WO2024256842A1

WO2024256842A1 - Therapeutic transfection

Info

Publication number: WO2024256842A1
Application number: PCT/GB2024/051542
Authority: WO
Inventors: Helen Horton; Stefania ZONA; Sarah MOORE
Original assignee: Touchlight IP Ltd
Current assignee: Touchlight IP Ltd
Priority date: 2023-06-16
Filing date: 2024-06-17
Publication date: 2024-12-19
Anticipated expiration: 2025-12-16

Abstract

The present invention concerns compositions and methods for the in vivo modification of cells of the immune system, in particular immune effector cells and immune memory cells to achieve therapeutic or prophylactic objectives. In particular, the novel method provides for the recruitment and in vivo modification of immune cells with a nucleic acid. Preferably, this enables the immune cells to express an antigen receptor. The novel method allows for increased yield of viable in vivo genetically modified immune cells, notably immune effector cells and/or immune memory cells, compared to prior art methods. Such transfected immune cells may have utility in treatment or prophylaxis.

Description

THERAPEUTIC TRANSFECTION

Field of the invention

The present invention relates to methods for the in vivo modification of immune cells, in particular immune effector cells and immune memory cells. In particular, the present invention relates to methods of improving the transfection of immune cells in vivo, circumventing the issues involved with ex vivo transfection, the steps of which include cell harvesting, expansion, transfection and re- introduction. Such transfected immune cells may have utility in treatment or prophylaxis.

Background to the invention

Adoptive immunotherapy or adoptive cell therapy (ACT) is a potent approach for the treatment of cancer, and other diseases such as infections and graft versus host disease. ACT is the passive transfer of ex vivo grown cells, most commonly immune-derived cells, into a host with the goal of transferring the immunologic functionality and characteristics of the transplant. ACT can be autologous, as is common in adoptive T-cell therapies, or allogeneic as is typical for treatment of infections or graft- versus-host disease. Clinically, common embodiments of this approach include transfer of either immune-promoting or tolerogenic cells such as lymphocytes to patients to either enhance immunity against viruses/infections and cancer or to promote tolerance in the setting of autoimmune disease, such as multiple sclerosis, type I diabetes or rheumatoid arthritis.

In ACT, the most commonly used cell types are T cells, in particular CD8+ T cells, but other immune cells such as CD4+ T cells, NK-cells, delta-gamma T cells, regulatory T cells, macrophages and peripheral blood mononuclear cells have also been employed. The immune cells can be unmodified and simply expanded to reach clinically relevant cell numbers, or alternatively, the cells can be engineered or reprogrammed prior to reinfusion. Typically, immune cells are genetically engineered ex vivo to express an antigen receptor to recognise a disease-associated antigen. However, other modifications may include introduction of accessory genes that provide new functions to immune cells and genetic engineering of intracellular pathways that modulate natural properties such as metabolism, survival, and proliferation.

In particular, ACT has been applied to the field of cancer treatment where cytotoxic T cells are genetically engineered to induce expression of novel genes that facilitate tumour recognition, enhance T-cell activation, induce tumour-specific cytotoxicity and augment immune memory. The two most common modifications used are expression of transgenes encoding a tumour-specific T cell receptor (TCR) or a chimeric antigen receptor (CAR). The ex vivo engineered cells are subsequently expanded and transplanted to a subject for the treatment of a disease. However, the complicated procedures and costs of ex vivo manipulation of T cells remains a major hurdle for the widespread adoption of ACT in the treatment of cancer and other diseases. In particular, clinical-scale manufacturing of T cells requires large starting cell numbers, involves elaborate protocols to isolate, genetically modify, and selectively expand the engineered cells, and can also lead to cell exhaustion. Moreover, semi-random integration of the viral vectors used to deliver the TCR or CAR transgene and irregular expression, result in a heterogenous pool of modified T cells. The subsequent reinfusion of engineered cells to a subject also requires patient pre-conditioning, including lymphodepleting chemotherapy, which can be expensive and time-consuming, and reinfusion of large quantities of cells can also have adverse effects on the subject. Due to this cumbersome procedure, the overall time between T cell harvest and patient infusion (the so-called "vein-to-vein time") can take several weeks. The complex processes of ACT also requires dedicated equipment and considerable technical expertise and training, further impeding implementation of ACT as a standard- of-care in the treatment of cancer and disease and limiting the scalability of this approach.

Moreover, in typical ex vivo workflows only a small fraction of T cells subsets are isolated from the circulation, thus T cells with a limited variety of differentiation states are modified. T cell differentiation states that are therapeutically favourable (e.g. tissue-resident memory T cells) may be present in a limited number in the sample taken, or not at all. Further, the T cell population is heterogenous as an array of endogenous TCRs with specificity for different target antigens will be present in the isolated population.

To circumvent some of the issues associated with ex v/vo-based ACT, in vivo modification of T cells using viral vectors and non-viral nucleic acid delivery platforms (such as lipid nanoparticles (LNPs)) has also been trialled, but with limited success. For example, Smith et al. (2017) (In situ programming of leukaemia-specific T cells using synthetic DNA nanocarriers. Nat Nanotechnol. 2017 Aug;12(8):813- 820. doi: 10.1038/nnano.2017.57) describe the use of polymeric nanocarriers bearing lymphocytetargeting ligands to deliver an integrating polynucleotide encoding a targeting agent to the cell of interest. However, nanomaterials are complex in design and contain a variety of chemical and bioactive components, making large-scale production of polymeric nanocarriers a challenge. Moreover, the interaction of nanomaterials and the immune system is still largely unclear.

Viral vectors such as lentiviruses and AAV have also been employed for in vivo engineering of CAR T cells. For example, Pfeiffer et al. (2018) (In vivo generation of human CD19-CAR T cells results in B-cell depletion and signs of cytokine release syndrome. EMBO Mol Med. 2018 Nov;10(ll):e9158. doi: 10.15252/emmm.201809158) reported lentiviral-mediated induction of in-situ CAR T cells in immunodeficient mice. However, the mice exhibited cytokine release syndrome, an undesirable effect seen in clinical practice. Moreover, viral vector based methods in general are non-specific, and there is an undesirable risk of random chromosomal insertion of genes and transgene integration into bystander cells.

Therefore, it would be desirable to provide an efficient method for engineering of, in particular, immune effector cells and immune memory cells that overcomes the standardisation and scale-up limitations of current methods of adoptive cell therapy. Moreover, it would be desirable to provide a strategy for immunotherapy wherein the modified immune cells have improved safety profiles, persistence, activity and/or proliferation.

Summary of the invention

The present invention provides compositions and methods that can effectively transfect cells of the immune system in vivo to achieve therapeutic or prophylactic objectives. In particular, the present invention relates to in vivo genetically modified immune cells for use in a method of treatment or therapy. The invention also relates to a novel method of in vivo modification of immune cells, which is suitable for use in treatment or therapy.

In particular, the novel method provides for the recruitment and in vivo modification of immune cells with a nucleic acid. Preferably, this enables the immune cells to express an antigen receptor. The novel method allows for increased yield of viable genetically modified immune cells compared to prior art methods. In particular, the novel method allows for increased yield of in vivo genetically modified immune cells, notably immune effector cells and/or immune memory cells compared to prior art methods.

The present invention provides:

A method of in vivo modification of an immune cell in a subject, said method comprising:

(a) administering an immune cell recruiting factor; and

(b) administering a nucleic acid encoding an antigen receptor.

According to an embodiment of the invention, the administration in steps (a) and (b) is in the same locality.

Thus, the present invention provides a modified immune cell for use in a method of therapy or treatment, wherein the immune cell is genetically modified in vivo to express an antigen receptor, the method comprising:

(a) administering an immune cell recruiting factor; and

(b) administering a nucleic acid encoding the antigen receptor. According to an embodiment of the invention, the administration in steps (a) and (b) is in the same locality.

Alternatively, the present invention provides a product or composition comprising:

(i) an immune cell recruiting factor; and

(ii) a nucleic acid encoding an antigen receptor as a combined preparation for simultaneous, separate or sequential use in a method of in vivo modification of immune cells.

Further alternatively, the present invention provides a product or composition comprising:

(i) an immune cell recruiting factor; and

(ii) a nucleic acid encoding an antigen receptor as a combined preparation for simultaneous, separate or sequential use in a method of therapy or treatment.

Further alternatively, the present invention provides an immune cell recruiting factor and a nucleic acid encoding an antigen receptor for use in a method of therapy or treatment in a subject, the method comprising:

(a) administering the immune cell recruiting factor; and

(b) administering the nucleic acid encoding an antigen receptor.

Thus, the use may be described as a dosage regimen of the products for use in therapy. As defined, the products or composition include an immune cell recruiting factor and a nucleic acid encoding an antigen receptor. According to any description of the invention, the products of the invention may be administered to a subject in need thereof in order to modify immune cells, preferably immune effector cells and/or immune memory cells. This administration may be simultaneous, separate or sequential. The administration may be made to the same locality simultaneously, separately or sequentially.

The methods or products of the invention may be utilised to modify immune cells in a subject. The immune cells may be any suitable immune cells, including but not limited to immune effector cell and/or immune memory cells.

The recruited and/or modified immune cell of the present invention is preferably a cytotoxic immune cell or an immune cell with cytotoxic potential. The immune cell may be a cytotoxic lymphocyte. The immune cell may be a T cell. The immune cell may be an effector T cell or a memory T cell. The immune cell may be a peripheral T cell. The peripheral T cell may be naive when recruited. Naive T cells have yet to acquire their designated function. Naive T cells require activation via presentation of their cognate antigen and the presence of co-stimulatory molecules. Naive T cells differ phenotypically from activated effector cells by expressions of many surface receptors but are generally distinguished by the absence of the activation protein, CD25. Naive T cells are therefore T cells with cytotoxic potential. The immune cell may be any one or more of a cytotoxic T cell, natural killer (NK) cell or NKT cell. Preferably, the immune cell is a CD8⁺T cell. The immune cell may be a CD8+ effector T cell or a CD8+ memory T cell. Preferably, the immune cell is a CD8+ memory T cell. The CD8+ memory T cell may be a CD8+ central memory T cell, CD8+ effector memory cell, CD8+ tissue-resident memory T cell or a CD8+ circulating memory T cell. Preferably, the immune cell is a CD8+ central memory T cell and/or CD8+ effector memory T cell.

The recruited and/or modified immune cell may be a regulatory T cell.

The recruited immune cell may be a memory T cell, or alternatively, may differentiate into a memory T cell. Some of the recruited T cells may differentiate into memory T cells before or after transfection with the nucleic acid encoding an antigen receptor. Activated effector T cells are short lived, but a proportion of these may survive as memory T cells. Memory T cells are more potent and persist longer than terminally differentiated effector cells. It is considered that memory T cells have a lower activation threshold than naive T cells, so they are more easily stimulated by antigen. Thus, the modified immune cell may be a memory T cell. In a preferred embodiment, the modified immune cell is a CD8+ memory T cell.

The immune cell recruiting factor is administered to the subject. Preferably this administration is to a defined location or locality.

The immune cell recruiting factor of the present invention recruits immune cells to the area or site of administration, for example to the location or locality of the administration.

The immune cell recruiting factor may recruit activated immune cells, and/or recruit and activate immune cells. By recruiting and/or activating immune cells, the immune cell recruiting factor primes or pre-conditions the locality around the administration site. In other words, the immune cell recruiting factor provides for the accumulation, aggregation or concentration of activated immune cells to a local site or area. The recruitment and/or activation of immune cells by the immune cell recruiting factor may be described as "priming" or "pre-conditioning". The immune cells recruited and/or activated by the immune cell recruiting factor may be described as "primed immune cells" or "activated immune cells". The immune cells, preferably the T cells recruited and/or activated by the immune cell recruiting factor may be memory T cells or may differentiate into memory T cells. Thus, the immune cell recruiting factor provides for the accumulation, aggregation or concentration of memory T cells to a local site or area. Alternatively, the immune cell recruiting factor provides for the accumulation, aggregation or concentration of immune cells capable of differentiating into memory T cells to a local site or area. As noted earlier, the cells that are capable of differentiation into memory T cells may be naive T cells or indeed effector T cells. By recruiting activated immune cells and/or recruiting and activating immune cells, the immune cell recruiting factor minimises off-target delivery of the nucleic acid.

The skilled person will appreciate that the administration of an immune cell recruiting factor will attract various subtypes of immune cells. For T cells, the immune cell recruiting factor is likely to attract naive T cells, effector T cells, memory T cells, natural killer T cells and regulatory T cells. Other immune cells will also be attracted, including but not limited to, mononuclear phagocytes and granulocytic cells, monocytes, natural killer cells (both naive and memory), dendritic cells, macrophages, B lymphocytes (including memory B lymphocytes). Various cell types and subsets thereof are likely to migrate to the locality of the administration as part of the normal functioning of the cell-based immune system.

The immune cell recruiting factor may be any suitable entity capable of raising an immune cell response. Suitable factors include, but are not limited to, any one or more of: a vaccine, an antigen, an epitope, a cytokine, a chemokine or a microbial product or toxin. The immune cell recruiting factor may be provided as a nucleic acid encoding said factor, such as a DNA or RNA.

Preferably, the immune cell recruiting factor is an antigen or epitope thereof recognised by an antigen receptor of the immune cell.

Preferably, the immune cell recruiting factor recruits and/or activates CD8+ T cells. Preferably, the immune cell recruiting factor recruits and/or activates CD8+ T cells to the locality or area of the administered factor. The CD8+ T cell may be a CD8+ naive T cell, a CD8+ effector T cell or a CD8+ memory T cell.

Since the immune cell recruiting factor may be a cognate antigen for an immune cell, the provision of the immune cell recruiting factor can also serve to activate a subset of the attracted cells. The activation will depend upon the antigen recognition of the recruited immune cells, where this is relevant (for example, for T cell activation). Immune cells may possess endogenous antigen receptors. T cells possess T cell receptors which have a cognate antigen, and presentation of the relevant antigen to the T cell will result in activation of that T cell. Thereby, the immune cell recruiting factor may be presented to T cells by an antigen-presenting cell.

Preferably, the immune cell recruiting factor is an antigen or epitope thereof that is recognised by and activates a cognate T cell receptor (TCR). In other words, the recruited and/or activated T cells express an endogenous T cell receptor (TCR) that is specific to the immune cell recruiting factor. The immune cell recruiting factor thereby may recruit and/or activate antigen-specific T cells. The recruited and/or activated antigen-specific T cells represent a safe and highly efficacious population of cells for transfection. Thereby, by recruiting T cells, unwanted modification of off-target cells or cells with undesirable properties is minimised.

Those modifying T-cell in vitro have discovered that the differentiation stage of T cells affects their proliferative and survival abilities. These characteristics strongly correlate with their anti-tumour activity, and it is thus beneficial if the T cells survive and proliferate. It has been indicated that longterm remission in ACT is related to the enrichment of T cells with memory-like features. Central memory (TCM), and stem-like memory (TSCM) lymphocytes are related to a good response due to their ability to proliferate and live longer.

Further, the present invention has the ability to select which T cells to attract, using the relevant immune cell recruiting factor, such as an antigen. Therefore, a population of T cells can be chosen, on the basis that they have previously been activated against an antigen that is foreign (not self). This helps to prevent unwanted effects of activating and modifying these cells, rather than attempting to modify any/all T cells whilst being unaware of their natural antigen specificity. Thus, T cells expressing a particular endogenous TCR with specificity for a particular antigen can be selected for.

The immune cell recruiting factor will also attract activated immune cells to the locality of administration. These activated immune cells have previously encountered their cognate antigen. According to the methods and products of the invention, once attracted/recruited, the immune cells are genetically modified, transfected or engineered, in vivo. In vivo modification circumvents the otherwise costly procedure of harvesting, culturing and modifying immune cells ex vivo, and subsequently reintroducing the modified cells to the subject.

Thus, according to the methods and products of the present invention, there is provided a nucleic acid encoding an antigen receptor. The nucleic acid encoding the antigen receptor may be DNA or RNA. The nucleic acid encoding the antigen receptor may be a vector, preferably an expression vector. The expression vector may be a closed linear DNA, a single-stranded circular DNA comprising at least one hairpin section, a plasmid, a minicircle, a messenger RNA (mRNA), a self-amplifying RNA (saRNA) vector, a circular RNA (circRNA), a guide RNA (gRNA) or any other suitable nucleic acid format. The expression vector may be a transient expression vector or an integrating expression vector. Transient expression may be preferred.

The nucleic acid may be a naked nucleic acid, for example the nucleic acid is not encapsulated in a virus or virus-like particle. However, the nucleic acid may be delivered in an encapsulated form, for example in a viral vector such as AAV or lentivirus. Alternatively, the nucleic acid may be encapsulated or formulated with transfection reagents such as lipids (liposomal reagents), nanoparticles, dendrimers, polymers and the like.

By recruiting and/or activating immune cells the immune cell recruiting factor primes or preconditions a location, site or area with activated immune effector cells and/or immune memory cells, thereby increasing the efficiency of in vivo transfection and modification of activated immune effector cells and/or immune memory cells. The recruited T cells may differentiate into memory T cells prior to, or after transfection with the nucleic acid encoding an antigen receptor. Compared to naive T cells, memory T cells respond more quickly to displayed cognate peptide/antigen with a shorter lag time for entering the cell cycle and exerting effector functions. Thus, in a preferred embodiment, the immune cell transfected with a nucleic acid encoding an antigen receptor is a memory T cell or a T cell capable of differentiating into a memory T cell. Thus, in a preferred embodiment the in vivo modified immune cell is a modified memory T cell. In a preferred embodiment, the in vivo modified immune cell is a CD8+ memory T cell.

The immune cell may be an effector memory T cell, a central memory T cell, a tissue-resident memory T cell or a circulating memory T cell. In a preferred embodiment, the immune cell is a central memory T cell and/or effector memory T cell.

