Liver-specific car for use against immunologic rejection

Abstract

The present invention relates to a CAR and to regulatory T-cells (Treg) expressing the CAR, especially for use in the treatment, especially for suppression, of adverse immune reactions against liver tissue, e.g. for use in the treatment of host-versus-graft disease (HvG) against a liver transplant, for use in the treatment of an autoimmune disease directed against liver tissue, or for use in the treatment of inflammatory liver disorder. The invention provides a chimeric antigen receptor (CAR) that comprises an scFv portion, which is specific for the asialoglycoprotein receptor (ASGPR).

Description

[0001] Liver-specific CAR for use against immunologic rejection

[0002] The present invention relates to a CAR and to regulatory T-cells (Treg) expressing the CAR, especially for use in the treatment, especially for suppression, of adverse immune reactions against liver tissue, e.g. for use in the treatment of host-versus-graft disease (HvG) against a liver transplant, for use in the treatment of an autoimmune disease directed against liver tissue, or for use in the treatment of inflammatory liver disorder. Treg of the invention that express the CAR induce immune tolerance specifically in liver tissue by liver-localized suppression of adverse immune reaction. Further, Treg of the invention that express the CAR migrate to the liver, allowing their use in the treatment by administration to a patient at a site spaced from the liver, e.g. administration by systemic injection, e.g. intra venous administration.

[0003] The Treg of the invention that express the CAR are suitable for use in suppressing adverse immune reactions directed against liver tissue, e.g. for use in the treatment of HvG, without specificity for a foreign antigen, especially without specificity for an immunologically incompatible HLA. Therefore, the Treg expressing the CAR of the invention are independent of a foreign antigen that is present in the liver but they are suitable for inducing immune tolerance that is essentially limited to liver tissue and depends of presence of liver cells, especially depends on the presence of hepatocytes only.

[0004] An advantage of embodiments of the CAR of the invention and of Treg expressing the CAR is that the CAR is liver-specifically effective both in humans and in rodents, e.g. mice and rats.

[0005] State of the art

[0006] WO 2021 / 038249 Al without any evidence for its therapeutic effect claims use in HvG of a Treg expressing a CAR having as an antigen-binding domain only a VH specific for asialoglycoprotein receptor with experimental in vitro data using effector T-cells that had been pre-activated with anti-CD3 / CD28 beads. The experiments show that CD69 is only upregulated in T-cells which in addition to the CAR also express GFP.

[0007] US 2018 / 0201902 Al as well as Chen et al., Cancer Immunol. Immunother. 2017, 475-489 for use in liver cancer therapy describe T-cells expressing both a CAR specific for GPC3 and a CAR specific for asialoglycoprotein receptor 1.

[0008] Rigopoulou et al., Autoimmunity Reviews 2012, 260-269 review asialoglycoprotein receptor (ASGPR) as a target autoantigen in liver autoimmunity, presence of anti-ASGPR autoantibodies during the course of autoimmunity disease and immunosuppressive treatments.

[0009] Saetzler et al., Cells, 12 (2023) 2115 for beta-amyloid-specific CAR-expressing Treg cells describe the FACS-assay used herein for detecting suppressive activity by measuring expression of CD69.

[0010] Tenspolde et al., Journal of Autoimmunity 103 (2019) 102289 and Pieper et al., Cells 2023, 12, 1453 describe the NF AT assay used herein for detecting suppressive activity by measuring expression as induction of GFP expression from an NF AT promoter.

[0011] Object of the invention

[0012] It is an object of the invention to provide a liver-specific immune suppression, which is limited to liver tissue and is preferably independent from presence of a heterologous antigen. Description of the invention

[0013] The invention achieves the object by the features of the claims, and especially provides a chimeric antigen receptor (CAR) that from N-terminus to C-terminus comprises or consists of an scFv portion, which is specific for the asialoglycoprotein receptor (ASGPR), especially for the human and / or for the murine and rat ASGPR, preferably a hinge region, e.g. a hinge comprising IgGl - IgGl CH2 - IgGl CH3 hinge, a transmembrane domain (TM), e.g. a CD4 TM or a CD4a TM, and intracellular signaling domains, e.g. a CD28 signaling domain and a CD3zeta signaling domain, wherein preferably the scFv portion has a pair of complementary domain regions 3 (CDR3) of the heavy and light chain, the pairs of CDR3 having an amino acid sequence which comprise or consist of amino acids No. 97..113 of SEQ ID NO: 28 and amino acids No. 89..97 of SEQ ID NO: 29, or which comprise or consist of amino acids No. 99..109 of SEQ ID NO: 30 and amino acids No. 91..101 of SEQ ID NO: 31, or which comprise or consist of amino acids No. 97..108 of SEQ ID NO: 32 and amino acids No. 92..100 of SEQ ID NO: 33, or which comprise or consist of amino acids No. 97..108 of SEQ ID NO: 34 and amino acids No. 92..100 of SEQ ID NO: 35, or which comprise or consist of amino acids No. 97..107 of SEQ ID NO: 36 and amino acids No. 89..97 of SEQ ID NO: 37.

[0014] Preferably, the scFv has a variable heavy chain (VH) comprising or consisting of amino acid sequence

[0015] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPGQGPEWMGIINPSG GSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARDAVGLRERYYYGM DVWGQGTTVTVSS (SEQ ID NO: 28, VH of G3) and a variable light chain (VL) comprising or consisting of amino acid sequence

[0016] DIVMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPFTFGGGTKVEIK (SEQ ID NO: 29, VL of G3), or a VH comprising or consisting of amino acid sequence

[0017] QIQLVQSGGGLVQPGGSLRLSCAGSGYTFSDHYMDWVRQAPGKGLEWVGRIRYKA NSYSTEYAASVKGRFTISRDDSRNSLFLQMNSLKTEDTAVYYCARIRDRYYFDYWGQ GALVTVSS (SEQ ID NO: 30, VH of Cl 1) and a variable light chain (VL) comprising or consisting of amino acid sequence QSVLTQPPSVSGAPGQRVTISCTGSSSNVGAGYDVHWYQQLPGAAPKLLIYGNSNRP SGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDDSLNGVVFGGGTKLTVL (SEQ ID NO: 31, VL of Cl l), or a VH comprising or consisting of amino acid sequence

[0018] QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWISAYN GNTNYAQKLQGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGYSYGDDFDYWG QGTLVTVSS (SEQ ID NO: 32, VH of E12) and a variable light chain (VL) comprising or consisting of amino acid sequence

[0019] NFMLTQPHSVSESPGKTVILSCTGTGGSIASNYVQWYQQRPGSAPTTVIYKDNERPSG VPDRFSGAIDSSSNSASLTISGLRTEDEADYYCQSYGSGNVVFGGGTKLTVL (SEQ ID NO: 33, VL of E12), or a VH comprising or consisting of amino acid sequence

[0020] QVTLKESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGS TYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDLKQWLEDYWGQGT LVTVSS (SEQ ID NO: 34, VH of C3) and a variable light chain (VL) comprising or consisting of amino acid sequence

[0021] DVVMTQSPSTLSASVGDRVTITCRASQSIGPWLAWYQQKPGEAPKLLLYKASILEGG

[0022] VPSRFSGRGSGTEFTLTISSLQPDDFATYYCQQYNTFGPTFGQGTKLEIK

[0023] (SEQ ID NO: 35, VL of C3), or a VH comprising or consisting of amino acid sequence

[0024] QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPN SGGTNYAQKFQGWVTMTRDTSISTAYMELSRLRSDDTAVYYCARKAVAADYFDYW GQGTLVTVSS (SEQ ID NO: 36, VH of BIO) and a variable light chain (VL) comprising or consisting of amino acid sequence

[0025] NFMLTQPHSVSASPGKTVTISCTGSGGSIASHYVQWYQQRPGSAPTSVIYEDDQRPSG VPDRF SGSID S S SNS ASLTISGLKTEDEAD YYCQ S YD S SHQ VFGGGTKLT VL (SEQ ID NO: 37, VL of BIO).

[0026] For localization of the CAR, its amino acid sequence preferably comprises at its N-terminus a signal peptide directing the CAR for transmembrane localization.

[0027] Generally preferred, the signal peptide which is arranged N-terminally of the scFv and the domains of the CAR which are arranged C-terminally of the scFv, e.g. a hinge, a transmembrane domain and signaling domains have sequences of human origin for use in the treatment of humans, especially in order to avoid immune reactions against these domains. Herein, domains or sections of a CAR having an amino acid sequence of human origin can be identified by a “h”, and domains or sections having a murine amino acid sequence can be identified by an “m”. For use in rodents, e.g. mice or rats, the signal peptide and the domains of the CAR which are arranged C-terminally of the scFv can have sequences of murine origin or, less preferred, of human origin.

[0028] Optionally, the CAR is expressed as a fusion protein with FOXP3, which for use in the treatment of a human preferably has the human amino acid sequence of FOXP3 or for use in an experimental has the autologous amino acid sequence, e.g. for use in mice has the murine amino acid sequence (Foxp3). Generally, the fusion protein from N-terminus to C-terminus may comprise or consist of the CAR and FOXP3, separated by a protease site, e.g. P2A, or consist of the CAR and optionally in addition deltaLNGFR. Suitably, the FOXP3 polypeptide encoded by a nucleic acid molecule, construct or vector as described herein may comprise or consist of the polypeptide sequence of a human FOXP3, such as UniProtKB accession Q9BZS1, or a functional fragment or variant thereof.

