U.S. patent application number 15/756473 was filed with the patent office on 2018-09-06 for compositions and methods of enhancing anti-tumor response using hybrid neutrophils.
The applicant listed for this patent is THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA. Invention is credited to Steven ALBELDA, Evgeniy ERUSLANOV.
Application Number | 20180250336 15/756473 |
Document ID | / |
Family ID | 58188221 |
Filed Date | 2018-09-06 |
United States Patent
Application |
20180250336 |
Kind Code |
A1 |
ERUSLANOV; Evgeniy ; et
al. |
September 6, 2018 |
COMPOSITIONS AND METHODS OF ENHANCING ANTI-TUMOR RESPONSE USING
HYBRID NEUTROPHILS
Abstract
The present invention relates to compositions and methods that
provide novel anti-tumor therapies in cancer. In one aspect, the
present invention features a hybrid neutrophil in a non-naturally
occurring container, wherein the hybrid neutrophil expresses at
least one neutrophil associated molecule selected from the group
consisting of: Arg1, MPO, CD66b, and CD15, and at least one
antigen-presenting cell (APC) associated molecule selected from the
group consisting of: CD14, HLA-DR, CD32, CD64, and CD89. In another
aspect, the present invention features methods of generating a
hybrid neutrophil. In still another aspect, the present invention
features methods of inhibiting tumor growth in a subject, treating
a tumor in a subject, and increasing efficacy of an antibody
against a tumor in a subject. The methods comprise (a)
administering to the subject an effective amount of an anti-tumor
antibody and (b) administering to or generating in the subject an
effective amount of a hybrid neutrophil.
Inventors: |
ERUSLANOV; Evgeniy;
(Havertown, PA) ; ALBELDA; Steven; (Philadelphia,
PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE TRUSTEES OF THE UNIVERSITY OF PENNSYLVANIA |
Philadelphia |
PA |
US |
|
|
Family ID: |
58188221 |
Appl. No.: |
15/756473 |
Filed: |
August 29, 2016 |
PCT Filed: |
August 29, 2016 |
PCT NO: |
PCT/US16/49205 |
371 Date: |
February 28, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 38/193 20130101;
A61K 35/15 20130101; C12N 2501/599 20130101; A61K 2300/00 20130101;
C12N 2501/22 20130101; A61P 35/00 20180101; A61K 2039/5158
20130101; A61K 45/06 20130101; A61K 2039/572 20130101; A61K
2039/585 20130101; C12N 5/0642 20130101; A61K 38/217 20130101; C12N
2501/24 20130101; A61K 39/39541 20130101; C12N 2502/30 20130101;
A61K 39/0011 20130101; A61K 31/454 20130101; A61K 38/193 20130101;
A61K 2300/00 20130101; A61K 38/217 20130101; A61K 2300/00 20130101;
A61K 31/454 20130101; A61K 2300/00 20130101 |
International
Class: |
A61K 35/15 20060101
A61K035/15; A61K 38/19 20060101 A61K038/19; A61P 35/00 20060101
A61P035/00; A61K 38/21 20060101 A61K038/21; A61K 39/00 20060101
A61K039/00; A61K 45/06 20060101 A61K045/06; A61K 39/395 20060101
A61K039/395 |
Goverment Interests
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under grant
number CA187392-01A1 NIH/NCI and awarded by LC140199 awarded by The
Department of Defense. The government has certain rights in the
invention.
Claims
1. A method of generating a hybrid neutrophil, the method
comprising contacting a composition comprising a bone marrow (BM)
immature CD15-positive (CD15.sup.+) cell with an amount of tumor
conditioned medium, wherein the hybrid neutrophil expresses at
least one neutrophil associated molecule selected from the group
consisting of: Arg1, MPO, CD66b, and CD15, and at least one
antigen-presenting cell (APC) associated molecule selected from the
group consisting of: CD14, HLA-DR, CD32, CD64, and CD89.
2. A method of generating a hybrid neutrophil, the method
comprising contacting a composition comprising a bone marrow (BM)
immature CD15-positive (CD15.sup.+) cell with an amount of
interferon .gamma. (IFN-.gamma.) and an amount of granulocyte
macrophage colony stimulating factor (GM-CSF), wherein the hybrid
neutrophil expresses at least one neutrophil associated molecule
selected from the group consisting of: Arg1, MPO, CD66b, and CD15,
and at least one antigen-presenting cell (APC) associated molecule
selected from the group consisting of: CD14, HLA-DR, CD32, CD64,
and CD89.
3. A method of generating a hybrid neutrophil, the method
comprising contacting a composition comprising a bone marrow (BM)
immature CD15-positive (CD15.sup.+) cell with an amount of an agent
that reduces the level of Ikaros polypeptide in the cell and an
amount of granulocyte macrophage colony stimulating factor
(GM-CSF), wherein the hybrid neutrophil expresses at least one
neutrophil associated molecule selected from the group consisting
of: Arg1, MPO, CD66b, and CD15, and at least one antigen-presenting
cell (APC) associated molecule selected from the group consisting
of: CD14, HLA-DR, CD32, CD64, and CD89.
4. A method of generating a hybrid neutrophil, the method
comprising contacting a composition comprising peripheral blood
immature neutrophils with an amount of tumor conditioned medium,
wherein the hybrid neutrophil expresses at least one neutrophil
associated molecule selected from the group consisting of: Arg1,
MPO, CD66b, and CD15, and at least one antigen-presenting cell
(APC) associated molecule selected from the group consisting of:
CD14, HLA-DR, CD32, CD64, and CD89.
5. A method of generating a hybrid neutrophil, the method
comprising contacting a composition comprising peripheral blood
immature neutrophils with an amount of interferon .gamma.
(IFN-.gamma.) and an amount of granulocyte macrophage colony
stimulating factor (GM-CSF), wherein the hybrid neutrophil
expresses at least one neutrophil associated molecule selected from
the group consisting of: Arg1, MPO, CD66b, and CD15, and at least
one antigen-presenting cell (APC) associated molecule selected from
the group consisting of: CD14, HLA-DR, CD32, CD64, and CD89.
6. The method of claim 5, wherein the peripheral blood immature
neutrophils are mobilized in peripheral blood by contacting
peripheral blood with an amount of granulocyte macrophage colony
stimulating factor (GM-CSF) or an amount of granulocyte colony
stimulating factor (G-CSF).
7. The method of claim 2, wherein the amount of granulocyte
macrophage colony stimulating factor (GM-CSF) or the amount of
interferon .gamma. (IFN-.gamma.) is at least about 50 pg/ml, at
least about 60 pg/ml, at least about 70 pg/ml, at least about 80
pg/ml, at least about 90 pg/ml, or at least about 100 pg/ml.
8. The method of claim 3, wherein the agent is lenalidomide.
9. The method of claim 4, wherein the amount of tumor conditioned
medium is about 50% v/v.
10. The method of claim 1, wherein the hybrid neutrophil further
expresses at least one molecule selected from the group consisting
of: MHC class I, MHC class II, OX40L, 4-1BBL, CD86, CD40, and
CCR7.
11. The method of claim 1, wherein the expression level of any one
of the molecules is low, intermediate, or high.
12. The method of claim 1, wherein the expression of any one of the
molecules is increased relative to expression of the molecule on a
canonical tumor-associated neutrophil (TAN).
13. The method of claim 1, wherein the hybrid neutrophil expresses
CD14, HLA-DR, CD32, CD64, and CD89.
14. The method of claim 10, wherein the hybrid neutrophil expresses
Arg1, MPO, CD66b, CD15, CD14, HLA-DR, MHC class I, OX40L, 4-1BBL,
CD86, CD40, CCR7, CD32, CD64, and CD89.
15. The method of claim 13, wherein the expression of CD32 and/or
CD64 and/or CD89 is high.
16. A method of inhibiting tumor growth in a subject, the method
comprising (a) administering to the subject an effective amount of
an anti-tumor antibody or an antigen-binding fragment thereof; and
(b) administering to or generating in the subject an effective
amount of a hybrid neutrophil, wherein the hybrid neutrophil
expresses at least one neutrophil associated molecule selected from
the group consisting of: Arg1, MPO, CD66b, and CD15, and at least
one antigen-presenting cell (APC) associated molecule selected from
the group consisting of: CD14, HLA-DR, CD32, CD64, and CD89,
thereby inhibiting tumor growth in the subject.
17. A method of increasing efficacy of an antibody against a tumor
in a subject, the method comprising (a) administering to the
subject an effective amount of an anti-tumor antibody or an
antigen-binding fragment thereof; and (b) administering to or
generating in the subject an effective amount of a hybrid
neutrophil, wherein the hybrid neutrophil expresses at least one
neutrophil associated molecule selected from the group consisting
of: Arg1, MPO, CD66b, and CD15, and at least one antigen-presenting
cell (APC) associated molecule selected from the consisting of:
CD14, HLA-DR, CD32, CD64, and CD89, thereby increasing efficacy of
the antibody against the tumor in the subject.
18. A method of treating a tumor in a subject, the method
comprising (a) administering to the subject an effective amount of
an anti-tumor antibody or an antigen-binding fragment thereof; and
(b) administering to or generating in the subject an effective
amount of a hybrid neutrophil, wherein the hybrid neutrophil
expresses at least one neutrophil associated molecule selected from
the group consisting of: Arg1, MPO, CD66b, and CD15, and at least
one antigen-presenting cell (APC) associated molecule selected from
the group consisting of: CD14, HLA-DR, CD32, CD64, and CD89,
thereby treating the tumor in the subject.
19. The method of claim 16, wherein the hybrid neutrophil further
expresses at least one molecule selected from the group consisting
of: MHC class I, MHC class II, OX40L, 4-1BBL, CD86, CD40, and
CCR7.
20. The method of claim 16, wherein the expression of any one of
the molecules is low, intermediate, or high.
21. The method of claim 16, wherein the expression of any one of
the molecules is increased relative to expression of the molecule
on a canonical tumor-associated neutrophil (TAN).
22. The method of claim 16, wherein the hybrid neutrophil expresses
CD14, HLA-DR, CD32, CD64, and CD89.
23. The method of claim 19, wherein the hybrid neutrophil expresses
Arg1, MPO, CD66b, CD15, CD14, HLA-DR, MHC class I, OX40L, 4-1BBL,
CD86, CD40, CCR7, CD32, CD64, and CD89 .
24. The method of claim 22, wherein the expression of CD32 and/or
CD64 and/or CD89 is high.
25. The method of claim 16, wherein the anti-tumor antibody is
selected from the group consisting of: anti-Her2/neu antibody,
rituximab, necitumumab, panitumumab, and cetuximab.
26. The method of claim 16, wherein the step of administering to
the subject an effective amount of a hybrid neutrophil increases
antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent
phagocytosis (ADP), or effector T cell response in the subject.
27. The method of claim 16, wherein the hybrid neutrophil is
generated ex vivo in a biological sample obtained from the
subject.
28. The method of claim 27, wherein the biological sample is blood
or bone marrow.
29. The method of claim 28, wherein the hybrid neutrophil
population is generated by a method according to claim 1.
30. The method of claim 16, wherein the hybrid neutrophil is
generated in situ in the subject.
31. The method of claim 30, wherein the hybrid neutrophil is
generated in situ by administering to the subject an amount of
granulocyte macrophage colony stimulating factor (GM-CSF) and at
least one agent selected from the group comprised of: IFN-.gamma.
and lenalidomide.
32. The method of claim 16, wherein the step of administering to or
generating in the subject an effective amount of a hybrid
neutrophil is followed by the step of administering to the subject
an effective amount of an anti-tumor antibody or an antigen-binding
fragment thereof.
33. The method of claim 16, wherein the step of administering to or
generating in the subject an effective amount of a hybrid
neutrophil is concurrent with the step of administering to the
subject an effective amount of an anti-tumor antibody or an
antigen-binding fragment thereof.
34. The method of claim 16, wherein the tumor comprises non-small
cell lung cancer (NSCLC).
35. The method of claim 16, wherein the subject is human.
36. A hybrid neutrophil in a non-naturally occurring container,
wherein the hybrid neutrophil expresses at least one neutrophil
associated molecule selected from the group consisting of: Arg1,
MPO, CD66b, and CD15, and at least one antigen-presenting cell
(APC) associated molecule selected from the group consisting of:
CD14, HLA-DR, CD32, CD64, and CD89.
37. The hybrid neutrophil of claim 36, wherein the hybrid
neutrophil further expresses at least one molecule selected from
the group consisting of: MHC class I, MHC class II, OX40L, 4-1BBL,
CD86, CD40, and CCR7.
38. The hybrid neutrophil of claim 36, wherein the expression of
any one of the molecules is low, intermediate, or high.
39. The hybrid neutrophil of claim 36, wherein the expression of
any one of the molecules is increased relative to expression of the
molecule on a canonical tumor-associated neutrophil (TAN).
40. The hybrid neutrophil of claim 36, wherein the hybrid
neutrophil expresses CD14, HLA-DR, CD32, CD64, and CD89.
41. The hybrid neutrophil of claim 37, wherein the hybrid
neutrophil expresses Arg1, MPO, CD66b, CD15, CD14, HLA-DR, MHC
class I, OX40L, 4-1BBL, CD86, CD40, CCR7, CD32, CD64, and CD89.
42. The hybrid neutrophil of claim 40, wherein the expression of
CD32 and/or CD64 and/or CD89 is high.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority under 35 U.S.C.
.sctn. 119(e) to U.S. Provisional Patent Application No.
62/212,279, filed Aug. 31, 2015, which is incorporated herein by
reference in its entirety.
BACKGROUND OF THE INVENTION
[0003] Neutrophils are antimicrobial effector cells equipped with
powerful killing machinery to respond to pathogens, especially to
opsonized bacteria. It is thought that therapeutic antibodies
against tumor antigens can direct and activate this cytotoxic
machinery against opsonized tumor cells through Fc receptors, a
process that is referred to as antibody-dependent cellular
cytotoxicity (ADCC) (Musolino et al., J Clin Oncol. 2008; 26(11):
1789-1796; Albanesi et al., Blood. 2013 Aug. 26 PMID23980063;
Hernandez-Ilizaliturri et al., Clin Cancer Res. 2003 Dec. 1; 9(16
Pt 1): 5866-5873). Unfortunately, the clinical efficacy of many
therapeutic antibodies is poor and needs to be enhanced (Liu et
al., Cancer Chemother Pharmacol. 2010 April; 65(5): 849-861; Fury
et al., Cancer Immunol Immunother. 2008 February; 57(2): 155-163;
Repp et al., Br J Cancer. 2003 Dec. 15; 89(12): 2234-2243). The
identification and administration of efficient effector subsets
responsible for mediating sufficient ADCC in humans could lead to
the development of more synergistic and combination therapies that
would enhance the effect of therapeutic antibodies.
[0004] Treatment of tumors with anti-tumoral antibodies such as
anti-Her2/neu, rituximab, necitumumab, panitumumab, or cetuximab in
combination with G-CSF/GM-CSF to induce the recruitment of effector
neutrophils from bone marrow was used in several clinical trials
(Repp et al., Br J Cancer. 2003 Dec. 15; 89(12): 2234-2243;
Pullarkat et al., Cancer Immunol Immunother. 1999 April; 48(1):
9-21; Cartron et al., J Clin Oncol. 2008 Jun. 1; 26(16): 2725-2731;
van der Kolk et al., Leukemia. 2003 August; 17(8): 1658-1664;
Niitsu et al., Clin Cancer Res. 2004 Jun. 15; 10(12 Pt 1):
4077-4082). However, these trials only showed limited therapeutic
effects, indicating that improvement of neutrophil-mediated Ab
therapy is required. Several Fc.gamma.R-bearing myeloid cell
populations have been proposed as a potential effector cells for
monoclonal antibody-mediated tumor regression, including natural
killer (NK) cells, monocytes, macrophages and neutrophils
(Hernandez-Ilizaliturri et al., Clin Cancer Res. 2003 Dec. 1; 9(16
Pt 1): 5866-5873; Repp et al., Br. J Cancer. 2003 Dec. 15; 89(12):
2234-2243; Gul et al., J Clin Invest. 2014 Feb. 3; 124(2): 812-823;
Hatjiharissi et al., Blood. 2007 Oct. 1; 110(7): 2561-2564;
Pullarkat et al., Cancer Immunol Immunother. 1999 April; 48(1):
9-21). The expansion and/or activation of these cells in a human
represents an attractive strategy to enhance the efficacy of
therapeutic antibodies through the induction of ADCC.
[0005] A need exists in the art for novel anti-tumor therapies,
especially for enhancing the efficacy of therapeutic anti-tumor
antibodies. The present invention satisfies this need.
SUMMARY OF THE INVENTION
[0006] As described herein, the present invention relates to
compositions, methods, and uses for hybrid neutrophils. In one
aspect, the invention includes a method of generating a hybrid
neutrophil. The method comprises contacting a composition
comprising a bone marrow (BM) immature CD15-positive (CD15) cell
with an amount of tumor conditioned medium. The hybrid neutrophil
expresses at least one neutrophil associated molecule selected from
the group consisting of: Arg1, MPO, CD66b, and CD15, and at least
one antigen-presenting cell (APC) associated molecule selected from
the group consisting of: CD14, HLA-DR, CD32, CD64, and CD89.
[0007] In another aspect, the invention includes a method of
generating a hybrid neutrophil comprising contacting a composition
comprising a bone marrow (BM) immature CD15-positive (CD15) cell
with an amount of interferon .gamma. (IFN-.gamma.) and an amount of
granulocyte macrophage colony stimulating factor (GM-CSF). The
hybrid neutrophil expresses at least one neutrophil associated
molecule selected from the group consisting of: Arg1, MPO, CD66b,
and CD15, and at least one antigen-presenting cell (APC) associated
molecule selected from the group consisting of: CD14, HLA-DR, CD32,
CD64, and CD89.
[0008] In yet another aspect, the invention includes a method of
generating a hybrid neutrophil comprising contacting a composition
comprising a bone marrow (BM) immature CD15-positive (CD15.sup.+)
cell with an amount of an agent that reduces the level of Ikaros
polypeptide in the cell and an amount of granulocyte macrophage
colony stimulating factor (GM-CSF). The hybrid neutrophil expresses
at least one neutrophil associated molecule selected from the group
consisting of: Arg1, MPO, CD66b, and CD15, and at least one
antigen-presenting cell (APC) associated molecule selected from the
group consisting of: CD14, HLA-DR, CD32, CD64, and CD89.
[0009] In still another aspect, the invention includes a method of
generating a hybrid neutrophil comprising contacting a composition
comprising peripheral blood immature neutrophils with an amount of
tumor conditioned medium. The hybrid neutrophil expresses at least
one neutrophil associated molecule selected from the group
consisting of: Arg1, MPO, CD66b, and CD15, and at least one
antigen-presenting cell (APC) associated molecule selected from the
group consisting of: CD14, HLA-DR, CD32, CD64, and CD89.
[0010] In another aspect, the invention includes a method of
generating a hybrid neutrophil comprising contacting a composition
comprising peripheral blood immature neutrophils with an amount of
interferon .gamma. (IFN-.gamma.) and an amount of granulocyte
macrophage colony stimulating factor (GM-CSF). The hybrid
neutrophil expresses at least one neutrophil associated molecule
selected from the group consisting of: Arg1, MPO, CD66b, and CD15,
and at least one antigen-presenting cell (APC) associated molecule
selected from the group consisting of: CD14, HLA-DR, CD32, CD64,
and CD89.
[0011] Another aspect of the invention includes a method of
inhibiting tumor growth in a subject. The method comprises (a)
administering to the subject an effective amount of an anti-tumor
antibody or an antigen-binding fragment thereof; and (b)
administering to or generating in the subject an effective amount
of a hybrid neutrophil. The hybrid neutrophil expresses at least
one neutrophil associated marker selected from the group consisting
of: Arg1, MPO, CD66b, and CD15, and at least one antigen-presenting
cell (APC) associated marker selected from the group consisting of:
CD14, HLA-DR, CD32, CD64, and CD89. The method thereby inhibits
tumor growth in the subject.
[0012] Yet another aspect of the invention includes a method of
increasing efficacy of an antibody against a tumor in a subject.
The method comprises (a) administering to the subject an effective
amount of an anti-tumor antibody or an antigen-binding fragment
thereof; and (b) administering to or generating in the subject an
effective amount of a hybrid neutrophil. The hybrid neutrophil
expresses at least one neutrophil associated molecule selected from
the group consisting of: Arg1, MPO, CD66b, and CD15, and at least
one antigen-presenting cell (APC) associated molecule selected from
the consisting of: CD14, HLA-DR, CD32, CD64, and CD89. The method
thereby increases efficacy of the antibody against the tumor in the
subject.
[0013] Still another aspect includes a method of treating a tumor
in a subject comprising (a) administering to the subject an
effective amount of an anti-tumor antibody or an antigen-binding
fragment thereof; and (b) administering to or generating in the
subject an effective amount of a hybrid neutrophil. The hybrid
neutrophil expresses at least one neutrophil associated molecule
selected from the group consisting of: Arg1, MPO, CD66b, and CD15,
and at least one antigen-presenting cell (APC) associated molecule
selected from the group consisting of: CD14, HLA-DR, CD32, CD64,
and CD89. The method thereby treats the tumor in the subject.
[0014] Another aspect of the invention includes a hybrid neutrophil
in a non-naturally occurring container. The hybrid neutrophil
expresses at least one neutrophil associated molecule selected from
the group consisting of: Arg1, MPO, CD66b, and CD15, and at least
one antigen-presenting cell (APC) associated molecule selected from
the group consisting of: CD14, HLA-DR, CD32, CD64, and CD89.
[0015] In various embodiments of the above aspects or any other
aspect of the invention delineated herein, the peripheral blood
immature neutrophils are mobilized in peripheral blood by
contacting peripheral blood with an amount of granulocyte
macrophage colony stimulating factor (GM-CSF) or an amount of
granulocyte colony stimulating factor (G-CSF). In another
embodiment, the amount of granulocyte macrophage colony stimulating
factor (GM-CSF) or the amount of interferon .gamma. (IFN-.gamma.)
is at least about 50 pg/ml, at least about 60 pg/ml, at least about
70 pg/ml, at least about 80 pg/ml, at least about 90 pg/ml, or at
least about 100 pg/ml.
[0016] In another embodiment, the agent that reduces the level of
Ikaros polypeptide in the cell is lenalidomide. In yet another
embodiment, the amount of tumor conditioned medium is about 50%
v/v.
[0017] In still another embodiment, the hybrid neutrophil further
expresses at least one molecule selected from the group consisting
of: MHC class I, MHC class II, OX40L, 4-1BBL, CD86, CD40, and CCR7.
In another embodiment, the expression level of any one of the
molecules is low, intermediate, or high. In yet another embodiment,
the expression of any one of the molecules is increased relative to
expression of the molecule on a canonical tumor-associated
neutrophil (TAN).
[0018] In another embodiment, the hybrid neutrophil expresses CD14,
HLA-DR, CD32, CD64, and CD89. In yet another embodiment, the hybrid
neutrophil expresses Arg1, MPO, CD66b, CD15, CD14, HLA-DR, MHC
class I, OX40L, 4-1BBL, CD86, CD40, CCR7, CD32, CD64, and CD89. In
still another embodiment, the expression of CD32 and/or CD64 and/or
CD89 is high.
[0019] In certain embodiments, the anti-tumor antibody is selected
from the group consisting of: anti-Her2/neu antibody, rituximab,
necitumumab, panitumumab, and cetuximab. In another embodiment, the
step of administering to the subject an effective amount of a
hybrid neutrophil increases antibody-dependent cellular
cytotoxicity (ADCC), antibody-dependent phagocytosis (ADP), or
effector T cell response in the subject. In another embodiment, the
step of administering to or generating in the subject an effective
amount of a hybrid neutrophil is followed by the step of
administering to the subject an effective amount of an anti-tumor
antibody or an antigen-binding fragment thereof. In yet another
embodiment, the step of administering to or generating in the
subject an effective amount of a hybrid neutrophil is concurrent
with the step of administering to the subject an effective amount
of an anti-tumor antibody or an antigen-binding fragment thereof.
In one embodiment, the subject is human. In another embodiment, the
tumor comprises non-small cell lung cancer (NSCLC).
[0020] In another embodiment, the hybrid neutrophil is generated ex
vivo in a biological sample obtained from the subject. In yet
another embodiment, the biological sample is blood or bone marrow.
In still another embodiment, the hybrid neutrophil is generated in
situ in the subject. In another embodiment, the hybrid neutrophil
is generated in situ by administering to the subject an amount of
granulocyte macrophage colony stimulating factor (GM-CSF) and at
least one agent selected from the group comprised of: IFN-.gamma.
and lenalidomide.
[0021] In yet another embodiment, the hybrid neutrophil population
is generated by a method according to any one of the claims of the
present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] For the purpose of illustrating the invention, there are
depicted in the drawings certain embodiments of the invention.
However, the invention is not limited to the precise arrangements
and instrumentalities of the embodiments depicted in the
drawings.
[0023] FIGS. 1A-1C are a series of plots showing tumor-associated
neutrophil (TAN) subsets in lung cancer. A single cell suspension
was obtained from freshly harvested tumor tissues and stained for
indicated markers. TANs were gated on live single
CD11b.sup.+CD15.sup.hiCD66b.sup.+Arg1.sup.+ cells (FIG. 1A, long
box) and further analyzed for the expression of HLA-DR, CD14, CD86,
CCR7, and CD206 by flow cytometry (FIG. 1B, boxes in upper right of
each plot). FIG. 1C shows the frequency of HLA-DR.sup.+ hybrid TANs
among all TANs in cancer patients (n=45) analyzed by flow cytometry
of tumor digests.
[0024] FIGS. 2A-2C are a series of plots and images showing
long-lived bone marrow (BM) immature neutrophils in vitro.
Neutrophils were purified from BM cell suspension using anti-CD15
magnetic beads. As shown in FIG. 2A, the purified BM CD15.sup.+
cells were MPO-positive "band-like" neutrophils. FIG. 2B shows bone
marrow neutrophils (BMN) that were purified and cultured in the
presence or absence of IFN-.gamma. and GM-CSF for 6 days at the
concentrations of 100 pg/ml. Fixable Viability dye FVD660 was used
to discriminate viable neutrophils in cell culture. In FIG. 2C,
purified BM CD15 neutrophils were cultured with hybrid-inducing TCM
for 5-7 days (top panel). Hybrid cells are highlighted in boxes. To
obtain canonical neutrophils, BM CD15 neutrophils were cultured
with TCM collected from patients where hybrid TANs were not found
(bottom panel). The indicated cell surface markers were analyzed by
flow cytometry on the gated CD11b.sup.+CD15.sup.+CD66b.sup.+ live
cells. Hybrid neutrophils were purified with HLA-DR.sup.+ beads,
spun on glass slides and stained with the Hema3 Stat Pack Kit.
