WO2024261027A1

WO2024261027A1 - Il-7-retargeting of adenoviruses for cell specific infection

Info

Publication number: WO2024261027A1
Application number: PCT/EP2024/067041
Authority: WO
Inventors: Vasileios ATSAVES; Bruno Loureiro CADILHA; Monique-Jacqueline KAUKE; Nicole KIRCHHAMMER; Hilary MERLINI; Jatina SCHUMACHER; Sebastian Weigert; Sheena Nicole SMITH
Original assignee: Vector Biopharma Ag
Current assignee: Vector Biopharma Ag
Priority date: 2023-06-19
Filing date: 2024-06-19
Publication date: 2024-12-26
Anticipated expiration: 2025-12-19

Abstract

Disclosed herein are recombinant adenoviruses, recombinant proteins and trimeric proteins that are useful for the transduction of human cells, specifically, immune cells, such as T cells, NK cells, monocytes, macrophages or dendritic cells. The present invention provides a versatile and highly specific system that is useful for numerous purposes, including the development of novel therapeutic approaches.

Description

IL-7-retargeting of adenoviruses for cell specific infection

Field of the invention

Background

Decades of research have aimed to genetically engineer cells for scientific and therapeutic purposes (Cell (2017) 168: 724-740; Nature (2018) 559: 405-9). Vast advances in cell therapy have been made using recombinant viral vectors, or adeno-associated vectors (Nature Methods (2019) 16: 247-54; Science (2015) 348: 62-8). These advances lead to a better understanding of required and distinct molecular mechanisms of action for effective therapeutics. Such molecular mechanisms benefit from precise tunability and controllability at the DNA level, thus leading to an increase of the required DNA complexity and unavoidably increasing the demand of base pairs to encode for said complexity (Molecular Cancer (2019) 18: 125). Currently used vectors do not allow insertion of large DNA payloads, being limited to payloads up around 7 kb (thousand base pairs) DNA. To circumvent viral production, non-viral delivery methods can be used for screening purposes. Circumventing the toxicity of the DNA, linear DNA transfection (Nature (2018) 559: 405-9; Vol. 23, Nature Medicine (2017) 23: 415-23), RNA transfection (Molecular Therapy (2006) 13: 151-9) or RNA-containing lipid nanoparticles (LNPs) (Science (2022) 375: 91-6) have been developed. However, not only remains the limitation of the deliverable DNA size as an obstacle, an incorporation of various payloads into a single vector with sufficient size also guarantees defined DNA ratios in the target cell and successful delivery of all fragments. Ideally a vector of choice would combine large packaging capacities in combination with effective transduction and reported safe delivery in mice and humans, allowing targeted transduction in an in vitro and in vivo setting.

Human adenovirus serotype 5 vectors (HAdV-C5) can not only be modified to high-capacity vectors (HC-AdVs) (36 kb packaging size) but are also validated for safety in various clinical trials and animal models (Virus Genes (2017) 53: 684-691; J Hematol Oncol (2020) 13: 84; Adenoviral Gene Therapy (2002) 7 , 46-59). Increasing the safety of human application further, HAdV genomes exist extra- chromosomally, minimizing the risk of unwanted insertional mutation and germline transmission. However, if desired, HC-AdVs have been reported as a single delivery entity combining donor DNA and a Cas9 system enabling site-specific insertion and deletion (Molecular Therapy: Methods and Clinical Development (2020) 17: 441-7), qualifying HAdV-C5 to an ideal and versatile vector overcoming limitations of currently available methods for the engineering of immune cells, such as for example immune cells.

Cellular entry of HAdV-C5 vectors can happen efficiently in a receptor-specific manner, but also at lower rates through unspecific and poorly characterized mechanisms. Receptor-specific cellular entry is mediated mostly through contact by the homotrimeric knob protein to the coxsackie and adenoviral receptors (CAR) and subsequent interaction with RGD binding integrins on the cell membrane. Both CAR and RGD binding integrins are unproven to be expressed by immune cells at high levels (Ann Rev Virol (2019) 6: 177-97; J Cell Sci (2003) 116: 4695-705; Int J Mol Sci (2018) 19: 485; J Virol (1995) 69: 2257-63). This renders immune cells, such as T cells or NK cells, hard to transduce by HAdV-C5 without engineering of vector or cells (PLoS ONE (2017) 12: 1-12). In the T cell engineering field, for instance, this has led to expression of either CAR (Proc Natl Acad Sci USA (1998) 95:13159-64) or aV 3/5 integrins (J Virol (1995) 69: 2257-63) making T cells more susceptible for adenoviral infections and the generation of chimeric adenoviral vectors such as Ad5/35 interacting with CD46 expressed on all nucleated human cells (Exp Hematol (2004) 32: 536-46; International J Biochem Cell Biol (1999) 31: 1255-60).

However, achieving transduction of immune cells by HAdV-C5 vectors in genetically unmodified cells would allow its application in various scientific and clinical settings. We hypothesized that a previously described trimeric adapter technology (J Mol Biol (2011) 405: 410-26; Proc Natl Acad Sci USA (2013) 110: E869-77) could be engineered to generate an easily applicable method to enable adenovirus mediated transduction, allowing universal usage with all currently developed adenoviral vectors. Additionally, direct in vivo engineering could be possible while being compatible with the previously reported shield design, increasing tissue specificity while reducing liver uptake.

The present invention makes use of specially designed adapter molecules that enable the specific targeting of immune cells, such as T cells, NK cells, monocytes, macrophages or dendritic cells, enabling transduction of these cell types, useful for ex vivo or in vivo applications. Summary of the invention

In certain embodiments, the present disclosure relates to a recombinant adenovirus displaying a functional interleukin 7 polypeptide.

In certain embodiments said functional interleukin 7 polypeptide is displayed on the knob of said adenovirus.

In certain embodiments, said functional interleukin 7 polypeptide is comprised in a recombinant protein comprising from the N- to the C-terminus. a) said functional interleukin 7 polypeptide, b) a designed ankyrin repeat domain which binds to the knob of the adenovirus, and c) a trimerization domain.

In certain embodiments, said functional interleukin 7 polypeptide comprises the amino acid sequence of SEQ ID No. 3 or 4.

In certain embodiments, said designed ankyrin repeat domain that binds to a knob of an adenovirus comprises the amino acid sequence of SEQ ID No. 2.

In certain embodiments, said trimerization domain is or is derived from the capsid protein SHP of lambdoid phage 21.

In certain embodiments, said trimerization domain comprises the amino acid sequence of SEQ ID No. 1.

In certain embodiments, said adenovirus is of adenovirus serotype 5 or wherein said adenovirus comprises a knob of an adenovirus of serotype 5.

In certain embodiments, the present disclosure relates to a eukaryotic cell expressing or producing aforementioned recombinant adenoviruses.

In certain embodiments, the present disclosure relates to a method for the transduction of immune cell, said method comprising a. contacting said immune cell with aforementioned recombinant adenoviruses, and b. incubating the mixture obtained in step a. for a time sufficient for transduction of said immune cells.

In certain embodiments, said immune cells are T cells. In certain embodiments, said T cells are CD4-positve T cells, CD8-positive T cells or Treg cells.

In certain embodiments, said immune cells are NK cells, monocytes, macrophages or dendritic cells.

In certain embodiments, said method is performed in vivo.

In certain embodiments, the present disclosure relates in step a. of said method said immune cells are additionally contacted with an agent capable of activating said immune cells.

In certain embodiments, said immune cells are contacted with aforementioned recombinant adenoviruses according and said agent capable of activating said immune cells at about the same time.

In certain embodiments, said immune cells are contacted with aforementioned recombinant adenoviruses according and said agent capable of activating said immune cells simultaneously.

In certain embodiments, said agent capable of activating said immune cells is selected from DynaBeads, TransAct, Polybrene and PMA (l-Methoxy-2-propylacetat).

In certain embodiments, said method is performed in vitro.

In certain embodiments, the present disclosure relates to a recombinant protein comprising from the N- to the C-terminus a) a functional interleukin 7 polypeptide, b) a designed ankyrin repeat domain which binds to the knob of an adenovirus, and c) a trimerization domain.

In certain embodiments, the present disclosure relates to a trimeric protein consisting of three of aforementioned recombinant proteins.

In certain embodiments, the present disclosure relates to a nucleic acid encoding aforementioned recombinant proteins.

In certain embodiments, the present disclosure relates to aforementioned recombinant adenoviruses, aforementioned recombinant proteins, or aforementioned trimeric protein for use in the transduction of immune cells, preferably T cells, NK cells, monocytes, macrophages or dendritic cells, or for use in medicine.

Figure legends Figure 1 shows an RNA expression dataset, highlighting in panel a clusters based on cell isolated from blood or lung tumors, in panel b several types of immune cells grouped in clusters, and in panel c the expression level of IL7R on RNA level across mentioned populations from panels a and b.

Figure 2 shows an RNA expression dataset, depicting expression of IL7R on RNA level across different organs and tissues.

Figure 3 shows the transduction efficiency of a GFP-VLP on the B cell lineage tumor cell line NALM-6 using an IL-7- retargeting adapter.

Figure 4 shows the transduction efficiency of a GFP-VLP on activated PBMCs using an IL-7-retargeting adapter.

Figure 5 shows the transduction efficiency of a GFP-VLP on CD4+ activated PBMCs using an IL-7- retargeting adapter.

Figure 6 shows the transduction efficiency of a GFP-VLP on CD8+ activated PBMCs using an IL-7- retargeting adapter.

Figure 7 shows the transduction efficiency of a GFP-VLP on activated T cells using an IL-7- retargeting adapter.

Figure 8 shows the transduction efficiency of a GFP-VLP on CD3+ non-activated PBMCs using an I L-7- retargeting adapter.

Definitions

The term "recombinant" as used in recombinant protein, recombinant protein domain and the like, means that said polypeptides are produced using recombinant DNA technologies well known by the practitioner skilled in the relevant art. For example, a recombinant DNA molecule (e.g. produced by gene synthesis) encoding a polypeptide can be cloned into a bacterial expression plasmid (e.g. pQE30, Qiagen) or a mammalian expression plasmid (e.g. pcDNA™3.1, ThermoFisher Scientific). When such a constructed recombinant expression plasmid is inserted into a host cell (e.g. E. coli, CHO, HEK), this host cell can produce the polypeptide encoded by this recombinant DNA. The correspondingly produced polypeptide is called a recombinant polypeptide.

The term "protein" as used herein refers to a polypeptide, wherein at least part of the polypeptide has, or can, acquire a defined three-dimensional arrangement by forming secondary, tertiary, or quaternary structures within and/or between its polypeptide chain(s). If a protein comprises two or more polypeptides, the individual polypeptide chains may be linked non-covalently or covalently, e.g. by a disulfide bond between two polypeptides.

