WO2023144404A1

WO2023144404A1 - Novel integrin associated protein (iap)

Info

Publication number: WO2023144404A1
Application number: PCT/EP2023/052233
Authority: WO
Inventors: Che YANG
Original assignee: Novo Nordisk A/S
Priority date: 2022-01-31
Filing date: 2023-01-31
Publication date: 2023-08-03

Abstract

The present invention relates to novel Integrin associated proteins, cells, compositions, and pharmaceutical products which improve the success of cellular therapy.

Description

DESCRIPTION

TITLE: NOVEL INTEGRIN ASSOCIATED PROTEIN (IAP)

TECHNICAL FIELD

The present invention is in the field of cellular therapy.

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, cells or tissues are grafted in the body of a host patient in need. This is an allograft if the grafted cells or tissue come from a donor, or an autograft if the grafted cells or tissue come from the recipient.

The success of cellular therapy depends on multiple factors, for example the survival of grafted cells, safety considerations, performance of the grafted cells. Upon grafting, the survival of the grafted cells is jeopardized by multiple elements and reactions from the host organism, such as the risk of rejection by the immune system of the host organism or the risk is ischemia.

Upon grafting, allograft cells are exposed the immune system of the host organism and usually get identified as non-self and rejected by way of an immune response.

Upon grafting, cells are often exposed to an ischemic environment and, subsequently, die due to lack of vascularisation.

Integrin associated protein (IAP) is a potent “don’t-eat-me” signal enabling the recognition between self-cells and immune cells such as macrophage cells. Cells normally express IAP as an anti-phagocytic signal to maintain tissue homeostasis.

Integrin associated protein (IAP) is a transmembrane protein. In humans, wild type IAP comprises an extracellular domain (ECD) comprising the amino acid sequence SEQ ID NO 1 and a transmembrane domain (TMD) comprising the amino acid sequence SEQ ID NO 2. IAP belongs to the immunoglobulin superfamily and partners with membrane integrins. IAP binds the ligands thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRPa). Recent research has shown that IAP is transiently upregulated during embryogenesis in mouse hematopoietic stem cells (HSCs) and progenitor cells just prior to migratory phase.

In cancer cells, it is known that overexpression of IAP avoids phagocytosis of cancer cells via engagement of IAP with SIRPa expressed on macrophage and other immune cells. Engagement of SIRPa leads to inhibition of phagocytosis.

SIRPa belongs to the signal regulatory protein family and is mainly expressed on myeloid and neuronal cells. The complex structure of SIRPa and IAP was previously determined and revealed a unique binding mode between the IgSF (immunoglobulin superfamily) domain of these two proteins, a binding mode that resembles an antibodyantigen interaction. lAP-SIRPa binding triggers the recruitment of SHP (Src (Sarcoma) Homology 2 Domain)-related phosphatases, leading in turn to myosin dephosphorylation and inhibition of phagocytosis of the lAP-expressing cell by immune cells.

Another biologically binding partner of IAP is thrombospondin-1 (TSP-1).

TSP-1 is a protein released by platelets. Via binding to IAP, TSP-1 can activate IAP, which leads to nitric oxide (NO) production and inhibition of signal transduction. TSP-1 binding to IAP promotes the production of reactive oxygen species which in turn causes vascular diseases. TSP-1 binding to IAP is also related to suppression of angiogenesis. TSP- 1 binding to IAP inhibits nitric oxide (NO)-stimulated responses in both endothelial and vascular smooth muscle cells.

TSP-1 exhibits multifaceted functionalities depending on local TSP-1 concentration as well as binding to different receptors. TSP-1 acts as a major driver to induce cell senescence by signalling through TSP1-IAP pathway.

Although the atomic structure of TSP-1 and IAP complex has not been solved, previous studies indicate that TSP-1 and SIRP-a compete for binding to IAP. Besides, binding assays have failed to detect simultaneous binding events of TSP-1 and to SIRPa to IAP.

A problem to be solved is to improve the success of cell therapy.

SUMMARY

In one aspect, the invention provides novel Integrin associated protein(s) (IAP(s)). In another aspect, the invention provides cells expressing said novel Integrin associated protein (IAP), compositions and pharmaceutical products comprising such cells and therapeutical uses thereof. In one aspect, the invention relates to an IAP(s) analogue comprising an extracellular domain, wherein said extracellular domain is an analogue of the Integrin associated protein extracellular domain (IAP-ECD) defined by SEQ ID NO 1 and comprises at least one, two or three of Y37W, R103L and K39V. In an embodiment, said IAP analogue further comprises a transmembrane domain (TMD). In an embodiment, said analogue of the IAP-ECD comprises at least one of Y37W and R103L. In an embodiment, said analogue of the IAP-ECD further comprises K39V. In an embodiment, said analogue of the IAP-ECD further comprises one, two, three or four of T49, L3, A30 and T102. In an embodiment, said analogue of the IAP-ECD has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97% homology with sequence SEQ ID NO 1.

In another aspect, the invention relates to a cell, or cells, expressing an IAP analogue as defined therein. In some embodiment, said cell(s) 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. In an embodiment, said cell(s) is a mammalian cell obtained from a donor or derived from a cell obtained from a donor.

In another aspect, the invention relates to a composition comprising a cell or cells as defined therein in a pharmaceutically acceptable medium.

In another aspect, the invention relates to a pharmaceutical product comprising a cell or cells as defined therein or a cell composition as defined therein.

In another aspect, the invention relates to a pharmaceutical product as defined therein for use in cell therapy.

