U.S. patent application number 17/260544 was filed with the patent office on 2022-05-05 for generating cik nkt cells from cord blood.
The applicant listed for this patent is NantKwest, Inc.. Invention is credited to Rohit DUGGAL, Ranjeet SINHA.
Application Number | 20220133789 17/260544 |
Document ID | / |
Family ID | 1000006151357 |
Filed Date | 2022-05-05 |
United States Patent
Application |
20220133789 |
Kind Code |
A1 |
DUGGAL; Rohit ; et
al. |
May 5, 2022 |
GENERATING CIK NKT CELLS FROM CORD BLOOD
Abstract
Provided herein are methods and customized media compositions
for culturing CIK NKT cells.
Inventors: |
DUGGAL; Rohit; (San Diego,
CA) ; SINHA; Ranjeet; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NantKwest, Inc. |
San Diego |
CA |
US |
|
|
Family ID: |
1000006151357 |
Appl. No.: |
17/260544 |
Filed: |
July 1, 2019 |
PCT Filed: |
July 1, 2019 |
PCT NO: |
PCT/US2019/040145 |
371 Date: |
January 14, 2021 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
62696131 |
Jul 10, 2018 |
|
|
|
Current U.S.
Class: |
424/93.71 |
Current CPC
Class: |
C12N 5/0646 20130101;
A61K 35/17 20130101; C12N 2501/515 20130101; C12N 2501/51 20130101;
C12N 2501/2302 20130101 |
International
Class: |
A61K 35/17 20060101
A61K035/17; C12N 5/0783 20060101 C12N005/0783 |
Claims
1. A population of CIK NKT cells, wherein greater than 50% of the
cells in the population express CD 56 and CD3 and less than 10% of
the cells in the population express Va24.
2. The population of CIK NKT cells of claim 1, wherein the CIK NKT
cells can kill a target cell in the absence of
alpha-galactosylceramide (Gal-Cer).
3. The population of CIK NKT cells of claim 1, wherein the target
cell is a cancer cell.
4. The population of CIK NKT cells of claim 1, wherein the cancer
cell line is selected from the group consisting of a myelogenous
leukemia cell, a medulloblastoma cell, and a monocytic cell.
5. The population of CIK NKT cells of claim 3, wherein the cancer
cell is selected from the group consisting of a K562 cell, a Daudi
cell, a DAOY cell, and a THP-1 cell.
6. The population of CIK NKT cells of claim 2, wherein the CIK NKT
cells kill a plurality of the target cells at an EC50 of between
1.0 and 10.0.
7. The population of CIK NKT cells of claim 6, wherein the CIK NKT
cells can kill the target cells at a EC50 that is no less than 90%
and no greater than 110% of the EC50 at which the CIK NKT cells
killing the target cells in the presence of Gal-Cer.
8. A composition comprising a plurality of CIK NKT cells from the
population of CIK NKT cells of claim 1, and a physiologically
acceptable excipient.
9. (canceled)
10. A method of enriching CIK NKT cells from a cord blood sample,
the method comprising: isolating mononuclear cells from the cord
blood sample; and contacting the isolated mononuclear cells with
one or more agents selected from the group consisting of IL-7,
ALT-803 or IL-15, FLT3 ligand, and Gal-Cer, whereby enriching CIK
NKT cells.
11. The method of claim 10, wherein the IL-7, if present, is in a
concentration ranging from 5 to 20 ng/mL.
12. The method of claim 10, wherein the ALT-803, if present, is in
a concentration ranging from 100 to 300 ng/mL.
13. The method of claim 10, wherein the FLT3 ligand, if present, is
in a concentration ranging from 5 to 20 ng/mL.
14. The method of claim 10, wherein the Gal-Cer is present in a
concentration ranging from 2 to 10 .mu.g/mL.
15. The method of claim 10, wherein the method further comprises
isolating the enriched CIK NKT cells from the rest of the cord
blood sample.
16. The method of claim 15, wherein the method further comprises
contacting the isolated CIK NKT cells with anti-CD3, anti-CD28, and
IL2 to expand the CIK NKT cells.
17. The method of claim 16, wherein the method further comprises
contacting the isolated CIK NKT cells with Gal-Cer.
18. The method of claim 17, wherein the Gal-Cer is a present in a
form of a Gal-Cer loaded CD1d tetramer.
19. The method of claim 16, wherein the anti-CD3 antibody is
present in an amount of 5 ng/mL to 60 ng/mL.
20. The method of claim 16, wherein the anti-CD28 antibody is
present in an amount of 0.1 .mu.g/mL to 2 .mu.g/mL.
21. The method of claim 16, wherein IL-2 is present in a
concentration of 50 ng/mL to 500 ng/mL.
22. The method of claim 16, wherein the production of CIK NKT cells
does not include interferon-gamma.
23. A method of treating cancer or viral infection in a patient in
need thereof, the method comprising administering to the patient a
therapeutically effective amount of CIK NKT cells from the
population of CIK NKT cells of claim 1, thereby treating
cancer.
24. The method of claim 23, wherein about 1.times.10.sup.8 to about
1.times.10.sup.11 cells per m.sup.2 of body surface area of the
patient are administered to the patient.
25. The method of claim 23, wherein the cancer is selected from the
group consisting of a leukemia, a lymphoma, polycythemia vera,
multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain
disease, a sarcoma and a carcinoma.
26. The method of claim 25, wherein the cells are administered to
the patient by a route selected from the group consisting of
intravenous, intraperitoneal, and subcutaneous.
27. The method of claim 23, wherein the method further comprises
administering an antibody.
28. A population of CIK NKT cells produced by the methods of claim
10.
29. The population of CIK NKT cells of claim 1, wherein the CIK NKT
cells express a CAR and/or a cytokine.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a national stage under 35 U.S.C. .sctn.
371 of PCT application no. PCT/US2019/040145, filed Jul. 1, 2019,
which claims priority benefit of U.S. Provisional Application No.
62/696,131 filed Jul. 10, 2018, each of which application is herein
incorporated by reference.
BACKGROUND
[0002] Natural killer T cells (NKT cells) represent a subset of T
lymphocytes that express natural killer (NK) cell surface markers.
A subset of NKT cells, termed invariant NKT cells (iNKT), express a
highly restricted T cell receptor (TCR). Although iNKT cells play
an important role in linking innate and adaptive immune responses
and have been implicated in various diseases, such as infectious
diseases, allergy, asthma, autoimmunity, and tumor surveillance
(Juno et al. PLoS Pathog. 2012; 8(8)), their activation typically
requires CD1d-restricted lipid ligands alpha-galactosylceramide
(Gal-Cer). Procedures necessary to introduce Gal-Cer (e.g., in the
form of a Gal-Cer/CD1d tetramer) could significantly increase the
cost of the treating patients using these NKT cells and in some
cases limit the scope of their therapeutical utility. Thus, a need
remains for a safe and cost-effective NKT cell therapy that can be
used to treat patients having a broad range of tissue types.
BRIEF SUMMARY
[0003] Provided herein are CIK NKT cells that can be activated in
the absence of Gal-Cer. In some embodiments, greater than 50% of
the cells in the CIK NKT cell population express CD56 and CD3 and
less than 10% of the cells in the population express Va24. Also
provided are compositions and kits comprising a plurality of CIK
NKT cells from the population. Methods of producing the CIK NKT
cells and using the cells to treat cancer are also provided.
[0004] This disclosure provides a population of CIK NKT cells,
wherein greater than 50% of the cells in the population express CD
56 and CD3 and less than 10% of the cells in the population express
Va24.
[0005] Optionally, the population of CIK NKT cells can kill a
target cell in the absence of alpha-galactosylceramide (Gal-Cer).
Optionally, the target cell is a cancer cell. The cancer cell line
may be selected from the group consisting of a myelogenous leukemia
cell, a medulloblastoma cell, and a monocytic cell. Optionally, the
cancer cell is selected from the group consisting of a K562 cell, a
Daudi cell, a DAOY cell, and a THP-1 cell.
[0006] Optionally, the CIK NKT cells kill a plurality of the target
cells at an EC50 of between 1.0 and 10.0. Optionally, the CIK NKT
cells can kill the target cells at a EC50 that is no less than 90%
and no greater than 110% of the IC50 at which the CIK NKT cells
killing the target cells in the presence of Gal-Cer.
[0007] This disclosure also provides a composition comprising a
plurality of CIK NKT cells from any of the populations of CIK NKT
cells described above, and a physiologically acceptable
excipient.
[0008] Also provided is a kit for treating cancer comprising a
plurality of CIK NKT cells from any of the populations of CIK NKT
cells described above and a container and/or a label indicating the
kit is for treating cancer.
