WO2010066858A1 - Procédés de traitement et de pronostic du cancer - Google Patents

Procédés de traitement et de pronostic du cancer Download PDF

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WO2010066858A1
WO2010066858A1 PCT/EP2009/066873 EP2009066873W WO2010066858A1 WO 2010066858 A1 WO2010066858 A1 WO 2010066858A1 EP 2009066873 W EP2009066873 W EP 2009066873W WO 2010066858 A1 WO2010066858 A1 WO 2010066858A1
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il4i1
cells
cancer
inhibitor
group
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PCT/EP2009/066873
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Valérie MOLINIER-FRENKEL
Fanette Lasoudris
Flavia Castellano
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INSERM (Institut National de la Santé et de la Recherche Médicale)
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/223—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin of alpha-aminoacids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4402—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 2, e.g. pheniramine, bisacodyl
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to methods for the treatment and the prognosis of cancer.
  • MDSC myeloid-derived suppressive cells
  • TAM tumor-associated macrophages
  • TAM and MDSC cancer promoting mechanisms consists of the expression of amino-acid catabolizing enzymes allowing tumor escape from the immune response 8 .
  • amino-acid catabolizing enzymes are Indoleamine-2,3-dioxygenase (IDO), Arginasel and Inducible Nitric Oxid Synthase, all associated with the inhibition of T cell responses.
  • IDO Indoleamine-2,3-dioxygenase
  • Arginasel Arginasel
  • Nitric Oxid Synthase Inducible Nitric Oxid Synthase
  • An object of the invention is thus to find alternative inhibitors of amino-acid catabolizing enzymes for use in methods of treatment of cancer.
  • the invention relates to the inhibition of an amino-acid catabolizing enzyme, called IL4I1 (Interleukin 4 Induced gene 1), which has been found by the inventors to be expressed in a large set of human cancers including lymphoma. Furthermore, the inventors have also shown that IL4I1 does not play the same role in all types of cancers. Indeed, they have shown that the expression of IL4I1 is indicative of poor prognosis in all cancers excepting follicular lymphoma, for which the expression of
  • IL4I1 is indicative of good prognosis.
  • the present invention thus provides a method for treating cancer displaying IL4I1- expressing cells provided that said cancer is not follicular lymphoma, comprising the step of administering a therapeutically effective amount of an inhibitor of IL4I1 to a subject in need thereof.
  • the invention in another aspect, relates to a method for treating Hodgkin lymphoma or follicular lymphoma, wherein said Hodgkin lymphoma or follicular lymphoma displays at most 10% of IL4I1 -expressing cells, said method comprising the step of administering a therapeutically effective amount of a mTOR inhibitor to a subject in need thereof.
  • the invention in another aspect, relates to a method for determining the prognosis of a subject suffering from a cancer provided that said cancer is not follicular lymphoma, comprising the step of detecting the presence of IL4I1 in a sample obtained from said subject, wherein the presence of IL4I1 indicates that the subject has a poor prognosis.
  • the invention relates to a method for determining the prognosis of a subject suffering from follicular lymphoma, comprising the step of detecting the presence of IL4I1 in a sample obtained from said subject, wherein the presence of IL4I1 indicates that the subject has a good prognosis.
  • the present invention provides a method for treating cancer displaying IL4I1- expressing cells provided that said cancer is not follicular lymphoma, comprising the step of administering a therapeutically effective amount of an inhibitor of IL4I1 to a subject in need thereof.
  • an inhibitor of IL4I1 for use in a method for treatment of cancer displaying IL4I1 -expressing cells in the human or animal body, provided that said cancer is not follicular lymphoma.
  • the present invention provides a method for treating cancer displaying IL4I1 -expressing cells provided that said cancer is not follicular lymphoma, comprising the step of administering a therapeutically effective amount of an inhibitor of IL4I1 to a subject in need thereof, wherein said inhibitor of IL4I1 is selected from the group consisting of
  • the invention provides a method for treating cancer displaying IL4I1 -expressing cells provided that said cancer is not follicular lymphoma, comprising the step of administering a therapeutically effective amount of an inhibitor of the enzymatic activity of IL4I1 of formula (I) to a subject in need thereof.
  • the inhibitors of the enzymatic activity of IL4I1 of formula (I) can bind IL4I1, as its natural substrate, phenylalanine, and inhibit its enzymatic activity.
  • the inhibitors of the enzymatic activity of IL4I1 of formula (I) have a K 1 (dissociation constant for binding of inhibitor to enzyme) lower or equal to 3mM, more preferably lower or equal to 30 ⁇ M, and more preferably lower or equal to lO ⁇ M.
  • the enzyme-inhibitor constant K 1 can be measured directly by various methods well known by the man skilled in the art; one accurate method is measurement of enzymatic activity, in the presence of the inhibitor, whereby the inhibitor is titrated into a solution of enzyme and the H ⁇ C ⁇ released by the enzymatic degradation of phenylalanine is measured by oxidation of colorimetric or fluorimetric substrate of peroxidase.
  • the invention thus relates to an inhibitor of the enzymatic activity of IL4I1 of formula (I)
  • - X is an aromatic moiety selected from the group consisting of phenyl and pyridyl; said aromatic moiety being optionally substituted with NH 2 or
  • - Y is a linear or branched C 1 -C 6 alkyl
  • - R 1 is selected from the group consisting of H, methyl, vinyl, propargyl, F and CN
  • - R 2 is selected from the group consisting of NH2, NH-Z, NH-Boc, NH- methyl, NH-Cl, NH-ter-butyl, NH-OH and NH-COCH 3 ,
  • R 3 is selected from the group consisting of COOH; COOR 4 , wherein R 4 is a linear or branched C 1 -C 6 alkyl; CONH 2 ; CO 2 H-P(OH) and CO 2 H-SO 3 H.
  • the invention also relates to an inhibitor of the enzymatic activity of IL4I1 of formula (I) wherein
  • - X is an aromatic moiety selected from the group consisting of phenyl and pyridyl; said aromatic moiety being optionally substituted with NH 2 or COOH,
  • - Y is a linear or branched C 1 -C 2 alkyl
  • R 1 is selected from the group consisting of H, methyl, vinyl, propargyl, F and CN,
  • - R 2 is selected from the group consisting of NH2, NH-Z, NH-Boc, NH- methyl, NH-Cl, NH-ter-butyl, NH-OH and NH-COCH 3 ,
  • R 3 is selected from the group consisting of COOH; COOR 4 , wherein R 4 is a linear or branched C 1 -C 6 alkyl; CONH 2 ; CO 2 H-P(OH) and CO 2 H-SO 3 H.
  • the invention further relates to an inhibitor of the enzymatic activity of IL4I1 of formula (I) wherein
  • - X is an aromatic moiety selected from the group consisting of phenyl and pyridyl; said aromatic moiety being optionally substituted with NH 2 or COOH, - Y is a linear or branched C 1 -C 2 alkyl,
  • R 1 is selected from the group consisting of H, methyl, vinyl, propargyl, F and CN,
  • R 2 is selected from the group consisting of NH2, NH-Z, NH-Boc, NH- methyl, NH-Cl, NH-ter-butyl, NH-OH and NH-COCH 3
  • - R 3 is selected from the group consisting of COOH; COOR 4 , wherein R 4 is a linear or branched C 1 -C 3 alkyl; CONH 2 ; CO 2 H-P(OH) and CO 2 H-SO 3 H.