A part of a protein, which individually has, or can acquire a defined three-dimensional arrangement by forming secondary or tertiary structures, is termed "protein domain" or "domain". Such protein domains are well known to the practitioner skilled in the art.

The term "polypeptide" as used herein refers to a molecule consisting of one or more chains of multiple, i.e. two or more, amino acids linked via peptide bonds. A polypeptide typically consists of more than twenty amino acids linked via peptide bonds.

The term "peptide" as used herein refers to a molecule consisting of one or more chains of multiple, i.e. two or more, amino acids linked via peptide bonds. A peptide typically consists of not more than twenty amino acids linked via peptide bonds.

The terms "designed ankyrin repeat protein", "designed ankyrin repeat domain" and "DARPin" as used herein refer to artificial polypeptides, comprising several ankyrin repeat motifs. These ankyrin repeat motifs provide a rigid interface arising from typically three repeated P-turns. DARPins usually carry two or three repeats corresponding to an artificial consensus sequence, wherein six positions per repeat are randomized, flanked by two capping repeats with a hydrophilic surface (Curr Olpin Chem Biol (2009) 13:245-55; WO 02/20565).

The term "protein scaffold" means a protein with exposed surface areas in which amino acid insertions, substitutions or deletions are highly tolerable. Examples of protein scaffolds that can be used to generate binding domains of the present invention are antibodies or fragments thereof such as single-chain Fv or Fab fragments, T cell receptor such as single chain T cell receptors, protein A from Staphylococcus aureus, the bilin binding protein from Pieris brassicae or other lipocalins, ankyrin repeat proteins, monobodies, human fibronectin, or antibodies from camelids, such as nanobodies. Protein scaffolds are known to the person skilled in the art (Curr Opin Biotechnol 22:849-57 (2011); Ann Rev Pharmacol Toxicol 60:391-415 (2020)). In certain embodiments of the present disclosure the protein scaffold is a polypeptide. In certain embodiments of the present disclosure the protein scaffold is a monomeric polypeptide. In certain embodiments of the present disclosure, the protein scaffold is an antibody fragment. In certain embodiments of the present disclosure the protein scaffold is a scFv. In certain embodiments of the present disclosure the protein scaffold is a single chain T cell receptor. In certain embodiments of the present disclosure the protein scaffold is a peptide. In certain embodiments of the present disclosure the protein scaffold is not a designed ankyrin repeat domain. The term "antibody" as used herein refers to a protein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, which interacts with an antigen. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FR's arranged from amino-terminus to carboxyterminus in the following order: FR1, CDR1 , FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes for example, monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies and chimeric antibodies. The antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl , lgG2, lgG3, lgG4, IgAl and lgA2) or subclass. Both the light and heavy chains are divided into regions of structural and functional homology.

The term "antibody fragment" as used herein refers to one or more portions of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing spatial distribution) an antigen. Examples of binding fragments include, but are not limited to, a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CHI domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as "single chain variable fragment", "single chain Fv" or "scFv"; see e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antibody fragment". These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies. Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23:1 126-1 136). Antibody fragments can be grafted into scaffolds based on polypeptides such as Fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies). Antibody fragments can be incorporated into single chain molecules comprising a pair of tandem Fv segments (VH-CH1 - VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen-binding sites (Zapata et al., (1995) Protein Eng. 8: 1057-1062; and U.S. Pat. No. 5,641 ,870).

The term "single chain TCR", "single chain T cell receptor" and "scTCR" as used herein refers to any construct containing the variable domains of a T cell receptor in single chain format. Such single chain formats include, but are not limited to the constructs described in (J Immunol Methods (1998) 221:59-76, Cancer Immunol Immunother (2002) 51:565-73, WO 2019/219709 and WO 2004/033685. In certain embodiments single chain TCR variable domains and/or single chain TCRs may have a disulfide bonds as described in WO 2004/033685.

The term "immunoglobulin" as used herein refers to any polypeptide or fragment thereof from the class of polypeptides known to the skilled person under this designation and comprising at least one antigen binding site. Preferably, the immunoglobulin is a soluble immunoglobulin from any of the classes IgA, IgD, IgE, IgG, or IgM, or a fragment comprising at least one antigen binding site derived thereof. Also comprised as immunoglobulins of the present invention are a bispecific immunoglobulin, a synthetic immunoglobulin, an immunoglobulin fragment, such as Fab, Fv or scFv fragments etc., a single chain immunoglobulin, and a nanobody. Further included are chemically modified derivatives of any of the aforesaid, e.g. PEGylated derivatives, as well as fusion proteins comprising any of the aforesaid immunoglobulins and fragments thereof. The immunoglobulin may be a human or humanized immunoglobulin, a primatized, or a chimerized immunoglobulin or a fragment thereof as specified above. Preferably, the immunoglobulin of the present invention is a polyclonal or a monoclonal immunoglobulin, more preferably a monoclonal immunoglobulin or a fragment thereof as specified above.

The terms "binds", "is specific" and "specifically binds" as used herein refers to a molecule, for example an antibody or an antibody fragment, which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. An antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more further species. Such cross-species reactivity does not itself alter the classification of an antibody as specific.

The term "nucleic acid" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically, or biochemically modified, nonnatural, or derivatized nucleotide bases.

The term "vector" as used herein means a construct, which is capable of delivering, and usually expressing or regulating expression of, one or more gene(s) or nucleic acid(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.

The term "linker" as used herein refers to a molecule or macromolecule serving to connect different moieties or domains of a peptide or a polypeptide or, a protein/polypeptide domain and a non- protein/non-polypeptide moiety. Linkers can be of different nature. Different domains or modules within proteins can be linked via peptide linkers. Linkers can also be generated chemically, for example to link small organic molecules or peptides to a protein.

The term "flexible linker" as used herein refers to a peptide linker linking two different domains or modules of a protein and providing a certain degree of flexibility. Preferably, the flexible linker is hydrophilic and does not interact with other surfaces. Commonly used flexible linkers are glycineserine linkers (Biochemistry 56(50):6565-6574 (2017)). Glycine and serine are flexible and the adjacent protein domains are free to move relative to one another. Such flexible linkers are referred to herein as "glycine-serine linkers". Other amino acids commonly used in respective linkers are proline, asparagine and threonine. Often the linker contains several repeats of a sequence of amino acids. A flexible linker used in the present disclosure is a (G ly₄Ser)4-lin ker, i.e. a linker containing four repeats of the sequence glycine- glycine- glycine- glycine- serine. Other linkers that could be used in accordance with the present disclosure include but are not limited to PAS linkers, i.e. linkers containing repeats of the sequence proline- alanine- serine (Protein Eng Des Sei (2013) 26, 489-501 and charged linkers.

The term "short linker" as used herein refers to a peptide linker linking two different domains or modules of a protein and which is no longer than four, preferably no longer than three amino acids long. More preferably the short linker is no longer than two amino acids long. Alternatively the short linker is only one amino acid long. Alternatively the short linker is a single glycine residue.

The term "amino acid mutation" refers to amino acid substitutions, deletions, insertions, and modifications, as well as combinations thereof. Amino acid sequence deletions and insertions include N-and/or C-terminal deletions and insertions of amino acid residues. Particular amino acid mutations are amino acid substitutions. Amino acid substitutions include replacement by non-naturally occurring amino acids or by naturally occurring amino acid derivatives of the twenty standard amino acids. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis and the like. It is contemplated that methods of altering the side chain group of an amino acid residue by methods other than genetic engineering, such as chemical modification, may also be useful.

The term "variant" as used herein refers to a polypeptide that differs from a reference polypeptide by one or more amino acid mutation or modifications.

The term "host cell" as used herein refers to any kind of cellular system which can be engineered to generate molecules according to the present disclosure. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein.

Host cells according to the present disclosure can be a "eukaryotic host cell" and include yeast and mammalian cells, including murine cells and from other rodents, preferably vertebrate cells such as those from a mouse, rat, monkey or human cell line, for example HKB11 cells, PERC.6 cells, HEL293T cells, CHO cells or any type of HEK cells, such as HEK293 cells or HEK 293 cells. Also suspension cell lines like CHO-S or HEK993 cells, or insect cell cultures like Sf9 cells may be used.

Host cells according to the present disclosure can also be "procaryotic cell" and include bacterial cells, such Escherichia coli. Certain strains of Escherichia coli may be particularly useful for expression of the molecules of the present disclosure, such as Escherichia coli strain DH5 (available from Bethesda Research Laboratories, Inc., Bethesda, Md/US).

The molecules of the present disclosure form trimers that are highly stable. Each monomer contains a domain responsible for the formation of trimers which is referred to herein as "trimerization domain". A preferred trimerization domain is the capsid protein SHP of lambdoid phage 21 (J Mol Biol; 344(1) :179-93; PNAS 110(10):E869-77 (2013)). SHP of lambdoid phage 21 has the following amino acid sequence:

VRIFAGNDPAHTATGSSGISSPTPALTPLMLDEATGKLWWDGQKAGSAVGILVLPLEGTETALTY

YKSGTFATEAIHWPESVDEHKKANAFAGSALSHAALP ( SEQ ID No . 1 )

The term "stable trimer" or "trimeric adapter" as used herein refers to a protein trimer by protein monomers comprising a trimerization domain, and wherein said trimer exhibits a stability which is higher than other, conventional protein trimers. For example, a stable trimer has a higher functional stability, a higher kinetic stability, or a higher half life for unfolding than other protein trimers. An example of a stable trimer is a trimer formed by monomers comprising the trimerization domain of the capsid protein SHP of lambdoid phage 21.

The term "derived from" in the context of an amino acid sequence refers to an amino acid sequence that is different to an original amino acid sequence, but maintains the function or activity of the original amino acid sequence.

The term "adenovirus" as used herein refers to any adenovirus, i.e. to human and non-human serotypes. The human isolates are classified into subgroups A-G. A preferred adenovirus of the present disclosure is adenovirus subtype 5 ("HAdV-C5"). HAdV-C5 includes modified version of the virus, such as replication-deficient HAdV-C5 version, e.g. containing an E1/E3 deletion and/or one or more of the 4 mutations in the HVR7 (I421G, T423N, E424S and L426Y) (Nat. Common. 9, 450 (2018)).

The terms "CAR" and "CXADR" as used herein refers to coxsackievirus and adenovirus receptor (UniProt: P78310). CAR is a type I membrane receptor for coxsackie viruses and adenoviruses.

