U.S. patent application number 16/301805 was filed with the patent office on 2019-12-26 for markers selectively deregulated in tumor-infiltrating regulatory t cells.
This patent application is currently assigned to CheckMab S.R.L.. The applicant listed for this patent is CheckMab S.R.L.. Invention is credited to Sergio Abrignani, Massimiliano Pagani.
Application Number | 20190391152 16/301805 |
Document ID | / |
Family ID | 58772864 |
Filed Date | 2019-12-26 |
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United States Patent
Application |
20190391152 |
Kind Code |
A1 |
Abrignani; Sergio ; et
al. |
December 26, 2019 |
MARKERS SELECTIVELY DEREGULATED IN TUMOR-INFILTRATING REGULATORY T
CELLS
Abstract
The present invention discloses a number of markers selectively
deregulated in tumor-infiltrating regulatory T cells. The invention
relates to molecules able to modulate the expression and/or
function of at least one such marker for use in the prevention
and/or treatment of the tumor. Preferably the molecule specifically
binds to the marker and induces antibody-dependent cell-mediated
cytotoxicity (ADCC). The invention further relates to a molecule
able to modulate the expression and/or function of at least one
such marker for use in a method for in vivo depleting
tumor-infiltrating regulatory T cell in a subject, or for use in a
method to enhance tumor immunity in a subject. Corresponding
pharmaceutical compositions are also contemplated.
Inventors: |
Abrignani; Sergio; (Rapolano
Terme, IT) ; Pagani; Massimiliano; (Trescore
Balneario, IT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CheckMab S.R.L. |
Milano |
|
IT |
|
|
Assignee: |
CheckMab S.R.L.
Milano
IT
|
Family ID: |
58772864 |
Appl. No.: |
16/301805 |
Filed: |
May 15, 2017 |
PCT Filed: |
May 15, 2017 |
PCT NO: |
PCT/EP2017/061642 |
371 Date: |
November 15, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
C07K 16/2866 20130101; G01N 33/57492 20130101; C12N 15/1138
20130101; C07K 2317/732 20130101; C12N 5/0637 20130101; A61K 45/06
20130101; C07K 14/4748 20130101; C07K 16/30 20130101; C12N 2310/14
20130101; A61P 43/00 20180101; C12N 15/1135 20130101; C07K 2317/76
20130101; A61P 37/04 20180101; A61P 35/04 20180101; C12N 2310/11
20130101 |
International
Class: |
G01N 33/574 20060101
G01N033/574; A61K 45/06 20060101 A61K045/06; C07K 16/28 20060101
C07K016/28; C07K 16/30 20060101 C07K016/30; C07K 14/47 20060101
C07K014/47; C12N 15/113 20060101 C12N015/113 |
Foreign Application Data
Date |
Code |
Application Number |
May 16, 2016 |
EP |
16169791.7 |
Nov 14, 2016 |
EP |
16198724.3 |
Claims
1.-26. (canceled)
27. A molecule able to modulate the expression and/or function of
at least one marker that is selectively deregulated in
tumor-infiltrating regulatory T cells, characterized in that: i)
the marker is selected from the group consisting of: LAYN, MAGEH1,
CCR8 and other proteins with upregulated expression in
tumor-infiltrating Treg cells, including BATF, CD30, IL-1RT,
IL-21R, PDL-1, PDL-2, LY75, CD177, SIRPG and CD7; ii) the molecule
targets a solid tumour selected from the group consisting of:
non-small cell lung cancer, colorectal cancer, breast cancer, and
gastric cancer or a metastatic cancer derived therefrom; and iii)
the molecule is for use in the prevention and/or treatment of said
tumor.
28. A molecule as claimed in claim 27, wherein the metastatic
cancer is a bone, brain, or liver metastasis.
29. A molecule as claimed in claim 27 for use in the treatment of
non-small cell lung cancer.
30. A molecule as claimed in claim 27 for use in the treatment of
colorectal cancer.
31. A molecule as claimed in claim 27, wherein the molecule is: a)
an antibody or a fragment thereof; b) a polypeptide; c) a small
molecule; d) a polynucleotide coding for said antibody or fragment
thereof or said polypeptide; e) a polynucleotide, which is an
antisense construct, antisense oligonucleotide, RNA interference
construct or siRNA; f) a vector comprising or expressing the
polynucleotide as defined in d) or e); or g) a host cell
genetically engineered expressing said antibody or fragment thereof
or polypeptide as defined in a) or b) or comprising the
polynucleotide as defined in d) or e).
32. A molecule as claimed in claim 31, wherein the molecule is: a)
an antibody or fragment thereof, d) a polynucleotide coding for
said antibody or a fragment thereof; or g) a host cell genetically
engineered expressing said antibody or fragment thereof as defined
in a) or comprising the polynucleotide as defined in d).
33. A molecule as claimed in claim 27, wherein the marker has a
sequence as defined by ENSEMBL_release 87 reference:
ENSG00000204381 (LAYN); ENSG00000179934 (CCR8); or ENSG00000187210
(MAGEH1).
34. A molecule as claimed in claim 33, wherein the marker has one
of a sequence LAYN (SEQ ID NOs:1-9), CCR8 (SEQ ID NOs:10-11) or
MAGEH1 (SEQ ID NOs: 708 or 709).
35. A molecule as claimed in claim 27, which is iii) for use in
depleting tumor-infiltrating regulatory T cells.
36. A molecule as claimed in claim 27, which is iii) for use in
enhancing tumor immunity.
37. A pharmaceutical composition comprising a molecule as claimed
in claim 27 and at least one pharmaceutically acceptable
carrier.
38. A pharmaceutical composition according to claim 37, wherein the
composition further comprises a further anti-tumoral agent.
39. An in vitro screening, monitoring, diagnostic or prognostic
method for assessing a solid tumor selected from the group
consisting of: non-small cell lung cancer, colorectal cancer,
breast cancer, gastric cancer, or a metastasis derived therefrom,
in a subject, the method comprising the steps of: a) obtaining an
isolated biological sample from the subject; b) detecting in the
isolated biological sample at least one marker from the group
consisting of: LAYN, MAGEH1 and CCR8; c) comparing said marker with
a control; and d) based on the comparison determining a diagnosis
or prognosis.
40. A method for identifying a molecule acting as an anti-tumoral
molecule, comprising the steps of: a) assaying candidate molecules
for their binding specificity to at least one marker from the group
consisting of: LAYN, MAGEH1 and CCR8; b) selecting molecules having
a specific binding specificity to the at least one marker; and c)
testing selected molecules for their capacity to inhibit
proliferation and/or induce an apoptotic response in a cell system
against a non-small cell lung cancer, colorectal cancer, breast
cancer, gastric cancer or a metastatic cancer derived
therefrom.
41. A method as claimed in claim 40 wherein step c) comprises
testing for selectively depleting tumor-infiltrating regulatory T
cells by inducing antibody-dependent cell-mediated cytotoxicity
(ADCC).
Description
RELATED APPLICATION
[0001] This application is a national stage filing under 35 U.S.C.
.sctn. 371 of International Application Serial No.
PCT/EP2017/061642, filed May 15, 2017, the contents of which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to a molecule able to modulate
the expression and/or function of at least one marker that is
selectively deregulated in tumor-infiltrating regulatory T cell or
to a molecule capable of specifically binding to at least one
marker that is selectively deregulated in tumor-infiltrating
regulatory T cell and inducing antibody-dependent cell-mediated
cytotoxicity (ADCC) for use in the prevention and/or treatment of
cancer or for use in a method for in vivo depleting
tumor-infiltrating regulatory T cell in a subject or for use in a
method to enhance tumor immunity in a subject and relative
pharmaceutical composition.
BACKGROUND OF THE INVENTION
[0003] The combination of genetic mutations and epigenetic
modifications that are peculiar to all tumors generate antigens
that T and B lymphocytes can use to specifically recognize tumor
cells (Jamal-Hanjani et al., 2013). It is increasingly clear that T
lymphocytes recognizing tumor derived peptides presented by major
histocompatibility complex (MHC) molecules play a central role in
immunotherapy and in conventional chemo-radiotherapy of cancer
(Galluzzi et al., 2015). In fact, anti-tumor T cell responses arise
in cancer patients but are disabled upon tumor progression by
suppressive mechanisms triggered by the interplay between malignant
cells and the tumor microenvironment (Munn and Bronte, 2015). The
tumor-dependent immunosuppressive mechanisms depend on the
integrated action of infiltrating leukocytes and lymphocytes that
upregulate a range of modulatory molecules, collectively called
immune checkpoints, whose function is only partially characterized
(Pardoll, 2012). Therefore, the search for agonists of
co-stimulatory complexes or antagonists of inhibitory molecules to
potentiate antigen-specific T cell responses is a primary goal of
current anti-tumor research (Sharma and Allison, 2015; Zitvogel et
al., 2013). Indeed, clinical trials have unequivocally shown that
the blockade of immune checkpoints unleashes the spontaneous
anti-tumor immune responses in such a powerful way that it has
created a paradigm shift in cancer therapy (Sledzinska et al.,
2015; Topalian et al., 2015).
[0004] Amongst the immune checkpoints targeted by blocking
strategies, CTLA-4 has been one of the first to be translated into
therapeutic applications.
[0005] Anti-CTLA-4 monoclonal antibodies (mAb) showed remarkable
success in metastatic melanoma, and more recently in non-small-cell
lung cancer, prostate cancer, renal cell carcinoma, urothelial
carcinoma and ovarian cancer (Carthon et al., 2010; Hodi et al.,
2010; van den Eertwegh et al., 2012; Yang et al., 2007). However,
the fraction of patients that do not respond remains high,
prompting a deeper investigation of the mechanisms underpinning the
modulation of immune responses by tumors. Recent experimental
evidence showed that anti-CTLA-4 mAb efficacy depends on Fc.gamma.R
mediated depletion of CD4.sup.+ regulatory T cells (Treg cells)
within the tumor microenvironment (Peggs et al., 2009; Selby et
al., 2013; Simpson et al., 2013; Twyman-Saint Victor et al., 2015).
Treg cells, which are physiologically engaged in the maintenance of
immunological self-tolerance and immune homeostasis (Josefowicz et
al., 2012; Sakaguchi et al., 2008), are potent suppressors of
effector cells and are found at high frequencies in various types
of cancers (Fridman et al., 2012; Nishikawa and Sakaguchi, 2010).
Interestingly, Treg cells adapt their transcriptional program to
the various cytokines to which they are exposed in the inflammatory
milieu (Campbell and Koch, 2011). This versatility is controlled by
transcription factors generally associated with the differentiation
of other effector CD4.sup.+ T cell subsets, resulting in various
Treg cell populations with unique features and immunomodulatory
functions (Duhen et al., 2012; Geginat et al., 2014). Moreover,
Treg cells infiltrating non-lymphoid tissues are reported to
exhibit unique phenotypes and transcriptional signatures, as they
can display functions beyond their well-established suppressive
roles, such as metabolic modulation in adipose tissue (Cipolletta
et al., 2012) or regulation of tissue repair in skeletal muscle
(Burzyn et al., 2013) and in lung tissue (Arpaia et al., 2015).
[0006] Treg cells depletion has been reported to increase
anti-tumor specific immune responses and to reduce tumor burden
(Marabelle et al., 2013; Teng et al., 2010; Walter et al., 2012).
Although promising clinical results have been achieved with Treg
cell depleting strategies, some relevant issues are to be
addressed, for a safer, more effective and wider clinical
application of these therapies. First, severe autoimmunity can
occur following systemic Treg cells depletion (Nishikawa and
Sakaguchi, 2010), which could be avoided if selective depletion of
tumor infiltrating Treg cells were feasible. A second issue
concerns the specificity of targeting, indeed Treg cells share with
effector lymphocytes most of the molecules targeted for therapy,
which can possibly deplete also the tumor-specific effector cells.
Therefore, the molecular characterization of Treg cells at
different tumor sites should help to better define therapeutic
targets through a better description of their signature molecules
and of the network that regulates Treg cell functions in the tumor
microenvironment.
[0007] Non-small-cell lung cancer (NSCLC) and colorectal cancer
(CRC) are the two most frequent cancers in both genders (Torre et
al., 2015). NSCLC has the worst prognosis due to its high mortality
rate even in early stages. Although CRC survival rate is highly
dependent on the tumor stage at diagnosis, about 50% of patients
will progress to metastatic cancer (Gonzalez-Pons and Cruz-Correa,
2015). Both tumors have been targeted with therapies based on
monoclonal antibodies to checkpoint inhibitors, but the outcomes
were different. While remarkable clinical success has been obtained
in NSCLC, evidence of durable response in CRC is scarce with the
exception of mismatch repair-deficient CRC lesions (Jacobs et al.,
2015; Kroemer et al., 2015; Le et al., 2015).
[0008] Then there is still need for agents that target tumor
infiltrating Treg cells for the treatment and/or prevention of
cancer.
SUMMARY OF THE INVENTION
[0009] Tumor-infiltrating regulatory T lymphocytes (Treg) can
suppress effector T cells specific for tumor antigens. Since new
anti-cancer immunotherapies aim at unleashing effector T cells by
targeting immune-checkpoints, deeper molecular definitions of
tumor-infiltrating-lymphocytes could offer new therapeutic
opportunities. Transcriptomes of T helper 1 (Th1), Th17 and Treg
cells infiltrating colorectal or non-small-cell lung cancers were
compared to transcriptomes of the same subsets from normal tissues,
and validated at the single cell level. The inventors found
tumor-infiltrating Treg cells are highly suppressive, upregulate
several immune-checkpoints, and express on the cell surface
specific signature molecules such as interleukin-1 receptor 2
(IL1R2), programmed death (PD)-1 Ligand1, PD-1 Ligand2, and CCR8
chemokine which were not previously described on Treg cells.
Remarkably, high expression in whole tumor samples of Treg
signature genes, such as LAYN, MAGEH1 or CCR8, correlated with poor
prognosis. The invention provides new insights into the molecular
identity and functions of human tumor-infiltrating Treg cells, and
define new potential targets for tumor immunotherapy.
[0010] In the present invention, the inventors provide a
comprehensive transcriptome analysis of human CD4.sup.+ Treg cells
and effector cells (Th1 and Th17) infiltrating NSCLC or CRC and
their matched normal tissues.
[0011] Inventors defined molecular signatures of tumor-infiltrating
Treg cells in these two cancer types and confirmed the relevance of
these signatures by single-cell analyses. These data could help a
better understanding of Treg functional role at tumor sites and
pave the way to the identification of therapeutic targets for more
specific and safer modulation of Treg cells in cancer therapy.
[0012] The inventors' findings provide new insights on the
inhibitory mechanisms of Treg cells and offer precise targets for
cancer immunotherapy.
[0013] Then the present invention provides a molecule able to
modulate the expression and/or function of at least one marker that
is selectively deregulated in tumor-infiltrating regulatory T cells
for use in the prevention and/or treatment of said tumor.
[0014] Preferably, the molecule according to the invention is
capable of specifically binding to said at least one marker and
inducing antibody-dependent cell-mediated cytotoxicity (ADCC).
[0015] Said molecule is preferably able to selectively deplete
tumor-infiltrating regulatory T cells. Said molecule is preferably
selected from the group consisting of:
[0016] a) an antibody or a fragment thereof;
[0017] b) a polypeptide;
[0018] c) a small molecule;
[0019] d) a polynucleotide coding for said antibody or polypeptide
or a functional derivative thereof;
[0020] e) a polynucleotide, such as antisense construct, antisense
oligonucleotide, RNA interference construct or siRNA,
[0021] e) a vector comprising or expressing the polynucleotide as
defined in d) or e);
[0022] f) a host cell genetically engineered expressing said
polypeptide or antibody or comprising the polynucleotide as defined
in d) or e).
[0023] Preferably, the marker is selected from the group consisting
of at least one marker disclosed in the following Table VIII.
TABLE-US-00001 TABLE VIII MARKER NAME ENSEMBL_release87
ENTREZ_IDrelease108 MARKER NAME ENSEMBL_release87
ENTREZ_IDrelease108 FUCA2 ENSG00000001036 2519 HADHB
ENSG00000138029 3032 ICA1 ENSG00000003147 3382 CEP55
ENSG00000138180 55165 TTC22 ENSG00000006555 55001 ENTPD1
ENSG00000138185 953 COX10 ENSG00000006695 1352 NAB1 ENSG00000138386
4664 IL32 ENSG00000008517 9235 HECW2 ENSG00000138411 57520 ETV7
ENSG00000010030 51513 CD27 ENSG00000139193 939 ATP2C1
ENSG00000017260 27032 CDH24 ENSG00000139880 64403 FAS
ENSG00000026103 355 RAB15 ENSG00000139998 376267 ARNTL2
ENSG00000029153 56938 ETFA ENSG00000140374 2108 IKZF2
ENSG00000030419 22807 KSR1 ENSG00000141068 8844 PEX3
ENSG00000034693 8504 PCTP ENSG00000141179 58488 MAT2B
ENSG00000038274 27430 SECTM1 ENSG00000141574 6398 TSPAN17
ENSG00000048140 26262 EVA1B ENSG00000142694 55194 COL9A2
ENSG00000049089 1298 WDTC1 ENSG00000142784 23038 TNFRSF9
ENSG00000049249 3604 CTTNBP2NL ENSG00000143079 55917 FOXP3
ENSG00000049768 50943 CASQ1 ENSG00000143318 844 NFE2L3
ENSG00000050344 9603 SNAP47 ENSG00000143740 116841 LIMA1
ENSG00000050405 51474 STAC ENSG00000144681 6769 TNIP3
ENSG00000050730 79931 ARL6IP5 ENSG00000144746 10550 LY75
ENSG00000054219 4065 ADPRH ENSG00000144843 141 ZNF280C
ENSG00000056277 55609 PAM ENSG00000145730 5066 YIPF1
ENSG00000058799 54432 RNF145 ENSG00000145860 153830 NFYC
ENSG00000066136 4802 TTBK1 ENSG00000146216 84630 ISOC1
ENSG00000066583 51015 TMEM140 ENSG00000146859 55281 PHKA1
ENSG00000067177 5255 CHST7 ENSG00000147119 56548 ACSL4
ENSG00000068366 2182 CHRNA6 ENSG00000147434 8973 MAST4
ENSG00000069020 375449 MKI67 ENSG00000148773 4288 LMCD1
ENSG00000071282 29995 PTPRJ ENSG00000149177 5795 TFRC
ENSG00000072274 7037 ZC3H12C ENSG00000149289 85463 PANX2
ENSG00000073150 56666 NCAM1 ENSG00000149294 4684 FNDC3B
ENSG00000075420 64778 INPP1 ENSG00000151689 3628 REXO2
ENSG00000076043 25996 JAKMIP1 ENSG00000152969 152789 TP73
ENSG00000078900 7161 GTF3C6 ENSG00000155115 112495 LXN
ENSG00000079257 56925 RHOC ENSG00000155366 389 CEACAM1
ENSG00000079385 634 SLC16A1 ENSG00000155380 6566 IL12RB2
ENSG00000081985 3595 BATF ENSG00000156127 10538 GSK3B
ENSG00000082701 2932 CXCL13 ENSG00000156234 10563 TDRD3
ENSG00000083544 81550 SH3RF2 ENSG00000156463 153769 RRAGB
ENSG00000083750 10325 NPTN ENSG00000156642 27020 STARD7
ENSG00000084090 56910 CCNB2 ENSG00000157456 9133 SSH1
ENSG00000084112 54434 RNF207 ENSG00000158286 388591 NCOA1
ENSG00000084676 8648 AHCYL2 ENSG00000158467 23382 MGST2
ENSG00000085871 4258 PTGIR ENSG00000160013 5739 ACOX3
ENSG00000087008 8310 CALM3 ENSG00000160014 808 AURKA
ENSG00000087586 6790 TMPRSS3 ENSG00000160183 64699 TPX2
ENSG00000088325 22974 FCRL3 ENSG00000160856 115352 ANKRD10
ENSG00000088448 55608 PAQR4 ENSG00000162073 124222 FKBP1A
ENSG00000088832 2280 ZG16B ENSG00000162078 124220 SIRPG
ENSG00000089012 55423 JAK1 ENSG00000162434 3716 BIRC5
ENSG00000089685 332 DIRAS3 ENSG00000162595 9077 RGS1
ENSG00000090104 5996 ACTG2 ENSG00000163017 72 DPYSL2
ENSG00000092964 1808 SGPP2 ENSG00000163082 130367 WHRN
ENSG00000095397 25861 NEURL3 ENSG00000163121 93082 CENPM
ENSG00000100162 79019 CTLA4 ENSG00000163599 1493 SEPT3
ENSG00000100167 55964 ICOS ENSG00000163600 29851 NCF4
ENSG00000100365 4689 RYBP ENSG00000163602 23429 CSF2RB
ENSG00000100368 1439 KIF15 ENSG00000163808 56992 IL2RB
ENSG00000100385 3560 TMEM184C ENSG00000164168 55751 CNIH1
ENSG00000100528 10175 C5orf63 ENSG00000164241 401207 ZMYND8
ENSG00000101040 23613 PTTG1 ENSG00000164611 9232 MAP1LC3A
ENSG00000101460 84557 MELK ENSG00000165304 9833 PIGU
ENSG00000101464 128869 FAAH2 ENSG00000165591 158584 NXT2
ENSG00000101888 55916 PRDX3 ENSG00000165672 10935 SMS
ENSG00000102172 6611 HPRT1 ENSG00000165704 3251 NDFIP2
ENSG00000102471 54602 CACNB2 ENSG00000165995 783 ACP5
ENSG00000102575 54 TPP1 ENSG00000166340 1200 NFAT5 ENSG00000102908
10725 AKIP1 ENSG00000166452 56672 CYB5B ENSG00000103018 80777 ACAA2
ENSG00000167315 10449 IL21R ENSG00000103522 50615 GNG8
ENSG00000167414 94235 LAPTM4B ENSG00000104341 55353 GNG4
ENSG00000168243 2786 IL7 ENSG00000104432 3574 CX3CR1
ENSG00000168329 1524 NCALD ENSG00000104490 83988 AHCYL1
ENSG00000168710 10768 ERI1 ENSG00000104626 90459 TSPAN5
ENSG00000168785 10098 EBI3 ENSG00000105246 10148 PGM2
ENSG00000169299 55276 PLA2G4C ENSG00000105499 8605 CRADD
ENSG00000169372 8738 CDK6 ENSG00000105810 1021 UGP2 ENSG00000169764
7360 HOXA1 ENSG00000105991 3198 ZNF282 ENSG00000170265 8427 GLCCI1
ENSG00000106415 113263 GLB1 ENSG00000170266 2720 MINPP1
ENSG00000107789 9562 SMAD1 ENSG00000170365 4086 ACTA2
ENSG00000107796 59 SPATA24 ENSG00000170469 202051 WSB1
ENSG00000109046 26118 PRKCDBP ENSG00000170955 112464 CLNK
ENSG00000109684 116449 TADA3 ENSG00000171148 10474 HTATIP2
ENSG00000109854 10553 RBKS ENSG00000171174 64080 CTSC
ENSG00000109861 1075 NETO2 ENSG00000171208 81831 VWA5A
ENSG00000110002 4013 LRG1 ENSG00000171236 116844 DCPS
ENSG00000110063 28960 FAM98B ENSG00000171262 283742 SLC35F2
ENSG00000110660 54733 CHST11 ENSG00000171310 50515 FOXM1
ENSG00000111206 2305 ECEL1 ENSG00000171551 9427 RAD51AP1
ENSG00000111247 10635 BCL2L1 ENSG00000171552 598 RASAL1
ENSG00000111344 8437 MALT1 ENSG00000172175 10892 VDR
ENSG00000111424 7421 ZMAT3 ENSG00000172667 64393 FAM184A
ENSG00000111879 79632 CORO1B ENSG00000172725 57175 DNPH1
ENSG00000112667 10591 CYP7B1 ENSG00000172817 9420 KIF20A
ENSG00000112984 10112 HPSE ENSG00000173083 10855 SEC24A
ENSG00000113615 10802 VANGL1 ENSG00000173218 81839 KAT2B
ENSG00000114166 8850 GLRX ENSG00000173221 2745 PPM1G
ENSG00000115241 5496 TRIB1 ENSG00000173334 10221 IL1R2
ENSG00000115590 7850 CD7 ENSG00000173762 924 IL1R1 ENSG00000115594
3554 HAP1 ENSG00000173805 9001 IL1RL2 ENSG00000115598 8808 FBXO45
ENSG00000174013 200933 IL1RL1 ENSG00000115602 9173 CHST2
ENSG00000175040 9435 UXS1 ENSG00000115652 80146 RMI2
ENSG00000175643 116028 SLC25A12 ENSG00000115840 8604 SLC35E3
ENSG00000175782 55508 THADA ENSG00000115970 63892 ZBTB38
ENSG00000177311 253461 PARK7 ENSG00000116288 11315 ZBED2
ENSG00000177494 79413 LEPR ENSG00000116678 3953 PARD6G
ENSG00000178184 84552 GADD45A ENSG00000116717 1647 GLDC
ENSG00000178445 2731 KIF14 ENSG00000118193 9928 AKAP5
ENSG00000179841 9495 MREG ENSG00000118242 55686 CCR8
ENSG00000179934 1237 HSDL2 ENSG00000119471 84263 PAK2
ENSG00000180370 5062 FLVCR2 ENSG00000119686 55640 YIPF6
ENSG00000181704 286451 CD274 ENSG00000120217 29126 TIGIT
ENSG00000181847 201633 SOCS2 ENSG00000120833 8835 CREB3L2
ENSG00000182158 64764 TNFRSF8 ENSG00000120949 943 XKRX
ENSG00000182489 402415 RDH10 ENSG00000121039 157506 CADM1
ENSG00000182985 23705 LAX1 ENSG00000122188 54900 LHFP
ENSG00000183722 10186 TWIST1 ENSG00000122691 7291 CSF1
ENSG00000184371 1435 ZWINT ENSG00000122952 11130 PTP4A3
ENSG00000184489 11156 CIT ENSG00000122966 11113 CDCA2
ENSG00000184661 157313 ACOT9 ENSG00000123130 23597 OSBP2
ENSG00000184792 23762 IKZF4 ENSG00000123411 64375 METTL7A
ENSG00000185432 25840 HJURP ENSG00000123485 55355 SPATC1
ENSG00000186583 375686 METTL8 ENSG00000123600 79828 TNFRSF4
ENSG00000186827 7293 TOX2 ENSG00000124191 84969 TNFRSF18
ENSG00000186891 8784 GTSF1L ENSG00000124196 149699 TMPRSS6
ENSG00000187045 164656 SOX4 ENSG00000124766 6659 GCNT1
ENSG00000187210 2650 TM9SF2 ENSG00000125304 9375 MAGEH1
ENSG00000187601 28986 HS3ST3B1 ENSG00000125430 9953 NHS
ENSG00000188158 4810 EML2 ENSG00000125746 24139 IL17REL
ENSG00000188263 400935 MGME1 ENSG00000125871 92667 ADAT2
ENSG00000189007 134637 IGFLR1 ENSG00000126246 79713 NEMP2
ENSG00000189362 100131211 DLGAP5 ENSG00000126787 9787 SPATS2L
ENSG00000196141 26010 HIVEP3 ENSG00000127124 59269 NTNG2
ENSG00000196358 84628 LRRC61 ENSG00000127399 65999 MYL6B
ENSG00000196465 140465 TST ENSG00000128311 7263 ARHGEF12
ENSG00000196914 23365 STRIP2 ENSG00000128578 57464 MAP3K5
ENSG00000197442 4217 MYO5C ENSG00000128833 55930 PDGFA
ENSG00000197461 5154 FOXA1 ENSG00000129514 3169 PDCD1LG2
ENSG00000197646 80380 ITFG1 ENSG00000129636 81533 TOR4A
ENSG00000198113 54863 KLHDC7B ENSG00000130487 113730 HIBCH
ENSG00000198130 26275 TRAF3 ENSG00000131323 7187 ZNF334
ENSG00000198185 55713 MCCC2 ENSG00000131844 64087 NTRK1
ENSG00000198400 4914 GRSF1 ENSG00000132463 2926 TMA16
ENSG00000198498 55319 SYT11 ENSG00000132718 23208 WDHD1
ENSG00000198554 11169 SLC41A1 ENSG00000133065 254428 FAM19A2
ENSG00000198673 338811 ATP13A3 ENSG00000133657 79572 F5
ENSG00000198734 2153 MICAL2 ENSG00000133816 9645 GK ENSG00000198814
2710 IL2RA ENSG00000134460 3559 INPP5F ENSG00000198825 22876
CABLES1 ENSG00000134508 91768 LAYN ENSG00000204381 143903 RFK
ENSG00000135002 55312 CARD16 ENSG00000204397 114769 HAVCR2
ENSG00000135077 84868 TBC1D8 ENSG00000204634 11138 CGA
ENSG00000135346 1081 CD177 ENSG00000204936 57126 FAIM2
ENSG00000135472 23017 LEPROT ENSG00000213625 54741 EGLN1
ENSG00000135766 54583 SEC14L6 ENSG00000214491 730005 ARHGEF4
ENSG00000136002 50649 TRIM16 ENSG00000221926 10626 SLC41A2
ENSG00000136052 84102 LTA ENSG00000226979 4049 FLNB ENSG00000136068
2317 PROB1 ENSG00000228672 389333 RCBTB1 ENSG00000136144 55213
AF165138.7 ENSG00000243440 NA TMOD1 ENSG00000136842 7111 USP51
ENSG00000247746 158880 TPMT ENSG00000137364 7172 CARD17
ENSG00000255221 440068 CASP1 ENSG00000137752 834 DOC2B
ENSG00000272636 8447 NUSAP1 ENSG00000137804 51203 C17orf96
ENSG00000273604 100170841 ADAM10 ENSG00000137845 102 SSTR3
ENSG00000278195 6753 ZNF280D ENSG00000137871 54816 AC019206.1
ENSG00000279229 NA
[0024] wherein each of said marker name is characterized by
"Ensembl gene id" and includes all of therein disclosed isoform
protein sequences.
