Cell-free method for recombinant antibody production

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 the modified system is engineered to express a variant target protein having a mutant epitope of a target epitope of an antibody.

Description

[0001] Related Applications

[0002] This application claims priority to U.S. Provisional Patent Application Serial No. 63 / 359,871 filed on July 10, 2022, which is hereby incorporated by reference 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 MBRACE003.xml, and is 35,964 bytes in size (as at 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 typically 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 and bind to almost any type of antigen with high affinity makes them attractive as a starting point for novel biopharmaceutical products and scientific research. Recently, since certain 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 are urgently needed to produce such antibodies, 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 making antibodies that bind to an intracellular target protein. The present disclosure also provides methods for producing a cell-free system for making antibodies that bind to an intracellular target protein.

[0009] In some embodiments, the present disclosure provides a method for recombinantly producing an antibody, the method comprising 1) expressing a nucleic acid encoding the antibody in a modified cell-free system, wherein the antibody specifically binds to a target epitope on a protein in the system, and wherein the modified cell-free system comprises a mutation of one or more amino acid residues of the target epitope on the protein in the system; and 2) initiating transcription and translation of the antibody in the cell-free system under conditions such that the antibody is produced.

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

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

[0012] In some embodiments, the present disclosure provides a method for recombinantly producing a monoclonal antibody, the method comprising 1) expressing a nucleic acid encoding an antibody in a modified eukaryotic cell-free system, wherein the antibody specifically binds to a target epitope on a protein in the system, and wherein the modified cell-free comprises a mutation in one or more amino acid residues of the target epitope on the protein in the system; and 2) initiating transcription and translation of the antibody in the cell-free system under conditions such that the antibody is produced.

[0013] In some embodiments, the present disclosure provides a method for recombinantly producing an antibody, the method comprising 1) modifying a 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; 2) creating 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 the antibody to the target protein; and 4) initiating transcription and translation from the nucleic acid template to produce the antibody in the cell-free antibody production system.

[0014] In some embodiments, the present disclosure provides a method for recombinantly producing a monoclonal antibody, the method comprising 1) modifying 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; 2) creating a cell-free antibody production system from the modified mammalian cell line; 3) introducing a nucleic acid template into the cell-free antibody production system, wherein the nucleic acid template encodes the antibody to the target protein; and 4) initiating transcription and translation from the nucleic acid template to produce the antibody in the cell-free antibody production system.

[0015] In some embodiments, the naturally expressed target protein is an intracellular protein in a secretory pathway. In some embodiments, the target protein is a stress protein. In some embodiments, the mutation is an inert mutation that retains the structure and function of the native target protein. In some embodiments, the mutation reduces or eliminates the binding of the antibody to the target epitope on the target protein.

[0016] In some embodiments, the target protein is expressed on an organelle of the cell line.

[0017] 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), heat shock protein 47 (HSP47), protein disulfide isomerase (PDI), calreticulin or GP94.

[0018] In a specific embodiment, the target protein is generally expressed on the Golgi apparatus. For example, the target protein can be a Golgi complex protein, for example, 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 protein 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).

[0019] In some embodiments, the target protein is associated with a membrane within the cell.

[0020] 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.

[0021] In some embodiments, antibodies are isolated from the cell-free system after production.

[0022] Additional features, advantages and aspects are described in greater detail below. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] Figure 4 Depicted are genomic sequence analysis of the interacting residues of anti-GRP78 clones B4 and F6 for residues located in exon 5 and flanking intronic residues in GRP78. Also shown is the amino acid sequence of full-length GRP78.

[0028] Figure 5A Depicted are the designs of dual gRNA and ssDNA donor templates for introducing exemplary mutations into GRP78 via HDR-based CRISPR gene editing.

[0029] Figure 5B Describes the verification Figure 5A Design of primer pairs for PCR amplification of gene editing as shown. DETAILED DESCRIPTION

[0030] 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 implemented without each of these specific features and functions.

[0031] 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 one or more embodiments in any suitable manner. Further, it is intended that each embodiment of the disclosed subject matter covers modifications and variations thereof.

[0032] 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.

[0033] In addition, the terms "approximately," "about," "close to," "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.

[0034] All features described in conjunction with one embodiment are intended to be applicable to the additional embodiments described below, unless expressly stated otherwise or the feature or feature is incompatible with the additional 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.

[0035] 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 as an intracellular protein in a cell. In some 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 some 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 a 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 a cancer cell but is not generally found on the surface of a normal cell. 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 a cancer cell, stress proteins exhibit the desired characteristics of a target protein used as a cancer therapy based on an antibody. Using stress proteins as a target protein based on an antibody therapy ensures that its surface expression in various cancers is upregulated, but it will not be expressed in normal tissues and cells, and reduces or minimizes off-target activity and / or toxicity.

[0036] Methods are needed to produce sufficient quantities of antibodies for use as therapeutic agents. 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.

[0037] Particularly desirable for recombinant production of human or humanized antibodies is a mammalian expression system. 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 recombinant proteins derived from HEK293 cells or CHO cells, glycosylation patterns similar to but not identical to those obtained from human cells can be obtained. Mammalian cell-free systems achieve very high product yields and are relatively robust to metabolic stress.

[0038] Although efforts have been made to produce antibodies against stress proteins, the antibodies have not been effectively produced in mammalian cells. For example, the observations herein demonstrate that antibodies against the exemplary stress protein GRP78 cannot be expressed in high yield from mammalian cells without using the methods 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, misfolded protein lack of degradation, calcium homeostasis disorders, and in some cases cell viability reduction or loss in the cell. The loss or reduction of this function affects the ability to produce antibodies in high yield.

[0039] Studies have shown that stress proteins (such as molecular chaperones) are necessary for normal function in cells during antibody production. For example, changes in the cellular abundance of secretory pathway proteins have been shown to be associated with the ability to produce large amounts of 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 are folded and assembled 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. 1999 Dec 3; 286: 1882-8; Helenius et al., Intracellular functions of N-linked glycans, Science. 2001 Mar 23; 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 & Furtak, K., Cell 74, 909-917 (1993); Ellis, RJ & Hemmingsen, SM, TiBS, 14, 339-342, (1989); Bukau, B., Hesterkamp, ​​T. & Luirink, J., Trends Cell Biol, 6, 480-486, (1996); Bukau, B., Deuerling, E., Pfund, C. & Craig, EA, Cell, 101, 119-122, (2000)). Secretory proteins also undergo various post-translational modifications, including glycosylation, during their passage through the Golgi complex.

[0040] 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.

[0041] The provided method relates to a modified cell line and a cell-free system for producing an antibody or antigen-binding fragment of an endogenous intracellular target protein (such as a target protein in a secretory pathway) in a targeted cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the target protein is a stress protein. In some embodiments, the provided method relates to a modified mammalian cell carrying a variant of a target protein by mutating the target epitope to a mutant epitope, so that the binding of the antibody to the endogenous target protein in the mammalian cell is reduced or eliminated. In a specific embodiment, the mutation is a genomic mutation of the target protein introduced by a gene editing method (such as gene editing mediated by a nuclease). In some embodiments, the mutation is introduced using an RNA-guided nuclease, a zinc finger nuclease, a large range of nucleases, or a transcription activator-like effector nuclease. In some cases, the mutation is introduced by homology-directed repair (HDR). In a specific embodiment, the mutation is an inert mutation, so that the variant target protein retains the structure and function of the endogenous target protein, but the epitope of the antibody is modified to eliminate the binding of the antibody to the target protein. In the provided methods, the modified cells can then be used to produce antibodies or antigen-binding fragments, such as by expressing a nucleic acid molecule encoding the antibody in the cell and culturing the cell under conditions that produce the antibody. In some embodiments, the produced antibodies can be isolated from the cells.

[0042] The results herein show that the provided methods can substantially improve protein production in mammalian cells. In some embodiments, the amount of antibodies produced by modified mammalian cells according to the provided methods is increased by 2 times or more compared to producing the same antibodies in natural mammalian cells in which the target protein has not mutated. In some embodiments, antibody production is increased by greater than or about 5 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times or more compared to producing the same antibodies in natural mammalian cells in which the target protein has not mutated. For example, in some cases, antibody production is increased by at least 20 times compared to natural mammalian cells in which the target protein has not mutated.

[0043] The production systems and methods provided are exemplified by antibodies targeting GRP78 and other intracellular target proteins. The systems and methods described herein can be extended to the production of 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 in 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.

[0044] 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.

[0045] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0046] Methods for cell generation in cell-free systems

[0047] A method for recombinantly producing an antibody is provided herein, wherein the antibody is combined with a target protein (or its homologue) that is usually naturally expressed as an intracellular protein. In the cell or cell-free system and method provided, the antibody is produced in a modified system (e.g., a mammalian cell line or a cell-free system), and the modified system is genetically altered to contain a variant of the target protein, wherein the antibody is reduced or eliminated from the combination of such variant target proteins. In some embodiments, the variant target protein is modified in a mammalian cell line for the preparation of a system so that the variant target protein contains a mutant epitope with a mutation of one or more amino acid residues of the target epitope of the antibody, to reduce or eliminate the recognition performed by the antibody or antigen-binding fragment. In a specific embodiment, the mutation of the variant target protein with a mutant epitope is an inert mutation that retains the structure and function of the natural intracellular protein, thereby ensuring the function of the target protein. Therefore, the method provided using a modified cell line or a cell-free system allows the production of antibodies in a manufacturing system at a high yield.

[0048] In some embodiments, the methods provided involve (a) expressing a nucleic acid encoding an antibody in a modified cell line, wherein the antibody is directed against a target protein or a homolog of the target protein that is naturally expressed as an intracellular protein in a cell, and wherein the modified cell is engineered with a variant target protein having a mutant epitope comprising a mutation of one or more amino acid residues of the target epitope of the antibody or antigen-binding fragment; and (b) culturing the modified cell line under conditions that allow the antibody to be produced. In some embodiments, the method may further include isolating the antibody from the culture supernatant.

[0049] In some embodiments, the methods provided involve (a) expressing a nucleic acid encoding an antibody in a modified mammalian cell line, wherein the antibody is directed against a target protein or a homolog of the target protein that is naturally expressed as an intracellular protein in a mammalian cell, and wherein the modified mammalian cell is engineered with a variant target protein having a mutant epitope comprising a mutation of 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 that allow the production of the antibody. In some embodiments, the method may further include, for example, isolating the antibody from the culture supernatant.

[0050] 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 hereby incorporated into the present application as a whole 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 (in some preferred embodiments, a mammalian cell line), wherein the antibody specifically binds to a target epitope on a target protein naturally expressed as an intracellular protein in a 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 (and in some preferred embodiments, separating the antibody) under conditions such that the antibody is produced. 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 intracellularly in a cell line (in some preferred embodiments, a mammalian cell line); b) identifying the epitope of the target protein to which the antibody binds; c) generating a modified cell line by mutating the epitope of the target protein in the cell line to reduce or eliminate 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 antibody to be produced by the expression vector; and f) expressing a 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 the 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.

[0051] A. Intracellular protein targets and their antibodies

[0052] 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 specific embodiments, 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 or in the endoplasmic reticulum. In some embodiments, the target protein is expressed on or in the Golgi apparatus.

[0053] 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.

[0054] 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 the production of biological agents that target endoplasmic reticulum chaperones as well as other identified targets without excessively compromising cell viability. Exemplary biologics may include, for example, biologics targeting HSP47 to treat 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) to treat central nervous system cancer, ovarian cancer, brain cancer, prostate cancer, and lung cancer (Xu Set 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 to treat various cancers (Zamanian M et al., Pathol Oncol Res 149-54 (2013); Gold LI et al., FASEB J 24:665-83 (2010)), biological agents targeting GP94 (Marzec M. et al., Biochim Biophys Acta. 2012 March; 1823(3):774-787)), and biological agents targeting GRP78 (Arap et al. Cancer Cell 6:275-284 (2004); Sato et al. Adv Genet 69:97-114 (2010)).

[0055] 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 generating 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) (same as above); 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).

[0056] 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 interacting factor 11 (TRIP11).

[0057] 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); anti-GRP78 antibodies described in the following documents: 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 against protein disulfide isomerase (PDI) include, but are not limited to, Invitrogen PDI monoclonal antibody clone 12 (Thermo Fisher Scientific catalog number MA5-43389) or Invitrogen PDI monoclonal antibody clone 2F6G12H2 (Thermo Fisher Scientific catalog number MA5-43389). Antibodies against calreticulin include, for example, mAb FMC 75 (Enzo Life Sciences, catalog number ADI-SPA-601) and mAb 16 (BD Transduction laboratories, catalog number 612137). Antibodies against GOLPH3 include, for example, Thermo Fisher Scientific 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.

[0058] In some 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 attack. 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.

[0059] In addition to using non-human monoclonal antibodies for humanization purposes, non-human antibodies can also be used as templates for selecting fully human or nearly fully 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 described 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 generated in which 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. Antibodies with desired specificity are selected by affinity with the selected epitope of organelle proteins.

[0060] 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.

[0061] The antibodies provided are monoclonal antibodies, including monoclonal antibody fragments.

[0062] B. Design of mutant epitopes on target proteins

[0063] In some embodiments, the method provided involves mutating the target protein to produce a variant target protein containing a mutant epitope to eliminate or reduce the binding by an antibody or antigen-binding fragment. In some embodiments, the mutant epitope is mutated by one or more amino acid residues of the target epitope of the antibody or antigen-binding fragment. Various methods can be used to map the epitopes of the monoclonal antibodies to be produced using the methods of the present disclosure to determine the edits that can be produced to produce such antibodies using the methods of the present disclosure. Exemplary methods are taught in U.S. Patent No. 11,174,479, entitled "Devices and methods for display of encoded peptides, polypeptides, and proteins on DNA", issued on November 16, 2021, and U.S. Patent No. 11,061,036, entitled "Epitope Mapping", issued on July 13, 2021, to Wilson et al.

[0064] 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, and then mutated. In some embodiments, the cDNA encoding the target protein is randomly mutated by method (such as by treating with chemical mutagens, irradiating during replication, by passage of error-induced (mutant gene) cell lines, or by oligonucleotide-directed random mutagenesis). In some embodiments, oligonucleotide-directed random mutagenesis is used, wherein the preselected area of ​​the targeted protein is to carry out random mutagenesis. In some embodiments, the preselected area for random mutagenesis is a region that is considered to contain a binding site or epitope. The preliminary assessment of the preselected area can be achieved by any method known in the art (including hydration analysis of protein sequences or by crystal structure analysis).