The term "knob" as used herein refers to a knob on the end of the adenovirus fiber (e.g. GenBank: AAP31231.1) that binds to the cellular receptor. The knob of adenovirus subtype 5 binds to CAR. Some adenoviruses carry mutations in the gene encoding the knob protein. Adenoviruses having a four- amino acid deletion within the FG loop of the knob (TAYT mutation) show a decreased ability of the mutated knob to bind to CAR (Science, 286: 1568-1571 (1999); J Mol Biol 405(2):410- 426). Adenoviruses carrying four amino acid mutations in the hypervariable region 7 (HVR7 mutation) show a strongly reduced binding to blood coagulation factor X (Nat Commun (2018) 9:450).

The molecules of the present invention contain a designed ankyrin repeat domain that binds to the knob of an adenovirus. A preferred designed ankyrin repeat domain that binds to a knob is DARPin 1D3. Another preferred designed ankyrin repeat domain that binds to a knob is DARPin lD3nc, a derivative of lD3nc containing a stabilized C-cap. DARPin 1D3 has the following amino acid sequence:

RSDLGKKLLEAARAGQDDEVRILMANGADANAYDHYGRTPLHMAAAVGHLEIVEVLLRNGADVNAV DTNGTTPLHLAASLGHLEIVEVLLKYGADVNAKDATGITPLYLAAYWGHLEIVEVLLKHGADVNAQ DKFGKTPFDLAIDNGNEDIAEVLQG ( SEQ ID No . 2 )

An "antigen-binding moiety" as used herein refers to a polypeptide that specifically binds to an antigenic determinant. Exemplary antigen-binding moieties include, protein scaffold and antibodies and antibody fragment, such as a single-chain Fv or a Fab fragment.

The term "interleukin 7", "IL7" or "IL-7" as used herein refers to human interleukin 7 (UniProt: P13232), a cytokine that plays an essential role in the development, expansion, and survival of naive and memory T-cells and B-cells thereby regulating the number of mature lymphocytes and maintaining lymphoid homeostasis. Human IL-7 has the following amino acid sequence:

MFHVSFRYI FGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVS IDQLLDSMKEIGSNCLNNEFNF FKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEA QPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH ( SEQ I D No . 3 )

The term "functional interleukin 7" or "functional IL-7" as used herein refers to a polypeptide which is a variant or a derivative of human interleukin 7 which retains the biological function of wild type IL-7. Such variant or derivative may have one or more mutations compared to wild type IL-7. Such variant or derivative also may contain additions or deletions of amino acid sequences, in particular at the N- terminal or C-terminal part of IL-7. The functional interleukin 7 may also be a variant of human IL-7 which retains the function of wild type human IL-7, such as a stability-enhanced variant of human IL-7. The functional interleukin 7 may also be a variant of human IL-7 with increase affinity to the IL-7 receptor. Exemplary variants of human IL-7 include those disclosed in W02007/010401 and WO2021/122866.

Exemplified herein is a functional interleukin 7 which is identical to the wild type sequence of human IL-7, except that the first methionine residue is missing, i.e. a molecule with the following amino acid sequence:

FHVSFRYI FGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVS IDQLLDSMKEIGSNCLNNEFNFF KRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQ PTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH ( SEQ ID No . 4 )

The exemplified functional interleukin 7 is encoded by the following nucleic acid sequence:

TTCCATGTTTCTTTTAGGTATATCTTTGGACTTCCTCCCCTGATCCTTGTTCTGTTGCCAGTAGCA TCAT CT GAT T GT GAT AT TGAAGGTAAAGAT GGCAAACAAT AT GAGAGT GT TC T AAT GGT C AGCAT C GATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTTT AAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCTGCTCGCAAGTTGAGG CAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAAGTTTCAGAAGGCACA ACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAA CCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTC CTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACAC ( SEQ ID No . 5 )

The term "displaying" as used herein refers to the presentation of a polypeptide on the outside of an entity, such as an adenovirus. The polypeptides so presented on the entity may be covalently or non-covalently attached to such entity. In the context of the present disclosure adapter molecules are recombinantly expressed and displayed on adenoviruses. This can be accomplished via a binding moiety or a scaffold, such as a designed ankyrin repeat domain that binds to the knob of an adenovirus. Alternatively, moiety or scaffold can also be genetically fused to an adenoviral protein, such as the hexon.

The term "adapter" as used herein refers to a recombinant protein which comprises a designed ankyrin repeat domain which binds to the knob of the adenovirus, a trimerization domain and a domain which specifically binds to a target protein. The domain which specifically binds to a target protein that is employed in the present invention is a functional interleukin 7 polypeptide. Through this domain, the adapter links the adenovirus to target cells which express a receptor for the interleukin 7 polypeptide. Certain other adapters are use in the present disclosure. Such other adapters comprise different domains which specifically binds to a target protein, for example an anti-CD3 scFv or an anti-CD28 scFv.

The term "about the same time" as used herein refers to a time frame in which certain entities, for example immune cells, a recombinant adenovirus displaying a functional interleukin 7 polypeptide and an agent capable of activating immune cells, are mixed within a short time interval. This time interval is typically less than 10 minutes, preferably less than 5 minutes, and more preferably at the same time. If the entities are mixed at the same time this is referred to as "simultaneous" or "simultaneously".

The term "agent capable of activating immune cells" as used herein refers to an agent that, when added to unactivated immune cells, such as T cells, activates such immune cells. Activated immune cells differ from unactivated immune cells by upregulation of markers such as CD25, CD69, PD-1, by a morphological change that encompasses an increase of diameter and increase of intracellular organelles and machinery, by an increased proliferation rate accompanied by doubling of cells up to every 6 hours. In the activated state such immune cells have an increase uptake or surrounding proteins and nutrients in response to the higher metabolic stress in preparation for expansion and cell division. Typical agents that are capable of activation immune cells are known to the skilled person and include DynaBeads (e.g. Dynabeads™ Human T-Expander CD3/CD28, Gibco, Catalogue number 11141D; Dynabeads™ Human T-Expander CD3/CD28/CD137, Gibco, Catalogue number 11162D), TransAct (T Cell TransAct™ human, Miltenyi, Catalogue number 130-111-160), Polybrene and PMA (1- Methoxy-2-propylacetat).

The term "extra corporeal" as used herein means refers to a procedure which is performed outside the body. Embodiments of the invention

Disclosed herein is an adenoviral-based system for targeted transduction of immune cells, such as T cells. The system can be used in field of medicine, particularly in T cell-based orT cell-related diseases and disorders. Cargo, such as nucleic acids, in particular nucleic acids encoding therapeutically active or therapeutically helpful proteins and peptides, can be delivered into immune cells in which they can exert their function. One advantage of the adenoviral system utilized here is the fact that it can encode cargo of a large size, i.e. up to 36 kb.

Specificity of the adenoviruses can conferred by adapter molecules as described in the present disclosure. The specificity of the system and the correlating transduction efficiency is considerably higher than the systems known in the art. High transduction efficiencies can however be also obtained without adapter molecules. The system is functional with adenoviruses of any kind, i.e. first-generation virus, as well as high-capacity, helper virus-dependent adenoviral systems. The system is also functional with other viruses, e.g. viruses that are engineered to carry a knob of an adenovirus of subtype 5.

The methods provided herein, allow for the efficient transduction of immune cells. Said immune cells may be activated immune cells or non-activated immune cells.

In certain embodiments, the present disclosure relates to a recombinant adenovirus displaying functional interleukin 7 polypeptides. In certain embodiments the recombinant adenovirus display said functional interleukin 7 polypeptides via recombinant adapter molecules.

In certain embodiments, the present disclosure relates to a recombinant adenovirus a functional interleukin 7 polypeptide, wherein said adenovirus is capable of transducing immune cells. In other embodiments, the present disclosure relates to a recombinant adenovirus a functional interleukin 7 polypeptide, wherein said adenovirus is capable of transducing T cells. Human T cells lack strong expression of coxsackie and adenoviral receptors (CAR) through which transduction of adenoviruses is typically mediated, as well as RGD binding integrins which are involved in subsequent interactions. Adenoviruses armored with adapters as described in the present disclosure overcome these hurdles by generating a sufficiently high specificity to the T cells that enables adenoviral infection of the cells.

In certain embodiments, the functional interleukin 7 polypeptide comprises the amino acid sequence of SEQ ID No. 3. In certain embodiments, the functional interleukin 7 polypeptide consists of the amino acid sequence of SEQ ID No. 3. In certain embodiments, the functional interleukin 7 polypeptide comprises the amino acid sequence of SEQ ID No. 3 with one, two, three, four or five amino acid substitutions, insertions or deletions. In certain embodiments, the functional interleukin 7 polypeptide is a variant of the polypeptide consisting of the amino acid sequence of SEQ ID No. 3, wherein said variant is functionally equivalent to the polypeptide consisting of the amino acid sequence of SEQ ID No. 3.

In certain embodiments, the functional interleukin 7 polypeptide comprises the amino acid sequence of SEQ ID No. 4. In certain embodiments, the functional interleukin 7 polypeptide consists of the amino acid sequence of SEQ ID No. 4. In certain embodiments, the functional interleukin 7 polypeptide comprises the amino acid sequence of SEQ ID No. 4 with one, two, three, four or five amino acid substitutions, insertions or deletions. In certain embodiments, the functional interleukin 7 polypeptide is a variant of the polypeptide consisting of the amino acid sequence of SEQ ID No. 4, wherein said variant is functionally equivalent to the polypeptide consisting of the amino acid sequence of SEQ ID No. 4.

In certain embodiments, the recombinant adenoviruses comprise recombinant polypeptides or proteins, that are displayed on said adenovirus. Said recombinant proteins comprises a functional interleukin 7 polypeptide. Said functional interleukin 7 polypeptide may be fused to the other parts of the recombinant protein in any order.

In certain embodiments, the present disclosure relates to a recombinant protein comprising a) a designed ankyrin repeat domain which binds to the knob of an adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide.

In other embodiments, the present disclosure relates to a recombinant protein comprising from the N- to the C-terminus a) a designed ankyrin repeat domain which binds to the knob of the adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide.

In specific embodiments, the present disclosure relates to a recombinant protein comprising from the N- to the C-terminus a) a functional interleukin 7 polypeptide, b) a designed ankyrin repeat domain which binds to the knob of the adenovirus, and c) a trimerization domain. In certain embodiments, the functional interleukin 7 polypeptide comprises the amino acid sequence of SEQ ID No. 3. In certain embodiments, the functional interleukin 7 polypeptide consists of the amino acid sequence of SEQ ID No. 3. In certain embodiments, the functional interleukin 7 polypeptide comprises the amino acid sequence of SEQ ID No. 3 with one, two, three, four or five amino acid substitutions, insertions or deletions. In certain embodiments, the functional interleukin 7 polypeptide is a variant of the polypeptide consisting of the amino acid sequence of SEQ ID No. 3, wherein said variant is functionally equivalent to the polypeptide consisting of the amino acid sequence of SEQ ID No. 3.