[0025] Each gene of table VIII is characterized by its Ensembl Gene
accession number (ENSG), retrievable in the public database EnsEMBL
(http://www.ensembl.org) and by its Entrez Gene ID, retrievable in
the public database NCBI (https://www.ncbi.nlm.nih.gov/), if
present.
[0026] Preferably the marker is selected from the group consisting
of: a transmembrane protein, a cytokine, an epigenetic factor, a
kinase phosphatase or a transcription factor.
[0027] More preferably, the marker is a transmembrane protein
selected from the group of SEQ ID NO:1-661, even more preferably,
the marker is selected from the group consisting of: LAYN (SEQ ID
NOs:1-9), CCR8 (SEQ ID Nos:10-11), IL21R (SEQ ID Nos: 12-14), IL1R2
(SEQ ID Nos:206-209), LY75 (SEQ ID NO: 78), SIRPG (SEQ ID
Nos:122-126), CD177 (SEQ ID Nos:651-653), CD7 (SEQ ID Nos:549-554),
FCRL3 (SEQ ID Nos:452-457), CADM1 (SEQ ID Nos: 570-583), NTNG2 (SEQ
ID Nos:621-622), CSF2RB (SEQ ID Nos:134-137), SECTM1 (SEQ ID Nos:
349-356), TSPAN5 (SEQ ID Nos:497-503), TMPRSS3 (SEQ ID
Nos:448-451), TMPRSS6 (SEQ ID Nos:605-611), METTL7A (SEQ ID
Nos:600-604), THADA (SEQ ID Nos: 237), NDFIP2 (SEQ ID Nos:148-151),
CHRNA6 (SEQ ID Nos:392-394), or from the group consisting of:
TABLE-US-00002 LAYN (SEQ ID NOs: 1-9
[>ENSG00000204381_ENST00000375614_ ENSP00000364764_LAYN (SEQ ID
NO: 1) MRPGTALQAVLLAVLLVGLRAATGRLLSGQPVCRGGTQRPCYKVIYFH
DTSRRLNFEEAKEACRRDGGQLVSIESEDEQKLIEKFIENLLPSDGDF
WIGLRRREEKQSNSTACQDLYAWTDGSISQFRNWYVDEPSCGSEVCVV
MYHQPSAPAGIGGPYMFQWNDDRCNMKNNFICKYSDEKPAVPSREAEG
EETELTTPVLPEETQEEDAKKTFKESREAALNLAYILIPSIPLLLLLV
VTTVVCWVWICRKRKREQPDPSTKKQHTIWPSPHQGNSPDLEVYNVIR
KQSEADLAETRPDLKNISFRVCSGEATPDDMSCDYDNMAVNPSESGFV
TLVSVESGFVTNDIYEFSPDQMGRSKESGWVENEIYGY*
>ENSG00000204381_ENST00000375615_ ENSP00000364765_LAYN (SEQ ID
NO: 2) MRPGTALQAVLLAVLLVGLRAATGRLLSASDLDLRGGQPVCRGGTQRP
CYKVIYFHDTSRRLNFEEAKEACRRDGGQLVSIESEDEQKLIEKFIEN
LLPSDGDFWIGLRRREEKQSNSTACQDLYAWTDGSISQFRNWYVDEPS
CGSEVCVVMYHQPSAPAGIGGPYMFQWNDDRCNMKNNFICKYSDEKPA
VPSREAEGEETELTTPVLPEETQEEDAKKTFKESREAALNLAYILIPS
IPLLLLLVVTTVVCWVWICRKRKREQPDPSTKKQHTIWPSPHQGNSPD
LEVYNVIRKQSEADLAETRPDLKNISFRVCSGEATPDDMSCDYDNMAV
NPSESGFVTLVSVESGFVTNDIYEFSPDQMGRSKESGWVENEIYGY*
>ENSG00000204381_ENST00000436913_ ENSP00000392942_LAYN (SEQ ID
NO: 3) MVTSGLGSGGVRRNKAIAQPARTFMLGLMAAYHNLEKPAVPSREAEGE
ETELTTPVLPEETQEEDAKKTFKESREAALNLAYILIPSIPLLLLLVV
TTVVCWVWICRKRKREQPDPSTKKQHTIWPSPHQGNSPDLEVYNVIRK
QSEADLAETRPDLKNISFRVCSGEATPDDMSCDYDNMAVNPSESGFVT
LVSVESGFVTNDIYEFSPDQMGRSKESGWVENEIYGY*
>ENSG00000204381_ENST00000525126_ ENSP00000434328_LAYN (SEQ ID
NO: 4) MRPGTALQAVLLAVLLVGLRAATGRLLSASDLDLRGGQPVCRGGTQRP
CYKVIYFHDTSRRLNFEEAKEACRRDGGQLVSIESEDEQKLIEKFIEN
LLPSDGDFWIGLRRREEKQSNSTACQDLYAWTDGSISQFRNWYVDEPS
CGSEVCVVMYHQPSAPAGIGGPYMFQWNDDRCNMKNNFICKYSDEKPA
VPSREAEGEETELTTPVLPEETQEEDAKKTFKESREAALNLAYILIPS
IPLLLLLVVTTVVCWVWICRKRQKTGAARP*
>ENSG00000204381_ENST00000525866_ ENSP00000434300_LAYN (SEQ ID
NO: 5) MRPGTALQAVLLAVLLVGLRAATGRLLSGQPVCRGGTQRPCYKVIYFH
DTSRRLNFEEAKEACRRDGGQLVSIESEDEQKLIEKFIENLLPSDGDF
WIGLRRREEKQSNSTACQDLYAWTDGSISQFRETSSSF*
>ENSG00000204381_ENST00000528924_ ENSP00000486561_LAYN (SEQ ID
NO: 6) MVTSGLGSGGVRRNKAIAQPARTFMLGLMAAYHNLEKPAVPSREAEGE
ETELTTPVLPEETQEEDAKKTFKESREAALNLAYILIPSIPLLLLLVV TTVVCWVWICRK
>ENSG00000204381_ENST00000530962_ ENSP00000431627_LAYN (SEQ ID
NO: 7) MYHQPSAPAGIGGPYMFQWNDDRCNMKNNFICKYSDEKPAVPSREAEG
EETELTTPVLPEETQEEDAKKTFKESREAALNLAYILIPSIPLLLLLV VTTVVCWVWICRK
>ENSG00000204381_ENST00000533265_ ENSP00000434972_LAYN (SEQ ID
NO: 8) MRPGTALQAVLLAVLLVGLRAATGRLLSGQPVCRGGTQRPCYKVIYFH
DTSRRLNFEEAKEACRRDGGQLVSIESEDEQKLIEKFIENLLPSDGDF
WIGLRRREEKQSNSTACQDLYAWTDGSISQFRNWYVDEPSCGSEVCVV
MYHQPSAPAGIGGPYMFQWNDDRCNMKNNFICKYSDEKPAVPSREAEG
EETELTTPVLPEETQEEDAKKTFKESREAALNLAYILIPSIPLLLLLV
VTTVVCWVWICRKRQKTGAARP* >ENSG00000204381_ENST00000533999_
ENSP00000432434_LAYN (SEQ ID NO: 9)
MYHQPSAPAGIGGPYMFQWNDDRCNMKNNFICKYSDEKPAVPSREAEG E]), CCR8 (SEQ ID
Nos: 10-11 [>ENSG00000179934_ENST00000326306_
ENSP00000326432_CCR8 (SEQ ID NO: 10)
MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNGKLLLAVFYCLLFVF
SLLGNSLVILVLVVCKKLRSITDVYLLNLALSDLLFVFSFPFQTYYLL
DQWVFGTVMCKVVSGFYYIGFYSSMFFITLMSVDRYLAVVHAVYALKV
RTIRMGTTLCLAVWLTAIMATIPLLVFYQVASEDGVLQCYSFYNQQTL
KWKIFTNFKMNILGLLIPFTIFMFCYIKILHQLKRCQNHNKTKAIRLV
LIVVIASLLFWVPFNVVLFLTSLHSMHILDGCSISQQLTYATHVTEII
SFTHCCVNPVIYAFVGEKFKKHLSEIFQKSCSQIFNYLGRQMPRESCE
KSSSCQQHSSRSSSVDYIL* >ENSG00000179934_ENST00000414803_
ENSP00000390104_CCR8 (SEQ ID NO: 11)
MDYTLDLSVTTVTDYYYPDIFSSPCDAELIQTNDLLSAGPVGVWDCNV
QSGVWLLLHWLLQQHVFHHPHECGQVPGCCPCRVCPKGEDDQDGHNAV
PGSMANRHYGYHPIASVLPSGL*]), IL21R (SEQ ID Nos: 12-14
[>ENSG00000103522_ENST00000337929_ ENSP00000338010_IL21R (SEQ ID
NO: 12) MPRGWAAPLLLLLLQGGWGCPDLVCYTDYLQTVICILEMWNLHPSTLT
LTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSV
NITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYED
PAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRK
DSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKEGWNPHLLL
LLLLVIVFIPAFWSLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDF
KKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQE
PAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDA
EGPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVS
AGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGS
PLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPP PLSSPGPQAS*
>ENSG00000103522_ENST00000395754_ ENSP00000379103_IL21R (SEQ ID
NO: 13) MPRGWAAPLLLLLLQGGWGCPDLVCYTDYLQTVICILEMWNLHPSTLT
LTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSV
NITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYED
PAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRK
DSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKEGWNPHLLL
LLLLVIVFIPAFWSLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDF
KKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQE
PAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDA
EGPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVS
AGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGS
PLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPP PLSSPGPQAS*
>ENSG00000103522_ENST00000564089_ ENSP00000456707_IL21R (SEQ ID
NO: 14) MPRGWAAPLLLLLLQGGWGCPDLVCYTDYLQTVICILEMWNLHPSTLT
LTWQDQYEELKDEATSCSLHRSAHNATHATYTCHMDVFHFMADDIFSV
NITDQSGNYSQECGSFLLAESIKPAPPFNVTVTFSGQYNISWRSDYED
PAFYMLKGKLQYELQYRNRGDPWAVSPRRKLISVDSRSVSLLPLEFRK
DSSYELQVRAGPMPGSSYQGTWSEWSDPVIFQTQSEELKEGWNPHLLL
LLLLVIVFIPAFWSLKTHPLWRLWKKIWAVPSPERFFMPLYKGCSGDF
KKWVGAPFTGSSLELGPWSPEVPSTLEVYSCHPPRSPAKRLQLTELQE
PAELVESDGVPKPSFWPTAQNSGGSAYSEERDRPYGLVSIDTVTVLDA
EGPCTWPCSCEDDGYPALDLDAGLEPSPGLEDPLLDAGTTVLSCGCVS
AGSPGLGGPLGSLLDRLKPPLADGEDWAGGLPWGGRSPGGVSESEAGS
PLAGLDMDTFDSGFVGSDCSSPVECDFTSPGDEGPPRSYLRQWVVIPP PLSSPGPQAS*]).
[0028] Said cytokine is preferably selected from the group of
consisting of: IL32 (SEQ ID Nos: 19-30), IL7 (SEQ ID Nos: 168-174),
EBI3 (SEQ ID NO: 175), SECTM1 (SEQ ID Nos: 349-356), CSF1 (SEQ ID
Nos: 585-592) and LTA (SEQ ID Nos: 657-658).
[0029] Said epigenetic factor is preferably selected from the group
of consisting of: TDRD3 (SEQ ID NO: 712-718), KAT2B (SEQ ID
NO:719), FOXA1 (SEQ ID Nos: 720-721) and RCBTB1 (SEQ ID Nos:
722-723).
[0030] Said kinase phosphatase is preferably selected from the
group of consisting of: GSK3B (SEQ ID Nos: 724-725), SSH1 (SEQ ID
NOS:111-112), CDK6 (SEQ ID Nos: 726-727), MINPP1 (SEQ ID
Nos:181-183), PTPRJ (SEQ ID Nos: 395-400), CALM3 (SEQ ID Nos:
728-734) and PTP4A3 (SEQ ID Nos: 593-598).
[0031] Said transcription factor is preferably selected from the
group of consisting of:
[0032] VDR (SEQ ID NO:204), ZNF334 (SEQ ID Nos: 736-741), CREB3L2
(SEQ ID Nos: 565-567), ETV7 (SEQ ID NO:31 or 32), SOX4 (SEQ ID
NO:735), TWIST1 (SEQ ID Nos: 743-745), TP73 (SEQ ID Nos: 746-756),
FOXP3, NFE2L3 (SEQ ID NO:76), ARNTL2 (SEQ ID Nos: 757-764), BATF
(SEQ ID Nos: 765-766), PTTG1 (SEQ ID Nos: 767-770), HIVEP3 (SEQ ID
Nos: 771-772), FOXA1 (SEQ ID Nos: 720-721), ZBTB38 (SEQ ID NO:561),
FOXM1 (SEQ ID Nos: 773-778), TADA3 (SEQ ID Nos: 779-782), NFAT5
(SEQ ID NO:160, 783-791, 742).
[0033] In a preferred embodiment, the marker is MAGEH1 (SEQ ID NO:
708 or 709)
TABLE-US-00003 [MAGEH1_Entrez: 28986_ENSG00000187601_
ENST00000342972_ENSP00000343706 (SEQ ID NO: 708)
ATGCCTCGGGGACGAAAGAGTCGGCGCCGCCGTAATGCGAGAGCCGCA
GAAGAGAACCGCAACAATCGCAAAATCCAGGCCTCAGAGGCCTCCGAG
ACCCCTATGGCCGCCTCTGTGGTAGCGAGCACCCCCGAAGACGACCTG
AGCGGCCCCGAGGAAGACCCGAGCACTCCAGAGGAGGCCTCTACCACC
CCTGAAGAAGCCTCGAGCACTGCCCAAGCACAAAAGCCTTCAGTGCCC
CGGAGCAATTTTCAGGGCACCAAGAAAAGTCTCCTGATGTCTATATTA
GCGCTCATCTTCATCATGGGCAACAGCGCCAAGGAAGCTCTGGTCTGG
AAAGTGCTGGGGAAGTTAGGAATGCAGCCTGGACGTCAGCACAGCATC
TTTGGAGATCCGAAGAAGATCGTCACAGAAGAGTTTGTGCGCAGAGGG
TACCTGATTTATAAACCGGTGCCCCGTAGCAGTCCGGTGGAGTATGAG
TTCTTCTGGGGGCCCCGAGCACACGTGGAATCGAGCAAACTGAAAGTC
ATGCATTTTGTGGCAAGGGTTCGTAACCGATGCTCTAAAGACTGGCCT
TGTAATTATGACTGGGATTCGGACGATGATGCAGAGGTTGAGGCTATC
CTCAATTCAGGTGCTAGGGGTTATTCCGCCCCTTAA (SEQ ID NO: 709)
MPRGRKSRRRRNARAAEENRNNRKIQASEASETPMAASVVASTPEDDL
SGPEEDPSTPEEASTTPEEASSTAQAQKPSVPRSNFQGTKKSLLMSIL
ALIFIMGNSAKEALVWKVLGKLGMQPGRQHSIFGDPKKIVTEEFVRRG
YLIYKPVPRSSPVEYEFFWGPRAHVESSKLKVMHFVARVRNRCSKDWP
CNYDWDSDDDAEVEAILNSGARGYSAP*].
[0034] In the present invention, the tumor is preferably a solid or
liquid tumor. Preferably, the solid tumor is selected from the
group consisting of: non-small cell lung cancer, colorectal cancer,
breast cancer, gastric cancer.
[0035] In a preferred embodiment of the invention, the tumor is a
metastasis, preferably a bone, a brain or a liver metastasis.
[0036] Preferably, the metastasis derives from colon rectal cancer
or non-small-cell lung cancer.
[0037] Another object of the invention is the above defined
molecule for use in a method for in vivo depleting
tumor-infiltrating regulatory T cells in a subject or for use in a
method to enhance tumor immunity in a subject.
[0038] Another object of the invention is a pharmaceutical
composition comprising the molecule as defined above and at least
one pharmaceutically acceptable carrier.
[0039] A further object of the invention is a pharmaceutical
composition comprising the molecule as above defined, for use in
the prevention and/or treatment of tumor or for use in a method for
in vivo depleting tumor-infiltrating regulatory T cell in a subject
or for use in a method to enhance tumor immunity in a subject.
[0040] The pharmaceutical composition according to the invention
may further comprise a therapeutic agent, preferably the
therapeutic agent in an anti-tumoral agent.
[0041] Another object of the invention is an in vitro method for
diagnosing and/or assessing the risk of developing and/or
prognosing and/or for monitoring the progression and/or for
monitoring the efficacy of a therapeutic treatment and/or for the
screening of a therapeutic treatment of a tumour in a subject
comprising the steps of:
[0042] a) detecting at least one of the marker as above defined in
an isolated biological sample obtained from the subject and
[0043] b) comparing with respect to a proper control.
[0044] Another object of the invention is an in vitro or ex-vivo
method for diagnosing and/or assessing the risk of developing
and/or prognosing and/or for monitoring the progression and/or for
monitoring the efficacy of a therapeutic treatment and/or for the
screening of a therapeutic treatment of a tumour in a subject as
above defined, wherein the marker to be detected is at least one of
the marker selected from the group consisting of: LAYN, MAGEH1 and
CCR8.
[0045] Preferably the above method is for prognosing of colorectal
cancer or non-small cell lung cancer in a subject and comprises the
steps of:
[0046] a) detecting at least one of the marker selected from the
group consisting of:
[0047] LAYN, MAGEH1 and CCR8
[0048] in an isolated biological sample obtained from the subject
and
[0049] b) comparing with respect to a proper control,
[0050] wherein an amount of said at least one marker in the
isolated biological sample obtained from the subject higher than
the control amount indicates that the subject has a poor
prognosis.
[0051] In the above method, preferably step a) comprises measuring
the amount of the marker or of fragments thereof or of the
polynucleotide coding for said protein (DNA or mRNA) or of
fragments thereof in said isolated biological sample obtained from
the subject and step b) comprises comparing the measured amount of
step a) with a proper control amount.
[0052] Preferably, the in vitro method for monitoring the
progression and/or for monitoring the efficacy of a therapeutic
treatment of a tumour, as above defined, comprises the steps
of:
[0053] a) measuring the alteration of the amount or the alteration
of the activity of the above markers or of fragments thereof or of
the polynucleotide coding for said protein or fragments thereof in
said isolated biological sample obtained from the subject and
[0054] b) comparing the measured alteration of step a) with a
proper control alteration.
[0055] Another object of the invention is a method for the
treatment and/or prevention of tumor comprising administering to a
subject the molecule as above defined.
[0056] A further object is a method for identifying a molecule
acting as an anti-tumoral, comprising the steps of: [0057] assaying
candidate molecules for their binding specificity to the at least
one marker as above defined; [0058] selecting molecules having a
specific binding activity to the at least one marker as above
defined; [0059] testing such specific binding molecules for their
capacity of inhibiting proliferation and/or inducing an apoptotic
response in a cell system,
[0060] preferably by selectively depleting tumor-infiltrating
regulatory T cell, more preferably by inducing antibody-dependent
cell-mediated cytotoxicity (ADCC).
[0061] Preferably, the biological sample is a fluid, a cell or a
tissue sample, more preferably said sample is plasma or serum.
[0062] The term "biological sample" encompasses a clinical sample,
and also includes tissue obtained by surgical resection, tissue
obtained by biopsy, cells in culture, cell supernatants, cell
lysates, tissue samples, organs, bone marrow, blood, plasma, serum,
and the like.
[0063] A "sample" in the context of the present teachings refers to
any biological sample that is isolated from a subject. A sample can
include, without limitation an aliquot of body fluid, whole blood,
serum, plasma, solid tissue samples such as tissue biopsies, or
tissue cultures or cells derived therefrom and the progeny thereof,
synovial fluid, lymphatic fluid, ascites fluid, and interstitial or
extracellular fluid. The term "sample" also encompasses the fluid
in spaces between cells, including gingival crevicular fluid, bone
marrow, cerebrospinal fluid (CSF), saliva, mucous, sputum, semen,
sweat, urine, or any other bodily fluids. "Blood sample" can refer
to whole blood or any fraction thereof, including serum and plasma.
Samples can be obtained from a subject by means including but not
limited to venipuncture, excretion, ejaculation, massage, biopsy,
needle aspirate, lavage, scraping, surgical incision, or
intervention or other means known in the art. The definition also
includes samples that have been manipulated in any way after their
procurement, such as by treatment with reagents; washed; or
enrichment for certain cell populations, such as cancer cells or
samples in which regulatory T cells, are isolated and then
analyzed. The definition also includes sample that have been
enriched for particular types of molecules, e.g., nucleic acids,
polypeptides, etc.
[0064] Another object of the invention is a kit for carrying out
the above methods, comprising [0065] means to measure the amount or
the activity of the above markers or of fragments thereof and/or
means to measure the amount of the polynucleotide coding for said
protein or of fragments thereof and optionally, [0066] control
means.
[0067] Any combination of the above markers is comprised within the
present invention. Preferred combinations of markers are LAYN and
MAGEH1; LAYN and CCR8; CCR8 and MAGEH1; LAYN, MAGEH1 and CCR8.
[0068] Preferably, the above polynucleotide is an RNAi inhibitor,
preferably selected from the group consisting of: siRNA, miRNA,
shRNA, stRNA, snRNA, and antisense nucleic acid, or a functional
derivative thereof.
[0069] A comparative analysis of gene expression arrays from CD4+ T
cells infiltrating NSCLC and CRC revealed Treg-specific expression
of 328 markers as listed in Table IV Manipulation of Treg cells via
these markers can therefore be used to enhance immunotherapy of
cancer.
[0070] The expression "molecule able to modulate" and "modulator"
are herein interchangeable. By the term "modulator" it is meant a
molecule that effects a change in the expression and/or function of
at least one marker as above defined.
[0071] The change is relative to the normal or baseline level of
expression and/or function in the absence of the modulator, but
otherwise under similar conditions, and it may represent an
increase (e.g. by using an inducer or activator) or a decrease
(e.g. by using a suppressor or inhibitor) in the normal/baseline
expression and/or function. In the context of the present
invention, a "modulator" is a molecule which may suppress or
inhibit the expression and/or function of at least one marker that
is selectively deregulated in tumor-infiltrating regulatory T cell
for use in the prevention and/or treatment of cancer.
[0072] By the term "suppressor or inhibitor" or a "molecule which
(selectively) suppresses or inhibits" it is meant a molecule that
effects a change in the expression and/or function of the
target.
[0073] In the context of the present invention, a "modulator" is a
molecule which may induce or activate the expression and/or
function of at least one marker that is selectively deregulated in
tumor-infiltrating regulatory T cell for use in the prevention
and/or treatment of cancer.
