CA3055200A1 - Compositions and methods for immunotherapy - Google Patents

Abstract

The present invention provides biocircuit systems, effector modules and compositions for cancer immunotherapy. Methods for inducing anti-cancer immune responses in a subject are also provided.

Description

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COMPOSITIONS AND METHODS FOR IMMUNOTHERAPY
CROSS REFERENCE TO RELATED APPLICATIONS
100011 This application claims priority to the US Provisional Patent Application No.
62/466,601, filed on March 3, 2017 entitled Compositions and Methods for Immunotherapy, US
Provisional Patent Application No. 62/484,063, filed on April 11, 2017 entitled Compositions and Methods for immunotherapy, and US Provisional Patent Application 62/542,402, filed on August 8, 2017 entitled Compositions and Methods for Immunotherapy, the contents of each of which are herein incorporated by reference in their entirety.
SEQUENCE LISTING
100021 The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 2095_1207PCT_SLixt, created on March 2, 2018, which is 1,566,189 bytes in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
100031 The present invention relates to compositions and methods for immunotherapy.
Provided in the present invention include polypeptides of biocircuit systems, effector modules, stimulus response elements (SREs) and immtmotherapeutic agents, polynucleotides encoding the same, vectors and cells containing the polypeptides and/or polynucleotides for use in cancer immunotherapy. In one embodiment, the compositions comprise destabilizing domains (DDs) which tune protein stability.
BACKGROUND OF THE INVENTION
100041 Cancer immunotherapy aims to eradicate cancer cells by rejuvenating the tumoricidal functions of tumor-reactive immune cells, predominantly T cells. Strategies of cancer immunotherapy including the recent development of checkpoint blockade, adoptive cell transfer (ACT) and cancer vaccines which can increase the anti-tumor immune effector cells have produced remarkable results in several tumors.
100051 The impact of host anti-tumor immunity and cancer inununotherapy is impeded by three major hurdles: 1) low number of tumor antigen-specific T cells due to clonal deletion; 2) poor activation of innate immune cells and accumulation of tolerogenic antigen-presenting cells in the tumor microenvironment; and 3) formation of an immunosuppressive tumor microenvironment. Particularly, in solid tumors the therapeutic efficacy of immunotherapeutic regimens remains unsatisfactory due to lack of an effective an anti-tumor response in the immunosuppressive tumor microenvironment. Tumor cells often induce immune tolerance or suppression and such tolerance is acquired because even truly foreign tumor antigens will become tolerated. Such tolerance is also active and dominant because cancer vaccines and adoptive transfer of pre-activated immune effector cells (e.g., T cells), are subject to suppression by inhibitory factors in the tumor microenvironment (TME).
100061 In addition, administration of engineered T cells could result in on/off target toxicities as well as a cytokine release syndrome (reviewed by Tey Clin. Transl.
lmmunol., 2014, 3: e 17 10.1038).
100071 Development of a tunable switch that can turn on or off the transgenic immunotherapeutic agent expression is needed in case of adverse events. For example, adoptive cell therapies may have a very long and an indefinite half-life. Since toxicity can be progressive, a safety switch is desired to eliminate the infused cells. Systems and methods that can tune the transgenic protein level and expression window with high flexibility can enhance therapeutic benefit, and reduce potential side effects.
100081 To develop regulatable therapeutic agents for disease therapy, in particular cancer immtmotherapy, the present invention provides biocircuit systems to control the expression of immunotherapeutic agents. The biocircuit system comprises a stimulus and at least one effector module that responds to the stimulus. The effector module may include a stimulus response element (SRE) that binds and is responsive to a stimulus and an inununotherapeutic agent operably linked to the SRE. In one example, a SRE is a destabilizing domain (DD) which is destabilized in the absence of its specific ligand and can be stabilized by binding to its specific ligand.
SUMMARY OF THE INVENTION
100091 The present invention provides compositions and methods for immunotherapy. The compositions relate to tunable systems and agents that induce anti-cancer immune responses in a cell or in a subject. The tunable system and agent may be a biocircuit system comprising at least one effector module that is responsive to at least one stimulus. The biocircuit system may be, but is not limited to, a destabilizing domain (DD) biocircuit system, a dimerization biocircuit system, a receptor biocircuit system, and a cell biocircuit system. These systems are further taught in co-owned U.S. Provisional Patent Application No. 62/320,864 filed April 11, 2016, 62/466,596 filed March 3, 2017 and the International Publication W02017/180587 (the contents each of which are herein incorporated by reference in their entirety).
100101 In some embodiments, the composition for inducing an immune response may comprise a first effector module. In some embodiments, the effector module may comprise a first

2

3 stimulus response element (SRE) operably linked to at least one payload. In one aspect, the payload may be an immunotherapeutic agent.
100111 In some embodiments, the immunotherapeutic agent may be selected from, but is not limited to a cytokine, a safety switch, a regulatory switch, a chimeric antigen receptor and combinations thereof 1001.21 In one aspect, the first SRE of the composition may be responsive to or interact with at least one stimulus.
100131 In some embodiments, the first SRE may comprise a destabilizing domain (DD). The DD may be derived from a parent protein or from a mutant protein having one, two, there, or more amino acid mutations compared to the parent protein. In some embodiments, the parent protein may be selected from, but is not limited to, human protein FKBP, comprising the amino acid sequence of SEQ. ID NO. 3; Inunan DHFR (hDHFR), comprising the amino acid sequence of SEQ. ID NO. 2; E. Coli DHFR, comprising the amino acid sequence of SEQ. ID
NO. 1;
PDE5, comprising the amino acid sequence of SEQ. ID NO. 4; PPAR, gamma comprising the amino acid sequence of SEQ. ID NO. 5; CA2, comprising the amino acid sequence of SEQ. ID
NO. 6; or NQ02, comprising the amino acid sequence of SEQ. ID NO. 7.
100141 In one aspect, the parent protein is hDHFR and the DD comprises a mutant protein having at least one mutation selected from Mldel,V2A, C7R, I8V, V9A, A 1 OT, AlOV, Q13R, N14S, G16S, 117N, 117V, K19E, N20D, G21T, G21E, D225, L235, P245, L28P, .N30D, N30H, N30S, E31G, E31D, F32M, R33G, R335, F35L, Q36R, Q365, Q36K, Q36F, R37G, M38V, M38T, T40A, V44A, K47R, N495, N49D, M53T, G54R, K56E, K56R, T57A, F595, I61.T, K64R, N65A, N655, N65D, N65F, L68S, K69E, K69R, R71G, I72T, I72A, I72V, N73G, L74N, V75F, R78G, L80P, K81R, E82G, H88Y, F89L, R92G, 593G, 593R, L94A, D96G, A97T, L985, K996, K99R, LlOOP, E1.02G, Q103R, P104S, E105G, A107T, A1.07V, NIO8D, K109E, K109R, V110A, DII1N, M112T, M112V, V113A, W114R, 1115V, V1161, G117D, V121A, Y122C, Y122D, Y1221, K123R, K123E, A125F, M1261, N127R, N1275, N127Y, H128R, H128Y, H131R, L132P, K133E, L134P, FI35P, F135L, F135S, F135V, V136M, T137R, R.138G, R1381, I139T, I139V, M1401, M140V, Q141.R, D142G, F143S, F143L, E144G, D146G, T147A, F1485, F148L, F149L, P150L, E151G, I152V, D153A, D153G, E155G, K156R, Y157R, Y157C, K158E, K158R, L15913, L160P, E162G, Y163C, V166A, S168C, D169G, VI70A, Q171R, E172G, E173G, E173A, K174R, I176A, I176F, I176T, K177E, K177R, Y178C, Y178H, F180L, E181G, V182A,Y183C, Y183H, E184R, E184G, K185R, K185del, K185E, N186S, N186D, D187G, and D187N.
100151 In one aspect, the stimulus of the SRE may be Trimethoprim or Methotrexate.

100161 In some embodiments, the immunotherapeutic agent may be a cytokine. In one aspect, the cytokine may be an interleukin, an interferon, a tumor necrosis factor, a transforming growth factor B, a CC chemokine, a CXC chemokine, a CX3C chemokine or a growth factor. In some embodiments, the cytokine is an interleukin. In some embodiments, the interleukin is selected from a group consisting of ILI, ILI-alpha, IL1-beta, IL1-delta, ILI-epsilon, IL! -eta, IL 1-zeta, IL-RA, 1L2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, ILI , ILIOC, ILIOD, ILI la, ILI
lb. IL13, IL14, IL16, IL17, IL-17A, IL17B, IL17C, IL17E, IL17F, IL18, IL19, IL20, IL2OL, IL21, IL22, IL23, IL23A, IL24, IL25, IL26, IL27, IL28, IL29, 1L30, IL31, IL32, IL33, IL34, IL36a, IL360, IL36y, IL36RN, IL37, IL37a, IL37b, IL37c, I137d, IL37e, and IL38.
100171 In one aspect, the interleukin may be IL2, comprising the amino acid sequence of SEQ
ID NO. 51.
100181 In one aspect, the immunotherapeutic agent may be a safety switch. In some embodiments, the safety switch may be selected from a Caspase 9, an inducible FAS (iFAS), an inducible caspase 9 (icasp9), a CD20/anti-CD20 antibody pair, a protein tag/anti-tag antibody, and a compact suicide gene (RQR8). In one aspect, the safety switch may be Caspase 9 comprising the amino acid sequence of SEQ ID NO. 65.
100191 in one aspect, the immunotherapeutic agent may encode a regulatory switch. In some embodiments, the regulatory switch may be selected from a FOXP3, a Nr4a, a FOXO, and a NF-KB. In one aspect, the regulatory switch may be a FOXP3, comprising the amino acid sequence of SEQ ID NO. 103-106.
100201 In one aspect, the immunotherapeutic agent may be a chimeric antigen receptor (CAR).
In some embodiments, the CAR may be selected from a GD2 CAR, a Her2 CAR, a BCMA
CAR, a CD33 CAR, an ALK CAR, a CD22 CAR, and a CD276 CAR. The CARS described herein may comprise an extracellular moiety, a transmembrane domain, an intracellular signaling domain, and optionally, one or more co-stimulatory domains.
100211 In one aspect, the CAR may be selected from, but is not limited, to a standard CAR, a split CAR, an off-switch CAR, an on-switch CAR, a first-generation CAR, a second-generation CAR, a third-generation CAR, or a fourth-generation CAR.
100221 In some embodiments, the extracellular target moiety of the CAR may be selected from, but is not limited to an Ig NAR, a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a F(ab)'3 fragment, an Fv, a single chain variable fragment (scFv), a bis-scFv, a (scFv)2, a minibody, a diabody, a triabody, a tetrabody, an intrabody, a disulfide stabilized FV protein (dsFv), a unibody, a nanobody, and an antigen binding region derived from an antibody that may

4 specifically bind to any of a protein of interest, a ligand, a receptor, a receptor fragment or a peptide aptamer.
100231 In some embodiments, the extracellular target moiety may be selected from an ALK
target moiety, comprising the amino acid sequence of SEQ ID NO. 242- 257 and 422-429, a CD22 target moiety, comprising the amino acid sequence of SEQ ID NO.258-262 and 430-432, a CD276 target moiety, comprising the amino acid sequence of SEQ TD NO. 263-270 and 433-436, a GD2 target moiety, comprising the amino acid sequence of SEQ ID NO.271-349 and 437-465, a CD33 target moiety, comprising the amino acid sequence of SEQ ID NO.
350-357, a BCMA target moiety, comprising the amino acid sequence of SEQ ID NO. 358-365, and a Her2 target moiety, comprising the amino acid sequence of SEQ ID NO. 366-421 and 466-473.
[0024] In some embodiments, the intracellular signaling domain of the CAR may be derived from T cell receptor CD3zeta or a cell surface molecule selected from the group consisting of FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d.
100251 In some embodiments, the CAR may comprise a co-stimulatory domain. The costimulatory, domain may be selected from the group consisting of 2B4, HVEM, ICOS, LAG3, DAPIO, DAP12, CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, ICOS
(CD278), glucocorticoid-induced tumor necrosis factor receptor (GITR), lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.
[0026] In one embodiment, the transmembrane domain of the CAR may be derived from a transmembrane domain. In one aspect, the transmembrane domain may comprise the amino acid sequence selected from, but not limited to SEQ ID NO. 527-624.
[0027] In some embodiments, the CAR of the effector module may further comprise a hinge region near the transmembrane domain. In one aspect, the hinge region may comprise an amino acid sequence selected from the group consisting of any of SEQ ID NOs. 628-694.
[0028] In one aspect, the first effector module may comprise an 1L2-DD, comprising the amino acid sequence of any of SEQ ID NOs. 52-54.
[0029] In one aspect, the first effector module may comprise a Caspase 9-DD, comprising the amino acid sequence of any of SEQ ID NOs. 72-80.
[0030] In one aspect, the first effector module may comprise a FOXP3-DD, comprising the amino acid sequence of any of SEQ ID NOs. 107-116.
[0031] In one aspect, the first effector module may comprise a BCMA CAR-DD, comprising the amino acid sequence of any of SEQ ID NOs. 775-777.

100321 In one aspect, the first effector module may comprise a HER2-DD, comprising the amino acid sequence of any of SEQ ID NO. 906.
100331 The present invention, also provides polynucleotides encoding the compositions of the invention.
100341 In one aspect, the polynucleotides may be a DNA or RNA molecule. In one aspect, the polynucleotides may comprise spatiotemporally selected codons. In some embodiments, the polynucleotides may be an RNA molecule. In one aspect, the RNA molecule may be a messenger molecule. In some embodiments, the RNA molecule may be chemically modified. In some embodiments, the polynucleotides may comprise spatiotemporally selected codons.
100351 In some embodiments, the polynucleotides may further comprise, at least one additional feature selected from, but not limited to, a promoter, a linker, a signal peptide, a tag, a cleavage site and a targeting peptide.
100361 The present invention also provides vectors comprising polynucleotides described herein. In one aspect, the vector may be a viral vector. In some embodiments, the viral vector may be a retroviral vector, a lentiviral vector, a gamma retroviral vector, a recombinant AAV
vector, an adeno viral vector, and an oncolytic viral vector.
100371 The present invention also provides immune cells for adoptive cell transfer (ACT) which may express the compositions of the invention, the polynucleotides described herein. In one aspect, the immune cells may be infected or transfected with the vectors described herein.
The immune cells for ACT may be selected from, but not limited to a CD8+ T
cell, a CD4+ T
cell, a helper T cell, a natural killer (NK) cell, a NKT cell, a cytotoxic T
lymphocyte (m), a tumor infiltrating lymphocyte (TIL), a memory T cell, a regulatory T (Treg) cell, a cytokine-induced killer (CIK) cell, a dendritic cell, a human embryonic stem cell, a mesenchymal stem cell, a hematopoietic stem cell, or a mixture thereof.
100381 In one aspect, the immune cell may comprise a destabilizing domain DD, wherein the DD is derived from human protein FKBP comprising the amino acid sequence of SEQ ID NO. 3, DHFR comprising the amino acid sequence of SEQ ID NO. 1-2, PDE5 comprising the amino acid sequence of SEQ ID NO. 4, PPAR gamma comprising the amino acid sequence of SEQ TD
NO. 5, CA2 comprising the amino acid sequence of SEQ ID NO. 6 and NQ02 comprising the amino acid sequence of SEQ ID NO. 7.
100391 In one aspect, the DD may be derived from a parent protein and the parent protein is hDHFR and the DD comprises a mutant protein having at least one mutation selected from Mldel, V2A, C7R, I8V, V9A, AlOT, AlOV, Q13R, N14S, G16S, I17N, 117V, K19E, N20D, G21T, G21E, D225, L235, P24S, L28P, N30D, N3OH, N30S, E31G, E31D, F32M, R33G, R33S, F35L, Q36R, Q36S, Q36K, Q36F, R37G, M38V, M38T, T40A, V44A, K47R, N49S, N49D, M53T, G54R, K56E, K56R, T57A, F59S, 16 IT, K64R, N65A, N65S, N65D, N65F, L68S, K69E, K69R, R71G, 172T, 172A, I72V, N73G, L74N, V75F, R786, L80P, K81.R, E82G, H88Y, F89L, R926, S93G, S93R, L94A, D96G, A97T, L98S, K99G, K99R, L100P, E102G, Q103R, P104S, E105G, A107T, A107V, NI08D, K109E, K109R, V110A, Di I IN, M112T, M112V, V1. 13A, W1. 14R, 111.5V, V1161, G117D, V1.21A, Y122C, Y1.22D, Y1221, K123R, K123E, A125F, M1261, N127R, N127S, N127Y, H128R, H128Y, H131R, L132P, K133E, L134P, F135P, F135L, F135S, F135V, V136M, T137R, R138G, R1381, I139T, 1139V, M1401, M140V, Q141R, DI42G, F143S, F143L, E144G, D146G, T147A, F148S, F148L, F149L, P1.50L, E151G, I152V, D153A, D153G, E1556, K1.56R, Y157R, Y157C, K158E, K158R, L159P, L160P, E162G,Y163C, V166A, S168C, D169G, V170A, Q171R, E172G, E1736, E173A, K174R, 1176A, 1176F, 1176T, K177E, K177R, Y178C, Y178H, F180L, E181G, V182A, Y183C, Y I83H, E184R, E184G, K185R, K185del, K185E, N186S, N186D, D187G, and D187N.
100401 In some embodiments, the immune cells may be autologous, allogeneic, syngeneic, or xenogeneic in relation to a particular individual subject.
100411 The present invention provides methods for reducing a tumor volume or burden in a subject comprising contacting the subject with the immune cells of the invention. Also provided herein, is a method for inducing an anti-tumor immune response in a subject, comprising administering the immune cells of the system to the subject.
[00421 Also provided herein, is a method for inducing an immune response in a subject, administering the compositions of the invention, the polynucleotides of the invention, and/or the immune cells of the invention to the subject.
100431 The present invention also provides methods for preventing or reversing T cell exhaustion in a subject in need thereof. Such methods may comprise administering to the subject, a therapeutically effective amount of compositions described herein, the polynucleotides of the invention, the vectors of the invention, or the immune cells described herein. Such methods may comprise an SRE that responds to a stimulus and tunes the expression and/or function of the inununotherapeutic agent, thereby preventing or reversing T
cell exhaustion.
100441 In some aspects, the inununotherapeutic agent is a chimeric antigen receptor. In some embodiments, the chimeric antigen receptor may be a GD2 CAR, a BCMA CAR, a CD33 CAR, a Her2 CAR, an ALK CAR, a CD22 CAR, or a CD276 CAR.
100451 Also provided herein, is a method for detecting cancer in a mammal, comprising the steps of (a) contacting a sample comprising one or more cells from the mammal with the compositions, the polynucleotides, the vector or the immune cells of the invention, and (b) detecting the complex, wherein the detection of the complex may be indicative of the presence of cancer in the mammal.
[0046] In some embodiments, the effector module comprises a stimulus response element (SRE) and at least one payload comprising a protein of interest (P01).
[0047] In some embodiments, the SRE may be a destabilizing domain (DD). In some examples, the DD is a mutant domain derived from a protein such as FKBP (FK506 binding protein), E. coli DHFR (Dihydrofolate reductase) (ecDHFR), human DHFR (hDHFR), or any protein of interest. In this context, the biocircuit system is a DD biocircuit system.
[0048] The payload may be any immunotherapeutic agent used for cancer immunotherapy such as a cytokine such as IL2, a safety switch such as Caspase 9, a regulatory switch encoding FOXP3, a chimeric antigen receptor such as BCMA CAR, CD33 CAR, GD2 CAR, Her2 CAR, ALK CAR, CD22 CAR, CD276 CAR or any agent that can induce an immune response.
The SRE and payload may be operably linked through one or more linkers and the positions of components may vary within the effector module.
[0049] In some embodiments, the effector module may further comprise of one or more additional features such as linker sequences (with specific sequences and lengths), cleavage sites, regulatory elements (that regulate expression of the protein of interest such as microRNA
targeting sites), signal sequences that lead the effector module to a specific cellular or subcellular location, penetrating sequences, or tags and biomarkers for tracking the effector module.
[0050] The invention provides isolated biocircuit polypeptides, effector modules, stimulus response elements (SREs) and payloads, as well as polynucleotides encoding any of the foregoing; vectors comprising polynucleotides of the invention; and cells expressing polypeptides, polynucleotides and vectors of the invention. The polypeptides, polynucleotides, viral vectors and cells are useful for inducing anti-tumor immune responses in a subject.
[0051] In some embodiments, the vector of the invention is a viral vector. The viral vector may include, but is not limited to a retroviral vector, an adenoviral vector, an adeno-associated viral vector, or a lentiviral vector.
[0052] In some embodiments, the vector of the invention may be a non-viral vector, such as a nanoparticles and liposomes.
[0053] The present invention also provides immune cells engineered to include one or more polypeptides, polynucleotides, or vectors of the present invention. The cells may be immune effector cells, including T cells such as cytotoxic T cells, helper T cells, memory T cells, regulatory T cells, natural killer (NK) cells, NK T cells, cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTLs), and tumor infiltrating lymphocytes (TILs). The engineered cell may be used for adoptive cell transfer for treating a disease (e.g., a cancer).
100541 The present invention also provides methods for inducing immune responses in a subject using the compositions of the invention. Also provided are methods for reducing a tumor burden in a subject using the compositions of the invention and methods for preventing or reversing T cell exhaustion.
BRIEF DESCRIPTION OF THE DRAWINGS
100551 Figure 1 shows an overview diagram of a biocircuit system of the invention. The biocircuit comprises a stimulus and at least one effector module responsive to a stimulus, where the response to the stimulus produces a signal or outcome. The effector module comprises at least one stimulus response element (SRE) and one payload.
100561 Figure 2 shows representative effector modules carrying one payload.
The signal sequence (SS), SRE and payload may be located or positioned in various arrangements without (A to F) or with (G to Z, and AA to DD) a cleavage site. An optional linker may be inserted between each component of the effector module.
100571 Figure 3 shows representative effector modules canying two payloads without a cleavage site. The two payloads may be either directly linked to each other or separated.
100581 Figure 4 shows representative effector modules carrying two payloads with a cleavage site. In one embodiment, an SS is positioned at the N-terminus of the construct, while other components: SRE, two payloads and the cleavage site may be located at different positions (A to L). In another embodiment, the cleavage site is positioned at the N-teminus of the construct (M
to X). An optional linker may be inserted between each component of the effector module.
100591 Figure 5 shows effector modules of the invention carrying two payloads, where an SRE
is positioned at the N-terminus of the construct (A to L), while SS, two payloads and the cleavage site can be in any configuration. An optional linker may be inserted between each component of the effector module.
100601 Figure 6 shows effector modules of the invention carrying two payloads, where either the two payloads (A to F) or one of the two payloads (G to X) is positioned at the N-terminus of the construct (A to L), while SS, SRE and the cleavage site can be in any configuration. An optional linker may be inserted between each component of the effector module.
100611 Figure 7 depicts representative configurations of the stimulus and effector module within a biocircuit system. A trans-membrane effector module is activated either by a free stimulus (Figure 7A) or a membrane bound stimulus (Figure 7B) which binds to SRE. The response to the stimulus causes the cleavage of the intracellular signal/payload, which activates down-stream effector/payload.
[0062] Figure 8 depicts a dual stimulus-dual presenter biocircuit system, where two bound stimuli (A and B) from two different presenters (e.g., different cells) bind to two different effector modules in a single receiver (e.g., another single cell) simultaneously and create a dual-signal to downstream payloads.
[0063] Figure 9 depicts a dual stimulus-single presenter biocircuit system, where two bound stimuli (A and B) from the same presenter (e.g., a single cell) bind to two different effector modules in another single cell simultaneously and create a dual-signal.
100641 Figure 10 depicts a single-stimulus-bridged receiver biocircuit system.
In this configuration, a bound stimulus (A) binds to an effector module in the bridge cell and creates a signal to activate a payload which is a stimulus (B) for another effector module in the final receiver (e.g., another cell).
[0065] Figure 11 depicts a single stimulus-single receiver biocircuit system, wherein the single receiver contains the two effector modules which are sequentially activated by a single stimulus.
[0066] Figure 12 depicts a biocircuit system which requires a dual activation.
In this embodiment, one stimulus must bind the transmembrane effector module first to prime the receiver cell being activated by the other stimulus. The receiver only activates when it senses both stimuli (B).
[0067] Figure 13 is a line graph depicting the effect of Shield-1 on DD-IL2 levels.
[0068] Figure 14 denotes the frequency of IFNgarnma positive T cells.
[0069i Figure 15A depicts IFN gamma production in T cells. Figure 15B depicts T cell expansion with IL15/1L15Ra treatment. Figure 15C is a dot plot depicting percentage human cells after in vivo cell transfer. Figure 15D is scatter plot depicting CD4+/CD8+ T cells.
[0070] Figure 16A is a western blot depicting luciferase levels in DD-luciferase expressing cells. Figure 16B depicts luciferase activity.
[0071] Figure 17A and Figure 17B are western blot depicting DD regulated expression of FOXP3.
[0072] Figure 18 is a bar graph representing the effect of promoters on transgene expression.
[0073] Figure 19 represents Shield-1 regulation of DD-IL2 secretion from HCT116 cells in vivo.
[0074] Figure 20 depicts the viability of T cells cultured with different ratios of CD3/CD8 beads.
[0075] Figure 21 represents the percentage BCMA CAR positive T cells with ligand treatment.

DETAILED DESCRIPTION OF THE INVENTION
[0076] The details of one or more embodiments of the invention are set forth in the accompanying description below. Although any materials and methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred materials and methods are now described. Other features, objects and advantages of the invention will be apparent from the description. In the description, the singular forms also include the plural unless the context clearly dictates otherwise. 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. In the case of conflict, the present description will control.
I. INTRODUCTION
100771 Cancer immunotherapy aims' at the induction or restoration of the reactivity of the immune system towards cancer. Significant advances in immunotherapy research have led to the development of various strategies which may broadly be classified into active immunotherapy and passive immunotherapy. In general, these strategies may be utilized to directly kill cancer cells or to counter the immunosuppressive tumor microenvironment. Active immunotherapy aims at induction of an endogenous, long-lasting tumor-antigen specific immune response. The response can further be enhanced by non-specific stimulation of immune response modifiers such as cytokines. In contrast, passive immunotherapy includes approaches where immune effector molecules such as tumor-antigen specific cytotoxic T cells or antibodies are administered to the host. This approach is short lived and requires multiple applications.
100781 Despite significant advances, the efficacy of current immunotherapy strategies is limited by associated toxicities. These are often related to the narrow therapeutic window associated with immunotherapy, which in part, emerges from the need to push therapy dose to the edge of potentially fatal toxicity to get a clinically meaningful treatment effect. Further, dose expands in vivo since adoptively transferred immune cells continue to proliferate within the patient, often unpredictably.
[0079] A major risk involved in immunotherapy is the on-target but off tumor side effects resulting from T-cell activation in response to normal tissue expression of the tumor associated antigen (TAA). Clinical trials utilizing T cells expressing T-cell receptor against specific TAA
reported skin rash, colitis and hearing loss in response to immunotherapy.
[0080] Immunotherapy may also produce on target, on-tumor toxicities that emerge when tumor cells are killed in response to the immunotherapy. The adverse effects include tumor lysis syndrome, cytokine release syndrome and the related macrophage activation syndrome.

Importantly, these adverse effects may occur during the destruction of tumors, and thus even a successful on-tumor immunotherapy might result in toxicity. Approaches to regulatably control immunotherapy are thus highly desirable since they have the potential to reduce toxicity and maximize efficacy.
100811 The present invention provides systems, compositions, immunotherapeutic agents and methods for cancer immunotherapy. These compositions provide tunable regulation of gene expression and function in immunotherapy. The present invention also provides biocircuit systems, effector modules, stimulus response elements (SREs) and payloads, as well as polynucleotides encoding any of the foregoing. In one aspect, the systems, compositions, immunotherapeutic agents and other components of the invention can be controlled by a separately added stimulus, which provides a significant flexibility to regulate cancer immunotherapy. Further, the systems, compositions and the methods of the present invention may also be combined with therapeutic agents such as chemotherapeutic agents, small molecules, gene therapy, and antibodies.
100821 The tunable nature of the systems and compositions of the invention has the potential to improve the potency and duration of the efficacy of immunotherapies.
Reversibly silencing the biological activity of adoptively transferred cells using compositions of the present invention allows maximizing the potential of cell therapy without irretrievably killing and terminating the therapy.
100831 The present invention provides methods for fme tuning of immunotherapy after administration to patients. This in turn improves the safety and efficacy of immunotherapy and increases the subject population that may benefit from immunotherapy.
II. COMPOSITIONS OF THE IN VENTION
100841 According to the present invention, biocircuit systems are provided which comprise, at their core, at least one effector module system. Such effector module systems comprise at least one effector module having associated, or integral therewith, one or more stimulus response elements (SREs). The overall architecture of a biocircuit system of the invention is illustrated in Figure 1. In general, a stimulus response element (SRE) may be operably linked to a payload construct which could be any protein of interest (POI) (e.g., an immunotherapeutic agent), to form an effector module. The SRE, when activated by a particular stimulus, e.g., a small molecule, can produce a signal or outcome, to regulate transcription and/or protein levels of the linked payload either up or down by perpetuating a stabilizing signal or destabilizing signal, or any other types of regulation. A much-detailed description of a biocircuit system are taught in co-owned U.S. Provisional Patent Application No. 62/320,864 filed April 11, 2016, 62/466,596 filed March 3, 2017 and the International Publication W02017/180587 (the contents each of which are herein incorporated by reference in their entirety). In accordance with the present invention, biocircuit systems, effector modules, SREs and components that tune expression levels and activities of any agents used for inununotherapy are provided.
[0085] As used herein, a "biocircuit" or "biocircuit system" is defined as a circuit within or useful in biologic systems comprising a stimulus and at least one effector module responsive to a stimulus, where the response to the stimulus produces at least one signal or outcome within, between, as an indicator of, or on a biologic system. Biologic systems are generally understood to be any cell, tissue, organ, organ system or organism, whether animal, plant, fungi, bacterial, or viral. It is also understood that biocircuits may be artificial circuits which employ the stimuli or effector modules taught by the present invention and effect signals or outcomes in acellular environments such as with diagnostic, reporter systems, devices, assays or kits. The artificial circuits may be associated with one or more electronic, magnetic, or radioactive components or parts.
[0086] In accordance with the present invention, a biocircuit system may be a destabilizing domain (DD) biocircuit system, a dimerization biocircuit system, a receptor biocircuit system, and a cell biocircuit system. Any of these systems may act as a signal to any other of these biocircuit systems.
Effector modules and SREs for immunotherapv [0087] In accordance with the present invention, biocircuit systems, effector modules, SREs, and components that tune expression levels and activities of any agents used for immunotherapy are provided. As non-limiting examples, an immunotherapeutic agent may be an antibody and fragments and variants thereof, a cancer specific T cell receptor (TCR) and variants thereof, an anti-tumor specific chimeric antigen receptor (CAR), a chimeric switch receptor, an inhibitor of a co-inhibitory receptor or ligand, an agonist of a co-stimulatory receptor and ligand, a cytokine, chemokine, a cytokine receptor, a chemokine receptor, a soluble growth factor, a metabolic factor, a suicide gene, a homing receptor, or any agent that induces an immune response in a cell and a subject.
[0088] As stated, the biocircuits of the invention include at least one effector module as a component of an effector module system. As used herein, an "effector module"
is a single or multi-component construct or complex comprising at least (a) one or more stimulus response elements (i.e. proteins of interest (POIs). As used herein a "stimulus response element (SRE)" is a component of an effector module which is joined, attached, linked to or associated with one or more payloads of the effector module and in some instances, is responsible for the responsive nature of the effector module to one or more stimuli. As used herein, the "responsive" nature of an SRE to a stimulus may be characterized by a covalent or non-covalent interaction, a direct or indirect association or a structural or chemical reaction to the stimulus.
Further, the response of any SRE to a stimulus may be a matter of degree or kind. The response may be a partial response. The response may be a reversible response. The response may ultimately lead to a regulated signal or output. Such output signal may be of a relative nature to the stimulus, e.g., producing a modulatory effect of between 1% and 100% or a factored increase or decrease such as 2-fold, 3-fold, 4-fold, 5-fold, 10-fold or more.
100891 In some embodiments, the present invention provides methods for modulating protein expression, function or level. In some aspects, the modulation of protein expression, function or level refers to modulation of expression, function or level by at least about 20%, such as by at least about 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100%, or at least 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-95%, 20-100%, 30-40%, 30-50%, 60%, 30-70%, 30-80%, 30-90%, 30-95%, 30-100%, 40-50%, 40-60%, 40-70%, 40-80%, 90%, 40-95%, 40-100%, 50-60%, 50-70%, 50-80%, 50-90%, 50-95%, 50-100%, 60-70%, 80%, 60-90%, 60-95%, 60-100%, 70-80%, 70-90%, 70-95%, 70-100%, 80-90%, 80-95%, 100%, 90-95%, 90-100% or 95-100%.
100901 In some embodiments, the present invention provides methods for modulating protein, expression, function or level by measuring the stabilization ratio and destabilization ratio. As used herein, the stabilization ratio may be defined as the ratio of expression, function or level of a protein of interest in response to the stimulus to the expression, function or level of the protein of interest in the absence of the stimulus specific to the SRE. In some aspects, the stabilization ratio is at least 1, such as by at least 1-10, 1-20, 1 -30, 1-40, 1-50, 1- 60, 1-70, 1-80, 1- 90, 1-100, 20-30, 20-40, 20-50, 20-60, 20-70, 20-80, 20-90, 20-95, 20-100, 30-40, 30-50, 30-60, 30-70, 30-80, 30-90, 30-95, 30-100, 40-50, 40-60, 40-70, 40-80, 40-90, 40-95, 40-100, 50-60, 50-70, 50-80, 50-90, 50-95, 50-100, 60-70, 60-80, 60-90, 60-95, 60-100, 70-80, 70-90, 70-95, 70-100, 80-90, 80-95, 80-100, 90-95, 90-100 or 95-100. As used herein, the destabilization ratio may be defined as the ratio of expression, function or level of a protein of interest in the absence of the stimulus specific to the effector module to the expression, function or level of the protein of interest, that is expressed constitutively and in the absence of the stimulus specific to the SRE.
As used herein "constitutively" refers to the expression, function or level of a protein of interest that is not linked to an SRE, and is therefore expressed both in the presence and absence of the stimulus. In some aspects, the destabilization ratio is at least 0, such as by at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or at least, 0-0.1, 0-0.2, 0 -0.3, 0-0.4, 0-0.5, 0-0.6, 0-0.7, 0-0.8, 0-0.9, 0.1-0.2, 0.1 -0.3, 0.1-0.4, 0.1-0.5, 0.1-0.6, 0.1-0.7, 0.1-0.8, 0.1-0.9, 0.2 -0.3, 0.2-0.4, 0.2-0.5, 0.2-0.6, 0.2-0.7, 0.2-0.8, 0.2-0.9, 0.3-0.4, 0.3-0.5, 0.3-0.6, 0.3-0.7, 0.3-0.8, 0.3-0.9, 0.4-0.5, 0.4-0.6, 0.4-0.7, 0.4-0.8, 0.4-0.9, 0.5-0.6, 0.5-0.7, 0.5-0.8, 0.5-0.9, 0.6-0.7, 0.6-0.8, 0.6-0.9,0.7-0.8, 0.7-0.9 or 0.8-0.9.
[0091] The SRE of the effector module may be selected from, but is not limited to, a peptide, peptide complex, peptide-protein complex, protein, fusion protein, protein complex, protein-protein complex. The SRE may comprise one or more regions derived from any natural or mutated protein, or antibody. In this aspect, the SRE is an element, when responding to a stimulus, can tune intracellular localization, intramolecular activation, and/or degradation of payloads.
100921 In some embodiments, effector modules of the present invention may comprise additional features that facilitate the expression and regulation of the effector module, such as one or more signal sequences (SSs), one or more cleavage and/or processing sites, one or more targeting and/or penetrating peptides, one or more tags, and/or one or more linkers. Additionally, effector modules of the present invention may further comprise other regulatory moieties such as inducible promoters, enhancer sequences, microRNA sites, and/or microRNA
targeting sites.
Each aspect or tuned modality may bring to the effector module or biocircuit a differentially tuned feature. For example, an SRE may represent a destabilizing domain, while mutations in the protein payload may alter its cleavage sites or dimerization properties or half-life and the inclusion of one or more microRNA or microRNA binding site may impart cellular detargeting or trafficking features. Consequently, the present invention embraces biocircuits which are multifactorial in their tenability. Such biocircuits may be engineered to contain one, two, three, four or more tuned features.
100931 In some embodiments, effector modules of the present invention may include one or more degrons to tune expression. As used herein, a "degron" refers to a minimal sequence within a protein that is sufficient for the recognition and the degradation by the proteolytic system. An important property of degrons is that they are transferrable, that is, appending a degron to a sequence confers degradation upon the sequence. In some embodiments, the degron may be appended to the destabilizing domains, the payload or both. Incorporation of the degron within the effector module of the invention, confers additional protein instability to the effector module and may be used to minimize basal expression. In some embodiments, the degron may be an N-degron, a phospho degron, a heat inducible degron, a photosensitive degron, an oxygen dependent degron. As a non-limiting example, the degron may be an Ornithine decarboxylase degron as described by Takeuchi et al. (Takeuchi J et at. (2008). Biochem J.
2008 Mar 1;410(2):401-7; the contents of which are incorporated by reference in their entirety). Other examples of degrons useful in the present invention include degrons described in International patent publication Nos. W02017004022, W02016210343, and W02011062962; the contents of each of which are incorporated by reference in their entirety.
100941 As shown in Figure 2, representative effector module embodiments comprising one payload, i.e. one immunotherapeutic agent are illustrated. Each components of the effector module may be located or positioned in various arrangements without (A to F) or with (G to Z, and AA to DD) a cleavage site. An optional linker may be inserted between each component of the effector module.
[00951 Figures 3 to 6 illustrate representative effector module embodiments comprising two payloads, i.e. two immunotherapeutic agents. In some aspects, more than two immunotherapeutic agents (payloads) may be included in the effector module under the regulation of the same SRE (e.g., the same DD). The two or more agents may be either directly linked to each other or separated (Figure 3). The SRE may be positioned at the N-terminus of the construct, or the C-terminus of the construct, or in the internal location.
100961 In some aspects, the two or more immunotherapeutic agents may be the same type such as two antibodies, or different types such as a CAR construct and a cytokine IL12. Biocircuits and components utilizing such effector molecules are given in Figures 7-12.
100971 in some embodiments, biocircuits of the invention may be modified to reduce their immunogenicity. Immunogenicity is the result of a complex series of responses to a substance that is perceived as foreign and may include the production of neutralizing and non-neutralizing antibodies, formation of immune complexes, complement activation, mast cell activation, inflammation, hypersensitivity responses, and anaphylaxis. Several factors can contribute to protein immunogenicity, including, but not limited to protein sequence, route and frequency of administration and patient population. In a preferred embodiment, compositions of the invention may be engineered to reduce the itrununogenicity of the compositions of the invention. In some embodiments, modifications to reduce immunogenicity may include modifications that reduce binding of the processed peptides derived from the parent sequence to major histocompatibility complex (IVIIIC) proteins. For example, amino acid modifications may be engineered such that the minimum munber of immune epitopes are available to bind with high affinity to any prevalent MHC alleles. Several methods of identifying MHC binding epitopes of known protein sequences are known in the art and may be useful in the present invention.
Such methods are disclosed in US Patent Publication No. US 20020119492, U S20040230380, and US
20060148009; the contents of each of which are incorporated by reference in their entirety.

100981 Epitope identification and subsequent sequence modification may be applied to reduce immunogenicity. The identification of immunogenic epitopes may be achieved using physical or computational methods. Physical methods of epitope identification may include, for example, mass spectrometry and tissue culture/cellular techniques. Computational approaches use antigen processing, loading and display information, structural and/or proteomic data to identify non-self-peptides produced by antigen processing with good MHC groove binding characteristics.
One or more mutations may be introduced into the biocircuits of the invention directing the expression of the protein, to maintain its functionality while simultaneously rendering the identified epitope less or non-immunogenic.
100991 Protein modifications may also be employed to interfere with antigen processing and peptide loading e.g. glycosylation and PEGylation. Compositions of the invention may also be engineered to include non-classical amino acid side chains to design less immunogenic compositions. Any of the methods discussed in International Patent Publication No.
W02005051975 for reducing immunogenicity may be useful in the present invention (the contents of which are incorporated by reference in their entirety).
1001001 In one embodiment, patients may also be stratified according to the immunogenic peptides presented by their immune cells and may be utilized as a parameter to determine suitable patient cohorts that may therapeutically benefit for the compositions of the invention.
[00101] in some embodiments, reduced immunogenicity may be achieved by limiting immunoproteasome processing. The proteasome is an important cellular protease that is found in two forms: the constitutive proteasome, which is expressed in all cell types and which contains active e.g. catalytic subunits and the immunoproteasome that is expressed in cell of the hematopoietic lineage, and which contains different active subunits termed low molecular weight proteins (LMP) namely LMP-2, LMP- 7 and LMP-10. Immunoproteasomes exhibit altered peptidase activities and cleavage site preferences that result in more efficient liberation of many MHC class I epitopes. A well described function of the immunoproteasome is to generate peptides with hydrophobic C terminus that can be processed to fit in the groove of MHC class I
molecules. Deol P et al. have shown that immunoproteasomes may lead to a frequent cleavage of specific peptide bonds and thereby to a faster appearance of a certain peptide on the surface of the antigen presenting cells; and enhanced peptide quantities (Deol P et al.
(2007) J Immunol 178 (12) 7557-7562; the contents of which are incorporated herein reference in its entirety). This study indicates that reduced inununoproteasome processing may be accompanied by reduced immunogenicity. In some embodiments, immunogenicity of the compositions of the invention may be reduced by modifying the sequence encoding the compositions of the invention to prevent immunoproteasome processing. Biocircuits of the present invention may also be combined with immunoproteasome-selective inhibitors to achieve the same effects. Examples of inhibitors useful in the present invention include UK-101 (Bli selective compound), IPSI-001, ONX 0914 (PR-957), and PR-924 (IPSI).
[00102] Another embodiment of the invention provides a method of detecting the presence of cancer in a mammal, comprising: (a) contacting a sample comprising one or more cells from the mammal with any of the CARS, nucleic acids, recombinant expression vectors, host cells, population of cells, or pharmaceutical compositions of the invention, thereby forming a complex, (b) and detecting the complex, wherein detection of the complex is indicative of the presence of cancer in the mammal. The sample may be obtained by any suitable method, e.g., biopsy or necropsy. A biopsy is the removal of tissue and/or cells from an individual.
Such removal may be to collect tissue and/or cells from the individual in order to perform experimentation on the removed tissue and/or cells. This experimentation may also be used to determine if the individual has and/or is suffering from cancer.
[00103] With respect to an embodiment of the inventive method of detecting the presence of cancer in a mammal, the sample comprising cells of the mammal can be a sample comprising whole cells, lysates thereof, or a fraction of the whole cell lysates, e.g., a nuclear or cytoplasmic fraction, a whole protein traction, or a nucleic acid fraction. If the sample comprises whole cells, the cells can be any cells of the mammal, e.g., the cells of any organ or tissue, including tumor cells. The contacting can take place in vitro or in vivo with respect, to the mammal. Preferably, the contacting is in vitro. Also, detection of the complex can occur through any number of ways known in the art. For instance, the inventive CARS, nucleic acids, recombinant expression vectors, host cells, populations of cells, or pharmaceutical compositions described herein can be labeled with a detectable label such as, for instance, a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phospho ace, horseradish peroxidase), and element particles (e.g., gold particles). Methods of testing the compositions of the invention for the ability to recognize target cells and for antigen specificity are known in the art. For instance, Clay et al, J. Immunol, 163: 507-51 3 (1999), teaches methods of measuring the release of cytoldnes (e.g., interferon-7, granulocyte/monocyte colony stimulating factor (GM-CSF), tumor necrosis factor a (TNF-a) or interleukin 2 (IL2)). In addition, anti-CD276 material function can be evaluated by measurement of cellular cytotoxicity, as described in Zhao et al., J. Immunol, 1 74: 44) 5-4423 (2005).
[00104] In some embodiments, the stimulus of the present invention maybe ultrasound stimulation. In some embodiments, the SREs of the present invention may derived from mechanosensitive proteins. In one embodiment, the SRE of the present invention may be the mechanically sensitive ion channel, Piezol.
[00105] Expression of the payload of interest in such instances is tuned by providing focused ultrasound stimulation. In other embodiments, the SREs of the present invention may be derived from calcium biosensors, and the stimulus of the present invention may calcium. The calcium may be generated by the ultrasound induced mechanical stimulation of mechanosensitive ion channels. The ultrasound activation of the ion channel causes a calcium influx thereby generating the stimulus. In one embodiment, the mechanosensitive ion channel is Piezo 1.
Mechanosensors may be advantageous to use since they provide spatial control to a specific location in the body.
1. Destabilizing domains (DDs) [00106] In some embodiments, biocircuit systems, effector modules, and compositions of the present invention relate to post-translational regulation of protein (payload) function anti-tumor immune responses of immunotherapeutic agents. In one embodiment, the SRE is a stabilizing/destabilizing domain (DD). The presence, absence or an amount of a small molecule ligand that binds to or interacts with the DD, can, upon such binding or interaction modulate the stability of the payload(s) and consequently the function of the payload.
Depending on the degree of binding and/or interaction the altered function of the payload may vary, hence providing a "tuning" of the payload function.
[00107] In some embodiments, destabilizing domains described herein or known in the art may be used as SREs in the biocircuit systems of the present invention in association with any of the immunotherapeutic agents (payloads) taught herein. Destabilizing domains (DDs) are small protein domains that can be appended to a target protein of interest. DDs render the attached protein of interest unstable in the absence of a DD-binding ligand such that the protein is rapidly degraded by the ubiquitin-proteasome system of the cell (Stankunas, K., et al., Mol. Cell, 2003, 12: 1615-1624; Banaszynski, etal., Cell; 2006,126(5): 995-1004; reviewed in Banaszynski, L.A., and Wandless, T.J. Chem. Biol.; 2006,13:11-21 and Rakhit R et al., Chem Biol. 2014;
21(9):1238-1252). However, when a specific small molecule ligand binds its intended DD as a ligand binding partner, the instability is reversed and protein function is restored. The conditional nature of DD stability allows a rapid and non-perturbing switch from stable protein to unstable substrate for degradation. Moreover, its dependency on the concentration of its ligand further provides tunable control of degradation rates.
[00108] In some embodiments, the desired characteristics of the DDs may include, but are not limited to, low protein levels in the absence of a ligand of the DD (i.e. low basal stability), large dynamic range, robust and predictable dose-response behavior, and rapid kinetics of degradation.
DDs that bind to a desired ligand but not endogenous molecules may be preferred.
[00109] Several protein domains with destabilizing properties and their paired small molecules have been identified and used to control protein expression, including FKBP/shield-1 system (Egelcr et al., J Bio/. Chem. 2011,286(36): 32328-31336; the contents of which are incorporated herein by reference in their entirety), ecDHFR and its ligand trimethoprim (TMP); estrogen receptor domains which can be regulated by several estrogen receptor antagonists (Miyazaki et al., J Am Chem. Soc., 2012, 134(9): 3942-3945; the contents of which are incorporated by reference herein in their entirety): and fluorescent destabilizing domain (FDD) derived from bilirubin-inducible fluorescent protein, UnaG and its cognate ligand bilirubin (BR) ( Navarro et al., ACS Chem Biol., 2016, June 6; the contents of which are incorporated herein by reference in their entirety).
[00110] Known DDs also include those described in U.S. Pat. NO. 8,173,792 and U.S. Pat. NO.
8,530,636, the contents of which are each incorporated herein by reference in their entirety.
[00111] In some embodiments, the DDs of the present invention may be derived from some known sequences that have been approved to be capable of post-translational regulation of proteins. For example, Xiong et al., have demonstrated that the non-catalytic N-terminal domain (54-residues) of ACS7 (1-aminocyclopropane-1-carboxylate synthase) in Arabidopsis, when fused to the 11-glucuronidase (GUS) reporter, can significantly decrease the accumulation of the GUS fusion protein (Xiong et al., J. Exp. Bot, 2014, 65(15): 4397-4408). Xiong et al. further demonstrated that both exogenous 1-aminocyclopropane-1 -carboxylic acid (ACC) treatment and salt can rescue the levels of accumulation of the ACS N-terminal and GUS
fusion protein. The ACS N-terminus mediates the regulation of ACS7 stability through the ubiquitin-proteasome pathway.
[00112] Another non-limiting example is the stability control region (SCR, residues 97-118) of Tropomyosin (Tm), which controls protein stability. A destabilizing mutation L110A, and a stabilizing mutation A109L dramatically affect Tropomyosin protein dynamics (Kinvan and Hodges, J. Biol. Chem., 2014, 289: 4356-4366). Such sequences can be screened for ligands that bind them and regulate their stability. The identified sequence and ligand pairs may be used as components of the present invention.
[00113] In some embodiments, the DDs of the present invention may be developed from known proteins. Regions or portions or domains of wild type proteins may be utilized as SREs/DDs in whole or in part. They may be combined or rearranged to create new peptides, proteins, regions or domains of which any may be used as SREs/DDs or the starting point for the design of further SREs and/or DDs.
[00114] Ligands such as small molecules that are well known to bind candidate proteins can be tested for their regulation in protein responses. The small molecules may be clinically approved to be safe and have appropriate pharmaceutical kinetics and distribution. In some embodiments, the stimulus is a ligand of a destabilizing domain (DD), for example, a small molecule that binds a destabilizing domain and stabilizes the POI fused to the destabilizing domain. In some embodiments, ligands, DDs and SREs of the present invention, include without limitation, any of those taught in Tables 2-4 of copending commonly owned U.S. Provisional Application NOs.
62/320,864, filed on 4/11/2016, or in US Provisional Application No.
62/466,596 filed March 3, 2017 and the International Publication W02017/180587, the contents of each which are incorporated herein by reference in their entirety. Some examples of the proteins that may be used to develop DDs and their ligands are listed in Table 1.
Table 1: Proteins and their binding ligands Protein Protein Sequence Protein Ligands SEQ ID
NOS.
E. coli MISLIAALAVDRVIGMENAMPWNLPADLAWFICRNT 1 Methotrexate Dihydrofolate LNICPVLMGRH1WESIGRPLPGRICNI1LSSOPGTDDRVT (MTX) reductase WVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQK Trimethoprim (ecDHER) LYLTHIDAEVEGDTHEPDYEPDDWESVESEFELDADA (TM?) (Uniprot ID: QNSHSYCFEILERR
POABQ4) Human MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQ 2 Methotrexate Dihyclrofolate RMTTTSSVEGKQNLVEMGKKTWFSIPEKNRPLKGRIN (MTX) reductase LVLSRELKEPPQGAHFLSRSLDDALKLTEQPELANKV Trimethoprim (hDHFR) DMVW1VGGSSVYKEAMNHPGHLKLEVTRIMQDFES (TM?) (Uniprot ID: DTFFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEV
P00374) YEKND
FK506 binding GVQVETESPGDGRTFPKRGQICVVHYTGMLEDGKKF 3 Shield-1 protein (FKBP) DSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQR
(Uniprot ID: AICLTISPDYAYGATGHPGIIPPHATLVEDVELLKLE
P62942) Phosphodiesterds MEETRELQSLAAAVVPSAQTLKITDFSFSDFELSDLET 4 Sildenatil;
e 5 (PDE5), ALCTIRMEIDLNLVQNFQMICHEVLCRWILSVICKNYR Vardenafil;
ligand binding KNVAYHNWRHAFNTAQCMFAALKAGKIQNKLTDL Tactalafil domain (Uniprot EILALLIAALSHDLDHRGVNNSYIQRSEHPLAQLYCH
ID: Uniprot ID SIMEHHHEDQCLMILNSPGNQILSGLSIEEYKITLKIIK
076074) QAILATDLALYIKRRGEFFELIRKNQFNLEDPHQKELF
LAMLMTACDLSAITKPWPIQQRIAELVATEFFDQGDR
ERKELNIEPTDLMNREKKNKIPSMQVGFIDAICLQLY
EALTHVSEDCFPLLDGCRICNRQKWQALAEQQ
PPAR gamma SVEAWEITEYAKSIPGFVNLDLNDQVTILKYGVHEII 5 Posiglitazone (PPARg), ligand YTMLASLMNKDGVLISEGQGFMTREFLKSLRKPFGD Pioglitazone binding domain FMEPKFEFAVICFNALELDDSDLAIFIAVIILSGDRPGL
(Uniprot ID: LN'VKPIEDIQDNLLQALELQLKLNHPESSQLFAKLLQ
P3 723 1. ; amino KMTDLRQIVTEHVQLLQVIKKTETDMSLHPLLQEIYK
acids 317-505) DLY

