Means and methods for delivery of agents to immune effector cells

Abstract

The current disclosure relates to targeted delivery of agents to T cells and / or NK cells. Highly suitable targeting domains were meticulously identified that allow for an agent, such as DNA and / or RNA, to be delivered to T cells and / or NK cells. As shown, when lipid nanoparticles were provided with such a targeting domain, nucleic acid comprised in said lipid nanoparticles was highly efficiently and specifically delivered. This allows for engineering of T cells and / or NK cells, highly useful in medical treatments for cancer.

Description

[0001] Title: Means and methods for delivery of agents to immune effector cells

[0002] Introduction

[0003] In recent years, pharmaceutical products have been developed for the delivery of agents such as nucleic acids to cells of a subject. For example, recently, vaccines for COVID-19 have been developed based on lipid nanoparticles comprising RNA agents, such as tozinameran. Approved products of lipid nanoparticles comprising siRNAs, such as patisiran have furthermore been developed. In addition to nanoparticles, gene therapy vector products have been made and approved for (stable) delivery of transgenes, based e.g. on AAV or lentivirus.

[0004] Hence, vehicles, such as lipid nanoparticles and viral vectors, have been developed that can carry an agent such as a DNA or an RNA molecule, which can be used to deliver such molecules to cells of a subject. Such delivery subsequently serves for evoking an immune response or treatment of a disorder in the subject, e.g. by being transcribed and / or translated into a therapeutic or antigenic protein, or, when an siRNA is delivered, by reducing expression of a target gene. Moreover, lipid nanoparticles are and may likewise be used to deliver further useful or therapeutic agents other than nucleic acids such as small molecules to cells of interest.

[0005] Nevertheless, delivery of an agent, such as a nucleic acid which includes an mRNA or DNA, remains a challenge. The delivery vehicles containing the agent, serve to protect the agents, e.g. from degradation, and are to facilitate uptake of the agent by cells and delivery thereof in the cell reaching the site within the cell where these are to have its action. For example, when the agent is a DNA encoding a transgene, the DNA is to be delivered across the cellular membrane and subsequently is to be transported to the nucleus where the transcription machinery of the cell resides. Delivery to the nucleus is a hurdle for compounds such as DNA. When the agent is to be released in the cytoplasm, such as with an mRNA, to have its action in the cytoplasm, the mRNA needs to be delivered across the cellular membrane and released, e.g. via endosomal escape, in the cytoplasm. Furthermore, delivery of agents to defined target cells remains to be a challenge as well. In spite of advancements made in the art, there is still a need in the art for improved delivery of agents to cells. There is a need in the art to provide for targeted delivery of agents to cells, in particular of nucleic acids such as DNA.

[0006] Summary

[0007] The current invention relates to targeted delivery of agents to T cells and / or NK cells. The current inventors identified highly suitable targeting domains that allows for an agent, such as DNA and / or RNA, to be delivered to T cells and / or NK cells. As shown in the examples, when lipid nanoparticles were provided with such a targeting domain, nucleic acid comprised in said lipid nanoparticles was highly efficiently and specifically delivered. This was achieved by meticulously selecting antigen binding regions that are capable of binding the human CD7 antigen. Delivery vehicles comprising a targeting moiety with such a binding region capable of binding human CD7 were found to be highly efficient in delivery of DNA and RNA to human target cells and allowing the DNA and RNA, and to have its intended function, i.e. expressing a transgene such as a CAR, TCR, or reporter gene. In particular, it was shown that such a targeting moiety with an antigen binding region capable of binding human CD7 was highly suitable for delivery of nucleic acid to resting T cells. This is highly advantageous, as primary T cells, in particular resting T cells, are difficult to effectively and specifically be transfected with e.g. a nucleic acid of interest. This is highly advantageous because this means it is not required to activate T cells prior to contacting the T cells with gene delivery vehicles. This in addition, also allows for a direct in vivo use of (lipid) nanoparticles, thereby avoiding ex vivo treatments of cells.

[0008] Figures

[0009] Figure 1. Schematic depicting exemplary immunoglobulins (a and b) and examplary fragments thereof (c and d).

[0010] Figure 2. GFP expression in T cells from mcDNA delivered by LNPs targeted with antibodies specific for different T cell antigens. GFP expression is plotted in percentage positive cells. Figure 3. VHH CD7 binding on Flp-ln™ 293 T-REx cell line stably expressing CD7. Left panel: VHH H7 binds to CD7 knock in T-rex cells but not wildtype (WT) or CD2 knock in Flp- ln™ 293 T-REx cell line, measured by increase in i Fluor 647 fluorescence intensity (MFI) using flow cytometry. Right panel: titration of VHH H7 on on Flp-ln™ 293 T-REx cell line stably expressing CD7 using flow cytometry.

[0011] Figure 4. EC50 of VHH H7 binding on primary human T cells determined using nonlinear curve fit of a flow cytometry titration binding assay.

[0012] Figure 5. GFP expression in WT and CD7 knock-in Flipin 293 T -rex cells delivered by either untargeted or CD7 VHH targeted LNPs. GFP expression is plotted as mean fluorescence intensity (MFI)

[0013] Figure 6. Specific binding / uptake of five different LNP formulations targeted using VHH H7 to T- and NK cells within peripheral blood mononuclear cells (PBMCs), compared to untargeted particles. LNPs were formulated using Cy5 labelled mRNA and targeted with the anti CD7 VHH. The LNPs were added to PBMCs isolated from healthy donors, the different Cy5 labeled positive cells were identified using specific markers in flow cytometry: T cells (CD4+ / CD8+), NK cells (CD56+), B cells (CD19+), monocytes (CD14+).

[0014] Figure 7. Specific transfection of NK and T cells with GFP encoding mRNA using different LNP formulations targeted with VHH H7 compared to untargeted particles. LNPs were formulated using GFP encoding mRNA and added to PBMCs isolated from healthy donors, The different GFP+ cells were identified using specific markers in flow cytometry: T cells (CD4 / CD8+), NK cells (CD56+), B cells (CD19+), monocytes (CD14+).

[0015] Figure 8. A. VHH H7 targeting enhances LNP mediated mRNA transfection of isolated activated and resting T cells. T cells were isolated from PBMCs, and either activated using CD3 / CD28 dynabeads (right panel, activated T cells) or used directly without adding activation beads (left panel, i.e. resting T cells). Transfection is measured by GFP expression after 24h incubation. B. VHH H7 targeting allows LNP mediated minicircle DNA transfection of T cells when combined with CD3 / CD28 activation. Transfection efficiency is measured by GFP expression after 96hour incubation with different nucleic acid doses.

[0016] Figure 9. CAR expression mediated by VHH H7 targeted LNPS. A) BMCA-CAR expression from two different CD7 H7 targeted LNP formulations loaded with mRNA encoding for BCMA- CAR in isolated resting T cells, and pre-activated using CD3 / CD28 dynabeads. LNP was added at one dose (500 ng) B) CD19-CAR expression from one CD7 H7 targeted LNP formulation loaded with mini-circle DNA encoding for CD19-CAR in isolated T cells. Cells were activated with CD3 / CD28 dynabeads at the same time as LNP transfection LNP was added at one dose (1200 ng)

[0017] Figure 10 . Binding of different VHH clones on Jurkat cell line. Jurkat cells were incubated with a titration 10x concentration range from 0.00001 - 1000 nM of the different VHH clones and subsequently with a fluorophore labeled anti-VHH antibody. Data is shown as (A) percentage of cells positive for VHH or (B) as mean fluorescence intensity (MFI) of the total cell population, as a measure for VHH binding. The VHH antibody clone of example 1 , H7 is, was also included in the experiments.

[0018] Figure 11 . Efficient stable integration of transgene encoding for CAR targeting CD19 (A) and efficient targeted cytotoxic killing of cells expressing CD19 (B), with subsequent cytokine release (C and D). Results are from results obtained with 3 batches of LNPs and 3 donors, i.e. n=9.

[0019] Figure 12. Targeting (A) and transfection (B) of T- B- and NK cells in vivo using CD7 targeted cy5 labeled, mRNA loaded tLNPs. CD34+ stem cell humanized mice were injected with CD7 targeted LNPs loaded with cy5 labeled siRNA (A) or mRNA encoding for CAR (B). Cy5 positiivey was assed after 1 day on circulating T- and B cells by flow cytometry. Expression of CAR mRNA was determined in different cell subsets in the spleen 48 hours after tLNP injection by flow cytometry. Figure 13. Efficient transfection of, and integration of transgenes in, NK cells. tLNPs loaded with mcDNA encoding either for CAR or for eGFP were used to transfect PBMCs. NK cells were grown out using K562 feeder cells and CAR or eGFP expression was assessed at day 5 (A) as measure for initial transfection efficiency and on day 11 (B) as measure for stable integration. NK cell proliferation was assessed by flow cytometry and plotted as percentage of total cells (C).

[0020] Figure 14. Functional assessment and comparison of CD7 targeting VHH clones. Isolated T cells were activated with CD3 / CD28 beads and transfected with tLNPs loaded with GFP encoding mcDNA and post inserted with different CD7 targeting VHH clones. eGFP expression was measured using flow cytometry (A). Conjugation efficiency of the different VHH clones was measured by SDS-Page and expressed as percentage (B).

[0021] Detailed description

[0022] In one embodiment, a nanoparticle for delivery of an agent to human T cells and / or human NK cells is provided, said nanoparticle comprising a targeting moiety and an agent, wherein said targeting moiety comprises an antigen binding region capable of binding to human CD7 and wherein the agent is a nucleic acid. The targeting moiety is of particular use for allowing the nanoparticle in accordance with the invention to deliver an agent to resting human T cells. Hence, the invention provides in particular and highly advantageously a nanoparticle for delivery of an agent to T cells and / or NK cells, including resting human T cells, said nanoparticle comprising a targeting moiety and agent, wherein said targeting moiety comprises an antigen binding region capable of binding to human CD7 and wherein said agent is a nucleic acid.

[0023] It is understood that in accordance with the invention, a nanoparticle refers to a particle of a size within the range of about 10 - 1000 nm, which may be useful to contain agents therein, and which can be provided with a targeting domain in accordance with the invention. Examples of nanoparticles include e.g. lipid nanoparticles which are composed of lipids and well known pharmaceutical formulations for drug delivery. A nanoparticle can hence have an envelope structure, e.g. comprising lipids, or a lipid bilayer, in which envelope agents can be contained. A nanoparticle may also have a solid structure. A nanoparticle may also comprise capsid structure or the like. Hence, any suitable nanoparticle may be contemplated in which agents can be contained and which can be provided with a targeting moiety in accordance with the invention.

[0024] T cells and / or NK cells are found in peripheral blood, and are peripheral blood mononuclear cells (PBMCs). PBMCs include lymphocytes (T cells, B cells, and NK cells), monocytes, and dendritic cells. In humans, the frequencies of cell populations in PBMCs can vary across individuals, lymphocytes may be in the range of 70-90 %, monocytes from 10 to 20 %, while dendritic cells are rare, accounting for only 1-2 %. T cells, also referred to as T lymphocytes are CD3+, i.e. positive for Cluster of Differentiation 3. T cells can be divided into helper T cells (CD3+CD4+) and cytotoxic T cells (CD3+CD8+), which function in the adaptive immune response. The frequencies of cell types within the lymphocyte population can be in the range of 70-85 % CD3+ T cells, 5-10 % B cells, and 5-20 % NK cells. NK cells are natural killer cells, and are a type of cytotoxic lymphocyte of the innate immune system. Both NK and T cells are of interest as these can be engineered to express a CAR, a chimeric antigen receptor (likewise of course a TCR, a T cell receptor) therewith specifically targeting malignant cells, e.g. in cancer.A targeting moiety in accordance with the invention relates to a functional group which serves to target or direct the nanoparticle to a particular cell type. In some embodiments, the targeting moiety is an antibody or a fragment thereof, a cell surface receptor ligand or fragment thereof, a peptide or a nucleic acid, such as e.g. an aptamer. Any molecule being suitable to function as a targeting moiety may be contemplated. For example, human CD7 ligands, or parts thereof, may be contemplated, which may have further modifications. The targeting moiety can be attached to the nanoparticle using suitable linking chemistry techniques known to those skilled in the art. In some embodiments, the targeting moiety is covalently bound to the nanoparticle. In other embodiments, the targeting moiety is associated with the nanoparticle by non-covalent bonding interactions such as ionic or by van der Waals forces.