The modified immune cell may be an effector memory T cell, a central memoryT cell, a tissue-resident memory T cell or a circulating memory T cell. In a preferred embodiment, the modified immune cell is a central memory T cell and/or effector memory T cell.

The resulting modified activated immune cells thereby express an antigen receptor specific to a target antigen. Without wishing to be bound by theory, the inventors postulate that, transfection in absence of the priming or pre-conditioning step would result in a significantly smaller pool of modified activated immune cells. Preferably, the modified immune cells are modified immune effector cells and/or modified immune memory cells. Thus, preferably, the immune cell recruiting factor and the nucleic acid encoding the antigen receptor are administered to the same locality. Wherein the immune cell recruiting factor is an antigen or an epitope thereof that is recognised by an antigen receptor, the resulting in vivo modified immune cells may be described as bispecific, as they express an antigen receptor specific to the immune cell recruiting factor (an endogenous antigen receptor) and an antigen receptor specific to the target antigen (the antigen receptor encoded on the nucleic acid).

In one aspect, the present invention provides an immune cell recruiting factor and a nucleic acid encoding an antigen receptor for use in a method of therapy or treatment in a subject, the method comprising:

(a) administering the immune cell recruiting factor; and

(b) administering the nucleic acid encoding an antigen receptor.

According to any aspect of the present invention, the immune cell recruiting factor and the nucleic acid encoding the antigen receptor comprise a therapeutic composition. The composition may be administered at the same locality or area.

Administration may be via any suitable route, such as but not limited to: intradermal, intramuscular, transmucosal, sub-cutaneous, inhalation, sub-lingual, intratumoural or intra-lymph node administration.

Administration may be simultaneous, separate or sequential. Administration may be separate or sequential. Administration of the immune cell recruiting factor may occur before the administration of the nucleic acid encoding an antigen receptor.

The immune cells of the present invention are in vivo genetically modified to express an antigen receptor. The antigen receptor may be described as being specific for or to a target antigen. The antigen to which the antigen receptor is targeted may comprise a naturally occurring antigen or a variant thereof, or a fragment of the naturally occurring antigen or variant thereof. The antigen may be a self-antigen or a non-self antigen.

Expression of the antigen receptor thereby enables targeting of the in vivo genetically modified immune cell to cells expressing the target antigen. The antigen receptor may be a T-cell receptor (TCR) or chimeric antigen receptor (CAR).

The target antigen may be a self-antigen or a fungal/bacterial/viral antigen. The target antigen may be a tumour antigen, neoantigen or tumour-associated antigen. Cells expressing the target antigen may be referred to as target cells. The target cells may express the antigen on the cell surface or may present an antigen fragment thereof, for example a peptide fragment.

Thus, the present invention relates to an immune cell in vivo modified to express an antigen receptor for use in treatment or therapy. In particular, the present invention generally relates to in vivo genetically modified immune cells for use in the treatment of diseases by targeting diseased cells expressing a target antigen. Such treatment provides for the selective elimination of target cells that express the target antigen, thereby minimising adverse effects to normal cells not expressing the target antigen.

The target antigen may be a tumour-associated antigen and the disease a cancer. Thus, the target cell population or target tissue may be tumour cells or tumour tissue. Thus, the present invention relates to the use of in vivo modified immune cells for the treatment of cancer, wherein the immune cells are genetically modified to express an antigen receptor that binds to a tumour-associated antigen or fragment thereof, thereby targeting the in vivo modified immune cell to cancer cells or cancer tissue expressing the tumour-associated antigen or fragment thereof.

In any aspect of the present invention, the nucleic acid encoding an antigen receptor may be provided or administered with a transfection reagent.

In any aspect of the present invention, the administration of the nucleic acid encoding an antigen receptor may be accompanied or followed by an electric pulse applied to the locality of administration. Alternatively, the nucleic acid may be administered using a biolistic particle delivery system.

The immune cell recruiting factor and nucleic acid encoding an antigen receptor may be administered simultaneously, sequentially, separately or independently. Preferably, sequential administration is utilised, and the immune cell recruiting factor is administered before the nucleic acid encoding an antigen receptor.

Following recruitment and in vivo genetic modification of immune cells, it may be desirable to expand the antigen receptor-engineered population of immune cells. The invention further relates to provision of the target antigen for the stimulation, priming and/or expansion of the in vivo genetically modified immune cells.

The target antigen is recognised by the antigen receptor expressed on the modified immune cells. Alternatively put, the modified immune cell specifically recognises the target antigen.

The target antigen may be provided by a vaccine. The vaccine may be provided in any suitable format. The vaccine may provide the target antigen or a variant thereof (e.g. a peptide or protein comprising an epitope of the target antigen), or may provide a nucleic acid encoding the target antigen or variant thereof. The vaccine may be any suitable vaccine, such as but not limited to any one or more of: inactivated vaccines, live-attenuated vaccines, subunit vaccines, recombinant vaccines, polysaccharide vaccines, conjugate vaccines, toxoid vaccines, a polypeptide vaccine, DNA vaccine, RNA vaccine or viral vector vaccine. The vaccine may include the target antigen presented on a carrier, such as a nano-cage or virus-like particle (VLP). Any appropriate presentation of the target antigen to the immune cell is covered with the term "vaccine".

In an alternative embodiment, the target antigen may be provided by a cell expressing the target antigen.

In an alternative embodiment, the target antigen may be provided by a virus expressing the target antigen. Any suitable virus may be used.

Thus, a vaccine providing the target antigen may be administered to the subject, thereby stimulating, priming and/or expanding the in vivo genetically modified immune cells. The vaccine providing the target antigen may be targeted to secondary lymphoid organs.

In any embodiment wherein the target antigen is a tumour-associated antigen and the disease a cancer, a vaccine may be administered, the vaccine providing the tumour-associated antigen or a fragment thereof recognised by the antigen receptor, thereby stimulating, priming and/or expanding the in vivo modified immune cells.

Thus, in a further aspect the present invention provides:

A method of in vivo modification and expansion of an immune cell in a subject, said method comprising:

(a) administering an immune cell recruiting factor;

(b) administering a nucleic acid encoding an antigen receptor; and

(c) administering a vaccine comprising a target antigen.

Thus, the present invention provides an expanded modified immune cell for use in a method of therapy or treatment, wherein the immune cell is genetically modified and expanded in vivo to express an antigen receptor, the method comprising:

(a) administering an immune cell recruiting factor;

(b) administering a nucleic acid encoding the antigen receptor; and (c) administering a vaccine comprising a target antigen.

In one aspect, the present invention provides an immune cell recruiting factor, a nucleic acid encoding an antigen receptor and a vaccine for use in a method of therapy or treatment in a subject, the method comprising:

(a) administering the immune cell recruiting factor;

(b) administering the nucleic acid encoding an antigen receptor; and

(c) administering a vaccine comprising a target antigen.

According to any one of the aspects of the invention pertaining to the use of a vaccine, the administration in steps (a) and (b) is in the same locality or area. The administration in step (c) may be to any locality or area. The method or mode of administration may be different or may be the same for each of the components. The method or mode of administration may be any suitable administration. The components are preferably administered separately or sequentially. The components are preferably administered in order (a), (b) and (c). The timing of administration may vary, with appropriate intervals between administrations as discussed further below.

Alternatively, the present invention provides a product or composition comprising

(i) an immune cell recruiting factor;

(ii) a nucleic acid encoding an antigen receptor; and

(iii) a vaccine comprising a target antigen as a combined preparation for simultaneous, separate or sequential use in a method of modifying and expanding immune cells.

Further alternatively, the present invention provides a product or composition comprising

(i) an immune cell recruiting factor;

(ii) a nucleic acid encoding an antigen receptor;

(iii) a vaccine comprising a target antigen as a combined preparation for simultaneous, separate or sequential use in prophylaxis or therapy.

The product or composition according to either aspect may be suitable for simultaneous, separate or sequential administration. The product or composition may be suitable for separate or sequential administration. The product or composition may be administered sequentially in the order of components (i), (ii) and (iii). The product or composition may be suitable for any type of administration. It is preferred that components (i) and (ii) are suitable for administration in the same locality or area. Component (iii) may be suitable for administration to any locality or area. The timing of administration may vary, with appropriate intervals between administrations as discussed further below.

Additionally or alternatively to a vaccine comprising a target antigen, a vaccine comprising the immune cell recruiting factor may be administered to the subject, thereby stimulating, priming and/or expanding the in vivo genetically modified immune cells. The vaccine providing the immune cell recruiting factor may be targeted to secondary lymphoid organs.

Thus, in a further aspect the present invention provides:

(a) administering an immune cell recruiting factor;

(b) administering a nucleic acid encoding an antigen receptor; and

(c) administering a vaccine comprising the immune cell recruiting factor.

In any aspect of the invention described above, for any one or more of steps (a) (b) and/or (c), or any of components (i), (ii) or (iii) the administration may be via any suitable means. Preferably, the administration is any one or more of intradermal, transdermal, transmucosal, sublingual, inhaled, intramuscular, sub-cutaneous, intratumoural or intra-lymph node.

In any aspect of the present invention, the components may be administered separately or sequentially. It may be preferred that there is an interval between step (a) and step (b) or between the administration of component (i) and component (ii). This interval may be any suitable interval.

The interval between step (a) and (b) is dependent on the timing of the recruitment of the immune cell. In particular, the interval between step (a) and (b) is dependent on the timing of the recruitment of immune cells. The immune cell recruiting factor and the nucleic acid encoding an antigen receptor may be are administered within about 1 hour to about 72 hours (e.g. within about 1, 2, 3, 6, 12, 24, 48, or 72 hours), within about 1 day to about 4 weeks (e.g. within about 1, 2 or 3 or 4 weeks), within about 1 week to about 3 weeks of each other or any range there between. The immune cell recruiting factor and the nucleic acid encoding an antigen receptor may be administered within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 days of each other. In any suitable aspect of the invention, the components/products may be administered separately or sequentially. It may be preferred that there is an interval between step (b) and step (c) or between the administration of component (ii) and component (iii). This interval may be any suitable interval.

The interval between step (b) and (c) should be sufficient to allow the immune cell to be modified to express the antigen receptor, and provide for expansion of the modified immune cell population so long as these cells persist. The interval between step (b) and (c) or between the administration of component (ii) and component (iii) may be within about 1 day to about 30 weeks (e.g. within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 weeks), or any range there between. The interval between these administrations may be selected as appropriate, but may be in the order of 1 day to 190 days, 10 to 180 days, 20 to 170 days, 30 to 160 days or any one or more of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 days apart.

The skilled person will appreciate that if the modified immune cells are memory cells, for example memory T cells, that these may persist for months or years in a subject. Thus, the interval between steps (b) and (c) may extend to several months or years.

In a preferred embodiment of the present invention, the immune cell is a cytotoxic T cell, the immune cell recruiting factor is an influenza vaccine, the antigen receptor is a T cell receptor (TCR) and the target antigen is a tumour-associated antigen.

The transfection and modification of the immune cells may be transient or permanent. Wherein the immune cell is transiently modified, the nucleic acid encoding an antigen receptor is not integrated into the immune cell's genome and is diluted out during cell division. Transient transfection may be preferable as the transfected nucleic acid is unable to replicate independently from the host cell's DNA, only persists for a shorter period of time and has an improved safety profile. Thus, the present invention relates to the recruitment, transient transfection and in vivo modification of immune cells.

In a preferred embodiment, the modified immune cells transiently express the antigen receptor. In other words, preferably transiently modified immune cells are generated. Following recruitment and transient in vivo genetic modification of immune cells, it may be desirable to expand the population of transiently modified immune cells expressing the antigen receptor. Thus, as discussed above, a vaccine comprising a target antigen may be provided for the stimulation, priming and/or expansion of the transiently modified immune cells. Thus, a vaccine providing the target antigen may be administered to the subject, thereby stimulating, priming and/or expanding the in vivo transiently modified immune cells.

In a further aspect of the present invention, steps (a) and (b) may be repeated, allowing further rounds of recruitment, in vivo transfection and genetic modification.

In a further aspect of the present invention the steps (a), (b) and/or (c) may be repeated, allowing further rounds of recruitment, in vivo transfection and genetic modification and expansion.

Thus, according to the present invention, one or more of steps (a), (b) and/or (c) may be repeated.

According to any aspect of the present invention, the method of therapy or treatment may be a method for treating cancer in a subject in need thereof.

According to any aspect of the present invention, the method of therapy or treatment may be a method of eliciting an immune response to a target antigen in a subject.

According to any aspect of the present invention, the method of therapy or treatment may be a method for treating an infection in a subject in need thereof. The infection may be fungal, bacterial or viral. The infection may be acute or chronic.

In a further aspect the present invention provides:

An immune cell recruiting factor and a nucleic acid encoding an antigen receptor for use in method of eliciting an immune response to a target antigen in a subject, said method comprising:

(a) administering an immune cell recruiting factor;

(b) administering a nucleic acid encoding an antigen receptor; and optionally,

(c) administering a vaccine comprising a target antigen.

According to a further aspect of the present invention, there is provided a kit comprising:

(a) an immune cell recruiting factor; and

(b) a nucleic acid encoding an antigen receptor; and optionally,

(c) a vaccine comprising a target antigen.

The invention further provides for the recruitment and in vivo modification of immune cells with a nucleic acid. Preferably, this enables the immune cells to express a transgene (i.e. a payload, such as a therapeutic payload). According to this further aspect of the present invention, there is provided:

(a) administering an immune cell recruiting factor; and

(b) administering a nucleic acid comprising a transgene.

Thus, the present invention provides a modified immune cell for use in a method of therapy or treatment, wherein the immune cell is genetically modified in vivo to express a transgene, the method comprising:

(a) administering an immune cell recruiting factor; and

(b) administering a nucleic acid comprising the transgene.

According to an embodiment of this definition of the invention, the administration in steps (a) and (b) is in the same locality.

(i) an immune cell recruiting factor; and

(ii) a nucleic acid comprising a transgene as a combined preparation for simultaneous, separate or sequential use in a method of in vivo modification of immune cells.

(i) an immune cell recruiting factor; and

(ii) a nucleic acid comprising a transgene as a combined preparation for simultaneous, separate or sequential use in a method of therapy or treatment.

The products or compositions of the invention may be administered to a subject in need thereof in order to modify immune cells, preferably immune effector cells and/or immune memory cells. This administration may be simultaneous, separate or sequential. The administration may be made to the same locality simultaneously, separately or sequentially.

The methods or products of the invention may be utilised to modify immune cells in a subject. The immune cells may be any suitable immune cells, including but not limited to T lymphocyte (T cell), B lymphocyte (B cell), macrophage, neutrophil, mast cell, basophil, eosinophil, monocyte or dendritic cell. The immune cell may be an immune effector T cell and/or immune memory T cell.

The recruited and/or modified immune cell of the present invention may be as previously described.

The immune cell recruiting factor is administered to the subject as previously described.

Thus, according to the methods and products of the present invention, there is provided a nucleic acid comprising a transgene. The nucleic acid comprising the transgene may be DNA or RNA. The nucleic acid comprising the transgene may be a vector, preferably an expression vector. The expression vector may be a closed linear DNA, a plasmid, a minicircle, a single-stranded circular DNA comprising at least one hairpin section, a messenger RNA (mRNA), a self-amplifying RNA (saRNA) vector, a circular RNA (circRNA), a guide RNA (gRNA) or any other suitable nucleic acid format.

The nucleic acid encoding a transgene may be a naked nucleic acid, for example the nucleic acid is not encapsulated in a virus or virus-like particle. However, the nucleic acid may be delivered in an encapsulated form, for example in a viral vector such as AAV or lentivirus.

Alternatively, the nucleic acid encoding a transgene may be encapsulated or formulated with transfection reagents such as lipids (liposomal reagents), nanoparticles, dendrimers, polymers and the like.

By recruiting and/or activating immune cells the immune cell recruiting factor primes or preconditions a location, site or area with activated immune effector cells and/or immune memory cells, thereby increasing the efficiency of in vivo transfection and modification of activated immune effector cells and/or immune memory cells. The recruited T cells may differentiate into memory T cells prior to, or after transfection with the nucleic acid comprising a transgene. Compared to naive T cells, memory T cells respond more quickly to displayed cognate peptide/antigen with a shorter lag time for entering the cell cycle and exerting effector functions. Thus, in a preferred embodiment, the immune cell transfected with a nucleic acid comprising a transgene is a memory T cell or a T cell capable of differentiating into a memory T cell. Thus, in a preferred embodiment the in vivo modified immune cell is a modified memory T cell. In a preferred embodiment, the in vivo modified immune cell is a CD8+ memory T cell.

The immune cell may be as described previously.

The resulting modified activated immune cells thereby express a transgene, for example an antigen receptor specific to a target antigen. Without wishing to be bound by theory, the inventors postulate that, transfection in absence of the priming or pre-conditioning step would result in a significantly smaller pool of modified activated immune cells. Preferably, the modified immune cells are modified immune effector cells and/or modified immune memory cells. Thus, preferably, the immune cell recruiting factor and the nucleic acid are administered to the same locality.

In one aspect, the present invention provides an immune cell recruiting factor and a nucleic acid comprising a transgene for use in a method of therapy or treatment in a subject, the method comprising:

(a) administering the immune cell recruiting factor; and

(b) administering the nucleic acid comprising a transgene.

According to any aspect of the present invention, the immune cell recruiting factor and the nucleic acid comprising a transgene may be administered at the same locality or area.

The method or mode of administration may be different or may be the same for each of the components. The method or mode of administration may be any suitable administration. The components are preferably administered separately or sequentially. The components are preferably administered in order (a), (b). The timing of administration may vary, with appropriate intervals between administrations as discussed further below.

Administration may be simultaneous, separate or sequential. Administration may be separate or sequential. Administration of the immune cell recruiting factor may occur before the administration of the nucleic acid comprising a transgene.

