipro are added on day 8, and AIM V media is used. In general,
REP expansion of CTL clones results in 50-200 fold expansion. Thus,
at least 2 REP cycles are required to generate sufficient cells for
patient treatment. If cells have grown to sufficient numbers for
patient treatment, a sample is collected from each flask for
microbiology tests 2-3 days before the beginning of CTL therapy
(the test takes 2 days). IL-2 is added to 50 CU/ml on day 14 and
every 3 days until the final product is prepared for infusion.
[0165] On day 14-20, get approval from the clinical team to proceed
with the cell harvest. Also, check the quality control tests that
are needed before infusion of the cells, as specified in the
Certificate of Analysis. The product for infusion is prepared by
harvesting and washing the cells in centrifuge tubes or in a
continuous centrifuge cell harvester system. Cell cultures in
flasks or a small number of Nexell culture bags, are transferred to
250 ml centrifuge tubes. These cells are centrifuged (400.times.g
for 15 min), and then resuspended in HBSS and combined in a single
250 ml tube. With about 4 liters or more of culture fluid in Nexell
culture bags, the cells are harvested with the Baxter/Fenwall
harvester system, the last step of which is a 2-liter wash with
0.9% sodium chloride. Cells from the continuous centrifuge harvest
are transferred from the harvest bag to 250 ml centrifuge tubes.
For the last step of both harvesting procedures, cells are
centrifuged and resuspended in 100-400 ml of 0.9% sodium chloride
containing 1) human albumin (25%) added to a final concentration of
2.5% and 2) recombinant human IL-2 at a final concentration of 50
CU/ml. The cell suspension is then transferred into the infusion
bag. The range of cells in the infusion bag is specified in the
clinical protocol. Aliquots are taken from the infusion bag for
viable cell counting, quality control testing, and cryopreservation
of cells. The product is then transferred to the clinical team for
infusion as soon as possible.
[0166] This example demonstrated a method of preparing cells for
administration to humans.
EXAMPLE 15
[0167] This example demonstrates a method of treating cancer with
the cells of the invention.
[0168] Peripheral blood lymphocytes (approximately 5.times.10.sup.9
cells)) are obtained by leukapheresis from patients with metastatic
melanoma. Whole PBMC will be cultured in the presence of anti-CD3
(OKT3), aldesleukin (IL-2), and gp100:154-162 in order to stimulate
T-cell growth. Donated whole blood and serum will be provided by
volunteers and obtained from the Department of Transfusion Medicine
in the NIH Clinical Center. The donated whole blood and serum will
be isolated and used in cell culture. In addition, volunteers will
undergo apheresis to obtain mononuclear cells which may be used as
feeder cells in cell culture. Separate consents will be obtained
from all blood and apheresis volunteers.
[0169] PBL will be assessed for tumor reactivity as specified in
Table 14.
TABLE-US-00014 TABLE 14 Re- Initials/ Test Method Limits sult Date
Cell trypan blue >70% viability.sup.1 exclusion Total visual
.gtoreq.1 .times. 10.sup.9 viable cell microscopic number.sup.1
count Tumor .gamma.-IFN >200 pg/ml antigen release vs
reactivity.sup.2 A2+/gp100+ tumor cell line Micro- gram
stain.sup.1,3 no micro- biological organisms studies seen aerobic
culture.sup.3,4 no growth fungal culture.sup.3,4 no growth
anaerobic culture.sup.3,4 no growth mycoplasma test.sup.2 no growth
Endotoxin.sup.1 limulus assay #5 E.U./kg .sup.1Performed on the
final product prior to infusion. Results are available at the time
of infusion. .sup.2Performed 2-10 days prior to infusion (test
performed prior to final manipulation). Results are available at
the time of infusion. .sup.3Performed 2-4 days prior to infusion.
Results are available at the time of infusion but may not be
definitive. .sup.4Sample for test collected on the final product
prior to infusion. Results will not be available before cells are
infused into the patient.
