Method for producing recombinant antibodies and related cell lines

Abstract

The present specification provides methods and cell lines for the production of recombinant antibodies intracellularly, where the antibodies are directed against target proteins that are naturally expressed as intracellular proteins within the cell. Further provided in the present specification are modified cell lines for use in such methods, where the modified cell lines are engineered to express a variant target protein having a mutant epitope of the antibody's target epitope.

Description

Technical Field

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 337,980, filed May 3, 2022, and U.S. Provisional Patent Application No. 63 / 359,541, filed Jul. 8, 2022, each of 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 MBRACE002.xml, created on May 2, 2023, and is 35,971 bytes in size (measured in the MS-Windows® operating system).

[0003] The present disclosure relates to methods and cell lines for the production of recombinant antibodies in cell lines, where the antibodies are against target proteins that are naturally expressed as intracellular proteins in the cell line. The present disclosure also provides modified cell lines for use in such methods, where the cell lines are engineered with variant target proteins having mutant epitopes of the antibody target epitope.

Background Art

[0004] Operated 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 virtually any type of antigen and bind with high affinity, 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 and their fragments that specifically recognize and bind to intracellular targets in mammalian cells.

Summary of the Invention

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

[0006] The present disclosure provides, in selected embodiments, methods and cell lines for producing antibodies that bind to intracellular target proteins. The present disclosure also provides methods for modifying cell lines to produce antibodies that bind to intracellular target proteins.

[0007] In some embodiments, the present disclosure provides a method for the recombinant production of an antibody, comprising the steps of expressing a nucleic acid encoding the antibody in a modified 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 modification of one or more amino acid residues of the target epitope, and culturing the modified cell line under conditions such that the antibody is produced.

[0008] In some embodiments, the present disclosure provides a method for recombinant production of an antibody, comprising the steps of expressing a nucleic acid encoding the antibody in a modified 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 mammalian cell line, the modified mammalian cell is engineered to express a variant target protein comprising a modification of one or more amino acid residues of the target epitope, and culturing the modified mammalian cell line under conditions such that the antibody is produced.

[0009] In some embodiments, the target protein that is naturally expressed is an intracellular protein in the secretory pathway of the cell line. In some embodiments, the target protein is a stress protein.

[0010] In some embodiments, the target protein that is naturally expressed as an intracellular protein within the cell is modified with an inactive mutation that retains the structure and function of the target protein within the cell. In some embodiments, the target protein that is naturally expressed as an intracellular protein within the cell is modified with one or more mutations that reduce or inhibit the binding of an antibody to the target epitope on the native target protein.

[0011] In some embodiments, the target protein associates with an intracellular membrane. In some embodiments, the membrane is an organelle membrane. In some embodiments, the membrane is a plasma membrane.

[0012] In some embodiments, the target protein is one involved in a signal transduction pathway, such as an intracellular signal transduction protein such as a kinase or a phosphatase.

[0013] In some embodiments, the target protein is expressed on an organelle of the cell line. In certain embodiments, the target protein is expressed on the endoplasmic reticulum. In certain embodiments, the target protein is expressed on the Golgi apparatus.

[0014] In some embodiments, the target protein is an endoplasmic reticulum protein, such as calreticulin, a heat shock protein, or an isomerase. In certain embodiments, the target protein is an endoplasmic reticulum chaperone, such as glucose-regulated protein 78 (GRP78), HSP47, protein disulfide isomerase (PDI), calreticulin or GP94.

[0015] In some embodiments, the target protein is a Golgi complex protein, such as golgin-2 (GOLPH2), golgin-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), transport protein particle complex subunit 11 (TRAPPC11), transport protein particle complex subunit 2 (TRAPPC2), or thyroid hormone receptor interactor 11 (TRIP11).

[0016] In some embodiments, the present disclosure provides a method for recombinantly producing an antibody that targets an epitope on a target protein found intracellularly in a cell line, the method comprising providing an antibody that selectively binds to an epitope on a target protein present intracellularly in the cell line, identifying the epitope of the target protein to which the antibody binds, modifying the epitope of the target protein in the cell line to reduce or inhibit binding of the antibody to the epitope on the target protein, introducing an expression vector encoding the antibody, and culturing the modified cell line under conditions that allow the modified cell line to produce the antibody from the expression vector.

[0017] In some embodiments, the present disclosure provides a method for the recombinant production of an antibody that targets an epitope on a target protein present within a mammalian cell line, the method comprising providing an antibody that selectively binds to an epitope on a target protein present within a mammalian cell line; identifying the epitope of the target protein to which the antibody binds; modifying the epitope of the target protein in the mammalian cell line to reduce or suppress binding of the antibody to the epitope on the target protein; introducing an expression vector encoding the antibody into the modified mammalian cell line; culturing the modified mammalian cell line under conditions that allow for the production of the antibody from the expression vector; and expressing the nucleic acid encoding the antibody in the modified mammalian cell line.

[0018] Examples of mammalian cell lines that can be used in the methods of the present disclosure include 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, and human cervical cancer HeLa cells.

[0019] In some embodiments, the method further comprises isolating the antibody from, for example, the culture supernatant.

[0020] In some embodiments, the present disclosure provides a method for expressing a nucleic acid encoding an antibody in a modified mammalian cell line, wherein the antibody selectively binds to GRP78, and the modified mammalian cell is engineered with a variant GRP78 having a mutant epitope comprising one or more amino acid residues of the target epitope of the antibody or antigen-binding fragment, and culturing the modified mammalian cell line under conditions such that an anti-GRP78 antibody is produced. In some embodiments, the method further comprises isolating the anti-GRP78 antibody from, for example, the culture supernatant.

[0021] Antibodies produced using the methods of the present disclosure can include human antibodies and humanized antibodies. In some embodiments, the antibody is a full-length antibody. In certain embodiments, the antibody is an antigen-binding fragment. In some embodiments, the antibody is an IgG, such as IgG1.

[0022] As will be apparent to those skilled in the art upon reading the present disclosure, exemplary embodiments can use a variety of methods for modifying cell lines. In some embodiments, mutations to the native target protein can be introduced using nuclease-based editing techniques, such as, for example, a nuclease selected from meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases. In some embodiments, the target protein is modified using nuclease-mediated homologous recombination repair (HDR), homologous recombination (HR), or combinations thereof.

[0023] In some embodiments, the cell line is modified using an RNA-guided nuclease. Such an editing system uses an RNA-guided nuclease, a guide RNA, and a homologous recombination repair (HDR) template (i.e., donor template), where the HDR template is homologous to the target protein and contains nucleotides encoding the mutant epitope of the target protein. In certain embodiments, the RNA-guided nuclease is a type II or type V RGN.

[0024] In some embodiments, the present disclosure provides a method of generating a modified mammalian cell line for recombinant antibody production, the method comprising introducing into a mammalian cell line a DNA target nuclease for inducing gene disruption at a target site within a gene encoding a target protein, wherein the target protein is a protein that is naturally expressed as an intracellular protein in the mammalian cell line, and introducing into the mammalian cell line a template polynucleotide, wherein the template polynucleotide is homologous to the gene encoding the target protein and comprises nucleotides encoding a mutant epitope comprising one or more amino acid residue mutations of a target epitope present in the target protein to which an antibody or antigen-binding fragment specifically binds, the template polynucleotide serving as a target for integration at or near at least one target site via nuclease-mediated homologous recombination repair (HDR), and after integration of the template polynucleotide into the gene, the gene encodes a variant target protein comprising the mutant epitope.

[0025] In some embodiments, the present disclosure provides a modified mammalian cell line comprising a variant target protein that is an intracellular protein. The variant target protein comprises a mutation of a native intracellular protein comprising a target epitope to which an antibody selectively binds, and the mutation of the variant target protein is an amino acid substitution of one or more amino acid residues for changing the target epitope of the antibody to a mutant epitope.

[0026] Other features, advantages, and aspects are described in more detail below.

[0027] 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 to assist in the description of the underlying features.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0029] 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.

[0030] As used herein, "one embodiment" or "an embodiment" means that the particular features, structures, or characteristics described in connection with that embodiment are 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 modifications and variations thereof.

[0031] When 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, when 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", "back", "side", "height", "length", "width", "upper", "lower", "inner", "outer", "inside", "outside", etc. as used in this specification merely describe a reference point and are not necessarily intended to 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, are not necessarily intended to limit the embodiments of the present disclosure to any particular configuration or orientation.

[0032] Further, terms such as "substantially", "about", "approximately", "minor variations", and similar terms generally refer to a range that includes the identified value within a margin of 20%, 10%, or preferably 5%, and any value between them.

[0033] 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 embodiments. 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, unless the feature or function is incompatible with that other embodiment.

