TITLE: A SIRP-ALPHA BINDING CHIMERIC PROTEIN

The present disclosure relates generally to chimeric proteins, cells, cell compositions or pharmaceutical products in the field of cellular therapy. In a further expect, the disclosure relates preparation methods and to medical uses of chimeric proteins, of cells, cell compositions or pharmaceutical products.

INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING

SEQUENCE LISTING

The present application is filed with a Sequence Listing in electronic form. The entire contents of the sequence listing are hereby incorporated by reference.

BACKGROUND

In the field of cellular therapy, a key challenge is the rejection of allogeneic cellular transplants in host patients. Researchers are working on various approaches to reduce this phenomenon and increase the success rate of cellular therapy.

An option is to express or overexpress CD47 in grafted cells. CD47 is a potent "don't-eat-me" signal enabling the recognition between a "self" cell and immune cells such as macrophage cell. All normal cells express CD47 on their cell surface as an antiphagocytic signal to maintain tissue homeostasis. Recent research has shown that CD47 was transiently upregulated on mouse hematopoietic stem cells (HSCs) and progenitors just prior to migratory phase to prevent immune cells to overreact against a fast-growing cell. In cancer cells, overexpression of CD47 is well known to avoid phagocytosis thereof by immune cells via binding of the SIRP-o (Signal regulatory protein a) expressed on macrophage and other immune cells with CD47.

There is a need to improve cellular therapy, to improve the success rate of allogeneic cellular transplantation in host patients and/or to reduce the "non self" recognition and rejection of grafted allogeneic cells by the immune system of host patients. SUMMARY

The present disclosure addresses the shortcomings identified above. In the present disclosure, novel chimeric proteins and cells expressing novel chimeric proteins are provided. The present disclosure provides a means of reducing or preventing non-self-recognition and/or rejection of grafted cells by the immune system of a host patient. The present disclosure provides a means of improving cell therapy e.g. by increasing the success rate of allogeneic cellular transplantation in host patients.

The novel chimeric proteins of the present disclosure are designed to enable self-cell recognition of grafted cells while not triggering CD47 downstream signaling.

The invention may also solve further problems that will be apparent from the disclosure of the exemplary embodiments.

In a first aspect of the present disclosure is provided a cell comprising a chimeric protein on its cell surface and/or comprising a nucleic acid encoding said chimeric protein, said chimeric protein comprising an extracellular domain, a transmembrane domain and a linker between said extracellular domain and said transmembrane domain, wherein - said extracellular domain comprises wild type CD47 ectodomain of sequence SEQ ID NO: 1 or an analogue thereof . The transmembrane domain may comprise the transmembrane domain of any one of CD3e, CD28, CD38 or CD80, or an analogue thereof. Said extracellular domain may comprise an analogue of wild type CD47 ectodomain of sequence SEQ ID NO: 1, said analogue having at least 90% homology and less than 100% homology with SEQ ID NO: 1. The chimeric protein might not comprise the amino acid sequence SEQ ID NO: 3 and/or might not comprise the amino acid sequence SEQ ID NO: 4. The transmembrane domain may comprise the transmembrane domain of CD3e of sequence SEQ ID NO: 5 or of a sequence a having at least 90% homology and less than 100% homology with SEQ ID NO: 5. The transmembrane domain may comprise the transmembrane domain of CD3e and the linker may comprise a sequence GX1GVSX2, wherein Xi can be S or P and X2 can be G or C and said extracellular domain may comprise an analogue of the ectodomain of wild type CD47, said analogue comprising Y113V and R114G compared to SEQ ID NO: 1. The transmembrane domain may comprise the transmembrane domain of CD28 of sequence SEQ ID NO: 6 or of a sequence having at least 90% homology with SEQ ID NO: 6. The transmembrane domain may comprise the transmembrane domain of CD28 and the linker may comprise sequence NCX3, wherein X3 can be P or A, and wherein said extracellular domain may comprise an analogue of the ectodomain of wild type CD47, said analogue comprising one or both of F14C and C15G compared to SEQ ID NO: l.The transmembrane domain may comprise the transmembrane domain of CD38 of sequence SEQ ID NO: 7 or of a sequence a having at least 90% homology with SEQ ID NO: 7. The transmembrane domain may comprise the transmembrane domain of CD38 and the linker may comprise a sequence X4X5X6X7X8X9, wherein X4 is E, Q or G, wherein X5 is E, D or S, Xe is absent or is G, X7 is absent or is A or S, Xs is C, G, S, P or D and X9 is P, G or C, and the extracellular domain may comprise an analogue of the ectodomain of wild type CD47, said analogue comprising one or more of F14C, F14A, F14G and C15G compared to SEQ ID NO: 1. The transmembrane domain may comprise the transmembrane domain of CD80 of sequence SEQ ID NO: 8 or of a sequence a having at least 90% homology with SEQ ID NO: 8. The transmembrane domain may comprise the transmembrane domain of CD80 and the linker may comprise sequence SDX10X11X12X13X14X15X16R, wherein X10 can be E or K, Xn can be E or T, X12 can be P or T, X13 can be absent or be P or D, X14 can be absent or A, X15 can be absent or T, Xie can be absent or C or G, and the extracellular domain may comprise an analogue of the ectodomain of wild type CD47, said analogue comprising C15G compared to SEQ ID NO: 1. The extracellular domain may comprise an analogue of the ectodomain of wild type CD47 of sequence SEQ ID NO: 1, and said analogue may comprise in its amino acid sequence one or more mutation selected from the group consisting of S64A, S64T, S64G, D62A, E69A, T61D, S85R, S89A and S89D compared to the sequence SEQ ID NO: 1. The chimeric protein may comprise or consist of anyone of sequences SEQ ID NO: 10, SEQ ID

NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO:

16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21,

SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and

SEQ ID NO: 27.

In a further aspect of the present disclosure is provided a cell comprising a nucleic acid sequence encoding a chimeric protein as described herein. The cell maybe a mammalian cell, such as a human cell, a beta cell, an INS+ and NKX6.1+ double positive cell or a C- peptide+/NKX6.1+ double positive cell, an insulin producing cell, an in vitro derived beta-like cell, a pancreatic endocrine cell or an endocrine cell, an endocrine progenitor cell or a NGN3+/NKX2.2+ double positive cell, a neural cell such as a neuron, an interneuron cell, an oligodendrocyte, an astrocyte or a dopaminergic cell, an exosome cell, an immune cell such as a T cell, a NK cell, a macrophage or a dendritic cell, a hepatocyte, a stellate cell, a fibroblast, a keratinocyte, a hair cell, an inner ear cell, an intestinal cell, an organoid cell, a nephroid cell or another kidney-related cell, a cortical neural progenitor cell, a cardiomyocyte, a retinal cell, a retinal pigment epithelium cell or a mesenchymal stem cell.

In a further aspect of the present disclosure is provided a pharmaceutical product comprising cells for use in cellular therapy of a patient in need thereof.

In further aspect of the present disclosure is provided a process for the preparation of a cell as described herein, said process comprising the step of inserting into a cell a nucleic acid sequence encoding a chimeric protein as described herein.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 (1) illustrates the structure model of wild type CD47 protein comprising wild type CD47 extracellular domain ("CD47 ectodomain" in the figure), wild type CD47 linker ("linker region" in the figure) and wild type CD47 transmembrane domain ("TM" in the figure).

Fig.l (2), (3), (4) and (5) illustrate the structure model of chimeric proteins comprising a modified CD47 ectodomain, i.e. with sequence modification compared to wild type CD47 ectodomain, and comprising a newly designed linker, i.e. with sequence modification compared to wild type CD47 linker, and comprising a single-pass transmembrane domain ("TM" in the figure) derived from either, respectively, CD28 (Fig. l (2)), CD3e (Fig.l (3)), CD80 (Fig.l (4)) or CD38 (Fig. l (5)).

Fig. 2 shows the amino acid sequence of 18 chimeric proteins comprising a functional CD47 ectodomain region or analogue thereof, the transmembrane domain from one of the protein (Fig.2(a)) CD3e, (Fig.2(b)) CD28, (Fig.2(c)) CD38 and (Fig.2(d)) CD80 and a linker in between. Fig. 2(a) shows the sequences for two such chimeric proteins comprising a transmembrane domain from the protein CD3e, said proteins being named "l.CD3e_0001" and "2.CD3e_0002" respectively. Fig. 2(b) shows the sequences for two such chimeric proteins comprising a transmembrane domain from the protein CD28, said proteins being named "l.CD28_0001" and "2.CD28_0002" respectively. Fig. 2(c) shows the sequences for eight such chimeric proteins comprising a transmembrane domain from the protein CD38, said proteins being named "l.CD38_0001", "2.CD38_0002", "3.CD38_0003", "4.CD38_0004", "5.CD38_0005", "6.CD38_0006", "7.CD38_0007" and "8.CD38_0008" respectively. Fig. 2(d) shows the sequences for six such chimeric proteins comprising a transmembrane domain from the protein CD80, said proteins being named "l.CD80_0001", "2.CD80_0002", "3.CD80_0003", "4.CD80_0004", "5.CD80_0005" and "6.CD80_0006" respectively. In Fig.2 (a) to (d), due to space constraints, each sequence is split in 3 sections and in the same row of said sections. Amino acid positions indicated above the first line of each section guides the reading of each sequence.

Fig. 3(a) shows in a graphical representation the FACS results of chimeric protein expression on the cells determined by APC signal as described in Example 3, for the chimeric proteins CD38_001 to CD38_008 (sequences SEQ ID NO: 14-21) in rows 4 to 11 respectively, and for wild type CD47 protein as reference in row 3.

Fig 3(b) shows information about the plasmids and chimeric protein used in the cells tested for chimeric protein expression and about the antibody (Abs) used for the cell surface expression measurement by FACS, and shows, in the column to the right, the median fluorescence signal of the FACS measurement (also reported in Fig.3(a)).

Fig. 4(a) shows in a graphical representation the FACS results of chimer protein expression on the cells as described in Example 3 for the chimeric proteins CD3e_0001, CD3e_0002, CD28_0001 and CD28_0002 (SEQ ID NO: 10-13) in rows 4 to 7 respectively, for wild type CD47 protein as reference in row 3. Fig 4(b) shows information about the plasmids and chimeric protein used in the cells tested in Fig.4(a), about the antibody (Abs) used for the cell surface expression measurement by FACS, and, in the column to the right, the median fluorescence signal of the FACS measurement (also reported in Fig.4(b)).

Fig. 5 shows in a graphical representation the FACS results of chimer protein expression on the cells as described in Example 3 for the chimeric proteins CD80_0001 to CD80_0006 (SEQ ID NO: 22-27) in rows 4 to 9 respectively, for wild type CD47 protein as reference in row 3. Fig 5(b) shows information about the plasmids and chimeric protein used in the cells tested in Fig.5(a), about the antibody (Abs) used for the cell surface expression measurement by FACS, and, in the column to the right, the median fluorescence signal of the FACS measurement (also reported in Fig.5(b)).

Fig. 6(a) shows in a graphical representation the FACS results of transfected cells' ability to bind to SIRP-o as described in Example 3, for cells transfected with the chimeric proteins CD3e_0001, CD3e_0002, CD28_0001 and CD28_0002 (SEQ ID NO: 10-13) in rows 3 to 6 and transfected with wild type CD47 protein as reference in row 2. Fig 6(b) shows information about the plasmids and chimeric protein used in the cells tested in Fig.6(a).

Fig. 7(a) and (b) show the FACS results of transfected cells' ability to bind to SIRP-o as described in Example 3, for cells transfected with the chimeric proteins CD38_001 to CD38_008 (sequences SEQ ID NO: 14-21) in rows 3 to 10 respectively, and for wild type CD47 protein as reference in row 2.

