WO2023110824A1 - Nouvelle protéine associée à l'intégrine (iap) - Google Patents

Nouvelle protéine associée à l'intégrine (iap) Download PDF

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Publication number
WO2023110824A1
WO2023110824A1 PCT/EP2022/085538 EP2022085538W WO2023110824A1 WO 2023110824 A1 WO2023110824 A1 WO 2023110824A1 EP 2022085538 W EP2022085538 W EP 2022085538W WO 2023110824 A1 WO2023110824 A1 WO 2023110824A1
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cell
iap
analogue
seq
extracellular domain
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PCT/EP2022/085538
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English (en)
Inventor
Che YANG
Gerd Schluckebier
Salka Elbøl RASMUSSEN
Nicolaj Strøyer CHRISTOPHERSEN
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Novo Nordisk A/S
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Priority to TW111148252A priority Critical patent/TW202325721A/zh
Publication of WO2023110824A1 publication Critical patent/WO2023110824A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention is 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.
  • 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.
  • a problem to be solved is to improve the success of cell therapy.
  • the invention provides novel Integrin associated protein(s) (IAP(s)).
  • IAP novel Integrin associated protein
  • the invention provides cells expressing said novel Integrin associated protein (IAP), compositions and pharmaceutical products comprising such cells and therapeutical uses thereof.
  • the invention relates to an Integrin associated protein (IAP) analogue comprising an extracellular domain and a transmembrane domain, wherein 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 said extracellular domain comprises one or more mutation selected from S64A, S64T, S64G, D62A, E69A, T61 D, S85R, S89A and S89D compared to the sequence SEQ ID NO 1.
  • IAP Integrin associated protein
  • IAP-ECD Integrin associated protein extracellular domain
  • the IAP analogue of the invention may further have at least 90% homology with SEQ ID NO 1; comprise a mutation selected from S64A, S64T or S64G compared to the sequence SEQ ID NO 1 ; comprise a mutation selected from S64T or S64G compared to the sequence SEQ ID NO 1 ; comprise one or both of the mutations D62A and E69A compared to the sequence SEQ ID NO 1 ; and/or comprise one or more of the mutations T61 D, S85R, S89A and S89D compared to the sequence SEQ ID NO 1.
  • the invention relates to IAP analogue(s) comprising an extracellular domain and a transmembrane domain, wherein said extracellular domain comprises an amino acid sequence of at least 90% homology with SEQ ID NO 1 and comprising the mutation S64A, S64T or S64G compared to the sequence SEQ ID NO 1.
  • said amino acid sequence comprises the mutation S64T or S64G compared to the sequence SEQ ID NO 1.
  • said amino acid sequence further comprises the mutation(s) D62A and/or E69A compared to the sequence SEQ ID NO 1.
  • said amino acid sequence further comprises one or more of the mutations T61 D, S85R, S89A and S89D compared to the sequence SEQ ID NO 1.
  • the invention relates to a cell, or cells, expressing the IAP analogue of the invention.
  • the invention relates to a cell, or cells, expressing an IAP analogue comprising an extracellular domain and a transmembrane domain, wherein said extracellular domain comprises an amino acid sequence of at least 90% homology with SEQ ID NO 1 , and comprising at least one of the following mutations: S64A, S64T or S64G, and/or D62A, E69A, T61 D, S85R, S89A, and/or S89D compared to SEQ ID NO 1.
  • Said cell(s) may be 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.
  • Said cell(s) may be a mammalian cell obtained from a donor or derived from a cell obtained from a donor.
  • the invention in another aspect, relates to a composition
  • a composition comprising a cell or cells as defined therein in a pharmaceutically acceptable medium.
  • the invention in another aspect, relates to a pharmaceutical product comprising a cell or cells as defined therein or a cell composition as defined therein.
  • the invention relates to a pharmaceutical product as defined therein for use in cell therapy.
  • 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.
  • novel lAPs, cells, compositions, and pharmaceutical products of the invention as defined herein improve the success of cellular therapy.
  • the invention provides improved cells survival upon cellular transplantation, 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.
  • NO nitric oxide
  • the invention may also solve further problems that will be apparent from the disclosure of the exemplary embodiments.
  • a cell “expressing” or that “expresses” a given protein means that the cell comprises the genetic material and transcription and/or translation machinery which together allow for and/or do produce said given protein.
  • stem cell is to be understood as an undifferentiated cell having differentiation potency and proliferative capacity, particularly self-renewal competence, but maintaining differentiation potency.
