[001] Introduction

[002] Cell-based cancer immunotherapies require repurposing cytotoxic T cells through engineered T cell receptors (TCRs) or chimeric antigen receptors (CARs). However, a drawback to this approach is the inability to modulate the extent and duration of T cell activation, which can lead to toxic immune responses in one extreme, or T-cell exhaustion in the other. A solution to these problems would be to modulate the level of engineered TCR or CAR expression using a small molecule that either prevents or enhances protein synthesis of the receptor. We describe protein fusions to engineered TCRs and CARs that can allow them to be upregulated or downregulated in response to orally bioavailable small molecules. An alternative to using engineered T cells for cance iimmunotherapy is to use natural killer (NK) cells expressing CARs. We describe protein fusions to engineered CARs in NK cells that can allow them to be upregulated or downregulated in response to orally bioavailable small molecules.

[003] It would be beneficial to control the expression of engineered immune cell receptors for use in cell -based cancer immunotherapy, known as adoptive cell therapy (ACT). In these therapies, immune cells such as T cells or natural killer (NK) cells are genetically modified to express an engineered cell surface receptor that directs these immune cells to tumor cells expressing a target ligand recognized by the receptor, thereby leading to tumor cell destruction. These receptors include, among others, T cell receptors (TCRs) and chimeric antigen receptors (CARs) as reviewed in the literature (Souza-Fonseca-Guimaraes, 2019; Lee, 2019; Paucek, 2019). However, it has been found that ACT can suffer from severe toxic side effects including cytokine release syndrome (CRS), graft-versus-host disease (GvHD), and neurotoxicity, in some cases leading to death of the patient. These toxicities arise due to overactivation of engineered immune cells used in ACT such as CAR T-cells, due to signaling by the engineered cell surface receptor. Conversely, overactive immune cells can become exhausted and lose efficacy over time. It would therefore be useful to be able to tune the activity of immune cells engineered for ACT, by either increasing or decreasing the expression of the engineered immune cell surface receptor, i.e. the engineered TCR or CAR.

[004] Relevant patent publications by Pfizer include US9,227,956; US20140315928;

US20160058768 and US20160102074. [005] Summary of the Invention

[006] The invention provides systems, such as engineered cells, and methods for small molecule-based control of expression of human proteins like immune cell receptors.

[007] In an aspect the invention provides an isolated, engineered human cell, the cell comprising a gene encoding an engineered fusion protein comprising a functional human protein domain and a ribosome modulation signal peptide, wherein translation of the protein is regulatable by a predetermined small molecule targeting the signal peptide.

[008] In embodiments:

[009] - the cell is configured for adoptive cell transfer;

[010] - the ribosome modulation signal peptide is a ribosome stalling signal peptide of the human protein CDH1, PCSK9 or USOl;

[Oil] - the ribosome modulation signal peptide is a ribosome enhancement signal comprising the amino acid sequence alanine-threonine -histidine-phenylalanine (ATHF);

[012] - the functional domain is a transmembrane receptor and the signal peptide is incorporated into the N-terminal, transmembrane, or C-terminal region of the receptor;

[013] - the cell is an immune cell for immunotherapy, the protein is an immune cell receptor and the functional domain is a T-cell receptor (TCR) or chimeric antigen receptor (CAR) domain, or an enzyme or hormone;

[014] - the fusion protein further comprises a ribosome stalling signal protector domain; and/or

[015] - the engineered fusion protein also comprises a signal protector domain selected from human protein CDH1, USOl, IFI30, RAB3GAP1, or rat protein Pigw, Pqlc3, Pcyoxll, Slcla3, Naga, Lyar, Steapl, Msln, Tfrc, Prssl2, Msh2, Kif23, Haplnl, Parpl4, Coxl5, or Ndc80.

[016] The invention provides methods of making and using the subject engineered cells.

[017] In an aspect the invention provides a method of modulating translation of an engineered fusion protein with a predetermined small molecule, comprising: (a) providing an engineered human cell comprising a gene encoding an engineered fusion protein comprising a functional domain and a ribosome modulation signal peptide wherein translation of the protein is regulatable by a predetermined small molecule targeting the modulation signal peptide; and (b) contacting the cell with the small molecule under conditions wherein the small molecule targets the modulation signal peptide and thereby modulates translation of the protein.

[018] In embodiments:

[019] - the cell is in a host and the contacting step comprises administering the small molecule to the host; [020] - the cell is in a host, the contacting step comprises administering the small molecule to the host, and the method further comprises the step of determining that the host is in need of adoptive cell transfer; and/or

[021] - the method further comprises the subsequent step of detecting a resultant modulation of translation of the protein.