In specific embodiments, the present disclosure relates to a recombinant protein comprising from the N- to the C-terminus a) a functional interleukin 7 polypeptide, b) a designed ankyrin repeat domain which binds to the knob of the adenovirus, and c) a trimerization domain. In certain embodiments, the functional interleukin 7 polypeptide comprises the amino acid sequence of SEQ ID No. 4. In certain embodiments, the functional interleukin 7 polypeptide consists of the amino acid sequence of SEQ ID No. 4. In certain embodiments, the functional interleukin 7 polypeptide comprises the amino acid sequence of SEQ ID No. 4 with one, two, three, four or five amino acid substitutions, insertions or deletions. In certain embodiments, the functional interleukin 7 polypeptide is a variant of the polypeptide consisting of the amino acid sequence of SEQ ID No. 4, wherein said variant is functionally equivalent to the polypeptide consisting of the amino acid sequence of SEQ ID No. 4.

The recombinant proteins of the present disclosure comprise a designed ankyrin repeat domain which binds to the knob of a virus or adenovirus. It will be appreciated that any designed ankyrin repeat domain with specificity for the knob of a virus or adenovirus may be used within the spirit of the present disclosure. Exemplified herein is a designed ankyrin repeat domain derived from DARPin 1D3 (Proc. Natl. Acad. Sci. 110, E869-E877 (2013)). DARPin 1D3 binds to the knob of an adenovirus and comprises the amino acid sequence of SEQ ID No. 2. Used herein is lD3nc, a derivative of 1D3 containing a stabilized C-cap.

It will also be understood that also variants of DARPin 1D3 may be used within the spirit of the present disclosure. In other words, the amino acid sequence of such modified DARPin 1D3 does not need to be identical to that of amino acid sequence of SEQ. ID No. 2, but may contain amino acids mutations, provided that the function of DARPin 1D3, i.e. binding to the knob of an adenovirus is preserved. Also, DARPins different than 1D3, but having the same target specificity, may be used within the scope of the present disclosure. Such new DARPin may for example be selected in a new screening campaign. Also binding entities different than DARPins, i.e. binders based on a different scaffold, but having the same target specificity as 1D3 might be used.

Therefore, in certain embodiments, the present disclosure relates to a recombinant protein comprising a) a designed ankyrin repeat domain which binds to the knob of an adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide.

In certain embodiments, the present disclosure relates to a recombinant protein comprising a) a designed ankyrin repeat domain which binds to the knob of an adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide, wherein said designed ankyrin repeat domain which binds to the knob of an adenovirus is DARPin 1D3.

In certain embodiments, the present disclosure relates to a recombinant protein comprising a) a designed ankyrin repeat domain which binds to the knob of an adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide, wherein said designed ankyrin repeat domain which binds to the knob of an adenovirus is or is derived from DARPin 1D3.

In certain embodiments, the present disclosure relates to a recombinant protein comprising a) a designed ankyrin repeat domain which binds to the knob of an adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide, wherein said designed ankyrin repeat domain which binds to the knob of an adenovirus is a variant of DARPin 1D3.

In certain embodiments, the present disclosure relates to a recombinant protein comprising a) a designed ankyrin repeat domain which binds to the knob of an adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide, wherein said designed ankyrin repeat domain which binds to the knob of an adenovirus comprises the amino acid sequence of SEQ. ID No. 2.

In certain embodiments, the present disclosure relates to a recombinant protein comprising a) a designed ankyrin repeat domain which binds to the knob of an adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide, wherein said designed ankyrin repeat domain which binds to the knob of an adenovirus comprising a variant of the amino acid sequence of SEQ ID No. 2.

In certain embodiments, the present disclosure relates to recombinant proteins comprising a designed ankyrin repeat domain that binds to the knob of an adenovirus. The present disclosure can however also be practiced with other viruses. If another virus is used a designed ankyrin repeat domain needs to be selected that binds to the knob of such virus. Therefore, in certain embodiments the present disclosure relates to recombinant proteins comprising a) a designed ankyrin repeat domain which binds to the knob of the adenovirus, b) trimerization domain, and c) a functional interleukin 7 polypeptide.

Viruses, other than adenoviruses of serotype 5, can also be used within the spirit of the present disclosure. For example, such viruses can be engineered to carry the knob of an adenovirus of serotype 5. The recombinant protein disclosed herein, in particular recombinant protein comprising DARPin 1D3, may then be used with such viruses. Therefore, in certain embodiments the present disclosure provides a recombinant virus or a set of recombinant viruses comprising a recombinant protein comprising: a) a functional interleukin 7 polypeptide, b) a designed ankyrin repeat domain which binds to the knob of an adenovirus, and c) a trimerization domain, wherein said designed ankyrin repeat domain binds to the knob of an adenovirus of subtype 5.

It will be understood that also other adenoviral serotypes may be used in the spirit of the present disclosure, including human adenovirus serotype c5 (HAdV-C5), HAd2, HAd3, HAdV-B35, HAdV-D26, as well as hybrids thereof. A list of adenoviruses can be found on the website of the Human Adenovirus Working group (http://hadvwg.gmu.edu). Also, non-human adenoviruses may be used within the scope of the present disclosure, such as the AstraZeneca vaccine chimpanzee adenovirus Y25 (CHAdY25), or non-human adenoviral vectors were developed from bovine (BAd), canine (CAd), chimpanzee (ChAd), ovine (OAd), porcine ( P Ad), or fowl (FAd).

A preferred virus to be used in the context of the present disclosure is adenovirus of serotype 5. Therefore, in certain embodiments the present disclosure relates to recombinant proteins comprising a) a designed ankyrin repeat domain which binds to the knob of an adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide, wherein said designed ankyrin repeat domain binds to the knob of an adenovirus of serotype 5.

In certain embodiments, the present disclosure relates to recombinant proteins or uses of such recombinant proteins that comprise a trimerization domain. The trimerization domain is responsible for the formation of trimers. Each monomer of the molecules of the present disclosure comprises a trimerization domain. Principally any trimerization domain may be used, provided it is stable and geometrically fits the knob of the adenovirus used. A preferred trimerization domain is the capsid protein SHP of lambdoid phage 21 (J Mol Biol; 344(l):179-93; PNAS 110(10):E869-77 (2013)).

Therefore, in certain embodiments the present disclosure relates to recombinant proteins comprising a) a designed ankyrin repeat domain which binds to the knob of an adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide, wherein said trimerization domain is the capsid protein SHP of lambdoid phage 21.

In other embodiments the present disclosure relates to recombinant proteins comprising a) a designed ankyrin repeat domain which binds to the knob of an adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide, wherein said trimerization domain is derived from the capsid protein SHP of lambdoid phage 21.

In other embodiments the present disclosure relates to recombinant proteins comprising a) a designed ankyrin repeat domain which binds to the knob of an adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide, wherein said trimerization domain comprises the amino acid sequence of SEQ. ID No. 1.

Also, other trimerization domains known to the skilled person may be used for the formation or trimers. Without being limited, other potential trimerization domains include the trimerization domain involved in collagen folding (Int J Biochem Cell Biol 44:21-32 (2012)), the trimerization domain of T4 phage fibritin (PLoS One 7:e43603 (2012)) or the GCN4-based isoleucine zipper (J Biol Chem 290: 7436- 42 (2015)).

The trimerization domain is responsible for the formation of the trimeric adapter molecules. The trimers disclosed herein are extraordinary stable (J Mol Biol (2004) 344:179-93; PNAS (2013) 1 10 E869-77). In certain embodiments the trimeric adapter molecules of the present disclosure remain intact in SDS gel electrophoresis. In other embodiments the trimeric adapter molecules are not denatured in SDS gel electrophoresis. In other embodiments the trimeric adapter molecules have a half-life in solution of at least one week, preferably at least two week and even more preferably at least one month.

Therefore, in certain embodiments the present disclosure relates to recombinant proteins comprising a) a designed ankyrin repeat domain which binds to the knob of an adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide, wherein said trimerization domain has a half-life in solution of at least one week, preferably at least two week and even more preferably at least one month.

The recombinant proteins of the present disclosure are encoded by nucleic acids. Vectors comprising these nucleic acids are used to transfect cells which express the recombinant proteins.

Therefore, in certain embodiments, the present disclosure relates to a nucleic acid encoding a recombinant protein of the present disclosure. The present disclosure also relates to a nucleic acid encoding a recombinant protein comprising a) a designed ankyrin repeat domain which binds to the knob of an adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide. In other embodiments, the present disclosure relates to a vector comprising a nucleic acid encoding a recombinant protein of the present disclosure. The present disclosure also relates to a vector comprising a nucleic acid encoding a recombinant protein comprising a) a designed ankyrin repeat domain which binds to the knob of an adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide.

In another embodiments, the present disclosure relates to an adenovirus comprising a nucleic acid encoding a recombinant protein of the present disclosure. In yet another embodiment, the present disclosure relates to an adenovirus comprising a vector comprising a nucleic acid encoding a recombinant protein of the present disclosure. In certain embodiments said adenovirus carries a TAYT mutation. In certain embodiments said adenovirus carries a HVR7 mutation.

In certain embodiments, the present disclosure also relates to an adenoviral vector comprising a nucleic acid encoding a recombinant protein comprising a) a designed ankyrin repeat domain which binds to the knob of an adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide.

The recombinant protein of the present disclosure may also comprise a flexible linker. If the recombinant protein comprising from the N- to the C-terminus a) a functional interleukin 7 polypeptide, b) a designed ankyrin repeat domain which binds to the knob of an adenovirus, and c) a trimerization domain, then said flexible linker is between said functional interleukin 7 polypeptide and said designed ankyrin repeat domain which binds to the knob of an adenovirus.

Therefore, in certain embodiments the present disclosure relates to a recombinant protein comprising from the N- to the C-terminus a) a functional interleukin 7 polypeptide, b) a flexible linker, c) a designed ankyrin repeat domain which binds to the knob of an adenovirus, and d) a trimerization domain.

If the recombinant protein comprising from the N- to the C-terminus a) a designed ankyrin repeat domain which binds to the knob of the adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide, then said flexible linker is between said trimerization domain and said functional interleukin 7 polypeptide.

Therefore, in certain embodiments the present disclosure relates to a recombinant protein comprising from the N- to the C-terminus a) a designed ankyrin repeat domain which binds to the knob of the adenovirus, b) a trimerization domain, c) a flexible linker, and d) a functional interleukin 7 polypeptide.

