Cell-free method for producing antibodies against intracellular targets

Abstract

Provided herein are methods and cell-free systems for producing recombinant antibodies, wherein the antibodies are directed against target proteins naturally expressed as intracellular proteins. Also provided herein are modified cell-free systems for use in such methods, wherein a cell-free protein expression system is modified by an agent that binds to an intracellular target protein or antibody complimentary and thus prevents binding of an antibody to a target protein.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 398,143 filed on August 15, 2022, which is hereby incorporated in its entirety into this application.

[0003] Incorporation of Sequence Listing

[0004] A computer readable form of the sequence listing is submitted electronically with this application and is hereby incorporated by reference in its entirety into this application. The sequence listing is contained in a file created on May 2, 2023, named MBRACE004.xml and is 23,837 bytes in size (as viewed in MS- The file is measured in the operating system. Technical Field

[0005] The present disclosure relates to methods and cell-free systems for producing recombinant antibodies directed against a target protein that is normally naturally expressed as an intracellular protein. Background Art

[0006] Engineered antibody molecules and fragments thereof are increasingly being used as scientific and clinical tools for the treatment and diagnosis of diseases. The unique ability of antibodies to specifically recognize almost any type of antigen and bind to it with high affinity makes it attractive as a starting point for novel biopharmaceutical products and scientific research. Recently, because some stress proteins that are usually present as intracellular proteins are usually not expressed on the surface of normal cells or are expressed at low levels on the surface of normal cells, such proteins have been identified as unique targets for antibody-based therapies for cancer and other pathological conditions. Improved methods for producing such antibodies are needed, including antibodies and fragments thereof that specifically recognize and bind to intracellular targets in mammalian cells. Summary of the invention

[0007] The foregoing general description of the illustrative embodiments and the following detailed description thereof are merely exemplary aspects of the teachings of the present disclosure and are not restrictive.

[0008] In selected embodiments, the present disclosure provides methods and cell-free systems for producing antibodies that bind to intracellular target proteins. The present disclosure also provides methods for producing cell-free systems for producing antibodies that bind to intracellular target proteins.

[0009] The present disclosure provides a method for producing an antibody using a cell-free production system and an agent that interferes with the binding between an antibody to be produced using the system and a protein in the cell-free system that is a target protein of the antibody. The binding of the antibody to a target epitope on the target protein in the cell-free system can be disrupted by: 1) binding of the agent to the target epitope on the target protein, thereby preventing the binding of the antibody paratope to the epitope, 2) binding of the agent to the antibody paratope, thereby blocking the binding of the antibody paratope to the target epitope, or 3) a combination thereof.

[0010] In some embodiments, the present disclosure provides a method for recombinantly producing an antibody, the method comprising 1) providing a cell-free protein expression system containing a target protein exhibiting 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 one or more nucleic acids encoding an antibody that binds to the antibody epitope into the cell-free system; and 4) initiating transcription and translation of the antibody in the cell-free system under conditions that allow production of the antibody.

[0011] In some embodiments, the present disclosure provides a method for recombinantly producing an antibody, the method comprising 1) providing a cell-free protein expression system containing a target protein exhibiting an antibody epitope; 2) modifying the cell-free system by introducing one or more agents that block the antibody paratope binding to the antibody epitope on the target protein; 3) introducing one or more nucleic acids encoding an antibody comprising an antibody paratope into the cell-free system; and 4) initiating transcription and translation of the antibody in the cell-free system under conditions that allow the production of the antibody. In a preferred embodiment, the method further provides 5) removing the agent that binds to the antibody paratope after the antibody is produced.

[0012] In some embodiments, the cell-free system comprises a cell lysate. In some embodiments, the cell-free system is a eukaryotic cell lysate.

[0013] In certain embodiments, the cell lysate is wheat germ lysate, insect cell lysate, reticulocyte lysate, keratinocyte lysate. In certain embodiments, the cell lysate is from mammalian cells, such as from CHO cells, HeLa cells, HEK293 cells, myeloma cells, hybridoma cells or cultured lymphoma cells.

[0014] Thus, in some embodiments, the present disclosure provides a method for recombinantly producing a monoclonal antibody, the method comprising 1) providing a mammalian cell-free protein expression system containing a target protein exhibiting 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 one or more nucleic acids encoding a monoclonal antibody that binds to the antibody epitope into the cell-free system; and 4) initiating transcription and translation of the monoclonal antibody in the mammalian cell-free system under conditions that allow production of the antibody.

[0015] In some embodiments, the cell-free system comprises a cell lysate. In some embodiments, the cell-free system is a eukaryotic cell lysate.

[0016] The present disclosure also provides a method for recombinant production of monoclonal antibodies, the method comprising 1) providing a mammalian cell-free protein expression system containing a target protein exhibiting an antibody epitope; 2) modifying the mammalian cell-free system by introducing one or more agents that block the antibody paratope binding to the antibody epitope on the target protein; 3) introducing one or more nucleic acids encoding a monoclonal antibody comprising an antibody paratope into the cell-free system; and, 4) initiating transcription and translation of the monoclonal antibody under conditions that allow the production of the antibody in the mammalian cell-free system. In a preferred embodiment, the method further provides 5) removing the agent bound to the antibody paratope after the production of the monoclonal antibody.

[0017] The agent that binds to the target epitope can be introduced at any time before 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 before the cell lysate is produced, or alternatively, the agent can be introduced into the cell lysate after the lysate has been prepared but before the transcription and translation of the antibody in the system is induced.

[0018] The agent that blocks the antibody epitope can be any agent that allows the target protein to retain sufficient function in the 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 activity of the target protein in the absence of the agent. In some embodiments, the protein activity is reduced, but it is sufficient to support effective antibody production in the 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.

[0019] The present disclosure also provides a method for recombinantly producing an antibody, the method comprising: 1) introducing an agent that selectively binds to a target epitope on an intracellular protein into a cell to produce a modified cell line; 2) generating a cell-free antibody production system from the modified cell line; 3) introducing a nucleic acid template into the cell-free antibody production system, wherein the nucleic acid template encodes an antibody that selectively binds to a target epitope on an intracellular protein; and 4) initiating transcription and translation from the nucleic acid template to produce the antibody in the cell-free antibody production system.

[0020] In some embodiments, the target protein is an intracellular protein in a secretory pathway. In some embodiments, the target protein is a stress protein. In some embodiments, the target protein is a signaling protein.

[0021] In some embodiments, the target protein is expressed on an organelle in the cell line.

[0022] In a specific embodiment, the target protein is a protein that is usually expressed on the endoplasmic reticulum. For example, the target protein can be an endoplasmic reticulum chaperone, such as calreticulin, heat shock protein or isomerase. Specifically, the target protein can be glucose-regulated protein 78 (GRP78), HSP47, protein disulfide isomerase (PDI), calreticulin or GP94.

[0023] In a specific embodiment, the target protein is usually 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), Golgi 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 interacting factor 11 (TRIP11).

[0024] In some embodiments, the target protein is associated with a membrane within a cell.

[0025] In some embodiments, the target protein is an intracellular signaling protein, such as a protein involved in a signal transduction pathway, for example, a kinase or a phosphatase.

[0026] In some embodiments, antibodies are isolated from the cell-free system following production.

[0027] Additional features, advantages and aspects are described in greater detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings, which are incorporated into and constitute a part of this specification, show one or more embodiments and, together with the description, explain these embodiments. The drawings are not necessarily drawn to scale. Any values ​​or dimensions shown in the accompanying tables 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 help describe essential features.

[0029] Figure 1 Depicted are SDS-PAGE analyses of purified supernatant samples following expression of anti-GRP78 antibody clones B4, D1, and F6 in CHO cells under reducing and non-reducing conditions.

[0030] Figure 2A and Figure 2B Depicted are flow cytometry results for binding of antibody clones B4 and F6, respectively, to GRP78 variants from an alanine scanning library. For each GRP78 variant, the mean fluorescence percentage for WT GRP78 binding plotted relative to control antibodies 1H11-1H7 is shown.

[0031] Figure 3 Depicted is a visualization of the major residues for binding of anti-GRP78 clones B4 and F6 to the GRP78 antibody, with exemplary major residues and other residues involved in binding indicated by arrows. DETAILED DESCRIPTION

[0032] The following description in conjunction with the accompanying drawings is intended to describe various illustrative embodiments of the disclosed subject matter. Specific features and functions are described in conjunction with each illustrative embodiment; however, it will be apparent to those skilled in the art that the disclosed embodiments can be practiced without each of these specific features and functions.

[0033] Throughout this specification, references to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment of the disclosed subject matter. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in various places throughout this specification do not necessarily all refer to the same embodiment. Further, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Further, it is intended that the various embodiments of the disclosed subject matter cover modifications and variations thereof.

[0034] It must be noted that, as used in the specification and the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly indicates otherwise. That is, unless otherwise indicated, the words "a / an" and "the" and the like used herein have the meaning of "one or more". In addition, it should be understood that terms such as "left", "right", "top", "bottom", "front", "back", "side", "height", "length", "width", "up", "down", "inside", "outside", "inside", "outside" and the like that may be used herein only describe reference points and do not necessarily limit the embodiments of the present disclosure to any particular orientation or configuration. In addition, terms such as "first", "second", "third" and the like only identify one of the many parts, components, steps, operations, functions and / or reference points as disclosed herein, and likewise do not necessarily limit the embodiments of the present disclosure to any particular configuration or orientation.

[0035] In addition, the terms “approximately,” “about,” “close,” “slightly varying,” and similar terms generally refer to a range including a certain value within a margin of 20%, 10%, or, in certain embodiments, preferably 5%, and any values ​​therebetween.

[0036] All features described in conjunction with one embodiment are intended to be applicable to another embodiment described below, unless expressly stated otherwise or the feature or feature is incompatible with the other embodiment. For example, where a given feature or feature is expressly described in conjunction with one embodiment, but is not expressly mentioned in conjunction with an alternative embodiment, it is understood that the inventors intend that the feature or feature may be deployed, utilized, or implemented in conjunction with the alternative embodiment, unless the feature or feature is incompatible with the alternative embodiment.

[0037] Provided herein is a method for recombinantly producing an antibody or its binding fragment, wherein the target protein of the antibody is also generally found in the cell as an intracellular protein. In certain embodiments, the target intracellular protein to which the antibody is bound is a protein related to a secretory pathway, such as a protein expressed on or in the endoplasmic reticulum (ER) or the Golgi apparatus. In certain embodiments, the target protein of the antibody is a stress protein, which is a protein that is generally used for ER or the Golgi apparatus or other intracellular organelles but may be regulated on the surface of the cell under cell stress conditions. For example, a stress protein (such as a protein related to an unfolded protein response (UFR)) is found to be regulated on the surface of cancer cells but is not generally found on the surface of normal cells. Since such proteins are generally not expressed or expressed at low levels on the surface of normal cells, and can be upregulated or overexpressed at the surface of cancer cells, stress proteins exhibit the desired characteristics of target proteins used as antibody-based cancer therapy. Using stress proteins as target proteins for antibody-based therapy ensures that their surface expression in various cancers is upregulated, but will not be expressed in normal tissues and cells, and reduces off-target activity and / or toxicity or minimizes it.

[0038] Methods for producing antibodies in sufficient quantities for use as therapeutic agents are needed. Several expression systems are available, both from prokaryotic and eukaryotic sources. The choice of system depends on many factors, including the molecular species being expressed and the precise sequence of the individual antibodies.

[0039] Recombinant production of human or humanized antibodies is particularly desirable in mammalian expression systems. For example, mammalian cells are able to perform the same desired protein folding and post-translational modifications as human systems. By expressing recombinant proteins in mammalian systems (such as those derived from HEK293 cells or CHO cells), glycosylation patterns similar but not identical to those obtained from human cells can be achieved. Mammalian cell-free systems allow very high product yields and are relatively robust to metabolic stress.