[022] The invention encompasses all combinations of the particular embodiments recited herein, as if each combination had been laboriously recited, such as wherein

[023] Brief Description of the Drawings

[024] Fig. la. Schematic representation of an engineered immune cell surface receptor.

[025] Fig. lb. Protein sequences from CDH1, PCSK9, and USOl useful for stalling.

[026] Fig. 2a Schematic representation of an engineered immune cell surface receptor.

[027] Fig. 2b. Sequences selected from mRNA libraries that can be used to inhibit engineered immune cell receptor translation.

[028] Fig. 2c. Sequence motif indicating the range of sequences that can be placed in the last 4 amino acids of the CDH1 sequence defined by the NC box.

[029] Fig. 3 a Schematic representation of an engineered immune cell surface receptor.

[030] Fig. 3b. Sequences selected from mRNA libraries that can be used to increase engineered immune cell receptor translation.

[031] Fig. 4. In vitro translation assays of the WT CDH1 stalling sequence, or with the ATHF or SRFD motif near the PTC, in the context of the CDH1 sequence. Reactions were carried out in triplicate, with standard deviation shown.

[032] Description of Particular Embodiments and Delivery Methods of the Invention

[033] Unless contraindicated or noted otherwise, in these descriptions and throughout this specification, the terms“a” and“an” mean one or more, the term“or” means and/or. The examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein, including citations therein, are hereby incorporated by reference in their entirety for all purposes.

[034] We disclose compositions and methods for selectively increasing or decreasing the protein synthesis of engineered immune cell surface receptors using orally bioavailable small molecules. Engineered TCRs and CARs are modular in nature, with an extracellular domain encoded at the N-terminus of the protein, followed by a transmembrane domain, and then followed C-terminally by signaling domains in the cytoplasm of the cell. See for example Figure 1 of (Lee, 2019). Modified receptors comprised of an additional domain encoded in either the N- terminal region (extracellular), as part of the transmembrane domain, or in the C-terminal region (intracellular) enable small-molecule mediated control of receptor expression. The modifications are further described herein, with mechanisms of action described in the cited references.

[035] We recently described orally- available small molecules that can inhibit protein production of specific polypeptides. See references: (Lintner, 2017; Londregan, 2018a;

Londregan, 2018b; Liaud, 2019). See also (Li, 2018).

[036] In an aspect this invention conceptually unites these small molecules with engineered immune cell receptors, incorporating polypeptide sequences, such as described in the above references, into the receptor, including the N-terminal, transmembrane, or C-terminal region of the receptor. Locations for inserting these sequences are informed by the length and composition of different polypeptide hinge and spacer regions described in, for example (Imai, 2005;

Almasbak, 2011; Almasbak, 2015; Oei, 2018). These polypeptide sequences can be encoded at the DNA level to enable genome engineering of T-cells or NK cells for ACT.

[037] Additional sequences can be incorporated into the engineered immune cell surface receptor to inhibit its protein synthesis by the action of one of the small molecules referenced above (Lintner, 2017; Londregan, 2018a; Londregan, 2018b; Liaud, 2019; Li, 2018) acting on the human ribosome. Alternatively, other sequences can be incorporated into the engineered immune cell surface receptor to increase its protein synthesis by the action of one of the small molecules referenced above acting on the human ribosome.

[038] In addition to the examples described in the references above, one of ordinary skill in the art can readily derive additional polypeptide sequences for related chemical species (See SMILES descriptions below, and Londgregan, 2018a; Londregan, 2018b) that can be incorporated into engineered TCRs or CARs. For example, one can use ribosome profiling to identify sequences in the human proteome, or in other eukaryotic proteomes, that elicit translation inhibition or translation increases in the presence of one of these compounds.

Methods for ribosome profiling used in such a way are included in the above references.

Additionally, one can use ribosome display of an mRNA library to identify protein sequences that either inhibit or increase translation of the polypeptide in the presence of one of these compounds; suitable methods are described, inter alia, in PCT/US 18/55262 and Li 2018 and Li 2019. The efficacy of these sequences can be verified using luciferase reporters as described in (Lintner, 2017; Li, 2018; Liaud, 2019).

[039] The use of engineered TCRs or CARs comprised of sequences sensitive to the small molecules referenced above, and with representative SMILES forms given below, involves genetically modifying T cells or NK cells with a DNA sequence encoding the engineered TCR or CAR. Many such methods have been described in the literature involving, for example, retroviral vectors (Imai, 2005; Liu, 2018), transposons (Kabriaei, 2016), and more recently the use of CRISPR-Cas9 (Roth, 2018). These methods are used to stably introduce DNA sequences into primary T cells, NK cells and induced pluripotent stem cells (iPSCs) that are subsequently differentiated into T cells (Nishimura, 2019) or NK cells (Bemareggi, 2019). These modified cells can then be expanded and used for ACT. Patients who receive these modified cells can then be treated with different doses of the compounds referenced above, and with representative SMILES forms given below, in order to decrease or increase the expression of the engineered immune cell surface receptors as needed for optimal therapeutic outcomes.