In another aspect, the invention relates to a pharmaceutical product as defined therein for use in the treatment, the cure, or the prevention of a chronic disease or of an acute disease.

The novel IPAs, cells, compositions, and pharmaceutical products as defined herein improve the success of cellular therapy. The invention provides improved cells survival upon cellular transplantation, reduced cells rejection by the immune system of the host organism, cells more silent to the immune system of the host organism, improved cells resistance to ischemia, reduced thrombospondin binding, reduced risk of suppressed angiogenesis, and/or reduced nitric oxide (NO)-stimulated responses in cells, reduced risk of TSP-1 related adverse effects, increased cells surviving in ischemic environments.

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

Express:

As used herein, a cell “expressing” or that “expresses” a given protein means that the cell comprises the genetic material and transduction and/or translation machinery which together allow for and do produce said given protein.

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.

Engineered cell:

The term “engineered” cell as used herein means a cell that has been artificially modified, for example genetically modified.

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.

Protein, peptide:

Unless otherwise specified, the terms “protein” and “peptide” refer to a functional version thereof.

Wild type and Analogue:

The term “wild type” as used herein in relation to a protein means that the amino acid sequence of said protein is the sequence which prevails among individuals in natural conditions, as distinct from an atypical variant thereof.

The term “analogue” as used herein in relation to a protein or peptide means that the amino acid sequence of said protein or peptide bears one or more modifications, such as one or more amino-acid mutations compared to a reference protein or peptide. The reference sequence may be a wild-type sequence.

Mutation:

The term “mutation” as used herein means the addition, deletion or replacement of one or more parts of a reference sequence. In relation to a protein or peptide, a mutation may be the addition, deletion and/or replacement of one or more amino-acids compared to a reference amino acid sequence. Mutations are herein defined according to conventional nomenclature.

IAP, IAP-ECD, IAP analogue, SEQ ID NO 1 and IAP(s) of the present invention:

The term “IAP” as used herein means Integrin associated protein. IAP may also be called CD47.

The term “IAP-ECD” as used herein means the extracellular domain of IAP.

“SEQ ID NO 1” is the amino acid sequence of wild-type IAP-ECD, which may also be called wild-type CD47 ECD.

“SEQ ID NO 12” is the amino acid sequence of full-length wild-type IAP, which may also be called wild-type CD47.

The IAP analogue(s) as defined herein comprise an IAP-ECD which is an analogue of wild-type IAP-ECD as defined by SEQ ID NO 1.

Pharmaceutically acceptable medium: As used herein, this term means any medium that is suitable for cells transplantation to a patient, especially in the context of cellular therapy.

Transmembrane domain (TMD): A transmembrane domain consists of amino acids that transverse the plasma membrane once or several times. These amino acids often form alpha helices and can include connecting loops on both sides of the membrane. BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 is a three-dimensional representation of IAP-ECD (IAP extracellular domain) of sequence SEQ ID NO 1 with identification of the amino acid positions which are subject of mutations as per the present invention. The key residues (colored in white with stick representation) of IAP-ECD (black) engage in the binding interface with SIRPa (colored in grey with surface representation).

Fig. 2(a), (b) and (c) show the FACS results of IAP protein expression (SEQ ID NO 1-21) on the cells surface as described in Example 5.

Fig. 3(a), (b) and (c) show the FACS results of binding to SIRP- a measurement on the cells as described in Example 5.

DESCRIPTION

A purpose of the present invention is to improve the success of cellular therapy.

A purpose of the present invention is to improve cells survival upon cellular transplantation, also called grafting, to a host organism. A more particular purpose of the present invention is to protect transplanted cells from rejection by the immune system of the host organism. Another purpose is to prolong the life cycle of the transplanted cells.

Another purpose of the present invention is to improve cells resistance to ischemia, to increase the chance of surviving ischemic environments for example in the context of cell transplantation, of cells in poorly vascularised tissue or of cells in encapsulation devices.

As part of the present invention, it has surprisingly been discovered certain mutations in the sequence of the Integrin Associated Protein (IAP) that reduce the risk of grafted cells being rejected by the immune system of the host organism. As part of the invention, it has surprisingly been discovered that said mutations may also increase the chance of grafted cells surviving ischemic environments for example in relation to cell transplantation and/or in poorly vascularised tissue or in encapsulation devices.

In one aspect of the present invention, the IAP analogue(s) of the present invention have increased binding ability to SIRPa compared to wild-type IAP and provide grafted cells that express said IAP analogue(s) a better survival chance upon cell transplantation. Advantageously, cells that express IAP analogue(s) of the present invention have a stronger ‘self’ signal and prolonged life cycle upon grafting into a host organism, are less prone of being identified as “non-self” and rejected by the immune system of the host organism and thereby more likely to remain intact and silent under the surveillance of immune system of the host organism.

Additionally, it was surprisingly found that the IAP analogue(s) of the present invention may have a reduced binding opportunity to TSP-1 and cells expressing said IAP analogue(s) have increased chance of surviving in TSP-1 surrounding environments e.g., in relation to cell transplantation and/or in poorly vascularised tissue or encapsulation devices. This might be due to IAP analogue(s) of the present invention staying in apo-form for TSP-1 binding for a shorter period of time compared to wild-type IAP.

In another aspect of the present invention, the new IAP analogue(s) provide grafted cells a better chance of not being rejected by the immune system of the host organism and of surviving ischemic environments upon cell transplantation and/or in poorly vascularised tissue or encapsulation devices.

IAP(s):

In one aspect, the invention provides an Integrin Associated Protein (IAP) analogue.

