Antiviral treatment

Abstract

The invention relates to a composition comprising a plurality of chromosome-free bacterial minicells and / or simple cells, wherein the minicells and / or simple cells comprise surface-displayed antigen-binding molecules that target at least one viral protein or epitope; and associated disease treatments.

Description

[0001] ANTIVIRAL TREATMENT

[0002] The present invention relates to bacteria derived minicells (mini-SimCells) and simple cells (SimCells), and their use in treating viral infections, and associated compositions, products and methods.

[0003] Viral infections carry significant burden in terms of both mortality and healthcare expenditure worldwide. Many viral infections have no cure, and although treatments (such as vaccines) have been designed for some viral infections, offering some degree of mitigation of the most severe impact, the ongoing emergence of variants and breakthrough infections for most viruses highlights the critical need for innovative strategies to address these pathogens. This pinpoints the urgent requirement for the development of effective prophylactics and therapeutic interventions to combat not only current pandemics but also future outbreaks. Respiratory tract infections (e.g flu), parasite-spread infections (e.g. West Nile fever), and sexually- and / or injection-transmitted diseases (e.g. AIDS, hepatitis C) are examples of diseases that can be caused by viral agents. One of the downsides of traditional antiviral approaches based on (small-molecule) inhibitors of viral enzymes is that viruses are prone to genetic drifting, which drastically reduces the utility lifespan of a given inhibitor. One possible strategy to target viruses is monoclonal antibodies, which may be able to recognise epitopes larger than traditional enzyme -inhibitor binding pockets.

[0004] A recently-arisen virus that results in respiratory tract infections is SARS-CoV-2, the aetiological agent of COVID-19. A number of monoclonal antibodies, such as sotrovimab, bebtelovimab and ronapreve, have been approved for effective neutralise SARS-CoV-2 and used to treat the infection. The cost of the antibody drugs ranges from £1,000 to £2,000 per treatment course. Unlike simpler, chemical drugs, monoclonal antibodies are complex and expensive to produce. Nanobodies™ (Nbs), which are the variable domains (VHH) of heavy chain-only antibodies derived from camelids, exhibit greater stability and reduced immunogenicity compared to full-length antibody sequences, making them more suitable for human administration. At only 15 kDa, Nbs offer enhanced potency, better recognition capabilities, and the ability to bind to epitopes that are inaccessible to traditional antibodies. Their capacity for engineering into multivalent forms allows for the simultaneous binding to multiple target sites, significantly boosting their effectiveness in neutralizing viruses and mitigating the riskof mutational escape by variants. Nbs can be tailored to specifically target and neutralize active sites of viruses, offering more effective neutralization than conventional antibodies.

[0005] Despite these advances in antibody technologies, there is a need to further develop alternative or improved antiviral therapeutics that are easier and cheaper to produce than antibodies, while retaining specificity and efficacy in targeting and neutralising the desired viral particles.

[0006] According to a first aspect, there is provided a composition comprising a plurality of chromosome-free bacterial minicells and / or simple cells, wherein the minicells and / or simple cells comprise surface-displayed antigen-binding molecules that target at least one viral protein or viral epitope.

[0007] The inventors have demonstrated that chromosome-free bacterial minicells (herein termed “mini-SimCells”) and simple cells (herein called SimCells) can be provided to target viral proteins or epitopes, for example for anti-viral therapy. Advantageously, SimCells and mini-SimCells are chromosome-free, non-replicating and reprogrammable bacterial-based chassis that function as “smart” living drugs. SimCells (size 1-2 pm) are created by degrading native chromosomes using specific endonuclease and nuclease, and are engineered to carry designed DNA to perform predefined functions44,47. Mini-SimCells (size 100-400 nm) are generated through asymmetric division of bacteria with a minD gene deletion48-50. These mini-SimCells are also programmed to contain designed DNA45,46and can be generated from various bacteria strains44,51, with E. coli BL21 (DE3) serving as an example chassis. Further advantageously, both SimCells and mini-SimCells cannot replicate but retain the machinery function of cells, making them highly controllable and easy to produce from engineered parental bacterial cells. In this study, the escape frequency of SimCells is below 10s. meeting the criteria with the NIH guidelines for clinical recombinant microorganisms52. Mini-SimCells are engineered minicells containing designed DNA. Clinical therapy based on minicells has been validated in both dogs and humans (phase I clinical trial)51,52, showing significant promise in safety and efficacy. Remarkably, minicell-based therapy has recently been granted the “Fast-Track” by the FDA53. Hence, SimCell and mini-SimCell platforms not only meet safety standards but also show great potential in biomedical applications, such as cancer treatment45,49,54,55. The invention strategy for the antibacterial therapy isengineering SimCells and mini-SimCells as smart ‘bio-particles’, to specifically attack viruses or virus infected cells.

[0008] The inventors have further demonstrated that chromosome-free bacterial minicells (herein termed “mini-SimCells”) can be provided in a composition in advantageously smaller sizes, whilst still being capable of maintaining complex and functional surface displayed molecules, such as antigen-binding molecule (e.g. nanobodies), on their surface. Minicells are chromosome-less, non-replicative bacterial cells that are smaller than regular (replicative) bacterial cells. They can be obtained from regular rod-shaped bacterial cells via a variety of methods, such as genomic minD or minC deletion, FtsZ overexpression, pomZ deletion, and / or growth of the parent bacterial cell in suboptimal (stress-inducing) conditions or any other knockout methods to produce minicells. Because a minicell is non-replicative, it does not pose any risks of infection if administered to a subject. Likewise, because it still comprises plasmids, proteins, and RNA inherited from its parent bacterial cell, a minicell may be employed as a chassis to produce proteins (such as antibodies or fragments thereof) in great quantities and relatively cheaply before being administered.

[0009] Further, the inventors have demonstrated that, despite the reduced i) overall size, ii) total surface area, and thus iii) number of antigen-binding molecules that can be displayed, the mini-SimCells of the invention can nonetheless bind, neutralise, and block viral particles of typical sizes. Surprisingly, the inventors have also demonstrated that despite its reduced size and lack of genetic circuitry enabling a sustained metabolism, the mini-SimCell of the invention nonetheless remains functional over time, and is able to synthesise a number of antigen-binding molecules sufficient to cause viral particle blocking, neutralisation, and agglutination. The mini-SimCells can be stored in PBS or other buffer at 4 °C for at least one month, and at -20 C for at least six months. The mini-SimCells can be freeze-dried, and remain functional. Advantageously, freeze-dried mini-SimCells can be stored at room temperature for at least one year.

[0010] Further advantageously, given their reduced overall size, the mini-SimCells of the composition can penetrate mucosal barriers effectively, and avoid rapid clearance by mucociliary mechanisms (such as those of mucous membranes lining the lungs), thereby enabling effective targeting of viral particles that cause airway infections in a subject.The mini-SimCells in the composition may have an average size from about 20 nm to about 1000 nm. Preferably, at least about 80% of the minicells are smaller than about 200 nm along their axis. The mini-SimCells in the composition may have an average size from about 20 nm to about 200 nm. The mini-SimCells in the composition may have an average size from about 100 nm to about 400 nm. The mini-SimCells in the composition may have an average size from about 100 nm to about 200 nm. The mini-SimCells in the composition may have an average size from about 20 nm to about 1000 nm. The mini-SimCells in the composition may have an average size from about 80 nm to about 1000 nm.

[0011] The mini-SimCells in the composition may have an average size that is suitable to enable them to penetrate mucosal barriers, such as airway epithelial cells. The mini-SimCells in the composition may have an average size that is suitable to enable them to avoid rapid clearance by mucociliary mechanisms, such as in a subject’s airways. The mini-SimCells in the composition may have an average size that is suitable to enable the mini-SimCells to penetrate at least about 5 pm, about 10 pm, about 15 pm, about 20 pm, about 25 pm, about 30 pm, about 35 pm, about 40 pm, about 45 pm, or more, into cell layers, such as into layers of airway epithelial cells.

[0012] The mini-SimCell may be derived from Escherichia spp. (e.g. E. coli). The mini-SimCell may be derived from Escherichia spp. (e.g. E. coli), Pseudomonas spp. (e.g. P. aeruginosa), Salmonella spp., Bordetella spp. (e.g. B. hronchiseptica), or other bacterial species.

[0013] The mini-SimCell may comprise LPS (lipid polysaccharide), or alternatively the mini-SimCell may comprise no LPS. In many embodiments, the mini-SimCell comprises LPS if the bacterial cell whence it is derived comprised LPS, and vice versa. For example, minicells formed from LPS-free bacteria can also be LPS-free. LPS may also be known (and referred to herein) as endotoxin.

[0014] The mini-SimCell may comprise no LPS, or may comprise a modified version of LPS wherein the polysaccharide side-chains are significantly reduced in their average molecular weight, such as a reduced number of O antigen moieties. The mini-SimCell may comprise a modified version of LPS wherein the Lipid A component consists of areduced number of acyl chains. The mini-SimCell may comprise a modified version of LPS which has no, or negligible, inflammatory and / or immunogenic properties in humans, such as no or negligible TLR4 / MD-2 recognition and / or NF-KB activation. In all such embodiments, and other embodiments only minimally functionally different therefrom, the mini-SimCell may be referred to herein as LPS mini-SimCell”.

[0015] The LPS mini-SimCell may be derived from a LPS-free bacteria ,such as ClearColi™ E. coli (or a genetic equivalent thereof), or from a genetically modified version thereof. The bacterial cell whence the mini-SimCell is derived may comprise ClearColi™ E. coli, or a genetically altered version thereof. The bacterial cell whence the mini-SimCell is derived may comprise one, or more (e.g. all), of the genetic mutations from the following list: msbA148, gutQ, kdsD, MpxL, MpxM, pagP, MpxP, eptA.

[0016] Surprisingly, the inventors have identified that when mini-SimCells are derived from lipopolysaccharide-free (LPS ) bacterial cells, at least about 80% of the LPS mini-SimCells thus obtained are smaller than about 200 nm along their axis, thereby providing a cheap and straightforward source of mini-SimCells that are smaller than normal minicell populations. Advantageously, LPS mini-SimCells can be used in vivo, such as for therapeutic purposes, with a reduced risk of inducing an undesirable immune response that is typically associated with LPS.

[0017] Prevention of functional minCIminD expression

[0018] In some embodiment, such as embodiments comprising LPS bacterial cells, the mini-SimCell is obtained by means of preventing the expression of the endogenous minC and / or minD genes. Prevention of functional minCIminD expression may be achieved in a variety of ways, for example by genetic knock-out such as may be achieved by employing a CRISPR-Cas system. Examples of such systems are presented in Jiang et al., “Multigene editing in the Escherichia coli genome via the CRISPR-Cas9 system”, Appl Environ Microbiol 81, 2506-2514 (2015), which publication is incorporated herein by reference in its entirety. Therefore, in some embodiments (such as some embodiments comprising LPS bacterial cells), the mini-SimCells according to the specification herein are obtained by a method comprising genetic deletion of the endogenous minC and / or minD locuses. The skilled person will be familiar with techniques to delete or mutate minC and / or minD to form minicells.Suboptimal bacterial cell culture

[0019] In some embodiments, the bacterial cell is cultured sub-optimally, so to obtain a smaller minicell 1 according to the specification instead of a normal size minicell. In some embodiments for producing a minicell, the cell is cultured sub-optimally by culturing in minimal growth medium (i.e. nutrient restriction), and / or at a temperature that is suboptimal for its growth, and / or with reduced agitation.

[0020] In some embodiments, the cell for producing a minicell is cultured sub-optimally in M9 minimal medium, e.g. a medium comprising or consisting of: about 47.8 mM Na2HPC>4, about 22.0 mM KH2PO4, about 18.7 mM NaCl, and about 9.4 mM NH4CI, in water. In some embodiments, the cell for producing a minicell is cultured in M9 minimal medium supplemented with trace elements, e.g. a medium comprising or consisting of: about 47.8 mM Na2HPC>4, about 22.0 mM KH2PO4, about 18.7 mM NaCl, about 9.4 mM NH4CI, about 134 pM EDTA, about 31 pM FeCh, about 6.2 pM ZnCh, about 0.76 pM CuCh, about 0.42 pM C0CI2, about 1.62 pM H3BO3, and about 81 nM MnCh, in water. In a preferred embodiment, the cell for producing a minicell is cultured in M9 minimal medium supplemented with trace elements, e.g. a medium comprising or consisting of about 47.8 mM Na2HPO4, about 22.0 mM KH2PO4, about 18.7 mM NaCl, about 9.4 mM NH4CI, about 134 pM EDTA, about 31 pM FeCh, about 6.2 pM ZnCh, about 0.76 pM CuCh, about 0.42 pM C0CI2, about 1.62 pM H3BO3, and about 81 nM MnCh, in water, and further supplemented with 1 mM MgSC , 0.3 mM CaCh, 0.4% w / v glucose, and 0.2% w / v casamino acids. “Water” as used herein may be, for example, doubledeionised H2O i.e. ddFhO. The cell for producing a minicell may be cultured sub-optimally, such as in minimal media, and supplemented with casamino acids. The casamino acids may be commercially sourced, and / or may be obtained by acid hydrolysis of casein (such as bovine casein).

[0021] In some embodiments, the bacterial cell for producing a minicell is cultured sub-optimally at a temperature below 35 °C, such as about 33 °C, about 30 °C, about 27 °C, or less. In one embodiment, the bacterial cell for producing a minicell is cultured at about 30 °C. In a preferred embodiment, the bacterial cell for producing a minicell is cultured at a temperature about from 10 to 16 °C. In one embodiment, the bacterial cell for producing a minicell is cultured with minimal growth media, and at a temperaturebelow 35°C, such as 10 to 16 °C. In another embodiment, the bacterial cell for producing a minicell is cultured with minimal growth media and a casamino acid supplement, and at a temperature below 35°C, such as 10 to 16 °C.

[0022] In some embodiments, the bacterial cell for producing a minicell is cultured sub-optimally in static conditions. In some embodiments, the bacterial cell is cultured sub-optimally by rotating the culture at less than 250 rpm, such as at about 200 rpm, about 170 rpm, about 150 rpm, about 120 rpm, about 100 rpm, about 50 rpm, or about 20 rpm.

[0023] In some embodiments, the bacterial cell for producing a minicell is cultured sub-optimally for 4-36 hr.

[0024] Another method of obtaining smaller minicells is to provide an minicell originating bacterial cell that is a naturally occurring small species or strain. Naturally small bacteria, such as those less than about 1000 nm in size, may be used to provide a minicell (reference to size is the average size of a population, and refers to the longest dimension of the cell). In particular, the methods described herein to produce a minicell, such as minC and / or a minD gene knockout, may be used on a naturally small bacterial species or strain. Additionally, the methods described herein to produce a smaller minicell, such as LPS-free strains and / or less optimal growth conditions, may be used in combination with a naturally small bacterial species or strain. The naturally small bacterial species or strain may be less than lOOOnm on average, or less than 800nm on average, preferably less than 500nm on average, more preferably less than 300nm on average. In one embodiment, the naturally small bacterial species or strain may be about 200nm on average. In one embodiment, naturally small bacterial species or strain may comprise Mycoplasmas sp., which can be about 200 nm in size. Mycoplasmas can be engineered to form minicells.

[0025] The chromosome-free bacterial simple cells (SimCells)

[0026] Chromosome-free bacterial simple cells (SimCells) are described in US patent application US2022259605A1, incorporated herein in its entirety by reference. The scientific publication of Fan et al. (Chromosome-free bacterial cells are safe and programmable platforms for synthetic biology, Proc. Natl. Acad. Sci. U.S.A. 117 (12) 6752-6761, https: / / doi.org / T0.1073 / pnas.1918859117. 2020), which is also incorporatedherein in its entirety by reference, also describes SimCells and their production. The person skilled in the art will in general realise that many aspects disclosed in that document may be easily applied to aspects and embodiments of the invention herein disclosed, occasionally with appropriate modifications that will appear plain to them.

[0027] Chromosome-free bacterial simple cells (SimCells) typically comprise a bacterial cell having its chromosome removed by degradation, and encoding a tightly regulated recombinant endonuclease (used for degrading the chromosomal DNA of the original bacterial cell) and optionally a recombinant energy pathway to enhance stability.

[0028] The endonuclease for degrading the chromosomal DNA may comprise a homing endonuclease. The endonuclease for degrading the chromosomal DNA may recognise and make double-stranded breaks (DSBs) in at least 1, 2, 3, 4, 5, 6 or 7 sites of the bacterial cell’s chromosomal DNA. In one embodiment, the endonuclease may recognise a 10-bp, or more, recognition sequence. In one embodiment, the endonuclease may recognise a 15 -bp, or more, recognition sequence. In another embodiment, the endonuclease may recognise a 20-bp, or more, recognition sequence. In another embodiment, the endonuclease may recognise a 26-bp, or more, recognition sequence. In one embodiment, the endonuclease may recognise a sequence that has a probability of being present in 1 in 4.5xl015, or more, of a genetic sequence, such as a gene, genecircuit or mini-genome.

[0029] The endonuclease for degrading the chromosomal DNA may recognise and cut a sequence present in 23 S bacterial ribosomal RNA encoding gene (rrl). In one embodiment, the endonuclease for degrading the chromosomal DNA recognises the sequence 5’- TAACTATAACGGTCCTAAGGTAGCGA -3’ (SEQ ID NO: 1), or a variant thereof consisting of 1, 2, 3, 4, or 5 nucleotide substitutions, additions or deletions of said sequence. In one embodiment, the endonuclease for degrading the chromosomal DNA is I-Ceul, or a homologue thereof. The I-Ceul may be a Chlamydomoncis spp. I-Ceul. In one embodiment, the I-Ceul may comprise the amino acid sequence of CAA78934.1 (European Nucleotide Archive: https: / / www.ebi.ac.uk / ena / data / view / CAA78934), In one embodiment, the I-Ceul may comprise the amino acid sequence of SEQ ID NO: 28, or variants thereof. In one embodiment, the I-Ceul may be encoded by a sequence of SEQ ID NO: 29, or variants thereof.The skilled person will recognise that nucleic acid sequences encoding the endonuclease for degrading the chromosomal DNA, such as I-Ceul, may be codon-optimized for expression in a bacterial cell species, such as Ralstonia spp.

[0030] The endonuclease, such as I-Ceul, may comprise a modified / engineered (e.g. mutated) form of a wild-type endonuclease. The modification may comprise one or more mutations which enables the endonuclease to recognise and cut at different sequences relative to wild-type and / or have different activity to wild-type. The mutation may be in the active site responsible for recognition of the nucleic acid sequence.

[0031] Advantageously, I-Ceul allows the degradation of most bacterial chromosomes as its activity creates double strand breaks in the 23 S bacterial ribosomal RNA (rRNA) encoding -gene (rrl).

[0032] The endonuclease may be encoded on a nucleic acid, and the SimCell may be produced by transformation of a bacterial cell with the nucleic acid encoding the endonuclease. In one embodiment, the endonuclease is encoded on a plasmid.

[0033] Expression of the endonuclease, such as I-Ceul, may be under the control of an inducible promoter or repressible promoter. In one embodiment, the expression of the endonuclease, such as I-Ceul, may be under the control of a repressible-promoter. Preferably the expression of the endonuclease, such as I-Ceul, is under tight regulation, in which there is no or an undetectable level of basal expression of the endonuclease in the absence of induction or de-repression of the promoter.

[0034] The promoter for the endonuclease, such as I-Ceul, may be under the control of the Tetracycline repressor (TetR) gene. In an embodiment wherein the bacterial cell is E. coli, the promoter for the endonuclease, such as I-Ceul, may be under the control of the Tetracycline repressor gene (TetR) gene. The expression of the endonuclease, such as I-Ceul, may be induced by the provision of anyhydrotetracycline.

[0035] The promoter for the endonuclease, such as I-Ceul, may be under the control of EilR. The repressible-promoter may comprise an EilR binding site. In one embodiment, the repressible-promoter comprises the ‘Jungle Express’ (JEx) repressor system, forexample as described in Ruegg et al. (Nature Communications volume 9, Article number: 3617 (2018)), which is herein incorporated by reference. A repressible-promoter comprising an EilR binding site, such as JEx, may be used in any bacterial cell. In another embodiment, a repressible-promoter comprising an EilR binding site, such as JEx, may be used in Pseudmonas spp. (e.g. Pseudmonas putida) and / or Ralstonia spp. (e.g. Ralstonia eutropha). In another embodiment, a repressible-promoter comprising an EilR binding site, such as JEx, may be used in Escherichia spp., such as E. coli.

[0036] Expressing the endonuclease in the bacterial cell may comprise exposing the bacterial cell to an inducer molecule. For example, where the promoter is under control of TetR, expression of the endonuclease, such as I-Ceul, may be induced by the provision of anyhydrotetracycline (ATc) as an inducer. In another example, where the promoter is under control of EilR, expression of the endonuclease, such as I-Ceul, may be induced by the provision of an EilR-binding molecule as an inducer. The EilR-binding molecule (i.e. inducer) may comprise a dye-molecule that is capable of binding to and antagonising EilR, such as crystal violet. The EilR-binding molecule (i.e. inducer) may comprise any one of crystal violet, malachite green, tetracycline and imidazolium-based ionic liquids such as imidazolium or pyridinium; or combinations thereof.

[0037] The creation of multiple DSBs is lethal to bacterial cells, therefore a strain encoding an endonuclease, such as I-Ceul, that recognises and cuts the cell’s chromosomal DNA, cannot survive unless the expression of the endonuclease is under control. Therefore, the transformation and selection of bacterial cell transformants encoding the endonuclease (e.g. to clone / engineer a plasmid encoding the endonuclease, and / or to introduce a plasmid encoding the endonuclease for subsequent SimCell creation) requires the expression of the endonuclease, such as I-Ceul, to be controlled. Advantageously, the tight regulation of the expression of the endonuclease ensures that the cellular defences of various bacteria that may inactivate the SimCell-forming machinery are avoided. Such defences include inactivation by transposable elements or other mutations (base pair change, deletions) made by the cell to inactivate the endonuclease gene, such as the I-Ceul gene, as discussed in Fan et al. (2019. ACS Synthetic Biology 8:2141-2151), which is incorporated herein by reference. Without being bound by theory, it is understood that if the endonuclease is present in the cell, even at low levels, the defence mechanism can be triggered and the gene might beinactivated. By having tight control, the cell will not be alerted that it is hosting something toxic and so the integrity of the gene will be preserved.

[0038] The endonuclease may be encoded on a nucleic acid for transformation of the bacterial cell. Nucleic acid encoding the endonuclease may be provided by recombining the sequence encoding the endonuclease gene into the nucleic acid for transformation of the bacterial cell. The sequence encoding the endonuclease gene may be a PCR product, or a product of an excision from another nucleic acid, such as a cloning plasmid. Therefore, the sequence encoding the endonuclease gene may be generated by PCR from a donor nucleic acid template, or by excision from a donor nucleic acid, such as a cloning plasmid.

