WO2024107119A1 - T3 vectors for recombinant protein production in mammalian cells - Google Patents

T3 vectors for recombinant protein production in mammalian cells Download PDF

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WO2024107119A1
WO2024107119A1 PCT/SG2023/050769 SG2023050769W WO2024107119A1 WO 2024107119 A1 WO2024107119 A1 WO 2024107119A1 SG 2023050769 W SG2023050769 W SG 2023050769W WO 2024107119 A1 WO2024107119 A1 WO 2024107119A1
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protein
recombinant
sequence
nucleic acid
binding molecule
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Martina MARIC
Alexandre Serge Stephan CHOJNOWSKI
Brian Edmund BURKE
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Agency For Science, Technology And Research
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/635—Externally inducible repressor mediated regulation of gene expression, e.g. tetR inducible by tetracyline
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
    • C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
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    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/61—Fusion polypeptide containing an enzyme fusion for detection (lacZ, luciferase)
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/80—Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C12N2830/003—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor tet inducible
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/20—Vector systems having a special element relevant for transcription transcription of more than one cistron

Definitions

  • the present disclosure relates generally to methods for producing recombinant proteins including expression systems, vectors and cells for producing the same and the recombinant proteins produced therefrom.
  • nucleoporins and antibodies are difficult to produce recombinantly in a sufficient amount or in a stable system.
  • nucleoporins expressed in expression systems such as BiolD express very low amounts of Nup133 (Kim et al. 2014). This low expression can also affect the localization of the nucleoporins complex which in turn affects the activity.
  • Antibodies represent essential tools of biological and biomedical research, with uses that run the gamut from basic science through diagnostics to therapeutics. Their escalating utility in recent decades has increased demands for improved specificity and reliability, particularly from established commercial sources, and has been associated with numerous innovations in the development of monoclonal and recombinant antibodies.
  • Antibody-based reagents are one of the mainstays of biomedical research, yet antibody production processes have inherent deficiencies that can severely impact their reliability and reproducibility.
  • polyclonal antibodies not only does their quality widely differ between animals, but their properties can also evolve over time within a single animal. Even under ideal circumstances, such antibodies represent a finite resource that is ultimately linked to the lifespan of the immunised animal, potentially hindering the reproducibility of published studies.
  • Validated antibodies can then be archived for posterity as sequence files devoid of supernumerary heavy and light chains, a strategy that may be applied to any antibodies.
  • a recent report by DeLuca et al. further demonstrated how one might reproduce already available antibodies in house through antibody sequencing (Deluca, K. F. et al. Elife 10, 1-23 (2021)), underlying the interest of such approaches.
  • Antibody production is a costly, non-trivial and time-consuming process. It is costly because it requires highly skilled people to prepare and maintain the antibodies over extended periods of time. It either necessitates the maintenance of native hybridoma cell lines or requires the repeated generation of producing cells, both of which have obvious downsides. Native hybridoma cell lines are hindered by cell culture costs, ease of maintenance, and can effectively be lost. Loss of hybridomas are unfortunately not that uncommon, and can occur for various reasons (Kromenaker, S. J. & Srienc, F. Biotechnol. Prog. 10, 299-307 (1994), and Dasch, J. R. & Dasch, A. L. Cold Spring Harb. Protoc. 2017, 810-813 (2017)) On the other hand, repeating the generation of temporary producing cells for each batch of antibody is necessarily detrimental in terms of cost, time and consistency.
  • Mammalian cells have generally been seen as a system of choice for antibody production as they contain a full range of pathways for the addition of appropriate post-translational modifications. N-glycosylation is of particular significance here, since it is required to facilitate folding of nascent proteins entering the secretory pathway (Kawabe, Y. et al., J. Biosci. Bioeng. 125, 599-605 (2016)).
  • the choice of a suitable expression system moves the main technical challenge of antibody production from animal work and hybridoma handling towards the generation and maintenance of antibody-producing cell lines.
  • CRISPR/Cas9 The development and improvement in methods of molecular biology and genetic engineering, as well as the rapid development of genomic editing methods using CRISPR/Cas9, allows researchers to set themselves the task of targeted delivery of target genes to almost any selected constitutively transcribed genome regions.
  • CRISPR-based knock-in strategies based on homology-independent repair pathways show varying efficiency for gene knock-in in mammalian cells.
  • some methods suffer from the probability of plasmid backbone insertion at the target site.
  • studies trying to combine the targeting ability of the Cas9 molecule and the excision/integration capacity of the PB transposase have shown random integrations.
  • a system, vector or recombinant cell for production of recombinant proteins and methods of detecting the same is envisaged.
  • an aspect of the invention refers to an expression system for producing recombinant proteins comprising a protein antigen-binding molecule, the system comprising: a vector comprising: an antibiotic selection cassette; a doxycycline inducible promoter; a cleavable sequence between two cistrons; comprising a nucleic acid expressing a detectable marker optionally selected from a fluorescent protein or a peroxidase, nucleic acid capable of expressing the protein antigen-binding molecule inserted between a set of homologous genomic locus targeting sequences; and a single guide RNA.
  • a vector comprising: an antibiotic selection cassette; a doxycycline inducible promoter; a cleavable sequence between two cistrons; comprising a nucleic acid expressing a detectable marker optionally selected from a fluorescent protein or a peroxidase, nucleic acid capable of expressing the protein antigen-binding molecule inserted between a set of homologous genomic locus targeting
  • a method for producing recombinant proteins comprising a protein antigen-binding molecule, comprising the steps of: a) integrating a nucleic acid sequence capable of expressing the protein antigenbinding molecule into a vector comprising an antibiotic selection cassette; a doxycycline inducible promoter; a cleavable sequence between two cistrons; comprising a nucleic acid expressing a detectable marker optionally selected from a fluorescent protein or a peroxidase, nucleic acid capable of expressing the protein antigen-binding molecule inserted between a set of homologous genomic locus targeting sequences; b) selecting transfected cells in the presence of an antibiotic in a cell culture media; c) inducing expression of the vector by supplementing the cell culture media with doxycycline; d) co-expressing a single guide RNA; and e) collecting the expressed protein antigen-binding molecule.
  • detectible recombinant protein comprising a recombinant protein co-expressed with a horseradish peroxidase detectable marker comprising an amino acid sequence set forth in SEQ ID NO. 7.
  • a vector comprising: an antibiotic selection cassette; a doxycycline inducible promoter; a cleavable sequence between two cistrons; comprising a nucleic acid expressing a detectable marker set forth in amino acid sequence SEQ ID NO. 7, nucleic acid expressing a recombinant protein inserted between a set of homologous genomic locus targeting sequences.
  • a recombinant cell line comprising the expression system as described herein above or a vector as described herein above.
  • a method of detecting a) a product derived from: i) the expression system as described herein above, ii) the vector as described herein above, iii) the recombinant cell line as described herein above; or b) the detectible recombinant protein as described herein above, in a single step without the use of a secondary labeling reagents by detecting the detectable marker or the horseradish peroxidase detectable marker.
  • FIG. 1 T3 vectors for stable and inducible gene expression.
  • A T3P vector map.
  • B FACS quantification showing doxycycline-dependent MitoTimer (green) fluorescence from two live 3T3 cells lines (3T3_L1 , 3T3_L2) stably expressing the MitoTimer reporter. Hedges’s g is indicated, n >50.103 cells per condition, AU: Arbitrary Units. The density curves are displayed in light grey with overlaid box plots showing the median and 25-75% interquartile range for each sample. See also Fig. 2A.
  • C Immunofluorescence microscopy of 0.47pm confocal sections of the 3T3_L1 stable cell line showing doxycycline-dependent expression of the MitoTimer reporter. Scale bar: 25pm.
  • FIG. 2 (A) FACS quantification showing doxycycline-dependent (red) fluorescence from the oxidized MitoTimer reporter shown in Figure 1 B. Hedges’s g is indicated, n >50.103 cells per condition, AU: Arbitrary Units. (B) Western blotting showing doxycycline dependent V5-LAP2p expression in 3T3 cells. V5-tagged LAP2p was probed using an anti-V5 antibody. LAP2p and GAPDH are indicated. (C) Immunofluorescence microscopy showing doxycycline-dependent expression of V5-tagged LAP2p (0.47pm confocal section of 3T3 cells). Scale bar: 50pm.
  • FIG. 3 Production of recombinant antibodies with the T3 vector.
  • A Vector map showing the structure of conventional rAbs produced with T3s. SP: Signal Peptide, LC: Light Chain, HC: Heavy Chain, F: Furin site, 2A: 2A peptide, mNG: mNeonGreen.
  • B Western-Blotting showing Lmnbl expression in mouse (Mm, C2C12) and human (Hs, 293T) cells using conventional rAb-LCHC-Lmnb1 antibodies. Lmnbl (top panel) and p- tubulin and are indicated.
  • C Immunofluorescence microscopy of 0.47pm confocal sections of HeLa cells using native Lmnbl antibodies (hybridoma, left panel), corresponding rAb-LCHC-Lmnb1 (middle panels) and rAb-HCLC-Lmnb1 (Right Panels), probed with anti-mouse Alexa488-conjugated secondary antibodies. Scale bar: 25pm.
  • D Immunofluorescence microscopy with mNG-tagged conventional rAb-LCHC-Lmnb1 antibody (projection of a 15pm optical section of 3T3 cells). Direct mNeonGreen fluorescence and anti-mouse secondary antibody (Alexa594-conjugated) are shown.
  • FIG. 4 (A) Immunofluorescence microscopy with conventional rAb-LCHC- Lmnb2 and rAb-
  • LCHC-Man1 antibodies projection of a 15pm optical section of 3T3 cells, left panels), with anti-mouse secondary antibody Alexa594-conjugated. Scale bar: 50pm.
  • Western-Blotting with the same primary antibodies showing Lmnb2 and Mani expression in mouse (Mm, 3T3) and human (Hs, 293T) cells. Lmnb2 and Mani are indicated.
  • Corresponding loading controls are shown panel (C), bottom panel.
  • (E) Growth curve of 3T3 stable cell lines expressing two different T3 constructs with (red) and without (blue) doxycycline supplementation (n 3, error bars represent standard deviation).
  • FIG. 5 High-resolution imaging with rAb-scFvs.
  • A Vector map showing the structure of single-chain rAb-scFvs. SP: Signal Peptide, scFv: single chain Fragment variable, mNG: mNeonGreen.
  • B Immunofluorescence microscopy of 0.47pm confocal sections of HeLa cells using mNG-tagged rAb-Lmnb1 , in single-chain (scFv, left panels) and conventional (LCHC, right panels) configuration. Cells were imaged without secondary antibody addition, directly using mNG fluorescence. Scale bar: 25pm.
  • FIG. 1 Western-Blotting showing Lmnbl expression in mouse C2C12 and human 293T (left and right lane, respectively) by single-chain rAb-scFv-Lmnb1-mNG, using an anti-mNeonGreen secondary antibody and an anti-mouse-HRP tertiary antibody. Lmnbl is indicated. See also Figure 2C for corresponding loading control.
  • D Representative images by super-resolution microscopy (3D-SIM) of HeLa cells (0.125pm-thick optical section with orthogonal views) using native Lmnbl antibodies (hybridoma, top panel) and corresponding rAb-Lmnb1 (bottom panels).
  • FIG. 6 (A) Immunofluorescence microscopy with rAb-scFv-Lmnb1-mNG (top and middle panels) and rAb-LCHC-Lmnb1-mNG (bottom panels) antibodies, projection of a 15pm optical section of 3T3 cells. Anti-mouse secondary antibody (Alexa594-conjugated) and direct mNeonGreen fluorescence are shown. For the top panels (rAb-scFv-Lmnb1- mNG), imaging intensities were kept identical to that used for Figure 2D.
  • imaging intensities for the Alexa594 were adjusted equally between LCHC- and scFv-Lmnb1 to allow visualisation of the Alexa594 signal from the rAb-scFv-Lmnb1 antibody.
  • Scale bar 50pm.
  • FIG. 7 (A-C) Immunofluorescence microscopy of 0.47pm confocal sections of HeLa cells using mNG-tagged single-chain (rAb-scFv-Lmnb1-mNG, top panels) and conventional (rAb-LCHC-Lmnb1-mNG, bottom panels) anti-Lmnb1 antibodies according to the permeabilization method.
  • Lmnbl green, directly imaged using the mNG signal
  • Lamin A red
  • Corresponding orthogonal views and corresponding maximum intensity projections of the whole nuclei (Yp and Xp) are shown.
  • Permeabilization is as follow: Triton+ SDS (left panels), Triton (middle panel) and Methanol (right panels). Scale bar: 10 pm. See also corresponding maximum intensity projections of the whole nuclei.
  • FIG. 8 Maximum intensity projections of the confocal imaging shown in Fig. 7. Lmnbl (green, directly imaged using the mNeonGreen signal) and Lamin A (red) are shown. Permeabilization is as follow: Triton+ SDS (left panels), Triton (middle panel) and Methanol (right panels). Scale bar: 10 pm.
  • FIG. 9 mHRP-conjugated recombinant antibodies with T3.
  • A Schematic representation of rAb-scFv-mHRP antibody and mutations included in mHRP.
  • SP Signal Peptide
  • scFv single chain Fragment variable.
  • IHC Immunohistochemical staining of FFPE human cancer sections using direct rAb-scFv-p53-mHRP staining (single-step IHC, no secondary antibodies, left panel) or native DO-1 p53 antibody (two steps IHC with HRP-conjugated secondary antibodies, right panel), with hematoxylin and eosin (H&E) counterstain.
  • H&E hematoxylin and eosin
  • Each core of the tissue microarray is colour-coded according to the p53 expression pattern. Green highlights: extensive p53 expression in multiple cells/areas, red: limited p53 expression (few cells I low intensity), blue: very limited p53 expression (very few cells I low intensity) for one or both antibodies.
  • FIG. 10 (A) Single-step Western-blotting with conventional rAb-LCHC-Lmnb1 antibodies conjugated to APEX (left panel) or mHRP (right panel) in mouse (C2C12) and human (U2OS) cells. Lmnbl is indicated. (B) Loading controls corresponding to Western Blotting of rAb-LCHC-Lmnb1 antibodies conjugated to APEX (left panel) or mHRP (right panel) in mouse (C2C12) and human (U2OS) cells, as shown in (A), p-actin is indicated.
  • FIG. 12 FACS quantification showing high production SP2 cells showing doxycycline-dependant expression of H2B-mScarlet (RFP). Hedges’s g is indicated. The density curves are displayed in light grey with overlaid box plots showing the median and 25-75% interquartile range for each sample. A fold change of at least 70 is always observed.
  • T3 is a vector expression system designed for the expression of recombinant proteins, particularly difficult proteins such as antibodies, in mammalian cells. It combines doxycycline-inducible expression, CRISPR-mediated stable genomic integration, and single construct design. This enables simple, flexible, efficient and stable recombinant antibody production in any mammalian cell line of choice. The cells can be stored and reused as required for production, in virtually unlimited quantities. The T3 system can equally be used for any other recombinant protein production, with identical advantages.
  • the T3 vector system enables the generation of stable, inducible mammalian cell lines to produce recombinant antibodies. It is composed of the following key advantages:
  • the single plasmid DNA design reduces the number of necessary cloning /DNA synthesis steps, enabling rapid and simple modifications of the protein e.g., recombinant antibodies (rAbts).
  • the single DNA construct design improves transfection efficiency during cell line generation when compared to the standard two DNA constructs strategy.
  • the vector uses a high expression / low background version of the doxycycline inducible promoter.
  • the genomic integration is performed using CRISPR in conjunction with homology-repair enhancing protein mutant.
  • the CRISPR targets known sites permitting stable protein expression.
  • the drug selection cassette of the vector allows for the generation of pure populations of producing cells.
  • the selection cassette is flanked by restriction sites which allows easy adjustments to the selection strategy.
  • the stable lines can be stored and reused indefinitely as they can be maintained without expressing the recombinant antibodies, and do not need to be generated each time a new batch of antibody needs to be produced.
  • the T3 expression vectors allow the creation of stable cell lines using CRISPR-mediated genomic integration.
  • the system enables the controlled regulation of recombinant antibody production by simply adding doxycycline into the cell culture media.
  • the system combines an efficient cloning strategy with a single vector design which allows quick and flexible modifications of the recombinant antibodies to be produced.
  • an expression system for producing recombinant proteins comprising a protein antigen-binding molecule
  • the system comprising: a vector comprising: an antibiotic selection cassette; a doxycycline inducible promoter; a cleavable sequence between two cistrons; comprising a nucleic acid expressing a detectable marker optionally selected from a fluorescent protein or a peroxidase, nucleic acid capable of expressing the protein antigen-binding molecule inserted between a set of homologous genomic locus targeting sequences; and a single guide RNA.
  • doxycycline inducible promoter allows for DOX-inducible capabilities and with a guide RNA for genomic integration for stable antibody production in any mammalian cell lines such as standard mammalian cell lines including mouse fibroblasts, human fibroblasts, iPSC’s, Chinese Hamster Overy (CHO) cell lines.
  • mammalian cell lines such as standard mammalian cell lines including mouse fibroblasts, human fibroblasts, iPSC’s, Chinese Hamster Overy (CHO) cell lines.
  • rAbt recombinant antibody
  • the technology is applicable to the production of recombinant monoclonal antibodies. It is also applicable to the production of any recombinant protein, particularly for proteins which require production in a mammalian cell line.
  • the term “antigen-binding molecule” and its plural form refers to one or more molecule which is capable of binding to a target antigen, and encompasses any type of recombinant protein capable of binding to a target antigen including antibody fragments (e.g. Fv, scFv, Fab, scFab, F(ab’)2, Fab2, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g. VhH), etc.), as long as the recombinant protein expressed displays binding to the relevant target molecule(s).
  • antibody fragments e.g. Fv, scFv, Fab, scFab, F(ab’)2, Fab2, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g. VhH), etc.
  • the protein antigen-binding molecule comprises a single chain fraction variable (scFv).
  • the protein antigen-binding molecule comprises a single chain fraction variable.
  • the protein antigen-binding molecule comprises a heavy chain sequence and a light chain sequence separated by a 2A cleavage sequence wherein the heavy chain sequence (HC) and the light chain sequence (LC) may be in either order around the 2A cleavage sequence.
  • the protein antigen-binding molecule comprises HC - 2A cleavage sequence - LC while in some other embodiments the protein antigen-binding molecule comprises LC - 2A cleavage sequence - HC.
  • the cleavage sequence is flanked by a furin site.
