CN116964199A

CN116964199A - Methods of identifying peptide therapies for treating various conditions

Info

Publication number: CN116964199A
Application number: CN202280018190.2A
Authority: CN
Inventors: 斯图尔特·考夫曼; 黄穗
Original assignee: Haopu Patent Co ltd
Current assignee: Haopu Patent Co ltd
Priority date: 2021-01-19
Filing date: 2022-01-18
Publication date: 2023-10-27

Abstract

The present disclosure provides methods of identifying peptide therapies for treating various conditions. In some embodiments, methods of identifying peptide therapies for treating a viral infection condition are described. In some embodiments, methods of identifying peptide therapies for treating cancer are described. Some embodiments relate to peptides identified by the methods described herein.

Description

Methods of identifying peptide therapies for treating various conditions

Cross Reference to Related Applications

The present application claims the benefit of U.S. provisional application No. 63/139,258 filed on day 19 at 1 in 2021 and U.S. provisional application No. 63/158,792 filed on day 9 at 3 in 2021, both of which are incorporated herein by reference in their entirety.

Background

Technical Field

The present technology relates generally to identifying peptide therapies.

Description of related Art

Covd-19 epidemic is global. Despite unprecedented efforts at multiple fronts of drug development, only one antiviral agent, the small molecule drug, adefovir, currently shows some (limited) clinical efficacy. In general, current strategies, including seeking rational design and/or large-scale screening of drugs with such activity based on molecular structure blocking key viral enzymes, or attempting to modulate pathogenic mechanisms in hosts using existing drugs with known molecular targets (drug reuse), have largely failed to identify compounds with meaningful beneficial effects in clinical control of covd-19.

One of the main reasons for the limited biological activity of small molecule drugs is that these drugs inherently have little molecular influence on the activity of any protein. Small molecule compounds work best when they are complexed into a folding pocket of a protein, typically the active site of an enzyme. In contrast, they are almost incapable of disrupting protein-protein interactions (PPI), which in disease mechanisms include key-lock perfect-mating molecular interactions that play a ubiquitous key role in microbial-host interactions by which infectious agents exert their virulence.

Most peptide drugs are rationally designed based on structural considerations to block the activity of specific proteins, or even PPIs, in order to disrupt the protein complex. Unlike small molecule drugs, peptides are screened primarily in vitro to identify peptides that bind to specific proteins (complexes), including peptides that bind to microbial antigens. Apart from binding assays and structure-directed designs, peptide libraries have not been routinely screened for function (phenotype), i.e., in bioassays that detect complex biological activities, such as inhibiting viral replication in cell culture or protecting host cells. In contrast, natural peptides have been found in the animal kingdom to have antiviral or antimicrobial activity, such as Mucroprin-M1 isolated from scorpions, or θ -defensin-1 found in macaques. See Mahendran a.karnan, et al The Potential of Antiviral Peptides as COVID-19Therapeutics,Frontiers in Pharmacology,2020, volume 11, page 1475; christine l.wohlford-Lenane, et al, rhesus Theta-Defensin Prevents Death in a Mouse Model of Severe Acute Respiratory Syndrome Coronavirus Pulmonary Disease, journal of Virology,2009,83 (21) 11385-11390;

Historically, phage (bacteria-infected viruses) have been used to identify peptides that have the ability to bind to specific target proteins in solution or on the cell surface, as phage display libraries that encode and express random peptides on their surface. Phage do not grow in mammalian cell culture and therefore phage display libraries are not suitable for phenotypic screening in mammalian cells.

Thus, new methods are needed to identify peptide-based therapies.

Disclosure of Invention

Some embodiments relate to methods of identifying bioactive peptides that confer a desired phenotype on an assay cell. The method comprises the following steps:

(a) Generating a DNA library of DNA sequences encoding a peptide library,

(b) Introducing the DNA sequence into an assay cell to express a peptide library,

(c) Optionally applying exogenous selection pressure to the assay cells,

(d) Selecting an assay cell exhibiting a desired phenotype, and

(e) Peptides are identified from a pool of peptides conferring a desired phenotype on a selected assay cell by sequencing DNA isolated from the assay cell.

a. generating a library of DNA sequences encoding a peptide library of 5-20 amino acids in length,

b. The library of DNA sequences is placed into a plasmid library,

c. construction of viral vectors for transfection or transduction of assay cells using plasmid libraries while minimizing loss of diversity,

d. transduction or transfection of viral vectors into assay cells capable of expressing the desired effect to be conferred by the peptides of the peptide pool,

e. selecting those assay cells that exhibit the desired phenotype via natural selection of cells or via physical sorting of cells, and

f. peptides conferring a desired phenotype to the cells of interest are identified by targeted sequencing of DNA isolated from the cells of interest.

b. the library of DNA sequences is placed into a plasmid library,

d. the viral vector is transduced or transfected into a plurality of cells,

e. combining one or more of the plurality of cells with one or more other cells not transduced or transfected with the viral vector alone to form a plurality of discrete micro-cultures, organoids, artificial organs or microdroplet cultures,

f. Selecting those micro-cultures, organoids, artificial organs or microdroplet cultures which exhibit one or more desired phenotypes, and

g. peptides conferring a desired phenotype are identified by targeted sequencing of DNA isolated from selected microcultures, organoids, artificial organs or microdroplet cultures.

Some embodiments relate to peptides identified by the methods described herein. Some embodiments relate to peptides comprising a peptide sequence having 90% identity to a peptide identified by the methods described herein. Some embodiments relate to compositions comprising a combination of 2 or more peptides identified by the methods described herein.