Fig. 8(a) and (b) show the FACS results of transfected cells's ability to bind to SIRP-o as described in Example 3, for cells transfected with the chimeric proteins CD80_0001 to CD80_0006 (SEQ ID NO: 22-27) in rows 3 to 8 respectively, and for wild type CD47 protein as reference in row 2.

Fig. 9 shows the median value of eGFP signal of transfected E1C3 cells ability to trigger the CD47-dependent signalling on THPl-NF-kB-GFP as described in Example 4, for cells transfected with the WT CD47 (SEQ ID 9, row 3), chimeric proteins CD28_0002 (SEQ ID 13, row 4), CD38_0003 (SEQ ID 16, row 5) or CD80_0006 (SEQ ID 27, row 6). hErbB2 was also used as a baseline control for the assay.

DESCRIPTION

The invention of present disclosure allows to reduce rejection of grafted cells by the immune system of host patients. The invention of present disclosure enables self-cell recognition of grafted cells by the immune system of a host patient while reducing or not triggering CD47 downstream signaling.

The invention of present disclosure relies upon novel chimeric proteins. The present disclosure provides a chimeric protein, the chimeric protein comprises an extracellular domain (ECD), a transmembrane domain (TMD) and a linker, such as a linker between said extracellular domain and said transmembrane domain.

The present disclosure provides a cell. In an aspect, the cell may comprise a chimeric protein as defined herein and/or a nucleic acid encoding a chimeric protein as defined herein.

In an aspect, the chimeric proteins of the invention advantageously present optimized performance such as compared to wild type CD47 protein. In an embodiment, the stability of the chimeric proteins of the invention on the cell surface membrane is on par with the stability of wild type CD47. In an embodiment, the stability of the chimeric proteins of the invention on the cell surface membrane is altered, such as compared to wild type CD47. In an embodiment, said stability is improved, such as compared to wild type CD47. In an embodiment, said stability is decreased, such as compared to wild type CD47. In an embodiment, the chimeric proteins of the invention have binding affinity to SIRP-o (Signal regulatory protein a), such as on par with or superior to the binding affinity of wild type CD47 to SIRP-o. In an embodiment, the chimeric proteins of the invention have binding affinity to TSP-1, such as on par with or inferior to the binding affinity of wild type CD47 to TSP-1.

As used herein, the term "chimeric protein", or"chimer protein", refers to a non-naturally occurring protein, typically a recombinant protein, comprising at least 2 parts from different sources.

As used herein, the term "CD47" refers to a protein also called Integrin-associated protein (IAP, CD47) and expressed on all mammalian cells, as further described by Brown, E. J. et al. (Integrin-associated protein: a 50 kDa plasma membrane antigen physically and functionally associated with integrins. J. Cell Biol. 1990, 2785-2794). The chimeric protein comprises an extracellular domain.

In an aspect, the extracellular domain comprises the ectodomain of wild type CD47. The sequence of wild type CD47 ectodomain is sequence SEQ ID NO: 1. In an aspect, said extracellular domain comprises sequence SEQ ID NO: 1.

As used herein, the term "ectodomain" means "extracellular domain" and refer to the domain of a membrane protein protruding from the outer membrane part of a cell or of a cell organelle into the space outside said cell or outside said organelle.

As used herein the term "wild type", or its abbreviation "wt", especially when referring to a gene or a protein, means that the nucleotide or amino acid sequence of said gene or protein is the sequence which prevails among individuals in natural conditions, as distinct from an atypical variant, or analogue, thereof.

In an aspect, the terms "wild type CD47" and "wild type CD47 ectodomain" refer to human wild type CD47 and human wild type CD47 ectodomain respectively.

In another aspect, the extracellular domain comprises an analogue of the ectodomain of human wild type CD47 of SEQ ID NO: 1.

As used herein in the context of a protein, the term "analogue" means a modified amino acid sequence wherein one or more amino acid residues of the sequence have been substituted by other amino acid residue(s) and/or wherein one or more amino acid residues have been deleted and/or wherein one or more amino acid residues have been added and/or inserted compared to the wild type sequence of said protein. In an embodiment, said "analogue" retains all or part of the functionality to the wild type protein.

In a particular embodiment, the ECD of the chimeric protein(s) of the invention, such as an "analogue" of wt CD47 ectodomain, has a binding affinity to SIRP-o that is on par with wild type CD47 ECD's binding affinity to SIRP-o. In a particular embodiment, the ECD of the chimeric protein(s) of the invention, such as an "analogue" of wt CD47 ectodomain, has a binding affinity to SIRP-o that is superior to wild type CD47 ECD's binding affinity to SIRP-o.

In an embodiment, said analogue of human wild type CD47 ectodomain may comprise one or more amino acid substitution, deletion and/or addition compared to SEQ ID NO: 1. In an embodiment, said analogue comprises up to 10 amino acid substitutions, deletions and/or additions, such as up to 9, 8, 7 or 6, especially such as up to 7, amino acid substitutions, deletions and/or additions, compared to SEQ ID NO: 1. In an embodiment, said analogue comprises up to 5 amino acid substitutions, deletions and/or additions, such as up to 4, 3, 2 or 1 amino acid substitution(s), deletion(s) and/or addition(s) compared to SEQ ID NO: 1. In an embodiment, said analogue comprises 1 amino acid substitution(s), deletion(s) and/or addition(s) compared to SEQ ID NO: 1.

In an embodiment, said extracellular domain comprises an analogue of the ectodomain of wild type CD47 of sequence SEQ ID NO: 1, and said analogue comprises in its amino acid sequence one or more mutation selected from the group consisting of S64A, S64T, S64G, D62A, E69A, T61D, S85R, S89A and S89D compared to the sequence SEQ ID NO: 1

In an embodiment, said analogue may comprise at least 80% homology, or at least 85%, 90%, 95% or 98% homology, and less than 100% or less than 99% homology compared to SEQ ID NO: 1. Said analogue may comprise between 80 and 99% homology, or between 85-99%, 85-98%, 90-99%, 90-98% or 80-95%, 85-90% homology compared to SEQ ID NO: 1. In an embodiment, said analogue comprises at least 95% homology compared to SEQ ID NO: 1, such as at least 95%, 96%, 97%, 98% or 99% homology compared to SEQ ID NO: 1.

In an embodiment, the extracellular domain comprises wild type CD47 ectodomain of sequence SEQ ID NO: 1 or an analogue thereof, such as an analogue having at least 90% homology and less than 100% homology with SEQ ID NO: 1.

As used herein, the term "functional ECD", especially when referring to the extracellular domain of a surface receptor, means an ECD that is able to recognize and bind to specific ligands, such as hormones, growth factors, or neurotransmitters, that are present in the extracellular space. This binding may or may not trigger a series of downstream intracellular signalling events that ultimately lead to a cellular response. As used herein, the term "variant", especially when referring to a gene or a protein, means that the nucleotide or amino acid sequence of said gene or protein bears modifications, such as additions, deletions or substitution of one or more parts of the sequence compared to the sequence which prevails among individuals in natural conditions. As used herein, the term "percent homology" refers to the percentage of sequence identity between two sequences, such as nucleotide or amino acid sequences. For illustration, an amino acid sequence A having "90% homology" with an amino acid sequence B means that 90% of the amino acids of sequence A sequentially align with the amino acids of sequence B.

In an embodiment, the extracellular domain does not comprise sequence(s) SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 4. In an embodiment, the extracellular domain does not comprise sequence(s) SEQ ID NO: 3 and/or SEQ ID NO: 4. In an embodiment, the extracellular domain comprises none of sequences SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

In an embodiment, the extracellular domain does not consist of at least one of sequence(s) SEQ ID NO: 3 and SEQ ID NO: 4. In an embodiment, the extracellular domain does not consist of any one of sequences SEQ ID NO: 3 and SEQ ID NO: 4.

In an embodiment, CD47 ectodomain analogue does not comprise sequences SEQ ID NO: 4.

In an embodiment, the chimeric protein does not comprise sequence(s) SEQ ID NO: 2, SEQ ID NO: 3 and/or SEQ ID NO: 4. In an embodiment, the chimeric protein comprises none of sequences SEQ ID NO: 3 and SEQ ID NO: 4. In an embodiment, the chimeric protein comprises none of sequences SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4.

In an embodiment, the extracellular domain has SIRP-alpha (SIRP-o) binding ability. The extracellular domain has been co-crystalized with SIRP-o and showing SIRP-o binding ability (Hatherley D et al., Mol Cell., 2008). In a preferred embodiment, the extracellular domain of the chimeric protein(s) of the invention binds to SIRP-o protein with at least the same affinity as wild type CD47, or with higher affinity than wild type CD47.

Binding affinity between the chimeric protein of the invention and SIPR-alpha can be determined by surface plasmon resonance (SPR) as described in "Polymorphisms in the Human Inhibitory Signal-regulatory Protein a Do Not Affect Binding to Its Ligand CD47", JBC 2014, vol.289, p.10024-28, or by similar binding kinetics measurement methods, such as by FACS as described in Example 3.

In a further embodiment, the novel chimeric proteins of the present disclosure may advantageously allow improved cell surface expression of CD47 extracellular domain. Cell surface expression may be determined by various methods, such as by FACS as described in Example 3. The chimeric protein comprises a transmembrane domain.

As used herein, the term "transmembrane domain" (TMD) refers to a membranespanning region of a protein. A TMD usually comprises mostly nonpolar amino acid residues and may traverse the lipid bilayer of a cell membrane once or multiple times, referring as single-pass TMD and multi-pass TMD respectively.

In an aspect, said transmembrane is not wild type CD47 transmembrane domain.

In an aspect, the transmembrane domain comprises one and no more than one helical structure within the cellular membrane. In an embodiment, it comprises only one helical structure. In an embodiment, it is a single-pass TMD.

In an aspect, the transmembrane domain of said chimeric protein comprises the transmembrane domain of any one of CD28, CD38, CD80 or CD3e. In an aspect, said TMD comprises or consists of one of the sequences SEQ ID NO: 5 to 8 as shown in Table 1.

The sequence of CD3e transmembrane domain is sequence SEQ ID NO: 5. The sequence of CD28 transmembrane domain is sequence SEQ ID NO: 6. The sequence of CD38 transmembrane domain is sequence SEQ ID NO: 7. The sequence of CD80 transmembrane domain is sequence SEQ ID NO: 8.

In another aspect, the transmembrane domain of said chimeric protein comprises an analogue of the transmembrane domain of any one of CD28, CD38, CD80 or CD3e. In an embodiment, the sequence of said TMD analogue has at least 60%, at least 70%, at least 80%, at least 90% or at least 95% and less than 100% homology with any one of sequences SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. In an embodiment, the sequence of said TMD analogue has at least 90% or at least 95% homology with sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. In an embodiment, the sequence of said TMD analogue comprises up to 3 amino acid substitutions, additions or deletions, such as 3, 2 or 1 amino acid substitution(s), addition(s) or deletion(s), such as 1 amino acid substitution, addition or deletion compared to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8.

In an embodiment, the transmembrane domain of said chimeric protein comprises the sequence SEQ ID NO: 5 (CD3e TMD), or a sequence comprising at least 90% or at least 95% homology with SEQ ID NO: 5.

In an embodiment, the transmembrane domain of said chimeric protein comprises a sequence comprising up to 3 amino acid substitutions, additions or deletions compared to SEQ ID NO: 5 (CD3e TMD), such as 3, 2 or 1 amino acid substitution(s), addition(s) or deletion(s), such as 1 amino acid substitution, addition or deletion.

