Synthetic her2 receptors to enrich, track, and / or eliminate modified cells

Abstract

The present disclosure is directed to synthetic receptor molecules comprising truncated versions of HER2 and CD8a for use in methods for enriching, tracking and / or eliminating genetically modified cells, including those used in cellular therapies.

Description

SYNTHETIC HER2 RECEPTORS TO ENRICH, TRACK, AND / OR ELIMINATE MODIFIED CELLS CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Application Serial No.63 / 564,147 filed on March 12, 2024. The disclosure of the prior application is considered part of the disclosure of this application and is incorporated in its entirety into this application. SEQUENCE LISTING

[0002] This application contains a Sequence Listing that has been submitted electronically as an XML file named “58666-0010WO1.XML.” The XML file, created on March 5, 2025, is 22,027 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety. GOVERNMENT SUPPORT

[0003] This invention was made with government support under 5-R01-CA207913 awarded by the National Cancer Institute. The government has certain rights in the invention. BACKGROUND

[0004] Adoptive cellular therapy utilizing patient-derived tumor infiltrating lymphocytes (TILs) has rapidly gained attention and interest as an appealing approach to treat refractory solid cancers, including melanoma, head and neck cancer, non-small cell lung cancer, and sarcoma, with over 300 clinical trials since 2010. Chimeric antigen receptor (CAR) and engineered T-cell receptor (TCR) engineered cell are among the most popular forms of cancer cell therapies. Although current immune cell therapies primarily use cytotoxic T cells, there are various types of immune (NK, macrophages, NTKs, etc.) and non-immune cells (stem cells, mesenchymal stem cells, etc.). In cancer patients, treatment with conventional TILs appears to be relatively safe, with incidences of adverse events being rarely observed. For example, TILs derived from melanoma patients have been observed to attack non-cancerous tissues such as melanocytes, leading to uveal depigmentation and vitiligo. In contrast, CAR T cells can lead to severe adverse events in patients due to expression of cytotoxic molecules, such as cytokines.

[0005] Therefore, there is a need to eliminate T cells efficiently and specifically from patients while sparing non-engineered immune cells. The present disclosure addresses this need byproviding synthetic HER2 polypeptides that can be used as a unique tag to eliminate engineered cells. Moreover, these synthetic HER2 polypeptides can also be used to enrich engineered cells during the manufacturing process, track cells within a patient, and identify cells within samples obtained from a patient. SUMMARY

[0006] The present disclosure provides synthetic HER2 polypeptides comprising, from N- terminus to C-terminus: i) a signal peptide; ii) an extracellular domain comprising the amino acid sequence of SEQ ID NO: 1; iii) a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 2; and iv) an intracellular domain comprising a single lysine residue.

[0007] In some aspects, the signal peptide comprises the amino acid sequence of SEQ ID NO: 3.

[0008] In some aspects, the synthetic HER2 polypeptide has the amino acid sequence of SEQ ID NO: 4.

[0009] The present disclosure provides nucleic acid molecules comprising a nucleic acid sequence encoding a synthetic HER2 polypeptide of the present disclosure.

[0010] In some aspects, the nucleic acid sequence encoding the synthetic HER2 polypeptide comprises the nucleic acid sequence of SEQ ID NO: 7.

[0011] In some aspects, the nucleic acid molecules of the present disclosure can further comprise a nucleic acid sequence encoding a CD8a:MYD88 fusion protein, wherein the CD8a:MYD88 fusion protein comprises, from N-terminus to C-terminus a first domain that comprises a CD8a polypeptide, a linker polypeptide, and a second domain that comprises an MYD88 polypeptide.

[0012] In some aspects, the nucleic acid sequence encoding a CD8a:MYD88 fusion protein comprises the nucleic acid sequence of SEQ ID NO: 8.

[0013] In some aspects, the nucleic acid molecules of the present disclosure comprise the nucleic acid sequence of SEQ ID NO: 11.

[0014] The present disclosure provides vectors comprising the nucleic acid molecule of the present disclosure. In some aspects, the vector is a viral vector. In some aspects, a viral vector is a lentiviral vector.

[0015] The present disclosure provides cells expressing the synthetic HER2 polypeptide of the present disclosure. In some aspects, the cells also express a CD8a:MYD88 fusion protein. In some aspects, the cells also express at least one of: i) a chimeric antigen receptor (CAR); and ii) an engineered T cell receptor (TCR).

[0016] In some aspects, the first domain of a CD8a:MYD88 fusion protein comprises the amino acid sequence of SEQ ID NO: 12.

[0017] In some aspects, the linker polypeptide of a CD8a:MYD88 fusion protein comprises the amino acid sequence of SEQ ID NO: 14.

[0018] In some aspects, the second domain of a CD8a:MYD88 fusion protein comprises the amino acid sequence of SEQ ID NO: 13.

[0019] In some aspects, a CD8a:MYD88 fusion protein has the amino acid sequence of SEQ ID NO: 15.

[0020] In some aspects, the cells are tumor infiltrating lymphocytes (TILs). In some aspects the TILS are T cells.

[0021] The present disclosure provides populations of the cells of the present disclosure.

[0022] The present disclosure provides methods of treating cancer in a subject, the methods comprising administering to the subject at least one cell population of the present disclosure.

[0023] In some aspects, the cancer is a solid tumor. In some aspects, the solid tumor is head and neck cancer, lung cancer, or a sarcoma.

[0024] In some aspects the methods of the present disclosure further comprise at a time point after administration of the cell population, administering to the subject at least one amount of at least one cytotoxic antibody that binds to the synthetic HER2 polypeptide, thereby reducing and / or eliminating the cell population and / or cells that result from in vivo expansion of the cell population.

[0025] In some aspects, the at least one cytotoxic antibody that binds to the synthetic HER2 polypeptide is ado-trastuzumab emtansine, fam-trastuzumab deruxtecan-nxki, margetuximab, trastuzumab, trastuzumab-anns, or a combination thereof.

[0026] In some aspects, the methods of the present disclosure can further comprise, at a time point after administration of the population of cells: i) administering to the subject at least one amount of an antibody that: a) binds to the synthetic HER2 polypeptide; and b) is linked to a detectable label; and ii) imaging the subject, or a portion thereof, with an imaging modality suitable to detect the detectable label, thereby identifying the presence and location of the therapeutic cells within the subject.

[0027] In some aspects, the methods of the present disclosure can further comprise, at a time point after administration of the population of cells: i) obtaining one or more biological samplesfrom the subject; ii) contacting the one more biological samples with at least one amount of an antibody that: a) binds to the synthetic HER2 polypeptides; and b) is linked to a detectable label; and iii) imaging the one or more biological samples with an imaging modality suitable to detect the detectable label, thereby identifying the presence and location of the therapeutic cells within the one or more biological samples. In some aspects, the one or more biological samples comprises a tumor sample, a blood sample, or a combination thereof.

[0028] The present disclosure provides methods of producing the population of the present disclosure, the methods comprising: a) obtaining a plurality of cells from a subject; and b) introducing into the plurality of cells a one or more nucleic acid molecules, wherein the one or more nucleic acid molecules comprise a nucleic acid sequence encoding the synthetic HER2 polypeptide. In some aspects, introducing into the plurality of cells one or more nucleic acid molecules comprises performing retroviral transduction. In some aspects, the plurality of cells obtained from the subject are peripheral blood mononuclear cells (PBMCs).

[0029] In some aspects, the methods of the present disclosure can further comprise, before step (b), after step (b), or both before step (b) and after step (b), expanding the cells.

[0030] In some aspects, the methods of the present disclosure can further comprise, after step (b), contacting the plurality of cells with an affinity reagent that binds to the synthetic HER2 polypeptide of the present disclosure, thereby enriching for cells that express the synthetic HER2 polypeptide.

[0031] Any of the above aspects can be combined with any other aspect.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In the Specification, the singular forms also include the plural unless the context clearly dictates otherwise; as examples, the terms “a,” “an,” and “the” are understood to be singular or plural and the term “or” is understood to be inclusive. By way of example, “an element” means one or more element. Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the statedvalue. Unless otherwise clear from the context, all numerical values provided herein are modified by the term “about.”

[0033] Although methods and materials similar or equivalent to those described herein can beused in the practice or testing of the present disclosure, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The references cited herein are not admitted to be prior art to the claimed invention. In the case of conflict, the present Specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the disclosure will be apparent from the following detailed description and claim. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The above and further features will be more clearly appreciated from the followingdetailed description when taken in conjunction with the accompanying drawings.

[0035] FIG. 1 is a schematic representation of expression cassettes for the constructs tested inthe experimental examples section of the present disclosure, including the MYC construct, the HER2FLconstruct, the HER2IVconstruct, and the HER2III&IVconstruct.

[0036] FIG. 2 shows the results of flow cytometry experiments described in more detail inExample 1 of the present disclosure.

[0037] FIG. 3 shows the results of flow cytometry experiments described in more detail inExample 1 of the present disclosure.

[0038] FIG. 4 shows the results from an in vitro depletion assay which is described in furtherdetail in Example 1 of the present disclosure.

[0039] FIG. 5 shows the results from an in vitro depletion assay which is described in furtherdetail in Example 1 of the present disclosure.

[0040] FIG. 6 shows the results from an in vivo depletion assay which is described in furtherdetail in Example 1 of the present disclosure.

[0041] FIGs. 7A-7L show the structure-function-directed design of a HER2-derived Kadcyla-operating safety switch. (A) Ribbon diagram of the extracellular regions of human HER2 and Herceptin Fab complex (PDB:1N8Z). HER2 domains , , , , and Herceptin Fab areindicated. (B) Schematic representation of the expression cassettes of the human HER2 truncatesin the retroviral vector pMSGV1. PEF1 (human EF1 promoter), LP (CD8 leader sequence),6myc (6x myc-tag EQKLISEEDL (SEQ ID NO: 16)), HER2 truncates: HER2FL(wild type, 1- 1255aa), HER2III&IV(342-676aa), HER2IV(511-676aa), HER2IV-S(563-676aa). (C) Representative flow plot showing cell-surface expression of the HER2 truncates in mouseprimary T cells. (D) Fold change of Mean Fluorescence Intensity (MFI) of the HER2 truncates(MYC (control), HER2FL, HER2III&IV, HER2IV, and HER2IV-S) between EGFP+and EGFP- cells.(E) Diagram of Trp460 / Trp499 - stacking interaction between human HER2 domain and. (F) Construct of myc-tagged HER2 variants (mycHER2III&IV, mycHER2IV, andmycHER2III&IVW499A) and representative MFI histograms showing the expression of myc-tagand HER2 variants in mouse primary T cells. (G) Fold change of MFI relative to mycHER2III&IV(n=3). (H) Fold change of EGFP% of mycCtl- or HER2 truncates- engineered mouse primary Tcells in the presence of ERBITUX®(cetuximab), KANJINTI®(trastuzumab), and KADCYLA®(trastuzumab DM1) (n=3). (I) Fold change of EGFP% of HER2III&IV-engineered mouse primary T cells in the presences of IgG control, KADCYLA®, ENHERTU®, or a combination ofKADCYLA®and ENHERTU® (n=6). (J) Fold change of CD3+EGFP+myc+ counts in thepresence of IgG control or KADCYLA®(n=3). (K) Representative flow plots illustrating the efficacy of MACS enrichment of HER2III&IV-expressing primary T cells using biotin-conjugated trastuzumab biosimilar. (L) Representative flow cytometry plot showing the ability to track T cells in mice. Antibody concentration and MFI values are indicated. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, by multiple unpaired, 2-tailed t test.

[0042] FIGs. 8A-8G show KADCYLA® not only kills HER2III-IV engineered T cells, but itimpairs their function. (A) Schematic representation of experiments to study cell death,proliferation, and cytokine production with KADCYLA® treatment in vitro. (B) Representativeflow plot showing the percentage of dead cells in CD4+EGFP+and CD8+EGFP+T cells. (C) Violin plots showing the distribution of dead cell percentage in CD4+EGFP+and CD8+EGFP+T cells (n=9). (D) Representative flow plot showing the percentage of poorly proliferated cells in CD4+EGFP+and CD8+EGFP+T cells. (E) Violin plots showing the distribution of percentage of poorly proliferated EGFP+cells in CD4+and CD8+T cells (n=18). (F) Representative flow plotshowing the percentage of IFN- -producing cells in CD4+ and CD8+ T cells. (G) Violin plotsshowing the distribution of percentage of IFN- -producing cells in CD4+EGFP+ and CD8+EGFP+T cells (n=17). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, by multiple unpaired, 2-tailed t test.

[0043] FIGs. 9A-9E show that Her2III-IV serves as a target to eliminate engineered cells. (A)Naïve mice underwent lymphodepletion (450 rads) followed by infusion of T cells engineered to express Her2III-IVon the cell surface followed by injection of IL-2 (10,000 U) and anti-EGFR antibody (ERBITUX®), anti-HER2 antibody (KANJINTI®), and anti-HER2 antibody-drug complex (ADC) (KADCYLA®). (B-C) Three days after antibody injection, the indicated tissues were collected and the frequency of engineered HER2III-IV+examined via FACS / flow cytometry. (D) Following the same schema described in FIG.9A, different doses of KADCYLA®were interrogated and the frequency of engineered T cells examined in the indicated tissues by FACS. (E) Following the same schema described in FIG.9A, mice were injected with 500 µg of KADCYLA®and the frequency of engineered T cells was examined in the indicated tissues at different times by flow cytometry.