[0034] The present specification provides a method for the recombinant production of an antibody or a binding fragment thereof within a cell, wherein the target protein of the antibody is also found as an intracellular protein within the cell. In some embodiments, the cell in which the antibody or binding fragment is produced is a mammalian 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 Golgi apparatus or a protein expressed on the endoplasmic reticulum (ER). In some embodiments, the target protein of the antibody is a stress protein, which typically functions in the ER or Golgi apparatus or other intracellular 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 in normal tissues and cells, is ensured, and off-target activity and / or toxicity are reduced or minimized.

[0035] 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.

[0036] 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 the folding and post-translational modification of the desired protein. By expressing recombinant proteins in mammalian cells such as 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 cells allow for very high product yields and are relatively resistant to metabolic stress.

[0037] 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, from the observations herein, it has been demonstrated 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.

[0038] Studies have demonstrated that stress proteins such as chaperones are required for proper functioning within cells 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 be folded in the endoplasmic reticulum (ER) compartment of the cell immediately after protein synthesis and 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. December 3, 1999; 286:1882-8; Helenius et al., Intracellular functions of N-linked glycans, Science. March 23, 2001; 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. and 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.

[0039] Alternatively, even when the binding of intracellular proteins by an antibody does not affect cell viability, the sequestration of products by intracellular proteins can affect antibody production. Simply put, intracellular proteins can act as a "sink" for the binding of antibodies or antigen-binding fragments, inhibiting secretion and thus limiting the yield of the desired product.

[0040] The provided method relates to a modified cell line for producing an antibody or antigen-binding fragment that targets an endogenous intracellular target protein in 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 method includes producing a modified mammalian cell that carries a variant of the target protein by mutating a target epitope to a variant epitope such that the 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. In some cases, 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 the binding of the antibody to the target protein. In the provided method, the modified cell 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 cell under conditions for antibody production. In some embodiments, the produced antibody can be isolated from the cell.

[0041] The results in this specification 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 two-fold compared to the production of the same antibody in natural mammalian cells where 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 where 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 where the target protein is not mutated.

[0042] The provided cell lines and methods are exemplified for antibodies targeting GRP78 and other intracellular target proteins. The cell lines and methods described herein can generally be generalized to the production of all or part of antibody-based biopharmaceuticals that selectively target proteins that are generally found intracellularly but can be found on the cell surface in certain pathological conditions. Thus, those skilled in the art will understand that this disclosure is illustrative and applicable to other cell lines and manufacturing methods for biopharmaceuticals targeting such other targets.

[0043] All publications, including patent documents, scientific papers, and databases referenced in this application, are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If the definitions set forth herein conflict with or are inconsistent with the definitions set forth in the patents, applications, published applications, and other publications incorporated by reference herein, the definitions set forth herein shall control over the definitions incorporated by reference herein.

[0044] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0045] Method for Producing Antibodies This specification provides a method for recombinantly producing an antibody that binds to a target protein (or a homolog thereof) that is naturally expressed as an intracellular protein in a normal 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 such that it includes a mutant epitope having a mutation in one or more amino acid residues of the target epitope of the antibody, thereby reducing or suppressing 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 intracellular protein, thereby ensuring the function of the target protein. Thus, the methods and systems provided using the modified cell line or cell-free system enable high-yield production of the antibody in the production system. These methods and systems disclosed herein can be used individually or in combination with all or certain aspects of other antibody production systems. For example, in some preferred embodiments, the method can utilize aspects of the cell-free system disclosed in U.S. Patent Application Publication No. 63 / 359,871, filed Jul. 10, 2022, and / or U.S. Patent Application Publication No. 63 / 398,143, filed Aug. 15, 2022, each of which is hereby incorporated by reference in its entirety for all purposes.

[0046] In some preferred embodiments, the cell-free system described in U.S. Patent Application Publication No. 63 / 359,871, filed on July 10, 2022, can include a method for recombinant production of an antibody by: a) specifically binding an antibody to a target epitope on a protein of a cell-free system (in some preferred embodiments, a eukaryotic cell-free system), and expressing a nucleic acid encoding the antibody in the system where the modified cell-free contains a mutation of one or more amino acid residues of the target epitope on the protein in the system; and b) initiating transcription and translation of the antibody in the cell-free system under conditions such that the antibody is produced. In some preferred embodiments, such a cell-free system can include a cell lysate (in some preferred embodiments, a eukaryotic cell lysate). In some preferred embodiments, the cell-free system can include: a) modifying a cell line (in some preferred embodiments, a mammalian cell line) to suppress the binding of an antibody to an endogenous protein within the cells of the cell line, where the modification results in the expression of a variant target protein containing 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 cell line; c) introducing a nucleic acid template encoding an antibody against the target protein into the cell-free antibody production system; and d) initiating transcription and translation from the nucleic acid template to produce (and, in some preferred embodiments, isolate) the antibody in the cell-free antibody production system.

[0047] In some preferred embodiments, the cell-free system of U.S. Patent Application Publication No. 63 / 398,143, filed on August 15, 2022, comprises: a) providing a cell-free protein expression system (in some preferred embodiments, a mammalian cell-free system) comprising a target protein exhibiting an antibody epitope; b) modifying the cell-free system by introducing one or more agents that block the antibody epitope on the target protein (or, in some preferred embodiments, the antibody paratope on the target protein) without eliminating the activity of the target protein within the cell-free system; c) introducing into the cell-free system one or more nucleic acids encoding an antibody that binds to the antibody epitope; 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 comprise: a) creating a modified cell line by introducing into a cell (in some preferred embodiments, a mammalian cell) 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 encoding an antibody that selectively binds to the target epitope on the intracellular protein; d) initiating transcription and translation from the nucleic acid template to produce an antibody in the cell-free antibody production system (and, in some preferred embodiments, isolating the antibody).

[0048] In some embodiments, the provided method comprises: (a) expressing a nucleic acid encoding an antibody in a modified cell line, wherein the antibody is against a target protein or a homolog of a target protein that is naturally expressed as an intracellular protein within the cell, and the modified cell is engineered with a variant target protein having a mutant epitope comprising one or more amino acid residues of the target epitope of the antibody or antigen-binding fragment; (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.

[0049] 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 homolog of the target protein, and the modified mammalian cell is engineered with a variant target protein having a mutant epitope comprising one or more amino acid residues of the target epitope of the antibody or antigen-binding fragment; (b) culturing the modified mammalian cell line under conditions such that the antibody is produced. In some embodiments, the method can further comprise, for example, isolating the antibody from the culture supernatant.

[0050] Target of intracellular protein and antibody against it In some embodiments, the provided cell lines and methods are useful for expressing an antibody intracellularly in a cell, such as a mammalian cell, where the target protein of the antibody is naturally expressed as an intracellular protein within the cell. In certain embodiments, the target protein is a protein that is aberrantly expressed on the cell surface in cancer and other diseases but is also required for normal cell function 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 a cell, such as a mammalian cell line. In some embodiments, the target protein is expressed on an organelle of a cell, 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.

[0051] 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. Various stress proteins are known, including proteins in the secretory pathway. Various stress proteins are known, including those resulting from ER stress or Golgi stress: Sasaki and Yoshida, J Biochem. 157:185, 2015; Sasaki and Yoshida, FEBS Letters, 593:2330-2340, 2019; Gao et al., Biofactors, 47:964-974, 2021; Li et al., Mol Neurobiol, 49:1449-59, 2014; Yadav et al., J Cancer Prev., 19:75-88, 2014; and Chen and Cubillos-Ruiz, Nature Reviews Cancer, 21:71-88, 2021.

[0052] 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 states. See, for example, Weirsma VR et al., Front. Oncol., Vol 5: Art. 7 (2015); Garg AD, Cancer Immunol Immunother 61:215 - 21 (2012). The disclosure of this embodiment and the application of its teachings enable the production of biologics that target endoplasmic reticulum chaperones and other identified targets without unduly compromising cell viability. Exemplary biologics include, for example, those 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)); biologics 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 biologics 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. March 2012;1823(3):774 - 787), and biologics targeting GRP78 (Arap et al. Cancer Cell 6:275 - 284 (2004); Sato et al. Adv Genet 69:97 - 114 (2010)).

[0053] 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. Such proteins include Golgi phosphoprotein 2 (GOLPH2) (Liu et al., Front Oncol 2021 Dec 7;11:78386); Golgi phosphoprotein 3 (GOLPH3) (Xing M, et al., Mol Biol Cell 27:3828-3840, 2016; Scott KL, et al., Nature 459:1085-1090, 2009); GM130 (Chang SH, et al., Mol Ther 20:2052-2063, 2012); ATPase H+ transporting V1 subunit A (ATP6V1A) (Van Damme T, et al., Am J Hum Genet100:216227, 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).

