AU2018227583B2 - CD19 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

[0001] 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 and the US Provisional Patent Application No. 62/484,052, filed on April 11, 2017 entitled Anti CD 19 compositions and methods for immunotherapy, the contents of each of which are herein incorporated by reference in their entirety.

[0002] Tlie present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 2095_1201PCT_SL.txt, created on March 2, 2018, which is 2, 116,001 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

[0003] 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 immunotlierapeutic 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

[0004] Cancer immunotherapy aims to eradicate cancer cells by rejuvenating tlie tumoricidal functions of tumor-reactive immune cells, predominantly T cells. Strategies of cancer immunotherapy including tlie recent development of checkpoint blockade, adoptive cell transfer (ACT) and cancer vaccines which can increase the anti-tumor immune effector ceils have produced remarkable results in several tumors.

[0005] The impact of host anti-tumor immunity and cancer immunotherapy 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 ceils 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 immunotlierapeutic 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

. I . become tolerated. Such tolerance is also active and dominant because cancer vaccines and adoptive transfer of pre-activated immune effector ceils (e.g., T cells), are subject to suppression by inhibitory factors in the tumor microenvironment (TME).

[0006] In addition, administration of engineered T cells could result in on/off target toxicities as well as a cytokine release syndrome (reviewed by ^"fey Clin. Trans 1. Immunol., 2014, 3: e l? 10.1038).

|0Θ07] 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 Song 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.

[0008] To develop regulatable therapeutic agents for disease therapy, in particular cancer immunotherapy, 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 immunotherapeutic 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 iigand and can be stabilized by binding to its specific ligand.

SUMMARY OF THE INVENTION

[0009] 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 1 1, 2016, 62/466,596 filed March 3, 2017 and the International Publication WO2017/180587 (the contents of each of which are herein incorporated by reference in their entirety).

[0010] In some embodiments, the composition for inducing an immune response may comprise an effector module. In some embodiments, the effector module may comprise a stimulus response element (SRE) operably linked to at least one payload. In one aspect, the payload may be an immunotherapeutic agent.

- Z ~ [0011] In some embodiments, the immunotherapeutic agent may be selected from, but is not limited to a chimeric antigen receptor (CAR) and an antibody.

[0012] In one aspect the SRE of the composition may be responsive to or interact with at least one stimulus.

[0013] In some embodiments, the 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 ammo acid sequence of SEQ ID NO. 3; human 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 ammo acid sequence of SEQ ID NO. 6; or NQ02, comprising the amino acid sequence of SEQ ID NO. 7.

[0014] In one aspect, the parent protein is hDHFR and the DD comprises a mutant protein. The mutant protein may comprise a single mutation and may be selected from, but not limited to hDHFR (I17V), hDHFR (F59S), hDHFR (N65D), hDHFR (K81R), hDHFR (A107V), hDHFR (Yl 221), hDHFR (Nl 27Y), hDHFR (Ml 401), hDHFR (K185E), hDHFR (Nl 86D), and hDHFR (Ml 401), hDHFR (Amino acid 2-187 of WT: N127Y), hDHFR (Amino acid 2-187 of WT; I17V), hDHFR (Amino acid 2-187 of WT; Y 122I), and hDHFR (Ammo acid 2-187 of WT: K185E). In some embodiments, the mutant protein may comprise two mutations and may be selected from, but not limited to, hDHFR (C7R, Y163C), hDHFR (A 10V, H88Y), hDHFR (Q36K, Y 122I), hDHFR (M53T, R138I), hDHFR (T57A, Γ72Α), hDHFR (E63G, I176F), hDHFR (G2IT, Y 122I), hDHFR (L74N, Y 122I), hDHFR (V75F, YI22I), hDHFR (L94A, T147A), DHFR (V121A, Y22I), hDHFR (Y122I, A125F), hDHFR (H131R, E144G), hDHFR (T137R, F143L), hDHFR (Y178H, E18IG), and hDHFR (Y183H, K185E), hDHFR (E162G, I176F) hDHFR (Amino acid 2-187 of WT; 117V, Y 122I), hDHFR (Amino acid 2-187 of WT; Y122I, M140I), hDHFR (Amino acid 2-187 of WT; 127Y, Y122I), hDHFR (Amino acid 2-187 of WT; E162G, I176F), and hDHFR (Ammo acid 2-187 of WT; H131 R, E144G), and hDHFR (Amino acid 2-187 of WT; Y122I, A125F). In some embodiments, the mutant may comprise three mutations and the mutant may be selected from hDHFR (V9A, S93R, P150L), hDHFR (I8V, K133E, Y 163C), hDHFR (L23S, V121A, Y157C), hDHFR (K19E, F89L, E181G), hDHFR (Q36F, N65F, Y 122I), hDHFR (G54R, M140V, S168C), hDHFR (VI 10A, V136M, K177R), hDHFR (Q36F, Y122I, A125F), hDHFR (N49D, F59S, DI53G), and hDHFR (G21E, I72V, I176T), hDHFR (Amino acid 2-187 of WT; Q36F, \ 1 221. A125F), hDHFR (Amino acid 2-187 of WT; Y 122L HI31R, E144G), hDHFR (Ammo acid 2-187 of WT; E31D, F32M, V I 161), and hDHFR (Amino acid 2-187 of WT; Q36F, N65F, Y122I). In some embodiments, the mutant may comprise four or more mutations and the mutant may be selected from hDHFR (V2A, R33G, Q36R, L100P, K185R), hDHFR (Amino acid 2-187 of WT: D22S, F32M, R33S, Q36S, N65S), hDHFR (ΙΓ7Ν, L98S, K99R, Ml 12T, E151G, E162G, E172G), hDHFR (G16S, 117V, F89L, D96G, K 123E, M14GV, D146G, K156R), hDHFR (K81 R, 99R, L100P, E102G, N108D, K123R, H128R, D142G, F180L, K185E), hDHFR (R138G, D 142G, F143S, K156R, K158E, E162G, V166A, K177E, Y 178C, K185E, N186S), hDHFR (N14S, P24S, F35L, M53T, K56E, R92G, S93G, N127S, H128Y, F135L, F143S, L159P, L16GP, El 73 A, F180L), hDHFR (F35L, R37G, N65A, L68S, 69E, R71G, L80P, 99G, G117D, L132P, I I 39V, Ml 401, D142G, D146G, E173G, D 187G), hDHFR (L28P, N30H, M38V, V44A, L68S, N73G, R78G, A97T, K99R, A107T, K109R, Dl l lN, I I 4 P. F135V, ΊΤ47Α, 1152V, K158R, E172G, V182A, E184R), hDHFR (V2A, I17V, N3GD, E31G, Q36R, F59S, K69E, I72T, H88Y, F89L, N108D, K109E, VI 10A, II 15V, Y122D, L132P, F135S, M140V, E144G, T147A, Y157C, V170A, K174R, N186S), hDHFR (L100P, E102G, Q103R, P104S, E105G, N 108D, V113A, W114R, Y122C, M126I, N127R, i 1 128V. L132P, F135P, I139T, F148S, F149L, I152V, D153A, D169G, VI 70 A, I176A, K177R, V182A, K 185R, N186S), and hDHFR (A 10T, Q13R, N14S, N20D, P24S, N30S, M38T, T40A, K47R, N49S, K56R, I61T, K64R, K69R, Γ72Α, R78G, E82G, F89L, D96G, N108D, Ml 12V, W114R, Y122D, K123E, 1139V, Q141R, D142G, F148L, E151G, E155G, Y157R, Q171R, Y183C, E184G, K185de3, D187N).

[0015] In one aspect, the stimulus of the SRE may be Trimethoprim or Methotrexate.

[0016] In some embodiments, the immunotherapeutic agent of the effector module is a chimeric antigen receptor (CAR). The chimeric antigen may comprise an extracellular target moiety; a transmembrane domain; an intracellular signaling domain; and optionally, one or more co-stimulatory domains.

[0017] 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-switeh CAR, a first-generation CAR, a second-generation CAR, a third-generation CAR, or a fourth-generation CAR.

[0018] 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 specifically bind to any of a protein of interest, a ligand, a receptor, a receptor fragment or a peptide aptamer.

[0019] In one aspect, the extracellular target moiety may be an scFv derived from an antibody. In one aspect, the scFv may specifically bind to a CD 19 antigen

[0020] In one aspect, the scFv of the CAR may be a CD 19 scFv, In some embodiments, the CD 19 scFv may comprise a heavy chain variable region having an amino acid sequence independently selected from the group consisting of SEQ ID NO: 49-80, and a light chain variable region having an amino acid sequence independently selected from the group consisting of any of SEQ ID NOs: 81-122. In some embodiments, the CD19 scFv may comprise an amino acid sequence selected from the group consisting of any of SEQ ID NOs: 123-267 and 624.

[0021] In some embodiments, the intracellular signaling domain of the CAR may be a signaling domain derived from T cell receptor CDSzeta. In some embodiments, the intracellular signaling domain may be selected from a cell surface molecule selected from the group consisting of FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD 22, CD79a, CD79b, and CD66d. In one aspect, the CAR may include a co-stimulatory domain. In some embodiments, the co-stimulatory domain may be selected from the group consisting of 2B4, HVEM, ICOS, LAG3, DAP10, DAP 12, CD27, CD28, 4-1BB (CD137), OX40 (CD 134), CD30, CD40, ICOS (CD278), glucocorticoid-induced tumor necrosis factor receptor (GITR), lymphocyte function-associated antigen- 1 (LFA-I), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0022] (b) the co-stimulatory domain is present and is selected from the group consisting of 2B4, HVEM, ICOS, LAG 3, DAP10, DAP 12, CD27, CD28, 4-1BB (CD 137), OX40 (CD134), CD30, CD40, ICOS (CD278), glucocorticoid-induced tumor necrosis factor receptor (GITR), lymphocyte function-associated antigen- 1 (LFA-i), CD2, CD7, LIGHT, NKG2C, and B7-H3.

[0023] In some embodiments, the intracellular signaling domain of the CAR may be a T cell receptor CD3zeta signaling domain, which may comprise the amino acid sequence of SEQ ID NO: 339.

[0024] In some embodiments, T cell receptor CD3zeta signaling domain of the CAR, comprising the amino acid sequence of SEQ ID NO: 626 may further comprise at least one co- stimulatory domain. The co-stimulatory domain may comprise an amino acid sequence of SEQ ID NOs: 268-374.

[0025] In one embodiment, the transmembrane domain of the CAR may be derived from a transmembrane region of an alpha, beta or zeta chain of a T-cell receptor. In one aspect, the transmembrane domain may be derived from the CD3 epsilon chain of a T-cell receptor. In one embodiment, the transmembrane domain may be derived from a molecule selected from CD4, CD5, CDS, CD8a, CD9, CD 16, CD22, CD33, CD28, CD37, CD45, CD64, CD80, CD86, CD I -18. DAP 10, EpoRI, GITR, LAG3, ICOS, Her2, OX40 (CD134), 4-1BB (CD137), CD152, CD 154, PD-1, or CTLA-4. In another embodiment, the transmembrane domain may be derived from an immunoglobulin selected from IgGl, IgD, IgG4, and an IgG4 Fc region. In one aspect, the transmembrane domain may comprise an am ino acid sequence selected from the group consisting of any of SEQ ID NOs: 375-425 and 897-907.

|0Θ26] 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: 426-504.

[0027] In some embodiments, the immunotherapeutic agent may be an antibody that is specifically immunoreactsve to an antigen selected from a tumor specific antigen (TSA), a tumor associated antigen (TAA), or an antigenic epitope.

[0028] In one aspect, the antigen may be an antigenic epitope. In some embodiments, the antigenic epitope may be CD 19.

[0029] In some embodiments, the antibody may comprise a heavy chain variable region having an amino acid sequence independently selected from, the group consisting of any of SEQ ID NOs: 49-80 and a light chain variable region having an amino acid sequence independently- selected from the group consisting of any of SEQ ID NOs: 81-122. In one aspect, the antibody may comprise an amino acid sequence selected from the group consisting of any of SEQ ID NOs: 123-267 and 624.

[0030] In one aspect, the first effector module may comprise the amino acid sequence of any of SEQ ID NO: 635-649, 1005-1010, 1015-1018 and 1215-1231.

[0031] In some embodiments, the first SRE of the effector module may stabilize the immunotherapeutic agent by a stabilization ratio of 1 or more, wherein the stabilization ratio may- comprise the ratio of expression, function or level of the immunotherapeutic agent in the presence of the stimulus to the expression, function or level of the immunotherapeutic agent in the absence of the stimulus.

[0032] In some embodiments, the SRE may destabilize the immunotherapeutic agent by a destabilization ratio between 0, and 0.09, wherein the destabilization ratio may comprise the ratio of expression, function or level of the immunotherapeutic agent in the absence of the stimulus specific to the SR to the expression, function or level of the immunotherapeutic agent that is expressed constitutive!}', and in the absence of the stimulus specific to the SRE.

[0033] The present invention also provides polynucleotides comprising the compositions of the invention. |0034] In one aspect, the polynucleotides may be a DNA or RNA molecule. In one aspect, the polynucleotides may comprise spatiotemporally selected codons. In one aspect, the

polynucleotides of the invention may be a DNA molecule. 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.

[0035] 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.

[0036] 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.

[0037] 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 (CTL), 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 ceil, a mesenchymal stem cell, a hematopoietic stem cell, or a mixture thereof.

[0038] In some embodiments, the immune cells may be autologous, allogeneic, syngeneic, or xenogeneic in relation to a particular individual subject.

[0039] In some embodiments, the immune cell may further express a composition comprising a second effector module, said second effector module comprising a second SRE linked to a second immunotherapeutic agent. In one aspect, the second immunotherapeutic agent may be selected from a cytokine, and a cytokine- cytokine receptor fusion.

[0040] In one aspect, the second immunothe apeutic agent may be a cytokine. In one aspect, the cytokine may be IL12 or IL15.

[0041] In one aspect, the second immunotherapeutic agent may be a cytokine- cytokine receptor fusion polypeptide.

[0042] In some embodiments, the cytokine-cytokine receptor fusion polypeptide may be selected from, but is not limited to a IL12-IL12 receptor fusion polypeptide, a IL15-IL15 receptor fusion polypeptide, and a IL15-IL15 receptor sushi domain fusion polypeptide. [0043] 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.

[0044] The present invention also provides methods for enhancing the expansion and/or survival of immune cells, comprising contacting the immune cells with the compositions of the invention, the polynucleotides of the invention, and/or the vectors of the invention.

[0045] 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.

[0046] The present invention also provides a method of identifying a domain of a CD 19 antigen which will not bind the FMC63 antibody (FMC63-distinct CD19 binding domain). The method may comprise (a) preparing a composition comprising a CD 19 antigen, (b) contacting the composition in (a) with saturating levels of FMC63 antibody, (c) contacting the composition of step (b) with one or more selected members of a librar - of potential CD 19 binders: and (d) identifying a binding domain on the CD 19 antigen based on the differential binding of the selected members of the library of CD19 binders compared to the binding of FMC63. In some embodiments, the binding domains of the library may be generated using phage display techniques with the CD 19 antigen as the seed sequence. In one aspect, the binding domain may be selected from 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 unibody, a nanobody, or an antigen binding region of an antibody, and an antibody fragment. In one aspect, the CD 19 antigen may be selected from a whole or a portion of a human CD 19 antigen, and a whole or a portion of a Rhesus CD 19 antigen.

[0047] The present invention also provides chimeric antigen receptors that may comprise the

FMC63 -distinct CD19 binding domain obtained according to the methods described herein.

Also, provided herein is a stimulus response element (SRE) operably linked to the chimeric antigen receptors that include the FMC63 -distinct CD19 binding domain.

[0048] In some embodiments, the effector module comprises a stimulus response element

(SRE) and at least one payload comprising a protein of interest (POI).

[0049] 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 protem), 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,

[0050] The payload may be any immunotherapeutic agent used for cancer immunotherapy such as a chimeric agent receptor (CAR) such as CD19 CAR that targets any molecule of tumor cells, an antibody, an antigen binding domain or combination of antigen binding domains, a cytokine such as 3L 12, TL 15 or IL 15/TL 15Ra fusion, 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.

[0051] 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.

[0052] In some embodiments, the DD may stabilize the immunotherapeutic agent with a stabilization ratio of at least one in the presence of the stimulus. According to the present invention, the DD may destabilize the immunotherapeutic agent in the absence of ligand with a destabilization ratio between 0, and 0.99.

[0053] 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.

[0054] 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 lenti viral vector.

[0055] In some embodiments, the vector of the invention may be a non- viral vector, such as a nanoparticles and liposomes.

[0056] 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 cvtotoxic 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). [0057] 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.

[0058] Also provided herein are methods for identifying FMC63 -distinct binding domains and using CD 19 antigens in which the FMC63 binding epitope is masked or absent. In some embodiments, the FMC63 binding domain may be included in the payloads and effector modules of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0059] 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 pay load.

[0060] 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.

[0061] Figure 3 shows representative effector modules carrying two payloads without a cleavage site. The two payloads may be either directly linked to each other or separated.

[0062] Figure 4 shows representative effector modules carrying two pay loads 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-terminus of the construct (M to X). An optional linker may be inserted between each component of the effector module.

[0063] 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.

[0064] 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.

[0065] 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 7 A) 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/payl oad .

[0066] 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.

|0Θ67] 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.

[0068] 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).

[0069] 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.

[0070] 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).

[0071] Figure 13 depicts a standard effector module of a chimeric antigen receptor (CAR) system which comprises an antigen binding domain as an SRE, and signaling domain(s) as payload.

[0072] Figure 14 depicts the structure design of a regulatabie CAR system, where the transmembrane effector modules comprise antigen binding domains sensing an antigen and a first switch domain and the intracellular module comprises a second switch domain and signaling domains. A stimulus (e.g., a dimerization small molecule) can dmierize the first and second switch domains and assemble an activated CAR system..

[0073] Figure 15 shows schematic representation of CAR systems having one (A) or two (B and C) SREs incorporated into the effector module.

[0074] Figure 16 depicts a split CAR design to control T cell activation by a dual stimulus (e.g., an antigen and small molecule). Figure 16A shows normal T cell activation which entails a dual activation of TCR and co-stimulatory receptor. The regular CAR design (Figure 16B) combines the antigen recognition domain with TCR signaling motif and co-stimulatory motif in a single molecule. The split CAR system separates the components of the regular CAR into two separate effector modules which can be reassembled when a heterodimerizing small molecule (stimulus) is present,

[0075] Figure 17 depicts the positive and negative regulation of CAR engineered T cell activation. The absence or presence of a second stimulus can negatively (A) or positively (B) control T cell activation.

[0076] Figure 18 shows schematic representation of gated activation of CAR engineered T cells. If a normal cell that has no stimulus (e.g., an antigen) (Figure 18A) or an antigen that cannot bind to the trans-membrane effector module (Figure 18B), or only an antigen that activates the trans-membrane effector module and primes the receiver T cell to express the second effector (Fig 18C), the receiver T cell remains inactive. When both stimuli (e.g. two antigens) that bind the trans-membrane effector module and the primed effector, are present on the presenter cell (e.g. a cancer cell), the T cell is activated (Figure 18D).

[0077] Figure 19A is a bar graph depicting IL12 levels in the various dilutions of media derived from cells expressing DD-IL12. Figure 19B is a bar graph depicting the Shield-1 dose responsive induction of DD~ 1L12. Figure 19C depicts plasma 1L12 levels in mice implanted with SKOV3 cells. Figure 19D depicts plasma IL12 levels in mice in response to different Shield- i dosing regimens.

[0078] Figure 20A is a western blot of IL15 protein levels in 293 cells. Figure 20B and 20C are histograms depicting surface expression of IL15 and lL15Ra. Figure 20 D is a western blot of 11,15 and hDHFR in HCT116 cells.

[0079] Figure 21 A and Figure 2 IB are western blots of depicting the protein levels of CD3 Zeta of the DD- CD 19 CAR construct and actin. Figure 21C shows the expression of CD 19 chimeric antigen receptors in a western blot using 4-1BB antibody. Figure 21D is a bar graph depicting the surface expression of CD 19 C AR.

[0080] Figure 22 denotes the frequency of IFNgamma positive T cells.

[0081] Figure 23A depicts 1FN gamma production in T cells. Figure 23B depicts T cell expansion with IL15/lL15R.a treatment. Figure 23C is a dot plot depicting percentage human cells after in vivo cell transfer. Figure 23D is scatter plot depicting CD4+/CD8+ T cells.

[0082] Figure 24A depicts T cell subpopulations expressing CD 19 CAR. Figure 28B depicts cell death caused by CD 19 CAR expressing T ceils.

[0083] Figure 25 A is a bar graph depicting lL15Ra positive cells with 24 hour TMP treatment. Figure 25B is a bar graph depicting IL15Ra positive cells with 48 hour TMP treatment. Figure 25C is a bar graph depicting IL15Ra positive cells in response to varying concentrations of TMP.

[0084] Figure 26 is a western blot of IL15Ra protein levels in HCT^'l 16 cells. |008S] Figure 27A represents percentage of human T cells blood with respect to mouse T cells. Figure 27B represents the number of T cells in blood. Figure 27C represents ratio of CD4 to CD8 cells in the blood. Figure 27D represents the percentage of IL 15 Ra positive CD4 and CDS T cells in the blood.

[0086] Figure 28 A depicts the expansion of T cells in response to cytokine treatment. Figure 28B, Figure 28C and Figure 28D depict the frequency of IFN gamma positive cells with IL12 treatment.

|0087] Figure 29 is a bar graph representing the effect of promoters on transgene expression.

