Method for cell-free production of recombinant antibodies

Abstract

Provided herein are methods and cell-free systems for the production of recombinant antibodies, where the antibodies are against target proteins that are naturally expressed as intracellular proteins. Also provided herein are modified cell-free systems for use in such methods, where the modified systems are engineered to express variant target proteins having mutant epitopes of the target epitopes of the antibodies.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 359,871, filed on July 10, 2022, which is hereby incorporated by reference in its entirety.

[0002] Incorporation of Sequence Listing The computer-readable form of the sequence listing is submitted with this application by electronic submission and is hereby incorporated by reference in its entirety. The sequence listing is contained in a file named MBRACE003.xml, created on May 2, 2023, and is 35,964 bytes in size (measured in the MS-Windows® operating system).

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

Background Art

[0004] Engineered antibody molecules and their fragments are increasingly being utilized as scientific and clinical tools for the treatment and diagnosis of diseases. Due to the unique ability of antibodies to specifically recognize and bind with high affinity virtually any type of antigen, antibodies have become attractive as starting points for new biological agents and scientific research. In recent years, certain stress proteins that normally exist as intracellular proteins have been identified as unique targets for antibody-based therapies in cancer and other pathological conditions because they are generally not expressed or are expressed at low levels on the surface of normal cells. There is a need for improved methods for producing antibodies, including antibodies and their fragments that specifically recognize and bind intracellular targets of mammalian cells.

Summary of the Invention

[0005] The above general description of the exemplary embodiments and the following detailed description are merely exemplary aspects of the teachings of the present disclosure and are not limiting.

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

[0007] In some embodiments, the present disclosure comprises: 1) 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 within the system, and the modified cell-free system comprises a mutation of one or more amino acid residues of the target epitope on the protein within the system; and 2) initiating transcription and translation of the antibody in the cell-free system under conditions in which the antibody is produced. The present disclosure provides a method for recombinant production of an antibody.

[0008] In some embodiments, the cell-free system comprises a cell lysate. In some embodiments, the cell-free system is a eukaryotic cell lysate.

[0009] In certain embodiments, the cell lysate is a wheat germ lysate, an insect cell lysate, a reticulocyte lysate, or a keratinocyte lysate. In some embodiments, the cell lysate is derived from mammalian cells such as CHO cells, HeLa cells, HEK293 cells, myeloma cells, hybridoma cells, or cultured lymphoma cells.

[0010] In some embodiments, the present disclosure comprises: 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 within the system, and the modified cell-free system comprises a mutation of one or more amino acid residues of the target epitope on the protein within the system; and 2) initiating transcription and translation of the antibody in the cell-free system under conditions in which the antibody is produced. The present disclosure provides a method for recombinant production of a monoclonal antibody.

[0011] In some embodiments, the present disclosure provides a method for recombinant production of an antibody, the method comprising: 1) modifying a cell line to inhibit binding of the antibody to an intracellular protein in the cell line, wherein the modification results in the expression of a variant target protein comprising a mutation of one or more amino acid residues of the target epitope of the antibody; 2) generating a cell-free antibody production system from the modified cell line; 3) introducing a nucleic acid template into the cell-free antibody production system, wherein the nucleic acid template encodes an antibody against 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.

[0012] In some embodiments, the present disclosure provides a method for recombinant production of a monoclonal antibody, the method comprising: 1) modifying a mammalian cell line to inhibit binding of the antibody to an intracellular protein in the cell line, wherein the modification results in the expression of a variant target protein comprising a mutation of one or more amino acid residues of the target epitope of the antibody; 2) generating 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 an antibody against 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.

[0013] In some embodiments, the naturally expressed target protein is an intracellular protein in the secretory pathway. In some embodiments, the target protein is a stress protein. In some embodiments, the mutation is an inactive mutation that retains the structure and function of the native target protein in the cell. In some embodiments, the mutation reduces or inhibits binding of the antibody to the target epitope on the target protein.

[0014] In some embodiments, the target protein is expressed on an organelle of the cell line.

[0015] In certain embodiments, the target protein is generally a protein expressed on the endoplasmic reticulum. For example, the target protein is an endoplasmic reticulum chaperone, such as calreticulin, a heat shock protein, or an isomerase. Specifically, the target protein is glucose-regulated protein 78 (GRP78), heat shock protein 47 (HSP47), protein disulfide isomerase (PDI), calreticulin, or GP94.

[0016] In certain embodiments, the target protein is generally expressed on the Golgi apparatus. For example, the target protein is a Golgi complex protein, such as Golgi phosphoprotein 2 (GOLPH2), Golgi phosphoprotein 3 (GOLPH3), GM130, ATPase H+ transporting V1 subunit A (ATP6V1A), ATPase H+ transporting V1 subunit E1 (ATPP6V1E1), ATPase H+ transporting V0 subunit A2 (ATP6VOA2), transmembrane protein 165 (TMEM165), Golgin B1 (GOLGB1), SCY1-like 1 binding protein 1 (SCYL1BP1), TRAPPC11 (TRAPPC11), transport protein particle complex subunit 2 (TRAPPC2), or thyroid hormone receptor interactor 11 (TRIP11).

[0017] In some embodiments, the target protein associates with intracellular membranes.

[0018] In some embodiments, the target protein is an intracellular signaling protein involved in a signaling pathway, such as a kinase or a phosphatase.

[0019] In some embodiments, the antibody is isolated from a cell-free system after production.

[0020] Other features, advantages, and aspects will be described in more detail below.

[0021] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one or more embodiments and, together with the description, explain these embodiments. The accompanying drawings are not necessarily drawn to scale. Any values or dimensions shown in the accompanying graphs and figures are for illustrative purposes only and may or may not represent actual values or preferred values or dimensions. In some cases, some or all of the features may not be illustrated in order to assist in the description of the underlying features.

Brief Description of the Drawings

[0022]

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Mode for Carrying Out the Invention

[0023] The following description, presented in connection with the accompanying drawings, is intended to be a description of various exemplary embodiments of the disclosed subject matter. Specific features and functions are described in connection with each exemplary embodiment. However, it will be apparent to those skilled in the art that the disclosed embodiments can be practiced without each of those specific features and functions.

[0024] As used herein, "one embodiment" or "an embodiment" means that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the disclosed subject matter. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Further, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments. Additionally, embodiments of the disclosed subject matter are intended to cover its modifications and variations.

[0025] As used in this specification and the appended claims, it should be noted that the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. That is, unless specifically identified otherwise, as used in this specification, terms such as "a", "an", "the", etc. have the meaning of "one or more". Further, terms such as "left", "right", "top", "bottom", "front", "rear", "side", "height", "length", "width", "upper", "lower", "inner", "outer", "inside", "outside", etc. as used in this specification merely describe a reference point and do not necessarily limit the embodiments of the disclosed subject matter to any particular orientation or configuration. Additionally, terms such as "first", "second", "third", etc. merely identify one of a number of parts, components, steps, operations, functions, and / or reference points disclosed in this specification and, likewise, do not necessarily limit the embodiments of the present disclosure to any particular configuration or orientation.

[0026] Further, terms such as "substantially", "about", "approximately", "minor variations" and similar terms generally refer, in a particular embodiment, to a range including the identified value within 20%, 10%, or preferably 5% and any value between them.

[0027] All features described in connection with one embodiment are intended to be applicable to the additional embodiments described below, unless explicitly stated otherwise or the feature or function is incompatible with the additional embodiment. For example, if a given feature or function is explicitly described in connection with one embodiment but not explicitly mentioned in connection with another embodiment, the inventor intends that the feature or function can be developed, utilized, or implemented in connection with that other embodiment, provided the feature or function is compatible with that other embodiment.

[0028] The present specification provides a method for the recombinant production of an antibody or a binding fragment thereof, wherein the target protein of the antibody is generally also found as an intracellular protein within a cell. In some embodiments, the target intracellular protein to which the antibody binds is a protein involved in the secretory pathway, such as a protein expressed on or in the endoplasmic reticulum (ER) or Golgi apparatus. In some embodiments, the target protein of the antibody is a stress protein, which usually functions in the ER or Golgi apparatus or other cellular organelles, but may be upregulated on the cell surface under cellular stress conditions. For example, stress proteins, such as those involved in the unfolded protein response (UPR), have been found to be upregulated on the surface of cancer cells, but are not normally found on the surface of normal cells. Stress proteins generally do not express or only express at low levels on the surface of normal cells, whereas they may be upregulated or overexpressed on the surface of cancer cells, thus showing desirable characteristics for use as target proteins for antibody-based cancer therapy. By using stress proteins as target proteins for antibody-based therapy, upregulation of surface expression in various cancers, rather than normal tissues and cells, is ensured, and off-target activity and / or toxicity are reduced or minimized.

[0029] There is a need for a method for producing an amount of antibody sufficient for use as a therapeutic agent. Several expression systems are available, both of prokaryotic and eukaryotic origin. The choice of system depends on many factors, including the molecular species to be expressed and the exact sequence of the individual antibody.

[0030] Particularly desirable for the recombinant production of human antibodies or humanized antibodies is a mammalian expression system. For example, mammalian cells, like human systems, can perform folding and post-translational modification of desirable proteins. By expressing recombinant proteins in mammalian systems derived from HEK293 cells or CHO cells, it is possible to achieve a glycosylation pattern that is similar but not identical to the glycosylation pattern obtained from human cells. Mammalian cell-free systems allow for very high product yields and are relatively resistant to metabolic stress.

[0031] Efforts have been made to produce antibodies against stress proteins, but it has not been possible to efficiently produce them in mammalian cells. For example, it has been demonstrated from the observations herein that antibodies against the exemplary stress protein GRP78 cannot be expressed at high yields from mammalian cells without using the methods of the present disclosure. Without being bound by theory, in certain situations, stress proteins are thought to be necessary for the cell viability or function of mammalian cells such that binding of intracellular proteins by antibodies during the process of recombinant antibody production becomes detrimental to the cell. For example, in some embodiments, binding of certain intracellular proteins inhibits the ability of the intracellular protein to perform its normal function within the cell, leading to protein misfolding within the cell, lack of degradation of misfolded proteins, inappropriate calcium homeostasis, and in some cases, a decrease or loss of cell viability. This loss or decrease in function affects the ability to produce antibodies at high yields.

[0032] Studies have demonstrated that stress proteins such as chaperones are required for proper intracellular functioning during antibody production. For example, changes in the cellular abundance of secretory pathway proteins have been shown to correlate with the ability to produce abundant recombinant monoclonal antibodies (see, for example, Lambert and Merten, 1997, Biotechnol. Bioeng., 54:165-180; Downham et al. 1996, Biotechnol. Bioeng., 51:691-696). Secretory proteins are known to fold in the endoplasmic reticulum (ER) compartment of the cell immediately after protein synthesis and to be assembled into higher order complexes. The ER consists of specific auxiliary assembly factors together with a quality control mechanism (Ellgaard et al., Quality control in the secretory pathway, Science. 1999 Dec 3; 286:1882-8; Helenius et al., Intracellular functions of N-linked glycans, Science. 2001 Mar 23;291:2364-9). Protein folding, and the refolding of misfolded soluble and aggregated proteins, are known to be mediated by a network of evolutionarily conserved protein molecules called chaperones (Haiti, F.U., Nature, 381, 571-580, (1996); Horwich, A.L., Brooks Low K., Fenton, W.A., Hirshfield, I.N. & Furtak, K., Cell 74, 909-917(1993); Ellis, R.J. and Hemmingsen, S.M., 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, E.A., Cell, 101, 119-122, (2000)). Secretory proteins also undergo various post-translational modifications, including glycosylation, as they pass through the Golgi complex.

[0033] Alternatively, even if the binding of an intracellular protein by an antibody does not affect cell viability, the sequestration of products by the intracellular protein can affect antibody production. Put simply, intracellular proteins can act as “sinks” for the binding of antibodies or antigen-binding fragments, inhibiting secretion and thus limiting the yield of the desired product.

[0034] The provided methods relate to modified cell lines and cell-free systems for producing antibodies or antigen-binding fragments that target endogenous intracellular target proteins within a cell, such as a target protein in the secretory pathway. In some embodiments, the cell is a mammalian cell. In some embodiments, the target protein is a stress protein. In some embodiments, the provided methods include producing a modified mammalian cell that possesses a variant of the target protein by mutating a target epitope to a variant epitope such that binding of an antibody to the endogenous target protein in the mammalian cell is reduced or suppressed. In certain embodiments, the mutation is a genomic mutation of the target protein introduced by a gene editing method such as nuclease-mediated gene editing. In some embodiments, the mutation is introduced using an RNA-guided nuclease, zinc finger nuclease, meganuclease, or transcription activator-like effector nuclease. Optionally, the mutation is introduced by homologous recombination repair (HDR). In certain embodiments, the mutation is an inactivating mutation such that the variant target protein retains the structure and function of the endogenous target protein, but the epitope of the antibody is modified to suppress binding of the antibody to the target protein. In the provided methods, the modified cells can then be used for the production of an antibody or antigen-binding fragment, such as by expressing a nucleic acid molecule encoding the antibody intracellularly and culturing the cells under conditions to produce the antibody. In some embodiments, the produced antibody can be isolated from the cells.

[0035] The results herein demonstrate that the provided method can substantially improve protein production in mammalian cells. In some embodiments, the amount of antibody produced from mammalian cells modified according to the provided method is increased by more than 2-fold compared to the production of the same antibody in natural mammalian cells in which the target protein is not mutated. In some embodiments, the improvement in antibody production is about 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold or more compared to the production of the same antibody in natural mammalian cells in which the target protein is not mutated. For example, in some cases, the improvement in antibody production is at least 20-fold compared to natural mammalian cells in which the target protein is not mutated.

[0036] The production systems and methods provided are illustrated for antibodies targeting GRP78 and other intracellular target proteins. The systems and methods described herein are generally applicable to the production of all or part of antibody-based biologic agents that selectively target proteins that are generally found intracellularly but may be found on the cell surface in certain disease states. Thus, those skilled in the art will understand that the present disclosure is illustrative and applicable to other cell lines and manufacturing methods for biologic agents targeting such other targets.

[0037] All publications, including patent documents, scientific papers, and databases referenced herein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were specifically and individually incorporated by reference. If the definitions set forth herein conflict with or are contrary to the definitions set forth in patents, applications, published applications, and other publications incorporated herein by reference, the definitions set forth herein shall control.

[0038] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0039] Cell-free system methods for cell production This specification provides a method for the recombinant production of an antibody that binds to a target protein (or a homolog thereof) that is normally expressed endogenously in a cell. In the provided cell line or cell-free system and method, the antibody is produced in a mammalian cell line or cell-free system that is genetically modified to include a variant of the target protein in a modified system, for example, a variant of the target protein in which binding of the antibody to the variant target protein is reduced or suppressed. In some embodiments, the variant target protein is modified in the mammalian cell line used to create the system to include a mutant epitope having a mutation in one or more amino acid residues of the target epitope of the antibody, so as to reduce or suppress recognition by the antibody or antigen-binding fragment. In certain embodiments, the mutation of the variant target protein having the mutant epitope is an inactivating mutation that retains the structure and function of the native endogenous protein, thereby ensuring the function of the target protein. Thus, the method provided using the modified cell line or cell-free system enables high-yield production of the antibody in the production system.

[0040] In some embodiments, the provided method comprises: (a) expressing a nucleic acid encoding the antibody in a modified cell line, wherein the antibody is against a target protein or a homolog of the target protein that is normally expressed endogenously in a cell, and the modified cell is engineered with a variant target protein having a mutant epitope that includes 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 cell line under conditions such that the antibody is produced. In some embodiments, the method can further comprise isolating the antibody from the culture supernatant.

[0041] In some embodiments, the provided method comprises: (a) expressing a nucleic acid encoding an antibody in a modified mammalian cell line, wherein the antibody is against a target protein that is naturally expressed as an intracellular protein or a homologue of the target protein, and the modified mammalian cell is engineered with a variant target protein having a mutated epitope comprising 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 antibody is produced. In some embodiments, the method can further comprise isolating the antibody, for example, from the culture supernatant.

[0042] These methods and systems disclosed herein can be used individually or in combination with all or certain aspects of other antibody production systems. In some preferred embodiments, the modified cell line can be produced using the methods disclosed in U.S. Patent Application Publication No. 63 / 337,980, filed on May 3, 2022, and / or U.S. Patent Application Publication No. 63 / 359,541, filed on July 8, 2022, each of these documents being incorporated herein by reference in its entirety for all purposes. For example, in some preferred embodiments, the methods of these applications involve: 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 that is naturally expressed as an intracellular protein within the cell, and the modified cell is engineered to express a variant target protein that contains a mutation in one or more amino acid residues of the target epitope; and b) culturing the modified cell line (and, in some preferred embodiments, isolating the antibody) under conditions such that the antibody is produced. In some preferred embodiments, the methods of these applications involve: 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 suppress the binding of the antibody to the epitope on the target protein; d) introducing an expression vector encoding the antibody into the modified cell line; e) culturing the modified cell line under conditions that allow the production of the antibody from the expression vector; f) expressing the nucleic acid encoding the antibody in the modified cell line (and, in some preferred embodiments, isolating the antibody).In some preferred embodiments, these modified mammalian cell lines of these applications contain a variant protein that is an intracellular protein in the secretory pathway, the variant protein contains a mutation of the native intracellular protein that is the target protein of the antibody, and the mutation is one or more amino acid substitutions of one or more amino acid residues for changing the target epitope of the antibody to a mutant epitope. In some preferred embodiments, the method comprises a) expressing a nucleic acid encoding an antibody in a modified mammalian cell line, wherein the antibody binds to GRP78, and the modified mammalian cell is engineered with a variant GRP78 having a mutant epitope containing a mutation of one or more amino acid residues of the target epitope of the antibody or antigen-binding fragment, and b) culturing this modified mammalian cell line under conditions such that an anti-GRP78 antibody is produced in the culture supernatant, thereby including the recombinant production of an antibody targeting GRP78.

[0043] A. Target of Intracellular Protein and Antibody Thereagainst In some embodiments, the provided cell lines and methods are useful for expressing an antibody whose target protein is naturally expressed as an intracellular protein within the cell. In certain embodiments, the target protein is abnormally expressed on the cell surface of cancer and other diseases, but is also necessary for the function of normal cells due to the role of the target protein in normal intracellular biological processes. In some embodiments, the target protein is a protein involved in the secretory pathway of the cell. In some embodiments, the target protein is expressed on an organelle such as a mammalian cell line. In some embodiments, the target protein is expressed on or within the endoplasmic reticulum. In some embodiments, the target protein is expressed on or within the Golgi apparatus.