[0074] The change is relative to the normal or baseline level of
expression and/or function in the absence of the modulator, but
otherwise under similar conditions, and it may represent an
increase (e.g. by using an inducer or activator) or a decrease
(e.g. by using a suppressor or inhibitor) in the normal/baseline
expression and/or function.
[0075] The suppression or inhibition of the expression and/or
function of the target may be assessed by any means known to the
skilled in the art. The assessment of the expression level or of
the presence of the target is preferably performed using classical
molecular biology techniques such as (real time Polymerase Chain
Reaction) qPCR, microarrays, bead arrays, RNAse protection analysis
or Northern blot analysis or cloning and sequencing.
[0076] The assessment of target function is preferably performed by
in vitro suppression assay, whole transcriptome analysis, mass
spectrometry analysis to identify proteins interacting with the
target.
[0077] In the context of the present invention, the target (or the
marker) may be the gene, the mRNA, the cDNA, or the encoded protein
thereof, including fragments, derivatives, variants, isoforms, etc.
Preferably, the marker is characterized by its Accession numbers
(i.e. NCBI Entrez ID; Ensembl Gene accession number (ENSG), Ensembl
transcript accession number (ENST) and Ensembl protein accession
number (ENSP), retrievable in the public database EnsEMBL
(http://www.ensembl.org) and/or amino acid and nucleotide
sequences, herein disclosed.
[0078] In the context of the present invention, the term "treat"
(or "treated", "treatment", etc.) when referred to CD4+ T cell,
means e.g. the exposure of the cell to an exogenous modulator as
above defined. The overexpression may be obtained e.g. by infecting
the cells with a viral vector expressing the molecule of the
invention. The inhibition of marker expression may e.g. by obtained
by transfection with polynucleotide, as e.g. with siRNAs. The term
"treat" may also mean that the cells are manipulated in order to
overexpress or silence the marker. The overexpression or the
silencing may be obtained e.g. by genetically modifying the
cells.
[0079] Control means can be used to compare the amount or the
increase of amount of the marker defined to a proper control. The
proper control may be obtained for example, with reference to known
standard, either from a normal subject or from normal population,
or from T cells different from tumour infiltrating regulatory T
cells or regulatory T cells.
[0080] The means to measure the amount of at least one marker as
above defined are preferably at least one antibody, functional
analogous or derivatives thereof. Said antibody, functional
analogous or derivatives thereof are specific for said marker.
[0081] In the context of the present invention, the antibody is
preferably selected from the group consisting of an intact
immunoglobulin, a Fv, a scFv (single chain Fv fragment), a Fab, a
F(ab')2, an "antibody-like" domain, an "antibody-mimetic domain", a
single antibody domain (VH domain or VL domains), a multimeric
antibody, recombinant or synthetic antigen-binding fragments, a
peptide or a proteolytic fragment containing the epitope binding
region. The terms "antibody" and "immunoglobulin" can be used
interchangeably and are herein used in the broadest sense and
encompass various antibodies and antibody mimetics structures,
including but not limited to monoclonal antibodies, polyclonal
antibodies, multispecific antibodies (e.g., bispecific antibodies),
chimeric antibodies, nanobodies, antibody derivatives, antibody
fragments, anticalins, DARPins, affibodies, affilins, affimers,
affitins, alphabodies, avimers, fynomers, monobodies and other
binding domains, so long as they exhibit the desired
antigen-binding activity.
[0082] The term immunoglobulin also includes "conjugate" thereof.
In the context of the present invention "conjugate" in relation to
the antibody of the invention includes antibodies (or fragments
thereof) conjugated with a substance (a compound, etc.) having a
therapeutic activity, e.g. anti-tumor activity and/or cell-killing
activity or a cytotoxic agents such as various A chain toxins,
ribosomes inactivating proteins, and ribonucleases; bispecific
antibodies designed to induce cellular mechanisms for killing
tumors (see, for example, U.S. Pat. Nos. 4,676,980 and 4,954,617).
The conjugate may be formed by previously preparing each of the
aforementioned antibody molecule and the aforementioned substance
having anti-tumor activity and/or cell-killing activity,
separately, and then combining them (immunoconjugate) or by
ligating a protein toxin used as such a substance having anti-tumor
activity and/or cell-killing activity to an antibody gene on a gene
according to a genetic recombination technique, so as to allow it
to express as a single protein (a fusion protein)
(immunotoxin).
[0083] An "antibody fragment" refers to a molecule other than an
intact antibody that comprises a portion of an intact antibody that
binds the antigen to which the intact antibody binds. Examples of
antibody fragments include but are not limited to Fv, Fab, Fab',
Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain
antibody molecules (e.g. scFv); and multispecific antibodies formed
from antibody fragments. VH or VL Fvs are also called
"Nanobodies".
[0084] The term "antibody mimetics" refers to those organic
compounds or binding domains that are not antibody derivatives but
that can bind specifically an antigen like antibodies do. They
include anticalins, DARPins, affibodies, affilins, affimers,
affitins, alphabodies, avimers, fynomers, monobodies and
others.
[0085] The term "chimeric" antibody refers to an antibody in which
a portion of the heavy and/or light chain is derived from a
particular source or species, while the remainder of the heavy
and/or light chain is derived from a different source or
species.
[0086] The terms "full length antibody," "intact antibody," and
"whole antibody" are used herein interchangeably to refer to an
antibody having a structure substantially similar to a native
antibody structure or having heavy chains that contain an Fc region
as defined herein.
[0087] In a preferred embodiment, the kit of the invention
comprises: [0088] a solid phase adhered antibody specific for said
compound; [0089] detection means of the ligand specific-marker
complex.
[0090] Alternatively, the reagents can be provided as a kit
comprising reagents in a suspension or suspendable form, e.g.
reagents bound to beads suitable for flow cytometry, preferably
magnetic beads coated with antibody capture. The instructions may
comprise instructions for conducting an antibody-based flow
cytometry assay.
[0091] Detection means are preferably means able to detect and/or
measure the amount of the described markers, e.g. means able to
detect the complex antigen-antibody, as enzyme conjugated secondary
antibodies, luminescent substrates, magnetic beads coated with
antibody capture, customized dried antibody cocktails and/or
columns with size filter cartridges and/or combined with specific
antibody filter (SAF).
[0092] In an embodiment, the method further comprises selecting a
therapeutic regimen based on the analysis. In an embodiment, the
method further comprises determining a treatment course for the
subject based on the analysis. Other means may be e.g. specific
primers and probes for RT PCR. The kits according to the invention
can further comprise customary auxiliaries, such as buffers,
carriers, markers, etc. and/or instructions for use. In the context
of the present invention the term "detecting" may be intended also
as "measuring the amount" or "measuring the alteration". In the
case of a method or a kit for assessing the risk and/or diagnosing
and/or prognosing of a tumour, the proper control may be a sample
taken from a healthy patient or from a patient affected by another
disorder or pathology, and the proper control amount or activity
may be the amount or activity of the same protein or polynucleotide
measured in a sample taken from a healthy patient or from a patient
affected by another disorder or pathology.
[0093] In the case of a method or a kit for monitoring the
progression of a tumour, the progress of the cancer is monitored
and the proper control may be a sample taken from the same subject
at various times or from another patient, and the proper control
amount or activity may by the amount or activity of the same
protein or polynucleotide measured in a sample taken from the same
subject at various times or from another patient.
[0094] In the case of a method or a kit for monitoring the efficacy
of a therapeutic treatment, the proper control may by a sample
taken from the same subject before initiation of the therapy or
taken at various times during the course of the therapy and the
proper control amount or activity may be the amount or activity of
the same protein or polynucleotide measured in a sample taken from
the same subject before initiation of the therapy or taken at
various times during the course of the therapy.
[0095] In the case of a method or a kit for the screening of a
therapeutic treatment, the proper control may be a sample taken
from subjects without treatment and from subjects treated with a
substance that is to be assayed or from subjects treated with a
reference treatment and the proper control amount or activity may
be the average of the amounts or activity of the same protein or
polynucleotide measured in samples taken from subjects without
treatment and from subjects treated with a substance that is to be
assayed or from subjects treated with a reference treatment. In
this case, if the amount or activity of MAGEH1 and/or LAYN and/or
CCR8 or polynucleotides thereof in the isolated biological sample
obtained from the subject is lower or equal than the control amount
or activity, it may indicate that the tested substance is effective
for the treatment of the tumour.
[0096] In the present invention, the expression "measuring the
amount" can be intended as measuring the amount (or the activity)
or concentration or level of the respective protein and/or mRNA
thereof and/or DNA thereof, preferably semi-quantitative or
quantitative. Measurement of a protein can be performed directly or
indirectly. Direct measurement refers to the amount or
concentration measure of the marker, based on a signal obtained
directly from the protein, and which is directly correlated with
the number of protein molecules present in the sample. This
signal--which can also be referred to as intensity signal--can be
obtained, for example, by measuring an intensity value of a
chemical or physical property of the marker. Indirect measurements
include the measurement obtained from a secondary component (e.g.,
a different component from the gene expression product) and a
biological measurement system (e.g. the measurement of cellular
responses, ligands, "tags" or enzymatic reaction products).
[0097] The term "amount", as used in the description refers but is
not limited to the absolute or relative amount of proteins and/or
mRNA thereof and/or DNA thereof, and any other value or parameter
associated with the same or which may result from these. Such
values or parameters comprise intensity values of the signal
obtained from either physical or chemical properties of the
protein, obtained by direct measurement, for example, intensity
values in an immunoassay, mass spectroscopy or a nuclear magnetic
resonance. Additionally, these values or parameters include those
obtained by indirect measurement, for example, any of the
measurement systems described herein. Methods of measuring mRNA and
DNA in samples are known in the art. To measure nucleic acid
levels, the cells in a test sample can be lysed, and the levels of
mRNA in the lysates or in RNA purified or semi-purified from
lysates can be measured by any variety of methods familiar to those
in the art. Such methods include hybridization assays using
detectably labeled DNA or RNA probes (i.e., Northern blotting) or
quantitative or semi-quantitative RT-PCR methodologies using
appropriate oligonucleotide primers. Alternatively, quantitative or
semi-quantitative in situ hybridization assays can be carried out
using, for example, tissue sections, or unlysed cell suspensions,
and detectably labeled (e.g., fluorescent, or enzyme-labeled) DNA
or RNA probes. Additional methods for quantifying mRNA include RNA
protection assay (RPA), cDNA and oligonucleotide microarrays,
representation difference analysis (RDA), differential display, EST
sequence analysis, and serial analysis of gene expression
(SAGE).
[0098] If by comparing the measured amount or activity of the above
markers or of the polynucleotide coding for said protein with the
amount or activity obtained from a control sample, the amount or
the activity of said marker in the sample isolated from the subject
corresponds to a higher value, the subject may present cancer or go
towards an aggravation of said disease.
[0099] If by comparing the measured amount or activity of the above
markers or of the polynucleotide coding for said protein with the
amount or the activity obtained from a control sample, the amount
or the activity of said marker in the sample isolated from the
subject corresponds to a similar or lower value, the subject may be
not affected by cancer or go toward an amelioration of cancer,
respectively.
[0100] Alternatively, the expression "detecting" or "measuring the
amount" is intended as measuring the alteration of the molecule.
Said alteration can reflect an increase or a decrease in the amount
or activity of the molecules as above defined. An increase of the
protein or of the activity of the marker or of the polynucleotide
coding for said marker can be correlated to an aggravation of
cancer. A decrease of the protein or of the activity of said marker
or of the polynucleotide coding for said protein can be correlated
to an amelioration of cancer or to recovery of the subject.
[0101] The expression "marker" is intended to include also the
corresponding protein encoded from said marker orthologous or
homologous genes, functional mutants, functional derivatives,
functional fragments or analogues, isoforms, splice variants
thereof.
[0102] When the expression "marker" is referred to genes, it is
intended to include also the corresponding orthologous or
homologous genes, functional mutants, functional derivatives,
functional fragments or analogues, isoforms thereof.
[0103] As used herein "fragments" refers to polynucleotides having
preferably a length of at least 1000 nucleotides, 1100 nucleotide,
1200 nucleotides, 1300 nucleotides, 1400 nucleotides, 1500
nucleotides.
[0104] As used herein "fragments" refers to polypeptides having
preferably a length of at least 10 amino acids, more preferably at
least 15, at least 17 amino acids or at least 20 amino acids, even
more preferably at least 25 amino acids or at least 37 or 40 amino
acids, and more preferably of at least 50, or 100, or 150 or 200 or
250 or 300 or 350 or 400 or 450 or 500 amino acids.
[0105] The term "polynucleotide" also refers to modified
polynucleotides.
[0106] As used herein, the term "vector" refers to an expression
vector, and may be for example in the form of a plasmid, a viral
particle, a phage, etc. Such vectors may include bacterial
plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived
from combinations of plasmids and phage DNA, viral DNA such as
vaccinia, adenovirus, lentivirus, fowl pox virus, and pseudorabies.
Large numbers of suitable vectors are known to those of skill in
the art and are commercially available.
[0107] The polynucleotide sequence, preferably the DNA sequence in
the vector is operatively linked to an appropriate expression
control sequence(s) (promoter) to direct mRNA synthesis. As
representative examples of such promoters, one can mention
prokaryotic or eukaryotic promoters such as CMV immediate early,
HSV thymidine kinase, early and late SV40, LTRs from retrovirus,
and mouse metallothionein-I. The expression vector may also contain
a ribosome binding site for translation initiation and a
transcription vector. The vector may also include appropriate
sequences for amplifying expression. In addition, the vectors
preferably contain one or more selectable marker genes to provide a
phenotypic trait for selection of transformed host cells such as
dihydro folate reductase or neomycin resistance for eukaryotic cell
culture, or such as tetracycline or ampicillin resistance in E.
coli.
[0108] As used herein, the term "host cell genetically engineered"
relates to host cells which have been transduced, transformed or
transfected with the polynucleotide or with the vector described
previously. As representative examples of appropriate host cells,
one can cite bacterial cells, such as E. coli, Streptomyces,
Salmonella typhimurium, fungal cells such as yeast, insect cells
such as Sf9, animal cells such as CHO or COS, plant cells, etc. The
selection of an appropriate host is deemed to be within the scope
of those skilled in the art from the teachings herein. Preferably,
said host cell is an animal cell, and most preferably a human
cell.
[0109] The introduction of the polynucleotide or of the vector
described previously into the host cell can be effected by method
well known from one of skill in the art such as calcium phosphate
transfection, DEAE-Dextran mediated transfection, electroporation,
lipofection, microinjection, viral infection, thermal shock,
transformation after chemical permeabilisation of the membrane or
cell fusion.
[0110] The polynucleotide may be a vector such as for example a
viral vector.
[0111] The polynucleotides as above defined can be introduced into
the body of the subject to be treated as a nucleic acid within a
vector which replicates into the host cells and produces the
polynucleotides or the proteins.
[0112] Suitable administration routes of the pharmaceutical
composition of the invention include, but are not limited to, oral,
rectal, transmucosal, intestinal, enteral, topical, suppository,
through inhalation, intrathecal, intraventricular, intraperitoneal,
intranasal, intraocular and parenteral (e.g., intravenous,
intramuscular, intramedullary, and subcutaneous). An additional
suitable administration route includes chemoembolization. Other
suitable administration methods include injection, viral transfer,
use of liposomes, e.g. cationic liposomes, oral intake and/or
dermal application.
[0113] In certain embodiments, a pharmaceutical composition of the
present invention is administered in the form of a dosage unit
(e.g., tablet, capsule, bolus, etc.).
[0114] For pharmaceutical applications, the composition may be in
the form of a solution, e.g. an injectable solution, emulsion,
suspension or the like. The carrier may be any suitable
pharmaceutical carrier. Preferably, a carrier is used which is
capable of increasing the efficacy of the RNA molecules to enter
the target cells. Suitable examples of such carriers are
liposomes.
[0115] The modulator as above defined is administered in a
pharmaceutically effective dosage, which in the case of
polynucleotides may be in the range of 0.001 pg/kg body weight to
10 mg/kg body weight depending on the route of administration and
the type or severity of the disease.
[0116] The term "pharmaceutical composition" refers to a
preparation which is in such form as to permit the biological
activity of an active ingredient contained therein to be effective,
and which contains no additional components which are unacceptably
toxic to a subject to which the formulation would be administered.
In the present invention the term "effective amount" shall mean an
amount which achieves a desired effect or therapeutic effect as
such effect is understood by those of ordinary skill in the art. In
the present invention, the antibody may be administered
simultaneously or sequentially with another therapeutic treatment,
that may be a chemotherapy or radiotherapy. The invention provides
formulations comprising a therapeutically effective amount of an
antibody as disclosed herein, a buffer maintaining the pH in the
range from about 4.5 to about 8.5, and, optionally, a surfactant.
The formulations are typically for an antibody as disclosed herein,
recombinant or synthetic antigen-binding fragments thereof of the
invention as active principle concentration from about 0.1 mg/ml to
about 100 mg/ml. In certain embodiments, the antibody, recombinant
or synthetic antigen-binding fragments thereof concentration is
from about 0.1 mg/ml to 1 mg/ml; preferably from 1 mg/ml to 10
mg/ml, preferably from 10 to 100 mg/ml.
[0117] Therapeutic formulations of the antibody/antibodies can be
prepared by mixing the antibody having the desired degree of purity
with optional physiologically acceptable carriers, excipients or
stabilizers (Remington's Pharmaceutical Sciences 16th edition,
Osol, A. Ed., 1980), in the form of lyophilized formulations or
aqueous solutions.
[0118] Pharmaceutical compositions containing the antibody of the
present invention may be manufactured by processes well known in
the art, e.g., using a variety of well-known mixing, dissolving,
granulating, levigating, emulsifying, encapsulating, entrapping or
lyophilizing processes. Proper formulation is dependent upon the
route of administration chosen. Parenteral routes are preferred in
many aspects of the invention.
[0119] For injection, including, without limitation, intravenous,
intramuscular and subcutaneous injection, the compounds of the
invention may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as physiological saline
buffer or polar solvents including, without limitation, a
pyrrolidone or dimethylsulfoxide.
[0120] Formulations for injection may be presented in unit dosage
form, e.g. in ampoules or in multi-dose containers. Useful
compositions include, without limitation, suspensions, solutions or
emulsions in oily or aqueous vehicles, and may contain adjuncts
such as suspending, stabilizing and/or dispersing agents.
Pharmaceutical compositions for parenteral administration include
aqueous solutions of a water soluble form, such as, without
limitation, a salt of the active compound. Additionally,
suspensions of the active compounds may be prepared in a lipophilic
vehicle. Suitable lipophilic vehicles include fatty oils such as
sesame oil, synthetic fatty acid esters such as ethyl oleate and
triglycerides, or materials such as liposomes. Aqueous injection
suspensions may contain substances that increase the viscosity of
the suspension, such as sodium carboxymethyl cellulose, sorbitol,
or dextran. Optionally, the suspension may also contain suitable
stabilizers and/or agents that increase the solubility of the
compounds to allow for the preparation of highly concentrated
solutions. Alternatively, the active ingredient may be in powder
form for constitution with a suitable vehicle, e.g., sterile,
pyrogen-free water, before use. For administration by inhalation,
the antibody of the present invention can conveniently be delivered
in the form of an aerosol spray using a pressurized pack or a
nebulizer and a suitable propellant. The antibody may also be
formulated in rectal compositions such as suppositories or
retention enemas, using, e.g., conventional suppository bases such
as cocoa butter or other glycerides. In addition to the
formulations described previously, the antibody may also be
formulated as depot preparations. Such long acting formulations may
be administered by implantation (for example, subcutaneously or
intramuscularly) or by intramuscular injection. The compounds of
this invention may be formulated for this route of administration
with suitable polymeric or hydrophobic materials (for instance, in
an emulsion with a pharmacologically acceptable oil), with ion
exchange resins, or as a sparingly soluble derivative such as,
without limitation, a sparingly soluble salt. Additionally, the
antibody may be delivered using a sustained-release system, such as
semi-permeable matrices of solid hydrophobic polymers containing
the therapeutic agent. Other delivery systems such as liposomes and
emulsions can also be used.
[0121] A therapeutically effective amount refers to an amount of
compound effective to prevent, alleviate or ameliorate cancer or
cancer recurrence symptoms. Determination of a therapeutically
effective amount is well within the capability of those skilled in
the art, especially in light of the disclosure herein. For any
antibody used in the invention, the therapeutically effective
amount can be estimated initially from in vitro assays. Then, the
dosage can be formulated for use in animal models so as to achieve
a circulating concentration range that includes the effective
dosage. Such information can then be used to more accurately
determine dosages useful in patients. The amount of the composition
that is administered will depend upon the parent molecule included
therein. Generally, the amount used in the treatment methods is
that amount which effectively achieves the desired therapeutic
result in mammals. Naturally, the dosages of the various compounds
can vary somewhat depending upon the compound, rate of in vivo
hydrolysis, etc. In addition, the dosage, of course, can vary
depending upon the dosage form and route of administration. The
range set forth above is illustrative and those skilled in the art
will determine the optimal dosing of the compound selected based on
clinical experience and the treatment indication. Moreover, the
exact formulation, route of administration and dosage can be
selected by the individual physician in view of the patient's
condition and of the most effective route of administration (e.g.,
intravenous, subcutaneous, intradermal). Additionally, toxicity and
therapeutic efficacy of the antibody and other therapeutic agent
described herein can be determined by standard pharmaceutical
procedures in cell cultures or experimental animals using methods
well-known in the art. It is contemplated that the treatment will
be given for one or more cycles until the desired clinical and
biological result is obtained. The exact amount, frequency and
period of administration of the compound of the present invention
will vary, of course, depending upon the sex, age and medical
condition of the patient as well as the severity and type of the
disease as determined by the attending clinician.
[0122] The modulator of the present invention may comprise a single
type of modulator or a plurality of different modulators.
[0123] The function of a regulatory T-cell may be inhibited by
inhibiting markers activity and/or expression or by decreasing the
number of cells positive for such markers in a T-cell population
(for example by binding at least one of the above marker and
inducing antibody-dependent cell-mediated cytotoxicity (ADCC)).
Inhibiting the function of regulatory T-cells in an organism may be
used to enhance the immune T-cell response in those circumstances
where such a response is desirable, such as in a patient suffering
from cancer.
[0124] When treating a cancer patient with an inhibitory agent that
binds to marker protein or mRNA, one may optionally co-administer
an anti-tumor vaccine or therapy. Such vaccines may be directed to
isolated antigens or to groups of antigens or to whole tumor cells.
It may be desirable to administer the inhibitory agent with
chemotherapeutic agents or together with radiotherapy.
[0125] Treatment with multiple agents need not be done using a
mixture of agents but may be done using separate pharmaceutical
preparations. The preparations need not be delivered at the same
exact time, but may be coordinated to be delivered to a patient
during the same period of treatment, i.e. within a week or a month
or each other.
[0126] Thus a composition comprising two active ingredients may be
constituted in the body of the patient. Any suitable anti-tumor
treatment can be coordinated with the treatments of the present
invention targeted to the markers. Similarly, if treating patients
with infections, other anti-infection agents can be coordinated
with the treatment of the present invention targeted to the
markers. Such agents may be small molecule drugs, vaccines,
antibodies, etc.
[0127] The number of marker+ cells in a T-cell population can be
modified by using an antibody or other agent that selectively binds
to the marker. marker+ cells represent an enriched population of
regulatory T-cells that can be introduced back into the original
source of the T-cells or into another compatible host to enhance
regulatory T-cell function. Alternatively, the marker-cells
represent a population of T-cells deficient in regulatory T-cell
activity that can be reintroduced into the original source of the
T-cells or another compatible host to inhibit or reduce regulatory
T-cell function while retaining general T-cell activity.
[0128] Any desired means for either increasing or decreasing
(modulating) marker activity can be used in the methods of the
invention. This includes directly modulating the function of marker
protein, modulating marker signal transduction, and modulating
expression of marker in T-cells by modulating either transcription
or translation or both. Those means which selectively modulate
marker activity are preferred over nonselective modulators. Also,
those inhibitory means which create a transient marker deficiency
in a population of T-cells which then return to normal levels of
marker activity may be preferred for treating a temporary T-cell
deficiency. The transiently deficient T-cells may be used to
reconstitute a diminished T-cell population with T-cells that will
be genetically normal with respect to the marker. Modulation of
marker activity can be performed on cells in vitro or in whole
animals, in vivo. Cells which are treated in vitro can be
administered to a patient, either the original source of the cells
or an unrelated individual. To inhibit the function of the marker
(antagonist), marker antibodies or small molecule inhibitors can be
used. Antibodies or antibody fragments that are useful for this
purpose will be those that can bind to the marker and block its
ability to function. Such antibodies may be polyclonal antibodies,
monoclonal antibodies, chimeric antibodies, humanized antibodies,
single-chain antibodies, soluble MHC class II molecules, antibody
fragments, etc.
[0129] Antibodies generated against marker polypeptides can be
obtained by direct injection of the marker polypeptides into an
animal or by administering marker polypeptides to an animal,
preferably a nonhuman. The antibody so obtained will then bind the
marker polypeptides itself. In this manner, even a sequence
encoding only a fragment of the marker polypeptide can be used to
generate antibodies binding the whole native marker
polypeptide.
[0130] For preparation of monoclonal antibodies, any technique
which provides antibodies produced by continuous cell line cultures
can be used. Examples include the hybridoma technique (Kohler and
Milstein, 1975, Nature, 256: 495-497), the trioma technique, the
human B-cell hybridoma technique (Kozbor et al., 1983, Immunology
Today 4: 72), and the EBV-hybridoma technique to produce human
monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies
and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Techniques
described for the production of single chain antibodies (U.S. Pat.
No. 4,946,778) can be readily used to produce single chain
antibodies to marker polypeptides. Also, transgenic mice may be
used to express humanized antibodies to immunogenic marker
polypeptides. To enhance or activate the function of the marker,
any agent which increases the level of the marker or the activity
of existing marker in the T-cell may be used. Such agents may be
identified using the screening assays described below. Expression
vectors encoding the marker can also be administered to increase
the gene dosage. The expression vectors can be plasmid vectors or
viral vectors, as are known in the art. Any vector can be chosen by
the skilled in the art for particularly desirable properties. In
the context of the present invention, the term "polynucleotide"
includes DNA molecules (e.g., cDNA or genomic DNA) and RNA
molecules (e.g., mRNA, siRNA, shRNA) and analogues of the DNA or
RNA generated using nucleotide analogues. The polynucleotide may be
single-stranded or double-stranded. The polynucleotide may be
synthesized using oligonucleotide analogues or derivatives (e.g.,
inosine or phosphorothioate nucleotides).
