Ligand-based chimeric antigen receptors and uses thereof

Abstract

A polynucleotide encoding a chimeric antigen receptor (CAR) is provided, The polynucleotide comprises from N to C: (i) an amino acid sequence of a ligand capable of binding a HER receptor, the ligand being selected from the group consisting of HRG-alpha proprotein (NRG-lalpha), neuregulin 1 type IV beta la (NRG-lbeta) and betacellulin (BTC); (ii) a hinge region; (iii) a transmembrane domain; and (iv) an intracellular cell signaling module comprising a CD3ζ activation signaling domain, and at least one costimulatory domain, such that upon binding of the amino acid sequence of the ligand to the receptor, the cell signaling module is activated. Also provided are cells expressing such polynucleotides and uses thereof.

Description

[0001] LIGAND-BASED CHIMERIC ANTIGEN RECEPTORS AND USES THEREOF

[0002] RELATED APPLICATIONS:

[0003] This Application claims the benefit of priority from U.S. Provisional Patent Application No. 63 / 538,316 filed September 14, 2023, which is hereby incorporated by reference in its entirety.

[0004] SEQUENCE LISTING STATEMENT

[0005] The XML file, entitled 101501.xml, created on September 13, 2024, comprising 48,337bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

[0006] FIELD AND BACKGROUND OF THE INVENTION

[0007] The present invention, in some embodiments thereof, relates to ligand-based chimeric antigen receptors and uses thereof.

[0008] HER2 is highly expressed in breast cancer patients. Although trastuzumab is he commonly used drug to treat HER2+patients, only a few subsets of patients respond to it. To elicit a proliferative signal, HER2 forms heterodimers with other members of the HER family of receptor tyrosine kinases (RTKs). Reports suggest that HER1, HER3, and HER4 prefer HER2 for heterodimer formation. HER2 heterodimers are particularly crucial in breast cancer, and their overexpression leads to increased signaling and cell proliferation, which can affect disease progression and response to treatment. HER3, a member of the HER family of RTKs and plays a key role in mediating signaling pathways involved in cell growth, differentiation, and survival. Emerging evidence suggests that HER3 upregulation is a frequent event in many types of cancer, and more importantly, HER3 upregulation has also been shown to limit the efficacy of multiple therapies used in the treatment of cancer, including chemotherapy, EGFR-targeted therapies, and other targeted therapies.

[0009] Chimeric antigen receptor (CAR)-based therapies are presented as innovative treatments for multiple malignancies. Despite their clinical success, there is scientific evidence of the limitations of these therapies mainly due to immunogenicity issues, toxicities associated with the infusion of the product, and relapses of the tumor. As a result, novel approaches are appearing aiming to solve and / or mitigate the harmful effects of CAR-T therapies. These include strategies based on the use of ligands as binding moi eties or ligand-based CART cells. Additional related background art:

[0010] Ramirez-Chaco (2022) Ligand-based CART cell: Different strategies to drive T cells in future new treatments. Front. Immunol. 13:932559;

[0011] WO2022 / 190099

[0012] SUMMARY OF THE INVENTION

[0013] According to an aspect of some embodiments of the present invention there is provided a polynucleotide encoding a chimeric antigen receptor (CAR) comprising from N to C:

[0014] (i) an amino acid sequence of a ligand capable of binding a HER receptor, the ligand being selected from the group consisting of HRG-alpha proprotein (NRG- 1 alpha), neuregulin 1 type IV beta la (NRG-lbeta) and betacellulin (BTC);

[0015] (ii) a hinge region;

[0016] (iii) a transmembrane domain; and

[0017] (iv) an intracellular cell signaling module comprising a CD3(^ activation signaling domain, and at least one costimulatory domain, such that upon binding of the amino acid sequence of the ligand to the receptor, the cell signaling module is activated.

[0018] According to an aspect of some embodiments of the present invention there is provided a nucleic acid expression construct comprising a nucleic acid sequence encoding the polynucleotide described herein under transcriptional control of a cis-acting regulatory element(s).

[0019] According to an aspect of some embodiments of the present invention there is provided an immune cell comprising the polynucleotide described herein or the nucleic acid construct described herein.

[0020] According to an aspect of some embodiments of the present invention there is provided a method of treating cancer which onset or progression depends on a HER protein, the method comprising administering to a subject in need thereof a therapeutically effective amount of the nucleic acid expression construct or immune cell described herein, thereby treating cancer.

[0021] According to an aspect of some embodiments of the present invention there is provided the nucleic acid expression construct or immune cell described herein for use in treating cancer which onset or progression depends on a HER protein.

[0022] According to some embodiments of the invention, the (i)-(iv), are amino acid sequences of human proteins.

[0023] According to some embodiments of the invention, the at least one co-stimulatory domain is selected from the group consisting of CD28, OX-40 and 4- IBB, ICOS, CD27, and CD40. According to some embodiments of the invention, the cell is an immune cell selected from the group consisting of a T-cell and a natural killer (NK) cell.

[0024] According to some embodiments of the invention, the cell is genetically modified to regulate an activity of the chimeric polypeptide.

[0025] According to some embodiments of the invention, the CAR is of a dual targeting CAR complex.

[0026] According to some embodiments of the invention, the method or use further comprises an additional agent being a chemotherapy.

[0027] According to some embodiments of the invention, the chemotherapy is lapatinib.

[0028] According to some embodiments of the invention, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), colorectal cancer, pancreatic cancer, head and neck cancer, breast cancer, endometrial cancer, ovarian cancer and gastric cancer.

[0029] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0030] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS )

[0031] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0032] In the drawings:

[0033] FIGs. 1 A-E shows the use of chimeric receptors according to embodiments of the invention in diagnotstics. (a) Sequence of NRGla, NRGlb, and BTC for the active part, (b) Construction of BW reporter. The sequences consist of selected amino acids of the ligands followed by (G4S)4 as a linker, MYC, human CD8, murine CD3 zeta transmembrane domain, and murine CD3 zeta cytoplasmic domain, (c) Detection of HER family homodimers / heterodimers and mIL-2 secretion upon binding to target cancer cells / BW standards, (d) Generation of BW standards expressing HER1, HER2, HER3, HER1+2, HER1+3, and HER2+3 (e) HER Expression Profile of mIL-2 on Standards with BW reporters.

[0034] FIGs.2A-B show the construction of CAR-T cells, (a) Construction of CAR-T. The sequences includes selected amino acids of the ligands followed by (G4S)4 as a linker, human hCD28 hinge, transmembrane domain (TMD) and cytoplasmic co-stimulatory domain (CD) human CD3 zeta and ZsGreen as the reporter (b) Selection of CAR-T cells after transduction.

[0035] FIGs. 3 A-C show the results or a degranulation assay, (a) ELISA for BW reporters against Cal33 (WT and KD), JIMT1 (WT and KO), and A375 (b) Degranulation assay for CAR-T against Cal33 (WT and KD for HER1), HSC3 (WT and KD for HER1), JIMT1 and A375 (c) Degranulation assay for CAR-T against BW standards.

[0036] FIGs. 4A-G shows a competition assay of BW reporters with soluble NRGlb. (a) mIL2 secretion by BW reporters with Cal33 WT in the presence of different concentrations of soluble NRGlb (b) mIL2 secretion by BW reporters with Cal33 KD in the presence of different concentrations of soluble NRGlb (c) mIL2 secretion by BW reporters with JIMT1 WT in the presence of different concentrations of soluble NRGlb (d) mIL2 secretion by BW reporters with JIMT1 KD in the presence of different concentrations of soluble NRGlb (e) mIL2 secretion by BW NRGla reporter with BW standards in the presence of soluble NRGlb (f) mIL2 secretion by BW NRGlb reporter with BW standards in the presence of soluble NRGlb (g) mIL2 secretion by BW BTC reporter with BW standards in the presence of soluble NRGlb.

[0037] FIGs. 5A-D show a competition assay of CAR-T cells with soluble NRGlb.

[0038] (a) Degranulation assay of BW standards with CAR-NRGla in presence of soluble NRGlb (b) Degranulation assay of BW standards with CAR-NRGlb in presence of soluble NRGlb (c) Degranulation assay cancer cells with CAR-NRGla in presence of soluble NRGlb (d) Degranulation assay of cancer cells with CAR-NRGlb in presence of soluble NRGlb

[0039] FIGs. 6A-B show the construction of dual CAR-T cells, (a) Construction of dual CAR-T. The sequences for NRGla and NRGlb comprises selected amino acids of the ligands followed by (G4S)4 as a linker, Myc, human CD8, human CD28 with transmembrane domain (TMD) and cytoplasmic domain (CD) , and 4- IBB as the co-stimulatory domain and mCHERRY as the reporter. BTC comprises selected amino acids of the ligands followed by (G4S)4 as a linker, human CD8, human CD3 with transmembrane domain (TMD) and cytoplasmic domain (CD), and ZsGreen as the reporter (b) Selection of dual CAR-T cells after transduction.

[0040] FIGs. 7A-D show activation of CAR-T-ligand cells by HER-expressing tumor cells. (A) Flow cytometry analysis of HER receptor expression (EGFR, HER2, HER3) on FaDu head and neck cancer cells. Cells were labeled with fluorescent antibodies specific to each HER receptor, and expression was quantified using the geometric mean fluorescence intensity. (B)Flow cytometry dot plots showing the activation of CAR-T cells. The upper panel represents CAR-T NRGlb, and the lower panel represents CAR-T-anti-HER2. For each, three conditions are shown: untreated, stimulated with 0KT3, and co-cultured with tumor cells. (C) CAR-T-ligand cell activation upon co-culture with FaDu cells. CAR-T-ligand cells expressing NRGlb, anti-HER2, or an untransduced control (UN) were co-cultured with FaDu cells for 4 hours, and CD 107a expression on CAR-T cells was measured by flow cytometry. The percentage of CD107a-positive cells indicates activation levels. (D) IFNy secretion by CAR-T-ligand cells upon co-culture with FaDu cells. IFNy levels were quantified in the supernatant.