[0044] In some embodiments, the intracellular protein target is a stress protein. Stress proteins include proteins involved in the autoregulation of organelles to maintain homeostasis and regulate the ability of organelles under stress conditions that may occur in the tumor environment. A variety of stress proteins are known, including proteins in the secretory pathway. A variety of stress proteins are known, including those resulting from ER stress or Golgi stress: Sasaki and Yoshida, J Biochem. 157:185, 2015; Sasaki and Yoshida, FEBS Letters, 593:2330-2340, 2019; Gao et al., Biofactors, 47:964-974, 2021; Li et al., Mol Neurobiol, 49:1449-59, 2014; Yadav et al., J Cancer Prev., 19:75-88, 2014; and Chen and Cubillos-Ruiz, Nature Reviews Cancer, 21:71-88, 2021.

[0045] For example, targets include endoplasmic reticulum chaperones (e.g., calreticulin, heat shock proteins, isomerases) that can move to the cytosol, and ultimately to the cell surface, particularly under stress conditions. These proteins have been found to be overexpressed in certain pathological conditions. See, e.g., Weirsma VR et al., Front. Oncol., Vol 5: Art. 7 (2015); Garg AD, Cancer Immunol Immunother 61:215-21 (2012). The disclosure of the present embodiment and the application of its teachings enable the production of biologic agents that target endoplasmic reticulum chaperones and other identified targets without unduly compromising cell viability. Exemplary biologic agents include, for example, those that target HSP47 for the treatment of cervical cancer, gastric cancer, or autoimmune diseases (Yokota S et al., Biochem Biophys Res Commun 303:413-8 (2003); Yamamoto N et al., Int J Oncol 43:1855-63 (2013)), biologic agents that target protein disulfide isomerase (PDI) for the treatment of central nervous system cancer, ovarian cancer, brain cancer, prostate cancer, and lung cancer (Xu S et al., Free Radic Biol Med 52:993-1002 (2012)); Zhang L et al., Cancer Invest 27:453-(2009); Pan Z et al., Int J Oncol 35:823-8 (2009)), and biologic agents that target calreticulin for the treatment of various cancers (Zamanian M et al., Pathol Oncol Res 149-54 (2013)); Gold LI et al., FASEB J 24:665-83 (2010), biologics targeting GP94 (Marzec M.et al., Biochim Biophys Acta.2012 March;1823(3):774-787), and biologic agents that target GRP78 (Arap et al. Cancer Cell 6:275-284 (2004); Sato et al. Adv Genet 69:97-114 (2010).

[0046] Various Golgi complex proteins are known to be involved in various pathological conditions, particularly cancer and autoimmune diseases, and monoclonal antibodies that selectively bind to these organelle proteins are suitable for the production of monoclonal antibodies using the methods disclosed herein.Examples of such proteins include, but are not limited to, golgi phosphoprotein 2 (GOLPH2) (Liu et al., Front Oncol., 2021 Dec 7;11:78386), golgin 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+ transporting V1 subunit A (ATP6V1A) (Van Damme T, et al., Am J Hum Genet 100:216-227, 2017), ATPase H+ transporting V1 subunit E1 (ATPP6V1E1) (ibid.), ATPase H+ transporting V0 subunit A2 (ATP6VOA2) (Kornak U, et al., Nat Genet 40:32-34, 2008), transmembrane protein 165 (TMEM165) (Rosnoblet C, et al., Hum Mol Genet 22:2914-2928, 2013), golgin B1 (GOLGB1) (Katayama K, et al., Biochem Biophys Res Commun 499:459-465, 2018), SCY1-like 1 binding protein 1 (SCYL1BP1) (Hennies HC, et al, Nat Genet 40:1410-1412, 2008), transport protein particle complex subunit 11 (TRAPPC11) (Larson AA, et al., 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 interactor 11 (TRIP11) (Smits P, N Engl J Med 362:206-216, 2010).

[0047] 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 gene is GP94. In some embodiments, the target protein is GOLPH2. In some embodiments, the target gene is GOLPH3. In some embodiments, the target gene is GM130. In some embodiments, the target gene is ATP6V1A. In some embodiments, the target protein is AATPP6V1E1. In some embodiments, the target protein is ATP6VOA2. In some embodiments, the target protein is transmembrane protein 165 (TMEM165). In some embodiments, the target protein is GOLGB1. In some embodiments, the target protein is SCYL1BP1. In some embodiments, the target protein is TRAPPC11. In some embodiments, the target protein is thyroid hormone receptor interactor 11 (TRIP11).

[0048] Those skilled in the art are familiar with antibodies against intracellular protein targets such as stress proteins. For example, antibodies against the GRP78 antibody include the 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 International Publication No. 2018 / 057703, International Publication No. 2014 / 153056, International Publication No. 2008 / 105560, US Patent Application No. 2010 / 0041074, US Patent No. 10,259,884, and US Patent No. 10,851,161. Antibodies against protein disulfide isomerase (PDI) include, but are not limited to, Invitrogen PDI monoclonal antibody clone 12 (Thermo Fisher Scientific catalog number MA5-43389) or Invitrogen PDI monoclonal antibody clone 2F6G12H2 (Thermo Fisher Scientific catalog number MA5-43389). Antibodies against calreticulin include, for example, mAb FMC75 (Enzo Life Sciences catalog number ADI-SPA-601) and mAb16 (BD Transduction laboratories catalog number 612137). Antibodies against GOLPH3 include, for example, the monoclonal antibody clone 905CT9.1.1 (Thermo Fisher Scientific catalog number MA5-37626) manufactured by Thermo Fisher. Each of these antibodies can function as a template for humanized antibodies using the methods as disclosed in more detail herein.

[0049] In some embodiments, the antibody is a human antibody. Human antibodies can be prepared by administering an immunogen to a transgenic animal modified to produce intact human antibodies or full antibodies having human variable regions in response to antigen challenge. Such animals typically contain all or part of the human immunoglobulin locus that replaces the endogenous immunoglobulin locus, or is present episomally or randomly integrated into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin locus is generally inactivated. Also, human antibodies can be derived from human antibody libraries including phage display and cell-free libraries that contain antibody coding sequences derived from the human repertoire.

[0050] In addition to using non-human monoclonal antibodies for humanization purposes, non-human antibodies can be used as templates for selecting fully human or nearly fully human antibodies. One particular method for selecting such human antibodies is the use of phage display technology. This method can use antibody screening techniques such as those described in, for example, International Publication No. WO 91 / 17271 by Dower et al. and International Publication No. WO 92 / 01047 by McCafferty et al., U.S. Patent Nos. 5,877,218, 5,871,907, 5,858,657, 5,837,242, 5,733,743, and 5,565,332. In these methods, a library of phages is produced in which various antibodies are displayed on the outer surface of the phage. The antibodies are typically displayed on the phage as Fv or Fab fragments. Antibodies having the desired specificity are selected by their affinity for selected epitopes of an organelle protein.

[0051] In certain exemplary methods, human antibodies that selectively bind to epitopes of organelle proteins can be produced using the techniques of Winter International Publication No. 92 / 20791. In this method, either the heavy or light chain variable region of a non-human monoclonal antibody is used. When the light chain variable region is selected as the starting material, a phage library is constructed in which the members display the light chain variable region of a non-human monoclonal antibody and different heavy chain variable regions. The heavy chain variable regions are obtained from a library of recombinant human heavy chain variable regions. Phages that show strong specific binding to the epitope of the organelle protein are selected. The human heavy chain variable region from this phage serves as the basis for constructing an optimized phage library in which each phage presents the same heavy chain variable region identified from the first display library and a different light chain variable region. The light chain variable regions are obtained from a library of recombinant human light chain variable regions. Phages that display the variable regions of a fully human antibody that shows strong specific binding to the epitope on the organelle protein are selected.

[0052] The antibodies provided include monoclonal antibodies, including monoclonal antibody fragments.

[0053] B. Design of Mutant Epitopes on the Target Protein In some embodiments, the provided method includes mutating a target protein to generate a variant target protein that includes a mutant epitope for suppressing or reducing binding by an antibody or antigen-binding fragment. In some embodiments, the mutant epitope is mutated by a change in one or more amino acid residues of the target epitope of the antibody or antigen-binding fragment. To determine the edits that can be made to produce an antibody using the methods of the present disclosure, the epitopes of monoclonal antibodies produced using the methods of the present disclosure can be mapped using various methods. Exemplary methods are taught in U.S. Patent No. 11,174,479 to Greenleaf, et al., entitled "Devices and methods for display of encoded peptides, polypeptides, and proteins on DNA," issued November 16, 2021, and U.S. Patent No. 11,061,036 to Wilson, et al., entitled "Epitope Mapping," issued July 13, 2021.

[0054] In some embodiments, residues within 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 methods such as treatment with chemical mutagens, irradiation during replication, passage through error-prone (mutator) cell lines, or oligonucleotide-directed random mutagenesis. In some embodiments, oligonucleotide-directed random mutagenesis is used where a preselected region of the protein is the target of random mutagenesis. In some embodiments, the preselected region for random mutagenesis is a region that is thought to include the binding site or epitope. A preliminary assessment of the preselected region can be accomplished by any method known in the art including hydrophobicity analysis of the protein sequence or crystal structure analysis.

[0055] In some embodiments, peptide-based approaches can be used to map the epitopes of antibodies. In one approach, there is peptide display technology that presents a library of protein fragments on the surface of a microarray or E. coli or phage, and finds out which the antibody binds to through microarray scanning or flow cytometry. In another approach, a series of overlapping peptides of the target protein or a pre-selected region thereof are screened for antibody binding by, for example, ELISA or other techniques for monitoring binding interactions.

[0056] In some embodiments, epitopes can be computationally predicted. In some embodiments, epitopes are predicted using the PEASE tool (Sela-Culang, J. et al. (2014) Structure 22(4):646-57; Sela-Culang et al. (2015) Bioinformatics 31(8):1313-5). In some embodiments, the predicted epitopes are experimentally verified. Methods for experimentally testing antibody epitopes are known in the art and include, for example, performing assays including screening a library using an antibody cross-blocking assay, performing mutagenesis analysis, performing deuterium exchange analysis, performing a peptide binding assay, and / or performing verification by X-ray crystallographic studies. In some embodiments, verification evaluates the importance of specific amino acids at specific positions for binding using a library and / or peptides derived from the target protein. In some embodiments, the library includes a library of mutations to a subset or all of the amino acid residues of the target protein. In some embodiments, the library is a yeast display library. In some embodiments, the library is a phage display library.

[0057] In some embodiments, to identify residues to be edited to reduce or completely inhibit the binding of an antibody to an intracellular target protein, it is desirable to start from the resolved crystal structure of the intracellular protein so that important contact residues can be identified and residues that disrupt antigen binding but do not interfere with its cellular function can be substituted. This resolved structure may be the resolved crystal structure of the intracellular protein alone, or alternatively, the resolved crystal structure of the intracellular protein bound to an antibody or antibody fragment.

[0058] However, in many cases, a good molecular model will be able to provide the necessary information. If the molecular model is insufficient, for example, if there is no suitable structural template in the structure database, amino acid exchange experiments can provide information about which residues may be targets for mutation. By alanine scanning mutagenesis (successively mutating each residue to Ala) through these regions, important residue(s) involved in antigen binding can be identified (Cunningham BC and Wells JA, Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3407-3411). If changing a single residue to Ala reduces but does not disrupt binding, that position can, if necessary, be targeted for more drastic mutations (e.g., substitutions that result in a charge difference) to further reduce binding.

[0059] The construction of comparative models provides a wide range of structural templates, and available computer programs ensure that the accuracy of the models is increasingly enhanced. For example, the resolved crystal structures of various proteins have been shown to be very close to the structures predicted by molecular modeling.

[0060] As shown in Example 3 for GRP78, once the structure of the protein target has been elucidated, one of ordinary skill in the art can identify residues that can be modified to reduce antibody binding to the epitope of the organelle protein while leaving sufficient function with respect to the cell viability of the organelle protein, as taught in more detail herein. Methods for making polypeptides containing one or more mutations are well known to those of ordinary skill in the art.

[0061] In some embodiments, one or more amino acid residues within or near an 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 generate a variant target protein. In some embodiments, one or more mutations generate mutant epitopes that disrupt one or more (or all) of the target epitopes on the target protein. In some embodiments, one or more mutations are within the target epitope of a particular antibody. In some embodiments, one or more mutations are conservative mutations. In some embodiments, one or more mutations are non-conservative mutations. In some embodiments, one or more mutations are a mixture of conservative and non-conservative mutations. In some embodiments, the mutation is a deletion or an insertion.

[0062] In some embodiments, the variant target protein comprises at least one amino acid substitution relative to the amino acid sequence of the epitope of the target protein of the antibody. In some embodiments, the variant target protein comprises 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more substitutions relative to the amino acid sequence of the epitope of the target protein of the antibody. In some embodiments, the variant target protein comprises one amino acid substitution relative to the amino acid sequence of the epitope of the target protein of the antibody. In some embodiments, the variant target protein comprises two amino acid substitutions relative to the amino acid sequence of the epitope of the target protein of the antibody. In some embodiments, the variant target protein comprises three amino acid substitutions relative to the amino acid sequence of the epitope of the target protein of the antibody. In some embodiments, the variant target protein comprises four amino acid substitutions relative to the amino acid sequence of the epitope of the target protein of the antibody. In some embodiments, the variant target protein comprises five amino acid substitutions relative to the amino acid sequence of the epitope of the target protein of the antibody.

[0063] In certain embodiments of the provided method, the mutations do not affect the function of the target protein. In some embodiments, the mutations are inactivating mutations that preserve the structure and function of the target protein. In some embodiments, one or more mutations do not disrupt the function of the polypeptide (e.g., the function of the variant target protein is not disrupted relative to the function of the corresponding non-mutated target protein). In some embodiments, one or more mutations do not disrupt the native protein-protein interactions of the protein (e.g., the variant protein retains the ability to form substantially the same protein-protein interactions as the corresponding non-mutated target protein). In some embodiments, one or more mutations do not disrupt the three-dimensional structure of the polypeptide (e.g., the variant target protein retains substantially the same three-dimensional structure as the corresponding non-mutated target protein). In some embodiments, one or more mutations do not disrupt the folding of the protein (e.g., the variant protein retains substantially the same protein folding as the corresponding non-mutated target protein). In some embodiments, one or more mutations do not interfere with the translation of the protein (e.g., the variant protein is translated at the same timing, at the same rate, at the same level, etc. as the corresponding non-mutated target protein). In some embodiments, one or more mutations do not disrupt the normal cellular localization of the protein (e.g., the variant target protein retains substantially the same cellular localization as the corresponding non-mutated target protein). In some embodiments, 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).

[0064] In some embodiments, 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 non-mutated target protein.

[0065] In some embodiments, the cell line is a mammalian cell line. In some embodiments, 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 maintained as compared to the mammalian cell line containing the non-mutated target protein.

[0066] In some embodiments, one or more mutations reduce or inhibit the binding of an antibody to a target protein. In some embodiments, the binding and / or reactivity of one or more antibodies to a variant target protein (e.g., one that includes a mutant epitope) can be evaluated. Methods for evaluating binding to a polypeptide are known in the art and can include performing an ELISA assay, performing a Western blot analysis, performing a radioimmunoassay (RIA), performing surface plasmon resonance (SPR), performing thermophoresis, performing a competitive assay, and performing isothermal titration calorimetry, but are not limited thereto. In some embodiments, the binding and / or reactivity of a variant target protein that includes one or more substitutions is 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% lower than the binding and / or reactivity to a target protein that lacks the one or more substitutions. In some embodiments, the binding and / or reactivity to a variant target protein that includes one or more substitutions is at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 100-fold, or at least about 1000-fold lower than the binding and / or reactivity to a target protein that does not include the one or more substitutions. In some embodiments, the binding and / or reactivity to a variant target protein is eliminated (e.g., the binding and / or reactivity is undetectable in a binding assay relative to a negative control such as an isotype control).

[0067] In some embodiments, variant target proteins with reduced or inhibited binding to an antibody exhibit a decrease in the affinity of the antibody for the variant target protein. In some embodiments, the affinity of the antibody for the variant target protein is 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% lower than 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 at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 100-fold, or at least about 1000-fold lower than 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., the binding of the antibody to the variant target protein is undetectable).

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

[0069] C. Generation of Modified Cell Lines Harboring Variant Target Proteins with Mutated Epitopes In some embodiments, the modified cell line is generated by mutating the epitope of a target protein in a mammalian cell line, such as reducing or suppressing the binding of an antibody to the epitope on the target protein. In some embodiments, the cell line is a mammalian cell line.

[0070] To produce the modified cell line, various mammalian cell culture systems can be used. Examples of suitable host cells include, but are not limited to, Chinese hamster ovary (CHO) cells, baby hamster kidney cells, NSO myeloma cells, monkey kidney COS cells, monkey kidney fibroblast CV-I 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 DHFR CHO cells that are auxotrophic for glycine, thymidine, and hypoxanthine. Other CHO cell lines with the same genomic background, such as CHO-kl, CHO-S, GS-CHO, are also suitable for the expression of recombinant proteins.

[0071] Methods for introducing nucleic acid mutations into target genes are well known in the art (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 Publication No. 2014085593, International Publication No. 2009071334, and International Publication No. 2011146121, U.S. Patent Nos. 8,771,945, 8,586,526, 6,774,279, and U.S. Patent Application Publication Nos. 20030232410, 20050026157, U.S. Patent No. 20060014264, the contents of which are incorporated herein by reference in their entirety), including targeted homologous recombination, site-specific recombinases, PB transposases, and genome editing by engineered nucleases. Agents for introducing nucleic acid modifications into target genes can be designed from publicly available sources or commercially obtained from Transposagen, Addgene, and Sangamo Biosciences.

[0072] Exemplary methods used to introduce nucleic acid mutations into target proteins include genome editing using endonucleases, meganucleases, zinc finger nucleases, and transcription activator-like effector nucleases (TALENs).

[0073] In some embodiments, methods for introducing nucleic acid mutations into a target protein include genome editing using engineered endonucleases. In some embodiments, this approach involves using artificially engineered nucleases to create specific double-strand breaks at desired location(s) within the genome, which are then repaired by endogenous cellular processes such as homologous recombination repair (HDR) and non-homologous end joining (NHEJ). NHEJ directly ligates the DNA ends of the double-strand break, whereas HDR utilizes homologous sequences as donor templates to regenerate the DNA sequences missing at the cleavage site. To introduce specific nucleotide modifications (such as mutations like amino acid substitutions) into genomic DNA, a DNA repair template containing the sequence of interest must be present during HDR. Most restriction enzymes recognize a few base pairs on DNA as targets, and the combinations of recognized base pairs are very likely to be found at many locations throughout the genome, resulting in multiple cleavage products where the desired location is not limited, so genomic editing cannot be carried out using conventional restriction endonucleases. To solve this problem and create site-specific single-strand or double-strand breaks, several different classes of nucleases have been discovered and engineered to date. These 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.

