U.S. patent application number 17/481404 was filed with the patent office on 2022-04-07 for tumor infiltrating lymphocytes and methods of therapy.
The applicant listed for this patent is Intima Bioscience, Inc., Regents of the University of Minnesota, The U.S.A., as Represented by the Secretary, Department of Health and Human Service. Invention is credited to Modassir Choudhry, Branden Moriarity, Douglas C. Palmer, Nicholas P. Restifo, Steven A. Rosenberg, Beau Webber.
Application Number | 20220105133 17/481404 |
Document ID | / |
Family ID | 1000006038560 |
Filed Date | 2022-04-07 |
View All Diagrams
United States Patent
Application |
20220105133 |
Kind Code |
A1 |
Moriarity; Branden ; et
al. |
April 7, 2022 |
TUMOR INFILTRATING LYMPHOCYTES AND METHODS OF THERAPY
Abstract
Genetically modified compositions, such as non-viral vectors and
tumor infiltrating lymphocytes, for the treatment of
gastrointestinal cancer are disclosed. Disclosed are methods of
utilizing a CRISPR system to generate genetically modified
compositions. Also disclosed are the methods of making and using
the genetically modified compositions for the treatment of
gastrointestinal cancer.
Inventors: |
Moriarity; Branden;
(Shoreview, MN) ; Webber; Beau; (Coon Rapids,
MN) ; Choudhry; Modassir; (New York, NY) ;
Rosenberg; Steven A.; (Potomac, MD) ; Palmer; Douglas
C.; (North Bethesda, MD) ; Restifo; Nicholas P.;
(Chevy Chase, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Regents of the University of Minnesota
Intima Bioscience, Inc.
The U.S.A., as Represented by the Secretary, Department of Health
and Human Service |
Minneapolis
New York
Bethesda |
MN
NY
MD |
US
US
US |
|
|
Family ID: |
1000006038560 |
Appl. No.: |
17/481404 |
Filed: |
September 22, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16885874 |
May 28, 2020 |
11154574 |
|
|
17481404 |
|
|
|
|
15947688 |
Apr 6, 2018 |
10912797 |
|
|
16885874 |
|
|
|
|
PCT/US17/57228 |
Oct 18, 2017 |
|
|
|
15947688 |
|
|
|
|
62452244 |
Jan 30, 2017 |
|
|
|
62409651 |
Oct 18, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/7158 20130101;
C07K 14/70521 20130101; C12N 9/22 20130101; A61K 9/0019 20130101;
C12N 2310/20 20170501; C12N 2510/00 20130101; C12N 15/113 20130101;
C07K 14/7051 20130101; A61K 45/06 20130101; A61K 2300/00 20130101;
C07K 14/4703 20130101; C07K 14/70503 20130101; C12N 15/79 20130101;
A61P 35/00 20180101; A61P 31/00 20180101; A61K 35/17 20130101; C12N
15/902 20130101; C12N 5/0636 20130101; A61K 9/0053 20130101 |
International
Class: |
A61K 35/17 20060101
A61K035/17; C12N 15/113 20060101 C12N015/113; C07K 14/705 20060101
C07K014/705; C07K 14/47 20060101 C07K014/47; A61P 31/00 20060101
A61P031/00; A61P 35/00 20060101 A61P035/00; A61K 9/00 20060101
A61K009/00; C07K 14/725 20060101 C07K014/725; C07K 14/715 20060101
C07K014/715; C12N 5/0783 20060101 C12N005/0783; C12N 9/22 20060101
C12N009/22; C12N 15/79 20060101 C12N015/79; C12N 15/90 20060101
C12N015/90 |
Goverment Interests
GOVERNMENT INTEREST STATEMENT
[0002] This invention was made with government support under
project numbers Z01BC010985 and Z01BC010763 by the National
Institutes of Health, National Cancer Institute. The government has
certain rights in the invention.
Claims
1.-193. (canceled)
194. A method of treating a cancer in a subject in need thereof,
the method comprising: obtaining a population of human immune
cells; obtaining from said subject a population of cancer cells
from said cancer; exomic sequencing said cancer cells to identify
mutations uniquely present in said cancer cells as compared to
normal cells of said subject; screening said human immune cells in
vitro for reactivity to said mutations, thereby identifying cancer
reactive immune cells; genetically modifying said cancer reactive
immune cells by disrupting a target sequence of an endogenous
cytokine inducible SH2-containing protein gene that suppresses
expression of a protein encoded by said cytokine inducible
SH2-containing protein gene, thereby obtaining a population of
genetically modified cancer reactive immune cells; administering to
said subject a preparative regime that comprises administration of
at least one immunosuppressant to said subject in an amount
sufficient to reduce an immune response in said subject; and a
pharmaceutical composition that comprises said population of
genetically modified cancer reactive immune cells.
195. The method of claim 194, further comprising administering an
antibiotic to the subject.
196. The method of claim 194, further comprising administering an
antifungal agent to the subject.
197. The method of claim 194, wherein said preparative regime
comprises administration of said immunosuppressant from about 14
days to about 24 hours before administering said genetically
modified cancer reactive immune cells.
198. The method of claim 194, wherein said disrupting is by a
system selected from the group consisting of CRISPR, Zinc Finger,
TALEN, and any combination thereof.
199. The method of claim 198, wherein said system is a CRISPR
system.
200. The method of claim 199, wherein said CRISPR system comprises
an endonuclease selected from the group consisting of Cas1, Cas1B,
Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2,
Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5,
Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14,
Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1,
c2c1, c2c3, and Cas9HiFi.
201. The method of claim 200, wherein said endonuclease is
Cas9.
202. The method of claim 194, wherein said disruption is within
about 5 base pairs of a protospacer adjacent motif (PAM).
203. The method of claim 194, wherein said pharmaceutical
composition comprises genetically modified cancer reactive immune
cells that are cryopreserved at a freeze density from about
7.0.times.10.sup.7 cells/mL to about 2.0.times.10.sup.8
cells/mL.
204. The method of claim 194, wherein said genetically modified
cancer reactive immune cells undergo pre-infusion testing prior to
said administering, and wherein said pre-infusion testing comprises
phenotypic testing, potency testing, endotoxin testing, viability
testing, and tumor cell testing.
205. The method of claim 204, wherein said phenotypic testing
comprises detecting a presence of CD3 on said genetically modified
cancer reactive immune cells.
206. The method of claim 204, wherein said potency testing
comprises detecting a level of IFN.gamma. upon anti-CD3 stimulation
of said genetically modified cancer reactive immune cells.
207. The method of claim 204, wherein said endotoxin testing
comprises performing a limulus assay.
208. The method of claim 204, wherein said tumor cell testing
comprises a cytopathology assay.
209. The method of claim 204, wherein said genetically modified
cancer reactive immune cells are administered to said subject when
at least 70% of the cell population is determined to be viable
cells in said viability testing.
210. The method of claim 204, wherein said genetically modified
cancer reactive immune cells are administered when said
pre-infusion testing is at least about 80% CD3 positive for said
phenotypic testing.
211. The method of claim 204, wherein said genetically modified
cancer reactive immune cells are administered when said
pre-infusion testing is at least about 200 pg/mL per 10.sup.5 cells
of IFN.gamma. upon anti-CD3 stimulation of said genetically
modified cancer reactive immune cells in said potency testing.
212. The method of claim 204, wherein said genetically modified
cancer reactive immune cells are administered when said
pre-infusion testing is negative for tumor cells per at least about
200 genetically modified cancer reactive immune cells examined in
said cytopathology testing.
Description
CROSS-REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 16/885,874, filed May 28, 2020, which is a continuation of U.S.
application Ser. No. 15/947,688, filed Apr. 6, 2018, now U.S. Pat.
No. 10,912,797, issued Feb. 9, 2021, which is a continuation of
International Application No. PCT/US17/57228, filed Oct. 18, 2017,
which claims priority to U.S. Provisional Patent Application No.
62/409,651, filed Oct. 18, 2016, and U.S. Provisional Patent
Application No. 62/452,244, filed Jan. 30, 2017, each of which is
entirely incorporated herein by reference for all purposes.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Sep. 1, 2021, is named 199827_719304_SL.txt and is 65,536 bytes
in size.
BACKGROUND
[0004] The adoptive transfer of tumor infiltrating lymphocytes
(TIL) has achieved considerable success in mediating durable
regression of metastatic melanoma. In spite of these successes, the
use of TIL therapy in the setting of other solid tumors has been
challenging. This is likely due to the suppressive effects exerted
by the tumor microenvironment and T-cell intrinsic impairments in
receptor signaling and acquisition of effector functions.
Monoclonal antibody-based immune-checkpoint inhibitors targeting
programmed cell death protein 1 (PD-1) and cytotoxic T
lymphocyte-associated protein (CTLA-4) function can sometimes
relieve T-cell extrinsic suppressive effects, but carry risk of
potentially lethal autoimmune side-effects resulting from their
systemic activity on non-tumor reactive T-cells. Recently there
have been significant advances in the genetic engineering of
lymphocytes to recognize molecular targets on tumors in vivo,
resulting in remarkable cases of remission of the targeted tumor.
However, these successes have been limited largely to hematologic
tumors, and more broad application to solid tumors is limited by
the lack of an identifiable molecule that is expressed by cells in
a particular tumor, and lack of a molecule that can be used to
specifically bind to the tumor target in order to mediate tumor
destruction. Some recent advances have focused on identifying
tumor-specific mutations that in some cases trigger an antitumor T
cell response. For example, these endogenous mutations can be
identified using a whole-exomic-sequencing approach. Tran E, et
al., "Cancer immunotherapy based on mutation-specific CD4+ T cells
in a patient with epithelial cancer," Science 344: 641-644
(2014).
SUMMARY OF THE INVENTION
[0005] Disclosed herein is a method of treatment, comprising:
administering to a subject in need thereof a preparative regime
that comprises administration of at least one immunosuppressant to
the subject in an amount sufficient to reduce an immune response in
the subject; a pharmaceutical composition that comprises an
antifungal agent in an amount sufficient to inhibit a fungal
infection in the subject; and a pharmaceutical composition that
comprises a plurality of tumor infiltrating lymphocytes (TILs) that
comprise a disruption of at least a portion of a cytokine inducible
SH2-containing protein (CISH) gene. In some cases, a method can
further comprise administering an antibiotic.
[0006] Disclosed herein is a method of treatment, comprising:
administering to a subject in need thereof a preparative regime
that comprises administration of at least one immunosuppressant to
the subject in an amount sufficient to suppress an immune response
in the subject; a pharmaceutical composition that comprises an
antibiotic in an amount sufficient to inhibit a bacterial infection
in the subject; and a pharmaceutical composition that comprises a
plurality of tumor infiltrating lymphocytes (TILs) that comprise a
disruption of at least a portion of a cytokine inducible
SH2-containing protein (CISH) gene. In some cases, a method can
further comprise administering an antifungal agent.
[0007] Disclosed herein is a method of treatment, comprising:
administering to a subject in need thereof: a pharmaceutical
composition that comprises cyclophosphamide and fludarabine in an
amount sufficient to reduce an immune response in the subject; a
pharmaceutical composition that comprises fluconazole in an amount
sufficient to inhibit a fungal infection in the subject; and a
pharmaceutical composition that comprises a plurality of tumor
infiltrating lymphocytes (TILs) that comprise a disruption of at
least a portion of a cytokine inducible SH2-containing protein
(CISH) gene. In some cases, a method can further comprise
administering a pharmaceutical composition that comprises
trimethoprim and sulfamethoxazole in an amount sufficient to
inhibit a bacterial infection in the subject.
[0008] Disclosed herein is a method of treatment, comprising:
administering to a subject in need thereof a pharmaceutical
composition that comprises cyclophosphamide and fludarabine in an
amount sufficient to reduce an immune response in the subject; a
pharmaceutical composition that comprises trimethoprim and
sulfamethoxazole in an amount sufficient to inhibit a bacterial
infection in said subject; and a pharmaceutical composition that
comprises a plurality of tumor infiltrating lymphocytes (TILs) that
comprise a disruption of at least a portion of a cytokine inducible
SH2-containing protein (CISH) gene. In some cases, a method can
further comprise administering a pharmaceutical composition that
comprises fluconazole in an amount sufficient to inhibit a fungal
infection in the subject.
[0009] Disclosed herein is a method for treating cancer,
comprising: administering to a subject in need thereof a
therapeutically effective amount of ex vivo engineered tumor
infiltrating lymphocytes (TILs), wherein the ex vivo engineered
TILs comprise: a disruption in a Cytokine Inducible SH2 Containing
Protein (CISH) gene that results in suppression of CISH protein
function in the ex vivo engineered TILs, wherein the disruption is
at a sequence that is bound by a guiding polynucleic acid of SEQ ID
NO: 68. In some cases, a preparative regime comprises
administration of an immunosuppressant from about 14 days to about
24 hours before administering TILs. In some cases, a preparative
regime comprises administration of an immunosuppressant from about
10 days to about 24 hours before administering TILs. A preparative
regime can comprise administration of an immunosuppressant from
about 7 days to about 24 hours before administering TILs. In some
cases, an immunosuppressant comprises a radiotherapeutic agent, a
biologic agent, or a chemical agent. An immunosuppressant can
comprise a chemical agent. A chemical agent can comprise at least
one member selected from the group consisting of: cyclophosphamide,
mechlorethamine, chlorambucil, melphalan, ifosfamide, thiotepa,
hexamethylmelamine, busulfan, fludarabine, nitrosoureas, platinum,
methotrexate, azathioprine, mercaptopurine, procarbazine,
dacarbazine, temozolomide, carmustine, lomustine, streptozocin,
fluorouracil, dactinomycin, anthracycline, mitomycin C, bleomycin,
and mithramycin. A chemical agent can comprise cyclophosphamide. A
chemical agent can be fludarabine. Cyclophosphamide can be
administered from about 40 mg/kg to about 50 mg/kg of a subject.
Cyclophosphamide can be administered to a subject over at least
about 2 days to about 5 days. In some cases, a cyclophosphamide can
be administered from about 10 mg/kg to about 15 mg/kg of a subject.
In some cases, cyclophosphamide can be administered to a subject
over at least about 7 days to about 10 days. In some cases,
cyclophosphamide can be administered from about 3 mg per kg to
about 5 mg per kg of a subject. In some cases, cyclophosphamide can
be administered from about 50 mg per kg to about 80 mg/kg of a
subject. In some cases, cyclophosphamide can be administered in
excess of 50 mg per kg. In some cases, cyclophosphamide can be
administered at about 60 mg per kg. In some cases, fludarabine can
be administered from about 20 mg/m.sup.2 to about 30 mg/m.sup.2 of
body surface area of a subject. In some cases, fludarabine can be
administered at about 25 mg/m.sup.2 of body surface area of a
subject. In some cases, a preparative regime comprises partial or
complete immunosuppression. An antifungal can be selected from a
group consisting of: polyene, azole, allylamine, and echinocandin.
An antifungal can be an azole. An azole can be selected from the
group consisting of: bifonazole, butoconazole, clotrimazole,
econazole, fenticonazole, isoconazole, ketoconazole, luliconazole,
miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole,
tioconazole, albaconazole, efinaconazole, epoxiconazole,
fluconazole, isavuconazole, itraconazole, posaconazole,
propiconazole, ravuconazole, terconazole, and voriconazole. An
antifungal that is an azole can be fluconazole. In some cases,
fluconazole can be administered from about 100 mg to about 800 mg.
Fluconazole can be administered at 400 mg. An antifungal can be
administered concurrent or sequential to TILs. An antifungal can be
administered from about day 0 to about day 4 after TILs. In some
cases, an antibiotic comprises least one of: a bacterial wall
targeting agent, a cell membrane targeting agent, a bacterial
enzyme interfering agent, a bactericidal agent, a protein synthesis
inhibitor, or a bacteriostatic agent. In some cases, an antibiotic
comprises a bactericidal agent. A bactericidal agent can be
cephalosporin or quinolone. In some cases, an antibiotic comprises
a bacteriostatic agent. A bacteriostatic agent can be administered
prophylactically. In some cases, a bacteriostatic agent can be
trimethoprim, sulfamethoxazole, or pentamidine. Trimethoprim,
sulfamethoxazole, or pentamidine can be administered from about 100
mg to about 1000 mg. In some cases, trimethoprim can be
administered at 160 mg. In some cases, sulfamethoxazole can be
administered at 800 mg. In some cases, pentamidine can be
administered at 300 mg. A bacteriostatic agent can be administered
prior to TILs, concurrent with TILs, or after TILs. A
bacteriostatic agent can be administered from about 14 days prior
to administration of TILs to about 6 months after administration of
TILs. In some cases, a bacteriostatic agent can be administered
from about 8 days prior to said TILs to at least 4 days after
TILs.
[0010] In some cases, administering comprises intravenous, oral,
intramuscular, intraperitoneal, or intrapleural administration. In
some cases, an immunosuppressant can be administered by infusion.
In some cases, cyclophosphamide can be at a dose of about 60 mg/kg
and is diluted in 250 ml 5% dextrose in water and infused over one
hour. In some cases, fludarabine can be at a dose of 25 mg/m.sup.2
in 100 ml 0.9% sodium chloride, USP and infused over about 15 to
about 30 minutes. A method can further comprise administering a
pharmaceutical composition that comprises an immunostimulant in an
amount sufficient to activate TILs in a subject. An immunostimulant
can comprise at least one member selected from the group consisting
of: vaccines, colony stimulating agents, interferons, interleukins,
viruses, antigens, co-stimulatory agents, immunogenicity agents,
immunomodulators, or immunotherapeutic agents. An immunostimulant
can comprise an interleukin An interleukin can be aldesleukin and
can be administered at a dose from about 550,000 to about 800,000
IU/kg. In some cases, aldesleukin can be administered at a dose of
about 720,000 IU/kg. In some cases, a method can further comprise
administering a pharmaceutical composition that comprises an
infection prophylaxis agent in an amount sufficient to prevent an
infection in a subject. In some cases, an infection prophylaxis
agent can be a herpes virus prophylactic agent. In some cases, a
subject can be HSV positive. In some cases, a herpes virus
prophylactic agent can be valacyclovir or acyclovir. In some cases,
a disruption can be induced by a system selected from the group
consisting of CRISPR, Zinc Finger, TALEN, and any combination
thereof. A system can be a CRISPR system. A CRISPR system can
comprise an endonuclease selected from the group consisting of
Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10,
Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3,
Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3,
Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2,
CsO, Csf4, Cpf1, c2c1, c2c3, and Cas9HiFi. An endonuclease can be
Cas9. In some cases, an endonuclease performs a disruption. A
disruption can comprise an exon or an intron of a gene. A
disruption can be in an exon of a gene. A disruption can be within
about 20 base pairs of a protospacer adjacent motif (PAM). A
disruption can be within about 10 base pairs of a protospacer
adjacent motif (PAM). A disruption can be within about 5 base pairs
of a protospacer adjacent motif (PAM). A disruption can be 3 base
pairs from a protospacer adjacent motif (PAM). In some cases, a
disruption is in an exon or intron of SEQ ID NO: 13. A disruption
can be in an exon of SEQ ID NO: 13. In some cases, a CRISPR system
comprises a guiding polynucleic acid. A guiding polynucleic can
comprise at least about 60% homology to SEQ ID NO: 68. In some
cases, a disruption comprises a double strand break. A double
strand break can occur at SEQ ID NO: 71 or SEQ ID NO: 77. In some
cases, TILs can be administered at a dose from about
1.times.10.sup.9 cells, 3.times.10.sup.9 cells, 1.times.10.sup.10
cells, 3.times.10.sup.10 cells, and to about 1.times.10.sup.11
cells. In some cases, TILs can be administered over about 30
minutes. In some cases, TILs can be administered intravenously.
Intravenous administration can comprise an infusion.
[0011] In some cases, a method can further comprise performing a
pre-infusion testing on TILs. A pre-infusion testing can comprise
at least one of: phenotypic testing, potency testing,
microbiological testing, endotoxin testing, viability testing, and
tumor cell testing. In some cases, a phenotypic testing comprises
detecting a presence of CD3 on TILs. In some cases, a potency
testing comprises detecting a level of IFN.gamma. upon anti-CD3
stimulation of TILs. In some cases, a microbiological testing
comprises detecting growth of an aerobic culture, anaerobic
culture, gram status, fungal status, or mycoplasma status. An
endotoxin testing can comprise performing a limulus assay. In some
cases, a viability testing comprises performing a trypan blue
exclusion assay. In some cases, a tumor cell testing comprises a
cytopathology assay. In some cases, TILs can be administered when a
pre-infusion testing can be negative for a microbiological testing.
In some cases, TILs can be administered when a pre-infusion testing
is over at least about 70% viable cells for said viability testing.
In some cases, TILs can be administered when a pre-infusion testing
is at least about 80% CD3 positive for a phenotypic testing. In
some cases, TILs can be administered when a pre-infusion testing is
at least about 200 pg/mL per 10.sup.5 cells of IFN.gamma. upon
anti-CD3 stimulation of TILs in a potency testing. In some cases,
TILs can be administered when a pre-infusion testing can be
negative for tumor cells per at least about 200 TILs examined in a
cytopathology testing.
[0012] Disclosed herein is a therapeutic product that comprises a
dosage form of a plurality of tumor infiltrating lymphocytes (TILs)
that comprise a disruption of at least a portion of a cytokine
inducible SH2-containing protein (CISH) gene; and a dosage form of
an antifungal agent.
[0013] Disclosed herein is a therapeutic product that comprises a
dosage form of a folate synthesis inhibitor or a nucleic acid
cross-linking agent in an amount sufficient to inhibit a fungal
infection in a subject; and a dosage form of a plurality of tumor
infiltrating lymphocytes (TILs) that comprise a disruption of at
least a portion of a cytokine inducible SH2-containing protein
(CISH) gene.
[0014] Disclosed herein is a therapeutic product that comprises a
dosage form of an anti-fungal agent selected from a polyene, azole,
allylamine, or echinocandins; and a dosage form of a plurality of
tumor infiltrating lymphocytes (TILs) that comprise a disruption of
at least a portion of a cytokine inducible SH2-containing protein
(CISH) gene.
[0015] Disclosed herein is a therapeutic product that comprises a
dosage form of a plurality of tumor infiltrating lymphocytes (TILs)
that comprise a disruption of at least a portion of a cytokine
inducible SH2-containing protein (CISH) gene wherein the TILs are
cryopreserved at a freeze density from about 7.0.times.10.sup.7
cells/mL to about 2.0.times.10.sup.8 cells/mL.
[0016] Disclosed herein is a therapeutic product that comprises: a
dosage form of an anti-fungal agent; a dosage form of an
immunosuppressant; a dosage form of an antibiotic; and a dosage
form of a plurality of tumor infiltrating lymphocytes (TILs) that
comprise a disruption of at least a portion of a cytokine inducible
SH2-containing protein (CISH) gene. In some cases, a therapeutic
product can further comprise a dosage form of an immunosuppressant.
An immunosuppressant can be formulated for administration to a
subject from about 14 days to about 24 hours before an
administration of TILs. In some cases, an immunosuppressant can be
formulated for administration to a subject from about 10 days to
about 24 hours before an administration of TILs. In some cases, an
immunosuppressant can be formulated for administration to a subject
from about 7 days to about 24 hours before an administration of
TILs. In some cases, an immunosuppressant comprises a
radiotherapeutic agent, a biologic agent, or a chemical agent. In
some cases, an immunosuppressant comprises a chemical agent. In
some cases, a chemical agent comprises at least one member selected
from the group consisting of: cyclophosphamide, mechlorethamine,
chlorambucil, melphalan, ifosfamide, thiotepa, hexamethylmelamine,
busulfan, fludarabine, nitrosoureas, platinum, methotrexate,
azathioprine, mercaptopurine, procarbazine, dacarbazine,
temozolomide, carmustine, lomustine, streptozocin, fluorouracil,
dactinomycin, anthracycline, mitomycin C, bleomycin, and
mithramycin. In some cases, a chemical agent comprises
cyclophosphamide. In some cases, a chemical agent comprises
fludarabine. In some cases, cyclophosphamide can be administered
from about 40 mg/kg to about 50 mg/kg of a subject. In some cases,
cyclophosphamide can be administered to a subject over at least
about 2 days to about 5 days. In some cases, cyclophosphamide can
be administered from about 10 mg/kg to about 15 mg/kg of a subject.
In some cases, cyclophosphamide can be administered to a subject
over at least about 7 days to about 10 days. In some cases,
cyclophosphamide can be administered from about 3 mg per kg to
about 5 mg per kg of a subject. In some cases, cyclophosphamide can
be administered from about 50 mg per kg to about 80 mg/kg of a
subject. In some cases, cyclophosphamide can be administered in
excess of 50 mg per kg. In some cases, cyclophosphamide can be
administered at about 60 mg per kg. In some cases, fludarabine can
be administered from about 20 mg/m.sup.2 to about 30 mg/m.sup.2 of
body surface area of a subject. In some cases, fludarabine can be
administered at about 25 mg/m.sup.2 of body surface area of a
subject. In some cases, an immunosuppressant produces a partial or
complete immunosuppression. In some cases, a dosage form of an
antifungal agent can be selected from a group consisting of:
polyene, azole, allylamine, and echinocandin. In some cases, an
antifungal agent can be an azole. An azole can be selected from the
group consisting of: bifonazole, butoconazole, clotrimazole,
econazole, fenticonazole, isoconazole, ketoconazole, luliconazole,
miconazole, omoconazole, oxiconazole, sertaconazole, sulconazole,
tioconazole, albaconazole, efinaconazole, epoxiconazole,
fluconazole, isavuconazole, itraconazole, posaconazole,
propiconazole, ravuconazole, terconazole, and, voriconazole. In
some cases, an azole can be fluconazole. In some cases, fluconazole
can be present in an amount from about 100 mg to about 800 mg. In
some cases, fluconazole can be present in an amount of 400 mg. In
some cases, a dosage form of an antifungal can be administered
concurrent or sequential to TILs. A dosage form of an antifungal
can be administered from about day 0 to about day 4 after TILs. In
some cases, a therapeutic product can further comprise a dosage
form of an antibiotic. An antibiotic can comprise at least one of:
a bacterial wall targeting agent, a cell membrane targeting agent,
a bacterial enzyme interfering agent, a bactericidal agent, a
protein synthesis inhibitor, and bacteriostatic agent. In some
cases, an antibiotic comprises a bactericidal agent. A bactericidal
agent can be cephalosporin or quinolone. In some cases, a dosage
form of an antibiotic can be a bacteriostatic agent. A
bacteriostatic agent can be formulated for prophylactic
administration. In some cases, a bacteriostatic agent can be
trimethoprim, sulfamethoxazole, or pentamidine In some cases,
trimethoprim, sulfamethoxazole, or pentamidine can be present in an
amount of from about 100 mg to about 1000 mg. In some cases,
trimethoprim can be present in an amount of 160 mg. In some cases,
sulfamethoxazole can be present in an amount of 800 mg. In some
cases, pentamidine can be present in an amount of 300 mg. In some
cases, a bacteriostatic agent can be administered prior to TILs,
concurrent with TILs, or after TILs. In some cases, a
bacteriostatic agent can be administered from about 14 days prior
to said administration of said TILs to about 6 months after said
administration of said TILs. In some cases, a bacteriostatic agent
can be administered from about 8 days prior to said TILs to at
least 4 days after said TILs. A therapeutic product can further
comprise administering a dosage form.
[0017] A therapeutic product can be formulated for administered by
intravenous, oral, intramuscular, intraperitoneal, or intrapleural
administration. In some cases, a dosage form of an
immunosuppressant can be formulated for administration by infusion.
In some cases, cyclophosphamide can be administered at a dose of
about 60 mg/kg and is diluted in 250 ml 5% dextrose in water and
infused over one hour. In some cases, fludarabine can be
administered at a dose of 25 mg/m.sup.2 in 100 ml 0.9% sodium
chloride, USP and infused over about 15 to about 30 minutes. In
some cases, an immunostimulant can be present in an amount
sufficient to activate TILs in a subject. An immunostimulant
comprises at least one member selected from the group consisting
of: vaccines, colony stimulating agents, interferons, interleukins,
viruses, antigens, co-stimulatory agents, immunogenicity agents,
immunomodulators, and immunotherapeutic agents. In some cases, a
dosage form of an immunostimulant comprises an interleukin. An
interleukin can be aldesleukin and can be administered at a dose
from about 550,000 to about 800,000 IU/kg. In some cases,
aldesleukin can be administered at a dose of about 720,000 IU/kg.
In some cases, a therapeutic product can further comprise a dosage
form of an infection prophylaxis agent in an amount sufficient to
prevent an infection in a subject. An infection prophylaxis agent
can be a herpes virus prophylactic agent. A herpes virus
prophylactic agent can be present in an amount effect to treat an
HSV positive subject. In some cases, a herpes virus prophylactic
agent can be valacyclovir or acyclovir. In some cases, a disruption
can be induced by a system selected from the group consisting of
CRISPR, Zinc Finger, TALEN, and any combination thereof. A system
can be a CRISPR system. A CRISPR system can comprise an
endonuclease selected from the group consisting of Cas1, Cas1B,
Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9, Cas10, Csy1, Csy2,
Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5,
Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14,
Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1,
c2c1, c2c3, and Cas9HiFi. In some cases, an endonuclease can be
Cas9. An endonuclease can perform a disruption. A disruption can
comprise an exon or an intron of a gene. In some cases, a
disruption can be in an exon of a gene. In some cases, a disruption
can be within about 20 base pairs of a protospacer adjacent motif
(PAM). In some cases, a disruption can be within about 10 base
pairs of a protospacer adjacent motif (PAM). In some cases, a
disruption can be within about 5 base pairs of a protospacer
adjacent motif (PAM). In some cases, a disruption can be 3 base
pairs from a protospacer adjacent motif (PAM). In some cases, a
disruption can be in an exon or intron of SEQ ID NO: 13. In some
cases, a disruption can be in an exon of SEQ ID NO: 13. In some
cases, a CRISPR system comprises a guiding polynucleic acid. A
guiding polynucleic can comprise at least about 60% homology to SEQ
ID NO: 68. A disruption can comprise a double strand break. In some
cases, a double strand break occurs at SEQ ID NO: 71 or SEQ ID NO:
77. In some cases, TILs can be present in an amount from about
1.times.10.sup.9 cells, 3.times.10.sup.9 cells, 1.times.10.sup.10
cells, 3.times.10.sup.10 cells, and to about 1.times.10.sup.11
cells. In some cases, TILs can be administered over about 30
minutes. In some cases, TILs can be administered intravenously. An
intravenous administration can comprise an infusion. In some cases,
a therapeutic product can further comprise performing a
pre-infusion testing on TILs. In some cases, pre-infusion testing
comprises at least one of: phenotypic testing, potency testing,
microbiological testing, endotoxin testing, viability testing, and
tumor cell testing. In some cases, phenotypic testing comprises
detecting a presence of CD3 on said TILs. In some cases, potency
testing comprises detecting a level of IFN.gamma. upon anti-CD3
stimulation of TILs. In some cases, microbiological testing
comprises detecting growth of an aerobic culture, anaerobic
culture, gram status, fungal status, or mycoplasma status. In some
cases, endotoxin testing comprises performing a limulus assay. In
some cases, a viability testing comprises performing a trypan blue
exclusion assay. In some cases, a tumor cell testing comprises a
cytopathology assay. In some cases, TILs can be administered when a
pre-infusion testing is negative for said microbiological testing.
In some cases, TILs can be administered when a pre-infusion testing
has over at least about 70% viable cells for a viability testing.
In some cases, TILs can be administered when a pre-infusion testing
is at least about 80% CD3 positive for a phenotypic testing. TILs
can be administered when a pre-infusion testing is at least about
200 pg/mL per 10.sup.5 cells of IFN.gamma. upon anti-CD3
stimulation of TILs in a potency testing. In some cases, TILs can
be administered when a pre-infusion testing can be negative for
tumor cells per at least about 200 TILs examined in a cytopathology
testing.
[0018] Disclosed herein is a nucleic acid composition that
comprises at least 60% sequence homology to any one of SEQ ID NO:
64 to SEQ ID NO: 69. In some cases, a sequence homology can be at
least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,
or up to about 100%.
[0019] Disclosed herein is a method of treating a condition such as
cancer comprising obtaining tumor infiltrating lymphocytes (TIL)
from a tumor sample, identifying a mutation reactive TIL, and
disrupting an endogenous gene or portion thereof with a CRISPR
nuclease. In some cases, a tumor sample can be subjected to
sequencing analysis. In some cases, sequencing analysis identifies
a mutation in a tumor sample and not in a non-tumor sample. In some
cases, sequencing analysis can comprise whole exomic sequencing,
transcriptome sequencing, or a combination thereof. Sequencing
analysis can be whole exomic sequencing.
[0020] In some cases, identifying can comprise introducing a TIL to
an antigen presenting cell (APC) expressing a peptide comprising a
mutation. In some cases, identifying can further comprise detecting
a presence of interferon y (IFNy) secreted by a TIL introduced to
an APC expressing a peptide comprising a mutation. A peptide can
comprise a length from about 15mer up to about 30mer. A peptide can
comprise a length of 25mers. In some cases, identifying can
comprise introducing a TIL to an antigen presenting cell (APC)
electroporated with a polynucleic acid comprising a mutation.
[0021] In some cases, identifying can further comprise detecting a
presence of interferon y (IFNy) secreted by a TIL introduced to an
APC. Disrupting can comprise a double strand break in a genome of a
TIL. A double strand break can be performed by a CRISPR nuclease.
In some cases, a CRISPR nuclease can be selected from the group
consisting of Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7,
Cas8, Cas9, Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2, Csa5,
Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6,
Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1,
Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, c2c1, c2c3, and Cas9HiFi.A
CRISPR nuclease can be Cas9.
[0022] In some cases, a method can further comprise expanding a
TIL. A TIL can be human. A method can further comprise
administering an anti-fungal to a subject. A method can further
comprise administering an antibiotic to a subject.
[0023] Disclosed herein is a method of treating gastrointestinal
cancer in a subject in need thereof comprising obtaining tumor
infiltrating lymphocytes (TIL) from a gastrointestinal tumor sample
of a subject in need thereof, identifying a mutation reactive TIL,
disrupting a CISH gene with a CRISPR nuclease in said mutation
reactive TIL and expanding said TIL to a dose of 1.times.10.sup.9
to about 2.times.10.sup.11; wherein said gastrointestinal tumor
sample is exomically sequenced to identify a mutation present in
said tumor sample and not in a healthy tissue sample and wherein
said identifying comprises presenting said mutation on a surface of
an antigen presenting cell (APC) and culturing said APC with said
TIL to detect a level of IFN-.gamma.. In some cases, presenting can
comprise culturing an antigen presenting cell (APC) electroporated
with a polynucleic acid comprising a mutation with a TIL. In some
cases, presenting can comprise culturing an antigen presenting cell
(APC) pulsed with a peptide comprising a mutation with a TIL. A
cytokine can be detected in a mutation reactive TIL and not
detected in a non-mutation reactive TIL. A cytokine can be detected
in a mutation reactive TIL at a higher level than in a non-mutation
reactive TIL. A CRISPR nuclease can be Cas9. A TIL can be a T
cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] The novel features of the invention are set forth with
particularity in the appended claims. A better understanding of the
features and advantages of the present invention will be obtained
by reference to the following detailed description that sets forth
illustrative embodiments, in which the principles of the invention
are utilized, and the accompanying drawings of which:
[0025] FIG. 1 demonstrates the structures of four plasmids,
including Cas9 nuclease plasmid, HPRT gRNA plasmid, Amaxa EGFPmax
plasmid and HPRT target vector.
[0026] FIG. 2 shows % gene modification occurring by CRISPR gRNAs
at potential target sites.
[0027] FIG. 3 demonstrates CRISPR-induced DSBs in stimulated T
cells.
[0028] FIG. 4A and FIG. 4B show PD-1, CTLA-4, PD-1 and CTLA-2, or
CCR5, PD-1, and CTLA-4 expression on day 6 post transfection with
guide RNAs. Representative guides: PD-1 (P2, P6, P2/6), CTLA-4 (C2,
C3, C2/3), or CCR5 (CC2). A. shows percent inhibitory receptor
expression. B. shows normalized inhibitory receptor expression to a
control guide RNA.
[0029] FIG. 5A show CTLA-4 expression in primary human T cells
after electroporation with CRISPR and CTLA-4 specific guide RNAs,
guides #2 and #3, as compared to unstained and a no guide control.
FIG. 5B. shows PD-1 expression in primary human T cells after
electroporation with CRISPR and PD-1 specific guide RNAs, guides #2
and #6, as compared to unstained and a no guide control.
[0030] FIG. 6 shows FACs results of CTLA-4 and PD-1 expression in
primary human T cells after electroporation with CRISPR and
multiplexed CTLA-4 and PD-1 guide RNAs.
[0031] FIG. 7 A and FIG. 7 B show percent double knock out in
primary human T cells post treatment with CRISPR. FIG. 7A. shows
percent CTLA-4 knock out in T cells treated with CTLA-4 guides #2,
#3, #2 and #3, PD-1 guide #2 and CTLA-4 guide #2, PD-1 guide #6 and
CTLA-4 guide #3, as compared to Zap only, Cas9 only, and an all
guide RNA control. FIG. 7B. shows percent PD-1 knock out in T cells
treated with PD-1 guide#2, PD-1 guide #6, PD-1 guides #2 and #6,
PD-1 guide #2 and CTLA-4 guide #2, PD-1 guide #6 and CTLA-4 guide
#3, as compared to Zap only, Cas9 only, and an all guide RNA
control.
[0032] FIG. 8 shows T cell viability post electroporation with
CRISPR and guide RNAs specific to CTLA-4, PD-1, or
combinations.
[0033] FIG. 9 results of a CEL-I assay showing cutting by PD-1
guide RNAs #2, #6, #2 and #6, under conditions where only PD-1
guide RNA is introduced, PD-1 and CTLA-4 guide RNAs are introduced
or CCR5, PD-1, and CLTA-4 guide RNAs, Zap only, or gRNA only
controls.
[0034] FIG. 10 results of a CEL-I assay showing cutting by CTLA-4
guide RNAs #2, #3, #2 and #3, under conditions where only CLTA-4
guide RNA is introduced, PD-1 and CTLA-4 guide RNAs are introduced
or CCR5, PD-1, and CLTA-4 guide RNAs, Zap only, or gRNA only
controls.
[0035] FIG. 11 results of a CEL-I assay showing cutting by CCR5
guide RNA #2 in conditions where CCR5 guide RNA is introduced, CCR5
guide RNA, PD-1 guide RNA, or CTLA-4 guide RNA, as compared to Zap
only, Cas 9 only, or guide RNA only controls.
[0036] FIG. 12 shows knockout of TCR alpha, as measured by CD3 FACs
expression, in primary human T cells utilizing optimized CRISPR
guide RNAs with 2' O-Methyl RNA modification at 5 micrograms and 10
micrograms.
[0037] FIG. 13 depicts a method of measuring T cell viability and
phenotype post treatment with CRISPR and guide RNAs to CTLA-4.
Phenotype was measured by quantifying the frequency of treated
cells exhibiting a normal FSC/SSC profile normalized to frequency
of electroporation alone control. Viability was also measured by
exclusion of viability dye by cells within the FSC/SSC gated
population. T cell phenotype is measured by CD3 and CD62L.
[0038] FIG. 14 shows method of measuring T cell viability and
phenotype post treatment with CRISPR and guide RNAs to PD-1, and
PD-1 and CTLA-4. Phenotype was measured by quantifying the
frequency of treated cells exhibiting a normal FSC/SSC profile
normalized to frequency of electroporation alone control. Viability
was also measured by exclusion of viability dye by cells within the
FSC/SSC gated population. T cell phenotype is measured by CD3 and
CD62L.
[0039] FIG. 15 shows results of a T7E1 assay to detect CRISPR gene
editing on day 4 post transfection with PD-1 or CTLA-4 guide RNA of
primary human T cells and Jurkat control. NN is a no T7E1 nuclease
control.
[0040] FIG. 16 shows results of a tracking of indels by
decomposition (TIDE) analysis. Percent gene editing efficiency as
shows to PD-1 and CTLA-4 guide RNAs.
[0041] FIG. 17 shows results of a tracking of indels by
decomposition (TIDE) analysis for single guide transfections.
Percent of sequences with either deletions or insertions are shown
for primary human T cells transfected with PD-1 or CTLA-1 guide
RNAs and CRISPR.
[0042] FIG. 18 shows PD-1 sequence deletion with dual
targeting.
[0043] FIG. 19 shows sequencing results of PCR products of PD-1
sequence deletion with dual targeting. Samples 6 and 14 are shown
with a fusion of the two gRNA sequences with the intervening 135bp
excised.
[0044] FIG. 20 shows dual targeting sequence deletion of CTLA-4.
Deletion between the two guide RNA sequences is also present in the
sequencing of dual guide targeted CTLA-4 (samples 9 and 14). A T7E1
Assay confirms the deletion by PCR.
[0045] FIG. 21 A and FIG. 21B show CTLA-4 FACs analysis of CTLA-4
positive human T cells post transfection with anti-CTLA-4 guide
RNAs and CRISPR. B. shows CTLA-4 knock out efficiency relative to a
pulsed control in human T cells post transfection with anti-CTLA-4
guide RNAs and CRISPR.
[0046] FIG. 22 depicts modified sgRNA for CISH, PD-1, CTLA4 and
AAVS1.
[0047] FIG. 23A shows percent PD-1 expression post transfection
with an anti-PD-1 CRISPR system. FIG. 23B. shows percent PD-1 knock
out efficiency as compared to Cas9 only control.
[0048] FIG. 24 depicts quantification data from a FACs analysis of
CTLA-4 stained human T cells transfected with CRISPR and
anti-CTLA-4 guide RNAs. Day 6 post transfection data is shown of
percent CTLA-4 expression and percent knock out.
[0049] FIG. 25 shows FACs analysis of PD-1 stained human T cells
transfected with CRISPR and anti-PD-1 guide RNAs. Day 14 post
transfection data is shown of PD-1 expression (anti-human CD279
PerCP-Cy5.5)
[0050] FIG. 26 shows percent PD-1 expression and percent knock out
of PD-1 compared to Cas9 only control of human T cells transfected
with CRISPR and anti-PD-1 guide RNAs.
[0051] FIG. 27 shows day 14 cell count and viability of transfected
human T cells with CRISPR, anti-CTLA-4, and anti-PD-1 guide
RNAs.
[0052] FIG. 28 shows FACs data for human T cells on day 14 post
electroporation with CRISPR, and anti-PD-1 guide #2 alone,
anti-PD-1 guide #2 and #6, or anti-CTLA-4 guide #3 alone. The
engineered T cells were re-stimulated for 48 hours to assess
expression of CTLA-4 and PD-1 and compared to control cells
electroporated with no guide RNA.
[0053] FIG. 29 shows FACs data for human T cells on day 14 post
electroporation with CRISPR, and anti-CTLA-4 guide #2 and #3,
anti-PD-1 guide #2 and anti-CTLA-4 guide #3, or anti-PD-1 guide #2
and #6, anti-CTLA-4 guide #3 and #2. The engineered T cells were
re-stimulated for 48 hours to assess expression of CTLA-4 and PD-1
and compared to control cells electroporated with no guide RNA.
[0054] FIG. 30 depicts results of a surveyor assay for CRISPR
mediated gene-modification of the CISH locus in primary human T
cells.
[0055] FIG. 31 shows that somatic mutational burden varies among
tumor type. Tumor-specific neo-antigen generation and presentation
is theoretically directly proportional to mutational burden.
[0056] FIG. 32 shows pseudouridine-5'-Triphosphate and
5-Methylcytidine-5-Triphosphate modifications that can be made to
nucleic acid.
[0057] FIG. 33 depicts duplicate experiments of densitometry
analysis for 293T cells transfected with CRISPR and CISH gRNAs
1,3,4,5 or 6.
[0058] FIG. 34A and FIG. 34B show duplicate TIDE analysis of CISH
gRNA 1.
[0059] FIG. 35A and FIG. 35B show duplicate TIDE analysis of CISH
gRNA 3.
[0060] FIG. 36A and FIG. 36B show duplicate TIDE analysis of CISH
gRNA 4.
[0061] FIG. 37A and FIG. 37B show duplicate TIDE analysis of CISH
gRNA 5.
[0062] FIG. 38A and FIG. 38B show duplicate TIDE analysis of CISH
gRNA 6.
[0063] FIG. 39 shows a western blot showing loss of CISH protein
after CRISPR knock out in primary T cells.
[0064] FIG. 40A and FIG. 40B show absolute cell count pre and post
stimulation of human TILs. FIG. 40A. shows a first donor's cell
count pre- and post-stimulation cultured in either RPMI media or ex
vivo media. FIG. 40B. shows a second donor's cell count pre- and
post-stimulation cultured in RPMI media.
[0065] FIG. 41A and FIG. 41B show cellular expansion of human tumor
infiltrating lymphocytes (TILs) electroporated with a CRISPR system
targeting PD-1 locus or controls cells FIG. 41A shows expansion
with the addition of autologous feeders or FIG. 41B shows expansion
without the addition of autologous feeders.
[0066] FIG. 42 depicts production of CISH KO TIL Interleukin-2
expanded TILs are stimulated by incubation with immobilized
anti-CD3 and soluble anti-CD28 for four days. On day 0, TILs are
collected and CRISPR/Cas9 reagents targeting CISH are delivered by
electroporation. Following electroporation, the modified TILs are
transferred to G-Rex flasks in the presence of irradiated
peripheral blood mononuclear cell feeders and IL-2 for subsequent
rapid expansion. During the first cell count and upon subculture at
day (+)7, a small aliquot of cells is collected to determine
insertion/deletion (indel) frequency by sequencing. TILs are
subsequently harvested at day (+) 14 at which point samples are
taken for quality control assessment and are subsequently
cryopreserved within infusion-ready bags. FIG. 43A, FIG. 43B, and
FIG. 43C depict TiDE analysis of CISH loci after CRISPR/Cas9
editing. After 14 days of rapid expansion, genomic DNA was isolated
and PCR was run across the CRISPR target region within PDCD1 and
CISH. PCR amplicons were subjected to TiDE analysis. Plots indicate
the total frequency of indels, and the distribution of insertions
and deletions based on their size in base pairs lost or gained for
subjects PV1, PV2, and PV3.
[0067] FIG. 44 depicts a summary of TiDE analysis of CISH loci
after CRISPR/Cas9 editing for subjects PV1, PV2, and PV3.
[0068] FIG. 45A and FIG. 45B show loss of CISH protein expression
after CRISPR/Cas9 editing. Fourteen days after electroporation, PB
T-cells and TILs were re-stimulated or not re-stimulated for 48
hours to induce CISH expression. Control indicates that cells
received no CRISPR/Cas9 components. Cas9+gRNA indicates cells that
received Cas9 mRNA and a gRNA designed to target exon 3 of the CISH
locus.
[0069] FIG. 46A, FIG. 46B, and FIG. 46C show growth and viability
of T-cells after CRISPR editing. FIG. 46A shows viability at day 6
post-electroporation. FIG. 46B shows total cell numbers at day 12
post electroporation beginning with 3.times.10.sup.6 cells.
Cells=no manipulation; ZAP=electroporation only. FIG. 46C shows
total cell numbers of TIL over 11 days in standard tissue culture
flasks beginning with 3.times.10.sup.6 cells for each
condition.
[0070] FIG. 47A and FIG. 47B show SPICE plot of cytokine production
in CRISPR/Cas9 edited T-cells and TILs. Fourteen days after
electroporation, T-cells and TILs were re-stimulated using
plate-bound anti-CD3 and soluble anti-CD28 antibody and assayed for
cytokine production by intracellular staining and flow
cytometry.
[0071] FIG. 48A depicts loss of PD-1 protein expression after
CRISPR/Cas9 editing. Fourteen days after electroporation,
peripheral blood T-cells or TILs were re-stimulated for 72 hours to
induce PD-1 expression. PD-1 expression in re-stimulated T-cells.
FIG. 48B shows PD-1 expression in re-stimulated TILs. Percent loss
is indicated in red.
[0072] FIG. 49 shows absolute cell number of control and
CISH-modified tumor infiltrating lymphocytes.
[0073] FIG. 50A shows control and CISH knockout TIL cultured in the
presence of IL-2 at day 7. FIG. 50B shows control and CISH knockout
TIL cultured in the absence of IL-2 at day 7.
[0074] FIG. 51A shows off-target sites for PDCD1. FIG. 51B shows
CISH gRNA identified by GUIDE-Seq.
[0075] FIG. 52 shows frequency of targeted indels at the CISH locus
across the GUIDE-Seq and GMP PQ manufacturing runs (ns,
P=0.93).
DETAILED DESCRIPTION OF THE DISCLOSURE
[0076] The following description and examples illustrate
embodiments of the invention in detail. It is to be understood that
this invention is not limited to the particular embodiments
described herein and as such can vary. Those of skill in the art
will recognize that there are numerous variations and modifications
of this invention, which are encompassed within its scope.
Definitions
[0077] The term "about" and its grammatical equivalents in relation
to a reference numerical value and its grammatical equivalents as
used herein can include a range of values plus or minus 10% from
that value. For example, the amount "about 10" includes amounts
from 9 to 11. The term "about" in relation to a reference numerical
value can also include a range of values plus or minus 10%, 9%, 8%,
7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.
[0078] The term "activation" and its grammatical equivalents as
used herein can refer to a process whereby a cell transitions from
a resting state to an active state. This process can comprise a
response to an antigen, migration, and/or a phenotypic or genetic
change to a functionally active state. For example, the term
"activation" can refer to the stepwise process of T cell
activation. For example, a T cell can require at least two signals
to become fully activated. The first signal can occur after
engagement of a TCR by the antigen-MHC complex, and the second
signal can occur by engagement of co-stimulatory molecules.
Anti-CD3 can mimic the first signal and anti-CD28 can mimic the
second signal in vitro.
[0079] The term "adjacent" and its grammatical equivalents as used
herein can refer to right next to the object of reference. For
example, the term adjacent in the context of a nucleotide sequence
can mean without any nucleotides in between. For instance,
polynucleotide A adjacent to polynucleotide B can mean AB without
any nucleotides in between A and B.
[0080] The term "antigen" and its grammatical equivalents as used
herein can refer to a molecule that contains one or more epitopes
capable of being bound by one or more receptors. For example, an
antigen can stimulate a host's immune system to make a cellular
antigen-specific immune response when the antigen is presented, or
a humoral antibody response. An antigen can also have the ability
to elicit a cellular and/or humoral response by itself or when
present in combination with another molecule. For example, a tumor
cell antigen can be recognized by a TCR. An antigen, such as a
Neoantigen, can be associated with tumors of high mutational
burden, FIG. 31.