Principally any flexible linker can be used within the spirit of the present disclosure. Certain preferred flexible linkers are glycine-serine linkers. A particularly preferred flexible linker is a (Gly4Ser)4- linker.

Therefore, in certain embodiments the present disclosure relates to a recombinant protein comprising from the N- to the C-terminus a) a functional interleukin 7 polypeptide, b) a flexible linker, c) a designed ankyrin repeat domain which binds to the knob of an adenovirus, and d) a trimerization domain, wherein said flexible linker is a glycine-serine linker.

In other embodiments the present disclosure relates to a recombinant protein comprising from the

N- to the C-terminus a) a functional interleukin 7 polypeptide, b) a flexible linker, c) a designed ankyrin repeat domain which binds to the knob of an adenovirus, and d) a trimerization domain, wherein said flexible linker is a (Gly4Ser)4-linker.

The recombinant protein of the present disclosure may also comprise a short linker. The short linker is located between the designed ankyrin repeat domain which binds to the knob of an adenovirus and the trimerization domain. Therefore, in certain embodiments the present disclosure relates to a recombinant protein comprising from the N- to the C-terminus a) a functional interleukin 7 polypeptide, b) a designed ankyrin repeat domain which binds to the knob of an adenovirus, c) a short linker, and d) a trimerization domain.

In other embodiments the present disclosure relates to a recombinant protein comprising from the N- to the C-terminus a) a designed ankyrin repeat domain which binds to the knob of the adenovirus, b) a short linker, c) a trimerization domain, and d) a functional interleukin 7 polypeptide.

The short linker does not necessarily be present. Possible short linkers of the present disclosure are linkers which are no longer than four, no longer than three, no longer than two or only one amino acid long. A preferred short linker is glycine. Another preferred short linker is glycine-alanine. Most preferably the short linker is absent.

In certain embodiments, the present disclosure relates to a trimeric protein consisting of three recombinant proteins described herein above.

The recombinant proteins of the present disclosure can be expressed in prokaryotic cells, such as Escherichia coli, and in eukaryotic cells. Preferred eukaryotic cells are CHO cells. Other preferred eukaryotic cells are HEK293 cells, HEK293-T cells, HEK293-F cells, CHO-S cells and Sf9 cells. Therefore, in certain embodiments the present disclosure provides a eukaryotic cell expressing the recombinant protein of the present disclosure. In certain other the present disclosure provides a CHO cell expressing the recombinant protein of the present disclosure. In certain embodiments, the present disclosure relates to a eukaryotic cell expressing a recombinant protein comprising a) a functional interleukin 7 polypeptide, b) a designed ankyrin repeat domain which binds to the knob of the adenovirus, and c) a trimerization domain.

In certain embodiments, the present disclosure relates to a CHO cell expressing a recombinant protein comprising a) a functional interleukin 7 polypeptide, b) a designed ankyrin repeat domain which binds to the knob of the adenovirus, and c) a trimerization domain.

Aforementioned recombinant adenoviruses displaying a functional interleukin 7 polypeptide are used for the transduction of immune cells. Therefore, in certain embodiments the present disclosure relates to a method for the transduction of immune cells, said method comprising a. contacting said immune cells with i. a recombinant adenovirus displaying a functional interleukin 7 polypeptide, and ii. an agent capable of activating said immune cells, and b. incubating the mixture obtained in step a. for a time sufficient for transduction of said immune cells.

In other embodiments, the present disclosure relates to a method for the transduction of immune cells, said method comprising a. contacting said immune cells at about the same time with i. a recombinant adenovirus displaying a functional interleukin 7 polypeptide, and ii. an agent capable of activating said immune cells, and b. incubating the mixture obtained in step a. for a time sufficient for transduction of said immune cells.

In other embodiments, the present disclosure relates to a method for the transduction of immune cells, said method comprising a. contacting said immune cells simultaneously with i. a recombinant adenovirus displaying a functional interleukin 7 polypeptide, and ii. an agent capable of activating said immune cells, and b. incubating the mixture obtained in step a. for a time sufficient for transduction of said immune cells.

The method disclosed herein can be employed used for the transduction of any immune cells. Such immune cells include T cells, NK cells, monocytes, macrophages or dendritic cells. Said T cells may be CD4-positve T cells or CD8-positive T cells. Said immune cells may also be CD4-positve T cells which are CD25^hlgh and CD127¹⁰”, cells known as T regulatory cell (Treg cells). Therefore, in certain embodiments, the present disclosure relates to a method for the transduction of T cells, NK cells, monocytes, macrophages or dendritic cells, said method comprising a. contacting said T cells, NK cells, monocytes, macrophages or dendritic cells at about the same time with i. a recombinant adenovirus displaying a functional interleukin 7 polypeptide, and ii. an agent capable of activating said immune cells, and b. incubating the mixture obtained in step a. for a time sufficient for transduction of said immune cells .

In other embodiments, the present disclosure relates to a method for the transduction of T cells, said method comprising a. contacting said T cells at about the same time with i. a recombinant adenovirus displaying a functional interleukin 7 polypeptide, and ii. an agent capable of activating said immune cells, and b. incubating the mixture obtained in step a. for a time sufficient for transduction of said T cells.

In other embodiments, the present disclosure relates to a method for the transduction of CD4- positive T cells, said method comprising a. contacting said CD4-positive T cells at about the same time with i. a recombinant adenovirus displaying a functional interleukin 7 polypeptide, and ii. an agent capable of activating said immune cells, and b. incubating the mixture obtained in step a. for a time sufficient for transduction of said CD4- positive T cells.

In other embodiments, the present disclosure relates to a method for the transduction of CD8- positive T cells, said method comprising a. contacting said CD8-positive T cells at about the same time with i. a recombinant adenovirus displaying a functional interleukin 7 polypeptide, and ii. an agent capable of activating said immune cells, and b. incubating the mixture obtained in step a. for a time sufficient for transduction of said CD8- positive T cells.

In other embodiments, the present disclosure relates to a method forthe transduction ofTreg cells, said method comprising a. contacting said Treg cells at about the same time with i. a recombinant adenovirus displaying a functional interleukin 7 polypeptide, and ii. an agent capable of activating said immune cells, and b. incubating the mixture obtained in step a. for a time sufficient for transduction of said Treg cells.

In other embodiments, the present disclosure relates to a method for the transduction of NK cells, said method comprising a. contacting said NK cells at about the same time with i. a recombinant adenovirus displaying a functional interleukin 7 polypeptide, and ii. an agent capable of activating said immune cells, and b. incubating the mixture obtained in step a. for a time sufficient for transduction of said NK cells.

In the method of the present disclosure, immune cells are activated at about the same time or simultaneously to the transduction with a recombinant adenovirus displaying a functional interleukin 7 polypeptide. Said activation is brought about by an agent capable of activating said immune cells. Such agents include but are not limited to e.g. DynaBeads (e.g. Dynabeads™ Human T-Expander CD3/CD28, Gibco, Catalogue number 11141D; Dynabeads™ Human T-Expander CD3/CD28/CD137, Gibco, Catalogue number 11162D), TransAct (T Cell TransAct™ human, Miltenyi, Catalogue number 130-111-160), Polybrene and PMA (l-Methoxy-2-propylacetat). These substances lead to an activation of several immune cells.

Therefore, in certain embodiments, the present disclosure relates to a method for the transduction of immune cells, said method comprising a. contacting said immune cells at about the same time with i. a recombinant adenovirus displaying a functional interleukin 7 polypeptide, and ii. an agent capable of activating said immune cells, and b. incubating the mixture obtained in step a. for a time sufficient for transduction of said immune cells, wherein said agent capable of activating said immune cells is DynaBeads.

In other embodiments, the present disclosure relates to a method for the transduction of immune cells, said method comprising a. contacting said immune cells simultaneously with i. a recombinant adenovirus displaying a functional interleukin 7 polypeptide, and ii. an agent capable of activating said immune cells, and b. incubating the mixture obtained in step a. for a time sufficient for transduction of said immune cells, wherein said agent capable of activating said immune cells is TransAct.

In other embodiments, the present disclosure relates to a method for the transduction of immune cells, said method comprising a. contacting said immune cells simultaneously with i. a recombinant adenovirus displaying a functional interleukin 7 polypeptide, and ii. an agent capable of activating said immune cells, and b. incubating the mixture obtained in step a. for a time sufficient for transduction of said immune cells, wherein said agent capable of activating said immune cells is Polybrene.

In other embodiments, the present disclosure relates to a method for the transduction of immune cells, said method comprising a. contacting said immune cells simultaneously with i. a recombinant adenovirus displaying a functional interleukin 7 polypeptide, and ii. an agent capable of activating said immune cells, and b. incubating the mixture obtained in step a. for a time sufficient for transduction of said immune cells, wherein said agent capable of activating said immune cells is PMA.

The method disclosed herein above can be performed in vivo or in vitro. The recombinant proteins of the present disclosure, the nucleic acids encoding the recombinant proteins and trimeric proteins of the present disclosure, the vectors containing the nucleic acids of the present disclosure and the recombinant adenoviruses containing the recombinant proteins, the nucleic acids or the vectors of the present disclosure have numerous uses, such as the use in an adenoviral delivery system. Therefore, in certain embodiments the present disclosure provides the use of the recombinant proteins of the present disclosure in an adenoviral delivery system. In other embodiments the present disclosure provides the use of the nucleic acids encoding the recombinant proteins of present disclosure in an adenoviral delivery system. In other embodiments the present disclosure provides the use of the vectors containing the nucleic acids of the present disclosure in an adenoviral delivery system. In other embodiments the present disclosure provides the use of the adenoviruses containing the recombinant proteins, the nucleic acids, or the vectors of the present disclosure in an adenoviral delivery system. Preferably, in said adenoviral delivery system immune cell, are contacted about the same time with a recombinant adenovirus displaying a functional interleukin 7 polypeptide and an agent capable of activating said cells.

In certain embodiments the present disclosure provides a method to treat a patient, said method comprising administering to a patient in need of a recombinant protein of the present disclosure. In certain embodiments the present disclosure provides a method to treat a patient, said method comprising administering to a patient in need of a nucleic acid encoding a recombinant protein of the present disclosure. In certain embodiments the present disclosure provides a method to treat a patient, said method comprising administering to a patient in need thereof a vector containing a nucleic acid of the present disclosure. In certain embodiments the present disclosure provides a method to treat a patient, said method comprising administering to a patient in need thereof a recombinant adenovirus containing a recombinant protein, a nucleic acid, or a vector of the present disclosure. Preferably, in said method to treat a patient immune cell, such as T cells, are contacted about the same time with a recombinant adenovirus displaying a functional interleukin 7 polypeptide and an agent capable of activating said T cells or NK cells. More preferably, said immune cell, such as T cells or NK cells, are contacted simultaneously with a recombinant adenovirus displaying a functional interleukin 7 polypeptide and an agent capable of activating said T cells or NK cells.