The transgene may encode an antigen receptor.

The transgene may encode a therapeutic payload. Thus, the immune cells of the present invention may be in vivo genetically modified to express a therapeutic payload.

The therapeutic payload may be: an antigen receptor, a therapeutic protein (such as an antigen, antibody, cytokine) an onco-suppressor protein (e.g. natural/unmutated p53), a differentiation factor (i.e. to modulate cell fate), or a protein with nucleic acid editing or gene activity modulation function (e.g. DNA or RNA editing, or transcriptional regulation). Thus, the transgene may encode a therapeutic protein, onco-suppressor protein, differentiation factor, or protein with a gene activity modulation function. In any aspect of the present invention, the nucleic acid may be provided or administered with a transfection reagent. The nucleic acid may be administered as discussed previously in relation to the nucleic acid encoding the antigen receptor.

The transfection and modification of the immune cells may be transient or permanent. Wherein the immune cell is transiently modified, the transgene (for example, the transgene encoding an antigen receptor) is not integrated into the immune cell's genome and is diluted out during cell division. Thus, the present invention relates to the recruitment, transient transfection and in vivo modification of immune cells.

In a further aspect the present invention provides:

An immune cell recruiting factor and a nucleic acid comprising a transgene for use in method of eliciting an immune response to a target antigen in a subject, said method comprising:

(a) administering the immune cell recruiting factor;

(b) administering the nucleic acid comprising a transgene.

In a further aspect the present invention provides:

(a) administering an immune cell recruiting factor;

(b) administering a nucleic acid encoding an antigen receptor.

(a) an immune cell recruiting factor; and (b) a nucleic acid comprising a transgene; and optionally,

(c) a vaccine comprising a target antigen.

Further embodiments are described below and in the claims. Further advantages are described below.

BRIEF DESCRIPTION OF FIGURES

Figure 1. Immune cells recruitment to the muscle following Cal09 vaccination. Representative images of H&E staining of muscle sections showing immune-cells infiltration 2 days and 4 days post Cal09 vaccination, circle C. The presence of infiltrates progressively decreases at later time points, day 7, 10 and 14.

Figure 2. Cal09 vaccine specific immune response. Splenocytes were isolated from mice at multiple timepoints, and immune response was measured by ICS for IFNy and TNFa. Antigen-specific Cal09 response by ICS was detected in the spleen at day 10 and day 14. Each symbol represents an individual sample with the error bars representing the s.e.m.

Figure 3. In vivo transfection of GFP in infiltrating T-cells following Cal09 vaccination. GFP expression was detected in T-cells recruited in the muscle by the Cal09 vaccine. Co-localisation of GFP and CD3 marker in T-cells, is shown by the circle; single stained cells, either for GFP or CD3, are indicated by the arrow.

Figure 4. In vivo transfection of infiltrating T and NK cells.

(A) GFP expression was detected in different types of immune cells, including CD3, CD8 and NK cells present in the muscles following Cal09 vaccination.

(B) Images from the control group, stimulated with Cal09 and transfected with PBS instead of GFP. T cells stained with CD3 did not show any positive GFP staining.

Figure 5. Detection of GFP transfected T cells in the spleen. FACS analysis of isolated splenocytes from day 2 post-transfection. Different DNA delivery systems were used for the in vivo transfection of immune cells recruited in the muscles. GFP expression was detected in the splenocytes, specifically in CD3, CD4 and CD8 T-cells proving that the transfected T-cells can re-circulate in the blood and spleen.

Figure 6. Transfection of effector/memory T cells. FACS analysis of isolated splenocytes from day 2 post-transfection (Fig. 6A). The majority GFP positive cells were of the effector cells, CD8 positive cells with a memory phenotype. They are CD44+CD62L+ antigen-experienced memory cells. Most naive cells were GFP negative (Fig. 6B).

Detailed Description of the Invention The present invention relates to methods and products to provide in vivo modified immune cells, which are suitable for use in therapy or treatment. In particular, the present invention relates to products with components capable of the recruitment and/or activation of immune cells, and products with components capable of the transfection and engineering of activated immune cells, and, optionally, the products with components for the subsequent expansion of in vivo modified immune cells.

The in vivo modified immune cells and methods disclosed herein provide many advantages over the current state of the art for both ex vivo and in v/vo-based engineering methods for adoptive cell therapy (ACT). For example, the methods of the present invention and the products of the present invention are suitable for in vivo modification of host cells, circumventing the need for patient-specific isolation, culturing and testing as is required by many adoptive cell therapies. Further, in vivo based modification does not require pre-treatment such as lymphodepleting chemotherapy, which is associated with significant toxicities. Moreover, the products and methods disclosed herein provide for the in vivo activation (if required) and engineering of immune cells, yielding modified immune cells primed for effector function. In comparison, incumbent procedures for ACT involve the isolation of peripheral blood lymphocytes from patients, wherein the majority of T lymphocytes are naive T cells and thus are not primed for cytotoxic activity. By recruiting and/or activating immune cells prior to transfection, the present invention allows for modification of immune cells with high functional potency and improved safety profiles. For example, the immune cell recruiting factor may be an antigen or epitope thereof that can recruit and activate T cell subsets that are therapeutically favourable (e.g. memory T cells).

Furthermore, the products and preparations can be manufactured on a large scale in a stable form with a long shelf life rendering them compatible with widespread distribution and inexpensive administration to large patient populations, compared to typical ex vivo ACT which requires patientspecific isolation and culturing. The resulting in vivo modified immune cells can selectively destroy target cells, whilst leaving healthy cells undamaged. Further, the products and preparations can be administered in booster doses to reinforce immune cell targeting and function.

Thus, the present invention provides logistical manufacturing improvements over current ex vivo and in vivo immune cell engineering strategies, and also provides therapeutic advantages through generation of safer and more effective modified immune cells.

Immune Cell The present invention provides products comprising components and methods that can rapidly and selectively direct cells of the immune system to achieve therapeutic objectives. Such cells may be referred to as "immune effector cells", "immune memory cells" or "immunoreactive cells." In particular embodiments, the compositions and methods involve in vivo modification of cells of the immune system, such as T cells, natural killer T cells (NKT) or natural killer (NK) cells, to target and destroy unwanted cell types. According to the invention, the term "immune cell" also includes a progenitor cell which can mature into an immune cell (such as T cell, in particular T helper cell, or cytolytic T cell) with suitable stimulation.

The immune cell of the present invention may be an immune effector cell or immune memory cell.

Preferably, the immune cells are cytotoxic immune cells, in particular cytotoxic cells such as cytotoxic T cells, natural killer T cells (NKT), natural killer (NK) cells, and lymphokine-activated killer (LAK) cells. Upon activation, each of these cytotoxic cells triggers the destruction of target cells. For example, cytotoxic T cells trigger the destruction of target cells by either or both of the following means. First, upon activation T cells release cytotoxins such as perforin, granzymes, and granulysin. Perforin and granulysin create pores in the target cell, and granzymes enter the cell and trigger a caspase cascade in the cytoplasm that induces apoptosis (programmed cell death) of the cell. Second, apoptosis can be induced via Fas-Fas ligand interaction between the T cells and target cells. The cells used in connection with the present invention will preferably be autologous cells. In a preferred embodiment, the immune cell is a CD8+ T-cell.

The immune cell may be a CD8+ effector T cell or a CD8+ memory T cell. Preferably, the immune cell is a CD8+ memory T cell. The CD8+ memory T cell may be a CD8+ central memory T cell, CD8+ effector memory cell, CD8+ tissue-resident memory T cell or a CD8+ circulating memory T cell.

The immune cell may be a macrophage, neutrophil, mast cell, basophil, eosinophil or dendritic cell.

The immune cell may be a T cell. The T cell can be any suitable T cell subset, but preferably it may be a memory T cell, or alternatively, may be a cell capable of differentiating into a memory T cell. Suitable cells which can differentiate into a memory T cell include naive T cells or effector T cells. Some of the recruited T cells may differentiate into memory T cells prior to, or after transfection with the nucleic acid encoding an antigen receptor. Thus, the modified immune cell may be a modified memory T cell. T cell memory follows initial antigenic exposure and priming, where naive T cells respond to antigenic peptides. Upon exposure to their target antigen, the memory T cells are quickly converted into large numbers of effector T cells (for example, cytotoxic T cells), thus providing a rapid response to the target. Compared to naive T cells, memory T cells respond more quickly to presented cognate antigen peptide with a shorter lag time for entering the cell cycle and exerting effector functions. Thus, in a preferred embodiment, the modified immune cell is a memory T cell. In a preferred embodiment, the modified immune cell is a CD8+ memory T cell.

Immune cells may be selected based upon particular effector functions. The term "effector functions" in the context of the present invention includes any functions mediated by components of the immune system that result, for example, in the killing of diseased cells such as tumour cells, or in the inhibition of tumour growth and/or inhibition of tumour development, including inhibition of tumour dissemination and metastasis. Preferably, the effector functions in the context of the present invention are T cell mediated effector functions. Such functions comprise in the case of a helper T cell (CD4+ T cell) the release of cytokines and/or the activation of CD8+ T lymphocytes (CTLs) and/or B cells, and in the case of CTLthe elimination of cells, i.e., cells characterised by expression of an antigen, for example, via apoptosis or perforin-mediated cell lysis, production of cytokines such as IFN-g and TNF-a, and specific cytolytic killing of antigen expressing target cells.

The term "antigen-specific T cell" or similar terms relate to a T cell which recognises the antigen to which the T cell is targeted and preferably exerts effector functions of T cells. T cells are considered to be specific for antigen if the cells kill target cells expressing an antigen. T cell specificity may be evaluated using any of a variety of standard techniques, for example, within a chromium release assay or proliferation assay. Alternatively, synthesis of lymphokines (such as interferon-g) can be measured. T cells recognise a peptide from their cognate antigen which is presented via other cells (such as dendritic cells) on their MHC molecules.

The terms "T cell" and "T lymphocyte" are used interchangeably herein. According to the invention, the term "T cell" also includes a cell which can mature into a T cell with suitable stimulation. Several different subsets of T cells have been discovered, each with a distinct function.

CD8+ T cells, also referred to as "cytotoxic T lymphocytes" (CTLs) or "cytotoxic T cells" destroy virally infected cells and tumour cells, and are also implicated in transplant rejection. CD8+ T cells express the CD8 glycoprotein on their cell surface and recognise their targets by binding to antigen associated with MHC class I, which is present on the surface of nearly every cell of the body. Generally, CD8+ T cells express the biomarkers CD8, CD3, CXCR3, CCR4, and CCR6.

"Regulatory T cells" or "Tregs" are a subpopulation of T cells that modulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune disease, Tregs are immunosuppressive and generally suppress or downregulate induction and proliferation of effector T cells. Generally, Tregs express the biomarkers CD3, CD4, and CD25. As used herein, the term "memory T cells" refers to a subgroup or subpopulation of T cells that have previously encountered and responded to their cognate antigen. At a second encounter with the antigen, memory T cells can reproduce to mount a faster and stronger immune response than the first time the immune system responded to the antigen. Memory T cells may be either CD4+ or CD8+ and usually express CD45RO. Particularly relevant to the present invention are CD8+ memory T cells.

As used herein, the term "naive T cell" refers to mature T cells that, unlike activated T cells or memory T cells, have not encountered their cognate antigen within the periphery. Naive T cells are commonly characterised by the surface expression of L-selectin (CD62L), the absence of the activation markers CD25, CD44 or CD69 and the absence of the memory CD45RO isoform. Table 1 - Examples of T Cell Subset Markers

* Some proteins are protein isoforms or multi-subunit protein complexes composed of several distinct genes.

As used herein, the term "activated immune cell" refers to an immune cell that has encountered its cognate antigen and is no longer naive, most notably NK cells, T cells and B cells. Natural killer cells (also known as NK cells, K cells, and killer cells) are activated in response to interferons or macrophage-derived cytokines. They serve to contain viral infections while the adaptive immune response is generating antigen-specific cytotoxic T cells that can clear the infection.

Natural killer T (NKT) cells, are a distinct population of T cells that express an ap T-cell receptor (TCR) and a number of cell surface molecules in common with natural killer (NK) cells. Although NKT cells are rare, comprising just 0.01-1% of peripheral blood mononuclear cells (PBMCs), they are important immunoregulatory cells rapidly producing large amounts of cytokines that can influence other immune cells.

Macrophages are specialised immune cells involved in the detection, phagocytosis and destruction of bacteria, cellular debris and other harmful particles. They also play an important role in both the initiation and resolution of inflammation. Macrophages can be in vivo modified to express an antigen receptor, thereby targeting the macrophage to eliminate cells or tissue expressing the target antigen through phagocytosis. In an embodiment of the present invention, a macrophage may be in vivo modified to express a tumour-specific TCR or CAR, thereby directing the macrophage to tumour sites.

In other embodiments, the compositions and methods can be used to modify cells of the immune system, such as monocytes/macrophages, to target and destroy viruses before they infect cells and/or to target bacteria, viruses or fungi. In additional embodiments, the compositions and methods modify cells of the immune system, such as immunosuppressive regulatory T cells to target and protect, rather than destroy, cell types.

In aspects of the invention wherein the nucleic acid comprises a transgene, the immune cell may be a T lymphocyte, a B lymphocyte, a natural killer (NK) cell, a natural killer T (NKT) cell, a phagocyte, a macrophage, a dendritic cell, a granulocyte (such as a neutrophil, eosinophil, basophil or mast cell). The immune cell may be an immune effector cell, an immune memory cell, a cytotoxic immune cell, or a regulatory T cell.

In relation to the aspects of the invention wherein the nucleic acid comprises a transgene, the immune cell may be a B lymphocyte. The terms "B lymphocyte" and "B cell" are used interchangeably. In general, a "B lymphocyte" is a type of white blood cells of the lymphocyte subtype. A major function of a B lymphocyte is to secrete antibodies. Accordingly, B lymphocytes belong to the humoral component of the adaptive immune system. In addition, B lymphocytes can present antigens and secrete cytokines. B lymphocytes express B cell receptors (BCRs) on their cell membrane. BCRs allow the B cell to bind to a specific antigen, against which it will initiate an antibody response. In relation to the aspects of the invention wherein the nucleic acid comprises a transgene, the immune cell may be a phagocyte. Phagocytes include monocytes and macrophages, granulocytes and dendritic cells. Phagocytes are characterised in their ability to phagocytose microbes and kill them through multiple bactericidal pathways.

In relation to the aspects of the invention wherein the nucleic acid comprises a transgene, the immune cell may be a macrophage. Macrophages are specialised immune cells involved in the detection, phagocytosis and destruction of bacteria, cellular debris and other harmful particles. They also play an important role in both the initiation and resolution of inflammation, and are involved in antigen presentation to T cells. Compared to neutrophils, macrophages are long-lived cells. Macrophages can be in vivo modified to express an antigen receptor, thereby targeting the macrophage to eliminate cells or tissue expressing the target antigen through phagocytosis. In an embodiment of the present invention, a macrophage may be in vivo modified to express a tumour-specific TCR or CAR, thereby directing the macrophage to tumour sites.

In relation to the aspects of the invention wherein the nucleic acid comprises a transgene, the immune cell may be a dendritic cell. The terms "dendritic cells" and "DCs" are used interchangeably. Dendritic cells phagocytose and function as antigen-presenting cells, initiating the acquired immune response and acting as important messengers between innate and adaptive immunity. DC may include, for example, "professional" antigen presenting cells, that are characterised in their high levels of surface MHC-class II expression and ability to present antigen to CD4+ and/or CD8+ T cells, particularly to naive T cells.

In relation to the aspects of the invention wherein the nucleic acid comprises a transgene, the immune cell may be a granulocyte. Neutrophil, eosinophils, basophils and mast cells are granulocytes.

In relation to the aspects of the invention wherein the nucleic acid comprises a transgene, the immune cell may be a neutrophil. Neutrophils are one of the first-responders of inflammatory cells to migrate towards the site of inflammation, and are usually recruited within minutes. In addition to their phagocytic properties, neutrophils contain granules and enzyme pathways that assist in the elimination of pathogenic microbes. Neutrophils are relatively short-lived cells.

In relation to the aspects of the invention wherein the nucleic acid comprises a transgene, the immune cell may be an eosinophil. Following activation, eosinophils typically release cationic granule proteins, reactive oxygen species, lipid mediators, growth factors and cytokines.

In relation to the aspects of the invention wherein the nucleic acid comprises a transgene, the immune cell may be a basophil. Basophils are typically present in peripheral blood. Basophils can be activated via antigen/lgE/FcsRI cross-linking to release molecules such as histamines, tryptase (particularly tryptase alpha), leukotrienes, and cytokines.

In relation to the aspects of the invention wherein the nucleic acid comprises a transgene, the immune cell may be a mast cell. A mast cell is a type of granulocyte immune cell. Mast cells are typically present in mucosal and epithelial tissues throughout the body. Mast cells contain cytoplasmic granules that store inflammatory mediators, including tryptase (particularly tryptase beta), histamine, heparin, and cytokines. Mast cells can be activated by antigen/lgE/FcsRI cross-linking, which can result in degranulation and release of inflammatory mediators.

Immune Cell Recruiting Factor

The immune cell recruiting factor of the present invention is effectively providing a non-self- provocation to the immune cells. The factor recruits immune cells to the locality, area or site of administration. The immune cell recruiting factor of the present invention recruits immune cells to the locality of administration. This recruitment is the primary aim of the factor - to attract immune cells to the locality of administration. These immune cells will be a homogenous population, both in terms of function and in terms of activation status (where relevant) to include naive, activated, effector and memory subsets. The immune cell recruiting factor may recruit activated immune cells, and/or activate the recruited immune cells. Thus, the immune cell recruiting factor serves to recruit and optionally activate immune cells to a locality, allowing subsequent transfection and in vivo modification of these immune cells. By recruiting and/or activating immune cells, the immune cell recruiting factor primes or pre-conditions a locality/site or area. In other words, the immune cell recruiting factor provides for the accumulation, aggregation or concentration of activated immune cells to a local site or area. The recruitment and/or activation of immune cells by the immune cell recruiting factor may be described as "priming" or "pre-conditioning". The immune cells recruited and/or activated by the immune cell recruiting factor may be described as "primed immune cells" or "activated immune cells".