[0040] Although efforts have been made to produce antibodies against stress proteins, it is not yet possible to effectively generate the antibodies in mammalian cells. For example, observations herein show 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 wishing to be bound by theory, in some cases, it is believed that stress proteins are necessary for the cell viability or function of mammalian cells, so that during the recombinant antibody production process, the binding of antibodies to intracellular proteins is harmful to cells. For example, in some embodiments, the binding of certain intracellular proteins inhibits the ability of intracellular proteins to perform their normal functions in cells, thereby causing protein misfolding in cells, lack of degradation of misfolded proteins, calcium homeostasis disorders, and in some cases cell viability reduction or loss. The loss or reduction of this function affects the ability to produce antibodies in high yield.

[0041] Studies have shown that stress proteins (such as chaperones) are necessary for normal function in cells during antibody production. For example, changes in the cell abundance of secretory pathway proteins have been shown to be associated with the ability to mass produce recombinant monoclonal antibodies (see, for example, Lambert and Merten, 1997, Biotechnol. Bioeng., 54: 165-180; Downham et al. 1996, Biotechnol. Bioeng., 51: 691-696). It is well known that secretory proteins will fold and assemble into higher-order complexes in the endoplasmic reticulum (ER) compartment of the cell shortly after protein synthesis. ER consists of specific auxiliary assembly factors and quality control mechanisms (Ellgaard et al., Quality control in the secretory pathway, Science. December 3, 1999; 286: 1882-8; Helenius et al., Intracellular functions of N-linked glycans, Science. March 23, 2001; 291: 2364-9). It is well known that the folding of proteins and the refolding of misfolded soluble and aggregated proteins are 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 and Furtak, K., Cell 74, 909-917 (1993); Ellis, RJ and Hemmingsen, SM, TiBS, 14, 339-342, (1989); Bukau, B., Hesterkamp, ​​T. and Luirink, J., Trends Cell Biol, 6, 480-486, (1996); Bukau, B., Deuerling, E., Pfund, C. and Craig, EA, Cell, 101, 119-122, (2000)). Secretory proteins also undergo various post-translational modifications, including glycosylation, during their passage through the Golgi complex.

[0042] Alternatively, even if antibody binding to intracellular proteins does not affect cell viability, it may affect antibody production due to sequestration of the product by intracellular proteins. In short, intracellular proteins can serve as a "sink" for antibody or antigen-binding fragment binding, thereby preventing secretion and thus limiting the yield of the desired production.

[0043] The provided methods relate to cell-free systems for producing antibodies or antigen-binding fragments that target endogenous intracellular target proteins in cells, such as target proteins in the secretory pathway.

[0044] The production systems and methods provided are exemplified as antibodies for targeting GRP78 and other intracellular target proteins. The systems and methods described herein can be extended to produce all or part of antibody-based biologics that selectively target proteins that are usually found in cells but can be found on the cell surface under certain pathologies. Therefore, it should be understood by those of ordinary skill in the art that the present disclosure is exemplary and can be applied to other cell systems and preparation methods for producing biologics targeting such other targets.

[0045] All publications (including patent documents, scientific papers, and databases) mentioned 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. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in patents, applications, published applications, and other publications incorporated herein by reference, the definition set forth herein takes precedence over the definition incorporated herein by reference.

[0046] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0047] Cell production methods for cell-free systems

[0048] Provided herein is a method for recombinantly producing an antibody that binds to a target protein (or a homolog thereof) that is typically naturally expressed as an intracellular protein. In the provided cell-free systems and methods, the antibody is produced in a modified system (e.g., a mammalian cell-free system) that is modified to contain an agent that selectively binds to an epitope on the target protein to which the antibody binds.

[0049] These methods and systems disclosed herein can be used alone or in combination with all or specific aspects of other antibody preparation systems. In some preferred embodiments, the modified cell line can be produced using the method disclosed in U.S. Serial No. 63 / 337,980 filed on May 3, 2022 and / or U.S. Serial No. 63 / 359,541 filed on July 8, 2022, each of which is incorporated in its entirety into this application for all purposes. For example, in some preferred embodiments, the methods of these applications may include: a) expressing a nucleic acid encoding an antibody in a modified cell line (a mammalian cell line in some preferred embodiments), wherein the antibody specifically binds to a target epitope on a target protein, and the target protein is naturally expressed as an intracellular protein in a cell; and wherein the modified cell is engineered to express a variant target protein, the variant target protein comprising a mutation of one or more amino acid residues of a target epitope; and, b) culturing a modified cell line (and, in some preferred embodiments, separating antibodies) under conditions that allow production of antibodies. In some preferred embodiments, the methods of these applications may include: a) providing an antibody that binds to an epitope on a target protein present in a cell line (in some preferred embodiments, a mammalian cell line) within a cell; b) identifying the epitope of the target protein to which the antibody binds; c) producing a modified cell line by mutating the epitope of the target protein in the cell line to reduce or eliminate the binding of the antibody to the epitope on the target protein; d) introducing an expression vector encoding the antibody into the modified cell line; e) culturing the modified cell line under conditions that allow the production of the antibody from the expression vector; and, f) expressing the nucleic acid encoding the antibody in the modified cell line (and, in some preferred embodiments, isolating the antibody). In some preferred embodiments, the modified mammalian cell line of these applications comprises a variant protein that is an intracellular protein in the secretory pathway, wherein the variant protein comprises a mutation of a natural intracellular protein that is a target protein of the antibody, and wherein the mutation is an amino acid substitution of one or more amino acid residues to change the target epitope of the antibody to a mutant epitope. In some preferred embodiments, the method comprises recombinantly producing an antibody targeting GRP78 by: a) expressing a nucleic acid encoding an antibody in a modified mammalian cell line, wherein the antibody binds to GRP78, and wherein the modified mammalian cell is engineered with a variant GRP78 having a mutant epitope comprising a mutation in one or more amino acid residues of the target epitope of the antibody or antigen-binding fragment; and, b) culturing the modified mammalian cell line under conditions such that the anti-GRP78 antibody is produced in the culture supernatant.

[0050] A. Intracellular protein targets and antibodies against them

[0051] In some embodiments, the systems and methods provided can be used to express antibodies, wherein the target protein of the antibody is naturally expressed as an intracellular protein in the cell. In a specific embodiment, the target protein is abnormally expressed on the surface of cells of cancer and other diseases, but due to the role of the target protein in the biological process in normal cells, it is also a protein necessary for the function of normal cells. In some embodiments, the target protein is a protein related to the secretory pathway of the cell. In some embodiments, the target protein is expressed on the organelles of the cell (such as in a mammalian cell line). In some embodiments, the target protein is expressed on the endoplasmic reticulum or therein. In some embodiments, the target protein is expressed on the Golgi apparatus or therein.

[0052] In some embodiments, the intracellular protein target is a stress protein. Stress proteins include proteins involved in organelle self-regulation to maintain homeostasis to regulate the ability of organelles under stress conditions (such as may occur in a tumor environment). Various stress proteins are known and include proteins in the secretory pathway. Various stress proteins are known, including those produced by 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.

[0053] For example, targets include endoplasmic reticulum chaperones (e.g., calreticulin, heat shock proteins, and isomerases) that can translocate to the cytosol and ultimately to the surface of the cell, especially during stress conditions. These proteins have been found to be overexpressed under 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 embodiments of the present disclosure and the applications taught therein allow for the production of biologics targeting endoplasmic reticulum chaperones as well as other identified targets without excessively compromising cell viability. Exemplary biologics may include, for example, biologics 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 Set 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 Mar; 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).

[0054] A variety of Golgi complex proteins are known to be involved in various pathological states, especially cancer and autoimmune diseases, and monoclonal antibodies that selectively bind to these organelle proteins are suitable for production of monoclonal antibodies using the methods as 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) (supra); 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); Golgi complex 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., Skelet 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 interacting factor 11 (TRIP11) (Smits P, N Engl J Med 362:206-216, 2010).

[0055] In some embodiments, the target protein 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 protein is GP94. In some embodiments, the target protein is GOLPH2. In some embodiments, the target protein is GOLPH3. In some embodiments, the target protein is GM130. In some embodiments, the target protein is ATP6V1A. In some embodiments, the target protein is AATPP6V1E1. In some embodiments, the target protein is ATP6VOA2. In some embodiments, the target protein is transmembrane protein 165 (TMEM165). In some embodiments, the target protein is GOLGB1. In some embodiments, the target protein is SCYL1BP1. In some embodiments, the target protein is TRAPPC11. In some embodiments, the target protein is thyroid hormone receptor interactor 11 (TRIP11).

[0056] Those skilled in the art are familiar with antibodies against intracellular protein targets such as stress proteins. For example, antibodies against GRP78 antibodies include: GRP78-specific mouse monoclonal IgG antibody MAb159 (Ojha and Amaravadi, Pharmacol. Res., 120: 258-266, 2017), PAT-SM6 (Ojha and Amaravadi, 2017); and anti-GRP78 antibodies described in: PCT Publication No. WO2018 / 057703, PCT Publication No. WO2014 / 153056; PCT Publication No. WO2008 / 105560; U.S. Publication No. US2010 / 0041074; U.S. Patent No. 10,259,884; and U.S. Patent No. 10 / 851,161. Antibodies to 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 to calreticulin include, for example, mAb FMC 75 (Enzo Life Sciences, catalog number ADI-SPA-601) and mAb 16 (BD Transduction laboratories, catalog number 612137). Antibodies to GOLPH3 include, for example, Thermo Fisher monoclonal antibody clone 905CT9.1.1 (Thermo Fisher Scientific catalog number MA5-37626). Each of these antibodies can be used as a template for humanized antibodies using methods such as those disclosed in more detail herein.

[0057] In certain embodiments, the antibody is a human antibody. Human antibodies can be prepared by administering an immunogen to a transgenic animal, which has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic stimulation. Such animals generally contain all or part of a human immunoglobulin locus, which replaces an endogenous immunoglobulin locus or exists outside a chromosome or is randomly integrated into the chromosome of the animal. In such transgenic animals, the endogenous immunoglobulin locus is generally inactivated. Human antibodies can also be derived from a human antibody library, including phage display and a cell-free library containing sequences encoding antibodies derived from a human library.

[0058] In addition to using non-human monoclonal antibodies for humanization purposes, non-human antibodies can also be used as templates for selecting full-human or near-full-human antibodies. A specific method for selecting such human antibodies is to use phage display technology. This method can use antibody screening technology, such as in Dower et al., WO 91 / 17271 and McCafferty et al., WO 92 / 01047, U.S. Patent Nos. 5,877,218, 5,871,907, 5,858,657, 5,837,242, 5,733,743 and 5,565,332. In these methods, a phage library is produced, wherein different antibodies are provided on the outer surface of the phage. Antibodies are usually displayed on phages in the form of Fv or Fab fragments. The antibody with the desired specificity is selected by affinity with the selected epitope of the organelle protein.

[0059] In a specific exemplary method, using the technology of Winter, WO 92 / 20791, human antibodies that selectively bind to the epitope of organelle proteins can be produced. In this method, the heavy chain variable region or light chain variable region of non-human monoclonal antibodies are used. If the light chain variable region is selected as the starting material, a phage library is constructed, wherein the members display the light chain variable region and different heavy chain variable regions of non-human monoclonal antibodies. Obtain the heavy chain variable region from the library of the rearranged human heavy chain variable region. Select the phage that shows strong specific binding to the epitope of organelle proteins. The human heavy chain variable region from this phage provides the basis for constructing an optimized phage library, wherein each phage shows the same heavy chain variable region and different light chain variable regions identified from the first display library. Obtain the light chain variable region from the library of the rearranged human variable light chain region. Select the phage that shows the variable region of the fully human antibody that shows strong specific binding to the epitope on the organelle protein.