These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and appended claims.

Drawings

Fig. 1 is a flow chart illustrating a general screening scheme.

FIG. 2 shows an embodiment of a method for identifying bioactive peptides that confer a desired phenotype on an assay cell against SARS-CoV-2. A in FIG. 2 shows the synthesis of code 10 ⁹ DNA sequences of a strip sequence or more random peptide libraries. B in fig. 2 shows that a plasmid for expressing random peptide was constructed. In FIG. 2 c shows the generation of viral vectors. D in FIG. 2 shows transduction of target cells with viral vectors. The exposure of target cells to SARS-CoV-2 virus is shown by e in FIG. 2. F in fig. 2 shows that cells with antiviral peptides survive viral infection and DNA sequences encoding such antiviral peptides are targeted sequenced; antiviral peptides can then be synthesized for drug lead development.

Detailed Description

Described herein are screening methods for identifying peptide therapies for treating various conditions. The method utilizes a phenotypic screening protocol whereby the test peptide is expressed in the same cells that exhibit the desired phenotype. Phenotypic screening protocols take advantage of the genetic encoding and biosynthesis of peptides by the very same cells that exhibit the desired phenotype that the new drug should elicit. As one non-limiting example, the desired phenotype may be a cell (e.g., airway epithelial cell) that inhibits replication of a pathogenic virus (e.g., SARS-CoV-2). In various embodiments, libraries of random peptides generated in new protocols that substantially increase diversity compared to the results achieved by current methods are screened. Genetic sequences encoding random peptides are introduced into target cells expressing them via expression vectors. Depending on the target disease state, the cells may then be exposed to a stimulus that causes the disease (e.g., a pathogenic virus). Cells exhibiting a desired phenotype (e.g., survival) can then be analyzed to determine the genetic sequence of the random peptide that elicited the phenotype. Further screening can be performed to verify these peptide hits, which can then be advanced to further studies for direct therapeutic applications, or as a lead to further optimization and identification of therapies.

Fig. 1 is a flow chart illustrating a general screening scheme. At step 100, a library of genetic sequences encoding random peptide sequences is generated. In step 102, a genetic sequence is introduced into a target cell. Each sequence of the random peptide is then expressed in a single or small branch of the target cell. At step 104, the cells are optionally exposed to exogenous selection pressure, such as a stimulus that causes a disease. Optionally, step 104 is not required if the cells are already in the desired screening state (e.g., if they are cancer cells). The procedure can also be used to enrich cells that make up secreted forms of random peptides that have the ability to affect other cells in the same micro-culture in a desired manner. At step 106, the cells exhibiting the desired phenotype are then genetically sequenced to determine which random peptides elicit the desired response. These hit peptides can then be directly developed into therapeutic agents or used as a lead for further therapeutic development at step 108. These steps will be described in more detail below.

Generation of DNA libraries encoding random peptide sequences

In some embodiments, a DNA library of DNA sequences encoding a peptide library is generated. In some embodiments, the DNA sequences in the DNA library are randomly generated. Non-limiting methods of generating random DNA libraries are described in U.S. patent No. 5,723,323, which is incorporated herein by reference in its entirety. The DNA sequence may be generated by known oligomerization techniques. The DNA sequence may then be integrated into a plasmid containing a protein expression cassette containing elements for driving expression of the DNA sequence. Elements for driving expression may include, for example, enhancers and promoters regulated by the target cell, transcribed but untranslated regions (UTRs) flanking the coding region, splice sites, poly (A) signals, and the like.

In some embodiments, the DNA sequence encodes a peptide that is 5-20 amino acids in length. In some embodiments, the peptide is 12-15 amino acids in length. In other embodiments, the peptide is 6-9 amino acids in length.

In some embodiments, randomness of a DNA sequence is constrained by probabilistic bias or deterministic modification in the composition of amino acid sequences encoded by the DNA sequence. In some embodiments, the DNA sequence is designed to encode a peptide that meets the desired physicochemical properties. For example, in some embodiments, the algorithm described in Smialowski, P., dose, G., tokker, P., kaufmann, S. & Frishman, D.PROSO II-a new method for protein solubility prediction.FEBS J279, 2192-2200 (2012), which is incorporated herein by reference in its entirety, may be used to design sequences encoding peptides that may have solubility. In some embodiments, the DNA sequence is designed as a peptide encoding CDRs (complementarity determining regions) of a mimetic antibody. For example, the techniques described in Sachdeva, s., joo, h., tsai, j., jami, B. & Li, x.arc Approach for Creating Peptides Mimicking Antibody binding.scientific reports 9,997 (2019) may be utilized, which are incorporated herein by reference in their entirety. In some embodiments, the DNA sequence is designed to encode a peptide that considers the genetic code and codon usage, for example, by reference to a codon usage table. In some embodiments, the DNA sequence is designed to encode a peptide containing a receptor binding motif.

In some embodiments, the DNA sequence is designed to encode a peptide having certain properties such as stability, cell permeability, or cell secretion.

In some embodiments, the randomly generated DNA sequence is fused to other DNA sequences encoding predetermined peptide sequences that confer a specific function. Specific functions may include, but are not limited to, cell Penetrating Peptides (CPPs), flanking sequences from introns for cyclization, and peptides allowing secretion of random peptides (i.e., signaling peptides). See Peng C, shi C, cao X, li Y, liu F, lu F.factors Influencing Recombinant Protein Secretion Efficiency in Gram-Positive Bacteria: signal Peptide and beyond Bioeng Biotechnol.2019;7:139, which is incorporated by reference herein in its entirety.