[0170] Cells will be expanded and considered for this trial if they
are reactive with the gp100:154-162 melanoma antigen. Once cells
have been deemed eligible for use in this trial, patients will be
consented on this study and enrolled. The patient must meet an
eligibility criteria prior to administration of the preparative
regimen. Patients who are otherwise eligible for cell
administration but who may not receive high dose aldesleukin
because of the presence of cardiovascular or respiratory system
medical illnesses will be eligible to receive low dose aldesleukin.
Growth and expansion of the final product will be performed after
the patient has consented to participate in this specific study.
Patients will receive up to 3.times.10.sup.11 gp100:154-162
reactive PBL. A minimum of approximately 1.times.10.sup.9 cells
will be given. In prior protocols over 3.times.10.sup.11 T cells
have been safely infused to cancer patients.
[0171] Once cells meet the reactivity requirements and are
projected to exceed the minimum number specified in Table 9, the
patient will receive the lymphocyte depleting preparative regimen
consisting of fludarabine and cyclophosphamide, followed by
infusion of up to 3.times.10.sup.11 lymphocytes and the
administration of either high-dose aldesleukin or low-dose
aldesleukin.
[0172] There will be two cohorts of patients depending on
eligibility to receive high-dose aldesleukin: 1) patients who are
eligible will receive high-dose aldesleukin; and 2) patients who
are not eligible to receive high-dose aldesleukin will receive low
dose aldesleukin.
[0173] Each cohort accrues independently of the other. Patients who
are not eligible to receive high-dose aldesleukin will be assigned
to receive cells (PBL) plus low-dose subcutaneous (SQ) aldesleukin.
The total number of such patients is projected to be too small
(<10) to have its own early stopping rule for accrual.
Therefore, if accrual ends for the patients who receive high-dose
aldesleukin, then no further patients receiving low-dose
aldesleukin will be entered. Patients will receive no other
experimental agents while on this protocol.
[0174] The protocol for drug administration will be as follows:
[0175] On Day -7 and -6 at 1 am: Hydrate: Begin hydration with 0.9%
Sodium Chloride Injection containing 10 meq/L of potassium chloride
at 2.6 ml/kg/hr (starting 11 hours pre-cyclophosphamide and
continue hydration until 24 hours after last cyclophosphamide
infusion).
[0176] On Day -7 and -6 at 11 am: Ondansetron (0.15 mg/kg/dose
[rounded to the nearest even mg dose between 8 mg and 16 mg based
on patient weight] IV every 8 hours.times.3 days) will be given for
nausea.
[0177] Also, Furosemide 10-20 mg iv will be given.
[0178] On Day -7 and -6 at 12 pm (NOON): Cyclophosphamide 60
mg/kg/day.times.2 days IV in 250 ml D5W with mesna 15
mg/kg/day.times.2 days over 1 hr.
[0179] On Day -7 and -6 at 1 pm: Begin to monitor potassium level
every 12 hours until hydration is stopped. KCl will be adjusted to
maintain serum potassium levels in the normal range.
[0180] Also, begin mesna infusion at 3 mg/kg/hour intravenously
diluted in a suitable diluent (see pharmaceutical section) over 23
hours after each cyclophosphamide dose.
[0181] On Day -5: Stop IV hydration (24 hours after last
cyclophosphamide dose). If urine output <1.5 ml/kg/hr, give
additional 20 mg furosemide iv. If body weight >2 kg over pre
cyclophosphamide value, give additional furosemide 20 mg iv.
[0182] On Day -5 to Day -1: Fludarabine 25 mg/m.sup.2/day IVPB
daily over 30 minutes for 5 days.
[0183] Cells are prepared as detailed in Example 14. Cells are
delivered to the patient care unit by a staff member from the Tumor
Immunology Cell Processing Laboratory. Prior to infusion, the cell
product identity label is double-checked by two authorized staff
(MD or RN), an identification of the product and documentation of
administration are entered in the patient's chart, as is done for
blood banking protocols. The cells are to be infused intravenously
over 20-30 minutes via non-filtered tubing, gently agitating the
bag during infusion to prevent cell clumping.