  • the invention also relates to an inhibitor of the enzymatic activity of IL4I1 of formula (I) wherein
  • - X is an aromatic moiety selected from the group consisting of phenyl and pyridyl; said aromatic moiety being optionally substituted with NH 2 or COOH,
  • - Y is CH 2
  • R 1 is selected from the group consisting of H, methyl, vinyl, propargyl, F and CN,
  • - R 2 is selected from the group consisting of NH2, NH-Z, NH-Boc, NH- methyl, NH-Cl, NH-ter-butyl, NH-OH and NH-COCH 3 ,
  • R 3 is selected from the group consisting of COOH, COOCH 2 CH 3, CONH 2 , CO 2 H-P(OH) and CO 2 H-SO 3 H.
  • the invention also relates to an inhibitor of the enzymatic activity of IL4I1 of formula (I) wherein - X is a phenyl optionally substituted with NH 2 or COOH,
  • - Y iS CH 2 , - R 1 is selected from the group consisting of H, methyl, vinyl, propargyl, F and CN,
  • - R 2 is selected from the group consisting of NH2, NH-Z, NH-Boc, NH- methyl, NH-Cl, NH-ter-butyl, NH-OH and NH-COCH 3
  • - R 3 is selected from the group consisting of COOH, COOCH 2 CH 3, CONH 2 ,
  • the invention further relates to an inhibitor of the enzymatic activity of IL4I1 of formula (I) wherein
  • - X is a pyridyl optionally substituted with NH 2 or COOH
  • - R 1 is selected from the group consisting of H, methyl, vinyl, propargyl, F and CN
  • - R 2 is selected from the group consisting of NH2, NH-Z, NH-Boc, NH- methyl, NH-Cl, NH-ter-butyl, NH-OH and NH-COCH 3 ,
  • R 3 is selected from the group consisting of COOH, COOCH 2 CH 3, CONH 2 , CO 2 H-P(OH) and CO 2 H-SO 3 H.
  • the compound of formula (I) is 3-(2-pyridyl)-alanine (or 2 '-aza-phenylalanine).
  • NH-Z and NH-Boc refer to the following protective groups:
  • the invention provides a method for treating cancer displaying IL4I1 -expressing cells provided that said cancer is not follicular lymphoma, comprising the step of administering a therapeutically effective amount of an antibody against IL4I1 or a fragment thereof which binds IL4I1 and wherein said antibody against IL4I1 or fragment thereof inhibits IL4I1 enzymatic activity.
  • an antibody against IL4I1 or a fragment thereof which binds IL4I1 and wherein said antibody against IL4I1 or fragment thereof inhibits IL4I1 enzymatic activity for use in a method for treatment of cancer displaying IL4I1- expressing cells in the human or animal body, provided that said cancer is not follicular lymphoma.
  • the person skilled in the art will be aware of standard methods for production of such specific antibody or fragment thereof.
  • specific antibodies or fragments thereof may be generated by immunizing an animal with IL4I1 and by selecting the antibodies, which inhibit the enzymatic activity of IL4I1.
  • the present invention therefore comprehends use of antibody specific for IL4I1 which includes F(ab') 2 , F(ab) 2 , Fab, Fv and Fd antibody fragments, chimeric antibodies in which one or more regions have been replaced by homologous human or non-human portions.
  • antibody specific for IL4I1 which includes F(ab') 2 , F(ab) 2 , Fab, Fv and Fd antibody fragments, chimeric antibodies in which one or more regions have been replaced by homologous human or non-human portions.
  • fragments such as for example ScFv fragments and divalent or multivalent ScFv-type molecules can be prepared using recombinant methods.
  • the inhibition of IL4I1 activity may be measured by any method known by the skilled person.
  • the inhibition of the enzymatic activity may be measured, as described in the experimental section, by quantification of H 2 O 2 produced by IL4I1 phenylalanine oxidative deamination in the presence of an inhibitor as described previously and compared to a control (i.e. without inhibitor).
  • the invention provides a method for treating cancer displaying IL4I1 -expressing cells provided that said cancer is not follicular lymphoma, comprising the step of administering a therapeutically effective amount of an agent down-regulating the expression of IL4I1 in said IL4I1 -expressing cells. Also provided is an agent down-regulating the expression of IL4I1 in IL4I1- expressing cells for use in a method for treatment of cancer displaying IL4I1- expressing cells in the human or animal body, provided that said cancer is not follicular lymphoma.
  • an agent down-regulating the expression of IL4I1 can be a nucleic acid, which interferes with the expression of IL4I1.
  • agents are antisense molecules or vectors comprising said antisense molecules.
  • Antisense molecules are complementary strands of small segments of mRNA. Methods for designing effective antisense molecules being well known (see for example US6165990), it falls within the ability of the skilled artisan to design antisense molecules able to downregulate the expression of IL4I1 in IL4I1 -expressing cells.
  • RNA interference (RNAi) molecules such as, for example, short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs).
  • RNAi refers to the introduction of homologous double stranded RNA to specifically target a gene's product, in the present case IL4I1, resulting in a null or hypomorphic phenotype.
  • Methods for designing effective RNAi molecules being well known (see for review ⁇ ), it falls within the ability of the skilled artisan to design RNAi molecules able to downregulate the expression of IL4I1 in IL4I1- expressing cells.
  • a particular agent down-regulating the expression of IL4I1 in IL4I1 -expressing cells is a siRNA comprising the nucleotide sequence as shown in SEQ ID NO: 1.
  • siRNA having a nucleotide sequence as shown in SEQ ID NO: 2 and a complementary nucleotide sequence as shown in SEQ ID NO: 3.
  • SEQ ID NO:2 [5'r(GGGUGGAGACGGCGGUCAA)d(TT)3']
  • SEQ ID NO:3 [5'r(UUGACCGCCGUCUCCACCC)d(AG)3']
  • This particular siRNA comprises a double stranded region of 19 nucleotides and a single stranded region of 2 nucleotides at the 3' end of each one of the strands of siRNA.
  • "r” means ribonucleotide
  • d means deoxyribonucleotide.
  • the down-regulation of the IL4I1 expression may be measured by various methods known by the skilled person, such as for example immunohistochemistry, binding assay, mRNA level for IL4I1 and radio-immunoassay or Enzyme link immunosorbent assay for IL4I1.
  • the down-regulation of the IL4I1 expression may further be detected indirectly by the measurement of its enzymatic activity since it was found by the inventors that enzymatic activity of IL4I1 paralleled IL4I1 expression.
  • the enzymatic activity of IL4I1 may be measured by quantification Of H 2 O 2 produced by IL4I1 phenylalanine oxidative deamination, as described in the experimental section.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising:
  • the invention relates to the pharmaceutical composition as defined previously for use in a method for treating cancer displaying IL4I1- expressing cells in the human or animal body, provided that said cancer is not follicular lymphoma.