[0074] In certain embodiments, including those disclosed herein, genome editing in exemplary embodiments utilizes clustered regularly interspaced short palindromic repeats (CRISPR) technology to edit specific target regions within the genome of an organism using an RGN. Standard CRIPSR systems for genome editing include separate components of a guide RNA (“gRNA”), a target region homologous to the target gene(s) including the edit(s) of interest (commonly referred to as the “homology arm” or “donor template”), and an RGN (Cas9, Cpf1, or MAD7). Delivery of the RGN, synthetic gRNA, and donor template into cells enables cleavage of the cell's genome at the desired location and editing of the target region of the genome. The guide RNA helps the RGN protein recognize and cleave the DNA of the target genomic region based on the recognized protospacer adjacent motif (PAM). Binding of the gRNA localizes the RGN to the genomic target sequence, causing the RGN to cleave both strands of the DNA to create a double-strand break. The double-strand break generated by the RGN can undergo homologous recombination repair or NHEJ.

[0075] A variety of RGN systems can be used in the cell lines and methods disclosed herein. Such systems include type II systems (e.g., those using an RGN such as Cas9) and type V systems (e.g., those using an RGN such as MAD7) (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)); RGN systems using class I CRISPR systems such as Cas3 (e.g., Morisaka H.et al., Nat Commun,;10(1):5302) or Cas10d, a functional nuclease in type I-D systems (e.g., Osakabe et al., Nucleic Acids Res. 2021 Jun 21;49(11):6347-6363), but are not limited thereto.

[0076] In some embodiments, the recombinant nuclease is Cas9. In some embodiments, Cas9 is derived from Streptococcus pyogenes (SpCas9). In some embodiments, Cas9 is derived from Staphylococcus aureus (SaCas9). In some embodiments, Cas9 is derived from Neisseria meningitidis (NmeCas9). In some embodiments, Cas9 is derived from Campylobacter jejuni (CjCas9). In some embodiments, Cas9 is derived from Streptococcus thermophilis (StCas9).

[0077] 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.

[0078] In some embodiments, the RGN comprises one or more mutations such that the RGN is converted into a nickase that lacks the ability to cleave both strands of a double-stranded DNA molecule. In some embodiments, the RGN comprises one or more mutations such that the RGN is converted into a nickase that can cleave only one strand of a double-stranded DNA molecule. For example, Cas9, which normally can induce double-strand cleavage, can be converted into a Cas9 nickase that can induce single-strand cleavage by mutating the RuvC domain or the NHN domain, which includes the RuvC I, RuvC II, and RuvC III motifs, which are one of the two Cas9 catalytic domains. In some embodiments, the type II RGN comprises one or more mutations in the RuvC catalytic domain or the HNH catalytic domain. In some embodiments, the recombinant nuclease is a recombinant nuclease modified to have nickase activity. In some embodiments, the recombinant nuclease cleaves the strand to which the guide RNA hybridizes but does not cleave the strand complementary to the strand to which the guide RNA hybridizes. In some embodiments, the recombinant nuclease does not cleave the strand to which the guide RNA hybridizes but cleaves the strand complementary to the strand to which the guide RNA hybridizes.

[0079] In some embodiments, the recombinant nuclease used for editing is a fusion protein that includes both nuclease activity and another enzymatic activity, such as reverse transcriptase activity, transposase activity, recombinase activity, or another activity that enhances the editing rate in cells. See, for example, Newby GA, Liu DR. Mol Ther. 2021 Nov 3;29(11):3107-3124. In some embodiments, the genome editing that introduces one or more desired edits into the target region is base editing that does not require double-strand cleavage or donor DNA. See 2021 Nov 3;2019 Dec;576(7785):149-157. doi:10.1038 / s41586-019-1711-4. Epub 2019 Oct 21. PMID:31634902.

[0080] In certain embodiments, RGN-directed genome editing introduces one or more desired edits into a target region and removes a protospacer motif (PAM) region from the target region, thereby preventing further genome editing in that target region. In this embodiment, for example, cells having the desired edits can be selected using an RGN that forms a complex with a synthetic gRNA complementary to the target region. Cells that have not undergone the first editing event will be cleaved and thus will not survive under appropriate selection criteria. Cells containing the mutation of interest will not be cleaved because they do not contain the required PAM site.

[0081] There are numerous publicly available tools that can be utilized to assist in the selection and / or design of target sequences, as well as bioinformatically determined lists of unique gRNAs for targeting specific genomic regions of various species using various RGNs, such as Target Finder from the Feng Zhang laboratory, Target Finder (E-CRISP) from the Michael Boutros laboratory, the RGEN tool: Cas-OFFinder, CasFinder: a flexible algorithm for identifying specific Cas9 targets within the genome, and CRISPR Optimal Target Finder; the Alt-R HDR design tool and templates provided by Integrated DNA Technologies (Coralville, Iowa), and MAD7 for gene editing design tools from Horizon (Cambridge, UK), among others.

[0082] In some embodiments, the RGN and gRNA are introduced into the cell as a ribonucleoprotein (RNP) complex. The RNP complex comprises a protein, such as a recombinant nuclease or a protein containing nuclease activity, and a guide RNA. In some embodiments, the recombinant nuclease is provided as a protein and the guide RNA is provided as a transcribed or synthesized RNA. In some embodiments, the guide RNA forms an RGN and RNP complex under appropriate conditions prior to delivery to the cell.

[0083] The examples described herein utilize an RGN-directed editing system, but those skilled in the art will appreciate upon reading this disclosure that various editing mechanisms can be used to create the disclosed cells, systems, and manufacturing methods. There are a number of different nuclease-based systems for providing edits to the genome of an organism, each of which can be used in either a single editing system, a sequential editing system (e.g., using different nuclease-directed systems sequentially to provide two or more genome edits within a cell), and / or a recursive editing system (e.g., using a single nuclease-directed system to introduce two or more genome edits within a cell). Thus, those skilled in the art will appreciate upon reading this disclosure that the various enzyme-directed editing systems are useful for the disclosed embodiments.

[0084] For example, in certain embodiments, the genomic alterations described herein can be introduced using zinc finger nuclease genome editing. Zinc finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA binding domain to a DNA cleavage domain. The zinc finger domain can be engineered to target specific regions within the genome of an organism (Urnov et al., Nature Reviews Genetics, 11:636-646 (2010); International Publication No. 2003 / 087341A2 of Carroll et al., filed Jan. 22, 2003). ZFNs can be used to utilize the organism's endogenous DNA repair machinery to precisely alter a target region of the genome. ZFNs can be used to produce a double-strand break (“DSB”) in the DNA of an allele that will be repaired by non-homologous end joining (NHEJ) in the absence of a homologous template. NHEJ repairs the DSB by ligating the ends together and typically does not generate additional mutations, although this is limited to cases where the cleavage is clean and not complex (Dural et al., Nucleic Acids Res., 33(18):5978-90 (2005)). This repair mechanism can be used to induce genome editing via indels or chromosomal rearrangements.

[0085] In selected embodiments, the genomic alterations 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 cleave specific sequences of DNA. They are created by fusing a TAL effector DNA-binding domain to a DNA cleavage domain. Because TALENs can be engineered to bind virtually any DNA sequence, when combined with a nuclease, they can cleave DNA at specific positions (see, e.g., Miller, et al., Nature Biotechnology, 29(2):143-8 (2011), Boch, Nature Biotechnology, 29(2):135-6 (2011), International Publication No. 2010 / 079430A1 of Bonas et al., filed Jan. 12, 2010, International Publication No. 2011 / 072246A2 of Voytas et al., filed Dec. 10, 2010).

[0086] Similar to ZFNs, TALENs can edit the genome by inducing DSBs. Site-specific DSBs created by TALENs at the target region are repaired through NHEJ or HDR, resulting in editing of the target genome. TALENs can be used to introduce indels, rearrangements, or introduce DNA into the genome via NHEJ in the presence of an exogenous double-stranded DNA fragment.

[0087] ZFN and TALEN restriction endonuclease technologies utilize a non-specific DNA cleavage enzyme that binds to a specific DNA binding domain (either a series of zinc finger domains or TALE repeats, respectively). Typically, a restriction enzyme is selected in which the DNA recognition site and the cleavage site are separated from each other. The cleavage moiety is isolated and then bound to the DNA binding domain, thereby generating an endonuclease with very high specificity for the target sequence. An exemplary restriction enzyme with such properties is FokI. Further, FokI has the advantage that dimerization is required to possess nuclease activity, which means that the specificity is dramatically increased because each nuclease partner recognizes a unique DNA sequence. To enhance this effect, FokI nucleases have been engineered to function only as heterodimers and to have increased catalytic activity. Heterodimer-functionalized nucleases avoid the possibility of unwanted homodimer activity, thereby increasing the specificity of double-strand cleavage.

[0088] Thus, for example, to target a specific site, ZFNs and TALENs are constructed as nuclease pairs, and each member of the pair is designed to bind to sequences flanking the target site. When transiently expressed in cells, the nucleases bind to their target sites, and the FokI domains dimerize heterologously to generate a double-strand break. Repair of these double-strand breaks via the non-homologous end joining (NHEJ) pathway most often results in small deletions or small sequence insertions. Since each repair performed by NHEJ is unique, the use of a single nuclease pair produces an allelic series with various deletions at the target site. The length of the deletions typically ranges from a few base pairs to several hundred base pairs, but the simultaneous use of two pairs of nucleases has successfully generated larger deletions in cell culture (Carlson et al., 2012; Lee et al., 2010). Furthermore, when a DNA fragment homologous to the target region is introduced in combination with the nuclease pair, the double-strand break can be repaired via homologous recombination repair to produce specific modifications (Li et al., 2011; Miller et al., 2010; Urnov et al., 2005).

[0089] The nuclease portions of both ZFN and TALEN have similar characteristics, but the difference between these artificial nucleases lies in the DNA recognition peptides. ZFN depends on Cys2-His2 zinc fingers, and TALEN depends on TALE. Both of these DNA recognition peptide domains are characterized by being found naturally in combinations in proteins. Cys2-His2 zinc fingers are typically found in repetitive sequences separated by 3bp and are found in various combinations with various nucleic acid interacting proteins. On the other hand, TALE is found in repetitive sequences with a 1:1 recognition ratio between the amino acids and the nucleotide pairs recognized. Since both zinc fingers and TALE occur in repetitive patterns, various combinations can be tried to create various sequence specificities. Approaches for creating site-specific zinc finger endonucleases include, among others, for example, modular assembly (where zinc fingers correlated with triplet sequences are joined in a row to cover the required sequences), OPEN (where peptide domain to triplet nucleotide is selected with low stringency and then peptide combinations to the final target in a bacterial system are selected with high stringency), and bacterial one-hybrid screening of zinc finger libraries. Also, ZFN can be designed and commercially obtained from, for example, Sangamo Biosciences (trademark) (Richmond, California).

[0090] Methods for designing and obtaining TALENs are described, for example, in 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 named Mojo Hand has been introduced by the Mayo Clinic for designing TAL and TALEN constructs for genome editing applications (accessible from www.talendesign.org). Also, TALENs can be designed and commercially obtained, for example, from Sangamo Biosciences (trademark), Richmond, Calif.

[0091] In selected embodiments, the genome modifications described herein can be introduced using meganuclease-directed editing. Meganucleases were identified in the 1990s, and subsequent studies have shown that meganucleases are particularly promising tools for genome editing because they can efficiently induce homologous recombination, generate mutations in the coding or non-coding regions of the genome, and alter the reading frame of the coding regions of the genome (see, for example, Epinat, et al., Nucleic Acids Research, 31(11):2952-2962, and U.S. Patent No. 8,921,332 to Choulika et al., issued Dec. 30, 2014). The high specificity of meganucleases results in high precision and very low cytotoxicity compared to other native restriction enzymes.

[0092] Meganucleases are generally classified into four families: the LAGLIDADG family, the GIY-YIG family, the His-Cys box family, and the 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 the conserved LAGLIDADG motif. The four families of meganucleases are widely separated from each other with respect to conserved structural elements, and as a result, DNA recognition sequence specificity and catalytic activity. Meganucleases are commonly found in microbial species and have the unique property of having very long recognition sequences (>14bp), making them highly specific for cleavage at natural desired positions. This can be utilized to perform site-specific double-strand breaks in genome editing. Those skilled in the art can use these native meganucleases, but the number of such native meganucleases is limited. To address this issue, mutagenesis and high-throughput screening methods have been used to generate meganuclease variants that recognize unique sequences. For example, various meganucleases have been fused to create hybrid enzymes that recognize new sequences. Alternatively, it is also possible to design sequence-specific meganucleases by modifying the DNA that interacts with the amino acids of meganucleases (see, for example, U.S. Patent No. 8,021,867). Meganucleases can be designed using, for example, the methods described in Certo, M T et al. Nature Methods (2012) 9:073-975, U.S. Patent 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, and the entire content of each of the above documents is hereby incorporated by reference in its entirety. Alternatively, nucleases with site-specific cleavage properties can be obtained using commercially available technologies, such as the Directed Nuclease Editor (trademark) genome editing technology manufactured by Precision Biosciences.

[0093] The introduction of the editing system can be carried out by any suitable delivery means. For example, delivery methods and / or vehicles for introduction or transfer into cells can use viral delivery, such as lentivirus, vectors, delivery vectors, or any known method and / or vehicle for delivering such agent(s). In some embodiments, one or more protein(s) and / or nucleic acids necessary for gene editing are introduced or delivered into cells using other physical delivery methods such as electroporation or microinjection, particle gun, calcium phosphate transfection, or cell compression or cell deformation (e.g., as described in Lee, et al., 2012, Nano Lett 12:6322-27).

[0094] In some embodiments, the methods provided herein include introducing or delivering a recombinant nuclease, optionally together with a gRNA, and simultaneously introducing or delivering a donor template. In some embodiments, the recombinant nuclease, gRNA, and donor template are introduced or delivered simultaneously. In some embodiments, the recombinant nuclease and gRNA, and the donor template are introduced or delivered sequentially, optionally together with the gRNA. In some embodiments, the recombinant nuclease and gRNA are introduced or delivered together prior to the introduction or delivery of the donor template.

[0095] In other aspects, genome editing of exemplary embodiments can utilize homologous recombination methods, including the cre-lox technology and FRET technology. Site-specific homologous recombination differs from general homologous recombination in that the short-chain specific DNA sequences required for recombinase recognition are the only sites where recombination occurs. Site-specific recombination requires specialized recombinases that recognize those sites and catalyze recombination at those sites. Many bacteriophage- and yeast-derived site-specific recombination systems, each containing a recombinase and specific cognate sites, have been shown to function in eukaryotic cells for the purpose of DNA integration and are thus applicable for use in the present invention, including the bacteriophage P1 Cre / lox, yeast FLP-FRT system, and Dre system of the tyrosine family of site-specific recombinases. Such systems and methods of use are described, for example, in U.S. Patent 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 lambda Int integrase, HK2022 integrase, etc.), and systems belonging to another serine family of recombinases (such as bacteriophage phiC31, R4Tp901 integrase, etc.) also function for the editing of mammalian cells using their respective recombination sites and are applicable for use in the present invention. Exemplary methods for homologous recombination are described in U.S. Patent Nos. 6,689,610, 6,204,061, 5,631,153, 5,627,059, 5,487,992, and 5,464,764.

[0096] 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 having 3' and 5' homology arms that include the mutation of interest, two Lox or FRT sequences, and a selectable cassette typically placed between the two Lox or FRT sequences. Positive selection is applied to identify homologous recombinants that contain the target mutation. By combining transient expression of Cre or Flp with negative selection, the selectable cassette is removed and cells lacking the cassette are selected. The final target allele contains the Lox or FRT scar of the exogenous sequence.

[0097] In some embodiments, a transposase system can be used. A transposon is a mobile genetic element that contains a nucleotide sequence that can move to different positions within the genome of a single cell. In the process, the transposon can cause mutations and / or change the amount of DNA within the cell's genome. A number of transposon systems that can transpose even within vertebrate cells have been isolated or engineered. For example, Sleeping Beauty (Izsvak and Ivics Molecular Therapy (2004) 9,147-156), PiggyBac (Wilson et al.Molecular Therapy(2007)15,139-145), Tol2 (Kawakami et al.PNAS(2000)97(21):11403-11408) or Frog Prince (Miskey et al.Nucleic Acids Res.Dec. 1,2003 31(23):6873-6881). Typically, the transposase system provides another means for removing the selectable cassette after homologous recombination, similar to the use of Cre / Lox or Flp / FRT. Thus, as an example, the PiggyBac (PB) transposase system includes the mutation of interest, two PB terminal repeats at the site of the endogenous TTAA sequence, and a selectable cassette located between the PB terminal repeats. ’ and 5 ’It includes the introduction of a targeting vector having identical arms. Positive selection is applied to identify homologous recombinants containing the target mutation. By combining transient expression of PBase with negative selection, the selection cassette is removed and cells lacking the cassette are selected. The final target allele contains an introduced mutation without foreign sequences. In some embodiments, there must be a native TTAA site relatively proximal to the position where a particular mutation is to be inserted in order to introduce sequence modification by a transposase system such as pB.

[0098] Methods for assaying effectiveness and detecting sequence modification are well known in the art and include, but are not limited to, DNA sequencing, electrophoresis, enzyme-based mismatch detection assays, and hybridization assays such as PCR, RT-PCR, RNase protection, in situ hybridization, primer extension, Southern blot, Northern blot, and dot blot analysis.

[0099] Sequence modification of a particular gene can also be determined at the protein level using, for example, chromatography, electrophoresis, immunoassay detection assays such as ELISA, Western blot analysis, and immunohistochemistry.

[0100] In some embodiments, positive selection markers and / or negative selection markers can be designed to efficiently select transformed cells that have undergone homologous recombination events with constructs, for example, by using knock-in / knock-out constructs. Positive selection provides a means of enriching a population of clones that have taken up foreign DNA. Examples of such positive markers include, but are not limited to, markers conferring antibiotic resistance such as glutamine synthetase, dihydrofolate reductase (DHFR), neomycin, hygromycin, puromycin, and blasticidin S resistance cassettes. Negative selection markers are necessary for selection against random integration and / or removal of marker sequences (such as positive markers). Examples of such negative markers include, but are not limited to, herpes simplex thymidine kinase (HSV-TK) that converts ganciclovir (GCV) to a cytotoxic nucleoside analog, hypoxanthine phosphoribosyltransferase (HPRT), and adenine phosphoribosyltransferase (ARPT).