[0025] A targeting moiety comprising an antigen binding region capable of binding to human CD7 refers to any moiety capable of binding to human CD7. Human CD7, i.e. Cluster of Differentiation 7, is a protein that in humans is encoded by the CD7 gene. This gene encodes a transmembrane protein which is a member of the immunoglobulin superfamily. This protein is a cell surface costimulatory molecule expressed on human T and natural killer (NK) cells and on cells in the early stages of T-, B-, and myeloid cell differentiation. A targeting moiety capable of binding human CD7 in accordance with the invention is capable of binding to the extracellular portion of a human CD7 protein, e.g., as set forth in GENBANK No.

[0026] AAH 13297.1.

[0027] Any agent may be contemplated to be comprised in the nanoparticle in accordance with the invention. It is understood that in accordance with the invention when reference is made to an agent, this is to refer to an agent that is to have an effect in the target cell once delivered thereto. It is understood that in accordance with the invention, a nanoparticle may comprise one or more agents. Agents that can be contemplated include peptides and nucleic acids. In particular, an agent that can be comprised in the nanoparticle in accordance with the invention is a nucleic acid, which is preferred.

[0028] Nucleic acid refers to any compound and / or substance that comprises a polymer of nucleotides (i.e. of nucleotide monomers). These polymers are referred to as polynucleotides. Thus, the terms "nucleic acid" and "polynucleotide" can be used interchangeably. Nucleic acids may be or may include, for example, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs). RNAs may include mRNAs or the like. DNAs may include double stranded DNA. It is understood that RNAs or RNAs may include artificial or modified nucleic acids, such as threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a [3-D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino-a-LNA having a 2'-amino functionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleic acids (CeNA), or chimeras or combinations thereof. The skilled artisan will appreciate that polynucleotide sequences may recite "T"s in a representative DNA sequence but where such a sequence represents RNA (e.g., mRNA), the "T"s would be substituted for "U"s. Thus, RNA polynucleotides encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each "T" of the DNA sequence is substituted with "U."

[0029] The nanoparticles in accordance with the invention preferably comprise an agent, which is as said highly preferably a nucleic acid. In accordance with the invention, it is understood that in particular the nucleic acid is a DNA or an RNA. In particular, as shown in the example section, an RNA that may be contemplated may include an mRNA, encoding for a protein of interest, and a DNA that may be contemplated may encode likewise for a gene of interest such as a protein as well.

[0030] As shown in the example section, highly advantageously, mRNA was highly efficiently transferred to resting T cells, and to NK cells as well. This was observed as Cy5 labelled mRNA was detected in resting T cells and NK cells, and by detected mRNA translated into GFP protein as well. The basic components of an mRNA molecule typically include at least one coding region, a 5' untranslated region (UTR), a 3' UTR, a 5' cap and a poly-A tail. Further regulatory sequences may be included and contemplated in mRNA molecules. Hence, the nanoparticles in accordance with the invention are in particular useful for delivery of RNA, in particular for delivery thereof to resting T cells and NK cells. Any RNA may be contemplated in accordance with the invention, including non-coding RNAs such as antisense RNA, siRNA, miRNA, and coding RNA, such as mRNA, which may be synthetized or isolated, and may comprise naturally occurring sequences or engineered sequences. RNA that may be contemplated may be selected from the group consisting of an mRNA, an siRNA and a microRNA. It may be preferred to select an mRNA. Hence, in some embodiments, a nucleic acid of the present invention may function as messenger RNA (mRNA). A messenger RNA (mRNA) refers to any polynucleotide that encodes a (at least one) polypeptide (a naturally-occurring, non-naturally-occurring, or modified polymer of amino acids) and can be translated to produce the encoded polypeptide in a cell. Polynucleotides of the present disclosure may function as mRNA but may be distinguished from wild-type mRNA in their functional and / or structural design features, which may serve to allow for effective polypeptide expression using nucleic-acid based therapeutics.

[0031] As likewise also shown in the example section, DNA was shown to be effectively transferred to T cells and to NK-cells as well . Such DNA may comprise e.g. an expression cassette for a gene of interest, such as a CAR. Such DNA may also comprise an expression cassette for a reporter gene, such as GFP. Preferably, such a DNA is provided as a double stranded DNA, for example as a minicircle DNA or the like. The DNA may be in the form of a double stranded DNA. The DNA furthermore can be in the form of a minicircle DNA. A minicircle DNA in accordance with the invention can be defined as having a circular structure and is double stranded. A minicircle DNA may typically and preferably not comprise any bacterial derived DNA sequences. A minicircle DNA may be derived from DNA, such as a plasmid DNA, comprising a selection marker and an origin of replication for production of the plasmid DNA in a suitable (bacterial) host. To provide for the minicircle DNA, the selection marker and origin of replication may be excised from the plasmid DNA and the circle religated to thereby providing a minicircle DNA without the (bacterial) selection marker and origin of replication sequences. Any length of DNA may be selected that is suitable to be comprised in an LNP. In one embodiment, the length of the DNA contemplated may depending on the size of the gene sequence of interest that is to be comprised in the DNA, and the the type of construct, i.e. elements that may need to be contained therein, that is to be delivered. As said, and as shown in the examples, in accordance with the invention the nucleic acid, i.e. a DNA such as a mcDNA, can highly advantageously encode for a CAR and / or a TCR.

[0032] With regard to CAR and / or a TCR, it is understood that a CAR is a chimeric antigen receptor that is to mimic a TCR or the like. CARs are engineered. The first generation of CAR were provided with an antigen recognition part often an antibody derived region (e.g. a scFv) fused to a transmembrane region and intracellular region of e.g. a CD3 chain. Later generations combined intracellular signalling domains from various costimulatory protein receptors (e.g., CD28, 41 BB, ICOS) incorporated in the cytoplasmic tail of the CAR to enhance signalling further. Further generations also incorporated in their design an inducible release of transgenic immune modifiers, such as IL-12, to shape the tumor environment by augmenting e.g. T-cell activation, attracting and activating innate immunity. As CARs often have antibody variable regions incorporated, these can target e.g. receptors themselves that are presented at the surface of a cell (e.g. Her2, CD19 etc.), or can also target antigens presented by MHC, derived e.g. from proteins intracellular processed by the ubiquitin- proteasome system. Such peptides presented by MHC include proteins that are processed internally and presented by MHC, which can be derived from receptors, secreted proteins, intracellular proteins or internalized proteins. Of course, instead of a CAR, likewise, identified suitable TCR receptors (humanized or human derived) may be selected instead, or any other suitable engineered receptor.

[0033] Nucleic acids in accordance with the invention, in some embodiments, are codon optimized. Codon optimization methods are known in the art and may be used as provided herein. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove / add post translation modification sites in encoded protein (e.g. glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art - non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and / or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms.

[0034] In some embodiments, the nanoparticle comprises a DNA which is to be integrated into the genome of T cells and / or NK cells. Such a DNA may e.g. comprise homology arms to allow for targeted integration. The nanoparticle may also comprise a means for enabling integration of the DNA. For example, an integrase. The nanoparticle may also comprise an mRNA that encodes for a protein facilitating DNA integration, such that upon delivery of the nanoparticle in T cells, such as resting T cells and / or NK cells, the DNA can be stably integrated into the genome of the T cells. The DNA may hence include suitable DNA sequences that accommodate integration, for example by interaction with the expressed protein. Integration of DNA may include homologous recombination and / or nicking of an endogenous target DNA sequence. Suitable (recombinant) DNA integration technologies are known in the art which may be employed. Such technologies include gene editing technology such as CRISPR / CAS, and Transposon technology such as sleeping beauty, piggyBac transposon vectors. In any case. The nanoparticlein accordance with the invention may include agents that provide for the necessary components to insert a transgene into the genome of the targeted cells, i.e. (resting) T cells and / or NK cells., or any other type of human CD7 expressing cell. Hence, preferably, in case the agent that is provided is a DNA with an expression cassette which is to be integrated further means can be comprised in the nanoparticle to facilitate integration, and the DNA may further comprise the necessary elements therefor. For example, in case of a transposon system, an mRNA encoding for a transposase may be included in the nanoparticle, and the DNA may have flanking its expression cassette inverted terminal repeats (ITRs) compatible with the selected transposase. Hence, it is understood that an LNP in accordance with the invention may comprise both an RNA, such as an mRNA and a DNA. In another embodiment, as outlined in the examples herein, the nanoparticle, such as an LNP, in accordance with the invention, comprises an mRNA encoding a transposase and a DNA encoding a CAR or a TCR with flanking ITRs compatible with the transposase, wherein preferably, the transposase is a sleeping beauty transposase.

[0035] In accordance with the invention, suitable nanoparticles in accordance with the invention may include non-viral vectors and viral vectors. Vectors are compositions of matter which comprise a nucleic acid and which can be used to deliver the nucleic acid to a cell. Vectors can be non-viral vectors and viral vectors. Viral vectors are based on viruses, utilizing the properties of the virus from which they are derived for delivery of a nucleic acid, which can be inserted in the viral vector genome. Viral vectors in accordance with the invention may include retroviral vectors and lentiviral vectors, and adenoviral vectors and adeno-associated viral vectors. Providing such viral vectors with a targeting moiety in accordance with the invention can be via covalent binding, or non-covalent interactions, which are well known in the art. For example, a targeting moiety may be comprised in a viral vector capsid, and / or may be associated with a viral envelop. It may be preferred in accordance with the invention to select non-viral vectors. As shown in the examples, nanoparticles in accordance with the invention which may in particular be suitable and that can be selected are lipid nanoparticles.

[0036] Preferably, the nanoparticle in accordance with the invention is a lipid nanoparticle. Lipid nanoparticles in accordance with the invention can be defined as comprising a lipid composition with an ionizable cationic lipid and / or a targeting moiety conjugated to a lipid. Lipids are fatty substances which may be naturally occurring or synthetic and include a broad group of molecules including fats, waxes, sterols, fat-soluble vitamins (such as vitamins A, D, E and K), fatty acids, and their derivatives, such as monoglycerides, diglycerides, triglycerides, and phospholipids. Lipids suitable for the preparation of lipid nanoparticles are widely available. Advantageous lipids to be included are ionizable lipids, which can be key components of lipid nanoparticles as they can encapsulate nucleic acid through electrostatic interactions. Preferably the lipid nanoparticles in accordance with the invention comprise the target moiety conjugated to a lipid such that the target moiety is presented at the surface of the lipid nanoparticle and capable of binding to its human CD7 target as presented on the surface of a T cell or the like.

[0037] The targeting moiety may be conjugated to a lipid via a linker molecule. Advantageous suitable lipids for conjugation of the targeting moiety thereto may be PEG- lipids. PEG-lipids can thus improve stability in vivo, and may advantageously allow for conjugation of the targeting moiety via a linker, i.e. a lipid PEG linker may be defined as [lipid] - (CH2 - CH2-O)n - [linker], wherein the - (CH2 - CH2-O)n - group refers to PEG, i.e. poly ethylene glycol. As shown in the examples, a variety of different lipid nanoparticle compositions were shown to be effective in accordance with the invention. Hence, the lipid nanoparticle can be understood not to be restricted by any defined lipid composition or lipid components, as long as the lipid nanoparticle can comprise the targeting moiety, i.e. present it at its surface, any lipid nanoparticle composition capable of transferring its contents to a cell may be contemplated in accordance with the invention. Although as shown in the examples, the delivery of nucleic acids to, T cells and NK cells, in particular to resting T cells, was found to be highly advantageous utilizing the lipid nanoparticles in accordance with the invention, other agents may be contemplated to be delivered to such cells utilizing the targeting moiety in accordance with the invention, and other suitable lipid nanoparticle compositions, e,g. suitable for another agent and / or nucleic acid, can be contemplated as well.

