Peptide conjugates and use thereof to promote cas nuclease immune tolerance in genome engineering gene therapy

Abstract

The present invention relates in general to the field of medicine. More particularly, it relates to polypeptide conjugates, compositions and methods for promoting immune tolerance to the CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein) system, for genome engineering gene therapy.

Description

[0001] DESCRIPTION

[0002] TITLE: PEPTIDE CONJUGATES AND THEIR USE TO PROMOTE IMMUNOTOLERANCY TO CAS NUCLEASES IN GENOME ENGINEERING GENE THERAPY

[0003] FIELD OF THE INVENTION

[0004] The present invention relates generally to the field of medicine. More particularly, it relates to polypeptide conjugates, compositions and methods for promoting immunotolerance to the CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated protein) system, for gene therapy by genome engineering.

[0005] PRIOR ART

[0006] Immunotolerance is a state of non-responsiveness of the immune system to substances or tissues that have the capacity to cause immune rejection in a healthy organism. It is induced by prior exposure to a specific antigen and contrasts with the conventional elimination of foreign bodies by the mechanisms of (innate) immunity. Immune tolerance is important for normal physiology. Central tolerance is the primary means by which the immune system learns to distinguish self from non-self. Peripheral tolerance is essential to prevent excessive reactivity of the immune system to various environmental entities (e.g., allergens, intestinal microbes, etc.) as well as to antigens produced by phagocytosis of dead body cells.Central or peripheral tolerance deficits are also at the origin of autoimmune diseases, leading to syndromes such as systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes or multiple sclerosis. However, recent discoveries establish that it is possible to induce peripheral immunotolerance, for example by targeting the entry of an antigen into antigen-presenting cells (APCs) through a receptor present on macrophages and dendritic cells (DCs) namely, the dendritic cell asialogly coprotein receptor (DC-ASGPR) (Li D et al., J Exp Med. 2012;209(l):109-121).

[0007] The CRISPR-Cas system of bacteria, archaea, and large bacteriophages is an adaptive defense system for the destruction of mobile genetic elements (Makarova, KS. et al. 2020. Nat Rev Microbiol 18(2):67-83), as well as the development of biotechnology tools for genome engineering of all cell types (Knott, GJ and Doudna, J. 2018 Science 361(6405):866-869). For this purpose, the use of different families of guide RNA-guided nucleases, such as Cas9 and Cas12 proteins, allows the recognition of a double-stranded DNA sequence and its cleavage at a specific location. Thus, nucleases programmed by a CRISPR-RNA or guide RNA for the recognition of a target sequence of nucleic acids are becoming one of the most effective biotechnological molecular compounds for genetic engineering and gene therapy for gene editing or the control of gene expression (FA Ran et al., Nature 520, 186-191 (2015); (Knott, GJ and Doudna, J. 2018 Science 361(6405):866-869). Cas nucleases cut DNA at a specific target sequence, guided by homology recognition between the guide RNA and a chromatin site. The DNA repair mechanism then makes it possible to modify a gene sequence for therapeutic purposes (FA Ran et al., Nature 520, 186-191 (2015)). In addition, some Cas nuclease mutants can act as a nickase, or lose their nuclease activity entirely and assume other functions such as chemical modification of DNA or gene transcription (Knott, GJ and Doudna, J. 2018 Science 361(6405):866-869). As CRISPR-Cas systems allow precise gene editing in eukaryotic cells, it opens up real prospects for in vivo gene repair and expression and makes it possible to envisage the cure of genetic or acquired diseases that are currently incurable.Thus, a first phase 11 trial has been launched, using Cas9 gene transfer into retinal photoreceptor cells with an AAV viral vector, to repair a mutant RPE65 gene causing Leber congenital amaurosis (A. Mullard, Nat. Rev. Drug Discov. 18, 656-656 (2019)). Furthermore, other clinical protocols for gene repair by in vivo nucleotide base editing are emerging to introduce a base change by a mutant Cas protein with nickase activity for the treatment of hyperlipidemia or sickle cell disease by modifying the Pcsk9 or hemoglobin genes, respectively (Ledford, H. Nature 2022). Finally, other clinical applications are also becoming possible with the use of Cas proteins without protease activity, but fused to a domain capable of directly modulating the transcription of target genes (Jensen, TI. 2021 Genome Res.31(11):2120-2130), or to an enzymatic domain that can modify the methylation of T DNA at a locus and abolish or durably activate its expression (Pulecio, J. 2017. Cell Stem Cell 21(4):431-447).

[0008] However, the use of Cas nucleases in medicine, for the genetic modification of organs in situ, is hampered by the prospect of mutagenesis following off-target Cas activity, or immune rejection of cells expressing this bacterial protein (CT Charlesworth, et al., Nat. Med. 25, 249-254 (2019)). To reduce off-target mutagenesis by Cas, high-fidelity variants of Cas9 and Casl2 have been developed (BP Kleinstiver, et al., Nature 529, 490-495 (2016) — IM Slaymaker, et al., Science 351, 84-8 (2016); Xiaoshu Xu, Augustine Chemparathy, et al. 2021. Mol Cell 81(20):4333-4345.e4; Pausch, P. Soczek, KM. 2021 Nat Struct & mol biol 28(8):652-661; Tsuchida, CA. Zhang, S. 2022 Mol Cell 82(6): 1199-1209. e6). However, the use of Cas proteins in medicine poses a major problem of immunogenicity, because approximately 80% of the population has humoral and cellular immunity against Cas9 orthologs of S. pyogenes and S.aureus, which are common human commensals (CT Charlesworth, et al, Nat. Med. 25, 249-254 (2019)). The risk of immune rejection is therefore not negligible and with it the failure of gene therapy based on the CRISPR-Cas system. This raises legitimate concerns about the use of Cas in gene therapy, since previous immunizations strongly compromise the effectiveness of in vivo genome editing in immunocompetent patients. Even if this disadvantage could be reduced through the occasional and ephemeral use of Cas proteins rarely in contact with humans, in the event of a second administration or a long-lasting expression of these proteins, the above problem would not be resolved.

[0009] BRIEF OVERVIEW OF THE INVENTION

[0010] Faced with this major challenge of being able to promise safe and effective in vivo gene therapy, the inventor has created new polypeptide conjugates, compositions and methods capable of inducing immune tolerance to the Cas protein, in particular Cas9, in the mammalian body. Thanks to these tools, it is now possible to prevent immune rejection of the body's cells expressing a Cas protein and to associate it with transient, inducible or long-lasting genetic expression of the Cas protein. Therefore, increasing immune tolerance to the Cas protein should lay the foundation for safe and effective protocols for gene repair or control of gene expression by the CRISPR-Cas system in humans.

[0011] DETAILED DESCRIPTION

[0012] POLYPEPTIDE CONJUGATES

[0013] According to a first aspect of the invention, the invention generally relates to a polypeptide conjugate comprising a first component which is an anti-antigen-presenting cell antibody which targets the asialoglycoprotein receptor present on APCs (anti-APC-ASGPR), or a fragment thereof, covalently or non-covalently linked to a second component which is a Cas protein, in particular a Cas9 protein, in particular that of Staphylococcus aureus (saCas9).The polypeptide conjugate of the invention having the capacity to induce immune tolerance to the Cas protein, in the organism of primates, one embodiment of the invention relates to a polypeptide conjugate comprising a first component which is an anti-antigen presenting cell antibody which targets the APC asialoglycoprotein receptor (anti-APC-ASGPR), or a fragment thereof capable of binding to the epitope recognized by the complete antibody, covalently or non-covalently linked to a second component which is a Cas protein, said polypeptide conjugate being capable of inducing immune tolerance to the Cas protein in the organism of mammals, and in particular in the organism of primates and humans.

[0014] In view of the above, it is also understood that according to a particular embodiment the invention relates to a polypeptide conjugate comprising a first component which is an anti-antigen presenting cell antibody which targets the APC asialoglycoprotein receptor (anti-APC-ASGPR), or a fragment thereof capable of binding to the epitope recognized by the full antibody, covalently or non-covalently linked to a second component which is a Cas9 protein, said polypeptide conjugate being capable of inducing immune tolerance to the Cas9 protein in the organism of mammals, and in particular in the organism of primates and humans. By "Cas protein" is generally meant the CRISPR associated protein (Clustered Regularly Interspaced Short Palindromic Repeats associated protein) and by "Cas9 protein" is meant the CRISPR associated protein 9 (Clustered Regularly Interspaced Short Palindromic Repeats associated protein 9).These correspond to the nucleases of the CRISPR-Cas system, which constitutes a prokaryotic adaptive defense mechanism for destroying invading foreign DNA. A great diversity of CRISPR-Cas systems exists among bacteria and archaea, and even within the same species. This implies divergence in sequence homology, organization and size of the genes and proteins constituting the CRISPR-Cas system and their ability to cleave single-stranded or double-stranded DNA and RNA. Nevertheless, the different elements of CRISPR-Cas systems and their functional logic are similar throughout the kingdom Prokaryota.

[0015] The CRISPR-Cas system is composed of a family of sequences present in the genomes of bacteria, archaea, and large bacteriophages that correspond to genes that perform adaptation, rcRNA processing, and interference functions. To interfere with the expression and replication of foreign DNA or RNA, the host expresses a Cas nuclease gene, the sequence motif in the exogenous DNA or RNA, acquired during previous encounters between bacteria and phages, plasmids, or mobile genetic elements, that encodes the non-coding rcRNA, and a sequence expressing the transactivating rcRNA (trcRNA). The processed rcRNA and trcRNA form a duplex guide RNA (gRNA) that guides the Cas nuclease to a target DNA sequence for double-strand cleavage. DNA cutting is limited by an adjacent DNA motif, called a protospacer adjacent motif (P AM), which is specific to each CRISPR-Cas system.

[0016] This functional framework has since been diverted from its initial function of prokaryotic immunity and is now applied in biotechnology for the targeting, processing, modification, destruction and programmed repair of genes. To date, several Cas proteins (Cas9, Casl2a, Casl2b, CasX or Casl2e, Casl2f or Casl4, Casl2j or Cas®) from different hosts have been identified and characterized, or even modified (e.g., improvement of their function), which can be implemented by the invention, for the engineering of mammalian genomes. In general, the Cas proteins mentioned above have decreasing sizes of 1,500 amino acids (AA) for Cas9, 1,000 AA for Casl2a and less than 1,000 AA for CasX, Casl2f and Casl2j. It is therefore understood that according to another embodiment, the subject of the invention is the polypeptide conjugate as described above, in which said Cas protein is chosen from:

[0017] - Cas9, Cas 12a, Cas 12b, CasX or Cas 12e, CRISPR type V, Cas 12j or Cas® nucleases;

[0018] - Cas9, Cas 12a, Cas 12b, CasX or Cas 12e, CRISPR type V, Cas 12j or Cas® orthologs; and

[0019] - Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas® mutants or functional variants.