  • suitable carriers Suitable formulations for administration by any desired route may be prepared by standard methods, for example by reference to well-known text such as Remington; The Science and Practice of Pharmacy.
  • the cancer to be treated according to the invention is selected from the group consisting of non-lymphoid cancers displaying IL4I1 -expressing cells and lymphomas displaying IL4I1 -expressing cells, provided that said lymphoma is not follicular lymphoma.
  • non lymphoid cancer means cancers, which are not derived from lymphoid cells.
  • lymphoid cells means lymphocytes and plasma cells.
  • non-lymphoid cancers are solid tumors.
  • non-lymphoid cancers are typically selected from carcinomas, sarcomas, mesotheliomas, blastomas and germ cell tumors.
  • non-lymphoid cancers are typically selected from the group consisting of mesotheliomas, non small-cell lung carcinomas, colon carcinoma, breast carcinoma, thyroid carcinoma, testicular germ cell tumors and ovarian carcinoma, displaying IL4I1 -expressing cells.
  • the cancer to be treated is selected from the group consisting of non-lymphoid cancers displaying IL4I1 -expressing cells, provided that the cancer is not breast carcinoma.
  • the cancer to be treated is selected from the group consisting of lymphomas displaying IL4I1 -expressing cells provided that said lymphoma is not follicular lymphoma.
  • Lymphomas are typically selected from B- cell lymphomas displaying IL4I1 -expressing cells provided that said lymphoma is not follicular lymphoma.
  • the cancer to be treated is selected from the group consisting of PMBL (Primary Mediastinal large B-cell Lymphoma), classical Hodgkin lymphomas (cHL), NLPHL (Nodular lymphocyte predominant Hodgkin's lymphoma), non-mediastinal Diffuse Large B-CeIl Lymphoma (DLBCL) and SLL/CLL (Small Lymphocytic Lymphoma / Chronic Lymphocytic Leukemia), displaying IL4I1 -expressing cells.
  • PMBL Primary Mediastinal large B-cell Lymphoma
  • cHL classical Hodgkin lymphomas
  • NLPHL Nodular lymphocyte predominant Hodgkin's lymphoma
  • non-mediastinal Diffuse Large B-CeIl Lymphoma DLBCL
  • SLL/CLL Mall Lymphocytic Lymphoma / Chronic Lymphocytic Leukemia
  • the cancer to be treated is selected from the group consisting of lymphomas displaying IL4I1 -expressing cells, provided that said cancer is not PMBL (Primary Mediastinal large B-cell Lymphoma) or follicular lymphoma.
  • PMBL Primary Mediastinal large B-cell Lymphoma
  • the cancer to be treated is selected from the group consisting of B-cell lymphomas displaying IL4I1 -expressing cells, provided that said cancer is not PMBL (Primary Mediastinal large B-cell Lymphoma) or follicular lymphoma.
  • PMBL Primary Mediastinal large B-cell Lymphoma
  • the invention also concerns mTOR inhibitors for use in methods for treating particular types of Hodgkin and follicular lymphomas. Indeed, it has been shown that the nature of the T lymphocyte populations present within the inflammatory infiltrate of Hodgkin lymphoma and follicular lymphoma is associated with the prognosis of these tumors 21 ' 22 . In contrast to what has been observed in non- lymphoid tumors, the magnitude of the regulatory T cell infiltrate is associated to a better prognosis, whereas the presence of cytotoxic T cells constitutes a pejorative factor.
  • the inventors have shown that both Hodgkin lymphoma and follicular lymphoma can express high levels of IL4I1 in the intratumoral macrophages or in the tumor cells.
  • the inventors have also shown that at least some of the toxicity mediated by IL4I1 on T cells is achieved via the antiproliferative effect of H 2 O 2 liberated by the enzymatic activity. Since regulatory T cells are more resistant than effector T cells to H 2 O 2 toxic effect 23 , the results of the inventors indicate that IL4I1 may participate to the selection of regulatory T cells in the tumor microenvironment.
  • T lymphocyte amino acid deficiency triggers a signaling pathway that inactivates the mTOR complex leading to inhibition of T lymphocyte proliferation (response to amino acid depletion by inactivation of the mTOR signaling pathway is for instance described by Cobbold SP et al. 24 ).
  • IL4I1 expression in Hodgkin lymphoma and follicular lymphoma participate to the enrichment in regulatory T cell populations at the expense of CD4 + and CD8 + effector T cells.
  • intratumor IL4I1 expression is low or negative in these pathologies (i.e. when said Hodgkin lymphoma or follicular lymphoma displays at most 10% of IL4I1 -expressing cells)
  • treatments using mTOR inhibitors such as rapamycin and its analogs could be considered to mimic the effect of amino acid depletion.
  • the invention thus concerns a method for treating Hodgkin lymphoma or follicular lymphoma, wherein said Hodgkin lymphoma or follicular lymphoma displays at most 10% of IL4I1 -expressing cells, said method comprising the step of administering a therapeutically effective amount of a mTOR inhibitor to a subject in need thereof.
  • a mTOR inhibitor for use in a method for treatment of Hodgkin lymphoma or follicular lymphoma in the human or animal body, wherein said Hodgkin lymphoma or follicular lymphoma displays at most 10% of IL4I1- expressing cells.
  • the percentage of IL4I1 -expressing cells in Hodgkin lymphoma or follicular lymphoma is typically measured by immunohistochemistry (immunostaining) performed on a sample obtained by biopsy.
  • samples obtained from the subjects are any type of tumor biopsy, including lymph nodes.
  • the percentage of IL4I1 -expressing cells may also be measured by immunological techniques such as ELISA on tumor cell suspensions or by the measurement of its enzymatic activity (by quantification of H 2 O 2 produced by IL4I1 phenylalanine oxidative deamination).
  • a mTOR inhibitor is a compound which targets intracellular mTOR ("mammalian Target Of Rapamycin").
  • mTOR is a family member of phosphatidylinositol 3- kinase(P 13 -kinase) related kinase.
  • the compound rapamycin and other mTOR inhibitors inhibit the mTOR pathway via a complex with its intracellular receptor FKBP 12 (FK506-binding protein 12).
  • FKBP 12 FK506-binding protein 12
  • mTOR modulates translation of specific mRNAs via the regulation of the phosphorylation state of several different translation proteins, mainly 4E-PB1 , P70S6K (p70S6 kinase 1 ) and eEF2.
  • a mTOR inhibitor of (according to) the present invention e.g. includes rapamycin, which is a known macrolide antibiotic produced by Streptomyces hygroscopicus, and rapamycin derivatives, e. g. rapamycin substituted in position 40 and/or 16 and/or 32, for example compounds of formula I disclosed in WO 2007/131689 (incorporated by reference), such as for example 40-O-(2-hydroxyethyl)- rapamycin (also known as everolimus), 32-deoxorapamycin, 16-0-substituted rapamycins such as 16-pent-2-ynyloxy-32-deoxorapamycin, 16-pent-2-ynyloxy-32 (S or R) -dihydro- rapamycin, 16-pent-2- ynyloxy-32 (S or R)-dihydro-40-0- (2-hydroxyethyl)- rapamycin, 40-[3- hydroxy-2-
  • mTOR inhibitors also include the so-called rapalogs, e. g. as disclosed in WO9802441 (incorporated by reference), WOOl 14387 (incorporated by reference) and WO0364383 (incorporated by reference), such as AP23573, e.g. 40-O-(dimethylphosphinoyl)- rapamycin, compounds as disclosed disclosed in WO2005047295 in Example 1 (incorporated by reference), also designated as biolimus A9 and compounds disclosed under the name TAFA-93.