[0038] In an embodiment, the lipid nanoparticle comprising the targeting moiety in accordance with the invention comprises 30 - 60 mole percent of ionizable lipid, 10 - 30 mole percent of helper lipid, 0.1 - 10 mole percent of PEG lipid, and 15 - 50 mole percent of a sterol. It is understood that these mole percentages are expressed relative to the constituents of ionizable lipid, helper lipid, PEG lipid and sterol, the sum of which surmounts to 100 mole percent. Preferably, in such compositions comprising a nucleic acid, the weight ratio of ionizable lipid to nucleic acid is in the range of 10 - 20 to 1. In another preferred embodiment, the lipid nanoparticle comprising the targeting moiety in accordance with the invention comprises 40-50 mole percent of ionizable lipid, 9 to 20 mole percent of helper lipid, 0.1 to 10 mole percent of PEG lipid, and 15 - 50 mole percent of a sterol. In a preferred embodiment, the sterol is cholesterol. Suitable ionizable lipids that may be contemplated in accordance with the invention can be selected from SM-102, MC3, KC2 Lipid A9 and C12- 200, preferably, SM-102 may be selected. In a highly preferred embodiment, the targeting moiety is conjugated to the PEG lipid.

[0039] Lipid nanoparticles may be prepared with methods and / or devices known in the art. As shown in the example section, LNPs can be prepared using microfluidic devices. The nucleic acid (or agent) can be provided e.g. in aqueous phase, and mixed with an organic phase comprising the lipid composition, e.g. the ionizable lipid, helper lipid, PEG lipid and sterol, and mixed with a microfluidic device. Lipid nanoparticles in accordance with the invention in one embodiment have a diameter in the range of 50 - 200 nm, allow for efficient encapsulation of >95% and / or can have a neutral charge at pH 7.4. Once the lipid nanoparticles have been prepared comprising the nucleic acid, a binding moiety can be subsequently applied thereto, e.g. via the post-insertion of a binder linked to a lipid anchor via aclick chemistry approach. Preferably this is via conjugation to a lipid-PEG. It is understood that the term lipid-PEG or PEG-lipid refers to the same type of molecule, i.e. a polymer of ethylene glycol, i.e. (CH2- CH2-O)n, covalently bound with a lipid. The PEG moiety may further comprise a linker molecule to which via appropriate reactions targeting moieties can be covalently bound thereto. As said, the structure of such a PEG lipid can be described by lipid-PEG-linker, i.e. [ lipid ] - (CH2 - CH2-O)n - [linker ]. When the targeting moiety is conjugated to a PEG-lipid this can be described by [lipid] - (CH2 - CH2-O)n - [linker] - [targeting moiety] , or, [lipid] - (CH2 - CH2-O)n - [targeting moiety]. For example, a human CD7 targeting moiety such as described in the example section having a VHH format or the like, may have a cysteine, e.g. a C-terminal cysteine, that can be conjugated with a lipid-PEG-maleimide compound via a maleimide-thiol reaction resulting in a stable thioether covalent bond. Such a specific conjugation may be preferred, however, it is understood that further types of conjugation, i.e. a reaction between a chemical group included in the targeting moiety and a linker moiety that results in the formation of a covalent bond, may be contemplated instead and such are known in the art.

[0040] It is hence understood that the LNP in addition to comprising PEG-lipid in accordance with the invention, thus further may comprise a lipid-PEG conjugated to a targeting moiety. In one embodiment, a range from 0.01 mole % - 5 mole %, preferably 0.02 mole % - 1 mole %, of lipid-PEG-(linker)-targeting moiety may be selected. Of course, any suitable mole % may be selected, and the skilled person is well equipped to optimize useful mole % for LNPs in accordance with the invention. More preferably, lipid nanoparticles in accordance with the invention comprise 0.025 - 0.5 mole percent of PEG-lipids conjugated with said targeting moiety. It is understood that the mole percentages are expressed relative to the lipid constituents of a lipid nanoparticle the sum of which surmounts to 100 mole percent, which lipid nanoparticle may thus comprise ionizable lipid, helper lipid, PEG lipid, PEG lipid targeting moiety, and sterol.

[0041] Hence, a lipid nanoparticle comprising the targeting moiety in accordance with the invention, preferably comprises 30 - 60 mole percent of ionizable lipid, 10 - 30 mole percent of helper lipid, 0.1 - 10 mole percent of PEG lipid, and 15 - 50 mole percent of a sterol, and 0.025 - 0.5 mole percent of lipids conjugated with said targeting moiety. It is understood that the lipid conjugated with the targeting moiety, preferably consists of a PEG-lipid conjugated with the targeting moiety. Hence, phrased differently, the lipid nanoparticle comprising the targeting moiety in accordance with the invention, preferably comprises 30 - 60 mole percent of ionizable lipid, 10 - 30 mole percent of helper lipid, 0.1 - 10 mole percent of PEG lipid, and 15 - 50 mole percent of a sterol, and 0.025 - 0.5 mole percent of PEG-lipids conjugated with said targeting moiety. It is understood that the PEG-lipids and PEG-lipids conjugated with said targeting moiety from the viewpoint of the PEG and / or lipid portion may be the same or different. For instance, as shown in the examples, a DSPE-PEG2000-Maleimide (i.e. lipid - PEG - linker) may be used for conjugation with the targeting moiety, whereas the lipid-PEG in a lipid nanoparticle may be DMG-PEG2000. Hence, the lipid portion of the PEG-lipid may be different, e.g. DMG, as compared with the lipid of the lipid-PEG-(linker)-targeting moiety, e.g. DSPE. Of course, conversely, the lipid of the PEG-lipid may also be the same as the lipid of the lipid-PEG-(linker)-targeting moiety. With regard to the PEG portion this may be the same (like PEG2000 used for both PEG-lipid and lipid-PEG-(linker)-targeting moiety), or this may be different. It is understood that the mole percentages of LNPs are expressed relative to the constituents of ionizable lipid, helper lipid, PEG lipid, PEG-lipid-(linker)-targeting moiety, and sterol, the sum of which surmounts to 100 mole percent. As outlined above, and as shown in the example section, it is preferred that the targeting moiety is conjugated to the nanoparticle. Conjugation is understood to encompass covalent binding with the nanoparticle, e.g. via a compound comprised in the nanoparticle such as to a Lipid-PEG. Conjugation of the targeting moiety can be well controlled, e.g. by conjugation of the targeting moiety to a compound to be comprised in the nanoparticle, before preparing the nanoparticle. Conjugation can also be performed by conjugation of the targeting moiety to already prepared nanoparticles. The targeting moiety conjugated with a compound, such as a lipid-PEG- targeting moiety as described herein, can also be inserted in the (lipid) nanoparticles after a (lipid) nanoparticle is produced. Methods for insertion of lipid-PEGs conjugated with a compound of interest, such as a targeting moiety as described herein, after production of a lipid nanoparticle are well known in the art, and such may be preferred. Hence, in any case, (lipid) nanoparticles with conjugated targeted moieties can be prepared with methods known in the art, involving well known established chemical reactions, and may also involve, e.g. a linker, to provide a targeting moiety conjugated with the nanoparticle, having the targeting moiety bind to the nanoparticle via a linker (as described above), including well known insertion methods for lipid nanoparticles. Nevertheless, as also shown in the example section, in other embodiments, the targeting moiety is associated with the nanoparticle by non-covalent bonding interactions such as ionic or by van der Waals forces, instead of covalent binding. Such an association may be via e.g. an adapter molecule covalently bound with the lipid nanoparticle, such as e.g. shown in the example section, wherein the adapter molecule has a specific non-covalent binding interaction with the targeting moiety. Such a specific non-covalent binding interaction may be e.g. an Fc-targeting moiety, targeting a an Fc-region covalently connected with a targeting moiety capable of binding with human CD7. Further specific non-covalent binding interactions may be contemplated and used instead of an Fc-targeting moiety - Fc-region interaction.

[0042] In one embodiment, the targeting moiety is an immunoglobulin or a fragment thereof. As shown in the examples, both a full length bivalent antibody (schematically depicted in Fig. 1a), targeting human CD7 bound to nanoparticles that carry an adaptor targeting the Fc- region of the CD7 antibody, and a CD7 antibody fragment, VHH (schematically depicted in Fig. 1c), conjugated to LNPs, was shown to be effective in targeting T cells. Hence, it is understood that the targeting moiety may be an immunoglobulin, such as an antibody, or a fragment thereof, and it is understood that the targeting moiety comprising the immunoglobulin or fragment thereof may be covalently linked or bound via noncovalent interactions with the nanoparticle. The term immunoglobulin as used herein refers to a class of structurally related glycoproteins involved in binding, recognition and adhesion. An immunoglobulin can be a T cell receptor, or an antibody. T cell receptor and antibodies generate diversity via genetic recombination of DNA-encoded segments in individual lymphocytes by somatic V(D)J recombination. The V(D)J segments are comprised in the variable region and interspersed with framework regions. In humans, a T cell receptor is composed of an alpha (VJ) and a beta (VDJ) chain, the combination of the variable regions forming an antigen binding domain. Antibodies in humans are composed of two pairs of heavy and light chains, all four interconnected by disulfide bonds. Such antibodies thus having two antigen binding domains, and thus are bivalent. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2, and CH3. The hinge region is the region between the CH1 and CH2 domains of the heavy chain and is highly flexible. Disulfide bonds in the hinge region are part of the interactions between two heavy chains in an IgG molecule. Each light chain typically is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL or CL). The light chain constant region is comprised of one domain, CL. The VH and VL regions may be further subdivided into regions of hypervariability (or hypervariable regions which may be hypervariable in sequence and / or form of structurally defined loops), also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J Mol Biol 1987;196:90117). Other species, such as camels, llamas and sharks, have a different antibody structure (See fig. 1 b) composed of only heavy chains. Hence, such antibodies are bivalent as well, and an antigen binding region of such an antibody is based on a variable region of the heavy chain only, such antigen binding regions referred to as VHH (see figure 1c). It is understood that an immunoglobulin, or a fragment thereof, in accordance with the invention also encompass alternative formats known in the art. Alternative formats may include single chain formats, such as scFv or the like, and may include heavy chain only or light chain only formats. A format with a single VH and a single VL, preferably in single chain format such as scFv can be contemplated. Provided of course Y1 such a format allows for binding to human CD7 and allows for delivery of an agent with a nanoparticle in accordance with the invention. The skilled person is well aware of means and methods for designing different immunoglobulin formats.

[0043] Preferably, the nanoparticle in accordance with the invention comprises an antigen binding region which is in a VHH format. Highly advantageously, and as shown in the examples, the antigen binding region preferably originates from a Llama antibody, i.e. a VHH such as described in the examples herein. VHHs with highly advantageous properties could be in particular be generated via LNPs, which were formulated to express full length human CD7, which were used to immunize llama’s, and subsequently selected from a phage library using human CD7 expressing cells. Such VHHs selected in this fashion were found to be highly useful for providing suitable antigen binding regions to be comprised in a targeting moiety in a nanoparticle in accordance with the invention.