[0054] In some embodiments, the target protein is GRP78. In some embodiments, the target protein is heat shock protein 47 (HSP47). In some embodiments, the target protein is protein disulfide isomerase (PDI). In some embodiments, the target protein is calreticulin. In some embodiments, the target protein is GP94. In some embodiments, the target protein is GOLPH2. In some embodiments, the target protein is GOLPH3. In some embodiments, the target protein is GM130. In some embodiments, the target protein is AATPP6V1A. In some embodiments, the target protein is ATPP6V1E1. 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).

[0055] A person skilled in the art is 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); International Publication No. 2018 / 057703, International Publication No. 2014 / 153056; International Publication No. 2008 / 105560; U.S. Patent Application No. 2010 / 0041074 Specification; U.S. Patent No. 10,259,884 Specification; and anti-GRP78 antibodies described in U.S. Patent No. 10,851,161 Specification. 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 methods as more specifically disclosed herein.

[0056] In some embodiments, the antibody is a human antibody. Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been 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 chromosomes of the animal. 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.

[0057] 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 generated 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 a selected epitope of an organelle protein.

[0058] 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 regions from this phage serve 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.

[0059] The antibodies provided include monoclonal antibodies, including monoclonal antibody fragments.

[0060] Design of Mutant Epitopes on Target Proteins 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 the 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., titled "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., titled "Epitope Mapping," issued July 13, 2021.

[0061] 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, radiation exposure 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 achieved by any method known in the art, including hydrophobicity analysis or crystal structure analysis of the protein sequence.

[0062] 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 a microarray or on the surface of 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 preselected region thereof are screened for antibody binding by, for example, ELISA or other techniques that monitor binding interactions.

[0063] 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 that include 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, the 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.

[0064] In some embodiments, to identify residues to be edited to reduce or suppress 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 contacting 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 the resolved crystal structure of the intracellular protein bound to an antibody or antibody fragment.

[0065] 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 structural database, amino acid exchange experiments can provide information about which residues may be targets for mutation. Alanine scanning mutagenesis (successively mutating each residue to Ala) through these regions has allowed the identification of important residue(s) involved in antigen binding (Cunningham BC and Wells JA, Proc Natl Acad Sci USA. April 15, 1991; 88(8):3407-3411). If changing a single residue to Ala decreased but did not disrupt binding, that position can, if necessary, be targeted for more radical mutations (e.g., substitutions that result in a charge difference) to further decrease binding.

[0066] 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.

[0067] 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 the binding of an antibody to an epitope of the organelle protein while leaving sufficient function for the cell viability of the organelle protein, as more particularly taught herein. Methods for making polypeptides containing one or more mutations are well known to those of ordinary skill in the art.

[0068] 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.

[0069] 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.

[0070] In certain embodiments of the provided methods, the mutations do not affect the function of the target protein. In some embodiments, the mutations are inactivating mutations in which the structure and function of the target protein are retained. 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 natural 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).

[0071] 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 as compared to the cell line containing the non-mutated target protein.

[0072] 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 compared to the mammalian cell line containing the non-mutated target protein.

[0073] In some embodiments, one or more mutations decrease 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 containing a mutant epitope) can be evaluated. Methods for evaluating binding to a polypeptide are known in the art and 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 containing 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 lacking the one or more substitutions. In some embodiments, the binding and / or reactivity to a variant target protein containing 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 not containing the one or more substitutions. In some embodiments, the binding and / or reactivity to the 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).

[0074] In some embodiments, a variant target protein with reduced or suppressed binding to an antibody exhibits 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 a 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 a 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).

[0075] 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.

[0076] Generation of a Modified Cell Line Harboring a Mutated Target Protein with a Mutated Epitope In some embodiments, the modified cell line is generated by mutating an 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.

[0077] To generate 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.

[0078] Methods for introducing nucleic acid mutations into target genes are well known in the art (see, for example, 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. 2014 / 085593, International Publication No. 2009 / 071334, and International Publication No. 2011 / 146121; U.S. Patent No. 8,771,945, U.S. Patent No. 8,586,526, U.S. Patent No. 6,774,279, and U.S. Patent Application Publication No. 2003 / 0232410, U.S. Patent Application Publication No. 2005 / 0026157, U.S. Patent Application Publication No. 2006 / 0014264; the contents of which are incorporated herein by reference in their entirety), and include genome editing by targeted homologous recombination, site-specific recombinases, PB transposases, and engineered nucleases. Agents for introducing nucleic acid modifications into target genes can be designed from publicly available sources or can be obtained commercially from Transposagen, Addgene, and Sangamo Biosciences.

[0079] 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).

[0080] 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 break point. 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 combination of recognized base pairs is very likely to be found at many locations throughout the genome, resulting in multiple cuts at non-limited desired locations. Therefore, genome editing cannot be performed 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.

[0081] In certain embodiments, including those disclosed herein, genome editing in exemplary embodiments utilizes clustered regularly interspaced short palindromic repeats (CRISPR) technology to edit a specific target region within the genome of an organism using an RGN. Standard CRIPSR systems for genome editing include a guide RNA ("gRNA"), a target region homologous to the target gene(s) including the edit(s) of interest (commonly referred to as a "homology arm" or "donor template"), and a separate component of the RGN (Cas9, Cpf1, or MAD7). Delivery of the RGN, synthetic gRNA, and donor template into a cell 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.

[0082] 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. October 22, 2015; 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, December 6, 2019; 10(1):5302) or Cas10d, a functional nuclease in type I-D systems (e.g., Osakabe et al., Nucleic Acids Res. June 21, 2021; 49(11):6347-6363), but are not limited thereto.

[0083] 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).

[0084] 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.

[0085] 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 induces single-strand cleavage by mutating the RuvC domain or the NHN domain, which are one of two Cas9 catalytic domains and contain the RuvCI, RuvCII, and RuvCIII motifs. In some embodiments, type II RGNs comprise 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.

[0086] In some embodiments, the recombinant nuclease used for editing is a fusion protein that comprises 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. November 3, 2021;29(11):3107-3124. In some embodiments, the genome editing that introduces one or more desired edits into a target region is base editing that does not require double-strand cleavage or donor DNA. See Anzalone AV, et al., Nature. December 2019;576(7785):149-157. doi:10.1038 / s41586-019-1711-4, Epub October 21, 2019 PMID:31634902.

[0087] 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 continue to survive under appropriate selection criteria. Cells containing the mutation of interest will not be cleaved because they do not contain the necessary PAM site.

[0088] 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 gRNA(s) for targeting specific genomic regions in 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 many others.

[0089] In some embodiments, the RGN and gRNA are introduced into the cell as a ribonucleoprotein (RNP) complex. The RNP complex includes 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 being delivered to the cell.

[0090] 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 multiple different nuclease-based systems for providing editing to an organism's genome, each of which can be used either in a single editing system, a sequential editing system (e.g., sequentially using different nuclease-directed systems to provide two or more genome edits within a cell), and / or a recursive editing system (e.g., utilizing a single nuclease-directed system to introduce two or more genome edits within a cell). Thus, those skilled in the art will recognize, upon reading this disclosure, that various enzyme-directed editing systems are useful for the disclosed embodiments.

[0091] 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 an organism's genome (Urnov et al., Nature Reviews Genetics, 11:636-646 (2010); International Publication No. WO 2003 / 087341 to Carroll et al., filed Jan. 22, 2003). ZFNs can be used to precisely alter a target region of the genome by utilizing an organism's endogenous DNA repair machinery. ZFNs can be used to generate double-strand breaks (“DSBs”) 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 DSBs by ligating the ends together and typically does not generate additional mutations, except when the cleavage is straightforward 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.

[0092] 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 cut 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 cut DNA at a specific location (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 / 079430 of Bonas et al., filed Jan. 12, 2010; International Publication No. 2011 / 072246 of Voytas et al., filed Dec. 10, 2010).

[0093] 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.

[0094] ZFN and TALEN restriction endonuclease technologies utilize non-specific DNA cleavage enzymes that are linked to specific DNA binding domains (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 linked 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. Additionally, FokI has the advantage that dimerization is required for nuclease activity, which means that 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.

[0095] Thus, for example, to target a particular 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 Fokl domains dimerize heterologously to create double-strand breaks. 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 made by NHEJ is unique, the use of a single nuclease pair generates 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, double-strand breaks can be repaired via homologous recombination repair to create specific modifications (Li et al., 2011; Miller et al., 2010; Urnov et al., 2005).

[0096] 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 have the feature of being found naturally in combinations in proteins. Cys2-His2 zinc fingers are typically found in repetitive sequences separated by 3 bp and in various combinations in diverse nucleic acid interacting proteins. On the other hand, TALE is found in repetitive sequences with a 1-to-1 recognition ratio between the amino acid and the nucleotide pair 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 ligated in a row to cover the required sequences), OPEN (where peptide domain to triplet nucleotide is selected with low stringency and then peptide combinations in a bacterial system to the final target are selected with high stringency), and bacterial one-hybrid screening of zinc finger libraries. Also, ZFN can be designed and commercially obtained, for example, from Sangamo Biosciences (trademark) (Richmond, California).