[0088] Figure 30A shows the expression of CD19 in parental K562 cells and K562-CD19 cells. Figure SOB shows the proliferation of K562 cells cocultured with T cells expressing DD regulated CAR constructs, in the presence or absence of ligand. Figure 30C shows the area of target cells killed by T cells expressing DD regulated CAR constructs, in the presence of ligand.

[0089] Figure 31A shows IFNgamma concentration. Figure 3 IB shows IL2 concentration.

[0090] Figure 32A provides the final IL12 concentration for each of the four groups tested. Figure 32B shows that 1L12 is detectable in kidney and Figure 32C shows that IL12 is detectable in tumor.

[0091] Figure 33A shows the regulation of IL12 over 24 hours. Figure 33B shows the regulation in the plasma and Figure 33C shows the detection of flexi-IL12 in the kidneys.

[0092] Figure 34A shows that restimulation increased the expression of IL12. Figure 34B and Figure 34C show that ligand increased production of IL12.

[0093] Figure 35A shows the concentration-dependent induction of IL12 secretion of TL 12 secretion from primary human T cells. Figure 35B shows the time course induction of IL12 secretion from primary human T ceils.

[0094] Figure 36A shows the dose response of Aquashield-Tnduced DD-TL12 regulation in vivo. Figure 36B shows that plasma levels of IL12 remain high in animals transplanted with constitutive IL12 transduced T cells.

[0095] Figure 37A and 37B show the expression of IL12 in vivo over 7 days. Figure 37C and 37D show the expression of TL 12 in vivo over 1 1 days. Figure 37E shows the Geometric MFI (GeoMFI) of Granzyme B (GrB) after 7 days in CD8+ T cells. Figure 37F shows the GeoMFI of Perforin at day 7 in CD 8+ T cells.

[0096] Figure 38A shows the regulation of IL12 with PGK and EFla promoters and FKBP domains. Figure 38B shows the relative expression of IL12.

[0097] Figure 39 depicts the kinetics of ILlSRa surface expression on CD4 T cells after IMP treatment. |0098] Figure 40 represents a western blot of IL15-IL15Ra protein in HCT116 tumors from mice treated with TMP for 17 days in xenograft assays,

[0099] Figure 41 is a graph of the results of the MSD assay of IL15 protein levels in HEK293 cells.

[00100] Figure 42A provides FACS plots showing the expression of membrane bound IL15 after a dose response study of TMP, Figure 42B is two graphs showing the dose and time of exposure of TMP in vitro influences membrane bound IL15 expression.

[00101] Figures 43A- 43C show the regulation of membrane bound 1L15 using IL15 (Figure 43A), IL15Ra (Figure 43B), or IL15/IL15Ra double ++ staining (Figure 43C). Figure 43D shows FACS plots of the expression of IL15. Figure 43E is a graph of the regulation of IL15 in blood and Figure 43F is a graph of the plasma TMP levels.

[00102] Figure 44 represents the regulation of membrane bound IL15 with PO or IP dosing of TMP,

DETAILED DESCRIPTION OF THE INVENTION

[00103] 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 tlie description. In tlie 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

[00104] 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 classifi ed 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. |0010S] 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.

|0Θ106] 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.

[00107] 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,

[00108] 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. Fuither, 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.

[00109] 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. fOOllO] The present invention provides methods for fine tuning of immunotherapy after administration to patients. This in turn improves the safely and efficacy of immunotherapy and increases the subject population that may benefit from immunotherapy.

II. COMPOSITIONS OF THE INVENTION

[00111] 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 can be found in U.S. Provisional Patent Application No. 62/320,864 filed April 11, 2016 or in US Provisional Application No. 62/466,596 filed March 3, 2017 and the International Publication

WO2017/180587 (the contents of 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 immunotherapy are provided.

[00112] 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.

[00113] 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 ceil biocircuit system. Any of these systems may act as a signal to any other of these biocircuit systems. Effector modules and SREs for immunotherapy

[00114] 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 chemokme 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.

[00115] 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'¹ 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 covaient or non-covalent interaction, a direct or indirect association or a stractural 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.

[00116] 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%, 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%. [00117] 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 herem, 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,

[00118] In some embodiments, the stimulus of the present invention maybe ultrasound stimulation. In some embodiments, the SREs of the present in vention may derived from mechanosensitive proteins. In one embodiment, the SRE of the present invention may be the mechanically sensitive ion channel, Piezol .

[00119] Expression of the payioad 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 tliereby 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.

[00120] 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 - protem 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.

[00121] 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 m odules of the present invention may further comprise other regulator}⁷ 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.

[00122] 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 instabilityto 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 al. (2008). Biochem J. 2008 Mar

l;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. WO2017004022, WO2016210343, and WO2011062962: the contents of each of which are incorporated by reference in their entirety.

[00123] As shown in Figure 2, representative effector module embodiments comprising one payload, i.e. one imniunotherapeutic 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.

[00124] 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-teiminus of the construct, or in the internal location.

[00125] 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.

[00126] 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, protem engineering may be used to reduce the immunogenicity 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 MHC proteins. For example, amino acid modifications may be engineered such that there are no or a minimal of number of immune epitopes that are predicted 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 used to score epitopes in the compositions of the present invention. Such methods are disclosed in US Patent Publication No. US 20020119492, US20040230380, and US 20060148009; the contents of each of which are incorporated by reference in their entirety, [00127] Epitope identification and subsequent sequence modification may be applied to reduce immunogenicity . The identification of immunogenic epitopes may be achieved either physically or computationally. Physical methods of epitope identification may include, for example, mass spectrometry and tissue culture/cellular techniques. Computational approaches thai utilize information obtained on antigen processing, loading and display, structural and/or proteomic data toward identifying non-self-peptides that may result from antigen processing, and that are likely to have good binding characteristics in the groove of the MHC may also be utilized. 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.

[00128] In some embodiments, protein modifications engineered into the structure of the compositions of the invention to interfere with antigen processing and peptide loading such as glycosylation and PEGylation, may also be useful in the present invention. Compositions of the invention may also be engineered to include non-classical ammo acid sidechains to design less immunogenic compositions. Any of the methods discussed in International Patent Publication No. WO2005051975 for reducing immunogenicity may be useful in the present invention (the contents of which are incorporated by reference in their entirety).

[00129] 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.

[00130] In some embodiments, reduced immunogenicity may be achieved by limiting immuproteasome 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, Imniunoproteasomes 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. Deoi 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 enlianced peptide quantities (Deoi 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 immunoproteasome 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), IPSl-001, ONX 0914 (PR-957), and PR-924 (IPSI). 1. Destabilizing domains (DDs)

[00131] In some embodiments, biocircuit systems, effector modules, and compositions of the present invention relate to post-translational regulation of protein (payload) function and -tumor immune responses of immuno therapeutic 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.

[00132] 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 protem 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 ubiquitm-proteasome system of the ceil (Stankunas, K., et ai., Mol. Cell, 2003, 12: 1615-1624; Banaszynski, et al., Cell; 2006, 126(5): 995-1004; reviewed in Banaszynski, L.A., and Wandless, TJ. Chem. Biol; 2006, 13: 11-21 and akhit 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 n on -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.

[00133] 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.

[00134] 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 (Egeier et al, J Biol. Chem. 2011, 286(36): 32328-31336; the contents of which are incorporated herein by reference in their entirety), ecDHFR and its ligand trimethoprim (IMP); 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 bilimbin-inducible fluorescent protein, UnaG and its cognate ligand bilirabin (BR) ( Navarro et al., ACS Chem Biol., 2016, June 6; the contents of which are incorporated herein by reference in their entirety),

[00135] 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.

[00136] 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 ai., have demonstrated that the non-catalytic -terminal domain (54-residues) of ACS7 (1-aminocyclopropane-l-carboxylate synthase) in Arabidopsis , when fused to the β-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-aminocyciopropane-l-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-26S proteasome pathway.

[00137] Another non-limiting example is the stability control region (SCR, residues 97-1 18) of Tropomyosin (Tm), which controls protein stability. A destabilizing mutation L110A, and a stabilizing mutation A109L dramatically affect Tropomyosin protein dynamics (Kirvvan 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.

[00138] 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.

[00139] 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 Patent Application No. 62/320,864 filed on 4/11/2016, or in US Provisional Application No. 62/466,596 filed

- T . March 3, 2017 and the International Publication WO2017/180587, the contents of each of 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.

ie 1 : Proteins and their binding ligands

[00140] In some embodiments, DDs of the invention may be FKBP DD or ecDHF DDs such as those listed in Table 2. The position of the mutated amino acid listed in Table 2 is relative to the ecDHFR (Unrprot ID: P0ABQ4) 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 FKBP DDs

[00141] 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.

[00142] 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 Mldei mutation.

[00143] In some embodiments, novel DDs derived from ecDHFR may comprise amino adds 2- 159 of the wild type ecDHFR sequence (also referred to as an Mldei mutation), and may include one, two, three, four, five or more mutations including, but not limited to, Mldei, R12Y, R12H, Y!OOI, and E129K.

[00144] 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 Mldei mutation.

[00145] In some embodiments, novel DDs derived from FKBP may comprise ammo acids 2- 107 of the wild type FBKP sequence (also referred to as an M!del mutation), and may include one, two, three, four, five or more mutations including, but not limited to, Mldei, E31G, F36V, R71G, K105E, and L106P.

[00146] 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 otliers 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 affinity of the DD. Additionally, such approaches can be used to minimize deleterious effects caused by off-target ligand binding.

[00147] 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.

[00148] In some embodiments, DDs of the invention may be derived from human dihydrofolate reductase (hDHFR). hDHFR 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 drags such as methotrexate (MTX), 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 hDHFR such as folate, TQD, Trimethoprim (TMP), epigallocatechin gallate (EGCG) and ECG (epicatechin gallate) can also bind to hDHFR mutants and regulates its stability in one aspect of the invention, the DDs of the invention may be hDHFR mutants including the single mutation hDHFR (Υ 122Ϊ), hDHFR (K81R), hDHFR (F59S), hDHFR (II TV), hDHFR (N65D), hDHFR (A 107V), hDHFR (N127Y), hDHFR

(K5 85E), hDHFR (N186D), and hDHFR (M140I); double mutations: hDHFR (M53T, R138I), hDHFR (V75F, Y 122I), hDHFR (A125F, YI221), hDHFR (L74N, YI221), hDHFR (L94A, T147A), hDHFR (G21T, Y122I), hDHFR (VI 21 A, Y 122I), hDHFR (Q36K, Y122I), hDHFR (C7R, Y 163C),hDHFR (Y178H, E18IG), hDHFR (A 10V, H88Y), hDHFR (T137R, F 143L), hDHFR (E63G, I1 76F), hDHFR (T57A, I72A), hDHFR (H 131 R, E144G), and hDHFR (Y183H, K185E); and triple mutations: hDHFR (Q36F, N65F, Y122I), hDHFR (G21E, 172V, ΙΓ76Τ), hDHFR (I8V, K 133E, Y163C), hDHFR (V9A, S93R, P150L), hDHFR (K19E, F89L, E 181G), hDHFR (G54R, M140V, S 168C), hDHFR (L23S, V 121A, Y157C), hDHFR (VI 10A, V 136M, K177R), and hDHFR (Η49Ό, F59S, D 153G).

[00149] In one embodiment, the stimulus is a small molecule that binds to a SRE to post- translationally regulate protein levels. In one aspect, DHF ligands: trimethoprim (TMP) and methotrexate (MTX) 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, "del" means that the mutation is the deletion of the amino acid at that position relative to the wild type sequence. Amino acid Sequence

hDHFR (I17V) MVGSLNCIVAVSQNMGVGKNGDLPWPPLRNEFRYFQR

MTTTSSVEGKQNLVIMGK TWFSIPEKNRPL GRINLVL

SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW

IVGGSSVTKEAMNWI iHL LFVTRIMQDFESDTFFPEID

LEKYta_^LPEYPGVLSDVQEEKGIKYKFEVYEKND hDHFR (F59S) MVGSLNCIVAVSQNMG1G NGDLPWPPLRNEFRYFQR

MTTTSSVEGKQNLVMGKKTWSSIPEKNRPLKGRINLVL

SREIJ EPPQGAHFLSRSLDDALKLTEQPELANKVDMVW

rVGGSSWKEAMNHPGHLKLFVTRMQDFESDTFFPEK)

LEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND

hDHFR (N65D) MVGSLNCIVAVSQNMGIGK GDLPWPPLRNEFRYFQR

MTTTSSVEGKQNLVIMGKKTWFSIPEKDRPLKGRI LVL

SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVD VW

IVGGSSVYKEAMNHPGHL LFV PJMQDFESDTFFPEID

LEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND

hDHFR (K81R) NIVGSLNCr AVSQNNlGIGKNGDLPWPPLR EFRYFQR

MTTTSSV¾GKQNLVB GKKTWFSIPEKNRPLKGRINL\⁷L SRELREPPQGAHFLSRSLDDALKLTEQPELA K >MVW IVGGSSVTKEAMNHPGHLKLFVTRIMQDFESDTFFPEID LEKYKLLPEYPGVLSDVQEEKGIKYKFEWEKND

hDHFR (A 107V) VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR

MTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVL

SRELKEPPQGAHFLSRSLDDALKLTEQPELVMCVDMVW

1VGGSSVYKEAMNHPGHLKLFVTR1MQDFESDTFFPEK)

LEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND

hDHFR (Y1221) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR

MTTTSSVEGKQNLVMGKKTWFSIPEKNRPLKGRINLVL SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW IVGGSSVIKEA^I HPGHLKLFVTTRIMQDFESDTFFPEIDL EKYKLLPEYPGVLSDVQEEKGIKYKFEVYEK D

hDHFR (N127Y) MVGSLNCIVAVSQNMGIGK GDLPWPPLRNEFRYFQR

MTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVL

SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMV¥/

IVGGSSWKEAMYHPGHLKLFVTRIMQDFESDTFFPEID LEKYKLLPEYPGVLSDVQEEKGIKYKFEWEKND

hDHFR ( 140I) MVGSLNCRVAVSQNMGIGKNGDLPWPPLRNEFRYFQR

MTTTSSVEGKQNLVBVIG TWSIPEKNRPLKGRINLV ,

SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW

RVGGSSVYKEANLNHPGHLKLFV RIIODFESDTFFPEIDL

EKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND

hDHFR (K 185E) MVGSLNCIVAVSQNMGIGK GDLPWPPLRNEFRYFQR

MTTTSSVEGKQNLVIMGKKTWFS1PEKNRPLKGRI LVL

SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW

IVGGSSVTKEAMNWI iHL LFVTRIMQDFESDTFFPEID

LEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEE

hDHFR (N186D) MVGSLNCIVAVSQNMG1G NGDLPWPPLRNEFRYFQR

MTTTSSVEGKQNLVMGKKTWFSIPEKNRPLKGRINLVL

SREIJ EPPQGAHFLSRSLDDALKLTEQPELANKVDMVW

rVGGSSWKEAMNHPGHLKLFVTRMQDFESDTFFPEK)

LEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKDD

hDHFR (C7R, Y163C) MVGSLNRIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR

MTTTSSWGKQNLVDvlGKKTWSIPEKNRPLKGRINLVL

SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVD VW

IVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFPEID

LEKYKLLPECPGVLSDVQEEKGIKYKFEVYEKND hDHFR (A10V, H88Y) MVGSLNCIVWSQNMGIGKNGDLPWPPLRNEFRYFQR 24

MTTTSSVEGKQNLVMGKKTWS1PEK PJ>!JCGR1NLVL

SRELKhP QGAYFLSRSLDDALKLTEOPELANKVDMVW

IVGGSSVTKEAMNWI iHL LFVTRIMQDFESDTFFPEID

LEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND

hDHFR (Q36K, Y122I) MVGSLNCIVAVSQ MGIGKNGDLPWPPLRNEFRYFKR 25

MTTTSSVEGKQNLVIMGKKTWFSIPEK RPLKGRINLVL SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW

lYGGSSVI EAMNHPGHLKLFVrRIMQDFESDTFFPEIDL EKYKLLPEYPG VL SDVQEEKG 1KYKFE VYE ND hDHFR M53T, R138I) MVGSLNCrVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 26

NITTTSSVEGKQNLVTTGKKTWFSIPEKNRPLKGRINLVL SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVD VW IVGGSSVYKEAMNHPGHLKLFVTilMODFESDTFFPEIDL EKYKLLPEYPGVLSDVQEEKGIKYKFE VYEKND

hDHFR (T57A, I72A) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 27

MTTTSSVEGKQNLVTMGKKAWFSIPEKNRPLKGRANLV

LSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMV

WIVGGSSVTKEANINHPGHLKLFV RIMQDFESDTFFPEI

DLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND

hDHFR (E63G, I176F) MVGSLNCIVAVSQNMG1GKNGDLPWPPLRNEFRYFQR 28

MTTTSSVEGKQNLVMGKKTWFSIPGKNRPLKGRINLVL SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW IVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFPEK⁾ LEKYKLLPEYPGVLSDVQEEKGFKYKFE VYEKND

hDHFR (G2 IT, Y 1221) MVGSLNCIVAVSQNMGIGKNTDLPWPPLRNEFRYFQRM 29

TTTSSVEGKQNLVEViGKKTWFSiPEKNRPLKGRINLVLS RELKEPPQG AHFL SR SLDD ALKLTEQPEL ANK VDMVWI VGGSSVIKEAMNHPGHLKiJ'VTRIMQDFESDTFFPEIDL EKYKLLPEYPGVLSDVQEEKGIKYKFE VYEKND

hDHFR (L74N, Y122I) MVGSLNCiVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 30

MTTTSSVEGKQNLmiGKKTWFSIPEKNRPLKGRINNVT. SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW

rVGGSSVIKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDL EKYKLLPEYPGVLSDVQEEKGIKYKFE VYEKND

hDHFR (V75F, Y 1221) MVGSLNC I V A VSQNMG I G KNGDLPWPPLRNEFR YFQR 33

MTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLFL SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW IVGGSSVIKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDL EKYKLLPEYPGVLSDVQEEKGIKYKFE VYEKND

hDHFR (L94A, T147A) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 32

MITrSSVEGKQNLA'TMGKKTWFSIPEKNRPLKGRlNL T. SRELJ^PPQGAHFLSRSADD ALKLTEQPEL ANK VT5MVW IVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDAFFPEID LEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND

DHFR (V121A, Y22I) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 33

MTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVL SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW

rVGGSSAIKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDL EKYKLLPEYPGVLSDVQEEKGIKYKFE VYEKND

hDHFR (Y122I, A125F) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 34

MTTTSSVEGKQNLVIMGKKTWSIPEKNRPLKGRINLVL SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW IVGGSSVIKEFMNHPGHLKLFVTRIMQDFESDTFFPEIDL EKYKLLPEYPGVLSDVQEEKGIKYKFE VYEKND

hDHFR (H13 1R, E144G) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 35

MTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVL

SRELKhPPQGAHFLSRSLDDALKLTEQPELANKVDMVW

IVGGSSV KEAM HPGRLKLFVTRIMQDFGSDTFFPEID

LEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND hDHFR (T137R, F143L) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 36

MTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGR1NLVL

SREL JiP QGAHFLSRSLDDALKLTEOPELANKVDMVW rVGGSSV KEAM HPGHLKLFVRRTMQDLESDTFFPE )

LEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND

hDHFR (Y178H, E18IG) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 37

MTTTSSVEGKONLVIMGKKTWFSIPEKi RPLKGRINLVL SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW IVGGS S VYKEAMNHPGHLKL F VTRIMQDFESDTFFPEID LEKYKLLPEYPGVLSDVOEEKGIKHKFGVYEKND

hDHFR (Y183H, K185E) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 38

MTTTSSVEGKQNLVIMG TWSIPEKNRPLKGRINLVL SRELKEPPOGAHFLSRSLDDALKLTEQPELAiNKVD VW IVGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFPEID LEK YKLLPE YP GVL SD VQEEKGIK YKFE VHEEND

hDHFR (V9A, S93R, P I SOL) MVGSLNCIAAVSQNMGIGKNGDLPWPPLRNEFRYFQR 39

MTTTS S VEGKQNL VIMGKKTWFS1 PEKNRPLK GRINLVL SRELKEPPQGAHFLSRRLDDALKLTEQPELANKVDMVW IVGGSSVTKEAMNHPGHLKLFVTRIMQDFESDTFFLEID LEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND

hDHFR (I8V, K133E, Y163C) MVGSLNCVVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 40

MTTTSSVEGKQNLVMGKKTWFSIPEKNRPLKGRINLVL SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW IVGGSSVYKEAMNHPGHLELFVTRIMQDFESDTFFPEID LEKYKLLPECPGVLSDVQEEKGIKY t- fc VYEKND hDHFR (L23S, V121A, Y157C) MVGSLNCIVAVSQNMGIGKNGDSPWPPLRNEFRYFQR 41

MTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVL SRELKEPPOGAHFLSRSLDDALKLTEQPELAINKVD VW IVGGSSAYKEAMNHPGHL LF VTRIMQDFESDTFFPEID LEKCKLLPEYPGVLSDVQEE GIKYKFEVYEK D

hDHFR (K19E, F89L, E181G) MVGSLNCIVAVSQNMGIGENGDLPWPPLRNEFRYFQRM 42

TTTSSVE-GKQNLVIMGKKTWFSIPEKNRPLKGRINLVL-S

RELKEPPQGAHLLSRSLDDALKLTEQPELANKVDMVWI

VGGSSVYKE INHPGHLKLFVTRIMQDFESDTFFPEIDL EKYKLLPEYPGVLSD VQEEKGIK YKFGVYEKND

hDHFR (Q36F, N65F, ΥΊ22Ι) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFFRM 43