[0039] The sequence encoding the endonuclease gene for recombining into the nucleic acid for transformation of the bacterial cell may additionally encode the promoter for expression, such as the repressible-promoter or inducible promoter described herein. Alternatively, the promoter for expression, such as the repressible-promoter or inducible promoter described herein, may be provided in the nucleic acid for transformation of the bacterial cell (i.e. the endonuclease gene is recombined into a nucleic acid that carries the appropriate promoter for expression of the endonuclease gene). The skilled person will recognise that the promoter for expression of the endonuclease gene should be operably linked to the endonuclease gene in order to effect expression of the endonuclease (which may also be dependent on inducement or derepression).

[0040] For provision of the sequence encoding the endonuclease gene, for example as a PCR product using a donor template, or a product of an excision from a donor nucleic acid, such as a cloning plasmid, the donor nucleic acid may encode the sequence of the endonuclease gene. The donor nucleic acid may encode the sequence of the endonuclease gene in a form that cannot be expressed. For example, the endonuclease gene may be promoterless, or at least not under the control of a functional promoter. Additionally or alternatively, the expression of the endonuclease gene may be prevented by a secondary structure in the donor nucleic acid, such as a hairpin loop. Such a secondary structure (e.g. a hairpin loop) may be provided when a promoter is provided with the endonuclease gene in the donor nucleic acid. The hairpin loop may be provided by a reverse complementary sequence in the donor nucleic acid. The reversecomplementary sequence in the donor nucleic acid may be reverse complementary to a sequence of the endonuclease gene and / or any associated promoter thereof, or to a sequence that is sufficiently close to the endonuclease gene and / or any associated promoter thereof, in order to prevent expression of the endonuclease gene. The hairpin loop may be arranged to form a hairpin loop in the endonuclease gene and / or any associated promoter thereof. In one embodiment, the secondary structure, such as the hairpin loop, is arranged to form a hairpin loop in the promoter of the endonuclease gene.

[0041] Expressing an endonuclease in the bacterial cell comprises the steps of:

[0042] i) providing a donor nucleic acid encoding the endonuclease gene, wherein the endonuclease gene is promoterless; or wherein the endonuclease gene is operably linked to a promoter, and the donor nucleic acid is further arranged to form a hairpin loop to prevent expression of the endonuclease;

[0043] ii) using the donor nucleic acid as a template to form PCR product encoding the endonuclease, and inserting the PCR product into a nucleic acid for transformation of the bacterial cell, wherein the endonuclease gene is inserted into a position such that it is under control of the inducible or repressible promoter; or

[0044] or recombining the endonuclease gene encoded on the donor nucleic acid with a nucleic acid for transformation of the bacterial cell, wherein the endonuclease gene is recombined into a position such that it is under control of the inducible or repressible promoter.

[0045] The skilled person will recognise that a secondary structure, such as a hairpin loop, can be created in a nucleic acid sequence by design of the sequence. A hairpin loop in double helix plasmids will be stable (i.e. hairpin loop formation is unlikely to occur). However, a hairpin loop may be formed in single stranded DNA or RNA, such that when transcription occurs, the hairpin loop can form to block further transcription or translation.

[0046] The reverse complementary sequence may be at least 6, 8, 10, 12 or 15 nucleotides in length. In another embodiment, the reverse complementary sequence may be about 10-1,000 nucleotides in length. In another embodiment, the reverse complementary sequence may be about 10-500 nucleotides in length. In another embodiment, the reverse complementary sequence may be about 10-100 nucleotides in length. In anotherembodiment, the reverse complementary sequence may be about 10-50 nucleotides in length. In another embodiment, the reverse complementary sequence may be about 10-20 nucleotides in length. In another embodiment, the reverse complementary sequence may be about 15 nucleotides in length. The reverse complementary sequence may be sufficiently long to be specific in the location of the secondary structure formation.

[0047] The secondary structure, such as the hairpin loop, may disrupt the expression of the endonuclease for example by ensuring that the sequence of the endonuclease gene and / or promoter is not accessible to mRNA polymerase or other enzymes or co-factors that may be required for transcription. Advantageously, ensuring the endonuclease cannot be expressed from the donor nucleic acid, for example by a hairpin loop, can ensure that the endonuclease is not detrimental, such as fatal, to a cell carrying or cloning the donor nucleic acid. The donor copy of the endonuclease gene may not be expressed in the template in order to maintain its integrity and allow long-term storage.

[0048] The nucleic acid, such as the plasmid, encoding the endonuclease may also encode a selection marker. The selection marker may comprise an antibiotic resistance gene to enable the identification of cells successfully transformed with the nucleic acid, such as the plasmid.

[0049] In an embodiment, the bacterial cell is transformed with nucleic acid encoding enzymes of one or more biochemical energy pathways, or parts thereof, before the induction of expression of the endonuclease. In an alternative embodiment, the bacterial cell is transformed with nucleic acid encoding enzymes of one or more biochemical energy pathways, or parts thereof, after the induction of expression of the endonuclease, for example after induction of the expression is stopped.

[0050] The biochemical energy pathway may be involved in, and capable of, the production of ATP and NAD(H), for example by converting glucose to pyruvate. Additionally, the biochemical energy pathway may be involved in, and capable of, the production of one or more molecules of NADPH, H+, GTP, and FADH2. In one embodiment, the biochemical energy pathway is the glycolysis pathway. The enzymes of the glycolysis pathway may be encoded in the glycolysis pathway encoding-sequence identified herein in SEQ ID NO: 30, or a variant thereof. In another embodiment, the biochemical energy pathway is the pentose phosphate pathway. Alternatively, the biochemical energypathway comprises a combination of the glycolysis and pentose phosphate pathways, or parts thereof. In one embodiment, at least part of the pentose phosphate pathway up to and including generation of Ru5P is provided.

[0051] The biochemical energy pathway may be an aerobic or anaerobic pathway. The biochemical energy pathway may be any pathway from a chemoautotroph or heterotroph that is capable of the production of ATP and NADH from sources of carbon, such as CO2, carbohydrate and / or monosaccharides.

[0052] Therefore, in one embodiment, the bacterial cell is transformed with nucleic acid encoding one or more, or all, enzymes of the glycolysis pathway. In one embodiment, the bacterial cell is transformed with nucleic acid encoding the upper and / or lower catabolic pathways of glycolysis. The complete glycolysis pathway may be provided. The biochemical energy pathway may be provided by transformation of bacterial cell with nucleic acid comprising or consisting of the sequence of SEQ ID NO: 30.

[0053] Reference to a “part thereof’ of a biochemical energy pathway is understood to mean that at least one, two, three, four, or more, enzymes that are critical for a biochemical energy pathway may be encoded or expressed. The skilled person will be familiar with biochemical energy pathways in bacteria, such as glycolysis, and the necessary components required for ATP and NAD(H) generation. Where a biochemical energy pathway, such as the glycolysis pathway, may classically comprise a number of enzymes, it will be recognised that some enzymes may not be critical for ATP and NAD(H) generation, or they may be exchanged with one or more alternative enzymes. Therefore, a biochemical energy pathway may be made up of any combination of enzymes arranged to generate at least ATP and NAD(H) from a carbon source. The enzymes in a biochemical energy pathway may also comprise membrane transporter proteins. Additionally or alternatively, the biochemical energy pathway may comprise the TCA cycle, a fermentation pathway, a respiration pathway, or parts thereof, or combinations thereof.

[0054] In one embodiment, the bacterial cell is transformed with nucleic acid encoding glyceraldehyde-3 -phosphate dehydrogenase and / or phosphoglycerate kinase. The skilled person will recognise that glyceraldehyde-3 -phosphate dehydrogenase and phosphoglycerate kinase are required to make NADH and ATP, respectively. Not everyenzyme may be required where the pathway may be supplemented with one or more biochemical energy pathway intermediates for the enzymes of the bacterial cell to use.

[0055] In an embodiment, the bacterial cell may be provided with nucleic acid encoding one, two, three, four, five, six, seven, eight, nine or ten biochemical energy pathways, or parts thereof, or hybrid combinations thereof.

[0056] The enzymes of the one or more biochemical energy pathways may be heterologous to the bacterial cell. In one embodiment, the enzymes are recombinant. The nucleic acid encoding enzymes of one or more biochemical energy pathways, or parts thereof, may be plasmid. In one embodiment, the enzymes of the entire biochemical energy pathway are encoded on a single nucleic acid, such as a plasmid. In another embodiment, the enzymes of the entire biochemical energy pathway are encoded on two or more nucleic acids, such as two or more plasmids. The upper and lower catabolic pathways of glycolysis may be encoded on a single nucleic acid, which may be a plasmid. The upper and lower catabolic pathways of glycolysis may be encoded on separate nucleic acids, which may be plasmids. Where two or more nucleic acids are provided for encoding the enzymes of the one or more biochemical energy pathways, or parts thereof, the method may comprise transforming the bacterial cell with the two or more nucleic acids.

[0057] The expression of the biochemical energy pathway(s) may be regulated. For example, the expression of the biochemical energy pathway(s) may be under the control of an inducible or repressible promoter. In another embodiment, the expression of the biochemical energy pathway(s) may be constitutive. In particular, a constitutive promoter may be encoded for promoting the expression of the components / enzymes or operon of the biochemical energy pathway(s). The expression of the entire biochemical energy pathway may be under control of a single promoter.

[0058] In an embodiment, the expression of one or more biochemical energy pathways, or parts thereof is under the control of an inducible promoter, but may not be induced. In particular, the inducible promoter may have a “leaky” basal level of expression, which may be sufficient to provide energy to the mini-SimCell.

[0059] In one embodiment, expression of the one or more biochemical energy pathways, or parts thereof, is under the control of the lac repressor (Lad). The Lad repressor maybe used with a Ptrc promoter. Expression of a promoter under LacI repression may be induced by the provision of IPTG (isopropyl P-D-l -thiogalactopyranoside). However, in one embodiment, the expression of the one or more biochemical energy pathways, or parts thereof, is not induced by IPTG, and the mini-SimCell relies on the non-induced (i.e. leaky) basal expression. The skilled person will recognise that the LacI repressor allows leaky expression due to poor binding kinetics. Equivalent inducible or repressible promoter systems may be used if they allow a basal (leaky) level of expression in the absence of an inducer. An inducible or repressible promoter system may be used if it allows a basal level of expression in the absence of an inducer, and the basal level of expression is at least a 10-fold lower relative to induced expression in the presence of an inducer.

[0060] In another embodiment, expression of the one or more biochemical energy pathways, or parts thereof, is under the control of pBAD (the arabinose promoter). In another embodiment, the nucleic acid encoding one or more enzymes of the biochemical energy pathways, or parts thereof, comprises one or more sequences encoding pBAD.

[0061] Advantageously basal expression (so called “leaky” expression) of the one or more biochemical energy pathways, or parts thereof, can be relied upon for the provision of energy to the mini-SimCell, without overloading the mini-SimCell and inducing premature death, or lack of stability or longevity. Such “leaky” expression may be defined as the detection of expression of the molecule encoded by the nucleic acid in the absence of the inducer, and in which the expression detected is higher than a strain (such as E. coli K-12) without the nucleic acid.

[0062] The Simcell may be further augmented by the reintroduction of one or more other energy generating pathways (e.g. TCA, pentose phosphate, fermentation, respiration, lightenergy pathways (e.g. utilising proteorhodopsin), DNA repairing pathways, ribosome regeneration pathways and nutrient supplementation, for example. In particular, the bacterial cell may be transformed with nucleic acid encoding enzymes of one or more other energy generating pathways, DNA repairing pathways, ribosome regeneration pathways. The SimCell may be transformed with nucleic acid to express photoactive retinylidene proteins. Nutrient supplementation into the media may also be provided to improve the longevity / stability of the SimCells. The media may be supplemented with one or more of nucleotides, amino acids, and cofactors.The antigen binding molecule

[0063] The antigen binding molecule may comprise an antibody or antigen-binding fragment thereof.

[0064] In one embodiment, the antigen binding molecule comprises a surface-displayed variable heavy chain-only single-domain antibody fragments (surface-displayed sdAbs), which may also be known in the art (and may alternatively be referred to herein throughout) as a single domain antibody (sdAb), as a sdAb fragment, or as a Nanobody™, which in each case is a single monomeric variable antibody domain. Like a whole antibody, a sdAb is able to bind selectively to a specific antigen, such as a viral antigen.

[0065] The bacterial cell, or the minicell and / or simple cell derived therefrom, may be transformed with nucleic acid encoding an antigen-binding molecule, such as a surface-displayed variable heavy chain-only antibody fragment.

[0066] The surface-displayed antigen-binding molecule may be capable of binding to a specific peptide, polypeptide, glycoprotein or glycolipid, for example on the surface of a viral particle. The surface-displayed antigen-binding molecule may have specific affinity for a peptide, polypeptide, glycoprotein or glycolipid, for example on the surface of a viral particle.

[0067] The target of the surface-displayed antigen-binding molecule may be a viral particle. The target of the surface-displayed antigen-binding molecule may be a cell. The target of the surface-displayed antigen-binding molecule may be a virally infected cell that displays an antigen (recognised by the surface-displayed antigen-binding molecule) that is known or predicted to be associated with, or mostly associated with, viral infection and / or viral particles.

[0068] In one embodiment, the surface-displayed antigen-binding molecule is capable of binding to a viral antigen on a cell surface. The surface-displayed antigen-binding molecule may have specific affinity for a viral antigen on a cell surface. The viral antigen may be a peptide, polypeptide, glycoprotein or glycolipid, that is present on thesurface of a cell. The viral antigen may be exclusively found on a virally-infected cell relative to an equivalent non-infected cell, or may be present at a higher level on the virally-infected cell relative to an equivalent non-infected cell. The viral antigen may have a different structure relative to an equivalent molecule on a non-infected cell. In one embodiment, the viral antigen is a SARS-CoV-2 antigen. In one embodiment, the viral antigen is a SARS-CoV-2 protein antigen, for example an antigen on the spike (S) glycoprotein, such as in its receptor-binding domain (RBD). In one embodiment, the viral antigen is an influenza or parainfluenza antigen, such as an Orthomyxoviridae antigen, for example an influenza haemagglutinin (HA) or neuraminidase (NA) antigen. In one embodiment, the viral antigen is a SARS-CoV-2, influenza, respiratory syncytial virus (RSV), Zaire ebolavirus (Ebola), human immunodeficiency virus (HIV), hepatitis (such as hepatitis C, HCV), Dengue virus, West Nile virus, Rotavirus, Norovirus, Astrovirus, Adenovirus, or Herpesvirus (such as Cytomegalovirus , Herpes simplex, or Varicella zoster) antigen.

[0069] The surface-displayed antigen-binding molecule may comprise or consist of TY1, NIH-CoV2nb-112, mNb6, V, and E described herein. The surface-displayed sdAb may comprise or consist of a fusion of sdAbs, such as VE described herein. In some embodiments, the surface-displayed antigen-binding molecule may comprise more than one antigen-binding molecule, i.e. at least two different surface-displayed antigenbinding molecules. In some embodiments, the minicell and / or simple cell comprises at least two different surface-displayed antigen-binding molecules, for example because the minicell and / or simple cell comprises nucleic acid(s) encoding at least two different surface-displayed antigen-binding molecules overall.

[0070] In one embodiment, the surface-displayed antigen-binding molecule comprises or consists of the amino acid sequence:

[0071] QVQLVETGGGLVQPGGSLRLSCAASGFTFSSVYMNWVRQAPGKGPEWVSRISPN SGNIGYTDSVKGRFTISRDNAKNTLYLQMNNLKPEDTALYYCAIGLNLSSSSVRG QGTQVTVSS (TY1) (SEQ ID NO. 2), or a variant thereof.

[0072] In one embodiment, the surface -displayed antigen-binding molecule is translated from a nucleic acid sequence that comprises the nucleotide sequence according to the following sequence:CAGGTGCAGCTGGTGGAAACCGGCGGCGGCCTGGTGCAGCCGGGCGGCAGCC TGCGCCTGAGCTGCGCGGCGAGCGGCTTTACCTTTAGCAGCGTGTATATGAAC TGGGTGCGCCAGGCGCCGGGCAAAGGCCCGGAATGGGTGAGCCGCATTAGCC CGAACAGCGGCAACATTGGCTATACCGATAGCGTGAAAGGCCGCTTTACCAT TAGCCGCGATAACGCGAAAAACACCCTGTATCTGCAGATGAACAACCTGAAA CCGGAAGATACCGCGCTGTATTATTGCGCGATTGGCCTGAACCTGAGCAGCA GCAGCGTGCGCGGCCAGGGCACCCAGGTGACCGTGAGCAGC (TY1) (SEQ ID NO. 3), or a variant thereof.

[0073] In one embodiment, the surface-displayed antigen-binding molecule comprises or consists of the amino acid sequence:

[0074] EVQLVESGGGSVQAGGSLRLSCAASGRYRMGWFRQAPGKEREFVAVISASGGST YYADSVKGRFTIARDNAKTMVYLQMNSLKPEDTAVYYCAAKVNYYGDYDLAQ NYDYWGQGTQVTVSS (NIH-CoV2nb-l 12) (SEQ ID NO. 4), or a variant thereof.

[0075] In one embodiment, the surface -displayed antigen-binding molecule is translated from a nucleic acid sequence that comprises the nucleotide sequence according to the following sequence:

[0076] GACGTACAGTTGCAGGAGAGTGGTGGCGGCCTGGTCCAGCCCGGCGGTTCTC TCCGGCTGTCATGTGCAGCGAGTGGTCTTACCTTAGATTACTACGCCATTGGC TGGTTTCGTCAAGCGCCTGGAAAAGAACGCGAAGGGGTGTCTTGCATCAGTTC GAGCGATGGAAGCACATACTATGCTGACTCCGTGAAAGGCCGCTTCACGACT AGCCGTGACAACGCTAAAAATACGGTTTATCTGCAGATGAACTCCCTGAAAC CGGAAGATACAGCCGTCTATTACTGCGCGGCAGTTCCATCGACCTATTACAGC GGGACTTACTATTATACGTGTCATCCGGGTGGGATGGATTATTGGGGCAAGGG TACCCAAGTGACCGTATCGTCA (NIH-CoV2nb-l 12) (SEQ ID NO. 5), or a variant thereof.

[0077] In one embodiment, the surface-displayed antigen-binding molecule comprises or consists of the amino acid sequence:QVQLVESGGGLVQAGGSLRLSCAASGYIFGRNAMGWYRQAPGKERELVAGITR RGSITYYADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCAADPASPAYGD YWGQGTQVTVSSHHHHHH (mNb6) (SEQ ID NO. 6), or a variant thereof.

[0078] In one embodiment, the surface -displayed antigen-binding molecule is translated from a nucleic acid sequence that comprises the nucleotide sequence according to the following sequence:

[0079] CAGGTGCAACTTGTCGAATCGGGAGGCGGCTTAGTTCAGGCGGGTGGATCTCT CCGCTTGTCCTGCGCAGCTAGCGGTTATATCTTTGGGCGAAATGCCATGGGTT GGTATCGTCAGGCTCCAGGGAAGGAGCGCGAACTGGTCGCCGGCATCACGCG GCGTGGCTCAATTACTTACTACGCGGACAGCGTAAAAGGCCGTTTCACGATTA GTCGCGATAATGCAAAAAACACAGTATATCTGCAAATGAACTCGCTGAAACC GGAAGATACCGCCGTGTATTACTGTGCAGCGGACCCGGCGTCCCCTGCGTACG GTGATTATTGGGGCCAGGGTACCCAGGTTACCGTGAGCAGTCATCATCATCAC CACCAC (mNb6) (SEQ ID NO. 7), or a variant thereof.

[0080] In one embodiment, the surface-displayed antigen-binding molecule comprises or consists of the amino acid sequence:

[0081] QVQLVETGGGLVQPGGSLRLSCAASGFTFSSYAMGWARQVPGKGLEWVSYIYS DGSTEYQDSVKGRFTISRDNAKSTVYLQMNSLKPEDTAVYYCATEGSLGGWGR DFGSWGQGTQVTVSSG (V) (SEQ ID NO. 8), or a variant thereof.

[0082] In one embodiment, the surface -displayed antigen-binding molecule is translated from a nucleic acid sequence that comprises the nucleotide sequence according to the following sequence:

[0083] CAGGTCCAGCTGGTTGAGACCGGGGGCGGTCTTGTTCAGCCAGGCGGTAGCC TGCGCCTGTCTTGCGCCGCGTCAGGGTTTACCTTCTCAAGCTACGCAATGGGA TGGGCCCGACAAGTACCAGGTAAAGGTCTCGAATGGGTGTCCTATATTTATTC GGACGGCAGTACGGAGTATCAAGATAGTGTGAAAGGTCGCTTCACAATCAGC CGTGATAACGCTAAAAGCACCGTGTACCTGCAAATGAACTCCCTGAAACCGG AAGATACTGCAGTTTATTATTGTGCTACCGAAGGGAGTCTGGGAGGGTGGGGCCGGGATTTTGGTTCGTGGGGACAGGGCACACAAGTAACGGTCTCTTCAGGC GG (V) (SEQ ID NO. 9), or a variant thereof.

[0084] In one embodiment, the surface-displayed antigen-binding molecule comprises or consists of the amino acid sequence:

[0085] QVQLVETGGGFVQPGGSLRLSCAASGVTLDYYAIGWFRQAPGKEREGVSCIGSS DGRTYYSDSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCALTVGTYYSGN YHYTCSDDMDYWGKGTQVTVSSGGYPYDVPDYAGHHHHHH (E) (SEQ ID NO.

[0086] 10), or a variant thereof.

[0087] In one embodiment, the surface -displayed antigen-binding molecule is translated from a nucleic acid sequence that comprises the nucleotide sequence according to the following sequence:

[0088] CAGGTCCAGTTAGTCGAAACGGGCGGTGGTTTCGTGCAACCCGGAGGCTCGC TGCGTCTTTCTTGCGCGGCGTCGGGCGTTACGTTAGATTATTACGCTATTGGCT GGTTTCGCCAGGCACCGGGCAAAGAAAGAGAGGGCGTGTCTTGCATTGGCTC CAGCGACGGCCGTACATACTATAGTGACAGCGTAAAGGGTCGTTTTACCATCT CCCGCGACAATGCGAAAAACACCGTTTACTTGCAGATGAATAGCCTAAAGCC GGAAGACACCGCCGTCTATTATTGTGCCTTGACCGTTGGTACTTATTACTCTG GCAATTATCACTATACTTGTAGTGATGACATGGATTATTGGGGAAAAGGGAC GCAGGTGACGGTGAGCAGCGGTGGGTACCCTTACGATGTGCCGGATTACGCG GGTCATCACCACCATCATCAT (E) (SEQ ID NO. 11), or a variant thereof.

