U.S. patent application number 17/060571 was filed with the patent office on 2021-01-28 for activation and expansion of nkg2c+ nk cells.
The applicant listed for this patent is Deutsches Rheuma-Forschungszentrum Berlin. Invention is credited to Quirin Hammer, Chiara Romagnani, Timo Ruckert.
Application Number | 20210023163 17/060571 |
Document ID | / |
Family ID | 1000005139000 |
Filed Date | 2021-01-28 |
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United States Patent
Application |
20210023163 |
Kind Code |
A1 |
Romagnani; Chiara ; et
al. |
January 28, 2021 |
ACTIVATION AND EXPANSION OF NKG2C+ NK CELLS
Abstract
The invention relates to an isolated peptide for use as a
medicament, wherein said peptide has 9 to 30 amino acids and
comprises or consists of an amino acid sequence according to SEQ ID
NO 1 (VMAPRTLXL), wherein X is an amino acid with a hydrophobic
side chain (A, I, L, F, V, P, G), preferably V, L, I or F. The
invention further relates to the peptide of the invention for use
as a medicament to expand and/or activate NKG2C+ natural killer
(NK) cells. The invention further relates to the peptide of the
invention for use in the treatment and/or prevention of a medical
condition associated with pathogenic cells expressing HLA-E and a
peptide comprising an amino acid sequence according to SEQ ID NO 1
or 2. Additionally, the invention relates to a genetically modified
virus encoding a peptide comprising or consisting of a polypeptide
of the invention for use as a medicament to expand and/or activate
NKG2C+ natural killer (NK) cells.
Inventors: |
Romagnani; Chiara; (Berlin,
DE) ; Ruckert; Timo; (Berlin, DE) ; Hammer;
Quirin; (Berlin, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Deutsches Rheuma-Forschungszentrum Berlin |
Berlin |
|
DE |
|
|
Family ID: |
1000005139000 |
Appl. No.: |
17/060571 |
Filed: |
October 1, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
16355577 |
Mar 15, 2019 |
10864245 |
|
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17060571 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 45/06 20130101;
C12N 5/0638 20130101; A61P 35/00 20180101; A61K 38/08 20130101;
A61K 38/208 20130101; A61P 31/20 20180101; A61P 35/02 20180101;
A61K 38/20 20130101; A61K 38/2086 20130101; A61K 35/17
20130101 |
International
Class: |
A61K 38/08 20060101
A61K038/08; A61P 31/20 20060101 A61P031/20; A61P 35/02 20060101
A61P035/02; A61P 35/00 20060101 A61P035/00; A61K 35/17 20060101
A61K035/17; A61K 38/20 20060101 A61K038/20; A61K 45/06 20060101
A61K045/06; C12N 5/0783 20060101 C12N005/0783 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 16, 2018 |
EP |
18162281.2 |
Claims
1. A method of treating a subject having or being at risk of
developing an active human cytomegalovirus (HCMV) infection, the
method comprising administering to said subject an effective amount
of an isolated peptide of 9 to 30 amino acids comprising an amino
acid sequence according to SEQ ID NO: 1 (VMAPRTLXL), wherein X is
an amino acid with a hydrophobic side chain (A, I, L, F, V, P, G),
wherein the method expands and/or activates NKG2C+ natural killer
(NK) cells.
2. The method according to claim 1, wherein said peptide comprises
an amino acid sequence consisting of SEQ ID NO 2 (VMAPRTLFL).
3. The method according to claim 1, wherein the treatment inhibits
reactivation of human HCMV.
4. The method according to claim 1, wherein the treatment reduces
viral titers in an individual infected with HCMV.
5. The method according to claim 1, wherein the subject has
leukemia and the treatment inhibits reactivation of HCMV infections
in subjects having received hematopoietic stem cell
transplantation.
6. The method according to claim 1, wherein the peptide is
administered in combination with an adjuvant that enhances
production of, or comprises, IL-15, IL-12 and/or IL-18.
7. The method according to claim 1, wherein the peptide is
administered in combination with a check point inhibitor.
8. The method according to claim 7, wherein the peptide is
administered in combination with an inhibitor of a receptor
selected from the group consisting of LILRB1, inhibitory KIRs,
NKG2A, PD-1, CTLA-4, TIM-3, TIGIT and LAG-3.
9. The method according to claim 1, wherein the peptide is
administered by a vector comprising or encoding the peptide
according to claim 1, wherein the peptide is encoded by a nucleic
acid molecule operably linked to a promoter for expression in
mammalian subjects.
10. The method according to claim 9, wherein the vector is a
genetically modified virus selected from the group consisting of
attenuated HCMV, vaccinia virus, adenovirus, adeno-associated
virus, retrovirus, and lentivirus.
11. The method according to claim 1, the method comprising
administering to said subject an effective amount of a genetically
modified virus encoding a peptide comprising or consisting of a
polypeptide according to claim 1.
Description
FIELD OF THE INVENTION
[0001] The invention relates to an isolated peptide for use as a
medicament, wherein said peptide has 9 to 30 amino acids and
comprises or consists of an amino acid sequence according to SEQ ID
NO 1 (VMAPRTLXL), wherein X is an amino acid with a hydrophobic
side chain (A, I, L, F, V, P, G), preferably V, L, I or F. The
invention further relates to the peptide of the invention for use
as a medicament to expand and/or activate NKG2C+ natural killer
(NK) cells. The invention further relates to the peptide of the
invention for use in the treatment and/or prevention of a medical
condition associated with pathogenic cells expressing HLA-E and a
peptide comprising an amino acid sequence according to SEQ ID NO 1
or 2. Additionally, the invention relates to a genetically modified
virus encoding a peptide comprising or consisting of a polypeptide
of the invention for use as a medicament to expand and/or activate
NKG2C+ natural killer (NK) cells.
REFERENCE TO SEQUENCE LISTING
[0002] A Sequence Listing submitted as an ASCII text file via
EFS-Web is hereby incorporated by reference in accordance with 35
U.S.C. .sctn. 1.52(e). The name of the ASCII 15 text file for the
Sequence Listing is 33616284_1, the date of creation of the ASCII
text file is Oct. 1, 2020, and the size of the ASCII text file is
14.1 KB.
BACKGROUND OF THE INVENTION
[0003] Natural killer (NK) cells are cytotoxic innate immune cells,
which contribute to early immune responses against viral infections
(1). Their role in host protection is highlighted by patients with
primary NK-cell deficiencies, who suffer from severe and
disseminated viral infections caused by herpesviruses such as human
cytomegalovirus (HCMV) (2); and further supported by studies of the
murine CMV (MCMV) infection model (3). HCMV has a high prevalence
in the adult human population and establishes livelong latency in
healthy individuals. The host innate and adaptive immune systems
jointly play a crucial role in restraining viral replication and
preventing disease but do not eliminate the virus, which in turn
engages in a dynamic interaction with the host, resulting in
drastically imprinted immune-cell repertoires (4).
[0004] In accordance with these findings, Ly49H+ NK cells from
C57BL/6 mice were shown to undergo expansion and adaptation in
response to MCMV (3). Similarly, HCMV-seropositivity is associated
with a skewed repertoire of human NK cells, and expression of the
activating receptor CD94/NKG2C (NKG2C) marks a well characterized
NK-cell subset adapted to HCMV infection, consequently termed
`adaptive NK cells` (5, 6, 7). Apart from NKG2C expression,
adaptive NKG2C+NK cells can be characterized by altered receptor
profiles and remodeled epigenetic landscapes compared to
conventional NK cells (5, 6, 7, 8, 9, 10, 11). In contrast to the
murine infection model, in which the MCMV protein m157 was
established as the ligand for Ly49H (12, 13), a HCMV ligand driving
the specific expansion and differentiation of human NKG2C+NK cells
has not been identified.
[0005] The non-classical MHC class I molecule HLA-E serves as
cognate ligand for NKG2C as well as its inhibitory counterpart
CD94/NKG2A (NKG2A) (14, 15, 16) and has been reported to elicit
effector functions in adaptive NKG2C+NK cells (6) as well as to
contribute to their expansion in vitro (17, 18). Cell surface
stabilization of HLA-E requires loading with peptides, which can be
derived from the signal sequences of MHC class I molecules (19) or
other proteins such as HSP60 (20) at steady state. In addition to
host peptides, the UL40 gene of HCMV was found to encode
HLA-E-stabilizing peptides, which partially mimic MHC class I
signal sequences (21, 22, 23, 24). Despite HCMV-mediated
down-regulation of HLA class I to evade recognition by CD8+ T
cells, UL40-derived peptides permit maintenance of HLA-E surface
expression on infected cells and thereby preserve inhibition of
NK-cell activation via engagement of NKG2A. Indeed, it was
demonstrated that co-transfection of UL40 and HLA-E confers
protection against NKG2A+NK-cell lines and infection of fibroblasts
with UL40-competent HCMV inhibits cytotoxic activity of NKG2A+NK
cells (21, 22, 23, 24).
[0006] However, whether NKG2C can recognize UL40 peptides during
HCMV infection and result in activation of NKG2C+NK cells remains
completely unclear. HLA-E-stabilizing nonameric peptides derived
from both MHC class I or UL40 share conserved residues at amino
acids 2 and 9, while mutations at positions 5 and 8 have been shown
to alter binding of HLA-E/peptide complexes to CD94 heterodimers
with NKG2A or NKG2C in structural and biochemical analyses (25, 26,
27, 28, 29, 30). Conversely, analysis of peptide impacting on
functional recognition of HLA-E-expressing cells by NKG2A and NKG2C
has been confined to NK-cell clones (28, 29, 30). It was shown that
CD94/NKG2C can be activated on an experimental cell line
(Jurkat-NKG2C+ reporter cells) by HLA-E displaying cells that were
pre-incubated with peptides VMAPRTLIL (SEQ ID NO. 3) or VMAPRTLFL
(SEQ ID NO. 2) (Pupuleku A et al: "Elusive Role of the CD94/NKG2C
NK Cell Receptor in the Response to Cytomegalovirus: Novel
Experimental Observations in a Reporter Cell System", FRONTIERS IN
IMMUNOLOGY, vol. 8, 24 Oct. 2017, (30)). These results demonstrate
the general peptide-dependency of the interaction of CD94/NKG2
receptors with HLA-E. However, these articles provide no
information about NKG2C receptor specificity towards peptides with
single amino acid differences and effects of the peptides on, for
example, NKG2A activation. They also do not assess the functional
consequences of this peptide specificity in terms of cytokine
production and most importantly the differential induction of
proliferation and specific expansion of NKG2C+NK cells by the
different peptides. Furthermore, a medical application of the
material and in particular the peptides is not described and no
conclusions about their potential use can be based on the data of
this article. The article completely focuses on experimentally
determining activation of NKG2C in an artificial reporter
system.
[0007] To which extent peptide recognition can impact on NK
cell-mediated immune responses and whether distinct peptides can
drive differential activation, expansion, differentiation, and
heterogeneity of adaptive NKG2C+NK cells during HCMV infection
remain outstanding questions.
[0008] Infection with human cytomegalovirus (HCMV) is widespread in
the general population, with the age-adjusted prevalence in Germany
being around 30%. HCMV is a major cause of morbidity and mortality
in immunocompromised individuals, especially patients undergoing
hematopoietic stem cell transplantation (HSCT), which are at large
risk for reactivating the virus with potentially lethal
consequences. Importantly, the immune system of these patients is
concomitantly challenged by HCMV and relapsing leukemia. Therefore,
a strategy aimed at controlling both viral infection and leukemia
relapse would be of great use.
[0009] Moreover, HCMV congenital infection is associated with
microcephaly, mental disabilities and hearing problems. About 1 in
100 to 500 babies is born with congenital HCMV, and of the 10-20%
symptomatic infections 30% are lethal, making this a large scale
global health problem. Accordingly, large efforts have been
invested into developing a vaccine against HCMV, but so far none of
these approaches has been of success.
[0010] While large scale efforts in prevention and significant
improvements in treatment strategies are bearing fruit in the last
years by reducing both cancer incidence and mortality rates,
especially advanced tumors still remain a challenge in modern
medicine. Increasing lifespans in industrialized and developing
countries mean that absolute incidence and mortality numbers are on
the rise, which opens opportunities for developing more specific
therapeutic approaches to treat subtypes of cancer. One
particularly successful concept, which quite recently has found its
way into the clinics with impressive results is cancer
immunotherapy, with checkpoint inhibition being named the
breakthrough of the year 2013. However, a great share of patients
remains unresponsive to checkpoint inhibition for various reasons,
and the widespread occurrence of autoimmunity further limits its
use. One way to circumvent this is to target more specific
tumor-associated ligands, as done by the recently FDA-approved CAR
T cell therapy for B cell leukemia. In sum, the identification of
new and more specific cancer immunotherapy targets bears great
potential to develop new and improve current treatment
regimens.
[0011] The use of HSP60-derived, HLA-E-binding nonameric peptides
for the treatment of tumors has been described in WO 03/011895 A2.
The invention described therein relates to modulation of CD94/NKG2
receptor function by HLA-E-bound peptides, which involves parallel
modulation of activation NKG2A and NKG2C. However, as also
disclosed in the context of the present invention, modifications of
single amino acids of a HLA-E-binding peptide can have a tremendous
impact on the activation of NK-cell receptors such as NKG2A and
NKG2C, so that effects of a specific peptide cannot be extrapolated
to apparently similar peptides that differ in one or more amino
acids from the peptides of WO 03/011895 A2.
[0012] The innate lymphocytes Natural killer (NK) cells expressing
the activating receptor CD94/NKG2C display adaptive features and
are stably expanded in a group of individuals who have been
infected with HCMV (41). It has been proposed that these cells have
beneficial effects against HCMV reactivation (46). Importantly,
correlation of reduced relapse rates in patients reactivating HCMV
and presenting with NKG2C+NK cell expansions has been reported,
pointing towards an anti-leukemic effect of these cells
highlighting their potential as an anti-cancer treatment (Cichocki
et al. Leukemia. 2016; 30(2):456-63). However, an ex vivo expansion
method for such protective CD56dim CD57+ NKG2C+NK cells has not
been described by Cichocki et al. and it is to be noted that ex
vivo manipulation and expansion of NKG2C+NK cells isolated form a
donor probably has an impact on the overall character of the cells,
such a modification of the epigenetic landscape, gene and surface
marker expression, morphology and/or other characteristics, so that
ex vivo expanded NKG2C+NK cells most likely differ significantly
from naturally occurring circulating NKG2C+NK cells.
[0013] However, the means by which NKG2C+NK cells can be expanded
and activated by HCMV or cancer cells remain unclear, thus limiting
their potential therapeutic use. WO2014037422A1 describes the ex
vivo expansion of NKG2C+NK cells for adoptive transfer. However,
this approach raises severe concerns with respect to safe use of
the cells, standardization of treatment and logistics. Besides this
method bearing these significant disadvantages, there are no means
known in the art to induce expansion and activation of NKG2C+
cells, in particular not for in vivo expansion and activation.
[0014] In light of the prior art, there remains a significant need
to provide means for harnessing the therapeutic use of NKG2C+ cells
in the treatment of HCMV infection and/or treatment of cancer.
SUMMARY OF THE INVENTION
[0015] In light of the prior art, the technical problem underlying
the present invention is to provide alternative and/or improved
means for expanding and/or activating NKG2C+ natural killer (NK)
cells. A further problem to be solved is the provision of means for
the treatment and/or prevention of a medical condition associated
with pathogenic cells expressing HLA-E and a peptide comprising an
amino acid sequence according to SEQ ID NO 1 or 2, such as HCMV
infection or cancer.
[0016] The technical problem underlying the present invention is
solved by the features of the independent claims. Preferred
embodiments of the present invention are provided by the dependent
claims.
[0017] The invention relates to an isolated peptide comprising or
consisting of an amino acid sequence according to SEQ ID NO 1
(VMAPRTLXL), wherein X is an amino acid with a hydrophobic side
chain (A, I, L, F, V, P, G), preferably V, L, I or F.
[0018] The invention further relates to an isolated peptide for use
as a medicament comprising or consisting of an amino acid sequence
according to SEQ ID NO 1 (VMAPRTLXL), wherein X is an amino acid
with a hydrophobic side chain (A, I, L, F, V, P, G), preferably V,
L, I or F.
[0019] The invention preferably relates to an isolated peptide for
use as a medicament, wherein said peptide has 9 to 30 amino acids
and comprises or consists of an amino acid sequence according to
SEQ ID NO 1 (VMAPRTLXL), wherein X is an amino acid with a
hydrophobic side chain (A, I, L, F, V, P, G), preferably V, L, I or
F.
[0020] The present invention is based on the entirely surprising
finding that peptides of the present invention specifically induce
the activation and expansion of NKG2C+NK cells. In particular,
presentation of the peptides of the present invention or fragments
thereof on a non-classical MHC class I molecule, such as preferably
HLA-E, leads to the activation of NKG2C+NK cells resulting in
expansion and/or activation of various effector functions such as
induction of cell death of the cell presenting the peptide of the
present invention on the non-classical MHC class I molecule,
preferably HLA-E, and secretion and/or expression of TNF-alpha,
IFN-gamma, CCL3 and/or CD107a. This was very surprising, since it
had been reported that presentation of such peptides on HLA-E
mainly display universal inhibitory effects on NK cells based on
their binding to the inhibitory receptor NKG2A. Contrastingly, some
of these peptides could induce preferential activation of NKG2C+NK
cells.
[0021] Furthermore, as disclosed herein, it was found out that the
peptides of the invention have a specific activating effect on the
subset of NKG2C+NK cells, but not for example on NKG2C- NK cells.
Additionally, the activating effect of the peptides of the
invention was receptor specific, since blockage of the NKG2C/CD94
heterodimers almost completely inhibited the activating effect of
the peptides of the present invention. Accordingly, the peptides of
the invention can be applied in the context of the treatment of
diseases associated with pathogenic cells expressing HLA-E and a
peptide of the present invention.
[0022] Furthermore, it was entirely unforeseen that, even though
the HLA-E binding peptides of the present invention do not differ
from other peptides by their binding efficiency to HLA-E, the
activation of NKG2C is peptide sequence specific. Indeed, other
peptides, which bind to and stabilize HLA-E with equal efficiency
as peptides of the present invention, lead to reduced or no
selective activation of NKG2C+NK cells. As shown in the example
disclosed herein, single amino acid exchanges within peptides
presented on HLA-E can be differentially recognized by adaptive
NKG2C+NK cells, resulting in differential activation of the cells.
This finding was very surprising because, in contrast, NKG2A
inhibition is less dependent on peptide sequence recognition and
NKG2A+NK cells are equally inhibited by recognition of HLA-E
complexed with the peptides according to SEQ ID NO 1.
[0023] It was completely unexpected that the complexes formed by
HLA-E and the peptides of the present invention engage
differentially with CD94/NKG2C and lead to specific activation of
this receptor complex on NK cells, whereas other receptors or
receptor complexes that can interact with HLA-E, such as NKG2A, do
not differentiate between specific peptide/HLA-E complexes.
Accordingly, the present invention unexpectedly enables efficient,
specific and selective activation of NKG2C+NK cells. Surprisingly,
the peptides of the present invention are comprised by the UL-40
protein of different HCMV strains as well as MHC Class I molecules,
in particular the signal sequence of HLA-G. HLA-G is a
non-classical MHC class I molecule absent in most healthy tissues
and is mainly expressed during pregnancy and in certain cancer
cells and is upregulated during inflammatory processes.
Accordingly, the peptide of the present invention can be used in
the context of treating diseases, which are associated with
pathogenic cells, which express HLA-E and peptides comprising an
amino acid sequence of a peptide of the present invention, for
example cells expressing HLA-G or UL-40 of HCMV.
[0024] In preferred embodiments, the peptide of the invention
consists of an amino acid sequence according to SEQ ID NO 2
(VMAPRTLFL). The peptide consisting of an amino acid sequence
according to SEQ ID NO2 shows a particularly efficient effect with
respect to activation and/or expansion of NKG2C+NK cells.
[0025] In further embodiments, the peptide of the invention
consists of an amino acid sequence according to SEQ ID NO 3
(VMAPRTLIL), SEQ ID NO 4 (VMAPRTLLL) or SEQ ID NO 5
(VMAPRTLVL).
[0026] Furthermore, the peptide of the present invention can be
used as a medicament to expand and/or activate NKG2C+ natural
killer (NK) cells in the treatment and/or prevention of a medical
condition associated with pathogenic cells expressing HLA-E and a
peptide comprising an amino acid sequence of a peptide of the
invention.
[0027] This aspect of the invention is based on the surprising
finding that cells presenting the complex formed by HLA-E and the
peptide of the present invention on their surface are recognized by
NKG2C+NK cells through engagement of the complex with the
CD94/NKG2C heterodimer. This leads to activation of the NKG2C+NK
cells resulting in secretion of several effector proteins and
induction of cytotoxicity towards the cells presenting the complex.
Such cells are in most cases pathogenic cells, such as tumor/cancer
cells expressing HLA-G, comprising a peptide of the present
invention in its leader/signal sequence, and HLA-E, or HCMV
infected cells comprising actively replicating HCMV expressing
UL-40. The pathogenic cells can be more efficiently fought by the
immune system after recognition by NKG2C+NK cells. Accordingly,
provision of an increased number of NKG2C+NK cells is beneficial
for the treatment of disease associated with pathogenic cells
expressing peptides comprising the amino acid sequence of a peptide
of the present invention an HLA-E. Such an increased number of
NKG2C+NK cells can be provided or achieved by administration of the
peptide of the present invention leading to in vivo expansion of
the NKG2C+NK cells, or administration of NKG2C+NK cells of the
present invention, which have been generated by the in vitro method
of the present invention.
[0028] In embodiments, the isolated peptide of the invention is
used as a medicament to expand and/or activate NKG2C+ natural
killer (NK) cells.
[0029] Furthermore, the isolated peptide can be used in the
treatment and/or prevention of a medical condition treatable by the
cytotoxic activity of said NKG2C+NK cells. Also, in embodiments,
the isolated peptide is used as a medicament to expand and/or
activate NKG2C+ natural killer (NK) cells in the treatment and/or
prevention of a medical condition treatable by the effector
function of said NKG2C+NK cells.
[0030] In embodiments of the present invention, the peptide is used
as a medicament to inhibit reactivation of human cytomegalovirus
(HCMV) infections and/or reduce viral titers in an individual
infected with HCMV. This embodiment is particularly relevant for
the treatment of subjects, which are at risk of reactivation of
HCMV or in which HCMV replication has been reactivated in some
cells already, and wherein a spreading of the active infection
should be prevented or suppressed.
[0031] In preferred embodiments of the invention, the peptide of
the invention is used to treat cancer, wherein said cancer
expresses HLA-G and HLA-E, preferably wherein the expression of
HLA-G and HLA-E is above levels in healthy control cells, and
wherein the cancer is preferably selected from the group consisting
of leukemia, melanoma, choriocarcinoma, breast cancer, endometrial
cancer, ovarian cancer, cervical cancer, esophageal squamous cell
carcinoma, colorectal cancer, gastric cancer, hepatocellular
carcinoma, glioblastoma, lung cancer, nasopharyngeal carcinoma,
pancreatic adenocarcinoma, thyroid carcinoma and renal
carcinoma.
[0032] The present invention is effective in the treatment of
diseases associated with pathogenic cells that present peptides of
the present invention on HLA-E on their surface. The non-classical
MHC class I molecule HLA-G comprises the peptide of the invention
in its signal sequence. HLA-G is expressed or upregulated in
particular cancer cells. Such cancer cells can be identified by
expression analysis of HLA-G and HLA-E, by various methods known to
the person skilled in the art, some of which are also disclosed in
the examples of the present patent application. Expression of HLA-G
has been reported for several cancers, which are preferably treated
with the peptide of the present invention, which comprise, without
limitation, leukemia, Melanoma, choriocarcinoma, breast cancer,
endometrial cancer, ovarian cancer, cervical cancer, esophageal
squamous cell carcinoma, colorectal cancer, gastric cancer,
hepatocellular carcinoma, glioblastoma, lung cancer, nasopharyngeal
carcinoma, pancreatic adenocarcinoma, thyroid carcinoma and renal
carcinoma. Cancers expressing HLA-G are known to the skilled person
and are disclosed in the art (see for example Curigliano G,
Criscitiello C, Gelao L, Goldhirsch A. Molecular pathways: human
leukocyte antigen G (HLA-G). Clin Cancer Res. 2013; 19(20):5564-71;
Lin A, Yan W H. HLA-G expression in cancers: roles in immune
evasion, metastasis and target for therapy. Mol Med. 2015; Seliger
B, Schlaf G. Structure, expression and function of HLA-Gin renal
cell carcinoma. Semin Cancer Biol. 2007; 17(6):444-50).
[0033] In embodiments of the invention, the peptide is used as a
medicament to treat a cancer associated with expression of HLA-G
and HLA-E, wherein the cancer is identified by [0034] a. providing
a sample comprising cancer cells from a patient and [0035] b.
determining expression of HLA-G and HLA-E in said sample.
[0036] Expression of HLA-G and HLA-E in cancer cells may be
determined on the protein or the nucleic acid level. For example,
mRNA expression levels of HLA-G and HLA-E encoding mRNA may be
determined by qRT-PCR or sequencing analysis, as known to the
person skilled in the art (see for example Paul, P., et al. (2000).
"HLA-G, -E, -F preworkshop: tools and protocols for analysis of
non-classical class I genes transcription and protein expression."
Human Immunology 61(11): 1177-1195). Furthermore, expression may be
analyzed on the protein level for example by cytometric analysis of
HLA-G and HLA-E expression on the cells surface.