Carbonic MSHHWGYCKHNGPEHWHKDEPTAKGERQSPVDIDT 6 Celecoxib anhydrase II HTAKYDPSLKPLSVSYDQATSLRILNNGHAFNVEFD Acetawlatnide (CA2) (Uniprot DSQDKAVLKGGPLDGTYRLIQFHFHWGSLDGQGSEH
ID: P00918) TVDKKKYAAELHLVHWNTKYGDFGKAVQQPDGLA
VLGIFLKVGSAKPGLQKVVDVIDSIKTKGKSADFTNE
DPRGLLPESLDYWTYPGSUITPPLLECVTWIVLKEPIS
VSSEQVLKFRKLNFNGEGEPEELMVDNWRPAQPLKN
RQIICASFK
NRH: Quinone MAGKKVLIVYAHQEPKSFNGSLKNVAVDELSRQGC 7 Imatinib oxidoreductase 2 TVTVSDLYAMNLEPRATDKDITGTLSNPEVFNYGVE Melatonin (NQ02) (Uniprot THEAYKQRSLASDITDEQKKVREADLVIFQFPLYWFS
ID: P16083) VPAILKGWMDRVLCQGFAFDIPGFYDSGLLQGKLAL
LSVTIOGTAEMYTKTGVNGDSRYFLWPLQHGTLHE

KEEPIPCTAHWHFGQ
Dipeptidyl MKTP'W'KVLLGLLGAAALVTIITVPVVLLNKGTDDAT 224 Sitagliptin, peptidases ADSRKTYTLTDYLKNTYRLKLYSLRWISDHEYLYKQ Saxagliptin, (DPPIV) (Uniprot ENNILVFNAEYGNSSVFLENSTFDEFGHSINDYSISPD Denagliptin ID: P27487) GQFILLEYNYVKQWRHSYTASYDIYDLNKRQLITEER
IPNNTQWVTWSPVGHKLAYVWNNDIYVKIEPNLPSY
RITWTGKEDIIYNGITDWVYEEEVFSAYSALWWSPN
GIFLAYAQFNDTEVPLIEYSFYSDESLQYPKTVRVPY
PKAGAVNPTVKFFVVNTDSLSSVTNATSIQITAPASM
LIGDHYLCDVTWATQERISLQWLRRIQNYSVMDICD
YDESSGRWNCLVARQHIEMSTTGWVGRFRPSEPITFT
LDGNSFYKIISNEEGYRHICYFQIDKKDCTFITKGTWE
VIGIEALTSDYLYYISNEYKGMPGGRNLYKIQLSDYT
KVTCLSCELNPERCQYYSVSFSKEAKYYQLRCSGPG
LPLYTLHSSVNDKGLRVLEDNSALDKMLQNVQMPS
KKLDFDLNETKEWYQMILPPHFDKSKKYPLLLDVYA
GPCSQKADTVFRLNWATYLASTENIIVASFDGRGSG
YQGDKIMHAINRRLGTFEVEDMEAARQFSKMGFVD
NKRIAIWGWSYGGYVTSMVLGSGSGVFKCGIAVAPV
SRWEYYDSVYTERYMGLPTPEDNLDHYRNSTVMSR
AENFKQVEYLLIHGTADDNVHFQQSAQISKALVDVG
VDFQAMWYTDEDHGIASSTAHQHIYTIEVISHFIKQCF
SLP
[00115] in some embodiments, DDs of the invention may be FKBP DD or ecDHFR DDs such as those listed in Table 2. The position of the mutated amino acid listed in Table 2 is relative to the ecDHFR. (Uniprot ID: POABQ4) of SEQ ID NO. 1 for ecDHFR DDs and relative to FKBP
(Uniprot ID: P62942) of SEQ ID NO. 3 for FKBP DDs.
Table 2: ecDHFR DDs and F KB? Dili, DD Sequencc SEQ ID
NO.
ecDHFR (R12Y, MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKP 8 Y1001) VIMGRHTWESIGRPLPGR KNI1LSSQPGTDDRWWVKSVDE
A IA ACGD VPE IM VI GGGRVIEQFLPKAQKLYLTHIDAEVEG
DTHEPD YEP DD WES VESEFH D.A D AQN SH SYCFEI LERR
ecDHFR (Amino ISLIAALAVDYVIGMENAIVIPWNLPADLAWFKRNTLNKPVI 9 acid 2-159 of WT) MGRHTWESIGRPLPGRKNDLSSQPGTDDRVIVVKSVDEAI
(R12Y, Y1001) AACGDVPEIMVIGGGRVIEQFLPKAQKLYLTHIDAEVEGDT
FIRDYEPDDWESVESEFFIDADAQNSHSYCFE ERR

ecDHFR (Amino 1SLIAALAVDH'VIGMENAMPWNLPADLA'WFKRNTLNKPVI 10 acid 2-159 of WI) MGRHTWESIORPLPGRKNIILSSQPGTDDRNITWVKSVDEA1 (R12H, E129K) AACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGD
THFPDYKPDDWESVFSEFFIDADAQNSHSYCFEILERR
FKBP (F36V, GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSR 11 L 106P) DRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPD
YAYGATGHPGIIPPHATLVFDVELLKPE
FKBP (E310, GVQVETISPGDGRIFPKROQICVVHYTGMLODOKKVDSSR 12 F36V, R710, DRNKPFKFMLGKQEVIROWEEGVAWSVOQGAKLTISPD
K105E) YAYGATGHPGIEPPHATLVFDVELLELE
[00116] Inventors of the present invention have tested and identified several candidate human proteins that may be used to develop destabilizing domains. As show in Table 2, these candidates include human DHFR (hDHFR), PDE5 (phosphodiesterase 5), PPAR gamma (peroxisome proliferator-activated receptor gamma), CA2 (Carbonic anhydrase II) and NQ02 (NRH:
Quinone oxidoreductase 2). Candidate destabilizing domain sequence identified from protein domains of these proteins (as a template) may be mutated to generate libraries of mutants based on the template candidate domain sequence. Mutagenesis strategies used to generate DD libraries may include site-directed mutagenesis e.g. by using structure guided information; or random mutagenesis e.g. using error-prone PCR, or a combination of both. In some embodiments, destabilizing domains identified using random mutagenesis may be used to identify structural properties of the candidate DDs that may be required for destabilization, which may then be used to further generate libraries of mutations using site directed mutagenesis.
[00117] In some embodiments, novel DDs derived from E.coli DHFR (ecDHFR) may comprise amino acids 2-159 of the wild type ecDHFR sequence. This may be referred to as an Mldel mutation.
[00118] In some embodiments, novel DDs derived from ecDHFR may comprise amino acids 2-159 of the wild type ecDHFR sequence (also referred to as an Mldel mutation), and may include one, two, three, four, five or more mutations including, but not limited to, Mldel, R12Y, R12H, Y100I, and E129K.
[00119] In some embodiments, novel DDs derived from FKBP may comprise amino acids 2-107 of the wild type FKBP sequence. This may be referred to as an Mldel mutation.
[00120] In some embodiments, novel DDs derived from FKBP may comprise amino acids 2-107 of the wild type FBKP sequence (also referred to as an Mldel mutation), and may include one, two, three, four, five or more mutations including, but not limited to, M
ldel, E31G, F36V, R71G, K105E, and 1,106P.
[00121] In some embodiments, DD mutant libraries may be screened for mutations with altered, preferably higher binding affinity to the ligand, as compared to the wild type protein. DD

libraries may also be screened using two or more ligands and DD mutations that are stabilized by some ligands but not others may be preferentially selected. DD mutations that bind preferentially to the ligand compared to a naturally occurring protein may also be selected.
Such methods may be used to optimize ligand selection and ligand binding affmity of the DD.
Additionally, such approaches can be used to minimize deleterious effects caused by off-target ligand binding.
[001221 In some embodiments, suitable DDs may be identified by screening mutant libraries using barcodes. Such methods may be used to detect, identify and quantify individual mutant clones within the heterogeneous mutant library. Each DD mutant within the library may have distinct barcode sequences (with respect to each other). In other instances, the polynucleotides can also have different barcode sequences with respect to 2,3,4,5,6,7,8,9,10 or more nucleic acid bases. Each DD mutant within the library may also comprise a plurality of barcode sequences.
When used in plurality may be used such that each barcode is unique to any other barcode.
Alternatively, each barcode used may not be unique, but the combination of barcodes used may create a unique sequence that can be individually tracked. The barcode sequence may be placed upstream of the SRE; downstream of the SRE, or in some instances may be placed within the SRE. DD mutants may be identified by barcodes using sequencing approaches such as Sanger sequencing, and next generation sequencing, but also by polymerase chain reaction and quantitative polymerase chain reaction. In some embodiments, polymerase chain reaction primers that amplify a different size product for each barcode may be used to identify each barcode on an agarose gel. In other instances, each barcode may have a unique quantitative polymerase chain reaction probe sequence that enables targeted amplification of each barcode.
[001231 In some embodiments, DDs of the invention may be derived from human dihydrofolate reductase (hDHFR). liDHFR is a small (18 kDa) enzyme that catalyzes the reduction of dihydrofolate and plays a vital role in variety of anabolic pathway.
Dihydrofolate reductase (DHFR) is an essential enzyme that converts 7,8-dihydrofolate (DHF) to

5,6,7,8, tetrahydrofolate (THF) in the presence of nicotinamide adenine dihydrogen phosphate (NADPH).
Anti-folate drugs such as methotrexate (M1'X), a structural analogue of folic acid, which bind to DHFR
more strongly than the natural substrate DHF, interferes with folate metabolism, mainly by inhibition of dihydrofolate reductase, resulting in the suppression of purine and pyrimidine precursor synthesis. Other inhibitors of hDH:FR such as folate, TQD, Trimethoprim (TMP), cpigallocatechin gallate (EGCG) and ECG (epicatechin gallate) can also bind to hDHFR mutants and regulates its stability.
[00124] In one aspect of the invention, the DHFR DDs of the invention may include mutations such as, but not limited to V2A, C7R, I8V, V9A, AlOT, AIOV, Q I3R, N14S, G16S, I l'7N, II7V, K19E, .N20D, G21T, G21E, D22S, L23S, P24S, L28P, N30D, N3OH, N30S, E31G, E31D, F32M, R33G, R33S, F35L, Q36R, Q36S, Q36K, Q36F, R37G, M38V, M38T, T40A, V44A, K47R, N49S, N49D, M53T, G54R, K56E, K56R, T57A, F59S, 16 IT, K64R, N65A, N65S, N65D, N65F, L68S, K69E, K69R, R71G, I72T, I72A, I72V, N73G, L74N, V75F, R78G, L80P, K81R, E82G, H88Y, F89L, R92G, S93G, S93R, L94A, D966, A97T, L98S, K99G, K99R, L1OOP, E102G, Q103R, P1.04S, E105G, A107T, A107V, NIO8D, K109E, KI09R, V110A, D111N, M112T, M112V, V113A, W114R, 1115V, V1161 ,G117D, V121A,Y122C,Y122D, Y1221, K123R, K123E, A125F, M1261, N127R, N127S,N127Y, H128R, H128Y, H131R, L132P, K133E, L134P, F135P, F135L, F135S, F135V, V136M, T137R, R138G, R1381, I139T, I139V, M1401, M140V, Q141R, D142G, F143S, F143L, E144G, D146G, T147A, F148S, F148L, F149L, P150L, E151G, I152V, D153A, D153G, E155G, K156R, Y157R, Y157C, K158E, K158R, L159P, L160P, E162G, Y163C, V166A, S168C, D169G, V170A, Q171R, E172G, E173G, E173A, K174R, I176A, I176F, I176T, K177E, K177R, Y178C, Y178H, F180L, E181G, V182A, Y183C, Y183H, E184R, E1846, K1.85R, K185del, K185E, N1.86S, N186D, D187G, and D187N.
[00125] In one embodiment, the stimulus is a small molecule that binds to a SRE in order to post-translationally regulate protein levels. In one aspect, DHFR ligands:
trimethoprim (TMP) and methotrexate (M'TX) are used to stabilize hDHFR mutants. The hDHFR based destabilizing domains are listed in Table 3. The position of the mutated amino acid listed in Table 3 is relative to the human DHFR (Uniprot ID: P00374) of SEQ ID NO. 2 for human DHFR. In Table 3, the mutations are underlined and in bold. In Table 3, "del" means that the mutation is the deletion of the amino acid at that position relative to the wild type sequence.
Table 3: Human DHFR mutants and novel destabilizine domains Mutants Amino acid Sequence SEQ
NO
hDHFR (II7V) MVGSLNCIVAVSQNMGVGKNGDLPWPPLRNEFRYFQR 13 SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW
IVGGSSVYKEAMNHPGFILKLFVTRLMQDFESDTFFPEID
LEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDHFR (F59S) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 14 SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW
IVGGSSVYKEAMNHPGHLKLFVTREVIQDFESDTFFPEID
LEKYKLLPEYPGVLSDVQEEKOKYKFEVYEKND
hDHFR (N65D) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 15 MTTTSSVEGKQNLVIMGKKIWFSIPEKDRPLKGRINLVL
SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW
IVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFPEID

hD1-1112 (K81R) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 16 SRELREPPQG AHFLSRSLDD A L. KLTEQPELA NK VDMVW

LEKYKLLPEYPGVLSDVQEEK.GTKYKFEVYEKND
hDHFR (A107V) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 17 MTTTS SVEGKQNLVIMGKICTWFSIPEKNRPLKGR INLVL
SRELICEPPQGAHFLSRSLDDALICLTEQPELVNKVDMVW
IVGGSSVYKEAMNHPGHLKLFVTR I MQDFESDTFFPEID
LEK YKLLPEYPGVLSDVQEEKGIKYK FENNEKND
hDHFR (Y1221) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 18 SRELKEPPQGAHFLSRSLDDALICLTEQPELANKVDMVW
IVGGSSVIKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDL
EKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDHFR (N1.27Y) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 19 MTTTSSVEGKQNLVIMGKKIWFSIPEKNRPLKGRINL VL
SRELICEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW
IVGGSSVYKEAMYHPGHLKLFVTRIMQDFESDTFFPEID

hDI-IFR (M1401) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 20 MITTSSVEGKQNLVIMGKICTWFSIPEKNRPLKGRINLVL
SRELICEPPQGAHFLSRSLDDALKUITHQPELANKVDMVW

EKYKLLPEYPGVLSDVQEEKGIKYKFENTYEKND
hDHFR (K185E) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 21 MITTS SVEGKQNLVIMGICKTWFSIPEKNR PLK GR INLVL
SRELICEPPQGAHFLSRSLDDALICLTEQPELANKVDMVW
IVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFPEID

hDHFR (3\1186D) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 22 MTITSSVEGKQNLV1MGICICTWF'S1PEKNRPLKGRINLVL
SRELKEPPQGAHFLSRSLDDALKLTEQPELANICVDMVW
IVGGSS'VYICEAMNHPGHLKLFVTRLMQDFESDTFFPEID
LEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKDD
13DI-IFR (C7R, Y163C) MVGSLNRIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 23 LEKYICLLPECPGVLSDVQEEKGIKYKFEVYEKND
1011FR (A WV, 1188Y) MVGSLNCIVVVSQNMGIGKNGDLPWPPLRNEFRYFQR 24 MffiSSVEGKQNLVINIGKKTWFS1PEKNRPLKGRINL VL
SRE LK EPPQGAYFL SRSL DD ALKLTEQPELANKVDMVW
IVGGSSVYKEAMNHPGHLKLINTRIMQDFESDTFFPEID
LEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDHFR (Q36K, Y1221) MVGSLNCIVAVSQNMGIGICNGDLPWPPLRNEFRYFKR 25 SRELKEPPQGAHFLSRSLDDALKLTEQPELANICVDMVW
IVGGSSVIICEAMNHPGHLKLYVTRIMQDFESDTFFPEIDL
EKYKLLPEYPGVL SDVQE EKG IKYKFE'VYEKND
hDHFR (M53T, R1381) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 26 SRELICEPPQGAHFLSRSLDDALKLTEQPELANICVDMVW
IVGGSSVYICEAMNFIPGHLKLFVTIIMQDFESDTFFPEDL

hDHFR (T57A, I72A) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 27 LSR ELK EPPQGAHFLSR SLDDALKLTEQPELAINIKVDMV
WIVGGssvyKEAMNHPGHLKLFVTRIMQDFESDTFFPEI
DLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND

hDHFR (E63G, I176F) MVGSLNCI VA VSQNMG IGICN GDL VW PPLR NEFRYFQR 28 11MTSSVEGKQNLVINIGKICT'WESIPGKNRPLKGRINLVL
SRELKEPPQGAHFLSRSLDD AL KLTEQPELANKVDMV'W
IVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFPEID
LEKYKLLPEYPGVLSDVQEEKGFKYKFEVYEKND
hDHFR (G21T, Y1221) MVGSLNCIVAVSQNIVIGIGICNTDLPWPPLRNEFRYFQRM 29 TTTSSVEGKQNLVLMGKKTWFSIPEKNRPLKGRINLVLS
RELKEPPQGAHFLSR SLDDALKLTEQPELANK'VDMVWI

FIC YK LLPEY PGVL SDVQE EKG IK YKFEVYEKND
hDHFR (L74N, Y1221) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 30 SRELKEPPQGAHFLSRSLDDALICLTEQPELANKVDMVW
IVGGSSVIKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDL
EKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDHFR (V75F, Y122I) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 31 SRELICEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW
IVGGSSVIKEAIVINHPGHLICLFVTRIMQDFESDTFFPEIDL
EKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDHFR (L94A, 1I47A) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 32 MITTSSVEGKQNLVIMGKICTWFSIPEKNRPLKGRINLVL
SRELKEPPQGAHFLSRSADD ALKLTEQPELANKVDMVW
WGGSSVYKEAMNHPGHLKLFVTRIMQDFESDAFFPEID
LEKYKLLPEYPGVLSDVQEEKGIKYK FEVYEKND
DHFR (V12IA, Y22I) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 33 SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW
IVGGSSATICEAMNHPGHLKLINTR IMQDFESDTFFPEIDL
EKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDHFR (Y 122!. A125F) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 34 MTITSSVEGKQNLVIMGICICTWF'S1PEKNRPLICGRINLVL
SRELICEPPQGAHFLSRSLDDALKLTEQPELANICVDMVW

EKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDH FR (H13 IR, E I44G) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 35 MTITSSVEGK QNLVIMGKKTWFSIPEKNRPLKGR INLVL
SRELKEPPQGAI-TFLSRSLDDALKLTEQPELANKVDMVW
IVGGSSVYKEAMNHPGRLKLFVTRIMQDFGSDTFFPEID

1011FR (r137R, F143L) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 36 MffiSSVEGKQNLVINIGKKTWFSIPEK NRPLKGRINLVL
SRE LKEPPQGAHFLSRSLDD ALKLTEQPELANKVDMV'W

LEKYKLLPEYPG VLSDVQEEKGIKYKFEVYEKND
hDHFR (Y178H, E18IG) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 37 SRELKEPPQGAHFLSRSLDDALKLTEQPELANICVDMVW

LEKYKLLPEYPGV L. SDVQ E EK GIKI-IK FGVYEKND
hDHFR (Y !83H, K185E) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR $8 IVITITSSVEGKQNLVIMGICKTWFSIPEKNRPLKGRINLVL
SRELICEPPQGAHFLSRSLDDALKLTEQPELANICVDMVW
IVGGSSVYICEAMNFIPGHLICLEVTRLMQDFESDTFFPEID

hDHFR (V9A, S93R, P1501.) MVGSLNCIAAVSQNMGIGKNGDLPWPPLRNEFRYFQR 39 MTTTSSVEGKQNLVIMGKKTWFSTPEKNRPLKGRINLVL
SRELKEPPQGAHFLSRRLDDALKLTEQPELANKVDMVVV
IVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFLEID

hDHFR (I8V, K133E, Y163C) MVGSLNC'VVAVSQNMGIGKNGDLRWPPLRNEFRYFQR 40 MTITSSVEGKQNLVIMGICKTW'ES1PEKNRPLKGRINLVL
SRELICEPPQGAHELSRSLDDALKLTEQPELANK VDMV'W
IVGGSSVYKE.AMNHPGHLELFVTRIMQDFESIYITEPEID
LEKYKLLPECPGVLSDVQEEK GiKYKFEVYEKNID
hDHFR (L23S, V121A, Y157C) MVGSLNCIVAVSQNMGIGKNGDSPWPPLRNEFRYFQR 41 MTITSSVEGKQNLVIMGKICTWFSIPEKNRPLKGRINLVL
SRELKEPPQGAHFLSRSLDDALICLTEQPELANICVDMVW
IVGGSSAYKEANINHPGHLKLEVTRIMQDFESDTFFPEID
LEKCKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDHFR (K19E, F89L, E18 1G) MVGSLNCIVAVSQNMGIGENGDLPWPPLRNEFRYFQRM 42 TITSSVEGKQNLVLMGKKTWFSIPEKNRPLKGRINLVLS

VGGSSVYKEAMNHPGHLKLEVTRIMQDFESDTFFPEDL
EKYKLLPEYPGVLSDVQEEKGIKYKFGVYEKND
hDHFR (Q36F, N65F, Y1221) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFFRM 43 ITTSSVEGKQNLVIMGKKTWFSIPEKFRPLKGRINLVLS

EKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
liDHER (G54R, M140V, S168C) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 44 MITTSSVEGKQNLVIMRICKTWFSIPEKNRPLKGRINLVL
SRELKEPPQGAHFLSRSLDDALKLTEQPELANICVDMVW
IVGGSSVYKEAMNHPGHLKLEVTRIVQDFESDIFFPEIDL
EKYKLLPEYPGVLCDVQEEKGI KFEVYEKND
hDHFR (V110A, V136M, K177R) MVGSLNCIVAVSQNMGIGKNGDLP'WPPLRNEFRYFQR 4 5 IvTITTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVL
SRELKEPPQGAHFLSRSLDDALKLTEQPELANKADMVW
IVGGSSVYKEAMNHPGH K LEMTRIMQDFESDIFFPEID
LEKYKLLPEYPGVLSDVQEEK GlRYKFEVYEKND
hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFFRMT 46 -Q36F, Y1221, A125F) TTSSVEGKQNLVIMGKKTWFS1PEKNRPLKGRINLVLSR
ELKEPPQGAHFLSRSLDDALICLTEQPELANKVDMVWIV
GGSSVIKEFMNHPGHLKLFVTRIMQDFESDTFFPEIDLEK
YICLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDHFR (N49D, F59S, D153G) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYEQR 47 MTTTSSVEGKQDLVIMGKKTWSSIPEKNRPLKGRINLVL
SRELKEPPQGAFTFLSRSLDDALKLTEQPELANKVDMVW
IVGGSSVYKEAMNHPGHLKLEVIREVIQDFESDTFFPEIG
LEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
1011FR (G21E, I72V, 11761) MVGSLNCIVAVSQNMGIGKNEDLPWPPLRNEFRYFQRM 48 TTTSSVEGKQNLVNIGKICTWFSIPEKNRPLKGRVNLVLS
RELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVWI
VGGSSVYK EAMNHPGHLKLEVTRIMQDFESDTEEPEIDL
EKYKLLPEYPGVLSDVQEEKUTKYKFEVYEKND
hDHFR (L100P, E102G, Q103R, MVGSLNCIVAVSQNMGIGKNGDLP'W'PPLRNEFRYFQR 225 P104S, E105G, NIO8D, VII3A, M1ITSSVEGKQNLVIMGKKTWFS1PEKNRPLKGRINLVL
W114R, Y122C, M126I, N127R, SRELKEPPQGAHFLSRSLDDALKPTGRSGLADKVDMAR
H128Y, L132P, F135P, I139T, IVGGSSVCKEAIRYPGHPKLPVIRTMQDFESDTSLPEVA
F148S, F149L, I152V, D153A, LEKYKLLPEYPGVLSGAQEEKGARYKFEAYERSD
D169G, V170A, I176A, K177R, V182A. K185R. N186S) hDHFR (V2A, R33G, Q36R, MAGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFGYFRR 226 LlOOP, K185R) IvTITTSSVEGKQNLVIMGICKTWFS1PEKNRPLKGRINLVL
SRELKEPPQGAHFLSRSLDDALKPTEQPELANKVDMVW

hDHFR (G1.6S, I17V, F89L, MVGSLNCIVAVSQNMSVGKNGDLPWPPLRNEFRYEQR 227 D96G, K123E, M140V, D1;46G, MTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLK VI.
K156R) SRELKEPPQGAHLLSRSLDGALKLTEQPELANKVDMVW

TVGGSS'VYEEAMNHPGHLKLFVTRIVQDFESGIFFPEIDL
ERYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDHFR (F35L, R37G, N65A, IvIVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYLQG 228 L68S, K69E, R7IG, L80P, K99G, MITTSSVEGKQNLVIMGICKTWFSIPEKARPSEGGINLVL
G117D, L I32P, I139V, M140I, SREPICEPPQGAHFLSRSLDDALGLTEQPELANKVDMVW
D142G, D146G, E173G, D I87G) IVDGSSVYICEAMNHPGHPKLFVTRVIQGFESGTFFPEIDL
EKYKLLPEYPGVLSDVQEGKGIKYKFEVYEKNG .......................
hDHFR (II7N, L98S, K99R, MVGSLNCIVAVSQNMGNGKNGDLPWPPLRNEFRYFQR 229 M112T, E151G, E162G, E I72G) MTITSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVL
SRELKEPPQGAHFLSRSLDDASRLTEQPELANKVDTVWI
VGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFPGIDL
EKYKLLPGYPGVLSDVQGEKGIKYKFEVYEKND
hDHFR (R138G, D142G, FI43S, MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 230 K156R, KI58E, E162G, V1.66A, mTrrsSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVL
K177E, Y I78C, K 185E, NI86S) SRELKEPPQGAITFLSRSLDDALKLTEQPELANKVDMVW
IVGGSSVYKEAMNHPGHLKISVTGIMQGSESDTFFPEID
LERYELLPGYPGALSDVQEEKGIECKFEVYEESD
WHIM (K8IR, K99R, LlOOP, MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 231 E102G, N108D, K I23R, H128R, MTITSSVEGKQNLVIMGICKTWFSIPEKNRPLKGRINLVL
D142G, F180L, K185E) SRELREPPQGAHFLSRSLDDALRPTGQPELADKVDMVW
IVGGSSVYREAMNRPGHLKLFVTRIMQGFESDIFFPEFD
LEKYKLLPEYPGVLSDVQEEKGIKYKLEVYEEND
hDHFR (N14S, P24S, F35L, MVGSLNCIVAVSQSMGIGKNGDLSWPPLRNEFRYLQRM 232 M53T, K56E, R92G, S93G, TTTSSVEGKQNLVITGICETWFSIPEKNRPLKGRINLVLSR
N127S, H128Y, F I35L, FI43S, ELKEPPQGAHFLSGGLDDALICLTEQPELANKVDMVWIV
L159P, L160P, E173A, F180L) GGSSVYKEAMSYPGHLKLLVTRIMQDSESDTFFPEIDLE
KYKPPPEYPGVLSDVQEAKOKYKLEVYEKND
hDHFR (V2A, II7V, N30D, MAGSLNCIVAVSQNMGVGKNGDLPWPPLRDGFRYFRR 233 E3 1G, Q36R, F59S, K69E, I72T, MTITSSVEGKQNLVIMGKKTWSSIPEKNRPLEGRTNLV
H88Y, F89L, N108D, K109E, LSRELKEPPQGAYLLSRSLDDALKLTEQPELADEAGMV
V110A, 1115V, Y122D, L132P, WVVGGSSVDKEAKNHPGHPKLSVIRIVQDFGSDAFFPE
F135S, M140V, E144G, T147A, IDLEKCKLLPEYPGVLSDAQEERGIKYKFEVYEKSD
Y157C, V170A, K174R, NI86S) hDHFR (L28P, N3OH, M38V, MVGSLNCIVAVSQNMGIGKNGDLPWPPPRHEFRYFQRV 234 V44A, L68S, N73G, R78G, A97T, TTTSSAEGKQNLVIMGKKTWFSIPEKNRPSKGRIGLVLS
K99R, A107T, K109R, DI I IN, GELKEPPQGAHFLSRSLDDTLRLTEQPELTNRVNMVWI
L134P, F135V, T147A, II52V, VGGSSVYKEAMNHPGHLRPVVTRIMQDFESDAFFPEVD
K158R, E172G, V I 82A, E184R) LEKYRLLPEYPGVLSDVQGEK G I KYKFEAYRKND
hDHFR (AlOT, Q13R, N I4S, MVGSLNCIVTVSRSMGIGKDGDLS'WPPLRSEFRYFQRTT 235 N20D, P24S, N30S, M381, T40A, ATSSVEGRQSLVINIGKRTWFS'IPERNRPLRGRANLVLS
K47R, N49S, K56R, 1611, K64R, GELKGPPQGAHLLSRSLDGALKLTEQPELADKVDVVRI
K69R, r2A, R78G, E820, F89L, VGGSSVDEEAMNHPGHLKLEVTRVMRGFESDTLFPGID
D96G, N108D, M112V, W114R, LGICRKLLPEYPGVLSDVREEKOKYICLEVCGNN
Y122D, K123E, I139V, Q141R, D142G, F I 48L, El5 I G, E1.55G, Y157R, Q17IR, Y1.83C, E184G, K185del, D 1 87N) hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGVGKNGDLPWPPLRNEFRYFQRM 236 117V, Y122I) TTTSSVEGKQNLVLMGKKTWFSIPEKNRPLKGRINLVLS
RELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVWI
VGGSSVIKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDL
EKYKLLPEYPGVLSDVQEEKGIKYKFE'VYEKND
hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 237 Y1221, M140I) TTSSVEGKQNLVIMGICKTWFSIPEKNRPLKGRINLVLSR

GGSSVIKEAMNHPGHLKLFVTRIIQDFESDTFFPEIDLEK
YKLLPEYPGVLSDVQEEKGIKYK.FEVYEKND
hDHFR (Amino acid 2-187 of WT VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 238 N127Y, Y1221) TISSVEGKQNLVIMGKICIVFSIPEKNRPLKGRINLVLSR
ELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVWIV

GGSSVIKEAMYHPGHLKLEVTRIMQDFESDTITPEIDLE
KYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDHFR (Amino acid 2-187 of WT: VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 239 Y1221, H131R, E144G) TTSSVEGKQNLVIMGICKTWFSEPEKNRPLKGRINLVLSR

GGSSVIKEAMNHPGRLKLEVTRIMQDFGSDTFFPEIDLE
KYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQN1s/IGIGKNGSLPWPPLRNEMSYFSRMT 240 D22S, F32M, R33S, Q36S, N65S) TTSSVEGKQNLV1MGKKTWFSIPEKSRPLKGRINLVLSR

KYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDI.PWPPLRNDMRYFQRM 241 E3 ID, F32M, VI 16!) TTTSSVEGKQNLVIMGKKTWFSIPEKNRPLK GRINLVLS

GGSSVYKEAMNHPGHLKLEVTRIMQDFESD rJ PPS-1)LE
KYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
WIER (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 854 E162G, 1176F) TTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSR

GGSSVYKEAMNHPGHLKLFvrRIMQDFESDTFFPEIDLE
KYKLLPGYPGVLSDVQEEKGFKYKFEVYEKND
hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 855 K185E) TTSSVEGKQNLVIMGKKTWFSIPEICNRPLKGRINLVLSR

KYKLLPEYPGVLSDVQEEKOKYKFEVYEEND
hDHFR (Amino acid 2-187 of WI; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 856 Y1221, A125F) TTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSR
ELKEPPQGAHFLSRSLDDALICLTEQPELANKVDMVWIV
GGSSVIKEFMNHPGHLKLFVTRIMQDFESDTFFPEIDLEK
YKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFFRMT 857 Q36F, N65F, Y1220 usSVEGKQNINIMGKKTWFSIPEKFRPLKGRINLVLSR

GGSSVIKEAMNHF'GHLKLEVTRIMQDFESDTFFPEIDLE
KYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 858 N127Y) TTSSVEGKQNLVIMGICKTWFSIPEKNRPLKGRINLVLSR

GGSSVYK EAMYHPGHLKLEVTRIMQDFESDTFFPEIDLE
KYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 859 H131R, E144G) TTSSVEGKQNLVIMGICKTWFSIPEKNRPLKGRINLVLSR

GGSSVYICEAMNFEPGRLKLFVTRIMQDFGSDTFFPEIDLE
KYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGVGKNGDLPWPPLRNEFRYFQRM 860 II 7V) ITTSSVEGKQNLVEVIGKKTWFSIPEKNRPLKGRINLVLS
RELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVWI
VGGSSVYKEAMNHPGHLKLEVTRIMQDFESDTFFPEIDL
EKYKLLPEYPGVLSDVQEEKGEKYKFEVYEKND
hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 861 Y122I) TISSVEGKQNLVIMGKICTWESIPEKNRPLKGRINLVLSR

GGSSVIKEAMNHPGFILKLEVTRIMQDFESDIFEPEIDLE
KYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
hDHFR (E162G, 1176F) MVGSLNCIVAVSQNMGIGKNGDLP'WPPLRNEFRYFQR 862 MITTSSVEGKQNLVIMGICKTWFSIPEKNRPLKGRINLVL
SRELICEPPQGAHELSRSLDDALKLTEQPELANKVDMVW

TVGGSS'VYKEAMNHPG11 LK LIWIRIMQDFESDIFFPEID
LEKYKLLPGYPGVLSDVQEEKGFKYKFEVYEKND
hDHFR (Amino acid 2-187 of WT: VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFKRMT 863 Q36K, Y1221) usSVEGKQNLVIMGKKTWFSEPEKNRPLKGRINLVLSR
ELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVWIV
GGSSVIKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDLE
KYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
1001261 In some embodiments, DD mutations that do not inhibit ligand binding may be preferentially selected. In some embodiments, ligand binding may be improved by mutation of residues in DHFR.. Amino acid positions selected for mutation include aspartic acid at position 22 of SEQ ID NO. 2, glutamic acid at position 31 of SEQ ID NO. 2; phenyl alanine at position 32 of SEQ ID NO. 2; arginine at position 33 of SEQ ID NO. 2; glutamine at position 36 of SEQ
ID NO. 2; asparagine at position 65 of SEQ ID NO. 2; and valine at position 115 of SEQ ID NO.
2. In some embodiments, one or more of the following mutations may be utilized in the DDs of the present invention to improve TMP binding, including but not limited to, D225, E3 ID, F32M, R335, Q365, N655, and V116I. The position of the mutated amino acids is relative to the wildty-pe human DHFR (Uniprot ID: P00374) of SEQ ID NO. 2.
[00127] In some embodiments, novel DDs derived from human DHFR may include one, two, three, four, five or more mutations including, but not limited to, Mldel, V2A, C7R, I8V, V9A, A1OT, AlOV, Q I3R, N145, G165, I17N, II7V, K19E, N20D, G2IT, G21E, D225, L235, P24S, L28P, N30D, N3OH, N305, E3 IG, E3 ID, F32M, R33G, R335, F35L, Q36R, Q365, Q36K, Q36F, R37G, M38V, M38T, T40A, V44A, K47R, N495, N49D, M53T, G54R, K56E, K56R, T57A, F595, 161T, K64R, N65A, N655, N65D, N65F, L685, K69E, K69R, R7IG, I72T, I72A, I72V, N73G, L74N, V75F, R786, L80P, K81R, E826, H88Y, F89L, R92G, S93G, 593R, L94A, D96G, A97T, L985, K99G, K99R, LIOOP, E102G, Q103R, P1045, E105G, A107T, A107V, N108D, K109E, K109R, V110A, D111N, M112T, M112V, V113A, W114R,1115V,1115L, VI161, GI17D, V121A,Y122C,Y122D,Y1221, K123R, K123E, A125F, MI261, N127R, NI275, N127Y, H128R, H128Y, H131R, L132P, KI33E, L134P, F135P, FI35L, F1355, F135V, V136M, TI37R, RI38G, R1381, I139T, I139V, MI401, M140V, Q141R, D142G, F1435, F143L, E144G, DI46G, T147A, F1485, F148L, F149L, P150L, E151G, 1152V, DI53A, DI53G, E155G, K156R,Y157R, YI57C, K158E, KI58R, L159P, L160P, E162G, Y163C, V166A, 5168C, D1.69G, V170A, QI71.R, E172G, E173G, E173A, K1.74R, 1176A, 1176F, 11.76T, K177E, K177R,Y178C, Y178H, F180L, E18IG, VI82A,Y183C,Y183H, E184R, E184G, K185R, KI85del, KI85E, N1865, N186D, D I87G, and D187N.
[001.28] In some embodiments, novel DDs derived from human DHFR may comprise amino acids 2-187 of the wild type human DHFR sequence. This may be referred to as an Mldel mutation.

[00129] In some embodiments, novel DDs derived from human DHFR may comprise amino acids 2-187 of the wild type human DHFR sequence (also referred to as an Mldel mutation), and may include one, two, three, four, five or more mutations including, but not limited to, Mldel, V2A, C7R, I8V, V9A, A1OT, AlOV, Q13R, N14S, G16S, I17N, 117V, K19E, N20D, G21T, G21E, D22S, L23S, P24S, L28P, N30D, N3OH, N30S, E31G, E3 ID, F32M, R33G, R335, F35L, Q36R, Q36S, Q36K, Q36F, R37G, M38V, M38T, T40A, V44A, K47R, N49S, N49D, M53T, G54R, K56E, K56R, T57A, F59S, I6 1T, K64R, N65A, N65S, N65D, N65F, L68S, K69E, K69R, R71G, I72T, I72A, I72V, N73G, L74N, V75F, R78G, L8013, K81R, E82G, H88Y, F89L, R92G, S93G, 593R, L94A, D96G, A97T, L98S, K99G, K99R, L100P, E102G, Q103R, P104S, E105G, A107T, A107V, NI08D, K109E, KI09R, V1 10A, DI 11N, M112T, M112V, VI 13A, WI
14R, 1115V, 1115L, V1161, G117D, V121A, Y122C, Y122D, Y1221, K123R, K123E, A125F, M1261, N127R,N127S, N127Y, H128R, H128Y, H131R, L132P, K133E, L134P, F135P, F135L, F135S, F135V, V136M, TI37R, R138G, R1381, I139T, I139V, M1401, M140V, Q14IR, D142G, F143S, F143L, E1446, D146G, T147A, F148S, F148L, F149L, P150L, E151G, I152V, D153A, D153G, E155G, K156R, Y157R, Y157C, K158E, K158R, L159P, L160P, E162G, YI63C, V166A, S168C, D169G, V170A, Q171R, E172G, E173G, E173A, K174R, I176A, I176F, 1176T, K177E, K177R, Y178C, Y178H, F180L, E181G, V182A, Y183C, Y183H, E184R, E184G, K185R, K185del, K185E, N186S,N186D, D187G, and D187N.
2. Stimulus [00130] Biocircuits of the invention are triggered by one or more stimuli.
Stimuli may be selected from a ligand, an externally added or endogenous metabolite, the presence or absence of a defined ligand, pH, temperature, light, ionic strength, radioactivity, cellular location, subject site, microenvironment, the presence or the concentration of one or more metal ions.
[00131] In some embodiments, the stimulus is a ligand. Ligands may be nucleic acid-based, protein-based, lipid based, organic, inorganic or any combination of the foregoing. In some embodiments, the ligand is selected from the group consisting of a protein, peptide, nucleic acid, lipid, lipid derivative, sterol, steroid, metabolite derivative and a small molecule. In some embodiments, the stimulus is a small molecule. In some embodiments, the small molecules are cell permeable. Ligands useful in the present invention include without limitation, any of those taught in Table 2 of copending commonly owned US serial number 62/320,684, filed on 4/11/2016, or in US Provisional Application No. 62/466,596 filed March 3, 2017 and the International Publication W02017/180587, the contents of each of which are incorporated herein by reference in their entirety. In some embodiments, the small molecules are FDA-approved, safe and orally administered.
[00132] In some embodiments, the ligand binds to dihydrofolate reductase. In some embodiments, the ligand binds to and inhibits dihydrofolate reductase function and is herein referred to as a dihydrofolate inhibitor.
[00133] In some embodiments, the ligand may be a selective inhibitor of human DHFR.
Ligands of the invention may also be selective inhibitors of dihydrofolate reductases of bacteria and parasitic organisms such as Pneumocystis spp., Toxoplasma spp., Trypanosoma spp., Mycobacterium spp., and Streptococcus spp. Ligands specific to other DHFR may be modified to improve binding to human dihydrofolate reductase.
[00134] Examples of dihydrofolate inhibitors include, but are not limited to, Trimethopiim (TMP), Methotrexate (MTX), Pralatrexate, Piritrexim Pyrimethamine, Talotrexin, Chloroguanide, Pentamidine, Trimetrexate, aminopterin, Cl 898 trihydrochloride, Pemetrexed Disodium, Raltitrexed, Sulfaguanidine, Foloty-n, Iclaprim and Diaveridine.
Other examples of DHFR inhibitors include BAL0030543, BAL0030544 and BAL0030545, developed by Basillea Pharmaceuticals; as well as WR 99210, and P218. Any of the inhibitors described by Zhang Q et al. (2015) Int J Antimicrob Agents. 2015 Aug; 46(2): 174-182 (the contents of which are incorporated herein by reference in their entirety). Some inhibitors contain bulky benzyl groups that dramatically diminish binding to human DHFR. In some embodiments, the inhibitors may be designed without bulky benzyl groups to improve TMP binding.
[00135] In some embodiments, ligands of the present invention may be polyglutamate or non polyglutamylatable. Like naturally occurring folates, polyglutamatable folates also contain a glutamic acid residue and therefore undergo intracellular polyglutamylation.
In contrast, non-polyglutamatable antifolates are devoid of a glutamate residue and thus are not available for polyglutamylation. In some embodiments, polyglutamylatable ligands may be preferred to increase intracellular retention as they can no longer be exported out of the cell. In other embodiments, non polyglutamylatable ligands may be preferred to decrease intracellular retention.
[00136] In some embodiments, ligands of the present invention may include dihydrofolic acid or any of its derivatives that may bind to hiunan DHFR. In some embodiments, the ligands of the present invention, may be 2,4, diaminohetrocyclic compounds. In some embodiments, the 4-oxo group in dihydrofolate may be modified to generate DHFR inhibitors. In one example, the 4 -oxo group may be replaced by 4-amino group. Various diamino heterocycles, including pteridines, quinazolines, pyridopyrimidines, pyrimidines, and triazines, may also be used as scaffolds to develop DHFR inhibitors and may be used in the present invention. The crystal structure of DHFR in complex with known DHFR inhibitors may be utilized in the rational design of better DHFR ligands. The ligands used herein include a 2,4-diaminopyrimidine ring with a propargyl group linked to an optionally substituted aryl or heteromyl ring (as described in US Patent No.
US 8,426,432; the contents of which are incorporated herein by reference in their entirety).
[00137] In some embodiments, ligands include TMP- derived ligands containing portions of the ligand known to mediate binding to DHFR. Ligands may also be modified to reduce off-target binding to other folate metabolism enzymes and increase specific binding to DHFR.
3. Payloads: immunotherapeutic aeents 1001381 In some embodiments, payloads of the present invention may be immunotherapeutic agents that induce immune responses in an organism. The immunotherapeutic agent may be, but is not limited to a cytokine, a safety switch (e.g., a suicide gene), a regulatory switch, a chimeric antigen receptor, or any agent that induces an immune response. In one embodiment, the immunotherapeutic agent induces an anti-cancer immune response in a cell, or in a subject.
[00139] In some embodiments, the payload of the invention may be any of the co-stimulatory molecules and/or intracellular domains described herein. In some embodiments, one or more co-stimulatory molecules, each under the control of different SRE may be used in the present invention. SRE regulated co- stimulatory molecules may also be expressed in conjunction with a first generation CAR, a second generation CAR, a third generation CAR, a fourth generation, or any other CAR design described herein.
Cvtokines. chemokines and other soluble factors 1001401 In accordance with the present invention, payloads of the present invention may be cytokines, chemokines, growth factors, and soluble proteins produced by immune cells, cancer cells and other cell types, which act as chemical communicators between cells and tissues within the body. These proteins mediate a wide range of physiological functions, from effects on cell growth, differentiation, migration and survival, to several effector activities. For example, activated T cells produce a variety of cytokines for cytotoxic function to eliminate tumor cells.
[00141] In some embodiments, payloads of the present invention may be cytokines, and fragments, variants, analogs and derivatives thereof, including but not limited to interleukins, tumor necrosis factors (1'NFs), interferons (IFNs). TGF beta and chemokines.
It is understood in the art that certain gene and/or protein nomenclature for the same gene or protein may be inclusive or exclusive of punctuation such as a dash "-" or symbolic such as Greek letters.
Whether these are included or excluded herein, the meaning is not meant to be changed as would be understood by one of skill in the art. For example, IL2, IL2 and IL 2 refer to the same interleukin. Likewise, TNFalpha, TNFa, TNF-alpha, TNF-a, TNF alpha and TNF a all refer to the same protein. In some embodiments, payloads of the present invention may be cytokines that stimulate immune responses. In other embodiments, payloads of the invention may be antagonists of cytokines that negatively impact anti-cancer immune responses.
[00142] In some embodiments, payloads of the present invention may be cytokine receptors, recombinant receptors, variants, analogs and derivatives thereof; or signal components of cytokines.
[00143] In some embodiments, cytokines of the present invention may be utilized to improve expansion, survival, persistence, and potency of immune cells such as C.D8+TEm, natural killer cells and tumor infiltrating lymphocytes (TM) cells used for immunotherapy. In other embodiments, T cells engineered with two or more DD regulated cytokines are utilized to provide kinetic control of T cell activation and tumor microenvironment remodeling. In one aspect, the present invention provides biocircuits and compositions to minimize toxicity related to cytokine therapy. Despite its success in mitigating tumor burden, systemic cytokine therapy often results in the development of severe dose limiting side effects. Two factors contribute to the observed toxicity (a) Pleiotropism, wherein cytokines affect different cells types and sometimes produce opposing effects on the same cells depending on the context (b) Cytokines have short serum half-life and thus need to be administered at high doses to achieve therapeutic effects, which exacerbates the pleiotropic effects. In one aspect, cytokines of the present invention may be utilized to modulate cytokinc expression in the event of adverse effects. In some embodiments, cytokines of the present invention may be designed to have prolonged life span or enhanced specificity to minimize toxicity.
[00144] In some embodiments, the payload of the present invention may be an interleukin (IL) cytokine. Tnterleukins (ILs) are a class of glycoproteins produced by leukocytes for regulating immune responses. As used herein, the term "interleukin (IL)" refers to an interleukin polypeptide from any species or source and includes the full-length protein as well as fragments or portions of the protein. In some aspects, the interleukin payload is selected from ILI, ILlalpha (also called hematopoietin-1), ILlbeta (catabolin), IL 1 delta, IL I
epsi lon, ILleta, IL 1 zeta, interleukin-1 family member 1 to 11 (IL1F1 to IL1F11), interleukin-1 homolog 1 to 4 (IL1H1 to IL1H4), ILI related protein 1 to 3 (IL1RP1 to IL1RP3), IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, ILIO, IL1OC, IL10D, IL11, ILI la, ILI lb, IL12, IL13, IL14, IL15, IL16, IL17, IL17A, 1117B, IL17C, IL17E, IL17F, IL18, IL19, IL20, IL20 like (IL2OL), 1121, IL22, IL23, IL23A, IL23-p19, 1L23-p40, IL24, 1125, IL26, IL27, IL28A, IL2813, IL29, IL30, 1L31, IL32. 1L33, IL34, IL35, IL36 alpha, IL36 beta, IL36 gamma. IL36RN, IL37, IL37a, IL37b, IL37c, 11,37d, IL37e and IL38. In other aspects, the payload of the present invention may be an interleukin receptor selected from CD121a, CDw121b, IL2Ra/CD25, IL2R13/CD122, IL2Ry/CD132, CDw131, CD124, CD131, CDw125, CD126, CD130, CD127, CDw210, IL8RA, ILI1Ra, CD212, CD213a1, CD213a2, IL14R, IL15Ra, CDw217, IL18Ra, IL18Ri3, IL2ORa, and IL201213.
1001451 In one embodiment, the payload of the invention may comprise IL2. In one aspect, the effector module of the invention may be a DD-IL2 fusion polypeptide. The amino acid sequences corresponding to DD-IL2 and its components are listed in the Table 4. The amino acid sequences in Table 4 may comprise a stop codon which is denoted in the table with a "s" at the end of the amino acid sequence.
Table 4: DD-IL2 construct seauences Description - Amino Acid Sequence Amino Nucleic Acid SEQ Acid SEQ
ID NO ID NO/
Sequence IL2 signal MYRMQLLSCIALSLALVTNS 49 55-56, sequence 117-118 Linker EFSTEF 50 57 Linker MH ATGCAC

LTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNF
BLRPRDLISNINVIVLELKGSE'TTFMCEYADETATIVEFL
NRWITFCQSIISTLT
FKBP (F36V, GVQVETISPGDGRIFPICRGQTCVVHYTGMLEDGKKVDS 11 60, 878-LIO6P) SRDRNKPFICFMLGKQEVIRGWEEGVAQMSNIGQRAICLT1 882 SPDYAYGATGHPGIIPPHATINFDVELLKPE
ecDHFR (Amino ISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNKP 9 61.869-acid 2-159 of VIMGRHTWESIGRPLPGRICNIILSSQPGTDDRVTWVKSV 874 (R12Y, DEAIAACGDVPELMVIGGGRVIEQFLPKAQICLYLTHIDA
Y100I) EVEGDTHFPDYEPDOWESVFSEFHDADAQNSHSYCFElL
ERR
OT-IL2-001 (1L2 MYRMQLLSCIALSLALViNSEFSTEFGVQVETISPGDGR 52 62 Signal Sequence - TFPKRGQTCWHYTGMLEDGKKVDSSRDRNKPFKFML
Linker (EFSTEF)- GKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGH
FICBP (F36V, PGIIPPHATLVFDVELLKPEMHAPTSSSTKKTQLQLEHLL
LIO6P)- Linker LDLQMILNG1NNYKNPICLTRMLTFKFYMPKKATELICHL
(MH) - IL2- stop) QCLEEELICPLEEVLNLAQSKNFHLRPRDLISNINVIVLEL
KGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT
OT-IL2-002 (IL2 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLL 53 63 Signal Sequence - DLQMILNGINNYKNPKL1RMLTFKFYMPKKATELKHLQ
1L2- stop) CLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK
GSETTFMCEY.ADETATIVEFLNRWrrH:Qsi ESTLT
OT-11,2-003 (IL2 MYRMQLLSCIALSLALVINSEFSTEFISLIAALAVDYVIG 54 64 Signal Sequence - MENAMPWNLPADLAWFKRNTLNKPVIMGRHTWESIGR
Linker (EFSTEF)- PLPGRKNIILSSQPGIDDRVINVVKSVDEAIAACGDVPEI
ecDHFR (Amino MVIGGGRVIEQFLPKAQICLYLTHIDAEVEGDTHFPDYEP
acid 2-159 of DDWESVFSEFHDADAQNSHSYCFEILERRMHAPTSSS1X
WT) (RI 2Y, KTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYM
Y100I) - Linker PKICATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDL
(MH) - IL2- stop) ISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFC
QSIISTLT

[00146] In some aspects of the invention, an IL2 mutein may be used as a payload. As used herein, the term "mutein" is a construct, molecule or sequence of a mutation, change or alteration in a protein and hence is also known as a mutant, e.g., a protein mutant, mutein. Consequently, an "IL2 mutein" is an IL2 mutant. In some embodiments an IL2 mutein is a variant of wild type IL2 protein, where the wildtype IL2 consists of the amino acid sequence of SEQ
ID NO. 51. In some aspects, it refers to an IL2 variant which binds to and activates only cells expressing IL2Ra1iy, but does not significantly bind to or activate cell expressing only IL2Riiy. In some examples, an IL2 mutein may be an IL2 protein in which residues of IL2 responsible for binding to either IL2R13 or IL2Ry are substituted to abolish the interaction of IL2 with IL2R13 or IL2Ry.
In other examples, an IL2 mutein may be an IL2 protein comprising mutations conferring high affinity for IL2Ra. An IL2 mutein may be an IL2 selective agonist (IL2sA) which can preferentially activate the high affinity 1L2 receptor (i.e., IL2Rc4ry) which is necessary to selectively activate T cells with respect to NK cells. In some embodiments, the IL2 mutein may be IL2 protein which preferentially binds to the lower affinity IL2Riii but with reduced affinity to CD25.
[00147] In some embodiments, IL2 muteins may be used to preferentially expand or stimulate Treg cells. As used herein "preferentially expand or stimulate Treg cells"
means the IL2 muteins promote the proliferation, survival, activation and /or function of T
regulatory cells.
[00148] Exemplary IL2 muteins may include, but are not limited to, N88R
substitution (Shanafelt et al.. Nature Biotech., 2000, 18:1197-1202), an IL2 with a V91K
substitution (e.g., US Patent publication NO. U520140286898); V91K substitution, C125A
substitution, an IL2 with three mutations: V69A, N71R, Q74P; an IL2 mutein with high affinity for IL2Ra (N295, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P); an IL2 mutein with high affinity for IL2Ra and reduced signaling activity (N29S, Y31H, K35R, T37A, K48E, V69A, N71R, Q74P, N88D), and D2OH, D201, N88G, N88I, N88R, and Q126L substitutions as described in PCT
application NO. 1999060128; the contents of each of which are incorporated herein by reference in their entirety. In other aspects, IL2 muteins may include those described in US
Patent NOs. 4,518,584;
5,116,943; 5,206,344; 6,955,807; 7,105,653; 7,371,371; 7,803,361; 8,124,066;
8,349,311;
8,759,486; and 9,206,243: PCT patent publication NOs. W02005086751 and W02012088446;
European Patent N.s.: EP0234599 and EP0200280 and Sim, G.C. et al. (2016) Cancer Immunol Res; 4(11):983-994; the contents of each of which are incorporated herein by reference in their entirety.
[00149] in some aspects, the IL2 mutein may be fused to a polypeptide that extends the serum half-life of the IL2 mutein, such as an IgG Fe fragment. Preferred Fe regions are derived from human IgG, which includes IgGI, IgG2, IgG3, and IgG4. In other aspects, the payload of the invention may be an IL2 fusion protein comparing a second functional polypeptide. In a non-limiting example, an IL2 fusion protein may comprise an IL2 or IL2 mutein polypeptide fused with a pro-apoptotic Bc1-2 family polypeptide (such as Bad, Bik/Nbk, Bid, Bim/Bod, Hrk, Bak or Bax); such fusion protein may be capable of inhibiting cell survival, inhibiting cell proliferation, or enhancing cell death or apoptosis of a target cell expressing an IL2 receptor.
Alternatively, an IL2 or IL2 mutein polypeptide may be fused with an anti-apoptotic Bc1-2 family polypeptide (such as Bc1-xi, Bcl-w or Bc1-2). The fusion protein may be capable of enhancing cell survival, enhancing cell proliferation, or inhibiting cell death or apoptosis of a target cell expressing an IL2 receptor. See, e.g., US patent publication NOS.
US2016/0229901.
[00150] In addition, the IL2 fusion protein may be a IL2-GMCSF fusion protein which can promote cell-cell interaction; therefore, enhances anti-cancer immune responses (Wen et Translational Med., 2016, 14: 41).
Safety switch [00151] In some embodiments, payloads of the present invention may comprise SRE regulated safety switches that can eliminate adoptively transferred cells in the case of severe toxicity, thereby mitigating the adverse effects of T cell therapy. Adoptively transferred T cells in immunotherapy may attack normal cells in response to normal tissue expression of TAA. Even on-tumor target activity of adoptively transferred T cells can result in toxicities such as tumor lysis syndrome, cytokine release syndrome and the related macrophage activation syndrome.
Safety switches may be utilized to eliminate inappropriately activated adoptively transferred cells by induction of apoptosis or by immunosurveillance.
[00152] In some embodiments, payloads of the present invention may comprise inducible killer/suicide genes that acts as a safety switch. The killer/suicide gene when introduced into adoptively transferred immune cells, could control their alloreactivity. The killer/suicide gene may be an apoptotic gene (e.g., any Caspase gene) which allows conditional apoptosis of the transduced cells by administration of a non-therapeutic ligand of the SRE
(e.g., DD).
[00153] In some embodiments, the payload of the present invention may be Caspase 9. In some instances, Caspase 9 may be modified to have low basal expression and lacking the caspase recruitment domain (CARD) (SEQ ID NOS. 26 and SEQ ID NOS. 28 of US Patent No.
U59434935B2; the contents of which are incorporated by reference in their entirety).
[00154] In one embodiment, the payload of the present invention is a suicide gene system, iCasp9/Chemical induced dimerization (CID) system which consists of a polypeptide derived from the Caspase9 gene fused to a drug binding domain derived from the human FK506 protein.