[0029] Optionally, the CAR is expressed from an expression cassette which, under the control of an IRES, encodes a peptide label, which preferably for use in humans is deltaLNGFR (SEQ ID NO: 39, encoded by SEQ ID NO: 38), and for use in mice is Thy 1.1 (also termed CD90.1). The peptide deltaLNGFR (SEQ ID NO: 39), in addition to presenting a surface-bound label which is not immunogenic in humans and which can be detected by an antibody, e.g. a labelled antibody, deltaLNGFR also provides a peptide label suitable for specifically triggering the deletion of cells that express the CAR, e.g. using an anti -del taLNGFR-antibody. Preferably, Treg are genetically manipulated for expression of the CAR in combination with FOXP3, e.g. expression from one common nucleic acid, preferably from one common expression cassette, with an IRES arranged between the coding sequence encoding the CAR, a protease site, and optionally FOXP3, and the coding sequence (SEQ ID NO: 38) encoding deltaLNGFR. Genetic manipulation can be by transduction of T cells, preferably of Treg, by a viral particle, preferably a retroviral particle, especially an a-retroviral or a y-retroviral particle, the viral particle containing an expression cassette between its 5'LTR and its 3'LTR.

[0030] In embodiments of Treg that in addition to the CAR, preferably also Foxp3, also express Thy 1.1, deletion of these Treg can be triggered specifically by an anti -Thy 1.1 -antibody. In this embodiment, the Treg are suitable for use in the treatment of adverse immune reactions directed against liver tissue with the additional feature of terminating the activity of the Treg, e.g. deleting the Treg, by using an antibody specific for the marker peptide, herein represented by deltaLNGFR for use in humans, or Thy 1.1 for use in mice, which represents a suicide moiety for binding of an antibody.

[0031] The safety switch polypeptide provides a cell in or on which it is expressed with a suicide moiety. This is useful as a safety mechanism which allows a cell which has been administered to a subject to be deleted should the need arise, or indeed more generally, according to desire or need, for example once a cell has performed or completed its therapeutic effect.

[0032] A suicide moiety possesses an inducible capacity to lead to cellular death, or more generally to elimination or deletion of a cell. An example of a suicide moiety is a suicide protein, encoded by a suicide gene, which may be expressed in or on a cell alongside a desired transgene, in this case the CAR, which when expressed allows the cell to be deleted to turn off expression of the transgene (CAR). A suicide moiety herein is a suicide polypeptide that is a polypeptide that under permissive conditions, namely conditions that are induced or turned on, is able to cause the cell to be deleted.

[0033] The suicide moiety may be a polypeptide, or amino acid sequence, which may be activated to perform a cell-deleting activity by an activating agent which is administered to the subject, or which is active to perform a cell-deleting activity in the presence of a substrate which may be administered to a subject. In a particular embodiment, the suicide moiety may represent a target for a separate cell-deleting agent which is administered to the subject. By binding to the suicide moiety, the cell-deleting agent may be targeted to the cell to be deleted. In particular, the suicide moiety may be recognised by an antibody, and binding of the antibody to the safety switch polypeptide, when expressed on the surface of a cell, causes the cell to be eliminated, or deleted.

[0034] The suicide moiety may be HSV-TK or iCasp9. However, it is preferred for the suicide moiety to be, or to comprise, an epitope which is recognised by a cell-deleting antibody or other binding molecule capable of eliciting deletion of the cell. In such an embodiment, the safety switch polypeptide is expressed on the surface of a cell. The term “delete” as used herein in the context of cell deletion is synonymous with “remove” or “ablate” or “eliminate”. The term is used to encompass cell killing, or inhibition of cell proliferation, such that the number of cells in the subject may be reduced. 100% complete removal may be desirable but may not necessarily be achieved. Reducing the number of cells, or inhibiting their proliferation, in the subject may be sufficient to have a beneficial effect.

[0035] In particular, the suicide moiety may be a CD20 epitope which is recognised by the antibody Rituximab. Thus, in the safety switch polypeptide the suicide moiety may comprise a minimal epitope based on the epitope from CD20 that is recognised by the antibody Rituximab. Biosimilars for Rituximab are available and may be used. A person of skill in the art is readily able to use routine methods to prepare an antibody having the binding specificity of Rituximab using the available amino acid sequences therefor.

[0036] CAR-cells specific for ASGPR, which also express a safety switch polypeptide comprising this sequence can be selectively killed using the antibody Rituximab, or an antibody having the binding specificity of Rituximab. The safety switch polypeptide is expressed on the cell surface and when the expressed polypeptide is exposed to or contacted with Rituximab, or an antibody with the same binding specificity, death of the cell ensues.

[0037] Thus, Rituximab, or an antibody having the binding specificity thereof, may be provided for use in adoptive cell transfer (ACT) in combination with a cell of the invention. The cell or nucleic acid or vector or construct for production of the cell and the Rituximab or equivalent antibody may be provided in a kit, or as a combination product.

[0038] For example, the suicide constructs of WO2013 / 153391 or WO2021 / 239812 may be used in a cell or cell population (e.g., Treg or Treg population) as described herein.

[0039] It was found that the specificity of the scFv portion for ASGPR essentially depends on the CDR3 domains of the heavy chain and of the light chain of the scFv, and that this specificity can be maintained also in combination with other CDR1 and CDR2 domains that originate from a paratope, e.g. an scFv, which is not specific for ASGPR.

[0040] Generally preferred, the Treg are immunologically compatible to the recipient, and preferably the Treg originate from T cells originating from the recipient, i.e. the Treg are derived from autologous T cells, wherein they are derived by genetic manipulation to express the CAR, preferably in combination with FOXP3, and optionally express the peptide label, which preferably is CD20 or deltaLNGFR for use in humans, or Thy 1.1 for use in mice.

[0041] The signal peptide can e.g. have the amino acid sequence MDFQVQIFSFLLISASVIMSRT (SEQ ID NO: 1, murine) or MWWRLWWLLLLLLLLWPMVWA (SEQ ID NO: 2, murine), preferably MWWRLWWLLLLLLLLWPMVW (SEQ ID NO: 3, human), or MDFQVQIFSFLLISASVIMSR (SEQ ID NO: 4, human).

[0042] The hinge domain, which connects the scFv portion with the transmembrane domain, can e.g. have an amino acid sequence comprising or consisting of the IgGl hinge, the IgGl CH2 hinge and the IgGl CH3 hinge, the hinge region of CD28, of Cd8a, of CD4, of CD7, of CH2CH3, of an immunoglobulin, or a part or variant thereof, preferably the CD8a hinge domain or CH2CH3 hinge domain, all of human origin or all of murine origin, e.g.

[0043] VPRDGGCKPCICTVPEVS S VFIFPPKPKDVLTITLTPKVTC VVVDISKDDPEVQF SWF V DDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTIS KTKGRPKAPQVCTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNT QPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHS

[0044] PG (SEQ ID NO: 5, murine) which consists of the mlgGl hinge, the mlgGl CH2 hinge and the mlgGl CH3 hinge, or the CD8 hinge of human origin or of murine origin, e.g.

[0045] FSSVVPVLQKVNSTTTKPVLRTPSPVHPTGTSQPQRPEDCRPRGSVKGTGLDFACDIY (SEQ ID NO: 6, murine), or e.g.

[0046] FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEASRPAAGGAVHTRGLDFADIYIWAP LAGTCGVLLLSLVITLYCNHRQL (SEQ ID NO: 7, human) consisting of the hCD8 hinge and the hCD28 transmembrane domain (hCD8 TM), or the deltaFc Ig, which in the CAR has the functions of a hinge domain and a transmembrane domain, e.g. having the amino acid sequence

[0047] PAEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVDVSHEDPEV I<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVLHQDWLNGI<EYI<

[0048] CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAV EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV MHEALHNHYTQKSLSLSPGKK (SEQ ID NO: 8, human). A transmembrane domain may be selected from the transmembrane domains of CD28, of ICOS, of CD8a, of CD4, of CD134 (0X40), of CD137 (4-1BB), of CD3 zeta, of CD45, of CD9, of CD16, of CD22, of CD33, of CD64, of CD80, of CD86, of CD154, of CH2CH3, or a part or variant thereof. Preferably, the CAR comprises a transmembrane domain of CD8a or of CH2CH3. As examples, the transmembrane domain (TM) may be the CD4 TM, e.g. VFLACVLGGSFGFLGFLGLCILCCV (SEQ ID NO: 9, murine) or its human equivalent, or the CD8a transmembrane domain CD8a TM, e g. IWAPLAGICVALLLSLIITLICYHR (SEQ ID NO: 10, murine) or its human equivalent.

[0049] Preferably, the intracellular signaling domains comprise or consist of, from N-terminus to C- terminus, the CD28 signaling domain (CD28 ICD) and the CD3zeta signaling domain (CD3z ICD). The CD28 ICD can e.g. have the amino acid sequence QLWTNSRRNRLLQSDYMNMTPRRPGLTRKPYQPYAPARDFAAYRP (SEQ ID NO: 11, murine) and the CD3z ICD can e.g. have the amino acid sequence RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQ EGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDA LHMQTLAPR (SEQ ID NO: 12, murine), or the CD28 ICD can e.g. have the amino acid sequence WTNSRRNRLLQSDYMNMTPRRPGLTRKPYQPYAPARDFAAYRP (SEQ ID NO: 13, murine), the CD3z ICD can e.g. have the amino acid sequence

[0050] RAKFSRSAETAANLQDPNQLYNELNLGRREEYDVLEKKRARDPEMGGKQQRRRNPQ EGVYNALQKDKMAEAYSEIGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQTLA PR (SEQ ID NO: 14, murine).

