Cell-free method for producing antibodies against cell-intrinsic targets

Abstract

Provided herein are methods and cell-free systems for the production of recombinant antibodies, where the antibodies are directed against a target protein that is naturally expressed as a cell-resident protein. Also provided herein are modified cell-free systems for use in such methods, in which the cell-free protein expression system is modified with an agent that binds to the cell-resident target protein or the antibody paratope, thus preventing the antibody from binding to the target protein.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 398,143, filed August 15, 2022, which is incorporated herein in its entirety.

[0002] Sequence Listing Reference A computer-readable form of the Sequence Listing is being submitted with this application via electronic submission and is incorporated herein by reference in its entirety. The Sequence Listing is contained in a file named MBRACE004.xml, created on May 2, 2023, and is 23,837 bytes in size (as measured on an MS-Windows operating system).

[0003] The present disclosure relates to methods and cell-free systems for the production of recombinant antibodies, which are generally directed against target proteins that are naturally expressed as intracellular proteins. [Background technology]

[0004] Engineered antibody molecules and fragments thereof are increasingly being utilized as scientific and clinical tools for the treatment and diagnosis of disease. The unique ability of antibodies to specifically recognize and bind with high affinity to virtually any type of antigen makes them attractive as novel biologics and starting points for scientific research. In recent years, certain stress proteins, which are normally present as internalized proteins in cells but are generally not expressed or expressed at low levels on the surface of normal cells, have been identified as unique targets for antibody-based therapy in cancer and other pathological conditions. There is a need for improved methods for producing such antibodies, including antibodies and fragments thereof, that specifically recognize and bind to internalized targets in mammalian cells.

[0005] Summary of the Invention The foregoing general description and the following detailed description of exemplary embodiments are merely exemplary aspects of the teachings of the present disclosure and are not restrictive.

[0006] In selected embodiments, the present disclosure provides methods and cell-free systems for producing antibodies that bind to cell-resident target proteins. The present disclosure also provides methods for producing cell-free systems for producing antibodies that bind to cell-resident target proteins.

[0007] The present disclosure provides a method for producing antibodies using a cell-free production system and an agent that prevents binding of an antibody produced using the cell-free production system to a protein in the cell-free system that is the antibody's target protein. Binding of an antibody to a target epitope on a target protein in a cell-free system can be prevented by 1) binding of an agent to the target epitope on the target protein, thereby preventing binding of the antibody paratope to the epitope, 2) binding of an agent to the antibody paratope, thereby blocking binding of the antibody paratope to the target epitope, or 3) a combination thereof.

[0008] In some embodiments, the present disclosure provides methods for the recombinant production of antibodies, including: 1) providing a cell-free protein expression system containing a target protein that displays an antibody epitope; 2) modifying the cell-free system by introducing one or more agents that block the antibody epitope on the target protein but do not eliminate the activity of the target protein in the cell-free system; 3) introducing into the cell-free system one or more nucleic acids encoding an antibody that binds the antibody epitope; and 4) initiating transcription and translation of the antibody in the cell-free system under conditions such that the antibody is produced.

[0009] In some embodiments, the present disclosure provides a method for recombinant production of an antibody, comprising: 1) providing a cell-free protein expression system containing a target protein that displays an antibody epitope; 2) modifying the cell-free system by introducing one or more agents that block an antibody paratope that binds to the antibody epitope on the target protein; 3) introducing into the cell-free system one or more nucleic acids encoding an antibody that includes the antibody paratope; and 4) initiating transcription and translation of the antibody in the cell-free system under conditions such that the antibody is produced. In a preferred embodiment, the method further provides 5) removing the agent that binds the antibody paratope after production of the antibody.

[0010] In some embodiments, the cell-free system comprises a cell lysate. In some embodiments, the cell-free system is a eukaryotic cell lysate.

[0011] In certain embodiments, the cell lysate is a wheat germ lysate, an insect cell lysate, a reticulocyte lysate, a keratinocyte lysate, hi some embodiments, the cell lysate is from mammalian cells, such as CHO cells, HeLa cells, HEK293 cells, myeloma cells, hybridoma cells, or cultured lymphoma cells.

[0012] Thus, in some embodiments, the present disclosure provides methods for the recombinant production of a monoclonal antibody, comprising: 1) providing a mammalian cell-free protein expression system containing a target protein that displays an antibody epitope; 2) modifying the mammalian cell-free system by introducing one or more agents that block the antibody epitope on the target protein but do not eliminate the activity of the target protein in the cell-free system; 3) introducing into the cell-free system one or more nucleic acids encoding a monoclonal antibody that binds to the antibody epitope; and 4) initiating transcription and translation of the monoclonal antibody in the mammalian cell-free system under conditions such that the antibody is produced.

[0013] In some embodiments, the cell-free system comprises a cell lysate. In some embodiments, the cell-free system is a eukaryotic cell lysate.

[0014] The present disclosure also provides a method for recombinant production of a monoclonal antibody, comprising: 1) providing a mammalian cell-free protein expression system containing a target protein displaying an antibody epitope; 2) modifying the mammalian cell-free system by introducing one or more agents that block the antibody paratope that binds to the antibody epitope on the target protein; 3) introducing into the cell-free system one or more nucleic acids encoding a monoclonal antibody that includes the antibody paratope; and 4) initiating transcription and translation of the monoclonal antibody in the mammalian cell-free system under conditions such that the antibody is produced. In a preferred embodiment, the method further provides 5) removing the agent that binds the antibody paratope after production of the monoclonal antibody.

[0015] The agent that binds to the target epitope can be introduced at any time prior to the production of the antibody in the cell-free system. For example, if the cell-free system is a cell lysate, the agent can be introduced into the cells prior to the production of the cell lysate, or alternatively, the agent can be introduced into the cell lysate after the lysate is prepared but prior to the induction of transcription and translation of the antibody in the system.

[0016] The agent that blocks the antibody epitope can be any agent that allows the target protein to retain sufficient function in a cell-free system. In some embodiments, the agent is a peptide. In some embodiments, the agent is a small molecule. In some embodiments, the agent does not affect the activity of the target protein compared to the target protein activity in the absence of the agent. In some embodiments, the protein activity is reduced but sufficient to support efficient antibody production in a cell-free system. In some embodiments, the agent reduces the target protein activity by about 10% compared to the target protein activity in the cell-free system in the absence of the agent. In some embodiments, the agent reduces the target protein activity by about 50% compared to the target protein activity in the cell-free system in the absence of the agent.

[0017] The present disclosure also provides a method for the recombinant production of an antibody, the method comprising: 1) introducing into cells an agent that selectively binds to a target epitope on a cell-resident protein to generate a modified cell line; 2) generating a cell-free antibody production system from the modified cell line; 3) introducing into the cell-free antibody production system a nucleic acid template, wherein the nucleic acid template encodes an antibody that selectively binds to the target epitope on the cell-resident protein; and 4) initiating transcription and translation from the nucleic acid template to produce the antibody in the cell-free antibody production system.

[0018] In some embodiments, the target protein is a cell-resident protein in the secretory pathway. In some embodiments, the target protein is a stress protein. In some embodiments, the target protein is a signaling protein.

[0019] In some embodiments, the target protein is expressed on an organelle of the cell line.

[0020] In certain embodiments, the target protein is generally a protein expressed on the endoplasmic reticulum. For example, the target protein may be an endoplasmic reticulum chaperone, such as calreticulin, a heat shock protein, or an isomerase. Specifically, the target protein may be glucose-regulated protein 78 (GRP78), HSP47, protein disulfide isomerase (PDI), calreticulin, or GP94.

[0021] In certain embodiments, the target protein is generally expressed on the Golgi apparatus. For example, the target protein can be a Golgi complex protein, such as Golgi phosphoprotein 2 (GOLPH2), Golgi phosphoprotein 3 (GOLPH3), GM130, ATPase H+ transport V1 subunit A (ATP6V1A), ATPase H+ transport V1 subunit E1 (ATPP6V1E1), ATPase H+ transport V0 subunit A2 (ATP6VOA2), transmembrane protein 165 (TMEM165), golgin B1 (GOLGB1), SCY1-like 1-binding protein 1 (SCYL1BP1), transport protein particle complex subunit 11 (TRAPPC11), transport protein particle complex subunit 2 (TRAPPC2), or thyroid hormone receptor interactor 11 (TRIP11).

[0022] In some embodiments, the target protein is associated with a membrane within the cell.

[0023] In some embodiments, the target protein is one that is involved in a signal transduction pathway, for example, an intracellular signaling protein such as a kinase or phosphatase.

[0024] In some embodiments, antibodies are isolated from a cell-free system after production.

[0025] Other features, advantages, and aspects are described in more detail below.

[0026] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments and, together with the description, explain these embodiments. The accompanying drawings are not necessarily drawn to scale. Any values ​​or dimensions shown in the accompanying graphs and figures are for illustrative purposes only and may or may not represent actual or preferred values ​​or dimensions. Where applicable, some or all features may not be shown to aid in the explanation of underlying features. [Brief explanation of the drawings]

[0027] [Figure 1] 1 shows SDS-PAGE analysis of purified supernatant samples following expression of anti-GRP78 antibody clones B4, D1 and F6 in CHO cells under reducing and non-reducing conditions. [Figure 2A] Flow cytometry results for antibody clone B4 binding to GRP78 variants from the alanine-scan library are shown. For each GRP78 variant, the mean percent fluorescence of WTGRP78 binding is plotted against the control antibodies 1H11-1H7. [Figure 2B] Flow cytometry results for antibody clone F6 binding to GRP78 variants from the alanine-scan library are shown. For each GRP78 variant, the mean percent fluorescence of WTGRP78 binding is plotted against the control antibodies 1H11-1H7. [Figure 3] 1 shows a visual image of key residues for antibody binding to GRP78 for anti-GRP78 clones B4 and F6, with exemplary key residues and other residues involved in binding indicated by arrows. DETAILED DESCRIPTION OF THE INVENTION

[0028] The description set forth below in conjunction with the accompanying drawings is intended to be a description of various exemplary embodiments of the disclosed subject matter. In connection with each exemplary embodiment, specific features and functions are described. However, it will be apparent to one skilled in the art that the disclosed embodiments may be practiced without each of those specific features and functions.

[0029] As used herein, the term "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the disclosed subject matter. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Furthermore, the embodiments of the disclosed subject matter are intended to cover such modifications and variations.

[0030] It should be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. That is, unless expressly specified otherwise, when used herein, terms such as "a," "an," and "the" have the meaning "one or more." Furthermore, terms such as "left," "right," "top," "bottom," "front," "rear," "side," "height," "length," "width," "upper," "lower," "internal," "external," "inside," and "outside" that may be used herein are intended to describe points of reference only and do not necessarily limit embodiments of the disclosed subject matter to any particular orientation or configuration. Furthermore, terms such as "first," "second," and "third" merely identify one of the numerous parts, components, steps, operations, functions, and / or points of reference disclosed herein and, similarly, do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.

[0031] Furthermore, terms such as "approximately," "about," "approximately," "minor variations," and similar terms generally refer to ranges that include the specified value within a margin of, in certain embodiments, 20%, 10%, or preferably 5%, and any value therebetween.

[0032] All features described in connection with one embodiment are intended to be applicable to the additional embodiments described below, unless expressly stated otherwise or unless the feature or function is incompatible with the additional embodiments. For example, if a given feature or function is explicitly described in connection with one embodiment but not explicitly mentioned in connection with an alternative embodiment, it should be understood that the inventors intend that the feature or function can be developed, utilized, or implemented in connection with the alternative embodiment, unless the feature or function is incompatible with the alternative embodiment.

[0033] Provided herein are methods for recombinant production of antibodies or binding fragments thereof, wherein the antibody's target protein is generally found as an internalized intracellular protein within a cell. In some embodiments, the internalized intracellular protein to which the antibody binds is a protein involved in the secretory pathway, such as a protein expressed on or in the endoplasmic reticulum (ER) or Golgi apparatus. In some embodiments, the antibody's target protein is a stress protein, which normally functions in the ER, Golgi apparatus, or other intracellular organelles but may be upregulated on the cell surface under conditions of cellular stress. For example, stress proteins, such as those involved in the unfolded protein response (UFR), have been found to be upregulated on the surface of cancer cells but are not normally found on the surface of normal cells. Stress proteins generally exhibit desirable characteristics for use as target proteins for antibody-based cancer therapy because they are not expressed or expressed only at low levels on the surface of normal cells, whereas they may be upregulated or overexpressed on the surface of cancer cells. The use of stress proteins as target proteins for antibody-based therapies ensures upregulated surface expression in various cancers but not in normal tissues and cells, reducing or minimizing off-target activity and / or toxicity.

[0034] There is a need for methods for producing antibodies in sufficient quantities for use as therapeutics. Several expression systems are available, of both prokaryotic and eukaryotic origin. The choice of system depends on many factors, including the species to be expressed and the precise sequence of the particular antibody.

[0035] Mammalian expression systems are particularly desirable for recombinant production of human or humanized antibodies. For example, mammalian cells are capable of desirable protein folding and post-translational modifications, similar to human systems. By expressing recombinant proteins in mammalian systems, such as those derived from HEK293 or CHO cells, it is possible to achieve glycosylation patterns similar, but not identical, to those obtained from human cells. Mammalian cell-free systems allow for very high product yields and are relatively tolerant to metabolic stress.

[0036] While efforts have been made to produce antibodies against stress proteins, it has not been possible to efficiently produce them in mammalian cells. For example, observations herein demonstrate that antibodies against the exemplary stress protein GRP78 cannot be expressed in high yield from mammalian cells without using methods such as those disclosed herein. Without being bound by theory, it is believed that in certain circumstances, stress proteins are necessary for cell viability or function of mammalian cells, such that binding of the internalized protein by the antibody during the recombinant antibody production process is deleterious to the cell. For example, in some embodiments, binding of certain internalized proteins inhibits the ability of the internalized protein to perform its normal function within the cell, resulting in intracellular protein misfolding, lack of degradation of the misfolded protein, improper calcium homeostasis, and, in some cases, reduced or absent cell viability. Loss or impairment of this function impacts the ability to produce antibodies at high yields.