TTTSSVEGKQNLVIMGKKTWFSIPEKFRPLKGRINLVLS RELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVWI VGGSSVIKEAMNHPGHL-KLFVTR QDFESDTFFPEIDL

EKYKLLPEYPGVLSD VQEEKGIK YKFE\⁷YEKND hDHFR (G54R, M140V, S168C) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 44

MTTTS S VEGKQNL VDVIRKKTWFS iPEKNRPLKGR INI, VL SRELKEPPQGAHFLSRSLDDALKLTCQPELA KVDMVW IVGGS SV YKEAMNHPGHL LF VTRIVQDFE SDTFFPE IDL EK YKLLPEYPGVLCD VQE EKG IKYKFE VYEKND

ITDHFR (V110A, V136M, K177R) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 45

MTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVL SRELKEPPQGAHFLSRSLDDALKLTEQPELANKADMVW IVGGSSWKEAMNHPGHLKLFMTRiMQDFESDTFFPEID LEK YKLLPEYPGVLSDVQEEKGIR YKFE VYEKND

hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFFRMT 46 Q36F, Y122I, A125F) TTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSR

ELKEPPQGAHFLSRSLDDALKLTEQPELANKVDM IV GGSSVIKEFMNHPGHLKLFVTRIMQDFESDTFFPEIDLEK YKLLPEYPGVLSDVQEEKGIKYKFE VYEKND

hDHFR (N49D, F59S, D153G) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 47

MTTTS S VEGKQDL VIMGKKTWS Si PEKNRPLK GRINL VL SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVW

rVGGSSV KEAMNHPGHLKLFVTRIMQDFESDTFFPEIG LEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND hDHFR (G21E, 172 V, Π76Τ) MVGSLNCIVAVSQNMGIGKNEDLPWPPLRNEFRYFQRM 48

TTTSSVEGKQNLVMGKKTWFS!PEKNRPLKGRVNLVLS RELKEPPQGAHFL SRSLDD ALKLTEQPEL ANKVDMVWI VGG SS VYKEAMNHPGHT , T FVTRIMQDFESDTFFPEIDL E YKLLPEYPGVLSDVQEEKGTKY FEVYEKND

hDHFR (L100P, E102G, Q103R, MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 871 P104S, E105G, N 10SD, V113A, MTTTSSVEGKONLVIMGKKTWFSIPEKNRPLKGRINLVL Wl 14R, Y 122C, Ml 261, N127R, SRELKEPPQGAHFL SRSLDD ALKPTGR SGLADKVDMAR H128Y, L132P, F135P, I139T, rVGGSSVCKEAIRYPGHPKLPVTRTMQDFESDTSLPEVA F148S, F149L, 1152V, D153A, LEKYKLLPEYPGVLSGAQEEKGARYKFEAYERSD

D169G, V170A, I176A, K177R,

V182A, K185R, N186S)

hDHFR (V2A, R33G, Q36R, MAGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFGYFRR 872 L100P, K185R) MTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVL

SRELKEPPOGAHFLSRSLDDALKPTEQPELANKVDMVW IVGGSSVY EAMNHPGHLKLFVTRIMQDFESDTFFPEID LEK YKLLPEYP G VL SD VQEEKGIK YKFE VYERND

hDHFR (G16S, 117 V, F89L, MVGSLNCIVAVSQNMSVGKNGDLPWPPLRNEFRYFQR 873 D96G, K123E, M140V, D I46G, MTTTSS\¾GKQNLVDv GKKTWFSIPEKNRPLKGRINL\⁷L K156R) SRELKEPPOGAHL-LSRSLDGALKLTEQPELANKVDMVW

IVGGSSVYEEAM HPGHLKLF^'VTRIVQDFESGTFFPEIDL

ERYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND

hDHFR (F35L. R37G, N65A, MVGSLNCIVAVSQNMGIG NGDLPWPPLRNEFRYLQG 874 L68S, 69E, R71G, L80P, 99G, MTTTSSVEGKQNLVTMGKKTWFSIPEKARPSEGGINLVL Gl 17D, L132P, I139V, M140I, SREP EPPQGAHFL SRSLDD ALGLTEQPELANKVDMVW D142G, D146G, E173G, D 187G) iVDGSSVYKEAMNHPGHPKLFVTRVIQGFESGTFFPE;]DL

EKYKLLPEYPGVLSDVQEGKGIKYKFEVYEKNG

hDHFR (ΙΓ7Ν, L98S, K99R, MVGSLNCIVAVSQNMGNGKNGDLPWPPLRNEFRYFQR 875 M112T, E151G, E162G, E172G) MTTTSSWGKQNLVMGKKTWSIPEKNRPLKGRINLVL

SRELKEPPQGAHFLSRSLDDASRLTEQPELANKVDTVWI VGGSSVYKEANINHPGHLKLFVTRIMQDFESDTFFPGIDL EKYKLLPGYPGVL SD VQGEKGIKY KJr E VYEKND

hDHFR (R138G, D142G, F143S, MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 876 K156R, K158E, E162G, V166A, MTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVL K177E, Y178C, K185E, N186S) SRELKEPPQGAHFLSRSLDD ALKLTEQPEL ANKVDMVW

IVGGSSWKEAMNHPGHLKLFVTGiMQGSESDTFFPEiD LERY^rELLPGYPGAL,SDVQEEKGiECKFEWEESD

hDHFR (K83 R, K99R, L i OOP, MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 877 E102G, N108D, K123R, H128R, MTTTSSVEGKQNLVIMGKKTWSIPEKNRPLKGRINL^ D142G, F180L, K185E) SRELREPPQGAHFLSRSLDDALRPTGQPELADKVDMVW

IVGGSS^TIEAM RPGHLKLFWRIMQGFESDTFFPEID

LEKYKLLPEYPGVLSDVQEEKGKYKLE\TEEND hDHFR ( 14S, P24S, F35L, MVGSLNCIVAVSQSMGIGKNGDLSWPPLRNEFRYLQRM 878 M53T, K56E, R92G. S93G, TTTS S VEGKQNL V1TG KETWF SIPEKNRPL KGRINL VLS R N127S, H 128Y, F135L, F143S, ELKEPPQGAHFLSGGLDDALKLTEQPELANKVDMVWIV L159P, L160P, E173A, F380L) GGSSV KEAMSYPGHLKLLVTRIMQDSESDTFFPEIDLE

K YKPPPEYPG VLSD VQEAKGIK YKLEVYEKND

ITDHFR (V2A, I17V, N30D, MAGSLNCIVAVSQNMGVGKNGDLPWPPLRDGFRYFRR 879 E31G, Q36R, F59S, K69E, Γ72Τ, MTTTSSVEGKQNLVIMGKKTWSSIPEKNRPLEGRTNLV H88Y, F89L, N108D, 109E, LSRELKEPPQGAYLLSRSLDDALKLTEQPELADEAGMV V110A, 1115V, Y122D, L132P, WVVGGSSVDKEAM HPGHPKLSVTRrVQDFGSDAFFPE F135S, M140V, E144G, T147A, IDLEKCKLLPEYPGVLSDAQEERGIKYKFEVYEKSD

Y157C, V170A, K174R, 186S)

ITDHFR (L28P, N30H, M38V, MVGSLNCIVAVSQNMGIGKNGDLPWPPPRHEFRYFQRV 880 V44A, L68S, N73G, R78G, A97T, TTTSSAEGKQNLVIMGKKTWFSIPEKNRPSKGRIGLVLS K99R, A107T, K109R, D111N, GELKEPPQGAHFLSRSLDDTLRLTEQPELTNRVNMVWI L134P, F135V, T147A, I152V, VGG S S VYKEAMNHPGHT ,RP V VTRIMQDFE SD AFFPE YD K158R, E172G, V182A, E184R) LEKYRLLPEYPGVLSDVQGEKGIKYKFEAYRKND hDHFR (A10T, Q13R, N HS, MVGSLNCIVTVSRSMGIGKDGDLSWPPLRSEFRYFQRTT 881 N20D, P24S, N30S, M38T, T40A, ATSSVEGRQSLVmiGKRTWFSTPERNRPLRGRANLVLS K47R, N49S, K56R, 16 IT, K64R, GELKGPPQGAHLLSRSLDGALKLTEQPELADKVDWRI K69R, I72A, R78G, E82G, F89L, VGGSSVDEEAMNHPGHLKLFVTRVNIRGFESDTLFPGID D96G. N108D, M112V, Wi 14R, LGKRKLLPEYPGVLSDVREEKGIKYKLEVCGNN

Y122D, K123E, 1139V, Q141R,

D142G, F 148L, E153 G, E155G,

Y157R, Q171R, Y183C, E184G,

K185del, D187N)

hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGVGKNGDLPWPPLRNEFRYFQRM 882 117 V, Y122I) TTTSSVEGKQNLVTMGKKTWFSIPEKNRPLKGRINLVLS

RELKt QGAHFLSRSLDDALKLTEQPELANKVDMVWI VGGSSVKEAMNHPGHLKLFVTRI QDFESDTFFPEIDL EKYKLLPEYPG VL SDVQEEKG 1KYKFE VYE ND

hDHFR (Amino acid 2-187 of WT: VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 883 Y122L M140I) TTSSVEGKQNL-VIMGKKTWFSIPEKNRPLKGRINLVLSR

ELKEPPQGAHFLSRSLDD ALKLTEQPELANKVDMVWIV GGSSVIKEAMNHPGHLKLFVTRIIQDFESDTFFPEIDLEK YKLLPEYPGVLSDVOEEKGIKYKFE VYEKND

hDHFR (Amino acid 2-187 of WT; VGSLNCrVAVSQN GIGKNGDLPWPPLRNE-FRYFQRMT 884 N127Y, Y122I) TTSSVEGKQNLVIMGKKTWTSIPEKNRPLKGRINLVLSR

ELKEPPQGAHFLSRSLDDALKLTEQPELANKVDM IV GGSSVIKEAMYHPGHLKLFVTRIMQDFE-SDTFFPEIDLE KYKLLPEYPGVLSDVQEEKGIKYKFE VYEKND

hDHFR (Amino acid 2-187 of WT; VGSLNCiVAVSQNMGiGKNGDLPWPPLRNEFRYFQRMT 885 Y122L H 131R, E 144G) TTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSR

ELKEPPQGAHFLSRSLDDALKLTEQPELANKVD VWIV GGSSVTKEAMNHPGRLKLFVIRI QDFGSDTFFPEIDLE KYKL LPEYPG VL -SD VQEE KG TKYKFEVYEKND

hDHFR (Amino acid 2-187 of WT; VGSLNCrvAVSONMGIGKNGSLPWPPLRNEMSYFSRMT 886 D22S, F32M, R33S, Q36S, N65S) TTS S VEGKQNL VIMGKKT WF S 1PEK SRPL K GR1NL VL S R

ELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVWIV GGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDLE KYKLLPEYPGVLSDVQEEKGIKYKFE VYEKND

hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNDMRYFQRM 887 E31D, F32M, VI 161) TTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLS

RELKEPPQG AHFL SR SLDD ALKLTEQPEL ANK VDMVWII GGSSWKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDLE KYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND

hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 888 E162G, I176F) TTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSR

ELKEPPQGAHFLSRSLDD ALKLTEQPEL ANK VDM^'VWIV GGSSVYKEANiNHPGHLKLFWRIMQDFESDTFFPEIDLE KYKLLPGYPGVLSDVQEEKGFKYKFE VYEKND

hDHFR (Amino acid 2-187 of WT; VGSLNCrVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 889 K185E) TTSSVEGKQNLV1¾1GKKTWFS1PEKNRPLKGRINLVLSR

ELKEPPQGAHFLSRSLDD ALKLTEQPEL ANK VDMVWrV GGSSV KEAM HPGHLKLFVTRIMQDFESDTFFPEIDLE K YKLLPEYPG VLSI) VQEEKGIK YKFE VYEEND

hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 890 Y122I, A125F) TTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSR

ELKEPPQGAHFLSRSLDD ALKLTEQPELANKVDMVWIV GGSSVIKEFMNHPGHLKLFVTRIMQDFESDTFFPEIDLEK YKLLPEYPGVLSDVQEEKGIKYKFE VYEKND

hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFFRMT 891 Q36F, N65F, Y122I) TTSSVEGKQNLVIMGKKTWTSIPEKFRPLKGRINL^SR

ELKEPPQGAHFLSRSLDD ALKLTEQPELANKVDMVWIV GGSSVIKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDLE KYKLLPEYPGVLSDVQEEKGIK YKFE VYEKND

hDHFR (Amino acid 2-187 of WT, VGSLNCrVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 892 N127Y) T SSVEGKQNLVTMGKKTWFSIPEKNRPLKGRINLVLSR

ELKEPPQGAHFLSRSLDD ALKLTEQPEL ANK VDM 'WIV GGSS KEAMYHPGHLKLFVTR1MQDFESDTFFPEIDLE

KYKLLPEYPGVLSDVOEEKGIKYKFEVYEKND

hDHF (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 893 H131R, E144G) TTSSVEGKQNLVTMGKKTWFSIPEKNRPLKGRINLVLSR

ELKEPPQGAHFLSRSLDDALKLTEOPELA KVDMVWIV GGSSVYKEANfNHPGRLKLFVTRIMQDFGSDTFFPEIDLE

KYKLLPEYPGVLSDVQEEKGIKYKFEVYEK D

hDHFR (Amino acid 2-187 of WT; VGSLNCrVAVSQN GVGKNGDLPWPPLRNEFRYFQRM 894 117V) TTTSSVE-GKQNLVTMGKKTWFSIPEKNRPLKGRrNLVL-S

RELKEPPQG AHFL SR SLDD ALKLTEQPEL A K VDMVWI VGG S S VYKE AMNHPGHLKLF VTRIMQDFE SDTFFPEIDL EKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND

hDHFR (Amino acid 2-187 of WT; VGSLNCTVAVSQNMGTGKNGDLPWPPLRNEFRYFQRMT 895 Υ122Γ) TTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSR

ELKEPPQGAHFLSRSLDDALKLTEQPELANKVD VWIV

GGSSVTKEAMNHPGHLKLFVTRIMQDFESDTFFPEIDLE

KYKLLPEYPGVLSDVQEEKGTKYKFEVYEKND

hDHFR (E162G, 1176F) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 896

XITTTSSVEGKQNLVDVIGKKTWFSIPEKNRPLKGRINLVL

SRELKEPPQGAHFLSRSLDDALKLTEOPELANKVDMVW

WGGSSVYKEAMNHPGHLKLFVTRIMQDFESDTFFPEID

LEKYKLLPGYPGVLSDVQEEKGFKYKFEVYEKND

hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFKRMT 981 Q36K, Y122I) TTSSVEGKQNLVIMGKKTWSIPE RPLKGRINLVLSR

ELKEPPQGAHFLSRSLDDALKLTEQPELANKVDMVWIV

GGSSVIKEAM HPGHLKLFVTRIMQDFESDTFFPEIDLE

KYKLLPEYPGVLSDVOEEKGIKYKFEVYEK D

00150] 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; arginme 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, D22S, E31 D, F32M, R33S, Q36S, N65S, and VI 161. The position of the mutated amino acids is relative to the wildtype human DHFR (Umprot ID: P00374) of SEQ ID NO. 2.

[00151 ] 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, AIOT, A GV, Q13R, N14S, G16S, 1T7N, 117V, K19E, N20D, G21T, G21E, D22S, L23S, P24S, L28P, N30D, N30H, N30S, E31 G, 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, R.92G, S93G, S93R, L94A, D96G, A97T, L98S, K99G, K99R, L100P, E102G, Q103R, P104S, E105G, A107T, A107V, N108D, K109E, K1G9R, V110A, D111N, M112T, Ml 12V, V113A, W114R, I115V, 1115L, VI 161, G117D, V121A, Y122C, Y122D, Y122L K123R, K123E, A125F, M126I, N127R, N127S,N127Y, H128R, H128Y, H131R, L132P, K133E, L134P, 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, ΥΊ 57C, 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, 186D, D187G, and D187N.

[00152] 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 Ml del mutation.

[00153] 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 Ml del mutation), and may include one, two, three, four, five or more mutations including, but not limited to, Ml del, V2A, C7R, I8V, V9A, A10T, A10V, Q13R, N14S, G16S, I17N, 117V, K19E, 20D, G21T, G21E, D22S, L23S, P24S, 1.28 P. N30D, N30H, N30S, E31G, E31D, F32M, R33G, R33S, F35L, Q36R, Q36S, Q36K, Q36F, R37G, M38V, M38T, T40A, V44A, K47 . 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, D96G, A97T, L98S, K99G, K99R, L100P, E102G, Q103R, P104S, E105G, A107T, A107V, N108D, K109E, K109R, VI 10A, Dl 1 IN, Ml 12T, Ml 12V, VI 13A, Wl 14R, 1115V, I115L, VI 161, G117D, V121A, Y122C, Y122D, Y122I, K123R, K123E, A125F, M126I, N127R, N127S, N127Y, H128R, H128Y, H131R, L132P, K133E, L134P, F135P, F135L,

F135S, F135V, V136M, T137R, R138G, R138I, I139T, I139V, M140I, M140V, Q141R, D142G, F143S, F143L, E144G, D146G, T147A, F148S, F148L, F149L, P150L, E151G, 1152V, D153A, D153G, E155G, K156R, Y157R, Y157C, K158E, K158R, L159P, L160P, E162G, Y163C, V166A, S168C, D169G, V170A, Q171R, E172G, E173G, E173A, K174R, I176A, I176F, I176T, 177E, K177R, Y178C, Y178H, F180L, E181G, V182A, Y183C, Y183H, E184R, E184G, K185R, K185del, K185E, N186S, N186D, D187G, and D187N.

2. Payloads: Immuiiotherapeutic agents

[00154] 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, an antibody and fragments and variants thereof, a chimeric antigen receptor (CAR), a chimeric switch receptor, a cytokine, chemokine, a cytokine receptor, a chemokine receptor, a cytokine-cytokine receptor fusion polypeptide, 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.

Antibodies

[00155] In some embodiments, antibodies, fragments and variants thereof are payloads of the present invention.

[00156] 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 4/1 1 /2016, or in US Provisional Application No.

62/466,596 filed March 3, 2017 and the International Publication WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.

Antibody fragments and variants

[00157] 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 multi specific 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.

[00158] 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 Sight 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.

[00159] 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 (FVV) 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 (Strohi, 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 Kabai (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).

|00160] VH and VL domains have three CDRs each. VL CDRs are referred to herein as CDR- Ll, CDR-L2 and CDR-L3, in order of occurrence when moving from N- to C- terminus along the variable domain polypeptide. 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., Peer,!, 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).

[00161] 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 fl exible 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).

[00162] 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., IgGl, lgG2, IgG3, IgG4, IgA, and IgA2.

[00163] 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 scFv 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.

[00164] 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)." Blinatumomab is an anti-CD 19/anti-CD3 bispecific tascFv that potentiates T-cell responses to B-cell non-Hodgkin lymphoma in Phase 2, MT110 is an anti-EP-CA /anti-CD3 bispecific tascFv that potentiates T-cell responses to solid tumors in Phase 1. Bispecific, tetravalent "TandAbs" are also being researched by Λ filmed (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.

[00165] 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 et al., 2005. Acta Pharmacologica Sinica. 2005, 26(6): 649- 658 and Schaefer et al, PNAS. 201 1, 108(27): 11187-11192, the contents of each of which are herein incorporated by reference in their entirety.

[00166] 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 93/1 1161; and I loi linger 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.

[00167] The term "intrabody" refers to a form of antibody that is not secreted from a ceil 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 .

[00168] 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), eacli monoclonal antibody is directed against a single determinant on the antigen.

[00169] 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.

[00170] 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, affinity, 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. US201303G3399, the contents of which are herein incorporated by reference in its entirety.

[00171 ] As used herein, the term "antibody variant" refers to a modified antibody (in relation to a native or starting antibody) or a biomoiecuie 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, IgG l, IgG2, IgG3, IgG4, or IgM), humanized variants, optimized variants, multispecific antibody variants (e.g., bispecific variants), and antibody fragments.

[00172] 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, affiiins, affitins, anticalins, avimers, Centyrins, DARPINSTM, Fynomers and Kunitz and domain peptides. In other embodiments, antibody mimetics may include one or more non-peptide regions.

[00173] In one embodiment, the antibody may comprise a modified Fc region. As a non- limiting example, the modified Fc region may be made by the methods or may be any of the regions described in US Patent Publication NO. US20150065690, the contents of which are herein incorporated by reference in its entirety.

[00174] In some embodiments, payioads 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 optim ized by the methods described in International Patent Publication NO. WO2011109726 and US Patent Publication NO. US20150252119, 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.

[00175] 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 ai., PNAS. 2011, 108(27): 1 1 187-11192, the contents of each of which are herein incorporated by reference in their entirety . New generations of BsMAb, called 'Afunctional 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 Fc region (the foot) comprises the two heavy chains and forms the third binding site.

[00176] In some embodiments, payioads 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 m camels and llamas, which lack light chains (Nelson, A. L., MAbs.2010. Jan-Feb;

2(l):77-83).