[0009] Also provided is a method of enriching CIK NKT cells from a
cord blood sample, the method comprising: isolating mononuclear
cells from the cord blood sample; and contacting the isolated
mononuclear cells with one or more agents selected from the group
consisting of IL-7, ALT-803 or IL-15, FLT3 ligand, and Gal-Cer,
whereby enriching CIK NKT cells. IL-7, if present, may be in a
concentration ranging from 5 to 20 ng/mL. ALT-803, if present, may
be in a concentration ranging from 100 to 300 ng/mL. FLT3 ligand,
if present, may be in a concentration ranging from 5 to 20 ng/mL.
Gal-Cer, if present, may be in a concentration ranging from 2 to 10
.mu.g/mL.
[0010] Optionally, the method further comprises isolating the
enriched CIK NKT cells from the rest of the cord blood sample.
Optionally, the method further comprises contacting the isolated
CIK NKT cells with anti-CD3, anti-CD28, and IL2 to expand the CIK
NKT cells. Optionally, the method further comprises contacting the
separated CIK NKT cells with Gal-Cer. Optionally, the Gal-Cer is
used in a form of a Gal-Cer loaded CD1d tetramer. Optionally, the
anti-CD3 antibody may be present in an amount of 5 ng/mL to 60
ng/mL. Optionally, the anti-CD28 antibody is present in an amount
of 0.1 .mu.g/mL to 2 .mu.g/mL. Optionally, IL-2 is present in a
concentration of 50 ng/mL to 500 ng/mL. Optionally, enriching
and/or expansion of CIK NKT cells does not include
interferon-gamma.
[0011] Also provided is a population of CIK NKT cells produced by
the methods of enriching, isolating and expanding CIK NKT cells
described above.
[0012] Also provided is a method of treating cancer or viral
infection in a patient in need thereof, the method comprising
administering to the patient a therapeutically effective amount of
CIK NKT cells from any of the populations of CIK NKT cells as
described above, thereby treating cancer. Optionally,
1.times.10.sup.8 to about 1.times.10.sup.11 CIK NKT cells per
m.sup.2 of body surface area of the patient are administered to the
patient. Optionally, the cancer is selected from the group
consisting of a leukemia, a lymphoma, polycythemia vera, multiple
myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, a
sarcoma and a carcinoma. Optionally the cells are administered to
the patient by a route selected from the group consisting of
intravenous, intraperitoneal, and subcutaneous. Optionally, the
method further comprises administering an antibody.
[0013] Also provided is a population of CIK NKT cells of claim 1,
wherein the CIK NKT cells express a CAR and/or a cytokine, and
greater than 50% of the cells in the CIK NKT cell population
express CD56 and CD3 and less than 10% of the cells in the
population express Va24.
[0014] The foregoing general description and the following detailed
description are exemplary and explanatory and are intended to
provide further explanation of the disclosure. Other objects,
advantages and novel features will be readily apparent to those
skilled in the art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The objects, features and advantages will be more readily
appreciated upon reference to the following disclosure when
considered in conjunction with the accompanying drawings.
[0016] FIG. 1 is a schematic representation of the pathway which
CIK NKT cells employ to kill target cells.
[0017] FIG. 2A shows the results of flow cytometry analysis of CIK
NKT cells stained with CD3, CD56, and Va24. FIG. 2A is the forward
scatter and size scatter diagram; FIG. 2B shows CD3 and CD56
diagrams; and FIG. 2C shows the Va24 diagram.
[0018] FIG. 3A shows the killing of DAOY cells in the presence
(represented by circles) or absence (represented by squares) by the
cord blood CIK NKT cells. FIG. 3B shows the killing of
luciferase-expressing THP 1 cells by cord blood CIK NKT cells
(represented by squares) or peripheral blood iNKT cells
(represented by circles).
[0019] FIG. 4 shows the killing of K562 cells, DAOY cells, and
Daudi cells by cord blood CIK NKT cells.
DETAILED DESCRIPTION
Overview
[0020] This application provides cytokine induced killer NKT cells
(CIK NKT cells) that can kill target cells in a non-CD1d restricted
manner, i.e., independent of the formation of the Gal-Cer/CD1d
tetramer. The CIK NKT cells can be used to target a broad range of
target cells and will not trigger Graft-versus-host disease (GVHD).
GVHD occurs due to invasive ability of lymphocytes to infiltrate
and cause extensive inflammation in organs such as the gut, skin
and liver. It has been shown that CIK NKTs do not express chemokine
receptors important for targeting to GVHD organs but do express
receptors that facilitate homing to tumors, thus they will not
trigger GVHD. As compared to existing technologies of generating
CIK NKT cells, which normally produce a heterogenous population
consisting of CD3+, CD56+ single positive cells and CD3+/CD56+
double positive cells, and the double positive cells typically 30%
or less, the present methods produce a cell population that
consists of predominantly CD3+/CD56+ double positive cells. In one
aspect, at least 50% of the CIK NKT cells in the population express
both CD56 and CD3 and less than 10% of the cells in the population
express Va24. The method of producing CIK NKT cells do not require
the exposing the cells to interferon-gamma, which saves cost. Thus,
the application provides a safe and cost-effective NKT cell therapy
that can be used broadly to treat various diseases, e.g., cancers,
without causing clinically adverse symptoms such as GVHD.
[0021] The disclosure also provides compositions and kits
comprising a plurality of CIK NKT cells from the population.
Methods of producing the CIK NKT cells and using the CIK NKT cells
to treat cancer are also provided.
Terminology
[0022] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art.
[0023] In this specification and in the claims that follow,
reference will be made to a number of terms that shall be defined
to have the following meanings:
[0024] The terminology used herein is for the purpose of describing
particular embodiments only and is not intended to be limiting. As
used herein, the singular forms "a," "an" and "the" are intended to
include the plural forms as well, unless the context clearly
indicates otherwise. Thus, for example, reference to "a natural
killer cell" includes a plurality of natural killer cells.
[0025] All numerical designations, e.g., pH, temperature, time,
concentration, amounts, and molecular weight, including ranges, are
approximations which are varied (+) or (-) by increments of 0.1 or
1.0, where appropriate. It is to be understood, although not always
explicitly stated, that all numerical designations may be preceded
by the term "about."
[0026] As used herein, "+", when used to indicate the presence of a
particular cellular marker, means that the cellular marker is
detectably present in fluorescence activated cell sorting over an
isotype control; or is detectable above background in quantitative
or semi-quantitative RT-PCR.
[0027] As used herein, "-", when used to indicate the presence of a
particular cellular marker, means that the cellular marker is not
detectably present in fluorescence activated cell sorting over an
isotype control; or is not detectable above background in
quantitative or semi-quantitative RT-PCR.
[0028] As will be understood by one skilled in the art, for any and
all purposes, particularly in terms of providing a written
description, all ranges disclosed herein also encompass any and all
possible subranges and combinations of subranges thereof. Any
listed range can be easily recognized as sufficiently describing
and enabling the same range being broken down into at least equal
halves, thirds, quarters, fifths, tenths, etc. As a non-limiting
example, each range discussed herein can be readily broken down
into a lower third, middle third and upper third, etc. As will also
be understood by one skilled in the art all language such as "up
to," "at least," "greater than," "less than," and the like, include
the number recited and refer to ranges which can be subsequently
broken down into subranges as discussed above. Finally, as will be
understood by one skilled in the art, a range includes each
individual member. Thus, for example, a group having 1-3 cells
refers to groups having 1, 2, or 3 cells. Similarly, a group having
1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so
forth.
[0029] It is also to be understood, although not always explicitly
stated, that the reagents described herein are merely exemplary and
that equivalents of such are known in the art.
[0030] "Optional" or "optionally" means that the subsequently
described event or circumstance can or cannot occur, and that the
description includes instances where the event or circumstance
occurs and instances where it does not.
[0031] The term "comprising" is intended to mean that the
compositions and methods include the recited elements, but not
excluding others. "Consisting essentially of," when used to define
compositions and methods, shall mean excluding other elements of
any essential significance to the combination. For example, a
composition consisting essentially of the elements as defined
herein would not exclude other elements that do not materially
affect the basic and novel characteristic(s) of the claims.
"Consisting of" shall mean excluding more than trace amount of
other ingredients and substantial method steps. Embodiments defined
by each of these transition terms are within the scope of the
disclosure.