[0131] The RNAi inhibitors as above defined are preferably capable
of hybridizing to all or part of specific target sequence.
Therefore, RNAi inhibitors may be fully or partly complementary to
all of or part of the target sequence
[0132] The RNAi inhibitors may hybridize to the specified target
sequence under conditions of medium to high stringency.
[0133] An RNAi inhibitors may be defined with reference to a
specific sequence identity to the reverse complement of the
sequence to which it is intended to target. The antisense sequences
will typically have at least about 75%, preferably at least about
80%, at least about 85%, at least about 90%, at least about 95% or
at least about 99% sequence identity with the reverse complements
of their target sequences.
[0134] The term polynucleotide and polypeptide also includes
derivatives and functional fragments thereof.
[0135] In the context of the present invention, the at least one
gene or marker as above defined is preferably characterized by at
least one of the sequence identified by its Ensembl Gene ID or NCBI
Accession Numbers, as disclosed in Tables VIII or VI, or by at
least one of the SEQ ID No. 1-709.
[0136] The term gene herein also includes corresponding orthologous
or homologous genes, isoforms, variants, allelic variants,
functional derivatives, functional fragments thereof. The
expression "protein" is intended to include also the corresponding
protein encoded from a corresponding orthologous or homologous
genes, functional mutants, functional derivatives, functional
fragments or analogues, isoforms thereof.
[0137] The term "analogue" as used herein referring to a protein
means a modified peptide wherein one or more amino acid residues of
the peptide have been substituted by other amino acid residues
and/or wherein one or more amino acid residues have been deleted
from the peptide and/or wherein one or more amino acid residues
have been deleted from the peptide and or wherein one or more amino
acid residues have been added to the peptide. Such addition or
deletion of amino acid residues can take place at the N-terminal of
the peptide and/or at the C-terminal of the peptide.
[0138] A "derivative" may be a nucleic acid molecule, as a DNA
molecule, coding the polynucleotide as above defined, or a nucleic
acid molecule comprising the polynucleotide as above defined, or a
polynucleotide of complementary sequence. In the context of the
present invention the term "derivatives" also refers to longer or
shorter polynucleotides and/or polypeptides having e.g. a
percentage of identity of at least 41%, 50%, 60%, 65%, 70% or 75%,
more preferably of at least 85%, as an example of at least 90%, and
even more preferably of at least 95% or 100% with the sequences
herein mentioned or with their complementary sequence or with their
DNA or RNA corresponding sequence. The term "derivatives" and the
term "polynucleotide" also include modified synthetic
oligonucleotides. The modified synthetic oligonucleotide are
preferably LNA (Locked Nucleic Acid), phosphoro-thiolated oligos or
methylated oligos, morpholinos, 2'-O-methyl, 2'-O-methoxyethyl
oligonucleotides and cholesterol-conjugated 2'-O-methyl modified
oligonucleotides (antagomirs).
[0139] The term "derivative" may also include nucleotide analogues,
i.e. a naturally occurring ribonucleotide or deoxyribonucleotide
substituted by a non-naturally occurring nucleotide. The term
"derivatives" also includes nucleic acids or polypeptides that may
be generated by mutating one or more nucleotide or amino acid in
their sequences, equivalents or precursor sequences. The term
"derivatives" also includes at least one functional fragment of the
polynucleotide.
[0140] In the context of the present invention "functional" is
intended for example as "maintaining their activity".
[0141] In the context of the present invention, the vector as above
defined is preferably selected from the group consisting of:
plasmids, viral vectors and phages, more preferably the viral
vector is a lentiviral vector.
[0142] In the context of the present invention, the host cell as
above defined is preferably selected from the group consisting of:
bacterial cells, fungal cells, insect cells, animal cells, plant
cells, preferably being an animal cell.
[0143] Compositions comprising a mixture of antibodies which
specifically bind to the marker(s); and an anti-cancer vaccine can
be made in vitro. Preferably the composition is made under
conditions which render it suitable for use as a pharmaceutical
composition. Pharmaceutical compositions may be sterile and
pyrogen-free. The components of the composition can also be
administered separately to a patient within a period of time such
that they are both within the patient's body at the same time. Such
a time-separated administration leads to formation of the mixture
of antibodies and vaccine within the patient's body. If the
antibody and vaccine are to be administered in a time-separated
fashion, they may be supplied together in a kit. Within the kit the
components may be separately packaged or contained. Other
components such as excipients, carriers, other immune modulators or
adjuvants, instructions for administration of the antibody and the
vaccine, and injection devices can be supplied in the kit as well.
Instructions can be in a written, video, or audio form, can be
contained on paper, an electronic medium, or even as a reference to
another source, such as a website or reference manual.
[0144] Anti-marker antibodies of the invention can be used to
increase the magnitude of anti-cancer response of the cancer
patient to the anti-cancer vaccine or anti-cancer therapy. It can
also be used to increase the number of responders in a population
of cancer patients. Thus the antibodies can be used to overcome
immune suppression found in patients refractory to anti-cancer
vaccines or treatment. The anti-cancer vaccines can be any that are
known in the art, including, but not limited to whole tumor cell
vaccines, isolated tumor antigens or polypeptides comprising one or
more epitopes of tumor antigens.
[0145] Expression of marker in T-cells can be modulated at the
transcriptional or translational level. Agents which are capable of
such modulation can be identified using the screening assays
described below.
[0146] Translation of marker mRNA can be inhibited by using
ribozymes, antisense molecules, small interference RNA (siRNA; See
Elbashir, S. M. et al., "Duplexes of 21-nucleotide RNAs mediate RNA
interference in cultured mammalian cells", Nature 411: 494-498
(2001)) or small molecule inhibitors of this process which target
marker mRNA. Antisense technology can be used to control gene
expression through triple-helix formation or antisense DNA or RNA,
both of which methods are based on binding of a polynucleotide to
DNA or RNA. For example, the 5'coding portion of the polynucleotide
sequence, which codes for the mature polypeptides of the present
invention, is used to design an antisense RNA oligonucleotide of
from about 10 to 40 base pairs in length. A DNA oligonucleotide is
designed to be complementary to a region of the gene involved in
transcription (triple helix--see Lee et al., Nucl. Acids Res., 6:
3073 (1979); Cooney et al, Science, 241: 456 (1988); and Dervan et
al., Science, 251: 1360 (1991)), thereby preventing transcription
and the production of the marker. The antisense RNA oligonucleotide
hybridizes to the mRNA in vivo and blocks translation of the mRNA
molecule into the marker polypeptide (Antisense--Okano, J.
Neurochem., 56: 560 (1991); Oligodeoxynucleotides as Antisense
Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla. (1988)).
The oligonucleotides described above can also be delivered to cells
by antisense expression constructs such that the antisense RNA or
DNA may be expressed in vivo to inhibit production of the marker.
Such constructs are well known in the art. Antisense constructs,
antisense oligonucleotides, RNA interference constructs or siRNA
duplex RNA molecules can be used to interfere with expression of
the marker. Typically, at least 15, 17, 19, or 21 nucleotides of
the complement of marker mRNA sequence are sufficient for an
antisense molecule. Typically at least 19, 21, 22, or 23
nucleotides of marker are sufficient for an RNA interference
molecule. Preferably an RNA interference molecule will have a 2
nucleotide 3'overhang. If the RNA interference molecule is
expressed in a cell from a construct, for example from a hairpin
molecule or from an inverted repeat of the desired marker sequence,
then the endogenous cellular machinery will create the overhangs.
siRNA molecules can be prepared by chemical synthesis, in vitro
transcription, or digestion of long dsRNA by Rnase III or Dicer.
These can be introduced into cells by transfection,
electroporation, or other methods known in the art. (See Hannon, G
J, 2002, RNA Interference, Nature 418:244-251; Bernstein E et al.,
2002, The rest is silence. RNA 7:1509-1521; Hutvagner G et al. 9
RNAi: Nature harbors a double-strand. Curr. Opin. Genetics &
Development 12: 225-232, 2002, A system for stable expression of
short interfering RNAs in mammalian cells. Science 296: 550-553;
Lee N S, Dohjima T, Bauer G, Li H, Li M-J, Ehsani A, Salvaterra P,
and Rossi J. (2002). Expression of small interfering RNAs targeted
against HIV-1 rev transcripts in human cells. Nature Biotechnol.
20: 500-505; Miyagishi M, and Taira K. (2002). U6-promoter-driven
siRNAs with four uridine 3'overhangs efficiently suppress targeted
gene expression in mammalian cells. Nature Biotechnol. 20: 497-500;
Paddison P J, Caudy A A, Bernstein E, Hannon G J, and Conklin D S.
(2002). Short hairpin RNAs (shRNAs) induce sequence-specific
silencing in mammalian cells. Genes & Dev. 16: 948-958; Paul C
P, Good P D, Winer I, and Engelke D R. (2002). Effective expression
of small interfering RNA in human cells. Nature Biotechnol. 20:
505-508; Sui G, Soohoo C, Affar E-B, Gay F, Shi Y, Forester W C,
and Shi Y. (2002). A DNA vector-based RNAi technology to suppress
gene expression in mammalian cells. Proc. Natl. Acad. Sci. USA 99
(6): 5515-5520; Yu J-Y, DeRuiter S L, and Turner D L. (2002). RNA
interference by expression of short-interfering RNAs and hairpin
RNAs in mammalian cells. Proc. Natl. Acad. Sci. USA 99 (9):
6047-6052).
[0147] In addition to known modulators, additional modulators of
markers activity that are useful in the methods of the invention
can be identified using two-hybrid screens, conventional
biochemical approaches, and cell-based screening techniques, such
as screening candidate molecules for an ability to bind to marker
or screening for compounds which inhibit marker activity in cell
culture.
[0148] This provides a simple in vitro assay system to screen for
marker activity modulators. The method may identify agents that
directly interact with and modulate the marker, as well as agents
that indirectly modulate marker activity by affecting a step in the
marker signal transduction pathway.
[0149] Cell-based assays employing cells which express the marker
can employ cells which are isolated from mammals and which
naturally express the marker. Alternatively, cells which have been
genetically engineered to express the marker can be used.
Preferably the genetically engineered cells are T-cells.
[0150] Agents which modulate the marker activity by modulating the
marker gene expression can be identified in cell based screening
assays by measuring amounts of the marker protein in the cells in
the presence and absence of candidate agents. The marker protein
can be detected and measured, for example, by flow cytometry using
anti-marker specific monoclonal antibodies. Marker mRNA can also be
detected and measured using techniques known in the art, including
but not limited to Northern blot, RT-PCR, and array
hybridization.
[0151] In accordance with the teachings of the invention, marker
inhibitors may be administered to an organism to increase the
number of T-cells in the organism. This method may be useful for
treating organisms suffering from conditions resulting in a low
T-cell population. Such conditions include disorders involving
unwanted cellular invasion or growth, such as tumor growth or
cancer. Marker inhibitors may also be useful when administered in
combination with conventional therapeutics to treat T-cell
proliferation sensitive disorders. For instance, a tumor, which is
a T-cell proliferation sensitive disorder, is conventionally
treated with a chemotherapeutic agent which functions by killing
rapidly dividing cells. The marker inhibitors of the invention when
administered in conjunction with a chemotherapeutic agent enhance
the tumoricidal effect of the chemotherapeutic agent by stimulating
T-cell proliferation to enhance the immunological rejection of the
tumor cells. In accordance with the teachings of the invention,
marker activators (agonists) or expression enhancers may be
administered to an organism to decrease the number of T-cells, in
particular tumor-infiltrating regulatory T cells, in the organism
and thereby decrease deleterious T-cell activity. The methods of
the invention may be applied to any organism which contains T-cells
that express the marker. This includes, but is not limited to, any
mammal and particularly includes humans and mice.
[0152] When methods of the invention are carried out in vivo, the
effective amount of the marker modulator used will vary with the
particular modulator being used, the particular condition being
treated, the age and physical condition of the subject being
treated, the severity of the condition, the duration of the
treatment, the nature of the concurrent therapy (if any), the
specific route of administration and similar factors within the
knowledge and expertise of the health practitioner. For example, an
effective amount can depend upon the degree to which an individual
has abnormally depressed levels of T cells.
[0153] When administered, the pharmaceutical preparations of the
invention are applied in pharmaceutically-acceptable amounts and in
pharmaceutically-acceptably compositions. Such preparations may
routinely contain salt, buffering agents, preservatives, compatible
carriers, and optionally other therapeutic agents. When used in
medicine, the salts should be pharmaceutically acceptable, but
non-pharmaceutically acceptable salts may conveniently be used to
prepare pharmaceutically-acceptable salts thereof and are not
excluded from the scope of the invention. Such pharmacologically
and pharmaceutically-acceptable salts include, but are not limited
to those prepared from the following acids: hydrochloric,
hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic,
salicylic, citric, formic, malonic, succinic, and the like. Also,
pharmaceutically-acceptable salts can be prepared as alkaline metal
or alkaline earth salts, such as sodium, potassium or calcium
salts. Marker modulators may be combined, optionally, with a
pharmaceutically-acceptable carrier. The term
"pharmaceutically-acceptable carrier" as used herein means one or
more compatible solid or liquid filler, diluents or encapsulating
substances which are suitable for administration into a human. The
term "carrier" denotes an organic or inorganic ingredient, natural
or synthetic, with which the active ingredient is combined to
facilitate the application. The components of the pharmaceutical
compositions also are capable of being co-mingled with the
molecules of the present invention, and with each other, in a
manner such that there is no interaction which would substantially
impair the desired pharmaceutical efficacy.
[0154] The pharmaceutical compositions may contain suitable
buffering agents, including: acetic acid in a salt; citric acid in
a salt; boric acid in a salt; and phosphoric acid in a salt. The
pharmaceutical compositions also may contain, optionally, suitable
preservatives, such as: benzalkonium chloride; chlorobutanol;
parabens and thimerosal. Compositions suitable for parenteral
administration conveniently comprise a sterile aqueous preparation
of the anti-inflammatory agent, which is preferably isotonic with
the blood of the recipient. This aqueous preparation may be
formulated according to known methods using suitable dispersing or
wetting agents and suspending agents. The sterile injectable
preparation also may be a sterile injectable solution or suspension
in a non-toxic parenterally-acceptable diluent or solvent, for
example, as a solution in butane diol. Among the acceptable
vehicles and solvents that may be employed are water, Ringer's
solution, and isotonic sodium chloride solution. In addition,
sterile, fixed oils are conventionally employed as a solvent or
suspending medium. For this purpose any bland fixed oil may be
employed including synthetic mono- or di-glycerides. In addition,
fatty acids such as oleic acid may be used in the preparation of
injectables. Carrier formulation suitable for oral, subcutaneous,
intravenous, intramuscular, etc. administrations can be found in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa.
[0155] A variety of administration routes are available. The
particular mode selected will depend, of course, upon the
particular drug selected, the severity of the condition being
treated and the dosage required for therapeutic efficacy. The
methods of the invention, generally speaking, may be practiced
using any mode of administration that is medically acceptable,
meaning any mode that produces effective levels of the active
compounds without causing clinically unacceptable adverse effects.
Such modes of administration include oral, rectal, topical, nasal,
interdermal, or parenteral routes. The term "parenteral" includes
subcutaneous, intravenous, intramuscular, or infusion. Intravenous
or intramuscular routes are not particularly suitable for long-term
therapy and prophylaxis. They could, however, be preferred in
emergency situations. Oral administration will be preferred because
of the convenience to the patient as well as the dosing schedule.
The pharmaceutical compositions may conveniently be presented in
unit dosage form and may be prepared by any of the methods
well-known in the art of pharmacy. All methods include the step of
bringing the active agent into association with a carrier which
constitutes one or more accessory ingredients. In general, the
compositions are prepared by uniformly and intimately bringing the
active agent into association with a liquid carrier, a finely
divided solid carrier, or both, and then, if necessary, shaping the
product. Compositions suitable for oral administration may be
presented as discrete units, such as capsules, tablets, lozenges,
each containing a predetermined amount of the active agent. Other
compositions include suspensions in aqueous liquids or non-aqueous
liquids such as a syrup, elixir or an emulsion.
[0156] Other delivery systems can include time-release, delayed
release or sustained release delivery systems. Such systems can
avoid repeated administrations of the active agent, increasing
convenience to the subject and the physician. Many types of release
delivery systems are available and known to those of ordinary skill
in the art. They include polymer base systems such as poly
(lactide-glycolide), copolyoxalates, polycaprolactones,
polyesteramides, polyorthoesters, polyhydroxybutyric acid, and
polyanhydrides. Microcapsules of the foregoing polymers containing
drugs are described in, for example, U.S. Pat. No. 5,075,109.
Delivery systems also include non-polymer systems that are: lipids
including sterols such as cholesterol, cholesterol esters and fatty
acids or neutral fats such as mono-di- and tri-glycerides; hydrogel
release systems; sylastic systems; peptide based systems; wax
coatings; compressed tablets using conventional binders and
excipients; partially fused implants; and the like. Specific
examples include, but are not limited to: (a) erosional systems in
which the anti-inflammatory agent is contained in a form within a
matrix such as those described in U.S. Pat. Nos. 4,452,775,
4,667,014, 4,748,034 and 5,239,660 and (b) diffusional systems in
which an active component permeates at a controlled rate from a
polymer such as described in U.S. Pat. Nos. 3,832,253, and
3,854,480. In addition, pump-based hardware delivery systems can be
used, some of which are adapted for implantation. Use of a
long-term sustained release implant may be particularly suitable
for treatment of chronic conditions. Long-term release, are used
herein, means that the implant is constructed and arranged to
deliver therapeutic levels of the active ingredient for at least 30
days, and preferably 60 days. Long-term sustained release implants
are well-known to those of ordinary skill in the art and include
some of the release systems described above. While the invention
has been described with respect to specific examples including
presently preferred modes of carrying out the invention, those
skilled in the art will appreciate that there are numerous
variations and permutations of the above described systems and
techniques that fall within the spirit and scope of the invention
as set forth in the appended claims.
[0157] The invention will be illustrated by means of non-limiting
examples in reference to the following figures.
[0158] FIG. 1. Purification, functional characterization and
expression of immune checkpoints in tumor infiltrating cells.
[0159] (A) Representation of the sorting strategy of Treg cells
infiltrating tumor or normal tissue.
[0160] (B) Representative flow cytometry plots showing suppressive
activity of Treg cells isolated from tumor (NSCLC or CRC), normal
lung and blood of the same patient. 4.times.10.sup.5
carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled CD4+
naive T cells from healthy donors were cocultured with an equal
number of Treg cells for 4 days with a CD3-specific mAb and
CD1c+CD11c+ dendritic cells. Percentage of proliferating cells are
indicated. Data are representative of three independent
experiments.
[0161] (C) Z-score normalized RNA-seq expression values of immune
checkpoints genes are represented as a heatmap. Cell populations
are reported in the upper part of the graph, while gene names have
been assigned to heatmap rows. Hierarchical clustering results are
shown as a dendrogram drawn on the left side of the matrix. Colon
tissues are indicated as C, lung tissues as L and peripheral blood
as B. See also FIG. 6.
[0162] FIG. 2. Differential expression analysis identifies
co-regulated genes in tumor infiltrating Treg cells
[0163] Z-score normalized expression values of genes that are
preferentially expressed in tumor-infiltrating Tregs (Wilcoxon Mann
Whitney test p<2.2.times.10-16) over the listed cell subsets are
represented as boxed plots. Colon tissues are indicated as C, lung
tissues as L and peripheral blood as B.
[0164] FIG. 3. Single cell analysis of tumor infiltrating Treg
cells
[0165] (A) Schematic representation of the experimental workflow.
Experiments were performed on Treg cells infiltrating CRC, NSCLC,
or isolated from peripheral blood of healthy donors (PB); five
samples were collected for each tissue.
[0166] (B) Percentage of co-expression of signature genes with
FOXP3 and IL2RA is depicted.
[0167] (C) Expression levels of the signature genes classified by
the percentage of co-expression are represented as box plot.
[0168] (D) Expression distribution (violin plots) in Treg cells
infiltrating CRC, NSCLC or PB. Plots representing the ontology
classes of receptors, signaling and enzymatic activity, cytokine
activity and transcription factors are shown (Wilcoxon Mann Whitney
test p<0.05). Gray scale gradient indicates the percentage of
cells expressing each gene in Treg cells isolated from the three
compartments.
[0169] (E) Gene expression analysis of tumor Treg signature genes
in different tumor types. Expression values are expressed as log 2
(2{circumflex over ( )}-DCt).
[0170] FIG. 4. Expression of tumor-infiltrating Treg cells protein
signatures in CRC and NSCLC samples.
[0171] (A and B) Representative flow cytometry plots for tumor
normal tissue infiltrating Treg cells and peripheral blood Treg
cells analyzed for the expression of the indicated proteins.
[0172] FIG. 5. Prognostic value of signature transcripts of tumor
infiltrating Treg cells.
[0173] (A) Kaplan-Meier survival curve comparing the high and low
expression of the tumor Treg signature transcripts (CCR8, MAGEH1,
LAYN) normalized to the CD3G for the CRC (n=177) and NSCLC (n=263)
studies. Univariate analysis confirmed a significant difference in
overall survival curve comparing patients with high and low
expression. Statistical significance was determined by the log-rank
test. (CRC: p=0.05 for CCR8, p=1.48.times.10-3 for MAGEH1,
p=2.1.times.10-4 for LAYN; NSCLC: p=0.0125 for CCR8, p=0.035 for
MAGEH1, p=0.0131 for LAYN) Each table depicts the Kaplan Meier
estimates at the specified time points. (B) Expression
distributions of CCR8, MAGEH1 and LAYN according to tumor staging
at the time of surgery in the cohort of CRC patients. See also FIG.
9.
[0174] FIG. 6 related to FIG. 1. Transcriptome analysis of tumor
infiltrating lymphocytes.
[0175] (A) Representation of the sorting strategy of Treg cells
infiltrating colorectal tumor or normal tissue.
[0176] (B) RNA-seq expression values (normalized counts) of FOXP3,
TBX21 and RORC in CD4+ Th1, Th17 and Treg cells from CRC (C), NSCLC
(L) or peripheral blood (PB) of healthy donors.
[0177] (C) RNA-seq normalized counts data for selected immune
checkpoints and their ligands are shown as histogram plot. Cell
population names are reported in the lower part of each graph,
while gene names are shown in the upper part.
[0178] FIG. 7 related to FIG. 3. Single-cell analysis of tumor
infiltrating Treg cells.
[0179] Assessment of CD4+ Treg, Th1, Th17, Th2, CD8+ T cells and B
cell markers expression (percentage of expressing cells) in single
Treg cells purified from NSCLC and CRC.
[0180] FIG. 8 related to FIG. 4. Comparison of BATF expression in
CD4+ Treg vs Th17 cells.
[0181] BATF expression levels (RNA-seq normalized counts data) in
CD4+ Treg and Th17 subsets isolated from tumor tissue or peripheral
blood
[0182] FIG. 9 related to FIG. 5. Expression levels of
tumour-infiltrating Treg signature genes.
[0183] RNA-seq normalized counts data of three tumour-infiltrating
Treg signature genes (MAGEH1 (panel A), LAYN (panel B) and CCR8
(panel C)) across listed cell populations.
[0184] FIG. 10. Results of RT-PCR analysis done on cDNA from Tumor
infiltrating Treg cells (L=NSCLC, C=CRC, -=ntc) with specific
primers able to discriminate the different transcript isoforms
annotated for SIRPG.
DETAILED DESCRIPTION OF THE INVENTION
[0185] Experimental Procedures
[0186] Human Primary Tissues
[0187] Primary human lung or colorectal tumors and non-neoplastic
counterparts were obtained respectively from fifteen and fourteen
patients who underwent surgery for therapeutic purposes at
Fondazione IRCCS Ca' Granda, Policlinico or San Gerardo Hospitals
(Italy). Records were available for all cases and included
patients' age at diagnosis, gender, smoking habit (for lung cancer
patients), clinicopathological staging (Sobin et al., 2009), tumor
histotype and grade (Table II). No patient received palliative
surgery or neoadjuvant chemo- and/or radiotherapy. Informed consent
was obtained from all patients, and the study was approved by the
Institutional Review Board of the Fondazione IRCCS Ca' Granda
(approval n. 30/2014).
[0188] Non-small-cell lung cancer (NSCLC) were cut into pieces and
single-cell suspensions were prepared by using the Tumor
Dissociation Kit, human and the gentleMACS.TM. Dissociator
(Miltenyi Biotech cat. 130-095-929) according to the accompanying
standard protocol. Cell suspensions were than isolated by
ficoll-hypaque density-gradient centrifugation (Amersham
Bioscience). Colorectal cancer (CRC) specimens were cut into pieces
and incubated in DTT 0.1 mM (Sigma-Aldrich) for 10 min, then
extensively washed in HBSS (Thermo Scientific) and incubated in 1
mM EDTA (Sigma-Aldrich) for 50 min at 37.degree. C. in the presence
of 5% CO2. They were then washed and incubated in type D
collagenase solution 0.5 mg/mL (Roche Diagnostic) for 4 h at
37.degree. C. Supernatants containing tumor infiltrating
lymphocytes were filtered through 100 .mu.m cell strainer,
centrifuged and fractionated 1800.times.g for 30 min at 4.degree.
C. on a four-step gradient consisting of 100%, 60%, and 40% and 30%
Percoll solutions (Pharmacia). The T cell fraction was recovered
from the inter-face between the 60% and 40% Percoll layers.
[0189] CD4 T cell subsets were purified by FACS sorting using the
following fluorochrome conjugated antibodies: anti-CD4 APC/Cy7
(Biolegend clone OKT4), anti-CD27 Pacific Blue (Biolegend, clone
M-T271), anti-IL7R PE (Milteniy, clone MB15-18C9), anti-CD25 PE/Cy7
(eBioscience, clone BC96), anti-CXCR3 PE/Cy5 (BD, clone 1C6/CXCR3),
anti-CCR6 APC (Biolegend, clone G034E3) and anti-CCRS FITC
(Biolegend, clone j418F1) using a FACSAria II (BD).
