High-throughput methods for kinetic characterization, quantifying and optimizing antibodies and antibody fragments expression in bacteria

Abstract

Described herein is a method of determining affinity of an antigen binding protein binding to a target, the method comprising: a) expressing an antigen binding protein in a prokaryotic host cell in a multi-well container, b) lysing the prokaryotic cell to produce a prokaryotic cell lysate; c) affixing the prokaryotic cell lysate to a substrate; and d) determining the affinity of the antigen binding protein to the target. Also described herein are methods, reagents, principles, and strategies for engineering synthetic tags at optimal locations on an antigen binding protein (e.g., a VHH) to allow for efficient capture from lysate and high-throughput collection of kinetic binding data. A method of quantifying the expression of an antigen binding protein in a prokaryotic cell lysate and a method for screening optimal conditions for its expression are also disclosed.

Description

HIGH-THROUGHPUT METHODS FOR KINETIC CHARACTERIZATION, QUANTIFYING, AND OPTIMIZING ANTIBODIES AND ANTIBODY FRAGMENTS EXPRESSION IN BACTERIA

[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: 58928P1_SeqListing.XML; Size: 2,599 bytes; Created: September 5, 2023.BACKGROUND

[0002] Antibody production in prokaryotic systems is challenging. Traditionally, researchers relied on eukaryotic production and purification to study the antigen-binding properties of antibodies of interest. The overall process of antibody production and antigen binding analysis is tedious, costly and time consuming. These limitations hamper protein engineering efforts for designing and screening novel biotherapeutics. Thus, there remains a need in the art for high-throughput methods for determining antibody affinity. Also, there remains a need in the art for high-throughput methods for determining affinity of an antigen binding protein (e.g., a VHH) binding to a target directly from prokaryotic host cell lysate. Finally, there remains a need in the art for high-throughput methods for quantifying expression level and screening optimal conditions for expression of antigen binding proteins.SUMMARY

[0003] In one aspect, described herein is a method of determining affinity of an antigen binding protein binding to a target, the method comprising: a) expressing an antigen binding protein in a prokaryotic host cell; b) lysing the prokaryotic cell to produce a prokaryotic cell lysate; c) affixing the prokaryotic cell lysate to a substrate; and d) determining the affinity of the antigen binding protein to the target.

[0004] In aspects, the antigen binding protein is selected from the group consisting of an antibody or fragment thereof, a Fab, a Fv, a scFv, a di-scFv, a nanobody, a mAb, or a VHH. In aspect, the antigen binding protein is a VHH.

[0005] In aspect, the antigen binding protein comprises at least one tag. In aspect, the antigen binding protein comprises three tags. In aspect, the tag is a FLAG tag. In aspect, the tag is a C-terminal 3X FLAG tag.

[0006] In another aspect, described herein is a method of determining affinity of a multichain protein binding to a target, the method comprising: a) expressing a multi-chain protein in a prokaryotic host cell, b) lysing the prokaryotic cell to produce a prokaryotic cell lysate; c) affixing the prokaryotic cell lysate to a substrate; and d) determining the affinity of the multichain protein to the target.

[0007] In yet another aspect, described herein is a method of quantifying expression level of an antigen binding protein in a prokaryotic cell lysate, the method comprising: a) modifying at least one expression parameter of the antigen binding protein; b) expressing the antigen binding protein in a prokaryotic host cell in a multi-well container; c) lysing the prokaryotic cell to produce a prokaryotic cell lysate; d) affixing the prokaryotic cell lysate to a substrate; and e) quantifying the amount of expression of the antigen binding protein.

[0008] In yet another aspect, described herein is a method for screening optimal conditions for expression of an antigen binding protein, the method comprising: a) modifying at least one expression parameter of the antigen binding protein, b) expressing the antigen binding protein in a prokaryotic host cell in a multi-well container, c) lysing the prokaryotic cell to produce a prokaryotic cell lysate; d) affixing the prokaryotic cell lysate to a substrate; and e) quantifying the amount of expression of the antigen binding protein, thereby determining the optimal conditions for expression of the antigen binding protein.

[0009] In another aspect, the at least one expression parameter is selected from the group consisting of: cell growth time, incubation temperature, expression inducer concentration, chaperone concentration, a transcription control element sequence, a translation control element sequence, and a codon optimized sequence. In aspect, the expression parameter is a translation control element sequence, the expression parameter is a translation control element sequence. In aspect, the translation control element sequence is a ribosome binding site (RBS) sequence.

[0010] In some embodiments, the prokaryotic host cell comprises (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.BRIEF DESCIRPTION OF FIGURES

[0011] Figure 1 shows Protein A-anti-His monoclonal antibody capture of anti-HER2 VHH and kinetic analysis of HER2 binding. The Figure depicts the use of a capture method using a synthetic His Tag and a high affinity anti-His monoclonal antibody. FIG. 1 A demonstrates the ineffective capture of an anti-His Tag monoclonal antibody when using Protein Afollowed by immobilization of anti-HER2 VHHs. FIG. 1 B shows that the capture reagent performed poorly with obvious dissociation during buffer wash steps. FIG. 1 C shows reasonable binding signal and expected KD values with serial dilution of HER2 antigen. FIG. 1 D shows that, in some instances, inverted responses are obtained due to perturbations in the refractive index between reference and sample, i.e., antigen binds reference and sample. FIG. 1 E shows results from buffer controls, which depicts that binding inverted sensorgrams are indicative of changes in the reference. FIG. 1 F shows the Table of reagents used in the experiments depicted in FIGs. 1 A-E and the kinetic parameters obtained for each immobilized sample. Note that buffer samples produce kinetic parameters within 1 log of experimental anti-HER2 VHH.

[0012] Figure 2 depicts the use of amine coupling as an alternative capture method of FIG. 1 . FIG. 2A shows significant improvement of the approach shown in FIG. 1 by adopting a strategy of amine coupling the anti- His monoclonal antibody to the chip surface, thereby avoiding the use of Protein A. FIG. 2B shows kinetics sensorgrams when employing the strategy of FIG. 2A, showing quantitative binding to HER2 antigen by anti-HER2 VHH. FIG. 2B demonstrates that anti-HER2 VHH with a C-terminal His Tag can be efficiently captured from lysate onto a SPR chip and that HER2 binding can be directly measured without the need for a purification step. FIG. 2C shows results from the negative buffer controls, showing that no buffer background signal is observed in the experiments.

[0013] Figure 3 shows schematic of why an anti-His Tag capture would not work when both the antibody (e.g., a VHH) and the antigen contain His Tags. Since most antigens purchased commercially have a His Tag, an anti-His Tag capture method would not work efficiently in broad applications.

[0014] Figure 4 shows two capture methods to avoid issues of having the same tags (e.g., His Tags) on both the antigen and the antibody. FIG. 4A shows the first method which is designed using Protein A capture of VHHs directly and antigen-binding kinetics measured using BLI. FIG. 4B shows Protein A binding to VHHs weakly and significant dissociation occurring over time, suggesting that Protein A is not a suitable method for direct capture of VHHs. FIG. 4C shows results from an anti-VHH antibody cocktail which contains a collection of antibodies that bind various VHH germlines, indicating that anti-VHH cocktail capture displays better performance than Protein A capture. However, this capture method is not reliable and will show bias across various VHH germlines and engineering single domain human VH fragments.

[0015] Figure 5 shows a comparison of N-terminal and C-terminal His Tag anti-HER2 VHH. FIG. 5A shows a BLI sensorgram of anti-HER2 VHH with a C-terminal His Tagconstruct binding to HER2. FIG. 5B shows a BLI sensorgram of an anti-HER2 VHH with a C- terminal His Tag binding to HER. C-terminal signal produces better kinetic binding responses compared to N-terminal tag, which leads to poor signal (FIG. 5B).

[0016] Figure 6 shows the use of 1X FLAG tag as an alternate to His Tag and compares two commercial FLAG tag antibodies to see which is better at binding 1X FLAG tag. FIG. 6A shows binding results from using GenScript MonoRab™ DYKDDDDK (SEQ ID NO: 1 ) Tag Antibody, mAb, Rabbit, whereas FIG. 6B shows binding results from using THE™ DYKDDDDK (SEQ ID NO: 1) Tag Antibody, mAb, Mouse. MonoRab antibody shows slower Koff than mouse antibody indicating it would be more suitable for FLAG capture.