[0032] As used herein, the terms "cytotoxic" and "cytolytic", when
used to describe the activity of effector cells such as NK cells,
are intended to be synonymous. In general, cytotoxic activity
relates to killing of target cells by any of a variety of
biological, biochemical, or biophysical mechanisms. Cytolysis
refers more specifically to activity in which the effector lyses
the plasma membrane of the target cell, thereby destroying its
physical integrity. This results in the killing of the target cell.
Without wishing to be bound by theory, it is believed that the
cytotoxic effect of NK cells is due to cytolysis.
[0033] The term "kill" with respect to a cell/cell population is
directed to include any type of manipulation that will lead to the
death of that cell/cell population.
[0034] The term "cytokine" or "cytokines" refers to the general
class of biological molecules which effect cells of the immune
system. Exemplary cytokines include but are not limited to FLT3
ligand, interferons and interleukins (IL), in particular IL-2,
IL-12, IL-15, IL-18 and IL-21.
[0035] The terms "patient," "subject," "individual," and the like
are used interchangeably herein, and refer to any animal, or cells
thereof whether in vitro or in situ, amenable to the methods
described herein. In certain non-limiting embodiments, the patient,
subject or individual is a human.
[0036] The term "treating" or "treatment" covers the treatment of a
disease or disorder described herein, in a subject, such as a
human, and includes: (i) inhibiting a disease or disorder, i.e.,
arresting its development; (ii) relieving a disease or disorder,
i.e., causing regression of the disorder; (iii) slowing progression
of the disorder; and/or (iv) inhibiting, relieving, or slowing
progression of one or more symptoms of the disease or disorder. The
term "administering" or "administration" of a monoclonal antibody
or a natural killer cell to a subject includes any route of
introducing or delivering the antibody or cells to perform the
intended function. Administration can be carried out by any route
suitable for the delivery of the cells or monoclonal antibody.
Thus, delivery routes can include intravenous, intramuscular,
intraperitoneal, or subcutaneous delivery. In some embodiments the
CIK NKT cells are administered directly to the tumor, e.g., by
injection into the tumor. In some embodiments the modified CIK NKT
cells described herein are administered parenterally, e.g., by
injection, infusion or implantation (subcutaneous, intravenous,
intramuscular, intravesicularly, or intraperitoneal).
[0037] The term "expression" refers to the production of a gene
product.
[0038] As used herein, the terms "cytotoxic" when used to describe
the activity of effector cells such as NK cells, relates to killing
of target cells by any of a variety of biological, biochemical, or
biophysical mechanisms.
[0039] The terms "decrease," "reduced," "reduction," and "decrease"
are all used herein to refer to a decrease by at least 10% as
compared to a reference level, for example a decrease by at least
about 20%, or at least about 30%, or at least about 40%, or at
least about 50%, or at least about 60%, or at least about 70%, or
at least about 80%, or at least about 90% or up to and including a
100% decrease (i.e. absent level as compared to a reference
sample), or any decrease between 10-100% as compared to a reference
level.
[0040] The term "cancer" refers to all types of cancer, neoplasm,
or malignant tumors found in mammals, including leukemia,
carcinomas and sarcomas. Exemplary cancers include cancer of the
brain, breast, cervix, colon, head & neck, liver, kidney, lung,
non-small cell lung, melanoma, mesothelioma, ovary, sarcoma,
stomach, uterus and medulloblastoma. Additional examples include,
Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma,
neuroblastoma, ovarian cancer, rhabdomyosarcoma, primary
thrombocytosis, primary macroglobulinemia, primary brain tumors,
cancer, malignant pancreatic insulanoma, malignant carcinoid,
urinary bladder cancer, premalignant skin lesions, testicular
cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal
cancer, genitourinary tract cancer, malignant hypercalcemia,
endometrial cancer, adrenal cortical cancer, neoplasms of the
endocrine and exocrine pancreas, and prostate cancer.
[0041] The term "therapeutically effective amount" or "effective
amount" refers to the amount required to ameliorate the symptoms of
a disease relative to an untreated patient. The effective amount of
active compound(s) used to practice the present disclosure for
therapeutic treatment of a disease varies depending upon the manner
of administration, the age, body weight, and general health of the
subject. Ultimately, the attending physician or veterinarian will
decide the appropriate amount and dosage regimen. Such amount is
referred to as an "effective" amount.
[0042] Titles or subtitles may be used in the specification for the
convenience of a reader, which are not intended to influence the
scope of the present disclosure. Additionally, some terms used in
this specification are more specifically defined below.
Cytokine Induced Killer Natural Killer T Cells (CIK NKT Cells)
[0043] Natural killer T cells are a heterogeneous group of T cells
that share properties of both T cells and natural killer cells. For
example, NKT cells express an alpha beta T-cell receptor, but also
express a variety of molecular markers that are typically
associated with NK cells, such as NKp44, NKp46, and NKp30. NKT
cells constitute only about 0.1% of all peripheral blood T cells.
NKT cells have been implicated in suppression of autoimmunity and
graft rejection, promotion of resistance to pathogens, and
promotion of tumor immunity.
[0044] NKT cells are typically classified into type I and type II,
based on differences in T cell receptor (TCR) usage. Type 1 NKT
cells, also commonly referred to as invariant NKT cells, are NKT
cells that express a highly restricted T cell receptor, which
comprises an invariant TCR alpha chain, Va24.
[0045] Invariant NKT cells are typically activated by recognizing
lipid ligands alpha-galactosylceramide (Gal-Cer) presented by CD1d.
CD1d is a member of CD1 family of glycoproteins expressed on the
surface of various antigen presenting cells and are involved in the
presentation of lipid antigens. In contrast to class I and II major
histocompatibility complex (MHC) molecules that present protein
antigens to CD8+ and CD4+ T cells, respectively, CD1 molecules can
capture and process both foreign and self lipid antigens for
display to T cells. Gal-Cer are typically derived from pathogenic
cells, for example, bacteria.
[0046] As compared to Type I NKT cells, type II NKT cells have a
more diverse TCR repertoire and a higher sequence diversity. Type
II NKT cells do not respond to Gal-Cer, i.e., their activation is
independent of the presence of Gal-Cer.
[0047] The CIK NKT cells disclosed herein belong to the category of
type II NKT cells. The CIK NKT cells can be produced through
cytokine induction. In some instances, they are produced from e.g.,
cord blood, through cytokine induction. In some instances they are
produced during the process of enriching or isolating iNKT cells
from cord blood samples. However, CIK NKT cells differ from the
typical iNKT cells in several aspects. Phenotypically, unlike iNKT
cells, which express Va24, a marker for the alpha chain of a TCR
receptor, and the percentage of iNKT cells that express Va24 is can
be 70% or higher, CIK NKT cells have reduced expression of Va24.
For example, the percentage of CIK NKT cells that express Va24 may
be less than 10% of the total population of CIK NKT cells. In terms
of cytotoxicity, unlike iNKT cells, which are activated by
recognizing the alpha galactosyl ceramide (Gal-Cer), a glycolipid,
the CIK NKT cells do not need Gal-Cer to be activated and can kill
the cells in the absence of (Gal-Cer). See FIG. 3 and FIG. 4.
[0048] Thus, in terms of phenotypes, the CIK NKT cells provided
herein typically have high expression levels of CD56 and CD3 and
low expression of Va24. In some instances, at least 90% of the CIK
NKT population produced from the cord blood cells express CD56 and
CD3, and less than 10% of the cells in the population express
Va24.
Method of Isolating and Culturing CIK NKT Cells
Collecting Cord Blood
[0049] Human umbilical cord blood has high composition of
hematopoietic stem cells and can be used as a source for generating
CIK NKT cells. To collect cord blood, generally, a human placenta
is recovered shortly after its expulsion after birth. The placenta
can be transported in a sterile, thermally insulated transport
device (maintaining the temperature of the placenta between
20-28.degree. C.), for example, in a cord blood collection kit
substantially as described in U.S. Pat. No. 7,147,626. Preferably,
the placenta is delivered to the laboratory four to twenty-four
hours following delivery.
[0050] The placenta can be subjected to a conventional cord blood
recovery process. Typically a needle or cannula is used, with the
aid of gravity, to exsanguinate the placenta (see, e.g., Anderson,
U.S. Pat. No. 5,372,581; Hessel et al., U.S. Pat. No. 5,415,665).
The needle or cannula is usually placed in the umbilical vein and
the placenta can be gently massaged to aid in draining cord blood
from the placenta. Such cord blood recovery may be performed
commercially, e.g., LifeBank Inc., Cedar Knolls, N.J., ViaCord,
Cord Blood Registry and CryoCell. Preferably, the placenta is
gravity drained without further manipulation so as to minimize
tissue disruption during cord blood recovery.