[0044] FIGs. 10A-10C show that Her2III-IV serves as a target to eliminate engineered cells intumor-bearing mice. (A) Naïve mice underwent lymphodepletion (450 rads), followed by infusion of T cells engineered to express Her2III-IVon the cell surface, followed by injection of IL-2 (10,000 U) and the isotype antibody or KADCYLA®(anti-HER2 ADC; 500 µg). (B-C) One week after antibody injection, mice were euthanized, and the indicated tissues were collected and the frequency of engineered HER2III-IV+examined via flow cytometry. Each dot represents one mouse. ****p < 0.0001, by multiple unpaired, *p < 0.052- tailed t test.

[0045] FIG. 11 shows Her2III-IV serves as a target to eliminate T cell leukemia cells in the eventthat engineered T cells become malignant, as has been shown to occur in a number of patients that have received CAR T cells. Jurkat T cell leukemia cells were injected intravenously (i.v.) into NSG mice. Once luciferase signal was detected, mice were treated with KADCYLA®(500 µg; intraperitoneal) or isotype control. Leukemia progression was measured every 7 days. DETAILED DESCRIPTION

[0046] Synthetic HER2 polypeptides of the Present Disclosure

[0047] The present disclosure provides synthetic HER2 polypeptides comprising, from N-terminus to C-terminus, an extracellular domain, a transmembrane domain, and an intracellular domain.

[0048] In some aspects, the synthetic HER2 polypeptides can further comprise a signal peptide.Accordingly, the synthetic HER2 polypeptides of the present disclosure can comprise, from N-terminus to C-terminus, a signal peptide, an extracellular domain, a transmembrane domain, and an intracellular domain.

[0049] In some aspects, an extracellular domain can comprise, consist essentially of, or consist of one or more fragments of the extracellular domain of human epidermal growth factor receptor 2 (HER2 / ErbB2 / Neu) protein. As would be appreciated by the skilled artisan, the extracellular domain of HER2 is divided into four functional domains denoted Domain I, Domain II, Domain III, and Domain IV. An extracellular domain can comprise, consist essentially of, or consist of one or more of the extracellular domains of HER2. In some aspects, an extracellular domain can comprise, consist essentially of, or consist of Domain III of HER2 or a fragment or portion thereof. In some aspects, an extracellular domain can comprise, consist essentially of, or consist of Domain IV of HER2 or a fragment or portion thereof. In some aspects, an extracellular domain can comprise, consist essentially of, or consist of Domain III of HER2, or a fragment or portion thereof, and Domain IV of HER2, or a fragment or portion thereof.

[0050] In some aspects, an extracellular domain of a synthetic HER2 polypeptide of the present disclosure can comprise, consist essentially of, or consist of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 1.

[0051] In some aspects, a transmembrane domain of a synthetic HER2 polypeptide of the present disclosure can comprise, consist essentially of, or consist of the transmembrane domain of HER2, or a fragment or portion thereof.

[0052] In some aspects, a transmembrane domain of a synthetic HER2 polypeptide of the present disclosure can comprise, consist essentially of, or consist of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 2.

[0053] In some aspects, a signal peptide of a synthetic HER2 polypeptide of the present disclosure can comprise, consist essentially of, or consist of the signal peptide of T-cell surface glycoprotein CD8 alpha chain (CD8a). In some aspects, a signal peptide of a synthetic HER2 polypeptide of the present disclosure can comprise, consist essentially of, or consist of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 3.

[0054] In some aspects, an intracellular domain of a synthetic HER2 polypeptide of the present disclosure can comprise at least about one amino acid, at least about two amino acids, at least about three amino acids, at least about four amino acids, at least about four amino acids, at least about five amino acids, at least about six amino acids, at least about seven amino acids, at least about eight amino acids, at least about nine amino acids, or at least about ten amino acids.

[0055] In some aspects, an intracellular domain of a synthetic HER2 polypeptide of the present disclosure can comprise about one amino acid, about two amino acids, about three amino acids, about four amino acids, about four amino acids, about five amino acids, about six amino acids, about seven amino acids, about eight amino acids, about nine amino acids, or about ten amino acids.

[0056] In some aspects, an intracellular domain of a synthetic HER2 polypeptide of the present disclosure comprises, consists essentially of, or consists of a single lysine residue.

[0057] Accordingly, the present disclosure provides synthetic HER2 polypeptides comprising, from N-terminus to C-terminus, an extracellular domain comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, a transmembrane domain comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 2, and an intracellular domain comprising at least one lysine residue. The present disclosure also provides synthetic HER2 polypeptides comprising, from N-terminus to C-terminus, an extracellular domain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 1, a transmembrane domain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 2, and an intracellular domain comprising at least one lysine residue. The present disclosure also provides synthetic HER2 polypeptides comprising, from N-terminus to C-terminus, an extracellular domain comprising the amino acid sequence of SEQ ID NO: 1, a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 2, and an intracellular domain comprising at least one lysine residue.

[0058] Accordingly, the present disclosure provides synthetic HER2 polypeptides comprising, from N-terminus to C-terminus, a signal peptide comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 3, an extracellular domain comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 1, a transmembrane domain comprising an amino acid sequence that is at least 98% identical to SEQ ID NO: 2, and an intracellular domain comprising at least one lysine residue. The present disclosure also provides synthetic HER2 polypeptides comprising, from N-terminus to C-terminus, a signal peptide comprising an aminoacid sequence that is at least 99% identical to SEQ ID NO: 3, an extracellular domain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 1, a transmembrane domain comprising an amino acid sequence that is at least 99% identical to SEQ ID NO: 2, and an intracellular domain comprising at least one lysine residue. The present disclosure also provides synthetic HER2 polypeptides comprising, from N-terminus to C-terminus, a signal peptide comprising the amino acid sequence of SEQ ID NO: 3, an extracellular domain comprising the amino acid sequence of SEQ ID NO: 1, a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 2, and an intracellular domain comprising at least one lysine residue.

[0059] The present disclosure provides synthetic HER2 polypeptides comprising, consisting essentially of, or consisting of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 4.

[0060] Nucleic Acid Molecules of the Present Disclosure

[0061] The present disclosure provides nucleic acid molecules comprising one or more sequences encoding for a synthetic HER2 polypeptide of the present disclosure, or any portion or fragment thereof.

[0062] Accordingly, the present disclosure provides nucleic acid molecules comprising a nucleic acid sequence encoding for an extracellular domain of a synthetic HER2 polypeptide of the present disclosure. A nucleic acid sequence encoding for an extracellular domain of a synthetic polypeptide of the present disclosure can comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 5.

[0063] The present disclosure also provides nucleic acid molecules comprising a nucleic acid sequence encoding for a transmembrane domain of a synthetic HER2 polypeptide of the present disclosure. A nucleic acid sequence encoding for a transmembrane domain of a synthetic HER2 polypeptide of the present disclosure can comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 6.

[0064] The present disclosure also provides a nucleic acid molecule comprising a nucleic acid sequence encoding for an intracellular domain of a synthetic HER2 polypeptide of the present disclosure. In embodiments wherein the intracellular domain of the synthetic HER2 polypeptidecomprises a single lysine residue, the nucleic acid sequence encoding the intracellular domain can have the nucleic acid sequence AAG or the nucleic acid sequence AAG.

[0065] Accordingly, the present disclosure provides nucleic acid molecules comprising, consisting essentially of, or consisting of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 7.

[0066] The nucleic acids of the present disclosure can further comprise one or more nucleic acid sequences that encode for a CD8a:MYD88 fusion protein (CD8a:MYD88 fusion proteins are described in further detail herein). A nucleic acid sequence encoding a CD8a:MYD88 fusion protein can comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 8.

[0067] Accordingly, the present disclosure provides nucleic acid molecules comprising at least one nucleic acid sequence encoding for a synthetic HER2 polypeptide of the present disclosure and at least one nucleic acid sequence encoding for a CD8a:MYD88 fusion protein. These nucleic acid sequences can be in any order, i.e. the nucleic acids molecules can comprise, from 5’ to 3’, a nucleic acid sequence encoding a synthetic HER2 polypeptide of the present disclosure and a nucleic acid sequence encoding a CD8a:MYD88 fusion protein, or the nucleic acid molecules can comprise, from 5’ to 3’, a nucleic acid sequence encoding a CD8a:MYD88 fusion protein and a nucleic acid sequence encoding a synthetic HER2 polypeptide of the present disclosure.

[0068] In aspects wherein a nucleic acid molecule of the present disclosure comprises two or more nucleic acid sequences encoding for polypeptides (e.g. a nucleic acid sequence encoding for a CD8a:MYD88 fusion protein and a nucleic acid sequence encoding for a HER2 polypeptide of the present disclosure), the nucleic acid sequences encoding the different polypeptides can be separated by one or more nucleic acid sequences that encode a self-cleaving peptide.

[0069] As would be appreciated by the skilled artisan, the amino acid sequences of self-cleaving peptides and the nucleic acids sequences encoding said amino acid sequences, are known in the art. Non-limiting examples of self-cleaving peptides include 2A self-cleaving peptide sequences such as the T2A peptide, the E2A peptide, the F2A peptide and the P2A peptide.

[0070] In some aspects of the nucleic acid molecules of the present disclosure, the sequence encoding for a self-cleaving peptide is a nucleic acid sequence encoding for a P2A self-cleaving peptide. As would be appreciated by the skilled artisan, a P2A self-cleaving peptide can have an amino acid sequence that comprises, consists essentially of, or consist of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 9. Accordingly, a nucleic acid sequence encoding for a P2A self-cleaving peptide can comprise, consist essentially of, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 10.

[0071] The present disclosure provides nucleic acid molecules comprising a nucleic acid sequence that comprises, consists essentially or, or consist of a nucleic acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 11.

[0072] In some aspects, a nucleic acid molecule of the present disclosure can be a vector. In some aspects, the vector is a viral vector.

[0073] Cells of the Present Disclosure

[0074] The present disclosure provides cells that express the synthetic HER2 polypeptides of the present disclosure. These cells are also referred to herein as “therapeutic cells”.

[0075] The present disclosure provides pluralities of cells that express the synthetic HER2 polypeptides of the present disclosure. These pluralities of cells are also referred to herein as “pluralities of therapeutic cells”.

[0076] The present disclosure provides a plurality (also referred to as a “population”) of cells, wherein at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% of the cells in the plurality express the synthetic HER2 polypeptides of the present disclosure.

[0077] The present disclosure provides a plurality of cells, wherein about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 55%, or about 60%, or about 65%, or about 70%, or about 75%, or about 80%, orabout 85%, or about 90%, or about 95%, or about 97%, or about 99% of the cells in the plurality express the synthetic HER2 polypeptides of the present disclosure.

[0078] In some aspects, the cells of the present disclosure that express the synthetic HER2 polypeptides of the present disclosure can also express a CD8a:MYD88 fusion protein of the present disclosure.

[0079] Accordingly, the present disclosure provides pluralities of cells the express the synthetic HER2 polypeptides of the present disclosure and the CD8a:MYD88 fusion proteins of the present disclosure. In some aspects, at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% of the cells in the plurality express the synthetic HER2 polypeptides and the CD8a:MYD88 fusion proteins. In some aspects, about 10%, or about 15%, or about 20%, or about 25%, or about 30%, or about 35%, or about 40%, or about 45%, or about 50%, or about 55%, or about 60%, or about 65%, or about 70%, or about 75%, or about 80%, or about 85%, or about 90%, or about 95%, or about 97%, or about 99% of the cells in the plurality express the synthetic HER2 polypeptides and the CD8a:MYD88 fusion proteins.

[0080] Accordingly, the present disclosure also provides a plurality of cells comprising one or more of the nucleic acid molecules described herein, including those that encode for the synthetic HER2 polypeptides and / or CD8a:MYD88 fusion proteins described herein.

[0081] In some aspects, the cells described above can be immune cells. Examples of such immune cells include, but are not limited to, T cells (e.g., regulatory T cells, CAR T cells, CD8+ CAR T cells, CD4+ CAR T cells, CD4+ T cells, CD8+ T cells, peripheral blood (PB) derived T cells, umbilical cord blood (UCB) derived T cells, or gamma-delta T cells), NK cells, NK-like cells, invariant NK cells, NKT cells, cytokine induced killer (CIK) cells, stem cells (e.g., mesenchymal stem cells (MSCs), hematopoietic stem cells, hematopoietic progenitor cells, or induced pluripotent stem (iPSC) cells). In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and / or basophils. Also provided herein are methods of producing and engineering the immune cells and methods of using and administering the cells for adoptive cell therapy, inwhich case the cells may be autologous or allogeneic. Thus, the immune cells may be used as immunotherapy, such as to target cancer cells.

[0082] In some aspects, the cells described above can be tumor infiltrating lymphocytes (“TILs”). As would be appreciated by the skilled artisan, TILs refers to populations of white blood cells that have left the bloodstream of a subject and migrated into a tumor. Populations of TILs can include, but are not limited to, T helper 17 cells (Thl7, CD4+IL17+ T cells), cytotoxic T cells (Tc17, CD8+IL17+ T cells) and regulatory T cells (Treg, CD4+CD25+Foxp3+ T cells), natural killer (NK) cells, dendritic cells and M1 macrophages. As would be appreciated by the skilled artisan, TILs can generally be defined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and affect treatment. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR , CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, and CD25. Additionally and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a subject.

[0083] Immune cells may be enriched / purified from any tissue where they reside including, but not limited to, blood (including blood collected by blood banks or cord blood banks), spleen, bone marrow, tissues removed and / or exposed during surgical procedures, and tissues obtained via biopsy procedures. Tissues / organs from which the immune cells are enriched, isolated, and / or purified may be isolated from both living and non-living subjects, wherein the non-living subjects are organ donors. The isolated immune cells may be used directly, or they can be stored for a period of time, such as by freezing. In the case of TILs, the population of TILs can be enriched / purified from a tumor sample obtained from the subject.