[0037] Studies have demonstrated that stress proteins, such as chaperones, are required for proper intracellular function during antibody production. For example, changes in the cellular abundance of secretory pathway proteins have been shown to correlate with the ability to abundantly produce recombinant monoclonal antibodies (see, e.g., Lambert and Merten, 1997, Biotechnol. Bioeng., 54:165-180; Downham et al. 1996, Biotechnol. Bioeng., 51:691-696). Secretory proteins are known to be folded and assembled into higher-order complexes in the intracellular endoplasmic reticulum (ER) compartment of cells immediately after protein synthesis. The ER is composed of specific auxiliary assembly factors along with a quality control mechanism (Ellgaard et al., Quality control in the secretory pathway, Science. 1999 Dec 3;286:1882-8; Helenius et al., Intracellular functions of N-linked glycans, Science. 2001 Mar 23;291:2364-9). Protein folding and the refolding of misfolded, soluble, and aggregated proteins are known to be mediated by a network of evolutionarily conserved protein molecules called chaperones (Haiti, FU, Nature, 381, 571-580, (1996); Horwich, AL, Brooks Low K., Fenton, WA, Hirshfield, IN & Furtak, K., Cell 74, 909-917 (1993); Ellis, RJ & Hemmingsen, SM, TiBS, 14, 339-342, (1989); Bukau, B., Hesterkamp, ​​T. & Luirink, J., Trends Cell Biol, 6, 480-486, (1996), Bukau, B., Deuerling, E., Pfund, C. & Craig, EA, Cell, 101, 119-122, (2000)). Secretory proteins also undergo various post-translational modifications, including glycosylation, as they pass through the Golgi complex.

[0038] Alternatively, even if antibody binding to internal cellular proteins does not affect cell viability, it may affect antibody production by sequestering the product by the internal cellular proteins. Briefly, internal cellular proteins may act as a "sink" for antibody or antigen-binding fragment binding, inhibiting secretion and therefore limiting the yield of the desired product.

[0039] The methods provided relate to cell-free systems for the production of antibodies or antigen-binding fragments that target endogenous, cell-resident target proteins within cells, such as target proteins in the secretory pathway.

[0040] The provided production systems and methods are exemplified for antibodies targeting GRP78 and other intracellular target proteins. The systems and methods described herein are generally generalizable to the production of all or part of antibody-based biologics that selectively target proteins found intracellularly but that may be found on the cell surface in certain disease states. Thus, those skilled in the art will understand that the present disclosure is exemplary and applicable to production methods for biologics targeting other cell systems and such other targets.

[0041] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. To the extent that a definition set forth herein is contrary to or inconsistent with a definition set forth in a patent, application, published application, or other publication incorporated herein by reference, the definition set forth herein shall take precedence over the definition incorporated herein by reference.

[0042] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0043] Methods for cell production for cell-free systems Provided herein are methods for the recombinant production of antibodies, where the antibody binds to a target protein (or a homolog thereof) that is normally expressed naturally as an endogenous protein in a cell. In the provided cell-free systems and methods, the antibody is produced in an engineered system, e.g., a mammalian cell-free system, that has been engineered to contain an agent that selectively binds to the epitope on the target protein to which the antibody binds.

[0044] These methods and systems disclosed herein can be used individually or in combination with all or specific aspects of other antibody production systems. In some preferred embodiments, the modified cell line can be produced using the methods disclosed in U.S. Patent Application No. 63 / 337,980, filed May 3, 2022, and / or U.S. Patent Application No. 63 / 359,541, filed July 8, 2022, each of which is incorporated herein in its entirety for all purposes. For example, in some preferred embodiments, the methods of these applications can include: a) expressing nucleic acid encoding an antibody in an modified cell line (in some preferred embodiments, a mammalian cell line), where the antibody specifically binds to a target epitope on a target protein naturally expressed as an endogenous protein in cells, and the modified cell is engineered to express a variant target protein comprising mutations of one or more amino acid residues of the target epitope; and b) culturing the modified cell line under conditions such that the antibody is produced (and, in some preferred embodiments, isolating the antibody). In some preferred embodiments, the methods of these applications include: a) providing a cell line (in some preferred embodiments, a mammalian cell line) with an antibody that binds to an epitope on a target protein present within the cell; b) identifying the epitope of the target protein to which the antibody binds; c) generating an engineered cell line by mutating the epitope of the target protein in the cell line to reduce or inhibit binding of the antibody to the epitope on the target protein; d) introducing an expression vector encoding the antibody into the engineered cell line; e) culturing the engineered cell line under conditions that allow production of the antibody from the expression vector; and f) expressing the nucleic acid encoding the antibody in the engineered cell line (and, in some preferred embodiments, isolating the antibody).In some preferred embodiments, the modified mammalian cell lines of these applications comprise a variant protein that is a cell-resident protein in the secretory pathway, the variant protein comprising a mutation of a native cell-resident protein that is a target protein of an antibody, the mutation being an amino acid substitution(s) of one or more amino acid residues to alter the target epitope of the antibody to a variant epitope. In some preferred embodiments, the method comprises: a) expressing in a modified mammalian cell line a nucleic acid encoding an antibody, wherein the antibody binds to GRP78, the modified mammalian cell being engineered with a variant GRP78 having a variant epitope comprising mutations of one or more amino acid residues of the target epitope of the antibody or antigen-binding fragment, and b) recombinantly producing an antibody that targets GRP78 by culturing the modified mammalian cell line under conditions such that anti-GRP78 antibody is produced in the culture supernatant.

[0045] A. Cell-Internal Protein Targets and Antibodies Thereto In some embodiments, the provided systems and methods are useful for expressing antibodies in which the antibody's target protein is naturally expressed in cells as an internalized protein. In certain embodiments, the target protein is a protein that is aberrantly expressed on the cell surface in cancer and other diseases, but is also necessary for normal cell function due to the target protein's role in normal intracellular biological processes. In some embodiments, the target protein is a protein involved in the cell's secretory pathway. In some embodiments, the target protein is expressed on an organelle of a cell, such as in a mammalian cell line. In some embodiments, the target protein is expressed on or within the endoplasmic reticulum. In some embodiments, the target protein is expressed on or within the Golgi apparatus.

[0046] In some embodiments, the cell-intrinsic protein target is a stress protein. Stress proteins include proteins involved in organelle autoregulation to maintain homeostasis and regulate organelle performance under stress conditions that may occur in a tumor environment. Various stress proteins are known, including proteins in the secretory pathway. Various stress proteins are known, including those resulting from ER stress or Golgi stress: Sasaki and Yoshida, J Biochem. 157:185, 2015; Sasaki and Yoshida, FEBS Letters, 593:2330-2340, 2019; Gao et al., Biofactors, 47:964-974, 2021; Li et al., Mol Neurobiol, 49:1449-59, 2014; Yadav et al., J Cancer Prev., 19:75-88, 2014; and Chen and Cubillos-Ruiz, Nature Reviews Cancer, 21:71-88, 2021.

[0047] For example, targets include endoplasmic reticulum chaperones (e.g., calreticulin, heat shock proteins, and isomerases), which can translocate to the cytosol and ultimately to the surface of cells, particularly under stress conditions. These proteins have been shown to be overexpressed in certain pathological conditions. See, e.g., Weirsma VR et al., Front. Oncol., Vol 5:Art. 7 (2015); Garg AD, Cancer Immunol Immunother 61:215-21 (2012). The disclosure of the present embodiments and application of its teachings enable the production of biologics that target endoplasmic reticulum chaperones and other identified targets without unduly compromising cell viability. Exemplary biologics include, for example, those targeting HSP47 for the treatment of cervical cancer, gastric cancer, or autoimmune diseases (Yokota S et al., Biochem Biophys Res Commun 303:413-8(2003); Yamamoto N et al., Int J Oncol 43:1855-63(2013)), biologics targeting protein disulfide isomerase (PDI) for the treatment of central nervous system cancer, ovarian cancer, brain cancer, prostate cancer, and lung cancer (Xu S et al., Free Radic Biol Med 52:993-1002(2012); Zhang L et al., Cancer Invest 27:453-(2009); Pan Z et al., Int J Oncol 35:823-8(2009)), and biologics targeting calreticulin for the treatment of various cancers (Zamanian M et al., Pathol Oncol Res 149-54 (2013), Gold LI et al., FASEB J 24:665-83 (2010), biologics targeting GP94 (Marzec M. et al., Biochim Biophys Acta. 2012 March; 1823 (3):774-787), and biologics targeting GRP78 (Arap et al. Cancer Cell 6:275-284 (2004), Sato et al. Adv Genet 69:97-114 (2010) can be mentioned.

[0048] Various Golgi complex proteins are known to be involved in various pathological conditions, particularly cancer and autoimmune diseases, and monoclonal antibodies that selectively bind to these organelle proteins are suitable for production of monoclonal antibodies using the methods disclosed herein.Such proteins include, but are not limited to, Golgi phosphoprotein 2 (GOLPH2) (Liu et al., Front Oncol 2021 Dec 7;11:78386), Golgi phosphoprotein 3 (GOLPH3) (Xing M, et al., Mol Biol Cell 27:3828-3840, 2016; Scott KL, et al., Nature 459:1085-1090, 2009), GM130 (Chang SH, et al., Mol Ther 20:2052-2063, 2012), ATPase H+ transport V1 subunit A (ATP6V1A) (Van Damme T, et al., Am J Hum Genet 100:216-227, 2017), ATPase H+ transport V1 subunit E1 (ATPP6V1E1) (ibid.), ATPase H+ transport V0 subunit A2 (ATP6VOA2) (Kornak U, et al., Nat Genet 40:32-34, 2008), transmembrane protein 165 (TMEM165) (Rosnoblet C, et al., Hum Mol Genet 22:2914-2928, 2013), golgin B1 (GOLGB1) (Katayama K, et al., Biochem Biophys Res Commun 499:459-465, 2018), SCY1-like 1-binding protein 1 (SCYL1BP1) (Hennies HC, et al., Nat Genet 40:1410-1412, 2008), transport protein particle complex subunit 11 (TRAPPC11) (Larson AA, et al., Skeleton Muscle 8:17, 2018), transport protein particle complex subunit 2 (TRAPPC2) (Davis EE, et al., Clin Genet 85:359-364, 2014), and thyroid hormone receptor interactor 11 (TRIP11) (Smits P, N Engl J Med 362:206-216, 2010).

[0049] In some embodiments, the target gene is GRP78. In some embodiments, the target protein is heat shock protein 47 (HSP47). In some embodiments, the target protein is protein disulfide isomerase (PDI). In some embodiments, the target protein is calreticulin. In some embodiments, the target gene is GP94. In some embodiments, the target protein is GOLPH2. In some embodiments, the target gene is GOLPH3. In some embodiments, the target gene is GM130. In some embodiments, the target gene is ATP6V1A. In some embodiments, the target gene is AATPP6V1E1. In some embodiments, the target gene is ATP6VOA2. In some embodiments, the target protein is transmembrane protein 165 (TMEM165). In some embodiments, the target gene is GOLGB1. In some embodiments, the target gene is SCYL1BP1. In some embodiments, the target gene is TRAPPC11. In some embodiments, the target protein is thyroid hormone receptor interactor 11 (TRIP11).

[0050] Those skilled in the art are familiar with antibodies directed against intracellular protein targets, such as stress proteins. For example, antibodies directed against GRP78 include the GRP78-specific mouse monoclonal IgG antibody MAb159 (Ojha and Amaravadi, Pharmacol. Res., 120:258-266, 2017), PAT-SM6 (Ojha and Amaravadi, 2017), and the anti-GRP78 antibodies described in WO 2018 / 057703, WO 2014 / 153056, WO 2008 / 105560, U.S. Publication No. 2010 / 0041074, U.S. Patent No. 10,259,884, and U.S. Patent No. 10 / 851,161. Antibodies against protein disulfide isomerase (PDI) include, but are not limited to, Invitrogen PDI monoclonal antibody clone 12 (Thermo Fisher Scientific catalog number MA5-43389) or Invitrogen PDI monoclonal antibody clone 2F6G12H2 (Thermo Fisher Scientific catalog number MA5-43389). Antibodies against calreticulin include, for example, mAb FMC75 (Enzo Life Sciences catalog number ADI-SPA-601) and mAb 16 (BD Transduction Laboratories catalog number 612137). Antibodies against GOLPH3 include, for example, Thermo Fisher monoclonal antibody clone 905CT9.1.1 (Thermo Fisher Scientific catalog number MA5-37626). Each of these antibodies can serve as a template for humanized antibodies using methods as disclosed in more detail herein.

[0051] In some embodiments, the antibody is a human antibody. Human antibodies can be prepared by administering an immunogen to transgenic animals that have been engineered to produce intact human antibodies or complete antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or part of human immunoglobulin loci that replace endogenous immunoglobulin loci or that are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci are generally inactivated. Human antibodies can also be derived from human antibody libraries, including phage display and cell-free libraries, that contain antibody coding sequences derived from the human repertoire.

[0052] In addition to using non-human monoclonal antibodies for humanization purposes, non-human antibodies can be used as templates for selecting fully human or nearly fully human antibodies. One specific method for selecting such human antibodies is the use of phage display technology. This method can use antibody screening techniques such as those described in, for example, WO 91 / 17271 by Dower et al. and WO 92 / 01047 by McCafferty et al., U.S. Patent Nos. 5,877,218, 5,871,907, 5,858,657, 5,837,242, 5,733,743, and 5,565,332. These methods generate phage libraries in which various antibodies are displayed on the outer surface of the phage. Antibodies are usually displayed on the phage as Fv or Fab fragments. Antibodies with the desired specificity are selected by their affinity for selected epitopes of organelle proteins.

[0053] In a specific exemplary method, human antibodies that selectively bind to epitopes on organelle proteins can be produced using the technology of Winter, WO 92 / 20791. In this method, either the heavy or light chain variable region of a non-human monoclonal antibody is used. If a light chain variable region is selected as the starting material, a phage library is constructed whose members display the light chain variable region of the non-human monoclonal antibody and a different heavy chain variable region. The heavy chain variable region is obtained from a library of rearranged human heavy chain variable regions. Phages that show strong specific binding to the organelle protein epitope are selected. The human heavy chain variable region from this phage serves as the basis for constructing an optimized phage library, in which each phage displays the same heavy chain variable region and a different light chain variable region identified from the initial display library. The light chain variable region is obtained from a library of rearranged human light chain variable regions. Phages that display the variable region of a fully human antibody that shows strong specific binding to the organelle protein epitope are selected.