[0051] Methods for collecting cord blood cells are well known, for
example, as described in US20150225697. Cord blood mononuclear
cells (Comics) can be isolated from collected cord blood using
methods well known in the art, e.g., a density gradient method
using Ficoll-Paque. Reagents suitable for isolating Comics are
commercially available, e.g., from Stem cell Technology Inc.
Enriching CIK NKT Cells
[0052] Comics can be cultured for a period of time in the presence
of various cytokines in order to enrich for CIK NKT cells.
Enriching refers to increasing the percentage of number of target
cells in a heterogenous cell population (e.g., the Comics). The
enrichment period may be 2 days-3 weeks, e.g., 1-2 weeks, 5-10
days, or about 2 weeks. Various growth media can be used, for
example, Roswell Park Memorial Institute medium (RPMI), or
Dulbecco's modified eagle medium (DMEM). Optionally, the medium
further comprises human AB serum and/or Gal-Cer. Optionally, the
human AB serum is present in 5-15% v/v, e.g., about 10% v/v.
Optionally, the Gal-Cer is present in a concentration of 2-10
.mu.g/mL, e.g., about 5 .mu.g/mL. Suitable cytokines that can be
added to the medium may include one or more cytokines selected from
the group consisting of stem cell factor, FLT3 ligand, IL-7, and
ALT-803 or IL-15. In some embodiments, FLT3 ligand is present in a
concentration ranging from 5-20 ng/mL, e.g., 10 ng/mL; IL-7 is
present in a concentration ranging from 5-20 ng/mL, e.g., 10 ng/mL;
and/or ALT-803 is present in a concentration ranging from 100-300
ng/mL, e.g., about 175 ng/mL.
[0053] FLT3 ligand is a hematopoietic four helical bundle cytokine
and it is structurally homologous to stem cell factor (SCF) and
colony stimulating factor 1 (CSF-1). In synergy with other growth
factors, FLT3 ligand stimulates the proliferation and
differentiation of various blood cell progenitors. It is a major
growth factor stimulating the growth of dendritic cells.
[0054] ALT-803 is a complex consisting of human IL-15 mutant
IL-15N72D (residue substitution at position 72) and IL-15R.alpha.
sushi-Fc fusion protein (see Zhu et al. J. Immunol. 2009;
183:3598-607, the relevant disclosure is hereby incorporated by
reference).
[0055] CIK NKT cells can be isolated from the enriched culture
described above by methods well known in the art, for example,
incubating magnetic beads coupled with antibody against the
Va24-J18 chain of the TCR with the enriched culture so that the CIK
NKT cells will bind to the magnetic beads, and subsequently
isolating the CIK NKT cells that are bound to the beads in presence
of a magnetic field. One example of the suitable antibodies that
can be used is the 6B11 antibody, which are commercially available
for vendors such as Biolegend (San Diego, Calif.). Suitable
reagents for isolating CIK NKT cells are from Miltenyi Biotec,
Germany. CIK NKT cells are typically isolated in PBS-containing
serum (e.g., human AB serum). Optionally, the isolation solution
also contains EDTA.
[0056] Thus, provided herein is a method of enriching CIK NKT cells
from cord blood, the method comprising: from a cord blood sample,
the method comprising: isolating mononuclear cells from the cord
blood sample; and contacting the isolated monocytes with one or
more agents selected from the group consisting of IL-7, ALT-803,
FLT3 ligand, and Gal-Cer to enrich CIK NKT cells.
Expanding CIK NKT Cells
[0057] The CIK NKT cells as isolated above can be expanded in
suitable growth medium. Expanding refers to growing an isolated
population of target cells so that the target cells increase in
number. In some embodiments, the growth medium is the NK Macs
medium, available from Miltenyi Biotec, Germany. In some
embodiments, the growth medium is supplemented with IL-2, anti-CD3,
and/or anti CD28 antibodies in amounts suitable for NKT cell
growth. In some embodiments, the anti-CD3 antibody is present in a
concentration of 5 ng/mL to 60 ng/mL, e.g., 20 ng/mL. In some
embodiments, the anti-CD28 antibody is present in a concentration
of 0.1 .mu.g/mL to 2 .mu.g/mL, e.g., 0.5 .mu.g/mL. In some
embodiments, IL-2 is present in a concentration of 50 ng/mL to 500
ng/mL, e.g., 200 ng/mL. In some embodiments, the growth medium
comprises human AB serum (e.g., about 10% v/v). In some
embodiments, the growth medium further comprises a Gal-Cer loaded
CD1d tetramer. In some embodiments, the Gal-Cer loaded CD1d
tetramer is a pre-assembled tetramer that are commerically
available, e.g., from ProImmune (Oxford, UK). Methods for
assembling Gal-Cer loaded CD1d tetramer is well known. Typically,
Gal-Cer lipid is co-incubated with CD1d protein, which are
oligomerized on streptavidin surface to become tetramers. Upon
Gal-Cer binding to CD1d complex, it was column purified and used as
reagents for expansion. Some of the exemplary methods for preparing
the Gal-Cer loaded CD1d tetramers are described in
www.proimmune.com/ecommerce/pdf_files/PS_DE000-RPE_V1.1%20%28CD1d%20Tetra-
mer%20Empty%20%28R-PE%20Labeled%29%29.pdf and
proimmune.com/ecommerce/pdf_files/ST14.pdf. In some embodiments,
the Gal-Cer loaded CD1d tetramer is used at an amount such that the
concentration of the Gal-Cer in the growth medium is about 20-200
ng/mL, e.g., 50-150 ng/mL, or 80-120 ng/mL, or about 100 ng/mL of
Gal-Cer. In some embodiments, the CIK NKT cells are let grown and
expanded for over a few days or weeks to reach a suitable amount of
cells for various applications.
[0058] Accordingly, the disclosure provides a method of growing CIK
NKT cells from cord blood, the method comprising: from a cord blood
sample, the method comprising: isolating mononuclear cells from the
cord blood sample; and contacting the isolated monocytes with one
or more agents selected from the group consisting of IL-7, ALT-803,
FLT3 ligand, and Gal-Cer to enrich CIK NKT cells.
Phenotyping the CIK NKT Cells
[0059] In certain embodiments, a CIK NKT cell populations can be
assessed by detecting one or more functionally relevant markers,
for example, CD56 and CD3 (markers for NKT cells) and TCR receptor
Va24 (a high expression of which indicates the NKT cells are
invariant NKT cells).
[0060] In some embodiments, provided herein are a CIK NKT cell
population comprising a lower percentage of Va24+ cells as compared
to typical invariant NKT cells. The CIK NKT cell population
comprises about 4%, about 5%, about 6%, about 7%, about 8%, about
9%, or about 10% of Va24+ cells. In some embodiments, the CIK NKT
cell population comprises between 0-20%, 5-10%, 1-7%, or 4-8% Va24+
cells. In some embodiments, the CIK NKT cell population comprises
no more than 20%, no more than 15%, no more than 10% of the Va24+
cells.
[0061] In some embodiments, provided herein are a CIK NKT cell
population comprising a percentage of CD56+ cells that is
substantially similar to that in a typical invariant NKT cell
population. The CIK NKT cell population comprises at least about
50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, at least about 95%, at least about 96%, at least
about 97%, at least about 98%, or about 99% of CD56+ cells. In some
embodiments, the CIK NKT cell population comprises between 50-100%,
70-100%, 85-100%, 90-100%, 95-100%, or 98-100% CD56+ cells. In some
embodiments, the CIK NKT cell population comprises no less than
50%, no less than 70%, no less than 85%, no less than 90%, no less
than 93%, or no less than 95% of the CD56+ cells.
[0062] In some embodiments, provided herein are a CIK NKT cell
population comprising a percentage of CD3+ cells that is
substantially similar to that in a typical invariant NKT cell
population. The CIK NKT cell population comprises at least about
50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, at least about 95%, at least about 96%, at least
about 97%, at least about 98%, or about 99% of CD3+ cells. In some
embodiments, the CIK NKT cell population comprises between 50-100%,
70-100%, 85-100%, 90-100%, 95-100%, or 98-100% of the CD3+ cells.
In some embodiments, the CIK NKT cell population comprises no less
than 50%, no less than 70%, no less than 85%, no less than 90%, no
less than 93%, or no less than 95% of the CD3+ cells.