The immune cell recruiting factor may be any suitable non-self entity, including but not limited to: a vaccine, an antigen, an epitope, a cytokine, a chemokine or a microbial product or toxin. Preferably, the immune cell recruiting factor recruits and/or activates immune cells, preferably, T cells, NKT cells or NK cells. The immune cell recruiting factor may recruit naive T cells, and activate them inducing differentiation to effector T cells and/or memory T cells. Preferably, the immune cell recruiting factor recruits and/or activates CD8+ T cells. Preferably, the immune cell recruiting factor recruits and/or activates CD8+ T cells in the locality of administration. Preferably the immune cell recruiting factor is an antigen or epitope thereof that binds to and activates the T cell receptor (TCR) of a T cell. Various animal studies using ex vivo based adoptive cell therapy have shown memory-T cell subsets have a superior antitumour function following adoptive transfer (see for example: Berger, C. et al. (2008) Adoptive transfer of effector CD8+ T cells derived from central memory cells establishes persistent T cell memory in primates. Journal of Clinical Investigation 118, 294-305; and see also: Klebanoff, C. A. et al. (2005) Central memory self/tumor

It may be preferred that the immune cell recruiting factor used comprises a non-self entity that the subject has already encountered, for example via infection or immunisation. This would have an additional benefit that the immune cells had already encountered the non-self entity, and thus retain memory T cells for this entity.

The immune cell recruiting factor may be any substance capable of eliciting an adaptive immune response, otherwise known as an antigen (antibody generator).

The immune cell recruiting factor may be a commercially available vaccine. Any suitable vaccine format may be used, such that an antigen is provided. Thus, the immune cell recruiting factor may be any one or more of vaccines to bacterial, viral or fungal pathogens. Suitable vaccines include, but are not limited to those directed to antigens from: Hepatitis B, Influenza, Measles, Mumps, Rubella, MMR (Measles, Mumps and Rubella), Hepatitis A, Polio, Haemophilus influenza type b (Hib), Rabies, Typhoid fever, Pertussis, Tetanus, Meningococcal diseases (including A, B, C, W and/or Y), DPT (diphtheria, pertussis, and tetanus), Diphtheria, Rotavirus, Hepatitis E, Shingles/Chickenpox, Pneumococcal (PPV), Human papillomavirus (HPV), SARs and MERs viruses, Coronavirus (such as COVID-19), Dengue, Adenovirus, Anthrax, Cholera, Japanese Encephalitis (JE), Smallpox, Typhoid Fever, Yellow Fever, Malaria, Tick-borne encephalitis, Ebola, Q. fever.

The immune cell recruiting factor may be a microbial product or toxin. Toxins are potent molecules produced by a large variety of bacterial pathogens. Bacterial toxins can be divided in several groups regarding their nature and mode of action. Bacterial exotoxins are secreted by the pathogen include pertussis toxin (PT) and adenylate cyclase toxin (ACT) secreted by Bordetella pertussis, anthrax toxin from Bacillus anthracis, Clostridia C3 toxins, and Staphylococcus aureus leukotoxins. A further exotoxin is AIP56, a recently described toxin from Photobacterium damselae piscicida (Phdp). Further toxins include Phytohemagglutinin (PHA), Staphylococcal enterotoxin B (SEB), phorbol meristate acetate (PMA) and ionomycin. In addition, other microbial products not classified as toxins can also attract immune cells. For example, mycolactone, a polyketide molecule produced by Mycobacterium ulcerans, and S. aureus superantigens-like proteins (SSLs) and phenol-soluble modulins (PSMs), may also be used. The immune cell recruiting factor may be a cytokine or chemokine. Suitable chemokines include, but are not limited to I L12, CXCR3 ligands, CCL19 and CCL5.

The immune cell recruiting factor may be administered as a nucleic acid encoding the immune cell recruiting factor. The nucleic acid may be provided in any suitable format as discussed herein in relation to the antigen receptor encoding nucleic acid.

The immune cell recruiting factor is preferably administered to the subject in need thereof. This factor is preferably administered prior to any of the other step of the method, or prior to components of the product or composition. This is step (a) of the method, or component (i) of the product or composition. The immune cell recruiting factor is preferably administered to a location of said subject via any suitable means. For example, the immune cell recruiting factor may be administered to a particular muscle via intramuscular injection. The location of the intramuscular injection may be the deltoid muscle in the arm or the anterolateral aspect of the thigh. The locality in this instance is the area of muscle into which the immune cell recruiting factor is injected.

The immune cell recruiting factor may be formulated appropriately depending on route of administration, with any one or more of pharmaceutically acceptable excipients, diluents, binders, fillers, lubricants, solubilizers, stabilizers, buffers, tonicity modifiers, bulking agents, viscosity enhancers/reducers, surfactants, chelating agents, and/or adjuvants. Immunological adjuvants may be useful in the preparation of the immune cell recruiting factor, since an adjuvant is a substance that increases or modulates the immune response to an antigen. Many adjuvants are in widespread use, including aluminium salts, oils and virosomes.

When administered to a subject in a defined locality, the immune cell recruiting factor acts to signal to the immune system that a non-self entity is present in the locality. Generally, the innate immune system provides a first-line of defence. It is a rapid immune response, initiated within minutes or hours after administration of the factor, but is antigen-independent. An important function of innate immunity is the rapid recruitment of immune cells to sites of infection and inflammation through the production of cytokines and chemokines (small proteins involved in cell-cell communication and recruitment). The innate immune system involves several cell types, including phagocytes (macrophages and neutrophils), dendritic cells, mast cells, basophils, eosinophils, natural killer (NK) cells and innate lymphoid cells.

Several cells involved in the innate immune response are termed "antigen presenting cells" or APCs. Such cells include macrophages and dendritic cells, which express cell surface proteins for presenting antigens to the cells of the adaptive immune system. Cells of the adaptive immune system, generally lymphocytes, are recruited to the locality of the nonself entity by the innate immune system. This may take several hours or days to recruit the adaptive immune system cells to the locality. The timeframe for recruitment of immune cells is dependent on the immune cell recruiting factor administered. In general, it may take between 2 and 7 days, notably 3 to 6 days or 4 to 5 days for T lymphocytes to be recruited to the locality of administration.

Nucleic Acid Vector

In some embodiments of any of the present invention provided herein, the nucleic acid encoding the antigen receptor is administered prior to, simultaneously with and/or subsequent to initiation of administration of the immune cell recruitment factor. The nucleic acid encoding the antigen receptor may also be referred to as a "nucleic acid vector" or "polynucleotide". The term "polynucleotide" or "nucleic acid", as used herein, is intended to include DNA and RNA such as genomic DNA, cDNA, mRNA, saRNA. A nucleic acid may be single-stranded or double-stranded.

As used herein, the term "transgene" refers to a nucleic acid sequence that encodes an antigen receptor. This definition includes various sequence polymorphisms, mutations, and/or sequence variants wherein such alterations do not affect the function of the encoded antigen receptor. Thus, the nucleic acid of the present invention may comprise a transgene. The term "transgene" may include not only coding sequences but also regulatory regions such as promoters, enhancers, and termination regions. Wherein the nucleic acid of any aspect of the invention is a DNA vector, the DNA vector preferably includes a promoter or enhancer operably linked to the gene encoding the antigen receptor. In some embodiments, expression of the transgene is under the control of a conditional promoter or enhancer or transactivator. The term "transgene" further can include all introns and other DNA sequences spliced from the mRNA transcript, along with variants resulting from alternative splice sites. Nucleic acid sequences encoding the antigen receptor can be DNA or RNA that directs the expression of the antigen receptor. These nucleic acid sequences may be a DNA strand sequence that is transcribed into RNA or an RNA sequence that is translated into protein. The nucleic acid sequences include both the full-length nucleic acid sequences as well as non-full-length sequences derived from the full-length protein. The sequences can also include degenerate codons of the native sequence or sequences that may be introduced to provide codon preference in a specific immune cell.

As used herein, the term "encoding" refers to a property of sequences of nucleotides in a nucleic acid, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. A nucleic acid can, for example, encode a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Unless otherwise specified, nucleic acids having a sequence encoding an antigen receptor include all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The nucleic acid that encode proteins and RNA can also include introns.

The nucleic acid may be provided in any appropriate format or architecture.

In some embodiments, the nucleic acid may be a closed linear DNA, a plasmid, a minicircle, a linear DNA, a linear DNA with capped ends, a cDNA, an mRNA or saRNA. In some embodiments, the nucleic acid may be a closed linear DNA, a plasmid, a minicircle, a linear DNA, a linear DNA with capped ends, a cDNA, an mRNA or saRNA encoding the antigen receptor. The nucleic acid may be any other suitable format, for example the nucleic acid may be a mbDNA (as described in WO2021/058984) or a linear DNA with sequestered or capped ends (as described in WO2022/058755). In other words, the nucleic acid may be a closed linear DNA, a minicircle, a linear DNA, a linear DNA with capped ends, a plasmid, a cDNA, an mRNA or saRNA that includes a sequence (e.g. a gene) for expressing an antigen receptor. The nucleic acid can further comprise any additional sequence information to facilitate transfer of the genetic material (e.g. a sequence encoding an antigen receptor) to the immune cell. For example, the nucleic acid may comprise a promoter or enhancer operably linked to the sequence to be expressed. One or more promoter or enhancers may be used, as required. Any suitable promoters or enhancers can be used. A general promoter, tissue-specific promoter, cell-specific promoter, and/or promoters specific for the nucleus may be used. In a particular embodiment, the nucleic acid comprising a transgene may comprise a lymphocyte-specific promoter, thereby allowing selective expression of the transgene in lymphocytes. In a particular embodiment, the nucleic acid encoding an antigen receptor may comprise a lymphocyte-specific promoter, thereby allowing selective expression of the antigen receptor in lymphocytes. Suitable lymphocyte-specific promoters include, but are not limited to, I FNg- p, dLcK-p, and CD3d-p. The promoter may be a synthetic promoter. Promoters are generally well known in the art and can be prepared using conventional techniques.

The nucleic acid may further comprise one or more enhancer sequences. Suitable enhancer sequences include, but are not limited to S/MAR regions (e.g. from I FNb, ApoB, or HPRT genes), intron sequences (e.g. rabbit beta-globin intron, CMV intron A); and 5' and/or 3' untranslated regions (UTRs).

Closed linear DNA is generally understood to be double-stranded DNA covalently closed at each end. There are, therefore, no free 3' or 5' ends to the DNA. The double stranded DNA in the linear section is complementary in sequence. When denatured, closed linear DNA may form a single-stranded circle. The DNA may be closed at each end by any suitable sequence, forming any secondary structure, such as a hairpin or a hairpin loop, or more complex structures such as cruciform. The sequence at the closed ends of the linear DNA may be complementary or non-complementary. The closed linear DNA may be made by any suitable method. Given the use of the DNA vector in subjects, it may be preferred to ensure that the closed linear DNA is free from any prokaryotic DNA sequences, such as antibiotic resistance genes or origins of replication. Closed linear DNA vectors can be designed to be minimal vectors, including only the sequences necessary for their desired function and structure (i.e. the sequence they are delivering and a sequence encoding the closed ends, for example a cruciform, hairpin or hairpin loops at the end of the double stranded linear section). Unnecessary or extraneous sequences (also described as bacterial or viral sequences) that may be excluded from closed linear DNA vectors may include bacterial origins of replication, bacterial selection markers (e.g. antibiotic resistance genes), and unmethylated CpG dinucleotides. By not including such sequences, this enables the creation of a "minimal" vector which does not contain extraneous genetic material.

Closed linear DNA is generally understood to be double-stranded DNA covalently closed at each end. The double stranded section of the DNA is therefore complementary. When denatured, closed linear DNA may form a single stranded circle. The DNA may be closed at each end by any suitable structure, including a cruciform, a hairpin or a hairpin loop, depending on preference. The end of the closed linear DNA may be composed of a non-complementary sequence, thus forcing the DNA into a single stranded configuration at the cruciform, hairpin or hairpin loop. Alternatively, the sequence can be complementary. It may be preferred that the end is formed by a portion of a target sequence for a protelomerase enzyme. A protelomerase target sequence is any DNA sequence whose presence in a DNA template allows for the enzymatic activity of protelomerase, which cuts a double stranded section of DNA and re-ligates them, leaving covalently closed ends. In general, a protelomerase target sequence comprises any perfect palindromic sequence i.e. any double-stranded DNA sequence having two-fold rotational symmetry, or a perfect inverted repeat. The closed linear DNA may have a portion of a protelomerase target sequence at one or both ends. The protelomerase target sequence can have the same cognate protelomerase at each end, or require a different protelomerase for each end. Closed linear DNA constructed via the action of various protelomerase enzymes have been previously disclosed by the applicants in W02010/086626, W02012/017210 and WO2016/132129, all of which are incorporated by reference. Closed linear DNA constructed using in vitro DNA amplification followed by cleavage with a protelomerase enzyme has the advantage that the closed linear DNA is produced in an in vitro, cell-free environment, and can be scaled up for commercial production. These closed linear DNA vectors are known as Doggybone DNA or dbDNA™. It is preferred that the closed linear DNA vectors are made using the prior methods of the applicants, in an in vitro, cell-free manner based upon amplification of a DNA template with at least one protelomerase target sequence, and processing of the amplified DNA with a protelomerase to produce closed linear DNA. Closed linear DNA can be constructed by a conversion of a plasmid with the requisite protelomerase target sequences into a closed linear DNA vector, although this is not an efficient method of production.

Other closed linear DNA vectors have been constructed by various in vitro strategies including the capping of PCR products, and the "minimalistic immunogenic defined gene expression (MIDGE)" vectors. MIDGE is generated by the digestion of both prokaryotic and eukaryotic backbones after isolation of plasmid from bacterial cells, followed by ligation of the required DNA sequence into hairpin sequences for end-refilling.

Closed linear DNA is a transient expression vector.

DNA "ministrings", which are produced in an in vivo manner in cell culture, based upon the action of protelomerase, are also closed linear DNA vectors that would be suitable for use in the invention.

Other forms of closed linear DNA that may be suitable include those closed at the ends with cruciform structures, which can be manufactured in cell culture.

It may be preferred that the closed linear DNA is manufactured in a cell-free system, since this ensures purity of product, in the alternative, stringent purification of closed linear DNA made by cellular methods will be required by the regulatory authorities.

Minimally sized non-viral, non-integrating DNA vectors have been used to engineer human T lymphocytes in vitro, with the resulting CAR-T cells demonstrating enhanced anti-tumour activity both in vitro and in vivo (see for example: Bozza Met al. (2021) A nonviral, nonintegrating DNA nanovector platform for the safe, rapid, and persistent manufacture of recombinant T cells. Sciences Advances 7(16) doi: 10.1126/sciadv.abfl333).

The nucleic acid molecule can be any suitable structure or format. Discussed above are closed linear DNA molecules, but sequences can be provided in various different DNA or RNA architectures. Alternative formats include single stranded linear nucleic acid molecules, single or double stranded linear nucleic acids with capped ends or sequestered ends, linear nucleic acid molecules with structural elements (formed by complementary base pairing, such as hairpins, G-quadruplexes, inverted terminal repeats, pseudoknots, cruciform or stem-loops). Structural elements within or at the ends of such molecules can have a function, such as targeting of the nucleic acid molecule. For example, structural elements in nucleic acid can form aptamers, which can permit cell or nuclear specific targeting of the nucleic acid. Suitable exemplary architectures are disclosed in W02020/217057 and WO2022/058755. Alternative nucleic acid structures can be based upon single stranded circular architecture. The advantage of single stranded DNA architecture is that the nucleic acid is readily available for translation machinery and the like. A further advantage of circular structures is resistance to exonuclease attack. The single stranded structure can include regions of self-complementary sequence, such that structural elements (formed by complementary base pairing, such as hairpins, inverted terminal repeats, pseudoknots, cruciform or stem-loops) can be included in the architecture. Such structural elements can have a function (such as targeting by the provision of an aptamer). Suitable architectures are disclosed in WO2016/132129. Alternatively, the structural elements can form the target site for enzymes, such as nucleases involved with gene editing. Suitable gene editing architectures are disclosed in WO2021/058984.

In some embodiments of any of the present invention provided herein, the nucleic acid comprising the transgene is administered prior to, simultaneously with and/or subsequent to initiation of administration of the immune cell recruitment factor. The nucleic acid comprising the transgene may also be referred to as a "nucleic acid vector", "polynucleotide" or a "payload vector". The term "polynucleotide" or "nucleic acid", as used herein, is intended to include DNA and RNA such as genomic DNA, cDNA, mRNA, saRNA. A nucleic acid may be single-stranded or double-stranded.

As used herein, the term "transgene" may refer to a nucleic acid sequence that encodes a gene that it is desired for expression in the immune cell. The transgene may be any suitable gene for expression. Suitable transgenes are discussed herein in relation to therapeutic payloads.

Therapeutic Payload

According to one aspect of the invention, the immune cell may be modified with a nucleic acid encoding a transgene.

The transgene may be a sequence encoding a product which is useful in biology and medicine, such as a prophylactic or a therapeutic transgene, e.g. protein or non-protein encoding oligonucleotide. Therefore, the transgene may encode a therapeutic payload. Thus, the immune cells of the present invention may be in vivo genetically modified to express a therapeutic payload. Preferably, the therapeutic payload is distinct from the immune cell recruiting factor.