[0065] In some embodiments, peptide-based methods can be used to map the epitope of the antibody. In one method, peptide display technology displays a library of protein fragments on a microarray or on the surface of E. coli or phage, and finds out which fragments the antibody binds to by microarray scanning or flow cytometry. In another method, a series of overlapping peptides of the target protein or its preselected regions are screened for antibody binding, such as by ELISA or other techniques to monitor the binding interaction.

[0066] 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 epitope is verified by experiment. Methods for experimentally testing antibody epitopes are known in the art, including, for example, performing verification using an antibody cross-blocking assay, performing an assay (including screening a library), performing mutation analysis, performing a deuterium exchange analysis, performing a peptide binding assay, and / or performing an X-ray crystallographic study. In some embodiments, verification uses a library and / or peptide derived from a target protein to assess the importance of a specific amino acid in a specific position for binding. In some embodiments, the library comprises a subset or all of the mutants 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.

[0067] In some embodiments, in order to identify residues to be edited to reduce or eliminate the binding of the antibody to the target intracellular protein, it is desirable to start with a resolved crystal structure of the intracellular protein so that key contact residues that will disrupt antigen binding but not interfere with its cellular function can be identified and replaced. This resolved structure can be a resolved crystal structure of the intracellular protein alone, or a resolved crystal structure of the intracellular protein bound to an antibody or antibody fragment.

[0068] 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 (Cunningham BC and Wells JA, Proc Natl Acad Sci US A. 1991Apr 15; 88 (8): 3407-3411) related to antigen binding can be identified. If a single residue is changed to Ala, it will reduce but will not destroy binding, then the position can be targeted to carry out more intense mutations (e.g., substitutions created in charge difference), to further reduce binding (if desired).

[0069] 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.

[0070] As shown for GRP78 in Example 3 herein, once the structure of the target protein has been elucidated, one of ordinary skill in the art can identify residues that can be modified to reduce antibody binding to an epitope of an organelle protein while retaining sufficient function of the organelle protein to maintain cell viability, as taught in more detail herein. Methods for preparing polypeptides comprising one or more mutations are well known to those of ordinary skill in the art.

[0071] In some embodiments, one or more amino acid residues in or near the identified or known epitope of the target protein are mutated. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 or more amino acid residues of the target protein are mutated to produce a variant target protein. In some embodiments, the one or more mutations produce mutant epitopes that destroy one or more (or all) target epitopes on the target protein. In some embodiments, the one or more mutations are in the target epitope for a specific antibody. In some embodiments, the one or more mutations are conservative mutations. In some embodiments, the one or more mutations are non-conservative mutations. In some embodiments, the one or more mutations are a mixture of conservative mutations and non-conservative mutations. In some embodiments, mutations are substitutions, deletions or insertions.

[0072] In some embodiments, the variant target protein contains at least one amino acid substitution of the amino acid sequence of the epitope of the target protein of the antibody. In some embodiments, the variant target protein contains one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, 10 or more substitutions of the amino acid sequence of the epitope of the target protein of the antibody. In some embodiments, the variant target protein contains one amino acid substitution of the amino acid sequence of the epitope of the target protein of the antibody. In some embodiments, the variant target protein contains two amino acid substitutions of the amino acid sequence of the epitope of the target protein of the antibody. In some embodiments, the variant target protein contains three amino acid substitutions of the amino acid sequence of the epitope of the target protein of the antibody. In some embodiments, the variant target protein contains four amino acid substitutions of the amino acid sequence of the epitope of the target protein of the antibody. In some embodiments, the variant target protein contains five amino acid substitutions of the amino acid sequence of the epitope of the target protein of the antibody.

[0073] In a specific embodiment of the provided method, the mutation does not affect the function of the target protein. In some embodiments, the mutation is an inert mutation, in which the structure and function of the target protein are retained. In some embodiments, the one or more mutations will not destroy the function of the polypeptide (for example, relative to the function of the corresponding unmutated target protein, the function of the variant target protein will not be destroyed). In some embodiments, the one or more mutations will not destroy the natural protein-protein interaction of the protein (for example, the variant protein retains the ability to form a protein-protein interaction substantially the same as the corresponding unmutated target protein). In some embodiments, the one or more mutations will not destroy the three-dimensional structure of the polypeptide (for example, the variant target protein retains the three-dimensional structure substantially the same as the corresponding unmutated target protein). In some embodiments, the one or more mutations will not destroy the folding of the protein (for example, the variant protein retains the protein folding substantially the same as the corresponding unmutated target protein). In some embodiments, the one or more mutations will not destroy the translation of the protein (for example, with the same timing as the corresponding unmutated target protein, at the same rate, to the same level, etc. to translate the variant protein). In some embodiments, the one or more mutations will not destroy the normal cellular localization of the protein (for example, the variant target protein retains the cellular localization substantially the same as the corresponding unmutated target protein). In some embodiments, the one or more mutations do not disrupt any post-translational modifications on the target protein (e.g., the variant target protein retains substantially the same post-translational modification profile as the corresponding non-mutated target protein).

[0074] In some embodiments, the one or more mutations do not significantly affect the viability of the cell line. In some embodiments, the viability of the modified cell line containing the variant target protein under standard culture and passage conditions is retained compared to the cell line containing the unmutated target protein.

[0075] In some embodiments, the cell line is a mammalian cell line. In some embodiments, the one or more mutations do not significantly affect the viability of the mammalian cell line. In some embodiments, the viability of the modified mammalian cell line containing the variant target protein under standard culture and passage conditions is retained compared to a mammalian cell line containing an unmutated target protein.

[0076] In some embodiments, the one or more mutations reduce or eliminate the combination of the antibody and the target protein. In some embodiments, the combination and / or reactivity of one or more antibodies and variant target proteins (e.g., containing mutant epitopes) can be assessed. The method for assessing the combination with the polypeptide is known in the art, and may include but is not limited to performing ELISA assays, performing Western blot analysis, performing radioimmunoassay (RIA), performing surface plasmon resonance (SPR), performing thermophoresis, performing competitive assays, and performing isothermal titration calorimetry. In some embodiments, the binding and / or reactivity to a variant target protein comprising the one or more substitutions 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 binding and / or reactivity to the target protein lacking the one or more substitutions. In some embodiments, binding and / or reactivity to a variant target protein comprising one or more substitutions 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 binding and / or reactivity to a target protein lacking one or more substitutions. In some embodiments, binding and / or reactivity to a variant target protein is eliminated (e.g., binding and / or reactivity is not detected by a binding assay, e.g., relative to a negative control, such as an isotype control).

[0077] In some embodiments, the variant target protein with reduced or eliminated binding to the antibody exhibits a reduced affinity of the antibody to the variant target protein. In some embodiments, the affinity of the antibody to the variant 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 to the target protein lacking one or more mutations. In some embodiments, the affinity of the antibody for the variant 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 lacking one or more mutations. In some embodiments, the affinity of the antibody for the variant target protein is eliminated (e.g., no binding of the antibody to the variant target protein is detected).

[0078] 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.

[0079] C. Generation of modified cell lines carrying variant target proteins with mutant epitopes

[0080] In some embodiments, a modified cell line is generated by mutating an epitope of a target protein in a mammalian cell line to reduce or eliminate binding of the antibody to an epitope on the target protein. In some embodiments, the cell line is a mammalian cell line.

[0081] Various mammalian cell culture systems can be used to produce modified cell lines. Examples of suitable host cells include cell lines, including but not limited to Chinese hamster ovary (CHO) cells, baby hamster kidney cells, NSO myeloma cells, monkey kidney COS cells, monkey kidney fibroblast CV-1 cells, human embryonic kidney 293 (HEK293) cells, human breast cancer SKBR3 cells, human leukemia Jurkat T cells, dog kidney MDCK cells, human cervical cancer HeLa cells. In some embodiments, the mammalian cell line is a CHO cell line. An example of a CHO cell line is a DHFR CHO cell, which is auxotrophic for glycine, thymidine and hypoxanthine. Other CHO cell lines (such as CHO-k1, CHO-S, GS-CHO) with the same genomic background are also suitable for recombinant protein expression.

[0082] Methods for introducing nucleic acid mutations into a gene of interest are well known in the art (see, e.g., Menke D. Genesis (2013) 51:618; Capecchi, Science (1989) 244:1288-1292; Santiago et al. Proc Natl Acad Sci USA (2008) 105:5809-5814; International Patent Application Nos. WO 2014085593, WO 2009071334 and WO 2011146121; U.S. Patent Nos. 8,771,945, 8,586,526, 6,774,279 and UP Patent Application Publication Nos. 20030232410, 20050026157, US20060014264; the contents of which are incorporated by reference in their entirety), and include targeted homologous recombination, site-specific recombinases, PB transposases, and genome editing by engineered nucleases. Agents for introducing nucleic acid changes into a gene of interest can be designed from publicly available sources or commercially available from Transposagen, Addgene, and Sangamo Biosciences.

[0083] Exemplary methods for introducing nucleic acid mutations into a target protein include genome editing using endonucleases, meganucleases, zinc finger nucleases, and transcription activator-like effector nucleases (TALENs).

[0084] In some embodiments, the method for introducing a nucleic acid mutation into a target protein involves genome editing using an engineered nuclease. In some embodiments, this method involves cutting and producing specific double-strand breaks at the desired position in the genome using an artificially engineered nuclease, and then repairing the reverse genetic method by endogenous processes such as homology-directed repair (HDR) and non-homologous end joining (NHEJ). NHEJ directly joins the DNA ends in double-strand breaks, while HDR uses homologous sequences as donor templates for regenerating missing DNA sequences at breakpoints. In order to introduce specific nucleotide modifications (e.g., mutations, such as amino acid substitutions) into genomic DNA, a DNA repair template containing the desired sequence must be present during HDR. Genome editing cannot be performed using traditional restriction endonucleases, because most restriction enzymes recognize several base pairs on DNA as their targets, and the probability that the recognized base pair combinations will be found in many positions across the genome is very high, so that multiple cuts are not limited to the desired position. In order to overcome this challenge and produce site-specific single-strand or double-strand breaks, several different classes of nucleases have been found and bioengineered so far. Such nucleases include meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases (RGNs), such as type II and type V RGNs.

[0085] In certain aspects, including those disclosed herein, the genome editing of the illustrative embodiments utilizes clustered regularly interspaced short palindromic repeats (CRISPR) technology, wherein RGN is used to edit a specific target region in the genome of an organism. A typical CRISPR system for genome editing contains different components: a guide RNA ("gRNA"), a target region homologous to a gene of interest containing the intended editing (commonly referred to as a "homologous arm" or "donor template"), and RGN, such as Cas9, Cpf1, or MAD7. By delivering RGN, synthetic gRNA, and donor template into cells, the genome of the cell can be cut at a desired position, thereby allowing the target region of the genome to be edited. The guide RNA helps the RGN protein to recognize and cut the DNA of the target genomic region based on the identified protospacer adjacent motif (PAM). The binding of gRNA locates the RGN to the genomic target sequence so that the RGN can cut both chains of the DNA, thereby causing a double-strand break. The double-strand break produced by the RGN can be subjected to homology-directed repair or NHEJ.

[0086] Various RGN systems can be used in the cell systems and methods disclosed herein. These systems include, but are not limited to, type II systems (e.g., systems using RGNs such as Cas9) and type V systems (e.g., systems using RGNs such as MAD7 (e.g., Wierson WA et al., CRISPR J. 2(6): 417-433 (2019)) or Cpf1 (e.g., Zetsche et al., Cell. 2015 Oct 22; 163(3): 759-771; Gao L et al., Nat Biotechnol. 35(8): 789-792 (2017)); using type I CRISPR systems (e.g., Cas3 (e.g., Morisaka H. et al., Nat Commun, 2019 Dec 6; 10(1): 5302) or Cas10d, functional nucleases in type ID systems (e.g., Osakabe et al., Nucleic Acids Res. 2021 Jun 21;49(11):6347-6363) of the RGN system.

[0087] In some embodiments, the recombinant nuclease is Cas9. In some embodiments, Cas9 is from Streptococcus pyogenes (SpCas9). In some embodiments, Cas9 is from Staphylococcus aureus (SaCas9). In some embodiments, Cas9 is from Neisseria meningitidis (NmeCas9). In some embodiments, Cas9 is from Campylobacter jejuni (CjCas9). In some embodiments, Cas9 is from Streptococcus thermophilis (StCas9).

[0088] In some embodiments, the recombinant nuclease is Cpf1. In some embodiments, the recombinant nuclease is a modified Cpf1, such as the Alt-R Cas12a system (Integrated DNA Technologies, Coralville, Iowa). In some embodiments, the recombinant nuclease is MAD7.

[0089] In some embodiments, RGN comprises one or more mutations, so that RGN is converted into a nickase lacking the ability to cut two chains of a double-stranded DNA molecule. In some embodiments, RGN comprises one or more mutations, so that RGN is converted into a nickase capable of cutting only one chain of a double-stranded DNA molecule. For example, by mutating one catalytic domain in two Cas9 catalytic domains, i.e., a RuvC domain (which comprises RuvC I, RuvC II, and RuvC III motifs) or an NHN domain, Cas9 (which is generally capable of inducing double-strand breaks) can be converted into a Cas9 nickase (which is capable of inducing single-strand breaks). In some embodiments, type II RGN comprises one or more mutations in a RuvC catalytic domain or a HNH catalytic domain. In some embodiments, a recombinant nuclease is a recombinant nuclease that has been modified to have nickase activity. In some embodiments, a recombinant nuclease cuts a chain hybridized with a guide RNA, but does not cut a chain complementary to a chain hybridized with a guide RNA. In some embodiments, a recombinant nuclease does not cut a chain hybridized with a guide RNA, but cuts a chain complementary to a chain hybridized with a guide RNA.