[0044] As shown in the example section, such a nanoparticle comprising such a VHH, allowed for a substantial increase in the TNK / O ratio of GFP+ or Fluorescently labeled RNA+ cells. As shown in the example section, when comparing the TNK / O ratio of nanoparticles with and without the antigen binding region, a substantial increase of this ratio was observed, indicative of the selectivity that the binding region can provide, in addition to the high efficiency. Likewise, advantageously, providing the nanoparticles with the antigen binding region in accordance with the invention also allowed to reduce aspecific delivery (see Fig. 5). The TNK / O ratio as defined herein refers to as the number of T cells and NK cells, divided by the number of other cells, as present in isolated PBMCs, which have been found to express GFP or comprise Cy5 labelled siRNA, after isolated PBMCs were subjected to nanoparticles, such as lipid nanoparticles, comprising an mRNA encoding GFP or Cy5 labelled siRNA, respectively. It is understood that this ratio expresses the preference for a nanoparticle or lipid nanoparticle to deliver a nucleic acid, and a higher number indicates a higher preference for T cells and NK cells. Hence, when comparing TNK / O ratio for a lipid nanoparticle with (+) and without (-) a targeting moiety in accordance with the invention, it can be observed that the TNK / O ratio is substantially increased, i.e. there is a certain fold increase of this ratio, which fold increase is a measure of the specificity of a targeting moiety in accordance with the invention. While this measure is determined with an mRNA encoding GFP or a Cy5 labelled siRNA, this measure provides a suitable measure for specificity for T and NK cells, related to the targeting moiety and the LNP composition. Hence, nanoparticles, in particular lipid nanoparticles, as defined herein in accordance with the invention can be defined by this TNK / O ratio.

[0045] Hence, in one embodiment, a nanoparticle in accordance with the invention, preferably a lipid nanoparticle, is provided wherein of a corresponding nanoparticle containing GFP encoding mRNA, the fold increase in the TNK / O ratio of GFP positive cells for nanoparticles with the antigen binding region relative to nanoparticles without the antigen binding region, is at least 5-fold. It is understood that with corresponding is meant that the composition of the corresponding nanoparticle is identical to the nanoparticle the difference being the nucleic acid, which is in the corresponding nanoparticle a GFP encoding mRNA (or a Cy5 labelled siRNA). In another embodiment, the fold increase in TNK / O ratio is in the range of 5 - 40 fold. In one embodiment, a nanoparticle, preferably lipid nanoparticle, in accordance with the inventions is provided, wherein of a corresponding nanoparticle without the antigen binding region containing a GFP encoding mRNA, the TNK / O ratio of GFP positive cells for nanoparticles is in the range of 0.1 - 1 , and, wherein, of a corresponding nanoparticle with the antigen binding region and containing the GFP encoding mRNA, the TNK / O ratio of GFP positive cells for nanoparticles is at least about 2, or, in the range of 2-10. In another embodiment, a nanoparticle, preferably a lipid nanoparticle, in accordance with the invention is provided, wherein of a corresponding nanoparticle containing a fluorescently labelled RNA, the fold increase in the TNK / O ratio of fluorescent positive cells for nanoparticles with the antigen binding region relative to nanoparticles without the antigen binding region is at least is 5-fold. In a further embodiment, said ratio is in the range of 5 - 40 fold. In another embodiment, a nanoparticle, preferably a lipid nanoparticle, in accordance with the invention is provided, wherein of a corresponding nanoparticle without the antigen binding region containing a fluorescently labelled RNA, the TNK / O ratio of fluorescent cells for nanoparticles is in the range of 0.1 - 5, and wherein, of a corresponding nanoparticle with the antigen binding region containing the fluorescently labelled RNA, the TNK / O ratio of GFP positive cells for nanoparticles is at least about 10, or, in the range of 5 - 50.

[0046] It is understood that with regard to antigen binding, the CDR regions of antigen binding regions as part of an immunoglobulin, or fragment thereof, as comprised in a targeting moiety, are highly important for the binding of antigen binding region to a target antigen. As is understood, CDR sequences of antibodies comprising a heavy and a light chain can be defined in accordance with IMGT rules (Brochet X., Nucl Acids Res 2008;36: W503-508; Lefranc MP., Nucl Acids Res 1999;27:209-12; www.imgt.org / ). Likewise, CDR sequences of llama antibodies or the like can be determined as well. It is understood that determined CDR regions may vary depending on the algorithm used. Hence, in one embodiment, preferably, the nanoparticle according to the invention comprises a targeting moiety, said targeting moiety comprises an immunoglobulin or part thereof, comprising CDR regions derived from the VHH as defined by SEQ ID NO.1. In one embodiment, said immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.1. In a further embodiment, said CDR regions of SEQ ID NO.1 are CDR1 , CDR2 and CDR3 of SEQ ID NO.Ias defined by SEQ ID NO. 2, 3 and 4, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.1 was identified.

[0047] In another embodiment, the nanoparticle according to the invention comprises a targeting moiety, said targeting moiety comprises an immunoglobulin or part thereof, comprising CDR regions derived from the VHH as defined by SEQ ID NO. 1 ,

[0048] In one embodiment, said immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.5. In a further embodiment, said CDR regions of SEQ ID NO.1 are CDR1 , CDR2 and CDR3 of SEQ ID NO.5 as defined by SEQ ID NO. 6, 7 and 8, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.5 was identified.

[0049] In one embodiment, said immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.9. In a further embodiment, said CDR regions of SEQ ID NO.9 are CDR1 , CDR2 and CDR3 of SEQ ID NO.9 as defined by SEQ ID NO. 10, 11 , 12, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.9 was identified.

[0050] In one embodiment, said immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.13. In a further embodiment, said CDR regions of SEQ ID NO.13 are CDR1. CDR2 and CDR3 of SEQ ID NO.13 as defined by SEQ ID NO. 14, 15 and 16, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.13 was identified. In one embodiment, said immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.17. In a further embodiment, said CDR regions of SEQ ID NO.17 are CDR1 , CDR2 and CDR3 of SEQ ID NO.17 as defined by SEQ ID NO. 18, 19 and 20, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.17 was identified.

[0051] In one embodiment, said immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.21 . In a further embodiment, said CDR regions of SEQ ID NO.21 are CDR1 , CDR2 and CDR3 of SEQ ID NO.21 as defined by SEQ ID NO. 22, 23, and 24, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.21 was identified.

[0052] In one embodiment, said immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.25. In a further embodiment, said CDR regions of SEQ ID NO.25 are CDR1 , CDR2 and CDR3 of SEQ ID NO.25 as defined by SEQ ID NO. 26, 27 and 28, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.25 was identified.

[0053] In one embodiment, said immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.29. In a further embodiment, said CDR regions of SEQ ID NO.29 are CDR1 , CDR2 and CDR3 of SEQ ID NO.29 as defined by SEQ ID NO. 30, 31 , and 32, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.29 was identified.

[0054] In one embodiment, said immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.33. In a further embodiment, said CDR regions of SEQ ID NO.33 are CDR1 , CDR2 and CDR3 of SEQ ID NO.33 as defined by SEQ ID NO. 34, 35 and 36, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.33 was identified.

[0055] In addition, and as shown in the examples, of the MT-701 antibody, which is a mouse antibody capable of binding with human CD7, CDR regions derived therefrom may be contemplated to be comprised in a targeting moiety in accordance with the invention as well. In one embodiment, said targeting moiety which comprises an immunoglobulin, or a part thereof, comprises CDR regions of the MT-701 antibody. In a further embodiment, the nanoparticle according to the invention comprises a targeting moiety, comprising a humanized immunoglobulin, or part thereof, comprising CDR regions of the MT-701 antibody. Such an immunoglobulin or part thereof may be in a scFv format, conjugated to e.g. a lipid nanoparticle similar to the conjugation of a VHH, or a full length (humanized) antibody format may be contemplated, linked to e.g. a lipid nanoparticle using e.g. monobody approach, as described herein in the example section.

[0056] Suitable CDR sequences as provided from antibodies generated in animals may be used in the context of the framework regions in which they were identified. However, as in vivo administration of nanoparticles with targeting moieties may be contemplated, or of cells contacted with such nanoparticles, it may be preferred to have the CDR sequences in the context of human sequences, i.e. human framework regions, and, if applicable, human constant regions, as it is well established that when non-human antibodies are (repeatedly) administered to human subjects, this can evoke an immune response thereto. Hence, in order to avoid immunogenicity, humanization of e.g. VHH sequences such as SEQ ID NO.1 ,5, 9, 13, 17, 21 , 25, 29, or 33, is contemplated. A humanized immunoglobulin in accordance with the invention refers to a genetically engineered nonhuman immunoglobulin, which contains human constant domains and non-human variable domains modified to contain a high level of sequence homology to human variable domains. This can be achieved by grafting of the non-human antibody CDRs, which together form the antigen binding site, onto a homologous human acceptor framework region (FR) (see WO92 / 22653 and EP0629240). In order to fully reconstitute the binding affinity and specificity of the parental immunoglobulin, such as a mouse antibody or llama antibody or the like, the substitution of framework residues from the parental antibody (i.e., the non-human antibody) into the human framework regions (back-mutations) may be required. Structural homology modelling may help to identify the amino acid residues in the framework regions that are important for the binding properties of the antibody. Thus, a humanized immunoglobulin may comprise non- human CDR sequences, primarily human framework regions optionally comprising one or more amino acid back-mutations to the non-human amino acid sequence, and fully human constant regions. Optionally, additional amino acid modifications, which are not necessarily back-mutations, may be applied to obtain a humanized immunoglobulin with preferred characteristics, such as affinity and biochemical properties. In any case, the skilled person is well aware of means and methods for designing different immunoglobulin formats that are suitable for human use.

[0057] Alternatively, or in addition, it may also be contemplated to de-immunize the amino acid sequence of a targeting moiety in accordance with the invention. De-immunization involves the identification of T-cell or B-cell epitopes in amino acid sequence and removal thereof by amending the sequence. Such identification can be performed by in silico analysis of amino acid sequences, and de-immunization services are widely available. It is understood that humanization of immunoglobulin sequences may, and often will, include de- immunization, as the objective and preferred feature of humanization of non-human immunoglobulin sequences is to avoid or at least severely reduce (possible) an immune response against such a targeting moiety while at the same time retaining advantageous target binding properties.

[0058] Hence, in one embodiment, a nanoparticle in accordance with the invention is provided, wherein the amino acid sequence of said immunoglobulin or a fragment thereof as comprised in an antigen binding region, is humanized and / or de-immunized.

[0059] As shown in the examples, the EC50 of an immunoglobulin or part thereof, for binding with cells expressing human CD7, may be in the range of 1 pM - 1 mM. Such EC50 may preferably be in the range of 10-6to 10-12. More preferably, said EC50 of such an immunoglobulin is in the range of 10-6to 10’11, or 10-6to IO-10, or 10-7to 10-9. In one embodiment, the EC50 may be selected to be in the range of 10-7to 10-9. As is understood, in case of humanization of immunoglobulin sequences, such as of the VHH of SEQ ID NO.1 , the EC50 of such a humanized immunoglobulin preferably may be selected to be in these ranges.

[0060] As described herein and as shown in the examples, the nanoparticles in accordance with the invention, and in particular the lipid nanoparticles in accordance with the invention, are highly useful for delivery of a nucleic acid to a T cell. In particular, for delivery of a nucleic acid to a resting T cell. In particular, as shown in the examples, when e.g. an mRNA is delivered to a resting T cell, it is translated into a protein. In addition, DNA delivered to activated T cells, provided for provided for highly functional expression as well. Targeting nanoparticles, such as LNPs, provided for highly specific delivery and subsequent functional expression as compared with untargeted nanoparticles. Hence, in one embodiment, in accordance with the invention, a use is provided of the nanoparticle in accordance with the invention, for delivery of a nucleic acid to a T cell. Such use in a further embodiment, comprises a DNA or RNA. In one embodiment, in accordance with the invention, a use is provided of the nanoparticle in accordance with the invention, for delivery of a nucleic acid to a T cell, wherein when said nucleic acid is an mRNA, said mRNA is translated into a protein in a T cell, or a resting T cell. In another embodiment, a use is provided of the nanoparticle in accordance with the invention, for delivery of a nucleic acid to a T cell, wherein when said nucleic acid is a DNA encoding a gene expression cassette, for expression of the gene, said resting T cells are activated. Likewise, the lipid nanoparticles in accordance with the invention, are highly useful for delivery of a nucleic acid to an NK cell. Hence, one may isolate T cells, or NK cells, and subsequent contact T cells, or, NK cells, with lipid nanoparticles in accordance with the invention.

