Protein secretion regulator

Abstract

This disclosure provides a method for identifying protein secretion regulators. It also provides protein secretion regulators and their uses.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to European Patent Application No. 23315197.6, filed on 11 May 2023, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence List This application includes a sequence listing submitted electronically in XML format, which is incorporated herein by reference in its entirety. The XML copy (created on 3 May 2024) is named 122548WO034.xml and has a size of 60,335 bytes. [Background technology]

[0003] Protein secretion is a fundamental biological process underlying cell proliferation, movement, and communication. Numerous regulatory mechanisms act as quality controls for this process. Defects in protein folding and secretion can lead to various pathologies, such as cancer, as well as neurodegenerative diseases, metabolic disorders, and inflammatory diseases.

[0004] Understanding cellular secretion mechanisms is also crucial for the development of biological therapeutics. The number of protein therapies derived from cultured cells has increased dramatically over the past 20 years, now accounting for a quarter of all approved drugs, with approximately half of them being antibody therapies (Walsh and Walsh, Nat Biotechnol. (2022) 40:1722-60). Numerous approaches, including host cell selection, vector design, and culture medium formulation, have been attempted to optimize the titer of recombinant proteins.

[0005] Conversely, uncontrolled protein secretion can induce cellular dysfunction and disease. See, for example, Kuo et al., Cell Sys. (2021) 12(9):873-84) and Wang et al., Mol Neurodegen. (2014) 9:31). If the secreted molecules have harmful effects on adjacent cells, uncontrolled secretion can lead to cell death, which is a concern in bioproduction. [Overview of the project] [Problems that the invention aims to solve]

[0006] Therefore, there remains a need to identify approaches for regulating protein secretion and optimizing the production of biologics. [Means for solving the problem]

[0007] This disclosure provides regulators of protein secretion from eukaryotic cells. Accordingly, in one embodiment, this disclosure provides a method for inhibiting protein secretion from eukaryotic cells, comprising contacting cells with nigericin or tylphostin AG-879, or an analog thereof. In some embodiments, the cells are in a cell culture (i.e., in vitro). In some embodiments, contact comprises adding nigericin, tylphostin AG-879, or an analog thereof to the cell culture. In further embodiments, nigericin, tylphostin AG-879, or an analog thereof is added to the cell culture to reach a concentration of about 1 μg / mL to about 100 μg / mL, optionally about 5 μg / mL to about 50 μg / mL. In further embodiments, nigericin, tylphostin AG-879, or an analog thereof is added to the cell culture to reach a concentration of about 50 μg / mL. In further embodiments, nigericin, tilphostin AG0879, or an analog is added to the cell culture to reach a concentration of at least about 50 μg / mL.

[0008] In another embodiment, the present disclosure provides a method for increasing protein secretion from eukaryotic cells, comprising contacting cells with lycorine or its analogues. In some embodiments, eukaryotic cells are recombinant cells engineered to express recombinant proteins, e.g., enzymes for enzyme replacement therapy, or antibodies. In some embodiments, the cells are in a cell culture. In some embodiments, contact comprises adding lycorine or its analogues to the cell culture. In further embodiments, lycorine or its analogues are added to the cell culture to reach a concentration of about 0.1 μM to about 100 μM, optionally about 1 μM to about 50 μM. In further embodiments, lycorine or its analogues are added to the cell culture to reach a concentration of at least about 1 μM. In further embodiments, lycorine or its analogues are added to the cell culture to reach a concentration of about 1 μM. In further embodiments, lycorine or its analogues are added to the cell culture to reach a concentration of about 50 μM.

[0009] In some embodiments of this method, the eukaryotic cells are mammalian cells, insect cells, or yeast cells. In further embodiments, the mammalian cells are CHO cells, NS0 cells, BHK cells, 293 cells, HeLa cells, HEK cells, or Sp2 / 0 cells.

[0010] Other features, purposes, and advantages of the present invention will become apparent in the detailed description below. However, it should be understood that this detailed description, while illustrating embodiments and aspects of the present invention, is provided for illustrative purposes only and not limiting. From this detailed description, various modifications and alterations within the scope of the present invention will become apparent to those skilled in the art. [Brief explanation of the drawing]