[0041] FIGs. 8A-E show comparison of CAR-T-NRGlb and CAR-T -anti -HER2 activity in JIMT1 WT and HER2 knockout cells. (A) Flow cytometry analysis of HER1, HER2, and HER3 expression on JIMT1 WT and HER2 KO cells. HER2 expression is completely ablated in KO cells, while HER1 / EGFR expression is increased, and HER3 expression is maintained. (B) Degranulation assay showing CD 107a expression on CAR-T cells. CAR-T-NRGlb and CAR-T- anti-HER2 cells were co-cultured with JIMT1 WT or HER2 KO cells. While both CAR-T cells showed similar activation levels with JIMT1 WT cells, only CAR-T-NRGlb was activated in the presence of HER2 KO cells. OKT3 stimulation was used as a positive control. (C) IFNy secretion by CAR-T cells upon co-culture with JIMT1 cells. CAR-T-NRGlb secreted higher levels of IFNy compared to CAR-T-anti-HER2 in co-culture with HER2 KO cells, consistent with the degranulation results. (D-E) Killing assay using DiD-labeled JIMT1 tumor cells. Both CAR-T- NRGlb and CAR-T-anti-HER2 induced effective lysis of JIMT1 WT cells. However, only CAR- T-NRGlb showed significant lysis of JIMT1 HER2 KO cells, demonstrating its superior cytotoxicity against HER1 and HER3 -expressing cells.

[0042] FIG. 9 shows anti -tumor efficacy of CAR-T-ligands in JIMT1 WT tumor-bearing mice. NSG immunodeficient mice were injected subcutaneously with 3 million JIMT1 WT tumor cells. After 21 days, when tumors were established, mice received an intravenous injection of CAR-T cells under four different conditions: CAR-T UN (untransduced control), and 2ndgeneration (CD28+zeta in the cytoplasmic domain) CARs: CAR-T-NRGlb, CAR-T-NRGla, and CAR-T- anti-HER2. Tumor volume was measured over time using calipers and presented in cubic millimeters (mm3). (A) Tumor growth curves for each treatment arm, analyzed using Compare Groups of Growth Curves (CGGC).

[0043] FIGS. 10A-B show targeting of HER-expressing cells by CAR-NK-NRGlb cells. CAR-NK cells were generated by transducing NK92 cells with a CAR construct expressing NRGlb (CAR- NK-NRGlb) and compared to wild-type NK92 cells (NK92 WT). (A) IFNy secretion by CAR- NK-NRGlb and NK92 WT cells after co-culture with a panel of target cells overexpressing different combinations of HER receptors: HER1, HER2, HER3, HER1+2, HER1+3, and HER2+3. (B) Validation of CAR-NK-NRGlb specificity using additional tumor cell lines: JIMT1 WT, JIMT1 HER2 KO, and FaDU.

[0044] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0045] The present invention, in some embodiments thereof, relates to ligand-based chimeric antigen receptors and uses thereof.

[0046] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0047] To eliminate EGFR (HERl) / HER3 / HERl+3 / HER2+3 expressing cancer cells the present inventors have developed CAR-T / NK cells with the active portion of NRGla or NRGlb or BTC, used as the HER3 targeting moiety, fused within a 2nd generation CAR (CD28 and zeta in the cytoplasmic domain) molecule. These ligand-based CAR-T cells utilize the receptor-binding domain of soluble EGFR(HER1) / HER3 ligands (either for EGFR(HER1) / HER3 monomers / homodimers or heterodimers) to induce the antitumor response. Unlike scFv-based CAR-T cells, which recognize with high affinity and specificity protein expressed on tumor cells, ligand-based CAR-T / NK cells can take advantage of the multi-targeting capacity of ligands that can bind to different EGFR / HER monomers or heterodimer receptors.

[0048] Described herein are carefully selected ligand-based CAR molecules and cells expressing same which can be used to induce an anti-tumor response. The ligand is of a HER receptor and its recognition on the target cells is HLA-independent. The present inventors showed that these molecules are able to bind their cognate receptors on the cancer cells and induce cell killing.

[0049] CAR molecules according to some aspect of the invention are highly advantageous as 1) they are less likely to provoke an undesired anti-CAR immune response as compared to antireceptor ScFv-based CARs, as the ligands are derived from natural human sequences; 2) somatic hypermutation phenomena are not necessary, thus reducing anti-idiotype antibodies; 3) the present CARs are able to bind to multiple targets, thus reducing the potential for tumor escape; 4) the nature of the ligands and their binding to the receptor allow for a certain tuning capacity that reduces their sensitivity and enables therapies with a greater ability to discriminate between tumor tissues, which tend to overexpress the target, and healthy tissues; and 5) less tonic signaling and longer lasting functionality should be detected associated with a reduction in the probability of ligand aggregation.

[0050] Thus, according to an aspect of the invention there is provided a polynucleotide encoding a chimeric antigen receptor (CAR) comprising from N to C:

[0051] (i) an amino acid sequence of a ligand capable of binding a HER receptor, said ligand being selected from the group consisting of HRG-alpha proprotein (NRG- 1 alpha), neuregulin 1 type IV beta la (NRG-lbeta) and betacellulin (BTC);

[0052] (ii) a hinge region, said hinge region;

[0053] (iii) a transmembrane domain; and

[0054] (iv) an intracellular cell signaling module comprising a CD3(^ activation signaling domain, and at least one costimulatory domain, such that upon binding of the amino acid sequence of said ligand to said receptor, said cell signaling module is activated.

[0055] As used herein “chimeric antigen receptor”, “CAR” or “CARs” as used herein refers to engineered T cell receptors, which graft a ligand onto, e.g., T cells (for example naive T cells, central memory T cells, effector memory T cells or combinations thereof) or other effector cells such as natual killer (NK) cells or B cells. CARs are also known as artificial T-cell receptors, chimeric T-cell receptors or chimeric immunoreceptors.

[0056] A CAR places a chimeric extracellular target-binding domain (i.e., ligand) that specifically binds a target, i.e., a receptor, to be targeted for an immune response, and in this case a HER receptor onto a construct including a transmembrane domain, and intracellular domain(s) (including signaling domains) of a T cell receptor molecule. In one embodiment, the chimeric extracellular target-binding domain comprises the target-binding domain(s) of a ligand that specifically binds a receptor expressed on a cell to be targeted for an immune, e.g., a T cell response. The properties of the intracellular signaling domain(s) of the CAR can vary as known in the art and as disclosed herein, but the chimeric target-binding domains(s) render the receptor sensitive to signaling activation when the chimeric target binding domain binds the target on the surface of a targeted cell.

[0057] With respect to intracellular signaling domains, so-called “first-generation” CARs include those that solely provide, e.g., CD3zeta (CD3) signals upon antigen binding. So-called “second- generation” CARs include those that provide both co-stimulation (e.g., CD28 or CD 137) and activation (CD3) domains, and so-called “third-generation” CARs include those that provide multiple costimulatory (e.g., CD28 and CD137) domains and activation domains (e.g., CD3).

[0058] As used herein, a “CAR T cell” or “CAR-T” refers to a T cell which expresses a CAR. When expressed in a T cell, CARs have the ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner, exploiting the antigen-binding properties of monoclonal antibodies and in this case those of the ligand toward the receptor on the tumor cell. The non-MHC-restricted antigen recognition gives T-cells expressing CARs the ability to recognize a target independent of antigen processing, thus bypassing a major mechanism of tumor escape.

[0059] Ligand

[0060] As used herein, the term “extracellular target binding domain” refers to a polypeptide found on the outside of the cell sufficient to facilitate binding to a target. The extracellular target binding domain will specifically bind to its binding partner. According to a specific embodiment, the target binding domain is a soluble portion of the natural ligand (i.e., without the transmembrane domain).

[0061] According to a specific embodiment, the CAR is a dual CAR which can be used to improve specificity or affinity of the CAR.

[0062] Van der Schans Front. Oncol., 05 August 2020 Sec. Cancer Immunity and Immunotherapy describes dual CAR strategies to improve specificity of CAR T-cell therapy. (A) Split CAR T- cells, in which activation through CD3(^ and co-stimulation are split over a first-generation CAR and a chimeric co-stimulatory CAR (CCR), each targeting distinct tumor antigens. Binding of both CARs is required for full T-cell activation. Split CAR T-cells can be generated using a bicistronic vector (Al) or two separate CAR and CCR vectors (A2). (B) An inhibitory CAR (iCAR) binding a self-antigen inhibits T-cell activation of a second CAR targeting a tumor-associated antigen (TAA). In the absence of a self-antigen it functions as a conventional single CAR T-cell. CAR / iCAR combinations can also be made using a bicistronic vector (Bl) or two separate CAR and iCAR vectors (B2). Alternatively described are dual CAR strategies to improve efficacy of CAR T-cell therapy. (A) Mixture of two pools of single CAR T-cells in which each pool targets a different antigen. These can be co-infused or sequentially infused into a patient. (B) Two CARs targeting distinct antigens expressed on one T-cell through the use of co-transduction (Bl) or a bicistronic vector (B2). Binding of either one of the CARs is sufficient to activate the T- cell. (C) Tandem CAR T-cells, which contain two distinct binding domains linked to one receptor. Binding of either one of the domains is sufficient to activate the T-cell. (D) Ligand-based CAR T- cells, containing a ligand as binding domain that can recognize more than one antigen. Binding of either one of the antigens is sufficient to activate the T-cell. In the present case a specific embodiment of dual targeting contemplated i.e. One CAR (CAR1) targeting antigen 1 and having only a part of the signaling-co-stimulation machinery (e.g., only zeta) and a 2ndCAR (CAR2) targeting antigen 2 having the complimentary part of the signaling-costimulation machinery (e.g.,- co-stimulation). Both CAR1 and CAR2 are transduced to the same CART cells, thus each CART cell expresses both CARs. Target tumor cells are expected to express both antigen 1 and antigen 2 on their cell surface (either as heterodimers or not as heterodimers)

[0063] As used herein “NRGlalpha” or “NRG1A” refers to a soluble form of Pro-neuregulin-1, membrane-bound isoform HRG-alpha proprotein having the GenBank Accession of NCBI NP_039258.1.

[0064] As used herein “NRGlbeta” or “NRG1B” refers to a soluble form of neuregulin 1 type IV beta la having the GenBank Accession ADN85612.1.

[0065] As used herein “BTC” refers to a soluble form of Betacellulin, e.g., AAB25452.1.

[0066] According to a specific embodiment, the NRG-1A, and NRG-1B comprise less than 90 amino acids which constitute at least a portion of the EGF-like domain of the ligand and excludes the Ig domain. According to a specific embodiment, the ligand portion is 40-100, 40-90, 45-100, 45-90, 50-100, 55-90, 52-85, 45-90 amino acids in length. For example, the NRG-1A is 50-55 amino acids in length, the NRG-1B is 50-55 amino acids in length or the BTC is 75-85 amino acids in length.