  • Other mTOR inhibitors are e.g. disclosed in WO2004101583, WO9205179, WO9402136, WO9402385, WO9613273 (all incorporated by reference).
  • a diagnostic test may be performed in order to determine whether the cancer displays IL4I1 -expressing cells.
  • a pre-treatment diagnostic test it is possible to determine whether a subject would be responsive to a method of treatment according to the invention.
  • the invention also concerns a method for determining the responsiveness of a subject suffering from cancer to an inhibitor of IL4I1 as defined previously, comprising the step of detecting the expression of IL4I1 in a cancer sample obtained from said subject, provided that said cancer is not follicular lymphoma.
  • the cancer sample is a tumor biopsy. It falls within the ability of the skilled artisan to carry out such a diagnostic test, since detection of the expression of IL4I1 in the above mentioned cancers can be easily carried out by any usual method known by the skilled person.
  • IL4I1 expression may be measured through the detection of IL4I1- mRNA in the tumor cell lysate, for example by RT-PCR.
  • IL4I1 expression may also be measured by immunohistochemistry performed on a sample obtained by biopsy, as described in the experimental section. Examples of samples obtained from the subjects are any type of tumor biopsy, including lymph nodes, and optionally on whole blood sample.
  • the expression of IL4I1 may also be detected by immunological techniques such as ELISA and Western Blot on whole blood sample, plasma sample or serum sample.
  • the expression of IL4I1 may further be detected by the measurement of its enzymatic activity since it was found by the inventors that enzymatic activity of IL4I1 paralleled IL4I1 expression.
  • the enzymatic activity of IL4I1 may be measured by quantification of H 2 O 2 produced by IL4I1 phenylalanine oxidative deamination, as described in the experimental section. This test can be carried out on a sample obtained from the patient, such as for example any type of tumor biopsy, whole blood sample, plasma sample or serum sample or other biological fluids such as bronchoalveolar liquid.
  • the in vivo enzymatic activity of IL4I1 may also indirectly be measured by quantitation of phenylalanine and phenylpyruvate in biological fluids.
  • treating means reversing, alleviating, inhibiting the progress of, or preventing the disorder or condition to which such term applies, or reversing, alleviating, inhibiting the progress of, or preventing one or more symptoms of cancer.
  • subject refers to a human or animal that may benefit from the administration of an inhibitor of IL4I1, a composition or a method as recited herein. Most often, the subject will be a human but can be any mammals.
  • a “therapeutically effective amount" of a compound according to the invention an antibody against IL4I1, a fragment thereof which binds IL4I1 and wherein said antibody against IL4I1 or fragment thereof inhibits IL4I1 enzymatic activity, of an agent down-regulating the expression of IL4I1, or of a mTOR inhibitor, is meant a sufficient amount to treat cancer, at a reasonable benefit/risk ratio applicable to any medical treatment.
  • the specific therapeutically effective dose level for any particular subject in need thereof will depend upon a variety of factors including the stage of cancer being treated and the activity of the specific inhibitor/cytotoxic agent employed, the age, body weight, general health, sex and diet of the subject, the time of administration, route of administration, the duration of the treatment; drugs used in combination or coincidental with the and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved.
  • the methods for treatment of cancer according to the invention are applied to the human or animal body simultaneously, separately or sequentially with another method for treatment of cancer, said another method for treatment of cancer being preferably selected from the group comprising surgery, external radiotherapy, chemotherapy, hormone therapy and cytokine therapy.
  • the method of treatment according to the invention is combined with a chemotherapy, wherein said chemotherapy comprises the administration of at least one anti-cancer agent.
  • anti-cancer agent refers to compounds, which are used in the treatment of cancer.
  • Anti-cancer agents include but are not limited to fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5-fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinote
  • the anti-cancer agent is selected for the group consisting oftaxol; taxotere; platinum complexes such as cisplatin, carboplatin and oxaliplatin; doxorubicin; taxanes such as docetaxel and paclitaxel; vinca alkaloids such as vinblastine, vincristine and vinorelbine; genistein; erbstatin; and lavendustin.
  • the invention in another aspect, relates to a method for determining the prognosis of a subject suffering from cancer, provided that said cancer is not follicular lymphoma, comprising the step of detecting the presence of IL4I1 in a sample obtained from said subject, wherein the presence of IL4I1 indicates that the subject has a poor prognosis.
  • IL4I1 should preferentially affect the number and/or the functionality of anti-tumor T lymphocytes, facilitating tumor escape from immune surveillance. Consequently, a patient having a cancer expressing IL4I1 will have a poor prognosis. In line with this, the inventors have shown a correlation between IL4I1 expression and tumor escape in a murine model of cancer.
  • the invention relates to a method for determining the prognosis of a subject suffering from a follicular lymphoma, comprising the step of detecting the presence of IL4I1 in a sample obtained from said subject, wherein the presence of IL4I1 indicates that the subject has a good prognosis.
  • the malignant B cell depends upon signalling by its environment for survival. For example, maintenance of interactions between follicular lymphoma B cells and follicular T helper cells has been proposed to support proliferation of the malignant clone .
  • IL4I1 -mediated T cell inhibition in this setting may suppress the positive stimuli exerted by follicular T helper cells.
  • the inventors have shown a correlation between higher IL4I1 expression and clinical parameters indicative of good prognosis.
  • samples obtained from the subjects are any type of tumor biopsies, including lymph nodes, whole blood sample, plasma sample or serum sample.
  • the detection of the expression of IL4I1 may be carried out by detecting the presence of IL4I1 mRNAs in the tumor cells, notably by RT-PCR, or any other method known by the skilled person.
  • the detection of the presence of IL4I1 in the sample may be also notably carried out by immunochemistry techniques, using anti-IL4Il antibodies, optionally tagged, as disclosed previously.
  • the expression of IL4I1 may also be indirectly detected by the measurement of its enzymatic activity since it was found by the inventors that enzymatic activity of IL4I1 paralleled IL4I1 expression. Measurement of the enzymatic activity of IL4I1 is fully described in the experimental section. The in vivo enzymatic activity of IL4I1 may be also indirectly measured by quantitation of phenylalanine and phenylpyruvate in biological fluids.
  • detecting includes qualitative and/or quantitative detection (measuring levels) with or without reference to a control.
  • prognosis is used herein to refer to the prediction of the likelihood of cancer-attributable death or progression, including recurrence, metastatic spread, and drug resistance.
  • poor prognosis indicates an increased likelihood of cancer- attributable death or progression, including recurrence, metastatic spread, and drug resistance.
