Use of rage for priming a treatment for lung cancer and metastases to the lung

Abstract

Provided herein is an immune cell for killing cancer cells in the lung. In some embodiments, the cell may comprise: (a) a binding-triggered transcriptional switch (BTTS) that binds to RAGE (Receptor for Advanced Glycation Endproducts), (b) a nucleic acid sequence encoding an anti-cancer therapeutic; and (c) a regulatory sequence operably linked to (b) that is responsive to the binding-triggered transcriptional switch, wherein binding of the binding-triggered transcriptional switch to RAGE activates expression of the anti-cancer therapeutic. Also provided is a method for killing cancer cells in the lung that makes use of the cell.

Description

[0001] USE OF RAGE FOR PRIMING A TREATMENT FOR LUNG CANCER AND

[0002] METASTASES TO THE LUNG

[0003] CROSS-REFERENCING

[0004] This application claims the benefit of U.S. provisional application serial no. 63 / 639,421, filed on April 26, 2024, which application is incorporated by reference herein.

[0005] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0006] This invention was made with government support under grant no. D24AC00084-00 awarded by the Advanced Research Projects Agency for Health (ARPA-H). The government has certain rights in the invention.

[0007] INCORPORATION-BY-REFERENCE OF MATERIAL ELECTRONICALLY SUBMITTED

[0008] A Sequence Listing is provided herewith as a Sequence Listing XML, “UCSF- 797WO_SEQLIST” created on April 22, 2025, and having a size of 7,110 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.

[0009] INTRODUCTION

[0010] Although chimeric antigen receptor (CAR) T cell therapy has demonstrated remarkable therapeutic responses and benefits for patients with hematologic malignancies, development of effective CAR T therapies for solid cancers remains a challenge, in large part due to the difficulty in identifying optimal target surface antigens. Precise tumor recognition is limited by the problem that very few antigens are truly tumor-specific, and potential on-target, off-tumor cross-reaction with normal tissues can cause life-threatening or lethal toxicities. Toxic crossreaction has also been observed with several other T cells engineered to recognize solid tumor antigens. For example, CAR T cells specific for ERBB2 induced acute pulmonary toxicity resulting in death, likely due to the recognition of ERBB2 expressed at low levels on pulmonary epithelium.

[0011] Thus, there is a general need for new tumor recognition strategies that can create larger therapeutic windows. This disclosure provides a potential solution to this issue. SUMMARY

[0012] Provided herein is an immune cell for killing cancer cells in the lung. In some embodiments, the cell may comprise: (a) a binding-triggered transcriptional switch (BTTS) that binds to RAGE (Receptor for Advanced Glycation Endproducts), (b) a nucleic acid sequence encoding an anti-cancer therapeutic; and (c) a regulatory sequence operably linked to (b) that is responsive to the binding-triggered transcriptional switch, wherein binding of the binding- triggered transcriptional switch to RAGE activates expression of the anti-cancer therapeutic. Also provided is a method for killing cancer cells in the lung.

[0013] The present cell contains a molecular circuit that integrates the expression of RAGE (which may be rcl'crrcd to as a “priming antigen” herein) in the lung and an anti-cancer therapeutic (e.g., a pro-inflammatory cytokine, an immune receptor or an antibody such as a multi-specific antibody or a checkpoint inhibitor, etc.) which facilitates the killing of cancer cells in trans, i.e., where the expression of the therapeutic is activated by one cell (i.e., a potentially non-cancerous the cell in the lung that expresses RAG, or a “priming” cell) and kills a different cell that is nearby or proximal to the priming cell (i.e., the cancer cell or the “target” cell). This circuit restricts the killing action of the therapeutic cell to the lung, thereby avoiding collateral damage to other tissues.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figs. 1A-1E: Rationale for RAGE as a lung specific target and development of a RAGE targeting CAR and SynNotch receptor. Figs. 1A and IB: Human protein atlas data showing high expression of AGER mRNA only in the lung, single cell RNA sequencing data showing high expression of AGER mRNA only in type I alveolar epithelial cells in the lung. Fig. 1C: Flow cytometry showing cell death (DRAQ7+) in human- or mouse- RAGE expressing target 3T3 cells when cultured for 24h with anti-RAGE CAR-T cells. Fig. ID: BFP expression in anti- RAGE SynNotch T cells coculturcd with RAGE negative or RAGE positive K562s with myc bead stimulated T cells as a positive control. Fig. IE: BFP expression in anti-RAGE SynNotch T cells that have been cocultured with mouse alveolar epithelial cells grown in media that promotes an ATI -like state in which cells express endogenous RAGE. Figs. 2A-2G: anti-RAGE CAR enables T cells trafficking to and proliferation in the lung specifically. Fig. 2A: Schematic of experiment testing administration of control (anti-human CD19 CAR T cells) vs anti-RAGE CAR T cells in NSG mice. Fig. 2B: 6 day time course of in vivo bioluminescence of luciferase+ T cells. Fig. 2C: Ex-vivo luminescence of organs in control (top) vs anti-RAGE CAR (bottom) showing luminescence primarily in lung of anti-RAGE CAR mouse when compared to other tissues. Fig. 2D: Quantification of luminescence over time from Fig. 2B. Fig. 2E: Flow cytometry of lungs on day 7 after T cell administration. Y axis mCH+ T cells (Adoptively transferred T cells were transduced with mCH prior to administration). X axis IV CD45 to indicate intravascular T cells. Fig. 2F: mCH-i- T cell number in L lobe of lung in control vs anti-RAGE CAR-T cell groups. Fig. 2G: Cell count and differential in bronchoalveolar lavage fluid from control vs anti-RAGE CAR-T cell groups.

[0016] Figs. 3A-3F: anti-RAGE SynNotch gated CD19 CAR enables CD19 expressing tumor clearance specifically in the lung. Fig. 3A: Schematic of experiment in which mice were injected with tumors (NIH1975 lung cancer overexpressing hCD19) in both lung and flank and treated with T cells 7 days later. Mice were split into 3 groups: untransduced T cells (UnT), anti- RAGE SynNotch to hCD19 CAR circuit (SN), and constitutive CD 19 CAR (CAR). Fig. 3B: Survival curves for 3 groups. Fig. 3C: Representative H&E sections of lungs in each group with tumors (arrows) grossly evident in UnT group, small tumors (arrows) evident in CAR group, none in SN group. Fig. 3E: In vivo bioluminescence of luciferase+ tumor in lung fields of UnT, SN, and CAR groups on day 31 post tumor injection (top) with quantification of luminescence from day of T cell administration until 24 days post T cells (bottom). Error bars represent SEM. Fig. 3F: In vivo bioluminescence of luciferase+ tumor in flank of UnT, SN, and CAR groups on day 31 post tumor injection (top) with quantification of luminescence from day of T cell administration until 24 days post T cells (bottom). Error bars represent SEM.

[0017] DEFINITIONS

[0018] As used herein, the terms "treatment," "treating," “treat” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect and / or a response related to the treatment. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0019] A “therapeutically effective amount” or “efficacious amount” refers to the amount of an agent (including biologic agents, such as cells), or combined amounts of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated.

[0020] The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murines (e.g., rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), lagomorphs, etc. In some cases, the individual is a human. In some cases, the individual is a non-human primate. In some cases, the individual is a rodent, e.g., a rat or a mouse. In some cases, the individual is a lagomorph, e.g., a rabbit.

[0021] The term “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. Operably linked nucleic acid sequences may but need not necessarily be adjacent. For example, in some instances a coding sequence operably linked to a promoter may be adjacent to the promoter. In some instances, a coding sequence operably linked to a promoter may be separated by one or more intervening sequences, including coding and non-coding sequences. Also, in some instances, more than two sequences may be operably linked including but not limited to e.g., where two or more coding sequences are operably linked to a single promoter.

[0022] The terms “synthetic”, “chimeric” and “engineered” as used herein generally refer to artificially derived polypeptides or polypeptide encoding nucleic acids that arc not naturally occurring. Synthetic polypeptides and / or nucleic acids may be assembled de novo from basic subunits including, e.g., single amino acids, single nucleotides, etc., or may be derived from preexisting polypeptides or polynucleotides, whether naturally or artificially derived, e.g., as through recombinant methods. Chimeric and engineered polypeptides or polypeptide encoding nucleic acids will generally be constructed by the combination, joining or fusing of two or more different polypeptides or polypeptide encoding nucleic acids or polypeptide domains or polypeptide domain encoding nucleic acids. Chimeric and engineered polypeptides or polypeptide encoding nucleic acids include where two or more polypeptide or nucleic acid “parts” that are joined are derived from different proteins (or nucleic acids that encode different proteins) as well as where the joined parts include different regions of the same protein (or nucleic acid encoding a protein) but the parts are joined in a way that does not occur naturally.

[0023] The term "recombinant", as used herein describes a nucleic acid molecule, e.g., a polynucleotide of genomic, cDNA, viral, semisynthetic, and / or synthetic origin, which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide sequences with which it is associated in nature. The term recombinant as used with respect to a protein or polypeptide means a polypeptide produced by expression from a recombinant polynucleotide. The term recombinant as used with respect to a host cell or a virus means a host cell or virus into which a recombinant polynucleotide has been introduced. Recombinant is also used herein to refer to, with reference to material (e.g., a cell, a nucleic acid, a protein, or a vector) that the material has been modified by the introduction of a heterologous material (e.g., a cell, a nucleic acid, a protein, or a vector).

[0024] A "vector" or "expression vector" is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e. an "insert", may be attached so as to bring about the replication of the attached segment in a cell.