Cell Penetrating Peptides (CPPs) are short peptides that facilitate cellular uptake and absorption of molecules ranging from nanoscale particles to small compounds to large fragments of DNA. The "cargo" is bound to the peptide by chemical linking via covalent bonds (i.e., biosynthesized into a fusion peptide) or by non-covalent interactions. CPPs that may be used with the random peptides described herein include, but are not limited to, those described in Agrawal, P.et al. CPPSite 2.0:a repository of experimentally validated cell-perpendicular peptides, nucleic acids research 44, D1098-1103 (2016), which is incorporated herein by reference in its entirety. In some embodiments, cyclic CPP sequences are used, such as those described in Park, S.E., sajid, M.I., park, K. & Tiwari, R.K. cyclic Cell-Penetrating Peptides as Efficient Intracellular Drug Delivery tools.mol Pharm 16,3727-3743 (2019), which is incorporated herein by reference in its entirety.

In some embodiments, the Cell Penetrating Peptide (CPP) is an HIV TAT protein or a protein encoded by an antennapedia (Antennaepedia) gene, such as D Derossi, S Calvet, ATrembleau, ABrunissen, G Chassaing, AProchiantz, cell internalization of the third helix of the Antennapedia homeodomain is receptor-index, J Biol chem.1996Jul 26;271 (30) 18188-93, which is incorporated herein by reference in its entirety.

In some embodiments, flanking sequences from introns are incorporated to induce cyclization of random peptides, such as those described in Delivoria, D.C. et al, bacterial production and direct functional screening of expanded molecular libraries for discovering inhibitors of protein aggregation.Sci Adv 5, eaax5108 (2019), which is incorporated herein by reference in its entirety.

Additional sequences that may be added to or designed into random sequences to achieve desired properties are described in Lee, a.c., harris, j.l., khanna, K.K, & Hong, j.h.a Comprehensive Review on Current Advances in Peptide Drug Development and design.int J Mol Sci 20 (2019), which is incorporated herein by reference in its entirety.

In some cases, the diversity of the random sequence library is statistically reduced due to the bias distribution caused by the amplification step. In some embodiments, this decrease in diversity is offset by utilizing their faster hybridization kinetics. In some embodiments, approaching the theoretical maximum of sequence diversity is achieved by a process comprising the steps of:

(a) DNA containing the random sequence is denatured and re-hybridized,

(b) The re-annealing is monitored by spectroscopy, e.g. UV 260nm absorption,

(c) Selectively digesting some of the random sequences with a predetermined degree of re-annealing using a thermostable double-strand specific nuclease, an

(d) The nuclease is inactivated by heating and continuing to re-anneal the less abundant species.

Additional details regarding this procedure are described in Peterson, d.g., wessler, S.R, & Peterson, a.h. efficiency capture of unique sequences from eukaryotic genome, trends Genet 18,547-550 (2002), which is incorporated herein by reference in its entirety.

In some embodiments, loss of sequence diversity is reduced by performing in vitro recombination and mutagenesis of the DNA sequences generated as described above.

Introduction of DNA sequences into assay cells

In some embodiments, introducing the DNA sequence into the assay cell comprises a method selected from the group consisting of transformation, transduction, and transfection.

In some embodiments, one or more DNA sequences are placed into one or more expression cassettes in a plasmid prior to introduction into an assay cell. In some embodiments, the plasmid is an episomal plasmid. In some embodiments, more than one randomly generated DNA sequence may be placed in the same expression cassette in order to detect synergistic peptide combinations.

In some embodiments, the plasmid is introduced into a viral vector prior to introducing the DNA sequence into the assay cell. The viral vector may be a lentiviral adenovirus or a baculovirus vector. In some embodiments, the viral vector is labeled with fluorescence, so selection of virus-infected cells can be performed by FACS.

In some embodiments, the DNA sequence is integrated into the genome of the cellIs a kind of medium. In some embodiments, the integration is by nuclease-mediated site-specific integration, transposon-mediated gene delivery, or virus-mediated gene delivery. In some embodiments, nuclease-mediated site-specific integration is by CRISPR/Cas9 RNP. In some embodiments, virus-mediated gene delivery uses lentiviruses. In some embodiments, the method is performed by transfecting HEK 293T cells with the resulting plasmid (e.g., using Lenti-X ^TM 293T lentivirus production platform) to generate lentiviruses.

In some embodiments utilizing viral vectors, loss of sequence diversity is reduced by iteratively transfecting virus-producing cells with different samples from a plasmid library.

In embodiments where the target assay cell is a bacterial cell, the DNA sequence may be introduced into the bacterial cell using known phage technology. For example, in some embodiments, λgt11 phage can be used, such as i.m. chiu and k.lehtoma, direct cloning of cDNAinserts from lambda gt11 phage DNA into a plasmid vector by a novel and simple method, genet Anal Tech Appl, month 2 1990; 7 (1) 18-23, which are incorporated herein by reference in their entirety.

Determination of cell selection

In various embodiments, the target assay cell may be an animal or bacterial cell. In some embodiments, the target assay cell is a mammalian cell. In some embodiments, the target assay cell is a human cell. In some embodiments, the target assay cell is selected from the group consisting of a human airway cell, a human cancer cell, wherein a phenotypic change, such as differentiation of a cancer stem cell into a post-mitotic cell, can be monitored.