[0089] In one embodiment, the surface-displayed antigen-binding molecule comprises or consists of the amino acid sequence:

[0090] QVQLVETGGGLVQPGGSLRLSCAASGFTFSSYAMGWARQVPGKGLEWVSYIYS DGSTEYQDSVKGRFTISRDNAKSTVYLQMNSLKPEDTAVYYCATEGSLGGWGR DFGSWGQGTQVTVSSGRWLGWWGFRGRRIQVQLVETGGGFVQPGGSLRLSCAA SGVTLDYYAIGWFRQAPGKEREGVSCIGSSDGRTYYSDSVKGRFTISRDNAKNTV YLQMNSLKPEDTAVYYCALTVGTYYSGNYHYTCSDDMDYWGKGTQVTVSSGG YPYDVPDYAGHHHHHH (VE) with linker (underlined) (SEQ ID NO. 12), or a variant thereof.In one embodiment, the surface -displayed antigen-binding molecule is translated from a nucleic acid sequence that comprises the nucleotide sequence according to the following sequence:

[0091] CAGGTCCAGCTGGTTGAGACCGGGGGCGGTCTTGTTCAGCCAGGCGGTAGCC TGCGCCTGTCTTGCGCCGCGTCAGGGTTTACCTTCTCAAGCTACGCAATGGGA TGGGCCCGACAAGTACCAGGTAAAGGTCTCGAATGGGTGTCCTATATTTATTC GGACGGCAGTACGGAGTATCAAGATAGTGTGAAAGGTCGCTTCACAATCAGC CGTGATAACGCTAAAAGCACCGTGTACCTGCAAATGAACTCCCTGAAACCGG AAGATACTGCAGTTTATTATTGTGCTACCGAAGGGAGTCTGGGAGGGTGGGG CCGGGATTTTGGTTCGTGGGGACAGGGCACACAAGTAACGGTCTCTTCAGGC GGCGGTGGCTCGGGTGGTGGGGGTTCCGGGGGCGGCGGATCACAGGTCCAGT TAGTCGAAACGGGCGGTGGTTTCGTGCAACCCGGAGGCTCGCTGCGTCTTTCT TGCGCGGCGTCGGGCGTTACGTTAGATTATTACGCTATTGGCTGGTTTCGCCA GGCACCGGGCAAAGAAAGAGAGGGCGTGTCTTGCATTGGCTCCAGCGACGGC CGTACATACTATAGTGACAGCGTAAAGGGTCGTTTTACCATCTCCCGCGACAA TGCGAAAAACACCGTTTACTTGCAGATGAATAGCCTAAAGCCGGAAGACACC GCCGTCTATTATTGTGCCTTGACCGTTGGTACTTATTACTCTGGCAATTATCAC TATACTTGTAGTGATGACATGGATTATTGGGGAAAAGGGACGCAGGTGACGG TGAGCAGCGGTGGGTACCCTTACGATGTGCCGGATTACGCGGGTCATCACCAC CATCATCAT (VE) (SEQ ID NO. 13), or a variant thereof.

[0092] The surface-displayed antigen-binding molecule may comprise or consist of a antigenbinding molecule that competes for binding with TY1, NIH-CoV2nb-l 12, mNb6, V, E, and / or VE. The surface-displayed antigen-binding molecule may comprise or consist of a antigen-binding molecule that binds to the same epitope as TY1, NIH-CoV2nb-l 12, mNb6, V, E, and / or VE. The surface-displayed antigen-binding molecule may comprise or consist of a antigen-binding molecule that comprises the CDRs of TY1, NIH-CoV2nb-l 12, mNb6, V, E, and / or VE.

[0093] The variant may be a functional variant having the same or similar binding function. The variant may be capable of specifically binding to the same target as the surface-displayed nanobody described herein. The variant may have at least 90%, 95%, 98% or99% sequence identity to the antigen-binding molecule sequences of any of SEQ ID NOs: 2-12.

[0094] The skilled person will recognise that whilst TY1, NIH-CoV2nb-l 12, mNb6, V, E, and VE are exemplified herein, the surface-displayed antigen-binding molecule can be any known virus-targeting antigen-binding molecule, such as an antibody, or readily adapted from any virus targeting antigen-binding molecule, such as an antibody. The surface-displayed antigen-binding molecule may comprise or consist of an antigen-binding molecule that competes for binding with any known virus-targeting antibody or antibody fragment. The surface -displayed antigen-binding molecule may comprise or consist of an antigen-binding molecule that binds to the same epitope as any known virus-targeting antibody or antibody fragment. The surface-displayed antigen-binding molecule may comprise or consist of an antigen-binding molecule that comprises the VH of any known virus-targeting antibody or antibody fragment. The surface-displayed antigen-binding molecule may comprise or consist of an antigen-binding molecule that comprises at least three CDRs of any known virus-targeting antibody or antibody fragment. The surface-displayed antigen-binding molecule may comprise or consist of an antigen-binding molecule that comprises the VH and / or VL chain of any known virus-targeting antibody or antibody fragment. The surface-displayed antigen-binding molecule may comprise or consist of an antigen-binding molecule that comprises six CDRs of any known virus -targeting antibody or antibody fragment.

[0095] The antigen-binding molecule sequence may be optimised for expression in the minicells and / or simple cells. The antigen-binding molecule sequence may be codon optimised for bacterial expression.

[0096] The surface-displayed antigen-binding molecule may be displayed on the minicells and / or simple cells in a number of copies from about 500 to about 15,000, or more. The surface-displayed antigen-binding molecule may be displayed on the minicells and / or simple cells in a number of copies from about 5,000 to about 10,000, or more. The surface-displayed antigen-binding molecule may be displayed on the minicells and / or simple cells in about 8,000 copies.

[0097] The antigen-binding molecule may be expressed as a fusion peptide further comprising a membrane polypeptide (i.e. a polypeptide comprising a hydrophobic domain arrangedto be anchored in a membrane bilayer). The membrane polypeptide may be used for targeting the minicells and / or simple cells, for example, to certain tissues or cells in the body. Additionally or alternatively, the membrane polypeptide may be used as a surface marker to be able to locate the minicells and / or simple cells.

[0098] The surface-displayed antigen-binding molecule may be anchored to the outer membrane by a membrane anchor polypeptide, such as an integral membrane protein. In one embodiment, the membrane anchor polypeptide comprises a P-intimin domain, or a part thereof that is capable of membrane anchoring. The surface-displayed antigenbinding molecule may be anchored to the outer membrane by a P-intimin N-terminus domain. The surface-displayed antigen-binding molecule may be encoded and expressed as a fusion protein with the outer membrane by a membrane anchor polypeptide, such as P-intimin N-terminus domain. Pinero-Lambea et al. (ACS Synth. Biol. 2015, 4, 4, 463-473, Publication Date:July 21, 2014, https: / / doi.org / 10.1021 / sb50Q252a, which is herein incorporated by reference) describe membrane anchoring with synthetic adhesins which may be used in accordance with the invention to anchor the antigen-binding molecule to the chromosome-free bacterial minicell and / or simple cell surface.

[0099] The membrane anchor polypeptide may be any molecule capable of surface display of a polypeptide, such as the antigen-binding molecule. The surface-displayed antigenbinding molecule may be anchored to the outer membrane by a membrane anchor polypeptide, wherein the membrane anchor polypeptide comprises Lpp-OmpA, for example as described in Gallus et al. (Surface Display of Complex Enzymes by in Situ SpyCatcher-SpyTag Interaction, ChemBioChem 2020, 21, 212; https: / / doi.org / 10.1002 / cbic.202000102), which is herein incorporated by reference. This anchor consists of the transmembrane domain (amino acids 46-159) from outer membrane protein A (OmpA) as well as the signal peptide and the first 9 N-terminal amino acids of the E. colt lipoprotein (Lpp). For example, the antigen-binding molecule may be tagged, for example by fusion, with a tag arranged to bind to a tag on an outer membrane protein, such as Lpp-OmpA. The tags may comprise the SpyCatcher (a 113 aa polypeptide MSYYHHHHHHDYDIPTTENLYFQGAMVDTLSGLSSEQGQSGDMTIEEDSATHIK FSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPD GYEVATAITFTVNEQGQVTVNGKATKGDAHI SEQ ID NO: 13) and SpyTag (a 13 aa peptide RGVPHIVMVDAYKRYK SEQ ID NO: 14) units described by Gallus et al.(Surface Interaction, ChemBioChem 2020, 27, 212), or a functionally equivalent pair of polypeptide and peptide tags. The tags may be expressed as a fusion polypeptide with the outer membrane protein, such as Lpp-OmpA, and the antigen-binding molecule.

[0100] Alternatively, the antigen-binding molecule may be expressed as fusion with an outer membrane protein, such as Lpp-OmpA.

[0101] In some embodiment, the surface-displayed antigen-binding molecule may comprise a “tag” sequence, which may be exploited for e.g. protein purification and / or assaying protein expression levels, e.g. a myc tag, a His tag, a fluorescent protein tag. Therefore, in some embodiments, the bacterial cell, or the minicells and / or simple cells derived therefrom, may be transformed with nucleic acid encoding a surface-displayed antigenbinding molecule and a tag sequence.

[0102] In some embodiments, the minicells and / or simple cells express at least two different antigen-binding molecules. In some such embodiments, the antigen-binding molecules may target different epitopes on the same target molecule, and / or they may target different target molecules. In embodiments wherein the minicells and / or simple cells express at least two different antigen-binding molecules, the minicells and / or simple cells (and / or the bacterial cells they are derived from) may be transformed with one nucleic acid molecules, such as a plasmid, that encodes all antigen-binding molecules, or alternatively the minicells and / or simple cells (and / or the bacterial cells they are derived from) may be transformed with more than one nucleic acid molecule, such as more than one plasmid. By way of example, minicells and / or simple cells may express both anti-HA, and anti-NA, antigen-binding molecules; or two anti-HA (or anti-NA) antigen-binding molecules each of which targets a different HA (or NA, respectively) epitope. By way of further example minicells and / or simple cells may express two, different, anti-SARS-CoV-2 S glycoprotein antigen-binding molecules (such as two antigen-binding molecules directed at different RBD epitopes, or one antigen-binding molecule directed at a RBD epitope and another antigen-binding molecule directed at a non-RBD epitope on S, or two antigen-binding molecules directed at two epitopes neither of which is on RBD); or two anti-SARS-CoV-2 antigen-binding molecules each of which targets a different SARS-CoV-2 protein epitope. The person skilled in the art will realise that, in embodiments wherein the minicells and / or simple cells express at least two different antigen-binding molecules, any promoter and / or RBS sequences maybe independently chosen to determine the levels of expression of each antigen-binding molecule, that is to say: not all antigen-binding molecule-encoding sequences may necessarily be downstream of the same regulatory genetic elements.

[0103] It is a surprise that surface displayed proteins on minicells and / or simple cells contain sufficient number to effectively bind and neutralise the virus.

[0104] In some embodiments, an accessory product is expressed by the minicells and / or simple cells in addition to the surface-displayed antigen-binding molecules. In some embodiments, the bacterial cell, or the minicells and / or simple cells derived therefrom, may be transformed with nucleic acid encoding the accessory product. In some embodiments, a single nucleic acid encodes the surface-displayed antigen-binding molecules, the membrane anchor polypeptide (if present) of the surface-displayed antigen-binding molecules, and the accessory product; in alternative embodiments, the accessory product is encoded by a nucleic acid (such as a plasmid) that is different from the nucleic acid (such as a different plasmid) encoding the surface-displayed antigenbinding molecules and the membrane anchor polypeptide (if present) of the surface-displayed antigen-binding molecules. In some embodiments, the accessory product comprises more than one protein, for example the accessory product may be expressed as a fusion peptide comprising at least two different peptides, optionally separated by at least one linker (which may be a peptide linker). In some embodiments, at least one of the proteins of the accessory product is surface-displayed. In some embodiment, the accessory product may comprise a “tag” sequence, which may be exploited for e.g. protein purification and / or for assaying protein expression levels, e.g. a myc tag, a His tag, a fluorescent protein tag.

[0105] The accessory product may comprise an enzyme catalyst that is capable of producing a biochemical, such as a therapeutic drug. A plurality of enzyme catalysts may be provided for expression such that a multi-step reaction can be provided to produce a biochemical. The therapeutic drug may comprise a cytotoxic drug, such as catechol. The accessory product may comprise SalA, SalR, and / or NahG. In one embodiment, the accessory product comprises NahG. The skilled person will recognise that when salicylic acid (Aspirin) is present it combines with SalR to yield an active form SalR*, which then initiates transcription of salA and salR (positive feedback). Then SalA or salicylate hydroxylase converts salicylic acid to catechol in the presence of NADH. Theskilled person will similarly recognize that NahG directly catalyses the conversion of salicylic acid to catechol and hydrogen peroxide. In some embodiments that comprise administration of the minicells and / or simple cells to a subject, Aspirin is administered in combination with the minicells and / or simple cells (i.e. concurrently, prior to, or after the administration of the minicells and / or simple cells), for example orally, intravenously, transdermally, transnasally, or by localized injection.

[0106] Additionally or alternatively, salicylate hydroxylase (SalA or NahG) may be provided to produce chlorocatechol or hydroxyanthranilate.

[0107] The accessory product may be encoded by nucleic acid, such as a plasmid, with a nucleotide sequence comprising the sequence of SEQ ID NO: 15, or a functional variant thereof.

[0108] ATGAAGAATAACAAACTTGGGTTACGGATCGGTATCGTAGGGGGTGGCATTT CAGGTGTCGCCTTGGCATTGGAACTGTGCCGTTACTCACACATCCAGGTGCAA CTGTTCGAGGCAGCCCCGGCATTTGGGGAGGTCGGCGCCGGTGTCAGTTTCGG TCCGAACGCTGTGCGGGCCATTGTCGGCTTAGGCCTGGGGGAGGCATATTTAC AGGTGGCCGATCGTACATCAGAGCCATGGGAGGATGTGTGGTTTGAGTGGCG GCGTGGCTCAGACGCATCGTACCTTGGCGCGACAATTGCGCCAGGCGTAGGC CAGTCTAGCGTACACCGGGCAGATTTCATCGATGCGCTGGTAACGCATCTCCC AGAAGGTATTGCACAGTTCGGTAAGCGGGCAACGCAAGTGGAACAGCAGGGG GGCGAAGTACAAGTGTTATTTACAGACGGTACAGAATATCGTTGTGATTTGCT GATTGGGGCTGATGGTATCAAATCGGCTCTGCGGTCGCATGTACTTGAGGGGC AAGGGCTTGCGCCGCAGGTCCCACGCTTCTCTGGCACCTGCGCATATCGGGGG ATGGTGGACTCCTTGCATCTTCGTGAAGCCTATCGCGCTCATGGGATCGACGA ACATCTGGTTGACGTGCCTCAGATGTACCTGGGGCTCGATGGCCATATTTTAA CTTTCCCGGTCCGTAATGGTGGGATTATTAATGTCGTTGCATTCATCTCGGACC GGTCTGAGCCGAAACCGACGTGGCCTGCTGATGCGCCGTGGGTACGTGAAGC ATCCCAACGGGAGATGCTTGACGCTTTTGCCGGTTGGGGCGATGCTGCACGTG CCCTGTTAGAGTGTATTCCTGCCCCAACGCTGTGGGCATTACACGATCTTGCT GAACTTCCTGGGTACGTGCATGGCCGGGTGGTTCTCATTGGCGACGCGGCACA CGCAATGCTTCCACACCAAGGCGCCGGGGCTGGGCAAGGGCTGGAAGATGCC TACTTCCTCGCCCGCTTACTTGGTGACACACAAGCCGACGCCGGTAATCTTGC AGAACTTCTTGAGGCCTACGATGATTTACGCCGGCCACGCGCCTGTCGGGTACAACAAACGAGTTGGGAGACCGGCGAGTTATATGAGTTACGCGACCCTGTTGT GGGTGCAAACGAACAGCTTCTTGGCGAGAACTTAGCAACGCGGTTTGACTGG TTGTGGAATCACGACCTTGACACTGATTTGGCAGAAGCTCGCGCACGGTTGGG CTGGGAGCATGGTGGTGGTGGCGCCTTACGGCAGGGCTAA (SEQ ID NO: 15)

[0109] Alternatively or additionally, the accessory product may comprise a polypeptide with an amino acid sequence comprising the sequence of SEQ ID NO: 16, or a functional variant thereof.

[0110] MKNNKLGLRIGIVGGGISGVALALELCRYSHIQVQLFEAAPAFGEVGAGVSFGPN AVRAIVGLGLGEAYLQVADRTSEPWEDVWFEWRRGSDASYLGATIAPGVGQSS VHRADFIDALVTHLPEGIAQFGKRATQVEQQGGEVQVLFTDGTEYRCDLLIGAD GIKSALRSHVLEGQGLAPQVPRFSGTCAYRGMVDSLHLREAYRAHGIDEHLVDV PQMYLGLDGHILTFPVRNGGIINVVAFISDRSEPKPTWPADAPWVREASQREMLD AFAGWGDAARALLECIPAPTLWALHDLAELPGYVHGRVVLIGDAAHAMLPHQG AGAGQGLEDAYFLARLLGDTQADAGNLAELLEAYDDLRRPRACRVQQTSWETG ELYELRDPVVGANEQLLGENLATRFDWLWNHDLDTDLAEARARLGWEHGGGG ALRQG* (SEQ ID NO: 16)

[0111] In one embodiment, the accessory product, such as NahG, is also surface displayed on the minicells and / or simple cells. The accessory product may be arranged to be expressed on the surface (e.g. outer membrane). The accessory product may be surface displayed by binding it to a membrane anchor polypeptide, or providing the accessory product as a fusion polypeptide with an anchor polypeptide. The membrane anchor polypeptide may be any molecule capable of surface display of a polypeptide, such as the accessory product. The surface-displayed accessory product may be anchored to the outer membrane by a membrane anchor polypeptide, wherein the membrane anchor polypeptide comprises Lpp-OmpA, for example as described in Gallus et al. (Surface Display of Complex Enzymes by in Situ SpyCatcher-SpyTag Interaction, ChemBioChem 2020, 21 , 212; https: / / doi.org / 10.1002 / cbic.202000102), which is herein incorporated by reference. This anchor consists of the transmembrane domain (amino acids 46-159) from outer membrane protein A (OmpA) as well as the signal peptide and the first 9 N-terminal amino acids of the E. coli lipoprotein (Lpp). For example, the accessory product may be tagged, for example by fusion, with a tag arranged to bind to a tag on an outer membrane protein, such as Lpp-OmpA. The tagsmay comprise the SpyCatcher (a 113 aa polypeptide MSYYHHHHHHDYDIPTTENLYFQGAMVDTLSGLSSEQGQSGDMTIEEDSATHIK FSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPD GYEVATAITFTVNEQGQVTVNGKATKGDAHI) and SpyTag (a 13 aa peptide RGVPHIVMVDAYKRYK) units described by Gallus et al. (Surface Interaction, ChemBioChem 2020, 21, 212), or a functionally equivalent pair of polypeptide and peptide tags. The tags may be expressed as a fusion polypeptide with the outer membrane protein, such as Lpp-OmpA, and the accessory product.

[0112] Alternatively, the accessory product may be expressed as fusion with an outer membrane protein, such as Lpp-OmpA.

[0113] Advantageously, it is found that the minicells and / or simple cells, and sill further the smaller minicells described herein, are capable of functional surface display of large polypeptides, such as NahG or full-size antibodies.

[0114] In some embodiments wherein the minicells and / or simple cells are internalised by the targeted virally infected cell, the accessory product of the minicells and / or simple cells may provide further cytotoxic effects, such as by converting a prodrug to a toxic drug locally within the virally infected cell. Therefore, in some embodiments, there is provided a method of harming and / or killing a virally infected cell in a subject, comprising the step of administering to the subject minicells and / or simple cells comprising a surface-displayed, viral protein or epitope-targeting antigen-binding molecule and an accessory product according to this specification.

[0115] The expression of the antigen-binding molecule (and / or of the accessory product)

[0116] Two or more products for expression or replication may be encoded on the nucleic acid encoding the accessory product, or encoded on a plurality of nucleic acids for expression in the minicells and / or simple cells. For example, a membrane polypeptide may be encoded and expressed in the minicells and / or simple cells for targeting the minicells and / or simple cells to specific cells or tissues or viruses, together with encoding and expressing a biologically active polypeptide.The expression of the antigen-binding molecule and / or accessory product may be regulated. For example, the expression of the antigen-binding molecule and / or accessory product may be under the control of an inducible or repressible promoter. For example, the expression of the accessory product may be under the control of the MphR regulation system that is inducible by erythromycin. In another embodiment, the expression of the antigen-binding molecule and / or accessory product may be constitutive. In particular, a constitutive promoter may be encoded for promoting the expression of the antigen-binding molecule and / or accessory product. In one embodiment, the expression of the antigen-binding molecule and / or accessory product may be controlled by a strong promoter, such as a viral promoter. The promoter may comprise CMV promoter, SV40. In one embodiment, the expression of the antigenbinding molecule and / or accessory product may be controlled by any of the promoters listed in Table 1, which are also described in Davis et al. (Nucleic Acids Research, 2011, Vol. 39, No. 3, 1131-1141), which is incorporated herein by reference. In one embodiment, the promoter may comprise proD as described herein.

[0117] Table 1:

[0118]

[0119]

[0120] In some embodiments, the expression of the antigen-binding molecule and / or accessory product may be further or alternatively regulated by a ribosome-binding site (RBS). The RBS may be downstream of the promoter sequence, such as directly downstream, or alternatively there may be intervening genetic sequences between the promoter and the RBS (i.e. downstream of the promoter and yet upstream of the RBS). The sequence of the RBS may influence the level of expression of the genetic elements that follow it, including that of the antigen-binding molecule and / or accessory product. In some embodiments, the RBS sequence dictates the level of expression of the antigen-binding molecule and / or accessory product. In some embodiments, the interplay between the promoter (such as J23100) and the RBS dictates the level of expression of the accessory product. The RBS may be a weakly expressing RBS, such as BBa_B0034. Alternatively, the RBS may be RBS 12K (RBS 12K).

[0121] In a preferred embodiment comprising Clearcoli™ E. coli and minicells and / or simple cells derived therefrom, which express a VE sdAb and the P-intimin N-terminus domain as the outer-membrane anchor polypeptide, the promoter is j23100 and the downstream RBS (controlling expression of the sdAb-anchor polypeptide fusion) is RBS 12K.