[0084] In some aspects, in addition to expressing synthetic HER2 polypeptides of the present disclosure, or expressing both synthetic HER2 polypeptides of the present disclosure and CD8a:MYD88 fusion proteins, the cells of the present disclosure can be modified to additionally express a chimeric antigen receptor (CAR) and / or an engineered T cell receptor (TCR). In some aspects, the chimeric antigen receptor is in the format of a universal CAR.

[0085] CD8a:MYD88 Fusion Proteins

[0086] The CD8a:MYD88 fusion proteins used in the methods and compositions of the present disclosure can comprise, from N-terminus to C-terminus, a first domain that comprises a CD8a polypeptide and a second domain that comprises a Myeloid differentiation primary responseprotein (MYD88) polypeptide. In some aspects, the first domain and the second domain of the CD8a:MYD88 fusion proteins are connected together via a linker polypeptide.

[0087] In some aspects, the first domain that comprises a CD8a polypeptide can comprise, consist essentially of or consist of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 12.

[0088] In some aspects, the second domain that comprises a MYD88 polypeptide can comprise, consist essentially of, or consist of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 13.

[0089] In some aspects, the linker polypeptide can comprise, consist essentially of, or consist of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 14.

[0090] Accordingly, a CD8a:MYD88 fusion protein of the present disclosure can comprise, consist essentially of, or consist of an amino acid sequence that is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% (or any percentage in between) identical to SEQ ID NO: 15.

[0091] The CD8a:MYD88 fusion proteins used in the methods and compositions of the present disclosure can also be selected from any of the CD8a:MYD88 fusion proteins disclosed in PCT Publication No. WO / 2016 / 073875 and US Patent No.10,975,137.

[0092] Functional Variants of the Polypeptides of the Present Disclosure

[0093] The present disclosure also contemplates functional variants of the polypeptides disclosed and described herein. The term "functional variant" as used herein refers to a polypeptide having substantial or significant sequence identity or similarity to a parent polypeptide where the functional variant retains the biological activity of the parent polypeptide of which it is a variant. In reference to the parent polypeptide, the functional variant can, for instance, be at least about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to the parent CAR.

[0094] A functional variant can, for example, comprise the amino acid sequence of the parent polypeptide with at least one conservative amino acid substitution. Alternatively, or additionally, the functional variants can comprise the amino acid sequence of the parent polypeptide with atleast one non-conservative amino acid substitution. In this case, it is preferable for the non- conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent polypeptide.

[0095] A conservative substitution of an amino acid, i.e., replacing an amino acid with a different amino acid of similar properties (e.g., hydrophilicity, degree and distribution of charged regions) is recognized in the art as typically involving a minor change. These minor changes can be identified, in part, by considering the hydropathic index of amino acids, as understood in the art. Kyte et al., J. Mol. Biol.157: 105-132 (1982). The hydropathic index of an amino acid is based on a consideration of its hydrophobicity and charge. Amino acids of similar hydropathic indexes can be substituted and still retain protein function. In an aspect, amino acids having hydropathic indexes of ±2 are substituted. The hydrophilicity of amino acids can also be used to reveal substitutions that would result in proteins retaining biological function. A consideration of the hydrophilicity of amino acids in the context of a polypeptide permits calculation of the greatest local average hydrophilicity of that polypeptide, a useful measure that has been reported to correlate well with antigenicity and immunogenicity. U.S. Patent No.4,554,101, incorporated fully herein by reference.

[0096] Substitution of amino acids having similar hydrophilicity values can result in polypeptides retaining biological activity, for example immunogenicity. Substitutions can be performed with amino acids having hydrophilicity values within ±2 of each other. Both the hydrophobicity index and the hydrophilicity value of amino acids are influenced by the particular side chain of that amino acid. Consistent with that observation, amino acid substitutions that are compatible with biological function are understood to depend on the relative similarity of the amino acids, and particularly the side chains of those amino acids, as revealed by the hydrophobicity, hydrophilicity, charge, size, and other properties.

[0097] As used herein, “conservative” amino acid substitutions may be defined as set out in Tables A, B, or C below. In some aspects, fusion polypeptides and / or nucleic acids encoding such fusion polypeptides include conservative substitutions that have been introduced by modification of polynucleotides encoding polypeptides of the disclosure. Amino acids can be classified according to physical properties and contribution to secondary and tertiary proteinstructure. A conservative substitution is a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in Table A.

[0098] Table A -- Conservative Substitutions I

[0099] Alternately, conservative amino acids can be grouped as described in Lehninger,(Biochemistry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp.71-77) as set forth in Table B.

[0100] Table B -- Conservative Substitutions II

[0101] Alternately, exemplary conservative substitutions are set out in Table C.

[0102] Table C -- Conservative Substitutions III

[0103] It should be understood that the polypeptides of the disclosure are intended to include polypeptides bearing one or more insertions, deletions, or substitutions, or any combination thereof, of amino acid residues as well as modifications other than insertions, deletions, or substitutions of amino acid residues. Polypeptides or nucleic acids of the disclosure may contain one or more conservative substitutions.

[0104] As used throughout the disclosure, the term “more than one” of the aforementioned amino acid substitutions refers to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more of the recited amino acid substitutions. The term “more than one” may refer to 2, 3, 4, or 5 of the recited amino acid substitutions.

[0105] Methods of Treatment of the Present Disclosure

[0106] The present disclosure provides methods of treating cancer in a subject, the method comprising administering to the subject one or more pluralities of therapeutic cells of the present disclosure.

[0107] The present disclosure provides one or more pluralities of therapeutic cells of the present disclosure for use in the treatment of cancer in a subject.

[0108] The present disclosure provides the use of one or more pluralities of therapeutic cells of the present disclosure for use in the manufacture of a medicament for the treatment of cancer.

[0109] In some aspects, the plurality or pluralities of therapeutic cells are administered to the subject in a therapeutically effective amount. The therapeutically effective amount (e.g., the dose) of therapeutic cells will be dependent on the subject being treated, the severity and type of the affliction, and the manner of administration. In some aspects, doses that could be used in the treatment of human subjects range from at least 3.8x104, at least 3.8x105, at least 3.8x106, at least 3.8x107, at least 3.8x108, at least 3.8x109, or at least 3.8x1010 therapeutic cells cells / m2. In a certain aspect, the dose used in the treatment of human subjects ranges from about 3.8x109 toabout 8x1011therapeutic cells / m2(e.g., from about 3.8x109immune cells / m2to about 4x1011,about 3.8x109 to about 1x1011, about 3.8x109 to about 7x1010, about 3.8x109 to about 4x1010,about 3.8x109to about 1x1010, about 3.8x109to about 7x109, about 7x109to about 8x1011, about 1x1010to about 8x1011, about 4x1010to about 8x1011, about 7x1010to about 8x1011, about 1x1011to about 8x1011, about 4x1011to about 8x1011, about 7x109to about 4x1011, about 1x1010to about 1x1011, or about 4x1010to about 7x1010). In additional aspects, a therapeutically effective amount of therapeutic cells can vary from about 5x106cells per kg body weight to about 7.5x108cells per kg body weight, such as from about 2x107cells to about 5x108cells per kg body weight, or from about 5x107cells to about 2x108cells per kg body weight, or from about 5x106cells per kg body weight to about 1x107cells per kg body weight. In a certain aspect, a therapeuticallyeffective amount of therapeutic cells ranges from about 1x105 cells per kg body weight to about1x1010cells per kg body weight (e.g., from about 1x105cells per kg body weight to about 8.8x109, from about 1x105to about 7.5x109, from about 1x105cells per kg body weight to about 6.3x109, from about 1x105cells per kg body weight to about 5x109, from about 1x105cells per kg body weight to about 3.8x109, from about 1x105cells per kg body weight to about 2.5x109, from about 1x105cells per kg body weight to about 1.3x109, from about 1.3x109to about 1x1010, from about 2.5x109to about 1x1010, from about 3.8x109to about 1x1010, from about 5x109to about 1x1010, from about 6.3x109to about 1x1010, from about 7.5x109to about 1x1010, from about 1.3x109to about 8.8x109, from about 2.5x109to about 7.5x109, or from about 3.8x109toabout 6.3x109). The therapeutically effective amount of therapeutic cells provided herein canremain constant or can be variable during the duration of treatment. Various factors caninfluence the actual therapeutically effective amount used for a particular application. For example, the severity of cancer when treating a mammal having cancer, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic or prophylactic treatments such as use of other agents, and the judgment of the treating physician may require an increase or decrease in the actual therapeutically effective amount of therapeutic cells provided herein that is administered. The exact amount of therapeutic cells is readily determined by one of skill in the art based on the age, weight, sex, and physiological condition of the subject. Effective doses can be extrapolatedfrom dose-response curves derived from in vitro or animal model test systems.

[0110] In some aspects, the plurality or pluralities of therapeutic cells are administered to thesubject with a therapeutically effective frequency. The therapeutically effective frequency ofadministration of therapeutic cells will be dependent on the subject being treated, the severityand type of the affliction, and the manner of administration. In some aspects, a therapeuticallyeffective frequency can be from about twice daily to about once a year (e.g., from about twicedaily to about once a month, from about twice daily to about once a week, from about once daily to about once a month, or from one once daily to about once a week). The therapeutically effective frequency of administration of therapeutic cells provided herein can remain constant or can be variable during the duration of treatment. Various factors can influence the actual therapeutically effective frequency used for a particular application. For example, the severity of the cancer, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic or prophylactic treatments such as use of other agents, and the judgment of the treating physician may require an increase or decrease in the actual therapeutically effective frequency of administration of therapeutic cells provided herein.

[0111] In some aspects, the plurality or pluralities of therapeutic cells are administered to thesubject for a therapeutically effective duration. The therapeutically effective duration ofadministration of therapeutic cells will be dependent on the subject being treated, the severityand type of the affliction, and the manner of administration. In some aspects, a therapeuticallyeffective duration can vary from a single time point of administration to several weeks to severalmonths (e.g., 4 to 12 weeks). Multiple factors can influence the actual therapeutically effective duration used for a particular application. For example, the severity of the cancer, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic or prophylactic treatments such as use of other agents, and the judgment of the treating physician may require an increase or decrease in the actual therapeutically effective duration of administration of therapeutic cells provided herein.

[0112] In some aspects, the plurality or pluralities of therapeutic cells are administered byintravenous administration. In some aspects, the plurality or pluralities of therapeutic cells are administered by intratumoral administration. In some aspects, the plurality or pluralities of therapeutic cells are administered by intrapleural administration, intraperitoneal administration,or intrathoracic administration. In some aspects, the route and / or mode of administration of the plurality or pluralities of therapeutic cells can be adjusted for the subject being treated.

[0113] In some aspects, the plurality or pluralities of therapeutic cells are administered in combination with at least one additional therapy. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy. The additional therapy is the administration of small molecule enzymatic inhibitor or anti-metastatic agent. In some embodiments, the additional therapy is the administration of side effect limiting agents (e.g., agents intended to lessen the occurrence and / or severity of side effects of treatment, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy may be one or more of the chemotherapeutic agents known in the art.

[0114] A wide variety of chemotherapeutic agents may be used in accordance with the present embodiments. The term "chemotherapy" refers to the use of drugs to treat cancer. A "chemotherapeutic agent" is used to connote a compound or composition that is administered in the treatment of cancer. These agents or drugs are categorized by their mode of activity within a cell, for example, whether and at what stage they affect the cell cycle. Alternatively, an agent may be characterized based on its ability to directly cross-link DNA, to intercalate into DNA, or to induce chromosomal and mitotic aberrations by affecting nucleic acid synthesis.

[0115] Examples of chemotherapeutic agents include alkylating agents, such as thiotepa and cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines, including altretamine, triethylenemelamine, trietylenephosphoramide, triethiylenethiophosphoramide, and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; asarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, and uracil mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as the enediyne antibiotics (e.g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall); dynemicin, including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromophores, aclacinomysins, actinomycin, authrarnycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin (including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxy doxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, such as mitomycin C, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; anti-metabolites, such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues, such as denopterin, pteropterin, and trimetrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, decitabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens, such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals, such as mitotane and trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSKpolysaccharide complex; razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; taxoids, e.g., paclitaxel and docetaxel gemcitabine; 6-thioguanine; mercaptopurine; platinum coordination complexes, suchas cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine; vinorelbine; novantrone; teniposide; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; irinotecan (e.g., CPT-11); topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; carboplatin, procarbazine,plicomycin, gemcitabien, navelbine, farnesyl-protein tansferase inhibitors, transplatinum, and pharmaceutically acceptable salts, acids, or derivatives of any of the above.

[0116] In some aspects, the methods of treating cancer using one or more pluralities of cells of the present disclosure can further comprise steps to reduce and / or eliminate the plurality or pluralities of cells that were administered and / or reduce and / or eliminate any cells that result from in vivo expansion of the plurality or pluralities of therapeutic cells following administration. As would be appreciated by the skilled artisan, the ability to specifically reduce and / or eliminate these therapeutic cells can help to prevent adverse events in patients, such as when the plurality of cells administered to the subject begin to target non-cancerous cells and tissues.

[0117] Accordingly, the present disclosure provides methods of treating cancer in a subject, the method comprising administering to the subject one or more pluralities of cells of the present disclosure, wherein the method further comprises, at a time point after administration, administering to the subject at least one amount of a cytotoxic antibody that binds to the synthetic HER2 polypeptides of the present disclosure, thereby reducing and / or eliminating the plurality of cells and / or any cells that result from in vivo expansion of the plurality of cells. In some aspects, the administration of the cytotoxic antibody results in at least about a 5%, or at least a about a 10%, or at least about a 15%, or at least about a 20%, or at least about a 25%, or at least about a 30%, or at least about a 35%, or at least about a 40%, or at least about a 45%, or at least about a 50%, or at least about a 55%, or at least about a 60%, or at least about a 65%, or at least about a 70%, or at least about a 75%, or at least about an 80%, or at least about a 85%, or at least about a 90%, or at least about a 95%, or at least about a 99%, or at least about a 100% reduction in the number in of cells that express the synthetic HER2 polypeptide of the present disclosure (i.e. the therapeutic cells administered to the subject and / or any cells resulting from in vivo expansion of the therapeutic cells).