In an embodiment, the transmembrane domain of said chimeric protein comprises the sequence SEQ ID NO: 6 (CD28 TMD), or a sequence comprising at least 90% or at least 95% homology with SEQ ID NO: 6.

In an embodiment, the transmembrane domain of said chimeric protein comprises a sequence comprising up to 3 amino acid substitutions, additions or deletions compared to SEQ ID NO: 6 (CD28 TMD), such as 3, 2 or 1 amino acid substitution(s), addition(s) or deletion(s), such as 1 amino acid substitution, addition or deletion.

In an embodiment, the transmembrane domain of said chimeric protein comprises the sequence SEQ ID NO: 7 (CD38 TMD), or a sequence comprising at least 90% or at least 95% homology with SEQ ID NO: 7.

In an embodiment, the transmembrane domain of said chimeric protein comprises a sequence comprising up to 3 amino acid substitutions, additions or deletions compared to SEQ ID NO: 7 (CD38 TMD), such as 3, 2 or 1 amino acid substitution(s), addition(s) or deletion(s), such as 1 amino acid substitution, addition or deletion.

In an embodiment, the transmembrane domain of said chimeric protein comprises the sequence SEQ ID NO: 8 (CD80 TMD), or a sequence comprising at least 90% or at least 95% homology with SEQ ID NO: 8.

In an embodiment, the transmembrane domain of said chimeric protein comprises a sequence comprising up to 3 amino acid substitutions, additions or deletions compared to SEQ ID NO: 8 (CD80 TMD), such as 3, 2 or 1 amino acid substitution(s), addition(s) or deletion(s), such as 1 amino acid substitution, addition or deletion.

In an aspect, said transmembrane domain of said chimeric protein does not comprise or is not wild type CD64 transmembrane domain. In an aspect, the transmembrane domain does not comprise or does not consist of sequence SEQ ID NO: 85, which is the sequence of wild type CD64 transmembrane domain. In an aspect, the chimeric protein does not comprise or does not consist of sequence SEQ ID NO: 86, which is the sequence of wild type CD47 ECD and wild type CD47 linker fused with wild type CD64 transmembrane domain.

In an aspect, the transmembrane domain of said chimeric protein does not comprise or does not consist of sequence SEQ ID NO: 87, which is the sequence of wild type CD47 transmembrane domain. In an aspect, the transmembrane domain of said chimeric protein is the transmembrane domain of a protein other than CD47 or is derived from the transmembrane domain of a protein other than CD47.

The chimeric protein comprises a linker. The linker is located between the extracellular domain and the transmembrane domain of the chimeric protein. The linker may link directly or indirectly the ECD and the TDM.

In an embodiment, the linker is an amino acid sequence that is bound to the C-terminal end of the ECD and to the N-terminal end of the TMD of the chimeric protein(s) of the invention. In an embodiment, the N-terminal amino acid of the linker is bound via a peptide bond to the C-terminal amino acid of the ECD and the C-terminal amino acid of the linker is bound via a peptide bond to the N-terminal amino acid of the TMD of the chimeric protein(s) of the invention.

The length of the linker may be at least 1 amino acid. In an embodiment, the length of the linker may be comprised between 1 and 40 amino acids. In an embodiment, the length of the linker may be comprised between 1 and 30 amino acids. In an embodiment, the length of the linker may be comprised between 1 and 20 amino acids. In an embodiment, the length of said linker is comprised between 2 and 40 amino acids, or between 2 and 20, 3 and 30, 3 and 20 or 4 and 20 amino acids. In an embodiment, the linker is a non-native linker sequence. As used herein, the term "non-native linker" means a linker which does not occur in nature together with one or more of the other protein parts which it links together.

In an embodiment, the linker comprises sequences SEQ ID NO: 2. In an embodiment, the linker is modified compared to SEQ ID NO: 2. In an embodiment, said modified linker provides an optimized tightening between the TMD and the ECD of the chimeric protein of the invention.

In an embodiment, the linker does not comprise sequence SEQ ID NO: 2. In an embodiment, the linker does not consist of sequence SEQ ID NO: 2. In another embodiment, the linker does not comprise the amino acid sequence VV. In another embodiment, the linker does not consist of the amino acid sequence VV. In another embodiment, the linker does not consist of the amino acid sequence VV and does not consist of sequence SEQ ID NO: 2.

In an aspect, the transmembrane domains of the chimeric proteins of the present disclosure advantageously pass across the cellular lipid bilayer.

In another aspect, the linkers of the chimeric proteins of the present disclosure are advantageously selected to better connect with the transmembrane domains of the present disclosure. In particular embodiments, the different length and sequence of the linkers are designed to be compatible with geometrical constraints in the chimeric protein as a whole.

In another aspect, the four different transmembrane structures derived from CD3e, CD28, CD38 and CD80 advantageously present an optimal energy profile in the presence of CD47 ectodomain. In particular, the combination of the transmembrane domains disclosed herein with linkers of optimal length and sequence as disclosed herein optimally combine with modified CD47 ectodomain.

In another aspect, the chimeric proteins of the invention are advantageously stable.

In an embodiment, the term "stable" as used herein refers to the protein's half-life on the cell membrane. In an embodiment, the chimeric protein of the invention is stable of the cell's surface membrane. This property may be measured as described by Zhang et al. in High resolution measurement of membrane receptor endocytosis in Journal of Biological Methods, 2018, Vol. 5(4), el05.

In an embodiment, the term "stable" refers to the protein's intrinsic biophysical features such as thermostability and/or pH resistance. In an embodiment, the chimeric protein of the invention is thermostable on the cell membrane. This property may be measured as described by Magnani et al. in Nature Protocols 11, 1554-1571 (2016), in A mutagenesis and screening strategy to generate optimally thermostabilized membrane proteins for structural studies.

In an embodiment, the entire chimeric protein has less than 80% homology with entire wild type CD47 protein. The sequence of wild type CD47 entire protein is SEQ ID NO: 9. In a preferred embodiment, the chimeric protein has less than 70%, less than 60% or less than 50% compared to wild type CD47 protein. In a preferred embodiment, the chimeric protein has more than 30% homology compared to wild type CD47. In a preferred embodiment, the entire chimeric protein has between 30 and 70% homology, more preferably between 40 and 60% homology, 40 and 50%, 43 and 47%, or between 45 and 55% homology compared to wild type CD47 protein.

As used herein, expressions like "transmembrane domain of CD3e" and "CD3e transmembrane domain" may be used interchangeably. Similarly, expressions like "the ectodomain of wild type CD47" and "wild type CD47 ectodomain" may be used interchangeably.

In an aspect, the transmembrane domain comprises or is CD3e transmembrane domain of sequence SEQ ID NO: 5 or an analogue thereof and the linker links said CD3e transmembrane domain or analogue thereof to the extracellular domain of the chimeric protein. In an embodiment, the length of said linker is comprised between 3 and 20 amino acids, 4 and 16 amino acids, 4 and 12 amino acids or 6 and 10 amino acids or is of 8 amino acids. In further embodiments, the length of said linker is comprised between 2 and 20 amino acids, 2 and 16 amino acids, 2 and 12 amino acids, 2 and 10 amino acids, 3 and 20 amino acids, 3 and 16 amino acids, 3 and 12 amino acids, 3 and 10 amino acids, 3 and 8 amino acids, 4 and 20 amino acids, 4 and 16 amino acids, 4 and 12 amino acids, 4 and 10 amino acids, 4 and 8 amino acids, 5 and 20 amino acids, 5 and 16 amino acids, 5 and 12 amino acids, 5 and 10 amino acids, 5 and 8 amino acids, 6 and 20 amino acids, 6 and 16 amino acids, 6 and 12 amino acids, 6 and 10 amino acids, 6 and 8 amino acids, 2 and 8, 3 and 8, amino acids, or is of 8 amino acids. In a further embodiment, said linker comprises a sequence GX1GVSX2MD (SEQ ID NO: 89), wherein Xi can be S or P and X2 can be G or C. In a further embodiment, said extracellular domain of the chimeric protein comprises an analogue of wild type CD47 ectodomain, said analogue comprising Y113V and R114G compared to SEQ ID NO: 1. In a further embodiment, said linker comprises or is the amino acid sequence GSGVSGMD (SEQ ID NO: 28) or GPGVSCMD (SEQ ID NO: 29). In a particular embodiment, the transmembrane domain of the chimeric protein comprises or is CD3e transmembrane domain of sequence SEQ ID NO: 5, the extracellular domain of the chimeric protein comprises an analogue of wild type CD47 ectodomain, said analogue comprising Y113V and R114G compared to SEQ ID NO: 1 and linker connecting said CD3e transmembrane domain to said extracellular domain comprises or is any one of the amino acid sequences GSGVSGMD (SEQ ID NO: 28) and GPGVSCMD (SEQ ID NO: 29). In an aspect, the transmembrane domain comprises or is CD28 transmembrane domain of sequence SEQ ID NO: 6 or an analogue thereof and the linker links said CD28 transmembrane domain or analogue thereof to the extracellular domain of the chimeric protein. In an embodiment, said extracellular domain of the chimeric protein comprises an analogue of wild type CD47 ectodomain, said analogue comprising one or both of F14C and C15G compared to SEQ ID NO: 1. In an embodiment, the length of said linker is comprised between 2 and 20 amino acids, 2 and 16 amino acids, 2 and 10 amino acids, 2 and 6 amino acids, 3 and 20 amino acids, 3 and 16 amino acids, 3 and 10 amino acids or 3 and 6 amino acids or is of 3 amino acids. In further embodiments, the length of said linker is comprised between 2 and 20 amino acids, 2 and 16 amino acids, 2 and 12 amino acids, 2 and 10 amino acids, 2 and 8 amino acids, 2 and 6 amino acids, 2 and 4 amino acids, 3 and 20 amino acids, 3 and 16 amino acids, 3 and 12 amino acids, 3 and 10 amino acids, 3 and 8 amino acids, 3 and 6 amino acids, 3 and 4 amino acids, 2 and 3 amino acids, or is of 3 amino acids. In a further embodiment, said linker comprises sequence NCX3, wherein X3 can be P or A. In a further embodiment, said linker comprises or is the amino acid sequence NCP or NCA. In a particular embodiment, the transmembrane domain of the chimeric protein comprises or is CD28 transmembrane domain of sequence SEQ ID NO: 6, the extracellular domain of the chimeric protein comprises an analogue of wild type CD47 ectodomain sequence SEQ ID NO: 1, said analogue comprising one or both of F14C and C15G compared to SEQ ID NO: 1 and the linker linking said CD28 transmembrane domain to said extracellular domain comprises or is any one of the amino acid sequences NCP or NCA.

