Peptide conjugates and their use to promote CAS nuclease immune tolerance in genomic engineered 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 CRISPR-Cas (clustered regularly spaced short palindromic repeats-CRISPR associated proteins) systems for use in genomic engineered 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 immune tolerance to the CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR-associated protein) system for use in genome-engineered gene therapy. Background Art

[0002] Immune tolerance is a state in which the immune system does not react to substances or tissues that can provoke immune rejection in healthy organisms. Immune tolerance is caused by previous exposure to specific antigens and is different from the traditional elimination of foreign substances by (innate) immune mechanisms. Immune tolerance is important for normal physiology. Central tolerance is the main way by which the immune system learns to distinguish between self and non-self. Peripheral tolerance is essential to prevent the immune system from overreacting to various environmental entities (such as allergens, intestinal microorganisms, etc.) and antigens produced by phagocytic dead cells of the body. Central or peripheral tolerance defects are also the root of autoimmune diseases, leading to syndromes such as systemic lupus erythematosus, rheumatoid arthritis, type 1 diabetes and multiple sclerosis. However, recent findings have shown that it is possible to induce peripheral immune tolerance, for example by targeting the entry of antigens into antigen presenting cells (APCs) with the help of receptors present on macrophages and dendritic cells (DCs), namely dendritic cell asialoglycoprotein receptors (DC-ASGPR) (Li D et al., J Exp Med. 2012; 209 (1): 109-121).

[0003] CRISPR-Cas systems in bacteria, archaea, and large phages are adaptive defense systems that enable the destruction of mobile genetic elements (Makarova, KS. et al., 2020. Nat Rev Microbiol 18(2): 67-83), as well as the development of biotechnology tools for engineering the genomes of all cell types (Knott, GJ and Doudna, J. 2018 Science 361(6405): 866-869). For this purpose, RNA-guided nucleases of different families (e.g., Cas9 and Cas12 proteins) are used to enable recognition of double-stranded DNA sequences and their cutting at specific locations. Therefore, CRISPR-RNA or guide RNA programming nucleases for identifying target nucleic acid sequences are becoming one of the most effective biotechnology molecular compounds for genetic engineering and gene therapy for gene editing or gene expression control (FARan et al., Nature 520, 186-191 (2015); Knott, GJ and Doudna, J. 2018 Science 361 (6405): 866-869). Cas nucleases cut DNA at specific target sequences under the guidance of homology recognition between guide RNA and chromatin sites. Then, DNA repair mechanisms make it possible to modify gene sequences for therapeutic purposes (FARan et al., Nature 520, 186-191 (2015)). In addition, some Cas nuclease mutants can act as nickases, or completely lose their nuclease activity and assume other functions, such as chemical modification of DNA or gene transcription (Knott, GJ and Doudna, J. 2018 Science 361 (6405): 866-869). Since the CRISPR-Cas system enables precise gene editing in eukaryotic cells, it opens up real prospects for in vivo repair and gene expression, and makes it possible to envision curing currently incurable genetic or acquired diseases. For example, an initial I / II phase trial has been launched, using AAV viral vectors to transfer the Cas9 gene into retinal photoreceptor cells to repair the mutated RPE65 gene that causes Leber's congenital amaurosis (A. Mullard, Nat. Rev. Drug Discov. 18, 656-656 (2019)). In addition, other in vivo nucleotide base editing gene repair clinical protocols are also emerging to introduce base conversions through mutant Cas proteins with nickase activity for the treatment of hyperlipidemia or sickle cell anemia by modifying the Pcsk9 or hemoglobin gene, respectively (Ledford, H. Nature 2022).Finally, other clinical applications become possible with the use of Cas proteins that lack protease activity but are fused to domains that can directly regulate transcription of target genes (Jensen, TI. 2021 Genome Res. 31(11): 2120-2130) or to enzymatic domains that can modify DNA methylation at the locus and permanently eliminate or activate its expression (Pulecio, J. 2017. Cell Stem Cell 21(4): 431-447).

[0004] However, the application of Cas nucleases in medicine for in situ genetic modification of organs is hampered by the prospect of mutagenesis following Cas off-target activity or immune rejection of cells expressing this bacterial protein (CT Charlesworth et al., Nat. Med. 25, 249-254 (2019)). In order to reduce off-target mutagenesis of Cas, high-fidelity variants of Cas9 and Cas12 have been developed (BP Kleinstiver et al., Nature 529, 490-495 (2016)-IM Slaymaker et al. in Science 351, 84-8 (2016); Xiaoshu Xu, Augustine Chemparathy et al. 2021. Mol Cell 81(20):4333-4345.e4; Pausch, P. Soczek, KM. 2021 Nat Struct&mol biol 28(8):652-661; Tsuchida, CA. Zhang, S. 2022 Mol Cell 82(6):1199-1209.e6). However, the application of Cas proteins in medicine has a major immunogenicity problem, because about 80% of the population has humoral and cellular immunity to Cas9 orthologs of Streptococcus pyogenes (S. pyogenes) and Staphylococcus aureus (S. aureus), and these two bacteria are common human commensal bacteria (CT Charlesworth et al., Nat. Med. 25, 249-254 (2019)). Therefore, the risk of immune rejection cannot be ignored, and gene therapy based on the CRISPR-Cas system has failed. This has raised reasonable concerns about the use of Cas in gene therapy, because previous immunizations have severely compromised the effectiveness of genome editing in patients with normal immune function. Even if this disadvantage can be reduced by using Cas proteins that rarely come into contact with humans on a timely and short-term basis, the above problems cannot be solved if these proteins are administered again or expressed continuously. Summary of the invention

[0005] Faced with this major challenge to ensure safe and effective in vivo gene therapy, the inventors have created new polypeptide conjugates, compositions and methods that can induce immune tolerance to Cas proteins (especially Cas9) in mammalian organisms. With these tools, it is now possible to prevent immune rejection of somatic cells expressing Cas proteins and link it to transient, inducible or persistent gene expression of Cas proteins. Therefore, increasing immune tolerance to Cas proteins should lay the foundation for safe and effective protocols for gene repair or control of gene expression by CRISPR-Cas systems in humans. DETAILED DESCRIPTION

[0006] Peptide conjugates

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

[0008] In view of the above, it should also be understood that, according to a specific embodiment, the present invention relates to a polypeptide conjugate, comprising a first component covalently or non-covalently linked to a second component, the first component being an antigen presenting cell antibody targeting APC asialoglycoprotein receptor (anti-APC-ASGPR), or a fragment thereof capable of binding to an epitope recognized by the intact antibody, and the second component being a Cas protein, and the polypeptide conjugate is capable of inducing immune tolerance to the Cas protein in mammalian organisms, and in particular in primates and human organisms.

[0009] "Cas protein" generally refers to CRISPR-associated protein (clustered regularly interspaced short palindromic repeats-associated protein), while "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, which is an adaptive defense mechanism of prokaryotes for destroying invading foreign DNA. There is a wide diversity of CRISPR-Cas systems between bacteria and archaea, and even within the same species. This means that there are 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 cut single-stranded or double-stranded DNA and RNA. Despite this, the various components of the CRISPR-Cas system and their functional logic are similar throughout the prokaryotic kingdom.

[0010] The CRISPR-Cas system is composed of a family of sequences found in the genomes of bacteria, archaea, and large phages, which correspond to genes with adaptation, crRNA maturation, and interference functions. In order to interfere with the expression and replication of foreign DNA or RNA, the host expresses the Cas nuclease gene, i.e., the sequence motifs of foreign DNA or RNA obtained in the previous contact of bacteria with phages, plasmids, or removable genetic elements, which encode non-coding crRNA and express trans-activating crRNA (tracrRNA) sequences. The converted crRNA and tracrRNA form a duplex guide RNA (gRNA), which guides the Cas nuclease to the target DNA sequence for double-stranded cutting. DNA cutting is limited by the adjacent DNA motifs known as the protospacer adjacent motifs (PAM), which are unique to each CRISPR-Cas system.

[0011] This functional framework has deviated from its original prokaryotic immune function and is currently being used in biotechnology for targeting, processing, modification, destruction and programmed repair of genes. So far, a variety of Cas proteins (Cas9, Cas12a, Cas12b, CasX or Cas12e, Cas12f or Cas14, Cas12j or CasΦ) from different hosts have been identified and characterized, or even modified (e.g., to improve their function), which can be achieved by the present invention for the engineering of mammalian genomes. In general, the size of the above-mentioned Cas proteins decreases successively, Cas9 is 1,500 amino acids (AA), Cas12a is 1,000 AA, and CasX, Cas12f and Cas12j are less than 1,000 AA.

[0012] Therefore, it should be understood that, according to another embodiment, the present invention relates to a polypeptide conjugate as described above, wherein the Cas protein is selected from:

[0013] - Cas9, Cas12a, Cas12b, CasX or Cas12e, V-type CRISPR, Cas 12j or CasΦ nuclease;

[0014] - Orthologs of Cas9, Cas12a, Cas12b, CasX or Cas12e, V-type CRISPR, Cas 12j or CasΦ; and

[0015] - Functional mutants or variants of Cas9, Cas12a, Cas12b, CasX or Cas12e, V-type CRISPR, Cas 12j or CasΦ.

[0016] "Cas9 nuclease" refers in particular to those of Streptococcus pyogenes (S.pyogenes), Staphylococcus aureus (S.aureus), Corynebacterium diphtheriae (C.diphtheriae), Neisseria meningitidis (N.meningitidis), Streptococcus canis (S.canis), Streptococcus macacae (S.macacae), Francisella tularensis (F.tularensis), Acidaminococcus (Acidaminococcus), Campylobacter jejuni (C.jejuni), Streptococcus pneumoniae (S.pneumoniae) and Streptococcus thermophilus (S.thermophilus). More particularly, Cas9 having sequences SEQ ID NOs: 87 to 95 and mutants thereof are contemplated. In view of the foregoing, it will be appreciated that, according to another embodiment, the present invention relates to a polypeptide conjugate as described above, wherein the Cas protein is a Cas9 protein selected from the following:

[0017] Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, Corynebacterium diphtheriae, Neisseria meningitidis, Streptococcus canis, Streptococcus simianus, Francisella tularensis, Acidaminococcus sp., Campylobacter jejuni, Streptococcus pneumoniae, and Streptococcus thermophilus; and

[0018] - Cas9 orthologs and Cas9 mutants or functional variants derived from these organisms,

[0019] And in particular, the Cas9 protein is selected from sequences SEQ ID NOs: 87 to 95 and mutants thereof.

