Peptide conjugates and their use to promote immune tolerance of Cas nucleases in genome engineering gene therapy

Abstract

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

Description

[Technical Field]

[0001] The present invention relates generally to the field of medicine. More particularly, the present invention relates to polypeptide conjugates, compositions and methods for promoting CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins)-based immune tolerance for genome engineering gene therapy. [Background technology]

[0002] Immune tolerance is a state in which the immune system does not respond to substances or tissues that have the potential to cause immune rejection in healthy organisms. This state is induced by prior exposure to specific antigens and contrasts with traditional foreign body clearance by (innate) immune mechanisms. Immune tolerance is important for normal physiological function. Central immune tolerance is the primary means by which the immune system learns to distinguish between self and nonself. Peripheral immune tolerance is essential for preventing the immune system from overreacting to various environmental entities (e.g., allergens, intestinal microorganisms) as well as antigens produced by phagocytosis of dead body cells. Deficiencies in central or peripheral immune tolerance also contribute to autoimmune diseases, leading to syndromes such as systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes, and multiple sclerosis. However, recent discoveries have shown that peripheral immune tolerance can be induced, for example, by targeting the entry of antigens into antigen-presenting cells (APCs) via a receptor present on macrophages and dendritic cells (DCs), namely, the dendritic cell asialoglycoprotein receptor (DC-ASGPR) (Li D et al., J Exp Med. 2012;209(1):109-121).

[0003] CRISPR-Cas systems in bacteria, archaea, and large bacteriophages are adaptive defense systems that enable the destruction of mobile genetic elements (Makarova, K.S. et al., 2020. Nat Rev Microbiol 18(2):67-83) and the development of biotechnology tools for engineering the genomes of all cell types (Knott, G.J. and Doudna, J., 2018. Science 361(6405):866-869). To this end, different families of RNA-guided nucleases, such as Cas9 and Cas12 proteins, are used to recognize double-stranded DNA sequences and cleave them at specific locations. Therefore, CRISPR-RNA or guide RNA programmed nucleases for the recognition of target nucleic acid sequences are becoming one of the most effective biotechnological molecular compounds for genetic engineering and gene therapy for gene editing or gene expression control (FA Ran et al., Nature 520, 186-191 (2015); (Knott, GJ and Doudna, J. 2018 Science 361 (6405): 866-869). Cas nucleases are guided by homology recognition between guide RNA and chromatin sites to cleave DNA at specific target sequences. DNA repair mechanisms then allow the gene sequence to be corrected, for example for therapeutic purposes (FA Ran et al., Nature 520, 186-191 (2015)). Furthermore, some Cas nuclease mutants can act as nickases or completely lose nuclease activity, acquiring other functions such as chemical modification of DNA or gene transcription (Knott, GJ and Doudna, J. 2018 Science 361(6405):866-869). As the CRISPR-Cas system enables precise gene editing in eukaryotic cells, it opens up a real vision for in vivo repair and gene expression, making it possible to attempt to treat currently untreatable genetic or acquired diseases.For example, early phase I / II trials have been undertaken using Cas9 gene delivery into retinal photoreceptor cells with AAV viral vectors to repair the mutant RPE65 gene that causes Leber congenital amaurosis (A. Mullard, Nat. Rev. Drug Discov. 18, 656–656 (2019)). Furthermore, other in vivo nucleotide base gene editing repair clinical protocols are emerging to introduce base switching with nickase-active mutant Cas proteins for the treatment of hyperlipidemia or sickle cell anemia by modifying the Pcsk9 or hemoglobin genes, respectively (Ledford, H. Nature 2022). Finally, other clinical applications are also becoming possible involving the use of Cas proteins fused to domains lacking protease activity but capable of directly modulating the transcription of target genes (Jensen, TI. 2021. Genome Res. 31(11):2120-2130) or to enzymatic domains that can modify DNA methylation at gene loci and permanently disable or activate their expression (Pulecio, J. 2017. Cell Stem Cell 21(4):431-447).

[0004] Yet, the use of Cas nucleases in medicine for in situ genetic modification of organs has been hampered by the prospect of mutagenesis associated with off-target activity of Cas or immune rejection of cells expressing this bacterial protein (C.T. Charlesworth et al., Nat. Med. 25, 249–254 (2019)). To reduce off-target mutagenesis by Cas, high-fidelity variants of Cas9 and Cas12 have been developed (B.P. Kleinstiver, et al., Nature 529, 490-495 (2016) - I.M. 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, K.M. 2021. Nat Struct & Mol Biol 28(8):652-661; Tsuchida, C.A. Zhang, S. 2022. Mol Cell 82(6):1199-1209.e6). However, the use of Cas proteins in medicine poses a significant immunogenicity problem, as approximately 80% of the population has humoral and cellular immunity to the Cas9 orthologs of the common human resident bacteria S. pyogenes and S. aureus (CT Charlesworth, et al., Nat. Med. 25, 249-254 (2019)). Therefore, the risk of immune rejection is not negligible, leading to the failure of CRISPR-Cas-based gene therapy. This raises legitimate concerns about the use of Cas in gene therapy, as previous immunizations have significantly impaired the efficacy of in vivo genome editing in immunocompetent patients. Even if this disadvantage could be mitigated through the temporary point-use of Cas proteins, which rarely come into contact with humans, the aforementioned issues would likely persist if these proteins were administered repeatedly or continuously. [Prior art documents] [Non-patent literature]

[0005] [Non-licensed document 1] Li Dら、J Exp Med.2012;209(1):109~121 [Non-licensed document 2] Makarova,KS.ら, 2020. Nat Rev Microbiol 18(2):67~83 [Non-licensed document 3] Knott,GJ Doudna,J.2018 Science 361(6405):866~869

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[0006] Faced with this significant challenge of promising safe and effective in vivo gene therapy, the inventors have created novel polypeptide conjugates, compositions, and methods capable of inducing immune tolerance to Cas proteins, particularly Cas9, in mammalian organisms. With these tools, it is now possible to prevent immune rejection of somatic cells expressing Cas proteins and couple it to transient, inducible, or sustained gene expression of Cas proteins. The resulting increased immune tolerance to Cas proteins should lay the foundation for safe and effective protocols for gene repair or control of gene expression using CRISPR-Cas systems in humans. [Means for solving the problem]

[0007] [Polypeptide conjugates] According to a first aspect of the present invention, a general subject of the present invention is a polypeptide conjugate comprising a first component which is an antigen-presenting cell antibody targeting a receptor for asialoglycoprotein 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 the Cas9 protein of Staphylococcus aureus (saCas9). Since the polypeptide conjugates of the present invention are capable of inducing immune tolerance to Cas proteins in primate organisms, one embodiment of the present invention provides a polypeptide conjugate comprising a first component which is an antigen-presenting cell antibody targeting an APC asialoglycoprotein receptor (anti-APC-ASGPR), or a fragment thereof capable of binding to an epitope recognized by the full-length antibody, covalently or non-covalently linked to a second component which is a Cas protein, The present invention relates to polypeptide conjugates capable of inducing immune tolerance to Cas proteins in mammalian organisms, particularly in primate and human organisms.

[0008] In view of the above, according to certain embodiments, the present invention provides a polypeptide conjugate comprising a first component that is an antigen-presenting cell antibody that targets the APC asialoglycoprotein receptor (anti-APC-ASGPR), or a fragment thereof capable of binding to an epitope recognized by the intact antibody, covalently or non-covalently linked to a second component that is a Cas9 protein, It is understood to also relate to polypeptide conjugates that have the ability to induce immune tolerance to the Cas9 protein in mammalian organisms, and in particular in primate and human organisms.

[0009] The term "Cas protein" generally refers to a CRISPR-associated protein (Clustered Regularly Interspaced Short Palindromic Repeats-associated protein), and the term "Cas9 protein" refers to CRISPR-associated protein 9 (Clustered Regularly Interspaced Short Palindromic Repeats-associated protein 9). The latter corresponds to the nuclease of the CRISPR-Cas system, a prokaryotic adaptive defense mechanism for destroying invading foreign DNA. Various CRISPR-Cas systems exist within bacteria and archaea, and even within the same species. This implies differences in the sequence homology, organization, and size of the genes and proteins that make up the CRISPR-Cas system, as well as their ability to cleave single- or double-stranded DNA and RNA. Nevertheless, the various components of the CRISPR-Cas system and their functional logic are similar throughout the prokaryotic kingdom.

[0010] CRISPR-Cas systems consist of a family of sequences found in the genomes of bacteria, archaea, and large bacteriophages that correspond to genes involved in adaptation, rRNA maturation, and interference functions. To interfere with the expression and replication of foreign DNA or RNA, the host expresses a Cas nuclease gene, a sequence motif from the exogenous DNA or RNA acquired during a previous encounter between the bacterium and the phage, plasmid, or mobile genetic element that encodes a non-coding rcRNA, and a sequence that expresses a transcription-activating rcRNA (rctraRNA). The transformed rcRNA and rctraRNA form a duplex guide RNA (gRNA) that guides the Cas nuclease to the target DNA for double-strand cleavage. DNA cleavage is restricted by an adjacent DNA motif, called a protospacer adjacent motif (PAM), which is specific to each CRISPR-Cas system.

[0011] This functional framework has since been derived from its original function in prokaryotic immunity and is now being applied in biotechnology for gene targeting, processing, modification, destruction, and programmed repair. To date, several Cas proteins (Cas9, Cas12a, Cas12b, CasX or Cas12e, Cas12f or Cas14, Cas12j or CasΦ) from different hosts have been identified, characterized, or modified (e.g., their functions improved), which can be implemented by the present invention for mammalian genome engineering. Generally speaking, the above-mentioned Cas proteins have reduced sizes: 1,500 amino acids (AA) for Cas9, 1,000 AA for Cas12a, and less than 1,000 AA for CasX, Cas12f, and Cas12j.