[0122] In one embodiment wherein the antigen-binding molecule comprises VE and the accessory product comprises NahG, expression of the antigen-binding molecule and of the accessory product may be achieved by transformation of the bacterial cell or of theminicells and / or simple cells with the plasmids comprising the sequences according to SEQ ID NO: 18. Alternatively, the expression may be achieved by transformation with one plasmid which comprises both the “VE” and “NahG” inserts, for example comprising the following sequence:

[0123] acccgacaccatcgaatggcgcaaaacctttcgcggtatggcatgatagcgcccggaagagagtcaattcagggtggtga atgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggcc agccacgtttctgcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcaca acaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgcaaattgtcgcg gcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaa gcggcggtgcacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgt ggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacagtattattttctcccatgaag acggtacgcgactgggcgtggagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgt ctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaaggcga ctggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgttcccactgcgatgctggttgccaacg atcagatggcgctgggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggacatctcggtagtgggatac gacgataccgaagacagctcatgttatatcccgccgttaaccaccatcaaacaggattttcgcctgctggggcaaaccagcg tggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaa ccaccctggcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttccc gactggaaagcgggcagtgagcggtacccgataaaagcggcttcctgacaggaggccgttttgttttgcagcccacctcaa cgcaattaatgtgagttagctcactcattaggcaccccaggcttgacggctagctcagtcctaggtacagtgctagcAAA GGAAATCTAatgattactcatggttgttatacccggacccggcacaagcataagctaaaaaaaacattgattatgctta gtgctggtttaggattgtttttttatgttaatcagaattcatttgcaaatggtgaaaattattttaaattgggttcggattcaaaactgt taactcatgatagctatcagaatcgccttttttatacgttgaaaactggtgaaactgttgccgatctttctaaatcgcaagatatta atttatcgacgatttggtcgttgaataagcatttatacagttctgaaagcgaaatgatgaaggccgcgcctggtcagcagatca ttttgccactcaaaaaacttccctttgaatacagtgcactaccacttttaggttcggcacctcttgttgctgcaggtggtgttgctg gtcacacgaataaactgactaaaatgtccccggacgtgaccaaaagcaacatgaccgatgacaaggcattaaattatgcgg cacaacaggcggcgagtctcggtagccagcttcagtcgcgatctctgaacggcgattacgcgaaagataccgctcttggtat cgctggtaaccaggcttcgtcacagttgcaggcctggttacaacattatggaacggcagaggttaatctgcagagtggtaata actttgacggtagttcactggacttcttattaccgttctatgattccgaaaaaatgctggcatttggtcaggtcggagcgcgttac attgactcccgctttacggcaaatttaggtgcgggtcagcgttttttccttcctgcaaacatgttgggctataacgtcttcattgat caggatttttctggtgataatacccgtttaggtattggtggcgaatactggcgagactatttcaaaagtagcgttaacggctattt ccgcatgagcggctggcatgagtcatacaataagaaagactatgatgagcgcccagcaaatggcttcgatatccgttttaatg gctatctaccgtcatatccggcattaggcgccaagctgatatatgagcagtattatggtgataatgttgctttgtttaattctgata agctgcagtcgaatcctggtgcggcgaccgttggtgtaaactatactccgattcctctggtgacgatggggatcgattaccgtcatggtacgggtaatgaaaatgatctcctttactcaatgcagttccgttatcagtttgataaatcgtggtctcagcaaattgaacc acagtatgttaacgagttaagaacattatcaggcagccgttacgatctggttcagcgtaataacaatattattctggagtacaag aagcaggatattctttctctgaatattccgcatgatattaatggtactgaacacagtacgcagaagattcagttgatcgttaaga gcaaatacggtctggatcgtatcgtctgggatgatagtgcattacgcagtcagggcggtcagattcagcatagcggaagcca aagcgcacaagactaccaggctattttgcctgcttatgtgcaaggtggcagcaatatttataaagtgacggctcgcgcctatg accgtaatggcaatagctctaacaatgtacagcttactattaccgttctgtcgaatggtcaagttgtcgaccaggttggggtaa cggactttacggcggataagacttcggctaaagcggataacgccgataccattacttataccgcgacggtgaaaaagaatg gggtagctcaggctaatgtccctgtttcatttaatattgtttcaggaactgcaactcttggggcaaatagtgccaaaacggatgc taacggtaaggcaaccgtaacgttgaagtcgagtacgccaggacaggtcgtcgtgtctgctaaaaccgcggagatgacttc agcacttaatgccagtgcggttatattttttgatggtgcgccggtgccgtatccggatccgctggaaccggcccagccggcc CAGGTGCAGCTGCAGGAAAGCGGTGGTGGTTCAGTGCAAGCAGGAGGCAGCC TGAAACTCACCTGCGCGGCCTCTGGCTACATTTTTAACTCGTGTGGAATGGGG TGGTACCGCCAGAGCCCGGGCCGCGAGCGTGAATTAGTCTCGCGTATTTCTGG CGATGGCGATACGTGGCATAAAGAAAGTGTCAAAGGTCGGTTTACAATCTCA CAGGATAACGTAAAAAAGACTCTGTATCTTCAAATGAATAGTCTGAAACCAG AGGACACCGCTGTTTATTTCTGTGCGGTGTGCTACAATTTGGAAACCTATTGG GGGCAAGGTACTCAGGTAACGGTTTCCTCCtaatcacacaggaaacctactaaATGAAGAA TAACAAACTTGGGTTACGGATCGGTATCGTAGGGGGTGGCATTTCAGGTGTCG CCTTGGCATTGGAACTGTGCCGTTACTCACACATCCAGGTGCAACTGTTCGAG GCAGCCCCGGCATTTGGGGAGGTCGGCGCCGGTGTCAGTTTCGGTCCGAACG CTGTGCGGGCCATTGTCGGCTTAGGCCTGGGGGAGGCATATTTACAGGTGGCC GATCGTACATCAGAGCCATGGGAGGATGTGTGGTTTGAGTGGCGGCGTGGCT CAGACGCATCGTACCTTGGCGCGACAATTGCGCCAGGCGTAGGCCAGTCTAG CGTACACCGGGCAGATTTCATCGATGCGCTGGTAACGCATCTCCCAGAAGGTA TTGCACAGTTCGGTAAGCGGGCAACGCAAGTGGAACAGCAGGGGGGCGAAGT ACAAGTGTTATTTACAGACGGTACAGAATATCGTTGTGATTTGCTGATTGGGG CTGATGGTATCAAATCGGCTCTGCGGTCGCATGTACTTGAGGGGCAAGGGCTT GCGCCGCAGGTCCCACGCTTCTCTGGCACCTGCGCATATCGGGGGATGGTGGA CTCCTTGCATCTTCGTGAAGCCTATCGCGCTCATGGGATCGACGAACATCTGG TTGACGTGCCTCAGATGTACCTGGGGCTCGATGGCCATATTTTAACTTTCCCG GTCCGTAATGGTGGGATTATTAATGTCGTTGCATTCATCTCGGACCGGTCTGA GCCGAAACCGACGTGGCCTGCTGATGCGCCGTGGGTACGTGAAGCATCCCAA CGGGAGATGCTTGACGCTTTTGCCGGTTGGGGCGATGCTGCACGTGCCCTGTT AGAGTGTATTCCTGCCCCAACGCTGTGGGCATTACACGATCTTGCTGAACTTC CTGGGTACGTGCATGGCCGGGTGGTTCTCATTGGCGACGCGGCACACGCAATGCTTCCACACCAAGGCGCCGGGGCTGGGCAAGGGCTGGAAGATGCCTACTTC CTCGCCCGCTTACTTGGTGACACACAAGCCGACGCCGGTAATCTTGCAGAACT TCTTGAGGCCTACGATGATTTACGCCGGCCACGCGCCTGTCGGGTACAACAAA CGAGTTGGGAGACCGGCGAGTTATATGAGTTACGCGACCCTGTTGTGGGTGC AAACGAACAGCTTCTTGGCGAGAACTTAGCAACGCGGTTTGACTGGTTGTGG AATCACGACCTTGACACTGATTTGGCAGAAGCTCGCGCACGGTTGGGCTGGG AGCATGGTGGTGGTGGCGCCTTACGGCAGGGCTAAAAAGAGGAGAAAGGTAC CATGAGCAAAGGAGAAGAACTTTTCACTGGAGTTGTCCCAATTCTTGTTGAAT TAGATGGTGATGTTAATGGGCACAAATTTTCTGTCCGTGGAGAGGGTGAAGGT GATGCTACAAACGGAAAACTCACCCTTAAATTTATTTGCACTACTGGAAAACT ACCTGTTCCGTGGCCAACACTTGTCACTACTCTGACCTATGGTGTTCAATGCTT TTCCCGTTATCCGGATCACATGAAACGGCATGACTTTTTCAAGAGTGCCATGC CCGAAGGTTATGTACAGGAACGCACTATATCTTTCAAAGATGACGGGACCTA CAAGACGCGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATCGTATCG AGTTAAAGGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAACT CGAGTACAACTTTAACTCACACAATGTATACATCACGGCAGACAAACAAAAG AATGGAATCAAAGCTAACTTCAAAATTCGCCACAACGTTGAAGATGGTTCCGT TCAACTAGCAGACCATTATCAACAAAATACTCCAATTGGCGATGGCCCTGTCC TTTTACCAGACAACCATTACCTGTCGACACAATCTGTCCTTTCGAAAGATCCC AACGAAAAGCGTGACCACATGGTCCTTCTTGAGTTTGTAACTGCTGCTGGGAT TACACATGGCATGGATGAGCTCTACAAAaagcttgacctgtgaagtgaaaaatggcgcacattgtg cgacattttttttgtctgccgtttaccgctactgcgtcacggatccccacgcgccctgtagcggcgcattaagcgcggcgggt gtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgcc acgttcgccggctttccccgtcaagctctaaatcggggcatccctttagggttccgatttagtgctttacggcacctcgacccc aaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacg ttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccga tttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttcaggtg gcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgtcgagacgtt gggtgaggttccaactttcaccataatgaaataagatcactaccgggcgtattttttgagttatcgagattttcaggagctaagg aagctaaaatggagaaaaaaatcactggatataccaccgttgatatatcccaatggcatcgtaaagaacattttgaggcatttc agtcagttgctcaatgtacctataaccagaccgttcagctggatattacggcctttttaaagaccgtaaagaaaaataagcaca agttttatccggcctttattcacattcttgcccgcctgatgaatgctcatccggagttccgtatggcaatgaaagacggtgagct ggtgatatgggatagtgttcacccttgttacaccgttttccatgagcaaactgaaacgttttcatcgctctggagtgaataccac gacgatttccggcagtttctacacatatattcgcaagatgtggcgtgttacggtgaaaacctggcctatttccctaaagggttta ttgagaatatgtttttcgtctcagccaatccctgggtgagtttcaccagttttgatttaaacgtggccaatatggacaacttcttcgcccccgttttcaccatgggcaaatattatacgcaaggcgacaaggtgctgatgccgctggcgattcaggttcatcatgccgtc tgtgatggcttccatgtcggcagaatgcttaatgaattacaacagtactgcgatgagtggcagggcggggcgtaatttttttaa ggcagttattggtgcccttaaacgcctggtgctacgcctgaataagtgataataagcggatgaatggcagaaattcgaaagc aaattcgacccggtcgtcggttcagggcagggtcgttaaatagccgcttatgtctattgctggtttaccggtttattgactaccg gaagcagtgtgaccgtgtgcttctcaaatgcctgaggccagtttgctcaggctctccccgtggaggtaataattgctcgacat gaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttt tttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaact ctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttc aagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtctt accgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagccca gcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggaga aaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctg gtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatgg aaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatg (SEQ ID NO: 18).

[0124] The Viral Protein or Epitope

[0125] The viral protein (e.g. comprising the viral epitope) that is specifically bound by the surface-displayed antigen-binding molecule may be selected from e.g. SARS-CoV-2 S protein (e.g. the RBD domain of the S protein), SARS-CoV-2 E protein, SARS-CoV-2 M protein, SARS-CoV-2 N protein, influenza HA, influenza NA, RSV F protein, Zaire ebolavirus glycoprotein, HIV gpl20, HIV gp41, HCV El, HCV E2, Dengue E protein, and / or West Nile Virus E protein. In one embodiment, the viral antigen that is specifically bound by the surface-displayed antigen-binding molecule is SARS-CoV-2 RBD in the S protein. The viral antigen (comprising the viral epitope) that is specifically bound by the surface-displayed antigen-binding molecule may be selected from SARS-CoV-2 S protein (e.g. the RBD domain of the S protein), SARS-CoV-2 E protein, SARS-CoV-2 M protein, and / or SARS-CoV-2 N protein.

[0126] The viral protein (comprising the viral epitope) may be any antigen that is known or predicted to be associated and / or viral infection, either because it is only expressed on virions and / or virally infected cells, or because it is expressed (other than on virions) in virally infected cells in a greater proportion than in non-infected tissue. The term “viral protein” may be used interchangeably with the term “viral antigen” herein.The antigen-binding molecule may bind to a viral epitope. The viral epitope may be a SARS-CoV-2 S glycoprotein epitope, such as an epitope on the RBD domain of SARS-CoV-2 S. The viral epitope may comprise or consist of an epitope that is targeted by the antigen-binding molecules identified as TY1, NIH-CoV2nb-l 12, mNb6, V, E, and / or VE (wherein VE identifies a fusion of the V and E sdAbs). The viral epitope may comprise or consist of a SARS-CoV-2 epitope that is not targeted by one or more of (or none of) TY1, NIH-CoV2nb-l 12, mNb6, V, E, and VE. The viral epitope may comprise or consist of an epitope that is targeted by the antigen-binding molecule identified as VE. The person skilled in the art recognises that epitopes may be linear or more complex, such as in cases where a single antibody or antigen-binding molecule recognises a three-dimensional epitope wherein residues that are not linearly arranged (primary sequence) may nonetheless be form part of the epitope-paratope binding surface given the folding pattern of the target / epitope polypeptide (tertiary structure). The viral epitope may comprise, at least partly, one or more (such as all) of the following SARS-CoV-2 S glycoprotein residues: Y369, N370, S371, F374, T376, F377, K378, Y380, R408, S435, N437, V503, Q506, Y508 (sdAb V epitope). The viral epitope may comprise, at least partly, one or more (such as all) of the following SARS-CoV-2 S glycoprotein residues: Y351, R403, K444, G446, G447, Y449, L452, 1468, E484, T470, F490, L492, N493, S494, Y495, G496, Q498, N501 (sdAb E epitope). The viral epitope may comprise one or more (such as all) of the following SARS-CoV-2 S glycoprotein residues: Y369, N370, S371, F374, T376, F377, K378, Y380, R408, S435, N437, V503, Q506, Y508, Y351, R403, K444, G446, G447, Y449, L452, 1468, E484, T470, F490, L492, N493, S494, Y495, G496, Q498, N501 (sdAb fusion VE combined epitope). SARS-CoV-2 residues numbering refers to the SARS-CoV-2 / human / China / Wuhan-Hu-l / 2019 isolate (GenBank: QHD43416.1, herein incorporated by reference), and it is expressly envisaged herein that the person skilled in the art would easily target equivalent residues in other SARS-CoV-2 isolates / strains / variants, which may be easily identified by employing common sequence alignment programmes (such as BLAST).

[0127] The viral epitope may be an influenza epitope. The viral epitope may comprise or consist of an epitope that is targeted by navivumab and / or firivumab. The viral epitope may comprise or consist of an influenza epitope that is not targeted by bevacizumab, ranibizumab, and / or aflibercept.The viral epitope may be a RSV epitope. The viral epitope may comprise or consist of an epitope that is targeted by nirsevimab and / or palivizumab. The viral epitope may comprise or consist of a RSV epitope that is not targeted by nirsevimab and / or palivizumab.

[0128] The viral epitope may be an Ebola epitope. The viral epitope may comprise or consist of an epitope that is targeted by atoltivimab, maftivimab, and / or odesivimab. The viral epitope may comprise or consist of an Ebola epitope that is not targeted by atoltivimab, maftivimab, and / or odesivimab.

[0129] The viral epitope may be a HIV epitope. The viral epitope may comprise or consist of an epitope that is targeted by ibalizumab, semzuvolimab, and / or leronlimab. The viral epitope may comprise or consist of a HIV epitope that is not targeted by ibalizumab, semzuvolimab, and / or leronlimab.

[0130] The viral epitope may be a HCV epitope. The viral epitope may comprise or consist of an epitope that is targeted by one or both of the antibodies known as HCV1 and 95-2. The viral epitope may comprise or consist of a HCV epitope that is not targeted by either or neither of the antibodies known as HCV1 and 95-2.

[0131] The viral epitope may be a Dengue epitope. The viral epitope may comprise or consist of an epitope that is targeted by the antibody known as VIS513. The viral epitope may comprise or consist of a Dengue epitope that is not targeted by the antibody known as VIS513.

[0132] The viral epitope may be a West Nile virus epitope. The viral epitope may comprise or consist of an epitope that is targeted by the antibody known as WNV-86. The viral epitope may comprise or consist of a West Nile virus epitope that is not targeted by the antibody known as WNV-86.

[0133] The skilled person will recognise that targeting a viral epitope also includes targeting the protein in which the epitope is located.

[0134] The compositionThe composition comprising a plurality of chromosome-free bacterial minicells and / or simple cells may be a pharmaceutically acceptable composition. The composition may comprise one or more pharmaceutically acceptable excipients. The composition may comprise an aqueous carrier, such as water, in which the minicells and / or simple cells are suspended. The composition may comprise a buffer, such as PBS, in which the minicells and / or simple cells are suspended.

[0135] The composition comprising a plurality of chromosome-free bacterial minicells and / or simple cells may be formulated into a nasal spray or a formulation suitable for a nebulizer.

[0136] The composition may comprise a concentration of from 10 / mL to 10E13 / mL of the minicells and / or simple cells. The composition may comprise about 10E13 / mL of the minicells and / or simple cells.

[0137] The Bacterial Cell

[0138] The bacterial cell from which the minicells and / or simple cells are derived may comprise any suitable bacterial species. The bacterial cell from which the minicells and / or simple cells are derived may comprise any cell whose genome comprises a minC and / or a minD gene. In one embodiment, the bacterial cell from which the minicells and / or simple cells are derived is selected from the bacterial species of Escherichia spp., Pseudmonas spp., and Ralstonia spp. In one embodiment, the bacterial cell from which the minicells and / or simple cells are derived is Escherichia spp. The Escherichia spp. may be Escherichia coli. In another embodiment, the bacterial cell from which the minicells and / or simple cells are derived is Pseudmonas spp. The Pseudmonas spp. may be Pseudmonas putida. In another embodiment, the bacterial cell from which the minicells and / or simple cells are derived is Ralstonia spp. The Ralstonia spp. may be Ralstonia eutropha. In some embodiments wherein the bacterial cell from which the minicells and / or simple cells are derived is LPS , the bacterial cell may be ClearColi™ E. coli.

[0139] In one embodiment, the method of minicells and / or simple cells production comprises the production of a population (i.e. a plurality) of bacterial cells. In particular apopulation of bacterial cells may be provided and converted to minicells and / or simple cells by the methods herein.

[0140] The skilled person will appreciate that nucleic acid, such as plasmids, may be provided or transformed into the bacterial cell according to aspects of the invention in a number of ways. For example, heat-shock transformation or electroporation may be used to introduce nucleic acid, such as plasmids to bacterial cells. Different such methods may be employed sequentially, to transform different plasmids into the same cell. By way of non-limiting example, in embodiments comprising LPS cells wherein the minD locus is knocked out with a CRISPR-Cas system, the bacterial cells may first be made chemically competent for (heat-shock transformation with) a Cas-expressing plasmid and, subsequently, the transformed cells may be rendered electrocompetent for (electroporation with) a m / w / )-targcting (s)gRNA-expressing plasmid; the mini-SimCells thus derived may themselves be rendered chemically competent for a surface-displayed antigen-binding molecule expression plasmid and / or an accessory product expression plasmid.

[0141] Other aspects

[0142] The minicells and / or simple cells according to the specification may be derived through a variety of methods, non-limiting examples of which are reported herein throughout. Particular methods of minicell and / or simple cell derivation may be more appropriate for a given bacterial cell. Without wishing to be bound by theory, for example, a method of derivation of a mini-SimCell from a LPS bacterial cell, the method comprising prevention of functional MinD expression (such as by minD knock-out), may not require additional endonuclease activity to destroy the bacterial cell genome. Conversely, still without wishing to be bound by theory, a method of derivation of a mini-SimCell from a LPS+bacterial cell may comprise culturing of the cell in suboptimal conditions; and / or prevention of functional MinD expression such as by minD knock-out.

[0143] According to another aspect, there is provided a method for obtaining a chromosome-free bacterial cell (mini-SimCell) from a LPS bacterial cell, wherein the LPS mini-SimCell is smaller than about 200 nm along its axis, and optionally wherein the LPS mini-SimCell has surface-displayed antigen-binding molecules that target at least one viral protein or epitope, the method comprising the steps of:i) culturing the LPS bacterial cell and preventing functional expression in the LPS bacterial cell of at least one of the endogenous genes selected from: minD and minC, thereby forming a LPS mini-SimCell; and

[0144] ii) optionally transforming the LPS mini-SimCell with recombinant nucleic acid for expression of at least one fusion protein comprising or consisting of a antigenbinding molecule that targets at least one viral epitope or protein, and an outermembrane anchor polypeptide.

[0145] According to another aspect, there is provided a method for obtaining a population of chromosome-free bacterial minicells (mini-SimCells) from LPS+bacterial cells, wherein the LPS+mini-SimCells are smaller than about 200 nm along their axis, and wherein the LPS+mini-SimCells have surface-displayed antigen-binding molecules that target at least one viral epitope, the method comprising the steps of:

[0146] i) preventing functional expression in the LPS+bacterial cells of at least one of the endogenous genes selected from: minD and minC, whilst

[0147] culturing the LPS+bacterial cells in suboptimal growth conditions, to reduce the average size of the resulting LPS+mini-SimCells to less than about 200 nm along their axis; and

[0148] iii) transforming the LPS+mini-SimCells with recombinant nucleic acid for expression of at least one fusion protein comprising or consisting of an antigen-binding molecule that targets at least one viral protein or epitope, and an outer-membrane anchor polypeptide.

[0149] Suboptimal growth conditions may comprise the culture of the LPS+bacterial cells in a minimal growth medium (i.e. a nutrient restricted medium) and / or at a temperature that is suboptimal for its growth. Suboptimal growth conditions may be provided as described herein.

[0150] Killing and control of chromosome-containing or actively dividing bacterial cells

[0151] In an embodiment, the method of minicells and / or simple cells production may further comprise inducing the death (killing) of any bacterial cells in which the chromosomal DNA remains intact. In another embodiment, the method may further comprise inducing the death (killing) of any actively dividing bacterial cells. In an embodiment, the killing may comprise treatment of the bacterial cell with an agent capable of killing activelydividing cells, such as D-cycloserine. Suitably, the cell or population of cells, or the composition, may be treated with D-cycloserine between 16 and 24 hours after using an endonuclease. The skilled person will recognise that the agent capable of killing actively dividing cells, such as D-cycloserine, may be added at any time point, for example when the cell population reaches a desired concentration.

[0152] Alternatively or additionally, particularly but not exclusively in embodiments wherein the minicells and / or simple cells are not produced by means of an inducible nuclease (such as embodiments comprising LPS bacterial cells), the method of minicells and / or simple cells production may further comprise the death (killing) of any bacterial cells in which the chromosomal DNA remains intact, such as may arise due to suboptimal minD knockout. In other embodiments comprising no inducible nuclease, the method of minicells and / or simple cells production may further comprise the death (killing) of any actively dividing bacterial cells. In an embodiment, the killing may comprise treatment of the bacterial cell with at least one agent capable of killing actively dividing cells, such as D-cycloserine, cephalosporins (e.g. ceftriaxone and / or cefotaxime), or a penicillin (e.g. penicillin G). Suitably, the cell or population of cells, or the composition, may be treated with D-cycloserine and / or one or more cephalosporins and / or one or more penicillins between 16 and 24 hours after deletion of impairment of the minD locus. The skilled person will recognise that the agent capable of killing actively dividing cells, such as D-cycloserine and / or one or more cephalosporins and / or one or more penicillins, may be added at any time point, for example when the cell population reaches a desired concentration.

