WO2025194098A1

WO2025194098A1 - Engineered immune cells expressing chimeric antigen receptors targeting trop2, and methods of using the same

Info

Publication number: WO2025194098A1
Application number: PCT/US2025/020032
Authority: WO
Inventors: Eric Smith; Elliott BREA
Original assignee: Dana Farber Cancer Institute Inc
Current assignee: Dana Farber Cancer Institute Inc
Priority date: 2024-03-14
Filing date: 2025-03-14
Publication date: 2025-09-18
Anticipated expiration: 2026-09-14

Abstract

Provided herein are compositions comprising engineered immune cells, chimeric antigen receptors, pharmaceutical compositions thereof, and methods of using the same. In embodiments, the engineered immune cells bind TROP2. In embodiments, the engineered immune cells comprise more than one heavy chain variable region and binds more than one TROP2 epitope.

Description

ENGINEERED IMMUNE CELLS EXPRESSING CHIMERIC ANTIGEN RECEPTORS TARGETING TROP2, AND METHODS OF

USING THE SAME

RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63/565,297 filed on March 14, 2024 which is incorporated by reference in its entirety herein.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing that has been submitted electronically in XML format and is incorporated by reference in its entirety. Said XML copy, created on March 14, 2025 is named 91016-419723_SeqLising.xml and is 128,000 bytes in size.

FIELD

[0003] The disclosure herein relates to engineered immune cells, chimeric antigen receptors, pharmaceutical compositions thereof, and methods of using the same, particularly engineered immune cells that bind TROP2.

BACKGROUND

[0004] In embodiments, TROP2 was targeted utilizing CAR-T cellular therapy; TROP2 is a cell surface protein normally expressed during fetal development and at low levels in some normal adult tissue, but is overexpressed on a variety of solid tumors including non-small cell lung cancer (NSCLC). Previous approaches to target TROP2 have relied on scFv based antibody drug conjugates such as sacitizumab govitecan (scFv clone hRS7), datopotumab deruxtecan (scFv dato)(Nangalia et al., 2013; Heist et al., 2017; Bardia et al., 2021 ; Okajima et al., 2021), or early chimeric antigen receptors (c.g., U.S. Patent Pub. 20230383007). Resistance to ADC and other antibody-based approaches has emerged, including on-target mutations in TROP2 (Coates et al., 2021). Durable responses to antibody-based therapies against TROP2 have been rare in previous approaches. Therefore, antibody -based or antibody fragment-based therapies against TROP2 with improved durability are desirable. SUMMARY

[0005] Provided herein are engineered cells, comprising an immune cell, comprising a chimeric antigen receptor of formula I;

R1 - R2 - R3 (1), wherein:

R1 is an extracellular' domain, comprising one or more heavy chain variable region means;

R2 is a transmembrane domain; and

R3 is an intracellular signaling means, and optionally, a hinge region between R1 and R2.

[0006] Provided herein are engineered cells, comprising an immune cell, comprising a chimeric antigen receptor of formula II:

R1 - R2 - R3 (II), wherein:

R1 is an extracellular domain, comprising one or more heavy chain variable regions;

R2 is a transmembrane domain; and

R3 is an intracellular domain, and optionally, a hinge region between R1 and R2.

[0007] Provided herein are pharmaceutical compositions comprising the engineered cells described herein and one or more pharmaceutically acceptable carriers, diluents, or excipients. Provided herein are pharmaceutical compositions comprising two or more engineered cells described herein and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0008] Provided herein are methods of treating cancer, comprising administering to a subject with cancer: engineered cells described herein; or pharmaceutical compositions described herein. Provided herein are compositions for use in treating cancer, comprising administering to a subject with cancer: engineered cells described herein; or pharmaceutical compositions described herein.

Provided herein are uses of a composition for treating cancer, comprising administering to a subject with cancer: engineered cells described herein or pharmaceutical compositions described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] FIGS. 1A-1D show an embodiment in which TROP2 directed CAR-T are highly active against EGFRm NSCLC in multiple preclinical models. FIG. ID shows activity of TROP2 CAR-T in a highly refractory Osimertinib resistant model of EGFRm NSCLC. PC9 were engineered with Osimertinib resistant C797S mutation. Tumor cells were incubated with CAR-T cells at 1:1 effector to target ratio for 24 h. Viability was assessed via luciferase based assay and normalized to irrelevant targeting CAR-T (BCMA).

[0010] FIGS. 2A-2F show an embodiment in which TROP2 directed CAR-T are highly active against EGFRm NSCLC in multiple preclinical models. FIG. 2A shows design of second generation lentiviral vectors with scFv or TROP2 VH binders.

[0011] FIGS. 3A-3D show an embodiment in which TROP2 CAR-T maintain a high degree of activity in setting of low epitope or TOPI mutation known to cause resistance to ADC. FIG. 3 A shows flow data for PC9 engineered with TROP2 T256R mutant. FIG. 3A shows flow of PC9 with either WT, KO, or T256R mutant TROP2. TROP2 expression evaluated by flow cytometry with corresponding mean fluorescences intensity shown (MFI). FIG. 3B shows PC9 engineered with TROP2 ADC resistant mutations in T256R or TOPI E418K previously described. FIG. 3C shows activity of TROP2 CAR-T in models of resistance to ADC based therapy. PC9 harboring either WT or TROP2 mutant (T256R) were incubated with TROP2 CAR-T and viability was assessed at 24h. FIG. 3D shows Activity of TROP2 CAR-T in models of resistance to ADC based therapy. PC9 harboring either WT or TOPI mutant (E418K) were incubated with TROP2 CAR-T and viability was assessed at 24h.

[0012] FIGS. 4A-4B show an embodiment in which VH TROP2 binders show high activity in NSCLC as well as binding to distinct domains compared to scFv-based approaches.

[0013] FIGS. 5A-5C show an embodiment in which VH TROP2 binders show high activity in NSCLC as well as binding to distinct domains compared to scFv-based approaches; TROP2 VH CAR constructs show in vitro/vivo activity against TROP2 tumors. FIG. 5A shows a summary of in vitro cytotoxicity with TROP2 VH based CAR T constructs against PC9 TROP2+ NSCLC human cell line and tonic signaling of TROP2 CAR T VH constructs. Cytotoxicity was performed against PC9 similar to FIG. ID. For tonic signaling Jurkat with Nur77 GFP reporter were transduced with various CAR T constructs and assessed for GFP expression as a marker of tonic signaling. Transact was used as a positive control. FIG. 5B shows 2.5E5 PC9 with ffLuc was injected tail vein into NSG mice at d-- 14. Mice were then imaged at baseline and treated at d- l with 0.3E6 CAR-T. Mice were imaged and assessed for bioluminesence as a marker of PC9 tumor burden. FIG. 5C shows Survival of NSG mice with CAR T constructs of FIGS 2A-2F.

[0014] FIGS. 6A-6B show an embodiment in which VH TROP2 binders show high activity in NSCLC as well as binding to distinct domains compared to scFv-based approaches.

[0015] FIGS. 7A-7C show an embodiment in which biparatopic TROP2 CAR-T exhibit superior anti tumor efficacy against models where scFv based approaches are not effective; biparatopic CAR design and activity in models refractory to scFv based approaches are shown. FIG. 7A shows design of second generation biparatopic tandem VH CAR against TROP2. FIG. 7B shows cytotoxicity of TROP2 single VH vs. biparatopic VH against PC9 (NSCLC) either parental, TROP2 KO, mCRD and mCPD domains. FIG. 7C shows tonic signaling of TROP2 biparatopic CAR T as well as activation of TROP2 biparatopic CAR T against TROP2+ cell line.

[0016] FIGS. 8A-C show an embodiment in which TROP2 CAR T construct design and specificity is described. FIG. 8A. Second-generation TROP2 CAR construct design. scFv was derived from Sacituzumab. Expressed in lentiviral vector with CMV promoter, IgG Kappa signal peptide, CD28 transmembrane domain (TM), and 4- IBB and CD3z costimulatory domain followed by P2A and VexGFP. FIG. 8B. Cytotoxicity assay of TROP2 CARs against PC9 WT (EGFR exon 19 del) and PC9 clone with knockout of TROP2. Cells were incubated with E:T of 1:1 and incubated for 24 h before cell viability of PC9 was read. Viability reported normalized to irrelevant control BCMA CAR. FIG. 8C. In vitro cytotoxicity of HCC827GR6 and (NSCLC EGFR exon 19 del/MET amp) and MDA-MB-231 (TNBC). P values reported as follows compared to control: ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0017] FIGS. 9A-H show an embodiment in which TROP2-targeted CARs are highly effective in vitro and in vivo against TROP2+solid tumors. FIG. 9A. In vitro cytotoxicity of TROP2 CART constructs against PC9 (EGFR exon 19 del) and HCC70 (TNBC). BCMA- or CD19-targeted CAR were used as a negative control CARs. Cell lines were assessed for cytotoxicity via luminescence-based cytotoxicity and CARs were incubated at indicated E:T ratios with targets for 24 h. Viability reported relative to control CARs. FIG. 9B. TROP2 CARs maintain activity against PC9 with osimertinib-acquired resistant EGFR C797S mutation. Similar to (A), cell line targets were incubated with cither osimertinib at increasing concentrations or CARs. For viability, osimertinib was normalized to vehicle, where tumor viability for CARs was normalized to BCMA at indicated E:T. Measurement performed after 24 h of co-culture. C. In vitro cytotoxicity of TROP2 CARs against PC9 in 3D Matrigel-based assay. Red object area was plotted over time. D. In vivo activity of TROP2 CARs in NSCLC xenograft. PC9 was injected subcutaneously in NSG mice and after tumors reached -100 mm3, BCMA or TROP2 CAR T were administered at indicated doses at d=0 as indicated by arrow. E. Orthotopic model of PC9. Mice were injected with PC9 ffLuc+ and after engraftment confirmed given control BCMA CARs or TROP2 CARs at indicated doses. Tumor was measured subsequently by bioluminescence imaging. F. Activity of TROP2 CARs in an ex vivo organoid model utilizing microfluidic device against patient derived lung adenocarcinoma DFCI243 (EGFR exon 19 del/T790M). Cell markers indicated in figure. G. Percent live/dead cell analysis of (F) performed by measuring the total cell area of each dye. H. In vivo activity of TROP2 CARs PDX DFCI243 (EGFR exon 19 del/T790M) and (EGFR L858R/MET amp) as well as DFCI642 (TROP2- EGFRm NSCLC transformed to SCLC). CAR cells (3 to 6E5) were injected at d=0, as indicated by arrow, and tumor volumes measured. Each line represents an independent mouse tumor volume measurement, with magenta for BCMA irrelevant control and green for TROP2. P values reported as follows compared to control: ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P< 0.0001.

[0018] FIG. 10 is an embodiment describing in vivo activity of TROP2 CARs in NSCLC xenograft HCC827GR6. Tumors were injected subcutaneously in NSG mice and after tumors reached -100 mm3, BCMA or TROP2 CAR T was administered at indicated doses at d=0. Growth kinetics were measured via caliper measurement. N=5 mice per group.

[0019] FIG. 11 is an embodiment describing ex vivo microfluidic device of cell lines demonstrates active cytolytic response with corresponding IFN-gamma release. A. TROP2 CAR activity against cell line derived organotypic tumor spheroids from PC9 and HCC827GR6. Images acquired after 3 days of co-culture. B. Cell death of PC9 and HCC827GR6 measured as percent area dead cells (DRAQ7) divided by pan cell (Calcein). C. IFN-gamma levels measured in the supernatant of CAR-treated organotypic tumor spheroids after 3 days via collection of supernatant, ns, P > 0.05; *P < 0.05.

[0020] FIGS. 12A-E show an embodiment describing models of acquired resistance to TROP2 ADC can be overcome with TROP2 CARs. A. PC9 with knockout of TROP2 generated by CRISPR/CAS9 targeted guide RNA against TROP2 locus were reconstituted with either WT TROP2 or T256R mutation previously described to have developed in acquired resistance to TROP2 ADC. Mean fluorescence intensity after TROP2 staining by flow cytometry on viable (DAPI negative) cells shown. B. In vitro viability of constructs described in (A) after generation of spheroids and treatment in ultralow attachment 96-well plate with datopotumab deruxtecan (Dato- DxD). Cells were incubated with ADC for 6 days followed by viability assessment by luciferase assay. Viability was normalized to vehicle (0 /r g/mL) for each construct. C. In vitro viability of either WT TROP2 or T256R TROP2 with TROP2 CARs at 1:1 E:T. Assay after 24 h incubation. D. In vitro viability of either WT TOPI or E418K TOPI with TROP2 CARs after 24 h incubation. 2E. Model of resistance to TROP2 ADC by TROP2 T256R mutation or TOPI mutation and how TROP2 CARs are able to maintain cytolytic activity, created with Biorender. P values reported as follows compared to control: ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. [0021] FIGS. 13A-B show an embodiment describing activity of TROP2 VH based CAR against PC9 and HCC827GR6. A. Viability of benchmark hRS7-based CAR and novel TROP2 VH CAR binders against PC9. Cytotoxicity performed at E:T of 1:1 with coincubation for 24 h. Assessed for cytotoxicity via luminescence based cytotoxicity. Viability reported relative to control irrelevant BCMA CAR. B. Viability of benchmark hRS7-based CAR and novel TROP2 VH CAR against HCC827GR6. Cytotoxicity performed at E:T of 1:1 with coincubation for 24 h. Assessed for cytotoxicity via luminescence based cytotoxicity. Viability reported relative to control irrelevant BCMA CAR. P values reported as follows compared to BCMA control: ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P <0.000.

[0022] FIGS. 14A-B show an embodiment describing a VH-only binder discovery, CAR engineering, and screening results in identification of CARs with high efficacy and capable of binding unique epitopes A. In vivo activity of selected constructs from (S4) with orthotopic model of PC9. Mice were injected at d=-14 with PC9 ffLuc+ and, after engraftment confirmed, given control BCMA CAR or TROP2 CAR at 0.3E6 CAR+/mouse, as indicated by arrow. Tumor was measured subsequently by BLI. B. Survival of mice from 3D with statistics shown using Log-Rank (Mantel-Cox) of hRS7 to VH681. *P < 0.05 C. In silico protein-protein docking prediction of Sacituzumab, modeled from FASTA Primary Sequence using antibody homology modeling (Molecular Operating Environment (MOE)), in complex with TROP2. Inset shows putative key residues and their side chains participating in protein-protein contacts, namely van der Waals interactions (pink halos), at the epitope-partatope interface D. In vitro viability of TROP2 (hRS7 based) CARs against WT, KO, or murine Q237-252 substitution of TROP2. Performed at E:T of 2: 1 and cocultured for 24h. E. TROP2 VH bind to distinct epitopes compared to scFv-based CAR constructs. Heatmap demonstrating that selected TROP2 VH constructs have activity against a unique domain compared to scFv-based (hRS7 and dato) CAR constructs. Viability normalized to irrelevant control BCMA CAR for each construct. F. In silico protein-protein docking prediction of TR0P2 VH375 and VH681, modeled from FASTA Primary Sequence using antibody homology modeling (Molecular Operating Environment (MOE)), in complex with TROP2. Inset shows putative key residues and their side chains participating in protein-protein contacts, namely van der Waals interactions (pink halos), at the epitope-partatope interface. P values reported as follows compared to control: ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0023] FIG. 15 shows an embodiment describing the structure of Sacituzumab IgG in complex with TROP2. As with the scFv, Sacituzumab IgG binds to a similar, 15aa epitope on TROP2 with similar residues mediating protein-protein contact. In silico protein-protein docking prediction of Sacituzumab IgG, modeled from FASTA Primary Sequence using antibody homology modeling (Molecular Operating Environment (MOE)), in complex with TROP2. Inset shows putative key residues and their side chains participating in protein-protein contacts, namely van der Waals interactions (pink halos), at the epitope-partatope interface.

[0024] FIG. 16 shows an embodiment describing a sequence alignment of murine and human TROP2 show areas of homology. TACSTD2 (TROP2) from mouse and human aligned using NCBI BLAST search. Areas of interest noted. The alignment highlights the signal peptide region, as well as regions including TY, CRD, CPD, sacituzumab binding domain, and transmembrane domain, with comparative sequences and amino acid coordinates displayed for both species to demonstrate conservation and divergence across the alignment.