[0190] Flow Cytometry
[0191] To validate surface marker expression cells were directly
stained with the following fluorochrome-conjugated antibodies and
analyzed by flow cytometry: anti-CD4 (Biolegend, clone OKT4);
anti-PD-L2 (Biolegend, Clone CL24F.10C12); anti-CD127 (eBioscience,
clone RDR5); anti-BATF (eBioscience, clone MBM7C7), anti-GITR
(eBioscience, clone eBIOAITR), anti-CD25 (Miltenyi, clone 4E3) and
anti 4-1BB (eBioscience clone 4B4) anti CCR8 (Biolegend clone
L263G8) anti CD30 (eBioscience, clone Ber-H2) anti PD-L1 (Biolegend
clone 29E.2A3) anti TIGIT (eBioscience, clone MBSA43) anti IL1R2 (R
and D clone 34141) IL21R (Biolegend clone 2G1-K12) anti OX40
(Biolegend clone Ber-ACT35). Intracellular staining was performed
using eBioscience Foxp3 staining kit according to the
manufactured's protocol (eBioscience cat 00-5523-00). Briefly cells
were harvested and fixed for 30 min in fixation/permeabilization
buffer at 4.degree. C., and than stained with anti-FOXP3 antibody
(eBioscience, clone 236A/E7) and anti-BATF (eBioscience clone
MBM7C7) in permeabilisation buffer for 30 min at 4.degree. C. Cells
were then washed two times, resuspended in FACS washing buffer and
analyzed by flow cytometry.
[0192] Suppression Assay.
[0193] 4.times.10.sup.4 carboxyfluorescein diacetate succinimidyl
ester (CFSE)-labeled (1 .mu.M) responders Naive.sup.+ T cells from
healthy donors were cocultured with different E/T ratio with
unlabeled CD127.sup.-CD25.sup.lowCD4.sup.+ T cells sorted from TILs
or PBMCs of patients with CRC or NSCLC, using FACS Aria II (BD
Biosciences), in the presence of CD11c.sup.+CD1c.sup.+ dendritic
cells as antigen-presenting cells and 0.5 mg/ml anti-CD3 (OKT3)
mAb. Proliferation of CFSE-labeled cells was assessed by flow
cytometry after 96 hr culture.
[0194] RNA Isolation and RNA Sequencing
[0195] RNA from tumor-infiltrating lymphocytes was isolated using
mirVana Isolation Kit. Residual contaminating genomic DNA was
removed from the total RNA fraction using Turbo DNA-free (Thermo
Fisher). The RNA yields were quantified using the QuantiFluor RNA
System (Promega) and the RNA quality was assessed by the Agilent
2100 Bioanalyzer (Agilent). Libraries for Illumina sequencing were
constructed from 50 ng of total RNA with the Illumina TruSeq RNA
Sample Preparation Kit v2 (Set A). The generated libraries were
loaded on to the cBot (Illumina) for clustering on a HiSeq Flow
Cell v3. The flow cell was then sequenced using a HiSeq 2500 in
High Output mode (Illumina). A paired-end (2.times.125) run was
performed.
[0196] RNA-Seq Data Analysis
[0197] Raw .fastq files were analyzed using FastQC v0.11.3, and
adapter removal was performed using cutadapt 1.8. Cutadapt is run
both for reverse and forward sequences with default parameters
[--anywhere <adapter1>--anywhere <adapter2>--overlap
10--times 2--mask-adapter]. Adapter sequences used for libraries
preparation are
TABLE-US-00004 Adapter1: (SEQ ID NO: 710)
AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNATCTCGTA TGCCGTCTTCTGCTTG
Adapter2: (SEQ ID NO: 711)
AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCG CCGTATCATT
[0198] Trimming was performed on raw reads using Trimmomatic
(Bolger et al., 2014): standard parameters for phred33 encoding
were used: ILLUMINACLIP (LEADING:3 TRAILING:3 SLIDINGWINDOW:4:15),
MINLEN parameter was set to 50.
[0199] Mapping and quantification: reads mapping to the reference
genome (GRCh38) was performed on quality-checked and trimmed reads
using STAR 2.4.1c: [STAR--genomeDir <index_star>--runThreadN
<cpu_number>--readFilesIn <trimmed>_R1.fastq.gz
<trimmed>_R2_P.fastq.gz--readFilesCommand zcat]. The
reference annotation is Ensembl v80. The overlap of reads with
annotation features found in the reference .gtf was calculated
using HT-seq v0.6.1. The output computed for each sample (raw read
counts) was then used as input for DESeq2 analysis. Raw counts were
normalized using DESeq2's function `r log`, and normalized counts
were used to perform and visualize Principal Component Analysis
(PCA) results (using DESeq2's `plotPCA` function).
[0200] Differential expression analysis: differential expression
analyses of tumor-infiltrating CD4+ Treg/Th1/Th17 subsets vs. CD4+
Treg/Th1/Th17 from PBMC were performed using DESeq2.
Upregulated/downregulated genes were selected for subsequent
analyses if their expression values were found to exceed the
threshold of 0.05 FDR (Benjamini-Hochberg correction).
[0201] Capturing of Single Cells, Preparation of cDNA and
Single-Cell PCR
[0202] Treg cells from 5 CRC and 5 NSCLC specimens were isolated as
previously described (See also Table II). Single cells were
captured on a microfluidic chip on the C1 System (Fluidigm) and
whole-transcriptome amplified. cDNA was prepared on chip using the
SMARTer Ultra Low RNA kit (Clontech). Cells were loaded onto the
chip at a concentration of 3-5E5 cells/ml, stained for viability
(LIVE/DEAD cell viability assay; Thermo Fisher) and imaged by
phase-contrast and fluorescence microscopy to assess the number and
viability of cells per capture site. Only single, live cells were
included in the analysis. For qPCR experiments, harvested cDNA was
pre-amplified using a 0.2.times. pool of primers prepared from the
same gene expression assays to be used for qPCR. Pre-amplification
allows for multiplex sequence-specific amplification 78 targets. In
detail, a 1.25 .mu.l aliquot of single cell cDNA was pre-amplified
in a final volume of 5 .mu.l using 1 .mu.l of PreAmp Master Mix
(Fluidigm) and 1.25 .mu.l pooled TaqMan assay mix (0.2.times.).
cDNA went through amplification by denaturing at 95.degree. C. for
15 s, and annealing and amplification at 60.degree. C. for 4 min
for 20 cycles. After cycling, pre-amplified cDNA was diluted 1:5 by
adding 20 .mu.l TE Buffer to the final 5 .mu.l reaction volume for
a total volume of 25 .mu.l.
[0203] Single-cell gene expression experiments were performed using
the 96.times.96 quantitative PCR (qPCR) DynamicArray microfluidic
chips (Fluidigm). A 2.25 .mu.l aliquot of amplified cDNA was mixed
with 2.5 .mu.l of TaqMan Fast Advanced Master Mix (Thermo Fisher)
and 0.25 .mu.l of Fluidigm's "sample loading agent," then inserted
into one of the chip "sample" inlets. A 2.5 .mu.l aliquot of each
20.times. TaqMan assay was mixed with 2.5 .mu.l of Fluidigm's
"assay loading agent" and individually inserted into one of the
chip "assay" inlets. Samples and probes were loaded into
96.times.96 chips using an IFC Controller HX (Fluidigm), then
transferred to a BioMark real-time PCR reader (Fluidigm) following
manufacturer's instructions. A list of the 78 TaqMan assays used in
this study is provided below.
TABLE-US-00005 TABLE V Related to FIG. 3. List of TaqMan Probes and
assay number used in RT-qPCR single-cell experiments Taqman Assays
Numbers Gene Name Assay Number Gene Number Assay Number BCL2L1
Hs00236329_ml ACP5 Hs00356261_ml EOS Hs00223842_ml BATF
Hs00232390_ml AHCYL1 Hs00198312_ml SLC35F2 Hs00213850_ml NFE2L3
Hs00852569_gl LAX1 Hs00214948_ml IL12RB2 Hs00155486_ml CCR8
Hs00174764_ml CD177 Hs00360669_ml ADPRH Hs00153890_ml OX40
Hs00937194_gl IKZF2 Hs00212361_ml METTL7A Hs00204042_ml CSF2RB
Hs00166144_ml ENTPD1 Hs00969559_ml NDFIP2 Hs00324851_ml NFAT5
Hs00232437_ml CADM1 Hs00942508_ml CTSC Hs00175188_ml ICOS
Hs00359999_ml SSH1 Hs00368014_ml COL9A2 Hs00156712_ml TMEM184C
Hs00217311_ml LTA Hs00236874_ml HTATIP2 Hs01091727_ml MAGEH1
Hs00371974_sl HSDL2 Hs00953689_ml IL21R Hs00222310_ml FOXP3
Hs01085834_ml SSTR3 Hs01066399_ml IL2RA Hs00907778_ml RNF145
Hs01099642_ml LIMA1 Hs01035646_ml LAPTM4B Hs00363282_ml NAB1
Hs00428619_ml GRSF1 Hs00909877_ml ACSL4 Hs00244871_ml ANKRD10
Hs00214321_ml ERI1 Hs00405251_ml NPTN Hs01033353_ml FKBP1A
Hs00356621_gl HS3ST3B1 Hs00797512_sl LEPROT Hs00956627_sl TRAF3
Hs00936781_ml NETO2 Hs00983152_ml RRAGB Hs01099767_ml VDR
Hs00172113_ml ZBTB38 Hs00257315_sl CSF1 Hs00174164_ml TIGIT
Hs00545087_ml GITR Hs00188346_ml TFRC Hs00951083_ml IL1R2
Hs01030384_ml JAK1 Hs01026983_ml IL1R1 Hs00991010_ml KSR1
Hs00300134_ml LAYN Hs00379511_ml ZNF282 Hs00411965_ml THADA
Hs00736554_ml PTPRJ Hs01119326_ml CTLA4 Hs00175480_ml CHRNA6
Hs02563509_sl CHST2 Hs01921028_sl IL2RB Hs01081697_ml CHST7
Hs00219871_ml TBX21 Hs00203436_ml LRBA Hs01032231_ml RORC
Hs01076112_ml ETV7 Hs00903229_ml CXCR5 Hs00540548_sl LY75
Hs00982383_ml CD8A Hs00233520_ml ADAT2 Hs00699339_ml CD8B
Hs00174762_ml GCNT1 Hs00155243_ml PTGDR2 Hs00173717_ml CASP1
Hs00354836_ml CD19 Hs01047410_gl
[0204] Single-Cell Data Analysis:
[0205] The Quality Threshold in the BioMark.TM. Analysis software
is a qualitative tool designed to measure the "quality" of each
amplification curve. Basically, each amplification curve is
compared to an ideal exponential curve and as the quality score
approaches 1 the closer it is to ideal. The further the curve is
from ideal, its quality score approaches 0. The default cutoff of
0.65 is an arbitrary value set by Fluidigm. Any curve above 0.65
passes. Any curve below, fails. Baseline correction was set on
Linear (Derivative)[default]. Ct Threshold Method was set on Auto
(Detectors). This method independently calculates a threshold for
each detector on a chip. For clustering and downstream analysis,
raw Cts have been converted to Log 2Exp by using a Limit of
Detection (LOD) of 35, which corresponds to the last PCR cycle.
Co-expression analysis has been performed by considering both CRC
and NSCLC samples on those genes for which both FOXP3 and IL2RA
were co-expressed at least to 2%. Gene's levels above the
background were depicted as violin plots after log 2 scale
transformation by ggplot2 (v. 2.1.10). The violin color gradient is
the percentage of cells that are expressing the gene of interest
and the upper bound of the color scale is the maximum percentage of
cells that express a gene of the whole geneset.
[0206] Procedure for the Removal of Transcripts Whose Expression
Values are Affected by the `Dropout` Effect.
[0207] Single-cell qPCR data are inherently noisy, and due the
limitations of current technologies the expression patterns of a
certain number of genes may be affected by the `dropout effect`.
Inventors performed a gene selection procedure in order to take
into account this `dropout` effect and discard those genes whose
expression values cannot be reliably used in a binary comparison
(tumor-peripheral vs blood). Inventors fitted a number of
parametric distributions to the ratios of detected genes on the
total number of tumor cells (both NSCLC and CRC) and selected the
reciprocal inverse Gaussian continuous random variable as best
fit.
[0208] Inventors then calculated the median value of the fitted
distribution and discarded those genes whose detection ratio is
less than this threshold value (at least 8.4% of detection).
Inventors reasoned that these genes are more likely to be affected
by the `dropout` effect. With this threshold inventors selected 45
genes for which a non-parametric T-test (Wilcoxon Mann Whitney test
p<0.05) has been performed (by comparing tumor vs. peripheral
blood samples).
[0209] Meta Analysis Kaplan-Meier and Stage Correlation
[0210] Statistical analysis was performed by using the R survival
package (Therneau T. 2013). Survival times were calculated as the
number of days from initial pathological diagnosis to death, or the
number of days from initial pathological diagnosis to the last time
the patient was reported to be alive. The Kaplan-Meier (KM) was
used to compare the high and low expression levels of the
tumor-Treg cell signature transcripts in either CRC (GSE17536) and
NSCLC (GSE41271) patients. For both studies annotation was
normalized to four tumor stages (1, 2, 3, 4). For study GSE41271
five patients were excluded due to incomplete or inaccurate
annotation (GSM1012883, GSM1012884, GSM1012885, GSM1013100,
GSM1012888), retaining a total of two hundred and sixty three
patients. Patients from both studies were labeled as `High` `Low`
whether or not their relative expression values exceeded a decision
boundary (mean of the samples). Inventors define {umlaut over
(x)}.sub.ij to denote the relative expression of the gene i for the
n samples of the study normalized to the CD3 level:
x i , j = x i , j x CD 3 G , j : ##EQU00001##
i=(CCR8,MAGEH1,LAYN) j=1, 2, . . . n samples
[0211] To classify a patient, a threshold on the is required and
defined as
T ( Upper , Lower ) = median ( x i , j ) .+-. .sigma. ( x i , j )
10 ##EQU00002##
[0212] where T.sub.(Upper,Lower) represent the upper and lower
extreme of the decision boundary:
{ x i , j > T Upper High x i , j < T Lower Low T Upper
.ltoreq. x i , j .ltoreq. T Lower excluded ##EQU00003##
[0213] Inventors examined the prognostic significance of tumor Treg
cells transcripts by using log-rank statistics; a p-value of less
than 0.05 was considered statistically significant. Since the
log-rank test resulted in a p-value of less than 0.05, a post stage
comparison by means of box plot representation was performed in
order to evaluate the correlation degree between the expression
level of the transcripts and tumor stages in the cohort of CRC
patients. The annotation was normalized to four tumor stages (1, 2,
3, 4).
[0214] Accession Numbers
[0215] The accession numbers for the present data are as follows:
ENA: PRJEB11844 for RNA-seq tumor and tissue infiltrating
lymphocytes; ArrayExpress: E-MTAB-2319 for RNA-seq human
lymphocytes datasets; ArrayExpress: E-MTAB-513 for Illumina Human
BodyMap 2.0 project; GEO: GSE50760 for RNA-seq datasets CRC; GEO:
GSE40419 for RNA-seq datasets NSCLC; GEO: GSE17536 for CRC
expression profiling by array; and GEO: GSE41271 for NSCLC
expression profiling by array.
[0216] Prediction of Surface-Exposed and Membrane-Associated
Proteins
[0217] The probability of surface exposure of the proteins encoded
by the genes of interest was determined by a combination of four
different cell localization prediction algorithms: Yloc
(Briesemeister et al, 2010), TMHMM
(http://www.cbs.dtu.dk/services/TMHMM/), SignalP
(http://www.cbs.dtu.dk/services/SignalP/) and Phobius (Kall et. al,
2007). In particular Yloc is a interpretable system offering
multiple predictive models in animal version; inventors used both
YLoc-LowRes predicting into 4 location (nucleus, cytoplasm,
mitochondrion, secretory pathway) and Yloc-HighRes predicting into
9 locations (extracellular space, plasma membrane, nucleus,
cytoplasm, mitochondrion, endoplasmic reticulum, peroxisome, Golgi
apparatus, and lysosome).
[0218] TMHMM and SignalP were developed by the bioinformatic unit
of the technical University of Denmark for the prediction of
transmembrane helices and the presence and location of signal
peptide cleavage sites in amino acid sequences, respectively.
Phobius is a combined transmembrane topology and signal peptide
predictor.
[0219] RT-PCR Analysis of Transcript Isoforms Expressed by
Tumor-Infiltrating Regulatory T Cells (Treg Cells)
[0220] Total RNA was extracted from tumor Treg cells (NSCLC or CRC)
using miRCURY RNA isolation kit (Exiqon) and 1 .mu.g was reverse
transcribed with iScript reverse transcription supermix (BIORAD).
Afterwards, 25 ng of cDNA were amplified with DreamTaq Green PCR
Master Mix (ThermoScientific) using multiple gene-specific primers
able to discriminate the different isoforms. PCR products were run
on agarose gel. The expression of specific transcripts was assessed
based on the expected band size.
[0221] Results
[0222] Tumor Infiltrating Tregs Cells Upregulate Immune Checkpoints
and are Highly Suppressive
[0223] To assess the gene expression landscape of tumor
infiltrating CD4.sup.+ T cells, the inventors isolated different
CD4.sup.+ lymphocytes subsets from two different tumors, NSCLC and
CRC, from the adjacent normal tissues, and from peripheral blood
samples. From all these tissues, the inventors purified by flow
cytometry (FIGS. 1A and 6A and 6B) CD4.sup.+ Treg (36 samples from
18 individuals), Th1 (30 samples from 21 individuals) and Th17 (22
samples from 14 individuals) cells (Table I and Table II).
TABLE-US-00006 TABLE 1 Purification and RNA-Sequencing of Human
Primary Lymphocyte Subsets Sorting Number of Mapped Tissue Subset
Phenotype Samples Reads (M) NSCLC CD4+ Treg CD4.sup.+ CD127.sup.- 8
587 CD25.sup.+ CD4.sup.+ Th1 CD4.sup.+ CXCR3.sup.+ 8 409 CCR6.sup.-
CD4.sup.+ Th17 CD4.sup.+ CCR6.sup.+ 6 206 CXCR3.sup.- CRC CD4.sup.+
Treg CD4.sup.+ CD127.sup.- 7 488 CD25.sup.+ CD4.sup.+ Th1 CD4.sup.+
CXCR3.sup.+ 5 266 CCR6.sup.- CD4.sup.+ Th17 CD4.sup.+ CCR6.sup.+ 5
308 CXCR3.sup.- Lung CD4.sup.+ Treg CD4.sup.+ CD127.sup.- 1 (pool
73 (normal tissue) CD25.sup.+ of 6) CD4.sup.+ Th1 CD4.sup.+
CXCR3.sup.+ 1 (pool 76 CCR6.sup.- of 6) Colon CD4.sup.+ Treg
CD4.sup.+ CD127.sup.- 7 404 (normal tissue) CD25.sup.+ CD4.sup.+
Th1 CD4.sup.+ CXCR3.sup.+ 6 352 CCR6.sup.- CD4.sup.+ Th17 CD4.sup.+
CCR6.sup.+ 6 284 CXCR3.sup.- PB CD4.sup.+ Treg CD4.sup.+
CD127.sup.- 8 259 (healthy donor) CD25.sup.+ CD4.sup.+ Th1
CD4.sup.+ CXCR3.sup.+ 5 70 CCR6.sup.- CD4.sup.+ Th17 CD4.sup.+
CCR6.sup.+ 5 77 CXCR3.sup.- For each cell subsets profiled by
RNA-sequencing tissue of origin, surface marker combinations used
for sorting, number of profited samples, as well as number of
mapped sequencing reads are indicated. M, million; CRC, colorectal
cancer; NSCLC, non-small cell lung cancer, PB, peripheral
blood.
TABLE-US-00007 TABLE II related to Table I. Patients information
and histological analysis. For each cell subset profiled by
RNA-sequencing, patient records are shown including: age at
diagnosis, gender, smoking habit (for lung cancer patients),
clinicopathological staging (TNM classification) tumor histotype
and grade. For Treg cell isolated for qPCR experiment the same
information are available, including also the number of live cells
captured from each tumor and available for single-cell analysis.
CRC: colorectal cancer; NSCLC: non-small cell lung cancer; (T):
Tumor Sample; (H): Healthy Tissue; ADC: Adenocarcinoma; SCC:
Squamous Cell Carcinoma; MUC ADC: Mucinous Adenocarcinoma. NSCLC
PATIENTS LIST (RNA SEQUENCING) (T)Th1 (T)Th17 (T)Treg (H)Th1
(H)Th17 (H)Treg SMOKE HABIT STATUS PATIENT1 SQ_0342 PREVIOUS SMOKER
> 15 y ALIVE PATIENT2 SQ_0339 PREVIOUS SMOKER < 15 y ALIVE
PATIENT3 SQ_0365 SQ_0375 PREVIOUS SMOKER < 15 y ALIVE PATIENT4
SQ_0366 SQ_0374 SQ_0341 PREVIOUS SMOKER > 15 y ALIVE PATIENT5
SQ_0364 SQ_0373 SQ_0350 SQ_0351 SMOKER DEAD PATIENT6 SQ_0358
SQ_0336 SQ_0350 SQ_0351 PREVIOUS SMOKER < 15 y ALIVE PATIENT7
SQ_0363 SQ_0376 SQ_0334 SQ_0350 SQ_0351 PREVIOUS SMOKER < 15 y
ALIVE PATIENT8 SQ_0357 SQ_0337 SQ_0350 SQ_0351 PREVIOUS SMOKER <
15 y ALIVE WITH RELAPSE PATIENT9 SQ_0404 SQ_0408 SQ_0398 SQ_0350
SQ_0351 SMOKER ALIVE PATIENT10 SQ_0403 SQ_0407 SQ_0396 SQ_0350
SQ_0351 PREVIOUS SMOKER > 15 y ALIVE NSCLC PATIENTS LIST (RNA
HISTOTYPE ADCA SUBTYPE SEQUENCING) GENDER AGE(y) MAJOR
(PREDOMINANT) GRADE pTNM:T pTNM:N pTNM:M STAGE PATIENT1 M 84 SCC G3
2b 0 0 IIA PATIENT2 M 83 SCC G3 2a 0 0 IB PATIENT3 M 72 SCC G3 2 2
0 IIIA PATIENT4 M 79 SCC G3 2a 0 0 IB PATIENT5 M 66 SCC G3 3 2 0
IIIA PATIENT6 M 71 SCC G3 4 1 0 IIIA PATIENT7 M 78 SCC G3 2b 1 0 IB
PATIENT8 M 77 ADCA SOLID G3 2 2 0 IIIA PATIENT9 F 69 ADCA SOLID G3
1a 0 0 IA PATIENT10 F 77 ADCA AC NAR G3 1a 0 0 IA NSLC = Non Small
Cell Lung Cancer ADC = Adenocarcinoma SCC = Squamous Cell Carcinoma
(T) = Tumor Sample (H) = HealthyTissue TUMOR INFILTRATING TREG FROM
HISTOTY NSCLC (SINGLE PE ADCA SUBTYPE CELL qPCR SMOKE HABIT STATUS
GENDER AGE(y) MAJOR (PREDOMINANT) PATIENT1 NEVER ALIVE F 65 ADCA
ACINAR and SMOKER PAPILLARY PATIENT2 PREVIOUS ALIVE M 62 ADCA SOLID
SMOKER < 15 y PATIENT3 NEVER ALIVE F 63 ADCA ACINAR SMOKER
PATIENT4 SMOKER ALIVE M 66 SCC PATIENT5 SMOKER ALIVE M 68 SCC TUMOR
INFILTRATING TREG FROM CAPTURED NSCLC (SINGLE SINGLE CELL qPCR
GRADE pTNM:T pTNM:N pTNM:M STAGE CELLS PATIENT1 G2 2a 0 0 IB 71
PATIENT2 G2 1b 0 0 IA 61 PATIENT3 G1 1a 0 0 IA 44 PATIENT4 G2 2a 0
0 IB 55 PATIENT5 G3 1b 0 0 IA 55 NSLC = Non Small Cell Lung Cancer
ADC = Adenocarcinoma SCC = Squamous Cell Carcinoma (T) = Tumor
Sample (H) = HealthyTissue CRC PATIENTS LIST (RNA SEQUENCING) (T)
Th1 (T) Th17 (T) Treg (H) Th1 (H) Th17 (H) Treg GENDER AGE(y)
PATIENT1 SQ_0389 SQ_0386 SQ_0387 SQ_0388 M 76 PATIENT2 SQ_0427
SQ_0434 SQ_0418 F 68 PATIENT3 SQ_0423 SQ_0436 SQ_0411 M 80 PATIENT4
SQ_0426 SQ_0437 SQ_0413 SQ_0428 SQ_0439 SQ_0417 M 79 PATIENT5
SQ_0425 SQ_0412 SQ_0429 SQ_0441 SQ_0422 M 78 PATIENT6 SQ_0424
SQ_0415 SQ_0431 SQ_0442 SQ_0421 M 69 PATIENT7 SQ_0435 SQ_0416
SQ_0432 SQ_0438 SQ_0420 F 84 PATIENT8 SQ_0414 F 75 PATIENT9 SQ_0433
SQ_0430 SQ_0440 SQ_0419 M 54 CRC PATIENTS LIST (RNA HISTOTYPE
SEQUENCING) MAJOR GRADE pTNM:T pTNM:N pTNM:M STAGE PATIENT1 ADC G2
3 1A 0 IIIB PATIENT2 ADC G2 3 0 .sup. 0 IIA PATIENT3 ADC G2 .sup.
4B 1B 0 IIIB PATIENT4 ADC G2 3 1A 0 IIIB PATIENT5 ADC G2 3 0 .sup.