[0063] In some embodiments, provided herein are a CIK NKT cell
population comprising a percentage of CD56+ CD3+ cells that is
substantially similar to that in a typical invariant NKT cell
population. The CIK NKT cell population comprises at least about
50%, at least about 60%, at least about 70%, at least about 80%, at
least about 90%, at least about 95%, at least about 96%, at least
about 97%, at least about 98%, or about 99% of CD56+ CD3+ cells. In
some embodiments, the CIK NKT cell population comprises between
50-100%, 70-100%, 85-100%, 90-100%, 95-100%, or 98-100% of the
CD56+ CD3+ cells. In some embodiments, the CIK NKT cell population
comprises no less than 50%, no less than 70%, no less than 85%, no
less than 90%, no less than 93%, or no less than 95% of the CD56+
CD3+ cells.
Cytotoxicity of the CIK NKT Cells
[0064] Optionally, the cytotoxic activity isolated or enriched
natural killer cells can be assessed, e.g., in a cytotoxicity assay
using tumor cells, e.g., cultured K562, DAOY, THP-1, LN-18, U937,
WERI-RB-1, U-118MG, HT-29, HCC2218, KG-1, or U266 tumor cells, or
the like as target cells. CIK NKT cells disclosed herein can kill
target cells regardless of MHC type and regardless of the presence
of .alpha.Gal-Cer.
[0065] Assays for evaluating cytotoxicity are well known, for
example, MTT assay. This is a system based on the tetrazolium
compound MTT. Briefly, after the treatment period in which the
target cells are in contact with CIK NKT cells, 10 uL of a freshly
diluted MTT solution (2.5 mg mL.sup.-1) was added to each well, and
the plate was incubated at 37 C in a humidified 5% CO2 atmosphere
for 4 h. At the end of the incubation period, the medium was
removed, and the formazan product was dissolved in 100 .mu.L of
dimethyl sulfoxide. Cell viability was evaluated by measurement of
the absorbance at 570 nm, using a SUNRICE Tecan absorbance reader
(Schoeller). Compound concentrations that produce 50% cell growth
inhibition (IC50) were calculated from curves constructed by
plotting cell survival (%) versus drug concentration (.mu.M). The
reading values were converted to the percentage of the control
(percentage cell survival). Non-limiting methods of cell killing
assays include Sulphorhodamine B (SRB) assay, Neutral red (NR)
assay, such as those described in
www.rsc.org/suppdata/mt/c4/c4mt00112e/c4mt00112e1.pdf, herein
incorporated by reference in its entirety.
[0066] The efficacy of the CIK NKT cells on killing target cells
can be evaluated with an EC50. EC50 used in this disclosure refers
to the effector to target ratio used in an assay where 50% of
target cells are killed. In some embodiments, the CIK NKT cells is
able to kill a plurality of the target cells at an EC50 of 1-10,
e.g., 1-8, 2-6, 2-5.5, or 3-7. In some embodiments, the target cell
is THP-1 and the EC50 is 4.64. In some embodiments, the target cell
is DAOY and the IC50 is 3.69. In some embodiments, the target cell
is K562 and the IC50 is 2.6.
Modified CIK NKT Cells
Chimeric Antigen Receptor
[0067] The CIK NKT cells produced as above can be further
engineered to express a chimeric antigen receptor (CAR) on the cell
surface. Optionally, the CAR is specific for a tumor-specific
antigen. Tumor-specific antigens are described, by way of
non-limiting example, in US 2013/0189268; WO 1999024566 A1; U.S.
Pat. No. 7,098,008; and WO 2000020460 A1, each of which is
incorporated herein by reference in its entirety. Tumor-specific
antigens include, without limitation, NKG2D, CS1, GD2, CD138,
EpCAM, EBNA3C, GPA7, CD244, CA-125, ETA, MAGE, CAGE, BAGE, HAGE,
LAGE, PAGE, NY-SEO-1, GAGE, CEA, CD52, CD30, MUCSAC, c-Met, EGFR,
FAB, WT-1, PSMA, NY-ESO1, AFP, CEA, CTAG1B, CD19 and CD33.
Additional non-limiting tumor-associated antigens, and the
malignancies associated therewith, can be found in Table 1.
TABLE-US-00001 TABLE 1 Tumor-Specific Antigens and Associated
Malignancies Target Antigen Associated Malignancy .alpha.-Folate
Receptor Ovarian Cancer CAIX Renal Cell Carcinoma CD19 B-cell
Malignancies Chronic lymphocytic leukemia (CLL) B-cell CLL (B-CLL)
Acute lymphoblastic leukemia (ALL); ALL post Hematopoietic stem
cell transplantation (HSCT) Lymphoma; Refractory Follicular
Lymphoma; B-cell non-Hodgkin lymphoma (B-NHL) Leukemia B-cell
Malignancies post-HSCT B-lineage Lymphoid Malignancies post
umbilical cord blood transplantation (UCBT) CD19/CD20 Lymphoblastic
Leukemia CD20 Lymphomas B-Cell Malignancies B-cell Lymphomas Mantle
Cell Lymphoma Indolent B-NHL Leukemia CD22 B-cell Malignancies CD30
Lymphomas; Hodgkin Lymphoma CD33 AML CD44v7/8 Cervical Carcinoma
CD138 Multiple Myeloma CD244 Neuroblastoma CEA Breast Cancer
Colorectal Cancer CS1 Multiple Myeloma EBNA3C EBV Positive T-cells
EGP-2 Multiple Malignancies EGP-40 Colorectal Cancer EpCAM Breast
Carcinoma Erb-B2 Colorectal Cancer Breast Cancer and Others
Prostate Cancer Erb-B 2,3,4 Breast Cancer and Others FBP Ovarian
Cancer Fetal Acetylcholine Receptor Rhabdomyosarcoma GD2
Neuroblastoma GD3 Melanoma GPA7 Melanoma Her2 Breast Carcinoma
Ovarian Cancer Tumors of Epithelial Origin Her2/new Medulloblastoma
Lung Malignancy Advanced Osteosarcoma Glioblastoma IL-13R-a2 Glioma
Glioblastoma Medulloblastoma KDR Tumor Neovasculature k-light chain
B-cell Malignancies B-NHL, CLL LeY Carcinomas Epithelial Derived
Tumors L1 Cell Adhesion Molecule Neuroblastoma MAGE-A1 Melanoma
Mesothelin Various Tumors MUC1 Breast Cancer; Ovarian Cancer NKG2D
Ligands Various Tumors Oncofetal Antigen (h5T4) Various Tumors PSCA
Prostate Carcinoma PSMA Prostate/Tumor Vasculature TAA Targeted by
mAb IgE Various Tumors TAG-72 Adenocarcinomas VEGF-R2 Tumor
Neovasculature
[0068] In some embodiments, the CAR targets CD19, CD33 or
CSPG-4.
[0069] In examples, variant polypeptides are made using methods
known in the art such as oligonucleotide-mediated (site-directed)
mutagenesis, alanine scanning, and PCR mutagenesis. Site direct
mutagenesis (Carter, 1986; Zoller and Smith, 1987), cassette
mutagenesis, restriction selection mutagenesis (Wells et al., 1985)
or other known techniques can be performed on the cloned DNA to
produce CD16 variants (Ausubel, 2002; Sambrook and Russell,
2001).
[0070] Optionally, the CAR targets an antigen associated with a
specific cancer type. Optionally, the cancer is selected from the
group consisting of leukemia (including acute leukemias (e.g.,
acute lymphocytic leukemia, acute myelocytic leukemia (including
myeloblastic, promyelocytic, myelomonocytic, monocytic, and
erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic
(granulocytic) leukemia and chronic lymphocytic leukemia),
polycythemia vera, lymphomas (e.g., Hodgkin's disease and
non-Hodgkin's disease), multiple myeloma, Waldenstrom's
macroglobulinemia, heavy chain disease, solid tumors including, but
not limited to, sarcomas and carcinomas such as fibrosarcoma,
myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma,
chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,
lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's
tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,
pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,
squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,
sweat gland carcinoma, sebaceous gland carcinoma, papillary
carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary
carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma,
bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilm's tumor, cervical cancer, testicular tumor, lung
carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial
carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma,
ependymoma, pinealoma, hemangioblastoma, acoustic neuroma,
oligodendroglioma, menangioma, melanoma, neuroblastoma and
retinoblastoma.
[0071] In some embodiments, a polynucleotide encoding a CAR is
mutated to alter the amino acid sequence encoding for CAR without
altering the function of the CAR. For example, polynucleotide
substitutions leading to amino acid substitutions at
"non-essential" amino acid residues can be made in the CARs
disclosed above. CARs can be engineered as described, for example,
in Patent Publication Nos. WO 2014039523; US 20140242701; US
20140274909; US 20130280285; and WO 2014099671, each of which is
incorporated herein by reference in its entirety. Optionally, the
CAR is a CD19 CAR, a CD33 CAR or CSPG-4 CAR.