[0061] As said, and as described above, such delivery of a nucleic acid is highly selective for T cells and NK cells. Thus, in one embodiment, a use is provided of the nanoparticle in accordance with the invention, for selective delivery of a nucleic acid to T cells and NK cells. Such selective delivery is in particular useful when the nanoparticles, such a LNPs, are contacted with PBMCs, or with whole blood. Hence, in another embodiment, such use for selective delivery of a nucleic acid to T cells and NK cells is when the nanoparticle is contacted with PBMCs, or, wherein the nanoparticle is contacted with whole blood. In particular, such selective delivery of comprises a TNK / O ratio, or fold-increase in TNK / O ratio as defined herein.

[0062] It is understood that in particular, the use of the nanoparticles as defined herein for delivery of a nucleic acid, involves preferably a nucleic acid encoding a CAR or a TCR.

[0063] In any case, the nanoparticle in accordance with the invention, is highly suitable for delivery of an agent, such as RNA or DNA to T Cells, i.e. CD4+ and / or CD8+ T cells, also referred to as helper T cells and / or cytotoxic T cells. In particular, the nanoparticles with the human CD7 targeting moiety in accordance with the invention were shown to be highly efficient and specific for delivery of an agent, such as RNA DNA, to the nucleus and / or the cytoplasm of (resting) T cells and NK cells.

[0064] In yet a further embodiment, a method is provide for delivery of an agent to T cells and / or NK cells, comprising: Providing T cells and / or NK cells;

[0065] Providing a nanoparticle with a targeting moiety capable of binding human CD7 as defined herein in accordance with the invention;

[0066] Contacting said T cells and / or NK cells with said nanoparticle.

[0067] In another embodiment, a method is provide for delivery of an agent to resting cytotoxic T cells, comprising:

[0068] Providing resting cytotoxic T cells;

[0069] Providing a nanoparticle with a targeting moiety capable of binding human CD7 as defined herein in accordance with the invention;

[0070] Contacting said resting cytotoxic T cells with said nanoparticle.

[0071] In another embodiment, a method is provide for delivery of an agent to resting T cells, comprising:

[0072] Providing resting T cells;

[0073] Providing a nanoparticle with a targeting moiety capable of binding human CD7 as defined herein in accordance with the invention;

[0074] Contacting said resting T cells with said nanoparticle.

[0075] As shown herein, such a targeting moiety capable of binding human CD7 is highly specific with regard to delivery of the agent comprised in the nanoparticle to T cells and / or NK cells. Such a targeting moiety allows for preferred and specific delivery of the an agent to said T cells and / or NK cells when these cells are e.g. provided within a population of cells, such as PBMCs. Hence, alternatively, a method is provide for delivery of an agent to T cells and / or NK cells, comprising:

[0076] Providing PBMCs;

[0077] Providing a nanoparticle with a targeting moiety capable of binding human CD7 as defined herein in accordance with the invention;

[0078] Contacting said PBMCs with the provided nanoparticles.

[0079] Highly advantageously, the T cells may not be required to be activated in accordance with the invention. Such may be advantageous as this may allow for minimal manipulation of primary T cells, which can be highly desirable e.g. in a clinical setting, wherein e.g. isolated resting T cells of a human subject are contacted with a nanoparticle in accordance with the invention and subsequently reinfused back into the patient, e.g. in a scenario wherein the resting T cells are provided with a CAR or the like, capable of binding to a target antigen.

[0080] Because the targeting moiety capable of binding human CD7 is highly selective, it may not be required to isolate resting T cells, or resting cytotoxic T cells, or NK cells, and one may simply contact isolated PBMCs with the nanoparticle in accordance with the invention, provided with a CAR or the like, and readminister the cells back into the patient. One may contemplate isolation of resting T cells, or resting cytotoxic T cells, either before or after contacting the cells with the nanoparticle in accordance with the invention. One may also contemplate to isolate cells that are provided with the agent, in accordance with the invention, by including in the nanoparticle a marker (or nucleic acid encoding it) that allows for specific selection. Suitable agents that may be contemplated as a marker include e.g. GFP as used in the examples herein. Also markers such as RQR8 (see Mosti et al. Gene Therapy 2021 ; Philip et al., Blood, 2014 Aug 21 ; 124(8): 1277-87), a FLAG-tag (Berahovich et al., 2017, Frontiers in Bioscience-Landmark, 22(10), pp.1644-1654), c-Myc tag (Repellin, et al., 2020. Advanced biosystems, 4(1), p.1900224), or mCherry (Choi, et al., 2019, Nature biotechnology, 37(9), pp.1049-1058.) may be contemplated. Selectable markers may e.g. be included in the expressed CAR I TCR, e.g. because these comprise a selectable epitope (added or comprised in its sequence) based on which cells expressing the CAR I TCR may be conveniently selected. It is understood that of course the expressed CAR I TCR itself may function as a selectable marker. Thus, the nucleic acid encoding a selectable marker may be a separate nucleic acid or may be comprised in the nucleic acid sequence encoding the CAR / TCR.

[0081] Hence, in a further embodiment, a method is provided for delivery of an agent to T cells and / or NK cells, comprising:

[0082] Providing PBMCs;

[0083] Providing a nanoparticle with a targeting moiety capable of binding human CD7 as defined herein in accordance with the invention, and wherein the nanoparticle comprises an agent, and wherein the nanoparticle comprises a selectable marker or nucleic acid encoding a selectable marker;

[0084] Contacting said PBMCs with the provided nanoparticles; Subsequently, Isolating cells carrying the selectable marker, or expressing the selectable marker, therewith providing cells substantially consisting of T cells and / or NK cells provided with said agent.

[0085] Of course, one may contemplate to in addition, isolate resting cytotoxic T cells as well. It understood that the order of steps of the step of isolating resting cytotoxic T cells and the step of selecting cells with the selectable marker may be reversed. Of course, one may also provide resting T cytotoxic T cells to be contacted with the nanoparticle instead. Hence, alternatively, in a further embodiment, a method is provided for delivery of an agent to resting cytotoxic T cells, comprising:

[0086] Providing resting cytotoxic T cells;

[0087] Providing a nanoparticle with a targeting moiety capable of binding human CD7 as defined herein in accordance with the invention, and wherein the nanoparticle comprises an agent, wherein nanoparticle comprises a selectable marker or a nucleic acid encoding a selectable marker;

[0088] Contacting said resting cytotoxic T cells with the provided nanoparticles; Subsequently, Isolating cells carrying the selectable marker, therewith providing cells substantially consisting of resting T cells provided with said agent.

[0089] The above methods relate to providing resting cytotoxic T cells that are provided with the agent, e.g. such that the resting cytotoxic T cells express a CAR or the like, which is highly advantageous in the treatment of e.g. cancer in cell therapy approaches and the like. Likewise however, one may also contemplate to provide resting T cells, helper T cells, or NK cells that are provided with the agent such as a nucleic acid, e.g. such that these express a gene of interest or the like, which may be highly advantageous in a cell therapy treatment of conditions that may benefit from such modified cells. In any case, whichever method is employed, the methods as described above provide for T cells and / or NK cells, that are provided with the agent which is highly useful e.g. in therapeutic approaches, and of course, for basic research as well. It is understood that prepared modified cells, as obtained with the methods as described herein may be highly useful in the treatment of human subjects and may be administered to human subjects, e.g. by infusion.

[0090] As it may be preferred not to activate T cells in methods and uses as described herein, it nevertheless may be contemplated to activate the T cells, or isolated CD8+ cells. Methods for activation of T cells are well known in the art, and common methods for activation of T cells include providing an activation stimulus to activate the cells and trigger their expansion. A variety suitable activation stimuli are known in the art and common activation stimuli known in the art are phytohemagglutinin (PHA) and CD3 / CD28 antibody. For example, isolated CD8+ T cells, or isolated T cells, are seeded in an appropriate cell culture medium, and subjected to activation for 2 days by providing a a CD3 / CD28 activator and supplementing IL-2 in the medium. CD3 / CD28 activators can be provided as CD3 and CD28 targeting antibodies, e.g. coated on beads, such as described in the examples herein, which are widely used in the art. In one embodiment, in the means and methods in accordance with the invention, the activation stimulus that is provided is DynaBeadsTM Human T-Activator CD3 / CD28. Furthermore, a highly suitable interleukin may be added for culturing T cells, i.e. Interleukin 2, (also referred to as IL-2 or IL2) in an appropriate amount.

[0091] In any case, whichever method is used, by contacting cells, e.g. whole blood, PBMCs, or isolated T cells and / or NK cells, with nanoparticle in accordance with the invention, the agent is delivered to T cells and / or NK cells. This delivery to the cells highly advantageously includes delivery of the agent to the cytoplasm and / or the nucleus. Of course, the methods as outlined above include contacting steps ex vivo, i.e. outside of the human body. It may be contemplated to administer the nanoparticles in accordance with the invention in vivo, such that delivery of the agent occurs in vivo. As outlined above, the nanoparticles in accordance with the invention are thus highly advantageous for use in the treatment of conditions that may benefit from modified T cells and / or NK cells, such modification involving the step of delivery of the agent to such cells with (lipid) nanoparticles in accordance with the invention.

[0092] Thus, in one embodiment, a nanoparticle comprising an agent is provided with a targeting moiety with an antigen binding region capable of binding to human CD7, as defined in accordance with the invention, wherein said nanoparticle is for use in the treatment of a disease, wherein said treatment comprises administering the nanoparticle to the patient. In one embodiment, a nanoparticle comprising an agent is provided with a targeting moiety with an antigen binding region capable of binding to human CD7, as defined in accordance with the invention, wherein said nanoparticle is for use in the treatment of a disease, wherein said treatment comprises administering the nanoparticle to the patient, and wherein said agent is a nucleic acid, which is an mRNA as defined herein. In another embodiment, a nanoparticle comprising an agent is provided with a targeting moiety with an antigen binding region capable of binding to human CD7, as defined in accordance with the invention, wherein said nanoparticle is for use in the treatment of a disease, wherein said treatment comprises obtaining T cells and / or NK cells from the patient, contacting the nanoparticle with the obtained T cells and / or NK cells ex vivo and allowing delivery of the agent to the cells, and subsequently administering the T cells and / or NK cells to the patient. In one embodiment, a nanoparticle comprising an agent is provided with a targeting moiety with an antigen binding region capable of binding to human CD7, as defined in accordance with the invention, wherein said nanoparticle is for use in the treatment of a disease, wherein said treatment comprises obtaining T cells and / or NK cells from the patient, contacting the nanoparticle with the obtained T cells and / or NK cells ex vivo and allowing delivery of the agent to the cells, and subsequently administering the T cells and / or NK cells to the patient, wherein said agent is a nucleic acid, which is an mRNA as defined herein. In another embodiment, a nanoparticle comprising an agent is provided with a targeting moiety with an antigen binding region capable of binding to human CD7, as defined in accordance with the invention, wherein said nanoparticle is for use in the treatment of a disease, wherein said treatment comprises obtaining T cells and / or NK cells from the patient, contacting the nanoparticle with the obtained T cells and / or NK cells ex vivo and allowing delivery of the agent to the cells, and subsequently administering the T cells and / or NK cells to the patient, wherein said agent is a nucleic acid as defined herein, and wherein said T cells are activated prior to administering the T cells to the patients.