[0025] FIGS. 3A-3F are a series of plots showing the
differentiation of human long-lived bone marrow (BM) immature
neutrophils into the hybrid neutrophils. Neutrophils were purified
from BM cell suspension using anti-CD15 magnetic beads. FIG. 3A
shows the differentiation of CD15.sup.+ bone marrow neutrophils
(BMNs) into HLA-DR.sup.+CD14.sup.+ hybrid neutrophils in the
presence of tumor conditioned media (TCMs) collected from cancer
patients or with IFN-.gamma. and GM-CSF at a concentration of 100
pg/ml. The expression of CD14 and HLA-DR was analyzed on gated live
CD11b.sup.+CD15.sup.hiCD66b.sup.+ cells by flow cytometry on day 7
of treatment. FIG. 3B shows the development of HLA-DR.sup.+
CD14.sup.+ hybrid neutrophils in the presence of lenalidomide (10
.mu.M) and/or GM-CSF (100 pg/ml). Expression of HLA-DR and CD14
molecules was analyzed on gated live CD11b.sup.+CD15.sup.+ BMNs at
day 6 of differentiation. In FIG. 3C, low density neutrophils were
isolated from G-CSF treated cancer patients by gradient separation
and cultured in the presence of IFN-.gamma. and GM-CSF at
concentration 100 pg/ml for 7 days. The expression of HLA-DR.sup.+
and CD14.sup.+ was measured on gated live
CD11b.sup.+CD66b.sup.+CD15.sup.- cells. In FIGS. 3D-3E, neutrophils
were isolated from peripheral blood (PB) and bone marrow (BM) using
anti-CD15 beads and stained for CD16 and CD10 markers. FIG. 3F
shows BM CD15 cells treated with TCMs collected from different
patients (top panel) or with IFN-.gamma. and GM-CSF at
concentration 20 pg/ml (low panel). Five days later, expression of
CD14 and HLA-DR was analyzed on gated CD11b.sup.+CD15.sup.+ live
cells by flow cytometry.
[0026] FIG. 4 is a heat map depicting the phenotype of canonical
and hybrid neutrophils. The heat map compares the phenotypes of
bone marrow neutrophils ("BMN"), peripheral blood neutrophils
("PBN"), canonical tumor-associated neutrophils ("Canonical TAN"),
hybrid tumor-associated neutrophils ("Hybrid TAN") and bone marrow
derived hybrid neutrophils ("BM Hybrid"). Neutrophils were gated on
live CD11b.sup.+CD15.sup.hiCD66b.sup.+ cells (PBN, BMN, canonical
TAN) and CD11b.sup.+CD15.sup.hiCD66b.sup.+HLA-DR.sup.+CD14.sup.+
cells (hybrid neutrophils) and further analyzed for the expression
of indicated markers by flow cytometry. Expression of each marker
was analyzed at least in 7 patients. The intensity key for the heat
map is shown in the top left corner of FIG. 4.
[0027] FIGS. 5A-5D are a series of plots and images showing the
functional characterization of the canonical and hybrid
neutrophils. FIG. 5A shows bone marrow (BM) derived hybrid (black
line) and canonical (grey line) neutrophils incubated with
pHrodo.TM. Red E. coli BioParticles.RTM. for 45 minutes to allow
phagocytosis (internalized particles become fluorescent red). In
FIG. 5B, BM derived canonical and hybrid neutrophils were incubated
with Cetuximab opsonized A431 tumor cell line labeled with DIO dye
for 4 hrs. Cell cultures were collected and stained for CD66b to
visualize neutrophils. Antibody dependent phagocytosis (ADP) was
evaluated by flow cytometry as a percentage of double positive
cells. To confirm ADP, stained cells were spun on glass slides and
examined for double positive cells. In FIG. 5C, non-activated or
phorbol myristate acetate (PMA) (40 ng/ml) activated peripheral
blood neutrophil (PBN), tumor-associated neutrophil (TAN), hybrid
and canonical bone marrow neutrophils (BMNs) were incubated with
adherent GFP-A549 tumor cells for 24 hours in 96 Well Black Flat
Bottom Microplate (Corning.RTM.) that has low fluorescent
background. To induce ADCC by neutrophils, A549 tumor cells were
opsonized with anti-EGFR monoclonal antibodies (Cetuximab), 1
.mu.g/ml for 30 min at 4C. These opsonized cells were incubated
with neutrophils for 24 hours. Tumor cell cytotoxicity was
calculated by comparing the remaining cell-associated GFP
fluorescence of adherent tumor cells cultured with neutrophils to
control wells (tumor cells without neutrophils). FIG. 5D shows
canonical and hybrid neutrophils differentiated from immature BM
CD15 cells. The expression of Fc.gamma. receptors (Fc.gamma.Rs) was
analyzed on gated live canonical and hybrid neutrophils by flow
cytometry.
[0028] FIGS. 6A-6M are a series of plots and images showing the
effect of canonical and hybrid neutrophils on T cell responses.
FIG. 6A shows the effect of BM-derived canonical HLA-DW and hybrid
HLA-DR.sup.+ neutrophils on T cell proliferation (top panel) and
IFN-.gamma. by activated T cells (bottom panel). CFSE-labeled
autologous peripheral blood mononuclear cells (PBMCs) were
stimulated with plate-bound anti-CD3 antibodies and mixed with
canonical and hybrid neutrophils at a 1:1 ratio for 4 days. To
measure intracellular IFN-.gamma. in CD3 cells, the autologous
PBMCs were stimulated with plate-bound anti-CD3 and CD28 antibodies
and mixed with canonical and hybrid neutrophils at a 1:1 ratio for
48 hrs. In FIG. 6B, the effect of BM-derived HLA-DR.sup.+ hybrid
neutrophils on NY-ESO-specific effector T cell responses is shown.
Human TCR-transfected CD8 effector T cells (Ly95 cells) that
recognize a HLA-A*02-restricted peptide of NY-ESO-1 were stimulated
with genetically modified A549 tumor cell line expressing NY-ESO-1
and HLA-A*02. The percentage of IFN-.gamma. (top panel) and
Granzyme B (low panel) positive TCR V.beta. 13.1-transfected CD8
cells (Ly95 cells) cultured in the presence of BM-derived HLA-DW or
HLA-DR.sup.+ neutrophils was measured by intracellular staining at
24 hours after stimulation. FIG. 6C shows autologous T cells
isolated from PBMC and co-cultured with BM-derived canonical HLA-DW
and hybrid HLA-DR.sup.+ neutrophils that had been pulsed with a
mixture of viral T cell epitopes for 2 hours. The number of
IFN-.gamma.-producing T cells was determined in three independent
ELISpot assays. Error bars represent mean.+-.SEM from 3 independent
experiments (*p.ltoreq.0.01, Mann-Whitney test). FIG. 6D shows the
ability of BM-derived canonical HLA-DW or hybrid HLA-DR.sup.+
neutrophils to trigger NYESO-specific effector T cell responses.
HLA-A2.sup.+ canonical or hybrid neutrophils were pulsed with
NY-ESO-1 peptide for 1 hour, washed and cultured with Ly95 cells at
a 1:1 ratio for 24 hrs. Antigen-specific activation of the Ly95
cells was assessed by measuring intracellular IFN-.gamma.. In FIGS.
6E-6H, TAN subsets were isolated by flow cytometry sorting based on
the phenotype of canonical (CD11b.sup.+CD66b.sup.+CD15.sup.+) and
hybrid (CD11b.sup.+CD66b.sup.+CD15.sup.+HLA-DR.sup.-CD14.sup.+)
TANs. BM-derived hybrid neutrophils were differentiated with
hybrid-inducing TCM. CFSE-labeled PBMC isolated from a healthy
donor were stimulated with plate-bound anti-CD3 antibodies and
mixed with canonical, hybrid TAN and BM hybrid cells at ratio 1:1
for 4 days. Numbers on histograms represent the percentage of
proliferating T cells (FIGS. 6E-6H). The percentage of IFN-.gamma.
(FIGS. 61-6J) and Granzyme B (FIGS. 6K-6L) positive TCR V.beta.
13.1-transfected CD8 cells cultured in the presence or absence BM
hybrid neutrophils was measured by intracellular staining at 24
hours after stimulation with A2/ESO A549 cells. FIG. 6M shows
antigen-presenting activity of canonical and hybrid neutrophils and
cross-presentation of viral epitopes. Tumor or BM-derived hybrid
and canonical neutrophils were pulsed with peptide pool of viral
antigens (CD8 epitopes from human CMV, Epstein-Barr, flu viruses,
and tetanus toxoid from all the common HLA types) and co-cultured
with autologous T cells for 24 hrs. IFN-.gamma. production was
assessed by ELISpot. Hybrid, canonical BMNs and monocyte-derived DC
(Mo-DC) were incubated with DQ-OVA for 30 min at 37.degree. C.
(black) or at 4.degree. C. (grey-tinted) (lower panel). DQ-OVA
exhibits bright green fluorescence upon proteolytic
degradation.
[0029] FIGS. 7A-7B are plots showing the expression of Fc.gamma.RI
(CD64) and Fc.gamma.RII (CD32) and neutrophil tumoricidal activity.
In FIG. 7A, the expression of Fc.gamma.Rs was analyzed on gated
live canonical CD11b.sup.+ CD15.sup.hiCD66b.sup.+ HLA-DR.sup.-
cells and hybrid neutrophils CD1 1 b.sup.+ CD15.sup.hiCD66b.sup.+
HLA-DR.sup.+ by flow cytometry. Tumor derived (TAN, top panel) and
BM-derived neutrophils (BMN, bottom panel). In FIG. 7B,
non-activated or PMA (40 ng/ml) activated PBN, TAN, hybrid and
canonical BMNs were incubated with adherent GFP-A549 tumor cells
for 24 hours in 96 Well Black Flat Bottom Microplate (Corning.RTM.)
that has the low fluorescent background. To induce ADCC by
neutrophils, A549 tumor cells were opsonized with anti-EGFR
monoclonal antibodies (mAbs) (Cetuximab), 1 .mu.g/ml for 30 min at
4.degree. C. These opsonized cells were incubated with neutrophils
for 24 hours. Tumor cell cytotoxicity was calculated by comparing
the remaining cell-associated GFP fluorescence of adherent tumor
cells cultured with neutrophils to control wells (tumor cells
without neutrophils).
[0030] FIGS. 8A-8B are plots showing results of a murine model to
study the human neutrophils mediated ADCC in vivo. In FIGS. 8A and
8B, five million human BM neutrophils (BMN) were intratumorally
injected into established human lung cancer cell line-derived
tumors (A549 lung cancer xenografts) in NSG mice. One and four days
later, tumors were harvested, enzymatically digested and the
presence of neutrophils in xenografts was detected by flow
cytometry (boxes in upper right of plots in FIG. 8B).
[0031] FIG. 9 is a graph showing the cytotoxic activity of
neutrophils. A431, A549 and Daudi tumor cell lines were labeled
with cell dye PKH67 and opsonized with cetuximab and rituximab at a
concentration of 1 ug/ml. PBN, hybrid and canonical BMNs were
incubated with cetuximab-opsonized A431 cell line,
cetuximab-opsonized A549 cell line and rituximab-opsonized Daudi
cell line at a ratio of 10:1. Sixteen hours later, floating and
adherent cells were collected using trypsin and stained with a
viability dye FVD eFluor.RTM. 660. Tumor cell cytotoxicity was
calculated as a percent of PKH.sup.+FVD660.sup.+ cells.
[0032] FIG. 10 is a set of graphs showing tumoricidal activity of
hybrid BM neutrophils in vivo. A549 tumor cells were injected
subcutaneously (2.times.10.sup.6 cells/mouse) or intraperitoneally
(1.times.10.sup.6 cells/mouse). Arrows show when hybrid neutrophils
were injected intratumorally. Sizes of subcutaneous tumors were
measured with calipers. The tumor growth in the peritoneum was
monitored by measuring bioluminescence (BLI) following
intraperitoneal transplantation of Luciferase-expressing A549 tumor
cells.
[0033] FIGS. 11A-11O are a series of graphs and images showing a
subset of TANs with hybrid characteristics of neutrophils and APCs.
FIG. 11A is a photograph of an excised lung showing the location of
tumor and distant adjacent tissues used for experiments, as well as
dot plots representing the frequency of live
CD11b.sup.+CD15.sup.hiCD66b.sup.+ TANs (inset boxes) in digested
tumor tissue. FIGS. 11B-11F show the expression of HLA-DR (FIG.
11B), CD14 (FIG. 11C), CD86 (FIG. 11D), CCR7 (FIG. 11E), and CD206
(FIG. 11F) on gated CD11b.sup.+CD15.sup.hiCD66b.sup.+ TANs (tumor),
distant lung neutrophils (distant), and PBNs (PB). Top panels show
summary of all patient data. Data are presented as the percentage
of cells among all TANs. Error bars represent mean.+-.SEM, 1-way
ANOVA with Tukey's multiple comparison test. Bottom panels show
representative dot plots. TANs were defined in (FIG. 11A) as live
D11b.sup.+CD15.sup.hiCD66b.sup.+ cells. FIG. 11G shows the presence
of APC-like hybrid neutrophils in the regional lymph nodes (LNs) of
lung cancer patients. LNs were mashed through the cell strainer and
single cell suspension was stained for indicated markers. Cells
were gated on live CD11b.sup.+CD15.sup.hi (black box) and further
analyzed for the expression of CD66b, CD14, and HLA-DR.
Representative dot plots are shown. The error bars represent the
mean.+-.SEM, n=10. FIG. 11H shows the frequency of live APC-like
hybrid TANs among all nucleated cells in (tumor), distant lung
(distant) and peripheral blood (PB). Cumulative results from 50
independent experiments are shown in the scatter plots. The error
bars represent the mean.+-.SEM. Statistical analyses were performed
with repeated measures one-way ANOVA with Tukey's multiple
comparison test. FIGS. 11I-11L show the frequency of APC-like
hybrid TANs in patients with NSCLC with different tumor type (FIG.
11I) (AD-adenocarcinoma, SCC-squamous cell carcinoma), stage (FIG.
11J), smoking history (FIG. 11K) and size (FIG. 11L). The error
bars represent the mean.+-.SEM, unpaired t test for FIG. 11I and
FIG. 11J, Kruskal-Wallis multiple comparison test for FIG. 11K,
nonparametric Spearman correlation for FIG. 11L. FIG. 11M shows
phagocytic activity of hybrid and canonical TANs. TANs were
isolated from tumor and incubated with pHrodo.TM. Red E. coli
BioParticles.RTM. for 45 min to allow phagocytosis (internalized
particles become fluorescent [red]). The level of phagocytosis was
measured in gated HLA-DW canonical (grey line) and HLA-DR.sup.+
hybrid TANs (black line). Representative results of 1 of 4
experiments are shown. FIGS. 11N-11O show the gating strategy for
sorting of canonical HLA-DW and hybrid HLA-DR.sup.+ TANs by flow
cytometry. A single cell suspension was obtained from freshly
harvested tumor, stained for indicated markers and sorted based on
the phenotype of canonical (CD11b.sup.+CD66b.sup.+CD15
.sup.hiHLA-DR.sup.-) and hybrid
(CD11b.sup.+CD66b.sup.+CD15.sup.hiHLA-DR.sup.+) TANs.
[0034] FIG. 12 is a table showing characteristics of the patients
taking part in the study (n=109).
[0035] FIGS. 13A-13G are a series of graphs and images showing a
subset of TANs with hybrid characteristics of neutrophils and APCs.
FIG. 13A shows a single-cell suspension was obtained from fresh
tumor and the expression of the indicated granulocytic markers was
analyzed by flow cytometry on gated live CD11b cells. Total TANs
are shown in inset boxes. FIG. 13B shows flow cytometric analysis
of the expression of APC markers on gated
CD11b.sup.+CD15.sup.hiCD66b.sup.+ TANs. The representative
cytomorphology of canonical (lower left inset boxes) and APC-like
hybrid TANs (upper right inset boxes) in NSCLC. Scale bar, 10
.mu.m. FIG. 13C shows the presence of APC-like hybrid TANs in tumor
detected by immunohistochemistry and immunofluorescence double
staining. Scale bar, 50 .mu.m (left image) and 10 .mu.m (other
images). FIG. 13D shows the frequency of APC-like hybrid
neutrophils in tumors, distant lung tissue, and peripheral blood
(PB) (right graph) and in tumors of different sizes (left graph)
(line represents mean.+-.SEM, n=50, one-way ANOVA test and unpaired
t test). APC-like hybrid TANs were defined as live
HLA-DR.sup.+CD11b.sup.+CD15.sup.hiCD66b.sup.+ cells. FIG. 13E shows
intracellular TNF-.alpha. and IL-12 production by HLA-DR.sup.+
hybrid or HLA-DW canonical TANs after stimulation with LPS. TANs
were gated on CD11b.sup.+CD15.sup.hiCD66b.sup.+ cells.
Representative results from one of five experiments are shown. FIG.
13F shows the proliferation of autologous CFSE-labeled PBMC
stimulated with plate-bound anti-CD3 Abs in the presence of hybrid
HLA-DR.sup.+ or canonical HLA-DW TANs. T cell stimulatory activity
was defined as the ratio CFSE.sup.lo (T cells+TANs)/CFSE.sup.lo (T
cells) (n=6, Wilcoxon matched-pairs rank test). FIG. 13G shows
autologous virus-specific memory T cell responses in the presence
of APC-like hybrid HLA-DR.sup.+ or canonical HLA-DW TANs.
IFN-.gamma.-ELISPOT assay (mean.+-.SEM, n=3, *p.ltoreq.0.01
canonical versus hybrid, Mann-Whitney test).
[0036] FIGS. 14A-14F are a series of graphs and images showing
tumor-derived factors differentiate long-lived immature BMNs into a
hybrid subset with a partial phenotype of dendritic cells and
macrophages. FIG. 14A shows fixable viability dye eFluor 660
(FVD660) was used to discriminate viable neutrophils in cell
culture. Representative dot plots from one of six experiments are
shown. FIG. 14B shows flow cytometric analysis of the expression of
MPO, CD66b, and CD15 markers on freshly isolated BMNs (day 0) and
BMNs cultured with (HLA-DR.sup.+ BMNs) or without hybrid-inducing
TCM (HLA-DW BMNs) for 7 days. Cytospins show the cytomorphology of
these BMNs. Scale bar, 10 .mu.m. FIG. 14C shows survival of BMNs in
cell culture in the presence or absence of TCM. Viability dye FVD
660 was used to discriminate viable BMNs in cell culture
(mean.+-.SEM, n=6, *p.ltoreq.0.01, Wilcoxon matched-pairs rank
test). FIGS. 14D-14F show flow cytometric analysis of the
expression of indicated APC markers on BM-derived hybrid
neutrophils (FIG. 14D) (inset boxes), dendritic cells (FIG. 14E),
and macrophages (FIG. 14F). Expression of APC markers was analyzed
by flow cytometry on gated CD11b.sup.+CD15.sup.hiCD66b.sup.+
BMNs.
[0037] FIGS. 15A-15F are a series of graphs showing tumor-derived
factors differentiate long-lived immature BMNs into a hybrid subset
with a partial phenotype of dendritic cells and macrophages. FIG.
15A shows flow cytometric analysis of the expression of CD15,
CD66b, CD11b, intracellular MPO, NE and Arg1 on freshly isolated
BMNs. Representative dot plots of 1 of 8 experiments are shown.
FIG. 15B shows BMN survival in vitro. BMNs were incubated with or
without hybrid-inducing TCM. Seven days later, BMNs were stained
with viability dye FVD 660 followed by staining for AnnexinV and
analyzed by flow cytometry. The error bars represent the
mean.+-.SEM, Wilcoxon matched-pairs rank test, n=4. FIG. 15C shows
BMNs were isolated from three different cancer patients and treated
with the same hybrid-inducing TCM collected from patient #78. The
expression of HLA-DR.sup.+ and CD14.sup.+ was measured on gated
live CD11b.sup.+CD66b.sup.+CD15.sup.hi BMNs by flow cytometry. FIG.
15D shows kinetics of indicated APC marker expression in BMNs
treated with hybrid-inducing TCM. The expressions of indicated
markers were assessed by flow cytometry on live
CD11b.sup.+CD66b.sup.+CD15.sup.hi BMNs for different time points.
Results represent 1 of 7 similar experiments. FIG. 15E shows
comparative analysis of IRF8 expression (presented as Mean
Fluorescence Intensity (MFI) histograms) in non-treated BMNs (BMNs
none) and BM-derived hybrid neutrophils differentiated with
IFN-.gamma. and GM-CSF (BMNs IFN-.gamma.+GM-CSF) or TCM (BMNs TCM).
BMNs neutrophils treated with M-CSF and GMCSF/IL-4 were used as
negative control whereas BM-derived macrophages (Mph) and dendritic
cells (DC) were used as positive control. Representative dot plots
from 1 of 6 experiments are shown. FIG. 15F shows proliferation of
HLA-DR.sup.- and HLA-DR.sup.+ BMNs in vitro in the presence of
hybrid-inducing TCM. BMNs were exposed to hybrid inducing TCM for 8
days. One, five and eight days later, the proliferation of
neutrophils was assessed by intracellular staining of incorporated
BrdU into DNA of HLA-DR.sup.- and HLA-DR.sup.+ BMNs. The expression
of HLA-DR was measured on gated live
CD11b.sup.+CD66b.sup.+CD15.sup.hi BMNs. Representative results from
1 of 3 experiments are shown.
[0038] FIGS. 16A-16F are a series of graphs showing tumor-derived
IFN-.gamma. and GM-SCF synergistically differentiate immature
neutrophils into a subset of APC-like hybrid neutrophils. FIG. 16A
shows flow cytometric analysis of CD14 and HLA-DR expression on
gated live CD11b.sup.+CD15 CD66b.sup.+ BMNs cultured in the
presence of hybrid-inducing TCM under normoxic and hypoxic cell
culture conditions. FIG. 16B shows flow cytometric analysis of CD14
and HLA-DR expression on gated live
CD11b.sup.+CD15.sup.hiCD66b.sup.-BMNs cultured in the presence of
different TCMs (upper panel) or with IFN-.gamma. and/or GM-CSF
(lower panel). FIG. 16C shows the effect of IFN-.gamma. and GM-CSF
blocking Abs (5 .mu.g/ml) in blunting the formation of
HLA-DR.sup.+CD14.sup.+ hybrid neutrophils in vitro (onset box).
FIG. 16D shows the expression of CD14 and HLA-DR markers on live
CD11b.sup.+CD15.sup.hiCD66b.sup.+ BMNs (upper panel) and PD-L1 on
gated HLA-DR.sup.+CD14.sup.+ hybrid neutrophils (lower panel)
differentiated with GM-CSF (50 pg/ml) and increasing doses of
IFN-.gamma. in vitro. FIGS. 16E-16F show levels of IFN-.gamma.
(FIG. 16E) and GM-CSF (FIG. 16F) in supernatants collected from the
cell culture of small-sized tumor digests where APC-like hybrid
TANs were or were not previously detected (set-off was >10%
among all TANs) (line represents mean.+-.SEM, n=10, Mann-Whitney
test for unpaired data). Lower panels represent the correlation
between the absolute levels of IFN-.gamma. and GM-CSF in the TCM,
with the frequency of hybrid neutrophils in each tumor shown in the
upper graphs. Non-parametric Spearman test was used to determine
the degree of correlation. Representative dot plots from one of
five experiments are shown in (FIG. 16A-16D).
[0039] FIGS. 17A-17B are a series of graphs and images showing
tumor-derived IFN-.gamma. and GM-SCF synergistically differentiate
immature neutrophils into a subset of APC-like hybrid neutrophils.
FIG. 17A shows flow cytometric analysis of the expression of CD14
and HLA-DR markers on gated live CD11b.sup.+CD66b.sup.+CD15.sup.hi
BMNs differentiated in the presence of IFN-.gamma. (50 pg/ml) and
GM-CSF (50 pg/ml) for 5 days. BMNs isolated from five different
lung cancer patients are shown. FIG. 17B shows the effect of TCMs
with different concentration of GM-CSF and IFN-.gamma. on the
formation of hybrid HLA-DR.sup.+CD14.sup.+ BMNs. BMNs were isolated
from one cancer patient and treated with hybrid-inducing TCM
collected from different patients (#58, #78, #41, #101). The
expression of HLA-DR and CD14 was measured on gated live
CD11b.sup.+CD66b.sup.+CD15 .sup.hi BMNs. Concentration of GM-CSF
and IFN-.gamma. in TCMs was quantified by ELISA.
[0040] FIGS. 18A-18D are a series of graphs and images showing
APC-like hybrid neutrophils originate from CD11b.sup.+CD15
.sup.hiCD66b.sup.+CD10.sup.-CD16.sup.1.degree. .sup.lo/int
progenitors. FIG. 18A shows flow cytometric analysis of the
expression of CD10 and CD16 on gated live CD11b.sup.-CD15
CD66b.sup.+ neutrophils isolated from peripheral blood (PBNs) and
bone marrow (BMNs) of cancer patients. FIG. 18B shows cytospins
were made from sorted BMNs at different stages of maturation and
stained with the Hema3 Stat Pack Kit (Wright-Giemsa-like stain).
FIG. 18C shows sorted BMNs at different stages of maturation were
differentiated in the presence of IFN-.gamma. (50 pg/ml) and GM-CSF
(50 pg/ml) in vitro. Expression of HLA-DR and CD14 markers was
analyzed by flow cytometry on CD11b.sup.+CD15 .sup.hiCD66b.sup.+
BMNs. FIG. 18D shows cytomorphology of APC-like HLA-DR.sup.+ hybrid
neutrophils differentiated from the sorted populations of BMNs at
different stages of maturation. Representative results from one of
four experiments are shown in (FIG. 18A-18D). Scale bar, 10
.mu.m.
[0041] FIGS. 19A-19C are a series of graphs and images showing
APC-like hybrid neutrophils originate from
CD11b.sup.+CD15.sup.hiCD66b.sup.+CD10-CD16.sup.lo/int progenitors.