[0081] The term "epitope" and its grammatical equivalents as used
herein can refer to a part of an antigen that can be recognized by
antibodies, B cells, T cells or engineered cells. For example, an
epitope can be a cancer epitope that is recognized by a TCR.
Multiple epitopes within an antigen can also be recognized The
epitope can also be mutated.
[0082] The term "autologous" and its grammatical equivalents as
used herein can refer to as originating from the same being. For
example, a sample (e.g., cells) can be removed, processed, and
given back to the same subject (e.g., patient) at a later time. An
autologous process is distinguished from an allogenic process where
the donor and the recipient are different subjects.
[0083] The term "cancer" and its grammatical equivalents as used
herein can refer to a hyperproliferation of cells whose unique
trait--loss of normal controls--results in unregulated growth, lack
of differentiation, local tissue invasion, and metastasis. With
respect to the inventive methods, the cancer can be any cancer,
including any of acute lymphocytic cancer, acute myeloid leukemia,
alveolar rhabdomyosarcoma, bladder cancer, bone cancer, brain
cancer, breast cancer, cancer of the anus, anal canal, rectum,
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,
fibrosarcoma, gastrointestinal cancer, Hodgkin lymphoma,
hypopharynx cancer, kidney cancer, larynx cancer, leukemia, liquid
tumors, liver cancer, lung cancer, lymphoma, malignant
mesothelioma, mastocytoma, 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, skin cancer, small intestine
cancer, soft tissue cancer, solid tumors, stomach cancer,
testicular cancer, thyroid cancer, ureter cancer, and/or urinary
bladder cancer. As used herein, the term "tumor" refers to an
abnormal growth of cells or tissues, e.g., of malignant type or
benign type.
[0084] The term "cancer neo-antigen" or "neo-antigen" or
"neo-epitope" and its grammatical equivalents as used herein can
refer to antigens that are not encoded in a normal, non-mutated
host genome. A "neo-antigen" can in some instances represent either
oncogenic viral proteins or abnormal proteins that arise as a
consequence of somatic mutations. For example, a neo-antigen can
arise by the disruption of cellular mechanisms through the activity
of viral proteins. Another example can be an exposure of a
carcinogenic compound, which in some cases can lead to a somatic
mutation. This somatic mutation can ultimately lead to the
formation of a tumor/cancer.
[0085] The term "cytotoxicity" as used in this specification,
refers to an unintended or undesirable alteration in the normal
state of a cell. The normal state of a cell may refer to a state
that is manifested or exists prior to the cell's exposure to a
cytotoxic composition, agent and/or condition. Generally, a cell
that is in a normal state is one that is in homeostasis. An
unintended or undesirable alteration in the normal state of a cell
can be manifested in the form of, for example, cell death (e.g.,
programmed cell death), a decrease in replicative potential, a
decrease in cellular integrity such as membrane integrity, a
decrease in metabolic activity, a decrease in developmental
capability, or any of the cytotoxic effects disclosed in the
present application.
[0086] The phrase "reducing cytotoxicity" or "reduce cytotoxicity"
refers to a reduction in degree or frequency of unintended or
undesirable alterations in the normal state of a cell upon exposure
to a cytotoxic composition, agent and/or condition. The phrase can
refer to reducing the degree of cytotoxicity in an individual cell
that is exposed to a cytotoxic composition, agent and/or condition,
or to reducing the number of cells of a population that exhibit
cytotoxicity when the population of cells is exposed to a cytotoxic
composition, agent and/or condition.
[0087] The term "engineered" and its grammatical equivalents as
used herein can refer to one or more alterations of a nucleic acid,
e.g., the nucleic acid within an organism's genome. The term
"engineered" can refer to alterations, additions, and/or deletion
of genes. An engineered cell can also refer to a cell with an
added, deleted and/or altered gene.
[0088] The term "cell" or "engineered cell" and their grammatical
equivalents as used herein can refer to a cell of human or
non-human animal origin.
[0089] The term "checkpoint gene" and its grammatical equivalents
as used herein can refer to any gene that is involved in an
inhibitory process (e.g., feedback loop) that acts to regulate the
amplitude of an immune response, for example, an immune inhibitory
feedback loop that mitigates uncontrolled propagation of harmful
responses. These responses can include contributing to a molecular
shield that protects against collateral tissue damage that might
occur during immune responses to infections and/or maintenance of
peripheral self-tolerance. Non-limiting examples of checkpoint
genes can include members of the extended CD28 family of receptors
and their ligands as well as genes involved in co-inhibitory
pathways (e.g., CTLA-4 and PD-1). The term "checkpoint gene" can
also refer to an immune checkpoint gene.
[0090] A "CRISPR," "CRISPR system," or "CRISPR nuclease system" and
their grammatical equivalents can include a non-coding RNA molecule
(e.g., guide RNA) that binds to DNA and Cas proteins (e.g., Cas9)
with nuclease functionality (e.g., two nuclease domains). See,
e.g., Sander, J. D., et al., "CRISPR-Cas systems for editing,
regulating and targeting genomes," Nature Biotechnology, 32:347-355
(2014); see also e.g., Hsu, P.D., et al., "Development and
applications of CRISPR-Cas9 for genome engineering," Cell
157(6):1262-1278 (2014).
[0091] The term "disrupting" and its grammatical equivalents as
used herein can refer to a process of modifying or altering a gene,
e.g., by cleavage, deletion, insertion, mutation, rearrangement, or
any combination thereof. A disruption can result in the knockout or
knockdown of protein expression. A knockout can be a complete or
partial knockout. For example, a gene can be disrupted by knockout
or knockdown. Disrupting a gene can partially reduce or completely
suppress expression of a protein encoded by the gene. Disrupting a
gene can also cause activation of a different gene, for example, a
downstream gene. In some embodiments, the term "disrupting" can be
used interchangeably with terms such as suppressing, interrupting,
or engineering.
[0092] The term "function" and its grammatical equivalents as used
herein can refer to the capability of operating, having, or serving
an intended purpose. Functional can comprise any percent from
baseline to 100% of normal function. For example, functional can
comprise or comprise about 5, 10, 15, 20, 25, 30, 35, 40, 45,
50,55, 60, 65, 70, 75, 80, 85, 90, 95, and/or 100% of normal
function. In some cases, the term functional can mean over or over
about 100% of normal function, for example, 125, 150, 175, 200,
250, 300% and/or above normal function.
[0093] The term "gene editing" and its grammatical equivalents as
used herein can refer to genetic engineering in which one or more
nucleotides are inserted, replaced, or removed from a genome. Gene
editing can be performed using a nuclease (e.g., a natural-existing
nuclease or an artificially engineered nuclease).
[0094] The term "mutation" and its grammatical equivalents as used
herein can include the substitution, deletion, and insertion of one
or more nucleotides in a polynucleotide. For example, up to 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 40, 50, or
more nucleotides/amino acids in a polynucleotide (cDNA, gene) or a
polypeptide sequence can be substituted, deleted, and/or inserted.
A mutation can affect the coding sequence of a gene or its
regulatory sequence. A mutation can also affect the structure of
the genomic sequence or the structure/stability of the encoded
mRNA.
[0095] The term "non-human animal" and its grammatical equivalents
as used herein can include all animal species other than humans,
including non-human mammals, which can be a native animal or a
genetically modified non-human animal The terms "nucleic acid,"
"polynucleotide," "polynucleic acid," and "oligonucleotide" and
their grammatical equivalents can be used interchangeably and can
refer to a deoxyribonucleotide or ribonucleotide polymer, in linear
or circular conformation, and in either single- or double-stranded
form. For the purposes of the present disclosure, these terms
should not to be construed as limiting with respect to length. The
terms can also encompass analogues of natural nucleotides, as well
as nucleotides that are modified in the base, sugar and/or
phosphate moieties (e.g., phosphorothioate backbones).
Modifications of the terms can also encompass demethylation,
addition of CpG methylation, removal of bacterial methylation,
and/or addition of mammalian methylation. In general, an analogue
of a particular nucleotide can have the same base-pairing
specificity, i.e., an analogue of A can base-pair with T.
[0096] The term "peripheral blood lymphocytes" (PBL) and its
grammatical equivalents as used herein can refer to lymphocytes
that circulate in the blood (e.g., peripheral blood). Peripheral
blood lymphocytes can refer to lymphocytes that are not localized
to organs. Peripheral blood lymphocytes can comprise T cells, NK
cells, B cell, or any combinations thereof
[0097] The term "phenotype" and its grammatical equivalents as used
herein can refer to a composite of an organism's observable
characteristics or traits, such as its morphology, development,
biochemical or physiological properties, phenology, behavior, and
products of behavior. Depending on the context, the term
"phenotype" can sometimes refer to a composite of a population's
observable characteristics or traits.
[0098] The term "protospacer" and its grammatical equivalents as
used herein can refer to a PAM-adjacent nucleic acid sequence
capable to hybridizing to a portion of a guide RNA, such as the
spacer sequence or engineered targeting portion of the guide RNA. A
protospacer can be a nucleotide sequence within gene, genome, or
chromosome that is targeted by a guide RNA. In the native state, a
protospacer is adjacent to a PAM (protospacer adjacent motif). The
site of cleavage by an RNA-guided nuclease is within a protospacer
sequence. For example, when a guide RNA targets a specific
protospacer, the Cas protein will generate a double strand break
within the protospacer sequence, thereby cleaving the protospacer.
Following cleavage, disruption of the protospacer can result though
non-homologous end joining (NHEJ) or homology-directed repair
(HDR). Disruption of the protospacer can result in the deletion of
the protospacer. Additionally or alternatively, disruption of the
protospacer can result in an exogenous nucleic acid sequence being
inserted into or replacing the protospacer.
[0099] The term "recipient" and their grammatical equivalents as
used herein can refer to a human or non-human animal The recipient
can also be in need thereof
[0100] The term "recombination" and its grammatical equivalents as
used herein can refer to a process of exchange of genetic
information between two polynucleic acids. For the purposes of this
disclosure, "homologous recombination" or "HR" can refer to a
specialized form of such genetic exchange that can take place, for
example, during repair of double-strand breaks. This process can
require nucleotide sequence homology, for example, using a donor
molecule to template repair of a target molecule (e.g., a molecule
that experienced the double-strand break), and is sometimes known
as non-crossover gene conversion or short tract gene conversion.
Such transfer can also involve mismatch correction of heteroduplex
DNA that forms between the broken target and the donor, and/or
synthesis-dependent strand annealing, in which the donor can be
used to resynthesize genetic information that can become part of
the target, and/or related processes. Such specialized HR can often
result in an alteration of the sequence of the target molecule such
that part or all of the sequence of the donor polynucleotide can be
incorporated into the target polynucleotide. In some cases, the
terms "recombination arms" and "homology arms" can be used
interchangeably.
[0101] The terms "target vector" and "targeting vector" are used
interchangeably herein.
[0102] The term "T cell" and its grammatical equivalents as used
herein can refer to a T cell from any origin. For example, a T cell
can be a primary T cell, e.g., an autologous T cell, a cell line,
etc. The T cell can also be human or non-human.
[0103] The term "TIL" or tumor infiltrating lymphocyte and its
grammatical equivalents as used herein can refer to a cell isolated
from a tumor. For example, a TIL can be a cell that has migrated to
a tumor. A TIL can also be a cell that has infiltrated a tumor. A
TIL can be any cell found within a tumor. For example, a TIL can be
a T cell, B cell, monocyte, natural killer (NK) cell, or any
combination thereof. A TIL can be a mixed population of cells. A
population of TILs can comprise cells of different phenotypes,
cells of different degrees of differentiation, cells of different
lineages, or any combination thereof
[0104] A "therapeutic effect" may occur if there is a change in the
condition being treated. The change may be positive or negative.
For example, a `positive effect` may correspond to an increase in
the number of activated T-cells in a subject. In another example, a
`negative effect` may correspond to a decrease in the amount or
size of a tumor in a subject. There is a "change" in the condition
being treated if there is at least 10% improvement, preferably at
least 25%, more preferably at least 50%, even more preferably at
least 75%, and most preferably 100%. The change can be based on
improvements in the severity of the treated condition in an
individual, or on a difference in the frequency of improved
conditions in populations of individuals with and without treatment
with the therapeutic compositions with which the compositions of
the present invention are administered in combination Similarly, a
method of the present disclosure may comprise administering to a
subject an amount of cells that is "therapeutically effective". The
term "therapeutically effective" should be understood to have a
definition corresponding to `having a therapeutic effect`.
[0105] The term "safe harbor" and "immune safe harbor", and their
grammatical equivalents as used herein can refer to a location
within a genome that can be used for integrating exogenous nucleic
acids wherein the integration does not cause any significant effect
on the growth of the host cell by the addition of the nucleic acid
alone. Non-limiting examples of safe harbors can include HPRT, AAVS
SITE (E.G. AAVS1, AAVS2, ETC.), CCRS, or Rosa26.
[0106] The term "sequence" and its grammatical equivalents as used
herein can refer to a nucleotide sequence, which can be DNA or RNA;
can be linear, circular or branched; and can be either
single-stranded or double stranded. A sequence can be mutated. A
sequence can be of any length, for example, between 2 and 1,000,000
or more nucleotides in length (or any integer value there between
or there above), e.g., between about 100 and about 10,000
nucleotides or between about 200 and about 500 nucleotides.
Overview
[0107] Disclosed herein are compositions and methods useful for
treating a disease or condition such as cancer. Disclosed herein
are also treatment regimes for the therapy of various diseases or
conditions such as cancer. A treatment regime can comprise
administering genetically modified cells, such as tumor
infiltrating lymphocytes for therapeutic applications. Effective
adoptive cell transfer-based immunotherapies (ACT) can be useful to
treat cancer (e.g., metastatic cancer) patients. For example, tumor
infiltrating lymphocytes can be modified to disrupt an immune
checkpoint gene. Cells
[0108] Compositions disclosed herein can utilize cells. Cells can
be primary cells. Cells can be recombinant cells. Cells can be
obtained from a number of non-limiting sources, including
peripheral blood mononuclear cells, bone marrow, lymph node tissue,
cord blood, thymus tissue, tissue from a site of infection,
ascites, pleural effusion, spleen tissue, and tumors. For example,
any T cell lines can be used. Alternatively, the cell can be
derived from a healthy donor, from a patient diagnosed with cancer,
or from a patient diagnosed with an infection. In another
embodiment, the cell can be part of a mixed population of cells
which present different phenotypic characteristics. A cell can also
be obtained from a cell therapy bank. Disrupted cells resistant to
an immunosuppressive treatment can be obtained. A desirable cell
population can also be selected prior to modification. A selection
can include at least one of: magnetic separation, flow cytometric
selection, antibiotic selection. The one or more cells can be any
blood cells, such as peripheral blood mononuclear cell (PBMC),
lymphocytes, monocytes or macrophages. The one or more cells can be
any immune cells such as lymphocytes, B cells, or T cells. Cells
can also be obtained from whole food, apheresis, or a tumor sample
of a subject. A cell can be a tumor infiltrating lymphocytes (TIL).
In some cases an apheresis can be a leukapheresis. Leukapheresis
can be a procedure in which blood cells are isolated from blood.
During a leukapheresis, blood can be removed from a needle in an
arm of a subject, circulated through a machine that divides whole
blood into red cells, plasma and lymphocytes, and then the plasma
and red cells are returned to the subject through a needle in the
other arm. In some cases, cells are isolated after an
administration of a treatment regime and cellular therapy. For
example, an apheresis can be performed in sequence or concurrent
with a cellular administration. In some cases, an apheresis is
performed prior to and up to about 6 weeks following administration
of a cellular product. In some cases, an apheresis is performed -3
weeks, -2 weeks, -1 week, 0, 1 week, 2 weeks, 3 weeks, 4 weeks, 1
month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months,
8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years,
4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or up to
about 10 years after an administration of a cellular product. In
some cases, cells acquired by an apheresis can undergo testing for
specific lysis, cytokine release, metabolomics studies,
bioenergetics studies, intracellular FACs of cytokine production,
ELISA-spot assays, and lymphocyte subset analysis. In some cases,
samples of cellular products or apheresis products can be
cryopreserved for retrospective analysis of infused cell phenotype
and function.
[0109] A TIL can be isolated from an organ afflicted with a cancer.
One or more cells can be isolated from an organ with a cancer that
can be a brain, heart, lungs, eye, stomach, pancreas, kidneys,
liver, intestines, uterus, bladder, skin, hair, nails, ears,
glands, nose, mouth, lips, spleen, gums, teeth, tongue, salivary
glands, tonsils, pharynx, esophagus, large intestine, small
intestine, rectum, anus, thyroid gland, thymus gland, bones,
cartilage, tendons, ligaments, suprarenal capsule, skeletal
muscles, smooth muscles, blood vessels, blood, spinal cord,
trachea, ureters, urethra, hypothalamus, pituitary, pylorus,
adrenal glands, ovaries, oviducts, uterus, vagina, mammary glands,
testes, seminal vesicles, penis, lymph, lymph nodes or lymph
vessels. One or more TILs can be from a brain, heart, liver, skin,
intestine, lung, kidney, eye, small bowel, or pancreas. TILs can be
from a pancreas, kidney, eye, liver, small bowel, lung, or heart.
TILs can be from a pancreas. The one or more cells can be
pancreatic islet cells, for example, pancreatic .beta. cells. In
some cases, a TIL can be from a gastrointestinal cancer. A TIL
culture can be prepared a number of ways. For example, a tumor can
be trimmed from non-cancerous tissue or necrotic areas. A tumor can
then be fragmented to about 2-3 mm in length. In some cases, a
tumor can be fragmented from about 0.5 mm to about 5 mm in size,
from about 1 mm to about 2 mm, from about 2 mm to about 3 mm, from
about 3 mm to about 4 mm, or from about 4 mm to about 5 mm. Tumor
fragments can then be cultured in vitro utilizing media and a
cellular stimulating agent such as a cytokine. In some cases, IL-2
can be utilized to expand TILs from a tumor fragment. A
concentration of IL-2 can be about 6000 IU/mL. A concentration of
IL-2 can also be about 2000 IU/mL, 3000 IU/mL, 4000 IU/mL, 5000
IU/mL, 6000 IU/mL, 7000 IU/mL, 8000 IU/mL, 9000 IU/mL, or up to
about 10000 IU/mL. Once TILs are expanded they can be subject to in
vitro assays to determine tumor reactivity. For example, TILs can
be evaluated by FACs for CD3, CD4, CD8, and CD58 expression. TILs
can also be subjected to cocultured, cytotoxicity, ELISA, or
ELISPOT assays. In some cases, TIL cultures can be cryopreserved or
undergo a rapid expansion. A cell, such as a TIL, can be isolated
from a donor of a stage of development including, but not limited
to, fetal, neonatal, young and adult. TILs can be isolated from an
adult human. A human from whom cells can be isolated can be under
the age of 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year(s). For example,
cells can be isolated from a human under the age of 6 years. Cells,
such as TILs, can also be isolated from a human under the age of 3
years. In some cases, a human donor can be an adult from at least
about 18 years of age. In some cases, a blood product may be
stored. For example, a cryostore freezing bag may be utilized to
store and freeze a blood product.
[0110] In some cases, a cell that can be utilized in a cellular
therapy or a cell that can be genomically disrupted can be positive
or negative for a given factor. In some embodiments, a cell may be
a CD3+ cell, CD3- cell, a CD5+ cell, CD5- cell, a CD7+ cell, CD7-
cell, a CD14+ cell, CD14- cell, CD8+ cell, a CD8- cell, a CD103+
cell, CD103- cell, CD11b+ cell, CD11b- cell, a BDCA1+ cell, a
BDCA1- cell, an L-selectin+ cell, an L-selectin- cell, a CD25+, a
CD25- cell, a CD27+, a CD27- cell, a CD28+ cell, CD28- cell, a
CD44+ cell, a CD44- cell, a CD56+ cell, a CD56- cell, a CD57+ cell,
a CD57- cell, a CD62L+ cell, a CD62L- cell, a CD69+ cell, a CD69-
cell, a CD45RO+ cell, a CD45RO- cell, a CD127+ cell, a CD127- cell,
a CD132+ cell, a CD132- cell, an IL-7+ cell, an IL-7- cell, an
IL-15+ cell, an IL-15- cell, a lectin-like receptor G1 positive
cell, a lectin-like receptor G1 negative cell, or an differentiated
or de-differentiated cell thereof. The examples of factors
expressed by cells is not intended to be limiting, and a person
having skill in the art will appreciate that a cell may be positive
or negative for any factor known in the art. In some embodiments, a
cell may be positive for two or more factors. For example, a cell
may be CD4+ and CD8+. In some embodiments, a cell may be negative
for two or more factors. For example, a cell may be CD25-, CD44-,
and CD69-. In some embodiments, a cell may be positive for one or
more factors, and negative for one or more factors. For example, a
cell may be CD4+ and CD8-. The selected cells can then be infused
into a subject. In some embodiments, the cells may be selected for
having or not having one or more given factors (e.g., cells may be
separated based on the presence or absence of one or more factors).
In some embodiments, the selected cells can also be expanded in
vitro. The selected cells can be expanded in vitro prior to
infusion. It should be understood that cells used in any of the
methods disclosed herein may be a mixture (e.g., two or more
different cells) of any of the cells disclosed herein. For example,
a method of the present disclosure may comprise cells, and the
cells are a mixture of CD4+ cells and CD8+ cells. In another
example, a method of the present disclosure may comprise cells, and
the cells are a mixture of CD4+ cells and naive cells. In some
cases, a cell can be a stem memory T.sub.SCM cell comprised of
CD45RO (-), CCR7(+), CD45RA (+), CD62L+ (L-selectin), CD27+, CD28+
and IL-7R.alpha.+, stem memory cells can also express CD95,
IL-2R.beta., CXCR3, and LFA-1, and show numerous functional
attributes distinctive of stem memory cells. Engineered cells can
also be central memory T.sub.CM cells comprising L-selectin and
CCR7, where the central memory cells can secrete, for example,
IL-2, but not IFN.gamma. or IL-4. Engineered cells can also be
effector memory T.sub.EM cells comprising L-selectin or CCR7 and
produce, for example, effector cytokines such as IFN.gamma. and
IL-4. In some cases a population of cells can be introduced to a
subject. For example, a population of cells can be a combination of
T cells and NK cells. In other cases, a population can be a
combination of naive cells and effector cells. A population of
cells can be TILs.
[0111] In particular, T cell populations can be stimulated in vitro
such as by contact with an anti-CD3 antibody or antigen-binding
fragment thereof, or an anti-CD2 antibody immobilized on a surface,
or by contact with a protein kinase C activator (e.g., bryostatin)
sometimes in conjunction with a calcium ionophore. For
co-stimulation of an accessory molecule on the surface of the T
cells, a ligand that binds the accessory molecule can be used. For
example, a population of T cells can be contacted with an anti-CD3
antibody and an anti-CD28 antibody, under conditions that can
stimulate proliferation of the T cells. In some cases, 4-1BB can be
used to stimulate cells. For example, cells can be stimulated with
4-1BB and IL-21 or another cytokine. To stimulate proliferation of
either CD4 T cells or CD8 T cells, an anti-CD3 antibody and an
anti-CD28 antibody can be used. For example, the agents providing a
signal may be in solution or coupled to a surface. The ratio of
particles to cells may depend on particle size relative to the
target cell. In further embodiments, the cells, such as T cells,
can be combined with agent-coated beads, where the beads and the
cells can be subsequently separated, and optionally cultured. Each
bead can be coated with either anti-CD3 antibody or an anti-CD28
antibody, or in some cases, a combination of the two. In an
alternative embodiment, prior to culture, the agent-coated beads
and cells are not separated but are cultured together. Cell surface
proteins may be ligated by allowing paramagnetic beads to which
anti-CD3 and anti-CD28 can be attached (3x28 beads) to contact the
T cells. In some cases cells and beads (for example, DYNABEADS.RTM.
M-450 CD3/CD28 T paramagnetic beads at a ratio of 1:1) are combined
in a buffer, for example, phosphate buffered saline (PBS) (e.g.,
without divalent cations such as, calcium and magnesium). Any cell
concentration may be used. The mixture may be cultured for or for
about several hours (e.g., about 3 hours) to or to about 14 days or
any hourly integer value in between. In another embodiment, the
mixture may be cultured for or for about 21 days or for up to or
for up to about 21 days. Conditions appropriate for T cell culture
can include an appropriate media (e.g., Minimal Essential Media or
RPMI Media 1640 or, X-vivo 5, (Lonza)) that may contain factors
necessary for proliferation and viability, including serum (e.g.,
fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-g
, IL-4, IL-7, GM-CSF, IL-10, IL-21, IL-15, TGF beta, and TNF alpha
or any other additives for the growth of cells. Other additives for
the growth of cells include, but are not limited to, surfactant,
plasmanate, and reducing agents such as N-acetyl-cysteine and
2-mercaptoethanol. Media can include RPMI 1640, A1 M-V, DMEM, MEM,
.alpha.-MEM, F-12, X-Vivo 1 , and X-Vivo 20, Optimizer, with added
amino acids, sodium pyruvate, and vitamins, either serum-free or
supplemented with an appropriate amount of serum (or plasma) or a
defined set of hormones, and/or an amount of cytokine(s) sufficient
for the growth and expansion of T cells. In some cases, an 865mL
bottle of RPMI may have 100 mL of human serum, 25 mL of Hepes 1M,
10 mL of Penicillin/streptomycin at 10,000U/mL and 10,000 .mu.g/mL,
and 0.2 mLof gentamycin at 50 mg/mL. After addition of additives an
RPMI media may be filtered using a 0.2 .mu.m.times.1 L filter and
stored at 4.degree. C. In some embodiments, antibiotics, e.g.,
penicillin and streptomycin, are included only in experimental
cultures but not in cultures of cells that are to be infused into a
subject. In some cases, human serum can be thawed in a 37 .degree.
C. water bath, and then heat inactivated (e.g., at 56 .degree. C.
for 30 min for 100 mL bottle). The sera can be filtered through a
0.8 .mu.m and 0.45 .mu.m filter prior to addition of medium.
[0112] The target cells can be maintained under conditions
necessary to support growth; for example, an appropriate
temperature (e.g., 37.degree. C.) and atmosphere (e.g., air plus 5%
CO.sub.2). In some instances, T cells that have been exposed to
varied stimulation times may exhibit different characteristics. In
some cases, a soluble monospecific tetrameric antibody against
human CD3, CD28, CD2, or any combination thereof may be used.
[0113] In some cases, cells to undergo disruption can be activated
or expanded by co-culturing with tissue or cells. A cell can be an
antigen presenting cell. An artificial antigen presenting cells
(aAPCs) can express ligands for T cell receptor and costimulatory
molecules and can activate and expand T cells for transfer, while
improving their potency and function in some cases. An aAPC can be
engineered to express any gene for T cell activation. An aAPC can
be engineered to express any gene for T cell expansion. An aAPC can
be a bead, a cell, a protein, an antibody, a cytokine, or any
combination. An aAPC can deliver signals to a cell population that
may undergo genomic transplant. For example, an aAPC can deliver a
signal 1, signal, 2, signal 3 or any combination. A signal 1 can be
an antigen recognition signal. For example, signal 1 can be
ligation of a TCR by a peptide--MHC complex or binding of agonistic
antibodies directed towards CD3 that can lead to activation of the
CD3 signal-transduction complex. Signal 2 can be a co-stimulatory
signal. For example, a co-stimulatory signal can be anti-CD28,
inducible co-stimulator (ICOS), CD27, and 4-1BB (CD137), which bind
to ICOS-L, CD70, and 4-1BBL, respectively. Signal 3 can be a
cytokine signal. A cytokine can be any cytokine. A cytokine can be
IL-2, IL-7, IL-12, IL-15, IL-21, or any combination thereof.
[0114] In some cases an artificial antigen presenting cell (aAPC)
may be used to activate and/or expand a cell population. In some
cases, an artificial may not induce allospecificity. An aAPC may
not express HLA in some cases. An aAPC may be genetically modified
to stably express genes that can be used to activation and/or
stimulation. In some cases, a K562 cell may be used for activation.
A K562 cell may also be used for expansion. A K562 cell can be a
human erythroleukemic cell line. A K562 cell may be engineered to
express genes of interest. K562 cells may not endogenously express
HLA class I, II, or CD1d molecules but may express ICAM-1 (CD54)
and LFA-3 (CD58). K562 may be engineered to deliver a signal 1 to T
cells. For example, K562 cells may be engineered to express HLA
class I. In some cases, K562 cells may be engineered to express
additional molecules such as B7, CD80, CD83, CD86, CD32, CD64,
4-1BBL, anti-CD3, anti-CD3 mAb, anti-CD28, anti-CD28mAb, CD1d,
anti-CD2, membrane-bound IL-15, membrane-bound IL-17,
membrane-bound IL-21, membrane-bound IL-2, truncated CD19, or any
combination. In some cases, an engineered K562 cell can expresses a
membranous form of anti-CD3 mAb, clone OKT3, in addition to CD80
and CD83. In some cases, an engineered K562 cell can expresses a
membranous form of anti-CD3 mAb, clone OKT3, membranous form of
anti-CD28 mAb in addition to CD80 and CD83.
[0115] In some cases, restimulation of cells can be performed with
antigen and irradiated, histocompatible antigen presenting cells
(APCs), such as feeder PBMCs. In some cases, cells can be grown
using non-specific mitogens such as PHA and allogenic feeder cells.
Feeder PBMCs can be irradiated at 40Gy. Feeder PBMCs can be
irradiated from about 10 Gy to about 15 Gy, from about 15 Gy to
about 20 Gy, from about 20Gy to about 25 Gy, from about 25 Gy to
about 30 Gy, from about 30 Gy to about 35 Gy, from about 35 Gy to
about 40 Gy, from about 40 Gy to about 45 Gy, from about 45 Gy to
about 50 Gy. In some cases, a control flask of irradiated feeder
cells only can be stimulated with anti-CD3 and IL-2.
[0116] An aAPC can be a bead. A spherical polystyrene bead can be
coated with antibodies against CD3 and CD28 and be used for T cell
activation. A bead can be of any size. In some cases, a bead can be
or can be about 3 and 6 micrometers. A bead can be or can be about
4.5 micrometers in size. A bead can be utilized at any cell to bead
ratio. For example, a 3 to 1 bead to cell ratio at 1 million cells
per milliliter can be used. An aAPC can also be a rigid spherical
particle, a polystyrene latex microbeads, a magnetic nano- or
micro-particles, a nanosized quantum dot, a 4,
polylactic-co-glycolic acid) (PLGA) microsphere, a nonspherical
particle, a 5, carbon nanotube bundle, a 6, ellipsoid PLGA
microparticle, a 7, nanoworms, a fluidic lipid bilayer-containing
system, an 8, 2D-supported lipid bilayer (2D-SLBs), a 9, liposome,
a 10, RAFTsomes/microdomain liposome, an 11, SLB particle, or any
combination thereof
[0117] In some cases, an aAPC can expand CD4 T cells. For example,
an aAPC can be engineered to mimic an antigen processing and
presentation pathway of HLA class II-restricted CD4 T cells. A K562
can be engineered to express HLA-D, DP .alpha., DP .beta. chains,
Ii, DM .alpha., DM .beta., CD80, CD83, or any combination thereof.
For example, engineered K562 cells can be pulsed with an
HLA-restricted peptide in order to expand HLA-restricted
antigen-specific CD4 T cells.
[0118] In some cases, the use of aAPCs can be combined with
exogenously introduced cytokines for T cell activation, expansion,
or any combination. Cells can also be expanded in vivo, for example
in the subject's blood after administration of genomically
transplanted cells into a subject.
[0119] In some cases, cells can be scaled up to yields achieved by
standard rapid expansion protocols (REP). An average fold expansion
of genetically modified TILs can be 1071. In some cases, an average
fold expansion can be from 500 to 2000. An average fold expansion
of genetically modified TILs can be from 500 to 600, from 600 to
700, from 700 to 800, from 800 to 900, from 900 to 1000, from 1000
to up to 2000 fold. In some cases, an engineered cellular dosage
can be from about 1.times.10.sup.10 knockout TIL for patient
infusion.
[0120] Cells (e.g., engineered cells or engineered primary T cells
or TILs) before, after, and/or during administration can be
functional. For example, cells can be functional for at least or at
least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 6, 27, 28, 29, 30, 40, 50, 60,
70, 80, 90, or 100 days after administration. Adoptively
transferred cells can be functional for at least or at least about
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after
administration. Administered cells can be functional for at least
or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30
years after infusion. In some cases, administered cells can be
functional for up to the lifetime of a recipient.
[0121] Further, adoptively transferred cells can function at 100%
of their normal intended operation. Cells can also function 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,
55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,
72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,
89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% of their normal
intended operation.
[0122] In long-term cultures, a genomically disrupted cell cannot
exhibit properties of stimulation-independent growth or
transformation. For example, in some cases, a genomically disrupted
cell may not expand when cultured without a cytokine, such as IL-2.
In some cases, a genomically disrupted cell may not be viable after
about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,
36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52,
53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,
87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 days after
contact with a cytokine such as IL-2.
[0123] Cellular compositions described herein can be cryopreserved.
A cryopreservation can be performed in, for example, a Cryostor
CS10 at 5% DMSO final concentration. A cryopreservation can be at a
freeze density from about 7.5.times.10.sup.7 cells/mL to about
1.5.times.10.sup.8 cells/mL. A freeze density can be from about
1.times.10.sup.7 cells/mL, 1.5.times.10.sup.7 cells/mL,
2.times.10.sup.7 cells/mL, 2.5.times.10.sup.7 cells/mL,
3.times.10.sup.7 cells/mL, 3.5.times.10.sup.7 cells/mL,
4.times.10.sup.7 cells/mL, 4.5.times.10.sup.7 cells/mL,
5.times.10.sup.7 cells/mL, 5.5.times.10.sup.7 cells/mL,
6.times.10.sup.7 cells/mL, 6.5.times.10.sup.7 cells/mL,
7.times.10.sup.7 cells/mL, 7.5.times.10.sup.7 cells/mL,
8.times.10.sup.7 cells/mL, 8.5.times.10.sup.7 cells/mL,
9.times.10.sup.7 cells/mL, 9.5.times.10.sup.7 cells/mL,
1.times.10.sup.8 cells/mL, 1.5.times.10.sup.8 cells/mL,
2.times.10.sup.8 cells/mL, 2.5.times.10.sup.8 cells/mL,
3.times.10.sup.8 cells/mL, 3.5.times.10.sup.8 cells/mL,
4.times.10.sup.8 cells/mL, 4.5.times.10.sup.8 cells/mL,
5.times.10.sup.8 cells/mL, 5.5.times.10.sup.8 cells/mL,
6.times.10.sup.8 cells/mL, 6.5.times.10.sup.8 cells/mL,
7.times.10.sup.8 cells/mL, 7.5.times.10.sup.8 cells/mL, or up to
about 8.times.10.sup.8 cells/mL.
[0124] For example, in some cases, a TIL can be harvested, washed,
and re-suspended in a buffer, such as Cryostor buffer. This
preparation can be mixed with an equal volume of Cryostore CS10. In
some cases, a cellular composition is thawed prior to an
introducing into a subject in need thereof.
[0125] Cellular viability can be determined by flow cytometry and
trypan blue exclusion. In some cases, a forward scatter and side
scatter on a flow cytometer can identify percent viable cells. In
other cases, cells can be stained with Annexin V to determine a
percent of dead/live cells. Trypan blue exclusion may also be
utilized to determine cellular viability with a hemocytometer. In
some cases, at least about 50% cells can be viable for
administration. In other cases from about 50%, 60%, 70%, 80%, 90%,
95%, or up to about 100% cells can be viable.
Cellular Targets
[0126] A cell such as a TIL can target an antigen. A cell can also
target an epitope. An antigen can be a tumor cell antigen. An
epitope can be a tumor cell epitope. Tumor infiltrating lymphocytes
(TIL) can be selected as clonal and/or oligoclonal subpopulations
possessing specific reactivity against subject-specific cancer
neoantigens. Specific reactivity can be determined using at least
one of: tumor whole-exome sequencing and tandem mini-gene/synthetic
long peptide screening approach, E. Tran et al., Cancer
immunotherapy based on mutation-specific CD4+ T cells in a patient
with epithelial cancer. Science 344, 641-645 (2014). A tumor cell
epitope may be derived from a wide variety of tumor antigens such
as antigens from tumors resulting from mutations (neo antigens or
neo epitopes), shared tumor specific antigens, differentiation
antigens, and antigens overexpressed in tumors. In some cases, a
neoantigen or neoepitope can be identified by 5'RACE and TCR-PCR.
Those antigens, for example, may be derived from alpha-actinin-4,
ARTC1, BCR-ABL fusion protein (b3a2), B-RAF, CASP-5, CASP-8,
beta-catenin, Cdc27, CDK4, CDKN2A, COA-1, dek-can fusion protein,
EFTUD2, Elongation factor 2, ETV6-AML1 fusion protein, FLT3-ITD,
FN1, GPNMB, LDLR-fucosyltransferase fusion protein, HLA-A2d, HLA-A1
1d, hsp70-2, KIAAO205, MART2, ME1, MUM-lf, MUM-2, MUM-3, neo-PAP,
Myosin class I, NFYC, OGT, OS-9, p53, pml-RARalpha fusion protein,
PRDXS, PTPRK, K-ras, N-ras, RBAF600, SIRT2, SNRPD1, SYT-SSX1- or
-SSX2 fusion protein, TGF-betaRII, triosephosphate isomerase,
BAGE-1, GAGE-1, 2, 8, Gage 3, 4, 5, 6, 7, GnTVf, HERV-K-MEL,
KK-LC-1, KM-HN-1, LAGE-1, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4,
MAGE-A6, MAGE-A9, MAGE-A10, MAGE-Al2, MAGE-C2, mucink, NA-88,
NY-ESO-1/LAGE-2, SAGE, Sp17, SSX-2, SSX-4, TAG-1, TAG-2, TRAG-3,
TRP2-INT2g, XAGE-lb, CEA, gp100/Pme117, Kallikrein 4,
mammaglobin-A, Melan-A/MART-1, NY-BR-1, OA1, PSA, RAB38/NY-MEL-1,
TRP-1/gp75, TRP-2, tyrosinase, adipophilin, AIM-2, ALDH1A1, BCLX
(L), BCMA, BING-4, CPSF, cyclin D1, DKK1, ENAH (hMena), EP-CAM,
EphA3, EZH2, FGFS, G250/MN/CAIX, HER-2/neu, IL13Ralpha2, intestinal
carboxyl esterase, alpha fetoprotein, M-CSFT, MCSP, mdm-2, MMP-2,
MUC1, p53, PBF, PRAME, PSMA, RAGE-1, RGSS, RNF43, RU2AS, secernin
1, SOX10, STEAP1, survivin, Telomerase, VEGF, and/or WT1, just to
name a few. Tumor-associated antigens may be antigens not normally
expressed by the host; they can be mutated, truncated, misfolded,
or otherwise abnormal manifestations of molecules normally
expressed by the host; they can be identical to molecules normally
expressed but expressed at abnormally high levels; or they can be
expressed in a context or environment that is abnormal.
Tumor-associated antigens may be, for example, proteins or protein
fragments, complex carbohydrates, gangliosides, haptens, nucleic
acids, other biological molecules or any combinations thereof. In
some cases, a target is a neo antigen or neo epitope. For example,
a neo antigen can be an E805G mutation in ERBB2IP. Neo antigen and
neo epitopes can be identified by whole-exome sequencing in some
cases. A neo antigen and neo epitope target can be expressed by a
gastrointestinal cancer cell in some cases. A neo antigen and neo
epitope can be expressed on an epithelial carcinoma.
[0127] An epitope can be a stromal epitope. An epitope can be on
the stroma of the tumor microenvironment. The antigen can be a
stromal antigen. An antigen can be on the stroma of the tumor
microenvironment. Those antigens and those epitopes, for example,
can be present on tumor endothelial cells, tumor vasculature, tumor
fibroblasts, tumor pericytes, tumor stroma, and/or tumor
mesenchymal cells, just to name a few. Those antigens, for example,
can comprise CD34, MCSP, FAP, CD31, PCNA, CD117, CD40, MMP4, and/or
Tenascin.
Genomic Disruptions
[0128] A genomic disruption can include exons or introns. In some
cases, a genomic disruption can be of a gene sequence. The
disruption of genes can be of any particular gene. It is
contemplated that genetic homologues (e.g., any mammalian version
of the gene) of the genes within this applications are covered.
Some genetic homologues are known in the art, however, in some
cases, homologues are unknown. However, homologous genes between
mammals can be found by comparing nucleic acid (DNA or RNA)
sequences or protein sequences using publically available databases
such as NCBI BLAST.
[0129] A genomic disruption can improve a function of a cell. For
example, a genomic disruption can enhance a cellular cytotoxicity
of a target. A genomic disruption can also enhance proliferation or
persistence of a cell. For example, a gene that can be disrupted
can improve therapeutic potential of cancer immunotherapy. A cell
can be engineered to knock out one or more endogenous genes.
Endogenous genes that can be knocked out can comprise immune
checkpoint genes. An immune checkpoint gene can be stimulatory
checkpoint gene or an inhibitory checkpoint gene Immune checkpoint
gene locations can be provided using the Genome Reference
Consortium Human Build 38 patch release 2 (GRCh38.p2) assembly. A
gene to be knocked out can be selected using a database. In some
cases, certain endogenous genes are more amendable to genomic
engineering. A database can comprise epigenetically permissive
target sites. A database can be ENCODE (encyclopedia of DNA
Elements) (http://www.genome.gov/10005107) in some cases. A
databased can identify regions with open chromatin that can be more
permissive to genomic engineering. A gene that can be disrupted can
be involved in attenuating TCR signaling, functional avidity, or
immunity to cancer. In some cases, a gene to be disrupted is
upregulated when a TCR is stimulated. A gene can be involved in
inhibiting cellular expansion, functional avidity, or cytokine
polyfunctionality. A gene can be involved in negatively regulating
cellular cytokine production. For example, a gene can be involved
in inhibiting production of effector cytokines, IFN-gamma and/or
TNF for example A gene can also be involved in inhibiting
expression of supportive cytokines such as IL-2 after TCR
stimulation.
[0130] A cell can have one or more disrupted genes. For example,
one or more genes whose expression is disrupted can be a checkpoint
gene, such as PD-1 or CISH. In some cases, one or more genes whose
expression can be disrupted are shown in Table 1. For example,
genes that can be disrupted can exhibit a certain identity and/or
homology to genes disclosed herein such as in Table 1, Therefore,
it is contemplated that a gene that exhibits or exhibits about 50%,
55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,
88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
homology (at the nucleic acid or protein level) of Table 1 can be
disrupted. It is also contemplated that a gene that exhibits or
exhibits about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
97%, 98%, 99%, or 100% identity (at the nucleic acid or protein
level) of Table 1 can be disrupted.
TABLE-US-00001 TABLE 1 Checkpoint gene summary NCBI number
(GRCh38.p2) SEQ Gene *AC010327.8 Original Original Location ID
Symbol Abbreviation Name ** GRCh38.p7 Start Stop in genome 1
ADORA2A A2aR; RDC8; adenosine A2a 135 24423597 24442360 22q11.23
ADORA2 receptor 2 CD276 B7H3; B7-H3; CD276 80381 73684281 73714518
15q23-q24 B7RP-2; 4Ig-B7-H3 molecule 3 VTCN1 B7X; B7H4; B7S1; V-set
domain 79679 117143587 117270368 1p13.1 B7-H4; B7h.5; containing T
VCTN1; PRO1291 cell activation inhibitor 1 4 BTLA BTLA1; CD272 B
and T 151888 112463966 112499702 3q13.2 lymphocyte associated 5
CTLA4 GSE; GRD4; cytotoxic T- 1493 203867788 203873960 2q33 ALPS5;
CD152; lymphocyte- CTLA-4; IDDM12; associated CELIAC3 protein 4 6
IDO1 IDO; INDO; IDO-1 indoleamine 3620 39913809 39928790 8p12-p11
2,3- dioxygenase 1 7 KIR3DL1 KIR; NKB1; killer cell 3811 54816438
54830778 19q13.4 NKAT3; NKB1B; immunoglobulin- NKAT-3; CD158E1;
like receptor, KIR3DL2; three domains, KIR3DL1/S1 long cytoplasmic
tail, 1 8 LAG3 LAG3;CD223 lymphocyte- 3902 6772483 6778455 12p13.32
activation gene 3 9 PDCD1 PD1; PD-1; CD279; programmed 5133
241849881 241858908 2q37.3 SLEB2; hPD-1; cell death 1 hPD-1; hSLE1
10 HAVCR2 TIM3; CD366; KIM- hepatitis A 84868 157085832 157109237
5q33.3 3; TIMD3; Tim-3; virus cellular TIMD-3; HAVcr-2 receptor 2
11 VISTA C10orf54, V-domain 64115 71747556 71773580 10q22.1
differentiation of immunoglobulin ESC-1 (Dies 1); suppressor of
platelet receptor T-cell Gi24 precursor; PD1 activation homolog
(PD1H) B7H5; GI24; B7-H5; SISP1; PP2135 12 CD244 2B4; 2B4; NAIL;
CD244 51744 160830158 160862902 1q23.3 Nmrk; NKR2B4; molecule,
SLAMF4 natural killer cell receptor 2B4 13 CISH CIS; G18; SOCS;
cytokine 1154 50606454 50611831 3p21.3 CIS-1; BACTS2 inducible SH2-
containing protein 14 HPRT1 HPRT; HGPRT hypoxanthine 3251 134452842
134500668 Xq26.1 phosphoribosyl transferase 1 15 AAV*S1 AAV adeno-
14 7774 11429 19q13 associated virus integration site 1 16 CCR5
CKR5; CCR-5; chemokine 1234 46370142 46376206 3p21.31 CD195; CKR-5;
(C--C motif) CCCKR5; receptor 5 CMKBR5; IDDM22; (gene/pseudogene)
CC-CKR-5 17 CD160 NK1; BY55; NK28 CD160 11126 145719433 145739288
1q21.1 molecule 18 TIGIT VSIG9; VSTM3; T-cell 201633 114293986
114310288 3q13.31 WUCAM immunoreceptor with Ig and ITIM domains 19
CD96 TACTILE CD96 molecule 10225 111542079 111665996 3q13.13-q13.2
20 CRTAM CD355 cytotoxic and 56253 122838431 122872643 11q24.1
regulatory T- cell molecule 21 LAIR1 CD305; LAIR-1 leukocyte 3903
54353624 54370556 19q13.4 associated immunoglobulin like receptor 1
22 SIGLEC7 p75; QA79; AIRM1; sialic acid 27036 51142294 51153526
19q13.3 CD328; CDw328; D- binding Ig like siglec; SIGLEC-7; lectin
7 SIGLECP2; SIGLEC19P; p75/AIRM1 23 SIGLEC9 CD329; CDw329; sialic
acid 27180 51124880 51141020 19q13.41 FOAP-9; siglec-9; binding Ig
like OBBP-LIKE lectin 9 24 TNFRSF10B DRS; CD262; tumor necrosis
8795 23006383 23069187 8p22-p21 KILLER; TRICK2; factor receptor
TRICKB; ZTNFR9; superfamily TRAILR2; member 10b TRICK2A; TRICK2B;
TRAIL- R2; KILLER/DR5 25 TNFRSF10A DR4; APO2; CD261; tumor necrosis
8797 23191457 23225167 8p21 TRAILR1; TRAILR- factor receptor 1
superfamily member 10a 26 CASP8 CAP4; MACH; caspase 8 841 201233443
201287711 2q33-q34 MCH5; FLICE; ALPS2B; Casp-8 27 CASP10 MCH4;
ALPS2; caspase 10 843 201182898 201229406 2q33-q34 FLICE2 28 CASP3
CPP32; SCA-1; caspase 3 836 184627696 184649475 4q34 CPP32B 29
CASP6 MCH2 caspase 6 839 109688628 109713904 4q25 30 CASP7 MCH3;
CMH-1; caspase 7 840 113679162 113730909 10q25 LICE2; CASP-7;
ICE-LAP3 31 FADD GIG3; MORT1 Fas associated 8772 70203163 70207402
11q13.3 via death domain 32 FAS APT1; CD95; FAS1; Fas cell surface
355 88969801 89017059 10q24.1 APO-1; FASTM; death receptor ALPS1A;
TNFRSF6 33 TGFBRII AAT3; FAA3; transforming 7048 30606493 30694142
3p22 LDS2; MFS2; RIIC; growth factor LDS1B; LDS2B; beta receptor II
TAAD2; TGFR-2; TGFbeta-RII 34 TGFBR1 AAT5; ALK5; transforming 7046
99104038 99154192 9q22 ESS1; LDS1; MSSE; growth factor SKR4; ALK-5;
beta receptor I LDS1A; LDS2A; TGFR-1; ACVRLK4; tbetaR-I 35 SMAD2
JV18; MADH2; SMAD family 4087 47833095 47931193 18q21.1 MADR2;
JV18-1; member 2 hMAD-2; hSMAD2 36 SMAD3 LDS3; LDS1C; SMAD family
4088 67065627 67195195 15q22.33 MADH3; JV15-2; member 3 HSPC193;
HsT17436 37 SMAD4 JIP; DPC4; MADH4; SMAD family 4089 51030213
51085042 18q21.1 MYHRS member 4 38 SKI SGS; SKV SKI proto- 6497
2228695 2310213 1p36.33 oncogene 39 SKIL SNO; SnoA; SnoI; SKI-like
proto- 6498 170357678 170396849 3q26 SnoN oncogene 40 TGIF1 HPE4;
TGIF TGFB induced 7050 3411927 3458411 18p11.3 factor homeobox 1 41
IL10RA CD210; IL10R; interleukin 10 3587 117986391 118001483 11q23
CD210a; receptor CDW210A; HIL- subunit alpha 10R; IL-10R1 42 IL10RB
CRFB4; CRF2-4; interleukin 10 3588 33266360 33297234 21q22.11
D21S58; D21S66; receptor CDW210B; IL-10R2 subunit beta 43 HMOX2
HO-2 heme 3163 4474703 4510347 16p13.3 oxygenase 2 44 IL6R IL6Q;
gp80; CD126; interleukin 6 3570 154405193 154469450 1q21 IL6RA;
IL6RQ; IL- receptor 6RA; IL-6R-1 45 IL6ST CD130; GP130; interleukin
6 3572 55935095 55994993 5q11.2 CDW130; IL-6RB signal transducer 46
CSK CSK c-src tyrosine 1445 74782084 74803198 15q24.1 kinase 47
PAG1 CBP; PAG phosphoprotein 55824 80967810 81112068 8q21.13
membrane anchor with glycosphingolipid microdomains 1 48 SIT1 SIT1
signaling 27240 35649298 35650950 9p13-p12 threshold regulating
transmembrane adaptor 1 49 FOXP3 JM2; AIID; IPEX; forkhead box
50943 49250436 49269727 Xp11.23 PIDX; XPID; P3 DIETER 50 PRDM1
BLIMP1; PRDI-BF1 PR domain 1 639 106086320 106109939 6q21 51 BATF
SFA2; B-ATF; basic leucine 10538 75522441 75546992 14q24.3 BATF1;
SFA-2 zipper transcription factor, ATF- like 52 GUCY1A2 GC-SA2;
GUC1A2 guanylate 2977 106674012 107018445 11q21-q22 cyclase 1,
soluble, alpha 2 53 GUCY1A3 GUCA3; MYMY6; guanylate 2982 155666568
155737062 4q32.1 GC-SA3; GUC1A3; cyclase 1, GUCSA3; soluble, alpha
3 GUCY1A1 54 GUCY1B2 GUCY1B2 guanylate 2974 50994511 51066157
13q14.3 cyclase 1, soluble, beta 2 (pseudogene) 55 GUCY1B3 GUCB3;
GC-SB3; guanylate 2983 155758973 155807642 4q31.3-q33 GUC1B3;
GUCSB3; cyclase 1, GUCY1B1; GC-S- soluble, beta 3 beta-1 56 TRA
IMD7; TCRA; T-cell receptor 6955 21621904 22552132 14q11.2 TCRD;
TRAalpha; alpha locus TRAC 57 TRB TCRB; TRBbeta T cell receptor
6957 142299011 142813287 7q34 beta locus 58 EGLN1 HPH2; PHD2; egl-9
family 54583 231363751 231425044 1q42.1 SM20; ECYT3; hypoxia-
HALAH; HPH-2; inducible factor HIFPH2; ZMYND6; 1 C1orf12; HIF-PH2
59 EGLN2 EIT6; PHD1; HPH- egl-9 family 112398 40799143 40808441
19q13.2 1; HPH-3; HIFPH1; hypoxia- HIF-PH1 inducible factor 2 60
EGLN3 PHD3; HIFPH3; egl-9 family 112399 33924215 33951083 14q13.1
HIFP4H3 hypoxia- inducible factor 3 61 PPP1R12C** p84; p85; LENG3;
protein 54776 55090913 55117600 19q13.42 MBS85 phosphatase 1
regulatory subunit 12C
[0131] A cell can have one or more suppressed genes. For example,
one or more genes whose expression is suppressed can comprise any
one of the genes in Table 1 as well as homologous or modified
versions thereof. Gene suppression can also be done in a number of
ways. For example, gene expression can be suppressed by knock out,
altering a promoter of a gene, and/or by administering interfering
RNAs. This can be done at an organism level or at a tissue, organ,
and/or cellular level. If one or more genes are knocked down in a
cell, tissue, and/or organ, the one or more genes can be suppressed
by administrating RNA interfering reagents, e.g., siRNA, shRNA, or
microRNA. For example, a nucleic acid which can express shRNA can
be stably transfected into a cell to knockdown expression.