In one aspect, the invention provides an Integrin Associated Protein (IAP) analogue comprising an extracellular domain, wherein said extracellular domain is an analogue of the Integrin Associated Protein extracellular domain (IAP-ECD) defined by SEQ ID NO 1 comprising at least one of the mutations Y37W, R103L and/or K39V.

In an embodiment, said analogue of the AIP-ECD comprises one, two or three of the mutations Y37W, R103L and/or K39V.

In an embodiment, the invention provides an Integrin Associated Protein (IAP) analogue comprising an extracellular domain, wherein said extracellular domain is an analogue of the Integrin Associated Protein extracellular domain (IAP-ECD) defined by SEQ ID NO 1 and comprises at least one of the mutations Y37W and R103L. In an embodiment, said analogue of IAP-ECD further comprises the mutation K39V.

In a further embodiment, said analogue of IAP-ECD further comprises one, two, three or four of the following (wild-type) amino-acids T49, L3, A30 and/or T102.

In other words, in an embodiment, the invention provides an IAP analogue comprising an extracellular domain, wherein said extracellular domain is an analogue of the IAP-ECD defined by SEQ ID NO 1, and: a) comprises: i) at least one or two of Y37W and R103L, and optionally further comprises K39V, or ii) at least one, two or three of Y37W, R103L and K39V, and b) optionally further comprises at least one, two, three or four of T49, L3, A30 and/or

T102.

In an embodiment, said IAP analogue further comprises a transmembrane domain.

In a further embodiment, said analogue of IAP-ECD has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97% homology with sequence SEQ ID NO 1 and comprises at least one of Y37W, R103L and/or K39V.

In an embodiment, the invention provides an IAP analogue comprising an extracellular domain and a transmembrane domain, wherein said extracellular domain is an analogue of IAP-ECD defined by SEQ ID NO 1 , has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97% homology with SEQ ID NO 1 and comprises at least one of the mutations Y37W, R103L and/or K39V compared to SEQ ID NO 1. In an embodiment, said analogue of IAP-ECD further comprises one, two, three or four of T49, L3, A30 and/or T102, which means no mutation in one, two, three or four of positions T49, L3, A30 and/or T102 compared to sequence SEQ ID NO 1.

In an embodiment, the invention provides an IAP analogue comprising an extracellular domain and a transmembrane domain, wherein said extracellular domain is an analogue of IAP-ECD defined by SEQ ID NO 1 , has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97% homology with SEQ ID NO 1 , and comprises at least one of the mutations Y37W and/or R103L compared to SEQ ID NO 1. In an embodiment, said analogue of IAP- ECD further comprises the mutation K39V compared to SEQ ID NO 1. In an embodiment, said analogue of IAP-ECD further comprises no mutation in one, two, three or four of positions T49, L3, A30 and/or T102 compared to sequence SEQ ID NO 01.

In an embodiment, the IAP analogues of the present invention comprise a transmembrane domain (TMD). In an embodiment, the IAP analogues of the present invention comprise a linker between their TMD and ECD. In an embodiment, the IAP analogues of the present invention comprise an intracellular domain (ICD).

In an embodiment, said TMD comprises the sequence of wild-type CD47 TMD, defined by sequence SEQ ID NO 13 or an analogue thereof. In an embodiment, said analogue has at least 90% homology to SEQ ID NO 13.

In an embodiment, said linker between IAP TMD and IAP ECD comprises the sequence of wild-type CD47 linker, defined by sequence SEQ ID NO 14 or an analogue thereof. In an embodiment, said analogue has at least 90% homology to SEQ ID NO 14. In an embodiment, said ICD comprises the sequence of wild-type CD47 ICD, defined by sequence SEQ ID NO 15 or an analogue thereof. In an embodiment, said analogue has at least 90% homology to SEQ ID NO 15.

In an embodiment, the IAP analogues of the present invention have increased binding affinity to SIRPa compared to wild-type IAP.

In an embodiment, the IAP analogues of the present invention have increased binding affinity to SIRPa and have same binding affinity to TSP-1 compared to wild-type IAP.

In another embodiment, the IAP analogues of the present invention have both increased binding affinity to SIRPa and decreased binding affinity to TSP-1 compared to wild-type IAP.

Cells:

In another aspect, the invention provides a cell expressing an IAP analogue as defined herein.

In an embodiment, the cell of the invention is an engineered cell.

In an embodiment, the cell of the invention is a mammalian cell. In an embodiment, it is human cell.

In an embodiment, the cell(s) of the invention is a stem cell. In an embodiment, said stem cell is an embryonic stem cell or a pluripotent stem cell. In another embodiment, said stem cell is an induced pluripotent stem cell (iPSC).

In an embodiment, the cell(s) of the invention is derived from a stem cell.

In another embodiment, the cell(s) of the invention is at a differentiated stage, such as a differentiated cell or mature cell. In an embodiment, the cell of the invention 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, a dopaminergic cell, an exosome cell, such as ESCs or NSCs, or an exosome cell derived from a ESC or NSC, an immune cell, such as a T cell, a NK cell, a macrophage, a dendritic cell, a hepatocyte, a stellate cell, a fibroblast, a keratinocyte or a hair cell, an inner ear cell, an intestinal cell or 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, a mesenchymal stem cell.

In an embodiment, the cell of the invention is obtained from a donor. In an embodiment, the cell of the invention is a differentiated cell obtained from a donor, such as cell of any type. In an embodiment, the cell of the invention is a differentiated cell derived from a cell obtained from a donor, such as a stem cell or a progenitor cell obtained from a donor. In an embodiment, the cell of the invention is a differentiated cell derived from an iPSC.