[0037] In embodiments, the isolated peptide of the invention is
used as a medicament to treat a cancer associated with elevated
expression of HLA-G compared to non-cancerous cells, preferably
with elevated expression of HLA-G and HLA-E compared to
non-cancerous cells, or a cancer susceptible to NKG2C+NK cell
cytotoxic activity. In the context of the method of the invention,
the expression level of HLA-E and/or HLA-G determined in a sample
comprising cancer cells from a subject may be compared to the
expression of HLA-E and/or HLA-G in a reference standard sample (as
in the disclosed example) and/or in a corresponding sample isolated
from a healthy individual, and/or in a corresponding sample that
does not comprise cancer cells. A corresponding sample may be a
sample that has been isolated form the same tissue or bodily fluid,
but does not comprise any cancer cells, for example because it has
been isolated from a healthy individual.
[0038] In embodiments, the peptide of the invention is used as a
medicament to treat a cancer associated with elevated expression of
HLA-G and HLA-E. In further embodiments, the peptide of the
invention is used as a medicament to treat a cancer susceptible to
NKG2C+NK cell cytotoxic activity.
[0039] In a preferred embodiment of the invention, the peptide is
used as a medicament to treat leukemia and inhibit reactivation of
HCMV infections in subjects having received hematopoietic stem cell
transplantation (HSCT). The activation and expansion of NKG2C+NK
cells by peptides of the present invention may be particular
advantageous in the context of HSCT, because NK cells are among the
first lymphocyte populations to recover after transplantation and
therefore can be targeted by the using the approach of the present
invention, thus potentially protecting against HCMV reactivation
and tumor relapse in leukemia patients after HSCT.
[0040] In a further preferred embodiment of the invention the
peptide is administered in combination with an adjuvant, preferably
selected from an adjuvant enhancing production of or comprising
IL-15, IL-12 and/or IL-18. In embodiments, the peptide of the
invention is administered in combination with IL-15, IL-12 and/or
IL-18. In preferred embodiments, the adjuvant is inducing or
enhancing the production of pro-inflammatory cytokines.
[0041] In embodiments, the peptides of the present invention are
administered in combination with one or more pro-inflammatory
cytokines. It was surprising, that combined stimulation of NKG2C+NK
cells with the complex consisting of HLA-E and the peptide of the
present invention and pro-inflammatory cytokines, such as for
example IL-15, IL-12 and/or IL-18, induces accumulation of NKG2C+NK
cells, also of NK cells from or in HCMV- individuals.
[0042] In preferred embodiments of the invention, the peptide is
administered in combination with a check point inhibitor,
preferably an inhibitor of an inhibitory receptor selected from the
group comprising LILRB1, inhibitory KIRs, NKG2A, PD-1, CTLA-4,
TIM-3, TIGIT and LAG-3.
[0043] Check point inhibitors have gained a lot of attention in the
context of cancer treatment, since it turned out that the
inhibition or blockage of inhibitory receptors expressed by immune
cells and in particular immune effector cells, such as for example
T cells, but also NK cells, enables robust activation of the
effector cells to elicit an effective immune response against for
example cancer cells. This is due to the fact that in many
pathological conditions, especially cancer and viral infections,
the pathogenic cells prevent an effective immune response by
activating inhibitory receptors on immune cells, thereby preventing
an effector response of the immune system against the pathogenic
cells. However, check point inhibitors make it possible to overcome
this pathological mechanism by preventing activation of the
inhibitory receptors and therefore enabling and potentiating the
activation of an effective immune response against the pathogenic
cells.
[0044] In the context of the present invention, it turned out that
combined administration of the peptides of the present invention
together with inhibitors of check point molecules, in particular
check point molecules that are expressed by NK cells, such as for
example LILRB1, inhibitory KIRs, NKG2A, PD-1, CTLA-4, TIM-3, TIGIT
and LAG-3, potentiates the activating effect of the peptides of the
present invention on NKG2C+NK cells. Preferably, such a combined
administration may be carried out in the context of the treatment
of cancer and HCMV infection.
[0045] In a preferred embodiment of the present invention, the
peptide is administered in combination with an activator of the
co-stimulatory receptor CD2. It can be advantageous to use the
peptides of the present invention in combination with an activator
or stimulator of CD2, which can act as a co-stimulatory receptor on
NK cells and particularly on NKG2C+NK cells, since surprisingly the
combined activation leads to an enhanced poly-functional response
of the NKG2C+NK cells including activation of cytotoxic activity as
well as secretion of inflammatory mediators such as CCL3, CD107a,
IFN-gamma and TNF-alpha, which cannot be explained by the addition
of the individual effects of the peptides and the CD2-activators,
but instead argue for the presence of a synergistic effect.
Accordingly, the engagement of the co-stimulatory receptor CD2 can
lower the activation threshold of NKG2C+NK cells by the peptides of
the present invention and therefore enable the peptides of the
present invention to optimally trigger multiple effector functions
in adaptive NKG2C+NK cells.
[0046] Further embodiments of the invention relate to
administration of the peptide in combination with IFN-alpha.
IFN-alpha is known to trigger antiviral NK-cell functions and in
the context of the present invention it was found that the combined
administration of the peptides of the invention and IFN-alpha leads
to an enhanced differential activation of NKG2C+NK cells.
[0047] In embodiments of the invention, the peptide for use as a
medicament is administered by a vector comprising or encoding the
peptide of the present invention.
[0048] This embodiment relates to the use of viral vectors or other
vectors, such as mammalian or prokaryotic cells or DNA molecules,
such as plasmids, which may be comprised in liposomes or other
suitable formulation for administration. The vectors used herein
may comprise the peptide of the invention. For example, a viral
vector comprising the proteins or peptides with the amino acid
sequence of the peptides of the invention may be administered.
Furthermore, cells expressing such peptides may be used as a
vector. Alternatively, the peptides are not present in the vector
at the time of administration. However, the vector may enable
expression of the peptide of the invention upon delivery to the
host, such as a patient suffering from HCMV or cancer, wherein
expression of the peptide is induced after administration. For
example, a cell carrying an exogenous nucleic acid molecule
comprising a sequence encoding the peptide of the present invention
under the control of a constitutive or inducible promoter may be
used as a vector to provide expression of the peptides of the
invention in a subject after administration of the cells.
[0049] Similarly, a viral vector may be used to infect cells of a
subject or patient in need of activation of NKG2C+NK cells. The
viral vector may comprise a nucleic acid molecule which enables
expression of the peptide of the invention in by a cell of the
subject upon infection with the viral vector. Alternatively, an
exogenous nucleic acid molecule, such as a DNA plasmid, may be
administered to a subject in need of activation of NKG2C+NK cells,
for example by means of a liposomal formulation, enabling delivery
of the plasmid to a host cell of the subject, which subsequently
expresses the peptide of the present invention. The person skilled
in the art is aware of further suitable vectors and means of
administering such vectors comprising or encoding the peptide of
the present invention.
[0050] In embodiments of the invention, the peptide is encoded by a
nucleic acid molecule operably linked to a promoter for expression
in mammalian, preferably human subjects. In further embodiments,
the nucleic acid molecule is a recombinant nucleic acid molecule.
It is particularly advantageous to use nucleic acid molecules
comprising promoters for expression of the peptide of the invention
in cells of the subject in need NKG2C+NK cell activation, since it
is possible to provide a source of renewed production of the
peptide of the present invention the subject with a single
administration. Use of a recombinant nucleic acid is advantageous,
since the peptide expression can be controlled by a suitable
promoter or promoter/enhancer combination, which is specifically
selected and suitable for the specific application. It is possible
to use controllable promoters, to be able to control expression
levels of the peptides of the invention.
[0051] According to embodiments of the invention, the vector is a
genetically modified virus selected from the group comprising
attenuated HCMV, vaccinia virus, adenovirus, adeno-associated
virus, retrovirus, or lentivirus.
[0052] The present invention also relates to an in vitro method for
cultivating and/or expanding NKG2C+ natural killer (NK) cells, said
method comprising: [0053] providing leukocyte cells from a donor,
wherein said leukocytes comprise NK cells; [0054] contacting said
NK cells with a peptide of the present invention; and [0055]
optionally isolating or enriching for NKG2C+NK cells.
[0056] Preferably, the method of the present invention comprises
contacting the NK cells with IL-15, IL-12 and/or IL18. Furthermore,
the cells may be contacted with an activator of CD2, such as LFA-3.
In the context of the method of the invention, contacting the cells
with an agent may relate to stimulation of the cells in cell
culture, for example by adding the respective agent to the cell
culture medium.
[0057] In embodiments, the leukocytes are purified CD56dim NK
cells, which may be characterized as CD56dim NKG2A- CD57+ NKG2C+ or
NKG2A- CD57+ KIR+ NKG2C+ NK cells. The leukocyte cells may be
contacted in cell culture with murine TAP-deficient RMA-S cells
transfected with human .beta.2-microglobulin and HLA-E
(RMA-S/HLA-E). Furthermore, the RMA-S/HLA-E may be transfected with
human LFA-3 (RMA-S/HLA-E/LFA-3). The leukocyte cells may also be
contacted with human K562 cells transfected with HLA-E
(K562/HLA-E). The RMA-S/HLA-E, RMA-S/HLA-E/LFA-3 and/or K562/HLA-E
may be pulsed with a peptide of the present invention and/or the
peptide of the invention may be added to the cell culture
directly.
[0058] The provision of IL-12 and/or IL-18 is particularly
advantageous in the context of the in vitro method of the present
invention since the presence of these cytokines in the culture
medium specifically accelerates proliferation of NKG2C+NK cells,
therefore leading to an expansion and competitive selection of the
cells in comparison to other cells present in the cell culture. As
shown in the example disclosed herein, the provision of
pro-inflammatory cytokines during the initial phase of culture
resulted in dramatically accelerated NKG2C+NK-cell division induced
by the peptide of the present invention.
[0059] The in vitro method of the invention may be used to generate
large amounts of patient specific NKG2C+NK cells ex vivo/in vitro.
These cells may be used for various purposes, such as for example
experimental use, screenings for example for the effectiveness of
compounds, such as drug candidates, and therapeutic use. For
example, the ex vivo expanded cells may be administered to a
patient, either as an autologous or allogenic transplant, wherein
the cells may be genetically modified or not.
[0060] Furthermore, the present invention also relates to an
isolated population of NKG2C+ natural killer (NK) cells produced by
the in vitro method of the present invention. The cells produced by
the method of the present invention display specific
characteristics, which make them suitable and preferable for
certain applications, including therapeutic applications such as
administration to patients in need of NKG2C+NK cells. Such patients
may suffer from a condition associated pathogenic cells expressing
HLA-E and a peptide comprising a sequence of the peptide of the
present invention, such as for example an HCMV infection or cancer,
wherein said cancer expresses HLA-G and HLA-E.
[0061] The cells of the present invention may be characterized by a
specific expression pattern and profile with respect to one or more
of the markers selected from the group comprising SIGLEC7, CD7,
SYK, CD2, LILRB1 (LIR-1/ILT2), NCR3 (NKp30), SH2DB1 (EAT2) and
ZBTB32 (PLZP), ZBTB16 (PLZF), ZBTB20, ITGAL, CRTAM, HLA-DR, TNFRSF9
(4-1 BB), LAGS, CTLA4, and PDCD1 (PD1) as well as of effector
functions including IFNG, TNF, CCL3, CCL4, IL8, CSF2, IL10, GZMB,
and TNFSF10 (TRAIL).
[0062] In particular, the cells of the invention may be
characterized by an up-regulation of one or more activation and
exhaustion markers, such as HLA-DR, TNFRSF9 (4-1 BB), LAGS, CTLA4,
PDCD1 (PD1), and of effector functions, such as IFNG, TNF, CCL3,
CCL4, IL8, CSF2, IL10, GZMB, and TNFSF10 (TRAIL) compared to NK
cells expanded in the absence of peptides of SEQ ID NO 1 and of
IL-12 and IL-18.
[0063] In particular, the cells of the invention may be
characterized by the epigenetic DNA demethylation of effector
cytokine genes, such as but not limited to IFNG, as in the
disclosed example.
[0064] It is a particular advantage of the in vitro method of the
present invention that the resulting expanded NK cells of the
present invention adapt an NKG2C+NK cells phenotype in comparison
to NK cells expanded according to methods known in the art, since
NKG2C+NK cells and also the cells of the present invention are
particularly beneficial for use in adoptive transfer and other
therapeutic approaches in patients suffering from conditions
associated with pathogenic cells expressing HLA-E and a peptide
comprising an amino acid sequence according to the peptides of the
present invention. The cells of the present invention can be
identified by specific remodeling of the epigenetic landscape, for
example the DNA methylation state of specific effector genes,
and/or by specific gene expression patterns with respect to
activation and exhaustion markers, effector functions and further
markers of NK cells and NK cell subsets.
[0065] In embodiments of the invention, the isolated population of
NKG2C+NK cells or the invention are for use as a medicament to
treat and/or prevent a medical condition associated with pathogenic
cells expressing HLA-E and a peptide of the present invention,
preferably a cancer associated with expression of HLA-G and
HLA-E.
[0066] Furthermore, the invention relates to a genetically modified
virus encoding a peptide comprising or consisting of a polypeptide
of the present invention for use as a medicament to expand and/or
activate NKG2C+NK cells in the treatment and/or prevention of a
medical condition associated with pathogenic cells expressing HLA-E
and a peptide of the present invention.
[0067] The preferred embodiments and associated advantages of the
peptide of the present invention for use as a medicament also
relate to the method and the cells of the present invention, and
the other way around.
BRIEF DESCRIPTION OF THE DRAWINGS
[0068] The invention is further described by the following figures.
These are not intended to limit the scope of the invention, but
represent preferred embodiments of aspects of the invention
provided for greater illustration of the invention described
herein.
Brief Description of the Figures:
[0069] FIG. 1. Sequence Variations in HCMV UL40-Encoded Peptides
Control the Activation of Adaptive NKG2C+NK Cells but Do Not
Differentially Affect Inhibition of NKG2C- NKG2A+NK Cells. (a-b)
PBMC of healthy HCMV- (n=20) and HCMV+ (n=40) individuals were
screened by flow cytometry. (a) Frequency of NKG2C+ cells within
the CD56dim population and (b) frequency of CD2+ Siglec-7- NKG2A-
FceR1g- cells within the CD56dim NKG2C+ population. CV, coefficient
of variation. Symbols indicate individual donors and lines median.
(c) Gating strategy for functional assays using HCMV+ donors with
adaptive NKG2C+NK cells. After culture of purified viable
CD3-CD56+NK cells with peptide-pulsed target cells, adaptive
NKG2C+NK cells were gated as viable single CD56dim NKG2A- CD57+
KIR+ NKG2C+ cells. Depending on the phenotype of the individual
donor, KIR were gated as KIR2DL1+, KIR2DL3+, or KIR3DL1+(d)
Purified NK cells from HCMV+ donors were used as effector cells in
cytotoxicity assays against labelled peptide-pulsed RMA-S/HLA-E and
% cytotoxicity was calculated as described in the Methods section.
Symbols and error bars indicate mean.+-.SEM (n=individual donors in
3 independent experiments). Two-way repeated-measure ANOVA with
Bonferroni correction between VMAPRTLIL (SEQ ID NO. 3) and
VMAPRTLFL (SEQ ID NO. 2). (e) RMA-S/HLA-E were pulsed with 300
.mu.M of the indicated peptides and geometric mean fluorescence
intensity (geoMFI) of HLA-E surface expression was detected (n=6
independent experiments). Horizontal lines depict median. Friedman
test with Dunn's post test. (f) Binding affinities were predicted
using the NetMHC4.0 algorithm. The HCMV pp65-derived
HLA-A2-resticted NLVPMVATV peptide serves as a non-HLA-E-binding
control. (g) RMA-S/HLA-E were pulsed with 300 .mu.M VMAPRTLIL (SEQ
ID NO. 3) or VMAPRTLFL (SEQ ID NO. 2) peptide followed by removal
of peptide and chase for 6 h. Decay in HLA-E surface expression was
calculated assuming first order kinetics (n=3 independent
experiments) and slopes compared using ANCOVA. (h) RMA-S/HLA-E were
pulsed with increasing concentrations of the indicated peptides and
geoMFI of HLA-E surface expression upon pulsing is displayed.
Symbols and error bars indicate mean.+-.SEM (n=6 independent
experiments). (i) Degranulation response of viable CD56dim NKG2C-
(triangles) or viable CD56dim NKG2A- CD57+ KIR+ NKG2C+NK cells
(circles) upon culture without or with VMAPRTLFL (SEQ ID NO.
2)-pulsed RMA-S/HLA-E. Connected symbols represent individual
donors (n=12 in 6 experiments). Two-tailed Wilcoxon test. (j)
Sorted viable CD56dim NKG2A- NKG2C+NK cells from HCMV+ donors were
treated with IgG1 isotype control or anti-CD94 blocking antibody
prior to culture without or with VMAPRTLFL (SEQ ID NO. 2)-pulsed
RMA-S/HLA-E. Summary of degranulation of viable CD56dim NKG2A-CD57+
NKG2C+NK cells is depicted. Connected symbols represent individual
donors (n=6 in 3 independent experiments). Two-tailed Wilcoxon
test. (k) Purified NK cells from HCMV+ donors were cultured with
K562/HLA-E pulsed with indicated peptides at indicated
concentrations. Summary of effector functions gated on viable
CD56dim NKG2A- CD57+ KIR+ NKG2C+NK cells (circles) or CD56dim
NKG2C- NKG2A+ cells (triangles). Symbols and error bars indicate
mean.+-.SEM (n=6 individual donors in 3 independent experiments).
Two-way repeated-measures ANOVA with Bonferroni correction between
VMAPRTLIL (SEQ ID NO. 3) and VMAPRTLFL (SEQ ID NO. 2). NS not
significant, *p<0.05, **p<0.01, ***p<0.005,
****p<0.0001.
[0070] FIG. 2. Sequence Variations in HCMV UL40-Encoded Peptides
Control the Activation of Adaptive NKG2C+NK Cells. (a-b) Integrated
analysis of 165 published and 52 newly determined HCMV UL40
sequences (VMAPRTLXL (SEQ ID NO: 1); VMAPRTLFL (SEQ ID NO: 2);
VMAPRTLIL (SEQ ID NO: 3); VMAPRTLLL (SEQ ID NO: 4); VMAPRTLVL (SEQ
ID NO: 5); and VMAPQSLLL (SEQ ID NO: 12). (a) Repertoire of peptide
sequences and (b) sequence logo (n=217 sequences). (c-d) Purified
NK cells from HCMV+ donors were cultured with RMA-S/HLA-E pulsed
with indicated peptides. (c) Representative FACS stainings and (d)
summary of effector functions gated on viable CD56dim NKG2A- CD57+
KIR+ NKG2C+NK cells (FIG. 1c for gating strategy). Symbols
represent individual donors (n=15 in 8 independent experiments) and
lines median. Friedman test with Dunn's post test. (e) Purified NK
cells from HCMV+ donors were cultured RMA-S/HLA-E pulsed with
increasing concentrations of the indicated peptides. Summary of
effector functions gated on viable CD56dim NKG2A-CD57+ NKG2C+NK
cells. Symbols and error bars indicate mean.+-.SEM (n=6 individual
donors in 3 independent experiments). Two-way repeated-measure
ANOVA with Bonferroni correction between VMAPRTLIL (SEQ ID NO. 3_)
and VMAPRTLFL (SEQ ID NO. 2). NS not significant, *p<0.05,
**p<0.01, ***p<0.001, ****p<0.0001.
[0071] FIG. 3. Co-Stimulation via LFA-3 Enhances Functional
Responses of Adaptive NKG2C+NK Cells. HCMV UL40 sequences:
VMAPRTLFL (SEQ ID NO: 2); VMAPRTLIL (SEQ ID NO: 3); VMAPRTLVL (SEQ
ID NO: 5); and VMAPQSLLL (SEQ ID NO: 12). (a) Purified NK cells
from HCMV+ donors were cultured with K562/HLA-E pulsed with
indicated peptides. Summary of effector functions gated on viable
CD56dim NKG2A-CD57+ KIR+ NKG2C+NK cells. Symbols indicate
individual donors (n=15 in 8 independent experiments) and lines
median. Friedman test with Dunn's post test. (b) K562/HLA-E were
examined for the expression of LFA-3 by flow cytometry.
Fluorescence minus one (FMO) control and stained condition gated on
viable cells. (c) Purified NK cells from HCMV+ donors were either
left untreated or treated with blocking anti-LFA-3 followed by
stimulation with VMAPRTLIL (SEQ ID NO. 3)-pulsed K562/HLA-E.
Effector functions gated on viable CD56dim NKG2A- CD57+ KIR+
NKG2C+NK cells. Connected symbols represent individual donors (n=9
in 5 independent experiments). Two-tailed Wilcoxon test.
*p<0.05, **p<0.01, ****p<0.0001.
[0072] FIG. 4. Co-Stimulatory Signals Are Required to Elicit
Polyfunctionality of Adaptive NKG2C+NK Cells Upon Engagement with
Sub-Optimal Peptides; HCMV UL40 sequences: VMAPRTLFL (SEQ ID NO:
2); and VMAPRTLIL (SEQ ID NO: 3). (a-d) Purified NK cells from
HCMV+ donors were cultured with peptide-pulsed RMA-S/HLA-E or
RMA-S/HLA-E/LFA-3. (a) Representative co-expression of CCL3 and TNF
as well as CD107a and IFN-.gamma. upon stimulation with VMAPRTLIL
(SEQ ID NO. 3)-pulsed RMA-S/HLA-E (left) or RMA-S/HLA-E/LFA-3
(right). Gated on viable CD56dim NKG2A-CD57+ KIR+ NKG2C+NK cells.
(b) SPICE charts depict pattern of 0 to 4 functions consisting of
combinatorial expression of CCL3, CD107a, IFN-.gamma., and TNF
gated on viable CD56dim NKG2A-CD57+ KIR+ NKG2C+NK cells. Pies and
arcs.
[0073] FIG. 5. Adaptive NKG2C+NK Cells Differentially Recognize
HCMV-Encoded Peptides during Infection. HCMV UL40 sequences:
VMAPRTLFL (SEQ ID NO: 2); VMAPRTLIL (SEQ ID NO: 3); and VMAPQSLLL
(SEQ ID NO: 12). (a) US2-6 genes were re-inserted into TB40 BAC4 to
generate repaired TB40 (TB40R) and nucleotide variations were
introduced within the UL40 sequence to encode the indicated
peptides. (b-c) HUVEC were either left uninfected or infected with
TB40R mutants. (b) Representative FACS staining of HCMV immediate
early antigen (HCMV-IE) in viable HUVEC 48 hours post infection and
(c) summary of infection rates. Symbols represent independent
experiments (n=10) and lines median. (d-e) Purified NK cells from
HCMV+ donors were overnight primed with IFN-.alpha., followed by
culture in medium or with virus-infected HUVEC. (d) Representative
FACS staining and (e) summary of effector functions gated on viable
CD56dim NKG2A-CD57+ KIR2DL1- KIR3DL1- KIR2DL3+ NKG2C+NK cells.
Connected symbols represent individual donors (n=12 in 3
independent experiments). Friedman test with Dunn's post test. NS
not significant, *p<0.05, ****p<0.0001.
[0074] FIG. 6. NKG2C- NK Cells Do Not Differentially Recognize
HCMV-Encoded Peptide Sequences During Infection. HCMV UL40
sequences: VMAPRTLFL (SEQ ID NO: 2); VMAPRTLIL (SEQ ID NO: 3); and
VMAPQSLLL (SEQ ID NO: 12). (a) HUVEC were infected with TB40R and
transcript levels of HCMV UL40 relative to human GAPDH were
determined by qPCR at indicated time points. Symbols indicate
independent experiments (n=4) and lines median. (b-c) HUVEC were
infected with TB40R mutants and analyzed by flow cytometry 48 h
post infection. (b) Representative FACS staining (left) of
uninfected and infected (HCMV-IE+) HUVEC compared to fluorescence
minus one (FMO) control and summary (right) of HLA class I
expression. Symbols indicate independent experiments (n=10) and
lines median. (c) Representative FACS staining (left) of uninfected
and infected (HCMV-IE+) HUVEC compared to FMO control and summary
(right) of HLA-E expression. Symbols indicate independent
experiments (n=9) and lines median. (d) Purified rested NK cells
from HCMV+ donors were cultured in medium or with virus-infected
HLA-C1 homozygous HUVEC for 6 h. Summary of effector functions
gated on viable CD56dim NKG2A-CD57+ KIR2DL1- KIR3DL1- KIR2DL3+
NKG2C+ adaptive NK cells. Connected symbols represent individual
donors (n=12 in 3 independent experiments). (e) Purified NK cells
from HCMV+ donors were primed with 25 ng/mL of IFN-.alpha. for 16 h
and subsequently cultured in medium or with virus-infected HLA-C1
homozygous HUVEC for 6 h. Summary of effector functions gated on
viable CD56dim KIR2DL1- KIR3DL1- KIR2DL3+ NKG2C- NK cells.
Connected symbols represent individual donors (n=12 in 3
independent experiments). Friedman test with Dunn's post test. NS
not significant, *p<0.05, **p<0.01, ***p<0.005,
****p<0.0001.
[0075] FIG. 7. Co-Stimulation via LFA-3 Enhances Proliferation of
NKG2C+NK Cells from HCMV- donors. HCMV UL40 sequences: VMAPRTLFL
(SEQ ID NO: 2); VMAPRTLIL (SEQ ID NO: 3); VMAPRTLVL (SEQ ID NO: 5);
and VMAPQSLLL (SEQ ID NO: 12). (a-b) Purified CD56dim NK cells from
HCMV- donors were cultured for 7 days with peptide-pulsed
RMA-S/HLA-E in the presence of IL-15. (a) Proliferation indices and
(b) replication indices of NKG2C+NK cells were normalized to
NKG2C-NK cells. Connected symbols represent individual donors (n=8
in 3 independent experiments). Friedman test with Dunn's post test.