[0118] In some aspects, a cytotoxic antibody that binds to the synthetic HER2 polypeptide can comprise an anti-HER2 antibody conjugated to a cytotoxic compound (i.e. an antibody-drug conjugate). In some aspects, a cytotoxic antibody that binds to the synthetic HER2 polypeptidecan comprise an anti-HER2 antibody conjugated to an antibody or antigen binding domain that can target and bind to an antigen (e.g., CD3) expressed on the surface of a cell (e.g., a T cell or an NK cell). For example, a cytotoxic antibody that binds to the synthetic HER2 polypeptide can be in the form of a cell engager (e.g., a bispecific cell engager such as a bispecific T cell engager (BiTE)). In some aspects, an anti-HER2 antibody conjugated to an antibody or antigen binding domain that can target and bind to an antigen (e.g., CD3) expressed on the surface of a cell (e.g., a T cell or an NK cell) can link a therapeutic cell of the present disclosure (e.g., a cell that expresses the synthetic HER2 polypeptide of the present disclosure) to another cell (e.g., a T cell or an NK cell).

[0119] Non-limiting examples of cytotoxic antibodies that bind to the synthetic HER2 polypeptides of the present disclosure include ado-trastuzumab emtansine (KADCYLA®), fam- trastuzumab deruxtecan-nxki (ENHERTU®), Margetuximab (MARGENZA®), trastuzumab (HERCEPTIN®), and trastuzumab-anns (KANJINTI®).

[0120] In some aspects of the cell reduction methods described above, the cytotoxic antibody can be replaced with an aptamer-drug conjugate, wherein the aptamer-drug conjugate comprises an aptamer the binds to a synthetic HER2 polypeptide of the present disclosure conjugated to a cytotoxic compound. Non-limiting examples of such aptamer-drug conjugates for use in the methods presented herein include those put forth in Jeong et al. Development of HER2-Specific Aptamer-Drug Conjugate for Breast Cancer Therapy. Int. J. Mol. Sci.2020, 21, 9764.

[0121] Accordingly, the present disclosure provides methods of treating cancer in a subject, the method comprising administering to the subject one or more pluralities of cells of the present disclosure, wherein the method further comprises, at a time point after administration, administering to the subject at least one amount of a aptamer-drug conjugate that binds to the synthetic HER2 polypeptides of the present disclosure, thereby reducing and / or eliminating the plurality of cells and / or any cells that result from in vivo expansion of the plurality of cells.

[0122] In some aspects, the methods of treating cancer of the present disclosure can further comprise tracking the therapeutic cells administered to the subject and / or any cells that result from the in vivo expansion of the therapeutic cells. That is, the expression of the synthetic HER2 polypeptides of the present disclosure in the therapeutic cells allows for their tracking within the subject’s body or a sample obtained from the subject through the use of antibodies that: (a) are linked to a detectable label; and (b) that bind to the synthetic HER2 polypeptides.

[0123] Accordingly, the present disclosure provides methods of treating cancer in a subject, the methods comprising administering to the subject one or more pluralities of cells of the present disclosure, wherein the method further comprises, at a time point after administration: i) administering to the subject at least one amount of an antibody that binds to the synthetic HER2 polypeptides of the present disclosure and that is linked to a detectable label; and ii) imaging the subject or a portion thereof with an imaging modality suitable to detect the detectable label, thereby identifying the presence and location of the therapeutic cells within the subject.

[0124] The present disclosure also provides methods of treating cancer in a subject, the methods comprising administering to the subject one or more pluralities of cells of the present disclosure, wherein the method further comprises, at a time point after administration: i) obtaining one or more biological samples from the subject; ii) contacting the one more biological samples with at least one amount of an antibody that binds to the synthetic HER2 polypeptides of the present disclosure and that is linked to a detectable label; and iii) imaging the one or more biological samples with an imaging modality suitable to detect the detectable label, thereby identifying the presence and location of the therapeutic cells within the one or more biological samples.

[0125] In some aspects, the sample is a tumor sample. In some aspects, the sample is a blood sample.

[0126] Examples of antibodies that bind to the synthetic HER2 polypeptides of the present disclosure include, but are not limited to, ado-trastuzumab emtansine (KADCYLA®) and fam- trastuzumab deruxtecan-nxki (ENHERTU®), Margetuximab (MARGENZA®), trastuzumab (HERCEPTIN®), trastuzumab-anns (KANJINTI®).

[0127] A detectable label can be any detectable label known in the art, including, but not limited to paramagnetic particles, radioisotopes, CT contrast agents, MRI contrast agents, microbubbles, fluorescent moieties, or any combination thereof.

[0128] Examples of suitable imaging modalities include, but are not limited to, positron emission tomography (PET), magnetic resonance imaging (MRI), ultrasound imaging, and fluorescence imaging.

[0129] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.

[0130] In some aspects, the cancer can be a solid tumor. Exemplary solid tumors can include, but are not limited to, a tumor of an organ selected from the group consisting of pancreas, colon,cecum, stomach, brain, head, neck, ovary, kidney, larynx, sarcoma, lung, bladder, melanoma, prostate, and breast.

[0131] In some aspects, the cancer is head and neck cancer.

[0132] In some aspects, the cancer is lung cancer. In some aspects, the lung cancer is non-small cell lung cancer.

[0133] In some aspects, the cancer is a sarcoma.

[0134] In some aspects, the cancer is a hematological cancer. Exemplary hematological tumors include but are not limited to tumors of the bone marrow, T or B cell malignancies, myeloid malignancies, leukemias, lymphomas, blastomas, and myelomas.

[0135] Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma.

[0136] The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma;cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; paget's disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; Sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra- mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo malignant melanoma; acral lentiginous melanomas; nodular melanomas; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; T lymphoblastic leukemia; T lymphoblastic lymphoma; B cell leukaemia; Hodgkin's disease; Hodgkin’s lymphoma; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-Hodgkin's lymphomas; B cell lymphoma; low grade / follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediategrade / follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); chronic myeloid leukemia, acute lymphoblastic leukemia (ALL); acute lymphoblastic lymphoma; acute myeloid leukemia (AML); myelodysplastic syndrome (MDS); myeloproliferative neoplasms; chronic myeloblasts leukemia; diffuse large B cell lymphoma (DLBCL); peripheral T cell lymphoma (PTCL); or anaplastic large cell lymphoma (ALCL). In some embodiments, the cancer comprises a liquid tumor. In some embodiments, the liquid tumor is a leukemia or a lymphoma. In some embodiments, the leukemia or lymphoma is B cell leukemia or B cell lymphoma.

[0137] The terms “subject” and “patient” are used interchangeably herein. The subject treated in accordance with the methods described herein can be any appropriate subject. In some embodiments, the subject treated in accordance with the methods described herein is a human patient, e.g., a human adult. In some embodiments, the subject treated in accordance with the methods described herein can be a non-human primate (e.g., monkey), horse, bovine species, porcine species, dog, cat, mouse, or rat.

[0138] As used herein, the term “treating” or “treat” describes the management and care of a patient for the purpose of combating a disease, condition, or disorder and includes the administration of a compound of the present disclosure, or a pharmaceutically acceptable salt, polymorph or solvate thereof, to alleviate the symptoms or complications of a disease, condition, or disorder, or to eliminate the disease, condition, or disorder. The term “treat” can also include treatment of a cell in vitro or an animal model.

[0139] The terms “effective amount” and “therapeutically effective amount” of an agent or compound are used in the broadest sense to refer to a nontoxic but sufficient amount of an active agent or compound to provide the desired effect or benefit.

[0140] The term "benefit" is used in the broadest sense and refers to any desirable effect and specifically includes clinical benefit as defined herein. Clinical benefit can be measured byassessing various endpoints, e.g., inhibition, to some extent, of disease progression, including slowing down and complete arrest; reduction in the number of disease episodes and / or symptoms; reduction in lesion size; inhibition (i.e., reduction, slowing down or complete stopping) of disease cell infiltration into adjacent peripheral organs and / or tissues; inhibition (i.e. reduction, slowing down or complete stopping) of disease spread; decrease of auto-immune response, which may, but does not have to, result in the regression or ablation of the disease lesion; relief, to some extent, of one or more symptoms associated with the disorder; increase in the length of disease-free presentation following treatment, e.g., progression-free survival; increased overall survival; higher response rate; and / or decreased mortality at a given point of time following treatment.

[0141] Methods of Cell Production of the Present Disclosure

[0142] The present disclosure provides methods of producing the therapeutic cells and pluralities of therapeutic cells of the present disclosure.

[0143] Accordingly, the present disclosure provides a method of producing a plurality of cells expressing the synthetic HER2 polypeptides of the present disclosure, the method comprising: a) obtaining a plurality of cells from a subject; and b) introducing into the plurality of cells one or more nucleic acid molecules of the present disclosure, wherein the one or more nucleic acid molecules comprise a nucleic acid sequence encoding for a synthetic HER2 polypeptide of the present disclosure.

[0144] As discussed herein, the one or more nucleic acid molecules of the present disclosure can also include additional nucleic acid sequences encoding other polypeptides, including, but not limited to, CD8a:MYD88 fusion proteins, CARs (including universal CARs), and engineered TCRs.

[0145] The cells obtained from the subject can be any of the cells discussed herein, for example, TILs. In some aspects, the cells obtained from the patient can be peripheral blood mononuclear cells (PBMCs).

[0146] In some aspects, PBMCs are used directly for genetic modification with the immune cells (such as CARs or TCRs) using methods as described herein. In certain embodiments, after isolating the PBMCs, T lymphocytes are further isolated, and both cytotoxic and helper T lymphocytes are sorted into naive, memory, and effector T cell subpopulations either before or after genetic modification and / or expansion.

[0147] In some aspects, CD8+ cells are further sorted into naive, central memory, and effector cells by identifying cell surface antigens that are associated with each of these types of CD8+ cells. In some embodiments, the expression of phenotypic markers of central memory T cells includes CCR7, CD3, CD28, CD45RO, CD62L, and CD127 and are negative for granzyme B. In some embodiments, central memory T cells are CD8+, CD45RO+, and CD62L+ T cells. In some embodiments, effector T cells are negative for CCR7, CD28, CD62L, and CD127 and positive for granzyme B and perforin. In certain embodiments, CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens.

[0148] In applications wherein cells are taken from a subject and then eventually transplanted back into the subject therapeutically, these cells are said to be “autologous”. In aspects wherein cells are taken from a first subject and then eventually transplanted into a different, second subject, these cells are said to be “allogeneic”.

[0149] The term "introducing" is intended to include presenting to the cell, the nucleic acid molecules in such a manner that the nucleic acid molecule gains access to the interior of the host cell. The methods of the present disclosure do not depend on a particular method for introducing a nucleic acid molecule into a host cell, only that the polynucleotide construct gains access to the interior of one cell of the host. Methods for introducing nucleic acid molecules (e.g. vectors) into bacteria, plants, fungi, and animals are known in the art including, but not limited to, stable transformation methods, transient transformation methods, and virus-mediated methods. For example, lentiviral and retroviral transduction methods can be used. As would be appreciated by the skilled artisan, these methods can comprise the use of retronectin to enhance efficiency of transduction.

[0150] The methods of cell production described above can further comprise, before step (b), after step (b), or both before step (b) and after step (b), expanding the cells. Cell expansion can be accomplished using any cell expansion method known in the art.

[0151] The methods of cell production described above can further comprise, before step (b), after step (b), or both before step (b) and after step (b), culturing the cells. Cell culturing can be accomplished using any cell culturing method known in the art.

[0152] The manufacturing methods described above can further comprise, after step (b), enriching for cells that express the synthetic HER2 polypeptide of the present disclosure. Theenrichment can be accomplished by contacting the plurality of cells with an affinity reagent that binds to the synthetic HER2 polypeptide of the present disclosure, thereby enriching for cells that express the synthetic HER2 polypeptide of the present disclosure. In some aspects, the affinity reagent is an antibody that binds to the synthetic HER2 polypeptide of the present disclosure. In some aspects, following enrichment, at least about 10%, or at least about 15%, or at least about 20%, or at least about 25%, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45%, or at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99%, or at least about 100% of the cells express the synthetic HER2 polypeptide of the present disclosure.

[0153] General Methods and Definitions

[0154] Methods of Gene Delivery and Cell Modification

[0155] One of skill in the art would be well-equipped to construct a vector through standardrecombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al, 1996, bothincorporated herein by reference) for the expression of the antigen receptors of the present disclosure. Vectors include but are not limited to, plasmids, cosmids, viruses (bacteriophage, animal viruses, and plant viruses), and artificial chromosomes (e.g., YACs), such as retroviral vectors (e.g. derived from Moloney murine leukemia virus vectors (MoMLV), MSCV, SFFV, MPSV, SNV etc), lentiviral vectors (e.g. derived from HIV-1, HIV-2, SIV, BIV, FIV etc.), adenoviral (Ad) vectors including replication competent, replication deficient and gutless forms thereof, adeno-associated viral (AAV) vectors, simian virus 40 (SV-40) vectors, bovine papilloma virus vectors, Epstein-Barr virus vectors, herpes virus vectors, vaccinia virus vectors, Harvey murine sarcoma virus vectors, murine mammary tumor virus vectors, Rous sarcoma virus vectors, parvovirus vectors, polio virus vectors, vesicular stomatitis virus vectors, maraba virus vectors, and group B adenovirus enadenotucirev vectors.