In an aspect, the transmembrane domain comprises or is CD38 transmembrane domain of sequence SEQ ID NO: 7 or an analogue thereof and the linker links said CD38 transmembrane domain of CD38 or analogue thereof to said extracellular domain. In an embodiment, said extracellular domain of the chimeric protein comprises an analogue of wild type CD47 ectodomain, said analogue comprising one or more of F14C, F14A, F14G and C15G compared to SEQ ID NO: 1. In an embodiment, the length of said linker is comprised between 2 and 20 amino acids, 2 and 16 amino acids, 2 and 10 amino acids, 2 and 6 amino acids, 3 and 20 amino acids, 3 and 16 amino acids, 3 and 10 amino acids or 4 and 6 amino acids or is of 4 or 6 amino acids. In an embodiment, said linker comprises a sequence X4X5X6X7X8X9, wherein X4 is E, Q or G, wherein X5 is E, D or S, Xe is absent or is G, X7 is absent or is A or S, Xs is C, G, S, P or D and X9 is P, G or C. In a further embodiment, said linker comprises a sequence EEXeX7XsP, wherein Xe is absent or is G, X7 is absent or is A and Xs is C, G or S. In a further embodiment, said linker comprises a sequence QDX6X7PX9, wherein Xe is absent or is G, X7 is absent or is S and X9 is C or G. In a further embodiment, said linker comprises a sequence GSDX9, wherein X9 is C or G. In a further embodiment, said linker comprises or is any one of the amino acid sequences EECP (SEQ ID NO: 90), EEGP (SEQ ID NO: 91), EEGASP (SEQ ID NO: 30), QDPC (SEQ ID NO: 92), QDPG (SEQ ID NO: 93), QDGSPG (SEQ ID NO: 31), GSDC (SEQ ID NO: 94) and GSDG (SEQ ID NO: 95). In a further embodiment, the transmembrane domain of the chimeric protein comprises or is CD38 transmembrane domain of sequence SEQ ID NO: 7 and the extracellular domain of the chimeric protein comprises an analogue of wild type CD47 ectodomain sequence SEQ ID NO: 1, said analogue comprising one or more of F14C, F14A, F14F and/or C15G compared to SEQ ID NO: 1 and the linker linking said CD38 transmembrane domain to said extracellular domain comprises or is any one of the amino acid sequences EECP (SEQ ID NO: 90), EEGP (SEQ ID NO: 91), EEGASP (SEQ ID NO: 30), QDPC (SEQ ID NO: 92), QDPG (SEQ ID NO: 93), QDGSPG (SEQ ID NO: 31), GSDC (SEQ ID NO: 94) and GSDG (SEQ ID NO: 95).

In an aspect, the transmembrane domain comprises or is CD80 transmembrane domain of sequence SEQ ID NO: 8 or an analogue thereof and the linker links said CD80 transmembrane domain of CD80 or analogue thereof to said extracellular domain. In an embodiment, said extracellular domain of the chimeric protein comprises an analogue of wild type CD47 ectodomain, said analogue comprising C15G compared to SEQ ID NO: 1. In an embodiment, the length of said linker is comprised between 2 and 20 amino acids, 2 and 16 amino acids, 2 and 12 amino acids, 3 and 20 amino acids, 3 and 16 amino acids, 4 and 12 amino acids or 6 and 10 amino acids or is of 6, 8 or 10 amino acids. In an embodiment, said linker comprises sequence SDX10X11X12X13X14X15X16R, wherein X10 can be E or K, Xu can be E or T, X12 can be P or T, X13 can be absent or be P or D, X14 can be absent or A, X15 can be absent or T, Xi6 can be absent or C or G. In a further embodiment, said linker comprises a sequence (SEQ ID NO: 32) SDEEPX13X16R, wherein X13 is absent or is P and X16 is absent or is C or G. In a further embodiment, said linker comprises a sequence (SEQ ID NO: 33) SDKTTDX14X15X16R, wherein Xi4 is absent or is A, X15 is absent or is T and X16 is C or G. In a further embodiment, said linker comprises or is any one of the amino acid sequences SDEEPPCR (SEQ ID NO: 34), SDEEPPGR (SEQ ID NO: 35), SDEEPR (SEQ ID NO: 36), SDKTTDCR (SEQ ID NO: 37), SDKTTDGR (SEQ ID NO: 38) and SDKTTDATGR (SEQ ID NO: 39). In a further embodiment, the transmembrane domain of the chimeric protein comprises or is CD80 transmembrane domain of sequence SEQ ID NO: 8 and the extracellular domain of the chimeric protein comprises an analogue of wild type CD47 ectodomain, said analogue comprising C15G compared to sequence SEQ ID NO: 1 and the linker linking said CD80 transmembrane domain to said extracellular domain comprises or is any one of the amino acid sequences SDEEPPCR (SEQ ID NO: 34), SDEEPPGR (SEQ ID NO: 35), SDEEPR (SEQ ID NO: 36), SDKTTDCR (SEQ ID NO: 37), SDKTTDGR (SEQ ID NO: 38) and SDKTTDATGR (SEQ ID NO: 39).

In an aspect, the linker comprises or is any one of the following sequences: - GSGVSGMD (SEQ ID NO: 28) or GPGVSCMD (SEQ ID NO: 29) provided that the transmembrane domain comprises CD3e transmembrane domain and the extracellular domain comprises an analogue of CD47 wild type ectodomain, said analogue comprising Y113V and R114G compared to SEQ ID NO: 1,

NCP or NCA provided that the transmembrane domain comprises CD28 transmembrane domain and the extracellular domain comprises an analogue of CD47 wild type ectodomain, said analogue comprising one or both of F14C and C15G compared to SEQ ID NO: 1,

- EECP (SEQ ID NO: 90), EEGP (SEQ ID NO: 91), EEGASP (SEQ ID NO: 30), QDPC (SEQ ID NO: 92), QDPG (SEQ ID NO: 93), QDGSPG (SEQ ID NO: 31), GSDC (SEQ ID NO: 94) or GSDG (SEQ ID NO: 95) provided that the transmembrane domain comprises CD38 transmembrane domain and the extracellular domain comprises an analogue of CD47 wild type ectodomain, said analogue comprising one or more of F14C, F14A, F14G and C15G compared to SEQ ID NO: 1, and

- SDEEPPCR (SEQ ID NO: 34), SDEEPPGR (SEQ ID NO: 35), SDEEPR (SEQ ID NO: 36), SDKTTDCR (SEQ ID NO: 37), SDKTTDGR (SEQ ID NO: 38), SDKTTDATGR (SEQ ID NO: 39) provided that said transmembrane domain comprises CD80 transmembrane domain and the extracellular domain comprises an analogue of CD47 wild type ectodomain, said analogue comprising C15G compared to SEQ ID NO: 1.

In an aspect, the chimeric protein comprises an intracellular domain such as a functional ICD, such as an ICD which has ability to trigger CD47 intracellular pathway, such as

CD47 wild type ICD.

In an embodiment, the chimeric protein comprises an intracellular domain with an ability to trigger CD47 intracellular pathway that is reduced compared to CD47 wild type ICD.

In an aspect, the chimeric protein comprises no intracellular domain (ICD), thereby has no ability to trigger CD47 intracellular pathway.

In another aspect, the chimeric protein comprises an intracellular domain which has no ability to trigger CD47 intracellular pathway. In an embodiment, presence or absence of ability to trigger CD47 intracellular pathway may be determined by reported gene assay. Gene reported gene assay may be performed as described by Battin C et al. in PLos One 24 May 2017, A human monocytic NF-KB fluorescent reporter cell line for detection of microbial contaminants in biological samples or adapted therefrom to measure the activation of CD47 in connection with NfkB signalling. Presence or absence of ability to trigger CD47 intracellular pathway is determined relative to the control as the reporter assay usually have a background signal.

The present disclosure provides a cell.

In an aspect, the cell comprises a chimeric protein as defined herein. In a preferred embodiment, the chimeric protein is on the cell surface, preferably the transmembrane domain of the chimeric protein is anchored in the cellular membrane, the extracellular domain of the chimeric protein is in the extracellular space, and the intracellular domain, if there is one, is located in the cellular cytoplasm. In a preferred embodiment, the chimeric protein is able to bind with SIRP-a.

In another aspect, the cell may comprise a nucleic acid encoding a chimeric protein as defined herein. The chimeric protein can be expressed from said nucleic acid. The term "expressed" means "transcribed and/or translated" into the encoded protein of interest, depending on the nature of the nucleic acid. For example, a nucleic acid in the form of DNA can be transcribed and translated, and a nucleic acid in the form of mRNA can be translated into the protein of interest. The nucleic acid may be inserted into the cell by any mean and in any form. For example, the nucleic acid may be a plasmid transfected into the cellular cytoplasm, or it may be a transgene inserted into the cellular genome for example by means of a gene editing technique for site directed transgenesis, i.e. nucleic acid insertion in a specific locus of the cellular genome. In a preferred embodiment, the nucleic acid is a transgene inserted into the cellular genome.

In an embodiment, the cell is a mammalian cell, preferably a human cell.

The cell may be a various development stage. In an embodiment, the cell is a stem cell, such as an embryonic stem cell, a pluripotent stem cell or an induced pluripotent stem cell (iPSC). In another embodiment, the cell is derived from a stem cell and may be at any differentiation stage therefrom. In another embodiment, the cell is a mature cell.

In an embodiment, the cell is derived from a pluripotent stem cell, such as any cell type derived from a pluripotent stem cell. In an embodiment, the cell is a differentiated cell and is obtained by culturing a pluripotent cell under differentiation conditions, such as in presence of differentiation factors. In another embodiment, the cell is a non-natural cell, such as a differentiated cell obtained by genome engineering of a pluripotent cell.

The cell may be selected from:

- a beta cell, an INS+ and NKX6.1+ double positive cell or a C-peptide+/NKX6.1+ double positive cell, an insulin producing cell, an in vitro derived beta-like cell, a pancreatic endocrine cell or an endocrine cell, an endocrine progenitor cell or a NGN3+/NKX2.2+ double positive cell,

- a neural cell such as a neuron, an interneuron cell, an oligodendrocyte, an astrocyte or a dopaminergic cell,

- an exosome cell,

- an immune cell such as a T cell, a NK cell, a macrophage or a dendritic cell,

- a hepatocyte, a stellate cell, a fibroblast, a keratinocyte, a hair cell, an inner ear cell, an intestinal cell, an organoid cell, a nephroid cell or another kidney-related cell, a card io myocyte, a retinal cell, a retinal pigment epithelium cell

- a cortical neural progenitor cell,

- a mesenchymal stem cell.

Stem cell:

As used herein, the term "stem cell" is to be understood as an undifferentiated cell having differentiation potency and proliferative capacity, particularly self-renewal competence, but maintaining differentiation potency. The term "stem cell" includes subpopulations such as pluripotent stem cell (PSC), multipotent stem cell, unipotent stem cell and the like according to the differentiation potency.

Pluripotent stem cell, also known as pluripotent cell, or pluripotent SC, or PSC:

As used herein, these terms refer to a stem cell capable of being cultured in vitro and having a potency to differentiate into any cell lineage belonging to three germ layers (ectoderm, mesoderm, endoderm). A PSC can be induced from fertilized egg, clone embryo, germ stem cell, stem cell in a tissue, somatic cell and the like. Examples of the PSC include embryonic stem cell (ESC), induced pluripotent stem cell (iPSC), embryonic germ cell (EG cell) and the like. Muse cell (Multi-lineage differentiating stress enduring cell) obtained from mesenchymal stem cell (MSC), and germline stem cell (GS cell) produced from reproductive cell (e.g., testis) are also encompassed in the PSC term. The pluripotent stem cells used in the present invention can thus be embryonic stem cells prepared from blastocysts, as described in e.g. WO 03/055992 and WO 2007/042225, or be commercially available cells or cell lines. ES cell lines can also be derived from single blastomeres without the destruction of ex utero embryos and without affecting the clinical outcome (Chung et al. (2006) and Klimanskaya et al. (2006)). Embryonic stem cells may also be derived from parthenotes as described in e.g. WO 2003/046141. Additionally, embryonic stem cells can be produced from a single blastomere or by culturing an inner cell mass obtained without the destruction of the embryo. Embryonic stem cells are available from given organizations and are also commercially available.

Induced pluripotent stem cell, iPS, iPSCs:

As used herein, the term "induced pluripotent stem cell" (also known as iPS cells or iPSCs) means a type of PSC that can be generated directly from adult cells by a process commonly known as reprogramming. By the introduction of products of specific sets of pluripotency-associated genes adult cells can be converted into PSCs

Preferably, the methods and products of the present invention are based on hPSCs, i.e. stem cells derived from either iPSCs or embryonic stem cells, including parthenotes.