[0067] According to a specific embodiment, the NRG-1A is as set forth in SEQ ID NO: 1. The core domain was taken from NCBI NP_039258.1 is 177-229 = 53 AA. It will be appreciated that amino acids coordinates 177 to 181 (5 AA) are not considered as part of the EGF-like protein and thus are from a disordered domain.

[0068] 1 MSERKEGRGK GKGKKKERGS GKKPESAAGS QSPALPPRLK EMKSQESAAG SKLVLRCETS

[0069] 61 SEYSSLRFKW FKNGNELNRK NKPQNIKIQK KPGKSELRIN KASLADSGEY MCKVISKLGN

[0070] 121 DSASANITIV ESNEIITGMP ASTEGAYVSS ESPIRISVST EGANTSSSTS

[0071] TSTTGTSHLV

[0072] 181 KCAEKEKTFC VNGGECFMVK DLSNPSRYLC KCQPGFTGAR CTENVPMKVQ NQEKAEELYQ

[0073] 241 KRVLTITGIC IALLVVGIMC VVAYCKTKKQ RKKLHDRLRQ SLRSERNNMM NIANGPHHPN

[0074] 301 PPPENVQLVN QYVSKNVISS EHIVEREAET SFSTSHYTST AHHSTTVTQT PSHSWSNGHT

[0075] 361 ESILSESHSV IVMSSVENSR HSSPTGGPRG RLNGTGGPRE CNSFLRHARE

[0076] TPDSYRDSPH 421 SERYVSAMTT PARMSPVDFH TPSSPKSPPS EMSPPVSSMT VSMPSMAVSP FMEEERPLLL

[0077] 481 VTPPRLREKK FDHHPQQFSS FHHNPAHDSN SLPASPLRIV EDEEYETTQE YEPAQEPVKK

[0078] 541 LANSRRAKRT KPNGHIANRL EVDSNTSSQS SNSESETEDE RVGEDTPFLG IQNPLAASLE

[0079] 601 ATPAFRLADS RTNPAGRFST QEEIQARLSS VIANQDPIAV (Whole sequence is SEQ ID NO: 2 and the NRG-1 A core used in the chimeric protein is SEQ ID NO: 1).

[0080] According to a specific embodiment, the NRG-1B is as set forth in SEQ ID NO: 3. The core domain is taken from ADN85612.1 and is 122-174 = 53 AA.

[0081] 1 MGKGRAGRVG TTALPPRLKE MKSQESAAGS KLVLRCETSS EYSSLRFKWF KNGNELNRKN

[0082] 61 KPQNIKIQKK PGKSELRINK ASLADSGEYM CKVISKLGND SASANITIVE SNATSTSTTG

[0083] 121 TSHLVKCAEK EKTFCVNGGE CFMVKDLSNP SRYLCKCPNE FTGDRCQNYV MASFYKHLGI

[0084] 181 EFMEAEELYQ KRVLTITGIC IALLVVGIMC VVAYCKTKKQ RKKLHDRLRQ SLRSERNNMM

[0085] 241 NIANGPHHPN PPPENVQLVN QYVSKNVISS EHIVEREAET SFSTSHYTST AHHSTTVTQT 301 PSHSWSNGHT ESILSESHSV IVMSSVENSR HSSPTGGPRG RLNGTGGPRE CNSFLRHARE

[0086] 361 TPDSYRDSPH SERYVSAMTT PARMSPVDFH TPSSPKSPPS EMSPPVSSMT VSMPSMAVSP

[0087] 421 FMEEERPLLL VTPPRLREKK FDHHPQQFSS FHHNPAHDSN SLPASPLRIV EDEEYETTQE

[0088] 481 YEPAQEPVKK LANSRRAKRT KPNGHVANRL EVDSNTSSQS SNSESETEDE RVGEDTPFLG

[0089] 541 IQNPLAASLE ATPAFRLADS RTNPAGRFST QEEIQARLSS VIANQDPIAV

[0090] (Whole sequence is SEQ ID NO: 4 and the NRG-1B core used in the chimeric protein is SEQ ID NO: 3).

[0091] According to a specific embodiment, the BTC is as set forth in SEQ ID NO: 5. The core domain is taken from AAB25452.1 32-111 = 80 AA.

[0092] 1 MDRAARCSGA SSLPLLLALA LGLVILHCVV ADGNSTRSPE TNGLLCGDPE 61 KRKGHFSRCP KQYKHYCIKG RCRFVVAEQT PSCVCDEGYI

[0093] GARCERVDLF YLRGDRGOIL

[0094] 121 VICLIAVMVV FIILVIGVCT CCHPLRKRRK RKKKEEEMET LGKDITPINE DIEETNIA (Whole sequence is SEQ ID NO: 6 and the BTC core used in the chimeric protein is SEQ ID NO: 5).

[0095] Also contemplated herein are homologous sequences which are at least 90 %, 91 %, 92 %, 93 %, 94 % 95 %, 96 %, 97 %, 99 %, 99 % or more identical to the contemplated sequences , e.g., SEQ ID Nos: 1, 3 and 5 as determined using Blast such as by using default parameters. Any contemplated homolog is capable of binding the cognate receptor in a manner which is about the same (or less affinity) as that of the natural binding. Methods of measuring binding are well known in the art and described herein in the Examples section which follows. Affinity can be measured using methods which are well known in the art, for example, solution equilibrium titration (SET) and surface plasmon resonance (SPR).

[0096] In various embodiments, the CAR is selected to have high affinity or avidity for the target — for example, the ligand-receptor binding may have higher affinity than would the natural pair (e.g., below KD 1 nM).

[0097] In other embodiments affinities lower than those of the natural pair are envisaged too such as KD above 1 pM but below 1 nM.

[0098] The different CARs contemplated herein have different abilities of binding HER receptors in terms of binding affinity.

[0099] Thus, the following is envisaged:

[0100] NRG1 A-binds HER2+HER3>HER1>HER3

[0101] NRGIB-binds HER1+HER3>HER3>HER2+HER3

[0102] BTC-binds HERl+HER3>HERl>HERl+2

[0103] Thus the selection of CAR or dual CAR will depend on the HER presentation on the treated tumor.

[0104] The epidermal growth factor receptor (EGFR) family encompasses four receptor proteins, namely ErbB- l / E’G / -7 - l to -4 (also called herein HER 1-4) that are expressed on cell surface and exhibit tyrosine kinase activities. These proteins have similar structures and are comprised of three domains: an extracellular domain with ligand binding site, a transmembrane domain, and an intracellular domain with kinase activity . There are 11 different growth factors, each possessing a conserved EGF domain that can bind with those four receptors. Upon ligand binding, the receptors form homo-or hetero-dimers, promoting activation, relaying signals for proliferation, survival, migration and differentiation and thus playing major roles in cancer progression . Overexpression and / or gene amplification of EGFR confer malignancy to diverse tissues. Moreover, constitutively active mutants of EGFR are found in different cancers, where they are often associated with poor prognosis.

[0105] The ability of the proteins in this family to form dimers and heterodimers affect their functionality. For instance, the heterodimer of ErbB-2 / ErbB-3 is particularly oncogenic and hence determining its presence and / or level is of high significance. Methods of determining receptor expression are well known in the art and can be done on cells or tissue biopsies by immunostaining, western blotting and ELISA as well as other methods at the protein level or by PCT (e.g., RT- PCR) at the RNA level. Sometimes determination of the gene, i.e., at the DNA level such as for gene amplification is contemplated. Also determining mutations associated with cancer in these genes is contemplated herein. It will be appreciated that activity assays can also be used to measure expression (such as proliferation assays) but with the proviso that they do not use a reporter cell expressing the chimeric protein as a sensor.

[0106] Hinge and Transmembrane Domains

[0107] Each CAR as described herein necessarily includes a transmembrane domain that joins the extracellular target-binding domain (i.e., ligand) to the intracellular signaling domain.

[0108] Accordingly, in some embodiments, the binding domain of the CAR is optionally followed by one or more “hinge domains,” which plays a role in positioning the target binding domain away from the immune cell surface to enable proper cell / cell contact, target binding and activation. A CAR optionally comprises one or more hinge domains between the binding domain and the transmembrane domain (TM). The hinge domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source. The hinge domain can include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region. Illustrative hinge domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8 (e.g., CD8a), CD4, CD28, 4-1 BB, and CD7, which may be wild-type hinge regions from these molecules or may be altered (e.g., at least 90 % identity to the native sequence). In some embodiments, the hinge region is derived from the hinge region of an immunoglobulin-like protein (e.g., IgA, IgD, IgE, IgG, or IgM), CD28, or CD8. In one embodiment, the hinge domain comprises a CD8a hinge region.

[0109] A hinge can also be used as an alternative to or an addition to a linker.

[0110] Thus, the components can be linked directly or via a linker (e.g., amino acid linker, e.g. GGGGSGGGGSGGGGSGGGGS SEQ ID NO:31), and / or a “hinge” region (e.g, amino acid hinge, e.g. CD8a hinge region) that is located between the transmembrane domain and recognition moiety.

[0111] Non-limiting examples of polypeptide linkers include linkers having the sequence LE, GGGGS (SEQ ID NO: 18), (GGGGS)n (n=l -4) (SEQ ID NO: 19), GGGGSGGGG (SEQ ID NO:20), (GGGGS)x2 (SEQ ID NO:21), (GGGGS)x2+GGGG (SEQ ID NO:22), (Gly)8 (SEQ ID NO:32), (Gly)6 (SEQ ID NO:33), (EAAAK)n (n=l -3) (SEQ ID NO:23), A(EAAAK)nA (n = 2-5) (SEQ ID NO:24), AEAAAKEAAAKA (SEQ ID NO:25), A(EAAAK)4ALEA(EAAAK)4A (SEQ ID NO:26), PAPAP (SEQ ID NO:27), KESGSVSSEQ LAQFRSLD (SEQ ID NO:28), EGKSSGSGSESKST (SEQ ID NO:29), GSAGSAAGSGEF (SEQ ID NO:30), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:31).

[0112] According to some embodiments the linker is as set forth in

[0113] GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:31).