[0156] Viral Vectors

[0157] Viral vectors encoding an antigen receptor, a cytokine, and / or a functional effectorelement may be provided in certain aspects of the methods of the present disclosure. In generating recombinant viral vectors, non-essential genes are typically replaced with a gene or coding sequence for a heterologous (or non-native) protein. A viral vector is a kind of expressionconstruct that utilizes viral sequences to introduce nucleic acid and possibly proteins into a cell. The ability of certain viruses to infect cells or enter cells via receptor mediated-endocytosis, and to integrate into host cell genomes and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign nucleic acids into cells (e.g., mammalian cells). Non-limiting examples of virus vectors that may be used to deliver a nucleic acid of certain aspects of the present invention are described below.

[0158] In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and / or a genomic editing construct into an immune cell ex vivo, in vivo, in vitro, or in situ comprises a viral vector. In some embodiments, the viral vector is a non-integrating non- chromosomal vector. Exemplary non-integrating non-chromosomal vectors include, but are not limited to, adeno-associated virus (AAV), adenovirus, and herpes viruses. In some embodiments, the viral vector is an integrating chromosomal vector. Integrating chromosomal vectors include, but are not limited to, adeno-associated vectors (AAV), lentiviruses, and gamma-retroviruses.

[0159] In some embodiments of the methods of the disclosure, introducing a nucleic acid sequence and / or a genomic editing construct into an immune cell ex vivo, in vivo, in vitro, or in situ comprises a combination of vectors. Exemplary, non-limiting vector combinations include: viral and non-viral vectors, a plurality of non-viral vectors, or a plurality of viral vectors. Exemplary but non-limiting vector combinations include: a combination of a DNA-derived and an RNA-derived vector, a combination of an RNA and a reverse transcriptase, a combination of a transposon and a transposase, a combination of a non-viral vector and an endonuclease, and a combination of a viral vector and an endonuclease.

[0160] In some embodiments of the methods of the disclosure, genome modification comprising introducing a nucleic acid sequence and / or a genomic editing construct into an immune cell ex vivo, in vivo, in vitro, or in situ stably integrates a nucleic acid sequence, transiently integrates a nucleic acid sequence, produces site-specific integration of a nucleic acid sequence, or produces a biased integration of a nucleic acid sequence. In some embodiments, the nucleic acid sequence is a transgene.

[0161] In some embodiments of the methods of the disclosure, genome modification comprising introducing a nucleic acid sequence and / or a genomic editing construct into an immune cell ex vivo, in vivo, in vitro, or in situ stably integrates a nucleic acid sequence. In some embodiments, the stable chromosomal integration can be a random integration, a site-specific integration, or abiased integration. In some embodiments, the site-specific integration can be non-assisted or assisted. In some embodiments, the assisted site-specific integration is co-delivered with a site- directed nuclease. In some embodiments, the site-directed nuclease comprises a transgene with 5’ and 3’ nucleotide sequence extensions that contain a percentage homology to upstream and downstream regions of the site of genomic integration. In some embodiments, the transgene with homologous nucleotide extensions enables genomic integration by homologous recombination, microhomology-mediated end joining, or nonhomologous end-joining. In some embodiments the site-specific integration occurs at a safe harbor site. Genomic safe harbor sites are able to accommodate the integration of new genetic material in a manner that ensures that the newly inserted genetic elements function reliably (for example, are expressed at a therapeutically effective level of expression) and do not cause deleterious alterations to the host genome that cause a risk to the host organism. Potential genomic safe harbors include, but are not limited to, intronic sequences of the human albumin gene, the adeno-associated virus site 1 (AAVS1), a naturally occurring site of integration of AAV virus on chromosome 19, the site of the chemokine (C-C motif) receptor 5 (CCR5) gene, and the site of the human ortholog of the mouse Rosa26 locus.

[0162] In some embodiments, the site-specific transgene integration occurs at a site that disruptsexpression of a target gene. In some embodiments, disruption of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements. In some embodiments, exemplary target genes targeted by site-specific integration include but are not limited to any immunosuppressive gene and genes involved in allo-rejection.

[0163] In some embodiments, the site-specific transgene integration occurs at a site that resultsin enhanced expression of a target gene. In some embodiments, enhancement of target gene expression occurs by site-specific integration at introns, exons, promoters, genetic elements, enhancers, suppressors, start codons, stop codons, and response elements.

[0164] Regulatory Elements

[0165] Expression cassettes included in vectors useful in the present disclosure in particularcontain (in a 5'-to-3' direction) a eukaryotic transcriptional promoter operably linked to a protein- coding sequence, splice signals including intervening sequences, and a transcriptional termination / polyadenylation sequence. The promoters and enhancers that control thetranscription of protein encoding genes in eukaryotic cells are composed of multiple genetic elements. The cellular machinery is able to gather and integrate the regulatory information conveyed by each element, allowing different genes to evolve distinct, often complex, patterns of transcriptional regulation. A promoter used in the context of the present disclosure includes constitutive, inducible, and tissue-specific promoters.

[0166] Origins of Replication

[0167] In order to propagate a vector in a host cell, it may contain one or more origins of replication sites (often termed "ori"), for example, a nucleic acid sequence corresponding to oriP of EBV as described above or a genetically engineered oriP with a similar or elevated function in programming, which is a specific nucleic acid sequence at which replication is initiated. Alternatively, a replication origin of another extra-chromosomally replicating virus as described above or an autonomously replicating sequence (ARS) can be employed.

[0168] Other Methods of Nucleic Acid Delivery

[0169] In addition to viral delivery of the nucleic acids encoding the antigen receptor, the following are additional methods of recombinant gene delivery to a given cell, (e.g. an NK cell) and are thus considered in the present disclosure.

[0170] Introduction of a nucleic acid molecule, such as DNA or RNA, into the immune cells of the current disclosure may use any suitable methods for nucleic acid delivery for transformation of a cell, as described herein or as would be known to one of ordinary skill in the art. Such methods include, but are not limited to, direct delivery of DNA such as by ex vivo transfection, by injection, including microinjection; by electroporation; by calcium phosphate precipitation; by using DEAE-dextran followed by polyethylene glycol; by direct sonic loading; by liposome mediated transfection and receptor-mediated transfection; by lipid nanoparticle transfection; by microprojectile bombardment; by agitation with silicon carbide fibers; by Agrobacterium- mediated transformation; by desiccation / inhibition-mediated DNA uptake, and any combination of such methods. Through the application of techniques such as these, organelle(s), cell(s), tissue(s) or organism(s) may be stably or transiently transformed.

[0171] Generally, the gene transfer system can include a transposon-based or a viral-based integration system.

[0172] In some embodiments, the gene transfer system comprises a transposon system. DNA transposons can translocate via a non-replicative “cut-and-paste” mechanism. This mechanismrequires recognition of the two inverse terminal repeats (ITRs) by a catalytic enzyme, i.e., transposase, which can cleave its target and consequently release the DNA transposon from its donor template. Upon excision, the DNA transposons may subsequently integrate into the acceptor DNA that is cleaved by the same transposase. In some of their natural configurations, DNA transposons are flanked by two ITRs and may contain a gene encoding a transposase that catalyzes transposition. As would be appreciated by the skilled artisan, transposon systems offer many advantages for nucleic acid integration, e.g., as compared to viral vectors. For example, transposons can carry larger cargos which can be advantageous for delivering one or more of the CARs, functional effector elements, and / or cytokines disclosed herein, to an immune cell (e.g., an NK cell). Further, transposons may comprise, for example, CRISPR tools (e.g., along with cargo), and thereby allow multiplex engineering of a cell.

[0173] Chimeric Antigen Receptors

[0174] A "chimeric antigen receptor" is also known as an artificial cell receptor, a chimeric cell receptor, or a chimeric immunoreceptor. As would be appreciated by the skilled artisan, Chimeric antigen receptors (CARs) are engineered receptors, which graft a selected specificity onto an immune effector cell. CARs typically have an extracellular domain (ectodomain), a transmembrane domain, and an intracellular (endodomain) domain. In some embodiments, the ectodomain comprises an antigen-binding domain and a stalk region, wherein the antigen- binding domain specifically binds to an antigen that is of particular interest in the treatment of a specific disease or disorder (e.g. an antigen that is located on particular cancer cells or an antigen located on an infected cell). In some embodiments, the antigen is a protein expressed on the surface of cells (e.g., on the surface of a cancer cell, or an infected cell).

[0175] As would be appreciated by the skilled artisan, CARs are available in various different “formats”, sometimes also referred to as different “generations” of CARs (see e.g. Hiltensperger M, Krackhardt AM. Current and future concepts for the generation and application of genetically engineered CAR-T and TCR-T cells. Front Immunol.2023 Mar 6;14:1121030. doi: 10.3389 / fimmu.2023.1121030. PMID: 36949949; PMCID: PMC10025359). Accordingly, the term engineered CAR as used herein encompasses any of the formats / generations known in the art.

[0176] In a non-limiting example, the CAR can be in the format of a universal CAR, such as those disclosed in PCT Publication No. WO / 2012 / 082841 and US Patent Nos.9,233,125 and10,973,893. As would be appreciated by the skilled artisan, in universal CAR systems, the extracellular domain of the CAR specifically binds to a common “tag” molecule. Such CARs can be referred to as “anti-tag CARs”. These tag molecules can then be fused to antigen-targeting molecules (e.g. antibodies), such that the anti-tag CARs bind to a target cell of interest indirectly through the tag-fusion protein. In this way, subjects can be administered a single population of cells expressing an anti-tag CAR and then a variety of different tag-fusion proteins that can specifically direct the anti-tag CAR cells to target different cells.

[0177] Engineered T cell Receptors

[0178] As would be appreciated by the skilled artisan, a T cell Receptor (TCR) is a heterodimeric cell surface protein of the immunoglobulin super-family, which is associated with invariant proteins of the CD3 complex involved in mediating signal transduction. TCRs exist as and heterodimers, which are structurally similar but have quite distinct anatomical locations and probably functions. The extracellular portion of native heterodimeric PTCR consists of two polypeptide chains, each of which has a membrane-proximal constant domain and a membrane-distal variable domain. Each of the constant and variable domains includes an intra-chain disulfide bond. The variable domains contain the highly polymorphic loops analogous to the complementarity determining regions (CDRs) of antibodies. As used herein, the term “engineered T cell receptor” refers to TCRs that have been designed to specifically bind to an antigen that is of particular interest in the treatment of a specific disease or disorder (e.g. an antigen that is located on particular cancer cells or an antigen located on an infected cell). In some embodiments, the antigen is a protein expressed on the surface of cells (e.g., on the surface of a cancer cell, or an infected cell). As would be appreciated by the skilled artisan, engineered TCRs are available in various different “formats” (see e.g. Hiltensperger M, Krackhardt AM. Current and future concepts for the generation and application of genetically engineered CAR-T and TCR-T cells. Front Immunol.2023 Mar 6;14:1121030. doi: 10.3389 / fimmu.2023.1121030. PMID: 36949949; PMCID: PMC10025359). Accordingly, the term engineered TCR as used herein encompasses any of the formats known in the art.

[0179] Definitions

[0180] As used herein, the term "antigen" is a molecule capable of being bound by an antibody, T cell receptor, Chimeric Antigen Receptor, and / or engineered immune receptor. An antigenmay generally be used to induce a humoral immune response and / or a cellular immune response leading to the production of B and / or T lymphocytes.

[0181] The terms "tumor-associated antigen," "tumor antigen," and "cancer cell antigen" are used interchangeably herein. In each case, the terms refer to proteins, glycoproteins, or carbohydrates that are specifically or preferentially expressed by cancer cells.

[0182] As used herein, the term “portion” when used in reference to a polypeptide or a peptide refers to a fragment of the polypeptide or peptide. In some embodiments, a “portion” of a polypeptide or peptide retains at least one function and / or activity of the full-length polypeptide or peptide from which it was derived. For example, in some embodiments, if a full-length polypeptide binds a given ligand, a portion of that full-length polypeptide also binds to the same ligand.

[0183] The terms “protein” and “polypeptide” are used interchangeably herein.

[0184] The term "exogenous," when used in relation to a protein, gene, nucleic acid, or polynucleotide in a cell or organism refers to a protein, gene, nucleic acid, or polynucleotide that has been introduced into the cell or organism by artificial or natural means; or in relation to a cell, the term refers to a cell that was isolated and subsequently introduced into a cell population or to an organism by artificial or natural means. An exogenous nucleic acid may be from a different organism or cell, or it may be one or more additional copies of a nucleic acid that occurs naturally within the organism or cell. An exogenous cell may be from a different organism, or it may be from the same organism. By way of a non-limiting example, an exogenous nucleic acid is one that is in a chromosomal location different from where it would be in natural cells or is otherwise flanked by a different nucleic acid sequence than that found in nature. The term “exogenous” is used interchangeably with the term “heterologous”.

[0185] The terms "expression construct" and "expression cassette" are used to mean a nucleic acid molecule that is capable of directing transcription. An expression construct includes, at a minimum, one or more transcriptional control elements (such as promoters, enhancers, or a structure functionally equivalent thereof) that direct gene expression in one or more desired cell types, tissues, or organs. Additional elements, such as a transcription termination signal, may also be included.