  • stem cell includes subpopulations such as pluripotent stem cell (PSC), multipotent stem cell, unipotent stem cell and the like according to the differentiation potency.
  • PSC pluripotent stem cell
  • multipotent stem cell multipotent stem cell
  • unipotent 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:
  • 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 also known as iPS cells or iPSCs
  • iPS cells iPSCs
  • reprogramming By the introduction of products of specific sets of pluripotency-associated genes adult cells can be converted into PSCs
  • 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 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.
  • the term “endocrine progenitor cell” refers to a cell characterised by expression of markers NGN3, NeuroD and NKX2.2.
  • the term “NGN3+/NKX2.2+ double positive cell” refers to a cell that co-express the two markers NGN3 and NKX2.2.
  • 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
  • NKX2.2” and “NKX6.1” refer to members of the NKX transcription factor family.
  • INS+ is a cell that produces insulin.
  • 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.
  • tissue specific cell e.g. a cardiomyocyte.
  • differentiated and undifferentiated refer to the stage of differentiation of a cell in the cellular differentiation process.
  • 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.
  • protein and “peptide” refer to a functional version thereof.
  • 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.
  • wild type or its abbreviation “wt”, as used herein 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 thereof.
  • variant as used herein 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 replacement of one or more parts of the sequence compared to the sequence which prevails among individuals in natural conditions.
  • variant and “analogue” are interchangeable with each other.
  • percent homology refers to the percentage of sequence identity between two sequences, such as nucleotide or amino acid sequences.
  • 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.
  • Extra-cellular domain (ECD):
  • ECD refers 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.
  • the ECD may also be called ectodomain and is usually involved in contact with surfaces thereby leading to signal transduction.
  • Transmembrane domain TM or TMD:
  • TMD refers to a membrane-spanning region of a protein. It 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 TM and multi-pass TM respectively.
  • Fig. 1 shows a three-dimensional representation of an IAP-ECD (IAP extracellular domain) of sequence SEQ ID NO 1.
  • Fig. 2 shows the FACS results for IAP (wild type or analogue) cell surface expression on the cells as described in Example 3, in a graph format (Fig. 2(a)) and in a numerical format (Fig. 2(b)).
  • Fig.3 shows the FACS results for SIRP-alpha binding to cells’ surface as described in Example 3, in a graph format (Fig. 3(a)) and in a numerical format (Fig. 3(b)).
  • 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 improve cells resistance to ischemia, to increase the chance of surviving ischemic environments for example for example in the context of cell transplantation, of cells in poorly vascularised tissue or of cells in encapsulation devices.
  • donor cells Upon grafting, donor cells are often exposed to an ischemic environment and, subsequently, die due to lack of vascularisation.
  • IAP Integrin Associated Protein
  • Fig. 1 is a three dimensions 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 amino acid S64 is herein proposed as a key binding site of TSP-1.
  • mutations of residues located close to S64 are also proposed such as T61, S89, S85 and nearby negatively charged residues, D62 and E69 to reduce the surface charge patch that may be involved in electrostatical interaction with TSP-1.
  • the present invention provides a novel IAP with reduced thrombospondin binding, reduced risk of suppressed angiogenesis, and/or reduced nitric oxide (NO)-stimulated responses in cells such as endothelial and vascular smooth muscle cells.
  • the invention provides cells expressing IPA analogues with said beneficial mutations or comprising genes encoding such IAP, as well as compositions of such cells.
  • the cells and cell compositions are for use as a medicament, for use in cell therapy, and/or for use in the treatment of chronic diseases such as 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 in treatment acute diseases such as lung infections, such as ventilator acquired bacterial pneumonia and hospital acquired bacterial pneumonia.
  • chronic diseases such as 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
  • treatment acute diseases such as lung infections, such as ventilator
  • the mutations in the sequence of IAP modulate the expression of IAP protein in the cell, reduce IAP interaction with thrombospondin-1 (TSP-1) and limit the risk of TSP-1 related adverse effects.
  • the new IAP analogue(s) have reduced or inhibited binding ability to TSP-1 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.
  • Integrin associated protein is a transmembrane protein.
  • 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.
  • ECD extracellular domain
  • TMD transmembrane domain
  • 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).
  • 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. Overexpression of IAP is well known in cancer cells to avoid phagocytosis. This is due to engagement of the SIRP-a of macrophages and other immune cells by IAP. Engagement of SIRP-a leads to inhibition of phagocytosis.
