WO2025144700A1 - Nanobody library screening using bacterial surface display - Google Patents

Abstract

The present disclosure provides materials and assays, herein referred to as surface display ACE (surfACE), that use E. coli strains expressing, in one embodiment, nanobodies (VHHs) on the outer cell membrane as a solution to increase de novo screening throughput and efficiency using a combination of MACS and FACS based enrichment strategies.

Description

NANOBODY LIBRARY SCREENING USING BACTERIAL SURFACE DISPLAY

[0001] This application contains, as a separate part of the disclosure, a Sequence Listing in computer-readable form which is incorporated by reference in its entirety and identified as follows: 59168_SeqListing.xml; Size: 3,776 bytes; Created: December 13, 2024.

BACKGROUND

[0002] Antibodies have become the predominant class of therapeutics over the past two decades. However, antibody drug discovery and manufacturing remain slow and costly. Traditionally, antibody drug discovery workflows require selection from either naive or immunized libraries derived from animal models or through synthetic methods such as phage display, yeast display, or in vitro display. Overall, these methods have been successful in bringing over 100 monoclonal antibody therapies to market with over 500 currently in clinical trials. Nevertheless, there are several limitations with current technologies including high cost, limited diversity, limited target screening, prolonged workflows, suboptimal throughput cell fixation and permeabilization requirements, and technical issues related to enriching internally expressed nanobody (VHH) libraries.

SUMMARY

[0003] The present disclosure provides materials and techniques that address the aforementioned needs in the art.

[0004] In one embodiment of the present disclosure, an expression cassette is provided comprising: (i) a nucleic acid encoding an intimin autotransporter or functional fragment thereof, (ii) a nucleic acid encoding a cleavage site, (iii) at least one nucleic acid encoding at least one epitope tag, (iv) a nucleic acid encoding a cargo protein, and (v) at least one nucleic acid encoding at least one purification tag cargo protein. In some embodiments, the epitope tag is a HA tag as described herein, and in other embodiments, the purification tag is a 6x His tag as described herein.

[0005] In one embodiment, an aforementioned expression cassette is provided wherein the cargo protein is selected from the group consisting of: an antibody, a Fab, a scFab, an Fv, an scFv, a di-scFv, a nanobody, a VHH, or fragments of any of the above. In one embodiment, the cargo protein is a VHH. In one embodiment, an aforementioned expression cassette is provided wherein the cleavage site is a TEV protease cleavage site. In another embodiment, an aforementioned expression cassette is provided wherein the expression cassette comprises one epitope tag and one purification tag. In one embodiment, the epitope tag is N-terminal and the purification tag is C-terminal to the cargo protein. In still another embodiment, the epitope tag is selected from a hemagglutinin (HA) tag and a Flag tag, and the purification tag is selected from a Histidine (His) tag. In one embodiment, the His tag is a 6x-His tag.

[0006] The present disclosure also provides, in one embodiment, an aforementioned expression cassette wherein the intimin autotransporter is selected from the group consisting of an invasion from Yersenia spp. or an intimin from pathogenic E. coli.

[0007] In one embodiment, an aforementioned expression cassette is provided further comprising an inducible promoter capable of promoting expression of a fusion protein comprising an intimin autotransporter or functional fragment thereof, a cleavage site, at least one epitope tag, a cargo protein, and at least one purification tag. In some embodiments, the inducible promoter is selected from the group consisting of an arabinose-inducible promoter, a propionate-inducible promoter, and a rhamnose-inducible promoter. In one embodiment, the inducible promoter is an arabinose-inducible promoter.

[0008] The present disclosure also provides, in one embodiment, an expression cassette comprising: (i) a nucleic acid encoding an intimin autotransporter or functional fragment thereof, (ii) a nucleic acid encoding a TEV protease cleavage site, (iii) at least one nucleic acid encoding at least one HA tag, (iv) a nucleic acid encoding a scFab, and (v) at least one nucleic acid encoding at least one His tag.

[0009] In still another embodiment, an expression vector comprising an aforementioned expression cassette is provided further comprising a nucleic acid encoding a protein that increases expression vector retention and/or stability. In one embodiment, the protein that increases expression vector retention and/or stability is ParB.

[0010] In one embodiment, an aforementioned expression cassette is provided further comprising a strong RBS (sRBS) or a weak/moderate RBS (wRBS) that is upstream of the nucleic acid encoding the intimin autotransporter or functional fragment thereof.

[0011] In still another embodiment, a host cell is provided comprising an aforementioned expression cassette or an aforementioned expression vector. In another embodiment, the host cell is a prokaryotic cell comprising one or more or all of: (a) an alteration of gene function of at least one gene encoding a transporter protein for an inducer of at least one inducible promoter; (b) a reduced level of gene function of at least one gene encoding a protein that metabolizes an inducer of at least one inducible promoter; (c) a reduced level of gene function of at least one gene encoding a protein involved in biosynthesis of an inducer of at least one inducible promoter; (d) an altered gene function of a gene that affects the reduction/oxidation environment of the host cell cytoplasm; (e) a reduced level of gene function of a gene that encodes a reductase; (f) at least one expression construct encoding at least one disulfide bond isomerase protein; (g) at least one polynucleotide encoding a form of DsbC lacking a signal peptide; and/or (h) at least one polynucleotide encoding Ervlp. In some embodiments, the host cell is derived from an Enterobacterial species. In one embodiment, the host cell is an Escherichia co// host cell. In still another embodiment, the Escherichia coli is a SoluPro cell. In some embodiments, the host cell further comprises a deletion of a periplasmic protease. In one embodiment, the periplasmic protease is DegP.

[0012] Methods are also provided by the present disclosure. In one embodiment, the present disclosure provides a method of screening for expression of a cargo protein on the surface of a host cell, the method comprising: a) preparing a library of aforementioned host cells; b) culturing the cells; c) inducing expression of the cargo protein; and d) screening for expression of the cargo protein; wherein said screening comprises a method selected from the group consisting of magnetic activated cell sorting (MACS), and Fluorescence- Activated Cell Sorting (FACS). Other techniques known in the art are also contemplated. In another embodiment, the aforementioned method is provided further comprising the step of recovering a host cell or host cells following the screening step (d) by plating recovered host cells on a growth medium, thereby enriching for said host cell or host cells. In another embodiment, the aforementioned method is provided further comprising the step of a second screening to assess cargo protein expression or cargo protein EC50, thereby screening for expression of a cargo protein capable of binding to a target with a desired EC50.

[0013] A method of determining affinity of a cargo protein to a target is also provided by the present disclosure, the method comprising: a) preparing a library of aforementioned host cells; b) culturing the cells; c) inducing expression of the cargo protein; and d) screening for expression of the cargo protein; optionally further comprising the step of recovering a host cell or host cells following the screening step (d) by plating recovered host cells on a growth medium; optionally further comprising the step of a second screening to assess cargo protein expression or cargo protein EC50; and e) determining the affinity of the cargo protein to the target. In one embodiment, the affinity is determined by a method selected from the group consisting of surface plasmon resonance (SPR) and BioLayer Interferometry.

[0014] A method for determining the sequence of a nucleic acid encoding a cargo protein capable of binding to a target with a desired affinity is also provided, the method comprising: a) preparing a library of aforementioned host cells; b) culturing the cells; c) inducing expression of the cargo protein; and d) screening for expression of the cargo protein; optionally further comprising the step of recovering a host cell or host cells following the screening step (d) by plating recovered host cells on a growth medium; optionally further comprising the step of a second screening to assess cargo protein expression or cargo protein EC50; e) determining the affinity of the cargo protein to the target; and f) sequencing the nucleic acid encoding said cargo protein. In another embodiment an aforementioned method is provided wherein the method does not require cell fixation. In another embodiment an aforementioned method is provided wherein the method does not require cell permeabilization. In still another embodiment an aforementioned method is provided wherein intracellular chaperones are not required for proper protein folding. As described herein, this embodiment allows for use of smaller plasmid size and increasing plasmid stability.

[0015] The present disclosure also provides an aforementioned method, as described herein and will eb appreciated by those in the art, wherein the method can increase signal-to-noise ratio by using a large and/or bright fluorophore; and/or wherein the large and/or bright fluorophore is selected from phycoerythrin (PE) and quantum dots; and/or wherein enrichment is carried out using a combination of magnetic activated cell sorting (MACS) and Fluorescence-Activated Cell Sorting (FACS) as described herein; and/or wherein the EC50 measurements correlate with affinities more closely than intracellular counterparts; and/or wherein the method allows for screening against membrane protein targets.

BRIEF DESCIRPTION OF FIGURES

[0016] FIG. 1 illustrates a schematic representation of a surface expression cassette design. In FIG. 1 , surface-displayed proteins are expressed as C-terminal fusions on the intimin autotransporter. A TEV cleavage site is included N-terminal to the VHH to allow controlled release from the cell surface and a 6x-His tag is included for purification of cleaved proteins. Both HA and 6x-His tags are included for flow cytometry staining.

[0017] FIG. 2 shows a schematic of a surface display proof of concept plasmid design 1 , which has the following features and expression cassettes: a repressor protein (ROP) to modulate ~ 50 copies per cell, an origin of replication (pBR322) to modulate expression levels, ParB to increase plasmid retention and stability, and a strong ribosome binding site (sRBS) to modulate expression levels N-terminal to the autotransporter and cargo protein. The benefits of the design shown in FIG. 2 include low strain aggregation, no known plasmid instability between rounds of selection and during expression, higher expression, higher fold enrichment, and less susceptibility to polyclonality. A drawback of the design shown in FIG. 2 includes lower overall levels of cell viability as measured by post-FACS plating and liquid growth, thereby, likely requires re-cloning post-sort rather than direct growth in most sorting scenarios and apparent growth rate effects between discrete strains, which may lead to issues in libraries.

[0018] FIG. 3 shows a schematic of a surface display proof of concept plasmid design 2, which has the following features and expression cassettes to generate high copy number (-500 copies per cell) and weak to moderate RBS (wRBS): the origin of replication (pBR322) to modulate expression levels and ParB to increase plasmid retention and stability. The benefits of the design are shown in FIG. 3 include low strain aggregation, no known plasmid stability issues between rounds of selection and during expression, high viability pre- and post-sorting (MACS/FACS) which yields quick cell recovery, fold-enrichment comparable to multiple rounds of ACE sorts observed in FACS and MACS, and no observed growth rate differences between discrete strains. Some potential drawbacks of the design are shown in FIG. 3 include polyclonality effects when transforming highly diverse libraries, lower overall expression levels leading to lower confidence when distinguishing low-affinity variants and negative controls, and lower fold-enrichment in MACS/FACS than when compared to low copy number and strong RBS plasmid construct.

[0019] FIGs. 4A-4B show validation of the expression and functionality of surface displayed VHHs in Design 1 and 2 plasmid backbones FIG. 4A shows the gMFI of expression stain (anti-VHH iFluor647) for HER2 VHH standards in the Design 1 backbone (orange) and the Design 2 backbone (blue). FIG. 4B shows the gMFI of antigen stain (50 nM HER2-PE) for HER2 VHH standards in the Design 1 backbone (orange) and the Design 2 backbone (blue)

[0020] FIG. 5 depicts a proposed workflow to utilize the surface display construct to identify target molecules.

[0021] FIG. 6 shows an enrichment validation of FACS and MACS. The results show an overall higher fold enrichment than other plasmid backbones.

[0022] FIGs. 7A-7C show initial validations of VHH surface display affinity curves. The results show that the EC50S obtained from surface display antigen titrations correlate with affinities more closely than intracellular counterparts. ECso’s determined by flow cytometry for Her2-binding constructs displayed on the surface of SoluPro. All three constructs have affinities for Her2 in the 1 to 2 nM range. FIG. 7A shows Her2-binding VHH (EC50 = 7.0 ± 2.1 nM); FIG. 7B shows Trastuzumab scFv (EC50 = 5.4 ± 1 .5 nM). FIG. 7C shows Trastuzumab scFab (EC50 = 2.4 ± 2.8 nM).

[0023] FIGs. 8A-8B shows microscopy images of SoluPro displaying intimin-VHH. Stained with 50 nM HER-PE (red) and DAPI (blue). FIG. 8A depicts the entire field, whereas FIG. 8B depicts a zoomed-in image of the region highlighted in FIG. 8A. [0024] FIG. 9 illustrates buffer composition's impact on scFab aggregation, displaying SoluPro cells as determined by a cell counter. The percentage of cells in aggregates is defined as 100 x (number of cells with size > 1 nm / total number of cells counted). Many cells are counted as aggregates under typical staining conditions (i.e., PBS or PBS/EDTA). The addition of cations such as magnesium and calcium seems to reduce aggregation in SoluPro.

[0025] FIGs. 10A-10B show a validation of additional surfACE probe VEGF by comparing the expression levels of anti-VHH iFluor647 staining (FIG. 10A) and 367 nM VEGF-PE (FIG. 10B). The samples tested are as follows: NC (Empty SoluPro negative control), NC2 (VHH negative control strain), Nb42 (nanobody from citation below with VEGF affinity ~60 nM), Nb35 (nanobody from citation below with VEGF affinity ~45 nM), Nb23 (nanobody from citation below with VEGF affinity ~10 nM), and Nb22 (nanobody from citation below with VEGF affinity ~1 nM). The results from FIGs. 10A-10B show that Nb23 expresses well as a fusion to intimin and has a VEGF binding signal well above the negative control.

[0026] FIG. 11A-11C illustrates the effects of DsbC chaperone on intimin-VHH fusions. FIG. 11 depicts the comparisons of viability (SYTOX Blue staining) (FIG. 11 A), expression levels (anti-VHH iFluor647) (FIG. 11 B), and ligand binding levels (50 nM HER2-PE) (FIG. 11C) for several HER2-binding intimin-VHH strains that were expressed in the presence or absence of DsbC. + indicates that DsbC is present, whereas - indicates that DsbC is absent.

DETAILED DESCRIPTION

[0027] The present disclosure provides materials and assays, herein referred to as surface display ACE (surfACE), that use E. co// strains expressing nanobodies (VHHs) on the outer cell membrane as a solution to increase de novo screening throughput and efficiency using a combination of MACS and FACS based enrichment strategies. As described herein, surfACE has several advantages over other assays including, for example, increased screening throughput via magnetic activated cell sorting (MACS, ~ 1 x10⁹ cells/column/day); reduced turnaround time between rounds of enrichment (FACS and/or MACS); no required fixation/permeabilization which permits the use of large/bright fluorophores (e.g., phycoerythrin (PE) and quantum dots) to increase signal-to-noise ratio; EC50 measurements obtained from surface display antigen titrations correlate with affinities more closely than intracellular counterparts (Figure 7); intracellular chaperones are not required for proper protein folding which allows the use of smaller plasmid sizes and consequently increases plasmid stability (Figure 11 ); and the ability to screen against membrane protein targets. [0028] An activity-specific cell-enrichment (ACE) assay that identifies host cells that express active gene product of interest (e.g., biomolecules, as used herein) rather than inactive material has been described in WO 2021/146626, incorporated herein in the relevant part. Active gene products can be distinguished from inactive material by the ability of active gene products to specifically bind a binding partner molecule, or by the ability of gene products to participate in a chemical or enzymatic reaction, as examples. The presence of properly formed disulfide bonds in a polypeptide gene product is an indication that it is correctly folded and presumptively active. In the cell-enrichment methods, the active gene product of interest is detected by utilizing an appropriate labeling complex that specifically binds to the active gene product of interest, such as a labeled antigen if the gene product of interest is an antibody, the fragment antigen-binding (Fab), or VHH; or a labeled ligand if the gene product of interest is a receptor or a receptor fragment, where the ligand specifically binds to an active conformation of the receptor; or a labeled substrate or a labeled substrate analog if the gene product of interest is an enzyme, as examples. For any gene product of interest, if there is an available antibody or antibody fragment that specifically binds to the active gene product and not to the inactive gene product, that antibody or antibody fragment can be used to label the active gene product of interest when attached to a detectable moiety.

[0029] Another assay - quantitative affinity ACE (qaACE) is a method for sampling the affinity of antibody variants at high throughput using flow cytometry and next-generation sequencing to generate a qaACE score that correlates with K_D. The main goal of this method is to generate highly quantitative high throughput training data for an Al model to perform sequence-based affinity predictions. This method can be applied to any antibody format, mAbs, Fabs, scFv, scFab, VHH, nanobody, etc., and could conceivably be applied to other binding drug formats as well. The qaACE method is described in PCT/US23/72153 and is incorporated herein in the relevant part.

[0030] Still, another assay, the transform and repeat ACE (trACE) assay, can expedite the antibody drug discovery process by performing iterative rounds of ACE to enrich high- diversity antibody libraries for antigen-binding variants. The trACE assay begins with a high- diversity antibody library (e.g., > 10⁵ diversity), and the ACE assay is performed as described to isolated antigen-binding variants. The enriched library DNA is re-cloned into an expression vector and re-expressed in the host strain for subsequent rounds of ACE to further enrich and isolate antigen-binding variants. Enrichment is verified by flow cytometry, NGS, and/or other methods known in the art. Antigen-binding antibody variants are confirmed by BLI or SPR assays and sequences identified by NGS. Plasmids and expression constructs

[0031] The present disclosure provides, in various embodiments, plasmids and expression vectors capable of expressing, for example, cargo proteins. As described herein, cargo proteins such as antibodies or antibody fragments (e.g., nanobodies) are expressed/displayed on the cell surface in certain embodiments.