[0025] FIG. 17 shows an embodiment describing TROP2 sacituzumab- and datopotamab- based CAR T bind to similar regions in the CPD domain. In vitro cytotoxicity assay demonstrates that both sacituzumab (hRS7) and datopotamab (dato) CAR have similar high activity against WT TROP2 and lack activity against KO or mTROP2 as well as mQ237-252 substitution. Assay performed with E:T 2: 1 with coculture for 24 h with viability normalized to irrelevant BCMA control.

[0026] FIG. 18A-H show an embodiment describing rational epitope binding-based design of biparatopic CARs leveraging single domain VH-only binders overcomes models of resistance to single epitope-targeted approaches. A. Schematic of biparatopic TROP2 VH-based CARs in second-generation CARs vector with linker between anti-CRD VH and anti-CPD VH, as well as hinge/transmembrane domain (H/TM), 4-1BB costimulatory domain, and CD3z signaling domain. B. In vitro cytotoxicity of TROP2 scFv, VH, and biparatopic VH CAR against PC9 with various substitutions of TROP2 domains with murine domains as indicated. Performed at 2: 1 E:T and cocultured for 24 h before viability was assessed via luciferase assay and normalized to irrelevant BCMA control. Data are representative of two different experiments with different donors C. In silico protein protein docking prediction of biparatopic TR0P2 VH681_375, modeled from FASTA Primary Sequence using antibody homology modeling (Molecular’ Operating Environment (MOE)), in complex with TROP2. D. In vitro activity in live-cell imaging assay of TROP2 CARs against PC9. PC9 TROP2 knock-out was stably transduced with murine CPD domain and mCherry and live cell imaging performed on Incucyte with counting of red objects. Cytotoxicity index is red cell object count relative to t=0 when CARs were applied at E:T of 0.25: 1 . Data are representative of two different experiments with different donors E. Similar to (D), with PC9 TROP2 murine CRD clone and transduced with GFP with counting of green objects. Data are representative of two different experiments with different donors F. PC9 harboring murine Q237-252 (CPD) or murine CRD tumors were injected subcutaneously into separate flanks of NSG mice and after tumors reached -100 mm3, CD 19 irrelevant control or TROP2 CARs were administered at a one-time dose of 2.5E6 CAR+ cells via tail vein, as indicated by arrow. Tumors measured by caliper measurement of mQ237-252 tumor on left flank with individual (dim) and mean (solid) measurements shown for groups. Data are representative of two different experiments with different donors G. Tumors measured by caliper measurement of mCRD tumor on right flank with individual (dim) and mean (solid) measurements shown for groups. Data are representative of two different experiments with different donors H. Survival of mice from (4FG) demonstrates prolonged survival with biparatopic TROP2 CAR (VH681_375) compared to benchmark hRS7 CAR. Survival statistics reported by Log-Rank (Mantel Cox). P values reported as follows compared to control: ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

[0027] FIG. 19A-C show an embodiment describing transduction and phenotyping of CAR T cells. CAR T were generated and on day 5 post transduction, cells were washed with FACS buffer, and stained with antibodies as indicated. A. Representative flow cytometry plots showing transduction efficiency of CAR T generated by gamma retroviral transduction of activated T-cells. Transduction efficiency shown by LssOrange+ expression across various conditions, including untransduced (UTD), CD 19, hRS7, VH375, VH681, and VH681_375, as well as PBMCs B. of CD4+ and CD8+ populations in different CAR T cell groups C. Assessment of T-cell memory of CAR T products via staining of CCR7 and CD45RA across the indicated conditions. Data are representative of multiple independent experiments with different donors.

[0028] FIG. 20 shows an embodiment describing in vitro live-cell imaging of PC9 WT treated with various TROP2 CAR constructs. In vitro activity in live-cell imaging assay of TROP2 CARs against PC9 WT. PC9 was transduced with GFP and live-cell imaging performed on Incucyte with counting of green objects. Cytotoxicity index is green cell object count relative to t=0 when CARs were applied at E:T of 0.25:1. Data are representative of multiple independent experiments with different donors.

[0029] FIG. 21 shows an embodiment describing in vivo kinetics of CAR infiltration into PC9 subcutaneous tumor. PC9 tumors once established at -100 mm3 were injected with either 2.5E6 control CD 19 or TROP2 CAR+ cells expressing NanoLuciferase, which allows for in vivo tracking of CAR. Mice were imaged after injecting with FFz substrate at indicated time points post CAR engraftment, with right flank representing PC9 mCRD and left flank PC9 mCPD tumor inoculation site.

[0030] FIG. 22 shows an embodiment describing the body weight monitoring of mice treated with CAR T cells. Mice bearing PC9 CPD or CRD tumors were treated with various TROP2 CAR T or control CD19 CAR T cells at a dose of 2.5E6 CAR+ via i.v., and body weights were measured at indicated time points to assess treatment-related toxicity. Data are presented as mean + SEM. Two-way ANOVA showed no significant differences in body weight between treatment groups and the control CD19 CAR groups.

DETAILED DESCRIPTION

[0031] The compositions and methods provided herein include embodiments in which immune cells, such as T cells, NK cells, macrophages, dendritic cells, hematopoietic stem cells (HSC), induced pluripotent stem cells, cord blood stem cells, and/or derivatives thereof, are engineered to express chimeric antigen receptors (CARs). In embodiments, the engineered immune cells express a CAR that binds TROP2. In embodiments, the engineered immune cells express a CAR that binds TROP2, wherein the CAR expresses more than one heavy chain variable region. In embodiments, the engineered immune cells express a CAR that binds TROP2, wherein the CAR expresses more than one heavy chain variable region, wherein a heavy chain variable region binds TROP2 cysteine-rich domain and a heavy chain variable region binds TROP2 cysteine-poor domain. In embodiments, engineered T cells express a CAR that binds TROP2, wherein the CAR expresses more than one heavy chain variable region, wherein a heavy chain variable region binds TROP2 cysteine-rich domain and a heavy chain variable region binds TROP2 cysteine-poor domain. In embodiments, engineered CD8+ T cells and/or CD4+ T cells express a CAR that binds TROP2, wherein the CAR expresses more than one heavy chain variable region, wherein a heavy chain variable region binds TROP2 cysteine-rich domain and a heavy chain variable region binds TROP2 cysteine-poor domain. Without being bound by theory, engineered immune cells that bind more than one TROP2 epitope (such as both the cysteine-rich domain and the cysteine-poor domain) maintain activity or efficacy against cancers where previous therapies are or become less active, less effective, inactive, or ineffective against treatment-resistant cancers or chemotherapyresistant cancers.

Terms

[0032] The engineered cells and pharmaceutical compositions provided herein can be administered to subjects or patients. Herein, “administration” refers to the act of the attending physician or caregiver, prescribing the agent for administration and thereby causing the application of an agent to a subject, through ingestion, infusion, injection, or any other means, whether selfadministered or administered by a clinician or other qualified care giver. Herein, a “subject” includes both human patients and veterinary subjects, including human and non-human mammals. In embodiments, the subject or patient has, or has a risk of, lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, or gastrointestinal cancer.

[0033] Used herein, an "antibody" is an immunoglobulin molecule comprising two heavy chains (HCs) and two light chains (LCs) interconnected by disulfide bonds. The amino terminal portion of each LC and HC includes a variable region of about 100-120 amino acids responsible for antigen recognition via the complementary determining region (CDRs) contained therein. The CDRs are interspersed with regions that are well-known and generally conserved among and between species (e.g., mouse and human), which are termed framework regions (FRs). In embodiments, the CDRs are interspersed with FRs. Antibodies disclosed herein have four FRs, termed FR1-FR4. In embodiments, the FRs are human FRs (e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)).

[0034] The three CDRs of the HC are referred to as "HCDR1, HCDR2, and HCDR3." The functional ability of an antibody to bind a particular antigen is determined by the CDRs. Assignment of amino acids to CDR domains within the light chain variable regions (LCVRs) and heavy chain variable regions (HCVRs) of the antibodies of the present disclosure is based on the well-known Kabat numbering conventions (Andrew Martin, Protein Sequence and Structure Analysis of Antibody Variable Domains in Antibody Engineering (Roland Kontermann and Stefan Diibel eds., 2d ed. 2010)).

[0035] The term “heavy chain variable region” (HCVR) refers to an antibody fragment, the heavy chain variable region, that binds TROP2, such as at the TROP2 cysteine-rich domain or at the TROP2 cysteine-poor domain. Heavy chain variable regions within the scope of heavy chain variable region means disclosed herein are the disclosed heavy chain variable regions and functional equivalents thereto. Functionally equivalent heavy chain variable regions comprise different specific amino acid residues but bind the TROP2, such as at the TROP2 cysteine-rich domain or at the TROP2 cysteine-poor domain. Functionally equivalent heavy chain variable regions may differ insubstantially in binding TROP2, such as at the TROP2 cysteine-rich domain or at the TROP2 cysteine-poor domain, and have a therapeutic effect. Methods of making these heavy chain variable region antibody fragments are routine (see, e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)).

[0036] The antibody fragments disclosed herein are derived from nanobodies. Nanobodies can be produced, for example, by transgenic mice. Transgenic mice can generate nanobodies where murine VH/VL has been knocked out and human VH-only has been knocked in (see, e.g., Generation of heavy-chain-only antibodies in mice, PNAS 103(41): 15130-15135 (2006)). Single B cells from transgenic mice can be used to generate specific and productive nanobody clones for single cell sequencing of the DNA encoding the variable domains. A variety of well-known methods and tools can be used for producing and purifying the antibody fragments disclosed herein, including vectors, for example, plasmids, virus, or other vehicles for polynucleotide insertion or expression, and hosts, for example, microbial, yeast, insect, and mammalian organisms (see, e.g., Process Scale Purification of Antibodies (Uwe Gottschalk, ed., 2d ed. 2017)).

[0037] Further antibodies having a complementary binding means can be prepared and screened by well-known methods, such as hybridoma, transgenic animals, and phage or yeast display (see, e.g., Monoclonal Antibodies: Methods and Protocols (Vincent Ossipow and Nicolas Fischer, eds., 2d ed. 2014)). Antibodies having equivalent complementary binding means may differ in their amino acid sequence but perform the same function of binding the target through CDR-target interaction acting as (inhibitor/agonist/antagonist) to achieve the same result (inhibiting tumor growth). Preferably, the complementary binding means functions through the same epitope as the disclosed heavy chain variable regions, such as a TROP2 epitope, for example, at the TROP2 cysteine-rich domain or at the TROP2 cysteine-poor domain. [0038] Herein, “binding” (or “binds”) refers to the well-understood interaction between a heavy chain variable region and a target protein, peptide, or polysaccharide (e.g., TROP2, for example, at the TROP2 cysteine-rich domain or at the TROP2 cysteine-poor domain). Binding can be measured in a variety of ways (see, e.g., Antibody Engineering: Methods and Protocols (Damien Nevoltris and Patrick Chames eds., 3d ed. 2018)), for example, by activity (such as cancer cell viability) or immunoassays (such as ELISA or western blotting). A particular heavy chain variable region antibody fragment or protein binds to a particular target protein, peptide, or polysaccharide (e.g., TROP2, for example, at the TROP2 cysteine-rich domain or at the TROP2 cysteine-poor domain) and does not bind in a significant amount to other proteins or polysaccharides present in a sample or subject disclosed herein. Binding occurs between the disclosed heavy chain variable region antibody fragment and epitopes of TROP2, such as TROP2 cysteine-rich domain and TROP2 cysteine poor domain. Herein, "epitope" refers to discrete sites of an antigen (e.g., TROP2, for example, at the TROP2 cysteine-rich domain or at the TROP2 cysteine-poor domain) recognized by the disclosed heavy chain variable region. Epitopes may be lineal’ or three- dimensional. A heavy chain variable region antibody fragment binds to a target protein when the interaction has a KD of less than 10‘⁶ molar, such as less than 10‘⁷ molar, less than 10'⁸ molar, less than 10'⁹ molar, less than IO'¹⁰ molar, less than 10’¹¹ molar, or less than 10'¹² molar.

[0039] “Chimeric antigen receptors” (CARs) are receptor proteins that have been engineered for expression on an immune cell and to target a specific antigen (“target antigen”) as well as activate the immune cell. CARs are used in therapies, such as immune cell therapy, including T and NK cell therapy. Virus or RNA encoding CARs can used for in vivo generation of CAR effector cells. CARs can be engineered into allogeneic immune cells (i.e., immune cells from a donor are engineered) or autologous immune cells (i.e., immune cells from a patient or subject that are re-introduced after engineering). CARs typically include (1) an extracellular antigenbinding motif (e.g., antibody or fragment thereof, such as heavy chain variable region(s)), (2) linking/transmembrane motifs (e.g., hinge and/or transmembrane regions), and (3) an intracellular domain (e.g., including a signaling domain (also known as an activating domain) derived from CD247 (CD3Q, optionally comprising a costimulatory domain, such as a costimulatory domain derived from CD137 (4-IBB)). In embodiments, the CAR expresses CD8+ or CD4+ on the immune cell surface (such as an CAR-NK or CAR-T cell surface).

[0040] In embodiments, the target antigen (e.g., TROP2, for example, at the TROP2 cysteine-rich domain or at the TROP2 cysteine-poor domain) is expressed or overexpressed on cancer cells, but not healthy cells. In embodiments, the target antigen is expressed or overexpressed on a tumor selected from the list consisting of: lung cancer, non- small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer.

[0041 ] The engineered cells of the present disclosure can also be used in combination therapy. In embodiments, the subject is treated with engineered cells disclosed herein in combination with one or more additional therapies to treat cancer, for example, radiation, surgery, bone marrow transplantation, chemotherapy, immunotherapy, hormone therapy, targeted therapy (such as tyrosine kinase inhibitors, TKIs), or other cell or engineered cell therapies. Use of the engineered cells of the present disclosure in combination with chemotherapy, TKIs, or other immunotherapy is contemplated. In embodiments, the additional treatment is directed to targeting similar or the same antigens (e.g., one or more engineered cells of provided herein can be used in combination, such as engineered cells expressing CD8+ and engineered cells expressing CD4+ at the cell surface). As used herein, “combination” therapy or “use in combination” refers to the administration of the engineered cells of the present disclosure to a patient in conjunction with (i.e., before, simultaneously, or following) any number of relevant treatments.

[0042] The phrase “complementary determining means” as used herein describes one or more of the complimentary determining regions (CDRs) that form specific interactions with the target antigen. CDRs within the scope of complementary determining means are the disclosed CDRs and functional equivalents thereto. Functional equivalent CDRs comprise different specific amino acid residues but bind TROP2 (e.g., the cysteine rich domain or the cysteine poor domain within). Functionally equivalent CDRs would differ insubstantially to bind (e.g., the cysteine rich domain or the cysteine poor domain within) and have a therapeutic effect.

[0043] Herein, an “effective amount” is a quantity sufficient to achieve a desired effect in a subject. For instance, this can be the amount necessary to prevent, treat, or ameliorate a disease, for example, inhibiting or suppressing lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer. In embodiments, an effective amount is the amount necessary to eliminate, reduce the size, or prevent metastasis of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer. Efficacy is first evident in the cellular response, for which a variety of in vitro and cell-based assays to measure are well-known. Kristina V. Kitaeva et al., Cell Culture Based In vitro Test Systems for Anticancer Drug Screening, 8 Front. Bioeng. Biotechnol. 322(2020)). In embodiments, an effective amount is the amount necessary to significantly inhibit or reduce cancer cell proliferation or migration, invasion, or adhesion. A cellular response manifests as significantly reduced tumor size, reduced or inhibited disease progression, and improvement in survival in a subject or patient. More particularly, an effective amount provides improvement in important cancer endpoints, Overall Survival (OS), Disease-Free Survival (DFS), Objective Response Rate, Complete Response Rate or Progression Free Survival (PFS). See Dept, of Health and Human Services, Food and Drug Admin, Clinical Trial Endpoints for the Approval of Cancer Drugs and Biologies: Guidance for Industry (2018); E.A. Eisenhauer et al., New Response Evaluation Criteria in Solid Tumours: Revised RECIST Guideline (Version 1.1), 45 Eur. J. Cancer 228 (2009).