Additional Modifications--Cytokines
[0072] In some embodiments, CAR-expressing CIK NKT cells are
further modified to express at least one cytokine. In specific
embodiments, the at least one cytokine is IL-2, IL-12, IL-15,
IL-18, IL-21 or a variant thereof. In preferred embodiments, the
cytokine is IL-12. A representative polypeptide of IL-12 comprises
or consists of an amino acid sequence set forth in Accession No.
IF45_A (https://www.ncbi.nlm.nih.gov/protein/1F45_A) and an amino
acid sequence set forth in Accession No. IF45_B
(https://www.ncbi.nlm.nih.gov/protein/1F45_B).
Therapeutic Applications
[0073] This disclosure also provides a method to treat any type of
cancer in a subject at any stage of the disease. Non-limiting
examples of the suitable cancers include carcinoma, melanoma, or
sarcoma. In some embodiments, the invention is used to treat cancer
of hemopoietic origin such as leukemia or lymphoma. In some
embodiments, the cancer is a solid tumor.
[0074] In some embodiments, the method to treat any type of cancer
in a subject comprises administering to the patient a
therapeutically effective amount of CIK NKT cells, wherein the
thereby treating cancer. The CIK NKT cells are from a population of
CIK NKT cells, wherein greater than 90% of the cells in the
population express CD 56 and CD3 and less than 10% of the cells in
the population express Va24.
[0075] The disclosure also provides a method to treat any type of
viral infection, the method comprising administering to the patient
a therapeutically effective amount of CIK NKT cells, wherein the
thereby treating cancer. The CIK NKT cells are from a population of
CIK NKT cells, wherein greater than 90% of the cells in the
population express CD 56 and CD3 and less than 10% of the cells in
the population express Va24.
[0076] Also provided are methods of treating a subject in need
thereof with CIK NKT cells as described herein. In some
embodiments, the subject or patient is suffering from cancer or an
infectious disease, such as a viral infection.
[0077] The CIK NKT cells can be administered to an individual by
absolute numbers of cells, e.g., said individual can be
administered from about 1000 cells/injection to up to about 10
billion cells/injection, such as at about, at least about, or at
most about, 1.times.10.sup.8, 1.times.10.sup.7, 5.times.10.sup.7,
1.times.10.sup.6, 5.times.10.sup.6, 1.times.10.sup.5,
5.times.10.sup.5, 1.times.10.sup.4, 5.times.10.sup.4,
1.times.10.sup.3, 5.times.10.sup.3 (and so forth) CIK NKT cells per
injection, or any ranges between any two of the numbers, end points
inclusive. Therefore, this disclosure also provides a composition
comprising a plurality of CIK NKT cells, wherein the number of
cells are 1.times.10.sup.8, 1.times.10.sup.7, 5.times.10.sup.7,
1.times.10.sup.6, 5.times.10.sup.6, 1.times.10.sup.5,
5.times.10.sup.5, 1.times.10.sup.4, 5.times.10.sup.4,
1.times.10.sup.3, or 5.times.10.sup.3 (and so forth).
[0078] In other embodiments, said individual can be administered
from about 1000 cells/injection/m.sup.2 to up to about 10 billion
cells/injection/m.sup.2, such as at about, at least about, or at
most about, 1.times.10.sup.8/m.sup.2, 1.times.10.sup.7/m.sup.2,
5.times.10.sup.7/m.sup.2, 1.times.10.sup.6/m.sup.2,
5.times.10.sup.6/m.sup.2, 1.times.10.sup.5/m.sup.2,
5.times.10.sup.5/m.sup.2, 1.times.10.sup.4/m.sup.2,
5.times.10.sup.4/m.sup.2, 1.times.10.sup.3/m.sup.2,
5.times.10.sup.3/m.sup.2 (and so forth) CIK NKT cells per
injection, or any ranges between any two of the numbers, end points
inclusive.
[0079] In other embodiments, CIK NKT cells can be administered to
such individual by relative numbers of cells, e.g., said individual
can be administered about 1000 cells to up to about 10 billion
cells per kilogram of the individual, such as at about, at least
about, or at most about, 1.times.10.sup.8, 1.times.10.sup.7,
5.times.10.sup.7, 1.times.10.sup.6, 5.times.10.sup.6,
1.times.10.sup.5, 5.times.10.sup.5, 1.times.10.sup.4,
5.times.10.sup.4, 1.times.10.sup.3, or 5.times.10.sup.3 (and so
forth) CIK NKT cells per kilogram of the individual, or any ranges
between any two of the numbers, end points inclusive.
[0080] In other embodiments, the total dose may be calculated by
m.sup.2 of body surface area, including about 1.times.10.sup.11,
1.times.10.sup.10, 1.times.10.sup.9, 1.times.10.sup.8,
1.times.10.sup.7, per m.sup.2, or any ranges between any two of the
numbers, end points inclusive. The average person is about 1.6 to
about 1.8 m.sup.2. In a preferred embodiment, between about 1
billion and about 3 billion CIK NKT cells are administered to a
patient. In other embodiments, the amount of CIK NKT cells injected
per dose may calculated by m.sup.2 of body surface area, including
1.times.10.sup.11, 1.times.10.sup.10, 1.times.10.sup.9,
1.times.10.sup.8, 1.times.10.sup.7, per m.sup.2. The average body
surface area for a person is 1.6-1.8 m.sup.2.
[0081] In other embodiments, CIK NKT cells can be administered to
such individual by relative numbers of cells, e.g., said individual
can be administered about 1000 cells to up to about 10 billion
cells per kilogram of the individual, such as at about, at least
about, or at most about, 1.times.10.sup.8, 1.times.10.sup.7,
5.times.10.sup.7, 1.times.10.sup.6, 5.times.10.sup.6,
1.times.10.sup.5, 5.times.10.sup.5, 1.times.10.sup.4,
5.times.10.sup.4, 1.times.10.sup.3, or 5.times.10.sup.3 (and so
forth) CIK NKT cells per kilogram of the individual, or any ranges
between any two of the numbers, end points inclusive.
[0082] CIK NKT cells can be administered once to a patient with
cancer or they can be administered multiple times, e.g., once every
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22 or 23 hours, or once every 1, 2, 3, 4, 5, 6 or 7 days,
or once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more weeks during
therapy, or any ranges between any two of the numbers, end points
inclusive.
[0083] In some embodiments, CIK NKT cells are administered in a
composition comprising the CIK NKT cells and a medium, such as
human serum or an equivalent thereof. In some embodiments, the
medium comprises human serum albumin. In some embodiments, the
medium comprises human plasma. In some embodiments, the medium
comprises about 1% to about 15% human serum or human serum
equivalent. In some embodiments, the medium comprises about 1% to
about 10% human serum or human serum equivalent. In some
embodiments, the medium comprises about 1% to about 5% human serum
or human serum equivalent. In a preferred embodiment, the medium
comprises about 2.5% human serum or human serum equivalent. In some
embodiments, the serum is human AB serum. In some embodiments, a
serum substitute that is acceptable for use in human therapeutics
is used instead of human serum. Such serum substitutes may be known
in the art, or developed in the future. Although concentrations of
human serum over 15% can be used, it is contemplated that
concentrations greater than about 5% will be cost-prohibitive. In
some embodiments, CIK NKT cells are administered in a composition
comprising CIK NKT cells and an isotonic liquid solution that
supports cell viability. In some embodiments, CIK NKT cells are
administered in a composition that has been reconstituted from a
cryopreserved sample.
[0084] Pharmaceutically acceptable compositions comprising the CIK
NKT cells can include a variety of carriers and excipients. A
variety of aqueous carriers can be used, e.g., buffered saline and
the like. These solutions are sterile and generally free of
undesirable matter. Suitable carriers and excipients and their
formulations are described in Remington: The Science and Practice
of Pharmacy, 21st Edition, David B. Troy, ed., Lippincott Williams
& Wilkins (2005). By pharmaceutically acceptable carrier is
meant a material that is not biologically or otherwise undesirable,
i.e., the material is administered to a subject without causing
undesirable biological effects or interacting in a deleterious
manner with the other components of the pharmaceutical composition
in which it is contained. If administered to a subject, the carrier
is optionally selected to minimize degradation of the active
ingredient and to minimize adverse side effects in the subject. As
used herein, the term pharmaceutically acceptable is used
synonymously with physiologically acceptable and pharmacologically
acceptable. A pharmaceutical composition will generally comprise
agents for buffering and preservation in storage and can include
buffers and carriers for appropriate delivery, depending on the
route of administration.