[0032] As described herein, in some embodiments a plasmid used in the surfACE assay contains the following genetic elements/expression cassette features: arabinose induction machinery for the expression of surface display fusion proteins; an intimin autotransporter N- terminally fused to a cargo protein (here, VHHs); a TEV protease cleavage site and a hemagglutinin (HA) tag N-terminal to the cargo protein for cleavage of the cargo protein and stain detection, respectively; a 6x-His tag C-terminal to the cargo protein for purification and stain detection; ParB to increase plasmid retention and stability; a ribosome binding site (RBS) of desired strength (e.g., strong and moderate/weak) to modulate expression levels; an origin of replication to modulate expression levels (for example, pBR322 (high copy number, -500 copies per cell), pBR322 + ROP (-50 copies per cell), and psc101 (-5 copies per cell). Thus, in some embodiments, surface-displayed proteins (e.g., VHHs) are expressed as C-terminal fusions to the intimin autotransporter. A TEV cleavage site is included in the N-terminal of the VHH to allow controlled release from the cell surface, and a 6x His tag is included for the purification of cleaved proteins. Both HA and 6x-His tags are included for flow cytometry staining.

[0033] Specific sequences and features of the plasmids and expression cassettes described herein can be tailored as described herein. In one embodiment, a first design includes the following features and benefits: low copy number (-50 copies per cell) + strong RBS (sRBS); low strain aggregation; no known plasmid stability issues between rounds of selection/during expression; high expression levels yields high confidence when distinguishing negative controls and lower affinity variants (e.g., Kd >150 nM); overall higher fold-enrichment than other plasmid backbones; less susceptible to polyclonality effects.

[0034] In another embodiment, a second design includes the following features and benefits: High copy number (-500 copies per cell) + weak/moderate RBS (wRBS); low strain aggregation; no known plasmid stability issues between rounds of selection/during expression; high viability pre- and post-sorting (MACS/FACS) which yields quick cell recovery; fold-enrichment comparable to trACE observed in FACS and MACS; no observed growth rate differences between discrete strains.

[0035] Additional features of expression construct suitable for use in the assays provided herein are also contemplated. [0036] In some embodiments, a prokaryotic cell described herein comprises one or more expression constructs that may optionally include one or more inducible promoters to express an antigen-binding protein of interest.

[0037] The term "expression construct," as used herein, refers to polynucleotides designed for the expression of one or more antigen-binding proteins of interest, and thus are not naturally occurring molecules. Expression constructs can be integrated into a host cell chromosome or maintained within the host cell as polynucleotide molecules replicating independently of the host cell chromosome, such as plasmids or artificial chromosomes. An example of an expression construct is a polynucleotide resulting from the insertion of one or more polynucleotide sequences into a host cell chromosome, where the inserted polynucleotide sequences alter the expression of chromosomal coding sequences. An expression vector is a plasmid expression construct specifically used for the expression of one or more antigen-binding proteins. One or more expression constructs can be integrated into a host cell chromosome or be maintained on an extrachromosomal polynucleotide such as a plasmid or artificial chromosome. The following are descriptions of particular types of polynucleotide sequences that can be used in expression constructs for the expression or coexpression of gene products, including fusion proteins as described herein.

[0038] Origins of replication. Expression constructs must comprise an origin of replication, also called a replicon, in order to be maintained within the host cell as independently replicating polynucleotides. Different replicons that use the same mechanism for replication cannot be maintained together in a single host cell through repeated cell divisions. As a result, plasmids can be categorized into incompatibility groups depending on the origin of replication that they contain, as shown in Table 2 of International Publication No. WO 2016/205570. Origins of replication can be selected for use in expression constructs on the basis of incompatibility group, copy number, and/or host range, among other criteria. As described above, if two or more different expression constructs are to be used in the same host cell for the coexpression of multiple gene products, it is best if the different expression constructs contain origins of replication from different incompatibility groups: a pMBI replicon in one expression construct and a pl5A replicon in another, for example. The average number of copies of an expression construct in the cell, relative to the number of host chromosome molecules, is determined by the origin of replication contained in that expression construct. Copy numbers can range from a few copies per cell to several hundred (Table 2 of WO/2016/205570). In some embodiments, different expression constructs are used, which comprise inducible promoters that are activated by the same inducer, but which have different origins of replication. By selecting origins of replication that maintain each different expression construct at a certain approximate copy number in the cell, it is possible to adjust the levels of the overall production of a gene product expressed from one expression construct relative to another gene product expressed from a different expression construct. As an example, to co-express subunits A and B of a multimeric protein, an expression construct is created which comprises the colEI replicon, the am promoter, and a coding sequence for subunit A expressed from the am promoter: 'colEI- Para-A’.

[0039] Another expression construct is created comprising the pl 5A replicon, the am promoter, and a coding sequence for subunit B: 'pl5A-Para-B'. These two expression constructs can be maintained together in the same host cells, and expression of both subunits A and B is induced by the addition of one inducer, arabinose, to the growth medium. If the expression level of subunit A needed to be significantly increased relative to the expression level of subunit B, in order to bring the stoichiometric ratio of the expressed amounts of the two subunits closer to a desired ratio, for example, a new expression construct for subunit A could be created, having a modified pMB 1 replicon as is found in the origin of replication of the pUC9 plasmid ('pUC9ori') : pUC9ori-Para-A. Expressing subunit A from a high-copy-number expression construct such as pUC9ori-Para-A should increase the amount of subunit A produced relative to the expression of subunit B from pl5A-Para-B. In a similar fashion, the use of an origin of replication that maintains expression constructs at a lower copy number, such as pSOOl (WO/2016/205570), could reduce the overall level of a gene product expressed from that construct. The selection of an origin of replication can also determine which host cells can maintain an expression construct comprising that replicon. For example, expression constructs comprising the colEI origin of replication have a relatively narrow range of available hosts, species within the Enterobacteriaceae family, while expression constructs comprising the RK2 replicon can be maintained in E. coli, Pseudomonas aeruginosa, Pseudomonas putida, Azotobacter vinelandii, and Alcaligenes eutrophus, and if an expression construct comprises the RK2 replicon and some regulator genes from the RK2 plasmid, it can be maintained in host cells as diverse as Sinorhizobium meliloti, Agrobacterium tumefaciens, Caulobacter crescentus, Acinetobacter calcoaceticus, and Rhodobacter sphaeroides (Kiies and Stahl, Microbiol Rev 1989 Dec; 53(4):491 -516).

[0040] Similar considerations can be employed to create expression constructs for inducible expression or coexpression in eukaryotic cells. For example, the 2-micron circle plasmid of Saccharomyces cerevisiae is compatible with plasmids from other yeast strains, such as pSRI (ATCC Deposit Nos. 48233 and 66069; Araki et al., J Mol Biol 1985 Mar 20; 182(2):191 -203) and pKDI (ATCC Deposit No. 37519; Chen et al, Nucleic Acids Res 1986 Jun 11 ; 14(11):4471-81 ). [0041] In some embodiments, the expression construct comprises a selection gene. A “selection gene”, also termed a selectable marker, encodes a protein necessary for the survival or growth of a host cell in a selective culture medium. Host cells not containing the expression construct comprising the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics or other toxins, or that complement auxotrophic deficiencies of the host cell. One example of a selection scheme utilizes a drug, such as an antibiotic, to arrest the growth of a host cell. Those cells that contain an expression construct comprising the selectable marker produce a protein conferring drug resistance and survive the selection regimen. Some examples of antibiotics that are commonly used for the selection of selectable markers (and abbreviations indicating genes that provide antibiotic resistance phenotypes) are ampicillin (AmpR), chloramphenicol (CmIR or CmR), kanamycin (KanR), spectinomycin (SpcR), streptomycin (StrR), and tetracycline (TetR). Many of the plasmids in Table 2 of WO/2016/205570 comprise selectable markers, such as pBR322 (AmpR, TetR); pMOB45 (CmR, TetR); pACYCIW (AmpR, KanR); and pGBMI (SpcR, StrR). The native promoter region for a selection gene is usually included, along with the coding sequence for its gene product, as part of a selectable marker portion of an expression construct. Alternatively, the coding sequence for the selection gene can be expressed from a constitutive promoter.

[0042] Exemplary selectable markers include, but are not limited to, neomycin phosphotransferase (npt II), hygromycin phosphotransferase (hpt), dihydrofolate reductase (dhfr), zeocin, phleomycin, bleomycin resistance gene (ble), gentamicin acetyltransferase, streptomycin phosphotransferase, mutant form of acetolactate synthase (als), bromoxynil nitrilase, phosphinothricin acetyltransferase (bar), enolpyruvylshikimate-3-phosphate (EPSP) synthase (aro A), muscle specific tyrosine kinase receptor molecule (MuSK-R), copper-zinc superoxide dismutase (sod1 ), metallothionein’s (cup1 , MT1 ), beta-lactamase (BLA), puromycin N-acetyl-transferase (pac), blasticidin acetyl transferase (bls), blasticidin deaminase (bsr), histidinol dehydrogenase (HDH), N-succinyl-5-aminoimidazole-4- carboxamide ribotide (SAICAR) synthetase (ade1), argininosuccinate lyase (arg4), betaisopropylmalate dehydrogenase (Ieu2), invertase (suc2), orotidine-5'-phosphate (OMP) decarboxylase (ura3), and orthologs of any of the foregoing.

[0043] Inducible promoter. As described herein, there are several different inducible promoters that can be included in expression constructs as part of the inducible coexpression systems of the disclosure. In some embodiments, inducible promoters share at least 80% polynucleotide sequence identity (more preferably, at least 90% identity, and most preferably, at least 95% identity) to at least 30 (more preferably, at least 40, and most preferably, at least 50) contiguous bases of a promoter polynucleotide sequence as defined in Table 1 of International Publication No. WO 2016/205570 by reference to the E. coli K-I Z substrain MG1655 genomic sequence, where percent polynucleotide sequence identity is determined using the methods of Example 11 of WO/2016/205570. Under 'standard' inducing conditions (see Example 5 of International Publication No. WO 2016/205570), preferred inducible promoters have at least 75% (more preferably, at least 100%, and most preferably, at least 110%) of the strength of the corresponding 'wild-type' inducible promoter of E. coli K-12 substrain MG1655, as determined using the quantitative PCR method of De Mey et al. (Example 6 of International Publication No. WO 2016/205570). Within the expression construct, an inducible promoter is placed 5' to (or 'upstream of’) the coding sequence for the gene product that is to be inducibly expressed, so that the presence of the inducible promoter will direct transcription of the gene product coding sequence in a 5' to 3' direction relative to the coding strand of the polynucleotide encoding the gene product.

[0044] Ribosome binding site. For polypeptide gene products, the nucleotide sequence of the region between the transcription initiation site and the initiation codon of the coding sequence of the gene product that is to be inducibly expressed corresponds to the 5' untranslated region ('UTR') of the mRNA for the polypeptide gene product. Preferably, the region of the expression construct that corresponds to the 5' UT comprises a polynucleotide sequence similar to the consensus ribosome binding site (RBS, also called the Shine- Dalgarno sequence) that is found in the species of the host cell. In prokaryotes (archaea and bacteria), the RBS consensus sequence is GGAGG (SEQ ID NO: 1) or GGAGGU (SEQ ID NO: 2), and in bacteria such as E. coli, the RBS consensus sequence is AGGAGG (SEQ ID NO: 3) or AGGAGGU (SEQ ID NO: 4). The RBS is typically separated from the initiation codon by 5 to 10 intervening nucleotides. In expression constructs, the RBS sequence is preferably at least 55% identical to the AGGAGGU (SEQ ID NO: 5) consensus sequence, more preferably at least 70% identical, and most preferably at least 85% identical, and is separated from the initiation codon by 5 to 10 intervening nucleotides, more preferably by 6 to 9 intervening nucleotides, and most preferably by 6 or 7 intervening nucleotides. The ability of a given RBS to produce a desirable translation initiation rate can be calculated at the website salis.psu.edu/software/RBSLibraryCalculatorSearchMode, using the RBS Calculator; the same tool can be used to optimize a synthetic RBS for a translation rate across a 100,000+ fold range (Salis, Methods Enzymol 2011 ; 498:19-42).

[0045] Multiple cloning site. A multiple cloning site (MCS), also called a polylinker, is a polynucleotide that contains multiple restriction sites in close proximity to or overlapping with each other. The restriction sites in the MCS typically occur once within the MCS sequence and preferably do not occur within the rest of the plasmid or other polynucleotide construct, allowing restriction enzymes to cut the plasmid or other polynucleotide construct only within the MCS. Examples of MCS sequences are those in the pBAD series of expression vectors, including pBAD18, pBAD18-Cm, pBAD18-Kan, pBAD24, pBAD28, pBAD30, and pBAD33 (Guzman et al., J Bacteriol 1995 Jul; 177(14):4121 -30); or those in the pPRO series of expression vectors derived from the pBAD vectors, such as pPR018, pPR018-Cm, pPR018- Kan, pPR024, pPRO30, and pPR033 (U.S. Patent No. 8,178,338 B2; May 15, 2012; Keasling, Jay). A multiple cloning site can be used in the creation of an expression construct: by placing a multiple cloning site 3' to (or downstream of) a promoter sequence, the MCS can be used to insert the coding sequence for a gene product to be expressed or coexpressed into the construct, in the proper location relative to the promoter so that transcription of the coding sequence will occur. Depending on which restriction enzymes are used to cut within the MCS, there may be some part of the MCS sequence remaining within the expression construct after the coding sequence or other polynucleotide sequence is inserted into the expression construct. Any remaining MCS sequence can be upstream of, downstream of, or on both sides of the inserted sequence. A ribosome binding site can be placed upstream of the MCS, preferably immediately adjacent to or separated from the MCS by only a few nucleotides, in which case the RBS would be upstream of any coding sequence inserted into the MCS. Another alternative is to include a ribosome binding site within the MCS, in which case the choice of restriction enzymes used to cut within the MCS will determine whether the RBS is retained and in what relation to the inserted sequences. A further alternative is to include an RBS within the polynucleotide sequence that is to be inserted into the expression construct at the MCS, preferably in the proper relation to any coding sequences to stimulate initiation of translation from the transcribed messenger RNA.

[0046] Expression from constitutive promoters. Expression constructs of the disclosure can also comprise coding sequences that are expressed from constitutive promoters. Unlike inducible promoters, constitutive promoters initiate continual gene product production under most growth conditions. One example of a constitutive promoter is that of the Tn3 bla gene, which encodes beta-lactamase and is responsible for the ampicillin-resistance (AmpR) phenotype conferred on the host cell by many plasmids, including pBR322 (ATCC 31344), pACYCIW (ATCC 37031), and pBAD24 (ATCC 87399). Another constitutive promoter that can be used in expression constructs is the promoter for the E. coli lipoprotein gene, Ipp, which is located at positions 1755731 -1755406 (plus strand) in

2 substrain MG1655 (Inouye and Inouye, Nucleic Acids Res 1985 May 10; 13(9):3101 -10). A further example of a constitutive promoter that has been used for heterologous gene expression in E. coli s the trpLEDCBA promoter, located at positions 1321169-1321133 (minus strand) in E. coli K-12 substrain MG1655 (Windass et al., Nucleic Acids Res 1982 Nov 11 ;

10(21 ):6639-57). Constitutive promoters can be used in expression constructs for the expression of selectable markers, as described herein, and also for the constitutive expression of other gene products useful for the coexpression of the desired product. For example, transcriptional regulators of the inducible promoters, such as AraC, PrpR, RhaR, and XylR, if not expressed from a bidirectional inducible promoter, can alternatively be expressed from a constitutive promoter on either the same expression construct as the inducible promoter they regulate or a different expression construct. Similarly, gene products useful for the production or transport of the inducer, such as PrpEC, AraE, or Rha, or proteins that modify the reduction-oxidation environment of the cell, as a few examples, can be expressed from a constitutive promoter within an expression construct. Gene products useful for the production of coexpressed gene products and the resulting desired product also include chaperone proteins, cofactor transporters, etc.

[0047] Signal peptides. Polypeptide gene products expressed or coexpressed by the methods of the disclosure can contain signal peptides or lack them, depending on whether it is desirable for such gene products to be exported from the host cell cytoplasm into the periplasm, or to be retained in the cytoplasm, respectively. Signal peptides (also termed signal sequences, leader sequences, or leader peptides) are characterized structurally by a stretch of hydrophobic amino acids, approximately five to twenty amino acids long and often around ten to fifteen amino acids in length that tends to form a single alpha-helix. This hydrophobic stretch is often immediately preceded by a shorter stretch enriched in positively charged amino acids (particularly lysine). Signal peptides that are to be cleaved from the mature polypeptide typically end in a stretch of amino acids that are recognized and cleaved by signal peptidase. Signal peptides can be characterized functionally by the ability to direct transport of a polypeptide, either co-translationally or post-translationally, through the plasma membrane of prokaryotes (or the inner membrane of gram-negative bacteria like E. coli) or into the endoplasmic reticulum of eukaryotic cells. The degree to which a signal peptide enables a polypeptide to be transported into the periplasmic space of a host cell like E. coli, for example, can be determined by separating periplasmic proteins from proteins retained in the cytoplasm, using a method such as described in Example 12 of International Publication No. WO 2016/205570.

[0048] Examples of inducible promoters and related genes are unless otherwise specified, from Escherichia coli (E. coli) strain MG1655 (American Type Culture Collection deposit ATCC 700926), which is a substrain

2 (American Type Culture Collection deposit ATCC 10798). Table 1 of International Publication No. WO 2016/205570 lists the genomic locations, in E. coli MG1655, of the nucleotide sequences for these examples of inducible promoters and related genes. Nucleotide and other genetic sequences, referenced by genomic location as in Table 1 of International Publication No. WO 2016/205570, are expressly incorporated by reference herein. Additional information about E. co// promoters, genes, and strains described herein can be found in many public sources, including the online EcoliWiki resource, located at ecoliwiki.net.