Administration of bioinert, small molecule AP1903(rimiducid), induces cross linking of the drug binding domains and dimerization of the fusion protein and in turn the dimerization of Caspase 9. This results in the activation of downstream effector Caspase 3 and subsequent induction of cellular apoptosis (Straathof et al., Blood, 2005, 105: 4247-4254;
incorporated herein by reference in its entirety). Preclinical trials using CART including an iCasp9 gene have shown effective elimination of CAR. T cells in vivo in mouse models and demonstrate the potential efficacy of this approach. (Budde et al, Plos One, 2013, 8: e82742.10.1371;
Hoyos et al., Leukemia, 2010; 24(6):1160-1170). In one embodiment; the payload of the invention may comprise Caspase 9. In one aspect, the effector module of the invention may be a DD-Caspase9 fusion polypeptide. The DD-Caspase 9 may comprise the amino acid sequences provided in Table 5. The amino acid sequences in Table 5 may comprise a stop codon which is denoted in the table with a "s" at the end of the amino acid sequence Table 5: DD-Casoase 9 constructs Description/ Amino acid sequence Amino Nucleic Construct ID Acid Acid SEQ
SEQ ID ID NO./
NO. Sequence Caspase 9 DEADRRLLRRCRLRLVEELQVDQLVvDALLSRELFRPH 65 81-82 MIEDIQRAGSGSRRDQARQUIDLETRGSQALPLFISCLE
DTGQDNILASFLRTNRQAAICLSICPTLENLTPVVLRPEER
ICPEVLRPETPRPVDIGSGGFGDVGALESLRGNADLAYI
LSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRR
FSSLHFMVEVKGDLTAKICMVLALLELAQQDHGALDC
CVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIF
NGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPED
ESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSY
STFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSL
LLRVANAVSVKGIYKQMPGCFNFLRICKLFFKTS
Caspase delta GVDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNV- - 66 83 CD NFCRESGLIURTGSNIDCEKLRRRFSSLHFMVEVKGDL

QFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFF
IQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQE
GLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGS
WYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYK
QMPGCFNFLRKKLFFKTS
Linker VDYPYDVPDYALD 67 84 Linker SGGGS 68 85.69,86 Linker QLIGMLQGLMRDL 908 909 Linker SG AGCGGC
FKBP (F36V, GVQVETISPGDGRTFPKRGQTCVVHYTGNILEDGKKVD 11 60, 878-L106P) SSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAK 882 LTISPDYAYGATGHPGIIPPHATLVFDVELLKPE
FKBP (F36V) GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKK VD 70 87 SSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAK
LTISPDYAYGATGHPGIIPPHATLVFDVELLKLE

FKBP (E31G, GVQ'VETISPGDGRTFPKRGQTCWHYTGMLGDGICKV 12 88, 883-F36V. R71G. DSSRDRNKPFKFMLGKQEVIRG'WEEGVAQMSVGQGA 889 K105E) KLTISPDYAYGATGIIPGITPPHATI, VFLYVELLELE
ecDHFR MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTL 8 89 (R12Y, Y100I) NKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTW
VKSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQICLYL
THIDAEVEGDTHFPDYEPDDWESVFSEFFIDADAQNSH
SYCFEILERR
ccDHFR 1SLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNK 71 90 (Amino acid 2- PVEVIGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWK
159 of WT) SVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQICLYLTH
(R 12Y, IDAEVEGDTHFPDYKPDDWESVFSEFHDADAQNSHSY
E129K) CFE1LERR
ecDHFR ISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNK 9 61,869-(Amino acid 2- PVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWVK 874 159 of WI) SVDEALAACGDVPEIMVIGGGRVIEQFLPKAQKLYLTHE
(R12Y, Y1001) DAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYC
FEILERR =
hDHFR MVGSLNC1VAVSQNMGIGKNGDLP'WPPLRN EFRYFQR 18 91 (Y1221) MTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLV
LSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDM

EIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
liDHFR MVGSLNCIVAVSQNMGIGKNGDLP'WPPLRNEFRYFQR 31 92 (V75F, Y1221) MTITSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLF
LSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDM

EIDLEKYICLLPEYPGVLSDVQEEKOKYKFEVYEKND
hDHFR MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 12 91 (L94A, MTITSSVEGKQNLVIMGKKTWFS1PEKNRPLKGRINLV
T147A) LSRELKEPPQGAHFLSRSADDALKLTEQPELANKVDM
VWIVGGSSVYKEAKNHPGHLKLFVTRIMQDFESDAFF

001 (Met ¨ VDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQR
FKBP (F36V, AKLTISPDYAYGATGHPGIIPPHATLVFDVELLKPESGG
L 1 06P) -- GSDEADRRLLRRCRLRLVEELQVDQLWDALLSRELFR
Linker PHMIEDIQRAGSGSRRDQARQLI1DLETRGSQALPLFISC
(SGGGS) ¨ LEDTGQDMLASFLRTNRQAAKLSKPTLENLTPWLRPE
Caspase 9- 1RKPEVLRPETPRPVDIGSGGFGDVGALESLRGNADLA
stop) YILSMEPCGHCLBNINIVNFCRESGLRTRTGSNIDCEKLR
RRFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGAL

NIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSP
EDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDTV

QSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS*

002 (ecDHFR NKPVIMGRHTWESIGRPLPGRKNI1LSSQPGTDDRVTW
(R12Y, Y1001) VKSVDEA1AACGDVPEIMVIGGGRVIEQFLPKAQKLYL
¨ Linker TH1DAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSH
(SGGGS) - SYCFEILERRSGGGSDEADRRLLRRCRLRLVEELQVDQ
Caspase 9- LWDALLSRELFRPFLMIEDIQRAGSGSRRDQARQLIIDLE
stop) TRGSQALPLFISCLEDTGQDMLASFLRTNRQAAKLSKP
TLENLTPVVLRPEIRICPEVLRPETPRPVDIGSGGFGDVG
ALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRT

LELAQQDHGALDCCVVVILSHGCQASHLQFPGAVYGT
DGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQK
DHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLD

AISSLPTHDIFV SY STFPGFVSWRDPK SOS WY v-gruDDJ
FEQWAHSED LQSLLLRVANAVSVKG IYKQMPGCF NT:
RKKLFFICTS*

003 (11DHFR MTITSSVEGKQNLVIMGKKTWFS1PEKNRPLKGRINLV
(Y122I) ¨ LSREL10EPPQGAHFLSRSLDDALKLTEQPELANKVDM
Linker VWIVGGSSVIKEAMNHPGHLKLFVTRLMQDFESDITFP
(SGGGS) - EIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKNDS
Caspase 9- GGGSDEADRRLLRRCRLRLVEELQVDQLWDALLSREL
stop) FRPHMIEDIQRAGSGSRRDQARQLDDLETRGSQALPLFI
SCLEDTGQDMLASFLRTNRQAAKLSKPTLENLTPVVLR
PEIRKPEVLRPETPRPVDIGSGGFGDVGALESLRGNADL
AY IL SMEPCGHCL TINNVNFCRESGLRTRTGSNIDCEKL
RRRFS SLHFMVEVKGDLTAKKMVLALLELAQQDHGA
LDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEK IV
NIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVA STSP
EDESPG SNPEPDATPFQEGLRTFDQLDA ISSLPTPSDIFV
SY STFPGFVSWRDPKSG SWYVETLDDIFEQWAHSEDL
QSLLLRVANAVSVKGIYKQNPGCFNFLRKKLFFKTS*
=
OT-CASP9- MVGSLNCIVAVSQNMGIGKNGDLP'WPPLRNEFRYFQR 75 97 004 (hDHFR MTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLF
(V75F, Y1221) LSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDM
¨ Linker VWIVGG SSVIKEAMNHPGHLKLFVTRIMQDFESDTFFP
(SGGGS) - EIDLEKYKLLPEYPGVL SD VQEEKGI( YKFE VYEKNDS
Caspase 9- GGGSDEADRRLLRRCRLRLVEELQVDQLWDALLSREL
stop) FRPHMIEDIQRAGSGSRRDQARQUIDLETRGSQALPLF1 SCLEDTGQDMLASFLRTNRQAAKLSKPTLENLTPVVLR
PEIRKPEVLRPETPRPVINGSGGFGDVGALESLRGNADL
AYILSMEPCGHCL FINN VNFCRESGLRTRTGSNIDCEKL
RRRFS SLHFMVEVKGDLTAKICMVLALLELAQQDHGA

NIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSP
EDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFV
SYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDL
QSLLLRVANAVSVKGIYKQNPGCFNFLRKKLFFKTS*

005 (hDHFR MMSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLV
(L94A. LSRELKEPPQGAHFLSRSADDALKLTEQ PEL ANKVDM
T147A) VWIVGG SSVYKEAMNHPGHLKLFVTRIMQDFESDAFF
Linker PEIDLEKYKLLPEYPGVLSDVQEEKG1KYKFEVYEKND
(SGGGS) - SGGGSDEADRRLLRRCRLRLVEELQVDQLWDALLSRE
Caspase 9- LFRPHMIEDIQRAGSGSRRDQARQLI1DLETRGSQALPL
stop) FISCLEDTGQDMLASFLRTNRQAAKLSKPTLENLTPVV

KLRRRFSSLHFMVEVKGDLTAKKMVLALLELAQQDH
GALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVE
KIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVAS
TSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSD

DLQSLLLRVANAVSVKGIYKQMPGCFNFLRKICLFFKTS

006 (Met ¨ KVDSSRDRNKPFICFMLGKQEVIRGWEEGVAQMSVGQ
Leu ¨ Glu - RAKLTISPDYAYGATGHF'GIIPPHATLVFDVELLKLESG
FKBP (F36V) GGSGVDGFGDVGALESLRGNADLAYILSMEPCGHCLII
¨ Linker NNVNFCRESGLRTRTGSN1DCEKLRRRFSSLHFMVEVK
(SGGGS) - GDLTAKKMVLALLELARQDHGALDCCVVV1LSHGCQ
Caspase Delta ASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGK
CD ¨ Linker PKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDAT

(QLIGNILQG PFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGINSWRDP
LMRDL) - KSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVK
stop) GIYKQMPGCFNFIRKKLIFFKTSQL.IGMLQGLMRDL*
OT-CASP9- MDEADRRLLRRCRLRLVEELQVDQL'WDALLSRELFRP 78 IOU
007 (Met - HMIEDIQRAGSGSRRDQARQUIDLETRGSQALPLFISCL
Caspase 9¨ EDTGQDMLASFLRTNRQAAKLSKPTLENLTPVVLRPEI
Linker (SG) ¨ RKPEVLRPETPRPVDIGSGGFGDVGALESLRGNADLAY
FKBP (E31G, ILSNIEPCGHCLIININVNFCRESGLWIRTGSNIDCEKLRR
F36V, R7 1G, RFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALD
K105E) - stop) CC VVVILSHGCQ ASHLQFPGA VYGTDGCPVS VEKIVN1 FNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPE
DESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVS
YSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQ
SLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSSGG
VQVETISPGDGRTFPKRGQTCWHYTGMLGDGKKVDS
SRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQGAKL
TISPDYAYGAIGHPGIIPPHATLVFDVELLELE*

008 (Met - HMLEDIQRAGSGSRRDQARQUIDLETRGSQALPLFISCL
Caspase 9¨ EDTGQDMLASFLRTNRQAAKLSKPTLENLTPVVLRPEI
Linker (SG) - RKPEVLRPETPRPVDIGSGGFGDVGALESLRGNADLAY
ecDHFR ILSNIEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRR
(Amino acid 2- RFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALD
159 of WT) CCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNI
(RI 2Y, YI00I) FNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPE
- stop DESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVS
YSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQ
SLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSSGI
SLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLNK
PVLMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWVK
SVDEAIAACGDVPEINIVIGGGRVIEQFLPKAQKLYLTHI
DAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYC
FEILERR*
OT-CASP9- MDEADRRLLRRCRLRLVEELQVDQUWDALLSRELF'RP 80 102 009 (Met - HMIEDIQRAGSGSRRDQARQL EIDLETRGSQALPLFISCI., Caspase 9- EDTGQDMLASFLRTNRQAAKLSKPTLENLTPVVLRPEI
ecDIER RKPEVLRPETPRPVDIGSGGFGDVGALESLRGNADLAY
(Amino acid 2- ILSMEPCGHCLIINNVNFCRESGLRTRTGSNTDCEKLRR
159 of WI) RFSSLHFMVEVKGDLTAKKMVLALLELAQQDHGALD
(R12Y, CC VVVILSHGCQ ASHLQFPGA VYGTDGCPVS VEK IVNI
E129K) - stop) FNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPE
DESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVS
YSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQ
SLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSSGI
SLIAALAVDYVIGNIENAMPWNLPADLAWFKRNTLNK
PVLMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWVK
SVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTH
IDAEVEGDTHFPDYKPDDWESVFSEFFIDADAQNSHSY
CFEILERR*
1001551 In some instances, the iCasp9/CID system has been shown to have a basal rate of dimerization even in the absence of rimiducid, resulting in unintended cell death. Regulating the expression levels of iCasp9/CID is critical for maximizing the efficacy of iCasp9/CID system.
Biocircuits of the present invention and/or any of their components may be utilized in regulating or tuning the iCasp9/CID system to optimize its utility. Other examples of proteins used in dimerization-induced apoptosis paradigm may include, but are not limited to Fas receptor, the death effector domain of Fas-associated protein, FADD, Caspase 1, Caspase 3, Caspase 7 and Caspase 8. (Belshaw P.J. et al, Chem BioL , 996,3:731-738; MacCorkle R.A. et al, Proc Natl Acad Sci, 1998, 95:3655-3660; Spencer, D.M. et al., Curr Biol. 1996; 6:839-847; the contents of each of which are incorporated herein by reference in their entirety).
1001561 In some embodiments, the safety switch of the present invention may comprise a metabolic enzyme, such as herpes simplex virus thymidine kinase (HSV-TK) and cytosine deaminase (CD). HSV-TK phosphoiylates nucleoside analogs, including acyclovir and ganciclovir (GCV) to generate triphosphate form of nucleosides. When incorporated into DNA, it leads to chain termination and cell death. Unlike the mammalian thymidine kinase, HSV-TK
is characterized by 1000-fold higher affinity to nucleoside analogs such as GCV, making it suitable for use as a suicide gene in mammalian cells. Cytosine deaminase (CD) can converts 5-fluorocytosine (5-FC) into the cytotoxic 5-fluorouracil (5-FU) (Tiraby et al., FEMS Lett., 1998, 167: 41-49).
[00157] In some embodiments, the safety switch of the present invention may comprise a CYP4B1 mutant (as suicide gene), which may be co-expressed in a CAR engineered T cells (Roellecker et al., Gen Ther., 2016, May 19, doi: 10.1038/gt.2016.38.).
[00158] In some embodiments, the payload of the present invention may comprise a fusion construct that can induce cell death, for example, a polypeptide with the formula of St-RI-Si-Q-52-R2, wherein the St is a stalk sequence, R1/2 and Q are different epitopes;
and SI/2 are optional spacer sequences (See International patent publication NOS.
W02013153391; the content of which are incorporated herein by reference in their entirety).
[00159] In some embodiments, safety switch may be mediated by therapeutic antibodies which specifically bind to an antigen that is expressed in the plasma membrane of adoptively transferred cells. The antigen-antibody interaction allows cell removal after administration of a specific monoclonal antibody against the antigen. As non-limiting examples, payloads of the present invention may comprise the antigen and antibody pair used to mediate safety switch such as CD20 and anti-CD20 antibody (Griffioen et al., Haematologica, 2009, 94:1316-1320), a protein tag and anti-tag antibody (Kieback et al., NatL Acad. Sci. US.A., 2008, 105: 623-628), a compact suicide gene (RQR8) combining epitopes from CD34 (as a marker moiety) and CD20 (as a suicide moiety) which enables CD34 selection, cell tracking, as well as cell deletion after anti-CD20 monoclonal antibody administration (Philip et al., Blood, 2014, 124:
1277-1287);
truncated Inunan EGFR polypeptide and anti-EGFR monoclonal antibody (Wang et al., Blood, 2011, 118:1255-1263); and a compact poly-peptide safety switch having a structural formula as discussed in U.S Patent Application Publication NOS. US20150093401; the contents of each of which are incorporated herein by reference in their entirety.
Regulatory switch [00160j The utility of adoptive cell therapy (ACT) has been limited by the high incidence of graft versus host disease (GVHD). GVHD occurs when adoptively transferred T
cells elicit an immune response resulting in host tissue damage. Recognition of host antigens by the graft cells triggers a proinflammatoiy cytokine storm cascade that signifies acute GVHD.
GVHD is characterized as an imbalance between the effector and the regulatory arms of the immune system. In some embodiments, the payloads of the present invention may be used as regulatory switches. As used herein "regulatory switch" refers proteins, which when expressed in target cells increase tolerance to the graft by enhancing the regulatory arm of the immune system.
1001611 in one embodiment, regulatory switches may include payloads that preferentially promote the expansion of regulatory T (Treg cells). Tregs are a distinct population of cells that are positively selected on high affinity ligands in the thymus and play an important role in the tolerance to self-antigens. In addition, T regs have also been shown to play a role in peripheral tolerance to foreign antigens. Since Tregs promote immune tolerance, expansion of Tregs with the compositions of the invention may be desirable to limit GVHD.
1001621 In some embodiments, the regulatory switch may include, but is not limited to T regs activation factors such NFKB, FOXO, nuclear receptor Nr4a, Retinoic acid receptor alpha, NFAT, AP-1 and SMAD. Such factors can result in the expression of Fork headbox P3 (FOXP3) in T cells resulting in the activation of the regulatory T cell program and the expansion of T
cells.
1001631 In one embodiment, the regulatory switch may be FOXP3, a transcriptional regulator in T cells. A function of FOXP3 is to suppress the function of NFAT, which leads to the suppression of expression of many genes including IL2 and effector T-cell cytokines. FOXP3 acts also as a transcription activator for genes such as CD2S, Cytotoxic T-Lymphocyte Antigen Cytotoxic T-Lymphocyte Antigen 4 (CTLA4), glucocorticoid-induced TNF receptor family gene (GITR) and folate receptor 4. FOXP3 also inhibits the differentiation of IL 1 7 producing helper T-cells (Th17) by antagonizing RORC (RAR related orphan receptor C). Isoforms of FOXP3 lacking exon2 (FOXP3 delta 2), or exon 7 (FOXP3 delta 7) may also be used as regulatory switches. In one aspect, the effector module of the invention may be a DD-FOXP3 fusion polypeptide. The DD-FOXP3 may comprise the amino acid sequences provided in Table 6. The amino acid sequences in Table 6 may comprise a stop codon which is denoted in the table with a "*" at the end of the amino acid sequence.

Table 6: DD-FOXP3 constructs Construct/ Amino Acid sequence Amino Nucleic Description Acid SEQ Acid SEQ
ID NO. ID NO.
Linker SGGGS 68 85,69, 86 Linker SG AGCGGC
FKBP (F36V, GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVD 11 60, 878-L106P) SSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAK 882 LTISPDYAYGATGHPGIIPPHATLVFDVELLKPE
FKBP (E3 1G, GVQVETISPGDGRTFPKRGQTCVVHYTGMLGDGKKV 12 88, 883-F36V, R71G, DSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQGA 889 K105E) KLTISPDYAYGATGHPGIIPPHATLVFDVELLELE
eeDHFR MISLIAALAVDYVIGMENAMP'W'NLPADLAWFICRNTL 8 89 (R12Y, NKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTW
Y100I) VKSVDEAIAACGDVPEIMVIGGGRVIEQFLPICAQKLYL
THIDAEVEGDTHFPDYEPDDWESVFSEFFIDADAQNSH
SYCFEILERR
ccDHFR ISLIAALAVDYVIGMENAMPWNLPADLAWFICRNTLNK 9 61,869-(Amino acid PVEVIGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWITK 874 2-159 of WT) SVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYLTHI
(R 12Y, DAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYC
YlOOD FElLERR
FOXP3 full MPNPRPGKPS A PSLA LOPSPGASPSWRAAPKASULLGA 103 911 length RGPGGIFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPL
VMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAH
ARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPP
GINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSS
YPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEK
GRAQCLLQREMVQSLEQQLVLEKEICLSAMQAHLAGK
MALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPRE
APDSLFAVRRHLWGSHGNSTFPEFLHNNIDYFKFHNNIR
PPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRN
HPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEF
EtKKRSQRPSRCSNPIT'GP*
Amino Acid PNPRPOKPSAPSLALGPSPGASPSWRA.APKASOLLGAR 104 912 2-431 of GPGGTFQGRDLRGGAHASSSSLN PMPPSQLQLPTLPLV
FOXP3 full MVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAHAR
length TPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPPGI
NVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSSY
PLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEKG
RAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGKM
ALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPREAP
DSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFI-LNMRPP
FTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRNH

KKRSQRPSRCSNPTPGP
FOXP3 della MPNPRPGK PS A PSLALGPSPGASPSWRAAPKASULLGA 105 913 HARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLP
PGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQS
SYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDE
KGRAQCLLQREh4VQSLEQQLVLEKEKLSAMQAHLAG
ICMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPR
EAPDSLFANTRRHLWGSHGNSTFPEFLHNNIDYFKFHNM
RPPFITATURWAILEAPEKORTLNEIYHWFIRMFAFFR

NH PATWIC.N AIRHNL SLHKCFVRVESEKG A'VWTVDELE
FRKKRSQRPSRCSNPTPGP
FOX P3 delta PNPRPGK PS APS L A LGPSPG .A SPSWR.A APK.A SDLLG A R 106 914 HARTPVLQVHPLESPAMISLTPPITATGVFSLKARPGLP
PGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQS
SYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDE
KGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAG
KMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPR
EAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFFLNM
RPPFTYATL1RWAlLEAPEKQRTLNEIYHWFIRMFAFFR
NHPATWKNAIRHNL SLHKCFVRVESEKGAVWTVDELE
FRK KR SQRPSRCSNPTPGP

001 (FoxP3 - RGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPL
stop) VMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAH

GINVA SLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSS
YPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEK
GRAQCLLQREMVQSLEQQLVLEKEICLSAMQAHLAGK
MALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPRE
APDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMR

HPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEF
RK KR SQRPSRCSNPTF'GP*

002 (FoxP3 RGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLSTVDA
Delta 2- HARTPVLQVHPLESPAM1SLTPP11'ATGVFSLICARPGLP
stop) PGINVA SLEWVSREPALLCTFPNPSAPRKDSTLSAVPQS
SYPLLANG VCKWPGCEKVFE EPED FLKHCQADHLLDE
KGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAG
KMALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPR
EAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNM
RPPFTYATL1RWAlLEAPEKQRTLNEIYHWFTRMFAFFR
NHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELE
FRK KR SQRPSRCSNPTPGP*

003 (Met - VDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQR
FKBP (F36V, AKLTISPDYAYGATGHPGITPPHATLVFDVELLKPESGG
L106P) - GSPNPRPGKPSAPSLALGPSPGASPSWR AA PK A SDLLG
Linker ARGPGGTFQGRDLRGGA HA SSSSLNPMPPSQLQLPTLP
(SGGGS) - LVMVAPSGARLGPLPFILQALLQDRPHFMHQLSTVDAH
Amino Acid ARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPP
2431 of GINVA SLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSS
FOXT3 full YPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEK
length - stop) GRAQCLLQREMVQSLEQQLVLEKEICLSAMQAHLAGK
MALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPRE
APDSLFAVRRHLWGSHGNSTFPEFLHNMDYFICFHNMR
PPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRN
HPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEF
RKICRSQRPSRCSNPTPGP*
OT-FOXP3- MISLI A ALAVD Y Vl G MEN AMPWNLPADL AWFKRNTL 110 196 004 (ecDHF'R NKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTW
(R 12Y, VKSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYL
Y100I) ¨ THIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSH
Linker SYCFEILERRSGGGSPNPRPGKPSAPSLALGPSPGASPS
(SGGGS) - WRAAPKASDLLGARGPGGTFQGRDLRGGAHASSSSLN
Amino Acid PMPPSQLQLPTLPLVMVAPSGARLGPLPHLQALLQDRP
2-431 of HFMHQLSTVDAHARTPVLQVHPLESPAMISLTPPTTAT
GVFSLKARPGLPPONVASLEWVSREPALLCTFPNPSAP

FO XP3 full RKDSTLSAVPQS SYPLLANGVCKWPGCEKVFEEPEDFL
length - stop) KHCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKE
KLSAMQAHL AGKMALTKA SSVAS SDKGSCCIVAAGSQ
GPVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFL
FINMDYFKFHNMRPPFTYATLIRWAILEAPEKQRTLNEI
YHWFTRMFAFFRNHPATWKNAIRHNLSLHKCFVRVES
EKGAVWTVDELEFRKKR SQRPSRCSNPTPGP*

005 (FoxP3 ¨ RGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLPTLPL
Linker (SG) - VMVAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAH
FKBP (E31G, ARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPP
F36V, R710, (31NVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSS
K105E) - YPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEK
stop) GRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGK
MALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPRE
APD SLFA VRR HLWG SHGNSTFPEFLHNMDYFICRIN MR
PPFTYATLIRWAILEAPEKQRTLNEWHWFTRMFAFFRN
HPATWKNAIRHNLSLHKCFVRVESEK G AVWTVD EL E
RKKRSQRPSRCSNPTPGPSGGVQVETISPGDGRTFPKRG
QTCWHYTGMLGDGKKVDS SRDRNKPFKFMLGKQEV
IRGWEEGVAQMSVGQGAKLTISPDYAYGATGHPGIIPP
HATLVFDVELLELE*

VM.VAPSGARLGPLPHLQALLQDRPHFMHQLSTVDAH
(FoxP3 ¨ ARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPP
Linker (SG) - GINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQSS
ecDHFR YPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEK
(Amino ac id GRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGK
2-159 of WT) MALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPRE
(R 12Y, APDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNMR
Y100I) - PPFTYATURWAILEAPEKQRTLNEIYHWFTRMFAFFRN
stop) HPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELEF
RKKRSQRPSRCSNPTPGPSGISLIAALAVDYVIGMENA
NTWNLPADLAWFKRNTLNKPVIMGRHTWESIGRPLPG

GGGRVIEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDD
WESVFSEFHDADAQNSHSYCFEILERR*

007 (Met - VDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQR
FKBP (F36V, AKLTISPDYAYGATGHPGUPPHATLVFDVELLKPESGP
L106P) ¨ NPRPGKPSAPSLALGPSPGASPSWRAAPKASDLLGARG
Linker (SG) - PGGTFQGRDLRGGAHASSSSLNPMPPSQLQLSTVDAH
Amino Acid ARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLPP
2-396 of GINVA SLEWVSREPALLCTFPNPSAPRKDSTLSAVPQS S
FOXP3 delta YPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDEK
2- stop) GRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAGK
MALTKASSVASSDKGSCCIVAAGSQGPVVPAWSGPRE
APDSLFAVRRHLWGSHGNSTFPEFLHNMDYFICFHNMR
PPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFRN
HPATWKN A IRHNLSLHKCFVRVESEKGAVWTVDELEF
RK KR SQR PSRCSNPTPG P*

008 (ecDHFR NKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTW
(R 12Y, VKSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYL
Y100I) ¨ THIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSH
Linker (SG) - SYCFEILERRSGPNPRPGKPSAPSLALGPSPGASPSWRA
Amino Acid APKASDLLGARGPGGTFQGRDLRGGAHASSSSLNPMP
2-396 of PSQLQLSTVDAHARTPVLQVHPLESPAMISLTPPTTATG
VFSLICARPGLPPGINVASLEWVSREPALLCTFPNPSAPR

FOXP3 delta KDslisAVPQSSYPLIANGNICKWPGCEKVFEEPEDFLK
2 - stop) HCQADHLLDEKGRAQCLLQREMVQSLEQQLVLEKEK
LSAMQAHLAGICNIALTICASSVASSDKGSCCIVAAGSQG
PVVPAWSGPREAPDSLFAVRRHLWGSHGNSTFPEFLH
NMDYFKFFINMRPPFTYATLIRWAILEAPEKQRTLNEIY
HWFIRMFAFFRNHPATWKNAIRHNLSLHKCFVRVESE
KG AVWTVDELE FR KKRSQRPSRCSNPTPGP*

009 (FoxP3 RGPGGTFQGRDLRGGAHASSSSLNPMPPSQLQLSTVDA
Delta 2- HARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLP
Linker (SG) - PGINVASLEWVSREPALICTFPNPSAPRKDSTLSAVPQS
FKBP (E31(3, SYPLLANGVCKWPGCEKVFEEPEDFLKHCQADHLLDE
F36V, R71(3, KGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAG
K105E) - KMALTKA SSVA S SDK GSCCIVA AGSQGPVVPAWSGPR
stop) EAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFHNM
RPPFTYATLIRWAILEAPEKQRTLNEIYHWFTRMFAFFR
NHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELE
FRKKRSQRPSRCSNPTPGPSGGVQVETISPGDGRTFPKR
GQTCVVHYTGMLGDGICKVDSSRDRNICPFKFMLGKQE
NIRGWEEGVAQMSVGQGAICLTISPDYAYGATGHPGIIP
PHATLVFDVELLELE*

010 (FoxP3 RGPGGTFQGRDLRGGAHASSSSLNPN1PPSQLQLSTVDA
Delta 2- HARTPVLQVHPLESPAMISLTPPTTATGVFSLKARPGLP
Linker (SG) - PGINVASLEWVSREPALLCTFPNPSAPRKDSTLSAVPQS
ecDHFR SYPLLANGVCKWPGCEKVFEEPEDFLKHCQADFILLDE
(Amino acid KGRAQCLLQREMVQSLEQQLVLEKEKLSAMQAHLAG
2-159 of WT) KMALTICA SSVA S SDK GSCCIVA AGSQGPVVPAWSGPR
(1212Y, EAPDSLFAVRRHLWGSHGNSTFPEFLHNMDYFKFFINM
Y100I) - RPPFTYATLIRWAILEAPEKQRTLNEIYHWFMNIFAFFR
stop) NHPATWKNAIRHNLSLHKCFVRVESEKGAVWTVDELE
FRICICRSQRPSRCSNPTPGPSGISLIAALAVDYVIGMENA
MPWNLPADLAWFICRNTLNICPVIMGRHTWESIGRPLPG
RICNDLSSQPGTDDRVTWVKSVDEAIAACGDVPDMVI
GGGRVIEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDD
WESVFSEFHDADAQNSHSYCFEILERR*
Antibodies [001641 In some embodiments, antibodies, fragments and variants thereof are payloads of the present invention.
[00165] In some embodiments, antibodies of the present invention, include without limitation, any of those taught in Table 5 of copending commonly owned U.S. Provisional Patent Application No. 62/320,864, filed on 4/11/2016, or in US Provisional Application No.
62/466,596 filed March 3, 2017 and the International Publication W02017/180587, the contents of which are incorporated herein by reference in their entirety.
Antibody fragments and variants [00166] in some embodiments, antibody fragments and variants may comprise antigen binding regions from intact antibodies. Examples of antibody fragments and variants may include, but are not limited to Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules such as single chain variable fragment (scFv); and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each with a single antigen-binding site. Also produced is a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking with the antigen. Pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention may comprise one or more of these fragments.
1001671 For the purposes herein, an "antibody" may comprise a heavy and light variable domain as well as an Fc region. As used herein, the term "native antibody" usually refers to a heterotetrameric glycoprotein of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Genes encoding antibody heavy and light chains are known and segments making up each have been well characterized and described (Matsuda et al., The Journal of Experimental Medicine. 1998, 188(11): 2151-62 and Li et al.. Blood, 2004, 103(12): 4602-4609; the content of each of which are herein incorporated by reference in their entirety). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains.
Each light chain has a variable domain at one end (VL) and a constant domain at its other end;
the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain.
1001681 As used herein, the term "variable domain" refers to specific antibody domains found on both the antibody heavy and light chains that differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen.
Variable domains comprise hypervariable regions. As used herein, the term "hypervariable region" refers to a region within a variable domain comprising amino acid residues responsible for antigen binding. The amino acids present within the hypervariable regions determine the structure of the complementarity determining regions (CDRs) that become part of the antigen-binding site of the antibody. As used herein, the term "CDR" refers to a region of an antibody comprising a structure that is complimentary to its target antigen or epitope.
Other portions of the variable domain, not interacting with the antigen, are referred to as framework (FW) regions.
The antigen-binding site (also known as the antigen combining site or paratope) comprises the amino acid residues necessary to interact with a particular antigen. The exact residues making up the antigen-binding site are typically elucidated by co-crystallography with bound antigen, however computational assessments based on comparisons with other antibodies can also be used (Strohl, W.R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA.
2012. Ch. 3, p47-54, the contents of which are herein incorporated by reference in their entirety).
Determining residues that make up CDRs may include the use of numbering schemes including, but not limited to, those taught by Kabat (Wu et al., JEM, 1970, 132(2):211-250 and Johnson et al., Nucleic Acids Res. 2000, 28(1): 214-218, the contents of each of which are herein incorporated by reference in their entirety), Chothia (Chothia and Lesk, J.
MoL Biol. 1987, 196, 901, Chothia et al., Nature, 1989, 342, 877, and Al-Lazikani et al., J MoL
Biol. 1997, 273(4):
927-948, the contents of each of which are herein incorporated by reference in their entirety), Lefranc (Lefranc et al., Immunome Res. 2005, 1:3) and Honegger (Honegger and Pluckthun, J.
MoL Biol. 2001, 309(3): 657-70, the contents of which are herein incorporated by reference in their entirety).
[00169] VH and VL domains have three CDRs each. VL CDRs are referred to herein as CDR-L 1 , CDR-L2 and CDR-L3, in order of occurrence when moving from N- to C-terminus along the variable domain poly-peptide. VH CDRs are referred to herein as CDR-H1, CDR-H2 and CDR-H3, in order of occurrence when moving from N- to C- terminus along the variable domain polypeptide. Each of CDRs has favored canonical structures with the exception of the CDR-H3, which comprises amino acid sequences that may be highly variable in sequence and length between antibodies resulting in a variety of three-dimensional structures in antigen-binding domains (Nikoloudis, et al., PeerJ. 2014, 2: e456). In some cases, CDR-H3s may be analyzed among a panel of related antibodies to assess antibody diversity. Various methods of determining CDR sequences are known in the art and may be applied to known antibody sequences (Strohl, W.R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA.
2012. Ch. 3, p47-54, the contents of which are herein incorporated by reference in their entirety).
[00170] As used herein, the term "Fv" refers to an antibody fragment comprising the minimum fragment on an antibody needed to form a complete antigen-binding site. These regions consist of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. Fv fragments can be generated by proteolytic cleavage, but are largely unstable.
Recombinant methods are known in the art for generating stable Fv fragments, typically through insertion of a flexible linker between the light chain variable domain and the heavy chain variable domain (to form a single chain Fv (scFv)) or through the introduction of a disulfide bridge between heavy and light chain variable domains (Strohl, W.R.
Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 3, p46-47, the contents of which are herein incorporated by reference in their entirety).

[00171] As used herein, the term "light chain" refers to a component of an antibody from any vertebrate species assigned to one of two clearly distinct types, called kappa and lambda based on amino acid sequences of constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be assigned to different classes. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGI, IgG2, IgG3, IgG4, IgA, and IgA2.
[00172] As used herein, the term "single chain Fv" or "scFv" refers to a fusion protein of VH
and VL antibody domains, wherein these domains are linked together into a single polypeptide chain by a flexible peptide linker. In some embodiments, the Fv polypeptide linker enables the say to form the desired structure for antigen binding. In some embodiments, scFvs are utilized in conjunction with phage display, yeast display or other display methods where they may be expressed in association with a surface member (e.g. phage coat protein) and used in the identification of high affinity peptides for a given antigen.
[00173] Using molecular genetics, two scFvs can be engineered in tandem into a single polypeptide, separated by a linker domain, called a "tandem scFv" (tascFv).
Construction of a tascFv with genes for two different scFvs yields a "bispecific single-chain variable fragments"
(bis-scFvs). Only two tascFvs have been developed clinically by commercial firms; both are bispecific agents in active early phase development by Micromet for oncologic indications, and are described as "Bispecific T-cell Engagers (BiTE)." Blinattunomab is an anti-CD19/anti-CD3 bispecific tascFv that potentiates T-cell responses to B-cell non-Hodgkin lymphoma in Phase 2.
MT110 is an anti-EP-CAM/anti-CD3 bispecific tascFv that potentiates T-cell responses to solid tumors in Phase 1. Bispecific, tetravalent "TandAbs" are also being researched by Affimed (Nelson, A. L., MAbs., 2010, Jan-Feb; 2(1):77-83). maxibodies (bivalent scFv fused to the amino terminus of the Fc (CH2-CH3 domains) of IgG may also be included.
[00174] As used herein, the term "bispecific antibody" refers to an antibody capable of binding two different antigens. Such antibodies typically comprise regions from at least two different antibodies. Bispecific antibodies may include any of those described in Riethmuller, G. Cancer Immunity. 2012, 12:12-18, Marvin etal., 2005. Acta Pharmacologica Sinica.
2005, 26(6): 649-658 and Schaefer et al., PNAS. 2011, 108(27):11187-11192, the contents of each of which are herein incorporated by reference in their entirety.
[00175] As used herein, the term "diabody" refers to a small antibody fragment with two antigen-binding sites. Diabodies are functional bispecific single-chain antibodies (bscAb).
Diabodies comprise a heavy chain variable domain 'VH connected to a light chain variable domain VL in the same polypeptide chain. By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites.
Diabodies are described more fully in, for example, EP 404,097; WO 93/11161; and Hollinger et al. (Hollinger, P. et al., "Diabodies": Small bivalent and bispecific antibody fragments.
PNAS, 1993. 90: 6444-6448); the contents of each of which are incorporated herein by reference in their entirety.
[00176] The term "intrabody" refers to a form of antibody that is not secreted from a cell in which it is produced, but instead targets one or more intracellular proteins.
Intrabodies may be used to affect a multitude of cellular processes including, but not limited to intracellular trafficking, transcription, translation, metabolic processes, proliferative signaling and cell division. In some embodiments, methods of the present invention may include intrabody-based therapies. In some such embodiments, variable domain sequences and/or CDR
sequences disclosed herein may be incorporated into one or more constructs for intrabody-based therapy.
1001771 As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous cells (or clones), i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variants that may arise during production of the monoclonal antibodies, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
[00178] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. The monoclonal antibodies herein include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.
1001791 As used herein, the term "humanized antibody" refers to a chimeric antibody comprising a minimal portion from one or more non-human (e.g., murine) antibody source(s) with the remainder derived from one or more human immunoglobulin sources. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region from an antibody of the recipient are replaced by residues from the hypervariable region from an antibody of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affmity, and/or capacity. In one embodiment, the antibody may be a humanized full-length antibody. As a non-limiting example, the antibody may have been humanized using the methods taught in US Patent Publication NO.
US20130303399, the contents of which are herein incorporated by reference in its entirety.
[00180] As used herein, the term "antibody variant" refers to a modified antibody (in relation to a native or starting antibody) or a biomolecule resembling a native or starting antibody in structure and/or function (e.g., an antibody mimetic). Antibody variants may be altered in their amino acid sequence, composition or structure as compared to a native antibody. Antibody variants may include, but are not limited to, antibodies with altered isotypes (e.g., IgA, IgD, IgE, IgGl, TgG2, IgG3, IgG4, or TgM), humanized variants, optimized variants, multispecific antibody variants (e.g., bispecific variants), and antibody fragments.
[00181] in some embodiments, pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention may be antibody mimetics. As used herein, the term "antibody mimetic" refers to any molecule which mimics the function or effect of an antibody and which binds specifically and with high affinity to their molecular targets. In some embodiments, antibody mimetics may be monobodies, designed to incorporate the fibronectin type III domain (Fn3) as a protein scaffold (US

6,673,901; US 6,348,584). In some embodiments, antibody mimetics may be those known in the art including, but are not limited to affibody molecules, affilins, affitins, anticalins, avimers, Centyrins, DARPINSTM, Fynomers and Kunitz and domain peptides. In other embodiments, antibody mimetics may include one or more non-peptide regions.
1001821 In one embodiment, the antibody may comprise a modified Fc region. As a non-limiting example, the modified Fe region may be made by the methods or may be any of the regions described in US Patent Publication NO. U520150065690, the contents of which are herein incorporated by reference in its entirety.
[00183] in some embodiments, payloads of the invention may encode multispecific antibodies that bind more than one epitope. As used herein, the terms "multibody" or "multispecific antibody" refer to an antibody wherein two or more variable regions bind to different epitopes.
The epitopes may be on the same or different targets. In one embodiment, the multispecific antibody may be generated and optimized by the methods described in International Patent Publication NO. W02011109726 and US Patent Publication NO. U520150252119, the contents of which each of which are herein incorporated by reference in their entirety.
These antibodies are able to bind to multiple antigens with high specificity and high affinity.

[00184] In certain embodiments, a multi-specific antibody is a "bispecific antibody" which recognizes two different epitopes on the same or different antigens. In one aspect, bispecific antibodies are capable of binding two different antigens. Such antibodies typically comprise antigen-binding regions from at least two different antibodies. For example, a bispecific monoclonal antibody (BsMAb, BsAb) is an artificial protein composed of fragments of two different monoclonal antibodies, thus allowing the BsAb to bind to two different types of antigen. Bispecific antibody frameworks may include any of those described in Riethmuller, G., 2012. Cancer immunity, 2012, 12:12-18; Marvin et al., Acta Pharmacologica Sinica. 2005, 26(6):649-658; and Schaefer et al., PNAS. 2011, 108(27): 11187-11192, the contents of each of which are herein incorporated by reference in their entirety. New generations of BsMAb, called "trifunctional bispecific" antibodies, have been developed. These consist of two heavy and two light chains, one each from two different antibodies, where the two Fab regions (the arms) are directed against two antigens, and the Fe region (the foot) comprises the two heavy chains and forms the third binding site.
[00185] In some embodiments, payloads may encode antibodies comprising a single antigen-binding domain. These molecules are extremely small, with molecular weights approximately one-tenth of those observed for full-sized mAbs. Further antibodies may include "nanobodies"
derived from the antigen-binding variable heavy chain regions (VHHs) of heavy chain antibodies found in camels and llamas, which lack light chains (Nelson, A. L., MAbs.2010.
Jan-Feb;
2(1):77-83).
[00186] In some embodiments, the antibody may be "miniaturized". Among the best examples of mAb miniaturization are the small modular inununopharmaceuticals (SMIPs) from Trubion Pharmaceuticals. These molecules, which can be monovalent or bivalent, are recombinant single-chain molecules containing one VL, one VH antigen-binding domain, and one or two constant "effector" domains, all connected by linker domains. Presumably, such a molecule might offer the advantages of increased tissue or tumor penetration claimed by fragments while retaining the immune effector functions conferred by constant domains. At least three "miniaturized" SMIPs have entered clinical development. TRU-015, an anti-CD20 SMIP developed in collaboration with Wyeth, is the most advanced project, having progressed to Phase 2 for rheumatoid arthritis (RA). Earlier attempts in systemic lupus er}rthrematosus (SLE) and B cell lymphomas were ultimately discontinued. Trubion and Facet Biotechnology are collaborating in the development of TRU-016, an anti-CD37 SMIP, for the treatment of CLL and other lymphoid neoplasias, a project that has reached Phase 2. Wyeth has licensed the anti-CD20 SMIP SBI-087 for the treatment of autoimmune diseases, including RA, SLE and possibly multiple sclerosis, although these projects remain in the earliest stages of clinical testing. (Nelson, A.
L., MAbs, 2010. Jan-Feb; 2(1):77-83).
[00187] On example of miniaturized antibodies is called "unibody" in which the hinge region has been removed from IgG4 molecules. While IgG4 molecules are unstable and can exchange light-heavy chain heterodimers with one another, deletion of the hinge region prevents heavy chain-heavy chain pairing entirely, leaving highly specific monovalent light/heavy heterodimers, while retaining the Fc region to ensure stability and half-life in vivo. This configuration may minimize the risk of immune activation or oncogenic growth, as IgG4 interacts poorly with FcRs and monovalent unibodies fail to promote intracellular signaling complex formation (see, e.g., Nelson, A. L., MAbs, 2010. Jan-Feb; 2(1):77-83).
[00188] In some embodiments, payloads of the invention may encode single-domain antibodies (sdAbs, or nanobodies) which are antibody fragment consisting of a single monomeric variable antibody domain. Like a whole antibody, it is able to bind selectively to a specific antigen. In one aspect, a sdAb may be a "Camel 1g or "camelid VHH". As used herein, the term "camel Ig"
refers to the smallest known antigen-binding unit of a heavy chain antibody (Koch-No lte, et al, FASEB J., 2007, 21: 3490- 3498). A "heavy chain antibody" or a "camelid antibody" refers to an antibody that contains two VH domains and no light chains (Riechmann L. et al, .1. hnmunol.
Methods, 1999, 231: 25-38; International patent publication NOs. W01994/04678 and W01994/025591; and U.S. Patent No. 6,005,079). In another aspect, a sdAb may be a "immunoglobulin new antigen receptor" (IgNAR). As used herein, the term "immunoglobulin new antigen receptor" refers to class of antibodies from the shark immune repertoire that consist of homodimers of one variable new antigen receptor (VNAR) domain and five constant new antigen receptor (CNAR) domains. IgNARs represent some of the smallest known immunoglobulin-based protein scaffolds and are highly stable and possess efficient binding characteristics. The inherent stability can be attributed to both (i) the underlying Ig scaffold, which presents a considerable number of charged and hydrophilic surface exposed residues compared to the conventional antibody VH and VL domains found in murine antibodies; and (ii) stabilizing structural features in the complementary determining region (CDR) loops including inter-loop disulphide bridges, and patterns of intra-loop hydrogen bonds.
[00189] In some embodiments, payloads of the invention may encode intrabodies.
Intrabodies are a form of antibody that is not secreted from a cell in which it is produced, but instead targets one or more intracellular proteins. Intrabodies are expressed and function intracellularly, and may be used to affect a multitude of cellular processes including, but not limited to intracellular trafficking, transcription, translation, metabolic processes, proliferative signaling and cell division. In some embodiments, methods described herein include intrabody-based therapies. In some such embodiments, variable domain sequences and/or CDR sequences disclosed herein are incorporated into one or more constructs for intrabody-based therapy. For example, intrabodies may target one or more glycated intracellular proteins or may modulate the interaction between one or more glycated intracellular proteins and an alternative protein.
[00190] The intracellular expression of intrabodies in different compartments of mammalian cells allows blocking or modulation of the function of endogenous molecules (Biocca, et al., EMBO J. 1990, 9: 101-108; Colby et al., Proc. Natl. Acad. Sci. USA. 2004, 101:
17616-17621).
Intrabodies can alter protein folding, protein-protein, protein-DNA, protein-RNA interactions and protein modification. They can induce a phenotypic knockout and work as neutralizing agents by direct binding to the target antigen, by diverting its intracellular trafficking or by inhibiting its association with binding partners. With high specificity and affinity to target antigens, intrabodies have advantages to block certain binding interactions of a particular target molecule, while sparing others.
[00191] Sequences from donor antibodies may be used to develop intrabodies.
intrabodies are often recombinantly expressed as single domain fragments such as isolated VH
and VL domains or as a single chain variable fragment (scFv) antibody within the cell. For example, intrabodies are often expressed as a single polypeptide to form a single chain antibody comprising the variable domains of the heavy and light chains joined by a flexible linker polypeptide.
Intrabodies typically lack disulfide bonds and are capable of modulating the expression or activity of target genes through their specific binding activity. Single chain intrabodies are often expressed from a recombinant nucleic acid molecule and engineered to be retained intracellularly (e.g., retained in the cytoplasm, endoplasmic reticulum, or periplasm).
Intrabodies may be produced using methods known in the art, such as those disclosed and reviewed in: (Marasco et al., PNAS, 1993, 90: 7889-7893; Chen et al., Hum. Gene Ther. 1994, 5:595-601;
Chen et al., 1994, PNAS, 91: 5932-5936; Maciejewski et al., 1995, Nature Med., 1: 667-673;
Marasco, 1995, Immunotech, 1: 1-19; Mhashilkar, et al., 1995, EMBO J. 14: 1542-51; Chen et al., 1996, Hum.
Gene Therap., 7: 1515-1525; Marasco, Gene Ther. 4:11-15, 1997; Rondon and Marasco, 1997, Amiu. Rev. Microbiol. 51:257-283; Cohen, et al., 1998, Oncogene 17:2445-56;
Proba et al., 1998, J. Mol. Biol. 275:245-253; Cohen et al., 1998, Oncogene 17:2445-2456;
Hassanzadeh, et al., 1998, FEBS Lett. 437:81-6; Richardson et al., 1998, Gene Ther. 5:635-44;
Ohage and Steipe, 1999, J. Mol. Biol. 291:1119-1128; Ohage etal., 1999, J. Mol. Biol. 291:1129-1134; Wirtz and Steipe, 1999, Protein Sci. 8:2245-2250; Zhu et al., 1999, J. Immunol. Methods 231:207-222;
Arafat et al., 2000, Cancer Gene Ther. 7:1250-6; der Maur et al., 2002, J.
Biol. Chem.