[0114] As used herein, “transmembrane domain” (TM domain) refers to the generally hydrophobic region of the CAR which crosses the plasma membrane of a cell. The TM domain can be the transmembrane region or fragment thereof of a transmembrane protein (for example a Type I transmembrane protein or other transmembrane protein), an artificial hydrophobic sequence, or a combination thereof. While specific examples are provided herein and used in the Examples, other transmembrane domains will be apparent to those of skill in the art and can be used in connection with alternate embodiments of the technology. A selected transmembrane region or fragment thereof would preferably not interfere with the intended function of the CAR. As used in relation to a transmembrane domain of a protein or polypeptide, “fragment thereof’ refers to a portion of a transmembrane domain that is sufficient to anchor or attach a protein to a cell surface.

[0115] In one embodiment, the transmembrane domain or fragment thereof of any of the CAR polypeptides described herein comprises a transmembrane domain selected from the transmembrane domain of, hCD28, CD8 or 4-1 BB. In an alternate embodiment of any aspect, the transmembrane domain or fragment thereof of the CAR described herein comprises a transmembrane domain selected from the transmembrane domain of an alpha, beta or zeta chain of a T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, KIRDS2, 0X40, CD2, CD27, LFA-1 (CDl la, CD18), ICOS (CD278), 4-1 BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRFI), CD 160, CD 19, IL2R beta, IL2R gamma, IL7Ra, ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 Id, ITGAE, CD 103, ITGAL, CD1 la, LFA-1, ITGAM, CD11 b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD 18, LFA-1, ITGB7, TNFR2, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, LylO8), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, and / or NKG2C.

[0116] 4-1 BB is a membrane receptor protein, also known as CD137, and is a member of the tumor necrosis factor (TNF) receptor superfamily. 4-1 BB is expressed on activated T lymphocytes. 4-1 BB sequences are known for a number of species, e.g., human 4-1 BB, also known as TNFRSF9 (NCBI Gene ID: 3604) and mRNA (NCBI Reference Sequence: NM 001561.5). 4-1 BB can refer to human 4-1 BB, including naturally occurring variants, molecules, and alleles thereof. In some embodiments of any of the aspects, e.g., in veterinary applications, 4-1 BB can refer to the 4-1 BB of, e.g., dog, cat, cow, horse, pig, and the like. Homologs and / or orthologs of human 4-1 BB are readily identified for such species by one of skill in the art, e.g., using the NCBI ortholog search function or searching available sequence data for a given species for sequence similar to a reference 4-1 BB sequence.

[0117] According to a specific embodiment, the hinge and transmembrane region are derived from hCD28, (see Figure 2A).

[0118] The sequence of human CD28 (hinge + TM) is IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVA FIIFWV (SEQ ID NO: 34)

[0119] The sequence of human CD28 (hinge-TM-cyto) is (cyto is the underline): lEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVA FIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 35).

[0120] The transmembrane domain is followed by an intracellular (cytoplasmic) domain. Such a domain typically comprises a CD3zeta and optionally a co-stimulatory domain.

[0121] As used herein, an “intracellular domain” refers to an amino acid sequence fully comprised within a cell. In one embodiment, the intracellular domain refers to the intracellular domain of a receptor. An intracellular domain can interact with the interior of a cell and elicit signaling. With respect to the intracellular domain of a receptor, the intracellular domain can function to relay a signal transduced. An intracellular domain of a receptor can comprise enzymatic activity.

[0122] Co-Stimulatory Domain

[0123] Each CAR described herein optionally comprises an intracellular domain of a costimulatory molecule, or co-stimulatory domain. As used herein, the term “co-stimulatory domain” refers to an intracellular signaling domain of a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes upon binding to antigen. Illustrative examples of such co-stimulatory molecules include CD137 (4-1BB), CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (0X40), CD150 (SLAMF1), CD 152 (CTLA4), CD223 (LAGS), CD270 (HVEM), CD273 (PD-L2), CD274 (PD- Ll), CD278 (ICOS), DAP10, LAT, NKD2C SLP76, TRIM, and ZAP70. In one embodiment, the intracellular domain is the intracellular domain of 4-1 BB, CD27, CD28, or 0X40.

[0124] In one embodiment, the intracellular domain is the intracellular domain of a 4-1 BB.

[0125] Intracellular Signaling molecule

[0126] CARs as described herein comprise an intracellular signaling molecule. An “intracellular signaling domain” or an “intracellular signaling molecule” refers to the part of a CAR polypeptide that participates in transducing the message of effective CAR binding to a target receptor into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and cytotoxic activity, including the release of cytotoxic factors to the CAR-bound target cell, or other cellular responses elicited following antigen binding to the extracellular target binding domain of the CAR.

[0127] CD3 is a T cell co-receptor that facilitates T lymphocytes activation when simultaneously engaged with the appropriate co-stimulation (e.g., binding of a co-stimulatory molecule). A CD3 complex consists of 4 distinct chains; mammal CD3 consists of a CD3y chain, a CD36 chain, and two CD3s chains. These chains associate with a molecule known as the T cell receptor (TCR) and the CD3(^ to generate an activation signal in T lymphocytes. A complete TCR complex comprises a TCR, CD3(^, and the complete CD3 complex.

[0128] In some embodiments of any aspect, a CAR polypeptide described herein comprises an intracellular signaling domain that comprises an Immunoreceptor Tyrosine-based Activation Motif or ITAM from CD3 zeta (CD3Q, ITAM-mutated CD3(^, CD3r|, or CD39. In some embodiments of any aspect, the ITAM comprises three motifs of ITAM of CD3(^ (ITAM3). In some embodiments of any aspect, the three motifs of ITAM of CD3^ are mutated.

[0129] IT AMS are known as a primary signaling domains regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary signaling domains that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or ITAMs. Non-limiting examples of ITAM containing intracellular signaling domains that are of particular use in the technology include those derived from TCF^, FcRy, FcRp, CD3y, CD36, CD38, CD3r|, CD3s, CD3< CD22, CD79a, CD79b, and CD66d. One skilled in the art will be capable of introducing mutations into the nucleic acid sequence of a gene or gene product, for example IT AM, using standard techniques. For example, point mutations can be introduced via site-directed point mutagenesis, a PCR technique. Site- directed mutagenesis kits are commercially available, for instance, through New England Biolabs; Ipswich, Mass. Non-limiting examples of alternative methods to introduce point mutations to the nucleic acid sequence of a gene or gene product include cassette mutagenesis or whole plasmid mutagenesis.

[0130] In one embodiment, the ITAM utilized in the CAR is based on alternatives to CD3(^, including mutated ITAMs from CD3(^ (which contains 3 ITAM motifs), truncations of CD3(^, and alternative splice variants known as CD3s, CD3q, CD39, and artificial constructs engineered to express fusions between CD3s, CD3r|, or CD39 and CD3(^.

[0131] In some embodiments, a CAR polypeptide described herein comprises the intracellular signaling domain of CD3(^ (including variants of CD3(^, e.g., ITAM-mutated CD3Q, CD3r|, or CD30.

[0132] According to a specific embodiment, the amino acid sequence of the CD3(^ is as set forth in SEQ ID NO: 14, homologous sequences which are able to transmit the CD3zeta signaling are also contemplated, e.g., at least 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 % 96 %, 97 %, 98 %, 99 % or more identical to:

[0133] RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA LPPR* SEQ ID NO: 14.

[0134] A more detailed description of CARs and CART cells can be found in Ramirez-Chacon (2022) Ligand-based CART cell: Different strategies to drive T cells in future new treatments. Front. Immunol. 13:932559, Maus et al. Blood 2014 123:2624-35; Reardon et al. Neuro-Oncology 2014 16: 1441-1458; Hoyos et al. Haematologica 2012 97: 1622; Byrd et al. J Clin Oncol 2014 32:3039-47; Maher et al. Cancer Res 200969:4559-4562; and Tamada et al. Clin Cancer Res 2012 18:6436-6445; each of which is incorporated by reference herein in its entirety.

[0135] In one embodiment, the CAR polypeptide further comprises a signal (leader) peptide, which is typically removed during presentation.

[0136] In an embodiment the leader sequence is MDMRVPAQLLGLLLLWLSGARC (SEQ ID NO: 17). Homologous leader sequences are also contemplated, e.g., at least 80 %, 85 %, 90 %, 91 %, 92 %, 93 %, 94 %, 95 % 96 %, 97 %, 98 %, 99 % identical to SEQ ID NO: 17 as long as their function in membrane presentation is maintained. In some embodiments, a CAR polypeptide as described herein includes a signal peptide. Signal peptides can be derived from any protein that has an extracellular domain or is secreted. A CAR polypeptide as described herein may include any signal peptides known in the art. In some embodiments, the CAR polypeptide includes a CD8 signal peptide.

[0137] In one embodiment, the CAR further comprises additional linker domains.

[0138] In one embodiment, a CAR as described herein further comprises a reporter molecule, e.g., to permit for non-invasive imaging (e.g., positron-emission tomography PET scan). In a bispecific CAR that includes a reporter molecule, the first extracellular binding domain and the second extracellular binding domain can include different or the same reporter molecule. In a bispecific CAR T cell, the first CAR and the second CAR can express different or the same reporter molecule. In another embodiment, a CAR as described herein further comprises a reporter molecule (for example hygromycin phosphotransferase (hph)) that can be imaged alone or in combination with a substrate or chemical (for example 9-[4-[18F]fluoro-3-(hydroxymethyl)butyl]guanine ([18F]FHBG)). In another embodiment, a CAR as described herein further comprises nanoparticles at can be readily imaged using non-invasive techniques (e.g., gold nanoparticles (GNP) functionalized with64Cu2+). Labeling of CAR T cells for non-invasive imaging is reviewed, for example in Bhatnagar P, et al. Integr Biol. (Camb). 2013 January; 5(1): 231-238, and Keu K V, et al. Sdci Transl Med. 2017 Jan. 18; 9(373), which are incorporated herein by reference in their entireties.

[0139] GFP and mCherry are demonstrated herein as fluorescent tags useful for imaging a CAR expressed on a T cell (e.g., a CAR T cell, see Figure 2A). It is expected that essentially any fluorescent protein known in the art can be used as a fluorescent tag for this purpose. For clinical applications, the CAR need not include a fluorescent tag or fluorescent protein.

[0140] In some embodiment, the CAR polypeptide is as set forth in SEQ ID NO: 2, 4 or 6, with or without the reporter molecules.