[0049] Arabinose promoter. (As used herein, ‘arabinose’ means L-arabinose.) Several E. coli operons involved in arabinose utilization are inducible by arabinose — araBAD, araC, arciE, and araFGH — but the terms ‘arabinose promoter’ and ‘ara promoter’ are typically used to designate the araBAD promoter. Several additional terms have been used to indicate the E. co// araBAD promoter, such as Para, ParaB, ParaBAD, and PBAD-. The use herein of ‘ara promoter’ or any of the alternative terms given above, means the E. coli araBAD promoter. As can be seen from the use of another term, ‘araC-araBAD promoter’, the araBAD promoter is considered to be part of a bidirectional promoter, with the araBAD promoter controlling expression of the araBAD operon in one direction, and the araC promoter, in close proximity to and on the opposite strand from the araBAD promoter, controlling expression of the araC coding sequence in the other direction. The AraC protein is both a positive and a negative transcriptional regulator of the araBAD promoter. In the absence of arabinose, the AraC protein represses transcription from PBAD, but in the presence of arabinose, the AraC protein, which alters its conformation upon binding arabinose, becomes a positive regulatory element that allows transcription from PBAD- The araBAD operon encodes proteins that metabolize L-arabinose by converting it, through the intermediates L-ribulose and L-ribulose-phosphate, to D-xylulose-5-phosphate. For the purpose of maximizing induction of expression from an arabinose-inducible promoter, it is useful to eliminate or reduce the function of AraA, which catalyzes the conversion of L- arabinose to L-ribulose, and optionally to eliminate or reduce the function of at least one of AraB and AraD, as well. Eliminating or reducing the ability of host cells to decrease the effective concentration of arabinose in the cell by eliminating or reducing the cell's ability to convert arabinose to other sugars allows more arabinose to be available for induction of the arabinose-inducible promoter. The genes encoding the transporters that move arabinose into the host cell are araE, which encodes the low-affinity L-arabinose proton symporter, and the araFGH operon, which encodes the subunits of an ABC superfamily high-affinity L- arabinose transporter. Other proteins which can transport L-arabinose into the cell are certain mutants of the LacY lactose permease: the LacY(AIWC) and the LacY(AIWV) proteins, having a cysteine or a valine amino acid instead of alanine at position 177, respectively (Morgan-Kiss et al., Proc Natl Acad Sci USA 2002 May 28; 99(11):7373-77). In order to achieve homogeneous induction of an arabinose-inducible promoter, it is useful to make the transport of arabinose into the cell independent of regulation by arabinose. This can be accomplished by eliminating or reducing the activity of the AraFGH transporter proteins and altering the expression of araE so that it is only transcribed from a constitutive promoter. Constitutive expression of araE can be accomplished by eliminating or reducing the function of the native araE gene and introducing into the cell an expression construct that includes a coding sequence for the AraE protein expressed from a constitutive promoter. Alternatively, in a cell lacking AraFGH function, the promoter controlling expression of the host cell's chromosomal araE gene can be changed from an arabinose-inducible promoter to a constitutive promoter. In a similar manner, as additional alternatives for homogenous induction of an arabinose-inducible promoter, a host cell that lacks AraE function can have any functional AraFGH coding sequence present in the cell expressed from a constitutive promoter. As another alternative, it is possible to express both the araE gene and the araFGH operon from constitutive promoters, by replacing the native araE and araFGH promoters with constitutive promoters in the host chromosome. It is also possible to eliminate or reduce the activity of both the AraE and the AraFGH arabinose transporters, and in that situation to use a mutation in the LacY lactose permease that allows this protein to transport arabinose. Since expression of the lacY gene is not normally regulated by arabinose, the use of a LacY mutant, such as LacY(A177C) or LacY(A177V), will not lead to the 'all or none' induction phenomenon when the arabinose-inducible promoter is induced by the presence of arabinose. Because the LacY(A177C) protein appears to be more effective in transporting arabinose into the cell, the use of polynucleotides encoding the LacY(A177C) protein is preferred to the use of polynucleotides encoding the LacY(A177V) protein.

[0050] Propionate promoter. The 'propionate promoter' or 'prp promoter' is the promoter for the E. coli prpBCDE operon. Like the ara promoter, the prp promoter is part of a bidirectional promoter, controlling the expression of the prpBCDE operon in one direction and with the prpR promoter controlling the expression of the prpR coding sequence in the other direction. The PrpR protein is the transcriptional regulator of the prp promoter and activates transcription from the prp promoter when the PrpR protein binds 2-methylcitrate ('2-MC'). Propionate (also called propanoate) is the ion, CH3CH2COO-, of propionic acid (or 'propanoic acid') and is the smallest of the 'fatty' acids having the general formula H(CH₂)nCOOH that shares certain properties of this class of molecules: producing an oily layer when salted out of water and having a soapy potassium salt. Commercially available propionate is generally sold as a monovalent cation salt of propionic acid, such as sodium propionate (CH₃CH₂COONa), or as a divalent cation salt, such as calcium propionate (Ca(CH₃CH₂COO)₂). Propionate is membrane-permeable and is metabolized to 2-MC by conversion of propionate to propionyl-CoA by PrpE (propionyl-CoA synthetase), and then conversion of propionyl-CoA to 2-MC by PrpC (2-methylcitrate synthase). The other proteins encoded by the prpBCDE operon, PrpD (2-methylcitrate dehydratase) and PrpB (2- methylisocitrate lyase), are involved in further catabolism of 2-MC into smaller products such as pyruvate and succinate. In order to maximize induction of a propionate-inducible promoter by propionate added to the cell growth medium, it is therefore desirable to have a host cell with PrpC and PrpE activity, to convert propionate into 2-MC, but also having eliminated or reduced PrpD activity, and optionally eliminated or reduced PrpB activity as well, to prevent 2-MC from being metabolized. Another operon encoding proteins involved in 2-MC biosynthesis is the scpA-argK-scpBC operon, also called the sbm-yg/DGH operon. These genes encode proteins required for the conversion of succinate to propionyl-CoA, which can then be converted to 2-MC by PrpC. Elimination or reduction of the function of these proteins would remove a parallel pathway for the production of the 2-MC inducer and thus might reduce background levels of expression of a propionate-inducible promoter and increase the sensitivity of the propionate-inducible promoter to exogenously supplied propionate. It has been found that a deletion of sbm-ygfD-ygfG-ygfH-ygfl was introduced into E. coli BL21 (DE3) to create strain JSB (Lee and Keasling, "A propionate-inducible expression system for enteric bacteria", Appl Environ Microbiol 2005 Nov; 71 (11 ):6856-62), was helpful in reducing background expression in the absence of exogenously supplied inducer, but this deletion also reduced overall expression from the prp promoter in strain JSB. It should be noted, however, that the deletion sbm-ygfD-ygfG-ygfH-ygfl also apparently affects ygfl, which encodes a putative LysR-family transcriptional regulator of unknown function. The genes sbm-yg/DGH are transcribed as one operon, and ygfl is transcribed from the opposite strand. The 3' ends of the ygfti and ygfl coding sequences overlap by a few base pairs, so a deletion that takes out all of the sbm- yg/DGH operon apparently takes out ygfl coding function as well. Eliminating or reducing the function of a subset of the sbm- ygfDGH gene products, such as YgfG (also called ScpB, methylmalonyl-CoA decarboxylase), or deleting the majority of the sbm-yg/DGH (or scpA-argK-scpBC) operon while leaving enough of the 3' end of the ygfli (or scpC) gene so that the expression of ygfl is not affected, could be sufficient to reduce background expression from a propionate- inducible promoter without reducing the maximal level of induced expression.

[0051] Rhamnose promoter. (As used herein, 'rhamnose' means L-rhamnose.) The 'rhamnose promoter' or 'rha promoter', or PrhaSR, is the promoter for the E. coli rhaSR operon. Like the ara and prp promoters, the rha promoter is part of a bidirectional promoter, controlling the expression of the rhaSR operon in one direction, and with the rhaBAD promoter controlling the expression of the rhaBAD operon in the other direction. The rha promoter, however, has two transcriptional regulators involved in modulating expression: RhaR and RhaS. The RhaR protein activates the expression of the rhaSR operon in the presence of rhamnose, while the RhaS protein activates the expression of the L-rhamnose catabolic and transport operons, rhaBAD and rhaT, respectively (Wickstrum et al., J Bacteriol 2010 Jan; 192(1):225-32). Although the RhaS protein can also activate expression of the rhaSR operon, in effect RhaS negatively autoregulates this expression by interfering with the ability of the cyclic AMP receptor protein (CRP) to coactivate expression with RhaR to a much greater level. The rhaBAD operon encodes the rhamnose catabolic proteins RhaA (L-rhamnose isomerase), which converts L-rhamnose to L-rhamnulose; RhaB (rhamnulokinase), which phosphorylates L-rhamnulose to form L-rhamnulose-1 -P; and RhaD (rhamnulose-1 -phosphate aldolase), which converts L-rhamnulose-1-P to L-lactaldehyde and DHAP (dihydroxy acetone phosphate). To maximize the amount of rhamnose in the cell available for induction of expression from a rhamnose-inducible promoter, it is desirable to reduce the amount of rhamnose that is broken down by catalysis, by eliminating or reducing the function of RhaA, or optionally of RhaA and at least one of RhaB and RhaD. E. co// cells can also synthesize L-rhamnose from alpha-D-glucose-1-P through the activities of the proteins RmlA, RmlB, RmIC, and RmID (also called RfbA, RfbB, RfbC, and RfbD, respectively) encoded by the rmIBDACX (or rfbBDACX) operon. To reduce background expression from a rhamnose-inducible promoter, and to enhance the sensitivity of induction of the rhamnose-inducible promoter by exogenously supplied rhamnose, it could be useful to eliminate or reduce the function of one or more of the RmlA, RmlB, RmIC, and RmID.

[0052] RmID proteins. L-rhamnose is transported into the cell by RhaT, the rhamnose permease, or L-rhamnose: proton symporter. As noted above, the expression of RhaT is activated by the transcriptional regulator RhaS. To make expression of RhaT independent of induction by rhamnose (which induces expression of RhaS), the host cell can be altered so that all functional RhaT coding sequences in the cell are expressed from constitutive promoters. Additionally, the coding sequences for RhaS can be deleted or inactivated, so that no functional RhaS is produced. By eliminating or reducing the function of RhaS in the cell, the level of expression from the rhaSR promoter is increased due to the absence of negative autoregulation by RhaS, and the level of expression of the rhamnose catalytic operon rhaBAD is decreased, further increasing the ability of rhamnose to induce expression from the rha promoter.

[0053] Xylose promoter. (As used herein, ‘xylose’ means D-xylose.) The xylose promoter, or xyl promoter’, or PxyiA, means the promoter for the E. coll xylAB operon. The xylose promoter region is similar in organization to other inducible promoters in that the xylAB operon and the xylFGHR operon are both expressed from adjacent xylose-inducible promoters in opposite directions on the E. co// chromosome (Song and Park, J Bacteriol. 1997 Nov; 179(22):7025-32). The transcriptional regulator of both the PxyiA and PxyiF promoters is XylR, which activates the expression of these promoters in the presence of xylose. The xylR gene is expressed either as part of the xylFGHR operon or from its own weak promoter, which is not inducible by xylose, located between the xylH and xylR proteincoding sequences. D-xylose is catabolized by XylA (D-xylose isomerase), which converts D- xylose to D-xylulose, which is then phosphorylated by XylB (xylulokinase) to form D- xylulose-5-P. To maximize the amount of xylose in the cell available for induction of expression from a xylose-inducible promoter, it is desirable to reduce the amount of xylose that is broken down by catalysis, by eliminating or reducing the function of at least XylA, or optionally of both XylA and XylB. The xylFGHR operon encodes XylF, XylG, and XylH, the subunits of an ABC super-family high-affinity D-xylose transporter. The xylE gene, which encodes the E. co// low-affinity xylose-proton symporter, represents a separate operon, the expression of which is also inducible by xylose. To make expression of a xylose transporter independent of induction by xylose, the host cell can be altered so that all functional xylose transporters are expressed from constitutive promoters. For example, the xylFGHR operon could be altered so that the xylFGH coding sequences are deleted, leaving XylR as the only active protein expressed from the xylose inducible PxyiF promoter, and with the xylE coding sequence expressed from a constitutive promoter rather than its native promoter. As another example, the xylR coding sequence is expressed from the PxyiA or the promoter in an expression construct, while either the xylFGHR operon is deleted and xylE is constitutively expressed, or alternatively a xylFGH operon (lacking the xylR coding sequence since that is present in an expression construct) is expressed from a constitutive promoter and the xylE coding sequence is deleted or altered so that it does not produce an active protein.

[0054] Lactose promoter. The term 'lactose promoter' refers to the lactose-inducible promoter for the lacZYA operon, a promoter which is also called lacZpl; this lactose promoter is located at ca. 365603-365568 (minus strand, with the NA polymerase binding ('-35') site at ca. 365603-365598, the Pribnow box ('-10') at 365579-365573, and a transcription initiation site at 365567) in the genomic sequence of the E. coli K-12 substrain MG1655 (NCBI Reference Sequence NC 000913.2, 11 -JAN-2012). In some embodiments, inducible coexpression systems of the disclosure can comprise a lactose-inducible promoter such as the lacZYA promoter. In other embodiments, the inducible coexpression systems of the disclosure comprise one or more inducible promoters that are not lactose-inducible promoters.

[0055] Alkaline phosphatase promoter. The terms ‘alkaline phosphatase promoter’ and ‘phoA promoter’ refer to the promoter for the phoApsiF operon, a promoter that is induced under conditions of phosphate starvation. The phoA promoter region is located at ca.

401647-401746 (plus strand, with the Pribnow box ('-1 O') at 401695-401701 (Kikuchi et al., Nucleic Acids Res 1981 Nov 11 ; 9(21 ):5671 -78)) in the genomic sequence of the E. coli K- 12 substrain MG1655 (NCBI Reference Sequence NC 000913.3, 16-DEC-2014). The transcriptional activator for the phoA promoter is PhoB, a transcriptional regulator that, along with the sensor protein PhoR, forms a two-component signal transduction system in E. coli. PhoB and PhoR are transcribed from the phoBR operon, located at ca. 417050-419300 (plus strand, with the PhoB coding sequence at 417,142-417,831 and the PhoR coding sequence at 417,889-419,184) in the genomic sequence of the E. coli K-12 substrain MG1655 (NCBI Reference Sequence NC 000913.3, 16-DEC-2014). The phoA promoter differs from the inducible promoters described above in that it is induced by the lack of a substance-e.g., intracellular phosphate-rather than by the addition of an inducer. For this reason, the phoA promoter is generally used to direct the transcription of gene products that are to be produced at a stage when the host cells are depleted for phosphate, such as the later stages of fermentation. In some embodiments, inducible coexpression systems of the disclosure can comprise a phoA promoter. In other embodiments, the inducible coexpression systems of the disclosure comprise one or more inducible promoters that are not phoA promoters.

[0056] As described herein, it may be advantageous or desirable to remove (e.g., by way of an inducible or constitutive "curing" mechanism) an expression construct described herein, e.g., if the cell line harboring the expression construct is or will be used for commercial purposes. Thus, in some embodiments, the expression construct may comprise a "kill switch." For example, in embodiment, the expression construct includes a temperature-sensitive origin of replication. Additional curing methods are known in the art and include using detergents and intercalating agents, drugs, and antibiotics (Buckner, M.M.C., et al., FEMS Microbiology Reviews, fuy031 ,42, 2018, 781 -804).

[0057] Intimin autotransporter. The term “Intimin autotransporter” refers to the coding region of an outer membrane protein from enterohemorrhagic E. coli 0157:H7 (EHEC) that mediates attachment to host cells. The coding region of the intimin autotransporter contains the following: an N-terminal signal sequence, LysM and p-domains, Ig-like domain DO, and the C-terminal passenger domain. In some embodiments, a functional fragment or portion of the intimin transported is contemplated herein.

Host Cells

[0058] The methods described herein comprise expressing an antibody or an antigenbinding protein in a host cell. A variety of host cells are suitable for expressing an antibody, and these may be selected from, for example, prokaryotic cells, yeast cells, insect cells, mammalian cells, or transgenic animals or plants. In one embodiment, the host cells are E. co// cells. As described herein, in another embodiment the SoluPro E. co// strain is contemplated (See, e.g., WO/2014/025663 and WO/2017/106583). In one embodiment, provided herein is a SoluPro strain (EB128) containing a deletion of a periplasmic protease (DegP).

[0059] Non-limiting examples of suitable mammalian host cells include but are not limited to, Chinese hamster ovary cells (CHO); monkey kidney CV1 cells transformed by SV440 (COS cells, COS-7, ATCC CRL-1651); human embryonic kidney cells (e.g., 293 cells); baby hamster kidney cells (BHK, ATCC CCL-10); monkey kidney cells (CV1 , ATCC CCL-70); African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); mouse Sertoli cells; human cervical carcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); and mouse mammary tumor cells (MMT 060562, ATCC CCL-51).

[0060] In an embodiment, the host cell is prokaryotic. Prokaryotic cells can include archaea (such as Haloferax volcanii, Sulfolobus solfataricus), Gram-positive bacteria (such as Bacillus subtilis, Bacillus licheniformis, Brevibacillus choshinensis, Lactobacillus brevis, Lactobacillus buchneri, Lactococcus lactis, and Streptomyces lividans), or Gram-negative bacteria, including Alphaproteobacteria (Agro bacterium tumefaciens, Caulobacter crescentus, Rhodobacter sphaeroides, and Sinorhizobium meliloti), Betaproteobacteria (Alcaligenes eutrophus), and Gammaproteobacteria (Acinetobacter calcoaceticus, Azotobacter vinelandii, Escherichia coll, Pseudomonas aeruginosa, and Pseudomonas putida). Exemplary host cells include Gammaproteobacteria of the family Enterobacteriaceae, such as Enterobacter, Erwinia, Escherichia (including E. coll), Klebsiella, Proteus, Salmonella (including Salmonella typhimurium), Serratia (including Serratia marcescens), and Shigella.

[0061] As described in International Publication No. WO 2017/106583, incorporated by reference in its entirety herein, producing an antigen binding protein at commercial scale and in soluble form is addressed by providing suitable host cells capable of growth at high cell density in fermentation culture, and which can produce soluble gene products in the oxidizing host cell cytoplasm through highly controlled inducible gene expression.