Differentiated cells:

As used herein, the term "differentiated cell" means a cell which does not have the potency to differentiate into any cell lineage. Differentiated cells may be obtained from stem cells or the like upon exposure to appropriate differentiation culture conditions. Differentiated cells may be obtained from donors.

Endocrine progenitor cell:

As used herein, the term "endocrine progenitor cell" refers to a cell characterised by expression of markers NGN3, NeuroD and NKX2.2. As used herein, the term "NGN3+/NKX2.2+ double positive cell" refers to a cell that co-express the two markers NGN3 and NKX2.2. As used herein, the term "NeuroD" refers to a member of the NeuroD family of basic helix-loop-helix (bHLH) transcription factors, the term "NGN3" refers to a member of the neurogenin family of basic loop- helix-loop transcription factors, and the terms "NKX2.2" and "NKX6.1" refer to members of the NKX transcription factor family.

An "INS+" cell as used herein is a cell that produces insulin.

The terms "differentiation" or "cell differentiation", "differentiating", as used herein refer to cellular differentiation. Cellular differentiation is the process in which a cell changes from one cell type to another, typically from a less specialized type, such as a stem cell, to a more specialized type, such as a tissue specific cell, e.g. a cardiomyocyte. The terms "differentiated" and "undifferentiated" refer to the stage of differentiation of a cell in the cellular differentiation process.

Mammalian cell: The term "mammalian cell" as used herein means a cell originating from a mammalian living organism, such as a mammalian animal cell or a human cell. The mammalian cell may be at an undifferentiated stage, for example at a pluripotent or multipotent stage, or at a differentiated stage, such as a fully mature stage, or at an intermediate stage of differentiation. The mammalian cell, whether differentiated or undifferentiated, may come from a donor, such as tissue stem cells, or may be derived from a cell coming from a donor. Terms as used herein to designate genes or proteins are meant to designate said human genes or proteins in the context of a human cell, and to designate the corresponding genes or proteins in the context of a non-human mammalian cell, especially in case of genes or proteins that might be named differently in a given mammalian species compared to the corresponding gene or protein in a human cell.

In a preferred embodiment, the cell is a stealth cell. The term "stealth" means that the cell is not subject to rejection by the immune system of a host upon allogeneic transplantation or is at least less subject to such rejection than the same cell without stealth modification. The stealth cell may comprise genetic modifications to improve its stealth property. The stealth properties may comprise changes in the HLA-class I and II expression.

In an embodiment, the stealth cell is B2M-/-. In an embodiment, the stealth cell is CIITA- /-. In an embodiment, the stealth cell comprises a B2M-HLA genetic fusion protein on its cell surface or a nucleic acid sequence encoding a B2M-HLA genetic fusion protein. In a preferred embodiment, the stealth cell is B2M-/-, CIITA-/- and may further comprise a B2M-HLA genetic fusion protein on its cell surface or a nucleic acid sequence encoding a B2M-HLA genetic fusion protein, such as B2M-HLA-E and/or B2M-HLA-G genetic fusion protein.

As used herein, the term "B2M -/-” means a cell in which the endogenous B2M gene is disrupted or otherwise inactivated, and the term "B2M" stands for [32 microglobulin. In a B2M deficient cell, endogenous HLA class I proteins cannot translocate to the cell surface. As used herein, the term "CIITA-/-" means a cell in which the endogenous CIITA gene is disrupted or otherwise inactivated, and the term CIITA stands for "class II, major histocompatibility complex, transactivator".

As used herein, "B2M-HLA fusion protein", also called "B2M-HLA genetic fusion protein", mean a protein comprising a B2M part and a HLA part fused into one same protein. Further definitions and embodiments, including sequences, for "B2M-HLA", "B2M-HLA-E" and "B2M-HLA-G" can be found in WO/2022/129472 and WO/2022/136215 and are incorporated herein by reference. As used herein, the term "HLA" stands for human Leucocyte Antigen and refers to the well-known HLA system responsible for the regulation of the immune system in mammalians. Class I HLA proteins comprise HLA-A, HLA-B and HLA-C proteins, which are highly polymorphic, and HLA-E, HLA-F and HLA-G proteins, which are less polymorphic. Class II HLAs proteins comprise HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR. All these specific HLA proteins are well defined in the art.

The stealth cells may additionally be engineered to have reduced or increased expression of additional immune evasive proteins on the cell surface. These could be, but is not limited to, PDL1, complement factors like, CD55, CD46, CD59, CD35, and/or other immune evasive ligands known in the art. PDL1 and CD55 are defined in the art. PDL1 wild type sequence is referenced as NP_001134823.1 in NCBI, CD55 wild type sequence is referenced as P08174-1 in Uniprot.

In a further aspect of the present invention, the extracellular domain of the chimeric protein may further comprise mutations which modulate the expression of said chimeric protein in the cell, reduce interaction of said chimeric protein with thrombospondin-1 (TSP-1) and/or limit the risk of TSP-1 related adverse effects. In one embodiment, the chimeric protein has reduced or inhibited binding ability to TSP-1 compared to wild type CD47 and provide grafted cells a better chance of surviving ischemic environments for example in relation to cell transplantation and/or in poorly vascularised tissue or encapsulation devices.

It has been surprisingly found that it is possible to modify the ectodomain of wild type CD47 such as to reduce its interaction with TSP-1 while maintaining its interaction with SIRP-o intact and/or to increase its interaction with SIRP-o. It has been shown that TSP- 1 can displace the SIRP-o binding to CD47 wild type ectodomain. Displacement of SIRP-o would be detrimental to the inhibition of phagocytosis which in turn might lead to the loss of transplanted cells. By reducing TSP-1 binding to CD47 ectodomain, the proposed modifications advantageously reduce the risk of TSP-1 related adverse effects in the context of cellular transplantation while preventing phagocytosis of transplanted cells by immune cells, said phagocytosis being inhibited by engagement of the ectodomain of the chimeric protein with SIRP-o from macrophages and other immune cells.

In one aspect, the invention provides a chimeric protein comprising an extracellular domain and a transmembrane domain, wherein said extracellular domain is an analogue of CD47 ectodomain defined by the amino acid sequence SEQ ID NO: 1, and wherein said extracellular domain comprises, in its amino acid sequence, one or more mutation selected from S64A, S64T, S64G, D62A, E69A, T61D, S85R, S89A and S89D compared to the sequence SEQ ID NO: 1.

In an embodiment, the ECD of the chimeric protein of the invention has at least 90% homology with SEQ ID NO: 1. In a particular embodiment, the ECD of the chimeric protein of the invention has between 90% and 99% homology with SEQ ID NO: 1.

In an embodiment, the ECD of the chimeric protein of the invention comprises a mutation selected from S64A, S64T or S64G compared to the sequence SEQ ID NO: 1. In a particular embodiment, the ECD of the chimeric protein of the invention comprises a mutation selected from S64T or S64G compared to the sequence SEQ ID NO: 1.

In an embodiment, the ECD of the chimeric protein of the invention comprises one of or both mutations D62A and E69A compared to the sequence SEQ ID NO: 1.

In an embodiment, the ECD of the chimeric protein of the invention comprises one or more of the mutations T61D, S85R, S89A and S89D compared to sequence SEQ ID NO: 1.

In one aspect, the invention provides a chimeric protein comprising an extracellular domain and a transmembrane domain, wherein said extracellular domain comprises one mutation selected from S64A, S64T and S64G, and further comprises at least mutation selected from D62A, E69A, T61D, S85R, S89A, S89D compared to SEQ ID NO: 1.

In a further aspect of the present invention, the extracellular domain of the chimeric protein may further comprise mutations which optimize the performance of the chimeric protein, such as its stability on the cell surface and/or its interaction with SIRP-o. In an embodiment, the ECD of the chimeric protein of the invention comprises one or more mutations selected from Y113V, R114G, F14C, F14A, F14G and/or C15G compared to SEQ ID NO: 1.

The present disclosure provides a cell composition. In an aspect, the composition comprises cells as defined herein in a pharmaceutically acceptable medium. As used herein, the term "pharmaceutically acceptable medium" refers to a medium suitable for maintaining cells alive, for example during storage and/or transportation, or a medium suitable for medical use of the cells, such as safe administration to a patient. Non limiting examples include PBS (Phosphate Buffer Saline) or cell culture media.

The present disclosure provides a pharmaceutical product. In an aspect, the pharmaceutical product comprises cells as defined herein, or a cell composition as defined herein. The present disclosure provides medical uses of the cells, cell composition and/or pharmaceutical product as defined herein. In an aspect, the cells, cell composition and/or pharmaceutical product as defined herein are for use in cellular therapy of a patient in need thereof. In an embodiment, the use in cellular therapy is for the treatment, the cure, or the prevention of a chronic disease or of an acute disease. In an embodiment, the chronic disease is selected from diabetes, type 1 diabetes, type 2 diabetes, dry macular degeneration, retinitis pigmentosa, neurological disease, Parkinson's disease, heart disease, tissue fibrosis, cirrhosis, hearing loss, corneal blindness, stroke, chronic heart failure, chronic kidney disease or cancer. In an embodiment, the acute disease is selected from bacterial lung infections, such as ventilator acquired bacterial pneumonia or hospital acquired bacterial pneumonia.

The present disclosure provides a process for the preparation of a cell as defined herein. In an aspect, the process comprises the step of inserting into a cell a nucleic acid sequence encoding a chimeric protein as defined herein.

For insertion of a nucleic acid sequence into the cell, any known transfection method may be used, for stable or transient expression. For insertion of a nucleic acid sequence into the genome of a cell, especially for stable expression thereof, any known gene editing tool and/or method may be used, for example a nuclease based gene editing tool such as CRISPR or TALEN.

Table 1 - Amino acid sequences

In an aspect, the transmembrane domains of the chimeric proteins of the present disclosure advantageously pass across the cellular lipid bilayer.

In another aspect, the linkers of the chimeric proteins of the present disclosure are advantageously selected to better connect with the transmembrane domains of the present disclosure. In particular embodiments, the different length and sequence of the linkers are designed to be compatible with geometrical constraints in the chimeric protein as a whole.

In another aspect, the four different transmembrane structures derived from CD3e, CD28, CD38, and CD80 advantageously present an optimal energy profile in the presence of CD47 ectodomain. In particular, the combination of the transmembrane domains disclosed herein with linkers of optimal length and sequence as disclosed herein optimally combine with modified CD47 ectodomain.

In another aspect, the chimeric proteins of the present invention are advantageously stable.

Non-limiting embodiments according to invention disclosed herein

1. A cell comprising a chimeric protein on its cell surface and/or comprising a nucleic acid encoding said chimeric protein, said chimeric protein comprising an extracellular domain, a transmembrane domain and a linker, such as a linker between said extracellular domain and said transmembrane domain, wherein

- said extracellular domain comprises wild type CD47 ectodomain of sequence SEQ ID NO: 1 or an analogue thereof.

2. A cell according to embodiment 1, wherein said transmembrane domain does not consist of sequence SEQ ID NO: 85 (CD64 TMD).

3. A cell according to embodiment 1, wherein said transmembrane domain does not comprise sequence SEQ ID NO: 85 (CD64 TMD).