[0060] The antibodies provided are monoclonal antibodies, including monoclonal antibody fragments.

[0061] B. Identification of binding epitopes on target proteins

[0062] In some embodiments, the residues in the target protein that are important for antibody binding can be mapped to define or identify the epitope or binding domain of the antibody, thereby designing and / or developing the medicament that blocks the epitope in the methods and systems disclosed herein. In some embodiments, the cDNA encoding the target protein is randomly mutated by a method (such as by treating with a chemical mutagen, irradiating during replication, by passage of a wrongly induced (mutated gene) cell line, or by oligonucleotide directed random mutagenesis). In some embodiments, oligonucleotide directed random mutagenesis is used, wherein the preselected region of the targeted protein is used for random mutagenesis. In some embodiments, the preselected region for random mutagenesis is considered to contain a binding site or epitope. The preliminary assessment of the preselected region can be achieved by any method known in the art (including hydration analysis of protein sequences or by crystal structure analysis).

[0063] In certain embodiments, the epi-position of antibody can be mapped using peptide-based methods. In one method, peptide display technology displays a library of protein fragments on the surface of a microarray or Escherichia coli or phage, and finds out which fragments the antibody is combined with by microarray scanning or flow cytometry. In another method, a series of overlapping peptides in a target protein or its preselected region are screened to carry out antibody binding, such as monitoring binding interactions by ELISA or other techniques. These technologies have additional benefits, namely they can also identify peptides that can potentially be used as a medicament for combination or as the basis for developing this medicament in the method and system of the present invention.

[0064] In some embodiments, epitopes can be predicted by calculation. 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, the predicted epitopes are verified experimentally. Methods for experimentally testing antibody epitopes are known in the art, including, for example, using antibody cross-blocking assays, by performing assays (including screening libraries), by performing mutation analysis, by deuterium exchange analysis, by peptide binding assays and / or by X-ray crystallography studies to perform verification. In some embodiments, verification uses libraries and / or peptides derived from target proteins to assess the importance of specific amino acids in specific positions for binding. In some embodiments, the library comprises a subset or all of the mutant libraries 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.

[0065] In some embodiments, in order to identify the residues in the epitope on the target intracellular protein to be blocked during the production of the antibody, it is necessary to start with a solved crystal structure of the intracellular protein so that the key contact residues between the antibody and the target protein can be identified. This solved structure can be a solved crystal structure of the intracellular protein alone, or a solved crystal structure of the intracellular protein combined with an antibody or antibody fragment.

[0066] However, in many cases, a good molecular model can provide the necessary information. In the case where the molecular model is insufficient, for example, if there is no appropriate structural template in the structural database, an amino acid exchange experiment can provide information on which residues can be targeted to mutate. By carrying out alanine scanning mutagenesis (mutating each residue into Ala in turn) in these regions, key residues involved in antigen binding can be identified (Cunningham BC and Wells JA, Proc Natl Acad Sci US A. April 15, 1991; 88 (8): 3407–3411). If a single residue is changed to Ala to reduce binding but not destroy binding, the position can be used for targets blocked with agents.

[0067] Comparative model building provides a wide range of structural templates, and available computer programs ensure that the models are increasingly accurate. For example, the solved crystal structures of various proteins have been shown to be very close to the structures predicted by molecular modeling.

[0068] As shown herein for GRP78, once the structure of the target protein has been elucidated, one of ordinary skill in the art can identify residues that can be bound to reduce antibody binding to an epitope of an organelle protein while retaining sufficient function of the organelle protein, and thus can block the epitope while retaining protein function, as taught in more detail herein. Methods for preparing blocking polypeptides and / or other agents are well known to one of ordinary skill in the art.

[0069] In some embodiments, the pharmaceutical agent used is introduced into the cells and does not significantly affect the viability of the cell line used to produce the cell-free system. In some embodiments, the viability of the modified cell line containing the pharmaceutical agent is retained under standard culture and passage conditions compared to the cell line without the pharmaceutical agent.

[0070] In some embodiments, binding of the agent to a target epitope on the target protein blocks binding of that epitope to the antibody to be produced, but has no measurable effect on the function of the target protein in the cell-free system.

[0071] In some embodiments, binding of the agent to the target epitope reduces the activity of the target protein 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 a molecular chaperone protein 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 an intracellular kinase can be assessed using methods such as Haubrich et al. * and Swinney Curr Drug Discov Technol. 2016;13(1):2-15.

[0072] In some embodiments, binding of the agent to the target paratope on the antibody blocks binding of the paratope to the target epitope on the target protein, but has no measurable effect on the function of the target protein in the cell-free system.

[0073] In some embodiments, the binding of the antibody produced after modification in the cell-free system to the target protein is reduced by about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 5.5 times, at least about 6 times, at least about 6.5 times, at least about 7 times, at least about 7.5 times, at least about 8 times, at least about 8.5 times, at least about 9 times, at least about 9.5 times, at least about 10 times, at least about 100 times, or at least about 1000 times compared to the binding of the antibody to the target protein produced in the cell-free system in the absence of the agent.

[0074] In some embodiments, the agent reduces the affinity of the antibody for the target protein. In some embodiments, 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 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 5.5 times, at least about 6 times, at least about 6.5 times, at least about 7 times, at least about 7.5 times, at least about 8 times, at least about 8.5 times, at least about 9 times, at least about 9.5 times, at least about 10 times, at least about 100 times, or at least about 1000 times relative to the affinity of the antibody for the target protein in the absence of the agent. In some embodiments, in the presence of the agent, the affinity of the antibody for the target protein is eliminated (e.g., the binding of the antibody to the target protein is undetectable).

[0075] Methods for measuring antibody affinity for 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.

[0076] Modification of Antibody Paratopes in Cell-Free Systems

[0077] In one aspect of the present disclosure, the provided cell-free system utilizes one or more agents that reversibly bind to the paratope of the antibody to be produced, thereby effectively reducing or eliminating the 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 compromising the ability of the antibody to be used for its intended purpose after production. In specific aspects, 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.

[0078] An 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 slight changes in these conditions. Certain monoclonal antibodies are pH-dependent and are able to bind antigen at a neutral pH of 7-7.4 but release antigen under slightly more acidic conditions. See, e.g., Bonvin et al., mAbs 7:2, 294-302; March / April 2015; Biochim Biophys Acta. November 2014; 1844(11): 1943-1950.

[0079] In another example, calcium-dependent antigen binding can be used to dissociate antigen 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 based on their ability to bind to the target protein in the presence of calcium ions and to elute in the absence of calcium ions. This property can be used to bind the antibody to the pharmaceutical agent in the cell-free system of the present disclosure, and once production is complete, the pharmaceutical agent can be removed from the antibody by removing the calcium ions from the system or isolating the antibody and eluting it to calcium-free or low calcium concentration conditions.

[0080] These and other exemplary reversible antibody-antigen pairs will be apparent to those of ordinary skill in the art upon reading this disclosure.

[0081] In certain embodiments, blocking peptides are used as agents that bind to the antibody paratope. In such embodiments, an excess of peptide is added to the cell-free system to achieve a peptide concentration estimated to be 100 to 500 times greater relative to the predicted antibody yield.

[0082] Improved mammalian cell-free system for anti-Grp78 antibody production

[0083] 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. Antibodies may include full-length antibodies or antigen-binding fragments thereof. GRP78 (also referred to as heat shock protein-5 (Bspa5 / BiP)) is a part of the stress response mechanism that provides evolutionary conservative, ER-connected cell survival signals during environmental and physiological stress. As a molecular component of the ER chaperone network, GRP78 is generally involved in the processing of unfolded proteins, however, updated insights also place proteins on the cell surface that may affect signal transduction (Lee, 2014, Nature Rev Cancer 1.4 (4); 263-276). Retrospective IHC studies have shown that GRP78 expression is positively correlated with poor survival rates of advanced breast cancer (Lee et al., 2006, Cancer Research 66 (16): 7849-7853) and recurrence of prostate cancer patients (Daneshmand et al., 2007, Human Pathology 38 (10): 1547-1552). Upregulation of GRP78 promotes survival and chemoresistance of both proliferating and dormant breast cancer cells. Synthetic peptides consisting of the GRP78 binding motif bind to cell death-inducing peptides to promote apoptosis in cancer cells, demonstrating their accessibility in vivo (Arap et al., 2004, Cancer Cell 6(3):275-284).

[0084] In some embodiments, the antibody comprises an antibody or an antigen-binding fragment thereof. In some embodiments, the antigen-binding fragment is selected from the group consisting of: Fab, single-chain variable fragment (scFv), single domain antibody and nanobody. In other embodiments, the antibody is a full-length antibody. In some embodiments, the anti-GRP78 antibody is a humanized antibody. In some embodiments, the anti-GRP78 antibody is a human antibody.

[0085] In some embodiments, the antibody is an antibody described in PCT Publication No. WO2018057703. In some embodiments, the anti-GRP78 antibody is an antibody named B4. In some embodiments, the anti-GRP78 antibody is an antibody named D1. In some embodiments, the anti-GRP78 antibody is an antibody named F6. In any of these embodiments, the antibody is a full-length antibody or an antigen-binding fragment thereof of any of the above antibodies. In some embodiments, the anti-GRP78 antibody is a full-length antibody.

[0086] In some embodiments, the anti-GRP78 antibody includes a variable heavy (VH) chain and a variable light (VL) chain. In some embodiments, the VH chain comprises the VH CDR1 shown in SEQ ID NO: 1, the VH CDR2 shown in SEQ ID NO: 2, and the VH CDR3 shown in SEQ ID NO: 3; and the VL chain comprises the VL CDR1 shown in SEQ ID NO: 4, the VLCDR2 shown in SEQ ID NO: 5, and the VL CDR3 shown in SEQ ID NO: 6. In some embodiments, the VH chain has an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity with the sequence shown in SEQ ID NO: 19, and the VL chain has an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity with the sequence shown in SEQ ID NO: 20. In some embodiments, the VH chain is shown in SEQ ID NO: 19, and the VL chain is shown in SEQ ID NO: 20.

[0087] In some embodiments, the VH chain comprises the VH CDR1 shown in SEQ ID NO: 7, the VH CDR2 shown in SEQ ID NO: 8, the VH CDR3 shown in SEQ ID NO: 9; and the VL chain comprises the VL CDR1 shown in SEQ ID NO: 10, the VL CDR2 shown in SEQ ID NO: 11, and the VL CDR3 shown in SEQ ID NO: 12. In some embodiments, the VH chain has an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the sequence shown in SEQ ID NO: 21, and the VL chain has an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the sequence shown 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.

[0088] In some embodiments, the VH chain comprises the VH CDR1 shown in SEQ ID NO: 13, the VH CDR2 shown in SEQ ID NO: 14, the VH CDR3 shown in SEQ ID NO: 15; and the VL chain comprises the VLCDR1 shown in SEQ ID NO: 16, the VL CDR2 shown in SEQ ID NO: 17, and the VL CDR3 shown in SEQ ID NO: 18. In some embodiments, the VH chain has an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the sequence shown in SEQ ID NO: 23, and the VL chain has an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the sequence shown 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.

[0089] In some 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 acids set forth in SEQ ID NO:25.

[0090] In some embodiments, the agent and 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 shown in SEQ ID NO:25.

[0091] 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 from R261, H265, H329, K271, K272, and D333 with reference to the residue numbering of human GRP78 as shown 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 as shown in SEQ ID NO: 25.

[0092] In some embodiments, both the agent and the anti-GPR78 antibody bind to a target epitope of GRP78 comprising one or more amino acid residues R261, R279, K113, K268, and K271 with reference to the residue numbering of human GRP78 as shown 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 shown in SEQ ID NO: 25.

