Suicide EGFR-iii compositions and methods of use thereof

Abstract

Lentiviral vectors are provided that incorporate a nucleic acid construct encoding a modified EGFR. The nucleic acid constructs encode a modified EGFR that lacks EGFR domains I, II, and IV and that includes domain III and the EGFR transmembrane domain. Also provided are pharmaceutical compositions including the lentiviral vectors or cells transduced with the lentiviral vectors, and methods of treating a patient having cancer by administering the compositions and optionally subsequently administering to the patient an EGFR antibody to target the destruction of the cells expressing the modified EGFR.

Description

Attorney Docket No.: 090723-1514847-MDA23-090PCTSUICIDE EGFR-III COMPOSITIONS AND METHODS OF USE THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 679,800, filed August 6, 2024, the entire contents of which are incorporated herein by this reference.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted herewith and is hereby incorporated by reference in its entirety. The Sequence Listing XML, created on August 01, 2025, is named MDA23-090PCT-090723-1514847 and is 15,535 bytes in size.BACKGROUND

[0003] Viral vectors have quickly evolved into routine tools for gene therapy and cell therapy. Integration of retroviral and lentiviral vectors into the host genome allows stable expression of transferred genes that can be passed on to the daughter cells. There has been a general preference to change from retroviral vectors toward lentiviral vectors for several reasons including, lower risk in terms of insertional mutagenesis and higher vector titer. The optimal packaging capacity of these viral vectors (approximately 8 kb) allows for the delivery of sophisticated elements such as chimeric antigen receptors (CARs) or T cell receptors (TCRs). However, therapy delivering these elements (e.g., CAR-T cell therapy, CAR-NK cell therapy, CAR-NKT cell therapy, TCR-T cell therapy) can result in toxicity to the patient. Therefore, suicide receptors have been introduced in some viral vectors to allow for the selective, in vivo ablation of cells expressing the suicide receptors, for safety control. The suicide design can allow physicians and clinicians to scale back the cells expressing the suicide receptors in patients. By reducing or omitting these cell populations, the side effects of highly amplified CAR or TCR cells in patients can be avoided.SUMMARY

[0004] The terms “invention,” “the invention,” “this invention,” “the present invention,” “this disclosure,” and the “the present disclosure” as used in this document, are intended toAttorney Docket No.: 090723-1514847-MDA23-090PCT refer broadly to all of the subject matter of this patent application and the claims below. Statements containing these terms should be understood not to limit the subject matter described herein or to limit the meaning or scope of the patent claims below. Covered embodiments of the invention are defined by the claims, not this summary. This summary is a high-level overview of various aspects of the invention and introduces some of the concepts that are described and illustrated in the present document and the accompanying figures. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. The subject matter should be understood by reference to appropriate portions of the entire specification, any or all figures and each claim. Some of the exemplary embodiments of the present disclosure are discussed below.

[0005] The Summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter.

[0006] In one aspect, provided herein is a nucleic acid construct comprising a polynucleotide sequence that encodes a modified epidermal growth factor receptor (EGFR), wherein the modified EGFR comprises an EGFR domain III and an EGFR transmembrane domain, and wherein the modified EGFR comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 1. In some embodiments, the nucleic acid construct comprises a nucleic acid sequence that encodes a signal domain. In some embodiments, the modified EGFR polypeptide comprises SEQ ID NO: 1. In some embodiments, the EGFR Domain III comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 2. In some embodiments, the EGFR Domain III comprises SEQ ID NO: 2. In some embodiments, the EGFR transmembrane (TM) domain comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 3. In some embodiments, the EGFR TM Domain comprises SEQ ID NO: 3.

[0007] In some embodiments, the modified EGFR polypeptide specifically binds an anti- EGFR antibody. In some embodiments, the anti-EGFR antibody is selected from a group consisting of cetuximab, matuzumab, necitumumab, panitumumab, zalutumumab, and nimotuzumab.Attorney Docket No.: 090723-1514847-MDA23-090PCT

[0008] In some embodiments, the nucleic acid construct further comprises a promoter operably linked to the nucleic acid sequence encoding the signal domain. In some embodiments, the promoter is a eukaryotic promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter comprises an elongation factor la (EFla) promoter. In certain embodiments, the promoter comprises SEQ ID NO: 4.

[0009] In some embodiments, the nucleic acid construct further comprises a central polypurine tract / central termination sequence (CPPT / CTS) positioned upstream of the promoter, wherein the CPPT / CTS comprises SEQ ID NO: 5.

[0010] In another aspect, provided herein is a vector comprising the nucleic acid construct of any one of the embodiments disclosed herein. In some embodiments, the nucleic acid sequence of the vector has at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, the nucleic acid sequence of the vector comprises SEQ ID NO: 6. In some embodiments, the vector comprises a viral promoter, a viral enhancer, a nucleic acid sequence encoding an antibiotic resistance polypeptide, a regulatory protein binding site, and a recruitment site. In some embodiments, the antibiotic resistance polypeptide confers resistance to ampicillin or kanamycin. In some embodiments, the viral enhancer comprises a woodchuck hepatitis virus post translational regulatory element (WPRE) or cytomegalovirus (CMV) enhancer. In some embodiments, the recruitment site is an internal ribosome entry site (IRES) or 2A peptide binding site. In some embodiments, the viral promoter is a CMV promoter or an RSV promoter. In some embodiments, the regulatory protein binding site is a rev response element (RRE) or a constitutive transport element (CTE). In some embodiments, the vector is a lentiviral vector, an adenoviral vector, a retroviral vector, or an adeno- associated viral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector further comprises a transgene. In some embodiments, the transgene encodes a chimeric antigen receptor or a T cell receptor.

[0011] In yet another aspect, provided herein is a genetically modified cell comprising the nucleic acid construct or the vector of any one of the embodiments described herein. In some embodiments, the genetically modified cell is a eukaryotic cell. In some embodiments, the genetically modified cell is a human T cell. In some embodiments, the genetically modified cell is a human NK cell or a human NKT cell. In some embodiments, the nucleic acid construct is integrated into a genome of the cell. In some embodiments, the geneticallyAttorney Docket No.: 090723-1514847-MDA23-090PCT modified cell expresses the polypeptide encoded by the transgene and the modified EGFR polypeptide encoded by the nucleic acid construct on the cell surface.

[0012] In one aspect, provided herein is a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the vector or the genetically modified cell of any one of the embodiments described herein.

[0013] In another aspect, provided herein is a method of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition comprising a pharmaceutically acceptable carrier and the vector or the genetically modified cell of any one of the embodiments described herein. In some embodiments, the pharmaceutical composition comprises the genetically modified cell of any one of the embodiments described herein, and the genetically modified cells are allogeneic cells. In some embodiments, the pharmaceutical composition comprises the genetically modified cell of any one of the embodiments described herein, and the genetically modified cells are autologous cells. In some embodiments, the genetically modified cells are HLA matched to the subject. In some embodiments, the subject has a cancer. In some embodiments, the subject has an autoimmune disease or condition.

[0014] In yet another aspect, provided herein is a method for killing the genetically modified cells of any one of the embodiments described herein in a subject. In some embodiments, the methods comprise administering to the subject an effective amount an anti- EGFR antibody. In some embodiments, the anti-EGFR antibody is selected from a group consisting of cetuximab, matuzumab, necitumumab, panitumumab, zalutumumab, and nimotuzumab. In some embodiments, the anti-EGFR antibody that is administered to the subject is cetuximab.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The present application includes the following figures. The figures are intended to illustrate certain embodiments and / or features of the compositions and methods, and to supplement any description of the compositions and methods. The figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case.Attorney Docket No.: 090723-1514847-MDA23-090PCT

[0016] FIGS. 1A-1B provide the plasmid design for an exemplary vector comprising an EGFR-III for targeted depletion of cells that express the EGFR-III. FIG. 1A is a vector map of pORBIT, and FIG. IB is a table showing the features of the pORBIT plasmid.

[0017] FIG. 2A is a schematic drawing showing the 3D structures from 6ARU for wild type human EGFR (WT HuEGFR), a previously described truncated human EGFR (HuEGFRt), and the human EGFR-III (HuEGFR-III) polypeptide described herein, bound to the surface of a cell and further bound to cetuximab. FIG. 2B is a schematic diagram of the components of the three EGFR polypeptides in FIG. 2A (WT EGFR, HuEGFRt, and the HuEGFR-III as described herein). The EGFR polypeptides include the signal peptide (SP) at the N-terminus of the polypeptides, one or more of EGFR Domains I-IV, and a transmembrane domain (TM) and linker at the C-terminus. The disclosed HuEGFR-III includes the amino acids between the arginine at position 309 (R309) and the lysine at position 513 (K513) of EGFR.