In some embodiments, the assay cell is a human airway cell and the exogenous selection pressure is a viral infection. In some embodiments, the virus is SARS-Cov2. In some embodiments, the desired phenotype of the cell performance is survival after SARS-Cov2 infection. In some embodiments, the virus may be labeled with fluorescence, so that the frequency or fraction of infection of infected cells may be measured. In some embodiments, FACS can be used to select for infected cells. In some embodiments, surviving assay cells are naturally enriched in the population of cells to provide self-selection of cells that exhibit the desired phenotype and contain the hit peptide.

In some embodiments, the assay cell is a cancer cell and the desired phenotype the assay cell has is cell death or switching of a specific marker gene indicative of conversion to a differentiated cell, as originally proposed by Stuart Kauffman in the article S.Kauffman.S. differentiation of malignant to benign cells J.Theor.biol.31,429-451 (1971).

In some embodiments, the assay cell is a microbial cell and the desired phenotype of the assay cell is cell death. For example, the microbial cells may be bacterial cells or fungal cells.

In various embodiments, determining a desired phenotype elicited by a peptide in a cell includes, but is not limited to:

a. protecting cells from viral infection;

b. microbial death or growth inhibition;

c. inducing a state transition in mammalian cells comprising

i. Enter an undesired state of apoptosis pathway cell death (cancer, inflammation, etc.),

differentiation from a cancer stem cell-like state to a post-mitotic state,

differentiation from one cell type into one or more other cell types; and

d. the cell morphology was altered in the indicated manner.

In some embodiments, the desired phenotype is altering the cell population structure in a desired manner. For example, the proportion of cellular phenotype in a particular cell population may be varied to different desired proportions.

As described above, in some embodiments, selection of an assay cell that exhibits a desired phenotype occurs naturally via enrichment of cells that exhibit the desired phenotype (e.g., cell survival). Thus, in these embodiments, no further cell selection is required. For example, in the case of infection with SARS-CoV-2 or other viruses, the virus replicates and kills the cells and propagates in the cell population, except in a minority of cells carrying the transgene expressing the peptide form that confers antiviral activity. These cells will resist the virus, remain viable and continue to proliferate. Their fraction increasingly dominates the surviving cell population. Their corresponding gene sequences for antiviral peptides ("hits") are also enriched.

In other embodiments, the method of selecting an assayed cell that exhibits a desired phenotype includes FACS, biopanning, magnetic bead cell separation, or other cell sorting techniques. For example, in embodiments that utilize viral infection (e.g., SARS-CoV-2), the virus can be fluorescently labeled. In other embodiments, lentiviral vectors may be labeled with fluorescence. In some embodiments, the cells are assayed with fluorescent reporter protein markers for monitoring cell viability and viral infection. In some embodiments, these techniques may be used in combination with each other and with the natural cell selection described above.

In some embodiments, the above assay is modified to identify peptides that cause the cells to induce some changes in other cells. Thus, in some embodiments, the desired property is exhibited by cells other than those expressing the random peptide. In some embodiments, the methods comprise the steps of:

b. the library of DNA sequences is placed into a plasmid library,

d. The viral vector is transduced or transfected into a plurality of cells,

e. combining one or more of the plurality of cells with one or more other cells not transduced or transfected with the viral vector alone to form a plurality of discrete micro-cultures, organoids, artificial organs, or microdroplet cultures,

In these embodiments, the random peptide is secreted by fusion with the signal peptide, and the selection unit is not a single cell carrying the gene of the random peptide, but rather the whole cell population in a micro-culture (e.g., a well in a multi-well cell culture plate, a cell culture droplet), an organoid, or an artificial organ. In this context, only a small fraction of the cells in the population contain genes expressing random peptides, and their ability to rescue the entire population is a target phenotype for screening.

In some embodiments, once the hit peptides are identified in the micro-culture, organoid, artificial organ, or microdroplet culture, any combined subset of hit peptides can be screened for the desired property.

Identification of hit peptides

Once cells exhibiting the desired phenotype are isolated or enriched, they can be analyzed to identify sequences of hit peptides that induce the desired phenotype. In some embodiments, DNA sequencing is used to identify sequences.

In some embodiments, the method of DNA sequencing is a next generation gene sequencing technique, including but not limited to targeted sequencing (e.g., using flanking sequences introduced into a randomly generated sequence as discussed above). Discovery of target ("hit") peptides occurs in a large population of human cells using next generation sequencing; this eliminates the need for high throughput parallel minicell culture assays, as is the case for small molecule drug screening.

In some cases, random libraries generated as described above may encounter "invisible species" problems. In particular, any particular sample of a library used in the assay procedures described herein may not include many sequences within the generated sequence space. Aspects of this problem are described in Raghunathan, a., valiant, G. & Zou, j. Timing the unseen from multiple displacements, proceedings of the 34th International Conference on Machine Learning,Sydney,Australia 70, (2017), which is incorporated herein by reference in its entirety. To address this problem, in some embodiments, the assays are performed iteratively or in a massively parallel manner. By using multiple runs of assays from different samples of the randomly generated library, more sequence space will be screened. Thus, for example, in addition to the above removal of the most abundant sequences by self-hybridization kinetics to prevent loss of diversity, plasmid libraries generated as described above can be sampled and analyzed by algorithms to cover more sequence space. Sampling optimisation is limited by the maximum number of clones that can be handled given by the number of cells producing the viral vector and can be determined by simulation and verified empirically by sequencing alone under the guidance of the coret rule, as described in Fisher, r.a., coret, A.S & Williams, c.b. the relation between the number of species and the number of individuals in a random sample of an Animal pore plan j Animal chemistry 12,42-58 (1943), which is incorporated herein by reference in its entirety.