[0011] [Figure 1A]Figure 1A is a schematic diagram illustrating the principle of the RUSH assay for antibody secretion. The fusion protein (in this case, an antibody fused to a streptavidin-binding domain (SBD) separated by a linker containing a furin cleavage site) is retained in the endoplasmic reticulum (ER) lumen via the interaction between the SBD and the streptavidin-KDEL hook protein. After biotin addition, the interaction between the hook and the fusion protein is competitively disrupted, thereby freeing the fusion protein to move to the Golgi apparatus via the classical secretory pathway, where furin proteases cleave the linker between the SBD and the antibody. The resulting portion is then secreted into the extracellular environment. The GFP site allows for monitoring of protein trafficking by fluorescence microscopy. [Figure 1B-1C] Figure 1B is a schematic diagram showing an exemplary RUSH antibody. The antibody consists of IgG fused to a peptide sequence containing a linker cleavable with furin at its heavy chain C-terminus, green fluorescent protein (GFP), and streptavidin-binding peptide (SBP). Figure 1C is a panel of photographs of immunostained, fixed cells showing co-localization of streptavidin-KDEL hooks and calreticulin in the ER. Pearson correlation coefficient (PCC) = 0.69. Surface overlap coefficient (SOC) = 0.45. [Figure 2A-2B]Figures 2A–2F illustrate the characterization of the anti-PD-L1 antibody RUSH system. Figure 2A shows time-lapse microscopy of clone #7 cells incubated, fixed, and stained with DAPI in the absence or presence of 40 μM biotin. Secretion rates can be estimated from the decrease in intracellular GFP intensity after biotin addition to the cell culture (Figure 2A). Figure 2B shows immunostaining of fixed cells incubated for 4 hours in the absence or presence of biotin, showing co-localization of streptavidin-KDEL hook and GFP-tagged antibody in the absence of biotin and loss of co-localization 4 hours after biotin addition. PCC=0.51 in the absence of PCC=biotin, 0.29 in the presence of PCC=biotin. Western blots of cell lysates (Figure 2C) or concentrated supernatants (Figures 2D-2F) of clone #7 incubated for 4 hours in the absence of biotin, in the presence of 40 μM biotin, or in the presence of 40 μM biotin and 100 μM furin inhibitor I. The membrane was probed with the indicated antibody to detect proteins containing GFP sites (Figures 2C, 2D), SBP sites (Figure 2E), or epitopes from immunoglobulin heavy and light chains (Figure 2F). Molecular weight standards (kDa) are shown on the left in panel Figure 2D. The structures of proteins detected by immunoblotting are shown based on the scheme shown in Figure 1B and include antibody heavy and light chains, GFP, SBP, and a linker with a furin cleavage site. The cleavage shape at the antibody C-terminus represents the linker residue remaining after furin cleavage. The results are representative of at least three independent experiments. [Figure 2C-2F] Same as above. [Figure 3A-3B]Figures 3A–F characterize the binding ability of anti-PD-L1 RUSH antibodies released via the biotin-activated RUSH system (Figures 3A, 3C, and 3E). Schematic diagrams of antibodies binding to PD-L1-positive cells and detection by flow cytometry. Figure 3B shows a FACS histogram plot of purified native recombinant anti-PD-L1 antibody. FACS histogram plots of treated and untreated RUSH anti-PD-L1 antibodies released by clone #7 in the presence or absence of biotin and furin inhibitors, respectively (Figures 3D, 3F). Dark gray indicates binding to PD-L1-positive CHO cells (CHO-PDL1); light gray indicates binding to PD-L1-negative parental CHO cells (CHO-K1). Antibody binding is detected using indirect immunofluorescence assay with a secondary anti-Fc APC conjugate or by direct measurement of GFP fluorescence. [Figure 3C-3D] Same as above. [Figure 3E-3F] Same as above. [Figure 4A]Figures 4A–4C show high-throughput screening of chemical compound libraries for the identification of protein secretion regulators. Figure 4A shows the screening workflow. U2OS clone #7 was seeded in a 384-well plate, incubated for 24 hours, and then treated with two concentrations (low and high, with a 10-fold difference) of compounds from Prestwick, ICCB Known Bioactives, or the Autophagy Compound Library. Biotin was added after 4 hours, and cells were washed and fixed 1 hour after biotin addition and processed for automated imaging of intracellular Hoechst33324 and GFP fluorescence. Figure 4B shows a dot plot of the drugs based on their effects on GFP release (cytoplasmic GFP signaling) and nuclear integrity (viability). Figure 4C shows the Z-score ranking of cytoplasmic GFP intensity after treatment. Results of Prestwick library screening at low drug concentrations are plotted. Brefelzin A was used as a positive control for secretion inhibition. Compounds that induce cytoplasmic GFP intensity lower than that observed in cells treated with biotin alone are potential enhancers of protein secretion. Drugs that induce higher GFP fluorescence levels than those measured in the presence of biotin alone are potential inhibitors of protein secretion. [Figure 4B-4C] Same as above. [Figure 5A-5B] Figures 5A–5C show the selection of potential enhancers of protein secretion. Figures 5A and 5B show dot plots of drug efficiency based on their Z-scores measured at low and high drug concentrations (Figure 5A, Prestwick Library; Figure 5B, ICCB Known Bioactives and TargetMol Autophagy Library). Compounds selected for further characterization are listed (Figure 5C). Clone #7 was treated, fixed, and analyzed with the indicated compounds as described in the legend of Figure 4A. [Figure 5C] Same as above. [Figure 6A]Figures 6A-6C show the verification of an enhancer of protein secretion. Clone #7 cells were treated with the indicated compounds as shown in the legend of Figure 4A and analyzed. The drugs were used at low concentration (Figure 6A) or high concentration (Figure 6B) as indicated by the letters, and the intracellular fluorescence after treatment was normalized to that measured in mock-treated cells (without biotin). Figure 6C shows an ELISA assay with immobilized PD-L1. [Figure 6B] Figures 6A-6C show the verification of an enhancer of protein secretion. Clone #7 cells were treated with the indicated compounds as shown in the legend of Figure 4A and analyzed. The drugs were used at low concentration (Figure 6A) or high concentration (Figure 6B) as indicated by the letters, and the intracellular fluorescence after treatment was normalized to that measured in mock-treated cells (without biotin). Figure 6C shows an ELISA assay with immobilized PD-L1. [Figure 6C] Figures 6A-6C show the verification of an enhancer of protein secretion. Clone #7 cells were treated with the indicated compounds as shown in the legend of Figure 4A and analyzed. The drugs were used at low concentration (Figure 6A) or high concentration (Figure 6B) as indicated by the letters, and the intracellular fluorescence after treatment was normalized to that measured in mock-treated cells (without biotin). Figure 6C shows an ELISA assay with immobilized PD-L1. [Figures 7A-7B] Figures 7A and 7B show the verification of an inhibitor of protein secretion. Figure 7A shows an ELISA assay with immobilized PD-L1. Clone #7 cells were treated with the indicated compounds and the supernatant was assayed for PD-L1 binding. Brefeldin A was used as a positive control for protein secretion inhibitor. Figure 7B shows that the cells treated in Figure 7A were subjected to a cytotoxicity assay. Hoechst 33324 fluorescence was measured, which made it possible to count the adherent live cells in each well.

Mode for Carrying Out the Invention

[0012] The present disclosure provides a method for identifying modulators of antibody secretion from recombinant host cells by utilizing a Retention Using Selective Hooks (RUSH) system that uses selective hooks in medium- to high-throughput drug library screening.

[0013] The RUSH system is based on retaining a protein of interest at a specific intracellular location in the secretory pathway until its induced release. The system utilizes a two-state system in which a hook protein fused to core streptavidin is firmly immobilized in a donor compartment and reversibly interacts with a reporter protein fused to a streptavidin-binding peptide (SBP). After addition of biotin, the reporter protein is simultaneously released from the hook protein. See, for example, Boncompain et al., Nat Methods. (2012) 9:493-98; Zhao et al., Sci Rep. (2018) 8:14966; International Publication No. 2022 / 101482 pamphlet.

[0014] Thus, the present disclosure provides a method for identifying a compound that modulates antibody secretion from eukaryotic cells, the method comprising: (i) a hook protein retained in the endoplasmic reticulum of the cell, the hook protein comprising a biotin-binding domain (e.g., streptavidin), and (ii) providing a cell engineered to express a bait protein comprising an antibody with a heavy chain C-terminus fused to a hook protein-binding peptide (e.g., streptavidin-binding peptide); contacting the cell with a candidate compound (e.g., a member of a small molecule library) and biotin, and determining the amount of the antibody secreted from the cell, wherein a modified amount of the antibody secreted from the cell as compared to a reference cell not contacted with the candidate compound indicates that the candidate compound is a compound capable of modulating antibody secretion from the cell.

[0015] The inventors have developed an antibody-based RUSH system that enables screening for regulators of protein secretion (e.g., antibodies, enzymes, or other protein biopharmaceuticals) from cells engineered to produce recombinant proteins (e.g., antibodies, enzymes, or other protein biopharmaceuticals). Screening a small library of compounds led to the discovery of lycorine, anisomycin, cycloheximide, digoxigenin, methenamine, ropinirole, pinosembrin, and pyrityldione as secretion enhancers; and nigericin and tilphostine AG-879 as secretion inhibitors. These compounds can be used to directly regulate protein secretion by adding them to cell culture media. For example, protein secretion enhancers can be used to increase the yield of recombinant protein production, while protein secretion inhibitors can be used to control the timing of protein secretion or to prevent or reduce the secretion of harmful proteins.

[0016] I.RUSH powder This disclosure provides a cell-based assay (RUSH system) for identifying regulators of antibody secretion. In certain embodiments, the cell-based assay utilizes cell lines co-expressing hook proteins and bait proteins to aid in the identification of small molecule regulators of protein secretion and to monitor the dynamics of protein secretion.

[0017] The cell line selected for the RUSH assay can be any eukaryotic cell line. The RUSH cell line may be the same as the cell line intended for the production of the target antibody. In some embodiments, the eukaryotic cell is a yeast cell. In some embodiments, the yeast cell is S. cerevisiae, P. pastoris, etc. In some embodiments, the eukaryotic cell is an insect cell. In some embodiments, the eukaryotic cell is a mammalian cell. In further embodiments, the mammalian cell is a CHO cell, HEK cell, BHK cell, 293 cell, HeLa cell, NS0 cell, Sp2 / 0 cell, U2OS cell, etc.

[0018] A. Hook protein In some embodiments, the hook proteins provided herein include biotin-binding proteins fused to peptides capable of sequestering the hook protein in the endoplasmic reticulum (ER). Exemplary ER-retaining peptides are shown in the table below.