In an embodiment, the cell of the invention expresses an IAP analogue comprising an extracellular domain and a transmembrane domain, wherein said extracellular domain is an analogue of IAP-ECD defined by SEQ ID NO 1 , has at least 90% homology with sequence SEQ ID NO 1 and comprises at least one, two or three mutation(s) selected from Y37W, R103L and K39V.

In an embodiment, the cell of the invention expresses an IAP analogue comprising an extracellular domain and a transmembrane domain, wherein said extracellular domain is an analogue of IAP-ECD defined by SEQ ID NO 1 , has at least 90% homology with sequence SEQ ID NO 1 and comprises at least one or two of Y37W and R103L. In an embodiment, said analogue of IAP-ECD further comprises K39V.

In a further embodiment, said analogue of IAP-ECD further comprises one, two, three or four of T49, L3, A30 and/or T102 In a further embodiment, said analogue of IAP- ECD has at least 91%, 92%, 93%, 94%, 95%, 96% or 97% homology with SEQ ID NO 1.

In a further embodiment, the cell(s) of the present invention do not express wild-type IAP, i.e. do not express IAP comprising unmodified amino acid sequence SEQ ID NO 1.

In a preferred embodiment, the cell comprises further genetic modifications to improve its stealth property. The term “stealth” means being protected from rejection by the immune system of a host upon allogeneic transplantation, being either not subject or at least less subject to such rejection than the same cell without stealth modification. The cell may comprise a decrease in the HLA-class I and II proteins expression.

In an embodiment, the cell of the invention is B2M-/-. The term “B2M-/-“ as used herein means that the cell comprises the knock-out of B2M endogenous gene(s).

In an embodiment, the cell of the invention is CIITA-/-. The term “CIITA-/-“ as used herein means that the cell comprises the knock-out of CIITA endogenous gene(s).

In an embodiment, the cell of the invention further expresses a B2M-HLA fusion protein, such as B2M-HLA-E and/or B2M-HLA-G genetic fusion protein. Said fusion protein expression may be achieved by insertion in the cell of a transgene encoding a B2M-HLA fusion protein, such as B2M-HLA-E and/or B2M-HLA-G. Said transgene may be inserted into the cell’s genome or may be located on a vector transfected in the cell. In a preferred embodiment, the cell of the invention further comprises a nucleic acid sequence encoding a B2M-HLA fusion protein, such as B2M-HLA-E and/or B2M-HLA-G genetic fusion protein. In a preferred embodiment, the cell of the invention is B2M-/-, CIITA-/- and further comprises a nucleic acid sequence encoding a B2M-HLA fusion protein, such as B2M-HLA-E and/or B2M-HLA-G genetic fusion protein.

The cell of the invention may be further engineered to have reduced or increased expression of additional immune evasive proteins on the cell surface such as, PDL1 , complement factors like, CD55, CD46, CD59, and/or other immune evasive ligands known in the art. In an embodiment, the cell comprises increased expression of at least one of CD47, HLA-G, HLA-E, HLA-F, PD-L1 , CD55 proteins. In an embodiment, the cell comprises a transgene encoding said protein.

Cell composition and pharmaceutical products

In another aspect, the invention provides a cell composition, said composition comprising cells as defined herein in a pharmaceutically acceptable medium.

In another aspect, the invention provides a pharmaceutical product comprising cell(s) or a cell composition as defined herein. In an embodiment, said pharmaceutical product is a cell therapy product.

Use, medical indication and method of treatment

In another aspect, the invention provides a cell(s), a cell composition or a pharmaceutical product as defined herein for use as a medicament.

In another aspect, the invention provides a cell(s), a cell composition or a pharmaceutical product as defined herein for use in cell therapy, such as for use in allogenic cell therapy.

In another aspect, the invention provides a cell(s), a cell composition or a pharmaceutical product as defined herein for use in the treatment, the cure, or the prevention of a chronic disease or of an acute disease.

In one embodiment said chronic disease comprises or is selected from the group consisting of 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, and cancer.

In one embodiment, said acute disease comprises bacterial lung infections, such as ventilator acquired bacterial pneumonia and hospital acquired bacterial pneumonia.

In another aspect, the invention provides a method of treatment, cure or prevention, wherein patients in need thereof are administered a cell, a cell composition or a pharmaceutical product as defined herein. In an embodiment, said method is a method of treatment, cure or prevention by cell therapy. In an embodiment, said method is a method of treatment, cure or prevention of a chronic disease or of an acute disease.

In another aspect, the invention provides a method for the preparation of a medicament for the treatment, cure or prevention of a chronic disease or of an acute disease, wherein patients in need thereof are administered a cell, a cell composition or a pharmaceutical product of the invention as defined herein.

Method

The IAP analogues and cell(s) as defined herein may be obtained by any conventional method.

In another aspect, the invention provides a method of preparing a cell expressing an IAP analogue as defined herein, said method comprising the following steps:

• culturing a cell,

• inserting into said cell a vector encoding an IAP analogue as defined herein or inserting into the genome of said cell a transgene encoding an IAP analogue as defined herein.

In an embodiment, the invention relates to an IAP analogue comprising an extracellular domain and a transmembrane domain, wherein said extracellular domain is an analogue of the IAP-ECD defined by SEQ ID NO 1 , and: a) comprises: i) at least one or two of Y37W and R103L, and optionally further comprises K39V, or ii) at least one, two or three of Y37W, R103L and K39V, and b) optionally further comprises at least one, two, three or four of T49, L3, A30 and/or

T102.