Pilot optimization of hit peptides

Once the hit peptide is identified using the procedure described above, it can be used as a starting point for identifying other peptides with increased potency to elicit the desired phenotype. In some embodiments, multiple rounds of screening are performed to drive evolutionary selection of more effective peptides. In some embodiments, genetic recombination of two or more identified peptides is used to generate a recombinant novel random peptide library, which is then screened for efficacy. In some embodiments, where the identified peptide is a secretory peptide, two or more of the identified peptides are combined to screen for efficacy. In some embodiments, the hit peptides used for recombination are selected as a set of hits exhibiting sequence similarity, which may indicate that the set of hits bind to the same cellular target. In some embodiments, the hit peptide for recombination is selected to have greater than 80%, 85%, 90%, 95%, 98%, or 99% homology. In various embodiments, 2, 3, 4, 5, 6, 7 or more hit peptides are selected for recombination.

In another embodiment, different hit peptides are selected for recombination, indicating that they can bind to different cellular targets. Thus, for example, hit peptides having less than 50%, 70%, 80%, 85%, 90%, 95%, 98% or 99% homology are selected and then recombined to generate a new library of peptides for screening. In various embodiments, 2, 3, 4, 5, 6, 7 or more hit peptides are selected for recombination.

In other embodiments, point mutations are introduced into hit peptides to generate a set of similar peptides. For example, mutations may be introduced at distances distant from 1, 2, or 3 hamming in sequence space to generate "quasi-species" populations. This new population of sequences can then be screened using the techniques described herein. In addition to and after the recombination techniques described above, available point mutations are utilized.

In some embodiments, the mutation methods described above may be performed iteratively to identify sequences with increasingly higher potency. The iteration may be repeated until a sufficient level of effectiveness is reached or no improvement in effectiveness is achieved.

Other known techniques may be used to further develop hit peptides into therapeutic agents. In some embodiments, the hit peptide is chemically modified to alter the physicochemical and pharmacokinetic properties of the peptide. For example, in embodiments as discussed above in which the CPP sequence has not been incorporated into a random sequence, the CPP sequence may be added to facilitate cell entry upon therapeutic use. In other embodiments, the peptide sequence is engineered to achieve a desired property, such as a desired physicochemical property or cyclization of the peptide. In some embodiments, one or more amino acids in the sequence are changed to D-amino acids to increase in vivo stability.

Other embodiments include incorporating the identified therapeutic peptides into formulation packages for delivery, such as into liposomes or other lipid nanoparticles.

Some embodiments relate to peptides identified by any of the methods described herein. Some embodiments relate to peptides comprising a combination of 2 or more peptides identified by the methods described herein. Some embodiments relate to peptides comprising a peptide sequence having 90% identity to a peptide described herein.

In some embodiments, hit peptides are further screened. In some embodiments, the hit peptide is synthetically prepared. The synthetic hit peptides can then be introduced into cells to test whether they can render the cells into the desired phenotype.

Selection of hit peptides effective against viral mutations

As discussed above, some embodiments include identifying peptides that are effective as antiviral drugs. In some embodiments, the collection of hit peptides identified as discussed above that exhibit antiviral properties is further screened to identify a subset of hits or a combination of hits that are most effective against the mutant virus. Such a subset or combination of hits may then be used to combat a particular circulating virus, with the expectation that the subset or combination will continue to be effective against the mutant strain of the virus. Such therapies are also expected to inhibit the accumulation of viral mutations in the host treated with the therapy.

In some embodiments, the additional screening comprises infecting all or a portion of the cells within a human cell culture with a lentivirus expressing a randomly selected single or combination hit peptide identified as described above. In some embodiments, one or more lentiviruses expressing 2, 3, 4, 5, 6, 7 or more hit peptides are used. In some embodiments, the cell fraction is produced by infecting a first set of cells, and then adding uninfected cells. After a sufficient growth period (e.g., 6 hours), the cells are infected with a target virus (e.g., SARS-CoV 2). In some embodiments, cells and/or target viruses may be labeled to allow for monitoring of infection. For example, cells may be labeled with green fluorescent protein, and viruses may be fluorescently labeled. The ratio of cell signal to viral signal allows testing of hit peptides for their ability to protect infected cells. Accumulation of mutations in the virus can be monitored by sequencing the virus at various (e.g., predetermined) time points (e.g., using targeted sequencing or deep sequencing).

In one variant, portions of the initial cell culture are transferred to different culture vessels, and the process may be repeated with successive transfers of cell portions. In some embodiments, the number of cells and the rate of transfer may vary between successive transfers. Viruses within each culture vessel or subset of culture vessels may be sequenced (e.g., using targeted sequencing or deep sequencing) to determine the presence of viral mutations.

Sequencing can be used to obtain the number of viral variants, which are still transmitting, and which are extinct, the copy number of each variant, and the cumulative mutation number of each variant. In some embodiments, these variables may be used to determine when to stop the experiment.

The experiment can be repeated with different randomly selected hit peptides or combinations of hit peptides. Hit peptides or combinations of peptides that reduce or slow the incidence of viral evolution can then be selected for development and use as therapeutic agents. This process can also be used to identify possible mutations in the virus. These mutant strains can then be used in the initial hit peptide identification process described above.