0 IIA PATIENT6 MUC ADC -- 3 1B 0 IIIB PATIENT7 ADC G2 .sup. 4B 0
.sup. 0 IIC PATIENT8 MUC ADC -- 3 1C 0 IIIB PATIENT9 ADC G2 2 0
.sup. 0 I ADC = Adenocarcinoma MUC ADC = Mucinous Adenocarcinoma
CRIB ADC = Cribrous Adenocarcinoma (T) = Subsets purified from
Tumor Sample (H) = Subsets purified from Healthy Tissue TUMOR
INFILTRATING TREG FROM CRC HISTOTY (SINGLE CELL PE CAPTURED qPCR)
GENDER AGE(y) MAJOR GRADE pTNM:T pTNM:N pTNM:M STAGE SINGLE CELLS
PATIENT1 M 64 ADC 2 3 0 0 IIA 62 PATIENT2 M 59 CRIB ADC -- 3 0 0
IIA 66 PATIENT3 F 75 MUC ADC -- .sup. 4A .sup. 2B 0 IIIC 65
PATIENT4 M 71 ADC 1 3 0 0 IIA 63 PATIENT5 M 64 ADC 2 3 0 0 IIA 64
ADC = Adenocarcinoma MUC ADC = Mucinous Adenocarcinoma CRIB ADC =
Cribrous Adenocarcinoma (T) = Subsets purified from Tumor Sample
(H) = Subsets purified from Healthy Tissue
[0224] To assess Treg cell function, inventors tested their
suppressor activity and showed that Treg cells infiltrating either
type of tumor tissues have a remarkably stronger suppressive
activity in vitro compared to Treg cells isolated from the adjacent
normal tissue and peripheral blood of the same patients (FIG.
1B).
[0225] The polyadenylated RNA fraction extracted from the sorted
CD4+ Treg, Th1, and Th17 cells was then analyzed by pair-end RNA
sequencing obtaining about 4 billion mapped "reads" (Table I).
First, inventors interrogated RNA-sequencing data of CD4+ T cells
infiltrating both CRC and NSCLC and their matched normal tissues,
to quantitate mRNA expression of known immune checkpoints and their
ligands. Second, inventors analyzed RNA-seq data of CRC and NSCLC,
as well as of normal colon and lung samples. Inventors found that
several immune checkpoints and their ligands transcripts were
strikingly upregulated in tumor infiltrating Treg cells compared to
both normal tissue and peripheral blood-derived Treg cells, as well
as to T and B lymphocyte subsets purified from peripheral blood
mononuclear cells (PBMCs) (FIGS. 1C and 6C and Table III).
TABLE-US-00008 TABLE III related to FIG. 1. Expression levels of
immune checkpoints genes in all the subsets analyzed. GENE
Treg_Tumor_Infiltrating Treg_Tumor_Infiltrating
Treg_Tissue_Infiltrating Treg_Tissue_Infiltrating Treg healthy NAME
CRC NSCLC Colon Lung Peripheral Blood ADORA2A 14.69 24.06 17.97
44.84 18.52 BTLA 554.04 742.11 389.51 208.76 108.2 BTNL2 0 0.14
0.29 0 0.75 (BTLN2) C10orf54 779.38 872.36 555.47 1405.63 1111.37
(VISTA) CD160 58.39 38.24 51.87 34.54 36.55 CD200 268.39 283.21
282.05 104.64 99.59 CD200R1 95.89 136.08 81.36 349.99 59.03 CD244
34.46 31.21 29.59 128.35 47.8 CD27 710.13 1068.55 583.58 496.38
468.93 CD274 1050.94 645.66 576.59 390.71 120.19 (PD-L1) CD276
16.85 72.3 10.44 65.98 3.61 CD28 4770.41 4585.17 5446.29 3687.01
5179.32 CD40 112.04 161.29 80.64 93.3 34.71 CD40LG 135.51 143.07
360.09 418.55 104.22 CD44 13049.36 8518.98 13513.69 19851 16013.71
CD48 346.61 489.78 494.58 594.83 1523.63 CD70 426.35 269.38 318.97
249.48 101.67 CD80 632.12 483.34 318.48 269.06 114.41 CD86 29.52
78.86 52.72 278.86 3.87 CTLA4 6798.82 10378.3 4810.74 5340.06
4806.23 HAVCR2 577.57 633.27 265.84 487.62 49.81 (TIM-3) HHLA2 3.41
3.66 4.47 9.28 12.7 ICOS 6830.94 7339.08 4119.2 5211.71 3398.28
ICOSLG 58.02 8.86 59.13 33.5 76.5 (B7RP1) IDO1 3.86 83.81 9.51 5.15
2.36 IDO2 0.22 2.25 1.41 5.15 1.58 KIR3DL1 0.38 0.43 0.28 4.64 0.9
(KIR) LAG3 705.14 1956.22 2181.52 1505.63 127.02 LAIR1 277.06
194.09 551.94 874.72 346.22 LGALS9 1175.81 1530.47 1160.89 1593.26
592.56 (Galectin-9) NRP1 7.38 36.24 8.89 106.7 8.59 PDCD1LG2 214.51
223.04 61.89 25.77 12.12 (PD-L2) PDCD1 467.22 496.56 405.01 676.27
111.26 (PD1) TIGIT 14821.45 14747.79 10986.74 4901.41 4611.14
TMIGD2 28.38 16.64 78.3 75.77 71.27 TNFRSF14 2230.85 2677.32
2297.43 2675.7 2274.82 (HVEM) TNFRSF18 4038.86 4078.14 2871.78
3071.57 333.36 (GITR) TNFRSF25 5236.86 4188.61 4986.56 5111.71
3587.58 TNFRSF4 4222.16 4642.56 2873.16 2992.18 400.56 (OX40)
TNFRSF8 155.59 430.23 115.57 208.24 30.89 (CD30) TNFRSF9 2921.72
3128.82 898.69 1739.13 502.86 (4-1BB) TNFSF14 148.57 183.77 223.49
421.12 105.12 (LIGHT) TNFSF15 1.58 3.75 0.89 25.77 1.23 TNFSF18 0.4
1.11 0.53 0 0.45 TNFSF4 110.82 136.82 100.95 98.97 16.33 (OX40LG)
TNFSF9 26.79 19.48 19.72 29.9 7.41 (CD137L) VTCN1 1.12 4.49 1.48
1.55 2.65 (B7-H4)
[0226] RNA-seq normalized counts data for selected immune
checkpoints genes and their ligands in all the subsets
analyzed.
[0227] These findings highlight the specific expression patterns of
immune checkpoints and their ligands in tumor infiltrating Treg and
effector cells and suggest that their functional relevance should
be investigated directly at tumor sites.
[0228] Tumor-Infiltrating Treg Cells Express a Specific Gene
Signature
[0229] The inventors then asked whether tumor infiltrating Treg
cells could be defined by specific gene expression patterns.
[0230] To identify signature transcripts of tumor-infiltrating Treg
cells, the inventors included in the expression pattern analyses
the transcriptome dataset they previously obtained from different T
and B lymphocyte subsets purified from PBMCs (Ranzani et al.,
2015). In so doing, the inventors obtained a signature of 328
transcripts whose expression is higher in tumor infiltrating Treg
cells (Wilcoxon Mann Whitney test p<2.2.times.10-16) (FIG. 2,
and Table IV compared to the other lymphocyte subsets purified from
non-tumoral tissues and from PBMCs of healthy or neoplastic
patients.
TABLE-US-00009 TABLE IV related to FIG. 2. Expression levels of
tumor-infiltrating Treg gene signatures in all the subsets
analysed. Normalized expression values of tumour-infiltrating Treg
signature genes across listed cell populations. Gene
Treg_Tumor_Infiltrating Treg_Tumor_Infiltrating
Treg_Tissue_Inflitrating Treg_Tissue_Inflitrating Treg healthy Name
CRC NSCLC Colon Lung Peripheral Blood AC019206.1 15.41 8.72 12.89
12.04 29.46 ACAA2 305.76 499.02 497.41 526.58 614.28 ACOT9 918.3
803.71 1361.82 2180.66 1272.07 ACOX3 183.48 384.73 469.06 506.97
439.27 ACP5 267.7 837.72 859.77 1872.29 1483.27 ACSL4 1154.87
1384.88 1903.56 2170.94 2043.91 ACTA2 86.65 270.74 108.76 234.86
232.15 ACTG2 10.69 6.16 22.68 21.11 36.14 ADAM10 2378.26 3051.7
2545.29 3600.38 3167.56 ADAT2 927.45 1272.17 1214.4 2094.25 3103.21
ADPRH 136.34 460.61 352.57 836.7 718.74 AHCYL1 914.19 1271.5
1269.55 1835.94 1711.94 AHCYL2 305.15 570.67 525.24 790.1 856.25
AKAP5 174.24 264 358.75 709.28 535.97 AKIP1 261.47 273.85 225.25
436.84 360.48 ANKRD10 2251.92 3433.73 2805.08 4192.8 4672.81
ARHGEF12 1371.05 2064.05 1536.04 3069.77 2637.79 ARHGEF4 19.42
71.47 28.87 195.02 252.84 ARL6IP5 3008.69 4385.74 4051.43 4983.16
4712.48 ARNTL2 20.4 201.3 281.95 560.77 445.13 ATP13A3 3776.14
4020.7 4688.02 6688.94 6967.94 ATP2C1 1491.87 1399.81 1553.57
2029.41 1819.78 AURKA 24.56 50.12 79.89 66.37 87.07 BATF 820.97
3325.93 1698.92 5052.64 2727.65 BCL2L1 212.64 478.8 537.61 554.11
892.28 BIRC5 14.74 20.27 20.62 25.03 44.99 C17orf96 19 174.31
159.79 239.88 377.03 C5orf63 146.45 201.44 112.88 228.2 357.09
CABLES1 59.04 196.68 125.77 473.94 386.73 CACNB2 67.43 50.49 40.21
169.83 105.62 CADM1 113.76 602.72 115.46 1766.12 901.32 CALM3
2474.48 2829.3 2675.18 2954.03 4107.03 CARD16 370.31 696.36 493.29
1220.7 823.89 CARD17 41.87 96.94 54.12 101.19 132.95 CASP1 925.29
1453.84 1521.09 2028.95 1980.45 CASQ1 52.11 31.21 24.74 135.08
174.95 CCNB2 18.28 27.62 34.02 51.57 58.08 CCR8 255.66 578.27
1355.63 3127.33 2069.11 CD177 2.36 204.74 299.99 718.58 470.27 CD27
468.93 583.58 496.38 710.13 1068.55 CD274 120.19 576.59 390.71
1050.94 645.66 CD7 1622.12 6900.01 2829.82 9053.96 6919.59 CDCA2
19.24 35.09 49.48 68.21 49.95 CDH24 57.67 57.11 89.69 148.93 105.02
CDK6 602.97 2175.36 2463.85 3580.4 3238.58 CEACAM1 360.01 340.84
326.28 381.79 732.86 CENPM 43.72 39.12 61.85 72.94 61.32 CEP55
56.18 88.17 223.71 220.17 273.64 CGA 1.08 13.59 22.68 334.28 9.73
CHRNA6 14.46 218.49 67.52 336.38 504.28 CHST11 1822.7 2085.92
2806.11 2790.19 2535.23 CHST2 75.46 218.75 156.7 458.24 604.97
CHST7 141.3 341.87 426.79 1087.21 333.3 CIT 89.25 105.13 155.15
150.2 262.67 CLNK 153.06 288.36 248.96 340.12 528.54 CNIH1 1028.31
1005.46 935.03 2336.95 1101.87 COL9A2 149.87 278.77 357.72 889.47
805.72 CORO1B 481.34 667.37 861.83 774.65 1040.47 COX10 305.31
399.33 397.93 447.17 612.29 CRADD 77.04 155.66 277.31 394.31 306.61
CREB3L2 739.04 1289.66 1415.94 2984.54 2590.37 CSF1 313.09 1629.13
1609.75 2204.79 3288.67 CSF2RB 1069.75 1275.49 1290.69 2036.76
2531.99 CTLA4 4806.23 4810.74 5340.06 6798.82 10378.3 CTSC 1026.76
2196.93 2514.88 3030.74 2767.27 CTTNBP2NL 85 200.53 248.45 500.75
267.16 CX3CR1 9.57 63.99 123.71 341.79 293.28 CXCL13 1.07 255.23
1145.33 1270.98 11433.26 CYB5B 714.26 1129.39 947.4 1156.4 1221.22
CYP7B1 9.83 210.33 29.38 186.99 161.17 DCPS 153.25 210.26 210.82
191.31 271.71 DFNB31 561.87 1636.56 1727.79 4251.83 2526.15 DIRAS3
1.9 4.59 3.61 26.01 35.64 DLGAP5 7.89 14.46 20.62 27.41 49.7 DNPH1
160.15 650.05 321.13 683.55 576.77 DOC2B 10.47 3.42 5.15 14.23
238.86 DPYSL2 208.98 189.08 580.4 591.32 618.42 EBI3 7.47 103.59
56.7 148.96 200.74 ECEL1 3.7 150.7 34.02 199.17 794.51 EGLN1 977.29
969.32 1021.11 1381.2 1271.06 EML2 861.51 1601.25 1643.25 2156.04
1957.43 ENTPD1 752.88 2078.17 1447.38 4321.79 4162.57 ERI1 354.33
862.86 932.45 1200.06 1070.15 ETFA 414.08 586.15 534.01 615.35
689.14 ETV7 93.62 511.26 361.85 728.85 1111.55 EVA1B 21.39 35.63
26.8 42.86 47.36 F5 2343.39 2346.94 2499.41 4868.41 4729.97 FAAH2
244.19 431.76 209.27 737.44 699.42 FAIM2 15.05 33.47 57.21 69.26
117.28 FAM184A 192.41 742.47 525.24 706.33 891.02 FAM19A2 311.38
204.56 302.57 264.46 748.09 FAM98B 314.26 664.69 491.22 698.92
657.42 FAS 2337.14 5167.46 2712.81 5982.39 3656.21 FBXO45 460.56
783.06 631.43 964.13 894.23 FCRL3 1161.64 1997.02 938.63 3281.36
2699.01 FKBP1A 733.83 1240.62 1174.19 1377.67 1578.09 FLNB 1671.04
1363.04 1394.81 3395.38 2307.44 FLVCR2 69.84 579.55 388.13 744.8
528.01 FNDC3B 377.47 501.27 506.17 1111.07 531.12 FOXA1 2.7 11.87
17.01 70.68 18.22 FOXM1 56.39 74.94 108.24 88.16 125.31 FOXP3
6586.98 10713.12 6060.66 13483.77 11472.41 FUCA2 107.56 175.46
160.82 249.54 315.45 GADD45A 745.14 1431.9 884.51 3681.24 1396.98
GCNT1 99.22 632.16 608.75 1133.62 845.83 GK 637.31 1994.73 2430.34
5200.55 2065.35 GLB1 563.96 819.22 873.17 1077.84 854.94 GLCCI1
1557.57 3211.73 1753.04 3189.77 2909.06 GLDC 19.25 20.56 25.26
31.21 74.61 GLRX 1213.06 1251.64 1512.85 1764.61 1872 GNG4 5.08
79.18 64.43 197.1 343.93 GNG8 11.94 63.28 10.82 67.63 175.16 GRSF1
1277.4 1725.67 1397.9 2899.76 2343.4 GSK3B 1099.5 1267.18 1208.73
1333.16 1454.67 GTF3C6 313.17 579.04 445.86 617.48 597.55 GTSF1L
13.67 20.36 15.46 44.6 99.03 HADHB 1179.61 1207.14 1287.59 1396.89
1521.16 HAP1 92.39 180.51 74.22 292.97 577 HAVCR2 49.81 265.84
487.62 577.57 633.27 HECW2 17.63 98.93 38.66 111.21 177.5 HIBCH
124.32 290.04 226.8 348.34 332.88 HIVEP3 358.34 649.68 893.27
1091.96 1316.89 HJURP 8.55 18.52 15.98 27.13 39.99 HOXA1 16.66
15.22 14.95 25.57 44.75 HPRT1 442.58 532.66 542.25 811.75 724.15
HPSE 248.88 676.54 515.45 674.09 754.04 HS3ST3B1 1222.43 1930.88
1980.87 2609.49 2431.83 HSDL2 242.56 611.72 285.56 785.27 921.97
HTATIP2 567.61 1439.29 997.4 3285.86 1576.24 ICA1 94.65 371.57
113.91 487.68 411.64 ICOS 3398.28 4119.2 5211.71 6830.94 7339.08
IGFLR1 67.43 78.13 92.78 108.12 185.13 IKZF2 6061.48 6317.6 4919.45
9983.52 8551.49 IKZF4 1422.66 2362.49 1258.21 3745.25 3958.19
IL12RB2 120.8 369.84 509.78 835.92 877.51 IL17REL 9.74 23.21 34.02
52.62 57.04 IL1R1 506.51 9670.81 2766.42 7852.18 5585.89 IL1R2
41.72 1225.4 526.79 2117.34 1793.21 IL1RL1 17.37 135.26 44.33
715.42 71.67 IL1RL2 8.65 76.53 28.35 74.81 59.47 IL21R 708.61
1355.83 1715.93 3092.3 3514.36 IL2RA 5244.31 9685.38 5627.68
11454.42 12731.31 IL2RB 6716.4 14249.6 12502.75 17733 18564.35 IL32
4332.08 13202.73 9755.92 11766.98 13883.45 IL7 117.66 230.78 165.97
257.71 178.1 INPP1 124.25 497.01 312.88 458.2 487.93 INPP5F 787.92
2172.55 830.9 2189.48 1549.46 ISOC1 233.44 329.49 400.5 514.43
335.93 ITFG1 313.34 324.11 402.05 396.94 511.86 JAK1 10779.78
11919.66 10072.4 17755.9 11521.32 JAKMIP1 291.14 387.49 1063.89
756.36 953.47 KAT2B 3145.05 3910.01 4756.57 5520.88 4632.76 KIF14
20.18 25.43 31.96 36.73 59.61 KIF15 20.64 29.67 51.03 41.9 68.63
KIF20A 9.84 14.93 7.22 20.97 32.72 KLHDC7B 131.39 211.42 188.65
245.3 394.73 KSR1 837.87 1569.86 1176.77 2241.36 1847.72 LAPTM4B
86.42 369.78 181.44 938.88 738.38 LAX1 1135.24 1155.91 1406.15
1721.7 1854.78 LAYN 441.73 796.76 859.25 2650.24 1681.25 LEPR 58.77
130.22 129.38 137.47 237.88 LEPROT 614.73 860.55 676.79 1044.66
1296.13 LHFP 1.58 10.38 9.79 18.09 63.16 LIMA1 404.55 727.57 1017.5
1064.46 1570.15 LMCD1 115.76 104.74 112.37 257.92 404.7 LOC388813
7.42 45.99 28.87 86.3 60.63 LRG1 17.67 61.54 46.39 71.6 78.3 LRRC61
98.78 291.45 138.66 292.51 314.79 LTA 214.07 516.57 270.61 351.26
747.01 LXN 67.37 91.06 75.77 114.23 133.43 LY75 249.92 970.85
680.91 1302.79 1624.82 MAGEH1 461.13 1349.51 448.96 2800.36 3719.29
MALT1 3362.14 3568.46 2743.74 5892.86 4776.24 MAP1LC3A 70.92 110.44
119.07 272.07 169.3 MAP3K5 1865.12 2189.99 1787.06 2822.55 2265.54
MAST4 1053.08 2239.36 2198.39 3373.36 1855.42 MAT2B 2305.62 4050.5
2959.2 4435.41 4159.25 MCCC2 737.75 875.78 873.69 1018.1 1245.79
MELK 28.77 50.08 83.5 72.28 83.06 METTL7A 280.99 442.99 385.04
845.09 1671.74 METTL8 318.99 882.21 377.82 880.99 1413.12 MGME1
236.76 332.08 342.77 400.19 552.69 MGST2 54.22 87.18 69.59 147.04
148.13 MICAL2 354.6 1601.79 1813.35 1910.22 3188.92 MINPP1 85.19
204.32 211.85 243.22 290.02 MKI67 192.68 206.77 518.03 372.61
650.04 MREG 120.75 119.91 226.28 229.41 325.33 MYL6B 122.13 182.71
107.73 174.22 252.52 MYO5C 95.68 122.36 157.21 130.81 347.49 NAB1
508.21 973.74 1261.31 1831.77 1227.51 NCALD 111.73 163.32 272.67
283.43 370.26 NCAM1 7.88 58.27 39.69 207.45 213.23 NCF4 509.63
630.55 880.39 894.67 1176.84 NCOA1 2088.38 2062.57 1941.7 2367.54
2618.11 NDFIP2 77.99 529.73 618.54 829.53 987.25 NEMP2 382.56 478.4
475.76 565.18 634.41 NETO2 145.84 559.95 773.69 1490.82 1137.73
NEURL3 4.04 29.74 12.37 24.02 35.49 NFAT5 2075.17 3880.92 3923.6
4786.04 5295.06 NFE2L3 279.28 590.19 560.29 743.24 1114.26 NFYC
588.49 713.51 756.16 733.52 798.27 NHS 7.27 18.73 55.15 60.16
159.44 NPTN 525.86 838.02 897.91 1007.87 969.1 NTNG2 117.04 296.81
534.52 669.43 1001.58 NTRK1 20.85 27.9 155.15 88.29 161.78 NUSAP1
199.28 266.11 445.86 635.51 365.17 NXT2 221.6 263.39 226.8 285.15
302.01 OSBP2 111.03 89.82 127.83 195.47 244.93 PAK2 4621.62 6173.86
5024.6 7194.78 6376.28 PAM 582.52 904.05 1069.56 1365.03 1631.64
PANX2 3.7 76.02 15.46 97.12 71.72 PAQR4 16.99 46.54 62.37 92.6
65.27 PARD6G 55.86 172.18 249.99 546.52 182.4 PARK7 1271.06 1563.96
1283.47 1764.8 1764.91 PCTP 49.2 173.47 163.4 253.27 270.62
PDCD1LG2 12.12 61.89 25.77 214.51 223.04 PDGFA 6.19 38.74 159.79
154.17 153.03 PEX3 179.31 239.78 205.66 326.61 291.17 PGM2 316.91
419.51 454.63 471.89 487.85 PHKA1 8.59 19.98 28.87 107.79 109.7
PIGU 147.54 205.18 184.53 220.25 265.12 PLA2G4C 22.16 128.81 65.98
245.65 159.6 PPM1G 1974.96 2324.16 2563.85 2751.69 2598.5 PRDX3
466.56 854.12 745.34 890.58 1052.67 PRKCDBP 4.45 6.8 19.07 28.51
27.92 PROB1 53.7 140.39 109.79 177.19 272.89 PTGIR 96.17 147.61
107.21 214.61 449.25 PTP4A3 134.06 262.63 463.39 340.08 667.84
PTPRJ 2654.92 3999.84 5584.38 6101.63 7239.3 PTTG1 211.97 198.56
236.59 302.53 335.68 RAB15 160.6 470.25 302.05 420.06 519.4
RAD51AP1 29.89 46.33 40.21 49.23 51.73 RASAL1 18.87 53.37 50 87.38
238.78 RBKS 67.62 56.45 133.5 141.16 85.46 RCBTB1 1154.33 1312.01
1131.41 1960.76 1384.84 RDH10 194.04 311.58 467.51 658.5 1448.57
REXO2 487.9 832.35 648.44 852.58 987.43 RFK 378.31 396.91 292.26
460.78 452.8 RGS1 16547.6 15176.27 18057.75 23425.18 17168.17 RHOC
78.07 230.17 207.21 317.85 290.86
RMI2 19.46 76.58 39.69 70.44 73.47 RNF145 1625.11 3074.78 2117.47
4417.29 3266.94 RNF207 41.75 469.3 314.94 723.56 765.87 RRAGB
281.49 274.98 196.9 384.81 506.1 RYBP 1861.27 2273.72 2496.32
3178.31 2818.02 SEC14L6 6.42 86.23 27.32 179.47 274.97 SEC24A 718
917.25 1157.7 1259.04 1062.95 SECTM1 69.01 1347.35 725.75 2354.1
1511.04 SEPT3 15.6 59.23 49.48 149.11 244.4 SGPP2 428.14 656.73
364.94 1001.71 809.92 SH3RF2 20.9 18.3 65.98 98.4 196.34 SIRPG
433.99 605.49 317 575.41 1245.12 SLC16A1 947.47 1385.08 1532.43
2050.74 1460.73 SLC25A12 246.72 323.6 423.18 406.15 498.91 SLC35E3
385.3 451.16 370.09 582.86 653.13 SLC35F2 378.22 795.55 688.64
1130.81 880.5 SLC41A1 1194.29 1119.86 1164.92 1401.41 1630.88
SLC41A2 13.45 356.73 114.95 482.48 395.27 SMAD1 15.34 53.93 30.41
63.54 87.46 SMS 565.6 760.65 719.57 818.12 735.99 SNAP47 310.71
503.77 577.82 690.31 696.18 SOCS2 245.77 405.76 463.39 605.25
611.78 SOX4 128.76 244.57 218.04 1205.78 715.01 SPATA24 38.86 77.02
36.6 66.43 94.41 SPATC1 7.97 10.96 19.59 61.51 55.84 SPATS2L 366.98
891.61 1172.13 1430.11 1531.61 SSH1 1890.01 3432.55 2771.06 4390.36
4552.26 SSTR3 230.28 248.12 341.74 240.77 901.25 STAC 11.63 48.36
39.69 75.94 71.4 STARD7 2415.01 3185.95 3024.66 3809.46 3445.47
STRIP2 103.39 1002.96 540.19 716.49 1192.77 SYT11 1078.51 1733.37
2080.36 2110.18 2818.39 TADA3 677.14 893.74 852.04 880.43 1189.01
TBC1D8 53.89 374.1 265.97 817.36 1087.39 TDRD3 461.34 383.25 520.09
584.64 643.84 TFRC 3608.04 4612.18 5640.05 8107.35 10082.21 THADA
1102.51 1505.13 1467.48 3472.21 3171.99 TIGIT 4611.14 10986.74
4901.41 14821.45 14747.79 TM9SF2 2048.03 2689.14 2665.91 2935.98
3358.4 TMA16 172.88 180.92 137.11 304.24 192.53 TMEM140 273.98
640.28 574.73 917.16 691 TMEM184C 520.19 508.83 599.98 1170.37
519.43 TMOD1 14.75 72.22 32.47 150.93 89.62 TMPRSS3 70.84 352.78
321.64 540.8 1106.85 TMPRSS6 113.53 548.87 265.97 698.41 985.34
TNFRSF18 333.36 2871.78 3071.57 4038.86 4078.14 TNFRSF4 400.56
2873.16 2992.18 4222.16 4642.56 TNFRSF8 30.89 115.57 208.24 155.59
430.23 TNFRSF9 502.86 898.69 1739.13 2921.72 3128.82 TNIP3 28.73
485.83 213.91 324.53 419.8 TOR4A 141.27 291.3 346.9 358.98 326.51
TOX2 237.46 860.48 490.71 861.08 1264.13 TP73 7.86 31.27 39.69
78.27 93.99 TPMT 357.13 354.93 305.66 480.15 519.82 TPP1 2589.92
6024.92 4380.81 7164.96 6236.83 TPX2 106.25 89.08 184.02 150.35
202.77 TRAF3 1140.85 3231.25 2706.11 4078.84 3554.01 TRIB1 927.27
1820.64 1482.95 2402.58 1469.85 TRIM16 160.05 115.2 121.13 240.55
210.13 TSPAN17 709.59 1721.26 1322.64 1685.38 1865.69 TSPAN5 372.4
1167.46 723.69 1230.67 1398.7 TST 3.8 26.32 26.8 39.78 41.65 TTBK1
13.41 164.27 99.48 380.69 460.64 TTC22 237.9 386.91 323.19 483.96
451.61 TWIST1 4.21 94.46 21.65 95.32 195.78 UGP2 1950.41 3283.79
2562.82 3399.18 2864.71 USP51 48.1 133.95 28.87 233.48 291.46 UXS1
1661.1 2156.16 1600.47 2614.66 1914.74 VANGL1 97.19 192.58 248.96
263.46 289.05 VDR 123 992.41 1771.6 2616.68 3656.18 VWA5A 426.29
550.67 373.7 604.53 739.57 WDHD1 101.74 126.37 140.2 136.76 193.58
WDTC1 1220.3 3855.35 2029.33 4398.54 3774.61 WSB1 2837.49 3876.77
4697.29 5090.18 5383.33 XKRX 16.06 71.84 90.2 115.05 101.81 YIPF1
310.29 351.68 285.04 354.44 456.27 YIPF6 342.01 687.07 705.14
1078.09 793.2 ZBED2 87.53 94.86 522.15 230.51 1238.63 ZBTB38
1986.89 5405.41 3134.97 6174.05 4680.43 ZC3H12C 123.76 159.39
518.54 1191.95 985.54 ZG16B 3.42 17.03 15.46 32.31 32.59 ZMAT3
529.91 925.46 822.66 1077.17 1234.3 ZMYND8 585.94 675.31 711.84
850.29 1131.01 ZNF280C 181.86 444.81 326.28 635.21 467.78 ZNF280D
698.54 973.93 616.48 1061.55 1290.04 ZNF282 374.36 1273.4 2253.55
2562.43 3165.99 ZNF334 6.95 26.52 17.53 40.03 100.33 ZWINT 60.55
73.28 101.03 87.1 105.4
[0231] Altogether, the data show that Treg cells display the most
pronounced differences in transcripts expression among CD4.sup.+ T
cell subsets infiltrating normal and tumor tissues. The inventors
defined a subset of signature genes that describe the specific gene
expression profile of tumor infiltrating Treg cells.