[0051] The human CD28 ICD can e.g. have the amino acid sequence WVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 15, human), the human CD3z can have the amino acid sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA LHMQALPPRSSR (SEQ ID NO: 16, human), or the human CD28 ICD can have the amino acid sequence DPKFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKH YQAYAAARDFAAYRSL (SEQ ID NO: 17, human), and the human CD28 ICD can have the amino acid sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDA

[0052] LHMQALPPR (SEQ ID NO: 18, human).

[0053] The CAR preferably has an intracellular domain (ICD) comprising or consisting of one or more intracellular signaling domains selected from the group consisting of the CD3zeta signaling domain or any of its homologs, the signaling domain of a CD3 polypeptide, of a syk family tyrosine kinase, of a src family tyrosine kinase, of CD2, of CD5, and of CD8, or a signaling domain of a part or variant thereof. Preferably, the CAR comprises the CD3 zeta signaling domain. Generally preferred, the CAR comprises or consists of a human signal peptide, an scFv, human a CD28 hinge and a human CD28 transmembrane domain, a human CD28 ICD and a human CD3z ICD, or the CAR comprises or consists of a human signal peptide, an scFv, a human delta Fc Ig as a hinge and transmembrane domain, a human CD28 ICD and a human CD3z ICD, especially for use in the treatment of adverse immune reactions directed against the liver in a human.

[0054] The CAR may comprise one or more co-stimulatory domains. The one or more costimulatory domains may be selected from the intracellular domains of CD28, of ICOS, of CD134 (0X40), of CD137 (4-1BB), of CD27, or of TNFRSF25, or of a part or variant thereof. Preferably, the CAR may comprise a CD28 co-stimulatory domain. The one or more co-stimulatory domain can be arranged C-terminally to the transmembrane domain.

[0055] The CAR, especially the intracellular domains, also referred to as endodomains, of the CAR which are arranged C-terminally to the transmembrane domain, may comprise one or more intracellular signaling domains selected from the group consisting of the CD3 zeta signaling domain or any of its homologs, a CD3 polypeptide signaling domain, a syk family tyrosine kinase signaling domain, a src family tyrosine kinase signaling domain, CD2 signaling domain, CD5 signaling domain, and CD8 signaling domain, or a part or variant thereof. Preferably, the CAR may comprise the CD3zeta signaling domain.

[0056] The CAR may comprise one or more co-stimulatory domains, e.g. arranged C-terminally to the transmembrane domain of the CAR. The one or more co-stimulatory domains may be selected from the intracellular domains of CD28, of ICOS, of CD134 (0X40), of CD137 (4- 1BB), of CD27, or of TNFRSF25, or a part or variant thereof. Preferably, the CAR may comprise a CD28 co-stimulatory domain. The CAR preferably comprises or consists of a signal peptide, an scFv, a hinge comprising Ig G1 hinge, Ig G1 CH2 hinge and Ig G1 CH3 hinge, a CD4 transmembrane domain, a CD28 ICD and a CD3zeta ICD, or the CAR comprises or consists of a signal peptide, an scFv, a CD8 hinge, a CD8a transmembrane domain, a CD28 ICD and a CD3z ICD, for use in humans in each case preferably all of human origin, for use in mice preferably all of murine origin. The CAR is preferably encoded as a fusion protein, in which C-terminally to the CAR, a protease site, e.g. P2A, and FOXP3 are linked. The P2A protease site can have the amino acid sequence ATNFSLLKQAGDVEENPGP (SEQ ID NO: 19), FOXP3 can have the amino acid sequence

[0057] MPNPRPAKPMAPSLALGPSPGVLPSWKTAPKGSELLGTRGSGGPFQGRDLRSGAHTS SSLNPLPPSQLQLPTVPLVMVAPSGARLGPSPHLQALLQDRPHFMHQL STVDAHAQTPVLQVRPLDNPAMISLPPPSAATGVFSLKARPGLPPGINVASLEWVSRE PALLCTFPRSGTPRKDSNLLAAPQGSYPLLANGVCKWPGCEKVFEEPEE FLKHCQADHLLDEKGKAQCLLQREVVQSLEQQLELEKEKLGAMQAHLAGKMALAK APSVASMDKSSCCIVATSTQGSVLPAWSAPREAPDGGLFAVRRHLWGSH GNSSFPEFFHNMDYFKYHNMRPPFTYATLIRWAILEAPERQRTLNEIYHWFTRMFAY FRNHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDEFEFRKKRSQRP NKCSNPCP (SEQ ID NO: 20, murine) or

[0058] ATNFSLLKQAGDVEENPGPSMPNPRPAKPMAPSLALGPSPGVLPSWKTAPKGSELLG TRGSGGPFQGRDLRSGAHTSSSLNPLPPSQLQLPTVPLVMVAPSGARLGPSPHLQALL QDRPHFMHQLSTVDAHAQTPVLQVRPLDNPAMISLPPPSAATGVFSLKARPGLPPGI NVASLEWVSREPALLCTFPRSGTPRKDSNLLAAPQGSYPLLANGVCKWPGCEKVFEE PEEFLKHCQADHLLDEKGKAQCLLQREVVQSLEQQLELEKEKLGAMQAHLAGKMA LAKAPSVASMDKSSCCIVATSTQGSVLPAWSAPREAPDGGLFAVRRHLWGSHGNSSF PEFFHNMDYFKYHNMRPPFTYATLIRWAILEAPERQRTLNEIYHWFTRMFAYFRNHP ATWKNAIRHNLSLHKCFVRVESEKGAVWTVDEFEFRKKRSQRPNKCSNPCP (SEQ ID NO: 21, murine).

[0059] Preferably FOXP3 (human) can have an amino acid sequence comprising or consisting of SEQ ID NO: 22.

[0060] An exemplary coding sequence for the CAR in the embodiment of the scFv domain being Cl 1 with the other domains in the embodiment of murine CD8 CAR according to Fig. 2 is SEQ ID NO: 40, including a coding sequence for a P2A site followed by FoxP3, and followed an optional IRES controlling the coding sequence for Thy 1.1.

[0061] An exemplary coding sequence for the CAR in the embodiment of the scFv domain being Cl 1 with the other domains in the embodiment of human CD8 CAR according to Fig. 3 is SEQ ID NO: 41, including a coding sequence for a P2A site followed by FoxP3, and followed an optional IRES controlling the coding sequence for LNGFR (delta LNGFR, or ALNGFR).

[0062] The optional marker deltaLNGFR or CD20 for use in humans, or Thy 1.1 for use in mice, can e.g. be encoded by a nucleic acid sequence that is connected in 3' to the nucleic acid sequence encoding the CAR or preferably a fusion protein of the CAR and FOXP3 by an interstitial IRES. The expression of FOXP3 in combination with expression of the CAR maintains the suppressor activity of regulatory T cells (Treg).

[0063] The CAR is preferably expressed in regulatory T cells, which are characterized by expressing the markers CD4+CD25+CD127low. Preferably, the regulatory T cells are immunologically compatible with the human to be treated, more preferably the regulatory T cells originate from the human to be treated who, after genetic manipulation of the T cells such that they express the CAR, can receive the T cells expressing the CAR.

[0064] The scFv domain of the CAR molecules of the invention were generated by phage display using a human naive phage display library HAL9 / 10. A cell panning approach was performed by panning against recombinant murine or human ASGPR. The phage display library or binding scFv of consecutive panning round were incubated with either murine or human ASGPR, immobilized for selecting phage displaying scFv. From eluted phage, the scFv were amplified and used for a next panning round. Panning was conducted for 3 consecutive rounds. After the last panning round, individual scFv were initially screened first for recognition of ASGPR. For screening, soluble scFv, provided with a His-Tag and a Myc-Tag were produced in E.coli and used to stain murine and human liver sections, and secondary staining was performed by PE staining the His-Tag of soluble scFv. Cells were measured in flow cytometry for PE (scFv binding).

[0065] The scFv that were identified as being soluble were used in liver section staining. Cryo IHC was performed to prove islet recognition by selected scFv molecules in liver tissues. For this, fresh livers of C57B1 / 6 mice were embedded in Tissue-Tek® Cryomold® and quick frozen in liquid nitrogen. 5 qm sections were prepared in a cryostat-microtome. Autofluorescence of sections was quenched by incubation in Glycin buffer. Blocking was performed by incubation in 2% BSA. Consecutively, sections were stained with rabbit a- Myc-Tag antibody, recognising the Myc Tag of soluble scFvs. Tertiary staining was performed by incubation with FITC-labelled goat a-rabbit antibody. Sections were embedded with Fluoromount-G™ Mounting Medium containing DAPI and analysed by fluorescence microscopy.

[0066] Cloning of murine and human scFvs into murine and human retroviral vectors, respectively, was performed to generate CARs with ASGPR specificity. The CARs either had a murine CD8a hinge and transmembrane domain (short hinge) or a murine IgG hinge domain and CD4 transmembrane domain (long hinge). The sequence of the CD8a hinge is shown in SEQ ID NO: 6. Retroviral vectors containing LTR flanked 2nd generation CAR scaffolds were used for cloning. Cloning was performed by digestion of vectors with Ncol / Notl restriction enzymes and subsequent ligation of the scFv encoding nucleic acid sequence into the CAR backbone. Murine CAR backbones contained an additional Foxp3 expression cassette allowing converted Tregs (cTreg) generation out of CD4+ T cells but lacked such a cassette for generation of CAR Teffs or natural Tregs (nTregs). Vectors also contained Thy 1.1. (CD90.1) as a CAR expression marker.