[0097] Methods for designing and obtaining TALENs are described, for example, in Reyon et al., Nature Biotechnology, May 2012; 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). TALENs can also be designed and obtained commercially, for example, from Sangamo Biosciences (trademark), Richmond, California.

[0098] In selected embodiments, the genome modifications described herein can be introduced using meganuclease-directed editing. Meganucleases were identified in the 1990s, and subsequent research has shown that meganucleases are particularly promising tools for genome editing because they can efficiently induce homologous recombination, generate mutations in both the coding and non-coding regions of the genome, and change 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 December 30, 2014). The high specificity of meganucleases results in high precision and very low cytotoxicity compared to other naturally occurring restriction enzymes.

[0099] 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 (>14 bp), 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 generate hybrid enzymes that recognize new sequences. Alternatively, DNA that interacts with the amino acids of meganucleases can be modified to design sequence-specific 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 No. 8,304,222; 8,021,867; 8,119,381; 8,124,369; 8,129,134; 8,133,697; 8,143,015; 8,143,016; 8,148,098; or 8,163,514, the entire content of each of the above documents being incorporated herein by reference.Alternatively, a gannuclease having site-specific cleavage characteristics can be obtained using commercially available techniques, such as the Directed Nuclease Editor™ genome editing technology manufactured by Precision Biosciences.

[0100] 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 proteins 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).

[0101] In some embodiments, the methods provided herein include, optionally, introducing or delivering a recombinant nuclease 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 donor template are introduced or delivered sequentially, optionally together with a gRNA. In some embodiments, the recombinant nuclease and gRNA are introduced or delivered together prior to the introduction or delivery of the donor template.

[0102] In other aspects, genome editing of the exemplary embodiments can utilize homologous recombination methods, including the cre-lox technology and the 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 these sites and catalyze recombination at these 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.

[0103] 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 containing 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.

[0104] 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 genome of the cell. A number of transposon systems that can transpose even within vertebrate cells have been isolated or designed. 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. December 1, 2003 31(23):6873-6881). Typically, the transposase system provides another means for removing the selection cassette after homologous recombination, similar to the use of Cre / Lox or Flp / FRT. Thus, as an example, the PiggyBac (PB) transposase system involves the introduction of a target vector having a 3' and 5' homologous arm that includes the mutation of interest, two PB terminal repeats at the site of the endogenous TTAA sequence, and a selection cassette located between the PB terminal repeats. 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 the 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.

[0105] Methods for validating and detecting array modifications 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.

[0106] Sequence modifications 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.

[0107] 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 a construct, 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).

[0108] 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, Inc. (Mountain View, CA), Oxford Nanopore Technologies (Oxford, UK), and Element Biosciences, Inc. (San Diego, CA).

[0109] Expression and culture 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 the cell line with an antibody-producing gene. 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, R. J. 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 capable of forming 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, MD) can be introduced.

[0110] In some embodiments, a nucleic acid sequence encoding an antibody can be constructed within, or inserted into, an expression vector. In some embodiments, a modified mammalian cell line is transfected with nucleic acids encoding the heavy (H) and light (L) chains of the antibody either separately or simultaneously. For example, in one embodiment, a nucleic acid encoding an antibody or an antigen-binding fragment thereof is constructed in a separate expression vector and then used for co-transfection into the modified cell line. Alternatively, nucleic acids encoding the H and L chains can 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.

[0111] Stable expression can be used to produce recombinant antibodies at high yields over long periods. For example, cell lines that stably express an antibody molecule can be used in the methods of the 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 within 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.

[0112] Expression vectors can include selectable markers, origins 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 skilled 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 in no way be construed as limiting: 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.

[0113] 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., 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 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.Viral vectors based on USA (1997) 94:10319-23; Takahashi et al., J. Virol. (1999) 73:7812-7816); or retroviral vectors (e.g., vectors derived from retroviruses such as murine leukemia virus, spleen necrosis virus, and Rous sarcoma virus, Harvey sarcoma virus, avian leukemia virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus), but are not limited thereto.

[0114] 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), as well as in other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

[0115] 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 U.S. Patent No. 6,326,193).

[0116] To evaluate the expression of a polypeptide (e.g., an antibody) or a portion thereof, the expression vector to be introduced into cells may contain either a selectable marker gene or a reporter gene, or both, to 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 for expression in the host cell may be proximal to both the selectable marker and the reporter gene. Useful selectable markers include, but are not limited to, antibiotic resistance genes.

[0117] In some embodiments, the 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 include 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 the drug (or selection agent) is added exogenously to the cell culture.

[0118] 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.

[0119] 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, differential solubility, or any other standard technique for protein purification. In many embodiments, the antibody is secreted from the cells into the culture medium and recovered from the medium.

[0120] Modified mammalian cells for producing anti-GRP78 antibodies In some embodiments, the provided methods and cell lines 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-related stress response mechanism that provides cell survival signals under environmental and physiological constraints. GRP78 classically participates in the processing of non-classically folded proteins as a molecular component of the ER chaperoning network, but more recent insights suggest that this protein can be located on the cell surface and may affect signal transduction (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 quiescent 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).

[0121] 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 an IgG antibody. In some embodiments, the anti-GRP78 antibody is a human antibody.

[0122] 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 the antibody designated as B4. In some embodiments, the anti-GRP78 antibody is the antibody designated as D1. In some embodiments, the anti-GRP78 antibody is the antibody designated as F6. In any of such embodiments, the antibody is either 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.

[0123] 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 shown in SEQ ID NO: 20. In some embodiments, the VH chain is shown in SEQ ID NO: 19 and the VL chain is shown in SEQ ID NO: 20.

[0124] 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 shown in SEQ ID NO: 22. In some embodiments, the VH chain is shown in SEQ ID NO: 21 and the VL chain is shown in SEQ ID NO: 22.

[0125] 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 shown in SEQ ID NO: 24. In some embodiments, the VH chain is shown in SEQ ID NO: 23 and the VL chain is shown in SEQ ID NO: 24.

[0126] 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.

[0127] 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.

[0128] 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 numbering of human GRP78 residues 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 numbering of human GRP78 residues shown in SEQ ID NO: 25.

[0129] 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 numbering of human GRP78 residues 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 numbering of human GRP78 residues shown in SEQ ID NO: 25.

[0130] 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 for 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), 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 for binding to human GRP78, and thus is considered to bind to the same epitope of human GRP78.

[0131] In some embodiments, the modified mammalian cell line is engineered with a variant GRP78 having a mutant epitope that includes mutations of 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 includes 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acid substitutions of the target epitope of the target protein.

[0132] In some embodiments, the modified mammalian cell line is engineered with a variant GRP78 having an amino acid sequence that includes at least one amino acid substitution as compared to the sequence of human GRP78 shown in SEQ ID NO: 25. In some embodiments, the modified cell line is engineered with a variant GRP78 having at least 85% and less than 100% sequence identity to the sequence of GPR78 shown in SEQ ID NO: 25. In some embodiments, the modified cell line is engineered with a variant GRP78 having at least 90% and less than 100% sequence identity to the sequence of GPR78 shown in SEQ ID NO: 25. In some embodiments, the modified cell line is engineered with a variant GRP78 having at least 95% and less than 100% sequence identity to the sequence of GPR78 shown 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 shown in SEQ ID NO: 25.

[0133] 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.

[0134] 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.

[0135] Provided herein are modified mammalian cell lines engineered with variant GRP78 represented by SEQ ID NO: 26.

[0136] Provided herein are modified mammalian cell lines engineered with variant GRP78 represented by SEQ ID NO: 27.

[0137] In some embodiments, mammalian cell lines may include 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 line is a CHO cell line. In some embodiments, the cell line is a HEK293 cell line.

[0138] In some embodiments, the anti-GRP78 antibody exhibits reduced or inhibited binding to variant GRP78 that contains 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 that does not contain 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 that does not contain 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.

[0139] In some embodiments, the anti-GRP78 antibody exhibits a reduced affinity for variant GRP78 relative to human GRP78 without one or more substitutions, such as that set forth in 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 relative to its affinity for human GRP78 lacking one or more amino acid substitutions, such as that set forth in 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 relative to its affinity for human GRP78 lacking one or more amino acid substitutions, such as that set forth in SEQ ID NO: 25. Any method described herein can be used as a method for measuring affinity.

[0140] 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.

[0141] Definitions Unless otherwise defined, all technical terms, notations, and other technical, scientific, or specialized terms used in this specification 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 in this specification for clarity and / or ease of reference, and the inclusion of such definitions in this specification should not necessarily be construed as representing a substantial difference from what is commonly understood in the art.

[0142] As used in this specification, 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 the aspects and variations described herein include aspects and variations "consisting of" and / or "consisting essentially of".