Prokaryotic cells with these qualities are produced by combining some or all of the following characteristics: (1) The host cells are genetically modified to have an oxidizing cytoplasm, by increasing the expression or function of oxidizing polypeptides in the cytoplasm, and/or by decreasing the expression or function of reducing polypeptides in the cytoplasm. Specific examples of such genetic alterations are provided herein. Optionally, host cells can also be genetically modified to express chaperones and/or cofactors that assist in the production of the desired gene product(s), and/or to glycosylate polypeptide gene products. (2) The host cells comprise one or more expression constructs designed for the expression of one or more gene products of interest; in certain embodiments, at least one expression construct comprises an inducible promoter and a polynucleotide encoding a gene product to be expressed from the inducible promoter. (3) The host cells contain additional genetic modifications designed to improve certain aspects of gene product expression from the expression construct(s). In some embodiments, the host cell strain lack an oxidizing cytoplasm. In particular embodiments, the host cells (A) have an alteration of gene function of at least one gene encoding a transporter protein for an inducer of at least one inducible promoter, and as another example, wherein the gene encoding the transporter protein is selected from the group consisting of araE, araE, araG, araH, rhaT, xylF, xylG, and xylH, or particularly is araE, or wherein the alteration of gene function more particularly is expression of araE from a constitutive promoter; and/or (B) have a reduced level of gene function of at least one gene encoding a protein that metabolizes an inducer of at least one inducible promoter, and as further examples, wherein the gene encoding a protein that metabolizes an inducer of at least one said inducible promoter is selected from the group consisting of araA, araB, araD, prpB, prpD, rhaA, rhaB, rhaD, xylA, and xylB; and/or (C) have a reduced level of gene function of at least one gene encoding a protein involved in biosynthesis of an inducer of at least one inducible promoter, which gene in further embodiments is selected from the group consisting of scpA/sbm, argK/ygfD, scpB/ygfG, scpC/ygfH, rmlA, rmlB, rmIC, and rmID.

[0062] Prokaryotic Cells with Oxidizing Cytoplasm. Examples of host cells are provided that allow for the efficient and cost-effective expression of gene products, including components of multimeric products. Host cells can include, in addition to isolated cells in culture, cells that are part of a multicellular organism, or cells grown within a different organism or system of organisms. In certain embodiments of the disclosure, the host cells are microbial cells such as yeasts (Saccharomyces, Schizosaccharomyces, etc.) or bacterial cells, or are gram-positive bacteria or gram-negative bacteria, or are E. coli, or are E. coli B strain, or are E. coli (B strain) EB0001 cells (also called E. coli ASE(DGH) cells) or are E. coli (B strain) EB0002 cells. In growth experiments with E. co// host cells having oxidizing cytoplasm, specifically the E. coli B strains SHuffle® Express (NEB Catalog No. C3028H) and SHuffle® T7 Express (NEB Catalog No. C3029H) and the E. coli K strain SHuffle® T7 (NEB Catalog No. C3026H), these E. coli B strains with oxidizing cytoplasm are able to grow to much higher cell densities than the most closely corresponding E. coli K strain (International Publication No. WO 2017/106583).

[0063] Certain alterations can be made to the gene functions of host cells comprising inducible expression constructs, to promote efficient and homogeneous induction of the host cell population by an inducer. In some embodiments, the combination of expression constructs, host cell genotype, and induction conditions results in at least 75% (more preferably at least 85%, and most preferably, at least 95%) of the cells in the culture expressing gene product from each induced promoter, as measured by the method of Khlebnikov et al. described in Example 9 of International Publication No. WO 2017/106583. For host cells other than E. coli, these alterations can involve the function of genes that are structurally similar to an E. co// gene, or genes that carry out a function within the host cell similar to that of the E. co// gene. Alterations to host cell gene functions include eliminating or reducing gene function by deleting the gene protein-coding sequence in its entirety, or deleting a large enough portion of the gene, inserting sequence into the gene, or otherwise altering the gene sequence so that a reduced level of functional gene product is made from that gene. Alterations to host cell gene functions also include increasing gene function by, for example, altering the native promoter to create a stronger promoter that directs a higher level of transcription of the gene, or introducing a missense mutation into the protein-coding sequence that results in a more highly active gene product. Alterations to host cell gene functions include altering gene function in any way, including, for example, altering a native inducible promoter to create a promoter that is constitutively activated. In addition to alterations in gene functions for the transport and metabolism of inducers, as described herein with relation to inducible promoters, and/or an altered expression of chaperone proteins, it is also possible to alter the reduction-oxidation environment of the host cell.

[0064] Host cell reduction-oxidation environment. In bacterial cells such as E. coli, proteins that need disulfide bonds are typically exported into the periplasm, where disulfide bond formation and isomerization are catalyzed by the Dsb system, comprising DsbABCD and DsbG. Increased expression of the cysteine oxidase DsbA, the disulfide isomerase DsbC, or combinations of the Dsb proteins, which are all normally transported into the periplasm, has been utilized in the expression of heterologous proteins that require disulfide bonds (Makino et al., Microb Cell Fact 2011 May 14;10:32). It is also possible to express cytoplasmic forms of these Dsb proteins, such as a cytoplasmic version of DsbA and/or of DsbC ('cDsbA' or 'cDsbC'), that lacks a signal peptide and therefore is not transported into the periplasm. Cytoplasmic Dsb proteins such as cDsbA and/or cDsbC are useful for making the cytoplasm of the host cell more oxidizing and thus more conducive to the formation of disulfide bonds in heterologous proteins produced in the cytoplasm. The host cell cytoplasm can also be made less reducing and thus more oxidizing by altering the thioredoxin and the glutaredoxin/glutathione enzyme systems directly: mutant strains defective in glutathione reductase (gor) or glutathione synthetase (gshB), together with thioredoxin reductase (trxB), render the cytoplasm oxidizing. These strains are unable to reduce ribonucleotides and, therefore, cannot grow in the absence of exogenous reductants, such as dithiothreitol (DTT). Suppressor mutations (such as ahpC* and ahpCA, Lobstein et al., Microb Cell Fact 2012 May 8; 11 :56; doi: 10.1186/1475-2859-11 -56) in the gene ahpC, which encodes the peroxiredoxin AhpC, convert it to a disulfide reductase that generates reduced glutathione, allowing the channeling of electrons onto the enzyme ribonucleotide reductase and enabling the cells defective in gor and trxB, or defective in gshB and trxB, to grow in the absence of DTT. A different class of mutated forms of AhpC can allow strains, defective in the activity of gamma-glutamylcysteine synthetase (gshA) and defective in trxB, to grow in the absence of DTT ; these include AhpC V164G, AhpC S71 F, AhpC E173/S71 F, AhpC E171Ter, and AhpC dupl62-169 (Faulkner et al., Proc Natl Acad Sci USA 2008 May 6; 105(18): 6735-40, Epub 2008 May 2). In such strains with oxidizing cytoplasm, exposed protein cysteines become readily oxidized in a process that is catalyzed by thioredoxins, in a reversal of their physiological function, resulting in the formation of disulfide bonds. Other proteins that may be helpful to reduce the oxidative stress effects in host cells of an oxidizing cytoplasm are HPI (hydroperoxidase I) catalase-peroxidase encoded by E. coll katG and HPII (hydroperoxidase II) catalase-peroxidase encoded by E. coll katE, which disproportionate peroxide into water and 02 (Farr and Kogoma, Microbiol Rev. 1991 Dec; 55(4):561-585; Review). Increasing levels of KatG and/or KatE protein in host cells through induced coexpression or through elevated levels of constitutive expression is an aspect of some embodiments of the disclosure.

[0065] Another alteration that can be made to host cells is to express the sulfhydryl oxidase Ervlp from the inner membrane space of yeast mitochondria in the host cell cytoplasm, which has been shown to increase the production of a variety of complex, disulfide-bonded proteins of eukaryotic origin in the cytoplasm of E. coll, even in the absence of mutations in gor or trxB (Nguyen et al, Microb Cell Fact 2011 Jan 7; 10:1).

[0066] Host cells comprising expression constructs preferably also express cDsbA and/or cDsbC and/or Ervlp; are deficient in trxB gene function; are also deficient in the gene function of either gor, gshB, or gshA; optionally have increased levels of katG and/or katE gene function; and express an appropriate mutant form of AhpC so that the host cells can be grown in the absence of DTT.

[0067] In one embodiment, the materials and assays described herein do not require a chaperone. In other embodiments, the host cell does not have an oxidizing environment.

[0068] ParB. ParB refers to the hok/sok system, a type I toxin-antitoxin pair that acts as a post-segregational killing mechanism in E. coll. The hok gene encodes a toxic protein and the sok gene is an anti-sense RNA that binds to hok mRNA, leading to rapid degradation. In cells lacking ParB-containing plasmids, the hok mRNA, which is much more stable than sok, gets translated into a toxic, cell death-inducing protein. This eliminates any cells that lack a plasmid. In this embodiment, ParB is used to stabilize plasmid retention during the induction of surfACE constructs.

[0069] Chaperones. In some embodiments, desired gene products are coexpressed with other gene products, such as chaperones, that are beneficial to the production of the desired gene product. Chaperones are proteins that assist the non-covalent folding or unfolding, and/or the assembly or disassembly, of other gene products, but do not occur in the resulting monomeric or multimeric gene product structures when the structures are performing their normal biological functions (having completed the processes of folding and/or assembly). Chaperones can be expressed from an inducible promoter or a constitutive promoter within an expression construct or can be expressed from the host cell chromosome; preferably, expression of chaperone protein(s) in the host cell is at a sufficiently high level to produce coexpressed gene products that are properly folded and/or assembled into the desired product. Examples of chaperones present in E. coli host cells are the folding factors DnaK/DnaJ/GrpE, DsbC/DsbG, GroEL/GroES, IbpA/IbpB, Skp, Tig (trigger factor), and FkpA, which have been used to prevent protein aggregation of cytoplasmic or periplasmic proteins. DnaK/DnaJ/GrpE, GroEL/GroES, and CIpB can function synergistically in assisting protein folding and therefore expression of these chaperones in combinations has been shown to be beneficial for protein expression (Makino et al., Microb Cell Fact 2011 May 14;10:32). When expressing eukaryotic proteins in prokaryotic host cells, a eukaryotic chaperone protein, such as protein disulfide isomerase (PDI) from the same or a related eukaryotic species, is in certain embodiments of the disclosure coexpressed or inducibly coexpressed with the desired gene product.

[0070] One chaperone that can be expressed in host cells is a protein disulfide isomerase from Humicola insolens, a soil hyphomycete (soft-rot fungus). An amino acid sequence of Humicola insolens PDI is shown as SEQ ID NO: 1 of International Publication No. WO 2017/106583; it lacks the signal peptide of the native protein so that it remains in the host cell cytoplasm. The nucleotide sequence encoding PDI was optimized for expression in E. coli; the expression construct for PDI is shown as SEQ ID NO: 2 of International Publication No. WO 2017/106583. SEQ ID NO: 2 contains a GCTAGC Nhel restriction site at its 5' end, an AGGAGG ribosome binding site at nucleotides 7 through 12, the PDI coding sequence at nucleotides 21 through 1478, and a GTCGAC Sall restriction site at its 3' end. The nucleotide sequence of SEQ ID NO: 2 was designed to be inserted immediately downstream of a promoter, such as an inducible promoter. The Nhel and Sall restriction sites in SEQ ID NO: 2 can be used to insert it into a vector multiple cloning sites, such as that of the pSOL expression vector (SEQ ID NO: 3 of International Publication No. WO 2017/106583), described in published U.S. Patent Application Publication No. 2015/353940A1 , which is incorporated by reference in its entirety herein. Other PDI polypeptides can also be expressed in host cells, including PDI polypeptides from a variety of species (Saccharomyces cerevisiae (UniProtKB PI 7967), Homo sapiens (UniProtKB P07237), Mus musculus (UniProtKB P09103), Caenorhabditis elegans (UniProtKB Q 17770 and Q 17967), Arabdopsis thaliana (UniProtKB 048773, Q9XI01 , Q9S G3, Q9LJU2, Q9MAU6, Q94F09, and Q9T042), Aspergillus niger (UniProtKB Q12730) and also modified forms of such PDI polypeptides. In certain embodiments of the disclosure, a PDI polypeptide expressed in host cells of the disclosure shares at least 70%, 80%, 90%, or 95% amino acid sequence identity across at least 50% (or at least 60%, or at least 70%, or at least 80%, or at least 90%) of the length of SEQ ID NO: 1 of International Publication No. WO 2017/106583, where amino acid sequence identity is determined according to Example 10 of International Publication No. WO 2017/106583.

[0071] Cellular transport of cofactors. Common cofactors include ATP, coenzyme A, flavin adenine dinucleotide (FAD), NAD+/NADH, and heme. Polynucleotides encoding cofactor transport polypeptides and/or cofactor synthesizing polypeptides can be introduced into host cells, and such polypeptides can be constitutively expressed, or inducibly coexpressed with the gene products to be produced by methods of the disclosure.

[0072] Glycosylation of polypeptide gene products. Host cells can have alterations in their ability to glycosylate polypeptides. For example, eukaryotic host cells can have eliminated or reduced gene function in glycosyltransferase and/or oligo saccharyltransferase genes, impairing the normal eukaryotic glycosylation of polypeptides to form glycoproteins. Prokaryotic host cells such as E. coll, which do not normally glycosylate polypeptides, can be altered to express a set of eukaryotic and prokaryotic genes that provide a glycosylation function (DeLisa et al., WO 2009/089154A2, 2009 Jul 16).

[0073] Available host cell strains with altered gene functions. To create preferred strains of host cells to be used in the expression systems and methods of the disclosure, it is useful to start with a strain that already comprises desired genetic alterations (Table A; International Publication No. WO 2017/106583).

[0074] Table A. Exemplary host cell strains

Biomolecules and cargo proteins

[0075] In some embodiments, the methods of the current disclosure comprise the expression of a biomolecule variant library, including cargo proteins, in host cells. In some embodiments, the biomolecules provided in the methods herein are biomolecules from Al- designed libraries.

[0076] As used herein, the term "biomolecule" or "biological molecule" refers to a molecule that is generally found in a biological organism. Typical biomolecules include, but are not limited to, RNA, DNA, peptides, polypeptides or proteins, lipids, carbohydrates, or other organic molecules. In some embodiments, the surface displayed antibody is a biomolecule. In some embodiments, the intimin transporter is a biomolecule. In some embodiments, the regulatory proteins expressed in the host cells are biomolecules. The term “biomolecule variants” as used herein, refers to new biomolecules whose sequences differ from the sequence of a parental biomolecule through mutations that are introduced according to the methods of the disclosure. In some embodiments, the biomolecule comprises a surface displayed antibody, an intimin transporter and optionally a regulatory protein.

[0077] As used herein, the term “parental polypeptide,” “parental polynucleotide,” “parent nucleic acid,” and “parent” are generally used to refer to the wild-type polypeptide, wild-type polynucleotide, or a variant used as a starting point in a diversity generation procedure such as a directed evolution. In some embodiments, the parent itself is produced via shuffling or other diversity generation procedure. In some embodiments, mutants used in directed evolution are directly related to a parent polypeptide. In some embodiments, the parent polypeptide is stable when exposed to extremes of temperature, pH and/or solvent conditions and can serve as the basis for generating variants for shuffling. In some embodiments, the parental polypeptide is not stable to extremes of temperature, pH and/or solvent conditions, and the parental polypeptide is evolved to make robust variants. In some embodiments, the parental polypeptide is any reference antibody displayed that is known to bind the antigen of interest for library screening. In some embodiments, the reference antibody displayed is a positive control.

[0078] As used herein, the term "directed evolution" or "artificial evolution" refers to the modification and improvement of biomolecule function by mimicking "Darwinian selection" through iterations of mutation and screening or selection for improved properties. After each step, the most promising candidates are used as templates for a new round of mutation and screening or selection. This strategy can be repeated until the desired features are obtained.

[0079] As used herein, the term “rational design” or “structure-based design” refers to the modification and improvement of biomolecule, such as an antibody, for targeting specific epitope by modifying specific amino acid(s) to alter the structure of the biomolecule based on available structural information.

[0080] As used herein, the term “Al-assisted design” refers to the modification and improvement of biomolecule for targeting specific epitope by using artificial intelligence to predict the modifications to be made to the specific biomolecule, thereby, improving targeting capability of the biomolecule to the target epitope.

[0081] In some embodiments, the biomolecule is a cargo protein such as an antibody, an enzyme, a hormone, a cytokine, a growth factor, a clotting factor, an anticoagulation factor, albumin, an antigen, an adjuvant, a transcription factor, or a cellular receptor.

[0082] Cytokines include but are not limited to, chemokines, interferons, interleukins, lymphokines, and tumor necrosis factors. Cellular receptors, such as cytokine receptors, are also contemplated. Examples of cytokines and cellular receptors include, but are not limited to, tumor necrosis factor alpha and beta and their receptors; lipoproteins; colchicine; corticotropin; vasopressin; somatostatin; lypressin; pancreozymin; leuprolide; alpha-1 - antitrypsin; atrial natriuretic factor; thrombin; enkephalinase; RANTES (regulated on activation normally T-cell expressed and secreted); human macrophage inflammatory protein (MIP-1 -alpha); cell determinant proteins such as CD-3, CD-4, CD-8, and CD-19; erythropoietin; interferon-alpha, -beta, -gamma, -lambda; colony-stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; IL-1 , 2, 3, 4, 5, 6, 7, 8, 9 and/or IL-10; T-cell receptors; and prostaglandin.

[0083] Examples of hormones include, but are not limited to, antidiuretic hormone (ADH), oxytocin, growth hormone (GH), prolactin, growth hormone-releasing hormone (GHRH), thyroid stimulating hormone (TSH), thyrotropin-release hormone (TRH), adrenocorticotropic hormone (ACTH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), luteinizing hormone-releasing hormone (LHRH), thyroxine, calcitonin, parathyroid hormone, aldosterone, cortisol, epinephrine, glucagon, insulin, estrogen, progesterone, and testosterone.