[040] Protein sequences sensitive to the compounds referenced above that can be inserted into engineered immune cell surface receptors include, but are not limited to, the following examples. These sequences require relevant cell surface receptor polypeptide sequences as referenced above located N-terminal and/or C-terminal to those shown. Additionally, one can add polypeptide linkers of various lengths between the compound-sensitive sequences and the N-terminal or C-terminal flanking sequences. These include, for example poly-(GS) sequences.

[041] Example 1. Sequences that can be used to inhibit engineered immune cell receptor translation. Fig. la is a schematic representation of the engineered immune cell surface receptor comprised of a PF-06446846 sensitive sequence. CDH1 sequence comprised of the CDH1-V domain followed by the small-molecule sensitive sequence. See Uniprot ID: P12830 and PCT/US 18/55262. Fig 1 b shows protein sequences from CDH1, PCSK9, and USOl useful for stalling (sequences in NC box). The arrow indicates the PF-06446846-dependent stalling site predicted from ribosome profiling data in (Lintner, 2017). On can add flexible polypeptide linkers of suitable length N-terminal and C-terminal to sequences defined by the NC box, and the CDH1-V box.

[042] Example 2. Additional chimeric polypeptides stalled by PF-06446846. Based on results in (Lintner, 2017), PF-06446846 is highly selective and stalls translation of just 18 polypeptides, of those that could be assayed using ribosome profiling (Figure 6 of Lintner,

2017). Of these 18, 3 have polypeptide sequences that would extend beyond the ribosome exit tunnel, which can enclose -35-40 amino acids at most due to its -100 A length (Wilson, 2011). In Example 1 we showed that CDH1, which in its cellular context stalls at amino acid 729, can be dissected to isolate a single domain that enables robust stalling of RNCs, with engineered N- terminal extensions and alternate stall sequences appended C-terminal to the Cadherin-5 (CDH1-V) domain. Such engineering can readily be applied to other sequences identified in Lintner, 2017. For example, the N-terminal sequence of USOl up to amino acid 298 can be used to stall RNCs, with additional sequences appended N-terminal and C-terminal to this segment of USOl. Alternatively, the N-terminal 92 amino acids of IFI30 can be used to form stalled RNCs, with additional sequences appended N-terminal and C-terminal to this segment of IFI30.

[043] The sequences of USOl or IFI30 needed to elicit sensitivity to PF-06446846 can be further minimized using structure prediction algorithms, for example Phyre2 (Kelley, 2015),. Furthermore, the USOl or IFI30 domain structure, coupled with information on other stalled sequences that reside solely in the exit tunnel (i.e. PCSK9, RPL27, MDK in Fig. 6 of Lintner, 2017), enable the design of other protein sequences that will be sensitive to PF-06446846. One can readily alter the linker length between the Cadherin-5 folding domain in CDH1, the N- terminal domain of USOl, or of IFI30 and any of the stalling sequences in Figure 6 of (Lintner, 2017) linked to the C-terminus. Also for any alternative chemical compound that induces stalling of ribosome nascent chains, i.e. compounds with the SMILES forms listed below, one can use ribosome profiling to identify the full spectrum of protein sequences stalled by the compound, as described in (Lintner, 2017). These alternative stalling sequences can similarly be appended to the Cadherin-5 domain of CDH1, or the folding domains of USOl or IFI30, analogously to the results in Example 1. Ribosome profiling with these alternative compounds can be used to identify additional proteins that extend beyond the ribosome exit tunnel, i.e. whose stall site resides ~40 or more amino acids from the N-terminus, and these can then be used to make engineered immune cell surface receptors whose translation is sensitive to the presence of the new compound.

[044] Example 3. Sequences selected from mRNA libraries that can be used to inhibit engineered immune cell receptor translation. mRNA libraries encoding randomized codons can be used to select for protein sequences that can be inhibited by compounds like PF- 06446846; see, PCT/US 18/55262 and Li, 2018. Fig. 2a shows the sequence context, and examples selected in the context of the CDH1 stalling peptide are shown in Fig. 2b - sequences in location of the NC box. Flexible polypeptide linkers of suitable length N-terminal and C- terminal can be added to sequences defined by the NC box and the CDH1-V domain. Fig. 2c is a sequence motif indicating the range of sequences that can be placed in the last 4 amino acids of the CDH1 sequence defined by the NC box.