[00177] In some embodiments, the antibody may be "miniaturized". Among the best examples of mAb miniaturization are the small modular immunopharmaceuticals (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 SMTP developed in collaboration with Wyeth, is the most advanced project, having progressed to Phase 2 for rheumatoid arthritis (RA). Earlier attempts in systemic lupus erythrematosus (SLE) and B ceil 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 SB1-087 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).

[00178] 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. ! ... MAbs, 2010. Jan-Feb; 2(l):77-83).

[00179] In some embodiments, pay loads of the invention may encode single-domain antibodies (sdAbs, or nanobodies) which are antibody fragment consisting of a single monomelic 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 Ig 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, J. Immunol. Methods, 1999, 231 : 25-38; international patent publication NOs. WO 1994/04678 and WO 1994/025591; 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 complementar - determining region (CDR) loops including inter-loop disulphide bridges, and patterns of intra-loop hydrogen bonds.

[00180] 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.

[001811 The intracellular expression of intrabodies in different compartments of mammalian cells allow s 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. U.S.A . 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.

[00182] 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 compri sing the variable domains of the heavy and light chains joined by a flexible linker polypeptide, interbodies typically lack disulfide bonds and are capable of m odulating the expression or activity of target genes through their specific binding activity. Single chain mtrabodies are often expressed from a recombinant nucleic acid molecule and engineered to be retained intracellulariy (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 ui., 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, Rum. Gene Therap., 7: 1515-1525; Marasco, Gene Ther. 4: 1 1 -15, 1997; Rondon and Marasco, 1997, Annu. Rev. Microbiol. 51 :257-283; Cohen, et al., 1998, Oncogene 17:2445-56; Proba et al., 1998, 1. 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 : 1 1 19-1128; Ohage et al., 1999, J. Mol. Biol. 291 : 1 129- 134; 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).

[00183] In some aspects, payioads 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.

[00184] In some embodiments, payioads 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. |0018S] 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. WO2015175375 and WO2016036916 and US Patent Pubhcation No. US20140378660, the contents of each of which are incorporated herein by reference in their entirety.

Antibody preparations

[00186] 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, 1 99 and "Therapeutic Antibody Engineering: Current and Future Advances Driving the Strongest Growth Area in the Pharmaceutical Industry" Woodhead Publishing, 2012.

[00187] 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: ( 1) 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 VHl , CHI , CH2, CH3 domains, a linker and the light chain or (6) the VHl, 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.

[00188] 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 |00189] In some embodiments, payloads of the present invention may be antibodies, fragments and variants thereof which are specific to tumor speci fic antigens (T^'SAs) 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 tumor 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.

[00190] 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.

[00191] In one embodiment, the antigen is CD 19, a B-cell surface protein expressed throughout B-cell development. CD19 is a well-known B cell surface molecule, which upon B cell receptor activation enhances B-celi antigen receptor induced signaling and expansion of B cell populations. CD19 is broadly expressed in both normal and neoplastic B cells. Malignancies derived from B cells such as chronic lymphocytic leukemia, acute lymphocytic leukemia and many non-Hodgkin lymphomas frequently retain CD 19 expression. This near universal expression and specificity for a single cell lineage has made CD 19 an attractive target for immunotherapies. Human CDI9 has 14 exons wherein exon 1-4 encode the extracellular portion of the CD 19, exon 5 encodes the transmembrane portion of CD19 and exons 6-14 encode the cytoplasmic tail.

[00192] In one embodiment, payloads of the present invention may be antibodies, fragments and variants thereof which are specific to CD 19 antigen.

[00193] In one embodiment, the payload of the invention may be a FMC63 antibody, antibody fragment of variant. FMC63 is an IgG2a mouse monoclonal antibody clone specific to the CD 19 antigen that reacts with CD19 antigen on cells of the B cell lineage. The epitope of CD19 recognized by the FMC63 antibody is in exon 2 (Sotillo et al (2015) Cancer Discov ;5(12): 1282- 95: the contents of which are incorporated by reference in their entirety). In some embodiments, the payload of the invention may be other CD 19 monoclonal antibody clones including but not limited to 4G7, SJ25C1, CVID3/429, CVID3/I55, HIB19, and J3-119.

[00194] In some embodiments, the payloads of the present invention may include variable heavy chain and variable light chain comprising the amino acid sequences selected from those in Table 4.

Table 4: Variable Heavy and Light Chain Sequences

CD 19 VK 82 SEQ Π) NO: 6 in US20160319020

CD19 VL 83 SEQ ID NO: 27 in WO2016168773 A3

CD19 VL 84 SEQ ID NO: 31 in WO2016168773 A3

CD 19 VL 85 SEQ ID NO: 49 in WO2016187349 Al

CD 19 VL 86 SEQ ID NO. 1 1 in WO2016134284

CD19 VL 87 SEQ ID NO. 194 in US20140134142A1

CD 19 VL 88 SEQ ID NO. 54 in WO2016120216

CD 19 VL. 89 SEQ ID NO. 56 in WO2016120216

CD 19 VL 90 SEQ ID NO: 13 in US20160152723

CD19 VL 91 SEQ ID NO: 14 in US20160152723

CD 19 VL 92 SEQ ID NO: 15 in US20160152723

CD 19 VL 93 SEQ ID NO: 16 in US20160152723

CD 19 VL 94 SEQ ID NO: 17 in US20160152723

CD19 VL. 95 SEQ ID NO: 186 in US20160152723

CD 19 VL 96 SEQ ID NO: 187 in US20160152723

CD 19 VL 97 SEQ ID NO: 188 US20160152723

CD 19 VL 98 SEQ ID NO: 389 in US20160152723

CD19 VL 99 SEQ ID NO: 192 in US20160152723

CD 19 VL 100 SEQ ID NO: 196 in US20160152723

CD 19 VL 101 SEQ ID NO: 197 in US20160152723

CD! 9 VL 102 SEQ ID NO: 198 in US20160152723

CD19 VL 103 SEQ ID NO: 199 in US20160152723

CD! 9 VL 104 SEQ ID NO: 200 in US20160152723

CD 19 VL 105 SEQ ID NO: 201 in US20160152723

CD 19 VL 106 SEQ ID NO: 202 in US20160152723

CD19 VL 107 SEQ ID NO: 203 in US20160152723

CD 19 VL 108 SEQ ID NO: 204 in US20160152723

CD 19 VL 109 SEQ ID NO: 205 in US20160152723

CD 19 VL 1 10 SEQ ID NO: 22 in US20160039942

CD19 VL. 111 SEQ ID NO: 63 in WO2016097231

CD 19 VL 112 SEQ ID NO: 64 in US20160152723

CD19 VL. 113 SEQ ID NO: 66 in US20160152723

CD 19 VL 114 SEQ ID NO: 67 in IJS20160352723

CD19 VL 115 SEQ ID NO: 68 in US20160152723

CD 19 VL 116 SEQ ID NO: 69 in US20160152723

CD19 VL 117 SEQ ID NO: 70 in US20160152723

CD 19 VL 1 18 SEQ ID NO: 73 in US20160152723

CD 19 VL 119 SEQ ID NO: 91 in US20160152723

CD 19 VL 120 SEQ ID NO. 3 in US20160145337A1

CD19 VL 121 SEQ ID NO: 112 in US20160333114A1

CD 19 VL 122 SEQ ID NO: 114 in US201603331 14A l |0019S] A tumor specific antigen (TSA) may be a tumor neoantigen. A neoantigen is a mutated antigen 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). In the context of CD 19, neoantigens such as a transcript variant of CD 19 lacking exon 2 or lacking exon 5-6 or both have been described (see International paieni publication No. WO2016061368; the contents of which are incorporated herein by reference in their entirety). Since FMC63 binding epitope is in exon 2. CD 19 neoantigen lacking exon 2 is not recognized by FMC63 antibody. Thus, in some embodiments, payloads of the invention may include FMC63-distinct antibodies, or fragments thereof. As used herein 'TMC63 -distinct" refers, to an antibody or fragment thereof that is immunologically specific and binds to an epitope of the CD 19 antigen that is different or unlike the epitope of CD 19 antigen that is bound by FMC63. In some instances, antibodies of the invention may include CD19 antibodies, antibody fragments or variants that recognize CD 19 neoantigens including the CD 19 neoantigen lacking exon2. In one embodiment, the antibody or fragment thereof is immunologically speci fic to the CD 19 encoded by exon 1, 3 and/or 4. In one example, the antibody or fragment thereof is specific to the epitope that bridges the portion of CD 19 encoded by exon 1 and the portion of CD 19 encoded by exon 3.

[00196] Chimeric antigen receptors (CARs)

[00197] 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 redirect the immune cells against the target (e.g., a tumor cell) which expresses a molecule recognized by the extracellular target moiety of the CAR.

[00198] 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€ϋ3ζ), and/or one or more costimulator signaling domains, such as those from CD28, 4-1BB (CDI37) and OX-40 (CD134). For example, a "first-generation CAR" only has the 033ζ 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 CD3 ignal domain plus one costimulatory signaling domain, and third generation CARs having 0Ο3ζ signal domain plus two or more costimulatory 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.

[00199| 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 costimulatory domains. The CA T cell, once activated, can destroy the target cell.

[00200] 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 AP21 67. 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).

[00201] 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 CD 19 CAR construct. The CD 19 CAR is activated only in the presence of doxycycline (Dox). Sakemura reported that Tet-CD19CAR T cells in the presence of Dox were equivalent!}' 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 Immuno. Res., 2016, Jun 21, Epub ahead of print). In one example, this Tet-CAR may be the payioad 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 CA R expression in transduced T cells. |00202] 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.

[00203] 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 in vivo.

[00204] 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 antitumor efficacy may prevent ( 1) On target off tumor toxicity caused by antigen expression in normal tissue, (2) antigen independent activation in vivo.

[00205] In one embodiment, selection of a CAR with a lower affinity may provide more T cell signaling and less toxicity.

Extracellular targeting domain/moiety

[00206] 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 ceils, 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.

[00207] 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 unithody, 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 CAR T 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 scFv can be generated using routine recombinant DNA technology techniques and is discussed in the present invention.

[00208] In one embodiment, the targeting moiety of the CAR may recognize CD19. CD19 is a well-known B cell surface molecule, which upon B cell receptor activation enhances B-cell antigen receptor induced signaling and expansion of B cell populations. CD 19 is broadly expressed in both normal and neoplastic B cells. Malignancies derived from B cells such as chronic lymphocytic leukemia, acute lymphocytic leukemia and many non-Hodgkin lymphomas frequently retain CD 19 expression. This near universal expression and specificity for a single cell lineage has made CD19 an attractive target for immunotherapies. Human CD 19 has 14 exons wherein exon 1-4 encode the extracellular portion of the CD 19, exon 5 encodes the transmembrane portion of CD19 and exons 6-14 encode the cytoplasmic tail. In one

embodiment, the targeting moiety may comprise scFvs derived from the variable regions of the FMC63 antibody. FMC63 is an IgG2a mouse monoclonal antibody clone specific to the CD 19 antigen that reacts with CD19 antigen on cells of the B lineage. The epitope of CD19 recognized by the FMC63 antibody is in exon 2 (Sotillo et al (2015) Cancer Discov ;5(12): 1282-95; the contents of which are incorporated by reference in their entirety ). In some embodiments, the targeting moiety of the CAR may be derived from the variable regions of other CD 19 monoclonal antibody clones including but not limited to 4G7, SJ25C 1 , CVID3/429, CVID3/155, AB 19, and J3-119.

[00209] 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). In the context of CD 19, TSAs may include a transcript variant of human CD 19 lacking exon 2 or lacking exon 5-6 or both (see International patent publication No. WO2016061368; the contents of which are incorporated herein by reference in their entirety). Since FMC63 binding epitope is in exon 2, CD 19 lacking exon 2 is not recognized by FMC63 antibody. Thus, in some embodiments, the targeting moiety of the CAR may be an FMC63 -distinct scFV. As used herein "FMC63-distinct" refers, to an antibody, scFv or a fragment tliereof that is immunologically specific and binds to an epitope of the CD19 antigen that is different or unlike the epitope of CD 19 antigen that is bound by FMC63. In some instances, targeting moiety may recognize a CD 19 antigen lacking exon2. In one embodiment, the targeting moiety recognizes a fragment of CD19 encoded by exon 1 , 3 and/or 4. In one example, the targeting moiety recognizes the epitope that bridges the portion of CD 19 encoded by exon 1 and the portion of CD 19 encoded by exon 3.

[00210] In some embodiments, the targeting moieties of the present invention may be scFv comprising the amino acid sequences in Table 5.

5: scFv sequences

CD 19 scFv 153 SEQ ID NO. 4 in WO2016033570

CD 19 scFv 154 SEQ ID NO. 45 in WO2016033570

CD 19 scFv 155 SEQ ID NO. 47 in WO2016033570

CD 19 scFv 156 SEQ ID NO. 49 in WO2016033570

^: CD 19 scFv 157 SEQ ID NO. 5 in WO2015 155341 Al

CD 19 scFv 158 SEQ ID NO. 5 in WO2015157252

CD 19 scFv 159 SEQ ID NO. 5 1 in WO2016033570

CD 19 scFv 160 SEQ ID NO. 53 in WO2016033570

CD 19 scFv 161 SEQ ID NO. 55 in WO2016033570

CD 19 scFv 162 SEQ ID NO. 57 in WO2016033570

CD 19 scFv 163 SEQ ID NO. 59 in WO2015157252

^: CD 19 scFv 164 SEQ ID NO. 59 in WO2016033570

CD 19 scFv 165 SEQ ID NO. 6 in WO2015157252

CD 19 scFv 166 SEQ ID NO. 6 in WO2016033570

CD 19 scFv 167 SEQ ID NO. 7 in WO2014184143

CD 19 scFv 168 SEQ ID NO. 7 in WO2015 157252

CD 19 scFv 169 SEQ ID NO. 8 in WO2015157252

CD 19 scFv 170 SEQ ID NO. 8 in WO2016033570 ^h CD 19 scFv 171 SEQ ID NO. 87 in WO2016033570

CD 19 scFv 172 SEQ ID NO. 9 in WO2015157252

CD 19 scFv 173 SEQ ID NO.9 in WO2016139487

CD 19 scFv 174 SEQ ID NO. 10 in US20160152723

CD 19 scFv 175 SEQ ID NO. 2 in US20160152723

CD 1 scFv 176 SEQ ID NO. 206 in US20160152723

CD 19 scFv 177 SEQ ID NO. 207 in US20160152723 ^h CD 19 scFv 178 SEQ ID NO. 208 in US20160152723

CD 19 scFv 179 SEQ ID NO. 209 in US20160152723

CD 19 scFv 180 SEQ ID NO. 210 in US20160152723

CD 19 scFv 181 SEQ ID NO. 211 in US20160152723

CD 19 scFv 182 SEQ ID NO. 212 in US20160152723

CD 1 scFv 183 SEQ ID NO. 213 in US20160152723

CD 19 scFv 184 SEQ ID NO. 214 in US20160152723 ^h CD 19 scFv 185 SEQ ID NO. 215 in US20160152723

CD 19 scFv 186 SEQ ID NO. 216 in US20160152723

CD 19 scFv 187 SEQ ID NO. 217 m US20160152723

CD 19 scFv 188 SEQ ID NO. 2,18 in US20160152723

CD 19 scFv 189 SEQ ID NO. 219 in US20160152723

CD 19 scFv 190 SEQ ID NO. 220 in US20160152723

CD 19 scFv 191 SEQ ID NO. 221 in US20160152723 ^h CD 19 scFv 192 SEQ ID NO. 222 in US20160152723

CD 19 scFv 193 SEQ ID NO. 223 in US20160152723

CD 19 scFv 194 SEQ ID NO. 224 m US20160 152723

CD 19 scFv 195 SEQ ID NO. 225 in US20160152723

CD 19 scFv 196 SEQ ID NO. 32 in EP3083691A2

CD 19 scFv 197 SEQ ID NO. 35 in EP3083691A2

CD 19 scFv 198 SEQ ID NO. 38 in EP3083691A2

^: CD 19 scFv 199 SEQ ID NO. 4 in US20160152723

CD 19 scFv 200 SEQ ID NO. 45 in US20160152723

CD 19 scFv 201 SEQ ID NO. 47 in US20160152723

CD 19 scFv 202 SEQ ID NO. 49 in US20160152723

CD 19 scFv 203 SEQ ID NO. 51 in US20160152723

CD 19 scFv 204 SEQ ID NO. 53 in US20160152723

CD 19 scFv 205 SEQ ID NO. 55 in US20160152723 ^h CD 19 scFv 206 SEQ ID NO. 57 in US20160152723

CD 19 scFv 207 SEQ ID NO. 59 in US20160152723

CD 19 scFv 208 SEQ ID NO. 6 in IJS20160152723

CD 19 scFv 209 SEQ ID NO. 8 in US20160152723

CD 19 scFv 210 SEQ ID NO. 87 in US20160152723 CD 19 scFv 21 1 SEQ ID NO. 89 in US20160152723

CD 19 scFv 212 SEQ ID NO. 39 in WO2016109410

CD 19 scFv 213 SEQ ID NO. 37 in EP3083671A 1

CD 19 scFv 214 SEQ ID NO. 174 in WO2016115482

^: CD 19 scFv 215 SEQ ID NO. 20 in WO2012079000

CD 19 scFv 216 SEQ ID NO. 32 in WO2015092024

CD 19 scFv 217 SEQ ID NO. 33 in WO2015092024 A2

CD 19 scFv 218 SEQ ID NO. 35 in WO2015092024A2

CD 19 scFv 219 SEQ ID NO. 38 in WO2015092024A2

CD 19 scFv 220 SEQ ID NO. 40 in WO2016 109410

CD 19 scFv 221 SEQ ID NO. 41 in WO2016109410

^: CD 19 scFv 222 SEQ ID NO. 42 in WO2016109410

CD 19 scFv 223 SEQ ID NO. 43 in WO2016109410

CD 19 scFv 224 SEQ ID NO. 44 in WO2016 109410

CD 19 scFv 225 SEQ ID NO. 45 in WO2016109410

CD 19 scFv 226 SEQ ID NO. 46 in WO2016109410

{ ^' P^; scFv 227 SEQ ID NO. 47 in WO2016 109410

CD 19 scFv 228 SEQ ID NO. 48 in WO2016109410 ^h CD 19 scFv 229 SEQ ID NO. 49 in WO2016109410

CD 19 scFv 230 SEQ ID NO. 5 in WO2015155341A1

CD 19 scFv 231 SEQ ID NO. 50 in WO2016 109410

CD 19 scFv 232 SEQ ID NO. 51 in WO2016109410

CD 19 scFv 233 SEQ ID NO. 7 in US20160145337Al

CD 1 scFv 234 SEQ ID NO. 9 in US20160145337A1

CD 19 scFv 235 SEQ ID NO.20 in US9499629B2 ^h CD 19 scFv 236 SEQ ID ΝΌ.6 in WO2015155341A1

CD 19 scFv 237 SEQ ID ΝΌ.73 in WO2016164580

CD 19 scFv 238 SEQ ID NO. 10 US20160152723

CD 19 scFv 239 SEQ ID NO. 2 in US20160152723

CD 19 scFv 240 SEQ ID NO. 206 in US20160152723

CD 1 scFv 241 SEQ ID NO. 207 in US20160152723

CD 19 scFv 242 SEQ ID NO. 209 in US20160152723 ^h CD 19 scFv 243 SEQ ID NO. 210 in US20160152723

CD 19 scFv 244 SEQ ID NO. 212 in US20160152723

CD 19 scFv 245 SEQ ID NO. 216 m US20160 152723

CD 19 scFv 246 SEQ ID NO. 218 in US20160152723

CD 19 scFv 247 SEQ ID NO. 219 in US20160152723

CD 19 scFv 248 SEQ ID NO. 220 in US20160152723

CD 19 scFv 249 SEQ ID NO. 221 in US20160152723 ^h CD 19 scFv 250 SEQ ID NO. 222 in US20160152723

CD 19 scFv 251 SEQ ID NO. 223 in US20160152723

CD 19 scFv 252 SEQ ID NO. 224 m US20160 152723

CD 19 scFv 253 SEQ ID NO. 225 in US20160152723

CD 19 scFv 254 SEQ ID NO. 4 in US20160152723

CD 19 scFv 255 SEQ ID NO. 45 in US20160152723

CD 19 scFv 256 SEQ ID NO. 47 in US20160152723

^: CD 19 scFv 257 SEQ ID NO. 49 in US20160152723

CD 19 scFv 258 SEQ ID NO. 51 in US20160152723

CD 19 scFv 259 SEQ ID NO. 53 in US20160152723

CD 19 scFv 260 SEQ ID NO. 55 in US20160152723

CD 19 scFv 261 SEQ ID NO. 57 in US20160152723

CD 19 scFv 262 SEQ ID NO. 59 in US20160152723

CD 19 scFv 263 SEQ ID NO. 6 in US20160152723 ^h CD 19 scFv 264 SEQ ID NO. 8 in US20160152723

CD 19 scFv 265 SEQ ID NO. 87 in US20160152723

CD 19 scFv 266 SEQ ID NO. 89 in US20160152723

CD 19 scFv 267 SEQ ID NO. 5 in WO2016055551 Intracellular signaling domains

[00211] The intracellular domain of a CAR fusion polypeptide, 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 cytolytic activity (e.g., cytokine secretion) or helper activity. Therefore, the intracellular domain comprises an "intracellular signaling domain" of a T cell receptor ( ICR)

[00212] 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 as long as it transduces the effector function signal.