[0019] [Table 1]

[0020] In some embodiments, the ER-retaining peptide comprises the KDEL (SEQ ID NO: 3), K(X)KXX (SEQ ID NO: 10), RR, RXR, or RXXR (SEQ ID NO: 12) motif (where X is any amino acid residue). In further embodiments, the ER-retaining peptide comprises or consists of KDEL (SEQ ID NO: 3).

[0021] In some embodiments, the biotin-binding protein is selected from avidin, streptavidin, tamavidin, bladavidin, extraavidin, rizavidin, and their derivatives. In further embodiments, the biotin-binding protein is streptavidin. An exemplary sequence of streptavidin is shown below: [ka] Variant proteins that are at least 90% (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to this exemplary sequence may also be used, as long as they retain the ability to bind biotin or its derivatives.

[0022] The percentage of identity between two amino acid sequences (or two nucleic acid sequences) can be obtained, for example, by BLAST® using default parameters (available on the website of the U.S. National Library of Medicine's National Center for Biotechnology Information). In some embodiments, the length of the reference sequence aligned for comparison is at least 30%, (e.g., at least 40, 50, 60, 70, 80, or 90% of the reference sequence).

[0023] In a given embodiment, the hook protein contains streptavidin fused to the KDEL (SEQ ID NO: 3) peptide.

[0024] Hook proteins can be introduced into host cells by transfecting them with an expression cassette encoding the hook protein. The expression cassette may contain a promoter that is active in the host cell. For mammalian host cells, for example, promoters selected from CMV, SFFV, CAG, EFI, EFIA, GALI, GALI0, GPD, ADH, and GAP promoters may be used.

[0025] B. Bait Protein The bait protein used in this method includes an antibody fused to a peptide capable of binding to a hook protein, or another multimeric protein. In some embodiments, the hook protein-binding peptide is a streptavidin-binding peptide (SBP) when the hook protein contains streptavidin. Examples of SBPs are shown in the table below. In some embodiments, the SBP includes or consists of SEQ ID NO: 32.

[0026] [Table 2]

[0027] The multimeric protein may be an antibody (e.g., IgG1, IgG2, IgG3, or IgG4) in which the heavy chain C-terminus is fused to an SBP. The antibody may bind to any antigen of interest, including, but is not limited to, cytokines (e.g., TNF-alpha, IL-6, or TGF-beta), tumor antigens (e.g., AFP, BCMA, CD19, CD20, CD22, CD123, EGFR, EpCAM, GPC2, GPC3, HER2, MUC16, ROR1, or ROR2), or immune checkpoints (e.g., PD-1, PD-L1, PD-L2, CTLA-4, TIGIT, TIM-3, or LAG-3).

[0028] In some embodiments, the bait protein may further include a fluorescent signaling domain (e.g., a green fluorescent protein (GFP) domain or enhanced GFP (eGFP)) domain to enable monitoring of intracellular trafficking of the bait protein. In such embodiments, the fluorescent signaling domain may be linked to a multimeric protein (e.g., an antibody) via a linker cleavable by a protease in an intracellular compartment, such as furin located in the Golgi apparatus. The furin cleavage site may be RX1X2R (X1 = any naturally occurring amino acid, and X2 = R or K (SEQ ID NO: 62)).

[0029] Bait proteins can be introduced into host cells by transfecting them with an expression cassette encoding the bait protein. The expression cassette may contain a promoter that is active in the host cell. For mammalian host cells, for example, promoters selected from vav, PGK, SV40, thymidine kinase promoter (TK), MSCV, and UbC promoter may be used.

[0030] C. peptide linker In hook proteins and bait proteins, various functional domains can be linked via peptide linkers. The peptide linker can be a flexible linker that allows for proper folding, movement, and interaction of the linked domains. In some embodiments, the flexible peptide linkers described herein mainly consist of small amino acids (e.g., Gly, Ser, or Thr). In some embodiments, the peptide linkers described herein consist mainly of Gly and Ser residues (e.g., more than 50% of the residues) ("GS" linkers). As described above, such peptide linkers are (G4S) n (Sequence ID 1) may be included. By adjusting the copy number "n", the linker length can be adjusted to achieve a desired distance between the connected functional domains. In some embodiments, n may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In further embodiments, n may be 2 or 3. In some embodiments, the peptide linker may contain additional amino acids such as Thr and Ala to maintain flexibility, and polar amino acids such as Lys and Glu to improve solubility. See, for example, Chen et al., Adv Drug Deliv Rev. (2013) 65(10):1357-69.

[0031] In some embodiments of the hook protein, the streptavidin domain and the ER-retaining domain (e.g., KDEL peptide) are linked to a GS linker, e.g., (G4S) n (Sequence 1) (n can be, for example, 2 or 3 (i.e., GGGGSGGGGS or GGGGSGGGGSGGGGS, respectively)) can be concatenated.

[0032] In some embodiments of the bait protein, IgG is fused to the SBP at its C-terminus via a peptide linker. In further embodiments, the peptide linker is cleavable. In a given embodiment, the peptide linker includes a furin cleavage site and is cleavable by furin. In a given embodiment, the cleavable (e.g., furin-cleavable) linker links IgG to the fluorescent signal peptide and the SBP.

[0033] D. Biotin and its derivatives Biotin is added to RUSH cells to interfere with the binding between the hook protein and the bait (antibody) protein. In some embodiments, biotin derivatives may be used. In some embodiments, the biotin derivative has the structure of formula (I): [ka] (In the formula, X is selected from H2, O, S, Se, SO, and SO2. Y is selected from CONH(CH2)4CH(NH2)COOH, COOH, and OH. n is 1, 2, or 3. z has (1 or 2).

[0034] In one embodiment, the biotin derivative is selected from the group consisting of biocitin, dethiobiotin, selenobiotin, biotin sulfoxide, oxybiotin, biotinol, norbiotin, homobiotin, alpha-dehydrobiotin, and biotin sulfone.

[0035] II. Identification of Protein Secretion Regulators This disclosure provides methods for screening for regulators (agonists or antagonists) of protein (e.g., multimeric proteins, e.g., antibodies) secretion. In these methods, candidate compounds (e.g., those in a small molecule library) are added to cells containing the antibody RUSH system described herein. Upon addition of biotin (or a derivative thereof) to the cells, the antibody bait is released from its isolation state in the ER. Measurement of antibody secretion may be performed (e.g., by ELISA) to determine the modification of antibody secretion compared to reference cells not treated with the candidate compound. The candidate compound may be added before, simultaneously with, or after the addition of biotin to the cell culture.

[0036] The screening method of this disclosure makes it possible to identify compounds (e.g., small molecule compounds) as enhancers or inhibitors of protein secretion, such as antibody secretion.

[0037] III. Small molecule regulators of antibody secretion This disclosure also provides enhancers and inhibitors of protein secretion (e.g., monomeric proteins or polymeric proteins, e.g., antibodies) identified by this screening method.