[0141] In some embodiments, a CAR polypeptide described herein comprises fully human sequences. Such polypeptides are produced according to methods known in the art.

[0142] Another aspect of the invention described herein relates to a polypeptide complex comprising two or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of any of the CAR polypeptides described herein or combinations with other CARs which can target any target expressed polyeptpides, e.g., of the HER family or others. In one embodiment, the polypeptide complex comprises one, two or three of any of the CAR polypeptides described herein. Nucleic Acids and Cells

[0143] In some embodiments, any of the CAR polypeptides described herein are encoded by a polynucleotide comprised in a viral vector. Optionally, a polynucleotide encoding a CAR polypeptide as described herein can be codon-optimized to enhance expression or stability. Codon optimization may be performed according to any standard methods known in the art.

[0144] According to some embodiments, retroviruses, such as lentiviruses, provide a convenient platform for delivery of nucleic acid sequences encoding a gene, or chimeric gene of interest. A selected nucleic acid sequence can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells, e.g. in vitro or ex vivo. Retroviral systems are well known in the art and are described in, for example, U.S. Pat. No. 5,219,740; Kurth and Bannert (2010) “Retroviruses: Molecular Biology, Genomics and Pathogenesis” Calster Academic Press (ISBN:978-l-90455-55-4); and Hu and Pathak Pharmacological Reviews 2000 52:493-512; which are incorporated by reference herein in their entirety. Lentiviral system for efficient DNA delivery can be purchased from OriGene; Rockville, Md. In alternative embodiments, the CAR polypeptide of any of the CARs described herein are expressed in the mammalian cell via transfection or electroporation of an expression vector comprising nucleic acid encoding the CAR. Transfection or electroporation methods are known in the art.

[0145] Efficient expression of the CAR polypeptide of any of the CAR polypeptides described herein can be assessed using standard assays that detect the mRNA, DNA, or gene product of the nucleic acid encoding the CAR, such as RT-PCR, FACS, northern blotting, western blotting, ELISA, or immunohistochemistry.

[0146] In one embodiment, the CAR polypeptide of any of the CAR polypeptides described herein is constitutively expressed. In one embodiment, the CAR polypeptide of any of the CAR polypeptides described herein is encoded by recombinant nucleic acid sequence.

[0147] Another aspect of the invention relates to a mammalian cell comprising any of the CAR polypeptides described herein; or a nucleic acid encoding any of the CAR polypeptides described herein. In one embodiment, the mammalian cell comprises an antibody, antibody reagent, antigenbinding portion thereof, or any of the CAR polypeptides described herein, or a nucleic acid encoding such an antibody, antibody reagent, antigen-binding portion thereof, or any of the CAR polypeptides described herein. The mammalian cell or tissue can be of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog or cat origin, but any other mammalian cell may be used. In a preferred embodiment of any aspect, the mammalian cell is human. In one embodiment, the cell is a T cell or a natural killer (NK) cell. In alternate embodiments of any aspect, the cell is an immune cell. As used herein, “immune cell” refers to a cell that plays a role in the immune response. Immune cells are of hematopoietic origin, and include lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes. In some embodiments, the cell is a T cell; a NK cell; a NKT cell; lymphocytes, such as B cells and T cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0148] The immune cell can be obtained from a subject having or diagnosed as having cancer. For example, the immune cell can be obtained from a subject having a cancer. Examples of cancer include but are not limited to glioma, melanoma mesothelioma, lung cancers of non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), colorectal cancer, pancreatic cancer, head and neck cancer, breast cancer, endometrial cancer, ovarian cancer, gastric cancer, prostate cancer, bladder cancer, renal cell cancer, all skin cancers, cervical and anal cancers, and rare cancers line salivary glands, rhabdiod cancer and others. Moreover, liquid cancers such as leukemia, B cell lymphoma, chronic and acute myeloid leukemia are also contemplated. According to a specific embodiment the cancer is non-small cell lung cancer (NSCLC), colorectal cancer, pancreatic cancer, head and neck cancer, breast cancer, endometrial cancer, ovarian cancer and gastric cancer.

[0149] According to a specific embodiment the cancer is selected as over-expressing EGFR and / or HER3 (as determined at the genome level (e.g., gene amplification), mRNA level or protein level).

[0150] HER2 therapy-resistant cancers due to mutations / deletions / alternative splicing and masking by other molecules of anti-HER2 binding mAbs used for CAR therapy or other type of therapy (eg ADC)

[0151] According to a specific embodiment, the cancer is triple negative breast cancer (TNBC).

[0152] For example, quantitative level of expression of these receptors on the surface of cancer cells compared to normal cells may be determined. These levels are often measured using various laboratory techniques such as immunohistochemistry (IHC), quantitative PCR, or fluorescence in situ hybridization (FISH).

[0153] HER1 (EGFR) overexpression is typically measured in terms of the number of receptors per cell. In normal cells, the level of EGFR is generally low (e.g., about 20,000 to 100,000 molecules per cell). In cancer cells with EGFR overexpression, the number can increase significantly, such as reaching over 1 million receptors per cell. IHC is a common method used to assess EGFR expression levels. The results are often graded on a scale from 0 to 3+, with 3+ indicating strong overexpression. Likewise, for HER3 overexpression: HER3 expression levels in normal tissues are relatively low, but in overexpressing cancer cells, these levels can increase significantly. HER3 overexpression is often evaluated by IHC, with a similar scoring system. High HER3 expression is typically indicated by a score of 2+ or 3+ Clinical Relevance: overexpression is often defined as 2+ or 3+ in IHC scoring, where 3+ indicates high expression and may correlate with increased signaling activity that drives cancer progression.

[0154] In some embodiments, the immune cell is obtained from a subject resistant to chemotherapy. Immune cells can also be obtained from allogeneic donors, which are non- genetically identical individuals of the same species as the intended recipients of the cells.

[0155] Immune cells (e.g., human immune cells) that can be used in the invention include autologous cells, obtained from the subject to whom the cells are later to be administered, after ex vivo modification and expansion. For example, the immune cells can be obtained from an individual having or diagnosed as having cancer, such as described above. Immune cells can also be obtained from allogeneic donors, which are non-genetically identical individuals of the same species as the intended recipients of the cells. Immune cells useful for the invention include T cells and NK cells.

[0156] Methods for obtaining T cells and NK are known in the art and can be useful for the engineered immune cells described herein. T cells and NK cells are typically obtained from peripheral blood that is collected from a subject by, e.g., venipuncture or withdrawal through an implanted port or catheter. Optionally, the blood can be obtained by a process including leukapheresis, in which white cells are obtained from the blood of a subject, while other blood components are returned to the subject. Blood or leukapheresis product (fresh or cryopreserved) is processed to enrich for T cells or NK cells using methods known in the art. For example, density gradient centrifugation (using, e.g., Ficoll) and / or counter-flow centrifugal elutriation can be carried out to enrich for mononuclear cells (including T cells or NK cells). In one example, for T cells, a T cell stimulation step employing, e.g., CD3 / CD28 antibodies coated on magnetic beads or artificial antigen presenting cells (aAPCs) expressing, e.g., cell surface-bound anti-CD3 and anti-CD28 antibody fragments (see below), can further be carried out in order to stimulate T cells and to deplete other cells, e.g., B cells. The T cells of enriched T cell preparations can then be subject to genetic modification.

[0157] As an alternative to peripheral blood, tissues including bone marrow, lymph nodes, spleen, and tumors can be used as a source for T cells and NK cells. The T cells and NK cells can be of human, primate, hamster, rabbit, rodent, cow, pig, sheep, horse, goat, dog, or cat origin, but any other mammalian cell may be used. In certain embodiments of any aspect, the T or NK cell is human.

[0158] An immune cell, e.g., a T cell or NK cell, can be engineered to comprise any of the CAR polypeptides described herein; or a nucleic acid encoding any of the CAR polypeptides described herein.

[0159] Activation of the CAR-T / NK cells can be measured using methods which are well known in the art, such as CD 107 expression or IFNy secretion.

[0160] The invention furthermore provides compositions and methods for treating and preventing diseases and conditions including, e.g., cancer. These methods include the use of an immune cell (e.g., a T cell or an NK cell) including a CAR polypeptide, or a nucleic acid encoding said CAR, as described herein, and administering the modified immune cell to a subject to treat, e.g., cancer. In some embodiments of any of the aspect, the modified immune cell (e.g., a T cell or an NK cell including one or more additional modification as described herein) is stimulated and / or activated prior to administration to the subject.

[0161] Therapeutic Methods

[0162] Thus, according to an aspect of the invention there is provided a method of treating cancer which onset or progression depends on a HER protein, the method comprising administering to a subject in need thereof a therapeutically effective amount of the nucleic acid expression construct or immune cell, thereby treating cancer.

[0163] Alternatively or additionally, there is provided the nucleic acid expression construct or immune cell as described herein for use in treating cancer which onset or progression depends on a HER protein.

[0164] Such cancers express higher levels of a HER protein, e.g., HER1, HER2, HER3 or HER4 that its level in a non-cancerous tissue.

[0165] Typically, the cancers that express high levels of EGFR are brain tumors like glioma, solid cancers like lung, head and neck, stomach renal urothelial breast and cervical, For HER2- Most dominant cancer are breast, urothelial, stomach esophagus, colorectal and lung, For HER3- high levels are melanoma breast, stomach, lung, colorectal.

[0166] According to a specific embodiment, the method comprises using an additional agent being a chemotherapy or an antibody.

[0167] According to a specific embodiment, the chemotherapy is lapatinib. Examples of chemotherapies include, but are not limited to, Lapatinib, Afatinib, TAK-285, TKI HER2 / EGFR specific like Neratinib, or EGFR specific inhibitor like Erlotinib. Moreover, antibodies that block EGFR like - Cetuximab, anti-HER2- like Trastuzumab or Pertuzumab, anti-HER3- like patritumab or any bi-specific antibody that binds HER- members, with or without conjugation of ADC (Antibody-drug-conjugates). Moreover, small molecules that inhibit HER downstream signaling like - inhibitors of the PI3K, AKT, mTOR, and MAPK pathways, such as Trametinib, Rapamycin, Alphelisib and others which are well known in the art.

[0168] According to a specific embodiment, the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), colorectal cancer, pancreatic cancer, head and neck cancer, breast cancer, endometrial cancer, ovarian cancer and gastric cancer. Other types of cancer are described hereinabove.