[0067] Production of recombinant CAR expressing retroviral virus and transduction of T cells and cell lines:

[0068] Retroviral particles allowing for CAR transduction of T cells were produced in HEK293T cells. They were K73 ecotropic-pseudotyped for murine constructs and VSV-G pantropic- pseudotyped for human constructs, respectively. For CAR transduction, CD4+ cells were enriched from murine spleenocytes by magnetic bead separation and activated by addition of anti-CD3 / anti-CD28 beads for 2 days, followed by spin-transduction with virus particles and protaminsulfate and then underwent continued cultivation for 1 - 4 days. For a CAR activation assay, NFAT-GFP reporter murine T cell hybridoma cells were transduced in an analogous manner.

[0069] Hybridoma NF AT activation assay:

[0070] Murine T cell hybridoma cell line reporting NF AT activation by GFP expression were transduced with CAR vector as described above. In parallel, hepatocytes were purified that all express the target antigen ASGPR. After 48 h both cells of the cell line and hepatocytes were cultivated for 24 h to allow for CAR activation. In a parallel approach, ASGPR peptide (human or murine) that was directly coated on wells of a microtiter plate was used to stimulate CAR transduced hybridoma cells. Wells that were directly coated with collagenase protein and untreated wells served as controls. Cells were stained with anti-Fab antibody binding to the CAR domains for detecting CAR expression. The level of CAR activation reported by GFP and CAR expression was measured by flow cytometry. Herein, this T cell hybridoma cell line is also referred to as the reporter cell line or as reporter cells, which is used in the examples, unless indicated otherwise.

[0071] The invention is now described in greater detail by way of examples and with reference to the figures, which show in

[0072] - Fig. 1 a schematic arrangement of a nucleic acid coding sequence for domains of a fusion protein of the CAR (mlg) and F0XP3 with the coding sequence for Thy 1.1 connected by an IRES,

[0073] - Fig. 2 a schematic arrangement of a nucleic acid coding sequence for domains of a fusion protein of the CAR (mCD8) and F0XP3 with the coding sequence for Thy 1.1 connected by an IRES,

[0074] - Fig. 3 a schematic arrangement of a nucleic acid coding sequence for domains of a fusion protein of the CAR (hCD8) and F0XP3 with the coding sequence for an exemplary marker protein (dLNGFR) connected by an IRES,

[0075] - Fig. 4 a schematic arrangement of a nucleic acid coding sequence for domains of a fusion protein of the CAR (hlg) with the coding sequence for an exemplary marker protein (dLNGFR) connected by an IRES,

[0076] - Fig. 5 A flow cytometry results for control Fibroblast cell line SCI, and Fig. 5B-F show flow cytometry results for SCI expressing a CAR of the invention,

[0077] - Fig. 6A flow cytometry results for activation of control T cells,

[0078] - Fig. 6D flow cytometry results for activation of T cells expressing the CAR but without stimulation,

[0079] - Fig. 6B, Fig. 6C, Fig. 6E and Fig 6F flow cytometry results for T cells expressing a CAR of the invention with stimulation,

[0080] - Fig. 7A flow cytometry results for control Treg, and Fig. 7B-E show flow cytometry results for Treg expressing a CAR of the invention, - Fig. 8A flow cytometry results for control Treg, and Fig. 8B-E show flow cytometry results for Treg expressing a CAR of the invention,

[0081] - Fig. 9A flow cytometry results for control Treg, and Fig. 9B-F show flow cytometry results for Treg expressing a CAR of the invention,

[0082] - Fig. 10A-F flow cytometry results for Treg expressing a CAR of the invention with immune staining for the CAR and for the reporter Thy 1.1, expressed as a portion of a fusion protein CAR-P2A-Thyl. l,

[0083] - Fig. 11A-F flow cytometry results of the distribution of Treg expressing an exemplary CAR after administration to a mouse,

[0084] Fig. 11G a graph summarizing in vivo results for homing of murine Treg into liver,

[0085] - Fig. 12A-C flow cytometry results for the activation of Treg expressing a CAR of the invention specifically by presence of hepatocytes,

[0086] - Fig. 13 A-B flow cytometry results for the background activity of Treg expressing a CAR of the invention,

[0087] - Fig. 13C-D flow cytometry results for the activation of Treg expressing a CAR of the invention specifically by purified ASGPR protein,

[0088] - Fig. 13E-J flow cytometry results for the activation of Treg expressing a CAR of the invention specifically by cells presenting ASGPR.

[0089] - Fig. 14A-B flow cytometry results for the background activity of Treg expressing a CAR of the invention,

[0090] - Fig. 14C-D flow cytometry results for the activation of Treg expressing a CAR of the invention specifically by purified ASGPR protein,

[0091] - Fig. 14E-J flow cytometry results for the activation of Treg expressing a CAR of the invention specifically by cells presenting ASGPR.

[0092] - Fig. 15 A-B flow cytometry results for the background activity of Treg expressing a CAR of the invention,

[0093] - Fig. 15C-D flow cytometry results for the activation of Treg expressing a CAR of the invention specifically by purified ASGPR protein,

[0094] - Fig. 15E-J flow cytometry results for the activation of Treg expressing a CAR of the invention specifically by cells presenting ASGPR.

[0095] - Fig. 16A-B flow cytometry results for the background activity of Treg expressing a CAR of the invention,

[0096] - Fig. 16C-D flow cytometry results for the activation of Treg expressing a CAR of the invention specifically by purified ASGPR protein,

[0097] 15

[0098] SUBSTITUTE SHEET (RULE 26) - Fig. 16E-J flow cytometry results for the activation of Treg expressing a CAR of the invention specifically by cells presenting ASGPR.

[0099] - Fig. 17A-D flow cytometry results for the background activity of Treg expressing a CAR of the invention,

[0100] - Fig. 17E-F flow cytometry results for the background activity of Treg expressing a PE-specific CAR for comparison to Fig. 17A-D,

[0101] - Fig. 18A-D flow cytometry results for the background activity of Treg expressing a CAR of the invention,

[0102] - Fig. 18E-F flow cytometry results for the background activity of Treg expressing a PE-specific CAR for comparison to Fig. 18A-D,

[0103] - Fig. 19A-D flow cytometry results for the activation of Treg expressing a CAR of the invention by HEK cells expressing human ASGPR,

[0104] - Fig. 19E-F flow cytometry results for the background activity of Treg expressing a PE-specific CAR for comparison to Fig. 19A-D,

[0105] - Fig. 20A-D flow cytometry results for the activation of Treg expressing a CAR of the invention by HEK cells expressing murine ASGPR,

[0106] - Fig. 20E-F flow cytometry results for the background activity of Treg expressing a PE-specific CAR for comparison to Fig. 20A-D,

[0107] - Fig. 21 A-D flow cytometry results for the activation of Treg expressing a CAR of the invention by purified human ASGPR,

[0108] - Fig. 21E-F flow cytometry results for the background activity of Treg expressing a PE-specific CAR for comparison to Fig. 21 A-D,

[0109] - Fig. 22A-D flow cytometry results for the activation of Treg expressing a CAR of the invention by purified murine ASGPR,

[0110] - Fig. 22E-F flow cytometry results for the background activity of Treg expressing a PE-specific CAR for comparison to Fig. 22A-D,

[0111] - Fig. 23 A-D flow cytometry results for the activation of Treg expressing a CAR of the invention by human liver cells HepG2,

[0112] - Fig. 23E-F flow cytometry results for the background activity of Treg expressing a PE-specific CAR for comparison to Fig. 23 A-D,

[0113] - Fig. 24A-D flow cytometry results for the activation of Treg expressing a CAR of the invention by human liver cells Huh7,

[0114] - Fig. 24E-F flow cytometry results for the background activity of Treg expressing a PE-specific CAR for comparison to Fig. 24A-D, and Fig. 25 a summary of the results for ASGPR-specific activation of suppressive activity in Treg cells expressing a CAR of the invention.

[0115] In the examples, the following CARs are used:

[0116] Signal peptide (SEQ ID NO: 1) - scFv-hinge of Ig G1 and IgGl CH2 and IgGl CH3 (SEQ ID NO: 5) - CD4 TM (SEQ ID NO: 9) - CD28 ICD (SEQ ID NO: 11) - CD3z ICD (SEQ ID NO: 12) - P2A (SEQ ID NO: 19) - FOXP3 (SEQ ID NO: 20), of murine origin and designated murine Ig-construct (mlg), which is schematically shown in Fig. 1,

[0117] Signal peptide (SEQ ID NO: 2) - scFv - CD8 hinge (SEQ ID NO: 6) - CD8a TM (SEQ ID NO: 10) - CD28 ICD (SEQ ID NO: 13) - CD3z ICD (SEQ ID NO: 14) - P2A - FOXP3 (SEQ ID NO: 21), of murine origin and designated murine CD8 construct (mCD8), which is schematically shown in Fig. 2,

[0118] Signal peptide (SEQ ID NO: 3) - scFv - CD8 hinge and CD8 TM (SEQ ID NO: 7) - CD28 ICD (SEQ ID NO: 15) - CD3z ICD (SEQ ID NO: 16) - P2A - FOXP3, of human origin and designated human CD8 construct (hCD8), which is schematically shown in Fig. 3,

[0119] Signal peptide (SEQ ID NO: 4) - scFv - deltaFc Ig (SEQ ID NO: 8) as hinge and transmembrane domain - CD28 ICD (SEQ ID NO: 17) - CD3z ICD (SEQ ID NO: 18), of human origin and designated human Ig construct (hlg), which is schematically shown in Fig. 4, with the specific scFv attached to the designation of the construct. Accordingly, the designations as of human origin or of murine origin generally only refer to the functional elements and domains except for the scFv, and independent the scFv.

[0120] The scFv sections have been generated by selection of scFv fragments contained in a phage display library according to affinity to immobilized ASGPR.