277:45075-85; Mhashilkar et al., 2002, Gene Ther. 9:307-19; and Wheeler et al., 2003, FASEB
J. 17: 1733-5; and references cited therein).
[00192] In some aspects, payloads of the invention may encode biosynthetic antibodies as described in U.S. Patent No. 5,091,513, the contents of which are herein incorporated by reference in their entirety. Such antibody may include one or more sequences of amino acids constituting a region which behaves as a biosynthetic antibody binding site (BABS). The sites comprise 1) non-covalently associated or disulfide bonded synthetic VH and VL
dimers, 2) VH-VL or VL-VH single chains wherein the 'VH and VL are attached by a polypeptide linker, or 3) individuals VH or VL domains. The binding domains comprise linked CDR and FR
regions, which may be derived from separate immunoglobulins. The biosynthetic antibodies may also include other polypeptide sequences which function, e.g., as an enzyme, toxin, binding site, or site of attachment to an immobilization media or radioactive atom. Methods are disclosed for producing the biosynthetic antibodies, for designing BABS having any specificity that can be elicited by in vivo generation of antibody, and for producing analogs thereof.
[00193] In some embodiments, payloads may encode antibodies with antibody acceptor frameworks taught in U.S. Patent No. 8,399,625. Such antibody acceptor frameworks may be particularly well suited accepting CDRs from an antibody of interest.
[00194] In one embodiment, the antibody may be a conditionally active biologic protein. An antibody may be used to generate a conditionally active biologic protein which are reversibly or irreversibly inactivated at the wild type normal physiological conditions as well as to such conditionally active biologic proteins and uses of such conditional active biologic proteins are provided. Such methods and conditionally active proteins are taught in, for example, International Publication No. W02015175375 and W02016036916 and US Patent Publication No. US20140378660, the contents of each of which are incorporated herein by reference in their entirety.
Antibody preparations [00195] The preparation of antibodies, whether monoclonal or polyclonal, is known in the art.
Techniques for the production of antibodies are well known in the art and described, e.g. in Harlow and Lane "Antibodies, A Laboratory Manual", Cold Spring Harbor Laboratory Press, 1988; Harlow and Lane "Using Antibodies: A Laboratory Manual" Cold Spring Harbor Laboratory Press, 1999 and "Therapeutic Antibody Engineering: Current and Future Advances Driving the Strongest Growth Area in the Pharmaceutical Industry" Woodhead Publishing, 2012.
[00196] The antibodies and fragments and variants thereof as described herein can be produced using recombinant polynucleotides. In one embodiment, the polynucleotides have a modular design to encode at least one of the antibodies, fragments or variants thereof As a non-limiting example, the polynucleotide construct may encode any of the following designs:
(I) the heavy chain of an antibody, (2) the light chain of an antibody, (3) the heavy and light chain of the antibody, (4) the heavy chain and light chain separated by a linker, (5) the VH1, CHI, CH2, CH3 domains, a linker and the light chain or (6) the VH I, CHI, CH2, CH3 domains, VL region. and the light chain. Any of these designs may also comprise optional linkers between any domain and/or region. The polynucleotides of the present invention may be engineered to produce any standard class of immunoglobulins using an antibody described herein or any of its component parts as a starting molecule.
1001971 Recombinant antibody fragments may also be isolated from phage antibody libraries using techniques well known in the art and described in e.g. Clackson et al., 1991, Nature 352:
624-628; Marks et al., 1991, J. Mol. Biol. 222: 581-597. Recombinant antibody fragments may be derived from large phage antibody libraries generated by recombination in bacteria (Sblattero and Bradbury, 2000, Nature Biotechnology 18:75-80; the contents of which are incorporated herein by reference in its entirety).
Antibodies used for immunotherapy [00198] In some embodiments, payloads of the present invention may be antibodies, fragments and variants thereof which are specific to tumor specific antigens (TSAs) and tumor associated antigens (TAAs). Antibodies circulate throughout the body until they find and attach to the TSA/TAA. Once attached, they recruit other parts of the immune system, increasing ADCC
(antibody dependent cell-mediated cytotoxicity) and ADCP (antibody dependent cell-mediated phagocytosis) to destroy tumor cells. As used herein, the term "tumor specific antigen (TSA)"
means an antigenic substance produced in ttunor cells, which can trigger an anti-tumor immune response in a host organism. In one embodiment, a TSA may be a tumor neoantigen. The tumor antigen specific antibody mediates complement-dependent cytotoxic response against tumor cells expressing the same antigen.
[00199] In some embodiments, the tumor specific antigens (TSAs), tumor associated antigens (TAAs), pathogen associated antigens, or fragments thereof can be expressed as a peptide or as an intact protein or portion thereof. The intact protein or a portion thereof can be native or mutagenized. Antigens associated with cancers or virus-induced cancers as described herein are well-known in the art. Such a TSA or TAA may be previously associated with a cancer or may be identified by any method known in the art.
[00200] in one embodiment, the antigen may be GD2 ganglioside. In one embodiment, payloads of the present invention may be antibodies, fragments and variants thereof which are specific to GD2 antigen. Gangliosides expressed on the tumor cell surface can be targets for cancer immunotherapy. GD2 is a disialoganglioside with a molecular formula of C74H134N4032. Gangliosides are acidic glycosphingolipids found on the outer surface of most cell membranes. They are ideal targets for immunotherapy because of the high antigen density, lack of modulation, relative homogeneity in many tumors and the possibility of up regulation by cytokines. Many tumors have abnormal glycolipid composition and structure. GD2 has been found in a wide spectrum of human tumors, including those of neuroectodermal or epithelial origin, virtually all melanomas, and approximately 50% of tumor samples from osteosarcoma and soft tissue sarcoma. Antibodies with high affinity for GD2 include, but not limited to 1B7, 2H12, 1G2, 1E9, 1H3, 2F5, 2F7, 31F9, 31F9V2, 32E2, ch14.18, hu14.18, 3F8, 8B6, 4B5, IA7, A1G4, GD2 mimotopes, hu14.18K322A, 5F11, 3G6, 14g2a, and 14.18. In one embodiment, the GD antibody is the 14g2a antibody (Mujoo K., et al. (1989) Cancer Res.
1;49(11):2857-61; the contents of which are incorporated herein by reference in its entirety). Any of the GD2 antibodies described in Long A.H. et al. (2015) Nat Med. 2I(6):581-90; the contents of which are incorporated by reference in their entirety).
[00201] In one embodiment, the antigen is HER2 antigen. In one embodiment, payloads of the present invention may be antibodies, fragments and variants thereof which are specific to HER2 antigen. HER2 is the oncogene product of human epidermal cell growth factor receptor 2 related oncogenes and is a transmembrane receptor protein having a molecular weight of 185 kDa and having a tyrosine kinase domain. HER2 is a member of the EGFR family consisting of HER!
(EGFR, ERBB I), HER2 (neu, ERBB-2), HER2 (ErbB-3), and Her4 (ErbB-4) and is known to be autophosphorylated at intracellular tyrosine residues by its homodimer formation or heterodimer formation with another EGFR receptor HER!, HER3, HER4 and is activated in this manner.
Thereby playing an important role in cell growth, differentiation, and survival in normal cells and tumor cells. In some embodiments, HER2 antibodies useful in the present invention may include 3B5 (from Oncogene Science/BAYER), 2C4 (ATCC HB-12697), 7C2 (ATCC HB-12215), ApoB17F/ocHER2, 8A4 (ATCC PTA-4565), A10Al2 (ATCC PTA- 4566), 9G6, 7H4, AIOE9, A I2D6, A6B12, Al0E11, B3G4, A5C7, 13A11, 11C11, 13E11, Her2Bi (OKT3 x 9184), Her2Bi (OK'T3 x Here), 7F3 (ATCC HB-12216), huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-6, huMAb4D5-7, 520C9, CB-11 (from Novocastra Laboratories), NCLB12 (from Novocastra Laboratories), humanized 2C4 mutant 560, humanized 2C4 mutant 561, humanized 2C4 mutant 562, humanized 2C4 mutant 568, humanized 2C4 mutant 569, humanized 2C4 mutant 570, humanized 2C4 mutant 571, humanized 2C4 mutant 56869, 3E8, 3H4, Cl!! (NeoMarkers), HER-81, 452F2, 736G9, 741F8, 758G5, 761B10 anti-p185HER2/FcTRIII (CD16) , anti-CD3/anti-p185HER2 , Hu4D5-8 and variants, 4D5-H, CB11 (from Ventana Medical Scientific Instruments), 6E9, 2H11, 5B8, 7D3, HERM), HER66, HER70, scFv C6.5, scFv C6ML3-9 (ML3.9 or C6ML3.9), scFv C6N1H3-B1 (B1 or C6NfF13.B1), scFv C6-B1D2, (B1D2 or C6MH3-B1D2), ALM, L87, N28 , N12 , MGr6 , 9GG.10 (Neomarkers), MGFc-5 (V379M), MGFc-9 (F243I, V379L), MGFc-10 (1(288N, A3305, P396L), MGFc-13 (K334E, T359N, T3665), MGFc-27 (G316D, A378V, D399E), MGFc-37 (K248M), MGFc-39 (E293V Q295E, A3271), MGFc-38 (K392T, P396L), MGFc-41 (H268N, P396L), MGFc-23 (K334E, R292L), MGFc-44, MGFc-45, MDX-210, 17.6.4 , HER2-PY1248, MAb74, FRP5, TAb250, HER-81, PN2A, mAb 191924 (R&D systems), IDM1, scFv23, Ab-3, Ab-5 , 2502A, Rexomun, MAB-1129 (R&D systems), and MM-111. In one embodiment, antibodies with high affinity may be derived from any of the HER2 antibody heavy and light chain variables described in Table 7.
[00202] In one embodiment of the present invention, the antigen is CD33. In one embodiment, payloads of the present invention may be antibodies, fragments and variants thereof which are specific to CD33 antigen. Acute myeloid leukemia (AML) is the second most common acute leukemia in the United States. The commonly applied therapy of leukemic disease includes irradiation and/or chemotherapy. However, very often 65-80% of patients receiving treatment relapse because the cells that survived the chemotherapy are enriched in AMC, leukemia stem cells (AML-LSCs), and constitute a reservoir of cells capable of re-expanding and causing a relapse. AML-LSCs express a characteristic set of cell surface antigens including among other CD33. CD33 (Sialic acid binding Ig-like lectin 3) or SIGLEC3(UNTPROT ID:
P20138) is a transmembrane receptor expressed on cells of myeloid lineage. It is usually considered myeloid specific, but it can also be found on some lymphoid cells. It binds to sialic acid, therefore is a member of the SIGLEC family of lectins. Exemplary antibodies targeting CD33 may include, but are not limited to M195, M2H12, DRB2, My 9-6. In one embodiment, the antibody is derived from My9.6. In some embodiments, antibodies with high affinity may be derived from any of the CD33 antibody heavy and light chain variables described in Table 7.
[00203] In one embodiment, the antigen of the present invention is a BCMA (B-cell maturation antigen), also referred to as the CD269. In one embodiment, payloads of the present invention may be antibodies, fragments and variants thereof which are specific to BCMA
antigen. BCMA
antigen (UNIPROT ID: Q02223) is encoded by the gene, TNFRS17. BCMA is a member of the TNF receptor super family. It binds to B cell activating factor (BAFF) and a proliferation inducing ligand (APRIL). Among non-malignant cells, BCMA has been reported to be expressed mostly by plasma cells and subsets of mature B cells, but not T cells and NK
cells. Therefore, BCMA represents a suitable therapeutic candidate in the treatment of multiple myeloma.
Exemplary antibodies targeting BCMA include, but are not limited to BCMA 50, BCMA30, C 1 1D5.3 and C13F12.1 In one embodiment, the antibody is derived from C
11D5.3. In some embodiments, antibodies with high affinity may be derived from any of the BCMA
antibody heavy and light chain variables described in Table 7.
[00204] In one embodiment, the antigen of the present invention is a CD276 (also known as B7-H3). In one embodiment, payloads of the present invention may be antibodies, fragment, and variants thereof which are specific to CD276. CD276 is expressed in a variety of human tumors, including pediatric solid tumors and adult carcinomas. Any of the CD276 antibodies taught in International Patent publications W02017044699 and W02014160627 (the contents of which are incorporated herein by reference in their entirety), may be useful in the present invention. In some embodiments, antibodies with high affinity may be derived from any of the antibody heavy and light chain variables described in Table 7.
[00205] In one embodiment, the antigen of the present invention is a ALK
protein. The developmentally-regulated cell surface receptor tyrosine kinase, ALK is known to be expressed as a tumor associated antigen as a fusion protein resulting from a chromosomal translocation.
Cancer associated ALK was first described as a 2;5 translocation associated with nucleophosphomin (NPM) in anaplastic large cell leukemia. The fusion protein is composed of intracellular component of NPM fused to ALK. In some embodiments, the ALK
antigen may be the extracellular portion of the protein. Any of the antibodies, fragment and variants specific to ALK may be useful in the present invention. In one embodiment, the ALK
antibodies described in the International Patent Publication, W02015069922 (the contents of which are incorporated by reference herein in its entirety). In some embodiments, antibodies with high affinity may be derived from any of the ALK antibody heavy and light chain variables described in Table 7.
(002061 In one embodiment, the antigen of the present invention is a CD22 antigen. In one embodiment, payloads of the present invention may be antibodies, fragment, and variants thereof which are specific to CD22. CD22 is a lineage restricted B cell antigen belonging to the immunoglobulin (Ig) superFamily. CD22 is expressed in 60-70% of B cell lymphomas and leukemias (e.g. B-chronic lymphocytic leukemia, hairy cell leukemia, acute lymphocytic leukemia (ALL) and Burkit's lymphoma) and is not present on the cell surface in early stages of B cell development or on to stem cells. In some embodiments, the antibodies, fragments, and variants thereof may be any of those taught in International Patent Publications, W02016149578, W02014065961, and W02013059593A1(the contents of each of which are incorporated by reference in its entirety). In some embodiments, antibodies with high affinity may be derived from any of the CD22 antibody heavy and light chain variables described in Table 7.
[00207] In some embodiments, the payloads of the present invention may include an antigen binding region comprising variable heavy chain and variable light chains with the amino acid sequences selected from those in Table 7.
Table 7: Variable Heavy and Light Chain Sequences Target Description and Clone Source Antibody SEQ
name chain ID
NO.
ALK ALKI5 VH SEQ ID NO. 1 in W02015069922 VH 242 ALK ALK48 VH SEQ ID NO. 3 in W02015069922 VH 243 ALK ALK53 VH SEQ ID NO. Sin W02015069922 VH 244 ALK ALK58 VH SEQ ID NO. 7 in 'W02015069922 VH 245 ALK humanized ALK15 VH SEQ ID NO. 9 in W02015069922 VH 246 ALK humanized ALK48 VH SEQ ID NO. 11 in W02015069922 VH 247 ALK humanized AI.K53 VH SEQ ID NO. 13 in W02015069922 VH 248 ALK humanized ALK58 VII SEQ ID NO. 15 in W02015069922 VH 249 ALK ALK15 VL SEQ ID NO. 2 in W02015069922 VL 250 ALK ALK48 VL SEQ ID NO. 4 in W02015069922 VL 251 ALK ALK53 VL SEQ ID NO. 6 in W02015069922 VL 252 ALK ALK58 VL SEQ ID NO. 8 in W02015069922 VL 253 ALK humanized ALK15 VL SEQ ID NO. 10 in W02015069922 VL 254 ALK humanized ALK48 VL SEQ ID NO. 12 in W02015069922 VL 255 ALE: humanized AL.K53 VI.. SEQ ID NO. 14 in W02015069922 VL 256 ALK humanized A1,K58 VI, SEQ ID NO. 16 in W02015069922 s VL 257 CD22 CD22 VL SEQ ID NO. 14 in W02016149578 VL 258 CD22 CD22 (HA22 or BL22) SEQ ID NO. 1 in W02013059593 VL 259 VL
CD22 CD22 VH SEQ ID NO. 13 in W02016149578 VH 260 CD22 CD22 (HA22 or BL22) SEQ ID NO. 3 in W02013059593 VH 261 VH
CD22 CD22 (HA22 or BL22) SEQ ID NO. 4 in W02013059593 VH 262 VH
-CD276 --+
CD276 VH SEQ ID NO. 7 in W02017044699 VH 263 CD276 CD276 VII (CD276.6 SEQ ID NO. 7 in W02014160627 VH 264 (m856)) CD276 CD276 VH (CD276.1. SEQ ID NO. 17 in W02014160627 VH 265 (m851)) CD276 CD276 VH (CD276.17 SEQ ID NO. 26 in W02014160627 VH 266 (m8517)) CD276 CD276 VL SEQ ID NO. 8 in W02017044699 VL 267 CD276 CD276 VL (CD276.6 SEQ ID NO. 8 in W02014160627 VL 268 (m856)) CD276 CD276 VL (CD276.1 SEQ ID NO. 18 in W02014160627 VL 269 (m851)) CD276 CD276 VL (CD276.17 SEQ ID NO. 27 in W02014160627 VL 270 (m8517)) GD2 3F8 heavy chain variable SEQ ID No. 1 in W02011160119 VH

GD2 3F8 light chain variable SEQ ID No. 2 in GD2 3F8 heavy chain variable SEQ ID No. 3 in W02011160119 VH

602 humanized 3F8 heavy SEQ ID No. 4 in chain variable GD2 humanized 31:8 light SEQ ID No. 5 in chain variable GD2 humanized 3F8 heavy SEQ ID No. 6 in chain 2 variable GD2 humanized 3F8 light SEQ No. 7 in chain 2 variable GD2 humanized 3F8 heavy SEQ ID No. 8 in chain variable GD2 Human GD2 heavy SEQ ID No. 16 in VH 279 chain variable W02010002822A1 GD2 Human GD2 light chain SEQ ID No. 32 in VL 280 variable W02010002822A1 GD2 chimeric Ch3F8 heavy Cheung et al., Oncoimmunology, VH 281 chain-gamma 1 2012, 1(4): 477-486 GD2 chimeric Ch3F8 light Cheung et al., Oncoimmunology, VL 282 chain-kappa 2012, 1(4). 477-486 GD2 humanized Hu3F8 heavy Cheung et al., Oncoirtimunology, VH

chain-gammal 2012, 1(4). 477-486 GD2 humanized HOB light Cheung et al., Oncoirtimunology, VI., 284 chain-kappa 2012, 1(4) 477-486 01)2 Chimeric Ch3F8 heavy Cheung et at., Oncoimmunology, VH 285 chain-gamma4 2012, 1(4): 477-486 GD2 humanized Hu3F8 heavy Cheung et at., Oncoimmunology, VL

chain-gamma4 2012, 1(4): 477-486 01)2 GD2 VH SEQ ID NO. 17 in W02016134284 VH 287 GD2 GD2 VL SEQ ID NO. 18 in W02016134285 VL 288 0132 Murine KM666 VH SEQ ID NO. 9 in W02015132604 VH 289 (heavy chain variable region) sequence GD2 Humanized KM666 VH SEQ 11) NO. 10 in W02015132604 VH 290 sequence GD2 Marine KM666 VL SEQ ID NO. 11 in W02015132604 VL 291 (light chain variable region) sequence GD2 Humanized KM666 VL SEQ ID NO. 12 in W02015132604 VL 292 .
sequence GD2 GD2 VL SEQ ID NO. 1 in US20040203100 VL 293 61)2 GD2 VH SEQ ID NO. 2 in US20040203100 VH 294 01)2 Marine KM666 VH SEQ ID NO. 9 in US20170066838 VH 295 (heavy chain variable region) sequence GD2 Humanized KM666 VH SEQ ID NO. 10 in US20170066838 VH 296 sequence GD2 Marine KM666 VL SEQ ID NO. 11 in US20170066838 VL 297 (light chain variable region) sequence GD2 Humanized KM666 VL SEQ ID NO. 12 in US20170066838 VL 298 sequence GD2 GD2 VL SEQ ID NO.3 in US20160304620 VL 299 GD2 GD2 VH SEQ ID NO. 4 in US20160304620 VH 300 GM 0D2 VH SEQ ID NO. 2 in US20150353645 VH 301 .
61)2 01)2 VL. SEQ ID NO. 4 in US20150353645 VL. 302 01)2 0D2 VH SEQ ID NO. 6 in US20150353645 VH 303 GD2 GD2 VL SEQ ID NO. 8 in US20154)353645 VL. 304 GD2 GD2 VH SEQ ID NO. 10 in US20150353645 VH 305 -GD2 GD2 VL SEQ ID NO. 12 in US20150353645 VL 306 GD2 GD2 VH SEQ ID NO. 14 in US20150353645 VH 307 0D2 0D2 VL SEQ ID NO. 16 in US20150353645 VL 308 .
GD2 GD2 VH SEQ ID NO. 18 in U520150353645 VH 309 GD2 0D2 VL SEQ ID NO. 20 in US20150353645 VL 310 GD2 GD2 VH SEQ ID NO. 22 in U520150353645 VH 311 GD2 GD2 VL SEQ ID NO. 24 in US20150353645 VL 312 GD2 GD2 VH SEQ ID NO. 26 in US20150353645 VH 313 GD2 GD2 VL SEQ ID NO. 28 in US20150353645 VL 314 GD2 GD2 VH SEQ ID NO. 30 in US20150353645 VH 315 GD2 GD2 VL SEQ ID NO. 32 in U S20150353645 VL 316 GD2 0D2 VH SEQ ID NO. 34 in US20150353645 VH 317 ¨
G1)2 01)2 VII SEQ ID NO. 36 in US20150353645 VII 318 GD2 GD2 VL SEQ ID NO. 38 in US20150353645 VL 319 GD2 01)2 VEI SEQ ID NO. 40 in US20150353645 VH 320 GD2 GD2 VL SEQ ID NO. 42 in US20150353645 VL 321 GD2 GD2 VL SEQ ID NO. 3 in US20150139942 VL 322 GD2 GD2 VH SEQ ID NO. 4 in US20150139942 ' VH 323 ..................................................... + .....
G1)2 (31)2 VL SEQ ID NO. 7 in US20150139942 I VL 324 G1)2 GD2 VH SEQ ID NO. 8 in US201.50139942 VII 325 GD2 0D2 VH SEQ ID NO. 16 in U520130287691 VH 326 GD2 GD2 VL SEQ ID NO. 32 in US20130287691 VL 327 GD2 GD2 VH SEQ ID NO. 40 in U520130287691 VH 328 GD2 01)2 VL SEQ ID NO. 42 in US20130287691 VL 329 GD2 GD2 VL SEQ ID NO. 3 in US20140134162 ' VL 330 ..................................................... + .....
GD2 G1)2 VT-I SEQ ID NO. 4 in US20140134162 VII 331.
602 GD2 VII SEQ ID NO. 20 in W02017055385 VII 332 GD2 0D2 VL SEQ ID NO. 20 in W02017(155385 VL 333 GD2 GD2 VH SEQ ID NO. 3 in W02013189516 VH 334 GD2 GD2 VL SEQ ID NO. 4 in W02013189516 VL 335 GD2-0- KM8B6 GD2-0- SEQ ID No. 1 in W02008043777 VH 336 acetylated acetylated heavy chain variable GD2-0- KM8B6 GD2-0- SEQ ID No. 2 in W02008043777 VL 337 acetylated acetylated light chain variable GD2-0- 0-acetylated-GD2 SEQ ID No. 6 in W02015067375 VL 338 acetylated ganglioside light chain variable region GD2-0- 0-acetylated-GD2 SEQ ID No. 7 in W02015067375 VH 339 acct ) lated ganglioside heavy chain variable region GD2-0- GD2 VL SEQ ID NO.7 in US20160068608 VL 340 acetylated _________________________________________________________________ ..

GD2-0- GD2 VH SEQ ID NO.8 in US20160068608 VH 341 acetylated GD2-0- GD2 VL (8136) SEQ ID NO. 7 in VL 342 acetylated W02014177271A1 GD2-0- GD2 VH (8B6) SEQ ID NO. 8 in VII 343 acetylated W02014177271A1 GD2-0- GD2 VL SEQ ID NO. 9 in Vi 344 acetylated W02014177271A1 GD2-0- GD2 VH SEQ ID NO. 10 in VH 345 acetylated W02014177271A1 Gangliosides GMab 1 -VH SEQ ID No. 11 in W02012071216 VH 346 (including GD2) Gangliosides GMab !NH SEQ ID No. 12 in W02012071216 VL 347 (including GD2) Gangliosides SEQ ID No. 13 in W02012071216 VH 348 (including GD2) Gangliosides GMab2-VH SEQ ID No. 14 in W02012071216 VL 349 (including GD2) CD33 Anti CD33 VH (Clone SEQ ID NO. 11 in W02015150526 VH 350 M195) CD33 Anti CD33 VL (Clone SEQ ID NO. 12 in W02015150526 VL 351 M I 95) CD33 Anti CD33 VH (Clone SEQ ID NO. 13 in W02015150526 VH 352 M2H12) CD33 Anti CD33 VL (Clone SEQ ID NO. 14 in W02015150526 VL 353 M2H12) CD33 Anti CD33 VH (Clone SEQ ID NO. 15 in W02015150526 VH 354 DRB2) CD33 Anti CD33 Vi (Clone SEQ ID NO. 16 in W02015150526 VL 355 DRB2) CD33 Anti CD33 VH (Clone SEQ ID NO. 17 in W02015150526 VH 356 My9-6) CD33 Anti CD33 VL (Clone SEQ ID NO. 18 in W02015150526 VI.: 357 My9-6) BCMA BCMA VH (Clone SEQ ID NO. 11 in W02015158671 VH 358 BCMA-50) BCMA BCMA VL (Clone SEQ ID NO. 12 in W02015158672 VL 359 BCMA-50) BCMA BCMA VH (Clone SEQ ID NO. 13 in W02015158673 I VH 360 BCMA-30) BCMA BCMA VL (Clone SEQ ID NO. 14 in W02015158674 VL 361 BCMA-30) BCMA BCMA VH (Clone SEQ ID NO. 15 in W02015158675 VH 362 CI ID5.3) BCMA BCMA 'VL (Clone SEQ ID NO. 16 in Cl1D5.3) BCMA BCMA VH (Clone SEQ ID NO. 17 in Cl3F12.1) BCMA BCMA VL (Clone SEQ ID NO. 18 in Cl3F12.1) Her2 Trastuzumab (Herceptin) SEQ ID NO. 1 in W02017093844 VH

Her2 Trastuzumab (Herceptin) SEQ ID NO. 7 in W02017093844 VL

Her2 huMAb4D5-5 SEQ ID NO. 1 in US 8,075,890 VL 368 Her2 IniMAb4D5-5 SEQ H) NO. 2 in US 8,075,890 V1-1 369 Her2 a consensus antibody SEQ ID NO. 3 in US
8,075,890 VL ' 370 variable domain Her2 a consensus antibody SEQ ID NO. 4 in US
8,075,890 VH 371 variable domain Her2 iniiMAb4D5 SEQ ID NO. 5 in US 8,075,890 VL 372 Her2 muMAb4D5 SEQ ID NO. 6 in US 8,075,890 VH 373 Her2 N29 No SEQ ID in W01993003741 VII 374 Her2 N29 No SEQ ID in W01993003741 VL 375 Her2 2C4 SEQ ID NO. 1 in US 7,981,418 VL 376 Her2 2C4 SEQ ID NO. 2 in US 7,981,418 VH 377 Her2 variant 574/Pertuzumab SEQ ID NO. 3 in US 7,981,418 VL
378 .
Her2 variant 574/Pertuzumab SEQ ID NO. 4 in US

7,981,418 VH 379 Her2 human VL consensus SEQ ID NO. 5 in US
7,981,418 'VL 380 (hum. kappa.1, lighi.
kappa subgroup 1) Her2 human 'VII consensus SEQ ID NO. 6 in US
7,981,418 VH 381 (humIII, heavy subgroup HI) Her2 Pertuzumab SEQ ID NO. 13 in US 7,981,418 'VL 382 Her2 Pertuzurnab - SEQ ID NO. 14 in US 7,981.418 VH 383 Her2 trastuzurnablituinMAb4 SEQ ID NO. 15 in US
7,981,418 VL 384 Her2 trastuzurnablituinMAb4 SEQ ID NO. 16 in US
7,981,418 VH 385 Her2 a variant Pertuzumab SEQ ID NO. 17 in US
7,981,418 VL 386 light chain sequence Her2 a variant Pertuzumab SEQ ID NO. 18 in US
7,981,418 VH 387 heavy chain sequence Her2 3. F2 monoclonal SEQ ID NO. 2 in W02001009187 VH 388 antibody Her2 3. F2 monoclonal SEQ 1D NO. 4 in W02001009187 VL 389 antibody Her2 1. D2 monoclonal SEQ ID NO. 6 in W02001009187 VH 390 antibody -Her2 1. D2 monoclonal SEQ ID NO. 8 in W02001009187 VL 391 antibody Her2 2. E8 monoclonal SEQ ID NO. 10 in antibody Her2 2. E8 monoclonal SEQ ID NO. 12 in antibody Her2 2C4 SEQ ID NO. 4 in US 7,097,840 VL 394 Her2 variant 574/Pertuzumab SEQ ID NO. 5 in US 7,097,840 VL

Her2 human VL subgroup SEQ ID NO. 6 in US 7,097,840 VL 396 Her2 4D5 SEQ ID NO. 14 in Her2 Hu4D5-8 SEQ ID NO. 1 in W02003087131 VL 398 Her2 rhuMAb SEQ ID NO. 50 in Her2 rhuMAb SEQ ID NO. 52 in US20040254108 VI.. 400 Her2 her2VHCH - SM5-1 SEQ ID NO. 54 in VH; human kappa chain constant (CH) Her2 her2VLCL - SM5-I VL; SEQ ID NO. 56 in US20040254108 VI.. 402 human kappa chain constant (CL) Her2 her2VH/Fc/FL- rhuMAb SEQ ID NO. 58 in U520040254108 VH 403 VH; IgG1 Fc; F1t3 ligand extracellular region (hFLex) Her2 her2VH/Fc/Link/FL - SEQ ID NO. 60 in rhuMAb; IgG1 Fc;
linker, F113 ligand extracellular region (hFLex) Her2 Herceptin Fab SEQ ID NO. 9 in VL. 405 S200502607I IA!
Her2 Herceptin Fab SEQ ID NO. 10 in VH 46-6 Her2 Pertuzumab with a signal SEQ ID NO. 17 in US20060018899 VL

peptide sequence Her2 Pertuzumab with a signal SEQ ID NO. 18 in U520060018899 VH

peptide sequence Her2 Periplasmic Fab-4D5 SEQ ID NO. 30 in US
7,632,924 VL 409 Her2 Periplasmic Fab-4D5 SEQ ID NO. 31 in US
7,632,924 VH 410 Her2 trastuzutnab A88C SEQ ID NO. 6 in US7521541 VH 411 Her2 trastuittmab A121C SEQ ID NO. 7 in Her2 trastuzumab \' 10C SEQ ID NO. 8 in Her2 B1D2 SEQ ID NO. 42 in US7,332,585 VH 414 Her2 B ID2 SEQ ID NO. 47 in US7,332,585 VL 415 Her2 Fab63 SEQ ID NO. 7 in US20100047230 VH 416 I4er2 Fab63 SEQ ID NO. 8 in US20100047230 VL 417 Her2 Herceptin SEQ H) NO. 3 in US20160256561 VH 418 f-{cr2 anti-her2/ricti antibody SEQ ID NO. 1 in US
9,534,057 VII 419 with a signal peptide Her2 anti-her2incii antibody SEQ ID NO. 2 in US
9,534,057 VL 420 with a signal peptide anti-Her2Ineti anti-I-Tea/mu - anti-CD3 SEQ ID NO. 3 in VII 421 anti-CD3 bispecific antibody 'VH W02014079000A
Chimeric antigen receptors (CARs) [00208] In some embodiments, payloads of the present invention may be a chimeric antigen receptors (CARS) which when transduced into immune cells (e.g., T cells and NK
cells), can re-direct the immune cells against the target (e.g., a tumor cell) which expresses a molecule recognized by the extracellular target moiety of the CAR.
[00209] As used herein, the term "chimeric antigen receptor (CAR)" refers to a synthetic receptor that mimics TCR on the surface of T cells. In general, a CAR is composed of an extracellular targeting domain, a transmembrane domain/region and an intracellular signaling/activation domain. In a standard CAR receptor, the components: the extracellular targeting domain, transmembrane domain and intracellular signaling/activation domain, are linearly constructed as a single fusion protein. The extracellular region comprises a targeting domain/moiety (e.g., a scFv) that recognizes a specific tumor antigen or other tumor cell-surface molecules. The intracellular region may contain a signaling domain of TCR
complex (e.g., the signal region of CD30, and/or one or more costimulatory signaling domains, such as those from CD28, 4-1BB (CD137) and OX-40 (CD134). For example, a "first-generation CAR"
only has the CD3C signaling domain. In an effort to augment T-cell persistence and proliferation, costimulatory intracellular domains are added, giving rise to second generation CARS having a CD3Csignal domain plus one costimulatory signaling domain, and third generation CARS having CD3 signal domain plus two or more costimulatoiy signaling domains. A CAR, when expressed by a T cell, endows the T cell with antigen specificity determined by the extracellular targeting moiety of the CAR. Recently, it is also desirable to add one or more elements such as homing and suicide genes to develop a more competent and safer architecture of CAR, so called the fourth-generation CAR.
[00210] In some embodiments, the extracellular targeting domain is joined through the hinge (also called space domain or spacer) and transmembrane regions to an intracellular signaling domain. The hinge connects the extracellular targeting domain to the transmembrane domain which transverses the cell membrane and connects to the intracellular signaling domain. The hinge may need to be varied to optimize the potency of CAR transformed cells toward cancer cells due to the size of the target protein where the targeting moiety binds, and the size and affinity of the targeting domain itself. Upon recognition and binding of the targeting moiety to the target cell, the intracellular signaling domain leads to an activation signal to the CAR T cell, which is further amplified by the "second signal" from one or more intracellular costimulatoiy domains. The CAR T cell, once activated, can destroy the target cell.
[00211] in some embodiments, the CAR of the present invention may be split into two parts, each part is linked a dimerizing domain, such that an input that triggers the dimerization promotes assembly of the intact functional receptor. Wu and Lim recently reported a split CAR
in which the extracellular CD19 binding domain and the intracellular signaling element are separated and linked to the FKBP domain and the FRB* (T2089L mutant of FKBP-rapamycin binding) domain that heterodimerize in the presence of the rapamycin analog AP21967. The split receptor is assembled in the presence of AP21967 and together with the specific antigen binding, activates T cells (Wu et al., Science, 2015, 625(6258): aab4077).
1002121 In some embodiments, the CAR of the present invention may be designed as an inducible CAR. Sakemura et al recently reported the incorporation of a Tet-On inducible system to the CD19 CAR construct. The CD19 CAR is activated only in the presence of doxycycline (Dox). Sakemura reported that Tet-CD I 9CAR T cells in the presence of Dox were equivalently cytotoxic against CD19 cell lines and had equivalent cytokine production and proliferation upon CD19 stimulation, compared with conventional CD19CAR T cells (Sakemura et al., Cancer Imintino. Res.. 2016, Jun 21, Epub ahead of print). In one example, this Tet-CAR may be the payload of the effector module under the control of SREs (e.g., DDs) of the invention. The dual systems provide more flexibility to turn-on and off of the CAR expression in transduced T cells.

[00213] According to the present invention, the payload of the present invention may be a first-generation CAR, or a second-generation CAR, or a third-generation CAR, or a fourth-generation CAR. Representative effector module embodiments comprising CAR constructs are illustrated in Figures 13-18. In some embodiments, the payload of the present invention may be a full CAR
construct composed of the extracellular domain, the hinge and transmembrane domain and the intracellular signaling region. In other embodiments, the payload of the present invention may be a component of the full CAR construct including an extracellular targeting moiety, a hinge region, a transmembrane domain, an intracellular signaling domain, one or more co-stimulatory domain, and other additional elements that improve CAR architecture and functionality including but not limited to a leader sequence, a homing element and a safety switch, or the combination of such components.
[00214] CARS regulated by biocircuits and compositions of the present invention are tunable and thereby offer several advantages. The reversible on-off switch mechanism allows management of acute toxicity caused by excessive CAR-T cell expansion.
Pulsatile CAR
expression using SREs of the present invention may be achieved by cycling ligand level. The ligand conferred regulation of the CAR may be effective in offsetting tumor escape induced by antigen loss, avoiding functional exhaustion caused by tonic signaling due to chronic antigen exposure and improving the persistence of CAR expressing cells invivo.
[00215] In some embodiments, biocircuits and compositions of the invention may be utilized to down regulate CAR expression to limit on target on tissue toxicity caused by tumor lysis syndrome. Down regulating the expression of the CARS of the present invention following anti-tumor efficacy may prevent (1) On target off tumor toxicity caused by antigen expression in normal tissue. (2) antigen independent activation in vivo.
Extracellular targeting domain/Moiety [00216] In accordance with the invention, the extracellular target moiety of a CAR may be any agent that recognizes and binds to a given target molecule, for example, a neoantigen on tumor cells, with high specificity and affinity. The target moiety may be an antibody and variants thereof that specifically binds to a target molecule on tumor cells, or a peptide aptamer selected from a random sequence pool based on its ability to bind to the target molecule on tumor cells, or a variant or fragment thereof that can bind to the target molecule on tumor cells, or an antigen recognition domain from native T- cell receptor (TCR) (e.g. CD4 extracellular domain to recognize HIV infected cells), or exotic recognition components such as a linked cytokine that leads to recognition of target cells bearing the cytokine receptor, or a natural ligand of a receptor.

[00217] In some embodiments, the targeting domain of a CAR may be a Ig NAR, a Fab fragment, a Fab' fragment, a F(ab)'2 fragment, a F(ab)'3 fragment, Fv, a single chain variable fragment (scFv), a bis-scFv, a (scFv)2, a minibody, a diabody, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a unitbody, a nanobody, or an antigen binding region derived from an antibody that specifically recognizes a target molecule, for example a tumor specific antigen (TSA). In one embodiment, the targeting moiety is a scFv antibody. The scFv domain, when it is expressed on the surface of a CART cell and subsequently binds to a target protein on a cancer cell, is able to maintain the CAR T cell in proximity to the cancer cell and to trigger the activation of the T cell. A say can be generated using routine recombinant DNA
technology techniques and is discussed in the present invention.
[00218] In some embodiments, natural ligands may be used as the targeting moieties of the CARS of the present invention. Such natural ligands may be capable of binding to the antigens with affinity in the range of the scFvs and can redirect T cells specificity and effector functions to target cells expressing the complementary receptor. In some embodiments, the targeting moiety of the CAR may be neuregulin-1 (NRG1) which is a natural ligand for HER3 and HER4;
VEGF which is a natural ligand of VEGFR; IL13 wildtype protein or IL13 mutein e.g. E13Y
which binds to ILI3Ra2; NKG2D ligand, which is a natural ligand of NKG2D
receptor; CD70 which is ligand of CD27; and a proliferation-inducing ligand (APRIL) which is a natural high affmity ligand for BCMA8 and transmembrane activator and CAML interactor (TACI). Any of the ligand based BCMA CARS taught in the US Patent Publication No.
U520160362467A1, the contents of which are incorporated by reference in their entirety.
[00219] In one embodiment, the targeting moiety of the CAR may recognize antigen such as, but not limited to a ganglioside, a growth factor receptor, a lectin or any other cell surface antigen. In some embodiments, any of the sequences described in Table 7 or Table 8 may be useful in the present invention.
[002201 In some embodiments, the targeting moiety of a CAR may recognize a tumor specific antigen (TSA), for example a cancer neoantigen that is only expressed by tumor cells because of genetic mutations or alterations in transcription which alter protein coding sequences, therefore creating novel, foreign antigens. The genetic changes result from genetic substitution, insertion, deletion or any other genetic changes of a native cognate protein (i.e. a molecule that is expressed in normal cells [00221] In some embodiments, the targeting moieties of the present invention may be scFv comprising the amino acid sequences in Table 8.

'Fable 8: scFv sequences Target Description and Clone name Son me SEQ
ID
NO.
ALK ALK 15 saw SEQ ID NO. 17 in W02015069922 422 ALK ALK48 scFv SEQ ID NO. 18 in W02015069922 423 ALK ALK53 scFv SEQ ID NO. 19 in W02015(169922 424 ALK ALK58 scFv SEQ ID NO. 20 in W02015069922 425 ALK humanized ALK15 scFv SEQ ID NO. 21 in W02015069922 426 ALK humanized ALK48 scFv SEQ ID NO. 22 in W02015069922 427 ALK humanized ALK53 scFv SEQ ID NO. 23 in W02015069922 428 ALK humanized ALK58 say SEQ ID NO. 24 in W02015069922 429 CD22 CD22 (m971) scFv SEQ ID NO. 9 in W02014065961 430 CD22 CD22 (HA22 or BL22) scFv SEQ ID NO.
Sin W02013059593 431 CD22 CD22 (HA22 or BL22) scFv SEQ ID NO.
6 in W02013059593 432 CD276 CD276 scFv SEQ ID NO. 21 in W02017044699 433 CD276 CD276 scFv (CD276.6) SEQ ID NO. 10 in W02014160627 434 CD276 CD276 scFv (CD276.1) SEQ ID NO. 19 in W02014160627 435 CD276 CD276 scFv (CD276.17) SEQ ID NO. 28 in W02014160627 GD2 hu3F8/huOKT3 scFv SEQ ID No. 23 in W02011160119 437 GD2 hu3F8/C8.2.5 scFv SEQ ID No. 24 in W02011160119 438 Gangliosides DMab14-86184 say SEQ ID No. 6 in W02012071216 439 including GD2 Gangliosides GMabl scFv SEQ ID No. 20 in W02012071216 440 including GD2 Gangliosides GMab2 scFV SEQ ID No. 21 in W02012071216 441 including GD2 Gangliosides DMabl4 scFV SEQ ID No. 22 in W02012071216 442 including 01)2 GD2 GD2 scFv SEQ ID NO. 19 in W02016134286 443 GD2 GD2 scFv SEQ ID NO. 20 in W02016134287 444 GD2 GD2 scFv SEQ ID NO. 21 in W02016134288 445 0D2 Murine KM666 sequence SEQ ID NO. 7 in W02015132604 446 GD2 Humanized KM666 sequence SEQ ID NO.

8 in W02015132604 447 ¨0D2 GD2 scFv SEQ ID NO. 11 in US20160032009 448 GD2 say SEQ ID NO. 12 in US20160032009 449 GD2 GD2 scFv SEQ ID NO. 13 in US20160032009 450 GD2 GD2 scFv SEQ ID NO. 14 in US20160032009 451 -'0D2 GD2 scFv SEQ ID NO. 15 in US20160032009 452 GD2 GD2 scFv SEQ ID NO. 16 in US20160032009 453 002 GD2 scFv SEQ H) NO.
17 in US20160032009 454 GD2 002 scFv SEQ ID NO.
18 in US20160032009 455 002 002 scFv SEQ ID NO.
19 in US20160032009 456 GD2 GD2 scFv SEQ ID NO.
20 in US20160032009 457 GD2 0D2 scFv SEQ ID NO.
21 in US20160032009 458 GD2 GD2 scFv SEQ ID NO.
22 in US20160032009 459 GD2 GD2 scFv SEQ ID NO.
23 in US20160032009 460 GD2 GD2 scFv SEQ H) NO.
24 in US20160032009 461 GD2 GD2 scFv SEQ ID NO.
25 in U S20160032009 462 002 Murine KM666 scFv sequence SEQ ID NO.
7 in US20170066838 463 0D2 Humanized KM666 scFv SEQ ID NO. 8 in US20170066838 464 sequence GD2 GD2 (clone 1A7) scFv SEQ ID NO. 1 in US 465 Hed F5 SEQ ID NO. 1 in US 9,388,244 466 Her2 Cl SEQ ID NO. 2 in US 9,388,244 467 anti-Her2/neu - anti- anti-Her2/neu - anti-CD3 SEQ ID NO.
1 in W02014079000A1 468 CD3 bispecific antibody scFv Her2 F5 SEQ ID NO. 1 inUS7,332.580 469 Her2 HER3.B12 SEQ ID NO. 6 in US7,332,580 470 Her2 FL/Fc/HER2Fv - F1t3 ligand SEQ ID NO.
62 in U520040254108 471 extracellular region (hFLex); IgG1 Fc; rhuMAb ScFv Her2 Periplasmic 6 x-His (SEQ ID NO. SEQ ID NO. 26 in US
7,632,924 472 933) C terminal scFv-4D5 Herr/ Periplasmic 6x-His (SEQ ID NO. SEQ NO. 28 in US 7,632,924 933) N terminal scFv-4D5 Intracellular signaling domains [002221 The intracellular domain of a CAR. fusion poly-peptide, after binding to its target molecule, transmits a signal to the immune effector cell, activating at least one of the normal effector functions of immune effector cells, including cytoly-tic activity (e.g., cytokine secretion) or helper activity. Therefore, the intracellular domain comprises an "intracellular signaling domain" of a T cell receptor (TCR).

[00223] In some aspects, the entire intracellular signaling domain can be employed. In other aspects, a truncated portion of the intracellular signaling domain may be used in place of the intact chain if it transduces the effector function signal.
[00224] In some embodiments, the intracellular signaling domain of the present invention may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (ITAMs). Examples of ITAN1 containing cytoplasmic signaling sequences include those derived from TCR CD3zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In one example, the intracellular signaling domain is a CD3 zeta (CD30 signaling domain.
[00225] In some embodiments, the intracellular region of the present invention further comprises one or more costimulatory signaling domains which provide additional signals to the immune effector cells. These costimulatory signaling domains, in combination with the signaling domain can further improve expansion, activation, memory, persistence, and tumor-eradicating efficiency of CAR engineered immune cells (e.g., CART cells). In some cases, the costimulatory signaling region contains 1, 2, 3, or 4 cytoplasmic domains of one or more intracellular signaling and /or costimulatory molecules. The costimulatory signaling domain may be the intracellular/cytoplasmic domain of a costimulatory molecule, including but not limited to CD2, CD7, CD27, CD28, 4-1BB (CD137), 0X40 (CD134), CD30, CD40, ICOS (CD278), GITR
(glucocorticoid-induced tumor necrosis factor receptor), LFA-1 (lymphocyte function-associated antigen- 1), LIGHT, NKG2C, B7-H3. In one example, the costimulatory signaling domain is derived from the cytoplasmic domain of CD28. In another example, the costimulatory signaling domain is derived from the cytoplasmic domain of 4-1BB (CD137). In another example, the co-stimulatory signaling domain may be an intracellular domain of GITR as taught in U.S. Pat.
NOS. 9, 175, 308; the contents of which are incorporated herein by reference in its entirety.
[00226] In some embodiments, the intracellular region of the present invention may comprise a functional signaling domain from a protein selected from the group consisting of an MHC class I
molecule, a TNF receptor protein, an inununoglobulin-like protein, a cytokine receptor, an integrin, a signaling lymphocytic activation protein (SLAM) such as CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME,CD2F-10, SLAMF6, SLAMF7, an activating NK cell receptor, BTLA, a Toll ligand receptor, 0X40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD! la/CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, IL-15Ra, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD! Id, ITGAE, CD103, ITGAL, CD1 la, LFA-1, ITGAM, CD! lb, ITGAX, CD! lc, ITGB1, CD29, 1TGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, NI(D2C SLP76, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, CD270 (HVEM), GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds with CD83, DAP 10, TRIM, ZAP70, Killer immunoglobulin receptors (KIRs) such as KIR2DL1, K1R2DL2/L3, K1R2DL4, K1R2DL5A, KIR2DL5B, K1R2DS1, K1R2DS2, KIR2DS3, KIR2DS4, KIR2DS5, KIR3DLI/S1, KIR3DL2, KIR3DL3, and KIR2DP1; lectin related NK cell receptors such as Ly49, Ly49A, and Ly49C.
[00227] In some embodiments, the intracellular signaling domain of the present invention may contain signaling domains derived from JAK-STAT. In other embodiments, the intracellular signaling domain of the present invention may contain signaling domains derived from DAP-12 (Death associated protein 12) (Topfer et al., Immunol., 2015, 194: 3201-3212;
and Wang et al., Cancer Immunol., 2015, 3: 815-826). DAP-12 is a key signal transduction receptor in NK cells.
The activating signals mediated by DAP-12 play important roles in triggering NK cell cytotoxicity responses toward certain tumor cells and virally infected cells.
The cytoplasmic domain of DAP12 contains an Immunoreceptor Tyrosine-based Activation Motif (ITAM).
Accordingly, a CAR containing a DAP12-derived signaling domain may be used for adoptive transfer of NK cells.
[00228] In some embodiments, T cells engineered with two or more CARs incorporating distinct co-stimulatory domains and regulated by distinct DD may be used to provide kinetic control of downstream signaling.
[00229] In some embodiments, the intracellular domain of the present invention may comprise amino acid sequences of Table 9.
Table 9: Intracellular si2naling and co-stimulatory Domain Sequence SEQ
ID
NO
2B4 co-stimulatory VIRRICRICEKOSETSPICEFLTIYEDVKDLKTRRNHEQEQTFPGGGSTIYSMIQ 474 domain SQSSAPTSQEPAYTLYSLIQPSRKSGSRKRNIISPSFNSTIYEVIGKSQPKAQ
NPARLSRKELENFDVYS
CD27 co- HORRKYRSNKGESPVEPAEPCRYSCPREEEGSTIPIQEDYRKPEPACSP 475 stimulatory domain CD272 (BMA I) RRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDN 476 co-stimulatory DPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAP
domain TEYASICVRS

CD272 (BTLA1) CCLRRHQGKQNELSDTA GREINLVDAHLKSEQTE ASTRQNSQVLLSETGI 477 co-stimulatoly YDNDPDLCFRMQEGSE VYSNPCLEENKPGIVYASLNHSVIGPNSRL ARNV
domain KEAPTEYASICVRS
CD28 co- FWVLWVGGVLACYSLLVIVAFIIFWV 478 stimulatory CD28 co- KRCiRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL 479 stimulatory domain CD28 co- FWVRSICRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 480 stimulatory domain CD28 co- RSKRSRGGHSDYIVINMTPRRPGPTRKHYQPYAPPRDFAAYRS 481 stimulatory domain CD28 co- RSICRSRGGHSDYIVINMTPRRPGPTRKHYQPYAPPRIWA AYRS 482 stimulatory domain CD28 co- IVILRLLLALNLFPSIQVTGNICILVKQSPMLVAYDNAVNLSCKYSYNLFSRE 483 stimulatoiy FRASLHKGLDSAVEVCWYGNYSQQLQVYSKTGFNCDGKLGNESVTFYL
signaling region QNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTI1HVKGKHLCPSPLFPGPS
KPFWVLVVVGG VLACYSLLVTVAFILFWVRSKRSRLLHSDYMNMTPRRP
GPTRKHYQPYAPPRDFAAYRS
CD30 co- RRACRKRIRQKLHLCYPVQTSQPICLELVDSRPRRSSTQLRSGASVTEPVAE 484 stimulatory domain ERGLMSQPLMETCHSVGAAYLESLPLQDASPAGGPSSPRDLPEPRVSTEHT
NNKIEKIYIMKADTVIVGTVKAELPEGRGLAGPAEPELEEELEADHTPHYP
EQETEPPLGSCSDVML SVEEEGKEDPLPTAASGK
GITR co- HIWQLRSQCNIWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEEKGRLG 485 stimulatory domain DLWV
HVEM co- CVKRRKPRGDWKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETI 486 stimulatory domain PSFTGRSPNH
ICOS co- TKICKYSSSVHDPNGEYMFMRAVNTAKKSRLTDVTL 487 stimulatory domain ICOS co- C'WLTICICKYSSSVHDPNGEYMFMR AVNTAKK SRLTD'VTL 488 stimulatory signaling domain LAG-3 co- HLWRRQVIRPRRFSALEQUHPPQAQSKTEELEQEPEPEPEPEPEPEPEPEPE 489 stimulatory region QL
0X40 co- ALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQ.AD Ali STLAKI 490 stimulatory domain 0X40 co- RRDQRLPPDAFIKPPGGGSFRTPIQEEQADAHSTLAKI 491 stimulatory domain 4- EBB intracellular KRGRKKLLYlFKQPFMRPVQTIQEEDGCSCRFPEEEEGGCEL 492 domain 4-1BB signaling KRGRKKLLYIFKQPFMRPVQ'TTQEEDGCSCRFPEEEEGGYEL 491 domain 4-1BB-CD3Zeta TGTTTPAPRPPTPAPTIASQPL SLRPEACRPAAGGAVHTRGLDFACDIYIWA 494 intracellular PLAGTCGVLLLSLVITLYCKRGRICKLLYTKQPFMRPVQTTQEEDGCSCRF
domain PEEEEGGCELRVICFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDICRR
GRDPEMGGKPRRKNPQEGLYNELQKDICMAEAYSEIGMKGERRRGKGHD
GLYQGLSTATKDTYD HMQALPPR

endodomain fusion APAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG
LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH
MQALPPR
CD 127 KR rKpi VWPSLPDHKKTLEHLCKKPRKNLNVSFNPESFLDCQIHRVDDIQA 496 intracellular RDEVEGFLQDTFPQQLEESEKQRLGGDVQSPNCPSED VVITPESFGRDSSL
domain TCLAGNVSACDAPILSSSRSLDCRESGKNGPHVYQDLLLSLGTTNSTLPPPF
SLQSGTLTLNPVAQGQPILTSLGSNQFEAYVTMSSFYQNQ

intracellular domain intracellular DLKLVGISQPKYAAELAENRGKNRYNNVLPYDISRVKLSNIQTHSTDDYIN
domain ANYMPGYHSKKDFIATQGPLPNTLKDFWRMVWEKNVYAIIMLTKCVEQ
GRTKCEEYWPSKQAQDYGDITVAMTSEIVLPEWTIRDFTVKNIQTSESHPL
RQFHFTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPILVHCSAGVGR
TGIFIA IDRLIYQTENENTVD VYGIVYDLRMHRPLMVQTEDQYVFLNQCV
LDIVRSQKDSKVDLIYONTTAMTIYENLAPVITFGKTNGYIA
C1)27 intracellular QRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP 499 domain CD28 intracellular FAAYRS 500 domain CD28 signaling FWVLVVVGGVLACYSLLVTVAPII FWVR SK R SR LLHSDYMNMTPRRPGPT 501 chain RKITYQPYAPPRDFA.AYRS
CD28 signaling RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 502 domain C1)28 signaling SKRSRLLHSDYMNNITPRRPGPIRKHYQPYAPPRDFAAYRS 503 domain CD28 signaling IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLNIVVGGVLA 504 domain CYSLLVTVAF1IFWRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDF
AAYRS
CD28, 4-1BB, RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRFSVVKRG 505 and/or CDR', RKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPA
signaling domain YQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRICNPQEGLYN
ELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQA
LPPR