[0153] The agent capable of killing actively dividing bacterial cells, such as D-cycloserine, may be further dosed into the culture of minicells and / or simple cells to ensure that any actively dividing bacterial cells, or bacterial cells capable of actively dividing, are supressed or killed. In one embodiment, the agent capable of killing actively dividing bacterial cells, such as D-cycloserine, may be continuously or periodically dosed into the culture of minicells and / or simple cells to ensure that any actively dividing bacterial cells, or bacterial cells capable of actively dividing, are supressed or killed.

[0154] The agent capable of killing actively dividing bacterial cells may comprise an antibiotic capable of inhibiting bacterial cell-wall biosynthesis, such as D-cycloserine. The agent capable of killing actively dividing bacterial cells may comprise an antibiotic that is nota porin. The skilled person will be familiar with a range of known antibiotics that may be used to prevent the growth of, or kill, actively dividing bacterial cells.

[0155] The size of the minicells may be selected, for example by filtration. For example, larger cells greater than 200nm may be removed.

[0156] According to another aspect of the present invention, there is provided a chromosome-free bacterial minicell and / or simple cell, wherein the minicell and / or simple cell comprises no lipopolysaccharide (LPS), and wherein the minicell and / or simple cell comprises surface-displayed antigen-binding molecules that target at least one viral protein or epitope.

[0157] Methods of production for SimCells (which are larger than mini-SimCells) are described in US patent application US2022259605A1, incorporated herein in its entirety by reference. Minicells (also larger than mini-SimCells) are described in e.g. Seung-Jin Kim, Woojin Chang, Min-Kyu Oh, "Escherichia coli minicells with targeted enzymes as bioreactors for producing toxic compounds”, Metabolic Engineering, 73, 2022, 214-224, https: / / doi.Org / 10.1016 / j .ymben.2022.08.006. incorporated herein in its entirety by reference. The person skilled in the art will in general realise that many aspects disclosed in those documents may be easily applied to aspects and embodiments of the invention herein disclosed, occasionally with appropriate modifications that will appear plain to them.

[0158] According to another aspect, the invention provides a minicell and / or simple cell or a population of minicells and / or simple cells having surface-displayed antigen-binding molecules produced by the method of the invention herein.

[0159] According to another aspect, the invention provides a minicell and / or simple cell or a population of minicells and / or simple cells produced by the method of the invention herein.

[0160] According to another aspect, the invention provides a minicell and / or simple cell or a population of minicells and / or simple cells, comprising nucleic acid encoding a fusion protein comprising or consisting of a antigen-binding molecule and an outer-membrane anchor polypeptide.The population of mini-SimCells may have an average size of less than 200 nm. The population of mini-SimCells may not comprise minicells greater than 200 nm in size.

[0161] The chromosome-free bacterial minicell(s) and / or simple cell(s) may further comprise nucleic acid encoding an accessory product. The accessory product may be an enzyme, such as a prodrug-converting enzyme, for example, NahG.

[0162] According to another aspect, the invention provides a composition comprising a population of minicells and / or simple cells according to the invention, or made according to the method of the invention.

[0163] The composition may be a pharmaceutical composition. The composition may comprise one or more pharmaceutically acceptable excipients. In one embodiment, the composition comprises buffer or saline. The composition may be sterile (i.e. free of living / replicating organisms).

[0164] According to another aspect, the invention provides a minicell and / or simple cell or a population of minicells and / or simple cells according to the invention, or a composition according to the invention, for use as a medicament.

[0165] According to another aspect, the invention provides a minicell and / or simple cell or a population of minicells and / or simple cells according to the invention, or a composition according to the invention, for use in treating or preventing a disease or condition in a subject.

[0166] According to another aspect, the invention provides a minicell and / or simple cell or a population of minicells and / or simple cells according to the invention, or a composition according to the invention, for use in the manufacture of a medicament, for example for treating or preventing a disease or condition in a subject.

[0167] According to another aspect, the invention provides a method of treatment or prevention of disease or condition, the method comprising the administration of a minicell and / or simple cell or a population of minicells and / or simple cells according to the invention, or a composition according to the invention to a subject in need thereof.Administration of the minicells and / or simple cells or composition may be supplemented by administration of other medicaments and / or pro-drugs, such as antiviral therapy and / or aspirin, either concurrently, before, or after minicells and / or simple cells are administered. Administration may be performed transnasally, such as by aerosol. The methods of this invention may be practiced using any mode of administration that is medically acceptable, and produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include, but are not limited to, oral, buccal, sublingual, inhalation, mucosal, rectal, intranasal (or transnasal), topical, ocular, periocular, intraocular, transdermal, subcutaneous, intra-arterial, intravenous, intracerebroventricular, intramuscular, parenteral, or infusion methodologies. In a specific embodiment, it may be desirable to administer the pharmaceutical complexes of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion, by injection, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibres.

[0168] The subject may be a mammal and is preferably a human, but may alternatively be a monkey, ape, cat, dog, sheep, cow, horse, rabbit or rodent.

[0169] In one embodiment, the disease to be treated or prevented comprises COVID-19. In one embodiment, the disease to be treated or prevented comprises flu. In one embodiment, the disease to be treated or prevented comprises one or more of COVID-19, flu, hepatitis, West Nile fever, Dengue fever, Ebola infection, AIDS, RSV infection, bronchitis, rhinitis, a cold, laryngitis, sinus infection (e.g. sinusitis), nasopharyngitis, pharyngitis, epiglottitis, laryngotracheitis, or tracheitis.

[0170] The use as a medicament may be for treatment of COVID-19. The use as a medicament may be for treatment of the flu. The use as a medicament may be for treatment of a cold. The use as a medicament may be for treatment of one or more of COVID-19, flu, hepatitis, West Nile fever, Dengue fever, Ebola infection, AIDS, RSV infection, bronchitis, rhinitis, a cold, laryngitis, sinus infection (e.g. sinusitis), nasopharyngitis, pharyngitis, epiglottitis, laryngotracheitis, or tracheitis.In embodiments wherein the composition or population of minicells and / or simple cells is used as a medicament for the treatment of respiratory infections, such as COVID-19, administration of the composition may be performed transnasally or by inhalation i.e. the composition may be formulated as an aerosol.

[0171] According to another aspect, the invention provides the use of a minicell and / or simple cell or a population of minicells and / or simple cells having surface-displayed antigenbinding molecules according to the invention for targeting a cell, such as a virally infected cell, for binding with the minicells and / or simple cells.

[0172] The cell may be targeted for diagnosis, such as using the minicells and / or simple cells as a marker for imaging.

[0173] The minicell and / or simple cell or a population of minicells and / or simple cells having surface-displayed antigen-binding molecules according to the invention may be used as a biosensor of an analyte. The analyte may be a cell, such as a virally infected cell. The analyte / cells, such as virally infected cells, may be targeted for agglutination by crosslinking the cells with the minicells and / or simple cells.

[0174] In another embodiment, cells, such as virally infected cells, may be targeted for killing, for example by targeted drug delivery. For example, the minicells and / or simple cells may further comprise or express an accessory product, such as NahG, which can convert a pro-drug to a cytotoxic drug. For example, in the case of NahG, the pro-drug may be aspirin which is converted to catechol, which is toxic to cells. The minicells and / or simple cells can be localised / targeted, for example to a virally infected cell, by the surface anchored antigen-binding molecule.

[0175] In one embodiment, the accessory product may be expressed in the bacterial cell prior to conversion into minicells and / or simple cells. For example, prior to chromosome degradation.

[0176] According to another aspect, the invention provides a method of producing a product (i.e. an accessory product for expression as described herein), and / or a biochemical, the method comprising use of the minicells and / or simple cells according to the invention, or a composition according to the invention, to express an accessory product.The method of producing an accessory product, and / or a biochemical may be in vitro or in vivo. The method of producing an accessory product and / or a biochemical may comprise incubating the minicells and / or simple cells according to the invention, or a composition according to the invention under conditions to promote expression of the product for expression. In an embodiment where the expression of the accessory product is regulated, the method may comprise the use of an inducer. For the production of a biochemical, a precursor or intermediate molecule may be provided for conversion to the biochemical. The skilled person will readily apply appropriate conditions, such as the appropriate temperature, gas, media, nutrients, energy sources, and carbon sources, for the minicells and / or simple cells to be active and capable of product expression.

[0177] In an embodiment wherein the minicells and / or simple cells according to the invention, or a composition according to the invention, is used as a biosensor, the minicells and / or simple cells may be arranged to express a polypeptide or peptide capable of binding to a target molecule to be detected.

[0178] Where reference is made to a variant polypeptide or nucleotide sequence, the skilled person will understand that one or more amino acid residue or nucleotide substitutions, deletions or additions, may be tolerated, optionally two substitutions may be tolerated in the sequence, such that it maintains its function. The skilled person will appreciate that 1, 2, 3, 4, 5 or more amino acid residues or nucleotides may be substituted, added or removed without affecting function References to sequence identity may be determined by BLAST sequence alignment (www.ncbi.nlm.nih.gov / BLAST / ) using standard / default parameters. For example, the sequence may have 99% identity and still function according to the invention. In other embodiments, the sequence may have 98% identity and still function according to the invention. In another embodiment, the sequence may have 95% identity and still function according to the invention. In another embodiment, the sequence may have 90%, 85%, or 80% identity and still function according to the invention. In one embodiment, the variation and sequence identity may be according to the full length sequence. In other embodiments, the variation may be limited to non-conserved sequences and / or sequences outside of active sites, such as binding domains. Therefore, an active site or binding site of a protein may be 100% identical, whereas the flanking sequences may comprise the stated variations in identity. Such variants may be termed “conserved active site variants”.Amino acid substitutions may be conservative substitutions. For example, a modified residue may comprise substantially similar properties as the wild-type substituted residue. For example, a substituted residue may comprise substantially similar or equal charge or hydrophobicity as the wild-type substituted residue. For example, a substituted residue may comprise substantially similar molecular weight or steric bulk as the wild-type substituted residue. With reference to “variant” nucleic acid sequences, the skilled person will appreciate that 1, 2, 3, 4, 5 or more codons may be substituted, added or removed without affecting function. For example, conservative substitutions may be considered.

[0179] By “antibody” we include substantially intact antibody molecules, as well as chimeric antibodies, human antibodies, humanised antibodies (wherein at least one amino acid is mutated relative to the naturally occurring human antibodies), single chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and / or light chains, and antigen binding fragments and derivatives of the same. In particular, the term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen, whether natural or partly or wholly synthetically produced. The term also covers any polypeptide or protein having a binding domain which is, or is homologous to, an antibody binding domain. These can be derived from natural sources, or they may be partly or wholly synthetically produced. Examples of antibodies are the immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD and IgA) and their isotypic subclasses; fragments which comprise an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies may be polyclonal or monoclonal. A monoclonal antibody may be referred to as a “mAb”.

[0180] It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which retain the specificity of the original antibody. Such techniques may involve introducing DNA encoding the immunoglobulin variable region, or the CDRs, of an antibody to the constant regions, or constant regions plus framework regions, of a different immunoglobulin. See, for instance, EP-A-184187, GB 2188638A or EP-A-239400, incorporated herein by reference. A hybridoma or other cell producing an antibody maybe subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.

[0181] As antibodies can be modified in a number of ways, the term “antibody” should be construed as covering any specific binding member or substance having a binding domain with the required specificity. Thus, this term covers antibody fragments, derivatives, functional equivalents and homologues of antibodies, humanised antibodies, including any polypeptide comprising an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023, incorporated herein by reference. A humanised antibody may be a modified antibody having the variable regions of a non-human, e.g., murine, antibody and the constant region of a human antibody. Methods for making humanised antibodies are described in, for example, US Patent No. 5225539, incorporated herein by reference.

[0182] The antibodies of the present disclosure may be intact or engineered. For example, the antibody may be fully or partially glycosylated and / or selected for increased or diminished binding to human effector systems such as complement, FcR-bearing effectors, such as macrophages, or to extend or reduce half-life. These modifications can be made to improve effectiveness and potentially also reduce toxic side effects.

[0183] It has been shown that fragments of a whole antibody can perform the function of binding antigens. Examples of binding fragments of the invention are (i) the Fab fragment consisting of VL, VH, CL and CHI domains; (ii) the Fd fragment consisting of the VH and CHI domains; (iii) the Fv fragment consisting of the VL and VH domains of a single antibody; (iv) the dAb fragment which consists of a VH domain; (v) isolated CDR regions; (vi) F(ab’)2 fragments, a bivalent fragment comprising two linked Fab fragments; (vii) single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form an antigen binding site; (viii) bispecific single chain Fv dimers (PCT / US92 / 09965, incorporated herein by reference) and; (ix) “diabodies”, multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804, incorporated herein by reference).References to the use of the minicells and / or simple cells in therapy may refer t the use of a therapeutically effective amount. A ‘therapeutically effective amount’, or ‘effective amount’, or ‘therapeutically effective’, as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen. This is a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e. a carrier or administration vehicle. Further, it is intended to mean an amount sufficient to reduce and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. As is appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity. Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent. In the methods and use for manufacture of compositions of the invention, a therapeutically effective amount of the active component is provided. A therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.

[0184] The skilled person will appreciate that preferred features of any one embodiment and / or aspect of the invention may be applied to all other embodiments and / or aspects of the invention.

[0185] Brief Description of the Figures

[0186] Figure 1. (a) A schematic illustration demonstrating nanobody-expressing SimCells and mini-SimCells capturing SARS-CoV-2 viral particles, thereby blocking the interaction between the spike RBD and human ACE2 to prevent infection, (b) Architectures of constructs utilized in the flow cytometry experiment: pNV_VHH and pNV_negative control. The Nanobody display system includes Intimin as the outer membrane anchor and VHH as the nanobody used in this study, encompassing TY1, NIH-CoVnb-112, and the bispecific Nb6 VE for SARS-CoV-2 RBD binding. J23100 regulates the expression of the nanobody display system, while Myc serves as the tag for flow cytometry analysis, (c) Histograms from flow cytometry of E. coli ClearColi™ DE3carrying the pNV nanobody display system. Samples were treated with both primary anti-Myc antibody and secondary Alexa Fluor 488 antibody. The X-axis represents the FL1 channel (Alexa Fluor 488 antibody) in relation to the cell count on the Y-axis. Untransformed ClearColi™ DE3 and ClearColi™ transformed with the negative control plasmid pNV_ served as controls. Fluorescent values are reported as median fluorescence intensity (MFI).

[0187] Figure 2. (a) pNV_non-specific VHH as a negative control. The Nanobody display system includes Intimin as the outer membrane anchor and VHH as the nanobody, featuring TY1, NIH-CoVnb-112, and the bispecific Nb6 VE for SARS-CoV-2 RBD binding. Nanobody 2Rsl5d, targeting human epidermal growth factor receptor 2 (HER2), served as the non-specific control. J23100 controls the expression of the nanobody display system, with sfGFP acting as the fluorescence reporter for imaging, (b) Cell agglutination test of nanobodyexpressing strains: (i) TY1, NB6, and NIH112; (ii) VE; with pNV_antiHER2 VHH_sfGFP serving as the negative control (NC). Positive binding between the displayed nanobodies and the targeted antigen (SARS-CoV-2 RBD) results in a cloudy cell suspension, whereas no binding results in a cell pellet. Red boxes indicate positive agglutination (cloudy cell suspension). Images were captured using the VersaDoc Imaging System under the FITC channel, (c) 42 nM of SARS-CoV-2 RBD was affixed to the bottom of the well. OD600=1 washed pNV TYl sfGFP, and pNV_antiHER2_sfGFP (NC) were added to the well and incubated for 1 hour at room temperature. Images were taken with a BioTek Cytation5 imaging reader using the excitation / emission wavelength of 488 / 530 nm for sfGFP detection. Cell adhesion was observed with RBD-targeting pNV_TYl_sfGFP only; the NC did not exhibit any cell adhesion to the SARS-CoV-2 RBD-coated microplate.

[0188] Figure 3. SimCell protein-protein Sars-Cov-2 RBD blocking Assays (a) A schematic of the workflow for the RBD-hACE2 protein-protein neutralization assay. Purified nanobody-displaying SimCells were induced, purified, and diluted to OD600=2 with IX PBS. The diluted SimCells were pre-incubated with HRP-conjugated RBD for 1 hour statically at 37 °C. The pre-incubation mixture was then added to ACE2-coated 96-well microplates. Colorimetric measurement was performed using the chromogenic substrate 3,3',5,5'-tetramethylbenzidine(TMB) (Invitrogen), which reacts with HRP on the RBD. Subsequently, the stop solution was added to yield a yellow color, measurable at 450 nm using a Tecan Spark plate reader. A high OD450nm reading indicates significant binding of HRP-RBD to ACE2 on the plate, while a low or no OD450nm reading suggests minimal or no binding of HRP-RBD to ACE2. (b) Neutralization Assays with Wuhan variant RBD: (i) using pNV_Nb6 sfGFP whole-cell and pNV_Nb6 sfGFP SimCell, and (ii) pNV_VE sfGFP whole-cell and pNV_VE sfGFP SimCell; Neutralization was compared with controls showing no binding and non-specific counterparts anti-HER2, labelled as unspecific cells. Wuhan HRP-RBD concentrations of 0, 4.3, 5.4, 6.5, 8.1, 13, 16.2, and 32.4nM were used. Both nanobody-displaying whole cells and purified SimCells were washed and diluted with IxPBS, then HRP-RBD pre-incubated with the washed cells for 1 hour at 37°C before addition to the ACE2-coated plate for 1 hour at room temperature. The microplate was washed five times with 1XPBST to remove unbound HRP-RBD, followed by the sequential addition of an equal volume of TMB and stop solution to yield an OD450nm reading, indicative of RBD-hACE2 binding. Error bars represent the standard deviation from three biological replicates (n=3). (iii) Neutralization Assays with the South African (Beta) variant RBD using pNV_Nb6 sfGFP whole-cell and bispecific pNV_VE sfGFP whole-cell; blocking efficiency was compared with controls showing no binding and non-specific counterparts anti-HER2, identified as the unspecific cell. South African variant HRP-RBD concentrations of 0, 4.3, 5.4, 6.5, 8.1, 13, 16.2, and 32.4 nM were selected. Error bars represent the standard deviation from three biological replicates (n=3).

[0189] Figure 4. Neutralization curves of anti-S RBD nanobody displaying mini-SimCell. (a) Neutralization curves of NB6 anti-Spike RBD monomeric nanobody-expressing ClearColi™ mini-SimCells and VE anti-Spike RBD bivalent nanobody-expressing ClearColi™ mini-SimCells against the Victoria and B.1.351 (Beta) variants. Mini-SimCells expressing a non-binding (anti-HER2) nanobody served as a negative control. Mini-SimCells were serially diluted 10-fold dilution for 5 times, starting from 5x lOlo / mL cells down to 5x lO5 / mL cells. For the assay, 50 pL of the mini-SimCell samples were mixed with 200 foci / 25pL / well of viral particles. The XBB-9 antibody, known for its neutralizing capability against both Victoria and Beta variants, was used as apositive control. Error bars represent the standard error (S.E.M) from two replicates (n=2).

[0190] Figure 5. (a) OD600 growth curve of the ClearColi™ strain containing ICeuI, referred to as ClearColi™ 12x, for SimCell conversion. The purple arrow marks the addition of an inducer for ICeuI expression at the 3-hour time point. The blue line represents the growth curve of the uninduced ICeuI, while the red line depicts the growth curve following ICeuI induction and ClearColi™ SimCell conversion. Error bars indicate the standard deviation from three biological replicates, (b) 50 pL of either induced or uninduced ClearColi™ 12x culture from the growth curve experiment was spotted onto an LB -only agar plate and incubated overnight at 37 °C in a static incubator to assess SimCell purity.

[0191] Figure 6. Purification procedure and characterisation for ClearColi™ SimCells (a) Schematics of the purification workflow for ClearColi™ SimCell conversion utilizing either LB media or minimal media exchange to yield SimCells of high purity and uniform sizes, (b) Fluorescence microscopic images of the ClearColi™ cells before and after induction into SimCells, using different media types. The magnification is 20x, with a scale bar of 10pm presented for reference.

[0192] Figure 7. (a) Top-down image of pNV_Nb6 sfGFP whole-cell and pNV_Nb6 sfGFP SimCell neutralization assay plate. The neutralization assay was repeated for three biological replicates, (b) (i) Calibration standard curve for Wuhan HRP-RBD. The equation of the standard curve is displayed in the top-right corner of the graph: y (OD450nm) = 0.0367 x (HRP-RBD concentration), (ii) Calibration standard curve for the South African variant HRP-RBD. The equation of the standard curve is displayed in the top-right corner of the graph: y (OD450nm) = 0.1083 x (HRP-RBD concentration).

[0193] Figure 8. Neutralization curves of anti-S RBD nanobody NB6 displaying SimCell and mini-SimCell. This panel illustrates the neutralization curves of (a)NB6 anti-Spike RBD monomeric nanobody-expressing ClearColi SimCell and (b) mini-SimCells, and with non-binding (anti-HER2) nanobody-expressing SimCell / mini-SimCells serving as a negative control, against the Victoria andB.1.351 (Beta) variants. SimCells were serially diluted from 10A9 / mL to 10A4 / mL. Mini-SimCells underwent a 10-fold serial dilution five times, starting from 5><10A10 / mL to 5><10A5 / mL. For the assay, 50pL of SimCell / mini-SimCell samples were mixed with 200 viral foci in 25pL per well, (c) neutralization curves of the XBB-9 antibody, known for its neutralization capability against both the Victoria and Beta variants, was used as a positive control.

[0194] Figure 9. Plasmid map for expression of a nanobody and NahG.

[0195] Figure 10. OmpA system for surface display in minicells and simple cells. A - shows the Spycatcher / Spytag OmpA surface display system. B - shows results of display. Catechol production test - After aspirin was added to the flask to a final concentration 800 pM and cultured at 25 and 180 rpm for 16 hours, the surface display NahG in BL21 showed the color change due to the conversion to catechol from aspirin. Note: Error bars represent the standard deviation from 3 replicates. Statistical test performed is 1-way ANOVA,***p<l x 10’5.

[0196] Example

[0197] The COVID- 19 pandemic has had an unprecedent impact on global social life, healthcare systems, and economies, with a long term effects. The protection of COVID-19 vaccines has limited protection to prevent the infection, although they can significantly reduce the risk of severe illness and death. Hence, there is an urgent need for effective interventions to tackle the ongoing challenges posed by the COVID-19 pandemic and to better prepare for future pandemics. In this study, we have developed a genome-free platform - SimCells and mini-SimCells, to effectively neutralise SARS-CoV-2 virus. The SimCells and mini-SimCells are derived from an LPS-free E. coli strain (ClearColi™), have been engineered to display nanobodies on the surface, specifically targeting the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein — a key immunogenic fragment critical for viral entry into host cells. We initially assessed the nanobody display on the surface of ClearColi™ using immunostaining and flow cytometry. We then demonstrated that nanobody-expressing SimCells achieved over 90% neutralization efficiency against both the original Wuhan strain and the B.1.351 (Beta) variant using an in vitro protein-protein blocking assay. Finally, we showed the effective neutralization capability of NB6 nanobody-presenting mini-SimCells against live Victoria at IC50 values of 2.95 x 109± 1.40 x 108cells / mL and VE nanobody-presenting mini-SimCells against B.1.351 (Beta) variants of SARS-CoV-2 virus with IC50 of 5.68 x 109± 9.94 x 108mini-SimCells / mL. The results show that the SimCell-based neutralisation could be a promising strategy for both prevention and treatment of SARS-CoV-2, as well as other viral infections.