(c) Purified CD56dim NK cells from HCMV- donors were cultured for 7
days with either RMA-S/HLA-E or RMA-S/HLA-E/LFA-3 in the presence
of IL-15. Proliferation and replication indices were normalized as
in (a). Connected symbols represent individual donors (n=8 in 3
independent experiments). Twotailed Wilcoxon test. (d-f) Purified
CD56dim NK cells from HCMV- donors were cultured with
peptide-pulsed RMA-S/HLA-E/LFA-3 in the presence of IL-15. (d)
Absolute numbers of NKG2C+NK cells per .mu.L of culture medium and
(e) precursor frequency of NKG2C+NK cells over time. Symbols
indicate individual donors (n=8) and lines median. Two-way
repeated-measures ANOVA with Bonferroni correction. (f) Frequency
of NKG2C+NK cells after 14 days of culture. Symbols indicate
individual donors (n=18) and lines median. Friedman test with
Dunn's post test. NS not significant, *p<0.05, **p<0.01,
***p<0.005, ****p<0.0001.
[0076] FIG. 8. Peptide Recognition Controls The Extent of
NKG2C+NK-Cell Proliferation in HCMV-Individuals. HCMV UL40
sequences: VMAPRTLFL (SEQ ID NO: 2); VMAPRTLIL (SEQ ID NO: 3);
VMAPRTLVL (SEQ ID NO: 5); and VMAPQSLLL (SEQ ID NO: 12). (a-e)
Purified CD56dim NK cells were cultured in the presence of IL-15
and peptide-pulsed RMA-S/HLA-E. (a) Representative CellTrace
dilution of viable NKG2C+ and NKG2C- NK cells from a HCMV- donor
after 7 days of culture. (b-c) CellTrace dilution patterns were
analyzed using FlowJo to obtain (b) proliferation index as well as
(c) replication index of NKG2C+ normalized to NKG2C- NK cells after
7 days of culture. Connected symbols represent individual donors
(n=12 in 5 independent experiments). Friedman test with Dunn's post
test. (d-e) Absolute NKG2C+NK-cell numbers were determined at (d)
day 7 and (e) day 14. Symbols represent individual donors (n=8 in 2
independent experiments) and lines median. Friedman test with
Dunn's post test. NS not significant, *p<0.05, ***p<0.001,
****p<0.0001.
[0077] FIG. 9. Peptide Recognition Controls Accumulation of
NKG2C+NK Cells from HCMV-Individuals in the Presence of
Pro-Inflammatory Signals. HCMV UL40 sequences: VMAPRTLFL (SEQ ID
NO: 2); VMAPRTLIL (SEQ ID NO: 3); VMAPRTLVL (SEQ ID NO: 5); and
VMAPQSLLL (SEQ ID NO: 12). (a-f) Purified CD56dim NK cells from
HCMV- donors were cultured with peptide-pulsed RMA-S/HLA-E/LFA-3 in
the presence of IL-15 combined with IL-12/IL-18 treatment during
the initial 20 h of culture. (a) Representative FACS stainings of
NKG2C on NK cells in the indicated conditions and (b) summary of
the frequencies of NKG2C+ cells within viable NK cells after 14
days of culture. Symbols represent individual donors (n=18 in 7
independent experiments) and lines median. Friedman test with
Dunn's post test. (c) Summary of absolute NKG2C+NK-cell numbers
determined at day 14. Symbols represent individual donors (n=8 in 2
independent experiments) and lines median. (d-e) Cultures were
monitored at indicated time points for (d) absolute NKG2C+NK-cell
numbers as well as (e) frequencies of NKG2C+ NK cells. Symbols
Represent individual donors (n=6 in 2 independent experiments) and
lines median. Repeated two-way ANOVA with Bonferroni correction.
(f) Modified Gett/Hodgkin model describing NKG2C+ NK-cell
proliferation and accumulation dynamics. Symbols and error bars
indicate mean.+-.SEM of experimentally obtained absolute NKG2C+
NK-cell counts as in (d), after normalization to day 1 values (set
as 1). Lines indicate best-fit curves of the model. Precursor
frequencies were experimentally obtained (FIG. 10a) while division
times and death rates (both mean.+-.SEM) were inferred as best-fit
parameters by non-linear optimization. NS not significant,
*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.
[0078] FIG. 10. Analysis of NKG2C+ NK-cell proliferation. HCMV UL40
sequences: VMAPRTLFL (SEQ ID NO: 2); and VMAPQSLLL (SEQ ID NO: 12).
(a) Purified CD56dim NK cells from HCMV-donors were cultured with
peptide-pulsed RMA-S/HLA-E/LFA-3 in the presence of IL-15 combined
with IL-12/18 treatment during the initial 20 h of culture.
Precursor frequency of NKG2C+ NK cells over time is displayed.
Symbols indicate individual donors (n=6) and lines median Two-way
repeated-measures ANOVA with Bonferroni correction. (b-f)
Mathematical analysis of NKG2C+NK-cell proliferation dynamics.
(b-c) Symbols and error bars indicate mean.+-.SEM of experimentally
obtained precursor frequencies of NKG2C+NK cells (b) with (data
from FIG. 10a) or (c) without (data from FIG. 7e) IL-12/18
treatment during the initial 20 h of culture. Lines indicate
best-fit gamma distributions, which are used as input for FIG. 9f
and FIG. 10d. (d) Modified Gett/Hodgkin model describing
NKG2C+NK-cell proliferation and accumulation dynamics in the
absence of IL-12/18 treatment. Symbols and error bars indicate
mean.+-.SEM of experimentally obtained absolute NKG2C+ NK-cell
counts after normalization to day 1 values (set as 1); lines
indicate best-fit curves of the model. Precursor frequencies were
experimentally obtained (FIG. 7e, FIG. 10c), while division times
and death rates (both mean.+-.SEM) were inferred as best-fit
parameters by non-linear optimization. (e-f) Modified Gett/Hodgkin
models with fixed input parameters in the presence (e) or absence
(f) of IL-12/18 treatment. Symbols and error bars indicate
mean.+-.SEM of experimentally obtained absolute counts after
normalization to day 1 values (set as 1); lines indicate curves of
the model. Precursor frequencies were experimentally obtained;
division time and death rate values were inferred by non-linear
optimization for the VMAPQSLLL (SEQ ID NO: 4) peptide (as in FIG.
9f and FIG. 10d, respectively) and set as fixed parameters for both
peptides. NS not significant, ***p<0.005, ****p<0.0001.
[0079] FIG. 11. FIG. 6: Peptide Recognition and Pro-Inflammatory
Cytokines Co-Operate in Guiding the Differentiation of Adaptive
NKG2C+NK Cells in vitro. HCMV UL40 sequences: VMAPRTLFL (SEQ ID NO:
2); and VMAPQSLLL (SEQ ID NO: 12). (a-d) Purified CD56dim NK cells
from HCMV- donors were cultured with peptide-pulsed
RMA-S/HLA-E/LFA-3 in the presence of IL-15 alone or combined with
IL-12/IL-18 treatment during the initial 20 h of culture. (a)
Representative FACS stainings of CD2, Siglec-7, KIR2DL1, and
KIR2DL3 gated on viable NKG2C+NK cells cultured in the indicated
conditions. (b) Summary of frequencies of CD2+, Siglec-7+, and
educating KIR+ cells within viable NKG2C+NK cells after 14 days of
culture. Connected symbols represent individual donors (n=8 for
educating KIR, n=10 for CD2 and Siglec-7 in 2-5 independent
experiments). Friedman test with Dunn's post test. (c-d) After 7
days of culture in the indicated conditions, viable NKG2C+NK cells
were sorted and probed for DNA methylation levels of the IFNG
conserved non-coding sequence (CNS) 1. (c) VISTA browser alignment
of mouse Ifng and human IFNG indicating conserved regions with
>70% sequence identity (light red) as well as UTR (light blue).
Arrow indicates transcription direction of human IFNG. (d) Average
percentage of methylation at 6 CpG within IFNG CNS1 were determined
using NGS. Connected symbols represent individual donors (n=6 in 3
independent experiments). Friedman test with Dunn's post test. (e)
Differential gene expression analysis of sorted viable CD56+
NKG2C+NK cells cultured in the presence of VMAPQSLLL (SEQ ID NO.
4)-pulsed targets (n=3 donors) or VMAPRTLFL (SEQ ID NO. 2)+IL-12/18
(n=5 donors) for 7 days. Selected genes highlighted in black are
differentially expressed with absolute fold changes >1.3 (dashed
vertical lines) and adjusted p values<0.05 (dashed horizontal
line). Dot sizes were adjusted to mean normalized counts. NS not
significant, *p<0.05, ***p<0.001, ****p<0.0001.
[0080] FIG. 12. Phenotypic Alterations of NKG2C+NK Cells. HCMV UL40
sequences: VMAPRTLFL (SEQ ID NO: 2); and VMAPQSLLL (SEQ ID NO: 12).
(a-b) Purified CD56dim NK cells from HCMV- donors were cultured for
14 days with peptide-pulsed RMA-S/HLA-E/LFA-3 in the presence of
IL-15 alone or in combination with IL-12/18. (a) Summaries of Syk,
CD161, FceR1g, CD7, NKG2A, and DNAM-1 expression on viable NKG2C+NK
cells. Connected symbols represent individual donors (n=6 for
FceR1.gamma.; n=8 for CD161, CD7, and DNAM-1; n=10 for NKG2A; n=12
for Syk in 2-5 independent experiments). Friedman test with Dunn's
post test. (b) Comparison of NKG2C- and NKG2C+NK cells after 14
days of culture with VMAPRTLFL (SEQ ID NO. 2)-pulsed
RMA-S/HLA-E/LFA-3 in the presence of IL-15 and IL-12/18. Connected
symbols represent individual donors (n=6 for FceR1.gamma.; n=8 for
educating KIR, CD161, CD7, and DNAM-1; n=10 for CD2, Siglec-7, and
NKG2A; n=12 for Syk in 2-5 independent experiments). Two-tailed
Wilcoxon test. NS not significant, *p<0.05, **p<0.01,
***p<0.005. (c-d) Gene expression analysis of sorted viable
CD56+ NKG2C+NK cells cultured in the presence of VMAPQSLLL (SEQ ID
NO. 4)-pulsed targets (n=3 donors) or VMAPRTLFL (SEQ ID NO.
2)+IL-12/18 (n=5 donors) for 7 days. Heatmaps of selected (c)
adaptive NKcell signature genes and (d) activation and exhaustion
markers based on zscores of rlog-transformed read counts clustered
by Pearson correlation and Ward minimum variance. Asterisk-marked
genes indicate adjusted p<0.05.
[0081] FIG. 13. Analysis of the Phenotype of Adaptive NKG2C+NK
cells Upon HCMV Infection in vivo. HCMV UL40 sequences: VMAPRTLFL
(SEQ ID NO: 2); and VMAPRTLIL (SEQ ID NO: 3). (a) Study design. (b)
Expression of CD2, Siglec-7, FceR1g, and NKG2A by NKG2C+ and NKG2C-
NK cells. Symbols represent individual patients (white circles,
HCMV- without reactivation, n=10; blue circles, HCMV reactivation
with VMAPRTLIL (SEQ ID NO. 3) peptide, n=10; red circles, HCMV
reactivation with VMAPRTLFL (SEQ ID NO. 2) peptide, n=2) and lines
depict median. (c) Frequency of NKG2C+ cells within the CD3-
CD56dim compartment over time. Black arrow heads indicate time
points of initial HCMV detection.
[0082] FIG. 14. Analysis of the Phenotype of Adaptive NKG2C+NK
cells Upon HCMV Infection in vivo. HCMV UL40 sequences: VMAPRTLFL
(SEQ ID NO: 2); and VMAPRTLIL (SEQ ID NO: 3). (a-d) Patients
undergoing alloHSCT were monitored for HCMV reactivation to
sequence the HCMV UL40-encoded peptide and determine the NK-cell
phenotype. (a) Representative FACS staining of (left) NKG2C gated
on viable CD14- CD19- CD3- CD56dim NK cells and of (right)
Siglec-7, NKG2A, CD2, and Fc.epsilon.R1.gamma. gated on CD56dim
NKG2C+NK cells. (b) Summary of the frequencies of NKG2C+ cells
within CD56dim NK cells as well as (c) summary of the frequencies
of CD2+ Siglec-7- NKG2A- Fc.epsilon.R1.gamma.- cells within NKG2C+
and NKG2C- NK cells. Symbols represent patients (white symbols,
HCMV-, n=10; blue symbols, HCMV reactivation with VMAPRTLIL (SEQ ID
NO. 3) peptide, n=10; red symbols, HCMV reactivation with VMAPRTLFL
(SEQ ID NO. 2) peptide, n=2) and lines median. (d) Frequencies of
CD2+ Siglec-7- NKG2A-Fc.epsilon.R1.gamma.- cells within NKG2C+(blue
circles, HCMV reactivation with VMAPRTLIL (SEQ ID NO. 3) peptide;
red circles, HCMV reactivation with VMAPRTLFL (SEQ ID NO. 2)
peptide) and NKG2C- (grey triangles) NK cells were monitored over
time. Black arrowheads indicate time point of initial HCMV
detection.
[0083] FIG. 15. NKG2C+NK cells preferentially recognize HLA-G/HLA-E
expressing tumor cells. CD56dim NK cells were co-cultured with
721.221 cells untransfected (221) or transfected with HLA-G (221 G)
in the presence or absence of a blocking antibody against LILRB1
(aLIR). (A) Quantification of degranulation as measured by CD107a
and production of IFN.gamma. and TNF by NKG2C+ NK cells (n=10). (B)
Direct comparison of degranulation and IFN.gamma. production
NKG2C+(2C+) and NKG2C- (2C-) NK cells, both pre-gated as LILRB1+ to
account for differences in expression of this receptor between the
two subsets. (C) Sorted NKG2C+NK cells were co-cultured with 221G
cells in the presence or absence of blocking antibodies against
LILRB1 (aLIR) and CD94 (aCD94) and their activation analyzed in
terms of degranulation and IFNg production (n=4). Multiple measures
ANOVA with Bonferroni post test (A-B) or Wilcoxon test (C). NS not
significant, *p<0.05, ***p<0.001, ****p<0.00011.
[0084] FIG. 16. NKG2C+NK cells preferentially recognize HLA-G/HLA-E
expressing tumor cells. 721.221 cells untransfected (221) or
transfected with HLA-G (221G) or a fusion protein driving
expression of HLA-E (221.AEH) were stained for HLA-E and HLA-G and
analyzed by flow cytometry.
DETAILED DESCRIPTION OF THE INVENTION
[0085] All cited documents of the patent and non-patent literature
are hereby incorporated by reference in their entirety.
[0086] The present invention relates to an isolated peptide for use
as a medicament, wherein said peptide has 9 to 30 amino acids and
comprises or consists of an amino acid sequence according to SEQ ID
NO 1 (VMAPRTLXL), wherein X is an amino acid with a hydrophobic
side chain (A, I, L, F, V, P, G), preferably V, L, I or F.
[0087] Amino acid sequences of preferred polypeptides of the
present invention are listed under Table 1.
TABLE-US-00001 TABLE 1 Amino acid sequences of preferred peptides
of the invention. SEQ ID NO 1 VMAPRTLXL Therein, X may be an amino
acid with a hydrophobic side chain (A, I, L, F, V, P, G),
preferably V, L, I or F. SEQ ID NO 2 VMAPRTLFL SEQ ID NO 3
VMAPRTLIL SEQ ID NO 4 VMAPRTLLL SEQ ID NO 5 VMAPRTLVL
[0088] In one embodiment the invention therefore encompasses a
polypeptide as described herein comprising or consisting of an
amino acid sequence selected from the group consisting of: a) an
amino acid sequence comprising or consisting of an amino acid
sequence according to SEQ ID NO 1-5; wherein the polypeptide is
preferably no longer than 100, 90, 80, 70, 60, 50 or 40, preferably
30, more preferably 20, most preferably no longer than 10 or 9
amino acids; b) an amino acid sequence comprising or consisting of
an amino acid sequence according to SEQ ID NO 1-5, wherein the
length of the amino acid molecule is between 5 and 300 amino acids,
6 and 200 amino acids, 7 and 100, 8 and 50, preferably between 9
and 30 amino acids, wherein the surrounding sequences are
preferably provided as UL-40 sequences flanking the amino acid
sequences according to SEQ ID NO 1-5, or as sequences from MHC
class I molecules, preferably non-classical MHC class I molecules,
most preferably HLA-G, preferably flanking the signal peptide (also
called leader sequence or leader peptide or signal sequence) of the
respective MHC class I molecule.
[0089] c) an amino acid sequence having sufficient sequence
identity to be functionally analogous/equivalent to an amino acid
sequence according to a), comprising preferably a sequence identity
to an amino acid sequence according to a) of at least 70%, 80%,
preferably 90%, more preferably 95%; and
[0090] d) an amino acid sequence of a), b) or c) which is modified
by deletions, additions, substitutions, translocations, inversions
and/or insertions and functionally analogous/equivalent to an amino
acid sequence according to a), b) or c).
[0091] Functionally analogous sequences refer preferably to the
ability to induce to induce expansion and/or activation of NKG2C+
natural killer (NK) cells.
[0092] Embodiments of the invention may comprise a polypeptide as
described herein comprising or consisting of an amino acid sequence
SEQ ID NO 1-5, or variants of these sequences, wherein the sequence
variant may comprise a sequence identity to SEQ ID NO 1-5 of 50,
55, 60, 65, 70, 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98 or 99%. Sequence identity may be
determined using methods known to one skilled in the art, such as
BLAST or other sequence alignment tools.
[0093] In further preferred embodiments, the invention relates to a
polypeptide comprising or consisting of an amino acid sequence
derived from the UL-40 protein of HCMV. In further embodiments, the
invention relates to a polypeptide comprising or consisting of an
amino acid sequence derived from the signal sequence of a MHC class
I molecule, preferably a non-classical MHC class I molecules, most
preferably HLA-G.
[0094] Preferably, the amino acid sequence of the peptide has a
length of at least 7 amino acids, more preferably 8 amino acids,
most preferably 9 amino acids. Sequence homology refers to a
sequence identity of more than 65%, preferably more than 70%.
[0095] Protein modifications to the polypeptides of the present
invention, which may occur through substitutions in amino acid
sequence, and nucleic acid sequences encoding such molecules, are
also included within the scope of the invention. Substitutions as
defined herein are modifications made to the amino acid sequence of
the protein, whereby one or more amino acids are replaced with the
same number of (different) amino acids, producing a protein which
contains a different amino acid sequence than the primary protein.
In some embodiments this amendment will not significantly alter the
function of the protein. Like additions, substitutions may be
natural or artificial. It is well known in the art that amino acid
substitutions may be made without significantly altering the
protein's function. This is particularly true when the modification
relates to a "conservative" amino acid substitution, which is the
substitution of one amino acid for another of similar properties.
Such "conserved" amino acids can be natural or synthetic amino
acids which because of size, charge, polarity and conformation can
be substituted without significantly affecting the structure and
function of the protein. Frequently, many amino acids may be
substituted by conservative amino acids without deleteriously
affecting the protein's function. In general, the non-polar amino
acids Gly, Ala, Val, lie and Leu; the non-polar aromatic amino
acids Phe, Trp and Tyr; the neutral polar amino acids Ser, Thr,
Cys, Gin, Asn and Met; the positively charged amino acids Lys, Arg
and His; the negatively charged amino acids Asp and Glu, represent
groups of conservative amino acids. This list is not exhaustive.
For example, it is well known that Ala, Gly, Ser and sometimes Cys
can substitute for each other even though they belong to different
groups.
[0096] In embodiments of the invention, the peptide is used as a
medicament to expand and/or activate NKG2C+ natural killer (NK)
cells in the treatment and/or prevention of a medical condition
associated with pathogenic cells expressing HLA-E and a peptide
comprising an amino acid sequence according to SEQ ID NO 1 or
2.
[0097] Natural killer cells (NK cells) are cytotoxic lymphocytes of
the innate immune system. NK cells provide rapid responses to
viral-infected cells, acting at around 3 days after infection, and
respond to tumor formation. Typically, immune cells detect major
histocompatibility complex (MHC) presented on infected cell
surfaces, triggering cytokine release, causing lysis or apoptosis.
NK cells are unique, however, as they have the ability to recognize
stressed cells in the absence of antibodies and MHC, allowing for a
much faster immune reaction. They were named "natural killers"
because of the initial notion that they do not require activation
to kill cells that are missing "self" markers of MHC class 1. This
role is especially important because harmful cells that are missing
MHC I markers cannot be detected and destroyed by other immune
cells, such as T lymphocyte cells.
[0098] CD94/NKG2 is a family of C-type lectin receptors which are
expressed predominantly on the surface of NK cells. These receptors
stimulate or inhibit cytotoxic activity of NK cells, therefore they
are divided into activating and inhibitory receptors according to
their function. CD94/NKG2 recognize non-classical MHC glycoproteins
class I. CD94/NKG2 family includes seven members: NKG2A, B, C, D,
E, F and H. NKG2 receptors are transmembrane proteins type II which
dimerize with CD94 molecule. CD94 contains a short cytoplasmic
domain and it is responsible for signal transduction. Therefore
NKG2 receptors form disulfide bonded heterodimers with CD94. NKG2D
represent an exception, since it predominantly forms a
homodimer.
[0099] NKG2A and NKG2B receptors transmit inhibitory signal. They
contain two immuno-receptor tyrosine-based inhibitory motives
(ITIM) in their cytoplasmic tail, which transduces the signal upon
engagement of a ligand through Src family kinases, and the tyrosine
phosphatase SHP-1, SHP-2 or SHIP. As a result, NK cell activation
is suppressed.
[0100] NKG2C, NKG2E and NKG2H are activating receptors. Ligand
binding enables interaction between receptor and the ITAM-bearing
adaptor protein DAP12. Subsequent signaling through Src family
kinases, the tyrosine kinases Syk and ZAP-70 can lead to release
cytotolytic granules containing perforin and granzyme and
production of many cytokines and chemokines. NKG2D is activating
receptor as well but it couples with adaptor protein DAP10 and
triggers actin reorganization (cell polarization) and degranulation
upon ligand engagement. The function of NKG2F receptor is not
clear.
[0101] Receptors of CD94/NKG2 family bind non-classical MHC
glycoproteins class I. Non-classical MHC glycoproteins class I are
structurally similar to classical MHC class I molecules, but they
present mainly peptides derived from the signal peptides of MHC
class I. Therefore NK cells can indirectly monitor the expression
of classical MHC class I molecules through the interaction of
CD94/NKG2 with HLA-E.
[0102] Non-classical MHC class I molecules comprise HLA-G, HLA-E
and HLE-F. For HLA-G, 7 protein isoforms have been described. Four
of these isoforms are membrane-bound (HLA-G 1-4) while 3 of them
lack exons 5-7, hence and existing as secreted forms (HLA-G 5-7).
Of all membrane-bound HLA-G variants, HLA-G1 represents the sole
full-length version of the molecule. Conversely, HLA-G2 does not
contain exon 3, HLA-G3 is missing exons 3 and 4, and HLA-G4 does
not include exon 4. The soluble isoforms of HLA-G (namely, HLA-G5,
HLA-G6, and HLA-G7) contain part of intron 4, harboring a stop
codon. This results in the expression of truncated proteins lacking
exon 5, which encodes the transmembrane domain. HLA-G5, -G6, and
-G7 represent the soluble counterparts of HLA-G1, G2, and-G3,
respectively. HLA-E consists of 8 exons, wherein the first encodes
the leader peptide sequence, exons 2, 3 and 4 encode the MHC
immunoglobulin-like a domains 1, 2, and 3, respectively, exon 5
encodes the transmembrane domain and exons 6 and 7 encode the
cytoplasmic tail. Similar to HLA-G, HLA-E forms a complex with
.beta.2 microglobulin. HLA-E consists of 8 exons. Of these, the
first encodes the leader peptide sequence, exons 2, 3 and 4 encode
the MHC immunoglobulin-like a domains 1, 2, and 3, respectively,
exon 5 encodes the transmembrane domain and exons 6 and 7 encode
the cytoplasmic tail. Similar to HLA-G, HLA-E forms a complex with
.beta.2 microglobulin.
[0103] HLA-G, -E, and -F are important regulators of the immune
system and the upregulation of HLA-G, -E, and -F following
IFN.gamma. stimulation suggests that non-classical MHC class I
molecules may be involved in negative feedback responses to
potentially harmful pro-inflammatory responses. While inflammatory
responses are required to eliminate cancer cells, they also trigger
strong immuno-regulatory mechanisms that limit the recognition of
malignant cells by the immune system, hence favoring tumor
progression. Non-classical MHC class I molecules constitute means
whereby malignant cells escape immuno-surveillance. Indeed, these
molecules inhibit the activity of the immune system by binding to
inhibitory receptors expressed by effector cells, hence suppressing
their functions or inducing their apoptotic demise (Kochan et al.
Oncoimmunology. 2013 Nov. 1; 2(11): e26491; Smyth et al.
Oncoimmunology. 2013 Mar. 1; 2(3): e23336). HLA-E has a very
specialized role in cell recognition by NK cells by binding a
restricted subset of peptides derived from signal peptides of
classical and non-classical MHC class I molecules, such as HLA-A,
B, C, G. These peptides are released from the membrane of the
endoplasmic reticulum (ER) by the signal peptide peptidase, trimmed
by the cytosolic proteasome, transport into the ER lumen by the
transporter associated with antigen processing (TAP) and
subsequently bound to the groove on the HLA-E molecule. This allows
HLA-E to assemble correctly and to be stabilized, leading to
expression on the cell surface. NK cells recognize the complex
formed by HLA-E+peptide using the heterodimeric inhibitory
receptors CD94/NKG2A, B and/or C. When CD94/NKG2A or CD94/NKG2B is
engaged, it produces an inhibitory effect on the cytotoxic activity
of the NK cell to prevent cell lysis, whereas binding of HLA-E to
CD94/NKG2C results in NK cell activation. This interaction has been
shown to trigger expansion of NK cell subsets in antiviral
responses.