PSP LFPGP SKP FWV L 'ATV G G 506 VLACYSLLVTVAFIlFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAP
PRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG
RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG
LYQGLSTATKDTYDALHMQALPPR
CD28-0)CZ RSKRSRLLHSDYNMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAH 507 intracellular KPPGGGSFRTPIQEEQADAHSTLAKIRVKFSR SADAPAYQQGQNQLYNEL
domain NLGRREEYDVLDKRRGRDPEIVIGGKPRRKNPQEGLYNELQKDKMAEAYS
EIGMKGERRRGKGHDGLYQGLSTATKDTYDALIIMQALPPR

intracellular TQEEDGCSCRFPEEEEGGCEL
domain intracellular CYSLLVTVAFIIFWVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEE
domain EGGCEL
CD28-CD3 Zeta RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSA 510 intracellular DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE
domain GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKUTYDAL
HMQALPPR
CD28-CD3Zeta KRSRLLHSDYKNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADA Ill intracellular PAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGL
domain YNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM
QALPPR
CD3 delta chain MEHSTFLSGLVLATLLSQVSPFKIP1EELEDRVFVNCNTSITWVEGTVGTLL 512 intracellular SDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPAT
signaling domain VAGIIVTDVIATLLLALGVFCFAGHETGRLSGAADTQALLRNDQVYQPLR
DRDDAQYSHLGGNWARNK
CD3 delta chain MEHSTFLSGLVLATLLSQVSPFKTPIEELEDRVFVNCNTSITWVEGTVGTLL 513 intracellular SDTTRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRTADTQALLRND
signaling domain QVYQPLRDRDDAQYSHLGGNWARNK
CD3 delta chain DQVYQPLRDRDDAQYSHLGGN 514 intracellular signaling domain CD3 delta MEHSTELSGLVLATLLSQVSPFKIPIEELEDRVEVNCNTSITWVEGTVGTLL 515 intracellular SDITRLDLGKRILDPRGIYRCNGTDIYKDKESTVQVHYRMCQSCVELDPAT
domain VAGIIVTDVIATLLLALGVECFAGHETGRLSGAADTQALLRNDQVYQPLR
DRDDAQYSHLGGNWARNK
CD3 delta MEHSTFLSGLVLATLL SQVSPFKIPEEELEDRVFVNCNTSITWVEGTVGTLL 516 intracellular SDITRLDLGKRILDPRGIYRCNGTDEYKDKESTVQVHYRTADTQALLRND
domain QVYQPLRDRDDAQYSHLGGNWARNK
CD3 epsilon MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCP 517 intracellular QYPGSEILWQHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYWYP
domain RGSKPEDANFYLYLRARVCENCMEMDVMSVATIVIVDICITGGLLLLVYY
WSKNRKAKAKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGQRDLY
SGLNQRRI
CD3 epsilon NPDYETIRKGQRDINSGLNQR
ininicellular domain CD3 gamma MEQGKGLAVLILAILLLQGTLAQSIKGNHLVKVYDYQEDGSVLLTCDAEA 519 intracellular ICNITWFKDGICMIGELTEDKKKWNLGSNAKDPRGMYQCKGSQNKSKPLQ
domain VYYRMCQNCIELNAATISGELFAEIVSIFVLAVGVYFIAGQDGVRQSRASD

CD3 gamma DQLYQPLKDREDDQYSHLQGN 520 intracellular domain CD3 zeta MKWKALFTAAILQAQLPrrEAQsFGLLDPKLCYLLDGILFIYGVILTALFLR 521 intracellular VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP
domain QRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA
TKDTYDALHMQALPPR
CD3 zeta NQLYNELNLGRREEYDVLDKR 522 intracellular domain CD3 zeta domain 2 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLD1CRRGRDPEMGGK 523 (NM_000734.3) PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA
TKDTYDALHMQALPPR
CD3 zeta DGLYQGLSTATKDTYDALHMQ 524 int racell War domain CD3 zeta RVKFSRSAEPPAYQQGQNQLYNELNLGRREEYDVLDICRRGRDPEMGGKP 525 intracellular RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT
domain KDTYDALI1MQALPPR
CD3 zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK 526 intracellular PQRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST
domain ATKDTYDALHMQALPPR
C1)3 zeta RSR VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMG 527 intracellular GKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLS
domain TATKDTYDALHMQALPPR
CD3 zeta RVICFSRSADAPAYQQGEYDVLDKRRGRDPENIGGKPRRKNPQEGLYNEL 528 intracellular QKDICMAEAYSEIGMKGERRRGKGFIDGLYQGLSTATKDTYDALHMQALP
domain PR
CD3 zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEVDVLDKRRGRDPEMGGK 529 intracellular PRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA
domain TKDTYDALHMQALPPR
CD3 zeta MIPAVVLLLLLLVEQAAALGEPQLCYILDAILELVGIVLTLINCRLKIQVRIC 530 intracellular AAUSYEKSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG
domain RDPEMGGKPRRKNPQEGLYNELQKDKMAEAVSEIGMKGERRRGKGHDG
LYQGLSTATKDIYDALHWALPPR
CD3 zeta LRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG 531 intracellular KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST
domain ATKDTYDALHMQALPPR
CD3 zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK 532 intracellular PQRRKNPQEGLY
domain CD3 zeta LR'VKFSRSADAPAYQQGQNQINNELNLGRREEYDVI,DKRRGRDPEMGG 533 intracellular ICPQRRKNPQEGLYNELQICDKMAEAYSEIGME.GERRRGKGHDGLYQGLS
domain TATKDTYDALHMQALPPR
CD3 zeta RRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDICRRGRDPEMGG 534 intracellular KPRRICNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLST
domain ATKDTYDALHMQALPPR
CD3 zeta EGLYNELQKDKMAEAYSEIGMK 535 intracellular domain CD3 zeta R'VICFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGK 536 intracellular PRRKNPQEGLYNELQKDICMAEAYSEIGMKGERRRGKGHDGLYQGLSTA
domain TKUTYDALEMQALPPR
CD3 zeta RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDICRRGRDPEMGGIK 537 intracellular PRRKNPQEGLYNELQICDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA
domain TICDTYDALHMQALP
C1)3 zeta DPKLCYLLDGILFIYGVILTALFERVKFSRSADAPAYQQGQNQLYNELNLG 538 intracellular RREEYDVLDKRRGRDPENIGGKPQRRKNPQEGLYNELQKDKMAEAYSEI
domain GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR
CD3 zeta MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFIYGVILTALFLR 339 intracellular VKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKP
domain RRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTAT
KDIYDALHMQALPPR
CD40 intracellular RSRDQFtLPPDAHICPPGGGSFRTPIQEEQADAHSTLAKI 540 domain CD79A MPGGPG'VLQALPATIFLLFLLSAVYLGPGCQALWMHKVPASLMVSLGED 541 intracellular AHFQCPHNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNGTLIIQNVNK
domain SHGGIYVCRVQEGNESYQQSCGTYLRVRQPPPRPFLDMGEGTKNRIITAEG
TILLFCAVVPGTLLLFRKRWQNEKLGLDAGDEYEDENLYEGLNLDDCSMY
EDISRGLQGTYQDVGSLNIGDVQLEKP

intracellular AFIFQCPITNSSNNANVTWWRVLHGNYTWPPEFLGPGEDPNEPPPRPFLDM
domain GEGTKNRIITAEGIILLFCAVVPGILLLFRKRWQNEKLGLDAGDEYEDENL
YEGLNLDDCSMYEDISRGLQGTYQDVGSLNIGDVQLEKP

intracellular domain CD8 intracellular FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF 544 domain ACDIYIWAPLAGTCGVLLLSLVITLYCNIIRNR
CD8 intracellular FVPVFLPAKPITTPAPRPPIPAPTIASQPLSLRPEACRPAAGGAVIIIRGLDF 545 domain ACDIYIWAPLAGTCGVLLLSLVITLYCNTIRNR
CD8a intracellular PMPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI 546 domain intracellular domain intracellular domain intracellular domain intracellular LVLTVLIALAVYFLGRLVPRGRGAAEANIRKQRITETESPYQELQGQRSD
domain VYSDLNTQRPYYK

intracellular LVLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESPYQELQGQRSDV
domain YSDLNTQRPYYK

intracellular FLGRLVPRGRG A AEAATRKQRITETESPYQELQGQR SDVY SDLNTQRPYY
domain intracellular FLGRLVPRGRGAAEATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK
domain intracellular LVLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSD
domain VYSDLNTQRPYYK

intracellular LVLTVLIALAVYFLGRINPRGRGAAEATRWRITETESPYQELQGQRSD V
domain YSDLNTQRPYYK

intracellular FLGRLVPRGRGAAEAATRKQRITETESPYQELQGQRSDVYSDLNTQRPYY
domain DAP! 2 MGG LEPCSRULLPLLLAVSDCSCSTVSPGVLAGIVMGDLVLIVLIA AVY 557 intracellular FLGRLVPRGRGAAEATRKQRITETESPYQELQGQRSDVYSDLNTQRPYYK
domain intracellular domain GITR intracellular R SQCNTWPR ETQLLL EVPPSTEDAR SCQFPEEER GER SAEEK GR LGDLW V

domain !COS intracellular TK K KY S S MTH DPNGEFMFMR A VNTAKKSRLTDvn, 560 domain IL- 15Ra KSRQTPPLASVEMEAMEALPµTWGTSSRDEDLENCSHHL 561 intracellular domain 0X40-CD3 Zeta RRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAICIRVKFSRSADAPAYQ 562 intracellular QGQNQLYNELNLGRREEYDVLDICRRGRDPEMGGKPRRKNPQEGLYNEL
domain QICDKMAEAYSEIGMKGERRRGKGIADGLYQGLSTATKDTYDALHMQALP
PR

intracellular VHDVRFHHFPIERQLNGTY Al AGG K AHC GPA ELC En( SRDPDGLPCNLRK
domain PCNRPSGLEPQPGVFDCLRDAMVRDYVRQTWKLEGEALEQAIISQAPQVE
KLIATTAFTERMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTYAL

KEACPNSSASNASGAAAPTLPAHPSTLTHPQRRIDTLNSDGYTPEPARITSP

GVCQAEALMLVMEMAGGGPLHKFLVGICREEIPVSNVAELLHQVSMGMK
YLEEKNFVHRDLAARNVLLVNRHYAKISDFGLSKALGADDSYYTARSAG
KWPLKWYAPECINFRKFSSRSDVWSYGVTMWEALSYGQKPYICKIVIKGPE
VMAFIEQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQRMRAC
YYSLASKVEGPPGSTQKAEAACA
CD28 intracellular MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSRE 564 domain FRA SLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYL
QNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPS
KPFWVLVVVGGVLACYSLLVTVAFILFWVR
4-113B intracellular MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPN 565 domain SFSSAGGQRTCDICRQCKGVFRTRICECSSTSNAECDCTPGFHCLGAGCSM
CEQDCKQGQELTKKGCICDCCFGTFNDQICRGICRP'WTNCSLDGK SVLVNG
TKERDVVCGPSPADL SPCA SSVTPPAPAREPGHSPQNSFFLALTSTALLFLL
FFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDG
Fc epsilon MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTLLYCRLKIQVRK 566 Receptor I gamma AAITSYEKSDGVYTGLSTRNQETYETLICHEKPPQ
chain intracellular domain Fc epsilon DGVYTGLSTRNQETYETLKHE 567 Receptor I gamma chain intracellular domain Fc epsilon DPKLCYILDAILFLYGIVLTLLYCRLKIQVRKAAITSYEKSDGVYTGLSTRN 568 Receptor I gamma QETYETLKHEKPPQ
chain intracellular domain CD28 intracellular SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS 569 domain CD28 signaling IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLA 570 domain CYSLLVIVAFIIFAVVR
CD8 signaling FVPVFLPAKP 1'11PAPRPPTPAPTIASQPLSLRPEACRPA AGGAVHTRGLDF 571 domain ACDIYIWAPLAGTCGVLLLSLVITLYCNHRNR
intracellular T cell IEVMYPPPYLDNEKSNGTEUIVKGKFILCPSPLFPGPSKPFWVLVVVGGVLA 572 signaling domain CYSLINTVAFITFWVR SKRSRLLHSDYMNNTI'PRRPGPIRKHYQPYAPPRD
comprising CD28 FAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP
and CD3 zeta EMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ
GLSTATKDTYDALHMQALPPR
intracellular T cell FVP'VFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF 573 signaling domain ACDIYIWAPLAGTCGVLLLSLVITLYCNI-IRNRSKRSRLLHSDYMNMTPRR
comprising CD28, PGPTRKHYQPYAPPRDFAAYRSRFSVVICRGRKKLLYTKQPFMRPVQIIQ
CD137, and CD3 EEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREE
zeta YDVLDK RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK GE
RRRGKGFIDGLYQGLSTATKUTYDALHMQALPPR
Transmembrane domains [00230] In some embodiments, the CAR of the present invention may comprise a transmembrane domain. As used herein, the term "Transmembrane domain (TM)"
refers broadly to an amino acid sequence of about 15 residues in length which spans the plasma membrane.
More preferably, a transmembrane domain includes at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41., 42, 43, 44, or 45 amino acid residues and spans the plasma membrane. In some embodiments, the transmembrane domain of the present invention may be derived either from a natural or from a synthetic source. The transmembrane domain of a CAR may be derived from any naturally membrane-bound or transmembrane protein.
For example, the transmembrane region may be derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T-cell receptor, CD3 epsilon, CD4, CDS, CD8, CD8a, CD9, CD16, CD22, CD33, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, or CD154.
[00231] Alternatively, the transmembrane domain of the present invention may be synthetic. In some aspects, the synthetic sequence may comprise predominantly hydrophobic residues such as leucine and valine.
[00232] In some embodiments, the transmembrane domain of the present invention may be selected from the group consisting of a CD8a transmembrane domain, a CD4 transmembrane domain, a CD 28 transmembrane domain, a CTLA-4 transmembrane domain, a PD-1 transmembrane domain, and a human Igo; Fc region. As non-limiting examples, the transmembrane domain may be a CTLA-4 transmembrane domain comprising the amino acid sequences of SEQ ID NOs. 1-5 of International Patent Publication NOS.
W02014/100385; and a PD-I transmembrane domain comprising the amino acid sequences of SEQ ID NOs. 6-8 of International Patent Publication NOS. W02014100385; the contents of each of which are incorporated herein by reference in their entirety.
1002331 In some embodiments, the CAR of the present invention may comprise an optional hinge region (also called spacer). A hinge sequence is a short sequence of amino acids that facilitates flexibility of the extracellular targeting domain that moves the target binding domain away from the effector cell surface to enable proper cell/cell contact, target binding and effector cell activation (Patel et al., Gene Therapy, 1999; 6: 412-419). The hinge sequence may be positioned between the targeting moiety and the transmembrane domain. The hinge sequence can be any suitable sequence derived or obtained from any suitable molecule. The hinge sequence may be derived from all or part of an immunoglobulin (e.g., IgGI, IgG2, IgG3, IgG4) hinge region, i.e., the sequence that falls between the CHI and CH2 domains of an immunoglobulin, e.g., an IgG4 Fc hinge, the extracellular regions of type 1 membrane proteins such as CD8a CD4, CD28 and CD7, which may be a wild type sequence or a derivative. Some hinge regions include an immunoglobulin CH3 domain or both a CH3 domain and a CH2 domain. In certain embodiments, the hinge region may be modified from an IgGl, IgG2, IgG3, or IgG4 that includes one or more amino acid residues, for example, 1, 2, 3, 4 or 5 residues, substituted with an amino acid residue different from that present in an unmodified hinge.
Table 10 provides various transmembrane regions that can be used in the CARs described herein.
Table 10: Transmembrane domains Tratismembrane domain Sequence SEQ Ill NO
CD8 Transme inbratie domain 11 1PAPRPPTPAPTIASQPLSLRPEACRPAAGGAV 574 HIRGLDFACDI
4-1BB Transmembrane domain IISFFLALTSTALLFILFFLTLRFSINKRGR 575 4-1BB Trans membrane domain IISFFLALTSTALLFLLITLTLRFSW 576 CD134 (0X40) Transmembrane VAAILGLGLVLGLLGPLAILLALYLL 577 domain CD148 Transmembrane and AVFOCIFGALVIVTVGGFIFWRKKRICDAKNNEVS 578 intracellular domain FSQIKPKKSKLIRVENFEAYFICKQQADSNCGFAEE
YEDLKINGISQPKYAAELAENROKNRYNNVLPY
DISIWKLSVQTHSTDDYINANYMPGYHSKKDFIA
TQGPLPNTLKINWRMVWEKNVYAIIMI.TKCVEQ
GRTKCEEYWPSKQAQDYGDITVAIVITSEIVI.PEW
Ti DFTVKNIQTSESFIPLA Q1-71-1FTSW PDHovpm-r DLLINFRYLVRDYNIKQSPPESPILVHCSAGVGRTG ............................... 1 TFIAIDRLIYQIENENTVDVYGIVYDLRMHRPLMV
QTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNTTA
MTIYENLAPVTTEGKINGYIA
Cl) 148 Tratismembiarie domain CD2 Transmembrane domain KEITNALETWGALGQDINLDIPSFQMSDDIDDIKW 580 EKTSDICKKIAQFRKEICETEKEKDTYKLEKNGTLK
IKHLKTDDQDIYKVSIYDTKGKNVLEKIFDLKIQE
RVSKPKISWTCINTTLTCEVNINGTDPELNLYQDG
KHLKLSQRN/TITIKWITSLSAKFKCTAGNKVSKES
SVEPVSCPEKGLD
CD28 Transmembrane and IEVMYPPPYLDNEKSNGTITHVKGICHLCPSPLFPG 581 intracellular domain PSKPFWVLYVVGGVLACYSLLVTVAIIIFWVRSK
RSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA
AYRS
CD28 Transmembrane domain FWVLVVVGGVLACYSLL'VTVAF1IFWV 582 CO28 Tra risme inb ninc domain IEVMYPPPYLDNEKSNGTIIHVKGKIILCPSPLFPGP 583 SKPFWVLVVVGGVLACYSLLVTVAFIIFWV
CD28 Transmembrane domain 1FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRR 584 CD28 Transmembrane domain FWVLVVVGGVLACYSLLVTVAFTIFWVRSKRSRL 585 LHSDYNINMTPRRPGPTRKHYQP
YAPPRDFAAYRS
CD28 Transmembrane domain NIFWVLVVVGGVLACYSLLVTVAFTIFWV 586 CD28 Transmembrane domain FWVLVVVGGVLACYSLLVINAFHFWV 587 CD28 Tramsmembrane domain NIFWVLVVVGGVLACYSGGVINAFIIFWV 588 CD28 Transinetribranc domain CD28 Tninsmerribnine domain PFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSR 590 LLHSDYMNMTPRRPGPMKHYQPYAPPRDFAAY
RS
CD28 Transmembrane domain and FWVLVVVGGVLACYSLLVTVAFIEFWVRSKRSRL 591 CD28 and CD3 Zeta intracellular LHSDININNTIT'RRPGPTRKHYQPYAPPRDFAAYR
domain SRVKFSRSADAPAYQQGQNQLYNELNLGRREEY
DVLDKRRGRDPEMGGKPRRICNPQEGLYNELQICD
ICMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK
DTYDALHMQALPPR
CD28 Transmembrane domain and FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRL 592 CD28, 0X40, and CO3 Zeta LHSDYMNNITPRRPGPIRICHYQPYAPPRDFAAYR
intracellular domain SRDQRLPPDAHKPPGGGSFRTPIQEEQADARSTLA
KIRVICFSRSADAPAYQQGQNQLYNELNLGRREEY
DVLDICRRGRDPEMGGKPRRICNPQEGLYNELQKD
KNIAEAYSEIGNIKGERRRGKGHDGLYQGLSTATK
DTYDALIIMQALPPR

CD28 Transmembrane domain and FWVLVVVGG'VLACYSLL'VTVAFTIFWVRR'VKFSR 593 CD3 Zeta intracellular domain SADAPAYQQGQNQLYNELNLGRREEYDVLDICRR
GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY
SEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL
HMQALPPR
CD28 transmembrane-CD3 zeta AAAIEVMYPPPYLDNEKSNGTIEFIVKGKHLCPSPL 594 signaling domain (28z") FPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV
RSICRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPR
DFAAYRSRVKFSRSADAPAYQQGQNQLYNELNL
GRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY
NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ
GLSTATICDTYDALHMQALPPR
CD3 zeta Transmembrane domain LCYLLDGILFIYGVILTALFLRV 595 CD3 zeta Transmembrane domain MK'WICALFTAAILQAQLPITEAQSFGLLDPKLCYL 596 CD3 zeta Transmetribrane domain LCYLLDGILFIYGVILTALFL 597 CD4 Transmembrane domain ALIVLGGVAGLLLFIGLGIFFCVRC 598 CD4 Transmembrane domain MALIVLGGVAGLLLFIGLGIFF 599 CD45 Transmembrane and ALIAFLAFLIIVTSIALLWLYKIYDLHKICRSCNLD 600 intracellular domain EQQELVERDDEKQLMINVENHADILLETYKRKIA

YVDILPYDYNRVELSEINGDAGSNYINASYIDGEK
EPRKYIAAQGPRDETVDDEWRMIWEQICATVIVM
VTRCEEGNRNKCAEYWPSMEEGTRAFGDVVVKI
NQHKRCPDYIIQKLNIVNKICEKATGREVTHIQFTS
WPDHGVPEDPHLLLKLRRRVNAFSNFFSGPIWHC
SAGVGRTGTYIGIDAMLEGLEAENKVDVYGYVV

NLSELHPYLHNMKKRDPPSEPSPLEAEFQRLPSYR
SWRTQHIGNQEENKSKNRNSNVIPYDYNRVPLKH
ELEMSKESEHDSDESSDDDSDSEEPSKYINASFIM
SYWICPEVMIAAQGPLKETIGDFWQMIFQRICVKVI
VMLTELKHGDQEICAQYWGEGKQTYGDIEVDLK
DTDKSSTYTLRVFELRHSKRICDSRTVYQYQYTN
WSVEQLPAEPKELISMIQWKQKLPQKNSSEGNKH
HKSTPLLIHCRDGSQQTGIFCALLNLLESAETEEW
DIFQWKALRKARPGMVSTFEQYQFLYDVIASTYP
AQNGQVKKNNHQEDKIEFDNEVDKVKQDANCV
NPLGAPEKLPEAKEQAEGSEPTSGTEGPEHSVNGP
ASPALNQGS
CD62L Transmembrane domain PLFIPVAVNIVTAFSGLAFIIWLA 601 CD7 Transmembrane domain ALPAALAVISFLLGLGLGVACVLA 602 CD8 Transmembrane domain MALPVTALLLPLALLLHAARP 603 CD8 Transmembrane domain and AAAFVPVFLPAICPTITPAPRPPTPAPTIASQPLSLR 604 CD28 signaling domain PEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG
VLILSLVITLYCNHRNRSKRSRILHSDYMNMTPR
RPGPTRKHYQPYAPPRDFAAYRSRFSVVICRGRICK
LINIFKOPFMRPVQTTQEEDGCSCRFPEEEEGGCE

LRVKFSRSADAPAYQQGQNQLYNELNLGRREEY
DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD
KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATK
DTYDALHMQALPPR
CD8 transniembralle domain- AAA111PAPRPPTPApTLASQPI_SLRPEAC7RPAAG 605 CD137 (4-1BB) signaling domain GAVHIRGLDFACDIYIWAPLAGTCGVLLLSLVITL
and CD3 zeta signaling domain YCKRGRKKLLY1FKQPFMRPVQTTQEEDGCSCRF
("BBz") PEEEEGGCELRVICFSRSADAPAYKQGQNQLYNEL
NLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG
LYNELQKDKMAEAYSEIGMKGERRRGKGHDGL
YQGLSTATKDTYDALHMQALPPR
CD8a Transmembrane domain FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEA 606 CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLL
LSLVITLYCNHRN
CD8a Transinembrane domain IWAPLAGTCGVLLLSLVITLYC 607 CD8a Ininsmembrane domain IYIWAPLAGTCGVI,IISLVITLYC 608 CD8a Tiaristnembrane domain CD8a Transmembrane domain IYIWAPLAGTCGVLLLSLVITLVCR 610 CD8a Transmembrane domain IYIWAPLAGTCGVLLLSLVIT 611 CD8a Transmembranc domain IYIWAPLAGTCGVLLLSLVITLY 612 CD8b Transmembrane domain LGLLVAGVLVLLVSLGVAIHLCC 613 EpoR Transmembrane domain APVGLVARLADESGHVVLR'WLPPPETPMTSHIRY 614 EVDVSAGNGAGSVQRVEILEGRTECVLSNLRGRT
RYTFAVRARMAEPSFGGFWSAWSEPVSLUPSD
FcERI a- Transmembrane domain MAPAMESPTLLCVALLFFAPDGVLAVPQKPKVSL 615 NPPWNRIFKGENVTLTCNGNNFFEVSSTKWFHNG
SLSEETNSSLNIVNAKFEDSGEYKCQHQQVNESEP
VYLEVFSDWLLLQASAEVVMEGQPLFLRCHGWR
NWDVYKVIYYKDGEALKYWYENHNISITNATVE
DSGTYYCTGKVWQLDYESEPLNITVIKAPREKW
LQFFIPLLVVILFAVDTGLFISTQQQVTFLLKIKRT
RKGFRLLNPHPKPNPKNN
FceRIa Transmembrane domain DIFIPLLVVILFAVDTGLFISTQQQVTFLLKIKRTRK 616 GFRLLNPHPKPNPKNNR
GITR Transmembrane domain PLGWLTVVLLAVAACVLLLTSAQLGLHIWQL 617 Her2 Transmembrane domain SIISAVVGILLVVVLGV4TGILII 618 Her2 Transmembrane domain CHPECQPQNGSVTCFGPEADQCVACAHYKDPPFC 619 VARCPSGVKPDLSYMPIWKFPDEEGACQPCPINC
THSCVDLDDKGCPAEQRASPLTSIISAVVGILLVV
VLGVVFGILI
(COS Tninsmerribmile domain IgG1Transinembwnc domain EPKSPDICITITCPPCPAPPVAGPSVFLFPFKPKDTL 621 MIARTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQD'WLNGK
EYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLP
PSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ
PENNYKTTPPVLDSDGSFTLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQK SLSLSPGKKD
0X40 Transmembrane domain VAAILGLGINLGLLGPLA ILL 622 Transmembrane domain IYIVi/APLAGTCGVLLLSLVITLYC 623 Transmembrane domain IYINVAPLAGTCGVLLLSLVITINC 624 CD28 transmembrane and IEVMYPPPYLDNEKSNGTIIH=VKGICHLCPSPLFPGP 625 signaling domains SKPFWVL VVVGGVLACY SLL VIVAFIIFWVRSKR
SRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFA
A'YRS
CD28 Transmembrane domain VMYPPPYLDNEK SNGTI IIINKGKHLCPSPLFPGPS 626 KPFWVLVVVGGVLACYSLINTVAFIEFWVR
CD8 Transmembrane domain TTIPAPRPPIPAPTIASQPISLRPEACRPAAGGAV 627 wrizaDFACDIYIWAPLAGTCGVILLSLVITLYC
1002341 Hinge region sequences useful in the present invention are provided in Table 11.
Table 11: Hinge regions Hinge Domain Sequence SEO ID
NO
Hinge DK.THT 628 Hinge CPPC7 629 Hinge CPEPKSCDTPPPCPR 630 Hinge ELKTPLGDTTHT 631 Hinge KSCDKTHTCP 632 Hinge KCCVDCP 633 Hinge KYGPPCP 634 C233P Hinge VEPKSPDKTHTCPPCP 635 C233S Hinge LDPKSSDKIIITCPPCP 636 CD28 Hinge IEVMYPPPYLDNEKSNGT11HVKGKHLCPSPLFPGPSKP 637 CD8a Hinge GGAVITIRGLDFA 638 CD8a Hinge I PA PRPPTPAPTIA SQPL SLR PEA CR PA AGGA VEITR GL DFA CD 639 CD8a Hinge AKPITTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD 640 CD8a Hinge TITPAPRPPTPAPTIA SQPLSLRPEACRPAAGGAVHTRGLDFACDEPKSP 641 DKIIITCPPCPAPPVAGPSVFLITPKPKDT
CD8a Hinge PAKPTTTF'APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI 642 CD8a Hinge *M'PAPRPPTPAPTIASQPLSLRPEACRPAAGGA VHTRGLDFACDIYIWA 643 PLAGTCGVLLLSINITLYC
CD8a Hinge TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD 644 ¨CD8a Hinge r111'APRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY 645 Delia5 Hinge IDK-111.1"CPPCP 646 EpoR Hinge APVGLVARLADESGHWLRWLPPPETPMTSHIRYEVDVSAGNGAGSV 647 QRVEILEGRIECVLSNLRGRTRYTFAVRARMAEPSFGGFWSAWSEPVS
LLTPSD
FCRHa Hinge GLAVSTISSFFPPGYQ 648 Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKE 649 KEICEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVV
GSDLKDAHLTWEVAGKVPTGG VEEGLLERHSNGSQSQHSRLTLPRSL
WNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEA
ASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSITFWA
WSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH
Hinge YVTVSSQDPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISR 650 TPEVTCVVVDVSHEDPEVICFNWYVDGVEVI-LxIAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGICEYKCKVSNKALPAPEEKTISICAICGQPREPQV
YTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYICITPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL
SPGKICDPK
Hinge KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA 651 Hinge LEPKSCDKTHTCPPCP 652 Hinge KPl'i'l PAPRPPTPApTIASQPLSLRPEACRPAAGGAVITTRGLD 653 Hinge ELKTPLGDIFITCPRCP 654 Hinge EPKSCDTPPPCPRCP 655 Hinge ESKYGPPCPSCP 656 Hinge ERKCC'VECPPCP 657 Hinge (CH2- ESKYOPPCPPCPAPEFLGGPSVFLFPPKPKDILMISRITEVICVVVDVSQ 658 CH3) EDPENTQFNWYVDGVEVHNAKTKPREEQFN STYRVVSVLTVLHQDWL
NGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ
VSLICLVKGFYPSDIAVEWESNGQPENNYKTITPVLDSDGSFFLYSRLT
VDKSRWQEGNVTSCSVMHEALHNHYTQKSLSLSLGK
Hinge (CH3) ESKYGPPCPPCPWPREPQ'VYTLPPSQEEMTKNQVSLTCLVKGFYPSDI 659 AVEWESNGQPENNYKTIPPVLDSDGSFFLYSRLIVDKSRWQEGNWS
CSVMHEALHNHYTQKSLSLSLGK
IgD Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKE 660 KEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFW
GSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSL
WNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEA
ASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTITWA
WSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH
IgD Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPAT1RNTGRGG EEKK K E 661 KEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFW
GSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSL
WNAGTSVTCTLHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEAA

SVLRVPAPPSPQPATYTCWSHEDSRTLLNASRSLEVSYVTDH
IgD Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEICKKE 662 KEKEEQEERETKTPECPSITTQPLGVYLLTPAVQDLWLRDKATFTCFVV
GSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSL
WNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEA
ASWLLCEVSGFSPPNILLMVVLEDQREVNTSGFAPARPPPQPGSTITWA
WSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH
IgD Hinge ESPKAQASSVPTAQPQAEGSL AKATTAPATTRNTGRGGEEKKK EK EKE 663 EQEEREIKTP
IgD Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKICE 664 KEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVV
GSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSL
WNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEA
ASWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTITWA
WSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH

IgD Hinge RWPIHS PK AQ AS SVPTAQPQAEGSLAKATTAPATTRNTGRGORFKKKE 665 KEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVV
GSDLK DA IILTWEVAGKVPIGG VEEGLLERHSNGSQSQHSRLTLPRSL
WNAGTSVTC'TLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEA
A SWLLCEVSGFSPPNILLMWLEDQRE VNTSGFAPARPPPQPGS'ITFWA
WSVLRVPAPPSPQPATYTCVVSFIEDSRTLINASRSLEVSYVIDH
IgD Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPAITRNTGRGGEEKKKE 666 KEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVV
GSDLKDAHLTWEVAGKVPTGG VEEGLLERHSNGSQSQHSRLTLPRSL
WNAGTSVICTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEA
A SWLLCEVSGFSPPNILLMWLEDQREVNTSGFAPARPPQPGSTTFWAW
SVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH
IgD Hinge RWPESPKAQAS SVPTAQPQAEGSLAKATTAPATTRNTGRGGEEKKKE 667 KEKEEQEERETKTPECPSHTQPLGVYLLTPAVQDLWLRDKATFTCFVV
GSDLKDAHLTWEVAGKVPTGGVEEGLLERHSNGSQSQHSRLTLPRSL
WNAGTSVTCTLNHPSLPPQRLMALREPAAQAPVKLSLNLLASSDPPEA
ASWLLCEVSGFSPPNIUMWLEDQREVNTSGFAPARPPPQPGSTITWA
WSVLRVPAPPSPQPATYTCVVSHEDSRTLLNASRSLEVSYVTDH
IgG1 (CH2CH3) AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVV 668 Hinge domain DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL
YSKLTVDKSRWOQGNVFSCSVMHEALFLNHYTQKSLSLSPGKKD
IgG I Hinge AEPKSPDKTHTCPPCPKDPK 669 IgG I Hinge EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVD 670 VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEVKCKVSNKALPAPTEKTISKAKGQPREPQVYTLPPSRDELT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLV
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKKD ¨
IgG I Hinge SVFLIPPI(PKDIL 671 igGi Hinge EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCVVVD 672 VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
IgG1 Hinge EPKSPDKTHTCPPCPAPPVAGP S VFLFPPKPKDTLM i A RTPEVTCVVVD 673 VSHEDPEVKFNWYN/DGVEVEINAKTKPREEQYNSTYRVVSVLIVLHQ
DWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSR DELT
KNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY
SKLTVDK SR WQQGNVFSCSVMHEALHNHYTQK SLSLSPGKKDPK
IgG I Hinge VECPPCPAPPVAGPSVFLFPPKPICDTLMISRTPEVICVVVDVSHEDPEV 674 KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGICE
YKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTC
LVKGFYPSDIAVF,Vv'ESNWPENNYICTIPPVLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
IgG1 Hinge DPAEPK SPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTPEVTCV 675 (CH2CH3 VVDVSHEDPEVICFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV
domain) LHQDWLNGICEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRD
ELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF
FLYSKLINDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKIK
IgG3 Hinge ELKTPLGDITEITCPRCP 676 IgG3 Hinge ELKTPLGDTHTCPRCPEPKSCDTF'PPCPRCPEPKSCDTPPPCPRCPEPKS 677 CDTPPPCPRCP
IgG4 (CH2 and ESKYGPPCPP(PAPEFI,GGPSVFLFPPKPKDTLIVISRTPEVTCVVVDVSQ 678 CH3) EDPEVQFNWYVDGVE'VHNAK TKPREEQFNSTYRVVSVLTVLHQDWL
NGKEYK CKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ
VSLICLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLT
VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKM

IgG4 (CH2 and ESKYGPPCPPCPAPEFEGGPSVFLEPPKPKWILMISRIPEVICVVN/DVSQ 679 CH3) EDPEVQFNWYVDG'VEVHNAKTKPREEQFQSTYRVVSVLTVLHQD'WL
NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ
VSLICLVKGFYPSDIAVEWESNGQPENNYKTITPVLDSDGSFELYSRLT
VDKSRWQEGNVESCSVMHEALHNHYTQKSLSLSLGKM
IgG4 Hinge SPNIvIVPHAHHAQ 680 IgG4 Hinge GQPREPQVYILPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE 681 NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH
YIQKSLSLSLGK
IgG4 Hinge ESKYGPPCPPCPGGGSSGGGSGCOPREPQVYTLPPSQEEMTKNQVSLT 682 CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSRLTVDK
SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK
I g64 Hinge ESKYGPPCPSCPAPEFEGGPSVFLEPPKPKDILMISRIPE'VTCVVVDVSQ 683 EDPEVQFNWYVDGVEVHQAKTKPREEQFNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ
VSLTCLVKGFVPSDIAVEVv'ESNGQPENNYKTTPPVLDSDGSFFLYSRLT
VDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK -IgG4 Hinge ESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQ 684 EIREVQFNWYVDGVEVHQAKTKPREEQFNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNK GLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ
VSLICLVKGFVPSDIAVEWESNGQPENNYKTITPVLDSDGSFELYSRLT
VDKSRWQEGNVESCSVMHEALHNHYTQKSLSLSLGK
IgG4 Hinge GAATCTAAGTACGGACCGCCCTGCCCCCMGCCCT 685 IgG4 Hinge ESKYGPPCPPCP 686 IgG4 Hinge YGPPCPPCP 687 IgG4 Hinge KYGPPCPPCP 688 IgG4 Hinge EVVKYGPPCPPCP 689 IgG4 Hinge ESKYGPPCPSCPAPEFLGGPS'VFLFPPKPICDTLMISRTPEVTCVVVDVSQ 690 EDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSEFLYSRLT
VDLSRWQEGNVESCSVMHEALHNHYTQKSLSLSLGK
IgG4 Hinge and ESKYGPPCPPCPGGGSSGGGSG 691 Linker Ig(31 Hinge EPKSPDKIHTCPPCPAPPVAGPSVFLEPPKPKDTLMIARTPEVICVVVD 692 VSFIEDPEVKENWYVDGVEVHNAKTKPREEQYNSTYRVVSVLIVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELT
KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFELYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
lgG1Hinge EPKSPDKTITTCPPCPAPPVAGPSVFLEPPKPKDTLMIARTPEVTCVVVD 693 VSHEDPEVKFNWYVDGVEVEINAKTKPREEQYNSTYRVVSVLTVLHQ
DWLNGKEYKCKVSNKALPAPIEKTISICAKGQPREPQVYTLPPSRDELT
KNQVSLTCLVKGFYPSDIAVENVESNGQPENNYKTIPPVLDSDGSFELYS
KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
CH2CH3 spacer EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV 694 domain DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH
QD'WLNGKEYKCK'VSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTIPPVLDSDGSFEL
YSKLINDKSRWQQGNVFSCSVMHEALIINHYTQKSLSLSPGICKDPK
[00235] In some embodiments, the CAR of the present invention may comprise one or more linkers between any of the domains of the CAR. The linker may be between 1-30 amino acids long. In this regard, the linker may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. In other embodiments, the linker may be flexible.
[00236] In some embodiments, the CH2CH3 may be preferentially excluded from the chimeric antigen structure to elicit a higher TNFa response as disclosed in W02016149578 (the contents of which are herein incorporated by reference). In some constructs a CH2CH3 structural domain is included. This domain extends the scFV away from the plasma membrane extracellular surface, and allows for the efficient detection of transduced T cells with anti-IgG Fc-specific antibody. CAR constructs which include CH2CH3 domain are disclosed in (the contents of which are incorporated herein by reference in its entirety).
[00237] In some embodiments, the components including the targeting moiety, transmembranc domain and intracellular signaling domains of the present invention may be constructed in a single fusion polypeptide. The fusion polypeptide may be the payload of an effector module of the invention. In some embodiments, more than one CAR fusion polypeptides may be included in an effector module, for example, two, three or more CARs may be included in the effector module under the control of a single SRE (e.g., a DD). Representative effector modules comprising the CAR payload are illustrated in Figures 2-6.
[00238] In some embodiments, the CAR sequences may be selected from Table 12.
Table 12: CAR sequences Description Source Target SEQ
ID
NO.
ALK CAR SEQ ID NO. 37 in W02015069922 ALK 695 ALK CAR SEQ ID NO. 39 in W02015069922 ALK 696 ALK CAR SEQ ID NO. 41 in W02015069922 ALK 697 ALK CAR SEQ ID NO. 43 in W02015069922 ALK 698 ALK CAR SEQ ID NO. 44 in W02015069922 ALK 699 ALK CAR SEQ ID NO. 45 in W02015069922 ALK 700 ALK CAR SEQ ID NO. 46 in W02015069922 ALK 701 ALK CAR SEQ D NO. 47 in W02015069922 ALK 702 ALK CAR SEQ ID NO. 48 in W02015069922 ALK 703 ALK CAR SEQ ID NO. 49 in W02015069922 ALK 704 .ALK CAR SEQ ID NO. 50 in W02015069922 ALK 705 ALK CAR SEQ ID NO. 51 in W02015069922 ALK 706 ALK CAR SEQ ID NO. 52 in W02015069922 ALK 707 ALK CAR SEQ ID NO. 53 in W02015069922 ALK 708 ALK CAR SEQ ID NO. 54 in W02015069922 ALK 709 .ALK CAR SEQ ID NO. 55 in W02015069922 ALK 710 ALK CAR SEQ ID NO. 56 in W02015069922 ALK 711 ALK CAR SEQ ID NO. 57 in W02015069922 ALK 712 ALK CAR SEQ ID NO. 58 in W02015069922 ALK 713 ALK CAR SEQ ID NO. 59 in W02015069922 ALK 714 ALK CAR SEQ ID NO. 60 in 'W02015069922 ALK 715 ALK CAR SEQ ID NO. 61 in W02015069922 ALK 716 .ALK CAR SEQ ID NO. 62 in W02015069922 ALK 717 ALK CAR SEQ ID NO. 63 in W02015069922 ALK 718 ALK CAR SEQ ID NO. 64 in W02015069922 ALK 719 ALK CAR SEQ ID NO. 65 in W02015069922 ALK 720 .
ALK CAR SEQ ID NO. 66 in W02015069922 ALK 721 ALICEAR SEQ ID NO. 67 in W02015069922 ALK 722 ALK CAR SEQ ID NO. 68 in W02015069922 ALK 723 ALK CAR SEQ ID NO. 69 in W02015069922 ALK 724 ALK CAR SEQ ID NO. 70 in W02015069922 ALK 725 ALK CAR SEQ ID NO. 71 in W02015069922 ALK 726 ALK CAR SEQ ID NO. 72 in W02015069922 ALK 727 ALK CAR SEQ ID NO. 73 in 'W02015069922 ALK 728 ALK CAR SEQ ID NO. 74 in W02015069922 ALK 729 .ALK CAR SEQ ID NO. 75 in W02015069922 ALK 730 ALK CAR SEQ ID NO. 76 in W02015069922 ALK 731 ALK CAR SEQ ID NO. 77 in W02015069922 ALK 732 ALK CAR SEQ ID NO. 78 in W02015069922 ALK 733 .
ALK CAR SEQ ID NO. 79 in W02015069922 ALK 734 .ALK CAR SEQ ID NO. 80 in W02015069922 ALK 735 ALK CAR SEQ ID NO. 81 in 'W02015069922 ALK 736 ALK CAR SEQ ID NO. 82 in W02015069922 ALK 737 ALK CAR SEQ ID NO. 83 in W02015069922 ALK 738 .
ALK CAR SEQ ID NO. 84 in W02015069922 ALK 739 ALK CAR SEQ ID NO. 85 in W02015069922 ALK 740 ALK CAR SEQ ID NO. 86 in 'W02015069922 ALK 741 ALK CAR SEQ ID NO. 87 in W02015069922 ALK 742 .ALK CAR SEQ ID NO. 88 in W02015069922 ALK 743 ALK CAR SEQ ID NO. 89 in W02015069922 ALK 744 ALK CAR SEQ ID NO. 90 in W02015069922 ALK 745 CD22 (m971) third generation CAR SEQ ID NO. 22 in W02014065961 CD22 746 .
CO22 (m971) third generation CAR SEQ ID NO. 23 in CD22 (m971) third generation CAR SEQ ID NO. 24 in W02014065961 CD22 748 CD22 (CARsHA22 28z) CAR SEQ ID NO. 15 in W02013059593 CD22 749 CD22 (HA22 28BBz) CAR SEQ ID NO. 16 in W02013059593 CD22 750 CD22 (HASH22 28z) CAR SEQ ID NO. 17 in W02013059593 CD22 751 CO22 (HASH22 28BBz) CAR SEQ ID NO. 18 in W02013059593 CD22 752 CD22 (BL22 28z) CAR SEQ ID NO. 19 in W02013059593 CD22 753 CD22 (BL22 28BBz) CAR SEQ ED NO. 20 in 'W02013059593 CD22 754 CO22 (HA22SH-CAR-second SEQ ID NO. 32 in W02013059593 CD22 755 generation, version 2) CAR

CD276 CAR (CD276.6 second SEQ ID NO. 39 in W02014160627 CD276 756 generation) CD276 CAR (CD276.I second SEQ ID NO. 42 in W02014160627 CD276 757 generation, version I) CD276 CAR (CD276.17 second SEQ ID NO. 45 in W02014160627 CD276 758 generation, version 1) CD276 CAR (CD276.6 CAR second SEQ ID NO. 122 in W02014160627 CD276 759 generation, version 1) CD276 CAR (CD276.6 CAR second SEQ ID NO. 123 in W02014160627 CD276 760 general ion, version 2) CD276 CAR (CD276.6 CAR third SEQ ID NO. 124 in W02014160627 CD276 761 generation) CD276 CAR (CD276.1 CAR second SEQ ID NO. 125 in W02014160627 CD276 762 ...generation, version I) (:1)276 CAR (CD276. 1 CAR second SEQ ID NO. 126 in W02014160627 CD276 763 generation. version 2) CD276 CAR (CD276.I CAR third SEQ ID NO. 127 in W02014160627 CD276 764 generation) CD276 CAR (CD276.17 CAR SEQ ID NO. 128 in W02014160627 CD276 765 second generation, version 1) CD276 CAR (CD276.17 CAR SEQ ID NO. 129 in W02014160627 .. CD276 766 second generation, version 2) CD276 CAR (CD276.17 CAR third SEQ ID NO. 130 in W02014160627 CD276 767 generation) CD 276 CAR SEQ ID NO. 20 in W02017044699 CD276 768 CD276.MG.BB.Z CAR SEQ ID NO. 12 in W02017044699 CD276 769 [00239] In one embodiment of the present invention, the payload of the invention is a CD33 specific CAR. The CD33 heavy and light chain may be combined with any of the signal peptides, transmembrane domains, costimulatory domains, intracellular domains and destabilizing domains described herein.
[00240] In one embodiment of the present invention, the payload of the invention is a GD2 specific CAR. The GD2 heavy and light chain may be combined with any of the signal peptides, transmembrane domains, costimulatory domains, intracellular domains and destabilizing domains described herein.
[00241] In one embodiment of the present invention, the payload of the invention is a Her2 specific CAR. The Her2 heavy and light chain may be combined with any of the signal peptides, transmembrane domains, costimulatory domains, intracellular domains and destabilizing domains described herein. Exemplary BCMA CAR sequences and its components are described in Table I 3A. The amino acid sequences in Table 13A may comprise a stop codon which is denoted in the table with a "s" at the end of the amino acid sequence Table 13A: 1)1)-Her2 construct sequences Description Amino Acid Sequence Amino Nucleic Acid SEQ Acid SEQ
ID NO ID NO/
Sequence GMCSF Leader LLLVTSLLLCELPHPAIILIP 778 922 Linker ASFE 920 921 Linker GS GGTTCC, GGATCC
Linker TS ACTAGT
Linker HM ATGCAC
4-1}3B KRGRKICLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGG 773 919 Intracellular CEL
Domain 4D5 scFV DIQM'TQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQ 923 924 KPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQ
PEDFATYYCQQHYTTPPTFGQGTKVEIKGSTSGSGKPGS
GEGSGEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYI
HWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISA
DTSKNTAYLQKNSLRAEDTAVYYCSRWGGDGFYAMD
VWGQGTINTSISS
CD8a Hinge 1T1'PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL 925 926 DFACD
Transinetribrane IYIWAPLAGICGVLLLSLVITLYC 927 928 Domain CD3 Zeta RV1CFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDK 772 918 signaling Domain RRGRDPEMGGKPRRICNPQEGLYNELQICD1(114AEAYSEI
GMKGERRRGKGHDGLYQGL STATICDTYDALHWALPP
hDHFR (Amino VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRM'r 861 903-907 acid 2-187 of WT; TTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSR
Y1221) ELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVWIV
GGSSVIKEAMNIMGHLKLFVTRIMQDFESDTFFPEIDLEK
YKLLPEYPGVLSDVQEEKGIKYKFEVYEKND
OT-Her2-004 MLLLVTSLLLCELPHPAFLL IPDIQM'TQSPSSLSASVGDR 906 907 (Met - GMCSF VTITCRASQDVNTAVAWYQQKPGICAPKLUYSASFLYS
Leader - 4D5 GVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPP
scFv - Linker TFGQGTKVEIKGSTSGSGKPGSGEGSGEVQLVESGGGLV
(ASFE) - CD8a QPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVAR
hinge - Linker IYPINGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAE
(GS) - DTAVYYCSRWGGDGFYAMDVWGQGTLVTVSSASFETT
Transmembrane TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF
Domain - Linker ACDGSIYIWAPLAGTCGVLLLSLVITLYCTSKRGRICKLL
(FS) - 4-1BB YIFKQPFIVIRPVQTTQEEDGCSCRFPEEEEGGCELHMRVK
intracellular FSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG
signaling domain RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK
- Linker (117V1) - GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGS
CD3 zeta - Linker VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT
(GS) - hDHFR TTSSVEGKQNLVIMGICKTWFSIPEKNRPLKGRINLVLSR
(Amino acid 2- ELKEPPQGAHFLSRSLDDALICLTEQPELANKVDMVWIV
187 of WT; GGSSVIICEAKNIIPGHLKLFVTRIMQDFESDTFFPEIDLEK
Y1221) - stop) YKLLPEYPGVLSDVQEEKG1KYKFEVYBKND*
[00242] In one embodiment, the CAR of the present invention is a BCMA (B-cell maturation antigen) CAR, also referred to as the CD269. The BCMA heavy and light chains may be combined with any of the signal peptides, transmembrane domains, costimulatory domains, intracellular domains and destabilizing domains described herein. Exemplary BCMA CAR
sequences and its components are described in Table 13B. The amino acid sequences in Table 13B may comprise a stop codon which is denoted in the table with a "*" at the end of the amino acid sequence.
Table 1.3B: BCMA CAR
Description Amino Acid Sequence Amino Nucleic Acid SEQ Acid SEQ
ID NO. ID NO.
BCMA scFy (C11D5.3) DIVLTQSPASLAMSLGKRATISCRASESVSVIGAHL 770 916 IHWYQQKPGQPPKLLIYLASNLETGVPARFSGSGS
GTDFTLTIDPVEEDDVAIYSCLQSRIFPRTFGGGTK