[0090] In some embodiments, the recombinant nuclease used for editing is a fusion protein containing both nuclease activity and another enzymatic activity (e.g., reverse transcriptase activity, transposase activity, recombinase activity, or other activity that enhances the editing rate in the cell). See, for example, Newby GA, Liu DR. Mol Ther. 2021 Nov 3; 29(11): 3107-3124. In some embodiments, genome editing that introduces one or more desired edits into the target region is base editing that does not require double-strand breaks or donor DNA. Anzalone AV, et al., Nature. 2019 Dec; 576(7785): 149-157. doi: 10.1038 / s41586-019-1711-4. Epub 2019 Oct 21. PMID: 31634902.

[0091] In certain aspects, RGN directed genome editing introduces one or more desired edits into the target region and removes the original spacer motif (PAM) region from the target region, thereby excluding any additional editing of the genome at the target region. In this regard, for example, RGNs compounded with synthetic gRNAs complementary to the target region can be used to select cells with desired edits. Cells that have not undergone the first editing event will be cut, and therefore will not be able to continue to survive under appropriate selection criteria. Cells containing desired mutations will not be cut because the cells will no longer contain the necessary PAM sites.

[0092] There are many publicly available tools that can be used to help select and / or design target sequences and lists of unique gRNAs for targeting specific genomic regions in different species determined bioinformatically using different RGNs, such as the Target Finder from Feng Zhang's laboratory, the Target Finder (E-CRISP) from Michael Boutros' laboratory, the RGEN Tools: Cas-OFFinder, CasFinder: A Flexible Algorithm for Identifying Specific Cas9 Targets in the Genome, and the CRISPR Optimal Target Finder; the Alt-R HDR Design Tool and Templates provided by Integrated DNA Technologies (Coralville, IA), and the MAD7 Gene Editing Design Tool from Horizon (Cambridge, UK).

[0093] In some embodiments, RGN and gRNA are introduced into cells in the form of a ribonucleoprotein (RNP) complex. The RNP complex comprises a protein (such as a recombinant nuclease or a protein comprising nuclease activity) and a guide RNA. In some embodiments, the recombinant nuclease is provided in the form of a protein, and the guide RNA is provided in the form of a transcribed or synthetic RNA. In some embodiments, the guide RNA forms an RNP complex with the RGN under appropriate conditions before delivery to the cell.

[0094] Although the examples described herein utilize RGN directed editing systems, it will be apparent to those skilled in the art upon reading this disclosure that various editing mechanisms may be used to generate the disclosed cells, systems, and preparation methods. There are a variety of different nuclease-based systems to provide editing to the genome of an organism, and each system may be used for a single editing system, a sequential editing system (e.g., using different nuclease directed systems in sequence to provide two or more genome edits in a cell) and / or a recursive editing system (e.g., using a single nuclease directed system to introduce two or more genome edits into a cell). Therefore, those skilled in the art upon reading this disclosure will recognize that various enzyme directed editing systems are useful for the disclosed embodiments.

[0095] For example, in certain embodiments, zinc finger nuclease genome editing can be used to introduce genome changes described herein. "Zinc finger nuclease" (ZFN) is an artificial restriction enzyme produced by fusing a zinc finger DNA binding domain with a DNA cleavage domain. The zinc finger domain can be engineered into a target-specific region in the genome of an organism. (Urnov et al., Nature Reviews Genetics, 11: 636-646 (2010); International patent application published WO 2003 / 087341 A2 to Carroll et al., filed on January 22, 2003). Using the endogenous DNA repair mechanism of an organism, ZFN can be used to accurately change the target region of the genome. ZFN can be used to produce double-strand breaks ("DSBs") in DNA in alleles, and in the absence of homologous templates, the DSBs will be repaired by non-homologous end joining (NHEJ). NHEJ repairs DSBs by joining two ends together, and generally does not produce additional mutations, provided that the cutting is clean and not complicated. (Dural et al., Nucleic Acids Res., 33(18):5978-90 (2005)). This repair mechanism can be used to induce edits in the genome through indels or chromosomal rearrangements.

[0096] In selected embodiments, the genome changes described herein can be introduced using transcription activator-like effector nuclease editing. Transcription activator-like effector nucleases (TALENs) are restriction enzymes that can be engineered to cut specific DNA sequences. TALENs are produced by fusing TAL effector DNA binding domains with DNA cleavage domains. TALENs can be engineered to bind to almost any desired DNA sequence, so when bound to nucleases, DNA can be cut at specific locations. (See, for example, Miller, et al., Nature Biotechnology, 29 (2): 143-8 (2011); Boch, Nature Biotechnology, 29 (2): 135-6 (2011); International patent application published WO 2010 / 079430 A1 to Bonas et al., filed on January 12, 2010; International patent application published WO 2011 / 072246 A2 to Voytas et al., filed on December 10, 2010).

[0097] Like ZFNs, TALENs can edit the genome by inducing DSBs. Site-specific DSBs generated by TALENs at the target region are repaired by NHEJ or HDR, resulting in targeted genome editing. TALENs can be used to introduce indels, rearrangements, or introduce DNA into the genome by NHEJ in the presence of exogenous double-stranded DNA fragments.

[0098] ZFN and TALEN restriction endonuclease technology utilizes non-specific DNA cleavage enzymes connected to specific DNA binding domains (respectively a series of zinc finger domains or TALE repeats). Generally, a restriction enzyme whose DNA recognition site and cleavage site are separated from each other is selected. The cleavage portion is separated and then connected to the DNA binding domain, thereby producing a nuclease with very high specificity for the desired sequence. An exemplary restriction enzyme with such properties is Fok1. In addition, the advantage of Fok1 is that dimerization is required to have nuclease activity, and this means that specificity increases significantly as each nuclease partner recognizes a unique DNA sequence. In order to enhance this effect, Fok1 nuclease has been engineered to only act as a heterodimer and have an increased catalytic activity. The nuclease of heterodimer function avoids the possibility of undesirable homodimer activity and thus increases the specificity of double-strand breaks.

[0099] Therefore, for example, in order to target specific sites, ZFN and TALEN are constructed as nuclease pairs, wherein each member of the pair is designed to bind adjacent sequences at the target site. When transiently expressed in cells, the nuclease binds to its target site, and the Fok1 domain heterodimerizes to produce double-strand breaks. The repair of these double-strand breaks by non-homologous end joining (NHEJ) pathways most commonly produces less deletions or less sequence insertions. Because each repair performed by NHEJ is unique, a single nuclease pair can be used to produce an allele series with a series of different deletions at the target site. The range of the length of the deletion is generally a few base pairs to hundreds of base pairs elsewhere, but in cell culture, larger deletions have been successfully produced by using two nuclease pairs simultaneously (Carlson et al., 2012; Lee et al., 2010). In addition, when a fragment of DNA with homology to the targeted region is introduced in combination with a nuclease pair, double-strand breaks can be repaired by homology-directed repair to produce specific modifications (Li et al., 2011; Miller et al., 2010; Urnov et al., 2005).

[0100] Although the nuclease parts of both ZFN and TALEN have similar properties, the difference between these engineered nucleases lies in their DNA recognition peptides. ZFN relies on Cys2-His2 zinc fingers, and TALEN relies on TALE. These two DNA recognition peptide domains have the property that they are naturally present in their proteins in a combined form. Cys2-His2 zinc fingers are usually present in repetitive sequences separated by 3bp, and are present in a variety of nucleic acid interaction proteins in the form of different combinations. On the other hand, TALE is present in a repetitive sequence with a one-to-one recognition ratio between amino acids and recognized nucleotide pairs. Because both zinc fingers and TALE occur in a repetitive pattern, different combinations can be tried to produce various sequence specificities. Methods for preparing site-specific zinc finger endonucleases include, for example, modular assembly (wherein zinc fingers associated with triplet sequences are connected in rows to cover the desired sequence), OPEN (low stringency selection of peptide domains for nucleotide triplets followed by high stringency selection of peptide combinations for the final target in bacterial systems) and bacterial single hybrid screening of zinc finger libraries, etc. ZFN can also be designed and obtained from, for example, Sangamo Biosciences TM (Richmond, CA).

[0101] Methods for designing and obtaining TALEN are described in, for example, Reyon et al. Nature Biotechnology 2012 May; 30(5): 460-5; Miller et al. Nat Biotechnol. (2011) 29: 143-148; Cermak et al. Nucleic Acids Research (2011) 39(12): e82 and Zhang et al. Nature Biotechnology (2011) 29(2): 149-53. A recently developed web-based program called Mojo Hand was introduced by the Mayo Clinic to design TAL and TALEN constructs for genome editing applications (accessible via www.talendesign.org). TALEN can also be designed and obtained from, for example, Sangamo Biosciences TM (Richmond, CA).

[0102] In selected embodiments, meganucleases can be used to introduce genome changes described herein for directed editing. Meganucleases were identified in the 1990s, and subsequent work has shown that meganucleases are particularly promising tools for genome editing because they can effectively induce homologous recombination, produce mutations in the coding or non-coding regions of the genome, and change the reading frame of the coding region of the genome. (See, e.g., Epinat, et al., Nucleic Acids Research, 31 (11): 2952-2962; and U.S. Pat. No. 8,921,332 issued to Choulika et al. on December 30, 2014). The high specificity of meganucleases makes them highly accurate and much less cytotoxic than other naturally occurring restriction enzymes.

[0103] Meganucleases are generally divided into four families: LAGLIDADG family, GIY-YIG family, His-Cys box family and HNH family. These families are characterized by structural motifs that affect catalytic activity and recognition sequences. For example, members of the LAGLIDADG family are characterized by having one or two copies of a conservative LAGLIDADG motif. The four families of meganucleases are significantly different from each other in terms of conservative structural elements, and therefore, significantly different in terms of DNA recognition sequence specificity and catalytic activity. Meganucleases are generally found in microbial species, and their unique characteristics are to have very long recognition sequences (>14bp), thereby making them naturally specific for cutting at the desired position. This meganuclease can be used to produce site-specific double-strand breaks in genome editing. Those skilled in the art can use these naturally occurring meganucleases, but the number of such naturally occurring meganucleases is limited. In order to overcome this challenge, mutagenesis and high-throughput screening methods have been used to produce meganuclease variants that recognize unique sequences. For example, a variety of meganucleases have been fused to produce hybrid enzymes that recognize new sequences. Alternatively, the DNA-interacting amino acids of the meganuclease can be altered to design sequence-specific meganucleases (see, e.g., U.S. Pat. No. 8,021,867). Meganucleases can be designed using methods described in, e.g., Certo, MT et al. Nature Methods (2012) 9:073-975; U.S. Pat. Nos. 8,304,222; 8,021,867; 8,119,381; 8,124,369; 8,129,134; 8,133,697; 8,143,015; 8,143,016; 8,148,098; or 8,163,514, the contents of each of which are incorporated herein by reference in their entirety. Alternatively, commercially available technology (e.g., Directed Nuclease Editor from Precision Biosciences) can be used to design meganucleases. TM Genome editing technology) to obtain large-range nucleases with site-specific cleavage properties.

[0104] The introduction of the editing system can be carried out by any suitable delivery means. For example, the delivery method and / or medium for introducing or transferring to the cell can be delivered using a virus, for example, a slow virus, a vector, a delivery vector, or any known method and / or medium for delivering such agents. In some embodiments, electroporation or other physical delivery methods (such as microinjection, a particle gun, calcium phosphate transfection, or cell compression or extrusion (such as described in Lee, et al, 2012, Nano Lett 12: 6322-27) are used to introduce or deliver one or more proteins and / or nucleic acids required for gene editing to cells.

[0105] In some embodiments, the method provided herein includes introducing or delivering a recombinant nucleic acid enzyme, and having gRNA in some cases, and introducing or delivering a donor template simultaneously. In some embodiments, introducing or delivering a recombinant nucleic acid enzyme, gRNA and a donor template simultaneously. In some embodiments, introducing or delivering a recombinant nucleic acid enzyme in sequence, and having gRNA and a donor template in some cases. In some embodiments, introducing a recombinant nucleic acid enzyme and gRNA together, and introducing or delivering a recombinant nucleic acid enzyme and gRNA before introducing or delivering a donor template.

[0106] In other respects, the genome editing of the illustrative embodiments can utilize homologous recombination methods, including cre-lox technology and FRET technology. The difference between site-specific homologous recombination and general homologous recombination is that the short specific DNA sequence required for recombinase recognition is the only site where recombination occurs. Site-specific recombination requires a special recombinase to recognize the site and catalyze the recombination at these sites. Many phage and yeast-derived site-specific recombination systems (each system comprising a recombinase and a specific homologous site) have been shown to work in eukaryotic cells for DNA integration, and are therefore suitable for use in the present invention, and these systems include phage P1 Cre / lox, yeast FLP-FRT systems and the Dre system of the tyrosine family of site-specific recombinases. Such systems and methods of use are described in the following documents: for example, U.S. Pat. Nos. 7,422,889; 7,112,715; 6,956,146; 6,774,279; 5,677,177; 5,885,836; 5,654,182; and 4,959,317. Other systems of the tyrosine family (such as bacteriophage λInt integrase, HK2022 integrase) and systems belonging to the independent serine family of recombinases (such as bacteriophage phiC31, R4Tp901 integrase) can also edit mammalian cells using their corresponding recombination sites, and may also be suitable for use in the present invention. Exemplary homologous recombination methods are described in the following documents: U.S. Pat. Nos. 6,689,610; 6,204,061; 5,631,153; 5,627,059; 5,487,992; and 5,464,764.

[0107] In some embodiments, the Cre / lox system is used as a site-specific recombinase. In some embodiments, the FLP-FRT site-specific recombinase system is used. In some embodiments, Cre / Lox and Flp / FRT recombination involves the introduction of a targeting vector with 3' and 5' homology arms, the targeting vector containing a mutation of interest, two Lox or FRT sequences and an optional box usually placed between the two Lox or FRT sequences. Positive selection is applied and homologous recombinants containing targeted mutations are identified. The transient expression of Cre or Flp combined with negative selection results in excision of the selection box, and cells in which the box has been lost are selected. The final targeted allele contains a Lox or FRT scar of an exogenous sequence.