[0198] To neutralize SARS-CoV-2 fusing to the host cell, we targeted the RBD region of spike protein, a critical region that allows the virus to gain entry to the host cell. Studies identified three classes of SARS-CoV-2 neutralizing nanobodies: Class 1 nanobodies physically block the binding between spike protein and hACE-2 receptor with competitive binding. Class 2 nanobodies recognise a highly conserved epitope on RBD, which are often inaccessible by conventional antibodies. Class 3 nanobodies stabilize spike protein in the post-fusion conformation, preventing binding to the hACE2. In this study, we developed the modular nanobody display platform to readily express RBD-neutralising nanobodies on the surface of the SimCell chassis. We selected four nanobody candidates: TY1, NIH-CoV2nb-l 12, mNb6 and bivalent nanobody VE. TY1 is a monomeric nanobody isolated from immunized Alpaca, which showed high affinity towards the SARS-CoV-2 RBD (described in Lan, J., Ge, J., Yu, J. et al. Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor. Nature 581, 215-220 (2020). https: / / doi.org / 10.1038 / s41586-020-2180-5. which is incorporated herein by reference). TY1 competitively binds to SARS-CoV-2, physically hindering the interaction between the virus and the human receptor. NIH-CoVnb-112 from the phage display library exhibited competitive binding to RBD and showed high neutralization efficiency against pseudo-typed lentivirus. mNB6 is a monomeric nanobody isolated from the yeast display library, which showed high sensitivity and efficient neutralization at a low nanomolar range. Both mNb6 and TY1 can stabilize spikes in the post-fusion form, preventing the interaction between spike protein and hACE2 from cell fusion. Lastly, bivalent nanobody VE consists of nanobody V and nanobody E joint by a neutral protein linker. V and E each bind to a distinct epitope on RBD, one diverse and one conserved. VE showed promising results for neutralising SARS-CoV-2 variants and simultaneously targeting two epitopes, effectively preventing the emergence of mutational escape.

[0199] In our research, we developed LPS-free and non-replicating bacterial chassis, SimCells (1-2 pm) and mini-SimCells (100-400 nm), as safe carriers for displaying anti-SARS-CoV-2 RBD nanobodies in a ‘plug-and-play’ manner. We initially verified the blocking efficiency of nanobody-expressing SimCells through an in vitro blocking assay. We thenconfirmed the safety of LPS -negative mini-SimCells through intranasal administration in mouse models and validated the neutralization efficiency of mini-SimCells against live Victoria and Beta variants using a live virus neutralization assay. Our aim was to explore the therapeutic potential of nanobodies in conjunction with the biological benefits of mini-SimCells to forge a highly effective, stable, cost-efficient, and safe 'smart particle.' This innovative strategy holds promise as both a preventive and neutralizing agent, applicable not only to SARS-CoV-2 infections but also to other viral infectious diseases.

[0200] Results

[0201] Confirmation of nanobody surface display on E. coli ClearColi™

[0202] The specific LPS-free E. coli mutant ClearColi™ has been chosen to express surface display of nanobodies, because LPS-free ClearColi™ avoids the unnecessary stimulation of TCR in immunity system, and the smooth surface of ClearColi™ enables the small nanobody (~2-4 nm) exposed to the targeted antigen. We have adopted the pNV surface display system driven by a strong promoter J23100 (http: / / parts.igem.org / Catalog) to express SARS-CoV-2 RBD binding nanobodies, TY1, NIH-112, NB6, and a bivalent nanobody VE (Fig. la). We constructed all plasmids using NEB HiFi assembly, and we used immunofluorescence followed by flow cytometry to confirm the surface display of nanobodies on Escherichia coli ClearColi™. For immunofluorescence tagging, we used a primary anti-Myc tag antibody to bind Myc-tagged nanobodies, followed by the detection using secondary Alexa Fluor 488 conjugated antibody to produce a fluorescence signal. The immunofluorescence samples were analysed using a fluorescent flow cytometer FACS (Fig. lb), which showed stronger fluorescence signals in the nanobody expressing Escherichia coli ClearColi™ (DE3) strains: pNV_TYl, pNV_NB6, pNV_NIH112 and pNV_VE (Table SI) compared to the control of ClearColi™ (DE3) WT. The results suggest that the nanobodies should have been successfully displayed on the surface of ClearColi™.

[0203] <

[0204]

[0205]

[0206]

[0207] Table SI.

[0208] Tables S2 and S3 list primers and DNA gblocks (respectively) used in this Example.

[0209] >

[0210]

[0211] Table S2.

[0212]

[0213]

[0214]

[0215] Table S3.

[0216] Nanobodies displaying on E. coli ClearColi™ binding to SARS-CoV-2 RBD

[0217] Next, we conducted a cell agglutination test to confirm ClearColi™ expressing nanobodies binding to the SARS-CoV-2 RBD and to evaluate the multivalency of the nanobodies. This cell agglutination test, serving as a biological analog to the well-established in vitro latex agglutination test, involves the formation of cross-links between multivalent nanobodies displaying on the surface of bacterial cells and thetarget antigen. This interaction results in cell agglutination and shows a visible cloudy suspension. In contrast, in the absence of the target antigen, the cells settle at the bottom of the well, forming a pellet. A GFP reporter gene was cloned into all pNV_ constructs at this stage for better visualisation (shown as pNV_sfGFP, Figure 2a). We selected the RBD antigen concentration ranging from 0.83 to 42 nM. For the negative control, we used the anti-HER2 nanobody, 2Rsl5d, known for its specific binding to human epidermal growth factor receptor 2 (HER2) which is unspecific to RBD antigen. The antigens were mixed with an equal concentration of cell cultures in a round bottom well plate and incubated the plate overnight at room temperature. We observed agglutination across all concentrations with pNVNB6_sfGFP, while pNV_NIHl 12_sfGFP only exhibited binding at the highest concentration of 42 nM RBD. The pNVTYl sfGFP and the negative control pNV_antiHER2_sfGFP showed no agglutination for all of the RBD concentrations (Fig. 2b). The results demonstrated the selective RBD binding function of NB6 and NIH112, with NB6 detecting antigens at the lower molar concentrations. Therefore, we concluded that pNVNB6_sfGFP had the highest sensitivity targeting against SARS-CoV-2 RBD. We confirmed the surface expression of a 30 kDa bispecific nanobody on the surface, VE, consisting of nanobody V and nanobody E joint by a neutral protein linker using flow cytometry (Figure 1c). In the cell agglutination assay, pNV VE sfGFP could detect RBD antigen up to 4.2nM (Fig. 2b). To further validate the binding of pNVTYl sfGFP to SARS-CoV-2 RBD, we performed whole-cell ELISA by fixing the SI RBD antigen onto microplates and incubating pNVTYl sfGFP in the RBD coated plate. Fluorescence microscopic images captured by BioTek Cytation5 imaging reader showed that only pNVTYl_sfGFP expressing cells could adhere onto the SARS-CoV-2 RBD coated plate, whilst the negative control pNV antiHER2_sfGFP cells were washed away, unable to bind to the plate. This further underscored the specific binding of pNVTYl_sfGFP to its target RBD (Fig. 2c). This result also suggests that TY1 nanobody is not multivalent as the ClearColi™ displaying TY1 failed to form cross-links in the agglutination assay. Hence, we selected ClearColi™ displaying NB6 and VE as our candidates for further study.

[0218] SimCell mediated protein-protein blocking assay to SARS-COV-2 RBD

[0219] To investigate neutralization efficacy, we focused on two hypotheses involving NB6 and VE. NB6 is known for its highly sensitive binding to the RBD at low concentrations, while VE can bind to two distinct RBD epitopes, potentially mitigating viral mutational escape. We utilized an in vitro neutralization assay that replicates the hACE2-RBDprotein-protein interaction to mimic the hACE2 receptor - virus interaction. This assay tested the neutralization efficiency of pNV_NB6 sfGFP and pNV_VE sfGFP SimCells against the Wuhan SARS-CoV-2 variant RBD and the South African (Beta) SARS-CoV-2 variant RBD, both sourced from GenScript, in their binding to hACE2 provided by Thermo Fisher (Fig. 3a). The negative control for the assay was pNV_anti-HER2, a nanobody targeting HER2, which lacks specificity for the SARS-CoV-2 RBD. After washing with IX PBST to remove any unbound HRP-RBD, a significant decrease in the colorimetric signal was observed with NB6-expressing ClearColi™ and its SimCells, indicating a highly efficient blockade of the RBD-hACE2 interaction (Figure S3a). At the highest concentration of the RBD protein, NB6 whole cells achieved an average blocking efficiency of 95.4% ±0.07%while NB6 SimCells reached 98.7% ±0.18% for the Wuhan variant RBD, confirming the effective blocking of RBD binding to hACE-2 by NB6-expressing ClearColi™ and its SimCells (Figure 3b(i)) . Additionally, pNV_VE sfGFP ClearColi™ exhibited significant blocking against the Wuhan variant, with 96.6%% ±0.07% efficiency in whole cells and 98.6%% ±0.16% in VE-expressing SimCells (Figure 3b(ii)). VE-expressing SimCells also neutralized 94.4% ±0.10% of the South African variant RBD, which NB6 only showed a 53.7%% ±1.39% blocking effect (Fig. 3b(iii)). These results support our hypotheses and suggest that monomeric NB6 should specifically target a unique epitope on the RBD, while VE could bind to two distinct epitopes, one of which is shared between the Wuhan and South African (Beta) variants.

[0220] SimCells and mini-SimCells neutralisation of live virus

[0221] While we have successfully demonstrated that NB6 and VE displaying SimCells effectively bind the Spike RBD in a protein-protein in vitro blocking assay, we intended to further evaluate the neutralization efficacy of our mini-SimCells against live SARS-CoV-2 variants. We conducted neutralization assays using NB6 and VE expressing mini-SimCells against the Victoria strain (SARS-CoV-2 / human / AUS / VIC01 / 2020) and the South African B.1.351 variant (Figure 4a). The live virus neutralization assay performed with SARS-CoV-2 Victoria on Vero E6 cells, demonstrating neutralizing activity for both NB6 and VE displaying mini-SimCells, with IC50 values of 2.95 x 109± 1.40 x 108cells / mL and 3.60 x 109± 1.53 x 109cells / mL, respectively. When assessing the neutralization against the South African B.1.351 variant using the live virus neutralisation assay, VE displaying mini-SimCells yielded an IC50 of 5.68 x 109± 9.94X 108cells / mL, while NB6 displaying mini-SimCells showed no neutralization effect (Table 2). These findings are consistent with our earlier protein-protein blocking assays, underscoring the targeted neutralizing ability of VE displaying mini-SimCells against different variants. In addition, the neutralization effect of NB6 SimCells was tested, yielding an IC50 of 4.78xl08cells / mL, which is shown in Figure S4 and Table S3.

[0222] Table 2 shows the half-maximal inhibitory concentration (IC50) of each experimental group against the Victoria and Beta viral variants. IC50 (number of cells / mL)

[0223] >

[0224]

[0225] > >

[0226] Table 2.

[0227] Discussion

[0228] As of 2024, the economic and social repercussions of the COVID-19 pandemic continue to affect human society. The emergence of more transmissible and potentially vaccineresistant variants underscores the reality that it requests cost-effective prevention and treatment strategies along with vaccines to confront with future viral threats. Neutralizing nanobodies have emerged as promising contenders in this scenario, demonstrating significant preclinical efficacy. They offer a versatile approach to combat variants that could diminish vaccine effectiveness.

[0229] In response to this challenge, our study explored the use of a modular nanobody display system in LPS-free ClearColi™. We demonstrated the system's flexibility by readily expressing four distinct SARS-CoV-2 nanobodies (TY1, NIH-CoVnb-112, Nb6, and the bivalent nanobodies VE) in a ‘plug-and-play’ fashion. Our simple cell agglutination assay confirmed the engineered bacteria's ability to bind the desired antigen, SARS-CoV-2's receptor-binding domain (RBD), with pNV_Nb6 sfGFP detecting antigen concentrations as low as 0.83 nM (Fig. 2b). Notably, the multivalent nanobody VE showed binding capabilities up to 4.2 nM of RBD (Fig. 2b), showing the potential for broad-spectrum neutralization.To simulate virus-host interactions, we developed an in vitro protein-protein blocking assay using human ACE2 and HRP-conjugated RBDs. The nanobody-displaying SimCells showed highly effective blocking capabilities, with NB6 and VE displaying SimCells inhibiting the interactions between the SARS-CoV-2 RBD and hACE2 by 98.7% and 98.6%, respectively. Furthermore, the bivalent nanobody VE achieved more than 94.4% neutralization against the RBD of the South African variant B.1.351. To evaluate the neutralization efficiency of our nanosized mini-SimCells, we conducted a live virus neutralization assay. The IC50 values of NB6 and VE expressing mini-SimCells were determined to be 2.95 x 109± 1.40 x 108and 3.60 x 109± 1.53 x 109mini-SimCells / mL against the Victoria variant, respectively. VE also demonstrated a neutralization effect against the Beta variant with an IC50 of 5.68 x 109± 9.94 x 108mini-SimCells / mL. Given the monoclonal antibody's molecular weight 150 kDa, the IC50 of XBB-9 monoclonal antibody is comparable, about 4 x 109XBB-9 / mL (Fig. 4 and Table 2). These results demonstrate the modularity and potential of nanobodydisplaying SimCells and mini-SimCells as powerful tools for neutralizing SARS-CoV-2, including its emerging variants.

[0230] Given the dynamic nature of the pandemic, our findings underscore the critical role of innovative therapeutic strategies such as nanobody-displaying mini-SimCells. Since nanobody-expressing SimCells and mini-SimCells can be readily produced without the need for a purification process, the cost of production can be significantly lower than that of purified antibody drugs. These platforms offer promising avenues for developing prophylactic and therapeutic interventions against SARS-CoV-2, particularly as components of a broader strategy that includes vaccination, public health measures, and global cooperation to combat current and future threats posed by this virus.

[0231] Materials and Methods

[0232] Strains, Cell Cultures and DNA manipulation. In this study, we utilized competent Escherichia coli DH5a strains for routine cloning and plasmid maintenance. For the expression of nanobodies and subsequent experiments, Escherichia coli ClearColi™ (DE3) strains were employed, unless specified otherwise.

[0233] All Escherichia coli cells were cultured in LB Miller broth (Sigma Aldrich) and plated on LB Miller agar (Sigma Aldrich), followed by incubation at 37 °C with shaking at250 rpm to ensure proper aeration. For SimCell conversion, we utilized M9 minimal medium. The 5X M9 minimal salts (Sigma Aldrich) composition included KH2PO4 (15 g / L), NaCl (2.5 g / L), Na2HPO4(33.9 g / L), and NH4C1 (5 g / L). The IX M9 medium was supplemented with IX trace elements (prepared according to the recipe available at: https: / / static.igem.org / mediawiki / 2019 / 2 / 20 / T— Tuebingen— M9 recipe.pdf). 1 mM MgSCh, 0.3 mM CaCh, 0.4% (w / v) glucose, and 0.2% (w / v) casamino acids. The antibiotics used were carbenicillin at 100 pg / mL, chloramphenicol at 34 pg / mL, and kanamycin at 50 pg / mL.

[0234] All enzymes required for molecular cloning were sourced from New England Biolabs (NEB). Plasmid construction was carried out using NEBuilder HiFi DNA Assembly, and DNA amplification for assembly purposes was conducted with Q5 high-fidelity DNA polymerase.

[0235] Escherichia coli competent cell preparation and transformations. General molecular cloning was performed using NEB 5 -a high-efficiency competent cells. To prepare competent E. coli cells (DH5a / ClearColi™ BL21(DE3)), we initiated the process by growing an overnight culture in LB medium. The following day, we diluted 1 mb of the overnight culture into 50 mb of LB broth in a sterile 250 mb Erlenmeyer flask and incubated the culture at 37 °C with shaking at 150 rpm until the OD600 reached 0.5-0.6. During this growth period, we prepared and filter-sterilized 25 mb each of 0.1 M CaCh and 0.1 M MgCh solutions, keeping them on ice. After the desired OD600 was achieved, the culture was cooled on ice for 30 minutes, then transferred to a 50 mb Falcon tube and centrifuged at 1,000g for 10 minutes at 4 °C. The supernatant was discarded, and the cell pellet was gently resuspended in chilled 25 mb of 0.1 M MgCh. This centrifugation step was repeated, and the pellet was resuspended in chilled 25 mb of 0.1 M CaCh. Following the final centrifugation, the supernatant was discarded, and the cells were resuspended in 900 pL of CaCh. We then added 600 pL of CaCh and 400 pL of 50% (v / v) glycerol solution, using 50% w / v sterile glycerol to achieve the correct concentration. The final suspension of competent cells was now ready for transformations or storage for later use.

[0236] For heat-shock transformation, we combined 50 pL of chemically competent cells with either 100 ng of plasmid DNA or 5 pL of a HiFi assembly product, mixing by flicking. The transformation protocol was as follows: incubation at 4 °C on ice for 30 minutes, a42 °C heat-shock in a water bath for 45 seconds, followed by a 2-minute rest on ice. Then, we added 950 pL of LB or SOC media to the cells and allowed them to recover at 37 °C for 1 hour. Post-recovery, the transformed cells were centrifuged at 8,000 rpm for 5 minutes, resuspended in 100 pL of LB, and plated onto LB agar plates with the appropriate antibiotics.

[0237] For strains carrying two plasmids, chemically competent cells harboring the first plasmid were prepared and used for sequential heat-shock transformation with the second plasmid.

[0238] In cases of difficult transformation with ClearColi™ BL21(DE3), we utilized electrocompetent cells purchased from LCG Biosearch Technologies and followed a standard electroporation protocol.

[0239] Immunostaining and Flow Cytometry. Flow cytometry was used to verify the pNV nanobody presentation on the surface of bacterial strains. Overnight bacterial culture carrying nanobody expression system was centrifuge at 4 °C, 2,000 g for 5 minutes. 1 mL non-fat milk blocking buffer (1 %) in PBS was added to resuspend the cell pellet and incubated at room temperature for 1 hour. Blocked culture was transferred to a 1.5 mL Eppendorf tube and were centrifuged at 4,000 g for 2 minutes. The pellet was washed twice and resuspended in 1 mL of PBS. 2 pL of primary anti-Myc antibody (Abeam, ab9106) was added to the 1 mL cell suspension and incubated at room temperature for 1.5 hours. The suspension was centrifuge at 1,000 g for 5 minutes and washed twice with 1 mL of PBS. The pellet was resuspended in 500 pL of PBS and added with 0.5 pL of secondary Alexa Fluor 488 conjugated antibody (Abeam, abl50077). Culture was incubated at room temperature for 1 hour. Finally, the culture was centrifuged at 1,000 g for 5 minutes, washed twice and resuspended in 1 mL of PBS.

[0240] Cell fluorescence was measured using an FACS Calibur (BD Biosciences) with FL1 filter to detect Alexa Fluor 488 has an excitation / emission at wavelength 488 / 530 nm. Fluorescence data was collected from 100,000 viable cells for each experiment using CellQuest and analysed using Flow Jo software.Cell Agglutination Assay. Overnight cultures of pNV_sfGFP nanobody-displaying strains were centrifuged at 2,000g at 4 °C for 5 minutes and washed three times with IX PBS, followed by dilution in IX PBS to an OD600 of 0.8. 100 pL of this diluted culture was then added to a U-shaped, round-bottom 96-well plate containing varying concentrations of SARS-CoV-2 RBD (Thermo Fisher). The plate was incubated statically overnight at room temperature. Subsequently, a top-view image was captured using the VersaDoc Imaging System (Bio-Rad) under the FITC channel.

[0241] Cell Adhesion Assay. ELISA plates (Nunc, MaxiSorp) were coated with 100 pL per well of RBD antigen diluted in PBS (pH 7.4) to the desired concentration and incubated overnight at 4 °C. The plates were washed three times with a washing buffer consisting of 0.05% Tween-20 in PBS. Overnight pNV_sfGFP cultures were centrifuged at 2,000g at 4 °C for 5 minutes and washed three times with IxPBS. The resulting cell pellet was resuspended in 600 pL of 1% skimmed milk in PBST and incubated at room temperature for 1 hour. The blocked cultures were then centrifuged and washed with IxPBS, followed by dilution in IxPBS to an OD600 of approximately 1. We added 100 pL of the diluted cultures into individual wells and incubated them at room temperature for 1 hour. After incubation, the cell cultures were removed, and the wells were washed four times with PBST. Finally, a fluorescence image was taken using a BioTek imager.

[0242] Batch Production of Pure SimCell. Overnight cultures of ClearColi™ (DE3) harbouring the endonuclease ICeuI were prepared in 5ml volumes and subsequently diluted 1:100 in 50ml of M9 minimal media, supplemented with 0.4% glucose and 0.2% casamino acids, in an Erlenmeyer flask. This flask was incubated in a shaking incubator at 37 °C and 250 rpm overnight. Following this incubation, 50ml of the culture was centrifuged at 1,000g for 10 minutes and washed three times with minimal media, also supplemented with 0.2% casamino acids. The cell pellet was then resuspended in minimal media containing 0.2% casamino acids and IpM Crystal Violet, serving as the inducer for ICeuI expression to initiate SimCell conversion. This resuspension was transferred to an Erlenmeyer flask and incubated at 37°C, 250 rpm overnight before the addition of antibiotics: cefotaxime (100 pg / mL) and penicillin G (100 pg / mL) to eradicate dividing parental cells. The purified SimCell culture underwent further incubation at 37 °C and 250 rpm for 24 hours. Our findings indicate that the ClearColi™ SimCells generated using this method result in uniformly sized SimCells, optimizing therapeutic outcomes and reducing the occurrence of filamentous chromosome-lessSimCells. The resultant genome-less SimCells can be stored at 4 °C for later use or, for immediate application, centrifuged at 1,000g for 10 minutes at 4 °C and washed three times with IX PBS. The PBS-washed culture is then ready for immediate use or can be stored at 4 °C for later use. Detailed protocol and characterisation are shown in Figure 6. To assess the purity of the SimCell, we plated 2 pL of both uninduced ClearColi™ 12x and induced ClearColi™ SimCell cultures on LB agar plates for CFU counting. No colonies were observed for the induced SimCell culture after 72 hours (Figure 5b).

[0243] Characterisation of SimCells conversion. ClearColi (DE3) was transformed with the ICeuI plasmid, referred to as 12x. Glycerol stocks of strains containing the 12x plasmid were streaked onto LB-agar plates containing antibiotics and incubated overnight at 37 °C. Single colonies were selected from these plates and inoculated into 5 mL of LB medium supplemented with the appropriate antibiotic, then incubated overnight at 37 °C at 250 rpm. 2pL of these overnight cultures were reinoculated into M9 medium supplemented with 0.4% glucose and 0.2% casamino acids, and incubated at 37 °C, 250 rpm. 2pL of the overnight culture was then diluted in 198pL of M9 medium supplemented with 0.4% glucose, 0.2% casamino acids, and the corresponding antibiotics. These cultures were transferred to the wells of a 96-well microplate (Costar), which was sealed with a Breathe-Easy® sealing membrane. The plate was placed in a Synergy2 microplate reader (BioTek) and incubated at 37 °C with orbital shaking at l,000rpm for 5 hours. The 96-well plate was subsequently removed, and IpM of Crystal Violet, along with penicillin G (lOOpg / mL), was added to each well. The plate was then returned to the incubator for an additional 40 hours (Figure 5a).