  • the protein antigen-binding molecule may comprises the variable regions of any known antibody.
  • the protein antigen-binding molecule comprises a heavy chain sequence and a light chain sequence separated by a 2A cleavage sequence.
  • the 2A cleavage sequence comprises the nucleic acid sequence set forth in SEQ ID NO. 19.
  • the 2A cleavage sequence is flanked by a furin site.
  • the furin site sequence comprises the nucleic acid sequence set forth in SEQ ID NO. 18.
  • the nucleic acid expressing a detectable marker expresses a fluorescent protein.
  • the fluorescent protein may be any fluorescent protein known in the art.
  • the fluorescent protein may comprise green fluorescent protein (GFP) originally isolated from jellyfish or known genetically modified variants of GFP such as EGFP (has an excitation peak at 488 nm and an emission peak at 509 nm).
  • GFP green fluorescent protein
  • the fluorescent protein may comprise mNeonGreen a fluorescent protein derived from the lancelet Branchiostoma lanceolatum comprising multimeric yellow fluorescence protein.
  • the fluorescence protein mNeonGreen has an excitation maximum at 506 nm and an emission maximum at 517 nm.
  • the fluorescent protein may comprise mScarlet.
  • the nucleic acid expressing a detectable marker expresses a peroxidase.
  • the nucleic acid expressing a detectable marker expresses an enhanced detectable marker.
  • the detectable marker comprises an enhanced detectable marker wherein the detectible recombinant protein expressed in the system has an enhanced detection activity compared to other fluorescent protein detectable markers or other common peroxidase detectable markers.
  • the detectible recombinant protein expressed is to be used directly without the use of a secondary antibody in most cases the detectible maker should have an enhanced detection activity to allow a strong and easily detected signal.
  • the enhanced detectable marker expresses a fluorescent protein comprising mNeonGreen. In various embodiments, the enhanced detectable marker expresses a fluorescent protein comprising mScarlet
  • the fluorescent protein may comprise green fluorescent protein (GFP) originally isolated from jellyfish or known genetically modified variants of GFP such as EGFP (has an excitation peak at 488 nm and an emission peak at 509 nm).
  • GFP green fluorescent protein
  • the fluorescent protein may comprise mNeonGreen a fluorescent protein derived from the lancelet Branchiostoma lanceolatum comprising multimeric yellow fluorescence protein.
  • the fluorescence protein mNeonGreen has an excitation maximum at 506 nm and an emission maximum at 517 nm.
  • the expression product or the fluorescent protein comprising mNeonGreen has amino acid sequence set forth in SEQ ID NO. 6 - MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFS PWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYE GSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRY RSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK.
  • the protein having enhanced detection activity compared to GFP.
  • any nucleic acid sequence able to express mNeonGreen set forth in amino acid sequence SEQ ID NO. 6 may be used.
  • nucleic acids GCU, GCC, GCA and GCG will each result in the expression of the amino acid alanine. Consequently, a definable range of nucleic acid sequences will be able to express mNeonGreen set forth in amino acid sequence SEQ ID NO. 6 which will be easily be able to be discernible to a person skilled in the art.
  • the enhanced detectable marker expresses a peroxidase comprising a high activity variant of horseradish peroxidase.
  • the peroxidase protein comprising an enhanced horseradish peroxidase or high activity variant of horseradish peroxidase has an amino acid sequence set forth in SEQ ID NO. 7 QLTPTFYDNSCPDVSNIVRDIIVNELRSDPRIAASILRLHFHDCFVNGCDASILLDNTTSFRT EKDAFGNANSASGFSVIDRMKAAVESACPGTVSCADLLTIAAQQSVTLAGGPSWRVPLG RRDSLQAFLDLANANLPAPFFTLPQLKDSFRNVGLNRSSDLVALSGGHTFGKSQCRFIMD RLYNFSNTGLPDPTLNTTYLQTLRGLCPLNGNLSALVDFDLRTPTIFDNKYYVNLEEQKGLI QSDQELFSSPDATDTIPLVRSFADSTQTFFNAFVEAMDRMGNITPLTGTQGQIRLNCRW NSNS wherein the underlined amino acids are each point mutations of the wild type being N13D, T211, N75S, P78S,
  • any nucleic acid sequence able to express the variant of horseradish peroxidase set forth in amino acid sequence SEQ ID NO. 7 may be used.
  • the nucleic acid sequence able to express the variant of horseradish peroxidase set forth in amino acid sequence SEQ ID NO. 7 may be a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO. 11 wherein the resulting protein is that set forth in amino acid sequence SEQ ID NO. 7.
  • a sequence identity of at least 80% may comprise a sequence identity of 80%, or 81 %, or 82%, or 83%, or 84% or 85%, or 86%, or 87%, or 88%, or 89%, or 90%, or 91 %, or 92%, or 93%, or 94% or 95%, or 96%, or 97%, or 98%, or 99% provided it is able to express the variant of horseradish peroxidase set forth in amino acid sequence SEQ ID NO. 7 of the protein that has demonstrated an enhanced effect.
  • the nucleic acid sequence able to express the variant of horseradish peroxidase set forth in amino acid sequence SEQ ID NO. 7 may be a nucleic acid sequence set forth in SEQ ID NO. 11 .
  • FP fluorescent protein
  • mHRP modified horseradish peroxidase
  • the enhanced detectable marker expresses peroxidase comprising an ascorbate peroxidase, APEX2.
  • the peroxidase protein comprising an ascorbate peroxidase APEX2 is site a directed modification of APEX ascorbate peroxidase isolated from pea or soyabean plants.
  • APEX2 expresses an amino acid sequence set forth in SEQ ID NO.
  • any nucleic acid sequence able to express the variant of ascorbate peroxidase APEX2 set forth in amino acid sequence SEQ ID NO. 8 may be used.
  • the single guide RNA is expressed in conjunction with an adenoviral E1 B55K mutant protein.
  • the single guide RNA is housed in an adenoviral vector co-expressing an adenoviral E1 B55K mutant.
  • an adenoviral vector co-expressing an adenoviral E1 B55K mutant.
  • the single guide RNA comprises a sequence having 95% or more sequence identity with the nucleic acid sequence set forth in SEQ ID NO. 1 .
  • the single guide RNA is for integration into any mammalian cell genome. In various embodiments, the single guide RNA is for integration into a mouse genome. In various embodiments, the single guide RNA is for integration into a human genome. In various embodiments, the single guide RNA comprises a sequence having 95% or more sequence identity with the nucleic acid sequence set forth in SEQ ID NO. 1 . In various embodiments, the single guide RNA comprises a sequence having 96% or more sequence identity with the nucleic acid sequence set forth in SEQ ID NO. 1 . In various embodiments, the single guide RNA comprises a sequence having 97% or more sequence identity with the nucleic acid sequence set forth in SEQ ID NO. 1.
  • the single guide RNA comprises a sequence having 98% or more sequence identity with the nucleic acid sequence set forth in SEQ ID NO. 1. In various embodiments, the single guide RNA comprises a sequence having 99% or more sequence identity with the nucleic acid sequence set forth in SEQ ID NO. 1. In various embodiments, the single guide RNA comprises a sequence having a nucleic acid sequence set forth in SEQ ID NO. 1 .
  • sequence for the tetracycline-trans-activator comprises the nucleic acid sequence set forth in SEQ ID NO. 9: 5’ CGGCCACGAGTTTGAGCAGATGTTTACCTGGCCG 3’
  • the inducible expression of genes from the TRE tetracycline response element, also called tetO
  • tetO tetracycline response element
  • rtTA Tet-On
  • the set of homologous genomic locus targeting sequences comprise homologous sequences targeting a mouse TIGRE genomic locus.
  • the mouse TIGRE locus is situated on chromosome 9 of the mouse genome between the AB124611 (HIDE1) and Carmi loci. This has the advantage of having tightly regulated high level expression of the detectable recombinant protein such as a protein antigen-binding molecule.
  • a method for producing recombinant proteins comprising a protein antigen-binding molecule, comprising the steps of: integrating a nucleic acid sequence capable of expressing the protein antigen-binding molecule into a vector comprising an antibiotic selection cassette; a doxycycline inducible promoter; a cleavable sequence between two cistrons; comprising a nucleic acid expressing a detectable marker optionally selected from a fluorescent protein or a peroxidase, nucleic acid capable of expressing the protein antigen-binding molecule inserted between a set of homologous genomic locus targeting sequences; selecting transfected cells in the presence of an antibiotic in a cell culture media; inducing expression of the vector by supplementing the cell culture media with doxycycline; co-expressing a single guide RNA; and collecting the expressed protein antigen-binding molecule.
  • the method comprises determining the nucleic acid sequence capable of expressing the protein antigen-binding molecule by sequencing the variable region of an antibody.
  • detectible recombinant protein comprising a recombinant protein co-expressed with a horseradish peroxidase detectable marker comprising an amino acid sequence set forth in SEQ ID NO. 7.
  • the detectable recombinant protein comprises a protein antigen-binding molecule.
  • the protein antigen-binding molecule of the detectable recombinant protein comprises a single chain fraction variable.
  • the protein antigen-binding molecule of the detectable recombinant protein comprises a heavy chain sequence and a light chain sequence separated by a 2A cleavage sequence.
  • the 2A cleavage sequence comprises the nucleic acid sequence set forth in SEQ ID NO. 19.
  • the 2A cleavage sequence is flanked by a furin site.
  • the furin site sequence comprises the nucleic acid sequence set forth in SEQ ID NO. 18.
  • the protein antigen-binding molecule of the detectable recombinant protein comprises anti-Lamin.
  • the anti-Lamin comprises the nucleic acid set forth in SEQ ID NO. 12.
  • the anti- Lamin comprises the nucleic acid set forth in SEQ ID NO. 13.
  • the anti-Lamin comprises the nucleic acid set forth in SEQ ID NO.
  • the detectable recombinant protein comprises the modified horseradish peroxidase (mHRP) expressed amino acid SEQ ID NO. 7.
  • the anti-Lamin comprises the nucleic acid set forth in SEQ ID NO. 15 wherein the detectable recombinant protein comprises the modified Neon green (mNeonGreen) expressed as amino acid SEQ ID NO. 6.
  • the protein antigen-binding molecule of the detectable recombinant protein comprising anti-Lamin is selected from the group consisting of any one of nucleic acid sequences set forth in SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16 and SEQ ID NO. 17.
  • the protein antigen-binding molecule of the detectable recombinant protein comprises anti-Lamin variable regions.
  • anti-Lamin variable regions of clone BBLB1 c7 comprising anti Lmnbl variable regions.
  • the nucleic acid sequence set forth in SEQ ID NO. 16 expresses anti-Lmnb1 variable regions of clone BBLB1 wherein the detectable recombinant protein comprises the modified Neon green (mNeonGreen) expressed as amino acid SEQ ID NO. 6.
  • the nucleic acid sequence set forth in SEQ ID NO. 16 expresses anti-Lmnb1 variable regions of clone BBLB1 wherein the detectable recombinant protein comprises the modified horseradish peroxidase (mHRP) expressed amino acid SEQ ID NO. 7.
  • the protein antigen-binding molecule of the detectable recombinant protein comprises anti-p53 variable regions.
  • anti-p53 variable regions comprise anti-p53 variable regions of clone DO-1.
  • nucleic acid sequence set forth in SEQ ID NO. 20 expresses anti-p53 variable regions of clone DO-1
  • a method of using the detectable marker by detecting the detectible recombinant protein described herein above in a single step without the use of a secondary labeling reagents.
  • the detectible marker is detected on a western blot.
  • the detectible marker is immunohistochemically detected in formalin fixed tissue samples.
  • a vector comprising: an antibiotic selection cassette; a doxycycline inducible promoter; a cleavable sequence between two cistrons; comprising a nucleic acid expressing a detectable marker set forth in amino acid sequence SEQ ID NO. 7, nucleic acid expressing a recombinant protein inserted between a set of homologous genomic locus targeting sequences.
  • the recombinant protein expressed comprises an antigen-binding molecule comprising a single chain fraction variable (scFv).
  • scFv single chain fraction variable
  • the recombinant protein expressed comprises an antigen-binding molecule comprising a heavy chain sequence and a light chain sequence separated by a 2A cleavage sequence.
  • the 2A cleavage sequence comprises the nucleic acid sequence set forth in SEQ ID NO. 19.
  • the 2A cleavage sequence is flanked by a furin site.
  • the furin site sequence comprises the nucleic acid sequence set forth in SEQ ID NO. 18.
  • a recombinant cell line comprising the expression system as described herein above or a vector as described herein above.
  • the recombinant cell line expresses the detectable recombinant protein as described herein above.
  • the recombinant cell line is a mouse fibroblast cell line comprising the expression system as described herein above or a vector as described herein above.
  • the set of homologous genomic locus targeting sequences comprise homologous sequences targeting a mouse TIGRE genomic locus.
  • the mouse TIGRE locus is situated on chromosome 9 of the mouse genome between AB124611 (HIDE1) and Carmi loci. This has the advantage of having tightly regulated high level expression of the detectable recombinant protein such as a protein antigen-binding molecule.
  • a method of detecting a) a product derived from: i) the expression system as described herein above, ii) the vector as described herein above, iii) the recombinant cell line as described herein above; or b) the detectible recombinant protein as described herein above, in a single step without the use of a secondary labeling reagents by detecting the detectable marker or the horseradish peroxidase detectable marker.
  • the method of detecting the detectible marker is detected via a western blot.
  • the method of detecting the detectible marker is immunohistochemically detected in formalin fixed tissue samples.
  • nucleic acid sequence capable of expressing the protein antigenbinding molecule into a vector comprising an antibiotic selection cassette; a doxycycline inducible promoter; a cleavable sequence between two citrons; comprising a nucleic acid expressing a detectable marker optionally selected from a fluorescent protein or a peroxidase, nucleic acid capable of expressing the protein antigen-binding molecule inserted between a set of homologous genomic locus targeting sequences;
  • the method further comprises determining the nucleic acid sequence capable of expressing the protein antigen-binding molecule by sequencing the variable region of an antibody and using this nucleic acid sequence capable of expressing the protein antigen-binding molecule to integrate into the vector. This allows the method to easily be used to express detectable recombinant protein for any protein antigen-binding molecule.
  • a detectible recombinant protein comprising a recombinant protein fused to a horseradish peroxidase detectable marker comprising an amino acid sequence set forth in SEQ ID NO. 7.
  • the detectible recombinant protein is derivable from or derived from the expression of the expression system described herein above.
  • the detectible recombinant protein is derivable from or derived from the expression of the vector described herein above.
  • the detectible recombinant protein is derivable from or derived from expression of the expression system or the vector in the recombinant cell line described herein.
  • the high activity variant of recombinant horseradish peroxidase detectable marker permits enhanced detection activity compared to wild type horseradish peroxidase or other known horseradish peroxidase mutations.
  • the detectible recombinant protein comprises a protein antigen-binding molecule.
  • the detectible recombinant protein antigen-binding molecule comprises a single chain fraction variable.
  • examples of the detectible recombinant protein antigen-binding molecule comprising a single chain fraction variable sequence are set forth in any one of SEQ ID NOS. 16, 17 and 20.
  • the detectible recombinant protein antigen-binding molecule comprises a heavy chain sequence and a light chain sequence separated by a 2A cleavage sequence.
  • the 2A cleavage sequence is flanked by a furin site.
  • examples of the detectible recombinant protein antigenbinding molecule comprising a heavy chain sequence and a light chain sequence are set forth in any one of SEQ ID NOS. 12 to 15.
  • nucleic acids 30 to 33 of the sequence set forth in SEQ ID NO. 10 may be replaced with the any one of the nucleic acid sequences set forth in SEQ ID NO.1 1 , or SEQ ID NO.12, or SEQ ID NO. 13, or SEQ ID NO. 14, or SEQ ID NO. 15, or SEQ ID NO. 16, or SEQ ID NO. 17, or SEQ ID NO. 20.
  • the detectible recombinant protein antigen-binding molecule comprises an anti-p53 variable region. In various embodiments, the detectible recombinant protein antigen-binding molecule comprises an anti-p53 clone DO-1 variable regions. These provide the advantage of being able to detect the antibody without the use of any secondary labeling reagents.
  • the detectible recombinant protein may be used for detecting the detectable marker in a single step without the use of a secondary labeling reagents. In various embodiments, the use further comprises detecting the detectable marker in a single step without the use of a secondary antibodies. In various embodiments, the detectible recombinant protein is used to be detected in any known method for detecting the presence of an antigen with a protein antigen-binding molecule.
  • the use of the detectible recombinant protein may be detected in any one of the methods selected from the group consisting of western blotting; immunohistochemistry (IHC), super-resolution imaging, enzyme-linked immunosorbent assay (ELISA), immunofluorescence microscopy, and Fluorescence activated cell sorting (FACS).
  • the detectible recombinant protein is used for detecting the presence of a protein on a western blot without the use of a secondary antibody.
  • the detectible recombinant protein may be used for detecting molecule such as a protein immunohistochemically in tissue samples without the use of a secondary labelling reagents.
  • a vector comprising: a) an antibiotic selection cassette; b) a doxycycline inducible promoter; c) a cleavable sequence between two cistrons; comprising a nucleic acid expressing a detectable marker set forth in amino acid sequence SEQ ID NO. 7, nucleic acid expressing a recombinant protein inserted between a set of homologous tetracycline-inducible genomic locus targeting sequences.
  • the recombinant protein comprises an antigenbinding molecule comprising a single chain fraction variable.
  • the recombinant protein comprises an antigen-binding molecule comprises a heavy chain sequence and a light chain sequence separated by a 2A cleavage sequence.
  • the 2A cleavage sequence is flanked by a furin site.
  • a recombinant cell line comprising the expression system according to any embodiment of the system described herein above or a vector according any embodiments of the vector described.
  • the recombinant cell line is able to express the detectable recombinant protein according to any embodiment of the detectable recombinant protein described herein above.
  • a method of detecting a) a product derived from i) the expression system described hereinabove; ii) the vector described hereinabove; or iii) the recombinant cell line described hereinabove; or b) the detectible recombinant protein described hereinabove in a single step without the use of a secondary labeling reagents by detecting the detectable marker or the horseradish peroxidase detectable marker.
  • the method comprises detecting the detectable marker in a single step without the use of a secondary antibodies.
  • the lack of a secondary labeling reagents comprises the lack of a secondary antibody.
  • the detectible marker is detected in any known method for detecting the presence of an antigen with a protein antigen-binding molecule.