[0012] Thus, according to another embodiment, the present invention provides a method for the preparation of a Cas protein comprising: - Cas9, Cas12a, Cas12b, CasX or Cas12e, CRISPR type V, Cas12j or CasΦ nuclease; - Cas9, Cas12a, Cas12b, CasX or Cas12e, CRISPR type V, Cas12j or CasΦ orthologs; and - Cas9, Cas12a, Cas12b, CasX or Cas12e, CRISPR type V, Cas12j or CasΦ functional mutants or variants; It is understood that the present invention relates to a polypeptide conjugate as described above, which is selected from among:

[0013] By "Cas9 nuclease" is meant, inter alia, 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. More specifically, it encompasses Cas9s of SEQ ID NOs: 87-95 and mutants thereof. In view of the above, in another embodiment, the present invention provides a method for the preparation of a Cas protein comprising: - 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 - Cas9 orthologues and Cas9 mutants or functional variants derived from these organisms; a Cas9 protein selected from And in particular, the Cas9 protein is selected from the sequences of SEQ ID NOs: 87 to 95 and mutants thereof; The present invention relates to a polypeptide conjugate as described above.

[0014] By "Cas12a (Cpf1) nuclease" is meant in particular that of a wild-type or improved Lachnospiraceae bacterium or Acidaminococcus sp. carrying the mutations E174R / S542R or E174R / S542R / K548R. More specifically, it corresponds to Cas12a of the sequences set forth in SEQ ID NOs: 105 and 106 and mutants thereof.

[0015] "Cas12b nuclease" refers in particular to those derived from Alicyclobacillus kakegawensis (AkCas12b) or Bacillus hisashii (BhCas12b), and the improved mutant version (K846R / S893R / E837G) of BhCas12b for gene editing. More specifically, it corresponds to the Cas12b sequence of SEQ ID NO: 107 and its mutants.

[0016] By "CasX or Cas12e nuclease" is meant, in particular, those derived from Deltaproteobacteria (DpbCasX) or Planctomycetes (PlmCasX) that are wild-type or inactivated by N672A, E769A, and N935A mutations, or that have been improved for gene editing in mammalian cells, such as the DpbCasX_R3V2 or PlmCasX_R1V2 versions. More specifically, it corresponds to Cas12e of the sequences set forth in SEQ ID NOs: 112 and 113, and mutants thereof.

[0017] Type V CRISPR nucleases are smaller (400-700 AA) in size than Cas9, Cas12a, and CasX nucleases. These include Cas12f (or Cas14) proteins from Uncultured Archaea (Un1Cas12f1), Syntrophomonas palmitatica (SpCas12f1), or Acidibacillus sulfuroxidans (AsCas12f1), which are either wild-type or carry one or more mutations that abolish catalytic activity (D225A and E324A) or partially induce nickase production (R383A and D401A). More specifically, they correspond to Cas12f or Cas14 and their mutants in sequences 108-111.

[0018] "Cas12j or CasΦ nuclease" refers specifically to those of the Biggiephage clade that are also type V CRISPR-Cas.

[0019] In view of the above, according to another embodiment, the present invention provides a method for producing a Cas protein comprising: - Cas9, Cas12a, Cas12b, CasX or Cas12e, CRISPR type V (Cas12f or Cas14), Cas12j or CasΦ nuclease; - Cas9, Cas12a, Cas12b, CasX or Cas12e, CRISPR type V (Cas12f or Cas14), Cas12j or CasΦ orthologs; and - Cas9, Cas12a, Cas12b, CasX or Cas12e, CRISPR V type (Cas12f or Cas14), Cas12j or CasΦ functional mutants or variants, is selected from and in particular, the Cas protein is selected from the sequences of SEQ ID NOs: 87 to 95 and 105 to 113 and mutants thereof; Concerning polypeptide conjugates.

[0020] By "ortholog" is meant a similar Cas protein present in two or more different species.

[0021] "Mutant" refers to a Cas protein into which one or more mutations, including deletion, substitution, and / or addition of one or more amino acids, have been introduced, which can enhance or abolish nuclease activity or increase or decrease the fidelity of target DNA recognition.

[0022] A "functional mutant" is a Cas protein that has been modified by artificial intervention (e.g., genetic engineering) for the purpose of, for example, increasing the activity of the Cas.

[0023] "Functional variants" are Cas proteins that have been naturally modified through evolution, e.g., with increased (or decreased) activity.

[0024] In particular, the present invention relates to a method for producing a Cas protein comprising: - 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; Cas12a nucleases from Lachnospiraceae bacterium and Acidaminococcus sp; Cas12b nucleases from Alicyclobacillus kakegawensis and Bacillus hisashii; CasX or Cas12e nucleases from Deltaproteobacteria and Planctomycetes; CRISPR type V nucleases from Syntrophomonas palmitatica and Acidibacillus sulfuroxidans; Cas12j or CasΦ nucleases from the Biggiephage clade; - Cas9, Cas12a, Cas12b, CasX or Cas12e, CRISPR type V, Cas12j or CasΦ orthologs derived from these organisms; and - mutants or functional variants of Cas9, Cas12a, Cas12b, CasX or Cas12e, CRISPR V-type, Cas12j or CasΦ derived from these organisms, is selected from In particular, the Cas protein is selected from SEQ ID NOs: 87 to 95 and 105 to 113, and mutants thereof. It relates to a polypeptide conjugate as described above.

[0025] In particular, the present invention relates to a polypeptide conjugate as described above, wherein said Cas protein is selected from among the sequences of SEQ ID NOs: 87 to 95 and 105 to 113 and mutants thereof. In particular, the present invention relates to a polypeptide conjugate as described above, wherein said Cas9 protein is selected from among the sequences of SEQ ID NOs: 87 to 95 and mutants thereof. Even more particularly, the present invention relates to a polypeptide conjugate as described above, wherein said Cas protein is selected from among the sequences of SEQ ID NOs: 87, 88, 89, 90, 91, 92, 93, 94, 95, 105, 106, 107, 108, 109, 110, 111, 112 and 113.

[0026] According to another embodiment, the present invention provides a method for the preparation of a Cas protein comprising: - Cas9 nucleases derived from S. pyogenes, S. aureus, C. diphtheriae, N. meningitidis, S. canis, S. macacae, F. tularensis, Acidaminococcus, C. jejuni, S. pneumoniae and S. thermophilus; - Cas12a nuclease from Lachnospiraceae bacterium, Cas12b from Bacillus hisashii, Cas12f (Cas14) from uncultured archaea and Cas12j (CasΦ) from the Baggiephage clade; and - Cas9, Cas12a, Cas12b, Cas12f and Cas12j orthologues, and Cas9, Cas12a, Cas12b, Cas12f and Cas12j mutants or functional variants derived from these organisms; is selected from And in particular, the Cas protein is selected from SEQ ID NOs: 87 to 95 and 105 to 113 and mutants thereof; It relates to a polypeptide conjugate as described above.

[0027] For example, mutants (variants) of S. pyogenes Cas9 (spCas9), of which SEQ ID NO: 87 is a representative sequence, exist in which amino acids have been modified at specific positions to abolish nuclease activity and increase the fidelity of target DNA sequence recognition. These mutations can be as follows: substitution of amino acids D10, E762, D839, H840, H863, H983, and / or D986 with different amino acids, such as alanine, or with non-natural amino acids, thereby reducing, substantially eliminating, or inhibiting nuclease activity. Other amino acid substitutions increase target DNA sequence recognition specificity and reduce off-target binding to other DNA sequences that share some homology with the targeted sequence. In the case of spCas9, these mutations can occur at one, two, three, four, five, six, and / or all seven of the following positions: L169, Y450, N497, R661, Q695, Q926, and / or D1135, generating so-called high-fidelity Cas9 (hifi spCas9) (Kleinstiver, B.P. et al., Nature. 2016;529(7587):490-495). Other distinct mutations have also been shown to increase spCas9 specificity, including 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 mutants also significantly increases the precision of spCas9 binding to target DNA sequences; these combine the substitutions N692A, M694A, Q695A, and H698A to form the ultra-precise HyppaCas9 (Chen, JS et al., Nature. 2017;550(7676):407-410). Furthermore, all mutations that abolish the catalytic activity of spCas9 can be associated with this increased specificity of DNA sequence recognition.

[0028] Similarly, mutants of S. aureus Cas9 (saCas9), represented by SEQ ID NO: 88, exist, in which amino acid changes D10A or N580A inactivate the RuvC and HNH nuclease domains, respectively, converting saCas9 into a nickase (Friedland, AE et al., Genome Biol. 2015;16:257). High-fidelity saCas9 can be obtained by mutagenesis and mutation of Y211A, Y212A, W229A, Y230, R245A, T392A, N413A, N419A, Y651A, and R654A, either alone or in combination, favoring a higher on- / off-target ratio compared to wild-type saCas9 (Tan, Y et al., Proc Natl Acad Sci USA. 2019;116(42):20969-20976). Furthermore, the saCas9 E782K / N968K / R1015H and E782K / K929R / R1015H triple mutants exhibit broader site recognition by being able to cleave sequences followed by the simpler "NNNRRT" PAM instead of the "NNGRRT" motif. Finally, combinations of the above mutations may lead to mixed functional improvements, such as high-fidelity saCas9 nucleases or nickases with broader PAM recognition.