[0143] Throughout this disclosure, various aspects of the claimed subject matter are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the claimed subject matter. Accordingly, a recitation of a range should be considered to have specifically disclosed all the possible subranges and 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 to be understood as being included within the claimed subject matter. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also to be included within the claimed subject matter, subject to any specifically excluded limits within the stated range. When the stated range includes one or both of the limiting values, ranges excluding one or both of those included limiting values are also to be included within the claimed subject matter. This applies regardless of the breadth of the range.

[0144] As used herein, the term "about" refers to the normal error range for each value that is readily known. References to "about" a value or parameter in this specification include (and describe) embodiments directed to the value or parameter itself. For example, a recitation of "about X" includes a description of "X".

[0145] As used herein, the term "antibody" is used in its broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies as well as functional (antigen-binding) antibody fragments such as fragment antigen-binding (Fab) fragments, F(ab') 2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, heavy chain variable (V H ) regions capable of specifically binding to an antigen, single-chain variable fragments (scFv), and single-chain antibody fragments including single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. This term includes intracellular antibodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific antibodies such as bispecific or trispecific antibodies, diabodies, triabodies, and tetra-bodies, tandem di-scFv, tandem tri-scFv, and other forms of immunoglobulins modified by genetic engineering and / or other methods.

[0146] Unless otherwise specified, the term "antibody" should be understood to expressly include its functional antibody fragments, which are also referred to herein as "antigen-binding fragments". This term also includes intact antibodies or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD.

[0147] 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 regions 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).

[0148] 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 schemes 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 ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding site topography", J. Mol. Biol. 262, 732-745; ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains", Dev Comp Immunol, January 2003; 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, June 8, 2001; 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. July 2013;41(Web Server issue):W34-40, (the "IgBLAST" numbering scheme).

[0149] 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 of 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 have different numberings. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many respects. The AbM scheme is a compromise between the Kabat and Chothia definitions based on the definitions used in the Oxford Molecular AbM antibody modeling software. The IgBLAST scheme is based on matching to germline V, D, and J genes and can be determined using the IgBLAST tool of the National Center for Biotechnology Information (NCBI) of the United States.

[0150] Table 1 below lists exemplary position boundaries of CDR-L1, CDR-L2, CDR-L3 and CDR-H1, CDR-H2, CDR-H3, respectively, as defined by the Kabat, Chothia, AbM, and Contact schemes. For CDR-H1, residue numbering is listed using both the Kabat and Chothia numbering schemes. FRs are located between 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 loop length. [Table 1] 1 - Kabat et al. (1991), "Sequences of Proteins of Immunological Interest", 5th Ed., Public Health Service, National Institutes of Health, Bethesda, Maryland. 2 - Al-Lazikani et al., (1997) JMB 273, 927 - 948.

[0151] Under the Kabat numbering scheme, in some embodiments, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 26-35 (HCDR1), 50-65 (HCDR2), and 95-105 (HCDR3), and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under the Chothia numbering scheme, in some embodiments, the CDR amino acids in VH are numbered 26-35 (HCDR1). In a numbering scheme that combines Kabat and Chothia, in some embodiments, the CDR corresponds to amino acid residues that are Kabat CDR, Chothia CDR, or a portion of both. For example, in some embodiments, the CDR corresponds to amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-105 (HCDR3) in VH, and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in VL.

[0152] 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 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 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 scheme or any other known numbering scheme.

[0153] Similarly, unless otherwise specified, the FR of such a region or an individually specified FR (e.g., FR-H1, FR-H2, FR-H3, FR-H4) such as a given antibody or variable region 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.

[0154] Permissible variations of CDR sequences will be known to those of skill 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.

[0155] As used herein, the term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding to an antigen of the antibody. 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 contains 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 be isolated using the 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).

[0156] As used herein, the terms "culture," "culturing," "growing," "growing up," "maintaining," "maintenance," "expanding," "expansion," 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 cell growth, differentiation, or division by culturing.

[0157] 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 an amino acid, sugar side chain, phosphoryl group, or sulfonyl group. 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. An epitope formed from contiguous residues (e.g., amino acids) is typically retained even when exposed to a denaturing solvent, whereas an epitope formed by tertiary folding is typically lost upon treatment with a denaturing solvent. 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.

[0158] 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 the epitope recognized by an antibody. In some embodiments, the mutation is one or more amino acid substitutions of amino acid residues that constitute the epitope recognized by the antibody.

[0159] As used herein, the terms "expression" or "expressed" with respect to a gene refer to the transcriptional and / or translational products 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).

[0160] 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 non-translated RNAs such as rRNA, tRNA, guide RNAs (e.g., small guide RNAs), or microRNAs.

[0161] "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 contain 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 contains 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 contains a heavy chain variable (V H ) region and / or a light chain variable (V L ) region such as scFv.

[0162] A single-domain antibody (sdAb) is an antibody fragment that includes 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.

[0163] 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 includes fragments with non-naturally occurring arrangements, such as fragments having two or more antibody regions or chains linked by a synthetic linker, e.g., a peptide linker, and / or recombinant-produced fragments such as those produced by enzymatic digestion of a native full antibody. In some aspects, the antibody fragment is a scFv.

[0164] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or an antibody encoded by a sequence utilizing a human antibody repertoire or library of other human antibodies, excluding 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.

[0165] The term "humanized" is used to describe antibodies in which complementarity-determining regions (CDRs) from a mammal, such as a mouse, are joined to a human framework region. Often, a polynucleotide encoding the isolated CDRs 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., humanized or fully human), an antibody fragment, etc. Further, a "humanized" antibody may 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.

[0166] 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 Winter's U.S. Patent No. 5,225,539. 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 shows 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 a 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 effect on the CDR conformation and / or binding to the antigen. Investigation of such potential effects is done by modeling, examining the properties of amino acids at specific positions, or empirical observation of the effect of substitution or mutagenesis of specific amino acids.

[0167] 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.

[0168] 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 from the equivalent position of the mouse donor antibody, or amino acids 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.

[0169] 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 example and not limitation, the heavy chain variable VH region is cloned by RT-PCR using mRNA prepared from hybridoma cells. Consensus primers are used as the 5’ primer for the VH 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 VH region can also be determined or confirmed by sequencing the VH fragment obtained by 5’ RACE RT-PCR and 3’ g2b-specific primers.

[0170] The light chain variable V of a mouse monoclonal antibody L region can be cloned in a manner similar to the V H region. In a first 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 clone the cDNA encoding V L . The cloned sequence is then ligated to a sequence encoding a human constant region.

[0171] In one approach, the variable regions of the heavy and light chains are 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.

[0172] The heavy and light chain variable regions of chimeric and / or humanized antibodies can be linked to at least a portion of a selected human constant region. The selection of the constant region may depend on the desired mechanism of action of the antibody, e.g., 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 joined via a linker.

[0173] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies or an antibody within such a population, i.e., the individual antibodies making up the population are identical except for natural variations or variants that may occur during the production of the monoclonal antibody preparation, and such variants generally exist in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies against 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.

[0174] 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, for a longer duration, and / or with a higher affinity than it does with another protein. It should also be 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.

[0175] 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) or 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.

[0176] 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, this term includes 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 includes its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the explicitly recited sequence.

[0177] As used herein, the terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The polymers may be linear, cyclic, or branched, may include 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, amino addition via transfer RNA to a protein, 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.

[0178] "Vector" refers to a nucleic acid molecule that carries an inserted nucleic acid molecule into and / or between host cells, preferably a self-replicating nucleic acid molecule. "Expression vector" refers to a polynucleotide sequence that can be transcribed and translated into a polypeptide(s) when introduced into a suitable host cell. "Expression system" generally means a suitable host cell composed of an expression vector that can function to produce a desired expression product.

[0179] The term "recombinant" as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction, and / or ligation steps, and other procedures that result in a construct different from a polynucleotide found in nature.

[0180] 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 a polypeptide when expressed as a precursor involved in the secretion of the polypeptide. Generally, being functionally linked means being contiguous.

[0181] 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 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 comprising a mutation of one or more amino acid residues of the target epitope, and culturing the modified cell line under conditions such that the antibody is produced. In some preferred embodiments, the method further comprises isolating the antibody from the culture supernatant. In some 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.