[0038] A. Protein secretion enhancers In some embodiments, the enhancer of protein (e.g., antibody) secretion is lycorine (3,12-didehydro-2'H-[1,3]dioxolo[4',5':9,10]galantane-1α,2β-diol). Lycorine is a natural alkaloid extracted from plants of the Amaryllidaceae family. Lycorine is a potent, orally active SCAP inhibitor. Lycorine downregulates SCAP (SREBP cleavage-activated protein; SREBP, sterol regulatory element-binding protein) protein levels without altering its transcription. Lycorine has shown potential as a melanoma angiogenesis inhibitor and has been useful in the study of metabolic diseases and prostate cancer. See, for example, Roy et al., Biomed Pharmacother. (2018) 107:615-24. Lycorine is identified by CAS number 476-28-8 and has the following chemical structure: [ka]

[0039] In some embodiments, the enhancer of protein (e.g., antibody) secretion is anisomycin ([(2R,3S,4S)-4-hydroxy-2-[(4-methoxyphenyl)methyl]pyrrolidine-3-yl]acetate). Anisomycin is a potent inhibitor of protein synthesis, interfering with protein and DNA synthesis by inhibiting peptidyltransferase or the 80S ribosomal system. Anisomycin acts as a JNK activator, increasing phospho-JNK levels. Anisomycin is also a bacterial antibiotic. Anisomycin is identified by CAS number 22862-76-6 and has the following chemical structure: [ka]

[0040] In some embodiments, the enhancer of protein (e.g., antibody) secretion is cycloheximide (4-[(2R)-2-[(1S,3S,5S)-3,5-dimethyl-2-oxocyclohexyl]-2-hydroxyethyl]piperidine-2,6-dione). Cycloheximide is a dicarboxymide, 4-(2-hydroxyethyl)piperidine-2,6-dione, in which one of the hydrogens bonded to the carbon having a hydroxyl group is replaced by a 3,5-dimethyl-2-oxocyclohexyl group. It is an antibiotic produced by the fungus Streptomyces griseus and functions as a bacterial metabolite, protein synthesis inhibitor, neuroprotective agent, anticoronavirus agent, and ferroptosis inhibitor. It is a type of piperidone, a piperidine antibiotic, an antibacterial agent, a dicarboxymide, a secondary alcohol, and a cyclic ketone, and is functionally related to piperidine-2,6-dione. Cycloheximide is identified by CAS number 17974-04-8 and has the following chemical structure: [ka]

[0041] In some embodiments, the enhancer of protein (e.g., antibody) secretion is digoxigenin (3-[(3S,5R,8R,9S,10S,12R,13S,14S,17R)-3,12,14-trihydroxy-10,13-dimethyl-1,2,3,4,5,6,7,8,9,11,12,15,16,17-tetradecahydrocyclopenta[a]heterocyclyl-17-yl]-2H-furan-5-one). Digoxigenin is a hydroxysteroid consisting of a 5-beta-cardanolide having a double bond at the 20(22) position and hydroxyl groups at the 3-beta, 12-beta, and 14-beta positions. It has been isolated from plant species of the genus Digitalis and functions as a hapten and plant metabolite. Digoxigenin is identified by CAS number 1672-46-4 and has the following chemical structure: [ka]

[0042] In some embodiments, the enhancer of protein (e.g., antibody) secretion is methenamine (1,3,5,7-tetrazatricyclo[3.3.1.13,7]decane). Methenamine is a heterocyclic organic compound with antibiotic activity. In the body, methenamine is converted to formaldehyde, which is a nonspecific bactericide. Methenamine is typically used long-term to treat chronic urinary tract infections and to prevent recurrence of infections. Methenamine is identified by CAS number 37604-90-3 and has the following chemical structure: [ka]

[0043] In some embodiments, the enhancer of protein (e.g., antibody) secretion is ropinirole (4-[2-(dipropylamino)ethyl]-1,3-dihydroindole-2-one). Ropinirole (SKF101468) is a potent D3 / D2 receptor agonist that is active orally but does not have affinity for the D1 receptor. Ropinirole is identified by CAS number 91374-21-9 and has the following chemical structure: [ka]

[0044] In some embodiments, the enhancer of protein (e.g., antibody) secretion is pinosembrin (5,7-dihydroxy-2-phenyl-2,3-dihydro-4H-chromen-4-one). Pinosembrin is a compound found in lodgepole pine (Pinus contorta var. latifolia) and pepper (Piper nigrum). Pinosembrin is identified by CAS number 68745-38-0 and has the following chemical structure: [ka]

[0045] In some embodiments, the enhancer of protein (e.g., antibody) secretion is pyrityldione (3,3-diethyl-1H-pyridine-2,4-dione). Pyrityldione is a psychoactive drug, identified by CAS number 77-04-3, and has the following chemical structure: [ka]

[0046] Analogues of the enhancers exemplified above may also be used. “Analog” means a compound that is functionally equivalent to a reference compound and shares the same or similar core chemical structure. Analogues of a compound include, for example, pharmaceutically acceptable salts or esters of that compound.

[0047] In some embodiments, the enhancer of this specification (e.g., lycorine or its analog) is added to the cell medium to reach a concentration of about 0.1 μM to about 1000 μM (e.g., about 0.5 μM to about 100 μM, or about 1 μM to about 50 μM). In some embodiments, lycorine (or its analog) is added to the cell medium to reach a concentration of about 1 μM or about 50 μM. In some embodiments, lycorine (or its analog) is added to the cell medium to reach a concentration of about 1 μM. In some embodiments, lycorine (or its analog) is added to the cell medium to reach a concentration of about 50 μM. In some embodiments, lycorine (or its analog) is added to the cell medium to reach a concentration of at least about 1 μM.

[0048] B. Inhibitors of protein secretion In some embodiments, the inhibitor of protein (e.g., antibody) secretion is nigericin. Nigericin is an antibiotic derived from Streptomyces hygroscopicus that functions as a K+ / H+ ionophore, promoting K+ / H+ exchange across the mitochondrial membrane. Nigericin can also act as an NLRP3 (nucleotide-binding domain leucine-rich [LRR] and pyrine-containing receptor 3) activator, resulting in the release of IL-1β. Nigericin has been found to promote elliptosis, a process associated with ROS formation and partially attributable to oxidative stress induction. Nigericin also induces apoptosis. Nigericin is identified by CAS number 28380-24-7 and has the following chemical structure: [ka]

[0049] In some embodiments, the inhibitor of protein (e.g., antibody) secretion is tilphostin AG879, i.e., "AG879". AG879 is a tyrosine kinase inhibitor that effectively inhibits TrKA phosphorylation and does not inhibit TrKB and TrKC. This compound is also a selective inhibitor of ErbB2 tyrosine kinase, exhibiting at least 500-fold higher specificity for ErbB2 than for EGFR. AG879 has also demonstrated anticancer activity. AG879 is identified by CAS number 148741-30-4 and has the following chemical structure: [ka]

[0050] Analogues of the inhibitors exemplified above may also be used. In some embodiments, the inhibitors of this specification (e.g., nigericin or AG879, or their analogues) are added to the cell medium to reach a concentration of about 0.5 μg / mL to about 500 μg / mL (e.g., about 1 μg / mL to about 100 μg / mL, about 5 μg / mL to about 50 μg / mL, about 10 μg / mL to about 100 μg / mL, or about 10 μg / mL to about 50 μg / mL). In some embodiments, nigericin (or its analogues) are added to the cell medium to reach a concentration of about 50 μg / mL. In some embodiments, nigericin (or its analogues) are added to the cell medium to reach a concentration of at least about 50 μg / mL. In some embodiments, AG879 (or its analogues) are added to the cell medium to reach a concentration of about 50 μg / mL. In some embodiments, AG879 (or its analogues) is added to the cell culture medium to reach a concentration of at least about 50 μg / mL.