Furthermore, a nucleic acid which can express shRNA can be inserted
into the genome of a T cell, thus knocking down a gene within the T
cell.
[0132] In some instances, a gene that can be disrupted or
suppressed can be CISH. A CISH gene can be a member of a
cytokine-induced STAT inhibitor (CIS), also known as suppressor of
cytokine signaling (SOCS) or STAT-induced STAT inhibitor (SSI),
protein family (see e.g., Palmer et al., Cish actively silences TCR
signaling in CD8+ T cells to maintain tumor tolerance, The Journal
of Experimental Medicine 202(12), 2095-2113 (2015)). A gene can be
part of a SOCS family of proteins that can form part of a classical
negative feedback system that can regulate cytokine signal
transduction. CISH can be involved in negative regulation of
cytokines that signal through the JAK-STAT5 pathway such as
erythropoietin, prolactin or interleukin 3 (IL-3) receptor. A gene
can inhibit STAT5 trans-activation by suppressing its tyrosine
phosphorylation. CISH family members are known to be
cytokine-inducible negative regulators of cytokine signaling.
Expression of a gene can be induced by IL2, IL3, GM-CSF or EPO in
hematopoietic cells. Proteasome-mediated degradation of a gene
protein can be involved in the inactivation of an erythropoietin
receptor. In some cases, a gene to be targeted can be expressed in
tumor-specific T cells. A gene to be targeted can increase
infiltration of an engineered cell into antigen-relevant tumors
when disrupted.
[0133] Improving the functional avidity of effector T cells can be
critical in overcoming inhibitory factors within the tumor
microenvironment and eliciting tumor regression. In some cases,
Cish (Cytokine-induced SH2 protein), a member of the Suppressor of
Cytokine Signaling (SOCS) family, can be induced by T cell receptor
(TCR) stimulation in CD8.sup.+ T cells, can be expressed in
tumor-resident T cells and can inhibit their functional avidity
against tumor(s). Genetic deletion of Cish in tumor-specific
CD8.sup.+ T cells can enhance their expansion, functional avidity
and cytokine polyfunctionality, resulting in pronounced and durable
regression of established tumors. Cish physically interacts with
the critical TCR signaling intermediate, Phospholipase C-.sub.11
(PLC-.sub.11), targeting it for proteasomal degradation following
TCR stimulation. These findings established a novel targetable
interaction that regulates the functional avidity of tumor-specific
CD8.sup.+ T cells and can be manipulated to improve ACT cancer
immunotherapy. In some cases, Cish knockout or knockdown may result
in an increase in cytokine levels. Increased cytokine levels may
comprise increases in IFN-.gamma., TNF-.alpha., IL-2, or a
combination thereof in a supernatant as compared to supernatants of
wildtype counterpart cells. In other cases, knockout or knockdown
of a gene, such as Cish, can comprise an increase in antigen
sensitivity. An increase in antigen sensitivity may be measured by
total cytokine expression levels in some cases, an increase in
antigen sensitivity may be from 40 to 100 fold. An increase in
antigen sensitivity may be from 40 to 50, from 50 to 60, from 60 to
70, from 70 to 80, from 80 to 90, from 90 to 100 fold as compared
to control counterpart cells.
[0134] In some cases, Cish knockout or knockdown may result in an
increase in a maximal amount of cytokine release as measured by
IFN-.gamma., TNF-.alpha. and/ or IL-2 levels. In some cases, an
ELISA can measure total cytokine levels in a supernatant and may
not directly measure cytokine-production on a sub-population or
cellular level. To evaluate if different subpopulations or
individual T cells may be responsible for an increase in cytokine
production, CD8.sup.+ T may be selected, stimulated, and co-stained
for intracellular IFN-.gamma., TNF-.alpha. and IL-2 and evaluated
with flow cytometry. In some cases, Cish can negatively regulate
both total effector cytokine production and polyfunctionality in
tumor-specific T cells. The genetic whole-body deletion of Cish can
enhance functional avidity and licenses CD8.sup.+ T cells into
long-lasting tumor killers which may have implications in memory
responses to a relapsing tumor.
[0135] One or more genes in a T cell can be knocked out or
disrupted using any method. For example, knocking out one or more
genes can comprise deleting one or more genes from a genome of a T
cell. Knocking out can also comprise removing all or a part of a
gene sequence from a T cell. It is also contemplated that knocking
out can comprise replacing all or a part of a gene in a genome of a
T cell with one or more nucleotides. Knocking out one or more genes
can also comprise inserting a sequence in one or more genes thereby
disrupting expression of the one or more genes. For example,
inserting a sequence can generate a stop codon in the middle of one
or more genes. Inserting a sequence can also shift the open reading
frame of one or more genes. It is also contemplated that any
combinations of knockout technology can be combined. For example,
tissue specific knockout or cell specific knockout can be combined
with inducible technology, creating a tissue specific or cell
specific, inducible knockout. Furthermore, other systems such
developmental specific promoter, can be used in combination with
tissues specific promoters, and/or inducible knockouts.
[0136] Knocking out technology can also comprise gene editing. For
example, gene editing can be performed using a nuclease, including
CRISPR associated proteins (Cas proteins, e.g., Cas9), Zinc finger
nuclease (ZFN), Transcription Activator-Like Effector Nuclease
(TALEN), and meganucleases. Nucleases can be naturally existing
nucleases, genetically modified, and/or recombinant. Gene editing
can also be performed using a transposon-based system (e.g.
PiggyBac, Sleeping beauty). For example, gene editing can be
performed using a transposase.
[0137] In some cases, a cell that is engineered or contains a
disrupted gene or portion thereof can undergo pre-infusion testing.
Pre-infusion testing can include at least one of phenotypic
testing, potency testing, microbiological testing, endotoxin
testing, viability testing, and tumor cell testing of a culture of
engineered cells. A phenotypic testing can comprise detecting a
presence of CD3, CD4, CD8, CD56, CD45RA, CD45RO, IL-7 receptor
alpha, CD28, to name a few. In some cases, a level of a marker on
an engineered cell is over at least about 20%, 30%, 40%, 50%, 60%,
70%, 80%, 90%, 95%, or up to about 100%. TILs can be administered
when pre-infusion testing can be at least about 80% CD3 positive
for a phenotypic testing.
[0138] In some cases, potency testing of TILs can include detecting
a level of IFN.gamma. upon anti-CD3 stimulation of a TIL culture.
In some cases, a level of FN.gamma. upon anti-CD3 stimulation of a
TIL culture is significantly greater than a comparable control
population of cells. In some cases, TILs are administered when a
pre-infusion testing yields at least about 200 pg/mL per 10.sup.5
cells of IFN.gamma. upon anti-CD3 stimulation of TILs in a potency
test. In some cases, TILs are administered when a pre-infusion
testing yields at least about 50 pg/mL per 10.sup.5 cells, 75 pg/mL
per 10.sup.5 cells, 100 pg/mL per 10.sup.5 cells, 150 pg/mL per
10.sup.5 cells, 200 pg/mL per 10.sup.5 cells, 250 pg/mL per
10.sup.5 cells, 300 pg/mL per 10.sup.5 cells, or up to about 350
pg/mL per 10.sup.5 cells of IFN.gamma. upon anti-CD3 stimulation of
TILs in a potency test.
[0139] In some cases, a population of TILs is tested for the
presence of tumor cells in a composition. TILs that can be
administered can be negative for tumor cells per at least about 200
TILs examined in a cytopathology testing. In other cases, less than
1% of tumor cells exist per at least about 200 TILs examined in a
cytopathology testing. In other cases, less than 2% of tumor cells
exist per at least about 200 TILs examined in a cytopathology
testing. In other cases, less than 3% of tumor cells exist per at
least about 200 TILs examined in a cytopathology testing. In other
cases, less than 4% of tumor cells exist per at least about 200
TILs examined in a cytopathology testing. In other cases, less than
5% of tumor cells exist per at least about 200 TILs examined in a
cytopathology testing.
CRISPR System
[0140] Methods described herein can take advantage of a CRISPR
system. There are at least five types of CRISPR systems which all
incorporate RNAs and Cas proteins. Types I, III, and IV assemble a
multi-Cas protein complex that is capable of cleaving nucleic acids
that are complementary to the crRNA. Types I and III both require
pre-crRNA processing prior to assembling the processed crRNA into
the multi-Cas protein complex. Types II and V CRISPR systems
comprise a single Cas protein complexed with at least one guiding
RNA.
[0141] The general mechanism and recent advances of CRISPR system
is discussed in Cong, L. et al., "Multiplex genome engineering
using CRISPR systems," Science, 339(6121): 819-823 (2013); Fu, Y.
et al., "High-frequency off-target mutagenesis induced by
CRISPR-Cas nucleases in human cells," Nature Biotechnology, 31,
822-826 (2013); Chu, V T et al. "Increasing the efficiency of
homology-directed repair for CRISPR-Cas9-induced precise gene
editing in mammalian cells," Nature Biotechnology 33, 543-548
(2015); Shmakov, S. et al., "Discovery and functional
characterization of diverse Class 2 CRISPR-Cas systems," Molecular
Cell, 60, 1-13 (2015); Makarova, KS et al., "An updated
evolutionary classification of CRISPR-Cas systems,", Nature Reviews
Microbiology, 13, 1-15 (2015). Site-specific cleavage of a target
DNA occurs at locations determined by both 1) base-pairing
complementarity between the guide RNA and the target DNA (also
called a protospacer) and 2) a short motif in the target DNA
referred to as the protospacer adjacent motif (PAM). For example,
an engineered cell can be generated using a CRISPR system, e.g., a
type II CRISPR system. A Cas enzyme used in the methods disclosed
herein can be Cas9, which catalyzes DNA cleavage. Enzymatic action
by Cas9 derived from Streptococcus pyogenes or any closely related
Cas9 can generate double stranded breaks at target site sequences
which hybridize to 20 nucleotides of a guide sequence and that have
a protospacer-adjacent motif (PAM) following the 20 nucleotides of
the target sequence.
[0142] In some cases, a CRISPR system may introduce a mutation. A
mutation can be an insertion or deletion. For example a CRISPR
system may introduce a lbp insertion comprising at least a portion
of CISH. In some cases, manipulation of TIL with a CRISPR system
may not have a negative effect on TIL expansion
post-electroporation of a CRISPR system. In some cases, to
determine whether an observed knockout frequency at the genetic
level correlates with loss of protein, the expression of protein,
such as CISH protein, after CRISPR knockout can be evaluated. For
example, peripheral blood (PB) T-cells and TILs can be
re-stimulated at day 14 post-electroporation using plate bound
anti-CD3 and soluble anti-CD28 antibody and the loss of protein,
for example CISH protein, by flow cytometry and western blot can be
assessed. In some cases, CRISPR modified PB T-cells and TILs can be
expanded for 14 days and then re-stimulated for 48hrs. Cells can be
collected and extracts analyzed by western blot as CISH is an
intracellular protein. Consistent with a high rate of knockout by
TiDE analysis of genomic modification, a protein, such as CISH, can
be essentially absent or reduced in knockout circulating T-cells
and TILs.
[0143] I. Cas Protein
[0144] A vector can be operably linked to an enzyme-coding sequence
encoding a CRISPR enzyme, such as a Cas protein (CRISPR-associated
protein). Non-limiting examples of Cas proteins can include Cas1,
Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known
as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csel, Cse2, Csc1, Csc2,
Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5,
Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3,
Csx1, Csx1S, Csf1, Csf2, CsO, Csf4, Cpf1, c2c1, c2c3, Cas9HiFi,
homologues thereof, or modified versions thereof. In some cases a
catalytically dead Cas protein can be used, for example a dCas9. An
unmodified CRISPR enzyme can have DNA cleavage activity, such as
Cas9. A CRISPR enzyme can direct cleavage of one or both strands at
a target sequence, such as within a target sequence and/or within a
complement of a target sequence. For example, a CRISPR enzyme can
direct cleavage of one or both strands within or within about 1, 2,
3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more
base pairs from the first or last nucleotide of a target sequence.
A vector that encodes a CRISPR enzyme that is mutated with respect
to a corresponding wild-type enzyme such that the mutated CRISPR
enzyme lacks the ability to cleave one or both strands of a target
polynucleotide containing a target sequence can be used. A Cas
protein can be a high fidelity Cas protein such as Cas9HiFi. In
some cases, a Cas protein can be modified. For example, a Cas
protein can be N7-Methyl-Gppp (2'-O-Methyl-A). In some cases, a Cas
protein, such as a Cas9 protein, can be sequenced prior to clinical
use. For example, a purified in vitro transcription product can be
assessed by polyacrylamide gel electrophoresis to verify no other
mRNA species exist within a clinical product other than Cas9.
Additionally, purified mRNA encoding a Cas protein, such as Cas9,
can undergo a validation by reverse-transcription followed by
sequencing to verify an identity at a nucleotide level. A Cas
sequence can contain a nuclear localization sequence (NLS). A
nuclear localization sequence can be from SV40. An NLS can be from
at least one of: SV40, nucleoplasmin, importin alpha, C-myc,
EGL-13, TUS, BORG, hnRNPA1, Mata2, or PY-NLS. An NLS can be on a
C-terminus or an N-terminus of a Cas protein. In some cases, a Cas
protein may contain from 1 to 5 NLS sequences. A Cas protein can
contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 NLS sequences. A Cas
protein, such as Cas9 may contain two NLS sequences. A Cas protein
may contain a SV40 and nuceloplasmin NLS sequence. A Cas protein
may also contain at least one untranslated region.
TABLE-US-00002 TABLE 2 Streptococcus pyogenes Cas9 (SpCas9) SEQ ID
Sequence 62 ATGGACTATAAGGACCACGAC GGAGACTACAAGGATCATGA
TATTGATTACAAAGACGATG ACGATAAGATGGCCCCAAAG AAGAAGCGGAAGGTCGGTAT
CCACGGAGTCCCAGCAGCCG ACAAGAAGTACAGCATCGGC CTGGACATCGGCACCAACTC
TGTGGGCTGGGCCGTGATCA CCGACG
TABLE-US-00003 TABLE 3 Modified Streptococcus pyogenes Cas9 mRNA
SEQ ID Sequence 63 GGAAATAAGAGAGAAAAGAAG AGTAAGAAGAAATATAAGAG
CCACCATGGCCCCAAAGAAG AAGCGGAAGGTCGGTATCCA CGGAGTCCCAGCAGCCGACA
AGAAGTACAGCATCGGCCTG GACATCGGCACCAACTCTGT GGGCTGGGCCGTGATCACCG
ACGAGTACAAGGTGCCCAGC AAGAAATTCAAGGTGCTGGG CAACACCGACCGGCACAGCA
TCAAGAAGAACCTGATCGGA GCCCTGCTGTTCGACAGCGG CGAAACAGCCGAGGCCACCC
GGCTGAAGAGAACCGCCAGA AGAAGATACACCAGACGGAA GAACCGGATCTGCTATCTGC
AAGAGATCTTCAGCAACGAG ATGGCCAAGGTGGACGACAG CTTCTTCCACAGACTGGAAG
AGTCCTTCCTGGTGGAAGAG GACAAGAAGCACGAGAGACA CCCCATCTTCGGCAACATCG
TGGACGAGGTGGCCTACCAC GAGAAGTACCCCACCATCTA CCACCTGAGAAAGAAACTGG
TGGACAGCACCGACAAGGCC GACCTGAGACTGATCTACCT GGCCCTGGCCCACATGATCA
AGTTCAGAGGCCACTTCCTG ATCGAGGGCGACCTGAACCC CGACAACAGCGACGTGGACA
AGCTGTTCATCCAGCTGGTG CAGACCTACAACCAGCTGTT CGAGGAAAACCCCATCAACG
CCAGCGGCGTGGACGCCAAG GCTATCCTGTCTGCCAGACT GAGCAAGAGCAGAAGGCTGG
AAAATCTGATCGCCCAGCTG CCCGGCGAGAAGAAGAACGG CCTGTTCGGCAACCTGATTG
CCCTGAGCCTGGGCCTGACC CCCAACTTCAAGAGCAACTT CGACCTGGCCGAGGATGCCA
AACTGCAGCTGAGCAAGGAC ACCTACGACGACGACCTGGA CAACCTGCTGGCCCAGATCG
GCGACCAGTACGCCGACCTG TTCCTGGCCGCCAAGAACCT GTCTGACGCCATCCTGCTGA
GCGACATCCTGAGAGTGAAC ACCGAGATCACCAAGGCCCC CCTGAGCGCCTCTATGATCA
AGAGATACGACGAGCACCAC CAGGACCTGACCCTGCTGAA AGCTCTCGTGCGGCAGCAGC
TGCCTGAGAAGTACAAAGAA ATCTTCTTCGACCAGAGCAA GAACGGCTACGCCGGCTACA
TCGATGGCGGCGCTAGCCAG GAAGAGTTCTACAAGTTCAT CAAGCCCATCCTGGAAAAGA
TGGACGGCACCGAGGAACTG CTCGTGAAGCTGAACAGAGA GGACCTGCTGAGAAAGCAGA
GAACCTTCGACAACGGCAGC ATCCCCCACCAGATCCACCT GGGAGAGCTGCACGCTATCC
TGAGAAGGCAGGAAGATTTT TACCCATTCCTGAAGGACAA CCGGGAAAAGATCGAGAAGA
TCCTGACCTTCAGGATCCCC TACTACGTGGGCCCCCTGGC CAGAGGCAACAGCAGATTCG
CCTGGATGACCAGAAAGAGC GAGGAAACCATCACCCCCTG GAACTTCGAGGAAGTGGTGG
ACAAGGGCGCCAGCGCCCAG AGCTTCATCGAGAGAATGAC AAACTTCGATAAGAACCTGC
CCAACGAGAAGGTGCTGCCC AAGCACAGCCTGCTGTACGA GTACTTCACCGTGTACAACG
AGCTGACCAAAGTGAAATAC GTGACCGAGGGAATGAGAAA GCCCGCCTTCCTGAGCGGCG
AGCAGAAAAAGGCCATCGTG GACCTGCTGTTCAAGACCAA CAGAAAAGTGACCGTGAAGC
AGCTGAAAGAGGACTACTTC AAGAAAATCGAGTGCTTCGA CTCCGTGGAAATCTCCGGCG
TGGAAGATAGATTCAACGCC TCCCTGGGCACATACCACGA TCTGCTGAAAATTATCAAGG
ACAAGGACTTCCTGGATAAC GAAGAGAACGAGGACATTCT GGAAGATATCGTGCTGACCC
TGACACTGTTTGAGGACCGC GAGATGATCGAGGAAAGGCT GAAAACCTACGCTCACCTGT
TCGACGACAAAGTGATGAAG CAGCTGAAGAGAAGGCGGTA CACCGGCTGGGGCAGGCTGA
GCAGAAAGCTGATCAACGGC ATCAGAGACAAGCAGAGCGG CAAGACAATCCTGGATTTCC
TGAAGTCCGACGGCTTCGCC AACCGGAACTTCATGCAGCT GATCCACGACGACAGCCTGA
CATTCAAAGAGGACATCCAG AAAGCCCAGGTGTCCGGCCA GGGCGACTCTCTGCACGAGC
ATATCGCTAACCTGGCCGGC AGCCCCGCTATCAAGAAGGG CATCCTGCAGACAGTGAAGG
TGGTGGACGAGCTCGTGAAA GTGATGGGCAGACACAAGCC CGAGAACATCGTGATCGAGA
TGGCTAGAGAGAACCAGACC ACCCAGAAGGGACAGAAGAA CTCCCGCGAGAGGATGAAGA
GAATCGAAGAGGGCATCAAA GAGCTGGGCAGCCAGATCCT GAAAGAACACCCCGTGGAAA
ACACCCAGCTGCAGAACGAG AAGCTGTACCTGTACTACCT GCAGAATGGCCGGGATATGT
ACGTGGACCAGGAACTGGAC ATCAACAGACTGTCCGACTA CGATGTGGACCATATCGTGC
CTCAGAGCTTTCTGAAGGAC GACTCCATCGATAACAAAGT GCTGACTCGGAGCGACAAGA
ACAGAGGCAAGAGCGACAAC GTGCCCTCCGAAGAGGTCGT GAAGAAGATGAAGAACTACT
GGCGACAGCTGCTGAACGCC AAGCTGATTACCCAGAGGAA GTTCGATAACCTGACCAAGG
CCGAGAGAGGCGGCCTGAGC GAGCTGGATAAGGCCGGCTT CATCAAGAGGCAGCTGGTGG
AAACCAGACAGATCACAAAG CACGTGGCACAGATCCTGGA CTCCCGGATGAACACTAAGT
ACGACGAAAACGATAAGCTG ATCCGGGAAGTGAAAGTGAT CACCCTGAAGTCCAAGCTGG
TGTCCGATTTCCGGAAGGAT TTCCAGTTTTACAAAGTGCG CGAGATCAACAACTACCACC
ACGCCCACGACGCCTACCTG AACGCCGTCGTGGGAACCGC CCTGATCAAAAAGTACCCTA
AGCTGGAAAGCGAGTTCGTG TACGGCGACTACAAGGTGTA CGACGTGCGGAAGATGATCG
CCAAGAGCGAGCAGGAAATC GGCAAGGCTACCGCCAAGTA CTTCTTCTACAGCAACATCA
TGAACTTTTTCAAGACCGAA ATCACCCTGGCCAACGGCGA GATCAGAAAGCGCCCTCTGA
TCGAGACAAACGGCGAAACC GGGGAGATCGTGTGGGATAA GGGCAGAGACTTCGCCACAG
TGCGAAAGGTGCTGAGCATG CCCCAAGTGAATATCGTGAA AAAGACCGAGGTGCAGACAG
GCGGCTTCAGCAAAGAGTCT ATCCTGCCCAAGAGGAACAG CGACAAGCTGATCGCCAGAA
AGAAGGACTGGGACCCCAAG AAGTACGGCGGCTTCGACAG CCCTACCGTGGCCTACTCTG
TGCTGGTGGTGGCTAAGGTG GAAAAGGGCAAGTCCAAGAA ACTGAAGAGTGTGAAAGAGC
TGCTGGGGATCACCATCATG GAAAGAAGCAGCTTTGAGAA GAACCCTATCGACTTTCTGG
AAGCCAAGGGCTACAAAGAA GTGAAAAAGGACCTGATCAT CAAGCTGCCTAAGTACTCCC
TGTTCGAGCTGGAAAACGGC AGAAAGAGAATGCTGGCCTC TGCCGGCGAACTGCAGAAGG
GAAACGAGCTGGCCCTGCCT AGCAAATATGTGAACTTCCT GTACCTGGCCTCCCACTATG
AGAAGCTGAAGGGCAGCCCT GAGGACAACGAACAGAAACA GCTGTTTGTGGAACAGCATA
AGCACTACCTGGACGAGATC ATCGAGCAGATCAGCGAGTT CTCCAAGAGAGTGATCCTGG
CCGACGCCAATCTGGACAAG GTGCTGTCTGCCTACAACAA GCACAGGGACAAGCCTATCA
GAGAGCAGGCCGAGAATATC ATCCACCTGTTCACCCTGAC AAACCTGGGCGCTCCTGCCG
CCTTCAAGTACTTTGACACC ACCATCGACCGGAAGAGGTA CACCAGCACCAAAGAGGTGC
TGGACGCCACCCTGATCCAC CAGAGCATCACCGGCCTGTA CGAGACAAGAATCGACCTGT
CTCAGCTGGGAGGCGACAAG AGACCTGCCGCCACTAAGAA GGCCGGACAGGCCAAAAAGA
AGAAGTGAGCGGCCGCTTAA TTAAGCTGCCTTCTGCGGGG CTTGCCTTCTGGCCATGCCC
TTCTTCTCTCCCTTGCACCT GTACCTCTTGGTCTTTGAAT AAAGCCTGAGTAGGAAGAAA
AAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAA
AAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAA
[0145] Cas9 can refer to a polypeptide with at least or at least
about 50%, 60%, 70%, 80%, 90%, 100% sequence identity and/or
sequence similarity to a wild type exemplary Cas9 polypeptide
(e.g., Cas9 from S. pyogenes). Cas9 can refer to a polypeptide with
at most or at most about 50%, 60%, 70%, 80%, 90%, 100% sequence
identity and/or sequence similarity to a wild type exemplary Cas9
polypeptide (e.g., from S. pyogenes). Cas9 can refer to the wild
type or a modified form of the Cas9 protein that can comprise an
amino acid change such as a deletion, insertion, substitution,
variant, mutation, fusion, chimera, or any combination thereof.
[0146] A polynucleotide encoding an endonuclease (e.g., a Cas
protein such as Cas9) can be codon optimized for expression in
particular cells, such as eukaryotic cells. This type of
optimization can entail the mutation of foreign-derived (e.g.,
recombinant) DNA to mimic the codon preferences of the intended
host organism or cell while encoding the same protein.
[0147] A vector that encodes a CRISPR enzyme comprising one or more
nuclear localization sequences (NLSs), such as more than or more
than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, NLSs can be used. For
example, a CRISPR enzyme can comprise more than or more than about
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, NLSs at or near the ammo-terminus,
more than or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, NLSs at
or near the carboxyl-terminus, or any combination of these (e.g.,
one or more NLS at the ammo-terminus and one or more NLS at the
carboxyl terminus). When more than one NLS is present, each can be
selected independently of others, such that a single NLS can be
present in more than one copy and/or in combination with one or
more other NLSs present in one or more copies.
[0148] An NLS can be monopartite or bipartite. In some cases, a
bipartite NLS can have a spacer sequence as opposed to a
monopartite NLS. An NLS can be from at least one of: SV40,
nucleoplasmin, importin alpha, C-myc, EGL-13, TUS, BORG, hnRNPA1,
Mata2, or PY-NLS. An NLS can be located anywhere within the
polypeptide chain, e g., near the N- or C-terminus. For example,
the NLS can be within or within about 1, 2, 3, 4, 5, 10, 15, 20,
25, 30, 40, 50 amino acids along a polypeptide chain from the N- or
C-terminus. Sometimes the NLS can be within or within about 50
amino acids or more, e.g., 100, 200, 300, 400, 500, 600, 700, 800,
900, or 1000 amino acids from the N- or C-terminus.
[0149] An endonuclease can comprise an amino acid sequence having
at least or at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%,
99%, or 100%, amino acid sequence identity to the nuclease domain
of a wild type exemplary site-directed polypeptide (e.g., Cas9 from
S. pyogenes).
[0150] While S. pyogenes Cas9 (SpCas9), Table 2, is commonly used
as a CRISPR endonuclease for genome engineering, it may not be the
best endonuclease for every target excision site. For example, the
PAM sequence for SpCas9 (5' NGG 3') is abundant throughout the
human genome, but a NGG sequence may not be positioned correctly to
target a desired gene for modification. In some cases, a different
endonuclease may be used to target certain genomic targets. In some
cases, synthetic SpCas9-derived variants with non-NGG PAM sequences
may be used. Additionally, other Cas9 orthologues from various
species have been identified and these "non-SpCas9s" bind a variety
of PAM sequences that could also be useful for the present
invention. For example, the relatively large size of SpCas9
(approximately 4 kb coding sequence) means that plasmids carrying
the SpCas9 cDNA may not be efficiently expressed in a cell.
Conversely, the coding sequence for Staphylococcus aureus Cas9
(SaCas9) is approximately 1 kilo base shorter than SpCas9, possibly
allowing it to be efficiently expressed in a cell Similar to
SpCas9, the SaCas9 endonuclease is capable of modifying target
genes in mammalian cells in vitro and in mice in vivo.
[0151] Alternatives to S. pyogenes Cas9 may include RNA-guided
endonucleases from the Cpf1 family that display cleavage activity
in mammalian cells. Unlike Cas9 nucleases, the result of
Cpf1-mediated DNA cleavage is a double-strand break with a short 3'
overhang. Cpfl's staggered cleavage pattern may open up the
possibility of directional gene transfer, analogous to traditional
restriction enzyme cloning, which may increase the efficiency of
gene editing. Like the Cas9 variants and orthologues described
above, Cpf1 may also expand the number of sites that can be
targeted by CRISPR to AT-rich regions or AT-rich genomes that lack
the NGG PAM sites favored by SpCas9.
[0152] Any functional concentration of Cas protein can be
introduced to a cell. For example, 15 micrograms of Cas mRNA can be
introduced to a cell. In other cases, a Cas mRNA can be introduced
from 0.5 micrograms to 100 micrograms. A Cas mRNA can be introduced
from 0.5, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, or 100 micrograms.
[0153] In some cases, a dual nickase approach may be used to
introduce a double stranded break or a genomic break. Cas proteins
can be mutated at known amino acids within either nuclease domains,
thereby deleting activity of one nuclease domain and generating a
nickase Cas protein capable of generating a single strand break. A
nickase along with two distinct guide RNAs targeting opposite
strands may be utilized to generate a DSB within a target site
(often referred to as a "double nick" or "dual nickase" CRISPR
system). This approach may dramatically increase target
specificity, since it is unlikely that two off-target nicks will be
generated within close enough proximity to cause a DSB.
[0154] A nuclease, such as Cas9, can be tested for identity and
potency prior to use. For example, identity and potency can be
determined using at least one of spectrophotometric analysis, RNA
agarose gel analysis, LC-MS, endotoxin analysis, and sterility
testing. In some cases, identity testing can determine an
acceptable level for clinical/therapeutic use. For example, an
acceptable spectrophotometric analysis result can be 105.+-.10
.mu.L/vial at 1.0.+-.0.1 mg/mL. An acceptable spectrophotometric
analysis result can also be from about 90-120.+-.10 .mu.L/vial at
1.0.+-.0.1 mg/mL or from about 90-120.+-.10 .mu.L/vial at about 0.1
to 5.0.+-.0.1 mg/mL.
[0155] In some cases, a nuclease, such as Cas9, can have a
UV260/280 ratio of about 1.0.+-.0.1. A UV260-280 ratio can be from
about 1.0-5.0.+-.0.1.
[0156] In some cases, a nuclease, such as Cas9, can have an
integrity/size measured by RNA agarose gel analysis. A size of a
Cas9 can be about 4500 bases. A size of a Cas9 can be from about
4000 to about 8000 bases. A size of a Cas9 can be from about 4000
to about 5000 bases, from about 5000 to about 6000 bases, from
about 6000 to about 7000 bases, from about 7000 to about 8000
bases. In some cases, a bioanalyzer can be utilized to determine a
size of a nuclease sequence, such as Cas9. A bioanalyzer can
determine a size of a Cas9 sequence that can be from about 4000 to
about 5000 bases, from about 5000 to about 6000 bases, from about
6000 to about 7000 bases, from about 7000 to about 8000 bases.
[0157] In some cases, an endotoxin level of a nuclease, such as
Cas9, can be determined. An endotoxin testing can be a limulus
assay. A clinically/therapeutically acceptable level of an
endotoxin can be less than 3 EU/mL. A clinically/therapeutically
acceptable level of an endotoxin can be less than 10 EU/mL. A
clinically/therapeutically acceptable level of an endotoxin can be
less than 8 EU/mL. A clinically/therapeutically acceptable level of
an endotoxin can be less than 5 EU/mL. A clinically/therapeutically
acceptable level of an endotoxin can be less than 4 EU/mL. A
clinically/therapeutically acceptable level of an endotoxin can be
less than 3 EU/mL. A clinically/therapeutically acceptable level of
an endotoxin can be less than 2 EU/mL. A clinically/therapeutically
acceptable level of an endotoxin can be less than 1 EU/mL. A
clinically/therapeutically acceptable level of an endotoxin can be
less than 0.5 EU/mL.
[0158] In some cases a nuclease, such as Cas9, can undergo
sterility testing. A clinically/therapeutically acceptable level of
a sterility testing can be 0 or denoted by no growth on a culture.
A clinically/therapeutically acceptable level of a sterility
testing can be less than 0.5% growth. A clinically/therapeutically
acceptable level of a sterility testing can be less than 1%
growth.
[0159] In some cases, a nuclease sequence, such as a Cas9 sequence
can be sequenced to confirm its identity. For example, an input
Cas9 mRNA transcription template can be sequenced at about a 4-fold
coverage prior to a production of a Cas9 mRNA lot. A purified in
vitro transcription produce can be assessed by polyacrylamide gel
electrophoresis (PAGE) to verify that an mRNA is the size expected
for Cas9 and no other mRNA species exist within a clinical or
therapeutic product. In some cases, a purified mRNA will undergo a
validation by reverse transcriptase (RT)-mediated
reverse-transcription followed by DNA sequencing to verify identity
at the nucleotide level.
[0160] In some cases, a potency of nuclease functionality can be
tested with a trial run. For example, a Cas9 can be tested for
functional potency in primary human T cells derived from three
independent donors. Reagent delivery can be performed in the same
fashion as for a patient sample. Potency can be determined by
sequencing of the target genomic locus and indel frequency may meet
or exceed 50% in each donor. In some cases, potency can be
determined by sequencing of the target genomic locus and indel
frequency may meet or exceed 60% in each donor. In some cases,
potency can be determined by sequencing of the target genomic locus
and indel frequency may meet or exceed 65% in each donor. In some
cases, potency can be determined by sequencing of the target
genomic locus and indel frequency may meet or exceed 70% in each
donor. In some cases, potency can be determined by sequencing of
the target genomic locus and indel frequency may meet or exceed 75%
in each donor.
[0161] II. Guiding Polynucleic Acid
[0162] A guiding polynucleic acid can be DNA or RNA. A guiding
polynucleic acid can be single stranded or double stranded. In some
cases, a guiding polynucleic acid can contains regions of single
stranded areas and double stranded areas. A guiding polynucleic
acid can also form secondary structures. As used herein, the term
"guide RNA (gRNA)," and its grammatical equivalents can refer to an
RNA which can be specific for a target DNA and can form a complex
with a Cas protein. A guide RNA can comprise a guide sequence, or
spacer sequence, that specifies a target site and guides an RNA/Cas
complex to a specified target DNA for cleavage. For example, a
guide RNA can target a CRISPR complex to different genes and
perform a targeted double strand break. Site-specific cleavage of a
target DNA occurs at locations determined by both 1) base-pairing
complementarity between a guide RNA and a target DNA (also called a
protospacer) and 2) a short motif in a target DNA referred to as a
protospacer adjacent motif (PAM). In some cases, gRNAs can be
designed using an algorithm which can identify gRNAs located in
early exons within commonly expressed transcripts. Candidate gRNAs
can be ranked by off-target potential using a scoring system that
can take into account: (a) the total number of mismatches between
the gRNA sequence and any closely matching genomic sequences; (b)
the mismatch position(s) relative to the PAM site which correlate
with a negative effect on activity for mismatches falling close to
the PAM site; (c) the distance between mismatches to account for
the cumulative effect of neighboring mismatches in disrupting
guide-DNA interactions; and any combination thereof. In some cases,
a greater number of mismatches between a gRNA and a genomic target
site can yield a lower potential for CRISPR-mediated cleavage of
that site. In some cases, a mismatch position is directly adjacent
to a PAM site. In other cases, a mismatch position can be from 1
nucleotide up to 100 kilobases away from a PAM site. Candidate
gRNAs comprising mismatches may not be adjacent to a PAM in some
cases. In other cases, at least two candidate gRNAs comprising
mismatches may bind a genome from 1 nucleotide up to 100 kilobases
away from each other. A mismatch can be a substitution of a
nucleotide. For example, in some cases a G will be substituted for
a T. Mismatches between a gRNA and a genome may allow for reduced
fidelity of CRISPR gene editing. In some cases, a positive scoring
gRNA can be about 110 nucleotides in length and may contain no
mismatches to a complementary genome sequence. In other cases, a
positive scoring gRNA can be about 110 nucleotides in length and
may contain up to 3 mismatches to a complementary genome sequence.
In other cases, a positive scoring gRNA can be about 110
nucleotides in length and may contain up to 20 mismatches to a
complementary genome sequence. A guiding polynucleic acid can have
at least or at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
95%, 98%, or up to about 100% sequence identity and/or sequence
similarity to any of the sequences of Table 4. In some cases, a
guiding polynucleic acid can contain internucleotide linkages that
can be phosphorothioates. Any number of phosphorothioates can
exist. For example from 1 to about 100 phosphorothioates can exist
in a guiding polynucleic acid sequence. In some cases from 1 to 10
phosphorothioates are present. In some cases, 0, 1, 2, 3, 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20
phosphorothioates exist in a guiding polynucleic acid sequence.
TABLE-US-00004 TABLE 4 Sequence listings for modified gRNAs
targeting the PD-1, CTLA-4, AAVS1, or CISH genes. SEQ ID gRNA
Sequence 64 PD-1 gRNA CCUGCUCGUGGUGACCGAAGGU #2
UUUAGAGCUAGAAAUAGCAAGU UAAAAUAAGGCUAGUCCGUUAU
CAACUUGAAAAAGUGGCACCGA GUCGGUGCUUUU 65 PD-1 gRNA
ACGGAAGCGGCAGUCCUGGCGU #6 UUUAGAGCUAGAAAUAGCAAGU
UAAAAUAAGGCUAGUCCGUUAU CAACUUGAAAAAGUGGCACCGA GUCGGUGCUUUU 66 CTLA4
CUAGAUGAUUCCAUCUGCACGU gRNA #3 UUUAGAGCUAGAAAUAGCAAGU
UAAAAUAAGGCUAGUCCGUUAU CAACUUGAAAAAGUGGCACCGA GUCGGUGCUUUU 67 CTLA4
GUGCGGCAACCUACAUGAUGGU gRNA #2 UUUAGAGCUAGAAAUAGCAAGU
UAAAAUAAGGCUAGUCCGUUAU CAACUUGAAAAAGUGGCACCGA GUCGGUGCUUUU 68 CISH
gRNA GGGUUCCAUUACGGCCAGCGGU #2 UUUAGAGCUAGAAAUAGCAAGU
UAAAAUAAGGCUAGUCCGUUAU CAACUUGAAAAAGUGGCACCGA GUCGGUGCUUUU 69 AAVS1
GUCACCAAUCCUGUCCCUAGGU UUUAGAGCUAGAAAUAGCAAGU
UAAAAUAAGGCUAGUCCGUUAU CAACUUGAAAAAGUGGCACCGA GUCGGUGCUUUU
[0163] In some cases, top scoring gRNAs can be designed and
selected and an on-target editing efficiency of each can be
assessed experimentally in patient derived TIL and peripheral
blood-derived T cells. In some cases, an editing efficiency as
determined by TiDE analysis can exceed at least about 20%. In other
cases, and editing efficiency can be from about 20% to from about
50%, from about 50% to from about 80%, from about 80% to from about
100%. In some cases, editing efficiency as measured by TiDE can be
85% for PD-1 and 90% for CISH. In some cases, a percent indel can
be determined in a trial GMP run. For example, a final cellular
product can be analyzed for on-target indel formation by Sanger
sequencing and TIDE analysis. Genomic DNA can be extracted froma
bout 1.times.10.sup.6 cells from both a control and experimental
sample and subjected to PCR using primers flanking a gene that has
been disrupted, such as CISH. Sanger sequencing chromatogramps can
be analyzed using a TIDE software program that can quantify indel
frequency and size distribution of indels by comparison of control
and knockout samples.
[0164] A method disclosed herein also can comprise introducing into
a cell or embryo at least one guide RNA or nucleic acid, e.g., DNA
encoding at least one guide RNA. A guide RNA can interact with a
RNA-guided endonuclease to direct the endonuclease to a specific
target site, at which site the 5' end of the guide RNA base pairs
with a specific protospacer sequence in a chromosomal sequence.
[0165] A guide RNA can comprise two RNAs, e.g., CRISPR RNA (crRNA)
and transactivating crRNA (tracrRNA). A guide RNA can sometimes
comprise a single-guide RNA (sgRNA) formed by fusion of a portion
(e.g., a functional portion) of crRNA and tracrRNA. A guide RNA can
also be a dual RNA comprising a crRNA and a tracrRNA. A guide RNA
can comprise a crRNA and lack a tracrRNA. Furthermore, a crRNA can
hybridize with a target DNA or protospacer sequence.
[0166] As discussed above, a guide RNA can be an expression
product. For example, a DNA that encodes a guide RNA can be a
vector comprising a sequence coding for the guide RNA. A guide RNA
can be transferred into a cell or organism by transfecting the cell
or organism with an isolated guide RNA or plasmid DNA comprising a
sequence coding for the guide RNA and a promoter. A guide RNA can
also be transferred into a cell or organism in other way, such as
using virus-mediated gene delivery.
[0167] A guide RNA can be isolated. For example, a guide RNA can be
transfected in the form of an isolated RNA into a cell or organism.
A guide RNA can be prepared by in vitro transcription using any in
vitro transcription system. A guide RNA can be transferred to a
cell in the form of isolated RNA rather than in the form of plasmid
comprising encoding sequence for a guide RNA.
[0168] A guide RNA can comprise a DNA-targeting segment and a
protein binding segment. A DNA-targeting segment (or DNA-targeting
sequence, or spacer sequence) comprises a nucleotide sequence that
can be complementary to a specific sequence within a target DNA
(e.g., a protospacer). A protein-binding segment (or
protein-binding sequence) can interact with a site-directed
modifying polypeptide, e.g. an RNA-guided endonuclease such as a
Cas protein. By "segment" it is meant a segment/section/region of a
molecule, e.g., a contiguous stretch of nucleotides in an RNA. A
segment can also mean a region/section of a complex such that a
segment may comprise regions of more than one molecule. For
example, in some cases a protein-binding segment of a DNA-targeting
RNA is one RNA molecule and the protein-binding segment therefore
comprises a region of that RNA molecule. In other cases, the
protein-binding segment of a DNA-targeting RNA comprises two
separate molecules that are hybridized along a region of
complementarity.
[0169] A guide RNA can comprise two separate RNA molecules or a
single RNA molecule. An exemplary single molecule guide RNA
comprises both a DNA-targeting segment and a protein-binding
segment.
[0170] An exemplary two-molecule DNA-targeting RNA can comprise a
crRNA-like ("CRISPR RNA" or "targeter-RNA" or "crRNA" or "crRNA
repeat") molecule and a corresponding tracrRNA-like ("trans-acting
CRISPR RNA" or "activator-RNA" or "tracrRNA") molecule. A first RNA
molecule can be a crRNA-like molecule (targeter-RNA), that can
comprise a DNA-targeting segment (e.g., spacer) and a stretch of
nucleotides that can form one half of a double-stranded RNA (dsRNA)
duplex comprising the protein-binding segment of a guide RNA. A
second RNA molecule can be a corresponding tracrRNA-like molecule
(activator-RNA) that can comprise a stretch of nucleotides that can
form the other half of a dsRNA duplex of a protein-binding segment
of a guide RNA. In other words, a stretch of nucleotides of a
crRNA-like molecule can be complementary to and can hybridize with
a stretch of nucleotides of a tracrRNA-like molecule to form a
dsRNA duplex of a protein-binding domain of a guide RNA. As such,
each crRNA-like molecule can be said to have a corresponding
tracrRNA-like molecule. A crRNA-like molecule additionally can
provide a single stranded DNA-targeting segment, or spacer
sequence. Thus, a crRNA-like and a tracrRNA-like molecule (as a
corresponding pair) can hybridize to form a guide RNA. A subject
two-molecule guide RNA can comprise any corresponding crRNA and
tracrRNA pair.
[0171] A DNA-targeting segment or spacer sequence of a guide RNA
can be complementary to sequence at a target site in a chromosomal
sequence, e.g., protospacer sequence such that the DNA-targeting
segment of the guide RNA can base pair with the target site or
protospacer. In some cases, a DNA-targeting segment of a guide RNA
can comprise from or from about 10 nucleotides to from or from
about 25 nucleotides or more. For example, a region of base pairing
between a first region of a guide RNA and a target site in a
chromosomal sequence can be or can be about 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, 20, 22, 23, 24, 25, or more than 25 nucleotides in
length. Sometimes, a first region of a guide RNA can be or can be
about 19, 20, or 21 nucleotides in length.
[0172] A guide RNA can target a nucleic acid sequence of or of
about 20 nucleotides. A target nucleic acid can be less than or
less than about 20 nucleotides. A target nucleic acid can be at
least or at least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 30 or more nucleotides. A target nucleic acid can be at
most or at most about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23,
24, 25, 30 or more nucleotides. A target nucleic acid sequence can
be or can be about 20 bases immediately 5' of the first nucleotide
of the PAM. A guide RNA can target the nucleic acid sequence. A
guiding polynucleic acid, such as a guide RNA, can bind to a
genomic sequence with at least or at least about 50%, 60%, 65%,
70%, 75%, 80%, 85%, 90%, 95%, 98%, or up to about 100% sequence
identity and/or sequence similarity to any of the sequences of
Table 5 or Table 6. In some cases, a guiding polynucleic acid, such
as a guide RNA, can bind a genomic region from about 1 basepair to
about 20 basepairs away from a PAM. A guide can bind a genomic
region from about 1, 2, 3, 4, 5 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18, 19, or up to about 20 base pairs away from a PAM.
TABLE-US-00005 TABLE 5 Engineered CISH guide RNA (gRNA) target
sequences SEQ gRNA Target ID No. Exon 5'-3' 70 1 2 TTGCTGGCTG
TGGAGCGGAC 71 2 2 GACTGGCTTG GGCAGTTCCA 72 3 2 TGCTGGGGCC
TTCCTCGAGG 73 4 2 CCGAAGGTAG GAGAAGGTCT 74 5 2 ATGCACAGCA
GATCCTGCTC 75 6 2 AGAGAGTGAG CCAAAGGTCC 76 1 3 GGCATACTCA
ATGCGTACAT 77 2 3 GCGTFCCATT ACGGCCAGCG 78 3 3 AAGGCTGACC
ACATCCGGAA 79 4 3 TGCCGACTCC AGCTTCCGTC 80 5 3 CTGTCAGTGA
AAACCACTCG 81 6 3 CGTACTAAGA ACGTGCCTTC
[0173] Genomic sequences that are targeted by engineered gRNAs are
shown in Table 5 and Table 6. FIG. 22 shows modified gRNA targeting
the CISH gene.
TABLE-US-00006 TABLE 6 AAVS1 gRNA target sequence SEQ ID Gene gRNA
Sequence (5' to 3') 82 AAVS1 GTCACCAATCCTGTCCCTAG-
[0174] A guide nucleic acid, for example, a guide RNA, can refer to
a nucleic acid that can hybridize to another nucleic acid, for
example, the target nucleic acid or protospacer in a genome of a
cell. A guide nucleic acid can be RNA. A guide nucleic acid can be
DNA. The guide nucleic acid can be programmed or designed to bind
to a sequence of nucleic acid site-specifically. A guide nucleic
acid can comprise a polynucleotide chain and can be called a single
guide nucleic acid. A guide nucleic acid can comprise two
polynucleotide chains and can be called a double guide nucleic
acid.
[0175] A guide nucleic acid can comprise one or more modifications
to provide a nucleic acid with a new or enhanced feature. A guide
nucleic acid can comprise a nucleic acid affinity tag. A guide
nucleic acid can comprise synthetic nucleotide, synthetic
nucleotide analog, nucleotide derivatives, and/or modified
nucleotides.
[0176] A guide nucleic acid can comprise a nucleotide sequence
(e.g., a spacer), for example, at or near the 5' end or 3' end,
that can hybridize to a sequence in a target nucleic acid (e.g., a
protospacer). A spacer of a guide nucleic acid can interact with a
target nucleic acid in a sequence-specific manner via hybridization
(i.e., base pairing). A spacer sequence can hybridize to a target
nucleic acid that is located 5' or 3' of a protospacer adjacent
motif (PAM). The length of a spacer sequence can be at least or at
least about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30
or more nucleotides. The length of a spacer sequence can be at most
or at most about 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
30 or more nucleotides.
[0177] A guide RNA can also comprises a dsRNA duplex region that
forms a secondary structure. For example, a secondary structure
formed by a guide RNA can comprise a stem (or hairpin) and a loop.