[0104] HLA-G may play a role in immune tolerance in pregnancy,
being expressed in the placenta by extravillous trophoblast cells
(EVT), while the classical MHC class I genes (HLA-A and HLA-B) are
not expressed. HLA-G is a ligand for NK cell inhibitory receptor
KIR2DL4, and therefore expression of this HLA by the trophoblast
defends it against NK cell-mediated death. Aberrant induction of
HLA-G expression has been observed in various malignancies and
strongly associated with tumor immune escape, metastasis and poor
prognosis. HLA-G, membrane-bound or soluble, strongly binds its
inhibitory receptors on immune cells (NK, T, B, monocytes/dendritic
cells), inhibits the functions of these effectors, and so induces
immune inhibition. HLA-G function may therefore be beneficial and
protective when expressed by a fetus or a transplant, but
deleterious when expressed by a tumor or cancer cells, because it
also protects malignant cells from antitumor immunity. Accordingly,
HLA-G can be classified as an checkpoint molecule.
[0105] The terms signal peptide, leader peptide, leader sequence
and signal sequence are used interchangeably in the context of the
present invention and refer to is a short peptide of up to around
30 amino acids length present at the N-terminus of the majority of
newly synthesized proteins that are destined towards the secretory
pathway. These proteins include those that reside either inside
certain organelles (the endoplasmic reticulum, golgi or endosomes),
secreted from the cell, or inserted into most cellular membranes.
In particular, the terms may be used herein when referring to the
signal peptides comprised by classical and non-classical MHC class
I molecules.
[0106] As used herein, expansion and/or activation of NKG2C+NK
cells refers to the stimulation of NKG2C+NK cells with an
activating signal leading to the execution of effector functions,
such as release of cytotoxic granules and production of
pro-inflammatory cytokines and chemokines, and/or the induction of
survival and/or proliferation of the cells.
[0107] The term "medical conditions associated with pathogenic
cells expressing HLA-E and a peptide comprising an amino acid
sequence according to SEQ ID NO 1 or 2" relates to several
pathologies that share the common feature of the presence of
pathological cells expressing HLA-E as well as a peptide of the
present invention, wherein these cells are involved in the
pathological mechanisms.
[0108] Such pathologies include, for example, the situation of
active HCMV infection, which may be a new infection or a
reactivation of a latent infection, wherein the UL-40 protein of
HCMV is expressed in a host cell that expresses HLA-E. Furthermore,
such pathologies include certain cancers, wherein the cancer cells
express HLA-G. The signal sequence of HLA-G comprises an amino acid
sequence corresponding to the peptide of the present invention, and
the signal sequence gets processed inside the cancer as described
above. Cancers expressing both, HLA-E and HLA-G, have been
described in the art for melanoma, choriocarcinoma, breast cancer,
endometrial cancer, ovarian cancer, cervical cancer, esophageal
squamous cell carcinoma, colorectal cancer, gastric cancer,
hepatocellular carcinoma, glioblastoma, lung cancer, nasopharyngeal
carcinoma, pancreatic adenocarcinoma, thyroid carcinoma and renal
carcinoma (Curigliano G, Criscitiello C, Gelao L, Goldhirsch A.
Molecular pathways: human leukocyte antigen G (HLA-G). Clin Cancer
Res. 2013; 19(20):5564-71; Lin A, Yan W H. HLA-G expression in
cancers: roles in immune evasion, metastasis and target for
therapy. Mol Med. 2015; Seliger B, Schlaf G. Structure, expression
and function of HLA-G in renal cell carcinoma. Semin Cancer Biol.
2007; 17(6):444-50), and methods and techniques for determining the
expression of HLA-E and HLA-G in a pathological cell are known to
the skilled person and have are described in the example below.
[0109] Medical conditions and cancers associated with pathogenic
cells expressing HLA-E and a peptide comprising an amino acid
sequence according to SEQ ID NO 1 or 2, that are treatable by the
effector function of said NKG2C+NK cells, and/or that are
susceptible to NKG2C+NK cell cytotoxic activity comprise, without
limitation, melanoma, choriocarcinoma, breast cancer, endometrial
cancer, ovarian cancer, cervical cancer, esophageal squamous cell
carcinoma, colorectal cancer, gastric cancer, hepatocellular
carcinoma, glioblastoma, lung cancer, nasopharyngeal carcinoma,
pancreatic adenocarcinoma, thyroid carcinoma and renal carcinoma,
and in particular cancer types previously described to be
susceptible to immunotherapy, such as melanoma, renal cell
carcinoma and hematological malignancies.
[0110] The invention is based on the surprising finding that the
peptides of the present invention can induce expansion and
activation of NKG2C+NK cells in vivo an in vitro. Accordingly, the
peptides can be used as a medicament in the treatment of medical
condition treatable by the effector function of said NKG2C+NK
cells. The receptor complex of CD94/NKG2C is an activating receptor
of NKG2C+NK cells and accordingly, the cells are useful in the
treatment of diseases that are associated with pathological cells
expressing the ligand of this receptor on their surface, such as
the complex of HLA-E with the bound peptide of the present
invention.
[0111] In addition to cancer cells, pathogenic cells expressing
HLA-E and peptides resembling SEQ ID NO 1, could plausibly
represent immune effector cells exacerbating pathology during
inflammatory disorders, such as but not limited to rheumatic
diseases and other autoimmune conditions.
[0112] In embodiments of the present invention, the peptide is used
as a medicament to inhibit reactivation of human cytomegalovirus
(HCMV) infections and/or reduce viral titers in an individual
infected with HCMV.
[0113] Human cytomegalovirus (HCMV) is a species of the
Cytomegalovirus genus of viruses, which in turn is a member of the
viral family known as Herpesviridae or herpesviruses. It is
typically abbreviated as HCMV or, commonly but more ambiguously, as
CMV. It is also known as human herpesvirus-5 (HHV-5). HCMV
infection is typically unnoticed in healthy people, but can be
life-threatening for the immune-compromised, such as HIV-infected
persons, organ transplant recipients, or newborn infants.
Congenital cytomegalovirus infection can lead to significant
morbidity and even death. After infection, HCMV remains latent
within the body throughout life and can be reactivated at any time.
Eventually, it may cause mucoepidermoid carcinoma and possibly
other malignancies such as prostate cancer.
[0114] UL-40 is protein of 221 amino acids of HCMV, which plays a
role in viral immune evasion. Human CMV has evolved multiple
strategies to interfere with immune recognition of the host. A
variety of mechanisms target Ag presentation by MHC class I
molecules resulting in a reduced class I cell-surface expression.
This down-regulation of class I molecules can trigger NK
cytotoxicity, which would have to be counteracted by the virus to
establish long-term infection. The UL-40 protein of HCMV, which is
encoded by the open reading frame UL-40, encodes a canonical ligand
for HLA-E, and expression of UL-40 in HLA-E-positive target cells
is thought to induce resistance to NK cell lysis via the CD94/NKG2A
receptor. However, as disclosed herein, UL-40 can comprise the
peptides of the present invention and therefore HCMV infected cells
may express the complex of HLA-E and the peptide of the present
invention on their surface. Accordingly, such cells can be
recognized by CD94/NKG2C, which leads to activation of NKG2C+NK
cells.
[0115] Accordingly, the present invention can be used to inhibit
reactivation of human cytomegalovirus (HCMV) infections and/or
reduce viral titers in an individual infected with HCMV. By means
of the present invention, the number of NKG2C+NK cells in a host
can be increased, either by administration of the peptide of the
present invention or by administering in vitro expanded NKG2C+ NK
cells of the present invention. The invention can be applied to
patients that are newly infected with HCMV and suffer from an
active infection to reduce the viral load and to stop or
accelerated the containment of the active infection. Furthermore,
the invention can be applied in the context of a reactivation of
HCMV infection in a latently infected individual. Additionally, the
invention can be applied to prevent clinical reactivation in
individuals at risk, for example because they are latently infected
or receive a transplant from a latently infected donor.
[0116] In further embodiments, the invention relates to the
treatment of cancer associated with elevated expression of HLA-G
compared to non-cancerous cells, preferably with elevated
expression of HLA-G and HLA-E compared to non-cancerous cells. The
expression of HLA-G and HLA-E can be determined by well-known
techniques, such as the nucleic acid and protein detection
techniques based on qPCR and flow cytometry, as described in the
examples below. Accordingly, it is possible to determine the
expression level of HLA-G and/or HLA-E in a sample comprising
pathological cells from a patient to a corresponding sample from a
healthy individual or to reference values generated from
corresponding samples. Such a comparison represents a routine
analysis for a person skilled in the art. By means of such a
comparison, it is possible to identify cancers that are susceptible
to NKG2C+NK cell cytotoxic activity.
[0117] Medical conditions associated with pathogenic cells
expressing HLA-E and a peptide comprising an amino acid sequence
according to SEQ ID NO 1 or 2 can be identified by a skilled person
by standard laboratory methods. For example, HLA-E expression on
the pathogenic cells can by analyzed by flow cytometry using HLA-E
specific antibodies. The additional presence of peptides according
to SEQ ID NO1 or 2 can be determined for example by mass
spectrometry or antibody mediated techniques. Furthermore, the
presence of proteins that lead to the generation of these peptides,
such as HLA-G or UL-40 of HCMV, can be determined by antibody based
techniques such as ELISA or flow cytometry or even by RT-PCR
detection expression of proteins encoding such leader peptides.
[0118] In the context of the present invention, the term "treatment
of a tumor" relates to the treatment of all kinds of cancer,
independent of whether the cancer is associated with the formation
of a solid tumor or whether the cancer cells do not form a solid
tumor, as it is the case for certain leukemias.
[0119] Cancer comprises a group of diseases that can affect any
part of the body and is caused by abnormal cell growth and
proliferation. These proliferating cells have the potential to
invade the surrounding tissue and/or to spread to other parts of
the body where they form metastasis. Worldwide, there were 14
million new cases of cancer and 8.2 million cancer related deaths
in 2012 (World Cancer Report 2014). The majority of cancers is
caused by environmental signals involving tobacco use, obesity and
infections among others, while around 5-10% are genetic cases.
Cancers can be classified into subcategories based on the cell of
origin. The most common subcategories are carcinomas from
epithelial cells, sarcomas from connective tissue and lymphomas and
leukemias from hematopoietic cells. Cancer is associated with a
high variety of local and systemic symptoms and cannot be cured in
many cases. In light of the high number of new cancer patients and
cancer related deaths novel treatment strategies are required.
Cancer according to the present invention refers to all types of
cancer or neoplasm or malignant tumors found in mammals, including
leukemias, sarcomas, melanomas and carcinomas. Either solid tumors
and/or liquid tumors (such as leukemia or lymphoma) may be
treated.
[0120] Leukemias include, but are not limited to acute
nonlymphocytic leukemia, chronic lymphocytic leukemia, acute
granulocytic leukemia, chronic granulocytic leukemia, acute
promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia,
a leukocythemic leukemia, basophylic leukemia, blast cell leukemia,
bovine leukemia, chronic myelocytic leukemia, leukemia cutis,
embryonal leukemia, eosinophilic leukemia, Gross' leukemia,
hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic
leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic
leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic
leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid
leukemia, lymphosarcoma cell leukemia, mast cell leukemia,
megakaryocytic leukemia, micromyeloblastic leukemia, monocytic
leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid
granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia,
plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia,
Rieder cell leukemia, Schilling's leukemia, stem cell leukemia,
subleukemic leukemia, and undifferentiated cell leukemia.
[0121] Sarcomas include, but are not limited to a chondrosarcoma,
fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma,
osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma,
alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma,
chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor
sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma,
fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma,
granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple
pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells,
lymphoma, immunoblastic sarcoma of T-cells, Jensen's sarcoma,
Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma,
malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic
sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and
telangiectaltic sarcoma.
[0122] Melanomas include, but are not limited to include, for
example, acral-lentiginous melanoma, amelanotic melanoma, benign
juvenile melanoma, Cloudman's melanoma, S91 melanoma,
Harding-Passey melanoma, juvenile melanoma, lentigo maligna
melanoma, malignant melanoma, nodular melanoma, subungal melanoma,
and superficial spreading melanoma. Carcinomas include, but are not
limited to acinar carcinoma, acinous carcinoma, adenocystic
carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum,
carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell
carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid
carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma,
bronchiolar carcinoma, bronchogenic carcinoma, cerebriform
carcinoma, cholangiocellular carcinoma, chorionic carcinoma,
colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform
carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical
carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma
durum, embryonal carcinoma, encephaloid carcinoma, epiermoid
carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma,
carcinoma exulcere, carcinoma fibrosum, gelatiniform carcinoma,
gelatinous carcinoma, giant cell carcinoma, carcinoma
gigantocellulare, glandular carcinoma, granulosa cell carcinoma,
hair-matrix carcinoma, hematoid carcinoma, hepatocellular
carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypernephroid
carcinoma, infantile embryonal carcinoma, carcinoma in situ,
intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's
carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma,
lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma,
lymphoepithelial carcinoma, carcinoma medullare, medullary
carcinoma, melanotic carcinoma, carcinoma molle, mucinous
carcinoma, carcinoma muciparum, carcinoma mucocellulare,
mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma,
carcinoma myxomatodes, nasopharyngeal carcinoma, oat cell
carcinoma, carcinoma ossificans, osteoid carcinoma, papillary
carcinoma, periportal carcinoma, preinvasive carcinoma, prickle
cell carcinoma, pultaceous carcinoma, renal cell carcinoma of
kidney, reserve cell carcinoma, carcinoma sarcomatodes,
schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti,
signet-ring cell carcinoma, carcinoma simplex, small-cell
carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle
cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous
cell carcinoma, string carcinoma, carcinoma telangiectaticurn,
carcinoma telangiectodes, transitional cell carcinoma, carcinoma
tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma
villosum.
[0123] Additional cancers include, but are not limited to Hodgkin's
Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma,
breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma,
primary thrombocytosis, primary macroglobulinemia, small-cell lung
tumors, primary brain tumors, stomach cancer, colon cancer,
malignant pancreatic insulanoma, malignant carcinoid, urinary
bladder cancer, premalignant skin lesions, testicular cancer,
lymphomas, thyroid cancer, esophageal cancer, genitourinary tract
cancer, malignant hypercalcemia, cervical cancer, endometrial
cancer, adrenal cortical cancer, and prostate cancer.
[0124] In some embodiments, "tumor" shall include, without
limitation, a prostate tumor, a pancreatic tumor, a squamous cell
carcinoma, a breast tumor, a melanoma, a basal cell carcinoma, a
hepatocellular carcinoma, a choloangiocellular carcinoma,
testicular cancer, a neuroblastoma, a glioma or a malignant
astrocytic tumor such as glioblastma multiforme, a colorectal
tumor, an endometrial carcinoma, a lung carcinoma, an ovarian
tumor, a cervical tumor, an osteosarcoma, a rhabdo/leiomyosarcoma,
a synovial sarcoma, an angiosarcoma, an Ewing sarcoma/PNET and a
malignant lymphoma. These include primary tumors as well as
metastatic tumors (both vascularized and non-vascularized).
[0125] In embodiment of the invention the peptide is administered
in combination with an adjuvant. Preferably, the adjuvant enhances
the production of pro-inflammatory cytokines. In embodiments, the
peptide of the invention is administered in combination with the
peptide is administered in combination with pro-inflammatory
cytokines.
[0126] As used herein, the term "adjuvant" relates to a compound or
composition that is administered in combination with the peptide of
the present invention, to enhance the effectiveness of the peptide.
In general, an adjuvant is an agent that is given in addition to
the primary or initial therapy to maximize its effectiveness. In
the context of the present invention, the adjuvant is to be
understood as an immunologic adjuvant. Adjuvants in immunology are
often used to modify or augment the effects of a compound that
modifies the immune system, such as the peptide of the present
invention or a vaccine. In embodiments, the peptide of the
invention may be regarded as a peptide vaccine. An immunological
adjuvant stimulates the immune system to respond more vigorously to
an immunological treatment. As a consequence, the combined
treatment with an adjuvant provides increased immunity to a
particular disease. It is believed that adjuvants accomplish this
task by mimicking specific sets of evolutionarily conserved
molecules, so called PAMPs, which include liposomes,
lipopolysaccharide (LPS), molecular cages for antigen, components
of bacterial cell walls, and endocytosed nucleic acids such as
double-stranded RNA (dsRNA), single-stranded DNA (ssDNA), and
unmethylated CpG dinucleotide-containing DNA. Because immune
systems have evolved to recognize these specific antigenic
moieties, the presence of an adjuvant can greatly increase the
innate immune response to the antigen by augmenting the activities
of dendritic cells (DCs), lymphocytes, and macrophages by mimicking
a natural infection. Furthermore, the use of such adjuvants that
are mimicking PAMPs leads to the production of pro-inflammatory
cytokines.
[0127] Immunological adjuvants for use in the context of the
present invention comprise, without limitation, inorganic
adjuvants, such as aluminium salts (aluminium phosphate and
aluminium hydroxide), squalene, AS02, AS03, AS04, oil-based
adjuvants (emulsions), MF59, QS21, cytokines, virosomes, pathogen
components, such as monophosphryl lipid A, Poly(IC:C) and CpG DNA
adjuvants.
[0128] As known in the art, a pro-inflammatory cytokine or an
immune response-stimulating cytokine is to be understood as a
cytokine that leads to or produces either directly or indirectly
the induction, activation and/or enhancement of an immune response,
preferably directed against an antigen, for example a tumor
antigen.
[0129] Cytokines are a diverse group of non-antibody proteins that
act as mediators between cells. Cytokines are currently being
clinically used as biological response modifiers for the treatment
of various disorders. The term cytokine is a general term used to
describe a large group of proteins. Particular kinds of cytokines
may include Monokines, namely cytokines produced by mononuclear
phagocytic cells, Lymphokines, namely cytokines produced by
activated lymphocytes, especially Th cells, Interleukins, namely
cytokines that act as mediators between leukocytes and Chemokines,
namely small cytokines primarily responsible for leucocyte
migration. Cytokine signaling is flexible and can induce both
protective and damaging responses. They can produce cascades, or
enhance or suppress production of other cytokines. Despite the
various roles of cytokines, a skilled person is aware of which
cytokines may be considered as immune response stimulating and
therefore applied in the treatment of a tumor disease as described
herein. Cytokines have the ability to modulate immune responses and
are often utilized by a tumor to allow it to grow and manipulate
the immune response. These immune-modulating effects allow them to
be used as drugs to provoke an immune response against the tumor.
Chemokines refer to a sub-group of cytokines (signaling proteins)
secreted by cells. Chemokines have the ability to induce directed
chemotaxis in nearby responsive cells; they are chemotactic
cytokines. Immune-response stimulatory or immune
response-modulatory cytokines and chemokines comprise, without
limitation, type 1 interferons (IFN alpha and IFN beta), type 2
(IFN gamma), type III interferons (IFN lambda), IFN gamma,
TNF-alpha, IL-1, IL-2, IL-12, IL-18, IL-23, IL-15 and IL-21,
CXCL-8, CCL2, CCL3, CCL4, CCL5, CCL11 and CXCL10, CXCL1, CXCL2,
CCL2, CCL1, CCL22, CCL17, CXCL13, CX3CL1, SDF-1, CXCL12, CCL23,
MIP-3, MPIF-1, CCL19, MIP-3-beta and MIP-1.beta..
[0130] The terms "stimulation" and "activation" of the "immune
system" or of an "immune response" may be used interchangeably.
[0131] In preferred embodiments of the invention, the peptide is
administered in combination with a check point inhibitor,
preferably an inhibitor of a receptor selected from the group
comprising LILRB1, inhibitory KIRs, NKG2A, PD-1, CTLA-4, TIM-3,
TIGIT and LAG-3.
[0132] Immune checkpoint molecules are molecules in the immune
system that either turn up a signal (co-stimulatory molecules) or
turn down a signal provided to immune effector cells. Thus, immune
checkpoint molecules can be subdivided into co-stimulatory
checkpoint molecules or co-inhibitory checkpoint molecules.
Co-stimulatory checkpoint molecules include co-stimulatory
lymphocyte receptors, which are lymphocyte surface-receptors that
can lead to an activation or stimulation of lymphocyte effector
functions. Co-inhibitory checkpoint molecules include co-inhibitory
lymphocyte receptors, which are lymphocyte surface-receptors that
can lead to an inhibition of lymphocyte effector functions.
[0133] An inhibitor of a receptor prevents the generation of a
signal by the respective receptor. Accordingly, an inhibitor of a
co-inhibitory lymphocyte receptor is a molecule that prevents the
activation of the respective receptor and thereby prevents the
generation of an inhibitory signal. Conversely, an activator of a
receptor induces the generation of a signal by the respective
receptor and an activator of a co-stimulatory lymphocyte receptor
leads to the generation of a stimulatory signal. Checkpoint
modulators are molecules that interfere with the activity of immune
checkpoint molecules, either by stimulating or inhibiting the
activity of immune checkpoint molecules.
[0134] Lymphocyte-stimulating checkpoint modulators are molecules
that lead to an activation of lymphocytes, preferably effector T
cells, either through activation of a co-stimulatory checkpoint
molecule, or through inhibition of a co-inhibitory checkpoint
molecules. Checkpoint modulators can be naturally occurring
molecules or engineered molecules with the respective function
interfering with or modulating the activity of an immune checkpoint
molecule. Checkpoint modulators include, for example, antibodies or
antibody-fragments activity directed against immune checkpoint
molecule with agonistic or antagonistic, and ligands or modified
ligands of immune checkpoint molecules.
[0135] Co-inhibitory checkpoint molecules comprise, without
limitation, LILRB1, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR,
LAGS, PD-1, TIM-3, TIGIT and VISTA.
[0136] Leukocyte immunoglobulin-like receptor subfamily B member 1
(LILRB1) is a protein that in humans is encoded by the LILRB1 gene.
The protein belongs to the subfamily B class of LIR receptors which
contain two or four extracellular immunoglobulin domains, a
transmembrane domain, and two to four cytoplasmic immunoreceptor
tyrosine-based inhibitory motifs (ITIMs). The receptor is expressed
on immune cells where it binds to MHC class I molecules on
antigen-presenting cells and transduces a negative signal that
inhibits stimulation of an immune response. It is involved in the
control of inflammatory responses and cytotoxicity to help focus
the immune response and limit autoreactivity.
[0137] A2AR (Adenosine A2A receptor) is regarded as an important
checkpoint in cancer therapy because adenosine in the immune
microenvironment, leading to the activation of the A2a receptor, is
negative immune feedback loop and the tumor microenvironment has
relatively high concentrations of adenosine.
[0138] B7-H3, also called CD276, was originally understood to be a
co-stimulatory molecule but is now regarded as co-inhibitory.
MacroGenics is working on MGA271 (Enoblituzumab), which is an
Fc-optimized monoclonal antibody that targets B7-H3.
[0139] B7-H4 (or VTCN1) is expressed by tumor cells and
tumor-associated macrophages and plays a role in tumor evasion.
[0140] BTLA (B and T Lymphocyte Attenuator, also called CD272) is a
co-inhibitory receptor, which has HVEM (Herpesvirus Entry Mediator)
as its ligand. Surface expression of BTLA is gradually
downregulated during differentiation of human CD8+ T cells from the
naive to effector cell phenotype, however tumor-specific human CD8+
T cells express high levels of BTLA.
[0141] CTLA-4 (Cytotoxic T-Lymphocyte-Associated protein 4, also
called CD152) is expressed on Treg cells and serves to control T
cell proliferation. CTLA-4 (CD152) is a protein receptor
functioning as an immune checkpoint and is expressed by activated T
cells and transmits an inhibitory signal to T cells. CTLA4 is
homologous to the T-cell co-stimulatory protein CD28, and both
molecules bind to CD80 and CD86 (B7-1 and B7-2 respectively), on
antigen-presenting cells. CTLA-4 has a greater affinity and avidity
to CD80 and CD86 with than CD28. CTLA4 transmits an inhibitory
signal to T cells. Antagonistic antibodies directed against CTLA4
include ipilimumab and tremelimumab.
[0142] IDO (Indoleamine 2,3-dioxygenase) is a tryptophan catabolic
enzyme with immune-inhibitory properties. Another important
molecule is TDO, tryptophan 2,3-dioxygenase. IDO is known to
suppress T and NK cells, generate and activate Tregs and
myeloid-derived suppressor cells, and promote tumor
angiogenesis.
[0143] KIR (Killer-cell Immunoglobulin-like Receptor) is a receptor
for MHC Class I molecules on Natural Killer cells. Lirilumab is a
monoclonal antibody to KIR.
[0144] LAG-3 (Lymphocyte Activation Gene-3) works to suppress an
immune response by action to Tregs as well as direct effects on
CD8+ T cells.
[0145] PD-1 (Programmed Death 1, or CD279) is a cell surface
receptor that plays an important role in down-regulating the immune
system and promoting self-tolerance by suppressing T cell
inflammatory activity. PD-1 has two ligands, PD-L1 and PD-L2. An
advantage of targeting PD-1 is that it can restore immune function
in the tumor microenvironment. PD-L1, the ligand for PD1, is highly
expressed in several cancers and can lead to the inhibition of
anti-cancer immune response by T cells. A number of cancer
immunotherapy agents that target the PD-1 receptor have been
developed, including the antagonistic antibodies nivolumab,
(Opdivo--Bristol Myers Squibb), Pembrolizumab (Keytruda, MK-3475,
Merck), Pidilizumab (CT-011, Cure Tech) and BMS-936559 (Bristol
Myers Squibb). Both Atezolizumab (MPDL3280A, Roche) and Avelumab
(Merck KGaA, Darmstadt, Germany & Pfizer) are monoclonal
antibodies directed against PD-L1, the ligand of PD-1.
[0146] TIM-3 (T-cell Immunoglobulin domain and Mucin domain 3)
expresses on activated human CD4+ T cells and regulates Th1 and
Th17 cytokines. TIM-3 acts as a negative regulator of Th1/Th17
function by triggering cell death upon interaction with its ligand,
galectin-9.
[0147] VISTA (V-domain Ig suppressor of T cell activation) is a
protein that is primarily expressed on hematopoietic cells so that
consistent expression of VISTA on leukocytes within tumors may
allow VISTA blockade to be effective across a broad range of solid
tumors.