[045] Example 4. Sequences selected from mRNA libraries that can be used to increase engineered immune cell receptor translation. mRNA libraries encoding randomized codons can be used to select for protein sequences that can increase translation in the presence of compounds like PF-06446846; see, PCT/US 18/55262 and Li, 2018.. Fig. 3a shows the sequence context of PF-06446846 sensitive sequences. Fig. 3b shows example sequences that can be used to increase translation due to the action of PF-06446846, when located in the position of the NC sequence in Fig. 3a. Flexible polypeptide linkers of suitable length N-terminal and C-terminal can be added to sequences defined by the NC box and the CDH1-V domain.

[046] Example 5. Sequences sensitive to PF -06446846 and/or compound 71 in rat intestinal cells (Londregan et aL, 2018a). Compound 71 is a next- generation selective translation inhibitor related to PF-06446846 and is described in (Londregan, 2018a). These sequences are readily screened with differing polypeptide start and end points for sensitivity to the compounds with SMILES codes provided below, using luciferase reporters as described in (Lintner, 2017; Li, 2018; Liaud, 2019). Suitable sequences with the main stall position induced by compounds 7f (PF-06446846) and/or 71 located within 40 amino acids (i.e. codons in the table) from the N-terminus can include amino acids 1 to the Dmax position (i.e. amino acids 1- 41 of Car9, amino acids 1-28 of Rpl27, or amino acids 1-40 of Cyp2sl, etc.). As shown in Example 1 and Example 2, sequences whose main stall position occurs in a more C-terminal position, i.e. after the first 40 amino acids, can also be used. The N-terminal boundary of the sensitive sequence may be determined using structure prediction algorithms such as Phyre2 (Kelley, 2015). The Dmax position is a suitable location for the C-terminus of the compound- sensitive sequence. Sequences in this category may be comprised of polypeptides derived from amino acids 1-361 of Pigw, amino acids 1-119 of Pqlc3, etc.

[047]

LOC102554034 ENSRN OTOOOOOO 14916 27 152

Pttglip ENSRNOTOOOOOOO 1638 27 42

Steapl ENSRN OTOOOOOOOOO 18 207 208

Msln ENSRNOT00000026395 449 451

Dadl ENSRN OTOOOOOO 12233 49 49

Sema7a ENSRNOTOOOOOO 10620 ^a Undet. 108

Tusc3 ENSRNOT00000085300 37 50

Pcbp3 ENSRN OTOOOOOOO 1675 31 45

Arfl ENSRN OTOOOOOO 80028 25 27

Efemp2 ENSRNOT00000080520 37 39

Spdll ENSRNOT00000009648 41 44

Pari ENSRN OT00000071076 76 75

Tfrc ENSRN OT00000002407 157 160

Plekhh3 ENSRN OT00000027452 38 46

Prssl2 ENSRN OT00000021116 72 125

Msh2 ENSRN OT00000021538 428 457

Sumfl ENSRN OT00000009008 31 43

Kif23 ENSRNOT00000037028 317 320

Haplnl ENSRN OT00000042958 142 144

Acadm ENSRN OTOOOOOO 13238 23 91

Parpl4 ENSRN OT00000046888 598 598

Torlb ENSRNOTOOOOOO66618 103 105

Ddrgkl ENSRNOT00000030192 59 68

Brwdl ENSRN OT00000002231 37 277

Haxl ENSRNOT00000076325 29 40

Rbm8a ENSRNOT00000028807 90 90

Nubp2 ENSRNOT00000020504 38 40

Igf2r ENSRNOT00000021840 24 ^b N/A

Slc9a8 ENSRNOTOOOOOO 12175 415 423

Atplb3 ENSRN OTOOOOOO 15476 19 110

Emc7 ENSRNOT00000008364 16 ^b N/A

Fbxo31 ENSRNOT00000079651 33 141

Ash21 ENSRNOT00000086891 203 213

Usol ENSRN OT00000003277 300 367

Dpp7 ENSRNOTOOOOOO 17271 42 42

[048] ^a Undetermined. Insufficient read depth to determine the stall position. ^b Dmax Z-score <

2.0; stalls were determined by examination of readmapsA

[049] SMILES codes for compounds useful for selective stalling or enhancement of translation. Codes taken from (Londregan et al , 2018).

[050] Example 6. Amino acid changes in stalled sequences can actually enhance rather that repress translation in a PF846-dependent manner. In the context of the CDH1 NC, sequences SFRD and ATHF increase overall translation in a PF846-dependent manner (Fig. 4), with ATHF increasing translation ~4-fold.

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