[0121] SEQ ID NO: 23 comprises embodiment G3 of the anti-ASGPR scFv at amino acids No. 1- 251, and C-terminally a portion of the phage protein at amino acids No. 252-259,

[0122] SEQ ID NO: 24 comprises embodiment Cl 1 of the anti-ASGPR scFv at amino acids No. 21- 287, an N-terminal secretory signal peptide at amino acids No. 1-22, and C-terminally a myc tag at amino acids No. 289-301 and a poly-His tag at amino acids No. 302-307. SEQ ID NO: 25 comprises embodiment E12 of the anti-ASGPR scFv at amino acids No. 23- 285, an N-terminal secretory signal peptide at amino acids No. 1-22, and C-terminally a myc tag at amino acids No. 289-301 and a poly-His tag at amino acids No. 302-307.

[0123] SEQ ID NO: 26 consists of embodiment C3 of the anti-ASGPR scFv at amino acids No. 1- 292. An N-terminal secretory signal peptide is not included.

[0124] SEQ ID NO: 27 consists of embodiment BIO of the anti-ASGPR scFv at amino acids No. 1- 303. An N-terminal secretory signal peptide is not included.

[0125] Preferably, in the CAR, the scFv is preceded by an N-terminal secretory signal peptide and is devoid of additional tags, e.g. devoid of a myc tag or of a poly-His tag. Generally preferred, the CAR does not contain and is not expressed in combination with a binding motif for STAT5, for JAK1, nor for JAK2.

[0126] For use in humans, the CAR preferably has an scFv in the embodiment of C3, Cl 1, G3 or BIO, for use in rodents, especially in mice or rats, the CAR preferably has an scFv in the embodiment of C3, Cl 1, E12 or G3. For use in both humans and rodents, the CAR preferably has an scFv in the embodiment of C3, Cl 1 or G3, wherein the other portions of the CAR for use in humans are of human origin, and the other portions of the CAR for use in rodents are murine portions.

[0127] Example 1 : Expression of ASGPR-specific CAR and marker protein

[0128] Several CARs, containing one of the scFv of SEQ ID NO: 23 (G3), SEQ ID NO: 24 (Cl 1) which includes a C-terminal His-tag, SEQ ID NO: 25 (E12) which includes a C-terminal His- tag, SEQ ID NO: 26 (C3), and SEQ ID NO: 27 (BIO), were expressed in HEK cells that were transduced by nucleic acid constructs containing expression cassettes for one of the CARs. Each CAR had the structure as schematically shown in Fig. 1, encoding the CAR containing mlg as a hinge as a fusion protein with P2A and murine Foxp3 (mFoxp3), and with a separate internal ribosome entry site (IRES) encoding Thy 1.1.

[0129] For analysis, cells that were transduced with a nucleic acid construct encoding one of the CARs were contacted with fluorescence-labelled anti-human-IgG-antibody for detecting the CAR and fluorescence-labelled anti-Thyl.l antibody. Fig. 5 depicts the signals detected by flow cytometry, showing in comparison with Fig. 5A for non-transduced cells without staining as background control, and in Fig. 5B to 5F with immune-staining for the marker Thy 1.1 and immune-staining for the CAR. In relation to the cells detected in Fig. 5A as background, expressing both the marker and the CAR 9.58 % of cells for the scFv of SEQ ID NO: 24 (Fig. 5B), 10.05% of cells for the scFv of SEQ ID NO: 25 (Fig. 5C), 6.87% of cells for the scFv of SEQ ID NO: 26 (Fig. 5D), 13.29% of cells for the scFv of SEQ ID NO: 27 (Fig. 5E), 10.37% of cells for the scFv of SEQ ID NO: 26 (Fig. 5F). This shows that each of the CAR molecules is expressed and that Thy 1.1 from the IRES is expressed at an approximately equimolar ratio. This shows that the CAR for each of the scFv variants is expressed and that the nucleic acid construct is suitable for co-expressing the marker protein under the control of an IRES.

[0130] Example 2: Activation of T cells expressing a CAR having the scFv portion of SEQ ID NO: 24 (CI D by ASGPR

[0131] CAR molecules that had the same scFv portion of SEQ ID NO: 24 (Cl 1) were expressed in the reporter cell line from a nucleic acid construct that was transduced into the cells. The CARs had one of the structures of the CAR containing a hlg hinge (schematically shown in Fig. 4), the structure of the CAR containing a hCD8 hinge (schematically shown in Fig.3), the structure of the CAR containing a mlg hinge (schematically shown in Fig. 1), and with the structure of the CAR containing a mCD8 hinge (schematically shown in Fig. 2).

[0132] Fig. 6A shows flow cytometry results for non-transduced reporter cells in the absence of stimulation by ASGPR, indicating a background of 99.7% cells in quadrant 4 (Q4). Fig. 6D shows flow cytometry results for reporter cells that were transduced with a nucleic acid construct encoding the CAR in the absence of stimulation, indicating 55.2% of nonexpressing cells in Q4 and 42.7% cells expressing the CAR (QI, quadrant 1), as detected by the anti-human IgG-antibody that is specific for the CAR. Background is indicated at 1.00% (quadrant 2, Q2) and 1.02% (quadrant 3, Q3) for GFP expression.

[0133] These reporter cells were activated by contacting with ASGPR protein of the human amino acid sequence (Fig. 6B and 6C) or with ASGPR of the murine amino acid sequence (Fig. 6D, Fig. 6E and Fig. 6F) and analyzed by flow cytometry for presence of the CAR by labelled anti-human IgG-antibody and analyzes for activation by detecting the GFP signal as this is induced by the T cell activation via NF AT. For activation by human ASGPR, Fig. 6B indicates an activation of 1.82% in Q2 for cells expressing the CAR and showing activation as measured by the GFP signal. Fig. 6C for human ASGPR indicates an activation of 1.31% in Q2 and of 11.6% in Q3, in total 12.91%, for CAR-expressing cells as measured by the GFP signal. In view of the controls without activation of Fig. 6A and Fig. 6D showing low background for non-transduced cells and for CAR-expressing cells, activation above background is indicated by cells in one of Q2 and Q3. These results show that the CAR having the scFv of SEQ ID NO: 24 both in the embodiment of the hinge being the hlg hinge and in the embodiment having the CD8 hinge recognizes human ASGPR and results in activation of the T cells.

[0134] Fig. 6E and Fig. 6F shows flow cytometry results for activation by murine ASGPR, in Fig. 6E indicating 3,41% (Q2) and 9.86% (Q3) activated cells, in total 13.27% for the embodiment of the hinge being murine Ig hinge, and in Fig. 6F indicating 4.04% (Q2) and 5.40% (Q3) activated cells, in total 9.44% for the embodiment of the hinge being murine CD8 hinge. These results show that the CAR having the scFv of SEQ ID NO: 24 both in the embodiment of the hinge being the murine hlg hinge and in the embodiment having the murine CD8 hinge recognizes murine ASGPR and results in activation of the T cells.

[0135] Further, the results show that this scFv is specific for both human and murine ASGPR and that a CAR containing this scFv can be used both in humans and in rodents, especially in mice and rats.

[0136] Example 3: Expression of CAR having the scFv portion of SEQ ID NO: 25 (E12) CAR molecules that had the same scFv portion of SEQ ID NO: 25 (E12) were expressed in reporter cells from a nucleic acid construct that was transduced into the cells. The CARs had one of the structures of the structure of CAR containing a hlg hinge (schematically shown in Fig. 4), the structure of hCD8 (schematically shown in Fig.3), the structure of the CAR containing a mlg hinge (schematically shown in Fig. 1), and with the structure of CAR containing a mCD8 hinge (schematically shown in Fig. 2).

[0137] The cells were activated by contacting with ASGPR protein of the human amino acid sequence (Fig. 7B and 7C) or with ASGPR of the murine amino acid sequence (Fig. 7E and Fig. 7F) and analyzed by flow cytometry. Fig. 7A shows flow cytometry results for non-transduced reporter cells in the absence of stimulation by ASGPR, indicating a background of 99.7% cells in quadrant 4 (Q4). Fig. 7D shows flow cytometry results for reporter cells that were transduced with a nucleic acid construct encoding the CAR in the absence of stimulation, indicating 56.5% of nonexpressing cells in Q4 and 43.1% cells expressing the CAR (QI, quadrant 1), as detected by the anti-human IgG-antibody that is specific for the CAR. Background is indicated at 0.27% (quadrant 2, Q2) and 0.13% (quadrant 3, Q3) for GFP expression.

[0138] For activation by human ASGPR, Fig. 7B and Fig. 7C indicate only background for CAR- expressing cells as measured by the GFP signal, indicating this scFv is not activated by human ASGPR, both in the embodiment of the hinge being the hlg hinge and in the embodiment having the CD8 hinge.

[0139] Fig. 7E and Fig. 7F shows flow cytometry results for activation by murine ASGPR, in Fig. 7E indicating 14.3% (Q2) and 7.68% (Q3) activated cells, in total 21.98% for the embodiment of the hinge being murine Ig hinge, and in Fig. 7F indicating 7.37% (Q2) and 9.13% (Q3) activated cells, in total 16.5% for the embodiment of the hinge being murine CD8 hinge. These results show that the CAR having the scFv of SEQ ID NO: 25 both in the embodiment of the hinge being the murine hlg hinge and in the embodiment having the murine CD8 hinge recognizes murine ASGPR and results in activation of the T cells.

[0140] The results show that in comparison to transduced cells that express the CAR but were not stimulated due to absence of ASGPR (Fig. 7D), each of the CARs were activated by presence of murine ASGPR but not by human ASGPR. The results show that a CAR having this scFv is highly specific for murine ASGPR and that a CAR containing this scFv can be used in rodents, especially in mice and rats.