In an embodiment, the invention relates to a cell, said cell expressing an IAP analogue comprising an extracellular domain and a transmembrane domain, wherein:

1. said extracellular domain is an analogue of the IAP-ECD defined by SEQ ID NO 1 , and: a) comprises: i) at least one or two of Y37W and R103L, and optionally further comprises K39V, or ii) at least one, two or three of Y37W, R103L and K39V, b) optionally further comprises one, two, three or four of T49, L3, A30 and/or T102, and

2. said cell is an engineered cell, such as an engineered human cell, and

3. said cell is optionally selected from a stem cell, such as an embryonic stem cell or a pluripotent stem cell, an induced pluripotent stem cell (iPSC), a cell derived from a stem cell, a cell that is at a differentiated stage, such as a differentiated cell or mature 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, a dopaminergic cell, an exosome cell, such as ESCs or NSCs, or an exosome cell derived from a ESC or NSC, an immune cell, such as a T cell, a NK cell, a macrophage, a dendritic cell, a hepatocyte, a stellate cell, a fibroblast, a keratinocyte or a hair cell, an inner ear cell, an intestinal cell or 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, a mesenchymal stem cell.

In an embodiment, the invention relates to a cell composition comprising a cell and a pharmaceutically acceptable medium, said cell expressing an IAP analogue comprising an extracellular domain and a transmembrane domain, wherein:

1. said extracellular domain is an analogue of the IAP-ECD defined by SEQ ID NO 1 , and a) comprises: i) at least one or two of Y37W and R103L, and optionally further comprises K39V, or ii) at least one, two or three of Y37W, R103L and K39V, b) optionally further comprises one, two, three or four of T49, L3, A30 and/or T102, and

2. said cell is an engineered cell, such as an engineered human cell.

In an embodiment, the invention relates to a pharmaceutical product comprising a cell or comprising a cell composition comprising a cell and a pharmaceutically acceptable medium, said cell expressing an IAP analogue comprising an extracellular domain and a transmembrane domain, wherein said extracellular domain is an analogue of the IAP-ECD defined by SEQ ID NO 1 , and:

1) comprises: a) at least one or two of Y37W and R103L, and optionally further comprises K39V, or b) at least one, two or three of Y37W, R103L and K39V, and

2) optionally further comprises one, two, three or four of T49, L3, A30 and/or T102.

In an embodiment, the invention relates to a cell composition for use in cellular therapy, said composition comprising a cell and a pharmaceutically acceptable medium, said cell expressing an IAP analogue comprising an extracellular domain and a transmembrane domain, 1. wherein said extracellular domain is an analogue of the IAP-ECD defined by SEQ ID NO

1 , and: a) comprises: i) at least one or two of Y37W and R103L, and optionally further comprises K39V, or ii) at least one, two or three of Y37W, R103L and K39V, b) optionally further comprises one, two, three or four of T49, L3, A30 and/or T102, and

2. wherein said cell is an engineered cell, such as an engineered human cell.

In an embodiment, the invention relates to a cell composition for use in the treatment, the cure, or the prevention of a chronic disease or of an acute disease, said composition comprising a cell and a pharmaceutically acceptable medium, said cell expressing an IAP analogue comprising an extracellular domain and a transmembrane domain, wherein:

1. said extracellular domain is an analogue of the IAP-ECD defined by SEQ ID NO 1 , and: a) comprises: i) at least one or two of Y37W and R103L, and optionally further comprises K39V, or ii) at least one, two or three of Y37W, R103L and K39V, b) optionally further comprises T49, L3, A30 and/or T102, and

2. said cell is an engineered cell, such as an engineered human cell, and,

3. said chronic disease is optionally 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, and cancer, and/or said acute disease is optionally selected from bacterial lung infections, such as ventilator acquired bacterial pneumonia and hospital acquired bacterial pneumonia.

Non-limiting embodiments as per the present invention are disclosed herein:

1. An Integrin associated protein (IAP) analogue comprising an extracellular domain, wherein said extracellular domain is an analogue of the Integrin associated protein extracellular domain (IAP-ECD) defined by SEQ ID NO 1 and comprises one or more mutations selected from Y37W, R103L and K39V compared to the sequence SEQ ID NO 1.

2. An IAP analogue according to embodiment 1 further comprising a transmembrane domain. 3. An IAP analogue according to any of embodiments 1 or 2, wherein said analogue of the IAP-ECD comprises at least one of the mutations Y37W and R103L.

4. An IAP analogue according to any of embodiment 3, wherein said analogue of the IAP- ECD further comprises the mutation K39V compared to sequence SEQ ID NO 1.

5. An IAP analogue according to any of embodiments 1 to 4, wherein said analogue of the IAP-ECD comprises one, two, three or four of T49, L3, A30 and T102.

6. An IAP analogue according to any of embodiments 1 to 5, wherein said extracellular domain has at least 90% homology with SEQ ID NO 1.

7. An IAP analogue according to any of embodiments 1 to 6, wherein said extracellular domain has between 90% and 99% homology with SEQ ID NO 1.

8. An IAP analogue according to any of embodiments 1 to 7, wherein said analogue of the IAP-ECD has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97% homology with sequence SEQ ID NO 1.

9. An IAP analogue according to any of embodiments 2 to 8, wherein said transmembrane domain comprises sequence SEQ ID NO 13 or an analogue thereof.