[0025] The term “heterologous”, as used herein, means a nucleotide or polypeptide sequence that is not found in the native (e.g., naturally-occurring) nucleic acid or protein, respectively. Heterologous nucleic acids or polypeptide may be derived from a different species as the organism or cell within which the nucleic acid or polypeptide is present or is expressed. Accordingly, a heterologous nucleic acids or polypeptide is generally of unlike evolutionary origin as compared to the cell or organism in which it resides.

[0026] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0027] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited.

[0028] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the cell” includes reference to one or more cells and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0029] 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. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0030] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0031] DETAILED DESCRIPTION

[0032] As summarized above, provided herein is an immune cell for killing cancer cells in the lung. In some embodiments, the cell may comprise: (a) a binding-triggered transcriptional switch (BTTS) that binds to RAGE (Receptor for Advanced Glycation Endproducts), (b) a nucleic acid sequence encoding an anti-cancer therapeutic; and (c) a regulatory sequence operably linked to (b) that is responsive to the binding-triggered transcriptional switch, wherein binding of the binding-triggered transcriptional switch to RAGE activates expression of the anticancer therapeutic.

[0033] BTTSs

[0034] A “binding-triggered transcriptional switch” or “BTTS” is a polypeptide or complex of the same that is capably of transducing a specific binding event on the outside of the cell (by binding of an extracellular domain of the BTTS to an antigen on another cell or extracellular matrix) that activates a recombinant promoter within the nucleus of the cell. Many BTTSs work by releasing a transcription factor that activates the promoter. In these embodiments, the BTTS is made up of one or more polypeptides that undergo proteolytic cleavage upon binding to the antigen to release a gene expression regulator that activates the recombinant promoter. For example, a BTTS may comprise (i) an extracellular domain comprising the antigen binding region of an antigen-specific antibody; (ii) a proteolytically cleavable sequence comprising one or more proteolytic cleavage sites; and (iii) an intracellular domain, wherein binding of the antigen binding region to the antigen induces cleavage of the sequence at the one or more proteolytic cleavage sites, thereby releasing the intracellular domain and wherein the intracellular domain activates transcription of an expression cassette that encodes the anti-cancer therapy. A BTTS can be based on Notch, A2, MESA, SNIPR or a force receptor, for example, although others are known (e.g., see Zhu et al 2022 Cell. 185: 1431-1443 and WO2021061856). In any embodiment, the BTTS may be a cleavable fusion protein that contains: (a) an extracellular binding domain comprising a protein binding domain (e.g., scFv or nanobody) that binds to RAGE, (b) a transmembrane domain, and (c) an intracellular domain comprising a transcriptional activator, where binding of the binding domain to RAGE on the surface of another cell induces proteolytic cleavage of the BTTS to release the transcriptional activator that, in turn, induces the expression of the anti-cancer therapy. In some embodiments, the BTTs may additional contain an extracellular force sensing region between regions (a) and (b) and (d) one or more force-dependent cleavage sites in (c) that are cleaved when the force sensing region is activated.

[0035] In this molecular switch, the fusion protein is cleaved to release the intracellular domain when the extracellular domain of the fusion protein engages with RAGE on another cell. In some cases, the fusion protein may contain a force sensing region (which is typically in the extracellular domain) and one or more force-dependent cleavage sites that are cleaved when the force sensing region is activated. The position of the force-dependent cleavage sites may vary and, in some embodiments the fusion protein may contain at least two cleavage sites. In some cases, one of the cleavage sites may be extracellular and the other may be in the transmembrane domain or within 10 amino acids of the transmembrane domain in the intracellular domain. In any embodiment, the force sensing region and / or the one or more force-dependent cleavage sites may be from a Delta / Serrate / Lag2 (DSL) superfamily protein, as reviewed by Pintar et al (Biology Direct 2007 2: 1-13). For example, the force sensing region and / or the one or more force-dependent cleavage sites may be from Notch (see Morsut Cell. 2016 164: 780-91), von Willebrand Factor (vWF), amyloid-beta, CD 16, CD44 , Delta, a cadherin , an ephrin-type receptor or ephrin ligand, a protocadherin, a filamin, a synthetic E cadherin, interleukin- 1 receptor type 2 (IL1R2), major prion protein (PrP), a neuregulin or an adhesion-GPCR. Several other examples of this type of protein are known and listed in Pintar, supra. Many members of this family appear to share a similar architecture a region that unfolds and opens up a protease cleavage site (e.g., EGF-likc repeats; sec Cordle ct al Nat. Struct. Mol. Biol. 2008 15: 849-857), a trans-membrane segment, and a relatively short (-100-150 amino acids) intracellular domain. These sequences permit the binding-triggered release of a transcriptional activator from the membrane in their natural environment and can be readily adapted herein.

[0036] In some cases, the one or more ligand-inducible proteolytic cleavage sites are selected from SI, S2, and S3 proteolytic cleavage sites. In some cases, the SI proteolytic cleavage site is a furin-like protease cleavage site comprising the amino acid sequence Arg-X-(Arg / Lys)-Arg, where X is any amino acid. In some cases, the S2 proteolytic cleavage site ADAM-17-type protease cleavage site comprising an Ala- Vai dipeptide sequence. In some cases, the S3 proteolytic cleavage site is a y-secretase cleavage site comprising a Gly-Val dipeptide sequence. The S3 proteolytic cleavage site is in the transmembrane domain. In many cases, the shear force generated by binding of the extracellular domain of this fusion protein to another cells unfolds the force sensing region (which, in the case of Notch contains EGF-like repeats whereas in other protein is made up of other sequences such as the A2 domain in vWF (see, e.g., I Thromb Haemost. 2009 7:2096-105, Lippok Biophys J. 2016 110: 545-54, Lynch Blood. 2014 123: 2585-92, Crawley, Blood. 2011 118:3212-21 and Xy J Biol Chem. 2013 288:6317-24) or modified A2 domain that has, e.g., the R1597W, E1638K and I1628T substitutions.

[0037] The architecture of such proteins is described in, e.g., Morsut et al, Cell. 2016 164: 780- 91, WO2016138034 and WO2019099689, among other places).

[0038] In some cases, the fusion protein includes an SI ligand-inducible proteolytic cleavage site. An SI ligand-inducible proteolytic cleavage site can be located between the HD-N segment and the HD-C segment. In some cases, the SI ligand- inducible proteolytic cleavage site is a furin-like protease cleavage site. A furin-like protease cleavage site can have the canonical sequence Arg-X-(Arg / Lys)-Arg, where X is any amino acid; the protease cleaves immediately C-terminal to the canonical sequence. For example, in some cases, an amino acid sequence comprising an SI ligand- inducible proteolytic cleavage site can have the amino acid sequence GRRRRELDPM (SEQ ID NO:1), where cleavage occurs between the “RE” sequence. As another example, an amino acid sequence comprising an S 1 ligand-inducible proteolytic cleavage site can have the amino acid sequence RQRRELDPM (SEQ ID NO:2), where cleavage occurs between the “RE” sequence.

[0039] In some cases, the fusion protein polypeptide includes an S2 ligand- inducible proteolytic cleavage site. An S2 ligand-inducible proteolytic cleavage site can be located within the HD-C segment. In some cases, the S2 ligand-inducible proteolytic cleavage site is an ADAM-17-typc protease cleavage site. An ADAM-17-type protease cleavage site can comprise an Ala-Vai dipeptide sequence, where the enzyme cleaves between the Ala and the Vai. For example, in some cases, amino acid sequence comprising an S2 ligand-inducible proteolytic cleavage site can have the amino acid sequence KIEAVKSE (SEQ ID NOG), where cleavage occurs between the “AV” sequence. As another example, an amino acid sequence comprising an S2 ligand- inducible proteolytic cleavage site can have the amino acid sequence KIEAVQSE (SEQ ID NO:4), where cleavage occurs between the “AV” sequence.

[0040] In some cases, the fusion protein includes an S3 ligand-inducible proteolytic cleavage site. An S3 ligand-inducible proteolytic cleavage site can be located within the TM domain. In some cases, the S3 ligand-inducible proteolytic cleavage site is a gamma- secretase (y-secretase) cleavage site. A y- secretase cleavage site can comprise a Gly-Val dipeptide sequence, where the enzyme cleaves between the Gly and the Vai. For example, in some cases, an S3 ligandinducible proteolytic cleavage site has the amino acid sequence VGCGVLLS (SEQ ID NO:5), where cleavage occurs between the “GV” sequence. In some cases, an S3 ligand-inducible proteolytic cleavage site comprises the amino acid sequence GCGVLLS (SEQ ID NO:6).

[0041] In some cases, the fusion protein polypeptide lacks an SI ligand-inducible proteolytic cleavage site. In some cases, the BETS lacks an S2 ligand-inducible proteolytic cleavage site. In some cases, the BTTS lacks an S3 ligand-inducible proteolytic cleavage site. In some cases, the BTTS lacks both an SI ligand-inducible proteolytic cleavage site and an S2 ligand- inducible proteolytic cleavage site. In some cases, the BTTS includes an S3 ligand-inducible proteolytic cleavage site; and lacks both an SI ligand- inducible proteolytic cleavage site and an S2 ligandinducible proteolytic cleavage site.

[0042] In some embodiments, the fusion protein may have an vWF A2 sequence or a variation thereof, an ADAMTS13 cleavage site (which may be described by the consensus sequence HEXXHXXGXXHD (SEQ ID NO:7); Crawley, Blood. 2011 118:3212-21), and an S3 or y- secretase cleavage site, although many other arrangements exist. In some embodiments, the switch may contain components that are borrowed from Notch. In other embodiments, the switch may not contain components that are from Notch.