[0054] The antibodies provided include monoclonal antibodies, including monoclonal antibody fragments.

[0055] B. Identification of Binding Epitopes on Target Proteins In some embodiments, residues in a target protein important for antibody binding can be mapped to define or identify the epitope or binding domain of the antibody, and agents that block the epitope in the disclosed methods and systems can be designed and / or developed. In some embodiments, the cDNA encoding the target protein is randomly mutated by methods such as treatment with chemical mutagens, irradiation during replication, passage through an error-inducing (mutator) cell line, or oligonucleotide-directed random mutagenesis. In some embodiments, oligonucleotide-directed random mutagenesis is used, in which a preselected region of the protein is targeted for random mutagenesis. In some embodiments, the preselected region for random mutagenesis is a region believed to contain the binding site or epitope. Preliminary evaluation of the preselected region can be achieved by any method known in the art, including hydrophobicity analysis or crystal structure analysis of the protein sequence.

[0056] In some embodiments, peptide-based approaches can be used to map antibody epitopes. One approach is peptide display technology, which displays libraries of protein fragments on microarrays or the surface of E. coli or phage, and discovers which antibodies bind to through microarray scanning or flow cytometry. Another approach screens a series of overlapping peptides of the target protein or preselected regions thereof for antibody binding, such as by ELISA or other techniques that monitor binding interactions. These techniques have the added advantage of also identifying peptides that can potentially be used as agents for binding in the methods and systems of the present invention, or as the basis for drug development.

[0057] In some embodiments, epitopes can be computationally predicted. In some embodiments, epitopes are predicted using the PEASE tool (Sela-Culang, J. et al. (2014) Structure 22(4):646-57; Sela-Culang et al. (2015) Bioinformatics 31(8):1313-5). In some embodiments, predicted epitopes are experimentally validated. Methods for experimentally testing antibody epitopes are known in the art, including, for example, performing assays involving screening libraries using antibody cross-blocking assays, performing mutational analysis, deuterium exchange analysis, peptide binding assays, and / or X-ray crystallography. In some embodiments, validation uses libraries and / or peptides derived from the target protein to assess the importance of specific amino acids at specific positions for binding. In some embodiments, the library comprises a library of mutations for a subset or all of the amino acid residues of the target protein. In some embodiments, the library is a yeast display library. In some embodiments, the library is a phage display library.

[0058] In some embodiments, to identify residues within the epitope on the target cell-resident protein that are blocked during antibody generation, it is desirable to start with a solved crystal structure of the cell-resident protein so that the key contact residue(s) between the antibody and the target protein can be identified. This solved structure can be the solved crystal structure of the cell-resident protein alone, or the solved crystal structure of the cell-resident protein bound to an antibody or antibody fragment.

[0059] However, in many cases, a good molecular model will be able to provide the necessary information. If a molecular model is not sufficient, for example, if there is no suitable structural template in the structural database, amino acid exchange experiments can provide information on which residues can be targeted for mutation. Alanine scanning mutagenesis (mutating each residue successively to Ala) through these regions can identify the key residue(s) involved in antigen binding (Cunningham BC and Wells JA, Proc Natl Acad Sci US A. 1991 Apr 15;88(8):3407-3411). If changing a single residue to Ala reduces but does not destroy binding, that position can be targeted for blocking with a drug.

[0060] Comparative model building provides a wide range of structural templates, and available computer programs ensure that the models are increasingly accurate: for example, solved crystal structures of various proteins have been shown to be very close to structures predicted by molecular modeling.

[0061] Once the structure of a protein target is elucidated, as shown herein for GRP78, one of skill in the art can identify residues that can be linked to reduce antibody binding to epitopes of the organelle protein while leaving sufficient function of the organelle protein intact, and thus epitopes that can be blocked while retaining protein function, as taught in more detail herein. Methods for making blocking polypeptides and / or other agents are well known to those of skill in the art.

[0062] In some embodiments, the agent used does not significantly affect the viability of the cell line introduced into the cells and used to produce the cell-free system, hi some embodiments, the viability of the modified cell line containing the agent is maintained under standard culture and passaging conditions compared to the cell line without the introduced agent.

[0063] In some embodiments, binding of an agent to a target epitope on a target protein blocks the epitope from binding to the antibody produced, but has no measurable effect on the function of the target protein in a cell-free system.

[0064] In some embodiments, binding of the agent to the target epitope reduces target protein activity by only about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% compared to the activity of the target protein in a cell-free system in the absence of the agent. For example, the activity of chaperone proteins such as GRP78 can be assessed using methods such as those taught in Hristozova N, et al., PLoS One. 2016;11(8):e0161970 and Mymrikov E et al., J Biol Chem. 2017 Jan 13;292(2):672-684. The activity of intracellular kinases can be assessed using methods such as those taught in Haubrich * and Swinney Curr Drug Discov Technol. 2016;13(1):2-15.

[0065] In some embodiments, binding of an agent to a target paratope on an antibody blocks binding of the paratope to a target epitope on a target protein, but does not significantly affect the function of the target protein in a cell-free system.

[0066] In some embodiments, binding of the produced antibody to the target protein after modification of the cell-free system is reduced by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 100-fold, or at least about 1000-fold compared to binding of the produced antibody to the target protein in the cell-free system in the absence of the agent.

[0067] In some embodiments, the agent results in a decrease in the affinity of the antibody for the target protein, ie, the affinity of the antibody for the target protein is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% relative to the affinity of the antibody for the target protein lacking one or more mutations. In some embodiments, the affinity of the antibody for the target protein is reduced by at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 100-fold, or at least about 1000-fold relative to the affinity of the antibody for the target protein in the absence of the agent. In some embodiments, the affinity of the antibody for the target protein is eliminated in the presence of the agent (e.g., binding of the antibody to the target protein is undetectable).

[0068] Methods for measuring antibody affinity to a polypeptide are known in the art and may include, but are not limited to, performing an ELISA assay, performing a radioimmunoassay (RIA), performing surface plasmon resonance (SPR), performing thermophoresis, performing a competition assay, and performing isothermal titration calorimetry.

[0069] Engineering antibody paratopes in a cell-free system In one embodiment of the present disclosure, the cell-free system provided utilizes one or more agents that reversibly bind to the paratope of the antibody being produced, thus effectively reducing or eliminating binding of the antibody to its target protein in the cell-free system. The agent can be any agent that can be removed from the antibody after production without impairing the ability of the antibody to be used for its intended purpose after production. In certain embodiments, the agent used to bind to the paratope of the antibody in the cell-free system is all or a portion of the antigen used to initially produce or discover the antibody.

[0070] One example of such an antibody that can be used with a reversible paratope binder is a monoclonal antibody that binds to its target epitope under certain physiological conditions but releases its target protein under mild changes in these conditions. Certain monoclonal antibodies are pH-dependent and can bind to antigens at a neutral pH of 7-7.4 but release antigens under slightly more acidic conditions. See, e.g., Bonvin et al., mAbs 7:2, 294-302; March / April 2015; Biochim Biophys Acta, 2014 Nov; 1844(11):1943-1950.

[0071] In another example, calcium-dependent antigen binding can be used for antigen dissociation after antibody production in the cell-free system of the present disclosure. See, e.g., Hironiwa et al., MAbs. 2016 Jan;8(1):65-73. Such antibodies can be selected for their ability to bind to target proteins in the presence of calcium ions and elute in the absence of calcium ions. Ibid. This property can be used to bind antibodies to drugs in the cell-free system of the present disclosure, and once production is complete, the drug can be removed from the antibody by removing calcium ions from the system or isolating the antibody and eluting it in calcium-free or low calcium conditions.

[0072] These and other exemplary reversible antibody-antigen pairs will be apparent to those of skill in the art upon reading the present disclosure.

[0073] In certain embodiments, blocking peptides are used as agents for binding to antibody paratopes. In such embodiments, excess peptide is added to the cell-free system to a concentration of peptide estimated to be 100-500 times higher than the expected antibody production.

[0074] Modified mammalian cell-free system for anti-Grp78 antibody production In some embodiments, the provided methods and cell-free systems can be used to produce antibodies against GRP78, also referred to as anti-GRP78 antibodies. The antibodies can include full-length antibodies or antigen-binding fragments thereof. GRP78, also known as heat shock protein 5 (Bspa5 / BiP), is part of an evolutionarily conserved ER-associated stress response mechanism that confers cell survival signals under environmental and physiological constraints. As a molecular component of the ER chaperoning network, GRP78 is classically involved in the processing of unfolded proteins, although newer insights suggest that this protein may be located on the cell surface and influence signal transduction (Lee, 2014, Nature Rev Cancer 1.4(4);263-276). Retrospective IHC studies have demonstrated that GRP78 expression is positively correlated with decreased survival in advanced breast cancer (Lee et al., 2006, Cancer Research 66(16):7849-7853) and recurrence in prostate cancer patients (Daneshmand et al., 2007, Human Pathology 38(10):1547-1552). Upregulation of GRP78 promotes survival and chemotherapy resistance in both proliferating and dormant breast cancer cells. The in vivo accessibility of a synthetic peptide composed of a GRP78-binding motif linked to a cell death-inducing peptide to promote apoptosis in cancer cells has been demonstrated (Arap et al., 2004, Cancer Cell 6(3):275-284).

[0075] In some embodiments, the antibody comprises an antibody or antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment is selected from the group consisting of a Fab, a single-chain variable fragment (scFv), a single-domain antibody, and a nanobody. In further embodiments, the antibody is a full-length antibody. In some embodiments, the anti-GRP78 antibody is an IgG antibody. In some embodiments, the anti-GRP78 antibody is a human antibody.

[0076] In some embodiments, the antibody is an antibody as described in International Patent Application Publication No. WO 2018 / 057703. In some embodiments, the anti-GRP78 antibody is the antibody designated B4. In some embodiments, the anti-GRP78 antibody is the antibody designated D1. In some embodiments, the anti-GRP78 antibody is the antibody designated F6. In any of such embodiments, the antibody is any of the full-length antibodies described above, or an antigen-binding fragment thereof. In some embodiments, the anti-GRP78 antibody is a full-length antibody.

[0077] In some embodiments, the anti-GRP78 antibody comprises a variable heavy (VH) chain and a variable light (VL) chain. In some embodiments, the VH chain comprises a VH CDR1 set forth in SEQ ID NO: 1, a VH CDR2 set forth in SEQ ID NO: 2, and a VH CDR3 set forth in SEQ ID NO: 3, and the VL chain comprises a VL CDR1 set forth in SEQ ID NO: 4, a VL CDR2 set forth in SEQ ID NO: 5, and a VL CDR3 set forth in SEQ ID NO: 6. In some embodiments, the VH chain has a sequence of amino acids having at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 19, and the VL chain has at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 20. In some embodiments, the VH chain is set forth in SEQ ID NO: 19 and the VL chain is set forth in SEQ ID NO: 20.

[0078] In some embodiments, the VH chain comprises a VH CDR1 set forth in SEQ ID NO:7, a VH CDR2 set forth in SEQ ID NO:8, and a VH CDR3 set forth in SEQ ID NO:9, and the VL chain comprises a VL CDR1 set forth in SEQ ID NO:10, a VL CDR2 set forth in SEQ ID NO:11, and a VL CDR3 set forth in SEQ ID NO:12. In some embodiments, the VH chain has a sequence of amino acids having at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO:21, and the VL chain has at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO:22. In some embodiments, the VH chain is set forth in SEQ ID NO:21 and the VL chain is set forth in SEQ ID NO:22.

[0079] In some embodiments, the VH chain comprises a VH CDR1 set forth in SEQ ID NO: 13, a VH CDR2 set forth in SEQ ID NO: 14, and a VH CDR3 set forth in SEQ ID NO: 15, and the VL chain comprises a VL CDR1 set forth in SEQ ID NO: 16, a VL CDR2 set forth in SEQ ID NO: 17, and a VL CDR3 set forth in SEQ ID NO: 18. In some embodiments, the VH chain has a sequence of amino acids having at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 23, and the VL chain has at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 24. In some embodiments, the VH chain is set forth in SEQ ID NO: 23 and the VL chain is set forth in SEQ ID NO: 24.

[0080] In some of the optional embodiments, both the agent and the anti-GRP78 antibody bind to the same epitope on human GRP78. In some embodiments, the human GRP78 protein comprises the amino acid sequence set forth in SEQ ID NO:25.

[0081] In some embodiments, both the agent and the anti-GPR78 antibody bind to a target epitope of GRP78 that includes one or more amino acid residues K113, R261, H265, K268, K271, K272, R279, E329, or D333, with reference to the residue numbering of human GRP78 as set forth in SEQ ID NO: 25.

[0082] In some embodiments, both the agent and the anti-GPR78 antibody bind to a target epitope of GRP78 that comprises one or more amino acid residues from R261, H265, H329, K271, K272, and D333, with reference to the residue numbering of human GRP78 as set forth in SEQ ID NO: 25. In some embodiments, the target epitope comprises amino acid residues R261, H265, H329, K271, K272, and D333, with reference to the residue numbering of human GRP78 as set forth in SEQ ID NO: 25.

[0083] In some embodiments, both the agent and the anti-GPR78 antibody bind to a target epitope of GRP78 that includes one or more amino acid residues R261, R279, K113, K268, and K271, with reference to the residue numbering of human GRP78 set forth in SEQ ID NO: 25. In some embodiments, the target epitope includes amino acid residues R261, H265, H329, K271, K272, and D333, with reference to the residue numbering of human GRP78 set forth in SEQ ID NO: 25.

[0084] In some embodiments, the agent is an antibody that binds to the same epitope on human GRP78 as an antibody comprising any of the above sequences (e.g., a peptide that has the ability to cross-compete with any of the above anti-GRP78 antibodies for binding to human GRP78). For example, Biacore analysis, ELISA assays, or flow cytometry can be used to demonstrate cross-competition with any of the described antibodies. The ability of the peptide to inhibit binding to human GRP78 as an anti-GRP78 antibody demonstrates that the peptide can compete with the anti-GRP78 antibody for binding to human GRP78 and is therefore likely to bind to the same epitope on human GRP78.