  • NO nitric oxide
  • TSP-1 can displace the SIRP-a binding to CD47 wild type ectodomain. Displacement of SIRP-a would be detrimental to the inhibition of phagocytosis which in turn might lead to the loss of transplanted cells.
  • the proposed modifications advantageously reduce the risk of TSP-1 related adverse effects in the context of cellular transplantation as used in cellular therapy while preventing phagocytosis of transplanted cells by immune cells, said phagocytosis being inhibited by engagement of IAP extracellular domain with SIRP-a from macrophages and other immune cells,.
  • cells expressing IAP analogue will have the ability to maintain inhibition of phagocytosis by SIRP-a engagement without the risk of TSP-1 binding compromising the survival of a transplanted cell product.
  • the cells of the present invention have better chance of surviving ischemic environments, and immune related reactions e.g., in relation to cell transplantation and/or in poorly vascularised tissue or encapsulation devices.
  • the invention provides an Integrin associated protein (IAP) analogue comprising an extracellular domain and a transmembrane domain, wherein 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 selected from S64A, S64T, S64G, D62A, E69A, T61D, S85R, S89A and S89D compared to the sequence SEQ ID NO 1.
  • IAP Integrin associated protein
  • IAP-ECD Integrin associated protein extracellular domain
  • the ECD of the IAP analogue of the invention has at least 90% homology with SEQ ID NO 1. In a particular embodiment, the ECD of the IAP analogue of the invention has between 90% and 99% homology with SEQ ID NO 1.
  • the ECD of the IAP analogue 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 IAP analogue of the invention comprises a mutation selected from S64T or S64G compared to the sequence SEQ ID NO 1.
  • the ECD of the IAP analogue of the invention comprises one or both of the mutations D62A and E69A compared to the sequence SEQ ID NO 1.
  • the ECD of the IAP analogue of the invention comprises one or more of the mutations T61 D, S85R, S89A and S89D compared to sequence SEQ ID NO 1.
  • the ECD of the IAP analogue of the invention comprises a mutation selected from S64A, S64T or S64G compared to the sequence SEQ ID NO 1 , and further comprises one or more of the mutations D62A, E69A, T61 D, S85R, S89A and S89D compared to the sequence SEQ ID NO 1.
  • the invention provides Integrin Associated Protein (IAP) analogue(s) comprising an extracellular domain and a transmembrane domain, wherein said extracellular domain comprises an amino acid sequence of sequence SEQ ID NO 1 and at least one of the following mutations:
  • IAP Integrin Associated Protein
  • the invention provides IAP analogue(s) comprising an extracellular domain and a transmembrane domain, wherein said extracellular domain comprises an amino acid sequence, of sequence SEQ ID NO 1 and at least one of the following mutations:
  • the invention provides an Integrin Associated Protein (IAP) analogue comprising an extracellular domain and a transmembrane domain, wherein said extracellular domain comprises an amino acid sequence having at least 90% homology with SEQ ID NO 1, and comprising at least one of the following mutations:
  • IAP Integrin Associated Protein
  • the invention provides an Integrin Associated Protein (IAP) analogue comprising an extracellular domain and a transmembrane domain, wherein said extracellular domain comprises an amino acid sequence having at least 90% homology with SEQ ID NO 1, and comprising at least one of the following mutations:
  • IAP Integrin Associated Protein
  • the invention provides an Integrin Associated Protein (IAP) analogue comprising an extracellular domain and a transmembrane domain, wherein said extracellular domain comprises an amino acid sequence having at least 90% homology with SEQ ID NO 1, comprising at least one of the following mutations: • S64A, S64T or S64G, and further comprising at least one of the following mutations:
  • IAP Integrin Associated Protein
  • SEQ ID NO 1 is the sequence of wild-type IAP-ECD (IAP extracellular domain) sequence.
  • the IAP(s) of the present invention is(are) a variant(s) IAP, also called IAP analogue herein.
  • the IAP analogue(s) of the present invention has (have) decreased binding affinity to TSP-1 compared to wild-type IAP of sequence SEQ ID NO 1.
  • the IAP analogue(s) of the present invention has(have) increased binding affinity to SIRP-alpha compared to wild-type IAP of SEQ ID NO 1.
  • the IAP analogue (s) of the invention comprise an IAP-ECD amino acid sequence of at least 90% homology with SEQ ID NO 1 , wherein said amino acid sequence comprises the mutation S64A, S64T or S64G compared to SEQ ID NO 1 , or comprise the mutation S64T or S64G compared to SEQ ID NO 1.