[0093] It is understood that the nanoparticles, such as LNPs, as described herein, may find in particular use in the treatment of diseases such as cancer, such as a cancer of blood cells and / or such as a B-cell lymphoma. Hence, the invention provides for nanoparticles, such as LNPs as described herein and shown in the examples, which preferably include an mRNA or DNA encoding a CAR or a TCR, for use in the treatment of cancer. More preferably, nanoparticles, such as LNPs as described herein are provided, comprises an mRNA encoding a transposase and a DNA encoding a CAR or a TCR with flanking ITRs compatible with the transpose, for use in the treatment of cancer. Such treatment of cancer preferably comprising the treatment of a cancer of blood cells, such as leukemia. Such treatment of cancer can also be of a B-cell cancer or B-cell malignancy such as a B-cell lymphoma. In the treatment of a B-cell cancer or malignancy, such as a B-cell lymphoma, the CAR or TCR preferably targets CD19 CD20, and / or CD22.

[0094] In one embodiment, an immunoglobulin is provided, or a fragment of an immunoglobulin, wherein said immunoglobulin or said fragment thereof comprises CDR regions derived from the VHH as defined by SEQ ID NO.1. In a further embodiment, said immunoglobulin, or a part thereof, comprises the CDR regions of SEQ ID NO.1. In yet a further embodiment, said CDR regions of SEQ ID NO.1 are CDR1 , CDR2 and CDR3 of SEQ ID NO.1 as defined by SEQ ID NO. 2, 3 and 4, respectively.

[0095] In one embodiment, an immunoglobulin is provided, or a fragment of an immunoglobulin, comprising the CDR regions of SEQ ID NO.5. In a further embodiment, said CDR regions of SEQ ID NO.1 are CDR1 , CDR2 and CDR3 of SEQ ID NO.5 as defined by SEQ ID NO. 6, 7 and 8, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.5 was identified.

[0096] In one embodiment, an immunoglobulin is provided, or a fragment of an immunoglobulin, comprising the CDR regions of SEQ ID NO.9. In a further embodiment, said CDR regions of SEQ ID NO.9 are CDR1 , CDR2 and CDR3 of SEQ ID NO.9 as defined by SEQ ID NO. 10, 11 , 12, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.9 was identified.

[0097] In one embodiment, an immunoglobulin is provided, or a fragment of an immunoglobulin, comprising the CDR regions of SEQ ID NO.13. In a further embodiment, said CDR regions of SEQ ID NO.13 are CDR1 , CDR2 and CDR3 of SEQ ID NO.13 as defined by SEQ ID NO. 14, 15 and 16, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.13 was identified.

[0098] In one embodiment, an immunoglobulin is provided, or a fragment of an immunoglobulin, comprising the CDR regions of SEQ ID NO.17. In a further embodiment, said CDR regions of SEQ ID NO.17 are CDR1 , CDR2 and CDR3 of SEQ ID NO.17 as defined by SEQ ID NO. 18, 19 and 20, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.17 was identified.

[0099] In one embodiment, an immunoglobulin is provided, or a fragment of an immunoglobulin, comprising the CDR regions of SEQ ID NO.21 . In a further embodiment, said CDR regions of SEQ ID NO.21 are CDR1 , CDR2 and CDR3 of SEQ ID NO.21 as defined by SEQ ID NO. 22, 23, and 24, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.21 was identified.

[0100] In one embodiment, an immunoglobulin is provided, or a fragment of an immunoglobulin, comprising the CDR regions of SEQ ID NO.25. In a further embodiment, said CDR regions of SEQ ID NO.25 are CDR1 , CDR2 and CDR3 of SEQ ID NO.25 as defined by SEQ ID NO. 26, 27 and 28, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.25 was identified.

[0101] In one embodiment, an immunoglobulin is provided, or a fragment of an immunoglobulin, comprising the CDR regions of SEQ ID NO.29. In a further embodiment, said CDR regions of SEQ ID NO.29 are CDR1 , CDR2 and CDR3 of SEQ ID NO.29 as defined by SEQ ID NO. 30, 31 , and 32, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.29 was identified. Such as

[0102] In one embodiment, an immunoglobulin is provided, or a fragment of an immunoglobulin, comprising the CDR regions of SEQ ID NO.33. In a further embodiment, said CDR regions of SEQ ID NO.33 are CDR1 , CDR2 and CDR3 of SEQ ID NO.33 as defined by SEQ ID NO. 34, 35 and 36, respectively. It is understood that such CDR sequences are preferably comprised in a VHH format, such as the antibody from which SEQ ID NO.33 was identified.

[0103] In one embodiment, the immunoglobulin or immunoglobulin fragment comprises or consists of SEQ ID NO.1. In one embodiment, the immunoglobulin or immunoglobulin fragment comprises or consists of SEQ ID NO.5. In one embodiment, the immunoglobulin or immunoglobulin fragment comprises or consists of SEQ ID NO.9. In one embodiment, the immunoglobulin or immunoglobulin fragment comprises or consists of SEQ ID NO.13. In one embodiment, the immunoglobulin or immunoglobulin fragment comprises or consists of SEQ ID NO.17. In one embodiment, the immunoglobulin or immunoglobulin fragment comprises or consists of SEQ ID NO.21 . In one embodiment, the immunoglobulin or immunoglobulin fragment comprises or consists of SEQ ID NO.25. In one embodiment, the immunoglobulin or immunoglobulin fragment comprises or consists of SEQ ID NO.29. In one embodiment, the immunoglobulin or immunoglobulin fragment comprises or consists of SEQ ID NO.33. In yet a further embodiment, a lipid-PEG is provided to which such an immunoglobulin or immunoglobulin fragment is conjugated. In still another embodiment, a nanoparticle is provide comprising such an such an immunoglobulin or immunoglobulin fragment, or immunoglobulin conjugated to a lipid-PEG or an immunoglobulin fragment conjugated to a lipid-PEG, as defined herein throughout. Such immunoglobulins, immunoglobulin fragments, which may be conjugated or not with a lipid-PEG, are in a further embodiment for use in the preparation of nanoparticles, including lipid nanoparticles as described herein. In a further embodiment, said use is for the preparation of (lipid ) nanoparticles for delivery of an agent to T cells or NK cells. Preferably, such T cells are resting T cells.

[0104] In a further embodiment, a nucleic acid is provided encoding a targeting moiety, an immunoglobulin, or an immunoglobulin fragment, as defined herein. In particular and preferably, such nucleic acid encoding an immunoglobulin or immunoglobulin fragment, comprises and encodes for the CDR regions of SEQ ID NO.1 , 5, 9, 13, 17, 21 , 25, 29, or 33, such as defined by herein. In yet a further embodiment, the use is provided of such a nucleic acid encoding an immunoglobulin, or immunoglobulin fragments, as defined herein, for the manufacturing of a nanoparticle for delivery of an agent to T cells or NK cells. Preferably, such T cells are resting T cells. In yet another further embodiment, a cell is provided comprising a nucleic acid encoding a targeting moiety, an immunoglobulin, or an immunoglobulin fragment, as defined herein. In particular and preferably, such a cell with a nucleic acid encoding an immunoglobulin or immunoglobulin fragment, comprises and encodes for the CDR regions of SEQ ID NO.1 , 5, 9, 13, 17, 21 , 25, 29, or 33 as defined herein. In yet a further embodiment, the use is provided of such a nucleic acid, or a cell with such a nucleic acid, encoding an immunoglobulin, or immunoglobulin fragments, as defined herein, for the manufacturing of a nanoparticle for delivery of an agent to T cells or NK cells. Preferably, such T cells are resting T cells. Examples

[0105] Example 1

[0106] Material and Methods

[0107] Llama immunization and Phage display

[0108] 2 Llama’s were immunized with LNPs constructed with SM-102 ionizable lipid, containing cap1 capped, N1 -methyl-pseudo uridine modified, 120 polyA mRNA encoding full length human CD7 antigen as set forth in GENBANK Accession No. AAH13297.1 Peripheral blood lymphocytes were isolated from Llama blood, and Ig-H cDNA fragments were amplified and used to construct a VCSM12 bacteriophage library expressing the VHHs via fusion to the minor coat protein PHI. For panning, phages were first incubated with CD7-expressing Flp-in T rex cells, after washing, phages were eluted from the panning cells. Eluted phages were then depleted with Flp-in T rex cells, the unbound phages were further incubated with CD7- expression Flp-in T rex cells again before elution. After recovery of the phages from the panning step, master plates are created with individual clones and further validated with production with IPTG induction. Flp-ln T-Rex 293 cells were obtained from Thermo Fisher. A panning construct fragment consisting of human CD7 fused to intracellular GFP was obtained as a gBIock from IDT. This panning construct was cloned into the pcDNA5 / FRT / TO plasmid compatible with the Flp-ln T Rex 293 system. A stable panning line was then generated using the Flp-ln T rex 293 system according to manufacturer's instructions. The VHH sequences of clone H7 used in this example corresponding with the CD7 nanobody binding human CD7 is listed herein in table 4, i.e. SEQ ID NO. 1 corresponds with the VHH sequence, and SEQ ID NO. 2-4 correspond with CDR1-3 based on IMGT repertoire protein display of Llama Glama. VHH H7 was fused to a C-terminal tag including a cysteine for conjugation to PEG-lipid and an EPEA sequence for affinity purification and was produced in yeast cells (Saccharomyces cerevisiae).

[0109] VHH H7 flow cytometry binding Cells were stained with VHH H7 at indicated concentration ( if not indicated 1 pg / ml was used) for 30 min at room temperature in PBS with 2% FCS. Cells were washed and stained with MonoRab Rabbit Anti-Camelid VHH Ab-iF647 (Genscript) for 30 minutes at room temperature. Cells were measured on FACs Canto (BD) and results were analysed using FlowJo software.

[0110] LNP production

[0111] LNP formulations were prepared using a nanoassemblr microfluidic device. Nucleic acid was diluted in 100mM acetate buffer at pH 4 and mixed with an organic phase containing ionizable lipid, helper lipid, PEG lipid and cholesterol at a flow rate ratio of 2:1 (aqueous : lipid phase). Ionizable lipids used were SM-102 (2089251-47-6), DLin-DMA-MC3 (1224606-06-7) , DLin-KC2-DMA (1190197-97-7) Lipid A9 (2036272-50-9) and C12-200 (1220890-25-4), as indicated. If not specified SM-102 was used. DSPC was used as a helper lipid and DMG- PEG-2000 was used as a PEG lipid. Particles were targeted via conjugation of a C-terminal cysteine on the VHH binder to a reactive maleimide group on DSPE-PEG-2000-maleimide at a molar percentage of 0.05% total lipid. Lipid conjugate was post-inserted into the LNP after LNP production according to a procedure described (Swart et al. Int J Pharm, 2022, May 25, Vol. 620 121741). We tested a range of binder densities from 0.025 to 1 mole% of total lipid and evidence of increased nucleic acid delivery and targeting as compared to an untargeted control LNP was observed at each density tested within this range. Within this range an optimal binder density was identified at 0.05%. It is of interest to note that this optimal binder density is considerably lower than the optimal binder densities found for antibody-mediated targeting. For addition of targeting antibodies to LNPs we made use of a monobody adapter approach as described previously by C. Albert et al. Antibodies used for targeting were: CD2: clone RPA-2.10 (BioLegend), CD3: clone UCHT1 (BioLegend), CD5: clone UCHT2 (BioLegend), CD7: clone M-T701 (BD), CD8 clone SK1 (BioLegend) CD28: clone 15E8 (Miltenyi Biotec). LNPs were produced by mixing a lipid containing organic phase and a nucleic acid containing aqueous phase using a Nanoassemblr microfluidic device.