[0108] In some embodiments, a transposase system can be used. A transposon is a mobile genetic element comprising a nucleotide sequence that can be moved around to different locations within the genome of a single cell. In the process, the transposon can cause mutations and / or change the amount of DNA in the genome of the cell. Many transposon systems that can be transposed in cells (e.g., vertebrates) have been isolated or designed, such as Sleeping Beauty (Sleeping Beauty) (Izsvák and Ivics Molecular Therapy (2004) 9, 147-156), piggyBac (Wilson et al. Molecular Therapy (2007) 15, 139-145), To12 (Kawakami et al. PNAS (2000) 97 (21): 11403-11408) or Frog Prince (Frog Prince) (Miskey et al. Nucleic Acids Res. Dec. 1, 2003 31 (23): 6873-6881). In general, the transposase system provides an alternative means similar to the use of Cre / Lox or Flp / FRT for removing the selection box after homologous recombination. Therefore, as an example, the piggyback (PB) transposase system involves the introduction of a targeting vector with 3' and 5' homology arms, the targeting vector containing the mutation of interest, two PB terminal repeats at the site of the endogenous TTAA sequence and a selection box placed between the PB terminal repeats. Positive selection is applied and homologous recombinants containing targeted mutations are identified. The transient expression of PBase removal is combined with negative selection to excise the selection box, and cells in which the box has been lost are selected. The final targeted allele contains a mutation that does not have an introduction of an exogenous sequence. In some embodiments, for the introduction of sequence changes by the same transposase system as pB, there must be a natural TTAA site that is relatively close to the position of the insertion into a specific mutation.

[0109] Methods for quantifying therapeutic efficacy and detecting sequence changes are well known in the art and include, but are not limited to, DNA sequencing, electrophoresis, enzyme-based mismatch detection assays, and hybridization assays (e.g., PCR, RT-PCR, RNase protection, in situ hybridization, primer extension, Southern blotting, Northern blotting, and dot blot analysis).

[0110] Sequence changes in specific genes can also be determined at the protein level using, for example, chromatography, electrophoresis, immunoassays (such as ELISA and Western blot analysis), and immunohistochemistry.

[0111] In some embodiments, positive and / or negative selection markers (such as by using knock-in / knock-out constructs) can be designed to effectively select transformed cells with homologous recombination events occurring with constructs. Positive selection provides means for enrichment of clone colonies that have absorbed foreign DNA. The limiting examples of this positive marker include glutamine synthetase, dihydrofolate reductase (DHFR), markers that confer antibiotic resistance, such as neomycin, hygromycin, puromycin and blasticidin S resistance box. Negative selection markers need to be selected for random integration and / or elimination of marker sequences (for example, positive markers). The limiting examples of this type of negative markers include herpes simplex thymidine kinase (HSV-TK), hypoxanthine phosphoribosyltransferase (HPRT) and adenine phosphoribosyltransferase (ARPT) that ganciclovir (ganciclovir, GCV) is converted into cytotoxic nucleoside analogs.

[0112] In some embodiments, the variant target protein or the polynucleotide encoding the variant target protein is sequenced to identify or confirm one or more substitutions thereof. Methods for sequencing polynucleotides and / or polypeptides are known in the art, including using sequencing systems from Illumina (San Diego, California), Pacific Biosciences (Mountain View, California), Oxford Nanopore Technologies (Oxford, England), and Element Biosciences (San Diego, California).

[0113] D. Expression and culture of modified cell lines

[0114] In some embodiments, there is provided a method for producing antibodies by transfecting a cell line with a gene producing an antibody using a modified cell line (such as a mammalian cell line) according to the provided embodiments. In some embodiments, a nucleic acid encoding an antibody is introduced into a modified mammalian cell line to express the antibody in a cell. Methods for introducing nucleic acids into cells are well known to those skilled in the art, and include various art-recognized protocols for introducing foreign DNA into a host cell (see Kaufman, RJ. Meth. Enzymology 185: 537 (1988)). The selection of transformation or transfection protocols will depend on the properties of the host cell and the transgene and protein product. Common methods for introducing foreign DNA into host cells include calcium phosphate precipitation or DEAE-dextran-mediated transfection. Alternatively, electroporation can be used to introduce DNA into the cytoplasm of the host cell, or reagents (e.g., Reagents or The agent, Gibco BRL, Gaithersburg, MD, facilitates the uptake of nucleic acids by host cells when the complex is applied to cultured cells.

[0115] In some embodiments, the nucleic acid sequence encoding the antibody can be assembled or inserted into an expression vector. In some embodiments, the modified mammalian cell line is transfected with nucleic acids encoding the heavy (H) chain and light (L) chain of the antibody alone, or co-transfected with nucleic acids encoding H and L chains. For example, in one embodiment, nucleic acids encoding antibodies or antigen-binding fragments thereof are assembled in a separate expression vector that is then used to co-transfect the modified cell line. Alternatively, nucleic acids encoding H and L chains can be assembled on the same expression vector. The modified mammalian cells are cultured under conditions that allow the expression of the incorporated genes, and the expressed immunoglobulin chains or complete antibodies or fragments are recovered from the culture.

[0116] In order to produce recombinant antibodies for a long time and in high yield, stable expression can be used.For example, the cell line of stably expressed antibody molecules can be used for the method of the present disclosure.Host cells can be transformed with immunoglobulin expression cassettes and selectable markers, rather than using expression vectors containing viral replication sources.After introducing foreign DNA, engineered cells can be grown in the culture medium of enrichment such as 1-2 days, and then switched to selective culture medium.The selectable marker in the recombinant plasmid confers resistance to selection, and allows cells to stably integrate plasmids into chromosomes and grow to form a focus, and then the focus can be cloned and expanded in the cell line.

[0117] The expression vector may include a selection marker, an origin of replication, and other features that provide for replication and / or maintenance of the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like. A large number of suitable vectors and promoters are known to those skilled in the art; many are commercially available for use in generating the subject recombinant constructs. The following vectors are provided by way of example and are not to be construed as limiting in any way: Bacteria: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a; pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5. Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG, pSVK3, pBPV, pMSG, and pSVL.

[0118] The expression vectors usually have convenient restriction sites located near the promoter sequence to provide for the insertion of the nucleic acid sequence encoding the heterologous protein. Optional selectable markers that are operative in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g., vaccinia virus-based viral vectors; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35:2543-2549; Borras et al., Gene Ther. (1999) 6:515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92:7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5:1088-1097; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); adeno-associated virus (see, e.g., Ali et al., Invest. Opthalmol. Vis. Sci. (1994) 35:2543-2549; Borras et al., Gene Ther. (1999) 6:515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92:7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5:1088-1097; WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655); al., Hum. Gene Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857-2863; Jomary et al., Gene Ther. (1997) 4:683 690, Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava, WO 93 / 09239, Samulskiet al.,J.Vir.(1989)63:3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319-23; Takahashi et al., J. Virol.(1999) 73:7812-7816); or retroviral vectors (e.g., murine leukemia virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus).

[0119] Other expression vectors suitable for use are, for example, but not limited to, lentiviral vectors, gamma retroviral vectors, foamy virus vectors, adeno-associated virus vectors, adenoviral vectors, poxvirus vectors, herpesvirus vectors, engineered hybrid virus vectors, transposon-mediated vectors, etc. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY, and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

[0120] In general, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites and one or more selectable markers, (see, e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0121] In order to evaluate the expression of polypeptide (for example, antibody) or its part, the expression vector to be introduced into the cell can also contain selectable marker gene or reporter gene or both, to promote identification and selection of expressing cells from the colony of cells attempted to be transfected. In some embodiments, selectable marker can be carried on a separate DNA fragment and used in a cotransfection program. Both selectable marker and reporter gene can be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selectable markers include but are not limited to antibiotic resistance genes.

[0122] In some embodiments, the expression vector encoding the antibody includes a selectable marker to further amplify the expression of the expressed antibody. In some embodiments, the method of amplifying the gene of interest is also desirable, and is generally related to the use of a selection gene that grants an optional phenotype. In general, a "selection gene" is a gene that grants a phenotype on a cell in which a gene is expressed as a detectable protein. Common examples of selection genes include, but are not limited to, antibiotic resistance genes. For example, useful dominant selectable markers include antibiotic resistance genes derived from microorganisms, and when a drug (or selection agent) is exogenously added to a cell culture, the gene grants neomycin, kanamycin or hygromycin resistance.

[0123] In some embodiments, mammalian cells provide post-translational modifications to immunoglobulin molecules, including leader peptide removal, folding and assembly of heavy and light chains, glycosylation of antibody molecules, and secretion of functional antibody proteins.

[0124] Once the antibodies have been produced, the antibody molecules can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., ion exchange, affinity (particularly affinity for specific antigens after protein A) and fractionated column chromatography), centrifugation, differential solubilization, or by any other standard technique for protein purification. In many embodiments, the antibodies are secreted from the cells into the culture medium and collected from the culture medium.

[0125] Exemplary methods for producing antibodies

[0126] In some preferred embodiments, the cell-free system of U.S. Serial No. 63 / 398,143, filed on August 15, 2022 (which is hereby incorporated in its entirety into this application for all purposes) may include: a) providing a cell-free protein expression system (in some preferred embodiments, a mammalian cell-free system) containing a target protein exhibiting an antibody epitope; b) modifying the cell-free system by introducing one or more agents that block the antibody epitope on the target protein (or in some preferred embodiments, and the antibody paratope 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 an antibody that binds to the antibody epitope; and d) initiating transcription and translation of the antibody in the cell-free system under conditions such that the antibody is produced. In some preferred embodiments, the cell-free system may comprise: a) introducing into cells (in some preferred embodiments, mammalian cells) an agent that selectively binds to a target epitope on an intracellular protein to create a modified cell line; b) creating a cell-free antibody production system from the modified cell line; c) introducing into the cell-free antibody production system a nucleic acid template, wherein the nucleic acid template encodes an antibody that selectively binds to the target epitope on the intracellular 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).

[0127] Modified mammalian cell-free system for producing anti-Grp78 antibodies

[0128] In some embodiments, the provided method and cell-free system can be used to produce antibodies against GRP78, also referred to as anti-GRP78 antibodies. The antibody may include a full-length antibody or an antigen-binding fragment thereof. GRP78, also referred to as heat shock protein-5 (Bspa5 / BiP), is part of an evolutionarily conservative ER-related stress response mechanism that provides cell survival signals under environmental and physiological stresses. As a molecular component of the ER chaperone network, GRP78 is generally involved in the processing of unfolded proteins, however, updated insights also place the protein at 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 in advanced breast cancer (Lee et al., 2006, Cancer Research 66(16):7849-7853) and recurrence in prostate cancer patients (Daneshmand et al., 2007, Human Pathology 38(10):1547-1552). Upregulation of GRP78 promotes survival and chemotherapy resistance in both proliferating and dormant breast cancer cells. Synthetic peptides composed of GRP78 binding motifs bind to cell death-inducing peptides to promote apoptosis in cancer cells, thereby demonstrating their accessibility in vivo (Arap et al., 2004, Cancer Cell 6(3):275-284).

[0129] 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.

[0130] 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 such 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.

[0131] 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 VL CDR2 shown in SEQ ID NO: 5, and the VL CDR3 shown in SEQ ID NO: 6. In some embodiments, the VH chain has a sequence of amino acids having at least 85%, at least 90%, or at least 95% sequence identity with the sequence shown in SEQ ID NO: 19, and the VL chain has 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.

[0132] 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 a sequence of amino acids having at least 85%, at least 90%, or at least 95% sequence identity with the sequence shown in SEQ ID NO:21, and the VL chain has at least 85%, at least 90%, or at least 95% sequence identity with the sequence shown in SEQ ID NO:22. In some embodiments, the VH chain is shown in SEQ ID NO:21, and the VL chain is shown in SEQ ID NO:22.

[0133] 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 a sequence of amino acids having at least 85%, at least 90%, or at least 95% sequence identity with the sequence shown in SEQ ID NO: 23, and the VL chain has at least 85%, at least 90%, or at least 95% sequence identity with the sequence shown in SEQ ID NO: 24. In some embodiments, the VH chain is shown in SEQ ID NO: 23, and the VL chain is shown in SEQ ID NO: 24.

[0134] In some embodiments, the anti-GRP78 antibody binds to human GRP78. In some embodiments, the human GRP78 protein comprises the amino acids set forth in SEQ ID NO:25.

[0135] In some embodiments, the anti-GPR78 antibody binds 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.

[0136] In some embodiments, the anti-GPR78 antibody binds 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.

[0137] In some embodiments, the anti-GPR78 antibody binds to a target epitope of GRP78 that includes one or more amino acid residues R261, R279, K113, K268, and K271 with reference to the residue numbering of human GRP78 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.

[0138] In some embodiments, the anti-GRP78 antibody is an antibody that binds to the same epitope on human GRP78 as an antibody containing any of the above sequences (i.e., an antibody that has the ability to cross-compete with any of the anti-GRP78 antibodies described to bind to human GRP78). In some embodiments, the reference antibody or antibody fragment used for cross-competition studies can be one of the antibodies or antibody fragments described herein, because equivalent molecules (e.g., Fab to Fab, full-length antibody to full-length antibody, etc.) are used for comparison. For example, Biacore analysis, ELISA assays, or flow cytometry can be used to demonstrate cross-competition with any of the described antibodies. The ability of the test antibody to inhibit binding to human GRP78 indicates that the test antibody can compete with the reference antibody for binding to human GRP78, and is therefore considered to bind to the same epitope of human GRP78.

[0139] In some embodiments, the modified mammalian cell-free system is engineered to contain a variant GRP78 having a mutant epitope comprising a mutation of one or more amino acid residues of a target epitope of an antibody or antigen-binding fragment. In some embodiments, the mutation is an amino acid substitution of one or more amino acid residues to change the target epitope. In some embodiments, the mutant epitope comprises 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions of a target epitope of a target protein.

[0140] In some embodiments, the modified mammalian cell-free system is engineered to contain a variant GRP78 having an amino acid sequence that contains at least one amino acid substitution compared to the human GRP78 sequence shown in SEQ ID NO: 25. In some embodiments, the modified cell line is engineered with a variant GRP78 having at least 85% and less than 100% identity to the GPR78 sequence shown in SEQ ID NO: 25. In some embodiments, the modified cell line is engineered with a variant GRP78 having at least 90% and less than 100% identity to the GPR78 sequence shown in SEQ ID NO: 25. In some embodiments, the modified cell line is engineered with a variant GRP78 having at least 95% and less than 100% identity to the GPR78 sequence shown in SEQ ID NO: 25. In some embodiments, the modified cell line is engineered with a variant GRP78 having the last 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions compared to the human GRP78 sequence shown in SEQ ID NO:25.