[0029] There are also organisms in nature that encode Cas nucleases smaller than Cas9, Cas12a (Cpf1), or Cas12b (1,000-1,500 AA). This is the case for CasX or Cas12e nucleases (less than 1,000 AA), Cas12f or Cas14 (400-700 AA), and Cas12j or CasΦ (700-800 AA), which provide a natural reservoir of compact Cass that allows 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(1):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 Planctomyces (PlmCasX), have been modified for gene editing, such as their wild-type versions, or DpbCasX-R3 (a chimeric DpbCasX harboring the R3 loop of PlmCasX) or PlmCasX-R1 (a chimeric PlmCasX harboring the R1 loop of DpbCasX) versions, or versions of CasX inactivated by the N672A, E769A, and N935A mutants, alone or in combination, to efficiently program mammalian cells to induce DNA cleavage or 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(1):94-102; Liu, Jun-Jie, et al., 2019 Nature.566(7743):218~223).Cas12f nuclease. Type V CRISPR nucleases are smaller in size (400-700 AA) than Cas9, Cas12a, and CasX nucleases; for example, Cas12f (or Cas14) proteins from uncultured archaea (Un1Cas12f1), Syntrophomonas palmitatica (SpCas12f1), or Acidibacillus sulfuroxidans (AsCas12f1), whether wild-type or carrying one or more mutations, either completely abolish the catalytic activity of Cas12f nuclease (D225A and E324A for Un1Cas12f1, or D326A and / or D510A for Un1Cas12f1) or partially abolish the nuclease activity leading to the production of nickases (R383A and D401A for AsCas12f1) (Do Yon et al., 2014). Kim, et al., 2022, Nature Biotechnology. 40(1):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 Cas12f can be fused not only to transcriptional activator or inhibitor domains to program the Cas12f protein for targeted expression of certain cellular genes, but also to base editor or deoxyadenosine deaminase enzyme domains to enable targeted transformation of A or T bases to G or C for repair of or introduction of point mutations into the genome of mammalian cells (Do Yon Kim, et al., 2022, Nature Biotechnology. 40(1):94-102; Xiaoshu Xu et al., 2021 Mol Cell. 81(20):4333-4345.e4). Bacteriophage Cas12j or CasΦ nucleases have the ability to introduce double-strand breaks in DNA sequences recognized by guide RNAs.The rate of this cleavage can be accelerated approximately 20-fold by mutations introduced into the α7 helix (E159A, S160A, S164A, D167A, E168A) or by replacing the negatively charged fragment with a short glycine-serine stretch (Pausch, P. et al., 2021, Nature Structural & molecular biology. 28(8):652-661).

[0030] Thus, in general terms, the invention may encompass a polypeptide conjugate comprising a first component that is an antigen-presenting cell antibody directed against the asialoglycoprotein receptor (anti-APC-ASGPR), or a fragment thereof, covalently or non-covalently linked to a second component that is one of the above-mentioned Cas proteins.

[0031] "Antigen-presenting cells (APCs)" refer to cells of the immune system that present cellular components to T lymphocytes. These are monocytes, macrophages, B lymphocytes, or dendritic cells. In particular, these are dendritic cells (DCs). Also, and according to certain embodiments, the present invention relates to polypeptide conjugates comprising a first component that is an anti-dendritic cell antibody targeting the asialoglycoprotein receptor (anti-DC-ASGPR), or a fragment thereof, covalently or noncovalently linked to a second component that is a Cas protein.

[0032] The "asialoglycoprotein receptor," abbreviated as ASGPR and also known as 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, for subsequent processing and presentation on major histocompatibility complex (MHC) molecules. Furthermore, some CLRs, including ASGPR, can initiate signaling cascades that modulate dendritic cell function and influence the elicited immune response. Unlike other Syk-related CLRs, activated ASGPR does not induce NF-κB activation but instead leads to CREB phosphorylation (Gu C et al., J Immunol. 2019;203(2):389-399).

[0033] Thus, the signaling cascade of activated ASGPR leads to the activation of Syk, PLCγ2, ΠKXδ, and then the MAPKs ERK1 / 2 and JNK, which, upon prolonged stimulation, leads to the phosphorylation of p90RSK and CREB, inducing the transcription of the anti-inflammatory cytokine IL-10, a specific end result of antigen endocytosis by DC-ASGPR.

[0034] "Antibody" refers to an immunoglobulin, a four-chain multimeric protein involved in acquired immune responses. Immunoglobulins are well known to those skilled in the art and are composed of an assembly of two dimers, each consisting of a heavy chain and a light chain. The multimeric complex is assembled by linking the light chain to the heavy chain through a disulfide bridge between two cysteines, and the two heavy chains are also linked together by two disulfide bridges.

[0035] The heavy and light chains each consist of a constant region and a variable region. The collection of chains that make up an antibody allows it to define a characteristic three-dimensional Y structure, in which: - the bases of Y correspond to the Fc constant region recognized by complement and Fc receptors, and - At the end of the Y arms there is a collection of variable regions, light and heavy chains respectively.

[0036] More specifically, each light chain comprises a variable region (V L ) and constant region (C L Each heavy chain is composed of a variable region (V H ) and three constant domains C H 1. C H 2 and C H It consists of a constant region consisting of domain C and domain 3. H 2 and C H The light chain variable region is composed of three antigen recognition regions (ARRs) surrounded by four framework domains. The heavy chain variable region is also composed of three complementarity 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, allowing them to form a specific structure that recognizes a given antigen.

[0037] The antibodies described in this invention are isolated and purified and belong to any isotype / class (e.g., IgG, IgE, IgM, IgD, IgA, and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) and are distinct from naturally occurring antibodies. These antibodies are mature, i.e., they have an ad hoc three-dimensional structure that allows antigen recognition, and they possess all the post-translational modifications essential for antigen recognition, including glycosylation and the formation of intra- and intermolecular disulfide bridges.

[0038] More specifically, these antibodies are "monoclonal antibodies," meaning that they recognize only one antigenic determinant on DC-ASGPR, which differs from polyclonal antibodies, which are a mixture of antibodies and can therefore recognize multiple antigenic determinants on the same protein.

[0039] By "fragment thereof" is meant any portion of an antibody of the present invention that retains the ability to bind to the epitope recognized by the intact antibody. Examples of such fragments include, but are not limited to, Fab, Fab' and F(ab'), Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-bridged Fv (dsFv), and V. L or V H Epitope-binding fragments, including single-chain antibodies, may be fragments comprising the hinge region, C, or C alone or in combination with other fragments. H 1. C H 2 and C H The fragments may contain the variable region in combination with all or some of the three domains. Such fragments may comprise either or both of the Fab fragment or the F(ab')2 fragment. Furthermore, the fragments may be members of any of the immunoglobulin classes, such as IgG, IgM, IgA, IgD, or IgE, and their subclasses, or may be combinations thereof.

[0040] Fab and F(ab')2 fragments can be produced by proteolytic cleavage using enzymes such as papain (Fab fragments) or pepsin (F(ab')2 fragments). "Single-chain Fv" ("scFv") fragments contain a light chain antibody variable region (V L ) linked to at least one fragment of an antibody variable region (V H ) are epitope-binding fragments that contain at least one fragment of the V L and V H It may be a short, flexible peptide selected to ensure that the correct three-dimensional folding of the region occurs, thereby maintaining the binding specificity for the target molecule of the whole antibody from which the single-chain antibody fragment is derived. L or V H The carboxyl terminus of the sequence is connected by a linker to the complementary V L or V H It may be covalently attached to the amino terminus of the sequence.

[0041] Furthermore, "antigen-presenting cell antibodies targeting APC asialoglycoprotein receptor (anti-DC-ASGPR) or fragments thereof" refers to proteins capable of specifically recognizing DC-ASGPR. These proteins include seven monoclonal antibodies that recognize human DC-ASGPR: 49C11, 49C11_bis, 1H11, 5F10, 4G2.2, 6.3H9.1D11, and 5H8.1D4.

[0042] By "49C11" antibody is meant an antibody that includes: a murine variable region targeting human DC-ASGPR of sequence SEQ ID NO: 4, comprising, from N-terminus to C-terminus, CDR1 of sequence SEQ ID NO: 1, CDR2 of sequence SEQ ID NO: 2 and CDR3 of sequence SEQ ID NO: 3; a fusion protein with the constant region of the human IgG4 heavy chain; a heavy chain of sequence SEQ ID NO: 10, which is a synthetic construct comprising: and a murine variable region targeting human DC-ASGPR of sequence SEQ ID NO: 19, comprising, from N-terminus to C-terminus, CDR1 of sequence SEQ ID NO: 16, CDR2 of sequence SEQ ID NO: 17 and CDR3 of sequence SEQ ID NO: 18; o a fusion protein with the constant region of the kappa chain of human IgG4; A synthetic construct comprising a light chain of sequence SEQ ID NO: 25.

[0043] The following five variants of the 49C11 antibody described above were also developed: a heavy chain of sequence SEQ ID NO: 11 comprising a variable region of sequence SEQ ID NO: 5, which comprises, from the N-terminus to the C-terminus, a CDR1 of sequence SEQ ID NO: 1, a CDR2 of sequence SEQ ID NO: 2 and a CDR3 of sequence SEQ ID NO: 3; and a light chain of sequence SEQ ID NO: 26 comprising a variable region of sequence SEQ ID NO: 20, which comprises, from the N-terminus to the C-terminus, a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence SEQ ID NO: 17 and a CDR3 of sequence SEQ ID NO: 18; Contains 49C11_var1; a heavy chain of sequence SEQ ID NO: 12 comprising a variable region of sequence SEQ ID NO: 6, which comprises, from the N-terminus to the C-terminus, a CDR1 of sequence SEQ ID NO: 1, a CDR2 of sequence SEQ ID NO: 2 and a CDR3 of sequence SEQ ID NO: 3; and a light chain of sequence SEQ ID NO: 27 comprising a variable region of sequence SEQ ID NO: 21, which comprises, from the N-terminus to the C-terminus, a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence SEQ ID NO: 17 and a CDR3 of sequence SEQ ID NO: 18; Contains 49C11_var2; a heavy chain of sequence SEQ ID NO: 13 comprising a variable region of sequence SEQ ID NO: 7, which comprises, from the N-terminus to the C-terminus, a CDR1 of sequence SEQ ID NO: 1, a CDR2 of sequence SEQ ID NO: 2 and a CDR3 of sequence SEQ ID NO: 3; and a light chain of sequence SEQ ID NO: 28 comprising a variable region of sequence SEQ ID NO: 22, which comprises, from the N-terminus to the C-terminus, a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence SEQ ID NO: 17 and a CDR3 of sequence SEQ ID NO: 18; Contains 49C11_var3; a heavy chain of sequence SEQ ID NO: 14 comprising a variable region of sequence SEQ ID NO: 8, which comprises, from the N-terminus to the C-terminus, a CDR1 of sequence SEQ ID NO: 1, a CDR2 of sequence SEQ ID NO: 2 and a CDR3 of sequence SEQ ID NO: 3; and a light chain of sequence SEQ ID NO: 29 comprising a variable region of sequence SEQ ID NO: 23, which comprises, from the N-terminus to the C-terminus, a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence SEQ ID NO: 17 and a CDR3 of sequence SEQ ID NO: 18; Contains 49C11_var4; a heavy chain of sequence SEQ ID NO: 15 comprising a variable region of sequence SEQ ID NO: 9, which comprises, from the N-terminus to the C-terminus, a CDR1 of sequence SEQ ID NO: 1, a CDR2 of sequence SEQ ID NO: 2 and a CDR3 of sequence SEQ ID NO: 3; and a light chain of sequence SEQ ID NO: 30 comprising a variable region of sequence SEQ ID NO: 24, which comprises, from the N-terminus to the C-terminus, a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence SEQ ID NO: 17 and a CDR3 of sequence SEQ ID NO: 18; Contains 49C11_var5;