[0085] These compositions for use in in vivo or in vitro may be
sterilized by sterilization techniques employed for cells. The
compositions may contain acceptable auxiliary substances as
required to approximate physiological conditions such as pH
adjusting and buffering agents, toxicity adjusting agents and the
like, for example, sodium acetate, sodium chloride, potassium
chloride, calcium chloride, sodium lactate and the like. The
concentration of cells in these formulations and/or other agents
can vary and will be selected primarily based on fluid volumes,
viscosities, body weight and the like in accordance with the
particular mode of administration selected and the subject's
needs.
[0086] In one embodiment, CIK NKT cells are administered to the
patient in conjunction with one or more other treatments or agent
for the cancer being treated. In some embodiments, the one or more
other treatments for the cancer being treated include, for example,
an antibody, radiation, chemotherapeutic, stem cell
transplantation, or hormone therapy.
[0087] In some embodiments, CIK NKT cells and the other cancer
agent/treatment are administered simultaneously or approximately
simultaneously (e.g., within about 1, 5, 10, 15, 20, or 30 minutes
of each other). In some embodiments, the CIK NKT cells and the
other cancer agent/treatment are administered sequentially. In some
embodiments, the other cancer treatment/agent is administered one,
two, or three days after the administration of the CIK NKT
cells.
[0088] In one embodiment, the other cancer agent is an antibody. In
one embodiment, CIK NKT cells are administered in conjunction with
an antibody targeting the diseased cells. In one embodiment, CIK
NKT cells and an antibody are administered to the patient together,
e.g., in the same formulation; separately, e.g., in separate
formulations, concurrently; or can be administered separately,
e.g., on different dosing schedules or at different times of the
day. When administered separately, the antibody can be administered
via any suitable route, such as intravenous or intra-tumoral
injection.
[0089] In some embodiments, CIK NKT cells of the present disclosure
are used in combination with therapeutic antibodies and/or other
anti-cancer agents. Therapeutic antibodies may be used to target
cells that express cancer-associated or tumor-associated markers.
Examples of cancer therapeutic monoclonal antibodies are shown in
Table 2.
TABLE-US-00002 TABLE 2 Illustrative therapeutic monoclonal
antibodies Examples of FDA-approved therapeutic monoclonal
antibodies Brand Indication Antibody name Company Target (Targeted
disease) Alemtuzumab Campath .RTM. Genzyme CD52 Chronic lymphocytic
leukemia Brentuximab Adcetris .RTM. CD30 Anaplastic large cell
vedotin lymphoma (ALCL) and Hodgkin lymphoma Cetuximab Erbitux
.RTM. Bristol-Myers epidermal growth Colorectal cancer, Head and
Squibb/Eli factor receptor neck cancer Lilly/Merck KGaA Gemtuzumab
Mylotarg .RTM. Wyeth CD33 Acute myelogenous leukemia (with
calicheamicin) Ibritumomab Zevalin .RTM. Spectrum CD20 Non-Hodgkin
tiuxetan Pharmaceuticals, lymphoma (with yttrium- Inc. 90 or
indium-111) Ipilimumab Yervoy .RTM. blocks CTLA-4 Melanoma
(MDX-101) Ofatumumab Arzerra .RTM. CD20 Chronic lymphocytic
leukemia Palivizumab Synagis .RTM. MedImmune an epitope of the
Respiratory Syncytial Virus RSV F protein Panitumumab Vectibix
.RTM. Amgen epidermal growth Colorectal cancer factor receptor
Rituximab Rituxan .RTM., Biogen CD20 Non-Hodgkin lymphoma Mabthera
.RTM. Idec/Genentech Tositumomab Bexxar .RTM. GlaxoSmithKline CD20
Non-Hodgkin lymphoma Trastuzumab Herceptin .RTM. Genentech ErbB2
Breast cancer Blinatunomab bispecific CD19- Philadelphia
chromosome- directed CD3 T- negative relapsed or cell engager
refractory B cell precursor acute lymphoblastic leukemia (ALL)
Avelumamab anti-PD-L1 Non-small cell lung cancer, metastatic Merkel
cell carcinoma; gastic cancer, breast cancer, ovarian cancer,
bladder cancer, melanoma, meothelioma, including metastatic or
locally advanced solid tumors Daratumumab CD38 Multiple myeloma
Elotuzumab a SLAMF7- Multiple myeloma directed (also known as CD
319) immunostimulatory antibody
[0090] Administration of such CIK NKT cells may be carried out
simultaneously with the administration of the monoclonal antibody,
or in a sequential manner. In some embodiments, the CIK NKT cells
are administered to the subject after the subject has been treated
with the monoclonal antibody. Alternatively, the CIK NKT cells may
be administered at the same time, e.g., within 24 hours, of the
monoclonal antibody.
[0091] In some embodiments, CIK NKT cells are administered
intravenously. In some embodiments the CIK NKT cells are infused
directly into the bone marrow.
[0092] Therefore, this disclosure provides a method of treating
cancer or viral infection in a patient in need thereof, the method
comprising administering to the patient a therapeutically effective
amount of CIK NKT cells from the population of CIK NKT cells using
the methods disclosed herein to thereby treating cancer
Kits
[0093] Also disclosed are kits for the treatment of cancer or an
infectious disease using compositions comprising an amount of CIK
NKT cells as described herein. In some embodiments, the kits of the
present disclosure may also include at least one monoclonal
antibody.
[0094] In certain embodiments, the kit may contain additional
compounds such as therapeutically active compounds or drugs that
are to be administered before, at the same time or after
administration of CIK NKT cells. Examples of such compounds include
an antibody, vitamins, minerals, fludrocortisone, ibuprofen,
lidocaine, quinidine, chemotherapeutic, etc.
[0095] In various embodiments, instructions for use of the kits
will include directions to use the kit components in the treatment
of a cancer or an infectious disease. The instructions may further
contain information regarding how to CIK NKT cells (e.g., thawing
and/or culturing). The instructions may further include guidance
regarding the dosage and frequency of administration.
[0096] In certain embodiments, the kit further comprises one or
more containers filled with one or more compositions described
herein, e.g., a composition comprising CIK NKT cells as described
herein. Optionally associated with such containers can be a label
indicating the kit is for treating a cancer, such as those
described herein. Optionally the label also includes a notice in
the form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
[0097] The present disclosure and the working examples exemplifies
producing and using CIK NKT cells derived from cord blood samples,
one or ordinary skill in the art would appreciate that CIK NKT
cells can also be generated from other hematopoietic progenitor
cell samples using similar approaches to the ones described
herein.
[0098] Disclosed are materials, compositions, and components that
can be used for, can be used in conjunction with, can be used in
preparation for, or are products of the disclosed methods and
compositions. These and other materials are disclosed herein, and
it is understood that when combinations, subsets, interactions,
groups, etc. of these materials are disclosed that while specific
reference of each various individual and collective combinations
and permutations of these compounds may not be explicitly
disclosed, each is specifically contemplated and described herein.
For example, if a method is disclosed and discussed and a number of
modifications that can be made to a number of molecules including
the method are discussed, each and every combination and
permutation of the method, and the modifications that are possible
are specifically contemplated unless specifically indicated to the
contrary. Likewise, any subset or combination of these is also
specifically contemplated and disclosed. This concept applies to
all aspects of this disclosure including, but not limited to, steps
in methods using the disclosed compositions. Thus, if there are a
variety of additional steps that can be performed, it is understood
that each of these additional steps can be performed with any
specific method steps or combination of method steps of the
disclosed methods, and that each such combination or subset of
combinations is specifically contemplated and should be considered
disclosed.
EXAMPLES
[0099] This disclosure comprises the following, non-limiting
embodiments.
Embodiment 1
[0100] A population of CIK NKT cells, wherein greater than 50% of
the cells in the population express CD 56 and CD3 and less than 10%
of the cells in the population express Va24.
Embodiment 2
[0101] The population of CIK NKT cells of embodiment 1, wherein the
CIK NKT cells can kill a target cell in the absence of
alpha-galactosylceramide (Gal-Cer).
Embodiment 3
[0102] The population of CIK NKT cells of embodiment 1, wherein the
target cell is a cancer cell.
Embodiment 4
[0103] The population of CIK NKT cells of embodiment 1, wherein the
cancer cell line is selected from the group consisting of a
myelogenous leukemia cell, a medulloblastoma cell, and a monocytic
cell.
Embodiment 5
[0104] The population of CIK NKT cells of embodiment 3, wherein the
cancer cell is selected from the group consisting of a K562 cell, a
Daudi cell, a DAOY cell, and a THP-1 cell.