[0085] In some embodiments, the agent binds to one or a combination of amino acids selected from the following: (i) K113Q, K113S, K113D, or K113T; (ii) R261Q, R261S, R261D, R261T, or R261A; (iii) H265Q, H256S, H265D, or H256T; (iv) K268Q, K268S, K268D, or or K268T, (v) K271Q, K271S, K271D, or K271S, (vi) K272Q, K272S, K271D, or K272S, (vii) R279Q, R279S, R279D, R279T, or R279A, (viii) E329Q, E329S, E329D, E329T, or E329A, or (ix) D333Q, D333S, or D333T. In some embodiments, the agent binds to one or a combination of amino acids selected from the following: (i) K113Q, (ii) R261Q or R261A, (iii) H265Q, (iv) K268Q, (v) K271Q, (vi) K272Q, (vii) R279Q or R279A, (viii) E329Q or E329A, or (ix) D333Q.

[0086] Cell-free systems for antibody production Numerous cell-free systems can be used as the basis for the cell-free production system of the present disclosure, including U.S. Patent Nos. 11,261,218, 10,774,354, 10,316,322, 10,308,716, 10,190,145, 9,951,366, 9,753,040, 9,617,533, and the like. 3, 9,175,327, 9,040,253, 8,778,631, 7,871,794, 7,118,883, 7,041,479, and U.S. Patent Application Nos. 2006 / 0233789 and 2004 / 0191858.

[0087] Cell-free systems can be based on either prokaryotic or eukaryotic sources. Among prokaryotic systems, E. coli-based extracts are routinely used and commercially available for the production of a diverse range of proteins, including antibodies. Systems based on Bacillus subtilis (Kelwick R, et al., Metab Eng. 2016;38:370-81), Pseudomonas putida (Wang H. Synth Biol. 2018), Streptomyces (Li J, et al., Biotechnol Bioeng. 2017;114:1343-53), and Vibrio parahaemolyticus (Dondapati SK, et al., Eng Life Sci. 2018;18:140-8) have been well optimized at the laboratory level due to the ease of preparing cell-free lysates. A wide range of detailed protocols for the preparation of E. coli-based lysates are now available. Among eukaryotic systems, extracts based on rabbit reticulocyte lysate (RRL), wheat germ, the insect Spodoptera frugiperda 21 (Sf21), Chinese hamster ovary (CHO), and cultured human cells are routinely used. For a review, see, e.g., Dondapati SK et al., BioDrugs (2020) 34:327-348.

[0088] For the production of complex proteins that require post-translational modifications, eukaryotic cell-free systems are often, but not always, preferred.

[0089] In some embodiments, cell-free protein synthesis systems can use crude cell extracts prepared from cells with edits as taught in this disclosure. These cells are grown to a suitable confluence, and their contents are removed by lysis, followed by numerous washing steps to remove cellular debris and genomic DNA (Jin X and Hong SH. Biochem Eng J. 2018;138:156-64; Gregorio NE et al., Methods Protoc. 2019;2:24). These cell extracts can be used immediately or stored for future use (e.g., frozen at -80°C and thawed before use). Such extracts contain all the key components required for transcription and translation, including aminoacyl-tRNA synthetases (AAS), ribosomes, and factors required for elongation, initiation, and transcription. Protein synthesis can be achieved by combining the cell extract with necessary substrates, such as amino acids, energy substrates, nucleic acid templates, cofactors, salts, and nucleotides. Cell-free protein synthesis is a fast protein production system because it does not require transfection or cell culture and lacks the constraints of cell viability.

[0090] In some embodiments, cell-free protein synthesis systems are created using transcription and translation-related factors derived from or based on genetic modifications to antibody epitopes in native proteins as taught herein. In these embodiments, the system contains only known proteins and substrates, such as those taught in Shimizu et al. (2001) Nat. Biotechnology, vol. 19, p. 751 and Shimizu et al. (2005) Methods, vol. 36, p. 299. These known components of the translation machinery are purified and individually added with a DNA template to produce proteins, resulting in a highly regulated system. Protein factors involved in the initiation, elongation, and termination of the protein synthesis process can be identified and individually tailored to the requirements of the system.

[0091] In some embodiments, the protein synthesis system is a microbial lysate system (prokaryotic or eukaryotic) with mammalian proteins added to allow for proper protein folding, e.g., folding and / or post-translational modifications. In such cases, recombinant proteins (e.g., mammalian proteins) can be added to the system and, if the protein is the target of a produced antibody, can be modified to suppress antibody binding. For example, if mammalian GRP78 is added to a yeast cell-free production system, the GRP78 can be modified prior to production of the recombinant protein to modify the epitope of a specific GRP78 antibody using mutations as taught in Example 3. Similarly, recombinant proteins of other cell-resident proteins added to a cell-free system can be modified so that antibodies produced using the system do not bind to the protein (or so that binding to the protein is reduced). Methods for producing recombinant proteins are well known in the art and are taught, for example, in the Protein Expression Handbook from Thermo Fisher Scientific, Waltham, MA.

[0092] Cell-free protein synthesis reaction format Cell-free synthesis using the disclosed system can be carried out in different formats. Successful synthesis of different antibody formats, including single-chain variable fragments (scFv), Fab fragments, and full IgG, has been demonstrated in E. coli (Groff D. MAbs. 2014, 6:671-8; Yin G et al., MAbs. 2012; 4:217-25), Sf21 (Jin X et al., Biochem Eng J. 2018, 138:156-64; Stech M et al., J Biotech-nol. 2012; 164:220-31), reticulocyte (Odegrip R et al., Proc Natl Acad Sci USA. 2004; 101:2806-10), wheat germ, and CHO cell-free systems (Thoring L., et al., Sci Rep. 2017; 7:17-12188; Stech M. et al., Sci Rep. 2017;7:17-12364; Martin RW et al., ACS Synth Biol. 2017;6:1370-9). Furthermore, upscaling of cell-free reactions to the liter scale (Zawada JF et al., Biotechnol Bioeng. 2011;108:1570-8; Yin G, Garces ED, Yang J, Zhang J, Tran C, Steiner AR, et al. MAbs. 2012;4:217-25) and downscaling (Norred SE et al., J Vis Exp. 2015;11:52616) and high-throughput applications (Contreras-Llano LE and Tan C. Synth Biol. 2018) have been demonstrated.

[0093] In some embodiments, the synthesis reaction format used is a batch format. Batch-based formats are the most commonly used method in both prokaryotic and eukaryotic systems. This method is relatively fast and inexpensive, allowing synthesis within 1.5 to 3 hours depending on the system. E. coli-based systems can provide protein yields ranging from 100 μg / mL to 2-3 mg / mL. While yields from batch-based eukaryotic systems are relatively low, membrane proteins automatically assemble into microsomal membranes and function can be addressed immediately after synthesis (Brodel AK et al., PLoS One. 2013;8:2013).

[0094] In another embodiment, to further scale up protein yields via batch-based eukaryotic systems, an iterative batch-based synthesis format has been proposed in which microsomes loaded with the desired MP produced in an initial synthesis reaction can be added to a fresh cell-free synthesis reaction that has been depleted of the microsomes (Thoring L. et al., PLoS One. 2016;11:2016; Zemella A, et al., Sci Rep. 2018;8:18-26936).

[0095] Another embodiment is a continuous exchange cell-free synthesis platform (CECFSP). In this format, a semipermeable dialysis membrane separates the reaction chamber and the feed chamber, allowing the feed chamber to provide fresh reaction components and enrich the reaction chamber. In exchange, inhibitors accumulated during the reaction are removed (Quast RB et al., Sci Rep. 2016;6:30399; Gurramkonda C et al., Biotechnol Bioeng. 2018;115:1253-64; Dondapati SK et al., PLoS One. 2019; Thoring L. et al., Sci Rep. 2017;7:17-12188). Typically, the CECFSP format extends reaction time and increases protein yield. To date, the CECFSP format has been used to double protein yields and is widely used as a cell-free platform.

[0096] In addition, advances in bioorthogonal reaction chemistry have paved the way for expanding the possibilities of ADC development. Site-specific introduction of non-canonical amino acids into genetically engineered sequences can be used to generate site-specifically labeled ADCs (Axup JY et al., Proc Natl Acad Sci USA. 2012;109:16101-6). Currently, several ADCs have been approved for therapy. To date, all of these ADCs have been generated by coupling mAbs to cytotoxic linker payloads via surface-exposed lysines or by partial disulfide reduction and conjugation to free cysteines, which typically results in controlled but heterogeneous ADC populations with varying numbers and positions of drug molecules attached to the mAb (Strop P et al., Chem Biol. 2013;20:161-7). From synthesis to functional testing, cell-free systems can accelerate the evaluation of antibody constructs through sequential or simultaneous screening. The introduction of non-canonical amino acids expands the chemical repertoire and therefore the possibilities to modify and improve antibody-based therapeutics. Advanced labeling techniques allow very fast qualitative analysis of drug-to-antibody ratio (DAR), linker, linker / position, drug, and drug / position (research applications), allowing full control of ADC design.

[0097] In some preferred embodiments, the cell-free system of U.S. Patent Application No. 63 / 359,871, filed July 10, 2022, which is incorporated herein in its entirety for all purposes, can include a method for recombinant production of an antibody by: a) expressing nucleic acid encoding an antibody in a modified cell-free system (in some preferred embodiments, a eukaryotic cell-free system), wherein the antibody specifically binds to a target epitope on a protein of the system, the modified cell-free system comprising mutations of one or more amino acid residues of the target epitope on the protein in the system, and initiating transcription and translation of the antibody in the cell-free system under conditions such that the antibody is produced. In some preferred embodiments, such a cell-free system can comprise a cell lysate (in some preferred embodiments, a eukaryotic cell lysate). In some preferred embodiments, the cell-free system of U.S. Patent Application No. 63 / 359,871, filed July 10, 2022, can include: a) modifying a cell line (in some preferred embodiments, a mammalian cell line) to inhibit binding of an antibody to a protein internal to cells of the cell line, wherein the modification results in expression of a variant target protein comprising mutations of one or more amino acid residues in a target epitope of the antibody; b) generating a cell-free antibody production system from the modified cell line; c) introducing a nucleic acid template into the cell-free antibody production system, wherein the nucleic acid template encodes an antibody to the target protein; and d) initiating transcription and translation from the nucleic acid template to produce the antibody in the cell-free antibody production system (and, in some preferred embodiments, isolating the antibody).

[0098] Unless otherwise defined, all technical terms, notations, and other technical and scientific or terminology used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms having a commonly understood meaning are defined herein for clarity and / or ease of reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0099] definition As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more." It is understood that embodiments and variations described herein include "consisting of" and / or "consisting essentially of" embodiments and variations.

[0100] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within that range. For example, when a range of values ​​is presented, it is understood that every value between the upper and lower limit of that range, and any other stated or intervening value in that stated range, is encompassed within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the claimed subject matter, subject to any explicitly excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the claimed subject matter. This applies regardless of the size of the range.

[0101] As used herein, the term "about" refers to a normal error range for each readily known value. Reference herein to "about" a value or parameter includes (and describes) embodiments directed to the value or parameter itself. For example, a description that refers to "about X" includes a description of "X."

[0102] As used herein, the term "agent" refers to any binding agent capable of selectively binding to and blocking an epitope on a target protein. Agents for use with the present disclosure include, but are not limited to, peptides, proteins, antibodies or fragments thereof, small molecules, aptamers, peptidomimetics, and pharmacological groups.

[0103] As used herein, the term "antibody" is used in the broadest sense and includes intact antibodies as well as fragments such as antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable (V) fragments capable of specifically binding to antigens. H (scFv) and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses polyclonal and monoclonal antibodies, including functional (antigen-binding) antibody fragments such as single-chain antibody fragments, including single-chain variable fragments (scFv), single-chain variable fragments (scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific antibodies, e.g., bispecific or trispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv, etc.

[0104] Unless otherwise specified, the term "antibody" should be understood herein to expressly encompass functional antibody fragments thereof, also referred to as "antigen-binding fragments." The term also encompasses complete or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0105] As used herein, the term "blocking peptide" refers to a peptide consisting of part or all of the amino acid sequence corresponding to an antibody epitope (i.e., the antigen recognized by an antibody). A blocking peptide specifically binds to a target antibody and prevents subsequent antibody binding to the target epitope. Incubation of an antibody with sufficient blocking peptide occupies the antibody binding site to the target protein epitope in a sample or system, preventing subsequent target protein binding to the antibody paratope in the sample or system.

[0106] The terms "complementarity-determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known to refer to non-contiguous sequences of amino acids in an antibody variable region that confer antigen specificity and / or binding affinity. Generally, each heavy chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light chain variable region has three CDRs (CDR-L1, CDR-L2, CDR-L3). The terms "framework region" and "FR" are known to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, each full-length heavy chain variable region has four FRs (FR-H1, FR-H2, FR-H3, and FR-H4), and each full-length light chain variable region has four FRs (FR-L1, FR-L2, FR-L3, and FR-L4).

[0107] The precise amino acid sequence boundaries of a given CDR or FR can be determined using the methods described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography," J. Mol. Biol. 262, 732-745 ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol. 2003 Jan;27(1):55-77 ("IMGT" numbering scheme), Honegger A and Plueckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol,2001 Jun 8;309(3):657-70, ("Aho" numbering scheme), Martin et al., "Modeling antibody hypervariable loops: a combined algorithm," PNAS,1989,86(23):9268-9272, ("AbM" numbering scheme), and Ye et al., "IgBLAST: an immunoglobulin variable domain sequence analysis tool," Nucleic Acids Res.This can be easily determined using any of several well-known schemes, including those described by 2013 Jul;41(Web Server issue):W34-40, ("IgBLAST numbering scheme").