[0051] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have meanings generally understood by those skilled in the art. Exemplary methods and materials are described below, but similar or equivalent methods and materials may also be used in the practice or testing of this disclosure. In case of any conflict, this specification, including definitions, shall prevail. Furthermore, unless otherwise required by context, singular terms shall include plural terms, and plural terms shall include singular terms. Throughout this specification and the embodiments, the terms “have” and “comprise,” or variations such as “has,” “having,” “comprises,” or “comprising,” shall be understood to include the integer or set of integers described, but not to exclude any other integer or set of integers. All publications and other references referenced herein are incorporated by reference as specifically and individually indicated, each individual reference being incorporated by reference as the whole. This specification references several documents, but such references do not constitute an endorsement that any of those documents form part of the common technical knowledge in the art. As used herein, the terms “approximately” or “about” refer to a value similar to the reference value described, when applied to one or more values ​​of interest. In certain embodiments, unless otherwise stated or otherwise evident from the context, the term refers to a range of values ​​that fall within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in either direction (greater than or less than) the reference value described.

[0052] According to this disclosure, backreferences in dependent claims mean abbreviated notation to any direct and obvious disclosure of any combination of claims indicated by that backreference. Furthermore, the headings in this specification are provided for ease of organization and are not intended to limit in any way the scope of the claimed invention.

[0053] To better understand the present invention, the following examples are provided. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way. [Examples]

[0054] A. Materials and Methods Cell culture, chemicals, and antibodies Human osteosarcoma U2OS cells obtained from the Kroemer Institute were maintained at 37°C in a humidified incubator with 5% CO2 in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 1% HEPES (Gibco, Carlsbad, CA). CHO-K1 (CCL-61) cells obtained from ATCC were cultured at 37°C in a humidified incubator with 5% CO2 in Nutrient Mixture F-12 Ham's (Sigma, Saint-Louis, MO) supplemented with 10% FBS and 2 mM L-glutamine (Gibco). PD-L1-expressing CHO cells were cultured under similar conditions in a medium supplemented with 7.5 μg / mL puromycin (Gibco). The Prestwick, BML-2840 ICCB Known Bioactives and Autophagy Compounds libraries were purchased from Prestwick Chemicals (Illkirch, France), Enzo Life Sciences (Farmingdale, NY), and TargetMol (Boston, MA), respectively. Anti-streptavidin (Santa Cruz Biotechnology, Dallas, TX), anti-calreticulin (Abcam, Cambridge, UK), anti-SBP (Santa Cruz Biotechnology), anti-human heavy and light IgG chain (Invitrogen), and anti-GFP (Cell Signaling, Danvers, MA) antibodies were used in Western blotting and immunofluorescence experiments at dilutions of 1:500, 1:200, 1:500, 1:5,000, and 1:1,000, respectively. The reference anti-human PD-L1 antibody used in the FACS experiment was generated from cells transfected with a plasmid construct based on publicly available atezolizumab heavy and light chain sequences, available at the Sanofi Institute on the IMGT website (imgt.org) (Lefranc et al., Methods in Molecular Biology (2018) 1827:35-69).Alexa Fluor568 and 488-conjugated anti-mouse and rabbit IgG secondary antibodies (Life Technologies, Carlsbad, CA, USA) were used in immunofluorescence and FACS experiments at dilutions of 1:1,000 and 1:500, respectively. HRP-conjugated goat anti-mouse and rabbit IgG secondary antibodies (Southern Biotech, Birmingham, AL) were used in Western blotting experiments at a dilution of 1:5000. Anti-human kappa light chain antibody (Thermo Fisher Scientific, Waltham, MA) was used in ELISA at a dilution of 1:4,000.

[0055] Construct used for the RUSH antibody secretion system The streptavidin-KDEL plasmid was described in Zhao et al., Sci Rep. (2018) 8:14966. Using the amino acid sequences of the heavy and light chains of the anti-PD-L1 atezolizumab antibody available at IMGT, nucleic acid sequences adapted for expression in human cells were generated. Sequences encoding a furin-cleavable site (AVSKERSKRSP) (SEQ ID NO: 63), linker (G4S)3 (SEQ ID NO: 1)-enhanced green fluorescent protein (eGFP), and streptavidin-binding peptide (SBP) were added to the 3' end of the anti-PD-L1 heavy chain coding sequence in this order. The anti-PD-L1 heavy and light chain constructs were manufactured by GeneArt (Ratisbonne, Germany) and provided in shuttle vectors. Next, the heavy and light chain sequences were cloned into multi-cloning sites 2 and 1, respectively, of the pVITRO1 plasmid (Invivogen, San Diego, CA) using the NEBuilder® assembly kit (New England Biolabs, Ipswich, MA). Subsequently, the integrity of the construct, referred to as the anti-PD-L1 IgG RUSH plasmid, was confirmed by sequencing (Eurofins, Luxemburg).

[0056] Establishment of RUSH-antibody cell lines U2OS cells were transfected with the streptavidin-KDEL plasmid using lipofectamine 2000 (Thermo Fisher Scientific) and selected for 2 weeks in medium supplemented with G418 (400 μg / mL, Gibco-Invitrogen). Limiting dilutions in 96-well plates were used for single-cell isolation from the selected pool. Clones were grown and selected based on streptavidin immunofluorescence. Clones #BD6, showing the highest streptavidin expression, were further grown in medium supplemented with G418 (200 μg / mL), transfected with the anti-PD-L1 IgG RUSH plasmid using lipofectamine 2000, and selected for 2 weeks in medium supplemented with G418 (200 μg / mL) and hygromycin (100 μg / mL, Santa Cruz Biotechnology). Limiting dilutions in 96-well plates were used for single-cell isolation from the selected pool derived from clone #BD6. The clones were grown and selected based on GFP fluorescence, and clone #7 was chosen for further characterization and screening campaigns.

[0057] Immunofluorescence U2OS clone #7 cells were seeded in a black 96-well imaging plate (Greiner bio one, Kremsmuenster, Austria). After 24 hours, the medium was removed, the cells were rinsed with PBS preheated to 37°C, and fixed at room temperature (RT) for 20 minutes under shaking in PBS supplemented with 4% paraformaldehyde (PFA, Sigma) and 1 μg / mL Hoechst (Life Technologies, Carlsbad, CA, USA). After rinsing the cells with PBS, a 5% FBS / 0.3% Triton X-100 / PBS permeabilization solution was added at room temperature under shaking for 30 minutes. The cells were rinsed with PBS and incubated overnight at 4°C with primary antibody in 1% BSA / PBS under shaking. The cells were washed with PBS and incubated at room temperature with secondary antibody in 1% BSA / PBS under shaking for 45 minutes. Finally, the cells were washed and processed for imaging using an IXM XL BioImager (Molecular Devices, Sunnyvale, CA).