[0169] According to a specific embodiment, the cancer is chemotherapy resistant.

[0170] According to a specific embodiment, the treatment is a first line treatment.

[0171] According to a specific embodiment, the treatment is not a first line treatment (e.g., 2nd, 3rd).

[0172] According to a specific embodiment, the cancer is resistant to anti HER therapy (e.g., by genetic mutations and / or cellular plasticity).

[0173] According to a specific embodiment, the cancer is resistant to anti EGFR, HER2 or HER3 therapy, such as by loss of EGFR, HER2 or HER3 associated tumor antigen presentation or by way of mutations in EGFR, HER2 or HER3 which render the receptor no longer recognizable by their cognate antibody.

[0174] According to a specific embodiment, the subject is selected for treatment using the reporter assay described in W02022 / 190099 or WO2024 / 057327, each of which is hereby incorporated by reference in its entirety.

[0175] Thus, a cell-based reporter system that can recognize the expression and availability of cancer-associated receptors on cancer cells and as such can be used in predicting disease outcome and selecting specific treatment modalities. In one embodiment, this system is referred to as “Targeted chemotherapy Artificial Reporter (TcAR)”.

[0176] Thus, there is provided a method of detecting presence and / or activity of a cancer-associated receptor in a cancer cell, the method comprising:

[0177] (a) contacting the cancer cell with a reporter cell comprising a polynucleotide encoding a chimeric polypeptide comprising an amino acid sequence of a ligand capable of binding a cancer- associated receptor, said amino acid sequence of said ligand being translationally fused to a heterologous amino acid sequence of a cell signaling module such that upon binding of the amino acid sequence of said ligand to said receptor, said cell signaling module is activated, wherein said ligand or cancer-associated receptor is not an immune checkpoint molecule; and (b) determining activation of said cell signaling module in the reporter cell, said activation being indicative of the presence and / or activity of the cancer-associated receptor or dimers or heterodimers thereof in the cancer cell.

[0178] Alternatively or additionally, there is provided a method of treating a subject diagnosed with cancer, the method comprising:

[0179] (a) detecting presence and / or activity of a cancer-associated receptor in a cancer cell as described; and

[0180] (b) treating the subject or selecting treatment with the nucleic acid expression construct or immune cell described throughout when the presence of HER1, HER3, is indicated.

[0181] As used herein the term “about” refers to ± 10 %.

[0182] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0183] The term “consisting of’ means “including and limited to”.

[0184] The term "consisting essentially of' means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0185] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0186] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0187] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween. As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0188] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0189] When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0190] It is understood that any Sequence Identification Number (SEQ ID NO) disclosed in the instant application can refer to either a DNA sequence or a RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or a RNA sequence format.

[0191] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0192] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

[0193] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non limiting fashion.

[0194] Materials and methods

[0195] Cell lines and culture media

[0196] Mouse BW5147 thymoma cells and their transfectants were cultured in RPMI 1640 (Gibco, MA, USA) with 10 % fetal bovine serum (Gibco, MA, USA), ImM Sodium pyruvate (Sartourious - Beit Haemek), 2 mM L-glutamine (Sartourious - Beit Haemek), 10 mM Hepes (Sartourious - Beit Haemek), 0.1 mM NEAA (non-essential amino acids) (Sartourious - Beit Haemek), and 1% Pen Strep (Gibco, MA, USA). JIMT1 WT, JIMT1 KO (KO for HER2). A375, HEK293T, HCT116, A549, and KYSE410 cell lines were grown in DMEM (Gibco, MA, USA) with 10 % FCS, HEPES, sodium pyruvate, pen-strep, L-glutamine, and non-essential amino acids. All the cell lines were maintained at 37°C with 5% CO2.

[0197] Cloning and construct design

[0198] The active part of the ligand consisting of 53 amino acids was selected from Jones et al. (1999) and cloned into a pHAGE2 Lenti-Vector. The NRGla and NRGip sequence differ in 11 amino acids. As a positive control, a-HER2 consisting of trastuzumab domain sequences was also designed. The sequences were ordered from HyLabs and amplified. The plasmid was cut with Sall and Notl and the sequences were inserted into the phage2 backbone. After transformation in DH10P, the plasmid was amplified and transfected to HEK293T cells using JetPrime® reagent (Polyplus).

[0199] For the construction of standards, the sequences for HER1 (Addgene 11011), HER2(addgen 9188), and HER3(addgene 23874) were obtained from Addgene and cloned into pHAGE2. The constructs were then transfected to HEK293T cells for Lentivirus (LV) production.

[0200] Lentivirus production, transduction and selection

[0201] After 2 days of transfection, the lentivirus was collected and centrifuged at 500 g for 5 minutes and the supernatant was filtered. The virus was concentrated by ultracentrifugation for 1.5 hours at 14,000 g in 4 °C, and used to transduce mouse BW5147 thymoma cells, which were cultured in RPMI 1640 with 10 % fetal calf serum, ImM Sodium pyruvate, 2 mM L-glutamine, 10 mM Hepes, 0.1 mM NEAA, 1 % Pen Strep, along with 10 ug / ml of puromycin or blasticidin for selection. The cells containing NRGla and NRGip sequences were sorted according to a- MYC positive staining. BW5147 was co-infected for preparing the heterodimer expressing cell lines (HER1+2, HER1+3, HER2+3) Cells were selected by puromycin or blasticidin and by sorting against PE-HER1 (biolegend), FITC- HER2 (biolegend), and APC-HER3 (biolegend) antibodies with a final concentration of 1 ug / ml. All the cell lines were maintained at 37 °C with 5 % CO2. Transduced BW cells were checked for expression by flow cytometry.

[0202] Isolation of functional primary T cells

[0203] 10 ml blood was withdrawn from healthy consenting donors for primary T cell isolation. The blood was diluted with an equal volume of 1 x PBS and added on top of Ficoll. Gradient centrifugation was performed at 1200 x g for 30 min at 16 °C. Mononuclear cells were collected and washed twice using 1 x PBS. and were plated in a 24-well plate in NutriT (Sartori ous) supplemented with HEPES, sodium pyruvate, pen-strep, L-glutamine, and non-essential amino acids (Sigma, MO, USA), lOO U / mL of rhIL2 (PeproTech) and lOO ng / mL of anti-human CD3 (OKT3) (BioLegend). After 24 h, the cells were washed and transduced with virus.

[0204] Designing and preparation of functional CAR-T cells

[0205] The NRGla, NRGip, BTC (SEQ ID Nos: 1, 3, 5), and trastuzumab ScFv-based a-HER2 sequences were cloned into a second-generation CAR-T plasmid co-expressing (TUN format) also ZsGreen in NRG1, BTC plasmids and Mcherry in a-HER2 plasmid and recombinant lentivirus containing these CARs were prepared (following transfection into HEK2993 cells) and purified by ultracentrifugation for 1.5 hours at 14,000 g in 4 °C two days post transfection thereby producing concentrated viruses.

[0206] Transduction of primary T cells

[0207] After 24 h of incubation with lOO U / mL rhIL2 and lOO ng / mL of anti -human CD3, the PBMCs were washed twice with IxPBS, and 0.5 x 106cells / well were transduced with the respective virus along with 6 nM of BX795(vendor)) and 5 nM of rosuvastatin in NutriT and 300 U / ml rhIL2. After 24h, the cells were again washed twice with 1 x PBS and re-plated in NutriT with 300 U / ml IL2. Primary T cells were maintained at 37 °C with 5 % CO2. 50,000 cells / well were then stained with DAPI (1 : 1000) and PE-conjugated anti-human CD3, to analyze the percent transduced cells by assessing ZsGreen and Mcherry signal through flow cytometry.

[0208] Flow cytometry (a-HERl, a-HER2, a-HER3, a-HER4 in cell lines)

[0209] The transduced BW-reporters were checked for the expression of MYC through flow cytometry. 50,000 cells / well were washed and seeded in a 96 well plate. T he cells were stained with a-MYC antibody (MABE282) (1 :250) and incubated for 30 minutes on ice. The cells were washed and APC-conjugated AffiniPure F(ab’)2 fragment goat anti-mouse IgG+IgM (H+l) (Jackson ImmunoResearch) (1 :500) was used as the secondary antibody for 30 minutes on ice. The cells were washed, and the dead cells were stained with DAPI (Biolegend) (1 : 1000).

[0210] To detect HER family's expression profile in cell lines, PDX suspension, and CDX suspension, 50,000 cells / well were washed with PAFX1 and seeded in a 96 well plate. The cells were stained with PE-EGFR (Biolegen;, cat: 352903), PE-CD340 (HER2, Biolegend; cat:324405), PE- HER3 (Biolegend; cat: 324705) and incubated for 30 minutes on ice. PE-mouse IgGl,k (Biolegend; CAT:400114) was used as an isotype control. The cells were washed twice with IX PAF, followed by addition of DAPI to stain the dead cells. All the samples were analyzed in Beckman CytoFLEX flow cytometer. For gating, isotype controls for each sample were analyzed and the experiment was performed in biological duplicates. Analysis was performed using Cytexpert software.

[0211] To detect the percentage of T cells expressing NRGla, NRGip, and a-HER2 after transduction, the cells were stained with PE-conjugated anti-human CD3 (UCHT1) (Biolegend). DAPI was used to stain dead cells. All the antibodies were used at a concentration of Ipg / ml.

[0212] ELISA for mIL2

[0213] 100,000 cancer cells / well or standards (target cells) were seeded in 96 U shape well plate along with 50,000 BW-reporter cells / well (effector cells) for 16-18 h at 37 °C with 5 % CO2 and incubated for 16-18 h at 37 °C, 5 % CO2. 2.5ug / ml of a-MYC (MABE282) was coated on the 96 U shape well plate as positive control along with addition of 50,000 BW-reporter cells / well. After the incubation, the supernatant was analyzed for murine IL2 secretion by standard ELISA (Kaufman B, Abramov O, levko A, Apple D, Shlapobersky M, Allon I, Greenshpan Y, Bhattachrya B, Cohen O, Charkovsky T, Gayster A, Shaco-Levy R, Rouvinov K, Livoff A, Elkabets M, Porgador A. Functional binding of PD1 ligands predicts response to anti-PDl treatment in patients with cancer. Science Advances. 2023 May 26;9(21):eadg2809. doi: 10.1126 / sciadv.adg2809.).