[0084] Examples of growth factors include, e.g., vascular endothelial growth factor (VEGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), transforming growth factor (TGF), bone morphogenic proteins (BMPs), and insulin-like growth factor-1 and -II (IGF-I and IGF-II).

[0085] Examples of clotting factors or coagulation factors include Factor I, Factor II, Factor III, Factor V, Factor VI, Factor VII, Factor VIII, Factor VIIIC, Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, von Willebrand factor (vWF), prekallikrein, heparin cofactor II, antithrombin III, and fibronectin.

[0086] Examples of enzymes include but are not limited to, angiotensin-converting enzyme, streptokinase, L-asparaginase, and the like. Other examples of enzymes include, e.g., nitrate reductase (NADH), catalase, peroxidase, nitrogenase, phosphatase (e.g., acid/alkaline phosphatases), phosphodiesterase I, inorganic diphosphatase (pyrophosphatase), dehydrogenase, sulfatase, arylsulfatase, thiosulfate sulfurtransferase, L- asparaginase/L-glutaminase, beta-glucosidase, aryl acylamindase, amidase, invertase, xylanase, cellulose, urease, phytases, carbohydrase, amylase (alpha-amylase/beta- amylase), arabinoxylanase, beta-glucanase, alpha-galactosidase, beta-mannanase, pectinase, non-starch polysaccharide degrading enzymes, endoproteases, exoproteases, lipases, cellulases, oxidoreductases, ligases, synthetases (e.g., aminoacyl-transfer RNA synthetase; glycyl-tRNA synthetase), transferases, hydrolases, lyase (e.g., decarboxylases, dehydratases, deaminases, aldolases), isomerases (e.g., triose phosphate isomerase), and trypsin. Further examples of enzymes include catalases (e.g., alkali-resistant catalases), alkaline amylase, pectinase, oxidase, laccases, proxidases, xylanases, mannanases, acylases, alcalase, alkylsulfatase, cellulolytic enzymes, cellobio-hydrolase, cellobiase, exo- 1 ,4-beta-D-glucosidase, chloroperoxidase, chitinase, cyanidase, cyanide hydratase, I- Galactono-lactone oxidase, lignin peroxidase, lysozyme, mn-peroxidase, muramidase, parathion hydrolase, pectinesterase, peroxidase, and tryosinase. Further examples of enzymes include nuclease (e.g., endonuclease, such as zinc finger nucleases, transcription activator-like effector nuclease, Cas nucleases, and engineered mega nucleases).

[0087] In an embodiment, the biomolecule or cargo protein is an antibody. The term "antibody" refers to an intact antigen-binding immunoglobulin. In various embodiments, an intact antibody comprises two full-length heavy chains and two full-length light chains. In a full-length antibody, each heavy chain consists of a heavy chain variable region (abbreviated herein VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains: CH1 , CH2, and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., lgG1 , lgG2, IgG 3, lgG4, lgA1 and lgA2) or subclass.

[0088] In an embodiment, the biomolecule is an "antigen-binding fragment" of an antibody. Examples of antigen-binding fragments of antibodies include, but are not limited to

(i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains;

(ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341 :544-46, Winter et al., PCT Publication No. WO 90/05144), which comprises a single variable domain. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423- 26; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-83). Such single-chain antibodies (scFv) are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. In some embodiments, the antigen-binding fragment to be used for surface display ACE (surfACE) is a VHH. In some embodiments, the antigen-binding fragment to be used for surface display ACE is a scFv. In some embodiments, the antigenbinding fragment to be used for surface display ACE is a scFab. In one embodiment, the cargo protein is a VHH or nanobody.

[0089] The architecture of antibodies has been exploited to create a growing range of alternative formats that span a molecular-weight range of at least about 12-150 kDa and have a valency (n) range from monomeric, to dimeric, to trimeric, to tetrameric, and potentially higher; such alternative formats are referred to herein as “antibody-like constructs.” Antibody-like protein constructs include those based on the full antibody structure and those that mimic antibody fragments that retain full antigen-binding capacity, e.g., scFvs, Fabs, and VHH. The smallest antigen-binding fragment that retains its complete antigen-binding site is the Fv fragment, which consists entirely of variable (V) regions. Other antibody-like protein constructs include disulfide-bond stabilized scFv (ds-scFv), single chain Fab (scFab), as well as di- and multimeric antibody formats like dia-, tria- and tetra-bodies, or minibodies (miniAbs) that comprise different formats consisting of scFvs linked to oligomerization domains. The smallest fragments are VHH/VH of camelid heavy chain Abs as well as single domain Abs (sdAb). A building block that is frequently used to create different antibody formats is the single-chain variable (V)-domain antibody fragment (scFv), which comprises V domains from the heavy and light chain (VH and VL domain) linked by a peptide linker of ~15 amino acid residues. A peptibody or peptide-Fc fusion is yet another antibody-like construct protein product. The structure of a peptibody consists of a biologically active peptide grafted onto an Fc domain. Peptibodies are described in the art. See, e.g., Shimamoto et al., mAbs 4(5): 586-91 (2012). Other antibody-like protein constructs include a single-chain antibody (SCA), a diabody, a triabody, a tetrabody, and the like.

[0090] In an embodiment, the biomolecule or cargo protein may be a multi-specific antibody (e.g., a bispecific antibody or trispecific antibody) having the CDR sequences set forth herein. Bispecific antibody products can be divided into five major classes: BsIgG, appended IgG, BsAb fragments (e.g., bispecific single chain antibodies), bispecific fusion proteins (e.g., antigen binding domains fused to an effector moiety), and BsAb conjugates. See, e.g., Spiess et al., Molecular Immunology 67 (2) Part A:97-106 (2015). Examples of bispecific antibody constructs include, but are not limited to, tandem scFvs and Fab2 bispecifics. See, e.g., Chames & Baty, 2009, mAbs 1 [6]:1 -9; and Holliger & Hudson, 2005, Nature Biotechnology 23[9]:1126-36; Wu et al., 2007, Nature Biotechnology 25[11 ]:1290-97; Michaelson et al., 2009, mAbs 1 [2]:128-41 ; International Patent Publication Nos. WO 2009032782 and WO 2006020258; Zuo et al., 2000, Protein Engineering 13[5]:361 -67; U.S. Patent Application Publication No. 20020103345; Shen et al., 2006, J Biol Chem 281 [16]:10706-14; Lu et al., 2005, J Biol Chem 280[20]:19665-72; and Kontermann, 2012 MAbs 4(2) : 182, all of which are expressly incorporated herein. Multispecific antibody constructs, such as trispecific antibody constructs (including three binding domains) or constructs having more than three (e.g., four, five, or more) specificities, also are contemplated.

[0091] The antibodies (or antigen-binding fragments thereof or antibody-like protein constructs) may be a human antibody (i.e., having one or more variable and constant regions derived from human immunoglobulin sequences), humanized (i.e., have a sequence that differs from the sequence of an antibody derived from a non-human species by one or more amino acid substitutions, deletions, and/or additions, such that the humanized antibody is less likely to induce an immune response, and/or induces a less severe immune response, as compared to the non-human species antibody, when it is administered to a human subject), or chimeric (i.e., containing one or more regions from one antibody and one or more regions from one or more other antibodies).

[0092] Suitable methods of making antibodies are known in the art. For instance, standard hybridoma methods are described in, e.g., Harlow and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988), and CA. Janeway et al. (eds.), IMMUNOBIOLOGY, 5th Ed., Garland Publishing, New York, NY (2001 )). Monoclonal antibodies for use in the methods of the disclosure may be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridoma technique originally described by Koehler and Milstein Nature 256:495-97, 1975), the human B-cell hybridoma technique (Kosbor et al., Immunol Today 4:72, 1983; Cote et al., Proc Natl Acad Sci 80:2026-30, 1983) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc, New York N.Y., pp 77-96, (1985). Alternatively, other methods, such as EBV-hybridoma methods (Haskard and Archer, J. Immunol. Methods, 74(2), 361 -67 (1984), and Roder et al., Methods Enzymol., 121 , 140-67 (1986)), and bacteriophage vector expression systems see, e.g., Huse et al., Science, 246, 1275-81 (1989)) are known in the art. Further, methods of producing antibodies in non-human animals are described in, e.g., U.S. Patent Nos.

5,545,806, 5,569,825, and 5,714,352, and U.S. Patent Application Publication No. 2002/0197266 A1 ). Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Orlandi et al {Proc Natl Acad Sci SQ 3833- 37; 1989), and Winter G and Milstein C {Nature 349:293-99, 1991 ). If the full sequence of the antibody or antigen-binding fragment is known, then methods of producing recombinant proteins may be employed. See, e.g., "Protein production and purification" Nat Methods 5(2):135-46 (2008). In some embodiments, the antibodies (or antigen-binding fragments) are isolated from cell culture or a biological sample {e.g., if generated in vivo). In preferred embodiments, the antibodies (or antigen-binding fragments) are expressed by E. coli strain(s) described herein.

[0093] Exemplary antibodies or antibody targets that can be used with the methods described herein include, but are not limited to, Activase® (Alteplase); alirocumab (anti- PCSK9 monoclonal antibody designated as H1 H316P, see U.S. Patent No. 8,062,640); Aranesp® (Darbepoetin-alfa), Epogen® (Epoetin alfa, or erythropoietin); Avonex® (Interferon P-la); Bexxar® (Tositumomab); Bseron® (Interferon-p); bococizumab (anti-PCSK9 monoclonal antibody designated as L1 L3, see U.S. Patent No. 8,080,243); Campath® (Alemtuzumab); Dynepo® (Epoetin delta); Velcade® (bortezomib); MLN0002 (anti-a4 7 mAb); MLN1202 (anti-CCR2 chemokine receptor mAb); Enbrel® (etanercept); Eprex® (Epoetin alfa); Erbitux® (Cetuximab); evolocumab (anti-PCSK9 monoclonal antibody designated as 21 B12, see U.S. Patent No. 8,030,467); Genotropin® (Somatropin); Herceptin® (Trastuzumab); Humatrope® (somatropin [rDNA origin] for injection); Humira® (Adalimumab); Infergen® (Interferon Alfacon-1 ); Natrecor® (nesiritide); Kineret® (Anakinra), Leukine® (Sargamostim); LymphoCide® (Epratuzumab); Benlysta™ (Belimumab); Metalyse® (Tenecteplase); Mircera® (methoxy polyethylene glycol-epoetin P); Mylotarg® (Gemtuzumab ozogamicin); Raptiva® (efalizumab); Cimzia® (certolizumab pegol); Soliris™ (Eculizumab); Pexelizumab (Anti-C5 Complement); MEDI-524 (Numax®); Lucentis® (Ranibizumab); Edrecolomab (Panorex®); Trabio® (lerdelimumab); TheraCim hR3 (Nimotuzumab); Omnitarg (Pertuzumab, 2C4); Osidem® (IDM-I); OvaRex® (B43.13); Nuvion® (visilizumab); Cantuzumab mertansine (huC242-DMI); NeoRecormon® (Epoetin P); Neumega® (Oprelvekin); Neulasta® (Pegylated filgastrim, pegylated G-CSF, pegylated hu-Met-G-CSF); Neupogen® (Filgrastim); Orthoclone OKT3® (Muromonab-CD3), Procrit® (Epoetin alfa); Remicade® (Infliximab), Reopro® (Abciximab), Actemra® (anti-l L6 Receptor mAb), Avastin® (Bevacizumab), HuMax-CD4 (zanolimumab), Rituxan® (Rituximab); Tarceva® (Erlotinib); Roferon-A®-(lnterferon alfa-2a); Simulect® (Basiliximab); Stelara™ (Ustekinumab); Prexige® (lumiracoxib); Synagis® (Palivizumab); 146B7-CHO (anti-IL15 antibody, see U.S. Patent No. 7,153,507), Tysabri® (Natalizumab); Valortim® (MDX-1303, anti-B. anthracis Protective Antigen mAb); ABthrax™; Vectibix® (Panitumumab); Xolair® (Omalizumab), ETI211 (anti-M RSA mAb), IL-I Trap (the Fc portion of human IgGI and the extracellular domains of both IL-I receptor components (the Type I receptor and receptor accessory protein)), VEGF Trap (Ig domains of VEGFRI fused to IgGI Fc), Zenapax® (Daclizumab); Zenapax® (Daclizumab), Zevalin® (Ibritumomab tiuxetan), Zetia (ezetimibe), Atacicept (TACI-lg), anti-a4p7-integrin mAb (vedolizumab); galiximab (anti-CD80 monoclonal antibody), anti-CD23 mAb (lumiliximab); BR2-Fc (huBR3 /huFc fusion protein, soluble BAFF antagonist); Simponi (Golimumab); Mapatumumab (human anti-TRAIL Receptor-1 mAb); Ocrelizumab (anti-CD20 human mAb); HuMax-EGFR (zalutumumab); M200 (Volociximab, anti-a5 1 integrin mAb); MDX-010 (Ipilimumab, anti-CTLA-4 mAb and VEGFR-I (IMC-18F1 ); anti-BR3 mAb; antiClostridium difficile Toxin A and Toxin B C mAbs M DX-066 (CDA-I) and MDX-1388); anti- CD22 dsFv-PE38 conjugates (CAT-3888 and CAT-8015); anti-CD25 mAb (HuMax-TAC); Adecatumumab (MT201 , anti-EpCAM-CD326 mAb); MDX-060, SGN-30, SGN-35 (anti-CD30 mAbs); M DX-1333 (anti- IFNAR); HuMax CD38 (anti-CD38 mAb); anti-CD40L mAb; anti- Cripto mAb; anti-CTGF Idiopathic Pulmonary Fibrosis Phase I Fibrogen (FG-3019); anti- CTLA4 mAb; anti-eotaxinl mAb (CAT-213); anti-FGF8 mAb; anti-ganglioside GD2 mAb; antiganglioside GM2 mAb; anti-GDF-8 human mAb (MYO-029); anti-GM-CSF Receptor mAb (CAM-3001 ); anti-HepC mAb (HuMax HepC); M EDI-545, MDX-1103 (anti-IFNa mAb); anti- IGFIR mAb; anti-IGF-IR mAb (HuMax-Inflam); anti-IL12/IL23p40 mAb (Briakinumab); anti-IL- 23pl9 mAb (LY2525623); anti-IL13 mAb (CAT-354); anti-IL-17 mAb (AIN457); anti-IL2Ra mAb (HuMax-TAC); anti-IL5 Receptor mAb; anti-integrin receptors mAb (MDX-018, CNTO 95); anti-IPIO Ulcerative Colitis mAb (MDX- 1100); anti-LLY antibody; BMS-66513; antiMannose Receptor/hCG mAb (MDX-1307); anti-mesothelin dsFv-PE38 conjugate (CAT-5001); anti-PDImAb (M DX-1 106 (ONO- 4538)); anti-PDGFRa antibody (IMC-3G3); anti-TGF mAb (GC-1008); anti-TRAIL Receptor-2 human mAb (HGS-ETR2); anti-TWEAK mAb; anti-VEGFR/Flt-1 mAb; anti- ZP3 mAb (HuMax-ZP3); NVS Antibody #1 ; and NVS Antibody #2.

[0094] Additional examples of antibodies (and antigen-binding fragments thereof) include; abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab, alacizumab, alacizumab pegol, ald518, alemtuzumab, alemtuzumab, alirocumab, altinumab, altumomab, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, aselizumab, atlizumab, atorolimiumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belimumab, benralizumab, bertilimumab, besilesomab, bevacizumab, bezlotoxumab, biciromab, bivatuzumab, bivatuzumab mertansine, blinatumomab, blosozumab, brentuximab vedotin, briakinumab, brodalumab, canakinumab, cantuzumab mertansine, cantuzumab mertansine, caplacizumab, capromab pendetide, carlumab, catumaxomab, cc49, cedelizumab, certolizumab pegol, cetuximab, citatuzumab bogatox, cixutumumab, clazakizumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, cr6261 , crenezumab, dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox, drozitumab, duligotumab, dupilumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, elotuzumab, elsilimomab, enavatuzumab, enlimomab pegol, enokizumab, enokizumab, enoticumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, exbivirumab, exbivirumab, fanolesomab, faralimomab, farletuzumab, fasinumab, fbta05, felvizumab, fezakinumab, ficlatuzumab, figitumumab, flanvotumab, fontolizumab, foralumab, foravirumab, fresolimumab, fulranumab, futuximab, galiximab, ganitumab, gantenerumab, gavilimomab, gemtuzumab ozogamicin, gevokizumab, girentuximab, glembatumumab vedotin, golimumab, gomiliximab, gs6624, ibalizumab, ibritumoma tiuxetan, icrucumab, igovomab, imciromab, i mgatuzumab, inclacumab, indatuximab ravtansine, infliximab, inolimomab, inotuzumab ozogamicin, intetumumab, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lebrikizumab, lemalesomab, lerdelimumab, lexatumumab, libivirumab, ligelizumab, lintuzumab, lirilumab, lorvotuzumab mertansine, lucatumumab, lumiliximab, mapatumumab, maslimomab, matuzumab, mavrilimumab, mepolizumab, metelimumab, milatuzumab, minretumomab, mitumomab, mogamulizumab, morolimumab, motavizumab, moxetumomab pasudotox, muromonab-cd3, nacolomab tafenatox, namilumab, naptumomab estafenatox, narnatumab, natalizumab, nebacumab, necitumumab, nerelimomab, nesvacumab, nimotuzumab, nivolumab, nofetumomab merpentan, ocaratuzumab, ocrelizumab, odulimomab, ofatumumab, olaratumab, olokizumab, omalizumab, onartuzumab, oportuzumab monatox, oregovomab, orticumab, otelixizumab, oxelumab, ozanezumab, ozoralizumab, pagibaximab, palivizumab, palivizumab, panitumumab, panobacumab, parsatuzuma pascolizumab, pateclizumab, patritumab, pemtumomab, perakizumab, pertuzumab, pexelizumab, pidilizumab, pintumomab, placulumab, ponezumab, priliximab, pritumumab, PRO 140, quilizumab, racotumomab, radretumab, rafivirumab, ramucirumab, ranibizumab, raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab, robatumumab, roledumab, romosozumab, rontalizumab, rovelizumab, ruplizumab, samalizumab, sarilumab, satumomab pendetide, secukinumab, seviruma sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab, tefibazumab, telimomab aritox, telimomab aritox, tenatumomab, tenatumomab, teneliximab, teplizumab, teprotumumab, TGN1412, ticilimumab, tigatuzumab, tildrakizumab, TNX-650, tocilizumab, tocilizumab, toralizumab, tositumomab, tralokinumab, trastuzumab, TRBS07, tregalizumab, tremelimumab, tremelimumab, tucotuzumab celmoleukin, tuvirumab, ublituximab, urelumab, urtoxazumab, ustekinumab, vapaliximab, vatelizumab, vedolizumab, veltuzumab, vepalimomab, vesencumab, visilizumab, volociximab, vorsetuzumab mafodotin, votumumab, zalutumumab, zanolimumab, zatuximab, ziralimumab, and zolimomab aritox.