  • the IAP analogue (s) of the invention comprise an IAP-ECD amino acid sequence of at least 90% homology with SEQ ID NO 1 , wherein said amino acid sequence comprises the mutation S64A, S64T or S64G compared to SEQ ID NO 1 , wherein said amino acid sequence further comprises the mutation S64T or S64G compared to SEQ ID NO 1.
  • the Integrin Associated Protein(s) analogue(s) of the invention comprise an IAP-ECD amino acid sequence of at least 90% homology with SEQ ID NO 1, wherein said amino acid sequence comprises the mutation(s) D62A and/or E69A compared to SEQ ID NO 1.
  • the IAP analogue(s) of the invention comprise an IAP-ECD amino acid sequence of at least 90% homology with SEQ ID NO 1 , wherein said amino acid sequence comprises one or more of the mutations T61D, S85R, S89A and S89D compared to SEQ ID NO 1.
  • the IAP analogue(s) of the invention comprise an IAP-ECD amino acid sequence of at least 90% homology with SEQ ID NO 1 , wherein said amino acid sequence comprises the mutation S64A, S64T or S64G and further comprises the mutation(s) D62A and/or E69A compared to SEQ ID NO 1.
  • the IAP analogue(s) of the invention comprise an IAP-ECD amino acid sequence of at least 90% homology with SEQ ID NO 1 , wherein said amino acid sequence comprises the mutation S64A, S64T or S64G and further comprises one or more of the mutations T61D, S85R, S89A and S89D compared to SEQ ID NO 1.
  • the IAP analogue(s) of the invention comprise an IAP-ECD amino acid sequence of at least 90% homology with SEQ ID NO 1 , wherein said amino acid sequence comprises the mutation S64A, S64T or S64G, further comprises the mutation(s) D62A and/or E69A, and further comprises one or more of the mutations T61 D, S85R, S89A and S89D compared to SEQ ID NO 1.
  • the invention relates to a cell expressing an IAP of the present invention as defined herein.
  • the cell of the invention expresses an IAP analogue comprising an IAP-ECD amino acid sequence of at least 90% homology with SEQ ID NO 1, and comprising at least one of the following mutations:
  • the cell of the invention comprises a gene sequence encoding an IAP analogue as per the present invention.
  • 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 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 selected from S64A, S64T, S64G, D62A, E69A, T61 D, S85R, S89A and S89D compared to the sequence SEQ ID NO 1.
  • said IAP-ECD has at least 90% homology with SEQ ID NO 1.
  • said IAP-ECD has between 90% and 99% homology with SEQ ID NO 1.
  • said IAP-ECD comprises a mutation selected from S64A, S64T or S64G compared to the sequence SEQ ID NO 1. In a particular embodiment, in the cell of the invention, said IAP-ECD comprises a mutation selected from S64T or S64G compared to the sequence SEQ ID NO 1. In an embodiment, in the cell of the invention, said IAP-ECD comprises one or both of mutations D62A and E69A compared to the sequence SEQ ID NO 1.
  • said IAP-ECD comprises one or more of the mutations T61 D, S85R, S89A and S89D compared to the sequence SEQ ID NO 1.
  • said IAP-ECD comprises a mutation selected from S64A, S64T or S64G compared to the sequence SEQ ID NO 1, and further comprises one or more of the mutations D62A, E69A, T61D, S85R, S89A and S89D compared to the sequence SEQ ID NO 1.
  • said IAP-ECD comprises one of the below listed groups of mutations compared to SEQ ID NO 1 :
  • T61D, D62A, S64G and S89A 131.
  • said IAP-ECD comprises an amino acid sequence selected from 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.
  • the invention provides a cell expressing an IAP analogue of the present invention as defined herein and expressing no IAP comprising SEQ ID NO 1.
  • the invention provides a cell expressing an IAP analogue of the present invention as defined herein for use as a medicament.
  • the invention provides a cell expressing an IAP analogue of the present invention as defined herein for use in cell therapy.
  • the invention provides a cell expressing an IAP analogue of the present invention as defined herein for use in the treatment, the cure, or the prevention of a chronic disease or of an acute disease.
  • this 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.
  • the acute disease comprises bacterial lung infections, such as ventilator acquired bacterial pneumonia and hospital acquired bacterial pneumonia.
  • the cell of the invention is a mammalian cell. In an embodiment, it is human cell.
  • the cell of the invention is a stem cell.
  • said stem cell is an embryonic stem cell or a pluripotent stem cell.