FIG. 19A shows the co-expression of CD15, CD66b, CD11 b, CD16 and
CD10 analyzed by flow cytometry on freshly isolated BMNs.
Representative dot plots from 1 of 5 experiments are shown. FIG.
19B is a schematic representation of the phenotype and nuclear
morphology of CD11b.sup.+CD15.sup.hi BMNs at different stages of
development. BMNs at different stages of maturation were isolated
by flow cytometry sorting and analyzed for the indicated surface
markers by flow cytometry. Cytospins were made from sorted BMNs and
stained with the Hema3 Stat Pack Kit (Wright-Giemsa-like stain).
FIG. 19C shows the formation of hybrid HLA-DR.sup.+CD14.sup.+
neutrophils from G-CSF mobilized low density immature PBNs. The
expression of HLA-DR and CD14 was measured on gated live
CD11b.sup.+CD66b.sup.+CD15.sup.hiPBNs after the treatment with
hybrid-inducing TCM or IFN-.gamma. (50 pg/ml) and GM-CSF (50
pg/ml). Representative dot plots from 1 of 4 similar experiments
are shown.
[0042] FIGS. 20A-20D are a series of graphs showing the
transcription factor Ikaros negatively regulates the
differentiation of hybrid neutrophils. FIG. 20A shows flow
cytometric analysis of the level of Ikaros and HLA-DR expression in
PBNs and BMNs at different stages of maturation. Results are shown
as mean fluorescence intensity (MFI). FIG. 20B shows flow
cytometric analysis of the level of Ikaros expression in the
HLA-DR.sup.+ hybrid and HLA-DR.sup.- canonical CD11b.sup.+CD15
.sup.hiCD66b.sup.+ BMNs. FIG. 20C shows flow cytometric analysis of
CD14 and HLA-DR expression on gated live
CD11b.sup.+CD15.sup.hiCD66b.sup.+ BMNs cultured in the presence of
lenalidomide (10 mM) and hybrid-inducing TCM (30% v/v) for 6 days.
FIG. 20D shows the effect of IFN-.gamma. (50 pg/ml) and GM-CSF (50
pg/ml) on the formation of HLA-DR.sup.+CD14.sup.+ hybrid
neutrophils in the absence (upper panel) or presence (lower panel)
of lenalidomide (10 mM) in vitro. The level of Ikaros expression
(MFI) in BMNs treated with IFN-g (50 pg/ml) and GM-CSF (50 pg/ml)
for 5 days is shown (mean.+-.SEM, n=3, *p.ltoreq.0.01, Wilcoxon
matched-pairs rank test). Representative dot plots from one of six
experiments are shown in (FIGS. 20A-20D).
[0043] FIGS. 21A-21D are a series of graphs and images showing
APC-like hybrid neutrophils are able to stimulate T cell responses.
FIG. 21A shows development of hybrid and canonical neutrophils. To
obtain hybrid HLA-DR.sup.+CD14.sup.+ neutrophils (top panel), BMNs
were treated with hybrid-inducing TCM collected from tumor digests
where the frequency of hybrid TANs was markedly elevated. To obtain
canonical HLA-DR-CD14.sup.- neutrophils (bottom panel), BMNs were
treated with TCM collected from tumor digests where hybrid TANs
were not detected. The expression of CD62L, CD54, HLA-DR and CD14
was measured by flow cytometry on gated live
CD11b.sup.+CD66b.sup.+CD15.sup.hi cells. Representative dot plots
from 1 of 12 experiments are shown. FIG. 21B shows expression of
the CD25, and CD69 markers on activated autologous T cells
co-incubated with BM-derived canonical and hybrid neutrophils. T
cells were isolated from PBMC, stimulated with plate-bound anti-CD3
Abs and incubated with BM-derived neutrophils at a 1:1 ratio for 24
hours. Error bars represent mean.+-.SEM from 6 independent
experiments (Wilcoxon matched-pairs rank test). FIG. 21C shows the
ability of hybrid HLA-DR.sup.+ or canonical HLA-DR.sup.- BMNs to
stimulate autologous virus-specific memory T cell response in an
IFN-.gamma. ELISPOT assay. Autologous T cells were isolated from
PBMC and co-cultured with BM-derived canonical HLA-DR- and hybrid
HLA-DR.sup.+ neutrophils that had been pulsed with a mixture of
viral T cell epitopes for 2 hours. The number of
IFN-.gamma.-producing T cells was determined in three independent
ELISpot assays. Error bars represent mean.+-.SEM from 3 independent
experiments (*p.ltoreq.0.01, Mann-Whitney test). FIG. 21D shows
flow cytometric analysis of IFN-.gamma. production by Ly95 T cells
stimulated with A2/NY-ESO A549 tumor cells in the presence of
hybrid HLA-DR.sup.+ BMNs using a transwell system. Activated Ly95 T
cells were mixed with HLA-DR.sup.+ BMNs at a 1:1 ratio (mix). To
separate T cells and BMNs, activated T cells were cultured in the
bottom chamber and HLA-DR.sup.+ BMNs were placed in the top chamber
of the 24-well flat-bottom transwell culture plate (TW).
Representative results from 1 of 3 experiments are shown.
[0044] FIGS. 22A-22D are a series of graphs showing APC-like hybrid
neutrophils stimulate antigen-nonspecific T cell responses. FIG.
22A shows the proliferation and IFN-.gamma. production of anti-CD3
Ab stimulated autologous T cells in the presence of BM-derived
canonical and hybrid neutrophils differentiated with
hybrid-inducing TCM or IFN-.gamma. (50 pg/ml) and GM-CSF (50
pg/ml). FIG. 22B shows summary results of autologous T cell
proliferation (upper graph) and IFN-.gamma. production (lower
graph) in the presence of canonical and hybrid neutrophils. Data
are presented as a ratio (CD3 cells+CD15.sup.hi)/(CD3) (n=8,
Wilcoxon matched-pairs rank test). FIG. 22C shows the proliferation
of CFSE-labeled autologous PBMCs cultured with hybrid BMNs with
different level of PD-L1 expression in the presence (lower panel)
or absence PD-L1 blocking Abs (5 mg/ml) (upper panel).
PD-L1.sup.-/lo/hi HLA-DR.sup.+ hybrid neutrophils were
differentiated with GM-CSF (50 pg/ml) and increasing doses of
IFN-.gamma.. FIG. 22D shows the proliferation of allogeneic T cells
from healthy donors in the presence of APC-like hybrid neutrophils
in a mixed-lymphocyte reaction. Representative results from one of
six experiments are shown in FIGS. 22C-22D.
[0045] FIGS. 23A-23E are a series of graphs and images showing
APC-like hybrid neutrophils are able to trigger and stimulate
NY-ESO-specific effector T cell responses. FIG. 23A shows
NY-ESO-specific Ly95 cells (TCR Vp13.1.sup.+CD8.sup.+) were
stimulated with A549 tumor cell line expressing NY-ESO-1 in the
context of HLA-A*02 (A2/NY-ESO-1 A549) in the presence of
BM-derived canonical and hybrid neutrophils. Intracellular
IFN-.gamma. and Granzyme B production was measured by flow
cytometry. FIG. 23B shows cumulative results of the Ly95 cell
stimulatory activity of canonical and hybrid neutrophils.
Stimulatory activity was defined as a ratio (Ly95
cells+A549-N-ESO+BMN)/(Ly95 cells+A549-NY-ESO) (n=6, Wilcoxon
matched-pairs rank test). FIG. 23C shows HLA-A02.sup.+ canonical or
hybrid neutrophils were pulsed with synthetic NY-ESO-1 peptide and
co-cultured with Ly95 cells for 24 hr. Intracellular IFN-.gamma.
was assessed by flow cytometry (mean.+-.SEM, n=6, *p<0.01,
Wilcoxon matched-pairs rank test). FIG. 23D shows DQ-OVA uptake and
processing by BM-derived canonical or hybrid neutrophils (open
histograms). Cells incubated at 4.degree. C. served as controls
(shaded histograms). FIG. 23E shows cross-presentation of NY-ESO-1
epitopes to Ly95 cells by HLA-A02.sup.+ canonical or hybrid
neutrophils preloaded with NY-ESO-1 protein, NY-ESO-1 peptide, or
NY-ESO-immune complex (IC). IFN-.gamma. ELISpot (mean.+-.SEM, n=6,
*p % 0.01 canonical versus hybrid, Wilcoxon matched-pairs rank
test).
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0046] 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 to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods and materials are described.
[0047] As used herein, each of the following terms has the meaning
associated with it in this section.
[0048] The articles "a" and "an" are used herein to refer to one or
to more than one (i.e., to at least one) of the grammatical object
of the article. By way of example, "an element" means one element
or more than one element.
[0049] As used herein when referring to a measurable value such as
an amount, a temporal duration, and the like, the term "about" is
meant to encompass variations of .+-.20% or within 10%, 9%, 8%, 7%,
6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the
specified value, as such variations are appropriate to perform the
disclosed methods. Unless otherwise clear from context, all
numerical values provided herein are modified by the term
about.
[0050] In this disclosure, "comprises," "comprising," "containing"
and "having" and the like can have the meaning ascribed to them in
U.S. Patent law and can mean " includes," "including," and the
like; "consisting essentially of" or "consists essentially"
likewise has the meaning ascribed in U.S. Patent law and the term
is open-ended, allowing for the presence of more than that which is
recited so long as basic or novel characteristics of that which is
recited is not changed by the presence of more than that which is
recited, but excludes prior art embodiments.
[0051] By "agent" is meant any small molecule chemical compound,
antibody, nucleic acid molecule, or polypeptide, or fragments
thereof. In some embodiments, contacting immature neutrophils or
immature CD15-positive cells with an agent stimulate generation of
hybrid neutrophils from these cells. The stimulating agent, for
example, may be INF-.gamma., GM-CSF, lenalidomide, or an analog
thereof.
[0052] As used herein, to "alleviate" a disease, disorder or
condition means reducing the severity of one or more symptoms of
the disease, disorder or condition.
[0053] By "analog" is meant a molecule that is not identical, but
has analogous functional or structural features.
[0054] The terms "binding," "bind," "bound" refer to an interaction
between two molecules. The interaction may include a covalent or
non-covalent bond. The interaction may also be reversible or
irreversible depending on the type of interaction, such as covalent
bond formation.
[0055] The term "antibody," as used herein, refers to an
immunoglobulin molecule which specifically binds with an antigen.
Antibodies can be intact immunoglobulins derived from natural
sources or from recombinant sources and can be immunoreactive
portions of intact immunoglobulins. Antibodies are typically
tetramers of immunoglobulin molecules. Tetramers may be naturally
occurring or reconstructed from single chain antibodies or antibody
fragments. Antibodies also include dimers that may be naturally
occurring or constructed from single chain antibodies or antibody
fragments. The antibodies in the present invention may exist in a
variety of forms including, for example, polyclonal antibodies,
monoclonal antibodies, Fv, Fab and F(ab') 2 , as well as single
chain antibodies (scFv), humanized antibodies, and human antibodies
(Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory Press, N.Y.; Harlow et al., 1989, In:
Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston
et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al.,
1988, Science 242:423-426).
[0056] The term "antibody fragment" refers to a portion of an
intact antibody and refers to the antigenic determining variable
regions of an intact antibody. Examples of antibody fragments
include, but are not limited to, Fab, Fab', F(ab') 2 , and Fv
fragments, linear antibodies, scFv antibodies, single-domain
antibodies, such as camelid antibodies (Riechmann, 1999, Journal of
Immunological Methods 231:25-38), composed of either a VL or a VH
domain which exhibit sufficient affinity for the target, and
multispecific antibodies formed from antibody fragments. The
antibody fragment also includes a human antibody or a humanized
antibody or a portion of a human antibody or a humanized
antibody.
[0057] An "antibody heavy chain," as used herein, refers to the
larger of the two types of polypeptide chains present in all
antibody molecules in their naturally occurring conformations.
[0058] An "antibody light chain," as used herein, refers to the
smaller of the two types of polypeptide chains present in all
antibody molecules in their naturally occurring conformations. k
and l light chains refer to the two major antibody light chain
isotypes.
[0059] By the term "synthetic antibody" as used herein, is meant an
antibody which is generated using recombinant DNA technology, such
as, for example, an antibody expressed by a bacteriophage as
described herein. The term should also be construed to mean an
antibody which has been generated by the synthesis of a DNA
molecule encoding the antibody and which DNA molecule expresses an
antibody protein, or an amino acid sequence specifying the
antibody, wherein the DNA or amino acid sequence has been obtained
using synthetic DNA or amino acid sequence technology which is
available and well known in the art.
[0060] The term "antigen" or "Ag" as used herein is defined as a
molecule that provokes an immune response. This immune response may
involve either antibody production, or the activation of specific
immunologically-competent cells, or both. The skilled artisan will
understand that any macromolecule, including virtually all proteins
or peptides, can serve as an antigen. Furthermore, antigens can be
derived from recombinant or genomic DNA. A skilled artisan will
understand that any DNA, which comprises a nucleotide sequences or
a partial nucleotide sequence encoding a protein that elicits an
immune response therefore encodes an "antigen" as that term is used
herein. Furthermore, one skilled in the art will understand that an
antigen need not be encoded solely by a full length nucleotide
sequence of a gene. It is readily apparent that the present
invention includes, but is not limited to, the use of partial
nucleotide sequences of more than one gene and that these
nucleotide sequences are arranged in various combinations to elicit
the desired immune response. Moreover, a skilled artisan will
understand that an antigen need not be encoded by a "gene" at all.
It is readily apparent that an antigen can be generated synthesized
or can be derived from a biological sample. Such a biological
sample can include, but is not limited to a tissue sample, a tumor
sample, a cell or a biological fluid.
[0061] By "anti-tumor antibody" or "therapeutic anti-tumor
antibody" is meant an antibody specifically binding to a tumor
antigen. Binding of the antibody to the tumor may effect an immune
response against the tumor. In some embodiments, hybrid neutrophils
of the invention increase efficacy of anti-tumor antibodies by
increasing antibody-dependent cellular cytoxicity (ADCC),
antibody-dependent phagocytosis (ADP), and/or effector T cell
response. Examples of anti-tumor antibodies include, without
limitation, anti-Her2/neu antibody, rituximab, necitumumab,
panitumumab, and cetuximab.
[0062] The term "anti-tumor effect" as used herein, refers to a
biological effect which can be manifested by a decrease in tumor
volume, a decrease in the number of tumor cells, a decrease in the
number of metastases, an increase in life expectancy, or
amelioration of various physiological symptoms associated with the
cancerous condition. An "anti-tumor effect" can also be manifested
by the ability of the peptides, polynucleotides, cells and
antibodies of the invention in prevention of the occurrence of
tumor in the first place.
[0063] As used herein, the term "autologous" is meant to refer to
any material derived from the same individual to which it is later
to be re-introduced into the individual.
[0064] "Allogeneic" refers to a graft derived from a different
animal of the same species.
[0065] "Xenogeneic" refers to a graft derived from an animal of a
different species.
[0066] By "antibody dependent cellular cytotoxicity" or "ADCC" is
meant a process whereby an effector cell lyses a target cell,
wherein the target cell is bound by antibodies recognizing antigens
on the surface of the target cell. By "antibody dependent
phagocytosis" or ADP is meant a process whereby a phagocytic cell
(e.g., a neutrophil) engulfs or phagocytoses a target cell, wherein
the target cell is bound by antibodies recognizing antigens on the
surface of the target cell. In some embodiments, hybrid neutrophils
of the invention increase effectiveness of ADCC and/or ADP in a
subject, particularly a subject having a tumor treated with
anti-tumor therapeutic antibodies (e.g., cetuximab). Without being
bound by theory, it is believed the hybrid neutrophils may increase
ADCC and/or ADP through Fc receptors (e.g., CD16, CD32, CD64 and
CD89) expressed by the hybrid neutrophils, enabling them to
recognize and bind to Fc portions of the antibodies bound to tumor
cells and thereby phagocytose and/or lyse the tumor cells. By an
"increase" or "enhancement" in ADCC and/or ADP is meant an increase
in the amount or frequency of these processes in a subject under
one condition relative to the amount or frequency of ADCC and/or
ADP processes in the subject in another condition (e.g. a control
condition such as an untreated subject or the subject treated only
with anti-tumor antibodies and not treated with hybrid
neutrophils). An "increase" or "enhancement" in ADCC and/or ADP is
expected to result in an increase in anti-tumor effect of
anti-tumor antibodies, increased inhibition of tumor cell growth,
and/or increased reduction in survival of tumor cells.
[0067] The term "cancer" as used herein is defined as disease
characterized by the rapid and uncontrolled growth of aberrant
cells. Cancer cells can spread locally or through the bloodstream
and lymphatic system to other parts of the body. Examples of
various cancers include but are not limited to, breast cancer,
prostate cancer, ovarian cancer, cervical cancer, skin cancer,
pancreatic cancer, colorectal cancer, renal cancer, liver cancer,
brain cancer, lymphoma, leukemia, lung cancer and the like. In
certain embodiments, the cancer is non-small cell lung cancer
(NSCLC).
[0068] By "effective amount" is meant the amount required to reduce
or improve at least one symptom related to the tumor or cancer in a
subject The effective amount of an anti-tumor antibody and/or the
effective amount of a composition comprising a hybrid neutrophil of
the present invention used for therapeutic treatment of a tumor
varies depending upon the manner of administration, the age, body
weight, and general health of the subject.
[0069] By "effector T cell response" is meant the process by which
effector T cells (e.g., effector CD8-positive T cells) recognize
peptide antigens presented by cell surface molecules (e.g., as MHC
class I molecules) expressed by antigen-presenting cells (APCs).
When effector T cells are presented with antigen, they become
activated and may begin to divide and/or secrete molecules (e.g.,
cytokines) that regulate or assist in the immune response.
[0070] Without being bound by theory, it is believed the hybrid
neutrophils may increase effector T cell response through their
APC-like characteristics (e.g., expression of MHC class I
molecules). This enables hybrid neutrophils to present antigens to
effector T cells such as CD8-positive T cells, thereby stimulating
an effector T cell response. By an "increase" or "enhancement" in
effector T cell response is meant an increase in the amount or
frequency of these effector T cell response processes in a subject
under one condition relative to the amount or frequency of these
processes in the subject in another condition (e.g. a control
condition such as an untreated subject or the subject treated only
with anti-tumor antibodies and not treated with hybrid
neutrophils). An "increase" or "enhancement" in effector T cell
response is expected to result in an increase in anti-tumor effect
of anti-tumor antibodies, increased inhibition of tumor cell
growth, and/or increased reduction in survival of tumor cells.
[0071] As used herein "endogenous" refers to any material from or
produced inside an organism, cell, tissue or system.
[0072] As used herein, the term "exogenous" refers to any material
introduced from or produced outside an organism, cell, tissue or
system.
[0073] The term "expression" as used herein is defined as the
transcription and/or translation of a particular nucleotide
sequence driven by its promoter. In some embodiments, "expression"
may refer to display of a polypeptide product of the transcription
and/or translation of the nucleotide on the surface of a cell. Such
polypeptides may be referred to as "cell surface markers" or "cell
surface molecules."
[0074] Different cell types (e.g., T cells, antigen-presenting
cells, or neutrophils) express molecules unique to the cell type.
Thus, these "cell molecules" typically serve as markers of specific
cell types. For example, a "neutrophil associated molecule" or
"neutrophil associated cell molecule" includes, without limitation,
Arg1, MPO, CD66b, and CD15. An "antigen-presenting cell (APC)
associated molecule" or "antigen-presenting cell (APC) associated
cell molecule" includes, without limitation, CD14, HLA-DR, CD32,
and CD64.
[0075] Cells of the invention may be characterized by expression of
cell molecules. A cell type expressing a cell molecule may be
classified as "positive" for the cell molecule. For example, a
neutrophil is Arg1-positive (Arg1.sup.+). Conversely, a cell type
that does not express a cell molecule may be classified as
"negative" for the cell molecule. Levels of expression of a cell
molecule may low, intermediate ("int"), or high ("hi"). For
example, neutrophils are CD15.sup.hi (i.e., express high levels of
CD15). Expression of cell molecules may be detected by any method
known to one of skill in the art (e.g., immunoassays using
antibodies against cell molecules).
[0076] As used herein, the term "fragment," as applied to a nucleic
acid, refers to a subsequence of a larger nucleic acid. A
"fragment" of a nucleic acid can be at least about 15 nucleotides
in length; for example, at least about 50 nucleotides to about 100
nucleotides; at least about 100 to about 500 nucleotides, at least
about 500 to about 1000 nucleotides; at least about 1000
nucleotides to about 1500 nucleotides; about 1500 nucleotides to
about 2500 nucleotides; or about 2500 nucleotides (and any integer
value in between).
[0077] As used herein, the term "fragment," as applied to a protein
or peptide, refers to a subsequence of a larger protein or peptide.
A "fragment" of a protein or peptide can be at least about 20 amino
acids in length; for example, at least about 50 amino acids in
length; at least about 100 amino acids in length; at least about
200 amino acids in length; at least about 300 amino acids in
length; or at least about 400 amino acids in length (and any
integer value in between).
[0078] As used herein, the terms "gene" and "recombinant gene"
refer to nucleic acid molecules comprising an open reading frame
encoding a polypeptide. Such natural allelic variations can
typically result in 1-5% variance in the nucleotide sequence of a
given gene. Alternative alleles can be identified by sequencing the
gene of interest in a number of different individuals. This can be
readily carried out by using hybridization probes to identify the
same genetic locus in a variety of individuals. Any and all such
nucleotide variations and resulting amino acid polymorphisms or
variations that are the result of natural allelic variation and
that do not alter the functional activity are intended to be within
the scope of the invention.
[0079] Moreover, nucleic acid molecules encoding proteins from
other species (homologs), which have a nucleotide sequence that
differs from that of the human proteins described herein are within
the scope of the invention. Nucleic acid molecules corresponding to
natural allelic variants and homologs of a cDNA of the invention
can be isolated based on their identity to human nucleic acid
molecules using the human cDNAs, or a portion thereof, as a
hybridization probe according to standard hybridization techniques
under stringent hybridization conditions.
[0080] By "granulocyte colony stimulating factor" or "G-CSF" is
meant a glycoprotein having growth factor activity and
immunomodulatory activities. G-CSF may stimulate bone marrow to
produce to produce granulocytes such as neutrophils, and may
promote proliferation and differentiation of neutrophil precursors.
In some embodiments, peripheral blood immature neutrophils are
mobilized in peripheral blood by contacting peripheral blood with
G-CSF and/or other agents.
[0081] By "granulocyte macrophage colony stimulating factor" or
"GM-CSF" is meant a glycoprotein having growth factor activity and
immunomodulatory activities. In particular, GM-CSF may stimulate
stem cells to produce granulocytes such as neutrophils. In some
embodiments, compositions comprising immature bone marrow
CD15-positive cells or peripheral blood immature neutrophils are
contacted with GM-CSF and/or other agents to stimulate generation
of hybrid neutrophils.
[0082] "Homologous" as used herein, refers to the subunit sequence
identity between two polymeric molecules, e.g., between two nucleic
acid molecules, such as, two DNA molecules or two RNA molecules, or
between two polypeptide molecules. When a subunit position in both
of the two molecules is occupied by the same monomeric subunit;
e.g., if a position in each of two DNA molecules is occupied by
adenine, then they are homologous at that position. The homology
between two sequences is a direct function of the number of
matching or homologous positions; e.g., if half (e.g., five
positions in a polymer ten subunits in length) of the positions in
two sequences are homologous, the two sequences are 50% homologous;
if 90% of the positions (e.g., 9 of 10), are matched or homologous,
the two sequences are 90% homologous.
[0083] As applied to a protein sequence, "homology" as used herein
refers to a protein sequence that has about 50% sequence
similarity. More preferably, the sequence has about 75% sequence
similarity, even more preferably, has at least about 80%, 85%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence
similarity.
[0084] "Identity" as used herein refers to the subunit sequence
identity between two polymeric molecules particularly between two
amino acid molecules, such as, between two polypeptide molecules.
When two amino acid sequences have the same residues at the same
positions; e.g., if a position in each of two polypeptide molecules
is occupied by an Arginine, then they are identical at that
position. The identity or extent that two amino acid sequences have
the same residues at the same positions in an alignment is often
expressed as a percentage. The identity between two amino acid
sequences is a direct function of the number of matching or
identical positions; e.g., if half (e.g., five positions in a
polymer ten amino acids in length) of the positions in two
sequences are identical, the two sequences are 50% identical; if
90% of the positions (e.g., 9 of 10), are matched or identical, the
two amino acids sequences are 90% identical. As applied to nucleic
acid sequences, "identity" as used herein refers to a sequence that
has about 50% sequence identity. More preferably, the homologous
sequence has about 75% sequence identity, even more preferably, has
at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% sequence identity.
[0085] As used herein, an "instructional material" includes a
publication, a recording, a diagram, or any other medium of
expression that can be used to communicate the usefulness of the
compositions and methods of the invention. The instructional
material of the kit of the invention may, for example, be affixed
to a container that contains the nucleic acid, peptide, and/or
composition of the invention or be shipped together with a
container that contains the nucleic acid, peptide, and/or
composition. Alternatively, the instructional material may be
shipped separately from the container with the intention that the
instructional material and the compound be used cooperatively by
the recipient.
[0086] By "interferon gamma," "interferon-.gamma.," or
"IFN-.gamma." is meant a cytokine belonging to the type II class of
interferons. IFN-.gamma. has antiviral activity and
immunoregulatory functions, such as activation of macrophages. In
some embodiments, compositions comprising immature bone marrow
CD15-positive cells or peripheral blood immature neutrophils are
contacted with IFN-.gamma. and/or other agents to stimulate
generation of hybrid neutrophils.