[0108] The boundaries of a particular CDR or FR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering in both the Kabat and Chothia schemes is based on the sequence length of the most common antibody regions, and in some antibodies, insertions and deletions corresponding to inserted letters (e.g., "30a") appear. In the two schemes, certain insertions and deletions ("indels") are located at different positions and are numbered differently. The Contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between the Kabat and Chothia definitions, based on the definitions used in Oxford Molecular's AbM antibody modeling software. The IgBLAST scheme is based on matching with germline V, D, and J genes and can be determined using the IgBLAST tool from the National Center for Biotechnology Information (NCBI).

[0109] Table 1 below lists exemplary boundary positions for CDR-L1, CDR-L2, CDR-L3, and CDR-H1, CDR-H2, and CDR-H3, as identified by the Kabat, Chothia, AbM, and Contact schemes, respectively. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between the CDRs; for example, FR-L1 precedes CDR-L1, FR-L2 is located between CDR-L1 and CDR-L2, and FR-L3 is located between CDR-L2 and CDR-L3. Note that in the Kabat numbering scheme shown, insertions are placed at H35A and H35B, so the end of the Chothia CDR-H1 loop when numbered using the Kabat numbering rules shown varies between H32 and H34 depending on the length of the loop. [Table 1]

[0110] Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 26-35 (HCDR1), 50-65 (HCDR2), and 95-105 (HCDR3), and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments, the CDR amino acids in the VH are numbered 26-35 (HCDR1). In a combined Kabat and Chothia numbering scheme, in some embodiments, the CDRs correspond to amino acid residues that are part of a Kabat CDR, a Chothia CDR, or both. For example, in some embodiments, the CDRs correspond to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-105 (HCDR3) in VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in VL.

[0111] Thus, unless otherwise specified, the "CDRs" or "complementarity determining regions" of a given antibody or region thereof, such as a variable region, or a particular individual CDR (e.g., CDR-H1, CDR-H2, CDR-H3) should be understood to encompass the complementarity determining regions defined (or specified) by any of the above schemes or other known schemes. For example, if a particular CDR (e.g., CDR-H3) is a CDR of a given V H or V L When a variable region amino acid sequence is described as comprising the amino acid sequence of a corresponding CDR in the variable region, it is understood that such CDR has the sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by any of the schemes described above or other known schemes. In some embodiments, specific CDR sequences are identified. While exemplary CDR sequences of the provided antibodies are described using various numbering schemes (see, e.g., Section II), it is understood that the provided antibodies can comprise CDRs as described according to any of the other numbering schemes described above or other known numbering schemes.

[0112] Similarly, unless otherwise specified, a FR or individual specified FR(s) (e.g., FR-H1, FR-H2, FR-H3, FR-H4) of a given antibody or region thereof, such as a variable region, should be understood to encompass one (or specified) framework region(s) defined by any of the known schemes. In some cases, a specific CDR, FR, or scheme for identifying FRs or CDRs is specified, such as CDRs defined by the Kabat, Chothia, AbM, IgBLAST, IMGT, or Contact method, or other known schemes. In other cases, the specific amino acid sequence of a CDR or FR is provided.

[0113] Acceptable variations in CDR sequences will be known to those of skill in the art. For example, in some embodiments, a polypeptide comprises a complementarity determining region (HCDR or LCDR) comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any CDR amino acid sequence.

[0114] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of a natural antibody (V H and V L ) have a generally similar structure, with each domain containing four conserved framework regions (FR) and three CDRs. (See, e.g., Kindt et al., Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single V H or V L The V domains may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen each contain a complementary V domain. L Domain or V H To screen a library of domains, V from an antibody that binds an antigen is selected. H Domain or V L The domains may be isolated using the antibody. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0115] As used herein, the terms "culture," "cultivating," "growth," "growing," "maintaining," "sustaining," "expanding," and the like, when referring to cell culture itself or the process of culturing, can be used interchangeably to mean that cells are maintained outside the body (e.g., ex vivo) under conditions suitable for their survival. Cultured cells are allowed to survive, and culturing may cause the cells to grow, differentiate, or divide.

[0116] As used herein, "epitope" refers to any polypeptide determinant capable of specific binding to an antibody. In certain embodiments, an epitope is a region of an antigen that is specifically bound by an antibody. In certain embodiments, an epitope can comprise a chemically active surface group of a molecule, such as an amino acid, a sugar side chain, a phosphoryl group, or a sulfonyl group. In certain embodiments, an epitope can have specific three-dimensional structural features (e.g., a "conformational" epitope) and / or specific charge characteristics. Epitopes can be formed from both contiguous residues and / or juxtaposed noncontiguous residues (e.g., amino acids) of a target molecule. Epitopes formed from contiguous residues (e.g., amino acids) are typically retained upon exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Epitopes can include, but are not limited to, at least 3, at least 5, or 8-10 amino acid residues. In some embodiments, the epitope is less than 20 amino acid residues in length, less than 15 residues in length, or less than 12 residues in length. If two antibodies exhibit competitive binding to an antigen, these antibodies may bind to the same epitope within the antigen. Epitopes can also be identified by a variety of scans; for example, alanine or arginine scans can reveal one or more residues with which antigen-binding molecules can interact.

[0117] The terms "expression" or "expressed," as used herein with respect to a gene, refer to the transcription and / or translation products of that gene. The level of expression of a DNA molecule in a cell is determined based either on the amount of corresponding mRNA that is present in the cell or on the amount of protein encoded by the DNA that is produced by the cell (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88).

[0118] The term "gene" refers to a segment of DNA involved in producing or encoding a polypeptide chain. It can include regions preceding and following the coding region (leader and trailer), and intervening sequences (introns) between individual coding segments (exons). Alternatively, the term "gene" can refer to a segment of DNA involved in producing or encoding a non-translated RNA, such as rRNA, tRNA, guide RNA (e.g., small guide RNA), or microRNA.

[0119] "Antibody fragment" or "antigen-binding fragment" refers to a molecule other than a complete antibody that contains a portion of the complete antibody that binds to the antigen to which the complete antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab'), diabodies, linear antibodies, variable heavy chain (V), and the like. H ) regions, single chain antibody molecules such as scFv, and V H These include, but are not limited to, single domain antibodies, which comprise only the variable heavy chain (V H ) region and the variable light chain (V L In certain embodiments, the antibody is or comprises an antibody fragment comprising a heavy chain variable (V) region, such as an scFv. H ) region and / or light chain variable (V L ) region.

[0120] A single domain antibody (sdAb) is an antibody fragment that contains all or part of the heavy chain variable region or all or part of the light chain variable region of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody.

[0121] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of whole antibodies as well as production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, such as a fragment containing a non-naturally occurring arrangement, such as a fragment having two or more antibody regions or chains joined by a synthetic linker, e.g., a peptide linker, and / or a fragment produced by enzymatic digestion of a native whole antibody. In some embodiments, the antibody fragment is an scFv.

[0122] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of a human or a non-human source that utilizes sequences encoding other human antibodies, including antibodies produced by a human cell or a human antibody repertoire or human antibody library. The term excludes humanized forms of non-human antibodies that contain non-human antigen-binding regions, such as those in which all or substantially all CDRs are non-human. The term encompasses antigen-binding fragments of human antibodies.

[0123] The term "humanized" is used to describe antibodies in which complementarity-determining regions (CDRs) derived from a mammal, e.g., a mouse, are combined with human framework regions. Often, isolated CDR-encoding polynucleotides will be grafted onto polynucleotides encoding suitable variable region frameworks (and optionally constant regions) to form polynucleotides encoding complete antibodies (e.g., humanized or fully human), antibody fragments, etc. Furthermore, "humanized" antibodies may be chimeric, human-like, humanized, or fully human antibodies to reduce potential antigenicity without reducing affinity for organelle protein targets. Chimeric, human-like, and humanized antibodies are commonly described in the art.

[0124] Humanized antibodies have variable region framework residues derived substantially from a human therapeutic antibody (referred to as the acceptor antibody) and complementarity-determining regions derived substantially from a mouse antibody (referred to as the donor immunoglobulin). See Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989), WO 90 / 07861, U.S. Pat. Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101, and Winter, U.S. Pat. No. 5,225,539. If present, the constant region(s) are also derived substantially or entirely from a human immunoglobulin. The human variable domain(s) are typically selected from human antibodies whose framework sequences exhibit a high degree of sequence identity with the mouse variable region domains from which the CDRs are derived. The heavy and light chain variable region framework residues can be derived from the same or different human therapeutic antibody sequences. The human therapeutic antibody sequence can be the sequence of a naturally occurring human antibody or a consensus sequence of several human antibodies. See WO 92 / 22653 by Carter et al. Specific amino acids from the human variable region framework residues are selected for substitution based on their potential effect on CDR conformation and / or binding to antigen. Examination of such potential effects is performed by modeling, examination of the properties of the amino acid at a particular position, or empirical observation of the effects of substituting or mutagenesing specific amino acids.

[0125] For example, if an amino acid differs between a murine variable region framework residue and a selected human variable region framework residue, the human framework amino acid should generally be substituted with the equivalent framework amino acid from the murine antibody if the amino acid is reasonably expected to: i) directly non-covalently bind to the antigen; ii) be adjacent to a CDR region; iii) otherwise interact with a CDR region (e.g., be within about 6 A of a CDR region); or iv) be involved in the VL-VH interface.

[0126] Other candidates for substitution are acceptor human framework amino acids that are rare for human antibodies at that position. These amino acids can be substituted with amino acids from the equivalent position in the mouse donor antibody or with amino acids from the equivalent position in a more typical human antibody. Other candidates for substitution are acceptor human framework amino acids that are rare for human antibodies at that position. Preferred variable region frameworks for humanized antibodies typically exhibit at least 75%, more preferably 80%, and even more preferably 85% sequence identity to a human variable region framework sequence or a consensus sequence of such sequences.

[0127] In certain instances, murine monoclonal antibodies can be used as the basis for producing human therapeutic biologics. In one method, by way of example and not limitation, the heavy chain variable V H The region was cloned by RT-PCR using mRNA prepared from hybridoma cells. The consensus primers were V, which contains the translation initiation codon as the 5' primer, and V, which contains the translation initiation codon as the 5' primer. H The g2b constant region-specific leader peptide and g2b constant region-specific 3' primers are used. Sequences from multiple independently derived clones can be compared to ensure that no changes were introduced during amplification. H The sequence of the region was determined by 5'RACE RT-PCR and 3'g2b-specific primers. H It can also be determined or confirmed by sequencing the fragment.

[0128] Light chain variable V of mouse monoclonal antibody L The region is V H In the first approach, the mouse V L The consensus primer set designed for amplification of the region V contains the translation initiation codon. L In the second approach, a 5' RACE RT-PCR method is used to identify the V region and a 3' primer specific for the mouse Ck region downstream of the VJ junction. LThe cDNA encoding the human constant region is then cloned. The cloned sequence is then combined with sequences encoding the human constant region.

[0129] In one approach, the heavy and light chain variable regions are reengineered to encode splice donor sequences downstream of their respective VDJ or VJ junctions and cloned into mammalian expression vectors such as pCMV-hγ1 for the heavy chain and pCMV-hk1 for the light chain. These vectors encode the human γ1 and Ck constant regions as exon fragments downstream of the inserted variable region cassettes. After sequence verification, the heavy and light chain expression vectors can be co-transfected into COS cells to produce chimeric antibodies. Conditioned medium is collected 48 hours after transfection and assayed for antibody production by Western blot analysis or antigen binding by ELISA. The chimeric antibodies are preferably humanized as described above.

[0130] The heavy and light chain variable regions of chimeric and / or humanized antibodies can be linked to at least a portion of a selected human constant region. The choice of constant region can depend on the desired mechanism of action of the antibody, for example, whether cell-mediated toxicity is desired. For example, isotypes IgG1 and IgG3 have antibody-dependent complement activity, while isotypes IgG2 and IgG4 do not. The light chain constant region can be lambda or kappa. Antibodies can be expressed as tetramers containing two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab', F(ab'), and Fv, or as single-chain antibodies in which the heavy and light chain variable domains are linked via a linker.

[0131] As used herein, the term "monoclonal antibody" refers to an antibody obtained from or within a population of substantially homogeneous antibodies. That is, the individual antibodies comprising the population are identical except for variants that may include natural mutations or arise during production of the monoclonal antibody preparation, and such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed against a single epitope on an antigen. This term should not be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be produced by a variety of techniques, including, but not limited to, generation from hybridomas, recombinant DNA methods, phage display, and other antibody display methods.

[0132] As used herein, the term "specifically binds" to a target protein or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or associates with a particular target antigen more frequently, more rapidly, with a longer duration, and / or with a higher affinity than it does with another protein. It is also understood that "specific binding" does not necessarily require (although it can include) exclusive binding. Generally, although not necessarily, reference to binding implies preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0133] As used herein, "stress proteins" refer to proteins that function in normal cells and may be present at elevated levels under stressful conditions such as hypoxia, nutrient deprivation, pH changes, oxidative stress, or other metabolic dysregulation of cells, such as those often encountered in cancer cells. Stress proteins include proteins whose expression increases when organelles such as the endoplasmic reticulum (ER) or Golgi apparatus become overwhelmed. ER stress proteins include proteins involved in the unfolded protein response (UPR), which act to alleviate ER stress and restore homeostasis, proteins involved in endoplasmic reticulum-associated protein degradation (ERAD), or proteins involved in ER stress-mediated apoptosis. Examples of ER stress proteins include calreticulin, heat shock proteins, and isomerases. Golgi stress proteins include proteins involved in post-translational modifications such as glycosylation or proteins involved in vesicle trafficking. Exemplary Golgi stress proteins include, for example, GOLPH3.

[0134] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single- or double-stranded form. Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized similarly to natural nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated.

[0135] The term "paratope," also known as "antigen-binding site," is the part of an antibody that recognizes and binds to an epitope.

[0136] As used herein, the terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear, cyclic, or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also encompasses amino acid polymers that have been modified, for example, through sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, transfer RNA-mediated amino addition to the protein, e.g., arginylation, ubiquitination, or conjugation with a labeling component. As used herein, the term "amino acid" refers to any natural and / or unnatural or synthetic amino acid, including glycine, both D- and L-optical isomers, and amino acid analogs.