MGWINTETREPAYAYDFRGRFAFSLETSASTAYL
QINNLKYEDTATYFCALDYSYAMDYWGQGTSVT
VSS
CD80. hinge¨TM ITTPAPRPPTF'APTIASQPLSLRPEACRPAAGGAVH 771 917 CD3 zeta signaling RVICIESR SA DAPAYKQGQNQLYNELNLGR REEYD 772 918 domain VLDKRRGRDPEMGGKPRR KNPQEGLYNEI-QKDK
MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD
TYDALHMQALPPR
4-EBB intracellular KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEE 773 919 signaliqg domain EEGGCEL
CD8ix leader MALPVTALLLPLALLLH AA RP 131 132-136, ecDHFR (Amino acid 2- ISLIAALAVDYVIGMENAMPWNLPADLAWFKRN 9 61,869-159 of WI) (R12Y, TLNKPVIMGRHTWESIGRPLPGRKNIILSSQPG1DD 874 YlOOD RVTWVKSNIDEAIAACGDVPEIMVIGGGRVIEQFLP
KAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSE
FFIDADAQNSHSYCFEILERR
FKBP (E31G, F36V. GVQVETISPGDGRTFPKRGQICVVHYTGMLGDG 12 88, 883-R71G, K105E) KKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQM 889 SVGQGAKLTISPDYAYGATGHPGIIPPHATLVFDV
ELLELE
OT-BCMA-001 (CD8a MALPVTALLLPLALLLHAARPDIVLTQSPASLAMS 775 833 leader- BCMA scFv - LGKRATISCRASESVSVIGAHLIHWYQQKPGQPPK
CD8a hinge¨Tm ¨4- LLIYLASNLETGVPARFSGSGSGTDFTLITDPVEED
1BB intracellular domain DVAIYSCLQSRIFPRTFGGGTKLEIKGSTSGSGKPG
¨ CD3 zeta - stop) SGEGSTKGQIQLVQSGPELKKPGETVKISCKASGY
11-c1DYSINWVKRAPGKGLKWMGWINTETREPAY

CALDYSYAMDYWGQGTSVTVSSITTPAPRPPTPA
PTIASQPLSLRPEACRPAAGGAVHIRGLDFACDIYI
WAPLAGTCGVLLL SLVITLYCKRGRKKLLYIFKQP
FMRPVQTTQEEDGCSCRFPEEEEGGCELRVICFSRS
ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG
RDPEMGGICPRRKNPQEGLYNELQICDICMAEAYSE
IGMKGERRRGKGHDGLYQGLSTATKDTYDALILM
QALPPR*
OT-BCMA-002 (CD8a MALPVTALLLPLALLLHAARPDIVLTQSPASLAMS 776 834 leader - BCMA scFv - LGKRATISCRASESVSVIGAHLIHWYQQKPGQPPK
CD8a hinge¨Tm ¨4- LLIYLASNLETGVPARFSGSGSGTDFTLT1DPVEED
1BB intracellular domain DVAIYSCLQSRIFPRTFGGGTICLEIKGSTSGSGKPG
- CD3 zeta - Linker (SG) SGEGSTKGQIQLVQSGPELIC1CPGETVKISCKASGY
- FKBP (E31G, F36V, -11-1DYSLNWVKRAPGKGLKWMGWINTETREPAY
R71G, K105E) - stop) AYDFRGRFAFSLETSASTAYLQINNLKYEDTATYF
CALDYSYAMDYWGQGTSVTVSSTTTPAPRPPTPA
PTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI

WAPLAGICGVLLLSLVITLYCKRGRICKLLYIFKQP
FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS
ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG
RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE
IGMKGERRRGKGHDGLYQGLSTATKDTYDALHM
QALPPRSGGVQVETISPGDGRTFPKRGQICVVHY
TGMLGDGKKVDSSRDRNKPFKFMLGKQEVIRGW
EEGVAQMSVGQGAKLTISPDYAYGATGHPGIIPPH
ATLVFDVELLELE*
OT-BCMA-003 (CD8a MALPVTALLLPLALLLHAARPDIVLTQSPASLAMS 777 835 leader - BCMA scEv - LGKRATISCRASESVSVIGAHLIHWYQQKPGQPPK
CD8a hinge¨Tm ¨4- LLIYLASNLETGVPARFSGSGSGTDFTLTIDPVEED
1BB intracellular domain DVAIYSCLQSRIFPRTFGGGTKLEIKGSTSGSGKPG
- CD3 zeta - Linker (SG) SGEGSTKGQIQLVQSGPELKKPGETVKISCKASGY
- ecDHFR (Amino acid 2- TFTDYSINWVKRAPGKGLKWMGWINTETREPAY
159 of WT) (R 12Y, AYDFRGRFAFSLETSASTAYLQINNLKYEDTATYF
)(100D - stop) CALDYSYAMDYWGQGTSVTVSSTTTPAPRPPTPA
PTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI
WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQP
FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS
ADAPAYKQGQNQLYNELNLGRREEYDVLDICRRG
RDPEMGGKPRRICNPQEGLYNELQICDKMAEAYSE
IGMKGERRRGKGHDGLYQGLSTATICDTYDALIEVI
QALPPRSGISLIAALAVDYVIGMENAMPWNLPAD
LAWFICRNTLNICPVIMGRHTWESIGRPLPGRICNDL
SSQPGTDDRVIWVICSVDEAIAACGDVPEIMVIGG
GRVIEQFLPKAQICLYLTHIDAEVEGDTHFPDYEPD
DWESVFSEFHDADAQNSHSYCFEILERR*
Tandem CAR (TanCAR) [00243] In some embodiments, the CAR of the present invention may be a tandem chimeric antigen receptor (TanCAR) which is able to target two, three, four, or more tumor specific antigens. In some aspects, The CAR is a bispecific TanCAR including two targeting domains which recognize two different TSAs on tumor cells. The bispecific CAR may be further defined as comprising an extracellular region comprising a targeting domain (e.g., an antigen recognition domain) specific for a first tumor antigen and a targeting domain (e.g., an antigen recognition domain) specific for a second tumor antigen. In other aspects, the CAR is a multispecific TanCAR that includes three or more targeting domains configured in a tandem arrangement. The space between the targeting domains in the TanCAR may be between about 5 and about 30 amino acids in length, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 amino acids.
Split CAR
[00244] In some embodiments, the components including the targeting moiety, transmembrane domain and intracellular signaling domains of the present invention may be split into two or more parts such that it is dependent on multiple inputs that promote assembly of the intact functional receptor. In one embodiment, the split synthetic CAR system can be constructed in which the assembly of an activated CAR receptor is dependent on the binding of a ligand to the SRE (e.g. a small molecule) and a specific antigen to the targeting moiety. As a non-limiting example, the split CAR consists of two parts that assemble in a small molecule-dependent manner; one part of the receptor features an extracellular antigen binding domain (e.g. scFv) and the other part has the intracellular signaling domains, such as the CDX
intracellular domain.
1002451 In other aspects, the split parts of the CAR system can be further modified to increase signal. In one example, the second part of cytoplasmic fragment may be anchored to the plasma membrane by incorporating a transmembrane domain (e.g., CD8a transmembrane domain) to the construct. An additional extracellular domain may also be added to the second part of the CAR
system, for instance an extracellular domain that mediates homo-dimerization.
These modifications may increase receptor output activity, i.e., T cell activation.
1002461 In some aspects, the two parts of the split CAR system contain heterodimerization domains that conditionally interact upon binding of a heterodimerizing small molecule. As such, the receptor components are assembled in the presence of the small molecule, to form an intact system which can then be activated by antigen engagement. Any known heterodimerizing components can be incorporated into a split CAR system. Other small molecule dependent heterodimerization domains may also be used, including, but not limited to, gibberellin-induced dimerization system (GID1-GAI), trimethoprim-SLF induced ecDHFR and FKBP
dimerization (Czlapinski et al., J Am Chem Soc., 2008, 130(40): 13186-13187) and ABA
(abscisic acid) induced dimerization of PP2C and PYL domains (Cutler et al., Annu Rev Plant Biol. 2010, 61:
651-679). The dual regulation using inducible assembly (e.g., ligand dependent dimerization) and degradation (e.g., destabilizing domain induced CAR degradation) of the split CAR system may provide more flexibility to control the activity of the CAR modified T
cells.
Switchable CAR
1002471 In some embodiments, the CAR of the invention may be a switchable CAR.
Juillerat et al (Juilerat et al., Sci. Rep., 2016, 6: 18950; the contents of which are incorporated herein by reference in their entirety) recently reported controllable CARS that can be transiently switched on in response to a stimulus (e.g. a small molecule). In this CAR design, a system is directly integrated in the hinge domain that separate the scFv domain from the cell membrane domain in the CAR. Such system is possible to split or combine different key functions of a CAR such as activation and costimulation within different chains of a receptor complex, mimicking the complexity of the TCR native architecture. This integrated system can switch the scFv and antigen interaction between on/off states controlled by the absence/presence of the stimulus.
Reversible CAR

1002481 In other embodiments, the CAR of the invention may be a reversible CAR
system. In this CAR architecture, a LID domain (ligand-induced degradation) is incorporated into the CAR
system. The CAR can be temporarily down-regulated by adding a ligand of the LID domain.
The combination of LID and DD mediated regulation provides tunable control of continuingly activated CAR T cells, thereby reducing CAR mediated tissue toxicity.
Activation-conditional C4 R
1002491 In some embodiments, payloads of the invention may be an activation-conditional chimeric antigen receptor, which is only expressed in an activated immune cell. The expression of the CAR may be coupled to activation conditional control region which refers to one or more nucleic acid sequences that induce the transcription and/or expression of a sequence e.g. a CAR
under its control. Such activation conditional control regions may be promoters of genes that are upregulated during the activation of the immune effector cell e.g. 1L2 promoter or NFAT binding sites. In some embodiments, activation of the immune cell may be achieved by a constitutively expressed CAR (International Publication NO. W02016126608; the contents of which are incorporated herein by reference in their entirety).
4. Additional effector module features 1002501 The effector module of the present invention may further comprise a signal sequence which regulates the distribution of the payload of interest, a cleavage and/or processing feature which facilitate cleavage of the payload from the effector module construct, a targeting and/or penetrating signal which can regulate the cellular localization of the effector module, a tag, and/or one or more linker sequences which link different components of the effector module.
Signal sequences 1002511 In addition to the SRE (e.g., DD) and payload region, effector modules of the invention may further comprise one or more signal sequences. Signal sequences (sometimes referred to as signal peptides, targeting signals, target peptides, localization sequences, transit peptides, leader sequences or leader peptides) direct proteins (e.g., the effector module of the present invention) to their designated cellular and/or extracellular locations. Protein signal sequences play a central role in the targeting and translocation of nearly all secreted proteins and many integral membrane proteins.
1002521 A signal sequence is a short (5-30 amino acids long) peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards a particular location.
Signal sequences can be recognized by signal recognition particles (SRPs) and cleaved using type I and type 11 signal peptide peptidases. Signal sequences derived from hiunan proteins can be incorporated as a regulatory module of the effector module to direct the effector module to a particular cellular and/or extracellular location. These signal sequences are experimentally verified and can be cleaved (Zhang et al., Protein Sci. 2004, 13:2819-2824).
1002531 In some embodiments, a signal sequence may be, although not necessarily, located at the N-terminus or C-terminus of the effector module, and may be, although not necessarily, cleaved off the desired effector module to yield a "mature" payload, i.e., an immunotherapeutic agent as discussed herein.
1002541 In some examples, a signal sequence may be a secreted signal sequence derived from a naturally secreted protein, and its variant thereof. In some instances, the secreted signal sequences may be cytokine signal sequences such as, but not limited to, IL2 signal sequence ( amino acid of SEQ ID NO. 49, encoded by the nucleic acid sequence of SEQ ID
NO. 55-56 and/or 117-118), p40 signal sequence (amino acid sequence of SEQ ID NO. 119, encoded by the nucleic acid sequence of SEQ ID NO. 120-128), or a GMCSF leader sequence (SEQ
ID NO. 778 (encoded by SEQ ID NO. 922), 779, 780).
1002551 In some instances, signal sequences directing the payload of interest to the surface membrane of the target cell may be used. Expression of the payload on the surface of the target cell may be useful to limit the diffusion of the payload to non-target in vivo environments, thereby potentially improving the safety profile of the payloads.
Additionally, the membrane presentation of the payload may allow for physiologically and qualitative signaling as well as stabilization and recycling of the payload for a longer half-life. Membrane sequences may be the endogenous signal sequence of the N terminal component of the payload of interest. Optionally, it may be desirable to exchange this sequence for a different signal sequence.
Signal sequences may be selected based on their compatibility with the secretory pathway of the cell type of interest so that the payload is presented on the surface of the T cell. In some embodiments, the signal sequence may be IgE signal sequence (amino acid SEQ ID NO. 129 and nucleotide sequence of SEQ ID NO. 130), CD8a signal sequence (also referred to as CD8a leader) (amino acid SEQ ID NO. 131 and nucleotide sequence of SEQ ID NO. 132-136, and/or 915) or an IL15Ra signal sequence (amino acid SEQ ID NO. 781, encoded by SEQ ID NO. 782).

1002561 Other examples of signal sequences include, a variant may be a modified signal sequence discussed in U.S. Pat. NOs. 8, 148, 494; 8,258,102; 9,133,265;

9,279,007; and U.S.
patent application publication NOS. 20070141666; and International patent application publication NOS. W01993018181: the contents of each of which are incorporated herein by reference in their entirety.
1002571 In other examples, a signal sequence may be a heterogeneous signal sequence from other organisms such as virus, yeast and bacteria, which can direct an effector module to a particular cellular site, such as a nucleus (e.g., EP 1209450). Other examples may include Aspartic Protease (NSP24) signal sequences from Trichoderma that can increase secretion of fused protein such as enzymes (e.g., U. S. Pat. NOS. 8,093,016 to Cervin and Kim), bacterial lipoprotein signal sequences (e.g., PCT application publication NOS.
W0199109952 to Lau and RiouN), E.coli enterotoxin II signal peptides (e.g., U.S. Pat. NOS. 6,605,697 to Kwon et al.), E. coil secretion signal sequence (e.g., U.S. patent publication NOS.
US2016090404 to Malley et al.), a lipase signal sequence from a methylotrophic yeast (e.g., U.S. Pat.
NOS. 8,975,041), and signal peptides for DNases derived from Coryneform bacteria (e.g., U.S. Pat.
NOS. 4,965,197);
the contents of each of which are incorporated herein by reference in their entirety.
[00258] Signal sequences may also include nuclear localization signals (NLSs), nuclear export signals (NESs), polarized cell tubulo-vesicular structure localization signals (See, e.g., U.S. Pat.
NOS. 8, 993,742; Cour et al., Nucleic Acids Res. 2003, 31(1): 393-396; the contents of each of which are incorporated herein by reference in their entirety, ),extracellular localization signals, signals to subcellular locations (e.g. lysosome, endoplasmic reticulum, golgi, mitochondria, plasma membrane and peroxisomes, etc.) (See, e.g., U.S. Pat. NOS. 7,396,811;
and Negi et al., Database, 2015, 1-7; the contents of each of which are incorporated herein by reference in their entirety).
[00259] In some embodiments, signal sequences of the present invention, include without limitation, any of those taught in Table 7 of copending commonly owned U.S.
Provisional Patent Application No. 62/320,864, filed on 4/11/2016, or in US Provisional Application No.
62/466,596 filed March 3, 2017 and the International Publication W02017/180587, the contents of which are incorporated herein by reference in their entirety.
Cleavage sites [00260] In some embodiments, the effector module comprises a cleavage and/or processing feature. The effector module of the present invention may include at least one protein cleavage signal/site. The protein cleavage signal/site may be located at the N-terminus, the C-terminus, at any space between the N- and the C- termini such as, but not limited to, half-way between the N-and C-termini, between the N-terminus and the half-way point, between the half-way point and the C-terminus, and combinations thereof.
[00261] The effector module may include one or more cleavage signal(s)/site(s) of any proteinases. The proteinases may be a serine proteinase, a cysteine proteinase, an endopcptidasc, a dipeptidase, a metalloproteinase, a glutamic proteinase, a threonine proteinase and an aspartic proteinase. In some aspects, the cleavage site may be a signal sequence of furin, actinidain, calpain-1, carboxypeptidase A, carboxypeptidase P. carboxypeptidase Y, caspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, cathepsin B, cathepsin C, cathepsin G. cathepsin H, cathepsin K, cathepsin L, cathepsin S, cathepsin V, clostripain, chymase, chymotrypsin, elastase, endoproteinase, enterokinase, factor Xa, formic acid, granzyme B, Matrix metallopeptidase-2, Matrix metallopeptidase-3, pepsin, proteinase K, SUMO protease, subtilisin, TEV protease. thermolysin, thrombin, tiypsin and TAGZyme.
1002621 In one embodiment, the cleavage site is a furin cleavage site comprising the amino acid sequence SARNRQKRS (SEQ ID NO. 137), encoded by nucleotide sequence of SEQ ID
NO.
138; or a revised furin cleavage site comprising the amino acid sequence ARNRQKRS (SEQ ID
NO. 139), encoded by nucleotide sequence of SEQ ID NO. 140: modified furin site comprising the amino acid sequence ESRRVRRNKRSK (SEQ ID NO. 141), encoded by nucleotide sequence of SEQ ID NO. 142-144; or a SGESRRVRRNKRSK (SEQ ID NO. 785), encoded by the nucleotide sequence of SEQ ID NO. 784. In some instances, the cleavage site is a P2A
cleavage site (ATNFSLLKQAGDVEENPGP (SEQ ID NO. 783), encoded by SEQ ID NO.
786, or GATNFSLLKQAGDVEENPGP (SEQ ID NO.. 864), encoded by SEQ ID NO. 865), wherein NPGP (SEQ ID NO. 866) is the P2A site.
1002631 In some embodiments, cleavage sites of the present invention, include without limitation, any of those taught in Table 7 of copending commonly owned U.S.
Provisional Patent Application No. 62/320,864, filed on 4/11/2016, or in US Provisional Application No.
62/466,596 filed March 3, 2017 and the International Publication W02017/180587, the contents of which are incorporated herein by reference in their entirety.
Protein tags 1002641 In some embodiments, the effector module of the invention may comprise a protein tag.
The protein tag may be used for detecting and monitoring the process of the effector module.
The effector module may include one or more tags such as an epitope tag (e.g., a FLAG or hemagglutinin (HA) tag). A large number of protein tags may be used for the present effector modules. They include, but are not limited to, self-labeling polypeptide tags (e.g., haloalkane dehalogenase (halotag2 or ha1otag7), ACP tag, clip tag, MCP tag, snap tag), epitope tags (e.g., FLAG, HA, His, and Myc), fluorescent tags (e.g., green fluorescent protein (GFP), red fluorescent protein (RFP), yellow fluorescent protein (YFP), and its variants), bioluminescent tags (e.g. luciferase and its variants), affinity tags (e.g., maltose-binding protein (MBP) tag, glutathione-S-transferase (GS'T) tag), immunogenic affinity tags (e.g., protein A/G, IRS, AU!, AU5, glu-glu, KT3, S-tag, HSV, VSV-G, Xpress and V5), and other tags (e.g., biotin (small molecule), StepTag (Strep11), SBP, biotin carboxyl carrier protein (BCCP), eXact, CBP, CYD, HPC, CBD intein-chitin binding domain, Trx, NorpA, and NusA.
1002651 In other embodiments, a tag may also be selected from those disclosed in U.S. Pat.
NOs. 8,999,897; 8,357,511; 7,094, 568; 5,011,912; 4,851,341; and 4,703,004;
U.S patent application publication NOs. U52013115635 and U52013012687; and International application publication NOS. W02013091661; the contents of each of which are incorporated herein by reference in their entirety.
[00266] In some aspects, a multiplicity of protein tags, either the same or different tags, may be used: each of the tags may be located at the same N- or C-terminus, whereas in other cases these tags may be located at each terminus.
[00267] In one embodiment, the protein tag is an HA tag. A non-limiting example of an HA tag is YPYDVPDYA (SEQ ID NO. 852, encoded by SEQ ID NO. 853, 867, and/or 868).
1002681 In some embodiments, protein tags of the present invention, include without limitation, any of those taught in Table 8 of copending commonly owned U.S. Provisional Patent Application No. 62/320,864, filed on 4/11/2016, or in US Provisional Application No.
62/466,596 filed March 3, 2017 and the International Publication W02017/180587, the contents of which are incorporated herein by reference in their entirety.
Targeting peptides [00269] In some embodiments, the effector module of the invention may further comprise a targeting and/or penetrating peptide. Small targeting and/or penetrating peptides that selectively recognize cell surface markers (e.g. receptors, trans-membrane proteins, and extra-cellular matrix molecules) can be employed to target the effector module to the desired organs, tissues or cells.
Short peptides (5-50 amino acid residues) synthesized in vitro and naturally occurring peptides, or analogs, variants, derivatives thereof, may be incorporated into the effector module for homing the effector module to the desired organs, tissues and cells, and/or subcellular locations inside the cells.
[00270] In some embodiments, a targeting sequence and/or penetrating peptide may be included in the effector module to drive the effector module to a target organ, or a tissue, or a cell (e.g., a cancer cell). In other embodiments, a targeting and/or penetrating peptide may direct the effector module to a specific subcellular location inside a cell.
[00271] A targeting peptide has any number of amino acids from about 6 to about 30 inclusive.
The peptide may have 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. Generally, a targeting peptide may have 25 or fewer amino acids, for example, 20 or fewer, for example 15 or fewer.

1002721 Exemplary targeting peptides may include, but are not limited to, those disclosed in the art, e.g., U.S. Pat. NOs. 9,206,231; 9,110,059; 8,706,219; and 8,772,449; and U.S. application publication NOs. 2016089447; 2016060296; 2016060314; 2016060312; 2016060311;
2016009772; 2016002613; 2015314011 and 2015166621; and International application publication NOs. W02015179691 and W02015183044; the contents of each of which are incorporated herein by reference in their entirety.
1002731 In some embodiments, targeting peptides of the present invention, include without limitation, any of those taught in Table 9 of copending commonly owned U.S.
Provisional Patent Application No. 62/320,864, filed on 4/11/2016, the contents of which are incorporated herein by reference in their entirety.
Linkers 1002741 in some embodiments, the effector module of the invention may further comprise a linker sequence. The linker region serves primarily as a spacer between two or more polypeptides within the effector module. The "linker" or "spacer", as used herein, refers to a molecule or group of molecules that connects two molecules, or two parts of a molecule such as two domains of a recombinant protein.
1002751 In some embodiments, "Linker" (L) or "linker domain" or "linker region" or "linker module" or "peptide linker" as used herein refers to an oligo- or polypeptide region of from about 1 to 100 amino acids in length, which links together any of the domains/regions of the effector module (also called peptide linker). The peptide linker may be 1-40 amino acids in length, or 2-30 amino acids in length, or 20-80 amino acids in length, or 50-100 amino acids in length. Linker length may also be optimized depending on the type of payload utilized and based on the crystal structure of the payload. In some instances, a shorter linker length may be preferably selected. In some aspects, the peptide linker is made up of amino acids linked together by peptide bonds, preferably from 1 to 20 amino acids linked by peptide bonds, wherein the amino acids are selected from the 20 naturally occurring amino acids:
Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I), Serine (S), Cysteine (C), Threonine (T), Methionine (M), Proline (P), Phenylalanine (F), Tyrosine (Y), Try, ptophan (W), Histidine (H), Lysine (K), Arginine (R), Aspartate (D), Glutamic acid (E), Asparagine (N), and Glutamine (Q). One or more of these amino acids may be glycosylated, as is understood by those in the art. In some aspects, amino acids of a peptide linker may be selected from Alanine (A), Glycine (G), Proline (P), Asparagine (R), Serine (S), Glutamine (Q) and Lysine (K).
1002761 in one example, an artificially designed peptide linker may preferably be composed of a polymer of flexible residues like Glycine (G) and Serine (S) so that the adjacent protein domains are free to move relative to one another. Longer linkers may be used when it is desirable to ensure that two adjacent domains do not interfere with one another. The choice of a particular linker sequence may concern if it affects biological activity, stability, folding, targeting and/or pharmacokinetic features of the fusion construct. Examples of peptide linkers include, but are not limited to: MH, SG, GGSG (SEQ ID NO. 145; encoded by the nucleotide sequence SEQ ID
NO. 146), GGSGG (SEQ ID NO. 147: encoded by any of the nucleotide sequences SEQ ID NO.
148-152), GGSGGG (SEQ ID NO. 153; encoded by any of the nucleotide sequences SEQ ID
NO. 154-155) SGGGS (SEQ ID NO. 68; encoded by the nucleotide sequence SEQ ID
NO. 85, 69, 86), GGSGGGSGG (SEQ ID NO. 156; encoded by the nucleotide sequence SEQ ID
NO.
157), GGGGG (SEQ ID NO. 158), GGGGS (SEQ ID NO. 159) or (GGGGS)n (n=1 (SEQ ID
NO. 159), 2 (SEQ ID NO. 160), 3 (SEQ ID NO. 161, encoded by 174, 175, 171, 219, 774, 837), 4 (SEQ ID NO. 162), 5 (SEQ ID NO. 163 ), or 6 (SEQ ID NO. 164)), SSSSG (SEQ ID
NO. 165) or (SSSSG)n (n=1 (SEQ ID NO. 165), 2 (SEQ ID NO. 166), 3 (SEQ ID NO. 167), 4 (SEQ ID
NO. 168), 5 (SEQ ID NO. 169), or 6 (SEQ ID NO. 170)), SGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO. 171; encoded by the nucleotide sequence SEQ ID NO. 172, 838-843), EFSTEF (SEQ ID NO. 50; encoded by any of the nucleotide sequences SEQ ID NO. 57, 173), GKSSGSGSESKS (SEQ ID NO. 176), GGSTSGSGKSSEGKG (SEQ ID NO. 177), GSTSGSGKSSSEGSGSTKG (SEQ ID NO. 178), GSTSGSGKPGSGEGSTKG (SEQ ID NO. 179), VDYPYDVPDYALD (SEQ ID NO. 67;
encoded by nucleotide sequence SEQ ID NO. 84), EGKSSGSGSESKEF (SEQ ID NO.
180), 503-(504)3-503-SLQ- YPYDVPDYA (SEQ ID NO. 787), encoded by the nucleotide sequence of SEQ ID NO. 788; DYKDDDDK (SEQ ID NO.789), encoded by the nucleotide sequence of SEQ ID NO. 790; SG3-(SG4)5-503-S (SEQ ID NO. 791), encoded by SEQ ID NO. 792;
SGGGSGGGGSGGGGSGGGGSYPYDVPDYASGGGS (SEQ ID NO. 793), encoded by SEQ
ID NO. 794; GSGATNFSLLKQAGDVEENPGP (SEQ ID NO. 795), encoded by SEQ ID
NO.796; SGGGSGGGGSGGGGSGGGGS (SEQ ID NO. 844), encoded by the nucleotide sequence of SEQ ID NO. 845: QLIGMLQGLMRDL (SEQ ID NO. 908), encoded by SEQ ID
NO. 909; ASFE (SEQ ID NO. 920), encoded by SEQ ID NO. 921; GS (encoded by GGTTCC), SG (encoded by AGCGGC), EF (encoded by GAGTTC), TS (encoded by ACTAGT), HM
(encoded by CACATG), MH (encoded by ATGCAC) or GSG (encoded by GGATCCGGA or GGATCCGGT).
[00277] In other examples, a peptide linker may be made up of a majority of amino acids that are sterically unhindered, such as Glycine (G) and Alanine (A). Exemplary linkers are polyglycines (such as (G)4 (SEQ ID NO. 929), (0)5 (SEQ ID NO. 930), (0)8 (SEQ
ID NO.

931)), poly(GA), and polyalanines. The linkers described herein are exemplary, and linkers that are much longer and which include other residues are contemplated by the present invention.
[00278] A linker sequence may be a natural linker derived from a multi-domain protein. A
natural linker is a short peptide sequence that separates two different domains or motifs within a protein.
[00279] In some aspects, linkers may be flexible or rigid. In other aspects, linkers may be cleavable or non- cleavable. As used herein, the terms "cleavable linker domain or region" or "cleavable peptide linker" are used interchangeably. In some embodiments, the linker sequence may be cleaved enzymatically and/or chemically. Examples of enzymes (e.g., proteinase/peptidase) useful for cleaving the peptide linker include, but are not limited, to Arg-C
proteinase, Asp-N endopeptidase, chymotiypsin, clostripain, enterokinase, Factor Xa, glutamyl endopeptidase, Granzyme B, Achromobacter proteinase I, pepsin, proline endopeptidase, proteinase K. Staphylococcal peptidase I, thermolysin, thrombin, tiypsin, and members of the Caspase family of proteolytic enzymes (e.g. Caspases 1-10). Chemical sensitive cleavage sites may also be included in a linker sequence. Examples of chemical cleavage reagents include, but are not limited to, cyanogen bromide, which cleaves methionine residues; N-chloro succinimide, iodobenzoic acid or BNPS-skatole (2-(2-nitrophenylsulfeny1)-3-methylindole), which cleaves tryptophan residues; dilute acids, which cleave at aspartyl-prolyl bonds; and e aspartic acid-proline acid cleavable recognition sites (i.e., a cleavable peptide linker comprising one or more D-P dipeptide moieties). The fusion module may include multiple regions encoding peptides of interest separated by one or more cleavable peptide linkers.
[00280] In other embodiments, a cleavable linker may be a "self-cleaving"
linker peptide, such as 2A linkers (for example T2A), 2A-like linkers or functional equivalents thereof and combinations thereof. In some embodiments, the linkers include the picomaviral 2A-like linker, CHYSEL sequences of porcine teschovirus (P2A), Thosea asigna virus (T2A) or combinations, variants and functional equivalents thereof. Other linkers will be apparent to those skilled in the art and may be used in connection with alternate embodiments of the invention.
1002811 In some embodiments, the biocircuits of the present invention may include 2A peptides.
The 2A peptide is a sequence of about 20 amino acid residues from a virus that is recognized by a protease (2A peptidases) endogenous to the cell. The 2A peptide was identified among picomaviruses, atypical example of which is the Foot-and Mouth disease virus (Robertson BH, et. al., J Virol 1985, 54:651-660). 2A-like sequences have also been found in Picomaviridae like equine rhinitis A virus, as well as unrelated viruses such as porcine teschovirus-1 and the insect Thosea asigna virus (TaV). In such viruses, multiple proteins are derived from a large polyprotein encoded by an open reading frame. The 2A peptide mediates the co-translational cleavage of this polyprotein at a single site that fonns the junction between the virus capsid and replication polyprotein domains. The 2A sequences contain the consensus motif D-V/I-E-X-N-P-G-P (SEQ ID NO. 932). These sequences are thought to act co-translationally, preventing the formation of a normal peptide bond between the glycine and last proline, resulting in the ribosome skipping of the next codon (Donnelly ML et al. (2001). J Gen Virol, 82:1013-1025).
After cleavage, the short peptide remains fused to the C -terminus of the protein upstream of the cleavage site, while the proline is added to the N-terminus of the protein downstream of the cleavage site. Of the 2A peptides identified to date, four have been widely used namely FMDV
2A (abbreviated herein as F2A): equine rhinitis A virus (ERAV) 2A (E2A);
porcine teschovirus-1 2A (P2A) and Thoseaasigna virus 2A (T2A). In some embodiments, the 2A
peptide sequences useful in the present invention are selected from SEQ ID NO.8-11 of International Patent Publication W02010042490, the contents of which are incorporated by reference in its entirety.
1002821 The linkers of the present invention may also be non-peptide linkers.
For example, alkyl linkers such as ¨NH¨(CH2) a-C(0)¨, wherein a=2-20 can be used. These alkyl linkers may further be substituted by any non-sterically hindering group such as lower alkyl (e.g., Ci-C6) lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc.
[00283] In some aspects, the linker may be an artificial linker from U.S. Pat.
NOs. 4,946,778; 5, 525, 491; 5,856;456; and International patent publication NOs. W02012/083424;
the contents of each of which are incorporated herein by reference in their entirety.
[00284] In some embodiments, linkers of the present invention, include without limitation, any of those taught in Table 11 of copending commonly owned U.S. Provisional Patent Application No. 62/320,864, filed on 4/11/2016, or in US Provisional Application No.
62/466,596 filed March 3, 2017 and the International Publication W02017/180587, the contents of which are incorporated herein by reference in their entirety.
[00285] In some embodiments, compositions of the invention may include optional proteasome adaptors. As used herein, the term "proteasome adaptor" refers to any nucleotide/ amino acid sequence that targets the appended payload for degradation. In some aspects, the adaptors target the payload for degradation directly thereby circumventing the need for ubiquitination reactions.
Proteasome adaptors may be used in conjunction with destabilizing domains to reduce the basal expression of the payload. Exemplary proteasome adaptors include the UbL
domain of Rad23 or hHR23b, HPV E7 which binds to both the target protein Rb and the S4 subunit of the proteasome with high affinity, which allows direct proteasome targeting;
bypassing the ubiquitination machinery; the protein gankyrin which binds to Rb and the proteasome subunit S6.
[00286] In one embodiment, the linker may be a spacer region of one or more nucleotides. Non-limiting examples of spacers are TCTAGATAATACGACTCACTAGAGATCC (SEQ ID NO.
846), TATGGCCACAACCATG (SEQ ID NO. 847), AATCTAGATAATACGACTCACTAGAGATCC (SEQ ID NO. 848), TCGCGAATG, TCGCGA, GCTTGCCACAACCCACAAGGAGACGACCTTCC (SEQ ID NO. 849), or ATNFSLLKQAGDVEENPGP (SEQ ID NO. 850, encoded by SEQ ID NO. 851).
[00287] In one embodiment, the linker may be a Bainfll site. As a non-limiting example, the BamHI site has the amino acid sequence GS and/or the DNA sequence GGATCC.
Embedded stimulus, signals and other regulatory features [00288] In some embodiments, the effector module of the present invention may further comprise one or more microRNAs, microRNA binding sites, promotors and tunable elements. In one embodiment, microRNA may be used in support of the creation of tunable biocircuits. Each aspect or tuned modality may bring to the effector module or biocircuit a differentially tuned feature. For example, a destabilizing domain may alter cleavage sites or dimerization properties or half-life of the payload, and the inclusion of one or more microRNA or microRNA binding site may impart cellular detargeting or trafficking features. Consequently, the present invention embraces biocircuits which are multifactorial in their tenability. Such biocircuits and effector modules may be engineered to contain one, two, three, four or more tuned features. In some embodiments, micro RNA sequences of the present invention, include without limitation, any of those taught in Table 13 of copending commonly owned U.S. Provisional Patent Application No.
62/320,864, filed on 4/11/2016, or in US Provisional Application No.
62/466,596 filed March 3, 2017 and the International Publication W02017/180587, the contents of which are incorporated herein by reference in their entirety.
Polvnucleotides [00289] The term "polynucleotide" or "nucleic acid molecule" in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides, e.g., linked nucleosides.
These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a fi- D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA

having a 2'-amino fimctionalization, and 2'-amino- a-LNA having a 2'-amino functionalization) or hybrids thereof 1002901 In some embodiments, polynucleotides of the invention may be a messenger RNA
(mRNA) or any nucleic acid molecule and may or may not be chemically modified.
In one aspect, the nucleic acid molecule is a mRNA. As used herein, the term "messenger RNA
(mRNA)" refers to any polynucleotide which encodes a poly-peptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo.
[00291] Traditionally, the basic components of an mRNA molecule include at least a coding region, a 5'UTR, a 3'UTR, a 5' cap and a poly-A tail. Building on this wild type modular structure, the present invention expands the scope of functionality of traditional mRNA
molecules by providing payload constructs which maintain a modular organization, but which comprise one or more structural and/or chemical modifications or alterations which impart useful properties to the polynucleotide, for example tenability of function. As used herein, a "structural" feature or modification is one in which two or more linked nucleosides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide without significant chemical modification to the nucleosides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide "ATCG" may be chemically modified to "AT-5meC-G". The same polynucleotide may be structurally modified from "ATCG" to "ATCCCG". Here, the dinucleotide "CC" has been inserted, resulting in a structural modification to the polynucleotide.
[00292] In some embodiments, polynucleotides of the present invention may harbor 5'UTR
sequences which play a role in translation initiation. 5'UTR sequences may include features such as Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of genes, Kozak sequences have the consensus XCCR(A/G) CCAUG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG) and X is any nucleotide. In one embodiment, the Kozak sequence is ACCGCC. By engineering the features that are typically found in abundantly expressed genes of target cells or tissues, the stability and protein production of the polynucleotides of the invention can be enhanced.
[00293] 'Further provided are polynucleotides, which may contain an internal ribosome entry site (IRES) which play an important role in initiating protein synthesis in the absence of 5' cap structure in the polynucleotide. An 1RES may act as the sole ribosome binding site, or may serve as one of the multiple binding sites. Polynucleotides of the invention containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes giving rise to bicistronic and/or multicistronic nucleic acid molecules.
[00294] In some embodiments, polynucleotides encoding biocircuits, effector modules, SREs and payloads of interest such as inununotherapeutic agents may include from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to

10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000 nucleotides). In some aspects, polynucleotides of the invention may include more than 10,000 nucleotides.
[00295] Regions of the polynucleotides which encode certain features such as cleavage sites, linkers, trafficking signals, tags or other features may range independently from 10-1,000 nucleotides in length (e.g., greater than 20, 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).
[00296] In some embodiments, polynucleotides of the present invention may further comprise embedded regulatory moieties such as microRNA binding sites within the 3'UTR
of nucleic acid molecules which when bind to microRNA molecules, down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation.
Conversely, for the purposes of the polynucleotides of the present invention, microRNA binding sites can be engineered out of (i.e. removed from) sequences in which they naturally occur in order to increase protein expression in specific tissues. For example, miR-142 and miR-146 binding sites may be removed to improve protein expression in the immune cells. In some embodiments, any of the encoded payloads may be may be regulated by an SRE and then combined with one or more regulatory sequences to generate a dual or multi-tuned effector module or biocircuit system.
[00297] In some embodiments, polynucleotides of the present invention may encode fragments, variants, derivatives of polypeptides of the inventions. In some aspects, the variant sequence may keep the same or a similar activity. Alternatively, the variant may have an altered activity' (e.g., increased or decreased) relative to the start sequence. Generally, variants of a particular polynucleotide or polypeptide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%
but less than 100% sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art. Such tools for alignment include those of the BLAST
suite (Stephen et al., Gapped BLAST and PSI-BLAST: a new generation of protein database search programs, Nucleic Acids Res., 1997, 25:3389-3402.) [00298] In some embodiments, polynucleotides of the present invention may be modified. As used herein, the terms "modified", or as appropriate, "modification" refers to chemical modification with respect to A, G, U (T in DNA) or C nucleotides.
Modifications may be on the nucleoside base and/or sugar portion of the nucleosides which comprise the polynucleotide. In some embodiments, multiple modifications are included in the modified nucleic acid or in one or more individual nucleoside or nucleotide. For example, modifications to a nucleoside may include one or more modifications to the nucleobase and the sugar.
Modifications to the polynucleotides of the present invention may include any of those taught in, for example, International Publication NO. W02013052523, the contents of which are incorporated herein by reference in its entirety.
[00299] As described herein "nucleoside" is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as "nucleobase"). As described herein, "nucleotide" is defined as a nucleoside including a phosphate group.
[00300] In some embodiments, the modification may be on the intemucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases "phosphate" and "phosphodiester" are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates). Other modifications which may be used are taught in, for example, International Application NO. W02013052523, the contents of which are incorporated herein by reference in their entirety.
[00301] Chemical modifications and/or substitution of the nucleotides or nucleobases of the polynucleotides of the invention which are useful in the present invention include any modified substitutes known in the art, for example, ( )1(2-Hydroxypropyl)pseudouridine TP, (2R)-1-(2-Hydroxypropyl)pseudouridine TP, 1-(4-Methoxy-phenyl)pseudo-UTPõ2'-O-dimethyladenosine, 1,2'-0-climethylguanosine, 1,2'-O-dimethylinosine, -Hexyl-pseudo-U'TP , 1-Homoallylpseudouridine TP, 1-Hydroxymethylpseudouridine TP, 1-iso-propyl-pseudo-UTP , 1-Me-2-thio-pseudo-UTP, 1-Me-4-thio-pseudo-UTP, 1-Me-alpha-thio-pseudo-UTP , 1-Me-GTP, 2'-Amino-2'-deoxy-ATP, 2'-Amino-2'-deoxy-CTP, 2'-Amino-2'-deoxy-G'TP, 2'-Amino-2'-deoxy-UTP, 2'-Azido-2'-deoxy-ATP, tubercidine, under modified hydroxywybutosine, uridine 5-oxyacetic acid, uridine 5-oxyacetic acid methyl ester, wybutosine, wyosine, xanthine, Xanthosine-5'-TP, xylo-adenosine, zebularine, a-thio-adenosine, a-thio-cytidine, a-thio-guanosine, and/or a-thio-uridine.
1003021 Polynucleotides of the present invention may comprise one or more of the modifications taught herein. Different sugar modifications, base modifications, nucleotide modifications, and/or internucleoside linkages (e.g., backbone structures) may exist at various positions in the polynucleotide of the invention. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a polynucleotide such that the function of the polynucleotide is not substantially decreased. A
modification may also be a 5' or 3' terminal modification. The poly-nucleotide may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10%
to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10%
to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20%
to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20%
to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50%
to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70%
to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90%
to 95%, from 90% to 100%, and from 95% to 100%).
[00303] In some embodiments, one or more codons of the polynucleotides of the present invention may be replaced with other codons encoding the native amino acid sequence to tune the expression of the SREs, through a process referred to as codon selection.
Since mRNA
codon, and tRNA anticodon pools tend to vary among organisms, cell types, sub cellular locations and overtime, the codon selection described herein is a spatiotemporal (SD codon selection.
[00304] in some embodiments of the invention, certain polynucleotide features may be codon optimized. Codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cell by replacing at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons of the native sequence with codons that are most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Codon usage may be measured using the Codon Adaptation Index (CAI) which measures the deviation of a coding polynucleotide sequence from a reference gene set. Codon usage tables are available at the Codon Usage Database (http://www.kazusa.or.jp/codon0 and the CAI can be calculated by EMBOSS CAI
program (http://emboss.sourceforge.net/). Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include to match codon frequencies in target and host organisms to ensure proper folding, bias nucleotide content to alter stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein signaling sequences, remove/add post translation modification sites in encoded protein (e.g. glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and degradation sites, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the poly-nucleotide. Codon optimization tools, algorithms and services are known in the art, and non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), OptimumGene (GenScript, Piscataway, NJ), algorithms such as but not limited to, DNAWorks v3.2.3, Mr.
Gene (GmBH, Regensburg, Germany) and/or proprietary methods. In one embodiment, a polynucleotide sequence or portion thereof is codon optimized using optimization algorithms.
Codon options for each amino acid are well-known in the art as are various species table for optimizing for expression in that particular species.
[00305] In some embodiments of the invention, certain polynucleotide features may be codon optimized. For example, a preferred region for codon optimization may be upstream (5') or downstream (3') to a region which encodes a polypeptide. These regions may be incorporated into the polynucleotide before and/or after codon optimization of the payload encoding region or open reading frame (ORF).
[00306] After optimization (if desired), the polynucleotide components are reconstituted and transfonned into a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.
[00307] Spatiotemporal codon selection may impact the expression of the polynucleotides of the invention, since codon composition determines the rate of translation of the mRNA species and its stability. For example, tRNA anticodons to optimized codons are abundant, and thus translation may be enhanced. In contrast, tRNA anticodons to less common codons are fewer and thus translation may proceed at a slower rate. Presnyak et al. have shown that the stability of an mRNA species is dependent on the codon content, and higher stability and thus higher protein expression may be achieved by utilizing optimized codons (Presnyak et al.
(2015) Cell 160, 1111-1124; the contents of which are incorporated herein by reference in their entirety). Thus, in some embodiments, ST codon selection may include the selection of optimized codons to enhance the expression of the SRES, effector modules and biocircuits of the invention. In other embodiments, spatiotemporal codon selection may involve the selection of codons that are less commonly used in the genes of the host cell to decrease the expression of the compositions of the invention. The ratio of optimized codons to codons less commonly used in the genes of the host cell may also be varied to tune expression.
[00308] In some embodiments, certain regions of the polynucleotide may be preferred for codon selection. For example, a preferred region for codon selection may be upstream (5') or downstream (3') to a region which encodes a polypeptide. These regions may be incorporated into the polynucleotide before and/or after codon selection of the payload encoding region or open reading frame (ORF).
[00309] The stop codon of the polynucleotides of the present invention may be modified to include sequences and motifs to alter the expression levels of the SREs, payloads and effector modules of the present invention. Such sequences may be incorporated to induce stop codon readthrough, wherein the stop codon may specify amino acids e.g.
selenocysteine or pyrrolysine.
In other instances, stop codons may be skipped altogether to resume translation through an alternate open reading frame. Stop codon read through may be utilized to tune the expression of components of the effector modules at a specific ratio (e.g.as dictated by the stop codon context).
Examples of preferred stop codon motifs include UGAN, UAAN, and UAGN, where N
is either C or U. Polynucleotide modifications and manipulations can be accomplished by methods known in the art such as, but not limited to, site directed mutagenesis and recombinant technology. The resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.
[00310] In some embodiments, polynucleotides of the invention may comprise two or more effector module sequences, or two or more payloads of interest sequences, which are in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times. In these patterns, each letter, A, B, or C
represent a different effector module component.
[00311] In yet another embodiment, polynucleotides of the invention may comprise two or more effector module component sequences with each component having one or more SRE
sequences (DD sequences), or two or more payload sequences. As a non-limiting example, the sequences may be in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times in each of the regions. As another non-limiting example, the sequences may be in a pattern such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than three times across the entire polynucleotide. In these patterns, each letter, A, B, or C represent a different sequence or component.
[00312] According to the present invention, polynucleotides encoding distinct biocircuits, effector modules, SREs and payload constructs may be linked together through the 3'-end using nucleotides which are modified at the 3'-terminus. Chemical conjugation may be used to control the stoichiometry of delivery into cells. Polynucleotides can be designed to be conjugated to other polynucleotides, dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, (MPEG)2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases, proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell, hormones and honnone receptors, non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, or a drug. As non-limiting examples, they may be conjugates with other immune conjugates.
1003131 In some embodiments, the compositions of the polynucleotides of the invention may be generated by combining the various components of the effector modules using the Gibson assembly method. The Gibson assembly reaction consists of three isothermal reactions, each relying on a different enzymatic activity including a 5' exonuclease which generates long overhangs, a polytnerase which fills in the gaps of the annealed single strand regions and a DNA
ligase which seals the nicks of the annealed and filled-in gaps. Polymerase chain reactions performed prior to Gibson assembly may be used to generate PCR products with overlapping sequence. These methods can be repeated sequentially, to assemble larger and larger molecules.
For example, the method can comprise repeating the steps described as above to join a second set of two or more DNA molecules of interest to one another, and then repeating the method again to join the first and second set DNA molecules of interest, and so on. At any stage during these multiple rounds of assembly, the assembled DNA can be amplified by transforming it into a suitable microorganism, or it can be amplified in vitro (e.g., with PCR).
1003141 In some embodiments, polynucleotides of the present invention may encode a fusion polypeptide comprising a destabilizing domain (DD) and at least one immunotherapeutic agent taught herein. The DD domain may be a FKBP mutant encoded by nucleotide sequence of SEQ
ID NOS. 60, 87-88, and/or 878-889, an ecDHFR mutant encoded by nucleotide sequence of SEQ
ID NO. 61, 89, 90, and/or 869-877, hDHFR mutant encoded by nucleotide sequence of SEQ ID
NO. 91-93, 182-192, SEQ ID NO. 797-832, and/or 890-905.
1003151 In some embodiments, the polynucleotides of the invention may encode effector modules comprising IL2 as the payload comprising the nucleotide sequence of SEQ ID NO. 62-64, or caspase 9 as the payload comprising the nucleotide sequence of SEQ ID
NO. 94-102, or FOXP3 as the payload, comprising the nucleotide sequence of SEQ ID NO. 193-202 or luciferase as the payload comprising the nucleotide sequence of SEQ ID NO. 203-208, or BCMA CAR as the payload comprising the nucleotide sequence of SEQ ID NO. 833-835 or Her2 as the payload comprising the nucleotide sequence of 907.
Cells 1003161 In accordance with the present invention, cells genetically modified to express at least one biocircuit, SRE (e. g, DD), effector module and immunotherapeutic agent of the invention, are provided. Cells of the invention may include, without limitation, immune cells, stem cells and tumor cells. In some embodiments, immune cells are immune effector cells, including, but not limiting to, T cells such as CD8+ T cells and CD4' T cells (e.g., 'Thl, Th2, Th17 , Foxp3+

cells), memory T cells such as T memory stem cells, central T memory cells;
and effector memory T cells, terminally differentiated effector T cells, natural killer (NK) cells, NK T cells, ttunor infiltrating lymphocytes (TILs), cytotoxic T lymphocytes (CTLs), regulatory T cells (Tregs), and dendritic cells (DCs), other immune cells that can elicit an effector function, or the mixture thereof. T cells may be Ta13 cells and Ty 5 cells. In some embodiments, stem cells may be from human embryonic stem cells, mesenchymal stem cells, and neural stem cells. In some embodiments, T cells may be depleted endogenous T cell receptors (See US Pat.
NOs. 9, 273, 283; 9; 181, 527; and 9,028, 812; the contents of each of which are incorporated herein by reference in their entirety).
[00317] In some embodiments, cells of the invention may be autologous, allogeneic, syngeneic, or xenogeneic in relation to a particular individual subject.
[003181 In some embodiments, cells of the invention may be mammalian cells, particularly human cells. Cells of the invention may be primary cells or immortalized cell lines.
[00319] In some embodiments, cells of the invention may be expanded using expansion factors to trigger proliferation and expansion of the cells. Exemplary expansion factors include RAS
such as KRAS, NRAS, RRAS, RRAS2, MRAS, ERAS, and HRAS, DIRAS such as DIRAS1, DIRAS2, and DIRAS3. NKTRAS such as NKTRAS I, and NKIRAS2, RAL such as RALA, and RALB, RAP such as RAP1A, RAP1B, RAP2A, RAP2B, and RAP2C, RASD such as RASD1, and RASD2. RASL such as RASL1OA, RASL10B, RASL11A, RASLI1B, and RASL12, REM
such as REM1, and REM2, GEM, RERG, RERGL, and RRAD.
[00320] Engineered immune cells can be accomplished by transducing a cell compositions with a polypeptide of a biocircuit, an effector module, a SRE and/or a payload of interest (i.e., immtmotherapeutic agent), or a polynucleotide encoding said polypeptide, or a vector comprising said polynucleotide. The vector may be a viral vector such as a lentiviral vector, a gamma-retroviral vector, a recombinant AAV, an adenoviral vector and an oncolytic viral vector.
In other aspects, non-viral vectors for example, nanoparticles and liposomes may also be used. In some embodiments, immune cells of the invention are genetically modified to express at least one immunotherapeutic agent of the invention which is tunable using a stimulus. In some examples, two, three or more inununotherapeutic agents constructed in the same biocircuit and effector module are introduced into a cell. In other examples, two, three, or more biocircuits, effector modules, each of which comprises an immunotherapeutic agent, may be introduced into a cell.
[00321] In some embodiments, T cells expressing Chimeric antigen receptors or T cells receptors may be further modified to express another one, two, three or more immunotherapeutic agents of the present invention. The immunotherapeutic agents may be another a cytokine such as IL2, IL12, IL15 and IL18; a regulatory switch: or a safety switch gene (e.g., a suicide gene) that kills activated T cells when a severe event is observed after adoptive cell transfer or when the transferred immune cells are no-longer needed. These molecules may be included in the same effector module or in separate effector modules.
[00322] In some embodiments, immune cells of the invention may be NK cells modified to express payloads of the invention.
[00323] Natural killer (NK) cells are members of the innate lymphoid cell family and characterized in humans by expression of the phenotypic marker CD56 (neural cell adhesion molecule) in the absence of CD3 (T-cell co-receptor). NK cells are potent effector cells of the innate immune system which mediate cytotoxic attack without the requirement of prior antigen priming, forming the first line of defense against diseases including cancer malignancies and viral infection.
[00324] Several pre-clinical and clinical trials have demonstrated that adoptive transfer of NK
cells is a promising treatment approach against cancers such as acute myeloid leukemia (Ruggeri et al., Science: 2002, 295: 2097-2100; and Geller et al., Immunotherapy, 2011, 3: 1445-1459).
[00325] NK cell activation is characterized by an array of receptors with activating and inhibitory functions. The important activation receptors on NK cells include CD94/NKG2C and NKG2D (the C-type lectin-like receptors), and the natural cytotoxicity receptors (NCR) NKp30, NKp44 and NKp46, which recognize ligands on tumor cells or virally infected cells. NK cell inhibition is essentially mediated by interactions of the polymorphic inhibitory killer cell immunoglobulin-like receptors (KIRs) with their cognate human-leukocyte-antigen (HLA) ligands via the alpha-1 helix of the HLA molecule. The balance between signals that are generated from activating receptors and inhibitory receptors mainly determines the immediate cytotoxic activation.
[00326] NK cells may be isolated from peripheral blood mononuclear cells (PBMCs), or derived from human embryonic stein (ES) cells and induced pluripotent stem cells (iPSCs). The primary NK cells isolated from PBMCs may be further expanded for adoptive immunotherapy.
Strategies and protocols useful for the expansion of NK cells may include interleukin 2 (IL2) stimulation and the use of autologous feeder cells, or the use of genetically modified allogeneic feeder cells. In some aspects, NK cells can be selectively expanded with a combination of stimulating ligands including IL15, IL21, IL2, 41BBL, IL12, IL18, MICA, 2B4, LFA-1, and BCM1/SLAMF2 (e.g., US patent publication NO. U520150190471).