[0214] Degranulation assay

[0215] For degranulation, 150,000 cells / well of JIMT1 WT or JIMT1 KO were seeded in 96 U shape well plate. 50,000 CAR-T cells / well and APC-conjugated anti-human CD 107a (Biolegend, San Diego, CA) were added. The cells were kept in a 37 °C, 5 % CO2 for 4 hours. The cells were stained with APC-conjugated anti-human CD107a for 30 minutes on ice after being washed. A flow cytometric analysis was conducted to assess degranulation. CAR-T cells alone were used as a negative control. A positive control of 1 pg / ml of anti-human CD3 (OKT3) was employed. Furthermore, internal controls were included in each group.

[0216] ELISA for IFNy

[0217] 100,000 / well of JIMT1 WT or KO were seeded in 96 U shape well plate along with 50,000 CAR-T cells and incubated for 18-20 h at 37 °C, 5 % CO2. After the incubation, the supernatant was analyzed for human IFNy secretion by standard ELISA (Edri, A. et al. The Ebola-gly coprotein modulates the function of natural killer cells. Front. Immunol. www(dot)doi(dot)org / l 0.3389 / fimmu(dot)2018(dot)01428 (2018)).

[0218] Killing assay

[0219] The Vybrant™ DiD cell-labeling solution (Invitrogen) (1 :250 dilution) was used to label 50,000 JIMT1 WT and KO cells for the killing assay. These cells were then cultured in 96 U shape well plates with varying ratios of CAR-T (Effector-E) (E:T ratios: 0: 1, 0.5: 1, 1 : 1, and 2: 1). The plate was incubated in 5 % CO2 for 3 hours at 37 °C. The cells were stained with DAPI (1 pg / ml) after incubation. The Beckman CytoFLEX flow cytometer was used and every sample was examined.

[0220] Ex-vivo assay

[0221] Cell-derived xenograft (CDX) of JIMT1 WT was prepared by injecting 1 million cells subcutaneously on the dorsal side of NSG mice. After the formation of the tumor, the CDX tumors were excised and cut into 2x2x2mm3pieces and incubated with 20,000 cells / well or 100,000 cells / well of CAR-T cells (U-96 well plate). The tumor chunks were incubated with the CAR-T cells for 18-20 h followed by IFNy detection in the supernatant by ELISA.

[0222] FFPE Block Preparation, and Tissue Microarray Construction

[0223] FFPE blocks of the tumor tissue explants of size 2x2x2 mm3was prepared using automated tissue processing machinery (Leica Biosystems, Nubloch, Germany). Subsequently, using 3-mm T-SueTM punch needles (Simport, Beloeil, QC, Canada), tissue microarray (TMA) blocks were prepared from the donor FFPE blocks. Each TMA block contained a maximum of 24 tissue explants. Paraffin-embedded tissue TMA blocks were then sectioned into 5 pm slices using a microtome.

[0224] Ki67 staining

[0225] The tissue sections were deparaffinized using xylene for a predetermined period. Antigen retrieval was conducted by immersing the cells in citrate buffer (pH 6) and subjecting them to heat in a water bath at 95 °C for 30 minutes. To counteract endogenous peroxidase activity, a 3 % hydrogen peroxide (H2O2) solution was applied to the sections for 20 minutes and then rinsed in water for 5 minutes. After antigen retrieval, the cells were blocked at room temperature for 1 hour using a blocking solution comprising phosphate-buffered saline (PBS) with 0.1 % Tween and 5 % bovine serum albumin (BSA). Ki67 anti-human primary antibody (Merck, Ref- 275R-14) (diluted 1:200), was incubated with the sections overnight at 4 °C. The dilution was prepared following the manufacturer's guidelines using the blocking solution. Following primary antibody incubation, an IHC ABC kit (VECTASTAIN Cat. VE-PK-6200) was employed for color detection. The ABC kit utilizes a peroxidase-based enzymatic reaction to visualize the antigen-antibody complex. The sections were counterstained with hematoxylin to enable visualization of cellular nuclei. Subsequently, the cells were mounted using VectaMount® permanent mounting medium to preserve the staining. High-resolution digital images of the stained slides were acquired using a Pannoramic Scanner (3DHISTECH, Budapest, Hungary). These scanned images were analyzed through Pannoramic Viewer software to assess and quantify immunohistochemical staining patterns.

[0226] EXAMPLE 1

[0227] Construction of NRGla, NRGlb, and BTC chimeras and BW reporters and standards Results

[0228] Initial experiments involved the construction of BW reporters by selecting the active part of NRGla (53 amino acids), NRGlb (53 amino acids), and BTC (81 amino acids) (Fig 1A, IB) and expressing them in BW 5147 mouse thymoma cells (Figure 1C) , and its efficacy in identifying the different HER expression patterns in cancer cells. Also developed were BW standards (HER1, HER2, HER3, HER1+2, HER1+3, and HER2+3) for normalizing the data (Figure ID). The present inventors observed that NRGla binds with greater affinity to HER2+3 heterodimer than HER3 homodimer, while NRGlb binds to HER3 homodimer with greater affinity than HER2+3 heterodimer. Similarly, the present inventors observed that BTC binds to HER1+3 heterodimer with greater affinity than HER1 homodimer (Figure IE).

[0229] EXAMPLE 2

[0230] Single CAR-T therapy

[0231] Results

[0232] Construction of CAR-T cells

[0233] The active part of NRGla (SEQ ID NO: 1), NRGlb (SEQ ID NO: 3), or BTC (SEQ ID NO: 5) was expressed in primary T cells (Figure 2A). For the construction of 2ndgeneration of CAR-T cells, HEK293T cells were plated in 10 cm plates with DMEM containing 10% fetal bovine serum, pen-strep, L-glutamate, HEPES, non-essential amino acids, and sodium pyruvate, and cultivated till 90% confluence. Transfection with the NRGla, NRGlb or BTC inserts in the phage2 backbone was performed using JetPrime® reagent (Polyplus). After 2 days of infection, the lentivirus was concentrated by ultracentrifugation at 17,000rpm for 90 minutes. The supernatant was discarded and 500ul of DMEM was added, aliquoted, and stored at -80°C for further use. PBMCs were isolated from a healthy donor and the T cells were activated for 48h with 2ug / ml of anti-human CD3 (OKT3) in NutriT media with 100 U / ml of rIL2. The T cells were then transduced with viruses using retronectin, BX795, and cristor. The transduced T cells were then checked by flow cytometry and maintained in NutriT media with 300U / ml rIL2. Figure 2B depicts the successful construction of CAR-T cells.

[0234] Degranulation assay with CAR-T

[0235] Following the ELISA results from BW-reporters (Figure 3 A, 1C), the present inventors checked the ability of CAR-T cells to degranulate the cancer cells, and the BW standards. For this, 300,000 target cells / ml were added to 96 U-Shape plate well along with 100,000 CAR-T cells / ml. 0.4 ul / well of APC-conjugated anti-human CD107a was added to the wells and incubated for 4 h. Separate wells, coated with 2 ug / ml of anti-human OKT3, were used as a positive control since the anti-hCD3 mAb OKT3 activates human T cells. After 4 h of incubation at 37 °C, 5 % CO2, the cells were washed and stained again with 0.2ul / well APC-conjugated anti -human CD 107 along with PE-conjugated anti-human CD3 for 30 minutes. The cells were washed twice, followed by the addition of DAPI to stain the dead cells. All the samples were analyzed in Beckman CytoFLEX flow cytometer. Percent degranulation was calculated and the data was normalized to degranulation by CAR-T cells only. It was observed that the degranulation response of the cancer cells (Figure 3B) and BW standards (Figure 3C) is similar to the IL-2-reporting assay with the BW-reporters (Figure 3A).

[0236] EXAMPLE 3 Competition assay for binding of BW reporter and CAR-T cells in the presence of soluble NRGlb (single CART therapy)

[0237] Results

[0238] To confirm the binding of constructed reporter system and CAR-T system to their respective HERs, the present inventors performed a competition assay with soluble NRGlb. Initially, the present inventors performed the assay with BW reporters where different concentrations (0-800ng / ml) of soluble NRGlb were added along with 100,000 cells / well BW reporters and 50,000 / well of target cells. The cells were incubated at 37 °C with 5 % CO2 for 16- 18h. After the incubation, the supernatant was analyzed for murine IL2 secretion by standard ELISA. It was observed that soluble NRGlb increased the response of BW-BTC reporter to cancer cells (Figure 4A,4B,4C,4D). While after 200ng / ml of soluble NRGlb, the response of BW-NRGlb reporter started to decline in JIMT1 WT (Figure 4C). Therefore, 200 ng / ml of soluble NRGlb was used to perform a competition assay against the BW reporters on the BW standards. It was observed that soluble NRGlb effectively reduces the binding of BW reporters to the BW standards (Figure 4E,4F,4G), thus confirming the binding efficacy of our BW reporter system.

[0239] Similarly, the CAR-T cells were analyzed to degranulate the cancer cells and BW standards in the presence of soluble NRGlb. For this, 300,000 target cells / ml were added to 96 U plate well along with 300ng / ml of soluble NRGlb. 100,000 CAR-T cells / ml were added with 0.4ul / well of APC-conjugated anti-human CD107a incubated for 4h. Wells coated with 2ug / ml of anti-human OKT3 was used as a positive control. After 4h of incubation at 37°C, 5% CO2, the cells were washed and stained again with 0.2ul / well APC-conjugated anti-human CD 107a along with PE- conjugated anti-human CD3 for 30 minutes. The cells were washed twice, followed by the addition of DAPI to stain the dead cells. All the samples were analyzed in Beckman CytoFLEX flow cytometer. Percent degranulation was calculated and the data was normalized to degranulation by CAR-T cells only. It was observed that the degranulation response of the CAR-T (Figure 5A-D) is similar to the functional assay with the BW reporters (Figure 4A-G). Thus, NRGla effectively recognizes the heterodimer 2+3 as compared to NRGlb which effectively recognizes the homodimer HER3 and also HER1+3 and HER2+3.