[0112] Formulations were based on the formulation of Onpattro / Patisaran and made use of various ionizable lipids. PBMC uptake or transfection

[0113] PBMCs were isolated from healthy donor blood using Fiqoll-PAque (GE healthcare) and SepMate PBMC isolation tubes (Stemcell) according to manufacturer’s protocol. For uptake and transfection experiments 200.000 PBMCs per well were plated in a 96 well plate and incubated with different LNP formulations (either untargeted or targeted with VHH H7) and loaded with GFP encoding cy5 labelled mRNA, a total nucleic acid dose of was used per condition. Cells were incubated with LNPs at 37 degrees for 4 hours for uptake and 24 hours for mRNA transfection experiments. Incubation was done in T cell medium (RPMI 1640 (Thermo Fisher) + 2.5% heath inactivated pooled human Serum (Sanquin), 1% Pen / Strep (Gibco) and 2mM 2-mercaptoethanol (Gibco) with addition of 1 ug / ml Apo-E (Abeam) for transfection only. After incubation the cells were incubated with Zombie Aqua viability dye (Biolegend) and FC-block (BD) in PBS for 20 minutes after which different cell subsets were stained with CD19-PE ( clone A3-B1 , Biolegend), CD4-PE-cy5 ( clone OKT4, Biolegend) CD8-Pe-cy7 (clone 3B5, Thermo scientific) CD2-BV605 (clone s5.2, BD), CD14-BV650 (clone M5E2 Biolegend) CD56-BV785 (clone NCAM16.2, BD). Cells were incubated with the Ab mix in PBS 2% FCS for 30 minutes and washed. Samples were acquired on LSRFortessa (BD) and results were analysed using FlowJo software.

[0114] Flp-ln-Trex 293 transfection

[0115] Flp-ln-Trex 293 cell line, WT or with CD7 knock-in, were seeded in a 96 well flat bottom plate, 7500 cells per well. Transfection with a dose range of untargeted and VHH CD7 targeted LNPs loaded with eGFP mcDNA was performed in DMEM 10% FCS (Biowest) 1% P / S (Gibco) and 1 ug / ml Apolipoprotein-E (Apo-E) (Abeam). Cells were incubated at 37 degrees 5% CO2 for 72hours, GFP expression was measured FACs Canto (BD) and analyzed using FlowJo software.

[0116] PBMC / T cell transfection

[0117] For transfection of T cells PBMCs were isolated from healthy donor blood using Fiqoll-PAque (GE healthcare) and SepMate PBMC isolation tubes (stemcell) according to manufacturer’s protocol. T cells (including CD3+CD4+ and CD3+CD8+ cells) were isolated from the PBMCs using EasySep Human T cell isolation kit (stemcell) using manufacturer’s protocol. T cells were plated 25.000 cells per well in a 96 well U-bottom plate in 100 ul T cell medium with 50 ill / ml IL2 (Miltenyi Biotec) and 1 ug / ml Apo lipoprotein-E ( Abeam), if indicated, the T cells were activated by CD3 / CD28 dynabeads (Gibco) at a 1 :1 bead to T cell ratio. LNPs were added at 250 ng total NA (nucleic acid)dose (if not indicated differently) and incubated for 24h for mRNA transfection and 96 hours for DNA transfection. Transfection efficiency was measured by flow cytometry. CAR was detected using CD19 CAR detection reagent (Miltenyi Biotec) and streptavidin-APC-cy7 (BD). Samples were measured on FACs Canto (BD) and results were analysed using FlowJo software.

[0118] Results

[0119] The use of CD7 as a target for efficient delivery of drugs to resting as well as activated T lymphocytes using a carrier vehicle was shown. Advantageously, the lipid nanoparticles in accordance with the invention were shown to effectively deliver nucleic acids to resting and activated T lymphocytes. We observed delivery of both DNA and RNA into resting T-cells as evidenced by expression of a reporter gene driven by mRNA and the fact that the two type of nucleic acids were co-loaded in a single LNP particle (data not shown). We here also demonstrated that the lipid nanoparticles in accordance with the invention are able to deliver DNA to activated T cells, resulting in expression of a gene cassette comprise in the DNA .

[0120] First of all, minicircle DNA was packaged into LNPs that were either non-functionalized or functionalized with monoclonal antibodies against different well known T cell antigens: CD2, CD3, CD5, CD7, CD8 and CD28. This was achieved by conjugation of an adaptor monobody to the LNPs that can bind to the Fc region of mAbs, as described in C. Albert et al., Nature Communications, (2022)13:5998 pp 1-13, published online 11 October 2022. As shown in Fig. 2, using this technology, only LNPs targeted to CD7 functionally transfected T cells with DNA, illustrated by 5% of the T lymphocytes showing GFP expression. Targeting to any of the other tested receptors did not result in GFP expression from DNA delivered by LNP.

[0121] To further explore transfection of T cells with nucleic acids (mRNA and DNA) using CD7 as targeting antigen, a novel anti-CD7 binder was generated by immunization of llama’s with LNPs loaded with CD7 encoding mRNA. After immunization, serum was collected from the llama’s and anti-CD7 nanobodies were identified by phage display. Specificity of one of the positive clone, H7, was first confirmed by flow cytometry binding assay on the CD7 knock-in Flp-ln-Trex 293 cell line, where specific binding was shown for the CD7 knock-in, but not CD2 knock-in or WT cell line (Figure 3). Binding EC50 of the anti CD7 VHH on primary T lymphocytes was determined to be 10.94 nM based on a flow cytometry titration assay (Figure 4).

[0122] In order to explore the potential of the identified CD7 specific VHH as a targeting moiety for nucleic acid, such as DNA, delivery by LNPs, the VHH was conjugated to a LNP loaded with GFP encoding DNA. First, the non-targeted and CD7 targeted LNPs were compared for transfection of knock-in Flp-ln-Trex 293 cell line with and without CD7 knock-in. When CD7 targeted particles were used for transfection of CD7 expressing Flipin-T rex cells, a large increase in transfection efficiency was seen compared to non-functionalized particles for most tested LNP doses, showing the added value of using CD7 as target for DNA delivery via LNP (Figure 5). Interestingly, on WT flipin-T rex cells (without CD7 expression) the CD7 VHH targeted LNPs induced less DNA transfection compared to untargeted particles. This indicates that the inclusion of the CD7 VHH on an LNP can reduce aspecific, untargeted, transfection of cells.

[0123] Next, CD7 VHH targeted LNPs were tested for binding and uptake in T lymphocytes. To this end, five different LNP formulations, made with different commercially available ionizable lipids, were prepared using Cy5 labelled siRNA, and incubated with freshly isolated peripheral blood mononuclear cells (PBMCs). Binding and / or uptake of the LNPs to the different PBMC subsets: B cells, T cells, monocytes, NK cells and other cells, was visualized using flow cytometry (See Table 1 and Figure 6). For all five tested formulations, addition of the anti- CD7 VHH increased the absolute amount of LNP binding and uptake in T lymphocytes and NK cells (Figure 6). This is also reflected by the big increase in TNK / O ratio of targeted particles when compared to the untargeted counterpart, calculated by dividing the number of cy5+ T- and NK cells by the total number of cy5 positive other cells (monocytes, B cells and further cells that are not classified as monocytes, B cells, T cells and NK cells). The fold increase in Cy5 TNK / O ratio’s comparing with and without CD7 VHH targeting was 7, 7, 8, 33, and 11 , respectively for LN P formulations with SM-102, MC3, KC2, Lipid A9 and C12-

[0124] 200, respectively (i.e. calculated by dividing the Cy5 TNK / O ratio of (+) by (-)).

[0125] Table 1 . Flow cytometry of Cy5 positive cells

[0126] Next, the assay was repeated with LNPs loaded with mRNA encoding eGFP to more specifically analyze transfection of the different PBMCs cell subsets. The absolute number (Figure 7, Table 2) of GFP expression in the different PBMC cellsubsets was determined using flow cytometry.

[0127] Table 2 Flow cytometry of GFP positive cells

[0128] For untargeted LNPS, 70 to 80% of the transfected cells were B cells and monocytes, while only a minor fraction was T and NK-cells. Addition of the aCD7 VHH binder strikingly increased the number of -T- and NK cells in the transfected cell populations (Figure 6B). The fold increase in GFP TNK / O ratio’s comparing with and without CD7 VHH targeting was 31 , 11 , 8, 23, and 16, respectively for LNP formulations with SM-102, MC3, KC2, Lipid A9 and 012-200, respectively (i.e. calculated by dividing the GFP+ TNK / O ratio of (+) by (-)). This shows that an aCD7 binder can be used to specifically deliver mRNA cargo to T lymphocytes and NK cells

[0129] To further explore the hypothesis that functionalization with the anti-CD7 VHH can enhance functional delivery of mRNA to T lymphocytes, isolated primary T lymphocytes were incubated with GFP mRNA loaded LNPs that were either untargeted or functionalized with anti-CD7 VHH. The T lymphocytes were either untreated, or pre-activated with CD3 / CD28 dynabeads and IL2. LNP formulation as described above with SM102 mediated mRNA transfection was determined by measuring GFP expression using flow cytometry (Figure 8A left panel). Transfection of both resting and pre-activated T lymphocytes with mRNA was enhanced by anti-CD7 VHH functionalization of the LNP, but the difference was most pronounced in resting T cells. This shows that while mRNA transfection of T-cells can be achieved by untargeted LNPs, the addition of CD7 targeting to LNPs can be of great value to achieve mRNA transfection of resting T cells. Functional delivery of DNA to T lymphocytes is another important hurdle to tackle, as nuclear delivery of the DNA is necessary, but for T lymphocytes a proven difficult barrier to cross. To address whether CD7 targeting with our anti-CD7 VHH can also enhance functional DNA delivery to T lymphocytes, LNPs loaded with minicircle DNA (mcDNA) encoding for eGFP were used to transfect isolated activated primary T lymphocytes (Figure 8A right panel).

[0130] Addition of VHH anti-CD7 targeting to the LNP greatly enhanced functional delivery of mcDNA, while there was no transfection of the T lymphocytes when using untargeted LNPs, LNPS functionalized with anti-CD7 VHH achieve efficient GFP expression efficiency in a dose depend manner (Figure 8B).

[0131] Lastly, LNPs formulated like as described above with SM102 targeted with anti-CD7 VHH were also used to induce chimeric antigen receptor (CAR) expression on resting and activated T cells, both from mRNA and from mcDNA (Figure 9), showing that with CD7 targeting, highly advantageously a functional gene encoding CAR was delivered to T lymphocytes.

[0132] Example 2

[0133] In addition to the previously described anti-CD7 VHH clone H7, eight additional CD7 binding VHH clones were identified from the same Phage library of which the original CD7 clone H7 originated (See Table 5 below). These VHHs were in a test assessed for binding on the T cell line Jurkat by flow cytometry, as described above for the H7 clone. Briefly, the VHH clones were incubated with Jurkat cells, which express CD7, after which VHH bound to the cells was detected using an anti-VHH secondary antibody which was fluorophore labeled. Figure 10 shows a titration of the different VHH clones in a flow cytometry binding assay, showing percentage VHH labeled positive cells (upper graph of Figure 10) or detected mean fluorescence (lower graph of Figure 10). These results show that while the different VHH clones do not show large differences marking cell populations positive at different VHH concentrations, the different VHH clones in this preliminary test appeared to plateau at different MFIs, indicating that these further candidates differ in binding characteristics (figure 10, and table 3 below).

[0134] Binding of the VH clones to primary T cells and CD7 knock-in Flp-ln-Trex 293 cell line, was also confirmed. None of the clones appeared to bind to PBMCs isolated from cynomolgus monkey blood.

[0135] Table 3. MFI of 10 nM datapoint shown in Figure 10, lower graph, with values ranked from high to low

[0136] Example 3

[0137] Transfection of T cells with LNPs targeted using CD7 VHH and loaded with minicircle DNA encoding for CAR DNA and mRNA encoding for SB100 mRNA leads to the generation of functional CAR-T cells

[0138] In this experiment, LNPs were generated as described herein above. Briefly, the ionizable lipid used was SM102., a CD7 VHH was conjugated to DSPE-PEG-2000-maleimide at a 2:1 lipid:protein ratio and post-inserted onto the particle at a molar percentage of 0.05% of total lipid. The LNPs were loaded with a minicircle DNA comprising a transposable transgene encoding for a CAR targeting CD19 and an mRNA encoding for an engineered transposase, Sleeping Beauty (SB100X) (Jin et al. Gene Ther. 2011 Sep;18(9):849-56.). The LNPs generated were used to transfect T-cells, isolated and activated as described above, and as a control were also transfected with LNPs without SB100x but a stuffer mRNA, or were left untreated.