[0020] By "Cas9 nucleases" is meant in particular those of S. pyogenes, S. aureus, C. diphtheriae, N meningitidis, S. canis, S. macacae, F. tularensis, Acidaminococcus, C. jejuni, S. pneumoniae and S. thermophilus. More particularly still, it is a question of the Cas9 of sequences SEQ ID NOs: 87 to 95 and the mutants thereof. In view of the above, it is understood that according to another embodiment, the invention relates to the polypeptide conjugate as described above, in which said Cas protein is a Cas9 portein chosen from:

[0021] - Cas9 nucleases from S. pyogenes, S. aureus, C. diphtheriae, N meningitidis, S. canis, S. macacae, F. tularensis, Acidaminococcus, C. jejuni, S. pneumoniae and S. thermophilus; And

[0022] - Cas9 orthologs and Cas9 mutants or functional variants derived from these organisms, and in particular said Cas9 protein is chosen from the sequences SEQ ID NOs: 87 to 95 and the mutants thereof.

[0023] By "Cas 12a (Cpfl) nucleases" is meant in particular those of Lachnospiraceae bacterium or Acidaminococcus sp, wild or improved, carrying the mutations E174R / S542R or E174R / S542R / K548R. More particularly, it is a question of Casl2a of sequences SEQ ID NOs: 105 and 106, and the mutants thereof.

[0024] By "Casl2b nucleases" is meant in particular those derived from Alicyclobacillus kakegawensis (AkCasl2b) or Bacillus hisashii (BhCasl2b), and an improved mutant version for gene editing of BhCasl2b (K846R / S893R / E837G). More particularly, it refers to Cas 12b of sequence SEQ ID NO: 107 and mutants thereof. By "CasX or Casl2e nucleases" is meant in particular those derived from Deltaproteobacteria (DpbCasX) or Planctomycetes (PlmCasX), wild type or deactivated by the mutations N672A, E769A and N935A, or improved for gene editing in mammalian cells such as the DpbCasX_R3 V2 or PlmCasX Rl V2 versions. More particularly, it is a question of Casl2e of sequences SEQ ID NOs: 112 and 113, and the mutants thereof.

[0025] By "Type V CRISPR nucleases" are meant nucleases having a reduced size (400 to 700 AA) compared to Cas9, Casl2a and CasX nucleases. These include the Casl2f (or Cas 14) proteins from uncultured archaea (UnlCasl2fl), Syntrophomonas palmitatica (SpCasl2fl) or Acidibacillus sulfuroxidans (AsCasl2fl), either wild or carrying one or more mutations abolishing the catalytic activity (D225A and E324A) or partially leading to the production of a nickase (R383A and D401 A). More particularly, this refers to Casl2f or Cas 14 of sequences SEQ ID NOs: 108 to 111 and the mutants thereof.

[0026] By "Cas 12j or CasP nucleases" we mean in particular those of the Biggiephage clade which is also a CRIPR-Cas type V.

[0027] In view of the above, it is also understood that according to another embodiment the subject of the invention is the polypeptide conjugate as described above, in which said Cas protein is chosen from:

[0028] - Cas9, Cas 12a, Cas 12b, CasX or Cas 12e, CRISPR type V (Casl2f or Cas 14), Cas 12j or Cas® nucleases;

[0029] - Cas9, Cas 12a, Cas 12b, CasX or Cas 12e, CRISPR type V (Casl2f or Cas 14), Cas 12j or Cas® orthologs; and

[0030] - the mutants or functional variants of Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V (Casl2f or Cas 14), Cas 12j or Cas®, and in particular said Cas protein is chosen from the sequences SEQ ID NOs: 87 to 95 and 105 to 113, and the mutants thereof.

[0031] Orthologs are similar Cas proteins found in two or more different species. Mutants are Cas proteins that have one or more mutations, including the deletion, substitution, and / or addition of one or more amino acids. These mutations can either increase or abolish nuclease activity, or increase or decrease the fidelity of target DNA recognition.

[0032] By "functional mutants" we mean Cas proteins modified by human intervention (e.g., by genetic engineering), in order to, / ?, e.g., increase Cas activity.

[0033] By "functional variants" we mean Cas proteins that have been naturally modified through evolution, which exhibit, / ?, e.g., increased (or decreased) activity.

[0034] In particular, the subject of the invention is the polypeptide conjugate as described above, in which said Cas protein is chosen from:

[0035] - Cas9 nucleases from S. pyogenes, S. aureus, C. diphtheriae, N. meningitidis, S. canis, S. macacae, F. tularensis, Acidaminococcus, C. jejuni, S. pneumoniae and S. thermophilus, ' Caslla nucleases from Lachnospiraceae bacterium and Acidaminococcus sp, ' Casllb nucleases from Alicyclobacillus kakegawensis and Bacillus hisashii, 'CasX or Cas 12e nucleases from Deltaproteobacteria and Planctomycetes,' type V CRISPR nucleases from Syntrophomonas palmitatica and Acidibacillus sulfuroxidans, 'Cas 12j or Cas® nucleases from the Biggiephage clade, -

[0036] - Cas9, Cas 12a, Cas 12b, CasX or Cas 12e, CRISPR type V, Cas 12j or Cas® orthologs derived from these organisms; and

[0037] - the Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas® mutants or functional variants derived from these organisms, and in particular said Cas protein is chosen from the sequences SEQ ID NOs: 87 to 95 and 105 to 113, and the mutants thereof.

[0038] In particular, the subject of the invention is the polypeptide conjugate as described above, in which said Cas protein is chosen from the sequences SEQ ID NOs: 87 to 95 and 105 to 113, and the mutants thereof. In particular, the subject of the invention is the polypeptide conjugate as described above, in which said Cas9 protein is chosen from the sequences SEQ ID NOs: 87 to 95 and the mutants thereof. More particularly still, the subject of the invention is a polypeptide conjugate as described above, in which said Cas protein is chosen from the sequences SEQ ID NOs: 87, 88, 89, 90, 91, 92, 93, 94, 95, 105, 106, 107, 108, 109, 110, 111, 112 and 113.

[0039] According to another embodiment, the subject of the invention is the polypeptide conjugate as described above, in which said Cas protein is chosen from:

[0040] - Cas9 nucleases from S. pyogenes, S. aureus, C. diphtheriae, N meningitidis, S. canis, S. macacae, F. tularensis, Acidaminococcus, C. jejuni, S. pneumoniae and S. thermophilus;

[0041] - the nucleases Casl2a from Lachnospiraceae bacterium, Casl2b from Bacillus hisashii, Casl2f (Cas 14) from uncultured Archaea and Cas 12j (Cas®) from the Baggiephage clade; And

[0042] - the orthologs Cas9, Cas 12a, Cas 12b Casl2f and Casl2j, and the mutants or functional variants Cas9, Casl2a, Casl2b, Casl2f and Casl2j derived from these organisms, and in particular said Cas protein is chosen from the sequences SEQ ID NOs: 87 to 95 and 105 to 113, and the mutants thereof.

[0043] For example, there are mutants (variants) of S. pyogenes Cas9 (spCas9), a representative sequence of which is SEQ ID NO: 87, in which amino acids at particular positions are modified, abolishing nuclease activity and increasing the fidelity of recognition of the target DNA sequence. These mutations may be the following: substitution of amino acids D10, E762, D839, H840, H863, H983 and / or D986 with a different amino acid such as alanine or with any other amino acid other than the native amino acid, which reduces, substantially eliminates or abolishes nuclease activity. Other amino acid substitutions may increase the specificity of recognition of the target DNA sequence, thereby decreasing off-target binding to other DNA sequences with some homology to the one being targeted.In spCas9, these mutations can be present at one, two, three, four, five, six, and / or all seven of the following positions: L169, Y450, N497, R661, Q695, Q926, and / or DI 135, which give rise to what is called a high-fidelity Cas9 (hifi spCas9) (Kleinstiver, BP et al., Nature. 2016;529(7587):490-495). Other different mutations have also been shown to increase the specificity of spCas9, namely the substitutions of amino acids K855, K810 / K1003 / R1060, or K848 / K1003 / R1060 with alanine (IM Slaymaker, et al., Science 351, 84-8 (2016)). Finally, another set of spCas9 mutations also significantly increase the accuracy of spCas9 binding to the target DNA sequence; these combine the substitutions N692A, M694A, Q695A, and H698A, and constitute the hyperprecise HyppaCas9 (Chen, JS et al., Nature. 2017;550(7676):407-410).Moreover, all mutations that abolish the catalytic activity of spCas9 can be associated with this increasing specificity of DNA sequence recognition.

[0044] Similarly, there are mutants (variants) of S. aureus Cas9 (saCas9), a representative sequence of which is SEQ ID NO: 88, in which amino acid changes, D10A or N580A, disable the RuvC and HNH nuclease domains, respectively, and convert saCas9 into a nickase (Friedland, AE et al., Genome Biol. 2015;16:257). High-fidelity saCas9 can be obtained by mutagenesis, and the mutations Y211A, Y212A, W229A, Y230, R245A, T392A, N413A, N419A, Y651A, R654A, alone or in combination, promote higher on / off-target ratios than wild-type saCas9 (Tan, Y et al., Proc Natl Acad Sci US A. 2019; 116(42):20969-20976). In addition, the saCas9 triple mutants E782K / N968K / R1015H and E782K / K929R / R1015H exhibit broader site recognition, as they can cut a sequence followed by a simpler PAM "NNNRRT", instead of the "NNGRRT" motif.Finally, combinations of the above-mentioned mutations may allow for mixed functional enhancements such as a high-fidelity saCas9 nuclease or nickase with broader PAM recognition.