[0184] Day 0 (one to four days after the last dose of fludarabine):
gp100:154-162 reactive PBL, from 1.times.10.sup.9 up to a maximum
of 3.times.10.sup.11 lymphocytes, will be infused intravenously
(i.v.) on the Patient Care Unit over 20 to 30 minutes (between one
and four days after the last dose of fludarabine). Cell infusions
will be given as an inpatient.
[0185] Aldesleukin will be administered as follows: (a) 720,000
IU/kg/dose IV (based on total body weight) over 15 minute every
eight hours beginning within 24 hours of cell infusion and
continuing for up to 5 days (maximum of 15 doses) or (b) 250,000
IU/kg/day subcutaneously (SQ) daily for five days in the first week
and then at a dose of 125,000 IU/kg/day for five days for five
weeks (two day break each week).
[0186] Day 1-4 (Day 0 is the day of cell infusion) and then as per
aldesleukin regimen: Start filgrastim at 10 mcg/kg/day daily on Day
1 or 2 subcutaneously until neutrophil count
>1.0.times.10.sup.9/L.times.3 days or >5.0.times.10.sup.9/L
(not to exceed 600 .mu.g/day). Aldesleukin will be administered as
follows: (a) 720,000 IU/kg/dose IV (based on total body weight)
over 15 minutes every eight hours beginning within 24 hours of cell
infusion and continuing for up to 5 days (maximum of 15 doses.) or
(b) 250,000 IU/kg/day subcutaneously (SQ) for five days in the
first week and then at a dose of 125,000 IU/kg/day for five days
for five weeks (two day break each week).
[0187] The protocol for drug administration is further depicted in
Table 15.
TABLE-US-00015 TABLE 15 Day Therapy -7 -6 -5 -4 -3 -2 -1 0.sup.1 1
2 3 4 Cyclo- X X phosphamide 60 mg/kg Fludarabine X X X X X 25
mg/m.sup.2 gp100:154-162 X reactive PBL Cells Aldesleukin.sup.3
X.sup.2 X X X X Filgrastim.sup.4 X X X X 10 mcg/kg/day
TMP/SMX.sup.5 X X X 160 mg/800 mg (example) Fluconazole.sup.6 X X X
X X 400 mg po Valacyclovir X X X X X po or Acyclovir IV.sup.7
.sup.1One to four days after the last dose of fludarabine
.sup.2Initiate within 24 hours after cell infusion
.sup.3Aldesleukin will be administered as follows: 720,000
IU/kg/dose IV (based on total body weight) over 15 minute every
eight hours beginning within 24 hours of cell infusion and
continuing for up to 5 days (maximum of 15 doses.) 250,000
IU/kg/day subcutaneously (SQ) for five days in the first week and
then at a dose of 125,000 IU/kg/day for five days for five weeks
(two day break each week). .sup.4Continue until neutrophils count
>1 .times. 10.sup.9/L for 3 consecutive days or >5 .times.
10.sup.9/L. .sup.5The TMP/SMX schedule should be adjusted to QD
three times per week (Monday, Wednesday, Friday) and continue for
at least six months and until CD4 >200 .times. 2 .sup.6Continue
until ANC >1000/mm.sup.3 .sup.7In patients positive for HSV,
continue until absolute neutrophil count is greater than
1000/mm.sup.3
[0188] Patients who are eligible to receive high-dose aldesleukin
will receive aldesleukin at a dose of 720,000 IU/kg (based on total
body weight) as an intravenous bolus over a 15 minute period every
eight hours beginning on the day of cell infusion and continuing
for up to 5 days (maximum 15 doses). Doses may be skipped depending
on patient tolerance. Doses will be skipped if patients reach Grade
III or IV toxicity due to aldesleukin except for the reversible
Grade III toxicities common to aldesleukin such as diarrhea,
nausea, vomiting, hypotension, skin changes, anorexia, mucositis,
dysphagia, or constitutional symptoms and laboratory changes.