10. An IAP analogue according to any of embodiments 9, wherein said transmembrane domain comprises sequence of at least 90% homology to SEQ ID NO 13.

11. An IAP analogue according to any of embodiments 1 to 10, wherein said IAP analogue further comprises an intracellular domain.

12. An IAP analogue according to embodiments 11 , wherein said intracellular domain comprises sequence SEQ ID NO 15 or an analogue thereof.

13. An IAP analogue according to embodiment 12, wherein said intracellular domain comprises sequence of at least 90% homology to SEQ ID NO 15.

14. An IAP analogue according to embodiment 2 to 13, wherein said IAP analogue further comprises a linker between said ECD and said TMD.

15. An IAP analogue according to embodiment 14, wherein said linker comprises sequence SEQ ID NO 14 or an analogue thereof of at least 90% homology to SEQ ID NO 14.

16. An IAP analogue according to any of embodiments 1 to 15, wherein said extracellular domain comprises a sequence selected from the list consisting of SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11.

17. An IAP analogue according to any of embodiments 1 to 15, wherein said IAP analogue comprises a sequence selected from the list consisting of 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 and SEQ ID NO 25. 18. A cell expressing an IAP analogue of any one of embodiments 1 to 17.

19. A cell comprising a gene sequence encoding an IAP analogue, wherein said IAP analogue is as defined in any one of embodiments 1 to 17.

20. A cell according to embodiments 18 or 19, wherein said cell is B2M-/-.

21. A cell according to embodiment 18 or 19 expressing an IAP analogue, wherein said cell is B2M-/-, wherein said IAP analogue comprises an extracellular domain and a transmembrane domain, said extracellular domain is an analogue of the Integrin associated protein extracellular domain (IAP-ECD) 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(s) selected from Y37W, R103L and K39V compared to the sequence SEQ ID NO 1.

22. A cell according to anyone of embodiments 18 to 21 , wherein said extracellular domain comprises no mutation in position T49, L3, A30 and/or T102.

23. A cell according to anyone of embodiments 18 to 22 wherein said cell is CIITA-/-.

24. A cell according to anyone of embodiments18 to 23 wherein said cell comprises a B2M- HLA fusion protein, such as B2M-HLA-E and/or B2M-HLA-G proteins, on its cell surface and/or said cell comprises a B2M-HLA encoding nucleic acid sequence, such as B2M-HLA-E and/or B2M-HLA-G encoding nucleic acid sequence.

25. A cell according to any of embodiments 18 to 24, wherein said extracellular domain has at least 90% homology with SEQ ID NO 1.

26. A cell according to any of embodiments 18 to 25, wherein said extracellular domain has between 90% and 99% homology with SEQ ID NO 1.

27. A cell according to anyone of embodiments 18 to 26, wherein said extracellular domain comprises an amino acid sequence selected from the group consisting of SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10 and SEQ ID NO 11.

28. A cell according to any of embodiments 18 to 26, wherein said transmembrane domain comprises sequence SEQ ID NO 13 or an analogue thereof of at least 90% homology to SEQ ID NO 13.

29. A cell according to any of embodiments 18 to 28, wherein said IAP analogue comprises a linker between said ECD and said TMD.

30. A cell according to embodiment 29, wherein said linker comprises sequence SEQ ID NO 14 or an analogue thereof of at least 90% homology to SEQ ID NO 14.

31. A cell according to any of embodiments 18 to 30, wherein said IAP analogue comprises an intracellular domain. 32. A cell according to embodiment 30, wherein said intracellular domain comprises sequence SEQ ID NO 15 or an analogue thereof of at least 90% homology to SEQ ID NO 15.

33. A cell according to any of embodiments 18 to 32, wherein the cell further comprises increased expression of at least one of CD47, HLA-G, HLA-E, HLA-F, PD-L1 , CD55 proteins.

34. A cell according to anyone of embodiments 18 to 33, wherein said cell comprises a PD-L1 encoding nucleic acid sequence and/or a PD-L1 protein on its cell surface.

35. A cell according to anyone of embodiments 18 to 34, wherein said cell comprises a CD55 encoding nucleic acid sequence and/or a CD55 protein on its cell surface.

36. A cell according to anyone of embodiments 18 to 35, wherein said cell comprises a PD-L1 and CD55 encoding nucleic acid sequence and/or a PD-L1 and CD55 protein on its cell surface.

37. A cell according to anyone of embodiments 18 to 36, wherein said cell is B2M-/-, CIITA-/- and further comprises a B2M-HLA-E and/or B2M-HLA-G encoding nucleic acid sequence and/or a B2M-HLA-E and/or B2M-HLA-G protein on its cell surface.

38. A cell according to anyone of embodiments 18 to 37, wherein said cell is B2M-/-, CIITA-/- and further comprises a PD-L1 and/or CD55 encoding nucleic acid sequence and/or a PD-L1 and/or CD55 protein on its cell surface.

39. A cell according to anyone of embodiments 18 to 38, wherein said cell is a stem cell, such as an embryonic stem cell, a pluripotent stem cell or an induced pluripotent stem cell (iPSC).

40. A cell according to anyone of embodiments 18 to 38, wherein said cell is a cell derived from a stem cell, such as an embryonic stem cell, a pluripotent stem cell or an induced pluripotent stem cell (iPSC).

41. A cell according to anyone of embodiments 18 to 40, wherein said cell is a mammalian cell, such as a mammalian cell obtained from a donor or derived from a cell obtained from a donor.