[0182] A method for recombinant production of an antibody comprises the steps of expressing a nucleic acid encoding the antibody in a modified mammalian cell line, wherein the antibody specifically binds to a target epitope on a target protein that is naturally expressed as an intracellular protein in the mammalian cell line, and the modified mammalian cell line is engineered to express a variant target protein comprising a mutation of one or more amino acid residues of the target epitope; and culturing the modified mammalian cell line under conditions such that the antibody is produced. In some preferred embodiments, the method further comprises isolating the antibody from the culture supernatant. In some preferred embodiments, the naturally expressed target protein is an intracellular protein in the secretory pathway of the mammalian cell line. In some preferred embodiments, the mutation is an 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 the antibody to the target epitope on the target. In some preferred embodiments, the target protein is a stress protein. In some preferred embodiments, the target protein is expressed on an organelle of the mammalian cell line. In some preferred embodiments, the organelle is the endoplasmic reticulum or the Golgi apparatus. In some preferred embodiments, the target protein is an endoplasmic reticulum chaperone. In some preferred embodiments, the endoplasmic reticulum chaperone is calreticulin, heat shock protein, or isomerase. In some preferred embodiments, the target protein is glucose-regulated protein 78 (GRP78), HSP47, PDI, calreticulin or GP94. In some preferred embodiments, the target protein is glucose-regulated protein 78 (GRP78). In some preferred embodiments, the target protein is a Golgi complex protein. In some preferred embodiments, the Golgi complex protein is GOLPH2, GOLPH3, GM130, ATP6V1A, ATP6V1E1, ATP6VOA2, TMEM165, GOLGB1, SCYL1BP1, TRAPPC11, TRAPPC2 or TRIP11.

[0183] In some preferred embodiments, the present disclosure provides a method for recombinantly producing an antibody that targets an epitope on a target protein present within a cell of a cell line, the method comprising: providing an antibody that binds to an epitope on a target protein present within a cell of the cell line; identifying the epitope of the target protein to which the antibody binds; 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; introducing an expression vector encoding the antibody into the modified cell line; culturing the modified cell line under conditions that allow for the production of the antibody from the expression vector; and expressing a nucleic acid encoding the antibody in the modified cell line. In some preferred embodiments, the produced antibody is secreted into the culture supernatant. In some preferred embodiments, the method further comprises isolating the antibody or antigen-binding fragment. In some preferred embodiments, the antibody is a humanized antibody. In some preferred embodiments, the antibody is a human antibody. In some preferred embodiments, the target protein present within the cell of the cell line is an intracellular protein in the secretory pathway of the cell line. In some preferred embodiments, the target protein is a stress protein.

[0184] In some preferred embodiments, the present disclosure provides a method for recombinant production of an antibody that targets an epitope on a target protein found within a mammalian cell line, the method comprising: providing an antibody that binds to an epitope on a target protein present within a mammalian cell line; identifying the epitope of the target protein to which the antibody binds; mutating the epitope of the target protein in the mammalian cell line to generate a modified cell line mutated by reducing or suppressing the binding of the antibody to the epitope on the target protein; introducing an expression vector encoding the antibody into the modified mammalian cell line; culturing the modified mammalian cell line under conditions that allow for the production of the antibody from the expression vector; and expressing a nucleic acid encoding the antibody in the modified mammalian cell line. In some preferred embodiments, the produced antibody is present in the culture supernatant. In some preferred embodiments, the method further comprises isolating the antibody or antigen-binding fragment. In some preferred embodiments, the antibody is a humanized antibody. In some preferred embodiments, the antibody is a human antibody. In some preferred embodiments, the target protein present within the mammalian cell line is an intracellular protein in the secretory pathway of the mammalian cell line. In some preferred embodiments, the target protein is a stress protein.

[0185] In some preferred embodiments, the present disclosure provides a method for recombinant production of an antibody that targets GRP78, the method comprising expressing, in a modified mammalian cell line, a nucleic acid encoding the antibody, wherein the antibody binds to GRP78 and the modified mammalian cell is engineered with a variant GRP78 having a mutant epitope comprising a mutation of one or more amino acid residues of the target epitope of the antibody or antigen-binding fragment; and culturing the modified mammalian cell line under conditions such that an anti-GRP78 antibody is produced in the culture supernatant. In some preferred embodiments, the method further comprises isolating the anti-GRP78 antibody from the culture supernatant. In some embodiments, the antibody is a humanized antibody. In some preferred embodiments, the antibody is a human antibody.

[0186] In some preferred embodiments, the present disclosure provides a modified mammalian cell line comprising a mutant protein that is an intracellular protein in the secretory pathway, the mutant protein comprising a mutation of a natural intracellular protein that is the target protein of an antibody, the mutation being 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 embodiments, the mutation is an inactivating mutation that retains the structure and function of the target protein of the mammalian cell. In some embodiments, the mutation reduces or suppresses the binding of the antibody to the variant protein. In some embodiments, the target protein is a stress protein. In some preferred embodiments, the present disclosure provides a modified mammalian cell line comprising a variant GRP78 comprising the sequence set forth in SEQ ID NO: 28. In some preferred embodiments, the present disclosure provides a variant GRP78 comprising the sequence set forth in SEQ ID NO: 29. In some preferred embodiments, the mammalian cell line is selected from the group consisting of 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 leukemia Jurkat T cells, canine kidney MDCK cells, human cervical cancer HeLa cells.

[0187] In some preferred embodiments, the present disclosure provides a method for recombinant production of an antibody, the method comprising the step of expressing a nucleic acid encoding the antibody in a modified mammalian cell line, wherein the antibody specifically binds to a target epitope on an intracellular signaling protein that is naturally expressed in the mammalian cell line, and the modified mammalian cell line is engineered to express a variant intracellular signaling protein comprising a mutation of one or more amino acid residues of the target epitope; and culturing the modified mammalian cell line under conditions such that the antibody is produced. In a preferred embodiment, the method comprises isolating the antibody from the culture supernatant. In a preferred embodiment, the intracellular signaling protein is a phosphatase. In some preferred embodiments, the intracellular signaling protein is a kinase.

[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 invention.

[0190] Example 1 Production and Purification of Anti-GPRP78 Antibody in Mammalian Cells The full-length human anti-GRP78 monoclonal antibody (mAb) formatted as a full-length IgG1 antibody (Table E1) was individually transfected into Chinese hamster ovary (CHO) cells by electroporation in a 6-well plate, replenished with fresh medium the next day, and incubated for 7 days after transfection. Transfection was performed using the Neon™ transfection system and Neon™ transfection system reagents (Thermo Fisher Scientific, Waltham, MA) with the pre-setting for CHO cells. The supernatant on day 7 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 day 5 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 intracellularly in the endoplasmic reticulum (ER), thereby inhibiting the role of the native chaperone intracellularly and potentially killing the cells before the antibodies are secreted into the supernatant for purification. The binding of GRP78 antibodies to native ER proteins has been demonstrated by experiments showing co-immunoprecipitation of the antibody and GRP78, as demonstrated 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). Also included in the experiment was a commercially available monoclonal antibody, 1H11-1H7, which has been 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 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 to be "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 predicted amino acids that constitute the GRP78 epitope binding to B4 mAb or F6 mAb. This structure was selected (PDB ID#6ASY, Yang et al., 2017) because it is the most complete among the experimentally determined structures of H. sapiens GRP78 (in the range of amino acids 25 - 633). 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 the 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 Strategies for producing and purifying antibodies from mammalian cells were developed, where the wild - type target of the antibody is generally 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 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, suppressing the binding of the produced antibodies. For the epitope residues identified for the anti-GRP78 antibodies in Table E3, the GRP78 mutation(s) were selected to (1) substitute one or more charged residues with uncharged residues; (2) ensure that the newly selected residues remained exposed to the solvent; and (3) prevent secondary structure changes and the potential for tertiary and quaternary structure changes to preserve the endogenous function and avoid cell death by ensuring that the α-helix was preserved. For amino acids with a high tendency to form this secondary structure, the α-helix is likely to be formed. Furthermore, polar or charged amino acids are likely to remain exposed to the solvent due to their interaction with water, although charged residues are likely to form interactions with antibodies, so polar amino acids are preferred for mutagenesis. The polar uncharged amino acids from the highest to the lowest tendency to form an α-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 acid with alanine (rank 1 as an α-helix tendency) (Pace and Scholtz, 1998) has a risk of changing the secondary and tertiary structure due to the hydrophobic R group, but is likely to increase the suppression of 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 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 generated structures, 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 changes in α-helices were 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 conservation of the secondary and tertiary structures of GRP78, which simultaneously promotes the retention of the natural function of GRP78 and the suppression of binding to the above mAbs.