[0044] The “pharmaceutically acceptable carriers” of use are conventional (e.g., as described in Remington, The Science and Practice of Pharmacy, 22nd Edition, Loyd V., ed., Pharmaceutical Press, 2012). In general, the nature of the carrier will depend on the mode of administration. For instance, parenteral formulations typically comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions or the like as a vehicle. Pharmaceutical compositions can additionally include minor amounts of non-toxic auxiliary substances for stability. In embodiments, the carrier may be sterile and/or suspended or otherwise contained in a unit dosage form including one or more measured doses of the composition suitable for administration to a subject of an effective amount of the engineered cells disclosed herein. Medications for use in treatment may also be included in embodiments. In embodiments, the unit dosage form may be in a sealed vial that contains sterile contents or a syringe for injection into a subject, lyophilized for subsequent solubilization and administration, or in a solid or controlled release dosage.

[0045] A pharmaceutical composition of the present disclosure contains an "effective" or "therapeutically effective" amount, as used interchangeably herein, of an engineered cell of the present disclosure. The dosages and dosage regimen to achieve the desired therapeutic result depending on the means of administration and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the engineered cell to elicit a desired response in the individual. An effective amount is also one in which any toxic or detrimental effects of the engineered cell of the present disclosure are outweighed by the therapeutically beneficial effects.

[0046] Further contemplated are variants of the disclosed amino acid and nucleic acid sequences. Herein, “sequence identity” is referred to as the similarity between amino acid or nucleic acid sequences, which is expressed as the similarity between the sequences. Sequence identity is frequently measured as percent identity, in which two sequences are considered more similar’ the higher the percentage. Homologs or variants of a polypeptide or nucleic acid molecule possess a relatively high degree of sequence identity when aligned using standard methods, which are well-known. Ceslovas Venclovas, Methods for Sequence-Structure Alignment in Homology Modeling: Methods and Protocols, 55-82 (Andrew Orry and Ruben Abagyan, eds., 2012)). Further contemplated are variants of the disclosed amino acid and nucleic acid sequences with a sequence identity of at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% of any one of SEQ ID NOs: 1-120. Herein, “sequence identity” is referred to as the similarity between amino acid or nucleic acid sequences, which is expressed as the similarity between the sequences. Sequence identity is frequently measured as percent identity, in which two sequences are considered more similar the higher the percentage. Homologs or variants of a polypeptide or nucleic acid molecule possess a relatively high degree of sequence identity when aligned using standard methods, which are well-known. Ceslovas Venclovas, Methods for Sequence-Structure Alignment in Homology Modeling: Methods and Protocols, 55-82 (Andrew Orry and Ruben Abagyan, eds., 2012)).

[0047] The term “therapeutic” in conjunction with engineered cells and pharmaceutical compositions disclosed herein refers to engineered cells suitable for use in human treatment of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer. Such engineered cells or pharmaceutical compositions express CAR (e.g., CAR for expressing one or more heavy chain variable regions) bind their cognate receptor (e.g., TROP2, such as the cysteine rich or cysteine poor domains of TROP2) with a KD of less than 10‘⁶ molar, such as less than 10‘⁷ molar, less than IO’⁸ molar, less than 10'⁹ molar, less than IO’¹⁰ molar, less than IO’¹¹ molar, or less than IO'¹² molar, and any toxic or detrimental effects of the engineered cells and pharmaceutical compositions disclosed herein are outweighed by the therapeutic beneficial effects.

[0048 J The engineered cells disclosed herein can be used in therapy. In embodiments, the engineered cells disclosed herein can be used to treat, prevent (such as through prophylactic treatment), or ameliorate a lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer. Herein, "preventing" a disease refers to inhibiting the full development of a disease, such as lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer. "Treating" refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in tumor burden or a decrease in the number of size of metastases. "Ameliorating" refers to the reduction in the number or severity of signs or symptoms of a disease, such as lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer. A "prophylactic" treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing a pathology, such as lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer.

[0049] Contemplated herein are “conservative variants” of the disclosed amino acid sequences herein. A protein is a conservative variant where it contains conservative amino acid substitutions that do not substantially affect or decrease the affinity of a protein. For example, a CAR (e.g., expressing one or more heavy chain variable regions) that binds its cognate antigen (e.g., TR0P2, such as the cysteine rich or cysteine poor domain of TROP2) can include at least 1, 2, 5, 10, or 15 conservative substitutions, for example, and bind the cognate antigen (e.g., TROP2, for example, at the TROP2 cysteine-rich domain or at the TROP2 cysteine-poor domain). Conservative amino acid substitution tables providing functionally similar amino acids are well-known to one of ordinary skill in the art. The following groups are examples of amino acids that are considered conservative substitutions for one another: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (T), leucine (L), methionine (M), valine (V); and 6) phenylalanine (F), tyrosine (Y), tryptophan (W). [0050] Herein, a "degenerate variant" refers to a polynucleotide encoding a polypeptide (such as a CAR) that includes a sequence that is degenerate based on the genetic code (i.e., the 20 natural amino acids can be specified by more than one codon). All degenerate nucleotide sequences encoding the disclosed CAR sequences are included.

[0051] “Vector”, as used herein, is an entity containing a nucleic acid molecule (such as a DNA or RNA molecule) comprising the coding sequence of a protein of interest and can express the coding sequence. In embodiments, vectors herein bear a promoter(s) that is operationally linked to the coding sequence of a protein of interest (e.g., the CAR sequences disclosed herein). Nonlimiting examples include a naked or packaged (lipid and/or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replication incompetent, or a virus or bacterium or other microorganism that may be replication-competent. A vector is sometimes referred to as a construct. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements. Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses. In some embodiments, a viral vector comprises a nucleic acid molecule encoding a disclosed CAR, such as a CAR expressing one or more heavy chain variable regions, such as a heavy chain variable region that binds TROP2, such as a heavy chain variable region that binds TROP2 cysteine rich domain and/or a heavy chain variable region that binds TROP2 cysteine poor domain. Other methods of engineering immune cells are understood in the ait, such as introducing RNA of adenovirus for CRISPR/Cas9 editing, such as for in vivo methods of engineering. 1 COMPOSITIONS

[0052] Provided in the present disclosure are engineered cells. In embodiments, the engineered cells comprise an immune cell, comprising a chimeric antigen receptor (CAR) of the formula:

Formula I. R1 - R2 - R3 wherein:

R1 is an extracellular domain, comprising one or more heavy chain variable region means;

R2 is a transmembrane domain; and

R3 is an intracellular signaling means.

In embodiments, the CAR of Formula I optionally comprises or consists of a hinge region between R1 and R2. In embodiments, R1 is an extracellular domain of the formula:

Formula III. R4 - R5 or R5 - R4 wherein:

R4 is a first heavy chain variable region means, and

R5 is a second heavy chain variable region means.

In embodiments, the extracellular domain of Formula III optionally comprises or consists of a peptide linker between R4 and R5.

In embodiments, the engineered cells comprise an immune cell, comprising a chimeric antigen receptor (CAR) of the formula:

Formula II. R1 - R2 - R3 wherein:

R2 is a transmembrane domain; and

R3 is an intracellular domain.

[0053] In embodiments, the CAR of Formula II optionally comprises or consists of a hinge region between R1 and R2. In other embodimentsof the engineered cell described herein, the one or more antibody heavy chain variable regions of R1 is the formula:

Formula IV. R4 - R5 or R5 - R4 wherein:

R4 is a first heavy chain variable region, and

R5 is a second heavy chain variable region. [0054] In embodiments, the extracellular domain of Formula IV optionally comprises or consists of a peptide linker between R4 and R5.

[0055] Various immune cells are contemplated for the engineered cells of the present disclosure. In embodiments, the immune cell is selected from the group consisting of T cells, natural killer (NK) cells, macrophages, dendritic cells, hematopoietic stem cells (HSC), induced pluripotent stem cells, cord blood stem cells, and/or derivatives thereof. Particularly, in embodiments, the immune cell is a T cell. Particularly, in embodiments, the immune cell is selected from the list consisting of: a cytotoxic lymphocyte, T cell, cytotoxic T cell (CD8⁺ T cell), T helper cell (CD4⁺ T cell), a T cell and/or y5 T cell, Thl7 T-cell, NK T (NKT) cell, and regulatory T (Treg) cell. Particularly, in embodiments, the immune cell is a CD8⁺ T cell. Particularly, in embodiments, the immune cell is a CD4⁺ T cell.

[0056] Various heavy chain variable region means are contemplated in Formulas I and III, and various heavy chain variable regions are contemplated in Formulas II and IV. In embodiments, Rl, R4, or R5 comprise the complementarity determining regions (CDRs): HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 1, the amino acid sequence of HCDR2 is SEQ ID NO:2, the amino acid sequence of HCDR3 is SEQ ID NO:3; the amino acid sequence of HCDR1 is SEQ ID NO:5, the amino acid sequence of HCDR2 is SEQ ID NO:6, the amino acid sequence of HCDR3 is SEQ ID NO:7; the amino acid sequence of HCDR1 is SEQ ID NO:9, the amino acid sequence of HCDR2 is SEQ ID NO: 10, the amino acid sequence of HCDR3 is SEQ ID NO: 11; the amino acid sequence of HCDR1 is SEQ ID NO: 13, the amino acid sequence of HCDR2 is SEQ ID NO: 14, the amino acid sequence of HCDR3 is SEQ ID NO: 15; the amino acid sequence of HCDR1 is SEQ ID NO: 17, the amino acid sequence of HCDR2 is SEQ ID NO: 18, the amino acid sequence of HCDR3 is SEQ ID NO: 19; the amino acid sequence of HCDR1 is SEQ ID NO:21, the amino acid sequence of HCDR2 is SEQ ID NO:22, the amino acid sequence of HCDR3 is SEQ ID NO:23; the amino acid sequence of HCDR1 is SEQ ID NO:25, the amino acid sequence of HCDR2 is SEQ ID NO:26, the amino acid sequence of HCDR3 is SEQ ID NO:27; the amino acid sequence of HCDR1 is SEQ ID NO:29, the amino acid sequence of HCDR2 is SEQ ID NO:30, the amino acid sequence of HCDR3 is SEQ ID NO:31; the amino acid sequence of HCDR1 is SEQ ID NO:33, the amino acid sequence of HCDR2 is SEQ ID NO:34, the amino acid sequence of HCDR3 is SEQ ID NO:35; the amino acid sequence of HCDR1 is SEQ ID NO:37, the amino acid sequence of HCDR2 is SEQ ID NO:38, the amino acid sequence of HCDR3 is SEQ ID NO:39; the amino acid sequence of HCDR1 is SEQ ID NO:41, the amino acid sequence of HCDR2 is SEQ ID NO:42, the amino acid sequence of HCDR3 is SEQ ID NO:43; the amino acid sequence of HCDR1 is SEQ ID NO:45, the amino acid sequence of HCDR2 is SEQ ID NO:46, the amino acid sequence of HCDR3 is SEQ ID NO:47; the amino acid sequence of HCDR1 is SEQ ID NO:49, the amino acid sequence of HCDR2 is SEQ ID NO:50, the amino acid sequence of HCDR3 is SEQ ID NO:51 ; the amino acid sequence of HCDR1 is SEQ ID NO:53, the amino acid sequence of HCDR2 is SEQ ID NO:54, the amino acid sequence of HCDR3 is SEQ ID NO:55; or the amino acid sequence of HCDR 1 is SEQ ID NO:57, the amino acid sequence of HCDR2 is SEQ ID NO:58, the amino acid sequence of HCDR3 is SEQ ID NO:59.

[0057] Particularly, in embodiments, Rl, R4, or R5 comprise a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 4, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 98; the amino acid sequence of the HCVR is SEQ ID NO: 8, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 99; the amino acid sequence of the HCVR is SEQ ID NO: 12, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 100; the amino acid sequence of the HCVR is SEQ ID NO: 16, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 101; the amino acid sequence of the HCVR is SEQ ID NO: 20, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 102; the amino acid sequence of the HCVR is SEQ ID NO: 24, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 103; the amino acid sequence of the HCVR is SEQ ID NO: 28, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 104; the amino acid sequence of the HCVR is SEQ ID NO: 32, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 105; the amino acid sequence of the HCVR is SEQ ID NO: 36, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 106; the amino acid sequence of the HCVR is SEQ ID NO: 40, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 107; the amino acid sequence of the HCVR is SEQ ID NO: 44, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 108; the amino acid sequence of the HCVR is SEQ ID NO: 48, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 109; the amino acid sequence of the HCVR is SEQ ID NO: 52, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 110; the amino acid sequence of the HCVR is SEQ ID NO: 56, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 111; or the amino acid sequence of the HCVR is SEQ ID NO: 60, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 112.

[0058] Particularly, in embodiments, Rl, R4, or R5 comprise the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO:9, the amino acid sequence of HCDR2 is SEQ ID NO: 10, the amino acid sequence of HCDR3 is SEQ ID NO: 11; the amino acid sequence of HCDR1 is SEQ ID NO: 13, the amino acid sequence of HCDR2 is SEQ ID NO: 14, the amino acid sequence of HCDR3 is SEQ ID NO: 15; or the amino acid sequence of HCDR1 is SEQ ID NO:25, the amino acid sequence of HCDR2 is SEQ ID NO:26, the amino acid sequence of HCDR3 is SEQ ID NO:27. Particularly, in embodiments, Rl, R4, or R5 comprise a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 12, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 100; the amino acid sequence of the HCVR is SEQ ID NO: 16, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 101; or the amino acid sequence of the HCVR is SEQ ID NO: 28, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 104.

[0059] Particularly, in embodiments, Rl, R4, or R5 comprise the complementarity determining regions (CDRs): HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO:25, the amino acid sequence of HCDR2 is SEQ ID NO:26, the amino acid sequence of HCDR3 is SEQ ID NO:27. Particularly, in embodiments, in embodiments, Rl, R4, or R5 comprise a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 28.

[0060] Particularly, in embodiments, Rl, R4, or R5 comprise the complementarity determining regions (CDRs): HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 9, the amino acid sequence of HCDR2 is SEQ ID NO: 10, the amino acid sequence of HCDR3 is SEQ ID NO: 11. Particularly, in embodiments, Rl, R4, or R5 comprise The engineered cell of any one of claims 1-13, wherein the Rl comprises a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 12.

[0061] Particularly, in embodiments, Rl, R4, or R5 comprise the complementarity determining regions (CDRs): HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 13, the amino acid sequence of HCDR2 is SEQ ID NO: 14, the amino acid sequence of HCDR3 is SEQ ID NO: 15. Particularly, in embodiments, Rl, R4, or R5 comprise a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 16.

[0062] Particularly, in embodiments, Rl comprises the complementarity determining regions (CDRs): HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO:25, the amino acid sequence of HCDR2 is SEQ ID NO:26, the amino acid sequence of HCDR3 is SEQ ID NO:27; and the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 9, the amino acid sequence of HCDR2 is SEQ ID NO: 10, the amino acid sequence of HCDR3 is SEQ ID NO: 11. Particularly, in embodiments, Rl comprises a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 28, and a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 12. Particularly, in embodiments, Rl comprises the amino acid sequence of SEQ ID NO: 62. Particularly, in embodiments, Rl comprises an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 113. Particularly, in embodiments, Rl comprises the amino acid sequence of SEQ ID NO: 63. Particularly, in embodiments, Rl comprises an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 114.

[0063] Particularly, in embodiments, Rl comprises the complementarity determining regions (CDRs): HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO:25, the amino acid sequence of HCDR2 is SEQ ID NO:26, the amino acid sequence of HCDR3 is SEQ ID NO:27; and the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 13, the amino acid sequence of HCDR2 is SEQ ID NO: 14, the amino acid sequence of HCDR3 is SEQ ID NO: 15. Particularly, in embodiments, Rl comprises a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 28, and a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 16. Particularly, in embodiments, Rl comprises the amino acid sequence of SEQ ID NO: 64. Particularly, in embodiments, Rl comprises an amino acid encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 115. Particularly, in embodiments, Rl comprises the amino acid sequence of SEQ ID NO: 65. Particularly, in embodiments, Rl comprises an amino acid encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 116. [0064] Various peptide linkers are contemplated for Formulas III and IV. A person of ordinary skill in the art understands that peptide linkers are typically about one to 100 amino acids in length (e.g., 1, 2, 3, 4, 5, 10, 12, 1-10, 1-12, or 1-20, or more amino acids) containing various types of amino acids (e.g., US Patent No. 11,041,023, incorporated by reference herein in its entirety on March 14, 2024). In embodiments, the amino acids include a combination of one or more glycine(s) and/or serine(s). In embodiments, the peptide linker of Formula III or IV comprises the amino acid sequence comprising GGGGS. Particularly, in embodiments, the peptide linker of Formula III or IV consists of the amino acid sequence GGGGS. In embodiments, the peptide linker of Formula III or IV comprises or consists of 2, 3, 4, 5, 6, or 7 consecutive repeats of the amino acid sequence GGGGS. Particularly in embodiments, the peptide linker of Formula III or IV comprises consists of the amino acid sequence GGGGSGGGGSGGGGS.