[0044] By "49C11_bis" antibody is meant an antibody that includes: - a heavy chain of sequence SEQ ID NO: 32 comprising a variable region of sequence SEQ ID NO: 31, which comprises, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 1, a CDR2 of sequence SEQ ID NO: 2 and a CDR3 of sequence SEQ ID NO: 3; and - a light chain of sequence SEQ ID NO: 34 comprising a variable region of sequence SEQ ID NO: 33 which comprises, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence SEQ ID NO: 17 and a CDR3 of sequence SEQ ID NO: 18.

[0045] By "1H11" antibody is meant an antibody that includes: - a heavy chain of sequence SEQ ID NO: 39 comprising a variable region of sequence SEQ ID NO: 38, which comprises, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 35, a CDR2 of sequence SEQ ID NO: 36 and a CDR3 of sequence SEQ ID NO: 37; and - a light chain of sequence SEQ ID NO: 44 comprising a variable region of sequence SEQ ID NO: 43, which comprises, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 40, a CDR2 of sequence SEQ ID NO: 41 and a CDR3 of sequence SEQ ID NO: 42.

[0046] By "5F10" antibody is meant an antibody comprising: - a heavy chain of sequence SEQ ID NO: 49 comprising a variable region of sequence SEQ ID NO: 48, which comprises, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 45, a CDR2 of sequence SEQ ID NO: 46 and a CDR3 of sequence SEQ ID NO: 47; and - a light chain of sequence SEQ ID NO: 54 comprising a variable region of sequence SEQ ID NO: 53, which comprises, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 50, a CDR2 of sequence SEQ ID NO: 51 and a CDR3 of sequence SEQ ID NO: 52.

[0047] By "4G2.2" antibody is meant an antibody that includes: - a heavy chain of sequence SEQ ID NO: 59 comprising a variable region of sequence SEQ ID NO: 58, which comprises, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 55, a CDR2 of sequence SEQ ID NO: 56 and a CDR3 of sequence SEQ ID NO: 57; and - a light chain of sequence SEQ ID NO: 64 comprising a variable region of sequence SEQ ID NO: 63, which comprises, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 60, a CDR2 of sequence SEQ ID NO: 61 and a CDR3 of sequence SEQ ID NO: 62.

[0048] By "6.3H9.1D11" antibody is meant an antibody that includes: - a heavy chain of sequence SEQ ID NO: 69 comprising a variable region of sequence SEQ ID NO: 68, which comprises, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 65, a CDR2 of sequence SEQ ID NO: 66 and a CDR3 of sequence SEQ ID NO: 67; and - a light chain of sequence SEQ ID NO: 74 comprising a variable region of sequence SEQ ID NO: 73, which comprises, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 70, a CDR2 of sequence SEQ ID NO: 71 and a CDR3 of sequence SEQ ID NO: 72.

[0049] By "5H8.1D4" antibody is meant an antibody that includes: - a heavy chain of sequence SEQ ID NO: 79 comprising a variable region of sequence SEQ ID NO: 78, which comprises, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 75, a CDR2 of sequence SEQ ID NO: 76 and a CDR3 of sequence SEQ ID NO: 77; and - a light chain of sequence SEQ ID NO: 84 comprising a variable region of sequence SEQ ID NO: 83, which comprises, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 80, a CDR2 of sequence SEQ ID NO: 81 and a CDR3 of sequence SEQ ID NO: 82.

[0050] In summary of the above, the anti-DC-ASGPR according to a particular embodiment of the present invention has the following sequence:

[0051] [Table 1]

[0052] Thus, according to another embodiment, the subject of the present invention is a polypeptide conjugate as described above, comprising a first component which is an antigen-presenting cell antibody targeting the antigen-presenting cell asialoglycoprotein receptor (anti-DC-ASGPR), or a fragment thereof capable of binding to an epitope recognized by the complete antibody, covalently or non-covalently linked to a second component which is a Cas protein, 1. The polypeptide conjugate having the ability to induce immune tolerance to a Cas protein in a mammalian organism, in particular in a primate or human organism, comprising: The anti-DC-ASGPR comprises: a heavy chain comprising, from the N-terminus to the C-terminus, a CDR1 having the sequence SEQ ID NO: 1, a CDR2 having the sequence SEQ ID NO: 2, and a CDR3 having the sequence SEQ ID NO: 3; and a light chain comprising, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence SEQ ID NO: 17 and a CDR3 of sequence SEQ ID NO: 18; an antibody comprising: a heavy chain comprising, from the N-terminus to the C-terminus, a CDR1 having the sequence of SEQ ID NO: 35, a CDR2 having the sequence of SEQ ID NO: 36 and a CDR3 having the sequence of SEQ ID NO: 37; a light chain comprising, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 40, a CDR2 of sequence SEQ ID NO: 41 and a CDR3 of sequence SEQ ID NO: 42; an antibody comprising: a heavy chain comprising, from the N-terminus to the C-terminus, a CDR1 of sequence SEQ ID NO: 45, a CDR2 of sequence SEQ ID NO: 46 and a CDR3 of sequence SEQ ID NO: 47; a light chain comprising, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 50, a CDR2 of sequence SEQ ID NO: 51 and a CDR3 of sequence SEQ ID NO: 52; an antibody comprising: a heavy chain comprising, from the N-terminus to the C-terminus, a CDR1 of the sequence SEQ ID NO: 55, a CDR2 of the sequence SEQ ID NO: 56 and a CDR3 of the sequence SEQ ID NO: 57; and a light chain comprising, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 60, a CDR2 of sequence SEQ ID NO: 61 and a CDR3 of sequence SEQ ID NO: 62; an antibody comprising: a heavy chain comprising, from the N-terminus to the C-terminus, a CDR1 of sequence SEQ ID NO: 65, a CDR2 of sequence SEQ ID NO: 66 and a CDR3 of sequence SEQ ID NO: 67; a light chain comprising, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 70, a CDR2 of sequence SEQ ID NO: 71 and a CDR3 of sequence SEQ ID NO: 72; an antibody comprising: a heavy chain comprising, from the N-terminus to the C-terminus, a CDR1 of the sequence SEQ ID NO: 75, a CDR2 of the sequence SEQ ID NO: 76 and a CDR3 of the sequence SEQ ID NO: 77; a light chain comprising, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 80, a CDR2 of sequence SEQ ID NO: 81 and a CDR3 of sequence SEQ ID NO: 82; an antibody comprising: It is understood that the polypeptide conjugate is selected from among:

[0053] According to another embodiment, the present invention relates to a method for treating DC-ASGPR comprising administering to a subject therapies comprising administering to said subject subject the anti-DC-ASGPR antibody: - an antibody comprising a heavy chain comprising a variable region of the sequence SEQ ID NO: 4, 5, 6, 7, 8, 9 or 31 and a light chain comprising a variable region of SEQ ID NO: 19, 20, 21, 22, 23, 24 or 33; - an antibody comprising a heavy chain comprising a variable region of sequence SEQ ID NO: 38 and a light chain comprising a variable region of sequence SEQ ID NO: 43; - an antibody comprising a heavy chain comprising a variable region of sequence SEQ ID NO: 48 and a light chain comprising a variable region of sequence SEQ ID NO: 53; - an antibody comprising a heavy chain comprising a variable region of sequence SEQ ID NO: 58 and a light chain comprising a variable region of sequence SEQ ID NO: 63; - an antibody comprising a heavy chain comprising a variable region of sequence SEQ ID NO: 68 and a light chain comprising a variable region of sequence SEQ ID NO: 73; - an antibody comprising a heavy chain comprising a variable region of sequence SEQ ID NO: 78 and a light chain comprising a variable region of sequence SEQ ID NO: 83; The present invention relates to a polypeptide conjugate as described above, selected from:

[0054] According to another embodiment, the present invention relates to a method for treating DC-ASGPR comprising administering to a subject therapies comprising administering to said subject subject the anti-DC-ASGPR antibody: - 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; - an antibody comprising a heavy chain of sequence SEQ ID NO: 39 and a light chain of sequence SEQ ID NO: 44; - an antibody comprising a heavy chain of sequence SEQ ID NO: 49 and a light chain of sequence SEQ ID NO: 54; - an antibody comprising a heavy chain of sequence SEQ ID NO: 59 and a light chain of sequence SEQ ID NO: 64; - an antibody comprising a heavy chain of sequence SEQ ID NO: 69 and a light chain of sequence SEQ ID NO: 74; - an antibody comprising a heavy chain of sequence SEQ ID NO: 79 and a light chain of sequence SEQ ID NO: 84; The present invention relates to a polypeptide conjugate as described above, selected from:

[0055] In this regard, and with respect to the sequences linked to antibodies 49C11, 49C11_var1, 49C11_var2, 49C11_var3, 49C11_var4, 49C11_var5 and 49C11_bis, the heavy and light chains share the same CDRs (see Table 1), making them interchangeable for the purpose of developing novel antibodies. Also, the phrase "an antibody comprising a heavy chain comprising a variable region of sequence SEQ ID NO: 4, 5, 6, 7, 8, 9 or 31 and a light chain comprising a variable region of sequence SEQ ID NO: 19, 20, 21, 22, 23, 24 or 33" is understood to mean - a heavy chain comprising a variable region of sequence SEQ ID NO: 4 and a light chain comprising a variable region of sequence SEQ ID NO: 19; - a heavy chain comprising a variable region of sequence SEQ ID NO: 5 and a light chain comprising a variable region of sequence SEQ ID NO: 20; - a heavy chain comprising a variable region of sequence SEQ ID NO: 6 and a light chain comprising a variable region of sequence SEQ ID NO: 21; - a heavy chain comprising a variable region of sequence SEQ ID NO: 7 and a light chain comprising a variable region of sequence SEQ ID NO: 22; - a heavy chain comprising a variable region of sequence SEQ ID NO: 8 and a light chain comprising a variable region of sequence SEQ ID NO: 23; - a heavy chain comprising a variable region of sequence SEQ ID NO: 9 and a light chain comprising a variable region of sequence SEQ ID NO: 24; and - a heavy chain comprising a variable region of sequence SEQ ID NO: 31 and a light chain comprising a variable region of sequence SEQ ID NO: 33; an antibody comprising for example: - a heavy chain comprising a variable region of sequence SEQ ID NO: 4 and a light chain comprising a variable region of sequence SEQ ID NO: 20; - a heavy chain comprising a variable region of sequence SEQ ID NO: 7 and a light chain comprising a variable region of sequence SEQ ID NO: 19; - a heavy chain comprising a variable region of sequence SEQ ID NO: 6 and a light chain comprising a variable region of sequence SEQ ID NO: 24; - a heavy chain comprising a variable region of sequence SEQ ID NO: 6 and a light chain comprising a variable region of sequence SEQ ID NO: 33; - etc; an antibody comprising It means both.

[0056] 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 antibodies comprising heavy and light chains of sequences SEQ ID NO: 10 and 25, 11 and 26, 12 and 27, 13 and 28, 14 and 29, 15 and 30 or 32 and 34, respectively, as well as antibodies comprising heavy and light chains such as SEQ ID NO: 10 and 30, 10 and 29, 13 and 25, 14 and 26, respectively.

[0057] Thus, generally speaking, the present invention may also cover a polypeptide conjugate comprising a first component which is an antigen-presenting cell antibody targeting the asialoglycoprotein 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 Cas proteins described above.

[0058] "Covalently linked" means that the polypeptide conjugate of the present invention can be covalently linked, i.e., the first and second components are linked by a covalent bond (with or without a linker), also known as a molecular bond, which is a strong chemical bond involving the sharing of electron pairs between atoms of the first and second components. Thus, a subject of the present invention is a polypeptide conjugate as described above, in which the anti-DC-ASGPR is covalently linked to the Cas protein. In particular, the present invention relates to a polypeptide conjugate as described above, in which the antigen-presenting cell antibody (anti-DC-ASGPR) targeting the asialoglycoprotein receptor 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 49C11_bis antibody, a subject of the present invention is a polypeptide conjugate as described above comprising the sequences of SEQ ID NO: 102 (heavy chain of 49C11_bis of sequence SEQ ID NO: 32 covalently linked at its C-terminus to saCas9 of sequence SEQ ID NO: 89) and SEQ ID NO: 34 (light chain of 49C11_bis). With regard to this non-limiting example, it should be pointed out that all structures possible from the above-mentioned antibody and the above-mentioned Cas that can be developed by a person skilled in the art are part of the present invention.

[0059] According to another embodiment, the present invention relates to a polypeptide conjugate as described above, wherein said anti-APC-ASGPR is covalently linked to said Cas protein, in particular wherein said polypeptide conjugate comprises the sequences of SEQ ID NOs: 102 and 34.

[0060] Advantageously, the present invention relates to a polypeptide conjugate as described above, wherein said anti-DC-ASGPR is covalently linked to said Cas protein by means of a linker, in particular a peptide linker.

[0061] It should be pointed out that among the available linkers, i.e., small molecules or peptides used to link the anti-DC-ASGPR and Cas proteins, some of which can incorporate glycosylation sites or introduce specific secondary structures, increase the expression efficiency or stability of the fusion protein and thus its efficiency. For the purposes of the present invention, the following peptide linkers are used: - QTPTNTISVTPTNNNSTPTNNSNPKPNPAS (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) It should be pointed out that this is not limited to:

[0062] According to another particular embodiment, the present invention provides a method for preparing a peptide linker comprising: - QTPTNTISVTPTNNNSTPTNNSNPKPNPAS (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), The present invention relates to a polypeptide conjugate as described above, selected from:

[0063] In particular, the subject of the present invention is a polypeptide conjugate as described above, in which an antigen-presenting cell antibody (anti-DC-ASGPR) targeting the APC asialoglycoprotein receptor as described above is covalently linked via its heavy chain to a second component, which is one of the above-mentioned Cas proteins, via a peptide linker. As an example and based on the 49C11_bis antibody, the subject of the present invention is a polypeptide conjugate as described above, comprising the sequences of SEQ ID NO: 101 (the heavy chain of 49C11_bis of sequence SEQ ID NO: 32 covalently linked at its C-terminus to a linker of sequence SEQ ID NO: 96, which is covalently linked to a Cas9 of sequence SEQ ID NO: 89) and SEQ ID NO: 34 (the light chain of 49C11_bis). With regard to this non-limiting example, it should be pointed out that all structures possible from the above-mentioned antibodies and the above-mentioned Cas proteins that can be developed by a person skilled in the art are part of the present invention.

[0064] According to another embodiment, the present invention provides a method for treating a steroid hormone comprising administering to a subject ... - QTPTNTISVTPTNNNSTPTNNSNPKPNPAS (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 of SEQ ID NOs: 101 and 34.

[0065] By "non-covalently bound" it is meant that the polypeptide conjugate of the present invention may also be non-covalently bound, i.e. the first and second components are linked by a weak bond, also called a non-covalent interaction, which does not involve the sharing of electrons between these first and second components. In particular, according to a second particular embodiment, it is understood that the present invention relates to a polypeptide conjugate as described above, wherein said anti-APC-ASGPR is non-covalently bound to said Cas protein.

[0066] For this purpose, it is possible to utilize non-covalent high affinity interactions that exist between two partners, known to those skilled in the art, such as, but not limited to, antibody / antigen interactions, receptor / ligand interactions, avidin / biotin interactions, cohesin / dockerin interactions, and barnase / barstar interactions. Also, according to another particular embodiment, the present invention provides a method for treating DC-ASGPR, wherein the anti-DC-ASGPR comprises: - Antibody / antigen interaction; - Receptor / ligand interactions; - Avidin / biotin interaction; - Cohesin / Dockerin interactions; and - Barnase / Barstar interaction; The present invention relates to a polypeptide conjugate as described above, which is non-covalently bound to said Cas protein using a high affinity interaction selected from the group consisting of:

[0067] In particular, the present invention also relates to a polypeptide conjugate as described above, wherein said high affinity interaction is a cohesin / dockerin interaction.

[0068] In particular, the present invention relates to a polypeptide conjugate as described above, in which an antigen-presenting cell antibody targeting 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. As an example and based on the 49C11_bis antibody, a subject of the present invention is a polypeptide conjugate as described above comprising the sequences SEQ ID NO: 103 or 86 (heavy chain of 49C11_bis of sequence SEQ ID NO: 32 covalently linked at its C-terminus to a dockerin), SEQ ID NO: 34 (light chain of 49C11_bis) and SEQ ID NO: 104 (Cas9 of sequence SEQ ID NO: 89 covalently linked at its N-terminus to a cohesin). With regard to this non-limiting example, it should be pointed out that all possible structures that a person skilled in the art can develop based on the above-mentioned antibodies, the above-mentioned Cas proteins and the above-mentioned means of non-covalent high-affinity interactions form part of the present invention. Similarly, if the above-mentioned structures show a dockerin linked to an antibody component and a cohesin linked to a Cas component, it is also possible, whatever the means of non-covalent high-affinity interaction used, to achieve the opposite, i.e., to place the cohesin on the antibody component and the dockerin on the Cas component of a polypeptide conjugate of the present invention.

[0069] According to another embodiment, the present invention relates to a polypeptide conjugate as described above, wherein said high affinity interaction is a cohesin / dockerin interaction, in particular wherein said polypeptide conjugate comprises the sequences of SEQ ID NOs: 103, 34 and 104 or the sequences of SEQ ID NOs: 85, 25 and 104.

[0070] [vector] According to a second aspect of the present invention, the present invention relates to a vector encoding a polypeptide conjugate of the present invention. In other words, the present invention comprises at least one nucleic acid comprising or consisting of a sequence encoding a first component, which is an antigen-presenting cell antibody targeting 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," we mean that the present invention may comprise two or three nucleic acids. For example, one nucleic acid encodes the anti-DC-ASGPR as described above or a fragment thereof as described above, and another nucleic acid encodes one of the aforementioned Cas proteins; or two nucleic acids encode the anti-DC-ASGPR as described above (e.g., one nucleic acid for the light chain and the second nucleic acid for the heavy chain), and a third nucleic acid encodes one of the aforementioned Cas proteins. However, it should be pointed out that the present invention, in its covalent configuration, preferably uses only one nucleic acid comprising or consisting of a sequence encoding a first component which is an antigen-presenting cell antibody targeting 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.

[0071] Thus, according to this second aspect, a subject of the present invention is an expression vector comprising at least one nucleic acid as defined above, said at least one nucleic acid being under the control of elements (promoters) allowing its expression.