The recombinant proteins of the present disclosure, the nucleic acids encoding the recombinant proteins of the present disclosure, the vectors containing the nucleic acids of the present disclosure, the recombinant adenovirus or set of recombinant adenoviruses displaying or containing the recombinant proteins, the nucleic acids or the vectors of the present disclosure, and the eukaryotic cells containing the recombinant adenovirus or set of recombinant adenoviruses of the present disclosure can be used in the treatment or prevention of any disease or disorder. In certain embodiments the recombinant adenovirus or set of recombinant adenoviruses display an antigen-binding moiety with specificity for an antigen expressed on T cells and a component which is involved in the activation of T cells, such as interleukin 7.

In certain embodiments of the present disclosure, the recombinant adenovirus or the set of recombinant adenoviruses are capable of transducing T cells. Human T cells poorly express coxsackie and adenoviral receptors (CAR) through which transduction of adenoviruses is typically mediated, as well as RGD binding integrins which are involved in subsequent interactions. Adenoviruses armored with adapters as described in the present disclosure overcome these hurdles by generating a sufficiently high specificity to the T cells that enables adenoviral infection of the cells.

In certain embodiments of the present disclosure, the recombinant adenovirus comprise a functional interleukin 7 polypeptide. In certain embodiments, the functional interleukin 7 polypeptide comprises the amino acid sequence of SEQ ID No. 3. In certain embodiments, the functional interleukin 7 polypeptide consists of the amino acid sequence of SEQ ID No. 3. In certain embodiments, the functional interleukin 7 polypeptide comprises the amino acid sequence of SEQ ID No. 3 with one, two, three, four or five amino acid substitutions, insertions, or deletions. In certain embodiments, the functional interleukin 7 polypeptide is a variant of the polypeptide consisting of the amino acid sequence of SEQ ID No. 3, wherein said variant is functionally equivalent to the polypeptide consisting of the amino acid sequence of SEQ ID No. 3. In certain embodiments, the functional interleukin 7 polypeptide comprises the amino acid sequence of SEQ ID No. 4. In certain embodiments, the functional interleukin 7 polypeptide consists of the amino acid sequence of SEQ ID No. 4. In certain embodiments, the functional interleukin 7 polypeptide comprises the amino acid sequence of SEQ ID No. 4 with one, two, three, four or five amino acid substitutions, insertions, or deletions. In certain embodiments, the functional interleukin 7 polypeptide is a variant of the polypeptide consisting of the amino acid sequence of SEQ ID No. 4, wherein said variant is functionally equivalent to the polypeptide consisting of the amino acid sequence of SEQ ID No. 4.

In specific embodiments, the present disclosure relates to a recombinant protein comprising from the N- to the C-terminus a) a functional interleukin 7 polypeptide, b) a designed ankyrin repeat domain which binds to the knob of the adenovirus, and c) a trimerization domain. In certain embodiments, the functional interleukin 7 polypeptide comprises the amino acid sequence of SEQ ID No. 3. In certain embodiments, the functional interleukin 7 polypeptide consists of the amino acid sequence of SEQ ID No. 3. In certain embodiments, the functional interleukin 7 polypeptide comprises the amino acid sequence of SEQ ID No. 3 with one, two, three, four or five amino acid substitutions, insertions, or deletions. In certain embodiments, the functional interleukin 7 polypeptide is a variant of the polypeptide consisting of the amino acid sequence of SEQ ID No. 3, wherein said variant is functionally equivalent to the polypeptide consisting of the amino acid sequence of SEQ ID No. 3. Said recombinant proteins may also comprise more than one of the entities selected from a functional interleukin 7 polypeptide.

In specific embodiments, the present disclosure relates to a recombinant protein comprising from the N- to the C-terminus a) a functional interleukin 7 polypeptide, b) a designed ankyrin repeat domain which binds to the knob of the adenovirus, and c) a trimerization domain. In certain embodiments, the functional interleukin 7 polypeptide comprises the amino acid sequence of SEQ ID No. 4. In certain embodiments, the functional interleukin 7 polypeptide consists of the amino acid sequence of SEQ ID No. 4. In certain embodiments, the functional interleukin 7 polypeptide comprises the amino acid sequence of SEQ ID No. 4 with one, two, three, four or five amino acid substitutions, insertions, or deletions. In certain embodiments, the functional interleukin 7 polypeptide is a variant of the polypeptide consisting of the amino acid sequence of SEQ ID No. 4, wherein said variant is functionally equivalent to the polypeptide consisting of the amino acid sequence of SEQ ID No. 4. Said recombinant proteins may also comprise more than one of the entities selected from a functional interleukin 7 polypeptide.

It will also be understood that also variants of DARPin 1D3 may be used within the spirit of the present disclosure. In other words, the amino acid sequence of such modified DARPin 1D3 does not need to be identical to that of amino acid sequence of SEQ ID No. 2, but may contain amino acids mutations, provided that the function of DARPin 1D3, i.e. binding to the knob of an adenovirus is preserved. Also, DARPins different than 1D3, but having the same target specificity, may be used within the scope of the present disclosure. Such new DARPin may for example be selected in a new screening campaign. Also binding entities different than DARPins, i.e. binders based on a different scaffold, but having the same target specificity as 1D3 might be used.

In certain embodiments, the present disclosure relates to recombinant proteins comprising a designed ankyrin repeat domain that binds to the knob of an adenovirus. The present disclosure can however also be practiced with other viruses. If another virus is used a designed ankyrin repeat domain needs to be selected that binds to the knob of such virus. Therefore, in certain embodiments the present disclosure relates to recombinant proteins comprising a) a designed ankyrin repeat domain which binds to the knob of the adenovirus, b) trimerization domain, and c) a said functional interleukin 7 polypeptide. Viruses, other than adenoviruses of serotype 5, can also be used within the spirit of the present disclosure. For example, such viruses can be engineered to carry the knob of an adenovirus of serotype 5. The recombinant protein disclosed herein, in particular recombinant protein comprising DARPin 1D3, may then be used with such viruses. Therefore, in certain embodiments the present disclosure provides a recombinant virus or a set of recombinant viruses comprising a recombinant protein comprising: a) a functional interleukin 7 polypeptide, b) a designed ankyrin repeat domain which binds to the knob of an adenovirus, and c) a trimerization domain, wherein said designed ankyrin repeat domain binds to the knob of an adenovirus of subtype 5.

Also, other trimerization domains known to the skilled person may be used for the formation or trimers. Without being limited other potential trimerization domains include the trimerization domain involved in collagen folding (Int J Biochem Cell Biol 44:21-32 (2012)), the trimerization domain of T4 phage fibritin (PLoS One 7:e43603 (2012)) or the GCN4-based isoleucine zipper (J Biol Chem 290: 7436-42 (201 5)).

Therefore, in certain embodiments, the present disclosure relates to a nucleic acid encoding a recombinant protein of the present disclosure. The present disclosure also relates to a nucleic acid encoding a recombinant protein comprising a) a designed ankyrin repeat domain which binds to the knob of an adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide.

In other embodiments, the present disclosure relates to a vector comprising a nucleic acid encoding a recombinant protein of the present disclosure. The present disclosure also relates to a vector comprising a nucleic acid encoding a recombinant protein comprising a) a designed ankyrin repeat domain which binds to the knob of an adenovirus, b) a trimerization domain, and c) a functional interleukin 7 polypeptide.

Therefore, in certain embodiments the present disclosure relates to a recombinant protein comprising from the N- to the C-terminus a) a functional interleukin 7 polypeptide, b) a flexible linker, c) a designed ankyrin repeat domain which binds to the knob of an adenovirus, and d) a trimerization domain, wherein said flexible linker is a glycine-serine linker. In other embodiments the present disclosure relates to a recombinant protein comprising from the

The recombinant protein of the present disclosure may also comprise a short linker. The short linker is located between the designed ankyrin repeat domain which binds to the knob of an adenovirus and the trimerization domain.

Therefore, in certain embodiments the present disclosure relates to a recombinant protein comprising from the N- to the C-terminus a) a functional interleukin 7 polypeptide, b) a designed ankyrin repeat domain which binds to the knob of an adenovirus, c) a short linker, and d) a trimerization domain.

The recombinant proteins of the present disclosure, the nucleic acids encoding the recombinant proteins and trimeric proteins of the present disclosure, the vectors containing the nucleic acids of the present disclosure and the recombinant adenoviruses or set of recombinant adenoviruses containing the recombinant proteins, the nucleic acids or the vectors of the present disclosure have numerous uses, such as the use in an adenoviral delivery system. Therefore, in certain embodiments the present disclosure provides the use of the recombinant proteins of the present disclosure in an adenoviral delivery system. In other embodiments the present disclosure provides the use of the nucleic acids encoding the recombinant proteins of present disclosure in an adenoviral delivery system. In other embodiments the present disclosure provides the use of the vectors containing the nucleic acids of the present disclosure in an adenoviral delivery system. In other embodiments the present disclosure provides the use of the adenoviruses containing the recombinant proteins, the nucleic acids or the vectors of the present disclosure in an adenoviral delivery system.

The recombinant proteins of the present disclosure, the nucleic acids encoding the recombinant proteins and trimeric proteins of the present disclosure, the vectors containing the nucleic acids of the present disclosure and the recombinant adenoviruses or set of recombinant adenoviruses containing the recombinant proteins, the nucleic acids or the vectors of the present disclosure can also be used for the transduction of immune cells. In certain embodiments the present disclosure provides the use of the recombinant proteins of the present disclosure for the transduction of immune cells. In certain embodiments said immune cells are T cells. In other embodiments said immune cells are NK cells, monocytes, macrophages or dendritic cells. In certain embodiments the present disclosure provides the use of the recombinant proteins of the present disclosure for the transduction of T cells. In other embodiments said immune cells are NK cells, monocytes, macrophages or dendritic cells. In certain other embodiments the present disclosure provides the use of the nucleic acids encoding the recombinant proteins of present disclosure for the transduction of T cells. In other embodiments said immune cells are NK cells, monocytes, macrophages or dendritic cells. In certain embodiments the present disclosure provides the use of the vectors containing the nucleic acids of the present disclosure for the transduction of T cells. In other embodiments said immune cells are NK cells, monocytes, macrophages or dendritic cells. In certain embodiments the present disclosure provides the use of the adenoviruses containing the recombinant proteins, the nucleic acids or the vectors of the present disclosure for the transduction of T cells. In other embodiments said immune cells are NK cells, monocytes, macrophages or dendritic cells.