1003271 In some embodiments, cells of the present invention may be dendritic cells that are genetically modified to express the compositions of the invention.
1003281 In some embodiments, cells of the invention may be Treg cells.
Payloads of the invention may be used to promote the proliferation, survival, activation and /or function of T
regulatory cells. Tregs are a distinct population of cells that are positively selected on high affinity ligands in the thymus and play an important role in the tolerance to self-antigens. In addition, T regs have also been shown to play a role in peripheral tolerance to foreign antigens.
The ability of Tregs to induce tolerance may be utilized to tune immune responses to the immunotherapeutic agents described herein. Methods for expanding Tregs for immunotherapy have been described by Tang et al., 2004, J. Exp. Med. 199: 1455-65; Battaglia et al., 2005, Blood 105: 4743-48; Earle et al., 2005, Clin. Immunol. 115: 3-9; Godfrey et al., 2004, Blood 104: 453-61; Hoffmann et al., 2004, Blood 104: 895-903.
III. PHARMACEUTICAL COMPOSITIONS AND FORMULATIONS
1003291 The present invention further provides pharmaceutical compositions comprising one or more biocircuits, effector modules, SREs (e.g., DDs), stimuli and payloads of interest (i.e., immunotherapeutic agents), vectors, cells and other components of the invention, and optionally at least one pharmaceutically acceptable excipient or inert ingredient.
[00330] As used herein the term "pharmaceutical composition" refers to a preparation of biocircuits, SREs, stimuli and payloads of interest (i.e., immunotherapeutic agents), other components, vectors, cells and described herein, or pharmaceutically acceptable salts thereof, optionally with other chemical components such as physiologically suitable carriers and excipients. The pharmaceutical compositions of the invention comprise an effective amount of one or more active compositions of the invention. The preparation of a pharmaceutical composition that contains at least one composition of the present invention and/or an additional active ingredient will be known to those skilled in the art in light of the present disclosure, as exemplified by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, incorporated herein by reference.
[00331] The term "excipient" or "inert ingredient" refers to an inactive substance added to a pharmaceutical composition and formulation to further facilitate administration of an active ingredient. For the purposes of the present disclosure, the phrase "active ingredient" generally refers to any one or more biocircuits, effector modules. SREs, stimuli and payloads of interest (i.e., immunotherapeutic agents), other components, vectors, and cells to be delivered as described herein. The phrases "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
[00332] In some embodiments, pharmaceutical compositions and formulations are administered to humans, human patients or subjects. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to any other animal, e.g., to non-human animals, e.g. non-human mammals. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, non-human mammals, including agricultural animals such as cattle, horses, chickens and pigs, domestic animals such as cats, dogs, or research animals such as mice, rats, rabbits, dogs and non-human primates. It will be understood that, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
[00333] A pharmaceutical composition and formulation in accordance with the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.
[00334] The compositions of the present invention may be formulated in any manner suitable for delivery. The formulation may be, but is not limited to, nanoparticles, poly (lactic-co-glycolic acid) (PLGA) microspheres, lipidoids, lipoplex, liposome, polymers, carbohydrates (including simple sugars), cationic lipids and combinations thereof.
[00335] In one embodiment, the formulation is a nanoparticle which may comprise at least one lipid. The lipid may be selected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG and PEGylated lipids. In another aspect, the lipid may be a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA and DODMA.
[00336] For polynucleotides of the invention, the formulation may be selected from any of those taught, for example, in International Application PCT/US2012/069610, the contents of which are incorporated herein by reference in its entirety.
[00337] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient or inert ingredient, and/or any additional ingredients in a pharmaceutical composition in accordance with the invention will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1 and 100, e.g., between 0.5 and 50, between 1-30, between 5-80, at least 80 (w/w) active ingredient.
1003381 Efficacy of treatment or amelioration of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of compositions of the present invention, "effective against"
for example a cancer, indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in disease load, reduction in tumor mass or cell numbers, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of cancer.
1003391 A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10 in a measurable parameter of disease, and preferably at least 20, 30, 40, 50 or more can be indicative of effective treatment. Efficacy for a given composition or formulation of the present invention can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant change is observed.
1003401 Preferably, the compositions of the invention are administered by injection, e.g., intravenously. When the inventive CAR material is a host cell (or a population thereof) expressing the inventive CAR, the pharmaceutically acceptable carrier for the cells for injection may include any isotonic carrier such as, for example, normal saline (about 0.90% w/v of NaC1 in water, about 300 mOsm/L NaCl in water, or about 9.0 g NaCl per liter of water), NORMOSOL R electrolyte solution (Abbott, Chicago, IL), PLASMA- LYTE A (Baxter, Deerfield, IL), about 5% dextrose in water, or Ringer's lactate. In an embodiment, the pharmaceutically acceptable carrier may be supplemented with human serum albumen. Any of the carriers taught in W02016149578A I may be useful in the present invention.

IV. APPLICATIONS
[00341] In one aspect of the present invention, methods for reducing a minor volume or burden are provided. The methods comprise administering a pharmaceutically effective amount of a pharmaceutical composition comprising at least one biocircuit system, effector module, DD, and/or payload of interest (i.e., an immunotherapeutic agent), at least one vector, or cells to a subject having a tumor. The biocircuit system and effector module having any immunotherapeutic agent as described herein may be in forms of a polypeptide, or a polynucleotide such as mRNA, or a viral vector comprising the polynucleotide, or a cell modified to express the biocircuit, effector module, DD, and payload of interest (i.e., immunotherapeutic agent).
[00342] In another aspect of the present invention, methods for inducing an anti-tumor immune response in a subject are provided. The methods comprise administering a pharmaceutically effective amount of a pharmaceutical composition comprising at least one biocircuit system, effector module, DD, and/or payload of interest (i.e., an immunotherapeutic agent), at least one vector, or cells to a subject having a tumor. The biocircuit and effector module having any immunotherapeutic agent as described herein may be in forms of a polypeptide, or a polynucleotide such as mRNA, or a viral vector comprising the polynucleotide, or a cell modified to express the biocircuit, effector module, DD, and payload of interest (i.e., immunotherapeutic agent).
[00343] The methods, according to the present invention, may be adoptive cell transfer (ACT) using genetically engineered cells such as immune effector cells of the invention, cancer vaccines comprising biocircuit systems, effector modules, DDs, payloads of interest (i.e., immunotherapeutic agents) of the invention, or compositions that manipulate the tumor immunosuppressive microenvironment, or the combination thereof. These treatments may be further employed with other cancer treatment such as chemotherapy and radiotherapy.
[00344] In some embodiments, the safety switches described herein may be useful in the treatment of diseases of protein proliferation and/or protein aggregation e.g.
renal diseases and/or neurological diseases such as Alzheimer's diseases, prior diseases etc. In one embodiment, safety switches of the present invention may be expressed in phagocytic cells that are engineered to target aggregated proteins such as amyloid proteins, wherein the safety switches described herein may be used to eliminate the phagocytic cells after the clearance of the aggregated proteins.

1. Adoptive cell transfer (adoptive inuntmotherapv) [00345] In some embodiments, cells which are genetically modified to express at least one biocircuit system, effector module, DD, and/or payload of interest (immunotherapeutic agent) may be used for adoptive cell therapy (ACT). As used herein, Adoptive cell transfer refers to the administration of immune cells (from autologous, allogenic or genetically modified hosts) with direct anticancer activity. ACT has shown promise in clinical application against malignant and infectious disease. For example, T cells genetically engineered to recognize CD19 have been used to treat follicular B cell lymphoma (Kochenderfer et al., Blood, 2010, 116:4099-4102; and Kochenderfer and Rosenberg, Nat Rev Clin Oncol.. 2013, 10(5): 267-276) and ACT
using autologous lymphocytes genetically-modified to express anti-tumor T cell receptors has been used to treat metastatic melanoma (Rosenberg and Dudley, Curr. Op/n. Immunol.
2009, 21: 233-240).
[00346] According to the present invention, the biocircuits and systems may be used in the development and implementation of cell therapies such as adoptive cell therapy. Certain effector modules useful in cell therapy are given in Figures 7-12. The biocircuits, their components, effector modules and their SREs and payloads may be used in cell therapies to regulate epitope tagged receptors, in APC platforms for stimulating T cells, as a tool to enhance ex vivo APC
stimulation, to improve methods of T cell expansion, in ex vivo stimulation with antigen, in TCR/CAR combinations, in the manipulation or regulation of TILs, in allogeneic cell therapy, in combination T cell therapy with other treatment lines (e.g. radiation, cytokines).
[00347] Provided herein are methods for use in adoptive cell therapy. The methods involve preconditioning a subject in need thereof, modulating immune cells with SRE, biocircuits and compositions of the present invention, administering to a subject, engineered immune cells expressing compositions of the invention and the successful engraftment of engineered cells within the subject.
[00348] in some embodiments, SREs, biocircuits and compositions of the present invention may be used to minimize preconditioning regimens associated with adoptive cell therapy. As used herein "preconditioning" refers to any therapeutic regimen administered to a subject to improve the outcome of adoptive cell therapy. Preconditioning strategies include, but are not limited to total body irradiation and/or lymphodepleting chemotherapy.
Adoptive therapy clinical trials without preconditioning have failed to demonstrate any clinical benefit, indicating its importance in ACT. Yet, preconditioning is associated with significant toxicity and limits the subject cohort that is suitable for ACT. In some instances, immune cells for ACT may be engineered to express cytokines such as IL2 as payload using SREs of the present invention to reduce the need for preconditioning.
1003491 In some embodiments, immune cells for ACT may be dendritic cells, T
cells such as CD8+ T cells and CD4+ T cells, natural killer (NK) cells, NK T cells, Cytotoxic T lymphocytes (CTLs), tumor infiltrating lymphocytes (TILs), lymphokine activated killer (LAK) cells_ memoiy T cells, regulatory T cells (Tregs), helper T cells, cytokine-induced killer (OK) cells, and any combination thereof. In other embodiments, immune stimulatory cells for ACT
may be generated from embryonic stem cell (ESC) and induced pluripotent stein cell (iPSC). In some embodiments, autologous or allogeneic immune cells are used for ACT.
1003501 In some embodiments, NK cells engineered to express the present compositions may be used for ACT. NK cell activation induces perforin/granzyme-dependent apoptosis in target cells.
NK cell activation also induces cytokine secretion such as IFN-y, TNF-a and GM-CSF. These cytokines enhance the phagocytic function of macrophages and their antimicrobial activity, and augment the adaptive immune response via up-regulation of antigen presentation by antigen presenting cells such as dendritic cells (DCs) (Reviewed by Vivier et al., Nat. Immunol., 2008, 9(5): 503-510).
1003511 NK cells may also be genetically reprogrammed to circumvent NK cell inhibitory signals upon interaction with tumor cells. For example, using CRISPR, ZFN, or TALEN to genetically modify NK cells to silence their inhibitory receptors may enhance the anti-tumor capacity of NK cells.
1003521 Immune cells can be isolated and expanded ex vivo using a variety of methods known in the art. For example, methods of isolating and expanding cytotoxic T cells are described in U.S. Pat. NOs. 6,805,861 and 6,531, 451; US Patent Publication NOs.
US20160348072A1 and International Patent Publication NO. W02016168595A1; the contents of each of which are incorporated herein by reference in their entirety. Isolation and expansion of NK cells is described in US Patent Publication NOS. US20150152387A1, U.S. Patent NOS.
7,435, 596, and Oyer, J.L. (2016). Cytotherapy.18(5):653-63; the contents of each of which are incorporated by reference herein in its entirety. Specifically, human primary NK cells may be expanded in the presence of feeder cells e.g. a myeloid cell line that has been genetically modified to express membrane bound IL15, 1L21, 1L12 and 4-1BBL.
1003531 In some instances, sub populations of immune cells may be enriched for ACT. Methods for inunune cell enrichment are taught in International Patent Publication NOS.
W02015039100A1. In another example, T cells positive for B and T lymphocyte attenuator marker BTLA) may be used to enrich for T cells that are anti-cancer reactive as described in U.S.

Pat. NOS. 9,512,401 (the content of each of which are incorporated herein by reference in their entirety).
[00354] In some embodiments, immune cells for ACT may be depleted of select sub populations to enhance T cell expansion. For example, immune cells may be depleted of Foxp3+
T lymphocytes to minimize the ant-tumor immune response using methods taught in US Patent Publication NOS. US 20160298081A the contents of which are incorporated by reference herein in their entirety.
[00355] In some embodiments, immune cells may be enriched for FOXP3+ cells to enrich for T
cells that are critical for immune tolerance to reduce graft versus host disease.
[00356] In some embodiments, activation and expansion of T cells for ACT is achieved antigenic stimulation of a transiently expressed Chimeric Antigen Receptor (CAR) on the cell surface. Such activation methods are taught in International Patent NOS.
W02017015427, the content of which are incorporated herein by reference in their entirety.
[00357] In some embodiments, immune cells may be activated by antigens associated with antigen presenting cells (APCs). In some embodiments, the APCs may be dendritic cells, macrophages or B cells that antigen specific or nonspecific. The APCs may autologous or homologous in their organ. In some embodiments, the APCs may be artificial antigen presenting cells (aAPCs) such as cell based aAPCs or acellular aAPCs. Cell based aAPCs are may be selected from either genetically modified allogeneic cells such as human erythroleukemia cells or xenogeneic cells such as murine fibroblasts and Drosophila cells.
Alternatively, the APCs maybe be acellular wherein the antigens or costimulatory domains are presented on synthetic surfaces such as latex beads, polystyrene beads, lipid vesicles or exosomes.
[00358] In some embodiments, cells of the invention, specifically T cells may be expanded using artificial cell platforms. In one embodiment, the mature T cells may be generated using artificial thymic organoids (AT0s) described by Seet CS et al. 2017. Na!
Methods. 14, 521-530 (the contents of which are incorporated herein by reference in their entirety). ATOs are based on a stromal cell line expressing delta like canonical notch ligand (DLL!). In this method, stromal cells are aggregated with hematopoietic stem and progenitor cells by centrifugation and deployed on a cell culture insert at the air¨fluid interface to generate organoid cultures. ATO-derived T
cells exhibit naive phenotypes, a diverse T cell receptor (TCR) repertoire and TCR-dependent function.
[00359] In some embodiments, the T cells of the invention may be separated from peripheral blood by a process known as apheresis, which separates lymphocytes from plasma, platelets and RBCs, and granulocytes. Lymphocyte in peripheral blood cells may further be separated from monocytes using a semi-automated elutriation device. T cells may also be enriched by magnetic selection with anti CD3/CD28 beads. In one embodiment, an additional step of using a plastic adherent surface to deplete monocytes from the PBMCs may be utilized. Methods of T cell enrichment are disclosed in Stroncek DF et al. (2017) Journal of Translational Medicine.15:59;
the contents of which are incorporated by reference in its entirety.
[00360] In some embodiments, adoptive cell therapy is carried out by autologous transfer, wherein the cells are derived from a subject in need of a treatment and the cells, following isolation and processing are administered to the same subject. In other instances, ACT may involve allogenic transfer wherein the cells are isolated and/or prepared from a donor subject other than the recipient subject who ultimately receives cell therapy. The donor and recipient subject may be genetically identical, or similar or may express the same HLA
class or subtype.
[00361] In some embodiments, the multiple immunotherapeutic agents introduced into the immune cells for ACT (e.g., T cells and NK cells) may be controlled by the same biocircuit system. In one example, a cytokine such as IL2 and a Caspase 9 are linked to the same hDHFR
destabilizing domain. The expression of IL2 and Caspase 9 is tuned using TMP
simultaneously.
In other embodiments, the multiple immunotherapeutic agents introduced into the immune cells for ACT (e.g., T cells and NK cells) may be controlled by different biocircuit systems. In one example, a cytokine such as IL2 and Caspase 9 constructs are linked to different DDs in two separate effector modules, and can be tuned separately using different stimuli. In another example, a suicide gene and a CAR construct may be linked to two separate effector modules.
[00362] Following genetic modulation using SREs, biocircuits and compositions of the invention, cells are administered to the subject in need thereof. Methods for administration of cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; US
Patent No.
4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85).
See, e.g.;
Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338;
the contents of each of which are incorporated herein by reference in their entirety.
1003631 In some embodiments, immune cells for ACT may be modified to express one or more immunotherapeutic agents which facilitate immune cells activation, infiltration, expansion, survival and anti-tumor functions.
[00364] In some embodiments, immune cells used for adoptive cell transfer can be genetically manipulated to improve their persistence, cytotoxicity, tumor targeting capacity, and ability to home to disease sites in vivo, with the overall aim of further improving upon their capacity to kill tumors in cancer patients. One example is to introduce effector modules of the invention comprising cytokines such as gamma-cytokines (IL2) into immune cells to promote immune cell proliferation and survival. Transduction of cytokine genes (e.g., gamma-cytokines IL2) into cells will be able to propagate immune cells without addition of exogenous cytokines and cytokine expressing NK cells have enhanced tumor cytotoxicity.
[00365] In some embodiments, biocircuits, their components, SREs or effector modules may be utilized to prevent T cell exhaustion. As used herein, "T cell exhaustion"
refers to the stepwise and progressive loss of T cell function caused by chronic T cell activation. T
cell exhaustion is a major factor limiting the efficacy of antiviral and antitumor immunotherapies.
Exhausted T cells have low proliferative and cytokine producing capabilities concurrent with high rates of apoptosis and high surface expression of multiple inhibitory receptors. T cell activation leading to exhaustion may occur either in the presence or absence of the antigen.
[00366] In some embodiments, effector modules of the present invention, useful for immtmotherapy may be placed under the transcriptional control of the T cell receptor alpha locus constant (TRAC) locus in the T cells. Eyquem et al. have shown that expression of the CAR
from the TRAC locus prevents T cell exhaustion and the accelerated differentiation of T cells caused by excessive T cell activation (Eyquem J. et al (2017) Nature.543(7643):113-117; the contents of which are incorporated herein by reference in their entirety).
[00367] In some embodiments, payloads of the invention may be used in conjunction with antibodies or fragments that target T cell surface markers associated with T
cell exhaustion. T-cell surface markers associated with T cell exhaustion that may be used include, but are not limited to, CTLA-1, PD-1; TGIT, LAG-3, 2B4, BTLA, TIM3, VISTA, and CD96.
[00368] T cells that are specific to certain tumor antigens, may be subject to chronic antigen exposure. Persistent antigen expression can lead to immune check-point expression, which in turn, induces a state of exhaustion among cognate antigen specific T cells.
Constant expression of the chimeric antigen receptors of the invention may result in chronic interaction with the antigen, which leads to exhaustion. The compositions disclosed herein may be used to prevent T
cell exhaustion by modulating surface CAR expression using the stimulus specific to the invention. In one embodiment, the SREs of the present invention may be used to achieve pulsatile expression of the compositions of the invention. As used here, "pulsatile" refers to a plurality of payload expression at spaced apart time intervals. Generally, upon administration of the stimulus, the expression of the payload is increased causing the first pulse; following the withdrawal of the stimulus, the expression of the payload decreases and this represents the interval time between the first exposure and the next exposure to the stimulus, after which the second exposure to the stimulus is initiated.
[00369] Also provided herein, is a method of preventing or reversing T cell exhaustion in a subject in need thereof, where the method comprising administering to the subject a therapeutically effective amount of a composition comprising at least one effector module. In some embodiments, the effector module includes a stimulus response element (SRE) operably linked to at least one immunotherapeutic agent, such that the SRE responds to a stimulus and tunes the expression and/or function of the immunotherapeutic agent, thereby preventing or reversing T cell exhaustion. In some embodiments, the immunotherapeutic agent may be a chimeric antigen receptor. Examples of chimeric antigen receptors include, but are not limited to GD2 CAR, BCMA CAR, CD33 CAR, Her2 CAR, ALK CAR, CD22 CAR, or a CD276 CAR. In some embodiments, the CAR may be a bispecific CAR comprising an extracellular domain which recognizes at least one antigen such as GD2, BCMA, CD33, Her2, ALK, CD22 or a CD276. In some embodiments, the methods described herein may include pulsatile expression of the compositions of the invention to prevent T cell exhaustion. In some instances. T cell exhaustion may be reversed by the addition of the stimulus. In other instances, T cell exhaustion may be reversed by the withdrawal of the stimulus.
[00370] In some embodiments, the compositions of the present invention may be utilized to alter TIL (tumor infiltrating lymphocyte) populations in a subject. In one embodiment, any of the payloads described herein may be utilized to change the ratio of CD4 positive cells to CD8 positive populations. In some embodiments, TILs may be sorted ex vivo and engineered to express any of the cytokines described herein. Payloads of the invention may be used to expand CD4 and/or CD8 populations of TILs to enhance TIL mediated immune response.
2. Cancer vaccines [00371] In some embodiments, biocircuits, effector modules, payloads of interest (immunotherapeutic agents), vectors, cells and compositions of the present invention may be used in conjunction with cancer vaccines. In one aspect, dendritic cells are modified to express the compositions of the invention and used as cancer vaccines.
[00372] In some embodiments, cancer vaccine may comprise peptides and/or proteins derived from tumor associated antigen (TAA). Such strategies may be utilized to evoke an immune response in a subject, which in some instances may be a cytotoxic T lymphocyte (cro response. Peptides used for cancer vaccines may also be modified to match the mutation profile of a subject. For example, EGFR derived peptides with mutations matched to the mutations found in the subject in need of therapy have been successfully used in patients with lung cancer (Li F et al. (2016) Oncoimmunology. Oct 7;5(12): e1238539; the contents of which are incorporated herein by reference in their entirety).
[00373] In one embodiment, cancer vaccines of the present invention may superagonist altered peptide ligands (APL) derived from TAAs. These are mutant peptide ligands deviate from the native peptide sequence by one or more amino acids, which activate specific CTL clones more effectively than native epitopes. These alterations may allow the peptide to bind better to the restricting Class I MHC molecule or interact more favorably with the TCR of a given tumor-specific CTL subset. APLs may be selected using methods taught in US Patent Publication NOS.
US20160317633A1, the contents of which are incorporated herein by reference in their entirety.
[00374] Relapse of hematologic malignancies is the primary cause of treatment failure after allogeneic hematopoietic stem cell transplantation (HC'T). The Wilm's tumor (WT1) gene product is a tumor associated antigen that is expressed in acute leukemia and other hematological malignancies, with limited expression in normal tissues. The compositions of the present invention may be co-administered with donor derived WT1 peptide loaded dendritic cell vaccine to prevent relapse of disease following immunotherapy (Shah NN et al. (2016) Biol Blood Marrow Transplant. 22(12):2149-215; the contents of which are incorporated herein by reference in their entirety.
3. Combination treatments 1003751 In some embodiments, it is desirable to combine compositions, vectors and cells of the invention for administration to a subject. Compositions of the invention comprising different immunotherapeutic agents may be used in combination or in conjunction with known immunotherapeutic agents for enhancement of immunotherapy.
[00376] In some embodiments, it is desirable to combine compositions of the invention with adjuvants, that can enhance the potency and longevity of antigen-specific immune responses.
Adjuvants used as inununostimulants in combination therapy include biological molecules or delivery carriers that deliver antigens. As non-limiting examples, the compositions of the invention may be combined with biological adjuvants such as cytokines, Toll Like Receptors, bacterial toxins, and/or saponins. In other embodiments, the compositions of the present invention may be combined with delivery carriers. Exemplary delivery carriers include, polymer microspheres, immune stimulating complexes, emulsions (oil-in-water or water-in-oil), aluminum salts, liposomes and virosomes.
1003771 In some embodiments, immune effector cells modified to express biocircuits, effector modules, DDs and payloads of the invention may be combined with the biological adjuvants described herein.

[00378] In some embodiments, immune effector cells modified to expressed biocircuits, effector modules, DDs and payloads of the invention may be combined with cancer vaccines.
[00379] In some embodiments, an effector module comprising a cytokine may be used in combination with an effector module encoding a safety switch or a regulatory switch.
[00380] In some embodiments, methods of the invention may include combination of the compositions of the invention with other agents effective in the treatment of cancers, infection diseases and other immunodeficient disorders, such as anti-cancer agents. As used herein, the term "anti-cancer agent" refers to any agent which is capable of negatively affecting cancer in a subject, for example, by killing cancer cells, inducing apoptosis in cancer cells, reducing the growth rate of cancer cells, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth; reducing the blood supply to a tumor or cancer cells, promoting an immune response against cancer cells or a tumor, preventing or inhibiting the progression of cancer, or increasing the lifespan of a subject with cancer.
[00381] In some embodiments, anti-cancer agent or therapy may be a chemotherapeutic agent, or radiotherapy, immunotherapeutic agent, surgery, or any other therapeutic agent which, in combination with the present invention, improves the therapeutic efficacy of treatment.
[00382] In some embodiments, compositions of the present invention may be used in combination with immunotherapeutics other than the inventive therapy described herein, such as antibodies specific to some target molecules on the surface of a tumor cell.
[00383] Exemplary chemotherapies include, without limitation, Acivicin;
Aclarubicin;
Acodazole hydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine;
Ambomycin;
Ametantrone acetate; Amsacrine; Anastrozole; Anthramycin; Asparaginase;
Asperrin, Sulindac, Curcumin, alkylating agents including: Nitrogen mustards such as mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas such as carmustine (BC U), lomustine (CCNU), and semustine (methyl-CC U);
thyleniminesimethylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethyhnelamine altretamine); alkyl sulfonates such as busulfan;
triazines such as dacarbazine (D'TIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrrolidine analogs such as 5- fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine), 5-azacytidine, 2,2'-difluorodeoxycidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin (pentostatin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (cladribine, 2- CdA); natural products including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate; epipodophylotoxins such as etoposide and teniposide;
antibiotics, such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mithrarnycin), mitomycinC, and actinomycin; enzymes such as L-asparaginase, cytokines such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, TNF-beta and GM-CSF, anti-angiogenic factors, such as angiostatin and endostatin, inhibitors of FGF or VEGF such as soluble forms of receptors for angiogenic factors, including soluble VGFNEGF receptors, platinum coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including N- methylhydrazine (MIFf) and procarbazine, adrenocortical suppressants such as mitotane (o,p'-DDD) and arninoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone/equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; non-steroidal antiandrogens such as flutamide; kinase inhibitors, histone deacetylase inhibitors, methylation inhibitors, proteasome inhibitors, monoclonal antibodies, oxidants, anti-oxidants, telomerase inhibitors, BH3 mimetics, ubiquitin ligase inhibitors, stat inhibitors and receptor tyrosin kinase inhibitors such as imatinib mesylate (marketed as Gleevac or Glivac) and erlotinib (an EGF receptor inhibitor) now marketed as Tarveca; anti-virals such as oseltarnivir phosphate, Amphotericin B, and palivizumab; Sdi 1 mimetics; Semustine; Senescence derived inhibitor 1; Sparfosic acid;
Spicamycin D;
Spiromustine; Splenopentin; Spongistatin 1; Squalamine; Stipiamide;
Stromelysin inhibitors;
Sulfmosine; Superactive vasoactive intestinal peptide antagonist; Velaresol;
Veramine; Verdins;
Verteporfm; Vinorelbine; Vinxaltine; Vitaxin; Vorozole; Zanoterone;
Zeniplatin; Zilascorb; and Zinostatin stimalamer; PI3Kil small-molecule inhibitor, GSK2636771; pan-PI3K
inhibitor (BKM120); BRAF inhibitors. Vemurafenib (Zelboraf) and dabrafenib (Taflnlar);
or any analog or derivative and variant of the foregoing.
1003841 Radiotherapeutic agents and factors include radiation and waves that induce DNA
damage for example, y-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, radioisotopes, and the like. Therapy may be achieved by irradiating the localized tumor site with the above described forms of radiations. It is most likely that all of these factors effect abroad range of damage DNA, on the precursors of DNA, the replication and repair of DNA, and the assembly and maintenance of chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200 roentgens for prolonged periods of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the half-life of the isotope, the strength and type of radiation emitted, and the uptake by the neoplastic cells.
1003851 In some embodiments, the chemotherapeutic agent may be an inununomodulatory agent such as lenalidomide (LEN). Recent studies have demonstrated that lenalidomide can enhance antitumor functions of CAR modified T cells (Otahal et al., Oncoimmunoingy, 2015, 5(4): el115940). Some examples of anti-tumor antibodies include tocilizumab, siltuximab.
1003861 Other agents may be used in combination with compositions of the invention may also include, but not limited to, agents that affect the upregulation of cell surface receptors and their ligands such as Fas/Fas ligand, DR4 or DR5/TRAIL and GAP junctions, cytostatic and differentiation agents, inhibitors of cell adhesion such as focal adhesion kinase (FAKs) inhibitors and Lovastatin, or agents that increase the sensitivity of the hyper proliferative cells to apoptotic inducers such as the antibody C225.
1003871 The combinations may include administering the compositions of the invention and other agents at the same time or separately. Alternatively, the present immunotherapy may precede or follow the other agent/therapy by intervals ranging from minutes, days, weeks to months.
1003881 In some embodiments, CAR-T cells of the invention may be co-administered with retinoids to eradicate myeloid derived suppressor cells. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of early myeloid progenitors, immature granulocytes, macrophages, and dendritic cells at different stages of differentiation. MDSCs have the capacity to suppress both the cytotoxic activities of natural killer (NK) and NKT
cells, and the adaptive immune response mediated by CD4+ and CD8+ T cells. Long AH et al. (2016) Cancer Immunol Res.;4(10):869-880 have described the co-treatment of CARs with all trans retinoic acid (A'TRA) for the successful treatment of solid tumors (the contents of which are incorporated by reference in its entirety).
1003891 Adjuvant therapy as used herein refers to the treatment that is given in addition to primary therapy to kill any cancer cells, even if the cancer is undetectable by standard laboratory tests. Experimental data have demonstrated that the lymphocyte depletion induced by cytotoxic regimens for the treatment of cancer could contribute to relapse. Relapse to cancer immunotherapy may be minimized by adjuvant immunotherapy. In some embodiments, adjuvant therapy may include the co-administration of recombinant human Interleukin2 in conjunction with dendritic cells pulsed with peptides derived from tumor cells. In some embodiments, dendritic cells pulsed with autologous tumor cell lysate and keyhole limpet hemocyanin (KLH).

Immune cells may be further depleted of CD25 positive T cells. Interleukin 7 may also be co-administered as immunotherapy. In some embodiments, the risk of reinfusing donor- derived tumor cells may be purged with monoclonal antibody 8H9, which interacts with tumor cell surface antigens. Any of the adjuvant therapy methods taught in Merchant et al. (2016), Clin Cancer Res. 1;22(13):3182-91 may be utilized (the contents of which are incorporated by reference in their entirety).
[00390] In some embodiments, compositions of the invention can be combined with CXCR2 inhibitors or anti - CXCR2 antibodies. Highfill SL et al. (2014) found that CXCR2 positive MDSC cells limit the efficacy of immunotherapy by mediating local immunosuppression (Highfill SL, et al. Sci Transl Med. 2014 May 21;6(237):237ra67; the contents of which are incorporated herein by reference in its entirety).
4. Diseases [00391] Provided in the present invention is a method of reducing a tumor volume or burden in a subject in need, the method comprising introducing into the subject a composition of the invention.
[00392] The present invention also provides methods for treating a cancer in a subject, comprising administering to the subject an effective amount of an immune effector cell genetically modified to express at least one effector module of the invention.
Cancer [00393] Various cancers may be treated with pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention. As used herein, the term "cancer" refers to any of various malignant neoplasms characterized by the proliferation of anaplastic cells that tend to invade surrounding tissue and metastasize to new body sites and also refers to the pathological condition characterized by such malignant neoplastic growths. Cancers may be tumors or hematological malignancies, and include but are not limited to, all types of lymphomas/leukemias, carcinomas and sarcomas, such as those cancers or tumors found in the anus, bladder, bile duct, bone, brain, breast, cervix, colon/rectum, endometrium, esophagus, eye, gallbladder, head and neck, liver, kidney, larynx, lung, mediastintun (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal marrow, tailbone, testicles, thyroid and uterus.
[00394] Types of carcinomas which may be treated with the compositions of the present invention include, but are not limited to, papilloma/carcinoma, choriocarcinoma, endodermal sinus tumor, teratoma, adenoma/adenocarcinoma, melanoma, fibroma, lipoma, leiomyoma, rhabdomyoma, mesothelioma, angioma, osteoma, chondroma, glioma, lymphoma/leukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinomas, basal cell carcinoma and sinonasal undifferentiated carcinoma.
[00395] Types of carcinomas which may be treated with the compositions of the present invention include, but are not limited to, soft tissue sarcoma such as alveolar soft part sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid tumor, desmoplastic small round cell tumor, extraskeletal chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma, hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma, malignant fibrous histiocytoma, neurofibrosarcoma, rhabdomyosarcoma, synovial sarcoma, and Askin's tumor, Ewing's sarcoma (primitive neuroectodermal tumor), malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and chondrosarcoma.
[00396] As a non-limiting example, the carcinoma which may be treated may be Acute granulocytic leukemia, Acute lymphocytic leukemia, Acute myelogenous leukemia, Adenocarcinoma, Adenosarcoma, Adrenal cancer, Adrenocortical carcinoma, Anal cancer, Anaplastic astrocytoma, Angiosarcoma, Appendix cancer, Astrocytoma, Basal cell carcinoma, B-Cell lymphoma), Bile duct cancer, Bladder cancer, Bone cancer, Bowel cancer, Brain cancer, Brain stem glioma, Brain tumor, Breast cancer, Carcinoid tumors, Cervical cancer, Cholangiocarcinoma, Chondrosarcoma, Chronic lymphocytic leukemia, Chronic myelogenous leukemia, Colon cancer, Colorectal cancer, Craniopharyngioma, Cutaneous lymphoma, Cutaneous melanoma, Diffuse astrocytoma, Ductal carcinoma in situ, Endometrial cancer, Ependymoma, Epithelioid sarcoma, Esophageal cancer, Ewing sarcoma, Extrahepatic bile duct cancer, Eye cancer, Fallopian tube cancer, Fibrosarcoma, Gallbladder cancer, Gastric cancer, Gastrointestinal cancer, Gastrointestinal carcinoid cancer, Gastrointestinal stromal tumors, General, Germ cell tumor, Glioblastoma multiforme, Glioma, Hairy cell leukemia, Head and neck cancer, Hemangioendothelioma, Hodgkin lymphoma, Hodgkin's disease, Hodgkin's lymphoma, Hypophalyneal cancer, Infiltrating ductal carcinoma, Infiltrating lobular carcinoma, Inflammatory breast cancer, Intestinal Cancer, lntrahepatic bile duct cancer, Invasive /
infiltrating breast cancer, Islet cell cancer, Jaw cancer, Kaposi sarcoma, Kidney cancer, Laryngeal cancer, Leiomyosarcoma. Leptomeningeal metastases, Leukemia, Lip cancer, Liposarcoma, Liver cancer, Lobular carcinoma in situ, Low-grade astrocytoma, Lung cancer, Lymph node cancer, Lymphoma, Male breast cancer, Medullary carcinoma, Medulloblastoma, Melanoma, Meningioma, Merkel cell carcinoma, Mesenchymal chondrosarcoma, Mesenchymous, Mesothelioma, Metastatic breast cancer. Metastatic melanoma, Metastatic squamous neck cancer, Mixed gliomas, Mouth cancer, Mucinous carcinoma, Mucosal melanoma, Multiple myeloma, Nasal cavity cancer, Nasophaiyngeal cancer, Neck cancer, Neuroblastoma, Neuroendocrine tumors, Non-Hodgkin lymphoma, Non-Hodgkin's lymphoma, Non-small cell lung cancer, Oat cell cancer, Ocular cancer, Ocular melanoma, Oligodendroglioma, Oral cancer, Oral cavity cancer, Orophary-ngeal cancer, Osteogenic sarcoma, Osteosarcoma, Ovarian cancer, Ovarian epithelial cancer, Ovarian germ cell tumor, Ovarian primary peritoneal carcinoma, Ovarian sex cord stromal tumor, Paget's disease, Pancreatic cancer, Papillary carcinoma, Paranasal sinus cancer, Parathyroid cancer, Pelvic cancer, Penile cancer, Peripheral nerve cancer, Peritoneal cancer, Pharyngeal cancer, Pheochromocytoma, Pilocytic astrocytoma, Pineal region tumor, Pineoblastoma, Pituitary gland cancer, Primary central nervous system lymphoma, Prostate cancer, Rectal cancer, Renal cell cancer, Renal pelvis cancer, Rhabdomyosarcoma, Salivary gland cancer, Sarcoma, Sarcoma, bone, Sarcoma, soft tissue, Sarcoma, uterine, Sinus cancer, Skin cancer, Small cell lung cancer, Small intestine cancer. Soft tissue sarcoma, Spinal cancer, Spinal column cancer, Spinal cord cancer. Spinal tumor, Squamous cell carcinoma, Stomach cancer, Synovial sarcoma, T-cell lymphoma), Testicular cancer, Throat cancer, 'Thymoma / thymic carcinoma, Thyroid cancer, Tongue cancer, Tonsil cancer, Transitional cell cancer, Transitional cell cancer, Transitional cell cancer, Triple-negative breast cancer, Tubal cancer, Tubular carcinoma, Ureteral cancer, Ureteral cancer, Urethral cancer, Uterine adenocarcinoma, Uterine cancer, Uterine sarcoma, Vaginal cancer, and Vulvar cancer.
1003971 In some embodiments, the CARS of the present invention may be a CAR
useful in the treatment of multiple myeloma such as a CS I CAR, a CD38 CAR, a CD138 CAR, and a BCMA
CAR. In some embodiments, the CARS of the present invention may be a CAR
useful in the treatment of acute myeloid leukemia such as a CD33 CAR, a CD123 CAR, and a CLL1 CAR. In some embodiments, the CARS of the present invention may be a CAR useful in the treatment of T cell leukemia such as a CD5 CAR, and a CD7 CAR. In some embodiments, the CARs of the present invention may be a CAR useful in the treatment of solid tumors such a mesothelin CAR, a GD2 CAR, a GPC3 CAR, a Her2 CAR, an EGFR CAR, a Mud l CAR, an EpCAM CAR, a PD-Li CAR, a CEA CAR, a Muc16 CAR, a CD133 CAR, a CD171 CAR, a CD70 CAR, a CLD18 CAR, a cMET CAR, a EphA2 CAR, a FAP CAR, a Folate Receptor CAR, an ILI3Ra2 CAR, an MG7 CAR, a PSMA CAR, a ROR1 CAR, and a VEGFR2 CAR.
infectious diseases 1003981 In some embodiment, biocircu its of the invention may be used for the treatment of infectious diseases. Biocircuits of the invention may be introduced in cells suitable for adoptive cell transfer such as macrophages, dendritic cells, natural killer cells, and or T cells. Infectious diseases treated by the biocircuits of the invention may be diseases caused by viruses, bacteria, fungi, and/or parasites. IL15-1L15Ra payloads of the invention may be used to increase immune cell proliferation and/or persistence of the immune cells useful in treating infectious diseases.
[00399] "Infection diseases" herein refer to diseases caused by any pathogen or agent that infects mammalian cells, preferably human cells and causes a disease condition. Examples thereof include bacteria, yeast, fungi, protozoans, mycoplasma, viruses, prions, and parasites.
Examples include those involved in (a) viral diseases such as, for example, diseases resulting from infection by an adenovirus, a herpesvirus (e.g., HSV-I, HSV-II, CMV, or VZV), a poxvirus (e-g-, an orthopoxvirus such as variola or vaccinia, or molluscum contagiosutn), a picomavirus (e.g., rhinovirus or enterovirus), an orthomyxovirus (e.g., influenzavirus), a paramyxovirus (e.g., parainfluenza virus, mumps virus, measles virus, and respiratory syricytial virus (RSV)), a coronavirus (e.g., SARS), a papovavirus (e.g., papillomaviruses, such as those that cause genital warts, common warts, or plantar warts), a hepadnavims (e.g., hepatitis B
virus), a flavivirus (e.g., hepatitis C virus or Dengue virus), or a retrovims (e.g., a lentivims such as HIV); (b) bacterial diseases such as, for example, diseases resulting from infection by bacteria of, for example, the genus Escherichia, Enterobacter. Salmonella, Staphylococcus. Shigella, Listeria, Aerobacter, Helicobacter, Klebsiella, Proteus, Pseudomonas, Streptococcus, Chlamydia, Mycoplasma, Pneumococcus, Neisseria, Clostridium, Bacillus, Corynebacterium, Mycobacterium.
Campylobacter. Vibrio, Serratia, Providencia, Chromobacterium, Brucella, Yersinia, Haemophilus, or Bordetella; (c) other infectious diseases, such chlamydia, fungal diseases including but not limited to candidiasis, aspergillosis, histoplasmosis, cryptococcal meningitis, parasitic diseases including but not limited to malaria, Pneumocystis carnii pneumonia, leishmaniasis, cryptosporidiosis, toxoplasmosis, and trypanosome infection and prions that cause human disease such as Creutzfeldt-Jakob Disease (CJD), variant Creutzfeldt-Jakob Disease (vCJD), Gerstmann-Straiissler-Scheinker syndrome, Fatal Familial Insomnia and kuru.
5. Microbiome 1004001 Alterations in the composition of the microbiome may impact the action of anti-cancer therapies. A diverse community of symbiotic, commensal and pathogenic microorganisms exist in all environmentally exposed sites in the body and is herein referred to as the "Microbiome."
Environmentally exposed sites of the body that may be inhabited by a microbiome include the skin, nasopharynx, the oral cavity, the respiratory tract, the gastrointestinal tract, and the reproductive tract.
[00401] In some embodiments, microbiome native or engineered with immunotherapeutic agents may be used to improve the efficacy of the anti-cancer immunotherapies.
Methods of using microbiome to improve responsive to immunotherapeutic agents have been described by Sivan et al. (Sivan A., et al.2015. Science; 350:1084-9; the contents of which are incorporated herein by reference in their entirety). In other embodiments, the microorganisms may be delivered along with immunotherapeutic compositions of the present invention to improve the efficacy of immunotherapy.
6. Tools and agents for making therapeutics [00402] Provided in the present invention are tools and agents that may be used in generating immunotherapeutics for reducing a tumor volume or burden in a subject in need.
A considerable number of variables are involved in producing a therapeutic agent, such as structure of the payload, types of cell, methods of gene transfer, method and time of ex vivo expansion, pre-conditioning and the amount and type of tumor burden in the subject. Such parameters may be optimized using tools and agents described herein.
Cell lines [00403] The present disclosure provides a mammalian cell that has been genetically modified with the compositions of the invention. Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include; but are not limited to Huinan embryonic kidney cell line 293, fibroblast cell line N1H 3T3, human colorectal carcinoma cell line HCT116, ovarian carcinoma cell line SKOV-3, immortalized T cell lines Jurkat cells and SupT1 cells, lymphoma cell line Raji cells, NALM-6 cells, K562 cells, HeLa cells, PC12 cells, HL-60 cells, NK cell lines (e.g., NKL, NK92, NK962, and YTS), REH, SEM; KOPN8, Daudi, Raji, and the like. In some instances, the cell is not an immortalized cell line, but instead a cell obtained from an individual and is herein referred to as a primary cell. For example, the cell is a T lymphocyte obtained from an individual. Other examples include, but are not limited to cytotoxic cells, stem cells, peripheral blood mononuclear cells or progenitor cells obtained from an individual.
Tracking SREs, biocircuits and cell lines [00404] In some embodiments, it may be desirable to track the compositions of the invention or the cells modified by the compositions of the invention. Tracking may be achieved by using payloads such as reporter moieties, which, as used herein, refers to any protein capable of creating a detectable signal, in response to an input. Examples include alkaline phosphatase, (3-galactosidase; chloramphenicol acetyltransferase, fi-glucuronidase, peroxidase, 0-lactamase, catalytic antibodies, bioluminescent proteins e.g. luciferase, and fluorescent proteins such as Green fluorescent protein (GFP).

[00405] Reporter moieties may be used to monitor the response of the DD upon addition of the ligand corresponding to the DD. In other instances, reporter moieties may be used to track cell survival, persistence, cell growth, and/or localization in vitro, in vivo, or ex vivo.
[00406] In some embodiments, the preferred reporter moiety may be luciferase proteins. In one embodiment, the reporter moiety is the Renilla luciferase, or a firefly luciferase. Table 14 provides the sequences of the reporter moieties. The amino acid sequences in Table 14 may comprise a stop codon which is denoted in the table with a "s" at the end of the amino acid sequence Table 14: DD-Iticiferase constructs Description Amino acid sequence Amino Nucleic Acid SEQ Acid SEQ
ID NO. ID NO.
Linker EF oAarre.
Linker SG AGCGGC
Renilla MTSKVYDPEQRICRIATFGPQWWARCKQKNVLDSFIN 209 217 luciferase YYDSEKHAENAVIFLHGNAASSYLWRHVVPH1EPVA
RCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELL

VI/DVIESWDEWPDIEEDIALIKSEEGEICMVLENNFFV

PREIFLVICGGKPDVVQIVRNYNAYLRASDDLPICMFIE
SDPGETSNAIVEGAICKFPNTEFVKVKGLHFSQEDAPD

Firefly MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMK RY AL 210 218 Luciferase VPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGL
NTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDW

KIIEVIDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPES

VVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKST
LIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGI

NALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKY

AAVVVLEHGKTMTEICEIVDYVASQVTTAKICLRGGV

FKBP (F36V, GVQVETISPGDGRTFPICRGQTCVVHYTGMLEDGKK 11 60, 878-LIO6P) VDSSRDRNKPFICFMLGKQEVIRGWEEGVAQMSVGQ 882 ____________ RAKLTISPDYAYGATGHPGIIPPHATINFDVELLKPE
FKBP (E31G, (3VQVETISPGDGRIFPKRGQICVVHYTGMLGDGICK 12 88, 883-F36V, R7 1G, VDSSRDRNKPFKFMLGKQEV1RGWEEGVAQMSVGQ 889 K105E) GAKLTESPDYAYGATGHPG1 EITHATLVFDVELLELE
eeDHFR MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNT 8 89 (R12Y, LNKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVT
Y100I) WVKSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQK
LYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADA
QNSHSYCFEILERR
OT-Rluc-001 MTSKVYDPEQRKRMITGPQWWARCKQKWLDSFIN 211 2t).3 (Renilla Luc - YYDSEKHAENAVIFLHGNAASSYLWRHVVPH1EPVA
stop) RCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELL
NLPKKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAES

VVDVIESWDEWPDIEEDIALIKSEEGEKMVLENNFFV
ETMLPSICIMRKLEPEEFAAYLEPFKEKGEVRR PTL S
PREIPL VKGGKPDVVQIVRNYNAYLR A SDDLPK MEI E
SDPGFF SNA IVEG AKKFPNTEFVKVKGLHFSQED APD
EMGKYIK SFVERVLKNEQ*
OT-F 1 uc-002 MGVQVETISPGDGRTFPKRGQTCVVHYTGML EDGK 212 204 (Met - FKBP KVDSSRDRNKPFICFMLGKQEVIRGWEEGVAQMSVG
(F36V, QRAKLTISPDYAY G ATGHPGI1PPH A TINFD VELLKPE
L106P) ¨ EIMEDAKNIKKGPAPFYPLEDGIAGEQLHKAMKRY
Linker (EF) ¨ ALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRY
Firefly Luc - GLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPAN
stop) DIY NERELLN SMGISQPTVVFVSICKGLQKILNVQICKL
PIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDF
VPESFDRUKTIALIMNSSGSTGLPKGVALPHRTACVR
FSHARDPIFGNQ IIPDTA SVVITHHGEGMETTLGYLI
CGFRVVLMYR FEEELFLRSLQDYK IQ SAL L VPTLF SFF
AKSTLIDKYDLSNLHELASGGAPLSKEVGEAVAKREH
LPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFE
AKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNP
EATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLI

ELPA AVVVLEHGKTMTEKEIVDY VA SQVTTAKKLR
GGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKSK
OT-Rluc-003 MGVQVE11SPGDGRTF PK RGQTCVVHYTGMLEDGK 213 205 (Met - FKBP KVDSSRDRNKPFKFMLGKQEV1RGWEEGVAQMSVG
(F36V, QR AK LTISPDYA YGATGHPGIIPPHATL VFDVELLKPE
L106P) ¨ SGTSKVYDPEQRKRMITGPQWWAR CKQMNVLDSFI
Linker (SG) - NYYDSEKHAENAVIELHGNAASSYLWRHVVPHIEPV
Amino acid 2 ARCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFE
¨311 of LLNLPKKIIFVGHDWGACLAFHYSYEHQDICHCAIVHA
Renilla ESVI/DVIESWDEWPDIEEDIALIKSEEGEKMVLENNE
Luciferase ¨ FVETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTL
stop) SWPREIPLVKGGKPDVVQIVRNYNAYLRASDDLPKM
FIESDPGFFSNAIVEGAKKFPNTEFVKVKGLHFSQED
APDEMGKYIKSFVERVLKNEQ*
OT-Rluc-004 MISLIAALAVDYVIGMENAMPWNLPADLAWFKRNT 214 206 (ecDHFR LNKPVIMGRHTWE SIGRPLPGRK N 'IL SSQPGTDDRVT
(R 12Y, WVK S VDEA I AACGDVPEIMVIGGGRVIEQFLPKAQK
Y100I) ¨ LYLTHIDAEVEGDTHFPDYEPDDWESVESEEHDADA
Linker (SG) - QNSHSYCFEILERRSGTSKVYDPEQRKRMITGPQWW
Amino acid 2 ARCKQMNVLDSFINYYDSEKHAENAVIFLHGNAASS
¨311 of YLWRHVVPHIEPVARCI1PDLIGMGKSGKSGNGSYRL
Renilla LDHYKYLTAWFELLNLPKKIIFVGHDWGACLAFHYS
Luciferase - YEHQDK1KAIVHAESVI/DVIESWDEWPDIEEDIAL1KS
stop) EEGEKMVLENNFFVETMLPSKIMRICLEPEEFAAYLEP
FKEKGEVRRPTLSWPREIPLVKGGKPDVVQIVRNYN
AYLRASDDLPKMFIESDPGFFSNAIVEGAKKFPNTEF
VKVKGLHFSQEDAPDEMGKYI1CSFVERVLKNEQ*
OT-Rhic-005 MTSKVYD PEQRKR MITGPQWWARCKQMNVLDSFIN 215 207 (Renilla Luc - YYDSEKHAENAVIFLHGNAASSYLWRHVVPHIEPVA
Linker (SG) - RCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELL
FKBP (E31G, NLPICKIIFVGHDWGACLAFHYSYEHQDKIKAIVHAES
F36V, R71G, VVDVIESWDEWPDIEEDIAL1KSEEGEKMVLENNFEV
K105E) - ETMLPSKIMRKLEPEEFAAYLEPFKEKGEVRRPTLSW
stop) PREIPLVKGGKPDVVQIVRNYNAYLRASDDLPKMFIE
SDPGFFSNAIVEGAKKFPNTEFVKVKGLHFSQEDAPD

RGQTCVVHYTGMLGDGKKVDSSRDRNICPFKFMLGK _____________ QEVIRGWEEGVAQMSVGQGAKLIISPDYAYGATGH
PGI1PPHATLVFDVELLELE*
OT-R1uc-006 MTSKVYDPEQRKRMITGPQWWARCKQIVLNVLDSFIN 216 208 (Renilla Luc - YYDSEKHAENAVIFLHGNAASSYLWRHVVPHIEPVA
Linker (SG) - RCIIPDLIGMGKSGKSGNGSYRLLDHYKYLTAWFELL
ecDHFR NLPICKBFVGHDWGACLAFHYSYEHQDKIKAIVHAES
(Amino acid VVDVIESWDEWPDIEEDIALIKSEEGEKMVLENNFFV
2-159 of WT) ETMLPSICIMRKLEPEEFAAYLEPFICEKGEVRRPTLSW
(R12Y, PRE IPL VKGGKPDVVQIVRNYNAYLRASDDLPICMFIE
Y1001) - SDPGFFSNAIVEGAKICFPNTEFVICVKGLHFSQEDAPD
stop) EMGKYIKSFVERVLKNEQSGISLIAALAVDYVIGMEN
AMPWNLPADLAWFKRNTLNICPVIMGRHTWESIGRP
LPGRKNIILSSQPGIDDRVIWVKSVDEAIAACGD VP E

YEPDDWESVFSEFHDADAQNSHSYCFEILERR*
Animal models 1004071 The utility and efficacy of the compositions of the present invention may be tested in vivo animal models, preferably mouse models. Mouse models used to may be syngeneic mouse models wherein mouse cells are modified with compositions of the invention and tested in mice of the same genetic background. Examples include pMEL-1 and 4T1 mouse models.
Alternatively, xenograft models where human cells such as tumor cells and immune cells are introduced into immunodeficient mice may also be utilized in such studies.
Immunodeficient mice used may be CByj.Cg-Foxnlnu/J, B6;129S7-RaglmilmmIJ, B6.129S7-Rag/im/mm/J, B6.
CB17-PrkdcwialSzJ, NOD.129S7(B6)-Rag/"Imma, NOD.Cg-Raglft"1"ffinPrflni zisdziszt NOD.CB17-Prkdc-ccid,oa, NOD.Cg-PrkdeidB2mwilunclJ, NOD-scid IL2Rgnum, Nude (nu) mice, SCID mice, NOD mice, RAG1/RAG2 mice, NOD-Scid mice, IL2rgnu// mice,b2mnull mice, NOD-scid IL2rfnull mice, NOD-scid-B2mnull mice, and HLA transgenic mice.
Cellular assays [00408] In some embodiments, the effectiveness of the compositions of the inventions as immunodierapeutic agents may be evaluated using cellular assays. Levels of expression and/or identity of the compositions of the invention may be determined according to any methods known in the art for identifying proteins and/or quantitating proteins levels.
In some embodiments, such methods may include Western Blotting, flow cytometry, and immunoassays.
[00409] Provided herein are methods for functionally characterizing cells expressing SRE, biocircuits and compositions of the invention. In some embodiments, functional characterization is carried out in primary immune cells or immortalized immune cell lines and may be determined by expression of cell surface markers. Examples of cell surface markers for T
cells include, but are not limited to, CD3, CD4, CD8, CD 14, CD20, CD! lb, CD16, CD45 and HLA-DR, CD 69, CD28, CD44, IFNganuna, PD!, TIM3, LAG3. Examples of cell surface markers for antigen presenting cells include, but are not limited to, NIHC class I. MHC Class 11, CD40, CD45, B7-1, B7-2, IFN-y receptor and 1L2 receptor, ICAM-1 and/or Fey receptor. Examples of cell surface markers for dendritic cells include, but are not limited to, MHC class I, MHC
Class II, B7-2, CD18, CD29, CD31, CD43, CD44, CD45, CD54, CD58, CD83, CD86, CMRF-44, CMRF-56, DCIR and/or Dectin-1 and the like; while in some cases also having the absence of CD2, CD3, CD4, CD8, CD14, CD15, CD16, CD 19, CD20, CD56, and/or CD57. Examples of cell surface markers for NK cells include, but are not limited to, CCL3, CCL4, CCL5, CCR4, CXCR4, CXCR3, NKG2D, CD71, CD69, CCR5, Phospho JAK/STAT, phospho ERK, phospho p38/
MAPK, phospho AKT, phospho STAT3, Granulysin, Granzyme B, Granzyme K, ILI , 1L22, IFNg, LAP, Perforin, and TNFa.
V. DELIVERY MODALITIES AND/OR VECTORS
Vectors [00410] The present invention also provides vectors that package polynucleotides of the invention encoding biocircuits, effector modules, SREs (DDs) and payload constructs, and combinations thereof. Vectors of the present invention may also be used to deliver the packaged polynucleotides to a cell, a local tissue site or a subject. These vectors may be of any kind, including DNA vectors, RNA vectors, plasmids, viral vectors and particles.
Viral vector technology is well known and described in Sambrook et at (2001, Molecular Cloning: A
Laboratory Manual, Cold Spring Harbor Laboratory, New York). Viruses, which are useful as vectors include, but are not limited to lentiviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, herpes simplex viral vectors, retroviral vectors, oncolytic viruses, and the like.
[00411] In general, vectors contain an origin of replication functional in at least one organism, a promoter sequence and convenient restriction endonuclease site, and one or more selectable markers e.g. a drug resistance gene.
[00412] As used herein a promoter is defined as a DNA sequence recognized by transcription machinery of the cell, required to initiate specific transcription of the polynucleotide sequence of the present invention. Vectors can comprise native or non-native promoters operably linked to the polynucleotides of the invention. The promoters selected may be strong, weak, constitutive, inducible, tissue specific, development stage-specific, and/or organism specific. One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of polynucleotide sequence that is operatively linked to it.
Another example of a preferred promoter is the Elongation Growth Factor-1. Alpha (EF-1. alpha) promoter. Other constitutive promoters may also be used, including, but not limited to simian virus 40 (5V40) promoter, the mouse mammary tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) promoter, long terminal repeat (LTR), promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter as well as human gene promoters including, but not limited to the phosphoglycerate kinase (PGK) promoter, the actin promoter, the myosin promoter, the hemoglobin promoter, the Ubiquitin C
(Ubc) promoter, the human U6 small nuclear protein promoter and the creatine kinase promoter.
In some instances, inducible promoters such as but not limited to the metallothionine promoter, the glucocorticoid promoter, the progesterone promoter, and the tetracycline promoter may be used.
In some embodiments, the promoter may be selected from the SEQ ID NO. 220-222, SEQ ID
NO. 836.
[00413] In some embodiments, the optimal promoter may be selected based on its ability to achieve minimal expression of the SREs and payloads of the invention in the absence of the ligand and detectable expression in the presence of the ligand.
[00414] Additional promoter elements e.g. enhancers may be used to regulate the frequency of transcriptional initiation. Such regions may be located 10-100 base pairs upstream or downstream of the start site. In some instances, two or more promoter elements may be used to cooperatively or independently activate transcription.
[00415] Suitable vectors include those designed for propagation and expansion or for expression or both, such as plasmids and viruses. The vector can be selected from the group consisting of the pUC series (Fermentas Life Sciences, Glen Burnie, MD), the pBluescript series (Stratagene, La Jolla, CA), the pET series (Novagen, Madison, WI), the pGEX
series (Pharmacia Biotech, Uppsala, Sweden), and the pEX series (Clontech, Palo Alto, CA).
Bacteriophage vectors, such as XGT10, XGT1 1, A.ZapII (Stratagene), XEMBL4, and XNM1 149, also can be used. Examples of plant expression vectors include pBI01, pBI 1 01.2, pBT101.3, pBI 121 and pBI1sT19 (Clontech). Examples of animal expression vectors include pEUK-C1, pMAM, and pMAMneo (Clontech). The recombinant expression vector may be a viral vector, e.g., a retroviral vector or a lenti viral vector. In some embodiments, the vector can be a transposon.
Any of the vectors disclosed in the International Patent Publication W02014065961, may be useful in the present invention (the contents of which are incorporated herein by reference in their entirety).
[00416] In some embodiments, the recombinant expression vector may comprise regulatoiy sequences, such as transcription and translation initiation and termination codons, which are specific to the type of the host cell into which the vector is to be introduced.