[0232] Gene Signature of Tumor-Infiltrating Treg Cells is Present
in Primary and Metastatic Human Tumors
[0233] The inventors then looked at the single cell level for the
differential expression profile of signature genes of tumor
infiltrating Treg cells. The inventors isolated CD4.sup.+ T cells
from 5 CRC and 5 NSCLC tumor samples as well as from 5 PBMCs of
healthy individuals (Table II), purified Treg cells, and using an
automated microfluidic system (C1 Fluidigm) captured single cells
(a total of 858 Treg cells: 320 from CRC and 286 from NSCLC; 252
from PBMCs of healthy individuals). The inventors then assessed by
high throughput RT-qPCR (Biomark HD, Fluidigm) the expression of 79
genes selected among the highly expressed (>10 FKPM) tumor Treg
cell signature genes (FIGS. 3A, 3C and 7).
[0234] Notably, it was found that the vast majority (75 over 79;
95%) of the tumor-infiltrating Treg cell signatures were
co-expressed with bona fide Treg cell markers (i.e., FOXP3.sup.+
and IL2RA) (FIG. 3B). The percentage of co-expression between these
Treg cell markers and the 79 genes selected among the
tumor-infiltrating-Treg-cell signature genes ranged between 81% of
TIGIT and 0.59% of CGA (FIG. 3B). The expression of Treg signature
genes in the RNA-seq of the whole Treg cell population correlated
with the percentage of single cells expressing the different genes
(FIG. 3C). In order to reduce the "drop-out" effect of the single
cell data (i.e., events in which a transcript is detected in one
cell but not in another one because the transcript is `missed`
during the reverse-transcription step) (Kharchenko et al., 2014), a
threshold (median value t=8.4%) was defined based on the expression
distribution for each transcript and discarded genes below this
threshold. The forty-five signature transcripts of tumor
infiltrating Treg cells detected above this threshold were in most
cases significantly over-expressed in Treg cells from both tumors
(39 over 45, 87%; Wilcoxon Mann Whitney test p<0.05) or in one
tumor type (43 over 45, 96%; FIG. 3D). Homogeneity of the purified
tissue infiltrating Treg cells can be affected by the carry-over of
cells from other lymphocyte subsets. To quantitate this possible
contamination, the single cell RT-qPCR analyses of Treg cells was
performed including markers specific for other lymphocytes subsets
(i.e., Th1, Th2, Th17, Tfh, CD8 T cells, B cells) (FIG. 7). Our
data showed that only a very low fraction of the purified single
cells displayed markers of lymphocytes subsets different from Treg
cells (FIG. 7).
[0235] The overlap between the signature genes in the CRC and NSCLC
infiltrating Treg cells (FIG. 2) prompted us to assess whether this
signature were also enriched in Treg cells infiltrating other
tumors. RNA was thus extracted from Treg cells infiltrating breast
cancer, gastric cancer, brain metastasis of NSCLC, and liver
metastasis of CRC. It was found by RT-qPCR that tumor infiltrating
Treg signatures genes were mostly upregulated also in these tumors
(FIG. 3E).
[0236] Overall these data show that the tumor-infiltrating Treg
cell signature genes are co-expressed at single cell level with
FOXP3 and IL2RA and that several primary and metastatic human
tumors express the tumor-infiltrating Treg cell signature.
[0237] Gene Signature of Tumor Infiltrating Treg Cells is
Translated in a Protein Signature
[0238] The inventors then assessed at the single cell level by flow
cytometry the protein expression of ten representative signature
genes present in CRC and NSCLC infiltrating Treg cells, adjacent
normal tissues, and patients PBMCs. Of the ten proteins, two are
proteins (OX40 and TIGIT) whose relevance for Treg cells biology
has been demonstrated (Joller et al., 2014; Voo et al., 2013),
seven are proteins (BATF, CCR8, CD30, IL-1R2, IL-21R, PDL-1 and
PDL-2) whose expression has never been described in
tumor-infiltrating Treg cells, and one protein, 4-1BB, is a
co-stimulatory receptor expressed on several hematopoietic cells,
whose expression on Treg cells has been shown to mark
antigen-activated cells (Schoenbrunn et al., 2012). Our findings
showed that all these proteins were upregulated (FIGS. 4A and 4B),
at different extent, in tumor infiltrating Treg cells compared to
the Treg cells resident in normal tissues.
[0239] Altogether, our data show there is a molecular signature of
tumor infiltrating Treg cells, which can be detected both at the
mRNA and at the protein levels.
[0240] Expression of Tumor Treg Signature Genes is Negatively
Correlated with Patients Survival
[0241] In an attempt to correlate our findings with clinical
outcome, the inventors asked whether the expression of the
tumor-Treg signature transcripts correlated with disease prognosis
in CRC and NSCLC patients. The inventors therefore interrogated for
expression of Treg signature genes transcriptomic datasets obtained
from resected tumor tissues of a cohort of 177 CRC patients
(GSE17536 (Smith et al., 2010) and of a cohort of 263 NSCLC
patients (GSE41271--(Sato et al., 2013), and correlated high and
low gene expression levels with the 5-years survival data. Among
those genes whose expression is highly enriched in tumor
infiltrating Treg cells, LAYN, MAGEH1 and CCR8 were selected as
they are the three genes more selectively expressed (FIG. 9A-C). To
normalize for differences in T cell densities within the resected
tumor tissues, the inventors used the ratio between expression of
the selected signature genes and CD3G. Remarkably, it was found
that high expression of the three signature genes is in all cases
correlated with a significantly reduced survival (FIG. 5A).
Interestingly, it was also observed that expressions of the three
signature genes increased with tumor staging of CRC patients (FIG.
5B).
[0242] In conclusion, high expression in the whole tumor samples of
three genes (LAYN, MAGEH1 and CCR8) that are specifically and
highly expressed in tumor infiltrating Treg cells, correlates with
a poor prognosis in both NSCLC and CRC patients.
[0243] Selection of Potential Targets Specifically Over-Expressed
on the Surface of Tumor-Infiltrating Treg
[0244] All annotated protein isoforms encoded by the 328 genes and
retrievable in the public database EnsEMBL (http://www.ensembl.org)
were simultaneously analysed with the four prediction algorithms
and genes encoding at least one isoform predicted to be surface
exposed were considered as potential targets.
[0245] Out of 328 genes, 193 encode for at least one potential cell
surface protein isoform on the basis of at least one of the four
predictors. The list of protein isoforms predicted to be
membrane-associated is reported in Table VI.
TABLE-US-00010 TABLE VI SEQ ID No of the aa sequence of Gene ENSG
ID the protein name Description release87 ENST ID ENSP ID isoform
LAYN Layilin ENSG00000204381 ENST00000375614 ENSP00000364764 1
ENST00000375615 ENSP00000364765 2 ENST00000436913 ENSP00000392942 3
ENST00000525126 ENSP00000434328 4 ENST00000525866 ENSP00000434300 5
ENST00000528924 ENSP00000486561 6 ENST00000530962 ENSP00000431627 7
ENST00000533265 ENSP00000434972 8 ENST00000533999 ENSP00000432434 9
CCR8 C-C chemokine receptor type 8 ENSG00000179934 ENST00000326306
ENSP00000326432 10 ENST00000414803 ENSP00000390104 11 IL21R
Interleukin-21 receptor ENSG00000103522 ENST00000337929
ENSP00000338010 12 ENST00000395754 ENSP00000379103 13
ENST00000564089 ENSP00000456707 14 FUCA2 Plasma alpha-L-fucosidase
ENSG00000001036 ENST00000002165 ENSP00000002165 15 ENST00000451668
ENSP00000398119 16 ICA1 Islet cell autoantigen 1 ENSG00000003147
ENST00000407906 ENSP00000386021 17 COX10 Protoheme IX
farnesyltransferase, ENSG00000006695 ENST00000261643
ENSP00000261643 18 mit. IL32 Interleukin-32 ENSG00000008517
ENST00000008180 ENSP00000008180 19 ENST00000396890 ENSP00000380099
20 ENST00000525228 ENSP00000431740 21 ENST00000525377
ENSP00000433866 22 ENST00000530890 ENSP00000433747 23
ENST00000534507 ENSP00000431775 24 ENST00000548246 ENSP00000447979
25 ENST00000548476 ENSP00000449483 26 ENST00000548807
ENSP00000448354 27 ENST00000551513 ENSP00000449147 28
ENST00000552356 ENSP00000446978 29 ENST00000552936 ENSP00000447033
30 ETV7 Transcription factor ETV7 ENSG00000010030 ENST00000339796
ENSP00000342260 31 ENST00000627426 ENSP00000486712 32 ATP2C1
Calcium-transporting ATPase ENSG00000017260 ENST00000328560
ENSP00000329664 33 type 2C member 1 ENST00000359644 ENSP00000352665
34 ENST00000422190 ENSP00000402677 35 ENST00000428331
ENSP00000395809 36 ENST00000504381 ENSP00000425320 37
ENST00000504571 ENSP00000422489 38 ENST00000504612 ENSP00000425228
39 ENST00000504948 ENSP00000423330 40 ENST00000505072
ENSP00000427625 41 ENST00000505330 ENSP00000423774 42
ENST00000507194 ENSP00000427087 43 ENST00000507488 ENSP00000421326
44 ENST00000508297 ENSP00000421261 45 ENST00000508532
ENSP00000424783 46 ENST00000508660 ENSP00000424930 47
ENST00000509662 ENSP00000426849 48 ENST00000510168 ENSP00000427461
49 ENST00000513801 ENSP00000422872 50 ENST00000515854
ENSP00000422890 51 ENST00000533801 ENSP00000432956 52 FAS Fatty
acid synthase ENSG00000026103 ENST00000352159 ENSP00000345601 53
ENST00000355279 ENSP00000347426 54 ENST00000355740 ENSP00000347979
55 ENST00000357339 ENSP00000349896 56 ENST00000479522
ENSP00000424113 57 ENST00000484444 ENSP00000420975 58
ENST00000488877 ENSP00000425159 59 ENST00000492756 ENSP00000422453
60 ENST00000494410 ENSP00000423755 61 ENST00000612663
ENSP00000477997 62 PEX3 Peroxisomal biogenesis factor 3
ENSG00000034693 ENST00000367591 ENSP00000356563 63 ENST00000367592
ENSP00000356564 64 TSPAN17 Tetraspanin-17 ENSG00000048140
ENST00000298564 ENSP00000298564 65 ENST00000310032 ENSP00000309036
66 ENST00000503030 ENSP00000425975 67 ENST00000503045
ENSP00000425212 68 ENST00000504168 ENSP00000423957 69
ENST00000507471 ENSP00000423610 70 ENST00000508164 ENSP00000422053
71 ENST00000515708 ENSP00000426650 72 COL9A2 Collagen alpha-2(IX)
chain ENSG00000049089 ENST00000372736 ENSP00000361821 73
ENST00000372748 ENSP00000361834 74 ENST00000417105 ENSP00000388493
75 NFE2L3 Nuclear factor erythroid 2-related ENSG00000050344
ENST00000056233 ENSP00000056233 76 factor 3 TNIP3
TNFAIP3-interacting prot.3 ENSG00000050730 ENST00000515036
ENSP00000424284 77 LY75 Lymphocyte antigen 75 ENSG00000054219
ENST00000263636 ENSP00000263636 78 YIPF1 Protein YIPF1
ENSG00000058799 ENST00000072644 ENSP00000072644 79 ENST00000371399
ENSP00000360452 80 ENST00000412288 ENSP00000416507 81
ENST00000464950 ENSP00000432266 82 ISOC1 Isochorismatase domain-
containing ENSG00000066583 ENST00000173527 ENSP00000173527 83
protein 1 ENST00000514194 ENSP00000421273 84 ACSL4
Long-chain-fatty-acid-CoA ligase 4 ENSG00000068366 ENST00000340800
ENSP00000339787 85 ENST00000469796 ENSP00000419171 86
ENST00000469857 ENSP00000423077 87 ENST00000502391 ENSP00000425408
88 ENST00000504980 ENSP00000421425 89 ENST00000508092
ENSP00000425378 90 MAST4 Microtubule-assoc.serine/threonine-
ENSG00000069020 ENST00000434115 ENSP00000396765 91 protein kinase 4
LMCD1 LIM and cysteine-rich domains protein 1 ENSG00000071282
ENST00000456506 ENSP00000405049 92 TFRC Transferrin receptor
protein 1 ENSG00000072274 ENST00000360110 ENSP00000353224 93
ENST00000392396 ENSP00000376197 94 ENST00000421258 ENSP00000402839
95 ENST00000426789 ENSP00000414015 96 PANX2 Pannexin-2
ENSG00000073150 ENST00000159647 ENSP00000159647 97 ENST00000395842
ENSP00000379183 98 ENST00000402472 ENSP00000384148 99 FNDC3B
Fibronectin type III domain-containing ENSG00000075420
ENST00000336824 ENSP00000338523 100 protein 3B ENST00000415807
ENSP00000411242 101 ENST00000416957 ENSP00000389094 102
ENST00000421757 ENSP00000408496 103 ENST00000423424 ENSP00000392471
104 IL12RB2 Interleukin-12 receptor subunit beta-2 ENSG00000081985
ENST00000262345 ENSP00000262345 105 ENST00000371000 ENSP00000360039
106 ENST00000441640 ENSP00000400959 107 ENST00000541374
ENSP00000445276 108 ENST00000544434 ENSP00000442443 109 STARD7
StAR-related lipid transfer protein 7, ENSG00000084090
ENST00000337288 ENSP00000338030 110 mitochondrial SSH1 Protein
phosphatase Slingshot homolog 1 ENSG00000084112 ENST00000546697
ENSP00000446652 111 ENST00000548522 ENSP00000448586 112 MGST2
Microsomal glutathione S-transferase 2 ENSG00000085871
ENST00000265498 ENSP00000265498 113 ENST00000503816 ENSP00000423008
114 ENST00000506797 ENSP00000424278 115 ENST00000616265
ENSP00000482639 116 ACOX3 Peroxisomal acyl- coenzyme A oxidase 3
ENSG00000087008 ENST00000514423 ENSP00000427321 117 ANKRD10 Ankyrin
repeat domain- containing protein 10 ENSG00000088448
ENST00000603993 ENSP00000474638 118 FKBP1A Peptidyl-prolyl
cis-trans isomerase FKBP1A ENSG00000088832 ENST00000612074
ENSP00000480846 119 ENST00000614856 ENSP00000482758 120
ENST00000618612 ENSP00000478093 121 SIRPG Signal-regulatory protein
gamma ENSG00000089012 ENST00000216927 ENSP00000216927 122
ENST00000303415 ENSP00000305529 123 ENST00000344103 ENSP00000342759
124 ENST00000381580 ENSP00000370992 125 ENST00000381583
ENSP00000370995 126 WHRN Whirlin ENSG00000095397 ENST00000374059
ENSP00000363172 127 CENPM Centromere protein M ENSG00000100162
ENST00000215980 ENSP00000215980 128 ENST00000402338 ENSP00000384731
129 ENST00000402420 ENSP00000384132 130 ENST00000404067
ENSP00000384814 131 ENST00000407253 ENSP00000384743 132 NCF4
Neutrophil cytosol factor 4 ENSG00000100365 ENST00000447071
ENSP00000414958 133 CSF2RB Cytokine receptor common subunit beta
ENSG00000100368 ENST00000262825 ENSP00000262825 134 ENST00000403662
ENSP00000384053 135 ENST00000406230 ENSP00000385271 136
ENST00000421539 ENSP00000393585 137 CNIH1 Protein cornichon homolog
1 ENSG00000100528 ENST00000216416 ENSP00000216416 138
ENST00000395573 ENSP00000378940 139 ENST00000553660 ENSP00000452457
140 ENST00000554683 ENSP00000452466 141 ENST00000556113
ENSP00000451142 142 ENST00000557659 ENSP00000451640 143
ENST00000557690 ENSP00000451852 144 PIGU Phosphatidylinositol
glycan anchor ENSG00000101464 ENST00000217446 ENSP00000217446 145
biosynthesis class U protein ENST00000374820 ENSP00000363953 146
ENST00000438215 ENSP00000395755 147 NDFIP2 NEDD4 family-
interacting protein 2 ENSG00000102471 ENST00000218652
ENSP00000218652 148 ENST00000487865 ENSP00000419200 149
ENST00000612570 ENSP00000480798 150 ENST00000620924 ENSP00000480881
151 ACP5 Tartrate-resistant acid phosphatase type 5 ENSG00000102575
ENST00000218758 ENSP00000218758 152 ENST00000412435 ENSP00000392374
153 ENST00000433365 ENSP00000413456 154 ENST00000589792
ENSP00000468685 155 ENST00000590420 ENSP00000468509 156
ENST00000590832 ENSP00000465127 157 ENST00000591319 ENSP00000464831
158 ENST00000592828 ENSP00000468767 159 NFAT5 Nuclear factor of
activated ENSG00000102908 ENST00000567990 ENSP00000455115 160
T-cells 5 CYB5B Cytochrome b5 type B ENSG00000103018
ENST00000307892 ENSP00000308430 161 ENST00000512062 ENSP00000423679
162 ENST00000568237 ENSP00000464102 163 LAPTM4B
Lysosomal-associated transmembrane ENSG00000104341 ENST00000445593
ENSP00000402301 164 protein 4B ENST00000517924 ENSP00000429868 165
ENST00000521545 ENSP00000428409 166 ENST00000619747 ENSP00000482533
167 IL7 Interleukin-7 ENSG00000104432 ENST00000263851
ENSP00000263851 168 ENST00000379113 ENSP00000368408 169
ENST00000518982 ENSP00000430272 170 ENST00000520215 ENSP00000428364
171 ENST00000520269 ENSP00000427750 172 ENST00000520317
ENSP00000427800 173 ENST00000541183 ENSP00000438922 174 EBI3
Interleukin-27 subunit beta ENSG00000105246 ENST00000221847
ENSP00000221847 175 PLA2G4C Cytosolic phospholipase A2 gamma
ENSG00000105499 ENST00000595161 ENSP00000469528 176 ENST00000595487
ENSP00000471328 177 ENST00000596352 ENSP00000471759 178
ENST00000598488 ENSP00000468972 179 GLCCI1 Glucocorticoid-induced
transcript 1 ENSG00000106415 ENST00000430798 ENSP00000396171 180
protein MINPP1 Multiple inositol polyphosphate ENSG00000107789
ENST00000371994 ENSP00000361062 181 phosphatase 1 ENST00000371996
ENSP00000361064 182 ENST00000536010 ENSP00000437823 183 WSB1 WD
repeat and SOCS box-containing ENSG00000109046 ENST00000581440
ENSP00000462737 184 protein 1 ENST00000582208 ENSP00000463621 185
ENST00000583193 ENSP00000462595 186 ENST00000583742 ENSP00000462365
187 HTATIP2 Oxidoreductase HTATIP2 ENSG00000109854 ENST00000419348
ENSP00000392985 188 ENST00000530266 ENSP00000436548 189
ENST00000532081 ENSP00000432107 190 ENST00000532505 ENSP00000432338
191
CTSC Dipeptidyl peptidase 1 ENSG00000109861 ENST00000227266
ENSP00000227266 192 ENST00000524463 ENSP00000432541 193
ENST00000527018 ENSP00000432556 194 ENST00000528020 ENSP00000433229
195 ENST00000529974 ENSP00000433539 196 VWA5A von Willebrand factor
A domain- ENSG00000110002 ENST00000392744 ENSP00000376501 197
containing protein 5A ENST00000392748 ENSP00000376504 198
ENST00000456829 ENSP00000407726 199 SLC35F2 Solute carrier family
35 member F2 ENSG00000110660 ENST00000375682 ENSP00000364834 200
ENST00000525071 ENSP00000434307 201 ENST00000525815 ENSP00000436785
202 ENST00000532513 ENSP00000433783 203 VDR Vitamin D3 receptor
ENSG00000111424 ENST00000547065 ENSP00000449074 204 SEC24A Protein
transport protein Sec24A ENSG00000113615 ENST00000398844
ENSP00000381823 205 IL1R2 Interleukin-1 receptor type 2
ENSG00000115590 ENST00000332549 ENSP00000330959 206 ENST00000393414
ENSP00000377066 207 ENST00000441002 ENSP00000414611 208
ENST00000457817 ENSP00000408415 209 IL1R1 Interleukin-1 receptor
type 1 ENSG00000115594 ENST00000409288 ENSP00000386478 210
ENST00000409329 ENSP00000387131 211 ENST00000409589 ENSP00000386555
212 ENST00000409929 ENSP00000386776 213 ENST00000410023
ENSP00000386380 214 ENST00000413623 ENSP00000407017 215
ENST00000422532 ENSP00000390349 216 ENST00000424272 ENSP00000415366
217 ENST00000428279 ENSP00000410461 218 ENST00000430171
ENSP00000408101 219 ENST00000442590 ENSP00000393296 220
ENST00000450319 ENSP00000411627 221 ENST00000452403 ENSP00000401646
222 IL1RL2 Interleukin-1 receptor- like 2 ENSG00000115598
ENST00000264257 ENSP00000264257 223 ENST00000421464 ENSP00000387611
224 ENST00000441515 ENSP00000413348 225 IL1RL1 Interleukin-1
receptor- like 1 ENSG00000115602 ENST00000233954 ENSP00000233954
226 ENST00000311734 ENSP00000310371 227 ENST00000404917
ENSP00000384822 228 ENST00000409584 ENSP00000386618 229
ENST00000427077 ENSP00000391120 230 ENST00000447231 ENSP00000409437
231 UXS1 UDP-glucuronic acid decarboxylase 1 ENSG00000115652
ENST00000283148 ENSP00000283148 232 ENST00000409501 ENSP00000387019
233 ENST00000441952 ENSP00000416656 234 ENST00000457835
ENSP00000399316 235 SLC25A12 Calcium-binding mitochondrial
ENSG00000115840 ENST00000426896 ENSP00000413968 236 carrier protein
Aralar1 THADA Thyroid adenoma- associated protein ENSG00000115970
ENST00000403856 ENSP00000385469 237 LEPR Leptin receptor
ENSG00000116678 ENST00000344610 ENSP00000340884 238 ENST00000349533
ENSP00000330393 239 ENST00000371058 ENSP00000360097 240
ENST00000371059 ENSP00000360098 241 ENST00000371060 ENSP00000360099
242 ENST00000406510 ENSP00000384025 243 ENST00000616738
ENSP00000483390 244 MREG Melanoregulin ENSG00000118242
ENST00000263268 ENSP00000263268 245 ENST00000620139 ENSP00000484331
246 FLVCR2 Feline leukemia virus subgroup C ENSG00000119686
ENST00000238667 ENSP00000238667 247 receptor- related protein 2
ENST00000539311 ENSP00000443439 248 ENST00000553341 ENSP00000452584
249 ENST00000553587 ENSP00000451603 250 ENST00000554580
ENSP00000451781 251 ENST00000555027 ENSP00000452453 252
ENST00000555058 ENSP00000451104 253 ENST00000556856 ENSP00000452468
254 SOCS2 Suppressor of cytokine signaling 2 ENSG00000120833
ENST00000548537 ENSP00000448709 255 ENST00000549510 ENSP00000474888
256 RDH10 Retinol dehydrogenase 10 ENSG00000121039 ENST00000240285
ENSP00000240285 257 ENST00000519380 ENSP00000428132 258
ENST00000521928 ENSP00000429727 259 LAX1 Lymphocyte transmembrane
adapter 1 ENSG00000122188 ENST00000367217 ENSP00000356186 260
ENST00000442561 ENSP00000406970 261 ZWINT ZW10 interactor
ENSG00000122952 ENST00000489649 ENSP00000473330 262 ACOT9
Acyl-coenzyme A thioesterase 9, ENSG00000123130 ENST00000336430
ENSP00000336580 263 mitochondrial ENST00000379303 ENSP00000368605
264 ENST00000494361 ENSP00000420238 265 TM9SF2 Transmembrane 9
superfamily member 2 ENSG00000125304 ENST00000376387