[0065] Various hinge regions (also known as spacers) are contemplated for Formulas I and II. A person or ordinary skill in the art understands that hinge regions or spacers in a chimeric antigen receptor (CAR) typically sit between a target (e.g., antigen or epitope)-recognition domain or region (e.g., TROP2-binding heavy chain variable region) and the outer membrane of a cell in which the hinge region or spacer is expressed (e.g., an immune cell, such as a T cell or NK cell). Hinge regions or spacers typically vary in size and can be derived from a variety of extant proteins, such as extracellular proteins or protein domains (e.g., US Patent No. 10,597,456, incorporated herein by reference in its entirely on March 14, 2024). In embodiments, the hinge region comprises a hinge domain derived from CD3(^, CD4, CD8a, CD28, IgGl, IgG2, or IgG4. Particularly, in embodiments, the hinge region comprises an amino acid sequence selected from the list consisting of SEQ ID NOS: 66-72. Particularly, in embodiments, the hinge region comprises a hinge domain derived from CD28. Particularly, in embodiments, the hinge region comprises or consists of an amino acid sequence comprising or consisting of SEQ ID NO: 69, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 117.

[0066] Various transmembrane domains are contemplated for R2 of Formulas I and II. A person of ordinary skill in the art understands that transmembrane domains in a chimeric antigen receptor (CAR) typically sit between a hinge region or spacer and an intracellular domain. Transmembrane domains can vary in size and are often derived from extant transmembrane proteins or protein domains (e.g., US Patent No. 10,597,456, incorporated herein by reference in its entirely on March 14, 2024). In embodiments, R2 comprises a transmembrane domain derived from CD3^, CD4, CD8a, CD28, or CD137. Particularly, in embodiments, R2 comprises an amino acid sequence selected from the list consisting of SEQ ID NOS: 73-77. Particularly, in embodiments, R2 comprises a transmembrane domain derived from CD28. Particularly, in embodiments, R2 comprises or consists of an amino acid sequence comprising or consisting of SEQ ID NO: 76, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 118.

[0067] Various intracellular domains or intracellular signaling means are contemplated for R3 of Formulas I and II. A person or ordinary skill in the art understands that intracellular domains or intracellular signaling means in a chimeric antigen receptor (CAR) are typically linked to a transmembrane domain and are within the intracellular compartment of a cell in which the intracellular domain or intracellular signaling means is expressed (e.g., an immune cell, such as a T cell or NK cell). Intracellular domains or intracellular signaling means typically vary in size and and include a signaling domain and/or a costimulatory domain can be derived from a variety of extant proteins, such as intracellular, signaling, and/or costimulatory proteins or protein domains (e.g., US Patent No. 10,597,456, incorporated herein by reference in its entirely on March 14, 2024). Functionally equivalent intracellular domains comprising different specific amino acid residues exert increased immune potency, such as equivalent or increased cytolytic activity and/or cytokine or CAR expression. Functionally equivalent intracellular domains would differ insubstantially in immune potency and have a therapeutic effect. In embodiments, R3 comprises a signaling domain or a signaling domain and a costimulatory domain. In embodiments, R3 is an intracellular domain of the formula:

Formula V. R6 - R7 or R7 - R6 wherein R6 comprises a signaling domain, and R7 comprises a costimulatory domain.

[0068] Particularly, in embodiments, R3 or R6 comprise a signaling domain derived from CD3(^, CD27, CD28, CD40, KIR2DS2, MyD88, or 0X40. Particularly, in embodiments, R3 or R6 comprise a signaling domain comprising an amino acid sequence selected from the list consisting of SEQ ID NOS: 79-81, 84-86, and 94-96. Particularly, in embodiments, R3 or R6 comprise a signaling domain derived from CD3L) Particularly, in embodiments, R3 or R6 comprise or consist of an amino acid sequence comprising or consisting of SEQ ID NO: 79, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 120. [0069] Particularly, in embodiments, R3 or R7 comprises a costimulatory domain derived from of CD3y, CD38, CD3s, CD3^, CD27, CD40, CD28, CD72, CD80, CD86, CLEC-1, 4-1BB, TYROBP (DAP12), Dectin-1, FcaRI, FcyRI, FcyRII, FcyRIII, FceRI, IL-2RB, ICOS, KIR2DS2, MyD88, 0X40, and ZAP70. Particularly, in embodiments, R3 or R7 comprise a costimulatory domain comprising an amino acid sequence selected from the list consisting of SEQ ID NOS: 78- 97. Particularly, in embodiments, R3 or R7 comprise a costimulatory domain derived from 4- IBB. Particularly, in embodiments, R3 or R7 comprise a costimulatory domain comprising or consisting of an amino acid sequence comprising or consisting of SEQ ID NO: 78, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 119.

[0070] Particularly, in embodiments, chimeric antigen receptors provided herein are encoded with codon optimized nucleic acid sequences. Particularly, in embodiments, chimeric antigen receptors provided herein are encoded by a vector. Particularly, in embodiments, chimeric antigen receptors provided herein are encoded by a plasmid vector. Particularly, in embodiments, chimeric antigen receptors provided herein are encoded by a lentiviral plasmid vector. Other exemplary vectors are understood in the art, such as gamma retroviral, RNA, CRISPR template, transpose template, adeno-associated virus vectors.

[0071] Provided herein are engineered cells and and pharmaceutical compositions thereof. Engineered cells and pharmaceutical compositions made in a variety of ways understood by a person of ordinary skill in the art. In embodiments, a vector is transduced into an immune cell ex vivo, such as a viral or retroviral vector. Particularly, in embodiments, a lentiviral vector, is transduced ex vivo into an immune cell (see, e.g., Zhang et al., Engineering CAR-T cells, Biomark Res. 5:22 (2017)). Other methods of engineering immune cells are understood in the ait, such as introducing RNA of adenovirus for CRISPR/Cas9 editing, such as for in vivo methods of engineering (see, e.g., Dabiri et al., Site-specific transgene integration in chimeric antigen receptor (CAR) T cell therapies, Biomarker Research 11: 67 (2023)).

[0072] Particularly, in embodiments, pharmaceutical compositions provided herein comprise engineered cells provided herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients. Particularly, in embodiments, pharmaceutical compositions provided herein comprise engineered CD8⁺ T cells provided herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients. Particularly, in embodiments, pharmaceutical compositions provided herein comprise engineered CD4⁺ T cells provided herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients. Particularly, in embodiments, pharmaceutical compositions provided herein comprise two or more engineered cells provided herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients. Particularly, in embodiments, pharmaceutical compositions provided herein comprise two or more engineered cells provided herein, and one or more pharmaceutically acceptable carriers, diluents, or excipients, wherein the two or more engineered cells comprise an engineered CD8⁺ T cell and an engineered CD4⁺ T cell. [0073] Provided herein are engineered cells and pharmaceutical compositions. A person of ordinary skill in the art understands that engineered cells or pharmaceutical compositions provided herein can be formulated and administered in a variety of ways. In embodiments, engineered cells and pharmaceutical compositions provided herein are formulated for administration by any suitable route, such as intramuscular, intravenous, intradermal, intralesional, intraperitoneal injection, subcutaneous, intratumoral, epidural, nasal, oral, vaginal, rectal, topical, local, systemic, otic, inhalational, buccal (e.g., sublingual), and transdermal and can include additional agents that are biologically active, can facilitate or enhance delivery, or can control release. Other biologically active agents can also be administered sequentially, intermittently or simultaneously, such as in the same composition. Controlled release formulations and devices are contemplated, such as by pump. Additional means of formulation, administration, storage, preparation, manufacturing are contemplated (e.g., US Patent Pub. No. 20200215123, incorporated herein by reference in its entirety on March 6, 2024).

[0074] Particularly, in embodiments, engineered cells or pharmaceutical compositions provided herein can be formulated into suitable pharmaceutical preparations, such as solutions, suspensions, tablets, dispersible tablets, pills, capsules, powders, patches, or sustained release formulations. Particularly, in embodiments, engineered cells or pharmaceutical compositions provided herein are formulated in a dried or liquid form. Particularly, in embodiments, engineered cells or pharmaceutical compositions provided herein are formulated in a liquid form, for example, as a suspension for injection (direct administration) or frozen suspension that is thawed prior to use, dried soluble form, and emulsion. Particularly, in embodiments, injection or infusion administration is contemplated, for example, subcutaneous, intramuscular, intratumoral, intravenous, or intradermal administration is contemplated. Particularly, in embodiments, excipients, such as water, saline, dextrose, or glycerol, and carriers, such as a diluents, adjuvants, anti-adherents, binders, coatings, fillers, flavors, colors, lubricants, glidants, preservatives, detergents, sorbents or combinations thereof, are contemplated. Particularly, in embodiments, aqueous vehicles, nonaqueous vehicles, isotonic agents, buffers, antioxidants, local anesthetics, suspending agents, dispersing agents, emulsifying agents, sequestering agents, chelating agents, or combinations thereof are contemplated herein as excipients and carriers. Particularly, in embodiments, a therapeutically effective amount of engineered cells or pharmaceutical compositions provided herein are formulated, for example, as single-unit or multi-unit dosage formulations. Additional formulations and modes of administration are contemplated (e.g., US Patent Pub. No.

20200215123, incorporated herein by reference in its entirety on March 6, 2024).

SEQUENCES

Table 1. Sequences.

METHODS

[0075] Provided herein are engineered cells and pharmaceutical compositions comprising the engineered cells disclosed herein and one or more acceptable carriers, diluents, or excipients. Further provided herein are methods of treating cancer, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of cancer, such as lung cancer, nonsmall cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer) a pharmaceutical composition provided herein. Further provided herein are methods of treating cancer, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of cancer, such as lung cancer, non- small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer) an effective amount of an engineered cell provided herein. Particularly, provided herein are methods of treating cancer selected from the list consisting of: lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer) an effective amount of a pharmaceutical composition comprising an engineered cell provided herein. Particularly, the provided herein are methods of treating cancer selected from the list consisting of lung cancer, non- small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of cancer selected from the list consisting of: lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer) an effective amount of an engineered cell provided herein.

[0076] Further provided herein are methods of treating treatment-resistant or chemotherapyresistant cancer, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of treatment-resistant or chemotherapy-resistant cancer, such as lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, wherein the cancer is treatment-resistant or chemotherapy-resistant) a pharmaceutical composition provided herein. Further provided herein are methods of treating treatment-resistant or chemotherapy-resistant cancer, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of treatment-resistant or chemotherapy-resistant cancer, such as lung cancer, nonsmall cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, wherein the cancer is treatment- resistant or chemotherapy-resistant) an effective amount of an engineered cell provided herein. Particularly, provided herein are methods of treating treatment-resistant or chemotherapyresistant cancer selected from the list consisting of: lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, wherein the cancer is treatment-resistant or chemotherapyresistant, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of treatment-resistant or chemotherapy-resistant cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, wherein the cancer is treatment-resistant or chemotherapy-resistant) an effective amount of a pharmaceutical composition comprising an engineered cell provided herein. Particularly, the provided herein are methods of treating treatment-resistant or chemotherapy-resistant cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of treatment-resistant or chemotherapy-resistant cancer selected from the list consisting of: lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, wherein the cancer is treatment-resistant or chemotherapy-resistant) an effective amount of an engineered cell provided herein.

[0077] Further provided herein are pharmaceutical compositions provided herein for use in treating cancer, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer) a pharmaceutical composition provided herein. Further provided herein are engineered cells provided herein for use in treating cancer, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer) an effective amount of an engineered cell provided herein. Particularly, provided herein are pharmaceutical compositions provided herein for use in treating cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer) an effective amount of a pharmaceutical composition comprising an engineered cell provided herein. Particularly, provided herein are pharmaceutical compositions provided herein for use in treating cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer) an effective amount of an engineered cell provided herein. [0078] Further provided herein are pharmaceutical compositions provided herein for use in treating treatment-resistant or chemotherapy-resistant cancer, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, wherein the cancer is treatmentresistant or chemotherapy-resistant) a pharmaceutical composition provided herein. Further provided herein are engineered cells provided herein for use in treating treatment-resistant or chemotherapy-resistant cancer, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, wherein the cancer is treatment-resistant or chemotherapy-resistant) an effective amount of an engineered cell provided herein. Particularly, provided herein are pharmaceutical compositions provided herein for use in treating treatment-resistant or chemotherapy-resistant cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, wherein the cancer is treatment-resistant or chemotherapy-resistant, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of treatment-resistant or chemotherapy-resistant cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, wherein the cancer is treatment-resistant or chemotherapy-resistant) an effective amount of a pharmaceutical composition comprising an engineered cell provided herein. Particularly, provided herein are pharmaceutical compositions provided herein for use in treating treatment-resistant or chemotherapy-resistant cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, wherein the cancer is treatment-resistant or chemotherapy-resistant , comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of treatment-resistant or chemotherapy-resistant cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, wherein the cancer is treatment-resistant or chemotherapy-resistant) an effective amount of an engineered cell provided herein.

[0079] Further provided herein are uses of a pharmaceutical composition provided herein for treating cancer, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer) a pharmaceutical composition provided herein. Further provided herein are uses of an engineered cell provided herein for treating cancer, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer) an effective amount of an engineered cell provided herein. Particularly, provided herein are uses of a pharmaceutical composition provided herein for treating cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer) an effective amount of a pharmaceutical composition comprising an engineered cell provided herein. Particularly, provided herein are uses of an engineered cell provided herein for treating cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer) an effective amount of an engineered cell provided herein.

[0080] Further provided herein are uses of a pharmaceutical composition provided herein for treating treatment-resistant or chemotherapy-resistant cancer, comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, wherein the cancer is treatmentresistant or chemotherapy-resistant) a pharmaceutical composition provided herein. Further provided herein are uses of an engineered cell provided herein for treating treatment-resistant or chemotherapy-resistant cancer, comprising administering to a patient or subject in need thereof (e.g . , a patient or subject with or at risk of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, wherein the cancer is treatment-resistant or chemotherapy-resistant) an effective amount of an engineered cell provided herein. Particularly, provided herein are uses of a pharmaceutical composition provided herein for treating treatment-resistant or chemotherapyresistant cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, wherein the cancer is treatment-resistant or chemotherapyresistant , comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of treatment-resistant or chemotherapy-resistant cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, wherein the cancer is treatment-resistant or chemotherapy-resistant) an effective amount of a pharmaceutical composition comprising an engineered cell provided herein. Particularly, provided herein are uses of an engineered cell provided herein for treating treatment-resistant or chemotherapy-resistant cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, wherein the cancer is treatment-resistant or chemotherapy-resistant , comprising administering to a patient or subject in need thereof (e.g., a patient or subject with or at risk of treatment-resistant or chemotherapy-resistant cancer selected from the list consisting of lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer, wherein the cancer is treatment-resistant or chemotherapy-resistant) an effective amount of an engineered cell provided herein.

EMBODIMENTS

[0081 J Embodiments :

1. A nucleic acid encoding a chimeric antigen receptor of formula I:

R1 - R2 - R3 (I), wherein:

R2 is a transmembrane domain; and

2. The nucleic acid of embodiment 1, wherein R1 is an extracellular domain of formula III: R4

- R5 or R5 - R4 (III), wherein: a. R4 is a first heavy chain variable region means, and b. R5 is a second heavy chain variable region means, and optionally, a peptide linker between R4 and R5.

3. A nucleic acid encoding a chimeric antigen receptor of formula II:

R1 - R2 - R3 (II), wherein:

R2 is a transmembrane domain; and

4. The nucleic acid of embodiment 3, wherein R1 is an extracellular domain of the formula IV R4 - R5 or R5 - R4 (IV), wherein: a. R4 is a first heavy chain variable region, and b. R5 is a second heavy chain variable region, and optionally, a peptide linker between R4 and R5.