[0058] Characterization of antibody expression and secretion by Western blotting U2OS clone #7 cells were seeded in T-175 flasks and maintained at 37°C for 24 hours in a humidified incubator with 5% CO2. The following day, the supernatant was removed, the cells were rinsed with PBS preheated to 37°C, and fresh serum-free medium containing 40 μM biotin, 40 μM biotin, or 40 μM biotin along with 100 μM furin inhibitor I (Sigma) was added to the flasks for 4 hours. At the end of the incubation period, the cells were harvested and lysed in RIPA buffer (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitors (Roche, Basel, Switzerland). The cell culture supernatant was also collected and concentrated approximately 100-fold using an Amicon 50 kDa filter unit (Sigma) according to the manufacturer's instructions. Protein concentrations in the cell lysates and concentrated supernatants were determined using the BCA® protein assay kit (Thermo Fisher Scientific). 10 μg of total protein contents from the lysate and supernatant were separated under reducing conditions using NuPAGE® 4-12% Bis-Tris gel (Invitrogen, Carlsbad, CA, USA) and transferred to a nitrocellulose membrane (BioRad) using the BioRad system. The membrane was blocked with 0.01% Tween-20 / 5% skim milk powder / TBS for 1 hour, and then the primary antibody was added overnight under shaking conditions at 4°C. The membrane was rinsed with 0.01% Tween-20 / 5% skim milk powder / TBS and incubated with the secondary antibody under shaking conditions at 4°C for 1 hour. The membrane was then washed three times for 5 minutes with 0.01% Tween-20 / TBS, and peroxidase activity was evaluated using ImageQuant® LAS4000 (GE Healthcare) with Amersham ECL Primer Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK).

[0059] Flow cytometry validation of antibody reactivity The U2OS clone #7 cell culture supernatant was prepared as described above, and the binding of RUSH antibody to PD-L1 expressing cells was evaluated by flow cytometry. Three million CHO-K1 and CHO PD-L1 cells were harvested, rinsed with PBS, and incubated with either 150 μL of recombinant atezolizumab prepared at the Sanofi Laboratory or concentrated U2OS clone #7 cell culture supernatant in the dark with shaking for 30 minutes at 4°C. The cells were then rinsed with PBS, secondary stained with Alexa Fluor647-conjugated anti-human IgG antibody in the dark with shaking for 30 minutes at 4°C, and 100 μL of the solution was analyzed by flow cytometry using MACSQuant® (Miltenyi Biotec, Bergisch Gladbach, Germany).

[0060] High-throughput compound screening for protein secretion regulators U2OS clone #7 cells were seeded at 2,000 cells / well in a black 384-well imaging plate (Greiner bio one) and incubated at 37°C for 24 hours in a humidified incubator with 5% CO2. The following day, the cells were sampled using the Prestwick library (20 μM or 10 μM) and the ICCB Known Bioactives library (1 / 100). e or 1 / 1000 eCells were treated for 4 hours with either a compound from the autophagy compound library (10 μM or 1 μM) or from the autophagy compound library. At the end of the incubation time, biotin was added at 40 μM for 1 hour (untreated cells and DMSO-treated cells were used as controls). Cells were then fixed as described above and processed for subsequent automated imaging using a robot-assisted IXM XL BioImager (Molecular Devices, Sunnyvale, CA, USA) equipped with a Sola light source (Lumencor, Beaverton, OR, USA), fitted excitation and emission filters (Semrock), and a 16-bit monochrome sCMOS PCO.edge 5.5 camera (PCO, Kelheim, Germany). A 20X PlanAPO objective lens (Nikon, Tokyo, Japan) was used to acquire a minimum of four fields of view in each well. Images acquired using open-access R software (https: / / www.r-project.org) were processed using the freely available packages EBImage (available in the Bioconductor repository https: / / www.bioconductor.org) and RBioFormats (https: / / github.com / aoles / RBioFormats), as well as custom packages MetaxpR (https: / / github.com / asauvat / MetaxpR) and MorphR (https: / / github.com / kroemerlab / MorphR). Nuclei were first detected using the Hoechst33342 signal, which was then used as a marker to detect cytoplasmic regions based on the GFP signal, enabling evaluation of cytoplasmic GFP intensity. The data were then extracted and statistically evaluated using R (https: / / www.r-project.org). The data were normalized, compressed, and converted to Z-scores using negative and positive controls. The effects of the selected drugs on secretion were confirmed by repeating experiments manually on a low scale. In this second set of assays, U2OS clone #7 cells incubated with secretion inhibitors were fixed as described above and stained with Hoechst for imaging and viability determination based on cell count.

[0061] ELISA U2OS clone #7 cells were seeded in a 6-well plate and maintained at 37°C for 24 hours in a humidified incubator with 5% CO2. The following day, the supernatant was removed, the cells were rinsed with PBS preheated to 37°C, and fresh medium containing a chemical treatment agent was added for 4 hours. At the end of the incubation time, 40 μM biotin was added to the flask for 30 minutes (selected inhibitor molecule) or 1 hour (selected activator molecule), or no biotin was added. The supernatant was then collected and used for the ELISA assay as follows: A MaxiSorp 96-well plate (Thermo Fisher Scientific) was coated with 0.5 μg / mL hFc-human-PD-L1 (R&D Systems, Minneapolis, MN) in PBS for 5.5 hours at room temperature. The coating solution was then removed, and a 2% BSA / PBS blocking solution was added overnight at 4°C. The following day, the wells were washed three times with a 0.5% BSA / 0.05% Tween-20 / PBS solution. The supernatant was diluted to 1 / 3 with 0.5% BSA / 0.05% Tween-20 / PBS and added at room temperature for 2.3 hours. The wells were washed three times with 0.5% BSA / 0.05% Tween-20 / PBS solution. 1 / 4000 of the supernatant was added to 0.5% BSA / 0.05% Tween-20 / PBS. e Diluted anti-human kappa light chain HRP-conjugated antibody was added at room temperature for 2.3 hours. The wells were washed three times with 0.5% BSA / 0.05% Tween-20 / PBS solution and then incubated with the substrate reagent pack (R&D SYSTEMS) in the dark for 15 minutes. 50 μL of STOP solution (R&D SYSTEMS) was then added to each well, and the absorbance in each well was measured at 450 nm and 635 nm for background subtraction using VICTOR® X4 (PerkinElmer, Waltham, MA).

[0062] statistics Unless otherwise specified, data are reported as the mean ± SD of two replicates in at least three independent experiments. Statistical significance was evaluated using the Mann–Whitney U test.

[0063] B. Results Design of the antibody RUSH system The RUSH system consists of a hook that is streptavidin and a bait that is a protein fused to a streptavidin-binding peptide (SBP). When these two molecules are co-expressed, the bait is sequestered by the hook through high-affinity streptavidin / SBP interactions (K in the range of 10 -8 ~10 -9 M) (Barrette-Ng et al., Acta Crystallographica. Section D, Biological Crystallography (2013) 69:879-87) and is released upon addition of biotin (which outcompetes the SBP due to its higher affinity for streptavidin (K in the range of 10 D M)) (Delgadillo et al., PLoS One (2019) 14:e0204194). Here, this cell-based assay was used to monitor the secretion kinetics of antibodies (bait) and to screen a library of small compounds to identify secretion modulators. Since antibodies are secreted via the conventional pathway, the streptavidin hook was fused to a KDEL peptide to ensure its retention in the endoplasmic reticulum (ER) via interaction with the KDEL receptor present in the ER (Capitani, FEBS letters (2009) 583:3863-71).