[0093] In some embodiments, the agent binds to the same epitope on human GRP78 as an antibody containing any of the above sequences (i.e., an antibody having the ability to cross-compete with any of the described anti-GRP78 antibodies to bind 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 a peptide to inhibit binding to human GRP78 as an anti-GRP78 antibody indicates that the peptide can compete with an anti-GRP78 antibody for binding to human GRP78, and is therefore considered to bind to the same epitope of human GRP78.

[0094] In some embodiments, the agent is conjugated to one or a combination of amino acids selected from: (i) K113Q, K113S, K113D, or K113T; (ii) R261Q, R261S, R261D, R261T, or R261A; (iii) H265Q, H256S, H265D, or H256T; (iv) K268Q, K268S, K268D, 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 is conjugated to one or a combination of amino acids selected from: (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.

[0095] Cell-free systems for antibody production

[0096] Many cell-free systems can be used as the basis for the cell-free production systems of the present disclosure, including but not limited to those disclosed in: 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; 9,175,327; 9,040,253; 8,778,631; 7,871,794; 7,118,883; 7,041,479; and U.S. Patent Application Nos. 20060233789 and 20040191858.

[0097] Cell-free systems can be based on prokaryotic or eukaryotic origins. In prokaryotic systems, extracts based on Escherichia coli are often used and are commercially available for the production of various proteins, including antibodies. Based on Bacillus subtilis (Bacillus subtilis) (Kelwick R et al., Metab Eng. 2016; 38: 370–81), Pseudomonas putida (Pseudomonas putida) (Wang H. Synth Biol. 2018), Streptomyces (Streptomyces) (Li J et al., Biotechnol Bioeng. 2017; 114: 1343–53) and Vibrio parahaemolyticus (Vibrio parahaemolyticus) (Dondapati SK et al., Eng Life Sci. 2018; 18: 140–8) systems are well optimized at the laboratory level due to the preparation of cell-free lysates. Currently, a variety of detailed protocols can be used to prepare lysates based on Escherichia coli. In eukaryotic systems, extracts based on rabbit reticulocyte lysate (RRL), wheat germ, insect Spodoptera frugiperda 21 (Sf21), Chinese hamster ovary (CHO), and cultured human cells are often used. For a review, see, e.g., Dondapati SK et al., BioDrugs (2020) 34: 327–348.

[0098] For the production of complex proteins that require post-translational modifications, eukaryotic cell-free systems are often, but not always, preferred.

[0099] In some embodiments, the cell-free protein synthesis system can use a crude cell extract prepared by cells with an editor as taught in the present disclosure. These cells grow to an appropriate confluence, and the contents are removed by lysis, followed by many steps of washing to remove cell 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 major components required for transcription and translation, such as aminoacyl-tRNA synthetases (AAS), ribosomes, and factors required for extension, initiation, and transcription. Protein synthesis can be achieved by combining cell extracts with necessary substrates (such as amino acids, energy substrates, nucleic acid templates, cofactors, salts, and nucleotides). Cell-free protein synthesis is a rapid protein production system because it does not require transfection or cell culture, and lacks the limitation of cell viability.

[0100] In certain embodiments, a cell-free protein synthesis system is produced using transcription and translation-related factors derived from or based on the genetic modification of the antibody epitope in the natural protein taught herein. In these embodiments, the system only comprises known protein and substrate, 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 added separately with DNA template to produce protein, thereby forming a highly controlled system. The protein factors involved in the initiation, extension and termination of the protein synthesis process are identified, and the requirements of the system can be adapted separately.

[0101] In some embodiments, the protein synthesis system is a microbial lysate system (prokaryotic or eukaryotic) to which mammalian proteins are added to allow for appropriate protein folding processing, such as folding and / or post-translational modifications. In such cases, recombinant proteins (e.g., mammalian proteins) can be added to the system, and in the case where the protein is a target for producing antibodies, it can be modified to eliminate antibody binding. For example, if mammalian GRP78 is added to a yeast cell-free production system, GRP78 can be modified prior to producing the recombinant protein to modify the epitope of a specific GRP78 antibody using mutations as taught in Example 3. Similarly, recombinant proteins of other intracellular proteins added to the cell-free system can be modified so that the antibodies produced using the system will not bind to the protein (or will have reduced binding to the protein). 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.

[0102] Cell-free protein synthesis reaction format

[0103] Cell-free synthesis using the disclosed system can be performed in different formats. Successful synthesis of different antibody formats, including single-chain variable fragments (scFv), Fab fragments, and intact IgG, has been shown 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); reticulocytes (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). In addition, the upgrading of cell-free reactions to up-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 down-scale (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.

[0104] In some embodiments, the synthesis reaction format used is an intermittent format. The batch-based format is the most commonly used method in both prokaryotic and eukaryotic systems. The method is relatively fast and cheap, and depending on the system, synthesis can be carried out in 1.5-3 hours. Systems based on Escherichia coli can provide protein yields in the range of 100 μg / mL to 2–3 mg / ml. Although the yield of eukaryotic systems based on batches is relatively low, membrane proteins are automatically integrated into microsomal membranes, and function can be solved immediately after synthesis (Brodel AK et al., PLoS One. 2013; 8: 2013).

[0105] In other embodiments, to further scale up protein production via batch-based eukaryotic systems, a repetitive batch-based synthesis format has been proposed, in which microsomes spiked with the MP of interest produced in an initial synthesis reaction can be added to a fresh cell-free synthesis reaction that has been depleted of its microsomes (Thoring L. et al., PLoS One. 2016; 11:2016; Zemella A et al., Sci Rep. 2018; 8:18–26936).

[0106] In other embodiments, continuous exchange cell-free synthesis platform (CECFSP). In this format, a semipermeable dialysis membrane separates the reaction chamber and the feed chamber, so that the feed chamber provides fresh reaction components and enriches the reaction chamber. In exchange, the inhibitory components 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). Generally, the CECFSP format prolongs the reaction time and increases protein yield. So far, the CECFSP format has been used to increase protein yield several times, and is widely used as a cell-free platform.

[0107] In addition, the progress of bioorthogonal reaction chemistry has paved the way for the possibility of expanding ADC development. The site-specific introduction of non-standard amino acids into genetically engineered sequences can be used to produce site-specific labeled ADCs (Axup JY et al., Proc Natl Acad Sci USA. 2012; 109: 16101–6). At present, several ADCs are approved for therapy. So far, all these ADCs have been produced by coupling mAbs with cytotoxic linkers-payloads via surface-exposed lysines, or by reducing partial disulfides and conjugating to free cysteine, which typically produces controlled but heterogeneous ADC populations, wherein the number and position of drug molecules attached to mAbs vary (Strop P et al., ChemBiol. 2013; 20: 161-7). From synthesis to functional testing, cell-free systems can accelerate the evaluation of antibody constructs by sequential or simultaneous screening. The introduction of non-standard amino acids expands the chemical library, and therefore expands the possibility of modifying and improving antibody-based therapies. Advanced labeling techniques allow very rapid qualitative analysis of drug-to-antibody ratio (DAR), linker, linker / position, drug, drug / position (research applications) and allow full control over ADC design.

[0108] In some preferred embodiments, the cell-free system of U.S. Serial No. 63 / 359,871, filed on July 10, 2022, which is incorporated herein in its entirety for all purposes, may include a method for recombinantly producing an antibody by: a) expressing a 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, and wherein the modified cell-free comprises a mutation of one or more amino acid residues of a target epitope on a protein in the system; and initiating transcription and translation of the antibody in the cell-free system under conditions that allow the antibody to be produced. In some preferred embodiments, such a cell-free system may comprise a cell lysate (in some preferred embodiments, a eukaryotic cell lysate). In some preferred embodiments, the cell-free system of U.S. Serial No. 63 / 359,871 filed on July 10, 2022 can include: a) modifying a cell line (in some preferred embodiments, a mammalian cell line) to eliminate binding of an antibody to an intracellular protein in cells of the cell line, wherein the modification results in expression of a variant target protein comprising a mutation of one or more amino acid residues of 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 against 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).

[0109] Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some cases, terms with commonly understood meanings 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 generally understood in the art.

[0110] definition

[0111] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly indicates otherwise. For example, "a" or "an" means "at least one" or "one or more". It should be understood that the aspects and variations described herein include "consisting of" and / or "consisting essentially of" the aspects and variations.

[0112] Throughout this disclosure, various aspects of the claimed subject matter are presented in the form of ranges. It should be understood that the description in the form of ranges is only for convenience and brevity, and should not be interpreted as a hard limit to the scope of the claimed subject matter. Therefore, the description of the range should be regarded as having specifically disclosed all possible sub-ranges and individual numerical values ​​within the range. For example, in the case of providing a range of values, it should be understood that each intermediate value between the upper and lower limits of the range and any other specified value or intermediate value within the specified range are included in the claimed subject matter. The upper and lower limits of these smaller ranges can be independently included in the smaller range, and are also included in the claimed subject matter, subject to any specifically excluded limits within the stated range. In the case where the stated range includes one or two of the limits, the range excluding any one or two of the limits included is also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0113] As used herein, the term "about" refers to the common error range of the corresponding value that is easy to know. References to "about" values ​​or parameters herein include (and describe) embodiments for the value or parameter itself. For example, reference to a description of "about X" includes a description of "X".

[0114] As used herein, the term "agent" refers to any binding agent that can selectively bind to and block an epitope on a target protein. Agents used in the present disclosure include, but are not limited to, peptides, proteins, antibodies, or fragments thereof; small molecules; aptamers; peptide mimetics; and pharmacophores.

[0115] As used herein, the term "antibody" is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable regions (V H ), single-chain antibody fragments, including single-chain variable fragments (scFv) and single domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified immunoglobulin forms such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugate antibodies, multispecific (e.g., bispecific or trispecific) antibodies, bifunctional antibodies, trifunctional antibodies and tetrafunctional antibodies, tandem bi-scFv, tandem tri-scFv.

[0116] Unless otherwise indicated, the term "antibody" should be understood to explicitly encompass functional antibody fragments thereof, also referred to herein as "antigen-binding fragments." The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0117] As used herein, the term "blocking peptide" refers to a peptide consisting of part or all of an amino acid sequence corresponding to an antibody epitope (i.e., an antigen recognized by an antibody). The blocking peptide will specifically bind to the target antibody, preventing subsequent antibody binding to the target epitope. Incubating the antibody with sufficient blocking peptide will occupy the antibody binding site of the target protein epitope in the sample or system, preventing subsequent target protein binding to the antibody paratope in the sample or system.

[0118] The terms "complementarity determining region" and "CDR", which are synonymous with "hypervariable region" or "HVR", are known to refer to non-contiguous amino acid sequences within the variable region of an antibody that confer antigen specificity and / or binding affinity. Typically, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region, and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. "Framework region" and "FR" are known to refer to the non-CDR portions of the variable regions of the heavy and light chains. Typically, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.

[0119] The precise amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described by 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 Plückthun 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. 2013 Jul;41(Web Server Issue):W34-40 (“IgBLAST numbering scheme).

[0120] The boundary of a given CDR or FR can vary according to the scheme used for identification. For example, the Kabat scheme is based on structural alignment, and the Chothia scheme is based on structural information. The numbering of both Kabat and Chothia schemes is based on the most common antibody region sequence length, wherein insertion is regulated by the deletion that occurs in inserted letters (e.g., "30a") and some antibodies. Two schemes place certain insertions and deletions ("indels") at different positions, thereby producing different numberings. The Contact scheme is based on the analysis of complex crystal structures, and is similar to the Chothia numbering scheme in many respects. The AbM scheme is based on the compromise between the Kabat and Chothia definitions defined by the AbM antibody modeling software used by Oxford Molecular. The IgBLAST scheme is based on the matching with germline V, D and J genes, and can be determined using the IgBLAST tool of the National Center for Biotechnology Information (NCBI).