[0137] A "vector" is a nucleic acid molecule, preferably self-replicating, that transports an inserted nucleic acid molecule into and / or between host cells. An "expression vector" is a polynucleotide sequence that can be transcribed and translated into polypeptide(s) when introduced into an appropriate host cell. An "expression system" usually refers to a suitable host cell comprised of an expression vector that can function to produce a desired expression product.

[0138] The term "recombinant" when applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction and / or ligation steps, and other procedures, that result in a construct that differs from polynucleotides found in nature.

[0139] As used herein, the terms "operably linked" or "operably linked" are used to refer to DNA sequences juxtaposed in a relationship permitting the components so described to function in their intended manner. For example, a promoter is operably linked to a coding sequence if it controls the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation. DNA for a signal sequence (secretory leader) is operably linked to DNA for a polypeptide if it is expressed as a precursor that participates in the secretion of that polypeptide. Generally, operably linked means contiguous.

[0140] In some preferred embodiments, the present disclosure provides a method for recombinant production of an antibody, comprising providing a cell-free protein expression system containing a target protein that displays an antibody epitope; modifying the cell-free system by introducing one or more agents that block the antibody epitope on the target protein but do not eliminate the activity of the target protein in the cell-free system; introducing into the cell-free system one or more nucleic acids encoding an antibody that binds to the antibody epitope; and initiating transcription and translation of the antibody in the cell-free system under conditions such that the antibody is produced. In some preferred embodiments, the cell-free system comprises a cell lysate. In some preferred embodiments, the agent is introduced into the cells before creating the cell lysate. In some preferred embodiments, the agent is introduced into the cells after creating the cell lysate. In some preferred embodiments, the cell lysate is selected from the group consisting of wheat germ lysate, insect cell lysate, reticulocyte lysate, keratinocyte lysate, or a cell extract from CHO cells, HeLa cells, myeloma cells, hybridoma cells, and cultured lymphoma cells. In some preferred embodiments, the method further comprises isolating the antibody from the cell-free system. In some preferred embodiments, the antibody epitope-blocking agent is a peptide. In some preferred embodiments, the antibody-blocking agent is a small molecule. In some preferred embodiments, the target protein is a cell-resident protein in the secretory pathway. In some preferred embodiments, the target protein is a stress protein. In some preferred embodiments, the target protein is a cell signaling protein. In some preferred embodiments, the agent does not affect the activity of the target protein compared to the target protein activity in the absence of the agent. In some preferred embodiments, the target protein activity is reduced by about 10% compared to the target protein activity in a cell-free system in the absence of the agent. In some preferred embodiments, the target protein activity is reduced by about 50% compared to the target protein activity in a cell-free system in the absence of the agent.

[0141] In some preferred embodiments, the present disclosure provides methods for the recombinant production of antibodies, the method comprising: providing a cell-free protein expression system containing a target protein that displays an antibody epitope; modifying the cell-free system by introducing one or more agents that block an antibody paratope that binds to the antibody epitope on the target protein; introducing one or more nucleic acids encoding an antibody that includes the antibody paratope into the cell-free system; and initiating transcription and translation of the antibody in the cell-free system under conditions such that the antibody is produced. In some preferred embodiments, the method further comprises isolating the antibody from the cell-free system. In some preferred embodiments, the method further comprises removing the agent that binds to the antibody paratope after antibody production. In some preferred embodiments, the method further comprises isolating the antibody from the cell-free system. In some preferred embodiments, the antibody is isolated prior to removal of the agent from the antibody. In some preferred embodiments, the cell-free system comprises a cell lysate. In some preferred embodiments, the cell lysate is selected from the group consisting of wheat germ lysate, insect cell lysate, reticulocyte lysate, keratinocyte lysate, cell extract from CHO cells, HeLa cells, myeloma cells, hybridoma cells, and cultured lymphoma cells. In some preferred embodiments, the agent is introduced into the cell-free system before initiating antibody transcription and translation. In some preferred embodiments, the agent that blocks the antibody paratope is a peptide. In some preferred embodiments, the target protein is a cell-intrinsic protein in the secretory pathway. In some preferred embodiments, the target protein is a stress protein. In some preferred embodiments, the target protein is a cell signaling protein.

[0142] In some preferred embodiments, the present disclosure provides methods for the recombinant production of monoclonal antibodies, the method comprising: providing a mammalian cell-free protein expression system containing a target protein that displays an antibody epitope; modifying the mammalian cell-free system by introducing one or more agents that block the antibody epitope on the target protein but do not eliminate the activity of the target protein in the cell-free system; introducing into the cell-free system one or more nucleic acids encoding a monoclonal antibody that binds to the antibody epitope; and initiating transcription and translation of the monoclonal antibody in the mammalian cell-free system under conditions such that the antibody is produced. In some preferred embodiments, the mammalian cell-free system comprises a cell lysate. In some preferred embodiments, the method further comprises isolating the antibody from the mammalian cell-free system.

[0143] In some preferred embodiments, the present disclosure provides methods for recombinant production of monoclonal antibodies, including providing a mammalian cell-free protein expression system containing a target protein that displays an antibody epitope; modifying the mammalian cell-free system by introducing one or more agents that block an antibody paratope that binds to the antibody epitope on the target protein; introducing into the cell-free system one or more nucleic acids encoding a monoclonal antibody that includes the antibody paratope; and initiating transcription and translation of the monoclonal antibody in the mammalian cell-free system under conditions such that the antibody is produced. In some preferred embodiments, the method further includes removing the agent that binds to the antibody paratope after production of the monoclonal antibody. In some preferred embodiments, the method further includes isolating the monoclonal antibody from the cell-free system. In some preferred embodiments, the monoclonal antibody is isolated prior to removal of the agent from the monoclonal antibody.

[0144] In some preferred embodiments, the present disclosure provides a method for recombinant production of an antibody, the method comprising: introducing an agent that selectively binds to a target epitope on a cell-resident protein into a cell to generate an engineered cell line; generating a cell-free antibody production system from the engineered cell line; introducing a nucleic acid template into the cell-free antibody production system, wherein the nucleic acid template encodes an antibody that selectively binds to the target epitope on the cell-resident protein; and initiating transcription and translation from the nucleic acid template to produce the antibody in the cell-free antibody production system. In some preferred embodiments, the method further comprises isolating the antibody from the cell-free system. In some preferred embodiments, the cell line is a mammalian cell line. In some preferred embodiments, the naturally expressed target protein is a cell-resident protein in the secretory pathway of the mammalian cell line.

[0145] In some preferred embodiments, the modified cell line can be produced using the methods disclosed in U.S. Patent Application No. 63 / 337,980, filed May 3, 2022, and / or U.S. Patent Application No. 63 / 359,541, filed July 8, 2022, each of which is incorporated herein in its entirety for all purposes. For example, in some preferred embodiments, the methods of these applications can include: a) expressing nucleic acid encoding an antibody in an modified cell line (in some preferred embodiments, a mammalian cell line), where the antibody specifically binds to a target epitope on a target protein naturally expressed as an endogenous protein in cells, and the modified cell is engineered to express a variant target protein comprising mutations of one or more amino acid residues of the target epitope; and b) culturing the modified cell line under conditions such that the antibody is produced (and in some preferred embodiments, isolating the antibody). In some preferred embodiments, the methods of these applications include: a) providing a cell line (in some preferred embodiments, a mammalian cell line) with an antibody that binds to an epitope on a target protein present in the cells; b) identifying the epitope of the target protein to which the antibody binds; c) generating an engineered cell line by mutating the epitope of the target protein in the cell line to reduce or inhibit binding of the antibody to the epitope on the target protein; d) introducing an expression vector encoding the antibody into the engineered cell line; e) culturing the engineered cell line under conditions that allow production of the antibody from the expression vector; and f) expressing the nucleic acid encoding the antibody in the engineered cell line (and, in some preferred embodiments, isolating the antibody). In some preferred embodiments, the engineered mammalian cell line of these applications includes a variant protein that is a cell-resident protein in the secretory pathway, the variant protein comprising a mutation of a native cell-resident protein that is the target protein of the antibody, the mutation being an amino acid substitution(s) of one or more amino acid residues to change the target epitope of the antibody to the variant epitope.In some preferred embodiments, the method includes recombinant production of an antibody that targets GRP78 by: a) expressing a nucleic acid encoding an antibody in an engineered mammalian cell line, wherein the antibody binds to GRP78, and the engineered mammalian cell is engineered with a variant GRP78 having a mutant epitope comprising mutations of one or more amino acid residues of the target epitope of the antibody or antigen-binding fragment; and b) culturing the engineered mammalian cell line under conditions such that anti-GRP78 antibody is produced in the culture supernatant.

[0146] In some preferred embodiments of the methods disclosed herein, the target protein is a cellular protein in the secretory pathway of the cell line. In some preferred embodiments, the target protein is a stress protein. In some preferred embodiments, the target protein is a cell signaling protein. In some preferred embodiments, the target protein is expressed on an organelle of the cell line. In some preferred embodiments, the organelle is the endoplasmic reticulum or the Golgi apparatus. In some preferred embodiments, the target protein is an endoplasmic reticulum chaperone. In some preferred embodiments, the endoplasmic reticulum chaperone is calreticulin, a heat shock protein, or an isomerase. In some preferred embodiments, the target protein is glucose-regulated protein 78 (GRP78), HSP47, PDI, calreticulin, or GP94. In some preferred embodiments, the target protein is glucose-regulated protein 78 (GRP78). In some preferred embodiments, the target protein is a Golgi complex protein. In some preferred embodiments, the Golgi complex protein is GOLPH2, GOLPH3, GM130, ATP6V1A, ATP6V1E1, ATP6VOA2, TMEM165, GOLGB1, SCYL1BP1, TRAPPC11, TRAPPC2, or TRIP11.

[0147] As will be appreciated by those skilled in the art, other embodiments are contemplated herein. [Example]

[0148] The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0149] Example 1 Production and purification of anti-GPRP78 antibodies in mammalian cells Fully human anti-GRP78 monoclonal antibodies (mAbs) (Table E1) formatted as full-length IgG1 antibodies were individually transfected into Chinese hamster ovary (CHO) cells by electroporation in six-well plates. The cells were replenished with fresh medium the following day and incubated for 7 days post-transfection. Transfections were performed using the Neon™ Transfection System and Neon™ Transfection System Reagent (Thermo Fisher Scientific, Waltham, MA) pre-set for CHO cells. The supernatants 7 days post-transfection were loaded onto a 1 mL MabSelect PrisimA™ column to purify the antibodies. Antibody concentrations and IgG titers in the supernatants were determined using an Octet QK384 with a Protein A biosensor (Molecular Devices, Wokingham, Berkshire, UK) using an IgG1 antibody as a standard.

[0150] Transfected CHO cells showed decreased viability during culture and at harvest. The titers of all anti-GRP78 antibody supernatants produced from CHO cells were low, less than 5 μg / mL (Table E2). To confirm that the decreased titers were not specific to CHO cells, we repeated the transfection in human embryonic kidney 293 (HEK293) cells and purified antibodies from the day 5 supernatant using a similar method; the titers also remained low (Table E2).

[0151] These results are consistent with the observation that production of anti-GRP78 antibodies in mammalian cells, such as human cells, is toxic to the cells and impacts their ability to produce antibodies at high yields. It has been hypothesized that the antibodies bind to native GRP78 within the endoplasmic reticulum (ER), thereby inhibiting its natural chaperone function within the cell and potentially killing the cells before the antibodies can be secreted into the supernatant for purification. Binding of GRP78 antibodies to native ER proteins has been demonstrated by experiments showing co-immunoprecipitation of the antibody with GRP78, as demonstrated by SDS-PAGE analysis of purified samples performed under reducing and non-reducing conditions. As shown in Figure 1, all samples displayed a typical SDS-PAGE profile for monomeric IgG, although an extra band was present between 70 and 80 kDa, consistent with the presence of bound GRP78 antigen. [Table 2] [Table 3]

[0152] Example 2 Epitope mapping of anti-GRP78 antibodies To characterize the binding properties of the antibody described in Example 1 to GRP78, epitope mapping analysis was performed by alanine scanning mutagenesis. An alanine scanning library of GRP78 was constructed. Each anti-GRP78 antibody was then screened for binding to individual GRP78 variants, allowing the identification of target protein residues involved in antibody binding.

[0153] The binding of each test antibody to each GRP78 variant in the alanine scanning library was determined by high-throughput flow cytometry under high stringency conditions (e.g., increasing pH, increasing salt, increasing temperature, and / or increasing wash time). The experiment also included the commercially available monoclonal antibody 1H11-1H7, which was verified to bind to wild-type (WT) GRP78 on cells and was therefore determined to be a suitable positive control under these conditions (Thermo Fisher Scientific, Waltham, MA). Antibody binding to each GRP78 variant, as measured by fluorescence signal (raw fluorescence data minus background), was normalized to binding to WT GRP78. For each GRP78 variant, such normalized binding to the test antibody was plotted relative to binding to the control antibody. Exemplary results for two anti-GRP78 antibody clones are shown in Figures 2A and 2B. GRP78 variants that showed >70% binding to the control antibody but <20% binding to the test antibody were identified as the primary key binding residues for the test antibody. Other GRP78 variants that did not meet the above criteria but showed reduced binding activity (20%-30%) and proximity (based on the known 3D structure of the protein) to the primary residues identified above were also identified (and considered to be "other residues involved in antibody binding").

[0154] Table E3 shows residues involved in antibody binding for exemplary anti-GRP78 clones B4 and F6. [Table 4]

[0155] PDB ID 6ASY (Yang et al. Nature Communications. 2017;8(1):1-3) was tested for the identification of predicted amino acids constituting the GRP78 epitope that binds to B4 mAb or F6 mAb. This structure was chosen because it is the most complete of the experimentally determined structures of H. sapiens GRP78 (ranging from amino acids 25 to 633) (PDB ID #6ASY, Yang et al., 2017). Figure 3 shows a visualization of key residues for antibody binding, with exemplary key residues and other residues involved in binding indicated by arrows. According to the analyzed structure, all of the amino acids in the predicted epitope are contained in an α-helix or as part of the linker between two α-helices and can be separated by an α-helix (key residues identified by shotgun mutagenesis are underlined): (i) K113, (ii) K114, (iii) K115, (iv) K116, (v) K117, (vi) K118, (vii) K119, (viii ...iii) K119, (iv) K119, (viii) K119, (viii) K119, (viii) K119, (viii) K119, (iv) K119, (viii) K119, (viii) K119, (iv R261 , H265 ,K268,K271,K272,(iii) R279 , and (iv) E329 , D333.