[0101] In some embodiments, the variant target protein, or the polynucleotide encoding it, is sequenced to identify or confirm one or more of its substitutions. Methods for sequencing polynucleotides and / or polypeptides are known in the art, including using sequencing systems from Illumina, Inc. (San Diego, CA), Pacific Biosciences (Mountain View, CA), Oxford Nanopore Technologies (Oxford, UK), and Element Biosciences (San Diego, CA).

[0102] D. Expression and Cultivation of Modified Cell Lines In some embodiments, provided is the use of a modified cell line, such as a mammalian cell line according to the provided embodiments, for producing an antibody by transfecting an antibody-producing gene into the cell line. In some embodiments, a nucleic acid encoding an antibody is introduced into a modified mammalian cell line to express the antibody intracellularly. Methods for introducing nucleic acids into cells are well known to those skilled in the art and include various protocols recognized in the art for introducing foreign DNA into host cells (see Kaufman, RJ. Meth. Enzymology 185:537 (1988)). The choice of transformation or transfection protocol depends on the host cell and the nature of the transgene and protein product. Commonly used 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 a reagent that can form lipid-nucleic acid complexes or liposomes that promote the uptake of nucleic acids into host cells when applied to cultured cells (e.g., Lipofectin® reagent or Lipofectamine® reagent, Gibco BRL, Gaithersburg, Maryland) can be introduced.

[0103] In some embodiments, the nucleic acid sequence encoding the antibody can be constructed within or inserted into an expression vector. In some embodiments, a modified mammalian cell line is transfected with the nucleic acids encoding the heavy (H) and light (L) chains of the antibody separately, or the nucleic acids encoding the H and L chains are transfected simultaneously. For example, in one embodiment, the nucleic acid encoding the antibody or its antigen-binding fragment is constructed in separate expression vectors and then used for co-transfection into the modified cell line. Alternatively, the nucleic acids encoding the H and L chains can also be constructed on the same expression vector. The modified mammalian cells are cultured under conditions that allow expression of the integrated gene, and the expressed immunoglobulin chains or full antibody or fragment are recovered from the culture medium.

[0104] Stable expression can be used to produce recombinant antibodies in high yields over long periods. For example, cell lines that stably express antibody molecules can be used in the methods of the present disclosure. Instead of using an expression vector containing a viral origin of replication, host cells can be transformed with an immunoglobulin expression cassette and a selectable marker. After introduction of the foreign DNA, the engineered cells are grown in enriched medium for a period such as 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to the selection, allowing the cells to stably integrate the plasmid into the chromosome, grow and form integration sites, which can then be cloned and expanded into cell lines.

[0105] Expression vectors can contain a selectable marker, an origin of replication, and other features for replicating and / or maintaining the vector. Suitable expression vectors include, for example, plasmids, viral vectors, and the like. A number of suitable vectors and promoters are known to those of skill in the art. Many are commercially available for generating the recombinant constructs of interest. The following vectors are provided by way of example and should not 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. Eukaryotes: pWLneo, pSV2cat, pOG44, PXR1, pSG, pSVK3, pBPV, pMSG, and pSVL.

[0106] Expression vectors generally have appropriate restriction sites located near the promoter sequence for inserting nucleic acid sequences encoding heterologous proteins. There may also be a selectable marker that functions in the expression host. Suitable expression vectors include viral vectors (e.g., viral vectors based on vaccinia virus; poliovirus; adenovirus (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; International Publication No. 94 / 12649, International Publication No. 93 / 03769; International Publication No. 93 / 19191; International Publication No. 94 / 28938; International Publication No. 95 / 11984 and International Publication No. 95 / 00655; adeno-associated virus (e.g., Ali et 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 in International Publication No. 93 / 09239, Samulski et 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 (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), but are not limited thereto.

[0107] Additional expression vectors suitable for use include, for example, lentiviral vectors, gammaretroviral vectors, foamy virus vectors, adeno-associated virus vectors, adenoviral vectors, poxvirus vectors, herpesvirus vectors, genetically engineered hybrid virus vectors, transposon-mediated vectors, etc., but are not limited thereto. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volume 1-4, Cold Spring Harbor Press, NY), and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

[0108] Generally, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, appropriate restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584, WO 01 / 29058, and US Patent No. 6,326,193).

[0109] To evaluate the expression of a polypeptide (e.g., an antibody) or a portion thereof, an expression vector to be introduced into cells may contain either a selectable marker gene, a reporter gene, or both, which can facilitate the identification and selection of expressing cells from a population of cells to be transfected. In some embodiments, the selectable marker is carried on a separate DNA fragment and can be used in a co-transfection procedure. Appropriate regulatory sequences can be adjacent to both the selectable marker and the reporter gene to enable expression in the host cell. Useful selectable markers include, but are not limited to, antibiotic resistance genes.

[0110] In some embodiments, an expression vector encoding an antibody includes a selectable marker for further amplifying the expression of the expressed antibody. In some embodiments, methods for amplifying a gene of interest are also desirable and typically involve the use of a selectable gene that confers a selectable phenotype. Generally, a "selectable gene" is a gene that confers a phenotype on cells that express the gene as a detectable protein. Commonly used examples of selectable genes include, but are not limited to, antibiotic resistance genes. For example, useful dominant selectable markers include antibiotic resistance genes from microorganisms that confer resistance to neomycin, kanamycin, or hygromycin when a drug (or selection agent) is externally added to the cell culture.

[0111] In some embodiments, host mammalian cells provide post-translational modifications to immunoglobulin protein molecules, including removal of the leader peptide, folding and assembly of the heavy and light chains, glycosylation of the antibody molecule, and secretion of the functional antibody protein.

[0112] Once the antibody is produced, it can be purified by any method known in the art for the purification of immunoglobulin molecules, e.g., by chromatography (e.g., ion exchange, affinity, particularly affinity for a specific antigen after protein A, and sizing column chromatography), centrifugation, differences in solubility, or any other standard technique for protein purification. In many embodiments, the antibody is secreted from the cells into the medium and recovered from the medium.

[0113] Exemplary methods for producing antibodies In some preferred embodiments, the cell-free system of U.S. Patent No. 63 / 398,143, filed August 15, 2022, which is hereby incorporated by reference in its entirety for all purposes, can include: a) providing a cell-free protein expression system (in some preferred embodiments, a mammalian cell-free system) that includes a target protein that exhibits an antibody epitope; b) modifying the cell-free system by introducing one or more agents that block the antibody epitope (or, in some preferred embodiments, the antibody paratope) on the target protein but do not eliminate the activity of the target protein within the cell-free system; c) introducing into the cell-free system one or more nucleic acids that encode 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 can include: a) creating a modified cell line by introducing into the cells (in some preferred embodiments, mammalian cells) an agent that selectively binds to a target epitope on an intracellular protein; 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 that encodes an antibody that selectively binds to a target epitope on an 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).

[0114] Modified Mammalian Cell-Free Systems for the Production of Anti-Grp78 Antibodies In some embodiments, the provided methods and cell-free systems can be used to produce antibodies against GRP78, also referred to as anti-GRP78 antibodies. Antibodies can include full-length antibodies or antigen-binding fragments thereof. GRP78, also known as heat shock protein 5 (Bspa5 / BiP), is part of an evolutionarily conserved ER-bound stress response mechanism that provides cell survival signals under environmental and physiological constraints. GRP78 classically participates in the processing of proteins that are not folded, as a molecular component of the ER chaperoning network, but more recent insights suggest that this protein may be located on the cell surface and can influence signaling (Lee, 2014, Nature Rev Cancer 1.4(4));263-276). Retrograde IHC studies have demonstrated a positive correlation between GRP78 expression and decreased survival in advanced breast cancer (Lee et al., 2006, Cancer Research 66(16):7849-7853) and recurrence in prostate cancer patients (Daneshmand et al., 2007, Human Pathology 38(10):1547-1552). Upregulation of GRP78 promotes the survival and chemoresistance of both proliferating and dormant breast cancer cells. The in vivo accessibility of a synthetic peptide composed of a GRP78-binding motif conjugated to a cell death-inducing peptide to promote apoptosis in cancer cells has been demonstrated (Arap et al., 2004, Cancer Cell 6(3):275-284).

[0115] 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 further 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.

[0116] In some embodiments, the antibody is the antibody described in International Patent Application Publication No. WO 2018 / 057703. In some embodiments, the anti-GRP78 is an antibody designated B4. In some embodiments, the anti-GRP78 antibody is an antibody designated D1. In some embodiments, the anti-GRP78 antibody is an antibody designated F6. In any of such embodiments, the antibody is any of the full-length antibodies described above or an antigen-binding fragment thereof. In some embodiments, the anti-GRP78 antibody is a full-length antibody.

[0117] In some embodiments, the anti-GRP78 antibody comprises a variable heavy (VH) chain and a variable light (VL) chain. In some embodiments, the VH chain comprises VH CDR1 shown in SEQ ID NO: 1, VH CDR2 shown in SEQ ID NO: 2, and VH CDR3 shown in SEQ ID NO: 3, and the VL chain comprises VL CDR1 shown in SEQ ID NO: 4, VL CDR2 shown in SEQ ID NO: 5, and VL CDR3 shown in SEQ ID NO: 6. In some embodiments, the VH chain has an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to 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 to the sequence set forth 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.

[0118] In some embodiments, the VH chain comprises VH CDR1 shown in SEQ ID NO: 7, VH CDR2 shown in SEQ ID NO: 8, and VH CDR3 shown in SEQ ID NO: 9, and the VL chain comprises VL CDR1 shown in SEQ ID NO: 10, VL CDR2 shown in SEQ ID NO: 11, and VL CDR3 shown in SEQ ID NO: 12. In some embodiments, the VH chain has an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the sequence shown in SEQ ID NO: 21, and the VL chain has at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 22. In some embodiments, the VH chain is shown in SEQ ID NO: 21 and the VL chain is shown in SEQ ID NO: 22.

[0119] In some embodiments, the VH chain comprises VH CDR1 shown in SEQ ID NO: 13, VH CDR2 shown in SEQ ID NO: 14, and VH CDR3 shown in SEQ ID NO: 15, and the VL chain comprises VL CDR1 shown in SEQ ID NO: 16, VL CDR2 shown in SEQ ID NO: 17, and VL CDR3 shown in SEQ ID NO: 18. In some embodiments, the VH chain has an amino acid sequence having at least 85%, at least 90%, or at least 95% sequence identity to the sequence shown in SEQ ID NO: 23, and the VL chain has at least 85%, at least 90%, or at least 95% sequence identity to the sequence set forth in SEQ ID NO: 24. In some embodiments, the VH chain is shown in SEQ ID NO: 23 and the VL chain is shown in SEQ ID NO: 24.

[0120] In some of any embodiments, the anti-GRP78 antibody binds to human GRP78. In some embodiments, the human GRP78 protein comprises the amino acids shown in SEQ ID NO: 25.

[0121] In some embodiments, the anti-GPR78 antibody binds to a target epitope of GRP78 comprising 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.

[0122] 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 shown in SEQ ID NO: 25. In some embodiments, the target epitope includes the amino acid residues R261, H265, H329, K271, K272, and D333, with reference to the residue numbering of human GRP78 shown in SEQ ID NO: 25.

[0123] 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 shown in SEQ ID NO: 25. In some embodiments, the target epitope includes the amino acid residues R261, H265, H329, K271, K272, and D333, with reference to the residue numbering of human GRP78 shown in SEQ ID NO: 25.

[0124] In some embodiments, the anti-GRP78 antibody is an antibody that binds to the same epitope on human GRP78 as an antibody comprising any of the above sequences (i.e., an antibody having the ability to cross-compete with any of the above anti-GRP78 antibodies with respect to binding to human GRP78). In some embodiments, since the comparison is made using molecules of equal parity (e.g., Fab vs. Fab, full-length antibody vs. full-length antibody, etc.), the reference antibody or antibody fragment for the cross-competition study can be one of the antibodies or antibody fragments described herein. For example, surface plasmon resonance analysis, ELISA assay, 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 demonstrates that the test antibody can compete with the reference antibody with respect to binding to human GRP78 and is thus considered to bind to the same epitope of human GRP78.

[0125] In some embodiments, the modified mammalian cell-free system is engineered to include a variant GRP78 having a mutant epitope that contains mutations in one or more amino acid residues of the 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 alter the target epitope. In some embodiments, the mutant epitope contains 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions of the target epitope of the target protein.

[0126] In some embodiments, the modified mammalian cell-free system is engineered to include a variant GRP78 having an amino acid sequence that contains at least one amino acid substitution as compared to the sequence of human GRP78 set forth 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% sequence identity to the sequence of GPR78 set forth 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% sequence identity to the sequence of GPR78 set forth 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% sequence identity to the sequence of GPR78 set forth in SEQ ID NO: 25. In some embodiments, the modified cell line is engineered with a variant GRP78 having at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions as compared to the sequence of human GRP78 set forth in SEQ ID NO: 25.

[0127] In some embodiments, the mutation of the mutant epitope is an amino acid substitution selected from at least one of the following (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 the following (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.

[0128] In some embodiments, variant GRP78 comprises the mutation R261Q. In some embodiments, variant GRP78 comprises the mutation H265Q. In some embodiments, variant GRP78 comprises the mutation R279Q. In some embodiments, variant GRP78 comprises the mutation E329Q. In some embodiments, variant GRP78 comprises the mutations R261Q and H265Q. In some embodiments, variant GRP78 comprises the mutations R261Q, H265Q, R279Q, and E329Q. In some embodiments, variant GRP78 comprises the mutations R261Q, H265Q, and E329Q. In some embodiments, variant GRP78 comprises the mutations R261Q and R279Q. In some embodiments, variant GRP78 comprises the mutations R261Q, H265Q, K271Q, R279Q, and E329Q. In some embodiments, variant GRP78 comprises the mutations R261Q, H265Q, K271Q, K272Q, E329Q, and D333Q. In some embodiments, variant GRP78 comprises the mutations K113Q, R261Q, K268Q, K271Q, and R279Q. In some embodiments, variant GRP78 comprises the mutations K113Q, R261Q, H265Q, K268Q, K271Q, K272Q, R279Q, E329Q, and D333Q. In some embodiments, variant GRP78 comprises the mutations R261A, H265A, and E329A; R261A and R279A. In some embodiments, variant GRP78 comprises the mutations R261A, H265A, R279A, and E329A.

[0129] Provided herein is a modified mammalian cell-free system having variant GRP78 represented by SEQ ID NO: 26.

[0130] Provided herein is a modified mammalian cell-free system having variant GRP78 represented by SEQ ID NO: 27.

[0131] 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, simian kidney COS cells, simian kidney fibroblast CV-I cells, human embryonic kidney 293 (HEK293) cells, human breast cancer SKBR3 cells, human Jurkat T cells, canine kidney MDCK cells, and human cervical cancer HeLa cells. In some embodiments, the mammalian cell-free system is a CHO cell lysate.

[0132] In some embodiments, the anti-GRP78 antibody exhibits a reduction or suppression of binding to variant GRP78 containing a variant epitope. In some embodiments, the binding and / or reactivity of the anti-GRP78 antibody to variant GPR78 is 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% reduced compared to the binding and / or reactivity to human GRP78 without one or more substitutions, e.g., human GRP78 represented by SEQ ID NO: 25. In some embodiments, the binding and / or reactivity of the anti-GRP78 antibody to variant GPR78 is at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 100-fold, or at least about 1000-fold reduced compared to the binding and / or reactivity to human GRP78 without one or more substitutions, e.g., that represented by SEQ ID NO: 25. Methods for assessing binding can include any of the methods described herein.

[0133] In some embodiments, the anti-GRP78 antibody exhibits a reduced affinity for variant GRP78, for example, relative to human GRP78 without any substitutions, such as that represented by SEQ ID NO: 25. In some embodiments, the affinity of the anti-GRP78 antibody for variant GRP78 is 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% reduced, for example, relative to the affinity for human GRP78 lacking one or more amino acid substitutions, such as that represented by SEQ ID NO: 25. In some embodiments, the affinity of the anti-GRP78 antibody for variant GRP78 is at least about 1.5-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 100-fold, or at least about 1000-fold reduced, for example, relative to the affinity for human GRP78 lacking one or more amino acid substitutions, such as that represented by SEQ ID NO: 25. Methods for measuring affinity can include any method described herein.

[0134] In some embodiments, a modified mammalian cell line comprising variant GPR78 can be used to produce an anti-GRP78 antibody according to any of the provided methods.

[0135] Cell-free system for antibody production A number of cell-free systems can be used as the basis for the cell-free production systems of the present disclosure, including but not limited to those disclosed in U.S. Patent Nos. 11,261,218; 10,774,354; 10,316,322; 10,308,716; 10,190,145; 9,951,366; 9,753,040; 9,617,533; 9,175,327; 9,040,253; 8,778,631; 7,871,794; 7,118,883; 7,041,479; and U.S. Patent Applications Nos. 20060233789 and 20040191858.

[0136] The cell-free system can be based on either prokaryotic or eukaryotic origin. Among prokaryotic systems, extracts based on Escherichia coli are regularly used and are commercially available for the production of various proteins, including antibodies. Systems based on Bacillus subtilis (Kelwick R, et al., Metab Eng. 2016;38:370-81), Pseudomonas putida (Wang H. Synth Biol. 2018), Streptomyces (Li J, et al., Biotechnol Bioeng. 2017;114:1343-53), and Vibrio enteritis (Dondapati SK, et al., Eng LifeSci. 2018;18:140-8) are well-optimized at the laboratory level because the preparation of cell-free lysates is easy. Currently, a wide range of detailed protocols are available for the preparation of E. coli-based lysates. Among eukaryotic systems, extracts based on rabbit reticulocyte lysate (RRL), wheat germ, Spodoptera frugiperda 21 (Sf21), Chinese hamster ovary (CHO), and human cultured cells are regularly used. For reviews, see, for example, Dondapati Sk et al., BioDrugs (2020) 34:327-348.

[0137] For the production of complex proteins that require post-translational modification, eukaryotic cell-free systems are often, though not always, preferred.