[0018] FIGS. 3A-3D provide data regarding the assessment of the lentiviral vector according to certain aspects of the disclosure. FIG. 3A is a plot of the fluorescence-activated cell sorting (FACS) data of healthy donor T cells transduced with a lentivirus encoding Velo8-bbz CAR (cyclin El (CCNE1) monomer TCR mimic antibody, including stimulatory 4-1BB and CD3(^ domains, used herein as the tool antibody) and EGFR-III as disclosed herein. The cells were subsequently stained with an anti-human IgG secondary antibody labeled with phycoerythrin (a-hlgG-PE) (darker grey peak) or with cetuximab and the a- hlgG-PE secondary antibody (lighter grey peak). The left panel of FIG. 3B provides a schematic drawing for the antibody dependent cell cytotoxicity (ADCC) mechanism of cetuximab binding to a target cell and mediating NK killing of the target cell. The right panel of FIG. 3B is a graph of the live target cells (%) after NK cells from a healthy donor were cocultured with transduced T cells (expressing the CAR and the disclosed EGFR-III) and further incubated with cetuximab (left and middle bars) or normal human IgG (right bar) for four hours. The ratios of effector cells to target cells (E:R) are shown on the x axis. FIG. 3C provides plots of FACS data of primary T cells that were not transduced (top panel) or primary T cells transduced with a lentiviral vector as described herein encoding EGFR-III (middle panel), or a lentiviral vector encoding EGFRIII / IV (EGFRt) (bottom panel)). The cells were detected using cetuximab-AF747. FIG. 3D is a graph showing the percent lysis of primary T cells transduced with lentiviral vectors encoding a Velo8-bbz CAR and the EGFR- III described herein (circles and squares) or the previously described EGFRIII / IV (EGFRt)Attorney Docket No.: 090723-1514847-MDA23-090PCT(triangles and inverted triangles) in the presence of an anti-human IgG secondary antibody (circles and triangles) or cetuximab (squares and inverted triangles).

[0019] FIGs. 4A-4D demonstrate the in vivo depletion of CAR-T cells expressing a disclosed EGFR-III polypeptide through interaction with cetuximab. FIG. 4A is a graph showing the in vivo efficacy of CAR-T depletion, comparing mice treated with parental T cells (from Donor 332) (black line) to mice treated with CAR-T cells expressing an EGFR-III according to certain aspects of the disclosure (CCNE CAR-T (Donor 332)) (gray line). CCNE here refers to cells with human leukocyte antigen serotype A02:01 presenting cyclin El peptide. As indicated, mice were treated with cetuximab at days 21 and 24 to deplete CAR- T+ cells. FIG. 4B is a graph showing the percentage of live T cells in blood from the mice after 2 treatment doses of cetuximab (2 mice shown, squares) or a control (human IgGl, 2 mice shown, circles). FIG. 4C is a graph showing the depletion of CAR-T cells by cetuximab in the total human T cell population. FIG. 4D provides plots of FACS data of human CD3+ T cells after two treatments of cetuximab (bottom panels) or isotype IgG (top panels).DETAILED DESCRIPTIONI. Introduction

[0020] Lentiviral vectors have been shown to effectively deliver nucleic acid sequences encoding CARs or TCRs to produce engineered cells (e.g., CAR-T cells, TCR-T cells, CAR- NK cells, CAR-NKT cells). However, the therapeutic use of these engineered cells (e.g., CAR-T cell therapy, TCR-T therapy, CAR-NK cell therapy, CAR-NKT cell therapy) can result in toxicity to the patient. Provided herein are vectors including a novel truncated EGFR polypeptide and methods of use thereof. The vectors including the truncated EGFR domain may be used to address the production and safety issues of T cell, NK cell, or NKT adoptive immunotherapy by providing an efficient means of increasing the antibody-dependent cell cytotoxicity in the CAR-T, CAR-NK, CAR-NKT, or TCR-T cell populations, thus reducing the various degrees of toxicity experienced in a subject. The introduction of the modified EGFR sequence to the cells via the lentiviral vectors provides an inert binding protein, following expression, on the cell surface, without causing or inducing cell signaling after ligand or antibody binding. Thus, provided herein are novel lentiviral backbones that incorporate a nucleic acid construct encoding a modified EGFR that lacks EGFR domains I,II, and IV while maintaining domain III and the EGFR transmembrane domain, thusAttorney Docket No.: 090723-1514847-MDA23-090PCT providing a reduced distance between the cell membrane and an anti-EGFR antibody when bound to the modified EGFR on the cell surface. The literature is inconsistent with respect to which position is the last amino acid in domain III and the first amino acid in domain IV; however, wherever that position is, the present modified EGFR polypeptides (EGFR-III) lack all, or the majority, of domain IV.II. Definitions

[0021] A number of terms and concepts are discussed below. They are intended to facilitate the understanding of various embodiments of the present disclosure in conjunction with the rest of the present document and the accompanying figures. These terms and concepts may be further clarified and understood based on the accepted conventions in the fields of the present disclosure, as well as the description provided throughout the present document and / or the accompanying figures. Some other terms can be explicitly or implicitly defined in other sections of this document and in the accompanying figures and may be used and understood based on the accepted conventions in the fields of the present disclosure, the description provided throughout the present document and / or the accompanying figures. The terms not explicitly defined can also be defined and understood based on the accepted conventions in the fields of the present disclosure and interpreted in the context of the present document and / or the accompanying figures.

[0022] Unless otherwise defined, all terms of art, notations, and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference over the definition of the term as generally understood in the art.

[0023] Articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0024] The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of’ those certain elements. As used herein, “and / or” refers to and encompasses any and allAttorney Docket No.: 090723-1514847-MDA23-090PCT possible combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0025] As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the combination of features of the claimfs]. See, e.g., In re Herz, 537 F.2d 549, 551-52 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0026] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0027] The terms “about” and “approximately” as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20% (%); preferably, within 10%; and more preferably, within 5% of a given value or range of values. Any reference to “about X” or “approximately X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, expressions “about X” or “approximately X” are intended to teach and provide written support for a claim limitation of, for example, “0.98X.” Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated. When “about” is applied to the beginning of a numerical range, it applies to both ends of the range.

[0028] As used throughout, the terms “nucleic acid,” “nucleic acid sequence,” “oligonucleotide,” “nucleotides,” or other grammatical equivalents as used herein mean at least two nucleotides, either deoxyribonucleotides or ribonucleotides, or analogs thereof, covalently linked together. Polynucleotides are polymers of any length, including, e.g., 20,Attorney Docket No.: 090723-1514847-MDA23-090PCT50, 100, 200, 300, 500, 1000, 2000, 3000, 5000, 7000, 10,000 nucleotides, etc. A polynucleotide described herein generally contains phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have at least one different linkage, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages, and peptide nucleic acid backbones and linkages. Mixtures of naturally occurring polynucleotides and analogs can be made; alternatively, mixtures of different polynucleotide analogs, and mixtures of naturally occurring polynucleotides and analogs may be made. The following are non-limiting examples of polynucleotides: a gene or gene fragment, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, cRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also includes both double- and single-stranded molecules. Unless otherwise specified or required, the term polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. A polynucleotide is composed of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. Unless otherwise indicated, a particular polynucleotide sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues.

[0029] Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof, alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated.

[0030] The terms “polypeptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues in a single chain. The terms apply to amino acid polymers inAttorney Docket No.: 090723-1514847-MDA23-090PCT which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. Amino acid polymers may comprise entirely L-amino acids, entirely D-amino acids, or a mixture of L and D amino acids. The term “protein” as used herein refers to either a polypeptide or a dimer (i.e., two) or multimer (z.e., three or more) of single chain polypeptides. The single chain polypeptides of a protein may be joined by a covalent bond, e.g., a disulfide bond, or non-covalent interactions. The terms “portion” and “fragment” are used interchangeably herein to refer to parts of a polypeptide, nucleic acid, or other molecular construct.

[0031] The amino acids in the polypeptides described herein can be any of the 20 naturally occurring amino acids, D-stereoisomers of the naturally occurring amino acids, unnatural amino acids and chemically modified amino acids. Unnatural amino acids (that is, those that are not naturally found in proteins) are also known in the art, as set forth in, for example, Zhang et al. (2013, “Protein engineering with unnatural amino acids,” Curr. Opin. Struct. Biol. 23(4): 581-87); Xie et al. (2005, “Adding amino acids to the genetic repertoire,” Curr. Opin. Chem. Biol. 9(6): 548-54); and all references cited therein. Beta and gamma amino acids are known in the art and are also contemplated herein as unnatural amino acids.

[0032] As used herein, a chemically modified amino acid refers to an amino acid whose side chain has been chemically modified. For example, a side chain can be modified to comprise a signaling moiety, such as a fluorophore or a radiolabel. A side chain can also be modified to comprise a new functional group, such as a thiol, carboxylic acid, or amino group. Post-translationally modified amino acids are also included in the definition of chemically modified amino acids.