42. A cell according to anyone of embodiments 18 to 40, wherein said cell is a human cell.

43. A cell of any of embodiments 18 to 39 or 40 to 42, 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, a dopaminergic cell, an exosome cell, such as ESCs or NSCs, or an exosome cell derived from a ESC or NSC, an immune cell, such as a T cell, a NK cell, a macrophage, a dendritic cell, a hepatocyte, a stellate cell, a fibroblast, a keratinocyte or a hair cell, an inner ear cell, an intestinal cell or 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, a mesenchymal stem cell.

44. A composition comprising cells of any of embodiments 18 to 43 in a pharmaceutically acceptable medium.

45. A cell of any of embodiments 18 to 43 for use as a medicament.

46. A cell of any of embodiments 18 to 43 for use in cell therapy.

47. A cell of any of embodiments 18 to 43 for use in the treatment, the cure, or the prevention of a chronic disease or of an acute disease.

48. A cell of embodiment 47 for use in the treatment, the cure, or the prevention of a chronic disease 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, and cancer.

49. A cell of embodiment 47, for use in the treatment, the cure, or the prevention of an acute disease selected from lung infections, such as ventilator acquired bacterial pneumonia and hospital acquired bacterial pneumonia.

50. A composition of embodiment 44 for use as a medicament.

51. A cell composition of embodiment 44 for use in cell therapy.

52. A cell composition of embodiment 50 or 51 for use in the treatment, the cure, or the prevention of a chronic disease or of an acute disease.

53. A cell composition of embodiment 52 for use in the treatment, the cure, or the prevention of a chronic disease 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, and cancer.

54. A cell composition of embodiment 52, for use in the treatment, the cure, or the prevention of an acute disease selected from lung infections, such as ventilator acquired bacterial pneumonia and hospital acquired bacterial pneumonia.

55. A human cell for use in cellular therapy for the treatment, the cure, or the prevention of a chronic disease 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, and cancer, or for the treatment, the cure, or the prevention of an acute disease selected from lung infections, such as ventilator acquired bacterial pneumonia and hospital acquired bacterial pneumonia, said cell being B2M-/- and CIITA-/-, said cell and further comprising a B2M-HLA-E and/or B2M-HLA-G encoding nucleic acid sequence and/or a B2M-HLA-E and/or B2M-HLA-G protein on its cell surface, said cell expressing an IAP analogue and/or comprising a gene sequence encoding an IAP analogue, wherein said IAP analogue comprises an extracellular domain which comprises in its amino acid sequence one or more mutation(s) selected from Y37W, R103L, K39V compared to the sequence SEQ ID NO 1.

Examples

Example 1 : Wild-type IAP sequences

SEQ ID NO 1 is the sequence of wild type IAP-ECD: QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTD FSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRV

SEQ ID NO 12 is the sequence of full-length wild type IAP: QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTD FSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENI LIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGL GLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILAL AQLLGLVYMKFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE

SEQ ID NO 13 is the sequence of wild type IAP TMD:

N I LI VI FPI FAI LLFWGQFGI KTLKYRSGGM DEKTI ALLVAGLVITVI VI VGAI LFVPGEYSLKNATG LGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILA LAQLLGLVYM

SEQ ID NO 14 is the sequence of wild type IAP linker between wild type IAP TMD and wild type IAP ECD:

VSWFSPNE

SEQ ID NO 15 is the sequence of wild type IAP ICD:

KFVASNQKTIQPPRKAVEEPLNAFKESKGMMNDE Example 2: Analogue IAP-ECD sequences

Ten IAP-ECD sequences with improved binding affinity towards SIRPa are reported below as

SEQ ID NO 2 to 11 :

• SEQ ID NO 2:

RLVFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVWVVWKFKGRDIYTFDGALNKSTVPT

DFSSAKI EVSQLLKGDASLKM DKSDAVSHTGNYTCEVTELTLEGETI I ELKYRV

• SEQ ID NO 3:

RLLFNKTKSVEFTFCNDTVVIPCFVTNMESQNTTEVWVVWKFKGRDIYTFDGALNKSTVPTD

FSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELSLEGETIIELKYRV

• SEQ ID NO 4:

KLIFNKTKSVEFTFCNDTVVIPCFVTNMESQNTTEVWVVWKFKGRDIYTFDGALNKSTVPTD

FSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTLEGETIIELKYRV

• SEQ ID NO 5:

RLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVWVKWKFKGRDIYTFDGALNKSTVPTD

FSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELSLEGETIIELKYRV

• SEQ ID NO 6:

ALLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVWVKWKFKGRDIYSFDGALNKSTVPTD

FSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTLEGETIIELKYRV

• SEQ ID NO 7:

ALLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVWVVWKFKGRDIYTFDGALNKSTVPTD

FSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTLEGETIIELKYRV

• SEQ ID NO 8:

RLLFNKTKSVEFTFCNDTVVIPCFVTNMESQNTTEVWVKWKFKGRDIYTFDGALNKSTVPTD

FSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELSLEGETIIELKYRV

• SEQ ID NO 9:

RLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVWVVWKFKGRDIYTFDGALNKSTVPTD

FSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELSLEGETIIELKYRV

• SEQ ID NO 10:

KLIFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVWVVWKFKGRDIYTFDGALNKSTVPTD

FSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTLEGETIIELKYRV

• SEQ ID NO 11:

ALLFNKTKSVEFTFCNDTVVIPCFVTNMESQNTTEVWVVWKFKGRDIYTFDGALNKSTVPTD

FSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTLEGETIIELKYRV The common mutation profile amongst these 10 sequences compared to wild-type IAP-ECD is Y37W and R103L. Another beneficial mutation is K39V. It is also beneficial to have no mutation (/.e. same as wild-type) in position T49 and to have no mutations in positions L3, A30 and T102.