  • prognosis indicates a decreased likelihood of cancer- attributable death or progression, including recurrence, metastatic spread, and drug resistance.
  • the prognosis results obtained according to the method of the invention can also be correlated to, or serve as a basis for, a "risk classification” of the patients.
  • risk classification means the level of risk or the prediction that a subject will experience a particular clinical outcome.
  • a subject may be classified into a risk group or classified at a level of risk based on the predictive methods of the present invention.
  • a "risk group” is a group of subjects or individuals with a similar level of risk for a particular clinical outcome. Examples of risk classifications are the International Prognostic Index (IPI) and the Follicular Lymphoma International Prognostic Index (FLIPI, 13 ).
  • IPI International Prognostic Index
  • FLIPI Follicular Lymphoma International Prognostic Index
  • Figure 1 IL4I1 protein expression in non-lymphoid malignancies.
  • IL4I1 Ovarian carcinoma with granulomatous reaction.
  • Double staining using IL4I1 (brown) and CD 14 (red) antibodies shows IL4I1 expression in tumor cells (arrow) and in CD 14 positive reactive cells (arrow-head and small inset) (HPF x200, inset HFPxIOOO).
  • IL4I1 is strongly expressed in epithelioid cells of the granulomatous reaction (HPF X 200).
  • Thyroid carcinoma IL4I1 expression in tumor cells is restricted to an inflammatory area with calcifications (HPF X 400).
  • Lung adenocarcinoma Lung adenocarcinoma. IL4I1 is expressed in reactive cells: intra-alveolar macrophages (arrow) and histiocytic cells within the reactive infiltrate (arrow head) (HPF X 400).
  • FIG. 1 IL4I1 protein expression in reactive lymphoid tissue and lymphoid malignancies
  • IL4I1 is expressed in tingible bodies macrophages located in the germinal centers of lymphoid follicles (HPFxIOO, inset, HPFxIOOO).
  • PMBL All tumor cells display strong IL4I1 intracytoplasmic positivity (HPFx400).
  • DLBCL One of the 17% cases demonstrating occasional IL4I1 positive cells is shown. (HPFx400).
  • d&e Follicular lymphomas. In case d, IL4I1 positive tumor cells are located predominantly in the intrafollicular areas (HPFxIOO, inset HPFxIOOO).
  • Figure 3 IL4I1 enzymatic activity in human tumors and tumor cell lines
  • Figure 4 Relation between IL4I1 expression, regulatory T cell infiltrate and bone marrow involvement in follicular lymphoma patients
  • the 23 patients described in table 3 were classified according to the level of IL4I1 expression in tumor cells and TAM ( ⁇ 10 % or >10% positive cells among the corresponding cell population).
  • the association between IL4I1 expression in tumor cells (A and C) or TAM (Band D) and clinical parameters was analyzed using Fisher's exact test (p values are indicated).
  • a and B distribution of patients according to bone marrow involvement at diagnosis.
  • C and D distribution of patients according to detection of a relapse during thefollow-up (see table 3 for median follow-up).
  • E and F distribution of patients according to percentage of FoxP3 + T cells (Tregs) in the tumour biopsy at diagnosis.
  • FIG. 5 IL4I1 expression by monocytes inhibits T cell proliferation and inflammatory cytokine and chemokine secretion.
  • C PBMC were co- cultured as in A and day 1 and day 3 culture media analyzed respectively for IL2 and IFN ⁇ secretion by ELISA.
  • PBMC peripheral blood mononuclear cells
  • a and culture media harvested at day 3 analyzed on a Raybiotech cytokine array. Dot blots were quantified using the Labwork software (UVP, United Kingdom).
  • B, C and D white bars and gray bars represent results obtained with THPl and THP1-IL4I1 cells, respectively.
  • FIG. 6 Dichotomy of IL4I1 activity induction by IFN ⁇ and IL4.
  • A 10 5 monocytes, monocyte-derived M ⁇ , monocyte-derived DC and B cells were stimulated with IFN ⁇ or IL4 and IL4I1 enzymatic activity against phenylalanine measured after 48h. The mean of 3 experiments ⁇ SD is shown.
  • B Kinetics of IL4I1 enzymatic activity of IFN ⁇ -treated M ⁇ and DC (left and center) and Untreated B lymphocytes (right). Representative experiments are shown. Enzymatic activity is expressed as pmoles H 2 O 2 produced by 105 cells per hour using phenylalanine as substrate.
  • FIG. 7 Hodgkin cells instruct monocytes to produce IL4I1 through IFN ⁇ and IL13 secretion.
  • A Monocytes from the monocytic cell line THPl were grown in 48-h conditioned media either from two different Hodgkin lymphoma cell lines (KMH2 and L428) or from a large B cell lymphoma cell line (SU-DHL-4) or in unconditioned medium. IL4I1 activity was measured in the THPl cells 48 h later. Results are expressed as pmoles H 2 O 2 produced per hour by 10 5 cells.
  • THPl cells were grown as in A with addition of neutralizing antibodies against ILlO, IFN ⁇ and IL 13 or control IgG. Results are expressed as percent inhibition of IL4I1 activity compared to activity measured in cells cultivated with IgG control.
  • Figure 8 Measurement of the in vivo growth of tumors displaying IL4I1- expressing-cells in mice
  • mice were vaccinated with a melanoma tumor cell epitope. Seven (left graph) or 28 days (right graph) after vaccination, mice were challenged with tumor cells expressing murine IL4I1 (square) or transfected with control vector (circle), and then the percentage of tumor-free mice was determined.
  • B Mice were vaccinated as in (5). Seven days after vaccination, mice were challenged with tumor cells expressing murine IL4I1 (tumor 1, square, and tumor 2, triangle) or transfected with control vector (control tumor, circle), and then the percentage of tumor-free mice was determined.
  • C Mice were challenged with tumor cells expressing murine IL4I1 (tumor 1, square, and tumor 2, triangle) or transfected with control vector (control tumor, circle), and then the percentage of tumor- free mice was determined.
  • Figure 9 IL4I1 activity in the sera of mice challenged with IL4I1 expressing tumors
  • Mice vaccinated against the gp33 tumor epitope as in figure 8 were challenged 7 days after immunization with different proportions of an IL4I1 expressing tumor (tumor 2) admixed with the control tumor.
  • IL4I1 activity was measured on a pool of three sera, 30 days after tumor challenge, i. e. before tumor appearance.
  • FIG. 10 Antitumor immune response modulation by tumor IL4I1 expression.
  • Two days after adoptive transfer of splenocytes from transgenic mice expressing a gp33 specific T cell receptor (2x10 6 cells/mouse) mice were immunized against the gp33 tumor epitope as in figure 5.
  • Seven days after immunization, mice were challenged with a high number of cells from IL4I1- expressing tumor 1 and tumor 2 or control tumor (10 7 cells/mouse, n 3 per group, representative experiment out of 3). All mice developed tumors synchronously at day 10 post-challenge.
  • gp33 -41 -specific CD8 + T cells were measured in the blood, using H2 d -gp33 tetramers. Results are presented as % of H2 d -gp33 positive cells amongst the CD8 positive T cells.