[0033] The term “identity” or “substantial identity,” as used in the context of a polynucleotide or polypeptide sequence described herein, refers to a sequence that has at least about 60% sequence identity to a reference sequence (e.g., at least about 60% identity to any one of SEQ ID NOs: l-6). Alternatively, percent identity can be any integer from 60% to 100%. Exemplary embodiments include at least about: 60%, 65%, 70%, 75%, 80%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, as compared to a reference sequence using the programs described herein; preferably BLAST using standard parameters, as described below. One of ordinary skill in the art will recognize that these values can be appropriately adjusted to determine corresponding identity of proteins encoded by twoAttorney Docket No.: 090723-1514847-MDA23-090PCT nucleotide sequences by taking into account codon degeneracy, amino acid similarity, reading frame positioning and the like.

[0034] For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0035] A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith & Waterman, 1981, Add. APL. Math. 2:482, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. (U.S.A.) 85:2444, by computerized implementations of these algorithms (e.g., BLAST), or by manual alignment and visual inspection.

[0036] Algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1990, J. Mol. Biol. 215: 403-10 and Altschul et al., 1977, Nucleic Acids Res. 25: 3389-402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (NCBI) web site. The algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al. (1977)). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotideAttorney Docket No.: 090723-1514847-MDA23-090PCT sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word size (W) of 28, an expectation (E) of 10, M=l, N=-2, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word size (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989, roc. Natl. Acad. Sci. USA 89: 10915).

[0037] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, 1993, Proc. Nat’l. Acad. Sci. USA 90:5873-87). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.01, more preferably less than about 10'5, and most preferably less than about IO'20.

[0038] The term “variant” or “variants” is intended to mean substantially similar sequences. Percent sequence identity or similarity between any two polynucleotides can be calculated using sequence alignment algorithms and / or programs and parameters described elsewhere in this disclosure. Where any given pair of polynucleotides of the disclosure is evaluated by comparison of the percent sequence identity, the percent sequence identity or percent sequence similarity between the two substantially similar sequences is at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more. Variants may differ by as few as 1-15 nucleic acid residues, as few as 1-10, as few as 6-10, as few as 5, as few as 4, as few as 3, as few as 2, or as few as 1 nucleic acid residue. Variant polynucleotides can comprise a 3’ or a 5’ end truncation, which can comprise, for example, at least a deletion of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleic acids or more from either the 3’ or a 5’ end of the polynucleotide used for comparison. Variant polynucleotides similarly can comprise a 3’ or a 5’ end addition of 1, 2,Attorney Docket No.: 090723-1514847-MDA23-090PCT3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nucleic acids or more on either the 3’ or a 5’ end of the polynucleotide used for comparison.

[0039] A “recombinant nucleic acid” or “recombinant polynucleotide” comprises a combination of two or more chemically linked nucleic acid segments which are not found directly joined in nature. By “directly joined” is intended the two nucleic acid segments are immediately adjacent and joined to one another by a chemical linkage. Alternatively, the chemically-linked nucleic acid segment of the recombinant polynucleotide can be formed by deletion of a sequence. The additional chemically linked nucleic acid segment or the sequence deleted to join the linked nucleic acid segments can be of any length, including for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or greater nucleotides. Various methods for making such recombinant polynucleotides include chemical synthesis or by the manipulation of isolated segments of polynucleotides by genetic engineering techniques. A recombinant polynucleotide can comprise a recombinant DNA sequence or a recombinant RNA sequence. A “fragment of a recombinant polynucleotide or nucleic acid” comprises at least one of a combination of two or more chemically linked nucleic acid segments which are not found directly joined in nature.

[0040] The term “promoter” refers to a region or sequence located upstream and / or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A promoter directs expression of an operably linked polynucleotide in a host cell. As used herein, a promoter includes necessary nucleic acid sequences near the start site of transcription. A promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription. In some embodiments, the promoters are selected based on the desired outcome. For example, different promoters in an expression construct or vector may be used to modulate the timing, location, and / or level of expression of the polynucleotides. In some embodiments, the promoter may confer inducible, constitutive, environmentally- or developmentally-regulated, or cell- or tissue- specific / selective expression.

[0041] The term “operably linked” refers to a functional linkage, for example, between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequenceAttorney Docket No.: 090723-1514847-MDA23-090PCT directs transcription of the second nucleic acid sequence. In certain embodiments, operably linked refers to nucleic acid sequences that are adjacent in a nucleic acid construct.

[0042] The term “vector” refers generally to a polynucleotide that may be used to deliver one or more isolated nucleic acids included in the vector to the interior of a cell, either in vitro or in vivo. The vector may be, for example, a plasmid or viral vector. Vectors may be capable of replication in a host cell or may require integration into the genome of the host cell.

[0043] A “restriction site” refers to a region of a nucleic acid (for example, a vector) that is a sequence of nucleotides that is recognized by and cleaved by at least one restriction enzyme. The term “restriction site” can be used interchangeably with the term “cloning site.” A “multiple cloning site” as the term is used herein is a region of a nucleic acid or a vector that contains more than one restriction site.

[0044] An “antibiotic resistance marker” or “antibiotic resistance gene” refers to a polynucleotide sequence that encodes a protein that, when expressed in a living cell, confers to that cell the ability to live and grow in the presence of a particular antibiotic.III. Nucleic Acid Constructs and Vectors for Expression of EGFR-III

[0045] In one aspect, the present disclosure provides nucleic acid constructs for the expression of a modified EGFR polypeptide (EGFR-III). The modified EGFR polypeptide as disclosed herein includes EGFR domain III and the EGFR transmembrane domain, but lacks EGFR domains I, II, and IV. The modified EGFR polypeptide thus reduces the distance between the cell membrane and an anti-EGFR antibody when bound to the modified EGFR polypeptide. In some embodiments, the modified EGFR includes a signal peptide on the N- terminus of EGFR domain III. In some embodiments, the modified EGFR polypeptide includes a linker positioned at the C-terminus of the EGFR domain III. In some embodiments, the polypeptide includes a linker positioned at the C-terminus of the transmembrane domain (TM). In some embodiments, the linker is part of the natural sequence of the transmembrane domain of the EGFR protein. In some embodiments, the modified EGFR polypeptide may bind specifically to an anti-EGFR antibody. In some embodiments, the anti-EGFR antibody may be cetuximab, matuzumab, necitumumab, panitumumab, zalutumumab, or nimotuzumab. In certain embodiments, the antibody is cetuximab.Attorney Docket No.: 090723-1514847-MDA23-090PCT

[0046] In some embodiments, the nucleic acid construct comprises a polynucleotide sequence that encodes a modified EGFR polypeptide (EGFR-III) having an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In some embodiments, the nucleic acid construct encodes a modified EGFR comprising SEQ ID NO: 1. In some embodiments, the nucleic acid construct encodes a modified EGFR polypeptide of SEQ ID NO: 1. In some embodiments, the nucleic acid construct has a nucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequence at positions 3687-4460 of SEQ ID NO: 6. In some embodiments, the nucleic acid construct comprises the polynucleotide sequence at positions 3687-4460 of SEQ ID NO: 6.

[0047] In some embodiments, the nucleic acid construct comprises a polynucleotide sequence encoding a modified EGFR polypeptide that includes an EGFR domain III comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2. In some embodiments, the EGFR domain III has an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 2 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity).

[0048] In some embodiments, the nucleic acid construct comprises a polynucleotide sequence encoding a modified EGFR polypeptide that includes an EGFR transmembrane domain comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 3. In some embodiments, the EGFR transmembrane domain comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 3 (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity).

[0049] In some embodiments, the modified EGFR polypeptide specifically binds an anti- EGFR antibody or antigen-binding fragment thereof. In some embodiments, the anti-EGFR antibody is selected from a group consisting of cetuximab, matuzumab, necitumumab, panitumumab, zalutumumab, and nimotuzumab. In some embodiments, the antibody or antigen binding fragment thereof can include a heavy (H) chain variable domain sequence (abbreviated herein as VH or VH), and a light (L) chain variable domain sequence (abbreviated herein as VL or VL). In some embodiments, an antibody molecule comprises or consists of a heavy chain and a light chain (sometimes referred to as a half antibody). In another example, an antibody molecule includes two heavy (H) chain variable domainAttorney Docket No.: 090723-1514847-MDA23-090PCT sequences and two light (L) chain variable domain sequence, thereby forming two antigen binding sites, such as Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single chain antibodies (scFv, for example), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to bind specifically to their respective antigen. Antibodies and antibody fragments can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgGl, IgG2, IgG3, and IgG4) of antibodies. The preparation of antibody molecules can be monoclonal. An antibody molecule can also be a human antibody, humanized antibody, CDR-grafted antibody, or an in vitro generated antibody. The antibody can have a heavy chain constant region chosen from, e.g., IgGl, IgG2, IgG3, or IgG4. The antibody can also have a light chain chosen from either kappa or lambda light chains.

[0050] Antigen binding fragments of an antibody molecule are well known in the art, and include, for example, (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a cam elid or camelized variable domain; (vii) a single chain Fv (scFv) (See, e.g., Bird et al., 1988 Science 242:423-26; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-83); and (viii) a single domain antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0051] Nucleic acid constructs encoding the modified EGFR polypeptide and variants thereof as described herein can be provided in expression cassettes for expression in an organism of interest. The cassettes may include 5’ and / or 3’ regulatory sequences including a heterologous promoter operably linked to the modified EGFR nucleic acid construct that allows for expression of the modified EGFR. Such an expression cassette is provided with a plurality of restriction sites and / or recombination sites for insertion of the gene of interest to be under the transcriptional regulation of the regulatory regions.