[00213] 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 ITAM containing cytoplasmic signaling sequences include those derived from. TCR CD3zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CDS epsilon, CDS, CD22, CD79a, CD79b, and CD66d. In one example, the intracellular signaling domain is a CDS zeta (033ζ) signaling domain.

[00214] 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., CAR T cells). In some cases, the

costimulatory signaling region contains I , 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, 4-1 BB (CD 137), OX40 (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- IBB (CD 137). In another example, the costimulatory signaling domain may be an intracellular domain of GITR as taught in U.S. Pat. NO.: 9, 175, 308; the contents of which are incorporated herein by reference in its entirety.

[00215] 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 T F receptor protein, an immunoglobulin-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 Toil ligand receptor, GX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD1 la/CD 18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), G1TR, BAFFR, LIGHT, HVEM (LIGHTR), SLAMF7, N p80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CDSalpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ILlSRa, ITGA4, VLA 1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD! Id, ITGAE, CD103, ITGAL, CDl la, LFA-1, ITGAM, CDl lb, ITGAX, CDl lc, ITGBl, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, NKD2C SLP76, TNFR2, TRANCE RA KL, DNAMl (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAMl, CRTAM, Ly9 (CD229), CD160 (BY55), PSGLi, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl 08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, CD270 (HVEM), GADS, SLP-76, PAG/Cbp, CD 19a, a ligand that specifically binds with CD83, DAP 10, TRIM, ZAP70, Killer immunoglobulin receptors (KIRs) such as KIR2DL1 ,

K1R2DL2/L3, K1R2DL4, K1R2DL5A, KIR2DL5B, KIR2DS 1, KIR2DS2, KIR2DS3, KIR2DS4, K1R2DS5, KIR3DL1/S 1, KIR3DL2, KIR3DL3, and KIR2DP1; lectin related NK cell receptors such as Ly49, Ly49A, and Ly49C.

[00216] 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.

[00217] 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.

[00218] In some embodiments, the intracellular domain of the present invention may comprise amino acid sequences of Table 6. Description Amino Acid Sequence

2334 co-stimulatory WR KR EKQSETSPKEFLT1YEDV LKTRRNHEQEQTFP domain GGGSTIYSMlQSQSSAPTSQEPAYTLYSLiQPSRKSGSRKRN

HSPSFNST1YEVIGKSQPKAQNPARLSRKELENFDVYS

CD27 co-stimulatory HQRRKYRSN GESPVEPAEPCRYSCPREEEGSTiPIQEDYR domain KPEPACSP

CD272 (BTLA1) co- RRH QGKQNEL SDTAGREINL VD AHLK SEQTE ASTRQN SQ stimulatory domain VLLSETGI YD NDPD LCFRMQEG SEVYS NPCLE E NKPG VY A

SLNHSVIGPNSRLARNVKEAPTEYASICVRS

CD272 (BTLA1) co- CCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQ stimulatory domain NSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPG

IVYASLNHSVIGPNSRLAR V EAPTEYASICVRS

CD28 co-stimulatorv FWVLVWGGVLACYSLLVTVAFIIFWV

CD28 co-stimulatory KRGRKKLLYTFKQPFMRPVQTTQEEDGCSCRFPEEEEGGC domain EL

CD28 co-stimulatory FWVRSi )YMNMTPRRPGPTRKHYQPYAPPRDF domain AAYRS

CD28 co-stimulatory RSKRSRGGHSDYMNMTPRRPGPTR HYQPYAPPRDFAAY domain RS

CD28 co-stimulatoiy RSKRSRGGHSDYIVINMTPRRPGPTRKHYQPYAPPRDFAA domain YRS

CD28 co-stimulatory MI^LI ALNLFPSIQWGNKILVKQSPMLVAYDNAV LSC signaling region KYSYNLFSREFRASLHKGLDSAVEVCWYGNYSQQLQVY

SKTGFNCDGKLGISIESWFYLQNLYYNIQTDIYFCKIEVMYP PPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWG GVLACYSLLWVAFIIFWVRSKRSRLLHSDYMNrN rPRRPG PTRKHYQPYAPPRDF AAYRS

CD30 co-stimulatory RRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLR domain SGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQD

ASPAGGPSSPRDLPEPRVST TNNKIE IYIMKADTVIVG

TVKAELPEGRGLAGPAEPELEEELEADHTPHYPEQETEPPL

GSCSDVMLSVEEEGKEDPLPTAASGK

CD30 co-stimulatoiy RRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLR domain SGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQD

ASPAGGPSSPRDLPEPRVSTEHT iv!KIEKIYm KADTViVG TVKAELPEGR G L AGP/ ZQETEPPL

GSCSD ¾/[i,SWI-iEGKEDPLPTAASGK

GITR co-stimulatory HIWQLRSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGE domain RSAEEKGRLGDLWV

HVEM co-stimulatory CVKRRKPRGDWKVIVSVQRKRQEAEGEATVIEALQAPP domain DVTTVAVEETIPSFTGRSPNH

ICOS co-stimulatory TKKKYS S S VHDPNGEYMFMRAVNTAKKSRLTD VTL domain

ICOS co-stimulatory CWLTKKKYSSSVHDPNGEYMFMRAVNTAK SRLTDVTL signaling domain

LAG-3 co-stimulatory HLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEP regkm EPEPEPEPEPEQL

OX40 co-stimulatoiy ALYLLRRDORLPPDAHKPPGGGSFRTPIQEEOADAHSTLA domain KI

OX40 co-stimulatory RRDQRLPPDAHKPPGGGSFRTPTQEEQADAHSTLAKI domain

4- IBB intracellular KRGRKKLLYIFKQPFMRPVQTIQEEDGCSCRFPEEEEGGCE domain L

4- IBB signaling KRGRKKLLYIFKOPFMRPVQTTQEEDGCSCRFPEEEEGGY domain EL 4-lBB-CD3Zeta TGTTTPAPRPPTPAPT1ASQPLSLRPEACRPAAGGAVHTRG 289 intracellular domain LDFACD1YIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIF

KQPFMRP VQTTQEED GC S CRFPEEEEG GCELR VKF SR S AD APAYQQGQNQLYNELNLGRRFPYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR

4-1BB-Z endodomain KRGRKKLLYIFKQPFMRPVQTTOEEDGCSCRFPEEEEGGC 290 fusion ELRVKFSRSADAPAYQQGQNQLY ELNLGRREEYDVLDK

RRGRDPEMGGKPRRKNPQEGLYNELQKD MAEAYSEIG MKGERRRGKGHDGLYQGLSTAT DTY'DALHMQALPPR

CD 127 intracellular KRI PIVWPSLPDHKKTTEHLCKKPRKNLNVSFNPESFLDC 291 domain OIHRVDDIQARDEVEGFLQDTFPQOLEESEKORLGGDVQS

P CPSEDVVITPESFGRDSSLTCLAGNVSACDAPILSSSRSL DCRESGKNGPHVYQDLLLSLGTTNSTLPPPFSLQSGILTLN PVAQGQPILTSLGSNQEEAYVT SSFYQNQ

CD 137 intracellular RFSWKRGRKKLLY1FKQPFMRPVQTTQEEDGCSCRFPEE 292 domain EEGGCEL

CD 148 intracellular RKKRKDAKNNEVSFSQIKPK S LIRVENFEAYF KQQAD 293 domain SNCGFAEEYEDLKLVGISOPKYAAELAENRGKNRYN VL

P YD 1 S R VKL S VQTH STDD YIN AN YMPG YH SKKDF I ATQGP

LPNTLKDFWRMVWEKNVYA1IMLT CVEQGRTKCEEYW

P SKQ AQD Y GDIT V AMTSEI VL PEWT1RDFT VKMQT SE SHP

LRQFHFTSWPDHGVPDTTDLLINFRYLVRDYMKQSPPESPI

LVHCSAGVGRTGTFTATDRLIYQIENENTVDVYGIVYDLR

MHRPLMVQTEDQYVFLNQCVLDIVRSQKDSKVDLIYQNT

TAMTiYE LAP\ TFGKTNGYlA

CD27 intracellular QRRKYRSNKGESPVEPAEPCHYSCPREEEGST QEDYRK 294 domain PEPACSP

CD28 intracellular FAAYRS 295 domain

CD28 signaling chain FWVLVWGGVLACYSLLVWAFIIFWVRS RSRLLHSDY 296

MNMTPRRPGPTRKHYQPYAPPRDFAAYRS

CD28 signaling domain RSKRSRLLHSDYMNMTPRRPGPTOKFFYQPYAPPRDFAAY 297

RS

CD28 signaling domain SKRSRLLHSDYMN TPRRPGPTRKHYQPYAPPRDFAAYR 298

S

CD28 signaling domain ffiV YPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFW 299

VL V WGG VL AC Y SLL VT VAFIIF WR SKR SRL L H S D YMN M TPRRPGPTRKHYQPYAPPRDFAAYRS

CD28, 4- IBB, and/or RSKRSRLLHSDYMNMTPRRPGPTOKHYQPYAPPRDFAAY 300 CD3 signaling domain RSRFS\⁷VKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP

EEEEGGCELR VKF SR S AD AP A YQQGQNQL YNELNL GRRE

EYDVLDKPvRGRDPEMGGKPRRKNPQEGLYNELQKDKMA

EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHM

QALPPR

CD28/CD3C AAAJEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSK 301

PFWVLWVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDY

MNMTPRRPGPTRKFiYQPYAPPRDFAAYRSRVKFSRSADA

PAYQOGONQLYNELNLGRREEYDVLDKRRGRDPEMGGK

PRRKNPOEGLYNELQKDKMAEAYSEIGMKGERRRGKGH

DGLYQGLSTATKDTYDALHMQALPPR

CD28-0XZ intracellular RSKRSRLLHSDYNMTPRRPGPTRKHYQPYAPPRDFAAYRS 302 domain RDQRLPPDAH PPGGGSFRTPIQFFQADAHSTLA IRVKFS

RSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDP

EMGGKPRRKNPQEGLYNELQKD MAEAYSEIGMKGERR

RGKGHDGLYQGLSTAn DTYDALHMQALPPR

CD28-4-1BB MFWVLVWGGVLACYSLLVWAFIIFWVKRGRKKLLYIF 303 intracellular domain KQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD28-4-1BB lEVMYPPPYLDNEKSNGT!IHVKGKHLCPSPLFPGPSKPFW 304 intracellular domain VLVWGGVLACYSI VWAF1IFWVKRGRKKLLYIF QPF

MRPVQTTQEEDGCSCRFPEEEEGGCEL

CD28-CD3 Zeta RSKRSRLLHSDYMNMTPR PGPTRKHYQPYAPPRDFAAY 305 intracellular domain RSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDK

RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD28-CD3Zeta KRSRLLHSDYMNMTPRRPGPTOKHYQPYAPPRDFAAYRS 306 intracellular domain RVKFSRSADAPAYQOGONOLYNEL LGRREEYDVLDKR

RGRDPEMGGKPRRKNPOEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYOGLSTATKDTYDALHMOALPPR

CD3 delta chain ' MEHSTFLSGL^ATLLSQV^FKIPIEELE 307 intracellular signaling WVEGTVGTLLSDITRLDLG RILDPRG!YRCNGTDIYKDK domain ESTVQVHYRMCQSCVELDPATVAGTIVTDVIATLLLALGV

FCFAGHETGRLSGAADTQALLRNDQVYQPLRDRDDAQYS HLGGNWARNK

CD3 delta chain MEHSTTLSGLVLATLLSQVSPFKIPEELEDR VNCNTSIT 308 intracellular signaling WVEGWGTLLSDI RLDLGKRILDPRGIYRCNGTD!YKDK domain ESTVQVHYRTADTQALLR DQVYQPLRDRDDAOYSHLG

GNWARNK

CD3 delta chain DQVY QPLRDRDDAQYSHLGGN 309 intracellular signaling

domain

CD3 delta intracellular MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSIT 310 domain WVEGWGTLLSDITRLDLGKRILDPRGIYRCNGTD!Y DK

ESWQVHYRMCQSCVELDPATVAGIIVroVTATLLLALGV

FCFAGHE-TGRLSGAADTQALLRNDQVYQPLRDRDDAQYS

HLGGNWARNK

CD3 delta intracellular MEHSTFLSGLVLATLLSQVSPFKIPIEELEDRVFVNCNTSIT 311 domain WVEGTVGTLLSDITRLDLGKRILDPRGIYRCNGTDIYKDK

ESTVQVHYRTADTQALLRNDOVYQPLRDRDDAQYSHLG

GNWARNK

CDS delta intracellular DQVYQPLRDRDDAQYSHLGGN 312 domain

CD3 epsi!on MQS GTHWRVLGLCLL S VGVWGQT5G EEMGG I QTPYKV 313 intracellular domain SISGTTVILTCPQYPGSEILWQHNDKNTGGDEDDKNTGSDE

DHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVC

ENC IFMD SVATIVIVDICITGGLLLLWWSKNRKAK

AKPVTRGAGAGGRQRGQNKERPPPVPNPDYEPIRKGORD

LYSGLNQRRI

CD3 epsilon NPDYEPIRKGQRDLYSGLNQR 314 intracellular domain

CD3 gamma MEQGKGLAVLILAIILLQGTLAOSIKGNHLVKVYDYOEDG 315 intracellular domain SVLLTCDAEAK ITWFKDGKMIGFLTEDKKKWNLGSNAK

DPRGMYQCKG SQNK SKPLQ VYYRMCQN CIELN A ATI S GF

LFAEIVSIFVLAVGVYFIAGQDGVROSRASDKQTLLPNDO LYQPLKDREDDQYSHLOGNQLRRN,

CDS gamma DQLYQPLKDREDDQYSHLQGN 316 intracellular domain

CD3 gamma DQLYQPL KD REDDQ YSHLQGN 317 intracellular domain

CD3 gamma MEQGKGLA\TILAIILLQGTLAQSIKGNHLVKVYDYQEDG 318 intracellular domain SVLLTCDAEAKNITWFKDGKMIGFL-TE-DKKKWNLGSNAK

DPRGMYQCKG SQNK SKPLQ VYYRMCQN CIELN A ATI S GF LFAEIVSIFVLAVGVYFIAGQDGVRQSRASDKQTLLPNDQ LYQPLKDREDDQYSHLQGNQLRRN

CDS zeta intracellular MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFI 319 domain YGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRR

EEYT⁾\ ,DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKN'I AEAYSE1GMKGERRRG GHDGLYQGLSTATKDTYDALH MQALPPR

CDS zeta intracellular MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFI 320 domain YGmTALFLRVKFSRSAD APAYQQGQNQL YNELNL GRR

EEYDVLDKRRGRDPEMGGKPQR K PQEGLYNELQKDK

MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR

CD3 zeta intracellular MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFI 321 domain YGVILTALFLRM FSRSADAPAYQQGQNQLYNELNLGRR

EEYDVLDKRRGRDPEMGGKPORRKNPQEGLYNELQKDK MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR

CD3 zeta intracellular NQLYNELNLGRREEYDVLDKR 322 domain

CD3 zeta domain 2 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYD VLDKR 323 (NM 000734.3) RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM

KGERRRGKGHDGLY^"OGLSTATKDTYDALHMQALPPR

CDS zeta intracellular DGLYQGLSTATKDTYDALHMQ 324 domain

CD3 zeta intracellular R VKF SRS AEPP A YQQGQNQL YNELNL GRREEYD VLDKRR 325 domain GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK

GERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CDS zeta intracellular RVKFSRSADAP A YQQGQNQL YNELNL GPJ^EYD VLDKR 326 domain RGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSEIG

MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD3 zeta intracellular R S R VKF SRS AD A PA YQOGQNQL YNELNL GRREEYD VLD 327 domain RRGRDPEMGGKPRRKNPQEGLYNELOKDKMAEAYSEIG

MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD3 zeta intracellular RVKFSRS AD APAYQQGEYD VLDKRR GRDPEMGGKPRRK 328 domain NPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY

QGLSTATKDTYDALHMQALPPR

CDS zeta intracellular RVKFSRSADAPAYQOGONQLYNELNLGRREEVDVLDKR 329 domain RGRDPEMGGKPRRKNPOEGLYNELQKDKMAEAYSEIGM

KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD3 zeta intracellular MIP A WLLLLLL VEQ AA ALG EPQLC YILD AILFL VG I LTL 330 domain LVCRLKTOVRKAATTSYEKSRVKFSRSADAPAYOQGQNOL

Y'NELNLGRREEYD VLDKRR GRDPEMGGKPRRKNPQEGL YNELQKDKMAEAVSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPR

CD3 zeta intracellular LRVKFSRSADAP A YQQGQNQL YNELNLGRREEYDVLDKR 331 domain RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM

KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CDS zeta intracellular RVKFSRSADAP A YQQGQNQL YNELNL GRREEYD VLDKR 332 domain RGRDPEMGGKPQRRKNPQEGLY

CDS zeta intracellular LR VKF S R S AD AP AYQQG QNQL .YNF i NLGRREEYD VLDKR 333 domain RGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSETG

MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CD3 zeta intracellular RRVKFSRSADAP A YQQGQNQL YNELNLGRREEYDVLDK 334 domain RRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIG

MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

CDS zeta intracellular NQLYNELNLGRREEYDVLDKR 335 domain

CD3 zeta intracellular EGLYNELQKDKMAEAYSE IG MK 336 domain

CDS zeta intracellular DGLYQGLSTATKDTYDALHMQ 337 domain

CDS zeta intracellular RVKFSRSADAP A YQQGQNQLYNELNL GRREEYD VLDKR 338 domain RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAY'SEIGM

KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR CD3 zeta intracellular RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKR 339 domain RGRDPEMGGKPRRK PQEGLYNELQKDKMAEAYSEIGM

KGERRRG GHDGLYQGLSTATKDTYDALHMQALPPR

CD3 zeta intracellular RVKFSRSADAPAYQQGQNQLYNEL LGRREEYDVLDKR 340 domain RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM

KGERRRGKGHDGLYQGLSTATKDTYDALHMQALP

CDS zeta intracellular DPKLCYLLDGILFIYGVIL ALFLRVKFSRSADAPAYQOGO 341 domain NQLYNELNLGRREEYD^'V'LDKRRGRDPEMGGKPQRRKNP

QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQG

LSTATKDTYDALHMQALPPR

CD3 zeta intracellular MKWKALFTAArLQAOLPITEAQSFGT .T .OPKLCYLLDGILFI 342 domain YGViLTALFLRV FSRSADAPAYQQGQNQLYNELNLGRR

EEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM

AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH

MQALPPR

CD40 intracellular RSRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI 343 domain

CD7 A intracellular MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVP 344 domain ASLMVSLGEDAHFQCPH SSNNANVTWWRVLHGNYTWP

PEFLGPGEDPNGTL.nONVNKSHGGIYVCRVQEGNESYQQ

SCGTTLRWQPPPRPFLDMGEGTKNRIITAEGIILLFCAVW

GTLLLFRKRWQNEKLGLDAGDEYEDENLYEGLNLDDCS

MYEDI SRGLQGTYQD VG SL IGD VQLEKP

CD79A intracellular MPGGPGVLQALPAT LLFLLSAVYLGPGCQALWMHKVP 345 domain ASL VSLGEDAHFQCPHNSSN ANVTWWRVLHGNYTWP

PEFLGPGEDPNEPPPRPFLDMGEGTKNRirTAEGTlT T .FCAV VPGTLLLFRKRWQNEKLGLDAGDEYEDE LYEGL LDDC

SMYEDISRGLQGTYQDVGSLNIGD VQLEKP

CD79A intracellular MPGGPGVLQALPATTFLLFLLSAVYLGPGCQALWMHKVP 346 domain ASLMVSLGEDAHFQCPHNSSN ANVTWWRVLHGNYTWP

PEFLGPGEDPNGTLIIQNVNKSHGGIYVCRVQEGNESYQQ S C GT YLR VRQPPPRPFLDMG EGTK RI I T AE GI1LLFC A VVP GTLLLFRKRWQNEKLGLDAGDEYEDENLYEGLNLDDCS MYEDTSRGLQGTYQDVGSLNIGD VQLEKP

CD7 A intracellular ENLYEGL LDDCSMYEDISRG 347 domain

CDS intracellular FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEAC PAAG 348 domain GAVHTT GLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHR

NR

CDS intracellular FVPVFLPAKPnTPAPRPPTPAPTIASQPLSLRPEACRPAAG 349 domain GAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHR

NR

CDSa intracellular PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGL 350 domain DFACDi

CTLA4 intracellular AVSLSKML KRSPLTTGVFV MAPTEAECE QFQPYFIPI 351 domain N

CTLA4 intracellular A VSL SKMl KKRSPLTTG VY NMTPRRPECEKQFQPY APP 352 domain RDFAAYRS

DAP 10 intracellular RPRRSPAQDGKVYINMPGRG 353 domain

DAP 12 intracellular MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSP 354 domain GvLAG!vMGDLV^TVLIALAVYFLGRLVPRGRGAAEAAT

RKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

DAP 12 intracellular MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSP 355 domain GVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATR

KQRITETESPYQELQGQRSDVYSDLNTQRPYYK

DAP 12 intracellular MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVNIGDL 356 domain VLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPY

QELQGQRSD VY SDL TQRPYYK DAP 12 intracellular MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAG1VMGDL 357 domain VLTVLIALAVYFLGRLVPRGRGAAEATRKQRITETESPYQ

ELQGQRSDVYSDLNTQRPYYK

DAP 12 intracellular MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSP 358 domain GVXAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAAT

RKQRITETESPYQELQGQRSDVYSDLNTQRPYYK

DAP 12 intracellular MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSP 359 domain GVLAGI GDLVLTVLIALAVYT^GRLVPRGRGAAEATR

KQRITETESPYQELQGQRSDVYSDLNTQRPYYK;

DAP 12 intracellular MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAG1VMGDL 360 domain VLTVLIALAVYFLGRLVPRGRGAAEAATRKQRl^'lE'i'ESPY

QELQGQRSDVYSDLNTQRPYYK

DAP 12 intracellular MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDL 361 domain VLTVLIALAVYFLGRLVPRGRGAAEATRKQR1TETESPYQ

ELQGQRSDVYSDLNTQRPYYK

DAP 12 intracellular ESPYQELQGQRSDVYSDLNTQ 362 domain

DAP 12 intracellular ESPYQELQGQRSDVYSDLNTQ 363 domain

GITR intracellular RSQCMWPRETQLLLEVPPSTEDARSCQFPEEERGERSAEE 364 domain KGRLGDLWV

TCOS intracellular TKKKYSSSVHDPNGEFMFMRAVNTAKKSRLTDVTL 365 domain

IL15Ra intracellular KSRQTPPLASVEMEA EALPVTWGTSSRDEDLENCSHHL 366 domain

OX40-CD3 Zeta RRDQRLPPDAHKPPGGGSFRTPTQEEQADAHSTLAKIRVK 367 intracellular domain FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR

DPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

ZAP70 intracellular MPDPAAHLPFFYGSISRAEAEEHLKLAGMADGLFLLRQCL 368 domain RSLGGYVLSLVHDVRFHHFPIERQLNGTYAIAGGKAHCGP

AELCEFYSRDPDGLPCNLRKPCNRPSGLEPQPGVFDCLRD

AMVRDYVRQTWKLEGEALEQAIISQAPQVEKLIATTAHE

RMPWYHSSLTREEAERKLYSGAQTDGKFLLRPRKEQGTY

ALSLIYGKTVYHYLISQDKAGKYCIPEGTKFDTLWQLVEY

LKLKADGLrYCLKEACPNSSASNASGAAAPTLPAHPSTLT

HPQRRIDTLNSDGYTPEPARITSPDKPRPMPMDTSVYESPY

SDPEELKDKKLFLKRDNLLIADIELGCGNFGSVRQGVYRM

RKKQIDVAIKVLKQGTEKADTEEMMREAQIMHQLDNPYI

VRLIGVCQAEAL-N1LVMEMAGGGPLHKFLVGKREEIPVSN

VAELLHQVSMGMKYLEEKNFVHRDLAARNVLLVNRHYA

KISDFGLSKALGADDSYYTARSAGKWPLKWYAPECINFR

KFSSRSDVWSYGVTMWEALSYGQKPYKKMKGPEV AFI

EQGKRMECPPECPPELYALMSDCWIYKWEDRPDFLTVEQ

RMRACYYSLASKVEGPPGSTQKAEAACA

CD28 intracellular MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSC 369 domain KYSYNLFSREFRASLHKGLDSAVEVCWYGNYSQQLQVY

SKTGFNCDGKLGNES\ FYLQNLY\'NQTDriT KiEV¾'n⁷P PPYLDNEKSNGTIIH GKHLCPSPLFPGPSKPFW'v'LVVVG GVLACYSLLVTVAFIIFWVR

4- IBB intracellular MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNN 370 domain RNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSST

S N AEC DCTPGFH CLGA GC SMCEQDCK QGQELTKK G CKD

CCFGTFNDQKRGICRPWTNCSLDGKSVLVNG^'IKERDVVC

GPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLF

LLFFLTLRFSVWRGRKKLLYIFKQPFMRPVQTTQEEDG

Fc epsilon Receptor I MIPAVVLLLLLLVEQAAALGEPQLCYILDAILFLYGIVLTL 371 gamma chain LYCRLKIQ\'RKAAITSYEKSDGWTGLSTRNQETY^rETLKH intracellular domain EKPPQ Fc epsilon Receptor I DGVYTGLSTRNQETYETLKHE 72 gamma chain

intracellular domain

Fc epsilon Receptor I DPKLCYILDAILFLYGIVLTLLYCRLKIQWKAAITSYEKSD 373 gamma chain GVYTGL STRNQETYETLKHEKPPQ

intracellular domain

Fc epsilon Recepto I DGVYTGLSTRNQETYETLKHE 374 gamma drain

intracellular domain

Transmembrane domains

[00219] 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 ammo 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, CD 3 epsilon, CD4, CD5, CDS, CD8a, CD9, CD16, CD22, CD33, CD28, CD37, CD45, CD64, CD80, CD86, CD134, CD 137, CD 152, or CD154.

|00220] 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.

[00221] 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 IgG4 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 NO.: WO2014/100385; and a PD- 1 transmembrane domain comprising the amino acid sequences of SEQ ID NOs.: 6-8 of International Patent Publication NO.: WO2014100385; the concents of each of which are incorporated herein by reference in their entirety.

[00222] 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 ammo 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 a!., 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., IgGl, 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 7 provides various transmembrane regions that can be used in the CARs described herein.

Table 7: Transmembrane domains

€i)28 Transmembrane IFWVLVVVGGVLACYSLLVTVAFIiFWVRSKRR 385 domain

CD28 Transmembrane FWVLWVGG\T.ACYSLLVTVAFIIFWVRSKRSRLLHSDYM 386 domain NMTPRRPGPTRKHYQP YAPPRDFAAYRS

LD28 1 ransmeinbrane FWVL W VGG VL A C Y SLL VT V A FIIFW V 387 domain

CD28 Transmembrane FWVLVWGGVLACYSLLVTVAFHFWV 388 domain

CD28 Transmembrane FW^WVGGVXACYSLLVTVAFIIFWVT SKRSRLLHSDYM 425 domain NMTPRRPGPTRKHYQAYAAARDFAAYRS

CD28 Transmembrane lEWyPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWV 897 domain LWVGGVLACYSLLVTVAFITFAVVRSKRSRLLHSDYMNMTP

RRPGPTRKHYQ YAPPRDFAAY S

CD28 Transmembrane MFWVL WVGGVLACYSGGVTVAFIIFWV 389 domain

CD28 Transmembrane WVLVWGGVLACYSLLVTVAFIIFWV 390 domain

Ci)28 Transmembrane FWVL V VVGG VL ACY S LL VT VAFIIFW VR 898 domain

CD28 Transmembrane PFWVLV^VGGVLACYSLLVTVAFIIFW SKRSRLLHSDY 3 1 domain M MTPRRPGPTRKHYQPYAPPRDFAAYRS

CD28 Transmembrane FWVL VGGVLA^ S^ 392 domain and CD28 and NMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPA CD3 Zeta intracellular YQQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRR domain KNPQEGLYNELQKDKMAEAYSEI GMK GERRR GKGHDGLY

QGLSTATKDTYDALHMQALPPR

Ci)28 Transmembrane FWVLVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYM 393 domain and CD28, OX40, NMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKP and CD3 Zeta intracellular PGGGSFRTPlQEEQADAHSTLA IRVKt SRSADAPAYQOGQ domain NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE

GL YNELQKDKMAEAYS E I GMK GERRR GK GHDGL YQGLST ATKDTYDALHMQALPPR

CD28 Transmembrane FWVLV 'VGG\^rLACYSLLVTVAFIIFW\^rRRVKFSRSADAPA 394 domain and CDS Zeta YQQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRR intracellular domain KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY

QGLSTATKDTYDALHMQALPPR

CD28 transmembrane-CD3 AAAffiVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP 395 zeta signaling domain FW^WVGGVXACYSLLVTVAFIIFWVT SKRSRLLHSDYM ("28z") NMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPA

YQQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEI GMK GERRR GKGHDGLY QGLSTATKDTYDALHMQALPPR

CD3 zeta Transmembrane LC YLLDG ILFIYG VILTALFLR V 396 domain

CD3 zeta Transmembrane MKWKALFTAAILQAQLPiTEAQSFGT J ,DPKL C YLLD GILFI Y 397 domain GVILTALFL

CD3 zeta Transmembrane LCYLLDGILFIYGVILTALFL 398 domain

CD4 Transmembrane ALIVLGGVAGLLLFIGLGIFFCVRC 399 domain

CD4 Transmembrane MALIVLGGVAGT J T FIGLGIFF 400 domain

CD45 Transmembrane and ALIAFLAFLmn'SIALLVVLYKrYDLHKKRSCNLDEQQELV 401 intracellular domain ERDDEKQLMNVEPIHADILLETYKRKIADEGRLFLAEFQSIP

RV SKFPrKEARKPFNQNKNRYVDrLPYDYNRVELSElNGD

AG SN YIN A S YID GFKEPRKYI A AQGPRDETVDDF WRMIWE

QKATVI VTOCEEGNRNKCAEWPSMEEGTRAFGDVW

KINQHKRCPDYIIQKLNIVNKKEKATGREVTHIQFTSWPDH

GWEDPHLLLKLRRRWAFSNFFSGPIWHCSAGVGRTGTYI GIDAMLEGLEAENKVDVYGYVVKLRRQRCLMVQVEAQYI

LIHQALVEYNQFGETEVNLSELHPYLHNMKKRDPPSEPSPL

EAEFQRLPSYRSWRTQHIGNQEENKSKNRNSNV1PYDYNR

VPLKHELEMSKESEHDSDESSDDDSDSEEPSKYINASF3MSY

WPEVMIAAQGPLKETIGDFWQ TFQRKVKV1V 1LTELKH

GDQE IC AQY W GEGKQTY GDIEVDLKDTDK S STYTLR VFE L

RHSKRKDSRTVYQYQYTNWSVEQLPAEPKELISMIQWKQK

LPQKNSSEGNKHHKSTPLLIHCRDGSQQTGIFCALLNLLES

AETEEWDIFQWKALRKARPGMVSTFEQYQFLYDVIASTYP

AQNGQVKKNNHQEDKIEFDNEVDKVKQDANCVNPLGAPE

KLPEAKEQAEGSEPTSGTEGPEHSV GPASPALNQGS

CD62L Transmembrane PLFIPVAVMVTAFSGLAFIIWLA 402 domain

CD7 Transmembrane ALPAALAVISFLLGLGLGVACVLA 403 domain

CDS 1 ransmembrane ^h MALPWALLLPLALLLHAARP 404 domain

CDS Transmembrane AAAFVPVFLPAKPTTTPAPRPPTPAPTIASOPLSLRPEACRPA 405 domain and CD28 AGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCN signaling domain HRNRS RSRIlilSDYMNMTPRRPGPTRKHYQPYAPPRDFA

AYRSRFSVVKRGR KLLYIFKQPFMRPVQTTQEEDGCSCRF

PF.F.F.F.GGCELRVKFSRSADAPAYQQGQNOLYNELNLGRRE

EYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMA

EAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQ

ALPPR

CDS transmembrane AAATTTPAPRPPTPAPTIASOPLSLRPEACRPAAGGAVHTR 406 domain-CD 137 (4- IBB) GLDFACDIYrWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI signaling domain and CDS FKQPFMRP VQTTOEED GC S CRFPFFF F GG CELRV F S S AD zeta signaling domain APAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG C'BBz") KPRRKNPQEGLYNELOKDKMAEAYSEIGMKGERRRGKGH

DGLYQGLSTATKDTYDALHMQALPPR

CDSa Transmembrane F VFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG 407 domain AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRN

CDSa Transmembrane IW APL AGTC G VLLL S L VITL YC 408 domain

CDSa Transmembrane IYIWAPLAGTCGVLLLSLVITLYC 409 domain

CDSa Transmembrane IYIWAPLAGTCGVL.LLSLVITLY^'CR 410 domain

CDSa Transmembrane PTTTPAPRPPTPAPTIASQPL SLRPE ACRP A AG GA VHTRGLD 411 domain FACDIYIWAPLAGTCGVLLLSLVITLYCN

CDSa Transmembrane IYIWAPLAGTCGVLLLSL VITL VCR 412 domain

CDSa Transmembrane IYIW APL AGTCGVLLL SL VIT 413 domain

CDSa Transmembrane IYIWAPLAGTCGVLLLSL VITLY 414 domain

CDSa Transmembrane TPAPRPPTPAPTIASQPLSLRPEACRPAAGGAWTRGLDFAC 899 domain (NP 001 1393451) DIYIWAPLAGTCGVLLLSLVITLYCNHRNRRR

CDSb Transmembrane LGLLVAGVLVLLVSLGVAIHLCC 900 domain

DAP 10 Transmembrane ILLAGLVAADAVA Si , ί VG AVFLC ARR 901 domain

EpoR Transmembrane APVGLVARLADESGHVVLRWLPPre 415 domain GNGAGSVQRVEILEGRTECVLSNLRGRTRYTFAVRARMAE

PSFGGFWSAWSEPVSLLTPSD

FcERI a Transmembrane FFIPLLV^ILFAVDTGLFISTQQQVTFLL IKRTRKGFRLLNP 416 domain HPKPNPKN

Transmembrane domain FWALVWAGVLFCYGLLVTVALCV!WT 907

[00223] Hinge region sequences useful in the present invention are provided in Table 8 A.

Table §A: Hinge regions

CD28 Hinge IE YPPPYLDN¾KSNGTIIHVK;GKHLCPSPLFPGPS P 435

CD8a Hinge GGAVHTRGLDFA 436

CD8a Hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD 437

FACD

CD8a Hinge AKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTR 438

GLDFACD

CD8a Hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD 439

FACD

CD8a Hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD 440

FACD

CD8a Hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLD 441

FACDEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDT

CD8a Hinge PAKPTTTPAPRPPTPAPTIASOPLSLRPEACRPAAGGAVHT 442

RGLDFACDIY

CD8a Hinge TTTPAPRPPTPAPTIA SQPLSLRPEACRPA AGGA VHTRGLD 443

FACDIYTWAPLAGTCGVLLLSLVITLYC

CD8a Hinge TTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDF 444

ACD

CD8a Hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA TRGLD 445

FACDIY

DeltaS Hinge LDKTHTCPP CP 446

EpoR Hinge APVGLVARLADESGHWLRWLPPPETPMTSHIRYEVDVS 447

AGNGAGSVQRVE1LEGRTECVLSNLRGRTRYTFAVRARM

AEPSFGGFWSAWSEPVSLLTPSD

FCRIIa Hinge GLAVSTISSFFPPGYQ 448

FcyRIlIa Hinge GLAVSTISSFFPPGYQ 449

Hinge RWPESPKAQASSVPTAOPOAEGSLAKATTAPATTRNTGR 450

GGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV

QDLWLRDKATFTCFWGSDLKDAHLTWEVAGK TGGV

EEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPS

LPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCE

VSGFSPPNILL WLEDQRE TSGFAPARPPPQPGSTTFW

AWSVLRWAPPSPQPATYTCVVSHEDSRTLLNASRSLEVS

YVTDH

Hinge YVTVSSQDPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPK 451

PKDTL ISRTTEWCVVVTDVSHEDPEVKFNWYVDGVEVH NAK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SN ALPAPIE TISKAKGOPREPQVYTLPPSRDELTKNOVS LTCLVKGFYPSDIA\¾WESNGQPEN YKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPG DPK

Hinge KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG 452

LDFA

Hinge LEPKSCDKTHTCPPCP 453

Hinge KPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRG 454

LD

Hinge EPKS CDKTHTCPPCP 455

Hinge ELKTPLGDTHTCPRCP 456

Hinge EP SCDTPPPCPRCP 457

Hinge ESKYGPPCPSCP 458

Hinge ERKCCVECPPCP 459

Hinge (CHIESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT 460 CKS) CVVVDVSQEDPEVQFNWYVDGVEVH AKTKPREEQF S

TYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIE^SK

AKGQPREPQV TLPPSQEEMTKiv!QVSLTCL GFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFTLYSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGK

Hinge (CHS) ESKYGPPCPPCPGQPREPOVYTLPPSQEEMTKNQVSLTCL 461

WGFYPSDIAVEWESiv!GQPE N ^rKTTPPVLDSDGSFFLYS RLTVDK SRWQEGNVF S C S VMHE ALHNHYTQKSL SL SL GK

IgD Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 462

GGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV

QDLWLRDKATFTCFWGSDLKDAHLTWEVAGKVPTGGV

EEGLLERH SNG SQS QH SRLTLPR SL WN A GT S VTCTLNHP S

LPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCE

VSGFSPPNILLMWLEDQRE-VNTSGFAPARPPPOPGSTTFW

AWSVLRVPAPPSPOPATYTCWSHEDSRTLLNASRSLEVS

YVTDH

IgD Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 463

GGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV

QDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGV

EEGLLERHSNGSQSOHSRLTLPRSLWNAGTSVTCTLHPSL

PPORLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCEV

SGFSPP ILLMWLEDQREVNTSGFAPARPPPQPGS^' i' A

WSVLRVPAPPSPQPATYTCWSHEDSRTLLNASRSLEVSY

VTDH

IgD Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 464

GGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV

QDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGV

EEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPS

LPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCE

VSGFSPPNn_^LMVVLEDQREVNTSGFAPARPPPQPGSTTFW

AWSVLRVPAPPSPQPATYTCWSHEDSRTLLNASRSLEVS

YVTDH

IgD Hinge ESPK AQAS S VPTAQPQ AEG SL AK ATTAP ATTR TGRGG EE 465

KKKEKE EEQEERETKTP

IgD Hinge RWPESPKAQASSWTAQPQAEGSLAKATTAPATTRNTGR 466

G GEEKKKEKEKEEQEERETKTPECP SHTQPL G VYLLTP A V QDLWLRDKATFTCFWGSDLKDAHLTWEVAGKVPTGGV EEGLLERH SNG SQS QH SRL TLPR SLWN A GT S VTCTLNHP S LPPQRL ALREPAAQAPVKLSLNLLASSDPPEAASWLLCE VSGFSPPNILLMWLEDQREVNTSGFAPARPPPQPGSTr

AWSVLRVPAPPSPQPATYTCWSHEDSRTLLNASRSLEVS YVTDH

IgD Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 467

GGEEKKKEKEKEEQEERETKTPECPSHTQPLGVYLLTPAV

QDLWLRDKATFTCFWGSDLKDAHLTWEVAGKVPTGGV

EEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPS

LPPQRLMALREPAAOAPVKLSLNLLASSDPPEAASWLLCE

VSGFSPPNILLMWLE QREVNTSGFAPARPPPQPGS i i'F

AWSVLRVPAPPSPQPATYTCWSHEDSRTLLNASRSLEVS

YVTDH

IgD Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 468

GGEEKKKEKEKEEQE ERETKTPECP SHTQPLG VYLLTP A V

ODLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGV

EEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPS

LPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCE

VSGFSPPNILLMWLEbQREWTSGFAPARPPPQPGS^'l'r

AWSVLRWAPPSPQPATYTCWSHEDSRTLLNASRSLEVS

YVTDH

IgD Hinge RWPESPKAQASSVPTAQPQAEGSLAKATTAPATTRNTGR 469

GGEEKKKEKEKEEQE ERETKTPECP SHTQPLG VYLLTP A V QDLWLRDKATFTCFVVGSDLKDAHLTWEVAGKVPTGGV EEGLLERHSNGSQSQHSRLTLPRSLWNAGTSVTCTLNHPS LPPQRLMALREPAAQAPVKLSLNLLASSDPPEAASWLLCE VSGFSPPNILLMWLEDQREV TSGFAPARPPPQPGSTTFW AWSVLRVPAPPSPQPATYTCWSHEDSRTLLNASRSLEVS YVTDH

IgGl (CH2CH3) AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTL IART 470 Hinge domain PEWCVVVDVSHEDPEVKFNWYVDGVEVHNAK^'rKPREE

QY STYRWSVUTVLHQDWLNGKEYKCKVSNKALPAPIE

KTISKAKGQPREPQWTLPPSRDELTKNQVSLTCLVKGFY

PSD1AVEWESNGOPEN YKTTPPVLDSDGSFFLYSKLTVD

K S R WQQG N VF S C S VMHE ALHNHYTQKSL SL SPGKKD

IgGl (CH2CH3) AEPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIART 471 Hinge domain PEWCVWDVSHEDPEVKTN VDGVEVHNAKTKPREE

QY STYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQWTLPPSRDELTKNQVSLTCLVKGFY P SDI A VEWE SNGQPENN YKTTPP VLD SD GSFFL Y SKLTVD K SRWQQGN VF S C S VMHE ALHNHYTQKSL SL SPGKKD

IgGl Hinge AEPKSPDKTHTCPPCPKDPK 472

IgGl Hinge EPKSCDKTHTCPPCP 473

IgGl Hinge EPKSPDKTHTCPPCPAPPVAGPSVTLFPPKPKDTLMIARTP 474

EWCWVDVSHEDPEVKFN VDG^VHNAKTKPREEQ

YNSTTRWSVLTVLHQDWLNGKEVKCKVSNKALPAPIEK

TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS

DIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKS

R WQQGN VF S C S VMHE ALHNH YTQK SL S L SP GKKD

IgGl Hinge SVFLFPPKPKDTL 475

IgGl Hinge EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMIARTP 476

EVTCVWD SH Kl ⁾PEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQWTLPPSRDELTKNQVSLTCLVKGFYPS DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS R WQQGN VF S C S VMHE ALHNH YTQK S L SL S P GK