  • B Mice were sacrificed at day 13 and gp33-specific splenocytes were characterized for CD44 (memory marker) and surface CD 107a (marker of cytotoxic activity) expression and analyzed for IFN ⁇ production by ELISPOT.
  • the enzymatic activity of IL4I1 was measured in the presence of 0 mM (circle, control), 0.7 mM (triangle), 1.5 mM (square) or 5mM (lozenge) of N-acetyl- phenylalanine.
  • the enzymatic activity of IL4I1 was measured in the presence of 0 mM (circle, control), 0.6 mM (triangle), 1.2 mM (square) or 5mM (lozenge) of 2'-aza- phenylalanine.
  • Figure 14 Inhibition of IL4I1 activity in Hodgkin lymphoma cells by a specific siRNA.
  • the Hodgkin lymphoma cell line L428 (2x10 6 cells) was transfected with 6 ⁇ g of IL4I1 siRNA or control siRNA by nucleofection (Nucleofector technology®, Lonza). IL4I1 activity was measured 3, 5 and 7 days later.
  • the 194 cases of B- and T/NK-cell neoplasms comprised 28 PMBL (Primary Mediastinal large B-cell Lymphoma), 36 diffuse large B cell lymphoma (DLBCL) of germinal center and non-germinal center cell type, 10 small lymphocytic lymphoma/chronic lymphoid leukemia (SLL/CLL), 12 marginal zone lymphoma (MZL), 36 follicular lymphoma, 24 classical Hodgkin lymphoma (cHL) and 11 nodular lymphocyte predominant Hodgkin lymphoma (NLPHL).
  • PMBL Primary Mediastinal large B-cell Lymphoma
  • DLBCL diffuse large B cell lymphoma
  • SLL/CLL small lymphocytic lymphoma/chronic lymphoid leukemia
  • MZL marginal zone lymphoma
  • follicular lymphoma 24 classical Hodgkin lymphoma (cHL) and 11 nodular lymphocyte predominant
  • Antibodies, cytokines, peptide and siRNA Human recombinant IL4 (50ng/ml) was purchased from R&D systems (Lille, France) IFN ⁇ (50ng/ml), GM-CSF (50 mg/ml), M-CSF (lOOng/ml) were purchased from Preprotech France (Levallois Perret, France). Monoclonal functional grade anti-CD3 (OKT3) was purchased from eBioscience (San Diego, USA). Neutralising antibodies against IL 13 (clone B-B 13) was purchased from Abeam and those anti IFN ⁇ (clone MMHG-I) was purchased from Calbiochem.
  • the 33-41 amino acid peptide from the glycoprotein of lymphochoriomenengitis virus was purchased from Polypeptide group.
  • IL4I1 siRNA [5 'r(GGGUGGAGACGGCGGUCAA)d(TT)3 ' ] , [5 '(UUGACCGCCGUCUCC ACCC)d(AG)3 ' ] was purchased from Qiagen.
  • the Lymphoma cell lines L428, KM-H2 and SU-DHL-4 were purchased from DSMZ (Braunschweig, Germany). L428 were cultivated in ISCOVE supplemented with 20% fetal calf serum, L-glutamine, non-essential amino acids and antibiotics. KM-H2 and SU-DHL-4 were cultivated in RPMI 1640 supplemented with 20% fetal calf serum (FCS), L-glutamine and antibiotics. The monocytic THPl cell line was cultivated in DMEM and RPMI 1640 (Invitrogen, Cergy Pontoise France) containing 10% FCS.
  • PBMC peripheral blood mononuclear cells
  • EFS French Blood Bank
  • EFS French Blood Bank
  • EFS French Blood Bank
  • Human monocytes were enriched by adherence from PBMC.
  • DC were differentiated in AIM-V serum free medium (Invitrogen) supplemented with glutamine and 50ng/ml IL4 and 50 ng/ml GM-CSF. Cells obtained after 5-6 days in culture were designated as immature DC (iDC).
  • Mature DC were obtained by culturing I xIO 6 iDC with different maturation stimuli in AIM-V serum-free medium in 6-well plates for up to 48-72 h. Adherent mDC were removed by gentle scraping. Monocyte-derived macrophages were seeded in sealed gas-permeable bags (Baxter Healthcare corporation, Deerf ⁇ eld, USA) under non-adherent conditions at a density of IxIO 6 cells/ml containing 100ng/ml of M-CSF. After 6 days of culture, macrophages were recovered, counted and seeded in 6 well plates at IxIO 6 cells/ml and activated with different stimuli.
  • B lymphocytes were obtained from human tonsils obtained from tonsillectomies performed at the Intercommunal Hospital of Creteil with ethical approval. Tissue was cut into small pieces in RPMI 1640 containing 10% FCS and forced through a lOO ⁇ m mesh nylon screen to disrupt the pieces into singles cells. After two washes in RPMI/FCS, the cells were counted. The viability of all cells or cell lines was established by Trypan blue exclusion.
  • viable cryopreserved cells from 5 follicular lymphomas, 1 MZL, 2 traumatic normal spleens and three frozen biopsies of melanoma tumors with non invaded adjacent skin were thawed and used to generate whole cell lysate or submitted to magnetic cell sorting using anti-human CD 19 MACS beads according to the manufacturer's instructions (Miltenyi Biotec, Paris, France).
  • IL4I1 protein expression was analyzed in tumor and stromal cells.
  • a semi-quantitative evaluation was performed to assess the density of IL4I1 -positive tumor cells ( ⁇ 10%, 10-50%, >50% of total tumor cells) and the percentage of IL4I1 -positive stromal cells ( ⁇ 10%, 10-50%, >50% of total stromal cells, which were considered as TAM after CD68 or CD 14 staining). Cases with few IL4I1- positive cells were scored as negative.
  • a first antigen retrieval was performed by water bath heating in citrate buffer pH6 (Dako). Slides were then incubated 10 min with horse serum (Vectastain kit, Vector Laboratories, Burlingame USA), then incubated 1 hour with anti-CD14 (diluted 1/50, mouse monoclonal Ab-2, clone7, Labvision, Fremont, CA, USA), CD68 (diluted 1/50, clone KPl, Dako, Glustrup, Denmark) or CD30 (diluted 1/30, clone Ber-H2, Dako, Glustrup, Denmark) antibodies and labelled using the alkaline phosphatase-conjugated ABC procedure (Vectastain kit, Vector Laboratories, Burlingame USA).
  • IL4I1 activity was measured on 10 6 total lymph node cells, splenocytes or sorted cell populations. For cell lines, the activity was measured on 10 5 cells. Lysates in Phosphate Buffer Saline containing protease inhibitors (Complete mini, Roche, Meylan, France) were obtained by 3 freeze-thaw cycles. IL4I1 phenylalanine oxidative deamination was performed against phenylalanine as in 14 . Fluorimetric quantification of H 2 O 2 produced by the enzymatic reaction was performed by Ultra Amplex Red (Invitrogen, Cergy-Pontoise, France) oxidation analysis using an Optima Fluostar plate reader (BMG Labtech, Champigny, France). For secreted activity, cells were plated in PBS containing 5% fetal calf serum (2.5xlO 5 cells in 100 ⁇ l) and 1OX reaction mix was directly added to the cells. Follicular lymphoma clinical data and statistical analysis
  • Cytokine and chemokine secretion in proliferation experiments was detected using the human cytokine antibody array 1 from RayBiotech according to manufacturer's instructions.