A length of a loop and a stem can vary. For example, a loop can
range from about 3 to about 10 nucleotides in length, and a stem
can range from about 6 to about 20 base pairs in length. A stem can
comprise one or more bulges of 1 to about 10 nucleotides. The
overall length of a second region can range from about 16 to about
60 nucleotides in length. For example, a loop can be or can be
about 4 nucleotides in length and a stem can be or can be about 12
base pairs. A dsRNA duplex region can comprise a protein-binding
segment that can form a complex with an RNA-binding protein, such
as a RNA-guided endonuclease, e.g. Cas protein.
[0178] A guide RNA can also comprise a tail region at the 5' or 3'
end that can be essentially single-stranded. For example, a tail
region is sometimes not complementarity to any chromosomal sequence
in a cell of interest and is sometimes not complementarity to the
rest of a guide RNA. Further, the length of a tail region can vary.
A tail region can be more than or more than about 4 nucleotides in
length. For example, the length of a tail region can range from or
from about 5 to from or from about 60 nucleotides in length.
[0179] A guide RNA can be introduced into a cell or embryo as an
RNA molecule. For example, a RNA molecule can be transcribed in
vitro and/or can be chemically synthesized. A guide RNA can then be
introduced into a cell or embryo as an RNA molecule. A guide RNA
can also be introduced into a cell or embryo in the form of a
non-RNA nucleic acid molecule, e.g., DNA molecule. For example, a
DNA encoding a guide RNA can be operably linked to promoter control
sequence for expression of the guide RNA in a cell or embryo of
interest. A RNA coding sequence can be operably linked to a
promoter sequence that is recognized by RNA polymerase III (Pol
III).
[0180] A DNA molecule encoding a guide RNA can also be linear. A
DNA molecule encoding a guide RNA can also be circular. A DNA
sequence encoding a guide RNA can also be part of a vector. Some
examples of vectors can include plasmid vectors, phagemids,
cosmids, artificial/mini-chromosomes, transposons, and viral
vectors. For example, a DNA encoding a RNA-guided endonuclease is
present in a plasmid vector. Other non-limiting examples of
suitable plasmid vectors include pUC, pBR322, pET, pBluescript, and
variants thereof. Further, a vector can comprise additional
expression control sequences (e g., enhancer sequences, Kozak
sequences, polyadenylation sequences, transcriptional termination
sequences, etc.), selectable marker sequences (e.g., antibiotic
resistance genes), origins of replication, and the like.
[0181] When both a RNA-guided endonuclease and a guide RNA are
introduced into a cell as DNA molecules, each can be part of a
separate molecule (e.g., one vector containing fusion protein
coding sequence and a second vector containing guide RNA coding
sequence) or both can be part of a same molecule (e.g., one vector
containing coding (and regulatory) sequence for both a fusion
protein and a guide RNA).
[0182] A Cas protein, such as a Cas9 protein or any derivative
thereof, can be pre-complexed with a guide RNA to form a
ribonucleoprotein (RNP) complex. The RNP complex can be introduced
into primary immune cells. Introduction of the RNP complex can be
timed. The cell can be synchronized with other cells at Gl, S,
and/or M phases of the cell cycle. The RNP complex can be delivered
at a cell phase such that HDR is enhanced. The RNP complex can
facilitate homology directed repair.
[0183] A guide RNA can also be modified. The modifications can
comprise chemical alterations, synthetic modifications, nucleotide
additions, and/or nucleotide subtractions. The modifications can
also enhance CRISPR genome engineering. A modification can alter
chirality of a gRNA. In some cases, chirality may be uniform or
stereopure after a modification. A guide RNA can be synthesized.
The synthesized guide RNA can enhance CRISPR genome engineering. A
guide RNA can also be truncated. Truncation can be used to reduce
undesired off-target mutagenesis. The truncation can comprise any
number of nucleotide deletions. For example, the truncation can
comprise 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more
nucleotides. A guide RNA can comprise a region of target
complementarity of any length. For example, a region of target
complementarity can be less than 20 nucleotides in length. A region
of target complementarity can be more than 20 nucleotides in
length. A region of target complementarity can target from about 5
bp to about 20 bp directly adjacent to a PAM sequence. A region of
target complementarity can target about 13 bp directly adjacent to
a PAM sequence.
[0184] In some cases, a potency of a guiding polynucleic acid can
be tested with a trial run. For example, a guiding polynucleic acid
can be tested for functional potency in primary human T cells
derived from three independent donors. Reagent delivery can be
performed in the same fashion as for a patient sample. Potency can
be determined by sequencing of the target genomic locus and indel
frequency may meet or exceed 50% in each donor. In some cases,
potency can be determined by sequencing of the target genomic locus
and indel frequency may meet or exceed 60% in each donor. In some
cases, potency can be determined by sequencing of the target
genomic locus and indel frequency may meet or exceed 65% in each
donor. In some cases, potency can be determined by sequencing of
the target genomic locus and indel frequency may meet or exceed 70%
in each donor. In some cases, potency can be determined by
sequencing of the target genomic locus and indel frequency may meet
or exceed 75% in each donor.
[0185] A CRISPR system can be introduced into a cell or pluarality
of cells using any means. In some cases, a CRISPR system may be
introduced by electroporation or nucleofection. Electroporation can
be performed for example, using the Neon.RTM. Transfection System
(ThermoFisher Scientific) or the AMARA.RTM. Nucleofector
(AMARA.RTM. Biosystems) can also be used for delivery of nucleic
acids into a cell. Electroporation parameters may be adjusted to
optimize transfection efficiency and/or cell viability.
Electroporation devices can have multiple electrical wave form
pulse settings such as exponential decay, time constant and square
wave. Every cell type has a unique optimal Field Strength (E) that
is dependent on the pulse parameters applied (e.g., voltage,
capacitance and resistance). Application of optimal field strength
causes electropermeabilization through induction of transmembrane
voltage, which allows nucleic acids to pass through the cell
membrane. In some cases, the electroporation pulse voltage, the
electroporation pulse width, number of pulses, cell density, and
tip type may be adjusted to optimize transfection efficiency and/or
cell viability.
[0186] In some cases, a Neon transfection system may be utilized. A
Neon system can be a three-component electroporation apparatus
comprising a central control module, an electroporation chamber
that can be connected to a central control module by a 3-foot-long
electrical cord, and a specialized pipette. In some cases, a
specialized pipette can be fitted with exchangeable and/or
disposable sterile tips. In some cases, an electroporation chamber
can be fitted with exchangeable/disposable sterile electroporation
cuvettes. In some cases, standard electroporation buffers supplied
by a manufacturer of a system, such as a Neon system, can be
replaced with GMP qualified solutions and buffers. In some cases, a
standard electroporation buffer can be replaced with GMP grade
phosphate buffered saline (PBS). A self-diagnostic system check can
be performed on a control module prior to initiation of sample
electroporation to ensure the Neon system is properly functioning.
In some cases, a transfection can be performed in a class 1,000
biosafety cabinet within a class 10,000 clean room in a cGMP
facility. Trained medical technologists can use aseptic technique
throughout an entire manufacturing process, and the product can be
ultimately tested for sterility.
[0187] In some cases, electroporation pulse voltage may be varied
to optimize transfection efficiency and/or cell viability. In some
cases, the electroporation voltage may be less than about 500
volts. In some cases, the electroporation voltage may be at least
about 500 volts, at least about 600 volts, at least about 700
volts, at least about 800 volts, at least about 900 volts, at least
about 1000 volts, at least about 1100 volts, at least about 1200
volts, at least about 1300 volts, at least about 1400 volts, at
least about 1500 volts, at least about 1600 volts, at least about
1700 volts, at least about 1800 volts, at least about 1900 volts,
at least about 2000 volts, at least about 2100 volts, at least
about 2200 volts, at least about 2300 volts, at least about 2400
volts, at least about 2500 volts, at least about 2600 volts, at
least about 2700 volts, at least about 2800 volts, at least about
2900 volts, or at least about 3000 volts. In some cases, the
electroporation pulse voltage required for optimal transfection
efficiency and/or cell viability may be specific to the cell type.
For example, an electroporation voltage of 1900 volts may optimal
(e.g., provide the highest viability and/or transfection
efficiency) for macrophage cells. In another example, an
electroporation voltage of about 1350 volts may optimal (e.g.,
provide the highest viability and/or transfection efficiency) for
Jurkat cells or primary human cells such as T cells. In some cases,
a range of electroporation voltages may be optimal for a given cell
type. For example, an electroporation voltage between about 1000
volts and about 1300 volts may optimal (e.g., provide the highest
viability and/or transfection efficiency) for human 578T cells.
[0188] In some cases, electroporation pulse width may be varied to
optimize transfection efficiency and/or cell viability. In some
cases, the electroporation pulse width may be less than about 5
milliseconds. In some cases, the electroporation width may be at
least about 5 milliseconds, at least about 6 milliseconds, at least
about 7 milliseconds, at least about 8 milliseconds, at least about
9 milliseconds, at least about 10 milliseconds, at least about 11
milliseconds, at least about 12 milliseconds, at least about 13
milliseconds, at least about 14 milliseconds, at least about 15
milliseconds, at least about 16 milliseconds, at least about 17
milliseconds, at least about 18 milliseconds, at least about 19
milliseconds, at least about 20 milliseconds, at least about 21
milliseconds, at least about 22 milliseconds, at least about 23
milliseconds, at least about 24 milliseconds, at least about 25
milliseconds, at least about 26 milliseconds, at least about 27
milliseconds, at least about 28 milliseconds, at least about 29
milliseconds, at least about 30 milliseconds, at least about 31
milliseconds, at least about 32 milliseconds, at least about 33
milliseconds, at least about 34 milliseconds, at least about 35
milliseconds, at least about 36 milliseconds, at least about 37
milliseconds, at least about 38 milliseconds, at least about 39
milliseconds, at least about 40 milliseconds, at least about 41
milliseconds, at least about 42 milliseconds, at least about 43
milliseconds, at least about 44 milliseconds, at least about 45
milliseconds, at least about 46 milliseconds, at least about 47
milliseconds, at least about 48 milliseconds, at least about 49
milliseconds, or at least about 50 milliseconds. In some cases, the
electroporation pulse width required for optimal transfection
efficiency and/or cell viability may be specific to the cell type.
For example, an electroporation pulse width of 30 milliseconds may
optimal (e.g., provide the highest viability and/or transfection
efficiency) for macrophage cells. In another example, an
electroporation width of about 10 milliseconds may optimal (e.g.,
provide the highest viability and/or transfection efficiency) for
Jurkat cells. In some cases, a range of electroporation widths may
be optimal for a given cell type. For example, an electroporation
width between about 20 milliseconds and about 30 milliseconds may
optimal (e.g., provide the highest viability and/or transfection
efficiency) for human 578T cells.
[0189] In some cases, the number of electroporation pulses may be
varied to optimize transfection efficiency and/or cell viability.
In some cases, electroporation may comprise a single pulse. In some
cases, electroporation may comprise more than one pulse. In some
cases, electroporation may comprise 2 pulses, 3 pulses, 4 pulses, 5
pulses 6 pulses, 7 pulses, 8 pulses, 9 pulses, or 10 or more
pulses. In some cases, the number of electroporation pulses
required for optimal transfection efficiency and/or cell viability
may be specific to the cell type. For example, electroporation with
a single pulse may be optimal (e.g., provide the highest viability
and/or transfection efficiency) for macrophage cells. In another
example, electroporation with a 3 pulses may be optimal (e.g.,
provide the highest viability and/or transfection efficiency) for
primary cells. In some cases, a range of electroporation widths may
be optimal for a given cell type. For example, electroporation with
between about 1 to about 3 pulses may be optimal (e.g., provide the
highest viability and/or transfection efficiency) for human cells.
The efficiency of genomic disruption of cells with any of the
nucleic acid delivery platforms described herein, for example,
nucleofection or electroporation, can be or can be about 20%, 25%,
30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or more
than 99.9%.
[0190] In some cases, a starting cell density for electroporation
may be varied to optimize transfection efficiency and/or cell
viability. In some cases, the starting cell density for
electroporation may be less than about 1.times.10.sup.5 cells. In
some cases, the starting cell density for electroporation may be at
least about 1.times.10.sup.5 cells, at least about 2.times.10.sup.5
cells, at least about 3.times.10.sup.5 cells, at least about
4.times.10.sup.5 cells, at least about 5.times.10.sup.5 cells, at
least about 6.times.10.sup.5 cells, at least about 7.times.10.sup.5
cells, at least about 8.times.10.sup.5 cells, at least about
9.times.10.sup.5 cells, at least about 1.times.10.sup.6 cells, at
least about 1.5.times.10.sup.6 cells, at least about
2.times.10.sup.6 cells, at least about 2.5.times.10.sup.6 cells, at
least about 3.times.10.sup.6 cells, at least about
3.5.times.10.sup.6 cells, at least about 4.times.10.sup.6 cells, at
least about 4.5.times.10.sup.6 cells, at least about
5.times.10.sup.6 cells, at least about 5.5.times.10.sup.6 cells, at
least about 6.times.10.sup.6 cells, at least about
6.5.times.10.sup.6 cells, at least about 7.times.10.sup.6 cells, at
least about 7.5.times.10.sup.6 cells, at least about
8.times.10.sup.6 cells, at least about 8.5.times.10.sup.6 cells, at
least about 9.times.10.sup.6 cells, at least about
9.5.times.10.sup.6 cells, at least about 1.times.10.sup.7 cells, at
least about 1.2.times.10.sup.7 cells, at least about
1.4.times.10.sup.7 cells, at least about 1.6.times.10.sup.7 cells,
at least about 1.8.times.10.sup.7 cells, at least about
2.times.10.sup.7 cells, at least about 2.2.times.10.sup.7 cells, at
least about 2.4.times.10.sup.7 cells, at least about
2.6.times.10.sup.7 cells, at least about 2.8.times.10.sup.7 cells,
at least about 3.times.10.sup.7 cells, at least about
3.2.times.10.sup.7 cells, at least about 3.4.times.10.sup.7 cells,
at least about 3.6.times.10.sup.7 cells, at least about
3.8.times.10.sup.7 cells, at least about 4.times.10.sup.7 cells, at
least about 4.2.times.10.sup.7 cells, at least about
4.4.times.10.sup.7 cells, at least about 4.6.times.10.sup.7 cells,
at least about 4.8.times.10.sup.10 cells, or at least about
5.times.10.sup.7 cells. In some cases, the starting cell density
for electroporation required for optimal transfection efficiency
and/or cell viability may be specific to the cell type. For
example, a starting cell density for electroporation of
1.5.times.10.sup.6 cells may optimal (e.g., provide the highest
viability and/or transfection efficiency) for macrophage cells. In
another example, a starting cell density for electroporation of
5.times.10.sup.6 cells may optimal (e.g., provide the highest
viability and/or transfection efficiency) for human cells. In some
cases, a range of starting cell densities for electroporation may
be optimal for a given cell type. For example, a starting cell
density for electroporation between of 5.6.times.10.sup.6 and
5.times.10.sup.7 cells may optimal (e.g., provide the highest
viability and/or transfection efficiency) for human cells such as T
cells.
[0191] In some cases, a GUIDE-Seq analysis can be performed to
determine the specificity of engineered guide RNAs. The general
mechanism and protocol of GUIDE-Seq profiling of off-target
cleavage by CRISPR system nucleases is discussed in Tsai, S. et
al., "GUIDE-Seq enables genome-wide profiling of off-target
cleavage by CRISPR system nucleases," Nature, 33: 187-197 (2015).
To assess off-target frequencies by next generation sequencing
human primary T cells can be transfected with Cas9 mRNA and a
guiding RNA, such as anti-CISH gRNA. Genomic DNA can be isolated
from transfected cells from about 72 hours post transfection and
PCR amplified at potential off-target sites. A potential off-target
site can be predicted using the Wellcome Trust Sanger Insisute
Genome Editing database (WGE) algorithm. Candidate off-target sites
can be chosen based on sequence homology to an on-target site. In
some cases, sites with about 4 or less mismatches between a gRNA
and a genomic target site can be utilized. For each candidate
off-target site, two primer pairs can be designed. PCR amplicons
can be obtained from both untreated (control) and Cas9/gRNA-treated
cells. PCR amplicons can be pooled. NGS libraries can be prepared
using TruSeq Nano DNA library preparation kit (Illumina). Samples
can be analyzed on an Illumina HiSeq machine using a 250 bp
paired-end workflow. In some cases, from about 40 million mappable
NGS reads per gRNA library can be acquired. This can equate to an
average number of about 450,000 reads for each candidate off-target
site of a gRNA. In some cases, detection of CRISPR-mediated
disruption can be at a frequency as low as 1%, 0.9%, 0.8%, 0.7%,
0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% at a genomic locus.
[0192] Computational predictions can be used to select candidate
gRNAs likely to be the safest choice for a targeted gene, such as
PD-1 and/or CISH functional disruption. Candidate gRNAs can then
tested empirically using a focused approach steered by
computational predictions of potential off-target sites. In some
cases, an assessment of gRNA off-target safety can employ a
next-generation deep sequencing approach to analyze the potential
off-target sites predicted by the CRISPR design tool for each gRNA.
In some cases, gRNAs can be selected with fewer than 3 mismatches
to any sequence in the genome (other than the perfect matching
intended target). In some cases, a gRNA can be selected with fewer
than 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 mismatch(es) to any
sequence in a genome. In some cases, a computer system or software
can be utilized to provide recommendations of candidate gRNAs with
predictions of low off-target potential. In some cases, potential
off-target sites can be identified with at least one of: GUIDE-Seq
and targeted PCR amplification, and next generation sequencing. In
addition, modified cells, such as Cas9/gRNA-treated cells can be
subjected to karyotyping to identify any chromosomal
re-arrangements or translocations.
[0193] A gRNA can be introduced at any functional concentration.
For example, a gRNA can be introduced to a cell at 10 micrograms.
In other cases, a gRNA can be introduced from 0.5 micrograms to 100
micrograms. A gRNA can be introduced from 0.5, 5, 10, 15, 20, 25,
30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100
micrograms.
[0194] Disclosed herein is a method of making an engineered TIL
cell comprising: introducing at least one guide RNA (gRNA)
comprising at least one modification; and introducing at least one
endonuclease; wherein the gRNA comprises at least one sequence
complementary to at least one endogenous genome. In some cases, a
modification is on a 5' end, a 3' end, from a 5' end to a 3' end, a
single base modification, a 2'-ribose modification, or any
combination thereof. A modification can be selected from a group
consisting of base substitutions, insertions, deletions, chemical
modifications, physical modifications, stabilization, purification,
and any combination thereof
[0195] In some cases, a modification is a chemical modification. A
modification can be selected from 5'adenylate, 5'
guanosine-triphosphate cap, 5'N7-Methylguanosine-triphosphate cap,
5'triphosphate cap, 3'phosphate, 3'thiophosphate, 5'phosphate,
5'thiophosphate, Cis-Syn thymidine dimer, trimers, C12 spacer, C3
spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer
9,3'-3' modifications, 5'-5' modifications, abasic, acridine,
azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG,
desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, dual biotin, PC
biotin, psoralen C2, psoralen C6, TINA, 3'DABCYL, black hole
quencher 1, black hole quencher 2, DABCYL SE, dT-DABCYL, IRDye
QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linkers,
2'deoxyribonucleoside analog purine, 2'deoxyribonucleoside analog
pyrimidine, ribonucleoside analog, 2'-0-methyl ribonucleoside
analog, sugar modified analogs, wobble/universal bases, fluorescent
dye label, 2'fluoro RNA, 2'O-methyl RNA, methylphosphonate,
phosphodiester DNA, phosphodiester RNA, phosphothioate DNA,
phosphorothioate RNA, UNA, pseudouridine-5'-triphosphate,
5-methylcytidine-5'-triphosphate, 2-O-methyl 3phosphorothioate or
any combinations thereof.
[0196] The polynucleic acids as described herein can be modified. A
modification can be made at any location of a polynucleic acid.
More than one modification can be made to a single polynucleic
acid. A polynucleic acid can undergo quality control after a
modification. In some cases, quality control may include PAGE,
HPLC, MS, or any combination thereof. A modification can be a
substitution, insertion, deletion, chemical modification, physical
modification, stabilization, purification, or any combination
thereof. A polynucleic acid can also be modified by 5'adenylate, 5'
guanosine-triphosphate cap, 5'N.sup.7-Methylguanosine-triphosphate
cap, 5'triphosphate cap, 3'phosphate, 3'thiophosphate, 5'phosphate,
5'thiophosphate, Cis-Syn thymidine dimer, trimers, C12 spacer, C3
spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer
9,3'-3' modifications, 5'-5' modifications, abasic, acridine,
azobenzene, biotin, biotin BB, biotin TEG, cholesteryl TEG,
desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-Biotin, dual biotin, PC
biotin, psoralen C2, psoralen C6, TINA, 3'DABCYL, black hole
quencher 1, black hole quencher 2, DABCYL SE, dT-DABCYL, IRDye
QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linkers,
2'deoxyribonucleoside analog purine, 2'deoxyribonucleoside analog
pyrimidine, ribonucleoside analog, 2'-O-methyl ribonucleoside
analog, sugar modified analogs, wobble/universal bases, fluorescent
dye label, 2'fluoro RNA, 2'O-methyl RNA, methylphosphonate,
phosphodiester DNA, phosphodiester RNA, phosphothioate DNA,
phosphorothioate RNA, UNA, pseudouridine-5'-triphosphate,
5-methylcytidine-5'-triphosphate, or any combination thereof. A
representative 2'O-methyl RNA modified gRNA is shown in FIG. 22. In
some cases, a modification can be permanent. In other cases, a
modification can be transient. In some cases, multiple
modifications are made to a polynucleic acid. A polynucleic acid
modification may alter physio-chemical properties of a nucleotide,
such as their conformation, polarity, hydrophobicity, chemical
reactivity, base-pairing interactions, or any combination
thereof
[0197] A modification can also be a phosphorothioate substitute. In
some cases, a natural phosphodiester bond may be susceptible to
rapid degradation by cellular nucleases and; a modification of
internucleotide linkage using phosphorothioate (PS) bond
substitutes can be more stable towards hydrolysis by cellular
degradation. A modification can increase stability in a polynucleic
acid. A modification can also enhance biological activity. In some
cases, a phosphorothioate enhanced RNA polynucleic acid can inhibit
RNase A, RNase T1, calf serum nucleases, or any combinations
thereof. These properties can allow the use of PS-RNA polynucleic
acids to be used in applications where exposure to nucleases is of
high probability in vivo or in vitro. For example, phosphorothioate
(PS) bonds can be introduced between the last 3-5 nucleotides at
the 5'- or 3'-end of a polynucleic acid which can inhibit
exonuclease degradation. In some cases, phosphorothioate bonds can
be added throughout an entire polynucleic acid to reduce attack by
endonucleases.
[0198] In some cases, a modification can be screened. Screening can
include, but is not limited to, testing for immunogenicity, testing
for toxicity, testing for efficiency of transcription, testing for
efficiency of translation, or any combination thereof. In some
cases, a modification may not be immunogenic. A modification may
not be toxic. In some cases, candidate modifications are screened
prior to being incorporated into a polynucleic acid. In other
cases, polynucleic acids with different modifications are screened
to determine the level of immunogenicity, toxicity, efficacy, or
any combination of the added modifications. In some cases, a
modification is screened for its ability to support reverse
transcription of a polynucleic acid. In some cases, a modification
is a pseudouridine-5'-triphosphate (see e.g., FIG. 32). In other
cases a modification is a 5-methylcytidine-5'-triphosphate (see
e.g., FIG. 32). A modification can also include a change in
chirality.
[0199] Guiding polynucleic acids can be assembled by a variety of
methods, e.g., by automated solid-phase synthesis. A polynucleic
acid can be constructed using standard solid-phase DNA/RNA
synthesis. A polynucleic acid can also be constructed using a
synthetic procedure. A polynucleic acid can also be synthesized
either manually or in a fully automated fashion. In some cases, a
synthetic procedure may comprise 5'-hydroxyl oligonucleotides can
be initially transformed into corresponding 5'-H-phosphonate mono
esters, subsequently oxidized in the presence of imidazole to
activated 5'-phosphorimidazolidates, and finally reacted with
pyrophosphate on a solid support. This procedure may include a
purification step after the synthesis such as PAGE, HPLC, MS, or
any combination thereof
[0200] In some cases, a modification is a 2-O-methyl 3
phosphorothioate addition denoted as "m". A phosphothioate backbone
can be denoted as "(ps)." A 2-O-methyl 3 phosphorothioate addition
can be performed from 1 base to 150 bases. A 2-O-methyl 3
phosphorothioate addition can be performed from 1 base to 4 bases.
A 2-O-methyl 3 phosphorothioate addition can be performed on 2
bases. A 2-O-methyl 3 phosphorothioate addition can be performed on
4 bases. A modification can also be a truncation. A truncation can
be a 5 base truncation. In some cases, a modification may be at C
terminus and N terminus nucleotides, for example: 5'
[mG](ps)[mG](ps)[mG](ps) [mU](ps)UC CAU UAC GGC CAG CGG UUU UAG AGC
UAG AAA UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG
GCA CCG AGU CGG UG[mC](ps)[mU](ps)[mU](ps)[mU](ps) U 3'.
[0201] A guiding polynucleic acid can have any frequency of bases.
For example a guiding polynucleic acid can have 29 As, 17 Cs, 23
Gs, 23 Us, 3 mGs, 1 mCs, and 4 mUs. A guiding polynucleic acid can
have any ratio of nucleotide bases. For example, a guiding
polynucleic acid can have a percent adenine of 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,
20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,
33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 1-5%, 3-8%, 5-12%, 10-15%,
8-20%, 15-25%, 20-30%, 25-35%, or up to about 30-40%. A guiding
polynucleic acid can have a percent cytosine of 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,
20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,
33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 1-5%, 3-8%, 5-12%, 10-15%,
8-20%, 15-25%, 20-30%, 25-35%, or up to about 30-40%. A guiding
polynucleic acid can have a percent thymine of 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,
20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,
33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 1-5%, 3-8%, 5-12%, 10-15%,
8-20%, 15-25%, 20-30%, 25-35%, or up to about 30-40%. A guiding
polynucleic acid can have a percent guanine of 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,
20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,
33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 1-5%, 3-8%, 5-12%, 10-15%,
8-20%, 15-25%, 20-30%, 25-35%, or up to about 30-40%. A guiding
polynucleic acid can have a percent uracil of 1%, 2%, 3%, 4%, 5%,
6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%,
20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%,
33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 1-5%, 3-8%, 5-12%, 10-15%,
8-20%, 15-25%, 20-30%, 25-35%, or up to about 30-40%. A guiding
polynucleic acid can have from about 1 to about 100 nucleotides. A
guiding polynucleic acid can have from about 1 to 30 of a single
polynucleotide. A guiding polynucleic acid can have from about 1 to
10, 10 to 20, or from 20 to 30 of a single nucleotide.
[0202] A guiding polynucleic acid (also referred to as a guide
polynucleic acid) can be tested for identity and potency prior to
use. For example, identity and potency can be determined using at
least one of spectrophotometric analysis, RNA agarose gel analysis,
LC-MS, endotoxin analysis, and sterility testing. In some cases,
identity testing can determine an acceptable level for
clinical/therapeutic use. For example, an acceptable
spectrophotometric analysis result can be 14.+-.2 .mu.L/vial at
5.0.+-.0.5 mg/mL. an acceptable spectrophotometric analysis result
can also be from about 10-20.+-.2 .mu.L/vial at 5.0.+-.0.5 mg/mL or
from about 10-20.+-.2 .mu.L/vial at about 3.0 to 7.0.+-.0.5 mg/mL.
An acceptable clinical/therapeutic size of a guiding polynucleic
acid can be about 100 bases. A clinical/therapeutic size of a
guiding polynucleic acid can be from about 5 bases to about 150
bases. A clinical/therapeutic size of a guiding polynucleic acid
can be from about 20 bases to about 150 bases. A
clinical/therapeutic size of a guiding polynucleic acid can be from
about 40 bases to about 150 bases. A clinical/therapeutic size of a
guiding polynucleic acid can be from about 60 bases to about 150
bases. A clinical/therapeutic size of a guiding polynucleic acid
can be from about 80 bases to about 150 bases. A
clinical/therapeutic size of a guiding polynucleic acid can be from
about 100 bases to about 150 bases. A clinical/therapeutic size of
a guiding polynucleic acid can be from about 110 bases to about 150
bases. A clinical/therapeutic size of a guiding polynucleic acid
can be from about 120 bases to about 150 bases.
[0203] In some cases, a mass of a guiding polynucleic acid can be
determined. A mass can be determined by LC-MS assay. A mass can be
about 32,461.0 amu. A guiding polynucleic acid can have a mass from
about 30,000 amu to about 50,000 amu. A guiding polynucleic acid
can have a mass from about 30,000 amu to 40,000 amu, from about
40,000 amu to about 50,000 amu. A mass can be of a sodium salt of a
guiding polynucleic acid.
[0204] In some cases, an endotoxin level of a guiding polynucleic
acid can be determined. A clinically/therapeutically acceptable
level of an endotoxin can be less than 3 EU/mL. A
clinically/therapeutically acceptable level of an endotoxin can be
less than 10 EU/mL. A clinically/therapeutically acceptable level
of an endotoxin can be less than 8 EU/mL. A
clinically/therapeutically acceptable level of an endotoxin can be
less than 5 EU/mL. A clinically/therapeutically acceptable level of
an endotoxin can be less than 4 EU/mL. A clinically/therapeutically
acceptable level of an endotoxin can be less than 3 EU/mL. A
clinically/therapeutically acceptable level of an endotoxin can be
less than 2 EU/mL. A clinically/therapeutically acceptable level of
an endotoxin can be less than 1 EU/mL. A clinically/therapeutically
acceptable level of an endotoxin can be less than 0.5 EU/mL.
[0205] In some cases a guiding polynucleic acid can go sterility
testing. A clinically/therapeutically acceptable level of a
sterility testing can be 0 or denoted by no growth on a culture. A
clinically/therapeutically acceptable level of a sterility testing
can be less than 0.5% growth. A clinically/therapeutically
acceptable level of a sterility testing can be less than 1%
growth.
Treatment Regimes
[0206] Disclosed herein can be cells used in a treatment regime.
For example, a subject can receive engineered cells as part of a
treatment regime for treatment of a cancer or disease. Treatment
regimes can include: surgery, chemotherapy, radiation,
immunosuppressive agents, immunostimulatory agents, antifungals,
antivirals, antibiotics, or antiemetics, to name a few. In some
cases, cellular compositions can be administered to a subject in
conjunction with (e.g., before, simultaneously or following) bone
marrow transplantation, T cell ablative therapy using either
chemotherapy agents such as, fludarabine, external-beam radiation
therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or
CAMPATH. In some cases, expanded cells can be administered before
or following surgery. A surgery can be a tumor resection in some
cases. A surgery can be performed to isolate a TIL.
[0207] With the instant application in hand, the skilled worker can
determine a therapeutically effective amount of cells for
administration. In some cases, about 5.times.10.sup.10 cells are
administered to a subject. In some cases, about 5.times.10.sup.10
cells represent the median amount of cells administered to a
subject. In some embodiments, about 5.times.10.sup.10 cells are
necessary to effect a therapeutic response in a subject. In some
embodiments, at least about at least about 1.times.10.sup.6 cells,
at least about 2.times.10.sup.6 cells, at least about
3.times.10.sup.6 cells, at least about 4.times.10.sup.6 cells, at
least about 5.times.10.sup.6 cells, at least about 6.times.10.sup.6
cells, at least about 6.times.10.sup.6 cells, at least about
8.times.10.sup.6 cells, at least about 9.times.10.sup.6 cells,
1.times.10.sup.7 cells, at least about 2.times.10.sup.7 cells, at
least about 3.times.10.sup.7 cells, at least about 4.times.10.sup.7
cells, at least about 5.times.10.sup.7 cells, at least about
6.times.10.sup.7 cells, at least about 6.times.10.sup.7 cells, at
least about 8.times.10.sup.7 cells, at least about 9.times.10.sup.7
cells, at least about 1.times.10.sup.8 cells, at least about
2.times.10.sup.8 cells, at least about 3.times.10.sup.8 cells, at
least about 4.times.10.sup.8 cells, at least about 5.times.10.sup.8
cells, at least about 6.times.10.sup.8 cells, at least about
6.times.10.sup.8 cells, at least about 8.times.10.sup.8 cells, at
least about 9.times.10.sup.8 cells, at least about 1.times.10.sup.9
cells, at least about 2.times.10.sup.9 cells, at least about
3.times.10.sup.9 cells, at least about 4.times.10.sup.9 cells, at
least about 5.times.10.sup.9 cells, at least about 6.times.10.sup.9
cells, at least about 6.times.10.sup.9 cells, at least about
8.times.10.sup.9 cells, at least about 9.times.10.sup.9 cells, at
least about 1.times.10.sup.10 cells, at least about
2.times.10.sup.10 cells, at least about 3.times.10.sup.10 cells, at
least about 4.times.10.sup.10 cells, at least about
5.times.10.sup.10 cells, at least about 6.times.10.sup.10 cells, at
least about 6.times.10.sup.10 cells, at least about
8.times.10.sup.10 cells, at least about 9.times.10.sup.10 cells, at
least about 1.times.10.sup.11 cells, at least about
2.times.10.sup.11 cells, at least about 3.times.10.sup.11 cells, at
least about 4.times.10.sup.11 cells, at least about
5.times.10.sup.11 cells, at least about 6.times.10.sup.11 cells, at
least about 6.times.10.sup.11 cells, at least about
8.times.10.sup.11 cells, at least about 9.times.10.sup.11 cells, or
at least about 1.times.10.sup.12 cells are administered to a
subject. For example, about 5.times.10.sup.10 cells may be
administered to a subject. In another example, starting with
3.times.10.sup.6 cells, the cells may be expanded to about
5.times.10.sup.10 cells and administered to a subject. In some
cases, cells are expanded to sufficient numbers for therapy. For
example, 5.times.10.sup.7 cells can undergo rapid expansion to
generate sufficient numbers for therapeutic use. In some cases,
sufficient numbers for therapeutic use can be 5.times.10.sup.10.
Any number of cells can be infused for therapeutic use. For
example, a patient may be infused with a number of cells between
1.times.10.sup.6 to 5.times.10.sup.12, inclusive. A patient may be
infused with as many cells that can be generated for them. In some
cases, cells that are infused into a patient are not all
engineered. For example, at least 90% of cells that are infused
into a patient can be engineered. In other instances, at least 40%
of cells that are infused into a patient can be engineered. The
amount of cells that are necessary to be therapeutically effective
in a patient may vary depending on the viability of the cells, and
the efficiency with which the cells have been genetically modified.
In some cases, the product (e.g., multiplication) of the viability
of cells post genetic modification may correspond to the
therapeutic aliquot of cells available for administration to a
subject. In some cases, an increase in the viability of cells post
genetic modification may correspond to a decrease in the amount of
cells that are necessary for administration to be therapeutically
effective in a patient.
[0208] In some cases, a method can comprise calculating and/or
administering to a subject an amount of engineered cells necessary
to effect a therapeutic response in the subject. In some
embodiments, calculating the amount of engineered cells necessary
to effect a therapeutic response comprises determining the
viability of the engineered cells. In some embodiments, in order to
effect a therapeutic response in a subject, the cells administered
to the subject are viable cells. In some embodiments, in order to
effect a therapeutic response in a subject, at least about 95%, at
least about 90%, at least about 85%, at least about 80%, at least
about 75%, at least about 70%, at least about 65%, at least about
60%, at least about 55%, at least about 50%, at least about 45%, at
least about 40%, at least about 35%, at least about 30%, at least
about 25%, at least about 20%, at least about 15%, at least about
10% of the cells are viable cells. In some embodiments, in order to
effect a therapeutic response in a subject, at least about 95%, at
least about 90%, at least about 85%, at least about 80%, at least
about 75%, at least about 70%, at least about 65%, at least about
60%, at least about 55%, at least about 50%, at least about 45%, at
least about 40%, at least about 35%, at least about 30%, at least
about 25%, at least about 20%, at least about 15%, at least about
10% of the cells have had one or more endogenous genes or portion
thereof disrupted in a genome of a cell.
[0209] In some cases, adoptively transplanted cells can be
monitored by quantitative PCR (qPCR). A qPCR assay of adoptively
transplanted cells can indicate a level of modified cells that
exist in a subject after an introduction. In some cases, adoptively
transferred cells can be monitored using flow cytometry. For
example a flow cytometry assay may determine a level of 4-1BB vs
TCR. In some cases, a single-cell TCR PCR can be performed. Levels
of adoptively transferred cells can be identified on day 40 post
infusion. Levels of adoptively transferred cells, such as modified
cells, can be identified of day 5, 10, 15, 20, 25, 30, 35, 40, 45,
50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120,
125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175,180, 185,
190, 195, or up to day 200 post infusion.
[0210] I. Immunostimulants
[0211] In some cases, an immunostimulant can be introduced to cells
or a subject. An immunostimulant can be specific or non-specific. A
specific immunostimulant can provide antigenic specificity such as
a vaccine or an antigen. A non-specific immunostimulant can augment
an immune response or stimulate an immune response. A non-specific
immunostimulant can be an adjuvant Immunostimulants can be
vaccines, colony stimulating agents, interferons, interleukins,
viruses, antigens, co-stimulatory agents, immunogenicity agents,
immunomodulators, or immunotherapeutic agents. An immunostimulant
can be a cytokine such as an interleukin. One or more cytokines can
be introduced with cells of the invention. Cytokines can be
utilized to boost cytotoxic T lymphocytes (including adoptively
transferred tumor-specific cytotoxic T lymphocytes) to expand
within a tumor microenvironment. In some cases, IL-2 can be used to
facilitate expansion of the cells described herein. Cytokines such
as IL-15 can also be employed. Other relevant cytokines in the
field of immunotherapy can also be utilized, such as IL-2, IL-7,
IL-12, IL-15, IL-21, or any combination thereof In some cases,
IL-2, IL-7, and IL-15 are used to culture cells of the invention.
An interleukin can be IL-2, or aldeskeukin. Aldesleukin can be
administered in low dose or high dose. A high dose aldesleukin
regimen can involve administering aldesleukin intravenously every 8
hours, as tolerated, for up to about 14 doses at about 0.037 mg/kg
(600,000 IU/kg). An immunostimulant (e.g., aldesleukin) can be
administered within 24 hours after a cellular administration. An
immunostimulant (e.g., aldesleukin) can be administered in as an
infusion over about 15 minutes about every 8 hours for up to about
4 days after a cellular infusion. An immunostimulant (e.g.,
aldesleukin) can be administered at a dose from about 100,000
IU/kg, 200,000 IU/kg, 300,000 IU/kg, 400,000 IU/kg, 500,000 IU/kg,
600,000 IU/kg, 700,000 IU/kg, 800,000 IU/kg, 900,000 IU/kg, or up
to about 1,000,000 IU/kg. In some cases, aldesleukin can be
administered at a dose from about 100,000 IU/kg to 300,000 IU/kg,
from 300,000 IU/kg to 500,000 IU/kg, from 500,000 IU/kg to 700,000
IU/kg, from 700,000 IU/kg to about 1,000,000 IU/kg. An
immunostimulant (e.g., aldesleukin) can be administered from 1 dose
to about 14 doses. An immunostimulant (e.g., aldesleukin) can be
administered from at least about 1 dose, 2 doses, 3 doses, 4 doses,
5 doses, 6 doses, 7 doses, 8 doses, 9 doses, 10 doses, 11 doses, 12
doses, 13 doses, 14 doses, 15 doses, 16 doses, 17 doses, 18 doses,
19 doses, or up to about 20 doses. In some cases, an
immunostimulant such as aldesleukin can be administered from about
1 dose to 3 doses, from 3 doses to 5 doses, from 5 doses, to 8
doses, from 8 doses to 10 doses, from 10 doses to 14 doses, from 14
doses to 20 doses. In some cases, aldeskeukin is administered over
20 doses. In some cases, an immunostimulant, such as aldesleukin,
can be administered in sequence or concurrent with a cellular
administration. For example, an immunostimulant can be administered
from about day: -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4,
-3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or up to
about day 14. In some cases, an immunostimulant, such as
aldesleukin, is administered from day 0 to day 4 after
administration of a population of cells. In some cases, an
immunostimulant (e.g., aldesleukin) is administered over a period
of about 10 min, 15 min, 20 min, 30 min, 40 min, 50 min, 1 hour, 2
hours or up to about 3 hours. In some cases, an immunostimulant
(e.g., aldesleukin) can be administered from about 24 hours prior
to an administration of engineered cell to about 4 days after an
administration of engineered cells. An immunostimulant (e.g.,
aldesleukin) can be administered from day -7, -6, -5, -4, -3, -2,
-1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, or up to about 20 days after an administration of
engineered cells.
[0212] Immunostimulants such as aldesleukin can be provided as
single-use vials containing 22 million IU (-1.3 mg) IL-2 as a
sterile, white to off-white lyophilized cake plus 50 mg mannitol
and 0.18 mg sodium dodecyl sulfate, buffered with approximately
0.17 mg monobasic and 0.89 mg dibasic sodium phosphate to a pH of
7.5 (range 7.2 to 7.8). The vial can be reconstituted with 1.2 mL
of Sterile Water for Injection, USP, and the resultant
concentration is 18 million IU/ml or 1.1 mg/mL. Diluent should be
directed against the side of the vial to avoid excess foaming Since
vials contain no preservative, reconstituted solution should be
used with 24 hours. Reconstituted aldesleukin can be further
diluted with 50 mL of 5% Human Serum Albumin (HSA). The HSA should
be added to the diluent prior to the addition of RIL-2. Dilutions
of the reconstituted solution over a 1000-fold range (i.e., 1 mg/mL
to 1 mcg/mL) are acceptable in either glass bottles or polyvinyl
chloride bags. Aldesleukin is chemically stable for 48 hours at
refrigerated and room temperatures, 2.degree.-30.degree. C.
Administration of aldesleukin can be calculated based on total body
weight. The final dilution of aldesleukin can be infused over 15
minutes.
[0213] In some cases, an immunostimulant is a colony stimulating
factor. A colony stimulating factor can be G-CSF (filgrastim).
Filgrastim can be stored in 300 mcg/ml and 480 ug/1.6 ml vials.
Filgrastim can be administered daily as a subcutaneous injection. A
filgrastim administration can be from about 5 mcg/kg/day. A
filgrastim administration can be from about 1 mcg/kg/day, a
filgrastim administration can be from about 2 mcg/kg/day, a
filgrastim administration can be from about 3 mcg/kg/day, a
filgrastim administration can be from about 4 mcg/kg/day, a
filgrastim administration can be from about 5 mcg/kg/day, a
filgrastim administration can be from about 6 mcg/kg/day, a
filgrastim administration can be from about 7 mcg/kg/day, a
filgrastim administration can be from about 8 mcg/kg/day, a
filgrastim administration can be from about 9 mcg/kg/day, a
filgrastim administration can be from about 10 mcg/kg/day. In some
cases, Filgrastim can be administered at a dose ranging from about
0.5 mcg/kg/day to about 1.0 mcg/kg/day, from about 1.0 mcg/kg/day
to 1.5 mcg/kg/day, from about 1.5 mcg/kg/day to about 2.0
mcg/kg/day, from about 2.0 mcg/kg/day to about 3.0 mcg/kg/day, from
about 2.5 mcg/kg/day to about 3.5 mcg/kg/day, from about 3.5
mcg/kg/day to about 4.0 mcg/kg/day, from about 4.0 mcg/kg/day to
about 4.5 mcg/kg/day. Filgrastim administration can continue daily
until neutrophil count is at least about
1.0.times.10.sup.9/L.times.3 days or at least about
5.0.times.10.sup.9/L. An immunostimulant such as Filgrastim can be
administered from day -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5,
6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or up to about
20 days after an administration of engineered cells.
[0214] II. Chemotherapeutic Agents
[0215] A chemotherapeutic agent or compound can be a chemical
compound useful in the treatment of cancer. The chemotherapeutic
cancer agents that can be used in combination with the disclosed T
cell include, but are not limited to, mitotic inhibitors (vinca
alkaloids). These include vincristine, vinblastine, vindesine and
NavelbineTM (vinorelbine, 5'-noranhydroblastine). In yet other
cases, chemotherapeutic cancer agents include topoisomerase I
inhibitors, such as camptothecin compounds. As used herein,
"camptothecin compounds" include Camptosar.TM. (irinotecan HCL),
Hycamtin.TM. (topotecan HCL) and other compounds derived from
camptothecin and its analogues. Another category of
chemotherapeutic cancer agents that can be used in the methods and
compositions disclosed herein are podophyllotoxin derivatives, such
as etoposide, teniposide and mitopodozide. The present disclosure
further encompasses other chemotherapeutic cancer agents known as
alkylating agents, which alkylate the genetic material in tumor
cells. These include without limitation cisplatin,
cyclophosphamide, nitrogen mustard, trimethylene thiophosphoramide,
carmustine, busulfan, chlorambucil, belustine, uracil mustard,
chlomaphazin, and dacarbazine. The disclosure encompasses
antimetabolites as chemotherapeutic agents. Examples of these types
of agents include cytosine arabinoside, fluorouracil, methotrexate,
mercaptopurine, azathioprime, and procarbazine. An additional
category of chemotherapeutic cancer agents that may be used in the
methods and compositions disclosed herein include antibiotics.
Examples include without limitation doxorubicin, bleomycin,
dactinomycin, daunorubicin, mithramycin, mitomycin, mytomycin C,
and daunomycin. There are numerous liposomal formulations
commercially available for these compounds. The present disclosure
further encompasses other chemotherapeutic cancer agents including
without limitation anti-tumor antibodies, dacarbazine, azacytidine,
amsacrine, melphalan, ifosfamide and mitoxantrone.
[0216] The disclosed T cell herein can be administered in
combination with other anti-tumor agents, including
cytotoxic/antineoplastic agents and anti-angiogenic agents.
Cytotoxic/anti-neoplastic agents can be defined as agents who
attack and kill cancer cells. Some cytotoxic/anti-neoplastic agents
can be alkylating agents, which alkylate the genetic material in
tumor cells, e.g., cis-platin, cyclophosphamide, nitrogen mustard,
trimethylene thiophosphoramide, carmustine, busulfan, chlorambucil,
belustine, uracil mustard, chlomaphazin, and dacabazine. Other
cytotoxic/anti-neoplastic agents can be antimetabolites for tumor
cells, e.g., cytosine arabinoside, fluorouracil, methotrexate,
mercaptopuirine, azathioprime, and procarbazine. Other
cytotoxic/anti-neoplastic agents can be antibiotics, e.g.,
doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin,
mitomycin, mytomycin C, and daunomycin. There are numerous
liposomal formulations commercially available for these compounds.
Still other cytotoxic/anti-neoplastic agents can be mitotic
inhibitors (vinca alkaloids). These include vincristine,
vinblastine and etoposide. Miscellaneous cytotoxic/anti-neoplastic
agents include taxol and its derivatives, L-asparaginase,
anti-tumor antibodies, dacarbazine, azacytidine, amsacrine,
melphalan, VM-26, ifosfamide, mitoxantrone, and vindesine.
[0217] Anti-angiogenic agents can also be used. Suitable
anti-angiogenic agents for use in the disclosed methods and
compositions include anti-VEGF antibodies, including humanized and
chimeric antibodies, anti-VEGF aptamers and antisense
oligonucleotides. Other inhibitors of angiogenesis include
angiostatin, endostatin, interferons, interleukin 1 (including
.alpha. and .beta.) interleukin 12, retinoic acid, and tissue
inhibitors of metalloproteinase-1 and -2. (TIMP-1 and -2). Small
molecules, including topoisomerases such as razoxane, a
topoisomerase II inhibitor with anti-angiogenic activity, can also
be used.