[0186] A "gene," "polynucleotide," "coding region," "sequence," "nucleic acid sequence," "segment," "fragment," or "transgene" that "encodes" a particular protein, is a section of anucleic acid molecule that is transcribed and optionally also translated into a gene product, e.g., a polypeptide, in vitro or in vivo when placed under the control of appropriate regulatory sequences. The coding region may be present in either a cDNA, genomic DNA, or RNA form. When present in a DNA form, the nucleic acid molecule may be single-stranded (i.e., the sense strand) or double-stranded. The boundaries of a coding region are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A gene can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences. A transcription termination sequence will usually be located 3' to the gene sequence.

[0187] The term "cell" is herein used in its broadest sense in the art and refers to a living body that is a structural unit of tissue of a multicellular organism, is surrounded by a membrane structure that isolates it from the outside, has the capability of self-replicating, and has genetic information and a mechanism for expressing it. Cells used herein may be naturally-occurring cells or artificially modified cells (e.g., fusion cells, genetically modified cells, etc.).

[0188] "Antibody" as used herein refers to monoclonal or polyclonal antibodies. The term "monoclonal antibodies," as used herein, refers to antibodies that are produced by a single clone of B-cells and bind to the same epitope. In contrast, "polyclonal antibodies" refer to a population of antibodies that are produced by different B-cells and bind to different epitopes of the same antigen. A whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CHL CH2 and CH3) regions, and each light chain contains one N-terminal variable (VL) region and one C-terminal constant (CL) region. The variable regions of each pair of light and heavy chains form the antigen binding site of an antibody. The VH and VL regions have a similar general structure, with each region comprising four framework regions, whose sequences are relatively conserved. The framework regions are connected by three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2, and CDR3, form the "hypervariable region" of an antibody, which is responsible for antigen binding.

[0189] The term "T cell" refers to T lymphocytes, and includes, but is not limited to, / T cells, / T cells, NK T cells, CD4+ T cells and CD8+ T cells. CD4+ T cells include THO, Th1 and TH2 cells, as well as regulatory T cells (Treg). There are at least three types of regulatory T cells:CD4+ CD25+ Treg, CD25 TH3 Treg, and CD25 TR 1 Treg. "Cytotoxic T cell" refers to a T cell that can kill another cell. The majority of cytotoxic T cells are CD8+ MHC class I-restricted T cells, however some cytotoxic T cells are CD4+. In some embodiments, the T cell of the present disclosure is CD4+ or CD8+.

[0190] The activation state of a T cell defines whether the T cell is "resting" (i.e., in the Go phase of the cell cycle) or "activated" to proliferate after an appropriate stimulus such as the recognition of its specific antigen or by stimulation with OKT3 antibody, PHA or PMA, etc. The "phenotype" of the T cell (e.g., naive, central memory, effector memory, lytic effectors, help effectors (THI and TH2 cells), and regulatory effectors), describes the function the cell exerts when activated. A healthy donor has T cells of each of these phenotypes and which are predominately in the resting state. A naive T cell will proliferate upon activation, and then differentiate into a memory T cell or an effector T cell. It can then assume the resting state again, until it gets activated the next time, to exert its new function and may change its phenotype again. An effector T cell will divide upon activation and antigen-specific effector function.

[0191] "Natural killer T cells" (NKT cells), not to be confused with natural killer cells of the innate immune system, bridge the adaptive immune system with the innate immune system. Unlike conventional T cells that recognize peptide antigens presented by major histocompatibility complex (WIC) molecules, NKT cells recognize glycolipid antigen presented by a molecule called CD1d. Once activated, these cells can perform functions ascribed to both Th and Tc cells (i.e., cytokine production and release of cytolytic / cell killing molecules). They are also able to recognize and eliminate some tumor cells and cells infected with herpes viruses.

[0192] “Natural killer cells” (“NK cells”) are a type of cytotoxic lymphocyte of the innate immune system. In some instances, NK cells provide a first line defense against viral infections and / or tumor formation. NK cells can detect MHC presented on infected or cancerous cells, triggering cytokine release, and subsequently induce lysis and apoptosis. NK cells can further detect stressed cells in the absence of antibodies and / or MHC, thereby allowing a rapid immune response.

[0193] The term "culturing" refers to the in vitro maintenance, differentiation, and / or propagation of cells in suitable media. By "enriched" is meant a composition comprising cells present in a greater percentage of total cells than is found in the tissues where they are present in an organism.

[0194] As used throughout the disclosure, identity between two sequences may be determined by using the stand-alone executable BLAST engine program for blasting two sequences (bl2seq), which can be retrieved from the National Center for Biotechnology Information (NCBI) ftp site, using the default parameters (Tatusova and Madden, FEMS Microbiol Lett., 1999, 174, 247-250; which is incorporated herein by reference in its entirety). The terms "identical" or "identity" when used in the context of two or more nucleic acids or polypeptide sequences, refer to a specified percentage of residues that are the same over a specified region of each of the sequences. In some embodiments, the sequence identity is determined over the entire length of a sequence. The percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over the specified region, determining the number of positions at which the identical residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying the result by 100 to yield the percentage of sequence identity. In cases where the two sequences are of different lengths or the alignment produces one or more staggered ends and the specified region of comparison includes only a single sequence, the residues of single sequence are included in the denominator but not the numerator of the calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm such as BLAST or BLAST 2.0.

[0195] Exemplary Embodiments

[0196] Embodiment 1. A synthetic HER2 polypeptide comprising, from N-terminus to C- terminus: i) a signal peptide; ii) an extracellular domain comprising the amino acid sequence of SEQ ID NO: 1; iii) a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 2; and iv) an intracellular domain comprising a single lysine residue.

[0197] Embodiment 2. The synthetic HER2 polypeptide of embodiment 1, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO: 3.

[0198] Embodiment 3. The synthetic HER2 polypeptide of embodiment 1 or embodiment 2, wherein the synthetic HER2 polypeptide has the amino acid sequence of SEQ ID NO: 4.

[0199] Embodiment 4. A nucleic acid molecule comprising a nucleic acid sequence encoding the synthetic HER2 polypeptide of any one of embodiments 1-3.

[0200] Embodiment 5. The nucleic acid molecule of embodiment 5, wherein the nucleic acid sequence encoding the synthetic HER2 polypeptide comprises the nucleic acid sequence of SEQ ID NO: 7.

[0201] Embodiment 6. The nucleic acid molecule of embodiment 4 or embodiment 5, wherein the nucleic acid molecule further comprises a nucleic acid sequence encoding a CD8a:MYD88 fusion protein, wherein the CD8a:MYD88 fusion protein comprises, from N-terminus to C- terminus a first domain that comprises a CD8a polypeptide, a linker polypeptide, and a second domain that comprises an MYD88 polypeptide.

[0202] Embodiment 7. The nucleic acid molecule of embodiment 6, wherein: i) the first domain of the CD8a:MYD88 fusion protein comprises the amino acid sequence of SEQ ID NO: 12; ii) the linker polypeptide of the CD8a:MYD88 fusion protein comprises the amino acid sequence of SEQ ID NO: 14; and iii) the second domain of the CD8a:MYD88 fusion protein comprises the amino acid sequence of SEQ ID NO: 13, preferably wherein the CD8a:MYD88 fusion protein has the amino acid sequence of SEQ ID NO: 15.

[0203] Embodiment 8. The nucleic acid molecule of embodiment 6 or embodiment 7, wherein the nucleic acid sequence encoding a CD8a:MYD88 fusion protein comprises the nucleic acid sequence of SEQ ID NO: 8.

[0204] Embodiment 9. The nucleic acid molecule of any one of embodiments 6-8, wherein the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 11.

[0205] Embodiment 10. A vector comprising the nucleic acid molecule of any one of embodiments 4-9, preferably wherein the vector is a viral vector, preferably wherein the viral vector is a lentiviral vector.

[0206] Embodiment 11. A cell expressing the synthetic HER2 polypeptide of any one of embodiments 1-3.

[0207] Embodiment 12. The cell of embodiment 11, wherein the cell also expresses a CD8a:MYD88 fusion protein,wherein the CD8a:MYD88 fusion protein comprises, from N-terminus to C-terminus a first domain that comprises a CD8a polypeptide, a linker polypeptide, and a second domain that comprises an MYD88 polypeptide, i) the first domain of the CD8a:MYD88 fusion protein comprises the amino acid sequence of SEQ ID NO: 12; ii) the linker polypeptide of the CD8a:MYD88 fusion protein comprises the amino acid sequence of SEQ ID NO: 14; and iii) the second domain of the CD8a:MYD88 fusion protein comprises the amino acid sequence of SEQ ID NO: 13, preferably wherein the CD8a:MYD88 fusion protein has the amino acid sequence of SEQ ID NO: 15.

[0208] Embodiment 13. The cell of embodiment 11 or embodiment 12, wherein the cell alsoexpresses at least one of: i) a chimeric antigen receptor (CAR); and ii) an engineered T cell receptor (TCR).

[0209] Embodiment 14. The cell of any one of embodiments 10-12, wherein the cell is a tumorinfiltrating lymphocyte (TIL), preferably wherein the TIL is a T cell.

[0210] Embodiment 15. A population of the cells of any one of embodiments 11-14.

[0211] Embodiment 16. A method of treating cancer in a subject, the method comprisingadministering to the subject the cell population of embodiment 15.

[0212] Embodiment 17. The method of embodiment 16, wherein the cancer is a solid tumor,preferably wherein the solid tumor is head and neck cancer, lung cancer, or a sarcoma.

[0213] Embodiment 18. The method of embodiment 16 or embodiment 17, further comprising ata time point after administration of the cell population, administering to the subject at least one amount of at least one cytotoxic antibody that binds to the synthetic HER2 polypeptide, therebyreducing and / or eliminating the cell population and / or cells that result from in vivo expansion ofthe cell population.

[0214] Embodiment 19. The method of embodiment 18, wherein the at least one cytotoxicantibody that binds to the synthetic HER2 polypeptide is ado-trastuzumab emtansine, fam- trastuzumab deruxtecan-nxki, margetuximab, trastuzumab, trastuzumab-anns, or a combination thereof.

[0215] Embodiment 20. The method of any one of embodiments 16-19, further comprising, at atime point after administration of the population of cells: i) administering to the subject at least one amount of an antibody that: a) binds to the synthetic HER2 polypeptide; and b) is linked to a detectable label; and ii) imaging the subject, or a portion thereof, with an imaging modality suitable to detect the detectable label, thereby identifying the presence and location of the therapeutic cells within the subject.

[0216] Embodiment 21. The method of any one of embodiments 16-20, further comprising, at atime point after administration of the population of cells: i) obtaining one or more biological samples from the subject; ii) contacting the one more biological samples with at least one amount of an antibody that: a) binds to the synthetic HER2 polypeptides; and b) is linked to a detectable label; and iii) imaging the one or more biological samples with an imaging modality suitable to detect the detectable label, thereby identifying the presence and location of the therapeutic cells within the one or more biological samples, preferably wherein the one or more biological samples comprises a tumor sample, a blood sample, or a combination thereof.

[0217] Embodiment 22. A method of producing the population of cells of 14, the methodcomprising: a) obtaining a plurality of cells from a subject; and b) introducing into the plurality of cells a one or more nucleic acid molecules, wherein the one or more nucleic acid molecules comprise a nucleic acid sequence encoding the synthetic HER2 polypeptide, preferably wherein introducing into the plurality of cells one or more nucleic acid molecules comprises performing retroviral transduction.

[0218] Embodiment 23. The method of embodiment 22, wherein the plurality of cells obtainedfrom the subject are peripheral blood mononuclear cells (PBMCs).

[0219] Embodiment 24. The method of embodiment 22 or embodiment 23, further comprising,before step (b), after step (b), or both before step (b) and after step (b), expanding the cells.

[0220] Embodiment 25. The method of any one of embodiments 22-24, wherein the methodfurther comprises, after step (b), contacting the plurality of cells with an affinity reagent thatbinds to the synthetic HER2 polypeptide of the present disclosure, thereby enriching for cells that express the synthetic HER2 polypeptide.

[0221] Experimental Examples

[0222] Example 1

[0223] The following experimental example describes the testing of the synthetic HER2 polypeptides of the present disclosure and their use in several of the methods of the present disclosure.

[0224] FIG.1 shows a schematic diagram of the expression cassettes of the four constructs tested in this experimental example: MYC – a negative control construct comprising a construct with a signal peptide (denoted LP in FIG.1), a MYC-containing extracellular domain, a CD8a stalk, and a CD8a transmembrane domain. HER2FL – a construct comprising a signal peptide (denoted LP in FIG.1), followed by full length HER2. HER2IV – a construct comprising a signal peptide (denoted LP in FIG.1), followed by an extracellular domain comprising Domain IV of HER2, followed by the transmembrane domain of HER2. HER2III&IV – a construct comprising a signal peptide (denoted LP in FIG.1), followed by an extracellular domain comprising Domain III and Domain IV of HER2, followed by the transmembrane domain of HER2.

[0225] As shown in FIG.1, the expression cassettes also included a self-cleaving peptide sequence followed by GFP, which allows for the tracking of expression in transduced cells.

[0226] In a first experiment, the four expression cassettes shown in FIG.1 were introduced into PBMCs by retroviral transduction. The cells were then stained with Zombie aqua, PE-conjugated anti-human CD3 antibody, and Alexa Fluor 700-conjugated trastuzumab (an anti-HER2 antibody) biosimilar. Fluorescent flow cytometry analysis was performed in Cytek Aurora. Cells were gated on live CD3+ cells for plot generation. FIG.2 shows the results of the flow cytometry analysis, which indicate that the synthetic HER2 constructs were successfully expressed on the surface of the cells.