[0141] Example 4: Expression of CAR having the scFv portion of SEQ ID NO: 23 (G3) CAR molecules that had the same scFv portion of SEQ ID NO: 23 (G3) were expressed in reporter cells from a nucleic acid construct that was transduced into the cells. The CARs had one of the structures of the structure of CAR containing a hlg hinge (schematically shown in Fig. 4), the structure of CAR containing a mlg hinge (schematically shown in Fig. 1), and with the structure of CAR containing a mCD8 hinge (schematically shown in Fig. 2). Fig. 8A shows flow cytometry results for non-transduced reporter cells in the absence of stimulation by ASGPR, indicating a background of 99.7% cells in quadrant 4 (Q4). Fig. 8D shows flow cytometry results for reporter cells that were transduced with a nucleic acid construct encoding the CAR in the absence of stimulation, indicating 75.5% of nonexpressing cells in Q4 and 24.3% cells expressing the CAR (QI, quadrant 1), as detected by the anti-human IgG-antibody that is specific for the CAR. Background is indicated at 0.065% (quadrant 2, Q2) and 0.11% (quadrant 3, Q3) for activation as indicated by GFP expression.

[0142] The cells were activated by contacting with ASGPR protein of the human amino acid sequence (Fig. 8B) or with ASGPR of the murine amino acid sequence (Fig. 8D and Fig. 8E) and analyzed by flow cytometry.

[0143] For activation by human ASGPR, Fig. 8B indicates an activation of 14.6% in Q2 and of 55.2% in Q3, in total 69.8%, for CAR-expressing cells as measured by the GFP signal. In view of the controls without activation of Fig. 6A and Fig. 6D showing low background for non-transduced cells and for CAR-expressing cells, activation above background is indicated by cells in one of Q2 and Q3. These results show that the CAR having the scFv of SEQ ID NO: 23 both in the embodiment of the hinge being the hlg hinge and in the embodiment having the CD8 hinge recognizes human ASGPR and results in activation of the T cells.

[0144] Fig. 8D and Fig. 8E shows flow cytometry results for activation by murine ASGPR, in Fig. 8D indicating 13.9% (Q2) and 9.54% (Q3) activated cells, in total 23.44% for the embodiment of the hinge being murine Ig hinge, and in Fig. 8E indicating 6.80% (Q2) and 0.47% (Q3) activated cells for the embodiment of the hinge being murine CD8 hinge. These results show that the CAR having the scFv of SEQ ID NO: 23 both in the embodiment of the hinge being the murine hlg hinge and in the embodiment having the murine CD8 hinge recognizes murine ASGPR and results in activation of the T cells.

[0145] The results show that in comparison to transduced cells that express the CAR but were not stimulated due to absence of ASGPR (Fig. 8C), each of the CARs were activated by presence of human or murine ASGPR. This shows that the activation of the T cell due to expression of the CAR according to the invention was stimulated by presence of ASGPR. Further, the results show that this scFv is specific for both human and murine ASGPR and that a CAR containing this scFv can be used both in humans and in rodents, especially in mice and rats. Example 5: Expression of CAR having the scFv portion of SEP ID NO: 26 (C3)

[0146] CAR molecules that had the same scFv portion of SEQ ID NO: 26 (C3) were expressed in reporter cells from a nucleic acid construct that was transduced into the cells. The CARs had one of the structures of the structure of CAR containing a hlg hinge (schematically shown in Fig. 4), the structure of hCD8 (schematically shown in Fig.3), the structure of CAR containing a mlg hinge (schematically shown in Fig. 1), and with the structure of CAR containing a mCD8 hinge (schematically shown in Fig. 2).

[0147] Fig. 9A shows flow cytometry results for non-transduced reporter cells in the absence of stimulation by ASGPR, indicating a background of 99.7% cells in quadrant 4 (Q4). Fig. 9D shows flow cytometry results for reporter cells that were transduced with a nucleic acid construct encoding the CAR in the absence of stimulation, indicating 80.2% of nonexpressing cells in Q4 and 18.4% cells expressing the CAR (QI, quadrant 1), as detected by the anti-human IgG-antibody that is specific for the CAR. Background is indicated at 0.32% (quadrant 2, Q2) and 1.09 % (quadrant 3, Q3) for activation as indicated by GFP expression.

[0148] The cells were activated by contacting with ASGPR protein of the human amino acid sequence (Fig. 9B and 9C) or with ASGPR of the murine amino acid sequence (Fig. 9E and Fig. 9F) and analyzed by flow cytometry as described above.

[0149] For activation by human ASGPR, Fig. 9B indicates an activation of 33.4% in Q2 and 8.68 in Q3, in total 42.08% for cells expressing the CAR and showing activation as measured by the GFP signal. Fig. 9C for human ASGPR indicates an activation of 9.03% in Q2 and of 2.01% in Q3, in total 11.04%, for CAR-expressing cells as measured by the GFP signal. In view of the controls without activation of Fig. 9A showing low background for non-transduced cells and and Fig. 9D for CAR-expressing cells, activation above background is indicated by cells in one of Q2 and Q3. These results show that the CAR having the scFv of SEQ ID NO: 26 both in the embodiment of the hinge being the hlg hinge and in the embodiment having the CD8 hinge recognizes human ASGPR and results in activation of the T cells.

[0150] Fig. 9E and Fig. 9F shows flow cytometry results for activation by murine ASGPR, in Fig. 9E indicating 3.70% (Q2) and 2.36% (Q3), in total 6.06% activated cells for the embodiment of the hinge being murine Ig hinge, and in Fig. 9F indicating 2.75% (Q2) and 0.13% (Q3) activated cells, in total 2.88% for the embodiment of the hinge being murine CD8 hinge. These results show that the CAR having the scFv of SEQ ID NO: 26 both in the embodiment of the hinge being the murine hlg hinge and in the embodiment having the murine CD8 hinge recognizes murine ASGPR and results in activation of the T cells.

[0151] The results show that in comparison to transduced cells that were not stimulated due to absence of ASGPR (Fig. 9D), each of the CARs were activated by presence of human or murine ASGPR. This shows that the activation of the reporter cells due to expression of the CAR according to the invention was stimulated by presence of ASGPR. Further, the results show that this scFv is specific for both human and murine ASGPR and that a CAR containing this scFv can be used both in humans and in rodents, especially in mice and rats.

[0152] Example 6: Expression of CAR having the scFv portion of SEO ID NO: 27 (BIO) CAR molecules that had the same scFv portion of SEQ ID NO: 27 (BIO) were expressed in reporter cells from a nucleic acid construct that was transduced into the cells. The CARs had one of the structures of the structure of CAR containing a hlg hinge (schematically shown in Fig. 4), the structure of CAR containing a hCD8 hinge (schematically shown in Fig.3), the structure of CAR containing a mlg hinge (schematically shown in Fig. 1), and with the structure of CAR containing a mCD8 hinge (schematically shown in Fig. 2).

[0153] Fig. 10A shows flow cytometry results for non-transduced reporter cells in the absence of stimulation by ASGPR, indicating a background of 99.7% cells in quadrant 4 (Q4). Fig. 9D shows flow cytometry results for reporter cells that were transduced with a nucleic acid construct encoding the CAR in the absence of stimulation, indicating 55.0% of nonexpressing cells in Q4 and 43.0% cells expressing the CAR (QI, quadrant 1), as detected by the anti-human IgG-antibody that is specific for the CAR. Background for activation is indicated at 1.59% (quadrant 2, Q2) and 0.34 % (quadrant 3, Q3) as indicated by GFP expression.

[0154] The cells were activated by contacting with ASGPR protein of the human amino acid sequence (Fig. 10B and 10C) or with ASGPR of the murine amino acid sequence (Fig. Fig. 10E and Fig. 10F) and analyzed by flow cytometry for presence of the scFv portion.

[0155] For activation by human ASGPR, Fig. 10B indicates an activation of 7.39% in Q2 and 19.7 in Q3, in total 27.09% for cells expressing the CAR and showing activation as measured by the GFP signal. Fig. IOC for human ASGPR indicates an activation of 7.36% in Q2 and of 13.6% in Q3, in total 20.96%, for CAR-expressing cells as measured by the GFP signal. In view of the controls without activation of Fig. 10A showing low background for transduced cells and Fig. 10D for CAR-expressing cells, activation above background is indicated by cells in one of Q2 and Q3. These results show that the CAR having the scFv of SEQ ID NO: 27 both in the embodiment of the hinge being the hlg hinge and in the embodiment having the CD8 hinge recognizes human ASGPR and results in activation of the T cells.

[0156] Fig. 10E and Fig. 9F shows flow cytometry results for activation by murine ASGPR, in Fig. 10E indicating 1.61% (Q2) and 0.64% (Q3), in total 2.25% activated cells for the embodiment of the hinge being murine Ig hinge, and in Fig. 10F indicating 1.64% (Q2) and 0.55% (Q3) activated cells, in total 2.19% for the embodiment of the hinge being murine CD8 hinge.

[0157] These results show that the CAR having the scFv of SEQ ID NO: 27 is essentially not activated by murine ASGPR.

[0158] The results show that the reporter cells expressing this CAR were activated by presence of human ASGPR. The results show that this scFv is highly specific for human ASGPR and that a cell expressing a CAR containing this scFv can be used for the treatment especially in humans.

[0159] Example 7: In vivo activity of Treg expressing a CAR

[0160] As a representative of a CAR of the invention, a CAR containing the scFv of SEQ ID NO: 27 (Cl 1) in a CAR containing a mCD8 hinge (schematically shown in Fig. 2) was expressed in Treg cells that were transduced with a nucleic acid construct that also expressed FOXP3 in a fusion protein with the CAR.