[0121] Table 1 below lists exemplary position boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3 as identified by Kabat, Chothia, AbM and Contact schemes, respectively. For CDR-H1, the residue numbering is listed using both Kabat and Chothia numbering schemes. FRs are located between CDRs, e.g., in the case where FR-L1 is located before CDR-L1, FR-L2 is located between CDR-L1 and CDR-L2, FR-L3 is located between CDR-L2 and CDR-L3, and so on. It should be noted that because the Kabat numbering scheme shown places insertions at H35A and H35B, the ends of the Chothia CDR-H1 loop vary between H32 and H34, depending on the length of the loop, when numbering using the Kabat numbering convention shown.

[0122]

[0123] 1-Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD

[0124] 2-Al-Lazikani et al., (1997) JMB 273, 927-948

[0125] According to 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 acid residues in VH are numbered 26-35 (HCDR1). In the combined Kabat and Chothia numbering schemes, in some embodiments, a CDR corresponds to an amino acid residue that is 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.

[0126] Therefore, unless otherwise indicated, a "CDR" or "complementarity determining region" of a given antibody or region thereof (such as a variable region thereof) or a single specific CDR (e.g., CDR-H1, CDR-H2, CDR-H3) should be understood to encompass the (or specific) complementary determining regions defined by any of the above schemes or other known schemes. For example, in stating that a specific CDR (e.g., CDR-H3) comprises a given V H or V L In certain embodiments, the CDR sequences of the antibodies provided are described using various numbering schemes (see, e.g., Part II), although it is understood that the antibodies provided may include CDRs described according to any of the above-mentioned other numbering schemes or other known numbering schemes.

[0127] Likewise, unless otherwise indicated, the FRs or individual specific FRs (e.g., FR-H1, FR-H2, FR-H3, FR-H4) of a given antibody or region thereof (such as its variable region) should be understood to encompass (or specific) framework regions defined by any known scheme. In some cases, schemes for identifying specific CDRs, FRs, or FRs or CDRs are indicated, such as CDRs defined by Kabat, Chothia, AbM, IgBLAST, IMGT, or Contact methods or other known schemes. In other cases, specific amino acid sequences of CDRs or FRs are given.

[0128] Tolerable changes in CDR sequences will be known to those skilled in the art. For example, in some embodiments, the polypeptide comprises a complementary 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.

[0129] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that is involved in binding an antibody to an antigen. H and V L ) generally have a similar structure, wherein each domain includes four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007). A single V H or V L A domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen may use a V H or V L The domains bind to the antigen to select complementary V L or V H See, for example, Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0130] As used herein, the terms "culture / culturing", "grow / growing", "maintain / maintaining", "expand / expanding", etc., when referring to cell culture itself or the culture process, can be used interchangeably to mean maintaining cells outside the body (e.g., ex vivo) under conditions suitable for survival. The cultured cells are allowed to survive, and the culture can cause the cells to grow, differentiate or divide.

[0131] As used herein, "epitope" refers to a polypeptide determinant that can specifically bind to an antibody. In certain embodiments, an epitope is an antigenic region specifically bound by an antibody. In certain embodiments, an epitope may include 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 may have a specific three-dimensional structural property (e.g., a "conformation" epitope) and / or a specific charge property. An epitope may be formed by both continuous and / or juxtaposed non-continuous residues (e.g., amino acids) of a target molecule. An epitope formed by continuous residues (e.g., amino acids) is usually retained when exposed to a denaturing solvent, while an epitope formed by a tertiary folding is usually lost when treated with a denaturing solvent. An epitope may include, but is not limited to, at least 3, at least 5, or 8-10 amino acid residues. In certain embodiments, the length of an epitope is less than 20 amino acid residues, less than 15 residues, or less than 12 residues. If two antibodies exhibit competitive binding to an antigen, they may bind to the same epitope in the antigen. Epitopes can also be identified by various scans, for example, alanine or arginine scans can indicate one or more residues that can interact with the antigen binding molecule.

[0132] As used herein, the term "expression" or "expressed" with respect to a gene refers to the transcription and / or translation product of the gene. The expression level of a DNA molecule in a cell can be determined based on the amount of the corresponding mRNA present in the cell or the amount of protein encoded by the DNA produced by the cell (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88).

[0133] The term "gene" may refer to a segment of DNA involved in the production or encoding of a polypeptide chain. It may include regions before and after the coding region (leader and trailer) and intermediate sequences (introns) between separate coding segments (exons). Alternatively, the term "gene" may refer to a segment of DNA involved in the production or encoding of non-translated RNA, such as rRNA, tRNA, guide RNA (e.g., small guide RNA), or microRNA.

[0134] "Antibody fragment" or "antigen-binding fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; heavy chain variable (V H ) region, single-chain antibody molecules such as scFv and those containing only V H In some embodiments, the antibody is or comprises an antibody fragment comprising a variable heavy chain (V H ) and variable light chain (V L In certain embodiments, the antibody comprises a heavy chain variable (V H ) region and / or light chain variable (V L ) region, such as a single-chain antibody fragment, such as a scFv.

[0135] A single domain antibody (sdAb) is an antibody fragment comprising 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, the single domain antibody is a human single domain antibody.

[0136] Antibody fragments can be prepared by various techniques, including but not limited to proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, such as a fragment comprising 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 naturally occurring intact antibody. In some embodiments, the antibody fragment is a scFv.

[0137] A "human antibody" is an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell or a non-human source utilizing a human antibody library or other human antibody coding sequences including a human antibody library. The term does not include humanized forms of non-human antibodies comprising non-human antigen binding regions, such as humanized forms in which all or substantially all CDRs are non-human antibodies. The term includes antigen-binding fragments of human antibodies.

[0138] The term "humanization" is used to describe antibodies in which the complementary determining regions (CDRs) from mammals (e.g., mice) are combined with human framework regions. Typically, the polynucleotides encoding the separated CDRs are transplanted into 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. In addition, "humanized" antibodies can be chimeric antibodies, human antibodies, humanized antibodies, or fully human antibodies, to reduce their potential antigenicity without reducing their affinity for organelle protein targets. Chimeric antibodies, human antibodies, and humanized antibodies have been generally described in the art.

[0139] Humanized antibodies have variable region framework residues substantially from human therapeutic antibodies (referred to as acceptor antibodies) and complementary determining regions substantially from mouse antibodies (referred to as donor immunoglobulins). 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. The constant region (if present) is also substantially or entirely from human immunoglobulins. Human variable domains are generally selected from human antibodies whose framework sequences exhibit a high degree of sequence identity with the murine variable region domains from which the CDRs are derived. The heavy and light chain variable region framework residues may 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 can be a consensus sequence of several human antibodies. See Carter et al., WO 92 / 22653. Based on the possible impact on CDR conformation and / or antigen binding, some amino acids from human variable region framework residues are selected for substitution. The study of such possible impact is carried out by modeling, checking the characteristics of the amino acids at specific positions or empirically observing the replacement or mutagenic effects of specific amino acids.

[0140] For example, when there is an amino acid difference between a murine variable region framework residue and a selected human variable region framework residue, the human framework amino acid should generally be replaced with the equivalent framework amino acid from the mouse antibody if the amino acid can reasonably be expected to: i) directly bind antigen non-covalently, ii) be adjacent to a CDR region, iii) otherwise interact with a CDR region (e.g., within about 6 Å of a CDR region), or iv) participate in the VL-VH interface.

[0141] Other candidates for substitution are the uncommon acceptor human framework amino acids at this position of human antibodies. These amino acids can be substituted by amino acids at the equivalent position of the mouse donor antibody or amino acids at the equivalent position of a more typical human antibody. Other candidates for substitution are the uncommon acceptor human framework amino acids at this position of human antibodies. The preferred variable region framework of humanized antibodies generally shows at least 75%, more preferably 80%, and even more preferably 85% sequence identity with the consensus sequence of the human variable region framework sequence or such sequence.

[0142] In some instances, mouse monoclonal antibodies can be used as the basis for the production of human therapeutic biologics. In one approach, for purposes of illustration and not limitation, heavy chain variable V H A consensus primer was used for the V region covering the translation start codon. H The g2b constant region leader peptide was used as the 5' primer and the g2b constant region specific 3' primer. Sequences from multiple independently generated clones can be compared to ensure that changes have not been introduced during amplification. V sequences obtained by 5' RACE RT-PCR method and 3' g2b specific primers can also be used to identify the V sequences obtained by 5' RACE RT-PCR and 3' g2b specific primers. H The fragments were sequenced to determine or confirm V H area sequence.

[0143] Can be used with V H The variable V region of the light chain of mouse monoclonal antibody was cloned in a similar manner. L In one approach, a region designed to amplify murine V L The consensus primer set for the region was designed to be aligned with the V region covering the translation start codon. L The 5' RACE RT-PCR method was used to clone the VJ-encoding cDNA. L The cloned sequence is then combined with sequences encoding human constant regions.

[0144] In one approach, the heavy chain variable region and the light chain variable region are reengineered to encode the splicing donor sequence located downstream of the corresponding VDJ or VJ junction, and cloned into a mammalian expression vector, such as pCMV-hγ1 for the heavy chain and pCMV-hκ1 for the light chain. These vectors encode human γ1 and Ck constant regions as exon fragments located downstream of the inserted variable region box. After sequence verification, the heavy chain expression vector and the light chain expression vector can be co-transfected into COS cells to produce chimeric antibodies. Conditioned medium is collected 48 hours after transfection, and the conditioned medium is determined by Western blot analysis for antibody production or ELISA for antigen binding. As described above, chimeric antibodies are preferably humanized.

[0145] The heavy chain variable region and light chain variable region of chimeric antibodies and / or humanized antibodies can be connected to at least a portion of the human constant region of selection. The selection of constant region can be driven by 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, and isotypes IgG2 and IgG4 do not. The light chain constant region can be λ or κ. The antibody can be expressed as a tetramer containing two light chains and two heavy chains, a single heavy chain, a light chain, Fab, Fab'F(ab')2 and Fv, or a single-chain antibody in which the heavy chain variable domain and the light chain variable domain are connected by a linker.

[0146] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population or obtained within a substantially homogeneous antibody population, i.e., the single antibodies comprising the population are identical, except for possible variants containing naturally occurring mutations or produced during the production of monoclonal antibody preparations, such variants are generally present in small amounts. Compared with polyclonal antibody preparations that generally include different antibodies for different epitopes, each monoclonal antibody of a monoclonal antibody preparation is directed to a single epitope on an antigen. The term should not be interpreted as requiring the production of antibodies by any ad hoc method. Monoclonal antibodies can be prepared by a variety of techniques, including but not limited to being produced by hybridomas, recombinant DNA methods, phage display methods, and other antibody display methods.

[0147] As used herein, the term "specific binding" to a target protein or epitope is a well-known term 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 protein more frequently, more rapidly, for a longer period of time, and / or with greater affinity than the molecule reacts or associates with an alternative protein. It should also be understood that "specific binding" does not necessarily require (although it may include) exclusive binding. Typically, but not necessarily, reference to binding means preferential binding. "Specificity" refers to the ability of a binding protein to selectively bind to an antigen.

[0148] As used herein, "stress proteins" refer to proteins that function in normal cells and can exist at high levels under stress conditions, such as hypoxia, nutrient deprivation, pH changes, oxidative stress, or other metabolic disorders of cells (such as often occurring in cancer cells). Stress proteins include proteins whose expression increases when the capacity of organelles (such as endoplasmic reticulum (ER) or Golgi apparatus) becomes insufficient. ER stress proteins include proteins related to the unfolded protein response (UPR) that acts to reduce ER stress and restore homeostasis, proteins related to endoplasmic reticulum-associated protein degradation (ERAD), or proteins related to apoptosis mediated by ER stress. Exemplary ER stress proteins include calreticulin, heat shock proteins, and isomerases. Golgi stress proteins include proteins related to post-translational modifications (such as glycosylation) or to vesicle transport. Exemplary Golgi stress proteins include, for example, GOLPH3.