[0020] "Cas12a (Cpf1) nuclease" refers in particular to those of wild-type or modified Lachnospiraceae bacteria and Acidaminococcus sp carrying mutations E174R / S542R or E174R / S542R / K548R. More particularly, it corresponds to Cas12a sequences SEQ ID NOs: 105 and 106 and mutants thereof.

[0021] "Cas12b nuclease" refers in particular to those derived from Alicyclobacillus kakegawensis (AkCas12b) and Bacillus hisashii (BhCas12b), as well as improved mutant versions (K846R / S893R / E837G) for gene editing of BhCas12b. More particularly, it corresponds to Cas12b and mutants thereof having sequence SEQ ID NO: 107.

[0022] "CasX or Cas12e nuclease" refers in particular to those derived from Deltaproteobacteria (DpbCasX) or Planctomycetes (PlmCasX) (PlmCasX) wild type or inactivated by N672A, E769A and N935A mutations, or modified for gene editing in mammalian cells (e.g., DpbCasX_R3V2 or PlmCasX_R1V2 versions). More particularly, it corresponds to the Cas12e sequences SEQ ID NOs: 112 and 113 and mutants thereof.

[0023] Type V CRISPR nucleases are nucleases smaller than Cas9, Cas12a and CasX nucleases (400 to 700AA). These include wild-type, or one or more mutations (D225A and E324A) that eliminate catalytic activity or partially cause one or more mutations (R383A and D401A) that produce nickases from uncultured archaea (Un1Cas12f1), Syntrophomonas palmitatica (SpCas12f1) or Acidibacillus sulfuroxidans) (AsCas12f1) Cas12f (or Cas14) proteins. More specifically, it corresponds to Cas12f or Cas14 and mutants thereof with sequence SEQ ID NOs: 108 to 111.

[0024] "Cas 12j or CasΦ nuclease" refers specifically to those of the Biggiephage clade, which are also type V CRIPR-Cas.

[0025] In view of the above, it should also be understood that, according to another embodiment, the present invention relates to a polypeptide conjugate as described above, wherein the Cas protein is selected from:

[0026] - Cas9, Cas12a, Cas12b, CasX or Cas12e, V-type CRISPR (Cas12f or Cas14), Cas 12j or CasΦ nuclease;

[0027] - Orthologs of Cas9, Cas12a, Cas12b, CasX or Cas12e, type V CRISPR (Cas12f or Cas14), Cas 12j or CasΦ; and

[0028] - a functional mutant or variant of Cas9, Cas12a, Cas12b, CasX or Cas12e, type V CRISPR (Cas12f or Cas14), Cas 12j or CasΦ,

[0029] And in particular, the Cas protein is selected from sequences SEQ ID NOs: 87 to 95 and 105 to 113 and mutants thereof.

[0030] "Orthologs" refer to similar Cas proteins that exist in two or more different species.

[0031] "Mutant" refers to a Cas protein into which one or more mutations (including deletion, substitution and / or addition of one or more amino acids) are introduced. These mutations can increase or eliminate nuclease activity, or improve or reduce the recognition fidelity of target DNA.

[0032] "Functional mutant" refers to a Cas protein modified by human intervention (e.g., genetic engineering), for example to increase the activity of Cas.

[0033] A "functional variant" refers to a Cas protein that has been naturally modified by evolution, e.g., with increased (or decreased) activity.

[0034] In particular, the present invention relates to a polypeptide conjugate as described above, wherein the Cas protein is selected from:

[0035] - Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, Corynebacterium diphtheriae, Neisseria meningitidis, Streptococcus canis, Streptococcus macaques, Francisella tularensis, Acidinococcus, Campylobacter jejuni, Streptococcus pneumoniae, and Streptococcus thermophilus; Cas12a nucleases from Lachnospiraceae and Acidinococcus; Cas12b nucleases from Alicyclobacillus sakegawensis and Bacillus hisashii; CasX or Cas12e nucleases from Deltaproteobacteria and Planctomyces; V-type CRISPR nucleases from Syntrophomonas palmitatica and Acidibacillus sulfuroxidans; Cas 12j or CasΦ nucleases from the Biggiephage clade;

[0036] - a Cas9, Cas12a, Cas12b, CasX or Cas12e, V-type CRISPR, Cas 12j or CasΦ ortholog derived from these organisms; and

[0037] - a mutant or functional variant of Cas9, Cas12a, Cas12b, CasX or Cas12e, type V CRISPR, Cas 12j or CasΦ derived from these organisms,

[0038] And in particular, the Cas protein is selected from SEQ ID NOs: 87 to 95 and 105 to 113, and mutants thereof.

[0039] In particular, the present invention relates to a polypeptide conjugate as described above, wherein the Cas protein is selected from the sequence 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 the Cas9 protein is selected from the sequence SEQ ID NOs: 87 to 95 and mutants thereof. More particularly, the present invention relates to a polypeptide conjugate as described above, wherein the Cas protein is selected from the sequence SEQ ID NOs: 87, 88, 89, 90, 91, 92, 93, 94, 95, 105, 106, 107, 108, 109, 110, 111, 112 and 113.

[0040] According to another embodiment, the present invention relates to a polypeptide conjugate as described above, wherein the Cas protein is selected from:

[0041] - Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, Corynebacterium diphtheriae, Neisseria meningitidis, Streptococcus canis, Streptococcus simianus, Francisella tularensis, Acidaminococcus sp., Campylobacter jejuni, Streptococcus pneumoniae, and Streptococcus thermophilus;

[0042] - Cas12a nuclease from Lachnospiraceae, Cas12b from Bacillus hisashii, Cas12f (Cas14) from uncultured Archaea, and Cas 12j (CasΦ) from the Biggiephage clade; and

[0043] - Cas9, Cas12a, Cas12b, Cas12f and Cas12j orthologs, and Cas9, Cas12a, Cas12b, Cas12f and Cas12j mutants or functional variants derived from these organisms,

[0044] And in particular, the Cas protein is selected from SEQ ID NOs: 87 to 95 and 105 to 113 and mutants thereof.

[0045] For example, there are mutants (variants) of Streptococcus pyogenes Cas9 (spCas9) (whose representative sequence is SEQ ID NO: 87) in which the amino acids at specific positions are modified, thereby eliminating the nuclease activity and improving the fidelity of target DNA sequence recognition. These mutations can be as follows: amino acids D10, E762, D839, H840, H863, H983 and / or D986 are replaced with different amino acids (such as alanine) or any other amino acid other than natural amino acids, thereby reducing, substantially eliminating or suppressing nuclease activity. Other amino acid substitutions can increase the specificity of target DNA sequence recognition, thereby reducing off-target binding to other DNA sequences that have a certain homology to the target sequence. In the case of spCas9, these mutations may be present in one, two, three, four, five, six and / or all seven of the following positions: L169, Y450, N497, R661, Q695, Q926 and / or D1135, which results in a so-called high-fidelity Cas9 (hifi spCas9) (Kleinstiver, BP et al., Nature. 2016; 529(7587): 490-495). Other different mutations have also been shown to increase the specificity of spCas9, which are substitutions of amino acids K855, K810 / K1003 / R1060 or K848 / K1003 / R1060 for alanine (IM Slaymaker et al., Science 351, 84-8 (2016)). Finally, another group of spCas9 mutations also significantly improved the accuracy of spCas9 binding to target DNA sequences; these mutations combined substitutions N692A, M694A, Q695A and H698A and constituted the ultra-precise HyppaCas9 (Chen, JS et al., Nature. 2017; 550(7676): 407-410). In addition, all mutations that abolished the catalytic activity of spCas9 can be associated with this increased specificity of DNA sequence recognition.

[0046] Similarly, there are mutants (variants) of Staphylococcus aureus Cas9 (saCas9) (whose representative sequence is SEQ ID NO: 88), in which the amino acid changes D10A or N580A inactivate the RuvC and HNH nuclease domains, respectively, and convert saCas9 into a nickase (Friedland, AE et al., Genome Biol. 2015; 16: 257). High-fidelity saCas9 can be obtained by mutagenesis, and mutations Y211A, Y212A, W229A, Y230, R245A, T392A, N413A, N419A, Y651A, R654A alone or in combination obtain a higher on-target / off-target rate than wild-type saCas9 (Tan, Y et al., Proc Natl Acad Sci USA. 2019; 116(42): 20969-20976). In addition, the E782K / N968K / R1015H and E782K / K929R / R1015H triple mutants of saCas9 exhibited broader site recognition, as they could cleave sequences followed by a simpler 'NNNRRT' PAM instead of a 'NNGRRT' motif. Finally, combinations of the above mutations may lead to hybrid functional improvements, such as high-fidelity saCas9 nucleases or nickases with broader PAM recognition.

[0047] There are also organisms in nature that encode Cas nucleases that are smaller than Cas9, Cas12a (Cpf1), or Cas12b (1,000-1,500AA). This is the case with CasX or Cas12e (less than 1,000AA), Cas12f or Cas14 (400-700AA), and Cas12j or CasfΦ (700-800AA) nucleases, which provide a natural reservoir of compact Cas, allowing 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 et al. 2021 P et al. Science. 2020 Jul 17; 369(6501): 333-337). CasX nucleases, such as those derived from Delta-Proteobacteria (DpbCasX) or Planctomyces (PlmCasX), in their wild-type versions or modified versions for gene editing, such as versions DpbCasX-R3 (a chimeric DpbCasX containing the R3 loop of PlmCasX) or PlmCasX-R1 (a chimeric PlmCasX containing the R1 loop of DpbCasX), or CasX versions inactivated by N672A, E769A and N935A mutations alone or in combination, enable efficient programming of mammalian cells to induce DNA breaks or regulate 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. 2019Nature.566(7743):218-223).Cas12f nuclease type V CRISPR nucleases are smaller in size (400 to 700AA) than Cas9, Cas12a, and CasX nucleases; for example, Cas12f (or Cas14) proteins from uncultured archaea (Un1Cas12f1), Syntrophomonas palmitatica (SpCas12f1), or Acidibacillussulfuroxidans (AsCas12f1), whether wild-type or carrying one or more mutations that completely eliminate the catalytic activity of the Cas12f nuclease (D225A and E324A of Un1Cas12f1, or D326A and / or D510A of Un1Cas12f1) or partially eliminate the nuclease activity to produce one or more mutations of a nickase (R383A and D401A of AsCas12f1) (Do Yon Kim et al. 2022, Nature Biotechnology. 40(1):94-102; Wu et al. 2019). Zhaowei et al. 2021 Nat. Chem. Biol. 17 (11): 1132-1138; Xiaoshu Xu et al. 2021 Mol Cell. 81 (20): 4333-4345. e4). Therefore, the catalytic mutant of Cas12f can be fused with a transcriptional activation or repression domain to program the Cas12f protein for targeted expression of certain cell genes, or it can be fused with an enzymatic domain of a base editor or deoxyadenosine deaminase, thereby allowing targeted conversion of A or T bases to G or C to repair or introduce 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). Phage Cas12j or CasΦ nuclease is able to introduce double-strand breaks on a DNA sequence recognized by a guide RNA. The rate of this cleavage can be accelerated by about 20 times by introducing mutations in the α7 helix (E159A, S160A, S164A, D167A, E168A) or replacing the negatively charged fragment with a short sequence of glycine-serine (Pausch, P. et al. 2021, Nature Structural & molecular biology. 28(8): 652-661).