[0138] In some embodiments, the cell-free protein synthesis system can use crude cell extracts prepared from cells having the edits as taught in the present disclosure. These cells are grown to an appropriate confluence, the contents are removed by lysis, followed by performing a number of 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 necessary for transcription and translation, such as aminoacyl tRNA synthetases (AAS), ribosomes, factors required for elongation, initiation, and transcription. Protein synthesis can be achieved by combining the cell extract with the necessary substrates such as amino acids, energy substrates, nucleic acid templates, cofactors, salts, nucleotides, etc. Cell-free protein synthesis is a high-speed protein production system as it does not require transfection or cell culture and is not restricted by cell viability.

[0139] In some embodiments, the cell-free protein synthesis system is generated using transcription and translation related factors derived from, or based on, genetic modifications to antibody epitopes in the native proteins taught herein. In these embodiments, the system contains only known proteins and substrates as taught in Shimizu et al. (2001) Nat. Biotechnology, Vol. 19, page 751 and Shimizu et al. (2005) Methods, Vol. 36, page 299. These known components of the translation machinery are purified and added individually along with a DNA template to produce protein, resulting in a highly controlled system. Protein factors involved in the initiation, elongation, and termination of the protein synthesis process are identified and can be individually tailored to the requirements of the system. Any protein targeted by an antibody can be pre-modified such that the epitope to which the antibody binds is altered, suppressing antibody binding to its original target during production while retaining the function of the protein.

[0140] In some embodiments, the protein synthesis system is a microbial lysate system (prokaryotic or eukaryotic) to which mammalian proteins are added to enable proper protein folding processes, such as folding and / or post-translational modifications. In such cases, a recombinant protein (such as a mammalian protein) can be added to the system, and if the protein is a target for antibody production, the protein can be modified to abolish antibody binding. For example, when mammalian GRP78 is added to a yeast cell-free production system, GRP78 can be modified prior to production of the recombinant protein 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 antibodies produced using that system do not bind to the protein (or so that binding to the protein is reduced). Methods for producing recombinant proteins are well known in the art and are described, for example, in the "Protein Expression Handbook" by ThermoFisher Scientific of Waltham, Massachusetts.

[0141] Reaction formats for cell-free protein synthesis Cell-free synthesis using the systems of the present disclosure can be performed in different formats. The success of synthesizing various antibody formats, including single-chain variable fragments (scFv), Fab fragments, and even full IgG, has already been shown in Escherichia coli (Groff D. MAbs. 2014;6:671-8; Yin G et al., MAbs. 2012;4:217-25); in Sf21 (Jin X et al., Biochem Eng J. 2018;138:156-64; Stech M et al., J Biotech-nol. 2012;164:220-31); in reticulocytes (Odegrip R et al., Proc Natl Acad Sci USA. 2004;101:2806-10), in wheat germ and CHO cell-free systems (Thoring L., et al., Sci Rep. 2017;7:17-12188; Stech M.et al., Sci Rep. 2017;7:17-12364; Martin RW et al., ACS Synth Biol. 2017;6:1370-9).Furthermore, scale-up of cell-free reactions to the liter 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), downscaling (Norred SE et al., J Vis Exp. 2015;11:52616;), and high-throughput applications (Contreras-Llano LE, and Tan C. Synth Biol. 2018) have been demonstrated.

[0142] In some embodiments, the synthetic reaction format used is the batch format. The batch-based format is the most commonly used method in both prokaryotic and eukaryotic systems. This method is relatively fast and inexpensive, and synthesis can be carried out within 1.5 to 3 hours depending on the system. In an E. coli-based system, protein yields in the range of 100 μg / mL to 2 - 3 mg / mL can be obtained. Yields from batch-based eukaryotic systems are relatively low, but membrane proteins are automatically incorporated into microsomal membranes and can address functionality immediately after synthesis (Brodel AK et al., PLoS One. 2013;8:2013).

[0143] In other embodiments, to further scale up protein yields via a batch-based eukaryotic system, a repetitive batch-based synthesis format is proposed in which microsomes incorporating the MP of interest generated in the first synthesis reaction can be added to a fresh cell-free synthesis reaction depleted of microsomes (Thoring L. et al., PLoS One. 2016;11:2016; Zemella A, et al., Sci Rep. 2018;8:18-26936).

[0144] In other embodiments, a continuous exchange cell-free synthesis platform (CECFSP). In this format, a semipermeable dialysis membrane separates the reaction chamber from the feed chamber, whereby the feed chamber provides fresh reaction components and concentrates the reaction chamber. Instead, inhibitory components that accumulate 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). Typically, in the CECFSP format, the reaction time is extended and the protein yield increases. So far, the CECFSP format has been used to increase protein yields several-fold and is widely used as a cell-free platform.

[0145] Furthermore, the progress of bioorthogonal reaction chemistry has opened up a path to expand the possibilities of ADC development. Site-specifically labeled ADCs can be produced using site-specific introduction of non-standard amino acids into genetically engineered sequences (Axup JY et al., Proc Natl Acad Sci USA. 2012;109:16101-6). Currently, several ADCs have been approved for therapeutic use. To date, all of these ADCs have been generated by conjugation of mAbs to cytotoxic linker payloads via surface-exposed lysines, or by partial disulfide reduction and conjugation to free cysteine, which usually results in a heterogeneous population of ADCs with different numbers and positions of drug molecules conjugated to the mAb (Strop P et al., Chem Biol. 2013;20:161-7). From synthesis to functional testing, cell-free systems can accelerate the evaluation of antibody constructs by sequential or simultaneous screening. The introduction of non-standard amino acids expands the chemical repertoire and thus broadens the possibilities for modifying and improving antibody-based therapies. Advanced labeling techniques enable very rapid qualitative analysis of drug-to-antibody ratio (DAR), linker, linker / position, drug, drug / position (research applications), allowing complete control of ADC design.

[0146] Unless otherwise defined, all technical terms, notations, and other technical, scientific, or specialized terms used herein are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and / or ease of reference, and inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0147] Definitions As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, "a" or "an" means "at least one" or "one or more". It is understood that aspects and variations described herein include aspects and variations of "consisting of" and / or "consisting essentially of".

[0148] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. The description in range format is for convenience and brevity only and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, a description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. When a range of values is provided, each intervening value between the upper and lower limits of that range, as well as any other stated value or intervening value within the stated range, is encompassed by the claimed subject matter. It is understood that the upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the claimed subject matter, subject to any specifically excluded limit within the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the claimed subject matter. This applies regardless of the breadth of the range.

[0149] As used herein, the term "about" refers to the normal error range for each readily known value. References to "about" a value or parameter herein include (and describe) embodiments that are directed to that value or parameter itself. For example, a description that refers to "about X" includes a description of "X".

[0150] As used herein, the term "antibody" is used in its broadest sense and includes intact antibodies as well as fragment antigen-binding (Fab) fragments, F(ab’)2 fragments, Fab’ fragments, Fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable (V H)Polyclonal and monoclonal antibodies are included, including functional (antigen-binding) antibody fragments such as single-chain antibody fragments including single-chain variable fragments (scFv) and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. This term encompasses intracellular antibodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific antibodies such as bispecific or trispecific antibodies, diabodies, triabodies, and tetrabody, tandem di-scFv, tandem tri-scFv, and other forms of immunoglobulins modified by genetic manipulation and / or other methods.

[0151] Unless otherwise specified, the term "antibody" should be understood to explicitly include its functional antibody fragments, which are also referred to herein as "antigen-binding fragments." This term also encompasses whole antibodies or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0152] The terms "complementary determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and are known to refer to the discontinuous sequences of amino acids within the antibody variable region that confer antigen specificity and / or binding affinity. Generally, each heavy-chain variable region has three CDRs (CDR-H1, CDR-H2, CDR-H3), and each light-chain variable region has three CDRs (CDR-L1, CDR-L2, CDR-L3). The "framework region" and "FR" are known to refer to the non-CDR portions of the variable regions of the heavy and light chains. Generally, each full-length heavy-chain variable region has four FRs (FR-H1, FR-H2, FR-H3, and FR-H4), and each full-length light-chain variable region has four FRs (FR-L1, FR-L2, FR-L3, and FR-L4).

[0153] The exact amino acid sequence boundaries of a particular CDR or FR can be readily determined using any of a number of well-known schemes, including the scheme described in the following references: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (the “Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (the “Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography”, J. Mol. Biol. 262, 732-745.」("Contact numbering" scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains", Dev Comp Immunol, 2003 Jan; 27(1): 55-77 ("IMGT" numbering scheme); Honegger A and Plueckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool", J Mol Biol, 2001 Jun 8; 309(3): 657-70, ("Aho" numbering scheme); Martin et al., "Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23): 9268-9272, ("AbM" numbering scheme); and Ye et al., "IgBLAST: an immunoglobulin variable domain sequence analysis tool," Nucleic Acids Res. 2013 Jul; 41(Web server issue): W34-40, ("IgBLAST" numbering scheme).

[0154] The boundaries of specific CDRs or FRs may vary depending on 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 in both the Kabat and Chothia schemes is based on the sequence lengths of the most common antibody regions, and in some antibodies, insertions and deletions corresponding to inserted characters (e.g., "30a") appear. In the two schemes, specific insertions and deletions ("indels") are placed at different positions and result 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 a compromise between the Kabat and Chothia definitions, based on the definitions used in the AbM antibody modeling software from Oxford Molecular. The IgBLAST scheme is based on matching to germline V, D, and J genes and can be determined using the IgBLAST tool from the National Center for Biotechnology Information (NCBI).

[0155] Table 1 below lists exemplary position boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3, respectively, as specified by the Kabat, Chothia, AbM, and Contact schemes. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. The FRs are located between the CDRs. For example, FR-L1 is located before CDR-L1, FR-L2 is located between CDR-L1 and CDR-L2, and FR-L3 is located between CDR-L2 and CDR-L3. Note that in the shown Kabat numbering scheme, insertions are placed at H35A and H35B, so the end of the Chothia CDR-H1 loop when numbered using the shown Kabat numbering rules varies between H32 and H34 depending on the length of the loop. [Table 1] 1-Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD 2-Al-Lazikani et al., (1997) JMB 273, 927-948。

[0156] Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues within the heavy chain variable domain (VH) are numbered 26-35 (HCDR1), 50-65 (HCDR2), and 95-105 (HCDR3), and the CDR amino acid residues within the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments, the CDR amino acids within VH are numbered 26-35 (HCDR1). In a numbering scheme that combines Kabat and Chothia, in some embodiments, the CDR corresponds to the amino acid residues that are Kabat CDR, Chothia CDR, or a portion of both. For example, in some embodiments, the CDR corresponds to the amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-105 (HCDR3) within VH, and the amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) within VL.

[0157] Thus, unless otherwise specified, the “CDR” or “complementary determining region” of such a region, such as a given antibody or variable region, or a particular individual CDR (e.g., CDR-H1, CDR-H2, CDR-H3), should be understood to encompass the (or a particular) complementary determining region as defined by any of the above schemes or other known schemes. For example, a particular CDR (e.g., CDR-H3) of a given V H or V LWhen described as including the amino acid sequence of the corresponding CDR in the domain amino acid sequence, such a CDR is understood to have the sequence of the corresponding CDR (e.g., CDR-H3) within the variable region, as defined by either the above scheme or any other known scheme. In some embodiments, a particular CDR sequence is specified. Exemplary CDR sequences of the provided antibodies are described using various numbering schemes (see, e.g., Section II), but it is understood that the provided antibodies may include CDRs as described according to either the other above numbering schemes or any other known numbering schemes.

[0158] Similarly, unless otherwise specified, a FR of that region such as a given antibody or variable region, or an individual specified FR(s) (e.g., FR-H1, FR-H2, FR-H3, FR-H4) should be understood to encompass a given (or specific) framework region defined by any of the known schemes. In some cases, a scheme for identifying a particular CDR, FR, or FR or CDR, such as a CDR defined by the Kabat, Chothia, AbM, IgBLAST, IMGT, or Contact method, or any other known scheme, is specified. In other cases, a particular amino acid sequence of a CDR or FR is given.

[0159] Permissible variations of CDR sequences will be known to those skilled in the art. For example, in some embodiments, a polypeptide includes a complementarity determining region (HCDR or LCDR) having an amino acid sequence with 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.

[0160] As used herein, the terms "variable region" or "variable domain" refer to the domain of an antibody heavy or light chain that is involved in binding of the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ), respectively) generally have a similar structure, and each domain comprises four conserved framework regions (FRs) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007). A single V H domain or V L domain may be sufficient to confer antigen-binding specificity. Further, antibodies that bind a particular antigen may each be isolated using a V L domain or V H domain from an antibody that binds the antigen to screen a library of complementary V H domains or V L domains. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0161] As used herein, the terms "culture," "culturing," "growing," "grows," "maintaining," "maintains," "expanding," "expands," etc., when referring to cell culture itself or a process, may be used interchangeably to mean that cells are maintained in vitro (e.g., ex vivo) under conditions suitable for survival. Culturing cells allows the cells to survive and results in growth, differentiation, or division of the cells by culturing.

[0162] As used herein, the term "epitope" refers to any polypeptide determinant that can specifically bind to an antibody. In certain embodiments, an epitope is a region of an antigen that is specifically bound by an antibody. In certain embodiments, an epitope can include chemically active surface groups of a molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups. In certain embodiments, an epitope can have specific three-dimensional structural features (e.g., "conformational" epitopes) and / or specific charge features. An epitope can be formed from both contiguous residues and / or juxtaposed non-contiguous residues (e.g., amino acids) of a target molecule. Epitopes formed from contiguous residues (e.g., amino acids) are typically retained even when exposed to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost upon treatment with denaturing solvents. Examples of epitopes can include, but are not limited to, at least 3, at least 5, or 8-10 amino acid residues. In some embodiments, an epitope is less than 20 amino acid residues in length, less than 15 residues in length, or less than 12 residues in length. When two antibodies exhibit competitive binding to an antigen, these antibodies may bind to the same epitope within the antigen. Epitopes can also be identified by various scans; for example, alanine or arginine scans can identify one or more residues with which an antigen-binding molecule can interact.

[0163] As used herein, the term "mutated epitope" refers to an epitope that has been genetically engineered to contain one or more non-natural mutations that alter the sequence of an epitope recognized by an antibody. In some embodiments, the mutation is one or more amino acid substitutions of amino acid residues that constitute an epitope recognized by an antibody.

[0164] As used herein with respect to a gene, the terms "expression" or "expressed" refer to the transcript and / or the translation product of that gene. The expression level of a DNA molecule in a cell is determined based on either 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).

[0165] The term "gene" refers to a segment of DNA involved in producing or encoding a polypeptide chain. This can include regions before and after the coding region (leader and trailer), and intervening sequences (introns) between individual coding segments (exons). Alternatively, the term "gene" can also refer to a segment of DNA involved in producing or encoding a non-translated RNA such as rRNA, tRNA, guide RNA (e.g., small guide RNA), or microRNA.

[0166] "Antibody fragment" or "antigen-binding fragment" refers to a molecule other than a full antibody that includes a portion of a full antibody that binds to an antigen to which the full antibody binds. Examples of antibody fragments include Fv, Fab, Fab’, Fab’-SH, F(ab’)2; diabody; linear antibody; single-chain antibody molecules such as heavy chain variable (V H ) region, scFv, and single-domain antibodies that include only the V H region; and multispecific antibodies formed from antibody fragments, but are not limited thereto. In some embodiments, the antibody is or includes an antibody fragment that includes a variable heavy chain (V H ) region and a variable light chain (V L ) region. In certain embodiments, the antibody is a single-chain antibody fragment that includes a heavy chain variable (V H ) region and / or a light chain variable (V L ) region such as scFv.

[0167] A single-domain antibody (sdAb) is an antibody fragment that comprises all or part of the heavy-chain variable region of an antibody, or all or part of the light-chain variable region. In certain embodiments, the single-domain antibody is a human single-domain antibody.

[0168] Antibody fragments can be made by a variety of techniques including, but not limited to, proteolytic digestion of a full antibody as well as production by recombinant host cells. In some embodiments, the antibody is a fragment that includes non-naturally occurring arrangements, such as a fragment having two or more antibody regions or chains linked by a synthetic linker, e.g., a peptide linker, and / or a recombinantly produced fragment such as a fragment produced by enzymatic digestion of a native full antibody. In some embodiments the antibody fragment is a scFv.

[0169] A "human antibody" is an antibody having an amino acid sequence corresponding to an amino acid sequence of a non-human source that utilizes a sequence encoding an antibody produced by a human or human cell, or another human antibody including a human antibody repertoire or human antibody library. The term excludes humanized forms of non-human antibodies that include non-human antigen-binding regions, such as those in which all or substantially all of the CDRs are non-human. The term encompasses antigen-binding fragments of human antibodies.

[0170] The term "humanized" is used to describe antibodies in which complementarity determining regions (CDRs) derived from a mammal, such as a mouse, are joined to human framework regions. Often, a polynucleotide encoding an isolated CDR is grafted onto a polynucleotide encoding a suitable variable region framework (and optionally a constant region) to form a polynucleotide encoding a complete antibody, e.g., a humanized or fully human, antibody fragment, etc. Further, a "humanized" antibody can be a chimeric antibody, a human-like antibody, a humanized antibody, or a fully human antibody in order to reduce potential antigenicity without reducing the affinity for a cellular organelle protein target. Chimeric antibodies, human-like antibodies, and humanized antibodies are generally described in the art.

[0171] A humanized antibody has variable region framework residues substantially derived from a human therapeutic antibody (referred to as the acceptor antibody) and complementarity determining regions substantially derived from a mouse antibody (referred to as the donor immunoglobulin). See Queen et al., Proc. Natl. Acad. Sci. USA 86:10029-10033 (1989), International Publication No. 90 / 07861, U.S. Patent Nos. 5,693,762, 5,693,761, 5,585,089, 5,530,101, and U.S. Patent No. 5,225,539 to Winter. When present, the constant region(s) are also substantially or completely derived from human immunoglobulins. The human variable domain(s) are usually selected from human antibodies in which the framework sequence exhibits a high degree of sequence identity with the mouse variable region domain from which the CDRs are derived. The heavy and light chain variable region framework residues can be derived from the same or different human therapeutic antibody sequences. The human therapeutic antibody sequence can be the sequence of a native human antibody or a consensus sequence of several human antibodies. See Carter et al., International Publication No. 92 / 22653. Specific amino acids from the human variable region framework residues are selected for substitution based on the potential impact on the CDR conformation and / or binding to the antigen. Investigation of such potential impact is carried out by modeling, examining the properties of amino acids at specific positions, or empirical observation of the effects of substitution or mutagenesis of specific amino acids.