[0240] EXAMPLE 4

[0241] Dual CAR-T therapy

[0242] Results

[0243] Construction of CAR-T cells

[0244] The active part of NRGla, NRGlb, and BTC was expressed in primary T cells (Figure 6 A). The present inventors prepared 3rdgeneration CAR-NRGla-CD28-41BB and CAR-NRGlb- CD28-41BB intended to be co-expressed with 1stgeneration CAR-BTC-CD3^. For the construction of CAR-T cells, HEK293T cells were plated in 10 cm plates with DMEM containing 10% fetal bovine serum, pen-strep, L- glutamate, HEPES, non-essential amino acids, and sodium pyruvate, and cultivated till 90% confluence. Transfection with the NRGla-, NRGlb- or BTC- CAR vectors inserts in the phage2 backbone was performed using JetPrime® reagent (Polyplus). After 2 days of infection, the lentivirus was concentrated by ultracentrifugation at 17,000rpm for 90 minutes. The supernatant was discarded and 500ul of DMEM was added, aliquoted, and stored at -80°C for further use. PBMCs were isolated from a healthy donor and the T cells were activated for 48h with 2ug / ml of anti-human CD3 (OKT3) in NutriT media with 100 U / ml of rIL2. The T cells were then co-transduced with NRGla and BTC CAR viruses or with NRGlb and BTC CAR viruses using retronectin, BX795, and Crestor. The transduced T cells were then checked by flow cytometry and maintained in NutriT media with 300U / ml rIL2. Figure 6B depicts the successful construction of CAR-T cells expressing either NRGla+BTC CARs or NRGlb+BTC CARs (2ndgeneration).

[0245] EXAMPLE 5

[0246] Functional Activity of NRGip CART on Head and Neck Cancer Cells

[0247] To evaluate the expression of HER family receptors (EGFR, HER2, and HER3) on tumor cells, the present inventors utilized the FaDu head and neck cancer cell line. Tumor cells were labeled with fluorescent antibodies specific to each HER receptor, and expression levels were quantified using flow cytometry, with the geometric mean fluorescence intensity presented (Figure 7 A). FaDu cells expressed very high levels of EGFR (HER1), high levels of HER2 and medium levels of HER3. The present inventors then investigated whether the CAR-T cells, engineered to express specific HER ligand (the NRGip ligand described), could be activated upon recognizing HER3, HER2+3 or HER1+3 receptors on the FaDu cells. NRGip CAR-T-ligand cells were cocultured with tumor cells (i.e., FaDu) for 4 hours, and CD107a surface expression was measured as an activation marker. In flow cytometry dot plot analyses, CAR-T-NRGip cells showed specific activation when co-cultured with HER-expressing FaDu tumor cells, as evidenced by increased CD 107a expression in the GFP+ population (Car-positivecell s). Notably, the CAR-non- transfected T cells (GFP negative) cells did not show significant activation in the presence of tumor cells, indicating that the observed activation is ligand-receptor specific. In the positive control, where cells were stimulated with anti-CD3 antibody (OKT3), both GFP+ and GFP- populations exhibited CD 107a expression, confirming that the activation machinery in both populations was intact. Similar results were observed in CAR-T-anti-HER2 cells, where GFP+ cells were specifically activated upon recognition of HER2 on FaDu tumor cells (Figure 7C). The percentage of CD107a-positive CAR-T-ligand cells is presented in the results. The present data shows that CAR-T-ligand cells expressing NRGip were significantly more activated by the tumor cells compared to CAR-T cells expressing anti-HER2, even though HER2 expression is higher than HER3 expression on FaDu cells. To confirm the activity of the CAR-T-ligand cell activation, IFNy secretion was measured. As shown in Figure 7D, CAR-T-ligand cells expressing NRGip secreted higher levels of IFNy compared to those expressing anti-HER2 or the untransduced T cells control (UN). EXAMPLE 6

[0248] Functional activity of CAR-T cells on JIMT1 WT and KO for HER2

[0249] Evaluation of CAR-T-NRGlb Activity in HER2 knockout tumor cells was evaluated. To assess the superiority of CAR-T-NRGlb against tumor cells expressing HER1 and HER3, JIMT1 cells were employed, which are known to express high levels of HER2. Using CRISPR technology, the present inventors generated HER2 knockout (KO) JIMT1 cells (JIMT1-HER2- KO were stained negatively by trastuzumab as compared to positive staining of JIMT1-WT by the same antibody). First measured were the expression levels of HER1, HER2, and HER3 using flow cytometry. As expected, HER2 expression was completely ablated in the HER2 KO cells, while the expression levels of HER1 and HER3 were maintained, with an observed increase in HER1 / EGFR expression (Figure 8A). Then co-culture experiments with CAR-T cells expressing NRGlb or anti-HER2 were done. The degranulation of CAR-T cells was assessed by staining for CD 107a. In co-culture with JIMT1 WT cells, both CAR-T-NRGlb and CAR-T-anti-HER2 exhibited similar levels of degranulation . However, in a co-culture with JIMT1 HER2 KO cells, significant activation was detected in CAR-T-NRGlb, while CAR-T-anti-HER2 showed no activation, highlighting the ability of CAR-T-NRGlb to target HER1 and HER3 (Figure 8B). OKT3 stimulation served as a positive control, confirming the activation potential of both CAR-T cell populations. To further validate these findings, IFNy secretion was measured. The results mirrored those of the degranulation assay, with CAR-T-NRGlb secreting higher levels of IFNy in the presence of HER2 KO cells compared to CAR-T-anti-HER2 (Figure 8C). Lastly, a killing assay was performed using DiD-labeled tumor cells to evaluate the cytotoxic efficacy of the CAR-T cells. The assay was conducted over 3-4 hours at various target-to-effector ratios. Flow cytometry analysis revealed that both CAR-T-NRGlb and CAR-T-anti-HER2 effectively lysed JIMT1 WT cells. However, only CAR-T-NRGlb was able to induce significant tumor cell lysis in JIMT1 HER2 KO cells, demonstrating its superior ability to target HER1 and HER3 -expressing cells (Figure 8D).

[0250] EXAMPLE 7

[0251] Anti-Tumor Efficacy of CAR-T-Ligands in JIMT1 WT Cells

[0252] The present inventors evaluated the anti-tumor efficacy of CAR-T cells expressing various ligands (CAR-T-ligands) in NSG immunodeficient mice bearing JIMT1 WT tumors. The experiment included four treatment arms: CAR-T UN (untransduced control), and 2ndgeneration CAR (CD28+ zeta in the cytoplasmic domain) for the CARs: CAR-T-NRGip, CAR-T-NRGla, and CAR-T-anti-HER2. Tumors were established by subcutaneous injection of 3 million JIMT1 WT cells, and after 21 days, when the tumors were palpable, mice received an intravenous injection of CAR-T cells. Tumor volume was measured using calipers, and the data was presented in cubic millimeters (mm3). The results, analyzed using Compare Groups of Growth Curves (CGGC), which accounts for the kinetics of tumor growth, revealed distinct outcomes across the treatment arms. Mice treated with CAR-T-NRGla exhibited a statistically significant reduction in tumor growth compared to the control group (CAR-T UN), demonstrating a strong anti-tumor effect. CAR-T-NRGlb treated group also showed tumor growth delay.

[0253] EXAMPLE 8

[0254] Activity of CAR-NK-NRGlb

[0255] To explore the potential of using CAR-NK cells, the present inventors generated CAR-NK cells expressing NRGip (CAR-NK-NRGip) and compared their activity to that of wild-type NK92 cells (NK92 WT). Results are shown in Figure 10A, 10B. After confirming CAR expression by flow cytometry, the CAR-NK-NRGlb cells were co-cultured with a panel of standard cell lines engineered to overexpress specific HER receptors: HER1, HER2, HER3, HER1+2, HER1+3, and HER2+3. These standard cell lines served as target cells to assess the specificity and efficacy of the CAR-NK cells. IFNy secretion, as an indicator of NK cell activation, was measured after co-culturing. The results show that CAR-NK-NRGip cells specifically recognized and responded to HER3, both when expressed alone and in combination with HER1 or HER2. This specificity was evidenced by significantly elevated levels of IFNy compared to cocultures with NK92 WT cells, which did not show any notable activation. To further confirm these findings, the present inventors tested the CAR-NK-NRGip cells with additional tumor cell lines, including JIMT1 (WT and HER2 KO) and FaDU. Consistently with the previous observations, CAR-NK-NRGlp cells recognized and responded to JIMT1 WT, JIMT1 HER2 KO, and FaDU cells.

[0256] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0257] It is the intent of the Applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

Claims

WHAT IS CLAIMED IS:

1. A polynucleotide encoding a chimeric antigen receptor (CAR) comprising from N to C:(i) an amino acid sequence of a ligand capable of binding a HER receptor, said ligand being selected from the group consisting of HRG-alpha proprotein (NRG- 1 alpha), neuregulin 1 type IV beta la (NRG-lbeta) and betacellulin (BTC);(ii) a hinge region;(iii) a transmembrane domain; and(iv) an intracellular cell signaling module comprising a CD3(^ activation signaling domain, and at least one costimulatory domain, such that upon binding of the amino acid sequence of said ligand to said receptor, said cell signaling module is activated.

2. A nucleic acid expression construct comprising a nucleic acid sequence encoding the polynucleotide of claim 1 under transcriptional control of a cis-acting regulatory element(s).

3. An immune cell comprising the polynucleotide of claim 1.

4. A method of treating cancer which onset or progression depends on a HER protein, the method comprising administering to a subject in need thereof a therapeutically effective amount of the immune cell of claim 2, thereby treating cancer.

5. The immune cell of claim 2 for use in treating cancer which onset or progression depends on a HER protein.

6. The polynucleotide of claim 1, wherein said (i)-(iv), are amino acid sequences of human proteins.

7. The polynucleotide of claim 1, wherein said at least one co-stimulatory domain is selected from the group consisting of CD28, OX-40 and 4- IBB, ICOS, CD27, and CD40.

8. The immune cell of claim 3, wherein said cell is an immune cell selected from the group consisting of a T-cell and a natural killer (NK) cell.

9. The immune cell of claim 3, wherein the cell is genetically modified to regulate an activity of said chimeric polypeptide.

10. The cell of claim 3, wherein the CAR is of a dual targeting CAR complex.

11. The method of claim 4 further comprising an additional agent being a chemotherapy.

12. The method of claim 11, wherein said chemotherapy is lapatinib.

13. The method of claim 4, wherein said cancer is selected from the group consisting of non-small cell lung cancer (NSCLC), colorectal cancer, pancreatic cancer, head and neck cancer, breast cancer, endometrial cancer, ovarian cancer and gastric cancer.

Images