[0156] Example 3: Generation of a cell-free lysate system from engineered GRP78 cells The cell-free system is generated from CHO cells as taught in Stech M, Scientific Reports 7:12030 (2017), Brodel, A. K. et al., PLoS One 8, e82234 (2013), and Thoring, L. et al., PLoS One 11, e0163670 (2016) for the efficient production of anti-GRP78 antibodies. al., Biotechnology and Bioengineering 111, 25-36 (2013).

[0157] CHO lysates containing endogenous microsomal vesicles derived from the ER are prepared as previously described (Brodel AK, et al., (2013) PloS one 8(12), e82234; Thoring L., et al., (2016). PloS one 11(9), e0163670). Briefly, CHO modified as described herein are cultured in a bioreactor at 37°C in a chemically defined serum-free medium (PowerCHO™ 2CD medium, Lonza, Basel, Switzerland) for up to 18 × 10 6 The cells are exponentially grown to 1000 cells / ml. The cells are harvested by centrifugation at 200 × g for 15 minutes, pelleted, washed twice, and resuspended in a buffer containing 30 mM HEPES-KOH (pH 7.5) and 100 mM NaOAc. The cell suspension is then passed through a 20-gauge needle using a syringe, resulting in mechanical disruption of the cells. Nuclei and cell debris are removed by centrifugation at 6,500 × g for 10 minutes. The resulting supernatant is subjected to a gel filtration step using a Sephadex G-25 column (GE Healthcare, Freiburg, Germany) equilibrated in a buffer containing 30 mM HEPES-KOH (pH 7.5) and 100 mM NaOAc. The filtered supernatant is eluted in 1 ml fractions, and those with an RNA content greater than 100 absorbance at 260 nm are pooled. To remove endogenous mRNA, the cell lysate is treated with S7 micrococcal nuclease (Roche, Mannheim, Germany) (10 U / ml) and CaCl2 (1 mM) and incubated at room temperature (RT) for 20 minutes. Micrococcal S7 nuclease is inactivated by adding EGTA (6.7 mM). Optionally, the CHO lysate is further supplemented with creatine kinase (100 μg / ml). The lysate is shock-frozen in liquid nitrogen and then stored at -80°C until further use.

[0158] As taught in Example 2, GRP78 peptides of approximately 8-12 amino acids containing a GRP78 peptide binding motif that matches the binding epitopes of the B4 and H6 binding epitopes are identified using the panning and specificity assays of Blond-Elguindi S Cell 1993 Nov 19;75(4):717-28 and Arap et al., Cancer Cell. 2004 Sep;6(3):275-84.

[0159] GRP78 peptides are synthesized and isolated using methods well known in the art. For example, peptides can be ordered from Thermo Fisher Scientific's (Waltham, Massachusetts) custom peptide synthesis service. These peptides are introduced into a CHO cell-free lysate system at an appropriate concentration to provide the expected epitope saturation within the system. GRP78 activity in the system was assessed using the method of Hristozova N, et al., PLoS One. 2016;11(8):e0161970, and CHO lysates were confirmed to have B4 or H6 epitope blocking by treating aliquots of the peptide-treated lysate with the respective antibodies. The modified CHO lysate with the best combination of epitope blocking and GRP78 activity is selected for the production of GRP78 antibodies, as described below.

[0160] Example 4: Antibody production in a GRP78-modified cell-free system The coupled transcription-translation reaction is then carried out as described using the modified CHO lysate in Example 3. Thoring, supra. To allow for subsequent quantitative and qualitative analysis of cell-free synthesized proteins, the translation reaction consisted of 40% (v / v) S7 nuclease-treated CHO lysate containing ER-derived endogenous microsomal vesicles, HEPES-KOH (pH 7.6, 30 mM, BioMol GmbH, Hamburg, Germany), complete amino acids (100 μM), Mg(OAc) (3.9 mM), KOAc (135 mM, Merck, Darmstadt, Germany), spermidine (0.25 mM, Sigma-Aldrich, St. Louis, United States), energy components (1.75 mM ATP, 0.3 mM GTP, 0.3 mM CTP, 0.3 mM UTP), creatine phosphate (20 mM), T7 polymerase (1 U / μl) (Agilent Technologies, Santa Clara, United States), and ATP. 14 The plasmid is composed of C-leucine (to a final concentration of 30 μM), specific radioactivity 46.15 dpm / pmol (PerkinElmer LAS (Germany) GmbH, Rodgau, Germany). Protein synthesis is initiated by the addition of DNA template (60 ng / μL). The reaction is incubated for 3 h at 30 °C and 600 rpm in a standard thermomixer (Eppendorf Thermomixer Comfort). Background translation activity is monitored by running the translation reaction without supplementing the plasmid.

[0161] The coding sequence for the anti-GRP78 antibody was codon-optimized for Cricetulus griseus and equipped with the necessary regulatory sequences to enable in vitro transcription and translation according to Stech et al. (2017). Sci. Rep. 7(1), 12030. The essential elements were as follows: 5' untranslated region (UTR): T7 promoter sequence, multiple cloning site (MCS), internal ribosome entry site (IRES) from the intergenic region (IGR) of cricket paralysis virus (CrPV), GCT as the start codon; 3' UTR: T7 terminator sequence, MCS (ibid.). The DNA template was de novo synthesized (Agilent, San Jose, CA) and cloned into the appropriate vector (pUC57-1.8k) by Biocat GmbH (Biocat GmbH, Heidelberg). Plasmid preparations for cell-free protein synthesis are prepared using the PureLink® HiPure Plasmid Midiprep Kit (Thermo Fisher Scientific, Waltham, Mass.) according to the manufacturer's instructions, and then digested and sequenced to verify the correct DNA sequence using an Illumina Miseq™ system (Illumina, San Diego, Calif.).

[0162] To allow for subsequent quantitative and qualitative analysis of cell-free synthesized proteins, the translation reaction consisted of 40% (v / v) S7 nuclease-treated CHO lysate containing ER-derived endogenous microsomal vesicles, HEPES-KOH (pH 7.6, 30 mM, BioMol GmbH, Hamburg, Germany), complete amino acids (100 μM), Mg(OAc) (3.9 mM), KOAc (135 mM, Merck, Darmstadt, Germany), spermidine (0.25 mM, Sigma-Aldrich, St. Louis, United States), energy components (1.75 mM ATP, 0.3 mM GTP, 0.3 mM CTP, 0.3 mM UTP), creatine phosphate (20 mM), T7 polymerase (1 U / μl) (Agilent Technologies, Santa Clara, United States), and ATP. 14 The plasmid was composed of C-leucine (to a final concentration of 30 μM, specific activity 46.15 dpm / pmol (PerkinElmer LAS (Germany) GmbH, Rodgau, Germany)). Protein synthesis was initiated by the addition of DNA template (60 ng / μL). Reactions were incubated for 3 h at 30 °C and 600 rpm in a standard thermomixer (Eppendorf Thermomixer Comfort). Background translation activity was monitored by running the translation reaction without supplementing the plasmid.

[0163] After the translation reaction, the sample is centrifuged at 16,000 × g for 10 minutes at 4 °C to separate the microsomes from the soluble fraction of the translation mixture. The resulting supernatant (first supernatant, or SUP1) is transferred to a fresh reaction tube and stored on ice until further analysis. Meanwhile, the microsomal pellet is resuspended in 1 × PBS containing 0.2% n-dodecyl-β-D-maltoside (DDM) to allow for the release of translocated microsome-containing antibodies. The microsomes are manually resuspended by repeatedly pipetting up and down, followed by vortexing and rocking on an oscillator for approximately 45 minutes. A second centrifugation step is performed to separate the released proteins from microsomal membrane residues. The resulting supernatant (second supernatant, or SUP2) is transferred to a fresh reaction tube and stored on ice until further analysis. 14 C-leucine supplemented reactions are analyzed by SDS-PAGE followed by autoradiography and liquid scintillation counting, while non-radioactive samples are subjected to functional analysis by enzyme-linked immunosorbent assay (ELISA).

[0164] Example 5 Humanization of anti-GOLPH3 monoclonal mouse antibody GOLPH3 was initially identified as a peripheral membrane protein localized in the trans-Golgi network, but other researchers have reported it to be a mitochondrial protein that regulates mitochondrial mass through the regulation of the mitochondrial-specific phospholipid cardiolipin. GOLPH3 has since been implicated in the target of rapamycin (TOR) signaling pathway. GOLPH3-transfected cells exhibited enhanced S6 kinase activity in response to growth factor stimulation with EGF. Concomitantly, AKT phosphorylation was increased in these cells, but this phenomenon was suppressed in GOLPH3 siRNA-treated cells compared with control cells, suggesting that GOLPH3 can enhance signaling through the TOR-associated complex. Scott, KL et al., Nature. 2009 Jun 25;459(7250):1085-1090. These results suggest that GOLPH3 is a bona fide oncogene and may be a useful target for therapeutic strategies.

[0165] Thermo Fisher monoclonal antibody clone 905CT9.1.1 (Thermo Fisher Scientific catalog number MA5-37626, Thermo Fisher Scientific, Waltham, MA) (hereinafter referred to as "mGOLPH3") is a mouse IgG1 monoclonal antibody that selectively binds to purified human His-tagged GOLPH3 protein. Because GOLPH3 is primarily found in the Golgi apparatus and mitochondria, the disclosed methods are well suited for the efficient production of such antibodies in mammalian cells.

[0166] Humanized GOLPH3 antibodies for use in the present methods preferably comprise CDR sequences derived from or based on mGOLPH3, as described in more detail herein and in the incorporated references. H and V L The amino acid sequence of the region is determined using the REmAB® antibody sequencing service provided by Rapid Novor (Ontario, Canada). The CDRs of mGOLPH3 are determined using the IMGT numbering system, for example, as provided in Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003). After the CDRs are identified, the mouse monoclonal antibody is humanized using methods such as those disclosed in U.S. Patent No. 10,613,094 to Do Couto et al. (issued April 7, 2020) and U.S. Patent No. 8,673,593 to Chilcote et al. (issued March 18, 2014).

[0167] Example 6 Epitope mapping of the protein binding site of the anti-GOLPH3 humanized antibody against hGOLPH3 Various methods can be used for epitope mapping of the binding site of an anti-mGOLPH3 mAb on human hGOLPH3. Exemplary methods are taught in U.S. Patent No. 11,174,479 to Greenleaf et al. (issued November 16, 2021) and U.S. Patent No. 11,061,036 to Wilson et al. (issued July 13, 2021).

[0168] For example, the binding of mGOLPH3 mAb to biotinylated hGOLPH3 peptides spanning hGOLPH3 can be measured using a streptavidin biosensor. Biotinylated peptides spanning the hGOLPH3 protein are loaded at 5 μg / ml for 700 seconds, followed by a baseline recording for 300 seconds. The association and dissociation of mGOLPH3 mAb with each peptide is then measured for 600 seconds at multiple concentrations. A dual-reference sensor is used in all experiments to measure any observed background signal and subtract it from nonspecific binding or system noise.

[0169] Analysis is performed using ForteBio data analysis software (v8.2). After background subtraction, a 1:1 local kinetic model was fitted to the observed association and dissociation curves. Overall, K D , K. on , K. off , and R2 correlation coefficients are determined. Where possible, a global curve fit was also performed for multiple concentrations of antibody / analyte.

[0170] Example 7 Identification of hGOLPH3 Antibody Epitopes for Identifying Pharmacophore Binders The crystal structure of hGOLPH3 and its orthologues can be used to predict residues that are important for identifying binding factors to the target epitope of antibodies against hGOLPH3. Wood et al. reported the X-ray crystal structure of hGOLPH3 to 2.9 Å resolution (Wood et al., J Cell Biol. 2009 Dec 28;187(7):967-975) and conserved regions of activity in the structures of hGOLPH3 and its yeast orthologue, Vps74p (Wood et al., Journal of Cell Biology, 209 187:67-75. Additional structural aspects and residues required for specific functions of GOLPH3 can be found in Bergeron JJM et al., Mol Cell Proteomics. 2017 Dec;16(12):2048-2054 and Schmitz KR, et al., Dev. Cell 2008;14:523-534).

[0171] After predicting the residues of the target epitope, discovery methods such as those taught in U.S. Patent No. 8,019,550 are used to identify potential pharmacophore agents for use in the methods and systems of the present disclosure. Briefly, antibody epitopes within the protein structure and relevant binding residues within the epitope site (e.g., pharmacophore points) are identified. Pharmacophore agents are then designed that fit within the predicted epitope both geometrically and in terms of satisfying sufficient pharmacophore points. Various techniques may be used to improve the design process, for example, using concatenated structures (e.g., hyperpositions with the target as a reference), comparison of epitopes determined by simulation models with shapes determined by mapping processes, and / or empirical determination of epitopes using epitope mapping techniques such as those described above.

[0172] Example 8 Generation of a cell-free system for the production of anti-GOLPH3 humanized antibodies A cell-free system is then created from CHO cells for efficient production of anti-GOLPH3 antibodies, as taught in Stech M, Scientific Reports 7:12030 (2017), Brodel, A.K. et al., PLoS One 8, e82234 (2013), and Thoring, L. et al., PLoS One 11, e0163670 (2016). CHO lysate is prepared from cultured CHO-K1 cells as described in Brodel, A.K. et al., Biotechnology and Bioengineering 111, 25-36 (2013). The pharmacophore developed as taught in Example 7 is introduced into the CHO lysate at a concentration sufficient to bind to the anti-GOLPH3 antibody epitope while not eliminating the activity of GOLPH3 in the CHO lysate system.