5. The nucleic acid of any one of embodiments 1-4, wherein R1 comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein: a. the amino acid sequence of HCDR1 is SEQ ID NO: 1, the amino acid sequence of HCDR2 is SEQ ID NO:2, the amino acid sequence of HCDR3 is SEQ ID NO:3; b. the amino acid sequence of HCDR1 is SEQ ID NO: 5, the amino acid sequence of HCDR2 is SEQ ID NO:6, the amino acid sequence of HCDR3 is SEQ ID NO:7; c. the amino acid sequence of HCDR1 is SEQ ID NO:9, the amino acid sequence of HCDR2 is SEQ ID NO: 10, the amino acid sequence of HCDR3 is SEQ ID NO: 11 ; d. the amino acid sequence of HCDR1 is SEQ ID NO: 13, the amino acid sequence of HCDR2 is SEQ ID NO: 14, the amino acid sequence of HCDR3 is SEQ ID NO: 15; e. the amino acid sequence of HCDR1 is SEQ ID NO: 17, the amino acid sequence of HCDR2 is SEQ ID NO: 18, the amino acid sequence of HCDR3 is SEQ ID NO: 19; f. the amino acid sequence of HCDR1 is SEQ ID NO:21, the amino acid sequence of HCDR2 is SEQ ID NO:22, the amino acid sequence of HCDR3 is SEQ ID NO:23; g. the amino acid sequence of HCDR1 is SEQ ID NO:25, the amino acid sequence of HCDR2 is SEQ ID NO:26, the amino acid sequence of HCDR3 is SEQ ID NO:27; h. the amino acid sequence of HCDR1 is SEQ ID NO:29, the amino acid sequence of HCDR2 is SEQ ID NO:30, the amino acid sequence of HCDR3 is SEQ ID NO:31; i. the amino acid sequence of HCDR1 is SEQ ID NO:33, the amino acid sequence of HCDR2 is SEQ ID NO:34, the amino acid sequence of HCDR3 is SEQ ID NO:35; j. the amino acid sequence of HCDR1 is SEQ ID NO:37, the amino acid sequence of HCDR2 is SEQ ID NO:38, the amino acid sequence of HCDR3 is SEQ ID NO:39; k. the amino acid sequence of HCDR1 is SEQ ID NO:41, the amino acid sequence of HCDR2 is SEQ ID NO:42, the amino acid sequence of HCDR3 is SEQ ID NO:43; l. the amino acid sequence of HCDR1 is SEQ ID NO:45, the amino acid sequence of HCDR2 is SEQ ID NO:46, the amino acid sequence of HCDR3 is SEQ ID NO:47; m. the amino acid sequence of HCDR1 is SEQ ID NO:49, the amino acid sequence of HCDR2 is SEQ ID NO:50, the amino acid sequence of HCDR3 is SEQ ID NO:51; n. the amino acid sequence of HCDR1 is SEQ ID NO:53, the amino acid sequence of HCDR2 is SEQ ID NO:54, the amino acid sequence of HCDR3 is SEQ ID NO:55; or o. the amino acid sequence of HCDR1 is SEQ ID NO:57, the amino acid sequence of HCDR2 is SEQ ID NO:58, the amino acid sequence of HCDR3 is SEQ ID NO:59. The nucleic acid of any one of embodiments 1-5, wherein the R1 comprises a heavy chain variable region (HCVR), wherein: a. the amino acid sequence of the HCVR is SEQ ID NO: 4, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 98; b. the amino acid sequence of the HCVR is SEQ ID NO: 8, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 99; c. the amino acid sequence of the HCVR is SEQ ID NO: 12, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 100; d. the amino acid sequence of the HCVR is SEQ ID NO: 16, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 101; e. the amino acid sequence of the HCVR is SEQ ID NO: 20, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 102; f. the amino acid sequence of the HCVR is SEQ ID NO: 24, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 103; g. the amino acid sequence of the HCVR is SEQ ID NO: 28, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 104; h. the amino acid sequence of the HCVR is SEQ ID NO: 32, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 105; i. the amino acid sequence of the HCVR is SEQ ID NO: 36, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 106; j. the amino acid sequence of the HCVR is SEQ ID NO: 40, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 107; k. the amino acid sequence of the HCVR is SEQ ID NO: 44, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 108; l. the amino acid sequence of the HCVR is SEQ ID NO: 48, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 109; m. the amino acid sequence of the HCVR is SEQ ID NO: 52, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 110; n. the amino acid sequence of the HCVR is SEQ ID NO: 56, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 111 ; or o. the amino acid sequence of the HCVR is SEQ ID NO: 60, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 112. The nucleic acid of any one of embodiments 1-6, wherein R1 comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein: a. the amino acid sequence of HCDR1 is SEQ ID NO:9, the amino acid sequence of HCDR2 is SEQ ID NO: 10, the amino acid sequence of HCDR3 is SEQ ID NO: 11; b. the amino acid sequence of HCDR1 is SEQ ID NO: 13, the amino acid sequence of HCDR2 is SEQ ID NO: 14, the amino acid sequence of HCDR3 is SEQ ID NO: 15; or c. the amino acid sequence of HCDR1 is SEQ ID NO:25, the amino acid sequence of HCDR2 is SEQ ID NO:26, the amino acid sequence of HCDR3 is SEQ ID NO:27. The nucleic acid of any one of embodiments 1-6, wherein the R1 comprises a heavy chain variable region (HCVR), wherein: a. the amino acid sequence of the HCVR is SEQ ID NO: 12, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 100; b. the amino acid sequence of the HCVR is SEQ ID NO: 16, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 101; or c. the amino acid sequence of the HCVR is SEQ ID NO: 28, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 104. The nucleic acid of any one of embodiments 1-8, wherein R1 comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO:25, the amino acid sequence of HCDR2 is SEQ ID NO:26, the amino acid sequence of HCDR3 is SEQ ID NO:27. The nucleic acid of any one of embodiments 1-8, wherein the R1 comprises a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 28. The nucleic acid of any one of embodiments 1-8, wherein R1 comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 9, the amino acid sequence of HCDR2 is SEQ ID NO: 10, the amino acid sequence of HCDR3 is SEQ ID NO: 11. The nucleic acid of any one of embodiments 1-8, wherein the R1 comprises a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 12. The nucleic acid of any one of embodiments 1-8, wherein R1 comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 13, the amino acid sequence of HCDR2 is SEQ ID NO: 14, the amino acid sequence of HCDR3 is SEQ ID NO: 15. The nucleic acid of any one of embodiments 1-8, wherein the R1 comprises a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 16. The nucleic acid of any one of embodiments 1-12, wherein: a. R1 comprises the complementarity deteimining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO:25, the amino acid sequence of HCDR2 is SEQ ID NO:26, the amino acid sequence of HCDR3 is SEQ ID NO:27; and b. R1 comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 9, the amino acid sequence of HCDR2 is SEQ ID NO: 10, the amino acid sequence of HCDR3 is SEQ ID NO: 11. nucleic acid of any one of embodiments 1-12, wherein the R1 comprises: a. (i)a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 28; and (ii) a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 12; b. the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 113; or c. the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 114. nucleic acid of any one of embodiments 1-10 or 13-14, wherein: a. R1 comprises: the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO:25, the amino acid sequence of HCDR2 is SEQ ID NO:26, the amino acid sequence of HCDR3 is SEQ ID NO:27; and b. R1 comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 13, the amino acid sequence of HCDR2 is SEQ ID NO: 14, the amino acid sequence of HCDR3 is SEQ ID NO: 15. nucleic acid of any one of embodiments 1-10 or 13-14, wherein the R1 comprises: a. (i) a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 28; and (ii) a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 16; b. the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 115; or c. the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 116. The nucleic acid of any one of embodiments 2 and 4-18, wherein the peptide linker comprises the amino acid sequence comprising GGGGS. The nucleic acid of of any one of embodiments 2 and 4-19, wherein the peptide linker consists of the amino acid sequence GGGGS. The nucleic acid of of any one of embodiments 2 and 4-19, wherein the peptide linker comprises or consists of 2, 3, 4, 5, 6, or 7 consecutive repeats of the amino acid sequence GGGGS. The nucleic acid of of any one of embodiments 2, 4-19, and 21, wherein the peptide linker consists of the amino acid sequence GGGGSGGGGSGGGGS. The nucleic acid of of any one of claims 1-27, wherein the hinge region comprises a hinge domain derived from CD3q. CD4, CD8a, CD28, IgGl, IgG2, or IgG4. The nucleic acid of of any one of embodiments 1-23, wherein the hinge region comprises an amino acid sequence selected from the list consisting of SEQ ID NOS: 66-72. The nucleic acid of of any one of embodiments 1-24, wherein the hinge region comprises a hinge domain derived from CD28. The nucleic acid of of any one of embodiments 1-25, wherein the hinge region comprises or consists of an amino acid sequence comprising or consisting of SEQ ID NO: 69, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 117. The nucleic acid of of any one of embodiments 1-26, wherein R2 comprises a transmembrane domain derived from CD3(^, CD4, CD8a, CD28, or CD137. The nucleic acid of of any one of embodiments 1-27 wherein R2 comprises an amino acid sequence selected from the list consisting of SEQ ID NOS: 73-77. The nucleic acid of any one of embodiments 1-28, wherein R2 comprises a transmembrane domain derived from CD28. The nucleic acid of any one of embodiments 1-29, wherein R2 comprises or consists of an amino acid sequence comprising or consisting of SEQ ID NO: 76, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 118. The nucleic acid of any one of embodiments 1-30, wherein R3 comprises: a. a signaling domain; or b. a signaling domain and a costimulatory domain, optionally wherein R3 is of the formula V: R6 - R7 or R7 - R6 (V), wherein R6 comprises a signaling domain, and R7 comprises a costimulatory domain. The nucleic acid of any one of embodiments 1-31, wherein the signaling domain is derived from CD3 , CD27, CD28, CD40, KIR2DS2, MyD88, or 0X40. The nucleic acid of of any one of embodiments 1-32, wherein the signaling domain comprises an amino acid sequence selected from the list consisting of SEQ TD NOS: 79-81 , 84-86, and 94-96. The nucleic acid of any one of embodiments 1-33, wherein the signaling domain is derived from CD3 The nucleic acid of any one of embodiments 1-34, wherein R3 comprises or consists of an amino acid sequence comprising or consisting of SEQ ID NO: 79, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 120. The nucleic acid of any one of embodiments 1-35, wherein the costimulatory domain is derived from of CD3y, CD36, CD3s, CD3 , CD27, CD40, CD28, CD72, CD80, CD86, CLEC-1, 4-1BB, TYROBP (DAP12), Dectin-1, FcaRI, FcyRI, FcyRII, FcyRIII, FcsRI, IL- 2RB, ICOS, KIR2DS2, MyD88, 0X40, and ZAP70. The nucleic acid of any one of embodiments 1-36, wherein the costimulatory domain comprises an amino acid sequence selected from the list consisting of SEQ ID NOS: 78-97. The nucleic acid of any one of embodiments 1-37, wherein the costimulatory domain is derived from 4- IBB. The nucleic acid of any one of embodiments 1-38, wherein the costimulatory domain comprises or consists of an amino acid sequence comprising or consisting of SEQ ID NO: 78, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 119. The nucleic acid of any one of embodiments 1-39, wherein the chimeric antigen receptor is encoded by codon-optimized nucleic acid sequences. A vector comprising the nucleic acid of any one of embodiments 1-40, wherein the chimeric antigen receptor is encoded by a vector. A plasmid vector comprising the nucleic acid of any one of embodiments 1-40. A lentiviral plasmid vector comprising the nucleic acid of any one of embodiments 1-40. A nucleic acid as described herein. A pharmaceutical composition comprising the nucleic acid or vector of any one of embodiments 1-44, and one or more pharmaceutically acceptable carriers, diluents, or excipients. A pharmaceutical composition comprising the nucleic acid or vector of any one of embodiments 1-44, and one or more pharmaceutically acceptable carriers, diluents, or excipients. A method of treating cancer, comprising administering to a subject with cancer: a. the nucleic acid or vector of any one of embodiments 1-44; or b. the pharmaceutical composition of embodiment 45 or 46. The method of embodiment 47, wherein the cancer is selected from the list consisting of: lung cancer, non- small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer. The method of embodiment 47 or embodiment 48, wherein the cancer is treatment-resistant or chemotherapy-resistant. A composition for use in treating cancer, comprising administering to a subject with cancer: a. the nucleic acid or vector of any one of embodiments 1-44; or b. the pharmaceutical composition of embodiment 45 or 46. The composition for use of embodiment 50, wherein the cancer is selected from the list consisting of: lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer. The composition for use of embodiment 50 or embodiment 51, wherein the cancer is treatment-resistant or chemotherapy-resistant. Use of a composition for treating cancer, comprising administering to a subject with cancer: a. the nucleic acid or vector of any one of embodiments 1-44; or b. the pharmaceutical composition of embodiment 45 or 46. 54. The use of embodiment 53, wherein the cancer is selected from the list consisting of: lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer.

55. The use of embodiment 53 or embodiment 54, wherein the cancer is treatment-resistant or chemotherapy-resistant.

56. An amino acid sequence as provided herein.

57. An amino acid sequence comprising any one of SEQ ID NOs: 1-98.

EXAMPLES

[0082] The EXAMPLES show embodiments in which compositions and methods show a high degree of activity in both in vitro and in vivo models of NSCLC, including in the setting of resistance. The VH binders of the EXAMPLES allowed for targeting domains that differ from what have been targeted by previous, scFv-based approaches. TROP2 CAR-Ts of the EXAMPLES showed activity against cell lines engineered to have resistance to TROP2 ADC, yielding durable responses.

[0083] Compositions of the EXAMPLES showed activity against EGFR mutant NSCLC that is resistant to Osimertinib, which is the first line therapy in advanced/metastatic EGFRm NSCLC (FIG. ID). TROP2-targeted fully-human heavy chain (VH)-only binders were generated through immunization of transgenic mice knocked in for the human heavy chain antibody locus. A second generation lentiviral CAR-T construct was used to encode TROP2 fully human VH binders with 41BB costimulatory molecule and CD3z signaling domain (FIG. 2A). TROP2 VH binders showed activity against domains that differ from the previous approaches based on scFv TROP2 antibody therapies, such as sacitizumab govitecan and datopotumab deruxtecan (FIG. 6A). TROP2 CAR-Ts showed activity in models with resistance to TROP2 ADC, such as on-target mutations in TROP2 as well as off-target mutations in TOPI pathway genes (FIGS. 3A-3D). TROP2 fully human VH CAR-Ts showed high activity in vitro against two human NSCLC cell lines as well as low tonic signaling using a cut off of <5% (FIG. 5A). Compositions of the EXAMPLES showed activity in vivo in an orthotopic disseminated xenograft model of NSCLC using the TROP2 CAR-T provided herein, which showed high degrees of activity and durable suppression of tumors with VH304681 (FIGS. 5B-5C). Compositions of the EXAMPLES showed targeting of TROP2+ tumors with CAR- T, which yielded higher response rates and more durable responses. TROP2-directed CAR-T were operable with heterogenous expression compared with previous CAR-T cell therapy approaches and target low antigen density as well (Harrington et al., 2017; Majzner et al., 2020).

[0084] Biparatopic TROP2-directed CAR-T utilizing a combination of VH sequences were identified, for example, VH304375, VH304377, and VH304681. The biparatopic approach of the EXAMPLES showed equivalent activity to single binders as well as the ability to maintain activity when various substitutions of the TROP2 domains were made (FIG. 7B). Constructs showed low levels of tonic signaling in a similar assay to what was performed in FIG. 3A as well as a high degree of activation, a surrogate for cytotoxicity (FIG. 7C).

Cell lines

[0085] PC9 and HCC827GR6 were obtained from and authenticated by short tandem repeat genotyping. MDA-MB-231 and HCC70 were obtained from ATCC (Manassas, VA, USA). The PC9 C797S cell line was generated through drug selection protocols or CRISPR engineering as previously published (see, Haikala et al., (2022)). Jurkat T cells (clone E6-1) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained in RPMI 1640 medium (Gibco, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Gibco), 1% penicillin-streptomycin (Gibco), and 2 mM L-glutamine (Gibco). Cells were cultured at 37°C in a humidified atmosphere with 5% CO2 and passaged every 2-3 days to maintain logarithmic growth. T cells were isolated from PBMCs (STEMCELL Technologies, catalog no. 70025) using the EasySep Human T Cell Isolation Kit (STEMCELL Technologies, catalog no. 17953) following the manufacturer’s instructions. The cells were cultured in RPMI1640 supplemented with 10% human serum (Sigma-Aldrich, catalog no. H5667), lx penicillinstreptomycin, 2 mmol/L l-glutamine, and 100 lU/mL IL2, 25 ng/mL IL7, and 25 ng/mL IL15. The T cells were immediately activated with 1% T cell TransAct (Miltenyi Biotec, catalog no. 130- 128-758) after isolation. Mycoplasma infection was regularly checked by PCR using the conditioned media from each cell line. All experiments were performed within 10 passages from the original frozen stocks.