[0172] For example, if an amino acid differs between a mouse variable region framework residue and a selected human variable region framework residue, the human framework amino acid should usually be substituted with the equivalent framework amino acid of the mouse antibody if the amino acid is reasonably expected to i) bind non-covalently directly to the antigen, ii) be adjacent to the CDR region, iii) otherwise interact with the CDR region (e.g., be within about 6 Å of the CDR region), or iv) be involved in the VL-VH interface.

[0173] Other candidates for replacement are rare acceptor human framework amino acids for human antibodies at that position. These amino acids can be replaced with amino acids derived from the equivalent position of the mouse donor antibody or amino acids derived from the equivalent position of a more typical human antibody. Other candidates for replacement are rare acceptor human framework amino acids for human antibodies at that position. A preferred variable region framework of a humanized antibody typically exhibits at least 75%, more preferably 80%, even more preferably 85% sequence identity to a human variable region framework sequence or a consensus sequence of such sequences.

[0174] In certain examples, a mouse monoclonal antibody can be used as a basis for manufacturing a human therapeutic biologic. In one method, by way of illustration and not limitation, the heavy chain variable V H region is cloned by RT-PCR using mRNA prepared from hybridoma cells. Consensus primers are used as the 5’ primer for the V H region leader peptide containing the translation start codon and the 3’ primer specific for the g2b constant region. Sequences from multiple independently derived clones can be compared to ensure that no changes are introduced during amplification. The sequence of the V H region can also be determined or confirmed by sequencing the V H fragment obtained by 5’ RACE RT-PCR and 3’ g2b-specific primers.

[0175] The light chain variable V L region of the mouse monoclonal antibody can be cloned in a manner similar to the V H region. In one approach, a consensus primer set designed for amplification of the mouse V L region is designed to hybridize to the V L region containing the translation start codon and the 3’ primer specific for the mouse Ck region downstream of the V-J junction region. In a second approach, the 5’ RACE RT-PCR method is used to obtain the V LClone it. Then, combine the cloned array with the array encoding the human constant region.

[0176] In one approach, the variable regions of the heavy and light chains are re-engineered to encode splice donor sequences downstream of their respective VDJ or VJ junctions and cloned into mammalian expression vectors such as pCMV-hγ1 for the heavy chain and pCMV-hκ1 for the light chain. These vectors encode the human γ1 and Cκ constant regions as exon fragments downstream of the inserted variable region cassette. After sequence verification, the heavy and light chain expression vectors can be co-transfected into COS cells to produce chimeric antibodies. The conditioned medium is collected 48 hours after transfection and assayed by Western blot analysis for antibody production or by ELISA for antigen binding. The chimeric antibodies are preferably humanized as described above.

[0177] The heavy and light chain variable regions of chimeric and / or humanized antibodies can bind to at least a portion of the selected human constant region. The choice of constant region may depend on the desired mechanism of action of the antibody, for example, whether cell-mediated toxicity is desired. For example, isotypes IgG1 and IgG3 have antibody-dependent complement activity, while isotypes IgG2 and IgG4 do not. The light chain constant region can be lambda or kappa. The antibody can be expressed as a tetramer containing two light chains and two heavy chains, as separate heavy and light chains, as Fab, Fab’, F(ab’)2, and Fv, or as a single-chain antibody in which the variable domains of the heavy and light chains are linked via a linker.

[0178] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies or an antibody within such population, i.e., the individual antibodies making up the population are identical except for natural occurring variations or variants that may arise during the production of the monoclonal antibody preparation, and such variants are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies to different epitopes, each monoclonal antibody in a monoclonal antibody preparation is directed against a single epitope on an antigen. The term should not be construed as requiring production of the antibody by any particular method. Monoclonal antibodies can be made by a variety of techniques including, but not limited to, production from hybridomas, recombinant DNA methods, phage display methods, and other antibody display methods.

[0179] As used herein, the term "specifically binds to" a target protein or epitope is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule can be said to "specifically bind" if it reacts or associates with a particular target antigen more frequently, more rapidly, with greater duration, and / or with greater affinity than it does with another protein. It is also understood that "specific binding" does not necessarily require exclusive binding (although it can include exclusive binding). In general, but not necessarily, binding means preferential binding. "Specificity" refers to the ability of a binding protein to bind selectively to an antigen.

[0180] As used herein, the term "stress protein" refers to a protein that functions in normal cells and is present at high levels under stress conditions such as hypoxia, nutrient deprivation, pH changes, oxidative stress, or other metabolic dysregulations of cells, such as those that often occur in cancer cells. Examples of stress proteins include proteins whose expression increases when the capabilities of organelles such as the endoplasmic reticulum (ER) and Golgi apparatus are 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 ER-associated protein degradation (ERAD), or proteins related to ER stress-mediated apoptosis. Examples of ER stress proteins include calreticulin, heat shock proteins, and isomerases. Golgi stress proteins include proteins involved in post-translational modifications such as glycosylation or proteins involved in vesicular transport. An exemplary Golgi stress protein is, for example, GOLPH3.

[0181] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), and polymers thereof in either single-stranded or double-stranded form. Unless otherwise limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to natural nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly recited sequence.

[0182] As used herein, the terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer 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 includes, for example, amino acid polymers modified through sulfation, glycosylation, lipidation, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, addition of amino acids via transfer RNA to proteins, such as arginylation, ubiquitination, or conjugation with a labeling component. As used herein, the term "amino acid" refers to any natural and / or non-natural or synthetic amino acid, including glycine, both D and L optical isomers, and amino acid analogs.

[0183] A "vector" is a nucleic acid molecule that preferably self-replicates and carries an inserted nucleic acid molecule into and / or between host cells. An "expression vector" is a polynucleotide sequence that can be transcribed and translated into a polypeptide(s) when introduced into a suitable host cell. An "expression system" generally means a suitable host cell composed of an expression vector that can function to produce a desired expression product.

[0184] The term "recombinant" when applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction, and / or ligation steps, and other procedures that result in a construct different from a polynucleotide found in nature.

[0185] As used herein, the terms "functionally linked" or "operably linked" are used to refer to DNA sequences juxtaposed in such a way that the components so described are in a relationship that enables them to function in the intended manner. For example, when a promoter controls the transcription of a coding sequence, the promoter is functionally linked to the coding sequence. Or, when a ribosome binding site is positioned to enable translation, the ribosome binding site is functionally linked to the coding sequence. The DNA of a signal sequence (secretory leader) is functionally linked to the DNA of that polypeptide when expressed as a precursor involved in the secretion of the polypeptide. Generally, being functionally linked means being contiguous.

[0186] In some preferred embodiments, the present disclosure provides a method for recombinant production of an antibody, the method comprising 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, the modified cell-free system comprises a mutation of one or more amino acid residues of the target epitope on a protein in the system, and initiating transcription and translation of the antibody in the cell-free system under conditions such that the antibody is produced. In some preferred embodiments, 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 lysates 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 recombinant production of a monoclonal antibody, the method comprising expressing a nucleic acid encoding the antibody in a modified eukaryotic cell-free system, wherein the antibody specifically binds to a target epitope on a protein in the system, the modified cell-free system comprises a mutation of one or more amino acid residues of the target epitope on a protein in the system, and initiating transcription and translation of the antibody in the cell-free system under conditions such that the antibody is produced. In some preferred embodiments, 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 the recombinant production of an antibody, the method comprising modifying a cell line to suppress the binding of the antibody to an intracellular protein within the cell line, the modification resulting in the 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 the modified cell line; introducing a nucleic acid template into the cell-free antibody production system, the nucleic acid template encoding an antibody against the target protein; and initiating transcription and translation from the nucleic acid template to produce the antibody in the cell-free antibody production system. In some preferred embodiments, the method comprises (c) isolating the antibody from the 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 inactivating mutation that retains the structure and function of the native target protein within the cell. In some preferred embodiments, the mutation reduces or suppresses 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 the cell line. In some preferred embodiments, the organelle is the endoplasmic reticulum or the Golgi apparatus. In some preferred embodiments, the target protein is an endoplasmic reticulum chaperone. In some preferred embodiments, the endoplasmic reticulum chaperone is calreticulin, a heat shock protein, or an isomerase. In some preferred embodiments, the target protein is glucose-regulated protein 78 (GRP78), HSP47, PDI, calreticulin or GP94. In some preferred embodiments, the target protein is glucose-regulated protein 78 (GRP78). In some preferred embodiments, the target protein is a Golgi complex protein.In some preferred embodiments, the Golgi complex protein is GOLPH2, GOLPH3, GM130, ATP6V1A, ATP6V1E1, ATP6VOA2, TMEM165, GOLGB1, SCYL1BP1, TRAPPC11, TRAPPC2 or TRIP11. In some preferred embodiments, the naturally expressed target protein is an intracellular protein in the secretory pathway of a mammalian cell line. In some preferred embodiments, the mutation is an inactive mutation that retains the structure and function of the target protein in a mammalian cell line. In some preferred embodiments, the mutation reduces or inhibits the binding of an antibody to a target epitope on the target. In some preferred embodiments, the target protein is a stress protein.

[0187] In some preferred embodiments, the present disclosure provides a method for the recombinant production of monoclonal antibodies, the method comprising modifying a mammalian cell line to inhibit the binding of an antibody to an intracellular protein within the cells of the cell line, the modification resulting in the 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 the modified mammalian cell line; introducing a nucleic acid template into the cell-free antibody production system, the nucleic acid template encoding an antibody against the target protein; and initiating transcription and translation from the nucleic acid template to produce the antibody in the cell-free antibody production system. In some preferred embodiments, the method further comprises isolating the antibody from the cell-free system. In some preferred embodiments, the naturally expressed target protein is an intracellular protein in the secretory pathway of the cell line. In some preferred embodiments, the mutation is an inactivating mutation that retains the structure and function of the native target protein within the cell. In some preferred embodiments, the mutation reduces or inhibits 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 the cell line. In some preferred embodiments, the organelle is the endoplasmic reticulum or the Golgi apparatus. In some preferred embodiments, the target protein is an endoplasmic reticulum chaperone. In some preferred embodiments, the endoplasmic reticulum chaperone is calreticulin, a heat shock protein, or an isomerase. In some preferred embodiments, the target protein is glucose-regulated protein 78 (GRP78), HSP47, PDI, calreticulin or GP94. In some preferred embodiments, the target protein is glucose-regulated protein 78 (GRP78). In some preferred embodiments, the target protein is a Golgi complex protein.In some preferred embodiments, the Golgi complex protein is GOLPH2, GOLPH3, GM130, ATP6V1A, ATP6V1E1, ATP6VOA2, TMEM165, GOLGB1, SCYL1BP1, TRAPPC11, TRAPPC2 or TRIP11. In some preferred embodiments, the naturally expressed target protein is an intracellular protein in the secretory pathway of a mammalian cell line. In some preferred embodiments, the mutation is an inactivating mutation that retains the structure and function of the target protein in the mammalian cell line. In some preferred embodiments, the mutation reduces or inhibits the binding of an antibody to a 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 phosphatase, and further optionally, the phosphatase is regenerating liver phosphatase 3. In some preferred embodiments, the target protein is a chaperone protein.

[0188] As will be understood by those skilled in the art, other embodiments are also contemplated herein.

Examples

[0189] The following examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0190] Example 1 Production and Purification of Anti-GPRP78 Antibody in Mammalian Cells The full-length human anti-GRP78 monoclonal antibody (mAb) (Table E1) formatted as a full-length IgG1 antibody was individually transfected into Chinese hamster ovary (CHO) cells by electroporation in a 6-well plate, fresh medium was replenished the next day, and the cells were incubated for 7 days after transfection. The transfection was performed using the Neon™ Transfection System and Neon™ Transfection System Reagents (Thermo Fisher Scientific, Waltham, Massachusetts) with the pre-setting for CHO cells. The supernatant on the 7th day after transfection was loaded onto a 1 mL MabSelect PrisimA™ column to purify the antibody. The antibody concentration and IgG titer in the supernatant were determined using Octet QK384 with a Protein A biosensor (Molecular Devices, Wokingham, Berkshire, UK) using an IgG1 antibody as a reference.

[0191] During culture and collection, the transfected CHO cells showed a decrease in viability. The titer of the supernatant of all anti-GRP78 antibodies produced from CHO cells was low, less than 5 μg / mL (Table E2). To confirm that the decrease in titer was not specific to CHO cells, transfection was repeated in human embryonic kidney 293 (HEK293) cells, and the antibody was purified from the supernatant on the 5th day using the same method, but the titer remained low (Table E2).

[0192] These results are consistent with the observation that the production of anti-GRP78 antibodies in mammalian cells such as human cells is harmful to the cells and affects the ability to produce antibodies in high yields. It has been hypothesized that the antibodies bind to native GRP78 within the endoplasmic reticulum (ER) of the cell, thereby inhibiting the role of the native chaperone within the cell and potentially killing the cell before the antibody is secreted into the supernatant for purification. The binding of the GRP78 antibody to the native ER protein has been demonstrated by experiments showing co-immunoprecipitation of the antibody and GRP78, as evidenced by SDS-PAGE analysis of purified samples run under reducing and non-reducing conditions. As shown in Figure 1, all samples showed a typical SDS-PAGE profile for monomeric IgG, but there were extra bands between 70 and 80 kDa consistent with the presence of the bound GRP78 antigen.

Table 2

Table 3

[0193] Example 2 Epitope Mapping of Anti-GRP78 Antibodies To characterize the binding properties of the antibodies described in Example 1 to GRP78, epitope mapping analysis was performed by alanine-scan mutagenesis. An alanine-scan library of GRP78 was constructed. Next, each anti-GRP78 antibody was screened for binding to individual GRP78 variants, enabling the identification of target protein residues involved in antibody binding.

[0194] The binding of each test antibody to each GRP78 variant in the alanine scanning library was determined by high-throughput flow cytometry under high-stringency conditions (e.g., increasing pH, increasing salt concentration, increasing temperature, and / or increasing wash time). The experiment also included a commercially available monoclonal antibody 1H11-1H7, which was verified to bind to wild-type GFR78 on cells and was thus determined to be an appropriate positive control under these conditions (Thermo Fisher Scientific, Waltham, Massachusetts). Antibody binding to each GRP78 variant, measured by the fluorescence signal (background subtracted from the raw fluorescence data), was normalized to the binding to wild-type (WT) GRP78. For each GRP78 variant, the normalized binding to the test antibody was plotted relative to the binding to the control antibody. Exemplary results for two anti-GRP78 antibody clones are shown in FIGS. 2A and 2B. GRP78 variants that showed >70% binding to the control antibody but <20% binding to the test antibody were identified as primary major binding residues for the test antibody. Other GRP78 variants that did not meet the above criteria but showed a decrease in binding activity (20% - 30%) and proximity to the above-identified primary residues (based on the known 3D structure of the protein) were also identified (and considered "other residues involved in antibody binding").

[0195] Table E3 shows the residues involved in antibody binding for exemplary anti-GRP78 clones B4 and F6. [Table 4]

[0196] The PDB ID 6ASY (Yang et al. Nature Communications. 2017;8(1):1-3) was tested for the identification of the predicted amino acids that constitute the GRP78 epitope binding to the B4 mAb or F6 mAb. This structure was selected because it is the most complete among the experimentally determined structures of H. sapiens GRP78 (in the range of amino acids 25 - 633). (PDB ID #6ASY, Yang et al., 2017). Figure 3 shows the visualization of the major residues for antibody binding, with exemplary major residues and other residues involved in binding indicated by arrows. According to the analyzed structure, all of the amino acids of the predicted epitope are either contained within an α - helix or as part of a linker between two α - helices and may be separated by α - helices (major residues by shotgun mutagenesis are underlined): (i) K113; (ii) R261 , H265 , K268, K271, K272; (iii) R279 ; and (iv) E329 , D333.

[0197] Example 3 Generation of Engineered GRP78 Cell Lines Antibodies are generally produced and purified from mammalian cells where the wild - type target of the antibody is found intracellularly (such as within the membranes of organelles), but is a protein present on the cell surface in the patient's pathological state, such as on cancer cells or cells present in patients with autoimmune diseases. This method was exemplified using GRP78 and exemplary anti - GRP78 antibodies described in Examples 1 and 2.

[0198] A. Identification of Mutations in the Target Protein to Suppress Antibody Binding Based on the knowledge of the epitopes involved in antibody binding, mutagenesis was used to modify one or more of the residues while maintaining the structure and function of the protein, thereby suppressing the binding of the produced antibodies. For the epitope residues identified for the anti-GRP78 antibodies in Table E3, the mutation(s) of GRP78 were selected such that (1) one or more charged residues were replaced with uncharged residues, (2) it was confirmed that the newly selected residues remained exposed to the solvent, and (3) to prevent changes in secondary structure and the possibility of changes in tertiary and quaternary structures and to preserve the endogenous function and avoid cell death, the α-helix was ensured to be preserved. Among the amino acids with a high tendency to form this secondary structure, the α-helix is highly likely to be formed. Furthermore, polar or charged amino acids are more likely to remain exposed to the solvent due to their interaction with water. However, since charged residues are highly likely to form interactions with antibodies, polar amino acids are recommended for mutagenesis. The polar uncharged amino acids from the highest to the lowest tendency to form an alpha helix (due to helix penalty) are glutamine (position 6), serine (position 10), asparagine (position 15), and threonine (position 16) (Pace and Scholtz, Biophysical journal. 1998;75(1):422-7). Glutamine was selected as the amino acid to replace the proposed amino acids that interact with the mAb. Replacing the major interacting amino acids with alanine (ranked 1 in terms of α-helix tendency) (Pace and Scholtz, 1998) has a risk of changing the secondary and tertiary structures due to the hydrophobic R group, but is likely to suppress the binding to the mAb. Therefore, it was more strategic to incorporate alanine at a low frequency, but at the most important sites.

[0199] Table E4 identifies GRP78 mutations that suppress binding to the exemplary B4 and F6 mAbs.

Table 5

[0200] The proposed GRP78 variants were modeled using Phyre2 (intensive processing) (Kelley et al., Nature Protocols. 2015;10(6):845-58). The full-length GRP78 sequences (wild type and 10 variants) were input. The wild-type sequence was modeled as a control. For all 11 structures generated, the residues approximately 25 - 633 were listed with a confidence of 100%. The residues approximately 1 - 24 and approximately 634 - 654 were not important for the analysis but were modeled from the beginning.

[0201] Each of the mutant GRP78 structures generated by Phyre2 was compared with the wild-type GRP78 structure generated by Phyre2 via 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. Compared with the wild-type structure, the regions containing mutations in all mutant structures did not visually change, and no change in the α-helix was observed. Nevertheless, as shown in Table E5, the root mean square deviation (RMSD) values were calculated according to the pruned Cα atoms (654 possible pairs).