[0048] Therefore, in a broad sense, the present invention may encompass a polypeptide conjugate comprising a first component covalently or non-covalently linked to a second component, wherein the first component is an antigen presenting cell antibody targeting asialoglycoprotein receptor (anti-APC-ASGPR), or a fragment thereof, and the second component is one of the above-mentioned Cas proteins.

[0049] "Antigen presenting cell (APC)" refers to an immune system cell that presents part of a cellular element to a T lymphocyte. These cells may be monocytes, macrophages, B lymphocytes or dendritic cells. In particular, these cells are dendritic cells (DC). In addition, according to a specific embodiment, the present invention relates to a polypeptide conjugate comprising a first component covalently or non-covalently linked to a second component, wherein the first component is an anti-dendritic cell antibody targeting an asialoglycoprotein receptor (anti-DC-ASGPR), or a fragment thereof, wherein the first component and the second component are Cas proteins.

[0050] "Asialoglycoprotein receptor" is abbreviated as ASGPR, and is also called CLEC10A, which refers to a C-type lectin receptor (CLR) expressed on antigen presenting cells, such as human dendritic cells (DCs). CLR allows APCs to capture and internalize antigens, especially glycosylated antigens, so that they can be subsequently processed and presented to major histocompatibility complex (MHC) molecules. In addition, a variety of CLRs including ASGPR can initiate signal transduction cascades and regulate dendritic cell function, thereby affecting the induced immune response. Unlike other Syk-related CLRs, activated ASGPR does not induce NF-κB activation, but causes CREB phosphorylation (Gu C et al., J Immunol. 2019; 203 (2): 389-399).

[0051] Thus, the signaling cascade of activated ASGPR leads to the activation of Syk, PLCγ2, PKCδ, and then MAPKERK 1 / 2 and JNK, which, when the stimulation is prolonged, leads to the phosphorylation of p90RSK and CREB, inducing the transcription of the anti-inflammatory cytokine IL-10, a specific end result of DC-ASGPR endocytosis of antigens.

[0052] "Antibody" refers to an immunoglobulin involved in the acquired immune response, which is a multimeric protein composed of four chains. Immunoglobulins are well known to those skilled in the art and are composed of an assembly of two dimers, each dimer consisting of one heavy chain and one light chain. The multimeric complex is assembled by connecting the light chain and the heavy chain through a disulfide bridge between two cysteines, and the two heavy chains are also linked together by two disulfide bridges.

[0053] The heavy and light chains are each composed of a constant region and a variable region. The assembly of chains that make up the antibody allows the definition of a characteristic three-dimensional Y structure, in which:

[0054] -The base of Y corresponds to the Fc constant region recognized by complement and Fc receptors, and

[0055] - The ends of the Y arms are the respective assemblies of the variable regions of the light and heavy chains.

[0056] More specifically, each light chain consists of a variable region (V L ) and constant region (C L ). Each heavy chain consists of a variable region (V H ) and a constant region, which consists of three constant domains C H 1. C H 2 and C H 3. Domain C H 2 and C H 3 constitute the Fc domain.

[0057] The light chain variable region consists of three antigen recognition regions (CDRs) surrounded by four framework domains. The heavy chain variable region also consists of three complementarity determining regions (CDRs) surrounded by four framework domains. The three-dimensional folding of these variable regions allows all 6 CDRs to be exposed on the same side of the protein, allowing the formation of a specific structure that recognizes a given antigen.

[0058] The antibodies described in the present invention are isolated and purified and may 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 different from natural antibodies. These antibodies are mature, i.e., they have a specific three-dimensional structure that enables them to recognize antigens and have all post-translational modifications necessary for antigen recognition, including glycosylation and the formation of intramolecular and intermolecular disulfide bonds.

[0059] More specifically, they are "monoclonal antibodies," meaning that they recognize only one antigenic determinant of DC-ASGPR, unlike polyclonal antibodies, which are mixtures of antibodies and can therefore recognize multiple antigenic determinants of the same protein.

[0060] "Fragment thereof" refers to any portion of an antibody according to the present invention which retains the ability to bind to an epitope recognized by the intact antibody. Examples of such fragments include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fv (scFv), single-chain antibodies, disulfide-bridged Fv (dsFv) and V-containing fragments. L or V H Epitope-binding fragments (including single-chain antibodies) may comprise the variable region alone or in combination with all or part of the following: hinge region, C H 1. C H 2 and C H3 domains. Such fragments may comprise one or two Fab fragments or F(ab')2 fragments. In addition, the fragments may be or may combine members of any of the following immunoglobulin classes: IgG, IgM, IgA, IgD or IgE and their subclasses.

[0061] 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 are epitope-binding fragments that contain a light chain antibody variable region (V L ) at least one fragment of an antibody variable region (V H ) at least one fragment. The linker can be a short flexible peptide, which is selected to ensure that V L and V H The correct three-dimensional folding of the region of the antibody fragment is maintained, thereby maintaining the binding specificity to the target molecule of the whole antibody from which the single-chain antibody fragment is derived. L and V H The carboxyl terminus of the sequence can be covalently linked to a complementary V L and V H The amino acid end of the sequence.

[0062] In addition, "antigen presenting cell antibody targeting APC asialoglycoprotein receptor (anti-DC-ASGPR) or its fragment" refers to a protein that can specifically recognize DC-ASGPR. It includes seven monoclonal antibodies that recognize human DC-ASGPR: 49C11, 49C11_bis, 1H11, 5F10, 4G2.2, 6.3H9.1D11 and 5H8.1D4.

[0063] The "49C11" antibody refers to an antibody comprising:

[0064] - a heavy chain having the sequence SEQ ID NO: 10, which is a synthetic construct comprising:

[0065] o a murine variable region targeting human DC-ASGPR having the sequence SEQ ID NO:4, which comprises, from N-terminus to 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

[0066] o A fusion protein with the constant region of a human IgG4 heavy chain; and

[0067] - a light chain having the sequence SEQ ID NO: 25, which is a synthetic construct comprising:

[0068] o a murine variable region targeting human DC-ASGPR having the sequence SEQ ID NO: 19, which comprises, from N-terminus to C-terminus, a CDR1 having the sequence SEQ ID NO: 16, a CDR2 having the sequence SEQ ID NO: 17, and a CDR3 having the sequence SEQ ID NO: 18; and

[0069] o Fusion protein with the constant region of the kappa chain of human IgG4.

[0070] From the above 49C11 antibody, 5 variants were also developed, namely:

[0071] -49C11_var1, containing:

[0072] o a heavy chain having the sequence of SEQ ID NO: 11, comprising a variable region having the sequence of SEQ ID NO: 5, the variable region comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO: 1, a CDR2 having the sequence of SEQ ID NO: 2, and a CDR3 having the sequence of SEQ ID NO: 3; and

[0073] o a light chain having the sequence of SEQ ID NO:26, comprising a variable region having the sequence of SEQ ID NO:20, the variable region comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO:16, a CDR2 having the sequence of SEQ ID NO:17, and a CDR3 having the sequence of SEQ ID NO:18;

[0074] -49C11_var2, containing:

[0075] o a heavy chain having the sequence of SEQ ID NO: 12, comprising a variable region having the sequence of SEQ ID NO: 6, the variable region comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO: 1, a CDR2 having the sequence of SEQ ID NO: 2, and a CDR3 having the sequence of SEQ ID NO: 3; and

[0076] o a light chain having the sequence of SEQ ID NO:27, comprising a variable region having the sequence of SEQ ID NO:21, the variable region comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO:16, a CDR2 having the sequence of SEQ ID NO:17, and a CDR3 having the sequence of SEQ ID NO:18;

[0077] -49C11_var3, containing:

[0078] o a heavy chain having the sequence of SEQ ID NO: 13, comprising a variable region having the sequence of SEQ ID NO: 7, the variable region comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO: 1, a CDR2 having the sequence of SEQ ID NO: 2, and a CDR3 having the sequence of SEQ ID NO: 3; and

[0079] o a light chain having the sequence of SEQ ID NO:28, comprising a variable region having the sequence of SEQ ID NO:22, the variable region comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO:16, a CDR2 having the sequence of SEQ ID NO:17, and a CDR3 having the sequence of SEQ ID NO:18;

[0080] -49C11_var4, containing:

[0081] o a heavy chain having the sequence of SEQ ID NO: 14, comprising a variable region having the sequence of SEQ ID NO: 8, the variable region comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO: 1, a CDR2 having the sequence of SEQ ID NO: 2, and a CDR3 having the sequence of SEQ ID NO: 3; and

[0082] o a light chain having the sequence of SEQ ID NO:29, comprising a variable region having the sequence of SEQ ID NO:23, the variable region comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO:16, a CDR2 having the sequence of SEQ ID NO:17, and a CDR3 having the sequence of SEQ ID NO:18; and

[0083] -49C11_var5, containing:

[0084] o a heavy chain having the sequence of SEQ ID NO: 15, comprising a variable region having the sequence of SEQ ID NO: 9, said variable region including, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO: 1, a CDR2 having the sequence of SEQ ID NO: 2, and a CDR3 having the sequence of SEQ ID NO: 3; and

[0085] o A light chain having sequence SEQ ID NO:30, comprising a variable region having sequence SEQ ID NO:24, wherein the variable region comprises, from N-terminus to C-terminus, a CDR1 having sequence SEQ ID NO:16, a CDR2 having sequence SEQ ID NO:17, and a CDR3 having sequence SEQ ID NO:18.