Cloning of CAR vector

[0086] 3rd gen lentiviral eGFP expression vector, CMV promoter, Hygro#17446 were obtained from Addgene (Watertown, MA, USA) and the hygroR cassette was removed via digestion with EcoRV and Kpnl followed by Gibson ligation of a fragment containing CD28EC domain, CD28TM domain, 41BB, and CD3z as well as P2A Vex reporter. Gibson cloning of scFv derived from anti=BCMA CAR orva-cel or scFv derived from Sacituzumab was preformed. Gene fragments were ordered from IDT (Coralville, 1A, USA). Sequencing of plasmids was confirmed via Primordium sequencing and was performed by Plasmidsaurus (Oxford Nanopore Technology, Oxford, UK) with custom analysis and annotation with whole plasmid sequencing.

CRISPR/CAS9 KO

[0087] TACSTD2 knock-out in PC9 clones were performed by CRISPR/CAS9 genome editing using the Alt-R CRISPR-Cas9 System (Integrated DNA Technologies, IDT) and Lonza 4D- Nucleofector (Lonza) as previously described (See, Kurppa et al., (2020)). Three different guide sequences for TACSTD2 were designed using Alt-R CRISPR-Cas9 crRNAs design tool from IDT with the following sequence: 5’-GCAACCAGACGTCGGTGTGC-3’ (SEQ ID NO: 121), 5’- TCGGCTGCACCCCAAGTTCG-3’ (SEQ ID NO: 122), 5’- GCACACGGTCATCTTGTTGG-3’ (SEQ ID NO: 123). The Alt-R CRISPR Negative Control crRNA #1 was also purchased from IDT to generate wild type control clones 5’-CGTTAATCGCGTATAATACG-3’ (SEQ ID NO: 124). These guides were used simultaneously to ensure high knock-out efficiency to create TROP2 sgRNA PC9 clones and CTL sgRNA PC9 clones (40 pmol of each crRNA mix with 120 pmol of tracrRNA). After 72 h, the nucleofected cells were single-cell cloned, and loss of TROP2 protein expression was analyzed from the single-cell clones by flow cytometry and western blotting.

Cloning of TROP2 constructs

[0088] Human TROP2 sequence was obtained from Genecards and cloned into SFFV IRES Puromycin vector available from TWIST (See, Stelzer et al., (2024)). HA tag was inserted after the signal peptide of TROP2. Variants were generated by substitution of murine residues of TROP2 at indicated residues.

Table 2. Sequences Generation of CARs

[0089] For packaging and production of lentivirus particles, 293 Lenti-X packaging cells (Takara, San Jose, CA, USA) were seeded into a 6-well plate (1E6 cells/plate) for 24 h or until 90% confluence reached, then transfected with lentiviral plasmids encoding CAR (1.25 pg), pMD.2G encoding VSV-G envelope (0.25 pg), and a packaging vector psPAX2 (1 pg) using Minis 293T Transit transfection reagent. Virus supernatants were harvested at 24 and 48 h after transfection and filtered through a 0.45 mm PES membrane. Neat viral supernatant was used and cells were centrifuged in viral supernatant 2000 x g for 1 h with IX Lentiboost (Revvity Gene Delivery, Inc. Lawrence, MA, USA). After transduction, T cells were expanded with cytokines IL2 (10 ng/mL), IL7 (3 ng/mL; #130-095-361), and IL15 (10 ng/mL; Miltenyi Biotec #130-095-762), in RPMI1640 supplemented 10% FBS, and their transduction efficiency was determined by FACS 3 days after transduction using Vex reporter gene or LssOrange as a readout and zombie green or zombie IR as a viability dye (#423111, Biolegend, London, UK). CAR doses were calculated by percent transduction of viable cells.

[0090] CAR T cells were stained for surface markers using a 96-well U-bottom plate. Cells were washed with FACS buffer and blocked for 15 minutes on ice with human FcX block then incubated with fluorochrome-conjugated antibodies, including CCR7 PE-Dazzle 594, CD45RA-PE, CD4-PE-Cy7, and CD8a-Alexa 647, along with appropriate unstained controls. Cells were stained at 4°C for 30 minutes. After staining, cells were washed and resuspended in FACS buffer. Viability was assessed using Zombie NIR before acquisition. Flow cytometry was performed on 3 laser Cytek Northern Lights Flow cytometer (Fremont, California USA). Data were analyzed using FlowJo software (Ashland, OR USA).

Table 3.

Flow cytometry

[0091] For flow of TROP2 on PC9 or HA, antibodies shown in Table 3 were used. For flow cytometry, cells were collected via trypsinization, washed with FACS buffer (0.5% BSA in PBS) and stained with cither HA-PcrCP Cy5.5 (1:50) or TROP2-PE antibody (1:200). Cells were stained with Zombie green viability dye as above. Cells were stained for 45 min on ice, washed once with FACS buffer, resuspended in FACS buffer, and analyzed on a Northern Lights flow cytometer (Fremont, CA, USA).

Luciferase-based cytotoxicity

[0092] PC9, HCC70, and HCC827GR6 were stably transduced with luciferase, as described above. 10,000 target cells were plated in 96-well plates in triplicate with CAR+ T cells at the indicated effector-to-target (E:T) ratios; cells were then incubated for 24 h. Assays were performed without addition of cytokines. Cell viability was determined by an luciferase-dependent assay with OneGlo substrate E6110 (Promega, Madison, WI, USA), where % cytotoxicity = (BLI Control - BLISAMPLE)/BLIMAX; BLI Control = mean target cell alone value of that experiment (typically BCMA used as non-targeting control). Bioluminescence was read on an Agilent Cytation 5 (Santa Clara, CA, USA). Significance was determined by two-way ANOVA.

Incucyte-based cytotoxicity

[0093] Cells were stably transduced with either GFP-ffLuc fusion protein or mCherry as well as TROP2 with substitutions for murine CPD or murine CRD. Single cell clones were isolated and confirmed. Cells were then seeded at 10E3 cells/well in a 96-well clear bottom plate and allowed to settle for 6 h. Assays were performed without addition of cytokines. Baseline image were acquired with counting of green or red cells prior to addition of effectors. Effectors were added at indicated E:T and imaging was acquired every 6 h for counting of green or red objects (targets). Cytotoxicity index was plotted as object number at time x/object number at t=Oh. Images were acquired on Incucyte S3 Live Cell Analysis system (Goettingen, Germany).

In vitro ADC assay [0094] Target cells were seeded at a density of 5000 cells per well in a 96-well Clear Round Bottom Ultra-Low Attachment Microplate (Corning #7007). CAR T cells (CAR BCMA, TROP2- CAR) were added in triplicate at decreasing effector (E:T) ratios (0.25:1, 0.06:1, 0.25:1), with a final concentration of 500 U/mL IL-2 maintained throughout the experiment. Following a 4-day coculture period, the Bright-Glo™ Luciferase Assay System (Promega, #E2610) was added at a 1:10 ratio. Samples were incubated for 5 min at 23°C in the dark, with gentle shaking to ensure even distribution of the reagent and stable luminescence. Subsequently, the samples were transferred to a 96-well Black Polystyrene Microplate (Corning #CLS3904), and luciferase activity was measured using a Tecan Infinite M Plex plate reader. Relative tumor cell viability was determined by normalizing the luminescence of the treatment conditions to the tumor-only samples.

2D ADC cytotoxicity

[0095] Target cells were seeded at a density of 5000 cells per well in a 96-well Clear Round Bottom Ultra-Low Attachment Microplate (Corning #7007). ADC-based treatment was added in triplicate at increasing concentration (25 pg/mL, 50 pg/mL, 100 pg/mL), along with free payload (25 nM, 50 nM, 100 nM) used as a positive control, and incubated for 6 days. To measure target cells’ viability, Bright-Glo™ Luciferase Assay System (Promega, #E2610) was added at a 1:10 ratio. Samples were incubated for 5 min at 23°C in the dark, with gentle shaking to ensure even distribution of the reagent and stable luminescence. Subsequently, the samples were transferred to a 96-well Black Polystyrene Microplate (Corning #CLS3904), and luciferase activity was measured using a Tecan Infinite M Plex plate reader.

Binding kinetics of TROP2 human VH binders

[0096] Surface plasmon resonance (SPR) experiments were done on the Nicoya AltoSPR using PBS supplemented with 1% BSA and 0.1% Tween 20 running buffer (Nicoya Lifesciences PBS-T, Kitchener, ON, Canada). To generate a capture surface, Protein A (Nicoya Lifesciences ALTO-R-PROA-KIT) diluted in a sodium acetate buffer (pH 5.0) was amine coupled to the sensor surface of a 16-channel carboxyl cartridge (Nicoya Lifesciences, KC-CBX-PEG-16) via EDC and NHS. For kinetics experiments, twelve anti-TROP2 heavy chain only antibodies (HCAbs), previously generated in-house by Harbour BioMed and single-step purified by Protein A, as well as commercially available sacituzumab (MedChemExpress, HY-P99045, Monmouth Junction, NJ, USA), datopotamab (MedChemExpress, HY-P99843), and an irrelevantly-targeted IgG control were diluted to 15 pg/mL in running buffer and individually captured on sensor surfaces previously immobilized with Protein A. Recombinant human TROP2-his (Bio-Techne Corporation 11158-T2- 100, Minneapolis, MN, USA) was diluted to 1800 nM in running buffer and then serially diluted 3- fold for a total of five concentrations as well as a baseline control sample at 0 nM. Each dilution of rhTROP2 was flowed over the captured HCAbs from low to high concentration with a regeneration of 10 mM glycine-HCl pH 1.5 (Nicoya Lifesciences ALTO-R-GLYHC1-1.5) in between each cycle. Resulting sensorgrams were double referenced and, where applicable, fit to a 1 : 1 Langmuir binding model. All U_/(s, k_offs, and KDS are reported as mean ± standard deviation from a total of four separate experiments.

Structural modeling of TROP2 and scFv/VH binders

[0097] Computational studies aimed at modeling the structure and binding interactions of datopotamab and sacituzumab scFvs and three VH binders (VH304375, VH304377, and VH304681) to human TROP2 were performed using the MOE software package (MOE2022.02, Chemical Computing Group, Montreal, Canada). The receptor molecule, human TROP2, was prepared for docking by removing water and organic molecules from a previously determined crystal structure of the homotetramer (PDB ID: 7PEE) and isolating a single monomer for use as the docking receptor. The appropriate regions within TROP2 — CRD, CPD, TY domain, and the previously identified sacituzumab binding site — were further defined and annotated via the sequence editor in MOE2022.02. To generate models of each VH or scFv, the primary amino acid sequence of the binder was uploaded into the sequence editor in MOE2022.02 and annotated using the IMGT schema to identify the CDR and framework residues. A homology model of the variable region of each antibody was then generated using the Antibody Modeler application within MOE2022.02. Using methods previously described, a homology search was performed for each benchmark or VH binder to identify -1000 human antibody models in PDB that share high primary sequence similarity to the queried antibody sequence (42-44). For sacituzumab, 4D9Q was used to model the heavy and light chain framework regions, while for datopotamab, 2XTJ was used to model the framework regions of both chains. The frameworks of the three VHs (VH304375, VH304377, and VH304681) were modeled by 6023, 5N2K, and 5HI4, respectively. For the CDRs, a chimeric template was generated using human VH or scFv structures that shared >90% primary sequence similarity for CDR 1 & 2 and >70% primary sequence similarity for CDR3 to the target or queried sequence. For each antibody, the best scoring framework and chimeric CDR templates were used to build the variable region models. To generate biparatopic VH models, homology models of each binder were independently generated and linked via a prebuilt, conformation- searched G4S linker using the Linker Modeler application in MOE2022.02. Each model was then energy minimized with the Amber 10:EHT forcefield in MOE2022.02 to generate the completed model used for protein-protein docking.

[0098] To perform docking analysis, both the receptor and antibody model were energy minimized as previously described. The appropriate region of TROP2 (i.e., CRD, CPD, or the Sacituzumab binding site) was then defined as the docking pocket within the receptor and at least 1000 conformations were generated for the binder to this binding site using the Protein-Protein Docking Module. Energetically favored conformations of antibody-hTROP2 complexes for each scFv or VH binder were clustered to identify a preferred bin of conformations. The conformations were analyzed in terms of binding energy, the geometry of the binder, and the total number of interactions, as determined by the protein-ligand interaction fingerprint (PLIF), to identify the preferred conformations. For biparatopic VH models, docking of each binder was performed sequentially with the higher-affinity binder mediating the initial interaction with the hTROP2 receptor.

In vivo xenograft experiments

[0099] Female NSG mice, 6-weeks old were purchased from The Jackson Laboratory (Bar Harbor, ME USA). Animals were allowed to acclimate for at least 5 days before initiation of the study. All in vivo studies were conducted at Dana-Farber Cancer Institute with the approval of the Institutional Animal Care and Use Committee in an AAALAC accredited vivarium. The cells (PC-9 or HCC827GR6) were harvested, and 5 x 10⁶ cells with 50% Matrigel (Fisher Scientific) were implanted subcutaneously in the right flank of the NSG mice. Tumors were allowed to establish to an average size of 106.2 mm³ and 123.6 mm³ for PC-9 and HCC827GR6 tumors, respectively. The PDX tumors for DFCI-161 (EGFR L858R, MET amplified) were derived from pleural effusions collected from the patient as a part of clinical care. DFCL243 (EGFR del/T790M) and DFCL642 (SCLC) were derived from surgical biopsies and implanted directly into the sub-renal capsule of NSG mice for expansion. Following initial implantation, the PDX models were expanded and passaged continuously in NSG mice as subcutaneous tumors. Tumor-bearing mice were randomized using Studylog software (San Francisco, CA, USA) into various treatment groups. Mice were treated with single IV injection of either control BCM A CAR-T or hRS7 CAR-T cells. Tumor volumes were determined from caliper measurements by using the formula, Tumor volume = (length x width²)/2. Tumor volumes and body weights were measured twice weekly.

[0100] For the hematogenous tail vein metastatic model, 0.25E5 PC9 stable transduced with ffLuc were injected tail vein into NSG mice. After 14 days mice were imaged utilizing Bioluminescence imaging (BLI) to monitor tumor growth and response to therapy. Mice were anesthetized using 2-3% isoflurane in oxygen and placed on the imaging stage of a bioluminescence imaging system (IVIS Spectrum, Revvity Waltham MA USA). Beetle Luciferin, Potassium Salt D-luciferin E1605 (Promega Madison WI USA) was prepared at a concentration of 8 mg/mL in sterile phosphate-buffered saline (PBS) and administered via intraperitoneal (i.p.) injection at a dose of 1.6 mg per mouse 5 minutes prior to imaging to ensure maximal photon emission.

[0101] Images were acquired using the IVIS system under controlled conditions, with the field of view adjusted to include all mice in each imaging session. The system parameters, including exposure time (typically 1-60 seconds), binning factor, and f-stop, were optimized based on the intensity of the signal to avoid oversaturation. Luminescence was quantified as total flux (photons/second) within a manually defined region of interest (RO I) corresponding to the tumor site, using Living Image software (Revvity Waltham, MA USA). To maintain consistency across imaging sessions, all mice were imaged at the same time points relative to treatment initiation. Animals were monitored closely for signs of distress during imaging, and body temperature was maintained using a heated stage.

Bioluminescence Imaging of Nanoluc Transducer CAR T Cells

[0102] To assess CAR T cell localization and persistence, furimazine-based flash luciferase (FFz) imaging was performed using nanoluc-expressing CAR T cells. CAR T cells were engineered to express a LssOrange-NanoLuciferase linked via 2A to CAR vector and expanded ex vivo prior to in vivo administration. NSG mice engrafted with PC9 expressing either mCRD (right flnak) or mCPD (left flank) xenografts were injected subcutaneouslty. When tumors reached -100 mm³, mice were injected with 2.5E6 CAR+ cells. For imaging, mice were anesthetized with isoflurane and injected intraperitoneally with 40 ug FFz substrate (N4100, Promega Madison WI USA) diluted in sterile PBS at 0.2 mg/m2. Bioluminescence was captured using an IVIS Spectrum (PerkinElmer, Waltham MA USA) with an automatic imaging acquisition time of 1-30 seconds and medium binning. Images were analyzed using Living Image software (PerkinElmer, Waltham MA USA).