[0173] CHO lysates containing endogenous microsomal vesicles derived from the ER are prepared as previously described (Brodel AK, et al., (2013) PloS one 8(12), e82234; Thoring L., et al., (2016). PloS one 11(9), e0163670). Briefly, CHO modified as described herein are cultured in a bioreactor at 37°C in a chemically defined serum-free medium (PowerCHO™ 2CD medium, Lonza, Basel, Switzerland) for up to 18 × 10 6The cells are exponentially grown to 1000 cells / ml. The cells are harvested by centrifugation at 200 × g for 15 minutes, pelleted, washed twice, and resuspended in a buffer containing 30 mM HEPES-KOH (pH 7.5) and 100 mM NaOAc. The cell suspension is then passed through a 20-gauge needle using a syringe, resulting in mechanical disruption of the cells. Nuclei and cell debris are removed by centrifugation at 6,500 × g for 10 minutes. The resulting supernatant is subjected to a gel filtration step using a Sephadex G-25 column (GE Healthcare, Freiburg, Germany) equilibrated in a buffer containing 30 mM HEPES-KOH (pH 7.5) and 100 mM NaOAc. The filtered supernatant is eluted in 1 ml fractions, and those with an RNA content greater than 100 absorbance at 260 nm are pooled. To remove endogenous mRNA, the cell lysate is treated with S7 micrococcal nuclease (Roche, Mannheim, Germany) (10 U / ml) and CaCl2 (1 mM) and incubated at room temperature (RT) for 20 minutes. Micrococcal S7 nuclease is inactivated by adding EGTA (6.7 mM). Optionally, the CHO lysate is further supplemented with creatine kinase (100 μg / ml). The lysate is shock-frozen in liquid nitrogen and then stored at -80°C until further use.

[0174] The GOLPH3 pharmacophore developed in Example 7 is used to engineer a cell-free system for the production of GOLPH3 monoclonal antibodies. The activity of GOLPH3 in the system is assessed by determining its ability to regulate the phosphorylation state of mTOR substrates, as taught in Scott et al. (supra), and CHO lysates confirmed to have GOLPH3 antibody epitope blocking are identified by treating aliquots of peptide-treated lysates with each antibody. The engineered CHO lysate with the best combination of epitope blocking and GOLPH3 phosphorylation activity is selected for the production of GOLPH3 antibodies, as described below.

[0175] Example 9: Antibody production in a GOLPH3-modified cell-free system The coupled transcription-translation reaction is then carried out as described in Thoring, supra. To allow for subsequent quantitative and qualitative analysis of cell-free synthesized proteins, the translation reaction consisted of 40% (v / v) S7 nuclease-treated CHO lysate containing ER-derived endogenous microsomal vesicles, HEPES-KOH (pH 7.6, 30 mM, BioMol GmbH, Hamburg, Germany), complete amino acids (100 μM), Mg(OAc) (3.9 mM), KOAc (135 mM, Merck, Darmstadt, Germany), spermidine (0.25 mM, Sigma-Aldrich, St. Louis, United States), energy components (1.75 mM ATP, 0.3 mM GTP, 0.3 mM CTP, 0.3 mM UTP), creatine phosphate (20 mM), T7 polymerase (1 U / μl) (Agilent Technologies, Santa Clara, United States), and ATP. 14 The plasmid was composed of C-leucine (to a final concentration of 30 μM, specific activity 46.15 dpm / pmol (PerkinElmer LAS (Germany) GmbH, Rodgau, Germany)). Protein synthesis was initiated by the addition of DNA template (60 ng / μL). Reactions were incubated for 3 h at 30 °C and 600 rpm in a standard thermomixer (Eppendorf Thermomixer Comfort). Background translation activity was monitored by running the translation reaction without supplementing the plasmid.

[0176] The coding sequence for the anti-GOLPH3 antibody was codon-optimized for Cricetulus griseus and equipped with the necessary regulatory sequences to enable in vitro transcription and translation according to Stech et al. (2017). Sci. Rep. 7(1), 12030. The essential elements were as follows: 5' untranslated region (UTR): T7 promoter sequence, multiple cloning site (MCS), internal ribosome entry site (IRES) from the intergenic region (IGR) of cricket paralysis virus (CrPV), GCT as the start codon; 3' UTR: T7 terminator sequence, MCS (ibid.). The DNA template was de novo synthesized (Agilent, San Jose, CA) and cloned into an appropriate vector (pUC57-1.8k) by Biocat GmbH (Biocat GmbH, Heidelberg). Plasmid preparations for cell-free protein synthesis are prepared using the PureLink® HiPure Plasmid Midiprep Kit (Thermo Fisher Scientific, Waltham, Mass.) according to the manufacturer's instructions, and then digested and sequenced to verify the correct DNA sequence using an Illumina Miseq™ system (Illumina, San Diego, Calif.).

[0177] The coupled transcription-translation reaction is then carried out as described in Thoring, supra. To allow for subsequent quantitative and qualitative analysis of cell-free synthesized proteins, the translation reaction consisted of 40% (v / v) S7 nuclease-treated CHO lysate containing ER-derived endogenous microsomal vesicles, HEPES-KOH (pH 7.6, 30 mM, BioMol GmbH, Hamburg, Germany), complete amino acids (100 μM), Mg(OAc) (3.9 mM), KOAc (135 mM, Merck, Darmstadt, Germany), spermidine (0.25 mM, Sigma-Aldrich, St. Louis, United States), energy components (1.75 mM ATP, 0.3 mM GTP, 0.3 mM CTP, 0.3 mM UTP), creatine phosphate (20 mM), T7 polymerase (1 U / μl) (Agilent Technologies, Santa Clara, United States), and ATP. 14 The plasmid was composed of C-leucine (to a final concentration of 30 μM, specific activity 46.15 dpm / pmol (PerkinElmer LAS (Germany) GmbH, Rodgau, Germany)). Protein synthesis was initiated by the addition of DNA template (60 ng / μL). Reactions were incubated for 3 h at 30 °C and 600 rpm in a standard thermomixer (Eppendorf Thermomixer Comfort). Background translation activity was monitored by running the translation reaction without supplementing the plasmid.

[0178] After the translation reaction, the sample is centrifuged at 16,000 × g for 10 minutes at 4 °C to separate the microsomes from the soluble fraction of the translation mixture. The resulting supernatant (first supernatant, or SUP1) is transferred to a fresh reaction tube and stored on ice until further analysis. Meanwhile, the microsomal pellet is resuspended in 1 × PBS containing 0.2% n-dodecyl-β-D-maltoside (DDM) to allow for the release of translocated microsome-containing antibodies. The microsomes are manually resuspended by repeatedly pipetting up and down, followed by vortexing and rocking on an oscillator for approximately 45 minutes. A second centrifugation step is performed to separate the released proteins from microsomal membrane residues. The resulting supernatant (second supernatant, or SUP2) is transferred to a fresh reaction tube and stored on ice until further analysis. 14 C-leucine supplemented reactions are analyzed by SDS-PAGE followed by autoradiography and liquid scintillation counting, while non-radioactive samples are subjected to functional analysis by enzyme-linked immunosorbent assay (ELISA).

[0179] The present invention is not intended to be limited in scope to the specific disclosed embodiments, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the described compositions and methods will become apparent from the descriptions and teachings herein. Such variations can be made without departing from the true scope and spirit of the present disclosure and are intended to be included within the scope of the present disclosure. [Table 5-1] [Table 5-2]

Claims

1. A method for recombinant production of antibodies, a) To provide a cell-free protein expression system containing a target protein exhibiting an antibody epitope, b) Modifying the cell-free system by introducing one or more agents that block the antibody epitope on the target protein but do not eliminate the activity of the target protein in the cell-free system, c) Introducing one or more nucleic acids encoding antibodies that bind to the antibody epitope into the cell-free system, d) A method comprising initiating the transcription and translation of the antibody in the cell-free system under conditions that result in the production of the antibody.

2. The method according to claim 1, wherein the cell-free system includes a cell lysate.

3. The method according to claim 2, wherein the drug is introduced into the cells before the preparation of the cell lysate.

4. The method according to claim 2, wherein the drug is introduced into the cells after the preparation of the cell lysate.

5. The method according to claim 4, wherein the cell lysate is selected from the group consisting of wheat germ lysate, insect cell lysate, reticulocyte lysate, keratinocyte lysate, CHO cells, HeLa cells, myeloma cells, hybridoma cells, and cell extracts from cultured lymphoma cells.

6. e) The method according to claim 1, further comprising isolating the antibody from the cell-free system.

7. The method according to claim 1, wherein the agent that blocks the antibody epitope is a peptide.

8. The method according to claim 1, wherein the agent that blocks the antibody is a small molecule.

9. The method according to claim 1, wherein the target protein is an intracellular protein in the secretory pathway.

10. The method according to claim 1, wherein the target protein is a stress protein.

11. The method according to claim 1, wherein the target protein is a cell signaling protein.

12. The method according to claim 1, wherein the drug does not affect the activity of the target protein compared to the target protein activity in the absence of the drug.

13. The method according to claim 1, wherein the target protein activity is reduced by about 10% compared to the target protein activity in the cell-free system in the absence of the drug.

14. The method according to claim 1, wherein the target protein activity is reduced by about 50% compared to the target protein activity in the cell-free system in the absence of the drug.

15. A method for recombinant production of antibodies, a) To provide a cell-free protein expression system containing a target protein exhibiting an antibody epitope, b) Modifying the cell-free system by introducing one or more agents that block antibody paratopes that bind to the antibody epitope on the target protein, c) Introducing one or more nucleic acids encoding an antibody containing the antibody paratope into the cell-free system, d) A method comprising initiating the transcription and translation of the antibody in the cell-free system under conditions that result in the production of the antibody.

16. e) The method according to claim 1, further comprising isolating the antibody from the cell-free system.

17. The method according to claim 15, further comprising removing the agent bound to the antibody paratope after the production of the antibody.

18. The method according to claim 15, further comprising isolating the antibody from the cell-free system.

19. The method according to claim 17, wherein the antibody is isolated before the removal of the drug from the antibody.

20. The method according to claim 15, wherein the cell-free system includes a cell lysate.

21. The method according to claim 20, wherein the cell lysate is selected from the group consisting of wheat germ lysate, insect cell lysate, reticulocyte lysate, keratinocyte lysate, CHO cells, HeLa cells, myeloma cells, hybridoma cells, and cell extracts from cultured lymphoma cells.

22. The method according to claim 15, wherein the drug is introduced into the cell-free system before the transcription and translation of the antibody begins.

23. The method according to claim 15, wherein the agent that blocks the antibody paratope is a peptide.

24. The method according to claim 1, wherein the target protein is an intracellular protein in the secretory pathway.

25. The method according to claim 1, wherein the target protein is a stress protein.

26. The method according to claim 1, wherein the target protein is a cell signaling protein.

27. A method for recombinant production of monoclonal antibodies, wherein the method is a) To provide a mammalian cell-free protein expression system containing a target protein exhibiting an antibody epitope, b) Modifying a mammalian cell-free system by introducing one or more agents that block the antibody epitope on the target protein but do not eliminate the activity of the target protein in the cell-free system, c) Introducing one or more nucleic acids encoding monoclonal antibodies that bind to the antibody epitope into the cell-free system, d) A method comprising initiating the transcription and translation of the monoclonal antibody in the mammalian cell-free system under conditions that enable the production of the antibody.

28. The method according to claim 27, wherein the mammalian cell-free system includes a cell lysate.

29. e) The method according to claim 27, further comprising isolating the antibody from a mammalian cell-free system.

30. A method for recombinant production of monoclonal antibodies, a) To provide a mammalian cell-free protein expression system containing a target protein exhibiting an antibody epitope, b) Modifying a mammalian cell-free system by introducing one or more agents that block antibody paratopes that bind to the antibody epitope on the target protein, c) Introducing one or more nucleic acids encoding a monoclonal antibody containing the antibody paratope into the cell-free system, d) A method comprising initiating the transcription and translation of the monoclonal antibody in the mammalian cell-free system under conditions that enable the production of the antibody.

31. The method according to claim 30, further comprising removing the agent bound to the antibody paratope after the production of the monoclonal antibody.

32. The method according to claim 30, further comprising isolating the monoclonal antibody from the cell-free system.

33. The method according to claim 31, wherein the monoclonal antibody is isolated before the removal of the drug from the monoclonal antibody.

34. A method for recombinant production of antibodies, wherein the method is a) Creating modified cell lines by introducing drugs that selectively bind to target epitopes on intracellular proteins into cells, b) To produce a cell-free antibody production system from the modified cell line, c) Introducing a nucleic acid template into the cell-free antibody production system, wherein the nucleic acid template encodes an antibody that selectively binds to the target epitope on the intracellular protein. d) A method comprising initiating transcription and translation from the nucleic acid template to produce the antibody in the cell-free antibody production system.

35. e) The method according to claim 34, further comprising isolating the antibody from a cell-free system.

36. The method according to claim 34, wherein the target protein is an intracellular protein in the secretory pathway of the cell line.

37. The method according to claim 34, wherein the target protein is a stress protein.

38. The method according to claim 34, wherein the target protein is a cell signaling protein.

39. The method according to claim 34, wherein the target protein is expressed on an organelle of the cell line.

40. The method according to claim 39, wherein the organelle is the endoplasmic reticulum or the Golgi apparatus.

41. The method according to claim 40, wherein the target protein is an endoplasmic reticulum chaperone.

42. The method according to claim 41, wherein the endoplasmic reticulum chaperone is calreticulin, a heat shock protein, or an isomerase.

43. The method according to claim 41, wherein the target protein is glucose regulatory protein 78 (GRP78), HSP47, PDI, calreticulin, or GP94.

44. The method according to claim 41, wherein the target protein is glucose regulatory protein 78 (GRP78).

45. The method according to claim 39, wherein the target protein is a Golgi complex protein.

46. The method according to claim 45, wherein the Golgi complex protein is GOLPH2, GOLPH3, GM130, ATP6V1A, ATP6V1E1, ATP6VOA2, TMEM165, GOLGB1, SCYL1BP1, TRAPPC11, TRAPPC2, or TRIP11.

47. The method according to claim 34, wherein the cell line is a mammalian cell line.

48. The method according to claim 47, wherein the naturally expressed target protein is an intracellular protein in the secretory pathway of the mammalian cell line.

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