[0043] For simplicity, BTTSs, including but not limited to chimeric notch receptor polypeptides, are primarily single polypeptide chains. However, BTTSs, including chimeric notch receptor polypeptides, may be divided or split across two or more separate polypeptide chains where the joining of the two or more polypeptide chains to form a functional BTTS, c.g., a chimeric notch receptor polypeptide, may be constitutive or conditionally controlled. For example, constitutive joining of two portions of a split BTTS may be achieved by inserting a constitutive heterodimerization domain between the first and second portions of the split polypeptide such that upon heterodimerization the split portions are functionally joined. Useful BTTSs that may be employed in the subject methods include, but are not limited to modular extracellular sensor architecture (MESA) polypeptides. A MESA polypeptide comprises: a) a ligand binding domain; b) a transmembrane domain; c) a protease cleavage site; and d) a functional domain. The functional domain can be a transcription regulator (e.g., a transcription activator, a transcription repressor). In some cases, a MESA receptor comprises two polypeptide chains. In some cases, a MESA receptor comprises a single polypeptide chain. Non-limiting examples of MESA polypeptides are described in, e.g., U.S. Patent Publication No. 2014 / 0234851; the disclosure of which is incorporated herein by reference in its entirety.

[0044] Useful BTTSs that may be employed in the subject methods include, but are not limited to polypeptides employed in the TANGO assay. The subject TANGO assay employs a TANGO polypeptide that is a heterodimer in which a first polypeptide comprises a tobacco etch virus (Tev) protease and a second polypeptide comprises a Tev proteolytic cleavage site (PCS) fused to a transcription factor. When the two polypeptides are in proximity to one another, which proximity is mediated by a native protein-protein interaction, Tev cleaves the PCS to release the transcription factor. Non-limiting examples of TANGO polypeptides are described in, e.g., Barnea et al. (Proc Natl Acad Sci USA. 2008 Jan. 8; 105( 1 ):64-9); the disclosure of which is incorporated herein by reference in its entirety.

[0045] Useful BTTSs that may be employed in the subject methods include, but are not limited to von Willebrand Factor (vWF) cleavage domain-based BTTSs, such as but not limited to e.g., those containing a unmodified or modified vWF A2 domain. A subject vWF cleavage domainbased BTTS will generally include: an extracellular domain comprising a first member of a binding pair; a von Willebrand Factor (vWF) cleavage domain comprising a proteolytic cleavage site; a cleavable transmembrane domain and an intracellular domain. Non-limiting examples of vWF cleavage domains and vWF cleavage domain-based BTTSs are described in Langridge & Struhl (Cell (2017) 171(6): 1383- 1396); the disclosure of which is incorporated herein by reference in its entirety.

[0046] Useful BTTSs that may be employed in the subject methods include, but arc not limited to chimeric Notch receptor polypeptides, such as but not limited to e.g., synNotch polypeptides, non-limiting examples of which are described in PCT Pub. No. WO 2016 / 138034, U.S. Patent No. 9,670,281, U.S. Patent No.9,834,608, Roybal et al. Cell (2016) 167(2):419-432, Roybal et al. Cell (2016) 164(4):770-9, and Morsut et al. Cell (2016) 164(4):780-91 ; the disclosures of which are incorporated herein by reference in their entirety. The "SNIPR" switch is another example of a BTTS (see Zhu et al 2022 Cell. 185: 1431-1443 and WO2021061856), although others exist (see, e.g., WO2019099689) and / or can be readily designed.

[0047] Expression of the BTTS in the cell may be constitutive or inducible, e.g., by binding of another BTTS to an antigen on another cell. As such, in some embodiments, the second promoter may be constitutive in the cell. For example, the second promoter may be a CMV, EF- 1, hPGK or RPBSA promoter, although many other choices are available.

[0048] In any embodiment, the BTTS comprises: i. an extracellular binding domain that binds to RAGE, ii. a force sensing region, iii. a transmembrane domain, iv. one or more forcedependent cleavage sites that are cleaved when the force sensing region is activated, and v. an intracellular domain comprising a transcriptional activator, where binding of the extracellular binding domain to the RAGE induces proteolytic cleavage of the one or more force-dependent cleavage sites to release the transcriptional activator, and wherein the released transcriptional activator induces transcription of the first coding sequence which, in turn, results in expression of the anti-cancer therapeutic.

[0049] Examples of transcriptional activators that can be part of the fusion protein are numerous and include artificial transcription factors (ATFs) such as, e.g., Zinc-finger-based artificial transcription factors (including e.g., those described in Sera T. Adv Drug Deliv Rev. 2009 61(7- 8):513-26; Collins et al. Curr Opin Biotechnol. 2003 14(4):371-8; Onori et al. BMC Mol Biol. 2013 14:3. In some cases, the transcriptional activator may contain a GAL4 DNA binding domain, which binds to the Gal4 responsive UAS, which has been well characterized in the art. Examples of suitable transcriptional activators include GAL4-VP16 and GAL4-VP64, although many others could be used. As would be appreciated, the identity of the transcription activators may vary. In some embodiments, the transcription factor may have a DNA binding domain that binds to a corresponding promoter sequence and an activation domain. In many embodiments, the DNA binding domain transcription factor may be independently selected from Gal4-, LexA- , Tet-, Lac-, dCas9-, zinc-finger- and TALE-based transcription factors. TALE- and CRISPR / dCas9-bascd transcription factors arc described in Lcbar (Methods Mol Biol. 2018 1772: 191-203), among others. The binding sites for such domains are well known or can be designed at will. The transcriptional activator can have any suitable activation domain, e.g., VP16, VP64, Ela, Spl, VP16, CTF, GAL4 among many others. In these embodiments, the regulatory sequence to which the transcription activator binds should contain one or more copies of a binding site for the transcriptional activator, e.g., a GAL4 UAS. RAGE binding domains

[0050] In some embodiments, the extracellular domain of the BTTS may contain the antigen binding region of a RAGE-specific antibody, e.g., a scFv or a nanobody. Examples of anti- RAGE antibodies are described in a variety of publications, including: Matsumoto et al (Scientific Reports 2017 7: 12255), Gasparotto et al (Brain Behav. Immun. 2017 62: 124-136), Vugmeyster et al (MAbs. 20102: 571-575), Healy et al (J. for ImmunoTherapy of Cancer 2019 7: 280), US20100143349A1, US20100028359A1, US20170002075A1 and US9527927B2. In some embodiments extracellular domain of the BTTS may contain the antigen binding region of any of the RAGE-specific antibodies described in US20100028359. Making scFvs from separate heavy and light chain sequences is well understood.

[0051] New antigen binding domains may also be generated in the form of immunoglobulin single variable (ISV) domains. The ISV domains may be generated using any suitable method. Suitable methods for the generation and screening of ISVs include without limitation, immunization of dromedaries, immunization of camels, immunization of alpacas, immunization of sharks, yeast surface display, etc. Yeast surface display has been successfully used to generate specific ISVs as shown in McMahon et al. (2018) Nature Structural Molecular Biology 25(3): 289-296 which is specifically incorporated herein by reference.

[0052] In some embodiments, the generation of immunoglobulin single variable domains such as e.g., VHHs or ISV may involve selection from phage display or yeast display, for example ISV can be selected by utilizing surface display platforms where the cell or phage surface display a synthetic library of ISV, in the presence of tagged antigen. A fluorescent secondary antibody directed to the tagged antigen is added to the solution thereby labeling cells bound to antigen. Cells are then sorted using any cell sorting platform of interest e.g., magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS). Sorted clones are amplified, resulting in an enriched library of clones expressing ISV that bind antigen. The enriched library is then re-screened with antigen to further enrich for surface displayed antigen binding ISV. These clones can then be sequenced to identify the sequences of the ISV of interest and further transferred to other heterologous systems for large scale protein production.

[0053] In some embodiments, the antigen binding region of a RAGE-specific antibody may have HC and LC CDR1, 2 and 3 sequences that are identical to or similar (i.e., may contain up to 5 amino acid substitutions, e.g., up to 1, up to 2, up to 3, up to 4 or up to 5 amino acid substitutions, collectively) to the CDRs of any of mAb7F9, mAbl l E6, and mAbAE5, as described in US20100028359. In some embodiments, the anti-RAGE antibody may have an HC and LC variable domains that are at least 90%, at least 95%, at least 98% or at least 99% identical to a pair of HC and LC sequences described in US20100028359, e.g., the HC and LC sequences of the 7F9 antibody, which are defined by SEQ ID NOS 1 and 5 in US20100028359, the HC and LC sequences of the 11E6 antibody, which are defined by SEQ ID NOS 9 and 13 in US20100028359, or the HC and LC sequences of the 4E5 which are defined by which are defined by SEQ ID NOS 17 and 22 in US20100028359, or a humanized version of the same (several of which are disclosed in US20100028359).

[0054] Payloads

[0055] The anti-cancer therapeutic induced by activation of the BTTS may be a secreted protein (e.g., a pro -inflammatory cytokine or an antibody such as a multi- specific antibody or an immune checkpoint inhibitor for example) or localized to the surface of the cell (e.g., may be a recombinant immune receptor such as a CAR or engineered T cell receptor). In some embodiments, activation of the BTTS may induce expression of both a secreted protein and an immune receptor (e.g., by way of a divergent promoter or by using an IRES, for example). If the anti-cancer therapeutic is not a recombinant immune receptor then the cell may further comprise a recombinant immune receptor e.g., a CAR or engineered TCR, that that is activated by binding to a killing antigen expressed by the cancer cells. In these latter embodiments, the recombinant immune receptor may be inducibly expressed, or constitutive. In any embodiment, the antibody or immune receptor may bind to a killing antigen expressed by the cancer cells.