[0149] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides, which have binding properties similar to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a specific nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated.

[0150] The term "paratope", also called "antigen binding site", is the portion of an antibody that recognizes and binds to an epitope.

[0151] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymer 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, by sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, transfer of amino acids to proteins-RNA-mediated addition (such as arginylation), ubiquitination or any other manipulation (such as conjugation with a labeling component). As used herein, the term "amino acid" refers to natural and / or non-natural or synthetic amino acids, including glycine and both D or L optical isomers and amino acid analogs.

[0152] A "vector" is a preferably self-replicating nucleic acid molecule that transfers an inserted nucleic acid molecule into and / or between host cells. An "expression vector" is a polynucleotide sequence that, when introduced into an appropriate host cell, can be transcribed and translated into a polypeptide. An "expression system" generally implies a suitable host cell consisting of an expression vector that can be used to produce a desired expression product.

[0153] The term "recombinant" as 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 is different from the polynucleotide found in nature.

[0154] As used herein, the terms "operably linked" or "operably connected" are used to refer to DNA sequences that are juxtaposed in a manner that places the described components in a relationship that allows them to function in the intended manner. For example, a promoter is operably linked to a coding sequence if it controls transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to allow translation. A signal sequence (secretionary leader) is operably linked to the DNA of a polypeptide if its DNA is expressed as a precursor that participates in the secretion of the polypeptide. Generally, operably linked means contiguous.

[0155] In some preferred embodiments, the present disclosure provides a method for recombinantly producing an antibody, comprising: providing a cell-free protein expression system containing a target protein exhibiting 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 one or more nucleic acids encoding an antibody that binds to the antibody epitope into the cell-free system; and initiating transcription and translation of the antibody in the cell-free system under conditions that allow the production of the antibody. In some preferred embodiments, the cell-free system comprises a cell lysate. In some preferred embodiments, the agent is introduced into the cell before the cell lysate is produced. In some preferred embodiments, the agent is introduced into the cell after the cell lysate is produced. 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 extracts 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 agent that blocks the antibody epitope is a peptide. In some preferred embodiments, the agent of the blocking antibody is a small molecule. In some preferred embodiments, the target protein is an intracellular 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 activity of the target protein 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 the 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 the cell-free system in the absence of the agent.

[0156] In some preferred embodiments, the present disclosure provides a method for recombinantly producing an antibody, the method comprising: providing a cell-free protein expression system comprising a target protein exhibiting an antibody epitope; modifying the cell-free system by introducing one or more agents that block the antibody paratope binding to the antibody epitope on the target protein; introducing one or more nucleic acids encoding an antibody comprising an antibody paratope into the cell-free system; and initiating transcription and translation of the antibody in the cell-free system under conditions that allow the production of the antibody. In some preferred embodiments, the method further comprises separating 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 producing the antibody. In some preferred embodiments, the method further comprises separating the antibody from the cell-free system. In some preferred embodiments, the antibody is separated before the agent is removed 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 extracts 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 transcription and translation of the antibody. In some preferred embodiments, the agent that blocks the antibody paratope is a peptide. In some preferred embodiments, the target protein is an intracellular 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.

[0157] In some preferred embodiments, the present disclosure provides a method for recombinantly producing a monoclonal antibody, the method comprising: providing a mammalian cell-free protein expression system comprising a target protein exhibiting 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 one or more nucleic acids encoding a monoclonal antibody that binds to the antibody epitope into the cell-free system; and initiating transcription and translation of the monoclonal antibody under conditions that allow the production of antibodies in the mammalian cell-free system. 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.

[0158] In some preferred embodiments, the present disclosure provides a method for recombinantly producing a monoclonal antibody, comprising: providing a mammalian cell-free protein expression system comprising a target protein exhibiting an antibody epitope; modifying the mammalian cell-free system by introducing one or more agents that block the antibody paratope binding to the antibody epitope on the target protein; introducing one or more nucleic acids encoding a monoclonal antibody comprising an antibody paratope into the cell-free system; and initiating transcription and translation of the monoclonal antibody under conditions that allow the production of antibodies in the mammalian cell-free system. In some preferred embodiments, the method further comprises removing the agent bound to the antibody paratope after producing the monoclonal antibody. In some preferred embodiments, the method further comprises separating the monoclonal antibody from the cell-free system. In some preferred embodiments, the monoclonal antibody is separated before removing the agent from the monoclonal antibody.

[0159] In some preferred embodiments, the present disclosure provides a method for recombinantly producing an antibody, the method comprising: introducing an agent that selectively binds to a target epitope on an intracellular protein into a cell to produce a modified cell line; generating a cell-free antibody production system from the modified 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 a target epitope on an intracellular protein; and initiating transcription and translation from the nucleic acid template to produce an antibody in a 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 an intracellular protein in the secretory pathway of the mammalian cell line.

[0160] In some preferred embodiments, the modified cell line can be produced using the methods disclosed in U.S. Serial No. 63 / 337,980 filed on May 3, 2022 and / or U.S. Serial No. 63 / 359,541 filed on 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 may include: a) expressing a nucleic acid encoding an antibody in a modified cell line (a mammalian cell line in some preferred embodiments), wherein the antibody specifically binds to a target epitope on a target protein that is naturally expressed as an intracellular protein in the cell; and wherein the modified cell is engineered to express a variant target protein comprising a mutation of one or more amino acid residues of the target epitope; and, b) culturing the modified cell line under conditions that allow the production of the antibody (and, in some preferred embodiments, isolating the antibody). In some preferred embodiments, the methods of these applications may include: a) providing an antibody that binds to an epitope on a target protein present in a cell line (in some preferred embodiments, a mammalian cell line) within a cell; b) identifying the epitope of the target protein to which the antibody binds; c) producing a modified cell line by mutating the epitope of the target protein in the cell line to reduce or eliminate the binding of the antibody to the epitope on the target protein; d) introducing an expression vector encoding the antibody into the modified cell line; e) culturing the modified cell line under conditions that allow the production of the antibody from the expression vector; and, f) expressing the nucleic acid encoding the antibody in the modified cell line (and, in some preferred embodiments, isolating the antibody). In some preferred embodiments, the modified mammalian cell line of these applications comprises a variant protein that is an intracellular protein in the secretory pathway, wherein the variant protein comprises a mutation of a natural intracellular protein that is a target protein of the antibody, and wherein the mutation is an amino acid substitution of one or more amino acid residues to change the target epitope of the antibody to a mutant epitope. In some preferred embodiments, the method comprises recombinantly producing an antibody targeting GRP78 by: a) expressing a nucleic acid encoding an antibody in a modified mammalian cell line, wherein the antibody binds to GRP78, and wherein the modified mammalian cell is engineered with a variant GRP78 having a mutant epitope comprising a mutation in one or more amino acid residues of the target epitope of the antibody or antigen-binding fragment; and, b) culturing the modified mammalian cell line under conditions such that the anti-GRP78 antibody is produced in the culture supernatant.

[0161] In some preferred embodiments of the methods disclosed herein, the target protein is an intracellular 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, heat shock protein or 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.

[0162] As will be readily appreciated by one of ordinary skill in the art, other embodiments are also contemplated.

[0163] Examples

[0164] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. Reality Example 1 Production and purification of anti-GPRP78 antibodies in mammalian cells

[0165] Fully human anti-GRP78 monoclonal antibodies (mAbs) formatted as full-length IgG1 antibodies (Table E1) were individually transfected into Chinese hamster ovary (CHO) cells by electroporation in 6-well plates, filled with fresh medium the next day and incubated for 7 days post-transfection. TM Transfection system and Neon TM Transfection was performed using the pre-set settings for CHO cells using the Transfection System reagent (Thermo Fisher Scientific, Waltham, MA). The supernatant on day 7 after transfection was loaded into 1 mL of MabSelect Prisim A TM The antibody concentration and IgG titer in the supernatant were determined using an Octet QK 384 protein A biosensor (Molecular Devices, Wokingham, Berkshire, UK) using IgG1 antibody as a standard.

[0166] During the culture period and at the time of collection, the transfected CHO cells showed reduced viability. The supernatant titers of all anti-GRP78 antibodies produced by CHO cells were very low and less than 5 μg / mL (Table E2). To confirm that the reduced titers were not specific to CHO cells, the transfection was repeated in human embryonic kidney 293 (HEK293) cells, and the antibodies were purified from the supernatant on day 5 using a similar method, but the titers remained very low (Table E2).

[0167] These results are consistent with the observation that production of anti-GRP78 antibodies in mammalian cells, such as human cells, is harmful to the cells and affects the ability to produce antibodies in high yields. It is hypothesized that the antibody can bind to native GRP78 in the endoplasmic reticulum (ER) of the cell, thereby inhibiting its natural chaperone function in the cell and killing the cell before the antibody 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 antibodies with GRP78 as shown by SDS-PAGE analysis of purified samples under reducing and non-reducing conditions. Figure 1 As shown, all samples displayed the typical SDS-PAGE pattern of monomeric IgG, however there was an additional band between 70-80 kDa consistent with the presence of bound GRP78 antigen.

[0168]

[0169]

[0170] Example 2 Epitope mapping of anti-GRP78 antibodies

[0171] To characterize the binding properties of the antibodies against GRP78 described in Example 1, 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 each individual GRP78 variant, allowing identification of target protein residues involved in antibody binding.

[0172] Under high stringency conditions (e.g., elevated pH, elevated salinity, elevated temperature, and / or increased washing time), the binding of each test antibody to each GRP78 variant in the alanine scanning library was determined by high-throughput flow cytometry. Commercially available monoclonal antibodies 1H11-1H7, which were validated to bind to wild-type (WT) GRP78 on cells, and were therefore determined to be suitable positive controls under these conditions (Thermo Fisher Scientific, Waltham, MA) were also included in the experiment. The binding of the antibody to each GRP78 variant as measured by the fluorescence signal (raw fluorescence data minus background) was normalized relative to its binding to WT GRP78. For each GRP78 variant, such normalization of binding to the test antibody relative to its binding to the control antibody was plotted. Exemplary results for two anti-GRP78 antibody clones are shown in Figure 2A and Figure 2B GRP78 variants that exhibited >70% binding to the control antibody but <20% binding to the test antibody were identified as essential and major binding residues for the test antibody. Other GRP78 variants that did not meet the above criteria but showed reduced binding activity (20%-30%) and were in close proximity to the above identified major residues (based on the known 3D structure of the protein) were also identified (and were considered "other residues involved in antibody binding").

[0173] Table E3 shows the residues involved in antibody binding of exemplary anti-GRP78 clones B4 and F6.

[0174]

[0175] PDB ID 6ASY (Yang et al., Nature Communications. 2017; 8(1): 1-3) was examined to identify the predicted amino acids that constitute the GRP78 epitope that binds to B4 mAb or F6 mAb. This structure was selected because it is the most complete experimentally determined structure (spanning aa25-633) of H. sapiens GRP78. (PDB ID number 6ASY, Yang et al., 2017). Figure 3 Depicted is a visualization of the major residues for antibody binding, with exemplary major residues and other residues involved in binding indicated by arrows. Based on the analyzed structures, all amino acids of the predicted epitope are contained in an alpha helix or as part of a linker between two alpha helices and can be separated by an alpha helix (major residues based on shotgun mutagenesis are underlined): (i) K113; (ii) R261 , H265 , K268, K271, K272; (iii) R279and (iv) E329 、D333.