[0017] Figure 7 shows comparison of anti-FLAG-mAb from GenScript and Millipore for 1X FLAG capture from bacterial lysate. FIG. 7A shows schematic representations of anti-FLAG- mAb from GenScript and Millipore binding to 1X FLAG-tagged VHH in lysate during immobilization step. Note sloping effect at the washout step of immobilization which indicates inefficient capture. FIG. 7B shows kinetic data collected after immobilization of VHHs comparing between GenScript and Millipore anti-FLAG-mAb. Neither capture method produced binding to antigen due to poor immobilization.

[0018] Figure 8 shows comparison of 1X and 3X FLAG capture of an anti-HER2 VHH from lysate. FIG. 8A shows schematic representations of 1X FLAG-tagged VHH vs. 3X FLAG-tagged VHH using anti-FLAG antibody from Millipore. FIG. 8B shows kinetics binding data comparing 1X and 3X FLAG capture. No HER2 binding is observed with 1X FLAG tagged VHH due to ineffective capture whereas binding is observed for 3X FLAG tagged VHH in all replicates.

[0019] Figure 9 shows comparison of 3X FLAG capture across multiple VHH germlines. FIG. 9A shows schematic representations of 3X FLAG tag capture method for anti-HER2 VHH binding HER2, anti-spike RBD VHH binding to SARS-CoV2 spike protein RBD, and anti-CTLA-4 VHH binding CTLA-4. FIG. 9B shows binding kinetics from anti-HER2 VHH. FIG. 9C shows binding kinetics from anti-spike RBD VHH. FIG. 9D shows binding kinetics from anti-CTLA-4 VHH.

[0020] Figure 10 shows improvements to the capture method using biotinylated anti-FLAG monoclonal antibody M2 clone antibody immobilization on streptavidin chips. FIG. 10A shows schematic representation of how biotinylated anti-FLAG monoclonal antibody immobilizes on streptavidin chips. FIG. 10B shows binding kinetics when using biotinylated anti-FLAG antibody is used to capture 3X FLAG tag anti-spike RBD VHH purified by affinity chromatography using protein A resin. FIG. 10C shows binding kinetics when biotinylated anti-FLAG antibody is used to capture 3X FLAG tag anti-spike RBD VHH from crude lysate.FIG. 10D shows binding kinetics when biotinylated anti-FLAG antibody is used to capture 3X FLAG tag anti-spike RBD VHH purified by affinity chromatography using anti-FLAG resin. Figure 11 shows comparison of amine coupling and biotinylated capture. Figure 11 A shows immobilization responses when amine coupled anti-FLAG antibody from Millipore to capture FLAG tag anti-spike RBD VHH from lysate. Figure 11 B shows sensorgrams obtained when spike RBD is injected as an analyte. Figure 11 C shows immobilization responses when biotinylated anti-FLAG antibody from Millipore is used to capture FLAG tag anti-spike RBD VHH from lysate. Figure 11 D shows sensorgrams obtained when spike RBD is injected as an analyte.

[0021] Figure 12 depicts a nonlimiting example of the optimization of the screening strategy by modifying an expression parameter. Specifically, Figure 12 optimizes the expression or detection of antigen binding protein by modification of the ribosome binding site (RBS) sequence or growth media. VHH is, in one embodiment, the antigen binding protein of interest.

[0022] Figure 13 shows a nonlimiting illustrative depiction of SPR capture method for VHH quantitation.

[0023] Figure 14 shows the results of ribosome binding site (RBS) optimization for expression of antigen binding protein in three different media: IBM, CDM, and CDM without yeast extract. Figure 14A shows that the Response Units measured were significantly different between the three media. IBM produces the highest Response Units across all RBS variants. Figure 14B shows that the differences in Response Units may be due to significant differences in GD600 and cell growth between the media conditions.

[0024] Figure 15 shows the measurements of SPR Response Units of the three different VHHs when the expression parameter to be modified is the ribosome binding site (RBS) sequence and carried out in IBM (Figure 15A), CDM (Figure 15B), or CDM without yeast extract (Figure 15C) media. In Figure 15A (in IBM medium), trc99 and RBS76 across all three VHHs had the highest SPR Response Units. However, none of the experimental RBS tested in Figure 15A were statistically significant when compared to the positive control. In Figure 15B (in CDM medium), some differences in the top RBS strengths were observed between the three VHHs. However, none of the experimental RBS tested in Figure 15B were statistically significant when compared to the positive control. In Figure 15C (in CDM medium without yeast extract), the Response Units were much lower than those detected in IBM (Figure 15A) or CDM (Figure 15B) media. Figure 15C shows no clear RBS based improvement across the three VHHs. The results in Figure 15C may be due to poor growth of all strains in CDM without yeast extract medium.

[0025] Figure 16 shows OD600 normalized results across three different media: IBM, CDM, and CDM without yeast extract. The results under CDM and CDM without yeast extract show significant OD differences, which may be due to poor growth in each medium resulting in large amount of noise. From these results, only IBM OD normalized data was selected for further analysis.

[0026] Figure 17 shows OD600 normalized results of RBS optimization. Trc99 and RBS76 across all three VHHs had the highest SPR Response Units. These results are statistically significant. Note: EB114 was excluded due to low OD.

[0027] Figure 18 shows RBS optimization results under IBM OD normalized condition. Trc99 and RBS76 across all three VHHs were significantly better than the control after OD normalization. All VHH expression strains showed significantly higher SPR signal than the negative control, which is the empty Host EB329.

[0028] Figure 19 shows nonlimiting schematic representation of RBS optimization for Fab discovery screening.

[0029] Figure 20 shows individual capture responses without OD normalization obtain for VHH expressed under the control of a collection of RBS sequences in various growth conditions for each replicates. Experiments were carried out in three different media: IBM (Figure 20A), CDM (Figure 20B), and CDM without yeast extract (Figure 20C) and each RBS sequence were tested in 8 replicates. The results in Figure 20 show a broad distribution in capture responses for cultures grown in IBM media. Responses for CDM with and without yeast extract are indistinguishable from positive control and negative control.

[0030] Figure 21 shows individual capture responses with OD normalization obtain for VHH expressed under the control of a collection of RBS sequences in various growth conditions for each replicates. Experiments were carried out in three different media: IBM (Figure 21 A), CDM (Figure 21 B), and CDM without yeast extract (Figure 21 C) and each RBS sequence were tested in 8 replicates. The results in Figure 21 suggest that OD normalization improves the accuracy of capture responses for IBM-grown samples and reveals negligible difference in expression for experimental samples and controls for CDM and CDM with no yeast extract.DETAILED DESCRIPTION

[0031] The present disclosure is based on the development of a rapid and high- throughput method for analyzing the antigen-binding properties of antigen binding proteins (e.g., antibodies, Fabs, and VHHs) directly from bacterial lysate. As described in the Examples, clarified bacterial lysate is analyzed by high throughput analysis (e.g., surfaceplasmon resonance (SPR)) to determine kinetic parameters of antibody binding, which allows for the direct analysis of antigen binding in optimized prokaryotic strains designed for antibody production. The present disclosure further enhances the high throughput method by incorporating, for instance, a 3X FLAG tag into the antigen binding proteins (e.g., VHHs). The present disclosure also provides that various expression parameters (e.g., RBS sequence) can be optimized to further improve the high throughput method for analyzing the antigen-binding properties of various antigen binding proteins.

[0032] In some embodiments, the present disclosure provides a method of determining affinity of an antigen binding protein binding to a target, the method comprising: a) expressing an antigen binding protein in a prokaryotic host cell; b) lysing the prokaryotic cell to produce a prokaryotic cell lysate; c) affixing the prokaryotic cell lysate to a substrate; and d) determining the affinity of the antigen binding protein to the target. In embodiments, the expression of an antigen binding protein in a prokaryotic host cell of step (a) is in a multi-well container. In embodiments, the antigen binding protein is selected from the group consisting of an antibody or fragment thereof, a Fab, a Fv, a scFv, a di-scFv, a nanobody, a mAb, or a VHH (e.g., a nanobody). In some embodiments, the antibody is derived from isotype IgG, IgE, IgM, IgD, IgA, or IgY, lgG1 , lgG2, lgG3, or lgG4. In one embodiment, the antigen binding protein is a VHH. In some embodiments, the VHH originates from germlines known in the art. Exemplary VHH germlines include IGHV3S53, IGHV3-23, and IGHV3-3. Additional examples are known in the art (Piotr Deszyhski, et al. Nucleic Acids Research, Volume 50, Issue D1 , 7 January 2022, Pages D1273-D1281 ). All VHH germlines are thus contemplated herein, including camelid, llama, alpaca or VH domains which are engineered soluble, standalone versions of human antibodies.