[0218] Other anti-cancer agents that can be used in combination
with the disclosed engineered cells can include, but are not
limited to: acivicin; aclarubicin; acodazole hydrochloride;
acronine; adozelesin; aldesleukin; altretamine; ambomycin;
ametantrone acetate; aminoglutethimide; amsacrine; anastrozole;
anthramycin; asparaginase; asperlin; avastin; azacitidine; azetepa;
azotomycin; batimastat; benzodepa; bicalutamide; bisantrene
hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate;
brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone;
caracemide; carbetimer; carboplatin; carmustine; carubicin
hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin;
cisplatin; cladribine; crisnatol mesylate; cyclophosphamide;
cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride;
decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate;
diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride;
droloxifene; droloxifene citrate; dromostanolone propionate;
duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin;
enloplatin; enpromate; epipropidine; epirubicin hydrochloride;
erbulozole; esorubicin hydrochloride; estramustine; estramustine
phosphate sodium; etanidazole; etoposide; etoposide phosphate;
etoprine; fadrozole hydrochloride; fazarabine; fenretinide;
floxuridine; fludarabine phosphate; fluorouracil; flurocitabine;
fosquidone; fostriecin sodium; gemcitabine; gemcitabine
hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide;
ilmofosine; interleukin II (including recombinant interleukin II,
or rIL2), interferon alfa-2a; interferon alfa-2b; interferon
alfa-n1; interferon alfa-n3; interferon beta-I a; interferon
gamma-I b; iproplatin; irinotecan hydrochloride; lanreotide
acetate; letrozole; leuprolide acetate; liarozole hydrochloride;
lometrexol sodium; lomustine; losoxantrone hydrochloride;
masoprocol; maytansine; mechlorethamine hydrochloride; megestrol
acetate; melengestrol acetate; melphalan; menogaril;
mercaptopurine; methotrexate; methotrexate sodium; metoprine;
meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin;
mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone
hydrochloride; mycophenolic acid; nocodazole; nogalamycin;
ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin;
pentamustine; peplomycin sulfate; perfosfamide; pipobroman;
piposulfan; piroxantrone hydrochloride; plicamycin; plomestane;
porfimer sodium; porfiromycin; prednimustine; procarbazine
hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin;
riboprine; rogletimide; safingol; safingol hydrochloride;
semustine; simtrazene; sparfosate sodium; sparsomycin;
spirogermanium hydrochloride; spiromustine; spiroplatin;
streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan
sodium; tegafur; teloxantrone hydrochloride; temoporfin;
teniposide; teroxirone; testolactone; thiamiprine; thioguanine;
thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone
acetate; triciribine phosphate; trimetrexate; trimetrexate
glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard;
uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine
sulfate; vindesine; vindesine sulfate; vinepidine sulfate;
vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate;
vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin;
zinostatin; zorubicin hydrochloride. Other anti-cancer drugs
include, but are not limited to: 20-epi-1,25 dihydroxyvitamin D3;
5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol;
adozelesin; aldesleukin; ALL-TK antagonists; altretamine;
ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin;
amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis
inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing
morphogenetic protein-1; antiandrogen, prostatic carcinoma;
antiestrogen; antineoplaston; antisense oligonucleotides;
aphidicolin glycinate; apoptosis gene modulators; apoptosis
regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase;
asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2;
axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III
derivatives; balanol; batimastat; BCR/ABL antagonists;
benzochlorins; benzoylstaurosporine; beta lactam derivatives;
beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor;
bicalutamide; bisantrene; bisaziridinylspermine; bisnafide;
bistratene A; bizelesin; breflate; bropirimine; budotitane;
buthionine sulfoximine; calcipotriol; calphostin C; camptothecin
derivatives; canarypox IL-2; capecitabine;
carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN
700; cartilage derived inhibitor; carzelesin; casein kinase
inhibitors (ICOS); castanospermine; cecropin B; cetrorelix;
chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin;
cladribine; clomifene analogues; clotrimazole; collismycin A;
collismycin B; combretastatin A4; combretastatin analogue;
conagenin; crambescidin 816; crisnatol; cryptophycin 8;
cryptophycin A derivatives; curacin A; cyclopentanthraquinones;
cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor;
cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin;
dexamethasone; dexifosfamide; dexrazoxane; dexverapamil;
diaziquone; didemnin B; didox; diethylnorspermine;
dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl
spiromustine; docetaxel; docosanol; dolasetron; doxifluridine;
droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine;
edelfosine; edrecolomab; eflornithine; elemene; emitefur;
epirubicin; epristeride; estramustine analogue; estrogen agonists;
estrogen antagonists; etanidazole; etoposide phosphate; exemestane;
fadrozole; fazarabine; fenretinide; filgrastim; finasteride;
flavopiridol; flezelastine; fluasterone; fludarabine;
fluorodaunorunicin hydrochloride; forfenimex; formestane;
fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate;
galocitabine; ganirelix; gelatinase inhibitors; gemcitabine;
glutathione inhibitors; hepsulfam; heregulin; hexamethylene
bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene;
idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod;
immunostimulant peptides; insulin-like growth factor-1 receptor
inhibitor; interferon agonists; interferons; interleukins;
iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine;
isobengazole; isohomohalicondrin B; itasetron; jasplakinolide;
kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin;
lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia
inhibiting factor; leukocyte alpha interferon;
leuprolide+estrogen+progesterone; leuprorelin; levamisole;
liarozole; linear polyamine analogue; lipophilic disaccharide
peptide; lipophilic platinum compounds; lissoclinamide 7;
lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone;
lovastatin; loxoribine; lurtotecan; lutetium texaphyrin;
lysofylline; lytic peptides; maitansine; mannostatin A; marimastat;
masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase
inhibitors; menogaril; merbarone; meterelin; methioninase;
metoclopramide; MIF inhibitor; mifepristone; miltefosine;
mirimostim; mismatched double stranded RNA; mitoguazone;
mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast
growth factor-saporin; mitoxantrone; mofarotene; molgramostim;
monoclonal antibody, human chorionic gonadotrophin; monophosphoryl
lipid A+myobacterium cell wall sk; mopidamol; multiple drug
resistance gene inhibitor; multiple tumor suppressor 1-based
therapy; mustard anticancer agent; mycaperoxide B; mycobacterial
cell wall extract; myriaporone; N-acetyldinaline; N-substituted
benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin;
naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid;
neutral endopeptidase; nilutamide; nisamycin; nitric oxide
modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine;
octreotide; okicenone; oligonucleotides; onapristone; ondansetron;
ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone;
oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues;
paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic
acid; panaxytriol; panomifene; parabactin; pazelliptine;
pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin;
pentrozole; perflubron; perfosfamide; perillyl alcohol;
phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil;
pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A;
placetin B; plasminogen activator inhibitor; platinum complex;
platinum compounds; platinum-triamine complex; porfimer sodium;
porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2;
proteasome inhibitors; protein A-based immune modulator; protein
kinase C inhibitor; protein kinase C inhibitors, microalgal;
protein tyrosine phosphatase inhibitors; purine nucleoside
phosphorylase inhibitors; purpurins; pyrazoloacridine;
pyridoxylated hemoglobin polyoxyethylene conjugate; raf
antagonists; raltitrexed; ramosetron; ras farnesyl protein
transferase inhibitors; ras inhibitors; ras-GAP inhibitor;
retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin;
ribozymes; RII retinamide; rogletimide; rohitukine; romurtide;
roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU;
sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence
derived inhibitor 1; sense oligonucleotides; signal transduction
inhibitors; signal transduction modulators; single chain antigen
binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium
phenylacetate; solverol; somatomedin binding protein; sonermin;
sparfosic acid; spicamycin D; spiromustine; splenopentin;
spongistatin 1; squalamine; stem cell inhibitor; stem-cell division
inhibitors; stipiamide; stromelysin inhibitors; sulfinosine;
superactive vasoactive intestinal peptide antagonist; suradista;
suramin; swainsonine; synthetic glycosaminoglycans; tallimustine;
tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium;
tegafur; tellurapyrylium; telomerase inhibitors; temoporfin;
temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine;
thaliblastine; thiocoraline; thrombopoietin; thrombopoietin
mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan;
thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine;
titanocene bichloride; topsentin; toremifene; totipotent stem cell
factor; translation inhibitors; tretinoin; triacetyluridine;
triciribine; trimetrexate; triptorelin; tropisetron; turosteride;
tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex;
urogenital sinus-derived growth inhibitory factor; urokinase
receptor antagonists; vapreotide; variolin B; erythrocyte gene
therapy; velaresol; veramine; verdins; verteporfin; vinorelbine;
vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb;
and zinostatin stimalamer. Any of the aforementioned
chemotherapeutics can be administered at a clinically effective
dose. A chemotherapeutic can also be administered from about day:
-14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or up to about day 14 after
administration of a population of cells. In some cases, a subject
can have a refractory cancer that is unresponsive to a
chemotherapeutic.
[0219] III. Antifungal Agents
[0220] In some cases, an antifungal therapy is administered to a
subject receiving engineered cells. Antifungals can be drugs that
can kill or prevent the growth of fungi. Targets of antifungal
agents can include sterol biosynthesis, DNA biosynthesis, and
.beta.-glucan biosynthesis. Antifungals can also be folate
synthesis inhibitors or nucleic acid cross-linking agents. A folate
synthesis inhibitor can be a sulpha based drug. For example, a
folate synthesis inhibitor can be an agent that inhibits a fungal
synthesis of folate or a competitive inhibitor. A sulpha based
drug, or folate synthesis inhibitor, can be methotrexate or
sulfamethaxazole. In some cases, an antifungal can be a nucleic
acid cross-linking agent. A cross-linking agent may inhibit a DNA
or RNA process in fungi. For example, a cross-linking agent can be
5-fluorocytosine, which can be a fluorinated analog of cytosine.
5-fluorocytosine can inhibit both DNA and RNA synthesis via
intracytoplasmic conversion to 5-fluorouracil. Other anti-fungal
agents can be griseofulvin. Griseofulvin is an antifungal
antibiotic produced by Penicillium griseofulvum. Griseofulvin
inhibits mitosis in fungi and can be considered a cross linking
agent. Additional cross linking agent can be allylamines (naftifine
and terbinafine) inhibit ergosterol synthesis at the level of
squalene epoxidase; one morpholene derivative (amorolfine) inhibits
at a subsequent step in the ergosterol pathway.
[0221] In some cases, an antifungal agent can be from a class of
polyene, azole, allylamine, or echinocandin. In some embodiments, a
polyene antifungal is amphotericin B, candicidin, filipin, hamycin,
natamycin, nystatin, or rimocidin. In some cases, an antifungal can
be from an azole family. Azole antifungals can inhibit lanosterol
14 .alpha.-demethylase. An azole antifungal can be an imidazole
such as bifonazole, butoconazole, clotrimazole, econazole,
fenticonazole, isoconazole, ketoconazole, luliconazole, miconazole,
omoconazole, oxiconazole, sertaconazole, sulcoazole, or
tioconazole. An azole antifungal can be a triazole such as
albaconazole, efinaconazole, epoxiconazole, fluconazole,
isavuvonazole, itraconazole, posaconazole, propiconazole,
ravuconazole, terconazole, or voriconazole. In some cases an azole
can be a thiazole such as abafungin. An antifungal can be an
allylamine such as amorolfin, butenafine, naftifine, or
terbinafine. An antifungal can also be an echinocandin such as
anidulafungin, caspofungin, or micafungin. Additional agents that
can be antifungals can be aurones, benzoic acid, ciclopirox,
flucytosine, griseofulvin, haloprogin, tolnaftate, undecylenic
acid, cystal violet or balsam of Peru.
[0222] A person of skill in the art can appropriately determine
which known antifungal medication to apply based on the fungus
infecting the individual. In some cases, a subject will receive
fluconazole in combination with engineered TILs comprising a
genomic knockout of at least a portion of a gene, such as CISH. An
anti-fungal therapy can be administered prophalaytically.
[0223] Fluconazole can be available in 200 mg tablets. In some
cases, fluconazole can be administered as a 50 mg, 100 mg, 150 mg,
200 mg, 250 mg, 300 mg, 350 mg, or up to about 400 mg tablet. For
IV administration in subjects who cannot tolerate an oral
preparation, fluconazole comes in 2 MG/ML solution for injection.
It should be administered at a maximum IV rate of 200 mg/hr. In
some cases, an infusion rate can be from about 50 mg/hr to about
500 mg/hr. An infusion rate can also be from about 20 mg/hr to
about 30 mg/hr, from about 30 mg/hr to about 40 mg/hr, from about
40 mg/hr to about 50 mg/hr, from about 50 mg/hr to about 60 mg/hr,
from about 60 mg/hr to about 70 mg/hr, from about 70 mg/hr to about
80 mg/hr, from about 80 mg/hr to about 90 mg/hr, from about 90
mg/hr to about 100 mg/hr, from about 100 mg/hr to about 120 mg/hr,
from about 120 mg/hr to about 140 mg/hr, from about 140 mg/hr to
about 160 mg/hr, from about 160 mg/hr to about 180 mg/hr, from
about 180 mg/hr to about 200 mg/hr, from about 180 mg/hr to about
220 mg/hr, from about 220 mg/hr to about 240 mg/hr, from about 240
mg/hr to about 275 mg/hr.
[0224] Antifungals can be administered at therapeutically effective
doses. A therapeutically effective dose is a dose that treats or
prevents a fungal infection but that is not effective for treating
a cancer. For example an antifungal such as fluconazole can be
administered from about 10 mg to about 1000 mg. Fluconazole can be
administered from about 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg,
70 mg, 80 mg, 90 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225
mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg,
450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650
mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg,
875 mg, 900 mg, 925 mg, 950 mg, 975 mg, or up to about 1000 mg.
Fluconazole can be administered at 400 mg. In some cases, an
antifungal administration can be before a cellular therapy, during
a cellular therapy or after a cellular therapy has been
administered. For example a fluconazole administration can be from
about day 0 (day a cellular therapy is introduced into a subject)
to about day 4 after administration of a cellular therapy. An
antifungal can be administered from about 14 days leading up to a
cellular therapy administration to about 14 days after a cellular
therapy is completed. An antifungal can be administered from about
day: -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1,
0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or up to about day
14.
[0225] IV. Immunosuppressive Agents
[0226] In some cases, a subject may receive an immunosuppressive
agent as part of a therapy regime. An immunosuppressive agent can
refer to a radiotherapeutic, a biologic, or a chemical agent. In
some cases, an immunosuppressive agent can include a chemical
agent. For example, a chemical agent can comprise at least one
member from the group consisting of: cyclophosphamide,
mechlorethamine, chlorambucil, melphalan, ifosfamide, thiotepa,
hexamethylmelamine, busulfan, fludarabine, nitrosoureas, platinum,
methotrexate, azathioprine, mercaptopurine, procarbazine,
dacarbazine, temozolomide, carmustine, lomustine, streptozocin,
fluorouracil, dactinomycin, anthracycline, mitomycin C, bleomycin,
and mithramycin. A chemical agent can be cyclophosphamide or
fludarabine.
[0227] Additionally, immunosuppressive agents can include
glucocorticoids, cytostatic, antibodies, anti-immunophilins, or any
derivatives thereof. A glucocorticoid can suppress an allergic
response, inflammation, and autoimmune conditions. Glucocorticoids
can be prednisone, dexamethasone, and hydrocortisone
Immunosuppressive therapy can comprise any treatment that
suppresses the immune system Immunosuppressive therapy can help to
alleviate, minimize, or eliminate transplant rejection in a
recipient. For example, immunosuppressive therapy can comprise
immuno-suppressive drugs. Immunosuppressive drugs that can be used
before, during and/or after transplant, but are not limited to, MMF
(mycophenolate mofetil (Cellcept)), ATG (anti-thymocyte globulin),
anti-CD154 (CD4OL), anti-CD40 (2C10, ASKP1240, CCFZ533X2201),
alemtuzumab (Campath), anti-CD20 (rituximab), anti-IL-6R antibody
(tocilizumab, Actemra), anti-IL-6 antibody (sarilumab, olokizumab),
CTLA4-Ig (Abatacept/Orencia), belatacept (LEA29Y), sirolimus
(Rapimune), everolimus, tacrolimus (Prograf), daclizumab
(Ze-napax), basiliximab (Simulect), infliximab (Remicade),
cyclosporin, deoxyspergualin, soluble complement receptor 1, cobra
venom factor, compstatin, anti C5 antibody (eculizumab/Soliris),
methylprednisolone, FTY720, everolimus, leflunomide, anti-IL-2R-Ab,
rapamycin, anti-CXCR3 antibody, anti-ICOS antibody, anti-OX40
antibody, and anti-CD122 antibody. Furthermore, one or more than
one immunosuppressive agents/drugs can be used together or
sequentially. One or more than one immunosuppressive agents/drugs
can be used for induction therapy or for maintenance therapy. The
same or different drugs can be used during induction and
maintenance stages. In some cases, daclizumab (Zenapax) can be used
for induction therapy and tacrolimus (Prograf) and sirolimus
(Rapimune) can be used for maintenance therapy. Daclizumab
(Zenapax) can also be used for induction therapy and low dose
tacrolimus (Prograf) and low dose sirolimus (Rapimune) can be used
for maintenance therapy. Immunosuppression can also be achieved
using non-drug regimens including, but not limited to, whole body
irradiation, thymic irradiation, and full and/or partial
splenectomy.
[0228] In some cases, a cytostatic agent can be administered for
immunosuppression. Cytostatic agents can inhibit cell division. A
cytostatic agent can be a purine analog. A cytostatic agent can be
an alkylating agent, an antimetabolite such as methotrexate,
azathioprine, or mercaptopurine. A cytostatic agent can be at least
one of cyclophosphamide, mechlorethamine, chlorambucil, melphalan,
ifosfamide, thiotepa, hexamethylmelamine, busulfan, fludarabine,
nitrosoureas, platinum, methotrexate, azathioprine, mercaptopurine,
procarbazine, dacarbazine, temozolomide, carmustine, lomustine,
streptozocin, fluorouracil, dactinomycin, anthracycline, mitomycin
C, bleomycin, and mithramycin.
[0229] In some cases, an immunosuppressive agent such as
fludarabine can be administered as part of a treatment regime.
Fludarabine phosphate can be a synthetic purine nucleoside that
differs from physiologic nucleosides in that the sugar moiety can
be arabinose instead of ribose or deoxyribose. Fludarabine can be a
purine antagonist antimetabolite. Fludarabine can be supplied in a
50 mg vial as a fludarabine phosphate powder in the form of a
white, lyophilized solid cake. Following reconstitution with 2 mL
of sterile water for injection to a concentration of 25 mg/ml, the
solution can have a pH of 7.7. The fludarabine powder can be stable
for at least 18 months at 2-8.degree. C.; when reconstituted,
fludarabine is stable for at least 16 days at room temperature.
Because no preservative is present, reconstituted fludarabine will
typically be administered within 8 hours. Specialized references
should be consulted for specific compatibility information.
Fludarabine can be dephosphorylated in serum, transported
intracellularly and converted to the nucleotide fludarabine
triphosphate; this 2-fluoro-ara-ATP molecule is thought to be
required for the drug's cytotoxic effects. Fludarabine inhibits DNA
polymerase, ribonucleotide reductase, DNA primase, and may
interfere with chain elongation, and RNA and protein synthesis.
Fludarabine can be administered as an IV infusion in 100 ml 0.9%
sodium chloride, USP over 15 to 30 minutes. The doses will be based
on body surface area (BSA). If patient is obese (BMI>35) drug
dosage will be calculated using practical weight. In some cases, an
immunosuppressive agent such as fludarabine can be administered
from about 20 mg/m.sup.2 to about 30 mg/m.sup.2 of body surface
area of a subject. In some cases, an immunosuppressive agent such
as fludarabine can be administered from about 5 mg/m.sup.2 to about
10 mg/m.sup.2 of body surface area of a subject, from about 10
mg/m.sup.2 to about 15 mg/m.sup.2 of body surface area of a
subject, from about 15 mg/m.sup.2 to about 20 mg/m.sup.2 of body
surface area of a subject, from about 20 mg/m.sup.2 to about 25
mg/m.sup.2 of body surface area of a subject, from about 25
mg/m.sup.2 to about 30 mg/m.sup.2 of body surface area of a
subject, from about 30 mg/m.sup.2 to about 40 mg/m.sup.2 of body
surface area of a subject. In some cases, an immunosuppressive
agent such as fludarabine can be administered from about 1
mg/m.sup.2, 2 mg/m.sup.2, 3 mg/m.sup.2, 4 mg/m.sup.2, 5 mg/m.sup.2,
6 mg/m.sup.2, 7 mg/m.sup.2, 8 mg/m.sup.2, 9 mg/m.sup.2, 10
mg/m.sup.2, 11 mg/m.sup.2, 12 mg/m.sup.2, 13 mg/m.sup.2, 14
mg/m.sup.2, 15 mg/m.sup.2, 16 mg/m.sup.2, 17 mg/m.sup.2, 18
mg/m.sup.2, 19 mg/m.sup.2, 20 mg/m.sup.2, 21 mg/m.sup.2, 22
mg/m.sup.2, 23 mg/m.sup.2, 24 mg/m.sup.2, 25 mg/m.sup.2, 26
mg/m.sup.2, 27 mg/m.sup.2, 28 mg/m.sup.2, 29 mg/m.sup.2, 30
mg/m.sup.2, 31 mg/m.sup.2, 32 mg/m.sup.2, 33 mg/m.sup.2, 34
mg/m.sup.2, 35 mg/m.sup.2, 36 mg/m.sup.2, 37 mg/m.sup.2, 38
mg/m.sup.2, 39 mg/m.sup.2, 40 mg/m.sup.2, 41 mg/m.sup.2, 42
mg/m.sup.2, 43 mg/m.sup.2, 44 mg/m.sup.2, 45 mg/m.sup.2, 46
mg/m.sup.2, 47 mg/m.sup.2, 48 mg/m.sup.2, 49 mg/m.sup.2, 50
mg/m.sup.2, 51 mg/m.sup.2, 52 mg/m.sup.2, 53 mg/m.sup.2, 54
mg/m.sup.2, 55 mg/m.sup.2, 56 mg/m.sup.2, 57 mg/m.sup.2, 58
mg/m.sup.2, 59 mg/m.sup.2, 60 mg/m.sup.2, 61 mg/m.sup.2, 62
mg/m.sup.2, 63 mg/m.sup.2, 64 mg/m.sup.2, 65 mg/m.sup.2, 66
mg/m.sup.2, 67 mg/m.sup.2, 68 mg/m.sup.2, 69 mg/m.sup.2, 70
mg/m.sup.2, 71 mg/m.sup.2, 72 mg/m.sup.2, 73 mg/m.sup.2, 74
mg/m.sup.2, 75 mg/m.sup.2, 76 mg/m.sup.2, 77 mg/m.sup.2, 78
mg/m.sup.2, 79 mg/m.sup.2, 80 mg/m.sup.2, 81 mg/m.sup.2, 82
mg/m.sup.2, 83 mg/m.sup.2, 84 mg/m.sup.2, 85 mg/m.sup.2, 86
mg/m.sup.2, 87 mg/m.sup.2, 88 mg/m.sup.2, 89 mg/m.sup.2, 90
mg/m.sup.2, 91 mg/m.sup.2, 92 mg/m.sup.2, 93 mg/m.sup.2, 94
mg/m.sup.2, 95 mg/m.sup.2, 96 mg/m.sup.2, 97 mg/m.sup.2, 98
mg/m.sup.2, 99 mg/m.sup.2, up to about 100 mg/m.sup.2 of body
surface area of a subject. In some cases, an immunosuppressive
agent such as fludarabine is at a dose of 25 mg/m.sup.2 in 100 ml
0.9% sodium chloride, USP and infused over about 15 to about 30
minutes.
[0230] In some cases, an immunosuppressive agent such as
cyclophosphamide can be administered as part of a treatment regime.
Cyclophosphamide can be a nitrogen mustard-derivative alkylating
agent. Following conversion to active metabolites in the liver,
cyclophosphamide functions as an alkyating agent; the drug also
possesses potent immunosuppressive activity. The serum half-life
after IV administration ranges from 3-12 hours; the drug and/or its
metabolites can be detected in the serum for up to 72 hours after
administration.Following reconstitution as directed with sterile
water for injection, cyclophosphamide can be stable for 24 hours at
room temperature or 6 days when kept at 2-8.degree. C.
Cyclophosphamide can be diluted in 250 ml D5W and infused over one
hour. The dose will be based on a subject's body weight. If a
subject is obese (BMI>35) drug dosage will be calculated using
practical weight as described in. In some cases, an
immunosuppressive agent such as cyclophosphamide can be
administered from about lmg/kg to about 3 mg/kg, from about 3 mg/kg
to about 5 mg/kg, from about 5mg/kg to about 10 mg/kg, from about
10 mg/kg to about 20 mg/kg, 20 mg/kg to about 30 mg/kg, from about
30 mg/kg to about 40 mg/kg, from about 40 mg/kg to about 50 mg/kg,
from about 50 mg/kg to about 60 mg/kg, from about 60 mg/kg to about
70 mg/kg, from about 70 mg/kg to about 80 mg/kg, from about 80
mg/kg to about 90 mg/kg, from about 90 mg/kg to about 100 mg/kg. In
some cases, an immunosuppressive agent such as cyclophosphamide is
administered in excess of 50 mg/kg of a subject. In some cases, an
immunosuppressive agent such as cyclophosphamide can be
administered from about 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5
mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12
mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg, 18 mg/kg,
19 mg/kg, 20 mg/kg, 21 mg/kg, 22 mg/kg, 23 mg/kg, 24 mg/kg, 25
mg/kg, 26 mg/kg, 27 mg/kg, 28 mg/kg, 29 mg/kg, 30 mg/kg, 31 mg/kg,
32 mg/kg, 33 mg/kg, 34 mg/kg, 35 mg/kg, 36 mg/kg, 37 mg/kg, 38
mg/kg, 39 mg/kg, 40 mg/kg, 41 mg/kg, 42 mg/kg, 43 mg/kg, 44 mg/kg,
45 mg/kg, 46 mg/kg, 47 mg/kg, 48 mg/kg, 49 mg/kg, 50 mg/kg, 51
mg/kg, 52 mg/kg, 53 mg/kg, 54 mg/kg, 55 mg/kg, 56 mg/kg, 57 mg/kg,
58 mg/kg, 59 mg/kg, 60 mg/kg, 61 mg/kg, 62 mg/kg, 63 mg/kg, 64
mg/kg, 65 mg/kg, 66 mg/kg, 67 mg/kg, 68 mg/kg, 69 mg/kg, 70 mg/kg,
71 mg/kg, 72 mg/kg, 73 mg/kg, 74 mg/kg, 75 mg/kg, 76 mg/kg, 77
mg/kg, 78 mg/kg, 79 mg/kg, 80 mg/kg, 81 mg/kg, 82 mg/kg, 83 mg/kg,
84 mg/kg, 85 mg/kg, 86 mg/kg, 87 mg/kg, 88 mg/kg, 89 mg/kg, 90
mg/kg, 91 mg/kg, 92 mg/kg, 93 mg/kg, 94 mg/kg, 95 mg/kg, 96 mg/kg,
97 mg/kg, 98 mg/kg, 99 mg/kg, up to about 100 mg/kg of a subject.
In some cases, an immunosuppressive agent such as cyclophosphamide
can be administered over at least about 1 day to about 3 days, from
3 days to 5 days, from 5 days to 7 days, from 7 days to about 10
days, from 10 days to 14 days, from 14 days to about 20 days. In
some cases, cyclophosphamide can be at a dose of about 60 mg/kg and
is diluted in 250 ml 5% dextrose in water and infused over one
hour.
[0231] An immunosuppressive agent can be, for example, a regime of
cyclophosphamide and fludarabine. For example, a cyclophosphamide
fludarabine regimen can be administered to a subject receiving an
engineered cellular therapy. A cyclophosphamide fludarabine regimen
can be administered at a regime of 60 mg/kg qd for 2 days and 25
mg/m.sup.2 qd for 5 days. A chemotherapeutic regime, for example,
cyclophosphamide fludarabine, can be administered from 1 hour to 14
days preceding administration of engineered cells of the present
invention. A chemotherapy regime can be administered at different
doses. For example, a subject may receive a higher initial dose
followed by a lower dose. A subject may receive a lower initial
dose followed by a higher dose.
[0232] In some cases, an immunosuppressive agent can be an
antibody. An antibody can be administered at a therapeutically
effective dose. An antibody can be a polyclonal antibody or a
monoclonal antibody. A polyclonal antibody that can be administered
can be an antilymphocyte or antithymocyte antigen. A monoclonal
antibody can be an anti-IL-2 receptor antibody, an anti-CD25
antibody, or an anti-CD3 antibody. An anti-CD20 antibody can also
be used. B-cell ablative therapy such as agents that react with
CD20, e.g., Rituxan can also be used as immunosuppressive
agents.
[0233] An immunosuppressive can also be an anti-immunophilin
Anti-immunophilins can be ciclosporin, tacrolimus, everolimus, or
sirolimus. Additional immunosuppressive agents can be interferons
such as IFN-beta, opiods, anti-TNF binding agents, mycophenolate,
or fingolimod.
[0234] Immunosuppressive agents can also refer to
radiotherapeutics. Radiotherapy can include radiation. Whole body
radiation may be administered at 12 Gy. A radiation dose may
comprise a cumulative dose of 12 Gy to the whole body, including
healthy tissues. A radiation dose may comprise from 5 Gy to 20 Gy.
A radiation dose may be 5 Gy, 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 11 Gy,
12, Gy, 13 Gy, 14 Gy, 15 Gy, 16 Gy, 17 Gy, 18 Gy, 19 Gy, or up to
20 Gy. Radiation may be whole body radiation or partial body
radiation. In the case that radiation is whole body radiation it
may be uniform or not uniform. For example, when radiation may not
be uniform, narrower regions of a body such as the neck may receive
a higher dose than broader regions such as the hips. For example,
in one embodiment, subjects may undergo standard treatment with
high dose chemotherapy followed by peripheral blood stem cell
transplantation. In certain embodiments, following the transplant,
subjects receive an infusion of the expanded immune cells of the
present invention. The dosage of the above treatments to be
administered to a patient will vary with the precise nature of the
condition being treated and the recipient of the treatment. The
scaling of dosages for human administration can be performed
according to art-accepted practices, The dose for CAMPATH, for
example, will generally be in the range 1 to about 100 mg for an
adult patient, usually administered daily for a period between 1
and 30 days, The preferred daily dose is 1 to 10 mg per day
although in some instances larger doses of up to 40 mg per day may
be used (described in U.S. Pat. No. 6, 120,766).
[0235] V. Antibiotic Agents
[0236] An antibiotic can be administered to a subject as part of a
therapeutic regime. An antibiotic can be administered at a
therapeutically effective dose. An antibiotic can kill or inhibit
growth of bacteria. An antibiotic can be a broad spectrum
antibiotic that can target a wide range of bacteria. Broad spectrum
antibiotics, either a 3.sup.rd or 4.sup.th generation, can be
cephalosporin or a quinolone.
[0237] An antibiotic can also be a narrow spectrum antibiotic that
can target specific types of bacteria. An antibiotic can target a
bacterial cell wall such as penicillins and cephalosporins. An
antibiotic can target a cellular membrane such as polymyxins. An
antibiotic can interfere with essential bacterial enzymes such as
antibiotics: rifamycins, lipiarmycins, quinolones, and
sulfonamides. An antibiotic can also be a protein synthesis
inhibitor such as macrolides, lincosamides, and tetracyclines. An
antibiotic can also be a cyclic lipopeptide such as daptomycin,
glycylcyclines such as tigecycline, oxazolidiones such as
linezolid, and lipiarmycins such as fidaxomicin.
[0238] In some cases, an antibiotic can be 1.sup.st generation,
2.sup.bd generation, 3.sup.rd generation, 4th generation, or Stn
generation. A first generation antibiotic can have a narrow
spectrum. Examples of 1.sup.st generation antibiotics can be
penicillins (Penicillin G or Penicillin V), Cephalosporins
(Cephazolin, Cephalothin, Cephapirin, Cephalethin, Cephradin, or
Cephadroxin). In some cases, an antibiotic can be 2.sup.nd
generation. 2.sup.nd generation antibiotics can be a penicillin
(Amoxicillin or Ampicillin), Cephalosporin (Cefuroxime,
Cephamandole, Cephoxitin, Cephaclor, Cephrozil, Loracarbef). In
some cases, an antibiotic can be 3.sup.rd generation. A 3.sup.rd
generation antibiotic can be penicillin (carbenicillin and
ticarcillin) or cephalosporin (Cephixime, Cephtriaxone,
Cephotaxime, Cephtizoxime, and Cephtazidime). An antibiotic can
also be a 4.sup.th generation antibiotic. A 4.sup.th generation
antibiotic can be Cephipime. An antibiotic can also be 5.sup.th
generation. 5.sup.th generation antibiotics can be Cephtaroline or
Cephtobiprole.
[0239] In some cases, an antibiotic can be a bacterial wall
targeting agent, a cell membrane targeting agent, a bacterial
enzyme interfering agent, a bactericidal agent, a protein synthesis
inhibitor, or a bacteriostatic agent. A bacterial wall targeting
agent can be a penicillin derivatives (penams), cephalosporins
(cephems), monobactams, and carbapenems. .beta.-Lactam antibiotics
are bactericidal or bacteriostatic and act by inhibiting the
synthesis of the peptidoglycan layer of bacterial cell walls. In
some cases an antibiotic may be a protein synthesis inhibitor. A
protein synthesis inhibitor can be ampicillin which acts as an
irreversible inhibitor of the enzyme transpeptidase, which is
needed by bacteria to make the cell wall. It inhibits the third and
final stage of bacterial cell wall synthesis in binary fission,
which ultimately leads to cell lysis; therefore, ampicillin is
usually bacteriolytic. In some cases, a bactericidal agent can be
cephalosporin or quinolone. In other cases, a bacteriostatic agent
is trimethoprim, sulfamethoxazole, or pentamidine
[0240] In some cases, an agent for the prevention of PCP pneumonia
may be administered. For example, Trimethoprim and Sulfamethoxazole
can be administered to prevent pneumonia. A dose of trimethoprim
and sulfamethoxazole (TMP/SMX; an exemplary sulfa drug) can be 1
tablet PO daily three times a week, on non-consecutive days, on or
after the first dose of chemotherapy and continuing for at least
about 6 months and until a CD4 count is greater than 200 on at
least 2 consecutive lab studies. In some cases, trimethoprim can be
administered at 160 mg. Trimethoprim can be administered from about
100 to about 300 mgs. Trimethoprim can be administered from about
100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225mg, 250 mg, 275 mg, or
up to about 300 mg. In some cases, sulfamethoxazole is administered
at 800 mg. Sulfamethoxazole can be administered from about 500 mg
to about 1000 mg. Sulfamethoxazole can be administered from about
500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700
mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg,
925 mg, 950 mg, 975 mg, or up to about 1000 mgs. In some cases, a
TMP/SMX regime can be administered at a therapeutically effective
amount. TMP/SMX can be administered from about 1.times. to about
10.times. daily. TMP/SMX can be administered 1.times., 2.times.,
3.times., 4.times., 5.times., 6.times., 7.times., 8.times.,
9.times., 10.times., 11.times., 12.times., 13.times., 14.times.,
15.times., 16.times., 17.times., 18.times., 19.times., or up to
about 20.times. daily. In some cases, TMP/SMX can be administered
on a weekly basis. For example, TMP/SMX can be administered from
1.times., 2.times., 3.times., 4.times., 5.times., 6.times., or up
to about 7.times. a week. A TMP/SMX regime can be administered from
about day: -14, -13, -12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2,
-1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or up to about
day 14 after administration of a cellular therapy, such as
TILs.
[0241] In some cases, said bacteriostatic agent is administered
from about 8 days prior to TILs to at least 4 days after said
TILs.
[0242] In some cases, a subject that has a sulfa allergy may
receive pentamidine. Pentamidine can be administered by aerosol.
Pentamidine 300 mg per nebulizer within one week prior to admission
and continued monthly until the CD4 count is above 200 on two
consecutive follow up lab studies and for at least 6 months post
chemotherapy. Pentamidine can be used to prevent the occurrence of
PCP infections. It can be supplied in 300 mg vials of lyophilized
powder and will be administered via nebulizer. Pentamidine can be
administered from about 300 mg to about 500 mgs. In some cases,
petamidine can be administered from about 100 mg, 200 mg, 300 mg,
400 mg, 500mg, 600 mg, 700 mg, or up to about 800 mgs.
[0243] In some cases, a bacteriostatic agent, such as an antibiotic
can be administered prior to TILs, concurrent with said TILs, or
after said TILs. In some cases, a bacteriostatic agent can be
administered from about 14 days prior to an administration of said
TILs to about 6 months after said administration of said TILs.
[0244] VI. Anti-Viral Agents
[0245] In some cases, an anti-viral agent may be administered as
part of a treatment regime. In some cases, a herpes virus
prophylaxis can be administered to a subject as part of a treatment
regime. A herpes virus prophylaxis can be valacyclovir (Valtrex).
Valtrex can be used orally to prevent the occurrence of herpes
virus infections in subjects with positive HSV serology. It can be
supplied in 500 mg tablets. Valacyclovir can be administered at a
therapeutically effective amount. For example, valacyclovir can be
administered from about 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175
mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg,
400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600
mg, 625 mg, 650 mg, or up to about 700 mg tablets. Valacyclovir can
be started the day after the last dose of fludarabine at a dose of
500 mg orally daily if a subject is able to tolerate oral intake.
An antiviral therapy can be administered from about day: -14, -13,
-12, -11, -10, -9, -8, -7, -6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, 11, 12, 13, or up to about day 14 after
administration of a cellular therapy such as TIL therapy.
[0246] In some cases, a subject may not be able to take oral
medication for the prophylaxis of herpes. In those cases, acyclovir
can be administered. Acyclovir can be supplied as a powder for
injection in 500 mg/vials. In some cases, acyclovir can be
administered at a therapeutically effective amount. Acyclovir can
be administered orally from about 50 mg, 75 mg, 100 mg, 125 mg, 150
mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg,
375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575
mg, 600 mg, 625 mg, 650 mg, or up to about 700 mgs. Acyclovir can
be administered 1.times., 2.times., 3.times., 4.times., 5.times.,
6.times., or up to about 7.times. per day. Acyclovir can be
administered from about day: -14, -13, -12, -11, -10, -9, -8, -7,
-6, -5, -4, -3, -2, -1, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,
13, or up to about day 14 after administration of a cellular
therapy such as TIL therapy. In some cases, acyclovir can be
administered intravenously. For example, acyclovir can be
administered at 1 mg/kg to about 3 mg/kg, from about 3 mg/kg to
about 5 mg/kg, from about 5mg/kg to about 10 mg/kg, from about 10
mg/kg to about 20 mg/kg, 20 mg/kg to about 30 mg/kg, from about 30
mg/kg to about 40 mg/kg, from about 40 mg/kg to about 50 mg/kg,
from about 50 mg/kg to about 60 mg/kg, from about 60 mg/kg to about
70 mg/kg, from about 70 mg/kg to about 80 mg/kg, from about 80
mg/kg to about 90 mg/kg, from about 90 mg/kg to about 100 mg/kg. In
some cases, acyclovir is administered in excess of 50 mg/kg.
Acyclovir can be reconstituted in 10 mL of sterile water for
injection to a concentration of 50 mg/mL. Reconstituted solutions
should be used within 12 hours. IV solutions can be diluted to a
concentration of 7mg/mL or less and infused over 1 hour to avoid
renal damage.
Disease Parameters
[0247] In some cases, a level of disease can be determined in
sequence or concurrent with a treatment regime or cellular
administration. A level of disease on target lesions can be
measured as a Complete Response (CR): Disappearance of all target
lesions, Partial Response (PR): At least a 30% decrease in the sum
of the longest diameter (LD) of target lesions taking as reference
the baseline sum LD, Progression (PD): At least a 20% increase in
the sum of LD of target lesions taking as reference the smallest
sum LD recorded since the treatment started or the appearance of
one or more new lesions, Stable Disease (SD): Neither sufficient
shrinkage to qualify for PR nor sufficient increase to qualify for
PD taking as references the smallest sum LD. In other cases, a
non-target lesion can be measured. A level of disease of a
non-target lesion can be Complete Response (CR): Disappearance of
all non-target lesions and normalization of tumor marker level,
Non-Complete Response: Persistence of one or more non-target
lesions, Progression (PD): Appearance of one or more new lesions.
Unequivocal progression of existing non-target lesions.
[0248] In some cases, a subject that undergoes a treatment regime
and cellular administration can be evaluated for best overall
response. A best overall response can be the best response recorded
from the start of treatment until disease progression/recurrence
(taking as reference for progressive disease the smallest
measurements recorded since the treatment started). A subject's
best response assignment can depend on the achievement of both
measurement and confirmation criteria. The time to progression can
be measured from the date of randomization.
TABLE-US-00007 TABLE 7 Lesion evaluation Target Lesions Non-Target
Lesions New Lesions Overall Response CR CR No CR CR Non-CR/Non-PD
No PR PR Non-PD No PR SD Non-PD No SD PD Any Yes or No PD Any PD
Yes or No PD Any Any Yes PD
[0249] To be assigned a status of PR or CR, changes in tumor
measurements must be confirmed by repeat studies that should be
performed at least about 4 weeks after the criteria for response
are first met. In the case of SD, follow-up measurements must have
met the SD criteria at least once after study entry at a minimum
interval of 6-8 weeks. In some cases, a duration of overall
response can be measured from the time measurement criteria are met
for CR/PR (whichever is first recorded) until the first date that
recurrent or progressive disease is objectively documented (taking
as reference for progressive disease the smallest measurements
recorded since the treatment started). The duration of overall
complete response can be measured from the time measurement
criteria are first met for CR until the first date that recurrent
disease is objectively documented. Stable disease can be measured
from the start of the treatment until the criteria for progression
are met, taking as reference the smallest measurements recorded
since the treatment started.
[0250] In some cases, measurable disease can be taken and recorded
in metric notation using a ruler or calipers. All baseline
evaluations can be performed as closely as possible to the
beginning of treatment. A lesion can be considered measurable when
it is superficial (e.g., skin nodule and palpable lymph nodes) and
over at least about 10 mm in diameter using calipers. In some
cases, color photography can be taken.
[0251] In other cases, a computerized tomography scan (CT) can or
magnetic resonance imaging (MRI) can be taken. A CT can be taken on
a slice thickness of 5 mm or less. If CT scans have slice thickness
greater than 5 mm, the minimum size for a measurable lesion should
be twice the slice thickness. In some cases, an FDG-PET scan can be
used. FDG-PET can be used to evaluate new lesions. A negative
FDG-PET at baseline, with a positive FDG-PET at follow up is a sign
of progressive disease (PD) based on a new lesion. No FDG-PET at
baseline and a positive FDG-PET at follow up: if a positive FDG-PET
at follow-up corresponds to a new site of disease confirmed by CT,
this is PD. If a positive PDG-PET at follow up corresponds to a
pre-existing site of disease on CT that may not be progressing on a
basis of anatomic imagines, this may not be PD. In some cases,
FDG-PET may be used to upgrade a response to a CR in a manner
similar to biopsy in cases where a residual radiographic
abnormality is thought to represent fibrosis or scarring. A
positive FDG-PET scan lesion means one which is FDG avid with an
uptake greater than twice that of the surrounding tissue on an
attenuation corrected image.
[0252] In some cases an evaluation of a lesion can be performed. A
complete response (CR) can be a disappearance of all target
lesions. Any pathological lymph nodes (target or non-target) may
have reduction in short axis to less than 10 mm. A partial response
(PR) can be at least a 30% decrease in a sum of the diameters of
target lesions, taking as reference the baseline sum of diameters.
Progressive disease (PD) can be at least a 20% increase in the sum
of the diameters of target lesions, taking as reference the
smallest sum. In addition to the relative increase of 20%, the sum
must also demonstrate an absolute increase of at least 5 mm. Stable
disease (SD) can be neither sufficient shrinkage to quality for PR
nor sufficient increase to quality for PD, taking as reference the
smallest sum of diameters.
[0253] In some cases, non-target lesions can be evaluated. A
complete response of a non-target lesion can be a disappearance and
normalization of tumor marker level. All lymph nodes must be
non-pathological in size (less than 10 mm short axis). If tumor
markers are initially above the upper normal limit, they must
normalize for a patient to be considered a complete clinical
response. Non-CR/Non-PD is persistence of one or more non-target
lesions and or maintenance of tumor marker level above the normal
limit. Progressive disease can be appearance of one or more new
lesions and or unequivocal progression of existing non-target
lesions. Unequivocal progression should not normally trump target
lesion status.
[0254] In some cases, a best overall response can be the best
response recorded from the start of treatment until disease
progression/recurrence.
Toxicity Criteria
[0255] In some cases, toxicity to a treatment regime or cellular
administration can be determined. A toxicity determination can
encompass toxicity, immunologic effects and anti-tumor efficacy of
a treatment regimen. A toxicity study can utilize the CTCAE version
3.0 for toxicity and adverse event reporting. Early toxicities
related specifically to the infusion of engineered cells (those
which are seen immediately following cell infusion and prior to
aldesleukin administration) are generally mild and can include
fevers, chills, headache, and malaise. Toxicities which occur
following administration of aldesleukin but are thought to be
related to engineered cell administration can include immune
mediated events such as vitiligo, transient uveitis, hearing loss
and vestibular dysfunction. The use of a non-myeloablative regimen
prior to cell administration can increase the toxicity of treatment
as profound myelosuppression occurs in subjects. In some cases, the
standard approach to the administration of high-dose aldesleukin
can be to continue dosing until grade 3 or 4 events occur. The most
commonly seen grade 4 events are pulmonary and renal impairment,
and mental status changes. These toxicities may sometimes require
intubation for protection of a subject's airway. In some cases,
fatal complications can be possible and it can be appropriate to
carry out treatment in the context of life threatening metastatic
cancer.
[0256] In some cases, a subject treated with a treatment regime or
cellular product described herein can experience an adverse event
associated with the regime or cellular product. An adverse event
can be any reaction, side effect, or untoward event that occurs
during the course of the treatment associated with the use of a
drug in humans, whether or not the event is considered related to
the treatment or clinically significant. In some cases, an adverse
event can include events reported by a subject, as well as
clinically significant abnormal findings on physical examination or
laboratory evaluation. A new illness, symptom, sign or clinically
significant laboratory abnormality or worsening of a pre-existing
condition or abnormality can be considered an adverse event. All
adverse events, including clinically significant abnormal findings
on laboratory evaluations, regardless of severity, will be followed
until resolution to grade 2 or less with the exception of
lymphopenia and alopecia. If an adverse event is not expected to
resolve to grade 2 or less a subject may cease therapy.
[0257] In some cases, a treatment regime may be administered with
toxicity reducing agents. A toxicity reducing agent can be a fever
or vomit reducing agent. For example Mesna can be administered to
reduce toxicities such as nausea, vomiting, and diarrhea.
[0258] Mesna can be a diluted solution (1 to 20 mg/mL) and can be
physically and chemically stable for at least 24 hours under
refrigeration. Mesna can be chemically stable at room temperature
for 48-72 hours in D5W, 48-72 hour in D5W/0.45% NaCl, or 24 hours
in 0.9% NaCl. Mesna can be diluted to concentrations less than or
equal to 20 mg mesna/ml fluid in D5W or 0.9% NaCl and to be
administered intravenously as a continuous infusion. If patient is
obese (BMI>35) drug dosage will be calculated using practical
weight.
[0259] In other cases, additional support medications can include
Ondansetron hydrochloride. Ondansetron hydrochloride can be used to
control nausea and vomiting during the chemotherapy preparative
regimen. It can cause headache, dizziness, myalgias, drowsiness,
malaise, and weakness. Less common side effects include chest pain,
hypotension, pruritis, constipation and urinary retention. In other
cases, furosemide can also be administered. Furosemide can be used
to enhance urine output during the chemotherapy preparative regimen
with cyclophosphamide Adverse effects include dizziness, vertigo,
paresthesias, weakness, orthostatic hypotension, photosensitivity,
rash and pruritus.
Methods of Administration
[0260] Provided herein can be methods for administering a
therapeutic regime to a subject having a condition such as cancer.
In some instances, a cellular composition (for example, comprising
TILs such as TILs (including autologous TILs) with a CISH
disruption) can be provided in a unit dosage form. A cellular
composition (for example, comprising TILs (including autologous
TILs) such as TILs with a CISH disruption) can be resuspended in
solution and administered as an infusion. Provided herein can also
be a treatment regime that includes immunostimulants,
immunosuppressants, antibiotics, antifungals, antiemetics,
chemotherapeutics, radiotherapy, and any combination thereof. A
treatment regime that includes any of the above can be lyophilized
and reconstituted in an aqueous solution (e.g., saline solution).
In some instances, a treatment (for example, a cellular treatment
such as TILs, e.g., TILs (including autologous TILs) with a CISH
disruption) is administered by a route selected from subcutaneous
injection, intramuscular injection, intradermal injection,
percutaneous administration, intravenous ("i.v.") administration,
intranasal administration, intralymphatic injection, and oral
administration. In some instances, a subject is infused with a
cellular composition comprising TILs by an intralymphatic
microcatheter.
[0261] Many drugs can be administered orally as liquids, capsules,
tablets, or chewable tablets. Because the oral route is the most
convenient and usually the safest and least expensive, it is the
one most often used. However, it has limitations because of the way
a drug typically moves through the digestive tract. For drugs
administered orally, absorption may begin in the mouth and stomach.
However, most drugs are usually absorbed from the small intestine.
The drug passes through the intestinal wall and travels to the
liver before being transported via the bloodstream to its target
site. The intestinal wall and liver chemically alter (metabolize)
many drugs, decreasing the amount of drug reaching the bloodstream.
Consequently, these drugs are often given in smaller doses when
injected intravenously to produce the same effect.
[0262] For a subcutaneous route, a needle is inserted into fatty
tissue just beneath the skin. After a drug is injected, it then
moves into small blood vessels (capillaries) and is carried away by
the bloodstream. Alternatively, a drug reaches the bloodstream
through the lymphatic vessels. The intramuscular route is preferred
to the subcutaneous route when larger volumes of a drug product are
needed. Because the muscles lie below the skin and fatty tissues, a
longer needle is used. Drugs are usually injected into the muscle
of the upper arm, thigh, or buttock. How quickly the drug is
absorbed into the bloodstream depends, in part, on the blood supply
to the muscle: The sparser the blood supply, the longer it takes
for the drug to be absorbed. For the intravenous route, a needle is
inserted directly into a vein. A solution containing the drug may
be given in a single dose or by continuous infusion. For infusion,
the solution is moved by gravity (from a collapsible plastic bag)
or, more commonly, by an infusion pump through thin flexible tubing
to a tube (catheter) inserted in a vein, usually in the forearm. In
some cases, cells or therapeutic regimes are administered as
infusions. An infusion can take place over a period of time. For
example, an infusion can be an administration of a cell or
therapeutic regime over a period of about 5 minutes to about 5
hours. An infusion can take place over a period of about 5 min, 10
min, 20 min, 30 min, 40 min, 50 min, 1 hour, 1.5 hours, 2 hours,
2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or up to about 5
hours.
[0263] In some embodiments, intravenous administration is used to
deliver a precise dose quickly and in a well-controlled manner
throughout the body. It is also used for irritating solutions,
which would cause pain and damage tissues if given by subcutaneous
or intramuscular injection. An intravenous injection can be more
difficult to administer than a subcutaneous or intramuscular
injection because inserting a needle or catheter into a vein may be
difficult, especially if the person is obese. When given
intravenously, a drug is delivered immediately to the bloodstream
and tends to take effect more quickly than when given by any other
route. Consequently, health care practitioners closely monitor
people who receive an intravenous injection for signs that the drug
is working or is causing undesired side effects. Also, the effect
of a drug given by this route tends to last for a shorter time.
Therefore, some drugs must be given by continuous infusion to keep
their effect constant. For the intrathecal route, a needle is
inserted between two vertebrae in the lower spine and into the
space around the spinal cord. The drug is then injected into the
spinal canal. A small amount of local anesthetic is often used to
numb the injection site. This route is used when a drug is needed
to produce rapid or local effects on the brain, spinal cord, or the
layers of tissue covering them (meninges)--for example, to treat
infections of these structures.
[0264] Drugs administered by inhalation through the mouth can be
atomized into smaller droplets than those administered by the nasal
route, so that the drugs can pass through the windpipe (trachea)
and into the lungs. How deeply into the lungs they go depends on
the size of the droplets. Smaller droplets go deeper, which
increases the amount of drug absorbed. Inside the lungs, they are
absorbed into the bloodstream. Drugs applied to the skin are
usually used for their local effects and thus are most commonly
used to treat superficial skin disorders, such as psoriasis,
eczema, skin infections (viral, bacterial, and fungal), itching,
and dry skin. The drug is mixed with inactive substances. Depending
on the consistency of the inactive substances, the formulation may
be an ointment, cream, lotion, solution, powder, or gel
[0265] In some cases, a treatment regime may be dosed according to
a body weight of a subject. In subjects who are determined obese
(BMI>35) a practical weight may need to be utilized. BMI is
calculated by: BMI=weight (kg)/[height (m)].sup.2.
[0266] An ideal body weight may be calculated for men as 50
kg+2.3*(number of inches over 60 inches) or for women 45.5kg + 2.3
(number of inches over 60 inches). An adjusted body weight may be
calculated for subjects who are more than 20% of their ideal body
weight. An adjusted body weight may be the sum of an ideal body
weight+(0.4.times.(Actual body weight-ideal body weight)). In some
cases a body surface area may be utilized to calculate a dosage. A
body surface area (BSA) may be calculated by: BSA (m2)= Height
(cm)*Weight (kg)/3600.