[0086] The "49C11_bis" antibody refers to an antibody comprising:

[0087] - a heavy chain having the sequence of SEQ ID NO:32, comprising a variable region having the sequence of SEQ ID NO:31, said variable region comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO:1, a CDR2 having the sequence of SEQ ID NO:2 and a CDR3 having the sequence of SEQ ID NO:3; and

[0088] - a light chain having sequence SEQ ID NO:34, comprising a variable region having sequence SEQ ID NO:33, said variable region comprising, from N-terminus to C-terminus, a CDR1 having sequence SEQ ID NO:16, a CDR2 having sequence SEQ ID NO:17 and a CDR3 having sequence SEQ ID NO:18.

[0089] "1H11" antibodies are antibodies that contain:

[0090] - a heavy chain having sequence SEQ ID NO:39, comprising a variable region having sequence SEQ ID NO:38, said variable region comprising, from N-terminus to C-terminus, a CDR1 having sequence SEQ ID NO:35, a CDR2 having sequence SEQ ID NO:36 and a CDR3 having sequence SEQ ID NO:37; and

[0091] - a light chain having sequence SEQ ID NO:44, comprising a variable region having sequence SEQ ID NO:43, said variable region comprising, from N-terminus to C-terminus, a CDR1 having sequence SEQ ID NO:40, a CDR2 having sequence SEQ ID NO:41 and a CDR3 having sequence SEQ ID NO:42.

[0092] The "5F10" antibody refers to an antibody comprising:

[0093] - a heavy chain having sequence SEQ ID NO:49, comprising a variable region having sequence SEQ ID NO:48, said variable region comprising, from N-terminus to C-terminus, a CDR1 having sequence SEQ ID NO:45, a CDR2 having sequence SEQ ID NO:46 and a CDR3 having sequence SEQ ID NO:47; and

[0094] - a light chain having sequence SEQ ID NO:54, comprising a variable region having sequence SEQ ID NO:53, said variable region comprising, from N-terminus to C-terminus, a CDR1 having sequence SEQ ID NO:50, a CDR2 having sequence SEQ ID NO:51 and a CDR3 having sequence SEQ ID NO:52.

[0095] "4G2.2" antibodies refer to antibodies that contain the following:

[0096] - a heavy chain having sequence SEQ ID NO:59, comprising a variable region having sequence SEQ ID NO:58, said variable region comprising, from N-terminus to C-terminus, a CDR1 having sequence SEQ ID NO:55, a CDR2 having sequence SEQ ID NO:56 and a CDR3 having sequence SEQ ID NO:57; and

[0097] - a light chain having sequence SEQ ID NO:64, comprising a variable region having sequence SEQ ID NO:63, said variable region comprising, from N-terminus to C-terminus, a CDR1 having sequence SEQ ID NO:60, a CDR2 having sequence SEQ ID NO:61 and a CDR3 having sequence SEQ ID NO:62.

[0098] The "6.3H9.1D11" antibody refers to an antibody comprising:

[0099] - a heavy chain having sequence SEQ ID NO:69, comprising a variable region having sequence SEQ ID NO:68, said variable region comprising, from N-terminus to C-terminus, a CDR1 having sequence SEQ ID NO:65, a CDR2 having sequence SEQ ID NO:66 and a CDR3 having sequence SEQ ID NO:67; and

[0100] - a light chain having sequence SEQ ID NO:74, comprising a variable region having sequence SEQ ID NO:73, said variable region comprising, from N-terminus to C-terminus, a CDR1 having sequence SEQ ID NO:70, a CDR2 having sequence SEQ ID NO:71 and a CDR3 having sequence SEQ ID NO:72.

[0101] The "5H8.1D4" antibody refers to an antibody comprising:

[0102] - a heavy chain having sequence SEQ ID NO: 79, comprising a variable region having sequence SEQ ID NO: 78, said variable region comprising, from N-terminus to C-terminus, a CDR1 having sequence SEQ ID NO: 75, a CDR2 having sequence SEQ ID NO: 76 and a CDR3 having sequence SEQ ID NO: 77; and

[0103] - a light chain having sequence SEQ ID NO:84, comprising a variable region having sequence SEQ ID NO:83, said variable region comprising, from N-terminus to C-terminus, a CDR1 having sequence SEQ ID NO:80, a CDR2 having sequence SEQ ID NO:81 and a CDR3 having sequence SEQ ID NO:82.

[0104] In summary, according to a specific embodiment of the present invention, the anti-DC-ASGPR has the following sequence:

[0105] Table 1. Anti-DC-ASGPR and corresponding sequences

[0106]

[0107] It is therefore understood that, according to another embodiment, the subject of the present invention is a polypeptide conjugate as described above, comprising a first component covalently or non-covalently linked to a second component, said first component being 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 intact antibody, said second component being a Cas protein,

[0108] The polypeptide conjugate is capable of inducing immune tolerance to the Cas protein in mammalian organisms, particularly in primates and humans, and

[0109] The anti-DC-ASGPR is selected from:

[0110] - Antibodies containing:

[0111] o a heavy chain comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO: 1, a CDR2 having the sequence of SEQ ID NO: 2, and a CDR3 having the sequence of SEQ ID NO: 3; and

[0112] o a light chain comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO: 16, a CDR2 having the sequence of SEQ ID NO: 17, and a CDR3 having the sequence of SEQ ID NO: 18;

[0113] - Antibodies containing:

[0114] o a heavy chain comprising, from N-terminus to 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; and

[0115] o a light chain comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO: 40, a CDR2 having the sequence of SEQ ID NO: 41, and a CDR3 having the sequence of SEQ ID NO: 42;

[0116] - Antibodies containing:

[0117] o a heavy chain comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO: 45, a CDR2 having the sequence of SEQ ID NO: 46, and a CDR3 having the sequence of SEQ ID NO: 47; and

[0118] o a light chain comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO: 50, a CDR2 having the sequence of SEQ ID NO: 51, and a CDR3 having the sequence of SEQ ID NO: 52;

[0119] - Antibodies containing:

[0120] o a heavy chain comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO: 55, a CDR2 having the sequence of SEQ ID NO: 56, and a CDR3 having the sequence of SEQ ID NO: 57; and

[0121] o a light chain comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO: 60, a CDR2 having the sequence of SEQ ID NO: 61, and a CDR3 having the sequence of SEQ ID NO: 62;

[0122] - Antibodies containing:

[0123] o a heavy chain comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO: 65, a CDR2 having the sequence of SEQ ID NO: 66, and a CDR3 having the sequence of SEQ ID NO: 67; and

[0124] o a light chain comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO: 70, a CDR2 having the sequence of SEQ ID NO: 71, and a CDR3 having the sequence of SEQ ID NO: 72; and

[0125] - Antibodies containing:

[0126] o a heavy chain comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO: 75, a CDR2 having the sequence of SEQ ID NO: 76, and a CDR3 having the sequence of SEQ ID NO: 77; and

[0127] o A light chain comprising, from N-terminus to C-terminus, a CDR1 having the sequence of SEQ ID NO: 80, a CDR2 having the sequence of SEQ ID NO: 81, and a CDR3 having the sequence of SEQ ID NO: 82.

[0128] According to another embodiment, the present invention relates to a polypeptide conjugate as described above, wherein the anti-DC-ASGPR is selected from:

[0129] - an antibody comprising a heavy chain comprising a variable region having a sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9 or 31 and a light chain comprising a variable region having a sequence of SEQ ID NO: 19, 20, 21, 22, 23, 24 or 33;

[0130] - an antibody comprising a heavy chain comprising a variable region having the sequence of SEQ ID NO: 38 and a light chain comprising a variable region having the sequence of SEQ ID NO: 43;

[0131] - an antibody comprising a heavy chain comprising a variable region having the sequence of SEQ ID NO: 48 and a light chain comprising a variable region having the sequence of SEQ ID NO: 53;

[0132] - an antibody comprising a heavy chain comprising a variable region having the sequence of SEQ ID NO: 58 and a light chain comprising a variable region having the sequence of SEQ ID NO: 63;

[0133] - an antibody comprising a heavy chain comprising a variable region having the sequence of SEQ ID NO: 68 and a light chain comprising a variable region having the sequence of SEQ ID NO: 73; and

[0134] - An antibody comprising a heavy chain comprising a variable region having the sequence of SEQ ID NO: 78 and a light chain comprising a variable region having the sequence of SEQ ID NO: 83.

[0135] According to another embodiment, the present invention relates to a polypeptide conjugate as described above, wherein the anti-DC-ASGPR is selected from:

[0136] - an antibody comprising a heavy chain having the sequence of SEQ ID NO: 10, 11, 12, 13, 14, 15 or 32 and a light chain having the sequence of SEQ ID NO: 25, 26, 27, 28, 29, 30 or 34;

[0137] - an antibody comprising a heavy chain having the sequence of SEQ ID NO: 39 and a light chain having the sequence of SEQ ID NO: 44;

[0138] - an antibody comprising a heavy chain having the sequence of SEQ ID NO: 49 and a light chain having the sequence of SEQ ID NO: 54;

[0139] - an antibody comprising a heavy chain having the sequence of SEQ ID NO: 59 and a light chain having the sequence of SEQ ID NO: 64;

[0140] - an antibody comprising a heavy chain having the sequence of SEQ ID NO: 69 and a light chain having the sequence of SEQ ID NO: 74; and

[0141] - an antibody comprising a heavy chain having the sequence of SEQ ID NO: 79 and a light chain having the sequence of SEQ ID NO: 84.