[0203] The single mutation R261Q was selected for later experiments as a major residue for both B4 and F6 mAbs, as detailed in Section B below. Additionally, the double mutation R261Q / H265Q was also selected for later 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) these mutations change residues determined by epitope mapping to be potentially important for the epitope binding of the GFR78 antibody to the native intracellular protein mass, and 2) these mutations are close enough to fit on the same DNA donor template for CRISPR-based editing. Also, other combinations could be used and it was thought necessary to use multiple donor templates and / or transfections to introduce mutations into cells either 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 interacting residues on GRP78 are located within exon 5 as shown in Figure 4. The flanking introns are also shown in Figure 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, resulting in 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" orientation as shown in Figure 5A. The modified codons within the ssDNA oligodonor sequences for causing genomic modifications are shown in bold and italic 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 Antibodies in Engineered 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 incubation was carried out for 5 days after transfection. Transfection was performed using the Neon™ transfection system and Neon™ transfection system reagents (Thermo Fisher Scientific, Waltham, Massachusetts) with the 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 the production of these antibodies 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, manufactured by ThermoFisher Scientific, Waltham, Massachusetts). 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 pelleted and passaged at 125 rpm in a humidified incubator at 37°C with 8% CO 2 , and monitored daily by cell counting and viability. When the viability after passage was 95% or higher and the doubling time was about 24 - 26 hours, the cells were considered adapted to suspension.

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, for example, by specific upregulation 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, it has been reported that PRL-3 is overexpressed in up to 70% of primary gastric cancers, and the higher the expression of PRL-3, the shorter the postoperative survival period 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 methods and systems 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 by Thura M, et al. (JCI Insight. 2016;1(9):e87607). Briefly, the humanized anti-PRL-3 antibody (referred to as “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 Cell Lines for Purification of Anti-PRL-3 Humanized Antibody A. Identification of Mutations in Target Proteins 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 retaining the overall structure and function of the protein.

[0218] Specifically, mutations (multiple 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 in PRL-3 at important sites to alanine 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 the binding epitope in the target protein 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 to Garst et al., 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 methods for producing and purifying antibodies from mammalian cells, as disclosed in Example 3, are 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. The 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 β 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, January 22, 2018; 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, they are both potential candidates for humanization to generate 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 such antibodies in mammalian cells.

[0225] The humanized PDI antibodies for use in the methods preferably include 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 sequence of the region was determined using the REmAB® antibody sequencing service provided by Rapid Novor (Ontario, Canada). It 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, 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.

[0227] Example 7 Epitope mapping of the protein binding site of an anti-PDI humanized antibody against hPDI Various 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. Load the biotinylated peptide spanning the hPDI protein at 5 μg / ml for 700 seconds, record the baseline for 300 seconds for baseline recording, and then measure the association and dissociation of mPDI-1 or mPDI-2 with each peptide at multiple concentrations for 600 seconds each. Use a dual-reference sensor 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 the 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 on , K off , and the R2 correlation coefficient are determined. If possible, a global curve fit is also performed for antibodies / analytes at multiple concentrations.

[0230] Identification of hPDI Structure for Defining Residues Important for Mutagenesis in Example 8 To identify residues that are edited to suppress the binding of antibodies to hPDI, the crystal structure of S. cerevisiae (Tian G., et al., Cell. 2006;124:61 - 73), and the crystallization of human PDI in both oxidized and reduced states (Wang C, et al., Antioxid Redox Signal 19:36 - 45) can be used to deduce 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 Purification of Anti - PDI Humanized Antibodies A. Identification of Mutations in Target Proteins for Suppressing 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 suppress the binding of the produced antibodies while maintaining 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 preserve the endogenous function to prevent the possibility of changes in tertiary and quaternary structure to avoid cell death. Substitutions of PDI amino acids to alanine at important sites are predicted, and the proposed PDI variants in mammalian cells are further evaluated for survival rate and inhibition of binding of humanized mAbs based on mPDI-1 and mPDI-2 to intracellular proteins.

[0232] The proposed PDI variants 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 variants) 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 desirable mutant structures do not visually change, and there are no major changes in secondary structure.

[0233] B. Editing of the Binding Epitope in the Target Protein Retrieve the genomic sequence data of PDI in Chinese hamster cells from the NCBI Gene Database (Gene ID: 100766687) and use it as the reference sequence for gene editing. For the production of both PDI-1 mAb and PDI-2 mAb, use the homologous recombination repair (HDR) strategy and edit the wild-type PDI (SEQ ID NO: 34) in CHO cells using type V CRISPR gene editing technology to generate the desired single mutant and / or double mutant. Such an editing strategy is described in U.S. Patent No. 11,220,697 to Garst et al., 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 to be used with type V RGNs are available from companies such as Integrated DNA Technologies (Coralville, Iowa) and GenScript (Piscataway, New Jersey).

[0234] Isolate cells with the desired mutation by clonal serial dilution and confirm the presence of the point mutation by Sanger sequencing. After growing in culture, test the cell growth and viability. Test the control (wild-type CHO) and mutant PDI CHO cells and simultaneously select cells that reached >90% confluence (on day 4 after seeding) as those showing minimal changes in cell growth or viability. Calculate the cell confluence 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 clone CHO cells by electroporation in a 6-well plate, fresh medium was replenished the next day, and 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 are selected.

[0236] Example 10 Humanization of Anti-GOLPH3 Monoclonal Mouse Antibody GOLPH3 was initially identified as a peripheral membrane protein localized to the trans-Golgi network, but some researchers have reported that it is a mitochondrial protein that controls mitochondrial mass through the regulation of the mitochondrial-specific phospholipid cardiolipin. Since then, GOLPH3 has been thought to be involved in the targets 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 to 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 the antibody 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, for example, using 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 the mGOLPH3 mAb to biotinylated hGOLPH3 peptide across hGOLPH3 can be measured using a streptavidin biosensor. Load the biotinylated peptide across the hGOLPH3 protein at 5 μg / ml for 700 seconds and for 300 seconds for baseline recording, and then measure the association and dissociation of the mGOLPH3 mAb with each peptide at multiple concentrations for 600 seconds each. Use a double-reference sensor in all tests to measure any observed background signal and subtract it from non-specific binding or system noise.

[0241] Perform the analysis using ForteBio data analysis software (v8.2). After subtracting the background, fit a 1:1 local kinetics model to the observed binding and dissociation curves. Overall, determine K D 、K on 、K off 、and the R2 correlation coefficient. If possible, also perform a global curve fit for multiple concentrations of antibody / analyte.

[0242] Identification of the hGOLPH3 Structure for Defining Residues Important for Mutagenesis in Example 12 To identify the 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 a resolution of 2.9 Å (Wood et al., J Cell Biol. December 28, 2009; 187(7):967-975), and also reported the 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. December 2017; 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 an Anti-GOLPH3 Humanized Antibody A. Identification of Mutations in the Target Protein 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 structure of the protein, prevent changes in secondary structure and the possibility of changes in tertiary and quaternary structures, 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 variants in mammalian cells are further evaluated for inhibition of cell viability and binding of the humanized mAb based on mGOLPH3 mAb to intracellular proteins.

[0244] The proposed GOLPH3 variants 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 variants) are input, and the wild-type sequence is modeled as a control. Each of the variant 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. When compared with the wild-type structure, the regions containing mutations in the desirable variant structures do not visually change, and there are no major changes in secondary structure.

[0245] B. Editing of the Binding Epitope in the Target Protein Retrieve the genomic sequence data of GOLPH3 in Chinese hamster cells from the NCBI gene database (Gene ID: 100766687) and use it 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 an editing strategy is described in U.S. Patent No. 11,220,697 to Garst et al. 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 the type V RGN are available from companies such as Integrated DNA Technologies (Coralville, Iowa) and GenScript (Piscataway, New Jersey).

[0246] Isolate the cells with the desired mutations by clonal serial dilution and confirm the presence of point mutations by Sanger sequencing. After growing in culture, evaluate the cell growth and viability. Test the control (wild-type CHO) and mutant GOLPH3 CHO cells, and simultaneously select the cells that reached >90% confluence (on the 4th day after seeding) as those showing minimal changes in cell growth or viability. Calculate the cell confluence using the IncuCyte® live cell analysis system.

[0247] C. Production and Purification of Anti-Golph3 Antibody in Modified Cho Cells The humanized anti-GOLPH3 monoclonal antibody (mAb) formatted as a full-length IgG1 antibody was individually transfected into clonal CHO cells by electroporation in 6-well plates, fresh medium was replenished the next day, and 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 produced from CHO wild-type cells were selected.