[0045] Furthermore, there are organisms in nature encoding Cas nucleases smaller than Cas9, Casl2a (Cpfl) or Casl2b (1000-1500 AA). This is the case for CasX or Casl2e nucleases (less than 1,000 AA), Casl2f or Casl4 (400-700 AA) and Casl2j or Cas(|) (700-800 AA), providing a natural reservoir of compact Cas allowing the efficient engineering of mammalian cells (Tsuchida, CA, et al. 2022 Mol. Cell. 82(6): 1199-1209. e6; Do Yon Kim, et al. 2022, Nature Biotechnology. 40(l):94-102; Wu Zhaowei, et al. 2021 Nat. Chem. Biol. 17(11): 1132-1138; Xu X, Chemparathy A, et al. Mol Cell. 2021 Oct 21;81(20):4333-4345. e4. ; Pausch P, et al Science. 2020 Jul 17;369(6501):333-337.).CasX nucleases such as those derived from Deltaproteobacteria (DpbCasX) or Planctomycetes (PlmCasX), in their wild-type version, or improved for gene editing such as DpbCasX-R3 (chimeric DpbCasX containing the R3 loop of PlmCasX), or PlmCasX-Rl (chimeric PlmCasX containing the RI loop of DpbCasX), or even versions of CasX deactivated by the mutations N672A, E769A and N935A alone or in combination, allow efficient programmed engineering of mammalian cells, either to induce DNA breaks or to modulate the expression of target genes ((Tsuchida, CA, et al. 2022 Mol. Cell. 82(6): 1199-1209. e6 ; Do Yon Kim, et al. 2022, Nature Biotechnology. 40(l):94-102; Liu, Jun-Jie, et al. 2019 Nature. 566(7743):218-223).Casl2f nucleases CRISPR type V nucleases are smaller in size (400 to 700 AA) compared to Cas9, Casl2a and CasX nucleases; these are, for example, the Casl2f (or Cas 14) proteins of uncultured archaea (UnlCasl2fl), Syntrophomonas palmitatica (SpCasl2fl) or Acidibacillus sulfuroxidans (AsCasl2fl), either wild or carrying one or more mutations completely abolishing the catalytic activity of the Casl2f nuclease (D225A and E324A for Uni Cas 12fl, or D326A and / or D510A for UnlCasl2fl), or partially abolishing the nuclease activity leading to the production of a nickase (R383A and D401A for AsCasl2fl) (Do Yon Kim, et al. 2022, Nature Biotechnology. 40(l):94-102; Wu Zhaowei, et al. 2021 Nat. Chem. Biol. 17(11): 1132-1138; Xiaoshu Xu et al. 2021 Mol Cell. 81(20):4333-4345.e4).Thus, catalytic mutants of Casl2f can be fused to transcription activator or inhibitor domains to program the Casl2f protein for the targeted expression of certain cellular genes, but also to an enzymatic domain of a base editor or deoxyadenosine deaminase allowing the transformation of A or T bases into G or C in a targeted manner, for the repair or introduction of point mutations in the genome of a mammalian cell (Do Yon Kim, et al. 2022, Nature Biotechnology. 40(l):94-102; Xiaoshu Xu et al. 2021 Mol Cell. 81(20):4333-4345.e4). The bacteriophage Casl2j or Cas® nuclease is capable of introducing a double-strand break on a DNA sequence recognized by a guide RNA.The rate of this cleavage can be accelerated approximately 20-fold by mutations introduced into helix α 7 (E159A, S160A, S164A, D167A, E168A) or the substitution of a negatively charged fragment by a short succession of glycine-serines (Pausch, P. et al. 2021, Nature Structural & molecular biology. 28(8):652-661).

[0046] Generally, the invention may therefore relate to a polypeptide conjugate comprising a first component which is an anti-antigen presenting cell antibody which targets the asialoglycoprotein receptor (anti-APC-ASGPR), or a fragment thereof, covalently or non-covalently linked to a second component which is one of the aforementioned Cas proteins.

[0047] The term "antigen-presenting cell", abbreviated to APC (antigen-presenting cell), means a cell of the immune system that presents parts of cellular elements to T lymphocytes. These may be monocytes, macrophages, B lymphocytes or dendritic cells. In particular, they are dendritic cells (DC). Also and according to a particular embodiment, the subject of the invention is a polypeptide conjugate comprising a first component which is an anti-dendritic cell antibody that targets the asialoglycoprotein receptor (anti-DC-ASGPR), or a fragment thereof, covalently or non-covalently linked to a second component which is a Cas protein.

[0048] Asialoglycoprotein receptor, abbreviated ASGPR and also called CLEC10A, refers to a C-type lectin receptor (CLR) expressed on antigen-presenting cells (e.g., human dendritic cells (DCs)). CLRs enable APCs to capture and internalize antigens, particularly glycosylated antigens, allowing their further processing and presentation on major histocompatibility complex (MHC) molecules. In addition, several CLRs, including ASGPR, can initiate signaling cascades and modulate dendritic cell function, with consequences for the induced immune response. Unlike other Syk-associated CLRs, activated ASGPR does not induce NF-κB activation, but rather leads to CREB phosphorylation (Gu C et al., J Immunol. 2019;203(2):389-399).

[0049] Thus, the signaling cascade of activated ASGPR results in the activation of Syk, PLCy2, PKCô and then MAPK ERK 1 / 2 and JNK, which leads, when the stimulation is prolonged, to the phosphorylation of p90RSK and CREB, inducing the transcription of the anti-inflammatory cytokine IL-10, which is a particular terminal result of the endocytosis of an antigen by DC-ASGPR.

[0050] By "antibody" is meant an immunoglobulin, a multimeric protein consisting of 4 chains participating in the acquired immune response. Immunoglobulins are well known to those skilled in the art and consist of an assembly of two dimers each consisting of a heavy chain and a light chain. The multimeric complex is assembled by the bonding of a light chain and a heavy chain by a disulfide bridge between two cysteines, the two heavy chains also being linked together by two disulfide bridges.

[0051] Each of the heavy and light chains consists of a constant region and a variable region. The assembly of the chains that make up an antibody allows for the definition of a characteristic three-dimensional Y-shaped structure, where,

[0052] - the base of the Y corresponds to the Fc constant region which is recognized by complement and Fc receptors, and - the end of the arms of the Y correspond to the respective assembly of the variable regions, the light chain and the heavy chain.

[0053] More specifically, each light chain consists of a variable region (VL) and a constant region (CL). Each heavy chain consists of a variable region (Vu) and a constant region consisting of three constant domains: CH1, CH2, and CH3. The CH2 and CH3 domains make up the Fc domain.

[0054] The light chain variable region consists of three antigen recognition determining regions (CDRs) surrounded by four framework domains. The heavy chain variable region also consists of three antigen recognition determining regions (CDRs) surrounded by four framework domains. The three-dimensional folding of these variable regions is such that all six CDRs are exposed on the same side of the protein and allow the formation of a specific structure recognizing a specific antigen.

[0055] The antibodies described in the invention are isolated and purified, can belong to any isotype / class (e.g. IgG, IgE, IgM, IgD, IgA and IgY) or to a subclass (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) and are different from natural antibodies. These antibodies are mature, i.e. they possess an ad hoc three-dimensional structure allowing them to recognize the antigen, and possess all the / wV-translational modifications essential for their antigenic recognition, in particular glycosylation and the formation of intra- and intermolecular disulfide bridges.

[0056] More specifically, these are "monoclonal antibodies", meaning that they only recognize a single antigenic determinant of DC-ASGPR, unlike polyclonal antibodies which correspond to a mixture of antibodies, and therefore can recognize several antigenic determinants of the same protein.

[0057] By "fragment thereof" is meant any portion of the antibody of the invention that retains the ability to bind to the epitope recognized by the full-length antibody. Examples of such fragments include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFvs), single-chain antibodies, disulfide-linked Fvs (dsFvs), and fragments comprising the VL or VH region. Epitope-binding fragments, including single-chain antibodies, may comprise the variable region(s) alone or in combination with all or part of the following: hinge region, CHI, CH2, and CH3 domains. Such fragments may contain one or both Fab fragments or the F(ab')2 fragment. In addition, the fragments may be or may combine members of any of the following immunoglobulin classes: IgG, IgM, IgA, IgD or IgE and their subclasses.

[0058] Fab and F(ab')2 fragments can be produced by proteolytic cleavage, using enzymes such as papain (Fab fragment) or pepsin (F(ab')2 fragment). "Single-chain Fv" fragments ("scFv") are epitope-binding fragments that contain at least one fragment of an antibody variable region (VH) linked to at least one fragment of a light-chain antibody variable region (VL). The linker can be a short, flexible peptide chosen to ensure that the correct three-dimensional folding of the VL and VH regions occurs once they are linked, so as to maintain the target molecule-binding specificity of the entire antibody from which the single-chain antibody fragment is derived. The carboxyl terminus of the VL or VH sequence can be covalently linked by a linker to the amino acid terminus of a complementary VL or VH sequence.

[0059] Also, by "anti-antigen-presenting cell antibody that targets the APC asialoglycoprotein receptor (anti-DC-ASGPR), or a fragment thereof," we mean proteins capable of specifically recognizing DC-ASGPR. Among them, seven monoclonal antibodies recognizing human DC-ASGPR can be cited: 49C11, 49C1 I bis, lHl l, 5F10, 4G2.2, 6.3H9.1Dl l and 5H8.1D4.

[0060] By “49C11” antibody is meant an antibody comprising:

[0061] - a heavy chain of sequence SEQ ID NO: 10, which is a synthetic construct comprising: o the murine variable region targeting the human DC-ASGPR of sequence SEQ ID NO: 4 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 1, the CDR2 of sequence SEQ ID NO: 2 and the CDR3 of sequence SEQ ID NO: 3; and o a fusion protein with the constant region of the heavy chain of human IgG4; and

[0062] - a light chain of sequence SEQ ID NO: 25, which is a synthetic construct comprising: o the murine variable region targeting the human DC-ASGPR of sequence SEQ ID NO: 19 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 16, the CDR2 of sequence SEQ ID NO: 17 and the CDR3 of sequence SEQ ID NO: 18; and o a fusion protein with the constant region of the Kappa chain of human IgG4.