Biomolecule and cargo protein variant library

[0095] In some embodiments, the methods described herein comprise the generation, expression, and analysis of a biomolecule, including cargo protein, and variant library. As used herein, the term “library” or “population” refers to a collection of at least two different molecules, such as nucleic acid sequences (e.g., genes, oligonucleotides, etc.) or expression products (e.g., enzymes or other proteins) therefrom. A library or population generally includes a number of different molecules. For example, a library or population typically includes at least about 10 different molecules. Large libraries typically include at least about 100 different molecules, more typically at least about 1000 different molecules. For some applications, the library includes at least about 10000 or more different molecules. In certain embodiments, the library contains a number of variants or chimeric nucleic acids, or proteins produced by directed evolution, rational design, or Al-assisted design. In some embodiments, the library contains a number of variant or chimeric nucleic acids or proteins produced by an Al-assisted design. For example, in some embodiments the methods described in WO 2023/133462 and PCT Application No. PCT/US23/72153, incorporated by reference in its entirety herein, as well as WO 2023/154829, incorporated by reference in its entirety herein.

[0096] Directed evolution methods can be readily applied to polynucleotides to generate variant libraries that can be expressed, screened, and assayed. Mutagenesis and directed evolution methods are well known in the art (see, e.g., U.S. Patent Nos. 5,605,793, 5,830,721 , 6,132,970, 6,420,175, 6,277,638, 6,365,408, 6,602,986, 7,288,375, 6,287,861 , 6,297,053, 6,576,467, 6,444,468, 5,811238, 6,117,679, 6,165,793, 6,180,406, 6,291 ,242, 6,995,017, 6,395,547, 6,506,602, 6,519,065, 6,506,603, 6,413,774, 6,573,098, 6,323,030, 6,344,356, 6,372,497, 7,868,138, 5,834,252, 5,928,905, 6,489,146, 6,096,548, 6,387,702, 6,391 ,552, 6,358,742, 6,482,647, 6,335,160, 6,653,072, 6,355,484, 6,03,344, 6,319,713, 6,613,514, 6,455,253, 6,579,678, 6,586,182, 6,406,855, 6,946,296, 7,534,564, 7,776,598, 5,837,458, 6,391 ,640, 6,309,883, 7,105,297, 7,795,030, 6,326,204, 6,251 ,674, 6,716,631 , 6,528,311 , 6,287,862, 6,335,198, 6,352,859, 6,379,964, 7,148,054, 7,629,170, 7,620,500, 6,365,377, 6,358,740, 6,406,910, 6,413,745, 6,436,675, 6,961 ,664, 7,430,477, 7,873,499, 7,702,464, 7,783,428, 7,747,391 , 7,747,393, 7,751 ,986, 6,376,246, 6,426,224, 6,423,542, 6,479,652, 6,319,714, 6,521 ,453, 6,368,861 , 7,421 ,347, 7,058,515, 7,024,312, 7,620,502, 7,853,410, 7,957,912, 7,904,249, and all related non-US counterparts; Ling et al., Anal. Biochem, 254(2):157-78 [1997]; Dale et al., Meth. Mol. Biol, 57:369-74 [1996]; Smith, Ann. Rev. Genet, 19:423-62 [1985]; Botstein et al., Science, 229:1193-201 [1985]; Carter, Biochem. J., 237:1 -7 [1986]; Kramer et al., Cell, 38:879-87 [1984]; Wells et al., Gene, 34:315-23 [1985]; Minshull et al., Curr. Op. Chem. Biol, 3:284-90 [1999]; Christians et al, Nat. Biotechnol, 17:259-64 [1999]; Crameri et al., Nature, 391 :288-91 [1998]; Crameri, et al., Nat. Biotechnol, 15:436-38 [1997]; Zhang et al., Proc. Nat. Acad. Sci. U.S.A., 94:4504-09 [1997]; Crameri et al, Nat. Biotechnol, 14:315-19 [1996]; Stemmer, Nature, 370:389-91 [1994]; Stemmer, Proc. Nat. Acad. Sci. USA, 91 :10747-51 [1994]; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651 ; WO 01/75767; and

WO 2009/152336, all of which are incorporated herein by reference).

[0097] In certain embodiments, directed evolution methods generate protein variant libraries by recombining genes encoding variants developed from a parent protein, as well as by recombining genes encoding variants in a parent protein variant library. Two nucleic acids are “recombined” when sequences from each of the two nucleic acids are combined in a progeny nucleic acid. Two sequences are “directly” recombined when both of the nucleic acids are substrates for recombination. The methods may employ oligonucleotides containing sequences or subsequences encoding at least one protein of a parental variant library. Some of the oligonucleotides of the parental variant library may be closely related, differing only in the choice of codons for alternate amino acids selected to be varied by recombination with other variants. The method may be performed for one or multiple cycles until desired results are achieved. If multiple cycles are used, each typically involves a screening step to identify those variants that have acceptable or improved performance and are candidates for use in at least one subsequent recombination cycle. In some embodiments, the screening step involves a virtual protein screening system for determining the catalytic activity and selectivity of enzymes for desired substrates.

[0098] In some embodiments, the variant sequences can be generated by CRISPR/Cas9- mediated homology-directed repair (HDR).

[0099] In some embodiments, directed evolution methods generate protein variants by site-directed mutagenesis at defined residues. These defined residues are typically identified by structural analysis of binding sites, quantum chemistry analysis, sequence homology analysis, sequence activity models, etc. Some embodiments employ saturation mutagenesis, in which one tries to generate all possible (or as close to as possible) mutations at a specific site, or narrow region of a gene.

[0100] "Shuffling" and "gene shuffling" are types of directed evolution methods that recombine a collection of fragments of the parental polynucleotides through a series of chain extension cycles. In certain embodiments, one or more of the chain extension cycles is selfpriming, i.e., performed without the addition of primers other than the fragments themselves. Each cycle involves annealing single-stranded fragments through hybridization, subsequent elongation of annealed fragments through chain extension, and denaturing. Over the course of shuffling, a growing nucleic acid strand is typically exposed to multiple different annealing partners in a process sometimes referred to as "template switching," which involves switching one nucleic acid domain from one nucleic acid with a second domain from a second nucleic acid (i.e., the first and second nucleic acids serve as templates in the shuffling procedure).

[0101] Template switching frequently produces chimeric sequences, which result from the introduction of crossovers between fragments of different origins. The crossovers are created through template switched recombination’s during the multiple cycles of annealing, extension, and denaturing. Thus, shuffling typically leads to the production of variant polynucleotide sequences. In some embodiments, the variant sequences comprise a "library" of variants (i.e., a group comprising multiple variants). In some embodiments of these libraries, the variants contain sequence segments from two or more parent polynucleotides. When two or more parental polynucleotides are employed, the individual parental polynucleotides are sufficiently homologous that fragments from different parents hybridize under the annealing conditions employed in the shuffling cycles. In some embodiments, the shuffling permits the recombination of parent polynucleotides having relatively limited/low homology levels. Often, the individual parent polynucleotides have distinct and/or unique domains and/or other sequence characteristics of interest. When using parent polynucleotides having distinct sequence characteristics, shuffling can produce highly diverse variant polynucleotides.

[0102] Various shuffling techniques are known in the art. See, e.g., U.S. Patent Nos. 6,917,882, 7,776,598, 8,029,988, 7,024,312, and 7,795,030, all of which are incorporated herein by reference in their entireties.

[0103] Some directed evolution techniques employ "Gene Splicing by Overlap Extension" or "gene SOEing," which is a PCR-based method of recombining DNA sequences without reliance on restriction sites and of directly generating mutated DNA fragments in vitro. In some implementations of the technique, initial PCRs generate overlapping gene segments that are used as template DNA for a second PCR to create a full-length product. Internal PCR primers generate overlapping, complementary 3' ends on intermediate segments and introduce nucleotide substitutions, insertions, or deletions for gene splicing. Overlapping strands of these intermediate segments hybridize at 3' region in the second PCR and are extended to generate the full-length product. In various applications, the full-length product is amplified by flanking primers that can include restriction enzyme sites for inserting the product into an expression vector for cloning purposes. See, e.g., Horton, et al., Biotechniques, 8(5):528-35 [1990]. "Mutagenesis' is the process of introducing at least one mutation into a standard or reference sequence such as a parent nucleic acid or parent polypeptide. Site-directed mutagenesis is one example of a useful technique for introducing mutations, although any suitable method finds use. Thus, alternatively or in addition, the mutants may be provided by gene synthesis, saturating random mutagenesis, semisynthetic combinatorial libraries of residues, recursive sequence recombination ("RSR") (See, e.g., U.S. Patent Application Publication No. 2006/0223143, incorporated by reference herein in its entirety), gene shuffling, error-prone PCR, and/or any other suitable method.

[0104] One example of a suitable saturation mutagenesis procedure is described in U.S. Patent Application Publication No. 2010/0093560, which is incorporated herein by reference in its entirety. A "fragment" is any portion of a sequence of nucleotides or amino acids.

[0105] In one embodiment of the disclosure, the antibody or antibody fragment variant library comprises about 10⁷ to about 10²⁰ different antibody variants and/or polynucleotide sequences encoding the antibody variants of the library. In some embodiments, the libraries of the instant disclosure are designed to include 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 1 O¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, or 10²⁰ different (i.e., unique) antibody variants and/or polynucleotide sequences encoding the antibody variants. In certain embodiments, the libraries of the disclosure may comprise or encode about 10³ to about 10⁵, about 10⁵ to about 10⁷, about 10⁷ to about 10⁹, about 10⁹ to about 10¹¹, about 10¹¹ to about 10¹³, about 10¹³ to about 10¹⁵, about 10¹⁵ to about 10¹⁷, or about 10¹⁷ to about 10²⁰ different antibody variants. In certain embodiments of the disclosure, the diversity of the libraries may be characterized as being greater than or less than one or more of the diversities enumerated herein, for example greater than about 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, or 1 O²⁰ or less than about 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹², 10¹³, 10¹⁴, 10¹⁵, 10¹⁶, 10¹⁷, 10¹⁸, 10¹⁹, or 1 O²⁰.

[0106] The genetic diversity of the host cell population can be defined as the number of different genetic variants present in the host cell population (e.g., biomolecule variant library), the number of different genetic variants relative to a negative control, and/or the number of different genetic variants relative to a reference cell strain. The number of genetic variants may be the actual number of variants or a calculated (“target”) number of genetic variants in the host cell population. These variants may be the result of one or more genetic (e.g., nucleic acid sequence) differences in the host cell genome between cells, one or more genetic (e.g., nucleic acid sequence) differences in expression construct(s) between host cells, or a combination thereof. In some examples, the genetic differences include alteration, deletion, or insertion of one or more nucleotides of a sequence or insertion or deletion of one or more elements (such as one or more tags, domains, expression control sequences, and/or associated proteins).

[0107] In some embodiments, the genetic diversity of the host cell population is at least 500, at least 1000, at least 2000, at least 5000, at least 10,000, and least 50,000, at least 100,000, at least 200,000, at least 500,000, at least 1 ,000,000, at least 2,000,000, at least 5,000,000, at least 10,000,000, at least 100,000,000, at least 500,000,000, or at least 1 ,000,000,000. In other examples, the genetic diversity is about 1000-1 ,000,000,000, such as about 1000-10,000, about 5000-50,000, about 50,000-200,000, about 100, 000-500, 000, about 200,000-1 ,000,000, about 500,000-2,000,000, about 1 ,000,000-5,000,000, about 5,000,000-50,000,000, about 20,000,000-100,000,000, about 50,000,000-500,000,000, or about 500,000,000-1 ,000,000,000.

[0108] Any type of genetic diversity can be probed using the methods provided herein. In some embodiments, the genetic diversity includes one or more differences (including alteration or presence or absence) between a gene product of interest (including but not limited to coding sequence variants and codon-optimization), promoters (including constitutive and/or inducible promoters), chaperones, ribosome binding sequences, tags, nuclear localization signals, signal peptides, knockout or knock in of one or more genes, presence of one or more (such as 1 , 2, 3, or more) plasmids, or any combination thereof. In some examples, genetic diversity is generated by standard-directed genetic modification techniques. In other examples, genetic diversity is generated by random mutagenesis, error-prone PCR mutagenesis, or transposon mutagenesis (e.g., Tn5). A combination of techniques can also be used to generate additional levels of genetic diversity.

[0109] Additional methods for making alterations to host cell genomes or expression constructs in order to change nucleotide sequences and/or to eliminate, reduce, or change gene function are known in the art. Methods of making targeted disruptions of genes in host cells such as E. coll and other prokaryotes have been described (Muyrers et al., "Rapid modification of bacterial artificial chromosomes by ET-recombination", Nucleic Acids Res 1999 Mar 15; 27(6): 1555-57; Datsenko and Wanner, "One-step inactivation of chromosomal genes in Escherichia coll K-12 using PCR products", Proc Natl Acad Sci U SA 2000 Jun 6; 97(12):6640-45), and kits for using similar Red/ET recombination methods are commercially available (for example, the Quick & Easy E. coli Gene Deletion Kit from Gene Bridges GmbH, Heidelberg, Germany). Red/ET recombination methods can also be used to replace a promoter sequence with that of a different promoter, such as a constitutive promoter, or an artificial promoter that is predicted to promote a certain level of transcription (De Mey et al., "Promoter knock-in: a novel rational method for the fine-tuning of genes", BMC Biotechnol 2010 Mar 24; 10:26). The function of host cell genomes or expression constructs can also be eliminated or reduced by RNA silencing methods (Man et al, "Artificial trans-encoded small non-coding RNAs specifically silence the selected gene expression in bacteria", Nucleic Acids Res 2011 Apr; 39(8):e50, Epub 2011 Feb 3). The Gibson assembly method (Gibson, "Enzymatic assembly of overlapping DNA fragments", Methods Enzymol 2011 ; 498:349-61 ; doi:10.1016/B978-0-12-385120-8.00015-2) can also be used to make targeted changes in host cell genomes or expression constructs, such as insertions, deletions, and point mutations. Another method for making directed alterations in host cell genomes or expression constructs utilizes CRISPR (clustered regularly interspaced short palindromic repeats) nucleotide sequences and Cas9 (CRISPR-associated protein 9), which recognizes and cleaves nucleotide sequences that are complementary to CRISPR sequences. Further, changes to host cell genomes can be introduced through traditional genetic methods. Evaluating the expressed library

[0110] After generating biomolecules as described herein, including, for example, variant antibodies, the methods of the present disclosure further comprise screening the expressed variants for particular biological characteristics or functions as desired.

[0111] As used herein, the term "screening" refers to the process in which one or more properties of one or more biomolecules are determined. For example, typical screening processes include those in which one or more properties of one or more members of one or more libraries is/are determined.

[0112] Non limiting examples of measurements that can be assayed during the screening of a library include: Activity, Catalytic efficiency (k_cat/K_m), Catalytic rate constant (k_cat), Count/Number, EC50, Enrichment, Epistasis, Fitness, IC50, Inhibition constant ( ), Maximal rate (V_max), Michaelis constant (K_m), Relative activity, Specific activity, Association constant (Ka), Binding affinity, Count/Number, Dissociation constant (K_d), Equilibrium Constant (K_D), ELISA, Energy, Enrichment, Enthalpy of binding (AH), Entropy of binding (AS), Frequency of occurrence, Gibbs free energy of binding (AG), Inhibition constant ( ), Rate constant of association (k_on), Rate constant of dissociation (k_Off), Concentration, Energy, Enrichment, Frequency of occurrence, Minimum inhibitory concentration (MIC), Yield, Antimicrobial resistance, Energy, Enrichment, Frequency of occurrence, Optical density (OD), Bioavailability, EC50, Half-life (ti/2), IC50, Immunogenicity, Toxicity, Concentration, Energy, Fractional increase in solubility, Insoluble fraction, Oligomerization state, Soluble fraction, Energy, Frequency of occurrence, Relative activity, Relative affinity, Relative k_cat, Relative kcat/Km, Relative Kd, Brightness, Emission wavelength (A_em), Energy, Excitation wavelength (A_ex), Extinction coefficient, Fluorescence intensity, Maturation half-time, Photobleaching half-time, pKa, Quantum yield, Constant pressure heat capacity of unfolding (AC_P), Count/Number, Denaturant concentration at midpoint of unfolding transition (C_m), Energy, Enthalpy of unfolding (AH), Entropy of unfolding (AS), Equilibrium constant (K), Gibbs free energy of folding/unfolding (AG), Melting temperature (T_m), Rate of folding (k_F), Rate of unfolding (ku), Slope of chevron plot (m), Slope of the denaturant unfolding curve/cooperativity value (m), Temperature of maximum stability, Thermal tolerance, B- Tanford value, viscosity, and <t>-value. In some embodiments, the protein identifier is a name or a full-length protein sequence.