IgG l Hinge EPKSPDKTHTCPPCPAPPVAGPS LFPPKPKDTLMIARTP 477

EVTCVWD SH Kl ⁾PEVKFNWYVDGVEVHNAKTKPREEQ YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQWTLPPSRDELTKNQVSLTCLVKGFYPS DI AVEWESNGQPENNYKTTPP VLD SDG SFFL YSKLT VDKS R WQQGN VF S C S VMHE ALHNH YTQK SL SL SP GKKDPK

IgGl Hinge \^0^>PCPAPPVAGPS\HTJ^:PPKPKDT MISRTPEVTCVVVD 478

VSHEDPEVKFNWYVDGVE-VHNAKTKPREEQYNSTYRW SVLTVLHQDWLNGKEYKCKVSNKGLPSSIE-KTISKAKGQP REPQWTTJPPSREEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK

IgGl Hinge DPAEPKSPDKTHTCPPCPAPPVAGPS LFPPKPKDTLMIA 479

(CH2CH3 RTPEVTCVVVDVSHEDPEVKF WYVDGVEVTINAKTKPR domain) EEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPA

PIE TISKAKGQPREPQVTTLPPSRDELTKNQVSLTCLVKG

FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT

VDKSRWQQGNVFSCS VMHE ALHNH YTQKSLSLSPGKK

IgG2 Hinge ERKCCVECPPCP 480

IgG3 Hinge ELKTPL GDTTHTCPRCP 481

IgG3 Hinge ELKTPLGDTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPC 482

PRCPEPKSCDTPPPCPRCP

IgG4 (CH2 and ESKYGPPCTPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVT 483 CH3) CWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS

TYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK

AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTTPVLDSDGSFFLYSRLTVDKSRW

QEGNVFSCS VMHEALHNHYTQK SLSLSLGKM IgG4 (CH2 and ESKYGPPCPPCP APEFEGGPS LFPPKPKDTLM1SRTPEVT 484 CH3) CVVYDVSQEDPEVQFNWWDGVEVHNAKT PREEQFQS

TYR VV S VLT VLHQDWLNGKEY CK VSN KGLP S SIEKTI SK

AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA

VEWES GQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRW

QEGNVFSCSVMHEALHNHYTQKSLSLSLGKM

igG4 Hinge SPNMVPHAHHAQ 485

IgG4 Hinge GQPREPQVYTLPPSQEEMTK QVSLTCLVKGFYPSDIAVE 486

WESNGQPE NY TTPPVLDSDGSFFLYSRLTVDKSRWQE GN VF S C S VMHE ALHNHYTQKSL S L SL GK

igG4 Hinge ESKYGPPCPPCPGGGSSGGGSGGQPREPOVYTLPPSQEEM 487

TKNQVSLTCLVXGFYTSDIAVEWESNGQPENNYTiTTPPVL DSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYT QKSLSLSLGK

TgG4 Hinge ESKYGPPCPSCPAPEFEGGPSWLFPPKPKDTLMISRTPEVT 488

CV\^VSQEDPEVQFNWYVDGV¾VHQAKTKPREEQFNS

TYRWSVLTVL-HQDWLNGKEYKCKVSNKGLPSSIEK^SK

AKGQPREPQWTLPPSQEEMTKNQVSLTCLVKGFVPSDIA

VEWESNGQPENlsTV 1¹ i PP VLD SDGSFFL YSRLTVDKSRW

QEGNVFSCSVMHEALHNHYTQKSLSLSLGK

IgG4 Hinge ESi ^GPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVT 489

CWVDVSQEDPEVQF WYVDGVEVHQAKTKPREEQFNS TYR VVS VLT VI .HQD L NGKEYKCK VSNK G LPS STEKTISK AKGQPREPQVYTLPPSQEEMTKNOVSLTCLVKGFVPSDIA VEWESNGQPENlsTV 1¹ i PP VLD SDGSFFL YSRLTVDKSRW QEGNVF S C S VMHE ALHNHYTQK SL SL SLGK

IgG4 Hinge ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTIJVIISRTPEVT 490

CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TYR WS VLT VLHQDWL NGKEYKCKVSNKG LPS S IEKTISK AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRW QEGNVFSCSVMHEALHNHYTQKSLSLSLGKM

igG4 Hinge GAATCTAAGTACGGACCGCCCTGCCCCCCTTGCCCT 491

IgG4 Hinge ESKYGPPCPPCP 492

IgG4 Hinge ES YGPPCPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCL 493

VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

IgG4 Hinge ESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVT 494

CVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEOFNS

TYR VV S VLT VLHQDWLNGKEYK CK VSN KGLP S SIEKTI SK

AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA

VEWESNGQPENNYKTTPPVLDSDGSFTLYSRLTVDKSRW

QEGNVFSCSVMHEALHNHYTQKSLSLSLGK

igG4 Hinge ESKYGPPCPPCP APEFLGGPSVFLFPPKPKDTLMISRTPEVT 495

C\^VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS

TYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISK

AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA

\¾WESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRW

QEGNVFSCSVMHEALHNHYTQKSLSLSLGK

TgG4 Hinge ESKYGPPCPPCP 496 igG4 Hinge YGPPCPPCP 497

IgG4 Hinge KYGPPCPPCP 498

IgG4 Hinge EWKYGPPCPPCP 499

IgG4 Hinge ESKYGPPCPSCP APEFLGGPSVFLFPPKPKDTLMISRTPEVT 500

CVVVOVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS TYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSffiKTISK

AKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIA VEWESNGQPENNYKT PPVLDSDGSFFLYSRLTVDLSRW

QEGNVFSCSVMHEALHNHYTQKSLSLSLGK

igG4 Hinge and ESKYGPPCPPCPGGGSSGGGSG 501

Linker

lgGl Hinge EPKSPDKTHTCPPCPAPPVAGPSVFLFPPKPKDTL IARTP 502

EVTC VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEO

Y STYR SVLTVLHQDWLNGKEYKCKVSNKALPAPIEK

TISKAKGQPREPQWTLPPSRDELTKNQVSLTCLVKGFYPS

DIA\¾WESNGQPENN 1^'1PPVL.DSDGSFFLYSKLTVDKS

R WQQGN VF S C S VMHE ALHNHYTQK SL SL SP GK

igGi Hinge EPKSPDKTHTCPPCPAPPVAGPSYFLFPPKPKDTLMIARTP 503

EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ

Y STTR SVLTVLHQDWLNGKEYKCKVSNKALPAPIEK

TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPS

D!AWWESNGQPENNYKT!PPVLDSDGSFFLYS LTVDKS

R WQQGN VF S C S VMHE ALH HYTQK SL S L SP GK

IgGI Hinge EPKSPDKTHTCPPCPAPPVAGPS LFPPKPKDTLMIARTP 504

EVTCWVDVSHEDPEV FNWYVDGVEVHNAKTKPREEQ

Y^STTRWS^TVLHQDWLNGKEY CKVSNKALPAPIEK

TIS AKGQPREPQVYTLPPSRDELT NQVSLTCLVKGFYPS

DIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS

R WQQGN VF S C S VMHE ALHNHYTQK SL S L SP GK

[00224] Hinge and transmembrane region sequences useful in the present invention are provided in Table 8B.

~ ~ ^"iinee and Transmembrane res

[00225] 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.

[00226] In some embodiments, the components including the targeting moiety, transmembrane domain and intracellular signaling domains of the present invention may be constructed in a single fusion polypeptide. The fusion polypeptide may be the pay load 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.

[00227] In some embodiments, the CAR sequences may be selected from Table 9,

Table 9: CAR sequences

Description SEQ ID Source

NO

CD 19 CAR 510 SEQ ID NO: 12 in US9499629B2

CD 19 CAR 51 3 SEQ ID NO. 24 in US20160333108A l

CD 19 CAR 512 SEQ ID NO: 25 in US20160333108Al

CD 19 CAR ' 513 SEQ ID NO: 26 in US20160333308A1

CD 19 CAR 514 SEQ ID NO: 27 in US20160333108Al

CD 19 CAR 515 SEQ ID NO: 1 in EP2997134A4

CD 19 CAR 516 SEQ ID NO: 19 in EP3071687A1

CD 19 CAR 517 SEQ ID NO: 20 in EP3071687A1

CD 19 CAR 518 SEQ ID NO. 181 m WO2016168773 A3

CD 19 CAR 519 SEQ ID NO: 2 in WO2015157399A9

CD 19 CAR ' 520 SEQ 3D NO: 56 in WO2016174409A 3

CD 19 CAR 521 SEQ ID NO: 62 in WO2016174409A1

CD 19 CAR 522 SEQ ID NO: 145 in WO2016179319A 1

CD 19 CAR 523 SEQ ID NO: 293 in US20160311907A l

CD 19 CAR 524 SEQ ID NO: 294 in US20160311907A 3

CD 19 C AR 525 SEQ ID NO: 295 in US20160311907A1

CD 19 CAR 526 SEQ ID NO: 296 in US20160311907Al

CD 19 CAR ' 527 SEQ 3D NO: 297 in US2036031 3907 A 3

CD 19 CAR 528 SEQ ID NO: 298 in US20160311907Al

CD 19 CAR 529 SEQ ID NO. 73 in WO2013176915A1

CD 19 CAR 530 SEQ ID NO. 73 in WO2013176916A1

CD 19 CAR 531 SEQ 3D NO. 73 in US20130315884Al

CD 19 CAR 532 SEQ ID NO. 73 in US20140134142Al

CD 19 CAR 533 SEQ ID NO. 73 in US20150017136Al

CD19 CAR ' 534 SEQ ID NO. 73 in US20150203817A1

CD 19 CAR 535 SEQ ID NO. 73 in US2016012090 Al

CD 39 CAR 536 SEQ ID NO, 73 in US20160120906A l

CD 19 CAR 537 SEQ ID NO. 8 in WO2015124715

CD 19 CAR 538 SEQ ID NO. 5 in WO2015 124715

CD 19 CAR 539 SEQ ID NO. 73 in WO2014184744

CD 19 CAR 540 SEQ ID NO. 73 in WO2014184741

CD19 CAR ' 541 SEQ ID NO. 14 in US20160145337A1

CD 19 CAR 542 SEQ ID NO. 15 in US20160145337Al

CD 39 CAR 543 SEQ ID NO, 14 in WO2014184143

CD 19 CAR 544 SEQ ID NO. 15 in WO2014184143

CD 19 CAR 545 SEQ ID NO. 15 in WO2015075175

CD 19 CAR 546 SEQ ID NO. 16 in WO2015075175

CD 19 CAR 547 SEQ ID NO. 16 in US20160145337Al

CD19 CAR ' 548 SEQ ID NO. 16 in WO2014184143

CD 19 CAR 549 SEQ ID NO 12 in WO2012079000

CD 39 CAR 55(5 SEQ ID NO.31 in WO2016164580

CD 19 CAR 551 SEQ ID NO.32 in WO2016164580

CD 19 CAR 552 SEQ ID NO.33 in WO2016164580

CD 19 CAR 553 SEQ ID NO.34 in WO2016164580 CD 19 CAR 554 SEQ ID NO.35 in WO2016164580

CD 19 CAR 555 SEQ ID NO.36 in WO2016164580

CD19 CAR 556 SEQ ID NO.37 in WO2016164580

CD 19 CAR 557 SEQ ID NQ.38 in WO2016164580

CD 39 CAR 558 SEQ ID NO.39 in WO2016164580

CD 19 CAR 559 SEQ ID NO.40 in WQ2016164580

CD 19 CAR 560 SEQ ID NO.41 in WO2016164580

CD 19 CAR 561 SEQ ID NO.42 in WO2016164580

CD 19 CAR 562 SEQ ID NO.58 in WO2016164580

CD 19 CAR ' 563 SEQ ID NO: 14 in US20160296563A1

CD 19 CAR 564 SEQ ID NO: 15 in US20160296563 Al

CD 19 CAR 565 SEQ ID NO.31 in WO2015157252

CD 19 CAR 566 SEQ ID NO.32 in WO2015157252

CD 19 CAR 567 SEQ ID NO.33 in WO2015157252

CD 19 CAR 568 SEQ ID NO.34 in WO2015157252

CD 19 CAR 569 SEQ ID NO.35 in WO2015157252

CD19 CAR ' 570 SEQ ID NO.36 in WO20151 57252

CD 19 CAR 571 SEQ ID N0.37 in WO2015157252

CD 19 CAR 572 SEQ ID NO,38 in WO2015157252

CD 19 CAR 573 SEQ ID NO.39 in WO2015157252

CD 19 CAR 574 SEQ ID NO.40 in WO2015157252

CD 19 CAR 575 SEQ ID N0.41 in WO2015157252

CD 19 CAR 576 SEQ ID NO.42 in WO2015157252

CD 19 CAR 577 SEQ ID NO. 14 in WO2016139487

CD 19 CAR 578 SEQ ID N0.15 in WO2016139487

CD 19 CAR 579 SEQ ID NO. 53 in US20160250258A l

CD 19 CAR 580 SEQ ID NO: 54 in US20160250258Al

CD 19 CAR 581 SEQ ID NO: 55 in US20160250258A1

CD 19 C AR 582 SEQ ID NO: 56 in US20160250258A1

CD 19 CAR 583 SEQ ID NO: 57 in US20160250258Al

CD 19 CAR ' 584 SEQ ID NO: 58 in US20160250258A1

CD 19 CAR 585 SEQ ID NO. 1 in WO2015187528

CD 19 CAR 586 SEQ ID NO, 2 in WO2015187528

CD 19 CAR 587 SEQ ID NO. 3 in WO2015187528

CD 19 CAR 588 SEQ ID NO. 4 in WO2015 187528

CD 19 CAR 589 SEQ ID NO. 5 in WO2015187528

CD 19 CAR 590 SEQ ID NO. 6 in WO2015187528

CD 19 CAR 591 SEQ ID NO. 7 in WO2015187528

CD 19 CAR 592 SEQ ID NO. 8 in WO2015 187528

CD 19 CAR 593 SEQ ID NO, 9 in WO2015187528

CD 19 CAR 594 SEQ ID NO. 10 in WO2015187528

CD19 CAR 595 SEQ ID NO. 1 1 in WO2015187528

CD 19 CAR 596 SEQ ID NO. 12 in WO2015187528

CD 19 CAR 597 SEQ ID NO. 13 in WO2015187528

CD 19 CAR 598 SEQ ID. NO. 31 in WO2015157252

CD 19 CAR 599 SEQ ID. NO. 32 in WO2015157252

CD 19 CAR 600 SEQ ID. NO. 33 in WO2015157252

CD 19 CAR 601 SEQ ID. NO. 34 in WO2015157252

CD19 CAR ' 602 SEQ ID. NO. 35 in WO2015157252

CD 19 CAR 603 SEQ ID. NO. 36 in WO2015157252

CD 19 CAR 604 SEQ ID. NO. 37 in WO2015 157252

CD 19 CAR 605 SEQ ID. NO. 38 in WO2015157252

CD 19 CAR 606 SEQ ID. NO. 39 in WO2015157252

CD 19 CAR 607 SEQ ID. NO. 40 in WO2015 157252

CD 19 CAR 608 SEQ ID. NO. 41 in WO2015157252

CD19 CAR ' 609 SEQ ID. NO. 42 in WO2015157252

CD 19 CAR 610 SEQ ID. NO. 58 in WO2015157252

CD 19 CAR 61 1 SEQ ID NO. 31 in WO2014153270 CD 19 CAR 612 SEQ ID NO. 32 in WQ2014153270

CD 19 CAR 613 SEQ ID NO. 33 in WO2014153270

CD19 CAR ' 634 SEQ ID NO. 34 in WO2014153270

CD 19 CAR 615 SEQ ID NO. 35 in WO2014153270

CD 39 CAR 616 SEQ ID NO, 36 in WO2034153270

CD 19 CAR 617 SEQ ID NO. 37 in WO2014153270

CD 19 CAR 6 8 SEQ ID NO. 38 in WO2014153270

CD 19 CAR 619 SEQ ID NO. 39 in WQ2014153270

CD 19 CAR 620 SEQ ID NO. 40 in WO2014153270

CD19 CAR ' 621 SEQ ID NO. 41 in WO2014153270

CD 19 CAR 622 SEQ ID NO. 42 in WO2014153270

CD 39 CAR (Third generation) 623 SEQ ID NO, 13 in WO2016139487

[00228] In one embodiment of the present invention, the pay load of the invention is a CD 19 specific CAR targeting different B cell. In the context of the invention, an effector module may comprise a hDHFR DD, ecDHFR DD, or FKBP DD operably linked to a CD 19 CAR fusion construct. In some instances, the promoter utilized to drive the expression of the effector module in the vector may be a CMV promoter or an EFla. The efficiency of the promoter in dri ving the expression of the same construct may be compared. For example, two constructs that differ only by their promoter, CMV (in OT-CD19N-001 ) or EF 1 a promoter (in OT-CD19N-017) may be compared. The amino acid sequences of CD 19 CAR constructs and its components are presented in Table 10a and Table 10b. The amino acid sequences in Table 10a and/or Table 10b may comprise a stop codon which is denoted in the table with a "*" at the end of the amino acid sequence.

sequences of components of CD19 CARs

KAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSE

FHD AD AQN SH S Y CFEILERR

ecDHFR (Amino acid 2- ISLIAALAVDHVIG ENAMPWNLPADLAWFKRN 10 798, 815, 159 of WT) (R12H, TT.NKPVIMGRHTWESIGRPLPGRKNIILSSOPGTDD 993 E129K) RVTWVKSVDEAIAACGDVPEIMVIGGGRVYEOFL

PKAQKLYLTHIDAEVEGDTHFPDYKPDDWESVFS EFHDADAQNSHSYCFEILERR

hDHFR (Amino acid 2- VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 895 694, 995 187 of WT; Y122I) QRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLK

GRINLVLSRELKEPPQGAHFLSRSLDDALKLTEQP ELANK\T)MVWIVGGSSVIKEAMNHPGHT T FVT

RmiQDFESDTFFPEIDLEKYKLLPEYPGVLSDVOE

EKGIKYKFEVYEKND

hDHFR (Amino acid 2- VGSLNCIVAVSQ MGIGKNGDLPWPPLRNEFRYF 890 696, 973, 187 of WT; ΥΊ22Ι, QRMTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLK 974, 996 A125F) GR INLVLSRELKEPPQGAHFL SR S LDDALKLTEQP

ELA KVDMVWIVGGSSVlKEl- lVfNrlPGHLKLFVT

RmiQDFESDTFFPEIDLEKYKLLPEYPGVLSDVQE

EKGIKYKFEVYEKND

hDHFR (Amino acid 2- VGSLNC1VAVSQNMGIGKNGDLPWPPLRNEFRYF 981 698, 997 187 of WT; Q36K, KRMTTTS S VEGKQNLVIMGKKTWFSIPEKNRPLK

Y1221) GRINLVLSRELKEPPQGAHFLSRSLDDALKLTEQP

EL ANKVDM VWI VGGS S VIKEAMNHPGHLKLF VT RIMQDFESDTFFPEIDLEKYKLLPEYPGVLSDVQE EKGIKYKFEVYEKND

hDHFR (Q36F, N65F, VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 891 700, 975, Y122I) FRMTTTS S VEGKQNL VMGKKTWF SIPEKFRPLKG 976, 998

RINLVLSRELKEPPOGAHFLSRSLDDALKLTEQPE LANKVDMVWiVGGSSVIKEAMNHPGHLKLFVrRI MQDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEK G IKYKFE EKND

Description Amino Acid Sequence Ami uo Niicleic

Acid SEQ Acid SEQ I» NO ID NO

CD19 scFv DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNW 624 626, 650- YQQKPDGT VKLLIYHTSR LHS G VP SRF S G S G S GTD 654 YSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEI TGGGGSGGGGSGGGGSEVKLQESGPGLVAPSQSL SVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIW GSETTYYNS. LKSRLTIIKDNSKSQWLKMNSLQT DDTAIYYCAKHYYYGGSY IDYWGQGTSVTVS

s

CD8a hinge-TM TTTPAPRPPTPAPTIA SQPLSLRPEACRP A AGGAVH 625 627, 655,

TRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC 982-984

CD 8 a hinge TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGA 440 656-660

TRGLDFACD

CD3 zeta signaling RVKFSRSADA AYKQGQNQLYNELNLGRREEYD 339 661-665, domain VLDKRRGRDPEMGGKPRRKNPOEGLYNELQKDK 986

MAEAYSEIGMKGERRRGKGHDGLYQGLSTATKD TYDAL-HMQALPPR

4- IBB (4 IBB) KRGRKKLLY!FKOPFMRPVQTTQEEDGCSCRFPEE 273 666-670, intracellular signaling EEGGCEL 985 domain

CD8a Transmembrane lYPvVAPLAGTCGVLLLSLVITLYC 409 990, 992 domain

CD8a leader MALPVTALLLPLALLLHAARP ^l· 628 673 -675 p40 signal sequence MCHQQLVISWFSLVFLASPLVA 719 736-744 p40 iWELKKDWV\¾LDWYPDAPGEMVVLTCDTPEE 723 632-634,

DGITWTLDQS SEVLGS GKTLTIQWEFGD AGQYT 752-761 CHKGGEVLSHSLLLLHKKEDGIWSTDILKDQKEP PKAQKLYLTHJDAEVEGDTHFPDYKPDDWESVFS

EFHDADAQNSHSYCFE1LERR

hDHFR (Amino acid 2- VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 895 694, 995 187 of WT; Y122I) QRMTTTSSVEGKONLVIMGKKTWFSIPEK RPLK

GRINLVL SRELKEPPQGAHFL SRSLDD AL LTEQP ELANKVDMVWIVGGSS\TKEAMNHPGHLKLFVT

RIMQDFESDTFFPELDLEKYKLLPEYPGVLSDVQE EKGIKYKFEVYEKND

hDHFR (Amino acid 2- VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 890 696, 973, 187 of WT; Y122I, QRMTT SSVEGKQNLYDV1GKKTWFSIPEKNRPLK 974, 996 A125F) GRINLVLSRELKEPPQGAHFLSRSLDD ALKLTEQP

ELANKVDMVWIVGGSSV1KEFMNHPGHLKLFVT

RIMQDFESDTFFPELDLEKYKLLPEYPGVLSDVQE EKGIKYKFEVYEKND

hDHFR (Amino acid 2- VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 981 698, 997

187 ofWT; Q36K, RMTTTSSVEGKQNLVIMGK TWFSIPEKNRPL

Y122I) GRINLVLSRELKEPPQGAHFLSRSLDD ALKLTEQP

FLANKVDMVWIVGGSSVIKEAMNHPGHLKLFVT RIMQDFESDTFFPEIDLEKYKLLPEYPGVLSDVQE EKGIKYKFEVYEKND

hDHFR (Q36F, N65F, VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYF 891 700, 975, Y1221) FRMTTTS S VEGKQNL VIMGKK TWF SIPEKFR PLKG 976, 998

RINLVLSRELKtPPQGAHFLSRSLDDALKLTEQPE

LANKVDMVWIVGGSSVIKEA NHPGHLKLFVTRI

MQDFESDTFFPEIDLEKYKLLPEYPGVLSDVQEEK

GIKYKFEVYEKND

Table 10b: Sequences of CD19 CARs

Description Amino Acid Sequence Amino Nucleic

Acid SEQ Acid SEQ ID NO ID NO

OT-CD19 CAR-001 (OT- MALPVTALLLPLAl l LHAARPDIQMTQTTSSLSAS 635 70!