Example 3: IAP expression

Cells expressing wild-type IAP of sequence SEQ ID NO 12 or expressing an IAP analogue as listed in Table 1 have been transiently expressed on HEK293 cells. Full-length IAP analogues of sequences SEQ ID NO 16 to 25 respectively comprise an ECD of sequences SEQ ID NO 2 to 11 as listed in Example 2.

Table 1 :

The nucleic acid sequences encoding IAP analogues of Table 1 are respectively listed in Table 2. The nucleic acid sequences provided in Table 2 comprise a start codon, a signal peptide sequence, a full-length IAP sequence, and a stop codon. They have been synthesized by Twist Bioscience® and cloned into the commercial cloning vector pcDNA 3.1. Table 2:

Example 4: Production of lAPs and cells

Ten IAP analogues identified in Table 1 (comprising ECD identified in Example 2 respectively) are expressed in HEK-293T cell as model cell line. 3 ml of HEK293 cells with cell density of 1 million cells/ml was mixed with transfection mixture comprising of 3pg of synthesized nucleic acid (DNA) and 8.1 pl of transfection reagents (expifectamine, Invitrogen, A14524) for 10 minutes. Cells viability upon transfection has been determined by Vi-CELL XR Cell Viability Analyzer (Beckman®). The results are summarized in Table 3. The figures reported in the “Cell Viability” column are provided by Beckman apparatus and indicate the count of living cells after transfection expressed as percentage compared to the count of living cells before transfection.

Table 3:

Example 5. IAP protein expression and binding to SIRP-a

Flow cytometry based screening has been used to determine the expression yield of IAP and the binding ability to SIRP-a on the cell surface of transiently transfected cells. For each nucleic acid of Table 2, 200,000 transfected cells were stained with primary probe containing IAP-APC antibody (eBiosciences®, Cat# 17-0479-42 with 50-fold dilution) for 30 min at 4°C, washed three times with Phosphate-buffered saline containing — 2 % BSA (PBS) buffer at 4 C°. The cells were then exposed to secondary labelling with phycoerythrin (PE)- conjugated Human SIRPa /CD172a Protein, Fc Tag (Aero Biosystems®, Cat# SIA-HP252 with 100-fold dilution). Labelled cells were pelleted by centrifugation at 300g and 4°C for 5 min and resuspended in 500 pL of PBS. Quantification of IAP protein expression on the cells surface was performed by acquisition on a Becton Dickinson FACSymphony flow cytometer. Cells were gated for live (DRAQ7 low/negative, DRAQ7 is the dye used to label dead cells) and singlet cells, followed by analysis of cell surface protein expression determined by APC signal (emission wavelength at 660 nm) and binding to SIRP-a monitored by PE signal. The results of the cell surface protein expression measurement are shown in Fig. 2(a) to (c) and the results of the binding to SIRP- a measurement are shown in Fig. 3(a) to (c), where “Mock” relates to cells transfected with pcDNA plasmid only, carrying no IAP encoding sequence (negative control).

Fig. 2(a) to (c) show a fluorescence increase on the cell surface in the cell transfected with an IAP protein (peak 2 and 3) compared to the mock cell (peak 1). Fig. 3(a) to (c) indicates intensity of the fluorescence signals, thereby indicates the relative expression level of the protein on the cell surface. In the graphs in Fig.3, a fluorescence signal more to the right of the graph indicates a higher binding to SIRP- a compared to a signal more the left.

Claims

1. An Integrin associated protein (IAP) analogue comprising an extracellular domain, wherein said extracellular domain is an analogue of the Integrin associated protein extracellular domain (IAP-ECD) defined by SEQ ID NO 1 and comprises one, two or three mutations selected from Y37W, R103L and K39V.

2. An IAP analogue according to claim 1, further comprising a transmembrane domain.

3. An IAP analogue according to claim 1 or 2, wherein said analogue of the IAP-ECD comprises at least one of the mutations Y37W and R103L and, optionally, further comprises the mutation K39V.

4. An IAP analogue according to claim 3, wherein said analogue of the IAP-ECD comprises at least one of the mutations Y37W and R103L and further comprises the mutation K39V.

5. An IAP analogue according to any of claims 1 to 4, wherein said analogue of the IAP- ECD comprises one, two, three or four of T49, L3, A30 and T102.

6. An IAP analogue according to any of claims 1 to 5, wherein said analogue of the IAP- ECD has at least 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97% homology with sequence SEQ ID NO 1.

7. A cell expressing an IAP analogue of any one of claims 1 to 6.

8. A cell according to claim 7 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.

9. A cell according to claim 7 wherein said cell is a mammalian cell obtained from a donor or derived from a cell obtained from a donor.

10. A cell according to any of claims 7 to 9, wherein said cell is B2M-/-. A cell according to any of claims 7 to 10, wherein said cell is CIITA-/-. A cell according to any of claims 7 to 11 , wherein said cell further expresses a B2M- HLA fusion protein, such as B2M-HLA-E and/or B2M-HLA-G genetic fusion protein. A cell composition comprising cells according to any one of claims 7 to 12 in a pharmaceutically acceptable medium. A cell according to any one of claims 7 to 12 or a cell composition according to claim 13 for use in cell therapy. A cell or a cell composition according to claim 14 for use in the treatment, the cure, or the prevention of a chronic disease or of an acute disease.