[0142] In this regard, for the sequences connected to antibodies 49C11, 49C11_var1, 49C11_var2, 49C11_var3, 49C11_var4, 49C11_var5 and 49C11_bis, since the heavy chain and the light chain share the same CDR (see Table 1), they can be interchanged to develop new antibodies. In addition, the expression "an antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a variable region having a sequence of SEQ ID NO: 4, 5, 6, 7, 8, 9 or 31, and the light chain comprises a variable region having a sequence of SEQ ID NO: 19, 20, 21, 22, 23, 24 or 33" refers to an antibody comprising:

[0143] - a heavy chain comprising a variable region having the sequence of SEQ ID NO: 4 and a light chain comprising a variable region having the sequence of SEQ ID NO: 19;

[0144] - a heavy chain comprising a variable region having the sequence of SEQ ID NO: 5 and a light chain comprising a variable region having the sequence of SEQ ID NO: 20;

[0145] - a heavy chain comprising a variable region having the sequence of SEQ ID NO: 6 and a light chain comprising a variable region having the sequence of SEQ ID NO: 21;

[0146] - a heavy chain comprising a variable region having the sequence of SEQ ID NO: 7 and a light chain comprising a variable region having the sequence of SEQ ID NO: 22;

[0147] - a heavy chain comprising a variable region having the sequence of SEQ ID NO: 8 and a light chain comprising a variable region having the sequence of SEQ ID NO: 23;

[0148] - a heavy chain comprising a variable region having the sequence of SEQ ID NO: 9 and a light chain comprising a variable region having the sequence of SEQ ID NO: 24; and

[0149] - a heavy chain comprising a variable region having the sequence of SEQ ID NO: 31 and a light chain comprising a variable region having the sequence of SEQ ID NO: 33;

[0150] And for example, antibodies comprising:

[0151] - a heavy chain comprising a variable region having the sequence of SEQ ID NO: 4 and a light chain comprising a variable region having the sequence of SEQ ID NO: 20;

[0152] - a heavy chain comprising a variable region having the sequence of SEQ ID NO: 7 and a light chain comprising a variable region having the sequence of SEQ ID NO: 19;

[0153] - a heavy chain comprising a variable region having the sequence of SEQ ID NO: 6 and a light chain comprising a variable region having the sequence of SEQ ID NO: 24;

[0154] - a heavy chain comprising a variable region having the sequence of SEQ ID NO: 6 and a light chain comprising a variable region having the sequence of SEQ ID NO: 33;

[0155] -wait.

[0156] Similarly, the expression "an antibody comprising a heavy chain having the sequence of SEQ ID NO: 10, 11, 12, 13, 14, 15 or 32 and a light chain having the sequence of SEQ ID NO: 25, 26, 27, 28, 29, 30 or 34" refers to antibodies comprising heavy chains and light chains each having the sequence of SEQ ID NOs: 10 and 25, 11 and 26, 12 and 27, 13 and 28, 14 and 29, 15 and 30 or 32 and 34, respectively, as well as antibodies comprising heavy chains and light chains each having the sequence of SEQ ID NOs: 10 and 30, 10 and 29, 13 and 25, 14 and 26, etc.

[0157] In general, the present invention may therefore also encompass a polypeptide conjugate comprising a first component covalently or non-covalently linked to a second component, the first component being an antigen presenting cell antibody targeting asialoglycoprotein receptor (anti-DC-ASGPR) as described above, or a fragment thereof as described above, and the second component being one of the above-mentioned Cas proteins.

[0158] "Covalently linked" means that the polypeptide conjugate of the present invention can be covalently linked, i.e., the first component and the second component are linked by a covalent bond (with or without a linker), which is also called a molecular bond, which is a strong chemical bond involving the sharing of electron pairs between the atoms of the first component and the second component. Therefore, the subject of the present invention is a polypeptide conjugate as described above, wherein the anti-DC-ASGPR is covalently linked to the Cas protein. In particular, the present invention relates to a polypeptide conjugate as described above, wherein the antigen presenting cell antibody targeting the asialoglycoprotein receptor (anti-DC-ASGPR) as described above is covalently linked to the second component through its heavy chain, and the second component is one of the above-mentioned Cas proteins. For example, based on the 49C11_bis antibody, the subject of the present invention is a polypeptide conjugate as described above, which comprises the sequence SEQ ID NO: 102 (the heavy chain of 49C11_bis with the sequence SEQ ID NO: 32, the C-terminus of which is covalently linked to the saCas9 with the 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 noted that all possible constructs of the above-mentioned antibodies and of the above-mentioned Cas that a person skilled in the art is able to develop are part of the present invention.

[0159] According to another embodiment, the present invention relates to a polypeptide conjugate as described above, wherein the anti-APC-ASGPR is covalently linked to the Cas protein, and in particular, the polypeptide conjugate comprises sequences SEQ ID NOs: 102 and 34.

[0160] Advantageously, the present invention relates to a polypeptide conjugate as described above, wherein the anti-DC-ASGPR is covalently linked to the Cas protein via a linker, in particular a peptide linker.

[0161] Among the linkers that can be used, i.e., small molecules or peptides for linking the anti-DC-ASGPR and Cas proteins (which can incorporate glycosylation sites or introduce specific secondary structures), it should be noted that some linkers increase the expression efficiency or stability of the fusion protein, thereby improving the efficiency of the latter. For the purposes of the present invention, it should be noted that the following peptide linkers are used but are not limited to:

[0162] -QTPTNTISVTPTNNSTPTNNSNPKPNPAS (SEQ ID NO:96);

[0163] -SSVSPTTSVHPTPTSVPPTPTKSSP(SEQ ID NO:97);

[0164] -PTSTPADSSTITTPTATPTATPTIKG (SEQ ID NO:98);

[0165] -TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO:99); and

[0166] -TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO: 100).

[0167] Furthermore, according to another specific embodiment, the present invention relates to a polypeptide conjugate as described above, wherein the peptide linker is selected from:

[0168] -QTPTNTISVTPTNNSTPTNNSNPKPNPAS (SEQ ID NO:96);

[0169] -SSVSPTTSVHPTPTSVPPTPTKSSP(SEQ ID NO:97);

[0170] -PTSTPADSSTITTPTATPTATPTIKG (SEQ ID NO:98);

[0171] -TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO:99); and

[0172] -TNGSITVAATAPTVTPTVNATPSAA (SEQ ID NO: 100).

[0173] In particular, the subject of the present invention is a polypeptide conjugate as described above, wherein the antigen presenting cell antibody targeting the APC asialoglycoprotein receptor as described above (anti-DC-ASGPR) is covalently linked to a second component through its heavy chain via a peptide linker, and the second component is one of the above-mentioned Cas proteins. For example, based on the 49C11_bis antibody, the subject of the present invention is a polypeptide conjugate as described above, which comprises the sequence SEQ ID NO: 101 (a heavy chain of 49C11_bis with a sequence of SEQ ID NO: 32, its C-terminus is covalently linked to a linker with a sequence of SEQ ID NO: 96, and the linker is covalently linked to a Cas9 with a sequence of SEQ ID NO: 89) and SEQ ID NO: 34 (a light chain of 49C11_bis). With regard to this non-limiting example, it should be noted that all possible constructs of the above-mentioned antibodies and the above-mentioned Cas that can be developed by a person skilled in the art are part of the present invention.

[0174] According to another embodiment, the present invention relates to a polypeptide conjugate as described above, wherein the peptide linker is selected from:

[0175] -QTPTNTISVTPTNNSTPTNNSNPKPNPAS (SEQ ID NO:96);

[0176] -SSVSPTTSVHPTPTSVPPTPTKSSP(SEQ ID NO:97);

[0177] -PTSTPADSSTITTPTATPTATPTIKG (SEQ ID NO:98);

[0178] -TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO:99); and

[0179] -TNGSITVAATAPTVTPTVNATPSAA(SEQ ID NO:100),

[0180] And in particular, the polypeptide conjugate comprises sequences of SEQ ID NOs: 101 and 34.

[0181] "Non-covalent binding" means that the polypeptide conjugate of the present invention can also be non-covalently bound, that is, the first component and the second component are connected by a weak bond, which is also called a non-covalent interaction, which does not involve the sharing of electrons between the first component and the second component. In particular, it should be understood that according to a second specific embodiment, the present invention relates to a polypeptide conjugate as described above, wherein the anti-APC-ASGPR is non-covalently bound to the Cas protein.

[0182] For this purpose, non-covalent high affinity interactions between two partners known to those skilled in the art can be utilized, such as, but not limited to, antibody / antigen interactions, receptor / ligand interactions, avidin / biotin interactions, adhesion protein / dockerin interactions, and barnase / barstar interactions. In addition, according to another specific embodiment, the present invention relates to a polypeptide conjugate as described above, wherein the anti-DC-ASGPR is non-covalently bound to the Cas protein via a high affinity interaction selected from the following:

[0183] - Antibody / antigen interactions;

[0184] - Receptor / ligand interactions;

[0185] -Avidin / biotin interaction;

[0186] - cohesin / dockerin interactions; and

[0187] -Barnase / barstar interactions.

[0188] In particular, the present invention also relates to a polypeptide conjugate as described above, wherein the high affinity interaction is a cohesin / dockerin interaction.

[0189] In particular, the present invention relates to a polypeptide conjugate as described above, wherein the antigen presenting cell antibody targeting the asialoglycoprotein receptor as described above (anti-DC-ASGPR) is non-covalently linked to a second component via its heavy chain, the second component being one of the above-mentioned Cas proteins. For example, based on the 49C11_bis antibody, the subject of the present invention is a polypeptide conjugate as described above, the polypeptide conjugate comprising the sequence SEQ ID NO: 103 or 86 (the heavy chain of 49C11_bis with the sequence SEQ ID NO: 32, covalently linked to dockerin at its C-terminus), SEQ ID NO: 34 (the light chain of 49C11_bis) and SEQ ID NO: 104 (Cas9 with the sequence SEQ ID NO: 89, covalently linked to adhesion protein at its N-terminus). With regard to this non-limiting example, it should be noted that all possible constructs based on the above-mentioned antibodies, the above-mentioned Cas proteins and the above-mentioned means of non-covalent high-affinity interactions that can be developed by a person skilled in the art are part of the present invention. Similarly, if the construct described above exhibits a dockerin linked to an antibody component and a cohesin linked to a Cas component, the reverse result can be achieved by placing the cohesin on the antibody component of the polypeptide conjugate of the invention and the dockerin on the Cas component, regardless of which non-covalent high affinity interaction is used.

[0190] According to another embodiment, the present invention relates to a polypeptide conjugate as described above, wherein the high affinity interaction is a cohesin / dockerin interaction, and in particular the polypeptide conjugate comprises the sequences of SEQ ID NOs: 103, 34 and 104 or the sequences of SEQ ID NOs: 85, 25 and 104.

[0191] Carrier

[0192] According to a second aspect of the present invention, it relates to a vector encoding a polypeptide conjugate of the present invention. In other words, the present invention as a whole comprises at least one nucleic acid comprising or consisting of a sequence encoding a first component and / or a second component, the first component being an antigen presenting cell antibody targeting an asialoglycoprotein receptor of an APC as described above (anti-DC-ASGPR), or a fragment thereof as described above, and the second component being one of the above-mentioned Cas proteins. "At least one nucleic acid" means that the present invention may comprise two or three nucleic acids. For example, one encoding an anti-DC-ASGPR as described above, or a fragment thereof as described above, and another encoding one of the above-mentioned Cas proteins; or two encoding anti-DC-ASGPR as described above (e.g., the first encoding a light chain, and the second encoding a heavy chain), and the third encoding one of the above-mentioned Cas proteins. However, it should be noted that in its covalent configuration, the present invention preferably uses only one nucleic acid comprising or consisting of a sequence encoding a first component and a second component, the first component being an antigen presenting cell antibody targeting an asialoglycoprotein receptor as described above (anti-DC-ASGPR), or a fragment thereof as described above, and the second component being one of the above-mentioned Cas proteins.