[0056] Pro-inflammatory cytokines

[0057] Pro-inflammatory cytokines are secreted from the cell. In these embodiments, the circuit may comprise a nucleic acid containing a promoter that is activated by the released transcriptional activator, and a coding sequence encoding a pro-inflammatory protein. In this disclosure, the term “pro-inflammatory cytokine” is intended to encompass any cytokine that have a pro -inflammatory activity (e.g., IL-2 , CCL-21, IL-12, IL-7, IL-15 and IL-21, etc.), as well as non-natural or “engineered” cytokines that have pro-inflammatory activity such as super IL-2 (see, e.g., Levin et al Nature 2012 484: 529-533, which has the following amino acid substitutions L80F, R81D, L85V, I86V, and I92F relative to wild type), mini-TGF-Beta (which blocks TGF-Beta signaling) and DR- 18 (an IL- 18 variant), etc.). Engineered cytokines include superkines, which often have up to 10 amino acid substitutes relative to a natural cytokine, as well as natural cytokines that have been truncated, and dominant variants.

[0058] Cytokines of interest include selected from IL-2, IL-12, IL-15, IL-7, CD40L, or a non-natural variant of IL-2, IL-12, IL-15, IL-7, CD40L that has pro-inflammatory activity. Cytokines include "ortho" cytokines that can be paired with a receptor in the immune cell (see, e.g., Sockolosky et al. 2018).

[0059] Sources for exemplary pro-inflammatory proteins are listed below.

[0060] As would be appreciated, pro-inflammatory proteins are secreted from the cell and their coding sequence will encode a secretion signal.

[0061] In some embodiments the immune cell may additionally express a recombinant receptor for the pro-inflammatory protein, which may enhances the immune cell’s response. For example, if the pro-inflammatory protein is an “ortho2”, then the immune cell may additionally express a receptor for that pro-inflammatory protein.

[0062] Immune checkpoint inhibitors

[0063] Immune checkpoint inhibitors are also secreted from the cell. These molecules block interactions with PD1, CTLA4, BTLA, CD160, KRLG-1, 2B4, Lag-3, Tim-3 and other immune checkpoints. See, e.g., Odorizzi and Wherry (2012) J. Immunol. 188:2957; and Baitsch et al. (2012) PLoSOne 7: e30852. Exemplary immune checkpoint inhibitors include antibodies to CD27, CD28, CD40, CD122, CD96, CD73, CD47, 0X40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD 122, PD-1, PD-L1 and PD-L2. Many examples of such antibodies are known in the art. For example, anti-PDl antibodies include Pembrolizumab, Nivolumab, Cemiplimab, Dostarlimab, JTX-4014, Spartalizumab, Camrelizumab, Sintilimab, Tislelizumab, Toripalimab, INCMGA00012, AMP-224, and AMP- 514. Anti-PDLl antibodies include Atezolizumab, Avelumab, Durvalumab, KN035, CK-301, AUNP12, CA-170, and BMS-986189.

[0064] Multi-specific antibodies

[0065] In some embodiments, activation of the BTTS may induce expression of a multi- specific antibody. While such an antibody may perform other roles in killing cancer cells, in some embodiments, the antibody may be a a monoclonal antibody that stimulates ADCC, or an NK cell engager (NKCE), such as a BiKE (bispecific killer cell engager) or TriKE (trispecific killer cell engager). BiKEs and TriKEs are reviewed in Felices et al (Methods Mol Biol. 2016; 1441: 333-346). Such molecules tether NK cells to a tumor cell and induce their activation at that site. BiKEs and TriKEs are molecules that contain a single variable portion of an antibody linked to one (BiKE) or two (TriKE) variable portions from other antibodies of different specificity. See, e.g., Shanshal et al (Cancers (Basel). 2023 15: 2824)

[0066] Multispecific antibodies can be in a variety of different formats, including, but not limited to IgG-like antibody formats (including an Fc domain) and non-IgG-like antibody formats (without an Fc domain). Multispecific antibodies with IgG-like antibody formats can be in a variety of different formats, including, but not limited to knob-into-hole (KIH), TrioMab, Duobody, KA body, CrossMab, common light chain, strand exchange engineered domain bodies (SEEDBodies), Azymetric heterodimeric Fc, dual action Fab (DAF), dual-variable-domain immunoglobulin (DVD-Ig), IgG-scFv, Fab-Fab-Fc, DutaMab, and DutaFab.

[0067] Non-IgG-like antibody formats may lack an Fc region entirely. For example, Fab, Fv and VHH antibody regions may be genetically engineered and combined in various orientations and pairings. Multispecific antibodies in non-IgG-like formats can be in a variety of different formats, including, but not limited to bivalent dual-affinity re-targeting protein (DART), tetravalent DART, half-life extended bispecific T-cell engager (HLE-BiTE), bispecific T-cell engager (BiTE), immune mobilizing monoclonal T-cell receptor (ImmTAC), tandem diabody (TandAb), bispecific killer cell engager (BiKE), trispecific killer cell engager (TRiKE), multispecific scFV single-chain variable fragment, trispecific T-cell activation construct (TriTAC), bispecific nanobody, and cross-over dual variable region (CODV). Engineered immune receptors

[0068] In some embodiments, activation of the BTTS may induce expression of an engineered receptor or, alternatively, the cell may further a nucleic acid encoding an engineered immune receptor. In these embodiments the engineered immune receptor may be, e.g., a chimeric antigen receptor (CAR), engineered T cell receptor (TCR) (including an HLA Independent TCR (HIT)).

[0069] In these embodiments, the recombinant immune receptor, when it is expressed, will localized on the surface of the cell and will be activated by binding to an antigen that is expressed by the cancerous cells, e.g., by the malignant cells, e.g., any of the antigens listed below, for example.

[0070] Binding of the immune receptor to its cognate antigen activates the immune cell. In these embodiments, the receptor may be a chimeric antigen receptor (CAR), where the terms “chimeric antigen receptor” and “CAR”, used interchangeably herein, refer to artificial multimodule molecules capable of triggering the activation of an immune cell which generally but not exclusively comprise an extracellular domain (e.g., a ligand / antigen binding domain), a transmembrane domain and one or more intracellular signaling domains.

[0071] CARs can be designed in several ways (see, generally, e.g., Guedan et al, Methods and Clinical Development 2019 12: 145-156) and may include an extracellular domain that contains an antigen binding domain such as a scFv or nanobody, a hinge, a transmembrane region (which may be derived from CD4, CD8a, or CD28), a costimulatory signaling domain (which may be derived from the intracellular domains of the CD28 family (e.g., CD28 and ICOS) CD2 (US9783591B2) or the tumor necrosis factor receptor (TNFR) family of genes (e.g., 4-1BB, 0X40, or CD27)), and an IT AM domain, e.g., the signaling domain from the zeta chain of the human CD3 complex (CD3zeta). In practice, any of these domains may be a variation of a wild type sequence. In practice, any of these sequences may be a variant of a wild type sequence, e.g., a sequence that is at least 90%, 95%, or 98% identical to a sequence described in WO2014127261, for example. For example, a CAR may have a signaling domain from CD3^ in which two of the three ITAM motifs (the second and third IT AM motifs) have been altered to be non-functional. More specifically, both tyrosine (Y) phosphorylation sites in the second and third ITAMs may be substituted by phenylalanine, thereby rendering those sites incapable of being phosphorylated. This altered CD3^ signaling domain is described in Feucht et al (Nat Med. 2019 25: 82-88).

[0072] The term CAR is not limited specifically to CAR molecules but also includes CAR variants. CAR variants include split CARs wherein the extracellular portion (e.g., the ligand binding portion) and the intracellular portion (e.g., the intracellular signaling portion) of a CAR are present on two separate molecules. CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional heterodimerization of the two portions of the split CAR is pharmacologically controlled (e.g., as described in PCT publication no. WO 2014 / 127261 Al and US Patent Application No. 2015 / 0368342 Al, the disclosures of which are incorporated herein by reference in their entirety). CAR variants also include bispecific CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation. CAR molecules and derivatives thereof (i.e., CAR variants) are described, e.g., in PCT Application No. US2014 / 016527; Fedorov et al. Sci Transl Med (2013) ;5(215):215ral72; Glienke et al. Front Pharmacol (2015) 6:21; Kakarla & Gottschalk 52 Cancer J (2014) 20(2): 151-5; Riddell et al. Cancer J (2014) 20(2): 141-4; Pegram et al. Cancer J (2014) 20(2): 127-33; Cheadle et al. Immunol Rev (2014) 257(1 ):91- 106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388-98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety. Useful CARs also include the anti-CD19 — 4-1BB — CD3^ CAR expressed by lentivirus loaded CTL019 (Tisagenlecleucel-T) CAR-T cells as commercialized by Novartis (Basel, Switzerland).

[0073] The terms “T cell receptor” and “TCR” are used interchangeably and will generally refer to a molecule found on the surface of T cells, or T lymphocytes, that is responsible for recognizing fragments of antigen as peptides bound to major histocompatibility complex (MHC) molecules. The TCR complex is a disulfide-linked membrane- anchored heterodimeric protein normally consisting of the highly variable alpha (a) and beta (0) chains expressed as pail of a complex with CD3 chain molecules. Many native TCRs exist in heterodimeric a0 or y8 forms. The complete endogenous TCR complex in heterodimeric a0 form includes eight chains, namely an alpha chain (referred to herein as TCRa or TCR alpha), beta chain (referred to herein as TCRP or TCR beta), delta chain, gamma chain, two epsilon chains and two zeta chains. In some instance, a TCR is generally referred to by reference to only the TCRa and TCRP chains, however, as the assembled TCR complex may associate with endogenous delta, gamma, epsilon and / or zeta chains an ordinary skilled artisan will readily understand that reference to a TCR as present in a cell membrane may include reference to the fully or partially assembled TCR complex as appropriate.