  • the detectible marker is detected in any one of the methods selected from the group consisting of western blotting; immunohistochemistry (IHC), super-resolution imaging, enzyme-linked immunosorbent assay (ELISA), immunofluorescence microscopy, and Fluorescence activated cell sorting (FACS).
  • the detectible marker is detected on a western blot without the use of a secondary antibody. In various embodiments, the detectible marker is immunohistochemically detected in tissue samples without the use of a secondary labelling reagents. In various embodiments, the detectible marker is immunohistochemically detected in tissue samples without the use of a secondary antibody.
  • T3 To address the problems of current recombinant protein expression systems, an expression system was designed that combines independently developed features into one cohesive vector hereafter named T3.
  • the vector is first targeted to the mouse TIGRE locus.
  • the system is further organised around an efficient rtTA variant and allows highly inducible expression while minimizing leakage.
  • T3 vectors are shown to support efficient expression of a wide range of cDNAs, then demonstrate their ability to generate stable and inducible lines producing recombinant antibodies by simply adding doxycycline to the cell culture medium. These lines address several problems inherent to antibody production, while retaining the advantages of the T3 design.
  • an integrated system for the reformatting and synthesis of recombinant proteins such as monoclonal antibodies are described, with the ability to tailor each recombinant protein such as monoclonal antibodies to specific applications.
  • T3 vector system for the generation of inducible stable cell lines producing a range of recombinant antibodies and their derivatives.
  • Using an exemplary antibody against Lamin B1 it was demonstrated that the T3-based system embodies a flexible and efficient platform with which to produce a wide range of recombinant antibodies, while addressing several issues inherent to such processes. It is shown that these antibodies replicate qualities of hybridoma-derived antibodies in applications ranging from immunohistochemistry to super-resolution imaging, without the need for purification steps. When directly fused to detectable marker such as fluorescent proteins, these recombinant antibodies obviate the requirement for secondary labelling reagents. This permits speedier and more facile imaging while retaining the performance of the parental antibody.
  • T3 is a vector expression system designed for the expression of recombinant antibodies in mammalian cells. It combines doxycyclin-inducible expression, CRISPR-mediated stable genomic integration, and single construct design.
  • the T3 vector is built around a tetON inducible system, with optimal orientation of its various elements, puromycin selection cassette and multiple-cloning site including efficient ligation-free cloning. These elements are enclosed between homology arms targeting the chosen genomic location (Fig. 1A), here the mouse safe harbour TIGRE locus is used however another set of homologous genomic locus targeting sequences may also be used.
  • the associated sgRNA is cloned into a vector co-expressing the adenoviral E1 B55K mutant, shown to enhance homology-directed repair.
  • 3T3 cells with integrated MitoTimer reporter were treated with mock or 1 pg/mL doxycycline for 48 h before being trypsinized, resuspended in cell culture growth medium, filter through cell strainder (Corning Falcon, #352235) and analysed for fluorescence intensity on (BD LSR II) flow cytometer. More than 50 000 cells were analysed per condition. Data analysis was performed using Excel, Prism (Graphpad) and R software. Box plots indicate median with 10-90 percentile whiskers and g indicates Hedge’s g.
  • the T3 vector permits stable and doxycycline (DOX)- inducible expression of constructs, with very low background and high selection efficiency.
  • DOX doxycycline
  • FACS fluorescence-assisted cell sorting
  • Fig. 1 B and C, Fig. 2A immunofluorescence microscopy in mouse 3T3 fibroblasts
  • LBR-GFP lamin B receptor/green fluorescent protein fusion
  • Hybridoma cell lines were grown in RPMI 1640 supplemented with 4 mM L- glutamine, 100U/ml penicillin and streptomycin, 50pM 2-mercaptoethanol and 10% heat inactivated fetal bovine serum.
  • HeLa, 293T, C2C12 and U2OS cells were grown at 37°C in 5% CO 2 in standard DMEM high glucose with 10% foetal bovine serum, 100U/ml penicillin and streptomycin, with the addition of 1 mM sodium pyruvate and 2mM L-glutamine for NIH3T3.
  • hybridoma-derived antibodies described in this study are as follow: anti- Lmnbl (clone BBLB1 c7), anti-Sun2 (clone 3.1 E), anti-Lmnb2 (clone BBLB2 3.13), anti-Man1 (clone BBManl A22. and anti-mNeonGreen (clone BBneon N19.1).
  • Establishment of the hybridomas was performed using the same procedure as described earlier (Chai, R. J. et al. Nat. Commun. 12, 4722 (2021)).
  • the best hybridoma clones were sent for antibody sequencing (Absolute Antibody), and the resulting sequences of the variable regions of the heavy and light chains (with their respective signal peptides) were further sent for DNA synthesis (Integrated DNA Technologies). Both chains were then separately cloned into pFUSE-CHIg and pFUSE2-CLIg-mK expression vectors (InvivoGen), respectively.
  • HeLa cells were transiently transfected with both pFUSE-CHIg and pFUSE2-CLIg-mK at a ratio of 2:3, using Lipofectamine 2000. Growth media was then collected at 24-48h post transfection, and the specificity of the recombinant native antibodies was compared to their hybridoma equivalent by immunofluorescence microscopy and western blotting, using antigen overexpression /knockout /knockdown strategies as appropriate.
  • 3T3 cells were transfected at 80-90% confluency using PEImax 40kDa (Polyscience 24765-2) with a mix of Cas9+sgRNA plasmid and targeting T3 vector (see sequesnce listing) for 6h. Media was then replaced with standard growth medium supplemented with 1 pg/ml puromycin (Sigma #P9620) after 24h. Cells were selected for 1- 2 weeks before production of the antibodies was started upon addition of 1 pg/ml doxycycline (Clontech #631311). Cell culture supernatant was then collected 48h hours after doxycycline induction, filter-sterilized and stored at 4°C. Antibody containing media was then used without further purification or concentration.
  • T3 plasmids are described herein. Complete sequence and the cloning strategy are provided in the sequence listing.
  • a mix of Cas9+sgRNA plasmid and the targeting T3 vector comprising any one of SEQ ID NOS 2-4 and 10-20 alone or in the combinations mentioned throughout or depicted in Fig. 1A, Fig. 3A, Fig. 5A, Fig. 9A, Fig. 11A or 11 C
  • the transfection mixture was replaced with regular growth medium 6h after transfection and selection was started 24h later with addition of 1 pg/mL puromycin (Sigma; #P9620) to standard growth medium for duration of 1-2 weeks.
  • T3 vectors were then customised for the production of conventional recombinant antibodies (rAbs) containing both light- and heavy-chain segments (LCHC) as seen in any one of SEQ ID NOS. 12-15.
  • rAbs conventional recombinant antibodies
  • LCHC light- and heavy-chain segments
  • the choice of a single-vector setup necessitated the use of a 2A cleavage sequence (SEQ ID NO. 19) to separate heavy and light chain sequences, with a furin site (SEQ ID NO. 18) intended to minimize left-over amino acids from the 2A sequence (Fig. 3A).
  • SEQ ID NO. 19 2A cleavage sequence
  • SEQ ID NO. 18 furin site intended to minimize left-over amino acids from the 2A sequence
  • Fig. 3A The inducible design of T3 vectors permits the production of antibodies by simply harvesting the growth medium of any cell line after supplementation with doxycycline.
  • Mouse 3T3 fibroblasts were used here as vessels for rapid validation purposes, but a cornucopia of cell lines is evidently available for antibody production.
  • rAbs produced from the T3 vectors retained the labelling properties of their original hybridoma parent. This is illustrated here using a mouse anti-Lmnb1 antibody derived from its hybridoma clone (lgG2b), in both Western-Blotting and confocal microscopy (Fig. 3B-3C). This strategy was applicable for multiple other conventional antibodies, including anti-Lmnb2 and anti-Man1 (Fig. 4A). Reversing heavy and light chain order within the T3 vector construct appears to have little to no effect on labeling performance (Fig. 3C right panels, and Fig. 4C).
  • the T3 design conveniently allows for simple modification of the antibodies by fusing their heavy chain (or indeed light chain) to any tag of choice.
  • mNeonGreen28 mNG
  • Fig. 3D mNeonGreen28
  • tagged rAbs still retain their utility for conventional indirect imaging whenever necessary, as shown with anti-Lmnb1 (Fig. 3D, middle panel), anti-Lmnb2 and anti-Sun2 (Fig. 4B).
  • these antibodies display excellent detection capabilities even in demanding immunofluorescence assays, such as formalin-fixed paraffin-embedded (FFPE) tissue microarrays used for cross-reactivity antibody testing (Fig. 3E).
  • FFPE formalin-fixed paraffin-embedded
  • anti-rabbit immunoglobulins HRP (Dako; #P0448), anti-mouse immunoglobulins (Dako; #P0447).Vast majority of the rAbs were produced from stable NIH3T3 cell lines by adding 1 pg/mL doxycycline (dox) (Clontech; #631311) to the growth medium and collecting the cell culture supernatant 48h later.
  • dox doxycycline
  • the two anti-Lmnb1 rAbs LCHC-APEX2 and LCHC-mHRP were produced by transiently transfecting 293T cells with T3 plasmids using Lipofectamine 2000, followed by replacing the media with 1 pg/mL dox- containing growth and collecting the cell culture supernatant 48h later.
  • the antibodies- containing culture media were filter-sterilized and used without further purification or concentration.
  • the DO-1 antibody was a generous gift from David Lane.
  • Fluorescently tagged scFvs allow direct, high-resolution imaging
  • Single chain antibodies constitute a valuable development to the antibody toolkit.
  • scFv Single chain antibodies
  • T3 vectors described above provide rapid means for scFv production, by simply cloning the heavy and light chain variable regions in tandem (Fig. 5A).
  • the two chains are separated by a 15 amino acid residue linker (3x GGGGS), while only the N-terminal signal peptide of the heavy chain variable region is retained.
  • the conventional indirect labelling approach would require fluorescent secondary antibodies to detect the primary scFv.
  • the mNG-tagged scFvs can also be used for Western-blotting when combined with a triple sandwich strategy, by adding an intermediate anti-mNeonGreen labelling step to the detection procedure (Fig. 5C).
  • Fig. 5C an intermediate anti-mNeonGreen labelling step to the detection procedure.
  • this approach can be of use in specific Western-blotting setups, it hardly appears satisfactory when considering the resulting increase in incubation times and labelling procedures.
  • the highly fluorescent proteins needed for direct immunofluorescence assays was mirrored, and highly active recombinant peroxidases were made use of, as described in the next section.
  • FFPE formalin- fixed parrafin-embedded
  • the results demonstrate that the T3 vectors accommodate a wide range of constructs expressed in a stable and inducible manner, from single proteins to conjugated single-chain antibodies. They enable applications spanning standard immunofluorescence, super-resolution microscopy and rapid detection of targets in IHC experiments, as shown here with p53 expression in human cancer sections, while merely requiring the collection of media from standard cell culture experiments.
  • the slides were quenched for endogenous peroxidase activity by 1% H2O2 in PBS and subsequently blocked in 10% goat serum in PBS (Gibco; # 16210064). Slides were then stained for p53 with either scFv-mHRP for 2 h or hybridoma derived antibody for 2 h followed by 30 min incubation with goat antimouse IgG HRP (Promega; #W402B) for 30 min and visualization with Envision DAB substrate (DAKO) for 3 min. The tissues were quickly counterstained with hematoxylin, dehydrated and mounted in Richard-Allen Scientific Mounting Medium (Thermo Scientific; # 4112). The control staining consisted of slides being incubated for 30 min with secondary HRP antibody only.
  • FFPE slides Biomax
  • FDA999y multiple organ normal tissue microarray
  • BCN601 a Multiple organ carcinoma with normal tissue microarray
  • Human lip cancer whole tissue section Human lip cancer whole tissue section
  • Retroviral constructs are however notoriously prone to silencing, a less than desirable attribute for producing cells lines that can require extensive passaging (Yao, S. et al., Mol. Ther.10, 27-36 (2004)). Of particular difficulty is the expression of detrimental proteins or enzymes which can place an additional burden on the cellular machinery (May, D. G. et al, Cells 9, (2020)). This is compounded by the fact that some expressing lines can also be inherently unstable, as has been shown to be the case for many rabbit hybridomas (Weber, J. et al., Exp. Mol. Med. 49, (2017)).
  • Transgene expression can be triggered for protein production, in this case antibodies, by doxycycline supplementation, and supernatants harvested before cells become debilitated by demands placed on their protein synthesis and secretory machineries. Final expression levels obviously depend on a multitude of factors, but tet-on systems should essentially perform on par with the best conventional constitutive promoters.
  • NIH3T3 stable lines expressing Sun2 HCLC-mNG or p53 scFv-mHRP were seeded in 6-well plates at a density of 34 000 cells/well. The following day the cells were mock or dox-induced and incubated for 6 days. After the incubation the cells were collected and the cells number was determined (Bio-Rad TC20TM Automated Cell Counter). The experiment were conducted in triplicate at three different times.
  • T3 vectors also provide a simple system to produce single-chain antibodies (scFvs, nanobodies etc.) where cleavage sites are superfluous.
  • sequence optimization may prove beneficial with scFvs and scFv fusion proteins being tailored for specific applications, for instance IHC versus superresolution microscopy.
  • T3 ecosystem allows the inducible expression of any construct of choice, and in particular the production of tailor-made antibodies.
  • the system is compatible with both conventional and single chain antibodies, either of which can be modified with fluorescent protein tags or with high activity peroxidases, such as th newly developed mHRP described herein.
  • high activity peroxidases such as th newly developed mHRP described herein.
  • Image processing and quantification was performed using a mix of Imaged (Schindelin, J. et al. Nat. Methods 9, 676-682 (2012)), SoftWorX (GE Healthcare), Imaris (Bitplane), VSViewer (Metasystems) and Cell Profiler (McQuin, C. et al. PLoS Biol. 16, 1-17 (2016)).
  • Data analysis was performed using Excel, Prism (Graphpad) and R software (Team, R. C. R (2020)). Box plots indicate median with 10-90 percentile whiskers, g indicates Hedge’s g.

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Abstract

Reported is the construction of a T3 vector expression system for the generation of inducible stable cell lines producing a range of recombinant proteins such as antibodies and their derivatives. It combines doxycyclin-inducible expression, CRISPR-mediated stable genomic integration, and single construct design. The T3-based system embodies a flexible and efficient platform with which to produce a wide range of recombinant antibodies, while addressing several issues inherent to such processes. When directly fused to detectable marker such as fluorescent proteins, these recombinant antibodies obviate the requirement for secondary labelling reagents. Additionally, a novel, high-activity variant of recombinant horseradish peroxidase was generated, which can be used in single-step Western-blotting experiments. Moreover, coupling this modified HRP with a single chain anti-p53 antibody effectively allowed single-step detection of p53 expression, in cancer lesions from formalin-fixed human tissue samples.

Description

DESCRIPTION
TITLE OF INVENTION: [T3 VECTORS FOR RECOMBINANT PROTEIN PRODUCTION IN MAMMALIAN CELLS]
REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority to Singapore patent application No. 10202260123P, filed 17 November 2022, the contents of which are incorporated herein by reference.
TECHNICAL FIELD
[0002] The present disclosure relates generally to methods for producing recombinant proteins including expression systems, vectors and cells for producing the same and the recombinant proteins produced therefrom.
BACKGROUND
[0003] The following discussion of the background to the invention is intended to facilitate an understanding of the present invention only. It should be appreciated that the discussion is not an acknowledgement or admission that any of the material referred to was published, known or part of the common general knowledge of the person skilled in the art in any jurisdiction as at the priority date of the invention.
[0004] Many proteins such as nucleoporins and antibodies are difficult to produce recombinantly in a sufficient amount or in a stable system. Often nucleoporins expressed in expression systems such as BiolD express very low amounts of Nup133 (Kim et al. 2014). This low expression can also affect the localization of the nucleoporins complex which in turn affects the activity.
[0005] Antibodies represent essential tools of biological and biomedical research, with uses that run the gamut from basic science through diagnostics to therapeutics. Their escalating utility in recent decades has increased demands for improved specificity and reliability, particularly from established commercial sources, and has been associated with numerous innovations in the development of monoclonal and recombinant antibodies.
[0006] Antibody-based reagents are one of the mainstays of biomedical research, yet antibody production processes have inherent deficiencies that can severely impact their reliability and reproducibility. Of particular concern are polyclonal antibodies: not only does their quality widely differ between animals, but their properties can also evolve over time within a single animal. Even under ideal circumstances, such antibodies represent a finite resource that is ultimately linked to the lifespan of the immunised animal, potentially hindering the reproducibility of published studies.
[0007] The development of monoclonal antibodies, therefore, provided a major advance in researchers’ ability to standardise reagents and experiments. However, even here performance cannot always be guaranteed as hybridomas may suffer from genomic instability. More insidiously, they may produce additional unwanted immunoglobulin heavy or light chains, potentially introducing unwanted or spurious reactivities (Bradbury, A. R. M. et al. MAbs 10, 539-546 (2018). As pointed out by Bradbury and Pluckthun (Nature 518, 27-9 (2015)), these uncertainties can be addressed by sequencing heavy and light chain variable regions and confirming that recombinant versions of the antibody retain the desired specificity. Validated antibodies can then be archived for posterity as sequence files devoid of supernumerary heavy and light chains, a strategy that may be applied to any antibodies. A recent report by DeLuca et al. further demonstrated how one might reproduce already available antibodies in house through antibody sequencing (Deluca, K. F. et al. Elife 10, 1-23 (2021)), underlying the interest of such approaches.
[0008] Antibody production is a costly, non-trivial and time-consuming process. It is costly because it requires highly skilled people to prepare and maintain the antibodies over extended periods of time. It either necessitates the maintenance of native hybridoma cell lines or requires the repeated generation of producing cells, both of which have obvious downsides. Native hybridoma cell lines are hindered by cell culture costs, ease of maintenance, and can effectively be lost. Loss of hybridomas are unfortunately not that uncommon, and can occur for various reasons (Kromenaker, S. J. & Srienc, F. Biotechnol. Prog. 10, 299-307 (1994), and Dasch, J. R. & Dasch, A. L. Cold Spring Harb. Protoc. 2017, 810-813 (2017)) On the other hand, repeating the generation of temporary producing cells for each batch of antibody is necessarily detrimental in terms of cost, time and consistency.