[0161] These Treg cells were injected as a single dose of 3 million cells into NOD / Ltj mice by i. v. injection. Five days later, the mice were sacrificed and the presence of Treg expressing the CAR was analyzed by flow cytometry using immune-detection of Thy 1.1 as a representative of the co-expressed CAR and of CD4 as an indicator of activation of suppressor activity. Fig. 11 shows representative flow cytometry results in samples of axillary lymph nodes (ax. LN, Fig. 11 A), cervical lymph nodes (cerv. LN, Fig. 1 IB), inguinal lymph nodes (ing. LN, Fig. 11C), mesenteric lymph nodes (mes. LN, Fig. 1 ID), spleen (Fig. 1 IE), and liver (Fig. 1 IF). In liver tissue, approx. 3% of lymphocytes were ASGPR-specific Treg expressing the CAR of the invention, while the lymphocytes in the control organs only contained approx. 0.1 to 0.4% ASGPR-specific Treg expressing the CAR of the invention.

[0162] These results show that the Treg cells expressing the CAR preferentially migrated into the liver and that their suppressor activity was activated essentially in liver only.

[0163] In a further experiment for testing the migration of Treg cells, murine Treg cells expressing a CAR having an scFv in the embodiment of Cl 1 with the mlgG-Fc-CAR (Fig. 1) were administered at a dose of 3 million i.v. into 7 NOD / Lti mice. Fig. 11G depicts the summarized results, showing that the Treg cells expressing the CAR of the invention preferentially migrated into liver tissue, also called homing into the liver.

[0164] Example 8: In vitro activity of Treg expressing a CAR

[0165] As a representative of a CAR of the invention, a CAR containing the scFv of SEQ ID NO: 27 (Cl 1) in a CAR containing a hCD8 hinge (schematically shown in Fig. 3) was used in an activation assay. In short, reporter cells were transduced to express the CAR of the invention and stimulated by adding purified human hepatocytes.

[0166] Analysis was by flow cytometry with immune-staining using anti-humanCD8-antibody for detection of expression of the CAR and measuring GFP expression for detection of activation of suppressor activity. Fig. 12A for comparison shows untransduced cells without stimulation. Fig 12B for comparison shows the cells when exposed to collagen only, and Fig. 12C shows the cells when exposed to collagen and hepatocytes.

[0167] This result shows that Treg cells expressing the CAR and preferably also FOXP3 in a collagen-containing environment were activated for suppressor activity specifically by presence of hepatocytes.

[0168] The results also show that the specificity of the CAR is determined by its scFv and also show that expression of the CAR in the presence of ASGPR results in the activation of the suppressor activity of Treg. Example 8: In vitro activation by mCD8-Cl 1 CAR

[0169] The NFAT-GFP reporter murine T cell hybridoma cells were transduced with a nucleic acid construct to express a CAR containing the scFv of SEQ ID NO: 27 (Cl 1) with a murine CAR containing a mCD8 hinge (schematically shown in Fig. 2). The nucleic acid construct also expressed FOXP3 in a fusion protein with the CAR. This batch of reporter cells transduced to express the CAR was contacted in parallel with controls, with ASGPR and with cells expressing ASGPR. Fig. 13 shows the FACS results, namely expression of the CAR on the Y- axis (FL3-H::scFv APC-H) and on the X-axis activation (FL1-H::GFP FITC-H) as indicated by expression of GFP. The proportion of activated cells in this experiment is indicated in Q2. Fig. 13 A shows background over cell culture medium (Medium), Fig. 13B shows background in presence of HEK cells devoid of ASGPR. Fig. 13C shows activation by murine ASGPR (Murine Protein) added to the cultivated cells, Fig. 13D shows activation by human ASGPR (human Protein) added to the cultivated cells. Fig. 13E shows activation by HEK cells expressing murine ASGPR (HEK + mASGPRl), Fig. 13F shows activation by HEK cells expressing human ASGPR (HEK + hASGPRl). Fig. 13G shows activation by the human liver cell line HepG2, Fig. 13H shows activation by the human liver cell line Huh7. Fig. 131 shows activation by murine liver cells (murine Liver), Fig. 13 J shows activation by human liver cells (human Liver). These results qualitatively show that the CAR is not activated by medium nor by HEK cells devoid of ASGPR, but that the CAR is activated by the protein murine ASGPR (Fig. 13C) and by HEK expressing murine ASGPR (Fig. 13E) as well as by HEK expressing human ASGPR (Fig. 13F), and is activated stronger by human liver cells (Fig. 13G and Fig. 13H), and is also activated by murine liver cells (Fig. 131) or human liver cells (Fig. 13 J).

[0170] In a further experiment, a batch of NFAT-GFP reporter murine T cell hybridoma cells were transduced to express a CAR containing the scFv of SEQ ID NO: 27 (Cl 1) with a murine CAR containing a mlgG hinge (schematically shown in Fig. 1). Fig. 14 shows the FACS results, namely expression of the CAR on the Y-axis (FL3-H::scFv APC-H) and on the X-axis activation (FL1-H::GFP FITC-H) as indicated by expression of GFP. The proportion of activated cells in this experiment is indicated in Q2. Fig. 14A shows background over cell culture medium (Medium), Fig. 14B shows background in presence of HEK cells devoid of ASGPR. Fig. 14C shows activation by murine ASGPR (Murine Protein) added to the cultivated cells, Fig. 14D shows activation by human ASGPR (human Protein) added to the cultivated cells. Fig. 14E shows activation by HEK cells expressing murine ASGPR (HEK + mASGPRl), Fig. 14F shows activation by HEK cells expressing human ASGPR (HEK + hASGPRl). Fig. 14G shows activation by the human liver cell line HepG2, Fig. 14H shows activation by the human liver cell line Huh7. Fig. 141 shows activation by murine liver cells (murine Liver), Fig. 14J shows activation by human liver cells (human Liver). These results qualitatively show that the CAR is not activated by medium nor by HEK cells devoid of ASGPR, but that the CAR is activated by the protein murine ASGPR (Fig. 14C) and by HEK expressing murine ASGPR (Fig. 14E) as well as by HEK expressing human ASGPR (Fig. 14F), and is activated stronger by human liver cells (Fig. 14G and Fig. 14H), and is also activated by murine liver cells (Fig. 141) or by human liver cells (Fig. 14J).

[0171] In a further experiment, a batch of NFAT-GFP reporter murine T cell hybridoma cells were transduced to express a CAR containing the scFv of SEQ ID NO: 27 (Cl 1) with a human CAR containing a human CD8 hinge and CD8 TM (schematically shown in Fig. 3). Fig. 15 shows the FACS results, namely expression of the CAR on the Y-axis (FL3-H::scFv APC-H) and on the X-axis activation (FL1-H::GFP FITC-H) as indicated by expression of GFP. The proportion of activated cells in this experiment is indicated in Q2. Fig. 15A shows background over cell culture medium (Medium), Fig. 15B shows background in presence of HEK cells devoid of ASGPR. Fig. 15C shows activation by murine ASGPR (Murine Protein) added to the cultivated cells, Fig. 15D shows activation by human ASGPR (human Protein) added to the cultivated cells. Fig. 15E shows activation by HEK cells expressing murine ASGPR (HEK + mASGPRl), Fig. 15F shows activation by HEK cells expressing human ASGPR (HEK + hASGPRl). Fig. 15G shows activation by the human liver cell line HepG2, Fig. 15H shows activation by the human liver cell line Huh7. Fig. 151 shows activation by murine liver cells (murine Liver), Fig. 15J shows activation by human liver cells (human Liver). These results qualitatively show that the CAR is not activated by medium nor by HEK cells devoid of ASGPR, but that the CAR is activated by the protein murine ASGPR (Fig. 15C) and by HEK expressing murine ASGPR (Fig. 15E) as well as by HEK expressing human ASGPR (Fig. 15F), and is activated stronger by human liver cells (Fig. 15G and Fig. 15H), and is also activated by murine liver cells (Fig. 151) or by human liver cells (Fig. 15 J).

[0172] In a further experiment, a batch of NFAT-GFP reporter murine T cell hybridoma cells were transduced to express a CAR containing the scFv of SEQ ID NO: 27 (Cl 1) with a human CAR containing a hlgG hinge (schematically shown in Fig. 4). Fig. 16 shows the FACS results, namely expression of the CAR on the Y-axis (FL3-H::scFv APC-H) and on the X-axis activation (FL1-H::GFP FITC-H) as indicated by expression of GFP. The proportion of activated cells in this experiment is indicated in Q2. Fig. 16A shows background over cell culture medium (Medium), Fig. 16B shows background in presence of HEK cells devoid of ASGPR. Fig. 16C shows activation by murine ASGPR (Murine Protein) added to the cultivated cells, Fig. 16D shows activation by human ASGPR (human Protein) added to the cultivated cells. Fig. 16E shows activation by HEK cells expressing murine ASGPR (HEK + mASGPRl), Fig. 16F shows activation by HEK cells expressing human ASGPR (HEK + hASGPRl). Fig. 16G shows activation by the human liver cell line HepG2, Fig. 16H shows activation by the human liver cell line Huh7. Fig. 161 shows activation by murine liver cells (murine Liver), Fig. 16J shows activation by human liver cells (human Liver). These results qualitatively show that the CAR is not activated by medium nor by HEK cells devoid of ASGPR, but that the CAR is activated by the protein murine ASGPR (Fig. 16C) and by HEK expressing murine ASGPR (Fig. 16E) as well as by HEK expressing human ASGPR (Fig. 16F), and is activated stronger by human liver cells (Fig. 16G and Fig. 16H), and is also activated by murine liver cells (Fig. 161) or by human liver cells (Fig. 16J).