Table 6

[0202] In summary, the proposed GRP78 variants involve substituting the residues that interact with B4 mAb and / or F6 mAb (all of which were charged amino acids) with glutamine (a polar uncharged amino acid with the highest tendency to form an α-helix) or alanine (an amino acid with the highest tendency to form an α-helix overall despite being hydrophobic). According to the modeling, these variants ensure the preservation of the secondary and tertiary structures of GRP78, which simultaneously promotes the retention of the native function and the suppression of binding to the above-mentioned mAbs.

[0203] The single mutation R261Q was selected for subsequent experiments as a major residue of both the B4 and F6 mAbs, as detailed in Section B below. Additionally, the double mutation R261Q / H265Q was also selected for subsequent experiments detailed in Section B below, as this is a mutation found in a combination that modified two residues identified as part of its epitope and suppressed binding as shown in Table E4. The combination of mutations in the double mutant was specifically selected because 1) the residues determined to be potentially important for epitope binding of the GFR78 antibody to the native intracellular protein were altered by epitope mapping, and 2) the mutations were close enough to fit on the same DNA donor template for CRISPR-based editing. Other combinations could also be used, and it was considered necessary to use multiple donor templates and / or transfections to introduce mutations into the cells simultaneously or sequentially.

[0204] B. Editing of the Binding Epitope in the Target Protein Genomic sequence data for GRP78 was obtained from the NCBI Gene database (Gene ID: 3309) and used as the reference sequence for gene editing. For both the B4 mAb and the F6 mAb, the codons for the predicted interaction residues on GRP78 are located within exon 5 as shown in FIG. 4. The flanking introns are also shown in FIG. 4.

[0205] Using a homologous recombination repair (HDR) strategy, wild-type GRP78 (SEQ ID NO: 25) was edited using CRISPR / Cas9 technology to generate a single mutant GRP78 (SEQ ID NO: 26) with the mutation R261Q or a double mutant GRP78 (SEQ ID NO: 27) with the mutation R261Q / H265Q. See, for example, U.S. Patent No. 9,822,372 to Zhang et al., issued November 21, 2017.

[0206] The vectors, gRNAs, and donor templates used were identical for the generation of single and double mutant R261Q / H265Q clones, and individual clones containing either the single mutant R261Q or the double mutant R261Q / H265Q were identified. The donor template contained two mutations but gave rise to some cells with a single mutation and some cells with a double mutation.

[0207] R261Q single mutant cells and R261Q / H265Q double mutant cells were generated by introducing genomic nucleotide modifications of CGT→CAG (conversion from R to Q) at positions 2188 - 2190 in exon 5 and CAC→CAG (conversion from H to Q) at positions 2200 - 2202 in exon 5 (Figure 4). The gRNAs and ssDNA donor templates were designed to introduce mutations by HDR-based CRISPR gene editing (Table E6). The dual gRNAs were designed in the "PAM-out" direction as shown in Figure 5A. Modified codons within the ssDNA oligodonor sequences for causing genomic modifications are shown in bold and italics in Table E6. The nucleotides of the ssDNA were designed to remove the gRNA-related PAM site from the donor template and prevent cleavage of the donor template DNA while preserving the sequence encoding the wild-type amino acid (underlined in Table E6).

[0208] PCR amplification using the primer pairs shown in Table E7 was used to verify the gene editing (Figure 5B). The length of the resulting amplicon was 509 base pairs.

Table 7

Table 8

[0209] Cells with the desired mutation were isolated by clonal serial dilution, and the presence of point mutations was confirmed by Sanger sequencing. After growth in culture, cell growth and viability were tested. Control (HEK293), GRP78 R261Q (single mutation), and GRP78 R261Q H265Q (double mutation) cells reached >95% confluence simultaneously (at 4 days post-seeding), indicating no change in cell growth or viability. Cell confluence was calculated using the IncuCyte® live cell analysis system.

[0210] C. Production and purification of anti-Gprp78 antibody in modified Hek293 cells Full-length human anti-GRP78 monoclonal antibodies (mAbs) formatted as the full-length IgG1 antibodies listed in Table E1 were individually transfected into clonal HEK293 cells by electroporation in 6-well plates, fresh medium was replenished the next day, and incubated for 5 days post-transfection. Transfection was performed using the Neon® transfection system and Neon® transfection system reagents (Thermo Fisher Scientific, Waltham, MA) with pre-setting for HEK293 cells. HEK293 wild-type cells were used as a control. The presence of human IgG in the cell supernatant was measured by ELISA.

[0211] The results shown in Table E8 demonstrate that clonal cell populations with the single mutant R261Q or double mutant R261Q, H265Q of GRP78 can produce and secrete anti-GRP78 IgG antibodies that recognize epitopes containing substantially more native amino acids than the antibodies produced from HEK293 wild-type cells. Notably, antibodies against GRP78 that do not recognize this epitope cannot be produced in significant amounts in these cells because their production results in a substantial decrease in cell viability (data not shown).

[0212] The cloned cell line was adapted to grow in serum-free medium and suspension. For full-scale antibody production, suspension culture in serum-free medium is used. Briefly, the cells were passaged twice at increasing concentrations of serum-free medium (FreeStyle™ 293 Expression Medium, ThermoFisher Scientific, Waltham, MA). The dilution concentrations were, in order, 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 passaged after pelleting at 125 rpm in a humidified incubator at 8% CO2 and 37 °C. Adaptation and growth were monitored daily by cell counting and viability. The cells were considered adapted to suspension if the viability after passage was 95% or greater and the doubling time was approximately 24 - 26 hours.

Table 9

[0213] Example 4 Generation of a Cell Line for the Production of a Humanized Antibody against a Signal Transduction Protein A. Targeting of Intracellular Signal Transduction Proteins Proteins involved in intracellular signal transduction, such as kinases and phosphatases, can also become extracellular during cancer progression and are promising targets for various forms of immunotherapy. The production of antibodies against these targets can be enhanced using the methods and systems of the present disclosure. For example, the protein "phosphatase of regenerating liver 3" (or "PRL-3", also known as "PTP4A3") is a member of the PRL family of dual-specificity protein tyrosine phosphatases (Zeng Q et al., Biochem Biophys Res Commun. 1998;244(2):421 - 427). PRL-3 localizes to the cytoplasmic face of the cell membrane and endosomes via its prenylated C-terminus (Zeng Q et al., J Biol Chem. 2000;275(28):21444 - 21452).

[0214] PRL-3 has been identified as a metastasis-related phosphatase through specific upregulation, for example, in metastatic colorectal cancer (Saha S, et al. Science. 2001;294(5545):1343-1346), and increased PRL-3 expression has been shown to be a significant predictor of metastatic recurrence in patients with melanoma (Laurent C, et al. Cancer Res. 2011;71(3):666-674). Clinically, an increase in PRL-3 mRNA expression levels has been shown to correlate with increased metastatic potential and poor prognosis in multiple types of cancers, including gastric cancer (Bessette DC, et al., Cancer Metastasis Rev. 2008;27(2):231-252). Since then, PRL-3 has been reported to be overexpressed in up to 70% of primary gastric cancers, and higher PRL-3 expression correlates with shorter postoperative survival at all tumor stages in gastric cancer patients (Li ZR, et al. Surg Today. 2007;37(8):646-651, Dai N, et al., World J Gastroenterol. 2009;15(12):1499-1505).

[0215] Since intracellular PRL-3 antigen has been shown to be exported outside the cell during the cancer process and become extracellular, PRL-3 is a potential target for immunotherapy for treating various forms of cancer that exhibit extracellular PRL-3 expression. Therefore, the production method and system of the present invention are useful for the production of humanized or human PRL-3 antibodies for use as human therapeutics.

[0216] B. Generation of Engineered PRL3 Cell Lines The humanized anti-PRL3 antibody was engineered from the original framework of a previously characterized murine anti-PRL-3 antibody as described in Thura M, et al., JCI Insight. 2016;1(9):e87607. Briefly, the humanized anti-PRL-3 antibody (renamed "PRL3-zumab") was engineered from the original framework of a previously characterized murine anti-PRL-3 antibody clone. The CDRs of the heavy and light chains of the murine antibody were grafted onto human sequence frameworks selected by aligning the murine framework sequences against a database of human framework sequences to find the human homologs closest to each chain. In addition to the CDRs of the murine sequence, three amino acid positions of the murine sequence adjacent to the CDRs were also grafted onto the human acceptor sequence to preserve the integrity of the CDR structure of the original murine anti-PRL-3 antibody.

[0217] Example 5 Generation of a Cell Line for Purification of Anti-PRL-3 Humanized Antibody A. Identification of Mutations in the Target Protein to Suppress Antibody Binding The epitope of PRL3-zumab was shown to specifically recognize an epitope within the conserved C-terminal region that is conserved between both murine and human PRL-3, but not PRL-1 or PRL-2. Ibid. Combining the knowledge that the epitope is within the C-terminal region of PRL-3 with the crystal structure of PRL-3 (as shown by Kim KA et al., FEBS Lett (2004) 565 p. 181-7), mutagenesis was used to modify one or more residues in the conserved C-terminal region of the native PRL-3 protein in the production cell line to suppress the binding of the antibody produced while maintaining the overall structure and function of the protein.

[0218] Specifically, mutations (plural possible) in the C-terminal binding region of natural mammalian PRL-3 are selected to preserve the protein structure, prevent changes in secondary structure and the possibility of changes in tertiary and quaternary structures, preserve the endogenous function, and avoid cell death. Substitutions of amino acids of PRL-3 to alanine at important sites are predicted, and the proposed PRL-3 mutants in mammalian cells are further evaluated for inhibition of survival rate and binding of humanized mAb to intracellular proteins. Depending on the cell line selected for antibody production, the sequence to be modified can be based on the sequence of the epitope of the species of that cell line, provided that the epitope is generally conserved between mammalian species such as rodents and humans.

[0219] B. Editing of Binding Epitopes in Target Proteins Gene sequence data for human PRL-3 (Gene ID: 11156) and the corresponding sequence of PRL-3 from Chinese hamster (Gene ID: 100772907) are 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 wild-type PRL-3 in HEK293 cells (SEQ ID NO: 33) and CHO cells (SEQ ID NO: 34) is edited using the homologous recombination repair (HDR) strategy with type V CRISPR gene editing technology to generate the desired single mutants and / or double mutants. Such editing strategies are described in U.S. Patent No. 11,220,697 issued on January 11, 2022 and "Gene Editing with MAD7™ in Mammalian Cells: Quick Start Guide" available at www.inscripta.com. Additionally, tools for designing specific guide RNAs to be used with type V RGNs are available from companies such as Integrated DNA Technologies (Coralville, Iowa) and GenScript (Piscataway, New Jersey).

[0220] Cells having the desired mutation are isolated by clonal serial dilution, and the presence of point mutations is confirmed by Sanger sequencing. After growth in culture, cell growth and viability are tested. Control (wild-type CHO) and mutant PRL-3 CHO cells are tested, and cells that reach >90% confluence simultaneously (on day 4 after seeding) are selected as those showing minimal changes in cell growth or viability. Cell confluence is calculated using the IncuCyte® live cell analysis system.

[0221] C. Production and purification of anti-PrI3 antibody in modified cells Humanized anti-PRL-3 monoclonal antibody (mAb) is individually transfected into clonal CHO cells by electroporation in 6-well plates, fresh medium is replenished the next day, and incubated for 5 days after transfection. Wild-type HEK293 cells 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 substantially more anti-PRL-3 IgG antibody than the antibody produced when from HEK293 and / or CHO wild-type cells are selected.

[0222] Example 6 Humanization of anti-PDI monoclonal mouse antibody The disclosed method for producing and purifying antibodies from mammalian cells, disclosed in Example 3, is also useful for the production of mAbs that selectively bind to other intracellular proteins, and such methods can not only increase production by enhancing cell viability, but also increase the amount of recoverable antibody by making it available for purification rather than secreting the antibody or binding it to an intracellular target. This method is particularly useful for the production of antibodies that selectively bind to intracellular targets in mammals, including targets within the ER and Golgi. The following example discloses the production of a humanized antibody against the ER target protein disulfide isomerase (PDI).

[0223] PDI, also known as the beta subunit of prolyl 4-hydroxylase, is an enzyme encoded by the P4HB gene in humans. PDI acts as a chaperone that catalyzes the formation, cleavage, and rearrangement of disulfide bonds in the endoplasmic reticulum (ER). There is increasing evidence that PDI plays an important role in maintaining cellular homeostasis by mediating oxidative protein folding. The progression of several cancers, particularly brain tumors such as glioblastoma, is associated with increased PDI expression, and PDI inhibitors are being studied for the treatment of cancers such as glioblastoma. See, for example, Kyani et al., ChemMedChem,;13(2):164-177. PDI inhibition has been demonstrated to downregulate genes in the DNA repair pathway and the E2F pathway in glioblastoma cells, so PDI remains a very promising oncology target. Xu S.,et al.,Theranostics.2019;9(8):2282-2298.

[0224] The Invitrogen PDI monoclonal antibody clone 12 (Thermo Fisher Scientific catalog number MA5-43389, Thermo Fisher Scientific, Waltham, Massachusetts) (hereinafter referred to as "mPDI-1") is a mouse IgG1 monoclonal antibody that selectively binds to human PDI / P4HB ("hPDI"). The Invitrogen PDI monoclonal antibody clone 2F6G12H2 (Thermo Fisher Scientific catalog number MA5-43389, Thermo Fisher Scientific, Waltham, Massachusetts) (hereinafter referred to as "mPDI-2") is a distinct mouse IgG1 monoclonal antibody that selectively binds to hPDI. These antibodies were both selected using recombinant human PDI protein as an antigen in a mouse host and are both sensitive enough for use in ELISA assays. Thus, both are potential candidates for humanization to create therapeutic monoclonal antibodies targeting hPDI. Since PDI is mainly found in the ER, the methods of the present disclosure are well-suited for the efficient production of antibodies in mammalian cells.

[0225] Humanized PDI antibodies for use in this method preferably contain 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 can be humanized as taught in more detail herein.

[0226] Briefly, the V of mPDI-1 and mPDI-2 H and V LThe amino acid sequences of the regions were determined using the REmAB® antibody sequencing service provided by Rapid Novor (Ontario, Canada). The CDRs of mPDI-1 and mPDI-2 can be determined using the IMGT numbering system provided in Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003). Once the CDRs are identified, murine monoclonal antibodies can be humanized using methods such as those disclosed in U.S. Patent No. 10,613,094 to Do Couto et al., issued April 7, 2020, and U.S. Patent No. 8,673,593 to Chilcote et al., issued March 18, 2014.

[0227] Example 7 Epitope Mapping of the Protein Binding Site of an Anti-PDI Humanized Antibody Against hPDI A variety of methods can be used for epitope mapping of the binding sites of mPDI-1 and mPDI-2 mAbs on human hPDI. Exemplary methods are taught in U.S. Patent No. 11,174,479 to Greenleaf et al., issued November 16, 2021, and U.S. Patent No. 11,061,036 to Wilson et al., issued July 13, 2021.

[0228] For example, the binding of mPDI-1 and mPDI-2 mAbs to biotinylated PDI peptides spanning hPDI can be measured using a streptavidin biosensor. A biotinylated peptide spanning the hPDI protein is loaded at 5 μg / ml for 700 seconds, and the baseline is recorded for 300 seconds for baseline recording, after which the association and dissociation of mPDI-1 or mPDI-2 with each peptide are measured at multiple concentrations for 600 seconds each. A dual-reference sensor is used in all tests to measure any observed background signal and subtract it from non-specific binding or system noise.

[0229] The analysis is performed using ForteBio data analysis software (v8.2). After subtracting the background, a 1:1 local kinetics model is fitted to the observed binding and dissociation curves. Overall, K D K オン K オフ and the R2 correlation coefficient are determined. If possible, a global curve fit is also performed for antibodies / analytes at multiple concentrations.

[0230] Example 8 Identification of the hPDI Structure to Define Residues Important for Mutagenesis To identify residues that are edited to suppress the binding of antibodies to hPDI, the combination of the crystal structures of S. cerevisiae (Tian G., et al., Cell. 2006;124:61 - 73), and the crystallization of human PDI in both the oxidized and reduced states (Wang C, et al., Antioxid Redox Signal 19:36 - 45) can be used to infer the structure and function of human PDI. Large - scale molecular dynamics simulations starting from the crystal structures of oxidized and reduced human PDI (hPDI) demonstrate the intrinsic conformational dynamics. PDI is composed of four thioredoxin - like domains, and this protein can form at least four compact conformations. Yang S.et al., PLoS One. 2014;9(8):e103472.

[0231] Example 9 Generation of Cell Lines for Anti - PDI Humanized Antibody Purification A. Identification of Mutations in the Target Protein to Suppress Antibody Binding Based on the epitopes involved in the binding of humanized mAbs based on mPDI-1 and mPDI-2 to hPDI, and knowledge of the crystal structure and molecular modeling of the hPDI protein, mutagenesis is used to modify one or more residues to abolish the binding of the resulting antibody while retaining the overall structure and function of the protein. Mutations (s) in native mammalian PDI are selected to preserve the structure of the protein, prevent changes in secondary structure, and prevent the possibility of changes in tertiary and quaternary structure to preserve the endogenous function and avoid cell death. Substitutions of amino acids in PDI at important sites to alanine are predicted, and the proposed PDI mutants in mammalian cells are further evaluated for survival rate and loss of binding to intracellular proteins of humanized mAbs based on mPDI-1 and mPDI-2.

[0232] The proposed PDI mutants are modeled using Phyre2 (intensive processing) (Kelley et al., Nature protocols. 2015;10(6):845-58). The full-length PDI sequences (wild type and 10 mutants) are input, and the wild-type sequence is modeled as a control. Each of the mutant PDI structures generated by Phyre2 is compared with the wild-type PDI structure generated by Phyre2 via the matchmaker function of UCSF Chimera (Pettersen et al., Journal of computational chemistry. 2004;25(13):1605-12). The wild-type structure of hPDI is used as a reference for this function. Compared with the wild-type structure, the regions containing mutations in the desired mutant structures do not visually change, and there are no major changes in secondary structure.