In some embodiments, the results of the above experiments are used to generate a phylogenetic map. The effectiveness of any hit peptide, peptide combination, or hit peptide concentration can be assessed based on phylogenetic map studies. Slowing or stopping of viral evolution can be indicated by a decrease in the branching structure of the phylogenetic map. As the effectiveness of slowing evolution increases, fewer branches are found at comparable time points after infection.

In some embodiments, the results of the above experiments are used to determine the copy number of each viral variant, as these variants occur, persist and may be extinct during the treatment.

In some embodiments, a hit peptide or combination of peptides is selected that is effective: 1) preventing the appearance of any new strain, 2) causing any new strain to extinct within a predetermined incubation time and total number of cells in the system, or 3) not allowing any other new strain to appear after a predetermined point in time, and not allowing the initial virus to continue to spread in the population after that point in time.

In some embodiments, combinations of hit peptides with desired anti-mutation properties can be determined by testing all combinations and sub-combinations of a set of hit peptides using the above-described experiments. The best performance combination of hit peptides can then be used for therapeutic development. In another embodiment, combinations of hit peptides may be identified by starting from a first trial set of hit peptides that are a subset of the entire peptide set. The first subsequent collection may include adding or subtracting a hit peptide from the first test collection. All such first subsequent sets are tested and the best one is used as the new starting set from which the second subsequent set can be tested, including all combined variants that add or subtract one from the new starting set. This "hill climbing" process may continue until the desired level of efficacy is achieved. It will be appreciated that other hill climbing optimization algorithms may be used to identify the appropriate final hit peptide set in an efficient manner.

Identification of cellular targets

Some embodiments relate to methods of identifying cellular targets of peptides identified herein. Thus, such identification may provide a new pharmaceutically acceptable target for therapeutic intervention. Once the target is known, various methods can be utilized to identify therapies that hit the target, including traditional screening or pharmaceutical chemistry methods. Any known suitable method for identifying a peptide-hitting cellular target may be used, including affinity-based methods. In some embodiments, protein-protein interaction partners and cellular pathways are identified that are involved in the mechanism of action of the peptide.

To further illustrate the invention, the following examples are included. Of course, these examples should not be construed as limiting the invention in any way. Variations of these embodiments that are within the scope of the claims are within the purview of one skilled in the art and are considered to fall within the scope of the invention described and claimed herein. The reader will recognize that those skilled in the art and familiar with this disclosure are able to make and use the invention without an exhaustive example.

Example 1

An experiment was performed to identify peptides that confer resistance to SARS-CoV-2 infection. The experiment is shown in figure 2. In a in fig. 2, code 10 is synthesized ⁹ DNA sequences of a strip sequence or more random peptide libraries. In fig. 2 b, a plasmid containing a protein expression cassette was constructed. The plasmid comprises one or more expression cassettes comprising a DNA sequence encoding a peptide comprising a CPP sequence and regulatory sites for expression in target cells, as well as sequence elements (flanking LTR sequences, packaging signals) for the production of the viral vector. In fig. 2 c, 293T cells are used to generate lentiviral vectors. In d in FIG. 2, the target cells are transduced with a viral vector. Human lung epithelial cells sensitive to SARS-Cov2 virus are transduced with lentiviral vectors containing a gene expression cassette encoding a random peptide such that each cell in the cell culture (population of hundreds of millions of cells) expresses a different random peptide form. Transduced human lung epithelial cells were grown to confluence. In e in FIG. 2, target cells are exposed to SARS-Cov2 virus. Viruses replicate and kill cells and propagate in cell populations other than a few that carry DNA sequences that express peptides that confer antiviral activity. These cells will resist the virus, remain viable and continue to proliferate. Their proportion is increasingly dominant in surviving cell populations. Their respective gene sequences for antiviral peptides were also enriched and identified by sequencing the entire surviving cell culture simultaneously for the next generation DNA (nextgen DNA). In f of fig. 2, cells with antiviral peptides survive viral infection and the DNA sequence encoding such antiviral peptides is targeted sequenced; antiviral peptides can then be synthesized for drug lead development.

Example 2

This example shows one application of selecting hit peptides that are effective against or block mutations in SARS-CoV-2 virus.

Hit peptides having antiviral activity against SARS-CoV-2 virus were identified according to the procedure in example 1.

The DNA sequences encoding these antiviral peptides are inserted into lentiviral vectors. Lentiviruses were generated as described previously. Y.kong, et al, int.j.clin.exp.pathol, 2017;10 (6):6198-6209. Briefly, lentiviruses are generated by co-transfecting 293T cells with a 4-plasmid system comprisingLentiviral expression plasmids for antiviral peptides and methods of use in Lipofectamine ^TM 2000. The resulting culture supernatants containing lentiviral particles were collected 48h and 72h after transfection, and the combined supernatants were centrifuged via 50,000g ultracentrifugation for 2h (Optima L-90K, beckman, USA). The infection titer of the formulations was determined by detecting the percentage of GFP positive 293T cells after transfection with serial dilutions of concentrated lentivirus using an inverted fluorescence microscope (Ti-s, nikon, JPN).

Human lung cells were transfected with the resulting lentiviruses. After allowing the transfected cells to express the antiviral peptide for a period of time, the transfected cells were mixed with other human lung cells not transfected in the culture flask. The mixed cells were then infected with SARS-Cov2 virus.

The infected cells were then separated into different flasks and the process was repeated continuously. After several passages of the cells, targeted sequencing was performed to determine evolution and propagation of the SARS-Cov2 virus. Based on these results, one or more antiviral peptides effective against or blocking the viral mutation are selected.