All animal procedures were performed following institutional guidelines for animal care and were approved by the [Institutional Animal Care and Use Committee (1ACUC)] at Dana-Farber Cancer Institute.

Ex vivo microfluidic device

[0103] PDX-derived organotypic tumor spheroid (XDOTS) culture was carried out as previously described, with modifications (see Knelson et al., (2022)). Following harvesting, DFCI243 PDX tumor specimens were minced using sterile scissors in several exchanges of prewarmed (37°C) full media containing 100 U/mL collagenase type IV (Thermo Fisher Scientific, Waltham, MA, USA) and 50 mg/mL DNase I (Roche, Indianapolis, IN, USA). Freshly liberated fragments were transferred into cold full media, and new prewarmed digestion media was added to the residual tumor. Xenograft fragments were strained sequentially through 100-pm and 40-pm filters. Spheroids smaller than 100 pm but larger than 40 pm were pelleted and resuspended in type I rat tail collagen (Corning, final concentration of 2.8 mg/mL). Spheroids were loaded into the gel ports of the DAX-1 3D cell culture chip (AIM Biotech). The number of cells per device well was evaluated using a lOx objective on a brightfield microscope (Olympus CKX41). The corresponding amount of CAR T cells was resuspended in full media and added to the media ports of the chip after the collagen with DFCI243 spheroids had polymerized. Assays were performed without addition of cytokines. Fluorescent viability labeling was performed after 5 days of incubation by loading microfluidic devices with 10 pg/mL solution of Hoechst 33342 (Thermo Fisher Scientific #H3570), 1 pg/mL solution of Calcein AM (Thermo Fisher Scientific #C1430), and 1 pg/mL solution of PI (Thermo Fisher Scientific #P3566). Following incubation with the dyes for 45 min at 23°C in the dark, images were captured using 4x objective of a Nikon Eclipse 80i fluorescence microscope equipped with automated motorized stage (Proscan), Z-stack (Prior), and Zyla 5.5 sCMOS camera (Andor). Image capture and analysis were performed using NIS-Elements AR software package version 5.00.00 64-bit. Live, metabolically active, and dead cell quantitation was performed by measuring the total cell area of each dye. Cytokine assessment

[0104] Individual cytokine assays for IFNy (Cat# DIF50C) was conducted using enzyme- linked immunosorbent assays (ELISA) in accordance with the manufacturer’s instructions (R&D Systems). IFNy from the 3D coculture was collected at 72 h.

Statistical analysis

[0105] Data normalization: Cytotoxicity was measured as the viability of target cells after co-incubation with CAR T cells. The results were normalized to control CARs (BCMA or CD 19- targeted CARs). A Student's t-test or ANOVA with multiple comparisons was used to assess the differences in cytotoxicity between different CAR constructs and conditions. Data were represented as mean ± SEM. P-values are reported in figures as follows: ns (P > 0.05), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0106] For tumor volume analysis: Tumor volumes were measured over time and plotted as a function of days post-CAR treatment. The comparison between groups (e.g., BCMA control vs. TROP2 CAR) was made using two-way ANOVA with a mixed-effects model or a repeated- measures ANOVA, with tumor volume data as dependent variables. Tumor measurements were collected by blinded observers.

[0107] Survival analysis: Kaplan-Meier survival analysis with Log-Rank (Mantel-Cox) test was performed to evaluate differences in survival between treatment.

[0108] Live-cell imaging: Cytotoxicity index was analyzed using Incucyte live-cell imaging by counting cell object numbers (e.g., red or green objects) over time. Statistical comparisons of cytotoxicity indices between different CAR constructs and time points were performed using repeated measures ANOVA. As in previous assays, p-values were reported in figures using thresholds (ns, *P < 0.05, etc.).

[0109] Ex vivo organoid model: Percent live and dead cells were measured by quantifying total dye area in organoid models, and cytokine release (e.g., IFN-y) was measured using supernatant assays. An unpaired t-test or ANOVA with post-hoc correction was used to compare between groups (control vs. CAR-treated). P-values between conditions were reported similarly, with significance thresholds. Results

TROP2-directed CARs are highly effective in in vitro and in vivo TROP2+ solid tumors

[0110] Second-generation CAR vectors were designed utilizing the scFv derived from sacituzumab-govitecan (clone hRS7) along with 4- IBB costimulatory and CD3z signaling domains (Figure 8A). In vitro activity against TROP2+ cell lines, PC9 (NSCLC EGFR exon 19 del) and HCC70 (TNBC), was induced by TROP2-targeted CARs (Figure 9A). Cytotoxicity was abrogated on TROP2 knock-out lines, confirming specific tumor-cell killing induced by the CAR (Figure 8B). The activity of TROP2 CAR was evaluted in additional NSCLC and TNBC models, HCC827GR6 (EGFR exon 19 del with MET amplification resistant to gefitinib) and MDA-MB-231 (TNBC) (Figure 8C). While HCC827GR6 is known to be resistant to EGFRi, additional on-target mechanisms of resistance to current third-generation EGFRi Osimertinib were evalutaed. EGFR C797S is a known on-target mechanism of resistance to the third-generation EGFR inhibitor Osimertinib (see, Thress et al., 2015)). TROP2-targeted CAR T cells were demonstrated to bypass and maintain a high degree of cytotoxicity in the setting of acquired resistance to osimertinib in PC9 harboring EGFR T790M/C797S mutation (Figure 9B). To determine if TROP2-targeted CAR T cells maintain a high degree of in vitro activity in a more physiologic 3D culture, 3D Matrigel- based assay was utilized and demonstrated substantial anti-tumor activity against PC9 (Figure 9C). To further investigate activity in vivo, either PC9 or HCC827GR6 was engrafted in NSG mice subcutaneously. When tumors reached -100 mm³, mice were treated with a single dose of either control (irrelevantly targeted to BCMA) CAR T cells or TROP2-targeted CAR T cells at various CAR T cell doses. Tumor regression was observed in all TROP2-targeted CAR-treated mice, including at low doses. Responses were durable until the conclusion of experiments (>100 days; Figure 9D and Figure 10). An intravenous metastatic lung tropic model was established of PC9 transduced with firefly luciferase (FLuc) in NSG mice, which more accurately recapitulates the tumor burden and disease location seen in patients. A single low dose of CAR T cells was administered IV after tumor engraftment was confirmed; in nearly all mice receiving TROP2- targeted CAR T cell therapy, a high degree of anti-tumor activity was observed (Figure 9E).

[0111] Previously it was established that the use of primary organotypic tumor spheroids for assessing immune responses including in xenograft-derived and patient-derived samples (See Jenkins et al., 2018). An established patient-derived EGFR mutant NSCLC sample (DFCI 243 EGFR dell9/T790M) was utilized. Ex vivo tumor spheroids were derived, and TROP2-targeted CARs demonstrated substantial in vitro activity (Figure 9F-G). It was also demonstrated that TROP2-targeted CARs have a high degree of activity against both PC9 and HCC827GR6 cell line- derived organotypic tumor spheroids (Figure 11 A). IFN-gamma levels substantially rose, which was correlated with the cytotoxicity of the TR0P2 CARs in the organotypic tumor spheroid cytotoxicity experiment (Figure 11B-C).

[0112] It was then evaluted whether the TROP2-targeted CARs were active against patient- derived xenograft (PDX) models (EGFR mutant NSCLC). Efficacy was observed with even bulky primary tumor-derived models, with a single CAR dose leading to tumor regression in two EGFR mutant PDX models (DFCI243 and DFCI161; EGFR dell9/T790M and EGFR L858R/MET amplified, respectively). This was specific for TROP2+ tumors, as a transformed small cell lung cancer (DFCI642; TROP2 negative) did not show response (Figure 9H).

Decreased TROP2 expression observed in the setting of TROP2 ADC resistance remains targetable with TROP2 CARs

[0113] Mutations in TACSTD2 (TROP2) and the topoisomerase gene TOPI have previously been reported as resistance mechanisms to sacituzumab-govitecan in TNBC. The mutation in TROP2 resulting in a T256R amino acid change leads to decreased TROP2 expression and resistance to TROP2 ADC in TNBC (see, Coates et al., (2021)), while the TOPI E418K and other TOPI mutations cause resistance to the ADC payload (see, Abelman et al., (2024)). Utilizing the PC9 with TROP2 KO, the cell line was reconstituted with either TROP2 WT, TROP2 T256R, or TOPI E418K mutant. This clinical tumor cell phenotype was recapitulated and observed that the T256R TROP2 mutation led to decreased TROP2 cell surface expression in a NSCLC cell line (Figure 12A). It was demonstrated that the TROP2 T256R and TOPI E418K mutation led to acquired resistance to TROP2 ADC in vitro (Figure 12B). It was evaluated whether the CARs were efficacious in the presence of acquired resistance in TROP2 T256R. Surprisingly, even in this low antigen setting, and in contrast to the ADC, TROP2-targeted CAR T cells were able to maintain a high degree of cytotoxicity approaching what was observed in the TROP2 WT setting (Figure 12C).

Mutations in TOPI genes can lead to resistance to ADC which remain targetable by TROP2 CARs

[0114] Mutations in TOPI have been observed to cause resistance to ADC with TOPI inhibitor drug conjugates including in the setting of clinical TROP2 ADC use. Activity against TOPI E418K mutation with our TROP2 CARs was observed(Figure 12D). Taken together, on- target antigen and off-target antigen resistance to TROP2 ADC and can be overcome by CAR T cells in these contexts (Figure 12E).

Point mutations preventing TROP2 ADC efficacy remain targetable with rational design of fully- human VH-only based TROP2 CARs

[0115] While the first round of CARs to receive FDA approvals were scFv-bascd, several newer CAR constructs, including the FDA-approved BCMA-targeted CAR cilta-cel, have utilized VH-only binding domains. Advantages of VH-only domains include smaller size (an advantage for limited genetic size in highly efficient lentiviral vectors) and easier ability to link in daisy-chain format to target multiple epitopes without concern for VH-VL mispairing and with less concern for steric hindrance. A novel TROP2-targeted fully-human heavy chain (VH)-only binders were generated through immunization of transgenic mice knocked out for murine VH/VL chain loci and knocked in only for the human VH chain locus (heavy chain-only antibody (HCAb) mice; (see, Drabek et al., (2016)). While humans do not naturally generate highly functional single domain binder, mice engineered to generate VH only binders can produce, through murine B cell somatic hypermutation, single domain antibodies with high specificity and affinity. Single B/plasma cells producing highly active and specific nanobodies were identified and sequenced. These sequences were cloned into a 4-lBB/CD3z containing second-generation CAR construct. The activity of these novel TROP2-targeted VH-based CARs was profiled against NSCLC cell lines PC9 and HCC827GR6. Constructs with high cytotoxicity (>75% equivalent to the benchmark hRS7) were selected for further study (Figure 13). These candidates were further tested in an in vivo aggressive orthotopic model of NSCLC. Many of the CARs performed as well as the hRS7 CAR, with the VH681 -based CAR demonstrating superior tumor control and improved survival (Figure 14A-B). [0116] TROP2 is comprised of three extracellular domains, a cysteine-rich (CRD), thyroglobulin-like (TY), and cystcinc-poor domain (CPD). Through previous structure-based and in vitro binding analyses, sacituzumab has been identified to bind to TROP2 residues Q237-252 in the CPD (25). It was confiimed that the target epitope of sacituzumab scFv to TROP2, highlighting the domain bound by the scFv as in the CPD (Figure 14C). In silico structural models suggest that the scFv structure binds to TROP2 similarly to the full-length IgG (Figure 15). While the TROP2- targeted CAR based on sacituzumab clone (hRS7) was able to overcome resistance to the T256R mutation outside the binding epitope (that decreased antigen expression, above, Figure 12A, 12C), a model of TROP2 (hRS7) resistance was further developed via substitution in the hRS7 binding epitope. As hRS7 (as well as VH-only binders) were generated by immunization of mice using the hRS7 parental mouse antibody (clone: RS7-3G11), the fact that these binders do not have murine cross-reactivity to generate resistance was leveraged. Thereby, to generate this model a series of mutations that swapped human AAs that were not preserved in the murine sequence were engineered (mQ237-252; Figure 14D and Figure 16). Surprisingly, it was observed that CARs generated from the scFv from datopotamab, another TROP2 ADC moving forward in clinical trials in multiple solid tumors including NSCLC, also lost all activity against human/murine chimeric TROP2, including murine mQ237-252 (Figure 17), demonstrating that both of the most clinically advanced TROP2 ADCs bind the same region.

[0117] Given that sacituzumab and datopotamab target the same region of TROP2, it was determined whether the novel TROP2 VH-only binders described herein targeted to a similar or distinct domain. A similar model as shown in Figure 14B was generated, whereby overexpression of various substitutions of domains of human TROP2 with their murine counterpart were made. It was determined that the TROP2 VH681 binds a unique domain, the CRD, compared to the scFv- based sacituzumab and datopotamab, as well as other VH binders, which bind to the CPD domain (Figure 14E). Protein-protein interaction was computationally modeleed between each novel VH binder and TROP2 using Molecular Operating Environment (MOE), identifying the key putative residues on TROP2 that form the epitope for each novel VH binder, further supporting experimental observations of VH681’s distinct targeting (Figure 14F).

Development of biparatopic TROP2-targeted VH-based CARs

[0118] A central advantage of utilizing fully-human VH-only binders is the ability to easily link these single-domain binders for dual-targeting, which were leveraged here to engineer biparatopic CARs simultaneously targeting unique epitopes (Figure 18A). Both scFv, single VH, and biparatopic CARs were profiled for CD4/CD8 and T-cell memory function with no significant differences seen (Figure 19). In vitro cytotoxicity assays were performed with the TROP2 biparatopic VH binder CARs, comparing to the scFv and the single VH CAR constructs against PC9 with murine CPD and CRD domains (similar to Figure 14E). Uniquely, only the biparatopic VH CARs, and not the scFv nor single VH TROP2 binder CARs, maintained cytotoxicity in both contexts (Figure 18B). To further assess CAR functionality, a panel of cytokines was profiled using a Luminex assay which revealed that CAR T treated samples exhibited elevated levels of pro- inflammatory cytokines, including GM-CSF, IFN-y, IL-1 , IL-2, IL-6, IL-8, and TNF-a, compared to controls, while levels of IL-4, IL-5, and IL- 10 remained low, indicating a predominantly pro- inflammatory response (Figure S9).

[0119J Computationally modeling utilizing MOE, similar to Figure 1 F, of biparatopic CAR VH681_375 elucidates the CAR’s putative ability to target both the CRD and CPD domains simultaneously (Figure 18C). For better discrimination between CARs of diverse classes of binders, a live-cell imaging cytotoxicity assay was performed with a subset of TROP2-targeted CARs, including the hRS7 scFv clinical ADC benchmark, single VH binders, and a biparatopic VH binder, against PC9 cells expressing various murine substitutions of TROP2 domains. It was observed that resistance to cytotoxicity with TROP2 CAR constructs VH375 against PC9 expressing murine CPD, phenocopying what is observed with hRS7 (Figure 18D). The VH681 demonstrated cytotoxicity against PC9 expressing murine CPD, consistent with the in vitro data and in silico structural modeling (Figure 18D). The TROP2 scFv- and VH-based binders, including the biparatopic binder, showed high degrees of cytotoxicity against PC9 expressing WT TROP2 (Figure 20). Conversely, it was observed that against PC9 expressing murine CRD TROP2, only VH681 and control constructs lacked activity, consistent with prior mapping (Figure 18E). The biparatopic constructs maintained activity regardless of the CRD/CPD substitutions (Figure 18D- E). As a proof-of-concept experiment, PC9 with either mCRD or mQ237-252 (CPD) were engrafted in contralateral flanks of NSG mice. When tumors were engrafted to -100 mm3, they were treated with either control irrelevant CAR (CD 19), hRS7 (anti CPD), or biparatopic CAR (VH681_375). On the left flank harboring the PC9 mQ237-252, it was observed that the hRS7 CAR had no effect on PC9, while the biparatopic VH681_375 had durable and effective tumor control (Figure 18F). On the contralateral flank with mice harboring PC9 mCRD, the hRS7 and biparatopic VH681_375 demonstrated improved tumor control (Figure 18G). This translated to prolonged survival of the VH681_375 compared to the benchmark hRS7 CAR (Figure 18H). Kinetics of tumor uptake seemed to peak at 14 days post CAR injection, which parallels when response was observed in mice (Figure 21). Weights were measured as a surrogate for toxicity with no significant differences in mouse weights between the various CAR T treatment groups and the control CD 19 CAR group (Figure 22). EQUIVALENTS

[0120] Various aspects of the present disclosure may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and is, therefore, not limited in its application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

Claims

CLAIMS What is claimed:

1. An engineered cell, comprising an immune cell, comprising a chimeric antigen receptor of formula I:

R1 - R2 - R3 (I), wherein:

R1 is an extracellular’ domain, comprising one or more heavy chain variable region means;

R2 is a transmembrane domain; and

2. The engineered cell of claim 1 , wherein R 1 is an extracellular domain of formula ITT: R4 - R5 or R5 - R4, wherein: a. R4 is a first heavy chain variable region means, and b. R5 is a second heavy chain variable region means, and optionally, a peptide linker between R4 and R5.