[0141] In some embodiments, the mutation of the mutant epitope is an amino acid substitution selected from at least one of (i) to (ix) or a combination thereof: (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 mutation of the mutant epitope is an amino acid substitution from at least one of (i) to (ix) or a combination thereof: (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.

[0142] In some embodiments, variant GRP78 contains mutation R261Q. In some embodiments, variant GRP78 contains mutation H265Q. In some embodiments, variant GRP78 contains mutation R279Q. In some embodiments, variant GRP78 contains mutation E329Q. In some embodiments, variant GRP78 contains mutation R261Q and H265Q. In some embodiments, variant GRP78 contains mutation R261Q, H265Q, R279Q and E329Q. In some embodiments, variant GRP78 contains mutation R261Q, H265Q and E329Q. In some embodiments, variant GRP78 contains mutation R261Q and R279Q. In some embodiments, variant GRP78 contains mutation R261Q, H265Q, K271Q, R279Q and E329Q. In some embodiments, variant GRP78 contains mutations R261Q, H265Q, K271Q, K272Q, E329Q and D333Q. In some embodiments, variant GRP78 contains mutations K113Q, R261Q, K268Q, K271Q and R279Q. In some embodiments, variant GRP78 contains mutations K113Q, R261Q, H265Q, K268Q, K271Q, K272Q, R279Q, E329Q and D333Q. In some embodiments, variant GRP78 contains mutations R261A, H265A and E329A; R261A and R279A. In some embodiments, variant GRP78 contains mutations R261A, H265A, R279A and E329A.

[0143] Provided herein are modified mammalian cell-free systems having the variant GRP78 shown in SEQ ID NO:26.

[0144] Provided herein are modified mammalian cell-free systems having the variant GRP78 shown in SEQ ID NO:27.

[0145] In some embodiments, the modified mammalian cell-free system can be a lysate from mammalian cells, including but not limited to Chinese hamster ovary (CHO) cells, baby hamster kidney cells, NSO myeloma cells, monkey kidney COS cells, monkey kidney fibroblast CV-1 cells, human embryonic kidney 293 (HEK293) cells, human breast cancer SKBR3 cells, human Jurkat T cells, dog kidney MDCK cells, and human cervical cancer HeLa cells. In some embodiments, the mammalian cell-free system is a CHO cell lysate.

[0146] In some embodiments, the anti-GRP78 antibody exhibits reduced or eliminated binding to variant GRP78 containing mutant epitopes. In some embodiments, the binding and / or reactivity of anti-GRP78 to variant GRP78 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 binding and / or reactivity to human GRP78 lacking one or more substitutions (e.g., human GRP78 shown in SEQ ID NO: 25). In some embodiments, the binding and / or reactivity of anti-GRP78 to variant GPR78 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 binding and / or reactivity to human GRP78 lacking one or more substitutions (e.g., as shown in SEQ ID NO: 25). Methods for assessing binding can include any method described herein.

[0147] In some embodiments, the anti-GRP78 antibody exhibits reduced affinity for variant GRP78 relative to the binding and / or reactivity of human GRP78 lacking one or more substitutions (e.g., as shown in SEQ ID NO: 25). In some embodiments, the affinity of the anti-GRP78 antibody for variant GRP78 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 for human GRP78 lacking one or more amino acid substitutions (e.g., as shown in SEQ ID NO: 25). In some embodiments, the affinity of the anti-GRP78 antibody to the variant GRP78 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 to human GRP78 lacking one or more amino acid substitutions (e.g., shown in SEQ ID NO: 25). Methods for measuring affinity can include any method described herein.

[0148] In some embodiments, modified mammalian cell lines containing variant GPR78 can be used to produce anti-GRP78 antibodies according to any of the provided methods.

[0149] Cell-free systems for antibody production

[0150] 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 the cell-free systems 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.

[0151] Cell-free systems can be based on prokaryotes or eukaryotes. In prokaryotic systems, extracts based on Escherichia coli are often used and are commercially available for the production of various proteins, including antibodies. Due to the ease of preparing cell-free lysates, systems 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) have been well optimized at the laboratory level. There are currently a variety of detailed protocols for preparing lysates based on Escherichia coli. In eukaryotic systems, extracts based on rabbit reticulocyte lysate (RRL), wheat germ, insect Spodopterafrugiperda 21 (Sf21), Chinese hamster ovary (CHO), and cultured human cells are often used. For a review, see, for example, Dondapati Sk et al., BioDrugs (2020) 34: 327-348.

[0152] For the production of complex proteins requiring post-translational modifications, eukaryotic cell-free systems are often, but not always, preferred.

[0153] 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 washing steps 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 fast protein production system because it does not require transfection or cell culture, and lacks cell viability restrictions.

[0154] In some embodiments, a cell-free protein synthesis system is created using transcription and translation-related factors derived from or based on the genetic modification of antibody epitopes in natural proteins taught herein. In these embodiments, the system contains only known proteins and substrates, such as 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 the DNA template to produce proteins, thereby forming a highly controlled system. Protein factors involved in the initiation, extension and termination of the protein synthesis process are identified, and can be adjusted separately according to the requirements of the system. Any protein targeted by the antibody can be modified in advance to change the epitope to which the antibody binds, to retain the function of the protein, while eliminating the combination of the antibody with its natural target during production.

[0155] 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 these cases, recombinant proteins (e.g., mammalian proteins) can be added to the system, and in the case where the protein is a target for antibody production, 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 before the recombinant protein is produced to modify the epitope of a specific GRP78 antibody using the mutations 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 the binding to the protein will be reduced). Methods for producing recombinant proteins are well known in the art and are taught in, for example, the Protein Expression Handbook of Thermo Fisher Scientific, Waltham, MA.

[0156] Cell-free protein synthesis reaction format

[0157] 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 complete IgG, has been achieved 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., J Biotech-nol. 2012; 164: 220-31); al., SciRep. 2017; 7: 17-12364; Martin RW et al., ACS Synth Biol. 2017; 6: 1370-9). In addition, the scale-up of cell-free reactions to upgraded scale (Zawada JF, Yin G, Steiner AR, Yang J, Naresh A, Roy SM, et al. Microscale to manufacturing scale-up of cell-free cytokine production—a new approach for shortening protein production development timelines. Biotechnol Bioeng. 2011; 108: 1570-8; Yin G, Garces ED, Yang J, Zhang J, Tran C, Steiner AR, et al. Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system. MAbs. 2012; 4: 217-25) as well as reduction (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.

[0158] In some embodiments, the synthetic reaction form used is a batch form. The batch-based form is the most commonly used method in prokaryotic and eukaryotic systems. This method is relatively fast and cheap, and according to the system, synthesis can be completed in 1.5-3 hours. The system based on Escherichia coli can provide a protein yield ranging from 100 μg / mL to 2-3 mg / mL. Although the yield of the eukaryotic system based on batches is relatively low, membrane proteins are automatically integrated into the microsomal membrane, and their functions can be solved immediately after synthesis (Brodel AKet al., PLoS One.2013; 8:2013).

[0159] In other embodiments, in order to further scale up protein yields via batch-based eukaryotic systems, a repetitive batch-based synthesis format is proposed, in which microsomes incorporating the MP of interest generated in an initial synthesis reaction can be added to a fresh cell-free synthesis reaction that has been depleted of microsomes (Thoring L. et al., PLoS One. 2016; 11: 2016; Zemella A, et al., Sci Rep. 2018; 8: 18-26936).

[0160] In other embodiments, continuous exchange cell-free synthesis platform (CECFSP). In this form, a semi-permeable 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 t 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 form prolongs the reaction time and improves the protein yield. So far, the CECFSP form has been used to increase the protein yield several times, and is widely used as a cell-free platform.

[0161] In addition, the progress of bioorthogonal reaction chemistry has paved the way for the possibility of expanding ADC development. The introduction of non-standard amino acid sites into genetically engineered sequences can be used to create site-specific labeled ADCs (Axup JY et al., Proc Natl Acad Sci USA. 2012; 109: 16101-6). At present, a variety of ADCs are approved for therapy. So far, all of these ADCs are generated by surface-exposed lysine or partial disulfide bond reduction and conjugation with free cysteine, and mAb is coupled to a cytotoxic linker-payload, which usually produces a controlled but heterogeneous ADC population, wherein the number and position of the drug molecules attached to mAb are different (Strop P et al., Chem Biol. 2013; 20: 161-7). From synthesis to functional testing, cell-free systems can accelerate antibody construct evaluation by sequential or simultaneous screening. The introduction of non-standard amino acids expands the chemical library, thereby expanding the possibility of modifying and improving antibody-based therapeutic agents. Advanced labeling technologies allow very rapid qualitative analysis of drug-to-antibody ratio (DAR), linker, linker / position, drug, drug / position (research applications) and allow for complete control of ADC design.

[0162] Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms used herein have the same meaning as commonly understood by those 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.

[0163] definition

[0164] As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more". It should be understood that the aspects and variations described herein include "consisting of" and / or "consisting essentially of" the aspects and variations.

[0165] 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 stubborn limitation on the scope of the claimed subject matter. Therefore, the description of the range should be regarded as having clearly 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 special exclusions within the stated range. In the case where the stated range includes one or two limits in the limits, the range excluding any one or two limits in the included limits is also included in the claimed subject matter. Regardless of the breadth of the range, this applies.

[0166] 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 values ​​or parameters themselves. For example, references to a description of "about X" include a description of "X".

[0167] As used herein, the term "antibody" is used in the broadest sense and includes polyclonal and monoclonal antibodies, including whole antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab') fragments, 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.

[0168] 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.

[0169] 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.

[0170] 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 sitetopography," 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.2013Jul;41(Web Server issue):W34-40("IgBLAST" numbering scheme). .

[0171] 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 by inserting letters, such as the deletion regulation occurring in "30a" and some antibodies. The two schemes are placed in different positions with some insertions and deletions ("deletions"), resulting in different numbering. 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).

[0172] 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.

[0173]

[0174] 1-Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD

[0175] 2-Al-Lazikani et al., (1997) JMB 273,927-948

[0176] Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 26-35 (HCDR1), 50-65 (HCDR2), and 95-105 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments, the CDR amino 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.

[0177] Therefore, unless otherwise indicated, a "CDR" or "complementarity determining region" of a given antibody or region thereof (such as its variable region) 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 some embodiments, the CDR sequences of the antibodies provided are described using various numbering schemes (see, e.g., Section 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.

[0178] Likewise, unless otherwise indicated, the FR or individual specific FR (e.g., FR-H1, FR-H2, FR-H3, FR-H4) of a given antibody or region thereof (e.g., variable region thereof) 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.

[0179] 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.

[0180] 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 comprises four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 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).

[0181] 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., in vitro) under conditions suitable for survival. The cultured cells are allowed to survive, and the culture can cause the cells to grow, differentiate or divide.

[0182] 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 some 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.

[0183] As used herein, the term "mutant epitope" refers to an epitope that has been genetically engineered to include one or more non-naturally occurring mutations that alter the epitope sequence recognized by an antibody. In some embodiments, the mutation is one or more amino acid substitutions of amino acid residues that constitute the epitope recognized by the antibody.

[0184] 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 the protein encoded by the DNA produced by the cell (Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 18.1-18.88).

[0185] The term "gene" may refer to a segment of DNA involved in producing or encoding a polypeptide chain. It may include regions before and after the coding region (leader and tail regions) and insertion sequences (introns) between individual coding segments (exons). Alternatively, the term "gene" may refer to a segment of DNA involved in producing or encoding non-translated RNA, such as rRNA, tRNA, guide RNA (e.g., small guide gRNA), or microRNA.

[0186] "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 ; bifunctional antibody; linear antibody; heavy chain variable (V H ) region, single-chain antibody molecules (such as scFv) and 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 scFv.

[0187] 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.

[0188] 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.

[0189] 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 encoding sequences including a human antibody library. The term does not encompass humanized forms of non-human antibodies that include non-human antigen binding regions, such as antibodies in which all or substantially all CDRs are non-human. The term encompasses antigen-binding fragments of human antibodies.

[0190] 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.

[0191] Humanized antibodies have variable region framework residues substantially derived from human therapeutic antibodies (referred to as acceptor antibodies) and complementary determining regions substantially derived 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. Patent Nos. 5,693,762, 5,693,761, 5,585,089 and 5,530,101 and Winter, U.S. Patent No. 5,225,539. If present, one or more constant regions are also substantially or entirely derived from human immunoglobulins. Human variable domains are typically selected from human antibodies whose framework sequences exhibit a high degree of sequence identity to 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 acid at a specific position or empirically observing the replacement or mutagenic effect of a specific amino acid.

[0192] 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 could 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.

[0193] The other candidate for replacement is the uncommon acceptor human framework amino acid of human antibody at said position.These amino acid can be replaced by the amino acid of the equivalent position from mouse donor antibody or the amino acid of the equivalent position from more typical human antibody.The other candidate for replacement is the uncommon acceptor human framework amino acid of human antibody at said position.The preferred variable region framework of humanized antibody is usually shown with the consensus sequence of people's variable region framework sequence or such sequence at least 75%, more preferably 80%, even more preferably 85% sequence identity.

[0194] In certain instances, mouse monoclonal antibodies can be used as the basis for the generation 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.

[0195] 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.

[0196] 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.

[0197] 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 joint.

[0198] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population or within the population, i.e., the individual antibodies comprising the population are identical, except for containing naturally occurring mutations or possible variants 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 the antigen. The term should not be construed 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.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] 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.

[0203] 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 containing an expression vector that can be used to produce a desired expression product.

[0204] 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.

[0205] 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, if a promoter controls transcription of the sequence, the promoter is operably linked to a coding sequence; or if a ribosome binding site is positioned to allow translation, the ribosome binding site is operably linked to a coding sequence. If the DNA for a signal sequence (secretionary leader) is expressed as a precursor that participates in the secretion of a polypeptide, it is operably linked to the DNA for a polypeptide. Generally, operably linked means contiguous.