[0063] From the above 49C11 antibody 5 variants have also been developed namely:

[0064] - 49Cl l_varl comprising: o a heavy chain of sequence SEQ ID NO: 11 comprising the variable region of sequence SEQ ID NO: 5 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 1, the CDR2 of sequence SEQ ID NO: 2 and the CDR3 of sequence SEQ ID NO: 3; and o a light chain of sequence SEQ ID NO: 26 comprising the variable region of sequence SEQ ID NO: 20 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 16, the CDR2 of sequence SEQ ID NO: 17 and the CDR3 of sequence SEQ ID NO: 18;

[0065] - 49C1 l_var2 comprising: o a heavy chain of sequence SEQ ID NO: 12 comprising the variable region of sequence SEQ ID NO: 6 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 1, the CDR2 of sequence SEQ ID NO: 2 and the CDR3 of sequence SEQ ID NO: 3; and o a light chain of sequence SEQ ID NO: 27 comprising the variable region of sequence SEQ ID NO: 21 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 16, the CDR2 of sequence SEQ ID NO: 17 and the CDR3 of sequence SEQ ID NO: 18;

[0066] - 49Cl l_var3 comprising: o a heavy chain of sequence SEQ ID NO: 13 comprising the variable region of sequence SEQ ID NO: 7 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 1, the CDR2 of sequence SEQ ID NO: 2 and the CDR3 of sequence SEQ ID NO: 3; and o a light chain of sequence SEQ ID NO: 28 comprising the variable region of sequence SEQ ID NO: 22 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 16, the CDR2 of sequence SEQ ID NO: 17 and the CDR3 of sequence SEQ ID NO: 18;

[0067] - 49C1 l_var4 comprising: o a heavy chain of sequence SEQ ID NO: 14 comprising the variable region of sequence SEQ ID NO: 8 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 1, the CDR2 of sequence SEQ ID NO: 2 and the CDR3 of sequence SEQ ID NO: 3; and o a light chain of sequence SEQ ID NO: 29 comprising the variable region of sequence SEQ ID NO: 23 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 16, the CDR2 of sequence SEQ ID NO: 17 and the CDR3 of sequence SEQ ID NO: 18; and

[0068] - 49C1 l_var5 comprising: o a heavy chain of sequence SEQ ID NO: 15 comprising the variable region of sequence SEQ ID NO: 9 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 1, the CDR2 of sequence SEQ ID NO: 2 and the CDR3 of sequence SEQ ID NO: 3; and o a light chain of sequence SEQ ID NO: 30 comprising the variable region of sequence SEQ ID NO: 24 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 16, the CDR2 of sequence SEQ ID NO: 17 and the CDR3 of sequence SEQ ID NO: 18.

[0069] By “49C11 bis” antibody is meant an antibody comprising:

[0070] - a heavy chain of sequence SEQ ID NO: 32 comprising the variable region of sequence SEQ ID NO: 31 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 1, the CDR2 of sequence SEQ ID NO: 2 and the CDR3 of sequence SEQ ID NO: 3; and

[0071] - a light chain of sequence SEQ ID NO: 34 comprising the variable region of sequence SEQ ID NO: 33 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 16, the CDR2 of sequence SEQ ID NO: 17 and the CDR3 of sequence SEQ ID NO: 18.

[0072] By “1H11” antibody is meant an antibody comprising:

[0073] - a heavy chain of sequence SEQ ID NO: 39 comprising the variable region of sequence SEQ ID NO: 38 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 35, the CDR2 of sequence SEQ ID NO: 36 and the CDR3 of sequence SEQ ID NO: 37; and - a light chain of sequence SEQ ID NO: 44 comprising the variable region of sequence SEQ ID NO: 43 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 40, the CDR2 of sequence SEQ ID NO: 41 and the CDR3 of sequence SEQ ID NO: 42.

[0074] By “5F 10” antibody is meant an antibody comprising:

[0075] - a heavy chain of sequence SEQ ID NO: 49 comprising the variable region of sequence SEQ ID NO: 48 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 45, the CDR2 of sequence SEQ ID NO: 46 and the CDR3 of sequence SEQ ID NO: 47; and

[0076] - a light chain of sequence SEQ ID NO: 54 comprising the variable region of sequence SEQ ID NO: 53 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 50, the CDR2 of sequence SEQ ID NO: 51 and the CDR3 of sequence SEQ ID NO: 52.

[0077] By “4G2.2” antibody is meant an antibody comprising:

[0078] - a heavy chain of sequence SEQ ID NO: 59 comprising the variable region of sequence SEQ ID NO: 58 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 55, the CDR2 of sequence SEQ ID NO: 56 and the CDR3 of sequence SEQ ID NO: 57; and

[0079] - a light chain of sequence SEQ ID NO: 64 comprising the variable region of sequence SEQ ID NO: 63 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 60, the CDR2 of sequence SEQ ID NO: 61 and the CDR3 of sequence SEQ ID NO: 62.

[0080] By “6.3H9.1D11” antibody is meant an antibody comprising:

[0081] - a heavy chain of sequence SEQ ID NO: 69 comprising the variable region of sequence SEQ ID NO: 68 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 65, the CDR2 of sequence SEQ ID NO: 66 and the CDR3 of sequence SEQ ID NO: 67; and

[0082] - a light chain of sequence SEQ ID NO: 74 comprising the variable region of sequence SEQ ID NO: 73 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 70, the CDR2 of sequence SEQ ID NO: 71 and the CDR3 of sequence SEQ ID NO: 72. By antibody “5H8.1D4” is meant an antibody comprising:

[0083] - a heavy chain of sequence SEQ ID NO: 79 comprising the variable region of sequence SEQ ID NO: 78 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 75, the CDR2 of sequence SEQ ID NO: 76 and the CDR3 of sequence SEQ ID NO: 77; and

[0084] - a light chain of sequence SEQ ID NO: 84 comprising the variable region of sequence SEQ ID NO: 83 comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 80, the CDR2 of sequence SEQ ID NO: 81 and the CDR3 of sequence SEQ ID NO: 82. In summary of the above, said anti-DC-ASGPR according to a particular embodiment of the invention has the following sequences:

[0085] Table 1. Anti-DC-ASGPR & corresponding sequences

[0086] It is therefore understood that according to another embodiment, the subject of the invention is the polypeptide conjugate as described above comprising a first component which is an anti-antigen presenting cell antibody which targets the asialogly coprotein receptor of antigen presenting cells (anti-DC-ASGPR), or a fragment thereof capable of binding to the epitope recognized by the complete antibody, covalently or non-covalently bound to a second component which is a Cas protein, said polypeptide conjugate being capable of inducing immune tolerance to the Cas protein in the organism of mammals, in particular in the organism of primates and humans, and said anti-DC-ASGPR being chosen from:

[0087] - an antibody comprising: o a heavy chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 1, the CDR2 of sequence SEQ ID NO: 2 and the CDR3 of sequence SEQ ID NO: 3; and o a light chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 16, the CDR2 of sequence SEQ ID NO: 17 and the CDR3 of sequence SEQ ID NO: 18;

[0088] - an antibody comprising: o a heavy chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 35, the CDR2 of sequence SEQ ID NO: 36 and the CDR3 of sequence SEQ ID NO: 37; and o a light chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 40, the CDR2 of sequence SEQ ID NO: 41 and the CDR3 of sequence SEQ ID NO: 42;

[0089] - an antibody comprising: o a heavy chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 45, the CDR2 of sequence SEQ ID NO: 46 and the CDR3 of sequence SEQ ID NO: 47; and o a light chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 50, the CDR2 of sequence SEQ ID NO: 51 and the CDR3 of sequence SEQ ID NO: 52;

[0090] - an antibody comprising: o a heavy chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 55, the CDR2 of sequence SEQ ID NO: 56 and the CDR3 of sequence SEQ ID NO: 57; and o a light chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 60, the CDR2 of sequence SEQ ID NO: 61 and the CDR3 of sequence SEQ ID NO: 62;

[0091] - an antibody comprising: o a heavy chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 65, the CDR2 of sequence SEQ ID NO: 66 and the CDR3 of sequence SEQ ID NO: 67; and o a light chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 70, the CDR2 of sequence SEQ ID NO: 71 and the CDR3 of sequence SEQ ID NO: 72; and

[0092] - an antibody comprising: o a heavy chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 75, the CDR2 of sequence SEQ ID NO: 76 and the CDR3 of sequence SEQ ID NO: 77; and o a light chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 80, the CDR2 of sequence SEQ ID NO: 81 and the CDR3 of sequence SEQ ID NO: 82.

[0093] According to another embodiment, the subject of the invention is the polypeptide conjugate as described above, in which said anti-DC-ASGPR is chosen from:

[0094] - an antibody comprising a heavy chain comprising the variable region of sequence SEQ ID NO: 4, 5, 6, 7, 8, 9 or 31 and a light chain comprising the variable region of sequence SEQ ID NO: 19, 20, 21, 22, 23, 24 or 33;

[0095] - an antibody comprising a heavy chain comprising the variable region of sequence SEQ ID NO: 38 and a light chain comprising the variable region of sequence SEQ ID NO: 43;

[0096] - an antibody comprising a heavy chain comprising the variable region of sequence SEQ ID NO: 48 and a light chain comprising the variable region of sequence SEQ ID NO: 53;

[0097] - an antibody comprising a heavy chain comprising the variable region of sequence SEQ ID NO: 58 and a light chain comprising the variable region of sequence SEQ ID NO: 63;

[0098] - an antibody comprising a heavy chain comprising the variable region of sequence SEQ ID NO: 68 and a light chain comprising the variable region of sequence SEQ ID NO: 73; and an antibody comprising a heavy chain comprising the variable region of sequence SEQ ID NO: 78 and a light chain comprising the variable region of sequence SEQ ID NO: 83.

[0099] According to another embodiment, the subject of the invention is the polypeptide conjugate as described above, in which said anti-DC-ASGPR is chosen from:

[0100] - an antibody comprising a heavy chain of sequence SEQ ID NO: 10, 11, 12, 13, 14, 15 or 32 and a light chain of sequence SEQ ID NO: 25, 26, 27, 28, 29, 30 or 34;

[0101] - an antibody comprising a heavy chain of sequence SEQ ID NO: 39 and a light chain of sequence SEQ ID NO: 44;

[0102] - an antibody comprising a heavy chain of sequence SEQ ID NO: 49 and a light chain of sequence SEQ ID NO: 54;

[0103] - an antibody comprising a heavy chain of sequence SEQ ID NO: 59 and a light chain of sequence SEQ ID NO: 64;

[0104] - an antibody comprising a heavy chain of sequence SEQ ID NO: 69 and a light chain of sequence SEQ ID NO: 74; and

[0105] - an antibody comprising a heavy chain of sequence SEQ ID NO: 79 and a light chain of sequence SEQ ID NO: 84.