3. An engineered cell, comprising an immune cell, comprising a chimeric antigen receptor of formula II:

R1 - R2 - R3 (II), wherein:

R2 is a transmembrane domain; and

4. The engineered cell of claim 3, wherein R1 is an extracellular domain of the formula IV R4 - R5 or R5 - R4, wherein: a. R4 is a first heavy chain variable region, and b. R5 is a second heavy chain variable region, and optionally, a peptide linker between R4 and R5.

5. The engineered cell of any one of claims 1-4, wherein the immune cell is selected from the list consisting of: T cells, natural killer (NK) cells, macrophages, dendritic cells, hematopoietic stem cells (HSC), induced pluripotent stem cells, cord blood stem cells, and derivatives thereof.

6. The engineered cell of any one of claims 1-5, wherein the immune cell is a T cell.

7. The engineered cell of any one of claims 1-6, wherein the immune cell is selected from the list consisting of: a cytotoxic lymphocyte, T cell, cytotoxic T cell (CD8⁺ T cell), T helper cell (CD4⁺ T cell), aP T cell and/or y5 T cell, Thl7 T-cell, NK T (NKT) cell, and regulatory T (Treg) cell.

8. The engineered cell of any one of claims 1-7, wherein the immune cell is a CD8⁺ T cell.

9. The engineered cell of any one of claims 1-8, wherein the immune cell is a CD4⁺ T cell.

10. The engineered cell of any one of claims 1-9, wherein R1 comprises the complementarity determining regions (CDRs) HCDR1 , HCDR2 and HCDR3, wherein: a. the amino acid sequence of HCDR1 is SEQ ID NO: 1, the amino acid sequence of HCDR2 is SEQ ID NO:2, the amino acid sequence of HCDR3 is SEQ ID NO:3; b. the amino acid sequence of HCDR1 is SEQ ID NO:5, the amino acid sequence of HCDR2 is SEQ ID NO:6, the amino acid sequence of HCDR3 is SEQ ID NO:7; c. the amino acid sequence of HCDR1 is SEQ ID NO:9, the amino acid sequence of HCDR2 is SEQ ID NO: 10, the amino acid sequence of HCDR3 is SEQ ID NO: 11; d. the amino acid sequence of HCDR1 is SEQ ID NO: 13, the amino acid sequence of HCDR2 is SEQ ID NO: 14, the amino acid sequence of HCDR3 is SEQ ID NO: 15; e. the amino acid sequence of HCDR1 is SEQ ID NO: 17, the amino acid sequence of HCDR2 is SEQ ID NO: 18, the amino acid sequence of HCDR3 is SEQ ID NO: 19; f. the amino acid sequence of HCDR1 is SEQ ID NO:21, the amino acid sequence of HCDR2 is SEQ ID NO:22, the amino acid sequence of HCDR3 is SEQ ID NO:23; g. the amino acid sequence of HCDR1 is SEQ ID NO:25, the amino acid sequence of HCDR2 is SEQ ID NO:26, the amino acid sequence of HCDR3 is SEQ ID NO:27; h. the amino acid sequence of HCDR1 is SEQ ID NO:29, the amino acid sequence of HCDR2 is SEQ ID NO:30, the amino acid sequence of HCDR3 is SEQ ID NO:31; i. the amino acid sequence of HCDR1 is SEQ ID NO:33, the amino acid sequence of HCDR2 is SEQ ID NO:34, the amino acid sequence of HCDR3 is SEQ ID NO:35; j. the amino acid sequence of HCDR1 is SEQ ID NO:37, the amino acid sequence of HCDR2 is SEQ ID NO:38, the amino acid sequence of HCDR3 is SEQ ID NO:39; k. the amino acid sequence of HCDR1 is SEQ ID NO:41, the amino acid sequence of HCDR2 is SEQ ID NO:42, the amino acid sequence of HCDR3 is SEQ ID NO:43; l. the amino acid sequence of HCDR1 is SEQ ID NO:45, the amino acid sequence of HCDR2 is SEQ ID NO:46, the amino acid sequence of HCDR3 is SEQ ID NO:47; m. the amino acid sequence of HCDR1 is SEQ ID NO:49, the amino acid sequence of HCDR2 is SEQ ID NO:50, the amino acid sequence of HCDR3 is SEQ ID NO:51; n. the amino acid sequence of HCDR1 is SEQ ID NO:53, the amino acid sequence of HCDR2 is SEQ ID NO:54, the amino acid sequence of HCDR3 is SEQ ID NO:55; or o. the amino acid sequence of HCDR1 is SEQ ID NO:57, the amino acid sequence of HCDR2 is SEQ ID NO:58, the amino acid sequence of HCDR3 is SEQ ID NO:59.

11 . The engineered cell of any one of claims 1-10, wherein the R1 comprises a heavy chain variable region (HCVR), wherein: a. the amino acid sequence of the HCVR is SEQ ID NO: 4, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 98; b. the amino acid sequence of the HCVR is SEQ ID NO: 8, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 99; c. the amino acid sequence of the HCVR is SEQ ID NO: 12, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 100; d. the amino acid sequence of the HCVR is SEQ ID NO: 16, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 101; e. the amino acid sequence of the HCVR is SEQ ID NO: 20, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 102; f. the amino acid sequence of the HCVR is SEQ ID NO: 24, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 103; g. the amino acid sequence of the HCVR is SEQ ID NO: 28, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 104; h. the amino acid sequence of the HCVR is SEQ ID NO: 32, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 105; i. the amino acid sequence of the HCVR is SEQ ID NO: 36, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 106; j. the amino acid sequence of the HCVR is SEQ ID NO: 40, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 107; k. the amino acid sequence of the HCVR is SEQ ID NO: 44, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 108; l. the amino acid sequence of the HCVR is SEQ ID NO: 48, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 109; m. the amino acid sequence of the HCVR is SEQ ID NO: 52, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 110; n. the amino acid sequence of the HCVR is SEQ ID NO: 56, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 111 ; or o. the amino acid sequence of the HCVR is SEQ ID NO: 60, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 112.

12. The engineered cell of any one of claims 1-11, wherein R1 comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein: a. the amino acid sequence of HCDR1 is SEQ ID NO:9, the amino acid sequence of HCDR2 is SEQ ID NO: 10, the amino acid sequence of HCDR3 is SEQ ID NO: 11; b. the amino acid sequence of HCDR1 is SEQ ID NO: 13, the amino acid sequence of HCDR2 is SEQ ID NO: 14, the amino acid sequence of HCDR3 is SEQ ID NO: 15; or c. the amino acid sequence of HCDR1 is SEQ ID NO:25, the amino acid sequence of HCDR2 is SEQ ID NO:26, the amino acid sequence of HCDR3 is SEQ ID NO:27.

13. The engineered cell of any one of claims 1-11, wherein the R1 comprises a heavy chain variable region (HCVR), wherein: a. the amino acid sequence of the HCVR is SEQ ID NO: 12, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 100; b. the amino acid sequence of the HCVR is SEQ ID NO: 16, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 101; or c. the amino acid sequence of the HCVR is SEQ ID NO: 28, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 104.

14. The engineered cell of any one of claims 1-13, wherein R1 comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO:25, the amino acid sequence of HCDR2 is SEQ ID NO:26, the amino acid sequence of HCDR3 is SEQ ID NO:27.

15. The engineered cell of any one of claims 1-13, wherein the R1 comprises a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 28.

16. The engineered cell of any one of claims 1-13, wherein R1 comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 9, the amino acid sequence of HCDR2 is SEQ ID NO: 10, the amino acid sequence of HCDR3 is SEQ ID NO: 11.

17. The engineered cell of any one of claims 1-13, wherein the R1 comprises a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 12.

18. The engineered cell of any one of claims 1-13, wherein R1 comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 13, the amino acid sequence of HCDR2 is SEQ ID NO: 14, the amino acid sequence ofHCDR3 is SEQ ID NO: 15.

19. The engineered cell of any one of claims 1-13, wherein the R1 comprises a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 16.

20. The engineered cell of any one of claims 1-17, wherein: a. R1 comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO:25, the amino acid sequence of HCDR2 is SEQ ID NO:26, the amino acid sequence of HCDR3 is SEQ ID NO:27; and b. R1 comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 9, the amino acid sequence of HCDR2 is SEQ ID NO: 10, the amino acid sequence of HCDR3 is SEQ ID NO: 11.

21. The engineered cell of any one of claims 1-17, wherein the R1 comprises: a. (i)a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 28; and (ii) a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 12; b. the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 113; or c. the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 114.

22. The engineered cell of any one of claims 1-15 or 18-19, wherein: a. R1 comprises: the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO:25, the amino acid sequence of HCDR2 is SEQ ID NO:26, the amino acid sequence of HCDR3 is SEQ ID NO:27; and b. R1 comprises the complementarity determining regions (CDRs) HCDR1, HCDR2 and HCDR3, wherein the amino acid sequence of HCDR1 is SEQ ID NO: 13, the amino acid sequence of HCDR2 is SEQ ID NO: 14, the amino acid sequence of HCDR3 is SEQ ID NO: 15.

23. The engineered cell of any one of claims 1-15 or 18-19, wherein the R1 comprises: a. (i) a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 28; and (ii) a heavy chain variable region (HCVR), wherein the amino acid sequence of the HCVR is SEQ ID NO: 16; b. the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 115; or c. the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 116.

24. The engineered cell of any one of claims 2 and 4-23, wherein the peptide linker comprises the amino acid sequence comprising GGGGS.

25. The engineered cell of any one of claims 2 and 4-24, wherein the peptide linker consists of the amino acid sequence GGGGS.

26. The engineered cell of any one of claims 2 and 4-24, wherein the peptide linker comprises or consists of 2, 3, 4, 5, 6, or 7 consecutive repeats of the amino acid sequence GGGGS.

27. The engineered cell of any one of claims 2, 4-24, and 26, wherein the peptide linker consists of the amino acid sequence GGGGSGGGGSGGGGS.

28. The engineered cell of any one of claims 1-27, wherein the hinge region comprises a hinge domain derived from CD3< , CD4, CD8u, CD28, IgGl, IgG2, or IgG4.

29. The engineered cell of any one of claims 1-28, wherein the hinge region comprises an amino acid sequence selected from the list consisting of SEQ ID NOS: 66-72.

30. The engineered cell of any one of claims 1-29, wherein the hinge region comprises a hinge domain derived from CD28.

31. The engineered cell of any one of claims 1-30, wherein the hinge region comprises or consists of an amino acid sequence comprising or consisting of SEQ ID NO: 69, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 117.

32. The engineered cell of any one of claims 1-31, wherein R2 comprises a transmembrane domain derived from CD3< CD4, CD8a, CD28, or CD137.

33. The engineered cell of any one of claims 1-32 wherein R2 comprises an amino acid sequence selected from the list consisting of SEQ ID NOS: 73-77.

34. The engineered cell of any one of claims 1-33, wherein R2 comprises a transmembrane domain derived from CD28.

35. The engineered cell of any one of claims 1-34, wherein R2 comprises or consists of an amino acid sequence comprising or consisting of SEQ ID NO: 76, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 118.

36. The engineered cell of any one of claims 1-35, wherein R3 comprises: a. a signaling domain; or b. a signaling domain and a costimulatory domain, optionally wherein R3 is of the formula V: R6 - R7 or R7 - R6 (V), wherein R6 comprises a signaling domain, and R7 comprises a costimulatory domain.

37. The engineered cell of any one of claims 1-36, wherein the signaling domain is derived from CD3 , CD27, CD28, CD40, KIR2DS2, MyD88, or 0X40.

38. The engineered cell of any one of claims 1-37, wherein the signaling domain comprises an amino acid sequence selected from the list consisting of SEQ ID NOS: 79-81, 84-86, and 94- 96.

39. The engineered cell of any one of claims 1-38, wherein the signaling domain is derived from CD3

40. The engineered cell of any one of claims 1-39, wherein R3 comprises or consists of an amino acid sequence comprising or consisting of SEQ ID NO: 79, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 120.

41. The engineered cell of any one of claims 1-40, wherein the costimulatory domain is derived from of CD3y, CD35, CD3e, CD3^, CD27, CD40, CD28, CD72, CD80, CD86, CLEC-1, 4- 1BB, TYROBP (DAP12), Dectin-1, FcaRI, FcyRI, FcyRII, FcyRIII, FcsRI, IL-2RB, ICOS, KIR2DS2, MyD88, 0X40, and ZAP70.

42. The engineered cell of any one of claims 1-41, wherein the costimulatory domain comprises an amino acid sequence selected from the list consisting of SEQ ID NOS: 78-97.

43. The engineered cell of any one of claims 1-42, wherein the costimulatory domain is derived from 4- IBB.

44. The engineered cell of any one of claims 1-43, wherein the costimulatory domain comprises or consists of an amino acid sequence comprising or consisting of SEQ ID NO: 78, or an amino acid sequence encoded by a nucleic acid sequence comprising or consisting of SEQ ID NO: 119.

45. The engineered cell of any one of claims 1-44, wherein the chimeric antigen receptor is encoded by codon-optimized nucleic acid sequences.

46. The engineered cell of any one of claims 1-44, wherein the chimeric antigen receptor is encoded by a vector.

47. The engineered cell of any one of claims 1-44, wherein the chimeric antigen receptor is encoded by a plasmid vector.

48. The engineered cell of any one of claims 1-44, wherein the chimeric antigen receptor is encoded by a lentiviral plasmid vector.

49. An engineered cell as described herein.

50. A pharmaceutical composition comprising the engineered cell of any one of claims 1-49, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

51. A pharmaceutical composition comprising two or more engineered cells of any one of claims 1-45, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

52. A pharmaceutical composition comprising two or more engineered cells of any one of claims 1-45, and one or more pharmaceutically acceptable carriers, diluents, or excipients, wherein the two or more engineered cells comprise: a. an engineered cell of any one of claims 1-8 and 10-49, wherein the immune cell is a CD8⁺ T cell; and b. an engineered cell of any one of claims 1-7 and 9-49, wherein the immune cell is a CD4⁺ T cell.

53. A method of treating cancer, comprising administering to a subject with cancer: a. the engineered cell of any one of claims 1-49; or b. the pharmaceutical composition of any one of claims 50-52.

54. The method of claim 55, wherein the cancer is selected from the list consisting of: lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer.

55. The method of claim 53 or claim 54, wherein the cancer is treatment- resistant or chemotherapy-resistant.

56. A composition for use in treating cancer, comprising administering to a subject with cancer: a. the engineered cell of any one of claims 1-49; or b. the pharmaceutical composition of any one of claims 50-52.

57. The composition for use of claim 52, wherein the cancer is selected from the list consisting of: lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer.

58. The composition for use of claim 56 or claim 57, wherein the cancer is treatment-resistant or chemotherapy-resistant.

59. Use of a composition for treating cancer, comprising administering to a subject with cancer: a. the engineered cell of any one of claims 1-49; or b. the pharmaceutical composition of any one of claims 50-52.

60. The use of claim 59, wherein the cancer is selected from the list consisting of: lung cancer, non-small cell lung cancer, breast cancer, triple negative breast cancer, pancreatic cancer, bladder cancer, solid tumor cancer, oral cancer, neoplasm cancer, ovarian cancer, adenocarcinoma, thyroid cancer, epithelial cancer, cervical cancer, endometrial cancer, brain cancer, bile duct cancer, prostate cancer, leptomeningeal disease, and gastrointestinal cancer.

61. The use of claim 59 or 60, wherein the cancer is treatment-resistant or chemotherapyresistant.