4. A cell according to any of preceding embodiments, wherein said chimeric protein does not consist of sequence SEQ ID NO: 86 (CD47 wt ECD-linker + CD64 TMD). A cell according to any of preceding embodiments, wherein said chimeric protein does not comprise sequence SEQ ID NO: 86 (CD47 wt ECD-linker + CD64 TMD). A cell according to any of preceding embodiments, wherein the sequences of said extracellular domain and said linker do not together consist of the amino acid sequence SEQ ID NO: 3 (wt CD47 ECD+wt CD47 linker). A cell according to any of preceding embodiments, wherein the sequences of said extracellular domain and said linker do not together consist of the amino acid sequence SEQ ID NO: 4 (wt CD47 ECD+ part of wt CD47 linker). A cell according to any of preceding embodiments, wherein the sequence of said transmembrane domain does not comprise sequence SEQ ID NO: 87 (wt CD47 TMD). A cell according to any of preceding embodiments, wherein the sequence of said transmembrane domain does not consist of sequence SEQ ID NO: 87 (wt CD47 TMD).. A cell according to any of preceding embodiments, wherein said transmembrane domain is the transmembrane domain of a protein other than CD47. . A cell according to any of preceding embodiments, wherein said transmembrane domain is the transmembrane domain of a protein other than CD64. . A cell according to any of preceding embodiments, wherein said protein does not comprise the amino acid sequence SEQ ID NO: 3 and/or does not comprise the amino acid sequence SEQ ID NO: 4. . A cell according to any of preceding embodiments, wherein said protein does not comprise the amino acid sequence SEQ ID NO: 3. . A cell according to any of preceding embodiments, wherein said protein does not comprise the amino acid sequence SEQ ID NO: 4. . A cell according to any of preceding embodiments, wherein said linker does not consist of wild type CD47 linker sequence SEQ ID NO: 2. . A cell according to any of preceding embodiments, wherein said linker does not consist of the amino acid sequence VV. . A cell according to any of preceding embodiments, wherein said protein does not comprise the amino acid sequence SEQ ID NO: 2. . A cell according to any of preceding embodiments, wherein the chimeric protein sequence has less than 80% homology with wild type CD47 protein sequence SEQ ID NO: 9. . A cell according to any of preceding embodiments, wherein the chimeric protein sequence has less than 70%, less than 60% or less than 50% homology compared to wild type CD47 protein sequence. . A cell according to any of preceding embodiments, wherein the chimeric protein sequence has more than 30% homology compared to wild type CD47 protein sequence. . A cell according to any of preceding embodiments, wherein the chimeric protein has between 30 and 70% homology, more preferably between 40 and 60%, 40 and 50%, 43 and 47%, or between 45 and 55% homology compared to wild type CD47 protein sequence. . A cell according to any of preceding embodiments, wherein said linker is a nonnative linker sequence. . A cell according to any of preceding embodiments, wherein the length of said linker is at least 1 amino acid, for example between 1 and 40 amino acids. . A cell according to any of preceding embodiments, wherein the length of said linker is comprised between 1 and 30 amino acids, 1 and 20 amino acids, between 2 and 40 amino acids, or between 2 and 20, 3 and 30, 3 and 20 or 4 and 20 amino acids. . A cell according to any of preceding embodiments, wherein said linker does not consist of the amino acid sequence VV. . A cell according to any of preceding embodiments, wherein said extracellular domain has SIRP-o binding ability. . A cell according to any of preceding embodiments, wherein said transmembrane domain comprises the transmembrane domain of any one of CD28, CD38, CD80 or CD3e, or an analogue thereof. . A cell according to any of preceding embodiments, wherein said transmembrane domain comprises an analogue (TMD analogue) of the transmembrane domain of any one of CD28, CD38, CD80 or CD3e. . A cell according to embodiment 28, wherein the sequence of said analogue (TMD analogue) has at least 60%, at least 70%, at least 80%, at least 90% or at least 95% and less than 100% homology with any one of sequences SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8. . A cell according to embodiment 29, wherein the sequence of said analogue (TMD analogue) has at least 90% or at least 95% homology with sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. . A cell according to any of preceding embodiments, wherein the sequence of said analogue (TMD analogue) comprises up to 3 amino acid substitutions, additions or deletions, such as 3, 2 or 1 amino acid substitution(s), addition(s) or deletion(s). . A cell according to any of preceding embodiments, wherein said transmembrane domain comprises the transmembrane domain of CD3e of sequence SEQ ID NO: 5 or an analogue thereof, such as a sequence having at least 90% homology or at least 95% and less than 100% homology with SEQ ID NO: 5. . A cell according to any of preceding embodiments, wherein the transmembrane domain of said chimeric protein comprises a sequence comprising up to 3 amino acid substitutions, additions or deletions compared to SEQ ID NO: 5 (CD3e TMD), such as 3, 2 or 1 amino acid substitution(s), addition(s) or deletion(s), such as 1 amino acid substitution, addition or deletion. . A cell according to embodiment 32, wherein said transmembrane domain comprises the transmembrane domain of CD3e of sequence SEQ ID NO: 5 and said linker links said transmembrane domain of CD3e to said extracellular domain. . A cell according to any of embodiments 32 to 34, wherein the length of said linker is between 3 and 20 amino acids, 4 and 16 amino acids, 4 and 12 amino acids or 6 and 10 amino acids or is of 8 amino acids. . A cell according to any of embodiments 32 to 35, wherein said transmembrane domain comprises the transmembrane domain of CD3e and said linker comprises a sequence GX1GVSX2MD (SEQ ID NO: 89), wherein XI can be S or P and X2 can be G or C. . A cell according to any of embodiments 32 to 36, wherein said extracellular domain comprises an analogue of the ectodomain of wild type CD47, said analogue comprising Y113V and R114G compared to SEQ ID NO: 1. . A cell according to any of embodiments 32 to 38, wherein said linker comprises or consists of the amino acid sequence GSGVSGMD (SEQ ID NO: 28) or GPGVSCMD (SEQ ID NO: 29). . A cell according to any of embodiments 1 to 31, wherein said transmembrane domain comprises the transmembrane domain of CD28 of sequence SEQ ID NO: 6 or an analogue thereof, such as a sequence having at least 90% or at least 95% homology with SEQ ID NO: 6. . A cell according to embodiment 39, wherein said transmembrane domain comprises a sequence comprising up to 3 amino acid substitutions, additions or deletions compared to SEQ ID NO: 6 (CD28 TMD), such as 3, 2 or 1 amino acid substitution(s), addition(s) or deletion(s), such as 1 amino acid substitution, addition or deletion. . A cell according to any of embodiment 39 or 40, wherein said transmembrane domain comprises the transmembrane domain of CD28 of sequence SEQ ID NO: 6 and said linker links said transmembrane domain of CD28 to said extracellular domain.. A cell according to any of embodiments 39 to 41, wherein the length of said linker is between 2 and 20 amino acids, 2 and 16 amino acids, 2 and 10 amino acids, 2 and 6 amino acids, 3 and 20 amino acids, 3 and 16 amino acids, 3 and 10 amino acids or 3 and 6 amino acids or is of 3 amino acids. . A cell according to any of embodiments 39 to 42, wherein said transmembrane domain comprises the transmembrane domain of CD28 and said linker comprises sequence NCX3, wherein X3 can be P or A. . A cell according to any of embodiments 39 to 43, wherein said linker comprises or is the amino acid sequence NCP or NCA. . A cell according to any of embodiments 39 to 44, wherein said extracellular domain comprises an analogue of the ectodomain of wild type CD47, said analogue comprising one or both of F14C and C15G compared to SEQ ID NO: 1. . A cell according to any of embodiments 1 to 31, wherein said transmembrane domain comprises the transmembrane domain of CD38 of sequence SEQ ID NO: 7 or an analogue thereof, such as a sequence having at least 90% or at least 95% homology with SEQ ID NO: 7. . A cell according to embodiment 46, wherein said transmembrane domain comprises a sequence comprising up to 3 amino acid substitutions, additions or deletions compared to SEQ ID NO: 7 (CD38 TMD), such as 3, 2 or 1 amino acid substitution(s), addition(s) or deletion(s), such as 1 amino acid substitution, addition or deletion. . A cell according to any of embodiments 46 to 47, wherein said transmembrane domain comprises the transmembrane domain of CD38 of sequence SEQ ID NO: 7 and a linker, wherein said linker links said transmembrane domain of CD38 to said extracellular domain. . A cell according to any of embodiments 46 to 48, wherein the length of said linker is between 2 and 20 amino acids, 2 and 16 amino acids, 2 and 10 amino acids, 2 and 6 amino acids, 3 and 20 amino acids, 3 and 16 amino acids, 3 and 10 amino acids or 4 and 6 amino acids or is of 4 or 6 amino acids. . A cell according to any of embodiments 46 to 49, wherein said transmembrane domain comprises the transmembrane domain of CD38 and said linker comprises a sequence X4X5X6X7X8X9, wherein X4 is E, Q or G, wherein X5 is E, D or S, X6 is absent or is G, X7 is absent or is A or S, X8 is C, G, S, P or D and X9 is P, G or C. . A cell according to any of embodiments 46 to 50, wherein said transmembrane domain comprises the transmembrane domain of CD38 and said linker comprises a sequence EEX6X7X8P, wherein X6 is absent or is G, X7 is absent or is A and X8 is C, G or S. . A cell according to any of embodiments 46 to 50, wherein said transmembrane domain comprises the transmembrane domain of CD38 and said linker comprises a sequence QDX6X7PX9, wherein X6 is absent or is G, X7 is absent or is S and X9 is C or G. . A cell according to any of embodiments 46 to 50, wherein said transmembrane domain comprises the transmembrane domain of CD38 and said linker comprises a sequence GSDX9, wherein X9 is C or G. . A cell according to any of embodiments 46 to 50, wherein said linker comprises or is any one of the amino acid sequences EECP (SEQ ID NO: 90), EEGP (SEQ ID NO: 91), EEGASP (SEQ ID NO: 30), QDPC (SEQ ID NO: 92), QDPG (SEQ ID NO: 93), QDGSPG (SEQ ID NO: 31), GSDC (SEQ ID NO: 94) and GSDG (SEQ ID NO: 95). . A cell according to any of embodiments 46 to 54, wherein said extracellular domain comprises an analogue of the ectodomain of wild type CD47, said analogue comprising one or more of F14C, F14A, F14G and C15G compared to SEQ ID NO: 1.. A cell according to any of embodiments 1 to 31, wherein said transmembrane domain comprises the transmembrane domain of CD80 of sequence SEQ ID NO: 8 or an analogue thereof, such as a sequence having at least 90% or at least 95% homology with SEQ ID NO: 8. . A cell according to embodiment 56, wherein said transmembrane domain comprises a sequence comprising up to 3 amino acid substitutions, additions or deletions compared to SEQ ID NO: 8 (CD80 TMD), such as 3, 2 or 1 amino acid substitution(s), addition(s) or deletion(s), such as 1 amino acid substitution, addition or deletion. . A cell according to any of embodiments 56 to 57, wherein said transmembrane domain comprises the transmembrane domain of CD80 of sequence SEQ ID NO: 8 and said linker links said transmembrane domain of CD80 to said extracellular domain.. A cell according to any of embodiment s 56 to 58, wherein the length of said linker is between 2 and 20 amino acids, 2 and 16 amino acids, 2 and 12 amino acids, 3 and 20 amino acids, 3 and 16 amino acids, 4 and 12 amino acids or 6 and 10 amino acids or is of 6, 8 or 10 amino acids. . A cell according to any of embodiments 56 to 59, wherein said transmembrane domain comprises the transmembrane domain of CD80 and said linker comprises sequence SDX10X11X12X13X14X15X16R, wherein X10 can be E or K, Xll can be E or T, X12 can be P or T, X13 can be absent or be P or D, X14 can be absent or A, X15 can be absent or T, X16 can be absent or C or G. 1. A cell according to any of embodiments 56 to 59, wherein said transmembrane domain comprises the transmembrane domain of CD80 and said linker comprises a sequence SDEEPX13X16R (SEQ ID NO: 32), wherein X13 is absent or is P and X16 is absent or is C or G. 2. A cell according to any of embodiments 56 to 59, wherein said transmembrane domain comprises the transmembrane domain of CD80 and said linker comprises a sequence SDKTTDX14X15X16R (SEQ ID NO: 33), wherein X14 is absent or is A, X15 is absent or is T and X16 is C or G. 3. A cell according to embodiment 61, wherein said transmembrane domain comprises the transmembrane domain of CD80 and said linker comprises a sequence selected from SDEEPPCR (SEQ ID NO: 34), SDEEPPGR (SEQ ID NO: 35) and SDEEPR (SEQ ID NO: 36).. 4. A cell according to embodiment 62, wherein said transmembrane domain comprises the transmembrane domain of CD80 and said linker comprises a sequence selected from SDKTTDCR (SEQ ID NO: 37), SDKTTDGR (SEQ ID NO: 38) and SDKTTDATGR (SEQ ID NO: 39). 5. A cell according to any of embodiments 56 to 64, wherein said extracellular domain comprises an analogue of the ectodomain of wild type CD47, said analogue comprising C15G compared to SEQ ID NO: 1. 6. A cell according to an of embodiments 1 to 65, wherein said transmembrane domain comprises no more than one helix. 7. A cell according to anyone of embodiments 1 to 31, wherein said linker comprises or is of any one of the following sequences:

• GSGVSGMD or GPGVSCMD provided that said transmembrane domain comprises the transmembrane domain of CD3e and said extracellular domain comprises an analogue of CD47 wild type ectodomain which comprises Y113V and R114G compared to SEQ ID NO: 1,

• NCP or NCA provided that said transmembrane domain comprises the transmembrane domain of CD28 and said extracellular domain comprises an analogue of CD47 wild type ectodomain which comprises F14C and C15G compared to SEQ ID NO: 1,

• EECP, EEGP, EEGASP, QDPC, QDPG, QDGSPG, GSDC or GSDG provided that said transmembrane domain comprises the transmembrane domain of CD38 and the extracellular domain comprises an analogue of CD47 wild type ectodomain, said analogue comprising one or more of F14C, F14A, F14G and C15G compared to SEQ ID NO: 1, and

• SDEEPPCR, SDEEPPGR, SDEEPR, SDETTDCR, SDETTDGR, SDETTDATGR provided that said transmembrane domain comprises the transmembrane domain of CD80 and the extracellular domain comprises an analogue of CD47 wild type ectodomain, said analogue comprising C15G compared to SEQ ID NO: 1. 8. A cell according to any of embodiments 1 to 67, wherein said protein comprises an intracellular domain, such as an ICD which has ability to trigger CD47 intracellular pathway. 9. A cell according to any of embodiments 1 to 67, wherein said protein comprises no intracellular domain. 0. A cell according to any of embodiments 1 to 50, wherein said chimeric protein comprises an intracellular domain which has no ability to trigger CD47 intracellular pathway. 1. A cell according to any of embodiments 1 or 68 to 70, wherein said chimeric protein comprises or consists of anyone of sequences SEQ ID NO: 10, SEQ ID NO: 11,

SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ

ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID

NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID

NO: 27. 2. A cell according to anyone of embodiments 1 to 27, wherein said chimeric protein comprises or consists of anyone of amino acid sequences SEQ ID NO: 10 and SEQ ID NO: 11. 3. A cell according to anyone of embodiments 1 to 27, wherein said chimeric protein comprises or consists of anyone of amino acid sequences SEQ ID NO: 12 and SEQ ID NO: 13. 4. A cell according to anyone of embodiments 1 to 27, wherein said chimeric protein comprises or consists of anyone of amino acid sequences SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21. 5. A cell according to anyone of embodiments 1 to 27, wherein said chimeric protein comprises or consists of anyone of amino acid sequences SEQ ID NO: 22, SEQ ID NO:23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26 and SEQ ID NO: 27. 6. A cell according to anyone of embodiments 1 to 70, wherein said extracellular domain comprises an analogue of the ectodomain of wild type CD47 of sequence SEQ ID NO: 1, and said analogue comprises in its amino acid sequence one or more mutations selected from Y113V, R114G, F14C, F14A, F14G, C15G, S64A, S64T, S64G, D62A, E69A, T61D, S85R, S89A and/or S89D compared to the sequence SEQ ID NO: 1. . A cell according to anyone of embodiments 1 to 70, wherein said extracellular domain comprises an analogue of the ectodomain of wild type CD47 of sequence SEQ ID NO: 1, and said analogue comprises in its amino acid sequence one or more mutations selected from Y113V, R114G, F14C, F14A, F14G and/or C15G compared to SEQ ID NO: 1. . A cell according to anyone of embodiments 1 to 70, wherein said extracellular domain comprises an analogue of the ectodomain of wild type CD47 of sequence SEQ ID NO: 1, and said analogue comprises in its amino acid sequence one or more mutation selected from S64A, S64T, S64G, D62A, E69A, T61D, S85R, S89A and S89D compared to the sequence SEQ ID NO: 1. . A cell according to embodiment 78, wherein said extracellular domain comprises a mutation selected from S64A, S64T or S64G compared to the sequence SEQ ID NO: 1. . A cell according to embodiment 78, wherein said extracellular domain comprises a mutation selected from S64T or S64G compared to the sequence SEQ ID NO: 1.. A cell according to embodiment 78, wherein said extracellular domain comprises the mutation S64T compared to the sequence SEQ ID NO: 1 . A cell according to anyone of embodiments 78, wherein the amino acid sequence of said analogue comprises one or more of the mutations D62A, E69A T61D, S85R, S89A and S89D compared to the sequence SEQ ID NO: 1. . A cell according to embodiment 81, wherein said extracellular domain comprises one or both of mutations D62A and E69A compared to the sequence SEQ ID NO: 1 and/or comprises one or more of the mutations T61D, S85R, S89A and S89D compared to the sequence SEQ ID NO: 1. . A cell according to anyone of embodiment 78, wherein said extracellular domain comprises a mutation selected from S64A, S64T or S64G compared to the sequence SEQ ID NO: 1 and further comprises one or more mutation selected from D62A, E69A, T61D, S85R, S89A and S89D compared to the sequence SEQ ID NO: 1. . A cell according to anyone of embodiments 1 to 70, wherein said extracellular domain comprises an analogue of wild type CD47 ectodomain of sequence SEQ ID NO: 1 . A cell according to anyone of embodiments 1 to 70 or 85, wherein said extracellular domain comprises an analogue of wild type CD47 ectodomain of sequence SEQ ID NO: 1 and said analogue of the ectodomain comprises at least 80% homology compared to SEQ ID NO: 1, such as at least 85%, 90%, 95% or 98% homology, and less than 100% homology compared to SEQ ID NO: 1. . A cell according to any of embodiments 1 to 70 and 85 to 86 wherein the sequence of said analogue comprises between 80 and 99% homology compared to SEQ ID NO: 1, such as between 85-99%, 85-98%, 90-99%, 90-98% or 80-95%, 85- 90% homology compared to SEQ ID NO: 1. . A cell according to anyone of embodiments 1 to 70 and 85 to 87, wherein the sequence of said analogue has at least 90% homology with SEQ ID NO: 1, such as between 90% and 99% homology. . A cell according to anyone of embodiments 1 to 70 and 85 to 87, wherein said extracellular domain comprises wild type CD47 ectodomain of sequence SEQ ID NO: 1 or an analogue thereof, said analogue having at least 90% homology and less than 100% homology with SEQ ID NO: 1. . A cell according to anyone of embodiments 1 to 70 and 85 to 89, wherein the sequence of said analogue comprises at least 95% homology compared to SEQ ID NO: 1, such as at least 95%, 96%, 97%, 98% or 99% homology compared to SEQ ID NO: 1. . A cell according to anyone of embodiments 1 to 70 and 85 to 90, wherein the sequence of said analogue comprises up to 10 amino acid substitutions, deletions and/or additions, such as up to 9, 8, 7 or 6 amino acid substitution(s), deletion(s) and/or addition(s) compared to SEQ ID NO: 1. . A cell according to anyone of embodiments 1 to 70 and 85 to 91, wherein the sequence of said analogue comprises up to 5 amino acid substitutions, deletions and/or additions, such as up to 4, 3, 2 or 1 amino acid substitution(s), deletion(s) and/or addition(s) compared to SEQ ID NO: 1. . A cell according to anyone of embodiments 1 to 70 and 85 to 92, wherein said extracellular domain comprises an analogue of the ectodomain of wild type CD47 of sequence SEQ ID NO: 1, and said analogue comprises the amino acid sequence of said analogue having at least 90%, such as at least 95%, homology with SEQ ID NO: 1 and comprises a mutation selected from S64A, S64T and S64G, such as S64T, compared to the sequence SEQ ID NO: 1. . A cell according to anyone of preceding embodiments, wherein said extracellular domain has ability to bind to SIRP-a. . A cell according to anyone of preceding embodiments, wherein said extracellular domain has reduced ability to bind to TSP-1 compared to the same cell with wild type CD47 and without said chimeric protein.

96. A cell according to anyone of preceding embodiments, wherein said extracellular domain has increased ability to bind to SIRP-a compared to the same cell with wild type CD47 and without said chimeric protein. . A cell comprising a nucleic acid sequence encoding a chimeric protein as defined in anyone of embodiments 1 to 96. . A cell comprising on its cell surface a chimeric protein as defined in anyone of embodiments 1 to 97. . A cell according to anyone of preceding embodiments wherein said cell is a mammalian cell, such as a human cell. 0. A cell according to anyone of preceding embodiments wherein said cell is at a differentiated stage, such as a differentiated cell or mature cell. 1. A cell according to anyone of preceding embodiments wherein said cell is any cell type derived from a pluripotent stem cell. 2. A cell according to anyone of preceding embodiments wherein said cell is a nonnatural cell, such as a cell at a differentiated stage obtained by genome engineering of a pluripotent cell. 3. A cell according to anyone of preceding embodiments, wherein said cell is a beta cell, an INS+ and NKX6.1+ double positive cell or a C-peptide+/NKX6.1+ double positive cell, an insulin producing cell, an in vitro derived beta-like cell, a pancreatic endocrine cell or an endocrine cell, an endocrine progenitor cell or a NGN3+/NKX2.2+ double positive cell, a neural cell such as a neuron, an interneuron cell, an oligodendrocyte, an astrocyte or a dopaminergic cell, an exosome cell, an immune cell such as a T cell, a NK cell, a macrophage or a dendritic cell, a hepatocyte, a stellate cell, a fibroblast, a keratinocyte, a hair cell, an inner ear cell, an intestinal cell, an organoid cell, a nephroid cell or another kidney-related cell, a cortical neural progenitor cell, a cardiomyocyte, a retinal cell or a retinal pigment epithelium cell. . A cell according to anyone of embodiments 1 to 99 wherein said cell is a stem cell, such as an embryonic stem cell, a pluripotent stem cell or an induced pluripotent stem cell (iPSC), or is a cell derived from a stem cell. 5. A cell according to embodiment 104 to wherein said cell is a mesenchymal stem cell. 6. A cell according to anyone of preceding embodiments wherein said cell is B2M-/-. 7. A cell according to anyone of preceding embodiments wherein said cell is CIITA- /-. 8. A cell according to anyone of embodiments 106 to 107 wherein said cell further comprises a B2M-HLA genetic fusion protein on its cell surface or a nucleic acid sequence encoding a B2M-HLA genetic fusion protein, such as a B2M-HLA-E and/or B2M-HLA-G genetic fusion protein. 9. A cell according to anyone of embodiments 106 to 108 wherein said cell is B2M- /-, CIITA-/- and further comprises a B2M-HLA genetic fusion protein on its cell surface or a nucleic acid sequence encoding a B2M-HLA genetic fusion protein, such as a B2M- HLA-E and/or B2M-HLA-G genetic fusion protein. 0. A cell according to anyone of embodiments 106 to 109 wherein said cell is B2M- /-, CIITA-/- and further comprises a PD-L1 and/or CD55 encoding nucleic sequence and/or a PD-L1 and/or CD55 protein on its cell surface. 1. A cell according to anyone of embodiments 76 to 110, wherein said chimeric protein comprises or consists of anyone of amino acid sequences SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83 and SEQ ID NO: 84. 2. A composition comprising cells according to anyone of previous embodiments in a pharmaceutically acceptable medium. 3. A pharmaceutical product comprising cells according to anyone of embodiments 1 to 110 or a composition according to embodiment 112. 4. A cell according to anyone of embodiments 1 to 111, a composition according to embodiment 112 or a pharmaceutical product according to embodiment 112 for use in cellular therapy of a patient in need thereof. 5. A cell, a composition or a pharmaceutical product for use according to embodiment 114, for the treatment, the cure, or the prevention of a chronic disease or of an acute disease. 6. A cell, a composition or a pharmaceutical product for use according to embodiment 115, wherein said chronic disease is diabetes, type 1 diabetes, type 2 diabetes, dry macular degeneration, retinitis pigmentosa, neurological disease, Parkinson's disease, heart disease, tissue fibrosis, cirrhosis, hearing loss, corneal blindness, stroke, chronic heart failure, chronic kidney disease or cancer. 117. A cell, a composition or a pharmaceutical product for use according to embodiment 115, wherein said acute disease is bacterial lung infections, such as ventilator acquired bacterial pneumonia or hospital acquired bacterial pneumonia.