In the detailed description and in the following claims and in the drawings, reference is made to specific features of the invention, including method steps. It should be understood that the disclosure of the present invention in this specification includes all possible combinations of these particular features. For example, where a particular feature is disclosed in the context of a particular aspect or embodiment of the invention or a particular claim, that feature may also be combined with and/or used in the context of other particular aspects and embodiments of the invention, to the extent possible, and generally in the invention.

Where methods comprising two or more defined steps are mentioned herein, the defined steps may be performed in any order or simultaneously (except where the context excludes such a possibility), and the method may comprise one or more other steps performed before any one of the defined steps, between two of the defined steps, or after all of the defined steps (except where the context excludes such a possibility).

It is to be understood that the presently disclosed and claimed inventive concepts are not limited in their application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings, experiments and/or results. The disclosed and claimed inventive concepts are capable of other embodiments or of being practiced or of being carried out in various ways. Thus, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are intended to be exemplary, not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

Unless defined otherwise herein, scientific and technical terms used in connection with the inventive concepts of the present disclosure and/or claims should have meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. Generally, the terms used in connection with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization described herein, and techniques thereof, are those well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). The enzymatic reactions and purification techniques are performed according to manufacturer's instructions or as commonly done in the art or as described herein. The techniques and procedures described above are generally performed according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout the present specification. See, e.g., sambrook et al molecular Cloning, A Laboratory Manual (2 nd edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y. (1989)) and Coligan et al current Protocols in Immunology (Current Protocols, wiley lnterscience (1994)), which are incorporated herein by reference. The terms used in connection with analytical chemistry, synthetic organic chemistry, and pharmaceutical chemistry, as well as the laboratory procedures and techniques thereof, described herein are those well known and commonly used in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.

All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which the presently disclosed and/or claimed inventive concepts pertain. All patents, published patent applications, and non-patent publications cited in any section of this application are expressly incorporated herein by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.

In accordance with the present disclosure, all of the compositions and/or methods disclosed and/or claimed herein can be made and executed without undue experimentation. While the compositions and methods of this inventive concept have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the inventive concept disclosed and/or claimed. It will be apparent to those skilled in the art that all such similar substitutes and modifications are deemed to be within the spirit, scope and concept of the application as defined by the appended claims.

As utilized in accordance with the present disclosure, the following terms, unless otherwise indicated, shall be understood to have the following meanings:

the use of the terms "a" or "an" when used in conjunction with the term "comprising" in the claims and/or specification may mean "one" but it is also consistent with the meaning of "one or more", "at least one", and "one or more". The singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a compound" may refer to 1 or more, 2 or more, 3 or more, 4 or more, or a greater number of compounds. The term "plurality" means "two or more". The use of the term "or" in the claims is intended to mean "and/or" unless explicitly indicated to refer to alternatives only or that alternatives are mutually exclusive, although the disclosure supports definitions of alternatives and "and/or" only. Throughout the present application, the term "about" is used to indicate that the value includes the inherent error change of the device, the method used to determine the value, or the change present in the subject. For example, and not by way of limitation, when the term "about (about)" is utilized, the specified value may vary from the specified value by ±20% or ±10% or ±5% or ±1% or ±0.1%, as such variations are suitable for carrying out the disclosed methods and as understood by one of ordinary skill in the art. The use of the term "at least one" should be understood to include one and any amount exceeding one, including but not limited to 2, 3, 4, 5, 10, 15, 20, 30, 40, 50, 100, etc. The term "at least one" may extend to 100 or 1000 or more, depending on the term to which it is attached; in addition, an amount of 100/1000 should not be considered limiting, as higher limits may also yield satisfactory results. In addition, use of the term "at least one of X, Y and Z" will be understood to include any combination of X only, Y only, and Z only, as well as X, Y and Z. The use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) are used solely for the purpose of distinguishing between two or more items and not, for example, to imply any order or sequence or importance of one item relative to another, or any order of addition.

As used in this specification and the claims, the terms "comprises" (and any form of comprising, such as "comprises") and "comprising," "having" (and any form of having, such as "having" and "having"), "including" (and any form of comprising, such as "including" and "including") or "containing" (and any form of containing, such as "containing" and "containing"), are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

The term "cancer" means a malignancy that has undergone genetic, epigenetic or phenotypic changes with loss of differentiation, increased growth rate, invasion of surrounding tissues, and is capable of metastasis. The term "cancer" is understood to include diseases characterized by uncontrolled cell growth in a subject. In some embodiments, the terms "cancer" and "tumor" are used interchangeably. In some embodiments, the term "tumor" refers to benign or non-malignant growth.

Although the invention has been described with reference to embodiments and examples, it will be understood that many and various modifications may be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims

1. A method of identifying a bioactive peptide that confers a desired phenotype on an assay cell, the method comprising the steps of:

(f) Generating a DNA library of DNA sequences encoding a peptide library,

(g) Introducing said DNA sequence into an assay cell to express said peptide pool,

(h) Optionally applying exogenous selection pressure to the assay cells,

(i) Selecting an assay cell that exhibits the desired phenotype, and

(j) Identifying peptides from said pool of peptides conferring said desired phenotype to said assay cells by sequencing DNA isolated from said selected assay cells.

2. The method of claim 1, wherein the DNA sequences in the DNA library are randomly generated.

3. The method of claim 2, wherein the randomness of the DNA sequence is constrained by probabilistic bias or deterministic modification of the amino acid sequence composition encoded by the DNA sequence.