[0033] Surface Plasmon Resonance (SPR) binding analysis of the interactions between antibodies or antibody fragments and their target antigen requires immobilization of either antigen or antibody to the surface of a chip sensor for accurate determination of the kinetic parameters describing the association, dissociation, and thermodynamic equilibria. Often there is a demand for high-throughput SPR where thousands of antibodies can be screened in a single experiment, yielding quantitative information on antigen binding. Such protocols typically require direct analysis of samples in their source of production (e.g., media if mammalian or crude lysate if prokaryotic).

[0034] In applications where high-throughput analysis is desired, it is optimal to immobilize the antibody on chip surfaces and probe antigen as the analyte at various concentrations. High-affinity capture reagents that bind antibodies with slow dissociation rates are generally the best performing reagents deployed for high-throughput SPR analysis. These reagents are typically antibodies or VHHs that bind to specific immunoglobulindomains of antibodies. For instance, high performance capture can be achieved with high affinity antibodies or VHHs that bind to Fab regions or Fc regions of antibodies. These methods exploit conserved elements in the constant domain that allows for versatile capture of antibodies regardless of isotype or allotype. Alternatively, Protein G or Protein A can be used for antibody immobilization, but their performance is lower compared to immunoglobulin specific capture. Although monoclonal antibodies (mAbs) and Fab immobilization have been largely solved, VHH SPR remains challenging. The present disclosure provides a method for solving the problems associated with VHH SPR.

[0035] VHHs are derived from the variable domain of heavy chain only antibodies (from camelid or shark) where they are converted into single-domain antibodies that can bind to antigens without the need of a light chain. Since VHHs comprise only the variable domain of antibodies, it is understandable that VHHs that originate from different species and moreover different germlines will have unrelated sequences. The variability in VHHs sequences limits the design of versatile capture reagents that can be used for all or most VHHs. The best performing capture reagents are cocktails that target various camelid germlines. In most instances, these capture cocktails show differential performance based on VHH sequence and are not reliable for high-throughput analysis where capture directly from crude samples is required.

[0036] To circumvent issues with VHH capture, synthetic epitope-specific tags such as histidine tags covalently attached to the N-terminal or C-terminal of VHHs may be used as an alternative capture method. First an antibody that binds to the synthetic tag is immobilized on the surface of a sensor chip through chemical coupling methods. The derivatized chip can then be used to immobilize VHHs. In cases where capture from crude samples such as lysate or media is required, the synthetic tag capture reagent must display specific properties for optimal performance. The antibody must bind the synthetic tag with high specificity and should not interact with other endogenous proteins in crude samples. Furthermore, the antibody must display slow dissociation rates to synthetic tags to form a stable immobilization complex.

[0037] The present disclosure describes an efficient and universal method for capturing VHHs, Fabs, mAbs, and recombinant proteins (e.g., those that bind to molecular targets) directly from bacterial lysate. The present disclosure describes methods, reagents, principles, and strategies for engineering synthetic tags at optimal locations on recombinant proteins (e.g., those that bind to molecular targets) to allow for efficient capture from lysate and high-throughput collection of kinetic binding data (e.g., by using SPR).

[0038] The present disclosure describes optimal methods for direct measurement of kinetic binding data from lysate by SPR. Previous methods attempted to use His Tag or 1X FLAG tag to capture recombinant proteins (e.g., those that bind to molecular targets) to chip surfaces for binding analysis. However, both strategies have severe limitations and have not been shown to work in examples where binding analysis directly in lysate is required. Furthermore, 1X FLAG capture shows significant dissociation and washout of immobilized proteins overtime and, thereby, reduces signal.

[0039] In some embodiments, the antigen binding protein comprises at least one tag. In embodiments, the antigen binding protein comprises one tag, two tags, three tags, four tags, five tags, six tags, seven tags, eight tags, nine tags, or more tags. In embodiments, the antigen binding protein comprises three tags. In embodiments, the tag is a N-terminal tag or a C-terminal tag relative to the antigen binding protein. In embodiments, the tag is C-terminal to the antigen binding protein. In embodiments, the tag is selected from a His-tag or a FLAG tag. In embodiments, the tag is a FLAG tag. In embodiments, the tag is a 3X FLAG tag. In embodiments, the tag is a C-terminal 3X FLAG tag. Other tags are known in the art.

[0040] The present disclosure demonstrates an optimized protocol for sample preparation, chip surface derivatization, immobilization of recombinant proteins (e.g., those that bind to molecular targets) and direct kinetic analysis using a high-throughput workflow and engineered proteins containing a synthetic tag. The present disclosure provides details of engineering methods for incorporating a 3X FLAG tag at the C-terminal of VHHs that are known to bind to the antigens HER2 and the spike protein receptor-binding domain (RBD) of SARS-CoV-2. The optimized workflow of the present disclosure allows for rapid and sensitive binding of recombinant proteins expressed in bacterial expression systems. This allows for high-throughput screening of antigen binding by VHHs, Fabs, and mAbs expressed in bacteria.

[0041] Using a genetically engineered cell line for high protein production, recombinant protein with 3X FLAG tag can be manufactured and directly tested in SPR binding experiments using a high-throughput lysis and capture method. This method allows for a universal capture method where any recombinant proteins can be tested by SPR without changing capture reagents or recombinant tag.

[0042] The present disclosure demonstrates a method for engineering synthetic tags on recombinant proteins for the purpose of immobilization to SPR sensor chip surfaces for measuring the kinetics of antigen binding interactions conducted directly from bacterial lysate. The SPR method for direct binding analysis in lysate is previously described in PCT / US23 / 26332 and is incorporated by reference in its entirety. This protocol useschemical lysis conducted in a high-throughput fashion where samples are arrayed on 384- well blocks for SPR analysis on, for instance, a Carterra LSA, which is a high-throughput platform for the characterization of monoclonal antibodies and other large molecules. The lysis method can be carried out directly on 96-well plates using an efficient 20-minute lysis protocol, as previously described in PCT / US23 / 26332 that removes nucleic acids and preserved protein functionality by blocking the activity of endogenous bacterial proteases. High-throughput clarification of lysis reactions are carried out by mechanical vacuum filtration to remove cellular debris and inclusion bodies formed by misfolded and unfolded antibodies. Filtrate is then normalized for capture on chip surface using HEPES-based running buffer for SPR analysis. Consequently, SPR experiments are carried out using an optimized capture mechanism using gold-plated SPR chips that are covalently functionalized with streptavidin for binding kinetics analysis on Carterra LSA. The streptavidin coated chips are used to prepare a saturated lawn by flowing biotinylated anti-FLAG mAb (MilliPore Sigma) over the chip surface. The chip coated with anti-FLAG mAb is primed to capture VHHs from lysate. Once the VHHs are immobilized on the chip surface, the final step of the method of the present disclosure involves antigen binding analysis using microfluidic flow and detection on Carterra LSA using pure antigen at various concentrations.

[0043] The present disclosure uses VHHs that bind to the antigens HER2 and the spike protein receptor-binding domain of SARS-CoV-2 (spike-RBD) as proofs-of-concepts. Anti- HER2 and anti-Spike-RBD VHHs are optimized for expression in the engineered SoluPro host as described in U.S. 9,617,335, 11 ,317,048, and US2018 / 0282405, each of which is incorporated by reference in its entirety. The first step of the method involves the design of a suitable tag for capture from lysate which is the preliminary step prior to antigen binding analysis.

[0044] The present disclosure is based on the development of a rapid and high- throughput method for analyzing the antigen-binding properties of antigen binding proteins (e.g., antibodies and VHHs) directly from bacterial lysate. Clarified bacterial lysate is analyzed by high throughput analysis (e.g., surface plasmon resonance (SPR)) to determine kinetic parameters of antibody binding, which allows for the direct analysis of antigen binding in optimized prokaryotic strains designed for antibody production.