[0206] In some preferred embodiments, the present disclosure provides a method for recombinantly producing an antibody, the method comprising: expressing a nucleic acid encoding an antibody in a modified 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 the target epitope on the protein in the system; and starting transcription and translation of the antibody in the cell-free system under conditions that allow the antibody to be produced. In some preferred embodiments, the cell-free system comprises a cell lysate. In some preferred embodiments, the cell lysate is a eukaryotic 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 method further comprises isolating the antibody from the cell-free system. In some preferred embodiments, the present disclosure provides a method for recombinantly producing a monoclonal antibody, the method comprising: expressing a nucleic acid encoding an antibody in a modified 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 system comprises a mutation of one or more amino acid residues of the target epitope on the protein in the system; and starting transcription and translation 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 method further comprises isolating the antibody from the cell-free system. In some preferred embodiments, the present disclosure provides a method for recombinantly producing an antibody, the method comprising: modifying a cell line to eliminate the binding of an antibody to an intracellular protein in a cell of the cell line, wherein the modification causes expression of a variant target protein comprising a mutation of one or more amino acid residues of the target epitope of the antibody; creating a cell-free antibody production system from a modified cell line; introducing a nucleic acid template into the cell-free antibody production system, wherein the nucleic acid template encodes the antibody to the target protein; and starting transcription and translation from the nucleic acid template to produce the antibody in the cell-free antibody production system. In some preferred embodiments, the method comprises (c) isolating the antibody from a cell-free system. In some preferred embodiments of such methods, the naturally expressed target protein is an intracellular protein in the secretory pathway of the cell line. In some preferred embodiments, the mutation is an inert mutation that retains the structure and function of the native target protein in the cell. In some preferred embodiments, the mutation reduces or eliminates the binding of the antibody to the target epitope on the target protein. In some preferred embodiments, the target protein is a stress protein. In some preferred embodiments, the target protein is expressed on an organelle of a 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. In some preferred embodiments, the naturally expressed target protein is an intracellular protein in the secretory pathway of the mammalian cell line. In some preferred embodiments, the mutation is an inert mutation that retains the structure and function of the target protein in the mammalian cell line. In some preferred embodiments, the mutation reduces or eliminates the binding of the antibody to the target epitope on the target. In some preferred embodiments, the target protein is a stress protein.

[0207] In some preferred embodiments, the present disclosure provides a method for recombinantly producing a monoclonal antibody, the method comprising: modifying a mammalian cell line to eliminate the binding of an antibody to an intracellular protein in a cell of the cell line, wherein the modification causes the expression of a variant target protein comprising a mutation of one or more amino acid residues of a target epitope of the antibody; creating a cell-free antibody production system from a modified mammalian cell line; introducing a nucleic acid template into the cell-free antibody production system, wherein the nucleic acid template encodes the antibody to the target protein; and starting transcription and translation from the nucleic acid template to produce the antibody in the cell-free antibody production system. In some preferred embodiments, the method further comprises isolating the antibody from the cell-free system. In some preferred embodiments, the naturally expressed target protein is an intracellular protein in the secretory pathway of the cell line. In some preferred embodiments, the mutation is an inert mutation that retains the structure and function of the natural target protein in the cell. In some preferred embodiments, the mutation reduces or eliminates the binding of the antibody to the target epitope on the target protein. In some preferred embodiments, the target protein is a stress protein. In some preferred embodiments, the target protein is expressed on an organelle of a 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. In some preferred embodiments, the naturally expressed target protein is an intracellular protein in the secretory pathway of the mammalian cell line. In some preferred embodiments, the mutation is an inert mutation that retains the structure and function of the target protein in the mammalian cell line. In some preferred embodiments, the mutation reduces or eliminates the binding of the antibody to the target epitope on the target. In some preferred embodiments, the target protein is a stress protein. In some preferred embodiments, the target protein is a protein involved in intracellular signal transduction, optionally a kinase or a phosphatase, further optionally wherein the phosphatase is liver regeneration phosphatase 3. In some preferred embodiments, the target protein is a chaperone protein.

[0208] As will be readily appreciated by one of ordinary skill in the art, other embodiments are also contemplated.

[0209] Example

[0210] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.

[0211] Example 1 Production and purification of anti-GPRP78 antibodies in mammalian cells

[0212] 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). Supernatant from day 7 post-transfection was loaded onto 1 mL of MabSelect PrisimA TM The antibody concentration and IgG titer in the supernatant were determined using an Octet QK 384 with a protein A biosensor (Molecular Devices, Wokingham, Berkshire, UK) using IgG1 antibody as a standard.

[0213] 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 antibodies were purified from the supernatant on day 5 using a similar method, but the titers remained very low (Table E2).

[0214] These results are consistent with the observation that anti-GRP78 antibodies produced in mammalian cells (such as human cells) are harmful to cells and affect 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. As 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.

[0215]

[0216] Example 2 Epitope mapping of anti-GRP78 antibodies

[0217] 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.

[0218] 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 have been validated to bind to WT GFR78 on cells and were therefore determined to be suitable positive controls under these conditions (Thermo Fisher Scientific, Waltham, Massachusetts) 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 wild-type (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 1. 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").

[0219] Table E3 shows the residues involved in antibody binding of exemplary anti-GRP78 clones B4 and F6.

[0220]

[0221] 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 aa 25-633) of H. sapiens GRP78 (PDB ID#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 are 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) R279 and (iv) E329 、D333.

[0222] Example 3 Generation of edited GRP78 cell lines

[0223] A strategy for producing and purifying antibodies from mammalian cells was developed, wherein the wild-type target of the antibody is a protein that is normally found within the cell (e.g., in the membrane of an organelle) but is present on the cell surface under a patient's pathological state (e.g., on cancer cells or cells of a patient with an autoimmune disease). The method is exemplified with GRP78 and the exemplary anti-GRP78 antibodies described in Examples 1 and 2.

[0224] A. Identification of mutations in target proteins that abolish antibody binding

[0225] Given the knowledge of epitopes involved in antibody binding, mutagenesis is used to modify one or more residues to eliminate binding of the resulting antibody while retaining the structure and function of the protein. For the epitope residues identified for anti-GRP78 antibodies in Table E3, mutations in GRP78 were selected to (1) replace one or more charged residues with non-charged residues; (2) ensure that the newly selected residues remain solvent exposed; and (3) ensure that the alpha helix is ​​conserved to prevent secondary structural changes and the possibility of changes in tertiary and quaternary structures to retain endogenous function and avoid cell death. The formation of the alpha helix is ​​more likely to be associated with amino acids with a high propensity to form this secondary structure. In addition, polar or charged amino acids are more likely to remain solvent exposed due to interactions with water; however, polar amino acids are preferred for mutagenesis because charged residues will be more likely to form interactions with antibodies. The polar uncharged amino acids with the highest to lowest propensity to form alpha helices (according to the helix penalty) are glutamine (ranked 6th), serine (ranked 10th), asparagine (ranked 15th), and threonine (ranked 16th) (Pace and Scholtz, Biophysical journal. 1998; 75(1):422-7). Glutamine was selected as an amino acid to replace the proposed amino acid that interacts with mAb. Replacing the main interacting amino acid with alanine (ranked 1st in alpha helical propensity) (Pace and Scholtz, 1998) will be more likely to eliminate binding to mAb, even though there is a risk of altering secondary and tertiary structure due to its hydrophobic R group. Therefore, it would be more strategic to incorporate fewer alanines but usually at the most critical sites.

[0226] Table E4 identifies GRP78 mutations that abrogate binding to exemplary B4 and F6 mAbs.

[0227] Table E4

[0228]

[0229] The proposed GRP78 mutants were modeled using Phyre2 (intensive processing) (Kelley et al., Nature protocols. 2015; 10 (6): 845-58). The full-length GRP78 sequence (wild type and ten mutants) was input. The wild-type sequence was modeled as a control. For all eleven generated structures, the confidence of residues 25-633 was listed as 100%. Initial modeling was performed for residues 1-24 and 634-654 (which were not important for the analysis).

[0230] Each Phyre2-generated mutant GRP78 structure was compared to the wild-type GRP78 structure generated by Phyre2 using the Matchmaker function of UCSF Chimera (Pettersen et al., Journal of computational chemistry. 2004; 25(13): 1605-12). The wild-type structure was used as a reference for this feature. The regions containing the mutations in all mutant structures were visually unchanged compared to the wild-type structure, and no changes were noted in the α helix. Nevertheless, the root mean square deviation (RMSD) values ​​were calculated based on the trimmed Cα atoms (654 possible pairs) as shown in Table E5:

[0231]

[0232] In summary, the proposed GRP78 mutants involve replacing residues that interact with B4 mAb and / or F6 mAb (all charged amino acids) with glutamine (a polar non-charged amino acid with the greatest propensity to form an alpha helix) or alanine (an amino acid that, although hydrophobic, has the greatest propensity to form an alpha helix overall). According to modeling, these mutants ensure the maintenance of the secondary and tertiary structures of GRP78 while promoting the retention of native function and abolishing binding to the above mAbs.

[0233] Since the single mutation R261Q is a major residue for both B4 and F6 mAbs, it was selected for subsequent experiments as detailed in Section B below. In addition, the double mutation R261Q / H265Q was selected for subsequent experiments as detailed in Section B below because this double mutant cell modifies two residues identified as part of its epitope and is a mutation found in a combination that eliminates binding as shown in Table E4. The combination of mutations in the double mutant was specifically selected because: 1) the residue changes were determined by epitope mapping to be important for epitope binding of the GFR78 antibody to native intracellular proteins; 2) these mutations are close enough to be suitable for CRISPR-based editing in the same DNA donor template. Other combinations can also be used, and there will be multiple donor templates and / or transfections required for introducing mutations into cells simultaneously or sequentially.

[0234] B. Editing Binding Epitopes in Target Proteins

[0235] The genomic sequence data of GRP78 was obtained from the NCBI gene database (Gene ID: 3309) and used as a reference sequence for gene editing. For both B4 mAb and F6 mAb, the codons for the predicted interacting residues on GRP78 are located in exon 5, as Figure 4 The flanking introns are also shown in Figure 4 Shown in.

[0236] Homology-directed repair (HDR) strategy was used to edit wild-type GRP78 (SEQ ID NO: 25) using CRISPR / Cas9 technology to generate single mutant GRP78 with mutation R261Q (SEQ ID NO: 26) or double mutant GRP78 with mutation R261Q / H265Q (SEQ ID NO: 27). See, e.g., U.S. Pat. No. 9,822,372 issued to Zhang et al. on November 21, 2017.

[0237] The vectors, gRNAs and donor templates used were also used to generate single and double mutant R261Q / H265Q clones, and individual clones containing single or double mutations R261Q / H265Q were identified. The donor template (despite containing two mutations) generated some cells with a single mutation and some cells with a double mutation.

[0238] R261Q single mutant cells and R261Q / H265Q double mutant cells were prepared by introducing genomic nucleotide modifications at positions 2188-2190 of exon 5 of CGT→CAG (converting R to Q) and at positions 2200-2202 of exon 5 of CAC→CAG (converting H to Q) ( Figure 4 ). gRNA and ssDNA donor template were designed to introduce mutations via HDR-based CRISPR gene editing (Table E6). Dual gRNAs were designed with “PAM outward”, such as Figure 5A As shown. The modified codons in the ssDNA oligo donor sequence that affect genome modification are depicted in bold and italics in Table E6. The nucleotides of the ssDNA are designed to remove the gRNA-associated PAM site from the donor template, thereby preventing the donor template DNA from being cut, while maintaining the sequence encoding the wild-type amino acid (underlined in Table E6).

[0239] PCR amplification using the primer pairs shown in Table E7 was used to verify gene editing ( Figure 5B ). The length of the generated amplicon was 509 base pairs.

[0240]

[0241] Cells carrying the desired mutation were isolated by clonal serial dilution and the presence of the point mutation was confirmed by Sanger sequencing. After expansion in culture, the cells were tested for growth and viability. Control (HEK293), GRP78 R261Q (single mutation) and GRP78 R261Q H265Q (double mutation) cells reached >95% confluence at the same time (day 4 after seeding), indicating no alteration in cell growth or viability. Live cell analysis system to calculate cell confluence.

[0242] C. Production and purification of anti-Gprp78 antibodies in modified Hek293 cells

[0243] Fully human anti-GRP78 monoclonal antibodies (mAbs) formatted as full-length IgG1 antibodies as described in Table E1 were individually transfected into clonal HEK293 cells by electroporation in 6-well plates, filled with fresh medium the next day and incubated for 5 days post-transfection. TM Transfection system and Neon TM Transfection was performed using the pre-set settings for HEK293 cells using the transfection system reagent (Thermo Fisher Scientific, Waltham, MA). HEK293 wild-type cells were used as a control. The presence of human IgG in cell supernatants was measured by ELISA.

[0244] The results shown in Table E8 indicate that clonal cell populations carrying the GRP78 single mutant R261Q or double mutation R261Q H265Q can produce and secrete anti-GRP78 IgG antibodies that recognize an epitope containing natural amino acids, and the amount of the antibody is substantially higher than the amount of antibody produced by HEK293 wild-type cells. It is noteworthy that antibodies against GRP78 that could not produce any significant amount of this epitope in these cells were not recognized, because the production of these antibodies significantly reduced cell viability (data not shown).

[0245] The clones are suitable for growth in serum-free medium and in suspension. Suspension cultures in serum-free medium are used for full-scale antibody production. Briefly, cells are grown in increasing concentrations of serum-free medium (FreeStyle TM 2X in DMEM 293 Expression Medium (Thermo Fisher Scientific, Waltham, MA). The dilution concentrations were 25% serum-free medium + 75% (DMEM + 10% FBS); 50% serum-free medium + 50% (DMEM + 10% FBS); and 75% serum-free medium + 25% (DMEM + 10% FBS) and finally 100% serum-free medium. The cells were diluted into suspension flasks and incubated at 8% CO. 2The cells were then subcultured at 125 rpm in a humidified incubator at 37°C. The cells were then considered suitable for suspension when the viability after subculture was 95% or more and the doubling time was approximately 24-26 hours.

[0246]

[0247] Example 4 Generation of cell lines for the production of humanized antibodies against signaling proteins

[0248] A. Targeting of intracellular signaling proteins

[0249] Proteins involved in intracellular signal transduction (such as kinases and phosphatases) can also become extracellular proteins during cancer progression and are promising targets for various forms of immunotherapy. The methods and systems disclosed herein can be used to improve the production of antibodies against these targets. For example, the protein "phosphatase 3 that regenerates the liver" (or "PRL-3", also known as "PTP4A3") is a member of the PRL family of dual-specific protein tyrosine phosphatases (Zeng Q et al., Biochem Biophys Res Commun. 1998; 244 (2): 421-427.). It is localized to the cytoplasmic surface of the plasma membrane and endosomes by the prenylated C-terminal of PRL-3 (Zeng Q et al., J Biol Chem. 2000; 275 (28): 21444-21452).