[0074] Recombinant or engineered individual TCR chains and TCR complexes have been developed. References to the use of a TCR in a therapeutic context may refer to individual recombinant TCR chains. As such, engineered TCRs may include individual modified TCRa or modified TCRP chains as well as single chain TCRs that include modified and / or unmodified TCRa and TCRP chains that are joined into a single polypeptide by way of a linking polypeptide

[0075] Any engineered TCR having immune cell activation function can be induced using a method of the present disclosure. Such TCRs include, e.g., antigen-specific TCRs, Monoclonal TCRs (MTCRs), Single chain MTCRs, High Affinity CDR2 Mutant TCRs, CDl-binding MTCRs, High Affinity NY-ESO TCRs, VYG HLA-A24 Telomerase TCRs, including e.g., those described in PCT Pub Nos. WO 2003 / 020763, WO 2004 / 033685, WO 2004 / 044004, WO 2005 / 114215, WO 2006 / 000830, WO 2008 / 038002, WO 2008 / 039818, WO 2004 / 074322, WO 2005 / 113595, WO 2006 / 125962; Strommes et al. Immunol Rev. 2014; 257(1): 145-64; Schmitt et al. Blood. 2013; 122(3):348-56; Chapuls et al. Sci Transl Med. 2013; 5(174): 174ra27; Thaxton et al. Hum Vaccin Immunother. 2014; 10(11):3313-21 (PMID:25483644); Gschweng et al. Immunol Rev. 2014; 257(l):237-49 (PMID:24329801); Hinrichs et al. Immunol Rev. 2014; 257(l):56-71 (PMID:24329789); Zoete et al. Front Immunol. 2013; 4:268 (PMID:24062738); Marr et al. Clin Exp Immunol. 2012; 167(2) :216-25 (PMID:22235997); Zhang et al. Adv Drug Deliv Rev. 2012; 64(8):756-62 (PMID:22178904); Chhabra et al. Scientific World Journal. 2011; 11:121-9 (PMID:21218269); Boulter et al. Clin Exp Immunol. 2005; 142(3):454-60 (PMID: 16297157); Sami et al. Protein Eng Des Sei. 2007; 20(8):397-403; Boulter et al. Protein Eng. 2003; 16(9):707- 11; Ashfield et al. IDrugs. 2006; 9(8):554-9; Li et al. Nat Biotechnol. 2005; 23(3):349-54; Dunn et al. Protein Sci. 2006; 15(4):710-21; Liddy et al. Mol Biotechnol. 2010; 45(2); Liddy et al. Nat Med. 2012; 18(6):980-7; Oates, et al. Oncoimmunology. 2013; 2(2):e22891; McCormack, et al. Cancer Immunol Immunother. 2013 Apr;62(4):773-85; Bossi et al. Cancer Immunol Immunother. 2014; 63(5):437-48 and Oates, et al. Mol Immunol. 2015 Oct;67(2 Pt A):67-74; the disclosures of which are incorporated herein by reference in their entirety. HLA-Independent TCRs (Eyquem et al. 2022 Feb;28(2):345-352) are also included in this definition.

[0076] Killing antigens

[0077] In any embodiment that involves an antibody or recombinant immune receptor, the antibody or recombinant immune receptor may bind to a lung cancer antigen selected from: CEA, CD276, EGFR, HER2, MSLN, MUC1, PD-L1, ROR1, TnMUCl, GPC3, TGF0, aPDl, NY-ESO-1, EGFRVIII, MAGE-A1, MAGE-A4, GD2, PSCA, MUC1, GPC3, Lewis-Y, AXL, EGFR, and B7-H3. CAR T cells that have binding domains that target these antigens are currently in clinical trials for lung cancer in various centers around the world (see, e.g., Chen et al Thorac. Cancer. 2022 13: 889-899). Alternatively, other cancer antigen could be targeted, such as CA9, CA12, CXorf61, DSG3, FAT2, GPR87, KISS1R, LYPD3, SLC7A11 or TMPRSS4 (See, e.g., Cohen et al Oncotarget. 2017 Dec 26; 8(69): 113373-113402.).

[0078] Exemplary sources for the sequences of some binding domains can be found below. However, these targets are all well known, as are binding domains that target those antigens.

[0079] Any embodiment, the binding domain of the antibody or recombinant immune receptor may have heavy and light chains (or heavy chains only, if the antibody is a nanobody / VHH antibody) that has CDR1, 2 and 3 sequences that are identical to or similar (i.e., may contain up to 5 amino acid substitutions, e.g., up to 1, up to 2, up to 3, up to 4 or up to 5 amino acid substitutions, collectively) to the CDRs of any of the antibodies listed above, which publications are incorporated by reference for those sequences. As would be apparent, the framework sequence could be humanized, for example. In some embodiments, the binding domain of the antibody or recombinant immune receptor may have HC and LC variable regions that are at least 90%, at least 95%, at least 98% or at least 99% identical to heavy and light chains (or heavy chains only, if the antibody is a nanobody / VHH antibody) of any of the antibodies.

[0080] Cells Immune cells of the present disclosure include mammalian immune cells including, e.g., those that are genetically modified to produce the components of a circuit of the present disclosure or to which a nucleic acid, as described above, has been otherwise introduced. In some instances, the subject immune cells have been transduced with one or more nucleic acids and / or expression vectors to express one or more components of a circuit of the present disclosure.

[0081] Suitable mammalian immune cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include human cell lines, non-human primate cell lines, rodent (e.g., mouse, rat) cell lines, and the like. In some instances, the cell is not an immortalized cell line, but is instead a cell (e.g., a primary cell) obtained from an individual. For example, in some cases, the cell is an immune cell, immune cell progenitor or immune stem cell obtained from an individual. As an example, the cell is a lymphoid cell, e.g., a lymphocyte, or progenitor thereof, obtained from an individual. As another example, the cell is a cytotoxic cell, or progenitor thereof, obtained from an individual.

[0082] Such cells include, e.g., lymphoid cells, i.e., lymphocytes (T cells, B cells, natural killer (NK) cells), and myeloid-derived cells (neutrophil, eosinophil, basophil, monocyte, macrophage, dendritic cells). “T cell” includes all types of immune cells expressing CD3 including T-helper cells (CD4+ cells) and cytotoxic T-cells (CD8+ cells). A “cytotoxic cell” includes CD8+ T cells, natural-killer (NK) cells, and neutrophils, which cells are capable of mediating cytotoxicity responses.

[0083] Immune cells encoding a circuit of the present disclosure may be generated by any convenient method. Nucleic acids encoding one or more components of a subject circuit may be stably or transiently introduced into the subject immune cell, including where the subject nucleic acids are present only temporarily, maintained extrachromosomally, or integrated into the host genome. Introduction of the subject nucleic acids and / or genetic modification of the subject immune cell can be carried out in vivo, in vitro, or ex vivo.

[0084] In some cases, the introduction of the subject nucleic acids and / or genetic modification may be carried out ex vivo. For example, a primary T lymphocyte may be obtained from an individual; and the cell obtained from the individual is modified to express components of a circuit of the present disclosure. Other embodiments may use “off the shelf’, allogeneic cells.

[0085] Populations of immune cells A population of the immune cells (e.g., T cells) is also provided. In some embodiments, these cells may be present in vitro and may be progenitors of primary immune cells that have been genetically modified to contain the present circuit, as described above. As noted above, in some embodiments immune cells may be genetically modified to be allogeneic in a human host. In these embodiments, the cells may be frozen. The population may comprise any number of the CAR-T cells (e.g. 100,000-1 Bn cells). However, in some embodiments, the population may contain 1M-500M of the immune cells.

[0086] In some embodiments, the harvested cells may be cryopreserved, where the term “cryopreserved” refers to cells that have been preserved or maintained by cooling to low subzero temperatures, such as 77 K or -196 deg. C. (the boiling point of liquid nitrogen). At these low temperatures, any biological activity, including the biochemical reactions that would lead to cell death, is effectively stopped. Useful methods of cry opreservation and thawing cryopreserved cells, as well as processes and reagents related thereto, include but are not limited to e.g., those described in U.S. Patent Nos. 10370638; 10159244; 9078430; 7604929; 6136525; and 5795711, the disclosures of which are incorporated herein by reference in their entirety. In contrast, the term “fresh”, as used herein with reference to cells, may refer to cells that have not been cryopreserved and, e.g., may have been directly obtained and / or used (e.g., transplanted, cultured, etc.) following collection from a subject or organ thereof.

[0087] Harvested therapeutic cell populations produced by the methods as described herein and therapeutic or pharmaceutical compositions thereof may be present in any suitable container (e.g., a culture vessel, tube, flask, vial, cryovial, cryo-bag, etc.) and may be employed (e.g., administered to a subject) using any suitable delivery method and / or device. Such populations of cells and pharmaceutical compositions may be prepared and / or used fresh or may be cryopreserved. In some instances, populations of therapeutic cells and pharmaceutical compositions thereof may be prepared in a “ready-to-use” format, including e.g., where the therapeutic cells are present in a suitable diluent and / or at a desired delivery concentration (e.g., in unit dosage form) or a concentration that can be readily diluted to a desired delivery concentration (e.g., with a suitable diluent or media). Populations of therapeutic cells and pharmaceutical compositions thereof may be prepared in a delivery device or a device compatible with a desired delivery mechanism or the desired route of delivery, such as but not limited to e.g., a syringe, an infusion bag, or the like. In some instances, the present disclosure provides one or a plurality of cell therapy doses, e.g., each contained in suitable container. Cell therapy doses may be generated through a variety of methods. Aliquoting expanded populations of therapeutic cells into cell therapy doses may be performed by a variety of means.