[0009] In an attempt to overcome such problems recombinant antibodies in mammalian cells, Insect-, plant- or algae-based systems have also been devised for antibody production (Donini, M. & Marusic, C. Biotechnol. Lett. 41 , 335-346 (2019); Palmberger, D. et al. J. Biotechnol. 153, 160-166 (2011); Fujita, R. et al. Biochem. Biophys. Res. Commun. 529, 257-262 (2020); and Rosales-Mendoza, S. et al. Molecules 25, 1-25 (2020)), and robust yields have been achieved by addition of production- enhancing drugs to the culture media (Ha, T. K et al., Biotechnol. J. 14, 1-11 (2019); and Ling, W. L. W. et al. Biotechnol. Prog. 19, 158-162 (2003)), increasing the proportion of cells transiently producing the antibody (Korn, J. et al. Sci. Rep. 10, 1-10 (2020)) or stable antibody-expressing cells (Kawabe, Y. et al., J. Biosci. Bioeng. 125, 599-605 (2018); Dangi, A. K.,et al., Pharmacol. 9, 1-12 (2018); and Kost, T. A. et al., Nat. Biotechnol. 23, 567-575 (2005)). However, derivation of recombinant antibodies necessitates the availability of a reliable expression system. Mammalian cells have generally been seen as a system of choice for antibody production as they contain a full range of pathways for the addition of appropriate post-translational modifications. N-glycosylation is of particular significance here, since it is required to facilitate folding of nascent proteins entering the secretory pathway (Kawabe, Y. et al., J. Biosci. Bioeng. 125, 599-605 (2018)). The choice of a suitable expression system, however, moves the main technical challenge of antibody production from animal work and hybridoma handling towards the generation and maintenance of antibody-producing cell lines. Unfortunately, these tend to display poor survival properties associated with endoplasmic reticulum (ER) stress, a consequence of the burden placed on the secretory pathway by high levels of antibody synthesis (Du, Z. et al. Biotechnol. Bioeng. 110, 2184-2194 (2013)). In addition, production-enhancing compounds themselves can impose additional stress in terms of cell proliferation and toxicity (Ling, W. L. W. et al. Biotechnol. Prog. 19, 158-162 (2003)). As a result, antibodyproducing cell lines grown for extended periods of time are often prone to diminishing transgene expression (Chusainow, J. et al. Biotechnol. Bioeng. 102, 1182-1196 (2009)).
[0010] Strategies developed for the expression of recombinant proteins in mammalian cells include transfection, transduction, have been developed (Nayerossadat, N.,et al., Adv. Biomed. Res. 1 , 27 (2012); and Lino, C. A., et al., Drug Deliv. 25, 1234-1257 (2018)). In one example the capabilities of a lentiviral system was harnessed to enrich for transduced cells expressing proteins of interest under a doxinducible promoter (Chojnowski, A. et al. Elife 4, 1-21 (2015)). Retroviral constructs are however notoriously prone to silencing, a less than desirable attribute for producing cells lines that can require extensive passaging (Yao, S. et al. Mol. Ther. 10, 27-36 (2004); Elsasser, S. J. et al., Nature 522, 240-244 (2015); and Xia, X. et al., Stem Cells Dev. 16, 167-76 (2007)). Of particular difficulty is the expression of detrimental proteins or enzymes which can place an additional burden on the cellular machinery (May, D. G. et al., Cells 9, (2020)). This is compounded by the fact that some expressing lines can also be inherently unstable, as has been shown to be the case for many rabbit hybridomas (Weber, J. et al., Exp. Mol. Med. 49, (2017)).
[0011] Vectors using a tetON inducible system have been reported (T. Das, A. et al., Curr. Gene Ther. 16, 156-167 (2016)). The optimization varies with the system they are used in and the other elements of the vector used. tetON inducible system with multiplecloning site including efficient ligation-free cloning have been reported (Weissmann, F. et al., Proc. Natl. Acad. Sci. U. S. A. 113, E2564-9 (2016)).
[0012] The development and improvement in methods of molecular biology and genetic engineering, as well as the rapid development of genomic editing methods using CRISPR/Cas9, allows researchers to set themselves the task of targeted delivery of target genes to almost any selected constitutively transcribed genome regions. CRISPR-based knock-in strategies based on homology-independent repair pathways show varying efficiency for gene knock-in in mammalian cells. However, some methods suffer from the probability of plasmid backbone insertion at the target site. On the other hand, studies trying to combine the targeting ability of the Cas9 molecule and the excision/integration capacity of the PB transposase have shown random integrations. Some reports have shown to enhance homology-directed repair where the associated sgRNA is cloned into a vector co-expressing the adenoviral E1 B55K mutant, (Chu, V. T. et al., Nat. Biotechnol. 33, 543-548 (2015)).
[0013] There exists a need for tools and methods to stably express recombinant proteins such as antibodies and alleviate at least one of the aforementioned problems.
SUMMARY
[0014] A system, vector or recombinant cell for production of recombinant proteins and methods of detecting the same is envisaged.
[0015] Accordingly, an aspect of the invention refers to an expression system for producing recombinant proteins comprising a protein antigen-binding molecule, the system comprising: a vector comprising: an antibiotic selection cassette; a doxycycline inducible promoter; a cleavable sequence between two cistrons; comprising a nucleic acid expressing a detectable marker optionally selected from a fluorescent protein or a peroxidase, nucleic acid capable of expressing the protein antigen-binding molecule inserted between a set of homologous genomic locus targeting sequences; and a single guide RNA.
[0016] According to another aspect there is a method for producing recombinant proteins comprising a protein antigen-binding molecule, comprising the steps of: a) integrating a nucleic acid sequence capable of expressing the protein antigenbinding molecule into a vector comprising an antibiotic selection cassette; a doxycycline inducible promoter; a cleavable sequence between two cistrons; comprising a nucleic acid expressing a detectable marker optionally selected from a fluorescent protein or a peroxidase, nucleic acid capable of expressing the protein antigen-binding molecule inserted between a set of homologous genomic locus targeting sequences; b) selecting transfected cells in the presence of an antibiotic in a cell culture media; c) inducing expression of the vector by supplementing the cell culture media with doxycycline; d) co-expressing a single guide RNA; and e) collecting the expressed protein antigen-binding molecule.
[0017] According to another aspect there is a detectible recombinant protein comprising a recombinant protein co-expressed with a horseradish peroxidase detectable marker comprising an amino acid sequence set forth in SEQ ID NO. 7.
[0018] According to another aspect there is a vector comprising: an antibiotic selection cassette; a doxycycline inducible promoter; a cleavable sequence between two cistrons; comprising a nucleic acid expressing a detectable marker set forth in amino acid sequence SEQ ID NO. 7, nucleic acid expressing a recombinant protein inserted between a set of homologous genomic locus targeting sequences. [0019] According to another aspect there is a recombinant cell line comprising the expression system as described herein above or a vector as described herein above.
[0020] According to another aspect there is a method of detecting: a) a product derived from: i) the expression system as described herein above, ii) the vector as described herein above, iii) the recombinant cell line as described herein above; or b) the detectible recombinant protein as described herein above, in a single step without the use of a secondary labeling reagents by detecting the detectable marker or the horseradish peroxidase detectable marker.
[0021] Other aspects and features of the present invention will become apparent to those of ordinary skill in the art upon review of the following description of specific embodiments of the invention in conjunction with the accompanying figures.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] In the figures, which illustrate, by way of non-limiting examples only, embodiments of the present invention,
[0023] [Fig. 1]: T3 vectors for stable and inducible gene expression. (A) T3P vector map. (B) FACS quantification showing doxycycline-dependent MitoTimer (green) fluorescence from two live 3T3 cells lines (3T3_L1 , 3T3_L2) stably expressing the MitoTimer reporter. Hedges’s g is indicated, n >50.103 cells per condition, AU: Arbitrary Units. The density curves are displayed in light grey with overlaid box plots showing the median and 25-75% interquartile range for each sample. See also Fig. 2A. (C) Immunofluorescence microscopy of 0.47pm confocal sections of the 3T3_L1 stable cell line showing doxycycline-dependent expression of the MitoTimer reporter. Scale bar: 25pm.
[0024] [Fig. 2]: (A) FACS quantification showing doxycycline-dependent (red) fluorescence from the oxidized MitoTimer reporter shown in Figure 1 B. Hedges’s g is indicated, n >50.103 cells per condition, AU: Arbitrary Units. (B) Western blotting showing doxycycline dependent V5-LAP2p expression in 3T3 cells. V5-tagged LAP2p was probed using an anti-V5 antibody. LAP2p and GAPDH are indicated. (C) Immunofluorescence microscopy showing doxycycline-dependent expression of V5-tagged LAP2p (0.47pm confocal section of 3T3 cells). Scale bar: 50pm. (D) Live fluorescence microscopy showing doxycycline-dependant expression of H2B-mScarlet in 3T3 cells. Scale bar: 200pm. (E) Live fluorescence microscopy showing the induction of LBR-GFP following dox supplementation in 293T cells. Scale bar: 100pm.
[0025] [Fig. 3]: Production of recombinant antibodies with the T3 vector. (A) Vector map showing the structure of conventional rAbs produced with T3s. SP: Signal Peptide, LC: Light Chain, HC: Heavy Chain, F: Furin site, 2A: 2A peptide, mNG: mNeonGreen. (B) Western-Blotting showing Lmnbl expression in mouse (Mm, C2C12) and human (Hs, 293T) cells using conventional rAb-LCHC-Lmnb1 antibodies. Lmnbl (top panel) and p- tubulin and are indicated. (C) Immunofluorescence microscopy of 0.47pm confocal sections of HeLa cells using native Lmnbl antibodies (hybridoma, left panel), corresponding rAb-LCHC-Lmnb1 (middle panels) and rAb-HCLC-Lmnb1 (Right Panels), probed with anti-mouse Alexa488-conjugated secondary antibodies. Scale bar: 25pm. (D) Immunofluorescence microscopy with mNG-tagged conventional rAb-LCHC-Lmnb1 antibody (projection of a 15pm optical section of 3T3 cells). Direct mNeonGreen fluorescence and anti-mouse secondary antibody (Alexa594-conjugated) are shown. Scale bar: 50pm. (E) Immunofluorescence microscopy of FFPE human tissue sections using mNG-tagged rAb-Sun2 antibody. Areas of interest are indicated on the slide overview (left panel, white boxes. O: Oesophagus, B: Breast, T: Testis), with higher magnification shown on the corresponding right panels. Direct mNeonGreen fluorescence and anti-mouse secondary antibody (Alexa594-conjugated) both corresponding to rAb-Sun2 are shown.
[0026] [Fig. 4]: (A) Immunofluorescence microscopy with conventional rAb-LCHC- Lmnb2 and rAb-
[0027] LCHC-Man1 antibodies (projection of a 15pm optical section of 3T3 cells, left panels), with anti-mouse secondary antibody Alexa594-conjugated. Scale bar: 50pm. Western-Blotting with the same primary antibodies (right panels), showing Lmnb2 and Mani expression in mouse (Mm, 3T3) and human (Hs, 293T) cells. Lmnb2 and Mani are indicated. Corresponding loading controls are shown panel (C), bottom panel. (B) Immunofluorescence microscopy with mNG-tagged conventional rAb-LCHC-Lmnb2 and rAb-LCHC-Sun2 antibodies (projection of a 15pm optical section of 3T3 cells, left panels), with anti-mouse secondary antibody Alexa594-conjugated. Scale bar: 50pm. Western- Blotting with the same primary antibodies (right panels), showing Lmnb2 expression in mouse (Mm, 3T3) and human (Hs, 293T) cells, and Sun2 expression in mouse (Mm, C2C12) and human (Hs, 293T) cells. Lmnb2 and Sun2 are indicated. See also Figure 2B and panel (C) bottom panels for corresponding loading controls. (C) Western-Blotting showing Lmnbl expression in mouse (Mm, 3T3) and human (Hs, 293T) cells using conventional rAb-HCLC-Lmnb1 antibodies. Lmnbl (upper panel) and a-actinin (bottom panel) are indicated. (D) Immunofluorescence microscopy with mNG-tagged conventional rAb-Lmnb1 antibody using rabbit constant regions (projection of a 3pm optical section on HeLa cells). Direct mNeonGreen fluorescence and anti-rabbit secondary antibody Alexa594-conjugated are shown. Scale bar: 50pm. (E) Growth curve of 3T3 stable cell lines expressing two different T3 constructs with (red) and without (blue) doxycycline supplementation (n =3, error bars represent standard deviation).
[0028] [Fig. 5]: High-resolution imaging with rAb-scFvs. (A) Vector map showing the structure of single-chain rAb-scFvs. SP: Signal Peptide, scFv: single chain Fragment variable, mNG: mNeonGreen. (B) Immunofluorescence microscopy of 0.47pm confocal sections of HeLa cells using mNG-tagged rAb-Lmnb1 , in single-chain (scFv, left panels) and conventional (LCHC, right panels) configuration. Cells were imaged without secondary antibody addition, directly using mNG fluorescence. Scale bar: 25pm. (C) Western-Blotting showing Lmnbl expression in mouse C2C12 and human 293T (left and right lane, respectively) by single-chain rAb-scFv-Lmnb1-mNG, using an anti-mNeonGreen secondary antibody and an anti-mouse-HRP tertiary antibody. Lmnbl is indicated. See also Figure 2C for corresponding loading control. (D) Representative images by super-resolution microscopy (3D-SIM) of HeLa cells (0.125pm-thick optical section with orthogonal views) using native Lmnbl antibodies (hybridoma, top panel) and corresponding rAb-Lmnb1 (bottom panels). Native antibodies from hybridoma supernatant (top panel) and conventional T3s (middle panels) were labelled using anti-mouse Alexa488-conjugated secondary antibodies, while mNG-tagged scFv-antibodies were directly imaged without secondary antibody addition (bottom panels). Lmnbl (green) and Lamin A (red) are shown. Scale bar: 5 pm. See also [Fig.6] B for individual channels. (E) Corresponding maximum intensity projections of the same whole nuclei showing Lmnbl (green, left panels) and Lamin A (red, middle panels). Scale bar: 5 pm.
[0029] [Fig. 6]: (A) Immunofluorescence microscopy with rAb-scFv-Lmnb1-mNG (top and middle panels) and rAb-LCHC-Lmnb1-mNG (bottom panels) antibodies, projection of a 15pm optical section of 3T3 cells. Anti-mouse secondary antibody (Alexa594-conjugated) and direct mNeonGreen fluorescence are shown. For the top panels (rAb-scFv-Lmnb1- mNG), imaging intensities were kept identical to that used for Figure 2D. For the bottom two panels (rAb-scFv-Lmnb1-mNG and rAb-LCHC-Lmnb1-mNG), imaging intensities for the Alexa594 were adjusted equally between LCHC- and scFv-Lmnb1 to allow visualisation of the Alexa594 signal from the rAb-scFv-Lmnb1 antibody. Scale bar: 50pm. (B) Individual channel images corresponding to Figure 3D merged images. Lmnbl (green) and Lamin A (red) are shown. Scale bar: 5 pm. (C) Representative images by super-resolution microscopy of HeLa cells (0.125pm-thick optical section with orthogonal views) using conventional rAb-LCHC-Lmnb1-mNG (top panels), rAb-HCLC-Lmnb1 (bottom panels), and corresponding maximum intensity projections of the same whole nuclei. Lmnbl (green) and Lamin A (red) are shown. rAb-LCHCLmnb1-mNG was imaged without secondary antibody addition (top panels), while rAb-HCLC-Lmnb1 was imaged using anti-mouse Alexa488- conjugated secondary antibodies (bottom panels). Scale bar: 5 pm. (D) Individual channel images corresponding to Fig. 6C merged images. Lmnbl (green) and Lamin A (red) are shown. Scale bar: 5 pm.
[0030] [Fig. 7]: (A-C) Immunofluorescence microscopy of 0.47pm confocal sections of HeLa cells using mNG-tagged single-chain (rAb-scFv-Lmnb1-mNG, top panels) and conventional (rAb-LCHC-Lmnb1-mNG, bottom panels) anti-Lmnb1 antibodies according to the permeabilization method. Lmnbl (green, directly imaged using the mNG signal) and Lamin A (red) are shown. Corresponding orthogonal views and corresponding maximum intensity projections of the whole nuclei (Yp and Xp) are shown. Permeabilization is as follow: Triton+ SDS (left panels), Triton (middle panel) and Methanol (right panels). Scale bar: 10 pm. See also corresponding maximum intensity projections of the whole nuclei.
[0031] [Fig. 8]: Maximum intensity projections of the confocal imaging shown in Fig. 7. Lmnbl (green, directly imaged using the mNeonGreen signal) and Lamin A (red) are shown. Permeabilization is as follow: Triton+ SDS (left panels), Triton (middle panel) and Methanol (right panels). Scale bar: 10 pm.
[0032] [Fig. 9]: mHRP-conjugated recombinant antibodies with T3. (A) Schematic representation of rAb-scFv-mHRP antibody and mutations included in mHRP. SP: Signal Peptide, scFv: single chain Fragment variable. (B) Standard two-steps Western-Blotting analysis using native hybridoma-derived Lmnbl antibodies (Hyb, using standard antibody/HRP-secondary antibodies, leftmost panel), and single-step Western-Blotting using mHRP-conjugated rAb-Lmnb1 antibodies (scFv and LCHC, without HRP-secondary antibodies, middle and right panels respectively) in mouse (C2C12) and human cells (U2OS). Lmnbl (upper panels) and corresponding p-actin loading control (lower panels) are shown. (C) Single-step Western-Blotting analysis using mHRP-conjugated single chain anti-p53 antibody (rAb-scFv-p53-mHRP, without HRP secondary antibodies, left panel) and standard two-steps Western-Blotting using native hybridoma-derived p53 antibodies (DO1 , using standard antibody/HRP secondary antibodies, right panel), in mouse (C2C12) and human (293T) cells. p53 is indicated (upper panels). The loading control (Sun2) is shown in the bottom panels. (D) Immunohistochemical (IHC) staining of FFPE human cancer sections using direct rAb-scFv-p53-mHRP staining (single-step IHC, no secondary antibodies, left panel) or native DO-1 p53 antibody (two steps IHC with HRP-conjugated secondary antibodies, right panel), with hematoxylin and eosin (H&E) counterstain. Each core of the tissue microarray is colour-coded according to the p53 expression pattern. Green highlights: extensive p53 expression in multiple cells/areas, red: limited p53 expression (few cells I low intensity), blue: very limited p53 expression (very few cells I low intensity) for one or both antibodies. Unlabelled: no detectable p53 expression with both antibodies. Scale bar: 2mm. (E) High magnification microscopy of selected IHC staining of FFPE human cancer sections shown in (D) using direct rAb-scFv-p53-mHRP staining (single-step IHC, no secondary antibodies, left panel) or native DO-1 p53 antibody (two steps IHC with HRP-conjugated secondary antibodies, right panel), with H&E counterstain. Scale bar: 100pm. (F) IHC staining of FFPE sections from human lower lip squamous cell carcinoma (grade II), using direct rAb-scFv-p53-mHRP staining (single-step IHC, no secondary antibodies, left panel), or native DO-1 p53 antibody (two steps IHC with HRP- conjugated secondary antibodies, middle panel) with H&E counterstain. Control using antimouse lgG1 -specific antibody as primary antibody is shown. Scale bar: 50pm.