[0250] PRL-3 is identified as a metastasis-related phosphatase specifically upregulated in, for example, metastatic colorectal cancer (Saha S, et al.. Science. 2001; 294 (5545): 1343-1346), and elevated PRL-3 expression is shown to be a significant predictor of metastatic recurrence in patients with uveal melanoma (Laurent C, et al., Cancer Res. 2011; 71 (3): 666-674). Clinically, elevated PRL-3 mRNA expression is associated with a higher metastatic potential and poor prognosis in a variety of cancer types, including gastric cancer (Bessette DC, et al., Cancer Metastasis Rev. 2008; 27 (2): 231-252). Since then, it has been reported that PRL-3 is overexpressed in up to 70% of primary gastric cancers, where the higher the PRL-3 expression, the shorter the postoperative survival of gastric cancer patients in all tumor stages (Li ZR, et al. Surg Today. 2007; 37(8): 646-651; Dai Netal., World J Gastroenterol. 2009; 15(12): 1499-1505).

[0251] It has been shown that intracellular PRL-3 antigens are externalized to become extracellular antigens during the course of cancer, and therefore PRL-3 is a potential target for immunotherapy to treat various forms of cancer that exhibit extracellular PRL-3 expression. The production methods and systems of the present invention can therefore be used to produce humanized or human PRL-3 antibodies for use as human therapeutic agents.

[0252] B. Generation of edited PRL3 cell lines

[0253] Humanized anti-PRL3 antibodies were engineered from the original framework of previously characterized murine anti-PRL-3 antibodies, as described in Thura M, et al., JCI Insight. 2016; 1(9): e87607. In short, humanized anti-PRL-3 antibodies (referred to as "PRL3-zumab") were engineered from the original framework of previously characterized murine anti-PRL-3 antibody clones. The CDRs of the heavy and light chains of the mouse antibody were transplanted to the human sequence framework, which was selected by comparing the mouse framework sequence with the human framework sequence database to find the closest human homolog for each chain. In addition to the CDRs from the mouse sequence, three amino acid positions from the mouse sequence flanking the CDR were transplanted into the human receptor sequence to maintain the CDR structural integrity of the original murine anti-PRL-3 antibody.

[0254] Example 5 Generation of cell lines for purification of anti-PRL-3 humanized antibodies

[0255] A. Identification of mutations in target proteins that abolish antibody binding

[0256] The epitope of PRL3-zumab is shown to specifically recognize an epitope within the C-terminal region that is conserved between mouse and human PRL-3, but not PRL-1 or PRL-2. Same as above. Considering the knowledge that the epitope is in the C-terminal region of PRL-3, combined with the crystal structure of PRL-3 (as shown by Kim KA et al., FEBS Lett (2004) 565p.181-7), mutagenesis is used to modify one or more residues in this conserved C-terminal region of the natural PRL-3 protein in the production cell line to eliminate the binding of the produced antibody while retaining the overall structure and function of the protein.

[0257] Specifically, mutations in the C-terminal binding region of native mammalian PRL-3 were selected to preserve the structure of the protein to prevent secondary structural changes and the possibility of changes in tertiary and quaternary structures to maintain endogenous function and avoid cell death. The amino acids of PRL-3 were replaced with alanine at key sites, and the activity of the proposed PRL-3 mutants in mammalian cells and the elimination of binding of humanized mAbs to intracellular proteins were further evaluated. Although epitopes are generally conserved between mammalian species (such as rodents and humans), depending on the cell line selected for antibody production, the modified sequence can be based on the epitope sequence of the species of the cell line.

[0258] B. Editing Binding Epitopes in Target Proteins

[0259] Gene sequence data for PRL-3 in humans (Gene ID: 11156) and the corresponding sequence for PRL-3 from Chinese hamster (Gene ID: 100772907) were obtained from the NCBI gene database and used as reference sequences for gene editing in HEK293 cells and CHO cells, respectively. For the production of anti-PRL-3 antibodies, the homology-directed repair (HDR) strategy was used to edit wild-type PRL-3 in HEK293 cells (SEQ ID NO: 33) and CHO cells (SEQ ID NO: 34) using V-type CRISPR gene editing technology to produce desired single mutants and / or double mutants. In U.S. Patent No. 11,220,697 issued on January 11, 2022 by Garst et al. and “Using MAD7 TMSuch editing strategies can be found in Gene Editing in Mammalian Cells: A Quick Start Guide (which can be found at www.inscripta.com). In addition, tools for designing specific guide RNAs for use with type V RGNs can be found from companies such as Integrated DNA Technologies (Coralville, IA) and GenScript (Piscataway, NJ).

[0260] Cells carrying the desired mutation were isolated by cloning serial dilutions, and the presence of point mutations was confirmed by Sanger sequencing. After amplification in culture, the growth and viability of the cells were tested. Control (wtCHO) and mutant PRL-3CHO cells were tested, and cells that reached >90% confluence at the same time (4 days after inoculation) were selected as cells that showed minimal changes in cell growth or viability. Use Live cell analysis system to calculate cell confluence.

[0261] C. Production and purification of anti-Prl3 antibodies in modified cells

[0262] Humanized anti-PRL-3 monoclonal antibody (mAb) is transfected into cloned CHO cells separately by electroporation in 6-well plates, filled with fresh medium the next day and incubated for 5 days after transfection. Wild-type HEK293 or CHO cells are used as controls. The presence of human IgG in the cell supernatant is measured by ELISA. Cells that produce and secrete anti-PRL-3 IgG antibodies are selected, and the amount of the antibody is substantially higher than the amount of the antibody produced by HEK293 and / or CHO wild type.

[0263] Example 6 Humanization of anti-PDI monoclonal mouse antibody

[0264] As disclosed in Example 3, the disclosed methods for producing antibodies by mammalian cells and purifying antibodies from mammalian cells can also be used to produce mAbs that selectively bind to other intracellular proteins, and such methods can not only increase yields by improving cell viability, but also increase the amount of recoverable antibodies by allowing the antibodies to be secreted or otherwise available for purification rather than binding to intracellular targets. The methods are particularly useful for producing antibodies that selectively bind to intracellular mammalian targets, including, for example, targets in the ER and Golgi apparatus. The following example discloses the production of humanized antibodies against the ER target protein disulfide isomerase (PDI).

[0265] PDI (also known as the β subunit of prolyl 4-hydroxylase) is an enzyme encoded by the P4HB gene in humans. PDI is used as a molecular chaperone that catalyzes disulfide bond formation, breakage, and rearrangement in the endoplasmic reticulum (ER). Increasing evidence supports that PDI plays a key role in maintaining intracellular homeostasis by mediating oxidative protein folding. The progression of several cancers (and particularly brain cancers such as glioblastoma) is associated with increased expression of PDI, and PDI inhibitors have been studied for the treatment of cancers such as glioblastoma. See, for example, Kyani et al., ChemMedChem, 2018 Jan 22; 13 (2): 164-177. PDI inhibition has been shown to downregulate genes in the DNA repair pathway and the E2F pathway in glioblastoma cells, and therefore PDI remains a very promising tumor target. Xu S., et al., Theranostics. 2019; 9 (8): 2282-2298.

[0266] Invitrogen PDI monoclonal antibody clone 12 (Thermo Fisher Scientific Catalog No. MA5-43389, Thermo Fisher Scientific, Waltham, MA), hereinafter referred to as "mPDI-1", is a mouse IgG1 monoclonal antibody that selectively binds to human PDI / P4HB ("hPDI"). Invitrogen PDI monoclonal antibody clone 2F6G12H2 (Thermo Fisher Scientific Catalog No. MA5-43389, Thermo Fisher Scientific, Waltham, MA), hereinafter referred to as "mPDI-2", is a different mouse IgG1 monoclonal antibody that selectively binds to hPDI. Both of these antibodies were selected using recombinant human PDI protein as an antigen in a mouse host, and both are sufficiently sensitive for use in ELISA assays; therefore, both are potential candidates for humanization to generate therapeutic monoclonal antibodies targeting hPDI. Because PDI is primarily found in the ER, the methods of the present disclosure are well suited for efficient production of such antibodies in mammalian cells.

[0267] The humanized PDI antibodies used in the methods of the invention preferably comprise CDR sequences derived from or based on mPDI-1 or mPDI-2, as described in more detail herein and in the incorporated references.These antibodies may be humanized as taught in more detail herein.

[0268] Briefly, the DNA sequencing was performed using a DNA sequencing kit provided by Rapid Novor (Ontario, Canada). Antibody sequencing service to determine the V of mPDI-1 and mPDI-2 H and V LThe amino acid sequence of mPDI-1 and mPDI-2 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, methods (e.g., Do Couto et al., U.S. Pat. No. 10,613,094, issued on April 7, 2020, and Chilcote et al., issued on March 18, 2014, U.S. Pat. No. 8,673,593 disclosed in) can be used to humanize mouse monoclonal antibodies.

[0269] Example 7 Epitope mapping of the protein binding site of anti-PDI humanized antibody and hPDI

[0270] Various methods can be used to epitope map the binding sites of mPDI-1 and mPDI-2 mAbs on human hPDI. 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.

[0271] For example, streptavidin protein biosensors can be used to determine the binding of mPDI-1 and mPDI-2 mAbs to biotinylated PDI peptides across hPDI. The biotinylated peptides across the hPDI protein were loaded at 5 μg / ml for 700 seconds, 300 seconds for baseline recording, and then the association and dissociation of mPDI-1 or mPDI-2 with each peptide was measured at multiple concentrations for 600 seconds. Dual reference sensors were used in all tests to measure any observed background signals and to subtract any observed background signals from nonspecific binding or system noise.

[0272] 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 off and R2 correlation coefficients. Where possible, global curve fitting was also performed for multiple concentrations of antibody / analyte.

[0273] Example 8 Identification of hPDI structure to define key residues for mutagenesis

[0274] To identify residues to be edited to eliminate antibody binding to hPDI, a combination of crystal structures of S. cerevisiae (Tian G., et al., Cell. 2006; 124: 61-73) and crystals of human PDI in both oxidized and reduced states (Wang C, et al., Antioxid Redox Signal 19: 36-45) can be used to extrapolate the structure and function of human PDI. Large-scale molecular dynamics simulations starting from the crystal structures of human PDI (hPDI) in oxidized and reduced states indicate intrinsic conformational dynamics. PDI is composed of four thioredoxin-like domains, and the protein can form at least four compact structures. Yang S. et al., PLoS One. 2014; 9 (8): e103472.

[0275] Example 9 Generation of cell lines for purification of anti-PDI humanized antibodies

[0276] A. Identification of mutations in target proteins that abolish antibody binding

[0277] Considering the epitopes involved in the binding of humanized mAbs based on mPDI-1 and mPDI-2 to hPDI and the knowledge of the crystal structure and molecular modeling of the hPDI protein, mutagenesis was used to modify one or more residues to eliminate the binding of the generated antibodies while retaining the overall structure and function of the protein. Mutations in native mammalian PDI were selected to preserve the structure of the protein to prevent secondary structural changes and the possibility of changes in tertiary and quaternary structures to maintain endogenous function and avoid cell death. Amino acid substitutions of PDI with alanine at key sites were predicted, and the proposed PDI mutants were further evaluated in mammalian cells for viability and elimination of binding of humanized mAbs based on mPDI-1 and mPDI-2 to intracellular proteins.

[0278] The proposed PDI mutants were modeled using Phyre2 (intensive processing) (Kelley et al., Nature protocols. 2015; 10 (6): 845-58). Full-length PDI sequences (wild type and ten mutants) were input, and the wild-type sequence was modeled as a control. The mutant PDI structure generated by each Phyre2 was compared with the wild-type PDI structure generated by Phyre2 by the Matchmaker function of UCSF chimera (Pettersen et al., Journal of computational chemistry. 2004; 25 (13): 1605-12). The wild-type structure of hPDI was used as a reference for this feature. Compared with the wild-type structure, the region containing the mutation in the expected mutant structure is visually unchanged, with no significant changes in the secondary structure.

[0279] B. Editing Binding Epitopes in Target Proteins

[0280] The genomic sequence data of PDI in Chinese hamster cells was obtained from the NCBI gene database (gene ID: 100766687) and used as a reference sequence for gene editing. For the production of both PDI-1mAb and PDI-2mAb, the homology-directed repair (HDR) strategy was used to edit wild-type PDI in CHO cells (SEQ ID NO: 33) using V-type CRISPR gene editing technology to produce the desired single mutants and / or double mutants. In U.S. Patent No. 11,220,697 issued on January 11, 2022 by Garst et al. and “Using MAD7 TM Such editing strategies can be found in Gene Editing in Mammalian Cells: A Quick Start Guide (which can be found at www.inscripta.com). In addition, tools for designing specific guide RNAs for use with type V RGNs can be found from companies such as Integrated DNA Technologies (Coralville, Iowa) and GenScript (Piscataway, New Jersey).

[0281] Cells carrying the desired mutation were isolated by cloning serial dilutions, and the presence of point mutations was confirmed by Sanger sequencing. After amplification in culture, the growth and viability of the cells were tested. Control (wtCHO) and mutant PDICHO cells were tested, and cells that reached >90% confluence at the same time (day 4 after inoculation) were selected as cells that showed minimal changes in cell growth or viability. Live cell analysis system to calculate cell confluence.

[0282] C. Production and purification of anti-hPDI antibodies in modified Cho cells

[0283] Humanized anti-hPDI monoclonal antibodies (mAbs) formatted as full-length IgG1 antibodies were transfected individually into cloned CHO cells by electroporation in 6-well plates, filled with fresh medium the next day and incubated for 5 days after transfection. Wild-type CHO cells were used as controls. The presence of human IgG in the cell supernatant was measured by ELISA. Cells that produced and secreted anti-hPDI IgG antibodies were selected in an amount substantially higher than that produced by the CHO wild-type.