[0106] On this point and with regard to the sequences linked to the antibodies 49C11, 49Cl l_varl, 49Cl l_var2, 49Cl l_var3, 49Cl l_var4, 49Cl l_var5 and 49Cl l_bis, the heavy and light chains respectively sharing the same CDRs (see Table 1), it is possible to interchange them to develop new antibodies. Also, and by the expression "an antibody comprising a heavy chain comprising the variable region of sequence SEQ ID NO: 4, 5, 6, 7, 8, 9 or 31 and a light chain comprising the variable region of sequence SEQ ID NO: 19, 20, 21, 22, 23, 24 or 33", we mean both the antibody comprising:

[0107] - a heavy chain comprising the variable region of sequence SEQ ID NO: 4 and a light chain comprising the variable region of sequence SEQ ID NO: 19;

[0108] - a heavy chain comprising the variable region of sequence SEQ ID NO: 5 and a light chain comprising the variable region of sequence SEQ ID NO: 20;

[0109] - a heavy chain comprising the variable region of sequence SEQ ID NO: 6 and a light chain comprising the variable region of sequence SEQ ID NO: 21; - a heavy chain comprising the variable region of sequence SEQ ID NO: 7 and a light chain comprising the variable region of sequence SEQ ID NO: 22;

[0110] - a heavy chain comprising the variable region of sequence SEQ ID NO: 8 and a light chain comprising the variable region of sequence SEQ ID NO: 23;

[0111] - a heavy chain comprising the variable region of sequence SEQ ID NO: 9 and a light chain comprising the variable region of sequence SEQ ID NO: 24; and

[0112] - a heavy chain comprising the variable region of sequence SEQ ID NO: 31 and a light chain comprising the variable region of sequence SEQ ID NO: 33; that the antibody, for example, comprises:

[0113] - a heavy chain comprising the variable region of sequence SEQ ID NO: 4 and a light chain comprising the variable region of sequence SEQ ID NO: 20;

[0114] - a heavy chain comprising the variable region of sequence SEQ ID NO: 7 and a light chain comprising the variable region of sequence SEQ ID NO: 19;

[0115] - a heavy chain comprising the variable region of sequence SEQ ID NO: 6 and a light chain comprising the variable region of sequence SEQ ID NO: 24;

[0116] - a heavy chain comprising the variable region of sequence SEQ ID NO: 6 and a light chain comprising the variable region of sequence SEQ ID NO: 33;

[0117] - etc.

[0118] Equivalently, the expression "an antibody comprising a heavy chain of sequence SEQ ID NO: 10, 11, 12, 13, 14, 15 or 32 and a light chain of sequence SEQ ID NO: 25, 26, 27, 28, 29, 30 or 34" means both an antibody comprising the heavy and light chains of respective sequences SEQ ID NOs: 10 and 25, 11 and 26, 12 and 27, 13 and 28, 14 and 29, 15 and 30 or 32 and 34, and an antibody comprising the heavy and light chains of respective sequences SEQ ID NOs: 10 and 30, 10 and 29, 13 and 25, 14 and 26, etc.

[0119] In general, the invention may therefore also relate to a polypeptide conjugate comprising a first component which is an anti-antigen presenting cell antibody that targets the asialogly coprotein receptor (anti-DC-ASGPR) as described above, or a fragment thereof as described above, covalently or non-covalently linked to a second component which is one of the aforementioned Cas proteins. By "covalently linked" is meant that the polypeptide conjugate of the invention may be covalently linked, i.e. the first and second components are linked by a covalent bond (with or without a linker), also called a molecular bond, which is a strong chemical bond that involves the sharing of electron pairs between the atoms of said first and second components.The invention therefore relates to the polypeptide conjugate as described above, wherein said anti-DC-ASGPR is covalently linked to said Cas protein. In particular, the invention relates to the polypeptide conjugate as described above, wherein the anti-antigen presenting cell antibody that targets the asialoglycoprotein receptor (anti-DC-ASGPR) as described above is covalently linked via its heavy chain to a second component which is one of the aforementioned Cas proteins. By way of example and based on the 49C1 Ibis antibody, the subject of the invention is the polypeptide conjugate as described above comprising the sequences SEQ ID NO: 102 (heavy chain of 49C1 Ibis of sequence SEQ ID NO: 32 covalently linked at its C-terminal end to saCas9 of sequence SEQ ID NO: 89) and 34 (light chain of 49C1 Ibis).With regard to this non-limiting example, it should be noted that all of the possible constructions from the antibodies described above and the Cases described above, which a person skilled in the art is able to develop, are part of the invention.

[0120] According to another embodiment, the subject of the invention is the polypeptide conjugate as described above, in which said anti-APC-ASGPR is covalently linked to said Cas protein, and in particular said polypeptide conjugate comprises the sequences SEQ ID NOs: 102 and 34.

[0121] Advantageously, the subject of the invention is the polypeptide conjugate as described above, in which said anti-DC-ASGPR is covalently linked to said Cas protein by means of a linker, in particular a peptide linker.

[0122] Among the linkers that can be used, i.e. small molecules or small peptides used to link the anti-DC-ASGPR and the Cas protein, which may incorporate glycosylation sites or introduce a particular secondary structure, it should be noted that some increase the efficiency of expression or the stability of the fusion protein and, consequently, the effectiveness thereof. For the purposes of the invention, it should be noted that the following peptide linkers are used, but are not limited to:

[0123] - QTPTNTISVTPTNNSTPTNNSNPKPNPAS (SEQ ID NO: 96); - SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID NO: 97);

[0124] - PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO: 98);

[0125] - TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO: 99); And

[0126] - TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO: 100).

[0127] Also and according to another particular embodiment, the subject of the invention is the polypeptide conjugate as described above, in which said peptide linker is chosen from:

[0128] - QTPTNTISVTPTNNSTPTNNSNPKPNPAS (SEQ ID NO: 96);

[0129] - SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID NO: 97);

[0130] - PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO: 98);

[0131] - TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO: 99); And

[0132] - TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO: 100).

[0133] In particular, the subject of the invention is the polypeptide conjugate as described above, wherein the anti-antigen presenting cell antibody that targets the APC asialoglycoprotein receptor (anti-DC-ASGPR) as described above is covalently linked via its heavy chain to a second component that is one of the aforementioned Cas proteins via a peptide linker. By way of example and based on the 49Cl l_bis antibody, the subject of the invention is the polypeptide conjugate as described above comprising the sequences SEQ ID NOs: 101 (heavy chain of 49Cl l_bis of sequence SEQ ID NO: 32 covalently linked at its C-terminus to the linker of sequence SEQ ID NO: 96 covalently linked to Cas9 of sequence SEQ ID NO: 89) and 34 (light chain of 49Cl l_bis).With regard to this non-limiting example, it should be noted that all of the possible constructions from the antibodies described above and the Cases described above, which a person skilled in the art is able to develop, are part of the invention.

[0134] According to another embodiment, the subject of the invention is the polypeptide conjugate as described above, in which said peptide linker is chosen from:

[0135] - QTPTNTISVTPTNNSTPTNNSNPKPNPAS (SEQ ID NO: 96);

[0136] - SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID NO: 97);

[0137] - PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO: 98);

[0138] - TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO: 99); And

[0139] - TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO: 100), and in particular said polypeptide conjugate comprises the sequences SEQ ID NOs: 101 and 34. By "non-covalently linked" is meant that the polypeptide conjugate of the invention can also be linked non-covalently, that is to say that the first and second components are linked by weak bonds, also called non-covalent interactions, which do not involve the sharing of electrons of said first and second components. In particular, it is understood that according to a second particular embodiment, the subject of the invention is the polypeptide conjugate as described above, in which said anti-APC-ASGPR is non-covalently linked to said Cas protein.

[0140] For this, it is possible to take advantage of high affinity non-covalent interactions known to those skilled in the art which exist between two partners, such as, but not limited to, the antibody / antigen interaction, the receptor / ligand interaction, the avidin / biotin interaction, the cohesin / dockerin interaction and the barnase / barstar interaction. Also and according to another particular embodiment, the subject of the invention is the polypeptide conjugate as described above, in which said anti-DC-ASGPR is non-covalently bound to said Cas protein by means of high affinity interactions chosen from:

[0141] - antibody / antigen interactions;

[0142] - receptor / ligand interactions;

[0143] - avidin / biotin interactions;

[0144] - cohesin / dockerin interactions; and

[0145] - barnase / barstar interactions.

[0146] In particular, the invention also relates to the polypeptide conjugate as described above, in which said high affinity interactions are cohesin / dockerin interactions.

[0147] In particular, the invention relates to the polypeptide conjugate as described above, in which the anti-antigen presenting cell antibody that targets the asialoglycoprotein receptor (anti-DC-ASGPR) as described above is non-covalently linked via its heavy chain to a second component which is one of the aforementioned Cas proteins. By way of example and based on the 49Cl l_bis antibody, the subject of the invention is the polypeptide conjugate as described above comprising the sequences SEQ ID NOs: 103 or 86 (heavy chain of 49Cl l_bis of sequence SEQ ID NO: 32 covalently linked at its C-terminal end to dockerin), 34 (light chain of 49C1 l_bis) and 104 (Cas9 of sequence SEQ ID NO: 89 covalently linked at its N-terminal end to cohesin).In view of this non-limiting example, it should be noted that all possible constructions from the antibodies described above, the Cas proteins described above and the means of high-affinity non-covalent interactions described above, which a person skilled in the art is able to develop, are part of the invention. In the same way, if the above construction shows dockerin linked to the antibody component and cohesin linked to the Cas component, it is possible to carry out the reverse, namely, to put cohesin on the antibody component and dockerin on the Cas component of the polypeptide conjugate of the invention, regardless of the means of high-affinity non-covalent interactions used.

[0148] According to another embodiment, the subject of the invention is the polypeptide conjugate as described above, in which said high affinity interactions are cohesin / dockerin interactions, and in particular said polypeptide conjugate comprises the sequences SEQ ID NOs: 103, 34 and 104 or the sequences SEQ ID NOs: 85, 25 and 104.

[0149] VECTORS

[0150] According to a second aspect of the invention, the invention relates to a vector encoding a polypeptide conjugate of the invention. In other words, the invention comprises as a whole at least one nucleic acid comprising or consisting of a sequence encoding a first component which is an anti-antigen-presenting cell antibody that targets the APC asialoglycoprotein receptor (anti-DC-ASGPR) as described above, or a fragment thereof as described above, and / or a second component which is one of the aforementioned Cas proteins. By "at least one nucleic acid" is meant that the invention may comprise two or three nucleic acids. For example, one encoding anti-DC-ASGPR as described above, or a fragment thereof as described above, and the other one of the aforementioned Cas proteins; or two encoding anti-DC-ASGPR as described above (e.g.a first for the light chain and a second for the heavy chain) and the third one of the aforementioned Cas proteins. It should be noted, however, that in its covalent configuration, the invention preferably only uses a nucleic acid, which comprises or consists of a sequence encoding a first component which is an anti-antigen-presenting cell antibody which targets the asialoglycoprotein receptor (anti-DC-ASGPR) as described above, or a fragment thereof as described above, and a second component which is one of the aforementioned Cas proteins.

[0151] According to this second aspect, the invention therefore relates to an expression vector comprising at least one nucleic acid as defined above, said at least one nucleic acid being under the control of elements allowing its expression (promoter).