[0233] B. Editing of Binding Epitopes in Target Proteins The genomic sequence data of PDI in Chinese hamster cells is obtained from the NCBI Gene Database (Gene ID: 100766687) and used as the reference sequence for gene editing. For the production of both PDI-1 mAb and PDI-2 mAb, a homologous recombination repair (HDR) strategy is used to edit the wild-type PDI (SEQ ID NO: 33) in CHO cells using type V CRISPR gene editing technology to generate the desired single and / or double mutants. Such editing strategies are described in U.S. Patent No. 11,220,697 issued on January 11, 2022 and "Gene Editing with MAD7™ in Mammalian Cells: Quick Start Guide" available at www.inscripta.com. Additionally, tools for designing the specific guide RNAs used in type V RGNs are available from companies such as Integrated DNA Technologies (Coralville, Iowa) and GenScript (Piscataway, New Jersey).

[0234] Cells with the desired mutations are isolated by clonal serial dilution, and the presence of point mutations is confirmed by Sanger sequencing. After growing in culture, the cell growth and viability are tested. Control (wild-type CHO) and mutant PDI CHO cells are tested, and cells that reach >90% confluence simultaneously (on day 4 after seeding) are selected as those showing minimal changes in cell growth or viability. Cell confluence is calculated using the IncuCyte® live cell analysis system.

[0235] C. Production and Purification of Anti-hPDI Antibodies in Modified Cho Cells A humanized anti-hPDI monoclonal antibody (mAb) formatted as a full-length IgG1 antibody was individually transfected into cloned CHO cells by electroporation in a 6-well plate, fresh medium was replenished the next day, and the cells were incubated for 5 days after transfection. Wild-type CHO cells were used as a control. The presence of human IgG in the cell supernatant was measured by ELISA. Cells that produce and secrete substantially more anti-hPDI IgG antibody than the antibody produced when from CHO wild-type cells were selected.

[0236] Example 10 Humanization of Anti-GOLPH3 Monoclonal Mouse Antibody GOLPH3 was initially identified as a peripheral membrane protein localized in the trans-Golgi network, but some researchers have reported that it is a mitochondrial protein that controls mitochondrial mass through the regulation of cardiolipin, a mitochondrial-specific phospholipid. Since then, GOLPH3 has been thought to be involved in the target of the rapamycin (TOR) signaling pathway. Cells transfected with GOLPH3 enhanced S6 kinase activity in response to growth factor stimulation by EGF. At the same time, AKT phosphorylation increased in these cells, but these phenomena were suppressed in GOLPH3 siRNA-treated cells compared with control cells, suggesting that GOLPH3 can enhance signal transduction through the TOR-related complex. These results suggest that GOLPH3 may be a true oncogene and a useful target for therapeutic strategies.

[0237] Thermo Fisher monoclonal antibody clone 905CT9.1.1 (Thermo Fisher Scientific catalog number MA5-37626, Thermo Fisher Scientific, Waltham, Massachusetts) (hereinafter referred to as "mGOLPH3") is a mouse IgG1 monoclonal antibody that selectively binds to human purified His-tagged GOLPH3 protein. Since GOLPH3 is mainly found in the Golgi apparatus and mitochondria, the methods of the present disclosure are well suited for the efficient production of antibodies in mammalian cells.

[0238] The humanized GOLPH3 antibody for use in the method of the present invention preferably comprises CDR sequences derived from or based on mGOLPH3, as described in more detail herein and in the incorporated references. Briefly, the amino acid sequences of the V H and V L regions of mGOLPH3 are determined using the REmAB® antibody sequencing service provided by Rapid Novor (Ontario, Canada). The CDRs of mGOLPH3 can be determined using, for example, the IMGT numbering system provided in Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003). Once the CDRs are identified, mouse monoclonal antibodies can be humanized using methods such as those disclosed in U.S. Patent No. 10,613,094 to Do Couto et al., issued April 7, 2020, and U.S. Patent No. 8,673,593 to Chilcote et al., issued March 18, 2014.

[0239] Example 11 Epitope mapping of the protein binding site of an anti-GOLPH3 humanized antibody against hGOLPH3 Various methods can be used for epitope mapping of the binding site of an anti-mGOLPH3 mAb on human hGOLPH3. Exemplary methods are taught in U.S. Patent No. 11,174,479 to Greenleaf et al., issued November 16, 2021, and U.S. Patent No. 11,061,036 to Wilson et al., issued July 13, 2021.

[0240] For example, the binding of mGOLPH3 mAb to biotinylated hGOLPH3 peptide spanning hGOLPH3 can be measured using a streptavidin biosensor. The biotinylated peptide spanning hGOLPH3 protein is loaded at 5 μg / ml for 700 seconds and for baseline recording for 300 seconds, and then the association and dissociation of mGOLPH3 mAb with each peptide are measured at multiple concentrations for 600 seconds each. A dual-reference sensor is used in all tests to measure any observed background signal and subtract it from non-specific binding or system noise.

[0241] The analysis is performed using ForteBio data analysis software (v8.2). After subtracting the background, a 1:1 local kinetics model is fitted to the observed binding and dissociation curves. Overall, K D 、K オン 、K オフ 、and the R2 correlation coefficient are determined. If possible, global curve fitting is also performed for multiple concentrations of antibody / analyte.

[0242] Example 12 Identification of the hGOLPH3 Structure for Defining Residues Important for Mutagenesis To identify residues to be edited to suppress the binding of antibodies to hGOLPH3, the crystal structure of hGOLPH3 and its orthologs can be used. Wood et al. reported the x-ray crystal structure of hGOLPH3 at 2.9-Å resolution (Wood et al., J Cell Biol. 2009 Dec 28; 187(7):967-975), and also reported conserved regions of activity in the structures 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 specific functions of GOLPH3 are described 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).

[0243] Example 13 Generation of a cell line for purification of anti-GOLPH3 humanized antibody A. Identification of mutations in target proteins to suppress antibody binding Based on the epitope involved in the binding of a humanized mAb based on mGOLPH3mAb to hGOLPH3, and knowledge of the crystal structure and molecular modeling of the hGOLPH protein, one or more residues are modified using mutagenesis to suppress the binding of the generated antibody while maintaining the overall structure and function of the GOLPH3 protein. Mutations (s) in native mammalian cell GOLPH3 are selected to preserve the protein structure, prevent secondary structure changes and the possibility of tertiary and quaternary structure changes, preserve the endogenous function, and avoid negative manufacturing results, such as cell death, decreased cell viability and / or decreased antibody production. Substitution of amino acids in hGOLPH3 with alanine at important sites is predicted, and the proposed GOLPH3 mutants in mammalian cells are further evaluated for their survival rate and binding to intracellular proteins of humanized monoclonal antibodies based on the mGOLPH3 monoclonal antibody (mGOLPH3 mAb).

[0244] The proposed GOLPH3 mutants are modeled using Phyre2 (intensive processing) (Kelley et al., Nature protocols. 2015;10(6):845-58). The full-length GOLPH3 sequences (wild type and 10 mutants) are input, and the wild-type sequence is modeled as a control. Each of the mutant GOLPH3 structures generated by Phyre2 is compared with the wild-type GOLPH3 structure generated by Phyre2 via the matchmaker function of UCSF Chimera (Pettersen et al., Journal of computational chemistry. 2004;25(13):1605-12). The wild-type structure of hGOLPH3 is utilized as the reference for this function. Compared with the wild-type structure, the regions containing mutations in the desirable mutant structures do not visually change, and there are no major changes in the secondary structure.

[0245] B. Editing of Binding Epitopes in the Target Protein Genomic sequence data of GOLPH3 in Chinese hamster cells was obtained from the NCBI Gene database (Gene ID: 100766687) and used as the reference sequence for gene editing. For the production of humanized mGOLPH3 mAb, the wild-type GOLPH3 (SEQ ID NO: 36) in CHO cells was edited using the type V CRISPR gene editing technology with a homologous recombination repair (HDR) strategy to generate the desired variants (e.g., mutations of one or more residues). Such editing strategies are described in U.S. Patent No. 11,220,697 issued on January 11, 2022 and "Gene Editing with MAD7™ in Mammalian Cells: Quick Start Guide" available at www.inscripta.com. Furthermore, tools for designing specific guide RNAs used in type V RGNs are available from companies such as Integrated DNA Technologies (Coralville, Iowa) and GenScript (Piscataway, New Jersey).

[0246] Cells with the desired mutations are isolated by clonal serial dilution, and the presence of point mutations is confirmed by Sanger sequencing. After growth in culture, the cell growth and viability are evaluated. Control (wild-type CHO) and mutant GOLPH3 CHO cells are tested, and cells that reached >90% confluence simultaneously (on the 4th day after seeding) are selected as those showing minimal changes in cell growth or viability. Cell confluence is calculated using the IncuCyte® live cell analysis system.

[0247] C. Production and Purification of Anti-Golph3 Antibodies in Modified Cho Cells A humanized anti-GOLPH3 monoclonal antibody (mAb) formatted as a full-length IgG1 antibody was individually transfected into cloned CHO cells by electroporation in a 6-well plate, fresh medium was replenished the next day, and the cells were incubated for 5 days after transfection. Wild-type CHO cells were used as a control. The presence of human IgG in the cell supernatant was measured by ELISA. Cells that produce and secrete substantially more anti-hGOLPH3 IgG antibody than the antibody produced when from CHO wild-type cells were selected.

[0248] Example 14 Production of Antibodies in a Modified Cell-Free System The modified cells prepared as described in Examples 3 and 11 are also used to prepare a cell-free system for antibody production.

[0249] The coding sequences of the anti-GRP78 antibody and the anti-GOLPH3 antibody are codon-optimized for Cricetulus griseus and have the control sequences necessary to enable in vitro transcription and translation according to 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 the start codon, 3 ’UTR: T7 terminator sequence, MCS (same as above). The DNA template is newly synthesized (Agilent, San Jose, CA) and cloned into the appropriate vector (pUC57-1.8k) by Biocat GmbH (Biocat GmbH, Heidelberg). Plasmid preparations for cell-free protein synthesis are prepared using the PureLink® HiPure Plasmid Midiprep Kit (Thermo Fisher Scientific, Waltham, USA) according to the manufacturer's instructions, followed by control digestion and sequencing using the Illumina Miseq™ system (Illumina, San Diego, CA) to confirm the correct DNA sequence.

[0250] CHO lysate containing endogenous microsomal vesicles derived from the ER is prepared as previously described (Broedel A.K., et al., (2013) PloS one 8(12), e82234; Thoring L., et al., (2016). PloS one 11(9), e0163670). Briefly, CHO modified as described herein is grown in a chemically defined serum-free medium (PowerCHO® 2 CD medium, Lonza, Basel, Switzerland) at 37 °C in a bioreactor at 18 × 10 6Grow exponentially up to cells / ml. Centrifuge to collect the cells at 200×g for 15 minutes, pellet them, wash twice, and resuspend in a buffer containing 30 mM HEPES-KOH (pH 7.5) and 100 mM NaOAc. Subsequently, pass the cell suspension through a 20-gauge needle using a syringe to mechanically disrupt the cells. Nuclei and cell debris are removed by a centrifugation step at 6,500×g for 10 minutes. The resulting supernatant is subjected to a gel filtration step using a Sephadex G-25 column (GE Healthcare, Freiburg, Germany) equilibrated with a buffer containing 30 mM HEPES-KOH (pH 7.5) and 100 mM. Elute the filtered supernatant in 1-ml fractions and pool those containing an RNA content with an absorbance at 260 nm exceeding 100. To remove endogenous mRNA, treat the cell lysate with S7 micrococcal nuclease (Roche, Mannheim, Germany) (10 U / ml) and CaCl2 (1 mM) and incubate at room temperature (RT) for 20 minutes. Micrococcus S7 nuclease is inactivated by adding EGTA (6.7 mM). Optionally, further add creatine kinase (100 μg / ml) to the CHO lysate as taught by "rapidly freeze the lysate in liquid nitrogen and then store at -80°C until use".

[0251] Next, the transcription and translation reactions are carried out as described. Thoring, ibid. The translation reaction consists of 40% (v / v) S7 nuclease-treated CHO lysate containing endogenous microsomal vesicles 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, USA), 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, USA) and 14C-Leucine (final concentration 30 μM; specific radioactivity 46.15 dpm / pmol (PerkinElmer LAS (Germany) GmbH, Rodgau, Germany)) was included to enable quantitative and qualitative analysis of the subsequent cell-free synthesized proteins. Protein synthesis was initiated by the addition of a DNA template (60 ng / μL). The reaction was incubated at 600 rpm and 30 °C for 3 h in a standard thermomixer (Eppendorf Thermomixer Comfort). Background translational activity was monitored by performing the translation reaction without plasmid supplementation.

[0252] After the translation reaction, the samples were centrifuged at 16,000×g for 10 min at 4 °C to separate microsomes from the soluble fraction of the translation mixture. The resulting supernatant (first supernatant, or SUP1) was transferred to a new reaction tube and stored on ice until further analysis. On the other hand, the microsomal pellet was resuspended in 1xPBS containing 0.2% n-dodecyl-β-D-maltoside (DDM) to enable the release of the antibodies contained in the translocated microsomes. The microsomes were manually resuspended by repeatedly pipetting up and down, and then vortexed and shaken for approximately 45 min with a Vibrax. A second centrifugation step was performed to separate the released proteins from the microsomal membrane debris. The resulting supernatant (second supernatant, or SUP2) was transferred to a new reaction tube and stored on ice until further analysis. 14 The reaction with added C-Leucine was analyzed by autoradiography and liquid scintillation counting after SDS-PAGE analysis, and non-radioactive samples were subjected to functional analysis by enzyme-linked immunosorbent assay (ELISA).

[0253] The present invention is not intended to be limited in scope to the specific disclosed embodiments provided, for example, to illustrate various aspects of the present invention. Various modifications to the described compositions and methods will become apparent from the description and teachings herein. Such variations can be practiced without departing from the true scope and spirit of the present disclosure and are intended to be included within the scope of the present disclosure.

Table 10-1

Table 10-2

Table 10-3

Table 10-4

Claims

**Claim 1** A method for the recombinant production of an antibody, comprising: (a) 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 within the system, and the modified cell-free system contains a variant of the protein with one or more amino acid residue mutations of the target epitope on the protein within the system; (b) initiating transcription and translation of the antibody in the cell-free system under conditions such that the antibody is produced. **Claim 2** The method according to claim 1, wherein the cell-free system comprises a cell lysate. **Claim 3** The method according to claim 2, wherein the cell lysate is a eukaryotic cell lysate. **Claim 4** The method according to 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. **Claim 5** The method according to claim 4, further comprising (c) isolating the antibody from the cell-free system. **Claim 6** A method for the recombinant production of a monoclonal antibody, comprising: (a) expressing a nucleic acid encoding the antibody in a modified eukaryotic cell-free system, wherein the antibody specifically binds to a target epitope on a protein within the system, and the modified cell-free system contains a protein with one or more amino acid residue mutations of the target epitope on the protein within the system; (b) initiating transcription and translation of the antibody in the cell-free system under conditions such that the antibody is produced. **Claim 7** The method according to claim 1, wherein the cell-free system comprises a cell lysate. **Claim 8** The method according to claim 6, further comprising (c) isolating the antibody from the cell-free system. **Claim 9** A method for the recombinant production of an antibody, comprising: (a) modifying a cell line to eliminate binding of an antibody to an intracellular protein within the cells of the cell line, wherein the modification results in the expression of a variant target protein containing one or more amino acid residue mutations of the target epitope of the antibody; (b) creating a cell-free antibody production system from the modified cell line. (c) introducing a nucleic acid template into the cell-free antibody production system, wherein the nucleic acid template encodes the antibody against the target protein; (d) initiating transcription and translation from the nucleic acid template to produce the antibody in the cell-free antibody production system. A method comprising: **Claim 10** (c) The method according to claim 9, further comprising isolating the antibody from the cell-free system. **Claim 11** The method according to claim 10, wherein the naturally expressed target protein is an intracellular protein in the secretory pathway of the cell line. **Claim 12** The method according to claim 10, wherein the mutation is an inactivating mutation that retains the structure and function of the native target protein in the cell. **Claim 13** (d) The method according to claim 9, wherein the mutation reduces or inhibits the binding of the antibody to the target epitope on the target protein. **Claim 14** The method according to claim 9, wherein the target protein is a stress protein. **Claim 15** The method according to claim 9, wherein the target protein is expressed on an organelle of the cell line. **Claim 16** The method according to claim 15, wherein the organelle is the endoplasmic reticulum or the Golgi apparatus. **Claim 17** The method according to claim 16, wherein the target protein is an endoplasmic reticulum chaperone. **Claim 18** The method according to claim 17, wherein the endoplasmic reticulum chaperone is calreticulin, a heat shock protein or an isomerase. **Claim 19** The method according to claim 18, wherein the target protein is glucose-regulated protein 78 (GRP78), HSP47, PDI, calreticulin or GP94. **Claim 20** The method according to claim 18, wherein the target protein is glucose-regulated protein 78 (GRP78). **Claim 21** The method according to claim 16, wherein the target protein is a Golgi complex protein. **Claim 22** The method according to claim 21, wherein the Golgi complex protein is GOLPH2, GOLPH3, GM130, ATP6V1A, ATP6V1E1, ATP6VOA2, TMEM165, GOLGB1, SCYL1BP1, TRAPPC11, TRAPPC2 or TRIP11. **Claim 23** The method according to claim 16, wherein the naturally expressed target protein is an intracellular protein in the secretory pathway of the mammalian cell line.

24. The method according to claim 16, wherein the mutation is an inactivating mutation that retains the structure and function of the target protein in the mammalian cell line.

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

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

27. A method for the recombinant production of monoclonal antibodies, comprising: (a) modifying a mammalian cell line to inhibit the binding of an antibody to an intracellular protein within the cell of the cell line, wherein the modification results in the expression of a variant target protein comprising a mutation of one or more amino acid residues of the target epitope of the antibody; (b) creating a cell-free antibody production system from the modified mammalian cell line; (c) introducing a nucleic acid template into the cell-free antibody production system, wherein the nucleic acid template encodes the antibody against the target protein; (d) initiating transcription and translation from the nucleic acid template to produce the antibody in the cell-free antibody production system.

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

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

30. The method according to claim 28, wherein the mutation is an inactivating mutation that retains the structure and function of the native target protein within the cell.

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

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

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

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

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

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

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

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

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

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

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

42. The method according to claim 27, wherein the mutation is an inactive mutation that retains the structure and function of the target protein of the mammalian cell line.

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

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

45. The method according to claim 27, wherein the target protein is a protein involved in intracellular signal transduction, optionally a kinase or a phosphatase, and in some cases, the phosphatase is regenerating liver phosphatase 3.

46. The method according to claim 27, wherein the target protein is a chaperone protein.

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