There are a number of potential therapeutic payloads that an immune cell could be in vivo modified to express to achieve a therapeutic objective. For example, the therapeutic payload may include: an antigen receptor, a therapeutic protein (e.g. to induce an immunogenic response, for example an antigen, antibody, cytokine), onco-suppressor protein (e.g. natural/unmutated p53), a differentiation factor (i.e. to modulate cell fate), or a protein with nucleic acid editing or gene activity modulation function (e.g. DNA or RNA editing, or transcriptional regulation).

The transgene may encode an antibody. An "antibody" or "Ab" is an immunoglobulin molecule capable of recognising and binding to a specific target or antigen, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" can encompass any type of antibody, including but not limited to monoclonal antibodies, polyclonal antibodies, "antigen-binding fragments" (or portion), such as Fab, Fab', F(ab')2, Fd, Fv, Fc, etc., of intact antibodies that retain the ability to specifically bind to a given antigen (e.g. PTK7), an isolated complementarity determining region (CDR), bispecific antibodies, heteroconjugate antibodies, mutants thereof, fusion proteins having an antibody, or antigen-binding fragment thereof, (e.g., a domain antibody), single chain (ScFv) and single domain antibodies (e.g., shark and camelid antibodies), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis- scFv, humanised antibodies, chimeric antibodies and any other modified configuration of the immunoglobulin molecule that includes an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibodies may be murine, rat, human, or any other origin (including chimeric or humanised antibodies). In a particular embodiment, the immune cell may be in vivo modified to express anti-PD-1 or anti-PD-Ll blocking antibodies. Blockade of the PD-1/PD-L1 interaction can induce durable anti-tumour responses in a wide range of solid and haematological tumours.

The transgene may encode a cytokine. Functions of cytokines in the immune system include, promoting influx of circulating leukocytes and lymphocytes into the site of immunological encounter; stimulating the development and proliferation of B cells, T cells, peripheral blood mononuclear cells (PBMCs) and other immune cells; and providing antimicrobial activity. Exemplary immune cytokines, include but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-15, IL17A, IL- 17F, IL-18, IL-21, IL-22, interferon (including IFN alpha, beta, and gamma), tumour necrosis factor (including TNF alpha, beta), transforming growth factor (including TGF alpha, beta), granulocyte colony stimulating factor (GCSF), granulocyte macrophage colony stimulating factor (GMCSF) and thymic stromal lymphopoietin (TSLP In a particular embodiment, the transgene may encode a cytokine such as IL-12 which has been shown to have anticancer activity. IL-12 cause induction of IFN-y production by resting and activated CD4+ T cells, CD8+ T cells, and NK cells, as well as enhancing the proliferation of activated T and NK cells, increasing the lytic activity of NK/lymphokine-activated killer cells, and facilitating specific cytotoxic T lymphocyte (CTL) responses. However, IL-12 cannot be administered as a systemic treatment due to excessive toxicity, therefore a targeted therapy where expression only occurs within a target cell would enable the delivery of this payload.

In some embodiments, the immune cell may be in vivo modified to impact the expression, presence, or activity of a protein or other factor or molecule associated with one or more activities or outcomes or effects, and/or a nucleic acid encoding such protein or factor. For example, the transgene may disrupt normal function and expression of a gene or protein. In some embodiments, the protein or factor to be modulated may have immunosuppressive effects. Such effects may include inhibition or dampening of T cell activity or function and/or promotion of regulatory T cell function, levels, or activity.

The transgene may encode a differentiation factor (i.e. a factor to modulate cell fate). For example, the transgene may encode a factor that reprograms macrophages from a resting or M2 phenotype to an Ml phenotype. Macrophages, in particular tumour-associated macrophages (TAMs) of M2 phenotype stimulate tumour angiogenesis and enhance tumour cell growth, invasion and metastasis. In comparison, Ml macrophages are pro-inflammatory and produce immunogenic cytokines, playing an important role in tumour suppression. Thus, in vivo modification of TAMs to express a differentiation factor to reprogram TAMs from M2 to Ml differentiation status may be an effective cancer treatment strategy.

In an alternative embodiment, the transgene encodes a non-protein encoding oligonucleotide, such as an RNA (other than mRNA), for example miRNA, siRNA, shRNA or IncRNA. It will be understood that transgenes encoding non-coding RNAs may be used in methods of gene silencing.

Antigen Receptor

The transgene may encode an antigen receptor. The immune cells of the present invention are in vivo genetically modified to express an antigen receptor. The antigen receptor is specific for a target antigen. Expression of the antigen receptor thereby enables targeting of the in vivo genetically modified immune cell to cells expressing or presenting the target antigen or a fragment (such as a peptide) therefrom. The in vivo genetically modified immune cell may be used for the treatment of diseases by targeting cells expressing or presenting a target antigen or fragment thereof. The target cells may express the antigen on the cell surface or may present an antigen fragment thereof, such as a peptide. Such treatment provides for the selective eradication of cells that express the target antigen, thereby minimising adverse effects to normal cells not expressing or presenting the target antigen or fragment thereof.

The antigen receptor may be any suitable antigen receptor, either naturally derived or synthetic. In general, antigen receptors are multi-protein complexes made up of clonally variable antigenbinding chains that are associated with invariant accessory proteins. They are present on the cell surface and are membrane-bound. The invariant chains are required for the transport of the receptors to the cell surface and for initiating signalling when the receptors bind to an extracellular ligand. Antigen binding to the receptor via the antigen-binding chains generates signals that lead ultimately to the activation of new gene expression involved in the immune response and deactivation of genes typically expressed only in resting cells. Thus, for example, T cells are activated by the recognition of a cognate peptide by the T-cell antigen receptor; said peptide is displayed on a major histocompatibility complex molecule (MHC).

In general, antigen receptors have an antigen binding domain. An "antigen binding domain" describes a region of a polypeptide capable of binding to an antigen or fragment thereof under appropriate conditions. The antigen binding domain may be a single-chain variable fragment (scFv) based on one or more antibodies. Alternatively, the antigen binding domain may comprise a variable heavy (VH) region and a variable light (VL) region, with the VH and VL regions being on the same polypeptide.

The variable regions of the antigen-binding domains of the polypeptides of the disclosure can be modified by mutating amino acid residues within the VH and/or VL CDR 1, CDR 2 and/or CDR 3 regions to improve one or more binding properties (e.g., affinity) of the antibody. The term "CDR" refers to a complementarity-determining region that is based on a part of the variable chains in immunoglobulins (antibodies) and T cell receptors, generated by B cells and T cells respectively, where these molecules bind to their specific antigen. Since most sequence variation associated with immunoglobulins and T cell receptors is found in the CDRs, these regions are sometimes referred to as hypervariable regions. Mutations may be introduced by site-directed mutagenesis or PCR-mediated mutagenesis and the effect on antibody binding, or other functional property of interest, can be evaluated in appropriate in vitro or in vivo assays. Preferably conservative modifications are introduced and typically no more than one, two, three, four or five residues within a CDR region are altered. The mutations may be amino acid substitutions, additions or deletions.

The term "specifically binds" or "specificity", as used herein, refers to the ability of the antigen receptor to recognise a specific antigen or peptide derived from an antigen, but not substantially recognise or bind to other non-target antigens or molecules.

The antigen receptor may be a T cell receptor (TCR) or a derivative or modified version thereof.

ATCR refers to a molecule that contains variable alpha and beta chains (also known as TCRa and TCRP, respectively) or variable y and 6 chains (also known as TCRy and TCR6, respectively), that is capable of specifically binding to an antigen peptide bound to an MHC protein. The two variable chains are generated by a process of random recombination and selection of the relevant genes. The recombination gives rise to three hypervariable regions, the complementarity-determining regions— CDR1, CDR2, and CDR3. Thus, TCRs are encoded in gene segments that undergo somatic recombination during T cell development to generate antigen-binding diversity. The specificity of a given TCR is dependent upon the amino acid sequence of each chain, particularly the complementarity-determining regions (CDR1, CDR2, CDR3). In general, CDR3 directly interacts with the presented peptide, while CDR1 and CDR2 primarily interact with the MHC molecule

The TCR is recognises an antigen - more particularly it is recognises a peptide or non-peptide derived from an antigen. The MHC proteins present peptides or non-peptides derived from antigens. Each TCR has a cognate peptide or non-peptide to which it will bind. In general TCRs are specific for one antigen peptide or non-peptide presented via an MHC molecule. Specificity is precisely defined in biochemical interactions, such as enzyme-substrate or antibody-antigen interactions, which involve two molecules. An affinity matured antibody that binds its target 1000-fold more tightly than unrelated antigens is considered highly specific. However, TCRs and MHC proteins are embedded in membranes, which greatly influences the biophysics of protein interactions. Thus, the concept of a solution K_D, on which the biochemical definition of binding specificity is based, may not be applicable to TCRs. Some TCRs may be capable of cross-reacting with one or more alternate antigen peptides or non-peptides presented via MHC. It is generally accepted that peptide-specific TCRs exhibit crossreactivity. TCR recognition of peptide - MHC complexes is both cross-reactive, given the high number of total epitopes that could be bound, and at the same time, highly specific considering the low frequency of epitopes that can be recognized by a given TCR. As TCR affinity has been generally accepted as the central role in defining T cell specificity and sensitivity, selection for and generation of high affinity TCRs is one approach taken currently.

In general, the TCR is selected on the basis of being unlikely to have cross-reactivity with structurally similar peptide antigens (mimotopes) expressed by normal tissue. TCRs can be modified by different methods, including affinity maturation of their CDRs in order to increase their affinity for the target antigen, thereby reducing cross-reactivity. Since TCRs recognise a cognate peptide of the antigen bound to MHC proteins, the TCR must have high specificity for the appropriate MHC-peptide complex.

The antigen receptor may be a chimeric antigen receptor (CAR), also referred to as a "chimeric T cell receptor" or "artificial T cell receptor".

Wherein the antigen receptor is a CAR and the immune cell is a T cell, the modified immune cell may be referred to as a "CAR-T Cell". A CAR is an artificial receptor comprising a single molecule or a complex of molecules which recognises and binds to a target structure (e.g. an antigen). Thus, a CAR may confer specificity onto an immune cell such as a T cell expressing said CAR on the cell surface.

A CAR comprises a target-specific binding element otherwise referred to as an antigen binding moiety or antigen binding domain that is generally part of the extracellular domain of the CAR. The antigen binding domain recognises a ligand that acts as a cell surface marker on target cells associated with a particular disease state. A CAR may be specific for the target antigen. Generally, CARs exhibit less cross-reactive behaviour than TCRs, since they are based upon antibodies.

CARs may be designed in a modular fashion that typically consists of an extracellular antigen-binding domain (such as from an antibody), a hinge region, a transmembrane domain that anchors the CAR to the cell membrane, and one or more intracellular domains that transmit activation signals. Depending on the number of costimulatory domains, CARs can be classified into first (CD3z only), second (one costimulatory domain + CD3z), or third generation CARs (more than one costimulatory domain + CD3z).

The essential components of CARs are an extracellular antigen-targeting moiety, such as a single-chain variable fragment (scFv), a transmembrane and hinge domain that anchors the receptor on the cell surface and projects the scFv from the membrane, and intracellular signalling domains that are triggered on antigen engagement. The antigen specificity of a CAR is most often provided by a scFv module (minimum functional domain of a monoclonal antibody). The advantages of using scFvs as the CAR antigen-binding domain include their high specificity, the fact that they can be readily generated against most target antigens through well-established methods, and the ease of including them into the CAR design. However, alternative binding domains have been used in preclinical studies, including receptors, ligands, Fc receptor fragments, nanobodies, designed ankyrin repeat proteins, adnectins, peptides, cytokines, and variable lymphocyte receptors. Any suitable antigen binding domain could be present in the CAR.

The flexible hinge domain of a CAR is a short peptide fragment that provides conformational freedom to facilitate binding to the target antigen. It may be used alone or in conjunction with a spacer domain that projects the antigen binding domain away from the cell surface. The optimal length of the spacer depends on the proximity of the target antigen, and the location thereon of the epitope recognised, to the cell surface. Long spacers typically include the CH2CH3 domain (approximately 220 amino acids) of immunoglobulins G1 (IgGl) or lgG4, whereas the CH3 region can be used on its own to construct an intermediate spacer (of around 120 amino acids). Shorter spacers may be derived from segments (less than 60 amino acids) of CD28, CD8a, CD3 or CD4. The polypeptide spacer may comprise a modified lgG4, IgGl, or lgG2 hinge region, or a combination thereof. In instances, the hinge region may be linked to other amino acid sequences including but not limited to the CH2 or CH3 regions of the Ig Fc. Any suitable spacer may be used.

The transmembrane domain can potentially affect CAR expression and association with endogenous membrane proteins and should be selected appropriately. Transmembrane domains in CARs serve as a fulcrum for transducing ligand recognition signals to the intracellular cytoplasmic domain. The transmembrane domain may come from the same source as the hinge domain, for example CD28 CD8a, CD3 or CD4. A transmembrane domain may be a hydrophobic alpha helix that spans the membrane.

In addition to any of the domains mentioned above, CARs also incorporate one or more co-stimulatory domains to provide additional activating signals. The choice of co-stimulatory domain influences the phenotype and metabolic signature of the immune cells. For example, in T cells, CD28 co-stimulation yields a potent, but short-lived, effector-like phenotype, with high levels of interleukin-2 (IL-2) secretion, cytolytic capacity, and glycolysis. By contrast, T cells modified with CARs bearing 4-1BB co- stimulatory domains are less prone to exhaustion, expand and persist longer in vivo, have increased oxidative metabolism, and have an increased capacity to generate central memory T cells. A blend of these two (third generation CAR T cells) may be advantageous. Several other co-stimulatory domains are in evaluation, for example, 0X40 and ICOS, and any appropriate co-stimulatory domain may be used.

In a preferred embodiment, the antigen receptor of the present invention recognises a target antigen or a fragment thereof such as a tumour antigen expressed on a diseased cell such as tumour cell, or a fragment thereof displayed on the cell. By recognised, it is understood that the antigen receptor has a binding affinity for the target antigen (or fragment thereof) that is higher than for other antigens (or fragments). If the antigen receptor recognises the antigen, it will be understood by those skilled in the art that it has a binding affinity for an epitope of that antigen. Specificity involves both binding to a specific target antigen (or fragment thereof) and not binding to other entities. The strength of interaction between receptor and antigen at single antigenic sites can be described by the affinity of the receptor for the antigen (howsoever presented - alone or as a peptide associated with MHC). Within each antigenic site, the variable region antigen binding domain interacts through weak noncovalent forces with antigen at numerous sites. The greater the interaction, the stronger the affinity. "Avidity" may be a more useful measure of the overall stability or strength of the receptorantigen complex. It is controlled by three major factors: receptor: antigen affinity, the valence of both the antigen and receptor, and the structural arrangement of the interacting parts. Ultimately these factors define the specificity of the antigen receptor, that is, the likelihood that the particular receptor is binding to a precise antigen epitope or fragment thereof.

The binding affinity of the antigen binding region, such as the variable regions, or of the CDRs may be at least 10'⁵M, 1O'^SM, 10'⁷M, 10'⁸M, 10'⁹M, 10¹⁰M, lO ^M, 10¹²M, or 10¹³M. In some embodiments, the K_D of the antigen binding region, such as the variable regions, or of the CDRs may be at least 10’ ⁵M, 10^-sM, 10'⁸M, 10'⁸M, 10'⁹M, 1O ^1OM, 10 ^X1M, 10¹²M, or 10 ¹³M. Those skilled in the art will be aware of methods to determine binding affinity, or K_D, including but not limited to: surface plasmon resonance (SRP)-based biosensors, ELISA, and/or kinetic exclusion assay (KinExA).

In an embodiment, the nucleic acid may comprise more than one transgene. Alternatively, the immune cell may be transfected with more than one nucleic acids comprising different transgenes. As a result, the in vivo modified immune cell may express more than one transgene. For example, the in vivo modified immune cell may be engineered to express both a therapeutic payload (e.g. a cytokine such as IL-12 or IL-15) and an antigen receptor (e.g. CAR or TCR).

Target Antigen

The in vivo modified immune cells of the present invention are able to recognise and eradicate cells expressing or displaying the target antigen. Thus, in one aspect, the present invention relates to the treatment of diseases by targeting cells expressing a target antigen. The target cells may express the antigen on the cell surface or may present an antigen fragment thereof, such as a peptide. The target cells may present a digestion product of the antigen. The target cells may display an epitope of the antigen. Such treatment provides for the selective eradication of cells that express the target antigen, epitope or fragment thereof, thereby minimising adverse effects to cells not expressing the target antigen.

Natural TCRs on T cells can recognize 8-14 amino acid long peptides, which are derived from a variety of proteins via proteasomal digestion, and are bound to MHC class I or MHC class II molecules expressed on most cells in the body. The antigen receptors on helper T cells recognise antigenic peptides bound to MHC class II molecules, whereas the antigen receptors on cytotoxic T cells recognise antigen displayed on MHC class I molecules. Thus, a fragment of a target antigen may be a peptide which is suitably capable of binding to an MHC class I molecule, the peptide being preferably in the range of 8 to 14 amino acids in length. It is generally thought that peptides longer than 10 amino acids in length are of the most value to immune surveillance by T cells. Thus, the fragment may be at least 9 or 10 amino acids in length. However, if the antigen receptor is a CAR, these do not require the presentation of the target antigen as a peptide via a MHC class I molecule. CARs can bind to the target antigen expressed on the cell surface. Thus, this type of antigen receptor may not recognise fragments of the antigen, depending on the region to which they bind.

The target antigen may be an antigen which is expressed on a cancer cell. Such may be a tumour associated antigen or a neoantigen.