[0173] Example 9: in vitro activation by CAR as assayed by expression of CD69

[0174] In this example, embodiments of the CAR of the invention having the scFv of Cl 1, the scFv of C3, or as a negative control an scFv specific for phycoerythrin (PE), as a murine CD8 CAR (mCD8) according to Fig. 2, as a murine IgG CAR (mlgG-Fc-CAR) according to Fig. 1. Murine CD4+ T-cells were transduced with a nucleic acid construct encoding a CAR as indicated in Fig. 17 and encoding murine FOXP3 separated from the CAR by a P2A site. From the same nucleic acid construct, expression of Thy 1.1 as a tag was from a coding sequence preceded by an IRES. Herein, expression of FOXP3 resulted in converting the murine CD4+ T-cells to murine Treg cells, and accordingly activation shows suppressive activity of the Treg cells. The assay was performed as described by Saetzler et al., Cells, 12 (2023).

[0175] Fig. 17 shows FACS results with medium only (control 1 : Medium) as negative controls, the Y-axis showing expression of the CAR as detected by the tag Thy 1.1 and the X-axis showing activated cells detected as expressing CD69 (FL3-H:: CD69 APC-H), with Q2 indicating activated cells, herein showing background. The anti-PE CAR (PE) in the mCD8 embodiment (Fig. 17E) or in the mlgG embodiment (Fig. 17F) serves as a negative control as the scFv domain is the only difference to the CARs of the invention. The embodiment of the scFv being Cl 1 in the embodiment of the mCD8-CAR (Fig. 17A) or in the embodiment of the mlgG-CAR (Fig. 17B), the embodiment of the scFv being C3 in the embodiment of the mCD8-CAR (Fig. 17C) or in the embodiment of the mlgG-CAR (Fig. 17D) in the absence of the CAR-specific antigen show similar low proportions of activated cells expressing a CAR.

[0176] Fig. 18 for the same cells as in Fig. 17 shows a further negative control with HEK cells. Similar to the results of Fig. 17, Fig. 18 shows that the cells expressed a CAR but were activated only to low background proportions (Q2).

[0177] ASGPR-specific activation is shown in Fig. 19, using HEK293 cells expressing human ASGPR. Fig. 19A and Fig. 19B show ASGPR-specific activation (Q2) for both CAR- embodiments with Cl 1 as the scFv, Fig. 19C and Fig. 19D show ASGPR-specific activation for both CAR-embodiments with C3 as the scFv, whereas both embodiments of the CAR containing the PE-specific scFv (Fig. 19E, 19F) show only background.

[0178] For the murine ASGPR, ASGPR-specific activation is shown in Fig. 20, using HEK293 cells expressing murine ASGPR. Fig. 20A and Fig. 20B show ASGPR-specific activation (Q2) for both CAR-embodiments with Cl 1 as the scFv, Fig. 20C and Fig. 20D show ASGPR-specific activation for both CAR-embodiments with C3 as the scFv, whereas both embodiments of the CAR containing the PE-specific scFv (Fig. 20E, 20F) show only background.

[0179] ASGPR-specific activation is shown in Fig. 21, using addition of purified human ASGPR. Fig. 21 A and Fig. 21B show hASGPR-specific activation (Q2) for both CAR-embodiments with Cl 1 as the scFv, Fig. 21C and Fig. 2 ID show hASGPR-specific activation for both CAR-embodiments with C3 as the scFv, whereas both embodiments of the CAR containing the PE-specific scFv (Fig. 2 IE, 2 IF) show only background.

[0180] ASGPR-specific activation is shown in Fig. 22, using addition of purified murine ASGPR. Fig. 22A and Fig. 22B show hASGPR-specific activation (Q2) for both CAR-embodiments with Cl 1 as the scFv, Fig. 22C and Fig. 22D show hASGPR-specific activation for both CAR-embodiments with C3 as the scFv, whereas both embodiments of the CAR containing the PE-specific scFv (Fig. 22E, 22F) show only background.

[0181] Fig. 23 shows ASGPR-specific activation by the human liver cell line HepG2. Fig. 23 A and Fig. 23B show specific activation (Q2) for both CAR-embodiments with Cl 1 as the scFv, Fig. 23 C and Fig. 23D show specific activation for both CAR-embodiments with C3 as the scFv, whereas both embodiments of the CAR containing the PE-specific scFv (Fig. 23E, 23F) show only very low background.

[0182] Fig. 24 shows ASGPR-specific activation by the human liver cell line Huh7. Fig. 24A and Fig. 24B show specific activation (Q2) for both CAR-embodiments with Cl 1 as the scFv, Fig. 24C and Fig. 24D show specific activation for both CAR-embodiments with C3 as the scFv, whereas both embodiments of the CAR containing the PE-specific scFv (Fig. 24E, 24F) show only very low background.

[0183] The results show that the CARs of the invention only in the presence of their cognate antigen ASGPR result in activation of the T-cells expressing the CAR, whereas unspecific protein, represented by medium only, or cells that do not express the cognate antigen, herein represented by HEK cells, do not result in activation. Further, a CAR having the same domains with an scFv domain specific for PE does not activate cells in the presence of ASGPR. A graphic summary of the results is shown in Fig. 25.

Claims

Claims1. Liver-specific CAR for use in the treatment of an autoimmune disease directed against liver tissue, or for use in the treatment of inflammatory liver disorder, the CAR containing from N-terminus to C-terminus an scFv portion, a hinge, a transmembrane domain and at least one signaling domain, wherein the scFv portion is specific for the asialoglycoprotein receptor (ASGPR) and wherein the scFv portion contains a pair of CDR3 selected from amino acids No. 97..113 of SEQ ID NO: 28 and amino acids No. 89..97 of SEQ ID NO: 29, or amino acids No. 99..109 of SEQ ID NO: 30 and amino acids No. 91..101 of SEQ ID NO: 31, or amino acids No. 97..108 of SEQ ID NO: 32 and amino acids No. 92..100 of SEQ ID NO: 33, or amino acids No. 97..108 of SEQ ID NO: 34 and amino acids No. 92..100 of SEQ ID NO: 35, or amino acids No. 97..107 of SEQ ID NO: 36 and amino acids No. 89..97 of SEQ ID NO: 37.

2. Liver-specific CAR for use according to the preceding claim, characterized in that in a regulatory T cell (Treg) the CAR is produced as a fusion protein with FOXP3 with a protease site arranged between the CAR and FOXP3.

3. Liver-specific CAR for use according to one of the preceding claims, characterized in that the CAR is encoded by a nucleic acid construct which downstream from the CAR encoding section contains a sequence encoding deltaLNGFR or CD20 epitope as a marker, each under the control of an IRES.

4. Liver-specific CAR for use according to one of the preceding claims, characterized in that the scFv comprises a pair of variable chains selected from SEQ ID NO: 28 and SEQ ID NO: 29, SEQ ID NO: 30 and SEQ ID NO: 31, SEQ ID NO: 32 and SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, or SEQ ID NO: 36 and SEQ ID NO: 37.

5. Liver-specific CAR for use according to one of the preceding claims, characterized inthat the CAR at the C-terminus of the scFv portion contains a hinge having an amino acid sequence of SEQ ID NO: 5 or of SEQ ID NO: 6.

6. Liver-specific CAR for use according to one of the preceding claims, characterized in that the CAR at the C-terminus of the scFv portion contains a hinge and a transmembrane domain having an amino acid sequence of SEQ ID NO: 7 or of SEQ ID NO: 8.

7. Liver-specific CAR for use according to one of the preceding claims, characterized in that the scFv portion is comprised in SEQ ID NO: 23, in SEQ ID NO: 24, in SEQ ID NO: 25, in SEQ ID NO: 26, or in SEQ ID NO: 27.

8. Liver-specific CAR for use according to one of the preceding claims, characterized in that the CAR is for use in humans and the scFv portion is one of scFv selected from SEQ ID NO: 26 (C3), SEQ ID NO: 24 (Cl 1), SEQ ID NO: 27 (BIO) and SEQ ID NO: 23 (G3).

9. Liver-specific CAR for use according to one of the preceding claims, characterized in that the CAR is for use in rodents and the scFv portion is one of scFv selected from SEQ ID NO: 26 (C3), SEQ ID NO: 24 (Cl 1), SEQ ID NO: 25 (E12) and SEQ ID NO: 23 (G3).

10. Liver-specific CAR for use according to one of the preceding claims, characterized in that a secretory signal peptide is arranged at the N-terminus of the scFv portion.

11. Regulatory T cell expressing a CAR for use in the treatment of an autoimmune disease directed against liver tissue, or for use in the treatment of inflammatory liver disorder in a patient, wherein the T cell is immunologically compatible with the patient, characterized in that the regulatory T cell is genetically manipulated to express a CAR according to one of the preceding claims.

12. Regulatory T cell for use according to claim 11, wherein the T cell is an autologous cell originating from the patient to be treated or the T cell is derived from an autologous cell originating from the patient to be treated.

13. Regulatory T cell for use according to one of claims 11 to 12, characterized in that the T cell in the presence of liver tissue has immune suppressive activity.

14. In vitro process for producing a regulatory T cell according to one of claims 11 to 13, characterized by introducing into a T cell a nucleic acid construct encoding a CAR according to one of claims 1 to 10.

15. In vitro process according to claim 14, characterized in that the nucleic acid construct comprises a portion encoding FOXP3, and the nucleic acid construct is introduced into CD4+ T-cells for converting the CD4+ T-cells into regulatory T-cells expressing the CAR.

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