[0088] In certain embodiments, the compositions may include the therapeutic cells present in a liquid medium. The liquid medium may be an aqueous liquid medium, such as water, a buffered solution, or the like. One or more additives such as a salt (e.g., NaCl, MgCh, KC1, MgSO4), a buffering agent (a Tris buffer, N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES), 2-(N-Morpholino)ethanesulfonic acid (MES), 2-(N-Morpholino)ethanesulfonic acid sodium salt (MES), 3-(N-Morpholino)propanesulfonic acid (MOPS), N- tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS), etc.), a solubilizing agent, a detergent (e.g., a non-ionic detergent such as Tween-20, etc.), a nuclease inhibitor, glycerol, a chelating agent, and the like may be present in such compositions.

[0089] A population may include a therapeutically effective amount of the cells. By “therapeutically effective amount” it is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to affect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease or disorder associated, e.g., with the target cell or a population thereof, as compared to a control. An effective amount can be administered in one or more administrations.

[0090] A “therapeutically effective amount” of such cells may vary according to factors such as the disease state, age, sex, and weight of the subject, and the ability of the cells to elicit a desired response in the subject. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” a subject (e.g., a patient). When a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments, to be administered, can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). In certain embodiments, a therapeutically effective amount of T cells may be 100,000-50M of the T cells. However, in other embodiments, a therapeutically effective amount of T cells may be 50M- 500M cells. The cells of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. More particularly, the cells of the present disclosure can be formulated for administration by combination with appropriate excipients, diluents and / or the like.

[0091] Formulations of the cells suitable for administration to a patient (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.

[0092] The cells may be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration.

[0093] An aqueous formulation of the cells may be prepared in a pH-buffered solution, e.g., at a pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that arc suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.

[0094] A tonicity agent may be included in the formulation to modulate the tonicity of the formulation. Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.

[0095] In some embodiments, a composition includes cells of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m- cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w / v). Cell manufacture methods

[0096] A cell manufacture method is also provided. In some embodiments, the method may comprise expanding the immune cells ex vivo to produce expanded immune cells, and harvesting the expanded immune cells to produce an immune cells population, where the term “harvesting” is intended to refer to a step in which the cells are removed from the container(s) / bioreactor(s) in which the cells were expanded. In some embodiments, the cells may be concentrated if desired, e.g., by centrifugation, a suitable cell separation technique (e.g., magnetic beads), and / or the like.

[0097] The cells may be modified by any suitable method, e.g., by introducing a transgene into immune cells. The general procedure for manufacturing CAR-T cells typically includes isolation of peripheral blood mononuclear cells (PBMCs). Next, PBMCs or T cells that have been further enriched from PBMCs are stimulated with antibody-coated beads (e.g. Dynabeads) or plate-bound antibodies to induce T cell activation and then genetically modified using lentiviral vectors, gamma-retroviral vectors, or other delivery methods, to express the cell surface CAR molecule. Subsequently, these engineered T cells are expanded in culture in the presence of one or more cytokines (e.g., IL-2, IL- 15, IL-7 or any combination thereof (e.g., IL-2 and IL-15 or IL-7 and IL-15)) for several days to reach the required cell numbers for either experimental testing or clinical treatment.

[0098] In some embodiments, the T cells may be harvested within 5-20 (e.g. 8-16) days of the initiation of cell expansion. In these embodiments, the harvested T cell population may comprise 50M-lBn of the CAR-T cells, however more or less cells can be used. In some embodiments, the T cells may be harvested within 8-16 days of the initiation of cell expansion, in which case the population may comprise 50M-lBn cells.

[0099] A cell population manufactured according to this method is also provided.

[0100] Method for killing cancer cells

[0101] A method for killing cancer cells in the lung is also provided. This method may comprise: administering to a subject an immune cell as described above, wherein binding of the binding-triggered transcriptional switch to RAGE activates expression of the anti-cancer therapeutic and killing of the cancer cells. In these embodiments, the subject may have lung cancer, e.g., non-small cell lung cancer (NSCLC) or small cell lung lancer (SCLC). In some cases, the lung cancer may an adenocarcinoma, a squamous cell carcinoma or a large cell carcinoma. Alternatively, the subject may have a non-lung cancer that has metastasized to the lung, e.g., a sarcoma. In some embodiments, the method may be a method for treating a patient that has lung cancer or a non-lung cancer that has metastasized to the lung.

[0102] This method may comprise administering an effective amount a population of the T cells to a patient in need thereof where, in some embodiments, an effective amount may be in the range of 10M to 1 Bn cells. In any embodiment, the T cells may be autologous / autogeneic (“self”) or non-autologous (“non-self,” e.g., allogeneic, syngeneic or xenogeneic). “Autologous” as used herein, refers to cells obtained from the subject to whom the therapeutic cells are later administered. “Allogeneic” as used herein refers to cells obtained from a donor other than the subject to whom the therapeutic cells are administered. In some embodiments, the cells (e.g., T cells) are cells obtained from a mammalian subject. In certain embodiments, the mammalian subject is a primate. In some embodiments, the cells are obtained from a human. Cells are typically infused into the patient, although routes of administration can be used.

[0103] In any embodiment, the patient may be a cancer patient where the treatment may result in least an amelioration of one or more symptoms associated with the condition of the subject, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g., symptom, associated with the condition being treated. As such, treatment also includes situations where the condition, or at least one or more symptoms associated therewith, are completely inhibited, e.g., prevented from happening, or stopped, e.g., terminated, such that the subject no longer suffers from the condition, or at least the symptoms that characterize the condition.

[0104] Routes for administration include by intravenous injection, although any other suitable route may be used.

[0105] Combination therapies

[0106] In some cases, the immune cells (e.g., CAR T cells) may be administered along with at least one additional therapeutic agent or therapeutic treatment (together or sequentially). Suitable additional therapeutic agents include, but are not limited to, a small molecule cancer chemotherapeutic agent, and an immune checkpoint inhibitor. Suitable additional therapeutic treatments include, e.g., radiation, surgery (e.g., surgical resection of a tumor), and the like.

[0107] A treatment method of the present disclosure can comprise co-administration of the immune cells and at least one additional therapeutic agent. By “co-administration” it is meant that both the immune cells and at least one additional therapeutic agent are administered to an individual, although not necessarily at the same time, in order to achieve a therapeutic effect that is the result of having administered both the immune cells and the at least one additional therapeutic agent. The administration of the immune cells and the at least one additional therapeutic agent can be substantially simultaneous, e.g., the polypeptide can be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 4 hours, within about 8 hours, within about 12 hours, or within about 24 hours) of administration of the at least one additional therapeutic agent. In some cases, the immune cells of the present disclosure are administered to an individual who is undergoing treatment with, or who has undergone treatment with, the at least one additional therapeutic agent. The administration of the CAR-T cells can occur at different times and / or at different frequencies.

[0108] In some cases, the subject may be additionally treated with an immune checkpoint inhibitor. Exemplary immune checkpoint inhibitors include inhibitors that target an immune checkpoint polypeptide such as CD27, CD28, CD40, CD122, CD96, CD73, CD47, 0X40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2. In some cases, the immune checkpoint polypeptide is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, 0X40, GITR, CD 122 and CD137. In some cases, the immune checkpoint polypeptide is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, CD96, TIGIT and VISTA.

[0109] Co-therapies include for example, (a) anthracycline therapy (e.g., by administering daunomycin, doxorubicin, or mitoxantrone), (b) alkylating agent therapy (e.g., by administering mechlorethane, cyclophosphamide, ifosfamide, melphalan, cisplatin, carboplatin, nitrosourea, dacarbazine, procarbazine or busulfan), (c) topoisomerase II inhibitor therapy (e.g., by administering etoposide or teniposide), (d) bleomycin therapy, (e) anti-metabolite therapy (e.g., by administering methotrexate, 5-fluorocil, cytarabine, 6-mercaptopurine or 6-thioguanine), (f) vinca alkyloid therapy (e.g., by administering vincristine or vinblastine), (g) steroid therapy (e.g., by administering prednisone or dexamethasone and (h) radiation treatment, etc. Alternative therapies include targeted therapies and non-targeted chemotherapies, where targeted therapy includes treatment with erlotinib (Tarceva), afatinib (Gilotrif), gefitinib (Iressa) or osimertinib (Tagrisso) which may be administered to patients having an activating mutation in EGFR, crizotinib (Xalkori), ceritinib (Zykadia), alectinib (Alecensa) or brigatinib (Alunbrig) which may be administered to patients having an ALK fusion, crizotinib (Xalkori), entrectinib (RXDX-101), lorlatinib (PF-06463922), crizotinib (Xalkori), entrectinib (RXDX-101), lorlatinib (PF-06463922), ropotrectinib (TPX-0005), DS-6051b, ceritinib, ensartinib or cabozantinib which may be administered to patients having an ROS1 fusion, or dabrafenib (Tafinlar) or trametinib (Mekinist) which may be administered to patients having an activating mutation in BRAF. Many other actionable mutations are known. If the patient is going to be switched to a non-targeted chemotherapy, the therapy may be, for example, a platinum-based doublet chemotherapy (in which the platinum-based doublet chemotherapy may comprise a platinumbased agent selected from cisplatin (CDDP), carboplatin (CBDCA), and nedaplatin (CDGP)) and one third-generation agent (selected from docetaxel (DTX), paclitaxel (PTX), vinorelbine (VNR), gemcitabine (GEM), irinotecan (CPT-11), pemetrexed (PEM), and tegafur gimeracil oteracil (SI)).