[0052] In some embodiments, the expression cassette may include, in the 5 ’-3’ direction of transcription, an enhancer region, a transcriptional and translational initiation region (i.e., aAttorney Docket No.: 090723-1514847-MDA23-090PCT promoter), an element for improving vector integration and transduction efficiency, a second transcriptional and translational initiation region (i.e., a promoter), a gene (encoding the modified EGFR polypeptide, and a transcriptional and translational termination region (i.e., termination region) functional in the organism of interest. The promoters of the invention are capable of directing or driving expression of a gene in a host cell. One or more of the promoter, the translational termination region, and the gene may be endogenous or heterologous to the host cell or to each other. In some embodiments, at least one of the promoter, the translational termination region, and the gene is heterologous to the polynucleotide encoding the modified EGFR polypeptide. In some embodiments, the polynucleotide encoding the modified EGFR polypeptide includes a polynucleotide encoding a signal domain.

[0053] Additional regulatory signals include, but are not limited to, transcriptional initiation start sites, operators, activators, enhancers, other regulatory elements, ribosomal binding sites, an initiation codon, termination signals, and the like. Such regulatory signals are discussed generally in Sambrook et al., 1992, Molecular Cloning: A Laboratory Manual, ed. Maniatis et al., (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); Advanced Bacterial Genetics, ed. Davis et al., 1980 (Cold Spring Harbor Laboratory Press), Cold Spring Harbor, N.Y., and the references cited therein. Other such regulatory protein binding sites may include a rev response element (RRE) or a constitutive transport element (CTE).

[0054] In preparing the expression cassette, the various DNA fragments may be manipulated, so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, substitutions, e.g., transitions and transversions, may be involved.

[0055] Any number of promoters can be used in the disclosed expression constructs and vectors for the expression of one or more genes. The promoter may be, for example, a eukaryotic promoter, a bacterial promoter, or a viral promoter. In some embodiments, the promoter is selected from a group consisting of an Elongation Growth Factor-1 alpha (EF-1 alpha) promoter, a CMV (cytomegalovirus) promoter, a SFFV (Spleen Focus-Forming Virus)Attorney Docket No.: 090723-1514847-MDA23-090PCT promoter, a ubiquitin promoter, and a CAG promoter. In some embodiments, the promoters can be selected based on the desired expression profile. In some embodiments, the promoter is a heterologous promoter. The polynucleotide sequence encoding the modified EGFR can be combined with any of constitutive, inducible, tissue- specific, and / or other promoters for expression of the gene in the organism of interest. In some embodiments, the promoter is a eukaryotic promoter or a viral promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a strong and / or substantially ubiquitous promoter. In some embodiments, the promoter is a eukaryotic promoter such as a mammalian promoter. In some embodiments, the mammalian promoter is Elongation Growth Factor-1 alpha (EF-1 alpha). In certain embodiments, the promoter comprises SEQ ID NO: 4. Other exemplary mammalian promoters include, but are not limited to the phosphoglycerokinase (PGK) promoter (human and / or mouse), U6 promoter, P-actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. In another example, the promoter is a viral promoter. In some embodiments, the promoter is the immediate early cytomegalovirus (CMV) promoter sequence, which is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto. Other exemplary viral promoters include, but are not limited to, the Rous sarcoma virus promoter, spleen focus-forming virus (SFFV) promoter, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, and Epstein-Barr virus immediate early promoter.

[0056] The term “enhancer” generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5’ or 3’ to the transcription unit. Furthermore, enhancers can be within an intron as well as within the coding sequence itself. Enhancers are usually between 10 and 300 bp in length, and they usually function to increase transcription from nearby promoters. Enhancers can also contain response elements that mediate the regulation of transcription. While many enhancer sequences are known from mammalian genes (e.g., globin, elastase, albumin, fetoprotein, insulin), typically an enhancer from a eukaryotic cell virus will be used for general expression. In some embodiments, examples of an enhancer include the woodchuck hepatitis virus post translational regulatory element (WPRE) and the cytomegalovirus early promoter enhancer.

[0057] Also provided in this disclosure is a vector comprising the modified EGFR nucleic acid construct. The vector preferably includes a signal domain operably linked to theAttorney Docket No.: 090723-1514847-MDA23-090PCT modified EGFR nucleic acid construct. The vector can include any one or more of the elements and embodiments discussed above with respect to the expression cassette provided. In some embodiments, the vector comprises necessary elements enabling gene expression such as suitable promoters, enhancers, splice acceptor sequences, internal ribosome entry site sequences (IRES), and transcription stop sites. Suitable elements for enabling gene expression are well known to those skilled in the art. In some embodiments, the vector includes a 2A peptide binding site sequence as a recruitment site. Suitable elements for enabling gene expression can include the natural endogenous elements associated with the gene or may be heterologous elements used in order to obtain a different level or tissue distribution of gene expression compared to the endogenous gene expression. Preferably, the vector comprises a promoter operably linked to the modified EGFR nucleic acid construct. The promoter may be a natural endogenous promoter of the gene or may be a heterologous promoter as discussed above.

[0058] The vector may be any vector capable of transferring DNA to a cell. In some embodiments, the vector is an integrating vector or an episomal vector. In some embodiments, the integrating vector can be a recombinant lentivirus or lentiviral vector. A recombinant lentiviral vector will include DNA of at least a portion of a lentivirus genome, which is capable of infecting the target cells. The terms “infection,” “transduction,” or the like are used to refer to the process by which a virus transfers genetic material to its host or target cell. In some embodiments, the lentivirus is also rendered replication-defective to remove the effect of viral replication of the target cells. In such cases, the replication defective viral genome can be packaged by a helper virus in accordance with conventional techniques. Generally, any lentivirus meeting the above criteria of infectiousness and capability of functional gene transfer can be employed in the practice of the disclosed compositions and methods. Lentiviral vectors are described, for example, in Milone & O’Doherty, 2018, Leukemia 32: 1529-1541.

[0059] Different types of lentiviral vector systems have been developed that seek to improve lentiviral vector system safety and efficacy. Second generation lentiviral systems contain a single packaging plasmid encoding the Gag, Pol, Rev, and Tat genes. Without an internal promotor, transgene expression is driven by the genomic 5' LTR, which is a weak promotor and requires the presence of Tat to activate expression. Third generation systems improve on the safety of the second generation system in two ways. First, the packaging system is split into two packaging plasmids: one encoding Rev and one encoding Gag andAttorney Docket No.: 090723-1514847-MDA23-090PCTPol. Second, Tat is eliminated from the third generation system; expression of the transgene from this promoter is no longer dependent on Tat transactivation. A third generation transfer plasmid can be packaged by either a second or a third generation packaging system. While the second and third generation systems address concerns related to unintentional generation of replication-competent viruses, the systems are still vulnerable to causing mutagenesis and off target effects in transduced cells.

[0060] The disclosed vectors or expression cassettes also may include a selectable marker gene for the selection of transformed cells or tissues. A marker gene can confer a selectable phenotype, e.g., antibiotic resistance, on a cell. The marker product is used to determine if the vector has been delivered to the cell and, once delivered, is being expressed. Examples of selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hygromycin, puromycin, and blasticidin. When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure. Examples of other markers include, for example, the E. coli lacZ gene, green fluorescent protein (GFP), and luciferase. In addition, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as GFP, glutathione S-transferase (GST), polyhistidine, c- myc, hemagglutinin, or FLAG™ tag (Kodak; New Haven, CT) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide including at either the carboxyl or amino terminus. Additional selectable markers are known to those skilled in the art, and any suitable marker can be used.

[0061] Different components may be used in the lentiviral vectors described herein to improve transduction efficiency or promote various cellular activity. For example, the vectors may include a central polypurine tract / central termination sequence (cPPT / CTS). The cPPT / CTS sequence creates a “DNA flap” that increases nuclear importation of the viral genome during target cell infection. The cPPT / CTS element improves vector integration and transduction efficiency. In some embodiments, the cPPT / CTS sequence is positioned upstream of a promoter. In some embodiments, the cPPT / CTS sequence comprises a polynucleotide sequence having at least 80% sequence identity to SEQ ID NO: 5 (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity). In certain embodiments, the cPPT / CTS sequence in the vector comprisesAttorney Docket No.: 090723-1514847-MDA23-090PCTSEQ ID NO: 5. In some embodiments, other elements for improving transduction efficiency, known by those skilled in the art, may be used.

[0062] In some embodiments, the lentiviral vector includes a viral promoter, a viral enhancer, an antibiotic resistance gene, a regulatory protein binding site, and a recruitment site. In some embodiments, the vector includes a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 6. In some embodiments, the vector includes a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO: 6 (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity).