1. Lentiviral vectors [00417] In some embodiments, lentiviral vectors/particles may be used as vehicles and delivery modalities. Lentiviruses are subgroup of the Retroviridae family of viruses, named because reverse transcription of viral RNA genomes to DNA is required before integration into the host genome. As such, the most important features of lentiviral vehicles/particles are the integration of their genetic material into the genome of a target/host cell. Some examples of lentivirus include the Human Immunodeficiency Viruses: HIV-1 and HIV-2, the Simian Immunodeficiency Virus (SIV), feline immunodeficiency virus (Fly), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), equine infectious anemia virus, visna-maedi and caprine arthritis encephalitis virus (CAEV).
[00418] Typically, lentiviral particles making up the gene delivery vehicle are replication defective on their own (also referred to as "self-inactivating"). Lentiviruses are able to infect both dividing and non-dividing cells by virtue of the entry mechanism through the intact host nuclear envelope (Naldini L et al., Curr. Opin. Biotechnol, 1998, 9: 457-463).
Recombinant lentiviral vehicles/particles have been generated by multiply attenuating the HIV virulence genes, for example, the genes Env, Vif, Vpr, Vpu, Nef and Tat are deleted making the vector biologically safe. Correspondingly, lentiviral vehicles, for example, derived from HIV-1/HIV-2 can mediate the efficient delivery, integration and long-term expression of transgenes into non-dividing cells. As used herein, the term "recombinant" refers to a vector or other nucleic acid containing both lentiviral sequences and non-lentiviral retroviral sequences.
[00419] Lentiviral particles may be generated by co-expressing the virus packaging elements and the vector genome itself in a producer cell such as human HEK293T cells.
These elements are usually provided in three (in second generation lentiviral systems) or four separate plasmids (in third generation lentiviral systems). The producer cells are co-transfected with plasmids that encode lentiviral components including the core (i.e. structural proteins) and enzymatic components of the virus, and the envelope protein(s) (referred to as the packaging systems), and a plasmid that encodes the genome including a foreign transgene, to be transferred to the target cell, the vehicle itself (also referred to as the transfer vector). In general, the plasmids or vectors are included in a producer cell line. The plasmids/vectors are introduced via transfection, transduction or infection into the producer cell line. Methods for transfection, transduction or infection are well known by those of skill in the art. As non-limiting example, the packaging and transfer constructs can be introduced into producer cell lines by calcium phosphate transfection, lipofection or electroporation, generally together with a dominant selectable marker, such as neo, DHFR, Gin synthetase or ADA, followed by selection in the presence of the appropriate drug and isolation of clones.
[00420] The producer cell produces recombinant viral particles that contain the foreign gene, for example, the effector module of the present invention. The recombinant viral particles are recovered from the culture media and titrated by standard methods used by those of skill in the art. The recombinant lentiviral vehicles can be used to infect target cells.
[00421] Cells used to produce high-titer lentiviral particles may include, but are not limited to, HEK293T cells, 293G cells, STAR cells (Relander et al., MoL Ther., 2005, 11:
452-459), FreeStyleTM 293 Expression System (ThennoFisher, Waltham, MA), and other HEK293T-based producer cell lines (e.g., Stewart et al., Hum Gene Ther. 2011, 22(3):357-369;
Lee et al., Biotechnol Bioeng, 2012, 10996): 1551-1560; Throm et al., Blood. 2009, 113(21): 5104-5110;
the contents of each of which are incorporated herein by reference in their entirety).
[00422] In some aspects, the envelope proteins may be heterologous envelop proteins from other viruses, such as the G protein of vesicular stomatitis virus (VSV G) or baculoviral gp64 envelop proteins. The VSV-G glycoprotein may especially be chosen among species classified in the vesiculovirus genus: Carajas virus (CJSV), Chandipura virus (CHPV), Cocal virus (COCV), Isfahan virus (ISFV). Maraba virus (MARAV), Piry virus (P1RYV), Vesicular stomatitis Alagoas virus (VSAV), Vesicular stomafitis Indiana virus (VSIV) and Vesicular stomatitis New Jersey virus (VSNJV) and/or stains provisionally classified in the vesiculovirus genus as Grass carp rhabdovirus, BeAn 157575 virus (BeAn 157575), Boteke virus (BTKV), Calchaqui virus (CQIV), Eel virus American (EVA), Gray Lodge virus (GLOV),Jurona virus (JURY), Klamath virus (ICLAV), Kwatia virus (KWAV), La Joya virus (LJV), Malpais Spring virus (MSPV), Mount Elgon bat virus (MEBV). Perinet virus (PERV), Pike .fry rhabdovirus (PFRV), Porton virus (PORV), Radi virus (RADIV), Spring viremia of carp virus (SVCV), Tupaia virus (TUPV), Ulcerative disease rhabdovirus (UDRV) and Yug Bogdanovac virus (YBV).
The gp64 or other baculoviral env protein can be derived from Autographa californica nucleopolyhedrovirus (AcMNPV), Anagrapha falcifera nuclear polyhedrosis virus, Bombyx mori nuclear polyhedrosis virus, Choristoneura fitmiferana nucleopolyhedrovirus, Orgyia pseudotsugata single capsid nuclear polyhedrosis virus, Epiphyas posivittana nucleopolyhedrovirus, Hyphantria cunea nucleopolyhedrovirus, Galleria mellonella nuclear polyhedrosis virus, Dhori virus, Thogoto virus, Antheraea pemyi nucleopolyhedrovirus or Batken virus.
[00423] Additional elements provided in lentiviral particles may comprise retroviral LTR (long-terminal repeat) at either 5' or 3' tenninus, a retroviral export element, optionally a lentiviral reverse response element (RRE), a promoter or active portion thereof; and a locus control region (LCR) or active portion thereof. Other elements include central polypurine tract (cPPT) sequence to improve transduction efficiency in non-dividing cells, Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) which enhances the expression of the transgene, and increases titer. The effector module is linked to the vector.
[00424] Methods for generating recombinant lentiviral particles are discussed in the art, for example, U.S. Pat. NOs. 8, 846, 385; 7,745, 179; 7,629,153; 7,575,924; 7,179, 903; and 6, 808, 905; the contents of each of which are incorporated herein by reference in their entirety.
[00425] Lentivirus vectors used may be selected from, but are not limited to pLVX, pLenti, pLenti6, pLJM1, FUGW, pWPXL, pWPI, pLenti CMV puro DEST, pLIM1-EGFP, pULTRA, pInducer20, pHIV-EGFP, pCW57.1, pTRPE, pELPS, pRRL, and pLionII.
[00426] Lentiviral vehicles known in the art may also be used (See, U.S. Pat.
NOs. 9; 260, 725;
9,068,199: 9,023,646: 8,900,858; 8,748,169; 8,709,799; 8,420,104; 8,329,462;
8,076,106;
6,013,516: and 5,994,136; International Patent Publication NOS. W02012079000;
the contents of each of which are incorporated herein by reference in their entirety).
2. Retroviral vectors (7-retroviral vectors) [00427] in some embodiments, retroviral vectors may be used to package and deliver the biocircuits, biocircuit components, effector modules, SREs or payload constructs of the present invention. Retroviral vectors (RVs) allow the permanent integration of a transgene in target cells.
In addition to lentiviral vectors based on complex HIV-1/2, retroviral vectors based on simple gamma-retroviruses have been widely used to deliver therapeutic genes and demonstrated clinically as one of the most efficient and powerful gene delivery systems capable of transducing a broad range of cell types. Example species of Gamma retroviruses include the murine leukemia viruses (MLVs) and the feline leukemia viruses (FeLV).
[004281 In some embodiments, gamma-retroviral vectors derived from a mammalian gamma-retrovirus such as murine leukemia viruses (MLVs), are recombinant. The MLV
families of gamma retroviruses include the ecotropic, amphotropic, xenotropic and polytropic subfamilies.
Ecotropic viruses can infect only murine cells using mCAT-1 receptor. Examples of ecotropic viruses are Moloney MLV and AKV. Amphotropic viruses infect murine, human and other species through the Pit-2 receptor. One example of an amphotropic virus is the 4070A virus.
Xenotropic and polytropic viruses utilize the same (Xprl) receptor, but differ in their species tropism. Xenotropic viruses such as NZB-9-1 infect human and other species but not murine species, whereas polytropic viruses such as focus-forming viruses (MCF) infect murine, human and other species.

[00429] Gamma-retroviral vectors may be produced in packaging cells by co-transfecting the cells with several plasmids including one encoding the retroviral structural and enzymatic (gag-pol) polyprotein, one encoding the envelope (env) protein, and one encoding the vector mRNA
comprising polynucleotide encoding the compositions of the present invention that is to be packaged in newly formed viral particles.
1004301 In some aspects, the recombinant gamma-retroviral vectors are pseudotyped with envelope proteins from other viruses. Envelope glycoproteins are incorporated in the outer lipid layer of the viral particles which can increase/alter the cell tropism.
Exemplary envelop proteins include the gibbon ape leukemia virus envelope protein (GALV) or vesicular stomatitis virus G
protein (VSV-G), or Simian endogenous retrovirus envelop protein, or Measles Virus H and F
proteins, or Human immunodeficiency virus gp120 envelope protein, or cocal vesiculovirus envelop protein (See, e.g., U.S. application publication NOS. 2012/164118; the contents of which are incorporated herein by reference in its entirety). In other aspects, envelope glycoproteins may be genetically modified to incorporate targeting/binding ligands into gamma-retroviral vectors, binding ligands including, but not limited to, peptide ligands, single chain antibodies and growth factors (Waehler et al., Nat. Rev. Genet. 2007, 8(8):573-587; the contents of which are incorporated herein by reference in its entirety). These engineered glycoproteins can retarget vectors to cells expressing their corresponding target moieties. In other aspects, a "molecular bridge" may be introduced to direct vectors to specific cells. The molecular bridge has dual specificities: one end can recognize viral glycoproteins, and the other end can bind to the molecular determinant on the target cell. Such molecular bridges, for example ligand-receptor, avidin-biotin, and chemical conjugations, monoclonal antibodies and engineered fusogenic proteins, can direct the attachment of viral vectors to target cells for transduction (Yang et al., Biotechnot Bioeng., 2008, 101(2): 357-368; and Maetzig et al., Viruses, 2011, 3, 677-713; the contents of each of which are incorporated herein by reference in their entirety).
[00431] in some embodiments, the recombinant gamma-retroviral vectors are self-inactivating (SIN) gammaretroviral vectors. The vectors are replication incompetent. SIN
vectors may harbor a deletion within the 3' U3 region initially comprising enhancer/promoter activity. Furthermore, the 5' U3 region may be replaced with strong promoters (needed in the packaging cell line) derived from Cytomegalovirus or RSV, or an internal promoter of choice, and/or an enhancer element. The choice of the internal promoters may be made according to specific requirements of gene expression needed for a particular purpose of the invention.
[00432] in some embodiments, polynucleotides encoding the biocircuit, biocircuit components, effector module, and SRE are inserted within the recombinant viral genome. The other components of the viral mRNA of a recombinant gamma-retroviral vector may be modified by insertion or removal of naturally occurring sequences (e.g., insertion of an IRES, insertion of a heterologous polymicleotide encoding a polypeptide or inhibitory nucleic acid of interest, shuffling of a more effective promoter from a different retrovirus or virus in place of the wild-type promoter and the like). In some examples, the recombinant gamma-retroviral vectors may comprise modified packaging signal, and/or primer binding site (PBS), and/or 5'-enhancer/promoter elements in the U3-region of the 5'- long terminal repeat (LTR), and/or 3'-SIN elements modified in the U3-region of the 3'-LTR. These modifications may increase the titers and the ability of infection.
[00433] Gamma retroviral vectors suitable for delivering biocircuit components, effector modules, SREs or payload constructs of the present invention may be selected from those disclosed in U.S. Pat. NOs. 8,828,718; 7,585,676; 7,351,585; U.S. application publication NOS.
2007/048285; PCT application publication NOs. W02010/113037; W02014/121005:
W02015/056014; and EP Pat. NOs. EP1757702; EP1757703 (the contents of each of which are incorporated herein by reference in their entirety).
3. Adeno-associated viral vectors (AAV) [00434] In some embodiments, polynucleotides of present invention may be packaged into recombinant adeno-associated viral (rAAV) vectors. Such vectors or viral particles may be designed to utilize any of the known serotype capsids or combinations of serotype capsids. The serotype capsids may include capsids from any identified AAV serotypes and variants thereof, for example, AAV1, AAV2, AAV2G9, AAV3, AAV4, AAV4-4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12 and AAVrh10.
[00435] In one embodiment, the AAV serotype may be or have a sequence as described in United States Publication No. US20030138772, herein incorporated by reference in its entirety, such as, but not limited to, AAVI (SEQ ID NO. 6 and 64 of US20030138772), AAV2 (SEQ ID
NO. 7 and 70 of U520030138772), AAV3 (SEQ ID NO. 8 and 71 of U520030138772), (SEQ ID NO. 63 of US20030138772), AAV5 (SEQ ID NO. 114 of U520030138772), AAV6 (SEQ ID NO. 65 of US20030138772), AAV7 (SEQ ID NO. 1-3 of US20030138772), AAV8 (SEQ ID NO. 4 and 95 of US20030138772), AAV9 (SEQ ID NO. 5 and 100 of U520030138772), AAVIO (SEQ ID NO. 117 of U520030138772), AAVI1 (SEQ ID NO. 118 of U520030138772), AAV12 (SEQ ID NO. 119 of U520030138772), AAVrh10 (amino acids 1 to 738 of SEQ ID NO. 81 of US20030138772) or variants thereof. Non-limiting examples of variants include SEQ ID NOs. 9, 27-45, 47-62, 66-69, 73-81, 84-94, 96, 97, 99, 101-113 of US20030138772, the contents of which are herein incorporated by reference in their entirety.

[00436] In one embodiment, the AAV serotype may have a sequence as described in Pulicherla et al. (Molecular Therapy, 2011, 19(6):1070-1078), U.S. Pat. NOs. 6,156,303:
7,198,951; U.S.
Patent Publication NOs. US2015/0159173 and US2014/0359799; and International Patent Publication NOs. W01998/011244, W02005/033321 and W02014/14422; the contents of each of which are incorporated herein by reference in their entirety.
[00437] AAV vectors include not only single stranded vectors but self-complementary AAV
vectors (scAAVs). scAAV vectors contain DNA which anneals together to form double stranded vector genome. By skipping second strand synthesis, scAAVs allow for rapid expression in the cell.
[00438] The rAAV vectors may be manufactured by standard methods in the art such as by triple transfection, in sf9 insect cells or in suspension cell cultures of human cells such as HEK293 cells.
[00439] The biocircuits, biocircuit components, effector modules, SREs or payload constructs may be encoded in one or more viral genomes to be packaged in the AAV capsids taught herein.
[00440] Such vectors or viral genomes may also include, in addition to at least one or two ITRs (inverted terminal repeats), certain regulatory elements necessary for expression from the vector or viral genome. Such regulatory elements are well known in the art and include for example promoters, introns, spacers, stuffer sequences, and the like.
[00441] In some embodiments, more than one effector module or SRE (e.g. DD) may be encoded in a viral genome.
4. Oncolvtic viral vector 1004421 In some embodiments, polynucleotides of present invention may be packaged into oncolytic viruses, such as vaccine viruses. Oncolytic vaccine viruses may include viral particles of a thymidine kinase (TK)-deficient, granulocyte macrophage (GM)-colony stimulating factor (CSF)-expressing, replication-competent vaccinia virus vector sufficient to induce oncolysis of cells in the tumor (e.g., US Pat. NOS. 9,226,977; the contents of which are incorporated by reference in their entirety).
5. Messenger RNA (mRNA) [00443] In some embodiments, the effector modules of the invention may be designed as a messenger RNA (mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded poly:peptide of interest in vitro, in vivo, in situ or ex vivo. Such mRNA
molecules may have the structural components or features of any of those taught in International Application number PCT/US2013/030062, the contents of which are incorporated herein by reference in its entirety.
[00444] Polynucleotides of the invention may also be designed as taught in, for example, Ribostem Limited in United Kingdom patent application serial number 0316089.2 filed on July 9, 2003 now abandoned, PCT application number PCT/GB2004/002981 filed on July 9, 2004 published as W02005005622, United States patent application national phase entry serial number 10/563,897 filed on June 8, 2006 published as US20060247195 now abandoned, and European patent application national phase entry serial number EP2004743322 filed on July 9, 2004 published as EP1646714 now withdrawn; Novozymes, Inc. in PCT application number PCT/US2007/88060 filed on December 19, 2007 published as W02008140615, United States patent application national phase entry serial number 12/520,072 filed on July 2, 2009 published as US20100028943 and European patent application national phase entry serial number EP2007874376 filed on July 7, 2009 published as EP2104739; University of Rochester in PCT
application number PCT/US2006/46120 filed on December 4, 2006 published as W02007064952 and United States patent application serial number 11/606,995 filed on December 1, 2006 published as US20070141030; BioNTech AG in European patent application serial number EP2007024312 filed December 14, 2007 now abandoned, PCT
application number PCT/EP2008/01059 filed on December 12, 2008 published as W02009077134, European patent application national phase entry serial number EP2008861423 filed on June 2, 2010 published as EP2240572, United States patent application national phase entry serial number 12/,735,060 filed November 24, 2010 published as US20110065103, German patent application serial number DE 10 2005 046 490 filed September 28, 2005, PCT application PCT/EP2006/0448 filed September 28, 2006 published as W02007036366, national phase European patent published March, 21, 2012 and national phase US patent application serial number 11/992,638 filed August 14, 2009 published as 20100129877; Immune Disease Institute Inc.
in United States patent application serial number 13/088,009 filed April 15, 2011 published as and PCT application PCT/US2011/32679 filed April 15, 2011 published as W020110130624;
Shire Human Genetic Therapeutics in United States patent application serial number 12/957,340 filed on November 20, 2010 published as US20110244026; Sequitur Inc. in PCT
application PCT/U51998/019492 filed on September 18, 1998 published as W01999014346; The Scripps Research Institute in PCT application number PCT/US2010/00567 filed on February 24, 2010 published as W02010098861, and United States patent application national phase entry serial number 13/203,229 filed November 3, 2011 published as U520120053333; Ludwig-Maximillians University in PCT application number PCT/EP2010/004681 filed on July 30, 2010 published as W02011012316; Cellscript Inc. in United States patent number 8,039,214 filed June 30, 2008 and granted October 18, 2011, United States patent application serial numbers 12/962,498 filed on December 7, 2010 published as US20110143436, 12/962,468 filed on December 7, 2010 published as US20110143397, 13/237,451 filed on September 20, published as US20120009649, and PCT applications PCT/US2010/59305 filed December 7, 2010 published as W02011071931 and PCT/US2010/59317 filed on December 7, 2010 published as W02011071936; The Trustees of the University of Pennsylvania in PCT
application number PCT/US2006/32372 filed on August 21, 2006 published as W02007024708, and United States patent application national phase entiy serial number

11/990,646 filed on March 27, 2009 published as U520090286852: Curevac GMBH in German patent application serial numbers DE10 2001 027 283.9 filed June 5, 2001, DE10 2001 062 480.8 filed December 19, 2001, and DE 20 2006 051 516 filed October 31, 2006 all abandoned, European patent numbers EP1392341 granted March 30, 2005 and EP1458410 granted January 2, 2008, PCT
application numbers PCT/EP2002/06180 filed June 5, 2002 published as W02002098443, PCT/EP2002/14577 filed on December 19, 2002 published as W02003051401, PCT/EP2007/09469 filed on December 31, 2007 published as W02008052770, PCT/EP2008/03033 filed on April 16, 2008 published as W02009127230, filed on May 19, 2005 published as W02006122828, PCT/EP2008/00081 filed on January 9, 2007 published as W02008083949, and United States patent application serial numbers 10/729,830 filed on December 5, 2003 published as U520050032730, 10/870,110 filed on June 18, 2004 published as U520050059624, 11/914,945 filed on July 7, 2008 published as U520080267873, 12/446,912 filed on October 27, 2009 published as U52010047261 now abandoned, 12/522,214 filed on January 4, 2010 published as U520100189729,

12/787,566 filed on May 26, 2010 published as US20110077287, 12/787,755 filed on May 26, 2010 published as U520100239608, 13/185,119 filed on July 18, 2011 published as U520110269950, and

13/106,548 filed on May 12, 2011 published as US20110311472 all of which are herein incorporated by reference in their entirety.
[00445] In some embodiments, the effector modules may be designed as self-amplifying RNA.
"Self-amplifying RNA" as used herein refers to RNA molecules that can replicate in the host resulting in the increase in the amount of the RNA and the protein encoded by the RNA. Such self-amplifying RNA may have structural features or components of any of those taught in International Patent Application Publication No. W02011005799 (the contents of which are incorporated herein by reference in their entirety).

VI. DOSING, DELIVERY AND ADMINISTRATIONS
[00446] The compositions of the invention may be delivered to a cell or a subject through one or more routes and modalities. The viral vectors containing one or more effector modules. SREs, immunotherapeutic agents and other components described herein may be used to deliver them to a cell and/or a subject. Other modalities may also be used such as mRNAs, plasmids, and as recombinant proteins.
I. Delivery to cells [00447] In another aspect of the invention, polynucleotides encoding biocircuits, effector modules, SREs (e.g., DDs), payloads of interest (immunotherapeutic agents) and compositions of the invention and vectors comprising said polynucleotides may be introduced into cells such as immune effector cells.
[00448] In one aspect of the invention, polynucleotides encoding biocircuits, effector modules, SREs (e.g., DDs), payloads of interest (immunotherapeutic agents) and compositions of the invention, may be packaged into viral vectors or integrated into viral genomes allowing transient or stable expression of the polynucleotides. Preferable viral vectors are retroviral vectors including lentiviral vectors. In order to construct a retroviral vector, a polynucleotide molecule encoding a biocircuit, an effector module, a DD or a payload of interest (i.e.
an immunotherapeutic agent) is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. The recombinant viral vector is then introduced into a packaging cell line containing the gag, pol, and env genes, but without the LTR and packaging components. The recombinant retroviral particles are secreted into the culture media, then collected, optionally concentrated, and used for gene transfer.
Lentiviral vectors are especially preferred as they are capable of infecting both dividing and non-dividing cells.
[00449] Vectors may also be transferred to cells by non-viral methods by physical methods such as needles, electropomtion, sonoporation, hyrdoporation; chemical carriers such as inorganic particles (e.g. calcium phosphate, silica, gold) and/or chemical methods. In some embodiments, synthetic or natural biodegradable agents may be used for delivery such as cationic lipids, lipid nano emulsions, nanoparticles, peptide based vectors, or polymer based vectors.
[00450] In some embodiments, the polypeptides of the invention may be delivered to the cell directly. In one embodiment, the polypeptides of the invention may be delivered using synthetic peptides comprising an endosomal leakage domain (ELD) fused to a cell penetration domain (CLD). The polypeptides of the invention are co introduced into the cell with the ELD-CLD-synthetic peptide. ELDs facilitate the escape of proteins that are trapped in the endosome, into the cytosol. Such domains are derived proteins of microbial and viral origin and have been described in the art. CPDs allow the transport of proteins across the plasma membrane and have also been described in the art. The ELD-CLD fusion proteins synergistically increase the transduction efficiency when compared to the co-transduction with either domain alone. In some embodiments, a histidine rich domain may optionally be added to the shuttle construct as an additional method of allowing the escape of the cargo from the endosome into the cytosol. The shuttle may also include a cysteine residue at the N or C terminus to generate multimers of the fusion peptide. Multimers of the ELD-CLD fusion peptides generated by the addition of cysteine residue to the terminus of the peptide show even greater transduction efficiency when compared to the single fusion peptide constructs. The polypeptides of the invention may also be appended to appropriate localization signals to direct the cargo to the appropriate sub-cellular location e.g. nucleus. In some embodiments any of the ELDs, CLDs or the fusion ELD-CLD
synthetic peptides taught in the International Patent Publication;
W02016161516 and W02017175072 may be useful in the present invention (the contents of each of which are herein incorporated by reference in their entirety).
2. Dosing [00451] The present invention provides methods comprising administering any one or more compositions for immunotherapy to a subject in need thereof. These may be administered to a subject using any amount and any route of administration effective for preventing or treating a clinical condition such as cancer, infection diseases and other immunodeficient diseases.
[00452] Compositions in accordance with the invention are typically formulated in dosage unit form for ease of administration and unifonnity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, or prophylactically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, previous or concurrent therapeutic interventions and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
[00453] Compositions of the invention may be used in varying doses to avoid T
cell energy, prevent cytokine release syndrome and minimize toxicity associated with inununotherapy. For example, low doses of the compositions of the present invention may be used to initially treat patients with high tumor burden, while patients with low tumor burden may be treated with high and repeated doses of the compositions of the invention to ensure recognition of a minimal tumor antigen load. In another instance, the compositions of the present invention may be delivered in a pulsatile fashion to reduce tonic T cell signaling and enhance persistence in vivo. In some aspects, toxicity may be minimized by initially using low doses of the compositions of the invention, prior to administering high doses. Dosing may be modified if serum markers such as ferritin, serum C-reactive protein, IL-6, IFN-y, and TNT-a are elevated.
3. Administration [00454] In some embodiments, the compositions for immunotherapy may be administered to cells ex vivo and subsequently administered to the subject. Immune cells can be isolated and expanded ex vivo using a variety of methods known in the art. For example, methods of isolating cytotoxic T cells are described in U.S. Pat. NOs. 6,805,861 and 6,531,451; the contents of each of which are incorporated herein by reference in their entirety. Isolation of NK cells are described in U.S. Pat. NOs. 7,435,596; the contents of which are incorporated by reference herein in its entirety.
[00455] In some embodiments, compositions of the present invention, may be administered by any of the methods of administration taught in the copending commonly owned U.S. Provisional Patent Application No. 62/320,864, filed on 4/11/2016, or in US Provisional Application No.
62/466,596 filed March 3, 2017 and the International Publication W02017/180587, the contents of which are incorporated herein by reference in their entirety.
[00456] In some embodiments, depending upon the nature of the cells, the cells may be introduced into a host organism e.g. a mammal, in a wide variety of ways including by injection, transfusion, infusion, local instillation or implantation. In some aspects, the cells of the invention may be introduced at the site of the tumor. The number of cells that are employed will depend upon a number of circumstances, the purpose for the introduction, the lifetime of the cells, the protocol to be used, for example, the number of administrations, the ability of the cells to multiply, or the like. The cells may be in a physiologically-acceptable medium.
[00457] In some embodiments, the cells of the invention may be administrated in multiple doses to subjects having a disease or condition. The administrations generally effect an improvement in one or more symptoms of cancer or a clinical condition and/or treat or prevent cancer or clinical condition or symptom thereof [00458] In some embodiments, the compositions for immunotherapy may be administered in vivo. In some embodiments, polypeptides of the present invention comprising biocircuits, effector molecules, SREs, payloads of interest (immunotherapeutic agents) and compositions of the invention may be delivered in vivo to the subject. In vivo delivery of immunotherapeutic agents is well described in the art. For example, methods of delivery of cytokines are described in the E.P. Pat. NOs. EP0930892 Al, the contents of which are incorporated herein by reference.
[00459] In one embodiment, the payloads of the present invention may be administered in conjunction with inhibitors of SHP-1 and/or SHP-2. The tyrosine-protein phospho ace SHIP!
(also known as PTPN6) and SHP2 (also known as PTPN11) are involved in the Programmed Cell Death (PD1) inhibitory signaling pathway. The intracellular domain of PD1 contains an immunoreceptor tyrosine-based inhibitory motif (MM) and an iirununoreceptor tyrosine-based switch motif (1TSM). ITSM has been shown to recruit SHP-1 and 2. This generates negative costimulatory, micro clusters that induce the dephosphorylation of the proximal TCR signaling molecules, thereby resulting in suppression of T cell activation, which can lead to T cell exhaustion. In one embodiment, inhibitors of SHP-1 and 2 may include expressing dominant negative versions of the proteins in T cells, TILs or other cell types to relieve exhaustion. Such mutants can bind to the endogenous, catalytically active proteins, and inhibit their function. In one embodiment, the dominant negative mutant of SHP-1 and/ or SHP-2 lack the phosphatase domain required for catalytic activity. In some embodiments, any of the dominant negative SHP-1 mutants taught Bergeron S et al. (2011). Endocrinology. 2011 Dec;152(12):4581-8.; Dustin JB
et al. (1999) J Immunol. Mar 1;162(5):2717-24.; Berchtold S (1998) Mol Endocrinol.
Apr;12(4):556-67 and Schram et al. (2012) Am J Physiol Heart Circ Physiol.
1;302(1):H231-43.;
may be useful in the invention (the contents of each of which are incorporated by reference in their entirety).
Routes of delivery [00460] The pharmaceutical compositions, biocircuits, biocircuit components, effector modules including their SREs (e.g., DDs), payloads (i.e. immunotherapeutic agents), vectors and cells of the present invention may be administered by any route to achieve a therapeutically effective outcome.
[00461] These include, but are not limited to enteral (into the intestine), gastroenteral, epidural (into the dura matter), oral (by way of the mouth), transdermal, peridural, intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intravenous bolus, intravenous drip, intra-arterial (into an artery), intramuscular (into a muscle), intracranial (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intrasinal infusion, intravitreal, (through the eye), intravenous injection (into a pathologic cavity) intracavitary (into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), in ear drops, auricular (in or by way of the ear), buccal (directed toward the cheek), conjunctival, cutaneous, dental (to a tooth or teeth), electro-osmosis, endocervical, endosinusial, endotracheal, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intm-articular, intrabiliary, intrabronchial, intrabursal, intracartilaginous (within a cartilage), intracaudal (within the cauda equine), intracistemal (within the cisterna magna cerebellomedularis), intracomeal (within the cornea), dental intracornal, intracoronary (within the coronary arteries), intracorporus cavernosum (within the dilatable spaces of the corporus cavernosa of the penis), intradiscal (within a disc), intraductal (within a duct of a gland), intraduodenal (within the duodenum), intradural (within or beneath the dura), intraepidennal (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach), intragingival (within the gingivae), intraileal (within the distal portion of the small intestine), intralesional (within or introduced directly to a localized lesion), intraluminal (within a lumen of a tube), intralymphatic (within the lymph), intramedullary (within the marrow cavity of a bone), intrameningeal (within the meninges), intramyocardial (within the myocardium), intraocular (within the eye), intraovarian (within the ovary), intrapericardial (within the pericardium), intrapleural (within the pleura), intraprostatic (within the prostate gland), intrapulmonary (within the lungs or its bronchi), intrasinal (within the nasal or periorbital sinuses), intraspinal (within the vertebral column), intrasynovial (within the synovial cavity of a joint), intratendinous (within a tendon), intratesticular (within the testicle), intrathecal (within the cerebrospinal fluid at any level of the cerebrospinal axis), intrathoracic (within the thorax), intratubular (within the tubules of an organ), intratumor (within a tumor), intratympanic (within the aurus media), intravascular (within a vessel or vessels), intmventricular (within a ventricle), iontophoresis (by means of electric current where ions of soluble salts migrate into the tissues of the body), irrigation (to bathe or flush open wounds or body cavities), laryngeal (directly upon the larynx), nasogastric (through the nose and into the stomach), occlusive dressing technique (topical route administration which is then covered by a dressing which occludes the area), ophthalmic (to the external eye), oropharyngeal (directly to the mouth and pharynx), parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (within the respiratory tract by inhaling orally or nasally for local or systemic effect), retrobulbar (behind the pons or behind the eyeball), intramyocardial (entering the myocardium), soft tissue, subarachnoid, subconjunctival, DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
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Claims (47)

1. A composition for inducing an immune response in a cell or a subject comprising a first effector module, said effector module comprising a first stimulus response element (SRE) operably linked to at least one immunotherapeutic agent, wherein said at least one immunotherapeutic agent is selected from a cytokine, a safety switch, a regulatory switch, a chimeric antigen receptor and combinations thereof

2. The composition of claim 1, wherein said first SRE is responsive to or interacts with at least one stimulus.

3. The composition of claim 2, wherein said first SRE is a destabilizing domain (DD).

4. The composition of claim 3, wherein the DD is derived from a parent protein or a mutant protein having one, two, three or more amino acid mutations compared to said parent protein, wherein the parent protein is selected from:
(a) human protein FKBP comprising the amino acid sequence of SEQ ID NO. 3, (b) human DHFR (hDHFR) comprising the amino acid sequence of SEQ ID NO. 1, (c) E. coli DHFR (ecDHFR) comprising the amino acid sequence of SEQ ID NO. 2, (d) PDE5 comprising the amino acid sequence of SEQ ID NO. 4, (e) PPAR gamma comprising the amino acid sequence of SEQ ID NO. 5, (f) CA2 comprising the amino acid sequence of SEQ ID NO. 6, (g) NQO2 comprising the amino acid sequence of SEQ ID NO. 7, and (h) human DPPIV comprising the amino acid sequence of SEQ ID NO. 224.

5. The composition of claim 4, wherein the parent protein is hDHFR and the DD
comprises a mutant protein having at least one mutation selected from M1del, V2A, C7R, I8V, V9A, A10T, A10V, Q13R, N14S, G16S, I17N, I17V, K19E, N20D, G21T, G21E, D225, L23S, P24S, L28P, N30D, N30H, N30S, E31G, E31D, F32M, R33G, R33S, F35L, Q36R, Q36S, Q36K, Q36F, R37G, M38V, M38T, T40A, V44A, K47R, N49S, N49D, M53T, G54R, K56E, K56R, T57A, F59S, I61T, K64R, N65A, N65S, N65D, N65F, L68S, K69E, K69R, R71G, I72T, I72A, I72V, N73G, L74N, V75F, R78G, L80P, K81R, E82G, H88Y, F89L, R92G, S93G, S93R, L94A, D96G, A97T, L98S, K99G, K99R, L00P, E102G, Q103R, P104S, E105G, A107T, A107V, N108D, K109E, K109R, V110A, D111N, M112T, M112V, V113A, W114R, I115V, V116I, G117D, V121A, Y122C, Y122D, Y122I, K123R, K123E, A125F, M126I, N127R, N127S, NI27Y, H128R, H128Y, H131R, L132P, K133E, L134P, F135P, FI35L, F135S, F I35V, V136M, T137R, R138G, R138I, I139T, I139V, M140I, M140V, Q141R, D142G, F143S, F143L, E144G, D146G, T147A, F148S, F148L, F149L, P150L, E151G, I152V, D153A, D153G, E155G, K156R, Y157R, Y157C, K158E, K158R, L159P, L160P, E162G, Y163C, V166A, S168C, D169G, V170A, Q171R, E172G, E173G, E173A, K174R, I176A, I176F, I176T, K177E, K177R, Y178C, Y178H, F180L, E181G, V182A, Y183C, Y183H, E184R, E184G, K185R, K185del, K185E, N186S, N186D, D187G, and D187N.

6. The composition of claim 5, wherein the stimulus is selected from Trimethoprim (TMP) and Methotrexate (MTX).

7. The composition of claim 1, wherein the immunotherapeutic agent is a cytokine.

8. The composition of claim 7, wherein the cytokine is an interleukin, an interferon, a tumor necrosis factor, a transforming growth factor B, a CC chemokine, a CXC
chemokine, a CX3C
chemokine or a growth factor.

9. The composition of claim 8, wherein the cytokine is an interleukin and the interleukin is selected from a group consisting of ILL IL1-alpha, IL1-beta, IL1-delta, IL1-epsilon, IL1-eta, IL1-zeta, IL-RA, IL2, IL3, IL4, IL5, IL6, IL7, IL8, 1L9, IL10, IL10C, IL10D, IL11a, IL11b, IL13, IL14, IL16, IL17, IL-17A, IL17B, IL17C, IL17E, IL17F, IL18, IL19, IL20, IL20L, IL21, IL22, IL23, IL23A, IL24, IL25, IL26, IL27, IL28, IL29, IL30, IL31, IL32, IL33, IL34, IL36.alpha., IL36.beta., IL36.gamma., IL36RN, IL37, IL37a, IL37b, IL37c, I137d, IL37e, and IL38.

10. The composition of claim 9, wherein the interleukin is IL2, comprising the amino acid sequence of SEQ ID NO. 51.

11. The composition of claim 1, wherein the immunotherapeutic agent is a safety switch.

12. The composition of claim 11, wherein the safety switch is selected from a Caspase 9, an inducible FAS (iFAS), an inducible caspase 9 (icasp9), a CD20/anti-CD20 antibody pair, a protein tag/anti-tag antibody, and a compact suicide gene (RQR8).

13. The composition of claim 12, wherein the safety switch is a Caspase 9 comprising the amino acid sequence of SEQ ID NO. 65.

14. The composition of claim 1, wherein the immunotherapeutic agent encodes a regulatory switch.

15. The composition of claim 14, wherein the regulatory switch is selected from a FOXP3, a Nr4a, a FOXO, and a NF-.KAPPA.B.

16. The composition of claim 15, wherein the regulatory switch is a FOXP3, comprising the amino acid sequence of SEQ ID NO. 103-106.

17. The composition of claim 1, wherein the immunotherapeutic agent is a chimeric antigen receptor (CAR) and is selected from a GD2 CAR, a Her2 CAR, a BCMA CAR, a CD33 CAR, an ALK CAR, a CD22 CAR, and a CD276 CAR, each of which comprises an extracellular moiety, a transmembrane domain, an intracellular signaling domain, and optionally, one or more co-stimulatory domains.

18. The composition of claim 17, wherein the CAR is designed as a standard CAR, a split CAR, an off-switch CAR, an on-switch CAR, a first-generation CAR, a second-generation CAR, a third-generation CAR, or a fourth-generation CAR.

19. The composition of claim 18, wherein the extracellular target moiety is selected from any of i. an Ig NAR, ii. a Fab fragment, iii. a Fab' fragment, iv. a F(ab)'2 fragment, v. a F(ab)'3 fragment, vi. an Fv, vii. a single chain variable fragment (scFv), viii. a bis-scFv, a (scFv)2, ix. a minibody, x. a diabody, xi. a triabody, xii. a tetrabody, xiii. an intrabody, xiv. a disulfide stabilized Fv protein (dsFv), xv. a unibody, xvi. a nanobody, and xvii. an antigen binding region derived from an antibody that specifically binds to any of a protein of interest, a ligand, a receptor, a receptor fragment or a peptide aptamer.

20. The composition of claim 17, wherein the extracellular target moiety is selected from an ALK target moiety, comprising the amino acid sequence of SEQ ID NO. 242- 257 and 422-429, a CD22 target moiety, comprising the amino acid sequence of SEQ ID NO.258-262 and 430-432, a CD276 target moiety, comprising the amino acid sequence of SEQ ID NO.
263-270 and 433-436, a GD2 target moiety, comprising the amino acid sequence of SEQ ID
NO.271-349 and 437-465, a CD33 target moiety, comprising the amino acid sequence of SEQ ID
NO. 350-357, a BCMA target moiety, comprising the amino acid sequence of SEQ ID NO. 358-365, and a Her2 target moiety, comprising the amino acid sequence of SEQ ID NO. 366-421 and 466-473.

21. The composition of claim 17, wherein (a) the intracellular signaling domain of the CAR is the signaling domain derived from T
cell receptor CD3zeta or a cell surface molecule selected from the group consisting of FcR gamma, FcR beta, CD3 gamma. CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d; and (b) the co-stimulatory domain is present and is selected from the group consisting of 2B4, HVEM, ICOS, LAG3, DAP10, DAP12, CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, ICOS (CD278), glucocorticoid-induced tumor necrosis factor receptor (GITR), lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, and B7-H3.

22. The composition of claim 17, wherein the transmembrane domain comprises the amino acid sequence of SEQ ID NO. 527-624.

23. The composition of claim 17, wherein the transmembrane domain further comprises a hinge region comprising the amino acid sequence of SEQ ID NO.628-694.

24. The composition of any of claims 1-23 wherein said first effector module comprises one or more of:
(a) an IL2-DD, comprising the amino acid sequence of any of SEQ ID NOs. 52-54, (b) a Caspase 9-DD, comprising the amino acid sequence of any of SEQ ID NOs.
72-80, (c) a FOXP3-DD, comprising the amino acid sequence of any of SEQ ID NOs. 107-116, (d) a BCMA CAR-DD, comprising the amino acid sequence of any of SEQ ID NOs.

777, and (e) a HER2-DD, comprising the amino acid sequence of SEQ ID NO. 906.

25. A polynucleotide encoding any of the compositions of claims 1-24, wherein said at least one immunotherapeutic agent is selected from a cytokine, a safety switch, a regulatory switch, a chimeric antigen receptor and the combination thereof.

26. The polynucleotide of claim 25, wherein the polynucleotide is a DNA
molecule, or a RNA
molecule.

27. The polynucleotide of claim 26, wherein the polynucleotide is RNA and said RNA is a messenger RNA.

28. The polynucleotide of claim 27, which is chemically modified.

29. The polynucleotide of claim 26, which comprises spatiotemporally selected codons.

30. The polynucleotide of claim 27, wherein the polynucleotide encodes at least one additional feature selected from a promoter, a linker, a signal peptide, a tag, a cleavage site and a targeting peptide.

31. The polynucleotide of claim 25, wherein the chimeric antigen receptor is selected from a GD2 CAR, a Her2 CAR, a BCMA CAR, a CD33 CAR, an ALK CAR, CD22 CAR, and a CD276 CAR.

32. A vector comprising a polynucleotide of any of claims 25-31 wherein said at least one immunotherapeutic agent is selected from a cytokine, a safety switch, a regulatory switch, a chimeric antigen receptor and the combination thereof.

33. The vector of claim 32, wherein the vector is a viral vector, or a plasmid.

34. The vector of claim 33, wherein the vector is a viral vector and said viral vector is a retroviral vector, a lentiviral vector, a gamma retroviral vector, a recombinant AAV
vector, an adeno viral vector, or an oncolytic viral vector.

35. The vector of claim 34, wherein the polynucleotide encodes any of the compositions of claim 1-24.

36. An immune cell for adoptive cell transfer (ACT), which expresses any of the compositions of any of claims 1-24, the polynucleotides of any of claims 25-31, and/or is infected or transfected with the vector of any of claims 32-35.

37. The immune cell of claim 36, wherein the immune cell is a CD8+ T cell, a CD4+ T cell, a helper T cell, a natural killer (NK) cell, a NKT cell, a cytotoxic T
lymphocyte (CTL), a tumor infiltrating lymphocyte (Tit), a memory T cell, a regulatory T (Treg) cell, a cytokine-induced killer (CIK) cell, a dendritic cell, a human embryonic stem cell, a mesenchymal stem cell, a hematopoietic stem cell, or a mixture thereof.

38. The immune cell of claim 36, wherein the SRE is a destabilizing domain DD, wherein the DD is derived from human protein FKBP comprising the amino acid sequence of SEQ ID NO. 3, DHFR comprising the amino acid sequence of SEQ ID NO. 1-2, PDE5 comprising the amino acid sequence of SEQ ID NO. 4, PPAR gamma comprising the amino acid sequence of SEQ ID
NO. 5, CA2 comprising the amino acid sequence of SEQ ID NO. 6 and NQO2 comprising the amino acid sequence of SEQ ID NO. 7.

39. The immune cell of claim 38, wherein the DD is derived from a parent protein and the parent protein is hDHFR and the DD comprises a mutant protein having at least one mutation selected from M1del, V2A, C7R, I8V, V9A, A10T, A10V, Q13R, N14S, G16S, I17N, I17V, K19E, N20D, G21T, G21E, D22S, L23S, P24S, L28P, N30D, N30H, N30S, E31G, E31D, F32M, R33G, R33S, F35L, Q36R, Q36S, Q36K, Q36F, R37G, M38V, M38T, T40A, V44A, K47R, N49S, N49D, M53T, G54R, K56E, K56R, T57A, F59S, I61T, K64R, N65A, N65S, N65D, N65F, L68S, K69E, K69R, R71G, I72T, I72A, I72V, N73G, L74N, V75F, R78G, L80P, K81R, E82G, H88Y, F89L, R92G, S93G, S93R, L94A, D96G, A97T, L98S, K99G, K99R, L100P, E102G, Q103R, P104S, E105G, A107T, A107V, N108D, K109E, K109R, V110A, D111N, M112T, M112V, V113A, W114R, I115V, V116I, G117D, V121A, Y122C, Y122D, Y122I, K123R, K123E, A125F, M1261, N127R, N127S, N127Y, H128R, H128Y, H131R, L132P, K133E, LI34P, F135P, F135L, F135S, F135V, V136M, T137R, R138G, R138I, I139T, I139V, M140I, M140V, Q141R, D142G, F143S, F143L, E144G, D146G, T147A, F148S, F148L, F149L, P150L, E151G, I152V, D153A, D153G, E155G, K156R, Y157R, Y157C, K158E, K158R, L159P, L160P, E162G, Y163C, V166A, S168C, D169G, V170A, Q171R, E172G, E173G, E173A, K174R, I176A, I176F, I176T, K177E, K177R, Y178C, Y178H, F180L, E181G, V182A, Y183C, Y183H, E184R, E184G, K185R, K185del, K185E, N186S, N186D, D187G, and D187N.

40. The immune cell of claim 39, which is autologous, allogeneic, syngeneic, or xenogeneic in relation to a particular individual subject.

41. A method of reducing a tumor volume or burden in a subject comprising contacting the subject with compositions of any of claims 1-24, the polynucleotides of any of claims 25-31, the vector of any of claims 32-35or the immune cells of any of claims 36-40.

42. A method of providing an anti-tumor immune response in a subject comprising administering to the subject an effective amount of the compositions of any of claims 1-24, the polynucleotides of any of claims 25-31, the vector of any of claims 32-35 or the immune cells of any of claims 36-40.

43. A method of inducing an immune response in a subject comprising administering to the subject an effective amount of any of the compositions of claims 1-24, the polynucleotides of any of claims 25-31, the vector of any of claims 32-35 or the immune cells of any of claims 36-40.

44. A method of preventing or reversing T cell exhaustion in a subject in need thereof, the method comprising administering to the subject, a therapeutically effective amount of compositions of any of claims 1-24, the polynucleotides of any of claims 25-31, the vector of any of claims 32-35 or the immune cells of any of claims 36-40, wherein the SRE responds to a stimulus and tunes the expression and/or function of the immunotherapeutic agent, thereby preventing or reversing T cell exhaustion.

45. The method of claim 44, wherein the immunotherapeutic agent is a chimeric antigen receptor.

46. The method of claim 45, wherein the chimeric antigen receptor is a GD2 CAR, BCMA CAR, CD33 CAR, Her2 CAR, ALK CAR, CD22 CAR, or a CD276 CAR.

47. A method of detecting the presence of cancer in a mammal, comprising the steps of:
(a) contacting a sample comprising one or more cells from the mammal with the compositions of any of claims 1-24, the polynucleotides of any of claims 25-31, the vector of any of claims 32-35 or the immune cells of any of claims 36-40 and (b) detecting the complex, wherein the detection of the complex is indicative of the presence of cancer in the mammal.