The target antigen may be a tumour associated antigen (TAA). The target antigen may be any appropriate TAA, which the tumour expresses. TAA may be self-antigens. In particular, TAA may be encoded by an open reading frame of gene products that are differentially expressed by tumours, and not by normal tissues. They may also be encoded by intronic sequences, splice variants, gene fusions, mutated genes or translated alternative open reading frames, antisense strands, pseudogenes, or be the products of genetic translocations. TAA may be classified as oncoviral (encoded by tumourigenic transforming viruses such as HPV), oncofoetal (typically only expressed during foetal development and in cancerous cells), cancer-testis or cancer germline antigens (CGAs) (expressed only by cancer cells and adult reproductive tissues), overexpressed/ accumulated (expressed by both normal and cancerous tissue, highly elevated expression in cancerous cells), lineage-restricted (expressed largely by a single cancer histotype), mutated (cancer specific expression as a result of genetic mutation/transcription alteration), post-translationally altered (for example, tumour-associated alterations in glycosylation), or idiotypic (highly polymorphic genes where a tumour cell expresses a specific "clonotype", such as in leukaemia resulting from clonal abnormalities).

TAA can derive from any protein (including glycoproteins) synthesised by the tumour cell. They may be membrane-bound, cytoplasmic, nuclear-localized, or even secreted by the tumour cells. The TAA of particular interest in the present invention are differentially expressed compared to the corresponding normal tissue and allow for a preferential recognition of tumour cells by their specific antigen-receptor.

Identification of specific TAAs is fundamental to immunotherapy approaches for the treatment of cancer. Regardless of which identification method is used, immunogenicity of each newly discovered antigen should be validated in functional assays. This is accomplished by demonstrating that T cell activation occurs only upon encountering a specific epitope, but not the corresponding control (e.g., wild-type peptide for mutant antigens) that is bound to the same MHC molecule. TAA may be identified by cDNA expression library screening, next-generation sequencing-based screening methods or immunopeptidomics (direct interrogation of the tumour immunopeptidome; all endogenous peptides that are presented by MHC molecules on the cell surface). Examples of tumour associated antigens include, but are not limited to, cancer-testes antigens such as members of the MAGE family (MAGE 1, 2, 3 etc), NY-ESO-1 and SSX-2, differentiation antigens such as tyrosinase, gplOO, PSA, Her-2 and CEA, mutated self-antigens and viral tumour antigens such as E6 and/or E7 from oncogenic HPV types. Further examples of particular tumour antigens include MART- 1, Melan-A, p97, beta-HCG, GalNAc, MAGE-1, MAGE-2, MAGE-4, MAGE-12, MUC1, MUC2, MUC3,

MUC4, MUC18, CEA, DDC, P1A, EpCam, melanoma antigen gp75, Hker 8, high molecular weight melanoma antigen, K19, Tyrl, Tyr2, members of the pMel 17 gene family, c-Met, PSM (prostate mucin antigen), PSMA (prostate specific membrane antigen), prostate secretary protein, alpha-fetoprotein, CA 125, CA 19.9, TAG-72, BRCA-1 and BRCA-2 antigen. Without limitation, the following cancers can be treated by targeting the associated provided target antigens are listed in Table 1:

Table 1 - Examples of Tumour Associated Antigens

The target antigen may also be a tumour-specific antigen (neoantigen). These antigens result mainly from genomic perturbations (such as genomic mutation, dysregulated RNA splicing, disordered post- translational modification, and integrated viral open reading frames) that occur exclusively in tumour cells and can be detected by any of the identification methods described above. Unlike TAAs, neoantigens exhibit entirely novel amino acid sequences, which are rarely shared among subjects. Thus, these provide a target antigen for a personalised cancer immunotherapy.

The target antigen may be an infection-specific antigen. The infection can be caused by any microorganism including but not limited to bacteria, viruses, parasites or fungi. Thus, the target antigen can be derived from any microorganism. The target antigen may be externally displayed on the surface of the microorganism, or may be an internal antigen that is processed and presented on the surface of infected cells, or via APCs. Thus, the target antigen may be presented as peptide fragments via MHC class I (infected cell) or class II (APCs) molecules. MHC-I molecules, typically bind peptides of 8-14 amino acids, whereas MHC-II molecules typically bind peptides of 12-25 amino acids that extend beyond the ends of their open groove. Thus, the peptide fragment of the target antigen will vary by presentation mechanism. MHC molecules present the fragment of the target antigen to the TCR.

Infection causing bacteria, include but are not limited to: Staphylococcus spp, Streptococcus spp, Enterococcus spp, Gram-positive cocci, Moraxella spp, Neisseria spp, Gram-negative cocci, Corynebacterium spp., Bacillus spp., Lactobacillus spp, Listeria spp., Gram-positive bacilli, Citrobacter spp, Escherichia coii, Klebsiella spp, Proteus spp, Serratia spp., Hafnia spp., Morganella spp., Providencia spp., Salmonella spp., Shigella spp., Yersinia spp., Enterobacteriaceae, Acinetobacter spp., Pseudomonas aeruginosa , Stenotrophomonas maltophilia , Burkholderia cepacia, Pseudomonadaceae family, Haemophilus spp., Legionella spp., Achromobacter spp., Aeromonas spp., Alcaligenes spp., Campylobacter spp., Flavobacterium spp., Helicobacter pylori , Pasteurella spp., Gram-neg bacilli, Bacteroides sppr, Clostridium spp, Propionibacterium spp., Prevotella spp., Mycoplasma spp., Actinomyces spp., and/orNocardia spp.

Infection causing fungi include but are not limited to: Candida spp., Aspergillus spp., Cryptococcus spp, Blastomyces spp.,Histoplasma, Coccidioides spp, and/or yeasts.

Infection causing viruses include but are not limited to: Adenovirus, Astrovirus, B15 Parvovirus, Chicken Pox, Chikungunya virus Coxsackievirus A, Coxsackievirus B, Coltivirus, Coronavirus (COVID- 19), Cytomegalovirus (CMV), Dengue virus, Echoviruses, Epstein Bar virus (EBV), Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Herpes simplex virus, Herpes zoster virus, Human immunodeficiency virus (HIV), Ebola virus, Influenza (flu), Lassa virus, Marburg virus, Measles virus, Mumps Virus, Norovirus, Parainfluenzavirus, Polio virus, Rabies virus, Respiratory syncytial virus (RSV), Rotavirus, Rubella virus, Severe acute respiratory virus (SARS), smallpox virus, Varicella-zoster virus, West Nile virus, Yellow fever virus, or Zika virus. The target antigen may also be present on the subject's own immune cells if an autoimmune condition is to be treated. For example, Chimeric Auto-Antibody Receptors (CAAR) T cells have been used with a target antigen found on B lymphocytes, which has led to an improvement in the treatment of lupus. In this particular instance, the T cells were provided with CAARs specific for CD19. Therefore, the target antigen in some instances may be a self-antigen on immune cells where an autoimmune condition is to be treated.

Target Cell

A cell expressing the target antigen or displaying a peptide derived from the target antigen may be referred to as a target cell. The present invention relates to the treatment of diseases/disorders by targeting cells expressing or displaying an antigen or a fragment thereof, such as diseased cells, in particular tumour or cancer cells expressing a tumour antigen (neoantigen) or tumour-associated antigen. Other diseased cells may be infected cells or inappropriately directed self-cells, such as overactive B cells in autoimmune conditions.

Thus, the in vivo modified immune cells of the present invention are directed to the target antigen or a derivative peptide thereof, thereby directing the modified immune cell to a target cell, target cell population or target tissue expressing or displaying the antigen. The modified immune cells thereby recognise the target cell, target cell population or target tissue expressing the antigen.

An advantage of using a TCR, such as a natural TCR or a minimally modified TCR, is that the antigen a TCR recognises can be an antigen that is normally present in the cytoplasm, which has been processed into peptides and displayed on the cell surface via MHC proteins.

Upon recognition of the target antigen or fragment thereof, the response elicited depends on the antigen receptor and/or immune cells used. For target antigens that are related to tumours, cancers, infections or autoimmune disorders, lysis of the target cell is the ultimate aim.

T cells modified with TCRs can recognize MHC-peptide complexes on target cells and transmit antigenstimulating signals through phosphorylation of the immune tyrosine-based activation motif (ITAM), activating the immune effects of T cells to eliminate the target cells.

NK cells may also be used in antigen receptor-based therapy. NK cells are naturally cytotoxic against cancer and virus-infected cells and are not restricted by MHC. Inserting TCR complexes into NK cell lines leads to the MHC-restricted, antigen-specific killing of target cells. NK cells genetically modified with TCRs have demonstrated the capability to recognise and kill tumour cells. CARs transduce antigen recognition events into a signalling cascade that induces immune cell effector functions, including the secretion of cytotoxic factors and pro-inflammatory cytokines. The ability of a CAR to transduce a strong activating signal to the immune cell is influenced by many factors, including the binding affinity to the antigen, the level of expression and target antigen density on the target cell.

The methods and products of the present invention therefore provide for the selective eradication/lysis of target cells expressing the target antigen, minimising adverse effects to normal, non-target cells.

Alternatively, the present invention relates to in vivo modification of immunosuppressive immune cells to mediate protection of target cells. In this instance, the target cells may be described as "wanted cells". Wanted cells can include cells undergoing autoimmune attack. Exemplary wanted cells to protect from autoimmune attack include neurons in multiple sclerosis or amylotrophic lateral sclerosis; connective tissue in rheumatoid arthritis; colon epithelium in Chrohn's disease; and the pancreas in Diabetes mellitus type 1.

Immune cells, such as regulatory T cells, can be in vivo modified to express an antigen receptor that binds to target antigens expressed by wanted cells or cells in the vicinity of wanted cells. Examples of autoimmune target antigens include glutamic acid decarboxylase 65 (GAD 65), native DNA, myelin basic protein, myelin proteolipid protein, acetylcholine receptor components, thyroglobulin, and the thyroid stimulating hormone (TSH) receptor.

Once bound to a wanted cell or in the vicinity of a wanted cell, the in vivo modified immune cell can mediate protection of the wanted cell. In an embodiment of the present invention, a regulatory T cell may be in vivo genetically modified to express an antigen receptor to target a target cell requiring protection. The target cells may express the antigen on the cell surface or may present a peptide or non-peptide fragment derived from the antigen.

The person skilled in the art would be able to select appropriate cell-specific promoters or other means to enable selective expression of the antigen receptor in in vivo modified immune cells that would facilitate protection of target cells, for example, regulatory T cells.

Thus, the in vivo modified immune cell of the present invention may be used to protect target cells from autoimmune attack or to reduce immune system activity in an area.

In Vivo Transfection and Modification

According to the methods and products of the invention, once attracted/recruited, the immune cells are genetically modified, transfected or engineered, in vivo. In vivo modification circumvents the otherwise costly procedure of harvesting, culturing and modifying immune cells ex vivo, and subsequently reintroducing the modified cells to the subject.

Thus, according to some aspects of the methods and products of the present invention, there is provided a nucleic acid comprising a transgene. The nucleic acid comprising the transgene may be DNA or RNA. The nucleic acid comprising the transgene may be a vector, preferably an expression vector. The expression vector may be a closed linear DNA, a plasmid, a single-stranded circular DNA comprising at least one hairpin section, a minicircle, a messenger RNA (mRNA), a self-amplifying RNA (saRNA) vector, a circular RNA (circRNA), a guide RNA (gRNA) or any other suitable nucleic acid format.

The transgene may encode an antigen receptor. Thus, according to the methods and products of the present invention, there may be provided a nucleic acid encoding an antigen receptor. The nucleic acid encoding the antigen receptor may be DNA or RNA. The nucleic acid encoding the antigen receptor may be a vector, preferably an expression vector. The expression vector may be a closed linear DNA, a plasmid, a minicircle, a messenger RNA (mRNA), a self-amplifying RNA (saRNA) vector, a circular RNA (circRNA), guide RNA (gRNA) or any other suitable nucleic acid format.

Thus, according to the methods and products of the present invention, there is provided a nucleic acid encoding an antigen receptor. The nucleic acid encoding the antigen receptor may be DNA or RNA. The nucleic acid encoding the antigen receptor may be a vector, preferably an expression vector. The expression vector may be a closed linear DNA, a single-stranded circular DNA comprising at least one hairpin section, a plasmid, a minicircle, a messenger RNA (mRNA), a self-amplifying RNA (saRNA) vector, a circular RNA (circRNA), guide RNA (gRNA) or any other suitable nucleic acid format.

The nucleic acid may be a naked nucleic acid, for example the nucleic acid is not encapsulated in a virus or virus-like particle. However, the nucleic acid may be present in an encapsulated form, for example in a viral vector such as AAV or lentivirus. Viral vectors are well known in the art. Such viruses may be RNA and DNA viruses with either single-stranded (ss) or double-stranded (ds) genomes. For example, viral vectors include, but are not limited to, adenoviruses, adeno-associated viruses (AAV), alphaviruses, flaviviruses, herpes simplex viruses (HSV), measles viruses, rhabdoviruses, retroviruses, lentiviruses, Newcastle disease virus (NDV), poxviruses and picornaviruses. Insert capacity and tropism can vary, therefore a viral vector may be chosen based upon the intended application.

Alternatively, the nucleic acid may be encapsulated or formulated with transfection reagents such as lipids (liposomal reagents), nanoparticles, dendrimers, polymers and the like. Such reagents would need to be suitable for in vivo use. The term "genetic modification" includes the transfection of cells with nucleic acid. As described further herein, the nucleic acid can be used to transfect immune cells. Unless otherwise specified, the terms transfect, transfected, or transfecting can be used to indicate the introduction or presence of exogenous nucleic acids or the expressed polypeptide therefrom in an immune cell.

A number of vectors are known to be capable of mediating transfer of genes to immune cells, as is known in the art.

The in vivo modified immune cell of the present invention may be stably or transiently transfected with the nucleic acid encoding the antigen receptor. In other words, the nucleic acid encoding the antigen receptor is integrated or is not integrated into the genome of the immune cell. The term "genetically modified" or "genetic modification" can be used to describe transient or permanent expression and modification.

In some embodiments of the present invention, the nucleic acid introduced in the transfection process is not integrated into the nuclear genome of the immune cell, and the nucleic acid will be diluted through each cell division. Thus, transfection and modification of the immune cells is transient. A number of transient modification methods are known in the art. For example, closed linear DNA, mRNA or self-amplifying RNA may be used to induce transient expression of the antigen receptor. For example, closed linear DNA, mRNA or self-amplifying RNA may be used to induce transient expression of the transgene.

Transient transfection may be preferable as the transfected nucleic acid is unable to replicate independently from the host cell's DNA, only persists for a shorter period of time and has an improved safety profile.

In a preferred embodiment, the modified immune cells transiently express the antigen receptor. In other words, transiently modified immune cells are generated. Following recruitment and transient in vivo genetic modification of immune cells, it may be desirable to expand the population of transiently modified immune cells expressing the antigen receptor. Thus, as discussed above, a vaccine comprising a target antigen may be provided for the stimulation, priming and/or expansion of the transiently modified immune cells. Thus, a vaccine providing the target antigen may be administered to the subject, thereby stimulating, priming and/or expanding the in vivo transiently modified immune cells.

In vivo transient transfection of T cells using mRNA nanocarriers has been demonstrated by Parayath et al. (2020), whereby the in vivo engineered T cells were able to induce antitumour responses with similar efficacy to conventional ex vivo transduced and adoptively transferred T cells (Parayath, N.N et al. (2020) In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating ? cells in vivo. Nature Communications 11 https://doi.org/10.1038/s41467-020-19486-2).

In an alternative embodiment, the nucleic acid encoding the antigen receptor may be integrated into the nuclear genome of the immune cell. Such modification may be described as permanent or stable transfection, as it is not diluted through cell division.

A variety of well-known methods may be used to introduce antigen receptors such as TCR constructs into cells such as T cells to produce stably transfected cells in vivo genetically modified to express the antigen receptors. Such methods include non-viral-based DNA transfection, transposon-based systems, and viral-based systems. Non-viral-based DNA transfection has low risk of insertional mutagenesis.

The nucleic acid encoding the antigen receptor can be directed to the immune cell nucleus by, for example, the inclusion of peptides containing microtubule-associated sequences (MTAS) and nuclear localisation signals (NLSs). A transposon-based system can be used to integrate nucleic acid encoding the antigen receptor into the genome of the immune cell. For example, the inclusion of transposons flanking the nucleic acid encoding the antigen receptor, and provision of a transposase may allow for integration of the nucleic acid into the chromosome of the immune cell. The transposase may be provided on a separate nucleic acid or polynucleotide.

Alternatively, the CRISPR/Cas9 system may be used to integrate an antigen receptor coding sequence at a specific locus. For example, CRISPR/Cas9 can be used to knockout an endogenous T cell receptor of the immune cell, while knocking in the antigen receptor and placing it under the regulatory control of the endogenous promoter that would otherwise moderate expression of an endogenous TCR.

Accordingly, in addition to the nucleic acid encoding an antigen receptor, gene editing tools such as CRISPR/Cas9 (or related) or transposon systems such as sleeping beauty or piggy bac may also be delivered to the immune cell. Such tools for genomic integration and/or editing may be delivered as protein or coding nucleic acid (DNA or RNA).

In embodiments wherein the immune cell is stably transfected with a nucleic acid encoding the antigen receptor, it may be preferable for the integration of a suicide switch or suicide gene. A suicide gene is a genetically coded element that allows for the elimination of the modified immune cell in case of unexpected toxicity or unwanted effects. Several suicide switches are known, including but not limited to, HSV-TK, iCasp9, CD20 and EGFRt.

Viral vectors may be used to deliver the nucleic acid to the immune cell. Viral-based systems include the use of g-retroviruses and lentiviral vectors. g-Retroviruses are relatively easy to produce, efficiently and permanently transduce immune cells, and have preliminarily proven safe from an integration standpoint in primary human T cells. Lentiviral vectors may be used to efficiently and permanently transduce immune cells such as T cells. Lentiviral vectors are potentially safer than retrovirus based systems.