[0148] TIGIT (T cell immunoreceptor with Ig and ITIM domains, also
called WUCAM and Vstm3) is an immune receptor present on some T
cells and Natural Killer Cells and regulates T cell mediated
immunity. TIGIT could bind to CD155 on DCs and macrophages with
high affinity and to CD112 with lower affinity.
[0149] Co-stimulatory checkpoint molecules comprise, without
limitation, HVEM, CD27, CD40, OX40, GITR, CD137, CD28 and ICOS.
[0150] In preferred embodiments of the invention, the peptide is
administered in combination with an anti-cancer or an anti-viral
therapy.
[0151] Anti-cancer therapies of the present invention comprise,
without limitation, surgery, chemotherapy, radiotherapy,
irradiation therapy, hormonal therapy, targeted therapy,
immunotherapy, cell therapy and immune cell therapy.
[0152] In the context of the present invention, chemotherapy refers
to a category of cancer treatment that uses one or more anti-cancer
drugs (chemotherapeutic agents) as part of a chemotherapy regimen.
Irradiation or radiation therapy or radiotherapy in the context of
the present invention relates to a therapeutic approach using
ionizing or ultraviolet-visible (UV/Vis) radiation, generally as
part of cancer treatment to control or kill malignant cells such as
cancer cells or tumor cells. As used herein, "immunotherapy"
comprises any kind of therapeutic approach or treatment directed
against a tumor employing means of the immune system to negate or
destroy tumor material. This includes, without limitation, immune
checkpoint modulators, immune cell therapy, adoptive transfer of
immune cells or other cells that modulate the immune response,
modulation of the immune cells by small molecules or
biopharmaceuticals such as monoclonal antibodies, cytokines,
chemokines, and cancer treatment vaccines. Immunotherapies of the
present invention further comprise administration of an antibody
that binds specifically to a tumor-associated antigen, the
administration of a cytokine or chemokine, the administration of a
small molecule with anti-tumor immune-stimulating properties, the
administration of tumor antigens and/or the administration of
patient-derived tumor material.
[0153] In embodiments of the invention, the peptide for use as a
medicament is administered by a vector comprising or encoding the
peptide of the present invention. Therefore, the present invention
encompasses gene therapy comprising the administration of a
therapeutic gene encoding the polypeptide described herein.
[0154] The term gene therapy preferably refers to the transfer of
DNA into a subject in order to treat a disease. The person skilled
in the art knows strategies to perform gene therapy using gene
therapy vectors. Such gene therapy vectors are optimized to deliver
foreign DNA into the host cells of the subject. In a preferred
embodiment the gene therapy vectors may be a viral vector. Viruses
have naturally developed strategies to incorporate DNA in to the
genome of host cells and may therefore be advantageously used.
Preferred viral gene therapy vectors may include but are not
limited to retroviral vectors such as moloney murine leukemia virus
(MMLV), adenoviral vectors, lentiviral, adenovirus-associated viral
(AAV) vectors, pox virus vectors, vaccinia virus, herpes simplex
virus vectors or human immunodeficiency virus vectors (HIV-1).
Furthermore, the vector of the present invention may be an
attenuated HCMV virus or vector, which has been genetically
modified to be less harmful to the infected host than the
unmodified wild-type version of the virus. The viral vectors of the
invention are preferably genetically modified.
[0155] However also non-viral vectors may be preferably used for
the gene therapy such as plasmid DNA expression vectors driven by
eukaryotic promoters or liposomes encapsulating the transfer DNA.
Furthermore preferred gene therapy vectors may also refer to
methods to transfer of the DNA such as electroporation or direct
injection of nucleic acids into the subject. Moreover it may be
preferred that the gene therapy vectors for example a viral gene
therapy vector is adapted to target suitable cells of the body,
such as for example bone marrow cells, hematopoietic cells, or
immune cells or progenitor cells of immune cells, preferably NK
cells, NK cell progenitors or NK cell subsets, such as NKG2C+NK
cells. To this end the viral capsid may be conjugated with ligands
binding to the specific target cells, such as bone marrow cells,
such as hematopoietic cells, or immune cells or progenitor cells of
immune cells, preferably NK cells, NK cell progenitors or NK cell
subsets, such as NKG2C+NK cells, such as monoclonal antibodies. It
may also be preferred that the viral gene therapy vectors are
genetically modified using inducible promoters or promoters that
are specific for the target cells of interest, such as bone marrow
cells, such as hematopoietic cells, or immune cells or progenitor
cells of immune cells, preferably NK cells, NK cell progenitors or
NK cell subsets, such as NKG2C+NK cells, to enhance the expression
of the nucleic acid specifically in the target cells. Preferred
gene therapy vectors may therefore comprise vectors for an
inducible or conditional expression of the polypeptides. The person
skilled in the art knows how to choose preferred gene therapy
vectors according the need of application as well as the methods on
how to implement the nucleic acid into the gene therapy vector. (P.
Seth et al., 2005, N. Koostra et, al. 2009., W. Walther et al.
2000, Waehler et al. 2007).
[0156] The present invention may relate to a nucleic acid molecule
encoding a peptide of the invention. The nucleic acid according to
the invention and preferred embodiments thereof, in particular a
nucleic acid encoding a polypeptide of the present invention, is
particularly efficient for gene therapy due to a high therapeutic
potential at a small size. This ensures a stable integration at
high expression levels over extended periods of times.
[0157] In a further preferred embodiment the invention relates to a
cell for use as a medicament to expand and/or activate NKG2C+
natural killer (NK) cells in the treatment and/or prevention of a
medical condition treatable by the effector function of said
NKG2C+NK cells. Therein the cell may be a NKG2C+NK cell generated
by the method of the present invention for cultivating and/or
expanding NKG2C+ natural killer (NK) cells, or a cell, which is
genetically modified and comprises an exogenous nucleic acid region
encoding for a polypeptide according to the invention or preferred
embodiments thereof and wherein the exogenous nucleic acid region
is operably linked to a promoter.
[0158] The person skilled in the art knows how to genetically
modify cells in order to express the polypeptides according to the
inventions. Advantageously by expressing the therapeutically
effective polypeptides the cells may act as bio pump or drug
factory that continuously expresses and provides the polypeptides
to the subject. Thereby the amount of the polypeptides can be held
at a therapeutic level over long periods. The person skilled in the
art knows which cells may be preferably used to this end. In a
preferred embodiment the cells are stem cells, characterized by a
stable expression of the polypeptides. Stem cells may include but
are not limited to, embryonic stem cells such as early embryonic
stem cells and blastocyst embryonic stem cells; fetal stem cells;
umbilical cord stem cells; and adult stem cells such as mesenchymal
stem cells, hematopoietic stem cells, endothelial stem cells,
peripheral blood stem cells, and multipotent somatic stem
cells.
[0159] The cells may migrate to the site of NK cells, NK progenitor
cells or NKG2C+NK cells in order to locally express the
polypeptides in vicinity of the cells to be activated and/or
expanded. Advantageously the cells may however also be transplanted
at a different location as the polypeptides can also be transported
by the vascular system throughout the body of the subject. Local
administration of the cells e.g. by a subcutaneous injection may
therefore contribute in a systemic manner largely irrespective of
the location of the cells within the body of the subject. In a
further preferred embodiment the peptides for use as a medicament
as described herein are characterized by introducing a
therapeutically effective number of said peptide either directly or
comprised by a suitable vector as described herein, such as a viral
vector or a cell carrying a nucleic acid encoding the peptide of
the invention, to a subject within a biocompatible matrix.
Preferred materials for the biocompatible matrix are agarose,
carrageenan, alginate, chitosan, gellan gum, hyaluronic acid,
collagen, cellulose and its derivatives, gelatin, elastin, epoxy
resin, photo cross-linkable resins, polyacrylamide, polyester,
polystyrene and polyurethane or polyethylene glycol (PEG). It is
further preferred that the biocompatible matrix is a semi-permeable
hydrogel matrix and the peptides or vectors carrying the peptide
and/or a nucleic acid encoding the peptide are entrapped by said
matrix. Advantageously the biocompatible matrix allows for an
efficient diffusion of nutrients, oxygens and other biomolecules to
ensure a long lasting persistence of the peptides or vectors
carrying the peptide and/or a nucleic acid encoding the peptide,
while immobilizing the peptides or vectors carrying the peptide
and/or a nucleic acid encoding the peptide. Thereby the cells can
be concentrated at preferred locations within the subject. For
instance the peptides or vectors carrying the peptide and/or a
nucleic acid encoding the peptide cells can be transplanted
subcutaneously and/or in proximity of diseased regions of the
subject.
[0160] In a preferred embodiment the invention further relate to
pharmaceutical composition for use as a medicament, preferably to
expand and/or activate NKG2C+ natural killer (NK) cells in the
treatment and/or prevention of a medical condition associated with
pathogenic cells expressing HLA-E and a peptide comprising an amino
acid sequence according to SEQ ID NO 1 or 2 as described herein,
wherein the pharmaceutical composition comprises the polypeptide,
the nucleic acid, the gene therapy vector and/or the cell, and
optionally a pharmaceutically accepted carrier. Preferably the
pharmaceutical composition is administered to the subject at a
therapeutically effective amount at any administration route as
described herein. In the context of the present invention, a cell
comprising or encoding a peptide of the present invention may be
considered a vector.
[0161] In a preferred embodiment the pharmaceutical composition for
use as a medicament as described herein is administered by
introducing a therapeutically effective amount of the composition
into the blood stream of a subject. In a further preferred
embodiment the pharmaceutical composition for use as a medicament
as described herein is administered locally, for example by
administration to a site of the subject's body in proximity to a
site where pathogenic cells expressing HLA-E and a peptide
comprising an amino acid sequence according to SEQ ID NO 1 or 2 are
localized. As used herein, in "proximity with" a tissue/site
includes, for example, within 50 mm, 20 mm, 10 mm, 5 mm, within 1
mm of the tissue, within 0.5 mm of the tissue and within 0.25 mm of
the tissue/site.
[0162] As used herein, "nucleic acid" shall mean any nucleic acid
molecule, including, without limitation, DNA, RNA and hybrids or
modified variants thereof. An "exogenous nucleic acid" or
"exogenous genetic element" relates to any nucleic acid introduced
into the cell, which is not a component of the cells "original" or
"natural" genome. Exogenous nucleic acids may be integrated or
non-integrated, or relate to stably transfected nucleic acids.
[0163] As used herein, "polypeptide" shall mean both peptides and
proteins. In this invention, the polypeptides may be naturally
occurring or recombinant (i.e., produced via recombinant DNA
technology), and may contain mutations (e.g., point, insertion and
deletion mutations) as well as other covalent modifications (e.g.,
glycosylation and labelling (via biotin, streptavidin, fluorescein,
and radioisotopes)) or other molecular bonds to additional
components. For example, PEGylate proteins are encompassed by the
scope of the present invention. PEGylation has been widely used as
a post-production modification methodology for improving biomedical
efficacy and physicochemical properties of therapeutic proteins.
Applicability and safety of this technology have been proven by use
of various PEGylated pharmaceuticals for many years (refer Jevsevar
et al, Biotechnol J. 2010 January; 5(1):113-28). In some
embodiments the polypeptides described herein are modified to
exhibit longer in vivo half-lives and resist degradation when
compared to unmodified polypeptides. Such modifications are known
to a skilled person, such as cyclized polypeptides, polypeptides
fused to Vitamin B12, stapled peptides, protein lipidization and
the substitution of natural L-amino acids with D-amino acids (refer
Bruno et al, Ther Deliv. 2013 November; 4(11): 1443-1467).
[0164] In some embodiments of the invention the peptide, preferably
according to sequences disclosed herein, may comprise a 0 to 10
amino acid addition or deletion at the N and/or C terminus of a
sequence.
[0165] As used herein the term "a 0 to 10 amino acid addition or
deletion at the N and/or C terminus of a sequence" means that the
polypeptide may have a) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
additional amino acids at its N terminus and 0, 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 amino acids deleted at its C terminus or b) 0, 1, 2,
3, 4, 5, 6, 7, 8, 9 or 10 additional amino acids at its C terminus
and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides deleted at its N
terminus, c) 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additional amino
acids at its N terminus and 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10
additional amino acids at its N terminus or d) 0, 1, 2, 3, 4, 5, 6,
7, 8, 9 or 10 amino acids deleted at its N terminus and 0, 1, 2, 3,
4, 5, 6, 7, 8, 9 or 10 amino acids deleted at its C terminus.
[0166] Furthermore, in addition to the polypeptides described
herein, peptidomimetics are also contemplated. Peptide analogs are
commonly used in the pharmaceutical industry as non-peptide drugs
with properties analogous to those of the template peptide. These
types of non-peptide compound are termed "peptide mimetics" or
"peptidomimetics" (Fauchere (1986) Adv. Drug Res. 15: 29; Veber and
Freidinger (1985) TINS p. 392; and Evans et al. (1987) J. Med.
Chem. 30: 1229) and are usually developed with the aid of
computerized molecular modelling. Peptide mimetics that are
structurally similar to therapeutically useful peptides may be used
to produce an equivalent therapeutic or prophylactic effect. It may
be preferred in some embodiments to use peptide mimetics in order
to prolong the stability of the polypeptides, when administered to
a subject. To this end peptide mimetics for the polypeptides may be
preferred that are not cleaved by human proteasomes.
[0167] The polypeptides, nucleic acid molecules, gene therapy
vectors or cells described herein may comprise different types of
carriers depending on whether they are to be administered in solid,
liquid or aerosol form, and whether they need to be sterile for
such routes of administration as injection.
[0168] The active agent of present invention can be administered
intravenously, intradermally, intraarterially, intraperitoneally,
intralesionally, intracranially, intraarticularly,
intraprostaticaly, intrapleurally, intratracheally, intranasally,
intravitreally, intravaginally, intrarectally, topically,
intratumorally, intramuscularly, intraperitoneally, subcutaneously,
subconjunctival, intravesicularlly, mucosally, intrapericardially,
intraumbilically, intraocularally, orally, topically, locally,
inhalation (e.g., aerosol inhalation), injection, infusion,
continuous infusion, directly, via a catheter, via a lavage, in
cremes, in lipid compositions (e.g., liposomes), locally applied by
sponges or by other method or any combination of the forgoing as
would be known to one of ordinary skill in the art (see, for
example, Remington's Pharmaceutical Sciences, 18th Ed. Mack
Printing Company, 1990, incorporated herein by reference).
[0169] Such administering can also be performed, for example, once,
a plurality of times, and/or over one or more extended periods. A
single injection is preferred, but repeated injections over time
(e.g., quarterly, half-yearly or yearly) may be necessary in some
instances. Such administering is also preferably performed using an
admixture of polypeptides, nucleic acids, gene therapy vectors or
cells and a pharmaceutically acceptable carrier. Pharmaceutically
acceptable carriers are well known to those skilled in the art and
include, but are not limited to, 0.01-0.1 M and preferably 0.05 M
phosphate buffer or 0.8% saline.
[0170] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like. The use of such media and agents for
pharmaceutical active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
active ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients can also be
incorporated into the compositions.
[0171] Additionally, such pharmaceutically acceptable carriers can
be aqueous or non-aqueous solutions, suspensions, and emulsions,
most preferably aqueous solutions. Aqueous carriers include water,
alcoholic/aqueous solutions, emulsions and suspensions, including
saline and buffered media. Parenteral vehicles include sodium
chloride solution, Ringers dextrose, dextrose and sodium chloride,
lactated Ringers and fixed oils. Intravenous vehicles include fluid
and nutrient replenishers, electrolyte replenishers such as
Ringer's dextrose, those based on Ringer's dextrose, and the like.
Fluids used commonly for i.v. administration are found, for
example, in Remington: The Science and Practice of Pharmacy, 20th
Ed., p. 808, Lippincott Williams S-Wilkins (2000). Preservatives
and other additives may also be present, such as, for example,
antimicrobials, antioxidants, chelating agents, inert gases, and
the like.
[0172] The phrase "pharmaceutically acceptable" refers to molecular
entities and compositions that do not produce an allergic or
similar untoward reaction when administered to a human. The
preparation of an aqueous composition that contains a protein as an
active ingredient is well understood in the art. Typically, such
compositions are prepared as injectables, either as liquid
solutions or suspensions; solid forms suitable for solution in, or
suspension in, liquid prior to injection can also be prepared. The
preparation can also be emulsified.
[0173] The composition can be formulated in a neutral or salt form.
Pharmaceutically acceptable salts, include the acid addition salts
(formed with the free amino groups of the protein) and which are
formed with inorganic acids such as, for example, hydrochloric or
phosphoric acids, or such organic acids as acetic, oxalic,
tartaric, mandelic, and the like. Salts formed with the free
carboxyl groups can also be derived from inorganic bases such as,
for example, sodium, potassium, ammonium, calcium, or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, histidine, procaine and the like. Upon formulation,
solutions will be administered in a manner compatible with the
dosage formulation and in such amount as is therapeutically
effective. The formulations are easily administered in a variety of
dosage forms such as injectable solutions, drug release capsules
and the like. As used herein, a "therapeutically effective amount"
for the pharmaceutical composition includes, without limitation,
the following amounts and ranges of amounts:
[0174] For a composition comprising a polypeptide according to the
invention or preferred embodiment thereof: (i) from about
1.times.10.sup.-3 to about 1.times.10.sup.6 .mu.g/kg body weight;
(ii) from about 1.times.10.sup.-2 to about 1.times.10.sup.5
.mu.g/kg body weight; (iii) from about 1.times.10.sup.-1 to about
1.times.10.sup.4 .mu.g/kg body weight; (iv) from about
1.times.10.sup.-1 to about 1.times.10.sup.3 .mu.g/kg body weight;
(v) from about 1.times.10.sup.-1 to about 1.times.10.sup.2 .mu.g/kg
body weight; (vi) from about 1.times.10.sup.-1 to about
0.5.times.10.sup.2 .mu.g/kg body weight; (vii) about
1.times.10.sup.-2 .mu.g/kg body weight; (viii) about
1.times.10.sup.1 .mu.g/kg body weight; (ix) about 10 .mu.g/kg body
weight (x) about 1.times.10.sup.2 .mu.g/kg body weight; (xi) about
5.times.10.sup.3 .mu.g/kg body weight.
[0175] For a composition comprising cells according to the
invention or preferred embodiment thereof: (i) from about
1.times.10.sup.2 to about 1.times.10.sup.8 cells/kg body weight;
(ii) from about 1.times.10.sup.3 to about 1.times.10.sup.7 cells/kg
body weight; (iii) from about 1.times.10.sup.4 to about
1.times.10.sup.6 cells/kg body weight; (iv) from about
1.times.10.sup.4 to about 1.times.10.sup.5 cells/kg body weight;
(v) from about 1.times.10.sup.5 to about 1.times.10.sup.6 cells/kg
body weight; (vi) from about 5.times.10.sup.4 to about
0.5.times.10.sup.5 cells/kg body weight; (vii) about
1.times.10.sup.3 cells/kg body weight; (viii) about
1.times.10.sup.4 cells/kg body weight; (ix) about 5.times.10.sup.4
cells/kg body weight; (x) about 1.times.10.sup.5 cells/kg body
weight; (xi) about 5.times.10.sup.5 cells/kg body weight; (xii)
about 1.times.10.sup.6 cells/kg body weight; and (xiii) about
1.times.10.sup.7 cells/kg body weight.
[0176] Human body weights envisioned include, without limitation,
about 5 kg, 10 kg, 15 kg, 30 kg, 50 kg, about 60 kg; about 70 kg;
about 80 kg, about 90 kg; about 100 kg, about 120 kg and about 150
kg.
[0177] Dosages of the viral gene therapy vector will depend
primarily on factors such as the condition being treated, the
selected gene, the age, weight and health of the patient, and may
thus vary among patients. For example, a therapeutically effective
human dosage of the viral vectors may be preferably in the range of
from about 1 to about 1000 ml, preferably 10 to 100 ml, preferably
20 to 50 ml of saline solution containing concentrations of from
about 1.times.10.sup.5 to 1.times.10.sup.12 preferably
1.times.10.sup.6 to 1.times.10.sup.11 more preferably
1.times.10.sup.7 to 1.times.10.sup.10 plaque forming units (pfu)/ml
viruses. The dosage will be adjusted to balance the therapeutic
benefit against any side effects. The levels of expression of the
selected gene can be monitored to determine the selection,
adjustment or frequency of dosage administration.
[0178] As used herein "inducible expression" or "conditional
expression" relates to a state, multiple states or system of an
expression of the polypeptide, wherein the polypeptide is
preferably not expressed, or in some embodiments expressed at
negligible or relatively low levels, unless there is the presence
of one or more molecules (an inducer) or other set of conditions in
the cell that allows for polypeptide expression. Inducible
promoters may relate to either naturally occurring promoters that
are expressed at a relatively higher level under particular
biological conditions, or to other synthetic promoters comprising
any given inducible element. Inducible promoters may refer to those
induced by particular tissue- or micro-environments or combinations
of biological signals present in particular tissue- or
micro-environments, or to promoters induced by external factors,
for example by administration of a small drug molecule or other
externally applied signal. As used herein, "treatment" of a disease
or "treating" a subject afflicted with a disorder shall mean
slowing, stopping or reversing the disorder's progression. In the
preferred embodiment, treating a subject afflicted with a disorder
means reversing the disorder's progression, ideally to the point of
eliminating the disorder itself. As used herein, ameliorating a
disorder and treating a disorder are equivalent. The treatment of
the present invention may also, or alternatively, relate to a
prophylactic administration of the active agents described herein.
Such a prophylactic administration may relate to the prevention of
any given medical disorder, or the prevention of development of
said disorder, whereby prevention or prophylaxis is not to be
construed narrowly under all conditions as absolute prevention.
Prevention or prophylaxis may also relate to a reduction of the
risk of a subject developing any given medical condition,
preferably in a subject at risk of said condition.
[0179] "Combined administration" may relate to concurrent and/or
sequential administration of said polypeptide prior to, during
and/or subsequent to said adjuvant, check point inhibitor and/or
further treatment. Combined treatment shall also include a
combination treatment regimens comprising multiple administrations
of either therapeutic component of the treatment. Further
embodiments of combined administration are provided herein.
[0180] Combined administration encompasses simultaneous treatment,
co-treatment or joint treatment, and includes the administration of
separate formulations of the polypeptide of the present invention
with said adjuvant, check point inhibitor and/or further treatment,
whereby treatment may occur within minutes of each other, in the
same hour, on the same day, in the same week or in the same month
or within 3 months as one another. Sequential administration of any
given combination of combined agents is also encompassed by the
term "combined administration". A combination medicament,
comprising one or more of said polypeptide, said adjuvant, check
point inhibitor and/or further treatment, may also be used in order
to co-administer the various components in a single administration
or dosage.
[0181] The term "vaccine" in the context of the present invention
relates to a biological preparation that provides active acquired
immunity to a particular disease, such as cancer, a pathogen or an
infectious agent, such as bacteria or viruses. In the context of
the present invention, NKG2C+NK cells may be considered to provide
adapted or acquired immunity. A vaccine can contain an agent or
antigen that resembles or is derived from a disease-causing
microorganism. Vaccines can be made from weakened, attenuated,
mutated or killed forms of the pathogen, its toxins or one of its
surface proteins. The agent stimulates the body's immune system to
recognize the agent as a threat, destroy it, and recognize and
destroy any pathogens or structures comprising the agent or antigen
of the vaccine that it later encounters. Vaccines can be
prophylactic (example: to prevent or ameliorate the effects of a
future infection by a natural or "wild" pathogen), or therapeutic,
such as specific cancer vaccines.
[0182] As used herein, the term "sample" is a biological sample
that is obtained or isolated from the patient or subject. "Sample"
as used herein may, e.g., refer to a sample of bodily fluid or
tissue obtained for the purpose of diagnosis, prognosis, or
evaluation of a subject of interest, such as a patient. Preferably
herein, the sample is a sample of a bodily fluid, such as blood,
serum, plasma, cerebrospinal fluid, urine, saliva, sputum, pleural
effusions, cells, a cellular extract, a tissue sample, a tissue
biopsy, a stool sample and the like. In the context of the present
invention, any kind of sample comprising pathogenic cells
potentially expressing HLA-E and a peptide comprising an amino acid
sequence according to SEQ ID NO 1 or 2, such as cancer cells or
cells that may comprise reactivated HCMV.
EXAMPLES
[0183] The invention is further described by the following
examples. These are not intended to limit the scope of the
invention, but represent preferred embodiments of aspects of the
invention provided for greater illustration of the invention
described herein.
Methods Employed in the Examples
Human Subjects
[0184] All analyses of human data were carried out in compliance
with the relevant ethical regulations. Healthy blood donors gave
informed consent at DRK Dresden, Germany and buffy coats from
donors with defined HCMV-serostatus were obtained as approved by
Charite ethics committee (EA1/149/12). PBMCs were isolated by
density gradient centrifugation (Ficoll Paque Plus, GE Healthcare)
and PBMCs of HCMV.sup.+ donors were screened for the presence of
adaptive NKG2C.sup.+ NK cells as previously described.sup.56. In
brief, co-expression analysis was employed to detect adaptive
CD2.sup.+ CD57.sup.+ILT2.sup.+Siglec-7.sup.-NKp30.sup.-NKG2A.sup.-
NK cells within the CD56.sup.dm NKG2C.sup.+ population. CD56.sup.+
cells were MACS-enriched (CD56 MicroBeads, Miltenyi Biotec) and
either used directly or cryopreserved in fetal bovine serum (FBS;
Biowest) containing 10% DMSO (Sigma).
[0185] Patients undergoing allogeneic hematopoietic stem cell
transplantation either at the Department of Hematology, Oncology
and Tumor Immunology, Charite--Universit tsmedizin Berlin or at the
Department of Hematology, Hemostasis, Oncology and Stem Cell
Transplantation, Hannover Medical School gave informed consent
(Charite ethics committee approval EA1/1/169/14; Hannover Medical
School institutional review board approval #1303-2012, #2032-2013,
#2604-2014, and #2604-2015; the latter cohort has been described
previously.sup.57). PBMCs were isolated by density gradient
centrifugation on the day of blood donation and cryopreserved in
FBS containing 10% DMSO. Serum samples from the Charite cohort were
handled and stored at Labor Berlin--Charite Vivantes GmbH.