[0152] The term "expression vector" means a DNA (deoxyribonucleic acid) molecule that has elements allowing its replication (duplication) in at least one living organism. These elements allowing replication include yeast or bacterial replication origins, or virus replication control elements. The vectors according to the invention include plasmids, phages, yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), modified genomes of replicative viruses or integrative viruses, etc. Some vectors carry a viral (replicative) or virus-derived genome, but lacking viral genes (non-replicative), DNA (deoxyribonucleic acid), or RNA (ribonucleic acid). RNA genomes can be reverse transcribed into DNA by a retroviral reverse transcriptase or directly translated by the cellular machinery.Viral or virus-derived genomes, DNA or RNA, can be packaged within a protective structure of viral proteins and cell membranes. These recombinant virus or virus-derived particles allow the transduction of target cells for the expression of a Cas protein or anti-CD-ASGPR. This transgene expression can be stable, cyclic or transient, depending on the method or promoter used.

[0153] These vectors are called "expression" vectors because they have nucleotide sequences that allow the expression, that is to say the transcription into RNA (ribonucleic acid), of the nucleotide sequences that they control, or the translation of the RNA that they carry.

[0154] In the invention, said at least one nucleic acid contained in said vector is placed "under the control of the elements allowing its expression". This means that said expression vector has at least one transcription initiation sequence such as a promoter of a virus such as the early promoter of the simian virus SV40, or of Cytomegalovirus (CMV) or the promoter sequences of the Rous sarcoma virus (RSV), and in particular a sequence or promoter comprising a TATAA box. It may also be a human promoter of a housekeeping gene such as that of the phosphoglycerate kinase (PGK) gene or a human promoter known as tissue-specific, active only in certain subpopulations of cells in the organism.It may also be a synthetic promoter containing one or more response elements, binding one or more transcription factors; these promoters may be inducible by an exogenous stimulus such as a pharmacological molecule, a hormone, a deficiency or stress. In addition, said vector also has at least one transcription termination sequence and in particular a polyadenylation sequence derived from a mammalian gene, in particular a human gene.

[0155] In addition to these sequences essential for the expression of the nucleotide sequence contained in said vector, other sequences may be added to regulate or modulate the expression of said sequence. A non-exhaustive list includes: introns of mammalian genes, and in particular human genes, transcription regulation sequences of the enhancer type or transcribed but not translated sequences of mammalian genes, and in particular human genes.

[0156] HOST CELLS

[0157] According to a third aspect of the invention, the invention relates to a host cell or cell line transformed by a nucleic acid as described above and / or an expression vector as described above. In other words, the invention relates to a host cell or cell line capable of expressing (producing) a polypeptide conjugate as described above comprising a first component which is an anti-antigen presenting cell antibody which targets the asialogly coprotein receptor (anti-APC-ASGPR), or a fragment thereof, covalently or non-covalently linked to a second component which is a Cas protein.

[0158] COMPOSITIONS

[0159] According to another aspect, the invention relates to a pharmaceutical composition comprising as active ingredient a polypeptide conjugate as described above comprising a first component which is an anti-antigen presenting cell antibody which targets the APC asialoglycoprotein receptor (anti-DC-ASGPR), or a fragment thereof, covalently or non-covalently linked to a second component which is a Cas protein, in association with an acceptable pharmaceutical vehicle. By "pharmaceutical composition" is meant a particular packaging of the invention, which allows the pharmaceutical composition of the invention to be possibly administrable to animals and humans by oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local, inhaled or rectal route.Furthermore, this packaging also allows the administration of the active ingredient (or active substance), alone or in combination with another active ingredient, in unit form or in mixture with conventional pharmaceutical carriers. Suitable unit administration forms include:

[0160] - oral administration forms such as tablets, capsules, powders, granules and oral suspensions or solutions;

[0161] - sublingual and buccal administration forms, aerosols, implants;

[0162] - subcutaneous, transdermal, intradermal, intraperitoneal, intramuscular, intravenous, subcutaneous, transdermal, intratracheal and nasal administration forms; and

[0163] - forms of rectal administration.

[0164] “Acceptable pharmaceutical carrier” (or pharmaceutically acceptable carrier) means a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or animal or human organism. This may include:

[0165] - crystalloid solutes, for example sodium chloride, bicarbonate, glucose;

[0166] - cationic lipids;

[0167] - peptide compounds; or

[0168] - surfactants, for example polysorbates.

[0169] In all cases, whatever the formulation chosen, it must be sterile, stable under the manufacturing and storage conditions, and must be protected from any contamination by micro-organisms, such as bacteria and fungi.

[0170] According to another embodiment, the subject of the invention is the pharmaceutical composition as described above in unit form, in which the polypeptide conjugate of the invention is at a (unit) dose of from 0.1 to 1,000 pg or at a (unit) dose of from 0.1 to 1,000 mg / kg (based on a 70 kg human). By "0.1 to 1000 ug (or mg / kg)" is meant that the dose may be 10 to 1000, 100 to 900, 200 to 800, 300 to 700, 400 to 600, 0.1 to 500, 500 to 1000, 10 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900 or 900 to 1000 ug (or mg / kg).It is also understood that the dose can be 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 1 1.0, 1 1.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400,.

[0171] 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580,

[0172] 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770,

[0173] 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960,

[0174] 970, 980, 990, or 1000 pg (or mg / kg).

[0175] According to another embodiment, the invention relates to the pharmaceutical composition as described above, said pharmaceutical composition being formulated to be capable of being administered by one of the following routes: oral, parenteral, injectable, topical, by inhalation, subcutaneous, nasal or pulmonary.

[0176] According to another embodiment, the invention relates to the pharmaceutical composition as described above, in combination with a second active ingredient. In particular, the invention relates to the pharmaceutical composition as described above, further comprising an anti-OX40L antibody or a fragment thereof. By "anti-OX40L antibody or a fragment thereof" is meant an antibody or a fragment thereof capable of specifically recognizing the ligand of OX40 (also called CD134 or TNFRSF4), which is stably expressed on many antigen-presenting cells such as DC2 (a subtype of dendritic cells), macrophages and activated B lymphocytes.

[0177] USES & METHODS

[0178] According to another aspect, the invention relates to a polypeptide conjugate according to the invention for its use in promoting immunotolerance to an above-mentioned Cas protein. The invention also relates to a pharmaceutical composition according to the invention for its use in promoting immunotolerance to a Cas protein of bacterial, archaeal or phage origin.

[0179] By "promoting immunotolerance to a Cas protein" is meant that the administration of the polypeptide conjugate according to the invention and / or a pharmaceutical composition according to the invention to a mammal induces (triggers) an immune tolerance to the Cas protein, which inhibits any subsequent immune rejection towards the Cas protein concerned. In this way, the increase in immune tolerance to Cas allows for the implementation of safe and effective gene repair protocols in humans using the CRISPR-Cas system.

[0180] According to another embodiment, the invention relates to a polypeptide conjugate according to the invention for its use in promoting immunotolerance to a Cas protein, said polypeptide conjugate being administered to a mammal. The invention also relates to a pharmaceutical composition according to the invention for its use in promoting immunotolerance to said Cas protein, said pharmaceutical composition being administered to a mammal. By "mammal" is meant an animal organism such as primates or humans (men, women and children).

[0181] According to another embodiment, the subject of the invention is a polypeptide conjugate according to the invention for its use in promoting immunotolerance to a Cas protein, said polypeptide conjugate being at a (unit) dose of from 0.1 to 1,000 pg or at a (unit) dose of from 0.1 to 1,000 mg / kg (based on a 70 kg human). The subject of the invention is also a pharmaceutical composition according to the invention for its use in promoting immunotolerance to a Cas protein, said pharmaceutical composition comprising a (unit) dose of from 0.1 to 1,000 pg of the polypeptide conjugate according to the invention or at a (unit) dose of from 0.1 to 1,000 mg / kg (based on a 70 kg human) of the polypeptide conjugate according to the invention.By "0.1 to 1000 pg (or mg / kg)" is meant that the dose may be 10 to 1000, 100 to 900, 200 to 800, 300 to 700, 400 to 600, 0.1 to 500, 500 to 1000, 10 to 100, 100 to 200, 200 to 300, 300 to 400, 400 to 500, 500 to 600, 600 to 700, 700 to 800, 800 to 900 or 900 to 1000 pg (or mg / kg). It is also understood that the dose can be 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,.

[0182] 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,

[0183] 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 ,

[0184] 92, 93, 94, 95, 96, 97, 98, 99, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220,

[0185] 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410,

[0186] 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590,

[0187] 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780,

[0188] 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970,

[0189] 980, 990, or 1,000 pg (or mg / kg).

[0190] According to another embodiment, the invention relates to a polypeptide conjugate according to the invention for its use in promoting immunotolerance to a Cas protein, said polypeptide conjugate being administered by one of the following routes: oral, parenteral, injectable, topical, by inhalation, subcutaneous, nasal or pulmonary. The invention also relates to a pharmaceutical composition according to the invention for its use in promoting immunotolerance to said Cas protein, said pharmaceutical composition being administered by one of the following routes: oral, parenteral, injectable, topical, by inhalation, subcutaneous, nasal or pulmonary.

[0191] According to another embodiment, the invention relates to a polypeptide conjugate according to the invention for its use in promoting immunotolerance to a Cas protein, said polypeptide conjugate being administered in association with an anti-OX40L antibody or a fragment thereof. The invention also relates to a pharmaceutical composition according to the invention for its use in promoting immunotolerance to said Cas protein, said pharmaceutical composition further comprising an anti-OX40L antibody or a fragment thereof.

[0192] According to another embodiment, the subject of the invention is the polypeptide conjugate according to the invention for its use in promoting immunotolerance to a Cas protein, and in particular said polypeptide conjugate is administered in association with an anti-OX40L antibody or a fragment thereof.

[0193] The invention also relates to a pharmaceutical composition according to the invention for its use in promoting immunotolerance to said Cas protein, and in particular said pharmaceutical composition further comprises an anti-OX40L antibody or a fragment thereof.

[0194] Alternatively, the subject of the invention is a pharmaceutical composition for its use in promoting immunotolerance to a Cas protein, said composition comprising as active ingredient the polypeptide conjugate of the invention in association with an acceptable pharmaceutical vehicle, and in particular said pharmaceutical composition further comprising an anti-OX40L antibody or a fragment thereof.

[0195] Alternatively, the invention also relates to a method for promoting immunotolerance to a Cas protein in a patient in need thereof, said method comprising a step of administering a polypeptide conjugate according to the invention. The invention also relates to a method for promoting immunotolerance to said Cas protein in a patient in need thereof, said method comprising a step of administering a pharmaceutical composition according to the invention.