In an alternative embodiment, the nucleic acid comprising the transgene may be integrated into the nuclear genome of the immune cell. Such modification may be described as permanent or stable transfection, as it is not diluted through cell division.

A variety of well-known methods may be used to introduce transgene such as a transgene encoding an antigen receptors into cells such as T cells to produce stably transfected cells in vivo genetically modified to express the antigen receptor. Such methods include non-viral-based DNA transfection, transposon-based systems, and viral-based systems. Non-viral-based DNA transfection has low risk of insertional mutagenesis.

The nucleic acid comprising the transgene can be directed to the immune cell nucleus by, for example, the inclusion of peptides containing microtubule-associated sequences (MTAS) and nuclear localisation signals (NLSs). A transposon-based system can be used to integrate nucleic acid comprising the transgene into the genome of the immune cell. For example, the inclusion of transposons flanking the nucleic acid comprising the transgene, and provision of a transposase may allow for integration of the nucleic acid into the chromosome of the immune cell. The transposase may be provided on a separate nucleic acid or polynucleotide.

Alternatively, the CRISPR/Cas9 system may be used to integrate a transgene, such as an antigen receptor coding sequence at a specific locus. For example, CRISPR/Cas9 can be used to knockout an endogenous T cell receptor of the immune cell, while knocking in the antigen receptor and placing it under the regulatory control of the endogenous promoter that would otherwise moderate expression of an endogenous TCR.

Accordingly, in addition to the nucleic acid comprising a transgene, gene editing tools such as CRISPR/Cas9 (or related) or transposon systems such as sleeping beauty or piggy bac may also be delivered to the immune cell. Such tools for genomic integration and/or editing may be delivered as protein or coding nucleic acid (DNA or RNA).

In embodiments wherein the immune cell is stably transfected with a nucleic acid comprising the transgene, it may be preferable for the integration of a suicide switch or suicide gene. A suicide gene is a genetically coded element that allows for the elimination of the modified immune cell in case of unexpected toxicity or unwanted effects. Several suicide switches are known, including but not limited to, HSV-TK, iCasp9, CD20 and EGFRt.

Administration

The products or methods of the invention are administered to a subject.

Administration may be via any suitable route, such as but not limited to: intradermal, intramuscular, transmucosal, sub-cutaneous, inhalation, sub-lingual, intra-lymph node or intratumoural administration. It may be preferred that administration is intramuscular, via an appropriate injection. For example, the administration may be to a particular muscle via intramuscular injection. The location of the intramuscular injection may be the deltoid muscle in the arm or the anterolateral aspect of the thigh. The locality in this instance is the area of muscle into which the component is injected.

The immune cell recruiting factor is preferably administered to the subject. This factor is preferably administered prior to any of the other step of the method, or prior to other components of the product. This is step (a) of the method, or component (i) of the product. The immune cell recruiting factor is preferably administered to a location of said subject via any suitable means. Preferably, the immune cell recruiting factor is administered via intra-muscular injection to a locality of a muscle.

Administration of the various components/steps may be simultaneous, separate or sequential. Administration may be separate or sequential. Administration of the immune cell recruiting factor may occur before the administration of the nucleic acid encoding an antigen receptor. It is preferred that certain components or steps are administered to the same locality, such as for example, the same locality of a muscle.

It is preferred that the immune cell recruiting factor is administered before the administration of the nucleic acid encoding an antigen receptor. The interval between step (a) and (b) is dependent on the timing of immune effector/immune memory cell recruitment. For example, for T cells this is anticipated to be within 1-10 days. For antigen-presenting cells this is anticipated to be within 1-72 hours but could be longer dependent on the immune cell recruiting factor. For example, the adjuvant MF59 recruits immune cells to the lymph node within 3 hours and up to 11 days after administration. The person skilled in the art would be able to select a suitable interval between step (a) and (b) to allow for recruitment of immune cells, for example, T cells.

The interval between these administrations may be selected as appropriate, but may be in the order of 1 day to 14 days, 2 to 10 days, 3 to 8 days, or any one or more of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 days apart. The interval between these administrations may be within about 1 hour to about 72 hours (e.g. within about 1, 2, 3, 6, 12, 24, 48, or 72 hours), within about 1 day to about 4 weeks (e.g. within about 1, 2 or 3 or 4 weeks), within about 1 week to about 3 weeks of each other or any range there between. Any interval or period between these are covered. Without wishing to be bound by theory, the interval between the administration permits the attraction of immune cells to the locality of the administration.

It is preferred that the immune cell recruiting factor is administered before the administration of the nucleic acid comprising a transgene. The interval between step (a) and (b) is dependent on the timing of immune cell recruitment, for example immune effector or immune memory cell recruitment.

The immune cell recruitment factor may be administered to the subject through intramuscular injection. In a preferred embodiment, intramuscular injection and electroporation (IMEP) may be used. Electroporation enhances cellular uptake of exogenous molecules such as DNA, RNA, proteins or chemicals, thereby enabling increased levels of gene transfer and expression.

The nucleic acid encoding an antigen receptor may be administered to the subject through intramuscular injection. In a preferred embodiment, intramuscular injection and electroporation (IMEP) may be used.

The nucleic acid comprising a transgene may be administered to the subject through intramuscular injection. In a preferred embodiment, intramuscular injection and electroporation (IMEP) may be used.

Vaccine

Following recruitment and in vivo genetic modification of immune cells, it is desirable to expand the antigen receptor-engineered population of immune cells. In vivo expansion of in vivo modified immune cells provides for a larger population of immune cells carrying an antigen receptor recognising the target antigen. Therefore, provision of the target antigen enables the selective in vivo expansion of in vivo modified immune cells. Thus, the invention further relates to provision of the target antigen for the stimulation, priming and/or expansion of the in vivo genetically modified immune cells. Immune cells stimulated, primed and/or expanded in the subject are able to recognise and eradicate target cells expressing the target antigen.

The target antigen may be provided to a subject by administering said target antigen in the form of a vaccine. The vaccine may take any appropriate format, including cells expressing the antigen.

The vaccine may provide the target antigen or a variant thereof (e.g. a peptide or protein comprising an epitope of the target antigen), or may provide a nucleic acid encoding the target antigen or variant thereof. The vaccine may be a polypeptide vaccine, DNA vaccine, RNA vaccine or viral vector vaccine.

In an alternative embodiment, the target antigen may be provided by a cell expressing the target antigen. The vaccine may provide the target antigen or a variant thereof (e.g. a peptide or protein comprising an epitope of the target antigen), or may provide a nucleic acid encoding the target antigen or variant thereof. The vaccine may be any suitable vaccine, such as but not limited to any one or more of: inactivated vaccines, live-attenuated vaccines, subunit vaccines, recombinant vaccines, polysaccharide vaccines, conjugate vaccines, toxoid vaccines, a polypeptide vaccine, DNA vaccine, RNA vaccine or viral vector vaccine. The vaccine may include the target antigen presented on a carrier, such as a nano-cage or virus-like particle (VLP). Any appropriate presentation of the antigen to the immune cell is covered with the term "vaccine".

Thus, a vaccine providing the target antigen may be administered to the subject, thereby stimulating, priming and/or expanding the in vivo genetically modified immune cells. The vaccine may be administered by any suitable route, such as those described earlier. The vaccine may be administered to any suitable locality, it is not necessary that the locality is the same as the earlier administrations.

It may be preferred that the vaccine is administered to the subject following the administration of the nucleic acid encoding the antigen receptor. It may be preferred that the vaccine is administered after the administration of the nucleic acid encoding an antigen receptor. The interval between these administrations may be selected as appropriate, but may be in the order of 1 day to 50 days, 2 to 45 days, 3 to 40 days, or any one or more of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 days apart. Any interval or period between these are covered. Without wishing to be bound by theory, the interval between the administrations permits the transfected immune cells to express the antigen receptor, such that they are ready to be stimulated by the vaccine. Moreover, the vaccine may be administered so long as the in vivo modified immune cells persist, thereby expanding the cell population. In the case of modified effector and or modified memory cells, such as memory T cells or memory NK cells, these cells may persist for longer in the body - thus leaving a resident population which can be stimulated again by vaccination.

The vaccine may be formulated appropriately, and may include an adjuvant to further stimulate the immune response to the antigen.

In any embodiment wherein the target antigen is a tumour-associated antigen and the disease a cancer, a vaccine may be administered, the vaccine providing the tumour-associated antigen or a fragment thereof recognised by the antigen receptor, thereby stimulating, priming and/or expanding the modified immune cells. Wherein the disease is a cancer, the vaccine providing the tumour- associated antigen or a fragment thereof may be referred to as a "cancer vaccine".

Additionally or alternatively to a vaccine comprising a target antigen, a vaccine comprising the immune cell recruiting factor may be administered to the subject, thereby stimulating, priming and/or expanding the in vivo genetically modified immune cells. Wherein the immune cell recruiting factor is an antigen or epitope thereof, the in vivo genetically modified immune cells will express an antigen receptor that recognises the immune cell recruiting factor, and also the antigen receptor encoded by the nucleic acid. Thereby, the population of in vivo genetically modified immune cells can be stimulated, primed and/or expanded by the immune cell recruiting factor and/or the target antigen. The vaccine providing the immune cell recruiting factor may be targeted to secondary lymphoid organs.

Dosing

As used herein, the term "dosing regimen" refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. For example, the immune cell recruiting factor and/or nucleic acid encoding an antigen receptor and/or the vaccine may be administered one or more times. Alternatively, the immune cell recruiting factor and/or nucleic acid comprising a transgene may be administered one or more times.

Without wishing to be bound by theory, the inventors postulate that following in vivo expansion using a vaccine providing the target antigen, the in vivo modified immune cells will be active for several weeks. A further dose of the vaccine would allow for further immune cell stimulation, priming and/or expansion in the subject, thereby enabling further eradication of target cells expressing the target antigen.

Multiple rounds of administration of the immune cell recruiting factor and/or nucleic acid comprising a transgene may be used until the therapeutic objective, such as eradication of the target cell or target tissue is suitably achieved. Any appropriate period of time between doses may be selected. Administration of the immune cell recruiting factor and/or nucleic acid encoding a transgene may be repeated independently of one another.

Multiple rounds of administration of the immune cell recruiting factor and/or nucleic acid encoding an antigen receptor and/or the vaccine may be used until the target cell or target tissue is suitably eradicated. Any appropriate period of time between doses may be selected. Administration of the immune cell recruiting factor and/or nucleic acid encoding an antigen receptor and/or the vaccine may be repeated independently of one another.

In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).

A variety of methods are known in the art to measure human immune response, for example ELISpot can be used to detect immune cell recruitment following administration of the immune cell recruiting factor.

Immune Modulation

The present invention may be combined with any known ways of enhancing or modulating proliferation and or/activity of immune cells in an immunotherapy.

In some embodiments of the present invention, the immune cell recruiting factor, nucleic acid encoding an antigen receptor and/or vaccine, may be administered as part of a combination treatment, which can be administered simultaneously with or sequentially to, or in any order with, another therapeutic intervention. The use of the term "in combination" does not restrict the order in which prophylactic and/or therapeutic agents are administered to a subject with a disorder. The immune cell recruiting factor, nucleic acid encoding an antigen receptor and/or vaccine and additional therapeutic agent may be administered within time intervals that allow that the therapeutic agents show a cooperative e.g., synergistic, effect. In some embodiments, the immune cell recruiting factor, nucleic acid encoding an antigen receptor and/or vaccine, are co-administered with another therapy sufficiently close in time such that the in vivo modified cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the immune cell recruiting factor, nucleic acid encoding an antigen receptor and/or vaccine, are administered prior to the one or more additional therapeutic agents. In some embodiments, the immune cell recruiting factor, nucleic acid encoding an antigen receptor and/or vaccine, are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents may be administered prior to or after the administration of any one or more of the immune cell recruiting factor, the nucleic acid encoding an antigen receptor and/or vaccine. For example, the one or more additional therapeutic agents may be administered after the immune cell recruiting factor, but prior to the nucleic acid encoding an antigen receptor and/or the vaccine, and vice versa.

In some embodiments of the present invention, the immune cell recruiting factor and the nucleic acid comprising a transgene, may be administered as part of a combination treatment, which can be administered simultaneously with or sequentially to, or in any order with, another therapeutic intervention. The use of the term "in combination" does not restrict the order in which prophylactic and/or therapeutic agents are administered to a subject with a disorder. The immune cell recruiting factor, nucleic acid comprising a transgene and additional therapeutic agent may be administered within time intervals that allow that the therapeutic agents show a cooperative e.g., synergistic, effect. In some embodiments, the immune cell recruiting factor and nucleic acid comprising a transgene, are co-administered with another therapy sufficiently close in time such that the in vivo modified cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the immune cell recruiting factor and nucleic acid comprising a transgene, are administered prior to the one or more additional therapeutic agents. In some embodiments, the immune cell recruiting factor, nucleic acid comprising a transgene, are administered after the one or more additional therapeutic agents. In some embodiments, the one or more additional therapeutic agents may be administered prior to or after the administration of any one or more of the immune cell recruiting factor and the nucleic acid comprising a transgene. For example, the one or more additional therapeutic agents may be administered after the immune cell recruiting factor, but prior to the nucleic acid comprising a transgene, and vice versa.

In some embodiments of the present invention, immune checkpoint inhibitors are used in combination with other therapeutic agents described herein. As used herein, "immune checkpoint" refers to co-stimulatory and inhibitory signals that regulate the amplitude and quality of T cell receptor recognition of an antigen. In certain embodiments, the immune checkpoint is an inhibitory signal. In certain embodiments, the inhibitory signal is the interaction between PD-1 and PD-L1. In certain embodiments, the inhibitory signal is the interaction between CTLA-4 and CD80 or CD86 to displace CD28 binding. In certain embodiments the inhibitory signal is the interaction between LAG3 and MHC class II molecules. In certain embodiments, the inhibitory signal is the interaction between TIM3 and galectin 9.

As used herein, "immune checkpoint inhibitor" refers to a molecule that totally or partially reduces, inhibits, interferes with or modulates one or more checkpoint proteins. In certain embodiments, the immune checkpoint inhibitor prevents inhibitory signals associated with the immune checkpoint. In certain embodiments, the immune checkpoint inhibitor is an antibody, or fragment thereof that disrupts inhibitory signalling associated with the immune checkpoint. In certain embodiments, the immune checkpoint inhibitor is a small molecule that disrupts inhibitory signalling. In some embodiments, the immune checkpoint inhibitor suitable for use in the methods disclosed herein, is an antagonist of inhibitory signals, e.g., an antibody which targets, for example, PD-1 , PD-L1 , CTLA-4, LAG3, B7-H3, B7-H4, orTIM3. In some embodiments, the immune checkpoint inhibitor is an antibody, fragment thereof, or antibody mimic, that prevents the interaction between checkpoint blocker proteins, e.g., an antibody, or fragment thereof, that prevents the interaction between PD-1 and PD- Ll. In some embodiments, the immune checkpoint inhibitor is an antibody, or fragment thereof, that prevents the interaction between CTLA-4 and CD80 or CD86. In certain embodiments, the immune checkpoint inhibitor is an antibody, or fragment thereof, that prevents the interaction between LAG3 and its ligands, orTIM-3 and its ligands. The checkpoint inhibitor may also be in the form of the soluble form of the molecules (or variants thereof) themselves, e.g., a soluble PD-L1 or PD-L1 fusion. In some embodiments, the immune checkpoint inhibitor is a small molecule that disrupts or inhibits signalling from an inhibitory immunoregulator. In some embodiments, the nucleic acid comprising a transgene encodes a checkpoint inhibitor. In other words, the in vivo modified immune cell is engineered to express a checkpoint inhibitor.

In some embodiments of the present invention, cytokines are used in combination with other therapeutic agents described herein. In some embodiments, the methods described herein may comprise providing to a subject one or more cytokines, for example, by administering to the subject the one or more cytokines, a polynucleotide encoding the one or more cytokines or a host cell expressing the one or more cytokines. In some embodiments, the nucleic acid comprising a transgene encodes a cytokine. In other words, the in vivo modified immune cell is engineered to express a cytokine. Immunosuppressive cells play an active role in immunological self-tolerance, and thus suppress effective tumour immunity and protect cancer cells from immune attack. Immunosuppressive cells include, but are not limited to, regulatory ? cells, myeloid-derive suppressive cells (MDSCs), tumour- associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs). Thus, in some embodiments of the present invention, the method may further comprise administration of an agent for depletion or immunomodulation of immunosuppressive cells. The term "cytokine" as used herein includes naturally occurring cytokines and functional variants thereof (including fragments of the naturally occurring cytokines and variants thereof).

Chemotherapy

In some embodiments, additional treatments may be administered to a subject in combination with the methods and treatments described herein. Such additional treatments includes classical cancer therapy, e.g., radiation therapy, surgery, hyperthermia therapy and/or chemotherapy.

Traditional chemotherapeutic agents are cytotoxic by means of interfering with cell division (mitosis) but cancer cells vary widely in their susceptibility to these agents. Chemotherapeutic agents include alkylating agents, antimetabolites, anti-microtubule agents, topoisomerase inhibitors, and cytotoxic antibiotics.

In some embodiments of the present invention, the methods further include a lymphodepleting therapy, such as administration of a chemotherapeutic agent. In some embodiments, the methods do not include a lymphodepleting therapy.

Treatment

The present disclosure provides compositions and methods that can rapidly and selectively direct immune cells within the body to achieve therapeutic objectives. In particular embodiments, the compositions and methods modify cells of the immune system, such as T-cells, NKT cells or NK cells, to target and destroy target cells. The methods and products described herein are, in particular, useful for the treatment or therapy of diseases characterised by diseased cells expressing a target antigen.