[0176] Example 3 System for generating cell-free lysate from modified GRP78 cells

[0177] Cell-free systems were generated from CHO cells as taught in: Stech M, Scientific Reports 7: 12030 (2017); AK et al. PLoS One 8, e82234 (2013) and Thoring, L. et al. PLoS One 11, e0163670 (2016) for the efficient production of anti-GRP78antibodies.al., Biotechnology and Bioengineering 111, 25–36 (2013).

[0178] CHO lysate containing endogenous microsomal vesicles derived from the ER was prepared as described previously ( 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 was grown in a bioreactor at 37°C in a chemically defined serum-free medium (PowerCHO TM 2CD Medium, Lonza, Basel, Switzerland) with exponential growth, up to 18×10 6Cells / ml. Cells were collected by centrifugation at 200 × g for 15 min, precipitated, washed twice and resuspended in a buffer containing 30mM HEPES-KOH (pH 7.5) and 100mM NaOAc. Subsequently, a syringe was used to pass the cell suspension through a 20-gauge needle, which resulted in mechanical destruction of the cells. Nuclei and cell debris were removed by centrifugation at 6,500 × g for 10 min. The obtained supernatant was subjected to a gel filtration step using a Sephadex G-25 column (GE Healthcare, Freiburg, Germany) balanced in a buffer containing 30mM HEPES-KOH (pH 7.5) and 100mM. The filtered supernatant was eluted in 1 ml fractions, and those with an absorbance of RNA content higher than 100 at 260 nm were combined. To remove endogenous mRNA, cell lysates were treated with S7 micrococcal nuclease (Roche, Mannheim, Germany) (10 U / ml) and CaCl2 (1 mM) and incubated for 20 min at room temperature (RT). Micrococcal S7 nuclease was inactivated by adding EGTA (6.7 mM). Optionally, CHO lysates were further supplemented with creatine kinase (100 μg / ml). Lysates were snap frozen in liquid nitrogen and subsequently stored at -80°C until further use.

[0179] Panning and specificity assays using Blond-Elguindi S Cell 1993 Nov 19;75(4):717-28 and Arap et al., Cancer Cell. 2004 Sep;6(3):275-84 were used to identify GRP78 peptides of approximately 8-12 amino acids containing a GRP78 peptide binding motif that matched the binding epitopes of the B4 and H6 binding epitopes as taught in Example 2.

[0180] GRP78 peptides are synthesized and isolated using methods well known in the art. For example, peptides can be ordered from the custom peptide synthesis service of Thermo Fisher Scientific (Waltham, Mass). These peptides are introduced into the CHO cell-free lysate system at appropriate concentrations to provide the predicted saturation of the epitope in the system. The activity of GRP78 in the system is assessed using the method of Hristozova N et al., PLoS One. 2016; 11(8): e0161970, and the CHO lysate is confirmed to have B4 or H6 epitope blocking by treating aliquots of the peptide-treated lysate with the corresponding antibodies. Modified CHO lysate with the best combination of epitope blocking and GRP78 activity is selected for the production of GRP78 antibodies as described below

[0181] Example 4Antibody production in a GRP78-modified cell-free system

[0182] The modified CHO lysate of Example 3 was then used for coupled transcription-translation reactions. Thoring, supra. The translation reaction consisted of 40% (v / v) S7 nuclease-treated CHO lysate containing endogenous microsomal vesicles derived from the ER, HEPES-KOH (pH 7.6, 30 mM, BioMol GmbH, Hamburg, Germany), complete amino acids (100 μM), Mg(OAc)2 (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 mMUTP), creatine phosphate (20 mM), T7 polymerase (1 U / μl) (Agilent Technologies, Santa Clara, United States) and 14 C-leucine (to a final concentration of 30 μM); specific radioactivity 46.15 dpm / pmol (PerkinElmer LAS (Germany) GmbH, Rodgau, Germany) to allow subsequent quantitative and qualitative analysis of cell-free synthesized proteins. Protein synthesis was initiated by adding DNA template (60 ng / μL). The reaction was incubated at 30° C. for 3 h at 600 rpm in a standard thermomixer (Eppendorf Thermomixer Comfort). Background translation activity was monitored by performing the translation reaction without supplementing the plasmid.

[0183] According to Stech et al., (2017). Sci. Rep. 7(1), 12030, the coding sequence of the anti-GRP78 antibody was codon-optimized for Cricetulus griseus and equipped with the necessary regulatory sequences to enable in vitro transcription and translation. The basic elements are as follows: 5′ untranslated region (UTR): T7 promoter sequence, multiple cloning site (MCS), internal ribosome entry site (IRES) from the intergenic region (IGR) of the cricket paralysis virus (CrPV), GCT as the start codon; 3′UTR: T7 terminator sequence, MCS (same as above). The DNA template was synthesized de novo by Biocat GmbH (Biocat GmbH, Heidelberg) (Agilent, San Jose, CA) and cloned into an appropriate vector (pUC57-1.8k). The expression vector was cloned using the pUC57-1.8k construct according to the manufacturer's instructions. Plasmid preparations for cell-free protein synthesis were prepared using the HiPure Plasmid Midiprep Kit (Thermo Fisher Scientific, Waltham, Mass.) and subsequently expressed using the Illumina Miseq TM Control digestion and sequencing were performed using the Illumina® system (Illumina, San Diego, CA) to verify the correct DNA sequence.

[0184] The translation reaction consisted of 40% (v / v) S7 nuclease-treated CHO lysate containing endogenous microsomal vesicles derived from the ER, HEPES-KOH (pH 7.6, 30 mM, BioMol GmbH, Hamburg, Germany), complete amino acids (100 μM), Mg(OAc)2 (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 14 C-leucine (to a final concentration of 30 μM); specific radioactivity 46.15 dpm / pmol (PerkinElmer LAS (Germany) GmbH, Rodgau, Germany) to allow subsequent quantitative and qualitative analysis of cell-free synthesized proteins. Protein synthesis was initiated by adding DNA template (60 ng / μL). The reaction was incubated at 30°C for 3 h at 600 rpm in a standard thermomixer (Eppendorf Thermomixer Comfort). Background translation activity was monitored by performing the translation reaction without supplementing the plasmid.

[0185] After the translation reaction, the samples were centrifuged at 16,000×g for 10 min at 4°C in order to separate the microsomes from the soluble fraction of the translation mixture. The resulting supernatant (first supernatant or SUP1) was transferred to a fresh reaction tube and stored on ice until further analysis, while the microsomal pellet was resuspended in 1x PBS containing 0.2% n-dodecyl-β-D-maltoside (DDM) to enable release of the translocated and microsomal-containing antibodies. The microsomes were manually resuspended by repeated up and down pipetting, followed by vortexing and shaking on a vibrax for approximately 45 min. In order to separate the released proteins from the microsomal membrane remnants, a second centrifugation step was performed. The resulting supernatant (second supernatant or SUP2) was transferred to a fresh reaction tube and stored on ice until further analysis. Supplemented with 14 The reactions of C-leucine were analyzed by SDS-PAGE followed by autoradiography and liquid scintillation counting, while nonradioactive samples were subjected to functional analysis by enzyme-linked immunosorbent assay (ELISA).

[0186] Example 5 Humanization of anti-GOLPH3 monoclonal mouse antibody

[0187] GOLPH3 was originally identified as a peripheral membrane protein localized to the trans-Golgi network, but other reports suggest that it is a mitochondrial protein that regulates mitochondrial mass by regulating the mitochondrial-specific phospholipid cardiolipin. GOLPH3 has since been implicated in the Target of Rapamycin (TOR) signaling pathway. In response to growth factors stimulated by EGF, GOLPH3-transfected cells enhance S6 kinase activity. At the same time, AKT phosphorylation in these cells increased, while these events were eliminated in GOLPH3 siRNA-treated cells compared to control cells, indicating that GOLPH3 can enhance signaling through TOR-related complexes. Scott, KL et al., Nature. 2009 Jun 25; 459(7250): 1085-1090. These results suggest that GOLPH3 is a true oncogene and may be a useful target for therapeutic strategies.

[0188] 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 human purified His-tagged GOLPH3 protein. Because GOLPH3 is primarily found in the Golgi apparatus and mitochondria, the methods of the present disclosure are well suited for efficient production of such antibodies in mammalian cells.

[0189] The humanized GOLPH3 antibodies used in the methods of the invention preferably comprise CDR sequences derived from or based on mGOLPH3, as described in more detail herein and in the incorporated references. Briefly, humanized GOLPH3 antibodies provided by Rapid Novor (Ontario, Canada) were used. Antibody Sequencing Service to Determine the V of mGOLPH3 H and V L The amino acid sequence of the region. The CDRs of mGOLPH3 can be determined using, for example, the IMGT numbering system provided in Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003). Once the CDRs are identified, the mouse monoclonal antibody can be humanized using methods (e.g., methods disclosed in Do Couto et al., U.S. Pat. No. 10,613,094, issued on April 7, 2020, and Chilcote et al., U.S. Pat. No. 8,673,593, issued on March 18, 2014).

[0190] Example 6 Epitope mapping of the protein binding site of hGOLPH3 by anti-GOLPH3 humanized antibody

[0191] Various methods can be used to epitope map the binding sites of anti-mGOLPH3 mAbs on human hGOLPH3. Exemplary methods are taught in U.S. Pat. No. 11,174,479, issued on November 16, 2021 to Greenleaf et al. and U.S. Pat. No. 11,061,036, issued on July 13, 2021 to Wilson et al.

[0192] For example, a streptavidin biosensor can be used to determine the binding of mGOLPH3 mAb to biotinylated hGOLPH3 peptides spanning hGOLPH3. Biotinylated peptides spanning hGOLPH3 protein were loaded at 5 μg / ml for 700 seconds, 300 seconds for baseline recording, and then the association and dissociation of mGOLPH3 mAb to each peptide was measured at various concentrations for 600 seconds. A dual reference sensor was employed in all tests to measure any observed background signal and to subtract any observed background signal from nonspecific binding or system noise.

[0193] Analyses were 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, the K D , K on , K offWhere possible, global curve fitting was also performed for multiple concentrations of antibody / analyte.

[0194] Example 7 Identification of hGOLPH3 antibody epitopes for identification of pharmacophore binders

[0195] To predict residues important for identifying binders to the target epitope of an antibody against hGOLPH3, the crystal structure of hGOLPH3 and its orthologs can be used. Wood et al. reported the X-ray crystal structure of hGOLPH3 at a resolution of 2.9- (Wood et al., J Cell Biol. 2009 Dec 28;187(7):967–975), and the activity of conserved regions in the structure of hGOLPH3 and its yeast ortholog Vps74p (Wood et al., Journal of Cell Biology, 209187:67-75. Additional structural aspects and residues required for the 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).

[0196] Once the residues of the target epitope are predicted, a discovery method such as those taught in U.S. Pat. No. 8,019,550 can be used to identify potential pharmacophore agents for use with the methods and systems of the present disclosure. In short, an antibody epitope in a protein structure is identified, as well as the relevant binding residues in the epitope site (e.g., pharmacophore point). The pharmacophore agent is then designed to conform to the predicted epitope both geometrically and in terms of satisfying sufficient pharmacophore points. Various techniques can be used to improve the design process, such as using a link structure (e.g., a superimposed position with the target as a reference), comparing the epitope determined by the simulation model with the shape determined by the mapping process, and / or using an epitope mapping technique such as the epitope mapping technique described above to empirically determine the epitope.