The recombinant proteins of the present disclosure, the nucleic acids encoding the recombinant proteins and trimeric proteins of the present disclosure, the vectors containing the nucleic acids of the present disclosure and the recombinant adenoviruses or set of recombinant adenoviruses containing the recombinant proteins, the nucleic acids or the vectors of the present disclosure can also be used in medicine. Therefore, in certain embodiments the present disclosure provides the use of the recombinant proteins of the present disclosure in medicine. In other embodiments the present disclosure provides the use of the nucleic acids encoding the recombinant proteins of the present disclosure in medicine. In other embodiments the present disclosure provides the use of the vectors containing the nucleic acids of the present disclosure in medicine. In other embodiments the present disclosure provides the use of the adenoviruses containing the recombinant proteins, the nucleic acids or the vectors of the present disclosure in medicine.

In certain embodiments the present disclosure provides a method to treat a patient, said method comprising administering to a patient in need of a recombinant protein of the present disclosure. In certain embodiments the present disclosure provides a method to treat a patient, said method comprising administering to a patient in need of a nucleic acid encoding a recombinant protein of the present disclosure. In certain embodiments the present disclosure provides a method to treat a patient, said method comprising administering to a patient in need thereof a vector containing a nucleic acid of the present disclosure. In certain embodiments the present disclosure provides a method to treat a patient, said method comprising administering to a patient in need thereof a recombinant adenovirus or a set of recombinant adenoviruses containing a recombinant protein, a nucleic acid or a vector of the present disclosure.

Principally, the recombinant proteins of the present disclosure, the nucleic acids encoding the recombinant proteins of the present disclosure, the vectors containing the nucleic acids of the present disclosure, the recombinant adenovirus or set of recombinant adenoviruses displaying or containing the recombinant proteins, the nucleic acids or the vectors of the present disclosure, and the eukaryotic cells containing the recombinant adenovirus or set of recombinant adenoviruses of the present disclosure can be used in the treatment or prevention of any disease or disorder.

Examples

Example 1: General experimental procedures

Human blood samples

Buffy coats from human donors were acquired from the Blutspende Bern, Bern, Switzerland. After Ficoll-Paque (GE Healthcare) gradient separation PBMC cells were aliquoted and frozen to be thawed before each assay.

After thawing either PBMCs were directly used in assays or T cells were first isolated using a Pan-T cell isolation kit from Miltenyi Biotech (Cat number 130-096-535) as described by the manufacturer.

For activation of immune cells, cells were put in RPMI 1640 at 0.5 x 10⁶cells/ml and supplemented with a 3:1 ratio of Dynabeads Human T-Expander CD3/CD28 (ThermoFisher, Cat number 11141D) to T cells, 50 lU/ml human IL-2 IS (Miltenyi Biotech, Cat number 130-097-743), recombinant adenovirus displaying a functional interleukin 7 polypeptide and incubated for at least 48 hours thereafter, sometimes as long as 120 hours.

Viral vector generation

The replication-deficient HAdV-C5 helper virus (HV) contains an E1/E3 deletion, a loxP-flanked packaging signal and 4 mutations in the hypervariable region 7 (HVR7) of the hexon protein (I421G, T423N, E424S and L426Y). The HV was generated as previously described (Nat. Commun. 9, 450 (2018)) or ordered from Vector Biolabs (Malvern, PA/USA). The helper-dependent adenovirus genome containing no adenoviral genes, but the adenoviral packaging sequences was propagated using the above HV as described by Brucher et al. (Mol Ther Methods Clin Dev (2021) 20:572-86). In short, a Cre- expressing HEK293 cell line was transfected with the helper-dependent genomes and co-transduced with the helper HAdV-C5 vector for replication. Purification was performed via density separation. Similarly, the cell line 116 was transfected with the reporter plasmid containing the HAdV-C5 packaging signal and co-transduced with a helper HAdV-C5 for replication. Purification was performed via two CsCI gradients at 250,000 g. Expression and purification of the recombinant proteins and adapter molecules

The trimeric adapters were cloned into the mammalian expression plasmid pcDNA3.1. Alternatively other plasmids, such as pTwist CMV WPRE Neo can be used. DNA synthesis and expression plasmid construct assembly was carried out by a commercial vendor (TWIST, San Francisco, USA). Alternatively Gibson assembly strategies can be used (Gibson et al., Nat. Methods 343-5 (2009)). The adapter contained an N-terminal Ig kappa leader peptide, a TEV-cleavable Strep-tag II and His-tag. Generally any N- or C-terminal peptide affinity purification tag (e.g. polyhistidine-tag, Strep-tag) or an epitope tag (e.g. FLAG-tag, polyhistine-tag, Myc-tag), or a combination thereof, can be used. Likewise, any proteolytic cleavage site (e.g. for furin, thrombin, TEV, or the like) may be introduced for affinity purification or epitope tag removal. The adapters were cloned into pcDNA3.1, as previously described (Adv. Cancer Res. 115, 39-67 (2012)). The retargeting domain is flanked by a BamHI and an Hindlll site for ready exchange of the domain. Adapters were expressed in Expi293 HEK cells as described (Protein Expr. Purif. 92, 67-76 (2013)). Generally, any mammalian expression host can be used, e.g. HEK293. Following seven days expression, supernatants were 1:1000 dialyzed in PBS pH 7.4 using dialysis tubes with a MWCO cutoff of 12-14 kDa at 4°C. During 24 h, the buffer was exchanged four times 1:10. Dialyzed supernatants were subjected 2.5 ml equilibrated nickel-nitrilotriacetic acid (Ni-NTA) resin (Thermo Fisher) in a PD-10 column (Merck Millipore). All columns were washed with 5 column volumes 20 mM imidazole, 10% glycerol, PBS pH 8.0 and then additionally with 5 column volumes of 500 mM NaCI, 50 mM Tris HCI pH 8.0. The samples were then eluted using 0.7 M imidazole in PBS pH 8.0, followed by subsequent 3C cleavage (GenScript) of the tags during dialysis against 20 mM Hepes at pH 7.4. An additional purification step included an anion exchange chromatography using a MonoQ. column (GE Healthcare). Purified protein was dialyzed four times 1:100 in 24h in endotoxin-free PBS (Merck Millipore) and then shock frozen in liquid nitrogen and stored at -80°C until usage.

In vitro transduction

PBMCs, T cells orTreg cells were thawed and directly taken into experiments. Cells were distributed in flat bottom 96-well plates with 5 x 10⁴ cells/well in 100 pl RPMI 1640 supplemented with human IL- 2 (50 lU/ml). Adenoviral vectors were incubated for 1 h at 4°C with adapters coupled to IL-7 and/or a- CD28 scFv (termed "retargeted") or IL2 adapter, or a blocking adapter containing the consensus DARPin E2_5 (J Mol Biol (2003) 332: 489-503) (termed "blocking adapter"). Genomic viral particles were also variable depending on specific experiments. Activation of immune cells was performed on some experiments as mentioned on below on the further Examples given, according to the Human Blood samples experimental protocol. Transgene activity was determined by flow cytometry as early as 48 h after transduction.

Flow Cytometry

For in vitro assays cells were centrifuged at 500-750 g for 5 min and supernatant was discarded. The pellet was resuspended in PBS containing 2% FBS and 2mM EDTA (FACS buffer). Cells were then kept at 4°C in the darkfor 30 min and washed twice with PBS. Cells were then resuspended in PBS containing 2% PFA and fixed for 15 min at room temperature. Remaining PFA was then quenched by adding 5x of the fixation volume of FACS buffer.

Dead cells were stained using the fixable viability dye Zombie (BioLegend) for 15 min at room temperature, followed by blocking of Fc receptors with TruStain FcX (BioLegend) for 20 min at 4°C. Following this, cell surface proteins were stained for 20 min at 4°C. Nuclear proteins were stained for 60 min at room temperature after permeabilization and fixation (Regulatory T cell Staining Kit, eBioscience). Samples were analyzed on a BD LSRFortessa™ flow cytometer (BD Biosciences). Commercially available antibodies were used in FACS experiments. GFP was detected as the transgene transduced.

Recombinant adapter molecules

The following recombinant adapter molecules were used for exemplification of the present invention.

The recombinant adapter molecule comprising the functional interleukin 7 polypeptide has the following amino acid sequence:

METDTLLLWVLLLWVPGSTGRWSHPQFEKSHHHHHHHHENLYFQSGSFHVSFRYI FGLPPLILVLL PVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAAR KLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLND LCFLKRLLQEIKTCWNKILMGTKEHKLGGGGSGGGGSGGGGSGGGGSRSDLGKKLLEAARAGQDDE VRI LMANGADANAYDHYGRT PLHMAAAVGHLE I VEVLLRNGADVNAVDTNGT T PLHLAAS LGHLE I VEVLLKYGADVNAKDATGITPLYLAAYWGHLEIVEVLLKHGADVNAQDKFGKTPFDLAIDNGNEDI AEVLQGVRI FAGNDPAHTATGSSGI SSPTPALTPLMLDEATGKLWWDGQKAGSAVGILVLPLEGT ETALTYYKSGTFATEAIHWPESVDEHKKANAFAGSALSHAALP ( SEQ ID No . 6)

Aforementioned recombinant adapter molecule is encoded by a nucleic acid of the following sequence:

GCCACCATGGAAACGGATACTCTGCTGCTCTGGGTGTTGTTGCTTTGGGTTCCTGGATCAACGGGG AGATGGAGCCACCCCCAGTTCGAGAAGTCTCACCACCATCACCATCATCACCACGAGAACCTGTAC TTCCAGAGCGGATCCTTCCATGTTTCTTTTAGGTATATCTTTGGACTTCCTCCCCTGATCCTTGTT CT GT T GCCAGT AGCAT CAT C TGATT GT GAT AT T GAAGGT AAAGAT GGCAAAC AAT ATGAGAGT GT T CTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAAT GAATTTAACTTTTTTAAAAGACATATCTGTGATGCTAATAAGGAAGGTATGTTTTTATTCCGTGCT GCTCGCAAGTTGAGGCAATTTCTTAAAATGAATAGCACTGGTGATTTTGATCTCCACTTATTAAAA GTTTCAGAAGGCACAACAATACTGTTGAACTGCACTGGCCAGGTTAAAGGAAGAAAACCAGCTGCC CT GGGT GAAGCCCAACCAAC AAAGAGT T T GGAAGAAAAT AAAT CT T TAAAGGAACAGAAAAAACT G AAT GAC T T GT GT T T C C T AAAGAGAC T AT T ACAAGAG AT AAAAAC T T GT T GGAAT AAAAT T T T GAT G GGCACTAAAGAACACAAGCTTGGCGGCGGTGGCTCTGGCGGTGGTGGCTCTGGCGGTGGCGGTTCT GGCGGTGGTGGCTCTCGGAGCGACTTGGGAAAGAAACTCCTCGAAGCTGCTAGAGCAGGTCAAGAT GATGAGGTCCGAATACTGATGGCTAATGGAGCAGATGCCAATGCTTACGATCACTACGGAAGAACG CCCTTGCATATGGCTGCCGCCGTGGGCCATCTTGAAATTGTCGAGGTGTTATTGCGGAATGGGGCG GATGTGAATGCAGTGGATACAAACGGGACGACACCTCTCCACCTGGCCGCATCTCTTGGCCATCTG GAGATTGTGGAAGTACTCCTCAAATATGGAGCAGATGTCAATGCCAAAGATGCTACTGGAATCACC CCCTTATATTTGGCCGCGTACTGGGGCCATCTCGAAATTGTAGAGGTGCTGCTCAAACATGGAGCT GATGTCAATGCGCAAGATAAGTTTGGGAAAACTCCCTTTGATTTAGCGATAGATAATGGAAATGAA GATATCGCAGAGGTCCTTCAGGGCGTCCGTATCTTTGCAGGGAACGACCCAGCTCACACTGCAACC GGAAGTTCAGGCATTTCTAGCCCGACTCCCGCACTCACCCCGTTGATGTTAGACGAGGCTACAGGG AAGCTCGTTGTCTGGGATGGTCAAAAGGCGGGCTCTGCCGTGGGAATTTTAGTATTACCTCTGGAA GGTACCGAAACTGCACTGACTTACTATAAAAGTGGTACTTTCGCCACAGAAGCTATCCATTGGCCT GAGAGCGTCGACGAGCATAAGAAAGCAAATGCTTTCGCAGGATCAGCTCTCTCACATGCGGCCCTG CCTTAATGA ( SEQ ID No . 7 )

Example 2: Rational to use IL-7 as a targeting moiety

Various publicly available sources of genomic and transcriptional data were screened for molecules. As a first source, transcriptional data from Immunity (2019) 50:1317-34 were queried for different immune cell types and the IL7 receptor gene expression using the SPRING plot tools available at: https://kleintools.hms.harvard.edu/tools/springViewer_l_6_clev.html7clatasets/Zilionis2019/human /NSCLC_and_blood_immune

Results are shown in Figure 1. As can be seen, the IL7 gene is highly and mainly expressed in T cells and with discrete expression on NK cells, monocytes, macrophages and dendritic cells. As another source based on protein expression data, we consulted the human protein atlas, Figure 2, to understand the pattern of expression of the IL7 receptor across healthy tissues and understand what the potential off-targeting of the IL-7 adapter is. Querying the database for the IL-7 receptor it is possible to appreciate that lung and gastrointestinal systems have a moderate expression of this receptor, albeit significantly inferior to organs mostly composed by immune cells. This suggests that the IL7 receptor is a receptor plausible to be targeted to transduce immune cells in vivo.

Based on these analyses it was concluded that IL-7 is a molecule that might be suited for the transduction of human cells, specifically, immune cells, such as T cells, NK cells, monocytes, macrophages, or dendritic cells.

Example 3: Transduction of multiple cell types is possible using IL-7-retargeted virus like particles

In this series of experiments, the efficacy to transduce different cell types was tested by retargeting virus like particles with IL-7-retargeting adapters. Untargeted VLPs without any adapter were used as controls. Likewise, a non-treated condition, where cells were not contacted with neither VLP nor an adapter were also included as a control. The NALM-6 tumor cell line (ATCC: CRL-3273) was used as a surrogate for a B cell line. PBMCs activated with Dynabeads (Human T-Expander CD3/CD28; ThermoFisher) and human recombinant human IL-2 (50 IU per ml; Miltenyi Biotech) were simultaneously incubated with retargeted VLPs. PBMCs were also used in conditions without activation. In some conditions T cells were isolated from PBMCs and transduced without the presence of other immune cells.

Human Blood samples, in vitro transduction, flow cytometry and recombinant adapter molecules are as stated in Example 1. Activation was performed with a ratio of Dynabeads to T cells of 3 to 1. Adapter to knob ratio was of 32.5 to 1. Per cell a total of 100 genomic particles were used.

Measured was GFP by flow cytometry. Results are shown in Figure 3-8. Figure 3 shows the percentage of NALM-6 expressing GFP and it was observed that the IL-7 adapter improves retargeting discretely, albeit at very low percentages of total cells transduced. Figure 4 shows the percentage of activated PBMCs expressing GFP, with an effective retargeting leading to transduction of considerable numbers of total cells. Figure 5 shows the percentage of CD4+ activated PBMCs expressing GFP, with an effective retargeting leading to transduction of considerable numbers of total cells. Figure 6 shows the percentage of CD8+ activated PBMCs expressing GFP, with an effective retargeting leading to transduction of considerable numbers of total cells. Figure 7 shows the percentage of activated T cells expressing GFP, with an effective retargeting leading to transduction of considerable numbers of total cells. Figure 8 shows the percentage of CD3+ non-activated PBMCs expressing GFP, where the total number of transduced cells is discrete albeit a clear improvement to untargeted conditions, demonstrating the potential of this retargeting strategy to transduce T cells without the need for activation. Results show that the 11-7 retargeting adapter efficiently enables the transduction of immune cells.

Example 4: In vivo transduction of T cells in immunodeficient mice

In this study NSG mice will be used. NSG do not have an own immune system and do not carry any T cells. 4xl0⁶ human PBMCs and 8xlO¹⁰ retargeted viral particles are injected to the mice. Control mice are transduced with untargeted vector or are injected with PBMCs only. After 48 h the T cell population is analyzed. The T cell population will also separated into T helper cells (CD4+) and cytotoxic T cells (CD8+) which will be analyzed separately.

Example 5: List of other interleukins potentially capable of mediating retargeted entry of VLPs into cells.

Other interleukins, potentially capable of retargeting and entering the VLP into cells include:

• Interleukin 1 alpha - IL-la - ILIA

• Interleukin 1 beta - IL-lb — IL1B

• Interleukin 33 - IL-33 - IL33

• Interleukin 18 - IL-18 - IL18

• Interleukin 37 - IL-37 - IL37

• Interleukin 36 - IL-36 - IL36

• Interleukin 38 - IL-38 - IL38

• Interleukin 2 - IL-2 - IL2

• Interleukin 4 - IL-4 - IL4

• Interleukin 7 - IL-7 - IL7

Interleukin 9 - IL-9 - IL9 • Interleukin 15 - IL-15 — IL15

• Interleukin 21 - IL-21 - IL21

• Interleukin 3 - IL-3 - IL3

• Interleukin 5 - IL-5 - IL5

• Interleukin 6 - IL-6 - IL6

• Interleukin 11 - IL-11 — IL11

• Interleukin 31 - IL-31 - IL31

• Interleukin 10 - IL-10 - IL10

• Interleukin 19 - IL-19 - IL19

• Interleukin 20 - IL-20 - IL20

• Interleukin 22 - IL-22 - IL22

• Interleukin 24 - IL-24 - IL24

• Interleukin 26 - IL-26 - IL26

• Interleukin 12 - IL-12 - IL12

• Interleukin 23 - IL-23 - IL23

• Interleukin 27 (30) - IL-27 (30) - IL27 (30)

• Interleukin 35 - IL-35 - IL35

• Interleukin 17A/F - I L-17A/F - IL17A/F

• Interleukin 17B - IL-17B - IL17B

• Interleukin 17C - IL-17C - IL17C

• Interleukin 17D - IL-17D - IL17D

• Interleukin 17E - IL-17E — IL17E

• Interleukin 28A - IL-28A - IL28A

• Interleukin 29 - IL-29 - IL29

• Interleukin 8 - IL-8 - IL8

• Interleukin 14 alpha - IL-14a - IL14A

• Interleukin 14 beta - IL-14b - IL14B

• Interleukin 16 - IL-16 - IL16

• Interleukin 32 - IL-32 - IL32

• Interleukin 34 - IL-34 - IL34

• Interleukin 29 - IL-29 - IL29

Claims

1. A recombinant adenovirus displaying a functional interleukin 7 polypeptide.

2. A recombinant adenovirus according to claim 1, wherein said functional interleukin 7 polypeptide is displayed on the knob of said adenovirus.

3. A recombinant adenovirus according to claim 1 or 2, wherein said functional interleukin 7 polypeptide is comprised in a recombinant protein comprising from the N- to the C-terminus b) said functional interleukin 7 polypeptide, d) a designed ankyrin repeat domain which binds to the knob of the adenovirus, and e) a trimerization domain.

4. A recombinant adenovirus according to any one of claims 1-3, wherein said functional interleukin 7 polypeptide comprises the amino acid sequence of SEQ ID No. 3 or 4.

5. A recombinant adenovirus according to claim 3 or 4, wherein said designed ankyrin repeat domain that binds to a knob of an adenovirus comprises the amino acid sequence of SEQ ID No. 2, and/or wherein said trimerization domain is or is derived from the capsid protein SHP of lambdoid phage 21, preferably wherein said trimerization domain comprises the amino acid sequence of SEQ ID No. 1.

6. The recombinant adenovirus according to any one of the preceding claims, wherein said adenovirus is of adenovirus serotype 5 or wherein said adenovirus comprises a knob of an adenovirus of serotype 5.

7. A eukaryotic cell expressing or producing a recombinant adenovirus according to any one of the preceding claims.

8. A method for the transduction of immune cell, said method comprising a. contacting said immune cell with a recombinant adenovirus according to any one of claims 1-6, and b. incubating the mixture obtained in step a. for a time sufficient for transduction of said immune cells.

9. The method according to claim 8, wherein said immune cells are T cells, preferably wherein said T cells are CD4-positve T cells, CD8-positive T cells or Treg cells.

10. The method according to claim 8 or 9, wherein said method is performed in vivo.

11. The method according to any one of claims 8-10, wherein in step a. said immune cells are additionally contacted with an agent capable of activating said immune cells, preferably wherein said agent capable of activating said immune cells is selected from DynaBeads, TransAct, Polybrene and PMA (l-Methoxy-2-propylacetat).

12. A recombinant protein comprising from the N- to the C-terminus b) a functional interleukin 7 polypeptide, c) a designed ankyrin repeat domain which binds to the knob of an adenovirus, and d) a trimerization domain.

13. A trimeric protein consisting of three recombinant proteins according to claim 12.

14. A nucleic acid encoding a recombinant protein according to claim 12.

15. The recombinant adenovirus according to any one of claims 1-6, the recombinant protein according to claim 12 or the trimeric protein according to claim 13 for use in the transduction of immune cells, preferably T cells, NK cells, monocytes, macrophages or dendritic cells, or for use in medicine.