[0244] Production and purification of mini-SimCell. Overnight cultures containing plasmids for the ClearColi™ AminD strain were initiated from 5 mL samples, which were then diluted 1:100 into 50 mL of LB medium in an Erlenmeyer flask. This setup was incubated at 37°C with shaking at 250 rpm, reaching an optical density at 600 nm (OD600) between 2-5 overnight. Following incubation, the culture was transferred into a 50 mL Falcon tube and centrifuged at 2,000g for 10 minutes at 4°C, preserving the supernatant for a subsequent centrifugation step at 12,000g for 15 minutes at the same temperature. The pellet obtained was resuspended in 5 mL of fresh LB medium supplemented with ceftriaxone (100 pg / mL), penicillin G (100 pg / mL), and cefotaxime (100 pg / mL), and the mixture was incubated again at 37 °C with shaking at 250 rpm for a period ranging from 4 hours to overnight. To eliminate cell debris, the culture wasfirst centrifuged at 200g for 10 minutes at 4 °C, and the clear supernatant was collected for a final centrifugation at 16,000g for 15 minutes at 4 °C. The quantity of mini-SimCells was determined by measuring the OD600 with a NanoVue™ Plus Spectrophotometer (GE Healthcare), employing the following formula for precise quantification.

[0245] minicells ^600 * 5. Ox 10 IvtlL

[0246] The collected pellet was then redissolved in 1 m of phosphate-buffered saline (PBS) and kept at 4 °C for subsequent use. This refined preparation is identified in this Example as mini-SimCells.

[0247] RBD protein-protein in vitro blocking Assay. For this study, a MaxiSORP ELISA plate (Nunc) was coated with 100 ng of human ACE-2 (hACE-2) suspended in 50 pL of 100 mM carbonate-bicarbonate coating buffer (pH 9.6) and incubated at 4 °C overnight. After incubation, the plate was washed four times with IX PBST and blocked with SuperBlock™ (Thermo Fisher) blocking buffer. Once the blocking buffer had dried completely, the plate was ready for immediate use or could be stored at 4 °C for future experiments.

[0248] HRP-conjugated SARS-CoV-2 (GenScript) was quantified using the Thermo Fisher QuanT-iT protein quantification kit. In the no-binding assay, a dilution series of HRP-conjugated SARS-CoV-2 Wuhan and South African B.1.351 (Beta) variant RBDs (produced by GenScript) ranged from 4.3 nM to 32.4 nM. These dilutions were added to the hACE-2-coated plates in 50 pL of PBS for 1 hour at room temperature. Unbound HRP-conjugated RBD was then washed away with phosphate-buffered saline containing 0.05% Tween-20 (PBST) five times. For colourimetric measurement, 100 pL of TMB chromogenic substrate (Invitrogen) was added and incubated for 15 minutes to allow the enzymatic reaction with HRP to occur. The reaction was stopped using an equal volume of TMB stop solution, and absorbance at 450 nm was read using a Tecan Spark plate reader.

[0249] For the whole cell or SimCell nanobody-expressing blocking assay, the same SARS-CoV-2 HRP-RBD dilution series was pre-incubated with 50pL of PBS-diluted bacterial whole cells / SimCells at OD=2 for 1 hour at 37 °C. This mixture was then added to aMaxiSORP ELISA plate coated with hACE-2 (100 ng per well) and incubated for 1 hour at room temperature. After five PBST washes to remove unbound antigens, the inhibition efficiency was calculated using standard curves (see Figure 7b).

[0250] Example 2 - Surface display of NahG protein on BL21(DE3)

[0251] With reference to Figure 10.

[0252] Bacteria culture

[0253] Inoculate one colony to a 10 mb fresh LB medium with relevant antibiotics and cultured at 37 and 250 rpm overnight. 1:100 ratio of overnight culture was transferred to 50mL fresh LB medium flask with relevant antibiotics. The flask was cultured at 37 and 250 rpm for around 4 hours till the OD600 reached 0.4-0.6. The flask was then added L-Arabinose to a final concentration lOmM and cultured at 25 and 180 rpm 24 hours.

[0254] Table: Bacteria Final OD600

[0255] &

[0256]

[0257] ELISA to detect NahG on the surface of BL21(DE3):

[0258] Dilute bacteria culture with IxPBS till OD600 reached 0.2. Coated lOOuL bacteria to the 96 well plate overnight at room temperature. Washed 3 times with wash buffer. Block plates with 300uL of block buffer (1% BSA in PBS) and incubate 2 hours at room temperature. Washed 3 times with wash buffer. Add lOOuL anti-Myc antibody (conjugated with HRP, 1:1000 dilution with TBST) and incubate 2 hours at room temperature. Washed 3 times with wash buffer. Add lOOuL substrate solution to each well and incubate for around 9 minutes. Add 50uL of stop solution to each well and detect the plate in a microplate reader set to 450nm.

[0259] Test kit: DuoSet® ELISA DEVELOPMENT SYSTEM, Catalog number:DY202.

[0260] Conclusion• NahG can be displayed on the surface of BL21(DE3) via the dual system of Lpp-OmpA. The same display can be made on minicell and simple cell surfaces.

[0261] • On the cell surface, the NahG enzyme still functioned to convert aspirin into catechol.

[0262] SEQUENCES:

[0263] I-Ceul (CAA78934.1) amino acid sequence (SEQ ID NO: 28):

[0264] MSNFILKPGEKLPQDKLEELKKINDAVKKTKNFSKYLIDLRKLFQIDEVQVTSESK LFLAGFLEGEASLNISTKKLATSKFGLVVDPEFNVTQHVNGVKVLYLALEVFKTG RIRHKSGSNATLVLTIDNRQSLEEKVIPFYEQYVVAFSSPEKVKRVANFKALLELF NNDAHQDLEQLVNKILPIWDQMRKQQGQSNEGFPNLEAAQDFARNYKKGIK

[0265] DNA sequence encoding I-Ceul (CAA78934.1) (SEQ ID NO: 29):

[0266] ATGTCAAACTTTATACTTAAACCGGGCGAAAAACTACCCCAAGACAAACTAG AAGAATTAAAAAAAATTAATGATGCTGTTAAAAAAACGAAAAATTTCTCAAA ATACTTGATTGACTTAAGAAAACTTTTTCAAATTGACGAAGTCCAAGTAACTT CTGAATCAAAACTCTTTTTAGCTGGTTTTTTAGAAGGTGAAGCTTCTCTAAAT ATTAGCACTAAAAAGCTCGCTACTTCTAAATTTGGTTTGGTGGTTGATCCTGA ATTCAATGTGACTCAACATGTCAATGGGGTTAAAGTGCTTTATTTAGCATTAG AAGTATTTAAAACAGGGCGTATTCGTCATAAAAGTGGTAGTAATGCAACTTTA GTTTTAACTATTGACAATCGTCAAAGTTTGGAAGAAAAAGTAATTCCTTTTTA TGAACAATATGTTGTTGCCTTCAGTTCTCCAGAAAAAGTCAAACGTGTAGCTA ATTTTAAAGCTTTGTTAGAATTATTTAATAATGACGCTCACCAAGATTTAGAA CAATTGGTAAACAAAATCCTACCAATTTGGGATCAAATGCGTAAACAACAAG GACAAAGTAACGAAGGCTTTCCTAATTTAGAAGCAGCTCAAGACTTTGCTCGT AATTATAAAAAAGGTATAAAGTAG

[0267] pSEVA224-GB3 sequence (SEQ ID NO: 30) (the glycolysis pathway sequences are highlighted in bold and underline):