[0064] ​​​​The antibody was also modified to suit the purpose of the assay. A polycistronic vector encoding both the heavy and light (kappa) chains of a human PD-L1-specific antibody was used (Zhang et al., Oncotarget (2017) 8:90215-24). The C-terminus of the heavy chain was fused to a (Gly4Ser)3 linker, a furin-cleavable site, green fluorescent protein (GFP), and SBP (Figure 1B). The fusion molecule (hereafter referred to as the RUSH antibody) was assembled into a mature homodimer after protein synthesis and remained in the ER due to its interaction with the streptavidin KDEL hook until biotin was added, with biotin triggering the release of the RUSH antibody and maturation in the Golgi apparatus during its movement into the extracellular space (Figure 1A). Clones expressing both the hook and bait were obtained by transfecting U2OS cells with a construct encoding the hook, and by selecting clones with high hook expression levels to ensure efficient bait retention and minimize leakage in the absence of biotin. As shown in Figure 1C, staining of clone #BD6 with streptavidin-specific antibody revealed co-localization of the hook with the prototypical ER-adhering protein calreticulin (Nomura et al., Histochemistry and Cell Biology (2011) 135:531-8). Clone #BD6 was further transfected with a vector encoding the RUSH antibody, followed by single-cell cloning and selection of the hook / bait clone used in this study (clone #7). This clone was selected based on two criteria: (i) a bright GFP-dependent fluorescence signal indicating a high abundance of RUSH antibody, and (ii) individual localization of the GFP signal in the perinuclear region indicating ER retention (Figure 2A). Fluorescence imaging microscopy of cells from clone #7 revealed that GFP remained in the same intracellular compartment over a two-hour period of acquisition.

[0065] In contrast, biotin addition resulted in the transfer of GFP signaling to scattered sites corresponding to the Golgi apparatus within 40–120 minutes, and complete cytoplasmic GFP signal decay within 4 hours, most likely due to the release of fusion proteins into the extracellular space (Figure 2A). GFP signaling co-localized with streptavidin at baseline and upon biotin addition did not alter this pattern, confirming the retention of the hook in the ER regardless of the presence or absence of biotin in the culture medium. In contrast, GFP / streptavidin co-localization was lost 4 hours after biotin addition (Figure 2B).

[0066] These results confirm bait expression and its retention in the ER via hooks, suggesting its active release into the extracellular space after biotin addition.

[0067] Biochemical validation of the antibody RUSH system In the next step, clone #7 underwent biochemical validation before being used in the screening campaign. For this purpose, the molecular species synthesized and released by clone #7 cultured with or without biotin and furin protease inhibitors were characterized. As can be seen in Figure 2C, the GFP-specific antibody used in Western blotting revealed the presence of a major molecular species of approximately 80 kDa and two minor species of approximately 55 kDa and 35 kDa in the lysates of cells grown in the absence of biotin. The 80 kDa mass is compatible with the full-length heavy chain of the RUSH antibody (theoretical mass of 84 kDa), while the 35 kDa mass is compatible with the GFP-SBP C-terminal fragment of the heavy chain released after furin cleavage (theoretical mass of 32 kDa). Similar analyses performed on cell culture supernatants using GFP, SBP, and human IgG-specific antibodies revealed trace amounts of recombinant protein in the absence of biotin (Figures 2D-2F). This is consistent with the minor treatment observed in cell lysates and, therefore, the efficient retention of RUSH antibodies in the intracellular compartments preceding the Golgi apparatus, as demonstrated by immunofluorescence (Figures 2A and B).

[0068] When cells were cultured in the presence of biotin, the Western blot pattern in cell lysates remained unchanged except for a lower signal intensity, which was associated with the detection of recombinant species in the cell culture supernatant (Figures 2C-2F). This demonstrates that the intracytoplasmic GFP decay observed by immunofluorescence in Figures 2A and 2B is a direct consequence of protein release in the extracellular space. Furthermore, the molecular weight of the substance detected in the supernatant suggests almost complete processing of the released RUSH antibody. This is further confirmed by the similar patterns obtained in cell lysates and cell culture supernatant when cells were grown in the presence of a furin inhibitor. Therefore, it can be concluded that the RUSH antibody is released and processed via the Golgi network after the addition of biotin.

[0069] In summary, these findings demonstrate that clone #7 enables biotin-stimulated release of the RUSH antibody and furin-dependent proteolytic maturation.

[0070] Immunological validation of the antibody RUSH system In addition to verifying the mechanism of clone #7 in terms of antibody production, retention, maturation, and release after biotin addition, we also looked at the PD-L1 binding properties of the secreted RUSH antibody. ELISA demonstrated the specificity of the RUSH antibody against immobilized recombinant PD-L1 (not shown). We further investigated the binding properties of the RUSH antibody to PD-L1 in its native conformation when PD-L1 is presented on the cell membrane. For this purpose, clone #7 was incubated with biotin, and the cell culture supernatant was assayed by FACS for binding to PD-L1-positive and PD-L1-negative CHO cells. As can be seen in Figures 3A and 3B, binding of native recombinant anti-PD-L1 to PD-L1-positive (CHO-PDL1) cells rather than PD-L1-negative (CHO-K1) cells can be demonstrated, confirming the use of such a system for further characterization of the RUSH antibody. Similarly, the supernatant of clone #7 cultured in the presence of biotin demonstrated binding activity to CHO-PDL1 cells but not to CHO-K1 cells (Figures 3C and D), indicating that the released RUSH antibody preserved its antigen-binding properties despite the presence of excess amino acids at its C-terminus remaining after furin treatment. In particular, PD-L1 binding was also detectable in the supernatant of clone #7 cultured in the presence of biotin and a furin protease inhibitor (Figures 3E and 3F). Under these conditions, binding was detected through both the secondary staining antibody and the GFP site of the untreated RUSH antibody, the GFP site being undetectable with the native antibody (Figure 3B) and only very faintly detected in the supernatant of clone #7 cultured with biotin alone (Figure 3D).

[0071] In summary, these observations indicate that clone #7 is suitable for both the screening campaign and further antibody characterization in the PD-L1 binding assay.

[0072] Pharmacological screening of regulators of biotin-induced antibody release Next, we explored using clone #7 in a screening campaign to identify drugs capable of enhancing or inhibiting antibody secretion. For this purpose, clone #7 was cultured in 384-well plates, treated with drugs from a chemical library for 4 hours, followed by biotin addition for 1 hour, and intracellular GFP signal analysis was performed (Figure 4A). Cell counting was facilitated by nuclear counterstaining with Hoechst33324 to assess viability, differentiate drug effects on secretory kinetics from direct cytotoxicity, and select relevant drugs for further characterization. Two independent screening campaigns were conducted to test a total of 2,300 drugs at two different concentrations (i.e., low and 10-fold higher concentrations, referred to as “low” and “high” doses as described in the Materials and Methods section).

[0073] The reduction in biotin-induced cellular GFP signaling was affected by a small number of compounds from various chemical libraries, as shown by plots of primary data (Figure 4B) or Z-score analysis (Figure 4C). As an internal control, brefeldin A (Misumi et al., J Biol Chem. (1986) 261:11398-403), a conventional Golgi-dependent protein secretion inhibitor, inhibited the reduction in biotin-induced GFP signaling (Figures 4B and C). Plots of Z-scores obtained at low and high concentrations for each compound (Figures 5A and B) consistently reduced the fluorescence signal, leading to the identification of several compounds that act as secretion activators. To select candidates for further characterization, the 20 best activators identified at either low or high concentrations were listed, and among these, activators that functioned at both concentrations were selected as candidates.

[0074] In the Prestwick library, sodium balsalazid (currently not commercially available), methenamine, pyrityldione, cycloheximide, digoxigenin, verteporfin, and ropinirole met these criteria (Figure 5A). Verteporfin was excluded due to its phototoxicity, which causes GFP fading (data not shown). In a combined screening including compounds from two additional libraries (ICCB Known Bioactives and TargetMol Autophag), several additional compounds, namely anisomycin, emetine, and pinosembrin, were active at both low and high concentrations. Lycorine was not among the 20 most active compounds at high concentrations, but it was more efficient than all other compounds at low concentrations and was therefore selected (Figure 5B).