[0227] Fluorescent flow cytometry analysis was then repeated using Alexa Fluor 700-conjugated trastuzumab biosimilar varying with 2-fold serial dilution starting from 1024 ng / ml. Cells were gated on live CD3+ GFPhi / GFPlow cells for plot generation. This analysis is shown in FIG.3. As shown inFIG.3, the HER2 constructs exhibited strong expression, with the HERIII&IV construct exhibiting the highest expression levels based on the MFI of the Alexa Fluor 700-conjugated trastuzumab biosimilar. The higher levels of HERIII&IV were readily apparent within the GFP-low population of cells.

[0228] In another experiment, the in vitro depletion of PBMCs expressing the constructs of FIG. 1 was tested. Retroviral transduction was used to introduce the expression cassettes of FIG.1 into PBMCs. Cells were then treated with the cetuximab (anti-EGFR antibody), trastuzumab, or ado-trastuzumab emtansine (KADCYLA®; a trastuzumab ADC with cytotoxic effector) at concentrations with 2-fold serial dilution for 72 hours. Fold change of GFP percentage was analyzed by normalization to a group of cells that was not treated with any antibody. Approximately 40% of T cells were GFP positive. Accordingly, these GFP negative cells were used as internal controls. The results of the depletion experiment are shown in FIG.4. As shown in FIG.4, neither cetuximab treatment or trastuzumab treatment resulted in the depletion of PBMCs. However, treatment with ado-trastuzumab emtansine (KADCYLA®) resulted in depletion of cells expressing HER2IV and HER2III&IV, with the strongest depletion being observed for the HER2III&IV construct.

[0229] In another experiment, the in vitro depletion of cells expressing the HER2III&IV construct was tested using various different antibodies and antibody-drug-conjugates. Mouse T cells were isolated from spleen and lymph nodes and were engineered to express the HER2III&IV construct. Cells were treated with IgG control, Cetuximab, Trastuzumab, KADCYLA®, ENHERTU®, or combination of KADCYLA® and ENHERTU® at concentrations with 2-fold serial dilution for 3 days. FIG.5 shows results of this experiment, more specifically, the percentage of GFP positive relative to the frequency of non-engineered T cells. As shown in FIG.5, robust depletion was observed with KADCYLA® and the combination of KADCYLA® and ENHERTU®.

[0230] In a final experiment, the in vivo depletion of pmel-1 T cells expressing the HER2III&IV construct was tested. First, CD90.1+ TCR transgenic pmel-1 T cells were isolated from the spleen and lymph nodes of the mice and were then engineered to express the HER2III&IV construct.106 cells were transfer to C57BL / 6J mice of 6-8 weeks through intravenous injection.10,000 units of rhIL-2 was administrated intraperitoneally every day for 3 days after cell transfer. Antibodies were administrated via intraperitoneal injection at a dose of 200 g / mouse at day 7 and 10 after cell transfer. At day 12 after cell transfer, mice were euthanized, and T cells isolated from spleen were analyzed for GFP percentage in live CD3+CD8+CD90.1+ population. The results of this analysis are shown in FIG.6. More specifically, FIG.6 shows the relative fold change of GFP percentage plottedby normalization to the control cetuximab group. Each dot represents one mouse. As shown in FIG. 6, the use of KADCYLA® resulted in robust depletion of the HER2III&IVexpressing cells.

[0231] Without wishing to be bound by theory, the results presented in this example demonstratethat the synthetic HER2 polypeptides of the present disclosure, most notably those containing an extracellular domain comprising the HER2 III domain and the HER2 IV domain, are readily expressed in immune cells following retroviral transduction and these constructs can be used torobustly deplete cells both in vitro and in vivo.

[0232] Example 2

[0233] Methods

[0234] Reagents

[0235] The following antibodies and reagents were used for the experiments described herein:human ErbB2 / Her2 (research grade trastuzumab biosimilar) ALEXA FLUOR®647-conjugated antibody (#AB9589R-100UG), human ErbB2 / Her2 (research grade trastuzumab biosimilar) ALEXA FLUOR®700-conjugated antibody (#FAB9589N-100UG), ErbB2 / Her2 (research grade trastuzumab biosimilar) biotinylated antibody (#FAB9589B-100), BRILLIANT VIOLENT™60 anti-mouse CD3 (Biolegend #100237), ALEXA FLUOR®700 anti-mouse CD4 (Biolegend #100430), APC / FIRE™750 anti-mouse CD8a (Biolegend #100766), ALEXA FLUOR ® 647anti-c-Myc Biolegend#626810), PE / Cyanine7 anti-mouse IFN- (Biolegend #505826),cetuximab (ERBITUX®, Eli Lilly), trastuzumab biosimilar (KANJINTI®, Amgen), ENHERTU®(Daiichi Sankyo / AstraZeneca), KADCYLA®(ado-trastuzumab emtansine; Genentech), recombinant human interleukin-2 (Proleukin, Clinigen), IgG from human serum (Sigma #I4506- 100MG), ZOMBIE AQUA™Fixable Viability Kit (Biolegend# 423102), PE Annexin V (Biolegend# 640908), propidium iodide (ThermoFisher #P1304MP), EBIOSCIENCE™ Cell Proliferation Dye EFLUOR™450 (ThermoFisher #65-0842-90).

[0236] Cell culture

[0237] B16-F10 cells (CRL-6475, ATCC), Phoenix-ECO cells (CRL3214, ATCC), andPhoenix-AMPHO cells (CRL-3213, ATCC), were cultured in DMEM (10-017-CV, Corning) supplemented with 10% FBS (PS-100, Phoenix Scientific) and 100 U / mL penicillin and 100 g / mL streptomycin (15140-122, Gibco) (pen / strep). Jurkat cells were cultured in RPMI-1640 (CM058-050, GenDEPOT) supplemented with 10% fetal bovine serum (FBS). pmel-1splenocytes and transduced T cells were cultured in RPMI-1640 supplemented with 10% FBS, pen / strep, 50 M 2-mercaptoethanol (21-985-023, Gibco), and 100 U / mL IL-2.

[0238] Mice

[0239] C57BL / 6J (#000664), pmel-1 (#005023), and NSG (#005557) were purchased from TheJackson Laboratory and housed under specific-pathogen-free conditions. The mice were humanely euthanized using compressed CO2 gas for primary euthanasia. Cardiac perfusion or cervical dislocation were used as secondary euthanasia methods.

[0240] Plasmids

[0241] A bicistronic gene construct encoding either full-length human HER2 (HER2FL) or itstruncated variants, along with enhanced green fluorescent protein (EGFP), linked by a T2Asequence was cloned into the pMSGV1 vector under the control of the human EF1 promoter.The HER2 variants were designed to retain the transmembrane domain and specific extracellular regions, including domain III & IV (HER2III&IV, 342–676 aa), domain IV alone (HER2IV, 511– 676 aa), or a shorter version of domain IV (HER2IV-S, 563–676 aa).The HER2III&IVvariant was further engineered by fusing two copies of the Myc tag (EQKLISEEDL, SEQ ID NO: 16) at the N-terminus (MycHER2III&IV) and introducing an amino acid substitution at position 499, replacing tryptophan with alanine (MycHER2III&IVW499A). Similarly, a truncated CD8 construct (mycCtrl) containing six copies of the Myc tag at the N-terminus, along with the stalk and transmembrane domain, was used as a control. Additionally, a tricistronic construct (HER2III&IV-LUC-EGFP) linked by T2A sequences, encoding HER2III&IV, firefly luciferase(LUC), and EGFP, was generated to enable in vivo cell monitoring.

[0242] Cell lines and retrovirus transduction

[0243] Stable retrovirus-producing Phenix-ECO cell lines expressing the gene of interest weregenerated through Phenix-Ampho cell-mediated transduction. Briefly, the pCL-Ampho helper plasmid and the plasmid of interest were co-transfected into Phenix-Ampho cells using LIPOFECTAMINE™2000, according to the manufacturer's instructions. Viral supernatants were collected 48 hours post-transfection and used to transduce Phenix-ECO cells in RetroNectin- coated 24-well plates, following the manufacturer's protocol. The transduced Phenix-ECO cells were subsequently expanded and purified by MACS sorting using a biotin-conjugated anti-HER2 antibody, following the manufacturer's protocol. For certain experiments, viruses collected fromtransiently transfected Phenix-ECO cells were used. Similarly, a Jurkat-derived cell line expressing HER2III&IV-LUC-EGFP was generated.

[0244] For generating engineered mouse T cells, C57BL / 6 T cells were activated 48 hours priorto transduction using plate-bound anti-CD3 (1 µg / mL), soluble anti-CD28 (2 µg / mL), and 100 U / mL of IL-2. Efficiency was determined by assessing the frequency of GFP+cells by flow cytometry.

[0245] HER2 variants expression and ADC effects on T cells in vitro

[0246] For determining HER2 variant expression on the cell surface, cells were stained withserially diluted ALEXA FLUOR®647-conjugated anti-HER2 antibody and analyzed by flow cytometry. Mean fluorescence intensity (MFI) was used to quantify HER2 expression levels.

[0247] To study the ADC killing effects on HER2 variant-expressing T cells, engineered mouseT cells were plated in 96-well flat-bottom plates at a density of 10,000 cells per well in 100 µL of mouse T cell media. Antibodies were serially diluted two-fold in mouse T cell media and added to the culture. After 72 hours, changes in GFP percentage or absolute T cell counts were analyzed by flow cytometry.

[0248] To further characterize the effects of KADCYLA® on T cells, engineered mouse T cellswere labeled with 5 µM proliferation dye and cultured in Grex in the presence of 2 µg / mL of control antibody or KADCYLA®. After 48 hours, cells were collected and subjected to PI / Annexin V staining for apoptosis analysis by flow cytometry. After 72 hours, cells were analyzed for proliferation and cytokine production. All assays were performed according to the manufacturer's instructions.

[0249] In vivo T cell depletion by KADCYLA®

[0250] C57BL / 6J mice received 5 Gy X-ray irradiation on day -3. A total of 2x10 proliferationdye-labeled engineered C57BL / 6 T cells were injected intravenously via the tail vein on day -1. Mice received intraperitoneal injections of 500 µg / mouse of antibodies on day 0 and 10,000 Units of IL-2 for three consecutive days. On day 3, mice were euthanized for tissue collection, and GFP percentage in proliferation dye-labeled T cells was analyzed in blood, spleen, peripheral lymph nodes, bone marrow, and liver. Similarly, engineered pmel-1 T cells were used to determine the optimal dose and time points for KADCYLA®-mediated T cell depletion.

[0251] To study T cell depletion in tumor-bearing mice, 2x10 B16-F10 cells were injectedsubcutaneously into the flanks of 8–10-week-old C57BL / 6J female mice on day -14. Micereceived 5 Gy X-ray irradiation on day -5, followed by intravenous injection of 2x10 engineeredpmel-1 T cells on day -3, along with 10,000 Units of IL-2 for three consecutive days. On day 0, mice were administered 500 µg / mouse of either a control antibody or KADCYLA®. Betweenday 3 and day 7, mice were euthanized for tissue analysis, and the GFP percentage in CD90.1 Tcells was evaluated in blood, spleen, peripheral lymph nodes, bone marrow, liver, lungs, and tumor.

[0252] For in vivo depletion of the Jurkat-HER2III&IV-LUC-EGFP cell line, a total of 2x10Jurkat-HER2III&IV-LUC-EGFP cells were injected intravenously via the tail vein in NSG mice. Luciferase signal was detected using the IVIS Lumina III System (PerkinElmer, USA) following the manufacturer’s instructions. Once the luciferase signal was stably detected, mice received 500 µg / mouse of either a control antibody or KADCYLA®. The mice were monitored for luciferase signal by IVIS imaging weekly.

[0253] Results

[0254] Structure-function-driven design of a HER2-derived, KADCYLA®-activated safetyswitch for engineered T cells

[0255] The FDA-approved HER2-targeting therapeutic antibodies, including Herceptin(trastuzumab), KANJINTI®(trastuzumab-anns), and antibody-drug conjugates (ADCs) like KADCYLA®(ado-trastuzumab emtansine) and ENHERTU®(trastuzumab deruxtecan), share the same fragment antigen-binding (Fab) amino acid sequences and have been proposed to recognize the extracellular domain IV of HER2. These antibodies can inhibit or eliminate HER2- expressing cancer cells through a variety of mechanisms including by blocking receptor signaling, inducing antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC), or, when conjugated to a toxic agent, killing target cells (FIG.7A). This was exploited to generate a safety switch for engineered T cells, such as CAR T cells, by optimizing the truncation of HER2 and the application of FDA-approved HER2-targeting antibodies.

[0256] To evaluate this, a serial of DNA constructs expressing either wildtype full length HER2(HER2FL) or truncated versions that removed the intracellular region was generated. These truncates included HER2III&IV(extracellular domains III and IV), HER2IV(domain IV alone), and HER2IV-S(a short version of domain IV that binds Herceptin). As a control, a constructexpressing a myc-tagged truncated CD8 was used. All constructs expressed EGFP as asecondary reporter to track the engineered T cells (FIG.7B).

[0257] The cell-surface expression of truncated HER2 tags in engineered T cells was compared. It was found that all HER2 truncates were efficiently expressed on the surface of T cells and correlated with EGFP expression. However, HER2FL and HER2III&IV exhibited higher mean fluorescence intensity (MFI) than HER2IV-S and higher yet than HER2IV (FIG.7C). To get a better sense of how these structural changes to HER2 impacted antibody binding, the anti-HER2 antibody was titrated, and MFI changes were analyzed. As shown in FIG.7D, HER2FL demonstrated the greatest level of staining followed by HER2III&IV with AUC. The fold change peaked at 2048 ng / mL. HER2III&IV showed slightly lower fold change than HER2FL but approximately three times higher than HER2IV and two times higher than HER2IV-S. Consistently, across antibody concentrations ranging from 32 ng / mL to 4096 ng / mL, HER2III&IV showed significantly higher sensitivity to antibody detection than HER2IV and HER2IV-S (FIG. 7D).