[0267] In some cases, a pharmaceutical composition comprising a
cellular therapy can be administered either alone or together with
a pharmaceutically acceptable carrier or excipient, by any routes,
and such administration can be carried out in both single and
multiple dosages. More particularly, the pharmaceutical composition
can be combined with various pharmaceutically acceptable inert
carriers in the form of tablets, capsules, lozenges, troches, hand
candies, powders, sprays, aqueous suspensions, injectable
solutions, elixirs, syrups, and the like. Such carriers include
solid diluents or fillers, sterile aqueous media and various
non-toxic organic solvents, etc. Moreover, such oral pharmaceutical
formulations can be suitably sweetened and/or flavored by means of
various agents of the type commonly employed for such purposes.
[0268] In some cases, a therapeutic regime can be administered
along with a carrier or excipient. Exemplary carriers and
excipients can include dextrose, sodium chloride, sucrose, lactose,
cellulose, xylitol, sorbitol, malitol, gelatin, PEG, PVP, and any
combination thereof.
[0269] In some cases, an excipient such as dextrose or sodium
chloride can be at a percent from about 0.5%, 1%, 1.5%, 2%, 2.5%,
3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%,
9.5%, 10%, 10.5%, 11%, 11.5%, 12%, 12.5%, 13%, 13. 5%, 14%, 14. 5%,
or up to about 15%.
[0270] Described herein is a method of treating a disease (e.g.,
cancer, such as gastrointestinal cancer) in a recipient comprising
transplanting to the recipient one or more cells (including organs
and/or tissues) comprising engineered cells. Cells prepared by
intracellular genomic transplant can be used to treat cancer.
[0271] In some embodiments, a method of treating gastrointestinal
cancer comprises: a) obtaining tumor infiltrating lymphocytes (TIL)
from a tumor sample (e.g., a tumor sample from a subject having
gastrointestinal cancer); b) identifying a mutation reactive TIL;
and disrupting an endogenous gene or portion thereof with a Cas
nuclease in said mutation reactive TIL. In some embodiments, the
tumor sample is subjected to sequencing analysis, such as whole
exomic sequencing, transcriptome sequencing, or a combination
thereof. In some embodiments, the identifying comprises introducing
the TIL to antigen presenting cells (APCs) pulsed with peptides
comprising the mutation (e.g., from about 15mer mutant peptides up
to about 30mer mutant peptides, such as 25mer mutant peptides). In
some embodiments, the TIL are contacted with APCs expressing
peptides to determine reactivity of TIL. A peptide can comprise a
tumor mutation that may be targeted by a TIL. For example, an APC
may express a multitude of peptides, some encoding a tumor mutation
that may be utilized to identify tumor-reactive TILs. Those TILs
that are identified as having tumor reactivity may be isolated or
purified and utilized for genomic engineering. In some cases, a
tumor-reactive TIL may be genomically engineered with a CRISPR
system. A CRISPR system may be used to knock out an endogenous
gene, such as CISH, in a tumor-reactive TIL. A CRISPR system may be
used to knock out an endogenous gene, such as PD-1, in a
tumor-reactive TIL. In some embodiments, the identifying comprises
introducing said TIL to antigen presenting cells (APCs)
electroporated with a polynucleic acid comprising a mutation. In
some embodiments, the identifying further comprises detecting a
cytokine such as IL-2, IFN-gamma, IL-6, degranulation, cellular
proliferation, or the like.
EXAMPLES
Example 1
Identify gRNA with Highest Double Strand Break (DSB) Induction at
Each Gene Site
Design and Construction of Guide RNAs:
[0272] Guide RNAs (gRNAs) were designed to the desired region of a
gene. Multiple primers to generate gRNAs (shown in Table 1) were
chosen based on the highest ranked values determined by off-target
locations. The gRNAs were ordered in oligonucleotide pairs:
5'-CACCG-gRNA sequence-3' and 5'-AAAC-reverse complement gRNA
sequence-C-3' (sequences of the oligonucleotide pairs are listed in
Table 1).
TABLE-US-00008 TABLE 8 Primers used to generate the gRNAs (the
sequence CACCG is added to the sense and AAAC to the antisense for
cloning purposes). Sequence SEQ ID Primer Name 5'-3' 82 HPRT gRNA 1
Sense CACCG CACGT GTGAA CCAAC CCGCC 83 HPRT gRNA 1 Anti AAACG GCGGG
TTGGT TCACA CGTGC 84 HPRT gRNA 2 Sense CACCG AAACA ACAGG CCGGG
CGGGT 85 HPRT gRNA 2 Anti AAACA CCCGC CCGGC CTGTT GTTTC 86 HPRT
gRNA 3 Sense CACCG ACAAA AAAAT TAGCC GGGTG 87 HPRT gRNA 3 Anti
AAACC ACCCG GCTAA TTTTT TTGT 88 HPRT gRNA 4 Sense CACCG TAAAT TTCTC
TGATA GACTA 89 HPRT gRNA 4 Anti AAACT AGTCT ATCAG AGAAA TTTAC 90
HPRT gRNA 5 Sense CACCG TGTTT CAATG AGAGC ATTAC 91 HPRT gRNA 5 Anti
AAACG TAATG CTCTC ATTGA AACAC 92 HPRT gRNA 6 Sense CACCG GTCTC
GAACT CCTGA GCTC 93 HPRT gRNA 6 Anti AAACG AGCTC AGGAG TTCGA GACC
94 HPRT Cell For AGTGA AGTGG CGCAT TCTTG 95 HPRT Cell Rev CACCC
TTTCC AAATC CTCAG C 96 AAVSI gRNA 1 Sense CACCG TGGGG GTTAG ACCCA
ATATC 97 AAVS1 gRNA 1 Anti AAACG ATATT GGGTC TAACC CCCAC 98 AAVSI
gRNA 2 Sense CACCG ACCCC ACAGT GGGGC CACTA 99 AAVSI gRNA 2 Anti
AAACT AGTGG CCCCA CTGTG GGGTC 100 AAVSI gRNA 3 Sense CACCG AGGGC
CGGTT AATGT GGCTC 101 AAVSI gRNA 3 Anti AAACG AGCCA CATTA ACCGG
CCCTC 102 AAVSI gRNA 4 Sense CACCG TCACC AATCC TGTCC CTAG 103 AAVSI
gRNA 4 Anti AAACC TAGGG ACAGG ATTGG TGAC 104 AAVSI gRNA 5 Sense
CACCG CCGGC CCTGG GAATA TAAGG 105 AAVSI gRNA 5 Anti AAACC CTTAT
ATTCC CAGGG CCGGC 106 AAVSI gRNA 6 Sense CACCG CGGGC CCCTA TGTCC
ACTTC 107 AAVSI gRNA 6 Anti AAACG AAGTG GACAT AGGGG CCCGC 108 AAVSI
Cell For ACTCC TTTCA TTTGG GCAGC 109 AAVSI Cell Rev GGTTC TGGCA
AGGAG AGAGA 110 PD-1 gRNA 1 Sense CACCG CGGAG AGCTT CGTGC TAAAC 111
PD-1 gRNA 1 Anti AAACG TTTAG CACGA AGCTC TCCGC 112 PD-1 gRNA 2
Sense CACCG CCTGC TCGTG GTGAC CGAAG 113 PD-1 gRNA 2 Anti AAACC
TTCGG TCACC ACGAG CAGGC 114 PD-1 gRNA 3 Sense CACCG CAGCA ACCAG
ACGGA CAAGC 115 PD-1 gRNA 3 Anti AAACG CTTGT CCGTC TGGTT GCTGC 116
PD-1 gRNA 4 Sense CACCG AGGCG GCCAG CTTGT CCGTC 117 PD-1 gRNA 4
Anti AAACG ACGGA CAAGC TGGCC GCCTC 118 PD-1 gRNA 5 Sense CACCG
CGTTG GGCAG TTGTG TGACA 119 PD-1 gRNA 5 Anti AAACT GTCAC ACAAC
TGCCC AACGC 120 PD-1 gRNA 6 Sense CACCG ACGGA AGCGG CAGTC CTGGC 121
PD-1 gRNA 6 Anti AAACG CCAGG ACTGC CGCTT CCGTC 122 PD-1 Cell For
AGAAG GAAGA GGCTC TGCAG
123 PD-1 Cell Rev CTCTT TGATC TGCGC CTTGG 124 CTLA4 gRNA 1 Sense
CACCG CCGGG TGACA GTGCT TCGGC 125 CTLA4 gRNA 1 Anti AAACG CCGAA
GCACT GTCAC CCGGC 126 CTLA4 gRNA 2 Sense CACCG TGCGG CAACC TACAT
GATG 127 CTLA4 gRNA 2 Anti AAACC ATCAT GTAGG TTGCC GCAC 128 CTLA4
gRNA 3 Sense CACCG CTAGA TGATT CCATC TGCAC 129 CTLA4 gRNA 3 Anti
AAACG TGCAG ATGGA ATCAT CTAGC 130 CTLA4 gRNA 4 Sense CACCG AGGTT
CACTT GATTT CCAC 131 CTLA4 gRNA 4 Anti AAACG TGGAA ATCAA GTGAA CCTC
132 CTLA4 gRNA 5 Sense CACCG CCGCA CAGAC TTCAG TCACC 133 CTLA4 gRNA
5 Anti AAACG GTGAC TGAAG TCTGT GCGGC 134 CTLA4 gRNA 6 Sense CACCG
CTGGC GATGC CTCGG CTGC 135 CTLA4 gRNA 6 Anti AAACG CAGCC GAGGC
ATCGC CAGC 136 CTLA4 Cell For TGGGG ATGAA GCTAG AAGGC 137 CTLA4
Cell Rev AATCT GGGTT CCGTT GCCTA 138 CCR5 gRNA 1 Sense CACCG ACAAT
GTGTC AACTC TTGAC 139 CCR5 gRNA 1 Anti AAACG TCAAG AGTTG ACACA
TTGTC 140 CCR5 gRNA 2 Sense CACCG TCATC CTCCT GACAA TCGAT 141 CCR5
gRNA 2 Anti AAACA TCGAT TGTCA GGAGG ATGAC 142 CCR5 gRNA 3 Sense
CACCG GTGAC AAGTG TGATC ACTT 143 CCR5 gRNA 3 Anti AAACA AGTGA TCACA
CTTGT CACC 144 CCR5 gRNA 4 Sense CACCG ACACA GCATG GACGA CAGCC 145
CCR5 gRNA 4 Anti AAACG GCTGT CGTCC ATGCT GTGTC 146 CCR5 gRNA 5
Sense CACCG ATCTG GTAAA GATGA TTCC 147 CCR5 gRNA 5 Anti AAACG GAATC
ATCTT TACCA GATC 148 CCR5 gRNA 6 Sense CACCG TTGTA TTTCC AAAGT
CCCAC 149 CCR5 gRNA 6 Anti AAACG TGGGA CTTTG GAAAT ACAAC 150 CCR5
Cell For CTCAA CCTGG CCATC TCTGA 151 CCR5 Cell Rev CCCGA GTAGC
AGATG ACCAT
[0273] The gRNAs were cloned together using a target sequence
cloning protocol. Briefly, the oligonucleotide pairs were
phosphorylated and annealed together using T4 PNK (NEB) and
10.times. T4 Ligation Buffer (NEB) in a thermocycler with the
following protocol: 37.degree. C. 30 minutes, 95.degree. C. 5
minutes and then ramped down to 25.degree. C. at 5.degree.
C./minute. pENTR1-U6-Stuffer-gRNA vector was digested with
FastDigest Bbsl (Fermentas), FastAP (Fermentas) and 10.times. Fast
Digest Buffer were used for the ligation reaction. The digested
pENTR1 vector was ligated together with the phosphorylated and
annealed oligo duplex (dilution 1:200) from the previous step using
T4 DNA Ligase and Buffer (NEB). The ligation was incubated at room
temperature for 1 hour and then transformed and subsequently
mini-prepped using GeneJET Plasmid Miniprep Kit (Thermo
Scientific). The plasmids were sequenced to confirm the proper
insertion. Validation of gRNAs
[0274] HEK293T cells were plated out at a density of
1.times.10{circumflex over ( )}5 cells per well in a 24 well plate.
150 uL of Opti-MEM medium was combined with 1.5 ug of gRNA plasmid,
1.5 ug of Cas9 plasmid. Another 150 uL of Opti-MEM medium was
combined with 5 uL of Lipofectamine 2000 Transfection reagent
(Invitrogen). The solutions were combined together and incubated
for 15 minutes at room temperature. The DNA-lipid complex was added
dropwise to wells of the 24 well plate. Cells were incubated for 3
days at 37.degree. C. and genomic DNA was collected using the
GeneJET Genomic DNA Purification Kit (Thermo Scientific). Activity
of the gRNAs was quantified by a Surveyor Digest, gel
electrophoresis, and densitometry (FIG. 61) (Guschin, D. Y., et
al., "A Rapid and General Assay for Monitoring Endogenous Gene
Modification," Methods in Molecular Biology, 649: 247-256
(2010)).
Results
[0275] The efficiencies of Cas9 in creating double strand break
(DSB) with the assistance of different gRNA sequences are listed in
Table 9. The percentage numbers in Table 9 indicated the percent of
gene modifications in the sample.
TABLE-US-00009 TABLE 9 The efficiencies of Cas9/gRNA pair in
creating double strand break (DSB) at each target gene site. HPRT
AAVS1 CCR5 PD1 CTLA4 gRNA#1 27.85% 32.99% 21.47% 10.83% 40.96%
gRNA#2 30.04% 27.10% >60% >60% 56.10% gRNA#3 <1% 39.82%
55.98% 37.42% 39.33% gRNA#4 <5% 25.93% 45.99% 20.87% 40.13%
gRNA#5 <1% 27.55% 36.07% 30.60% 15.90% gRNA#6 <5% 39.62%
33.17% 25.91% 36.93%
[0276] DSB were created at all five tested target gene sites. Among
them, CCRS, PD1, and CTLA4 provided the highest DSB efficiency.
Other target gene sites, including hRosa26, are tested using the
same methods described herein.
[0277] The percent of double strand break compared to donor control
and Cas9 only controls are listed. A three representative target
gene sites (i.e., CCRS, PD1, and CTLA4) were tested.
Example 2
GUIDE-Seq Library Preparation
[0278] To examine off-target cutting of PD-1 gRNA known as
genome-wide, unbiased identification of double-strand breaks (DSBs)
is achieved by sequencing or GUIDE-seq. It is a method that uses
capture of double-stranded oligonucleotides into DSBs generated by
CRISPR/Cas9, followed by next-generation sequencing to determine
the location of the genetic modification.
[0279] Human T cells were isolated using solid-phase reversible
immobilization magnetic beads (Agencourt DNAdvance), were sheared
with a Covaris 5200 instrument to an average length of 500 bp,
end-repaired, A-tailed, and ligated to half-functional adapters,
incorporating a 8-nt random molecular index. Two rounds of nested
anchored PCR, with primers complementary to the oligo tag, were
used for target enrichment. End Repair Thermocycler Program:
12.degree. C. for 15 min, 37.degree. C. for 15 min; 72.degree. C.
for 15 min; hold at 4.degree. C.
[0280] Start sites of GUIDE-Seq reads mapped back to the genome
enable localization of the DSB to within a few base pairs. The
library was quantitated using Kapa Biosy stems kit for Illumina
Library Quantification kit, according to manufacturer instruction.
Using the mean quantity estimate of number of molecules per uL
given by the qPCR run for each sample, the total set of libraries
were normalized to 1.2 X 10{circumflex over ( )}10 molecules,
divided by the number of libraries to be pooled together for
sequencing. This gave a by molecule input for each sample, and also
a by volume input for each sample. Reads were mapped for the on-
and off-target sites of the three RGNs directed by truncated gRNAs
assessed by GUIDE-Seq. In all cases, the target site sequence has
the protospacer sequence to the left and the PAM sequence to the
right on the x-axis. The library was denatured and loaded onto the
Miseq according to Illumina's standard protocol for sequencing with
an Illumina Miseq Reagent Kit V2 - 300 cycle (2.times.150 bp paired
end).
[0281] In a second experiment, GUIDE-Seq analysis of the PD-1 gRNA
revealed only one reproducible off-target modification. This site
matches the single off-target location detected by the deep
sequencing of predicted sites demonstrating a correlation between
the two approaches. Thus, the NGS analysis of off-targets is
sensitive and comprehensive. The gRNA designs for PD-1 were highly
specific with only one detectable off target site which resides in
an inert section of the genome and which modification of would not
be expected to result in any adverse effects. The gRNA designs for
CISH were highly specific with no measurable off-target
activity.
Example 3
Genomic Engineering of TIL to Knock Out PD-1, CTLA-4, and CISH
[0282] Suitable tumors from eligible stage IIIc-IV cancer patients
are resected and cut up into small 3-5 mm.sup.2 fragments and
placed in culture plates or small culture flasks with growth medium
and high-dose (HD) IL-2. The TILs are initially expanded for 3-5
weeks during this "pre-rapid expansion protocol" (pre-REP) phase to
at least 50.times.10.sup.6 cells. TILs are electroporated using the
Neon Transfection System (100 uL or 10 ul Kit, Invitrogen, Life
Technologies). TILS are pelleted and washed once with T buffer.
TILs are resuspended at a density of 2.times.10.sup.5 cells in 10
uL of T buffer for 10 ul tip, and 3.times.10.sup.6 cells in 100 ul
T buffer for 100 ul tips. TILs are then electroporated at 1400 V,
10 ms, 3 pulses utilizing 15ug Cas9 mRNA, and 10-50 ug PD-1,
CTLA-4, and CISH gRNA-RNA (100 mcl tip). After transfection, TILs
are plated at 1000 cells/effectuL in antibiotic free culture media
and incubated at 30 C in 5% CO2 for 24 hrs. After 24 hr recovery,
TILs can be transferred to antibiotic containing media and cultured
at 37.degree. C. in 5% CO2.
[0283] The cells are then subjected to a rapid expansion protocol
(REP) over two weeks by stimulating the TILs using anti-CD3 in the
presence of PBMC feeder cells and IL-2. The expanded TIL (now
billions of cells) are washed, pooled, and infused into a patient
followed by one or two cycles of HD IL-2 therapy. Before TIL
transfer, a patient can be treated with a preparative regimen using
cyclophosphamide (Cy) and fludaribine (Flu) that transiently
depletes host lymphocytes "making room" for the infused TIL and
removing cytokine sinks and regulatory T cells in order to
facilitate TIL persistence. Subjects receive an infusion of their
own modified TIL cells over 30 minutes and remain in the hospital
to be monitored for adverse events until they have recovered from
the treatment. FIG. 40A and FIG. 40B show cellular expansion of TIL
of two different subjects. FIG. 41A and FIG. 41B show cellular
expansion of TIL electroporated with a CRISPR system, and anti-PD-1
guides and cultured with the addition of feeder cells or no
addition of feeder cells.
Example 4
gRNA Modification
Design and Construction of Modified Guide RNAs:
[0284] Guide RNAs (gRNAs) were designed to the desired region of a
gene. Multiple gRNAs (shown in Table 4) were chosen based on the
highest ranked values determined by off-target locations. The gRNAs
targeting PD-1, CTLA-4, and CISH gene sequences were modified to
contain 2-O-Methyl 3phosphorothioate additions, FIG. 22 and FIG.
32.
Example 5
Phase I Clinical Trial of TIL Therapy for Gastrointestinal
Cancer
[0285] A subject with evaluable gastrointestinal cancer will
undergo a resection of tumorous samples. Tumor infiltrating
lymphocytes (TIL) will be grown and expanded. Multiple individual
fragments or multiple individual cultures of tumor infiltrating
lymphocytes will be grown ex vivo. Individual cultures are
separately expanded and when a sufficient yield of TIL
(approximately 10.sup.8 cells) is expanded from each culture, the
TIL is cryopreserved and aliquots taken for immunologic testing. An
aliquot of the original tumor is subjected to exomic and, when
possible, transcriptome sequencing to identify mutations uniquely
present in the cancer compared to normal cells of the subject.
Aliquots of each cultured TIL preparation are tested for reactivity
against identified mutations using in vitro functionality testing.
In brief, the subjects's antigen presenting cells are pulsed with
25mer peptides including the mutated peptide in the center flanked
by 12 normal amino acids. Alternatively, in vitro transcribed RNA
encoding all mutations are electroporated into the patient's
antigen presenting cells. Using either or both of these techniques,
the previously cryopreserved TIL cultures are then tested for
reactivity to identify the exact mutations recognized The cultures
with high degrees of reactivity against individual cancer mutations
are then subjected to CRISPR knockout of the CISH gene. Up to five
different cultures can be selected for this procedure from each
patient. Cultures with demonstrated mutation reactivity are sent to
the good manufacturing practices (GMP) Cell Production Facility
where CRISPR technology is used to knockout the CISH gene. Knockout
of the CISH gene is determined by DNA sequencing based tracking of
indels by decomposition (TIDE) analysis. Cell populations with
successful knockout of the CISH gene are then rapidly expanded
utilizing OKT3 and feeder cells. Cryopreserved lymphocytes are then
utilized in patient treatment. In the first part of this clinical
trial protocol, a dose escalation is initiated utilizing one
subject per group starting at 10.sup.9 CISH knockout cells per
subject. Individual subjects are treated at half log increments and
if any toxicity higher than Grade 2 is seen due to the cell
infusion, that group is then expanded to additional patients. The
following doses are utilized: 1) 10.sup.9 cells 2) 3.times.10.sup.9
cells3) 10.sup.10 cells4) 3.times.10.sup.10 cells5)
3.times.10.sup.10 to 2.times.10.sup.11 cells. Patients receive a
lymphodepleting preparative regimen followed by infusion of
engineered cells and high dose IL-2. Patients are evaluated for
persistence of the cells as well as for clinical response
approximately 4 to 6 weeks following administration of the cell
product. Each patient receives only a single course of treatment
and patients are followed monthly with lymphocyte and serum
collections for immunologic studies.
[0286] The safety of the administration of mutation reactive
autologous lymphocytes with knockout of the CISH gene in patients
with refractory metastatic cancer is evaluated. The response rate
of the standard non-myeloablative conditioning regimen mutation
reactive TIL with knockout of CISH gene plus high dose IL-2 using
lymphocytes in patients with metastatic cancer also is determined
as well as the survival and persistence of CISH knockout mutation
reactive lymphocytes in subjects with metastatic gastrointestinal
cancer.
Example 6
CISH Knock Down in a Murine Model
[0287] To explore the role of Cish in CD8.sup.+ T cells, CD8.sup.+
T cells are isolated from wild type (WT) or Cish pmel-1 mice,
stimulated with peptide-pulsed splenocytes from C57BL/6 mice, and
examined for proliferation and cytokine production in vitro. Flow
cytometric analysis after CD8.sup.+ T cell isolation reveals that
the CD8.sup.+ T cell differentiation state remains unaltered in the
steady-state with or without Cish. Enumeration of CD8.sup.+ T cells
one week after in vitro priming reveals significantly more T cells
in the absence of Cish. Evaluation of apoptosis may account for
this increased in vitro T cell expansion. Primed T cells are
TCR-stimulated and stained with the nuclear stain, 7-AAD and
Annexin V, which binds to phosphatidylserine on the cell surface of
pre-apoptotic cells.
Example 7
Melanoma Murine Model of Cish-Deficiency
[0288] The in vivo functional significance of Cish-deficient
CD8.sup.+ T cells can be determined by using a pmel-1 melanoma
model. Melanoma-specific pmel-1 T cells with or without Cish is
adoptively transferred (ACT) into established B16 melanoma-bearing
C57BL/6 hosts and evaluated for tumor growth and in vivo
persistence by IVIS imaging of fluorescently labelled tumor cells
and flow cytometric analysis of transferred engineered cells. The
ACT of Cish-deficient pmel-1 T cells, results in a significant and
durable regression of large, established tumors as compared to WT T
cells. This profound regression also results in improved survival,
with the ACT of Cish.sup.-/- T cells extending the survival of
tumor-bearing mice for greater than 60 days. Serial sampling of
treated mice after the ACT of congenically-marked pmel-1 T cells
reveals a pronounced expansion and delayed contraction of
Cish-deficient T cells compared to T cells obtained from their WT
littermates.
Example 8
Melanoma Murine Model of Cish-Deficiency in Rag1.sup.-/-
[0289] Using tumor-bearing hosts lacking an adaptive immune system
Cish regulated CD8.sup.+ T cell-intrinsic in vivo tumor killing is
evaluated. Sub-therapeutic numbers of pmel-1 T cells with or
without Cish (2.5.times.10.sup.5) are adoptively transferred into
"empty" Rag1.sup.-/- B16 tumor-bearing hosts, with reduced
administration of vaccine (1.times.10.sup.7 PFU) and exogenous IL-2
(2.times.10.sup.4 IU), and then tumor growth is evaluated.
Long-term tumor "maintenance" or regression with the
sub-therapeutic administration of tumor-specific Cish-deficient but
not the WT CD8.sup.+ T cells is anticipated, with no progression of
palpable tumor masses for greater than 50 days. Whether
Cish.sup.-/- tumor-specific T cells maintain their increased
functional avidity in vivo will be determined. To this end,
congenically-marked pmel-1 T cells are ex vivo enriched from
splenocytes with magnetic beads seven days after transfer and
evaluated for IFN-.gamma. release against peptide-pulsed targets. A
significant increase in functional avidity of ex vivo stimulated
Cish.sup.-/- compared to WT T cells can result. This apparent
"maintenance" of enhanced functional avidity can be important when
targeting tumors that typically express low levels of antigen.
Cish.sup.-/- tumor-specific T cells are eliminated even 35 days
after ACT by CD8-depletion, followed by tumor growth analysis to
evaluate long-term tumor specificity.
Example 9
gRNA Safety Analysis
[0290] To assess off-target sites by next-generation deep
sequencing, human peripheral blood-derived T cells were
electroporated with CRISPR/Cas9 reagents targeting PD-1 or CISH,
and genomic DNA was harvested 72-hours post transfection. Each site
was amplified by PCR (250 nucleotides surrounding the predicted
Cas9 cleavage site) from the pool of targeted T cells and subjected
to deep sequencing using an Illumina Hi-Seq machine. This enabled
an incredibly deep analysis of sequence reads from the pool of PCR
products, with on average 450,000 sequence reads available for
detection for each off-target site. Therefore, even off-target
modifications with low abundance are readily detectible using this
method.
[0291] The deep sequencing output of each off-target site from T
cells transfected with the CRISPR/Cas9 reagents were compared to T
cells left untreated to identify sites, which have undergone
CRISPR-mediated modification. For PD-1, only 1 of the 71 candidate
sites displayed evidence of an off target modification frequency
higher than that observed in untreated control cells (see Table 10)
and none of the sites analyzed for CISH showed evidence of a
CRISPR-mediated modification (see Table 11). The off-target
activity uncovered for the PD-1 gRNA showed a disruption frequency
of less than 1% and this site maps to a sequence within the genome,
which does not fall within any known coding genes and therefore is
unlikely to have any influence on T cell function if modified.
Example 10
Identification and Isolation of Reactive TIL
[0292] TILs are isolated from a cancer lesion. ELISPOT assay of 24
individual TIL cultures after co-culture with autologous dendritic
cells transfected with an irrelevant tandem minigene (TMG) RNA, or
the indicated TMG-MCSP construct encoding the 61 mutations
identified by whole-exome and transcriptome sequencing is
performed. IFN-.gamma. production as determined by ELISPOT assay,
and flow cytometric analysis of 4-1BB or CD28 expression on CD8+
and/or CD4+T cells of TIL culture after co-culture with dendritic
cells (DCs) transfected with an irrelevant TMG RNA or TMG-MCSP, or
incubated overnight with the mutated long peptides encoded by
TMG-MCSP.
[0293] Flow cytometry based TCR-V.beta. spectratyping of TIL
culture showing reactivity to MCSP is performed. Magnetic
bead-based enrichment of the anti-MCSP TCR-V.beta. population is
performed using TCR-alpha and beta chain sequences from enriched
TCR-V.beta. population determined by 5' RACE and TCR-PCR.
TABLE-US-00010 TABLE 10 Predicted off-target sites for the PD-1
gRNA analyzed by deep sequencing. DNA sequences and their genomic
locations are indicated along with the number of mismatches between
the genomic site and gRNA. Where sites reside within genes, the
gene names are noted. A blank under mutation frequency means there
was no detectable frequency greater than observed in the untreated
control cells. Off target Mis Genomic Mutation ID sequence matches
location Gene frequency 158 CCTCCTCCTGGTGACCGGAGAGG 3
chr17:77041232- 77041254 159 CTTGCTGGTGGTGACAGAAGAGG 3
chr16:6995450- RBFOX1 6995472 160 ACTGCTTGTTGTGACCGATGGGG 4
chr6:168617770- SMOC2 168617792 161 CCAGCACATGGTGACCAAAGGAG 4
chr6:3146908- BPHL 3146930 162 CCTGCTTGTGGTGACCTAATAAG 3
chr5:144090947- 144090969 163 CCAGGTGGTGGTGAGCGAAGAGG 4
chr18:75064030- ZNF407 75064052 164 CCTAATCGTGGTCACCGAAAGAG 4
chr3:35529500- 35529522 165 CCTGCTGGTGGTGACCTATGAGG 3
chr6:148164881- 148164903 166 CCTGCTACTGGTGTCCGAAGTGG 3
chr4:63174604- 63174626 167 GCAGCTCGAGGTGACGGAAGAGG 4 chrY:7092092-
7092114 168 CATGCTCCTAGTGACAGAAGGGG 4 chr2:174733509- 174733531 169
CCCGCTGGTGGGAACCGAAGGAG 4 chr17:40648598- 40648620 170
CCTGGGCGTAGTGAGCGAAGCGG 4 chr17:79841725- 79841747 171
CCTGGCCGCGGTGACCGAGGCGG 4 chr19:36215042- ZNF146, 36215064 ZNF565
172 CCTGCCCAAGGTTACCGAAGTGG 4 chr2:144324103- GTDC1 144324125 173
CCTGCTCCAGTTGACCAAAGAGG 4 chr15:29622634- 29622656 174
ACTGCACGTGGTGAACCAAGAGG 4 chr19:11426472- CCDC151 11426494 175
GCTGCCCATGGTGACAGAAGGGG 4 chr13:26920698- 0.80% 26920720 176
ACTGCTGGTGGTGAACGAAAGGG 4 chr6:136920831- 136920853 177
GCTGCTAGTGGTGACGGAAAGAG 4 chrX:56147227- 56147249 178
TCTGCTATTGGTGTCCGAAGTGG 4 chr11:23650922- 23650944 179
CGAGCTCGTGGTTACAGAAGGGG 4 chr7:98230892- TECPR1 98230914 180
CCAGCTCGTCGGGACAGAAGAGG 4 chr1:1295498- ACAP3 1295520 181
CCACCTCGGGGTGGCCGAAGCGG 4 chr1:6428267- ESPN 6428289 182
CCAGCTAGTGTTGACTGAAGGGG 4 chr17:45123452- PLCD3 45123474 183
CCTGGCTGTGGTGACAGAAGGGG 4 chrX:7445082- 7445104 184
CCTGCACGTGATGACCTAACAGG 4 chr15:68344743- ITGA11 68344765 185
CCTGCTCGTGCTGTCCGCAGCAG 3 chr17:14206970- COX10 14206992 186
CCTGCTGGTGGTGACGGCAGCAG 3 chr6:40571706- LRFN2 40571728 187
GCTGCTGGTGGTGCCCGATGGGG 4 chr7:135152893- TMEM140 135152915 188
CCTGCTCGTGGTGATATGAGGGG 4 chr20:32138893- TM9SF4 32138915 189
CCAGCTGGTGGTGACTCAAGGAG 4 chr2:227246997- COL4A3 227247019 190
CCTTCTCGTGGAGACAGATGAGG 4 chr16:31360121- ITGAX 31360143 191
CCTTCTCGTGGTGCCTGGAGTGG 4 chr17:75077493- 75077515 192
CCTTCTCATGGTGGCGGAAGGAG 4 chr20:12304659- 12304681 193
CCTTCTCATGGTGACAGTAGAGG 4 chr10:15505067- 15505089 194
CCTGGTCGTGTTGAGTGAAGGGG 4 chr22:44286292- KIAA1644 44286314 195
CCTGGTAGTGGTGACCACAGTGG 4 chr14:93411523- UNC79 93411545 196
CCTGGTGGTGGTGACCATAGAGG 4 chr17:52593937- 52593959 197
CCTGCCCGTGGTAATCGAACCGG 4 chr16:55091717- 55091739 198
CCTGCCCGTGGTGAACGCTGTGG 4 chr1:11838633- CLCN6 11838655 199
CCTGCACGTGGTGACAGCATGGG 4 chr15:29685533- 29685555 200
CCTGCGTGTGGAGGCCGAAGGGG 4 chr20:62333572- LAMA5 62333594 201
CCTGCCGGTGGTGACCTATGTGG 4 chr11:45161912- PRDM11 45161934 202
CCTGCTGGTGCTGCCTGAAGGGG 4 chr10:70197667- 70197689 203
CCTGCTGGTGCTGGCCGATGGGG 4 chr13:28923534- 28923556 204
CCTGCTAGTGGAAACCGCAGGGG 4 chr16:85775103- 85775125 205
CCTGCTGGTGGTACCAGAAGAGG 4 chr16:69413041- 69413063 206
CCTGCTGGTGGTACCAGAAGAGG 4 chr22:38687937- JOSD1 38687959 207
CCTGCTGGTGGTGGCAGCAGTGG 4 chr10:117265710- SLC18A2 117265732 208
CCTGCTAGTGGTGGCAGTAGTGG 4 chr12:125804661- 125804683 209
CCTGCTTATGGGGACTGAAGGGG 4 chr10:103671399- SH3PXD2A 103671421 210
CCTGCTTCTGGTGTCAGAAGTGG 4 chr9:32579566- 32579588 211
CCTGCTGCTGGTGGCCGACGTGG 4 chr11:1017425- MUC6 1017447 212
CCTGCTGTTGGTGACTCAAGGAG 4 chr11:129696156- 129696178 213
CCTGCTGTTGGTGACTCAAGGAG 4 chr9:33720172- 33720194 214
CCTGCTTGGGCTGACCGTAGGGG 4 chr6:31860468- NEU1 31860490 215
CCTGCTAGGGGAGACAGAAGAGG 4 chr19:27868452- 27868474 216
CCTGCTCCTGCTGCACGAAGTGG 4 chr11:7884410- 7884432 217
CCTGCTCTTGGGGACTGAATTGG 4 chrX:89186332- 89186354 218
CCTGCTCCTGGTTCCCAAAGAGG 4 chr4:102987791- SLC9B1 102987813 219
CCTGCTCTTGGTCACTGCAGAGG 4 chr11:1977982- 1978004 220
CCTGCTCCTGGTGCAAGAAGGGG 4 chrX:115722874- 115722896 221
CCTGCTCCTGGTGCAAGAAGGGG 4 chrX:115840863- 115840885 222
CCTGCTCTTGGTGGAAGAAGTGG 4 chr14:56244618- PELI2 56244640 223
CCTGCTCCTGGTGTGGGAAGAGG 4 chr7:157720635- PTPRN2 157720657 224
CCTGCTCTTGGTGAACACAGGAG 4 chr2:74250861- SLC4A5 74250883 225
CCTGCTCAAGGAGACAGAAGTGG 4 chr22:32997494- SYN3 32997516 226
CCTGCTCCAGGTGAATGAAGAGG 4 chr15:95449375- 95449397 227
CCTGCTCTGGGTGACGGAGGGAG 4 chr16:89293161- ANKRD11 89293183 228
CCTGCTCGCGGTGGGCTAAGGGG 4 chr5:10250461- CCT5 10250483
TABLE-US-00011 TABLE 11 predicted off-target sites for the CISH
gRNA analyzed by deep sequencing. DNA sequences and their genomic
locations are indicated along with the number of mismatches between
the genomic site and gRNA. Where sites reside within genes, the
gene names are noted. A blank under mutation frequency means there
was no detecTABLE frequency greater than observed in the untreated
control cells and `TBD` denotes a site that requires follow-up
sequencing. Muta- Off tion target Genomic fre- se- Mis Loca- que-
ID quence matches tion Gene ncy 229 CAGT 3 chr11: TCCA 72372913-
TTAC 72372935 CGCC AGCG GGG 230 GGTT 3 chr2: TCCA 65436495- TTCC
65436517 TGCC AGCG GGG 231 GTGT 4 chr17: CCCG 28521552- TTAC
28521574 GGCC AGCT CGG 232 AGCT 4 chr9: TCCC 134261588- TTAG
134261610 GGCC AGCG AAG 233 GGGG 4 chr1: TCCC 109980531- TAAG
109980553 GGCC AGCG AGG 234 GCGT 4 chr21: CGCA 44899646- CTAC
44899668 GGCC AGCG AGG 235 GCGT 4 chr6: GCCA 44184900- CTAC
44184922 GGCC AGCT AAG 236 GGGT 4 chr19: CCCC 51490678- ATAG
51490700 GGCC AGCG AGG 237 GGCT 4 chr10: CCCA 2405356- TTGC 2405378
AGCC AGCG TGG 238 TTGT 4 chr13: TCCA 69635293- TTAT 69635315 GGCC
AGAG AAG 239 TTGT 4 chr2: TCCA 161519910- TTAT 161519932 GGCC AGAG
AAG 240 GGAT 4 chr20: ACCA 59259210- ATAC 59259232 GGCC AACG AGG
241 GGTT 4 chr15: TCCT 59996211- TTAC 59996233 TGCC GGCG AGG 242
GGGT 4 chr12: GCCA 111198693- GTGG 111198715 GGCC AGCG AGG 243 GGGT
4 chr19: TCCT 50418174- TCAG 50418196 GGCC AGAG AGG 244 GGGC 4
chr6: TCCA 160117643- TCAC 160117665 AGCC AGCA GGG 245 AGGT 4 chr2:
TCCA 68053890- GTTC 68053912 GGCC AGTG AAG 246 GTGT 4 chr13: TCCA
98047590- TTCC 98047612 GGCC CGCC AGG 247 GGCT 4 chr2: TCCA
85513577- ATAA 85513599 GGCC AACG AGG 248 GGGT 4 chr15: TCCA
30985934- GTGT 30985956 GGCC AGCA AGG 249 GGGT 4 chr3: RAP2B TCCT
153163818- TTTC 153163840 TGCC AGCT TGG 250 GGGT 4 chr19: TBD TCCT
56218323- TTTC 56218345 TGCC AGCT TGG 251 GGGT 4 chr19: TCCT
56233892- TTTC 56233914 TGCC AGCT TGG 252 GGGT 4 chr1: TCCT
23956834- TTAG 23956856 GGCC AGGA TGG 253 GGGT 4 chr4: TCTA
25743884- TTCC 25743906 AGCC AGCT TGG 254 GGGT 4 chrX: TTCA
102018328- GTAA 102018350 GGCC AGGG TGG 255 GGGC 4 chr12: CCCA
130872701- TTAC 130872723 AGCC ATCG TGG 256 GGGG 4 chr8: TCCA
62633692- TGAC 62633714 AGAC AGCG TGG 257 GGGG 4 chr7: TCCA
2382393- TTCC 2382415 CGCC AGAG TGG 258 GGGT 4 chr22: TACA
20208799- TCAA 20208821 GGCC ACCG AGG 259 GGGT 4 chr8: NDRG1 TCAA
133296918- TTAC 133296940 GGAA AGGG AGG 260 GGGT 4 chr14: DHRS4LI
TBD TCCA 24036459- CTCC 24036481 TTCC AGCG AGG 261 GGGT 4 chr3:
TCCA 120731615- TTAT 120731637 GACA AGCA TGG
262 GGGT 4 chr16: TCCA 73686902- TTCT 73686924 GGCA AGGG TGG 263
GGGT 4 chr1: TCCA 67494513- TTGC 67494535 TGGC AGAG TGG 264 GGGT 4
chr1: TCCA 92108114- TGAA 92108136 GGCC AAAG GGG 265 GGGT 4 chr11:
TCCA 3217536- ATCC 3217558 GGCC ACTG TGG 266 GGGT 4 chr1: TCCA
159529114- CTAC 159529136 TGCC TGCT TGG 267 GGGT 4 chr19: TCCG
32230844- TTCC 32230866 GGCC ACTG GGG 268 GGGT 4 chr16: TTCA
66480442- TTGG 66480464 AGCC AGCG TGG 269 GGGT 4 chr16: TTCA
8173059- TTAC 8173081 GCTC AGCT AAG 270 GGGT 4 chr1: TACC
121422833- TTAC 121422855 GACC TGCG TGG 271 GGGC 4 chr16: TCCA
55487566- TTCC 55487588 GGCC ATGG TGG
Example 10
A Phase I/II Trial in Patients with Metastatic Cancer of the
Adoptive Transfer of Tumor-Infiltrating Lymphocytes in which the
Gene Encoding CISH was Inactivated Using the CRISPR/Cas 9
System
Objectives:
[0294] Primary objectives are 1) to determine the safety of the
administration of mutation reactive autologous lymphocytes with
knockout of the CISH gene in patients with refractory metastatic
cancer; and 2) to determine the response rate of the standard
non-myeloablative conditioning regimen plus administration of
mutation reactive TIL with knockout of the CISH gene plus high dose
IL-2 in patients with metastatic cancer. A secondary objective is
to evaluate the survival and persistence of CISH knockout
mutation-reactive lymphocytes in patients with metastatic
cancer.
Eligibility
[0295] Subjects with an age greater than or equal to 18 and less
than or equal to 70 years are eligible. Subjects have evaluable
metastatic cancer refractory to standard chemotherapy, metastatic
cancer lesions suitable for surgical resection for the preparation
of TIL, no contraindications to high-dose aldesleukin
administration, and no concurrent major medical illnesses or any
form of immunodeficiency
Inclusion Criteria
[0296] Inclusion criteria include measurable cancer melanoma with
at least one lesion that is resectable for TIL generation, plus one
other lesion that can be measured and confirmation of diagnosis of
metastatic cancer. Patients with three or fewer brain metastases
that are less than 1 cm in diameter and that are asymptomatic are
eligible. Lesions that have been treated with stereotactic
radiosurgery must be clinically stable for 1 month after treatment
for the patient to be eligible. Patients with surgically resected
brain metastases are eligible. Progressive disease following at
least one first line standard therapy: six weeks must have elapsed
from the time of any of these prior antibody therapies that could
effect an anti-cancer immune response, at the time the patient
receives the preparative regimen to allow antibody levels to
decline. Patients who have previously received ipilimumab and have
documented GI toxicity must have a normal colonoscopy with normal
colonic biopsies. Patients are greater than or equal to 18 years of
age and less than or equal to 70 years of age, and have a clinical
performance status of ECOG 0 or 1. Patients have a life expectancy
of greater than three months. Patients of both genders must be
willing to practice birth control from the time of enrollment on
this study and for up to four months after treatment.
Serology
[0297] Patients are seronegative for HIV antibody. Patients who are
HIV seropositive can have decreased immune-competence and thus are
less responsive to the experimental treatment and more susceptible
to its toxicities. Patients are seronegative for hepatitis B
antigen, and seronegative for hepatitis C antibody. If hepatitis C
antibody test is positive, then patient must be tested for the
presence of antigen by RT-PCR and be HCV RNA negative. Women of
child-bearing potential must have a negative pregnancy test.
Hematology
[0298] Absolute neutrophil count greater than 1000/mm.sup.3 without
the support of filgrastim, WBC.gtoreq.3000/mm.sup.3, Platelet
count.gtoreq.100,000/mm.sup.3, Hemoglobin>8.0 g/dl
Chemistry
[0299] Patients have serum ALT/AST .ltoreq. to 2.5 times the upper
limit of normal, Serum Creatinine .ltoreq. to 1.6 mg/dl, and total
bilirubin .ltoreq. to 1.5 mg/dl, except in patients with Gilbert's
Syndrome who must have a total bilirubin less than 3.0 mg/dl. More
than four weeks have elapsed since any prior systemic therapy at
the time the patient receives the preparative regimen, and
patients' toxicities have recovered to a grade 1 or less (except
for toxicities such as alopecia or vitiligo). Patients have
progressive disease after prior treatment. Patients may have
undergone minor surgical procedures within the past 3 weeks, as
long as all toxicities have recovered to grade 1 or less. Six weeks
have elapsed from the time of any antibody therapy that could
effect an anti-cancer immune response, including anti-CTLA4
antibody therapy, at the time the patient receives the preparative
regimen to allow antibody levels to decline.
Exclusion Criteria
[0300] Excluded are women of child-bearing potential who are
pregnant or breastfeeding, or have any form of primary
immunodeficiency (such as Severe Combined Immunodeficiency
Disease). Excluded are patients with concurrent opportunistic
infections. Excluded are patients with active systemic infections
(e.g., requiring anti-infective treatment), coagulation disorders
or other active major medical illnesses of the cardiovascular,
respiratory or immune system, as evidenced by a positive stress
thallium or comparable test, myocardial infarction, cardiac
arrhythmias, obstructive or restrictive pulmonary disease. Excluded
are patients on concurrent systemic steroid therapy, with a history
of severe immediate hypersensitivity reaction to cyclophosphamide
or fludarabine, or a history of coronary revascularization or
ischemic symptoms. Patients have a documented LVEF of less than or
equal to 45%; testing is required in patients with: age >65
years old, clinically significant atrial and or ventricular
arrhythmias including but not limited to: atrial fibrillation,
ventricular tachycardia, second or third degree heart block.
Screening Evaluation
Within 4 Weeks Prior to Starting the Chemotherapy Regimen:
[0301] Complete history and physical examination, including weight,
ECOG, and vital signs, and eye exam noting in detail the exact size
and location of any lesions that exist is taken. (Note: patient
history may be obtained within 8 weeks), Chest x-ray, EKG, Baseline
CT of the chest, abdomen and pelvis, and brain MRI to evaluate the
status of disease. Additional scans and x-rays are performed if
clinically indicated based on patients' signs and symptoms.
Pulmonary Function Testing for patients with a prolonged history of
cigarette smoking (20 pack/year of smoking within the past 2 years)
or symptoms of respiratory dysfunction. (Note: are performed within
8 weeks of treatments). Cardiac Evaluation (stress thallium,
echocardiogram, MUGA, etc.) for patients who are greater than or
equal to age 60, or who have a history of ischemic heart disease,
chest pain, or clinically significant atrial and/or ventricular
arrhythmias including but not limited to: atrial fibrillation,
ventricular tachycardia, heart block. Patients with a LVEF of less
than or equal to 45% will not be eligible. Patients under the age
of 60 who present with cardiac risk factors may undergo cardiac
evaluation as noted above (e.g., diabetes, hypertension, and
obesity.) (Note: may be performed within 8 weeks of treatment). HIV
antibody titer and HbsAG determination, and anti HCV (may be
performed within 3 months of chemotherapy start date.) Anti CMV
antibody titer, HSV serology, and EBV panel. (Note, may be
performed within 3 months of chemotherapy start date; patients who
are known to be positive for any of the above do not need to be
retested.)
Within 14 Days Prior to Starting the Chemotherapy Regimen:
[0302] Baseline blood tests, Chem 20: (Sodium (Na), Potassium (K),
Chloride (Cl), Total CO.sup.2 (bicarbonate), Creatinine, Glucose,
Urea nitrogen (BUN), Albumin, Calcium total, Magnesiumtotal (Mg),
Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, AST/GOT, Total
Bilirubin, Direct Bilirubin, LD, Total Protein, Total CK, Uric
Acid), Thyroid Panel, CBC with differential and platelet count,
PT/PTT, Urinalysis and culture, if indicated
Within 7 Days Prior to Starting the Chemotherapy Regimen:
[0303] .beta.-HCG pregnancy test (serum or urine) on all women of
child-bearing potential, ECOG performance status of 0 or 1
Study Design
[0304] Patients with evaluable metastatic cancer undergo resection
of tumor. Lymphocytes from the tumor (TIL) are grown and expanded
according to the procedure shown in FIG. 42. Multiple individual
fragments or multiple individual cultures of tumor infiltrating
lymphocytes are grown. Individual cultures are separately expanded
and when a sufficient yield of TIL (approximately 10.sup.8 cells)
is expanded from each culture, the TIL are cryopreserved and
aliquots taken for immunologic testing. An aliquot of the original
tumor is subjected to exomic and, when possible, transcriptome
sequencing to identify mutations uniquely present in the cancer
compared to normal cells. Aliquots of each cultured TIL preparation
is tested for reactivity against these mutations using the
approaches previously described. In brief, the patient's antigen
presenting cells are pulsed with 25mer peptides including the
mutated peptide in the center flanked by 12 normal amino acids.
Alternatively, in vitro transcribed RNA encoding all mutations are
electroporated into the patient's antigen presenting cells. Using
either or both of these techniques the previously cryopreserved TIL
cultures are tested for reactivity against individual cancer
mutations are subjected to CRISPR knockout of the CISH gene. Up to
five different cultures are selected for this procedure from each
patient.
[0305] Cultures with demonstrated mutation reactivity are sent to a
GMP Cell Production Facility where CRISPR technology is used to
knockout the CISH gene under cGMP conditions. Knockout of the CISH
gene is determined by DNA sequencing based tracking of indels by
decomposition (TIDE) analysis. Cell populations with successful
knockout of the CISH gene are then rapidly expanded utilizing OKT3
and feeder cells. Cryopreserved lymphocytes are used in patient
treatment.
[0306] In the first part of this protocol a dose escalation is
initiated utilizing one patient per group starting at 10.sup.9 CISH
knockout cells per patient. Individual patients are treated at half
log increments and if any toxicity higher than Grade 2 is seen,
that group is expanded to additional patients. Thus the following
doses are utilized: 10.sup.9 cells, 3.times.10.sup.9 cells,
10.sup.10 cells, 3.times.10.sup.10 cells, and 3 to
10.times.10.sup.10 cells.
[0307] Patients receive a lymphodepleting preparative regimen
followed by infusion of the cells and high dose aldesleukin.
Patients are evaluated for persistence of the cells as well as for
clinical response approximately 4-6 weeks following administration
of the cell product. Each patient receives only a single course of
treatment and patients are followed monthly with lymphocyte and
serum collections for immunologic studies
Drug Administration
[0308] Preparative Regimen with Cyclophosphamide and
Fludarabine:
[0309] Times are offered as examples and may be changed as long as
a similar time relationship between administrations of the drugs is
maintained Study medication start times for drugs given once daily
should be given within 2 hours of the scheduled time. All other
medications should be given +/- one hour of the scheduled time; the
length of administration is all +/-15 minutes. Administration of
diuretics, electrolyte replacement, and hydration and monitoring of
electrolytes should all be performed as clinically indicated--the
doses and times noted below are offered only as examples. Infusions
may be slowed or delayed as medically indicated.