Without wishing to be bound by theory, the inventors hypothesise that the compositions and methods of the present invention provide for increased or prolonged expansion and/or persistence of modified immune cells in the subject as compared to a method in which the immune cell recruiting factor and/or the vaccine is not administered.

The therapeutic objective of the present invention may be to induce an immune response in the subject. The immune response may be a T cell-mediated immune response. The immune response may be an immune response to a target cell population or target tissue expressing a target antigen. The target cell population or target tissue may be cancer cells or cancer tissue. The cancer cells or cancer tissue may be a solid cancer. The target cells may be infected cells. The target cells may be overactive immune cells of the subject, in relation to autoimmune diseases.

The subject may have a haematological cancer, such as a leukaemia or lymphoma. The leukemia may be chronic lymphocytic leukemia (CLL), acute lymphocytic leukemia (ALL), or chronic myelogenous leukemia (CML). The lymphoma may be mantle cell lymphoma, non-Hodgkin's lymphoma or Hodgkin's lymphoma. The hematological cancer may be multiple myeloma.

Alternatively, the cancer may be a carcinoma comprising oral and pharynx cancer (tongue, mouth, pharynx, head and neck), digestive system cancers (oesophagus, stomach, small intestine, colon, rectum, anus, liver, intrahepatic bile duct, gallbladder, pancreas), respiratory system cancers (larynx, lung and bronchus), bones and joint cancers, soft tissue cancers, skin cancers (melanoma, basal and squamous cell carcinoma), paediatric tumours (neuroblastoma, rhabdomyosarcoma, osteosarcoma, Ewing's sarcoma), tumours of the central nervous system (brain, astrocytoma, glioblastoma, glioma), and cancers of the breast, the genital system (uterine cervix, uterine corpus, ovary, vulva, vagina, prostate, testis, penis, endometrium), the urinary system (urinary bladder, kidney and renal pelvis, ureter), the eye and orbit, the endocrine system (thyroid), and the brain and other nervous system, or any combination thereof.

In the context of cancers, therapeutically effective amounts of the modified immune cell can decrease the number of tumour cells, decrease the number of metastases, decrease tumour volume, increase life expectancy, induce apoptosis of cancer cells, induce cancer cell death, induce chemo- or radiosensitivity in cancer cells, inhibit angiogenesis near cancer cells, inhibit cancer cell proliferation, inhibit tumour growth, prevent metastasis, prolong a subject's life, reduce cancer-associated pain, reduce the number of metastases, and/or reduce relapse or re-occurrence of the cancer following treatment.

The subject may have an infection may be caused by any infectious agent. The infection may be fungal, bacterial or viral. Examples of infectious organisms have been described herein.

The subject may have an autoimmune disease caused by an overactive immune system response to self-antigen. Autoimmune diseases are characterised by an abnormal immune response involving either cells or antibodies, that are directed against normal tissues.

Autoimmune diseases in mammals can generally be classified in one of two different categories: cell- mediated disease (i.e. T-cell) or antibody-mediated disorders. Cell-mediated autoimmune diseases include but are not limited to multiple sclerosis, rheumatoid arthritis, autoimmune thyroiditis, diabetes mellitus (Juvenile onset diabetes) and autoimmune uveoretinitis. Antibody-mediated autoimmune disorders include myasthenia gravis and systemic lupus erythematosus (or SLE).

Autoimmune diseases include, without limitation, diabetes mellitus, multiple sclerosis, premature ovarian failure, scleroderm, Sjogren's disease, lupus, vilelego, alopecia, polyglandular failure, Grave's disease, hypothyroidism, polymyosititis, pempligus, Chron's disease, colititis, autoimmune hepatitis, hypopituitarism, myocardititis, Addison's disease, autoimmune skin diseases, uveititis, prericious anemia, hypoparathyroidism, and rheumatoid arthritis.

The invention will now be described with reference to non-limiting examples.

EXAMPLES

Example 1 - Pre-conditioning time-course in Balb/c mice

Figure 1 shows representative images of immune cell infiltration in the muscle where closed linear DNA (dbDNA) encoding Cal09 hemagglutinin (HA) of Influenza A (dbCalO9-HA) was used as an immune cell recruiting factor. Five Balb/c mice per group were administered dbCalO9-HA by intramuscular injection and electroporation (IMEP) at day 0 and then culled at different time points after immunisation (day 2-4-7-10-14). Morphological analysis of Haematoxylin & Eosin (H&E) stained muscle sections shows the presence of lymphocyte infiltrates (indicated in Figure 1 with the circle "C") at earlier time points from day 2 to day 4 while during later time points (day 7 to day 14) the lymphocyte infiltration was progressively reduced until being completely absent. The necrotic tissue is highlighted by the circle "B", the normal tissue is shown within the circle "A". The presence of histiocytes was also observed in the samples collected from day 2 until day 10, but not at day 14. Table 3 shows the semi-quantitative analysis of necrotic tissue and lymphocyte infiltration post immunisation. Figure 2 shows antigen-specific responses to dbCalO9-HA measured in splenocytes by intracellular cytokine staining (ICS) for INFy and TNFa. The CD4 and CD8 response in the spleen increased considerably at day 10 and 14 (Fig. 2). Thus, the present example demonstrates that an immune cell recruiting factor, such as CalO9-HA, allows for recruitment and localisation of immune cells. Table 3 - Semi quantitative analysis of necrotic tissue and lymphocyte infiltration post-immunisation

Example 2 - GFP expression in infiltrating immune cells in the muscle

Five Balb/c mice were administered with dbCalO9-HA (closed linear DNA (dbDNA) encoding Cal09 hemagglutinin (HA) of Influenza A) as an immune cell recruiting factor in one limb by intramuscular injection followed by intramuscular injection and electroporation (IMEP). Two days postimmunisation, four mice were transfected by electroporation at the site of injection with a nucleic acid encoding GFP (dbCAG-Lux-2A-eGFP). Figure 3 show co-localisation of GFP and T-cells markers, CD3 and CD8, in the treated mice but not in the control (Fig. 4B). The arrows indicate single stained T- cells and the circles in the overlayed images show double stained cells confirming the T cells (CD3- positive cells) were transfected with GFP. A more in depth analysis was performed by co-staining immune cells for GFP with the specific markers, CD3, CD8 and NKp46. Figure 4 shows that not only CD3-positive T-cells and CD8-positive T-cells, but also NK cells were successfully transfected with dbCAG-Lux-2A-eGFP by IMEP at the site of injection (NKp46-positive cells). Double stained cells are shown with the circle while single marker positive cells are indicated with the arrows (Fig. 4A). Thus, the present example demonstrates that recruitment of immune cells by an immune cell recruiting factor leads to increased in vivo modification of immune cells, compared to administration of the nucleic acid encoding the transgene without the recruitment step.

Example 3 -Transfected T cells can recirculate in the blood back to the spleen

Transfection of immune cells was evaluated in preconditioned C57BL/6 mice after prime and boost immunisation with dbDNA encoding for CalO9-HA by intramuscular injection and electroporation (IMEP) delivery. The immune cells were then transfected in vivo two days after boost vaccination, when most of the immune cells were recruited at the site of injection (data based on the Preconditioning time-course in Balb/c mice study, Table 3 and Fig. 3 and Fig. 4). The db-TE eGFP fluorescent reporter was transfected into the immune cells via three different delivery systems: intramuscular injection followed by electroporation (IMEP), formulated using biodegradable polymeric nanoparticles developed by 20Med Therapeutics, and intramuscular injection of DNA formulated using in vivo JetPEI® (Polyplus). The mice were culled 2 days post-transfection with db-TE- eGFP and the immune cells isolated from the spleen for FACS analysis. Fig. 5 shows that the immune cells, transfected at the site of injection recirculated back to the blood and into the spleen. GFP positive immune cells were detected using well-established T cell markers, CD3, CD4 and CD8 (Fig. 5).

Example 4 -Transfection of effector/memory T cells

Figure 6 shows that following the recruitment and activation of T cells at the site of immunisation, most of the immune cells transfected with GFP were effector T cells and memory T cells. Accordingly, 80% of the CD8+ cells transfected with GFP had a memory phenotype (CD44+ and CD62L+), while the majority of naive cells were GFP negative.

Example 5 - Expansion of Modified T Cells

Balb/c mice will be primed using dbCalO9-HA as described in Example 1 and 2, to recruit and activate immune cells. Two days post administration of dbCalO9-HA, mice will be transfected by electroporation at the site of injection with a nucleic acid encoding a T Cell Receptor (dbDNA-LNGFR- OT1-TCR). Three days or seven days post transfection, mice will be vaccinated with pDNA OVA (ovalbumin) by intramuscular injection followed by intramuscular injection and electroporation (IM EP) to provide the target antigen for the TCR. Recruitment, transfection and expansion will be evaluated using FACS staining (splenocytes (CD3-CD4-CD8-CD44-CD62L-LNGFR and/or pentamer/tetramer staining), and intracellular cytokine staining (ICS).

Clauses

A. A product comprising an immune cell recruiting factor and a nucleic acid comprising a transgene as a combined preparation for simultaneous, separate or sequential use in a method of therapy or treatment in a subject.

B. The product for use according to clause A, wherein said method comprises:

(a) administering the immune cell recruiting factor; and

(b) administering the nucleic acid comprising a transgene.

C. The product for use according to clause B, wherein the immune cell recruiting factor and the nucleic acid comprising a transgene are administered in the same locality.

D. The product for use according to any preceding clause, wherein the immune cell is a T lymphocyte, B lymphocyte, macrophage, neutrophil, mast cell, basophil, eosinophil, monocyte or dendritic cell.

E. The product for use according to any preceding clause, wherein the immune cell is a T lymphocyte and is an immune effector cell or an immune memory cell.

F. The product for use according to any preceding clause wherein the immune cell is a cytotoxic immune cell.

G. The product for use according to clause F wherein the cytotoxic immune cell is a cytotoxic lymphocyte.

H. The product for use according to clause G wherein the cytotoxic lymphocyte is a cytotoxic T cell, natural killer (NK) cell or NKT cell.

I. The product for use according to clause H wherein the cytotoxic T cell is a CD8+ T cell. J. The product for use according to clause I wherein the CD8+ T cell is a CD8+ effector T cell or a CD8+ memory T cell.

K. The product for use according to claim clause J wherein the CD8+ memory T cell is a CD8+ central memory T cell, CD8+ effector memory T cell, CD8+ tissue-resident memory T cell or a CD8+ circulating memory T cell.

L. The product for use according to any preceding clause, wherein the immune cell recruiting factor is a vaccine, an antigen, an epitope, a cytokine, chemokine or a microbial product or toxin.

M. The product for use according to any preceding clause, wherein the nucleic acid is DNA or RNA.

N. The product for use according to any preceding clause, wherein the nucleic acid is a vector, preferably an expression vector.

O. The product for use according to clause N, wherein the expression vector is a closed linear DNA, a plasmid, a minicircle, a messenger RNA (mRNA), a self-amplifying RNA (saRNA) vector, a viral vector or a single-stranded circular DNA comprising at least one hairpin section.

P. The product for use according to any preceding clause, wherein the transgene encodes a therapeutic payload.

Q. The product for use according to any preceding clause, wherein the transgene encodes a therapeutic protein, onco-suppressor protein, differentiation factor, or protein with a gene activity modulation function.

R. The product for use according to any preceding clause, wherein the transgene encodes an antigen receptor.

S. The product for use according to claim R, wherein the antigen receptor binds to, or preferably is specific to a target antigen or fragment thereof.

T. The product for use according to clause R or S, wherein the antigen receptor is a T-cell receptor (TCR) or chimeric antigen receptor (CAR).

U. The product for use according to any one of claims R to T, wherein the target antigen is a selfantigen, fungal antigen, bacterial antigen or a viral antigen.

V. The product for use according to any one of claims R to U, wherein the target antigen is a tumour antigen, neoantigen or tumour-associated antigen.

W. The product for use according to any preceding clause wherein the nucleic acid is provided with a transfection reagent. X. The product for use of according to any one of clauses B to W wherein accompanying or following administration of the immune cell recruiting factor and/or the nucleic acid an electric pulse is applied to the site of administration.

Y. The product for use according to any one of clauses B to X, wherein for any one or more of steps (a) and (b) the administration is intradermal, intramuscular, transmucosal, sub-cutaneous, inhalation, sub-lingual, intratumoural or intra-lymph node administration.

Z. The product for use according to any preceding one of clauses B to Y, wherein step (a) and (b) are carried out separately or sequentially.

AA. The product for use according to any preceding clause, wherein the immune cell is a cytotoxic T cell, the immune cell recruiting factor is an influenza vaccine, the transgene encodes a TCR and the target antigen is a tumour-associated antigen.

BB. The product for use according to any one of clauses B to AA, wherein one or more of steps (a) and (b) are repeated.

CC. The product for use according to any one of clauses B to BB wherein steps (a) and (b) are performed separately, preferably wherein step (b) is carried out within 1 hour to 720 hours of step (a).

DD.The product for use according to any preceding clause, wherein said method is a method for treating cancer in a subject.

EE. The product for use according to any preceding clause, wherein said method is a method for treating chronic infection in a subject.

FF. The product for use according to any preceding clause, wherein the method is a method of eliciting an immune response to a target antigen in a subject.

GG. A kit comprising:

(a) an immune cell recruiting factor; and

(b) a nucleic acid encoding a transgene.

Claims

1. An immune cell recruiting factor and a nucleic acid encoding an antigen receptor for use in a method of therapy or treatment in a subject, the method comprising:

(a) administering the immune cell recruiting factor; and

(b) administering the nucleic acid encoding an antigen receptor.

2. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to claim 1, wherein the immune cell recruiting factor and the nucleic acid encoding an antigen receptor are administered in the same locality.

3. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim, wherein the immune cell is an immune effector cell or an immune memory cell.

4. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim wherein the immune cell is a cytotoxic immune cell, which optionally can be any one or more of:

(i) a cytotoxic lymphocyte;

(ii) a cytotoxic T cell, natural killer (NK) cell or NKT cell;

(iii) a CD8+ T cell;

(iv) a CD8+ effector T cell or a CD8+ memory T cell; and/or

(v) a CD8+ central memory T cell, CD8+ effector memory T cell, CD8+ tissue-resident memory T cell or a CD8+ circulating memory T cell.

5. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim, wherein the immune cell recruiting factor is a vaccine, an antigen, an epitope, a cytokine, chemokine or a microbial product or toxin.

6. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim, wherein the nucleic acid encoding the antigen receptor is DNA or RNA.

7. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim, wherein the nucleic acid encoding the antigen receptor is a vector, preferably an expression vector, which optionally can be any one or more of a closed linear DNA, a plasmid, a minicircle, a messenger RNA (mRNA), a self-amplifying RNA (saRNA) vector, a viral vector or a single-stranded circular DNA comprising at least one hairpin section.

8. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to claim 7, wherein the expression vector is a transient expression vector.

9. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim, wherein the antigen receptor binds to, or preferably is specific to a target antigen or fragment thereof.

10. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim, wherein the antigen receptor is a T-cell receptor (TCR) or chimeric antigen receptor (CAR).

11. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim, wherein the target antigen is a self-antigen, fungal antigen, bacterial antigen, viral antigen, a tumour antigen, neoantigen or tumour-associated antigen.

12. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim wherein the nucleic acid is provided with a transfection reagent.

13. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim wherein accompanying or following administration of the immune cell recruiting factor and/or the nucleic acid an electric pulse is applied to the site of administration.

14. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim, further comprising a vaccine comprising a target antigen.

15. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim, wherein said method further comprises step (c):

(c) administering a vaccine comprising a target antigen.

16. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any one of claims 14 or 15, wherein the vaccine is an inactivated vaccine, live-attenuated vaccine, subunit vaccine, recombinant vaccine, polysaccharide vaccine, conjugate vaccine, toxoid vaccine, polypeptide vaccine, DNA vaccine, RNA vaccine or viral vector vaccine.

17. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim, wherein for any one or more of steps (a) (b) and/or (c) the administration is intradermal, intramuscular, transmucosal, sub-cutaneous, inhalation, sub-lingual, intratumoural or intra-lymph node administration.

18. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim, wherein step (a), (b) and/or (c) are carried out separately or sequentially.

19. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim, wherein the immune cell is a cytotoxic T cell, the immune cell recruiting factor is an influenza vaccine, the antigen receptor is a TCR and the target antigen is a tumour- associated antigen.

20. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim, wherein one or more of steps (a), (b) and/or (c) are repeated.

21. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any one of claims 15 to 20 wherein steps (a), (b) and (c) are performed separately, preferably wherein step (b) is carried out within 1 to 30 days of step (a), and step (c) is carried out within 1 to 210 days of step (b).

22. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim, wherein said method is a method for treating cancer in a subject.

23. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim, wherein said method is a method for treating chronic infection in a subject.

24. The immune cell recruiting factor and nucleic acid encoding an antigen receptor according to any preceding claim, wherein the method is a method of eliciting an immune response to a target antigen in a subject.

25. An immune cell recruiting factor, a nucleic acid encoding an antigen receptor and a vaccine for use in a method of therapy or treatment in a subject, the method comprising:

(a) administering the immune cell recruiting factor;

(b) administering the nucleic acid encoding an antigen receptor; and

(c) administering a vaccine comprising a target antigen.

26. A method for the treatment of a cancer, a chronic infection, or eliciting an immune response to a target antigen in a subject, comprising:

(a) administering an immune cell recruiting factor; and

(b) administering a nucleic acid encoding an antigen receptor; and optionally,

(c) administering a vaccine comprising a target antigen.

27. A kit comprising:

(a) an immune cell recruiting factor; and

(b) a nucleic acid encoding an antigen receptor; and optionally,

(c) a vaccine comprising a target antigen.