[0193] According to this second aspect, a subject of the present invention is therefore an expression vector comprising at least one nucleic acid as defined above, said at least one nucleic acid being under the control of an element enabling its expression (promoter).

[0194] "Expression vector" refers to a DNA (deoxyribonucleic acid) molecule with elements that enable it to replicate (multiply) in at least one living organism. These elements that enable it to replicate are, in particular, origins of replication in yeast or bacteria, or elements that control viral replication. The vector according to the present invention is particularly a modified genome of a plasmid, a bacteriophage, a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC), a replicating virus or an integrating virus, etc. Some vectors carry viral genomes (replicating) or genomes (non-replicating), DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) derived from viruses but lacking viral genes. The RNA genome can be reverse transcribed into DNA by the reverse transcriptase of a retrovirus, or directly translated by a cellular mechanism. The genome, DNA or RNA of a virus or viral origin can be wrapped in a protective structure of a cell membrane and viral proteins. These recombinant viruses or viral-derived particles allow transduction of target cells to express Cas proteins or anti-CD-ASGPR. This transgenic expression can be stable, cyclic or transient, depending on the process or promoter used.

[0195] These vectors are called "expression" vectors because they have nucleotide sequences capable of effecting expression (ie, transcription into RNA (ribonucleic acid)) of the nucleotide sequences they control or translation of the RNA they carry.

[0196] In the present invention, the at least one nucleic acid contained in the vector is placed "under the control of elements that enable it to be expressed". This means that the expression vector has at least one transcription initiation sequence, for example a viral promoter, such as the early promoter of the simian virus SV40 or cytomegalovirus (CMV) or the promoter sequence of the Rous sarcoma virus (RSV), and in particular a sequence or promoter comprising a TATAA box. It can also be a promoter of a human housekeeping gene, such as the promoter of the phosphoglycerate kinase (PGK) gene, or a so-called tissue-specific human promoter that is active only in certain cell subpopulations in the body. It can also be a synthetic promoter that contains one or more response elements and binds one or more transcription factors; these promoters can be induced by exogenous stimuli (such as pharmaceutical molecules, hormones, defects or stress). In addition, the vector also has at least one transcription termination sequence, and in particular a polyadenylation sequence derived from a mammalian gene (in particular a human gene).

[0197] These sequences are essential for the expression of the nucleotide sequence contained in the vector and may be supplemented by other sequences capable of regulating or modulating the expression of the sequence. A non-exhaustive list includes: transcriptional regulatory sequences of the intron, enhancer type or transcribed but untranslated sequences of mammalian (particularly human) genes.

[0198] Host cells

[0199] According to a third aspect of the present invention, the present invention relates to a host cell or cell line transformed by a nucleic acid as described above and / or an expression vector as described above. In other words, the subject of the present invention is a host cell or cell line capable of expressing (producing) a polypeptide conjugate as described above, wherein the polypeptide conjugate comprises a first component covalently or non-covalently linked to a second component, wherein the first component is an antigen presenting cell antibody targeting an asialoglycoprotein receptor (anti-APC-ASGPR) or a fragment thereof, and the second component is a Cas protein.

[0200] Composition

[0201] On the other hand, the present invention relates to a pharmaceutical composition comprising as an active ingredient a polypeptide conjugate as described above and an acceptable pharmaceutical excipient in combination therewith, wherein the polypeptide conjugate comprises a first component covalently or non-covalently linked to a second component, wherein the first component is an antigen presenting cell antibody targeting APC asialoglycoprotein receptor (anti-DC-ASGPR) or a fragment thereof, and the second component is a Cas protein.

[0202] "Pharmaceutical composition" refers to a specific packaging form of the present invention, which allows the pharmaceutical composition of the present invention to be administered to animals and humans by oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, topical, inhalation or rectal routes. In addition, such packaging also allows the active ingredient (or active substance) to be administered alone or in combination with another active ingredient in unit form or mixed with conventional pharmaceutical carriers. Suitable unit administration forms include:

[0203] - oral administration forms, such as tablets, capsules, powders, granules and oral suspensions or solutions;

[0204] - Sublingual and buccal administration, aerosols and implants;

[0205] - subcutaneous, transdermal, intradermal, intraperitoneal, intramuscular, intravenous, subcutaneous, transdermal, intratracheal and nasal administration forms; and

[0206] - Rectal administration.

[0207] An "acceptable pharmaceutical carrier" (or pharmaceutically acceptable excipient) is a non-toxic material that is compatible with biological systems (e.g., cells, cell cultures, tissues, or animals or humans). This may include:

[0208] -Crystalloid solutions, such as sodium chloride, bicarbonate, dextrose;

[0209] - cationic lipids;

[0210] - a peptide compound; or

[0211] - Surfactants, such as polysorbates.

[0212] In all cases, whatever preparation is chosen, it must be sterile, stable under the conditions of manufacture and storage, and must be preserved against the contaminating microorganisms such as bacteria and fungi.

[0213] According to another embodiment, the subject of the present invention is a pharmaceutical composition as described above in unit form, wherein the polypeptide conjugate of the present invention is at a (unit) dose of 0.1 to 1,000 μg or at a (unit) dose of 0.1 to 1,000 mg / kg (based on a 70 kg person). "0.1 to 1,000 μg (or mg / kg)" means that the dose can 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).It will also be understood that the dosage can be 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0, 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 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, 520 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1,000 μg (or mg / kg).

[0214] According to another embodiment, a subject of the invention is a pharmaceutical composition as described above, formulated so as to be able to be administered by one of the following routes: oral, parenteral, injection, topical, inhalation, subcutaneous, nasal or pulmonary.

[0215] 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. "Anti-OX40L antibody or a fragment thereof" refers to an antibody or a fragment thereof that is capable of specifically recognizing a ligand of OX40 (also known as CD134 or TNFRSF4) stably expressed on a variety of antigen presenting cells, such as DC2 (a subtype of dendritic cells), macrophages and activated B lymphocytes.

[0216] Uses and methods

[0217] According to another aspect, the present invention relates to a polypeptide conjugate according to the present invention for promoting immune tolerance to the above-mentioned Cas protein. The present invention also relates to a pharmaceutical composition according to the present invention for promoting immune tolerance to Cas proteins of bacterial, archaeal or phage origin.

[0218] "Promoting immune tolerance to Cas proteins" means that administering a polypeptide conjugate according to the present invention and / or a pharmaceutical composition according to the present invention to a mammal induces (triggers) immune tolerance to Cas proteins, which inhibits any subsequent immune rejection of the relevant Cas proteins. In this way, the increase in immune tolerance to Cas makes it possible to establish a safe and effective gene repair program in humans using the CRISPR-Cas system.

[0219] According to another embodiment, the present invention relates to a polypeptide conjugate according to the present invention for promoting immune tolerance to Cas proteins, wherein the polypeptide conjugate is administered to a mammal. The present invention also relates to a pharmaceutical composition according to the present invention for promoting immune tolerance to the Cas protein, wherein the pharmaceutical composition is administered to a mammal. "Mammal" refers to an animal organism, such as a primate or a human (male, female, and child).

[0220] According to another embodiment, the present invention relates to a polypeptide conjugate according to the present invention for promoting immune tolerance to Cas proteins, the polypeptide conjugate being at a (unit) dose of 0.1 to 1,000 μg or at a (unit) dose of 0.1 to 1,000 mg / kg (based on a 70 kg human). The present invention also relates to a pharmaceutical composition according to the present invention for promoting immune tolerance to Cas proteins, the pharmaceutical composition comprising a polypeptide conjugate according to the present invention at a (unit) dose of 0.1 to 1,000 μg or a polypeptide conjugate according to the present invention at a (unit) dose of 0.1 to 1,000 mg / kg (based on a 70 kg human). "0.1 to 1,000 μg (or mg / kg)" means that the dosage can 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).It will also be understood that the dosage can be 0.1, 0.2, 0.3, 0.4, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 12.5, 13.0, 13.5, 14.0, 14.5, 15.0, 15.5, 16.0, 16.5, 17.0, 17.5, 18.0, 18.5, 19.0, 19.5, 20.0 , 21, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88 ,89,90,91,92,93,94,95,96,97,98,99,100,110,120,130,140,150,160,170,180,190,200,210,220,230,240,250,260,270,280,290,300,310,320,330,340,350,360,370,380,390,400,410,420,430,440,441,450,460,470,480,490,500,510,5 940, 950, 960, 970, 980, 990, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000, 1,000

[0221] According to another embodiment, the present invention relates to a polypeptide conjugate according to the present invention for promoting immune tolerance to Cas proteins, wherein the 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 according to the present invention for promoting immune tolerance to the Cas protein, wherein the pharmaceutical composition is administered by one of the following routes: oral, parenteral, injection, topical, inhalation, subcutaneous, nasal or pulmonary.

[0222] According to another embodiment, the present invention relates to a polypeptide conjugate according to the present invention for promoting immune tolerance to Cas proteins, wherein the polypeptide conjugate is administered in combination with an anti-OX40L antibody or a fragment thereof. The present invention also relates to a pharmaceutical composition according to the present invention for promoting immune tolerance to the Cas protein, wherein the pharmaceutical composition further comprises an anti-OX40L antibody or a fragment thereof.

[0223] According to another embodiment, a subject of the present invention is a polypeptide conjugate according to the invention for promoting immune tolerance to Cas proteins, and in particular, said polypeptide conjugate is administered in combination with an anti-OX40L antibody or a fragment thereof.

[0224] The present invention also relates to a pharmaceutical composition according to the present invention for promoting immune tolerance to the Cas protein, and in particular, the pharmaceutical composition further comprises an anti-OX40L antibody or a fragment thereof.

[0225] Alternatively, the present invention relates to a pharmaceutical composition for promoting immune tolerance to Cas protein, the composition comprising the polypeptide conjugate of the present invention as an active ingredient and an acceptable pharmaceutical carrier in combination therewith, and in particular, the pharmaceutical composition further comprises an anti-OX40L antibody or a fragment thereof.