DAYS-7 and -6
[0310] 6 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). At any time
during the preparative regimen, if urine output <1.5 ml/kg/hr or
if body weight >2 kg over pre- cyclophosphamide value,
furosemide 10-20 mg IV can be administered. Serum potassium should
be monitored and treated as indicated following administration of
furosemide.
[0311] 4 PM: 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) is given for nausea.
[0312] 5 PM: 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. If patient
is obese (BMI>35) drug dosage is calculated using practical
weight.
[0313] 10 pm: Begin mesna infusion at 3 mg/kg/hour intravenously
diluted in a suitable diluent (see pharmaceutical section) over 23
hours after each cyclophosphamide dose. If patient is obese
(BMI>35) drug dosage is calculated using practical weight.
DAYS -7 to -3
[0314] Fludarabine 25 mg/m.sup.2/day IVPB daily over 30 minutes for
5 days. If patient is obese (BMI>35) drug dosage is calculated
using practical weight as described in Appendix 2. (The fludarabine
is started approximately 1-2 hours after the cyclophosphamide and
mesna on Days -7 and -6). Day 0 (two to four days after the last
dose of fludarabine):
[0315] Autologous TIL infusion is administered intravenously over
20 to 30 minutes via non-filtered tubing, gently agitating the bag
during infusion to prevent cell clumping.
[0316] Aldesleukin as described below. Day 1-4 (Day 0 is the day of
cell infusion):
[0317] Aldesleukin as described below. Beginning on day 1 or 2,
filgrastim may be administered subcutaneously at a dose of 5
mcg/kg/day (not to exceed 300 mcg/day). Filgrastim administration
continues daily until neutrophil count>1.0
.times.10.sup.9/L.times.3 days or >5.0 .times.10.sup.9/L.
TABLE-US-00012 TABLE 12 Treatment Timeline and administered
medicaments Day -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 Therapy
Cyclophosphamide (60 mg/kg) X X Fludarabine (25 mg/m.sup.2) X X X X
X TIL X.sup.1 Aldesleukin X.sup.2 X X X X Filgrastim.sup.3 (5
mcg/kg/day) X X X X TMP/SMX.sup.4 X X X X X X X X X X X X 160
mg/800 mg (example) Fluconazole.sup.5 (400 mg po) X X X X X
Valacyclovir po or Acyclovir IV.sup.6 X X X X X .sup.1Two to four
days after the last dose of fludarabine .sup.2Initiate within 24
hours after cell infusion .sup.3Continue until neutrophils count
> 1 .times. 10.sup.9/L for 3 consecutive days or > 5 .times.
10.sup.9/L. .sup.4The 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.5Continue
until ANC > 1000/mm.sup.36 In patients positive for HSV continue
until CD4 > 200 .times. 2
Aldesleukin: Intravenous Administration
[0318] Aldesleukin is administered at a dose of 720,000 IU/kg
(based on total body weight) as an intravenous bolus over a 15
minute period beginning within 24 hours of cell infusion and
continuing for up to 5 days (maximum 15 doses). Doses are
preferentially administered every eight hours; however, up to 24
hours may elapse between doses depending on patient tolerance.
Aldesleukin dosing is stopped if toxicities are not sufficiently
recovered with supportive measures within 24 hours of the last dose
of aldesleukin. Doses are delayed or stopped if patients reach
Grade 3 or 4 toxicity due to aldesleukin except for the reversible
Grade 3 toxicities common to aldesleukin such as diarrhea, nausea,
vomiting, hypotension, skin changes, anorexia, mucositis,
dysphagia, or constitutional symptoms and laboratory changes as
detailed in Appendix 3. Toxicities are managed. If these toxicities
can be easily reversed within 24 hours by supportive measures then
additional doses are given.
Prior to Starting the Preparative Regimen
[0319] Apheresis as indicated. Within 14 days prior to starting the
preparative regimen, patients have a complete blood count,
electrolytes, BUN, creatinine, liver function tests, TBNK, and
serum chemistries performed. If any results are beyond the criteria
established for eligibility, the patient does not proceed until the
abnormalities can be resolved.
During the Preparative Regimen: DAILY
[0320] Complete Blood Count, Chem 20 equivalent: Sodium (Na),
Potassium (K), Chloride (Cl), Total CO2 (bicarbonate), Creatinine,
Glucose, Urea nitrogen (BUN), Albumin, Calcium total, Magnesium
total (Mg), Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT,
AST/GOT, Total Bilirubin, Direct Bilirubin, LD, Total Protein,
Total CK, Uric Acid, Urinalysis.
After Cell Infusion
[0321] Vital signs are monitored hourly (+/-15 minutes) for four
hours and then routinely (every 4-6 hours) unless otherwise
clinically indicated. Once total lymphocyte count is greater than
200/mm.sup.3, TBNK for peripheral blood CD4 count is drawn weekly
(while the patient is hospitalized).
During Hospitalization Every 1-2 Days
[0322] A review of systems and physical exam as clinically
indicated, CBC, Chem 20 equivalent: Sodium (Na), Potassium (K),
Chloride (Cl), Total CO2 (bicarbonate), Creatinine, Glucose, Urea
nitrogen (BUN), Albumin, Calcium total, Magnesium total (Mg),
Inorganic Phosphorus, Alkaline Phosphatase, ALT/GPT, AST/GOT, Total
Bilirubin, Direct Bilirubin, LD, Total Protein, Total CK, Uric
Acid, Other tests are performed as clinically indicated.
Post Study Evaluation (Follow-Up)
[0323] All patients return to the hospital for evaluation 6 weeks
(+/-2 weeks) following administration of the cell product. At each
scheduled evaluation patients undergo: Physical examination, Chem
20 equivalent: Sodium (Na), Potassium (K), Chloride (Cl), Total CO2
(bicarbonate), Creatinine, Glucose, Urea nitrogen (BUN), Albumin,
Calcium total, Magnesium total (Mg), Inorganic Phosphorus, Alkaline
Phosphatase, ALT/GPT, AST/GOT, Total Bilirubin, Direct Bilirubin,
LD, Total Protein, Total CK, Uric Acid, Complete blood count,
Thyroid panel as clinically indicated, TBNK, until CD4
>200.times.2, Toxicity assessment, including a review of
systems. CT of the chest, abdomen and pelvis as clinically
indicated. If clinically indicated, other scans or x-rays may be
performed, e.g. brain MRI, bone scan. A 5 liter apheresis may be
performed at the first follow up visit, if the patient is unable to
undergo apheresis, approximately 96 ml of blood may be obtained.
Subsequently, approximately 60 ml of blood is obtained at follow up
visits for at least 3 months. Peripheral blood mononuclear cells
are cryopreserved so that immunologic testing may be performed.
Infection Prophylaxis
Pneumocystis Jirovecii Pneumonia
[0324] All patients receive the fixed combination of trimethoprim
and sulfamethoxazole [SMX] as double strength (DS) tab (DS tabs=TMP
160 mg/tab, and SMX 800 mg/tab) P.O. daily three times a week on
non-consecutive days, beginning between days -5 and -8. Pentamidine
is substituted for TMP/SMX-DS in patients with sulfa allergies. It
is administered aerosolized at 300 mg per nebulizer within one week
of chemotherapy start date.
Herpes Virus Prophylaxis
[0325] Patients with positive HSV serology are given valacyclovir
orally at a dose of 500 mg daily the day after chemotherapy ends,
or acyclovir, 250 mg/m.sup.2 IV q 12 hrs if the patient is not able
to take medication by. Reversible renal insufficiency has been
reported with IV but not oral acyclovir. Neurologic toxicity
including delirium, tremors, coma, acute psychiatric disturbances,
and abnormal EEGs have been reported with higher doses of
acyclovir. Should this occur, a dosage adjustment is made or the
drug is discontinued. Acyclovir is not used concomitantly with
other nucleoside analogs which interfere with DNA synthesis, e.g.
ganciclovir. In renal disease, the dose is adjusted as per product
labeling. Prophylaxis for Pneumocystis and Herpes will continue for
6 months post chemotherapy. If the CD4 count is less than 200 at 6
months post chemotherapy, prophylaxis will continue until the CD4
count is greater than 200 for 2 consecutive measures.
Fungal Prophylaxis (Fluconazole)
[0326] Patients start Fluconazole 400 mg p.o. the day after
chemotherapy concludes and continue until the absolute neutrophil
count is greater than 1000/mm.sup.3 The drug may be given IV at a
dose of 400 mg in 0.9% sodium chloride USP daily in patients unable
to take it orally.
Empiric Antibiotics
[0327] Patients start on broad-spectrum antibiotics, either a
3.sup.rd or 4.sup.th generation cephalosporin or a quinolone for
fever of 38.3.degree. C. once or two temperatures of 38.0.degree.
C. or above at least one hour apart, and an ANC<500/mm.sup.3
Infectious disease consultation is obtained for all patients with
unexplained fever or any infectious complications.
Blood Product Support
[0328] Using daily CBC's as a guide, the patient receives platelets
and packed red blood cells (PRBC's) as needed. All blood products
are irradiated. Leukocyte filters are utilized for all blood and
platelet transfusions to decrease sensitization to transfused WBC's
and decrease the risk of CMV infection.
Other Concomitant Medications to Control Side Effects
[0329] Concomitant medications to control side effects of therapy
may be given. Meperidine (25-50 mg) is given intravenously if
severe chilling develops. Other supportive therapy is given as
required and may include acetaminophen (650 mg q4h), indomethacin
(50-75 mg q6h) and ranitidine (150 mg g12h). If patients require
steroid therapy they are taken off treatment. Patients who require
transfusions will receive irradiated blood products. Ondansetron
0.15 mg/kg/dose IV every 8 hours is administered for nausea and
vomiting. Additional antiemetics are administered as needed for
nausea and vomiting uncontrolled by ondansetron. Antibiotic
coverage for central venous catheters may be provided at the
discretion of the investigator.
Tumor Biopsies
[0330] Biopsies of tumor tissue or lymph node may be performed but
are not required during the course of therapy. Studies may evaluate
the antigen expression by the tumor and to evaluate the reactivity
of lymphocytes grown from these biopsies. Biopsies may be performed
at baseline, after the course of therapy, and in the event of
response. These biopsies are only be performed if minimal morbidity
is expected based on the procedure performed and the granulocyte
and platelet count. Biopsies are only be allowed if the biopsy does
not require more than 1 day in the hospital
Immunological Testing:
[0331] Apheresis may be performed prior to and 6 weeks (+/-2 weeks)
following the administration of the cell product. At other time
points, patient peripheral blood lymphocytes (PBL) are obtained
from whole blood by purification using centrifugation on a Ficoll
cushion. Aliquots of these PBMC are cryopreserved for immunological
monitoring of cell function. A variety of tests including
evaluation of specific lysis and cytokine release, metabolomic and
bioenergetic studies (using Seahorse), intracellular FACS of
cytokine production, ELISA-spot assays, and lymphocyte subset
analysis may be used to evaluate the immunological correlates of
treatment. In general, differences of 2 to 3 fold in these assays
are indicative of true biologic differences. Samples of all infused
cell products are cryopreserved, and extensive retrospective
analysis of infused cell phenotype and function are performed to
attempt to find in vitro characteristics of the infused cells which
correlate with in vivo antitumor activity. Analyses of TIL samples
will include evaluation of the activity, specificity, and telomere
length of the infused TIL. Blood and tissue specimens collected in
the course of this research project may be banked and used in the
future to investigate new scientific questions.
Aldesleukin (Interleukin-2, Proleukin, Recombinant Human
Interleukin 2)
[0332] Formulation/Reconstitution: Aldesleukin is provided as
single-use vials containing 22 million IU (1.3 mg) IL-2 as a
sterile, white to off-white lyophilized cake plus 50 mg mannitol
and 0.18 mg sodium dodecyl sulfate, buffered with approximately
0.17 mg monobasic and 0.89 mg dibasic sodium phosphate to a pH of
7.5 (range 7.2 to 7.8). The vial is reconstituted with 1.2 mL of
Sterile Water for Injection, USP, and the resultant concentration
is 18 million IU/ml or 1.1 mg/mL. Diluent should be directed
against the side of the vial to avoid excess foaming. Swirl
contents gently until completely dissolved. Do not shake. Since
vials contain no preservative, reconstituted solution should be
used with 24 hours.
[0333] Dilution/Stability: Reconstituted aldesleukin should be
further diluted with 50 mL of 5% Human Serum Albumin (HSA). The HSA
is added to the diluent prior to the addition of RIL-2. Dilutions
of the reconstituted solution over a 1000-fold range (i.e., 1 mg/mL
to 1 mcg/mL) are acceptable in either glass bottles or polyvinyl
chloride bags. Aldesleukin is chemically stable for 48 hours at
refrigerated and room temperatures, 2.degree.-30.degree. C.
[0334] Administration: The dosage is calculated based on total body
weight. The final dilution of aldesleukin is infused over 15
minutes. Aldesleukin is administered as an inpatient.
Fludarabine
[0335] Administration: Fludarabine is administered as an IV
infusion in 100 ml 0.9% sodium chloride, USP over 15 to 30 minutes.
The doses are based on body surface area (BSA). If patient is obese
(BMI>35) drug dosage is calculated using practical weight.
Cyclophosphamide
[0336] Administration: cyclophosphamide is diluted in 250 ml D5W
and infused over one hour. The dose is based on the patient's body
weight. If patient is obese (BMI>35) drug dosage is calculated
using practical weight
Mesna (Sodium 2-mercaptoethanesulfonate, Mesnum, Mesnex,
NSC-113891)
[0337] Administration: Dilute to concentrations less than or equal
to 20 mg mesna/ml fluid in D5W or 0.9% NaCl and to be administered
intravenously as a continuous infusion. If patient is obese
(BMI>35) drug dosage is calculated using practical weight as
described in Appendix 2. Toxicities include nausea, vomiting and
diarrhea.
Filgrastim (Granulocyte Colony-Stimulating Factor, G-CSF,
Filgrastim, Neupogen)
[0338] Filgrastim is obtained commercially and is supplied in 300
ug/ml and 480 ug/1.6 ml vials. Filgrastim should be refrigerated
and not allowed. Filgrastim is given as a daily subcutaneous
injection.
Example 14
GMP Process Validation
[0339] A consideration for clinical translation of a CRISPR/Cas9
system is the capacity to scale up production of knockout TILs to
the yields achieved by standard rapid expansion protocols (REP).
Utilizing a modified REP strategy, following electroporation of
CRISPR/Cas9 reagents an average fold expansion of 1071 across 3
independent cGMP process validation runs was achieved (Table 13 and
FIG. 43A, FIG. 43B, FIG. 43C, and FIG. 44). As the capacity to
initiate multiple REP simultaneously is now possible, this system
will consistently produce cell yields in excess of
1.times.10.sup.10 knockout TIL for patient infusion.
Cyropreservation is also achievable, Table 14.
TABLE-US-00013 TABLE 13 Cell yields achieved during cGMP process
validation #modified (Starting #Post- Post- Post- # #Control- KO-
#Post- thaw #Post- rest #post- stim for D7 KO-D7 Control- D14 Expt
#/vi al thaw Viability rest Viability stim Viability REP) (FoldX)
(FoldX) D14(FoldX) (FoldX) PV1 2e7 1.16e7 69% 6.81e6 52% 1.01e6 99%
KO: 4e5 1.21e8 5.7e7 1.09e9 4.48e8 CON: (269) (130) (2620) (1028)
4e5 PV2 4e7 3.7e7 74% 2.68e7 85% 3.97e7 97% KO: 5e6 2.9e7 1.9e8
4.5e8 3.5e9 CON: (59) (38) (900) (701) 5e5 PV3 4e7 4.4e7 87% 4.4e7
87% 2.4e7 97% KO: 5e6 4.7e7 2.64e8 7.53e8 7.42e9 CON: (93) (53)
(1507) (1484) 5e5
TABLE-US-00014 TABLE 14 Cyropreservation achieved during cGMP
process validation Freeze #/140 ml % cell recovery 4 hours %Viable
4 hours density bag post thaw post thaw 7.5e7/ml 1.05e10 89% 82%
1.5e8/ml 2.1e10 90% 81%
Example 11
Detection of Genomic Disruption at the Protein Level
[0340] To determine whether observed knockout frequencies at the
genetic level correlate with loss of protein, the expression of
PD-1 and CISH protein after CRISPR/Cas9 knockout was assessed.
Peripheral blood (PB) T-cells and TILs were re-stimulated at day 14
post-electroporation using plate bound anti-CD3 and soluble
anti-CD28 antibody and assessed the loss of PD-1 and CISH protein
by flow cytometry and western blot, respectively. As shown in FIG.
48, PD-1 protein is lost in >90% of PB T-cells and TILs at day
14. These data confirm that knockout cells have lost target protein
expression after modification, and importantly, demonstrate that
knockout cells are retained, expand after modification, and are
viable, FIG. 46.
[0341] The same strategy was employed to determine the degree of
CISH knockout at the protein level. CRISPR/Cas9 modified PB T-cells
and TILs were expanded for 14 days and then re-stimulated for 48
hrs. Cells were subsequently collected and extracts analyzed by
western blot as CISH is an intracellular protein. Consistent with
the high rate of knockout by TiDE analysis of genomic modification,
CISH protein was essentially absent in knockout PB T-cells and TILs
(FIG. 45). These data demonstrate that this strategy for
CRISPR/Cas9 based gene disruption in primary human T-cells and TILs
results in targeted gene knockout with high efficiency.
Example 12
Simplified Presentation of Incredibly Complex Evaluations (SPICE)
Analysis
[0342] In addition to the maintenance of high cell viability and
proliferation after CRISPR/Cas9 editing, whether knockout T-cells
retain effector function upon activation was also sought to be
determined. Using intracellular staining and flow cytometry coupled
with Simplified Presentation of Incredibly Complex Evaluations
(SPICE) analysis, knockout T-cells were demonstrated to maintain
cytokine poly-functionality upon re-stimulation. This indicated
that the effector function of PB T-cells and TILs following
CRISPR/Cas9-mediated gene editing is preserved (FIG. 47A and FIG.
47B).
Example 13
Growth Kinetics of Good Manufacturing Practices TIL with and
without IL-2
[0343] Tumor infiltrating lymphocytes used in good manufacturing
practices runs were selected as oligoclonal subpopulations
possessing specific reactivity against patient-specific cancer
neoantigens using tumor whole-exome sequencing and tendem
mini-gene/synthetic long peptide screening. In long-term cultures,
cell expansion was observed in the presence of IL-2 (FIG. 50A).
Growth experiments in the absence of IL-2 at day 7 after IL-2
withdrawal, FIG. 50B. Without IL-2, cell number and viability
decreased and by day 7 there were only a few viable cells remaining
(FIG. 50B).
Example 14
Off-Target Frequency Detection by GUIDE-Seq
[0344] To assess off-target frequencies by GUIDE-Seq,
3.times.10.sup.6 human PBL derived T cells were electroporated
using the Neon Transfection system, 100 .mu.L tip, with 8 pmol or
16 pmol double stranded oligonucleotide (dsODN)
(5'GTTTAATTGAGTTGTCATATGTTAATAACGGTAT-3') in the presence or
absence of 15 .mu.g Cas9 mRNA and 10 .mu.g of CISH gRNA, FIG. 52.
Genomic DNA was harvested 72 hours after transfection and the
integration frequency of the dsODN at the CISH target site was
assessed by PCR and Sanger sequencing followed by TiDE analysis.
Genomic DNA was sheared to an average length of 500 bp using
Covartis S2 sonicator (Covartis). The sheared DNA was purified
using Agencourt AMPure XP bead purification kit (Beckman Coulter)
before conversion of any cohesive DNA ends to blunt
5'phosphorylated ends using the Anza DNA End Repair Kit (Thermo
Fisher). Barcoded sequencing adapters were then ligated to the
genomic fragments using PCR to generate the final libraries ready
for NGS analysis.
Example 15
Cytogenetic Analysis of CISH Disrupted TIL
[0345] Cytogenetic analysis was performed on CISH knockout mutation
reactive TIL produced during GMP manufacturing process
qualification runs. TIL were stimulated using anti-CD3 and
anti-CD28 antibodies followed by electroporation-based delivery of
Cas9 mRNA (15 .mu.g) and CISH gRNA (10 .mu.g) using the Neon
electroporation system. After electroporation, CISH disrupted
mutation reactive TIL underwent a REP in the presence of irradiated
feeders for 14 days, at which time a sample was analyzed for
cytogenetics. Cytogenetic analysis was performed on 5 samples: PQ1
CISH KO mutation reactive TIL, PQ2 CISH KO mutation reactive TIL,
PQ3 pre-treatment, PQ3 CISH KO mutation reactive TIL, and PQ3
control.
[0346] A 13.0 mL suspension culture of TIL underwent a 2.0 hr.
colcemid treatment, cells were harvested and 20 metaphases were
completely analyzed by G-banding at a 400 band level resolution.
Additional 30 metaphases were screened for a translocation between
chromosomes 4 and 9. Cytogenomics results indicated that:
[0347] PQ 3 Control: 46, XX and 46, XX. Findings indicate a normal
female karyotype. No translocations were observed.
TABLE-US-00015 TABLE 15 PQ3 Cytogenetics Summary Number of
Metaphase cells Analyzed 20 Number of Metaphase cells Screened 30
Number of Metaphase cells Karyotyped 2
[0348] PQ 3 Pretreatment: 46, XX and 46, XX, t (4; 9) (q31.3; q13)
[9]/46, XX. 9 of the 21 metaphase cells analyzed had a reciprogral
translocation between the long arm of a chromosome 4 at band 4q31.3
and the long arm of a chromosome 9 at band 9q13. Each of the
remaining 12 metaphases had a 46, XX karyotype with two normal #4
and #9 chromosomes.
TABLE-US-00016 TABLE 16 PQ3 Pretreatment Summary Number of
Metaphase cells Analyzed 21 Number of Metaphase cells Screened 0
Number of Metaphase cells Karyotyped 4
[0349] PQ1 Mutation reactive TIL: 46, XX. 45, X,-X,
add(1)(p13),der(14)t(1;14)(p13;p11.1),+21,i(21)(q10). 45, X,-X, add
(1) (p13), -7, +11, der (14) t (1; 14) (p13; p11.1). 43, X, dic(x;
12) (q21; p13), -17, -19, der (21; 22) (q10; q10), +22. 46, X,
Del(x) (q21). 45,XX,-7, -18, +r/46, XX,del(8)(p12), +14,
i(14)(q10). Six of the 23 metaphase cells analyzed had numerical
and or structural chromosomal abnormalities. All except one of
these cells had three or more abnormalities that preferentially
involved the X chromosome (4 cells) and rearrangements of the short
arms of the acrocentric chromosomes (13, 14, 15, 21, 22). Only two
of these six cells shared the same abnormalities, meeting the
criteria for the clone.
TABLE-US-00017 TABLE 17 PQ1 mutation reactive TIL Number of
Metaphase cells Analyzed 23 Number of Metaphase cells Screened 0
Number of Metaphase cells Karyotyped 4
[0350] PQ2 Mutation reactive TIL: 46, XX. 45, XX der (15; 22) (q10;
q10). One of the 50 metaphase cells analyzed had a translocation
between chromosome 15 and a chromosome 22.
TABLE-US-00018 TABLE 18 PQ2 Mutation reactive TIL Number of
Metaphase cells Analyzed 20 Number of Metaphase cells Screened 30
Number of Metaphase cells Karyotyped 2
[0351] PQ 3 Mutation Reactive TIL: 46, XX. 46, XX, t (4; 9) (q31.3;
q13). 46, XX, Del (7) (p11.2). Eight of the 20 metaphase cells
analyzed had a reciprocal translocation between the long arm of a
chromosome 4 at band 4q31.3 and the long arm of a chromosome 9 at
band 9q13. One additional cell had a deletion of the short arm of
chromosome 7 that was interpreted as a nonclonal event, likely
representing a random chromosomal break.
TABLE-US-00019 TABLE 19 PQ 3 Mutation Reactive TIL Number of
Metaphase cells Analyzed 20 Number of Metaphase cells Screened 0
Number of Metaphase cells Karyotyped 4
Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID
NOS: 293 <210> SEQ ID NO 1 <400> SEQUENCE: 1 000
<210> SEQ ID NO 2 <400> SEQUENCE: 2 000 <210> SEQ
ID NO 3 <400> SEQUENCE: 3 000 <210> SEQ ID NO 4
<400> SEQUENCE: 4 000 <210> SEQ ID NO 5 <400>
SEQUENCE: 5 000 <210> SEQ ID NO 6 <400> SEQUENCE: 6 000
<210> SEQ ID NO 7 <400> SEQUENCE: 7 000 <210> SEQ
ID NO 8 <400> SEQUENCE: 8 000 <210> SEQ ID NO 9
<400> SEQUENCE: 9 000 <210> SEQ ID NO 10 <400>
SEQUENCE: 10 000 <210> SEQ ID NO 11 <400> SEQUENCE: 11
000 <210> SEQ ID NO 12 <400> SEQUENCE: 12 000
<210> SEQ ID NO 13 <400> SEQUENCE: 13 000 <210>
SEQ ID NO 14 <400> SEQUENCE: 14 000 <210> SEQ ID NO 15
<400> SEQUENCE: 15 000 <210> SEQ ID NO 16 <400>
SEQUENCE: 16 000 <210> SEQ ID NO 17 <400> SEQUENCE: 17
000 <210> SEQ ID NO 18 <400> SEQUENCE: 18 000
<210> SEQ ID NO 19 <400> SEQUENCE: 19 000 <210>
SEQ ID NO 20 <400> SEQUENCE: 20 000 <210> SEQ ID NO 21
<400> SEQUENCE: 21 000 <210> SEQ ID NO 22 <400>
SEQUENCE: 22 000 <210> SEQ ID NO 23 <400> SEQUENCE: 23
000 <210> SEQ ID NO 24 <400> SEQUENCE: 24 000
<210> SEQ ID NO 25 <400> SEQUENCE: 25 000 <210>
SEQ ID NO 26 <400> SEQUENCE: 26 000 <210> SEQ ID NO 27
<400> SEQUENCE: 27 000 <210> SEQ ID NO 28 <400>
SEQUENCE: 28 000 <210> SEQ ID NO 29 <400> SEQUENCE: 29
000 <210> SEQ ID NO 30 <400> SEQUENCE: 30 000
<210> SEQ ID NO 31 <400> SEQUENCE: 31 000 <210>
SEQ ID NO 32 <400> SEQUENCE: 32 000 <210> SEQ ID NO 33
<400> SEQUENCE: 33 000 <210> SEQ ID NO 34 <400>
SEQUENCE: 34 000 <210> SEQ ID NO 35 <400> SEQUENCE: 35
000 <210> SEQ ID NO 36 <400> SEQUENCE: 36 000
<210> SEQ ID NO 37 <400> SEQUENCE: 37 000 <210>
SEQ ID NO 38 <400> SEQUENCE: 38 000 <210> SEQ ID NO 39
<400> SEQUENCE: 39 000 <210> SEQ ID NO 40 <400>
SEQUENCE: 40 000 <210> SEQ ID NO 41 <400> SEQUENCE: 41
000 <210> SEQ ID NO 42 <400> SEQUENCE: 42 000
<210> SEQ ID NO 43 <400> SEQUENCE: 43 000 <210>
SEQ ID NO 44 <400> SEQUENCE: 44 000 <210> SEQ ID NO 45
<400> SEQUENCE: 45 000 <210> SEQ ID NO 46 <400>
SEQUENCE: 46 000 <210> SEQ ID NO 47 <400> SEQUENCE: 47
000 <210> SEQ ID NO 48 <400> SEQUENCE: 48 000
<210> SEQ ID NO 49 <400> SEQUENCE: 49 000 <210>
SEQ ID NO 50 <400> SEQUENCE: 50 000 <210> SEQ ID NO 51
<400> SEQUENCE: 51 000 <210> SEQ ID NO 52 <400>
SEQUENCE: 52 000 <210> SEQ ID NO 53 <400> SEQUENCE: 53
000 <210> SEQ ID NO 54 <400> SEQUENCE: 54 000
<210> SEQ ID NO 55 <400> SEQUENCE: 55 000 <210>
SEQ ID NO 56 <400> SEQUENCE: 56 000 <210> SEQ ID NO 57
<400> SEQUENCE: 57 000 <210> SEQ ID NO 58 <400>
SEQUENCE: 58 000 <210> SEQ ID NO 59 <400> SEQUENCE: 59
000 <210> SEQ ID NO 60 <400> SEQUENCE: 60 000
<210> SEQ ID NO 61 <400> SEQUENCE: 61 000 <210>
SEQ ID NO 62 <211> LENGTH: 187 <212> TYPE: DNA
<213> ORGANISM: Streptococcus pyogenes <400> SEQUENCE:
62 atggactata aggaccacga cggagactac aaggatcatg atattgatta
caaagacgat 60 gacgataaga tggccccaaa gaagaagcgg aaggtcggta
tccacggagt cccagcagcc 120 gacaagaagt acagcatcgg cctggacatc
ggcaccaact ctgtgggctg ggccgtgatc 180 accgacg 187 <210> SEQ ID
NO 63 <211> LENGTH: 4478 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 63 ggaaataaga gagaaaagaa
gagtaagaag aaatataaga gccaccatgg ccccaaagaa 60 gaagcggaag
gtcggtatcc acggagtccc agcagccgac aagaagtaca gcatcggcct 120
ggacatcggc accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag
180 caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga
acctgatcgg 240 agccctgctg ttcgacagcg gcgaaacagc cgaggccacc
cggctgaaga gaaccgccag 300 aagaagatac accagacgga agaaccggat
ctgctatctg caagagatct tcagcaacga 360 gatggccaag gtggacgaca
gcttcttcca cagactggaa gagtccttcc tggtggaaga 420 ggacaagaag
cacgagagac accccatctt cggcaacatc gtggacgagg tggcctacca 480
cgagaagtac cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc
540 cgacctgaga ctgatctacc tggccctggc ccacatgatc aagttcagag
gccacttcct 600 gatcgagggc gacctgaacc ccgacaacag cgacgtggac
aagctgttca tccagctggt 660 gcagacctac aaccagctgt tcgaggaaaa
ccccatcaac gccagcggcg tggacgccaa 720 ggctatcctg tctgccagac
tgagcaagag cagaaggctg gaaaatctga tcgcccagct 780 gcccggcgag
aagaagaacg gcctgttcgg caacctgatt gccctgagcc tgggcctgac 840
ccccaacttc aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga
900 cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt
acgccgacct 960 gttcctggcc gccaagaacc tgtctgacgc catcctgctg
agcgacatcc tgagagtgaa 1020 caccgagatc accaaggccc ccctgagcgc
ctctatgatc aagagatacg acgagcacca 1080 ccaggacctg accctgctga
aagctctcgt gcggcagcag ctgcctgaga agtacaaaga 1140 aatcttcttc
gaccagagca agaacggcta cgccggctac atcgatggcg gcgctagcca 1200
ggaagagttc tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact
1260 gctcgtgaag ctgaacagag aggacctgct gagaaagcag agaaccttcg
acaacggcag 1320 catcccccac cagatccacc tgggagagct gcacgctatc
ctgagaaggc aggaagattt 1380 ttacccattc ctgaaggaca accgggaaaa
gatcgagaag atcctgacct tcaggatccc 1440 ctactacgtg ggccccctgg
ccagaggcaa cagcagattc gcctggatga ccagaaagag 1500 cgaggaaacc
atcaccccct ggaacttcga ggaagtggtg gacaagggcg ccagcgccca 1560
gagcttcatc gagagaatga caaacttcga taagaacctg cccaacgaga aggtgctgcc
1620 caagcacagc ctgctgtacg agtacttcac cgtgtacaac gagctgacca
aagtgaaata 1680 cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc
gagcagaaaa aggccatcgt 1740 ggacctgctg ttcaagacca acagaaaagt
gaccgtgaag cagctgaaag aggactactt 1800 caagaaaatc gagtgcttcg
actccgtgga aatctccggc gtggaagata gattcaacgc 1860 ctccctgggc
acataccacg atctgctgaa aattatcaag gacaaggact tcctggataa 1920
cgaagagaac gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggaccg
1980 cgagatgatc gaggaaaggc tgaaaaccta cgctcacctg ttcgacgaca
aagtgatgaa 2040 gcagctgaag agaaggcggt acaccggctg gggcaggctg
agcagaaagc tgatcaacgg 2100 catcagagac aagcagagcg gcaagacaat
cctggatttc ctgaagtccg acggcttcgc 2160 caaccggaac ttcatgcagc
tgatccacga cgacagcctg acattcaaag aggacatcca 2220 gaaagcccag
gtgtccggcc agggcgactc tctgcacgag catatcgcta acctggccgg 2280
cagccccgct atcaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa
2340 agtgatgggc agacacaagc ccgagaacat cgtgatcgag atggctagag
agaaccagac 2400 cacccagaag ggacagaaga actcccgcga gaggatgaag
agaatcgaag agggcatcaa 2460 agagctgggc agccagatcc tgaaagaaca
ccccgtggaa aacacccagc tgcagaacga 2520 gaagctgtac ctgtactacc
tgcagaatgg ccgggatatg tacgtggacc aggaactgga 2580 catcaacaga
ctgtccgact acgatgtgga ccatatcgtg cctcagagct ttctgaagga 2640
cgactccatc gataacaaag tgctgactcg gagcgacaag aacagaggca agagcgacaa
2700 cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcgacagc
tgctgaacgc 2760 caagctgatt acccagagga agttcgataa cctgaccaag
gccgagagag gcggcctgag 2820 cgagctggat aaggccggct tcatcaagag
gcagctggtg gaaaccagac agatcacaaa 2880 gcacgtggca cagatcctgg
actcccggat gaacactaag tacgacgaaa acgataagct 2940 gatccgggaa
gtgaaagtga tcaccctgaa gtccaagctg gtgtccgatt tccggaagga 3000
tttccagttt tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct
3060 gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa
gcgagttcgt 3120 gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc
gccaagagcg agcaggaaat 3180 cggcaaggct accgccaagt acttcttcta
cagcaacatc atgaactttt tcaagaccga 3240 aatcaccctg gccaacggcg
agatcagaaa gcgccctctg atcgagacaa acggcgaaac 3300 cggggagatc
gtgtgggata agggcagaga cttcgccaca gtgcgaaagg tgctgagcat 3360
gccccaagtg aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc
3420 tatcctgccc aagaggaaca gcgacaagct gatcgccaga aagaaggact
gggaccccaa 3480 gaagtacggc ggcttcgaca gccctaccgt ggcctactct
gtgctggtgg tggctaaggt 3540 ggaaaagggc aagtccaaga aactgaagag
tgtgaaagag ctgctgggga tcaccatcat 3600 ggaaagaagc agctttgaga
agaaccctat cgactttctg gaagccaagg gctacaaaga 3660 agtgaaaaag
gacctgatca tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg 3720
cagaaagaga atgctggcct ctgccggcga actgcagaag ggaaacgagc tggccctgcc
3780 tagcaaatat gtgaacttcc tgtacctggc ctcccactat gagaagctga
agggcagccc 3840 tgaggacaac gaacagaaac agctgtttgt ggaacagcat
aagcactacc tggacgagat 3900 catcgagcag atcagcgagt tctccaagag
agtgatcctg gccgacgcca atctggacaa 3960 ggtgctgtct gcctacaaca
agcacaggga caagcctatc agagagcagg ccgagaatat 4020 catccacctg
ttcaccctga caaacctggg cgctcctgcc gccttcaagt actttgacac 4080
caccatcgac cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca
4140 ccagagcatc accggcctgt acgagacaag aatcgacctg tctcagctgg
gaggcgacaa 4200 gagacctgcc gccactaaga aggccggaca ggccaaaaag
aagaagtgag cggccgctta 4260 attaagctgc cttctgcggg gcttgccttc
tggccatgcc cttcttctct cccttgcacc 4320 tgtacctctt ggtctttgaa
taaagcctga gtaggaagaa aaaaaaaaaa aaaaaaaaaa 4380 aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4440
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 4478 <210> SEQ ID
NO 64 <211> LENGTH: 100 <212> TYPE: RNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 64 ccugcucgug gugaccgaag
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 65
<211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 65 acggaagcgg caguccuggc
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 66
<211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 66 cuagaugauu ccaucugcac
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 67
<211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 67 gugcggcaac cuacaugaug
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 68
<211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 68 ggguuccauu acggccagcg
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 69
<211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 69 gucaccaauc cugucccuag
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 70
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 70 ttgctggctg tggagcggac 20
<210> SEQ ID NO 71 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 71 gactggcttg
ggcagttcca 20 <210> SEQ ID NO 72 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 72 tgctggggcc ttcctcgagg 20 <210> SEQ ID NO 73
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 73 ccgaaggtag gagaaggtct 20
<210> SEQ ID NO 74 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 74 atgcacagca
gatcctcctc 20 <210> SEQ ID NO 75 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 75 agagagtgag ccaaaggtgc 20 <210> SEQ ID NO 76
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 76 ggcatactca atgcgtacat 20
<210> SEQ ID NO 77 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 77 gggttccatt
acggccagcg 20 <210> SEQ ID NO 78 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 78 aaggctgacc acatccggaa 20 <210> SEQ ID NO 79
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 79 tgccgactcc agcttccgtc 20
<210> SEQ ID NO 80 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 80 ctgtcagtga
aaaccactcg 20 <210> SEQ ID NO 81 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 81 cgtactaaga acgtgccttc 20 <210> SEQ ID NO 82
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 82 caccgcacgt gtgaaccaac ccgcc 25 <210>
SEQ ID NO 83 <211> LENGTH: 25 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 83 aaacggcggg ttggttcaca
cgtgc 25 <210> SEQ ID NO 84 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 84
caccgaaaca acaggccggg cgggt 25 <210> SEQ ID NO 85 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 85 aaacacccgc ccggcctgtt gtttc 25 <210> SEQ ID NO
86 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
primer <400> SEQUENCE: 86 caccgacaaa aaaattagcc gggtg 25
<210> SEQ ID NO 87 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 87 aaaccacccg gctaattttt
ttgt 24 <210> SEQ ID NO 88 <211> LENGTH: 25 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic primer <400> SEQUENCE: 88 caccgtaaat
ttctctgata gacta 25 <210> SEQ ID NO 89 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 89
aaactagtct atcagagaaa tttac 25 <210> SEQ ID NO 90 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 90 caccgtgttt caatgagagc attac 25 <210> SEQ ID NO
91 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
primer <400> SEQUENCE: 91 aaacgtaatg ctctcattga aacac 25
<210> SEQ ID NO 92 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 92 caccggtctc gaactcctga
gctc 24 <210> SEQ ID NO 93 <211> LENGTH: 24 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic primer <400> SEQUENCE: 93 aaacgagctc
aggagttcga gacc 24 <210> SEQ ID NO 94 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 94
agtgaagtgg cgcattcttg 20 <210> SEQ ID NO 95 <211>
LENGTH: 21 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 95 caccctttcc aaatcctcag c 21 <210> SEQ ID NO 96
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 96 caccgtgggg gttagaccca atatc 25 <210>
SEQ ID NO 97 <211> LENGTH: 25 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 97 aaacgatatt gggtctaacc
cccac 25 <210> SEQ ID NO 98 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 98
caccgacccc acagtggggc cacta 25 <210> SEQ ID NO 99 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 99 aaactagtgg ccccactgtg gggtc 25 <210> SEQ ID NO
100 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
primer <400> SEQUENCE: 100 caccgagggc cggttaatgt ggctc 25
<210> SEQ ID NO 101 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 101 aaacgagcca cattaaccgg
ccctc 25 <210> SEQ ID NO 102 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 102
caccgtcacc aatcctgtcc ctag 24 <210> SEQ ID NO 103 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 103 aaacctaggg acaggattgg tgac 24 <210> SEQ ID NO
104 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
primer <400> SEQUENCE: 104 caccgccggc cctgggaata taagg 25
<210> SEQ ID NO 105 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 105 aaacccttat attcccaggg
ccggc 25 <210> SEQ ID NO 106 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 106
caccgcgggc ccctatgtcc acttc 25 <210> SEQ ID NO 107
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 107 aaacgaagtg gacatagggg cccgc 25
<210> SEQ ID NO 108 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 108 actcctttca tttgggcagc 20
<210> SEQ ID NO 109 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 109 ggttctggca aggagagaga 20
<210> SEQ ID NO 110 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 110 caccgcggag agcttcgtgc
taaac 25 <210> SEQ ID NO 111 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 111
aaacgtttag cacgaagctc tccgc 25 <210> SEQ ID NO 112
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 112 caccgcctgc tcgtggtgac cgaag 25
<210> SEQ ID NO 113 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 113 aaaccttcgg tcaccacgag
caggc 25 <210> SEQ ID NO 114 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 114
caccgcagca accagacgga caagc 25 <210> SEQ ID NO 115
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 115 aaacgcttgt ccgtctggtt gctgc 25
<210> SEQ ID NO 116 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 116 caccgaggcg gccagcttgt
ccgtc 25 <210> SEQ ID NO 117 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 117
aaacgacgga caagctggcc gcctc 25 <210> SEQ ID NO 118
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 118 caccgcgttg ggcagttgtg tgaca 25
<210> SEQ ID NO 119 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 119 aaactgtcac acaactgccc
aacgc 25 <210> SEQ ID NO 120 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 120
caccgacgga agcggcagtc ctggc 25 <210> SEQ ID NO 121
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 121 aaacgccagg actgccgctt ccgtc 25
<210> SEQ ID NO 122 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 122 agaaggaaga ggctctgcag 20
<210> SEQ ID NO 123 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 123 ctctttgatc tgcgccttgg 20
<210> SEQ ID NO 124 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 124 caccgccggg tgacagtgct
tcggc 25 <210> SEQ ID NO 125 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 125
aaacgccgaa gcactgtcac ccggc 25 <210> SEQ ID NO 126
<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 126 caccgtgcgg caacctacat gatg 24 <210>
SEQ ID NO 127 <211> LENGTH: 24 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 127 aaaccatcat gtaggttgcc
gcac 24 <210> SEQ ID NO 128 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 128
caccgctaga tgattccatc tgcac 25 <210> SEQ ID NO 129
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 129 aaacgtgcag atggaatcat ctagc 25
<210> SEQ ID NO 130 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 130 caccgaggtt cacttgattt
ccac 24 <210> SEQ ID NO 131 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 131
aaacgtggaa atcaagtgaa cctc 24 <210> SEQ ID NO 132 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 132 caccgccgca cagacttcag tcacc 25 <210> SEQ ID NO
133 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
primer <400> SEQUENCE: 133 aaacggtgac tgaagtctgt gcggc 25
<210> SEQ ID NO 134 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 134 caccgctggc gatgcctcgg
ctgc 24 <210> SEQ ID NO 135 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 135
aaacgcagcc gaggcatcgc cagc 24 <210> SEQ ID NO 136 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 136 tggggatgaa gctagaaggc 20 <210> SEQ ID NO 137
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 137 aatctgggtt ccgttgccta 20 <210> SEQ
ID NO 138 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
primer <400> SEQUENCE: 138 caccgacaat gtgtcaactc ttgac 25
<210> SEQ ID NO 139 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 139 aaacgtcaag agttgacaca
ttgtc 25 <210> SEQ ID NO 140 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 140
caccgtcatc ctcctgacaa tcgat 25 <210> SEQ ID NO 141
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 141 aaacatcgat tgtcaggagg atgac 25
<210> SEQ ID NO 142 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 142 caccggtgac aagtgtgatc
actt 24 <210> SEQ ID NO 143 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 143
aaacaagtga tcacacttgt cacc 24 <210> SEQ ID NO 144 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 144 caccgacaca gcatggacga cagcc 25 <210> SEQ ID NO
145 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
primer <400> SEQUENCE: 145 aaacggctgt cgtccatgct gtgtc 25
<210> SEQ ID NO 146 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 146 caccgatctg gtaaagatga
ttcc 24 <210> SEQ ID NO 147 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 147
aaacggaatc atctttacca gatc 24 <210> SEQ ID NO 148 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 148 caccgttgta tttccaaagt cccac 25 <210> SEQ ID NO
149 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
primer <400> SEQUENCE: 149 aaacgtggga ctttggaaat acaac 25
<210> SEQ ID NO 150 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 150 ctcaacctgg ccatctctga 20
<210> SEQ ID NO 151 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 151 cccgagtagc agatgaccat 20
<210> SEQ ID NO 152 <400> SEQUENCE: 152 000 <210>
SEQ ID NO 153 <400> SEQUENCE: 153 000 <210> SEQ ID NO
154 <400> SEQUENCE: 154 000 <210> SEQ ID NO 155
<400> SEQUENCE: 155 000 <210> SEQ ID NO 156 <400>
SEQUENCE: 156 000 <210> SEQ ID NO 157 <400> SEQUENCE:
157 000 <210> SEQ ID NO 158 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 158 cctcctcctg gtgaccggag agg 23 <210> SEQ ID NO
159 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 159 cttgctggtg gtgacagaag agg
23 <210> SEQ ID NO 160 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 160
actgcttgtt gtgaccgatg ggg 23 <210> SEQ ID NO 161 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 161 ccagcacatg gtgaccaaag gag 23 <210>
SEQ ID NO 162 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 162 cctgcttgtg
gtgacctaat aag 23 <210> SEQ ID NO 163 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 163 ccaggtggtg gtgagcgaag agg 23 <210> SEQ ID NO
164 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 164 cctaatcgtg gtcaccgaaa gag
23 <210> SEQ ID NO 165 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 165
cctgctggtg gtgacctatg agg 23 <210> SEQ ID NO 166 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 166 cctgctactg gtgtccgaag tgg 23 <210>
SEQ ID NO 167 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 167 gcagctcgag
gtgacggaag agg 23 <210> SEQ ID NO 168 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 168 catgctccta gtgacagaag ggg 23 <210> SEQ ID NO
169 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 169 cccgctggtg ggaaccgaag gag
23 <210> SEQ ID NO 170 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 170
cctgggcgta gtgagcgaag cgg 23 <210> SEQ ID NO 171 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 171 cctggccgcg gtgaccgagg cgg 23 <210>
SEQ ID NO 172 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 172 cctgcccaag
gttaccgaag tgg 23 <210> SEQ ID NO 173 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 173 cctgctccag ttgaccaaag agg 23 <210> SEQ ID NO
174 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 174 actgcacgtg gtgaaccaag agg
23 <210> SEQ ID NO 175 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 175
gctgcccatg gtgacagaag ggg 23 <210> SEQ ID NO 176 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 176 actgctggtg gtgaacgaaa ggg 23 <210>
SEQ ID NO 177 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 177 gctgctagtg
gtgacggaaa gag 23 <210> SEQ ID NO 178 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 178 tctgctattg gtgtccgaag tgg 23 <210> SEQ ID NO
179 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 179 cgagctcgtg gttacagaag ggg
23 <210> SEQ ID NO 180 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 180
ccagctcgtc gggacagaag agg 23 <210> SEQ ID NO 181 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 181 ccacctcggg gtggccgaag cgg 23 <210>
SEQ ID NO 182 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 182 ccagctagtg
ttgactgaag ggg 23 <210> SEQ ID NO 183 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 183 cctggctgtg gtgacagaag ggg 23 <210> SEQ ID NO
184 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 184 cctgcacgtg atgacctaac agg
23 <210> SEQ ID NO 185 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 185
cctgctcgtg ctgtccgcag cag 23 <210> SEQ ID NO 186 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 186 cctgctggtg gtgacggcag cag 23 <210>
SEQ ID NO 187 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 187 gctgctggtg
gtgcccgatg ggg 23 <210> SEQ ID NO 188 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 188 cctgctcgtg gtgatatgag ggg 23 <210> SEQ ID NO
189 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 189 ccagctggtg gtgactcaag gag
23 <210> SEQ ID NO 190 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 190
ccttctcgtg gagacagatg agg 23 <210> SEQ ID NO 191 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 191 ccttctcgtg gtgcctggag tgg 23 <210>
SEQ ID NO 192 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 192 ccttctcatg
gtggcggaag gag 23 <210> SEQ ID NO 193 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 193 ccttctcatg gtgacagtag agg 23 <210> SEQ ID NO
194 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 194 cctggtcgtg ttgagtgaag ggg
23 <210> SEQ ID NO 195 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 195
cctggtagtg gtgaccacag tgg 23 <210> SEQ ID NO 196 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 196 cctggtggtg gtgaccatag agg 23 <210>
SEQ ID NO 197 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 197 cctgcccgtg
gtaatcgaac cgg 23 <210> SEQ ID NO 198 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 198 cctgcccgtg gtgaacgctg tgg 23 <210> SEQ ID NO
199 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 199 cctgcacgtg gtgacagcat ggg
23 <210> SEQ ID NO 200 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 200
cctgcgtgtg gaggccgaag ggg 23 <210> SEQ ID NO 201 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 201 cctgccggtg gtgacctatg tgg 23 <210>
SEQ ID NO 202 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 202 cctgctggtg
ctgcctgaag ggg 23 <210> SEQ ID NO 203 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 203 cctgctggtg ctggccgatg ggg 23 <210> SEQ ID NO
204 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 204 cctgctagtg gaaaccgcag ggg
23 <210> SEQ ID NO 205 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 205
cctgctggtg gtaccagaag agg 23 <210> SEQ ID NO 206 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 206 cctgctggtg gtaccagaag agg 23 <210>
SEQ ID NO 207 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 207 cctgctggtg
gtggcagcag tgg 23 <210> SEQ ID NO 208 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 208 cctgctagtg gtggcagtag tgg 23 <210> SEQ ID NO
209 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 209 cctgcttatg gggactgaag ggg
23 <210> SEQ ID NO 210 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 210
cctgcttctg gtgtcagaag tgg 23 <210> SEQ ID NO 211 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 211 cctgctgctg gtggccgacg tgg 23 <210>
SEQ ID NO 212 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 212 cctgctgttg
gtgactcaag gag 23 <210> SEQ ID NO 213 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 213 cctgctgttg gtgactcaag gag 23 <210> SEQ ID NO
214 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 214 cctgcttggg ctgaccgtag ggg
23 <210> SEQ ID NO 215 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 215
cctgctaggg gagacagaag agg 23 <210> SEQ ID NO 216 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 216 cctgctcctg ctgcacgaag tgg 23 <210>
SEQ ID NO 217 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 217 cctgctcttg
gggactgaat tgg 23 <210> SEQ ID NO 218 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 218 cctgctcctg gttcccaaag agg 23 <210> SEQ ID NO
219 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 219 cctgctcttg gtcactgcag agg
23 <210> SEQ ID NO 220 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 220
cctgctcctg gtgcaagaag ggg 23 <210> SEQ ID NO 221 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 221 cctgctcctg gtgcaagaag ggg 23 <210>