[0196] Finally, it is understood that immunizing the patient in need of Cas (the one who suffers from a pathology whose treatment requires gene therapy based on the CRISPR-Cas system) using the invention offers him the possibility of benefiting from a safe and effective gene therapy protocol. To a certain extent, it is therefore understood that the invention implements a recombinant protein (anti-DC-ASGPR-Cas) and a vector encoding the Cas protein. The recombinant anti-DC-ASGPR-Cas protein is injected, in particular intradermally, to immunize the patient in need, and then the vector is administered to allow the expression of Cas in a given previously genetically modified tissue.

[0197] In other words, the invention also relates to a kit comprising:

[0198] - the polypeptide conjugate according to the invention, or the vector coding for the polypeptide conjugate of the invention, or the pharmaceutical composition according to the invention; and

[0199] - a vector encoding a Cas protein, in particular encoding the same Cas protein as that present in the polypeptide conjugate according to the invention.

[0200] On this point, it should be noted that the "vector encoding a Cas protein" of the kit does not correspond to the vector of the invention as described above, the purpose of the vector of the kit being to allow the expression of Cas for the purpose of gene therapy (and not to produce the polypeptide conjugate of the invention). In any respect, it should be noted that the different aspects of the invention, just like the different embodiments thereof, are interdependent. The latter can therefore be combined with each other as much as necessary to obtain preferred aspects and / or embodiments of the invention not explicitly described. This is also valid for all the definitions provided in the present description, which applies to all aspects of the invention and its embodiments.

[0201] Further, the present invention is illustrated, but not limited to, the following figures and examples.

[0202] LIST OF FIGURES

[0203] Figure 1. Experimental protocol for vaccination of a cynomolgus macaque with anti-DC-ASGPR-Cas9. The protocol lasted 42 days. Blood was sampled every 7 days. The first sample at D0 provided baseline levels for all subsequent measurements. The animal received three intradermal (ID) injections of anti-DC-ASGPR-Cas9 at one-week intervals each, on days 14, 21, and 28. Finally, the animal received an injection of Cas9 protein at D35.

[0204] Figure 2. Optimization of the assembly of the Anti-DC-ASGPR-dockerin and saCas9-cohesin moieties. The assembly of two molar ratios of the Anti-DC-ASGPR-dockerin and saCas9-cohesin proteins was analyzed by CAPE blot electrophoresis (Chretien P, et al. J Autoimmun. 1994 Jun;7(3):379-88. PMID: 7916909). Lane 1, deposition of saCas9-cohesin alone (20 mm migration). Lane 2, deposition of anti-DC-ASGPR-dockerin alone (8 mm migration). Lane 3, mix of saCas9-cohesin and anti-DC-ASGPR-dockerin at a molar ratio of 1:0.5 (12 mm smear and migration). Lane 4 mix of saCas9-cohesin and anti-DC-ASGPR-dockerin at a molar ratio of 1:2 (single band and 12 mm migration). The 1:2 molar ratio of Anti-DC-ASGPR-dockerin and saCas9-cohesin proteins is selected for anti-DC-ASGPR-saCas9 vaccination. Figure 3. Strategies for analyzing cell subtypes from cynomolgus macaque PBMCs by flow cytometry.

[0205] Figure 4. Strategy for analyzing the regulatory phenotype of CD4 lymphocytes + cynomolgus macaque, expressing CD25, FOXP3 and CD39 markers.

[0206] Figure 5. Cytometry analysis of CD4 lymphocytes + of interest in unstimulated (NS) conditions, or stimulated with Cas9 protein (saCas9) at D0 of the protocol. Dial numbers indicate cell proportions.

[0207] Figure 6. Cytometry analysis of CD4 lymphocytes + of interest in unstimulated (NS) conditions, or stimulated with the Cas9 protein (saCas9) at D21 of the protocol. The dial numbers indicate cell proportions.

[0208] Figure 7. Cytometry analysis of CD4 lymphocytes + of interest in unstimulated (NS) conditions, or stimulated with Cas9 protein (saCas9) at D35 of the protocol. The dial numbers indicate cell proportions.

[0209] Figure 8. Number of CD4+ regulatory T lymphocytes expressing FOXP3 and CD39 in the CD4+ CD25+ 0X40+ lymphocyte population (A) Proportion of CD4+ CD25+ OX40+ lymphocytes, seven days after one (D21) and three (D35) vaccinations with anti-DC-ASGPR-Cas9 (vertical dotted lines). (B) Number of Foxp3+ CD39- lymphocytes in the CD4+ CD25+ 0X40+ lymphocyte population (C) Number of Foxp3+ CD39+ lymphocytes in the CD4+ CD25+ 0X40+ lymphocyte population

[0210] Figure 9. Measurement of TGFbl in cynomolgus macaque serum after three vaccinations with anti-DC-ASGPR-Cas9 (D35) and one week after immunization with recombinant Cas9 protein. A significant increase in TGFbl level in the animal's serum one week after immunization with Cas9 protein is observed. Thin dotted line: vaccination with anti-DC-ASGPR-Cas9; thick dotted line: immunization with Cas9 protein.

[0211] Figure 10. Measurement of IL-10 in the culture supernatant of cynomolgus macaque PBMCs collected at D21, D28, D35 and D42 after the first rhMOG immunization. An increase in IL-10 secretion in PBMCs collected at D42 (one week after a second rhMOG immunization) stimulated by saCas9 (Cas9) is observed but not in the supernatant of unstimulated (NS) PBMCs.

[0212] EXAMPLES

[0213] PRECLINICAL STUDY: saCAS9 IMMUNOTOLERIZATION IN A CYNOMOLGUS MACAQUE VIA IMMUNIZATION WITH ANTI-APC-ASGPR-saCAS9

[0214] PRELIMINARY DATA

[0215] In vitro

[0216] Anti-APC-ASGPR-saCas9 was obtained. It was constructed based on monoclonal antibody 49C11 and S. aureus Cas9, and the resulting conjugate includes:

[0217] - the light chain of the anti-DC-ASGPR monoclonal antibody 49C11 L (SEQ ID NO: 34); and

[0218] - the heavy chain of the anti-DC-ASGPR monoclonal antibody 49C11_H which contains a dockerin domain (SEQ ID NO: 103) allowing its association with the saCas9 protein which contains a cohesin domain (SEQ ID NO: 89).

[0219] GENERAL EXPERIMENTAL DESIGN

[0220] A protocol was established to induce immune tolerance in an immunocompetent adult Cynomolgus macaque to the heterologous protein saCas9 (Fig. 1). For this purpose, a recombinant anti-DC-ASGPR-saCas9 protein obtained by combining an anti-DC-ASGPR-dockerin antibody and the saCas9-cohesin protein was used (Fig. 2). Then, it was determined whether this procedure allows the appearance of a population of regulatory CD4+ T lymphocytes specific for saCas9 and expressing the markers 0X40+ CD25+ FOXP3 and CD39+ (Fovet CM, et al. EBioMedicine. 2019 Sep;47:492-505. PMC6796575.). For this purpose, a blood mononuclear cell labeling strategy was established (Fig. 3). This allows the detection of circulating CD4+ lymphocytes, where the regulatory phenotype was determined by immunostaining of CD25+ FOXP3 and CD39+ antigens (Fig. 4).saCas9-specific regulatory CD4+ T cells (CD25+ FOXP3 and CD39- or CD39+) were detected by the presence of the activation marker 0X40, 44 h after priming with saCas9 protein (Fig. 5). During this protocol, 1 monkey was immunized by intradermal injections of an anti-APC-ASGPR-saCas9 antibody.

[0221] Vaccination protocol

[0222] The animal received an intradermal dose, in the back, of 250 pg of recombinant anti-APC-ASGPR-saCas9 protein every week for 3 weeks (Fig. 1).

[0223] Immunization with saCas9

[0224] On day 35 of the protocol, 7 days after receiving the last vaccine dose, the animal was immunized with 250 pg of saCas9 protein.

[0225] Pre- and post-vaccination immune analysis

[0226] From day 1, plasma and PBMCs were collected weekly to explore the CD4 response to saCas9 in vitro.

[0227] Blood samples (10 mL) were taken from the animal before the first vaccination and then at regular intervals to assess hematological and biological functions.

[0228] PBMCs from the animal were cultured. Cells were labeled with CFSE (Carboxyfluorescein succinimidyl ester) at day 0 and cultured, then stimulated with recombinant saCas9, or nothing. Labeled cells were collected after 44 h and processed for intracellular cytokine analysis and phenotype study by flow cytometry. A portion of these cells was used to evaluate the expression of CD4, CD8, CD25, 0X40, CD39, and Foxp3 markers after stimulation with saCas9.

[0229] The animal was examined daily and clinical signs were quantified using an assessment grid.

[0230] Moreover :

[0231] The anti-APC-ASGPR-saCas9 fusion protein was injected intradermally into the animal's back;

[0232] Recombinant saCas9 protein was injected intradermally into the animal's back;

[0233] Resiquimod was applied as a gel to the shaved skin surface and the fusion protein injection site. The gel application was performed immediately after the fusion protein injection and was followed by a gentle “skin massage” for 30 s;

[0234] At each antibody vaccination date, the animal received five injections of 50 pg of fusion protein (250 pg in total); - At the injection date of the recombinant saCas9 protein, the animal received 5 injections of 50 pg of saCas9 (250 pg in total); and

[0235] - Immunomonitoring included: a. Measurement of anti-saCas9 specific T cells (CD4 + and CD8 +) in the blood and TGF01 in the serum. b. Monitoring of markers of T and B cell activation in the blood, anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-CD95, anti-CD28, anti-CD69, anti-HLADR, anti-CD20, anti-CD27 and anti-IgD.

[0236] MATERIALS AND METHODS

[0237] Animals

[0238] An adult cynomolgus monkey (Macaca fascicularis) imported from Mauritius was included in this study. MHC class I and class II typing was determined.

[0239] Immunogens

[0240] Recombinant proteins were prepared by the Baylor Institute for Immunology Research (BUR). Anti-DC-ASGPR-dockerin (human) was combined with saCas9-cohesin extemporaneously at the time of intradermal injection by a mixture at a molar ratio of 1:2.

[0241] Recombinant saCas9 produced in E.Coli was obtained by BIIR.

[0242] Immunizations

[0243] The animal received injections in the back with a syringe for intradermal inoculation (ID). The injection site was shaved.

[0244] In addition, the animal received 5 injections of 100 μL of each anti-DC-ASGPR-saCas9 vaccine spaced 0.5 cm apart at each vaccination date.

[0245] Each injection contains 50 pg of antibody.