[0072] An "expression vector" refers to a DNA (deoxyribonucleic acid) molecule containing elements that allow replication (duplication) in at least one organism. These elements that allow replication are, in particular, origins of replication in yeast or bacteria, or elements that control viral replication. Vectors of the present invention include, inter alia, plasmids, phages, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), and modified genomes of replicating or integrating viruses. Some vectors carry viral genomes (replicating) or genomes derived from viruses but lacking viral genes (non-replicating), DNA (deoxyribonucleic acid) or RNA (ribonucleic acid). The RNA genome can be reverse transcribed into DNA by retroviral reverse transcriptase or directly translated by the cellular machinery. The viral genome or virus-derived genome, DNA or RNA, can be encapsidated within the protective structure of viral proteins and the cell membrane. These recombinant virus or virus-derived particles allow transduction of target cells for expression of Cas proteins or anti-CD-ASGPR. This transgenic expression can be stable, cyclic or transient, depending on the process or promoter used.

[0073] These vectors are known as "expression" vectors because they contain nucleotide sequences that enable the expression of nucleotide sequences that they control, i.e., the transcription into RNA (ribonucleic acid) or the translation of the RNA that they carry.

[0074] In the present invention, at least one nucleic acid contained in the vector is "under the control of elements enabling its expression." This means that the expression vector has at least one transcription initiation sequence, such as a viral promoter such as the simian virus SV40 or cytomegalovirus (CMV) early promoter or the Rous sarcoma virus (RSV) promoter sequence, and in particular a sequence or promoter containing a TATAA box. It may be a human promoter of a housekeeping gene, such as the human promoter of the phosphoglycerate kinase (PGK) gene, or a so-called tissue-specific human promoter that is active only in certain subpopulations of cells in the body. It may also be a synthetic promoter containing one or more response elements that bind one or more transcription factors; these promoters may be inducible by exogenous stimuli such as pharmacological molecules, hormones, deficiencies, or stress. Furthermore, the vector also has at least one transcription termination sequence, in particular a polyadenylation sequence derived from a mammalian gene, in particular a human gene.

[0075] These sequences, which are essential for the expression of the nucleotide sequence contained in the vector, may be supplemented by other sequences which allow for the regulation or modulation of the expression of said sequences. A non-exhaustive list includes introns of mammalian, especially human, genes, transcriptional regulatory sequences of the enhancer type or transcribed but untranslated sequences of mammalian, especially human, genes.

[0076] [Host cell] According to a third aspect, the present invention relates to a host cell or cell line transformed with a nucleic acid as defined above and / or an expression vector as defined above. In other words, a subject of the present invention is a host cell or cell line capable of expressing (producing) a polypeptide conjugate as defined above, comprising a first component which is an antigen-presenting cell antibody targeting the asialoglycoprotein receptor (anti-APC-ASGPR), or a fragment thereof, covalently or non-covalently linked to a second component which is a Cas protein.

[0077] [Composition] In another aspect, the present invention relates to a pharmaceutical composition comprising as an active ingredient a polypeptide conjugate as described above, comprising a first component which is an antigen-presenting cell antibody targeting 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 carrier.

[0078] "Pharmaceutical composition" refers to a specific packaged form of the present invention that allows for the administration of the pharmaceutical composition to animals and humans by oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical, inhalation, or rectal routes. Furthermore, the package also allows for the administration of the active ingredient (or active substance), either alone or in combination with another active ingredient, in unit form or mixed with a conventional pharmaceutical carrier. Suitable unit dosage forms include the following: - Oral dosage forms such as tablets, capsules, powders, granules and oral suspensions or solutions; - Sublingual and buccal administration, aerosols and implants; - subcutaneous, transdermal, intradermal, intraperitoneal, intramuscular, intravenous, intratracheal and intranasal administration forms; and - Rectal administration.

[0079] By "acceptable pharmaceutical vehicle" (or pharmaceutically acceptable vehicle) is meant 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: - Crystalloid solutions, e.g. sodium chloride, bicarbonate, glucose; - cationic lipids; - a peptide compound; or - Surfactants such as polysorbates.

[0080] In all cases, whatever formulation is selected must be sterile and stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0081] According to another embodiment, a subject of the present invention is a pharmaceutical composition as described above in unit form, in which the polypeptide conjugate of the invention is present in a (unit) dose of 0.1 to 1,000 μg or in a (unit) dose of 0.1 to 1,000 mg / kg (based on a 70 kg male). "0.1 to 1,000 μg (or mg / kg)" means that the dose may be 10 to 1,000, 100 to 900, 200 to 800, 300 to 700, 400 to 600, 0.1 to 500, 500 to 1,000, 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 1,000 μg (or mg / kg).Doses were 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, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0, 51.0, 52.0, 53.0, 54.0, 55.0, 56.0, 57.0, 58.0, 59.0, 60.0, 61.0, 62.0, 63.0, 64.0, 65 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, 9 3, 94, 95, 96, 97, 98, 99, 100, 110, 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, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560 , 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1,000 μg (or mg / kg).

[0082] According to another embodiment, the subject of the present invention is a pharmaceutical composition as described above, formulated to be administered by one of the following routes: oral, parenteral, injection, topical, inhalation, subcutaneous, nasal or pulmonary.

[0083] According to another embodiment, the present invention relates to a pharmaceutical composition as described above in combination with a second active ingredient. In particular, the present invention relates to a 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 known as CD134 or TNFRSF4), which is stably expressed on many antigen-presenting cells, such as DC2 (a subtype of dendritic cell), macrophages, and activated B lymphocytes.

[0084] [Use and Method] In another aspect, the present invention relates to a polypeptide conjugate according to the invention for use in promoting immune tolerance to the aforementioned Cas proteins. The present invention also relates to a pharmaceutical composition according to the invention for use in promoting immune tolerance to Cas proteins from bacteria, archaea or phages.

[0085] "Promoting immune tolerance to a Cas protein" means that administering a polypeptide conjugate of the present invention and / or a pharmaceutical composition of the present invention to a mammal induces (causes) immune tolerance to the Cas protein, thereby preventing any subsequent immune rejection of the associated Cas protein. In this way, increased immune tolerance to Cas enables the establishment of safe and effective gene repair protocols in humans using the CRISPR-Cas system.

[0086] In another embodiment, the present invention relates to a polypeptide conjugate of the present invention for use in promoting immune tolerance to a Cas protein, wherein the polypeptide conjugate is administered to a mammal. The present invention also relates to a pharmaceutical composition of the present invention for use in promoting immune tolerance to the Cas protein, wherein the pharmaceutical composition is administered to a mammal. By "mammal" is meant an animal organism, such as a primate or human (male, female, and child).

[0087] In another embodiment, the present invention relates to a polypeptide conjugate according to the present invention for use in promoting immune tolerance to a Cas protein, said polypeptide conjugate being present in a (unit) dose of 0.1 to 1,000 μg or a (unit) dose of 0.1 to 1,000 mg / kg (based on a 70 kg male). The present invention also relates to a pharmaceutical composition according to the present invention for use in promoting immune tolerance to a Cas protein, said pharmaceutical composition comprising a (unit) dose of 0.1 to 1,000 μg or a (unit) dose of 0.1 to 1,000 mg / kg (based on a 70 kg male) of the polypeptide conjugate according to the present invention. "0.1 to 1,000 μg (or mg / kg)" means that the dose may be 10 to 1,000, 100 to 900, 200 to 800, 300 to 700, 400 to 600, 0.1 to 500, 500 to 1,000, 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 1,000 μg (or mg / kg).Doses were 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, 21.0, 22.0, 23.0, 24.0, 25.0, 26.0, 27.0, 28.0, 29.0, 30.0, 31.0, 32.0, 33.0, 34.0, 35.0, 36.0, 37.0, 38.0, 39.0, 40.0, 41.0, 42.0, 43.0, 44.0, 45.0, 46.0, 47.0, 48.0, 49.0, 50.0, 51.0, 52.0, 53.0, 54.0, 55.0, 56.0, 57.0, 58.0, 59.0, 60.0, 61.0, 62.0, 63.0, 64.0, 65 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, 9 3, 94, 95, 96, 97, 98, 99, 100, 110, 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, 410, 420, 430, 440, 441, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560 , 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1,000 μg (or mg / kg).

[0088] In another embodiment, the present invention relates to a polypeptide conjugate of the present invention for use in promoting immune tolerance to a Cas protein, wherein said polypeptide conjugate is administered by one of the following routes: oral, parenteral, injection, topical, inhalation, subcutaneous, nasal, or pulmonary. The present invention also relates to a pharmaceutical composition of the present invention for use in promoting immune tolerance to a Cas protein, wherein said pharmaceutical composition is administered by one of the following routes: oral, parenteral, injection, topical, inhalation, subcutaneous, nasal, or pulmonary.

[0089] In another embodiment, the present invention relates to a polypeptide conjugate of the present invention for use in promoting immune tolerance to a Cas protein, wherein the polypeptide conjugate is administered in association with an anti-OX40L antibody or a fragment thereof. The present invention also relates to a pharmaceutical composition of the present invention for use in promoting immune tolerance to a Cas protein, wherein the pharmaceutical composition further comprises an anti-OX40L antibody or a fragment thereof.

[0090] According to another embodiment, the subject of the present invention is a polypeptide conjugate according to the invention for use in promoting immune tolerance to a Cas protein, in particular said polypeptide conjugate being administered in association with an anti-OX40L antibody or a fragment thereof.

[0091] The present invention also relates to a pharmaceutical composition according to the present invention for use in promoting immune tolerance to said Cas protein, in particular said pharmaceutical composition further comprising an anti-OX40L antibody or a fragment thereof.

[0092] Alternatively, the present invention relates to a pharmaceutical composition for use in promoting immune tolerance to a Cas protein, comprising as an active ingredient a polypeptide conjugate of the present invention in association with an acceptable pharmaceutical carrier, and particularly further comprising an anti-OX40L antibody or a fragment thereof.

[0093] Alternatively, the present invention relates to a method for promoting immune tolerance to a Cas protein in a patient in need thereof, said method comprising administering a polypeptide conjugate according to the invention. Another subject of the present invention is a method for promoting immune tolerance to said Cas protein in a patient in need thereof, said method comprising administering a pharmaceutical composition according to the invention.