  • said stem cell is an induced pluripotent stem cell (iPSC).
  • the cell of the invention is derived from a stem cell.
  • the cell of the invention is at a differentiated stage, such as a differentiated cell or mature cell.
  • 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 T cell, a
  • the cell of the invention is obtained from a donor or is derived from a cell 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.
  • the invention provides a composition comprising a cell of the present invention as defined herein.
  • the cell of the invention as defined herein may be obtained by any conventional gene modification method.
  • the invention provides a method of modifying a cell comprising the following steps:
  • the invention in another aspect, relates to a modified cell expressing an IAP analogue of the present invention as defined herein wherein said cell modification results in increased IAP expression compared to prior to said cell modification.
  • the invention in another aspect, relates to a pharmaceutical product comprising a cell of the invention as defined herein.
  • the invention in another aspect, relates to a cell composition
  • a cell composition comprising cells of the invention as defined herein in a pharmaceutically acceptable medium.
  • the invention relates to a pharmaceutical product comprising a cell that expresses an IAP analogue comprising an IAP-ECD amino acid sequence of at least 90% homology with SEQ ID NO 1 , and comprising at least one of the following mutations:
  • the invention in another aspect, relates to a pharmaceutical product comprising a cell of the invention as defined herein, or a cell composition as defined herein, for use in cell therapy.
  • the invention in another aspect, relates to a cell therapy product comprising a cell of the invention as defined herein or a cell composition as defined herein.
  • the invention relates to a pharmaceutical product comprising a cell of the invention as defined herein, or a cell composition as defined herein, for use in cell therapy for the treatment, the cure, or the prevention of a chronic disease or of an acute disease.
  • the invention relates to a pharmaceutical product comprising a cell of the invention as defined herein or a cell composition as defined herein, for use in the treatment, the cure, or the prevention of a chronic disease or of an acute disease.
  • the pharmaceutical product comprises cells of the invention obtained from a donor or derived from cells obtained from a donor for use in allogenic cell therapy.
  • the invention relates to a pharmaceutical product comprising a cell that expresses an IAP analogue comprising an IAP-ECD amino acid sequence of at least 90% homology with SEQ ID NO 1 , and comprising at least one of the following mutations:
  • the invention relates to an IAP analogue as defined herein, wherein said IAP presents a decreased binding affinity to TSP-1 compared to a wild-type IAP comprising sequence SEQ ID NO 1.
  • the invention relates to an IAP analogue as defined herein, wherein said IAP presents an increased binding affinity to SIRP-alpha compared to a wildtype IAP comprising sequence SEQ ID NO 1.
  • the invention relates to a method of treatment, cure or prevention, wherein patients in need thereof are administered a cell, a composition or a pharmaceutical product of the invention as defined herein.
  • said method is a method of treatment, cure or prevention by cell therapy.
  • said method is a method of treatment, cure or prevention of a chronic disease or of an acute disease.
  • IAP analogues as disclosed herein have been prepared. More specifically, HEK293 cells have been prepared to express IAP proteins as listed in Table 1. For each IAP protein of Table 1 , cells have been transiently transfected with a nucleic acid, as listed in Table 2 encoding said IAP protein. The associated nucleic acid sequences listed in Table 2 were designed and synthesized by Twist Bioscience® and cloned into the commercial cloning vector pcDNA 3.1.
  • Table 1 Table 2 - Nucleic acid sequences cloned into pcDNA 3.1 vector
  • 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.
  • DRAQ7 low/negative, DRAQ7 is the dye used to label dead cells
  • APC signal emission wavelength at 660 nm
  • SIRP- a binding to SIRP- a by analyzing the PE signal (wavelength at 561 nm).
  • Fig. 2and Fig. 3 where “Unstained” relates to unlabelled cells transfected with CD47 (negative control), “Mockrelates to cells transfected with pcDNA plasmid only, carrying no CD47 encoding sequence (negative control).
  • Fig. 2(a) shows a fluorescence increase in rows 4-6 (cells transfected with pcDNA carrying CD47 encoding sequences) compared to rows 2 and 3 (mock cell) which indicates an increase in IAP protein expression on the cell surface.
  • Fig. 2(b) indicates in the right column the median intensity of the fluorescence signals, thereby indicates the relative expression level of the protein on the cell surface.
  • Fig. 3(a) shows a fluorescence increase in rows 3-10 compared to rows 1 and 2 which indicates an increase in SIRP-a binding on cells comprising CD47 encoding sequences.