118. A process for the preparation of a cell according to anyone of embodiments 1 to 111, said process comprising the step of inserting into a cell a nucleic acid sequence encoding a chimeric protein as defined in anyone of embodiments 1 to 111.

119. A nucleic acid sequence encoding a chimeric protein as defined in anyone of embodiments 1 to 111.

120. A process according to claim 118, wherein said step of inserting into a cell said nucleic acid sequence is performed in vitro.

Examples

Example 1. Chimeric proteins and cells

Cells expressing chimeric protein as disclosed herein have been prepared. More specifically, HEK293 cells have been prepared to express chimeric proteins as listed in Table 2(a) and 2(b). For each tested chimeric protein, two cell versions have been prepared, one version has been transiently transfected with a nucleic acid expressing the chimeric protein, another version has been prepared for stable expression of the chimeric protein. The process for making said chimeric proteins and cells is further described in Example 2.

Table 2(a):

Table 2(b):

Example 2. Process for making chimeric proteins and cells

Nucleic acid sequences listed in Table 3 were designed and synthesized by Twist Bioscience®. These nucleic acid sequences comprise a start codon, a signal peptide sequence, an ectodomain which is the ectodomain of wild type CD47 or an analogue thereof, a linker sequence, the transmembrane domain sequence and a stop codon. Each of these sequences was cloned into the commercial cloning vector pcDNA 3.1. As a reference, the nucleic acid sequence SEQ ID NO: 9 encoding wild type CD47 protein was also cloned into a pcDNA 3.1 vector. Table 3. Nucleic acid sequences cloned into pcDNA 3.1 vector

For transient expression:

For each pcDNA 3.1 vector comprising a sequence of Table 3, as well as for the reference pcDNA 3.1 vector comprising wild type CD47 nucleic acid sequence, around 3 ml of HEK293 cells with cell density of 1 million cells/ml was mixed with 3pg of synthesized nucleic acid (cloned into pcDNA 3.1 vector) and 8.1 pl of transfection reagents (expifectamine, Invitrogen, A14524) for 10 minutes. A mock transfection was also prepared under the same conditions except for using 3pg of "empty" pcDNA 3.1 vector (i.e. comprising no cloned sequence in it) instead of 3pg of cloned pcDNA 3.1 vector. Cells viability upon transfection was determined by Vi-CELL XR Cell Viability Analyzer (Beckman®). The results are summarized in Table 4. The figure in the Cell Viability column (right column) is provided by Beckman apparatus and is the % of alive cells in the cell population exposed to the transfection protocol (sample thereof placed under a cover slip). Table 4. Nucleic acid sequences and cell survival upon transfection

Example 3. Process for confirming chimeric protein expression and SIRP-o binding ability

Upon transfection, the cells were tested for their expression of the chimeric protein and for their ability to bind to SIRP-o. FACS (FACSymphony cytometer) based screening was used to determine the binding capability of transiently transfected cells to SIRP- a. For each nucleic acid of Table 3 and wild type CD47 protein, 200,000 transfected cells were stained with primary probe containing CD47-APC (Allophycocyanin) antibody (Miltenyi Biotec®, Cat# 130-123-254 with 100-fold dilution) and washed three times with Phosphate-buffered saline (PBS) buffer. The cells were then exposed to secondary labelling with phycoerythrin (PE)-conjugated Human SIRPo /CD172a Protein, Fc Tag (Aero Biosystems®, Cat# SIA-HP252 with 100-fold dilution). The mixture of PE- conjugated SIRP-o and cells was then incubated at 4 C° for 60 mins and followed with 3 times washing steps to remove unbound PE-conjugated SIRP-o. The labelled cells were pelleted down by 300g at 4°C for 3 min and resuspended in 200 pL of PBS buffer supplemented with 1 % of Bovine Serum Albumin (BSA).

Quantification of the protein expression and binding ability was performed by FACS (FACSymphony cytometer) analysis. Cells were gated for live (DRAQ7 low/negative, DRAQ7 being a dye for staining dead cells) and singlet cells by low intensity of forward scatter (FSC) and side scatter (SCC), followed by analysis of signals from the blue laser (650 nm, CD47 signal) and yellow-green laser (561 nm, SIRP- a signal).

- The chimeric protein expression was determined by APC signal (emission wavelength at 660 nm) upon cells labelling with CD47-APC reagent. The results are shown in Fig. 3 - 5. In Fig.3-4, rows 1 and 2 show the CD47 expression on the surface of cells in which no CD47 encoding nucleic acid has been transfected (blank cells), rows 3 show the CD47 expression on the surface of cells in which wild type CD47 encoding nucleic acid has been transfected, and the following rows show the CD47 expression on the surface of cells transfected with CD47 chimeric protein encoding nucleic acid.

The results reported on Fig.3(a) indicate that proteins tested in row 3 to 11 are expressed on the cell surface of the transfected cells, and that the chimeric proteins tested in row 4 to 11 are expressed with an expression level at least on par with the expression level of wild type CD47 in row 3.

The results reported on Fig.4(a) indicate that proteins tested in row 3 to 7 are expressed on the cell surface of the transfected cells, and that the chimeric proteins tested in row 4 to 7 are expressed with an expression level at least on par with the expression level of wild type CD47 in row 3.

The results reported on Fig.5(a) indicate that proteins tested in row 3 to 9 are expressed on the cell surface of the transfected cells, and that the chimeric proteins tested in row 4 to 9 are expressed with an expression level at least on par with the expression level of wild type CD47 in row 3.

- Based on the median intensity unit at 570 nm, the binding affinity of designed chimeric protein to SIRP-o can be ranked (higher 570 nm emission signal represents higher binding affinity to SIRP- a). The results are shown in Fig. 6-8. (Row 1, blank cell with labelled SIRP-o-PE reagent; Row 2-6 (Fig. 6), Row 2-10 (Fig. 7) and Row 2-8 (Fig. 8) show the binding ability to SIRP-o of engineered CD47 variants).

The results reported on Fig.6(a) indicate that proteins tested in row 2 to 6 and expressed on the cell surface of the transfected cells do bind SIRP-o, and that the chimeric proteins tested in row 3 to 6 present an ability to bind SIRP- a which is at least on par with wild type CD47 in row 2.

The results reported on Fig.7(a) indicate that proteins tested in row 2 to 10 and expressed on the cell surface of the transfected cells do bind SIRP-o, and that the chimeric proteins tested in row 3 to 9 present an ability to bind SIRP- a which is at least on par with wild type CD47 in row 2.

The results reported on Fig.8(a) indicate that proteins tested in row 2 to 8 and expressed on the cell surface of the transfected cells do bind SIRP-o, and that the chimeric proteins tested in row 3 to 8 present an ability to bind SIRP- a which is at least on par with wild type CD47 in row 2.

Example 4. THP1 assay for CD47 transfected E1C3 cell-line

Preparation of transfected E1C3 cell:

The stable cell line of E1C3 human embryonic stem cell was generated by CRISPR-MAD7 method.

The E1C3 cells were cultured in NutriSter medium (from Sartorius®) until cell density reached 80 % of confluency. The cells were then incubated with 3 mL of Versene solution (Gibco™ Versene from ThermoFisher®, catalogue #15040066. Gibco™ Versene solution (0.48 mM) is formulated as 0.2 g EDTA(Na4) per liter of Phosphate Buffered Saline (PBS) for 20 min at 37 degree and were centrifuged down. The pelleted cells were resuspended in 70 pL of buffer R (from ThermoFisher®) at a final density of 1.0 x 10 ⁷ cells/mL. 10 pL of the cell mixture was incubated with 2 pL of RNPs (Ribonucleoproteins) mix comprising of 1 pL of MAD7 protein (1 mg/1 mL), 1 pL of gRNA (guide RNA) (75 pM) and 0.5 pg of plasmid DNA carrying the gene sequences for targeted insertion in the cells' genome:

Table 5. Nucleic acid sequence on plasmid for transfection into cells' genome

The transfection was performed by electroporation per standard Neon™ transfection protocol (from ThermoFisher®) by using 1100 Volt of pulse 2 with the pulse width of 20. After the transfection, the cells were transferred into recovering medium comprising iMatrix™ (3 mL NutriStem® + 6 pL ROCKi (ROCK inhibitor) + 5 pL iMatrix™ 511 (recombinant laminin 511)) and 1 mL of NutriStem®. The surviving clones were picked- up and split in 2 different duplicate plates, one for expansion and one for cell collection (QE). Sanger Sequencing was performed to confirm the integration of the plasmid and to verify whether the correct knock-in was biallelic or monoallelic.

THP1 assay:

Transfected E1C3 cells were seeded on a 48-well plate with a total number of 25,000 cells per well and cultured overnight with 1 mL NutriStem® medium. After overnight culture, 250 pL of transfected E1C3 cell was mixed with equal amount of THPl-NF-kB- GFP cells ("THP1 NF-KB GFP" cells are designed for monitoring the NF-KB signal transduction pathway in cell line of interest.) in presence of different stimuli for 24 hours. Those stimuli include anti-hErbB2 antibody (R&D Systems cat# MAB9589, 250 ng/mL) as a baseline control, anti-hErbB2 antibody together with anti-CD47 antibody (Bio X Cell cat# BE0283, 4 pg/mL), and anti-hErbB2 antibody together with IgGl isotype control (Bio X Cell cat#BE0083, 4 pg/mL). The cells were then harvested and eGFP expression was analysed by flow cytometry. Mean and standard deviation of the geometric mean of fluorescence intensity (gMFI) of the viable population of reporter cells was determined and reported in Fig.9(a).

The results reported on Fig.9(a) indicate that proteins tested in row 3 to 6 and expressed on the cell surface of the transfected cells are able to activate the NF-KB signal transduction pathway in THP1 NF-KB GFP cells, and that the chimeric proteins tested in row 4 to 6 present an ability to activate the NF-KB signal transduction pathway in THP1 NF-KB GFP cells which is higher than cells of row 1 and 2 and on par with cells of row 3, i.e. cells expressing wild type CD47.

While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.