[0094] Finally, it is understood that immunization of a patient in need (i.e., a patient suffering from a condition for which gene therapy based on the CRISPR-Cas system is indicated for treatment) with Cas according to the present invention provides the patient with the potential to benefit from a safe and effective gene therapy protocol. It is therefore understood that, to some extent, the present invention involves a recombinant protein (anti-DC-ASGPR-Cas) and a vector encoding the Cas protein. The recombinant anti-DC-ASGPR-Cas protein is injected, particularly intradermally, to immunize a patient in need thereof, after which the vector is administered to allow expression of Cas in a given genetically modified tissue.

[0095] In other words, the present invention also relates to a kit comprising: - a polypeptide conjugate of the invention or a vector encoding a polypeptide conjugate of the invention, or a pharmaceutical composition of the invention; and - a vector encoding a Cas protein, in particular encoding the same Cas protein as that present in the polypeptide conjugate according to the invention.

[0096] In this regard, it should be pointed out that the "vector encoding the Cas protein" in the kit does not correspond to the vector of the present invention as described above, and the purpose of the vector in the kit is to enable expression of Cas for gene therapy purposes (and not to generate the polypeptide conjugate of the present invention).

[0097] In any event, it should be pointed out that the various aspects of the invention, as well as the various embodiments thereof, are independent and can therefore be combined with one another as many times as necessary to obtain aspects and / or preferred embodiments of the invention not explicitly described. This also applies to all definitions provided herein that apply to all aspects and embodiments of the invention.

[0098] The present invention is further illustrated, but not limited to, by the following figures and examples. [Brief explanation of the drawings]

[0099] [Figure 1] The experimental protocol for vaccinating cynomolgus monkeys with anti-DC-ASGPR-Cas9 is shown. This protocol was carried out over a 42-day period. Blood samples were collected every 7 days. The first sample on day 0 (the same day) provided the baseline level for all subsequent measurements. Animals received three intradermal (ID) injections of anti-DC-ASGPR-Cas9 at weekly intervals on days 14, 21, and 28. Finally, animals received an injection of Cas9 protein on day 35. [Figure 2]Optimization of assembly of anti-DC-ASGPR-dockerin and saCas9-cohesin. Assembly of two molar ratios of 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). [Line 1]: Deposition of saCas9-cohesin alone (20 mm run). [Line 2]: Deposition of anti-DC-ASGPR-dockerin alone (8 mm run). [Line 3]: saCas9-cohesin and anti-DC-ASGPR-dockerin mixed at a molar ratio of 1:0.5 (smear and run 12 mm). [Line 4], saCas9-cohesin and anti-DC-ASGPR-dockerin mixed at a molar ratio of 1:2 (single band and 12 mm run). For anti-DC-ASGPR-saCas9 vaccination, anti-DC-ASGPR-dockerin and saCas9-cohesin proteins are used at a molar ratio of 1:2. [Figure 3] 1 shows a strategy for analyzing cell subtypes from cynomolgus monkey PBMCs by cytometry. [Figure 4] 1 shows a strategy for analyzing the regulatory phenotype of CD4+ lymphocytes from cynomolgus monkeys, which express the markers CD25, FOXP3, and CD39. [Figure 5] 1 shows cytometric analysis of CD4+ lymphocytes of interest under non-stimulated (NS) conditions or stimulated with Cas9 protein (saCas9) on protocol day 0. Dial numbers indicate the percentage of cells. [Figure 6] Cytometric analysis of the CD4+ lymphocytes of interest under non-stimulated (NS) conditions or stimulated with Cas9 protein (saCas9) on day 21 of the protocol is shown. Dial numbers indicate the percentage of cells. [Figure 7] 1 shows cytometric analysis of the CD4+ lymphocytes of interest under non-stimulated (NS) conditions or stimulated with Cas9 protein (saCas9) on day 35 of the protocol. The dial numbers indicate the percentage of cells. [Figure 8] The numbers of regulatory CD4+ T cells expressing FOXP3 and CD39 within the CD4+CD25+OX40+ cell population are shown. (A) Percentage of CD4+CD25+OX40+ lymphocytes (vertical dotted line) 7 days after one (day 21) and three (day 35) vaccinations with anti-DC-ASGPR-Cas9. (B) Number of Foxp3+CD39- lymphocytes within the CD4+CD25+OX40+ lymphocyte population. (C) Number of Foxp3+CD39+ lymphocytes within the CD4+CD25+OX40+ lymphocyte population. [Figure 9] Measurement of TGFb1 in serum of cynomolgus macaques after three vaccinations with anti-DC-ASGPR-Cas9 (day 35) and one week after immunization with recombinant Cas9 protein. A significant increase in TGFb1 levels in the serum of animals was observed one week after immunization with Cas9 protein. [Thin dotted line]: vaccination with anti-DC-ASGPR-Cas9; [Thick dotted line]: immunization with Cas9 protein. [Figure 10] Measurement of IL10 in culture supernatants of cynomolgus monkey PBMCs collected on days 21, 28, 35, and 42 after the first rhMOG immunization is shown. Increased IL10 secretion was observed in saCas9 (Cas9)-stimulated PBMCs collected on day 42 (1 week after the second rhMOG immunization), but not in the supernatants of non-stimulated (NS) PBMCs. [Example]

[0100] [Preclinical study: saCAS9 immune tolerization in cynomolgus monkeys via immunization with anti-APC-ASGPR-saCAS9] [Preliminary data] In vitro Anti-APC-ASGPR-saCas9 was obtained, which was constructed based on the monoclonal antibody 49C11 and S. aureus Cas9, and the resulting conjugate contained the following: - the light chain of the anti-DC-ASGPR monoclonal antibody 49C11_L (SEQ ID NO: 34): and - the heavy chain of the anti-DC-ASGPR monoclonal antibody 49C11_H (SEQ ID NO: 103) containing a dockerin domain that allows it to associate with the saCas9 protein (SEQ ID NO: 89) containing a cohesin domain.

[0101] [General experimental design] We established a protocol to induce immune tolerance in immunocompetent adult cynomolgus monkeys against the heterologous saCas9 protein (Fig. 1). We used a recombinant anti-DC-ASGPR-saCas9 protein obtained by combining an anti-DC-ASGPR-dockerin antibody with the saCas9-cohesin protein (Fig. 2). This procedure was then used to induce immune tolerance in OX40 + CD25 + FOXP3 and CD39 + saCas9-specific regulatory CD4 expressing markers + We determined whether this would allow the emergence of a population of T lymphocytes (Fovet CM, et al., BioMedicine. 2019 Sep;47:492-505. PMC6796575.). To this end, we established a blood mononuclear cell labeling strategy (Figure 3). This allowed us to identify circulating CD4 + This allows for the detection of lymphocytes, where the regulatory phenotype is CD25 + FOXP3 and CD39 + saCas9-specific regulatory CD4 T cells were identified by immunostaining for the antigen (Figure 4). 44 hours after priming with saCas9 protein, the presence of the activation marker OX40 confirmed the expression of saCas9-specific regulatory CD4 T cells. + T cells (CD25 + FOXP3 and CD39 - or CD39 + ) was detected (Figure 5).

[0102] In this protocol, one monkey is immunized by intradermal injection of anti-APC-ASGPR-saCas9 antibody.

[0103] [Vaccine Protocol] Animals received an intradermal dose of 250 μg of recombinant anti-APC-ASGPR-saCas9 protein on their backs weekly for 3 weeks (Figure 1).

[0104] [Immunization with saCas9] Seven days after receiving the final dose of vaccine, on day 35 of the protocol, animals were immunized with 250 μg of saCas9 protein.

[0105] [Immune analysis before and after vaccination] Starting on day 1, plasma and PBMCs were collected weekly to investigate CD4 responses to saCas9 in vitro.

[0106] Blood samples (10 mL) were taken from the animals before the first vaccination and at regular intervals thereafter to assess hematological and biological functions.

[0107] PBMCs from the animals were cultured. On the same day, cells were labeled with CFSE (carboxyfluorescein succinimidyl ester), cultured, and then stimulated with recombinant saCas9 or no stimulation at all. The labeled cells were collected 44 hours later and processed for analysis of intracellular cytokines and for studying their phenotype by flow cytometry. Some of these cells were used to assess the expression of CD4, CD8, CD25, OX40, CD39, and Foxp3 markers after stimulation with saCas9. Animals were examined daily, and clinical signs were quantified using a scoring grid.

[0108] moreover: -Animals were injected intradermally with anti-APC-ASGPR-saCas9 fusion protein on their backs; - The animals were injected intradermally with recombinant saCas9 protein on their backs; Resiquimod was applied as a gel to the shaved skin surface and the fusion protein injection site. The gel was applied immediately after the fusion protein injection, followed by a gentle "skin massage" for 30 seconds; - On each day of antibody vaccination, animals received five injections of 50 μg of fusion protein (total 250 μg); - On the day of injection of recombinant saCas9 protein, animals received five injections of 50 μg of saCas9 (total of 250 μg); -Immunosurveillance included: a. Anti-saCas9-specific T cells in the blood (CD4 + and CD8 + ) and measurement of TGFβ1 in serum. b. Monitoring of T and B cell activation markers 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.

[0109] [material and method] [animal] Adult cynomolgus monkeys (Macaca fascicularis) imported from Mauritius were included in this study. MHC class I and class II classification was determined.

[0110] [Immunogen] The recombinant proteins were prepared by the Baylor Institute for Immunology Research (BIIR). Anti-DC-ASGPR-dockerin (human) and saCas9-cohesin were combined at a 1:2 molar ratio for extemporaneous intradermal injection. Recombinant saCas9 produced in E. coli was obtained by BIIR.

[0111] [Immunization] The animals were injected into the back using an intradermal (ID) syringe, and the injection site was shaved.

[0112] Additionally, on each vaccination day, animals received five injections of 100 μL of each anti-DC-ASGPR-saCas9 vaccine, spaced 0.5 cm apart.

[0113] Each injection contains 50 μg of antibody.

[0114] [Lifelong observation] Animals were observed by CEA staff via webcam monitoring 7 days a week. Food and water consumption was recorded. Each time an animal was bled, a clinical examination was performed, and its weight and rectal temperature were recorded.