[0063] The vector may further comprise a transgene. In some embodiments, the transgene encodes a chimeric antigen receptor or a T cell receptor. Chimeric antigen receptors (CARs, also known as chimeric T cell receptors) are designed to be expressed in host effector cells, e.g., T cells or NK cells, and to induce an immune response against a specific target antigen and cells expressing that antigen. Adoptive T cell immunotherapy, in which a patient’s own T lymphocytes are engineered to express CARs, has shown great promise in treating hematological malignancies. CARs can be engineered and used as described, for example, in Sadelain et al., 2013, Cancer Discov. 3:388-98. A CAR typically comprises an extracellular target-binding module, a transmembrane (TM) domain, and an intracellular signaling domain (ICD). The CAR domains can be joined via flexible hinge and / or spacer regions. The extracellular target-binding module generally comprises an antibody or antigen binding fragment thereof. In some embodiments, multiple binding specificities can be included in the extracellular target-binding module. For example, multiple antibodies or antigen binding fragments thereof that target different antigens can be included to produce bi-specific, tri- specific, or quad-specific CARs.

[0064] For some embodiments, other vectors may be useful for the expression or transfer of nucleic acid sequences encoding the modified EGFR polypeptides disclosed herein. In certain embodiments, other suitable vectors may include, for example, adenovirus, adeno-associated virus, SV40 virus, vaccinia virus, HSV and pox virus vectors. In some embodiments, the vector is an adenovirus transfer vector. Adenovirus vectors are well-known to those skilled in the art and have been used to deliver genes to numerous cell types, including airway epithelium, skeletal muscle, liver, brain, and skin (see, e.g., Hitt et al., 1997, Advances in Pharmacology 40: 137-206; Anderson, 1998, Nature 392 (6679 Suppl):25-30). In someAttorney Docket No.: 090723-1514847-MDA23-090PCT embodiments, the vector is an adeno-associated (AAV) vector. AAV vectors are well-known to those skilled in the art and have been used to stably transduce human T-lymphocytes, fibroblasts, nasal polyp, skeletal muscle, brain, erythroid, and hematopoietic stem cells for gene therapy applications (Philip et al., 1994, Mol. Cell. Biol. 14:2411-2418; Russell et al., 1994, PNAS USA 91(19): 8915-8919: Flotte et al., 1993, PNAS USA 90(22): 10613-10617; Walsh et al., 1992, PNAS USA 89(15):7257-7261; Miller et al., 1994, PNAS USA 91(21): 10183-10187; Emerson, 1996, Blood 87:3082-3088; Naso et al., 2017, BioDrugs 31(4):317-334 (41)). Episomal vectors can include transient non-replicating episomal vectors and self-replicating episomal vectors with functions derived from viral origins of replication such as those from EBV, human papovavirus (BK) and BPV-1. In some embodiments, the vector may be a replicating episomal vector. Such vectors have a larger size capacity than many viral vectors and have less risk of insertional mutagenesis. Such integrating and episomal vectors are well-known to those skilled in the art. Certain suitable episomal vectors are described in Ehrhardt et al., 2008, Current Gene Therapy, 8(3): 147-161. In some embodiments, the vector is a mammalian artificial chromosome. The use of mammalian artificial chromosomes is discussed by Kazuki & Oshimura, 2011, Mol. Therapy 19(9): 1591- 1601.

[0065] In some embodiments, the vector is a plasmid. For example, the plasmid can be a non-replicating, non-integrating plasmid. The term “plasmid” as used herein refers to any nucleic acid encoding a gene and includes linear or circular nucleic acids and double or single stranded nucleic acids. The nucleic acid can be DNA or RNA and may comprise modified nucleotides or ribonucleotides, and may be chemically modified by Such means as methylation or the inclusion of protecting groups or cap- or tail structures. A non-replicating, non-integrating plasmid is a nucleic acid which, when transfected into a host cell, does not replicate and does not specifically integrate into the host cell's genome (i.e. does not integrate at high frequencies and does not integrate at specific sites). In some embodiments, the plasmid is a naked nucleic acid. As used herein, the term “naked” refers to a nucleic acid molecule that is free of direct physical associations with proteins, lipids, carbohydrates or proteoglycans, whether covalently or through hydrogen bonding. The term does not refer to the presence or absence of modified nucleotides or ribonucleotides, or chemical modification of the, all or a portion of, a nucleic acid molecule by such means as methylation or the inclusion of protecting groups or 5' cap and / or poly A elements.Attorney Docket No.: 090723-1514847-MDA23-090PCTIV. Engineered Cells Expressing a Modified EGER Polypeptide

[0066] Also provided herein are cells, tissues, and organisms comprising the nucleic acid constructs or the vectors encoding the modified EGFR polypeptide (EGFR-III) as described above. In some embodiments, host cells are provided in which a nucleic acid construct encoding the modified EGFR has been introduced. Nucleic acid constructs comprising polynucleotide sequences encoding the modified EGFR can be used to transform cells of organisms of interest. Methods for transformation involve introducing a nucleic acid construct into a host cell. The EGFR nucleic acid construct (e.g., alone or as part of an expression cassette or vector) is introduced into a host cell in such a manner that the construct gains access to the interior of the host cell. The disclosed methods do not require a particular method for introducing a nucleic acid construct to a cell, only that the nucleic acid construct gains access to the interior of the host cell or at least one cell of a host organism. Methods for introducing nucleic acid constructs into cells are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods.

[0067] The host cell may be any cell, such as bacterial cells or mammalian cells. For example, in some embodiments, the cells are eukaryotic cells. In some embodiments, the cells are transduced with a lentiviral vector that includes a nucleic acid construct that encodes a modified EGFR (e.g., EGFR-III). In some embodiments, the nucleic acid construct is integrated into a genome of the cell. In some embodiments, the modified cell expresses the modified EGFR polypeptide. In some embodiments, the cells are transduced with a lentiviral vector that includes both a polynucleotide sequence that encodes a transgene (e.g, a CAR) and a nucleic acid construct that encodes a modified EGFR. In some embodiments, the nucleic acid construct encoding the modified EGFR is integrated into a genome of the cell. In some embodiments, the modified cell expresses the polypeptide encoded by the transgene (e.g., the CAR) and the EGFR-III polypeptide.

[0068] In some embodiments, the engineered cells are immune cells (e.g., T cells) expressing the modified EGFR polypeptides described herein. In some embodiments, the immune cell expresses the EGFR on its surface. In certain embodiments, the cells further express a CAR or TCR on the cell surface. In some embodiments, the immune cell comprises a nucleic acid encoding the modified EGFR and / or the CAR or TCR, wherein the polypeptide(s) are expressed from the nucleic acid and localized to the immune cell surface. In some embodiments, the immune cell is a B lymphocyte, T lymphocyte, thymocyte,Attorney Docket No.: 090723-1514847-MDA23-090PCT dendritic cell, natural killer (NK) cell, monocyte, macrophage, granulocyte, eosinophil, basophil, neutrophil, myelomonocytic cell, megakaryocyte, peripheral blood mononuclear cell, myeloid progenitor cell, or a hematopoietic stem cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a cytotoxic T cell, a helper T cell, a natural killer T cell, a suppressor T cell, a CD8+T cell, a CD4+T cell, a CD8+ / CD4+T cell, y5 T cell, or a T-regulatory (T-reg) cell.

[0069] In some embodiments, immune cells are obtained from a subject and transduced with a lentiviral vector as described herein. Where the immune cells are used to treat (e.g., according to the treatment methods described herein below) the same subject from which they are obtained, they are referred to as autologous cells. Where the immune cells are obtained from a different subject, they are referred to as heterologous cells. Immune cells can be isolated from peripheral blood using techniques well known in the art, include Ficoll density gradient centrifugation followed by negative selection to remove undesired cells. In some embodiments, heterologous immune cells useful for the methods provided herein comprise allogeneic T cells, as described in, e.g., Bedoya et al.. 2021, Front. Immunol. 12:640082.V. Pharmaceutical Compositions and Formulations

[0070] In one aspect, pharmaceutical compositions are provided that include a nucleic acid construct or vector (e.g., lentiviral vector) that includes a nucleic acid sequence that encodes the modified EGFR polypeptide, or modified cells that express a CAR or TCR and the modified EGFR polypeptide, as described herein in combination with a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier (excipient) is a material that is not biologically or otherwise undesirable, i.e., the material is administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with the other components of the pharmaceutical composition in which it is contained. The carrier can be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject.

[0071] The term “pharmaceutically acceptable carrier” as used herein is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington’s Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.Attorney Docket No.: 090723-1514847-MDA23-090PCTExemplary carriers or diluents include, but are not limited to, water, saline, Ringer’s solutions, dextrose solution, and 5% human serum albumin. The use of such media and agents is well known in the art. Except insofar as any conventional media or agent is incompatible with the agents provided herein, use thereof in the composition is contemplated.