[0284] Example 10 Humanization of anti-GOLPH3 monoclonal mouse antibody

[0285] GOLPH3 was initially 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 in GOLPH3 siRNA-treated cells were all eliminated compared to control cells, indicating that GOLPH3 can enhance signaling through TOR-related complexes. These results suggest that GOLPH3 is a true oncogene and may be a useful target for therapeutic strategies.

[0286] Thermo Fisher Scientific monoclonal antibody clone 905CT9.1.1 (Thermo Fisher Scientific Catalog No. 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 the efficient production of such antibodies in mammalian cells.

[0287] 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, the humanized GOLPH3 antibodies provided by Fast Sequence Bio (Ontario, Canada) were used. Antibody Sequencing Service to Determine the V of mGOLPH3 H and V LThe amino acid sequence 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 U.S. Pat. No. 10,613,094, issued April 7, 2020, by Do Couto et al., and U.S. Pat. No. 8,673,593, issued March 18, 2014, by Chilcote et al.).

[0288] Embodiment 11 Epitope mapping of the protein binding site of anti-GOLPH3 humanized antibody and hGOLPH3

[0289] 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.

[0290] 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.

[0291] 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 off Where possible, global curve fitting was also performed for multiple concentrations of antibody / analyte.

[0292] Example 12 Identification of hGOLPH3 structure to define key residues for mutagenesis

[0293] To identify residues to be edited to eliminate antibody binding to hGOLPH3, the crystal structure of hGOLPH3 and its orthologs can be used. Wood et al. The x-ray crystal structure of hGOLPH3 has been reported at high resolution (Wood et al., J Cell Biol. 2009 Dec 28; 187(7): 967-975), as well as the activity of conserved regions in the structure of hGOLPH3 and its yeast ortholog Vps74p (Wood et al., Journal of Cell Biology, 209 187: 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.

[0294] Example 13 Generation of cell lines for purification of anti-GOLPH3 humanized antibodies

[0295] A. Identification of mutations in target proteins that abolish antibody binding

[0296] Given the knowledge of the epitopes involved in the binding of the mGOLPH3 mAb-based humanized mAb to hGOLPH3 and the crystal structure and molecular modeling of the hGOLPH protein, mutagenesis was used to modify one or more residues to eliminate the binding of the generated antibody while retaining the overall structure and function of the GOLPH3 protein. Mutations in native mammalian cell GOLPH3 were selected to preserve the structure of the protein to prevent secondary structural changes and the potential for changes in tertiary and quaternary structures to maintain endogenous function and avoid negative manufacturing outcomes, such as cell death, decreased cell viability, and / or decreased antibody production. Amino acid substitutions of hGOLPH3 with alanine at key sites were predicted and the proposed GOLPH3 mutants were further evaluated in mammalian cells for viability and elimination of humanized mAb-based mGOLPH3 mAb binding to intracellular proteins.

[0297] The proposed GOLPH3 mutants were modeled using Phyre2 (intensive processing) (Kelley et al., Nature protocols. 2015; 10 (6): 845-58). The full-length GOLPH3 sequence (wild type and ten mutants) was input, and the wild-type sequence was modeled as a control. Each Phyre2-generated mutant GOLPH3 structure was compared with the wild-type GOLPH3 structure generated by Phyre2 by the Matchmaker function of UCSF Chimera (Pettersen et al., Journal of computational chemistry. 2004; 25 (13): 1605-12). The hGOLPH3 wild-type structure was used as a reference for this feature. Compared with the wild-type structure, the region containing the mutation in the expected mutant structure is visually unchanged, with no significant changes in the secondary structure.

[0298] B. Editing Binding Epitopes in Target Proteins

[0299] The genomic sequence data of GOLPH3 in Chinese hamster cells was obtained from the NCBI gene database (gene ID: 100766687) and used as a reference sequence for gene editing. For the generation of humanized mGOLPH3 mAb, a homology-directed repair (HDR) strategy was used to edit wild-type GOLPH3 in CHO cells (SEQ ID NO: 36) using V-type CRISPR gene editing technology to generate the desired mutant (e.g., mutation of one or more residues). In Garst et al., U.S. Patent No. 11,220,697, issued on January 11, 2022, and “Using MAD7 TM Such editing strategies can be found in Gene Editing in Mammalian Cells: A Quick Start Guide (which can be found at www.inscripta.com). In addition, tools for designing specific guide RNAs for use with type V RGNs can be found from companies such as Integrated DNA Technologies (Coralville, Iowa) and GenScript (Piscataway, New Jersey).

[0300] Cells carrying the desired mutation were isolated by cloning serial dilutions, and the presence of point mutations was confirmed by Sanger sequencing. After amplification in culture, the growth and viability of the cells were evaluated. Control (wtCHO) and mutant GOLPH3CHO cells were tested, and cells that reached >90% confluence at the same time (day 4 after inoculation) were selected as cells that showed minimal changes in cell growth or viability. Live cell analysis system to calculate cell confluence.

[0301] C. Production and purification of anti-Golph3 antibodies in modified Cho cells

[0302] Humanized anti-GOLPH3 monoclonal antibodies (mAbs) formatted as full-length IgG1 antibodies were transfected individually into clonal CHO cells by electroporation in 6-well plates, filled with fresh medium the next day and incubated for 5 days after transfection. Wild-type CHO cells were used as controls. The presence of human IgG in the cell supernatant was measured by ELISA. Cells that produced and secreted anti-hGOLPH3 IgG antibodies were selected in an amount substantially higher than that produced by the CHO wild-type.

[0303] Embodiment 14 Antibody production in modified cell-free systems

[0304] The modified cells described in Examples 3 and 11 can also be used to create a cell-free system for producing antibodies.

[0305] The coding sequences of anti-GRP78 antibody and anti-GOLPH3 antibody were codon-optimized for Chinese hamster (Cricetulus griseus) and equipped with the regulatory sequences required for in vitro transcription and translation according to the following: Stech et al., (2017). Sci. Rep. 7(1), 12030. 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 cricket paralysis virus (CrPV), GCT as start codon; 3'UTR: T7 terminator sequence, MCS (same as above). The DNA template was synthesized de novo (Agilent, San Jose, CA) and cloned into an appropriate vector (pUC57-1.8k) by Biocat GmbH, Heidelberg. The pUC57-1.8k vector was used 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, USA) and subsequently analyzed using Illumina Miseq TM Control digestion and sequencing were performed using the BIOLUBLE® system (Illumina, San Diego, CA) to verify the correct DNA sequence.

[0306] CHO lysate containing endogenous ER-derived microsomal vesicles was prepared as previously described ( 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 2 CD medium (Lonza, Basel, Switzerland) and exponentially grew to a maximum of 18 × 10 6 Cells / ml. Collect cells by centrifugation at 200 × g for 15 minutes, precipitate, wash twice, and resuspend in a buffer containing 30mM HEPES-KOH (pH 7.5) and 100mM NaOAc. Subsequently, a syringe is used to pass the cell suspension through a 20-gauge needle, which mechanically ruptures the cells. Remove nuclei and cell debris by centrifugation at 6,500 × g for 10 minutes. Use a Sephadex G-25 column (GE Healthcare, Freiburg, Germany) balanced in a buffer containing 30mM HEPES-KOH (pH 7.5) and 100mM to make the supernatant obtained undergo a gel filtration step. The filtered supernatant is eluted into 1 ml fractions, and fractions with an absorbance of RNA content higher than 100 at 260nm are combined. To remove endogenous mRNA, S7 micrococcal nuclease (Roche, Mannheim, Germany) (10 U / ml) and CaCl 2 Cell lysates were treated with 5% paraformaldehyde (100 μg / ml) and incubated at room temperature (RT) for 20 minutes. Micrococcal S7 nuclease was inactivated by adding EGTA (6.7 mM). Optionally, CHO lysates were further supplemented with creatine kinase (100 μg / ml) as taught. Lysates were snap frozen in liquid nitrogen and subsequently stored at -80°C until further use.

[0307] 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 (final concentration 30 μM; specific radioactivity 46.15 dpm / pmol (PerkinElmer LAS (Germany) GmbH, Rodgau, Germany)) was added for subsequent quantitative and qualitative analysis of cell-free protein synthesis. 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 Comfort Thermomixer). Background translation activity was monitored by performing translation reactions without plasmid supplementation.

[0308] After the translation reaction, the samples were centrifuged at 16,000×g for 10 minutes 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 minutes. 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 new 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).

[0309] 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. These changes may be practiced without departing from the true scope and spirit of the present disclosure, and these changes are intended to fall within the scope of the present disclosure.

[0310] sequence

[0311]

[0312]

[0313]

Claims

1. A method for recombinantly producing an antibody, the method comprising: (a) expressing a nucleic acid encoding an antibody in a modified cell-free system, wherein the antibody specifically binds to a target epitope on a protein in the system, and wherein the modified cell-free system comprises a mutation in one or more amino acid residues of the target epitope on the protein in the system; and (b) initiating transcription and translation of the antibody in the cell-free system under conditions such that the antibody is produced.

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

4. The method of claim 3, 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.

5. The method of claim 4, further comprising (c) isolating the antibody from the cell-free system.

6. A method for recombinantly producing a monoclonal antibody, the method comprising: (a) expressing a nucleic acid encoding an antibody in a modified eukaryotic cell-free system, wherein the antibody specifically binds to a target epitope on a protein in the system, and wherein the modified cell-free system comprises a protein comprising a mutation in one or more amino acid residues of the target epitope on the protein in the system; as well as, (b) initiating transcription and translation in the cell-free system under conditions that allow production of the antibody.

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

8. The method of claim 6, further comprising (c) isolating the antibody from the cell-free system.

9. A method for recombinantly producing an antibody, the method comprising: (a) modifying a 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 in one or more amino acid residues of a target epitope of the antibody; (b) creating a cell-free antibody production system from modified cell lines; (c) introducing a nucleic acid template into the cell-free antibody production system, wherein the nucleic acid template encodes the antibody to the target protein; and (d) initiating transcription and translation from the nucleic acid template to produce the antibody in the cell-free antibody production system.

10. The method of claim 9, further comprising (c) isolating the antibody from the cell-free system. The method of claim 10 , wherein the naturally expressed target protein is an intracellular protein in the secretory pathway of the cell line.

12. The method of claim 10, wherein the mutation is a lazy mutation that retains the structure and function of the native target protein in the cell.

13. The method of claim 9, wherein the mutation reduces or eliminates binding of the antibody to the target epitope on the target protein. The method of claim 9 , wherein the target protein is a stress protein.

15. The method of claim 9, wherein the target protein is expressed on an organelle of the cell line.

16. The method of claim 15, wherein the organelle is the endoplasmic reticulum or the Golgi apparatus.

17. The method of claim 16, wherein the target protein is an endoplasmic reticulum chaperone.

18. The method of claim 17, wherein the endoplasmic reticulum chaperone is calreticulin, a heat shock protein, or an isomerase.

19. The method of claim 18, wherein the target protein is glucose-regulated protein 78 (GRP78), HSP47, PDI, calreticulin, or GP94.

20. The method of claim 18, wherein the target protein is glucose-regulated protein 78 (GRP78).

21. The method of claim 16, wherein the target protein is a Golgi complex protein.

22. The method of claim 21, wherein the Golgi complex protein is GOLPH2, GOLPH3, GM130, ATP6V1A, ATP6V1E1, ATP6VOA2, TMEM165, GOLGB1, SCYL1BP1, TRAPPC11, TRAPPC2, or TRIP11.

23. The method of claim 16, wherein the naturally expressed target protein is an intracellular protein in the secretory pathway of a mammalian cell line.

24. The method of claim 16, wherein the mutation is a lazy mutation that preserves the structure and function of the target protein in the mammalian cell line.

25. The method of claim 16, wherein the mutation reduces or eliminates binding of the antibody to the target epitope on a target.

26. The method of claim 16, wherein the target protein is a stress protein.

27. A method for recombinantly producing a monoclonal antibody, the method comprising: (a) modifying 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 in one or more amino acid residues of a target epitope of the antibody; (b) creating a cell-free antibody production system from modified mammalian cell lines; (c) introducing a nucleic acid template into the cell-free antibody production system, wherein the nucleic acid template encodes the antibody to the target protein; and, (d) initiating transcription and translation from the nucleic acid template to produce the antibody in the cell-free antibody production system.

28. The method of claim 27, further comprising (c) isolating the antibody from the cell-free system.

29. The method of claim 28, wherein the naturally expressed target protein is an intracellular protein in the secretory pathway of the cell line.

30. The method of claim 28, wherein the mutation is a lazy mutation that preserves the structure and function of the native target protein in the cell.

31. The method of claim 27, wherein the mutation reduces or eliminates binding of the antibody to the target epitope on the target protein.

32. The method of claim 27, wherein the target protein is a stress protein.

33. The method of claim 27, wherein the target protein is expressed on an organelle of the cell line.

34. The method of claim 33, wherein the organelle is the endoplasmic reticulum or the Golgi apparatus.

35. The method of claim 27, wherein the target protein is an endoplasmic reticulum chaperone.

36. The method of claim 35, wherein the endoplasmic reticulum chaperone is calreticulin, a heat shock protein, or an isomerase.

37. The method of claim 35, wherein the target protein is glucose-regulated protein 78 (GRP78), HSP47, PDI, calreticulin, or GP94.

38. The method of claim 35, wherein the target protein is glucose-regulated protein 78 (GRP78).

39. The method of claim 33, wherein the target protein is a Golgi complex protein.

40. The method of claim 39, wherein the Golgi complex protein is GOLPH2, GOLPH3, GM130, ATP6V1A, ATP6V1E1, ATP6VOA2, TMEM165, GOLGB1, SCYL1BP1, TRAPPC11, TRAPPC2, or TRIP11.

41. The method of claim 27, wherein the naturally expressed target protein is an intracellular protein in the secretory pathway of the mammalian cell line.

42. The method of claim 27, wherein the mutation is a lazy mutation that preserves the structure and function of the target protein in the mammalian cell line.

43. The method of claim 27, wherein the mutation reduces or eliminates binding of the antibody to the target epitope on a target.

44. The method of claim 27, wherein the target protein is a stress protein.

45. The method of claim 27, wherein the target protein is a protein involved in intracellular signal transduction, optionally a kinase or a phosphatase, further optionally wherein the phosphatase is liver regeneration phosphatase 3.

46. ​​The method of claim 27, wherein the target protein is a chaperone protein.

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