[0033] [Fig. 10]: (A) Single-step Western-blotting with conventional rAb-LCHC-Lmnb1 antibodies conjugated to APEX (left panel) or mHRP (right panel) in mouse (C2C12) and human (U2OS) cells. Lmnbl is indicated. (B) Loading controls corresponding to Western Blotting of rAb-LCHC-Lmnb1 antibodies conjugated to APEX (left panel) or mHRP (right panel) in mouse (C2C12) and human (U2OS) cells, as shown in (A), p-actin is indicated. (C) Western-blotting in denaturing and reducing condition showing heavy and light chains of recombinant antibodies used in (B), detected using anti-mouse HRP-conjugated secondary antibodies. Heavy Chain (HC) and Light Chain (LC) are indicated. (D) Full slide imaging (upper panels) and intermediate magnification (lower panels) of the selected areas (white boxes) of IHC staining of FFPE sections from human lower lip squamous cell carcinoma shown Figure 4F, using direct rAb-scFvp53-mHRP staining (single-step IHC, no secondary antibodies, left panel) or native DO-1 p53 antibody (two steps IHC with HRP- conjugated secondary antibodies, middle panels) with H&E counterstain. Control using anti-mouse lgG1 -specific antibody as primary antibody is shown. Scale bar: 100pm. [0034] [Fig. 11]: (A) Development and (B) performance of different vectors until (C) the advantageously allows stable recombinant protein production when used with a CRISPR guide RNA.
[0035] [Fig. 12]: FACS quantification showing high production SP2 cells showing doxycycline-dependant expression of H2B-mScarlet (RFP). Hedges’s g is indicated. The density curves are displayed in light grey with overlaid box plots showing the median and 25-75% interquartile range for each sample. A fold change of at least 70 is always observed.
DETAILED DESCRIPTION
[0036] Throughout this document, unless otherwise indicated to the contrary, the terms “comprising”, “consisting of’, “having” and the like, are to be construed as non-exhaustive, or in other words, as meaning “including, but not limited to”.
[0037] Furthermore, throughout the document, unless the context requires otherwise, the word “include” or variations such as “includes” or “including” will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.
[0038] T3 is a vector expression system designed for the expression of recombinant proteins, particularly difficult proteins such as antibodies, in mammalian cells. It combines doxycycline-inducible expression, CRISPR-mediated stable genomic integration, and single construct design. This enables simple, flexible, efficient and stable recombinant antibody production in any mammalian cell line of choice. The cells can be stored and reused as required for production, in virtually unlimited quantities. The T3 system can equally be used for any other recombinant protein production, with identical advantages.
[0039] The T3 vector system enables the generation of stable, inducible mammalian cell lines to produce recombinant antibodies. It is composed of the following key advantages:
1) The cloning of the sequence of the protein to produce is done using robust, restriction-enzyme free cloning. Cloning steps can easily be bypassed entirely by direct DNA synthesis.
2) The single plasmid DNA design reduces the number of necessary cloning /DNA synthesis steps, enabling rapid and simple modifications of the protein e.g., recombinant antibodies (rAbts). 3) The single DNA construct design improves transfection efficiency during cell line generation when compared to the standard two DNA constructs strategy.
4) The vector uses a high expression / low background version of the doxycycline inducible promoter.
5) The vector structure and orientation of its elements enhances inducibility and lowers background expression.
6) The genomic integration is performed using CRISPR in conjunction with homology-repair enhancing protein mutant.
7) The CRISPR targets known sites permitting stable protein expression.
8) The drug selection cassette of the vector allows for the generation of pure populations of producing cells. The selection cassette is flanked by restriction sites which allows easy adjustments to the selection strategy.
9) The inducibility of the protein production such as antibodies decouples the generation of the cell line from the production of the antibodies. This bypasses the toxicity and stability issues that are inherent to stable lines, enabling the growth of producing cells to any chosen final amounts before the production of antibodies is triggered.
10) The stable lines can be stored and reused indefinitely as they can be maintained without expressing the recombinant antibodies, and do not need to be generated each time a new batch of antibody needs to be produced.
11) The strategy is applicable to any producing line of choice.
[0040] Unless defined otherwise, all other technical and scientific terms used herein have the same meaning as is commonly understood by a skilled person to which the subject matter herein belongs.
[0041] As disclosed herein, the T3 expression vectors allow the creation of stable cell lines using CRISPR-mediated genomic integration. In various embodiments the system enables the controlled regulation of recombinant antibody production by simply adding doxycycline into the cell culture media. In addition, in various embodiments the system combines an efficient cloning strategy with a single vector design which allows quick and flexible modifications of the recombinant antibodies to be produced. [0042] According to various embodiments there is an expression system for producing recombinant proteins comprising a protein antigen-binding molecule, the system comprising: a vector comprising: an antibiotic selection cassette; a doxycycline inducible promoter; a cleavable sequence between two cistrons; comprising a nucleic acid expressing a detectable marker optionally selected from a fluorescent protein or a peroxidase, nucleic acid capable of expressing the protein antigen-binding molecule inserted between a set of homologous genomic locus targeting sequences; and a single guide RNA.
[0043] The availability of antibodies in recombinant format opens-up numerous avenues for the creation of novel protein binding reagents. In the examples, variable region sequencing of the monoclonal antibodies was applied and recombinant equivalents targeting nuclear proteins (including p53 and lamin B1) were created, in conventional and single chain versions. By including fluorescent protein (FP) tags such as mNeonGreen (mNG), The developed system is suitable for applications ranging from one-step immunofluorescence microscopy to more demanding assays such as fluorescent immunohistochemistry (IHC) on paraffin sections and 3D-Structured Illumination microscopy (3D-SIM). For the latter, it was additionally able to achieve a subtle yet clear superiority of the single chain (scFv) versus conventional antibody format for the nuclear envelope protein Lmnbl .
[0044] Using doxycycline inducible promoter allows for DOX-inducible capabilities and with a guide RNA for genomic integration for stable antibody production in any mammalian cell lines such as standard mammalian cell lines including mouse fibroblasts, human fibroblasts, iPSC’s, Chinese Hamster Overy (CHO) cell lines. The single vector construction brings together multiple independent features, giving the system unique capabilities and flexibility for recombinant antibody (rAbt) production. The technology is applicable to the production of recombinant monoclonal antibodies. It is also applicable to the production of any recombinant protein, particularly for proteins which require production in a mammalian cell line. [0045] Throughout the description, it is to be appreciated that the term “antigen-binding molecule” and its plural form refers to one or more molecule which is capable of binding to a target antigen, and encompasses any type of recombinant protein capable of binding to a target antigen including antibody fragments (e.g. Fv, scFv, Fab, scFab, F(ab’)2, Fab2, diabodies, triabodies, scFv-Fc, minibodies, single domain antibodies (e.g. VhH), etc.), as long as the recombinant protein expressed displays binding to the relevant target molecule(s).
[0046] In various embodiments, the protein antigen-binding molecule comprises a single chain fraction variable (scFv).
[0047] Surprisingly, scFvs fused to fluorescent proteins were found to mostly be as effective as their conventional counterparts used in conjunction with fluorescent secondary antibodies or better.
[0048] In various embodiments, the protein antigen-binding molecule comprises a single chain fraction variable. In various embodiments the protein antigen-binding molecule comprises a heavy chain sequence and a light chain sequence separated by a 2A cleavage sequence wherein the heavy chain sequence (HC) and the light chain sequence (LC) may be in either order around the 2A cleavage sequence. For example, in some embodiments the protein antigen-binding molecule comprises HC - 2A cleavage sequence - LC while in some other embodiments the protein antigen-binding molecule comprises LC - 2A cleavage sequence - HC. In various embodiments the cleavage sequence is flanked by a furin site. In various embodiments the protein antigen-binding molecule may comprises the variable regions of any known antibody.
[0049] In various embodiments, the protein antigen-binding molecule comprises a heavy chain sequence and a light chain sequence separated by a 2A cleavage sequence. In various embodiments the 2A cleavage sequence comprises the nucleic acid sequence set forth in SEQ ID NO. 19.
[0050] In various embodiments, the 2A cleavage sequence is flanked by a furin site. In various embodiments the furin site sequence comprises the nucleic acid sequence set forth in SEQ ID NO. 18.
[0051] In various embodiments, the nucleic acid expressing a detectable marker expresses a fluorescent protein. [0052] In various embodiments, the fluorescent protein may be any fluorescent protein known in the art. In various embodiments the fluorescent protein may comprise green fluorescent protein (GFP) originally isolated from jellyfish or known genetically modified variants of GFP such as EGFP (has an excitation peak at 488 nm and an emission peak at 509 nm). In various embodiments the fluorescent protein may comprise mNeonGreen a fluorescent protein derived from the lancelet Branchiostoma lanceolatum comprising multimeric yellow fluorescence protein. The fluorescence protein mNeonGreen has an excitation maximum at 506 nm and an emission maximum at 517 nm. In various embodiments the fluorescent protein may comprise mScarlet.
[0053] In various embodiments, the nucleic acid expressing a detectable marker expresses a peroxidase.
[0054] In various embodiments, the nucleic acid expressing a detectable marker expresses an enhanced detectable marker.
[0055] In various embodiments, the detectable marker comprises an enhanced detectable marker wherein the detectible recombinant protein expressed in the system has an enhanced detection activity compared to other fluorescent protein detectable markers or other common peroxidase detectable markers. As the detectible recombinant protein expressed is to be used directly without the use of a secondary antibody in most cases the detectible maker should have an enhanced detection activity to allow a strong and easily detected signal.
[0056] In various embodiments, the enhanced detectable marker expresses a fluorescent protein comprising mNeonGreen. In various embodiments, the enhanced detectable marker expresses a fluorescent protein comprising mScarlet
[0057] In various embodiments the fluorescent protein may comprise green fluorescent protein (GFP) originally isolated from jellyfish or known genetically modified variants of GFP such as EGFP (has an excitation peak at 488 nm and an emission peak at 509 nm). In various embodiments the fluorescent protein may comprise mNeonGreen a fluorescent protein derived from the lancelet Branchiostoma lanceolatum comprising multimeric yellow fluorescence protein. The fluorescence protein mNeonGreen has an excitation maximum at 506 nm and an emission maximum at 517 nm.
[0058] In various embodiments, the expression product or the fluorescent protein comprising mNeonGreen has amino acid sequence set forth in SEQ ID NO. 6 - MVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFS PWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYE GSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRY RSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYK. The protein having enhanced detection activity compared to GFP.
[0059] In various embodiments, any nucleic acid sequence able to express mNeonGreen set forth in amino acid sequence SEQ ID NO. 6 may be used. In is known in the art that there are several nucleic acids that are able to express the same amino acid. For example, nucleic acids GCU, GCC, GCA and GCG will each result in the expression of the amino acid alanine. Consequently, a definable range of nucleic acid sequences will be able to express mNeonGreen set forth in amino acid sequence SEQ ID NO. 6 which will be easily be able to be discernible to a person skilled in the art.
[0060] In various embodiments, the enhanced detectable marker expresses a peroxidase comprising a high activity variant of horseradish peroxidase.
[0061] In various embodiments, the peroxidase protein comprising an enhanced horseradish peroxidase or high activity variant of horseradish peroxidase has an amino acid sequence set forth in SEQ ID NO. 7 QLTPTFYDNSCPDVSNIVRDIIVNELRSDPRIAASILRLHFHDCFVNGCDASILLDNTTSFRT EKDAFGNANSASGFSVIDRMKAAVESACPGTVSCADLLTIAAQQSVTLAGGPSWRVPLG RRDSLQAFLDLANANLPAPFFTLPQLKDSFRNVGLNRSSDLVALSGGHTFGKSQCRFIMD RLYNFSNTGLPDPTLNTTYLQTLRGLCPLNGNLSALVDFDLRTPTIFDNKYYVNLEEQKGLI QSDQELFSSPDATDTIPLVRSFADSTQTFFNAFVEAMDRMGNITPLTGTQGQIRLNCRW NSNS wherein the underlined amino acids are each point mutations of the wild type being N13D, T211, N75S, P78S, R93G, N175S, N255D and N268D that collectively result in a protein having enhanced detection activity compared to the wild type and other known mutations. In various embodiments, any nucleic acid sequence able to express the variant of horseradish peroxidase set forth in amino acid sequence SEQ ID NO. 7 may be used. In various embodiments, the nucleic acid sequence able to express the variant of horseradish peroxidase set forth in amino acid sequence SEQ ID NO. 7 may be a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO. 11 wherein the resulting protein is that set forth in amino acid sequence SEQ ID NO. 7. In various embodiments a sequence identity of at least 80% may comprise a sequence identity of 80%, or 81 %, or 82%, or 83%, or 84% or 85%, or 86%, or 87%, or 88%, or 89%, or 90%, or 91 %, or 92%, or 93%, or 94% or 95%, or 96%, or 97%, or 98%, or 99% provided it is able to express the variant of horseradish peroxidase set forth in amino acid sequence SEQ ID NO. 7 of the protein that has demonstrated an enhanced effect. In various embodiments, the nucleic acid sequence able to express the variant of horseradish peroxidase set forth in amino acid sequence SEQ ID NO. 7 may be a nucleic acid sequence set forth in SEQ ID NO. 11 .
[0062] As a complement to fluorescent protein (FP) tags, a novel high-activity form of horse radish peroxidase variant of horseradish peroxidase or modified horseradish peroxidase (mHRP) which incorporates a number of activity-enhancing mutations have been generated. In Various embodiments, by fusing mHRP to single chain and conventional antibody species, a unique set of reagents have been created that can be employed for rapid Western-blot analyses, without the need for secondary antibodies. Strikingly, in an example where mHRP was coupled to single-chain antibodies against human p53 the rapid identification of p53 expression in cancer lesions in formalin-fixed paraffin-embedded (FFPE) sections from patient tissue samples was observed.
[0063] In various embodiments, the enhanced detectable marker expresses peroxidase comprising an ascorbate peroxidase, APEX2.
[0064] In various embodiments, the peroxidase protein comprising an ascorbate peroxidase APEX2 is site a directed modification of APEX ascorbate peroxidase isolated from pea or soyabean plants. In various embodiments the ascorbate peroxidase, APEX2 expresses an amino acid sequence set forth in SEQ ID NO. 8 - MGKSYPTVSPDYQDAIEKAKRKLRGFIAEKKCAPLILRLAFHSAGTFDSKTKTGGPFGTIKH QAELAHGANNGLDIAVRLLEPIKEQFPIVSYADFYQLAGVVAVEITGGPKVPFHPGREDKP EPPPEGRLPDPTKGSDHLRDVFGKAMGLSDQDIVALSGGHTIGAAHKERSGFEGPWTSN PLIFDNSYFTELLTGEKDGLLQLPSDKALLTDSVFRPLVEKYAADEDVFFADYAEAHLKLSE LGFAEA wherein the underlined amino acids are each point mutations of the wild type being K14D, W41 F, E112K and A134P that collectively result in a protein having enhanced detection activity compared to the wildtype. In various embodiments, any nucleic acid sequence able to express the variant of ascorbate peroxidase APEX2 set forth in amino acid sequence SEQ ID NO. 8 may be used.
[0065] In various embodiments, the single guide RNA is expressed in conjunction with an adenoviral E1 B55K mutant protein.
[0066] In various embodiments, the single guide RNA is housed in an adenoviral vector co-expressing an adenoviral E1 B55K mutant. Which has the ability to enhance the set of homologous tetracycline-inducible genomic locus targeting sequences to have tightly regulated high level expression of the detectable recombinant protein such as a protein antigen-binding molecule.
[0067] In various embodiments, the single guide RNA comprises a sequence having 95% or more sequence identity with the nucleic acid sequence set forth in SEQ ID NO. 1 .
[0068] In various embodiments, the single guide RNA is for integration into any mammalian cell genome. In various embodiments, the single guide RNA is for integration into a mouse genome. In various embodiments, the single guide RNA is for integration into a human genome. In various embodiments, the single guide RNA comprises a sequence having 95% or more sequence identity with the nucleic acid sequence set forth in SEQ ID NO. 1 . In various embodiments, the single guide RNA comprises a sequence having 96% or more sequence identity with the nucleic acid sequence set forth in SEQ ID NO. 1 . In various embodiments, the single guide RNA comprises a sequence having 97% or more sequence identity with the nucleic acid sequence set forth in SEQ ID NO. 1. In various embodiments, the single guide RNA comprises a sequence having 98% or more sequence identity with the nucleic acid sequence set forth in SEQ ID NO. 1. In various embodiments, the single guide RNA comprises a sequence having 99% or more sequence identity with the nucleic acid sequence set forth in SEQ ID NO. 1. In various embodiments, the single guide RNA comprises a sequence having a nucleic acid sequence set forth in SEQ ID NO. 1 .
[0069] In various embodiments, the sequence for the tetracycline-trans-activator (rtTA) comprises the nucleic acid sequence set forth in SEQ ID NO. 9: 5’ CGGCCACGAGTTTGAGCAGATGTTTACCTGGCCG 3’
[0070] In various embodiments, the inducible expression of genes from the TRE (tetracycline response element, also called tetO) promoter in response to rtTA (Tet-On) activation.
[0071] In various embodiments, where the system is expressed in mouse fibroblasts the set of homologous genomic locus targeting sequences comprise homologous sequences targeting a mouse TIGRE genomic locus. The mouse TIGRE locus is situated on chromosome 9 of the mouse genome between the AB124611 (HIDE1) and Carmi loci. This has the advantage of having tightly regulated high level expression of the detectable recombinant protein such as a protein antigen-binding molecule.
[0072] According to various embodiments there is a method for producing recombinant proteins comprising a protein antigen-binding molecule, comprising the steps of: integrating a nucleic acid sequence capable of expressing the protein antigen-binding molecule into a vector comprising an antibiotic selection cassette; a doxycycline inducible promoter; a cleavable sequence between two cistrons; comprising a nucleic acid expressing a detectable marker optionally selected from a fluorescent protein or a peroxidase, nucleic acid capable of expressing the protein antigen-binding molecule inserted between a set of homologous genomic locus targeting sequences; selecting transfected cells in the presence of an antibiotic in a cell culture media; inducing expression of the vector by supplementing the cell culture media with doxycycline; co-expressing a single guide RNA; and collecting the expressed protein antigen-binding molecule.