  • Fig. 3(b) indicates in the right column the median intensity of the fluorescence signals, thereby indicates the relative binding strength of the CD47 variants to SIRP-a.
  • SEQ ID NO 2 is the sequence of full-length wild type IAP:
  • SEQ ID NO 3 is the sequence of a IAP-ECD variant comprising S64A mutation:
  • SEQ ID NO 4 is the sequence of a IAP-ECD variant comprising S64T mutation:
  • SEQ ID NO 5 is the sequence of a IAP-ECD variant comprising S64G mutation:
  • SEQ ID NO 6 is the sequence of a IAP-ECD variant comprising D62A mutation:
  • SEQ ID NO 8 is the sequence of a IAP-ECD variant comprising T61 D mutation: QLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPDD FSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRV
  • SEQ ID NO 10 is the sequence of a IAP-ECD variant comprising S89A mutation: QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTD FSSAKIEVSQLLKGDASLKMDKSDAVAHTGNYTCEVTELTREGETIIELKYRV
  • SEQ ID NO 11 is the sequence of a IAP-ECD variant comprising S89D mutation: QLLFNKTKSVEFTFCNDTWIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTD FSSAKIEVSQLLKGDASLKMDKSDAVDHTGNYTCEVTELTREGETIIELKYRV.
  • SEQ ID NO 14 is a nucleotide sequence encoding full-length wild type IAP: see Table 2
  • SEQ ID NO 16 is a nucleotide sequence encoding full-length IAP variant comprising S64T mutation in IAP-ECD: see in Table 2
  • An IAP analogue of embodiment 1 wherein said extracellular domain has at least 90% homology with SEQ ID NO 1 , such as between 90% and 99% homology.
  • a cell comprising a gene sequence encoding an IAP analogue, wherein said IAP analogue is as defined in embodiment 7.
  • a cell of any of embodiments 7 to 12, wherein said extracellular domain comprises a mutation selected from S64T or S64G compared to the sequence SEQ ID NO 1.
  • a cell of embodiment 13, wherein said extracellular domain further comprises one or both of mutations D62A and E69A compared to SEQ ID NO 1.
  • a cell of embodiment 15, wherein said extracellular domain further comprises one or more of the mutations T61 D, S85R, S89A and S89D compared to SEQ ID NO 1.
  • a cell of embodiment 14, wherein said extracellular domain further comprises one or more of the mutations T61 D, S85R, S89A and S89D compared to SEQ ID NO 1.
  • a cell of embodiment 19, wherein said extracellular domain further comprises one or more of the mutations T61 D, S85R, S89A and S89D compared to SEQ ID NO 1.
  • a cell of embodiment 14, wherein said extracellular domain further comprises one or more mutation selected from D62A, E69A, T61 D, S85R, S89A and S89D compared to SEQ ID NO 1.
  • 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.
  • composition comprising cells of any of embodiments 7 to 25 in a pharmaceutically acceptable medium.
  • a cell of any of embodiments 7 to 25 for use as a medicament for use as a medicament.
  • 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.
  • composition of embodiment 26 for use as a medicament is a composition of embodiment 26 for use as a medicament.
  • a cell of embodiment 34 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.
  • a cell of embodiment 35 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.
  • Integrin associated protein comprising an extracellular domain and a transmembrane domain, wherein said extracellular domain comprises an amino acid sequence having at least 90% homology with SEQ ID NO 1 and comprises the mutation S64A, S64T or S64G compared to the sequence SEQ ID NO 1.
  • amino acid sequence further comprises the mutation(s) D62A and/or E69A compared to the sequence SEQ ID NO 1.
  • 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.
  • a cell according to embodiment 41 or 42 wherein said cell is a mammalian cell obtained from a donor or derived from a cell obtained from a donor.
  • composition comprising cells according to any one of embodiments 41 to 46 in a pharmaceutically acceptable medium.
  • a human cell for use in cellular therapy said cell expressing an IAP analogue and/or comprising a gene sequence encoding an IAP analogue, wherein said IAP analogue comprises an extracellular domain and a transmembrane domain, said extracellular domain comprising an amino acid sequence selected from the group consisting of 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.

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Abstract

La présente invention concerne de nouvelles protéines associées à l'intégrine, des cellules, des compositions et des produits pharmaceutiques qui améliorent la thérapie cellulaire.
PCT/EP2022/085538 2021-12-15 2022-12-13 Nouvelle protéine associée à l'intégrine (iap) WO2023110824A1 (fr)

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