[0113] In an embodiment, the screening method of the present disclosure measures binding affinities.

[0114] In further embodiments, the screening method measures expression levels. Screening and validation techniques

[0115] In another aspect, the present disclosure provides a method of sorting the host cell population based on the specific binding of the expressed biomolecule or cargo protein - e.g., an antibody or antibody fragment to a target antigen, comprising providing a diverse library of transformed host cells expressing a diverse library of biomolecules (e.g., binding proteins) as disclosed herein; contacting the host cells with the target antigen; and sorting host cells based on their binding to the target antigen, thereby identifying subpopulations of cells that specifically bind to a target antigen.

[0116] In another aspect, the disclosure provides a method of sorting the host cell population based on the specific binding of the expressed antibody or antibody fragment to a first target antigen probe and a second non-antigen probe simultaneously, the method comprising: providing a diverse library of transformed host cells expressing a diverse library of binding proteins disclosed herein; contacting the host cells with the first and second probes; and sorting host cells based on their binding to the first and second probes, thereby identifying subpopulations of cells that specifically bind to a first and a second probe simultaneously.

[0117] In certain embodiments of the methods disclosed herein, host cells that bind to the first and/or second probe are selected by Magnetic Activated Cell Sorting (MACS) using magnetically labeled antigens.

[0118] In certain embodiments of the methods disclosed herein, host cells that bind to the first and/or second probe are selected by Fluorescence Activated Cell Sorting (FACS) using fluorescently labeled antigens.

[0119] FACS is a powerful tool that allows the analysis of multiple individual cell parameters, providing the ability to separate a heterogeneous suspension of cells into a homogenous fraction of single cells based on fluorescence and light scattering properties. Instruments for carrying out flow cytometry are known to those of skill in the art and are commercially available to the public. Examples of such instruments include but are not limited to, BD FACSAria(TM)-llu instrument (Becton Dickinson), COULTER EPICS XL/XL- MCL (Coulter Epics Division), and MoFlo XDP (Beckman Coulter), Attune NxT Flow Cytometer (ThermoFisher). Once cells are sorted, gates or boundaries are placed around populations of cells with common characteristics, usually forward scatter (FSC), side scatter (SSC), and the fluorescence of the labels detecting expressed proteins or labeled DNA. FSC and SSC give an idea of the size and granularity of the cells respectively. By setting specific gates, the subpopulations of host cells can be separated and collected into a plurality of collection tubes for investigation and/or quantification of the subpopulations of interest. In some embodiments of the methods disclosed herein, host cells are gated according to antigen binding affinity and expression levels of the expressed antibodies or antibody fragments. In particular examples, the gating parameters also identify and exclude aggregated cells or non-cellular debris, in order to measure signal substantially only from single cells. This reduces artifacts of increased expression of the product of interest due to cell "clumping" rather than actual increase due to the particular genetic diversity of a cell.

[0120] In certain embodiments, the methods disclosed herein optionally comprise the rescreening of sorted host cell subpopulations from the plurality of collection tubes sorted by FACS to validate the calculated K_Ds an additional technique. As used herein, the term “optional” or “optionally” means that the subsequently described event, circumstance, or substituent may or may not occur and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0121] Suitable alternative methods for rescreening and measuring binding affinities are known in the art and can be selected from the group consisting of ELISA, Surface Plasmon Resonance (SPR), Biolayer Interferometry and flow cytometry derived binding curves.

[0122] In one embodiment, the rescreening is performed by SPR. A BIAcore-2000 or BIAcore-3000 real-time kinetic interaction analysis system (Biacore Inc., Piscataway, N.J.) may then be used to determine association (k_on) and dissociation (k_Off) constants (Karlsson, R., Michaelsson, A. & Mattsson, L., J Immunol Methods 145(1-2):229-40 (1991)) of the antibody fragments in binding interactions with immobilized antigen, according to the manufacturer’s instructions. In some embodiments, the rescreening is performed by Carterra SPR systems. The KD may be calculated from k₀ff/k_0n, as known in the art.

[0123] In some embodiments, the binding affinities of the antibodies described herein are measured by array surface plasmon resonance (SPR), according to standard techniques (Abdiche, et al. (2016) MAbs 8:264-77). Briefly, antibodies were immobilized on an HC 30M chip at four different densities/antibody concentrations. Varying concentrations (0-500 nM) of antibody targets are then bound to the captured antibodies. Kinetic analysis is performed using Carterra software to extract association and dissociation rate constants (k_a and k_d, respectively) for each antibody. Apparent affinity constants (KD) are calculated from the ratio of kd/k_a. In some embodiments, the Carterra LSA Platform is used to determine kinetics and affinity. In other embodiments, binding affinity can be measured, e.g., by surface plasmon resonance (e.g., BIAcore™) using, for example, the IBIS MX96 SPR system from IBIS Technologies or the Carterra LSA SPR platform, or by Bio-Layer Interferometry, for example using the Octet™ system from ForteBio. In some embodiments, binding affinity is measured by Gator BLI system. In some embodiments, a biosensor instrument such as Octet RED384, ProteOn XPR36, IBIS MX96 and Biacore T100 is used (Yang, D., et al., J. Vis. Exp., 2017, 122:55659).

[0124] KD is the equilibrium dissociation constant, a ratio of k₀ff/k_0n, between the antibody and its antigen. K_D and affinity are inversely related. The K_D value relates to the concentration of the antibody so the lower the KD value (lower concentration) and thus the higher the affinity of the antibody. Antibody, including reference antibody and variant antibody, KD according to various embodiments of the present disclosure can be, for example, in the micromolar range (10^-4 to 10^-6), the nanomolar range (10^-7 to 10^-9), the picomolar range (1 O'¹⁰ to 10^-12) or the femtomolar range (10^-13 to 10^-15). In some embodiments, antibody affinity of a variant antibody is improved, relative to a reference antibody, by approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50% or more. The improvement may also be expressed relative to a fold change (e.g., 2x, 4x, 6x, or 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-fold, or more improvement in binding activity, etc.) and/or an order of magnitude (e.g., 10⁷, 10⁸, 10⁹, etc.).

Next Generation Sequencing

[0125] In some embodiments, the subpopulations of host cells sorted into a plurality of collection tubes (i.e., “bins”) are further characterized to gain insight into possible mutational correlations or relationships that lead to a desired functional change. In some embodiments, further characterizing these subpopulations comprises analyzing variants individually through sequencing, to identify the specific mutation or mutations that are connected to the change in characteristic (such as a highly functional characteristic). Individual mutant variants of the biomolecule can be isolated through standard molecular biology techniques for later analysis of function.

[0126] The term "sequence" is used herein to refer to the order and identity of any biological sequences including but not limited to a whole genome, whole chromosome, chromosome segment, collection of gene sequences for interacting genes, gene, nucleic acid sequence, protein, peptide, polypeptide, polysaccharide, etc. In some contexts, a "sequence" refers to the order and identity of amino acid residues in a protein (i.e., a protein sequence or protein character string) or to the order and identity of nucleotides in a nucleic acid (i.e., a nucleic acid sequence or nucleic acid character string). A sequence may be represented by a character string. A "nucleic acid sequence" refers to the order and identity of the nucleotides comprising a nucleic acid. A "protein sequence" refers to the order and identity of the amino acids comprising a protein or peptide. "Codon" refers to a specific sequence of three consecutive nucleotides that is part of the genetic code and that specifies a particular amino acid in a protein or starts or stops protein synthesis. [0127] In some embodiments, further characterizing the host subpopulations comprises high throughput sequencing or next generation sequencing (NGS) of the plurality of host sub-populations comprising high binders, low binders, and everything in between. This approach may, in some embodiments, may allow for the rapid identification of mutations that are over-represented in one or more sub-populations.

[0128] As used herein, the terms "next generation sequencing (NGS)" and "high- throughput sequencing" are sequencing techniques that parallelize the sequencing process, producing thousands or millions of sequences at once. Examples of suitable nextgeneration sequencing methods include, but are not limited to, single-molecule real-time sequencing (e.g., Pacific Biosciences, Menlo Park, California), ion semiconductor sequencing (e.g., Ion Torrent, South San Francisco, California), pyrosequencing (e.g., 454, Branford, Connecticut), sequencing by ligation (e.g., SOLiD sequencing of Life Technologies, Carlsbad, California), sequencing by synthesis and reversible terminator (e.g., Illumina, San Diego, California), nucleic acid imaging technologies such as transmission electron microscopy, and the like.

[0129] NGS can produce high throughput data indicating the functional effect of the library members. In embodiments wherein one or more libraries represent every possible mutation of every monomer location, such high throughput sequencing can evaluate the functional effect of every possible mutation. Such sequencing can also be used to evaluate one or more highly or less functional sub-populations of a given library, which in some embodiments may lead to the identification of mutations that result in improved and decreased function, respectively.

[0130] In certain embodiments, the methods disclosed herein may comprise the amplification of DNA obtained from the sorted host cell subpopulations. In some embodiments, RNA can also be recovered from selected host cells and reverse-transcribed into DNA. DNA amplification is useful when the quantity of isolated DNA is inadequate for NGS. If the cells that were FACS sorted comprise cells that express the library of antibody or antibody fragment variants from a plasmid (for example, E. co// cells transformed with a plasmid expression vector), these plasmids can be isolated, for example through a miniprep. Conversely, if the library of biomolecule variants has been integrated into the genomes of the FACs sorted cells, this DNA region can be PCR amplified and, optionally, subcloned into a suitable vector for further characterization using methods known in the art. Thus, the end product of library screening is a DNA library representing the initial, or ‘naive,’ library, as well as one or more DNA libraries containing sub-populations of the naive library, which comprise highly functional mutant variants of the biomolecule identified by the screening processes described herein. [0131] An Example of one embodiment of the sRCA amplification technique is provided below.

[0132] In an embodiment, the DNA amplification step disclosed herein further comprises the addition of barcodes or Unique Molecular Indices (UMI) to the DNA isolated from the sorted host cell subpopulations.

[0133] As used herein, the term "barcode" refers to a nucleic acid sequence that is used to identify a single cell or a subpopulation of cells. Barcode sequences can be linked to a target nucleic acid of interest during amplification and used to trace back the amplicon to the cell from which the target nucleic acid originated. A barcode sequence can be added to a target nucleic acid of interest during amplification by carrying out PCR with a primer that contains a region comprising the barcode sequence and a region that is complementary to the target nucleic acid such that the barcode sequence is incorporated into the final amplified target nucleic acid product (i.e., amplicon). Barcodes can be included in either the forward primer or the reverse primer or both primers used in PCR to amplify a target nucleic acid. A barcode can be any number of nucleotides in length. A barcode can be 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or more than 30 nucleotides in length. In some cases, the barcode is more than 30 nucleotides in length. A barcode can be generated by degenerate oligonucleotide synthesis. A barcode can be rationally designed or user specified.

[0134] As used herein, the term “Unique Molecular Indices (UMI)” refers to randomized nucleotides sequences applied to or identified in DNA molecules that may be used to distinguish individual DNA molecules from one another. Since UMIs are used to identify DNA molecules, they are also referred to as unique molecular identifiers. See, e.g., Kivioja, Nature Methods 9, 72-74 (2012). UMIs may be sequenced along with the DNA molecules with which they are associated to determine whether the read sequences are those of one source DNA molecule or another. The term “UMI” is used herein to refer to both the sequence information of a polynucleotide and the physical polynucleotide per se.

[0135] The addition of UMIs (random molecular barcodes) to amplicons during the first few PCR cycles will allow to uniquely tag each template molecule. Down the line, when sequencing will yield identical reads, one will be able to disambiguate sequencing/PCR duplicates (not of interest, to be counted only once) from identical but molecularly independent templates (biologically interesting, each to be counted). UMIs are widespread in several modern molecular biology protocols leveraging PCR with downstream NGS endpoints. [0136] The amplification reaction, according to the present method, may be either a nonisothermal method or an isothermal method.

[0137] Suitable methods for non-isothermal amplification include polymerase chain reaction (PCR) (Saiki et al. Science (1985) 230:1350-54) and ligase chain reaction (LCR) (Landegren et al. Science (1988) 241 :1077-80).

[0138] "Polymerase chain reaction," or "PCR," means a reaction for the in vitro amplification of specific DNA sequences by the simultaneous primer extension of complementary strands of DNA. In other words, PCR is a reaction for making multiple copies or replicates of a target nucleic acid flanked by primer binding sites, such reaction comprising one or more repetitions of the following steps: (i) denaturing the target nucleic acid, (ii) annealing primers to the primer binding sites, and (iii) extending the primers by a nucleic acid polymerase in the presence of nucleoside triphosphates. Usually, the reaction is cycled through different temperatures optimized for each step in a thermal cycler instrument. Particular temperatures, durations at each step, and rates of change between steps depend on many factors well-known to those of ordinary skill in the art, e.g., exemplified by the references: McPherson et al, editors, PCR: A Practical Approach and PCR2: A Practical Approach (IRL Press, Oxford, 1991 and 1995, respectively).

[0139] Suitable isothermal amplification methods may be selected from the group of helicase-dependent amplification (HDA) (Vincent et al. EMBO Rep (2004) 5(8):795-800), thermostable HDA (tHDA) (An et al. J. Biol. Chem. (2005) 280(32) :28952-58), strand displacement amplification (SDA) (Walker et al. Nucleic Acids Res. (1992) 20(7):1691-96), multiple displacement amplification (MDA) (Dean et al. Proc. Natl. Acad. Sci. USA (2002) 99(8): 5261-66), selective rolling-circle amplification (sRCA, as described herein), restriction aided RCA (Wang et al. Genome Res (2004) 14:2357-66), single primer isothermal amplification (SPIA) (Dafforn et al. Biotechniques (2004), 37(5):854-57), transcription mediated amplification (TMA) (Vuorinen et al. J. Clin. Microbiol. (1995) 33:1856-59), nicking enzyme amplification reaction (NEAR) (Maples et al. U.S. Patent Application Publication No. 2009017453), exponential amplification reaction (EXPAR) (Van Ness et al. Proc. Natl. Acad. Sci. USA (2003) 100(8):4504-09), loop mediated isothermal amplification (LAMP) (Notomi et al. Nucleic Acids Res. (2000) 28(12):e63), recombinase polymerase amplification (RPA) (Piepenburg et al. PloS Biol. (2006) 4(7):1115-20), nucleic acid sequence based amplification (NASBA) (Kievits et al. J. Virol. Methods (1991) 35:273-86), smart-amplification process (SMAP) (Mitani et al. Nat. Methods (2007) 4(3):257-62).

[0140] In an embodiment, the amplification method is the selective rolling-circle amplification (sRCA) method. [0141] As used herein, the term “rolling circle amplification (RCA)” refers to an isothermal acid amplification reaction that amplifies a circular nucleic acid template (e.g., single/double stranded DNA circles) using a strand-displacing polymerase. Rolling circle amplification reaction is initiated by the hybridization of a primer to a circular, often single-stranded, nucleic acid template. The nucleic acid polymerase then extends the primer that is hybridized to the circular nucleic acid template by continuously progressing around the circular nucleic acid template to replicate the sequence of the nucleic acid template over and over again (rolling circle mechanism). The rolling circle amplification typically produces concatemers comprising tandem repeat units of the circular nucleic acid template sequence. The rolling circle amplification may be a linear RCA (LRCA), exhibiting linear amplification kinetics (e.g., RCA using a single, specific primer), or may be an exponential RCA (ERCA) exhibiting exponential amplification kinetics. Rolling circle amplification may also be performed using multiple primers (multiply primed rolling circle amplification or MPRCA) leading to hyper-branched concatemers. For example, in a double-primed RCA, one primer may be complementary, as in the linear RCA, to the circular nucleic acid template, whereas the other may be complementary to the tandem repeat unit nucleic acid sequences of the RCA product. Consequently, the double-primed RCA may proceed as a chain reaction with exponential amplification kinetics featuring a cascade in a series of multiple-hybridization, primer-extension, and strand-displacement events involving both the primers and both strands. This often generates a discrete set of concatemeric, double-stranded nucleic acid amplification products. The RCA may be performed in vitro under isothermal conditions using a suitable nucleic acid polymerase such as Phi29 DNA polymerase. Suitable polymerases possess strand displacement DNA synthesis ability. In some embodiments, the Phi29 DNA polymerase possesses a 70,000 base pair strand displacement capability that allows primers to bind in a relatively small portion of the template, while still effectively amplifying the entire sequence. In an embodiment, the rolling circle amplification employs primers designed to target conserved regions of antibiotic markers and their flanking regions in the template (selective RCA or sRCA). In further embodiments, the template is plasmid DNA. The sRCA primer design allows for the amplification of a plasmid carrying a specific resistance marker in cells containing plasmids carrying multiple other resistance markers, while avoiding off-target amplification of other plasmids or genomic DNA. In additional embodiments, sRCA primers may also be used in combination to amplify two or more plasmids from the same cell.

[0142] All patents and other publications identified are expressly incorporated herein by reference in their entirety or in relevant part, as would be apparent from the context of the citation, for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the information described herein.

[0143] The following examples are given merely to illustrate the present invention and not in any way to limit its scope.