Embodiment 6
[0105] The population of CIK NKT cells of embodiments 2-5, wherein
the CIK NKT cells kill a plurality of the target cells at an EC50
of between 1.0 and 10.0.
Embodiment 7
[0106] The population of CIK NKT cells of embodiments 6, wherein
the CIK NKT cells can kill the target cells at a EC50 that is no
less than 90% and no greater than 110% of the EC50 at which the CIK
NKT cells killing the target cells in the presence of Gal-Cer.
Embodiment 8
[0107] A composition comprising a plurality of CIK NKT cells from
the population of CIK NKT cells of any of embodiments 1-7, and a
physiologically acceptable excipient.
Embodiment 9
[0108] A kit for treating cancer comprising a plurality of CIK NKT
cells from the population of CIK NKT cells of any of embodiments
1-7, wherein the kit further comprises a container and/or a label
indicating the kit is for treating cancer.
Embodiment 10
[0109] A method of enriching CIK NKT cells from a cord blood
sample, the method comprising: isolating mononuclear cells from the
cord blood sample; and contacting the isolated mononuclear cells
with one or more agents selected from the group consisting of IL-7,
ALT-803 or IL-15, FLT3 ligand, and Gal-Cer, whereby enriching CIK
NKT cells.
Embodiment 11
[0110] The method of embodiment 10, wherein the IL-7, if present,
is in a concentration ranging from 5 to 20 ng/mL.
Embodiment 12
[0111] The method of embodiment 10 or 11, wherein the ALT-803, if
present, is in a concentration ranging from 100 to 300 ng/mL.
Embodiment 13
[0112] The method of any of embodiments 10-12, wherein the FLT3
ligand, if present, is in a concentration ranging from 5 to 20
ng/mL.
Embodiment 14
[0113] The method of any of embodiments 10-13, wherein the Gal-Cer
is present in a concentration ranging from 2 to 10 .mu.g/mL.
Embodiment 15
[0114] The method of embodiment 10, wherein the method further
comprises isolating the enriched CIK NKT cells from the rest of the
cord blood sample.
Embodiment 16
[0115] The method of embodiment 15, wherein the method further
comprises contacting the isolated CIK NKT cells with anti-CD3,
anti-CD28, and IL2 to expand the CIK NKT cells.
Embodiment 17
[0116] The method of embodiment 16, wherein the method further
comprises contacting the isolated CIK NKT cells with Gal-Cer.
Embodiment 18
[0117] The method of embodiment 17, wherein the Gal-Cer is a
present in a form of a Gal-Cer loaded CD1d tetramer.
Embodiment 19
[0118] The method of embodiment 16, wherein the anti-CD3 antibody
is present in an amount of 5 ng/mL to 60 ng/mL.
Embodiment 20
[0119] The method of any of embodiments 16-19, wherein the
anti-CD28 antibody is present in an amount of 0.1 .mu.g/mL to 2
.mu.g/mL.
Embodiment 21
[0120] The method of any of embodiments 16-20, wherein IL-2 is
present in a concentration of 50 ng/mL to 500 ng/mL.
Embodiment 22
[0121] The method of any of embodiments 16-21, wherein the
production of CIK NKT cells does not include interferon-gamma.
Embodiment 23
[0122] A method of treating cancer or viral infection in a patient
in need thereof, the method comprising administering to the patient
a therapeutically effective amount of CIK NKT cells from the
population of CIK NKT cells of any of embodiments 1-7, thereby
treating cancer.
Embodiment 24
[0123] The method of embodiment 23, wherein about 1.times.10.sup.8
to about 1.times.10.sup.11 cells per m.sup.2 of body surface area
of the patient are administered to the patient.
Embodiment 25
[0124] The method of embodiment 23, wherein the cancer is selected
from the group consisting of a leukemia, a lymphoma, polycythemia
vera, multiple myeloma, Waldenstrom's macroglobulinemia, heavy
chain disease, a sarcoma and a carcinoma.
Embodiment 26
[0125] The method of embodiment 25, wherein the cells are
administered to the patient by a route selected from the group
consisting of intravenous, intraperitoneal, and subcutaneous.
Embodiment 27
[0126] The method of any of embodiments 23-26, wherein the method
further comprises administering an antibody.
Embodiment 28
[0127] A population of CIK NKT cells produced by the methods of any
of embodiments 10-27.
Embodiment 29
[0128] The population of CIK NKT cells of embodiment 1, wherein the
CIK NKT cells express a CAR and/or a cytokine.
EXAMPLES
[0129] The following examples are for illustrative purposes only
and should not be interpreted as limitations. There are a variety
of alternative techniques and procedures available to those of
skill in the art which would similarly permit one to successfully
perform the examples below.
Example 1: Phenotypes of CIK-NKT Cells
[0130] Cord blood mononuclear cells (Comics) were isolated from
cord blood samples by density gradient method using Ficoll-Paque
and Sepmate.TM., from Stem cell Technology Inc. Comics were
incubated with Gal-Cer (5 .mu.g/mL), FLT3-L (10 ng/mL), IL-7 (10
ng/mL), and ALT-803 (175 ng/mL) for enrichment of NKT for 2 weeks
in RPMI medium with 10% Human AB serum. NKT cells were isolated by
affinity chromatography using reagents from Miltenyi Biotec,
Germany. Subsequently, isolated cells were expanded in the presence
of anti-CD3 antibody (20 ng/mL), anti-CD28 antibody (0.5 .mu.g/mL),
and IL2 (200 ng/mL) in the NK Macs medium with 10% Human AB serum
for overnight activation with Gal-Cer loaded CD1d tetramer. The
Gal-Cer loaded CD1d tetramer was from ProImmune, Oxford, UK and was
used in an amount such that the Gal-Cer was present in an amount of
100 ng/mL Upon a week of expansion, these cells were stained with
antibodies recognizing CD3, CD56, or Va24. The results show that a
majority of the cells (97.1%) were positive for both CD56 and CD3
(FIG. 2B) and a small percentage of cells (6.45%) were positive for
Va24 (FIG. 2C). This indicates that cord blood CIK NKT cells have
low Va24 expression but the expression of CD3 and CD56 remain
intact.
Example 2: Cytotoxicity of CIK NKT Cells is Independent of
Gal-Cer
[0131] The NKT-CIK cells prepared as described in Example 2 were
assessed for cytotoxicity against cancer cell line DAOY. DAOY cells
were incubated with Gal-Cer at 1 .mu.g/mL overnight. The Gal-Cer
treated DAOY Cells were then incubated with a fluorescent dye,
Calcien AM, for 30 min. The cells were washed and then incubated
with CB-NKT CIKs, at various effector to target ratios as indicated
(the highest ratio was 32:1), for 4 hours. The killing of the DAOY
cells was measured by the cell lysis, which is represented by the
amount of CalcienAM dye released from cells. FIG. 3A shows the
amount of CalcienAM dye released from DAOY cells caused by the
CB-NKT CIKs. Data were presented as % lysis of target cells. The
results show that there were significant difference in terms of
killing between the group in which target cell were loaded with
Gal-Cer and the group in which target cells were not loaded with
Gal-Cer, indicating that Gal-Cer treatment did not confer killing
specificity.
[0132] FIG. 3B compares the cytotoxicity of NKT-CIK cells derived
from cord blood as described above (CB-CIK NKT cells) versus PBiNKT
cells (iNKT cells isolated from peripheral blood) on a
luciferase-expressing THP1 cells. PBiNKT cells were isolated in the
same manner as the CB-CIK NKT cells (see Example 1) except that the
source is peripheral blood instead of cord blood. The THP1 cells
were co-cultured with CB-CIK NKT cells or PBiNKT cells at various
effector to target ratios as indicated, the highest ratio being
32:1. No Gal-Cer was used in this experiment. The results show that
CB-CIK NKT cells are more potent in killing THP-1 than do PBiNKT
cells.
Example 3: Cytotoxicity of CIK NKT Cells on K562 and Daudi
Cells
[0133] CB-NKT CIK cells obtained as above were assayed for their
ability to kill luciferase-expressing cancer cell lines, DAOY,
Daudi, and K562 (represented by triangles, squares, and circles,
respectively, in FIG. 4). Peripheral blood isolated iNKTs were used
as control (inverted triangles in FIG. 4). Cell killing assays were
performed after 4-hour of co-culturing of the cancer cells and
effector cells (CB CIK NKTs or PBiNKTs). The killing of the cancer
cells was measured by % of cell lysis, which was represented by the
loss of luciferase in these cancer cell lines. Various effector to
target ratios were used as indicated, the highest being 32:1. The
results indicate that CB-CIK NKT cells kill target cells in a non
CD1d/MHC restricted manner.
* * * * *
References