[0110] In some embodiments, the patient may receive platinum-based doublet chemotherapy, which is sometimes used for lung cancer (in which the platinum-based doublet chemotherapy may comprise a platinum-based agent selected from cisplatin (CDDP), carboplatin (CBDCA), and nedaplatin (CDGP)) and one third-generation agent (selected from docetaxel (DTX), paclitaxel (PTX), vinorelbine (VNR), gemcitabine (GEM), irinotecan (CPT-11), pemetrexed (PEM), and tegafur gimeracil oteracil (SI)) then, in these embodiments, pembrolizumab can be administered to the patient as an intravenous infusion (200 mg or 2 mg / kg, up to 200 mg) over 30 minutes, every three weeks. Dosages and timing for platinum-based doublet chemotherapy are also known but vary from combination to combination (see, e.g., Besse et al, Annals of Oncology 2005 16: 997-998 and Sangal et al Lung Cancer Management 2013 2: 5, among many others). As would be apparent, if a patient is switched onto treatment that includes pembrolizumab and a platinum-based doublet chemotherapy, then the treatment schedule for pembrolizumab (i.e., intravenous infusion over 30 minutes, every three weeks) may continue under the same schedule and the platinum-based doublet chemotherapy may be added. In these embodiments the platinum-based doublet chemotherapy may be added. Some platinum-based doublet chemotherapies are administered every three weeks. In these embodiments, the platinum-based doublet chemotherapy may be administered on the same day as the pembrolizumab. In other embodiments, the platinum-based doublet chemotherapy may be administered in between the pembrolizumab administrations.

[0111] Additional embodiments A recombinant nucleic acid encoding a binding-triggered transcriptional switch (BTTS) that binds to RAGE (Receptor for Advanced Glycation Endproducts), as described above, as well as an immune cell encoding the same and the protein encoded by the same, are also provided. The recombinant nucleic acid may be in a vector, such as a viral vector, e.g., an adenoviral vector, a retroviral vector, a lentiviral vector, an adeno-associated virus vector, an herpes simplex virus vector, etc. Replication-defective viruses can also be advantageous. Some vectors become incorporated into the nuclear genome of the host cell, whereas others do not. The use of viral vectors (particularly retroviral and lentiviral vectors) for expressing CARs in immune cells is known (see, e.g., Lanne et al Viruses. 2021 13: 1528) and Moco et al Methods Mol Biol 20202086: 69-76 among many others).

[0112] EMBODIMENTS

[0113] In any embodiment described above or below, Slc34a2, Grpc5a, Lamp3, Mucl or CD55 may be used instead of RAGE. RAGE is an example of an antigen to which the BTTS may bind.

[0114] Embodiment 1. An immune cell for the killing cancer cells in the lung, comprising:

[0115] (a) a binding-triggered transcriptional switch (BTTS) that binds to RAGE (Receptor for Advanced Glycation Endproducts),

[0116] (b) a nucleic acid sequence encoding an anti-cancer therapeutic; and

[0117] (c) a regulatory sequence operably linked to (b) that is responsive to the binding- triggered transcriptional switch; wherein binding of the binding-triggered transcriptional switch to RAGE activates expression of the anti-cancer therapeutic.

[0118] Embodiment 2. The immune cell of embodiment 1, wherein the anti-cancer therapeutic, when expressed by the cell, is secreted by the cell or localized to the surface of the cell.

[0119] Embodiment 3. The immune cell of embodiment 1 or 2, wherein anti-cancer therapeutic is a pro-inflammatory cytokine.

[0120] Embodiment 4. The immune cell of embodiment 1 or 2, wherein anti-cancer therapeutic is an antibody.

[0121] Embodiment 5. The immune cell of embodiment 4, wherein the antibody is a multispecific antibody or (e.g., a bispecific or trispecific antibody that engages NK cells) or an immune checkpoint inhibitor. Embodiment 6. The immune cell of embodiment 1, wherein the anti-cancer therapeutic is a recombinant immune receptor that is activated by binding to a killing antigen expressed by the cancer cells.

[0122] Embodiment 7. The immune cell of embodiment 5 or 7, wherein the recombinant immune receptor is a chimeric antigen receptor (CAR) or engineered T cell receptor (TCR).

[0123] Embodiment 8. The immune cell of any of embodiments 4-7, wherein the antibody or immune receptor binds to a killing antigen expressed by the cancer cells.

[0124] Embodiment 9. The immune cell of any of embodiments 1-5, wherein anti-cancer therapeutic is not a recombinant immune receptor and the cell further comprises a recombinant immune receptor that recognizes a killing antigen expressed by the cancer cells.

[0125] Embodiment 10. The immune cell of any of embodiments 4-9, wherein the antibody or immune receptor binds to a killing antigen for lung cancer selected from: CEA, CD276, EGER, HER2, MSLN, MUC1, PD-L1, ROR1, TnMUCl, GPC3 or TGF , aPDl and MSLN, NY-ESO- 1 or EGFRVIII, MAGE-A1, MAGE-A4, GD2„ PSCA, MUC1, GPC3, Lewis-Y, AXL, EGFR, and B7-H3.

[0126] Embodiment 11. The immune cell of embodiment 10, wherein the antibody or immune receptor binds to a killing antigen expressed by a non-lung cancer that has metastasized to the lung.

[0127] Embodiment 12. The immune cell of embodiment 11, wherein the non-lung cancer is sarcoma and the killing antigen is NY-ESO-1.

[0128] Embodiment 13. The immune cell of any prior embodiment, wherein the BTTS undergoes proteolytic cleavage upon binding to RAGE to release a gene expression regulator that induces expression of the anti-cancer therapeutic of (b) via the regulatory sequence of (c).

[0129] Embodiment 14. The immune cell of any prior embodiment, wherein the BTTS comprises:

[0130] (i) an extracellular domain comprising the antigen binding region of a RAGE-specific antibody;

[0131] (ii) a proteolytically cleavable sequence comprising one or more proteolytic cleavage sites; and

[0132] (iii) an intracellular domain; wherein binding of the antigen binding region to RAGE induces cleavage of the sequence at the one or more proteolytic cleavage sites, thereby releasing the intracellular domain and wherein the intracellular domain induces expression of the anti-cancer therapeutic of (b) via the regulatory sequence of (c).

[0133] Embodiment 15. The immune cell of any prior embodiment, wherein the cell is cytotoxic T cell.

[0134] Embodiment 16. A method for killing cancer cells in the lung, comprising: administering to a subject an immune cell of any prior embodiment: wherein binding of the binding-triggered transcriptional switch to RAGE activates expression of the anti-cancer therapeutic and killing of the cancer cells.

[0135] Embodiment 17. The method of embodiment 16, wherein the subject has lung cancer.

[0136] Embodiment 18. The method of embodiment 17, wherein the lung cancer is non- small cell lung cancer (NSCLC) or small cell lung lancer (SCLC).

[0137] Embodiment 19. The method of any of embodiments 16-18, wherein the lung cancer is an adenocarcinoma, a squamous cell carcinoma or a large cell carcinoma.

[0138] Embodiment 20. The method of embodiment 16, wherein the subject has a non-lung cancer that has metastasized to the lung.

[0139] Embodiment 21. The method of embodiment 20, wherein the non-lung cancer is a sarcoma.

[0140] Embodiment 22. The method of any of embodiments 16-21, wherein the method is a method of treatment.

[0141] EXAMPLES

[0142] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m., intramuscularly ); i.p., intraperitoneally ); s.c., subcutaneous(ly); and the like.

[0143] Cell based therapy has long been an area of interest in preclinical studies of acute lung injury treatment. In the following study, a new cell-based therapy was designed to target alveolar epithelial cells with therapeutics that will improve epithelial regeneration after injury. Receptors that enable T cell targeting to the lung and lung specific delivery of therapeutics were developed.

[0144] RAGE (receptor for advanced glycation endproducts) as a promising candidate cell surface protein expressed specifically on the lung epithelium. Retroviral vectors that express single chain variable fragments (scFv) from anti-RAGE antibodies as the receptor for a chimeric antigen receptor (CAR) and a synthetic notch receptor (SynNotch). SynNotch allows for transcription of a genetically encoded “payload” only then the receptor is bound to its target. These constructs were used transduce mouse T cells and cocultured these T cells for 24 to 48 hours with either cell lines that express RAGE protein or cultured primary mouse AT2s. For in vivo experiments, 5 million T cells were injected, either expressing luciferase only or luciferase plus antiRAGE CAR into cytoxan treated C57 / B16 mice and measured whole body luminescence by IVIS. anti-RAGE CAR T cells can recognize and kill target cells expressing RAGE but do not kill RAGE negative target cells. anti-RAGE SynNotch expressing cells activate expression of a fluorescent reporter when cocultured with cultured primary mouse lung cells to a similar degree as when cultured with positive control myc beads that are known to activate SynNotch signal transduction. When we adoptively transferred anti-RAGE CAR and luciferase-GFP expressing human T cells into NSG immunodeficient mice, we detected luciferase only in the lung fields of the mice. GFP+ cells were detected by flow cytometry in the mouse lungs but not mouse spleens at 1 week post injection. Histology of the mouse lungs showed lymphocyte infiltration into the lungs, and damage to the lung epithelium, suggesting that the CAR T cells did kill lung epithelial cells.