[0073] In various embodiments, the method comprises determining the nucleic acid sequence capable of expressing the protein antigen-binding molecule by sequencing the variable region of an antibody.
[0074] According to various embodiments there is a detectible recombinant protein comprising a recombinant protein co-expressed with a horseradish peroxidase detectable marker comprising an amino acid sequence set forth in SEQ ID NO. 7.
[0075] In various embodiments, the detectable recombinant protein comprises a protein antigen-binding molecule.
[0076] In various embodiments, the protein antigen-binding molecule of the detectable recombinant protein comprises a single chain fraction variable.
[0077] In various embodiments, the protein antigen-binding molecule of the detectable recombinant protein comprises a heavy chain sequence and a light chain sequence separated by a 2A cleavage sequence. In various embodiments the 2A cleavage sequence comprises the nucleic acid sequence set forth in SEQ ID NO. 19.
[0078] In various embodiments, the 2A cleavage sequence is flanked by a furin site. In various embodiments the furin site sequence comprises the nucleic acid sequence set forth in SEQ ID NO. 18. [0079] In various embodiments, the protein antigen-binding molecule of the detectable recombinant protein comprises anti-Lamin. In various embodiments the anti-Lamin comprises the nucleic acid set forth in SEQ ID NO. 12. In various embodiments the anti- Lamin comprises the nucleic acid set forth in SEQ ID NO. 13. In various embodiments the anti-Lamin comprises the nucleic acid set forth in SEQ ID NO. 14 wherein the detectable recombinant protein comprises the modified horseradish peroxidase (mHRP) expressed amino acid SEQ ID NO. 7. In various embodiments the anti-Lamin comprises the nucleic acid set forth in SEQ ID NO. 15 wherein the detectable recombinant protein comprises the modified Neon green (mNeonGreen) expressed as amino acid SEQ ID NO. 6. In various embodiments, the protein antigen-binding molecule of the detectable recombinant protein comprising anti-Lamin is selected from the group consisting of any one of nucleic acid sequences set forth in SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16 and SEQ ID NO. 17. In various embodiments, the protein antigen-binding molecule of the detectable recombinant protein comprises anti-Lamin variable regions. In various embodiments, anti-Lamin variable regions of clone BBLB1 c7 comprising anti Lmnbl variable regions. In various embodiments, the nucleic acid sequence set forth in SEQ ID NO. 16 expresses anti-Lmnb1 variable regions of clone BBLB1 wherein the detectable recombinant protein comprises the modified Neon green (mNeonGreen) expressed as amino acid SEQ ID NO. 6. In various embodiments, the nucleic acid sequence set forth in SEQ ID NO. 16 expresses anti-Lmnb1 variable regions of clone BBLB1 wherein the detectable recombinant protein comprises the modified horseradish peroxidase (mHRP) expressed amino acid SEQ ID NO. 7.
[0080] In various embodiments, the protein antigen-binding molecule of the detectable recombinant protein comprises anti-p53 variable regions.
[0081] In various embodiments, anti-p53 variable regions comprise anti-p53 variable regions of clone DO-1.
[0082] In various embodiments, the nucleic acid sequence set forth in SEQ ID NO. 20 expresses anti-p53 variable regions of clone DO-1
[0083] According to various embodiments there is a method of using the detectable marker by detecting the detectible recombinant protein described herein above in a single step without the use of a secondary labeling reagents. In other words, in various embodiments use of the detectible recombinant protein as described herein above, for detecting the detectable marker in a single step without the use of a secondary labeling reagents.
[0084] By reducing the number of labelling steps, sources of experimental error can be eliminated, speed of analyses increased, and potentially non-specific background reduced.
[0085] In various embodiments, the detectible marker is detected on a western blot.
[0086] In various embodiments, the detectible marker is immunohistochemically detected in formalin fixed tissue samples.
[0087] According to various embodiments there is a vector comprising: an antibiotic selection cassette; a doxycycline inducible promoter; a cleavable sequence between two cistrons; comprising a nucleic acid expressing a detectable marker set forth in amino acid sequence SEQ ID NO. 7, nucleic acid expressing a recombinant protein inserted between a set of homologous genomic locus targeting sequences.
[0088] In various embodiments, the recombinant protein expressed comprises an antigen-binding molecule comprising a single chain fraction variable (scFv).
[0089] In various embodiments, the recombinant protein expressed comprises an antigen-binding molecule comprising a heavy chain sequence and a light chain sequence separated by a 2A cleavage sequence. In various embodiments the 2A cleavage sequence comprises the nucleic acid sequence set forth in SEQ ID NO. 19.
[0090] In various embodiments, the 2A cleavage sequence is flanked by a furin site. In various embodiments the furin site sequence comprises the nucleic acid sequence set forth in SEQ ID NO. 18.
[0091] According to various embodiments there is a recombinant cell line comprising the expression system as described herein above or a vector as described herein above.
[0092] In various embodiments, the recombinant cell line expresses the detectable recombinant protein as described herein above. [0093] In various embodiments, where the recombinant cell line is a mouse fibroblast cell line comprising the expression system as described herein above or a vector as described herein above.
[0094] In various embodiments, in the mouse fibroblast cell the set of homologous genomic locus targeting sequences comprise homologous sequences targeting a mouse TIGRE genomic locus. The mouse TIGRE locus is situated on chromosome 9 of the mouse genome between AB124611 (HIDE1) and Carmi loci. This has the advantage of having tightly regulated high level expression of the detectable recombinant protein such as a protein antigen-binding molecule.
[0095] According to various embodiments there is a method of detecting: a) a product derived from: i) the expression system as described herein above, ii) the vector as described herein above, iii) the recombinant cell line as described herein above; or b) the detectible recombinant protein as described herein above, in a single step without the use of a secondary labeling reagents by detecting the detectable marker or the horseradish peroxidase detectable marker.
[0096] By reducing the number of labelling steps, sources of experimental error can be eliminated, speed of analyses can be increased, and potentially non-specific background can be reduced.
[0097] In various embodiments, the method of detecting the detectible marker is detected via a western blot.
[0098] In various embodiments, the method of detecting the detectible marker is immunohistochemically detected in formalin fixed tissue samples.
[0099] According to various embodiments, there is a method for producing recombinant proteins comprising a protein antigen-binding molecule, comprising the steps of:
(a) integrating a nucleic acid sequence capable of expressing the protein antigenbinding molecule into a vector comprising an antibiotic selection cassette; a doxycycline inducible promoter; a cleavable sequence between two citrons; comprising a nucleic acid expressing a detectable marker optionally selected from a fluorescent protein or a peroxidase, nucleic acid capable of expressing the protein antigen-binding molecule inserted between a set of homologous genomic locus targeting sequences;
(b) selecting transfected cells in the presence of an antibiotic in a cell culture media;
(c) inducing expression of the vector by supplementing the cell culture media with doxycycline;
(d) co-expressing a single guide RNA; and
(e) collecting the expressed protein antigen-binding molecule.
[00100] In various embodiments, the method further comprises determining the nucleic acid sequence capable of expressing the protein antigen-binding molecule by sequencing the variable region of an antibody and using this nucleic acid sequence capable of expressing the protein antigen-binding molecule to integrate into the vector. This allows the method to easily be used to express detectable recombinant protein for any protein antigen-binding molecule.
[00101] According to various embodiments, there is a detectible recombinant protein comprising a recombinant protein fused to a horseradish peroxidase detectable marker comprising an amino acid sequence set forth in SEQ ID NO. 7. In various embodiments, the detectible recombinant protein is derivable from or derived from the expression of the expression system described herein above. In various embodiments, the detectible recombinant protein is derivable from or derived from the expression of the vector described herein above. In various embodiments, the detectible recombinant protein is derivable from or derived from expression of the expression system or the vector in the recombinant cell line described herein. The high activity variant of recombinant horseradish peroxidase detectable marker permits enhanced detection activity compared to wild type horseradish peroxidase or other known horseradish peroxidase mutations.
[00102] In various embodiments, the detectible recombinant protein comprises a protein antigen-binding molecule. In various embodiments, the detectible recombinant protein antigen-binding molecule comprises a single chain fraction variable. In various embodiments, examples of the detectible recombinant protein antigen-binding molecule comprising a single chain fraction variable sequence are set forth in any one of SEQ ID NOS. 16, 17 and 20. In various embodiments, the detectible recombinant protein antigen-binding molecule comprises a heavy chain sequence and a light chain sequence separated by a 2A cleavage sequence. In various embodiments, the 2A cleavage sequence is flanked by a furin site. In various embodiments, examples of the detectible recombinant protein antigenbinding molecule comprising a heavy chain sequence and a light chain sequence are set forth in any one of SEQ ID NOS. 12 to 15.
[00103] In various embodiments, nucleic acids 30 to 33 of the sequence set forth in SEQ ID NO. 10 may be replaced with the any one of the nucleic acid sequences set forth in SEQ ID NO.1 1 , or SEQ ID NO.12, or SEQ ID NO. 13, or SEQ ID NO. 14, or SEQ ID NO. 15, or SEQ ID NO. 16, or SEQ ID NO. 17, or SEQ ID NO. 20.
[00104] In various embodiments, the detectible recombinant protein antigen-binding molecule comprises an anti-p53 variable region. In various embodiments, the detectible recombinant protein antigen-binding molecule comprises an anti-p53 clone DO-1 variable regions. These provide the advantage of being able to detect the antibody without the use of any secondary labeling reagents.
[00105] According to various embodiments, the detectible recombinant protein may be used for detecting the detectable marker in a single step without the use of a secondary labeling reagents. In various embodiments, the use further comprises detecting the detectable marker in a single step without the use of a secondary antibodies. In various embodiments, the detectible recombinant protein is used to be detected in any known method for detecting the presence of an antigen with a protein antigen-binding molecule. In various embodiments, the use of the detectible recombinant protein may be detected in any one of the methods selected from the group consisting of western blotting; immunohistochemistry (IHC), super-resolution imaging, enzyme-linked immunosorbent assay (ELISA), immunofluorescence microscopy, and Fluorescence activated cell sorting (FACS). In various embodiments, the detectible recombinant protein is used for detecting the presence of a protein on a western blot without the use of a secondary antibody. In various embodiments, the detectible recombinant protein may be used for detecting molecule such as a protein immunohistochemically in tissue samples without the use of a secondary labelling reagents.
[00106] According to various embodiments, there is a vector comprising: a) an antibiotic selection cassette; b) a doxycycline inducible promoter; c) a cleavable sequence between two cistrons; comprising a nucleic acid expressing a detectable marker set forth in amino acid sequence SEQ ID NO. 7, nucleic acid expressing a recombinant protein inserted between a set of homologous tetracycline-inducible genomic locus targeting sequences.
[00107] In various embodiments, the recombinant protein comprises an antigenbinding molecule comprising a single chain fraction variable. In various embodiments, the recombinant protein comprises an antigen-binding molecule comprises a heavy chain sequence and a light chain sequence separated by a 2A cleavage sequence. In various embodiments, the 2A cleavage sequence is flanked by a furin site.
[00108] According to various embodiments, there is a recombinant cell line comprising the expression system according to any embodiment of the system described herein above or a vector according any embodiments of the vector described.
[00109] In various embodiments, the recombinant cell line is able to express the detectable recombinant protein according to any embodiment of the detectable recombinant protein described herein above.
[00110] According to various embodiments, there is a method of detecting: a) a product derived from i) the expression system described hereinabove; ii) the vector described hereinabove; or iii) the recombinant cell line described hereinabove; or b) the detectible recombinant protein described hereinabove in a single step without the use of a secondary labeling reagents by detecting the detectable marker or the horseradish peroxidase detectable marker.
[00111] In various embodiments, the method comprises detecting the detectable marker in a single step without the use of a secondary antibodies. In various embodiments, the lack of a secondary labeling reagents comprises the lack of a secondary antibody. In various embodiments, the detectible marker is detected in any known method for detecting the presence of an antigen with a protein antigen-binding molecule. In various embodiments, the detectible marker is detected in any one of the methods selected from the group consisting of western blotting; immunohistochemistry (IHC), super-resolution imaging, enzyme-linked immunosorbent assay (ELISA), immunofluorescence microscopy, and Fluorescence activated cell sorting (FACS). In various embodiments, the detectible marker is detected on a western blot without the use of a secondary antibody. In various embodiments, the detectible marker is immunohistochemically detected in tissue samples without the use of a secondary labelling reagents. In various embodiments, the detectible marker is immunohistochemically detected in tissue samples without the use of a secondary antibody.
[00112] Examples
[00113] To address the problems of current recombinant protein expression systems, an expression system was designed that combines independently developed features into one cohesive vector hereafter named T3. For robust and stable expression of the transgene, the vector is first targeted to the mouse TIGRE locus. The system is further organised around an efficient rtTA variant and allows highly inducible expression while minimizing leakage. T3 vectors are shown to support efficient expression of a wide range of cDNAs, then demonstrate their ability to generate stable and inducible lines producing recombinant antibodies by simply adding doxycycline to the cell culture medium. These lines address several problems inherent to antibody production, while retaining the advantages of the T3 design. In total, an integrated system for the reformatting and synthesis of recombinant proteins such as monoclonal antibodies are described, with the ability to tailor each recombinant protein such as monoclonal antibodies to specific applications.
[00114] Reported is the construction of the T3 vector system for the generation of inducible stable cell lines producing a range of recombinant antibodies and their derivatives. Using an exemplary antibody against Lamin B1 it was demonstrated that the T3-based system embodies a flexible and efficient platform with which to produce a wide range of recombinant antibodies, while addressing several issues inherent to such processes. It is shown that these antibodies replicate qualities of hybridoma-derived antibodies in applications ranging from immunohistochemistry to super-resolution imaging, without the need for purification steps. When directly fused to detectable marker such as fluorescent proteins, these recombinant antibodies obviate the requirement for secondary labelling reagents. This permits speedier and more facile imaging while retaining the performance of the parental antibody. Additionally, a novel, high-activity variant of recombinant horseradish peroxidase was generated, which can be used in single-step Western-blotting experiments. Moreover, coupling this modified HRP with a single chain anti-p53 antibody effectively allowed single-step detection of p53 expression, in cancer lesions from formalin-fixed human tissue samples. Taken together, these results highlight the robustness and versatility of the T3 ecosystem for antibody production and emphasize the potential of tailored antibodies, from standard laboratory work to diagnostic and therapeutic applications. T3 is a vector expression system designed for the expression of recombinant antibodies in mammalian cells. It combines doxycyclin-inducible expression, CRISPR-mediated stable genomic integration, and single construct design. This enables simple, flexible, efficient and stable recombinant antibody production in any mammalian cell line of choice. The cells can be stored and reused as required for production, in virtually unlimited quantities. The T3 system can equally be used for any other recombinant protein production, with identical advantages.
[00115] Results
[00116] Sequence data for an entire monoclonal antibody collection was originally obtained, constituted of IgGs against nuclear, cytoskeletal, or fluorescent proteins, and a range of tags/epitope tags. While the initial experiments were designed to confirm antibody specificity using transient transfection, a more robust and adaptable system was obviously required for actual antibody production.
[00117] T3 vector design
[00118] The T3 vector is built around a tetON inducible system, with optimal orientation of its various elements, puromycin selection cassette and multiple-cloning site including efficient ligation-free cloning. These elements are enclosed between homology arms targeting the chosen genomic location (Fig. 1A), here the mouse safe harbour TIGRE locus is used however another set of homologous genomic locus targeting sequences may also be used. To maximize CRISPR-mediated integration, the associated sgRNA is cloned into a vector co-expressing the adenoviral E1 B55K mutant, shown to enhance homology-directed repair.
[00119] Testing the efficiency, stability and inducible protein expression of the T3 vector
[00120] Flow cytometry and analysis
[00121] 3T3 cells with integrated MitoTimer reporter were treated with mock or 1 pg/mL doxycycline for 48 h before being trypsinized, resuspended in cell culture growth medium, filter through cell strainder (Corning Falcon, #352235) and analysed for fluorescence intensity on (BD LSR II) flow cytometer. More than 50 000 cells were analysed per condition. Data analysis was performed using Excel, Prism (Graphpad) and R software. Box plots indicate median with 10-90 percentile whiskers and g indicates Hedge’s g.
[00122] As expected, the T3 vector permits stable and doxycycline (DOX)- inducible expression of constructs, with very low background and high selection efficiency. This is shown here with the mitochond ria-targeted MitoTimer (Hernandez, G. et al. Autophagy 9, 1852-1861 (2013)), by fluorescence-assisted cell sorting (FACS) and immunofluorescence microscopy in mouse 3T3 fibroblasts (Fig. 1 B and C, Fig. 2A). These results provide an expression clone fold change of 235 times (3T3J1) and 170 times (3T3_I2) which is a much higher expression compared to the next most efficient vectors EF-1a reported in Wang et al. (J. Cell. Mol. Med Vol 21 , No 1 , 2017 pp. 3044-3054) to have a 47.7 times fold change in high expression clones, a 27.9 times fold change in medium expression clones and 6.65 times fold change in low expression clones. This enhanced fold change was repeatedly observed to be above 70 and in most cases a fold change of at least 75 [Fig. 12], Appropriate expression and localisation were achieved for a broad spectrum of constructs, as illustrated here using the nuclear lamina-associated integral membrane protein LAP2p (Fig. 2B&C) and the nucleoplasmic protein Histone-H2B (Figure 2D, live cells). The targeting is adaptable to any locus of choice by simply selecting alternate combinations of homology arms/sgRNA. This is demonstrated here with the targeting of a lamin B receptor/green fluorescent protein fusion (LBR-GFP) to the AAVS1 safe harbour locus (Aschenbrenner, S. et al. Sci. Adv. 6, 1-12 (2020)) in human HEK293T cells (Fig. 2E, live cells).