SEQ ID NO 222 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 222 cctgctcttg
gtggaagaag tgg 23 <210> SEQ ID NO 223 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 223 cctgctcctg gtgtgggaag agg 23 <210> SEQ ID NO
224 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 224 cctgctcttg gtgaacacag gag
23 <210> SEQ ID NO 225 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 225
cctgctcaag gagacagaag tgg 23 <210> SEQ ID NO 226 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 226 cctgctccag gtgaatgaag agg 23 <210>
SEQ ID NO 227 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 227 cctgctctgg
gtgacggagg gag 23 <210> SEQ ID NO 228 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 228 cctgctcgcg gtgggctaag ggg 23 <210> SEQ ID NO
229 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 229 cagttccatt accgccagcg ggg
23 <210> SEQ ID NO 230 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 230
ggtttccatt cctgccagcg ggg 23 <210> SEQ ID NO 231 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 231 gtgtcccgtt acggccagct cgg 23 <210>
SEQ ID NO 232 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 232 agcttccctt
agggccagcg aag 23 <210> SEQ ID NO 233 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 233 ggggtcccta agggccagcg agg 23 <210> SEQ ID NO
234 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 234 gcgtcgcact acggccagcg agg
23 <210> SEQ ID NO 235 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 235
gcgtgccact acggccagct aag 23 <210> SEQ ID NO 236 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 236 gggtccccat agggccagcg agg 23 <210>
SEQ ID NO 237 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 237 ggctcccatt
gcagccagcg tgg 23 <210> SEQ ID NO 238 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 238 ttgttccatt atggccagag aag 23 <210> SEQ ID NO
239 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 239 ttgttccatt atggccagag aag
23 <210> SEQ ID NO 240 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 240
ggataccaat acggccaacg agg 23 <210> SEQ ID NO 241 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 241 ggtttccttt actgccggcg agg 23 <210>
SEQ ID NO 242 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 242 gggtgccagt
ggggccagcg agg 23 <210> SEQ ID NO 243 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 243 gggttccttc agggccagag agg 23 <210> SEQ ID NO
244 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 244 gggctccatc acagccagca ggg
23 <210> SEQ ID NO 245 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 245
aggttccagt tcggccagtg aag 23 <210> SEQ ID NO 246 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 246 gtgttccatt ccggcccgcc agg 23 <210>
SEQ ID NO 247 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 247 ggcttccaat
aaggccaacg agg 23 <210> SEQ ID NO 248 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 248 gggttccagt gtggccagca agg 23 <210> SEQ ID NO
249 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 249 gggttccttt tctgccagct tgg
23 <210> SEQ ID NO 250 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 250
gggttccttt tctgccagct tgg 23 <210> SEQ ID NO 251 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 251 gggttccttt tctgccagct tgg 23 <210>
SEQ ID NO 252 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 252 gggttccttt
agggccagga tgg 23 <210> SEQ ID NO 253 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 253 gggttctatt ccagccagct tgg 23 <210> SEQ ID NO
254 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 254 gggtttcagt aaggccaggg tgg
23 <210> SEQ ID NO 255 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 255
gggccccatt acagccatcg tgg 23 <210> SEQ ID NO 256 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 256 ggggtccatg acagacagcg tgg 23 <210>
SEQ ID NO 257 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 257 ggggtccatt
cccgccagag tgg 23 <210> SEQ ID NO 258 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 258 gggttacatc aaggccaccg agg 23 <210> SEQ ID NO
259 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 259 gggttcaatt acggaaaggg agg
23 <210> SEQ ID NO 260 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 260
gggttccact ccttccagcg agg 23 <210> SEQ ID NO 261 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 261 gggttccatt atgacaagca tgg 23 <210>
SEQ ID NO 262 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 262 gggttccatt
ctggcaaggg tgg 23 <210> SEQ ID NO 263 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 263 gggttccatt gctggcagag tgg 23 <210> SEQ ID NO
264 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 264 gggttccatg aaggccaaag ggg
23 <210> SEQ ID NO 265 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 265
gggttccaat ccggccactg tgg 23 <210> SEQ ID NO 266 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 266 gggttccact actgcctgct tgg 23 <210>
SEQ ID NO 267 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 267 gggttccgtt
ccggccactg ggg 23 <210> SEQ ID NO 268 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 268 gggtttcatt ggagccagcg tgg 23 <210> SEQ ID NO
269 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 269 gggtttcatt acgctcagct aag
23 <210> SEQ ID NO 270 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 270
gggttacctt acgacctgcg tgg 23 <210> SEQ ID NO 271 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 271 gggctccatt ccggccatgg tgg 23 <210>
SEQ ID NO 272 <211> LENGTH: 20 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 272 gtcaccaatc
ctgtccctag 20 <210> SEQ ID NO 273 <211> LENGTH: 96
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 273 caggccctgg aaccccccca ccttctcccc agccctgctc
gtggtgaccg aggactgccg 60 cttccgtgtc acacaactgc ccaacgggcg tgactt 96
<210> SEQ ID NO 274 <211> LENGTH: 119 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polynucleotide <400> SEQUENCE: 274 cggcaggctg
acagccaggt gactgaagtc tgtgcggcaa cctacatgat ggggaatgag 60
ttgaccttcc tagatgattc catctgcacg ggcacctcca gtggaaatca agtgaacct
119 <210> SEQ ID NO 275 <211> LENGTH: 81 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 275
cggcaggctg acagccaggt gactgaagtc tgtgcggcaa cctacatgca cgggcacctc
60 cagtggaaat caagtgaacc t 81 <210> SEQ ID NO 276 <211>
LENGTH: 101 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<220> FEATURE: <223> OTHER INFORMATION: Description of
Combined DNA/RNA Molecule: Synthetic oligonucleotide <400>
SEQUENCE: 276 gcctgctcgt ggtgaccgaa gguuuuagag cuagaaauag
caaguuaaaa uaaggcuagu 60 ccguuaucaa cuugaaaaag uggcaccgag
ucggugcuuu u 101 <210> SEQ ID NO 277 <211> LENGTH: 101
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <220> FEATURE:
<223> OTHER INFORMATION: Description of Combined DNA/RNA
Molecule: Synthetic oligonucleotide <400> SEQUENCE: 277
gctagatgat tccatctgca cguuuuagag cuagaaauag caaguuaaaa uaaggcuagu
60 ccguuaucaa cuugaaaaag uggcaccgag ucggugcuuu u 101 <210>
SEQ ID NO 278 <211> LENGTH: 100 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <220> FEATURE: <223> OTHER
INFORMATION: Description of Combined DNA/RNA Molecule: Synthetic
oligonucleotide <400> SEQUENCE: 278 gggttccatt acggccagcg
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 279
<211> LENGTH: 100 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic oligonucleotide
<400> SEQUENCE: 279 gtcaccaatc ctgtccctag guuuuagagc
uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu
ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 280 <211>
LENGTH: 101 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<220> FEATURE: <223> OTHER INFORMATION: Description of
Combined DNA/RNA Molecule: Synthetic oligonucleotide <400>
SEQUENCE: 280 gcctgctcgt ggtgaccgaa gguuuuagag cuagaaauag
caaguuaaaa uaaggcuagu 60 ccguuaucaa cuugaaaaag uggcaccgag
ucggugcuuu u 101 <210> SEQ ID NO 281 <211> LENGTH: 101
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <220> FEATURE:
<223> OTHER INFORMATION: Description of Combined DNA/RNA
Molecule: Synthetic oligonucleotide <400> SEQUENCE: 281
gctagatgat tccatctgca cguuuuagag cuagaaauag caaguuaaaa uaaggcuagu
60 ccguuaucaa cuugaaaaag uggcaccgag ucggugcuuu u 101 <210>
SEQ ID NO 282 <211> LENGTH: 100 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <220> FEATURE: <223> OTHER
INFORMATION: Description of Combined DNA/RNA Molecule: Synthetic
oligonucleotide <400> SEQUENCE: 282 gggttccatt acggccagcg
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 283
<211> LENGTH: 100 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic oligonucleotide
<400> SEQUENCE: 283 gtcaccaatc ctgtccctag guuuuagagc
uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu
ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 284 <211>
LENGTH: 101 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<220> FEATURE: <223> OTHER INFORMATION: Description of
Combined DNA/RNA Molecule: Synthetic oligonucleotide <400>
SEQUENCE: 284 gcctgctcgt ggtgaccgaa gguuuuagag cuagaaauag
caaguuaaaa uaaggcuagu 60 ccguuaucaa cuugaaaaag uggcaccgag
ucggugcuuu u 101 <210> SEQ ID NO 285 <211> LENGTH: 101
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <220> FEATURE:
<223> OTHER INFORMATION: Description of Combined DNA/RNA
Molecule: Synthetic oligonucleotide <400> SEQUENCE: 285
gctagatgat tccatctgca cguuuuagag cuagaaauag caaguuaaaa uaaggcuagu
60 ccguuaucaa cuugaaaaag uggcaccgag ucggugcuuu u 101 <210>
SEQ ID NO 286 <211> LENGTH: 100 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <220> FEATURE: <223> OTHER
INFORMATION: Description of Combined DNA/RNA Molecule: Synthetic
oligonucleotide <400> SEQUENCE: 286 gggttccatt acggccagcg
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 287
<211> LENGTH: 100 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic oligonucleotide
<400> SEQUENCE: 287 gtcaccaatc ctgtccctag guuuuagagc
uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu
ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 288 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION: a,
c, t, g, unknown or other <400> SEQUENCE: 288 cctgctcgtg
gtgaccgaag ngg 23 <210> SEQ ID NO 289 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <220> FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: a, c, t, g, unknown or
other <400> SEQUENCE: 289 gctgcccatg gtgacagaag ngg 23
<210> SEQ ID NO 290 <211> LENGTH: 23 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <220> FEATURE: <221>
NAME/KEY: modified_base <222> LOCATION: (21)..(21)
<223> OTHER INFORMATION: a, c, t, g, unknown or other
<400> SEQUENCE: 290 gggttccatt acggccagcg ngg 23 <210>
SEQ ID NO 291 <211> LENGTH: 100 <212> TYPE: RNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polynucleotide <400> SEQUENCE: 291 ggguuccauu
acggccagcg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID
NO 292 <211> LENGTH: 34 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 292 gtttaattga gttgtcatat
gttaataacg gtat 34 <210> SEQ ID NO 293 <211> LENGTH:
101 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <220> FEATURE:
<223> OTHER INFORMATION: Description of Combined DNA/RNA
Molecule: Synthetic oligonucleotide <400> SEQUENCE: 293
gcctgctcgt ggtgaccgaa gguuuuagag cuagaaauag caaguuaaaa uaaggcuagu
60 ccguuaucaa cuugaaaaag ugggaccgag ucggugcuuu u 101
1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 293
<210> SEQ ID NO 1 <400> SEQUENCE: 1 000 <210> SEQ
ID NO 2 <400> SEQUENCE: 2 000 <210> SEQ ID NO 3
<400> SEQUENCE: 3 000 <210> SEQ ID NO 4 <400>
SEQUENCE: 4 000 <210> SEQ ID NO 5 <400> SEQUENCE: 5 000
<210> SEQ ID NO 6 <400> SEQUENCE: 6 000 <210> SEQ
ID NO 7 <400> SEQUENCE: 7 000 <210> SEQ ID NO 8
<400> SEQUENCE: 8 000 <210> SEQ ID NO 9 <400>
SEQUENCE: 9 000 <210> SEQ ID NO 10 <400> SEQUENCE: 10
000 <210> SEQ ID NO 11 <400> SEQUENCE: 11 000
<210> SEQ ID NO 12 <400> SEQUENCE: 12 000 <210>
SEQ ID NO 13 <400> SEQUENCE: 13 000 <210> SEQ ID NO 14
<400> SEQUENCE: 14 000 <210> SEQ ID NO 15 <400>
SEQUENCE: 15 000 <210> SEQ ID NO 16 <400> SEQUENCE: 16
000 <210> SEQ ID NO 17 <400> SEQUENCE: 17 000
<210> SEQ ID NO 18 <400> SEQUENCE: 18 000 <210>
SEQ ID NO 19 <400> SEQUENCE: 19 000 <210> SEQ ID NO 20
<400> SEQUENCE: 20 000 <210> SEQ ID NO 21 <400>
SEQUENCE: 21 000 <210> SEQ ID NO 22 <400> SEQUENCE: 22
000 <210> SEQ ID NO 23 <400> SEQUENCE: 23 000
<210> SEQ ID NO 24 <400> SEQUENCE: 24 000 <210>
SEQ ID NO 25 <400> SEQUENCE: 25 000 <210> SEQ ID NO 26
<400> SEQUENCE: 26 000 <210> SEQ ID NO 27 <400>
SEQUENCE: 27 000 <210> SEQ ID NO 28 <400> SEQUENCE: 28
000 <210> SEQ ID NO 29 <400> SEQUENCE: 29 000
<210> SEQ ID NO 30 <400> SEQUENCE: 30 000 <210>
SEQ ID NO 31 <400> SEQUENCE: 31 000 <210> SEQ ID NO 32
<400> SEQUENCE: 32 000 <210> SEQ ID NO 33 <400>
SEQUENCE: 33 000 <210> SEQ ID NO 34 <400> SEQUENCE: 34
000 <210> SEQ ID NO 35 <400> SEQUENCE: 35 000
<210> SEQ ID NO 36 <400> SEQUENCE: 36 000 <210>
SEQ ID NO 37 <400> SEQUENCE: 37 000 <210> SEQ ID NO 38
<400> SEQUENCE: 38 000 <210> SEQ ID NO 39 <400>
SEQUENCE: 39 000 <210> SEQ ID NO 40 <400> SEQUENCE: 40
000 <210> SEQ ID NO 41 <400> SEQUENCE: 41 000
<210> SEQ ID NO 42 <400> SEQUENCE: 42 000 <210>
SEQ ID NO 43 <400> SEQUENCE: 43 000 <210> SEQ ID NO 44
<400> SEQUENCE: 44 000 <210> SEQ ID NO 45 <400>
SEQUENCE: 45 000 <210> SEQ ID NO 46 <400> SEQUENCE: 46
000 <210> SEQ ID NO 47 <400> SEQUENCE: 47 000
<210> SEQ ID NO 48 <400> SEQUENCE: 48 000 <210>
SEQ ID NO 49 <400> SEQUENCE: 49 000 <210> SEQ ID NO 50
<400> SEQUENCE: 50 000 <210> SEQ ID NO 51 <400>
SEQUENCE: 51 000 <210> SEQ ID NO 52 <400> SEQUENCE: 52
000 <210> SEQ ID NO 53 <400> SEQUENCE: 53 000
<210> SEQ ID NO 54 <400> SEQUENCE: 54 000 <210>
SEQ ID NO 55 <400> SEQUENCE: 55 000 <210> SEQ ID NO 56
<400> SEQUENCE: 56 000 <210> SEQ ID NO 57 <400>
SEQUENCE: 57 000 <210> SEQ ID NO 58 <400> SEQUENCE: 58
000 <210> SEQ ID NO 59 <400> SEQUENCE: 59 000
<210> SEQ ID NO 60 <400> SEQUENCE: 60 000 <210>
SEQ ID NO 61 <400> SEQUENCE: 61 000 <210> SEQ ID NO 62
<211> LENGTH: 187 <212> TYPE: DNA <213> ORGANISM:
Streptococcus pyogenes <400> SEQUENCE: 62 atggactata
aggaccacga cggagactac aaggatcatg atattgatta caaagacgat 60
gacgataaga tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc
120 gacaagaagt acagcatcgg cctggacatc ggcaccaact ctgtgggctg
ggccgtgatc 180 accgacg 187 <210> SEQ ID NO 63 <211>
LENGTH: 4478 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic polynucleotide
<400> SEQUENCE: 63 ggaaataaga gagaaaagaa gagtaagaag
aaatataaga gccaccatgg ccccaaagaa 60 gaagcggaag gtcggtatcc
acggagtccc agcagccgac aagaagtaca gcatcggcct 120 ggacatcggc
accaactctg tgggctgggc cgtgatcacc gacgagtaca aggtgcccag 180
caagaaattc aaggtgctgg gcaacaccga ccggcacagc atcaagaaga acctgatcgg
240 agccctgctg ttcgacagcg gcgaaacagc cgaggccacc cggctgaaga
gaaccgccag 300 aagaagatac accagacgga agaaccggat ctgctatctg
caagagatct tcagcaacga 360 gatggccaag gtggacgaca gcttcttcca
cagactggaa gagtccttcc tggtggaaga 420 ggacaagaag cacgagagac
accccatctt cggcaacatc gtggacgagg tggcctacca 480 cgagaagtac
cccaccatct accacctgag aaagaaactg gtggacagca ccgacaaggc 540
cgacctgaga ctgatctacc tggccctggc ccacatgatc aagttcagag gccacttcct
600 gatcgagggc gacctgaacc ccgacaacag cgacgtggac aagctgttca
tccagctggt 660 gcagacctac aaccagctgt tcgaggaaaa ccccatcaac
gccagcggcg tggacgccaa 720 ggctatcctg tctgccagac tgagcaagag
cagaaggctg gaaaatctga tcgcccagct 780 gcccggcgag aagaagaacg
gcctgttcgg caacctgatt gccctgagcc tgggcctgac 840 ccccaacttc
aagagcaact tcgacctggc cgaggatgcc aaactgcagc tgagcaagga 900
cacctacgac gacgacctgg acaacctgct ggcccagatc ggcgaccagt acgccgacct
960 gttcctggcc gccaagaacc tgtctgacgc catcctgctg agcgacatcc
tgagagtgaa 1020 caccgagatc accaaggccc ccctgagcgc ctctatgatc
aagagatacg acgagcacca 1080 ccaggacctg accctgctga aagctctcgt
gcggcagcag ctgcctgaga agtacaaaga 1140 aatcttcttc gaccagagca
agaacggcta cgccggctac atcgatggcg gcgctagcca 1200 ggaagagttc
tacaagttca tcaagcccat cctggaaaag atggacggca ccgaggaact 1260
gctcgtgaag ctgaacagag aggacctgct gagaaagcag agaaccttcg acaacggcag
1320 catcccccac cagatccacc tgggagagct gcacgctatc ctgagaaggc
aggaagattt 1380 ttacccattc ctgaaggaca accgggaaaa gatcgagaag
atcctgacct tcaggatccc 1440 ctactacgtg ggccccctgg ccagaggcaa
cagcagattc gcctggatga ccagaaagag 1500 cgaggaaacc atcaccccct
ggaacttcga ggaagtggtg gacaagggcg ccagcgccca 1560 gagcttcatc
gagagaatga caaacttcga taagaacctg cccaacgaga aggtgctgcc 1620
caagcacagc ctgctgtacg agtacttcac cgtgtacaac gagctgacca aagtgaaata
1680 cgtgaccgag ggaatgagaa agcccgcctt cctgagcggc gagcagaaaa
aggccatcgt 1740 ggacctgctg ttcaagacca acagaaaagt gaccgtgaag
cagctgaaag aggactactt 1800 caagaaaatc gagtgcttcg actccgtgga
aatctccggc gtggaagata gattcaacgc 1860 ctccctgggc acataccacg
atctgctgaa aattatcaag gacaaggact tcctggataa 1920 cgaagagaac
gaggacattc tggaagatat cgtgctgacc ctgacactgt ttgaggaccg 1980
cgagatgatc gaggaaaggc tgaaaaccta cgctcacctg ttcgacgaca aagtgatgaa
2040 gcagctgaag agaaggcggt acaccggctg gggcaggctg agcagaaagc
tgatcaacgg 2100 catcagagac aagcagagcg gcaagacaat cctggatttc
ctgaagtccg acggcttcgc 2160 caaccggaac ttcatgcagc tgatccacga
cgacagcctg acattcaaag aggacatcca 2220 gaaagcccag gtgtccggcc
agggcgactc tctgcacgag catatcgcta acctggccgg 2280 cagccccgct
atcaagaagg gcatcctgca gacagtgaag gtggtggacg agctcgtgaa 2340
agtgatgggc agacacaagc ccgagaacat cgtgatcgag atggctagag agaaccagac
2400 cacccagaag ggacagaaga actcccgcga gaggatgaag agaatcgaag
agggcatcaa 2460 agagctgggc agccagatcc tgaaagaaca ccccgtggaa
aacacccagc tgcagaacga 2520 gaagctgtac ctgtactacc tgcagaatgg
ccgggatatg tacgtggacc aggaactgga 2580 catcaacaga ctgtccgact
acgatgtgga ccatatcgtg cctcagagct ttctgaagga 2640 cgactccatc
gataacaaag tgctgactcg gagcgacaag aacagaggca agagcgacaa 2700
cgtgccctcc gaagaggtcg tgaagaagat gaagaactac tggcgacagc tgctgaacgc
2760 caagctgatt acccagagga agttcgataa cctgaccaag gccgagagag
gcggcctgag 2820 cgagctggat aaggccggct tcatcaagag gcagctggtg
gaaaccagac agatcacaaa 2880 gcacgtggca cagatcctgg actcccggat
gaacactaag tacgacgaaa acgataagct 2940 gatccgggaa gtgaaagtga
tcaccctgaa gtccaagctg gtgtccgatt tccggaagga 3000 tttccagttt
tacaaagtgc gcgagatcaa caactaccac cacgcccacg acgcctacct 3060
gaacgccgtc gtgggaaccg ccctgatcaa aaagtaccct aagctggaaa gcgagttcgt
3120 gtacggcgac tacaaggtgt acgacgtgcg gaagatgatc gccaagagcg
agcaggaaat 3180 cggcaaggct accgccaagt acttcttcta cagcaacatc
atgaactttt tcaagaccga 3240 aatcaccctg gccaacggcg agatcagaaa
gcgccctctg atcgagacaa acggcgaaac 3300 cggggagatc gtgtgggata
agggcagaga cttcgccaca gtgcgaaagg tgctgagcat 3360 gccccaagtg
aatatcgtga aaaagaccga ggtgcagaca ggcggcttca gcaaagagtc 3420
tatcctgccc aagaggaaca gcgacaagct gatcgccaga aagaaggact gggaccccaa
3480 gaagtacggc ggcttcgaca gccctaccgt ggcctactct gtgctggtgg
tggctaaggt 3540 ggaaaagggc aagtccaaga aactgaagag tgtgaaagag
ctgctgggga tcaccatcat 3600 ggaaagaagc agctttgaga agaaccctat
cgactttctg gaagccaagg gctacaaaga 3660 agtgaaaaag gacctgatca
tcaagctgcc taagtactcc ctgttcgagc tggaaaacgg 3720 cagaaagaga
atgctggcct ctgccggcga actgcagaag ggaaacgagc tggccctgcc 3780
tagcaaatat gtgaacttcc tgtacctggc ctcccactat gagaagctga agggcagccc
3840 tgaggacaac gaacagaaac agctgtttgt ggaacagcat aagcactacc
tggacgagat 3900 catcgagcag atcagcgagt tctccaagag agtgatcctg
gccgacgcca atctggacaa 3960 ggtgctgtct gcctacaaca agcacaggga
caagcctatc agagagcagg ccgagaatat 4020 catccacctg ttcaccctga
caaacctggg cgctcctgcc gccttcaagt actttgacac 4080 caccatcgac
cggaagaggt acaccagcac caaagaggtg ctggacgcca ccctgatcca 4140
ccagagcatc accggcctgt acgagacaag aatcgacctg tctcagctgg gaggcgacaa
4200 gagacctgcc gccactaaga aggccggaca ggccaaaaag aagaagtgag
cggccgctta 4260 attaagctgc cttctgcggg gcttgccttc tggccatgcc
cttcttctct cccttgcacc 4320 tgtacctctt ggtctttgaa taaagcctga
gtaggaagaa aaaaaaaaaa aaaaaaaaaa 4380 aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 4440 aaaaaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaa 4478 <210> SEQ ID NO 64
<211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 64 ccugcucgug gugaccgaag
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 65
<211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 65 acggaagcgg caguccuggc
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 66
<211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 66 cuagaugauu ccaucugcac
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 67
<211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 67 gugcggcaac cuacaugaug
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 68
<211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 68 ggguuccauu acggccagcg
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 69
<211> LENGTH: 100 <212> TYPE: RNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 69 gucaccaauc cugucccuag
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 70
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 70 ttgctggctg tggagcggac 20
<210> SEQ ID NO 71 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 71 gactggcttg
ggcagttcca 20 <210> SEQ ID NO 72 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 72 tgctggggcc ttcctcgagg 20 <210> SEQ ID NO 73
<211> LENGTH: 20 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 73 ccgaaggtag
gagaaggtct 20 <210> SEQ ID NO 74 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 74 atgcacagca gatcctcctc 20 <210> SEQ ID NO 75
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 75 agagagtgag ccaaaggtgc 20
<210> SEQ ID NO 76 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 76 ggcatactca
atgcgtacat 20 <210> SEQ ID NO 77 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 77 gggttccatt acggccagcg 20 <210> SEQ ID NO 78
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 78 aaggctgacc acatccggaa 20
<210> SEQ ID NO 79 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 79 tgccgactcc
agcttccgtc 20 <210> SEQ ID NO 80 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 80 ctgtcagtga aaaccactcg 20 <210> SEQ ID NO 81
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 81 cgtactaaga acgtgccttc 20
<210> SEQ ID NO 82 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 82 caccgcacgt gtgaaccaac
ccgcc 25 <210> SEQ ID NO 83 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 83
aaacggcggg ttggttcaca cgtgc 25 <210> SEQ ID NO 84 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 84 caccgaaaca acaggccggg cgggt 25 <210> SEQ ID NO
85 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
primer <400> SEQUENCE: 85 aaacacccgc ccggcctgtt gtttc 25
<210> SEQ ID NO 86 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 86 caccgacaaa aaaattagcc
gggtg 25 <210> SEQ ID NO 87 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 87
aaaccacccg gctaattttt ttgt 24 <210> SEQ ID NO 88 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 88 caccgtaaat ttctctgata gacta 25 <210> SEQ ID NO
89 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
primer <400> SEQUENCE: 89 aaactagtct atcagagaaa tttac 25
<210> SEQ ID NO 90 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 90 caccgtgttt caatgagagc
attac 25 <210> SEQ ID NO 91 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 91
aaacgtaatg ctctcattga aacac 25 <210> SEQ ID NO 92 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 92 caccggtctc gaactcctga gctc 24 <210> SEQ ID NO 93
<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 93 aaacgagctc aggagttcga gacc 24 <210>
SEQ ID NO 94 <211> LENGTH: 20 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 94 agtgaagtgg cgcattcttg 20
<210> SEQ ID NO 95 <211> LENGTH: 21 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 95 caccctttcc aaatcctcag c
21 <210> SEQ ID NO 96 <211> LENGTH: 25 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic primer <400> SEQUENCE: 96 caccgtgggg
gttagaccca atatc 25 <210> SEQ ID NO 97 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 97
aaacgatatt gggtctaacc cccac 25 <210> SEQ ID NO 98 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 98 caccgacccc acagtggggc cacta 25 <210> SEQ ID NO
99 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
primer <400> SEQUENCE: 99 aaactagtgg ccccactgtg gggtc 25
<210> SEQ ID NO 100 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 100 caccgagggc cggttaatgt
ggctc 25 <210> SEQ ID NO 101 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 101
aaacgagcca cattaaccgg ccctc 25 <210> SEQ ID NO 102
<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 102 caccgtcacc aatcctgtcc ctag 24 <210>
SEQ ID NO 103 <211> LENGTH: 24 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 103 aaacctaggg acaggattgg
tgac 24 <210> SEQ ID NO 104 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 104
caccgccggc cctgggaata taagg 25 <210> SEQ ID NO 105
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 105 aaacccttat attcccaggg ccggc 25
<210> SEQ ID NO 106 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 106 caccgcgggc ccctatgtcc
acttc 25 <210> SEQ ID NO 107 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 107
aaacgaagtg gacatagggg cccgc 25 <210> SEQ ID NO 108
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 108 actcctttca tttgggcagc 20 <210> SEQ
ID NO 109
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 109 ggttctggca aggagagaga 20 <210> SEQ
ID NO 110 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
primer <400> SEQUENCE: 110 caccgcggag agcttcgtgc taaac 25
<210> SEQ ID NO 111 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 111 aaacgtttag cacgaagctc
tccgc 25 <210> SEQ ID NO 112 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 112
caccgcctgc tcgtggtgac cgaag 25 <210> SEQ ID NO 113
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 113 aaaccttcgg tcaccacgag caggc 25
<210> SEQ ID NO 114 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 114 caccgcagca accagacgga
caagc 25 <210> SEQ ID NO 115 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 115
aaacgcttgt ccgtctggtt gctgc 25 <210> SEQ ID NO 116
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 116 caccgaggcg gccagcttgt ccgtc 25
<210> SEQ ID NO 117 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 117 aaacgacgga caagctggcc
gcctc 25 <210> SEQ ID NO 118 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 118
caccgcgttg ggcagttgtg tgaca 25 <210> SEQ ID NO 119
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 119 aaactgtcac acaactgccc aacgc 25
<210> SEQ ID NO 120 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 120 caccgacgga agcggcagtc
ctggc 25 <210> SEQ ID NO 121 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 121
aaacgccagg actgccgctt ccgtc 25 <210> SEQ ID NO 122
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 122 agaaggaaga ggctctgcag 20 <210> SEQ
ID NO 123 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
primer <400> SEQUENCE: 123 ctctttgatc tgcgccttgg 20
<210> SEQ ID NO 124 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 124 caccgccggg tgacagtgct
tcggc 25 <210> SEQ ID NO 125 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 125
aaacgccgaa gcactgtcac ccggc 25 <210> SEQ ID NO 126
<211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 126 caccgtgcgg caacctacat gatg 24
<210> SEQ ID NO 127 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 127 aaaccatcat gtaggttgcc
gcac 24 <210> SEQ ID NO 128 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 128
caccgctaga tgattccatc tgcac 25 <210> SEQ ID NO 129
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 129 aaacgtgcag atggaatcat ctagc 25
<210> SEQ ID NO 130 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 130 caccgaggtt cacttgattt
ccac 24 <210> SEQ ID NO 131 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 131
aaacgtggaa atcaagtgaa cctc 24 <210> SEQ ID NO 132 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 132 caccgccgca cagacttcag tcacc 25 <210> SEQ ID NO
133 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
primer <400> SEQUENCE: 133 aaacggtgac tgaagtctgt gcggc 25
<210> SEQ ID NO 134 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 134 caccgctggc gatgcctcgg
ctgc 24 <210> SEQ ID NO 135 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 135
aaacgcagcc gaggcatcgc cagc 24 <210> SEQ ID NO 136 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 136 tggggatgaa gctagaaggc 20 <210> SEQ ID NO 137
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 137 aatctgggtt ccgttgccta 20 <210> SEQ
ID NO 138 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
primer <400> SEQUENCE: 138 caccgacaat gtgtcaactc ttgac 25
<210> SEQ ID NO 139 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 139 aaacgtcaag agttgacaca
ttgtc 25 <210> SEQ ID NO 140 <211> LENGTH: 25
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 140
caccgtcatc ctcctgacaa tcgat 25 <210> SEQ ID NO 141
<211> LENGTH: 25 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic primer
<400> SEQUENCE: 141 aaacatcgat tgtcaggagg atgac 25
<210> SEQ ID NO 142 <211> LENGTH: 24 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 142 caccggtgac aagtgtgatc
actt 24 <210> SEQ ID NO 143 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 143
aaacaagtga tcacacttgt cacc 24 <210> SEQ ID NO 144 <211>
LENGTH: 25 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 144 caccgacaca gcatggacga cagcc 25
<210> SEQ ID NO 145 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 145 aaacggctgt cgtccatgct
gtgtc 25 <210> SEQ ID NO 146 <211> LENGTH: 24
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 146
caccgatctg gtaaagatga ttcc 24 <210> SEQ ID NO 147 <211>
LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 147 aaacggaatc atctttacca gatc 24 <210> SEQ ID NO
148 <211> LENGTH: 25 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
primer <400> SEQUENCE: 148 caccgttgta tttccaaagt cccac 25
<210> SEQ ID NO 149 <211> LENGTH: 25 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic primer <400> SEQUENCE: 149 aaacgtggga ctttggaaat
acaac 25 <210> SEQ ID NO 150 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic primer <400> SEQUENCE: 150
ctcaacctgg ccatctctga 20 <210> SEQ ID NO 151 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic primer <400>
SEQUENCE: 151 cccgagtagc agatgaccat 20 <210> SEQ ID NO 152
<400> SEQUENCE: 152 000 <210> SEQ ID NO 153 <400>
SEQUENCE: 153 000 <210> SEQ ID NO 154 <400> SEQUENCE:
154 000 <210> SEQ ID NO 155 <400> SEQUENCE: 155 000
<210> SEQ ID NO 156 <400> SEQUENCE: 156 000 <210>
SEQ ID NO 157 <400> SEQUENCE: 157 000 <210> SEQ ID NO
158 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 158 cctcctcctg gtgaccggag agg
23 <210> SEQ ID NO 159 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 159
cttgctggtg gtgacagaag agg 23 <210> SEQ ID NO 160 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 160 actgcttgtt gtgaccgatg ggg 23 <210>
SEQ ID NO 161 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 161 ccagcacatg
gtgaccaaag gag 23 <210> SEQ ID NO 162 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 162 cctgcttgtg gtgacctaat aag 23 <210> SEQ ID NO
163 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 163 ccaggtggtg gtgagcgaag agg
23 <210> SEQ ID NO 164 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 164
cctaatcgtg gtcaccgaaa gag 23 <210> SEQ ID NO 165 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 165 cctgctggtg gtgacctatg agg 23
<210> SEQ ID NO 166 <211> LENGTH: 23 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 166 cctgctactg
gtgtccgaag tgg 23 <210> SEQ ID NO 167 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 167 gcagctcgag gtgacggaag agg 23 <210> SEQ ID NO
168 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 168 catgctccta gtgacagaag ggg
23 <210> SEQ ID NO 169 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 169
cccgctggtg ggaaccgaag gag 23 <210> SEQ ID NO 170 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 170 cctgggcgta gtgagcgaag cgg 23 <210>
SEQ ID NO 171 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 171 cctggccgcg
gtgaccgagg cgg 23 <210> SEQ ID NO 172 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 172 cctgcccaag gttaccgaag tgg 23 <210> SEQ ID NO
173 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 173 cctgctccag ttgaccaaag agg
23 <210> SEQ ID NO 174 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 174
actgcacgtg gtgaaccaag agg 23 <210> SEQ ID NO 175 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 175 gctgcccatg gtgacagaag ggg 23 <210>
SEQ ID NO 176 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 176 actgctggtg
gtgaacgaaa ggg 23 <210> SEQ ID NO 177 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 177 gctgctagtg gtgacggaaa gag 23 <210> SEQ ID NO
178 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 178 tctgctattg gtgtccgaag tgg
23 <210> SEQ ID NO 179 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 179
cgagctcgtg gttacagaag ggg 23 <210> SEQ ID NO 180 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 180 ccagctcgtc gggacagaag agg 23 <210>
SEQ ID NO 181 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 181 ccacctcggg
gtggccgaag cgg 23 <210> SEQ ID NO 182 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 182 ccagctagtg ttgactgaag ggg 23 <210> SEQ ID NO
183 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 183
cctggctgtg gtgacagaag ggg 23 <210> SEQ ID NO 184 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 184 cctgcacgtg atgacctaac agg 23 <210>
SEQ ID NO 185 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 185 cctgctcgtg
ctgtccgcag cag 23 <210> SEQ ID NO 186 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 186 cctgctggtg gtgacggcag cag 23 <210> SEQ ID NO
187 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 187 gctgctggtg gtgcccgatg ggg
23 <210> SEQ ID NO 188 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 188
cctgctcgtg gtgatatgag ggg 23 <210> SEQ ID NO 189 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 189 ccagctggtg gtgactcaag gag 23 <210>
SEQ ID NO 190 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 190 ccttctcgtg
gagacagatg agg 23 <210> SEQ ID NO 191 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 191 ccttctcgtg gtgcctggag tgg 23 <210> SEQ ID NO
192 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 192 ccttctcatg gtggcggaag gag
23 <210> SEQ ID NO 193 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 193
ccttctcatg gtgacagtag agg 23 <210> SEQ ID NO 194 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 194 cctggtcgtg ttgagtgaag ggg 23 <210>
SEQ ID NO 195 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 195 cctggtagtg
gtgaccacag tgg 23 <210> SEQ ID NO 196 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 196 cctggtggtg gtgaccatag agg 23 <210> SEQ ID NO
197 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 197 cctgcccgtg gtaatcgaac cgg
23 <210> SEQ ID NO 198 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 198
cctgcccgtg gtgaacgctg tgg 23 <210> SEQ ID NO 199 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 199 cctgcacgtg gtgacagcat ggg 23 <210>
SEQ ID NO 200 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 200 cctgcgtgtg
gaggccgaag ggg 23 <210> SEQ ID NO 201 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 201
cctgccggtg gtgacctatg tgg 23 <210> SEQ ID NO 202 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 202 cctgctggtg ctgcctgaag ggg 23 <210>
SEQ ID NO 203 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 203 cctgctggtg
ctggccgatg ggg 23 <210> SEQ ID NO 204 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 204 cctgctagtg gaaaccgcag ggg 23 <210> SEQ ID NO
205 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 205 cctgctggtg gtaccagaag agg
23 <210> SEQ ID NO 206 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 206
cctgctggtg gtaccagaag agg 23 <210> SEQ ID NO 207 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 207 cctgctggtg gtggcagcag tgg 23 <210>
SEQ ID NO 208 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 208 cctgctagtg
gtggcagtag tgg 23 <210> SEQ ID NO 209 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 209 cctgcttatg gggactgaag ggg 23 <210> SEQ ID NO
210 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 210 cctgcttctg gtgtcagaag tgg
23 <210> SEQ ID NO 211 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 211
cctgctgctg gtggccgacg tgg 23 <210> SEQ ID NO 212 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 212 cctgctgttg gtgactcaag gag 23 <210>
SEQ ID NO 213 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 213 cctgctgttg
gtgactcaag gag 23 <210> SEQ ID NO 214 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 214 cctgcttggg ctgaccgtag ggg 23 <210> SEQ ID NO
215 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 215 cctgctaggg gagacagaag agg
23 <210> SEQ ID NO 216 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 216
cctgctcctg ctgcacgaag tgg 23 <210> SEQ ID NO 217 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 217 cctgctcttg gggactgaat tgg 23 <210>
SEQ ID NO 218 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 218 cctgctcctg
gttcccaaag agg 23 <210> SEQ ID NO 219 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 219 cctgctcttg gtcactgcag agg 23 <210>
SEQ ID NO 220 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 220 cctgctcctg
gtgcaagaag ggg 23 <210> SEQ ID NO 221 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 221 cctgctcctg gtgcaagaag ggg 23 <210> SEQ ID NO
222 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 222 cctgctcttg gtggaagaag tgg
23 <210> SEQ ID NO 223 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 223
cctgctcctg gtgtgggaag agg 23 <210> SEQ ID NO 224 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 224 cctgctcttg gtgaacacag gag 23 <210>
SEQ ID NO 225 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 225 cctgctcaag
gagacagaag tgg 23 <210> SEQ ID NO 226 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 226 cctgctccag gtgaatgaag agg 23 <210> SEQ ID NO
227 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 227 cctgctctgg gtgacggagg gag
23 <210> SEQ ID NO 228 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 228
cctgctcgcg gtgggctaag ggg 23 <210> SEQ ID NO 229 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 229 cagttccatt accgccagcg ggg 23 <210>
SEQ ID NO 230 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 230 ggtttccatt
cctgccagcg ggg 23 <210> SEQ ID NO 231 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 231 gtgtcccgtt acggccagct cgg 23 <210> SEQ ID NO
232 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 232 agcttccctt agggccagcg aag
23 <210> SEQ ID NO 233 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 233
ggggtcccta agggccagcg agg 23 <210> SEQ ID NO 234 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 234 gcgtcgcact acggccagcg agg 23 <210>
SEQ ID NO 235 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 235 gcgtgccact
acggccagct aag 23 <210> SEQ ID NO 236 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 236 gggtccccat agggccagcg agg 23 <210> SEQ ID NO
237 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide
<400> SEQUENCE: 237 ggctcccatt gcagccagcg tgg 23 <210>
SEQ ID NO 238 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 238 ttgttccatt
atggccagag aag 23 <210> SEQ ID NO 239 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 239 ttgttccatt atggccagag aag 23 <210> SEQ ID NO
240 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 240 ggataccaat acggccaacg agg
23 <210> SEQ ID NO 241 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 241
ggtttccttt actgccggcg agg 23 <210> SEQ ID NO 242 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 242 gggtgccagt ggggccagcg agg 23 <210>
SEQ ID NO 243 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 243 gggttccttc
agggccagag agg 23 <210> SEQ ID NO 244 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 244 gggctccatc acagccagca ggg 23 <210> SEQ ID NO
245 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 245 aggttccagt tcggccagtg aag
23 <210> SEQ ID NO 246 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 246
gtgttccatt ccggcccgcc agg 23 <210> SEQ ID NO 247 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 247 ggcttccaat aaggccaacg agg 23 <210>
SEQ ID NO 248 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 248 gggttccagt
gtggccagca agg 23 <210> SEQ ID NO 249 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 249 gggttccttt tctgccagct tgg 23 <210> SEQ ID NO
250 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 250 gggttccttt tctgccagct tgg
23 <210> SEQ ID NO 251 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 251
gggttccttt tctgccagct tgg 23 <210> SEQ ID NO 252 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 252 gggttccttt agggccagga tgg 23 <210>
SEQ ID NO 253 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 253 gggttctatt
ccagccagct tgg 23 <210> SEQ ID NO 254 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 254 gggtttcagt aaggccaggg tgg 23 <210> SEQ ID NO
255 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence:
Synthetic
oligonucleotide <400> SEQUENCE: 255 gggccccatt acagccatcg tgg
23 <210> SEQ ID NO 256 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 256
ggggtccatg acagacagcg tgg 23 <210> SEQ ID NO 257 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 257 ggggtccatt cccgccagag tgg 23 <210>
SEQ ID NO 258 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 258 gggttacatc
aaggccaccg agg 23 <210> SEQ ID NO 259 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 259 gggttcaatt acggaaaggg agg 23 <210> SEQ ID NO
260 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 260 gggttccact ccttccagcg agg
23 <210> SEQ ID NO 261 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 261
gggttccatt atgacaagca tgg 23 <210> SEQ ID NO 262 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 262 gggttccatt ctggcaaggg tgg 23 <210>
SEQ ID NO 263 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 263 gggttccatt
gctggcagag tgg 23 <210> SEQ ID NO 264 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 264 gggttccatg aaggccaaag ggg 23 <210> SEQ ID NO
265 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 265 gggttccaat ccggccactg tgg
23 <210> SEQ ID NO 266 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 266
gggttccact actgcctgct tgg 23 <210> SEQ ID NO 267 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 267 gggttccgtt ccggccactg ggg 23 <210>
SEQ ID NO 268 <211> LENGTH: 23 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 268 gggtttcatt
ggagccagcg tgg 23 <210> SEQ ID NO 269 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <400>
SEQUENCE: 269 gggtttcatt acgctcagct aag 23 <210> SEQ ID NO
270 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 270 gggttacctt acgacctgcg tgg
23 <210> SEQ ID NO 271 <211> LENGTH: 23 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Description of Artificial
Sequence: Synthetic oligonucleotide <400> SEQUENCE: 271
gggctccatt ccggccatgg tgg 23 <210> SEQ ID NO 272 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<400> SEQUENCE: 272 gtcaccaatc ctgtccctag 20 <210> SEQ
ID NO 273 <211> LENGTH: 96 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 273 caggccctgg
aaccccccca ccttctcccc agccctgctc gtggtgaccg aggactgccg 60
cttccgtgtc acacaactgc ccaacgggcg tgactt 96 <210> SEQ ID NO
274 <211> LENGTH: 119 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
polynucleotide <400> SEQUENCE: 274 cggcaggctg acagccaggt
gactgaagtc tgtgcggcaa cctacatgat ggggaatgag 60 ttgaccttcc
tagatgattc catctgcacg ggcacctcca gtggaaatca agtgaacct 119
<210> SEQ ID NO 275 <211> LENGTH: 81 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <400> SEQUENCE: 275 cggcaggctg
acagccaggt gactgaagtc tgtgcggcaa cctacatgca cgggcacctc 60
cagtggaaat caagtgaacc t 81 <210> SEQ ID NO 276 <211>
LENGTH: 101 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<220> FEATURE: <223> OTHER INFORMATION: Description of
Combined DNA/RNA Molecule: Synthetic oligonucleotide <400>
SEQUENCE: 276 gcctgctcgt ggtgaccgaa gguuuuagag cuagaaauag
caaguuaaaa uaaggcuagu 60 ccguuaucaa cuugaaaaag uggcaccgag
ucggugcuuu u 101 <210> SEQ ID NO 277 <211> LENGTH: 101
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <220> FEATURE:
<223> OTHER INFORMATION: Description of Combined DNA/RNA
Molecule: Synthetic oligonucleotide <400> SEQUENCE: 277
gctagatgat tccatctgca cguuuuagag cuagaaauag caaguuaaaa uaaggcuagu
60 ccguuaucaa cuugaaaaag uggcaccgag ucggugcuuu u 101 <210>
SEQ ID NO 278 <211> LENGTH: 100 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <220> FEATURE: <223> OTHER
INFORMATION: Description of Combined DNA/RNA Molecule: Synthetic
oligonucleotide <400> SEQUENCE: 278 gggttccatt acggccagcg
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 279
<211> LENGTH: 100 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic oligonucleotide
<400> SEQUENCE: 279 gtcaccaatc ctgtccctag guuuuagagc
uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu
ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 280 <211>
LENGTH: 101 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<220> FEATURE: <223> OTHER INFORMATION: Description of
Combined DNA/RNA Molecule: Synthetic oligonucleotide <400>
SEQUENCE: 280 gcctgctcgt ggtgaccgaa gguuuuagag cuagaaauag
caaguuaaaa uaaggcuagu 60 ccguuaucaa cuugaaaaag uggcaccgag
ucggugcuuu u 101 <210> SEQ ID NO 281 <211> LENGTH: 101
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <220> FEATURE:
<223> OTHER INFORMATION: Description of Combined DNA/RNA
Molecule: Synthetic oligonucleotide <400> SEQUENCE: 281
gctagatgat tccatctgca cguuuuagag cuagaaauag caaguuaaaa uaaggcuagu
60 ccguuaucaa cuugaaaaag uggcaccgag ucggugcuuu u 101 <210>
SEQ ID NO 282 <211> LENGTH: 100 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <220> FEATURE: <223> OTHER
INFORMATION: Description of Combined DNA/RNA Molecule: Synthetic
oligonucleotide <400> SEQUENCE: 282 gggttccatt acggccagcg
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 283
<211> LENGTH: 100 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <220> FEATURE: <223> OTHER INFORMATION:
Description of Combined DNA/RNA Molecule: Synthetic oligonucleotide
<400> SEQUENCE: 283 gtcaccaatc ctgtccctag guuuuagagc
uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac uugaaaaagu
ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 284 <211>
LENGTH: 101 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<220> FEATURE: <223> OTHER INFORMATION: Description of
Combined DNA/RNA Molecule: Synthetic oligonucleotide <400>
SEQUENCE: 284 gcctgctcgt ggtgaccgaa gguuuuagag cuagaaauag
caaguuaaaa uaaggcuagu 60 ccguuaucaa cuugaaaaag uggcaccgag
ucggugcuuu u 101 <210> SEQ ID NO 285 <211> LENGTH: 101
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <220> FEATURE:
<223> OTHER INFORMATION: Description of Combined DNA/RNA
Molecule: Synthetic oligonucleotide <400> SEQUENCE: 285
gctagatgat tccatctgca cguuuuagag cuagaaauag caaguuaaaa uaaggcuagu
60 ccguuaucaa cuugaaaaag uggcaccgag ucggugcuuu u 101 <210>
SEQ ID NO 286 <211> LENGTH: 100 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <220> FEATURE: <223> OTHER
INFORMATION: Description of Combined DNA/RNA Molecule: Synthetic
oligonucleotide <400> SEQUENCE: 286 gggttccatt acggccagcg
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100
<210> SEQ ID NO 287 <211> LENGTH: 100 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic oligonucleotide <220> FEATURE: <223> OTHER
INFORMATION: Description of Combined DNA/RNA Molecule: Synthetic
oligonucleotide <400> SEQUENCE: 287 gtcaccaatc ctgtccctag
guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60 cguuaucaac
uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID NO 288
<211> LENGTH: 23 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <220> FEATURE: <221> NAME/KEY:
modified_base <222> LOCATION: (21)..(21) <223> OTHER
INFORMATION: a, c, t, g, unknown or other <400> SEQUENCE: 288
cctgctcgtg gtgaccgaag ngg 23 <210> SEQ ID NO 289 <211>
LENGTH: 23 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Description of Artificial Sequence: Synthetic oligonucleotide
<220> FEATURE: <221> NAME/KEY: modified_base
<222> LOCATION: (21)..(21) <223> OTHER INFORMATION: a,
c, t, g, unknown or other <400> SEQUENCE: 289 gctgcccatg
gtgacagaag ngg 23 <210> SEQ ID NO 290 <211> LENGTH: 23
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <220> FEATURE:
<221> NAME/KEY: modified_base <222> LOCATION:
(21)..(21) <223> OTHER INFORMATION: a, c, t, g, unknown or
other <400> SEQUENCE: 290 gggttccatt acggccagcg ngg 23
<210> SEQ ID NO 291 <211> LENGTH: 100 <212> TYPE:
RNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Description of Artificial Sequence:
Synthetic polynucleotide <400> SEQUENCE: 291 ggguuccauu
acggccagcg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 100 <210> SEQ ID
NO 292 <211> LENGTH: 34 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Description of Artificial Sequence: Synthetic
oligonucleotide <400> SEQUENCE: 292 gtttaattga gttgtcatat
gttaataacg gtat 34 <210> SEQ ID NO 293 <211> LENGTH:
101 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Description of
Artificial Sequence: Synthetic oligonucleotide <220> FEATURE:
<223> OTHER INFORMATION: Description of Combined DNA/RNA
Molecule: Synthetic oligonucleotide <400> SEQUENCE: 293
gcctgctcgt ggtgaccgaa gguuuuagag cuagaaauag caaguuaaaa uaaggcuagu
60 ccguuaucaa cuugaaaaag ugggaccgag ucggugcuuu u 101
* * * * *
References