[0139] T cells transfected with CD7 targeted LNPs loaded with mcDNA CAR and SB100x transposase mRNA were shown to have stable CAR expression during a culture period of more than 3 weeks, while T cells transfection with targeted LNPs without SB100x mRNA only induced transient CAR expression (Figure 11 A). 22 days post-transfection mean CAR expression was about 2.3%, and approximately 30% of initially transfected cells had persistent CAR expression demonstrating stable integration.

[0140] The CD19 CAR-T cells generated using CD7 targeted LNPs were also shown to be functional in target cell killing, as evidenced by specific lysis of CD19 expressing Raji target cells and K562 cells engineered to express CD19 (Figure 11 B).This killing assay was performed after 15 days, demonstrating CAR expression was stable. The experiment was conducted without CD19-CAR-T cell enrichment. In this experiment, E:T ratios refer to the ratio of CAR positive T cells in the total T cell mix to target cells. T cells treated with non- transposase containing tLNPs were not included in this experiment as CAR expression was 0% at day 15. Furthermore, an upregulation of cytokines IFN-y and IL-2 after co-culture with CD19 positive target cells (K562+CD19 and Raji) was observed but not CD19 negative K562 cells (Figure 11C and D), demonstrating specificity.

[0141] Combined the results show that LNPs engineered to target CD7 can efficiently and stably deliver DNA encoding for a CAR targeting CD19 to T-cells, which are potent and specific in CD19+ cell killing.

[0142] Example 4

[0143] Mice experiments were conducted wherein huCD34+ humanized NSG mice (Walsh et al, Annu Rev Pathol. 2017 Jan 24; 12: 187-215) were injected with tLNPs targeted with CD7 VHH. In this experiment, LNPs used were generated as described herein above. Briefly, the ionizable lipid used was SM102 , and CD7 VHH was conjugated to DSPE-PEG-2000- maleimide and post-inserted onto LNPs at a molar percentage of 0.05% of total lipid. The LNPs were loaded with a Cy5 labelled mRNA or a CAR encoding mRNA.

[0144] It was observed that 60% of circulating T cells are positive for Cy5 1 day after LNP injection, with no signal in B cells (Fig. 12A). Distribution in organs 2 days after LNP injection shows Cy5 signal mainly in T- and NK-cells in blood, and to a lower extent also in the spleen and bone marrow T cells (not shown) whereas expression from mRNA as observed as percentage of CAR positive cells, was high in spleen cells (Fig.12B).

[0145] These results show that CD7 targeted LNPs specifically target T cells and not B cells in vivo, in the blood. Furthermore, CD7 targeted LNPs can functionally deliver nucleic acids, i.a. mRNA, to T- and NK cells in the spleen of humanized mice (panel B). The specificity of targeting was evidenced by the lack of both uptake and transfection of B cells.

[0146] Example 5

[0147] Engineering NK CARs - Transfection and Stable Integration of Primary NK Cells Using CD7-Targeted LNPs

[0148] Primary NK cells were transfected using LNPs targeted with CD7-specific VHH. The LNPs as used in this experiment were generated as described herein above. Briefly, the ionizable lipid used was SM102, and CD7 VHH was conjugated to DSPE-PEG-2000-maleimide and postinserted onto LNPs at a molar percentage of 0.05% of total lipid. The LNPs were loaded with a minicircle DNA comprising a transposable transgene encoding for a CAR targeting CD19 or reporter gene eGFP and an mRNA encoding for an engineered transposase, Sleeping Beauty (SB100x). The LNPs generated were used to transfect isolated PBMCs, as described above. After tLNP transfection, the PBMCs were cultured with K562 feeder cells to stimulate the outgrowth of NK cells from the PBMCs.

[0149] Flow cytometry analysis on day 5 demonstrated that up to 20% of NK cells (gated based on

[0150] CD56 positivity) expressed either eGFP or CD19-specific CAR, indicating successful transfection (Figure 13A). Notably, continued culture of these transfected cells over a two- week period revealed sustained expression of CAR or eGFP (Figure 13B), suggesting stable integration of the gene constructs mediated by the SB100X transposase. Importantly, LNP transfection did not impact expansion of NK cells in culture (Figure 13C).

[0151] This shows that targeting NK cells via CD7 provides for efficient generation of engineered NK cells stably expressing the gene of interest.

[0152] Example 6

[0153] Of the eight additional CD7 VHH clones, in addition to the H7 clone, as listed above in Table 3, five were selected for further functional testing. These VHHs were produced at lager scale and used to target tLNPs loaded with mcDNA encoding for eGFP as described above.

[0154] Briefly, the ionizable lipid used was SM102, and CD7 VHH was conjugated to DSPE-PEG- 2000-maleimide at a molar percentage of 0.05% of total lipid. The LNPs were loaded with a minicircle DNA comprising a transposable transgene encoding the reporter gene eGFP. A dose range of LNPs (expressed as total nucleic acid dose) was used to transfect isolated and activated T cells as described. Transfection efficiency was measured by eGFP expression. Figure 14A shows that all of the tested VHH clones boosted transfection of T cells with mcDNA when used to target the LNPs. There was variation observed in in the transfection efficiency. There was also variation observed in conjugation efficiency. Both are regarded to be highly important. The H7 VHH clone provided the best % of transfection, having a highly similar profile as the 2A-C6 and 5A-C6 clones, while having the highest percentage at the low dose of 75 ng. The conjugation efficiency of the H7 clone was the highest of all VHHs tested. Combined, this indicates that the H7 VHH clone can be preferred.

[0155] SEQUENCES

[0156] Table 4

[0157] Table 5

Claims

CLAIMS1 . A nanoparticle for delivery of an agent to human T cells and / or human NK cells, said nanoparticle comprising a targeting moiety and an agent, wherein said targeting moiety comprises an antigen binding region capable of binding to human CD7 and wherein the agent is a nucleic acid.

2. The nanoparticle according to claim 1 , wherein said nucleic acid is a DNA.

3. The nanoparticle according to claim 2, wherein said DNA is a minicircle DNA.

4. The nanoparticle according to claims 2 or claim 3, wherein the DNA is double stranded DNA.

5. The nanoparticle according to claim 1 , wherein said nucleic acid is an RNA, such as an mRNA.

6. The nanoparticle according to any one of claims of 1-5, wherein the nucleic acid encodes for a CAR and / or a TCR.

7. The nanoparticle according to any one of claims 1-6, wherein said nanoparticle is a lipid nanoparticle.

8. The nanoparticle according to claim 7, wherein said lipid nanoparticle comprises:(a) an ionizable lipid ;(b) a helper lipid ;(c) a PEG lipid ; and(d) a sterol.

9. The nanoparticle according to claim 8, wherein said lipid nanoparticle comprises 30- 60 mole % ionizable lipid, 0-30 mole % of helper lipid, 0-10 mole % of PEG lipid, and 15-50 mole percent of sterol.

10. The nanoparticle according to claim 8 or 9, wherein the ionizable lipid is selected from the group consisting of SM-102, MC3, KC2, Lipid A9 and C12-200.

11. The nanoparticle according to claim 8-10, wherein the weight ratio of ionizable lipid to agent is in the range of 10 - 20 : 1 .

12. The nanoparticle according to claim any of claims 7-11 , wherein the targeting moiety is conjugated to the PEG lipid, and, preferably, said lipid nanoparticle comprising 0.01 mole % - 5 mole % of PEG lipid conjugated with the targeting moiety.

13. The nanoparticle according to any of claims 1-12, wherein the antigen binding region is an immunoglobulin, such as an antibody, or a fragment thereof.

14. The nanoparticle according to claim 13, wherein the antigen binding region is in a VHH format.

15. The nanoparticle according to claim 13 or 14, wherein of a corresponding nanoparticle containing GFP encoding mRNA, the fold increase in the TNK / O ratio of GFP positive cells for nanoparticles with the antigen binding region relative to nanoparticles without the antigen binding region, is at least 5-fold.

16. The nanoparticle according to claim 13 or 14, wherein of a corresponding nanoparticle containing a fluorescently labelled RNA, the fold increase in the TNK / O ratio of fluorescent positive cells for nanoparticles with the antigen binding region relative to nanoparticles without the antigen binding region is at least is 5-fold.

17. The nanoparticle according to any of claims 13-16, wherein the antigen binding region comprises the CDR1 , CDR2, and CDR3 of SEQ ID NO. 1.

18. The nanoparticle according to any of claims 13-16, wherein the antigen binding region comprises the CDRI , CDR2, and CDR3 of SEQ ID NO. 1 , 5, 9, 13, 17, 21 , 25, 29, or 33.

19. The nanoparticle according to any of claims 13-18, wherein the amino acid sequence of said immunoglobulin or a fragment thereof, is humanized and / or de-immunized.

20. The nanoparticle according to any one of claims 1-19, wherein the EC50 of antigen binding region is in the range of 1 pM - 1 mM.

21. Use of the nanoparticle as defined in any of claims 1-20, for delivery of a nucleic acid to a T cell.

22. Use according to claim 21 , for delivery of a nucleic acid to a resting T cell.

23. Use according to claim 21 or 22, wherein said nucleic acid is a DNA or RNA.

24. Use according to claim 23, wherein when said nucleic acid is an mRNA, said mRNA is translated into a protein in a T cell, or a resting T cell.

25. Use according to claim 23, wherein when said nucleic acid is a DNA encoding a gene expression cassette, for expression of the gene, said resting T cells are activated.

26. Use of the nanoparticle as defined in any of claims 1-20, for selective delivery of a nucleic acid to T cells and NK cells.

27. Use of the nanoparticle as defined in claim 26, for selective delivery of a nucleic acid to T cells and NK cells when the nanoparticle is contacted with PBMCs.

28. Use of the nanoparticle as defined in claim 26, for selective delivery of a nucleic acid to T cells and NK cells wherein the nanoparticle is contacted with whole blood.

29. Use in accordance with claim 21 , wherein the selective delivery comprises a fold increase in TNK / O ratio as defined in claim 15 or 16.

30. Use in accordance with any of claims 21-29, wherein said nucleic acid encodes a CAR or a TCR.

31. A nanoparticle comprising an agent and a targeting moiety with an antigen binding region capable of binding to human CD7, as defined in accordance with any one of claims 1-20, wherein said nanoparticle is for use in the treatment of a disease, wherein said treatment comprises administering the nanoparticle to the patient.

32. A nanoparticle for use in the treatment of a disease in accordance with claim 31 , wherein said agent is a nucleic acid, which is an mRNA as defined in any of claims 5- 6.

33. A nanoparticle comprising an agent and a targeting moiety with an antigen binding region capable of binding to human CD7, as defined in accordance with any one of claims 1-20, wherein said nanoparticle is for use in the treatment of a disease, wherein said treatment comprises obtaining T cells and / or NK cells from the patient, contacting the nanoparticle with the obtained T cells and / or NK cells ex vivo and allowing delivery of the agent to the cells, and subsequently administering the T cells and / or NK cells to the patient.

34. A nanoparticle for use in the treatment of a disease in accordance with claim 33, wherein said agent is a nucleic acid, which is an mRNA as defined in any of claims 5- 6.

35. A nanoparticle for use in the treatment of a disease in accordance with claim 34, wherein said agent is a nucleic acid as defined in any of claims 2-6, and wherein said T cells are activated prior to administering the T cells to the patients.

36. An immunoglobulin or a fragment thereof, comprising the antigen binding region with the CDR1 , CDR2, and CDR3 of SEQ ID NO. 1 , 5, 9, 13, 17, 21 , 25, 29, or 33.

37. A nucleic acid encoding the immunoglobulin or a fragment thereof, as defined in claim36.

38. A cell comprising the nucleic acid of claim 37.

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