4. The method of claim 2 or 3, wherein the randomly generated DNA sequences are fused to other DNA sequences encoding a predetermined peptide sequence within a single fusion peptide encoded by polycistronic transcripts in the same or different expression cassettes or conferring specific functions as multiple peptides encoded by polycistronic transcripts in the same or different expression cassettes.

5. The method of claim 1, wherein the peptides in the library are 5-20aa long.

6. The method of claim 1, wherein introducing the DNA sequence into the assay cell comprises a method selected from the group consisting of transformation, transduction, and transfection.

7. The method of claim 1, wherein the one or more DNA sequences are placed into one or more expression cassettes in a plasmid prior to introduction into the assay cells.

8. The method of claim 7, wherein the diversity of sequences is increased by correcting for the off-set distribution of species frequencies caused by amplification using hybridization kinetics.

9. The method of claim 8, wherein the increase in sequence diversity is achieved by:

(a) Denaturing and re-hybridizing the DNA containing the random sequence,

(b) The re-annealing was monitored by spectroscopy,

(c) Selectively digesting some of the random sequences with a double-strand specific nuclease to a predetermined extent of re-annealing, an

(d) Inactivating the nuclease.

10. The method of claim 7, wherein the plasmid is introduced into a viral vector prior to introducing the DNA sequence into an assay cell.

11. The method of claim 10, wherein loss of sequence diversity is reduced by iterative transfection of cells producing the vector virus.

12. The method of claim 10, wherein loss of sequence diversity is reduced by performing recombination and mutagenesis of the DNA sequence in vitro.

13. The method of claim 1, wherein the DNA sequence is integrated into the genome of the cell.

14. The method of claim 13, wherein the integration is by nuclease-mediated site-specific integration, transposon-mediated gene delivery, or virus-mediated gene delivery.

15. The method of claim 14, wherein the nuclease-mediated site-specific integration is by CRISPR/Cas9 RNP.

16. The method of claim 14, wherein the virus-mediated gene delivery uses lentiviruses.

17. The method of claim 1, wherein the assay cell is selected from the group consisting of human airway cells, cancer cells, and bacterial cells.

18. The method of claim 17, wherein the assay cell is a human airway cell and the exogenous selection pressure is a viral infection.

19. The method of claim 18, wherein the virus is SARS-Cov2.

20. The method of claim 19, wherein the desired phenotype exhibited by the assay cells is survival following SARS-Cov2 infection.

21. The method of claim 1, wherein the assay cell is a cancer cell and the desired phenotype of the assay cell is cell death.

22. The method of claim 1, wherein said determining that the cell has a desired phenotype comprises transitioning from an undesired state to a desired state.

23. The method of claim 1, wherein the method of selecting an assay cell that exhibits the desired phenotype comprises natural selection or cell sorting.

24. The method of claim 1, wherein the method of sequencing the DNA is next generation targeted gene sequencing.

25. A peptide identified by the method of any one of claims 1-24.

26. A composition comprising a combination of 2 or more peptides identified by the method of any one of claims 1-24.

27. A peptide comprising a peptide sequence having 90% identity to the peptide of claim 25.

28. A method for identifying bioactive peptides from a random peptide library, said method comprising the steps of:

b. placing said library of DNA sequences into a plasmid library,

c. constructing viral vectors for transfecting or transducing assay cells with the plasmid library while minimizing loss of diversity,

d. Transduction or transfection of said viral vector into an assay cell capable of exhibiting the desired effect conferred by the peptides of said peptide pool,

f. peptides conferring the desired phenotype to the assay cells are identified by targeted sequencing of DNA isolated from the selected cells.

29. The method of claim 28, further comprising one of:

a. performing genetic recombination of two or more of the identified peptides to generate a novel pool of recombinant random peptides, and

b. point mutations were introduced into the identified peptides to generate a set of similar peptides.

30. The method of claim 1 or 28, wherein the desired phenotype conferred by the peptides of the peptide library comprises:

a. protecting cells from viral infection;

b. microbial death or growth inhibition;

c. inducing a state transition in mammalian cells; or alternatively

d. Altering the cell morphology.

31. The method of claim 1 or 28, further comprising identifying a protein-protein interaction partner or cellular pathway that is involved in the mechanism of action of the identified peptide.

32. A method for identifying bioactive peptides from a random peptide library, said method comprising the steps of:

b. placing said library of DNA sequences into a plasmid library,

d. transducing or transfecting the viral vector into a plurality of cells,

e. combining one or more of the plurality of cells alone with one or more other cells not transduced or transfected with the viral vector to form a plurality of discrete micro-cultures, organoids, artificial organs, or microdroplet cultures,

f. selecting those micro-cultures, organoids, artificial organs or microdroplet cultures that exhibit one or more desired phenotypes, and

g. peptides conferring the desired phenotype are identified by targeted sequencing of DNA isolated from selected microcultures, organoids, artificial organs or microdroplet cultures.

33. The method of claim 1 or 28, wherein the desired phenotype is antiviral effectiveness, and the method further comprises:

constructing a viral vector configured to deliver DNA expressing one or more of the identified peptides;

infecting a human cell culture or a portion of a human cell culture with a viral vector expressing one or more of the identified peptides;

Growing the human cell culture for a period of time;

infecting the human cell culture or a portion of the human cell culture with a virus for which antiviral effectiveness is desired;

DNA sequencing of a virus expected to have antiviral effectiveness is performed at a plurality of time points to determine the presence of viral mutations; and

one or more of the identified peptides is selected based on its ability to inhibit the development of viral mutations.