[00123] Western-blotting
[00124] Cells were harvested, washed once in PBS, denaturated in 2x Laemmli Sample Buffer (Bio-rad) at 85 °C for 10 min and briefly sonicated. Protein concentration was then determined using BCA protein Assay (Pierce/Thermo Fisher Scientific). Whole cell lysates were then reduced with 2.5% of 2-mercaptoethanol, and samples were separated on polyacrylamide gels, transferred to a nitrocellulose membrane and incubated in blocking buffer (0.1% Tween 20, 5% milk in PBS) for >30 min before addition of primary antibodies. Secondary HRP-conjugated antibodies goat-anti mouse (DAKO #P0447) and goat-anti rabbit (DAKO #P0448) were used as appropriate. Primary and secondary antibodies were diluted in blocking buffer (0.1 % Tween 20 in PBS) and left on the membrane at room temperature. The membranes were imaged after addition of Immobilon Forte Western HRP substrate (Millipore, #WBLUF0100). mHRP-conjugated antibodies were directly placed in ECL for visualization without secondary antibody step unless stated otherwise. [00125] Using the T3 vector for antibody production
[00126] Cell culture
[00127] Hybridoma cell lines were grown in RPMI 1640 supplemented with 4 mM L- glutamine, 100U/ml penicillin and streptomycin, 50pM 2-mercaptoethanol and 10% heat inactivated fetal bovine serum. HeLa, 293T, C2C12 and U2OS cells were grown at 37°C in 5% CO2 in standard DMEM high glucose with 10% foetal bovine serum, 100U/ml penicillin and streptomycin, with the addition of 1 mM sodium pyruvate and 2mM L-glutamine for NIH3T3.
[00128] Sequencing of monoclonal antibodies produced from hybridomas and validation of antibody sequences.
[00129] The hybridoma-derived antibodies described in this study are as follow: anti- Lmnbl (clone BBLB1 c7), anti-Sun2 (clone 3.1 E), anti-Lmnb2 (clone BBLB2 3.13), anti-Man1 (clone BBManl A22. and anti-mNeonGreen (clone BBneon N19.1). Establishment of the hybridomas was performed using the same procedure as described earlier (Chai, R. J. et al. Nat. Commun. 12, 4722 (2021)). The best hybridoma clones were sent for antibody sequencing (Absolute Antibody), and the resulting sequences of the variable regions of the heavy and light chains (with their respective signal peptides) were further sent for DNA synthesis (Integrated DNA Technologies). Both chains were then separately cloned into pFUSE-CHIg and pFUSE2-CLIg-mK expression vectors (InvivoGen), respectively.
[00130] To validate the resulting heavy and light variable chain sequences, HeLa cells were transiently transfected with both pFUSE-CHIg and pFUSE2-CLIg-mK at a ratio of 2:3, using Lipofectamine 2000. Growth media was then collected at 24-48h post transfection, and the specificity of the recombinant native antibodies was compared to their hybridoma equivalent by immunofluorescence microscopy and western blotting, using antigen overexpression /knockout /knockdown strategies as appropriate.
[00131] Western-blotting
[00132] Cells were harvested, washed once in PBS, denaturated in 2x Laemmli Sample Buffer (Bio-rad) at 85 °C for 10 min and briefly sonicated. Protein concentration was then determined using BCA protein Assay (Pierce/Thermo Fisher Scientific). Whole cell lysates were then reduced with 2.5% of 2-mercaptoethanol, and samples were separated on polyacrylamide gels, transferred to a nitrocellulose membrane and incubated in blocking buffer (0.1 % Tween 20, 5% milk in PBS) for >30 min before addition of primary antibodies. Secondary HRP-conjugated antibodies goat-anti mouse (DAKO #P0447) and goat-anti rabbit (DAKO #P0448) were used as appropriate. Primary and secondary antibodies were diluted in blocking buffer (0.1 % Tween 20 in PBS) and left on the membrane at room temperature. The membranes were imaged after addition of Immobilon Forte Western HRP substrate (Millipore, #WBLUF0100). mHRP-conjugated antibodies were directly placed in ECL for visualization without secondary antibody step unless stated otherwise.
[00133] Antibody production in NIH3T3 with the T3 vectors
[00134] 3T3 cells were transfected at 80-90% confluency using PEImax 40kDa (Polyscience 24765-2) with a mix of Cas9+sgRNA plasmid and targeting T3 vector (see sequesnce listing) for 6h. Media was then replaced with standard growth medium supplemented with 1 pg/ml puromycin (Sigma #P9620) after 24h. Cells were selected for 1- 2 weeks before production of the antibodies was started upon addition of 1 pg/ml doxycycline (Clontech #631311). Cell culture supernatant was then collected 48h hours after doxycycline induction, filter-sterilized and stored at 4°C. Antibody containing media was then used without further purification or concentration.
[00135] T3 plasmids and generation of stable cell lines
[00136] The key features of the T3 plasmids are described herein. Complete sequence and the cloning strategy are provided in the sequence listing. To generate stable 293T or NIH3T3 cell lines, cells were transfected at 80-90% confluency with a mix of Cas9+sgRNA plasmid and the targeting T3 vector (comprising any one of SEQ ID NOS 2-4 and 10-20 alone or in the combinations mentioned throughout or depicted in Fig. 1A, Fig. 3A, Fig. 5A, Fig. 9A, Fig. 11A or 11 C) sing PEImax 40kDa (Polyscience; #24765-2). The transfection mixture was replaced with regular growth medium 6h after transfection and selection was started 24h later with addition of 1 pg/mL puromycin (Sigma; #P9620) to standard growth medium for duration of 1-2 weeks.
[00137] The T3 vectors were then customised for the production of conventional recombinant antibodies (rAbs) containing both light- and heavy-chain segments (LCHC) as seen in any one of SEQ ID NOS. 12-15. The choice of a single-vector setup necessitated the use of a 2A cleavage sequence (SEQ ID NO. 19) to separate heavy and light chain sequences, with a furin site (SEQ ID NO. 18) intended to minimize left-over amino acids from the 2A sequence (Fig. 3A). The inducible design of T3 vectors permits the production of antibodies by simply harvesting the growth medium of any cell line after supplementation with doxycycline. Mouse 3T3 fibroblasts were used here as vessels for rapid validation purposes, but a cornucopia of cell lines is evidently available for antibody production. Crucially, rAbs produced from the T3 vectors retained the labelling properties of their original hybridoma parent. This is illustrated here using a mouse anti-Lmnb1 antibody derived from its hybridoma clone (lgG2b), in both Western-Blotting and confocal microscopy (Fig. 3B-3C). This strategy was applicable for multiple other conventional antibodies, including anti-Lmnb2 and anti-Man1 (Fig. 4A). Reversing heavy and light chain order within the T3 vector construct appears to have little to no effect on labeling performance (Fig. 3C right panels, and Fig. 4C). The T3 design conveniently allows for simple modification of the antibodies by fusing their heavy chain (or indeed light chain) to any tag of choice. Of particular interest is the use of highly fluorescent tags such as mNeonGreen28 (mNG), as this facilitates direct imaging without the need for a secondary antibody labeling step (Fig. 3D).
[00138] Nevertheless, tagged rAbs still retain their utility for conventional indirect imaging whenever necessary, as shown with anti-Lmnb1 (Fig. 3D, middle panel), anti-Lmnb2 and anti-Sun2 (Fig. 4B). Importantly, these antibodies display excellent detection capabilities even in demanding immunofluorescence assays, such as formalin-fixed paraffin-embedded (FFPE) tissue microarrays used for cross-reactivity antibody testing (Fig. 3E). Finally, the T3 design allows the replacement of heavy and light chain constant region sequences with those from other species, easily altering the species identity of the rAb. This is shown here using the mNG-tagged anti-Lmnb1 rAb described above, in which the mouse heavy and light chain constant regions were exchanged for their rabbit equivalents (Fig. 4D). With respect to the production of antibodies, no evidence of short-term growth defects once synthesis of rAbs was initiated by doxycycline supplementation was observed (Fig. 4E).
[00139] Commercial Antibodies and rAbs produced from the T3 vectors
[00140] Commercial primary and secondary antibodies used were as follow: anti - Lamin A (Abeam 26300), V5 (Invitrogen, 46-1157), GAPDH (Sigma G9545), p-tubulin (Sigma T4026), a-actinin (Cell Signalling Technology 3134), anti-mouse Alexa fluor 488, 594 conjugated (Invitrogen), anti-rabbit IgG Alexa Fluor 594 conjugated (Invitrogen) and Hoechst dye (Thermo #H3570). anti-rabbit immunoglobulins HRP (Dako; #P0448), anti-mouse immunoglobulins (Dako; #P0447).Vast majority of the rAbs were produced from stable NIH3T3 cell lines by adding 1 pg/mL doxycycline (dox) (Clontech; #631311) to the growth medium and collecting the cell culture supernatant 48h later. The two anti-Lmnb1 rAbs LCHC-APEX2 and LCHC-mHRP were produced by transiently transfecting 293T cells with T3 plasmids using Lipofectamine 2000, followed by replacing the media with 1 pg/mL dox- containing growth and collecting the cell culture supernatant 48h later. The antibodies- containing culture media were filter-sterilized and used without further purification or concentration. The DO-1 antibody was a generous gift from David Lane.
[00141] Fluorescently tagged scFvs allow direct, high-resolution imaging
[00142] Single chain antibodies (scFv) constitute a valuable development to the antibody toolkit. By linking heavy- and light- variable regions sequences in a single polypeptide, they offer smaller and more compact protein affinity reagents (29). The T3 vectors described above provide rapid means for scFv production, by simply cloning the heavy and light chain variable regions in tandem (Fig. 5A). In this design, the two chains are separated by a 15 amino acid residue linker (3x GGGGS), while only the N-terminal signal peptide of the heavy chain variable region is retained. For their use in immunofluorescence microscopy, the conventional indirect labelling approach would require fluorescent secondary antibodies to detect the primary scFv. This might however be ineffective: commercially available reagents are mainly directed against constant region epitopes which are omitted in scFvs (Fig. 6A). Therefore scFvs were used as direct labelling reagents. This is illustrated here with the single chain version of the rAb-Lmnb1-mNG antibody (scFv Lmnb1-mNG), which retained detection properties indistinguishable from its conventional counterpart when imaged directly using the 488nm channel (fig. 5B).
[00143] Next the suitability of direct imaging of scFvs fused to mNG in super resolution imaging studies employing 3D-Structured Illumination Microscopy (3DSIM) were assessed. In order to best preserve 3D ultrastructure formaldehyde fixation was used. This avoided solvent- and alcohol-based- permeabilization protocols. Both conventional anti-Lmnb1 antibodies, either hybridoma-derived or their rAb equivalent (rAb-LCHCLmnb1), displayed identical labelling patterns of the nuclear envelope (Fig. 5D and Fig. 5E, top rows, and Fig. 6B top rows). What was striking, however, was that neither antibody (with conventional H+L format) labelled the nuclear envelope that was closest to the coverslip. This contrasts with the conventional Lamin A-specific antibody (rabbit IgG) that uniformly labels the nuclear periphery (Fig. 5D, arrowheads). The dorsally-restricted lamin B1 labelling was independent of the secondary antibody step, since rAb-LCHC-Lmnb1 -mNG imaged via mNG fluorescence (Fig.6C and Fig. 6D, upper panels) displayed a similar restricted distribution. It is also, not surprisingly, unaffected by reversing the light and heavy chain positions in the T3 construct (Fig. 6C and Fig. 6D, bottom panels). Strikingly, the single chain version rAb-scFv- Lmnb1-mNG did not show such restricted labelling pattern and labelled the entire nuclear periphery, in a fashion very similarto that observed with the lamin A antibody (Fig. 5D bottom panels, and Fig. 6B bottom panels). These results could be replicated, albeit at lower resolution, employing orthodox confocal microscopy. Conventional anti-lamin B1 (LCHC) displayed reduced labelling of the ventral surface of nuclei, whereas antilamin B1 formatted as a scFv displayed uniform labelling of the nuclear envelope (Fig. 7A and Fig 8). This effect could still be observed with harsher permeabilization conditions, suggesting that conventional antibodies against lamin B1 have accessibility restrictions which are not evident with their corresponding scFv (Fig. 7A-C and Fig. 8). This could be a reflection of the overall size of conventional H+L antibody formats (~80kDa for the rAb-LCHCLmnb1) when compared with the smaller scFvs (~55kDa for the rAb-scFv-Lmnb1-mNG, including mNG).
[00144] The mNG-tagged scFvs can also be used for Western-blotting when combined with a triple sandwich strategy, by adding an intermediate anti-mNeonGreen labelling step to the detection procedure (Fig. 5C). Although this approach can be of use in specific Western-blotting setups, it hardly appears satisfactory when considering the resulting increase in incubation times and labelling procedures. To enable direct Western blotting and IHC, the highly fluorescent proteins needed for direct immunofluorescence assays was mirrored, and highly active recombinant peroxidases were made use of, as described in the next section.
[00145] mHRP-tagged T3Abs enable single-step Western-Blotting
[00146] The above findings indicate that rAbs, either conventional or scFv, can readily be employed in one step direct labelling protocols for immunofluorescence microscopy. Whether similar benefits could be obtained for rapid Western-Blotting was then tested, by conjugating rAbs with Horse Radish Peroxidase (HRP) (Fig. 9A). To compensate forthe lack of signal amplification resulting from the omission of a secondary antibody step, a new high- activity variant of HRP was first generated by combining several mutations separately reported to increase enzymic activity (Humer, D. & Spadiut, O. Int. J. Mol. Sci. 20, (2019); and Martell, J. D. et al., Nat. Biotechnol. 34, 774-780 (2016)). This modified sequence was then codon-optimized and resulted in a HRP variant thereafter named mHRP (Fig. 9A). The results show that both conventional and single-chain variants of rAb-Lmnb1 fused to mHRP support single-step Western-blotting, with the same specificity and similar sensitivity to the native hybridoma-derived antibody. The latter, of course, requires an HRP-conjugated secondary antibody (Fig. 9B and Fig. 10A). Fusion of APEX2, an ascorbate peroxidase from pea, to rAb-Lmnb1 is also effective although the signal obtained by Western- blotting is weaker than that seen with mHRP (Fig. 10A-C). This is consistent with previous reports on the relative kinetics of these enzymes (Charest-Morin, X. et al., Sci. Rep. 7, 1-14 (2017)). Importantly, the strategy is translatable to antibodies of diagnostic interest, provided they are available in recombinant form. As an example, an identical design strategy was employed to prepare an mHRP-coupled version of an anti-p53 scFv. This was derived from the very widely used anti-p53 monoclonal antibody produced by the DO-1 hybridoma clone. As can be seen in Fig. 9, the rAb-scFv-p53-mHRP efficiently detects p53 in single-step Western Blotting, similarto the native DO-1 employing standard, two steps Western Blotting (Fig. 9C).
[00147] mHRP-p53 antibodies for single-step detection of p53 expression in human cancers
[00148] Whether rAb-scFv-p53-mHRP could identify p53 expression patterns in formalin- fixed parrafin-embedded (FFPE) tissue sections of human cancers was then assessed. p53 protein remained undetectable in 41 out of the 60 cores of the tissue microarray for both native DO-1 and rAb-scFv-p53-mHRP, while 6 and 5 cores were characterized by a strong p53 staining in multiple areas for the DO-1 and the rAbscFv-p53-mHRP antibodies, respectively (Fig. 9D, green highlighted cores). The remaining cores showed no obvious signs of pronounced p53 presence, with either limited/faint (red highlights) or extremely limited/very faint (blue highlights) staining. The differences in overall staining intensity of the rAb-scFv-p53-mHRP compared to the native DO-1 antibody most probably arise from multiple factors, including the antibodies themselves, the lack of amplification consequential to the absence of secondary HRP-conjugated antibody step, but also from the evident differing quality and location of the tissue sections (Fig. 9E, F, or Fig 10D). At the cellular level, both antibodies yielded indistinguishable nuclear signals characteristic of p53, with identical areas of pancreas and lung carcinomas identified as p53 positives (Fig. 9E). In agreement with previous reports showing that squamous cell carcinomas tend to be associated with overexpression of p53 (Wang, X. et al., Medicine (Baltimore). 96, e6424 (2017); Benzerdjeb, N. et al. Histopathology 79, 381-390 (2021)), strong and widespread nuclear p53 expression in sections of squamous cell carcinoma of the lower lip for both rAb- scFv-p53-mHRP and DO-1 antibodies (Fig. 9F and Fig. 10D) was detected.
[00149] Taken together, the results demonstrate that the T3 vectors accommodate a wide range of constructs expressed in a stable and inducible manner, from single proteins to conjugated single-chain antibodies. They enable applications spanning standard immunofluorescence, super-resolution microscopy and rapid detection of targets in IHC experiments, as shown here with p53 expression in human cancer sections, while merely requiring the collection of media from standard cell culture experiments.
[00150] Immunohistochemistry [00151] Formalin fixed paraffin embedded (FFPE) tissue array sections were baked for 30 min at 60 °C, dewaxed in xylene and rehydrated through a graded series of ethanol dilutions. For the normal tissues array (Biomax; #FDA999y) immunofluorescently stained for Sun2, the antigen retrieval was performed in Tris/EDTA buffer (pH 9) using a pressure cooker (2100 Antigen Retriever). Slides were then placed for 30 min in 1 % Triton X-100/PBS solution, followed by quick incubation in TrueBlack/ 70% ethanol solution (Biotium; # 23007) to minimize background signal. Slides were stained using anti-Sun2 LCHC-mNG rAb for 3 h followed by 1 h incubation with goat-anti mouse IgG Alexa Fluor 594 together with Hoechst 33342. Prolong Diamond Antifade (Invitrogen; #P36970) was used for mounting. For multiple organ carcinoma with normal tissue microarray (Biomax; #BCN601 a) and human lip cancer whole tissue section (Biomax; #HuCAT506) chromogenically stained for p53, the antigen retrieval was performed in citrate buffer pH 6 (Electron Microscopy Science; #64142- 08) using a pressure cooker (2100 Antigen Retriever). The slides were quenched for endogenous peroxidase activity by 1% H2O2 in PBS and subsequently blocked in 10% goat serum in PBS (Gibco; # 16210064). Slides were then stained for p53 with either scFv-mHRP for 2 h or hybridoma derived antibody for 2 h followed by 30 min incubation with goat antimouse IgG HRP (Promega; #W402B) for 30 min and visualization with Envision DAB substrate (DAKO) for 3 min. The tissues were quickly counterstained with hematoxylin, dehydrated and mounted in Richard-Allen Scientific Mounting Medium (Thermo Scientific; # 4112). The control staining consisted of slides being incubated for 30 min with secondary HRP antibody only.