[0072] In certain embodiments, acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed. In certain embodiments, the formulation material(s) are for subcutaneous and / or intravenous administration. In certain embodiments, the formulation comprises an appropriate amount of a pharmaceutically- acceptable salt to render the formulation isotonic. In certain embodiments, the pharmaceutical composition can contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In certain embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen- sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta- cyclodextrin); fillers; monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate 80, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants. In certain embodiments, the optimal pharmaceutical composition is determined by one skilled in the art depending upon, for example, the intended route of administration, delivery format and desired dosage. See,Attorney Docket No.: 090723-1514847-MDA23-090PCT for example, Remington: The Science and Practice of Pharmacy, 22ndEdition, Lloyd V. Allen, Jr., ed., The Pharmaceutical Press (2014). In certain embodiments, such compositions may influence the physical state, stability, rate of in vivo release and / or rate of in vivo clearance of the polypeptide that comprises a signal domain, an EGFR domain III, and an EGFR transmembrane domain. In some embodiments, the nucleic acid sequence encoding the signal domain is operably linked to a nucleic acid sequence encoding the EGFR domain III.VI. Kits and Packaging

[0073] The vectors (e.g., lentiviral vectors) comprising the nucleic acid construct encoding the modified EGFR polypeptide described herein may be used for the preparation of a kit (e.g., a kit for producing a vector for the preparation of CAR-T cells, TCR-T cells, CAR-NK cells, or CAR-NKT cells for the treatment of a patient). For example, the kit may include a lentiviral vector as disclosed herein, as well as one or more buffers, restriction enzymes, and the like for cloning a transgene (e.g., transgene encoding a CAR or TCR) into the vector, as well as instructions. In some embodiments, kits are provided for carrying out any of the methods described herein. In some embodiments, the kits may comprise a carrier container being compartmentalized to receive in close confinement one or more containers such as vials, tubes, and the like, each of the containers comprising one of the separate elements to be used in the method.VII. Methods of Use for the Treatment of a Disease and Targeted Depletion of Cells

[0074] Provided herein are methods to treat, inhibit, a disease or disorder (e.g., cancer). The methods comprise administering to a subject a pharmaceutically effective amount of a nucleic acid construct, vector, or cell comprising a nucleic acid encoding a modified EGFR as described herein. In some embodiments, a cell expressing the modified EGFR is administered, wherein a nucleic acid encoding the modified EGFR is introduced into the cell by transduction with a viral vector comprising a modified EGFR as described herein. In some embodiments, the viral vector is a lentiviral vector comprising a transgene and a modified EGFR as described herein. In some embodiments, the transgene is a CAR or a TCR. In some embodiments, the methods further include the targeted depletion of cells expressing the modified EGFR.

[0075] As used throughout, subject can be a vertebrate, more specifically a mammal (e.g., a human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig), birds,Attorney Docket No.: 090723-1514847-MDA23-090PCT reptiles, amphibians, fish, and any other animal. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. As used herein, patient or subject may be used interchangeably, and the term patient or subject includes human and veterinary subjects. The EGFR-III nucleic acid constructs described herein are useful for treating cancer in humans, including, without limitation, pediatric and geriatric populations, and in animals, e.g., veterinary applications. In one embodiment, the subject is a human.

[0076] As used herein the terms “cancer” and “tumor” are used to indicate malignant tissue. The term, “cancer” is also used to refer to the disease associated with the presence of malignant tumor cells in an individual, and the term “tumor” is used to refer to a plurality of cancer cells that are physically associated with each other. Cancer cells are malignant cells that give rise to cancer, and tumor cells are malignant cells that can form a tumor and thereby give rise to cancer.

[0077] As used herein, an “effective amount” means the amount of an agent that is effective for producing a desired effect in a subject. The actual dose that comprises the effective amount may depend upon the route of administration, the size and health of the subject, the disorder being treated (e.g., cancer), and the like.

[0078] Methods of treatment as described herein can reduce one or more symptoms of a cancer (e.g., a cancerous or non-cancerous malignancy) by enhanced antibody dependent cell cytotoxicity (ADCC) as a result of administration of compositions as described herein comprising the nucleic acid construct or CAR constructs or TCR constructs described herein. As used herein the terms “cancer” and “tumor” are used to indicate malignant tissue. The term “cancer” is also used to refer to the disease associated with the presence of malignant tumor cells in an individual, and the term “tumor” is used herein to refer to a plurality of cancer cells that are physically associated with each other. Cancer cells are malignant cells that give rise to cancer, and tumor cells are malignant cells that can form a tumor and thereby give rise to cancer. The term “cancer,” as used herein, may be used to describe a solid tumor, metastatic cancer, or non-metastatic cancer. The term also encompasses a circulating tumor cell. In certain embodiments, the cancer may originate in the blood, bladder, bone, bone marrow, brain, breast, colon, esophagus, duodenum, small intestine, large intestine, gum, head, kidney, liver, lung, nasopharynx, neck, ovary, pancreas, prostate, rectum, skin, stomach, testis, tongue, or uterus.Attorney Docket No.: 090723-1514847-MDA23-090PCT

[0079] “ Treat,” “treatment,” and the like are used herein to generally mean obtaining a desired pharmacologic and / or physiologic effect. “Treating” or “treatment” may refer to any indicia of success in the treatment or amelioration of cancer. Treating or treatment of any disease or disorder refers to ameliorating a disease or disorder that exists in a subject or any one or more symptoms thereof. The term ameliorating refers to any therapeutically beneficial result in the treatment of a disease state, e.g., a cancer, lessening in the severity or progression, promoting remission or durations of remission, or curing thereof. Thus, treating or treatment includes ameliorating at least one physical parameter or symptom. Treating or treatment includes modulating the disease or disorder, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. “Treating” or “treatment” includes the administration of an agent to impede growth of a cancer, to do one or more of the following: cause a cancer to shrink by weight or volume (i.e., shrink from a first weight or volume to a second weight or volume, wherein the second weight or volume is less than the first), delay or prevent metastasis, extend the expected survival time of the subject, or extend the expected time to progression of the tumor, or the like. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of an established disease or condition or symptom of the disease or condition. For example, a method for treating a cancer in a subject by administering a pharmaceutical composition as described in this disclosure is considered to be a treatment if there is a 10% reduction in one or more symptoms of the cancer in a subject as compared to a control. Thus, the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. It is understood that treatment does not necessarily refer to a cure or complete ablation of the disease, condition, or symptoms of the disease or condition.

[0080] Also provided are methods of treatment to reduce one or more symptoms of an autoimmune condition or disorder, by administering a modified EGFR composition (e.g., an engineered cell expressing a modified EGFR) to a patient. In some embodiments, the inflammatory condition or disorder can be one selected from the group consisting of systemic lupus erythematosus (SLE), Sjogren’s syndrome, dermatitis, Type 1 diabetes, Type 2 diabetes, thyroiditis, Addison disease, pernicious anemia, autoimmune hepatitis, multipleAttorney Docket No.: 090723-1514847-MDA23-090PCT sclerosis, autoimmune encephalitis, rheumatoid arthritis, myasthenia gravis, neuritis, primary biliary cholangitis, Goodpasture’s disease, primary membranous nephropathy, interstitial cystitis, ovarian insufficiency, autoimmune orchitis, chronic obstructive pulmonary disease (COPD), pneumonitis, high blood pressure, heart disease, myositis, myocarditis, or inflammatory arteritis (Takayasu arteritis, giant cell arteritis). Other such autoimmune diseases are known in the art (see, e.g., Ludwig et al. (2017, Frontiers in Immunol 8:Article 603; Hofmann et al.. 2018, Frontiers in Immunol 9:Article 835). Other diseases may be treated by methods of administering a modified EGFR composition (e.g., an engineered cell expressing a modified EGFR) as described herein. For example, the methods may be used to reduce one or more symptoms of HIV / AIDS, other infectious diseases, or heart disease such as Hepatitis B, and cyto-megalovirus.