[0045] In one aspect, described herein is a method of determining affinity of an antigen binding protein binding to a target, the method comprising: a) expressing an antigen binding protein in a prokaryotic host cell in a multi-well container, b) lysing the prokaryotic cell to produce a prokaryotic cell lysate; c) affixing the prokaryotic cell lysate to a substrate; and d) determining the affinity of the antigen binding protein to the target.

[0046] In another aspect, described herein is a method of determining affinity of a multichain protein binding to a target, the method comprising: a) expressing an antigen binding protein in a prokaryotic host cell in a multi-well container, b) lysing the prokaryotic cell to produce a prokaryotic cell lysate; c) affixing the prokaryotic cell lysate to a substrate; and d) determining the affinity of the multi-chain protein to the target.

[0047] In yet another aspect, described herein is a method of quantifying expression level of an antigen binding protein in a prokaryotic cell lysate, the method comprising: a) modifying at least one expression parameter of the antigen binding protein; b) expressing the antigen binding protein in a prokaryotic host cell in a multi-well container; c) lysing the prokaryotic cell to produce a prokaryotic cell lysate; d) affixing the prokaryotic cell lysate to a substrate; and e) quantifying the amount of expression of the antigen binding protein.

[0048] In yet another aspect, described herein is a method for screening optimal conditions for expression of an antigen binding protein, the method comprising: a) modifying at least one expression parameter of the antigen binding protein, b) expressing the antigen binding protein in a prokaryotic host cell in a multi-well container, c) lysing the prokaryotic cell to produce a prokaryotic cell lysate; d) affixing the prokaryotic cell lysate to a substrate; and e) quantifying the amount of expression of the antigen binding protein, thereby determining the optimal conditions for expression of the antigen binding protein.

[0049] In another aspect, the methods described herein can also be used for increasing expression of antigen binding proteins. In another aspect, the methods described herein can also be used for improving the activity of the antigen binding protein being detected.

[0050] In some embodiments, the prokaryotic cells are lysed, insoluble debris removed, and the soluble fraction is used in a binding assay without any additional purification steps or with minimal purification. In such aspects, the antigen binding protein (or multi-chain protein) is only a fraction of the protein content within the sample. In some embodiments, the antigen binding protein (or multi-chain protein) is at most 60%, at most 55%, at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15% or at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% of the protein content within the sample. The antigen binding protein or multi-chain protein may be from 5% to 60%, 10% to 40%, or 15% to 30% of the protein content within the sample.

[0051] Binding assays, for example assays that measure protein-protein interactions, including antibody-antigen interactions and including measuring binding affinity, are well known in the art. By way of example, Surface plasmon resonance (SPR), Bio-layer interferometry (BLI), Dual polarisation interferometry (DPI), Static light scattering (SLS),Dynamic light scattering (DLS), Flow-induced dispersion analysis (FIDA), Fluorescence polarization / anisotropy, Fluorescence resonance energy transfer (FRET), Isothermal titration calorimetry (ITC), Microscale thermophoresis (MST), Single colour reflectometry (SCORE) are contemplated. Additionally, Bimolecular fluorescence complementation (BiFC), affinity electrophoresis, label transfer, phage display, Tandem affinity purification (TAP), crosslinking, Quantitative immunoprecipitation combined with knock-down (QUICK), and Proximity ligation assay (PLA) are other well-known assays that provide protein-protein interaction information.

[0052] In embodiments, the measured binding affinity measured by the method of the present disclosure is comparable to its published value.

[0053] In embodiments, the high-throughput screening method is surface plasmon resonation (SPR). In embodiments, the high-throughput screening method is Bio-Layer Interferometry (BLI). In embodiments, the high-throughput screening method is surface plasmon resonation (SPR) or Bio-Layer Interferometry (BLI).

[0054] Antigen binding proteins can be captured on any substrate for use in the methods described herein. Exemplary substrates (e.g., optionally in association with a capture agent as described herein) include, but are not limited to, metal surfaces coated with one or more of the following: covalent conjugation to polycarboxylate hydrogel and caboxymethyldextran matrices, protein A / G derivatived hydrogel and caboxymethyldextran matrices, Streptavidin derivatized hydrogel and caboxymethyldextran. Exemplary capture agents include, but are not limited to, kappa capture, lambda capture, FLAG tag capture, HIS tag capture, Protein A, Protein G, Protein L, and biotin / streptavidin.

[0055] In some embodiments, the binding affinities of antigen binding proteins are measured by array surface plasmon resonance (SPR), according to standard techniques (Abdiche, et al. (2016) MAbs 8:264-277). Briefly, antibodies were immobilized on a HC 30M chip at four different densities / antibody concentrations. Varying concentrations (0-500 nM) of antibody target are then bound to the captured antibodies. Kinetic analysis is performed using Carterra software to extract association and dissociation rate constants (kaand kd, respectively) for each antibody. Apparent affinity constants (KD) are calculated from the ratio of kd / ka. 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, a biosensor instrument suchas Octet RED384, ProteOn XPR36, IBIS MX96 and BIAcore T100 is used (Yang, D., et al., J. Vis. Exp., 2017, 122:55659).

[0056] KD is the equilibrium dissociation constant, a ratio of k0ff / k0n, between the antigen binding protein (e.g., antibody) and its antigen. KDand affinity are inversely related. The KDvalue relates to the concentration of antibody and 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 (10-4to 10-6), the nanomolar range (1 O'7to 10-9), the picomolar range (10-10to 10-12), or the femtomolar range (10-13to 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.).

[0057] Antigen binding proteins

[0058] Antigen binding proteins contemplated herein include full-length proteins, precursors of full-length proteins, biologically active subunits or fragments of full-length proteins, as well as biologically active derivatives and variants of any of these forms of therapeutic proteins. Thus, antigen binding proteins include those that (1 ) have an amino acid sequence that has greater than about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% or greater amino acid sequence identity, over a region of at least about 25, about 50, about 100, about 200, about 300, about 400, or more amino acids, to a polypeptide encoded by a referenced nucleic acid or an amino acid sequence described herein. According to the present disclosure, the term "recombinant protein" includes any protein obtained via recombinant DNA technology. In certain embodiments, the term encompasses proteins as described herein.

[0059] In some embodiments, the antigen binding protein is an antibody. The term “antibody” as used herein refers to whole antibodies that interact with (e.g., by binding, steric hindrance, stabilizing / destabilizing, spatial distribution) an epitope on a target antigen.

[0060] The term “multi-chain protein” as used herein refers to an antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as 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 as 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. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term “antibody” includes for example, monoclonal antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), Fab fragments, F (ab') fragments, and anti-idiotypic (anti-ld) antibodies (including, e.g., anti-ld antibodies to antibodies of the invention), and epitope-binding fragments of any of the above. The antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG 1 , lgG2, lgG3, lgG4, lgA1 , and lgA2) or subclass. The antibody or epitope-binding fragments may be, or be a component of, a multi-specific molecule.

[0061] Both the light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. In this regard, it will be appreciated that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light chain (CL) and the heavy chain (CH1 , CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention the numbering of the constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody. The N-terminus is a variable region and at the C-terminus is a constant region; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chain, respectively.

[0062] Exemplary antibodies include, but are not limited to, antibodies such as infliximab, bevacizumab, cetuximab, ranibizumab, palivizumab, abagovomab, abciximab, actoxumab, adalimumab, afelimomab, afutuzumab, alacizumab, alacizumab pegol, ald518, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab mafenatox, anrukinzumab, apolizumab, arcitumomab, aselizumab, altinumab, atlizumab, atorolimiumab, tocilizumab, 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, crenezumab, cr6261 , dacetuzumab, daclizumab, dalotuzumab, daratumumab, demcizumab, denosumab, detumomab, dorlimomab aritox, drozitumab, duligotumab, dupilumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, elotuzumab, elsilimomab, enavatuzumab, enlimomab pegol, enokizumab, enoticumab, ensituximab, epitumomab cituxetan, epratuzumab, erenumab, erlizumab, ertumaxomab, etaracizumab, etrolizumab, evolocumab, 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, ibritumomab tiuxetan, icrucumab, igovomab, imciromab, imgatuzumab, inclacumab, indatuximab ravtansine, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin, ipilimumab, iratumumab, itolizumab, ixekizumab, keliximab, labetuzumab, lebrikizumab, lemalesomab, lerdelimumab, lexatumumab, libivirumab, ligelizumab, lintuzumab, lirilumab, lorvotuzumab mertansine, lucatumumab, lumiliximab, mapatumumab, maslimomab, mavrilimumab, matuzumab, 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, panitumumab, panobacumab, parsatuzumab, 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, sevirumab, sibrotuzumab, sifalimumab, siltuximab, simtuzumab, siplizumab, sirukumab, solanezumab, solitomab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizumab, tanezumab, taplitumomab paptox, tefibazumab, telimomab aritox, tenatumomab, tefibazumab, teneliximab, teplizumab, teprotumumab, tezepelumab, TGN1412, tremelimumab, ticilimumab, tildrakizumab, tigatuzumab, TNX-650, tocilizumab, toralizumab, tositumomab, tralokinumab, trastuzumab,TRBS07, tregalizumab, 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.