  • Human IL2 and IFN ⁇ were measured with OptEIA ELISA kits from BD Biosciences (Le Pont de Claix, France) according to manufacturer's instructions.
  • mice C57BL/6 mice were vaccinated with the gp33-41 T cell epitope expressed by melanoma tumor cells (the model used is the B16 tumor cell line trans fected with the 33-41 amino acid peptide from the glycoprotein of lymphochoriomenengitis virus) emulsified in incomplete Freund adjuvant. Seven (left graph) or 28 days (right graph) after vaccination, mice were challenged with 10 5 tumor cells expressing murine IL4I1 or trans fected with control vector. The number of tested animals is indicated in parenthesis.
  • the percent of tumor-free mice is represented as Kaplan Meier's survival curves and statistical analysis was performed using the log rank test.
  • Figure 8B C57BL/6 mice were vaccinated as in figure 8A. Seven days after vaccination, mice were challenged with 10 5 tumor cells expressing murine IL4I1 (tumor 1 and tumor 2) or transfected with control vector (control tumor). The number of tested animals is indicated in parenthesis. The percent of tumor-free mice is represented as Kaplan Meier's survival curves and statistical analysis was performed using the log rank test comparing tumor 1 and tumor 2 with control.
  • Figure 8C C57BL/6 mice were challenged with 5xlO 4 tumor cells expressing murine IL4I1 (tumor 1 and tumor 2) or transfected with control vector (control tumor).
  • mice The number of tested animals is indicated in parenthesis. Data are expressed as percent of tumor-free mice represented as Kaplan Meier's survival curves and statistical analysis was performed using the log rank test. Monitoring of the mouse immune response with IFN ⁇ -ELISPOT and flow cytometry immunophenotyping
  • bood cells were incubated with gp33-41 tetramers and an anti-CD107a antibody (clone 1D4B, APC coupled) for 30 minutes as above, before a 5-hour culture at 37°C to allow cell stimulation by the tetramers. One hour after the beginning of the stimulation, 2 ⁇ M monensin were added. Then, CD8 labelling was performed for 15 minutes at room temperature, red blood cells were lyzed and cells were fixed as above.
  • an anti-CD107a antibody clone 1D4B, APC coupled
  • IFN ⁇ -ELISPOT ethanol treated PVDF microplates (Millipore) were coated in PBS with an anti-mouse IFN- ⁇ monoclonal antibody (clone AN-18 15 ⁇ g/ml). Duplicate aliquots of spleen cells (2 x 10 5 /well) were added together with the g ⁇ 33- 41 peptide or a control H2-D b restricted peptide from the influenza virus nucleoprotein (1 ⁇ g/ml) in culture medium supplemented with 30 U/ml of human rIL-2.
  • IL4I1 activity was measured on conditioned media from IL4I1 expressing and secreting HEK 293 cells preincubated 5 min on ice with the different inhibitors.
  • IL4I1 phenylalanine oxidative deamination was performed against phenylalanine (control) as in 14 , or against phenylalanine in presence of different concentrations of inhibitors of IL4I1 of formula (I).
  • Inhibitors tested are the folio wings:
  • Fluorimetric quantification of H 2 O 2 produced by the enzymatic reaction was performed by Ultra Amplex Red (Invitrogen, Cergy-Pontoise, France) oxidation analysis using an Optima Fluostar plate reader (BMG Labtech, Champigny, France). Global non linear regression analysis and computation of best fit Ki values was performed using the Prism 5 software package (Graphpad).
  • IL4I1 expression in non-lymphoid malignancies IL4I1 protein expression was analyzed in 121 cases of solid tumors (Table 1). Expression in tumor cells was observed in only 9 cases (7.4%), including 5/10 mesotheliomas, 2/13 non small-cell carcinomas, 1/6 thyroid carcinoma and 1/3 ovarian carcinoma. In tumor cells, IL4I1 immunoreactivity consisted of intracytoplasmic granular staining. Among these 9 cases, the percentage of positive tumor cells was heterogeneous. One mesothelioma and one ovarian carcinoma (figure Ia) showed high IL4I1 expression in more than 50% of the malignant cells whereas the other cases demonstrated usually less than 10% positive tumor cell expression.
  • IL4I1 staining was also detectable in a variable percentage of the infiltrating histiocytes.
  • the IL4I1 -positive ovarian carcinoma was characterized by an intense intratumoral granulomatous reaction and numerous IL4I1 -positive epithelioid and giant cells were observed (figure Ib).
  • IL4I1 expression in tumor cells was focal and restricted to an inflammatory area with calcifications (figure Ic). Both of these observations suggest a possible role for inflammatory processes in the induction of IL4I1 expression.
  • IL4I1 expression was observed in tingible bodies macrophages located in the germinal centers of lymphoid follicles (figure 2a). In addition, scattered positive histiocytes were found in interfollicular areas. In reactive lymph nodes with peripheral sinusal histiocytosis, numerous IL4I1 -positive histiocytes were observed in the sinuses (previously reported in ref 14 ). No expression was observed in centrocytes and centroblasts of the germinal centers. In the spleen, tingible bodies macrophages of germinal centers of lymphoid follicles located in the white pulp were positive as well as occasional histiocytes of the red pulp. In the thymus, scattered IL4I1 -positive histiocytes were observed in the medulla.
  • neoplastic cells of MZL, mantle cell lymphoma and plasmacytoma/myeloma did not express IL4I1, while 64% of follicular lymphomas were IL4I1 -positive with a percentage of positive tumor cells ranging from 10% to 80%.
  • Positive tumor cells were located predominantly in the intrafollicular areas (figure 2d) except for one case of follicular lymphoma with marginal zone differentiation where cells were mainly observed in the perifollicular marginal zone of neoplastic follicles (figure 2e). No correlation between IL4I1 staining and the histological grade of the follicular lymphomas was found.
  • NLPHL (91%) contained IL4I1- positive tumor cells (figure 2h).
  • cHL and NLPHL showed particularly high expression of IL4I1 in histiocytes, with a common strong granular staining.
  • Double immuno fluorescent staining with IL4I1 and CD5, CD20 or CD68 confirmed the exclusive labelling of macrophages (data not shown).
  • an inverse correlation was observed in cHL between IL4I1 expression in HRS and infiltrating TAM, respectively. Indeed, cHL with a high number of IL4I1- positive HRS displayed few positive TAM whereas cHL with low/negative HRSpresented many IL4I1 -positive TAM.
  • PeripheralT-cellunspecified 3 0 3 0 0 Angioimmunoblastic 6 0 3 1 2
  • Enteropathy-type 2 0 1 1 1 0
  • IL4I1 activity in reactive lymphoid tissues, lymphoid malignancies, melanoma biopsies and Hodgkin lymphoma-derived cell lines
  • IL4I1 activity in stromal cells can be detected in the CD 19 " cell population of both IL4I1 -positive or negative lymphoma.