[0087] The terms "isolated," "purified," or "biologically pure"
refer to material that is free to varying degrees from components
which normally accompany it as found in its native state. "Isolate"
denotes a degree of separation from original source or
surroundings. For example, in some embodiments, a hybrid neutrophil
is isolated from tumor tissues in a cancer patient. In some other
embodiments, immature CD15-positive cells are isolated from bone
marrow. "Purify" denotes a degree of separation that is higher than
isolation. A "purified" or "biologically pure" protein is
sufficiently free of other materials such that any impurities do
not materially affect the biological properties of the protein or
cause other adverse consequences. That is, a nucleic acid or
peptide is purified if it is substantially free of cellular
material, viral material, or culture medium when produced by
recombinant DNA techniques, or chemical precursors or other
chemicals when chemically synthesized. Purity and homogeneity are
typically determined using analytical chemistry techniques, for
example, polyacrylamide gel electrophoresis or high performance
liquid chromatography. The term "purified" can denote that a
nucleic acid or protein gives rise to essentially one band in an
electrophoretic gel. For a protein that can be subjected to
modifications, for example, phosphorylation or glycosylation,
different modifications may give rise to different isolated
proteins, which can be separately purified. "Purified" can also
refer to a molecule separated after a bioconjugation technique from
those molecules which were not efficiently conjugated.
[0088] An "isolated nucleic acid" refers to a nucleic acid segment
or fragment that has been separated from sequences that flank it in
a naturally occurring state, e.g., a DNA fragment that has been
removed from the sequences that are normally adjacent to the
fragment, e.g., the sequences adjacent to the fragment in a is
genome that it naturally occurs. The term also applies to nucleic
acids that have been substantially purified from other components
that naturally accompany the nucleic acid, e.g., RNA or DNA or
proteins, that naturally accompany it in the cell. The term
therefore includes, for example, a recombinant DNA that is
incorporated into a vector, into an autonomously replicating
plasmid or virus, or into the genomic DNA of a prokaryote or
eukaryote, or that exists as a separate molecule (e.g., as a cDNA
or a genomic or cDNA fragment produced by PCR or restriction enzyme
digestion) independent of other sequences. It also includes a
recombinant DNA that is part of a hybrid gene encoding additional
polypeptide sequence.
[0089] A "neutrophil" is a type of leukocyte which constitutes
about 50% to 80% of all leukocytes in the human body. Neutrophils
are generated from precursor cells in the bone marrow and have
phagocytic activity. Neutrophils display the cell surface markers
Arg1, MPO, CD66b, and CD15. Neutrophils may also infiltrate a tumor
microenvironment and mediate processes associated with tumor
progression. Such neutrophils are "tumor-associated neutrophils" or
"TANs." Tumor-recruited myeloid cells represent a significant
portion of inflammatory cells in the tumor microenvironment and
influence nearly all steps of tumor progression. Among these
myeloid cells, tumor-associated neutrophils (TANs) are present in
large numbers. The majority of TANs expressed classic neutrophil
markers ("canonical TANs").
[0090] In the context of the present invention, the following
abbreviations for the commonly occurring nucleic acid bases are
used. "A" refers to adenosine, "C" refers to cytosine, "G" refers
to guanosine, "T" refers to thymidine, and "U" refers to
uridine.
[0091] Unless otherwise specified, a "nucleotide sequence encoding
an amino acid sequence" includes all nucleotide sequences that are
degenerate versions of each other and that encode the same amino
acid sequence. The phrase nucleotide sequence that encodes a
protein or an RNA may also include introns to the extent that the
nucleotide sequence encoding the protein may in some version
contain an intron(s).
[0092] The term "oligonucleotide" typically refers to short
polynucleotides, generally, no greater than about 50 nucleotides.
It will be understood that when a nucleotide sequence is
represented by a DNA sequence (i.e., A, T, G, C), this also
includes an RNA sequence (i.e., A, U, G, C) that "U" replaces
"T."
[0093] "Pharmaceutically acceptable" refers to those properties
and/or substances that are acceptable to the patient from a
pharmacological/toxicological point of view and to the
manufacturing pharmaceutical chemist from a physical/chemical point
of view regarding composition, formulation, stability, patient
acceptance and bioavailability. "Pharmaceutically acceptable
carrier" refers to a medium that does not interfere with the
effectiveness of the biological activity of the active
ingredient(s) and is not toxic to the host to which it is
administered.
[0094] As used herein, the term "pharmaceutical composition" or
"pharmaceutically acceptable composition" refers to a mixture of at
least one compound or molecule or cell useful within the invention
with a pharmaceutically acceptable carrier. The pharmaceutical
composition facilitates administration of the compound or molecule
or cell to a patient. Multiple techniques of administering a
compound or molecule exist in the art including, but not limited
to, intravenous, oral, aerosol, parenteral, ophthalmic, pulmonary
and topical administration.
[0095] As used herein, the term "pharmaceutically acceptable
carrier" means a pharmaceutically acceptable material, composition
or carrier, such as a liquid or solid filler, stabilizer,
dispersing agent, suspending agent, diluent, excipient, thickening
agent, solvent or encapsulating material, involved in carrying or
transporting a compound, molecule, or cell useful within the
invention within or to the patient such that it may perform its
intended function. Typically, such constructs are carried or
transported from one organ, or portion of the body, to another
organ, or portion of the body. Each carrier must be "acceptable" in
the sense of being compatible with the other ingredients of the
formulation, including the compound useful within the invention,
and not injurious to the patient. Some examples of materials that
may serve as pharmaceutically acceptable carriers include: sugars,
such as lactose, glucose and sucrose; starches, such as corn starch
and potato starch; cellulose, and its derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa
butter and suppository waxes; oils, such as peanut oil, cottonseed
oil, safflower oil, sesame oil, olive oil, corn oil and soybean
oil; glycols, such as propylene glycol; polyols, such as glycerin,
sorbitol, mannitol and polyethylene glycol; esters, such as ethyl
oleate and ethyl laurate; agar; buffering agents, such as magnesium
hydroxide and aluminum hydroxide; surface active agents; alginic
acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl
alcohol; phosphate buffer solutions; and other non-toxic compatible
substances employed in pharmaceutical formulations. As used herein,
"pharmaceutically acceptable carrier" also includes any and all
coatings, antibacterial and antifungal agents, and absorption
delaying agents, and the like that are compatible with the activity
of the compound useful within the invention, and are
physiologically acceptable to the patient. Supplementary active
compounds may also be incorporated into the compositions. The
"pharmaceutically acceptable carrier" may further include a
pharmaceutically acceptable salt of the compound or molecule useful
within the invention. Other additional ingredients that may be
included in the pharmaceutical compositions used in the practice of
the invention are known in the art and described, for example in
Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing
Co., 1985, Easton, Pa.), which is incorporated herein by
reference.
[0096] The term "polynucleotide" as used herein is defined as a
chain of nucleotides. Furthermore, nucleic acids are polymers of
nucleotides. Thus, nucleic acids and polynucleotides as used herein
are interchangeable. One skilled in the art has the general
knowledge that nucleic acids are polynucleotides, which can be
hydrolyzed into the monomeric "nucleotides." The monomeric
nucleotides can be hydrolyzed into nucleosides. As used herein
polynucleotides include, but are not limited to, all nucleic acid
sequences that are obtained by any means available in the art,
including, without limitation, recombinant means, i.e., the cloning
of nucleic acid sequences from a recombinant library or a cell
genome, using ordinary cloning technology and PCR.TM., and the
like, and by synthetic means.
[0097] As used herein, the terms "peptide," "polypeptide," and
"protein" are used interchangeably, and refer to a compound
comprised of amino acid residues covalently linked by peptide
bonds. A protein or peptide must contain at least two amino acids,
and no limitation is placed on the maximum number of amino acids
that can comprise a protein's or peptide's sequence. Polypeptides
include any peptide or protein comprising two or more amino acids
joined to each other by peptide bonds. As used herein, the term
refers to both short chains, which also commonly are referred to in
the art as peptides, oligopeptides and oligomers, for example, and
to longer chains, which generally are referred to in the art as
proteins, that there are many types. "Polypeptides" include, for
example, biologically active fragments, substantially homologous
polypeptides, oligopeptides, homodimers, heterodimers, variants of
polypeptides, modified polypeptides, derivatives, analogs, fusion
proteins, among others. The polypeptides include natural peptides,
recombinant peptides, synthetic peptides, or a combination
thereof.
[0098] A "signal transduction pathway" refers to the biochemical
relationship between a variety of signal transduction molecules
that play a role in the transmission of a signal from one portion
of a cell to another portion of a cell. The phrase "cell surface
receptor" includes molecules and complexes of molecules capable of
receiving a signal and transmitting signal across the plasma
membrane of a cell.
[0099] As used herein, "sample" or "biological sample" refers to
anything, which may contain the cells of interest (e.g., immature
granulocytes). The sample may be a biological sample, such as a
biological fluid or a biological tissue. In one embodiment, a
biological sample is a tissue sample including pulmonary arterial
endothelial cells. Such a sample may include diverse cells,
proteins, and genetic material. Examples of biological tissues also
include organs, tumors, lymph nodes, arteries and individual
cell(s). Examples of biological fluids include urine, blood,
plasma, serum, saliva, semen, stool, sputum, cerebral spinal fluid,
tears, mucus, amniotic fluid or the like. In some embodiments, the
biological sample is blood. In some other embodiments, the
biological sample is bone marrow.
[0100] By the term "specifically binds," as used herein, is meant a
compound, e.g., a protein, a nucleic acid, an antibody, and the
like, which recognizes and binds a specific molecule, but does not
substantially recognize or bind other molecules in a sample.
[0101] The term "subject" is intended to include living organisms
that an immune response can be elicited (e.g., mammals). A
"subject" or "patient," as used therein, may be a human or
non-human mammal. Non-human mammals include, for example, livestock
and pets, such as ovine, bovine, porcine, canine, feline and murine
mammals. Preferably, the subject is human.
[0102] As used herein, a "substantially purified" cell is a cell
that is essentially free of other cell types. A substantially
purified cell also refers to a cell that has been separated from
other cell types that it is normally associated in its naturally
occurring state. In some instances, a population of substantially
purified cells refers to a homogenous population of cells. In other
instances, this term refers simply to cell that have been separated
from the cells that they are naturally associated in their natural
state. In some embodiments, the cells are cultured in vitro. In
other embodiments, the cells are not cultured in vitro.
[0103] The term "therapeutic" as used herein means a treatment
and/or prophylaxis. A therapeutic effect is obtained by
suppression, remission, or eradication of a disease state.
[0104] As used herein, a "therapeutic agent" is a molecule or atom
that is useful for therapy. Examples of therapeutic agents include
drugs, toxins, enzymes, hormones, cytokines, immunomodulators,
anti-tumor agents, chemotherapeutic agents, anti-cell proliferation
agents, boron compounds, and therapeutic radioisotopes.
[0105] The term "transfected" or "transformed" or "transduced" as
used herein refers to a process that exogenous nucleic acid is
transferred or introduced into the host cell. A "transfected" or
"transformed" or "transduced" cell is one that has been
transfected, transformed or transduced with exogenous nucleic acid.
The cell includes the primary subject cell and its progeny.
[0106] As used herein, the terms "treat," treating," "treatment,"
and the like refer to reducing or improving a disorder and/or
symptom associated therewith. It will be appreciated that, although
not precluded, treating a disorder or condition does not require
that the disorder, condition or symptoms associated therewith be
completely ameliorated or eliminated.
[0107] By "tumor conditioned medium" or "TCM" is meant a medium
obtained by collecting medium from a digested tumor, wherein the
tumor that was digested contained populations of tumor-associated
neutrophils exhibiting the hybrid neutrophil phenotype (i.e.,
positive for Arg1, MPO, CD66b, and CD15 and positive for CD14,
HLA-DR, CD32, CD64, and CD89).
[0108] Ranges provided herein are understood to be shorthand for
all of the values within the range. For example, a range of 1 to 50
is understood to include any number, combination of numbers, or
sub-range from the group consisting 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, or 50.
[0109] The recitation of an embodiment for a variable or aspect
herein includes that embodiment as any single embodiment or in
combination with any other embodiments or portions thereof. Any
compositions or methods provided herein can be combined with one or
more of any of the other compositions and methods provided
herein.
Composition and Methods of the Invention
[0110] The present invention relates to compositions and methods
that include novel anti-tumor therapies for cancer. The invention
is based, at least in part, on the discovery of a novel subset of
tumor-associated neutrophils exhibiting composite characteristics
of neutrophils and antigen-presenting cells. Described herein are
studies demonstrating that hybrid
CD14.sup.+HLA-DR.sup.+CD32.sup.hiCD64.sup.hi neutrophils can be
generated from bone marrow or peripheral blood immature
granulocytes. These differentiated cells efficiently phagocytose
bacteria, mediate a high level of antibody dependent phagocytosis
and stimulate the effector T cell responses in vitro. These
abilities of hybrid neutrophils provide new opportunities to boost
anti-tumor and anti-infectious immunity.
Hybrid Neutrophils
[0111] Neutrophils are a type of leukocyte and constitute about 50%
to 80% of all leukocytes in the human body. Neutrophils are
generated from precursor cells in the bone marrow. Within the body,
neutrophils migrate to areas of infection or tissue injury.
Neutrophils are antimicrobial effector cells equipped with powerful
killing machinery to respond to pathogens. For example, neutrophils
are phagocytic and may engulf bacteria or other microorganisms by
phagocytosis.
[0112] Neutrophils especially target opsonized bacteria or other
microorganisms (i.e., bacteria or microorganisms "marked" for
destruction by phagocytic cells). Neutrophils may target a
microorganism through a process referred to as antibody-dependent
cellular cytotoxicity (ADCC). In this process, antibodies bind to
antigens on the microorganism's cell membrane. The Fc portion of
the antibody is recognized by Fc receptors on neutrophils, thus
directing neutrophils to the microorganism.
[0113] Without intending to be bound by theory, it is hypothesized
that therapeutic antibodies against tumor antigens can direct and
activate neutrophils' cytotoxic machinery against opsonized tumors
through ADCC. Unfortunately, the clinical efficacy of many
therapeutic antibodies is poor and needs to be enhanced (Liu et
al., Cancer Chemother Pharmacol. 2010 April; 65(5): 849-861; Fury
et al., Cancer. Immunol Immunother. 2008 February; 57(2): 155-163;
Repp et al., Br J Cancer. 2003 Dec. 15; 89(12): 2234-2243). The
identification and administration of efficient effector subsets
responsible for mediating sufficient ADCC in humans could lead to
the development of more synergistic and combination therapies that
would enhance the effect of therapeutic antibodies (which may also
include antibodies directed at bacteria or other
microorganisms).
[0114] In some aspects, the invention described herein features a
novel approach to boost anti-tumor and anti-infectious immunity by
engaging a newly identified subpopulation of activated neutrophils
with the composite characteristics of neutrophils
(Arg1.sup.+MPO.sup.-CD66b.sup.+CD15.sup.hi) and antigen-presenting
cells (CD14.sup.+HLA-DR.sup.+CD32.sup.hiCD64.sup.hi). Heretofore
these cells are referred to as "hybrid neutrophils." The unique
phenotype of hybrid neutrophils described herein enables them to
more efficiently mediate antibody-dependent phagocytosis and
stimulate effector T cell responses. In some aspects, the hybrid
neutrophil expresses at least one neutrophil associated molecule
selected from the group consisting of: Arg1, MPO, CD66b, and CD15,
and at least one antigen-presenting cell (APC) associated molecule
selected from the group consisting of: CD14, HLA-DR, CD32, and
CD64. In certain embodiments, the hybrid neutrophil further
expresses at least one molecule selected from the group consisting
of: MHC class I, MHC class II, OX40L, 4-1BBL, CD86, CD40, CCR7, and
CD89. In certain embodiments, the expression of any one of the
molecules is low, intermediate, or high. In other embodiments, the
expression of any one of the molecules is increased relative to
expression of the molecule on a canonical tumor-associated
neutrophil (TAN). In particular embodiments, the hybrid neutrophil
expresses CD14, HLA-DR, CD32, CD64, CD89. In still other
embodiments, wherein the hybrid neutrophil expresses Arg1, MPO,
CD66b, CD15, CD14, HLA-DR, MHC class I, OX40L, 4-1BBL, CD86, CD40,
CCR7, CD32, CD64, and CD89. In particular embodiments, the
expression of CD32 and/or CD64 and/or CD89 is high.
[0115] The development of neutrophils exhibiting dual phenotype and
functionality of neutrophils and dendritic cells (DC) has recently
been described in mice (Matsushima et al., Blood. 2013 Mar. 7;
121(10): 1677-1689; Geng et al., Blood. 2013 Mar. 7; 121(10):
1690-1700), where the differentiation of bone marrow cells into
DC-neutrophil hybrids has been performed in the presence of GM-CSF.
However, the phenotype of hybrid neutrophils differentiated from
human bone marrow neutrophils with low concentrations of
IFN-.gamma. and GM-CSF is quite different from the phenotype of
those described previously. Human BM derived hybrid neutrophils
exhibit only partial phenotype of DC (MHC class II, CD86, CCR7);
however, they also acquire the partial phenotype of
monocyte/macrophages (CD14, CD206, CD64.sup.hi, CD32.sup.hi,
CD89.sup.hi). Importantly, it is believed that there is no report
of clinical use of human hybrid neutrophils.
[0116] In some aspects of the invention, the hybrid neutrophil is
in a container comprising at least one non-naturally occurring
component. The non-naturally occurring container may be any vessel
holding or capable of holding a hybrid neutrophil or composition
comprising a hybrid neutrophil. The non-naturally occurring
component may be, without limitation, glass, plastic, metal, or a
composite material. The non-naturally occurring container may be,
without limitation, a tube, capsule, dish, plate, flask, packet,
vial, pouch, jar, or bottle.
[0117] In certain aspects, the hybrid neutrophil expresses at least
one neutrophil associated molecule selected from the group
consisting of: Arg1, MPO, CD66b, and CD15, and at least one
antigen-presenting cell (APC) associated molecule selected from the
group consisting of: CD14, HLA-DR, CD32, CD64, and CD89. In another
aspect, the hybrid neutrophil further expresses at least one
molecule selected from the group consisting of: MHC class I, MHC
class II, OX40L, 4-1BBL, CD86, CD40, and CCR7. The expression level
of any one of the aforementioned molecules can be low,
intermediate, or high. In some embodiments, the expression of any
one of the molecules is increased relative to the expression of the
molecule on a canonical tumor-associated neutrophil (TAN).
[0118] In some embodiments, the hybrid neutrophil expresses CD14,
HLA-DR, CD32, CD64, and CD89. In some embodiments, the hybrid
neutrophil expresses Arg1, MPO, CD66b, CD15, CD14, HLA-DR, MHC
class I, OX40L, 4-1BBL, CD86, CD40, CCR7, CD32, CD64, and CD89. In
some embodiments, the expression of CD32 and/or CD64 and/or CD89 is
high.
Methods of Generating Hybrid Neutrophils
[0119] Hybrid neutrophils of the invention have been shown to
efficiently phagocytose bacteria, mediate a high level of antibody
dependent phagocytosis and stimulate the effector T cell responses
in vitro. These properties of hybrid neutrophils provide new
opportunities to boost anti-tumor and anti-infectious immunity.
[0120] In some aspects of the present invention, the hybrid
neutrophils are isolated from a tumor tissue of a cancer patient.
Hybrid neutrophils were found in tumor tissues of cancer patients,
although the frequency of these newly identified "hybrid" subset of
tumor associated neutrophils (TANs) varied widely in the tumor
tissues. It was found that these hybrid TANs comprised 0.5-25% of
all TANs.
[0121] Large numbers of hybrid neutrophils may be difficult to
obtain by isolation from tumor tissues. For therapeutic use of
hybrid neutrophils, it is desirable to generate large numbers of
these cells. Accordingly, provided herein are methods of generating
a hybrid neutrophil, particularly in large numbers. The studies
described herein demonstrated that hybrid
CD14.sup.+HLA-DR.sup.+CD32.sup.hiCD64.sup.hiCD89.sup.hi neutrophils
could be generated from bone marrow or peripheral blood immature
granulocytes. The methods herein feature identified conditions in
which bone marrow or peripheral blood immature granulocytes can be
differentiated into hybrid neutrophils in large numbers.
[0122] In one aspect, the present invention provides a method of
generating a hybrid neutrophil, the method comprising contacting a
composition comprising a bone marrow (BM) immature CD15-positive
(CD15+) cell with an amount of tumor conditioned medium (TMC). In
another aspect, the method comprises contacting a composition
comprising a bone marrow (BM) immature CD15-positive (CD15+) cell
with an amount of interferon g (IFN-.gamma.) and an amount of
granulocyte macrophage colony stimulating factor (GM-CSF). In yet
another aspect, the method comprises contacting a composition
comprising a bone marrow (BM) immature CD15-positive (CD15+) cell
with an amount of an agent that reduces the level of Ikaros
polypeptide in the cell and an amount of granulocyte macrophage
colony stimulating factor (GM-CSF). The agent reducing the level of
Ikaros may be lenalidomide, or an analog thereof (e.g.,
pomalidomide or thalidomide).
[0123] In still another aspect, the method comprises contacting a
composition comprising peripheral blood immature neutrophils with
an amount of tumor conditioned medium (TCM). In yet another aspect,
the method comprises contacting a composition comprising peripheral
blood immature neutrophils with an amount of interferon .gamma.
(IFN-.gamma.) and an amount of granulocyte macrophage colony
stimulating factor (GM-CSF). The tumor conditioned medium may be
obtained by collecting the medium from a digested tumor where
hybrid tumor-associated neutrophils (TANs) were previously
detected. The TCM may be added to the composition at about 50% v/v.
In various embodiments of any of the above aspects, the peripheral
blood immature neutrophils may be mobilized in peripheral blood by
contacting peripheral blood with an amount of GM-CSF or G-CSF.
[0124] In one aspect, the present invention provides a method of
generating a hybrid neutrophil comprising contacting a composition
comprising a bone marrow (BM) immature CD15-positive (CD15.sup.+)
cell with an amount of tumor conditioned medium, wherein the hybrid
neutrophil expresses at least one neutrophil associated molecule
selected from the group consisting of: Arg1, MPO, CD66b, and CD15,
and at least one antigen-presenting cell (APC) associated molecule
selected from the group consisting of: CD14, HLA-DR, CD32, CD64,
and CD89.
[0125] In another aspect, the present invention provides a method
of generating a hybrid neutrophil comprising contacting a
composition comprising a bone marrow (BM) immature CD15-positive
(CD15.sup.+) cell with an amount of interferon .gamma.
(IFN-.gamma.) and an amount of granulocyte macrophage colony
stimulating factor (GM-CSF), wherein the hybrid neutrophil
expresses at least one neutrophil associated molecule selected from
the group consisting of: Arg1, MPO, CD66b, and CD15, and at least
one antigen-presenting cell (APC) associated molecule selected from
the group consisting of: CD14, HLA-DR, CD32, CD64, and CD89.
[0126] In yet another aspect, the invention provides a method of
generating a hybrid neutrophil, comprising contacting a composition
comprising a bone marrow (BM) immature CD15-positive (CD15.sup.+)
cell with an amount of an agent that reduces the level of Ikaros
polypeptide in the cell and an amount of granulocyte macrophage
colony stimulating factor (GM-CSF), wherein the hybrid neutrophil
expresses at least one neutrophil associated molecule selected from
the group consisting of: Arg1, MPO, CD66b, and CD15, and at least
one antigen-presenting cell (APC) associated molecule selected from
the group consisting of: CD14, HLA-DR, CD32, CD64, and CD89. In one
embodiment, the agent reduces the level of Ikaros polypeptide in
the cell is lenalidomide.
[0127] Another aspect of the invention includes a method of
generating a hybrid neutrophil comprising contacting a composition
comprising peripheral blood immature neutrophils with an amount of
tumor conditioned medium, wherein the hybrid neutrophil expresses
at least one neutrophil associated molecule selected from the group
consisting of: Arg1, MPO, CD66b, and CD15, and at least one
antigen-presenting cell (APC) associated molecule selected from the
group consisting of: CD14, HLA-DR, CD32, CD64, and CD89. In certain
embodiments, the tumor conditioned medium is about 50% v/v.
[0128] Yet another aspect of the invention provides a method of
generating a hybrid neutrophil comprising contacting a composition
comprising peripheral blood immature neutrophils with an amount of
interferon .gamma. (IFN-.gamma.) and an amount of granulocyte
macrophage colony stimulating factor (GM-CSF), wherein the hybrid
neutrophil expresses at least one neutrophil associated molecule
selected from the group consisting of: Arg1, MPO, CD66b, and CD15,
and at least one antigen-presenting cell (APC) associated molecule
selected from the group consisting of: CD14, HLA-DR, CD32, CD64,
and CD89.
[0129] In some embodiments, the INF-.gamma. and/or GM-CSF is added
at concentrations of at least about 50 pg/ml, at least about 60
pg/ml, at least about 70 pg/ml, at least about 80 pg/ml, at least
about 90 pg/ml, or at least about 100 pg/ml. In particular
embodiments, GM-CSF is added at about 100 pg/ml. In particular
embodiments, IFN-.gamma. is added at about 100 pg/ml. Lenalidomide
may be added at a concentration of about 10 .mu.M.
[0130] Bone marrow (BM) immature CD15-positive cells may be
obtained from human bone marrow. The bone marrow may be obtained
from rib fragments removed from subjects during a surgery. An
enriched population of bone marrow neutrophils may be obtained by
using an anti-CD15 to isolate the CD15-positive cells. For example,
in one embodiment, bone marrow (BM) immature CD15 cells were
isolated using anti-CD15 magnetic beads.
[0131] Immature granulocytes may also be obtained from peripheral
blood after G-CSF or GM-CSF induced mobilization. In addition to
bone marrow origin, hybrid neutrophils may be differentiated from
peripheral blood immature neutrophils mobilized in peripheral blood
by an administration with G-SCF or GM-CSF. Immature neutrophils or
immature cells may then be collected and separated by any methods
known in the art, such as leukapheresis and/or
Fluorescence-Activated Cell Sorting (FACS). In a particular
embodiment, low-density immature neutrophils are isolated by
gradient separation.
[0132] In certain embodiments, peripheral blood immature
neutrophils are mobilized in peripheral blood by contacting
peripheral blood with an amount of granulocyte macrophage colony
stimulating factor (GM-CSF) or an amount of granulocyte colony
stimulating factor (G-CSF). In some embodiments, a subject is
treated with G-SCF or GM-CSF and peripheral blood low-density
immature neutrophils are isolated by gradient separation. In some
other embodiments, the peripheral blood immature neutrophils are
cultured in the presence of hybrid-inducing TCM (about 50% v/v) or
IFN-.gamma. and GM-CSF at concentrations of about 50-100 pg/ml. In
particular embodiments, at day 5, similar to BMNs, a significant
portion of G-CSF mobilized low-density PBNs acquire
HLA-DR.sup.+CD14.sup.+ phenotype.