Cells and Cell Lines
[0186] K562/HLA-E.sup.21 (kindly provided by E. Weiss, Ludwig
Maximilian University) and RMA-S/HLA-E.sup.58 (kindly provided by
J. Coligan, National Institutes of Health) cells were maintained in
complete medium (RPMI-1640 containing glutamine and supplemented
with 10% [v/v] FBS, 20 .mu.M .beta.-mercaptoethanol, and 100 U/ml
Penicillin-Streptomycin; all Thermo Fisher) in the presence of 400
.mu.g/ml Hygromycin B and 1 mg/ml G418 (both InvivoGen),
respectively. RMA-S/HLA-E were transfected by electroporation (Cell
Line Nucleofector Kit T, Lonza) with pUNO1-hLFA3a plasmid
(InvivoGen). RMA-S/HLA-E/LFA-3 were FACS-sorted for high LFA-3
expression and selected and maintained in complete medium
containing both 400 .mu.g/ml Hygromycin B and 25 .mu.g/ml
Blasticidin (InvivoGen). Primary human umbilical vein endothelial
cells (HUVECs) were purchased from Lonza and maintained in EGM-2
(Lonza) according to the manufacturer's instructions.
Amplification, Sequencing, and Analysis of UL40-Encoded Peptide
Sequences
[0187] DNA was extracted from serum or plasma samples using QIAmp
DNA Micro Kit (Qiagen) according to the manufacturer's
instructions. A partial HCMV UL40 sequence was amplified with a
nested PCR approach (Table 5 for primer sets) using the AccuPrime
high fidelity DNA Polymerase (ThermoFisher) and PCR products were
sequenced at Eurofins Genomics. At least 2 independent DNA
extractions, PCRs, and sequencing reactions were performed for each
sample. 165 published UL40 sequences were obtained from GenBank
(NCBI) and combined with 52 newly determined sequences from patient
samples. The integrated data set was analyzed using BioEdit (Ibis
Biosciences), SerialCloner (SerialBasics), and WebLogo.sup.59
(University of California).
HLA E Surface Stabilization
[0188] HLA-E surface stabilization was induced as described
previously.sup.30. In brief, 300 .mu.M synthetic peptides
(Peptides&Elephants) were added to target cells cultured at a
density of 2.times.10.sup.6 cells/ml in serum-free Opti-MEM
(ThermoFisher) and incubated for 16 h at 37.degree. C.
Peptide-pulsed cells were either stained for FACS analysis of HLA-E
surface expression or washed with complete media and used in
co-cultures. For pulse-chase experiments, cells were pulsed as
above, washed twice, and resuspended in Opti-MEM without peptide.
HLA-E surface expression was monitored by FACS analysis at
indicated time points.
In Vitro Stimulation of NK Cells
[0189] CD56.sup.+ MACS-enriched cells were stained with viability
dye, CD3, and CD56, FACS-sorted for viable CD3.sup.- CD56.sup.+ NK
cells, and rested overnight in complete medium. Purified NK cells
were stimulated by co-culture with peptide-pulsed (300 .mu.M)
irradiated (30 Gy) target cells at a 2:1 NK:target ratio for 6 h in
the presence of 300 .mu.M synthetic peptides. Anti-CD107a (Table 4)
antibody was added at the start of the assay and GolgiStop and
GolgiPlug (both BD Biosciences) were added 1 h after start of the
stimulation.
[0190] For blocking experiments, rested FACS-sorted viable
CD3.sup.- CD56.sup.dim NKG2A.sup.- NKG2C.sup.+ NK cells were
treated with 20 .mu.g/ml IgG1 isotype control or anti-CD94 antibody
(both RnD Systems) for 15 min prior to start of the co-culture.
[0191] For infection experiments, 2.5.times.10.sup.4 HUVEC
homozygous for both HLA-C1 and HLA-Bw4 seeded in 48-well plates
were infected with HCMV TB40R variants in serum-free media at a
multiplicity of infection of 3-5. After 24 h, HUVECs were washed
and further incubated in EGM-2. At 48 h post infection, HUVECs were
washed and co-cultured with 5.times.10.sup.4 rested or overnight
IFN-.alpha.-primed (25 ng/ml; Miltenyi) FACS-sorted viable
CD3.sup.- CD56.sup.+ NK cells for 6 h. Anti-CD107a antibody (Table
4) was added at the start of the assay and GolgiStop and GolgiPlug
(both BD Biosciences) were added 1 h after start of the
stimulation.
Cytotoxicity Assays
[0192] CD56.sup.+ MACS-enriched cells were stained with viability
dye, CD3, and CD56, FACS-sorted for viable CD3.sup.- CD56.sup.+ NK
cells, and rested overnight in complete medium. Varying numbers of
purified NK cells were co-cultured with 2.times.10.sup.4 CellTrace
violet (ThermoFisher)-labeled peptide-pulsed (300 .mu.M) target
cells resulting in NK:target ratios ranging from 0.1 to 10. To
control for spontaneous cell death, target cells were cultured in
the absence of NK cells. After 6 h, cell suspensions were stained
for viability of target cells using Fixable Viability Dye eFluor780
(ThermoFisher) and analyzed by flow cytometry. Cytotoxicity (%) was
calculated as follows: (% dead target cells in experimental
condition-% dead target cells in spontaneous control)/(100%-% dead
target cells in spontaneous control).times.100.
In Vitro Culture of NK Cells
[0193] CD56.sup.+ MACS-enriched cells from HCMV.sup.- donors were
stained with viability dye, CD3, and CD56, FACS-sorted for viable
CD3.sup.- CD56.sup.dim NK cells, and rested overnight in complete
medium. Purified CD56.sup.dim NK cells were labeled with 2 .mu.M
CellTrace violet (Thermo Fisher) according to the manufacturer's
instructions. 5.times.10.sup.5 labeled NK cells were cultured with
1.times.10.sup.5 peptide-pulsed (300 .mu.M) irradiated (30 Gy)
target cells in complete medium containing 10 ng/ml IL-15 (Miltenyi
Biotec) in U-bottom 96-well plates (Greiner Bio-One) for the
indicated times. Complete medium containing IL-15 was replaced
every third to fourth day. On days 4, 7, and 11 of culture,
1.times.10.sup.5 fresh peptide-pulsed irradiated target cells were
added to the cultures.
[0194] Where indicated, 10 ng/ml IL-12 (Miltenyi Biotec) and 100
ng/ml IL-18 (MBL) were added at the start of co-cultures. After 20
h of culture, the cells were washed 3 times and resuspended in
complete medium containing 10 ng/ml IL-15. Control wells without
IL-12/18 were treated equally. To determine NKG2C.sup.+ NK cell
numbers per .mu.l of culture medium, CountBright Absolute Counting
Beads (ThermoFisher) were used according to the manufacturer's
instructions.
[0195] Proliferation and replication indices.sup.60 of NKG2C.sup.+
cells were calculated using the proliferation analysis platform
included in FlowJo v9.9 (FlowJo LLC) and normalized to those of
NKG2C.sup.- cells in the same condition.
[0196] For assessment of KIR ligands present in individual donors
to determine educating KIR, DNA extracted from PBMCs was HLA-B and
HLA-C typed at low and high resolution, respectively, by the Center
for Transfusion Medicine and Cell Therapy,
Charite--Universitatsmedizin Berlin.
Flow Cytometry
[0197] Cell suspensions were stained in different combinations of
fluorochrome-conjugated antibodies (Table 4), following established
guidelines.sup.61. Dead cells were excluded using LIVE/DEAD Fixable
Violet Dead Cell Stain Kit, Fixable Viability Dye eFluor780 (both
ThermoFisher), or Zombie Aqua Fixable Viability Kit (BioLegend).
For intracellular staining, cells were fixed with 2% PFA (EMS
Sciences) and permeabilized with Permeabilizing Solution 2 (BD
Biosciences) according to the manufacturer's instructions. Staining
of HCMV-IE in HUVEC was performed using the Foxp3/Transcription
Factor Staining Buffer Set (eBioscience) according to the
manufacturer's instructions. Data were acquired on a LSR Fortessa
or FACSymphony (both BD Biosciences). FlowJo v9.9 and vX (FlowJo
LLC) as well as SPICE.sup.62 (NIAID) software were used for
analysis. ARIA, ARIA II, or Influx instruments (all BD Biosciences)
were used for cell sorting experiments.
Expression Analysis of HCMV UL40
[0198] RNA of HUVECs was extracted at indicated time points post
infection using the Nucleospin RNA kit (Macherey Nagel) according
to the manufacturer's instructions. Viral genomic DNA was
eliminated by in-solution rDNase digestion for 10 min at 37.degree.
C. RNA was re-purified by addition of 2.5 volumes ethanol (Sigma)
and 0.1 volume 3 M sodium acetate (Thermo Fisher) and incubation at
-20.degree. C. overnight. Precipitated RNA was washed with 70%
ethanol, dried, and resuspended in RNase-free H.sub.2O. 50 ng RNA
were reverse transcribed with TaqMan Reverse Transcription Reagents
(Applied Biosciences). Quantitative real-time PCR was performed
using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Fisher) in a
StepOnePlus system (Applied Biosciences) using UL40_nested and
hGAPDH primer pairs (Table 5). Specificity of amplification was
assessed by melting curve analysis, gel electrophoresis, and
controls without reverse transcription to confirm complete
digestion of genomic DNA.
Generation of HCMV Mutant Viral Strains
[0199] HCMV mutants encoding different UL40 peptides were
constructed using the bacterial artificial chromosome (BAC) TB40
BAC4.sup.63 containing the genome of the HCMV strain TB40/E. For
reinsertion of viral genes US2-US6 deleted in TB40 BAC4, two-step
replacement mutagenesis based on homologous recombination in E.
coli.sup.64 was performed utilizing the shuttle plasmid
pUH15.sup.65. The resulting BAC TB40R was further modified by
addition of the Cre gene and loxP sites flanking the BAC vector
sequences to allow self-excision upon introduction into
HCMV-permissive cells. A gpt gene in BAC TB40R was replaced with a
tetracycline resistance marker by en passant mutagenesis in E. coli
strain GS1783.sup.66 and utilizing a PCR fragment amplified with
primers US2-lox-tet.for (see Table 6 for primer sequences using
during mutagenesis) and G-lox-tet.rev and template plasmid
pori6K-Tet (M. Messerle, unpublished). The PCR product was
recombined with TB40R, giving rise to TB40R-lox-Tet, into which the
Cre recombinase sequences were inserted. To this end, a template
plasmid was generated containing sequences for a polyomavirus
promoter, an intron-containing Cre gene with internal insertion of
a kanamycin-resistance marker (kan.sup.R) and a loxP site. The
kan.sup.R marker (with an adjacent I-Scel restriction site) was
amplified with primers pIC1.for and MC-Cre.rev with plasmid
pori6K-RIT (M. Messerle unpublished) as template, and the PCR
fragment for the Cre gene was amplified with primers
kan.sup.R-ISce.for and pIC.rev and template plasmid pMC-Cre-Intron
(E. M. Borst, unpublished; pMC-Cre-Intron contains the Cre gene of
plasmid pGS403.sup.67, the latter kindly provided by G. Smith,
Northwestern University). The resulting PCR products were added to
vector pIC1.sup.68 next to a loxP site by Gibson assembly.sup.69
using the Gibson Assembly Master Mix according to the
manufacturer's instructions (NEB). Integrity of the plasmid
pIC-kan.sup.R-Cre-Intron was verified by restriction analysis and
sequencing. A PCR fragment was produced from
pIC-kan.sup.R-Cre-Intron with primer pair loxPUS2.for and
KanRITyellow.rev, and recombined with TB40R-lox-Tet, followed by
seamless excision of the kan.sup.R cassette as described.sup.66.
The final HCMV BAC, TB40R-Cre, served for mutagenesis of the UL40
sequences. To mutate the UL40 region, the following primer pairs
were used with pori6K-RIT as template: UL40_VMAPRTLIL.for and
UL40_VMAPRTLIL.rev, UL40_VMAPRTLFL.for and UL40_VMAPRTLFL.rev, and
UL40_VMAPQSLLL.for and UL40_VMAPQSLLL.rev. En passant mutagenesis
using the resulting PCR products and BAC TB40R-Cre, followed by
excision of the kan.sup.R marker as reported.sup.66, gave rise to
recombinant HCMV BAC genomes containing UL40 genes encoding for
VMAPRTLFL (SEQ ID NO. 2), VMAPRTLIL (SEQ ID NO. 3), or VMAPQSLLL
(SEQ ID NO. 4) peptides, respectively. Successful mutagenesis was
confirmed by restriction analysis and sequencing of the relevant
parts within the BAC genomes. Virus mutants were reconstituted by
transfection of human foreskin fibroblasts (HFF; Merck Millipore;
cultured as outlined elsewhere.sup.64) by adenofection as
described.sup.70. Virus stock was produced by seeding
1.times.10.sup.7 HFF into four T75 flasks, followed by infection
the next day in serum-free medium at a multiplicity of infection of
1, followed by centrifugal enhancement at 950 g for 30 min. Five
days post infection, supernatants were harvested, cleared from
cellular debris by centrifugation at 3,500 g for 45 min, virus was
pelleted by ultracentrifugation in an SW32 rotor at 143,000 g for
60 min, and stored in 50 mM Tris-HCl pH 7.8 containing 12 mM KCl, 5
mM Na.sub.2EDTA, and 20% FBS at -80.degree. C. Viral titers were
determined by plaque assay on HFF.sup.64.
Mathematical Model of NK Cell Proliferation
[0200] For mathematical analysis of the kinetic NK cell
proliferation data, the following mathematical model was used:
N 0 ( t ) = ( 1 - .PHI. ( t ) ) + .chi. e - k d t d N 1 d t = 2
.psi. ( t ) - .psi. ( t - t d i v ) e - k d t div - k d N 1 ( t ) ,
N 1 ( 0 ) = 0 N i ( t ) = ( 2 e - k d t div ) i - 1 N 1 ( t - [ i -
1 ] t d i v ) , N i ( t ) = 0 for t .ltoreq. 0 , i = 2 , , .infin.
( 1 ) ##EQU00001##
[0201] Here, N.sub.i(t) is the number of cells in the i-th
generation at time t, given as fold-change with respect to the cell
number at time 0. .chi. is the fraction of non-dividing cells,
.psi.(t) is a normalized probability density determining the time
until the first division of each cell (also known as precursor
frequency), and .phi.(t) is the cumulative probability distribution
to .psi.(t). Further, k.sub.d is the death rate, and t.sub.div is
the division time, that means the time required for completion of
each cell division after the first division, which is assumed to be
constant and deterministic. From Equation (1), we may compute the
total cell number at each time point as
C(t)=.SIGMA..sub.i=0.sup..infin.N.sub.i(t). Equation (1) is a
variant of the well-established Gett/Hodgkin model.sup.38, for
which a convenient analytical formulation was described.sup.71.
That analytical description considers the dynamics of dividing
cells after their first division (generations N.sub.1, N.sub.2, . .
. ). Since the cell number data in our study show substantial
initial cell death before the onset of clonal expansion, we
additionally consider generation N.sub.0. That generation consists
of dividing cells that have not yet divided (first term), and
non-dividing cells, which we take as decaying with the same death
rate as dividing cells (second term). To be consistent with our
observation that almost all cells present at day 7 have divided at
least once across conditions (FIG. 7 and FIG. 10a), in Equation
(1), we set the fraction of non-dividing cells to
.chi.=0.05/e.sup.-k.sup.d.sup.t.sup.pt.sub.p=6 days. Thus, in the
model, the non-dividing cells have dropped to 5% of their initial
population on the 6th day after stimulation. For precursor
frequencies, we have experimental measurements at days 1 through 7
from the CellTrace data analysis (FIG. 7e and FIG. 10a). The means
of measured values obtained from n=6 individual donors are well
described by the cumulative distribution function to the gamma
distribution
.psi. ( t ) = t .alpha. - 1 e - .beta.t .GAMMA. ( .alpha. ) .beta.
- .alpha. , ( Fig . 10 b , c ) ##EQU00002##
which were therefore used in simulations of Equation (1). Here,
.GAMMA.(x) is the Euler gamma function, and we determined the
parameters .alpha.,.beta. by nonlinear optimization. Thus, the only
free parameters were division time t.sub.div and death rate
k.sub.d. These values were obtained by non-linear optimization with
respect to experimentally measured cell numbers. For all curve
fitting procedures, the function fitnlm in Matlab, (Mathworks,
version R2017b) was used. Specifically, the distribution .phi.(t)
of first division times from the measured precursor frequencies,
and the division time t.sub.div as well as death rate k.sub.d were
determined by fitting Equation (1) to kinetic cell number data. In
both cases, experimentally obtained kinetic data was averaged
across donors and the variability between donors at each time-point
was considered by weighting the averaged data by the weights
w.sub.l=1/.sigma..sub.l.sup.2, where .sigma..sub.l is the standard
deviation across donors at the l-th measurement time-point.
Validation of Methylation Profiles Using Next Generation
Sequencing
[0202] For PCR amplicon design, locus-specific primers were
designed using an in-house bisulfite primer design tool (see Table
5 for primer sequences). To validate methylation profiles of IFNG
regulatory region CNS1 after in vitro culture of NK cells from
HCMV.sup.- donors, samples were sorted for viable CD56.sup.+
NKG2C.sup.+ cells at day 7 and DNA was isolated using QIAmp DNA
Micro kit (Qiagen) according to the manufacturer's instructions.
Genomic DNA was bisulfite converted using the EZ DNA Methylation
Gold Kit (Zymo Research) following the manufacturer's protocol and
PCR was performed with Hot FirePol DNA Polymerase and Buffer (both
Solis BioDyne) using the following program: 15 min 95.degree. C.;
40 cycles 1 min 95.degree. C., 2.5 min 56.degree. C., and 40 s
72.degree. C.; followed by 7 min 72.degree. C. PCR products were
purified using Agencourt AMPure XP Beads (Beckman Coulter).
Amplicon NGS tags were finalized with HotStartTaq polymerase and
buffer (both Qiagen) using the following program: 15 min 97.degree.
C.; 5 cycles 30 s 97.degree. C., 30 s 60.degree. C., and 30 s
72.degree. C. Purified samples were quantified by Qubit High
Sensitivity Assay (Life Technologies) and diluted to 10 nM.
Finally, all samples were pooled and amplicons were pair-end
sequenced (2.times.300 nt) with two index reads using a MiSeq
reagent kit V2 chemistry on Illumina MiSeq (both Illumina). Raw
sequencing data was quality controlled using FastQC (v0.10.3; on
the World-Wide-Web at:
bioinformatics.babraham.ac.uk/projects/fastqc/) and trimmed for
adaptors and low quality bases using cutadapt (v1.3; on the
internet at: code.google.com/p/cutadapt/) and Trim Galore! (v0.3.3;
on the World-Wide-Web at:
bioinformatics.babraham.ac.uk/projects/trim_galore/). Paired reads
were joined using FLASh (on the internet at:
ccb.jhu.edu/software/FLASH/). Reads were sorted by (i) the NGS
barcode adaptors to assign Sample ID and (ii) the initial 15 bp to
assign amplicon ID. Sorted data was loaded into BiQAnalyzer HiMod
software (on the internet at:
biq-analyzer-himod.bioinf.mpi-inf.mpg.de/) using the following
settings: analyzed methylation context set to "C", minimal sequence
identity set to 0.9 and minimal conversion rate set to 0.95.
Filtered high quality reads were used for methylation calls of the
respective CpG and analyzed using in-house R-scripts.
Gene Expression Analyses Using Next Generation Sequencing
[0203] To assess transcriptome profiles after in vitro culture of
NK cells from HCMV.sup.- donors, samples were sorted for viable
CD56.sup.+ NKG2C.sup.+ cells at day 7 and total RNA was isolated
using RNeasy Plus Micro kit (Qiagen) according to the
manufacturer's instructions. Illumina libraries were prepared using
Smart-Seq v4 mRNA Ultra Low Input RNA Kit (Clontech) and
Nextera.RTM. XT DNA Sample Preparation Kit (Illumina), with up to
10 ng of purified cDNA, according to the manufacturer's
instructions. Libraries were paired-end sequenced (2.times.75 bp)
on an Illumina NextSeq500 device. Raw sequences were mapped to
human GRCh37/hg19 genome with TopHat2.sup.72 in very-sensitive
settings for Bowtie2.sup.73 and GENCODE annotation release 19
(GRCh37.p13). Per gene counts were calculated using
featureCounts.sup.74 and gene expression analyses were performed
using DESeq2 1.18.sup.75. Heat maps for selected genes were
generated using z-scores of rlog-transformed read counts, clustered
by Pearson correlation and Ward minimum variance method.
Statistical Analysis
[0204] Statistical parameters including sample size, employed
statistical tests, and statistical significance are reported in the
Figure Legends. Two groups of paired samples were compared with
two-tailed Wilcoxon test, while three or more groups of paired
samples were analyzed using Friedman test with Dunn's post test to
control for multiple comparisons. Datasets of paired samples
containing two variables (e.g. different peptide sequences and
different concentrations) were compared with repeated-measures
two-way ANOVA with Bonferroni correction. First order kinetics of
decay in HLA-E surface expression obtained from pulse-chase
experiments were analyzed by linear regression and ANCOVA.
Statistical analyses were performed with GraphPad PRISM 7 (Graph
Pad Software) using a confidence level of 0.95 and P-values above
0.05 were considered not significant.
Method for Determining the Surface Expression of HLA-E
[0205] Tumor cells from patients are tested for the expression of
HLA-E by staining with the commercially available antibody clone
3D12 and analysis by flow cytometry.
Methods for Determining the Expression of HLA-G
[0206] Transcripts of HLA-G are detected by an isoform specific
RT-PCR. RNA from tumor cells is reversely transcribed to dDNA and
HLA-G transcripts amplified using the primers G.257F and G.936R
(Paul, P., et al. (2000). "HLA-G, -E, -F preworkshop: tools and
protocols for analysis of non-classical class I genes transcription
and protein expression." Human Immunology 61(11): 1177-1195).
Cycling Conditions:
TABLE-US-00002 [0207] Initial denaturation 94.degree. C. 120
seconds Denaturation 94.degree. C. 60 seconds 35 cycles Primer
annealing 60.degree. C. 60 seconds Primer elongation 72.degree. C.
90 seconds Final elongation 72.degree. C. 300 seconds Pause
4.degree. C.
[0208] Specific bands are detected at 848 bp (isoform G6), 726 bp
(G1, G4, G5), 450 bp (G3), 174 bp (G2). As a positive control, cDNA
produced from the HLA-G expressing cell line JEG-3 is used as a
standard for the specific bands.
[0209] For a quantitative assessment of the isoforms HLA-G1, -G4,
-G5 and G6, a quantitative RT-PCR is performed using the primers
GqF and GqR. Expected amplicon size: 123 bp
Cycling Conditions:
TABLE-US-00003 [0210] Initial denaturation 95.degree. C. 600
seconds Denaturation 94.degree. C. 15 seconds 40 cycles Primer
annealing 60.degree. C. 30 seconds Primer elongation 72.degree. C.
30 seconds
Primers:
TABLE-US-00004 [0211] G.257F 5'-GGAAGAGGAGACACGGAACA-3' (SEQ ID NO.
67) G.936R 5'-GCAGCTCCAGTGACTACAGC-3' (SEQ ID NO. 68) GqF
5'-GAGGAGACACGGAACACCAAG-3' (SEQ ID NO. 69) GqR
5'-GTCGCAGCCAATCATCCACT-3' (SEQ ID NO. 70)
[0212] To assess expression of HLA-G on the protein level, cells
are stained with the commercially available antibody clones MEM-G9
and 87G and analyzed by flow cytometry.
Patients
[0213] The characteristics of the patients used in the present
examples are summarized in Table 3. Abbreviations use in Table 3:
Abbreviations: AML, acute myeloid leukemia; ALL, acute lymphoid
leukemia; ATG, anti-thymocyte globulin; BCNU:
1,3-bis(2-chloroethyl)-1-nitrosourea [carmustine]; Bu, busulfan;
CML, chronic myeloid leukemia; CsA, cyclosporine A; Cy,
cyclophosphamide; FLAMSA, fludarabine-cytarabine-amsacrine; Flu,
fludarabine; GvHD, graft-versus-host disease; HCMV, human
cytomegalovirus; MDS, myelodysplastic syndrome; Mel, melphalan;
MMF: mycophenolate mofetil; MTX, methotrexate; TBI, total body
irradiation; Thio, thiopeta; Tx, transplantation.
Results of the Examples
Summary of the Results
[0214] Natural Killer (NK) cells are innate lymphocytes lacking
antigen-specific rearranged receptors, a hallmark of adaptive
lymphocytes. In some individuals infected by human cytomegalovirus
(HCMV), an NK-cell subset expressing the activating receptor NKG2C
undergoes clonal-like expansion, partially resembling anti-viral
adaptive responses. However, the viral ligand driving the
activation and differentiation of adaptive NKG2C+ NK cells remains
unclear. Here, we demonstrate that adaptive NKG2C+ NK cells
differentially recognize distinct HCMV strains encoding variable
UL40 peptides, which--in combination with pro-inflammatory
signals--control the expansion and differentiation of adaptive
NKG2C+ NK cells. Thus, we propose that polymorphic HCMV peptides
contribute to shape the heterogeneity of adaptive NKG2C+ NK-cell
populations among HCMV-seropositive individuals.