[0063] The phrase “antibody fragment”, as used herein, refers to one or more portions of an antibody that retain the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing / destabilizing, spatial distribution) a target epitope. Examples of binding fragments include, but are not limited to, a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1 domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., (1989) Nature 341 :544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR). 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 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antibody fragment”. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0064] Prokaryotic Cells

[0065] The methods described herein comprise expressing an antigen binding protein (or a multi-chain binding protein) in a prokaryotic host cell. 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). Preferred 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 marcescans), and Shigella.

[0066] 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, through 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 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.

[0067] 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 cellsare 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 an E. coli B strain, or are E. coli lB 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).

[0068] 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.

[0069] 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 is 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, hasbeen 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 reductant, 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): 6735-6740, 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. coli katG and HPII (hydroperoxidase II) catalase-peroxidase encoded by E. coli 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.

[0070] 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. coli, even in the absence of mutations in gor or trxB (Nguyen et al, Microb Cell Fact 2011 Jan 7; 10:1).

[0071] 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.

[0072] 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.

[0073] 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 Sail 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 Sail restriction sites in SEQ IDNO: 2 can be used to insert it into a vector multiple cloning site, such as that of the pSOL expression vector (SEQ ID NO: 3 of International Publication No. WO 2017 / 106583), described in published US patent application US 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%, or 80%, or 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.

[0074] 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.

[0075] 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).

[0076] 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).

[0077] Table A. Exemplary host cell strains

[0078] Expression constructs

[0079] In some embodiments, a prokaryotic cell described herein comprises one or more expression constructs may optionally include one or more inducible promoters to express an antigen binding protein of interest.

[0080] 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.

[0081] 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 thebasis 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 number 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 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 coexpress 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'.

[0082] 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 expression of subunit B from pl5A-Para-B. In a similar fashion, 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. 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 Alcaligeneseutrophus, 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).

[0083] 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): 4471-4481).

[0084] 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 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.

[0085] 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), gentamycin acetyltransferase, streptomycin phosphotransferase, mutant form of acetolactate synthase (als), bromoxynil nitrilase, phosphinothricin acetyl transferase (bar), enolpyruvylshikimate-3-phosphate (EPSP) synthase (aro A), muscle specific tyrosine kinase receptor molecule (MuSK-R), copper-zinc superoxide dismutase (sod1), metallothioneins (cup1 , MT1 ), beta-lactamase (BLA), puromycin N-acetyl-transferase (pac), blasticidin acetyl transferase (bls), blasticidindeaminase (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.

[0086] 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.-^ 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.

[0087] 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 or GGAGGU, and in bacteria such as E. coli, the RBS consensus sequence is AGGAGG or AGGAGGU. 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 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 interveningnucleotides. 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).

[0088] In certain embodiments, the sequence of the ribosome binding site (RBS) can be screened to select for optimal expression of a specific antigen binding protein. An RBS sequence is one parameter of the translation control element sequence. In embodiments, the translation control element sequence is an expression parameter than can be further optimized as described in the method of the present disclosure. In some embodiments, the translation control element sequence is a ribosome binding site (RBS) sequence.

[0089] 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 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 -4130); 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 (US Patent No. 8178338 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 or, or 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 expressionconstruct at the MCS, preferably in the proper relation to any coding sequences to stimulate initiation of translation from the transcribed messenger RNA.

[0090] 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 E.2 substrain MG1655 (Inouye and Inouye, Nucleic Acids Res 1985 May 10; 13(9): 3101 -3110). 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 ): 6639-6657). 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.

[0091] 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 has a tendency 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 cleavedfrom the mature polypeptide typically end in a stretch of amino acids that is 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. coll, 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.

[0092] 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 of E. coli K-12 (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.

[0093] 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 thepurpose 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 which 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): 7373-7377). In order to achieve homogenous induction of an arabinose-inducible promoter, it is useful to make 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 which 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 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, 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, use of polynucleotides encoding the LacY(A177C) protein is preferred to the use of polynucleotides encoding the LacY(A177V) protein.

[0094] Propionate promoter. The 'propionate promoter' or 'prp promoter' is the promoter for the E. coli prpBCDE operon, and is also called PPQB.-Like the ara promoter, the prp promoter is part of a bidirectional promoter, controlling expression of the prpBCDE operon in one direction, and with the prpR promoter controlling 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 sensitivity of the propionate-inducible promoter to exogenously supplied propionate. It has been found that a deletion of sbm-ygfD-ygfG-ygfH-ygfl, 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): 6856-6862), 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 adeletion 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.

[0095] 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 expression of the rhaSR operon in one direction, and with the rhaBAD promoter controlling 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 expression of the rhaSR operon in the presence of rhamnose, while RhaS protein activates expression of the L-rhamnose catabolic and transport operons, rhaBAD and rhaT, respectively (Wickstrum et al, J Bacteriol 2010 Jan; 192(1): 225-232). 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 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 byrhamnose (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.

[0096] Xylose promoter. (As used herein, ‘xylose’ means D-xylose.) The xylose promoter, or xyl promoter’, or PxyiA, means the promoter for the E. coli 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): 7025-7032). The transcriptional regulator of both the PxyiA and PxyiF promoters is XylR, which activates 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 protein-coding 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 an 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.

[0097] 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, 1 l-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.

[0098] Alkaline phosphatase promoter. The terms ‘alkaline phosphatase promoter’ and ‘phoA promoter’ refer to the promoter for the phoApsiF operon, a promoter which is induced under conditions of phosphate starvation. The phoA promoter region is located at ca.401647 - 401746 (plus strand, with the Pribnow box ('-10') at 401695 - 401701 (Kikuchi et al., Nucleic Acids Res 1981 Nov 11 ; 9(21 ): 5671 -5678)) in the genomic sequence of the E. coli K.-^ 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 - intracellular phosphate - rather than by the addition of an inducer. For this reason, the phoA promoter is generally used to direct 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.

[0099] 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 artand include using detergents and intercalating agents, drugs, and antibiotics (Buckner, et al., FEMS Microbiology Reviews, fuy031 ,42, 2018, 781 -804).

[0100] Expression parameters. The phrase “expression parameter(s)” as used throughout the disclosure refers to any variable that can be modified or optimized in order to improve the expression (or enhance an activity being detected, such as binding activity or enzymatic activity, and the like) of the desired antigen binding protein. Without limitations, some examples of expression parameters include cell growth time, incubation temperature, expression inducer concentration, the presence or absence of a chaperone, either endogenously / naturally expressed or exogenously provided and expressed from an expression vector and, when present, chaperone concentration, a transcription control element sequence, a translation control element sequence, and a codon optimized sequence. In one embodiment, the expression parameter is a translation control element sequence. In other embodiments, the translation control element sequence is a ribosome binding site (RBS) sequence. Such optimization of the expression (or SPR signal detection) of an antigen binding protein by screening for optimal RBS sequences is shown in Example 7 of the present disclosure. In some embodiments, any individual components of the expression construct can be considered an expression parameter to be modified for the purpose of optimizing the expression of the antigen binding protein. In some embodiments, promoters are expression parameters that can be modified according to the present disclosure. In other embodiments, the selection gene is an expression parameter that can be modified. In other embodiments, a signal peptide is an expression parameter that can be modified. In still other embodiments, the origin of replication is an expression parameter that can be modified according to the present disclosure.