ENSP00000365567 266 HS3ST3B1 Heparan sulfate glucosamine3-O-
ENSG00000125430 ENST00000360954 ENSP00000354213 267
sulfotransferase 3B1 ENST00000466596 ENSP00000436078 268 EML2
Echinoderm microtubule-associated protein-like 2 ENSG00000125746
ENST00000245925 ENSP00000245925 269 ENST00000586195 ENSP00000465339
270 ENST00000586405 ENSP00000465885 271 ENST00000586770
ENSP00000465786 272 ENST00000587152 ENSP00000468312 273
ENST00000587484 ENSP00000465994 274 ENST00000588272 ENSP00000466100
275 ENST00000588308 ENSP00000468329 276 ENST00000589876
ENSP00000464789 277 ENST00000590018 ENSP00000468373 278
ENST00000590043 ENSP00000464804 279 ENST00000590819 ENSP00000464950
280 ENST00000591721 ENSP00000468470 281 ENST00000592853
ENSP00000468383 282 ENST00000593255 ENSP00000467941 283 MGME1
Mitochondrial genome maintenance ENSG00000125871 ENST00000377704
ENSP00000366933 284 exonuclease 1 ENST00000377709 ENSP00000366938
285 ENST00000377710 ENSP00000366939 286 IGFLR1 IGF-like family
receptor 1 ENSG00000126246 ENST00000246532 ENSP00000246532 287
ENST00000588018 ENSP00000468545 288 ENST00000588992 ENSP00000465962
289 ENST00000591277 ENSP00000468644 290 ENST00000591748
ENSP00000476009 291 ENST00000592537 ENSP00000466181 292
ENST00000592693 ENSP00000474913 293 ENST00000592889 ENSP00000467750
294 MYO5C Unconventional myosin- Vc ENSG00000128833 ENST00000261839
ENSP00000261839 295 ITFG1 T-cell immunomodulatory protein
ENSG00000129636 ENST00000320640 ENSP00000319918 296 ENST00000544001
ENSP00000441062 297 ENST00000563730 ENSP00000455630 298
ENST00000565262 ENSP00000457665 299 ENST00000565940 ENSP00000459192
300 SYT11 Synaptotagmin-11 ENSG00000132718 ENST00000368324
ENSP00000357307 301 SLC41A1 Solute carrier family 41 member 1
ENSG00000133065 ENST00000367137 ENSP00000356105 302 ATP13A3
Probable cation- transporting ATPase 13A3 ENSG00000133657
ENST00000256031 ENSP00000256031 303 ENST00000429136 ENSP00000402550
304 ENST00000439040 ENSP00000416508 305 ENST00000446356
ENSP00000410767 306 ENST00000457986 ENSP00000406234 307
ENST00000619199 ENSP00000482200 308 MICAL2 Protein-methionine
sulfoxide ENSG00000133816 ENST00000379612 ENSP00000368932 309
oxidase MICAL2 CABLES1 CDK5 and ABL1 enzyme substrate 1
ENSG00000134508 ENST00000256925 ENSP00000256925 310 ENST00000579963
ENSP00000464435 311 HAVCR2 Hepatitis A virus cellular receptor 2
ENSG00000135077 ENST00000307851 ENSP00000312002 312 ENST00000522593
ENSP00000430873 313 CGA Chromogranin-A ENSG00000135346
ENST00000369582 ENSP00000358595 314 ENST00000610310 ENSP00000482232
315 ENST00000625577 ENSP00000486666 316 ENST00000627148
ENSP00000486024 317 ENST00000630630 ENSP00000487300 318 FAIM2
Protein lifeguard 2 ENSG00000135472 ENST00000320634 ENSP00000321951
319 ENST00000547871 ENSP00000449360 320 ENST00000550195
ENSP00000447715 321 ENST00000550635 ENSP00000449711 322
ENST00000550890 ENSP00000450132 323 ENST00000552669 ENSP00000446771
324 ENST00000552863 ENSP00000449957 325 ARHGEF4 Rho guanine
nucleotide exchange ENSG00000136002 ENST00000392953 ENSP00000376680
326 factor 4 SLC41A2 Solute carrier family 41 member 2
ENSG00000136052 ENST00000258538 ENSP00000258538 327 ENST00000437220
ENSP00000391377 328 NUSAP1 Nucleolar and spindle- associated
ENSG00000137804 ENST00000557840 ENSP00000453428 329 protein 1
ENST00000559046 ENSP00000452725 330 ADAM10 Disintegrin and
metalloproteinase ENSG00000137845 ENST00000260408 ENSP00000260408
331 domain-containing protein 10 ENST00000396136 ENSP00000456542
332 ENST00000402627 ENSP00000386056 333 ENST00000439637
ENSP00000391930 334 ENST00000461408 ENSP00000481779 335
ENST00000558004 ENSP00000452704 336 ENST00000559053 ENSP00000453952
337 ENST00000561288 ENSP00000452639 338 HADHB Trifunctional enzyme
subunit beta, ENSG00000138029 ENST00000545822 ENSP00000442665 339
mitochondrial CD27 CD27 antigen ENSG00000139193 ENST00000266557
ENSP00000266557 340 CDH24 Cadherin-24 ENSG00000139880
ENST00000267383 ENSP00000267383 341 ENST00000397359 ENSP00000380517
342 ENST00000487137 ENSP00000434821 343 ENST00000554034
ENSP00000452493 344 ENST00000610348 ENSP00000478078 345 ETFA
Electron transfer flavoprotein ENSG00000140374 ENST00000560044
ENSP00000452942 346 subunit alpha, mitochondrial ENST00000560309
ENSP00000453753 347 KSR1 Kinase suppressor of Ras 1 ENSG00000141068
ENST00000580163 ENSP00000463204 348 SECTM1 Secreted and
transmembrane protein 1 ENSG00000141574 ENST00000269389
ENSP00000269389 349 ENST00000580437 ENSP00000463904 350
ENST00000581691 ENSP00000463114 351 ENST00000581864 ENSP00000464111
352 ENST00000581954 ENSP00000464385 353 ENST00000582290
ENSP00000462294 354 ENST00000582563 ENSP00000463120 355
ENST00000583093 ENSP00000462563 356 EVA1B Protein eva-1 homolog B
ENSG00000142694 ENST00000270824 ENSP00000270824 357 CTTNBP2NL
CTTNBP2 N-terminal-like protein ENSG00000143079 ENST00000271277
ENSP00000271277 358 ENST00000441739 ENSP00000390976 359 CASQ1
Calsequestrin-1 ENSG00000143318 ENST00000368078 ENSP00000357057 360
ARL6IP5 PRA1 family protein 3 ENSG00000144746 ENST00000273258
ENSP00000273258 361 ENST00000478935 ENSP00000420138 362
ENST00000484921 ENSP00000419374 363 ENST00000485444 ENSP00000419021
364 ADPRH [Protein ADP- ribosylarginine] ENSG00000144843
ENST00000357003 ENSP00000349496 365 hydrolase ENST00000465513
ENSP00000417430 366 ENST00000478399 ENSP00000420200 367
ENST00000478927 ENSP00000417528 368 ENST00000481816 ENSP00000419703
369 PAM Peptidyl-glycine alpha- ENSG00000145730 ENST00000304400
ENSP00000306100 370 amidating monooxygenase ENST00000345721
ENSP00000302544 371 ENST00000346918 ENSP00000282992 372
ENST00000348126 ENSP00000314638 373 ENST00000438793 ENSP00000396493
374 ENST00000455264 ENSP00000403461 375 ENST00000504691
ENSP00000424203 376 ENST00000505654 ENSP00000421569 377
ENST00000506006 ENSP00000423611 378 ENST00000509832 ENSP00000423763
379 ENST00000511477 ENSP00000421823 380 ENST00000511839
ENSP00000426448 381 ENST00000512073 ENSP00000420851 382 RNF145 RING
finger protein 145 ENSG00000145860 ENST00000274542 ENSP00000274542
383 ENST00000424310 ENSP00000409064 384 ENST00000518802
ENSP00000430955 385 ENST00000519865 ENSP00000430397 386
ENST00000520638 ENSP00000429071 387 ENST00000521606 ENSP00000430753
388 ENST00000611185 ENSP00000482720 389 TMEM140 Transmembrane
protein 140 ENSG00000146859 ENST00000275767 ENSP00000275767 390
CHST7 Carbohydrate sulfotransferase 7 ENSG00000147119
ENST00000276055 ENSP00000276055 391 CHRNA6 Neuronal acetylcholine
receptor ENSG00000147434 ENST00000276410 ENSP00000276410 392
subunit alpha-6 ENST00000533810 ENSP00000434659 393 ENST00000534622
ENSP00000433871 394 PTPRJ Receptor-type tyrosine- protein
ENSG00000149177 ENST00000418331 ENSP00000400010 395 phosphatase eta
ENST00000440289 ENSP00000409733 396 ENST00000527952 ENSP00000435618
397 ENST00000534219 ENSP00000432686 398 ENST00000613246
ENSP00000477933 399 ENST00000615445 ENSP00000479342 400 NCAM1
Neural cell adhesion molecule 1 ENSG00000149294 ENST00000316851
ENSP00000318472 401 ENST00000401611 ENSP00000384055 402
ENST00000524916 ENSP00000478072 403 ENST00000526322 ENSP00000479687
404 ENST00000528158 ENSP00000486241 405 ENST00000528590
ENSP00000480269 406 ENST00000529356 ENSP00000482205 407
ENST00000531044 ENSP00000484943 408 ENST00000531817 ENSP00000475074
409 ENST00000533073 ENSP00000486406 410 ENST00000613217
ENSP00000479353 411 ENST00000615112 ENSP00000480797 412
ENST00000615285 ENSP00000479241 413 ENST00000618266 ENSP00000477835
414 ENST00000619839 ENSP00000480132 415 ENST00000620046
ENSP00000482852 416 ENST00000621128 ENSP00000481083 417
ENST00000621518 ENSP00000477808 418 ENST00000621850 ENSP00000480774
419 INPP1 Inositol polyphosphate 1-phosphatase ENSG00000151689
ENST00000413239 ENSP00000391415 420 ENST00000444194 ENSP00000404732
421 ENST00000451089 ENSP00000410662 422 ENST00000458193
ENSP00000412119 423 JAKMIP1 Janus kinase and
microtubule-interacting ENSG00000152969 ENST00000409021
ENSP00000386711 424 protein 1 ENST00000409371 ENSP00000387042 425
RHOC Rho-related GTP-binding protein ENSG00000155366
ENST00000468093 ENSP00000431392 426 RhoC ENST00000484280
ENSP00000434310 427 ENST00000528831 ENSP00000432209 428 SLC16A1
Monocarboxylate transporter 1 ENSG00000155380 ENST00000369626
ENSP00000358640 429 ENST00000429288 ENSP00000397106 430
ENST00000443580 ENSP00000399104 431 ENST00000458229 ENSP00000416167
432 ENST00000538576 ENSP00000441065 433 CXCL13 C-X-C motif
chemokine 13 ENSG00000156234 ENST00000286758 ENSP00000286758 434
SH3RF2 Putative E3 ubiquitin- protein ENSG00000156463
ENST00000359120 ENSP00000352028 435 ligase SH3RF2 ENST00000511217
ENSP00000424497 436 NPTN Neuroplastin ENSG00000156642
ENST00000345330 ENSP00000290401 437 ENST00000351217 ENSP00000342958
438 ENST00000562924 ENSP00000456349 439 ENST00000563691
ENSP00000457028 440 ENST00000565325 ENSP00000457470 441 AHCYL2
Adenosylhomocysteinase 3 ENSG00000158467 ENST00000466924
ENSP00000419346 442 PTGIR Prostacyclin receptor ENSG00000160013
ENST00000291294 ENSP00000291294 443 ENST00000594275 ENSP00000469408
444 ENST00000596260 ENSP00000468970 445 ENST00000597185
ENSP00000470566 446 ENST00000598865 ENSP00000470799 447 TMPRSS3
Transmembrane protease ENSG00000160183 ENST00000291532
ENSP00000291532 448 serine 4 ENST00000398397 ENSP00000381434 449
ENST00000398405 ENSP00000381442 450 ENST00000433957 ENSP00000411013
451 FCRL3 Fc receptor-like protein 3 ENSG00000160856
ENST00000368184 ENSP00000357167 452 ENST00000368186 ENSP00000357169
453 ENST00000477837 ENSP00000433430 454 ENST00000485028
ENSP00000434331 455 ENST00000492769 ENSP00000435487 456
ENST00000496769 ENSP00000473680 457 PAQR4 Progestin and adipoQ
receptor ENSG00000162073 ENST00000293978 ENSP00000293978 458 family
member 4 ENST00000318782 ENSP00000321804 459 ENST00000572687
ENSP00000459418 460 ENST00000574988 ENSP00000458683 461
ENST00000576565 ENSP00000460326 462 ZG16B Zymogen granule protein
16 homolog B ENSG00000162078 ENST00000382280 ENSP00000371715 463
ENST00000570670 ENSP00000460793 464 ENST00000571723 ENSP00000458847
465 ENST00000572863 ENSP00000461740 466 SGPP2
Sphingosine-1-phosphate phosphatase 2 ENSG00000163082
ENST00000321276 ENSP00000315137 467 NEURL3 E3 ubiquitin-protein
ligase NEURL1B ENSG00000163121 ENST00000310865 ENSP00000479456 468
ENST00000435380 ENSP00000480933 469 KIF15 Kinesin-like protein
KIF15 ENSG00000163808 ENST00000438321 ENSP00000406939 470 TMEM184C
Transmembrane protein 184C ENSG00000164168 ENST00000296582
ENSP00000296582 471 ENST00000505999 ENSP00000421159 472
ENST00000508208 ENSP00000425940 473 C5ORF63 Glutaredoxin-like
protein C5orf63 ENSG00000164241 ENST00000296662 ENSP00000453964 474
ENST00000508527 ENSP00000475157 475 ENST00000509733 ENSP00000475415
476 ENST00000535381 ENSP00000454153 477 ENST00000606042
ENSP00000475733 478 ENST00000606937 ENSP00000475810 479
ENST00000607731 ENSP00000476160 480 MELK Maternal embryonic leucine
ENSG00000165304 ENST00000495529 ENSP00000487536 481 zipper kinase
ENST00000536329 ENSP00000443550 482 ENST00000536987 ENSP00000439184
483 ENST00000543751 ENSP00000441596 484 ENST00000626154
ENSP00000486558 485 FAAH2 Fatty-acid amide hydrolase 2
ENSG00000165591 ENST00000374900 ENSP00000364035 486 TPP1
Alpha-tocopherol transfer protein ENSG00000166340 ENST00000299427
ENSP00000299427 487 ENST00000436873 ENSP00000398136 488
ENST00000528571 ENSP00000434647 489 ENST00000528657 ENSP00000435001
490 CX3CR1 CX3C chemokine receptor 1 ENSG00000168329
ENST00000358309 ENSP00000351059 491 ENST00000399220 ENSP00000382166
492 ENST00000412814 ENSP00000408835 493 ENST00000435290
ENSP00000394960 494 ENST00000541347 ENSP00000439140 495
ENST00000542107 ENSP00000444928 496 TSPAN5 Tetraspanin-5
ENSG00000168785 ENST00000305798 ENSP00000307701 497 ENST00000505184
ENSP00000423916 498 ENST00000508798 ENSP00000421808 499
ENST00000511651 ENSP00000426248 500 ENST00000511800 ENSP00000422548
501 ENST00000515287 ENSP00000423504 502 ENST00000515440
ENSP00000422351 503 UGP2 UTP-glucose-1-phosphate ENSG00000169764
ENST00000467999 ENSP00000418642 504 uridylyltransferase
ENST00000496334 ENSP00000420760 505 GLB1 Beta-galactosidase
ENSG00000170266 ENST00000307363 ENSP00000306920 506 ENST00000307377
ENSP00000305920 507 ENST00000399402 ENSP00000382333 508
ENST00000415454 ENSP00000411813 509 ENST00000436768 ENSP00000387989
510 ENST00000438227 ENSP00000401250 511 ENST00000440656
ENSP00000411769 512 ENST00000446732 ENSP00000407365 513
ENST00000450835 ENSP00000403264 514 SPATA24 Spermatogenesis-
associated ENSG00000170469 ENST00000514983 ENSP00000423424 515
protein 24 RBKS Ribokinase ENSG00000171174 ENST00000449378
ENSP00000413789 516 NETO2 Neuropilin and tolloid- like
ENSG00000171208 ENST00000303155 ENSP00000306726 517 protein 2
ENST00000562435 ENSP00000455169 518 ENST00000562559 ENSP00000454213
519 ENST00000563078 ENSP00000456818 520 ENST00000564667
ENSP00000457133 521 LRG1 Leucine-rich alpha-2-glycoprotein
ENSG00000171236 ENST00000306390 ENSP00000302621 522 FAM98B Protein
FAM98B ENSG00000171262 ENST00000491535 ENSP00000453166 523
ENST00000559431 ENSP00000453926 524 CHST11 Carbohydrate
sulfotransferase 11 ENSG00000171310 ENST00000303694 ENSP00000305725
525 ENST00000546689 ENSP00000448678 526 ENST00000547956
ENSP00000449093 527 ENST00000549260 ENSP00000450004 528 ECEL1
Endothelin-converting enzyme-like 1 ENSG00000171551 ENST00000304546
ENSP00000302051 529 ENST00000409941 ENSP00000386333 530 BCL2L1
Bcl-2-like protein 1 ENSG00000171552 ENST00000307677
ENSP00000302564 531 ENST00000376055 ENSP00000365223 532
ENST00000376062 ENSP00000365230 533 MALT1 Mucosa-associated
lymphoid ENSG00000172175 ENST00000345724 ENSP00000304161 534 tissue
ENST00000348428 ENSP00000319279 535 lymphoma translocation protein
1 ENST00000591792 ENSP00000467222 536 CYP7B1 25-hydroxycholesterol
7- ENSG00000172817 ENST00000310193 ENSP00000310721 537
alpha-hydroxylase HPSE Heparanase ENSG00000173083 ENST00000311412
ENSP00000308107 538 ENST00000405413 ENSP00000384262 539
ENST00000507150 ENSP00000426139 540 ENST00000508891 ENSP00000421827
541 ENST00000509906 ENSP00000421038 542 ENST00000512196
ENSP00000423265 543 ENST00000513463 ENSP00000421365 544 VANGL1
Vang-like protein 1 ENSG00000173218 ENST00000310260 ENSP00000310800
545 ENST00000355485 ENSP00000347672 546 ENST00000369509
ENSP00000358522 547 ENST00000369510 ENSP00000358523 548 CD7 T-cell
antigen CD7 ENSG00000173762 ENST00000312648 ENSP00000312027 549
ENST00000578509 ENSP00000464565 550 ENST00000581434 ENSP00000464546
551 ENST00000582480 ENSP00000464182 552 ENST00000583376
ENSP00000463489 553 ENST00000584284 ENSP00000463612 554 HAP1
Huntingtin-associated protein 1 ENSG00000173805 ENST00000455021
ENSP00000397242 555 FBXO45 F-box/SPRY domain-containing
ENSG00000174013 ENST00000440469 ENSP00000389868 556 protein 1 CHST2
Carbohydrate sulfotransferase 2 ENSG00000175040 ENST00000309575
ENSP00000307911 557 RMI2 RecQ-mediated genome instability
ENSG00000175643 ENST00000572173 ENSP00000461206 558 protein 2
SLC35E3 Solute carrier family 35 member E3 ENSG00000175782
ENST00000398004 ENSP00000381089 559 ENST00000431174 ENSP00000403769
560 ZBTB38 Zinc finger and BTB domain- ENSG00000177311
ENST00000503809 ENSP00000422051 561 containing protein 38 YIPF6
Protein YIPF6 ENSG00000181704 ENST00000374622 ENSP00000363751 562
ENST00000451537 ENSP00000401799 563 ENST00000462683 ENSP00000417573
564 CREB3L2 Cyclic AMP-responsive element- ENSG00000182158
ENST00000330387 ENSP00000329140 565 binding protein 3-like protein
2 ENST00000420629 ENSP00000402889 566 ENST00000456390
ENSP00000403550 567 XKRX XK-related protein 2 ENSG00000182489
ENST00000372956 ENSP00000362047 568 ENST00000468904 ENSP00000419884
569 CADM1 Cell adhesion molecule 1 ENSG00000182985 ENST00000331581
ENSP00000329797 570 ENST00000452722 ENSP00000395359 571
ENST00000536727 ENSP00000440322 572 ENST00000537058 ENSP00000439817
573 ENST00000540951 ENSP00000445375 574 ENST00000542447
ENSP00000439176 575 ENST00000542450 ENSP00000442001 576
ENST00000543540 ENSP00000439847 577 ENST00000545380 ENSP00000442387
578 ENST00000612235 ENSP00000483648 579 ENST00000612471
ENSP00000483793 580 ENST00000616271 ENSP00000484516 581
ENST00000621043 ENSP00000482840 582 ENST00000621709 ENSP00000482924
583 LHFP Lipoma HMGIC fusion partner ENSG00000183722
ENST00000379589 ENSP00000368908 584 CSF1 Macrophage colony-
stimulating ENSG00000184371 ENST00000329608 ENSP00000327513 585
factor 1 ENST00000357302 ENSP00000349854 586 ENST00000369801
ENSP00000358816 587 ENST00000369802 ENSP00000358817 588
ENST00000420111 ENSP00000407317 589 ENST00000488198 ENSP00000433837
590 ENST00000525659 ENSP00000431547 591 ENST00000527192
ENSP00000434527 592 PTP4A3 Protein tyrosine phosphatase type
ENSG00000184489 ENST00000329397 ENSP00000332274 593 IVA 3
ENST00000349124 ENSP00000331730 594 ENST00000520105 ENSP00000428758
595 ENST00000521578 ENSP00000428976 596 ENST00000523147
ENSP00000428725 597 ENST00000524028 ENSP00000430332 598 OSBP2
Oxysterol-binding protein 2 ENSG00000184792 ENST00000445781
ENSP00000411497 599 METTL7A Methyltransferase-like protein 7A
ENSG00000185432 ENST00000332160 ENSP00000331787 600 ENST00000547104
ENSP00000447542 601 ENST00000548553 ENSP00000448785 602
ENST00000550097 ENSP00000448286 603 ENST00000550502 ENSP00000450239
604 TMPRSS6 Transmembrane protease serine 6 ENSG00000187045
ENST00000346753 ENSP00000334962 605 ENST00000381792 ENSP00000371211
606 ENST00000406725 ENSP00000385453 607 ENST00000406856
ENSP00000384964 608 ENST00000423761 ENSP00000400317 609
ENST00000429068 ENSP00000392433 610 ENST00000442782 ENSP00000397691
611 GCNT1 Beta-1,3-galactosyl-O-glycosyl- ENSG00000187210
ENST00000376730 ENSP00000365920 612 glycoprotein beta-1,
ENST00000442371 ENSP00000415454 613
6-N-acetylglucosaminyltransferase ENST00000444201 ENSP00000390703
614 MAGEH1 Melanoma-associated antigen H1 ENSG00000187601
ENST00000342972 ENSP00000343706 615 NEMP2 Nuclear envelope integral
membrane ENSG00000189362 ENST00000343105 ENSP00000340087 616
protein 2 ENST00000409150 ENSP00000386292 617 ENST00000414176
ENSP00000404283 618 ENST00000421038 ENSP00000410306 619
ENST00000444545 ENSP00000403867 620 NTNG2 Netrin-G2 ENSG00000196358
ENST00000372179 ENSP00000361252 621 ENST00000393229 ENSP00000376921
622 PDGFA Platelet-derived growth factor ENSG00000197461
ENST00000354513 ENSP00000346508 623 subunit A ENST00000400761
ENSP00000383572 624 ENST00000402802 ENSP00000383889 625
ENST00000405692 ENSP00000384673 626 PDCD1LG2 Programmed cell death
1 ligand 2 ENSG00000197646 ENST00000397747 ENSP00000380855 627
TOR4A Torsin-4A ENSG00000198113 ENST00000357503 ENSP00000350102 628
HIBCH 3-hydroxyisobutyryl-CoA hydrolase, ENSG00000198130
ENST00000392333 ENSP00000376145 629 mitochondrial ENST00000414928
ENSP00000414820 630 NTRK1 High affinity nerve growth factor
ENSG00000198400 ENST00000358660 ENSP00000351486 631 receptor
ENST00000368196 ENSP00000357179 632 ENST00000392302 ENSP00000376120
633 ENST00000497019 ENSP00000436804 634 ENST00000524377
ENSP00000431418 635 FAM19A2 Protein FAM19A2 ENSG00000198673
ENST00000416284 ENSP00000393987 636 ENST00000548780 ENSP00000449310
637 ENST00000549379 ENSP00000447584 638 ENST00000549958
ENSP00000447280 639 ENST00000550003 ENSP00000449457 640
ENST00000551449 ENSP00000449632 641 ENST00000551619 ENSP00000447305
642 ENST00000552075 ENSP00000449516 643 F5 Coagulation factor V
ENSG00000198734 ENST00000367796 ENSP00000356770 644 ENST00000367797
ENSP00000356771 645 GK Glycerol kinase ENSG00000198814
ENST00000378943 ENSP00000368226 646 ENST00000427190 ENSP00000401720
647 ENST00000488296 ENSP00000419771 648 INPP5F
Phosphatidylinositide phosphatase SAC2 ENSG00000198825
ENST00000490818 ENSP00000487706 649 ENST00000631572 ENSP00000488726
650 CD177 CD177 antigen ENSG00000204936 ENST00000378012
ENSP00000367251 651 ENST00000607855 ENSP00000483817 652
ENST00000618265 ENSP00000479536 653 LEPROT Leptin Receptor
Overlapping Transcrip ENSG00000213625 ENST00000371065
ENSP00000360104 654 ENST00000613538 ENSP00000483521 655 TRIM16
Tripartite motif- containing protein 16 ENSG00000221926
ENST00000579219 ENSP00000463639 656 LTA Lymphotoxin-alpha
ENSG00000226979 ENST00000418386 ENSP00000413450 657 ENST00000454783
ENSP00000403495 658 PROB1 Proline-rich basic protein 1
ENSG00000228672 ENST00000434752 ENSP00000416033 659 SSTR3
Somatostatin receptor type 3 ENSG0000
References