[0226] Alternatively, the present invention also relates to a method for promoting immune tolerance to Cas proteins in patients in need, the method comprising the step of administering a polypeptide conjugate according to the present invention. Another subject of the present invention is a method for promoting immune tolerance to the Cas protein in patients in need, the method comprising the step of administering a pharmaceutical composition according to the present invention.

[0227] Finally, it will be understood that, thanks to the present invention, immunizing a patient in need (i.e., a patient suffering from a pathology that requires gene therapy based on the CRISPR-Cas system to be treated) with Cas makes it possible for him to benefit from a safe and effective gene therapy regimen. To a certain extent, it should therefore be understood that the present invention relates to a recombinant protein (anti-DC-ASGPR-Cas) and a vector encoding the Cas protein. The recombinant anti-DC-ASGPR-Cas protein is injected, in particular intradermally, to immunize a patient in need thereof, and then a vector is administered to allow Cas to be expressed in a given genetically modified tissue.

[0228] In other words, the present invention also relates to a kit comprising:

[0229] - a polypeptide conjugate of the present invention, or a vector encoding the polypeptide conjugate of the present invention, or a pharmaceutical composition of the present invention; and

[0230] - A vector encoding a Cas protein, in particular a vector encoding a Cas protein identical to the Cas protein present in the polypeptide conjugate according to the present invention.

[0231] In this regard, it should be noted that the "vector encoding the Cas protein" in the kit does not correspond to the vector of the present invention described above, and the purpose of the kit vector is to allow the expression of Cas for gene therapy purposes (rather than to produce the polypeptide conjugate of the present invention).

[0232] In any case, it should be noted that the various aspects of the invention (like its various embodiments) are interdependent. Therefore, they can be combined with each other as many times as necessary to obtain aspects and / or preferred embodiments of the invention that are not explicitly described. This also applies to all definitions provided in this specification, which apply to all aspects of the invention and its embodiments.

[0233] Furthermore, the present invention is described by the following figures and examples, but is not limited thereto. BRIEF DESCRIPTION OF THE DRAWINGS

[0234] Figure 1 . Experimental protocol for vaccination of cynomolgus macaques with anti-DC-ASGPR-Cas9. The protocol was carried out for 42 days. Blood samples were collected every 7 days. The first sample at D0 provided the baseline level for all subsequent measurements. Animals received three intradermal (ID) injections of anti-DC-ASGPR-Cas9 on days 14, 21 and 28, separated by one week. Finally, animals received an injection of Cas9 protein at D35.

[0235] Figure 2Optimization of the assembly of anti-DC-ASGPR-dockerin and saCas9-adhesion protein. The assembly of two molar ratios of anti-DC-ASGPR-dockerin and protein (saCas9-adhesion protein) was analyzed by CAPE blot electrophoresis (Chretien P et al. J Autoimmun. 1994 Jun; 7(3): 379-88. PMID: 7916909). Lane 1 , Deposition of saCas9-cohesin alone (migration 20 mm). Lane 2 , Deposition of anti-DC-ASGPR-dockerin alone (migration 8 mm). Lane 3 , a mixture of saCas9-cohesin and anti-DC-ASGPR-dockerin at a molar ratio of 1:0.5 (trailing and migration 12 mm). Lane 4 , a mixture of saCas9-cohesin and anti-DC-ASGPR-dockerin at a molar ratio of 1:2 (single band and migration 12 mm). Anti-DC-ASGPR-dockerin and protein (saCas9-cohesin) at a molar ratio of 1:2 were used for anti-DC-ASGPR-saCas9 vaccination.

[0236] Figure 3 .Strategy for analyzing cell subtypes of cynomolgus macaque PBMCs by cytometry.

[0237] Figure 4 . CD4 from macaques expressing markers CD25, FOXP3 and CD39 + Strategies for analyzing the regulatory phenotype of lymphocytes.

[0238] Figure 5 CD4 T cells of interest were cultured under unstimulated (NS) conditions or stimulated with Cas9 protein (saCas9) at D0 of the protocol. + Cytometry analysis of lymphocytes. The numbers on the scale indicate the cell proportion.

[0239] Figure 6 CD4 T cells of interest were cultured under unstimulated (NS) conditions or stimulated with Cas9 protein (saCas9) at D21 of the protocol. + Cytometry analysis of lymphocytes. The numbers on the scale indicate the cell proportion.

[0240] Figure 7 CD4 T cells of interest were cultured under unstimulated (NS) conditions or stimulated with Cas9 protein (saCas9) at D35 of the protocol. + Flow cytometric analysis of lymphocytes. The numbers on the scale indicate the cell proportion.

[0241] Figure 8 .Number of regulatory CD4+T cells expressing FOXP3 and CD39 in the CD4+CD25+OX40+ cell population. (A) Proportion of CD4+CD25+OX40+ lymphocytes seven days after one vaccination (vertical dashed line) with anti-DC-ASGPR-Cas9 (D21) and seven days after three vaccinations (D35). (B) Number of Foxp3+CD39- lymphocytes in the CD4+CD25+OX40+ lymphocyte population. (C) Number of Foxp3+CD39+ lymphocytes in the CD4+CD25+OX40+ lymphocyte population.

[0242] Fig. 9 Measurement of TGFb1 in the serum of cynomolgus monkeys after three vaccinations with anti-DC-ASGPR-Cas9 (D35) 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 dashed line : Immunization with Cas9 protein.

[0243] Fig.10 Measurement of IL10 in the culture supernatants of cynomolgus macaque PBMCs collected at D21, D28, D35, and D42 after the first rhMOG immunization. An increase in IL10 secretion was observed in saCas9 (Cas9)-stimulated PBMCs collected at D42 (one week after the second rhMOG immunization), but not in the supernatants of non-stimulated (NS) PBMCs.

[0244] Example

[0245] Preclinical studies: Immunization with anti-APC-ASGPR-saCAS9 in cynomolgus macaques Immune tolerance of saCAS9

[0246] Preliminary data

[0247] in vitro

[0248] Anti-APC-ASGPR-saCas9 was obtained. It was constructed based on monoclonal antibody 49C11 and Staphylococcus aureus Cas9, and the obtained conjugate contained:

[0249] - the light chain 49C11_L (SEQ ID NO: 34) of the anti-DC-ASGPR monoclonal antibody; and

[0250] - The heavy chain 49C11_H of the anti-DC-ASGPR monoclonal antibody, which comprises a dockerin domain (SEQ ID NO: 103) that enables it to associate with the saCas9 protein comprising a cohesin domain (SEQ ID NO: 89).

[0251] General Experimental Design

[0252] The following protocol was established to induce immune tolerance to heterologous saCas9 protein in immunocompetent adult cynomolgus macaques ( Figure 1 ). Using a recombinant anti-DC-ASGPR-saCas9 protein obtained by combining an anti-DC-ASGPR-dockerin antibody and a protein (saCas9-adhesin) ( Figure 2 ). Next, it was determined whether this procedure allowed the emergence of a saCas9-specific regulatory CD4+ T lymphocyte population expressing OX40+CD25+FOXP3 and CD39+ markers (Fovet CM et al. EBioMedicine. 2019 Sep; 47:492-505. PMC6796575). For this purpose, a blood monocyte labeling strategy was established ( Figure 3 This enables the detection of circulating CD4+ lymphocytes, where the regulatory phenotype is determined by immunostaining for CD25+FOXP3 and CD39+ antigens ( Figure 4 ). 44 hours after priming with saCas9 protein, saCas9-specific regulatory CD4+ T cells (CD25+FOXP3 and CD39- or CD39+) were detected by the presence of the activation marker OX40 ( Figure 5 ).

[0253] In this protocol, 1 monkey was immunized by intradermal injection of anti-APC-ASGPR-saCas9 antibody.

[0254] Vaccine regimen

[0255] Animals received intradermal doses of 250 μg of recombinant anti-APC-ASGPR-saCas9 protein weekly in the back for 3 weeks ( Figure 1 ).

[0256] Immunization with saCas9

[0257] On day 35 of the regimen, 7 days after receiving the last dose of vaccine, animals were immunized with 250 μg of saCas9 protein.

[0258] Pre- and post-vaccination immune analysis

[0259] Starting from day 1, plasma and PBMCs were collected weekly to explore CD4 responses to saCas9 in vitro.

[0260] Blood samples (10 mL) were collected from animals before the first vaccination and then at regular intervals to assess hematology and biological functions.

[0261] PBMCs from animals were cultured. Cells were labeled with CFSE (carboxyfluorescein succinimidyl ester) and cultured on day 0, then stimulated or not stimulated with recombinant saCas9. Labeled cells were collected and processed 44 hours later to analyze intracellular cytokines and study their phenotypes by flow cytometry. Some of these cells were used to evaluate the expression of CD4, CD8, CD25, OX40, CD39, and Foxp3 markers after stimulation with saCas9.

[0262] Animals were examined daily, and clinical signs were quantified using an assessment form.

[0263] also:

[0264] - Inject anti-APC-ASGPR-saCas9 fusion protein intradermally into the back of the animal;

[0265] - Inject recombinant saCas9 protein intradermally into the back of the animals;

[0266] -Apply resiquimod as a gel to the shaved skin surface and the fusion protein injection site. Apply the gel immediately after the fusion protein injection, followed by a gentle "skin massage" for 30 seconds;

[0267] - On each antibody vaccination date, animals received five injections of 50 μg of fusion protein (250 μg total);

[0268] - On the day of injection of recombinant saCas9 protein, animals received five injections of 50 μg saCas9 (250 μg total); and

[0269] -Immunity monitoring includes:

[0270] a. Measure anti-saCas9-specific T cells (CD4 + and CD8 + ) and TGFβ1 in serum.

[0271] b. Monitor T and B cell activation markers in the blood: anti-CD45, anti-CD3,

[0272] -CD4, anti-CD8, anti-CD95, anti-CD28, anti-CD69, anti-HLADR, anti-CD20, anti-CD27, and anti-IgD.

[0273] Materials and methods

[0274] animal

[0275] Adult macaques (Macaca fascicularis) imported from Mauritius were included in this study. MHC class I and class II typing was determined.

[0276] Immunogen

[0277] Recombinant proteins were produced by Baylor Institute for Immunology Research (BIIR). Anti-DC-ASGPR-dockerin (human) was combined with saCas9-cohesin extemporaneously by mixing at a 1:2 molar ratio during intradermal injection.

[0278] Recombinant saCas9 produced in Escherichia coli was obtained by BIIR.

[0279] Immunization

[0280] Animals were injected intradermally (ID) with a syringe in the back. The injection site was shaved.