[0197] Example 8 Production of a cell-free system for anti-GOLPH3 humanized antibody production

[0198] A cell-free system was then generated from CHO cells for efficient production of anti-GOLPH3 antibodies as taught in: Stech M, Scientific Reports 7: 12030 (2017); AK et al. PLoS One 8, e82234 (2013) and Thoring, L. et al. PLoS One 11, e0163670 (2016). CHO lysate was prepared from cultured CHO-K1 cells, such as As described in AK et al., Biotechnology and Bioengineering 111, 25-36 (2013). The pharmacophore developed as taught in Example 7 was introduced into CHO lysate at a sufficient concentration to bind to the anti-GOLPH3 antibody epitope while not eliminating the activity of GOLPH3 in the CHO lysate system.

[0199] CHO lysate containing endogenous microsomal vesicles derived from the ER was prepared as described previously ( 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 was grown in a bioreactor at 37°C in a chemically defined serum-free medium (PowerCHO TM 2CD Medium, Lonza, Basel, Switzerland) with exponential growth, up to 18×10 6 Cells / ml. Cells were collected by centrifugation at 200 × g for 15 min, precipitated, washed twice and resuspended in a buffer containing 30mM HEPES-KOH (pH 7.5) and 100mM NaOAc. Subsequently, a syringe was used to pass the cell suspension through a 20-gauge needle, which resulted in mechanical destruction of the cells. Nuclei and cell debris were removed by centrifugation at 6,500 × g for 10 min. The obtained supernatant was subjected to a gel filtration step using a Sephadex G-25 column (GE Healthcare, Freiburg, Germany) balanced in a buffer containing 30mM HEPES-KOH (pH 7.5) and 100mM. The filtered supernatant was eluted in 1 ml fractions, and those with an absorbance of RNA content higher than 100 at 260 nm were combined. To remove endogenous mRNA, cell lysates were treated with S7 micrococcal nuclease (Roche, Mannheim, Germany) (10 U / ml) and CaCl2 (1 mM) and incubated for 20 min at room temperature (RT). Micrococcal S7 nuclease was inactivated by adding EGTA (6.7 mM). Optionally, CHO lysates were further supplemented with creatine kinase (100 μg / ml). Lysates were snap frozen in liquid nitrogen and subsequently stored at -80°C until further use.

[0200] The GOLPH3 pharmacophore developed as in Example 7 was used to modify a cell-free system for producing a GOLPH3 monoclonal antibody. As taught by Scott et al. above, the activity of GOLPH3 in the system was assessed by identifying the ability of GOLPH3 in the system to regulate the phosphorylation state of mTOR substrates, and the CHO lysate was confirmed to have GOLPH3 antibody epitope blocking by treating an aliquot of the lysate with the corresponding antibody peptide. As described below, the modified CHO lysate with the best combination of epitope blocking and GOLPH3 phosphorylation activity was selected for the production of GOLPH3 antibodies.

[0201] Example 9 Antibody production in a GOLPH3-modified cell-free system

[0202] The coupled transcription-translation reaction was then performed as described. Thoring, supra. The translation reaction consisted of 40% (v / v) S7 nuclease-treated CHO lysate containing endogenous microsomal vesicles derived from the ER, HEPES-KOH (pH 7.6, 30 mM, BioMol GmbH, Hamburg, Germany), complete amino acids (100 μM), Mg(OAc)2 (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 14 C-leucine (to a final concentration of 30 μM); specific radioactivity 46.15 dpm / pmol (PerkinElmer LAS (Germany) GmbH, Rodgau, Germany) to allow subsequent quantitative and qualitative analysis of cell-free synthesized proteins. Protein synthesis was initiated by adding DNA template (60 ng / μL). The reaction was incubated at 30°C for 3 h at 600 rpm in a standard thermomixer (Eppendorf Thermomixer Comfort). Background translation activity was monitored by performing the translation reaction without supplementing the plasmid.

[0203] According to Stech et al., (2017). Sci. Rep. 7(1), 12030, the coding sequence of the anti-GOLPH3 antibody was codon-optimized for Vibrio parahaemolyticus and equipped with the necessary regulatory sequences to enable in vitro transcription and translation. The basic elements are as follows: 5′ untranslated region (UTR): T7 promoter sequence, multiple cloning site (MCS), internal ribosome entry site (IRES) from the intergenic region (IGR) of the cricket paralysis virus (CrPV), GCT as the start codon; 3′UTR: T7 terminator sequence, MCS (same as above). The DNA template was synthesized de novo by Biocat GmbH (Biocat GmbH, Heidelberg) (Agilent, San Jose, CA) and cloned into an appropriate vector (pUC57-1.8k). The vector was cloned using the pUC57-1.8k construct according to the manufacturer's instructions. Plasmid preparations for cell-free protein synthesis were prepared using the HiPurePlasmid Midiprep Kit (Thermo Fisher Scientific, Waltham, Mass.) and subsequently expressed using the Illumina Miseq TM Control digestion and sequencing were performed using the Illumina® system (Illumina, San Diego, CA) to verify the correct DNA sequence.

[0204] The coupled transcription-translation reaction was then performed as described. Thoring, supra. The translation reaction consisted of 40% (v / v) S7 nuclease-treated CHO lysate containing endogenous microsomal vesicles derived from the ER, HEPES-KOH (pH 7.6, 30 mM, BioMol GmbH, Hamburg, Germany), complete amino acids (100 μM), Mg(OAc)2 (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 14C-leucine (to a final concentration of 30 μM); specific radioactivity 46.15 dpm / pmol (PerkinElmer LAS (Germany) GmbH, Rodgau, Germany) to allow subsequent quantitative and qualitative analysis of cell-free synthesized proteins. Protein synthesis was initiated by adding DNA template (60 ng / μL). The reaction was incubated at 30°C for 3 h at 600 rpm in a standard thermomixer (Eppendorf Thermomixer Comfort). Background translation activity was monitored by performing the translation reaction without supplementing the plasmid.

[0205] After the translation reaction, the samples were centrifuged at 16,000×g for 10 min at 4°C in order to separate the microsomes from the soluble fraction of the translation mixture. The resulting supernatant (first supernatant or SUP1) was transferred to a fresh reaction tube and stored on ice until further analysis, while the microsomal pellet was resuspended in 1x PBS containing 0.2% n-dodecyl-β-D-maltoside (DDM) to enable release of the translocated and microsomal-containing antibodies. The microsomes were manually resuspended by repeated up and down pipetting, followed by vortexing and shaking on a vibrax for approximately 45 min. In order to separate the released proteins from the microsomal membrane remnants, a second centrifugation step was performed. The resulting supernatant (second supernatant or SUP2) was transferred to a fresh reaction tube and stored on ice until further analysis. Supplemented with 14 The reactions of C-leucine were analyzed by SDS-PAGE followed by autoradiography and liquid scintillation counting, while nonradioactive samples were subjected to functional analysis by enzyme-linked immunosorbent assay (ELISA).

[0206] The scope of the present invention is not intended to be limited to specific disclosed embodiments, which are provided, for example, to illustrate various aspects of the present invention. Various modifications to the described compositions and methods will become apparent from the description and teachings herein. Such changes may be practiced without departing from the true scope and spirit of the present disclosure, and such changes are intended to fall within the scope of the present disclosure.

[0207] sequence

[0208]

[0209]

[0210]

Claims

1. A method for recombinantly producing an antibody, the method comprising: a) providing a cell-free protein expression system containing a target protein displaying an antibody epitope; b) modifying the cell-free system by introducing one or more agents that block the antibody epitopes 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; as well as, d) initiating transcription and translation of said antibody in said cell-free system under conditions allowing production of said antibody.

2. The method of claim 1, wherein the cell-free system comprises a cell lysate.

3. The method of claim 2, wherein the agent is introduced into the cells prior to producing the cell lysate.

4. The method of claim 2, wherein the agent is introduced into the cells after the cell lysate is produced.

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

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

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

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

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

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

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

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

13. The method of 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 agent.

14. The method of 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 agent.

15. A method for recombinantly producing an antibody, the method comprising a) providing a cell-free protein expression system containing a target protein displaying an antibody epitope; b) modifying the cell-free system by introducing one or more agents that block the antibody paratope binding to the antibody epitope on the target protein; c) introducing one or more nucleic acids encoding antibodies comprising said antibody paratope into said cell-free system; as well as, d) initiating transcription and translation of said antibody in said cell-free system under conditions allowing production of said antibody.

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

17. The method of claim 15, further comprising removing the agent that binds to the antibody paratope after producing the antibody.

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

19. The method of claim 17, wherein the antibody is isolated prior to removing the agent from the antibody.

20. The method of claim 15, wherein the cell-free system comprises a cell lysate.

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

22. The method of claim 15, wherein the agent is introduced into the cell-free system prior to initiating transcription and translation of the antibody.

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

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

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

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

27. A method for recombinantly producing a monoclonal antibody, the method comprising: a) providing a mammalian cell-free protein expression system containing a target protein displaying an antibody epitope; b) modifying the mammalian cell-free system by introducing one or more agents that block the antibody epitopes on the target protein but do not eliminate the activity of the target protein in the cell-free system; c) introducing into the cell-free system one or more nucleic acids encoding monoclonal antibodies that bind to the antibody epitope; and, d) initiating transcription and translation of said monoclonal antibody in said mammalian cell-free system under conditions allowing production of said antibody.

28. The method of claim 26, wherein the mammalian cell-free system comprises a cell lysate.

29. The method of claim 26, further comprising (c) isolating the antibody from the mammalian cell-free system.

30. A method for recombinantly producing a monoclonal antibody, the method comprising a) providing a mammalian cell-free protein expression system containing a target protein displaying an antibody epitope; b) modifying said mammalian cell-free system by introducing one or more agents that block the antibody paratope binding to said antibody epitope on said target protein; c) introducing one or more nucleic acids encoding monoclonal antibodies comprising said antibody paratope into said cell-free system; and, d) initiating transcription and translation of said monoclonal antibody in said mammalian cell-free system under conditions allowing production of said antibody.

31. The method of claim 29, further comprising removing the agent that binds to the antibody paratope after producing the monoclonal antibody.

32. The method of claim 29, further comprising isolating the monoclonal antibody from the cell-free system.

33. The method of claim 30, wherein the monoclonal antibody is isolated prior to removing the agent from the monoclonal antibody.

34. A method for recombinantly producing an antibody, the method comprising: a) introducing into cells an agent that selectively binds to a target epitope on an intracellular protein to produce a modified cell line; b) generating a cell-free antibody production system from said 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; as well as, d) initiating transcription and translation from the nucleic acid template to produce the antibody in the cell-free antibody production system.

35. The method of claim 33, further comprising e) isolating the antibody from the cell-free system.

36. The method of claim 33, wherein the target protein is an intracellular protein in the secretory pathway of the cell line.

37. The method of claim 33, wherein the target protein is a stress protein.

38. The method of claim 33, wherein the target protein is a cell signaling protein.

39. The method of claim 33, wherein the target protein is expressed on an organelle of the cell line.

40. The method of claim 38, wherein the organelle is the endoplasmic reticulum or the Golgi apparatus.

41. The method of claim 39, wherein the target protein is an endoplasmic reticulum chaperone.

42. The method of claim 40, wherein the endoplasmic reticulum chaperone is calreticulin, a heat shock protein, or an isomerase.

43. The method of claim 40, wherein the target protein is glucose-regulated protein 78 (GRP78), HSP47, PDI, calreticulin, or GP94.

44. The method of claim 40, wherein the target protein is glucose-regulated protein 78 (GRP78).

45. The method of claim 38, wherein the target protein is a Golgi complex protein.

46. ​​The method of claim 44, wherein the Golgi complex protein is GOLPH2, GOLPH3, GM130, ATP6V1A, ATP6V1E1, ATP6VOA2, TMEM165, GOLGB1, SCYL1BP1, TRAPPC11, TRAPPC2, or TRIP11.

47. The method of claim 33, wherein the cell line is a mammalian cell line.

48. The method of claim 46, wherein the naturally expressed target protein is an intracellular protein in the secretory pathway of the mammalian cell line.

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