[0133] In certain embodiments for the generation of hybrid
neutrophils, the isolated or purified immature CD15-positive cells
or immature granulocytes may be incubated or cultured with tumor
conditioned medium (TCM), IFN-.gamma., GM-CSF, and/or lenalidomide
as described herein for at least about 5 days, at least about 6
days, or at least about 7 days. In some embodiments, about 40% of
bone marrow neutrophils (BMNs) could survive in cell culture for up
to one week (FIG. 2B). Thus, human BMNs have a prolonged lifespan
in vitro, providing large quantities of cells (>50 million
cells) that can be used to generate hybrid neutrophils.
[0134] In certain embodiments, the hybrid neutrophil further
expresses at least one molecule selected from the group consisting
of: MHC class I, MHC class II, OX40L, 4-1BBL, CD86, CD40, and CCR7.
In certain embodiments the expression level of any one of the
aforementioned molecules is low, intermediate, or high. In certain
embodiments the expression of any one of the molecules is increased
relative to expression of the molecule on a canonical
tumor-associated neutrophil (TAN).
[0135] In certain embodiments, the hybrid neutrophil expresses
CD14, HLA-DR, CD32, CD64, and CD89. In other embodiments, the
hybrid neutrophil expresses Arg1, MPO, CD66b, CD15, CD14, HLA-DR,
MHC class I, OX40L, 4-1BBL, CD86, CD40, CCR7, CD32, CD64, and CD89.
In certain embodiments, the expression of CD32 and/or CD64 and/or
CD89 is high.
Methods of Treatment
[0136] The present invention features methods for increasing
efficacy of an antibody against a tumor in a subject using hybrid
neutrophils. Hybrid neutrophils are ideal effector cells for
augmenting antibody-mediated immunotherapy of cancer or infectious
diseases. Hybrid neutrophils are ideal for augmenting
antibody-mediated immunotherapy for at least the following reasons:
(1) hybrid neutrophils can be generated from BM of cancer patients
in large numbers, (2) the most potent Fc receptors for triggering
ADCC (CD32, CD64, and CD89) are highly up-regulated on hybrid
neutrophils, and (3) hybrid neutrophils exhibit prolonged survival
times, and (4) hybrid neutrophils are able to phagocyte bacteria at
higher level than canonical neutrophils. In addition, the hybrid
neutrophils have characteristics of antigen-presenting cells (APC)
and thus may be able to more efficiently stimulate effector T
cells. The ability of hybrid neutrophils to mediate efficient ADCC
and augment effector T cell responses provides new opportunities to
boost anti-tumor and anti-infectious immunity
[0137] In some embodiments, the step of administering to the
subject an effective amount of a hybrid neutrophil increases
antibody-dependent cellular cytotoxicity (ADCC), antibody dependent
phagocytosis (ADP), or effector T cell response in the subject. As
described herein, the hybrid neutrophils provided by the present
invention have been demonstrated to efficiently phagocytose
bacteria, mediate a high level of antibody-dependent phagocytosis
(ADP), mediate antibody-dependent cellular cytotoxicity (ADCC), and
stimulate the effector T cell responses in vitro.
[0138] In patients with cancer or infectious diseases, enhancement
of ADCC or ADP may be achieved by collecting bone marrow, expanding
hybrid neutrophils ex vivo, and then reinfusing these cells in
combination with therapeutic antibodies. The present invention thus
features methods wherein the hybrid neutrophil is obtained by
expanding a hybrid neutrophil population ex vivo in a biological
sample obtained from the subject. In particular embodiments, the
biological sample is bone marrow. In other embodiments, the
biological sample is blood, particularly a blood sample comprising
peripheral blood immature neutrophils. Generation of hybrid
neutrophils from bone marrow (BM) immature CD15 cells in a bone
marrow sample or from immature neutrophils in a blood sample may be
accomplished by contacting the sample with tumor conditioned
medium, interferon .gamma. (IFN-.gamma.), lenalidomide and/or an
amount of granulocyte macrophage colony stimulating factor
(GM-CSF). Methods for generating hybrid neutrophils from bone
marrow or blood are further described elsewhere herein.
[0139] The present invention also features methods wherein the
hybrid neutrophil is generated in situ in the subject. Without
being bound by theory, it is also possible to generate a large
numbers of hybrid neutrophils "in situ" by currently approved drugs
(i.e., administration of lenalidomide or IFN-.gamma. followed by
GM-CSF). This could then be followed by therapeutic antibody
treatment. Also this technology would be amenable for use in
combination with conventional T cell immunotherapy to enhance and
support the effect of cytotoxic T cell response against malignant
or infected cells. Therapeutic antibodies used may be any antibody
specifically binding to a tumor antigen. In some embodiments, the
antibody is anti-Her2/neu antibody, rituximab, necitumumab,
panitumumab or cetuximab. In particular embodiments, the antibody
is cetuximab. In certain embodiments, the administration of the
hybrid neutrophil may be concurrent with or followed by
administration of the therapeutic antibody, anti-tumor antibody or
antigen-binding fragment thereof.
[0140] Without being bound by theory, it is expected that the
expansion of hybrid neutrophils ex vivo or in situ followed by the
administration of therapeutic antibodies will be a more effective
strategy to inhibit tumor growth for the following reasons: (1)
hybrid neutrophils can be generated from BM or G-CSF/GMCSF
mobilized peripheral blood neutrophils in large numbers; 2) the
most potent Fc receptors for triggering ADCC (CD32, CD64, and CD89)
are highly up-regulated on hybrid neutrophils; (3) hybrid
neutrophils exhibit a high phagocytic activity; (4) hybrid
neutrophils exhibit a prolonged survival time; and (5) ability of
hybrid neutrophils to phagocyte tumor cells and present tumor
antigens enables them to induce and support the cytotoxic T cell
response against malignant cells, pathogen infected cells, or tumor
cells. Example of tumors include, but are not limited to lung
cancer, liver cancer, breast cancer, kidney cancer, gastric cancer,
and pancreatic cancer. In some embodiments, the tumor comprises a
non-small cell lung cancer (NSCLC).
[0141] The hybrid neutrophils generated as described herein can be
administered to an animal, preferably a mammal, even more
preferably a human, to treat a tumor, suppress tumor formation, and
the like. In addition, the hybrid neutrophils of the present
invention can be used for the treatment of any condition in which a
tumor response, especially a cell-mediated immune tumor response,
is desirable to treat or alleviate the disease. In particular, the
administration of antibodies against tumor or pathogen-specific
antigens in combination with hybrid neutrophils represents an
effective strategy to inhibit tumor growth or infectious process.
Accordingly, the present invention features methods of inhibiting
tumor growth in a subject and treating a tumor in a subject, the
methods comprising (a) administering to the subject an effective
amount of an anti-tumor antibody or an antigen-binding fragment
thereof; and (b) administering to or generating in the subject an
effective amount of a hybrid neutrophil. In one aspect, the
invention includes treating a condition, such as a tumor, in a
subject, comprising administering to the subject a therapeutically
effective amount of a pharmaceutical composition comprising a
population of hybrid neutrophils.
[0142] Hybrid neutrophils of the invention and compositions
comprising the hybrid neutrophils can be administered in dosages
and routes and at times to be determined in appropriate
pre-clinical and clinical experimentation and trials. Cell
compositions may be administered multiple times at dosages within
these ranges. Administration of the cells of the invention may be
combined with other methods useful to treat the desired disease or
condition as determined by those of skill in the art. The cells of
the invention to be administered may be autologous, allogeneic or
xenogeneic with respect to the mammal undergoing therapy. In
particular embodiments, the cells are autologous.
[0143] The administration of the cells of the invention may be
carried out in any convenient manner known to those of skill in the
art. The cells of the present invention may be administered to a
mammal by aerosol inhalation, injection, ingestion, transfusion,
implantation or transplantation. The compositions described herein
may be administered to a patient transarterially, subcutaneously,
intradermally, intratumorally, intranodally, intramedullary,
intramuscularly, by intravenous (i. v.) injection, or
intraperitoneally. In other instances, the cells of the invention
are injected directly into a site of inflammation in the mammal, a
local disease site in the mammal, a lymph node, an organ, a tumor,
and the like.
[0144] In one aspect the invention includes a method of inhibiting
tumor growth in a subject. The method comprises (a) administering
to the subject an effective amount of an anti-tumor antibody or an
antigen-binding fragment thereof; and (b) administering to or
generating in the subject an effective amount of a hybrid
neutrophil, wherein the hybrid neutrophil expresses at least one
neutrophil associated marker selected from the group consisting of:
Arg1, MPO, CD66b, and CD15, and at least one antigen-presenting
cell (APC) associated marker selected from the group consisting of:
CD14, HLA-DR, CD32, CD64, and CD89, thereby inhibiting tumor growth
in the subject.
[0145] In another aspect, the invention includes a method of
increasing efficacy of an antibody against a tumor in a subject
comprising (a) administering to the subject an effective amount of
an anti-tumor antibody or an antigen-binding fragment thereof; and
(b) administering to or generating in the subject an effective
amount of a hybrid neutrophil, wherein the hybrid neutrophil
expresses at least one neutrophil associated molecule selected from
the group consisting of: Arg1, MPO, CD66b, and CD15, and at least
one antigen-presenting cell (APC) associated molecule selected from
the consisting of: CD14, HLA-DR, CD32, CD64, and CD89, thereby
increasing efficacy of the antibody against the tumor in the
subject.
[0146] In yet another aspect, the invention includes a method of
treating a tumor in a subject comprising (a) administering to the
subject an effective amount of an anti-tumor antibody or an
antigen-binding fragment thereof; and (b) administering to or
generating in the subject an effective amount of a hybrid
neutrophil, wherein the hybrid neutrophil expresses at least one
neutrophil associated molecule selected from the group consisting
of: Arg1, MPO, CD66b, and CD15, and at least one antigen-presenting
cell (APC) associated molecule selected from the group consisting
of: CD14, HLA-DR, CD32, CD64, and CD89, thereby treating the tumor
in the subject.
[0147] In one embodiment, the hybrid neutrophil further expresses
at least one molecule selected from the group consisting of: MHC
class I, MHC class II, OX40L, 4-1BBL, CD86, CD40, and CCR7. In
another embodiment, the expression of at least one of any one of
the molecules is low, intermediate, or high. In yet another
embodiment, the expression of at least one of any one of the
molecules is increased relative to expression of the molecule on a
canonical tumor-associated neutrophil (TAN).
[0148] In another embodiment, the hybrid neutrophil expresses CD14,
HLA-DR, CD32, CD64, and CD89. In yet another embodiment, the hybrid
neutrophil expresses Arg1, MPO, CD66b, CD15, CD14, HLA-DR, MHC
class I, OX40L, 4-1BBL, CD86, CD40, CCR7, CD32, CD64, and CD89. In
still another aspect the expression of CD32 and/or CD64 and/or CD89
is high.
[0149] In certain embodiments, the step of administering to the
subject an effective amount of a hybrid neutrophil increases
antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent
phagocytosis (ADP), or effector T cell response in the subject. In
other embodiments, the step of administering to or generating in
the subject an effective amount of a hybrid neutrophil is followed
by the step of administering to the subject an effective amount of
an anti-tumor antibody or an antigen-binding fragment thereof. In
other embodiments, the step of administering to or generating in
the subject an effective amount of a hybrid neutrophil is
concurrent with the step of administering to the subject an
effective amount of an anti-tumor antibody or an antigen-binding
fragment thereof.
Pharmaceutical Compositions
[0150] Pharmaceutical compositions of the present invention may
comprise hybrid neutrophils as described herein, in combination
with one or more pharmaceutically or physiologically acceptable
carriers, diluents or excipients. Such compositions may comprise
buffers such as neutral buffered saline, phosphate buffered saline
and the like; carbohydrates such as glucose, mannose, sucrose or
dextrans, mannitol; proteins; polypeptides or amino acids such as
glycine; antioxidants; chelating agents such as EDTA or
glutathione; adjuvants (e.g., aluminum hydroxide); and
preservatives. Compositions of the present invention are preferably
formulated for intravenous administration.
[0151] Pharmaceutical compositions of the present invention may be
administered in a manner appropriate to the disease to be treated
(or prevented). The quantity and frequency of administration will
be determined by such factors as the condition of the patient, and
the type and severity of the patient's disease, although
appropriate dosages may be determined by clinical trials.
[0152] When "an effective amount," "an immunologically effective
amount", "an anti-immune response effective amount", "an immune
response-inhibiting effective amount", or "therapeutic amount" is
indicated, the precise amount of the compositions of the present
invention to be administered can be determined by a physician with
consideration of individual differences in age, weight, immune
response, and condition of the patient (subject/mammal). It can
generally be stated that a pharmaceutical composition comprising
the hybrid neutrophils described herein may be administered at a
dosage of 10.sup.4 to 10.sup.9 cells/kg body weight, preferably
10.sup.5 to 10.sup.6 cells/kg body weight, including all integer
values within those ranges. Cell compositions may also be
administered multiple times at these dosages. The cells can be
administered by using infusion techniques that are commonly known
in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med.
319:1676, 1988). The optimal dosage and treatment regime for a
particular patient can readily be determined by one skilled in the
art of medicine by monitoring the patient for signs of disease and
adjusting the treatment accordingly.
[0153] In certain embodiments, it may be desired to draw a blood or
bone marrow sample from a subject, generate hybrid neutrophils
therefrom according to the present invention, and reinfuse the
patient with these cells. This process can be carried out multiple
times every few weeks. In certain embodiments, cells can be
obtained from blood draws of from 10 ml to 400 ml. In certain
embodiments, cells are obtained from blood draws of 20 ml, 30 ml,
40 ml, 50 ml, 60 ml, 70 ml, 80 ml, 90 ml, or 100 ml. Without being
bound by theory, using this multiple blood draw/multiple reinfusion
protocol, may select out certain populations of cells. In
particular embodiments, the cells of the invention are administered
in conjunction with (e.g., before, simultaneously or following)
therapeutic anti-tumor antibodies. Examples of anti-tumor
antibodies include, without limitation, anti-Her2/neu antibody,
rituximab, necitumumab, panitumumab and cetuximab.
[0154] In certain embodiments of the present invention, cells are
generated using the methods described herein, or other methods
known in the art where cells are obtained at therapeutic levels,
administered to a patient in conjunction with (e.g., before,
simultaneously or following) any number of relevant treatment
modalities, including but not limited to treatment with agents such
as antiviral therapy, cidofovir and interleukin-2, Cytarabine (also
known as ARA-C) or natalizumab treatment for MS patients or other
treatments for PML patients. In further embodiments, the cells of
the invention may be used in combination with chemotherapy,
radiation, immunosuppressive agents, such as cyclosporin,
azathioprine, methotrexate, mycophenolate, and FK506, antibodies,
or other immunoablative agents such as CAM PATH, anti-CD3
antibodies or other antibody therapies, cytoxin, fludaribine,
cyclosporin, FK506, rapamycin, mycophenolic acid, steroids,
FR901228, cytokines, and irradiation. These drugs inhibit either
the calcium dependent phosphatase calcineurin (cyclosporine and
FK506) or inhibit the p70S6 kinase that is important for growth
factor induced signaling (rapamycin). (Liu et al., Cell 66:807-815,
1991; Henderson et al., Immun. 73:316-321, 1991; Bierer et al.,
Curr. Opin. Immun. 5:763-773, 1993). In a further embodiment, the
cell compositions of the present invention are administered to a
patient 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 another embodiment, the cell
compositions of the present invention are administered following
B-cell ablative therapy such as agents that react with CD20, e.g.,
Rituxan. 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 hybrid neutrophils of the present invention. In an
additional embodiment, expanded cells are administered before or
following surgery.
[0155] In certain embodiments, the cells described herein may be
used for the manufacture of a medicament for the treatment of an
immune response in a subject in need thereof. In yet other
embodiments, the cells described herein may be used for the
manufacture of a medicament for the treatment of a cancer,
particularly a tumor, in a subject in need thereof.
[0156] 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).
[0157] It should be understood that the method and compositions
that would be useful in the present invention are not limited to
the particular formulations set forth in the examples. The
following examples are put forth so as to provide those of ordinary
skill in the art with a complete disclosure and description of how
to make and use the cells, expansion and culture methods, and
therapeutic methods of the invention, and are not intended to limit
the scope of what the inventors regard as their invention.
[0158] The practice of the present invention employs, unless
otherwise indicated, conventional techniques of molecular biology
(including recombinant techniques), microbiology, cell biology,
biochemistry and immunology, which are well within the purview of
the skilled artisan. Such techniques are explained fully in the
literature, such as, "Molecular Cloning: A Laboratory Manual",
fourth edition (Sambrook et al., (2012) Molecular Cloning, Cold
Spring Harbor Laboratory); "Oligonucleotide Synthesis" (Gait, M. J.
(1984). Oligonucleotide synthesis. IRL press); "Culture of Animal
Cells" (Freshney, R. (2010). Culture of animal cells. Cell
Proliferation, 15(2.3), 1); "Methods in Enzymology" "Weir's
Handbook of Experimental Immunology" (Wiley-Blackwell; 5 edition
(Jan. 15, 1996); "Gene Transfer Vectors for Mammalian Cells"
(Miller and Carlos, (1987) Cold Spring Harbor Laboratory, New
York); "Short Protocols in Molecular Biology" (Ausubel et al.,
Current Protocols; 5 edition (Nov. 5, 2002)); "Polymerase Chain
Reaction: Principles, Applications and Troubleshooting", (Babar,
M.,VDM Verlag Dr. Muller (Aug. 17, 2011)); "Current Protocols in
Immunology" (Coligan, John Wiley & Sons, Inc. Nov. 1,
2002).
[0159] The following examples further illustrate aspects of the
present invention. However, they are in no way a limitation of the
teachings or disclosure of the present invention as set forth
herein.
EXAMPLES
[0160] The invention is now described with reference to the
following Examples. These Examples are provided for the purpose of
illustration only and the invention should in no way be construed
as being limited to these Examples, but rather should be construed
to encompass any and all variations which become evident as a
result of the teaching provided herein.
[0161] Without further description, it is believed that one of
ordinary skill in the art can, using the preceding description and
the following illustrative examples, make and utilize the compounds
of the present invention and practice the claimed methods. The
following working examples therefore, specifically point out the
preferred embodiments of the present invention, and are not to be
construed as limiting in any way the remainder of the
disclosure.
[0162] The materials and methods employed in these experiments are
now described.
[0163] Study Design. A total of 109 patients with stage I-II lung
cancer, who were scheduled for surgical resection, consented to
tissue collection of a portion of their tumor and/or blood for
research purposes. All patients selected for entry into the study
met the following criteria: (i) histologically confirmed pulmonary
squamous cell carcinoma (SCC) or adenocarcinoma (AC), (ii) no prior
chemotherapy or radiation therapy within two years, and (iii) no
other active malignancy. Detailed characteristics of the patients
can be found in FIG. 12.
[0164] Reagents. The enzymatic cocktail for tumor digestion
consisted of serum-free Hyclone.TM. Leibovitz L-15 media
supplemented with 1% Penicillin-Streptomycin, Collagenase type I
and IV (170 mg/L=45-60 U/mL), Collagenase type II (56 mg/L=15-20
U/mL), DNase-I (25 mg/L), and Elastase (25 mg/L) (all from
Worthington Biochemical, N.J.). Complete cell culture media
DME/F-12 1:1 media (HyClone, Thermo Scientific) was supplemented
with 2.5 mM L-glutamine, 15 mM HEPES Buffer, 10% of Embryonic Stem
(ES) Cell Screened FBS (U.S.) (Thermo Scientific.TM. HyClone.TM.),
Penicillin (100 U/ml) and Streptomycin (100 .mu.g/mL).
HLA-A*0201-restricted NY-ESO-1 peptide was synthesized by AnaSpec,
Inc (Fremont, Calif.). Pierce.TM. NY-ESO-1 full-length recombinant
protein and anti-NY-ESO-1 monoclonal Abs (clone E978 IgG1) were
purchased from Thermo Scientific.TM.. The PepMixCEF-MHC class I
peptide pool (23 viral peptides) and The PepMixCEFT-MHC class II
peptide pool (14 viral peptides) were purchased from JPT Peptide
Technologies (Acton, Mass.). These peptide pools contain MHC class
I and class II-restricted T-cell epitopes from CMV, EBV and
Influenza virus, designed to stimulate T cells from donors with a
variety of HLA types. Human recombinant IFN-.gamma., GM-CSF, IL-4
and M-CSF were purchased from PeproTech, Inc.
[0165] Preparation of a Single-Cell Suspension from Tumor and
Adjacent Lung Tissue. Surgically-removed fresh lung tumors and
adjacent lung tissue were processed within 20 minutes of removal
from the patient. An optimized disaggregation method for human lung
tumors was used that preserves the phenotype and function of the
immune cells (Quatromoni et al., 2015. J. Leukoc. Biol. 1,
201-209). Briefly, under sterile conditions, all areas of tissue
necrosis were trimmed away. The tumor and adjacent uninvolved lung
tissue was sliced into 1-2 mm.sup.3 pieces with micro-dissecting
scissors equipped with tungsten carbide insert blades (Biomedical
Research Instruments, Inc. Silver Spring, Md.). For enzymatic
digestion, the pieces were incubated in a shaker for 45 minutes at
37.degree. C. in serum-free L-15 Leibovitz media (HyClone)
containing different enzymes at low concentrations and 1%
Penicillin-Streptomycin (Life Technologies, Carlsbad, Calif.). L-15
Leibovitz media was formulated for use in carbon dioxide-free
systems. After 45 minutes, any visible tumor pieces were vigorously
pipetted against the side of a 50 mL tube to enhance disaggregation
and then further incubated for 30-50 minutes under the same
conditions. Larger pieces of tumor tissue were permitted to settle
to the bottom of the tube and the supernatant was passed through a
70 .mu.M nylon cell strainer (BD Falcon). The remaining pieces in
the tube underwent further pipetting before being passed through
the same cell strainer. Typically, less than 5% of the tissue
(consisting of chiefly non-cellular connective tissue) remained on
the cell strainer. After filtration the red blood cells were lysed
using 1.times. Red Blood Cell (RBC) Lysis Buffer (Santa Cruz,
Dallas, Tex.). The remaining cells were washed twice in RPMI
supplemented with 2% FBS and re-suspended in the cell culture
media. Cell viability, as determined by trypan blue exclusion or
Fixable Viability Dye eFluor.RTM. 450 staining, was typically
>90%. If the viability of cells was less than 80%, dead cells
were eliminated using a "dead cell removal kit" (Miltenyi Biotec
Inc., Germany).
[0166] Tumor-Conditioned Media. A single-cell suspension was
obtained from lung tumors by enzymatic digestion as described
herein. After washing the cells with PBS, the single cell
suspensions were re-suspended in DMEM/F12 (HyClone) medium
supplemented with 5% FBS/antibiotics (penicillin/streptomycin,
HyClone) and placed in 175 mm.sup.2 flasks at a concentration of
2.times.10.sup.6 cells/mL. Twenty-four hours later, supernatant
(tumor-conditioned medium, TCM) was collected, filtered, aliquoted,
and frozen at -80.degree. C.
[0167] Neutrophil Isolation. TANs were isolated from tumor
single-cell suspensions using positive selection of CD15.sup.+ or
CD66b.sup.+ cells with microbeads as previously described
(Eruslanov et al., 2014. J. Clin. Invest. 12, 5466-5480). TAN
subsets were flow sorted based on the phenotype of canonical
(CD11b.sup.+CD66b.sup.+CD15.sup.hiHLA-DR.sup.-) and hybrid
(CD11b.sup.+CD66b.sup.+ CD15.sup.hiHLA-DR.sup.+) TANs. PBNs and
BMNs were isolated from EDTA anticoagulated peripheral blood and BM
single-cell suspension, respectively, using positive selection of
CD15.sup.+ or CD66b.sup.+ cells with microbeads.
[0168] Since temperature gradients can activate neutrophils, all
tissues and reagents were maintained at a constant temperature
during preparation. After tumor harvest, the neutrophil populations
used in this study were prepared at room temperature (RT) and
rapidly utilized. TANs were isolated from tumor single-cell
suspensions using positive selection of CD15.sup.+ or CD66b.sup.+
cells. In the rare instances when cellular aggregates formed, the
suspensions were passed through a 30 .mu.M pre-separation filter
(Miltenyi) before addition to the LS columns (Miltenyi). For
positive selection of TANs through engagement of the CD15
transmembrane protein, single cell suspensions were incubated with
anti-CD15 antibody (Ab)-conjugated magnetic microbeads (Miltenyi
Biotec) for 15 minutes. For positive selection of TANs through
engagement of the CD66b transmembrane protein, single cell
suspensions were first incubated with PEconjugated anti-CD66b Abs
(Biolegend) and then with anti-PE microbeads (Miltenyi Biotec). In
some experiments, TANs were isolated by flow cytometric
cell-sorting based on the phenotype of TANs as
CD11b.sup.+CD66b.sup.+CD15.sup.hi. Neutrophils from distant
non-involved lung tissue were isolated similarly to TANs.
[0169] TAN subsets were sorted based on the phenotype of canonical
(CD11b.sup.+CD66b.sup.+CD15.sup.hiHLA-DR.sup.-) and hybrid
(CD11b.sup.+CD66b.sup.+CD15.sup.hiHLA-DR.sup.+) TANs. Gating
strategy for flow cytometry sorting of canonical and hybrid TANs is
shown in Figure S1N and S10. CD11b.sup.+ myeloid cells are all
CD45.sup.+EpCam.sup.- cells. Sterile cell sorting was performed on
the BD FACSAria II (BD Biosciences) and MoFlo.RTM. Astrios.TM.
(Beckman Coulter).
[0170] PBNs were obtained from EDTA anti-coagulated peripheral
blood collected from lung cancer patients during surgery or from
healthy donors. The PBNs were obtained from Lymphoprep (Accu-Prep,
1.077 g/ml, Oslo, Norway) density gradient centrifugation followed
by erythrocyte lysis with 1.times. RBC Lysis Buffer. To account for
any possible effect of tissue digestion enzymes on the function
neutrophils, peripheral blood granulocytes were processed in a
similar manner. Specifically, peripheral blood granulocytes were
incubated with enzymatic cocktail before positive selection using
microbeads or flow cytometry.