EXAMPLES

[0144] Materials and Methods

[0145] 1 . Strain cultivation and protein expression

[0146] Plasmids were transformed into a SoluPro strain lacking DegP protease (Strain EB128) by electroporation and cultivated on LB agar plates supplemented with 1% glucose and 50 pg/mL kanamycin (Teknova cat #L5825). For protein expression, a single colony was picked from a freshly transformed plate and was used to inoculate LB supplemented with 50 pg/mL kanamycin. Cultures were grown overnight at 37°C, 250 RPM. In the morning, overnight cultures were used to inoculate LB with a 20% fill volume in ultra-yield shake flasks (Thomson Instrument Company) to an OD₆oo of -0.1 . Strains were grown at 37°C, 250 RPM until an OD₆oo of -0.4-0.6 was reached. Protein expression was induced by the addition of 0.004% (w/v) L-arabinose. Induced cultures were incubated at 30°C, 250 RPM for 3 hours. Cultures were then either placed at 4°C overnight for staining the following day or used immediately for flow cytometry or for the sorting of spike-in libraries.

[0147] 2. Generation of spike-in libraries

[0148] 2. Spike-in libraries were generated by first pelleting cells at 4000xg, 4°C for 5 minutes, discarding the supernatant, and washing pellets by resuspending in 5 mL PMC buffer (1X Phosphate Buffered Saline (PBS) supplemented with 20 mM MgCl2 and 100 pM CaCl2). The washed pellets were then normalized to an OD₆oo of 1 . Each of the strains that bind the antigen of interest was mixed in equal proportions to create the spike-in pool. The strain that does not bind the antigen of interest (referred to as the background strain) was used as the diluent to create a 1 :20 dilution of the spike-in pool. Ten-fold dilutions were then prepared from the 1 :20 spike-in stock using the background strain as the diluent.

[0149] 3. Staining for flow cytometry and sorting

[0150] Each experiment included a positive control and two negative controls in addition to the spike-in library (expression negative/antigen negative, and expression positive/antigen negative). All samples were OD normalized to an ODeoo of 1 after washing in PMC buffer. 500 pL of the OD normalized sample was aliquoted into 1 .4 mL matrix tubes and pelleted at 4000xg, 4°C for 5 minutes. The supernatant was aspirated, and pellets were resuspended in primary amine labeled biotinylated antigen-containing solution in PMC buffer (either HER2 (Aero Biosystems Cat # HE2-H822R) or VEGF (Aero Biosystems Cat # VES-H8210)). Samples were incubated at room temperature for 1 hour with 360-degree rotation. Samples were then pelleted at 4000xg, 4°C for 5 minutes and washed three times with 500 pL PMC buffer. Pellets were resuspended in a mixture containing a 1 :500 dilution of an anti-camelid VHH antibody conjugated to iFluor647 (GenScript Cat # A02019-200) and a 1 :200 dilution of streptavidin-PE (Invitrogen Cat # S-866). Cells were incubated at 4°C protected from light for 30 minutes with 360-degree rotation. Cells were then pelleted at 4000xg, 4°C for 5 minutes and the supernatant was aspirated. Cells were resuspended in 500 pL PMC buffer for flow cytometry and sorting.

[0151] 4. MACS staining and sorting

[0152] After OD normalization, 1 mL of the spike-in library was pelleted at 4000xg, 4°C for 5 minutes in an Eppendorf tube. The supernatant was aspirated, and the pellet was resuspended in 500 pL of the primary amine labeled biotinylated antigen diluted in PMC buffer. Cells were incubated with 360-degree rotation at room temperature for 1 hour. Cells were then pelleted at 4000xg, 4°C for 5 minutes. The supernatant was aspirated, and cells were washed three times with 500 pL of PMC buffer. The pellet was resuspended in 180 pL of PMC buffer and 20 pL of streptavidin-coated microbeads (Miltenyi Biotec Cat # 130-048- 101). Cells were incubated with beads for 20 minutes at 4°C with 360-degree rotation. After incubation, cells were pelleted at 4000xg, 4°C for 5 minutes. Cells were washed three times with 500 pL of PMC buffer. The pellet was resuspended in 500 pL PMC buffer and loaded onto an LS column (Miltenyi Biotec Cat # 130-042-401 ) that was equilibrated with 3 mL PMC buffer and mounted on a quadroMACS separator (Miltenyi Biotec Cat # 130-091 -051). The loaded column was washed three times with 3 mL PMC buffer. Cells were eluted from the column by removing the column from the quadroMACS separator, adding 5 mL PMC buffer, and using the plunger to recover the entire solution from the LS columns.

[0153] 5. Post-sorting sample preparation for seguencing

[0154] There were three post-sort sample processing methods tested: 1) direct cell amplification (direct DCA), 2) plating on LB agar plates supplemented with 1% glucose and 50 pg/mL kanamycin, and 3) growing in liquid LB media supplemented with 50 pg/mL kanamycin. For direct DCA, sorted samples (MACS or FACS) were pelleted at 6000xg, 4°C for 30 minutes. The supernatant was removed, and the pellets were resuspended in 20 pL sterile ddH₂O. Resuspended samples were amplified (forward: 5’- CAAGCAACGGGAGATGGTCGCA-3' (SEQ ID NO: 6) and reverse: 5’- CGAGCTAACGGTAACCTGGGTACCC-3' (SEQ ID NO: 7)) with an annealing temperature of 72°C. The amplicon DNA was AMPure bead purified (Beckman Coulter Inc. Cat # A63881 ) according to the manufacturers protocol and quantified with a Qubit Flex instrument to a minimum concentration of 10 ng/pL. For methods two and three, cells were recovered at 37°C overnight. The recovery was submitted to DCA to prepare amplicons for next generation sequencing (NGS) analysis.

[0155] 6. NGS Enrichment analyses

[0156] For all spike-in sorting experiments, the sorted libraries and the unsorted spike-in were submitted for NGS analysis to determine the initial frequency of each variant in the library and the post-sort of frequency in sorted pools. All sequences were subjected to a quality control step where sequences with less than 5 counts were omitted. Fold-enrichment (E) was calculated as follows:

[0157] E = (post-sort frequency) / (initial frequency).

[0158] Workflow

[0159] An exemplary workflow is shown in Figure 5.

[0160] Some designs of the surface expression cassette used for the experiments are described herein.

[0161] A schematic representation of a surface expression cassette design is shown in FIG. 1 . surface-displayed proteins are expressed as C-terminal fusions on the intimin autotransporter. A TEV cleavage site is included N-terminal to the VHH to allow controlled release from the cell surface and a 6x-His tag is included for purification of cleaved proteins. Both HA and 6x-His tags are included for flow cytometry staining.

[0162] A schematic of a surface display proof of concept plasmid design 1 is shown in FIG. 2. Design 1 has the following features and expression cassettes: a repressor protein (ROP) to modulate ~ 50 copies per cell, an origin of replication (pBR322) to modulate expression levels, ParB to increase plasmid retention and stability, and a strong ribosome binding site (sRBS) to modulate expression levels N-terminal to the autotransporter and cargo protein. The benefits of the design shown in FIG. 2 include low strain aggregation, no known plasmid instability between rounds of selection and during expression, higher expression, higher fold enrichment, and less susceptibility to polyclonality. A drawback of the design shown in FIG. 2 includes lower overall levels of cell viability as measured by post- FACS plating and liquid growth, thereby, likely requires re-cloning post-sort rather than direct growth in most sorting scenarios and apparent growth rate effects between discrete strains, which may lead to issues in libraries.

[0163] A schematic of a surface display proof of concept plasmid design 2 is shown in FIG. 3. Design 2 has the following features and expression cassettes to generate high copy number (-500 copies per cell) and weak to moderate RBS (wRBS): the origin of replication (pBR322) to modulate expression levels and ParB to increase plasmid retention and stability. The benefits of the design are shown in FIG. 3 include low strain aggregation, no known plasmid stability issues between rounds of selection and during expression, high viability pre- and post-sorting (MACS/FACS) which yields quick cell recovery, foldenrichment comparable to multiple rounds of ACE sorts observed in FACS and MACS, and no observed growth rate differences between discrete strains. Some potential drawbacks of the design are shown in FIG. 3 include polyclonality effects when transforming highly diverse libraries, lower overall expression levels leading to lower confidence when distinguishing low-affinity variants and negative controls, and lower fold-enrichment in MACS/FACS than when compared to low copy number and strong RBS plasmid construct.

Example 1 - Validation of expression and functionality of surface displayed VHH (HER2)

[0164] This Example validates the expression and functionality of surface displayed VHHs in Design 1 and 2 plasmid backbones described above. FIGs. 4A-4B show validation of the expression and functionality of surface displayed VHHs in Design 1 and 2 plasmid backbones FIG. 4A shows the gMFI of expression stain (anti-VHH iFluor647) for HER2 VHH standards in the Design 1 backbone (orange) and the Design 2 backbone (blue). FIG. 4B shows the gMFI of antigen stain (50 nM HER2-PE) for HER2 VHH standards in the Design 1 backbone (orange) and the Design 2 backbone (blue)

Example 2 - Enrichment validation - FACS and MACS

[0165] This experiment demonstrates an enrichment validation of FACS and MACS. The results shown in figure 6 demonstrate an overall higher fold enrichment than other plasmid backbones.

Example 3 - EC50 determination using flow cytometry

[0166] This experiment demonstrates initial validations of VHH surface display affinity curves. The results shown in figure 7 demonstrate that the EC50S obtained from surface display antigen titrations correlate with affinities more closely than intracellular counterparts. ECso’s determined by flow cytometry for Her2-binding constructs displayed on the surface of SoluPro. All three constructs have affinities for Her2 in the 1 to 2 nM range. FIG. 7A shows Her2-binding VHH (EC50 = 7.0 ± 2.1 nM); FIG. 7B shows Trastuzumab scFv (EC50 = 5.4 ± 1 .5 nM). FIG. 7C shows Trastuzumab scFab (EC50 = 2.4 ± 2.8 nM). [0167] Figures 8A-8B show microscopy images of SoluPro displaying intimin-VHH. Stained with 50 nM HER-PE (red) and DAPI (blue). FIG. 8A depicts the entire field, whereas FIG. 8B depicts a zoomed-in image of the region highlighted in FIG. 8A. Finally, Figure 9 illustrates buffer composition's impact on scFab aggregation, displaying SoluPro cells as determined by a cell counter. The percentage of cells in aggregates is defined as 100 x (number of cells with size > 1 nm / total number of cells counted). Many cells are counted as aggregates under typical staining conditions (i.e., PBS or PBS/EDTA). The addition of cations such as magnesium and calcium seems to reduce aggregation in SoluPro.

Example 4 - Validation of expression and functionality of surface displayed VHH (VEGF)

[0168] This experiment demonstrates the validation of additional surfACE probe VEGF by comparing the expression levels of anti-VHH iFluor647 staining (FIG. 10A) and 367 nM VEGF-PE (FIG. 10B). The samples tested are as follows: NC (Empty SoluPro negative control), NC2 (VHH negative control strain), Nb42 (nanobody from citation below with VEGF affinity ~60 nM), Nb35 (nanobody from citation below with VEGF affinity ~45 nM), Nb23 (nanobody from citation below with VEGF affinity ~10 nM), and Nb22 (nanobody from citation below with VEGF affinity ~1 nM). The results from FIGs. 10A-10B show that Nb23 expresses well as a fusion to intimin and has a VEGF binding signal well above the negative control.

Example 5 - Effects of DsbC chaperone on intimin-VHH fusions

[0169] This experiment demonstrates the effects of DsbC chaperone on intimin-VHH fusions. FIG. 11 depicts the comparisons of viability (SYTOX Blue staining) (FIG. 11 A), expression levels (anti-VHH iFluor647) (FIG. 11 B), and ligand binding levels (50 nM HER2- PE) (FIG. 11 C) for several HER2-binding intimin-VHH strains that were expressed in the presence or absence of DsbC. + indicates that DsbC is present, whereas - indicates that DsbC is absent.

Claims

What is claimed is:

1 . An expression cassette comprising: (i) a nucleic acid encoding an intimin autotransporter or functional fragment thereof, (ii) a nucleic acid encoding a cleavage site, (iii) at least one nucleic acid encoding at least one epitope tag, (iv) a nucleic acid encoding a cargo protein, and (v) at least one nucleic acid encoding at least one purification tag cargo protein.

2. The expression cassette of claim 1 , wherein the cargo protein is selected from the group consisting of: an antibody, a Fab, a scFab, an Fv, an scFv, a di-scFv, a nanobody, a VHH, or fragments of any of the above.

3. The expression cassette of claim 1 or claim 2, wherein the cargo protein is a VHH.

4. The expression cassette of any one of claims 1 -3, wherein the cleavage site is a TEV protease cleavage site.

5. The expression cassette of any one of claims 1-4, wherein the expression cassette comprises one epitope tag and one purification tag.

6. The expression cassette of claim 5, wherein the epitope tag is N-terminal and the purification tag is C-terminal to the cargo protein.

7. The expression cassette of any one of claims 5-6, wherein the epitope tag is selected from a hemagglutinin (HA) tag and a Flag tag, and the purification tag is selected from a Histidine (His) tag.

8. The surface expression cassette of claim 7, wherein the His tag is a 6x-His tag.

9. The expression cassette of any one of claims 1-8, wherein the intimin autotransporter is selected from the group consisting of an invasion from Yersenia spp. Or an intimin from a pathogenic E. coli.

10. The expression cassette of claim 9 wherein the intimin autotransporter is from a pathogenic E. coli or a functional fragment thereof.

11 . The expression cassette of any one of claims 1-10, further comprising an inducible promoter capable of promoting expression of a fusion protein comprising an intimin autotransporter or functional fragment thereof, a cleavage site, at least one epitope tag, a cargo protein, and at least one purification tag.

12. The expression cassette of claim 11 , wherein the inducible promoter is selected from the group consisting of an arabinose-inducible promoter, a propionate- inducible promoter, and a rhamnose-inducible promoter.

13. The expression cassette of claim 12 wherein the inducible promoter is an arabinose-inducible promoter.

14. An expression cassette comprising: (i) a nucleic acid encoding an intimin autotransporter or functional fragment thereof, (ii) a nucleic acid encoding a TEV protease cleavage site, (iii) at least one nucleic acid encoding at least one HA tag, (iv) a nucleic acid encoding a scFab, and (v) at least one nucleic acid encoding at least one His tag.

15. An expression vector comprising an expression cassette of any one of claims 1 -14, and further comprising a nucleic acid encoding a protein that increases expression vector retention and/or stability.

16. The expression vector of claim 14, wherein the protein that increases expression vector retention and/or stability is selected ParB.

17. The expression vector of claim 15, wherein the protein is ParB.

18. The expression vector of any one of claims 15-17, further comprising a strong RBS (sRBS) or a weak/moderate RBS (wRBS) that is upstream of the nucleic acid encoding the intimin autotransporter or functional fragment thereof.

19.. A host cell comprising the expression cassette of any one of claims 1 -14 or the expression vector of any one of claims 15-18.

20. The host cell of claim 18, wherein the host cell is a prokaryotic cell comprising one or more or all of:

(a) an alteration of gene function of at least one gene encoding a transporter protein for an inducer of at least one inducible promoter;

(b) a reduced level of gene function of at least one gene encoding a protein that metabolizes an inducer of at least one inducible promoter;

(c) a reduced level of gene function of at least one gene encoding a protein involved in biosynthesis of an inducer of at least one inducible promoter;

(d) an altered gene function of a gene that affects the reduction/oxidation environment of the host cell cytoplasm;

(e) a reduced level of gene function of a gene that encodes a reductase;

(f) at least one expression construct encoding at least one disulfide bond isomerase protein; (g) at least one polynucleotide encoding a form of DsbC lacking a signal peptide; and/or

(h) at least one polynucleotide encoding Ervlp.

21 . The host cell of claim 19 or claim 20, wherein the host cell is derived from an Enterobacterial species.

22. The host cell of claim 21 , wherein the host cell is an Escherichia coli host cell.

23. The host cell of claim 22, wherein the Escherichia coli is a SoluPro cell.

24. The host cell of any one of claims 20-23, wherein the host cell further comprises a deletion of a periplasmic protease.

25. The host cell of claim 24 wherein the periplasmic protease is DegP.

26. A method of screening for expression of a cargo protein on the surface of a host cell, the method comprising: a) preparing a library of host cells according to any one of claims 19-25; b) culturing the cells; c) inducing expression of the cargo protein; and d) screening for expression of the cargo protein; wherein said screening comprises a method selected from the group consisting of magnetic activated cell sorting (MACS), and Fluorescence-Activated Cell Sorting (FACS).

27. The method of claim 26, further comprising the step of recovering a host cell or host cells following the screening step (d) by plating recovered host cells on a growth medium, thereby enriching for said host cell or host cells.

28. The method of claim 27, further comprising the step of a second screening to assess cargo protein expression or cargo protein EC50, thereby screening for expression of a cargo protein capable of binding to a target with a desired EC50.

29. A method of determining affinity of a cargo protein to a target, the method comprising: a) preparing a library of host cells according to any one of claims 19-25; b) culturing the cells; c) inducing expression of the cargo protein; and d) screening for expression of the cargo protein; optionally further comprising the step of recovering a host cell or host cells following the screening step (d) by plating recovered host cells on a growth medium; optionally further comprising the step of a second screening to assess cargo protein expression or cargo protein EC50; and e) determining the affinity of the cargo protein to the target.

30. The method of claim 29, wherein the affinity is determined by a method selected from the group consisting of surface plasmon resonance (SPR) and BioLayer Interferometry.

31 . A method for determining the sequence of a nucleic acid encoding a cargo protein capable of binding to a target with a desired affinity, the method comprising: a) preparing a library of host cells according to any one of claims 19-25; b) culturing the cells; c) inducing expression of the cargo protein; and d) screening for expression of the cargo protein; optionally further comprising the step of recovering a host cell or host cells following the screening step (d) by plating recovered host cells on a growth medium; optionally further comprising the step of a second screening to assess cargo protein expression or cargo protein EC50; e) determining the affinity of the cargo protein to the target; and f) sequencing the nucleic acid encoding said cargo protein.

32. The method of any one of claims 26-31 , wherein the method does not require cell fixation.

33. The method of any one of claims 26-32, wherein the method does not require cell permeabilization.

34. The method of any one of claims 26-33, wherein intracellular chaperones are not required for proper protein folding.