[0101] Epitope tags. The first use of epitope tagging was described in Munro, S., & Pelham, H. R. "Use of peptide tagging to detect proteins expressed from cloned genes: deletion mapping functional domains of Drosophila hsp 70." The EMBO joumal3.13 (1984): 3087-3093. FLAG-tag, or FLAG octapeptide, or FLAG epitope, is a peptide protein tag that can be added to a protein using recombinant DNA technology. The peptide sequence of the FLAG-tag from the N-terminus to the C-terminus is: DYKDDDDK (SEQ ID NO: 1 ) (where D=aspartic acid, Y=tyrosine, and K=lysine). The size of a FLAG-tag is approximately 1012 dalton. Additionally, FLAG-tags may be used in tandem, commonly the 3xFLAG peptide: DYKDHD-G-DYKDHD-I-DYKDDDDK (SEQ ID NO: 2) (with the final tag encoding an enterokinase cleavage site). FLAG-tag can be fused to the C-terminus or the N-terminus of a protein, or inserted within a protein. Some commercially available antibodies (e.g., M1 / 4E11) recognize the epitope only when FLAG-tag is present at the N-terminus. However, other available antibodies (e.g., M2) are position-insensitive. The tyrosine residue in the FLAG-tagcan be sulfated when expressed on certain secreted proteins, which can affect antibody recognition of the FLAG epitope. The FLAG-tag can be used in conjunction with other affinity tags, for example a polyhistidine tag (His-tag), HA-tag, or myc-tag. In embodiments, the antigen binding protein of the present disclosure comprises a FLAG-tag in conjunction with other affinity tags.

[0102] VHHs. VHHs are the variable domains of heavy-chain antibodies (HCAb) found in Camelidae. This family is composed of Vicugna, Alpaca, Llama, Camel and Dromedary. As other mammals, they express conventional antibodies composed of two heavy chains and two light chains. In addition, they also express non-conventional antibodies named heavychain antibodies devoid of CH1 domain and light chain. HCAb are present for lgG2 and lgG3 isotypes. These antibodies are composed of two heavy chains divided in three domains each: CH3-CH2-VHH. The molecular weight of these HCAb is 90 kDa, smaller than the 150 kDa of conventional antibodies. Their small size allows them to diffuse more than four times better in tissues and tumors than antibodies. However, half-life-extension modules linked in some cases to the VHHs might obliterate their small size benefit and negatively impact this diffusion and internalization. Some VHHs have been shown to cross cell membranes and reach cytosolic targets. In this case, their internalization has been linked to their basic isoelectric point and positive surface charges.

[0103] 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 information described herein.

[0104] The following examples are given merely to illustrate the present invention and not in any way to limit its scope.EXAMPLES

[0105] Materials and Methods

[0106] Sample preparation: 200 pL Cell Cultures

[0107] 200 pL per well of cell cultures were transferred into a new 96-Well Sample plate. The 96-Well Sample containing the cell cultures was centrifuged at 3500xG for 10 minutes at 4°C. The supernatant was discarded, and the cell pellets remained in the plate.

[0108] Sample preparation: 200 pL Cell Pellets

[0109] Obtain a 96-Well plate or plates containing 200 pL cell pellets per well. Cell Lysis Buffer was performed by mixing BugBuster® Protein Extraction Reagent, Protease InhibitorCocktail, and Benzonase Nuclease. For a single 96-Well plate, 6.25 mL of BugBuster® Protein Extraction Reagent, 62.5 pL of Protease Inhibitor Cocktail, and 5 pL of Benzonase Nuclease were added into a 15 mL Conical-Bottom Centrifuge Tube. The tube was vortexed to mix the Cell Lysis Buffer thoroughly.

[0110] The 200 pL cell pellets were resuspended by adding 50 pL of Cell Lysis Buffer into each well. The resuspended samples in the 96-Well plate(s) (covered with 96-Well Plate Adhesive Sealing Film) were mixed for 20 minutes at 37°C in an incubator shaker.

[0111] After 20 minutes, the 96-Well plate(s) were removed from the shaker and 170 pL of 1 X HBSTE ((HEPES-buffered saline with 0.05% Tween20 and 3mM EDTA) buffer was added into each well. The 96-Well containing cell lysates at were centrifuged at 3500xG for 5 minutes. After centrifugation, the supernatant was transferred into 96-Well Filter Plate(s). The plate containing the pellets was discarded.

[0112] The mixture was filtered using the Pall or MultiScreen® Vacuum Manifold and a vacuum filtration system.Example 1 - Protein A is ineffective for detecting VHH scaffolds

[0113] This experiment demonstrates the use of a capture method using a synthetic His Tag and a high affinity anti-His mAb (Figure 1 A). The results demonstrate the ineffective capture of an anti-His Tag monoclonal antibody (mAb) using Protein A followed by immobilization of anti-HER2 VHHs (Figure 1). Capture reagents performed poorly with obvious dissociation during buffer wash steps (Figure 1 B). Reasonable binding signal and expected KD values with serial dilution of HER2 antigen were observed (Figure 1C). However, in some instances inverted responses are obtained due to perturbations in the refractive index between reference and sample, i.e., antigen binds reference and sample (Figure 1 D).Example 2 - His Tag works upon removal of Protein A / G capture and addition of amine coupling

[0114] This experiment shows improvement of the approach shown in FIG. 1 by adopting a strategy of amine coupling the anti-his mAb to the chip surface, thereby avoiding the use of protein A (Figure 2A). In kinetics experiments with this strategy, quantitative binding to HER2 antigen by the anti-HER2 VHH are shown (Figure 2B). This example demonstrates that anti- HER2 VHH with a C-terminal His Tag can be efficiently captured from lysate onto a SPR chip and HER2 binding directly measured without the need for a purification step (Figure 2B). The measured binding affinity is comparable to published values. Figure 2 shows a clear proof-of-principle that the disclosure described herein can be expanded to any recombinant protein that binds to a molecular target. However, there will be cases wherebinding partner, such as commercial antigens, will contain a His Tag. In this scenario the presence of the His Tag on the analyte (antigen) would bind to capture reagent anti-His Tag mAb and produce a false positive binding signal.Example 3 - Two possible methods to avoid the issue of having similar tags on antibody and antigen

[0115] When His tag is used, the method would not work efficiently if His Tag is used on both antibody and antigen in broad applications (Figure 3).

[0116] The example demonstrates two capture methods to avoid issues of similar tags on antigen and antibodies (Figure 4). The first method is designed using Protein A capture of VHHs directly and antigen-binding kinetics measured using BLI (Figure 4A). This interaction is weak and is dependent on the VHH germline. Figure 4B clearly demonstrates rapid dissociation within 2 minutes of dissociation phase for all VHHs tested. The result shows that protein A is not a suitable method for direct capture of VHHs.

[0117] Next, the example further tested an anti-VHH antibody cocktail which contains a collection of antibodies that bind various VHH germlines (Figure 4C). Anti-VHH cocktail capture displays better performance than protein A capture, however, there is obvious selectivity for some germlines over others based on the signal and differences in dissociation kinetics for each VHH tested (Figure 4C). This confirms that although anti-VHH cocktail capture works, it is not universal and likely will not be useful for affinity screening workflows required for a large collection of antibodies and antibody fragments.Example 4 - Universal capture using a 3X FLAG tag on the C-terminal of VHHs

[0118] The innovative solution to avoiding similar tags between antigen and recombinant protein (e.g., antibody) while developing a universal capture method is the use of a unique tag that when attached to recombinant proteins can be efficiently captured onto SPR chip surfaces. The present disclosure demonstrates a proof-of-concept for universal capture using a 3X FLAG tag on the C-terminal of VHHs. The C-terminal FLAG tag on VHH constructs were chosen because of better kinetic binding responses when comparing N- terminal and C-terminal His Tag anti-HER2 VHH (Figure 5).

[0119] Experiments were designed using commercial anti-FLAG antibodies that bound the best to a 1X FLAG tag (Figure 6). The result shows that lysate capture works best with anti- FLAG monoclonal antibody M2 clone from Millipore (F1084). Significant dissociation was observed with commercial antibodies when used to capture 1X FLAG tag VHH constructs, with no measurable affinity to target. This result can be interpreted as significant dissociation of captured FLAG tag VHH from the chip surface resulting with no measurable antigen binding at the analyte injection step. In additional full kinetic experiments using 1X Flagcapture produced no detectable binding after immobilization of VHH contain 1X Flag tag at its C-terminal. These results suggest that 1X FLAG tag is not a suitable capture method for measuring binding due to unstable capture.