[0258] To determine whether such differences arose from surface protein abundance or antibody affinity, constructs expressing myc-tagged HER2 truncations were generated, and the MFI of anti-HER2 staining in myc-positive T cells was compared with comparable anti-myc MFI levels. Despite comparable protein abundance on the T cell surface, as indicated by myc staining, HER2IV exhibited an MFI about half that of HER2III&IV (FIGs.7F-7G), suggesting that HER2III&IV has higher affinity for Herceptin Fab than HER2IV. Whether the interaction between HER2 domains III and IV contributed to this difference was investigated further by disrupting the - stacking interaction between these domains through a single amino acid substitution (Trp499 to Ala499). This substitution reduced the binding of HER2III&IV to Herceptin Fab while having a negligible effect on the binding of myc to its antibody, indicating that HER2 domain III stabilizes the binding of domain IV to Herceptin Fab (FIGs.7E-7G).

[0259] Next, to investigate whether binding ability influences antibody-mediated cytotoxicity, the susceptibility of T cells expressing HER2III&IV or HER2IV-S to the cytotoxic effects of KADCYLA® in vitro were evaluated by monitoring the percentage of EGFP-positive T cells in the presence of antibodies for 72 hours. As expected, ERBITUX® (cetuximab), an EGFR- targeting antibody, showed no effect on T cells, nor did KANJINTI®, a trastuzumab biosimilar. In contrast, KADCYLA® specifically reduced the percentage of HER2III&IV- or HER2IV-S- expressing T cells in a dose-dependent manner, as indicated by EGFP expression. Notably, HER2III&IV-expressing T cells were responsive to a 16-fold lower concentration (32 ng / mL vs512 ng / mL) of KADCYLA®compared to HER2IV-S-expressing T cells, indicating that HER2III&IVis an optimal HER2 truncate for a safety switch in T cells due to its high binding ability to KADCYLA®(FIG.7H). KADCYLA®was then compared with another FDA-approved anti-HER2 ADC, ENHERTU®, and the combination of the two drugs. Results showed that KADCYLA®was more efficient than ENHERTU®or the two-drug combination in reducing HER2III&IV-expressing T cells (FIG.7I). To further evaluate KADCYLA®’s toxicity, the absolute cell counts of T cells co-expressing myc and EGFP following KADCYLA®treatment were monitored. The results showed that KADCYLA®could eliminate over 90% of these T cells (FIG.7J). Moreover, HER2III&IVserved efficiently as a surface tag for T cells in MACS enrichment (FIG.7K).

[0260] Finally, whether HER2III&IV tag could be used to track the engineered T cells incirculation (in mice) was investigated. For these studies, mice were injected with T cells expressing HER2III&IVand EGFP. Blood was drawn ten days later and stained with anti-HER2 antibody. FIG.7L is a representative flow cytometry plot showing the ability to track T cells in mice. Collectively, these data suggest that HER2III&IVand KADCYLA®represent the optimal components for a safety switch system in T cell transfer-based therapy.

[0261] Kadcyla induces T cell death, inhibits cell proliferation, and suppresses cytokineproduction

[0262] To investigate the mechanisms of KADCYLA® toxicity to HER2III&IV-expressing T cells,cell death, proliferation, and cytokine production were monitored during expansion in Grex plates with KADCYLA®treatment (FIG.8A). After 48 hours of treatment, both the CD4+EGFP+and CD8+EGFP+populations in the KADCYLA®group showed a higher number of dead cells compared to the control group, as indicated by the Annexin V and propidium iodide double- positive population. Compared to CD4+T cells, CD8+T cells were generally more prone to cell death in culture and more sensitive to KADCYLA®toxicity (FIGs.8B-8C). Consistently, after 72 hours of treatment, there were more poorly proliferated cells in the KADCYLA®group in both CD4+EGFP+and CD8+EGFP+populations compared to the control group, as shown by the dilution of the proliferation dye. Moreover, as an internal control, the non-engineered cell population (EGFP negative) in the KADCYLA®group proliferated at a comparable level to the control group (FIGs.8D-8E). As expected, the CD8+T cell population was the major source ofIFN- -producing cells. Strikingly, in the same culture, KADCYLA® selectively inhibited theCD8+EGFP+ cell population from producing IFN- , while the CD8+EGFP- population remainedunaffected. A similar trend was observed in the CD4+T cells (FIGs.8F-8G). Together, these results reveal that KADCYLA®selectively delivered toxicity to HER2III&IV-expressing T cells in a mixed cell population through multiple mechanisms including the induction of cell death,inhibition of cell proliferation, and suppression of functional cytokine production, such as IFN- .

[0263] HER2III-IV serves as an effective target for elimination of engineered cells

[0264] The ability for KADCYLA® to target and selectively eliminate engineered T cells in vivowas investigated. A schematic flow chart describing the experimental design is shown in FIG. 9A. Three days after intravenous injection of T cells, mice were treated with control antibodies that target EGFR (ERBITUX®), KANJINTI®, an antibody that targets HER2, and KADCYLA®. To differentiate and track engineered T cells, they also were engineered to express a GFP reporter protein. FIG.9B shows representative flow cytometry histograms. The presence of endogenous (EGFP negative) and engineered T cells were evaluated in the indicated tissues (FIG.9B). Anti-EGFR antibodies had no impact on eliminating engineered T cells except in the bone marrow. Anti-HER2 antibody alone had moderate effects at depleting engineered T cells. This was attributed to the known antibody dependent cell cytotoxicity. However, in sharp contrast, KADCYLA®was highly effective at eliminating engineered HER2-expressing T cells (FIGs.9B-9C).

[0265] Next, the optimal dosing of HER2 antibody was investigated. It was found that asingle injection of 500 µg was sufficient to eliminate most engineered T cells (FIG.9D). How quickly ADC eliminated engineered T cells in the indicated tissues was also investigated. It was found that as early as day one after ADC injection, nearly 50% of engineered T cells were eliminated in the indicated tissues. Furthermore, most engineered T cells were depleted within three days of ADC treatment.

[0266] KADCYLA® effectively eliminates engineered T cells in various tissues, but spares Tcells in the tumor

[0267] FIG. 10A shows the experimental design for these studies. Engineered T cells wereintravenously injected into mice with established B16 melanoma tumors followed by treatment with KADCYLA®or isotype control antibody three days later. The presence of engineered HER2III-IVT cells was examined in the indicated tissues seven days after a single dose of KADCYLA®or isotype antibody treatment. While engineered T cells were clearly visible inmice treated with isotype antibody, KADCYLA®effective depleted most T cells in all tissues examined except for the tumor (FIGs.10B-10C). This suggested that tumor infiltrating lymphocytes could be spared and allowed to continue carrying out their antitumor effector function. That engineered (HER2III-IV, EGFP) T cells treated with KADCYLA®did not stain with the anti-HER flow cytometry antibody suggested that KADCYLA®could have remained bound to HER2III-IVon engineered T cells and therefore prevented the flow antibody from staining.

[0268] KADCYLA® treatment controls T cell lymphoma growth in vivo

[0269] Several cases have been reported in which engineered CAR T cells develop into leukemiaor lymphomas. Here, the ability of HERIII-IVto serve as a target to eliminate established human T cell lymphoma (Jurkat) in mice was examined. As shown in FIG.11, control antibody did not prevent Jurkat from progressing. In sharp contrast, mice treated with KADCYLA®showed tumor regression or delay in tumor growth. Repeated dosing with KADCYLA®may eliminate Jurkat lymphoma more efficiently.

[0270] Sequence Listing

Claims

What is claimed is:

1. A synthetic HER2 polypeptide comprising, from N-terminus to C-terminus: i) a signal peptide; ii) an extracellular domain comprising the amino acid sequence of SEQ ID NO: 1; iii) a transmembrane domain comprising the amino acid sequence of SEQ ID NO: 2; and iv) an intracellular domain comprising a single lysine residue.

2. The synthetic HER2 polypeptide of claim 1, wherein the signal peptide comprises the amino acid sequence of SEQ ID NO:

3.

3. The synthetic HER2 polypeptide of claim 1 or claim 2, wherein the synthetic HER2 polypeptide has the amino acid sequence of SEQ ID NO:

4.

4. A nucleic acid molecule comprising a nucleic acid sequence encoding the synthetic HER2 polypeptide of any one of claims 1-3.

5. The nucleic acid molecule of claim 4, wherein the nucleic acid sequence encoding the synthetic HER2 polypeptide comprises the nucleic acid sequence of SEQ ID NO:

7.

6. The nucleic acid molecule of claim 4 or claim 5, wherein the nucleic acid molecule further comprises a nucleic acid sequence encoding a CD8a:MYD88 fusion protein, wherein the CD8a:MYD88 fusion protein comprises, from N-terminus to C-terminus a first domain that comprises a CD8a polypeptide, a linker polypeptide, and a second domain that comprises an MYD88 polypeptide.

7. The nucleic acid molecule of claim 6, wherein: i) the first domain of the CD8a:MYD88 fusion protein comprises the amino acid sequence of SEQ ID NO: 12; ii) the linker polypeptide of the CD8a:MYD88 fusion protein comprises the amino acid sequence of SEQ ID NO: 14; andiii) the second domain of the CD8a:MYD88 fusion protein comprises the amino acid sequence of SEQ ID NO: 13, preferably wherein the CD8a:MYD88 fusion protein has the amino acid sequence of SEQ ID NO:

15.

8. The nucleic acid molecule of claim 6 or claim 7, wherein the nucleic acid sequence encoding a CD8a:MYD88 fusion protein comprises the nucleic acid sequence of SEQ ID NO:

8.

9. The nucleic acid molecule of any one of claims 6-8, wherein the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO:

11.

10. A vector comprising the nucleic acid molecule of any one of claims 4-9, preferably wherein the vector is a viral vector, preferably wherein the viral vector is a lentiviral vector.

11. A cell expressing the synthetic HER2 polypeptide of any one of claims 1-3.

12. The cell of claim 11, wherein the cell also expresses a CD8a:MYD88 fusion protein, wherein the CD8a:MYD88 fusion protein comprises, from N-terminus to C-terminus a first domain that comprises a CD8a polypeptide, a linker polypeptide, and a second domain that comprises an MYD88 polypeptide, wherein i) the first domain of the CD8a:MYD88 fusion protein comprises the amino acid sequence of SEQ ID NO: 12; ii) the linker polypeptide of the CD8a:MYD88 fusion protein comprises the amino acid sequence of SEQ ID NO: 14; and iii) the second domain of the CD8a:MYD88 fusion protein comprises the amino acid sequence of SEQ ID NO: 13, preferably wherein the CD8a:MYD88 fusion protein has the amino acid sequence of SEQ ID NO:

15.

13. The cell of claim 11 or claim 12, wherein the cell also expresses at least one of: i) a chimeric antigen receptor (CAR); andii) an engineered T cell receptor (TCR).

14. The cell of any one of claims 10-12, wherein the cell is a tumor infiltrating lymphocyte (TIL), preferably wherein the TIL is a T cell.

15. A population of the cells of any one of claims 11-14.

16. A method of treating cancer in a subject, the method comprising administering to the subject the cell population of claim 15.

17. The method of claim 16, wherein the cancer is a solid tumor, preferably wherein the solid tumor is head and neck cancer, lung cancer, or a sarcoma.

18. The method of claim 16 or claim 17, further comprising at a time point after administration of the cell population, administering to the subject at least one amount of at least one cytotoxic antibody that binds to the synthetic HER2 polypeptide, thereby reducing and / or eliminating thecell population and / or cells that result from in vivo expansion of the cell population.

19. The method of claim 18, wherein the at least one cytotoxic antibody that binds to the synthetic HER2 polypeptide is ado-trastuzumab emtansine, fam-trastuzumab deruxtecan-nxki, margetuximab, trastuzumab, trastuzumab-anns, or a combination thereof.

20. The method of any one of claims 16-19, further comprising, at a time point after administration of the population of cells: i) administering to the subject at least one amount of an antibody that: a) binds to the synthetic HER2 polypeptide; and b) is linked to a detectable label; and ii) imaging the subject, or a portion thereof, with an imaging modality suitable to detect the detectable label, thereby identifying the presence and location of the therapeutic cells within the subject.

21. The method of any one of claims 16-20, further comprising, at a time point after administration of the population of cells: i) obtaining one or more biological samples from the subject; ii) contacting the one more biological samples with at least one amount of an antibody that: a) binds to the synthetic HER2 polypeptides; and b) is linked to a detectable label; and iii) imaging the one or more biological samples with an imaging modality suitable to detect the detectable label, thereby identifying the presence and location of the therapeutic cells within the one or more biological samples, preferably wherein the one or more biological samples comprises a tumor sample, a blood sample, or a combination thereof.

22. A method of producing the population of cells of 14, the method comprising: a) obtaining a plurality of cells from a subject; and b) introducing into the plurality of cells a one or more nucleic acid molecules, wherein the one or more nucleic acid molecules comprise a nucleic acid sequence encoding the synthetic HER2 polypeptide, preferably wherein introducing into the plurality of cells one or more nucleic acid molecules comprises performing retroviral transduction.

23. The method of claim 22, wherein the plurality of cells obtained from the subject are peripheral blood mononuclear cells (PBMCs).

24. The method of claim 22 or claim 23, further comprising, before step (b), after step (b), or both before step (b) and after step (b), expanding the cells.

25. The method of any one of claims 22-24, wherein the method further comprises, after step (b), contacting the plurality of cells with an affinity reagent that binds to the synthetic HER2 polypeptide of the present disclosure, thereby enriching for cells that express the synthetic HER2 polypeptide.

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