Toxicities will be managed. If these toxicities can be easily
reversed within 24 hours by supportive measures then additional
doses may be given. Additional instances may arise when in the
clinical judgment of the attending physician, based on the
extensive clinical experience in the Surgery Branch with
aldesleukin, when doses of aldesleukin may be skipped. If greater
than 2 doses of aldesleukin are skipped, aldesleukin administration
will be stopped. Aldesleukin will be administered as an
inpatient.
[0189] Patients who are not eligible to receive high-dose
aldesleukin will receive low dose aldesleukin at a dose of 250,000
IU/kg subcutaneously daily for 5 days. After a two-day rest,
aldesleukin will be administered at a dose of 125,000 IU/kg
subcutaneously daily for 5 days for the next five weeks (2 days
rest per week). Doses may be skipped depending on patient
tolerance. Doses will be skipped if patients reach Grade III or IV
toxicity due to low dose aldesleukin except for the reversible
Grade III toxicities occurring with low dose aldesleukin such as
diarrhea, nausea, vomiting, hypotension, peripheral edema, changes
in level of consciousness, infection or laboratory changes. In
addition, local inflammation at the injection site with occasional
nodular induration has been seen, which is reversible over a period
of weeks to months. If the toxicity experienced while receiving low
dose aldesleukin is easily reversed by supportive measures, then
dosing may continue. Aldesleukin will be administered as an
outpatient after the patient or family member have been taught to
self-administer the subcutaneous injections.
[0190] This example demonstrates a method of treating cancer in
humans with the cells of the invention.
[0191] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0192] The use of the terms "a" and "an" and "the" and similar
referents 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. The terms "comprising,"
"having," "including," and "containing" are to be construed as
open-ended terms (i.e., meaning "including, but not limited to,")
unless otherwise noted. Recitation of ranges of values herein are
merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range,
unless otherwise indicated herein, and each separate 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 unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0193] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as 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.
Sequence CWU 1
1
2019PRTArtificialSynthetic 1Ile Thr Asp Gln Val Pro Phe Ser Val 1 5
29PRTArtificialSynthetic 2Lys Thr Trp Gly Gln Tyr Trp Gln Val 1 5
39PRTArtificialSynthetic 3Ala Ala Gly Ile Gly Ile Leu Thr Val 1 5
49PRTArtificialSynthetic 4Ile Leu Lys Glu Pro Val His Gly Val 1 5
59PRTArtificialSynthetic 5Gly Ile Leu Gly Phe Val Phe Thr Leu 1 5
69PRTArtificialSynthetic 6Ser Leu Leu Met Trp Ile Thr Gln Cys 1 5
721DNAArtificialSynthetic 7agctctgcat cgttttgggt t
21824DNAArtificialSynthetic 8gttccattat ccgctacatc tgaa
24925DNAArtificialSynthetic 9tcttggctgt tactgccagg accca
25109PRTArtificialSynthetic 10Lys Val Leu Glu Tyr Val Ile Lys Val 1
5 119PRTArtificialSynthetic 11Ser Leu Leu Phe Leu Leu Phe Ser Leu 1
5 129PRTArtificialSynthetic 12Phe Leu Leu Phe Ser Leu Gly Trp Val 1
5 139PRTArtificialSynthetic 13Asn Met Asn Gly Ser Glu Tyr Phe Val 1
5 149PRTArtificialSynthetic 14Val Leu Pro Leu Thr Val Ala Glu Val 1
5 159PRTArtificialSynthetic 15Leu Ile Phe Tyr Lys Lys Trp Glu Leu 1
5 169PRTArtificialSynthetic 16Leu Leu Ala Thr Gln Met Asp Arg Val 1
5 179PRTArtificialSynthetic 17Leu Leu Gly Phe Pro Cys Ala Glu Val 1
5 189PRTArtificialSynthetic 18Val Leu Leu Pro Arg Leu Val Ser Cys 1
5 199PRTArtificialSynthetic 19Leu Pro Leu Asp Leu Leu Leu Phe Leu 1
5 209PRTArtificialSynthetic 20Arg Leu Ser Glu Pro Pro Glu Asp Leu 1
5
* * * * *