[00152] For fluorescence imaging, slides were incubated in Tris/EDTA buffer (pH 9) using a pressure cooker for antigen retrieval. Subsequent steps were all performed at room temperature. Slides were then placed for 30 min in 1 % Triton X-100/PBS solution, then quickly soaked in TrueBlack I 70%Ethanol solution (Biotium, # 23007) to minimize background signal. Slides were then stained using rAb- LCHC-Sun2-mNG antibody for 3 h. A mix of goat-anti mouse Alexa594-conjugated (Invitrogen) with Hoechst 33342 (Invitrogen) was then added, and Prolong Diamond Antifade (Invitrogen, P36970) used for mounting. For chemiluminescence I HRP staining, slides were first incubated in Citrate Buffer pH 6 (Electron Microscopy Science 64142-08) for antigen retrieval, endogenous peroxidase was quenched using 1 % H2O2 in PBS, and slides blocked in 10% goat serum in PBS. Slides were then stained using either a mouse monoclonal anti-p53 from hybridoma supernatant (clone DO-1) for 2 h, followed by incubation with goat anti-mouse HRP conjugate (Promega, # W402B) for 30 min, or were simply incubated for 2h with rAb-scFv-p53-mHRP). Slides were then incubated in Envision DAB substrate (DAKO) for 3 min, followed by quick counterstain with haematoxylin for 30 seconds, dehydrated then mounted. The slides for mounted with in Richard-Allen Scientific Mounting Medium (Thermo Scientific; # 41 12).
[00153] The following FFPE slides (Biomax) were used: multiple organ normal tissue microarray (FDA999y), Multiple organ carcinoma with normal tissue microarray (BCN601 a), human lip cancer whole tissue section (HuCAT506).
[00154] Discussion
[00155] Strategies developed for the expression of recombinant proteins in mammalian cells include transfection, transduction, and rather more recently, CRISPR-mediated genome integration (Nayerossadat, N. et al., Adv. Biomed. Res. 1 , 27 (2012); and Lino, C. A. et al., Drug Deliv. 25, 1234-1257 (2018)). The capabilities of a lentiviral system was previously harnessed to enrich for transduced cells expressing proteins of interest under a dox-inducible promoter (Chojnowski, A. et al., Elife 4, 1-21 (2015)). Retroviral constructs are however notoriously prone to silencing, a less than desirable attribute for producing cells lines that can require extensive passaging (Yao, S. et al., Mol. Ther.10, 27-36 (2004)). Of particular difficulty is the expression of detrimental proteins or enzymes which can place an additional burden on the cellular machinery (May, D. G. et al, Cells 9, (2020)). This is compounded by the fact that some expressing lines can also be inherently unstable, as has been shown to be the case for many rabbit hybridomas (Weber, J. et al., Exp. Mol. Med. 49, (2017)).
[00156] To address these issues, a family of vectors that allow for stable, robust, and tightly inducible protein production was developed, effectively decoupling the expression of the constructs of interest from the generation of the producing cell lines. The use of CRISPR- based integration effectively sidesteps any potential viral silencing, and does away with virus production, mammalian- or insect-based, a process in and of itself time consuming and not necessarily inconsequential. This drug-selectable strategy requires limited numbers of cells and limited quantities of costly reagents to perform the initial genomic integration of the T3 constructs, while its inducibility provides unfettered cell growth until the desired production scale is reached. Transgene expression can be triggered for protein production, in this case antibodies, by doxycycline supplementation, and supernatants harvested before cells become debilitated by demands placed on their protein synthesis and secretory machineries. Final expression levels obviously depend on a multitude of factors, but tet-on systems should essentially perform on par with the best conventional constitutive promoters.
[00157] Cell growth curve [00158] NIH3T3 stable lines expressing Sun2 HCLC-mNG or p53 scFv-mHRP were seeded in 6-well plates at a density of 34 000 cells/well. The following day the cells were mock or dox-induced and incubated for 6 days. After the incubation the cells were collected and the cells number was determined (Bio-Rad TC20™ Automated Cell Counter). The experiment were conducted in triplicate at three different times.
[00159] In this paper it is demonstrated that the use of T3 vectors targeted to the TIGRE safe harbour locus in 3T3 cells. The same strategy can obviously be applied to any chosen locus, regardless of cell line or species since it merely requires modification of the recombination arms within the vector. CHO cells represent a common choice for antibody production, and the identification of appropriate safe harbours in these and other cell lines has been a focus of numerous studies (Hilliard, W. & Lee, K. H., Biotechnol. Bioeng. 118, 659-675 (2021); and Chi, X. et al., PLoS One 14, 1-14 (2019)). This inherent flexibility is portrayed in Fig. 2, with the use of the AAVS1 locus as a safe harbour for LBR-GFP expression in HEK293 cells. Moreover, while it is show here that multiple proteins can be produced within a single site by making use of 2A linkers, the availability of multiple targeting sites within a single cell means that these can be harnessed simultaneously, either to increase the copy number of the constructs or for expression of multi-component systems such as 2C-BiolD setups (Chojnowski, A. et al., iScience 10, 40-52 (2018)).
[00160] All variations of the Lmnbl antibody described here derive from rescued heavy and light chain mRNA sequences of a conventional hybridoma that was lost through liquid nitrogen failure. Loss of hybridomas are unfortunately not that uncommon, and can occur for various reasons (Kromenaker, S. J. et al., Biotechnol. Prog. 10, 299-307 (1994)). cDNA sequencing neatly sidesteps such potential disasters since variable region sequence data is the only information that is absolutely required for long-term preservation of the antibody. In this way, the need for the retention and distribution of viable hybridoma stocks can be eliminate and, ideally, distribution of sequence information would become the norm. With appropriate expression systems combined with rapid DNA synthesis, derivation of cell lines secreting recombinant antibody then becomes a simple matter. As described here for antibodies against LmnB1 , p53 and several others, inducible expression systems with integration into genomic DNA facilitates storage and subsequent thawing of antibody producing cell lines, eliminating concerns about long term viability and stability of expression. In summary, these strategies provide us with the ability to securely archive antibodies and their derivatives in the form of recombinant protein, inducible cell lines, plasmids, or simply as sequence files. [00161] All recombinant and reformatted antibodies described here mirrored the utility for microscopy modalities of their parental, hybridoma-derived forms. It came as no surprise, therefore, that the various rAb-Lmnb1 species were as effective in 3D-SIM imaging as their native hybridoma-derived counterparts. Intriguingly, all heavy and light chain formats, hybridoma-derived or rAb-produced, displayed little or no labelling of the region of the nuclear envelope closest to the coverslip, independent of the secondary antibody step. This phenomenon is exceedingly easy to miss, since only orthogonal views and to a much lesser extent some maximum-intensity Z-projections were able to reveal this localized reduction in signal. In double labelling experiments, polyclonal anti-lamin A antibodies suffered no such defect. At first sight, these results would imply that lamin B1 has a more restricted distribution within the nuclear envelope than lamin A. However, when the same experiment was carried out with scFvs fused to mNG, the discrepancy between lamin distributions disappeared. This implies that the apparently restricted localization of lamin B1 could arise from reduced access to epitopes by the native IgGs, and that scFvs provide a more accurate rendition of lamin B1 distribution. This would likely be a reflection of the substantial size difference between conventional IgGs and scFvs. Surprisingly, it was found that scFvs fused to fluorescent proteins were mostly as effective as their conventional counterparts used in conjunction with fluorescent secondary antibodies. It could be concluded that whatever scFvs lack in terms of avidity and signal amplification, they certainly make up in terms of enhanced sample penetration.
[00162] Taking advantage of the adaptability of the T3 vector design, fluorescent protein and HRP-tagged antibodies were generated, facilitating rapid immunofluorescence and IHC experiments that do not require the use of secondary antibodies. By reducing the number of labelling steps, sources of experimental error can be eliminated, increase speed of analyses, and potentially reduce non-specific background. As alluded to above, the downside of such single-step direct labelling strategies is the elimination of the signal amplification that is inherent to conventional indirect labelling employing primary/secondary antibody combinations. This results in some measure of sensitivity loss. However, the use of high- performance proteins such as mNeonGreen (Shaner, N. C. et al. Nat. Methods 10, 407-409 (2013)), mScarlet (Bindels, D. S. et al., Nat. Methods 14, 53-56 (2016)) and mHRP goes some way towards compensating for the loss of signal amplification.
[00163] Forthe purpose of this study, no attempt was made to further optimize production of conventionally formatted rAbs and neither was there any endeavour to purify the antibodies. Nevertheless, even in more demanding applications such as 3D-SIM and fluorescent IHC on human cancer tissue sections, the use of culture supernatants from cells expressing rAbs and their various derivatives proved to be efficient. Improving rAb recovery and fine-tuning the structure of the antibody constructs themselves should, however, still bring additional enhancements in performance (Ling, W. L. W. et al., Biotechnol. Prog. 19, 158-162 (2003); Fang, J. et al., Nat. Biotechnol. 23, 584-590 (2005); Bennett, B. T. et al, Methods 48, 63-71 (2009); Hong, J. K. et al., J. Biotechnol. 155, 225-231 (2011)). Design improvements to the various antibodies will depend on a multitude of factors and is likely to evolve with understanding of what constitutes a “best” configuration for any given purpose. For instance, previous reports showed that 2A and Furin processing can vary according to their flanking sequences, and are likely to be dependent also on the particular antibody/isotype as well as on the producing cell line (Chng, J. et al., MAbs 7, 403-412 (2015); Izidoro, M. A. et al., Arch. Biochem. Biophys. 487, 105-1 14 (2009); and Ueo, A. et al., J. Virol. 94, 1-12 (2020)) a phenomenon that was indeed observed. Optimizing the Furin and 2A cleavage sequences/environments may therefore prove valuable (Chng, J. et al., MAbs 7, 403-412 (2015)). Obviously, the T3 vectors also provide a simple system to produce single-chain antibodies (scFvs, nanobodies etc.) where cleavage sites are superfluous. However, even here, sequence optimization may prove beneficial with scFvs and scFv fusion proteins being tailored for specific applications, for instance IHC versus superresolution microscopy.
[00164] Several peroxidases were evaluated, including APEX2 and HRP, for use with rAbs, including scFvs. An attribute of these enzymes is that they fold correctly in the mammalian secretory pathway, and hence can acquire their functional prosthetic group (heme). The system allows antibodies to be engineered in a variety of formats that could be employed for immunohistochemistry as well as for western blot applications. mHRP, contains several activity enhancing modifications, and is efficiently secreted in active form by both 3T3 and HEK293 cells. As a proof of principle, mHRP fused to a reformatted version of DO- 1 was chosen to evaluate, a well characterized monoclonal antibody against human p53. This focus on p53 was predicated upon its function as a tumour suppressor and its potential interest for cancer diagnostics. Certainly, the evaluation of p53 mutations and expression levels as predictive cancer markers has been a subject of much scrutiny (Braunschmid, T. et al. Ann. N. Y. Acad. Sci. 1434, 46-53 (2018)), and its utility has been documented in the prognosis of squamous cell carcinomas (Wang, X. et al. Medicine (Baltimore). 96, e6424 (2017)). In western blotting, direct labelling with scFvs fused to mHRP gave very robust and specific signals with little to no detectable background. For IHC analyses of paraffin sections, direct staining using mHRP-tagged scFvs yielded signals marginally less intense than conventional indirect labelling employing primary/secondary antibody combinations, while fully retaining similar specificity. It is suspected that the trade-off between labelling time and signal intensity can significantly be addressed by increasing the scFv concentration or by simply increasing the number of mHRP fused to the scFvs. Both approaches are currently being examined in experiments that are underway.
[00165] In summary, establishment of the T3 ecosystem allows the inducible expression of any construct of choice, and in particular the production of tailor-made antibodies. The system is compatible with both conventional and single chain antibodies, either of which can be modified with fluorescent protein tags or with high activity peroxidases, such as th newly developed mHRP described herein. Considering the current demands and promises placed on antibody production, antibody-based assays, and antibody-based therapeutics, it is likely that homebrewing antibodies tailored for specific purposes will become an integral part of any respectable research toolbox.
[00166] Image processing, quantification, and statistical analysis
[00167] Image processing and quantification was performed using a mix of Imaged (Schindelin, J. et al. Nat. Methods 9, 676-682 (2012)), SoftWorX (GE Healthcare), Imaris (Bitplane), VSViewer (Metasystems) and Cell Profiler (McQuin, C. et al. PLoS Biol. 16, 1-17 (2018)). Data analysis was performed using Excel, Prism (Graphpad) and R software (Team, R. C. R (2020)). Box plots indicate median with 10-90 percentile whiskers, g indicates Hedge’s g.
[00168] It should be further appreciated by the person skilled in the art that variations and combinations of features described above, not being alternatives or substitutes, may be combined to form yet further embodiments falling within the intended scope of the invention.
[00169] As would be understood by a person skilled in the art, each embodiment, may be used in combination with other embodiment or several embodiments.

Claims

Claims
Claim 1 . An expression system for producing recombinant proteins comprising a protein antigen-binding molecule, the system comprising:
(a) a vector comprising: i. an antibiotic selection cassette; ii. a doxycycline inducible promoter;
Hi. a cleavable sequence between two cistrons; comprising a nucleic acid expressing a detectable marker optionally selected from a fluorescent protein or a peroxidase, nucleic acid capable of expressing the protein antigen-binding molecule inserted between a set of homologous genomic locus targeting sequences; and
(b) a single guide RNA.
Claim 2. The expression system according to claim 1 , wherein the protein antigenbinding molecule comprises a single chain fraction variable.
Claim 3. The expression system according to claim 1 , wherein the protein antigenbinding molecule comprises a heavy chain sequence and a light chain sequence separated by a 2A cleavage sequence.
Claim 4. The expression system according to claim 3, wherein the 2A cleavage sequence is flanked by a furin site.
Claim 5. The expression system according to any one of claims 1 to 4, wherein the nucleic acid expressing a detectable marker expresses a fluorescent protein.
Claim 6. The expression system according to any one of claims 1 to 4, wherein the nucleic acid expressing a detectable marker expresses a peroxidase.
Claim 7. The expression system according to any one of claims 1 to 6, wherein the nucleic acid expressing a detectable marker expresses an enhanced detectable marker.
Claim 8. The expression system according to claim 7, wherein the enhanced detectable marker expresses a fluorescent protein comprising mNeonGreen.
Claim 9. The expression system according to claim 7, wherein the enhanced detectable marker expresses a peroxidase comprising a high activity variant of horseradish peroxidase.
Claim 10. The expression system according to claim 7, wherein the enhanced detectable marker expresses peroxidase comprising an ascorbate peroxidase, APEX2.
Claim 11 . The expression system according to any one of claims 1 to 10, wherein the single guide RNA is expressed in conjunction with an adenoviral E1 B55K mutant protein.
Claim 12. The expression system according to any one of claims 1 to 11 , wherein the single guide RNA comprises a sequence having 95% or more sequence identity with the nucleic acid sequence set forth in SEQ ID NO. 1 .
Claim 13. A method for producing recombinant proteins comprising a protein antigenbinding molecule, comprising the steps of:
(a) integrating a nucleic acid sequence capable of expressing the protein antigen-binding molecule into a vector comprising an antibiotic selection cassette; a doxycycline inducible promoter; a cleavable sequence between two cistrons; comprising a nucleic acid expressing a detectable marker optionally selected from a fluorescent protein or a peroxidase, nucleic acid capable of expressing the protein antigen-binding molecule inserted between a set of homologous genomic locus targeting sequences;
(b) selecting transfected cells in the presence of an antibiotic in a cell culture media;
(c) inducing expression of the vector by supplementing the cell culture media with doxycycline;
(d) co-expressing a single guide RNA; and
(e) collecting the expressed protein antigen-binding molecule.
Claim14. The method according to claim 13, comprising determining the nucleic acid sequence capable of expressing the protein antigen-binding molecule by sequencing the variable region of an antibody.
Claim 15. A detectible recombinant protein comprising a recombinant protein coexpressed with a horseradish peroxidase detectable marker comprising an amino acid sequence set forth in SEQ ID NO. 7.
Claim 16. The detectible recombinant protein according to claim 15, wherein the recombinant protein comprises a protein antigen-binding molecule.
Claim 17. The detectible recombinant protein according to claim 16, wherein the protein antigen-binding molecule comprises a single chain fraction variable.
Claim 18. The detectible recombinant protein according to claim 16, wherein the protein antigen-binding molecule comprises a heavy chain sequence and a light chain sequence separated by a 2A cleavage sequence.
Claim 19. The detectible recombinant protein according to claim 18, wherein the 2A cleavage sequence is flanked by a furin site.
Claim 20. The detectible recombinant protein according to any one of claims 16 to 19, wherein the protein antigen-binding molecule comprises an anti-p53 variable regions.
Claim 21 . The detectible recombinant protein according to claim 20, wherein anti-p53 variable regions comprise anti-p53 variable regions of clone DO-1.
Claim 22. The detectible recombinant protein according to any one of claims 16 to 19, wherein the protein antigen-binding molecule comprises an anti-lamin variable regions.
Claim 23. A vector comprising: an antibiotic selection cassette; a doxycycline inducible promoter; a cleavable sequence between two cistrons; comprising a nucleic acid expressing a detectable marker set forth in amino acid sequence SEQ ID NO. 7, nucleic acid expressing a recombinant protein inserted between a set of homologous genomic locus targeting sequences.
Claim 24. The vector according to claim 23, wherein the recombinant protein comprises an antigen-binding molecule comprising a single chain fraction variable.
Claim 25. The vector according to claim 23, wherein the recombinant protein comprises an antigen-binding molecule comprising a heavy chain sequence and a light chain sequence separated by a 2A cleavage sequence.
Claim 26. The vector according to claim 23, wherein the 2A cleavage sequence is flanked by a furin site.
Claim 27. A recombinant cell line comprising the expression system according to any one of claims 1 to 12 or a vector according to any one of claims 23 to 25.
Claim 28. The recombinant cell line according to claim 27, expressing the detectable recombinant protein according to any one of claims 15 to 22.
Claim 29. A method of detecting: a) a product derived from: i) the expression system according to any one of claims 1-12, ii) the vector according to any one of claims 23-25, iii) the recombinant cell line according to claim 26 or 27; or b) the detectible recombinant protein according to any one of claims 15 to 21 , in a single step without the use of a secondary labeling reagents by detecting the detectable marker or the horseradish peroxidase detectable marker.
Claim 30. The method according to claim 29, wherein the detectible marker is detected on a western blot.
Claim 31 . The method according to claim 29, wherein the detectible marker is immunohistochemically detected in formalin fixed tissue samples.
PCT/SG2023/050769 2022-11-17 2023-11-17 T3 vectors for recombinant protein production in mammalian cells WO2024107119A1 (en)

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