[0115] [Table 2]

[0116] [Complete blood test] Complete blood work was performed using HMX A / L (Beckman Coulter®) according to procedure SOP#EXAM006_01. Serum transaminases as well as LDL and PCSK9 protein were measured weekly.

[0117] saCas9-specific T cell activation, phenotype, and cytokine production saCas9-specific autoreactivity and regulatory CD4 in circulating PBMCs + The level of lymphocytes has not been studied in animals after saCas9 gene transfer. Their stimulation and large-scale culture alter their original phenotype and function, preventing accurate study. To assess and characterize these cells in macaques, we used CD4 overexpressing CD140 (OX40) and CD25 (IL2R), which allows for 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)). + A recent assay for detecting T lymphocytes was used. Ex vivo stimulation of PBMCs with recombinant saCas9 protein resulted in the expression of saCas9-specific CD4 T lymphocytes. + Phenotypic polarization of T cells was assessed over a 44-hour period. Under these conditions, saCas9-specific CD4 +T cells overexpress OX40 and CD25 before they begin to rapidly proliferate (N. Seddiki, et al., Eur J Immunol 44, 1644-61 (2014)). Therefore, cultured PBMCs stimulated with saCas9 epitopes were labeled with antibodies directed against CD4, CD134, CD25, CD39, and FOXP3 to identify T cells. regs and T R1 (CD4 + , CD25 + , CD134 + , CD39 + ) were distinguished (N. Seddiki, et al., Eur J Immunol 44, 1644-61 (2014)). Different cell subtypes were identified, quantified, and sorted by cytometry.

[0118] This experiment, which requires only a small amount of blood, demonstrated the ability of circulating anti-saCas9CD4 antibodies at different time points after vaccination with anti-DC-ASGPR-saCas9 antibodies. + It provided basic quantitative and qualitative information about the level and phenotype of cells.

[0119] [Serum cytokines] TGFb1 was measured at different time points before and after vaccination and one week after immunization with saCas9.

[0120] [Cytokines in PBMC culture supernatant] IL10 was measured in culture supernatants of PBMCs collected at different time points after immunization with rhMOG and vaccination with anti-ASGPR-saCas9, with or without stimulation of the cultured PBMCs with saCas9.

[0121] [Device Storage] Biospecimens were stored frozen at −80°C (plasma, serum, RNA) or −135°C (PBMCs) for the duration of the study and for 2 years after the study completion.

[0122] [Data Analysis] Data were recorded and analyzed using an IDBS type data management system.

[0123] Statistical analysis was performed using nonparametric Mann-Whitney or Wilcoxon rank tests using PRISM® 8 (GraphPad®).

[0124] Flow cytometry analysis was performed using FlowJo® and SPICE® software.

[0125] [ethics] Animals were housed at the CEA facility in Fontenay-aux-Roses in accordance with institutional care standards and European Directive D8906, as well as the standards of the Office of Laboratory Animal Welfare (OLAW) of the National Institutes of Health (USA).

[0126] [result] Healthy adult cynomolgus macaques were treated to develop immune tolerance to the heterologous saCas9 protein using the vaccination protocol depicted in Figure 1 and described below. This was achieved using a recombinant anti-DC-ASGPR-saCas9 protein, obtained by combining two recombinant proteins: an anti-DC-ASGPR-dockerin antibody and a saCas9-cohesin protein (Figure 2). The animals then received an intradermal injection of anti-DC-ASGPR-saCas9. Concurrently, this procedure was performed on OX40 + CD25 + FOXP3 and CD39 + saCas9-specific regulatory CD4 expressing markers + To this end, a blood mononuclear cell labeling strategy was established (Figure 3), which was characterized by CD25 + OX40 + FOXP3 + and CD39 + Regulatory CD4 expressing markers +This allowed the detection of a novel population of T lymphocytes (Figure 4). These cells (i.e., saCas9-specific CD4 T cells) were not present prior to the vaccination protocol. + Regulatory T cells (CD25 + FOXP3 and CD39 - or CD39 + ) increased proportionally as the anti-DC-ASGPR-saCas9 vaccine regimen was completed (Figures 5-7). Moreover, total counts of these cells indicated that their absolute numbers were increased by activation with recombinant saCas9 protein (Figure 8). Finally, in animals that received three anti-DC-ASGPR-saCas9 injections, intradermal immunization with saCas9 induced the expression of the anti-inflammatory cytokines TGFb1 and IL10, as detected in the plasma or supernatants of cultured PBMCs, respectively (Figures 9 and 10).

[0127] Thus, the results demonstrate that vaccination with anti-DC-ASGPR-saCas9 protein leads to the emergence of a population of regulatory T lymphocytes, a hallmark of an anti-inflammatory immunoregulatory response in the presence of the saCas9 antigen.

Claims

1. 1. A polypeptide conjugate comprising a first component which is an antigen-presenting cell antibody targeting an antigen-presenting cell asialoglycoprotein receptor (anti-DC-ASGPR), or a fragment thereof capable of binding to an epitope recognized by the full-length antibody, covalently or non-covalently linked to a second component which is a Cas protein, Said polypeptide conjugate having the ability to induce immune tolerance to a Cas protein in a mammalian organism, in particular in a primate or human organism, The anti-DC-ASGPR comprises: a heavy chain comprising, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 1, a CDR2 of sequence SEQ ID NO: 2 and a CDR3 of sequence SEQ ID NO: 3; and a light chain comprising, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 16, a CDR2 of sequence SEQ ID NO: 17 and a CDR3 of sequence SEQ ID NO: 18; an antibody comprising: a heavy chain comprising, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 35, a CDR2 of sequence SEQ ID NO: 36 and a CDR3 of sequence SEQ ID NO: 37; a light chain comprising, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 40, a CDR2 of sequence SEQ ID NO: 41 and a CDR3 of sequence SEQ ID NO: 42; an antibody comprising: a heavy chain comprising, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 45, a CDR2 of sequence SEQ ID NO: 46 and a CDR3 of sequence SEQ ID NO: 47; a light chain comprising, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 50, a CDR2 of sequence SEQ ID NO: 51 and a CDR3 of sequence SEQ ID NO: 52; an antibody comprising: a heavy chain comprising, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 55, a CDR2 of sequence SEQ ID NO: 56 and a CDR3 of sequence SEQ ID NO: 57; and a light chain comprising, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 60, a CDR2 of sequence SEQ ID NO: 61 and a CDR3 of sequence SEQ ID NO: 62; an antibody comprising: a heavy chain comprising, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 65, a CDR2 of sequence SEQ ID NO: 66 and a CDR3 of sequence SEQ ID NO: 67; a light chain that comprises, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 70, a CDR2 of sequence SEQ ID NO: 71 and a CDR3 of sequence SEQ ID NO: 72; an antibody comprising: a heavy chain comprising, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 75, a CDR2 of sequence SEQ ID NO: 76 and a CDR3 of sequence SEQ ID NO: 77; a light chain comprising, from N-terminus to C-terminus, a CDR1 of sequence SEQ ID NO: 80, a CDR2 of sequence SEQ ID NO: 81 and a CDR3 of sequence SEQ ID NO: 82; an antibody comprising: A polypeptide conjugate selected from:

2. The Cas protein: -S. pyogenes, S. aureus, C. diphtheriae, N. Meningitidis, S. canis, S. macacae, F. tularensis, Acidaminococcus, C. jejuni, S. pneumoniae and S. pneumoniae. Cas9 nuclease from Thermophilus; Cas12a nuclease from Lachnospiraceae bacterium and Acidaminococcus sp.; Cas12b nuclease from Alicyclobacillus kakegawensis and Bacillus hisashii; CasX or Cas12e nuclease from Deltaproteobacteria and Plantomycetes; CRISPR from Syntrophomonas palmitatica and Acidibacillus sulfuroxidans Type V nuclease; Cas12j or CasΦ nuclease from the Biggiephage clade; Cas9, Cas12a, Cas12b, CasX or Cas12e, CRISPR type V, Cas12j or CasΦ orthologues derived from these organisms; and - Cas9, Cas12a, Cas12b, CasX or Cas12e, CRISPR type V, Cas12j or CasΦ mutants or functional variants derived from these organisms, is selected from In particular, the Cas protein is selected from among SEQ ID NOs: 87-95 and 105-113, and mutants thereof. The polypeptide conjugate of claim 1.

3. the anti-DC-ASGPR is covalently linked to the Cas protein; In particular, said polypeptide conjugate comprises the sequences of SEQ ID NOs: 102 and 34. A polypeptide conjugate according to claim 1 or 2.

4. The polypeptide conjugate of claim 3, wherein the anti-DC-ASGPR is covalently linked to the Cas protein by a linker, particularly a peptide linker.

5. The peptide linker is - QTPTNTISVTPTNNNSTPTNNSNPKPNPAS (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), is selected from 5. The polypeptide conjugate of claim 3 or 4, in particular, wherein said polypeptide conjugate comprises the sequences of SEQ ID NOs: 101 and 34.

6. 3. The polypeptide conjugate of claim 1 or 2, wherein the anti-DC-ASGPR is non-covalently bound to the Cas protein.

7. The anti-DC-ASGPR comprises: - antibody / antigen interactions; - receptor / ligand interactions; - avidin / biotin interaction; - cohesin / dockerin interactions; and - barnase / barstar interaction; 7. The polypeptide conjugate of claim 6, wherein the polypeptide conjugate is non-covalently bound to the Cas protein through a high affinity interaction selected from the group consisting of:

8. the high affinity interaction is a cohesin / dockerin interaction; 8. The polypeptide conjugate according to claim 6 or 7, in particular comprising the sequences of SEQ ID NOs: 103, 34 and 104 or the sequences of SEQ ID NOs: 85, 25 and 104.

9. A pharmaceutical composition for use in promoting immune tolerance to a Cas protein, comprising as an active ingredient a polypeptide conjugate described in any one of claims 1 to 8 in association with an acceptable pharmaceutical carrier, and in particular further comprising an anti-OX40L antibody or a fragment thereof.

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