[0281] In addition, on each vaccination date, animals received 5 injections of 100 μL of each anti-DC-ASGPR-saCas9 vaccine spaced 0.5 cm apart.

[0282] Each injection contained 50 μg of antibody.

[0283] Observations during life

[0284] Animals were observed by CEA staff via webcam monitoring 7 days a week. Food and water consumption was recorded. Each time the animals had their blood drawn, a clinical examination was performed, and their body weight and rectal temperature were recorded.

[0285] Table 2. Observations during life

[0286]

[0287] Complete blood test

[0288] According to procedure SOP#EXAMO006_01, use HMX A / L (Beckman ) Complete blood workup was performed. Serum transaminases, as well as LDL and PCSK9 protein were measured weekly.

[0289] saCas9-specific T cell activation, phenotype, and cytokine production

[0290] saCas9-specific autoreactive and regulatory CD4 in circulating PBMCs have not been studied in animals after saCas9 gene transfer. + The level of lymphocytes. Their stimulation and large-scale culture change their original phenotype and function and hinder their accurate study. In order to evaluate and characterize these cells in macaques, a recent assay was used, which detects CD4+T lymphocytes that overexpress CD140 (OX40) and CD25 (IL2R), because they enable the identification of cells that respond to antigen restimulation with a sensitivity far higher than that of intracellular cytokine (ICS) markers (JJ Zaunders et al. J Immunol 183, 2827-36 (2009)).

[0291] saCas9-specific CD4 + Phenotypic polarization of T cells. Under these conditions, before cells begin to proliferate, saCas9-specific CD4 + T cells overexpress OX40 and CD25 (N. Seddiki et al., Eur J Immunol 44, 1644-61 (2014)). Therefore, cultured PBMCs stimulated with saCas9 epitopes were labeled with antibodies against CD4, CD134, CD25, CD39, and FOXP3 to distinguish T cells from cultured PBMCs. regs and T R1 (N. Seddiki et al., Eur J Immunol 44, 1644-61 (2014)). Different cell subtypes were identified, quantified and sorted by cytometry.

[0292] This experiment, which requires a small amount of blood, provides essential quantitative and qualitative information on the levels and phenotype of circulating anti-saCas9 CD4+ cells at different times after vaccination with anti-DC-ASGPR-saCas9 antibodies.

[0293] Serum cytokines

[0294] TGFb1 was measured before and at different times after vaccination and one week after immunization with saCas9.

[0295] Cytokines in PBMC culture supernatant

[0296] IL10 was measured in culture supernatants of PBMCs collected at different times after immunization with rhMOG and vaccination with anti-ASGPR-saCas9. PBMCs in culture were stimulated with or without scCas9.

[0297] Equipment preservation

[0298] During the study and for 2 years after the end of the study, biological samples were stored frozen at -80°C (plasma, serum, RNA) or -135°C (PBMC).

[0299] Data analysis

[0300] Data were recorded and analyzed using an IDBS-based data management system.

[0301] use 8 Statistical analysis was performed using the nonparametric Mann & Whitney or Wilcoxon rank test.

[0302] use and Software for flow cytometry analysis.

[0303] ethics

[0304] Animals were housed at the CEA facility in Fontenay-aux-Roses in compliance with institutional standards of care and European Directive D8906, and the standards of the Office of Laboratory Animal Welfare (OLAW) of the National Institutes of Health (USA).

[0305] result

[0306] use Figure 1 Healthy adult cynomolgus monkeys were treated with the vaccination regimen described in and below to induce immune tolerance to the heterologous saCas9 protein. This was achieved using a recombinant anti-DC-ASGPR-saCas9 protein obtained by combining two recombinant proteins: the anti-DC-ASGPR-dockerin antibody and the protein saCas9-cohesin ( Figure 2 ). The animals then received an intradermal injection of anti-DC-ASGPR-saCas9. At the same time, it was determined whether this procedure allowed the emergence of a saCas9-specific regulatory CD4+ T lymphocyte population expressing OX40+CD25+FOXP3 and CD39+ markers. For this purpose, a blood monocyte labeling strategy was established ( Figure 3 ), which enables the detection of a new population of regulatory CD4+ T lymphocytes expressing CD25+OX40+FOXP3+ and CD39+ markers ( Figure 4 These cells (i.e., saCas9-specific CD4+ regulatory T cells (CD25+FOXP3 and CD39- or CD39+)) were not present prior to the vaccine regimen and increased proportionally with the completion of the anti-DC-ASGPR-saCas9 vaccine regimen ( Figures 5 to 7). In addition, total counts of these cells showed that their absolute number increased upon activation with recombinant saCas9 protein ( Figure 8 Finally, in animals that received three injections of anti-DC-ASGPR-saCas9, immunization by intradermal injection of saCas9 induced the expression of the anti-inflammatory cytokines TGFb1 and IL10, as detected in plasma or in the supernatant of cultured PBMCs, respectively ( Fig. 9 and Fig.10 ).

[0307] Therefore, these results indicate that vaccination with anti-DC-ASGPR-saCas9 protein induces the appearance of a population of regulatory T lymphocytes and, in the presence of saCas9 antigen, is a hallmark of an anti-inflammatory immunoregulatory response.

Claims

1. A polypeptide conjugate comprising a first component covalently or non-covalently linked to a second component, wherein the first component is an antigen presenting cell antibody targeting an asialoglycoprotein receptor of an antigen presenting cell (anti-DC-ASGPR), or a fragment thereof capable of binding to an epitope recognized by a full-length antibody, and the second component is a Cas protein, The polypeptide conjugate is capable of inducing immune tolerance to the Cas protein in mammalian organisms, and in particular in primates and humans, and The anti-DC-ASGPR is selected from: - Antibodies containing: o 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 o a light chain comprising, from N-terminus to C-terminus, a CDR1 of SEQ ID NO: 16, a CDR2 of SEQ ID NO: 17, and a CDR3 of SEQ ID NO: 18; - Antibodies containing: o a heavy chain comprising, from N-terminus to C-terminus, a CDR1 of SEQ ID NO: 35, a CDR2 of SEQ ID NO: 36, and a CDR3 of SEQ ID NO: 37; and o a light chain comprising, from N-terminus to C-terminus, a CDR1 of SEQ ID NO: 40, a CDR2 of SEQ ID NO: 41, and a CDR3 of SEQ ID NO: 42; - Antibodies containing: o 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; and o a light chain comprising, from N-terminus to C-terminus, a CDR1 of SEQ ID NO: 50, a CDR2 of SEQ ID NO: 51, and a CDR3 of SEQ ID NO: 52; - Antibodies containing: o a heavy chain comprising, from N-terminus to C-terminus, a CDR1 of SEQ ID NO: 55, a CDR2 of SEQ ID NO: 56, and a CDR3 of SEQ ID NO: 57; and o a light chain comprising, from N-terminus to C-terminus, a CDR1 of SEQ ID NO: 60, a CDR2 of SEQ ID NO: 61, and a CDR3 of SEQ ID NO: 62; - Antibodies containing: o 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; and o a light chain comprising, from N-terminus to C-terminus, a CDR1 of SEQ ID NO: 70, a CDR2 of SEQ ID NO: 71, and a CDR3 of SEQ ID NO: 72; and - Antibodies containing: o 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; and o A light chain comprising, from N-terminus to C-terminus, a CDR1 of SEQ ID NO: 80, a CDR2 of SEQ ID NO: 81, and a CDR3 of SEQ ID NO:

82.

2. The polypeptide conjugate according to claim 1, wherein The Cas protein is selected from: -Cas9 nucleases from Streptococcus pyogenes, Staphylococcus aureus, Corynebacterium diphtheriae, Neisseria meningitidis, Streptococcus canis, Streptococcus macaques, Francisella tularensis, Acidinococcus, Campylobacter jejuni, Streptococcus pneumoniae, and Streptococcus thermophilus; Cas12a nucleases from Lachnospiraceae and Acidinococcus; Cas12b nucleases from Alicyclobacillus kakegawensis and Bacillus hisashii; CasX or Cas12e nucleases from Delta-Proteobacteria and Planctomyces; Type V CRISPR nucleases from Syntrophomonas palmitatica and Acidibacillus sulfuroxidans; Cas 12j or CasΦ nucleases from the Biggiephage clade; - Cas9, Cas12a, Cas12b, CasX or Cas12e, V-type CRISPR, Cas 12j or CasΦ orthologs derived from these organisms; as well as - a mutant or functional variant of Cas9, Cas12a, Cas12b, CasX or Cas12e, type V CRISPR, Cas 12j or CasΦ derived from these organisms, And in particular, the Cas protein is selected from SEQ ID NO:87 to SEQ ID NO:95 and SEQ ID NO:105 to SEQ ID NO:113, and mutants thereof.

3. The polypeptide conjugate according to claim 1 or 2, wherein: The anti-DC-ASGPR is covalently linked to the Cas protein, And in particular, the polypeptide conjugate comprises the sequences of SEQ ID NO:102 and SEQ ID NO:

34.

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

5. The polypeptide conjugate according to claim 3 or 4, wherein: The peptide linker is selected from: -QTPTNTISVTPTNNSTPTNNSNPKPNPAS (SEQ ID NO:96); -SSVSPTTSVHPTPTSVPPTPTKSSP(SEQ ID NO:97); -PTSTPADSSTITTPTATPTATPTIKG(SEQ ID NO:98); -TVTPTATATPSAIVTTITPTATTKP (SEQ ID NO:99); and -TNGSITVAATAPTVTPTVNATPSAA(SEQ ID NO:100), And in particular, the polypeptide conjugate comprises the sequences of SEQ ID NO:101 and SEQ ID NO:

34.

6. The polypeptide conjugate according to claim 1 or 2, wherein: The anti-DC-ASGPR non-covalently binds to the Cas protein.

7. The polypeptide conjugate according to claim 6, wherein The anti-DC-ASGPR non-covalently binds to the Cas protein via a high affinity interaction selected from: - Antibody / antigen interactions; - Receptor / ligand interactions; -Avidin / biotin interaction; - cohesin / dockerin interactions; and -Barnase / barstar interactions.

8. The polypeptide conjugate according to claim 6 or 7, wherein: The high affinity interaction is a cohesin / dockerin interaction, And in particular, the polypeptide conjugate comprises the sequences of SEQ ID NO:103, SEQ ID NO:34 and SEQ ID NO:104 or the sequences of SEQ ID NO:85, SEQ ID NO:25 and SEQ ID NO:

104.

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

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