Example 1: Sequence Variations in HCMV UL40-Encoded Peptides
Control the Activation of Adaptive NKG2C+ NK Cells
[0215] Elevated frequency of NKG2C+NK cells as well as an altered
receptor repertoire, including preferential expression of CD2
combined with low levels of Siglec-7, NKG2A, and
Fc.epsilon.R1.gamma., is a central feature of the NK-cell
repertoire in healthy HCMV+ individuals (5, 6, 7, 8, 10). However,
both the percentage of NKG2C+NK cells and their CD2+
Siglec-7-NKG2A-Fc.epsilon.R1.gamma.- adaptive phenotype are
prominently heterogeneous, as indicated by the respective
coefficients of variation (CV) (FIG. 1a, b). This heterogeneity
suggests the involvement of variable host or HCMV factors in
driving the expansion and differentiation of adaptive NKG2C+NK
cells among different individuals. In quest of virus factors
driving this phenomenon, we performed an integrated analysis of 217
clinical isolates obtained by combining sequencing of viral UL40
DNA with a meta-analysis of reported HCMV isolates. The analysis
corroborated considerable heterogeneity within the UL40-encoded
peptide repertoire (30, 31) and the sequences VMAPRTLIL (SEQ ID NO.
3), VMAPRTLLL (SEQ ID NO. 4), and VMAPRTLVL (SEQ ID NO. 5), were
most commonly represented (FIG. 2a, b), while 31.2% of the strains
encoded alternative and not yet described HCMV UL40 sequences, such
as VMAPRTLLM (SEQ ID NO. 27), VMGPRTLLL (SEQ ID NO. 35), VMAPWTLLL
(SEQ ID NO. 17), or VMAPRTLFL (SEQ ID NO. 2) (FIG. 2a, Table 2). We
next asked whether high sequence variability within the HCMV UL40
region could impact on the capacity to stabilize HLA-E and to
activate NKG2C+NK cells. To address this question, murine
TAP-deficient RMA-S cells transfected with human
.beta.2-microglobulin and HLA-E (RMA-S/HLA-E) were pulsed with a
selection of peptides from the sequence analysis, namely VMAPRTLIL
(SEQ ID NO. 3), VMAPRTLVL (SEQ ID NO. 5), VMAPRTLFL (SEQ ID NO. 2,
and VMAPQSLLL (SEQ ID NO. 12), the latter serving as control due to
its poor interaction with NKG2 receptors (30). Stimulation of
purified NK cells with peptide-pulsed RMA-S/HLA-E revealed that
VMAPRTLVL (SEQ ID NO. 5), VMAPRTLIL (SEQ ID NO. 3), and VMAPRTLFL
(SEQ ID NO. 2) differentially triggered TNF, IFN-.gamma., CD107a,
and CCL3 expression by adaptive NKG2C+NK cells from healthy HCMV+
individuals, while no activation was detected when using VMAPQSLLL
(SEQ ID NO. 12)-pulsed targets. Notably, a gradient in the ability
of peptides to induce NKG2C+NK-cell effector functions could be
consistently observed (VMAPRTLFL (SEQ ID NO. 2)>VMAPRTLIL (SEQ
ID NO. 3)>VMAPRTLVL) (SEQ ID NO. 5) (FIG. 2c, d). Differential
activation was also detected in cytotoxicity assays (FIG. 1d).
Importantly, the ability of peptides to activate NKG2C+NK cells
with different efficiency did not depend on their intrinsic ability
to bind HLA-E: with the exception of VMAPQSLLL (SEQ ID NO. 12), the
analyzed UL40-encoded peptides equally stabilized HLA-E at
saturating concentrations (FIG. 1e), which is in line with
comparable binding affinities predicted by the NetMHC 4.0
algorithm32 (FIG. 1f). Moreover, pulse-chase experiments
demonstrated that the decay in surface HLA-E of VMAPRTLIL (SEQ ID
NO. 3)- and VMAPRTLFL (SEQ ID NO. 2)-pulsed cells followed
identical kinetics (FIG. 1g). Along this line, elevated activation
of adaptive NKG2C+ NK cells by VMAPRTLFL (SEQ ID NO. 2) was
consistent upon pulsing with sub-optimal peptide concentrations, at
which VMAPRTLIL (SEQ ID NO. 3) was more efficiently stabilizing
HLA-E (FIG. 2e; FIG. 1h). Further corroborating specific
receptor-mediated recognition, activation was restricted to
NKG2C+NK cells and entirely inhibited in the presence of an
anti-CD94 blocking antibody (FIG. 1i, j). Since both NKG2A and
NKG2C can recognize HLA-E/peptide complexes, we co-cultured
purified NK cells with K562 cells transfected with HLA-E
(K562/HLA-E) pulsed with varying peptide concentrations. As opposed
to murine RMA-S, human K562 represent susceptible targets for all
human NK cells, rendering this experimental platform optimally
tailored for side by side analysis of adaptive NKG2C+ cell
activation and conventional NKG2C- NKG2A+ cell inhibition. In
contrast to adaptive NKG2C+NK-cell activation, NKG2A+ NKG2C- NK
cells did not preferentially respond to VMAPRTLFL(SEQ ID NO.
2)-pulsed targets (FIG. 1k), suggesting different functional
abilities of NKG2C and NKG2A upon interaction with HLA-E/peptide
complexes. Together, these data demonstrate that HCMV isolates
encode for a broad range of HLA-E-stabilizing peptides and that
recognition of distinct HLA-E/peptide complexes controls the
activation of adaptive NKG2C+NK cells.
Example 2: Co-Stimulatory Signals are Required to Elicit
Polyfunctionality of Adaptive NKG2C+NK Cells Upon Engagement with
Sub-Optimal Peptides
[0216] Since NK cells generally rely on cross-linking of multiple
receptors for proficient activation, we next examined whether
differences in peptide recognition could be overcome by
co-engagement of additional NK-cell receptors. Stimulation with
peptide-pulsed K562/HLA-E, which express a broad range of ligands
for NK-cell receptors, resulted in overall amplified NKG2C+ cell
activation, while the hierarchy among peptides was maintained (FIG.
3a). Since effector functions were strongly decreased by blocking
the LFA-3-CD2 axis (11) (FIG. 3b, c), we next examined whether
peptides recognized with low or intermediate functional avidity
could preferentially benefit from CD2 co-stimulation by using
RMA-S/HLA-E transfected with human LFA-3. While stimulation with
VMAPRTLFL (SEQ ID NO. 2)-pulsed RMA-S/HLA-E was sufficient to
induce consistent co-expression of multiple effector functions
(CCL3+ CD107a+ IFN-.gamma.+ TNF+) even in the absence of
co-stimulation, engagement of CD2 was required to elicit
polyfunctional responses upon stimulation with the VMAPRTLIL (SEQ
ID NO. 3) variant (FIG. 4a-d). These findings highlight that
recognition of the VMAPRTLFL (SEQ ID NO. 2) peptide is sufficient
to induce maximal functionality of adaptive NKG2C+NK cells, but
that engagement of the co-stimulatory receptor CD2 can lower the
activation threshold, enabling peptides such as VMAPRTLIL (SEQ ID
NO. 3) to optimally trigger multiple effector functions in adaptive
NKG2C+NK cells.
Example 3: Adaptive NKG2C+NK Cells Differentially Recognize
HCMV-Encoded Peptides During Infection
[0217] To ascertain whether distinct UL40-encoded peptides could be
differentially recognized by adaptive NKG2C+NK cells during
infection, we re-inserted the immunomodulatory US2-6 genes into the
TB40 BAC4 and genetically modified the UL40 locus of the resulting
repaired TB40 (TB40R) HCMV strain to encode either VMAPQSLLL (SEQ
ID NO. 12), VMAPRTLIL (SEQ ID NO. 3), or VMAPRTLFL (SEQ ID NO. 2)
peptides (FIG. 5a). Upon infection of primary human umbilical vein
endothelial cells (HUVEC), UL40 transcripts were abundantly present
as early as 16 hours post infection (FIG. 6a) and the genetically
modified viruses were equally efficient in infecting HUVEC (FIG.
5b, c) as well as in modulating HLA class I levels (FIG. 6b). In
line with previous reports, HCMV infection of HUVEC did not result
in HLA-E up-regulation (33) (FIG. 6c). Importantly, adaptive
NKG2C+NK cells displayed significantly elevated TNF, IFN-.gamma.,
CD107 and CCL3 expression in response to HUVEC infected with
TB40R.sup.UL40_VMAPRTLFL compared to the other strains, while
TB40R.sup.UL40_VMAPRTLIL elicited slightly increased activation of
selected effector functions (FIG. 6d). Differential activation by
HUVEC infected with distinct HCMV strains was further enhanced by
IFN-.gamma. treatment, which can prime anti-viral NK-cell functions
(34) (FIG. 5d, e). In contrast, NKG2C- NK cells were not affected
by virus variants even after IFN-.alpha. priming (FIG. 6e). These
findings suggest that single amino acid exchanges within the UL40
protein can be differentially recognized by adaptive NKG2C+NK cells
during infection and imply that selected mutations in UL40 modulate
adaptive NKG2C+NK-cell responses.
Example 4: Peptide Recognition Controls Relative Accumulation of
NKG2C+NK Cells from HCMV- Individuals in the Presence of
Pro-Inflammatory Signals
[0218] To examine whether peptide recognition not only affects the
differential activation of adaptive NKG2C+NK cells from HCMV+
individuals, but could also influence the extent of NKG2C+NK-cell
proliferation from HCMV- individuals, purified CD56dim NK cells
from HCMV- donors were co-cultured with peptide-pulsed target cells
in the presence of IL-15. In the absence of co-stimulation, only
the VMAPRTLFL (SEQ ID NO. 2) peptide significantly induced
preferential cell division of NKG2C+NK cells (FIG. 7a-b). However,
co-engagement of CD2 synergized with peptide recognition and
enabled both the VMAPRTLIL (SEQ ID NO. 3) and the VMAPRTLFL (SEQ ID
NO. 2) peptides to drive consistently higher proliferation of
NKG2C+NK cells compared to VMAPQSLLL (SEQ ID NO. 12) (FIG. 8a-c,
FIG. 7c). Elevated cell division was reflected in increased
absolute counts, although not in frequency, of NKG2C+NK cells after
7 and 14 days of culture (FIG. 8d-e, FIG. 7d-e). In vivo, CMV
generates a systemic inflammatory milieu (35), which is required
for the generation of MCMV-specific adaptive Ly49H+ mouse NK cells
(36). Integration of pro-inflammatory signals by short-term
addition of IL-12 and IL-18 (IL-12/18) to VMAPRTLVL (SEQ ID NO.
5)-, VMAPRTLIL (SEQ ID NO. 3)-, or VMAPRTLFL (SEQ ID NO. 2)-pulsed
targets resulted in an increase of NKG2C+NK-cell absolute counts as
well as frequencies compared to the non-activating VMAPQSLLL (SEQ
ID NO. 12) peptide (FIG. 9a-c), indicating a permissive role for
pro-inflammatory cytokine signals in the accumulation of NKG2C+
cells in a peptide-dependent fashion. In the early culture period,
presence of IL-12/18 lead to progressive NK-cell loss independent
of peptide recognition, while at later time points, engagement of
NKG2C by VMAPRTLFL (SEQ ID NO. 2) rescued cell numbers, resulting
in increasing frequencies of NKG2C+NK cells (FIG. 9d, e, FIG. 10a).
In order to gain a quantitative understanding of NKG2C+NK-cell
proliferation and accumulation dynamics in this setting, we
modified the Gett-Hodgkin model (37) to explicitly take cell
division and cell death as well as non-dividing, dying cells into
account. Using experimentally determined precursor frequencies
(FIG. 10b, c), division times as well as death rates as fitting
parameters, the model could describe the data experimentally
obtained in the first week of culture, both in the presence (FIG.
9f) and absence of IL-12/18 signaling (FIG. 10d). Without taking
varying cell division times and death rates into account,
experimental differences in precursor frequencies alone could not
explain the observed dynamics of NKG2C+ NK cells (FIG. 10e, f). In
the absence of IL-12/18, model analysis revealed shortened division
times in the presence of VMAPRTLFL (SEQ ID NO. 2)--compared to
VMAPQSLLL (SEQ ID NO. 12)-pulsed targets, while inferred cell death
rates were similar (FIG. 10d). Provision of pro-inflammatory
cytokines during the initial phase of culture resulted in
dramatically accelerated NKG2C+NK-cell division induced by
VMAPRTLFL (SEQ ID NO. 2), while death rates were higher in the
presence of VMAPQSLLL (SEQ ID NO. 12) compared to VMAPRTLFL (SEQ ID
NO. 2) (FIG. 9f). Thus, mathematical model analysis suggests that
the experimentally observed fast proliferation onset and increased
absolute NKG2C+NK-cell numbers in response to VMAPRTLFL (SEQ ID NO.
2)-pulsed targets can be largely explained by accelerated cell
division; and presence of IL-12/18 results in slightly decreased
cell death upon pulsing with VMAPRTLFL (SEQ ID NO. 2) compared to
VMAPQSLLL (SEQ ID NO. 12).
[0219] Collectively, these data show that combined recognition of
distinct HCMV peptides and pro-inflammatory cytokines control the
relative accumulation of NKG2C+NK cells from HCMV- individuals,
potentially contributing to the variable size of the adaptive
NKG2C+NK-cell population observed in healthy HCMV+ individuals.
Example 5: Peptide Recognition and Pro-Inflammatory Cytokines
Co-Operate in Guiding the Differentiation of Adaptive NKG2C+ NK
Cells
[0220] Bac Remodeled receptor repertoires and epigenetic landscapes
are hallmarks of adaptive NKG2C+NK cells in HCMV+ individuals (5,
6, 7, 8, 9). Since peptide recognition was required to enable
relative accumulation of NKG2C+NK-cells from HCMV- individuals in
the presence of IL-12/18, we examined the individual and combined
contributions of the potent VMAPRTLFL (SEQ ID NO. 2) peptide and of
pro-inflammatory cytokines to initiate adaptive NK-cell
differentiation. While culture with VMAPRTLFL (SEQ ID NO. 2) alone
only had mild effects, the combination of VMAPRTLFL (SEQ ID NO. 2)
with IL-12/18 skewed expression of several markers including CD2,
Siglec-7, educating KIR, Syk, and CD161 preferentially in NKG2C+
cells (FIG. 11a-b, FIG. 12a). Moreover, short stimulation with
IL-12/18 played a predominant role in inducing down-regulation of
Fc.epsilon.R1.gamma. and CD7 in both NKG2C+ and NKG2C- cells (FIG.
12a, b), while other factors might control NKG2A and DNAM-1
dynamics, since their expression was variable after culture (FIG.
12a, b). Notably, levels of DNA methylation of the IFNG conserved
non-coding sequence (CNS1), which is hypo-methylated in adaptive NK
cells (9, 11), were strikingly reduced after combined treatment
with VMAPRTLFL (SEQ ID NO. 2) and IL-12/18 (FIG. 11c, d). Overall,
NKG2C engagement co-operated with pro-inflammatory cytokines in
guiding the differentiation of NKG2C+NK cells from HCMV-
individuals. As VMAPRTLFL (SEQ ID NO. 2)+IL-12/18 stimulation of
NKG2C+NK cells from HCMV- individuals appeared to most efficiently
favor the phenotypic skewing towards adaptive NK cells, we next
analyzed the global transcriptional imprinting induced by the
combined stimuli. Apart from SIGLEC7, CD7, SYK, and CD2, VMAPRTLFL
(SEQ ID NO. 2)+IL-12/18 resulted in consistent transcriptional
modulation of several other markers associated with adaptive NK
cells including NCR3 (NKp30), SH2DB1 (EAT2) and ZBTB32 (PLZP),
while the expression of other adaptive genes such as ZBTB16 (PLZF),
ZBTB20, ITGAL or CRTAM was altered only in some individuals (FIG.
11e; FIG. 12c) (5, 7, 38). Functionally, VMAPRTLFL (SEQ ID NO.
2)+IL-12/18 promoted sustained up-regulation of activation and
exhaustion markers such as HLA-DR, TNFRSF9 (4-1 BB), LAG3, CTLA4,
and PDCD1 (PD1) as well as of effector functions including IFNG,
TNF, CCL3, CCL4, IL8, CSF2, IL10, GZMB, and TNFSF10 (TRAIL) (FIG.
12d), indicating that the combination of these two stimuli promote
broad transcriptional imprinting of phenotypic and effector
features typical of adaptive NK cells.
[0221] Finally, to substantiate the data obtained from in vitro
systems, we monitored a cohort of hematopoietic stem cell
transplantation (HSCT) patients, which did or did not reactivate
HCMV (Table 3). Upon detection of HCMV reactivation, the
peptide-encoding UL40 region of the strain causing the acute
infection was sequenced. Next, the NK-cell phenotype was
comparatively analyzed after resolution of acute infection
selectively in patients infected with VMAPRTLFL (SEQ ID NO. 2)- or
VMAPRTLIL (SEQ ID NO. 3)-encoding viruses (FIG. 13a). In line with
HCMV causing the expansion of NKG2C+ NK cells (39, 40), percentages
of NKG2C-expressing CD56dim NK cells were elevated in patients with
HCMV reactivation, although this was not consistently impacted by
HCMV strains (FIG. 14a, b) and NKG2C+ NK-cell frequencies were
dynamic in time among patients infected with both VMAPRTLIL (SEQ ID
NO. 3) or VMAPRTLFL (SEQ ID NO. 2) HCMV variants (FIG. 13c).
Notably, frequencies of NK cells displaying the adaptive phenotype
CD2+ Siglec-7-NKG2A-Fc.epsilon.R1.gamma.-were elevated in NKG2C+ NK
cells derived from patients infected with VMAPRTLFL (SEQ ID NO.
2)-encoding viruses (FIG. 14c, FIG. 13b). Conversely, patients
infected with VMAPRTLIL (SEQ ID NO. 3) variants displayed varying
degrees of adaptive NK-cell differentiation (FIG. 14c, FIG. 13b).
Phenotypic alterations were acquired early after HCMV-reactivation
within the NKG2C+ compartment and remained relatively stable over
time (FIG. 14d), implying that this phenomenon is largely uncoupled
from NK-cell maturation after HSCT. Together with the data from in
vitro experiments, these findings imply that recognition of
distinct UL40-encoded peptides differentially contributes to the
accumulation and differentiation of adaptive NKG2C+NK cells during
infection.
[0222] In summary, our data provide evidence for sensitive
recognition of HCMV-encoded peptides by adaptive NKG2C+NK cells,
which--in co-operation with pro-inflammatory cytokine cues--drives
their activation and shapes their population size as well as their
phenotypic features in response to HCMV infection. Thus, we propose
that the diversity of the NK-cell repertoire among healthy HCMV+
individuals is impacted by the HCMV strain causing the
infection.
Discussion of Examples 1-5
[0223] HCMV-induced adaptive NKG2C+NK cells display several
features partially recapitulating aspects of T-cell biology,
including clonal-like expansion, reprogramming of their recognition
repertoire, and global epigenetic remodeling (41). In this study,
we propose that expression of NKG2C marks an additional adaptive
feature of innate lymphocytes, equipping NKG2C+NK cells with a germ
line-encoded receptor displaying fine peptide specificity and
enabling differential recognition of HCMV strains. These findings
are even more striking considering our previous identification of
CD8+ T cells bearing T-cell receptors (TCR) specific for UL40
peptide/HLA-E complexes, which indicates UL40 as a shared epitope
recognized by both NKG2C+NK cells and CD8+ T cells (42). It seems
counter-intuitive that HCMV has maintained an activating ligand for
NKG2C, despite extensive co-evolution with ist human host.
Inversely, UL40-encoded peptides were suggested to serve as an
immune evasion strategy by maintaining HLA-E surface expression in
the absence of HLA-A/B/C-derived peptides, thus preventing
activation of NK cells expressing the cognate inhibitory receptor
NKG2A22. Indeed, peptides unable to stabilize HLA-E are rarely
encoded by UL40, suggesting that expression of UL40 could be an
advantage for the virus. However, our data indicate that the
inhibitory function of NKG2A is only subtly tuned by recognition of
distinct peptides, unlikely explaining highly polymorphic UL40
sequences. Conversely, UL40 polymorphisms result in NKG2C-mediated
responses with different functional avidities: recognition of
potent HCMV peptides is sufficient for optimal activation of
NKG2C+NK cells, while peptides with lower potency rely more
stringently on co-stimulatory signals. Thus, peptides with lower
potency might provide a window of opportunity for the virus to
suppress activation of NKG2A+ NK cells and simultaneously allow a
certain degree of NKG2C+ NK-cell activation on the host side.
Therefore, we propose that immune pressure mediated by adaptive
NKG2C+NK cells could underlie the heterogeneity of UL40 sequences.
This immune pressure potentially explains the relative rareness of
the VMAPRTLFL (SEQ ID NO. 2) peptide--displaying potent functional
avidity for NKG2C- and the high frequency of UL40-encoded peptides
with low or intermediate avidity towards NKG2C.
[0224] Interestingly, commonly represented HCMV UL40 peptides with
low or intermediate avidity toward NKG2C, such as VMAPRTLIL (SEQ ID
NO. 3) and VMAPRTLVL (SEQ ID NO. 5), closely resemble self-peptides
derived from HLA class I alleles. We envisage that these peptides
would not promote expansion and differentiation of adaptive
NKG2C+NK cells at steady state. However, viral mimics with
identical sequences might enable activation and differentiation of
adaptive NKG2C+NK cells in the presence of pro-inflammatory
signaling and HLA class I down-regulation, which concomitantly
occur during HCMV infection. Conversely, the rare UL40-derived
VMAPRTLFL (SEQ ID NO. 2) peptide, which is particularly efficient
in driving adaptive NKG2C+NK-cell responses, mimics the signal
peptide of HLA-G, the expression of which is restricted to
pregnancy and up-regulated in tumors or during inflammation (43).
In light of these data, it is conceivable that activation and
expansion of adaptive NKG2C+NK cells from HCMV+ individuals
observed during heterologous infections (44, 45) could reflect
cross-reactivity of NKG2C towards other viral or even self-peptides
in the presence of co-stimulation and inflammatory conditions.
Structural analyses of CD94/NKG2 heterodimers have indicated that
the C-terminus of peptides presented on HLA-E primarily interacts
with CD94 (25, 26, 27). Intriguingly, our data imply that
engagement of CD94/NKG2A and CD94/NKG2C by HLA-E/peptide complexes
results in strikingly differential and peptide-dependent functional
consequences. Although NKG2A and NKG2C display high sequence
homology, several amino acids distinct between the two proteins are
located in close proximity to the interface with CD94 (25, 26).
Accordingly, it was suggested that slight differences in the amino
acid sequence between NKG2A and NKG2C could alter the conformation
or orientation of the CD94/NKG2 heterodimers (25, 26). Such
conformational changes could indirectly affect the recognition of
the C-terminal sequence of HLA-E-bound peptides by CD94, resulting
in distinct interaction strengths and functional avidities by the
two heterodimers.
[0225] Despite the pivotal role of NKG2C in activating NK cells
during infection and a case report suggesting that NKG2C+NK cells
can control HCMV infection in the absence of T cells in vivo (46),
individuals deficient for KLRC2 (encoding NKG2C) lack a clinical
phenotype (11, 47). These findings imply the presence of
compensatory mechanisms and suggest that NKG2C recognition of
HLA-E/UL40 peptide complexes does not represent the only
HCMV-derived signal driving adaptive responses of specific NK-cell
subsets. Indeed, adaptive NKG2C- NK cells expressing activating KIR
have been described in KLRC2-deficient and -sufficient humans (8,
11, 48) and recognition of yet unknown viral ligands by activating
KIR or other receptors could result in expansion and
differentiation of adaptive cells within the NKG2C- compartment.
Supporting this scenario, it was recently reported that selected
HCMV strains can modulate HLA-C on infected cells and thereby
induce activation of KIR2DS1+ NK cells (49). Similarly,
Ly49H-independent NK-cell control of MCMV has been demonstrated in
mouse strains other than C57BL/6 (50, 51), reinforcing the concept
that NK-cell subsets expressing distinct receptors provide multiple
and partially redundant recognition strategies of CMV-infected
cells in both humans and mice. Generation of murine adaptive Ly49H+
NK cells requires cross-linking of Ly49H (12, 13, 52) and is
enhanced by DNAM-1 co-stimulation53. Furthermore, IL-12 is
essential for the expansion and protective function of
virus-specific Ly49H+ NK cells (36). Our data indicate that
pro-inflammatory signaling via IL-12/18, which was shown to
generate memory-like NK-cell properties independent of CMV
infection (54, 55), results in fundamental phenotypic and
functional changes and initiates adaptive NK-cell differentiation.
However, provision of IL-12/18 is not sufficient to expand NKG2C+
NK cells form HCMV- individuals and induces sustained
activation-induced cell death. When exposed to IL-12/18, the
strength of peptide recognition by NKG2C correlated with the
numerical and relative expansion of NKG2C+ NK cells, implying that
the quality of peptide recognition contributes to configure
variable pools of differentiated adaptive NK cells in infected
individuals.
[0226] In conclusion, our data reveal an exquisite mechanism of
specific innate recognition of HCMV- infected cells as a novel
adaptive property of NKG2C+ NK cells. By recognizing variable
UL40-encoded peptides, NKG2C can contribute to dynamic shifts in
the NK-cell compartment, favoring the expansion and differentiation
of adaptive subsets based on the strength of peptide recognition.
On the pathogen side, variability in the UL40 peptide repertoire
can be regarded as an adaptation attempt of HCMV to modulate host
responses. Together, peptide recognition by NKG2C and sequence
variants of UL40-derived peptides further expose the well-adapted
host-pathogen interaction between NK cells and HCMV.
Example 6: Induction of a Protective Anti-Tumor Response
[0227] The HCMV peptide VMAPRTLFL is also found in the leader
sequence of HLA-G (Llano et al., 1998; European journal of
immunology 28, 2854-2863), another non-classical MHC class I
molecule which is not expressed in most healthy tissues but
frequently upregulated by tumors (Curigliano et al. 2013; Clin
Cancer Res. 2013; 19(20):5564-71). HLA-G inhibits immune cells by
directly binding to inhibitory receptors of the LIR-family, such as
LILRB1, which allows tumor cells to evade immune recognition. On
the other hand, VMAPRTLFL (SEQ ID NO. 2) is cleaved off the signal
sequence of HLA-G and presented an HLA-E (Llano et al., 1998;
European journal of immunology 28, 2854-2863), making HLA-G/HLA-E
coexpressing tumors potentially susceptible to recognition by
NKG2C+ NK cells.