[0145] RAGE is a surface marker that is relatively specific to the lung epithelium. CAR and SynNotch receptors that bind to RAGE can activate cell killing and transcription of a fluorescent reporter in vitro. An anti-RAGE CAR promotes trafficking of T cells to the lung fields in a mouse model. EXAMPLE 1

[0146] RATIONALE FOR RAGE AS A LUNG SPECIFIC TARGET AND DEVELOPMENT OF A RAGE TARGETING CAR AND SYNNOTCH RECEPTOR.

[0147] Materials and Methods: T cells from human donors were isolated using a magnetic bead based CD4 or CD8 isolation kit (StemCell Technologies). For CAR and SynNotch experiments, CD8+ T cells (CAR) or CD4+ T cells (SynNotch) were transduced with lentiviral vectors encoding anti-RAGE CAR, sorted 4 days after transduction, and cocultured with RAGE+ 3T3s, K562s, or mouse lung cells 7 days after sorting. 24 hours after coculture, cells were harvested for flow cytometry and stained with CD3-GFP and Draq7. CD3- cells in RAGE CAR experiment were 3T3s, and flow plots for Draq7 expression in CD3- cells are shown. CD3+ cells in RAGE SynNotch experiments were analyzed for BFP expression. Mouse lung cells were EPCAM+ MHC11+ sorted cells from C57 / B16 mice grown in 2D culture in DMEM + 10%FBS for 7 days.

[0148] Results: The gene encoding RAGE (AGER) is highly expressed in the human lung and in no other tissues (low expression in thyroid). See Figs. 1 A and IB. A CAR and SynNotch receptor able to recognize RAGE in vitro either in cell lines that overexpress RAGE or in mouse lung cells was generated. A nti-RAGE CAR T cells can kill RAGE expressing cells and when anti-RAGE SynNotch expressing cells are co-cultured with RAGE+ cells, they express their engineered transcriptional response element (BFP). See Figs. 1C-1E.

[0149] EXAMPLE 2

[0150] ANTI-RAGE CAR ENABLES T CELLS TRAFFICKING TO AND PROLIFERATION IN THE LUNG SPECIFICALLY.

[0151] Materials and Methods: T cells from human donors were isolated using a magnetic bead based CD4 or CD8 isolation kit (StemCell Technologies). CD8+ T cells and CD4+ T cells were transduced with lentiviral vectors encoding anti-RAGE CAR, luciferase, and mCherry. 4 days after transduction, they were sorted, and 7 days after sorting, 2e6 cells (le6 CD4 and le6 CD8) were injected into the tail vein of NSG mice. Live mice were injected with 3ug luciferin and live mice or dissected organs were analyzed on Xenogen IVIS 2000 10 minutes after intraperitoneal d-luciferin injection. For flow cytometry, mice were injected with IV CD45 prior to sacrifice and lungs were minced and digested with collagenase I, dispase, and DNAse. Cell counts were obtained using CountB right flow cytometry counting beads. Bronchoalveolar lavage was performed by instilling 1ml of PBS into mouse lungs 3 times, then centrifuging cells, resuspending in formalin, using cytospin to adhere cells to a slide, and analyzing percentage of lymphocytes, neutrophils, monocytes, and eosinophils after H&E staining.

[0152] Results: antiRAGE CAR-T cells traffick to the lung specifically and proliferate there over time when compared to a control CAR-T cell. The T cells migrate to the lung parenchyma and cause inflammation in the lung as evidenced by neutrophilia in BAL fluid. Ex vivo luminescence after luciferin injection shows no significant accumulation of T cells in any organ other than the lung, confirming that the anti-RAGE CAR allows for lung-specific T cell targeting. See Figs. 2A-2G.

[0153] EXAMPLE 3 ANTI-RAGE SYNNOTCH GATED CD19 CAR ENABLES CD19 EXPRESSING TUMOR CLEARANCE

[0154] SPECIFICALLY IN THE LUNG.

[0155] Materials and Methods: NIH1975 cells were transduced with human CD 19 and mCherry / f-lucif erase lentivirus and a pure population of hCD19+ mCH+ cells were sorted to purity and cultured. Ie6 hCD19+ mCH+ fLuc+ NIH1975 cells were injected into the right lungs and left flanks of NSG mice. CD4 and CD8 T cells were prepared as described above in 3 groups: Untransduced T cells, T cells transduced with antiRAGE SynNotch and human CD 19 CAR response element, and T cells transduced with the same hCD19 constitutively expressed CAR. 7 days after tumor injections, mice were intravenously injected (retro-orbital) with 6e6 total (3e6 CD4 and 3e6 CD8) T cells from each group. Mice were imaged with in vivo bioluminescence 10 minutes after d-luciferin injection as described above. The experiment was concluded after 31 days. At the endpoint, lungs were harvested and embedded in paraffin and flank tumors were harvested.

[0156] Results: When expression of a hCD19 CAR T cell is controlled by an anti-RAGE SynNotch receptor, hCD19+ tumor killing is restricted to the lung. Mice with CD 19+ tumors in both the lung and flank were treated with untransduced T cells “UnT”, antiRAGE SynNotch hCD19 CAR T cells (SN), or constitutive hCD19 CAR T cells (CAR). In the UnT group, neither lung nor flank tumors were controlled. In the CAR group, both lung and flank tumors were controlled. In the SN group, the lung tumors were controlled but flank tumors persisted, confirming that the anti-RAGE SynNotch enables lung-specific gene expression (in this case of the hCD19 CAR). See Figs. 3A-3F.

[0157] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Claims

CLAIMSWhat is claimed is:

1. An immune cell for the killing cancer cells in the lung, comprising:(a) a binding-triggered transcriptional switch (BTTS) that binds to RAGE (Receptor for Advanced Glycation Endproducts);(b) a nucleic acid sequence encoding an anti-cancer therapeutic; and(c) a regulatory sequence operably linked to (b) that is responsive to the binding- triggered transcriptional switch; wherein binding of the binding-triggered transcriptional switch to RAGE activates expression of the anti-cancer therapeutic.

2. The immune cell of claim 1, wherein the anti-cancer therapeutic, when expressed by the cell, is secreted by the cell or localized to the surface of the cell.

3. The immune cell of claim 1 or 2, wherein anti-cancer therapeutic is a pro-inflammatory cytokine.

4. The immune cell of claim 1 or 2, wherein anti-cancer therapeutic is an antibody.

5. The immune cell of claim 4, wherein the antibody is a multispecific antibody or (e.g., a bispecific or trispecific antibody that engages NK cells) or an immune checkpoint inhibitor.

6. The immune cell of claim 1, wherein the anti-cancer therapeutic is a recombinant immune receptor that is activated by binding to a killing antigen expressed by the cancer cells.

7. The immune cell of claim 5 or 6, wherein the recombinant immune receptor is a chimeric antigen receptor (CAR) or engineered T cell receptor (TCR).

8. The immune cell of any of claims 4-7, wherein the antibody or immune receptor binds to a killing antigen expressed by the cancer cells.

9. The immune cell of any of claims 1-5, wherein anti-cancer therapeutic is not a recombinant immune receptor and the cell further comprises a recombinant immune receptor that recognizes a killing antigen expressed by the cancer cells.

10. The immune cell of any of claims 4-9, wherein the antibody or immune receptor binds to a killing antigen for lung cancer selected from: CEA, CD276, EGFR, HER2, MSLN, MUC1, PD-L1, ROR1, TnMUCl, GPC3 or TGF , aPDl and MSLN, NY-ESO-1 or EGFRVIII, MAGE-A1, MAGE-A4, GD2„ PSCA, MUC1, GPC3, Lewis-Y, AXL, EGFR, and B7-H3.

11. The immune cell of claim 10, wherein the antibody or immune receptor binds to a killing antigen expressed by a non-lung cancer that has metastasized to the lung.

12. The immune cell of claim 11, wherein the non-lung cancer is sarcoma and the killing antigen is NY-ESO-1.

13. The immune cell of any prior claim, wherein the BTTS undergoes proteolytic cleavage upon binding to RAGE to release a gene expression regulator that induces expression of the anti-cancer therapeutic of (b) via the regulatory sequence of (c).

14. The immune cell of any prior claim, wherein the BTTS comprises:(i) an extracellular domain comprising the antigen binding region of a RAGE-specific antibody;(ii) a proteolytically cleavable sequence comprising one or more proteolytic cleavage sites; and(iii) an intracellular domain; wherein binding of the antigen binding region to RAGE induces cleavage of the sequence at the one or more proteolytic cleavage sites, thereby releasing the intracellular domain and wherein the intracellular domain induces expression of the anti-cancer therapeutic of (b) via the regulatory sequence of (c).

15. The immune cell of any prior claim, wherein the cell is cytotoxic T cell.

16. A method for killing cancer cells in the lung, comprising: administering to a subject an immune cell of any prior claim: wherein binding of the binding-triggered transcriptional switch to RAGE activates expression of the anti-cancer therapeutic and killing of the cancer cells.

17. The method of claim 16, wherein the subject has lung cancer.

18. The method of claim 17, wherein the lung cancer is non-small cell lung cancer (NSCLC) or small cell lung lancer (SCLC).

19. The method of any of claims 16-18, wherein the lung cancer is an adenocarcinoma, a squamous cell carcinoma or a large cell carcinoma.

20. The method of claim 16, wherein the subject has a non-lung cancer that has metastasized to the lung.

21. The method of claim 20, wherein the non-lung cancer is a sarcoma.

22. The method of any of claims 16-21, wherein the method is a method of treatment.

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