[0120] Figure 7 shows comparison of anti-FLAG-mAb from GenScript and Millipore for 1X FLAG capture from bacterial lysate. FIG. 7A shows schematic representations of anti-FLAG- mAb from GenScript and Millipore binding to 1X FLAG-tagged VHH in lysate during immobilization step. Note sloping effect at the washout step of immobilization which indicates inefficient capture. FIG. 7B shows kinetic data collected after immobilization of VHHs comparing between GenScript and Millipore anti-FLAG-mAb. Neither capture method produced binding to antigen due to poor immobilization. The results in Figure 7 show that 1X FLAG works for purified sample but not for capture from lysate. Because the SPR run takes approximately 19 hours, it requires a stable capture that slowly dissociates over time. The capture from the lysate shows too rapid of a dissociation s most of the captured VHH has dissociated by the time it reaches the analyte injection step which results in no antigen binding observed.

[0121] To improve on the capture technology, 3X FLAG tag was designed at the C- terminal of an anti-HER2 VHH. Binding kinetics of anti-HER VHH that contains either a 1X FLAG tag or 3X FLAG tag at the C-terminal were compared with HER2 using an anti-FLAG monoclonal antibody M2 clone antibody (Figure 8A). The results illustrate that 3X FLAG tag capture is better than 1X FLAG tag for producing accurate binding kinetics directly from lysate (Figure 8B).Example 5 - Anti-FLAG monoclonal antibody M2 clone to capture C-terminal FLAG tag VHHs is universal for VHH germlines

[0122] This example demonstrates that the capture method using anti-FLAG monoclonal antibody M2 clone to capture C-terminal FLAG tag VHHs is universal for VHH germlines. In this example, the capture method can be used to measure antigen binding by anti-HER VHH, anti-SARS-CoV-2 Spike RBD VHH, and an anti-CTLA4 VHH. Results were observed at nanomolar binding affinities that match published values (Figures 9A-D).Example 6 - Biotinylated anti-FLAG monoclonal antibody M2 clone antibody immobilization on streptavidin chips

[0123] This example provides additional methods, such a biotinylated anti-FLAG monoclonal antibody M2 clone antibody immobilization on streptavidin chips, that works with equivalent performance to amine coupling (Figures 10A-C).

[0124] Figure 11 shows comparison of amine coupling and biotinylated capture. Figure 11 A shows immobilization responses when amine coupled anti-FLAG antibody from Milliporeto capture FLAG tag anti-spike RBD VHH from lysate. Figure 11 B shows sensorgrams obtained when spike RBD is injected as an analyte. Figure 11 C shows immobilization responses when biotinylated anti-FLAG antibody from Millipore is used to capture FLAG tag anti-spike RBD VHH from lysate. Figure 11 D shows sensorgrams obtained when spike RBD is injected as an analyte.Example 7 - Improvement of expression and / or SPR signal of VHHs by modifying ribosome binding site (RBS) sequences

[0125] The present disclosure provides high-throughput methods for increasing the expression of antigen binding protein, improving the activity of antigen binding protein, screening for optimal conditions for expression of antigen binding protein, and measuring the titer of antigen binding protein. The high-throughput methods of the present disclosure are carried out directly from the lysates of prokaryotic cells that expressed the antigen binding protein.

[0126] Although multiple parameters that control the expression of antigen binding protein can be modified, this Example shows the modification of the ribosomal binding site (RBS) sequence as a proof-of-concept that other parameters can also be modified in a high- throughput manner to achieve the desired improved expression (or, for instance, improved SPR signal being detected).

[0127] The problem to be solved in this Example is whether the expression or SPR signal detection of antigen binding protein, specifically VHHs in this Example, can be improved by modifying one of the parameters. Specifically, this Example demonstrates the modification of RBS sequence on the plasmid backbone. The experiments were carried out using 12 different RBS sequences to express three VHHs that were previously shown to express poorly and / or yield low SPR signals. This Example shows that the expression or SPR signals of such VHHs can be improved by modifying the RBS sequence in a quick and high- throughput manner.

[0128] Plasmid / Host architecture to be used was PL5652 (PDI+PGdx) backbone in EB329. The three VHHs used in this Example were CTLA4-nb16_VHH1 (PL6417), CTLA4- nb30_VHH1 (PL6418), and VHH H11 -H4 (PL6281 ). Discrete cloning was carried out using Gibson assembly with single strand oligos. 12 RBS strengths were chosen based on Scon GFP experiments and OSTIR RBS strength prediction. Table B shows the cloning strategy for this RBS optimization experiment.

[0129] Table B. Cloning Strategy.strategy by modifying an expression parameter. Specifically, Figure 12 optimizes the expression or detection of antigen binding protein by modification of the ribosome binding site (RBS) sequence or growth media. VHH is the antigen binding protein of interest.

[0131] Table C shows the list of strains used in this Example.

[0132] Table C. Strain List.

[0133] Protocol. All Strains were seeded in LB + Kan50 in deep-well plate (DWP) and grown at 37°C, 80% humidity, and 1000 rpm. 50 pl of overnight seed culture was used to inoculate 500 pl of IBM, CDM, and CDM without Yeast Extract with 200 pM Arabinose and 1 mM Propionate in DWP. The strains were incubated for 21 hrs at 30°C, 80% humidity, 1000 rpm (EoF OD600 were measured in Envision plate reader in 96-well plates). 200 pl broth was harvested, pelleted and lysed with BugBuster + Benzonase lysis buffer for 30 min at 30°C. The lysate was pelleted, supernatant diluted in HBSTE buffer and filtered before use for SPR analysis.

[0134] Figure 13 shows a nonlimiting illustrative depiction of SPR capture method for VHH quantitation.

[0135] Figure 14 shows results from expressing in three different media: IBM, CDM, and CDM without yeast extract. The Response Units observed were significantly different between the 3 media tested. IBM clearly had the highest Response Units across all RBS variants. These differences maybe mainly due to significant differences in OD600 and cell growth between the media conditions.

[0136] Figure 15 shows results from three different media: IBM (Fig. 15A), CDM (Fig. 15B), and CDM without yeast extract (Fig. 15C). In the experiment using IBM (as depicted in Fig. 15A), trc99 and RBS76 across all three VHHs had the highest SPR Response Units. However, none of the experimental RBS tested here were statistically significant when compared to the positive control. In the experiment using CDM (as depicted in Fig. 15B), some differences in the top RBS strengths were observed between the 3 VHHs tested. However, none of the experimental RBS tested here were statistically significant when compared to the positive control. In the experiment using CDM without yeast extract (as depicted in Fig. 15C), The Response Units in CDM without yeast extract were much lower than those tested in IBM (Fig. 15A) and CDM (Fig. 15C). No clear RBS based improvement observed across the three VHHs tested. Without wishing to be bound by a specific theory, this result is possibly due to poor growth of all strains in CDM without yeast extract.

[0137] Figure 16 shows OD600 normalized results across three different media: IBM, CDM, and CDM without yeast extract. The large OD differences due to poor growth in CDM and CDM without yeast extract especially resulted in large amount of noise. Only IBM OD normalized data was used for further analysis.

[0138] Figure 17 shows OD600 normalized results of RBS optimization. Trc99 and RBS76 across all three VHHs had the highest SPR Response Units and the results were statistically significant. Note: EB114 was excluded due to unexpectedly low OD.

[0139] Figure 18 shows RBS optimization results under IBM OD normalized condition. Trc99 and RBS76 across all three VHHs were significantly better than the control after OD normalization. As expected, All VHH expression strains showed significantly higher SPR signal than the empty Host EB329.

[0140] Conclusions

[0141] Among the newly tested RBS variants, RBS_trc99 and RBS76 performed better across all three VHH molecules in IBM. There are large differences in SPR Response Units measurements across the three media tested with IBM producing the highest OD600 and highest Response Units in SPR.

[0142] Further study could be focused on fermentation media and growth condition optimization in further improvement in SPR signal for drug discovery projects. RBS76 will be used for future VHH experiments.

[0143] Figure 19 shows nonlimiting schematic representation of RBS optimization for Fab discovery screening.