[0246] Lifetime Observations

[0247] The animal was observed by CEA staff 7 days a week, including through webcam monitoring. Food and water consumption were recorded. At each bleed, a clinical examination was performed, and weight and rectal temperature were recorded.

[0248] Table 2. Lifetime observations

[0249] Complete hematology

[0250] Complete hematology was performed using an HMX A / L (Beckman Coulter®) according to procedure SOP#EXAM0006_01. Serum transaminases were measured weekly as well as LDL and PCSK9 protein.

[0251] SaCas9-specific T cell activation, phenotype and cytokine production.

[0252] CD4 lymphocyte count +The presence of saCas9-specific autoreactive and regulatory cells among circulating PBMCs has not been studied in animals after saCas9 gene transfer. Their large-scale stimulation and culture alters their original phenotype and function and prevents their precise study. To precisely evaluate and characterize these cells in macaques, a recent assay that detects CD4+ T cells overexpressing CD 140 (0X40) and CD25 (IL2R) was used, as they allow the identification of cells that respond to antigen restimulation with much greater sensitivity than intracellular cytokine (ICS) labeling (JJ Zaunders et al., J Immunol 183, 2827-36 (2009)).

[0253] Among PBMCs, the phenotypic polarization of CD4 T cells + specific for saCas9 was assessed by ex vivo stimulation of PBMCs with recombinant saCas9 protein for 44 hours. Under these conditions, CD4 lymphocytes +saCas9-specific T cells overexpress 0X40 and CD25 before cells begin to proliferate (N. Seddiki, et al., Eur J Immunol 44, 1644-61 (2014)). Thus, cultured PBMCs stimulated with saCas9 epitopes were labeled with antibodies against CD4, CD134, CD25, CD39, and FOXP3 to distinguish T reg s and TRI (CD4+, CD25+, CD134+, CD39+) (N. Seddiki, et al., Eur J Immunol 44, 1644-61 (2014)). The different cell subtypes were identified, quantified and sorted by cytometry. This experiment requiring small amounts of blood provided basic quantitative and qualitative information on the level and phenotype of circulating anti-saCas9 CD4+ cells at different times after vaccination with the anti-DC-ASGPR-saCas9 antibody.

[0254] Serum cytokines

[0255] TGFbl was measured at different times before and after vaccination as well as one week after saCas9 immunization.

[0256] Cytokines in PBMC culture supernatant

[0257] ILIO was measured in the culture supernatant of PBMCs collected at different times after immunization with rhMOG and vaccination with anti-ASGPR-saCas9. Cultured PBMCs were stimulated or not with scCas9.

[0258] Conservation of material

[0259] Biological samples were kept frozen at -80°C (plasma, serum, RNA) or -135°C (PBMC) for the duration of the study and 2 years after the end of the study.

[0260] Data analysis

[0261] The data were recorded and analyzed using data management systems such as IDBS.

[0262] Statistical analysis was performed using the non-parametric Mann & Whitney or Wilcoxon rank test using PRISM® 8 (GraphPad®).

[0263] Flow cytometry analyses were performed using FlowJo® and SPICE® software.

[0264] Ethics

[0265] The animal was housed in the CEA facilities in Fontenay-aux-Roses in accordance with the institutions' standards of care and European directive D8906, and those of the Office of Laboratory Animal Welfare (OLAW) of the National Institutes of Health (USA).

[0266] RESULTS

[0267] A healthy adult cynomolgus macaque was treated to make it immunotolerant to the heterologous saCas9 protein using the vaccination protocol described in Figure 1 and recalled below. For this purpose, a recombinant anti-DC-ASGPR-saCas9 protein was used, obtained by combining two recombinant proteins: an anti-DC-ASGPR-dockerin antibody and the saCas9-cohesin protein (Fig. 2). Subsequently, the animal received intradermal injections of the anti-DC-ASGPR-saCas9. At the same time, it was determined whether this procedure allows the appearance of a population of regulatory CD4+ T lymphocytes specific for saCas9 and expressing the 0X40+ CD25+ FOXP3 and CD39+ markers. For this purpose, a blood mononuclear cell labeling strategy was established (Fig. 3), which allowed the detection of a new population of regulatory CD4+ T lymphocytes expressing the markers CD25+ 0X40+ FOXP3+ and CD39+ (Fig. 4). These cells (i.e.saCas9-specific regulatory CD4+ T lymphocytes (CD25+ FOXP3 and CD39- or CD39+), non-existent before the vaccination protocol, increased proportionally as the vaccination regimen with anti-DC-ASGPR-saCas9 progressed (Fig. 5 to 7). In addition, the total count of these cells indicates that their absolute number increased by activation with the recombinant saCas9 protein (Fig. 8). Finally, in the animal that received the three injections of anti-DC-ASGPR-saCas9, immunization by intradermal injection of saCas9 induced the expression of the anti-inflammatory cytokines TGFbl and IL10 as detected respectively in the plasma or in the supernatant of cultured PBMCs (Fig. 9 and 10).

[0268] These results therefore demonstrated that vaccine treatment with the anti-DC-ASGPR-saCas9 protein allows the appearance of a population of regulatory T lymphocytes and are the mark of an anti-inflammatory immunomodulatory response in the presence of the saCas9 antigen.

Claims

CLAIMS Polypeptide conjugate comprising a first component which is an anti-antigen presenting cell antibody which targets the asialoglycoprotein receptor of antigen presenting cells (anti-DC-ASGPR), or a fragment thereof capable of binding to the epitope recognized by the full-length antibody, covalently or non-covalently linked to a second component which is a Cas protein, said polypeptide conjugate being capable of inducing immune tolerance to the Cas protein in the organism of mammals, in particular in the organism of primates and humans, and said anti-DC-ASGPR being selected from: - an antibody comprising: o a heavy chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 1, the CDR2 of sequence SEQ ID NO: 2 and the CDR3 of sequence SEQ ID NO: 3; and o a light chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 16, the CDR2 of sequence SEQ ID NO: 17 and the CDR3 of sequence SEQ ID NO: 18; - an antibody comprising: o a heavy chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 35, the CDR2 of sequence SEQ ID NO: 36 and the CDR3 of sequence SEQ ID NO: 37; and o a light chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 40, the CDR2 of sequence SEQ ID NO: 41 and the CDR3 of sequence SEQ ID NO: 42; - an antibody comprising: o a heavy chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 45, the CDR2 of sequence SEQ ID NO: 46 and the CDR3 of sequence SEQ ID NO: 47; and o a light chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 50, the CDR2 of sequence SEQ ID NO: 51 and the CDR3 of sequence SEQ ID NO: 52; - an antibody comprising: o a heavy chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 55, the CDR2 of sequence SEQ ID NO: 56 and the CDR3 of sequence SEQ ID NO: 57; and o a light chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 60, the CDR2 of sequence SEQ ID NO: 61 and the CDR3 of sequence SEQ ID NO: 62; - an antibody comprising: o a heavy chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 65, the CDR2 of sequence SEQ ID NO: 66 and the CDR3 of sequence SEQ ID NO: 67; and o a light chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 70, the CDR2 of sequence SEQ ID NO: 71 and the CDR3 of sequence SEQ ID NO: 72; and - an antibody comprising: o a heavy chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 75, the CDR2 of sequence SEQ ID NO: 76 and the CDR3 of sequence SEQ ID NO: 77; and o a light chain comprising from the N-terminal end to the C-terminal end the CDR1 of sequence SEQ ID NO: 80, the CDR2 of sequence SEQ ID NO: 81 and the CDR3 of sequence SEQ ID NO:

82.

2. Polypeptide conjugate according to claim 1, wherein said Cas protein is chosen from: - the Cas9 nucleases of S. pyogenes, S. aureus, C. diphtheriae, N meningitidis, S. canis, S. macacae, F. tularensis, Acidaminococcus, C. jejuni, S. pneumoniae and S. thermophilus , the Caslla nucleases of Lachnospiraceae and A. sp. the Casllb nucleases of Alicyclobacillus kakegawensis and Bacillus hisashii , the CasX or Casl2e nucleases of Deltaproteobacteria and Planctomycetes , the type V CRISPR nucleases of Syntrophomonas palmitatica and Acidibacillus , or the Casllb nucleases Cas® of the Biggiephage clade, ' - the Cas9, Cas 12a, Cas 12b, CasX or Cas 12e, CRISPR type V, Cas 12j or Cas® orthologs derived from these organisms; et - Cas9, Casl2a, Casl2b, CasX or Casl2e, CRISPR type V, Cas 12j or Cas® mutants or functional variants derived from these organisms, and in particular said Cas protein is selected from the sequences SEQ ID NOs: 87 to 95 and 105 to 113, and mutants thereof. Polypeptide conjugate according to claim 1 or 2, wherein said anti-DC-ASGPR is covalently linked to said Cas protein, and in particular said polypeptide conjugate comprises the sequences SEQ ID NOs: 102 and 34. Polypeptide conjugate according to claim 3, wherein said anti-DC-ASGPR is covalently linked to said Cas protein by means of a linker, in particular a peptide linker. Polypeptide conjugate according to claim 3 or 4, wherein said peptide linker is selected from: - QTPTNTISVTPTNNSTPTNNSNPKPNPAS (SEQ ID NO: 96); - SSVSPTTSVHPTPTSVPPTPTKSSP (SEQ ID NO: 97); - PTSTPADSSTITPTATPTATPTIKG (SEQ ID NO: 98); - TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO: 99); And - TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO: 100), and in particular said polypeptide conjugate comprises the sequences SEQ ID NOs: 101 and 34.

6. A polypeptide conjugate according to claim 1 or 2, wherein said anti-DC-ASGPR is non-covalently linked to said Cas protein.

7. Polypeptide conjugate according to claim 6, wherein said anti-DC-ASGPR is non-covalently linked to said Cas protein through high affinity interactions selected from: - antibody / antigen interactions; - receptor / ligand interactions; - avidin / biotin interactions; - cohesin / dockerin interactions; and - barnase / barstar interactions.

8. Polypeptide conjugate according to claim 6 or 7, wherein said high affinity interactions are cohesin / dockerin interactions, and in particular said polypeptide conjugate comprises the sequences SEQ ID NOs: 103, 34 and 104 or the sequences SEQ ID NOs: 85, 25 and 104.

9. Pharmaceutical composition for use in promoting immunotolerance to the Cas protein, said composition comprising as active ingredient a polypeptide conjugate according to any one of claims 1 to 8 in association with an acceptable pharmaceutical vehicle, and in particular said pharmaceutical composition further comprising an anti-OX40L antibody or a fragment thereof.

Images