[0248] 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 made without departing from the true scope and spirit of the present disclosure and are intended to be included within the scope of the present disclosure. [Table 10-1] [Table 10-2] [Table 10-3]

Claims

**Claim 1** (a) In a modified cell line, a step of expressing a nucleic acid encoding an antibody, wherein the antibody specifically binds to a target epitope on a target protein that is naturally expressed as an intracellular protein in the cell, and the modified cell is engineered to express a variant target protein comprising a mutation of one or more amino acid residues of the target epitope, and (b) culturing the modified cell line under conditions such that the antibody is produced, A method for recombinant production of an antibody, comprising: **Claim 2** The method according to claim 1, further comprising (c) isolating the antibody from the culture supernatant. **Claim 3** The method according to claim 1 or 2, wherein the target protein that is naturally expressed is an intracellular protein in the secretory pathway of the cell line. **Claim 4** The method according to any one of claims 1 to 3, wherein the mutation is an inactivating mutation that retains the structure and function of the native target protein in the cell. **Claim 5** The method according to any one of claims 1 to 4, wherein the mutation reduces or suppresses the binding of the antibody to the target epitope on the target protein. **Claim 6** The method according to any one of claims 1 to 5, wherein the target protein is a stress protein. **Claim 7** The method according to any one of claims 1 to 6, wherein the target protein is expressed on an organelle of the cell line. **Claim 8** The method according to claim 7, wherein the organelle is the endoplasmic reticulum or the Golgi apparatus. **Claim 9** The method according to any one of claims 1 to 8, wherein the target protein is an endoplasmic reticulum chaperone. **Claim 10** The method according to claim 9, wherein the endoplasmic reticulum chaperone is calreticulin, a heat shock protein or an isomerase. **Claim 11** The method according to any one of claims 1 to 10, wherein the target protein is glucose-regulated protein 78 (GRP78), HSP47, PDI, calreticulin or GP94. **Claim 12** The method according to any one of claims 1 to 11, wherein the target protein is glucose-regulated protein 78 (GRP78). **Claim 13** The method according to any one of claims 1 to 8, wherein the target protein is a Golgi complex protein. **Claim 14** The method according to claim 13, wherein the Golgi complex protein is GOLPH2, GOLPH3, GM130, ATP6V1A, ATP6V1E1, ATP6VOA2, TMEM165, GOLGB1, SCYL1BP1, TRAPPC11, TRAPPC2 or TRIP11.

15. (a) In a modified mammalian cell line, a step of expressing a nucleic acid encoding an antibody, wherein the antibody specifically binds to a target epitope on a target protein that is naturally expressed as an intracellular protein in the mammalian cell line, and the modified mammalian cell line is engineered to express a variant target protein comprising a mutation of one or more amino acid residues of the target epitope, and (b) A step of culturing the modified mammalian cell line under conditions such that the antibody is produced, a method for recombinant production of an antibody.

16. (c) The method according to claim 15, further comprising a step of isolating the antibody from the culture supernatant.

17. The method according to claim 15 or 16, wherein the target protein that is naturally expressed is an intracellular protein in the secretory pathway of the mammalian cell line.

18. The method according to any one of claims 15 to 17, wherein the mutation is an inactivating mutation that retains the structure and function of the target protein in the mammalian cell line.

19. The method according to any one of claims 15 to 18, wherein the mutation reduces or suppresses the binding of the antibody to the target epitope on the target.

20. The method according to any one of claims 15 to 19, wherein the target protein is a stress protein.

21. The method according to any one of claims 15 to 20, wherein the target protein is expressed on an organelle of the mammalian cell line.

22. The method according to claim 21, wherein the organelle is the endoplasmic reticulum or the Golgi apparatus.

23. The method according to any one of claims 15 to 22, wherein the target protein is an endoplasmic reticulum chaperone.

24. The method according to claim 23, wherein the endoplasmic reticulum chaperone is calreticulin, a heat shock protein or an isomerase.

25. The method according to any one of claims 15 to 24, wherein the target protein is glucose-regulated protein 78 (GRP78), HSP47, PDI, calreticulin or GP94.

26. The method according to any one of claims 15 to 25, wherein the target protein is glucose-regulated protein 78 (GRP78).

27. The method according to any one of claims 15 to 22, wherein the target protein is a Golgi complex protein.

28. The method according to claim 27, wherein the Golgi complex protein is GOLPH2, GOLPH3, GM130, ATP6V1A, ATP6V1E1, ATP6VOA2, TMEM165, GOLGB1, SCYL1BP1, TRAPPPC11, TRAPPPC2 or TRIP11.

29. A method for recombinantly producing an antibody targeting an epitope on a target protein found intracellularly in a cell line, the method comprising: (a) providing an antibody that binds to an epitope on a target protein present intracellularly in a cell line; (b) identifying the epitope of the target protein to which the antibody binds; (c) 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, thereby generating a modified cell line; (d) introducing an expression vector encoding the antibody into the modified cell line; (e) culturing the modified cell line under conditions that allow production of the antibody from the expression vector; and (f) expressing a nucleic acid encoding the antibody in the modified cell line.

30. The method according to claim 29, wherein the produced antibody is secreted into the culture supernatant.

31. The method according to claim 29 or 30, further comprising (g) isolating the antibody or antigen-binding fragment.

32. The method according to any one of claims 29 to 31, wherein the antibody is a humanized antibody.

33. The method according to any one of claims 29 to 31, wherein the antibody is a human antibody.

34. The method according to any one of claims 29 to 33, wherein the target protein present intracellularly in the cell line is an intracellular protein in the secretory pathway of the cell line.

35. The method according to any one of claims 29 to 34, wherein the target protein is a stress protein.

36. A method for recombinantly producing an antibody targeting an epitope on a target protein found intracellularly in a mammalian cell line, the method comprising: Step of providing an antibody that binds to an epitope on a target protein present inside a mammalian cell line Step of identifying the epitope of the target protein to which the antibody binds Step of generating a modified cell line by mutating the epitope of the target protein in the mammalian cell line to reduce or suppress the binding of an antibody to the epitope on the target protein Step of introducing an expression vector encoding the antibody into the modified mammalian cell line Step of culturing the modified mammalian cell line under conditions that allow the production of the antibody from the expression vector, and Step of expressing a nucleic acid encoding the antibody in the modified mammalian cell line, the method comprising the above steps. **Claim 37** The method according to claim 36, wherein the produced antibody is present in the culture supernatant. **Claim 38** The method according to claim 37, further comprising step (g) of isolating the antibody or antigen-binding fragment. **Claim 39** The method according to any one of claims 36 to 38, wherein the antibody is a humanized antibody. **Claim 40** The method according to any one of claims 36 to 38, wherein the antibody is a human antibody. **Claim 41** The method according to any one of claims 36 to 40, wherein the target protein present inside the mammalian cell line is an intracellular protein in the secretory pathway of the mammalian cell line. **Claim 42** The method according to any one of claims 40 to 41, wherein the target protein is a stress protein. **Claim 43** A method for recombinant production of an antibody targeting GRP78, the method comprising: (a) In a modified mammalian cell line, a step of expressing a nucleic acid encoding an antibody, wherein the antibody binds to GRP78, and the modified mammalian cell is engineered with a variant GRP78 having a mutated epitope comprising one or more amino acid residues of the target epitope of the antibody or antigen-binding fragment; and (b) A step of culturing the modified mammalian cell line under conditions such that an anti-GRP78 antibody is produced in the culture supernatant. **Claim 44** The method according to claim 43, further comprising step (c) of isolating the anti-GRP78 antibody from the culture supernatant. **Claim 45** The method according to claim 43 or 44, wherein the antibody is a humanized antibody. **Claim 46** The method according to claim 43 or 44, wherein the antibody is a human antibody.

47. A modified mammalian cell line comprising a variant protein that is an intracellular protein in the secretory pathway, wherein the variant protein comprises a mutation of a natural intracellular protein that is 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.

48. The modified mammalian cell line according to claim 47, wherein the mutation is an inactive mutation that retains the structure and function of the target protein of the mammalian cell.

49. The modified mammalian cell line according to claim 47 or 48, wherein the mutation reduces or suppresses the binding of the antibody to the variant protein.

50. The modified mammalian cell line according to any one of claims 47 to 49, wherein the target protein is a stress protein.

51. A modified mammalian cell line comprising variant GRP78 comprising the sequence shown in SEQ ID NO:

28.

52. A modified mammalian cell line comprising variant GRP78 comprising the sequence shown in SEQ ID NO:

29.

53. The modified mammalian cell line according to any one of claims 47 to 52, wherein the mammalian cell line is selected from the group consisting of Chinese hamster ovary (CHO) cells, baby hamster kidney cells, NSO myeloma cells, monkey kidney COS cells, monkey kidney fibroblast CV-I cells, human fetal kidney 293 (HEK293) cells, human breast cancer SKBR3 cells, human leukemia Jurkat T cells, dog kidney MDCK cells, and human cervical cancer HeLa cells.

54. (a) In a modified mammalian cell line, a step of expressing a nucleic acid encoding an antibody, wherein the antibody specifically binds to a target epitope on an intracellular signaling protein that is naturally expressed in the mammalian cell line, and the modified mammalian cell line is engineered to express a variant intracellular signaling protein comprising a mutation of one or more amino acid residues of the target epitope, and (b) A step of culturing the modified mammalian cell line under conditions such that the antibody is produced. A recombinant production method of an antibody comprising these steps.

55. The method according to claim 54, further comprising the step of isolating the antibody from the culture supernatant. **Claim 56** The method according to claim 54, wherein the intracellular signal transduction protein is a phosphatase. **Claim 57** The method according to claim 54, wherein the intracellular signal transduction protein is a kinase.

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