[0144] Figure 20 shows individual capture responses without OD normalization obtain for VHH expressed under the control of a collection of RBS sequences in various growth conditions for each replicates. Experiments were carried out in three different media: IBM (Figure 20A), CDM (Figure 20B), and CDM without yeast extract (Figure 20C) and each RBS sequence were tested in 8 replicates. The results in Figure 20 show a broad distribution in capture responses for cultures grown in IBM media. Responses for CDM with and without yeast extract are indistinguishable from positive control and negative control.

[0145] Figure 21 shows individual capture responses with OD normalization obtain for VHH expressed under the control of a collection of RBS sequences in various growth conditions for each replicates. Experiments were carried out in three different media: IBM (Figure 21 A), CDM (Figure 21 B), and CDM without yeast extract (Figure 21 C) and each RBS sequence were tested in 8 replicates. The results in Figure 21 suggest that OD normalization improves the accuracy of capture responses for IBM-grown samples and reveals negligible difference in expression for experimental samples and controls for CDM and CDM with no yeast extract.

Claims

What is claimed is:1 . A method of determining affinity of an antigen binding protein binding to a target, the method comprising: a) expressing an antigen binding protein in a prokaryotic host cell; b) lysing the prokaryotic cell to produce a prokaryotic cell lysate; c) affixing the prokaryotic cell lysate to a substrate; and d) determining the affinity of the antigen binding protein to the target.

2. The method of claim 1 , wherein the expression of an antigen binding protein in a prokaryotic host cell of step (a) is in a multi-well container.

3. The method of claim 1 or claim 2, wherein the antigen binding protein is selected from the group consisting of an antibody or fragment thereof, a Fab, a Fv, a scFv, a di-scFv, a nanobody, a mAb, or a VHH.

4. The method of claim 3, wherein the antibody is derived from isotype IgG, IgE, IgM, IgD, IgA, IgY, lgG1 , lgG2, lgG3, or lgG4.

5. The method of claim 3, wherein the antigen binding protein is a VHH.

6. The method of claim 5, wherein the VHH is a IGHV3S53, a IGHV3-23, or a IGHV3-3.

7. The method of any one of claims 1-6, wherein the antigen binding protein comprises at least one tag.

8. The method of claim 7, wherein the antigen binding protein comprises one tag, two tags, three tags, four tags, five tags, six tags, seven tags, eight tags, nine tags, or more tags.

9. The method of claim 7 or claim 8, wherein the antigen binding protein comprises three tags.

10. The method of any one of claims 7-9, wherein the tag is a N-terminal tag or a C-terminal tag relative to the antigen binding protein.11 . The method of claim 10, wherein the tag is C-terminal to the antigen binding protein.

12. The method of any one of claims 7-11 , wherein the tag is selected from a His- tag or a FLAG tag.

13. The method of claim 12, wherein the tag is a FLAG tag.

14. The method of any one of claims 7-13, wherein the tag is a 3X FLAG tag.

15. The method of claim 14, wherein the tag is a C-terminal 3X FLAG tag.

16. The method of any one of claims 1-15, wherein the prokaryotic host cell comprises 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.

17. The method of any one of claims 1-16, wherein the prokaryotic host cell is derived from an Enterobacterial species.

18. The method of claim 17, wherein host cell is Escherichia coli.

19. The method of any one of claims 1-18, wherein the affinity of the antigen binding protein to the target is determined by a high-throughput screening method.

20. The method of claim 19, wherein the high-throughput screening method is surface plasmon resonation (SPR) or Bio-Layer Interferometry (BLI).21 . The method of any one of claims 1-20, wherein the substrate is kappa capture, lambda capture, FLAG or HIS tag capture, Fc capture, Protein A, Protein G, Protein L, or biotin / streptavidin.

22. The method of any one of claims 1-21 , wherein the substrate comprises an anti-tag antibody.

23. The method of claim 22, wherein the anti-tag antibody displays slow dissociation rates to the tag.

24. The method of claim 22 or claim 23, wherein the anti-tag antibody is chemically coupled for binding to a chip surface.

25. The method of claim 24, wherein the anti-tag antibody is amine coupled for binding to the chip surface.

26. The method of claim 22 or claim 23, wherein the anti-tag antibody is biotinylated for binding to a chip surface.

27. The method of any one of claims 22-26, wherein the anti-tag antibody is an anti-FLAG antibody.

28. The method of any one of claims 1-27, wherein the capture agent is kappa capture.

29. The method of anyone of claims 1-28, wherein the antigen binding protein has a KD between 10 pM and 1for the target.

30. The method of any one of claims 1-29, wherein the antigen binding protein is a multi-chain protein.31 . The method of any one of claims 1-30, wherein the antigen binding protein is an antibody.

32. The method of any one of claims 1-30, wherein the antigen binding protein is an antigen binding fragment of an antibody.

33. A method of determining affinity of a multi-chain protein binding to a target, the method comprising: a) expressing a multi-chain protein in a prokaryotic host cell; b) lysing the prokaryotic cell to produce a prokaryotic cell lysate; c) affixing the prokaryotic cell lysate to a substrate; and d) determining the affinity of the multi-chain protein to the target.

34. The method of claim 33, wherein the prokaryotic host cell comprises 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.

35. The method of claim 33 or claim 34, wherein the prokaryotic host cell is derived from an Enterobacterial species.

36. The method of claim 35, wherein host cell is Escherichia coli.

37. The method of any one of claims 33-36, wherein the expressing step is performed in a multi-well container.

38. The method of any one of claims 33-37, wherein the affinity of the multi-chain protein to the target is determined by a high-throughput screening method.

39. The method of claim 38, wherein the high-throughput screening method is surface plasmon resonation (SPR) or Bio-Layer Interferometry (BLI).

40. The method of any one of claims 34-39, wherein the substrate is kappa capture, lambda capture, FLAG or HIS tag capture, Fc capture, Protein A, Protein G, Protein L, or biotin / streptavidin.41 . The method of claim 40, wherein the capture agent is kappa capture.

42. The method of anyone of claims 33-41 , wherein the multi-chain protein has a KD betweenr the target.

43. The method of any one of claims 33-42, wherein the antigen binding protein is an antibody.

44. The method of any one of claims 33-43, wherein the antibody has two heavy chains and two light chains.

45. A method of quantifying expression level of an antigen binding protein in a prokaryotic cell lysate, the method comprising: a) modifying at least one expression parameter of the antigen binding protein; b) expressing the antigen binding protein in a prokaryotic host cell in a multi-well container; c) lysing the prokaryotic cell to produce a prokaryotic cell lysate; d) affixing the prokaryotic cell lysate to a substrate; and e) quantifying the amount of expression of the antigen binding protein.

46. A method for screening optimal conditions for expression of an antigen binding protein, the method comprising: a) modifying at least one expression parameter of the antigen binding protein;b) expressing the antigen binding protein in a prokaryotic host cell in a multi-well container; c) lysing the prokaryotic cell to produce a prokaryotic cell lysate; d) affixing the prokaryotic cell lysate to a substrate; and e) quantifying the amount of expression of the antigen binding protein, thereby determining the optimal conditions for expression of the antigen binding protein.

47. The method of claim 45 or claim 46, wherein the antigen binding protein expression level is quantified by a high-throughput method.

48. The method of claim 47, wherein the high-throughput screening method is surface plasmon resonation (SPR) or Bio-Layer Interferometry (BLI).

49. The method of any one of claims 45-48, wherein the at least one expression parameter is selected from the group consisting of: cell growth time, incubation temperature, expression inducer concentration, chaperone concentration, a transcription control element sequence, a translation control element sequence, and a codon optimized sequence.

50. The method of claim 49, wherein the prokaryotic host cell is derived from an Enterobacterial species.51 . The method of claim 49, wherein the expression parameter is a translation control element sequence.

52. The method of claim 51 , wherein the translation control element sequence is a ribosome binding site (RBS) sequence.

53. The method of any one of claims 45-52, wherein the antigen binding protein is selected from the group consisting of an antibody or fragment thereof, a Fab, a Fv, a scFv, a di-scFv, a nanobody, a mAb, or a VHH.

54. The method of claim 53, wherein the antigen binding protein is a VHH.

55. The method of any one of claims 45-54, wherein the host cell comprises 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.

56. The method of any one of claims 45-55, further comprising the step of determining binding activity of the antigen binding protein.

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