[0171] BMNs were isolated from bone marrow cell suspensions using
positive selection of CD15.sup.- or CD66b.sup.+ cells with
microbeads according to the manufacturer's instructions (Miltenyi
Biotec, Auburn, Calif.). Bone marrow cell suspension was obtained
from the rib fragments that were removed from patients as part of
their lung cancer surgery. The single cell suspension was obtained
by vigorous pippeting of cells flushed from bone marrow and passing
the disaggregated cells through a 70 .mu.M nylon cell strainer. To
exclude the possible contamination of common progenitors,
neutrophils were isolated from a CD34-depleted population of bone
marrow cells. Anti-CD15 Ab-conjugated magnetic microbeads (Miltenyi
Biotec) or PEconjugated anti-CD66b Abs (Biolegend) and anti-PE
microbeads (Miltenyi Biotec) were used for positive selection.
Given that resting naive neutrophils do not tightly adhere to cell
culture plastic as opposed to macrophage and monocytes the bead
sorted BM neutrophils were additionally cultured in cell culture
dishes to exclude the possible contamination of BM
macrophages/monocytes. Two-four hours later, the floating cells
were removed and used for further experiments.
[0172] The purity and activation status of isolated TANs, BMNs and
PBNs were measured by flow cytometry for the granulocyte/myeloid
markers CD66b, CD15, arginase-1 (Arg), myeloperoxidase (MPO),
CD11b, and the activation markers CD62L and CD54 as described
(Eruslanov et al., 2014. J. Clin. Invest. 12, 5466-5480). All
neutrophil subsets demonstrated high cell viability with minimal
enzyme-induced premature cellular activation or cleavage of myeloid
cell markers. The purity of TANs, BMNs and PBNs was typically
higher than 94%. Isolates with less than 90% purity were discarded.
To evaluate the cytomorphology of isolated PBNs, BMNs, and TAN
subsets cells were spun on glass slides and stained with the Hema3
Stat Pack Kit (Fisher Scientific).
[0173] Lymphocyte isolation from Peripheral Blood. Standard
approaches were utilized. Peripheral blood mononuclear cells
(PBMCs) were separated by 1.077 g/ml Lymphoprep (Accu-Prep, Norway)
gradient density centrifugation of EDTA anti-coagulated whole blood
collected from cancer patients and healthy donors. T cells were
purified from the PBMC fraction using human T cell enrichment
columns (R&D Systems, Inc.) according to the manufacturer's
protocol.
[0174] Generation of BM-Derived Hybrid and Canonical Neutrophils.
To differentiate BMNs into cells that resemble canonical TANs,
purified long-lived BMNs were cultured for 7 days with a TCM (50%
v/v) collected from a patient's tumor digest where a large number
of hybrid TANs were previously unable to be identified by flow
cytometry. To differentiate BMNs into cells that resemble hybrid
TANs, purified long-lived BMNs were cultured for 5-7 days with a
TCM (50% v/v) collected from a tumor digest where the frequency of
hybrid TANs was markedly elevated (.gtoreq.15% of all TANs).
Alternatively, hybrid-neutrophils were differentiated from BMNs
with low doses IFN-.gamma. (50 pg/ml) and GM-CSF (50 pg/ml) for 7
days. If it was observed that the formation of hybrid BMNs was less
than 80% the HLA-DR+CD14+ hybrid cells from TCM treated BMNs were
enriched by positive selection using magnetic beads coated with
anti-HLA-DR antibodies or by flow cytometric cell sorting. The
proliferation of BMNs during the differentiation was assessed by
flow cytometry using BrdU Flow Kit (BD Pharmingen). BMNs were
exposed to bromo-deoxyuridine for 6 hours.
[0175] To test the effect of hypoxia on hybrid neutrophil
formation, BMNs were cultured for 6 days under normoxic and hypoxic
culture conditions maintained in a 37.degree. C. incubator
containing 5% CO.sub.2, and either atmospheric 21% O.sub.2 or 5%
O.sub.2 condition (Hypoxia Incubator Chamber, Stemcell Techology).
BMNs were also cultured in the presence of hybrid-inducing TCM and
cobalt chloride (25 .mu.M) (MP Biomedicals LLC), an agent that
induces the hypoxia-inducible factor-1.alpha., the main
transcriptional factor activated in hypoxic conditions.
[0176] To differentiate hybrid neutrophils from circulating
immature neutrophils, peripheral blood collected from healthy
donors who were treated with G-CSF (filgrastim) was used to
mobilize hematopoietic stem cells for allogeneic hematopoietic cell
transplantation. Peripheral blood mononuclear cells (PBMCs) were
separated by 1.077 g/ml Lymphoprep (Accu-Prep, Norway) gradient
density centrifugation of EDTA anti-coagulated whole blood
collected from G-CSF treated healthy donors. Low-density immature
neutrophils were isolated from PBMC using anti-CD15 microbeads and
cultured with hybrid-inducing TCM for 7 days.
[0177] Generation of BM-Derived Macrophages and Dendritic Cells.
BM-derived macrophages and dendritic cells were generated by
culturing CD15.sup.-CD11b.sup.+ BM cells with M-CSF or IL-4 and
GM-CSF, respectively. Macrophages and dendritic cells were
differentiated from myeloid C CD11b.sup.+ cells purified with CD11
b beads from CD15-depleted bone marrow cell suspensions. To obtain
BM-derived mature dendritic cells (DC), CD11b cells were cultured
in the presence of GM-CSF (25 ng/ml) and IL-4 (25 ng/ml) for 7 days
in the complete cell culture medium, as described in detail
elsewhere (Inaba, et al, 1992. J. Clin. Invest. 12, 5466-5480;
Lutz, et al, 1999. J. Immunol. Methods. 1, 77-92). Maturation
cocktail (LPS 100 ng/ml and sOX40L 50 ng/ml) was added during the
last 24 hours of cell culturing. To obtain BM-derived macrophages
(Mph), BM CD11b cells were cultured in the presence of M-CSF in the
complete cell culture medium for 7 days as described in detail
elsewhere (Manzanero, 2012. Methods Mol. Biol. 177-181).
[0178] Flow Cytometry. Flow cytometric analysis was performed
according to standard protocols. Negative gating was based on a
fluorescence minus one (FMO) strategy. To exclude dead cells from
analysis, cells were stained with the Fixable Viability Dye
eFluor.RTM. 450 (ebioscience), LIVE/DEAD.RTM. fixable dead cell
stains (Molecular probes, Life Technologies), or Zombie Yellow.TM.
Fixable Viability dye (Biolegend). To distinguish early-stage
apoptotic and late-stage apoptotic/necrotic cells, cells were first
stained with Fixable Viability Dye eFluor.RTM. 660 (eBioscience).
Then cells were washed with the AnnexinV-Binding Buffer and stained
with anti-AnnexinV Abs (FITC) in the AnnexinV-Binding Buffer for 10
min at RT. Cells were washed and analyzed by flow cytometry.
[0179] For intracellular staining, fixed cells stained for surface
markers were permeabilized with BD Perm/Wash.TM. Buffer (BD
Biosciences) and then stained with the following Abs for 45 minutes
at RT: antihuman Arg (R&D Systems), anti-human MPO
(e-bioscience), FITC-anti-human IFN-.gamma. (Biolegend, clone:
4S.B3), APC anti-human GranzymeB (Biolegend, clone GB11) or
PE-anti-human/mouse IRF8 (ebioscience, Clone: V3GYWCH). For NE
staining, fixed cells stained for surface markers were
permeabilized with BD Perm/Wash.TM. Buffer (BD Biosciences) and
then incubated with 1.times. PBS/10% normal goat serum (Abcam)/0.3M
glycine to block non-specific protein interactions followed by the
antihuman Neutrophil Elastase antibodies (EPR7479, Abcam) for 30
min at RT. The secondary Abs used were goat anti-rabbit (Abcam) at
1/2000 dilution for 30 min at RT. Isotype control Abs were rabbit
IgG used under the same conditions.
[0180] For transcription factor Ikaros staining, cells were stained
with fluorochrome-labeled primary Abs for 20 min on ice. After
washing in FACS buffer (BD Biosciences), cells were fixed with
Fix/Perm.TM. Buffer (BD Biosciences). Following fixation, cells
were permeabilized with Perm/Wash.TM. Buffer (BD Biosciences) and
incubated with rabbit anti-mouse Ikaros (ab26083, Abcam, Cambridge,
Mass,). Following staining with the Ikaros Ab, cells were washed
and then stained with a PE-labeled anti-rabbit secondary Ab.
[0181] For phenotypic and functional analysis PBNs, BMNs, and TANs
were gated on live CD11b.sup.+CD15.sup.hiCD66b.sup.+ cells. The
following cell surface antibodies were utilized: anti-CD11b
(Biolegend, clone: ICRF44), anti-CD15 (Biolegend, clone: HI98),
anti-CD66b (Biolegend, clone: G10F5), anti-CD54 (Biolegend, clone:
HA58), anti-CD62L Biolegend, (clone: DREG-56), anti-CCRS
(Biolegend, clone: HEK/1/85a), anti-CCR7 (Biolegend, clone:
G043H7), anti-CXCR1 (Biolegend, clone: 8F1/CXCR1), anti-CXCR2
(Biolegend, clone: 5e8/cxcr2), anti-PD-L1 (Biolegend, clone:M1H1),
anti-Gal-9 (Biolegend, clone: 9M1-3), anti-CD301 (Biolegend, clone:
H037G3), anti-CD200R, (Biolegend, clone: OX-108), anti-FASL
(Biolegend, clone: NOK-1), anti-TRAIL (Biolegend, clone: RIK2),
anti-TWEAK (clone: CARL-1), anti-CD86 (Biolegend, clone: IT2.2),
anti-CD80 (Biolegend, clone:2D10), anti-CD40 (Biolegend,
clone:5C3), anti-OX40L (Biolegend, clone:11C3-1), anti-4-1BBL
(Biolegend, clone:5F4), anti-HLA-A2 (Biolegend, clone: bb7.2),
anti-CD14 (Biolegend, clone: M5E2), anti-HLA-DR (BD Bioscience,
clone: G46-6), anti-CD206 (Biolegend, clone: 15-2), anti-CD115
(Biolegend, clone: 9-4D2-1E4), anti-CD83 (Biolegend, clone: HB15e),
anti-CD1c (Biolegend, clone: L161), anti-CD204 (Biolegend, clone:
7G5C33), anti-CD209 (Biolegend, clone: 9E9A8), anti-CD163
(Biolegend, clone: GHI/61). All data were acquired using the BD
FACSCalibur or BD LSRFortessa.TM. (BD Bioscience) flow cytometers
and analyzed using FlowJo software (TreeStar Inc.).
[0182] Antigen Non-specific T Cell Response. To induce antigen
non-specific T cell responses, PBMC or purified T cells were
stimulated with plate-bound anti-human CD3 and/or anti-CD28
antibodies. To evaluate the effects of different neutrophil subsets
on antigen non-specific autologous T cell response, several
parameters were measured: (i) T cell proliferation using standard
CFSE dilution method or BrdU incorporation assay, (ii) T cell
IFN-.gamma. production using intracellular cytokine staining, and
(iii) expression of T cell activation markers CD25 and CD69 using
flow cytometry.
[0183] PBMCs or purified T cells (responders) were labeled with 5
.mu.M of the fluorescent dye 5,6-carboxyfluorescein diacetate
succinimidyl ester (CFSE) (Invitrogen, Molecular Probe), according
to the manufacturer's instructions. CFSE-labeled PBMCs or T cells
were stimulated with plate-bound anti-human CD3 Ab or anti-human
CD3 (clone: OKT3) and anti-human CD28 (clone: CD28.2) Abs
(Biolegend), respectively. To coat the 96 U-bottom well plates with
Abs, anti-CD3 (1 .mu.g/ml) and/or anti-CD28 Abs (5 .mu.g/ml) were
added in 100 .mu.L of PBS per well and incubated for 4 hours at
37.degree. C. Wells were washed twice with PBS before the addition
of cells. CFSE-labeled responders (1.5.times.10.sup.5 cells/well)
were mixed with either different subsets of TANs or differentiated
BMNs or PBNs in a 1:1 ratio and co-cultured in CD3/CD28-coated
plates for 4 days in the complete cell culture media. The CFSE
signal was analyzed by flow cytometry on gated CD4 and CD8
lymphocytes. In other experiments, the proliferation of T cells was
assessed by flow cytometry using BrdU Flow Kit (BD Pharmingen).
Forty-eight hours after stimulation, T cells were exposed to
bromo-deoxyuridine for 12 hours.
[0184] To measure IFN-.gamma. production, 1.5.times.10.sup.5
autologous PBMCs stimulated with plate-bound antihuman CD3 Ab were
co-incubated with different neutrophil subsets in a 1:1 ratio for
48 hr in 96 well Ubottom plate in the complete cell culture media.
To accumulate intracellular IFN-.gamma., BD GolgiStop.TM. and BD
GolgiPlug.TM. were added into the cell cultures during the last 12
hr. The cells were collected, washed in Stain Buffer (BD
Biosciences) and stained for surface markers as described herein.
Surface stained cells were fixed with BD Cytofix.TM. Fixation
Buffer (BD Biosciences) for 20 minutes. The fixed cells were
permeabilized with BD Perm/Wash.TM. Buffer (BD Biosciences) and
then stained with the anti-human IFN-.gamma. (Biolegend, clone:
4S.B3). The percent of IFN-.gamma. positive CD4 and CD8 cells was
analyzed by flow cytometry.
[0185] To measure the expression of T cell activation markers,
purified autologous T cells stimulated with plate-bound anti-human
CD3 and CD28 Ab were co-incubated with different neutrophil subsets
at concentration 1.5.times.10.sup.5 cells/well (96 well U-bottom
plate) in a 1:1 ratio for 24 hr in the complete cell culture media.
The cells were collected, washed in Stain Buffer (BD Biosciences)
and stained for surface activation markers CD25 and CD69 as
described herein.
[0186] Virus-Specific Memory T Cell Response. Autologous T cells
purified from peripheral blood with human T cell enrichment columns
(R&D Systems, Inc.) were used as responders and co-cultured
with different subsets of neutrophils that had been pulsed with a
mixture of peptides from Cytomegalovirus, Epstein-Barr virus,
Influenza virus or Clostridium tetani with a broad array of HLA
types. Since most humans have been exposed to these antigens, these
peptide pools are good control antigens for eliciting a response
from antigen-specific memory T cells in PBMC samples. Specifically,
TAN subsets were sorted based on the phenotype of canonical
(CD11b.sup.+CD66b.sup.+CD15.sup.hiLA-DR.sup.-) and hybrid
(CD11b.sup.+CD66b.sup.+CD15.sup.hiHLA-DR.sup.+) TANs as described
herein. BM-derived canonical and hybrid neutrophils were
differentiated with different types of TCM as described herein.
Tumor and BM-derived canonical and hybrid neutrophils were
incubated with 2 .mu.g/ml of PepMixCEF-MHC class I or
PepMixCEFT-MHC class II peptide pools (JPT Peptide Technologies)
for 30 minutes. Neutrophil subsets incubated with irrelevant
mesothelin-derived peptides were used as a negative control to
define a background. Following extensive washing, 1.times.10.sup.4
of neutrophils pulsed with viral peptides were incubated with
5.times.10.sup.4 autologous T cells in 96-Well PVDF Membrane
ELISPOT Plate (Millipore) for 2 days. The T cell response was
quantified by human IFN-gamma ELISPOT (Ready-SET-Go!.RTM.,
ebioscience) according to the manufacturer's instructions.
IFN-.gamma. positive spots were counted and analyzed using
ImmunoSpot.RTM. S5 Micro Analyzer (Cellular Technology
Limited).
[0187] Generation of NY-ESO specific Ly95 T cells and
A549-NY-ESO-1-A2 target lung cancer cell line. The
NY-ESO-1-reactive Ly95 TCR construct is an affinity-enhanced
variant of the wild-type IG4 TCR identified from T cells
recognizing the HLA-A2 restricted NY-ESO-1:157-165 peptide antigen.
The generation of this Ly95 TCR construct and its packaging into a
lentiviral vector has been described in detail previously (Moon et
al., 2016. Clin. Cancer Res. 22, 436-447; Robbins et al., 2008. J.
Immunol. 9, 6116-6131). Human T cells were isolated from PBMC of
healthy volunteer donors by negative selection using RosetteSep
kits (Stem Cell Technologies, Vancouver, Canada). Isolated T cells
were stimulated with magnetic beads coated with anti-CD3/anti-CD28
at a 1:3 cell to bead ratio. T cells were transduced with
lentiviral vectors at an MOI of approximately 5. Cells were counted
and fed with complete cell culture medium every 2 days. A small
portion of expanded cells was stained for flow cytometry
confirmation of successful Ly95 transduction using the V.beta.13.1
TCR chain antibody (Beckman Coulter: clone IMMU 222). Transduction
of human T cells undergoing anti-CD3/CD28 bead activation with high
titer lentivirus that encodes the Ly95 TCR recognizing NY-ESO-1
resulted in approximately 20-50% of TCRVb13.1.sup.+ CD8 cells.
[0188] For target cells, the A549 human lung adenocarcinoma cell
line was genetically modified to express both NY-ESO-1 protein and
HLA-A*02 as described earlier (Moon et al., 2016. Clin. Cancer Res.
22, 436-447). Briefly, A549 cell line was transduced by a
retroviral vector encoding NY-ESO-1-T2A-HLA-A*02. The transduced
A549 cells were subjected to limiting dilution at 0.5 cell per well
in 96-well plates. Resulting clones were tested by flow cytometry
for HLA-A*02 expression using anti-HLA-A2 Abs (Biolegend, clone:
bb7.2). HLA-A2 positive clones were selected and tested in
co-culture with T cells expressing the NY-ESO-1 Ly95 TCR. The
clones expressing HLA-A2 that could stimulate NY-ESO-1 Ly95
TCR-expressing T cells to secrete IFN-.gamma. were pooled to
generate the A549-NY-ESO-1-A2 (A549-A2-ESO) cell line.
[0189] NY-ESO-Specific T Cell Response. To study the regulation of
antigen-specific effector T cell responses by neutrophil subsets,
TCR transduced T cells (Ly95 T cells) recognizing the HLA-A2
restricted NY-ESO-1:157-165 peptide antigen were used. In one set
of experiments, in order to stimulate the Ly95 T cell response, an
A549 human lung adenocarcinoma cell line that was genetically
modified to express both NY-ESO-1 protein and HLA-A*02 A549
(A2-NY-ESO-1 tumor cells) was used. The Ly95 T cells at
concentration 1.5.times.10.sup.5 cells/well (96 well-U-bottom
plate) were mixed with A549 A2-NY-ESO-1 tumor cells in the presence
of different neutrophil subsets at ratio 1:0.25:1 (Ly95 T
cells:A549 A2-NY-ESO-1:Neutrophils) for 18 hours in the complete
cell culture media. BD GolgiStop.TM. and BD GolgiPlug.TM. were
added into the cell cultures during the last 12 hr. The Ly95 T
cells co-cultured with NY-ESO-1 negative A549 tumor cells and
neutrophil subsets were used as a negative control to define the
level of allostimulation. The cells were collected, washed in Stain
Buffer (BD Biosciences) and stained for CD8 and Ly95 TCR surface
markers using anti-CD8 (Biolegend, clone: HIT8a) and
anti-TCRV.beta.13.1 (Beckman Coulter: clone IMMU 222) antibodies
with following intracellular staining for IFN-.gamma. as described
herein. The production of IFN-.gamma. and Granzyme B was analyzed
in gated CD8.sup.+TCRVP13.1.sup.+ cells by flow cytometry.
[0190] In several experiments, blocking Abs against CD86 (clone:
IT2.2), OX40L (clone: 11C3.1), 4-1BBL (clone: 5F4), CD54 (clone:
HCD54), (all from Biolegend) were added to the co-cultures of
hybrid neutrophils and Ly95 T cells activated with A549 A2-NY-ESO-1
tumor cells. The blocking Abs at the concentration 5 .mu.g/ml were
present in neutrophils/Ly95 cell co-culture for 18 hours, starting
from the beginning of the assay. Matched isotype antibodies were
used as controls. Transwell assays were performed using 24-well
flat-bottom Transwell culture plates (Corning) with inserts of 0.4
.mu.m membrane pore size (Corning). To separate Ly95 T cells and
neutrophil subsets, 0.5.times.10.sup.6 Ly95 T cells were mixed with
A549 A2-NY-ESO-1 tumor cells in ratio 1:0.25 and added to the
bottom chamber. BM-derived canonical and hybrid neutrophils were
placed in the top at a ratio of 1:1 (Ly95 cells:Neutrophils). Cells
were cultured in complete cell culture media for 24 hours and the
production of intracellular IFN-.gamma. was measured in gated
CD8.sup.+TCRV.beta.13.1.sup.+ cells by flow cytometry.
[0191] Antigen presenting cell functions of hybrid neutrophils. To
assess whether the hybrid neutrophils perform functions of APCs,
the effector Ly95 T cells were stimulated with different subsets of
HLA-A*02.sup.+ neutrophils pulsed with HLA-A*02-restricted NY-ESO-1
peptide. For this purpose, HLA-A*02 positive BM-derived canonical
and hybrid neutrophils were incubated with NY-ESO-1 peptide (1
.mu.g/ml) for 1 hour, washed three times with cell culture medium
and mixed with Ly95 T cells at concentration 1.5.times.10.sup.5
cells/well (96 well U-bottom plate) in ratio 1:1 in the complete
cell culture media. Eighteen hours later, NY-ESO-specific
activation of the Ly95 cells was assessed by measuring
intracellular IFN-.gamma. in gated CD8.sup.-TCRVP13.1.sup.+
cells.
[0192] To assess whether the hybrid neutrophils cross-present
NY-ESO epitopes, HLA-A*02 positive BM-derived canonical and hybrid
neutrophils were differentiated as described above but in AIM V
AlbuMAX.RTM. serum free cell culture medium. These neutrophil
subsets were incubated with free NY-ESO full-length protein (5
.mu.g/ml) or NY-ESO immune complex for 12 hours in AIM V
AlbuMAX.RTM. serum free cell culture medium prior Ly95 T cells
assays. NY-ESO Immune complexes were formed by incubating the
NY-ESO full-length protein (5 .mu.g/ml) with monoclonal anti-NY-ESO
Abs (clone E978, Thermo Scientific.TM.) for 30 minutes at
37.degree. C. Following extensive washing in serum free medium,
1.times.10.sup.5 neutrophils were mixed with 5.times.10.sup.3 Ly95
T cells (transduction efficiency: 20% of CD8.sup.+TCRVP13.1.sup.-
cells) in 96-Well PVDF Membrane ELISPOT Plate (Millipore) in AIM V
AlbuMAX.RTM. serum free cell culture. NY-ESO-free neutrophils
incubated with Ly95 T cells were used as a negative control to
define a background and level of allostimulation. Twenty four hours
later, the NY-ESO-specific production of IFN-.gamma. by Ly95 cells
was assessed by human IFN-.gamma. ELISPOT (Ready-SET-Go!.RTM.,
ebioscience) according to the manufacturer's instructions. IFN-Y
positive spots were counted and analyzed using ImmunoSpot.RTM. S5
Micro Analyzer (Cellular Technology Limited).
[0193] To determine the ability of canonical and hybrid neutrophils
to uptake and process an antigen, DQ-OVA (Molecular Probes) was
used which is a self-quenched conjugate of ovalbumin that exhibits
bright green fluorescence upon proteolytic degradation. Briefly,
BM-derived canonical and hybrid neutrophil subsets were incubated
with DQ-OVA (10 .mu.g/ml) at 37.degree. C. for 2 hours. Cells
incubated at 4.degree. C. served as controls. Neutrophils were
collected, washed with cold Stain Buffer (BD Biosciences) and
stained with APC-anti-HLA-DR Abs (BD Bioscience, clone: G46-6) at
4.degree. C. The green fluorescence was analyzed by flow cytometry
in canonical HLA-DW and hybrid HLA-DR.sup.+ neutrophils.
[0194] Allogeneic Mixed-Lymphocyte Reaction (MLR). Purified
allogeneic T cells from healthy donor PBMCs were used as responders
and reacted with 1.times.10.sup.5 BM-derived canonical or hybrid
neutrophils (inducers) from lung cancer patients at a ratio of 1:1
in 96-well round bottom plate (Corning.RTM.). Five days later, the
proliferation of CD4 and CD8 T cells was measured using a BrdU
incorporation assay (BD Pharmaingen) according to the
manufacturer's instructions.
[0195] Phagocytosis. The phagocytic activity of neutrophil subsets
was assayed with the pHrodo.TM. Red E. coli BioParticles.RTM.
Phagocytosis Kit for flow cytometry (Life Technologies.TM.),
according to the manufacturer's instructions. Briefly, TANs or
BM-derived hybrid neutrophils were incubated with pHrodo.TM. Red E.
coli for 1 hour at 37.degree. C. in 5% CO2. After incubation, the
neutrophils were washed twice with cold PBS and stained for the
surface HLA-DR to distinguish the canonical HLA-DW and hybrid
HLA-DR+ neutrophils. The level of phagocytosis was analyzed by flow
cytometry in gated HLA-DR.sup.+ and HLA-DR- cells.
[0196] Neutrophil survival in vitro. PBNs and BMNs were cultured at
concentration 1.times.10.sup.6/ml in the presence or absence of TCM
(50% v/v) in 24 well clear tissue culture-treated plate
(Corning.RTM.) in complete cell culture media. Three and 7 days
later neutrophil viability was analyzed by flow cytometry using
Fixable Viability dye FVD 660 (eBioscince).
[0197] Measurement of cytokines, chemokines and growth factors. The
levels of 30 cytokines/chemokines and growth factors were measured
in TCM using the Cytokine Human Magnetic 30-Plex Panel for the
Luminex.RTM. platform (Invitrogen), according to the manufacturer's
instructions. The concentration of IFN-.gamma. and GM-CSF in TCM
was measured with commercial ELISA kits purchased from BD
Bioscience. Standards and samples were analyzed in triplicates and
the mean value used for analysis.
[0198] TNF and IL-12 production by canonical and hybrid TANs were
measured by intracellular staining after 6 and 24 hours of LPS
stimulation (100 ng/ml), respectively. For intracellular cytokine
staining, fixed TANs stained for HLA-DR were permeabilized with BD
Perm/Wash.TM. Buffer (BD Biosciences) and then stained with the
following Abs for 45 minutes at room temperature: APC anti-human
TNF-.alpha. (Biolegend, clone: MAB11) and PE anti-human IL-12
(Biolegend, clone C 11.5).