[0228] Indeed, we could demonstrate this effect using a cell line
transfected with HLA-G. Compared to the untransfected control,
HLA-G-expressing cells specifically activated NKG2C+ NK cells, even
in the context of low HLA-E expression (FIG. 15a-b, FIG. 16). The
activation was blocked by an antibody against CD94 (FIG. 15c),
demonstrating its role in recognition. These data demonstrate the
reactivity of NKG2C+ NK cells against HLA G expressing targets.
Since in this system HLA-G is extrinsically overexpressed to very
high levels and the endogenous expression of HLA-E is low (Supp.
FIG. 1), we assessed the activation under blockade of the
inhibitory HLA-G receptor LILRB1. The blockade further increased
the activation, arguing that the in vivo NK cell response might be
even more pronounced, as endogenous expression of HLA-G will not be
that much higher than of HLA-E and therefore the inhibitory signal
through LILRB1 will be less dominant. Based on these findings, we
propose the in vivo expansion of NKG2C+ NK cells as an
immunotherapy to treat HLA-G/HLA-E co-expressing tumors.
[0229] To identify patients suitable for these treatment
strategies, tumor biopsies or circulating tumor cells can be tested
for HLA-G and HLA-E expression by flow cytometry or by RT-PCR.
Isolated tumor cells from biopsies can also be directly tested for
susceptibility to lysis by NKG2C+ adaptive NK cells in in vitro
cytotoxicity assays using adaptive NKG2C+ NK cells. Our approach of
NKG2C+ NK cell expansion might be especially valuable for patients
receiving HSCT for the treatment of leukemia. In contrast to
adaptive cell types targeted by classical vaccines, NK cells are
among the first lymphocyte populations to recover after
transplantation and therefore can be targeted early using our
approach, thus potentially protecting against HCMV reactivation and
tumor relapse. Secondly, an interesting correlation of reduced
relapse rates in patients reactivating HCMV and presenting with
adaptive NK cell expansions points towards an anti-leukemic effect
of adaptive NK cells (Elmagaacli et al., Blood. 2011;
118(5):1402-12; Cichocki et al. Leukemia. 2016; 30(2):456-63.)
Therefore, our approach might be of dual advantage for these
patients, combining protective effects against HCMV with an
enhanced graft-versus-leukemia response.
Tables
TABLE-US-00005 [0230] TABLE 2 Abundance of peptide identified
peptide sequences in clinical isolates. Identified Number of
Peptide clinical SEQ ID NO Sequence Isolates 3 VMAPRTLIL 88 4
VMAPRTLLL 35 5 VMAPRTLVL 27 6 VMAPRSLLL 7 7 VMAPRSLIL 6 8 VMTPRTLVL
6 2 VMAPRTLFL 3 9 VMAPRILIL 3 10 AMAPRTLIL 3 11 VIAPRTLVL 2 12
VMAPQSLLL 2 13 VMAPRTFVL 2 14 VMTPRTLIL 2 15 VTAPRTLIL 2 16
VTAPRTLLL 2 17 VMAPWTLLL 2 18 VMVPRSLIL 1 19 AMAPRTLVL 1 20
VIAPRTLIL 1 21 VIAPRTLLL 1 22 VLAPRTLIL 1 23 VMALRTLIL 1 24
VMAPRGLIL 1 25 VMAPRNLIL 1 26 VMAPRTLFV 1 27 VMAPRTLLM 1 28
VMAPRTLVM 1 29 VMAPRTSLL 1 30 VMAPRTSVL 1 31 VMAPWTLIL 1 32
VMAPWTLVL 1 33 VMDPRTLLL 1 34 VMGPRTLIL 1 35 VMGPRTLLL 1 36
VMVPQTLIL 1 37 VMVPRTLLL 1 38 VTAPRTLVL 1 39 VVAPRTLIL 1 40
VVAPRTLLL 1 41 VMVPRTLIL 1 42 VMATRTLLL 1 TOTAL 217
TABLE-US-00006 TABLE 3 Patient Characteristics HCMV Patient Gen-
GvHD serostatus ID Age der Diagnosis Conditioning Graft type
prophylaxis (pre Tx) 1636 69 Male AML BCNU/ 10/10 ATG/CsA/ Negative
Flu/Mel MMF 1555 66 Female AML FLAMSA/ 9/10 ATG/CsA/ Negative Bu/Cy
MMF 1637 62 Male AML BCNU/ 10/10 ATG/CsA/ Negative Flu/Mel MMF 1642
39 Male Hodgkin Cy/Flu/ Mismatched Cyclo/CsA/ Negative TBI sibling
MMF 1553 30 Female CML Bu/Cy 10/10 ATG/CsA/ Negative MTX #018 58
Female AML Bu/Flu/ 10/10 CsA/MMF Negative ATG sibling #050 52 Male
AML TBI/Flu/ 10/10 CsA/MMF Negative Cy/ATG #063 71 Male AML
TBI/Flu/ 9/10 CsA/MMF Negative Cy/ATG #071 60 Male AML Bu/Flu/
10/10 CsA/MMF Negative ATG sibling #078 72 Male AML TBI/Flu/ 10/10
CsA/MMF Negative ATG 1553 33 Female AML FLAMSA- 9/10 ATG/CsA/
Positive Bu MMF 1606 40 Female CML Bu/Cy 10/10 ATG/CsA/ Positive
MTX 1628 56 Male MDS FLAMSA/ 9/10 ATG/CsA/ Positive Bu MMF 1630 70
Female AML BCNU/ 10/10 ATG/CsA/ Positive Flu/Mel MMF 1640 29 Male
AML Thio/ Mismatched Cyclo/CsA/ Positive Bu/Flu sibling MMF #074 53
Female AML TBI/Cy/ 10/10 CsA/MTX Positive ATG #033 21 Male ALL
TBI/Cy/ 9/10 CsA/MTX Positive ATG #069 65 Female AML Bu/Flu/ 10/10
CsA/MTX Positive ATG #008 43 Male AML TBI/Cy/ 10/10 CsA/MTX
Positive ATG #023 52 Female AML Bu/Flu/ 9/19 CsA/MMF Positive ATG
#087 58 Male AML Bu/Flu/ 10/10 CsA/MMF Positive ATG #075 60 Male
MDS Bu/Flu/ 10/10 CsA/MMF + Positive ATG Basiliximab Patient
Characteristics HCMV reactivation Phenotype % CD2.sup.+ [days post
analysis % % % NKG2A.sup.- Tx] UL40 [days CD56.sup.bright
NKG2A.sup.+ NKG2C.sup.+ Siglec-7.sup.- Patient (detection peptide
post of CD3.sup.- of of FceR1g.sup.- ID method) sequence Tx]
CD56.sup.+ CD56.sup.dim CD56.sup.dim of 1636 Not detected N/A 180
7.59 45.1 7.26 0.57 1555 Not detected N/A 180 18.9 73.7 10.3 1.33
1637 Not detected N/A 180 3.88 36.5 9.32 0.30 1642 Not detected N/A
60 29.7 88.9 10.8 0.90 1553 Not detected N/A 60 38.6 69.6 6.05 0.76
#018 Not detected N/A 35 45.0 81.2 8.56 0.00 #050 Not detected N/A
119 72.9 94.1 9.73 0.67 #063 Not detected N/A 35 42.5 91.0 3.97
7.16 #071 Not detected N/A 153 29.7 88.9 8.49 2.75 #078 Not
detected N/A 101 57.3 56.5 7.05 0.77 1553 30-60 (pp65) VMAPRTLIL
120 5.44 33.7 38.7 5.49 1606 60 (pp65) VMAPRTLIL 180 27.1 41.6 37.9
9.89 1628 120 (pp65) VMAPRTLIL 180 5.41 27 25.1 22.4 1630 60 (pp65)
VMAPRTLIL 180 18.6 81.4 21.2 33.2 1640 30 (pp65) VMAPRTLIL 180 19.6
74.0 24.8 14.4 #074 12 (viremia) VMAPRTLIL 105 13.7 79.4 17.5 3.25
#033 25 (viremia) VMAPRTLIL 53 31.6 85.5 10.8 0.36 #069 31
(viremia) VMAPRTLIL 46 13.9 87.9 9.10 2.86 #008 12 (viremia)
VMAPRTLIL 55 13.9 91.2 8.61 2.66 #023 12 (viremia) VMAPRTLIL 215
9.31 53.1 10.8 9.74 #087 27 (viremia) VMAPRTLFL 190 11.4 26.4 49.9
46.9 #075 104 VMAPRTLFL 278 17.8 78.6 15.3 35.2 (viremia)
TABLE-US-00007 TABLE 4 Antibodies Used for Flow Cytometry and
Functional Assays. Catalogue Antibody Supplier number CD14 BV510
(clone: M5E2) BioLegend Cat#301842 CD19 BV510 (clone: HIB19)
BioLegend Cat#302242 CD2 PerCP-Cy5.5 BioLegend Cat#300216 (clone:
RPA-2.10) CD3 PE-Cy5 (clone: UCHT1) BioLegend Cat#300410 CD56
PE-Dazzle594 BioLegend Cat#318348 (clone: HCD56) CD57 Pacific Blue
(clone: HCD57) BioLegend Cat#322316 HLA class I Pacific Blue
BioLegend Cat#311418 (clone: W6/32) IgM BV605 (clone: RMM-1)
BioLegend Cat#406523 KIR3DL1 Alexa700 (clone: DX9) BioLegend
Cat#312712 KIR3DL1 PerCP-Cy5.5 BioLegend Cat#312718 (clone: DX9)
LFA-3 PE (clone: TS2/9) BioLegend Cat#330905 LFA-3 Purified (clone:
TS2/9) BioLegend Cat#330912 Streptavidin BV785 BioLegend Cat#405249
TNF BV605 (clone: Mab11) BioLegend Cat#502936 CD7 FITC (clone:
CD7-667) BioLegend Cat#982704 CD161 BV785 (clone: HP-3G10)
BioLegend) Cat#339930 CD3 Cy5 (clone: UCHT1) In house N/A
Fc.epsilon.R1.gamma. FITC (rabbit polyclonal) Merck Millipore
Cat#FCABS400F HCMV-IE Alexa488 (clone: 8B1.2) Merck Millipore
Cat#MAB810X DNAM-1 PE-Vio770 Miltenyi Cat#130-099-966 (clone: DX11)
CCL3 APC (clone: REA254) Miltenyi Biotec Cat#130-103-630 HLA-E PE
(clone: 3D12) Miltenyi Biotec Cat#130-096-849 IFN-.gamma. PE-Vio770
(clone: 45-15) Miltenyi Biotec Cat#130-096-752 KIR2DL1 APC (clone:
REA284) Miltenyi Biotec Cat#130-103-935 KIR2DL1 APC-Vio770 Miltenyi
Biotec Cat#130-103-937 (clone: REA284) KIR2DL3 FITC (clone: REA147)
Miltenyi Biotec Cat#130-100-125 NKG2A Biotin (clone: REA110)
Miltenyi Biotec Cat#130-098-819 NKG2A PE-Vio770 (REA110) Miltenyi
Biotec Cat#130-105-647 NKG2C PE (clone: REA205) Miltenyi Biotec
Cat#130-103-635 Siglec-7 APC-Vio770 Miltenyi Biotec Cat#130-101-009
(clone: REA214) Siglec-7 PE-Vio700 Miltenyi Biotec Cat#130-100-975
(clone: REA214) KIR2DL2/2DS2/2DL3 A. Morretta N/A Alexa700 (clone:
GL183) (in house conjugated) CD94 unconjugated RnD Systems
Cat#MAB1058 (clone: #131412) IgG1 isotype control unconjugated RnD
Systems Cat#MAB002 (clone: #11711) CD107a Alexa488 (clone: H3A4)
ThermoFisher Cat#65-0865-18 CD57 unconjugated (clone: TBO1)
ThermoFisher Cat#16-0577-85 Syk APC (clone: 4D10.1) ThermoFisher
Cat#17-6696-42
TABLE-US-00008 TABLE 5 Primer sequences used for nested PCR,
RT-qPCR and DNA methylation analysis. SEQ ID Primer Name No. Primer
Sequence UL40_forward 43 5'-GGCTCTGTCTCGTCGTCATT-3' UL40_reverse 44
5'-TAAGGGCACTCGTGAGGATG-3' UL40_nested_forward 45
5'-CAACAGTCGGCAGAATGAAC-3' UL40_nested_reverse 46
5'-CTGGAACACGAGCGGACATA-3' hGAPDH_forward 47
5'-TTCGTCATGGGTGTGAACCA-3' hGAPDH_reverse 48
5'-GGACTGTGGTCATGAGTCCTT-3' IFNG_CNS1_forward 49
5'-TCTTTCCCTACACGACGCTCTTCCGATCTATGAGTTAATATTGGATTTATGTTTTT-3'
IFNG_CNS1_reverse 50
5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATATAAATAACATACCTTCTATATTCTTT-3'
NGS_forward 51
5-CAAGCAGAAGACGGCATACGAGATXXXXXXGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3'
NGS_reverse 52
5'-AATGATACGGCGACCACCGAGATCTACACXXXXXXTCTTTCCCTACACGACGCTCTTCCGATC-3'
IFNG CNS1-specific oligos were designed to have a region-specific
3' part (bold) and a Next generation sequencing (NGS)-compatible 5'
tag. NGS oligos were employed to introduce unique DNA barcodes
(marked as X) to individual samples.
TABLE-US-00009 TABLE 6 Primer Sequences Used for HCMV Mutagenesis.
Primer Name SEQ ID No. Primer Sequence US2-lox-tet.for 53
5'-GATCACATCCCCTTGCAGTACCAGACGCAGGGCTATAACTTCGTATAATGTATGCTATACGAAGTTATGG-
CTGG TTTATGCATATCG-3' G-lox-tet.rev 54
5'-TGCATGCCATGGTACCCGGGAGCTCGAATTCGAAGCTTCTTTCCCTTTGTCAACAGCAA-3'
pIC1.for 55 5'-CTAGCAGATCTGCAGGGACGCATCGTGGCCGGAT-3' MC-Cre.rev 56
5'-TAAGGATGCATGTTTAAACGTGACCACGTCGTGGAATGC-3' KnR-ISce.for 57
5'-GTGGTCACGTTTAAACATGCATCCTTAATTAAGGCTGCGATCTATCGAGTTTTCCCAGTCACGACG-3'
pIC.rev 58 5'-TAGGAAGCTTGATATCGAACAAACGA000AACACC-3' loxPUS2.for 59
5'-GCCAGATCACATCCCCTTGCAGTACCAGACGCAGGGCTAGGACGGTATCGATAAGCTGGAT-3'
KanRITyellow.rev 60
5'-TGCATGCCATGGTACCCGGGAGCTCGAATTCGAAGCTTCTGACGCATCGTGGCCGGATCTC-3'
UL40_VMAPRTLI 61
5'-ACAGGAGTCCAAGCGTCAGAATTAAAGTCCGCGGAGCCATAACCGCGCAAGTGAAGCCGACGCATCGTGG-
CC L.for GGAT-3' UL40_VMAPRTLI 62
5'-AAATTCAGCAACACTCGTATCGGCTTCACTTGCGCGGTTATGGCTCCGCGGACTTTAATGGTGACCACGT-
CGT L.rev GGA-3' UL40_VMAPRTL 63
5'-TCCTCATACACAGGAGTCCAAGCGTCAGAAATAAAGTCCGCGGAGCCATAACCGCGCAAGGACGCATCGT-
GG FL.for CCGGAT-3' UL40_VMAPRTL 64
5'-AACACTCGTATCGGCTTCACTTGCGCGGTTATGGCTCCGCGGACTTTATTTCTGACGCTTTGGTGACCAC-
GTC FL.rev GTGGA-3' UL40_VMAPOSL 65
5'-TCCTCATACACAGGAGTCCAAGCGTCAGAAGTAAACTCTGCGGAGCCATAACCGCGCAAGGACGCATCGT-
GG LL.for CCGGAT-3' UL40_VMAPOSL 66
5'-AACACTCGTATCGGCTTCACTTGCGCGGTTATGGCTCCGCAGAGTTTACTTCTGACGCTTTGGTGACCAC-
GTC LL.rev GTGGA-3'
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Sequence CWU 1
1
7019PRTArtificial Sequencesynthesized peptideSITE(8)...(8)Xaa is an
amino acid with a hydrophobic side chain (Ala, Ile, Leu, Phe, Val,
Pro, Gly), preferably Val, Leu, Ile or Phe. 1Val Met Ala Pro Arg
Thr Leu Xaa Leu1 529PRTArtificial Sequencesynthesized peptide 2Val
Met Ala Pro Arg Thr Leu Phe Leu1 539PRTArtificial
Sequencesynthesized peptide 3Val Met Ala Pro Arg Thr Leu Ile Leu1
549PRTArtificial Sequencesynthesized peptide 4Val Met Ala Pro Arg
Thr Leu Leu Leu1 559PRTArtificial Sequencesynthesized peptide 5Val
Met Ala Pro Arg Thr Leu Val Leu1 569PRTArtificial
Sequencesynthesized peptide 6Val Met Ala Pro Arg Ser Leu Leu Leu1
579PRTArtificial Sequencesynthesized peptide 7Val Met Ala Pro Arg
Ser Leu Ile Leu1 589PRTArtificial Sequencesynthesized peptide 8Val
Met Thr Pro Arg Thr Leu Val Leu1 599PRTArtificial
Sequencesynthesized peptide 9Val Met Ala Pro Arg Ile Leu Ile Leu1
5109PRTArtificial Sequencesynthesized peptide 10Ala Met Ala Pro Arg
Thr Leu Ile Leu1 5119PRTArtificial Sequencesynthesized peptide
11Val Ile Ala Pro Arg Thr Leu Val Leu1 5129PRTArtificial
Sequencesynthesized peptide 12Val Met Ala Pro Gln Ser Leu Leu Leu1
5139PRTArtificial Sequencesynthesized peptide 13Val Met Ala Pro Arg
Thr Phe Val Leu1 5149PRTArtificial Sequencesynthesized peptide
14Val Met Thr Pro Arg Thr Leu Ile Leu1 5159PRTArtificial
Sequencesynthesized peptide 15Val Thr Ala Pro Arg Thr Leu Ile Leu1
5169PRTArtificial Sequencesynthesized peptide 16Val Thr Ala Pro Arg
Thr Leu Leu Leu1 5179PRTArtificial Sequencesynthesized peptide
17Val Met Ala Pro Trp Thr Leu Leu Leu1 5189PRTArtificial
Sequencesynthesized peptide 18Val Met Val Pro Arg Ser Leu Ile Leu1
5199PRTArtificial Sequencesynthesized peptide 19Ala Met Ala Pro Arg
Thr Leu Val Leu1 5209PRTArtificial Sequencesynthesized peptide
20Val Ile Ala Pro Arg Thr Leu Ile Leu1 5219PRTArtificial
Sequencesynthesized peptide 21Val Ile Ala Pro Arg Thr Leu Leu Leu1
5229PRTArtificial Sequencesynthesized peptide 22Val Leu Ala Pro Arg
Thr Leu Ile Leu1 5239PRTArtificial Sequencesynthesized peptide
23Val Met Ala Leu Arg Thr Leu Ile Leu1 5249PRTArtificial
Sequencesynthesized peptide 24Val Met Ala Pro Arg Gly Leu Ile Leu1
5259PRTArtificial Sequencesynthesized peptide 25Val Met Ala Pro Arg
Asn Leu Ile Leu1 5269PRTArtificial Sequencesynthesized peptide
26Val Met Ala Pro Arg Thr Leu Phe Val1 5279PRTArtificial
Sequencesynthesized peptide 27Val Met Ala Pro Arg Thr Leu Leu Met1
5289PRTArtificial Sequencesynthesized peptide 28Val Met Ala Pro Arg
Thr Leu Val Met1 5299PRTArtificial Sequencesynthesized peptide
29Val Met Ala Pro Arg Thr Ser Leu Leu1 5309PRTArtificial
Sequencesynthesized peptide 30Val Met Ala Pro Arg Thr Ser Val Leu1
5319PRTArtificial Sequencesynthesized peptide 31Val Met Ala Pro Trp
Thr Leu Ile Leu1 5329PRTArtificial Sequencesynthesized peptide
32Val Met Ala Pro Trp Thr Leu Val Leu1 5339PRTArtificial
Sequencesynthesized peptide 33Val Met Asp Pro Arg Thr Leu Leu Leu1
5349PRTArtificial Sequencesynthesized peptide 34Val Met Gly Pro Arg
Thr Leu Ile Leu1 5359PRTArtificial Sequencesynthesized peptide
35Val Met Gly Pro Arg Thr Leu Leu Leu1 5369PRTArtificial
Sequencesynthesized peptide 36Val Met Val Pro Gln Thr Leu Ile Leu1
5379PRTArtificial Sequencesynthesized peptide 37Val Met Val Pro Arg
Thr Leu Leu Leu1 5389PRTArtificial Sequencesynthesized peptide
38Val Thr Ala Pro Arg Thr Leu Val Leu1 5399PRTArtificial
Sequencesynthesized peptide 39Val Val Ala Pro Arg Thr Leu Ile Leu1
5409PRTArtificial Sequencesynthesized peptide 40Val Val Ala Pro Arg
Thr Leu Leu Leu1 5419PRTArtificial Sequencesynthesized peptide
41Val Met Val Pro Arg Thr Leu Ile Leu1 5429PRTArtificial
Sequencesynthesized peptide 42Val Met Ala Thr Arg Thr Leu Leu Leu1
54320DNAArtificial Sequencesynthesized peptide 43ggctctgtct
cgtcgtcatt 204420DNAArtificial Sequencesynthesized peptide
44taagggcact cgtgaggatg 204520DNAArtificial Sequencesynthesized
peptide 45caacagtcgg cagaatgaac 204620DNAArtificial
Sequencesynthesized peptide 46ctggaacacg agcggacata 204720DNAHomo
sapiens 47ttcgtcatgg gtgtgaacca 204821DNAHomo sapiens 48ggactgtggt
catgagtcct t 214956DNAHomo sapiens 49tctttcccta cacgacgctc
ttccgatcta tgagttaata ttggatttat gttttt 565063DNAHomo sapiens
50gtgactggag ttcagacgtg tgctcttccg atctatataa ataacatacc ttctatattc
60ttt 635164DNAArtificial Sequencemisc_feature(25)..(30)n is a, c,
g, or tsynthesized nucleotide sequence 51caagcagaag acggcatacg
agatnnnnnn gtgactggag ttcagacgtg tgctcttccg 60atct
645263DNAArtificial Sequencemisc_feature(30)..(35)n is a, c, g, or
tsynthesized nucleotide sequence 52aatgatacgg cgaccaccga gatctacacn
nnnnntcttt ccctacacga cgctcttccg 60atc 635387DNAArtificial
Sequencesynthesized nucleotide sequence 53gatcacatcc ccttgcagta
ccagacgcag ggctataact tcgtataatg tatgctatac 60gaagttatgg ctggtttatg
catatcg 875459DNAArtificial Sequencesynthesized nucleotide sequence
54tgcatgccat ggtacccggg agctcgaatt cgaagcttct ttccctttgt caacagcaa
595534DNAArtificial Sequencesynthesized nucleotide sequence
55ctagcagatc tgcagggacg catcgtggcc ggat 345639DNAArtificial
Sequencesynthesized nucleotide sequence 56taaggatgca tgtttaaacg
tgaccacgtc gtggaatgc 395766DNAArtificial Sequencesynthesized
nucleotide sequence 57gtggtcacgt ttaaacatgc atccttaatt aaggctgcga
tctatcgagt tttcccagtc 60acgacg 665835DNAArtificial
Sequencesynthesized nucleotide sequence 58taggaagctt gatatcgaac
aaacgaccca acacc 355961DNAArtificial Sequencesynthesized nucleotide
sequence 59gccagatcac atccccttgc agtaccagac gcagggctag gacggtatcg
ataagctgga 60t 616061DNAArtificial Sequencesynthesized nucleotide
sequence 60tgcatgccat ggtacccggg agctcgaatt cgaagcttct gacgcatcgt
ggccggatct 60c 616176DNAArtificial Sequencesynthesized nucleotide
sequence 61acaggagtcc aagcgtcaga attaaagtcc gcggagccat aaccgcgcaa
gtgaagccga 60cgcatcgtgg ccggat 766276DNAArtificial
Sequencesynthesized nucleotide sequence 62aaattcagca acactcgtat
cggcttcact tgcgcggtta tggctccgcg gactttaatg 60gtgaccacgt cgtgga
766378DNAArtificial Sequencesynthesized nucleotide sequence
63tcctcataca caggagtcca agcgtcagaa ataaagtccg cggagccata accgcgcaag
60gacgcatcgt ggccggat 786478DNAArtificial Sequencesynthesized
nucleotide sequence 64aacactcgta tcggcttcac ttgcgcggtt atggctccgc
ggactttatt tctgacgctt 60tggtgaccac gtcgtgga 786578DNAArtificial
Sequencesynthesized nucleotide sequence 65tcctcataca caggagtcca
agcgtcagaa gtaaactctg cggagccata accgcgcaag 60gacgcatcgt ggccggat
786678DNAArtificial Sequencesynthesized nucleotide sequence
66aacactcgta tcggcttcac ttgcgcggtt atggctccgc agagtttact tctgacgctt
60tggtgaccac gtcgtgga 786720DNAHomo sapiens 67ggaagaggag acacggaaca
206820DNAHomo sapiens 68gcagctccag tgactacagc 206921DNAHomo sapiens
69gaggagacac ggaacaccaa g 217020DNAHomo sapiens 70gtcgcagcca
atcatccact 20
* * * * *