[0268] TTAATTAATTGACACCATCGAATGGTGCAAAACCTTTCGCGGTATGGCATGAT AGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTT ATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGG TGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGC GATGGCGGAGCTGAATTACATTCCCAACCGCGTGGCACAACAACTGGCGGGC AAACAGTCGTTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCAACTGGGTGCCAGCG TGGTGGTGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGT GCACAATCTTCTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGG ATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTA TTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAA GACGGTACGCGACTGGGCGTGGAGCATCTGGTCGCATTGGGTCACCAGCAAA TCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCT GGCTGGCATAAATATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGG AAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAA TGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGG GCGCAATGCGCGCCATTACCGAGTCCGGGCTGCGCGTTGGTGCGGATATCTCG GTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAAC CACCATCAAACAGGATTTTCGCCTGCTGGGGCAAACCAGCGTGGACCGCTTGC TGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCTGTTGCCCGTCTCA CTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCC GCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAA AGCGGGCAGTGAGCGCAACGCAATTAATGTAAGTTAGCGCGAATTGATCTGG TTTGACAGCTTATCATCGACTGCACGGTGCACCAATGCTTCTGGCGTCAGGCA GCCATCGGAAGCTGTGGTATGGCTGTGCAGGTCGTAAATCACTGCATAATTCG TGTCGCTCAAGGCGCACTCCCGTTCTGGATAATGTTTTTTGCGCCGACATCAT AACGGTTCTGGCAAATATTCTGAAATGAGCTGTTGACAATTAATCATCCGGCT CGTATAATGTGTGGAATTGTGAGCGGATAACAATTTCACACCCTAGGAGGAG GAAAAACATATGACAAAGTATGCATTAGTCGGTGATGTGGGCGGCACCAA CGCACGTCTTGCTCTGTGTGATATTGCCAGTGGTGAAATCTCGCAGGCTA AGACCTATTCAGGGCTTGATTACCCCAGCCTCGAAGCGGTCATTCGCGTT TATCTTGAAGAACATAAGGTCGAGGTGAAAGACGGCTGTATTGCCATCGC TTGCCCAATTACCGGTGACTGGGTGGCGATGACCAACCATACCTGGGCGT TCTCAATTGCCGAAATGAAAAAGAATCTCGGTTTTAGCCATCTGGAAATT ATTAACGATTTTACCGCTGTATCGATGGCGATCCCGATGCTGAAAAAAGA GCATCTGATTCAGTTTGGTGGCGCAGAACCGGTCGAAGGTAAGCCTATTG CGGTTTACGGTGCCGGAACGGGGCTTGGGGTTGCGCATCTGGTCCATGT CGATAAGCGTTGGGTTAGCTTGCCAGGCGAAGGCGGTCACGTAGATTTT GCGCCGAATAGTGAAGAAGAGGCCATTATCCTCGAAATATTGCGTGCGG AAATTGGTCATGTTTCGGCGGAGCGCGTGCTTTCTGGCCCTGGGCTGGTG AATTTGTATCGCGCAATTGTGAAAGCTGACAACCGCCTGCCAGAAAATCTCAAGCCAAAAGATATTACCGAACGCGCGCTGGCTGACAGCTGCACCGATT GCCGCCGCGCATTGTCGCTGTTTTGCGTCATTATGGGCCGTTTTGGCGGC AATCTGGCGCTCAATCTCGGGACATTTGGCGGCGTGTTTATTGCGGGCGG TATCGTGCCGCGCTTCCTTGAGTTCTTCAAAGCCTCCGGTTTCCGTGCCG CATTTGAAGATAAAGGGCGCTTTAAAGAATATGTCCATGATATTCCGGTG TATCTCATCGTCCATGACAATCCGGGCCTTCTCGGTTCCGGTGCACATTT ACGCCAGACCTTAGGTCACATTCTGTAAGAATTCAGGAGGAAAAACATAT GAAAAACATCAATCCAACGCAGACCGCTGCCTGGCAGGCACTACAGAAA CACTTCGATGAAATGAAAGACGTTACGATCGCCGATCTTTTTGCTAAAGA CGGCGATCGTTTTTCTAAGTTCTCCGCAACCTTCGACGATCAGATGCTGG TGGATTACTCCAAAAACCGCATCACTGAAGAGACGCTGGCGAAATTACAG GATCTGGCGAAAGAGTGCGATCTGGCGGGCGCGATTAAGTCGATGTTCT CTGGCGAGAAGATCAACCGCACTGAAAACCGCGCCGTGCTGCACGTAGC GCTGCGTAACCGTAGCAATACCCCGATTTTGGTTGATGGCAAAGACGTAA TGCCGGAAGTCAACGCGGTGCTGGAGAAGATGAAAACCTTCTCAGAAGC GATTATTTCCGGTGAGTGGAAAGGTTATACCGGCAAAGCAATCACTGACG TAGTGAACATCGGGATCGGCGGTTCTGACCTCGGCCCATACATGGTGACC GAAGCTCTGCGTCCGTACAAAAACCACCTGAACATGCACTTTGTTTCTAA CGTCGATGGGACTCACATCGCGGAAGTGCTGAAAAAAGTAAACCCGGAA ACCACGCTGTTCTTGGTAGCATCTAAAACCTTCACCACTCAGGAAACTAT GACCAACGCCCATAGCGCGCGTGACTGGTTCCTGAAAGCGGCAGGTGAT GAAAAACACGTTGCAAAACACTTTGCGGCGCTTTCCACCAATGCCAAAGC CGTTGGCGAGTTTGGTATTGATACTGCCAACATGTTCGAGTTCTGGGACT GGGTTGGCGGCCGTTACTCTTTGTGGTCAGCGATTGGCCTGTCGATTGTT CTCTCCATCGGCTTTGATAACTTCGTTGAACTGCTTTCCGGCGCACACGC GATGGACAAGCATTTCTCCACCACGCCTGCCGAGAAAAACCTGCCTGTAC TGCTGGCGCTGATTGGCATCTGGTACAACAATTTCTTTGGTGCGGAAACT GAAGCGATTCTGCCGTATGACCAGTATATGCACCGTTTCGCGGCGTACTT CCAGCAGGGCAATATGGAGTCCAACGGTAAGTATGTTGACCGTAACGGT AACGTTGTGGATTACCAGACTGGCCCGATTATCTGGGGTGAACCAGGCAC TAACGGTCAGCACGCGTTCTACCAGCTGATCCACCAGGGAACCAAAATGG TGCCGTGCGATTTCATCGCTCCGGCTATCACCCATAACCCGCTCTCTGAT CATCACCAGAAACTGCTGTCTAACTTCTTCGCCCAGACCGAAGCGCTGGC GTTTGGTAAATCCCGCGAAGTAGTTGAGCAGGAATATCGTGATCAGGGTA AAGATCCGGCAACGCTTGACTACGTGGTGCCGTTCAAAGTATTCGAAGGTAACCGCCCGACCAACTCCATCCTGCTGCGTGAAATCACTCCGTTCAGCCT GGGTGCGTTGATTGCGCTGTATGAGCACAAAATCTTTACTCAGGGCGTGA TCCTGAACATCTTCACCTTCGACCAGTGGGGCGTGGAACTGGGTAAACAG CTGGCGAACCGTATTCTGCCAGAGCTGAAAGATGATAAAGAAATCAGCAG CCACGATAGCTCGACCAATGGTCTGATTAACCGCTATAAAGCGTGGCGCG GTTAAGAGCTCAGGAGGAAAAACATATGATTAAGAAAATCGGTGTGTTGA CAAGCGGCGGTGATGCGCCAGGCATGAACGCCGCAATTCGCGGGGTTGT TCGTTCTGCGCTGACAGAAGGTCTGGAAGTAATGGGTATTTATGACGGCT ATCTGGGTCTGTATGAAGACCGTATGGTACAGCTAGACCGTTACAGCGTG TCTGACATGATCAACCGTGGCGGTACGTTCCTCGGTTCTGCGCGTTTCCC GGAGTTCCGCGACGAAAACATCCGCGCCGTGGCTATCGAAAACCTGAAA AAACGTGGTATCGACGCGCTGGTGGTTATCGGCGGTGACGGTTCCTACAT GGGTGCAATGCGTCTGACCGAAATGGGCTTCCCGTGCATCGGTCTGCCG GGCACTATCGACAACGACATCAAAGGCACTGACTACACTATCGGTTTCTT CACTGCGCTGAGCACCGTTGTAGAAGCGATCGACCGTCTGCGTGACACCT CTTCTTCTCACCAGCGTATTTCCGTGGTGGAAGTGATGGGCCGTTATTGT GGAGATCTGACGTTGGCTGCGGCCATTGCCGGTGGCTGTGAATTTGTTGT GGTTCCGGAAGTTGAATTTAGCCGTGAAGACCTGGTAAACGAAATCAAAG CGGGTATCGCGAAAGGTAAAAAACACGCGATCGTGGCGATTACCGAACA TATGTGTGATGTTGACGAACTGGCGCATTTCATCGAGAAAGAAACCGGTC GTGAAACCCGCGCAACTGTGCTGGGCCACATCCAGCGCGGTGGTTCTCC GGTGCCTTACGACCGTATTCTGGCTTCCCGTATGGGCGCTTACGCTATCG ATCTGCTGCTGGCAGGTTACGGCGGTCGTTGTGTAGGTATCCAGAACGAA CAGCTGGTTCACCACGACATCATCGACGCTATCGAAAACATGAAGCGTCC GTTCAAAGGTGACTGGCTGGACTGCGCGAAAAAACTGTATTAAGGTACCA GGAGGAAAAACATATGTCTAAGATTTTTGATTTCGTAAAACCTGGCGTAA TCACTGGTGATGACGTACAGAAAGTTTTCCAGGTAGCAAAAGAAAACAAC TTCGCACTGCCAGCAGTAAACTGCGTCGGTACTGACTCCATCAACGCCGT ACTGGAAACCGCTGCTAAAGTTAAAGCGCCGGTTATCGTTCAGTTCTCCA ACGGTGGTGCTTCCTTTATCGCTGGTAAAGGCGTGAAATCTGACGTTCCG CAGGGTGCTGCTATCCTGGGCGCGATCTCTGGTGCGCATCACGTTCACCA GATGGCTGAACATTATGGTGTTCCGGTTATCCTGCACACTGACCACTGCG CGAAGAAACTGCTGCCGTGGATCGACGGTCTGTTGGACGCGGGTGAAAA ACACTTCGCAGCTACCGGTAAGCCGCTGTTCTCTTCTCACATGATCGACC TGTCTGAAGAATCTCTGCAAGAGAACATCGAAATCTGCTCTAAATACCTGGAGCGCATGTCCAAAATCGGCATGACTCTGGAAATCGAACTGGGTTGCAC CGGTGGTGAAGAAGACGGCGTGGACAACAGCCACATGGACGCTTCTGCA CTGTACACCCAGCCGGAAGACGTTGATTACGCATACACCGAACTGAGCAA AATCAGCCCGCGTTTCACCATCGCAGCGTCCTTCGGTAACGTACACGGTG TTTACAAGCCGGGTAACGTGGTTCTGACTCCGACCATCCTGCGTGATTCT CAGGAATATGTTTCCAAGAAACACAACCTGCCGCACAACAGCCTGAACTT CGTATTCCACGGTGGTTCCGGTTCTACTGCTCAGGAAATCAAAGACTCCG TAAGCTACGGCGTAGTAAAAATGAACATCGATACCGATACCCAGTGGGCA ACCTGGGAAGGCGTTCTGAACTACTACAAAGCTAACGAAGCGTATCTGCA AGGTCAGCTGGGTAACCCGAAAGGCGAAGATCAGCCGAACAAGAAATAC TACGATCCGCGCGTATGGCTGCGTGCCGGTCAGACTTCGATGATCGCTCG TCTGGAGAAAGCATTCCAGGAACTGAACGCGATCGACGTTCTGTAACCCG GGAGGAGGAAAAACATATGCGACATCCTTTAGTGATGGGTAACTGGAAA CTGAACGGCAGCCGCCACATGGTTCACGAGCTGGTTTCTAACCTGCGTAA AGAGCTGGCAGGTGTTGCTGGCTGTGCGGTTGCAATCGCACCACCGGAA ATGTATATCGATATGGCGAAGCGCGAAGCTGAAGGCAGCCACATCATGCT GGGTGCGCAAAACGTGGACCTGAACCTGTCCGGCGCATTCACCGGTGAA ACCTCTGCTGCTATGCTGAAAGACATCGGCGCACAGTACATCATCATCGG TCACTCTGAACGTCGTACTTACCACAAAGAATCTGACGAACTGATCGCGA AAAAATTCGCGGTGCTGAAAGAGCAGGGCCTGACTCCGGTTCTGTGCATC GGTGAAACCGAAGCTGAAAATGAAGCGGGCAAAACTGAAGAAGTTTGCG CACGTCAGATCGACGCGGTACTGAAAACTCAGGGTGCTGCGGCATTCGA AGGTGCGGTTATCGCTTACGAACCTGTATGGGCAATCGGTACTGGCAAAT CTGCAACTCCGGCTCAGGCACAGGCTGTTCACAAATTCATCCGTGACCAC ATCGCTAAAGTTGACGCTAACATCGCTGAACAAGTGATCATTCAGTACGG CGGCTCTGTAAACGCGTCTAACGCAGCTGAACTGTTTGCTCAGCCGGATA TCGACGGCGCGCTGGTTGGTGGTGCTTCTCTGAAAGCTGACGCCTTCGCA GTAATCGTTAAAGCAGCTGAAGCGGCTAAACAGGCTTAAGCTAGGCCGC GGCCGCGCGAATTCGAGCTCGGTACCCGGGGATCCAGGAGGAAAAACAT ATGACTATCAAAGTAGGTATCAACGGTTTTGGCCGTATCGGTCGCATTGT TTTCCGTGCTGCTCAGAAACGTTCTGACATCGAGATCGTTGCAATCAACG ACCTGTTAGACGCTGATTACATGGCATACATGCTGAAATATGACTCCACT CACGGTCGTTTCGACGGCACCGTTGAAGTGAAAGACGGTCATCTGATCGT TAACGGTAAAAAAATCCGTGTTACCGCTGAACGTGATCCGGCTAACCTGA AATGGGACGAAGTTGGTGTTGACGTTGTCGCTGAAGCAACTGGTCTGTTCCTGACTGACGAAACTGCTCGTAAACACATCACCGCTGGTGCGAAGAAAGT GGTTATGACTGGTCCGTCTAAAGACAACACTCCGATGTTCGTTAAAGGCG CTAACTTCGACAAATATGCTGGCCAGGACATCGTTTCCAACGCTTCCTGC ACCACCAACTGCCTGGCTCCGCTGGCTAAAGTTATCAACGATAACTTCGG CATCATCGAAGGTCTGATGACCACCGTTCACGCTACTACCGCTACTCAGA AAACCGTTGATGGCCCGTCTCACAAAGACTGGCGCGGCGGCCGCGGCGC TTCCCAGAACATCATCCCGTCCTCTACCGGTGCTGCTAAAGCTGTAGGTA AAGTACTGCCAGAACTGAATGGCAAACTGACTGGTATGGCGTTCCGCGTT CCGACCCCGAACGTATCTGTAGTTGACCTGACCGTTCGTCTGGAAAAAGC TGCAACTTACGAGCAGATCAAAGCTGCCGTTAAAGCTGCTGCTGAAGGCG AAATGAAAGGCGTTCTGGGCTACACCGAAGATGACGTAGTATCTACCGAT TTCAACGGCGAAGTTTGCACTTCCGTGTTCGATGCTAAAGCTGGTATCGC TCTGAACGACAACTTCGTGAAACTGGTATCCTGGTACGACAACGAAACCG GTTACTCCAACAAAGTTCTGGACCTGATCGCTCACATCTCCAAATAATCT AGAAGGAGGAAAAACATATGTCTGTAATTAAGATGACCGATCTGGATCTT GCTGGGAAACGTGTATTCATCCGTGCGGATCTGAACGTACCAGTAAAAGA CGGGAAAGTAACCAGCGACGCGCGTATCCGTGCTTCTCTGCCGACCATC GAACTGGCCCTGAAACAAGGCGCAAAAGTGATGGTAACTTCCCACCTGG GTCGTCCTACCGAAGGCGAATACAACGAAGAGTTCTCTCTGCTGCCGGTT GTTAACTACCTGAAAGACAAACTGTCTAACCCGGTTCGTCTGGTTAAAGA TTACCTCGACGGCGTTGACGTTGCTGAAGGTGAACTGGTTGTTCTGGAAA ACGTTCGCTTCAACAAAGGCGAGAAGAAAGACGACGAAACCCTGTCCAA AAAATACGCTGCACTGTGTGACGTGTTCGTAATGGACGCATTCGGTACTG CTCACCGCGCGCAGGCTTCTACTCACGGTATCGGTAAATTCGCTGACGTT GCGTGCGCAGGCCCGCTGCTGGCAGCTGAACTGGACGCGCTGGGTAAAG CACTGAAAGAACCTGCTCGCCCGATGGTGGCTATCGTTGGTGGTTCTAAA GTATCTACCAAACTGACCGTTCTGGACTCCCTGTCTAAAATCGCTGACCA GCTGATTGTTGGTGGTGGTATCGCTAACACCTTTATCGCGGCACAAGGCC ACGATGTGGGTAAATCCCTGTACGAAGCTGACCTGGTTGACGAAGCTAAA CGTCTGCTGACCACCTGCAACATCCCGGTTCCGTCTGATGTTCGCGTAGC AACCGAGTTCTCTGAAACTGCACCGGCTACCCTGAAATCTGTTAACGATG TGAAAGCTGACGAGCAGATCCTGGATATCGGTGATGCTTCCGCTCAGGAA CTGGCTGAAATCCTGAAGAATGCGAAAACCATTCTGTGGAACGGTCCGGT TGGCGTGTTCGAATTTCCGAACTTCCGCAAAGGTACTGAAATCGTGGCTA ACGCTATCGCAGACAGCGAAGCGTTCTCCATCGCTGGCGGCGGCGACACTCTGGCAGCAATCGACCTGTTCGGCATTGCTGACAAAATCTCCTACATCT CCACTGGCGGCGGCGCATTCCTCGAGTTCGTGGAAGGTAAAGTACTGCC TGCGGTAGCAATGCTCGAAGAACGCGCTAAGAAGTAAGTCGACAGGAGG AAAAACATATGGCTGTAACTAAGCTGGTTCTGGTTCGTCATGGCGAAAGT CAGTGGAACAAAGAAAACCGTTTCACCGGTTGGTACGACGTGGATCTGTC TGAGAAAGGCGTAAGCGAAGCAAAAGCAGCAGGTAAGCTGCTGAAAGAG GAAGGTTACAGCTTTGACTTTGCTTACACTTCTGTGCTGAAACGCGCTAT CCATACCCTGTGGAATGTGCTGGACGAACTGGATCAGGCATGGCTGCCC GTTGAGAAATCCTGGAAACTGAACGAACGTCACTACGGTGCGTTGCAGG GTCTGAACAAAGCGGAAACTGCTGAAAAGTATGGCGACGAGCAGGTGAA ACAGTGGCGTCGTGGTTTTGCAGTGACTCCGCCGGAACTGACTAAAGATG ATGAGCGTTATCCGGGTCACGATCCGCGTTACGCGAAACTGAGCGAGAA AGAACTGCCGCTGACGGAAAGCCTGGCGCTGACCATTGACCGCGTGATC CCTTACTGGAATGAAACTATTCTGCCGCGTATGAAGAGCGGTGAGCGCGT GATCATCGCTGCACACGGTAACTCTTTACGTGCGCTGGTGAAATATCTTG ATAACATGAGCGAAGAAGAGATTCTTGAGCTTAATATCCCGACTGGCGTG CCGCTGGTGTATGAGTTCGACGAGAATTTCAAACCGCTGAAACGCTATTA TCTGGGTAATGCTGACGAGATCGCAGCGAAAGCAGCGGCGGTTGCAAAC CAGGGTAAAGCGAAGTAACTGCAGAGGAGGAAAAACATATGTCCAAAAT CGTAAAAATCATCGGTCGTGAAATCATCGACTCCCGTGGTAACCCGACTG TTGAAGCCGAAGTACATCTGGAAGGTGGTTTCGTCGGTATGGCAGCTGCT CCGTCAGGTGCTTCTACTGGTTCCCGTGAAGCTCTGGAACTGCGCGATGG CGACAAATCCCGTTTCCTGGGTAAAGGCGTAACCAAAGCTGTTGCTGCGG TAAACGGCCCGATCGCTCAGGCGCTGATTGGCAAAGATGCTAAAGATCA GGCTGGCATTGACAAGATCATGATCGACCTGGACGGCACCGAAAACAAA TCCAAATTCGGCGCGAACGCAATCCTGGCTGTATCTCTGGCTAACGCCAA AGCTGCTGCTGCTGCAAAAGGTATGCCGCTGTACGAGCACATCGCTGAAC TGAACGGTACTCCGGGCAAATACTCTATGCCGGTTCCGATGATGAACATC ATCAACGGTGGTGAGCACGCTGACAACAACGTTGATATCCAGGAATTTAT GATTCAGCCGGTTGGCGCGAAAACTGTGAAAGAAGCCATCCGCATGGGT TCTGAAGTTTTCCATCACCTGGCAAAAGTTCTGAAAGCGAAAGGCATGAA CACTGCTGTTGGTGACGAAGGTGGCTATGCGCCGAACCTGGGTTCCAAC GCTGAAGCTCTGGCTGTTATCGCTGAAGCTGTTAAAGCTGCTGGTTATGA ACTGGGCAAAGACATCACTTTGGCGATGGACTGCGCAGCTTCTGAGTTCT ACAAAGATGGTAAATACGTTCTGGCTGGCGAAGGCAACAAAGCGTTCACCTCTGAAGAGTTCACTCACTTCCTGGAAGAACTGACCAAACAGTACCCGAT CGTTTCTATCGAAGACGGTCTGGACGAATCTGACTGGGACGGTTTCGCAT ACCAGACCAAAGTTCTGGGCGACAAAATCCAGCTGGTTGGTGACGACCT GTTCGTAACCAACACCAAGATCCTGAAAGAAGGTATCGAAAAAGGTATCG CTAACTCCATCCTGATCAAATTCAACCAGATCGGTTCTCTGACCGAAACT CTGGCTGCAATCAAGATGGCGAAAGATGCAGGCTACACTGCTGTTATCTC TCACCGTTCTGGCGAAACTGAAGACGCTACCATCGCAGACCTGGCTGTTG GTACTGCTGCTGGCCAGATCAAAACTGGTTCTATGAGCCGTTCTGACCGT GTTGCTAAATACAACCAGCTGATTCGTATCGAAGAAGCTCTGGGCGAAAA AGCACCGTACAACGGTCGTAAAGAAATCAAAGGCCAGGCATAAGCATGC AGGAGGAAAAACATATGAAAAAGACCAAAATTGTTTGCACCATCGGACCG AAAACCGAATCTGAAGAGATGTTAGCTAAAATGCTGGACGCTGGCATGAA CGTTATGCGTCTGAACTTCTCTCATGGTGACTATGCAGAACACGGTCAGC GCATTCAGAATCTGCGCAACGTGATGAGCAAAACTGGTAAAACCGCCGCT ATCCTGCTTGATACCAAAGGTCCGGAAATCCGCACCATGAAACTGGAAGG CGGTAACGACGTTTCTCTGAAAGCTGGTCAGACCTTTACTTTCACCACTG ATAAATCTGTTATCGGCAACAGCGAAATGGTTGCGGTAACGTATGAAGGT TTCACTACTGACCTGTCTGTTGGCAACACCGTACTGGTTGACGATGGTCT GATCGGTATGGAAGTTACCGCCATTGAAGGTAACAAAGTTATCTGTAAAG TGCTGAACAACGGTGACCTGGGCGAAAACAAAGGTGTGAACCTGCCTGG CGTTTCCATTGCTCTGCCAGCACTGGCTGAAAAAGACAAACAGGACCTGA TCTTTGGTTGCGAACAGGGCGTAGACTTTGTTGCTGCTTCCTTTATTCGT AAGCGTTCTGACGTTATCGAAATCCGTGAGCACCTGAAAGCGCACGGCG GCGAAAACATCCACATCATCTCCAAAATCGAAAACCAGGAAGGCCTCAAC AACTTCGACGAAATCCTCGAAGCCTCTGACGGCATCATGGTTGCGCGTGG CGACCTGGGTGTAGAAATCCCGGTAGAAGAAGTTATCTTCGCCCAGAAGA TGATGATCGAAAAATGTATCCGTGCACGTAAAGTCGTTATCACTGCGACC CAGATGCTGGATTCCATGATCAAAAACCCACGCCCGACTCGCGCAGAAGC CGGTGACGTTGCAAACGCCATCCTCGACGGTACTGACGCAGTGATGCTGT CTGGTGAATCCGCAAAAGGTAAATACCCGCTGGAAGCGGTTTCTATCATG GCGACCATCTGCGAACGTACCGACCGCGTGATGAACAGCCGTCTCGAGT TCAACAATGACAACCGTAAACTGCGCATTACCGAAGCGGTATGCCGTGGT GCCGTTGAAACTGCTGAAAAACTGGATGCTCCGCTGATCGTGGTTGCTAC TCAGGGCGGTAAATCTGCTCGCGCAGTACGTAAATACTTCCCGGATGCCA CCATCCTGGCACTGACCACCAACGAAAAAACGGCTCATCAGTTGGTACTGAGCAAAGGCGTTGTGCCGCAGCTTGTTAAAGAGATCACTTCTACTGATGA TTTCTACCGTCTGGGTAAAGAACTGGCTCTGCAAAGCGGTCTGGCACACA AAGGTGACGTTGTAGTTATGGTTTCTGGTGCACTGGTTCCGAGCGGCACT ACTAACACCGCATCTGTACACGTCCTGTAAAAGCTTGCGGCCGCGTCGTG ACTGGGAAAACCCTGGCGACTAGTCTTGGACTCCTGTTGATAGATCCAGT AATGACCTCAGAACTCCATCTGGATTTGTTCAGAACGCTCGGTTGCCGCC GGGCGTTTTTTATTGGTGAGAATCCAGGGGTCCCCAATAATTACGATTTA AATTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATAT CATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGT GTTATGAGCCATATTCAGCGTGAAACGAGCTGTAGCCGTCCGCGTCTGAA CAGCAACATGGATGCGGATCTGTATGGCTATAAATGGGCGCGTGATAAC GTGGGTCAGAGCGGCGCGACCATTTATCGTCTGTATGGCAAACCGGATG CGCCGGAACTGTTTCTGAAACATGGCAAAGGCAGCGTGGCGAACGATGT GACCGATGAAATGGTGCGTCTGAACTGGCTGACCGAATTTATGCCGCTGC CGACCATTAAACATTTTATTCGCACCCCGGATGATGCGTGGCTGCTGACC ACCGCGATTCCGGGCAAAACCGCGTTTCAGGTGCTGGAAGAATATCCGG ATAGCGGCGAAAACATTGTGGATGCGCTGGCCGTGTTTCTGCGTCGTCTG CATAGCATTCCGGTGTGCAACTGCCCGTTTAACAGCGATCGTGTGTTTCG TCTGGCCCAGGCGCAGAGCCGTATGAACAACGGCCTGGTGGATGCGAGC GATTTTGATGATGAACGTAACGGCTGGCCGGTGGAACAGGTGTGGAAAG AAATGCATAAACTGCTGCCGTTTAGCCCGGATAGCGTGGTGACCCACGGC GATTTTAGCCTGGATAACCTGATTTTCGATGAAGGCAAACTGATTGGCTG CATTGATGTGGGCCGTGTGGGCATTGCGGATCGTTATCAGGATCTGGCCA TTCTGTGGAACTGCCTGGGCGAATTTAGCCCGAGCCTGCAAAAACGTCTG TTTCAGAAATATGGCATTGATAATCCGGATATGAACAAACTGCAATTTCA TCTGATGCTGGATGAATTTTTCTAATAATTAATTGGACCGCGGTCCGCGC GTTGTCCTTTTCCGCTGCATAACCCTGCTTCGGGGTCATTATAGCGATTT TTTCGGTATATCCATCCTTTTTCGCACGATATACAGGATTTTGCCAAAGG GTTCGTGTAGACTTTCCTTGGTGTATCCAACGGCGTCAGCCGGGCAGGAT AGGTGAAGTAGGCCCACCCGCGAGCGGGTGTTCCTTCTTCACTGTCCCTT ATTCGCACCTGGCGGTGCTCAACGGGAATCCTGCTCTGCGAGGCTGGCC GTAGGCCGGCCGCGATGCAGGTGGCTGCTGAACCCCCAGCCGGAACTGA CCCCACAAGGCCCTAGCGTTTGCAATGCACCAGGTCATCATTGACCCAGG CGTGTTCCACCAGGCCGCTGCCTCGCAACTCTTCGCAGGCTTCGCCGACC TGCTCGCGCCACTTCTTCACGCGGGTGGAATCCGATCCGCACATGAGGCGGAAGGTTTCCAGCTTGAGCGGGTACGGCTCCCGGTGCGAGCTGAAATA GTCGAACATCCGTCGGGCCGTCGGCGACAGCTTGCGGTACTTCTCCCATA TGAATTTCGTGTAGTGGTCGCCAGCAAACAGCACGACGATTTCCTCGTCG ATCAGGACCTGGCAACGGGACGTTTTCTTGCCACGGTCCAGGACGCGGA AGCGGTGCAGCAGCGACACCGATTCCAGGTGCCCAACGCGGTCGGACGT GAAGCCCATCGCCGTCGCCTGTAGGCGCGACAGGCATTCCTCGGCCTTC GTGTAATACCGGCCATTGATCGACCAGCCCAGGTCCTGGCAAAGCTCGTA GAACGTGAAGGTGATCGGCTCGCCGATAGGGGTGCGCTTCGCGTACTCC AACACCTGCTGCCACACCAGTTCGTCATCGTCGGCCCGCAGCTCGACGCC GGTGTAGGTGATCTTCACGTCCTTGTTGACGTGGAAAATGACCTTGTTTT GCAGCGCCTCGCGCGGGATTTTCTTGTTGCGCGTGGTGAACAGGGCAGA GCGGGCCGTGTCGTTTGGCATCGCTCGCATCGTGTCCGGCCACGGCGCA ATATCGAACAAGGAAAGCTGCATTTCCTTGATCTGCTGCTTCGTGTGTTT CAGCAACGCGGCCTGCTTGGCTTCGCTGACCTGTTTTGCCAGGTCCTCGC CGGCGGTTTTTCGCTTCTTGGTCGTCATAGTTCCTCGCGTGTCGATGGTC ATCGACTTCGCCAAACCTGCCGCCTCCTGTTCGAGACGACGCGAACGCTC CACGGCGGCCGATGGCGCGGGCAGGGCAGGGGGAGCCAGTTGCACGCT GTCGCGCTCGATCTTGGCCGTAGCTTGCTGGACTATCGAGCCGACGGACT GGAAGGTTTCGCGGGGCGCACGCATGACGGTGCGGCTTGCGATGGTTTC GGCATCCTCGGCGGAAAACCCCGCGTCGATCAGTTCTTGCCTGTATGCCT TCCGGTCAAACGTCCGATTCATTCACCCTCCTTGCGGGATTGCCCCGGAA TTAATTCCCCGGATCGATCCGTCGATCTTGATCCCCTGCGCCATCAGATC CTTGGCGGCAAGAAAGCCATCCAGTTTACTTTGCAGGGCTTCCCAACCTT ACCAGAGGGCGCCCCAGCTGGCAATTCCGGTTCGCTTGCTGTCCATAAAA CCGCCCAGTCTAGCTATCGCCATGTAAGCCCACTGCAAGCTACCTGCTTT CTCTTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGACATT CATCCGGGGTCAGCACCGTTTCTGCGGACTGGCTTTCTACGTGGCTGCCA TTTTTGGGGTGAGGCCGTTCGCGGCCGAGGGGCGCAGCCCCTGGGGGGA TGGGAGGCCCGCGTTAGCGGGCCGGGAGGGTTCGAGAAGGGGGGGCAC CCCCCTTCGGCGTGCGCGGTCACGCGCACAGGGCGCAGCCCTGGTTAAA AACAAGGTTTATAAATATTGGTTTAAAAGCAGGTTAAAAGACAGGTTAGC GGTGGCCGAAAAACGGGCGGAAACCCTTGCAAATGCTGGATTTTCTGCCT GTGGACAGCCCCTCAAATGTCAATAGGTGCGCCCCTCATCTGTCAGCACT CTGCCCCTCAAGTGTCAAGGATCGCGCCCCTCATCTGTCAGTAGTCGCGC CCCTCAAGTGTCAATACCGCAGGGCACTTATCCCCAGGCTTGTCCACATCATCTGTGGGAAACTCGCGTAAAATCAGGCGTTTTCGCCGATTTGCGAGGC TGGCCAGCTCCACGTCGCCGGCCGAAATCGAGCCTGCCCCTCATCTGTCA ACGCCGCGCCGGGTGAGTCGGCCCCTCAAGTGTCAACGTCCGCCCCTCA TCTGTCAGTGAGGGCCAAGTTTTCCGCGAGGTATCCACAACGCCGGCGG CCCTACATGGCTCTGCTGTAGTGAGTGGGTTGCGCTCCGGCAGCGGTCCT GATCCCCCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTA CGGCGCGCCCAGCTGTCTAGGGCGGCGGATTTGTCCTACTCAGGAGAGC GTTCACCGACAAACAACAGATAAAACGAAAGGCCCAGTCTTTCGACTGAG CCTTTCGTTTTATTTGATGCCT

Claims

CLAIMS1. A composition comprising a plurality of chromosome-free bacterial minicells and / or simple cells, wherein the minicells and / or simple cells comprise surface-displayed antigen-binding molecules that target at least one viral protein or epitope.

2. The composition according to claim 1, wherein the minicells have an average size from 20 nm to 1000 nm.

3. The composition according to claim 1 or 2, wherein the minicells have an average size of less than 200 nm.

4. The composition according to any preceding claim, wherein at least 80% of the minicells are smaller than about 200 nm along their axis.

5. The composition according to any preceding claim, wherein minicells and / or simple cells are derived from Escherichia spp., Pseudomonas spp., Salmonella spp., or Bordetella spp.

6. The composition according to any preceding claim, wherein the minicells and / or simple cells do not comprise LPS or are formed from an LPS-free bacteria.

7. The composition according to any preceding claim, wherein the minicell is derived from a bacterial cell comprising one or more of the genetic mutations selected from msbA148, gutQ, Skdsl). lpxL, lpxM, Spa P. SlpxP, and SeptA.

8. The composition according to any preceding claim, wherein the minicell is derived from a bacterial cell comprising a minC and / or minD gene knockout.

9. The composition according to any preceding claim, wherein the target of the surface-displayed antigen-binding molecule is a viral particle or a virally infected cell that displays a viral antigen recognised by the surface-displayed antigen-binding molecule on its surface.

10. The composition according to any preceding claim, wherein the target of the antigen-binding molecule is a SARS-CoV-2, influenza, respiratory syncytial virus (RSV), Zaire ebolavirus (Ebola), human immunodeficiency virus (HIV), hepatitis, Dengue virus, West Nile virus, Rotavirus, Norovirus, Astrovirus, Adenovirus, or Herpesvirus antigen.

11. The composition according to any preceding claim, wherein the surface-displayed antigen-binding molecule comprises or consists of TY1, NIH-CoV2nb-l 12, mNb6, V, and E.

12. The composition according to any preceding claim, wherein an accessory product is expressed by the minicells and / or simple cells in addition to the surface-displayed antigen-binding molecule; optionally wherein the accessory product is surface displayed on the minicells and / or simple cells.

13. The composition according to any preceding claim, wherein the accessory product comprises an enzyme catalyst.

14. The composition according to any preceding claim, wherein the composition is formulated into a nasal spray or a formulation suitable for a nebulizer.

15. The composition according to any preceding claim, wherein the antigen-binding molecule comprises a single-domain antibody (sdAb); or wherein the antigen-binding molecule comprises an antibody, or antibody fragment thereof.

16. A method for obtaining a chromosome-free bacterial minicells from a EPS bacterial cell, wherein the EPS minicells are smaller than about 200 nm along their axis, and optionally wherein the EPS minicells have surface-displayed antigen-binding molecules that target at least one viral epitope, the method comprising the steps of: i) culturing the EPS bacterial cell and preventing functional expression in the EPS bacterial cell of at least one of the endogenous genes selected from: minD and minC, thereby forming EPS minicells; andii) optionally transforming the EPS minicells with recombinant nucleic acid for expression of at least one fusion protein comprising or consisting of a antigen-bindingmolecule that targets at least one viral epitope, and an outer-membrane anchor polypeptide.

17. The method of claim 15, further comprising culturing the LPS+bacterial cells in suboptimal growth conditions, to reduce the average size of the resulting LPS+minicells to less than about 200 nm along their axis.

18. The method according to claim 15 or 16, further comprising inducing the death of any bacterial cells in which the chromosomal DNA remains intact and / or actively dividing bacterial cells.

19. A chromosome-free bacterial minicell and / or simple cell, wherein the minicell and / or simple cell comprises no lipopolysaccharide (LPS), and wherein the minicell and / or simple cell comprises surface-displayed antigen-binding molecules that target at least one viral protein.

20. A minicell or a composition of minicells produced by the method according to any of claims 16-18.

21. A composition of minicells and / or simple cells according to any of claims 1-15, 19 or 20, for use as a medicament.

22. A composition of minicells and / or simple cells according to any of claims 1-15, 19 or 20, for use in treating or preventing a viral infection in a subject.

23. A composition of minicells and / or simple cells according to any of claims 1-15, 19 or 20, for use in the manufacture of a medicament, for example for treating or preventing a disease or condition in a subject.

24. A method of treatment or prevention of disease or condition, the method comprising the administration of a composition of minicells and / or simple cells according to any of claims 1-15, 19 or 20, to a subject in need thereof, optionally wherein the disease is a viral infection.

25. Use of a composition of minicells and / or simple cells having surface-displayed antigen-binding molecules according to any of claims 1-15, 19 or 20, for targeting a virally infected cell, for binding with the minicells and / or simple cells.

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