[0199] Immunohistochemistry. Formalin-fixed, paraffin-embedded
tumor specimens collected at the time of surgical resection were
co-stained for neutrophils (MPO) and antigen-presenting cells
(HLA-DR) using antibodies against HLA-DR (Biolegend; Clone L243,
1:12,000 dilution), CD66b (BD Biosciences: clone G10F5, 1:1000
dilution), and against human myeloperoxidase (MPO) (Dako;
Polyclonal; 1:6000 dilution). Secondary staining was done using
Leica Bond refine detection polymer (DAB) or Refine Red detection
(Alk Phosphatase). All staining was performed on an automated
stainer Bond III (Leica Biosystems Inc, Richmond Va.).
[0200] Statistics. All data were tested for normal distribution of
variables. Comparisons between two groups were assessed with a
two-tailed Student's t test for paired and unpaired data if data
were normally distributed. Non-parametric Wilcoxon matched-pairs
test and Mann-Whitney unpaired test were used when the populations
were not normally distributed. Likewise, multiple groups were
analyzed by one-way analysis of variance (ANOVA) with corresponding
Tukey's multiple comparison test if normally distributed, or by the
Kruskal-Wallis test with Dunn's multiple comparison test if not
normally distributed. Non-parametric Spearman test was used for
correlation analysis. All statistical analyses were performed with
GraphPad Prism 6. p-values less than 0.05 were considered
statistically significant.
[0201] The results of the experiments are now described.
Example 1
Identification of a Novel Subset of Tumor-Associated Neutrophils
(TANs) Exhibiting the Composite Characteristics of Neutrophils and
Antigen-Presenting Cells
[0202] Characterization of tumor-associated neutrophils (TANs)
revealed that the majority of TANs in early stage of non-small cell
lung cancer (NSCLC) express classic neutrophil markers
CD11b.sup.+CD15.sup.hiCD66b.sup.+MPO.sup.+Arg1.sup.+ ("canonical
TANs", FIG. 1B, boxes in lower left corner). However, another
subpopulation of TANs that displayed a combination of neutrophil
(Arg1.sup.+MPO.sup.+CD66b.sup.+CD15.sup.+) and antigen-presenting
cell (APC) (CD14.sup.+HLA-DR.sup.+CCR7.sup.-CD86.sup.+) markers was
identified. This subpopulation of TANs is hereinafter referred to
as "hybrid TANs" or "hybrid tumor-associated neutrophils." (FIG.
1B, boxes in upper right corner). The frequency of these newly
identified "hybrid" subset of TANs varied widely in tumor tissues
of cancer patients (FIG. 1C).
Example 2
Identification of Conditions in which the Immature Bone Marrow or
Peripheral Blood Granulocytes Could be Differentiated into Hybrid
Neutrophils in a Large Numbers
[0203] Using anti-CD15 magnetic beads, a highly enriched population
of human bone marrow neutrophils (BMNs) was obtained from rib
fragments that were removed from patients during routine lung
cancer surgery. It was found that these BMNs exhibited a prolonged
survival in vitro compared to peripheral blood neutrophils (PBNs).
These CD15.sup.+ BMNs expressed the myeloid/granulocytic specific
markers CD11b, CD66b, Arg1, myeloperoxidase (MPO) and were mostly
"band"-like immature neutrophils (FIG. 2A; FIG. 2C). Importantly,
unlike blood, about 40% of these BMNs could survive in cell culture
for up to 1 week (FIG. 2B). Thus, human BMNs have a prolonged
lifespan in vitro, providing large quantities of cells (>50
million cells) that can be used to differentiate immature
neutrophils into the hybrid neutrophils.
[0204] Several ways to differentiate immature granulocytes into the
hybrid CD14.sup.+HLA-DR.sup.+CD32.sup.hiCD64.sup.hiCD89.sup.hi
neutrophils that resemble hybrid TANs were developed. The various
methods are described herein.
Incubation of BM Immature CD15 Cells with Tumor Conditioned
Medium
[0205] As used herein, a "tumor conditioned medium" is medium
collected from digested tumors where a high frequency of hybrid
TANs was detected. In the studies described herein, tumor
conditioned medium (TCM) was collected from digested tumors where a
high frequency of hybrid TANs was previously detected by flow
cytometry (FIG. 3A).
[0206] To obtain TCM, a single cell suspension obtained from
digested tumors was cultured for 24 hours. Cell culture supernatant
was collected, filtered, aliquoted and frozen down. Bone marrow
granulocytes were isolated with anti-CD15 magnetic beads (FIGS.
3D-3E), washed and plated on Corning.RTM. Costar.RTM. Ultra-Low
attachment multi-well plates/6 well plates at a concentration of
1-2.times.10.sup.6/ml in complete cell culture medium supplemented
with TCM (50% v/v). Cells were cultured in the presence of
hybrid-inducing TCM for 5 days. The differentiated cells were then
collected, washed from TCM and analyzed for the markers of hybrid
neutrophils (HLA-DR, CD14, CD15, CD66b) to ensure that hybrid
neutrophils were formed (FIG. 3F).
Incubation of BM Immature CD15 cells with IFN-.gamma. and
GM-CSF
[0207] Bone marrow (BM) immature CD15 cells were incubated with IFN
at low concentrations (50-100 pg/ml) (FIG. 3A). Comparative
analysis of TCMs collected from digested tumors with or without
hybrid TANs revealed that IFN-.gamma. and GM-CSF are necessary
factors in the tumor microenvironment for the development of the
hybrid neutrophils. It was found that only IFN-.gamma. and GM-CSF
at the very low concentration of 50 pg/ml were able to induce
expression both CD14 and HLA-DR on the surface of BMNs in a
synergistic manner. Similar to differentiation of hybrid
neutrophils with TCM described above, CD15 immature granulocytes
were cultured for 5 days in the complete cell culture medium
supplemented with IFN-.gamma. and GM-CSF.
Incubation of BM Immature CD15 Cells with GM-CSF and
Lenalidomide
[0208] Bone marrow (BM) immature CD15 cells were incubated with
GM-CSF (50-100 pg/m1) along with the FDA approved drug
lenalidomide. Lenalidomide reduces the level of the transcriptional
factor Ikaros in BM immature neutrophils by inducing proteosomal
degradation of this protein (Kronke et al., Oncoimmunology. 2014
Jul. 3; 3(7): e941742) (FIG. 3B). Similar to differentiation of
hybrid neutrophils with TCM described above, the bone marrow
granulocytes were isolated with anti-CD15 magnetic beads, washed
and plated down to Corning.RTM. Costar.RTM. Ultra-Low attachment
multi-well plates/6 well plates at concentrations of 1-2 million/ml
in the complete cell culture medium supplemented with GM-CSF (100
ng/ml) and lenalidomide (30 .mu.M). Cells were cultured in the
presence of these factors for 6 days.
Differentiation from Peripheral Blood Immature Neutrophils
[0209] Hybrid neutrophils could be differentiated from peripheral
blood immature neutrophils mobilized in peripheral blood by an
administration with GM-SCF or G-CSF. Patients were treated with
GM-SCF or G-CSF and peripheral blood low-density immature
neutrophils were isolated by gradient separation. Cells were
cultured in the presence of hybrid-inducing TCM (50% v/v) or
IFN-.gamma. and GM-CSF at concentrations between 50-100pg/ml. At
day 5, similar to BMNs, a significant portion of GM-CSF or G-CSF
mobilized low-density PBNs acquired the HLA-DR.sup.+CD14.sup.+
phenotype (FIG. 3C).
[0210] The detailed phenotypic analysis described herein has
revealed that hybrid neutrophils were phenotypically different from
canonical neutrophils in multiple ways (summarized in FIG. 4). The
differences included (1) upregulation of the MHC class II and class
I molecules; (2) increased expression of T cell co-stimulatory
molecules OX40L, 4-1BBL, CD86, CD40 and chemokine receptor CCR7,
and (3) upregulation of the Fc.gamma.RI (CD64), Fc.gamma.RII (CD32)
and Fc.alpha.RI (CD89), which are the most potent Fc receptors for
triggering antibody-dependent cell cytotoxicity (ADCC).
Example 3
Hybrid CD14.sup.+HLA-DR.sup.+CD32.sup.hiCD64.sup.hi CD89.sup.hi
Neutrophils Efficiently Phagocytose Bacteria and Mediate a High
Level of Antibody Dependent Phagocytosis (ADP).
[0211] CD32.sup.hiCD64.sup.hi CD89.sup.hi hybrid neutrophils (which
could be generated in large numbers from immature bone marrow or
peripheral blood) are powerful effector cells that trigger
sufficient removal of tumor cells or infectious pathogens through
ADP or ADCC. The support for this claim comes from a comparative
analysis of canonical and hybrid neutrophils that revealed that
hybrid neutrophils are characterized by (1) augmented ability to
phagocytose bacteria (FIG. 5A); (2) expression of very high levels
of Fc.gamma.RI (CD64), Fc.gamma.RII (CD32) and Fc.alpha.R (CD89)
(FIG. 5D) (of note, the high affinity Fc.gamma.RI/CD64 represents
the most potent neutrophil Fc.gamma.R for induction of ADCC
(Valerius et al., Blood. 1993 Aug. 1; 82(3): 931-939)); (3)
increased ability to mediate the high level of antibody-dependent
phagocytosis (FIG. 5B); and (4) ability to mediate ADCC (FIG.
5C).
Example 4
Hybrid Neutrophils Trigger and Stimulate Effector T Cell
Responses
[0212] Results of the studies described herein demonstrate that
tumor or BM-derived hybrid neutrophils were able to: (1)
dramatically augment proliferation of naive resting T cells
stimulated with anti-CD3/CD28 Abs compared to canonical neutrophils
(FIG. 6A; FIGS. 6E-6H); (2) augment response of anti-tumoral
effector T cells (FIG. 6B; FIGS. 6I-6L); (3) present viral antigens
to autologous memory CD8 and CD4 cells (FIG. 6C); and, (4) cross
present tumor antigens to cytotoxic T cells (FIG. 6D; FIG. 6M).
Given these findings, it is expected that the expansion and use of
hybrid neutrophils in humans can significantly augment the efficacy
of therapeutic antibodies and boost anti-tumor and anti-infectious
immunity.
Example 5
Hybrid Neutrophil Cytotoxicity Triggered by Tumor Antigen Specific
Antibodies
[0213] Comparative analysis of BM and tumor-derived canonical and
hybrid neutrophils revealed that hybrid neutrophils are
characterized by expression of high levels of Fc.gamma.RI (CD64),
Fc.gamma.RII (CD32) and Fc.alpha.R (CD89) (FIG. 7A, FIG. 5D). It
was tested whether the elevated levels of these receptors,
particular the high affinity Fc.gamma.RI, significantly enhanced
ADCC. To study tumor cell killing by neutrophils in vitro, a GFP
expressing A549 tumor cell line (lung carcinoma) was generated.
Using this cell line as a target, it was found that PBNs, TANs and
canonical BMNs, were unable to directly kill tumor cells unless a
non-physiologic activator, such as phorbol ester was added (FIG.
7B). These cells were also not able to kill tumor cells opsonized
with the anti-EGFR monoclonal antibody (mAb) (cetuximab).
Interestingly, however, the BM-derived hybrid neutrophils showed
significant antitumor activity when cultured with anti-EGFR
mAbs-opsonized target cells (FIG. 7B, right bars).
[0214] To characterize the mechanisms of Cetuximab-induced tumor
cell killing by BM hybrid neutrophils, these cells were incubated
with A549 tumor cells opsonized with anti-EGFR Abs with or without
different inhibitors. Tumor cell death was quantified after 24
hours of co-culture as described in FIG. 7B.
Effector Mechanisms of Hybrid Neutrophil Mediated ADCC
[0215] To identify if antibody-dependent phagocytosis (ADP) by
hybrid neutrophils is involved in the elimination of opsonized
target cells, anti-EGFR Ab-coated GFP-A549 cells are co-cultured
with red-fluorescent dye Dil-labeled BM-derived hybrid neutrophils
for 4 and 18 hours. Phagocytosed A549 cells are identified as both
GFP+ and DiI+ cells by flow cytometry. To determine the role of
reactive oxygen species (ROS) dependent mechanisms, an inhibitor of
the NADPH oxidase complex (apocynin) is added to the cytotoxicity
assay. To assess the role of superoxide anion O.sub.2--, hydrogen
peroxide, or hypochlorous acid (HOC1) in hybrid neutrophil-mediated
killing, cytotoxic assays are performed in the presence of their
specific inhibitors: superoxide dismutase, catalase, and taurine,
respectively. The ability of each neutrophil subtype to produce ROS
following the incubation with opsonized A549 tumor cells using
Amplex Red or CM-H2DCFDA in parallel with the cytotoxicity assays
are compared as described (Eruslanov et al., 2010. Methods Mol
Biol. 594: 57-72). To identify whether the generation of reactive
nitrogen intermediates by neutrophils is involved in neutrophil
mediated tumor cell killing, the nitric oxide synthase inhibitor
L-NMMA is added to the cytotoxic assay.
Contribution of Fc.gamma.R Signaling to Hybrid Neutrophil Mediated
ADCC
[0216] It has been demonstrated that cross-linking of Fc receptors
triggers activation of the PI3K and RAS-ERK pathways which then
play a critical role during NK cell and macrophage-mediated ADCC
(Garcia-Garcia et al., J Immunol. 2009 Apr. 15; 182(8): 4547-4556;
Joshi et al., PLoS One. 2009; 4(1): e4208; Jiang et al., Nat
Immunol. 2000 November; 1(5): 419-425; Wei et al., J Exp Med. 1998
Jun. 1; 187(11): 1753-1765). Thus, the hypothesis that these
signaling pathways are activated and necessary for triggering the
tumoricidal activity of hybrid neutrophils can be tested.
Activation of RAS-ERK pathway is assessed by measuring the
phosphorylation of MEK1/2 and Erk1/2 in hybrid neutrophils
stimulated with opsonized GFP-A549 tumor cells for 15 minutes.
Cells are fixed with BD PhosFlow Fix Buffer.TM. and stained for
intracellular ERK1/2 (pT202/pY204) and MEK1 (pS218)/MEK2 (pS222)
Abs (BD Phosflow.TM.). Levels of MEK and ERK phosphorylation are
quantified by flow cytometry on gated GFP-negative neutrophils. In
order to examine whether the activation of the PI3K and RAS-ERK
pathways are important during neutrophil-mediated ADCC, the hybrid
neutrophils are pre-treated with the specific inhibitors wortmannin
(PI3K inhibitor), PD98059 (MEK1/2 inhibitor) or LY294002 (PI3K
inhibitor). In order to determine what class of Fc receptors on
hybrid neutrophils triggers the high level of ADCC, BM-derived
hybrid neutrophils are pre-incubated with blocking Abs against CD32
or CD64 (Biolegend) and added to opsonized A549 cells.
Hybrid TANs Killing by ADCC
[0217] To evaluate the ability of canonical and real hybrid TANs to
mediate ADCC, TAN subsets are isolated from tumors and mixed with
anti-EGFR Ab opsonized A549 tumor cells at different ratios, as
described above.
[0218] Without being bound by specific theory, it is expected that
BM and tumor-derived hybrid neutrophils will mediate high levels of
ADCC by antibody-dependent phagocytosis of opsonized A549 cells and
by subjecting them to oxidative damage. Given that the high
affinity Fc.gamma.RI/CD64 represents the most potent neutrophil
Fc.gamma.R for induction of ADCC, it is anticipated that triggering
of Fc.gamma.RI/CD64 signal pathway will lead to activation of
tumoricidal activity of hybrid neutrophils. If the hybrid
neutrophils are not able to phagocytose the opsonized A549 cells or
do not use ROS as their primary killing mechanism, other
extracellular cytotoxic mechanisms such as NET (neutrophil
extracellular traps) formation after binding opsonized tumor cells
will be investigated. NETs are visualized by fluorescence imaging
of extracellular DNA stained with Sytox Green. Without intending to
be bound by specific theory, it is also possible that alternative
non-oxidative pathways can be involved in tumor cell lysis by
hybrid cells. This can be explored by using inhibitors of different
serine proteinases and peptide defensins in the cytotoxic assay
performed. Antibodies that block possible death receptor/death
receptor ligands, including anti-TRAIL and anti-FASL antibodies,
will be used. It is possible that hybrid TANs will not show the
same type of high tumoricidal activity as BM hybrid cells during
ADCC. This will be useful and important information, however, it
will not diminish the potential clinical value of BM-derived hybrid
neutrophils that can be generated in large numbers from lung cancer
patients for potential treatment with therapeutic antibodies.
Example 6
Clinical Potential of Hybrid Cells Generated from BM to Mediate
ADCC In Vivo
[0219] Epidermal growth factor receptor (EGFR) is commonly
overexpressed in NSCLC (Brabender et al., Clin Cancer Res. 2001
July; 7(7): 1850-1855) and targeting this receptor is a validated
approach to treating cancer. However, the efficacy of therapeutic
anti-EGFR monoclonal antibody (mAbs) (cetuximab)-based monotherapy
is poor (Liu et al., Cancer Chemother Pharmacol. 2010 April; 65(5):
849-861. 47). Combined treatment of tumors with G-CSF/GM-CSF to
induce the recruitment of effector neutrophils from bone marrow and
therapeutic mAbs was used in several clinical trials to enhance the
efficacy of cetuximab through ADCC (Repp et al, Br J Cancer. 2003
Dec. 15; 89(12): 2234-2243; Pullarkat et al., Cancer Immunol
Immunother. 1999 April; 48(1): 9-21; Cartron et al., J Clin Oncol.
2008 Jun. 1; 26(16): 2725-2731). However, these trials only showed
limited therapeutic effects, indicating that improvement of
neutrophil-mediated Ab therapy is required (Fury et al, Cancer
Immunol Immunother. 2008 February; 57(2): 155-163; Repp et al., Br
J Cancer. 2003 Dec. 15; 89(12): 2234-2243). It was hypothesized
that CD64.sup.hiCD32.sup.hi hybrid neutrophils (which could be
generated in large numbers from bone marrow) are powerful effector
cells to trigger sufficient ADCC.
[0220] It has been difficult to study human neutrophils in any
animal models because after systemic injection of human peripheral
neutrophils, the cells are either rapidly destroyed or are trapped
in the lung and do not localize to the tumors. However, this
limitation was overcome herein by injecting 10.sup.6 human
neutrophils intratumorally into established human lung cancer cell
line-derived tumors (100mm.sup.3 A549 lung cancer xenografts) in
NOD/SCID/.gamma.-chain knockout (NSG) mice. It was found that
injected BM neutrophils (but not blood neutrophils) were still
present in A549 tumors 4 days post-injection (FIGS. 8A-8B). It has
been previously demonstrated that cetuximab is capable of
activating ADCC activity against A549 lung cancer cells (Kurai et
al., Clin Cancer Res. 2007 Mar. 1; 13(5): 1552-1561). Thus, this
model allowed the cetuximab-induced tumoricidal effect of hybrid
neutrophils to be studied in vivo (FIG. 10).
[0221] NSG mice with established A549 flank tumors (100-200
mm.sup.3) can be studied. The ability of BM-derived hybrid
neutrophils to mediate ADCC in vivo using the cetuximab can be
characterized. Briefly, cetuximab is injected IV into tumor-bearing
mice. Next, 10.sup.7 BM-derived hybrid or canonical neutrophils are
injected intratumroally (IT). All IV injections are performed 2
hours before the IT injections to allow antibody binding to tumors.
Tumor size over 5 days is measured. Table 1 shows the groups needed
in this study. Ten (10) mice per group (enough to enable detection
of 25% differences in tumor size-based on years of previous similar
studies and consultation with a biostatistician) are studied.
Compared to a control group (Group 1), little effect of antibody
alone is expected (Group 2). No effect from canonical BMN (groups 3
and 4) or the hybrid BMN without cetuximab is expected (Group 5).
If the hypothesis is correct, the most dramatic effects will be
observed in Group 6, where the hybrid BMN is highly active against
opsonized tumor and leads to tumor regression.
TABLE-US-00001 TABLE 1 Experimental groups to study ADCC in vivo IV
Injection IT injection Group 1 saline saline Group 2 cetuximab
saline Group 3 saline canon BMN Group 4 cetuximab canon BMN Group 5
saline hybrid BMN Group 6 cetuximab hybrid BMN
[0222] If ADCC is not observed in vivo, it will be confirmed first
that tumors are coated with cetuximab by harvesting tumors after
injection and staining with anti-human IgG to detect cetuximab. If
very low levels of Ab penetration are observed, cetuximab will be
injected intratumorally. Without being bound by specific theory,
binding of cetuximab to the receptor may result in sufficient
internalization of the antibody-receptor complex that leads to
downregulation of EGFR expression (Patel et al., Anticancer Res.
2007 September-October; 27(5A): 3355-336663). The dissociation from
FcRs is also possible. To determine these effects, the level of
bound cetuximab on EpCam+ cells in A549 lung cancer xenografts is
measured at different time points following IV and IT injection of
cetuximab in NSG mice. For this purpose, tumors at 1, 4, 6 and 24
hours are harvested and enzymatically digested. The tumor single
cell suspensions are stained for EpCam (to detect tumor cells) and
anti-human IgG (Fc) secondary Ab (to detect bound cetuximab). If
substantial downregulation of EGFR in tumors is observed, the
injections of cetuximab daily are repeated. Alternatively, other
therapeutic antibodies against EGFR, such as necitumumab and
panitumumab, that may better penetrate tumor tissue and have a
lower rate of antibody-receptor complex internalization, are
used.
Example 7
Early-Stage Human Lung Cancers Accumulate a Neutrophil Subset with
a Composite Phenotype of Granulocytes and APCs
[0223] The expression of APC surface markers was measured on
neutrophils from three locations: lung cancer tissue, adjacent
(within the same lobe) lung parenchyma (termed "distant tissue"),
and peripheral blood (FIG. 11A). Phenotypic analysis of 50 random
patients with stage I-II non-small cell lung cancer (NSCLC) was
performed. Detailed characteristics of all patients involved in
this study are shown in FIG. 12. Fresh tissue was digested using
defined conditions that minimize enzyme-induced ex vivo effects on
the viability, premature activation, phenotype, and function of
neutrophils (Quatromoni et al., J. Leukoc. Biol. 2015 1, 201-209).
TANs were previously characterized as
CD11b.sup.+CD15.sup.hiCD66b.sup.+MPO.sup.+Arg1.sup.+CD16.sup.intIL-5R.alp-
ha..sup.- cells (Eruslanov et al., 2014 J. Clin. Invest. 12,
5466-5480). Importantly, all CD66b.sup.-CD11b.sup.- cells also
expressed the other neutrophil/myeloid cell markers CD15, MPO
(myeloperoxidase), Arg-1(arginase-1), and NE (neutrophil elastase)
at very high levels (FIG. 13A, inset boxes) and thus could be
segregated from other CD15.sup.loMPO.sup.loNE.sup.loArg.sup.-
non-granulocytic CD11b.sup.+ myeloid cells. Since there was a high
concordance among the selected neutrophil markers, for the present
study TANs were defined as CD15.sup.hiCD66b.sup.+CD11b.sup.+ cells.
Analysis revealed that the majority of neutrophils from lung
tumors, termed "canonical TANs," expressed only these classic
neutrophil markers (FIG. 13A and FIG. 11A). However, TANs with
surface expression of additional markers normally expressed on APCs
were also identified, specifically human leukocyte antigen
(HLA)-DR, CD14, CD206, CD86, and CCR7 (FIGS. 11B-11F). These
receptors were completely absent in peripheral blood neutrophils
(PBNs). The "distant tissue" neutrophils also expressed these APC
markers, albeit at much lower levels in comparison with TANs.
[0224] Further analysis revealed that the APC markers
(CD14.sup.+HLA-DR.sup.+HLA-ABC.sup.hiCCR7.sup.+CD86.sup.+CD206.sup.+)
were co-expressed on a unique subset of
CD11b.sup.+CD66b.sup.+CD15.sup.hi TANs (FIG. 13B and FIG. 1B),
exhibiting a composite phenotype of canonical neutrophils and APCs.
As used herein, this subset is termed "APC-like hybrid TANs",
"hybrid TANs" or "hybrid tumor-associated neutrophils." This
population of hybrid TANs expressed some markers of the APC
phenotype (e.g., CD14, HLA-DR, CCR7, CD86, and CD206) but lacked
other defining markers of "professional APC" such as CD209, CD204,
CD83, CD163, CD1c, and CCR6. Of note, the expression of CD206,
CCR7, and CD86 varied, whereas there was a consistent co-expression
of HLA-DR and CD14 on hybrid TANs. Cytospins prepared from
flow-sorted HLA-DR.sup.- canonical and HLA-DR.sup.+ hybrid TANs
revealed that some of the hybrid TANs had round and oval nuclear
shapes in comparison with the classic nuclear segmentation of
canonical TANs (FIG. 13B). Histological review of lung tumors also
revealed "double-positive" MPO.sup.+HLA-DR+ and CD66b.sup.+
HLA-DR.sup.+ TANs that were scattered throughout lung tumors (FIG.
13C). Additionally, a small but clearly distinguishable population
of HLA-DR.sup.+CD15.sup.hiCD66b.sup.+CD11b+ cells were detected in
the draining lymph nodes of several lung cancer patients (FIG.
11G).
[0225] The frequency of this identified subset of TANs varied from
0.5% to 25% among all TANs (FIG. 13D) and from 0.1% to 4.3% among
all cells in tumor digests (FIG. 11H). The hybrid population was
significantly higher in patients with adenocarcinoma compared with
patients with squamous cell carcinoma (FIG. 11I). There were no
significant associations between the frequency of APC-like TANs and
tumor stage or smoking history (FIG. 11J-11K). Interestingly, a
significantly smaller percentage of HLA-DR.sup.+ hybrid neutrophils
among TANs in large tumors (diameter >3 cm) versus the small
tumors (diameter <3 cm) (FIG. 13D and FIG. 11L) was observed.
Thus, the hybrid population appears to decline as tumors enlarge,
and is completely absent in tumors greater than 5-7 cm in diameter.
Together, these data demonstrate that neutrophils in some
early-stage lung tumors undergo unique phenotypic changes, yielding
a subset of TANs with composite characteristics of neutrophils and
APC.