  • Data from Ma et al demonstrated that IL4I1 can be detected in the secretome of HL cell lines in vitro 16 .
  • Our immunohistochemistry results showed that HRS expresses variable levels of IL4I1.
  • IL4I1 a putative anti-inflammatory gene expressed by myeloid cells
  • IL4I1 The purified IL4I1 enzyme was able to inhibit T lymphocyte proliferation in vitro w .
  • THPl monocytic cell line to overexpress human IL4I1 (THP1-IL4I1), as IL4I1 expression is mainly detected in cells of myeloid origin in vitro and in vivo 14 ' 19 .
  • IL4I1 activity in the transfected cells was 895 ⁇ 363 pmoles H 2 O 2 ZhZlO 5 cells (mean from 5 independent tests).
  • THP1-IL4I1 cells were added to a culture of anti-CD3 stimulated PBMC, the level of PBMC proliferation was significantly decreased (37.6 ⁇ 13.9 %, mean of 5 different experiments) in accordance with a decrease in IL2 secretion at day 1 (Fig. 5A & C). Moreover, this inhibition was proportional to the number of THP1-IL4I1 cells added to the culture and detectable for THP 1-IL4I IZPBMC ratios of as little as 1 per 32 cells (Fig. 5A and data not shown). A similar effect was observed using PBMC stimulated with purified protein derivative (PPD, Fig. 5B).
  • IL4I1 has been originally described in B cells as an IL4-inducible gene 20 .
  • IL4 was able to induce a two-fold increase in IL4I1 activity in B lymphocytes, whereas IFN ⁇ did not have any effect.
  • IFN ⁇ was a strong inducer of IL4I1 activity in all the myeloid populations, particularly in monocytes, which have a low basal activity (5 folds).
  • IL4I1 can be induced both in B lymphocytes and myeloid cells, with IL4 and IFN ⁇ exerting dichotomous roles in these populations.
  • M ⁇ and DC display much higher levels of IL4I1 activity than B lymphocytes, with activities in a similar range of those measured in the THP1-IL4I1 cell line.
  • IL4I1 mRNA is known to be induced from the first hours post-IL4 stimulation in B cells, we determined the kinetics of IL4I1 production in myeloid cells. IL4I1 activity continued to increase after 48h of stimulation of DC, M ⁇ or B cells with their respective stimulating cytokine (Fig. 6B), indicating that IL4I1 protein accumulated over time.
  • the monocytic cell line THPl was cultivated for 48h in conditioned media from the Hodgkin lymphoma cell lines KM-H2 and L428 or from the Large B cell lymphoma cell line SU-HDL-4 as control or normal medium (Fig. 7).
  • the media from both Hodgkin cell lines were able to induce a level of IL4I1 expression that was comparable to stimulation of THPl cells by IFN ⁇ . This expression was partially blocked when neutralising antibodies against IL 13 or IFN ⁇ were added to the culture.
  • IL4I1 -expressing tumors escape the anti-tumoral immune response (Figure 8A): mice challenged with the IL4I1 + cell line develop tumors with a significantly higher frequency than mice challenged with control tumors in both experimental settings. These results indicate that IL4I1 expression by the tumor facilitate escape from the CD8 T cell response induced by the gp33-41 vaccination
  • mice challenged with the IL4I1 + cell line develop tumors with a significantly higher frequency than mice challenged with control tumors in both experimental settings.
  • IL4I1 expression by the tumor facilitate escape from the CD8 T cell response induced by the gp33-41 vaccination.
  • Tumors developed more rapidly in the group challenged with tumor 2 cells than in the group challenged with tumor 1 cells, although the difference was not statistically significant. This might be in accordance with tumor 2 displaying a higher IL4I1 activity in vitro.
  • the systemic immune response against the tumor epitope gp33-41 was evaluated in the blood and spleen of mice vaccinated with gp33-41 and challenged with high numbers of control tumor cells or IL4I1 expressing tumors (Figure 10).
  • the number of gp33-41 specific T cells increased rapidly (6 days) after control tumor injection, due to restimulation of memory cells (Figure 10A). In contrast, this increase was absent (tumor 2) or considerably less prominent (tumor 1) in mice challenged with the two IL4I1 expressing tumors. The importance of this effect correlated with the level of IL4I1 activity of these tummors.

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Abstract

L'invention porte sur un inhibiteur de IL4I1 destiné à être utilisé dans un procédé de traitement d'un cancer présentant les cellules exprimant IL4I1 dans le corps humain ou animal, à la condition que ledit cancer ne soit pas un lymphome folliculaire. Sous un autre aspect, l'invention porte sur un procédé de détermination du pronostic d'un sujet souffrant d'un cancer, à la condition que ledit cancer ne soit pas un lymphome folliculaire, comprenant l'étape consistant à détecter la présence de IL4I1 dans un échantillon obtenu auprès dudit sujet, la présence de IL4I1 indiquant que le pronostic du sujet est médiocre. L'invention porte également sur un procédé de détermination du pronostic d'un sujet souffrant d'un lymphome folliculaire, comprenant l'étape de détection de la présence de IL4I1 dans un échantillon obtenu auprès dudit sujet, la présence de IL4I1 indiquant que le pronostic du sujet est médiocre.
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WO2019185907A1 (fr) 2018-03-29 2019-10-03 Universite Paris Est Creteil Val De Marne Dérivés de phénylalanine destinés à être utilisés dans le traitement de cancers
EP3656794A1 (fr) 2018-11-23 2020-05-27 Centre National De La Recherche Scientifique Composition et procédés de détection du cancer
US11906520B2 (en) 2018-11-23 2024-02-20 Centre National De La Recherche Scientifique Composition and methods for detecting cancer
EP3721894A1 (fr) * 2019-04-10 2020-10-14 Deutsches Krebsforschungszentrum, Stiftung des öffentlichen Rechts Gène 1 induit par l'interleukine-4 (il4i1) en tant que biomarqueur et ses utilisations
WO2020208190A1 (fr) * 2019-04-10 2020-10-15 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Gène 1 induit par l'interleukine 4 (il4i1) en tant que biomarqueur et ses utilisations
EP3835432A1 (fr) 2019-12-10 2021-06-16 Deutsches Krebsforschungszentrum, Stiftung des öffentlichen Rechts Gène 1 induit par l'interleukine-4 (il4i1) et métabolites respectifs en tant que biomarqueurs pour le cancer
WO2021116357A1 (fr) 2019-12-10 2021-06-17 Deutsches Krebsforschungszentrum Stiftung des öffentlichen Rechts Gène 1 induit par l'interleukine 4 (il4i1) et métabolites respectifs en tant que biomarqueurs pour le cancer
WO2022232333A1 (fr) * 2021-04-30 2022-11-03 Merck Sharp & Dohme Llc Inhibiteurs d'il4i1 et procédés d'utilisation
WO2024051106A1 (fr) * 2022-09-09 2024-03-14 上海百英生物科技股份有限公司 Préparation et utilisation d'un nano-anticorps anti-il4i1

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