[0081] The term “administer,” as used herein, refers to a method of delivering agents, compounds, or compositions to the desired site of biological action. The pharmaceutical compositions (e.g., as described above) are prepared for administration in a number of ways, including but not limited to injection, ingestion, transfusion, implantation, or transplantation, depending on whether local or systemic treatment is desired, and on the area to be treated. The preparation of such pharmaceutically acceptable compositions is within the ability of one skilled in the art. The compositions are administered via any of several routes of administration, including topical, oral, parenteral, intravenous, intra-articular, intraperitoneal, intracerebral (intra-parenchymal), intracerebral, intraventricular, intramuscular, subcutaneous, intraarterial, intraportal, intracavity, intralesional, transdermal, intradermal, intrahepatical, intrathecal, intracranial, rectal, transmucosal, intestinal, intra-ocular or ocular, otic, nasal, inhalation, or intrabronchial delivery, or any other method known in the art. In some embodiments, the engineered lentivirus is administered intravenously, or through local injection. In certain embodiments, administered is by bolus injection, continuously by infusion, by sustained release system, or by implantation device. In certain embodiments, individual elements of a combination therapy (as discussed below) may be administered by different routes. In certain embodiments, the composition can be administered locally, e.g., during surgery or topically. Optionally local administration is via implantation of a membrane, sponge, or another appropriate material onto which the desired molecule has been absorbed or encapsulated. In certain embodiments, where an implantation device is used, the device can be implanted into any suitable tissue or organ, and delivery of the desired molecule can be via diffusion, timed-release bolus, or continuous administration.Attorney Docket No.: 090723-1514847-MDA23-090PCT

[0082] As used herein, the term “therapeutically effective amount” or effective amount refers to an amount of a therapeutic composition that, when administered to a subject, is effective to treat a disease or disorder such that the symptoms of the disease or disorder are ameliorated, or the likelihood of the disease or disorder developing or progressing is decreased. A therapeutically effective amount is not, however, a dosage so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like. A suitable dose of a therapeutic composition as described herein, which dose is capable of treating a subject, can depend on a variety of factors including the particular therapeutic composition used and whether it is used concomitantly with other therapeutic agents. For example, a different dose of a genetically modified cell or viral vector comprising a transgene and modified EGFR nucleic acid may be required to treat a subject. Other factors affecting the dose administered to the subject include, e.g., the type or extent of the disease, such as cancer. For example, a subject that has had a previous cancer (e.g., a subject with relapsed or recurrent cancer) may require administration of a different dosage of a genetically modified cell or viral vector comprising the transgene and modified EGFR nucleic acid than a subject who has not previously had cancer. Generally, a therapeutically effective amount may vary with the subject’s age, condition, and sex, as well as the extent of the disease in the subject and can be determined by one of skill in the art. Other factors can include, e.g., other medical disorders concurrently or previously affecting the subject, the age and general health of the subject, the genetic disposition of the subject, diet, time of administration, the route of administration, and the size (body weight, body surface, or organ size), the rate of excretion, drug combination, and any other additional therapeutics that are administered to the subject. It should also be understood that a specific dosage and treatment regimen for any particular subject also depends upon the judgment of the treating medical practitioner (e.g., doctor or nurse) as described above. A therapeutically effective amount is also one in which any toxic or detrimental effects of the composition are outweighed by the therapeutically beneficial effects. The dosage of the therapeutically effective amount may be adjusted by the individual physician or veterinarian in the event of any complication. In some embodiments, a therapeutically effective amount may vary from about 0.01 mg / kg to about 50 mg / kg, preferably from about 0.1 mg / kg to about 20 mg / kg, most preferably from about 0.2 mg / kg to about 2 mg / kg, in one or more dose administrations daily, for one or several days. In some embodiments, the doses can be about 1, about 0.5, about 0.1, about 0.05, or about 0.01 mg / kg, or any intervening dose between about 0.01 mg / kg and 1 mg / kg. In some embodiments, the genetically modified cell or vector comprising the transgene and modifiedAttorney Docket No.: 090723-1514847-MDA23-090PCTEGFR is administered for 2 to 5 or more consecutive days. In some embodiments, the genetically modified cell or vector comprising the transgene and modified EGFR nucleic acid is administered to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times.

[0083] In some embodiments, the nucleic acid constructs encoding the modified EGFR may be included in a vector described herein and may be administered to a subject who has been diagnosed with cancer or an autoimmune disease. In certain embodiments, the vectors described herein can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express the modified EGFR. In certain embodiments, such cells can be animal or human cells, and can be autologous, heterologous, or xenogeneic. In certain embodiments, the cells can be immortalized.

[0084] In yet another aspect, provided herein, is a method of killing genetically modified cells in a subject. The method includes administering to the subject an effective amount of an anti-EGFR antibody. In some embodiments, the therapeutically effective amount may vary from about 0.01 mg / kg to about 50 mg / kg, preferably from about 0.1 mg / kg to about 20 mg / kg, most preferably from about 0.2 mg / kg to about 2 mg / kg, in one or more dose administrations daily, for one or several days. In some embodiments, the doses can be about 1, about 0.5, about 0.1, about 0.05, or about 0.01 mg / kg, or any intervening dose between about 0.01 mg / kg and 1 mg / kg. In some embodiments, the anti-EGFR antibody is administered for 2 to 5 or more consecutive days. In some embodiments, the anti-EGFR antibody is administered to a subject at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times. In some embodiments, the anti-EGFR antibody may bind to the genetically modified cells that are coexpressing a CAR or TCR and the modified EGFR described herein. When the modified cells are exposed to an anti-EGFR antibody, the modified EGFR expressed on the modified cells may mediate the destruction of the cell by binding the antibody which then targets the destruction of the cell. In some embodiments, the modified EGFR as described herein may be incorporated into cancer treatments to improve the safety of cell-based therapeutics.EXAMPLES

[0085] The following examples are offered to illustrate, but not to limit, the present disclosure.Attorney Docket No.: 090723-1514847-MDA23-090PCTEXAMPLE 1. Design of the EGFR-III suicide gene.

[0086] The therapeutic use of engineered cells in cell therapy (e.g., CAR-T cell therapy, TCR-T therapy, CAR-NK cell therapy, and CAR-NKT cell therapy) can result in toxicity to the patient. Therefore, an efficient means of depleting those cell populations would be beneficial by reducing the related toxicity experienced by a patient. A modified EGFR was designed to be expressed on the therapeutic cell surface, bind a specific antibody, and target the cell population for destruction. The modified human EGFR polypeptide (EGFR-III or huEGFR-III) lacks the extracellular N-terminal ligand binding domains and intracellular receptor tyrosine kinase activity, but it retains the native amino acid sequence over certain portions of the polypeptide, retains type 1 transmembrane cell surface localization, and is a conformationally intact binding epitope for pharmaceutical-grade anti-EGFR antibody binding. The huEGFR-III polypeptide includes domain III and the transmembrane domain, but it does not include an intact domain IV, resulting in a reduced distance between the EGFR antibody to the cell membrane when bound to the huEGFR-III on the cell surface.

[0087] U.S. Patent Application Publication No. 2023 / 0174,621 describes another modified EGFR polypeptide (EGFRt) which lacks domains I and II of EGFR and still binds to the EGFR antibody cetuximab. However, when cetuximab binds to EGFRt on the cell surface, the distance from cetuximab to the cell surface was maintained at about 80 angstroms (FIG. 2A). Surprisingly and unexpectedly, a modified EGFR that lacks domains I, II, and IV (HuEGFR-III as described herein) was still capable of binding cetuximab, and the distance from cetuximab to the cell surface decreased to about 35 angstroms when bound to huEGFR- III (FIG. 2A). Further analysis of the differences in the wild type and modified EGFR polypeptides are shown in FIG. 2B, which identifies the domains present in each of the three EGFR polypeptides in FIG. 2A (WT EGFR, huEGFRt, and the huEGFR-III as described herein). Each of the EGFR polypeptides include the signal peptide (SP) at the N-terminus of the polypeptides, one or more of EGFR Domains I-IV, and a transmembrane domain (TM) at the C-terminus. The disclosed HuEGFR-III includes the amino acids between the arginine at position 309 (R309) and the lysine at position 513 (K513) of EGFR. Optionally, the polypeptides also include a linker (not shown).Attorney Docket No.: 090723-1514847-MDA23-090PCTEXAMPLE 2. In vitro ADCC killing of T cells expressing EGFR-III mediated by cetuximab.

[0088] The closer distance of cetuximab to the cell membrane when bound to EGFR-III may improve cetuximab’s depleting efficiency of cells expressing EGFR-III. Therefore, to assess the effect of EGFR-III on antibody dependent cell cytotoxicity (ADCC), lentiviral vectors as described herein that included a gene encoding EGFR-III (FIG. 1A-1B) were used. The lentiviral vectors also included a transgene encoding Velo8-bbz CAR (CAR including stimulatory 4-1BB and CD3(^ domains) to assess the ability of the vector to introduce both EGFR-III and a CAR to cells. Healthy donor T cells were transduced with the lentiviruses encoding Velo8-bbz CAR and EGFR-III. Three days after transduction, the cells were stained with an anti-human IgG secondary antibody labeled with phycoerythrin (a-hlgG-PE) (left plot) or cetuximab and / or a-hlgG-PE secondary antibody. Fluorescence-activated cell sorting (FACS) was performed on the stained cells (FIG. 3A). These data show that EGFR-III was expressed and could be readily detected by cetuximab.

[0089] The cells were analyzed for antibody dependent cell cytotoxicity (ADCC). The left panel of FIG. 3B shows the ADCC mechanism of cetuximab binding to a target cell and mediating NK killing of the target cell. Transduced T cells, expressing the disclosed EGFR- III, were labeled with carboxyfluorescein succinimidyl ester (CFSE) and then co-cultured with purified primary NK cells from a healthy donor with the indicted effector to target (E:T) ratio in the presence of cetuximab (3 g / ml or 1 pg / ml) or normal human IgG (3 pg / ml). Live T cells were detected and quantified by FACS as shown in the right panel of FIG. 3B. The number for T cells cultured alone was arbitrarily defined as 100%. NK cell killing of EGFR- III transduced T cells mediated by cetuximab could be clearly detected (left two bars shown for each E:T ratio).