Example 8
APC-like Hybrid TANs Stimulate and Support T Cell Responses
[0226] The functional activity of APC-like TANs was first evaluated
to ensure that these activated cells were not "exhausted" or
hypo-functional. TANs were thus isolated from tumors and stimulated
with lipopolysaccharide (LPS). After LPS stimulation, HLA-DR.sup.+
hybrid TANs produced much more tumor necrosis factor .alpha.
(TNF-.alpha.) and interleukin-12 (IL-12) when compared with
HLA-DR.sup.+ canonical TANs (FIG. 13E). Furthermore, HLA-DR.sup.+
hybrid TANs phagocytosed Escherichia coli bioparticles more
efficiently than HLA-DR.sup.- canonical TANs (FIG. 11M). These data
demonstrate that APC-like hybrid TANs are fully functional and, in
fact, perform major functions such as cytokine production and
phagocytosis superior to canonical TANs.
[0227] To determine the effect of APC-like hybrid TANs on T cell
responses, TAN subsets were isolated by flow cytometry cell sorting
(FIG. 11N-11O). Each sorted TAN subset was co-cultured with
autologous carboxyfluorescein succinimidyl ester (CFSE)-labeled
peripheral blood mononuclear cells (PBMCs) that had been stimulated
with plate-bound anti-CD3 antibodies (Abs) (FIG. 13F). The
proliferation of CD4 and CD8 cells after 4 days of stimulation was
markedly augmented after exposure to HLA-DR.sup.+ hybrid TANs
versus the HLA-DR.sup.- canonical TANs (FIG. 13F).
[0228] Experiments were performed to determine whether APC-like
hybrid TANs could trigger and sustain antigen-specific T cell
responses. Autologous T cells were co-cultured with TAN subsets
that had been pulsed with mixtures of overlapping peptides from
commercially available peptide pools. Each peptide pool
corresponded to defined HLA class I or II restricted T cell
epitopes from cytomegalovirus, Epstein-Barr virus, influenza virus,
or Clostridium tetani designed to stimulate T cells with a broad
array of HLA types. As shown in FIG. 13G, the HLA-DR.sup.+ hybrid
TANs efficiently triggered memory CD8 and CD4 T cell responses to
HLA class I and II restricted T cell epitopes, respectively.
Canonical TANs and PBNs induced only weak CD8 T cell responses and
did not trigger CD4 T cell responses. Together, these data
demonstrate that HLA-DR.sup.+ hybrid TANs are able to function as
efficient APCs.
Example 9
Long-Lived Immature Neutrophils Recapitulate the Phenotype of
APC-like Hybrid TANs in the Presence of Tumor-Derived Factors
[0229] Given the anti-tumor activity of APC-like TANs due to their
strong stimulatory effect on T cell responses, the mechanisms by
which these cells could originate and expand in the human tumor
microenvironment was investigated.
[0230] Tumor-conditioned media (TCM) was collected from digested
lung cancers that contained >15% of hybrid TANs among all TANs
(termed hybrid-inducing TCM). Purified PBNs were exposed to
hybrid-inducing TCM and it was discovered that PBNs did not
differ-entiate into the HLA-DR.sup.+CD14.sup.- neutrophils and died
within 3 days (FIG. 14A). To determine whether more immature
neutrophils with a higher degree of plasticity differentiate into
APC-like hybrid neutrophils, a highly enriched population of
immature human bone marrow neutrophils (BMNs) was obtained.
Isolated BMNs expressed the myeloid/granulocytic specific markers
CD11b, CD66b, CD15, Arg-1, NE, and MPO, and were mostly "band"-like
immature neutrophils in appearance (FIG. 14B and FIG. 15A). Of
note, the purified BMNs did not express HLA-DR and CD14 and were
not contaminated with macrophages and monocytes (FIG. 15A). Unlike
blood neutrophils, about 40% of these BMNs could survive in cell
culture for up to 1 week and their viability was dramatically
increased in the presence of TCM (FIGS. 14A, 14C, and 15B). Thus,
BMNs with a prolonged lifespan in vitro provided large quantities
of cells that could be used to model the origins and
differentiation process of neutrophils in the tumor
microenvironment.
[0231] After 7 days of incubation of BMNs with hybrid-inducing
TCMs, the formation of a cell subset that retained all its
granulocytic markers (FIG. 14B and FIG. 4) and acquired the same
phenotype as the tumor-derived hybrid TANs
(HLA-DR.sup.+CD14.sup.+CD86.sup.-CD206.sup.+CCR7.sup.+) was
observed (FIG. 14D). Similar to hybrid TANs, most of the BMNs also
changed their nuclear shape from band-like to oval when they
converted into hybrid BMNs (FIG. 14B). A detailed phenotypic
comparison of PBNs, BMNs, and bone marrow (BM)- and tumor-derived
hybrid neutrophils is summarized in FIG. 4. The differentiation of
BMNs into HLA-DR.sup.+CD14.sup.+ APC-like hybrid BMNs after
exposure to hybrid-inducing TCM was donor dependent and varied from
20% to 80% of the initial BMN population (FIG. 15C). BMNs began to
upregulate CD14 within 24 hr of co-culturing with hybrid-inducing
TCM, while the expression of HLA-DR, CD86, CCR7, and CD206 markers
did not appear until day 4 (FIG. 15D). This suggests that these
late APC markers are synthesized de novo.
[0232] Similar to hybrid TANs, differentiated hybrid BMNs acquired
only the partial phenotype of dendritic cells (DC) and macro-phages
(Mph) (HLA-DR.sup.+CD14.sup.-CD86.sup.+CD206.sup.+) (FIGS. 2E-2G).
The hybrid subset of BMNs and TANs differed from BM-derived DC and
Mph by absence of CD1c, CD83, CD163, and CD209 markers, and low
expression of CD40, CD86, CD115, and CCR7 (FIGS. 14D-14G). The
level of the transcription factor IRF8, which regulates monocyte/DC
lineage commitment (Yanez et al., 2015. Blood 9, 1452-1459), was
not dramatically changed in hybrid BMNs and was much lower than the
amount detected in BM-derived Mph and DC (FIG. 15E).
[0233] The ability of differentiated APC-like hybrid BMNs to
proliferate in the presence of hybrid-inducing TCM and thus
represent a self-maintained population of neutrophils was tested. A
bromodeoxyuridine (BrdU) incorporation assay revealed that within
24 hr of treatment with hybrid-inducing TCM, 10%-15% of BMNs begin
to synthesize DNA in vitro (FIG. 15F). As the differentiation
process progressed, a small proportion of HLA-DR.sup.- BMNs
continued to incorporate BrdU up to day 8, whereas the
differentiated HLA-DR.sup.+ neutrophils lost proliferative
potential (FIG. 15F).
[0234] Given that the frequency of hybrid TANs was reduced in large
tumors (FIG. 13D), it was hypothesized that hypoxia, which is
strongly associated with the tumor progression, may negatively
regulate the formation of hybrid neutrophils. Thus, BMNs were
cultured in the presence of hybrid-inducing TCM for 6 days under
normoxic (5% CO.sub.2 and 21% O.sub.2) and hypoxic (5% CO.sub.2 and
5% O.sub.2) cell culture conditions. BMNs were also cultured in the
presence of hybrid-inducing TCM and cobalt chloride, an agent that
induces hypoxia-inducible factor 1 .alpha. (HIF-1 .alpha.), the
main transcriptional factor activated in hypoxic conditions. The
development of hybrid CD14.sup.+HLA-DR.sup.+ neutrophils was
profoundly inhibited under these hypoxic and hypoxia-simulating
conditions (FIG. 16A).
Example 10
IFN-.gamma. and GM-CSF are Requisite Factors in the Tumor
Microenvironment for the Development of Hybrid Neutrophils
[0235] To determine the particular tumor-specific factors that
promote the formation of hybrid TANs, primary TCMs collected from
20 consecutive lung cancer patients were screened and categorized
based on their ability to induce: (1) the full phenotype of hybrid
cells (CD14.sup.+HLA-DR.sup.+CD11b.sup.+CD66b.sup.+CD15.sup.hi)
(FIG. 16B, example TCM #41); (2) the partial phenotype of hybrid
cells (CD14.sup.-HLA-DR.sup.-CD11b.sup.+CD66b.sup.+CD15.sup.hi)
(FIG. 16B, example TCM #63); or (3) no phenotypic changes (FIG.
16B, example TCM #58). Each TCM was evaluated using a multiplex
cytokine/chemokine bead assay. Those TCMs that induced
CD14.sup.+HLA-DR.sup.+ hybrid cells had increased amounts of
granulocyte-colony stimulating factor (G-CSF), IL-6, IL-15,
granulocyte-macrophage colony-stimulating factor (GM-CSF),
interferon-.gamma. (IFN-.gamma.), macrophage inflammatory
protein-1.alpha. (MIP-1.alpha.), TNF-.alpha., monocyte
chemoattractant protein-1 (MCP-1), and monokine induced by
IFN-.gamma. (MIG) compared with TCMs that did not induce hybrid
cells. The ability of each of these factors (at the low
concentrations found in the TCMs) to induce the
CD14.sup.+HLA-DR.sup.+ hybrid phenotype in BMNs was tested and only
IFN-.gamma. and GM-CSF were able to induce the phenotype, although
in a relatively low percentage of cells (FIG. 16B and FIG. 17A).
However, these factors worked in a synergistic manner: when
combined at very low concentrations of 50 pg/ml of each factor,
they induced expression of APC markers in a large proportion
(>40%) of the cells in a donor-dependent fashion (FIG. 3F and
FIG. 17A). The addition of neutralizing monoclonal antibodies for
either IFN-.gamma. or GM-CSF completely inhibited the formation of
BM hybrid cells in the presence of hybrid-inducing TCM (FIG. 16C),
thereby confirming that both IFN-.gamma. and GM-CSF play a key role
in the induction process. Interestingly, incubation of BMNs with a
low dose of GM-CSF (50 pg/ml) and increasing concentrations of
IFN-.gamma. (from 50 pg/ml to 20 ng/ml) resulted in the expansion
of CD14.sup.+HLA-DR.sup.+ BMNs from 40% to 96% among all BMNs (FIG.
16D, upper panel). However, the treatment of BMNs with IFN-.gamma.
at a concentration of more than 1 ng/ml gradually induced the
expression of PD-L1 on the HLA-DR.sup.+ BMNs (FIG. 16D, lower
panel), resulting in the formation of hybrid neutrophils with T
cell suppressive activity (described in detail herein).
[0236] The frequency of APC-like TANs in the tumor digests was
analyzed, and, in parallel, the concentration of IFN-.gamma. and
GM-CSF in the supernatants collected from digested autologous tumor
cell cultures was measured. FIGS. 16E-16F demonstrate that the
levels of IFN-.gamma. and GM-CSF were statistically higher in
tumors where there was a high proportion of hybrid TANs (>10% of
all TANs). However, the generation of hybrid neutrophils in vivo is
most likely more complex and not solely due to IFN-.gamma. and
GM-CSF levels, because the absolute levels of IFN-.gamma. and
GM-CSF in the TCM did not necessarily correlate with the frequency
of hybrid neutrophils (>10% of all TANs) in each tumor as shown
in FIGS. 16E-16F. Also, when BMNs from the same donor were exposed
to different hybrid-inducing TCMs containing variable
concentrations of IFN-.gamma. and GM-CSF, a clear relationship
between absolute levels of GM-CSF and IFN-.gamma. and the degree of
hybrid neutrophil formation was not observed (FIG. 17B). These data
suggest that there is a requisite threshold level of GM-CSF and
IFN-.gamma., and additional tumor-derived factors may contribute to
the process of hybrid neutrophil differentiation.
Example 11
CD11b.sup.+CD15.sup.hiCD10.sup.-CD16.sup.int/low Progenitors Give
Rise to APC-like Hybrid Neutrophils
[0237] The low frequency of APC-like hybrid TANs along with high
heterogeneity in their accumulation in cancer patients suggested
that there might be precursor cells that could differentiate into
this unique subset of neutrophils under specific favorable
conditions in some tumors. Therefore, whether the ability of
long-lived immature BMNs to develop hybrid neutrophils is either
shared by all immature subsets or limited to a specific
differentiation stage was investigated.
[0238] The combined expression of CD11b, CD15, CD10, CD49d, and
CD16 was used to distinguish the different maturational states of
BMNs (Elghetany, 2002. Blood Cells Mol. Dis. 2, 260-274).
CD11b.sup.+CD15.sup.hi BMNs consisted of a heterogeneous
combination of mature CD16.sup.hiCD10.sup.+CD49d.sup.- cells,
immature CD16.sup.intCD10.sup.-CD49d.sup.- band cells, and
CD16.sup.low/-CD10.sup.-CD49d.sup.+ metamyelocytes/myelocytes (FIG.
18A). Of note, all mature and immature BMNs express CD66b but at
slightly different levels (FIG. 19A). The detailed phenotype of
neutrophils at different maturation stages is summarized in FIG.
19B. BMNs were isolated at different stages of maturation by flow
cytometry sorting based on these phenotypes. Cytomorphology
confirmed that each population was associated with distinct
maturation stages (FIG. 18B). These sorted subsets of BMNs were
cultured in the presence of low concentration of IFN-.gamma. (50
pg/ml) and GM-CSF (50 pg/ml) for 6 days, after which the resulting
CD11b.sup.+ CD15.sup.hiCD66b.sup.+ neutrophil populations were
analyzed for surface expression of CD14 and HLA-DR (FIG. 18C). Data
revealed that CD14.sup.+HLA-DR.sup.+ hybrid neutrophils could be
generated from all immature stages of neutrophils except the
terminally differentiated, mature, segmented neutrophils. However,
the level of HLA-DR expression on these hybrid neutrophils was
affected by the degree of immaturity of the neutrophils prior to
exposure to IFN-.gamma. and GM-CSF: the more mature
CD15.sup.hiCD10'' CD 16.sup.int band cells gave rise to hybrid
neutrophils, with the highest expression of HLA-DR on the surface
when compared with hybrid neutrophils differentiated from
CD15.sup.hiCD10.sup.-CD16.sup.-/low myelocytes and
metamyelocytes/early bands (FIG. 18C). Interestingly, the majority
of the neutrophils differentiated from
CD15.sup.hiCD10.sup.-CD16.sup.int band cells were able to change
their nuclear contour from band-like to oval when compared with
neutrophils differentiated from myelocytes and metamyelocytes/early
bands (FIG. 18D).
[0239] Importantly, the circulating blood CD16.sup.int/loCD10.sup.-
immature neutrophils that could potentially traffic into tumors
were also able to differentiate into hybrid neutrophils in the
presence of hybrid-inducing TCM or IFN-.gamma. and GM-CSF (FIG.
19C).
Example 12
Ikaros Negatively Regulates the Development of APC-like Hybrid
Neutrophils
[0240] Murine models have shown that the transcription factor
Ikaros is involved in the control of neutrophil differentiation by
silencing specific pathways in common precursors that allow for
macrophage-monocyte development. Given that hybrid neutrophils
exhibit some characteristics of monocytic lineage cells, but can be
differentiated from granulocyte-committed precursors, it was
hypothesized that the hybrid-inducing ability of TCM may be due to
two possible synergistic effects on granulocyte progenitor cells:
(1) premature downregulation of Ikaros, thus allowing some degree
of monocyte differentiation to occur; and (2) the provision of the
appropriate macrophage stimulating factors (i.e., GM-CSF) to
activate the monocyte differentiation pathways.
[0241] The level of Ikaros expression were measured in BMNs at
different stages of maturation and Ikaros was upregulated in all
immature neutrophils (bands and metamyelocytes), with lower levels
in mature BMNs and PBNs (FIG. 20A). The analysis of BMNs treated
with hybrid-inducing TCM revealed that the Ikaros level was lower
in HLA-DR.sup.+ hybrid BMNs compared with HLA-DW canonical BMNs
(FIG. 20B). Thus hybrid-inducing TCM induced premature
downregulation of Ikaros in HLA-DR.sup.+ hybrid BMNs. BMNs were
cultured with hybrid-inducing TCM in the presence or absence of the
drug lenalidomide, which causes proteasomal degradation of the
human Ikaros proteins (Kronke et al., 2014. Oncoimmunology. 7,
e941742). The addition of lenalidomide to TCM-treated BM
neutrophils dramatically facilitated the development of
HLA-DR.sup.+CD14.sup.+ hybrid neutrophils (FIG. 20C). Together,
these data suggest that Ikaros negatively regulates this process in
the presence of tumor-derived factors.
[0242] The level of Ikaros was measured in BMN progenitors
incubated with or without low-dose IFN-.gamma. and/or GM-CSF at
days 1, 3, and 5. Downregulation of Ikaros was only observed when
both IFN-.gamma. and GM-CSF were present for at least 5 days,
confirming their synergistic effect in this process (FIG. 20D).
Next, Ikaros was downregulated in BMNs by adding lenalidomide and
culturing these cells with either IFN-.gamma. or GM-CSF. The
incubation of BMNs with the combination of GM-CSF and lenalidomide,
but not IFN-.gamma. and lenalidomide, resulted in efficient
development of HLA-DR.sup.+CD14.sup.+ hybrid cells (80%-90% among
all BMNs) (FIG. 20D). These data confirm the hypothesis that the
premature downregulation of Ikaros in concert with the macrophage
stimulatory factor GM-CSF are requisite for the development of
hybrid neutrophils from neutrophil progenitors.
Example 13
BM-Derived Hybrid Neutrophils Recapitulate the Function of APC-like
Hybrid TANs
[0243] It was investigated whether the BM-derived hybrid
neutrophils also functionally resemble hybrid TANs in their ability
to stimulate T cell responses. For this purpose, immature BMNs were
differentiated into activated canonical and hybrid neutrophils
(FIG. 21A) and co-cultured with autologous PBMCs stimulated with
plate-bound anti-CD3 Abs. Both subsets of neutrophils augmented the
expression of activation markers CD25 and CD69 on stimulated T
cells to the same degree (FIG. 21B). However, HLA-DR.sup.- hybrid
neutrophils exerted a significantly stronger stimulatory effect on
T cell proliferation and IFN-.gamma. production than the canonical
neutrophils (FIGS. 22A-22B). The BM-derived hybrid neutrophils
differentiated with low doses of IFN-.gamma. and GM-CSF also
recapitulated the T cell stimulatory activity of hybrid TANs (FIG.
22A). However, as described herein, the treatment of BMNs with a
low dose of GM-CSF and IFN-.gamma. at concentrations more than 1
ng/ml gradually induced the expression of PD-L1 on the HLA-DR+ BMNs
(FIG. 22D, lower panel). When PD-L1.sup.+HLA-DR.sup.+ BMNs were
co-cultured with autologous PBMCs stimulated with anti-CD3 Abs, T
cell proliferation was markedly suppressed (FIG. 22C, upper panel),
which was substantially inhibited by PD-L1 blocking Abs (FIG. 22C,
lower panel). Thus, high doses of IFN-.gamma. can convert the T
cell stimulatory HLA-DR.sup.+ BMNs into a suppressive population
via upregulation of PD-L1. These results demonstrate some
functional plasticity in the APC-like neutrophils.
[0244] To determine whether the hybrid neutrophils are able to
induce the proliferation of allogeneic T cells in a
mixed-lymphocyte reaction, BM-derived hybrid and canonical
neutrophils were co-cultured with allogeneic T cells purified from
the peripheral blood of healthy donors. BrdU incorporation assays
revealed that hybrid neutrophils, but not canonical neutrophils,
were able to initiate the allogeneic proliferation of both CD4 and
CD8 cells (FIG. 22D). In addition, similar to hybrid TANs,
BM-derived hybrid neutrophils pulsed with a peptide pool of viral
antigens were able to initiate the autologous memory CD8 and CD4
cell response more efficiently than canonical neutrophils (FIG.
22C). These data demonstrate the functional resemblance between
BM-derived and tumor-derived hybrid neutrophils, and justify the
use of this model to investigate additional functions of this rare
subset of TANs.
Example 14
APC-like Hybrid Neutrophils Stimulate and Augment Anti-Tumor
Effector T Cell Responses
[0245] The effect of canonical and hybrid neutrophils on anti-tumor
effector T cells was evaluated using a newly developed in vitro
model. Human T cells were transduced with a high-affinity
transgenic T cell receptor (TCR) called Ly95 that recognizes an
HLA-A*0201-restricted peptide sequence in the human cancer testis
antigen, NY-ESO-1 (Moon et al., 2016. Clin. Cancer Res. 22,
436-447). As target cells, a genetically modified A549 human lung
adenocarcinoma cell line expressing the NY-ESO-1 protein in the
context of HLA-A*0201 (A549 A2-NY-ESO-1 cells) (Moon et al., 2016.
Clin. Cancer Res. 22, 436-447) was used. Co-culturing of Ly95 T
cells with A549 A2-NY-ESO-1 tumor cells resulted in robust
production of IFN-.gamma. and Granzyme B in Ly95 T cells (FIG.
23A). When BM-derived hybrid neutrophils were added into this
system, the production of IFN-.gamma. and Granzyme B in Ly95 T
cells was markedly elevated (FIGS. 23A-22B) and increased compared
with canonical neutrophils. Of note, the addition of the hybrid
neutrophils into Ly95 T cells co-cultured with control A549 cells
did not induce the production of these factors, indicating that
hybrid neutrophil-mediated stimulation of Ly95 cells was NY-ESO-1
specific and not the result of allostimulation.
[0246] HLA-DR.sup.+ hybrid BMNs induced the stimulation of
IFN-.gamma. production by Ly95 T cells only when the cells were in
direct contact (FIG. 21D). Since hybrid BMNs are characterized by
increased expression of co-stimulatory molecules OX40L, 4-1BBL
CD86, and CD54 (FIGS. 14D-14E, and 15D), Ly95 T cells were
co-cultured with A549 A2-NY-ESO-1 tumor cells and with hybrid BMNs
in the presence of blocking Abs to these upregulated co-stimulatory
molecules. FIG. 23A shows a representative experiment in which the
stimulatory effect of hybrid neutrophils was partially abrogated in
the presence of anti-CD54, 4-1BBL, OX-40L, and CD86 blocking Abs
(FIG. 23A). Next it was determined whether APC-like hybrid
neutrophils could directly trigger NY-ESO-1 specific response of
Ly95 cells. Given that Ly95 cells specifically recognize the
HLA-A*02-restricted peptide of NY-ESO-1, HLA-A*02.sup.- BM-derived
canonical and hybrid neutrophils were pulsed with the NY-ESO-1
(157-165) peptide and then co-cultured with Ly95 T cells for 24 hr.
Hybrid HLA-A* 02.sup.+HLA-DR.sup.+ hybrid neutrophils preloaded
with the peptide triggered IFN-.gamma. production in Ly95 T cells
more effectively than peptide-loaded canonical neutrophils (FIG.
23C). These data demonstrate that hybrid neutrophils can trigger
and significantly augment the activation of antigen-specific
effector T cells.
Example 15
APC-like Hybrid Neutrophils are Able to Cross-Present Tumor
Antigens
[0247] Hybrid neutrophils were able to take up and process
ovalbumin (DQ-OVA) to a higher degree than canonical neutrophils
(FIG. 23D). To evaluate whether hybrid neutrophils are able to
present extracellular protein to effector CD8 cells
(cross-presentation), HLA-A*02-positive BM-derived hybrid and
canonical neutrophils were preloaded with full-length NY-ESO-1
protein and mixed with Ly95 cells for 24 hr (FIG. 23E). These
canonical and hybrid neutrophils were not sufficient to trigger
Ly95 T cell response. Ly95 T cells mixed with control, unloaded
neutrophils generated a low background of IFN-.gamma.-positive
spots due to endogenous activity of Ly95 cells from the prior CD3
stimulation required for expansion of these cells after TCR
transduction (FIG. 23E). Next, the Fc receptors (FcgR) that are
highly expressed on hybrid neutrophils (FIG. 2D) were employed and
the NY-ESO-1 protein delivered as an immunoglobulin G (IgG)-immune
complex to trigger the more efficient FcgR-mediated antigen uptake
and presentation. For this purpose, the neutrophil subsets were
pre-exposed to NY-ESO-1 immune complexes formed by incubating the
NY-ESO-1 protein with anti-NY-ESO-1 monoclonal Abs and mixed them
with Ly95 cells for 24 hr. Under these conditions, hybrid
neutrophils, but not canonical neutrophils, were able to
cross-present NY-ESO epitopes and induce low-level, but
NY-ESO-specific, production of IFN-.gamma. by Ly95 T cells (FIG.
7E). These data demonstrate that hybrid neutrophils have the
ability to take up and cross-present exogenous tumor antigens.
Example 16
Hybrid CD14.sup.+HLA-DR.sup.+CD32.sup.hiCD64.sup.hi Neutrophils
Efficiently Phagocytose Bacteria and Mediate a High Level of
Antibody Dependent Cell Cytotoxicity
[0248] CD64.sup.hiCD32.sup.hi hybrid neutrophils (which could be
generated in large numbers from immature bone marrow or peripheral
blood) are powerful effector cells that trigger sufficient removal
of tumor cells or infectious pathogens through ADP or ADCC. Support
for this claim comes from comparative analysis described herein of
canonical and hybrid neutrophils that revealed that hybrid
neutrophils are characterized by: 1) augmented ability to
phagocytose bacteria (FIG. 5A), 2) expression of very high levels
of FcRI (CD64) and FcRII (CD32) (FIG. 5D). Of note, the high
affinity Fc.gamma.RI/CD64 represents the most potent neutrophil
Fc.gamma.R for induction of ADCC, 3) increased ability to mediate
the high level of antibody-dependent phagocytosis/trogocytosis
(FIG. 5B), 4) ability to mediate the ADCC towards different types
of cancer: (1) human epidermoid carcinoma (A431 cell line), (2)
adenocarcinomic human alveolar basal epithelial cells (A549 cell
line), and (3) B lymphoblasts (Daudi cell line) (FIGS. 9), and 5.)
ability to inhibit A549 tumor growth in NOD scid gamma (NSG) mice
(FIG. 10).
[0249] The disclosures of each and every patent, patent
application, and publication cited herein are hereby incorporated
herein by reference in their entirety. While the present invention
has been disclosed with reference to specific embodiments, it is
apparent that other embodiments and variations of the present
invention may be devised by others skilled in the art without
departing from the true spirit and scope of the invention. The
appended claims are intended to be construed to include all such
embodiments and equivalent variations.
* * * * *