[0075] We also identified several drugs that inhibited biotin-induced cytoplasmic GFP signal attenuation and thus acted as potential secretion inhibitors. Among these, nigericin and tilphostine AG-879 were selected for further characterization because they had not been previously identified (Zhao et al., Sci Rep. (2018) 8:14966) (Figure 5B).

[0076] Verification of stimulants and inhibitors of biotin-induced antibody release Medium- and high-throughput screenings often result in false-positive and false-negative hits, requiring independent validation in low-throughput experiments using orthogonal techniques. In the first round of experiments, the effects of selected candidate compounds with potential antibody secretion-enhancing activity were evaluated. Compounds identified in the screening campaign were retested in clone #7 at low concentrations (Figure 6A) or 10-fold higher concentrations (Figure 6B) for their ability to increase antibody secretion. This experiment showed that all selected hits were active at the highest concentrations, confirming the results of the screening (Figure 6B). These hits were further characterized in PD-L1-conjugated ELISA. In this system, sodium butyrate induced a 2-fold increase in RUSH antibody release after biotin addition, as expected from its effect on protein expression (Jiang and Sharfstein, Biotechnol Bioeng. (2008) 100:189-94; Mimura et al., J Imm Methods (2001) 247:205-16). Of the drugs tested, only lycorine (tested at both 1 μM and 50 μM) appeared to be active, but its effect was milder than that of sodium butyrate, while all the other compounds showed no antibody secretion-stimulating activity (Figure 6B).

[0077] Similarly, potential secretion inhibitors identified during the screening campaign were also tested in specific assays. Both nigericin and tilphostin AG-879 (used at 50 μg / mL) were found to be as potent as brefelzin A in suppressing antibody secretion without affecting cell viability (Figure 7B) (Figure 7A). Cell imaging further confirmed the secretion-activating effect of lycorine as well as the inhibitory effects of nigericin and tilphostin AG-879 (Figure 5C).

[0078] C.Overview This study provides evidence that a small library of chemical compounds was successfully screened using an antibody-compatible RUSH system with a high-throughput fluorescence imaging system for identifying conventional antibody secretion regulators. Here, we report one novel activator and two novel inhibitors of antibody secretion validated by ELISA antibody titration.

[0079] The RUSH system configuration requires the addition of SBP to the antibody for reversible ER targeting and a fluorescent protein (e.g., GFP) for automated imaging microscopy monitoring of antibody secretion. This presents a challenge for the antibody because immunoglobulins and GFP do not typically originate from the same cellular compartment, and the addition of exogenous sequences to antibodies has been shown to interfere with their secretion and functionality (Haas et al., Methods Mol Biol. (2012) 901:265-76; Luria et al., MAbs. (2012) 4:373-84). The mechanism of IgG heavy and light chain assembly before mature antibody secretion presented another challenge. In fact, the antibody heavy chain requires assembly with the light chain for secretion, but the free light chain can be secreted. Therefore, to ensure reliable monitoring of the secretion of a well-assembled antibody, we decided to retain the RUSH antibody via its heavy chain. Considering all constraints, the RUSH mechanism was added to the C-terminus of the anti-PD-L1 heavy chain. A furin cleavage site was also added between the heavy chain and the RUSH construct to remove the SBP and GFP sequences after passing through the Golgi network (otherwise, they could affect antibody function). The data show that the uncleaved full-length antibody retained its specificity and was detectable via its GFP site.

[0080] Of the two unique compounds that caused retention of RUSH antibodies in the cytoplasm, tilphostin AG-879 was identified as a tyrosine kinase inhibitor (IC). 50Characterized as (=10 μM), it was confirmed to be an inhibitor of antibody secretion when used at a concentration of 50 μg / mL. Nigericin was also confirmed to be a secretion inhibitor when used at a concentration of 50 μg / mL.

[0081] The screening campaign also led to the identification of candidate molecules that increased antibody secretion in the biotin-stimulated RUSH system (because they reduced intracellular GFP fluorescence signaling), of which eight compounds (anisomycin, cycloheximide, digoxigenin, lycorine, methenamine, ropinirole, pinosembrin, and pyritildione) were selected for further characterization by ELISA. This assay was used to measure the binding of RUSH antibodies to their immobilized PD-L1 target and to measure antibody titers in cells treated with the candidate molecules. Unexpectedly, of these eight compounds that consistently reduced intracellular GFP fluorescence, which was interpreted as accelerated antibody release, only lycorine was shown to increase RUSH antibody titers in the treated cell culture supernatant. In contrast, the fact that lycorine was validated in both assays and in several independent experiments strongly suggests its stimulating effect on antibody secretion in the system.

[0082] Lycorine likely has multiple effects on cell biology involving several cellular targets, as well as proposed utility for a wide range of indications, including cancer (Roy et al., Biomedicine & Pharmacotherapy (2018) 107:615-24), fungal infections (Zhao et al., Biotechnology Letters (2021) 43:1503-12), and Alzheimer's disease (Kola et al., Int J Mol Sci. (2023) 24(3):2500); however, its effects on secretion have not been published to our knowledge.

Claims

1. A method for inhibiting protein secretion from eukaryotic cells, comprising contacting the cells with nigericin or tilphostin AG-879, or an analog thereof.

2. The method according to claim 1, wherein the cells are in a cell culture.

3. The method according to claim 2, wherein the contact includes adding nigericin, tilphostin AG-879, or an analog to the cell culture.

4. The method according to claim 3, wherein the nigericin, tilphostin AG0879, or analog is added to the cell culture to reach a concentration of approximately 1 μg / mL to approximately 100 μg / mL, optionally, approximately 5 μg / mL to approximately 50 μg / mL.

5. The method according to claim 3, wherein the nigericin, tilphostin AG0879, or analog is added to the cell culture to reach a concentration of at least about 50 μg / mL; optionally, the nigericin, tilphostin AG0879, or analog is added to the cell culture to reach a concentration of about 50 μg / mL.

6. A method for increasing protein secretion from eukaryotic cells, comprising contacting the cells with lycorine or an analogue thereof.

7. The method according to claim 5, wherein the eukaryotic cells are recombinant cells that have been modified to express recombinant proteins.

8. The method according to claim 6, wherein the recombinant protein is an enzyme or antibody for enzyme replacement therapy.

9. The method according to any one of claims 5 to 7, wherein the cells are in a cell culture.

10. The method according to claim 8, wherein the contact includes adding lycorine or an analog to the cell culture.

11. The method according to claim 9, wherein the lycorine or analog is added to the cell culture to reach a concentration of approximately 0.1 μM to approximately 100 μM, optionally, approximately 1 μM to approximately 50 μM.

12. The method according to claim 9, wherein the lycorine or analog is added to the cell culture to reach a concentration of at least about 1 μM.

13. The method according to claim 9, wherein the lycorine or analog is added to the cell culture to reach a concentration of about 1 μM or about 50 μM.

14. The method according to any one of claims 1 to 13, wherein the eukaryotic cell is a mammalian cell, an insect cell, or a yeast cell.

15. The method according to claim 11, wherein the cells are mammalian cells, optionally CHO cells, NS0 cells, BHK cells, 293 cells, HeLa cells, HEK cells, or Sp2 / 0 cells.

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