[0090] Primary T cells were transduced with lentiviruses encoding Velo8-bbz CAR and EGFR-III or EGFRt. After three days, cells were stained with cetuximab (AF747), and transduced cells were sorted, achieving approximately 95% purity. The sorted cells exhibited very similar levels of expression of surface EGFR-III and EGFRt (FIG. 3C). Sorted T cells were labeled with CFSE, and then co-cultured with purified primary NK cells from a healthy donor with indicated E:T ratio of 2:1, with presence of different concentrations of cetuximab or normal human IgG for 4 hours (FIG. 3D). Live T cells were then detected and quantified by FACS to calculate killing. While normal IgG did not induce T cell killing by NK cells (circles and triangles), cetuximab mediated NK killing on both T cells expressing EGFR-IIIAttorney Docket No.: 090723-1514847-MDA23-090PCT(squares) and on T cells expressing EGFRt (inverted triangles). However, the killing was greater on T cells expressing EGFR-III, indicating a more efficient depletion of T cells by EGFR-III as compared to EGFRt.EXAMPLE 3. In vivo depletion of CAR-T cells expressing EGFR-III by cetuximab.

[0091] The in vivo effect of EGFR-III on T cell depletion was examined in a mouse model. NSG mice were inoculated with patient derived acute myeloid leukemia cells (SMIP92 (PDX of AML)), and then treated with parent T cells or CCNE CAR-T cells expressing EGFR-III (FIG. 4A). Blood was collected on the indicated days to detect tumor cells (hCD45+hCD3-) by FACS. Treatment with the CCNE CAR-T cells clearly inhibited tumor growth as opposed to parental T cells. To study if cetuximab could deplete CCNE CAR-T cells in vivo, 4 mice from the CAR-T group were randomly divided into two groups of two, treated with either normal IgGl or cetuximab at 20 mg / kg (IP) on Day 21 and Day 24. Blood was collected 3 days and 7 days after the second antibody treatment, and totally T cells were quantified (FIG. 4B) The live T cell percentages increased for both IgGl treated mice (circles), while cetuximab treatment inhibited T cell growth (squares). Peripheral blood mononuclear cells (PBMCs) from the blood were stained with a-hCD3 and A2 / CCNE tetramer to quantify total T cells and CAR-T cells. FIG. 4C shows CAR-T cell percentage increased for both IgG treated mice, yet cetuximab treatment dramatically depleted CAR-T cells in the total T population. FIG. 4D shows raw FACS data for CAR-T cell plotting. Cetuximab treated mice blood had significantly lower CAR-T cells in total T cells (bottom panel) than the IgG treated group (top panel).SEQUENCESAttorney Docket No.: 090723-1514847-MDA23-090PCTAttorney Docket No.: 090723-1514847-MDA23-090PCTAttorney Docket No.: 090723-1514847-MDA23-090PCTAttorney Docket No.: 090723-1514847-MDA23-090PCT

[0092] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

Attorney Docket No.: 090723-1514847-MDA23-090PCTWHAT IS CLAIMED:

1. A nucleic acid construct, comprising a polynucleotide sequence that encodes a modified epidermal growth factor receptor (EGFR), wherein the modified EGFR comprises an EGFR domain III and an EGFR transmembrane domain, and wherein the modified EGFR comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 1.

2. The nucleic acid construct of claim 1, wherein the nucleic acid comprises a polynucleotide sequence that encodes a signal domain.

3. The nucleic acid construct of claim 1 or 2, wherein the modified EGFR polypeptide comprises SEQ ID NO: 1.

4. The nucleic acid construct of any one of claims of claim 1-3, wherein the EGFR Domain III comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 2.

5. The nucleic acid construct of any one of claims 1-4, wherein the EGFR Domain III comprises SEQ ID NO: 2.

6. The nucleic acid construct of any one of claims 1-5, wherein the EGFR transmembrane (TM) domain comprises an amino acid sequence that has at least 95% sequence identity to SEQ ID NO: 3.

7. The nucleic acid construct of any one of claims 1-6, wherein the EGFR TM Domain comprises SEQ ID NO: 3.

8. The nucleic acid construct of any one of claims 1-7, wherein the modified EGFR polypeptide specifically binds an anti-EGFR antibody.

9. The nucleic acid construct of claim 8, wherein the anti-EGFR antibody is selected from a group consisting of cetuximab, matuzumab, necitumumab, panitumumab, zalutumumab, and nimotuzumab.

10. The nucleic acid construct of any one of claims 1-9, further comprising a promoter operably linked to the nucleic acid construct.Attorney Docket No.: 090723-1514847-MDA23-090PCT11. The nucleic acid construct of claim 10, wherein the promoter is a eukaryotic promoter.

12. The nucleic acid construct of claim 10 or 11, wherein the promoter is a constitutive promoter.

13. The nucleic acid construct of any one of claims 10-12, wherein the promoter comprises an elongation factor la (EFla) promoter.

14. The nucleic acid construct of any one of claims 10-13, wherein the promoter comprises SEQ ID NO: 4.

15. The nucleic acid construct of any one of claims 1-14 , further comprising: a central polypurine tract / central termination sequence (CPPT / CTS) positioned upstream of the promoter, wherein the CPPT / CTS comprises SEQ ID NO: 5.

16. A vector comprising the nucleic acid construct of any one of claims 1-15 .

17. The vector of claim 16, wherein the nucleic acid sequence of the vector has at least 80% sequence identity to SEQ ID NO: 6.

18. The vector of claim 16 or 17, wherein the nucleic acid sequence of the vector comprises SEQ ID NO: 6.

19. The vector of any one of claims 16-18, comprising: a viral promoter; a viral enhancer; a nucleic acid sequence encoding an antibiotic resistance polypeptide; a regulatory protein binding site; and a recruitment site.

20. The vector of claim 19, wherein the antibiotic resistance polypeptide confers resistance to ampicillin or kanamycin.

21. The vector of claim 19 or 20, wherein the viral enhancer comprises a woodchuck hepatitis virus post translational regulatory element (WPRE) or cytomegalovirus (CMV) enhancer.Attorney Docket No.: 090723-1514847-MDA23-090PCT22. The vector of any one of claims 19-21, wherein the recruitment site is an internal ribosome entry site (IRES) or 2A peptide binding site.

23. The vector of any one of claims 19-22, wherein the viral promoter is a CMV promoter or a respiratory syncytial virus (RSV) promoter.

24. The vector of any one of claims 19-23, wherein the regulatory protein binding site is a rev response element (RRE) or a constitutive transport element (CTE).

25. The vector of any one of claims 16-24, wherein the vector is a lentiviral vector, an adenoviral vector, a retroviral vector, or an adeno-associated viral vector.

26. The vector of any one of claims 16-25, wherein the vector is a lentiviral vector.

27. The vector of any one of claims 16-26, wherein the vector further comprises a transgene.

28. The vector of claim 27, wherein the transgene encodes a chimeric antigen receptor or a T cell receptor.

29. A genetically modified cell comprising the nucleic acid construct of any one of claims 1-15 or the vector of any one of claims 16-28.

30. The genetically modified cell of claim 29, wherein the genetically modified cell is a eukaryotic cell.

31. The genetically modified cell of claim 29 or 30, wherein the genetically modified cell is a human T cell.

32. The genetically modified cell of any one of claims 29 or 30, wherein the genetically modified cell is a human NK cell or a human NKT cell.

33. The genetically modified cell of any one of claims 29-32, wherein the nucleic acid construct is integrated into a genome of the cell.

34. The genetically modified cell of any one of claims 29-33, wherein the genetically modified cell expresses the polypeptide encoded by the transgene and the modified EGFR polypeptide encoded by the nucleic acid construct on the cell surface.Attorney Docket No.: 090723-1514847-MDA23-090PCT35. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the vector of any one of claims 16-28 or the genetically modified cell of any one of claims 29-34.

36. A method of treating a cancer or an autoimmune disease or condition in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 35.

37. The method of claim 36, wherein the pharmaceutical composition comprises the genetically modified cell of any one of claims 29-34, and the genetically modified cells are allogeneic cells.

38. The method of claim 36, wherein the pharmaceutical composition comprises the genetically modified cell of any one of claims 29-34, and the genetically modified cells are autologous cells.

39. The method of claim 36, wherein the pharmaceutical composition comprises the genetically modified cell of any one of claims 29-34, and the genetically modified cells are HLA matched to the subject.

40. The method of any one of claims 36-39, wherein the subject has a cancer.

41. The method of any one of claims 36-39, wherein the subject has an autoimmune disease or condition.

42. A method for killing the genetically modified cell of any one of claims 29-34 in a subject, comprising administering to the subject an effective amount an anti-EGFR antibody.

43. The method of claim 42, wherein the anti-EGFR antibody is selected from a group consisting of cetuximab, matuzumab, necitumumab, panitumumab, zalutumumab, and nimotuzumab.

44. The method of claim 42 or 43, wherein the anti-EGFR antibody that is administered to the subject is cetuximab.

Images