CD 39c-001) (CD8a LGDRVnSCRASQDISKYLNWYQQKPDGTVKLLI

leader -CD 19 scFV - YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

CD8a-Trn -4 i BB - TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG

CD3zeia - stop) GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

YGVSWSRQPPRI GLE iJjVIWGSE'i lYYNSALKS

RLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY

YYGGSYAMD GQGTSVTVSSTTTPAPRPPTPAP

TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI

WAPL AGTC GVLLL SL\TTLYCKRGRKKLLYIFKQP

FMRP VQTTQEED GC S CRFPEEEEGGCELR VKF SRS

ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG

RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE

IGMKGERRRGKGHDGLYQGLSTATKDTYDALHM

QALPPR*

OT-CD19 CAR-002 (OT- MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS 636 702 CD19c-002) (CD8a LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLI

leader - CD 19 scFV - YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

FKBP (F36V, L106P) - ^'lYFCQQG TLPYTFGGGTKLEITGGGGSGGGGSG

CD8a-Tm - 41BB - GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

CD3zeta - stop) YGVSWIRQPPRKGLEWLGVIWGSETIYYNSALKS

RLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY

YYGGSYAMDYWGQGTSVTVSSGVQVETISPGDG

RTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP

FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPD

YAYGATGHPGnPPHATLVFDVELLKPETTTPAPRP

PTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFA CDIYIWAPLAGTCGVLLLSLVITLYC RGRKKLLY

IFKQPFMRPVQTTQEEDGCSCRFPKFFPGGCELRV FSRS AD AP AYKQGQN QL YNELNL GRREEYD VL

DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM

AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT

YDALHMQALPPR*

OT-CD19 CAR-003 (OT- MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS 637 703 CD 19c-003) (CD8a LGDRVnSCRASQDISKYLNWYQOKPDGTVKLLI

leader - CD 19 scFV - H1 SRLHSG SRFSGSGSGTDYSLTISNLEQEDIA

ecDHFR - CD8a-Tm - TYFCQOG TLPYTFGGGT LEITGGGGSGGGGSG

4 IBB - CDS zeta - stop) GGGSEVKLQESGPGLVAPSOSLSVTCTVSGVSLPD

YG VS WIRQPPR GLEWLGVIWGbE'l T YYN S ALKS

RLTIIKDNSKSOVFLKMNSLQTDDTAiYYCAKHY

YYGGSYAMDYWGQGTSVTVSSISLIAALAVDYVI

GMENAMPWNLPADLAWFKRNTLNKPVIMGRHT

WESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEA

lAACGDVPElMVIGGGRVIEQFLPKAQKLYLTHID

AEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHS

YCFF.TT FRRTTTPAPRPPTPAPT1ASQPLSLRPEACR

PAAGGAVH RGLDFACDIYIWAPLAGTCGVLLLS

LV!TLYCKRGRK i f .YIFKQPFMRPVQTTQEEDGC

SCRFPF.FFPGGCELRVKFSRSADAPAYKQGQNQL

YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKN

PQEGLYNELQKDKMAEAYSE1GMKGERRRGKGH

DGLYQGLSTATKDTYDALHMQALPPR*

OT-CD19 CAR-004 (OT- MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS 638 704

CD 39c-004) (CD8a LGDRVnSCRASODISKYLNWYQQKPDGTVKLLI

leader - CD 19 scFV - YHTSRLHSG SRFSGSGSGTDYSLTIS LEQEDIA

CD8a Hinge - FKBP TYFCQOG TLPYTFGGGT LEITGGGGSGGGGSG

(F36V, L106P) -CD8a GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

Transmembrane domain- YGVSWIRQPPRKGLEWLGVIWGSETTYY SALKS

4 IBB - CDS zeta - stop) RL IIKDNSKSOVFLKIVINSLOTDDTAIYYCAKHY

WGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAP

TTASQPLSLRPEACRPAAGGAVHTRGLDFACDGV

Q\¾TISPGDGRTFP RGQTC\⁷VHYTGMLEDGKKV

DSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVG

QRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL

KPEIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLL

YIFKQPFMRP VQTTQEEDGC SCRFPFFF FGGCELR

VKFSRSADAPAYKQGQNQLYNELNLGRREEYDV

U3KRRGRDPEMGGKPRRKNPQEGLYNELQKDKM

AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT

YDALHMQALPPR*

OT-CD19 CAR -005 (OT- MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS 639 705 CD19c-005) (CD8a LGDRVTISCRASQD1SKYLNWYQQKPDGTVKLLI

leader - CD19 scFV - YHI^RLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

CD8a Hinge - ecDHFR TYFCQOGNTLPYTFGGGTKLE!TGGGGSGGGGSG

(Amino acid 2-159 of GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

WT) (R12Y, Y 100I) - YGVSWIRQPPRKGLEWLGVIWGSE¹1 1¹ YYN S ALKS

CD8a Transmembrane RLllU JNSkSQVFL.KMNSLQTDDTAIYYCAKHY

domain -4 IBB -CD3zeta - YYGGSYAMDYWGQGTSVTVSST1 PAPRPPTPAP

stop) TIASOPLSLRPEACRPAAGGAVHTRGLDFACDISLI

AALA\DYVIGMENAMP LPADLA\WKRNTLN

KP VIMGRHTWESIGRPLPGRKNIIL S SQPGTDDR V

TWVKSVDEAIAACGDVPEIMVIGGGRVIEQFLPK

AQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEF

HDADAQNSHSYCFEILERRIYIWAPLAGTCGVLLL SI ^TLYC RGRKKLLYIFKQPFMRPVQTTOtEDG

CSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQ LYNELNLGRREEYDVLDKRRGRDPEMGGKPRRK NPQEGLYNELQKDKMAEAYSE1GMKGERRRGKG

HD GL YQGL S T ATKDT YD ALHMQ ALPPR *

OT-CD19c-006 (CD8a MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS 640 706 leader-CD19 scFV - LGDRVnSCRASQDISKYLNWYOQKPDGTVKLLI

CD8a-Tm - 41BB - YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

CDSzeta -linker TYFCQQGNTLPYTFGGGTKLEiTGGGGSGGGGSG

(GGSGG) - ecDHFR GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

(Amino acid 2-159 of YG VS WIRQPPRKGLE WLG VTWG SETTYYN S ALK S

WT) (R12H, E129K) - RLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY

stop) YYGGS Y AMDYWGQGTS VTVS S 1 Ί TP APRPPTP AP

TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI

WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKOP

FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS

ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG

RDPEMGGKPRRKNPQEGLYNELQ DKMAEAYSE

IGMKGERRRGKGHDGLYQGLSTATKDTYDAL

QALPPRGGSGGISLIAALAVDHVIGMENAMPWNL

PADLAWFKR TLNKPVDV1GRHTWESIGRPLPGRK

NIILSSQPGTDDRVTWVKSVDEA!AACGDVPEIMV

IGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPD

YKPDDWESVFSEFHDADAQNSHSYCFETLERR*

OT-CDl 9c-007 (CD8a MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS 641 707 leader - CD 19 scFV- LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLI

CD8a-Tm - 43 BB - YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

CDSzeta - linker TYFCQQGNTLPYTFGGGTKLEiTGGGGSGGGGSG

(GGSGG) -FKBP (E31G, GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

F36V, R71G, K105E) - YGVS WIRQPPRKGLE WLG VTWG SETTYYN S ALK S

stop) RLTnKDNSkSQVFL-KMNSLQTDDTAIYYCAKHY

YYGGSY AMD YWGQGTS VTVSS^'IT'¹ Ρ APRPPTP AP

TIASOPLSLRPEACRPAAGGAVHTRGLDFACDrYI

WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKOP

FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS

ADAPAYKQGONQLYNELNLGRREEYDVLDKRRG

RDPEMGGKPRRKNPQEGLYNEEQKDKMAEAYSE

IGMKGERRRGKGHDGLYQGLSTATKDTYDALHM

QALPPRGGSGGGVQVETISPGDGRTFPKRGQTCV

VHYTGMLGDGKKVDSSRDRNKPFKFMLGKQEVI

RGWEEGVAQMSVGQGAKLT1SPDYAYGATGHPG

I1PPHATLVFDVELLELE*

OT-CDl 9c-008 (CD8a MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS 642 708 leader - CD19 scFV - LGDRVTTSCRASQDISKYLNVVYQQKPDGTV LLi:

CD8a-Tm -41BB - YHTSRLHSGVPSRFSGSGSGTDYSLTiSNLEQEDIA

CD3zeta - linker TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG

(GGSGG) - hDHFR GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

(Amino acid 2-187 of YGVS WIRQPPRKGLE WLG VTWG SETT ΥΎΝ S ALK S

WT; Υ122Γ) - stop) RLTIIKDNSKSQWLKMNSLQTDDTArYYCAKHY

YYGGSY AMD YWGQGTS VTVS STTTP APRPPTP AP

TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI

WAPL AGTC GVLLL SLWLYCKRGRKKLLYIFKQP

FMRP VQTTQEED GC S CRFPEEEEGGCELR VKF SRS

ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG

RDPEMGGKPRRKNPOEGLYNELQKDKMAEAYSE

IGMKGERRRGKGHDGLYQGLSTATKDTYDALHM

QALPPRGGSGGVGSLNCIVAVSONMGIGKNGDLP WPPLR EFRYFQRMITTSSVEGKQNLVIMGKKT

WFSIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRS

LDDALKLTEQPELAN VDMV'WIVGGSSVIKEAM

NHPGHLKLFVTRI QDFESDTFFPEIDLEKYKLLP

EYPGVLSDVQEEKGIKYKFEVYEKND*

OT-CD19c-009 (CD8a MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS 643 709 leader - CD 19 scFV - LGDRVnSCRASQDISKYLNWYOQKPDGTVKLLI

CD8a-Tm - 4 IBB - YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

CDSzeta -linker TYFCQQG TLPYTFGGGTKLEITGGGGSGGGGSG

(GGSGG) - hDHFR GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

(Amino acid 2-187 of YGVSWrRQPPRKGLEWLGVIWGSETTYY SALKS

WT; Y122I, A125F) - RLTIIKDNSKSQVFLK NSLQTDDTAIYYCAKHY

stop) YYGGSYAMDYWGQGTSVTVSSTi PAPRPPTPAP

TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI

WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKOP

FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS

ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG

RDPEMGGKPRRKNPQEGLYNELQ DKMAEAYSE

IGMKGERRRGKGHDGLYQGLSTATKDTYDALHM

QALPPRGGSGGVGSLNCIVAVSQNMG1GKNGDLP

WPPLRNEFR YFQRMTTTS S VEGKQNLV GKKT

WFSIPEK^PLKGRINLVLSRELKEPPQGAHFLSRS

LDDALKLTCQPELANKVDMVWIVGGSSVIKEFM

NHPGHLKLFVTRIMQDFESDTFFPEIDLEKYKI .1 P

EYPG VL SDVQEEK G IKYKFE VYEK ND *

OT-CD19C-010 (CD8a MALPVTALLLPLAl l LHAARPDIQM QTTSSLSAS 644 710 leader - CD19 scFV - LGDRVnSCRASODISKYLNWYQQKPDGTVKLLI

CD8a-Tm - 41BB - YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

C^'D3zeta - linker TYFCQOG TLPYTFGGGT LEITGGGGSGGGGSG

(GGSGG) -hDHFR GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

(Amino acid 2-187 of YGVSWTRQPPRKGLEWLGVIWGSETTYY SALKS

WT; Q36K, Y122I) - PJ-TIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY

stop) YYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAP

TiASQPLSLRPEACRPAAGGAWTRGLDFACDiYi

WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQP

FMRP VQTTQEED GC S CRFPEEEEGGCELR VKF SRS

ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG

RDPEMGGKPRRKNPOEGLY ELQKDKMAEAYSE

IGMKGERRRGKGHDGLYQGLSTATKDTYDALHM

QALPPRGGSGGVGSLNCIVAVSQNMGIGKNGDLP

\ TPLR EFRYF RMTTTSSVEGKQNLVIMGK T

WFSIPEKNRPL GRINLVLSREL EPPQGAHFLSRS

LDDALKLTCQPELAN VDMV'WIVGGSSVIKEAM

NHPGHLKLFVTRIMQDFESDTFFPEIDLEKYKLLP

EYPGVLSDVQEEKGIKYKFEVYEKND*

OT-CD19C-01 1 (CD8a MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS 645 71 1 leader -CD 19 scFV - LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLI

CD8a-Tm - 4 IBB - YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

CD3zeta -linker TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG

(GGSGG) - hDHFR GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

(Amino acid 2-187 of YGVSWrRQPPRKGLEWLGVIWGSETTYY SALKS

WT; Q36K,N65F, Y122I) RLTIKDNSkSQVFL.KMNSLQTDDTAIYYCAKHY

-stop) YYGGSYAMDYWGQGTSVTVSST TPAPRPPTPAP

TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI

WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQP

FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS

ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG RDPEMGGKPRRKNPQEGLYNELQ DKMAEAYSE

IGMKGERRRGKGHDGLYQGLSTATKDTYDALH

QALPPRGGSGGVGSLNCIVAVSQNMG1GKNGDLP

WPPLRNEFRYFFRMITTSSVEGKQNLVIMGKKTW

FSIPEKFRPLKGRINLVLSRELKEPPQGAilFLSRSL

DDALKLTEQPELANKVDMVWIVGGSSVIKEAMN

HPGHLKLFVTRIMQDFESDTFFPEIDLEKYKLLPE

YPG VL S DVQEEK G IK YKFE VYEKND *

OT-CD 1911-012 (CD8a MALP VT ALLLPL ALLLH AARP S GG VQ VETISP GD G 646 712

Leader - Linker (SG)- RTFPKR GQTC WH YTGMLED GKK VD S SRDRNKP

FKBP (F36V, L106P) - FKFMLGKQE\TRGVVEEGVAQMSVGQRAKLTISPD

Furin Site -CD 19 scFV - YAYGATGHPG11PPHATLVFDVELLKPEESRR\⁷RR

CD8a-Tm - 41BB - NKRSKDIQMTQTTSSLSASLGDRVTISCRASQDIS

CDSzeia - stop) KYLNWYQQKPD GTV T J T YHT SRLH S G VP SRF S G

SGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFG

GGTKLEITGGGGSGGGGSGGGGSEVKLQESGPGL

VAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLE

WLGVIWGSEl'i'YYNSALKSRLTIIKDNSKSQVFLK

MNSLQTDDTArt'YCAKHYYYGGSYAMDYWGQG

TS VT V SS I J J ^'PAPRPPTPAPTIASQPLSLRPEACRPA

AGG A VHTRGLD F ACDI YI W APL AGTC G VLLL S L V

lTL YC RGRKKL L YIFKQPFMRP VQTTQE EDG C S C

RFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYN

ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ

EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDG

LYQGLSTATKDTYDALHMQALPPR*

OT-CD19n-013 (CD8a MALPVTALLLPLALLLHAARPSGISLIAALAVDYVI 647 713 leader - Linker (SG)- G IENA^IPWNLPADLAWFKRNTL-NKPVIMGRHT

ecDHF (Amino acid 2- WESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEA

L59 of WT) (R12Y, IAACGD EIMVLGGGRVIEQFLPKAQKLYLTHID

Y LOOI) - Furin Site - AEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHS

CD 19 scFV -CD8a-Tm - YCFEILERRESRRVRR KRSKDIQMTQTTS SL S AS

4 IBB - CD3zeta - stop) LGDRVnSCRASODISKYLNWYQOKPDGTVKLLI

YHTSRLHSGVPSRFSGSGSGTDYSLTIS LEQEDIA

TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG

GGGSEVKLQESGPGLVAPSOSLSVTCTVSGVSLPD

YGVSWIRQPPRKGLEWLGVIWGSE'l'lY^'YNSALKS

RLTIIKDNSKSOVFLKMNSLQTDDTAIYYCAKHY

YYGGSYAMDYWGOGTSVTVSSTTTPAPRPPTPAP

TI A SQPL SLRPE ACRP A AGGA VHTRGLDF ACDI YI

W APL AGTC G VLLL. SL VITL YCKRGRKKLL Y IFKQP

FMRPVQTTQEEDGCSCRFPF.EEEGGCELRVKFSRS

ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG

RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE

1G MKGERRRG GHD GL Y QGL ST ATKDT YD ALHM

QALPPR*

OT-CD19n-0l4 (CD8a MALPVTALLLPLALLLHAARPSGVGSLNCIVAVSO 648 714

Leader - Linker (SG)~ MGIGKNGDLPWPPLRNEFR YFQRM^' Γ1 Ί 'S S VEGK

hDHFR (Amino acid 2- QNLVD lGKKTWFSIPEKNRPLKGRINLVLSRELKE

187 of WT; Y122I, PPQGAHFL-SRSLDDALKLTE-QPELA KVDMVWIV

A125F) - Furin Site - GG S S VIKEFM HPGHLKLF VTRINIQDFE SDTFFPEI

CD 19 scFV - CD8a-Tm - DLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKN

4 IBB -CD3zeta - stop) DESRRVRRNKRSKDIQMTQTTSSLSASLGDRVTIS

CRASODISKYLNWYQQKPDGTVKLLIYHTSRLHS

G SRFSGSGSGTDYSLTISNLEQEDIATYFCQQG

NTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVK

LQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIR QPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDN

SKSQVFLKMNSLQTDDTA!YYCAKHYYYGGSYA

MDYWGQGTSVTVSS 1 ' 1 1 P APRPPTP APT! ASQPL S

I J3PEA CRPA AGGA VHTRGLDF A CDIYTW APL AGT

CGVLLLSLVTTLYCKRGRKKLLYTFKQPFMRPVQT TOEEDGCSCRFPFFF.EGGCELRVKFSRSADAPAY

KQGQNQLYNELNLG RREEYD VLD KRRGRDPEMG GKPRRKNPQEGLY ELQKDKMAEAYSEIGMKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

*

OT-CD 1911-015 (CD8a MALP VT ALLLPL ALLLH AARP S G VG SLNCTVA VSQ 649 715 leader - Linker (SG)- NMGIGKNGDLPWPPLRNEFRYFKRMTTTSSVEGK

liDHFR (Amino acid 2- ONLVLMGKKTWFSIPEKNRPLKGRINLVLSRELKE

187 of WT; Q36K, PPQGAHFLSRSLDDALKLTEQPELANKVDMVWIV

Υ122Γ) -Furin Site - GGSSVT EAMNHPGHLKXFVTRIMQDFESDTFFPE

CD19 scFV - CD8a-Tm - IDLEKYKLLPEYPGVLSDVQEEKGD YKFEVYEKN

4 IBB - CD3zeta -stop) DESRRVRRNKRSKDIQMTQTTSSLSASLGDRVTIS

CRASQDISKYL Vv'^'YQQKPDGTVKLLIYHTSRLHS

GVPSRFSGSGSGTDYSLTISNLEOEDIATYFCQQG

NTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVK

LQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIR

QPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDN

SKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYA

MDYWGQGTSVTVSS i ! ! APRPPTP APTIASQPLS

LRPEACRPAAGGAVHTRGLDFACDTYIWAPLAGT

CGVLLLSLVITLYCKRGRKKLLYTFKQPFMRPVQT

TQ EEDGC SCRFPF F EEGGCELR VKF SRS AD AP A Y

KQGQNQLYNELNLG RREEYD VLDKRRGRDPEM G

GKPRRK PQEGLY ELQKDKMAEAYSEIGMKGE

RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

OT-CD19-056 (CD8a MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS 1005 1022 Leader - GDI 9 scFV - LGDRVnSCRASQDISKYLNWYQQKPDGTVKLLI

CD8a-Tm - 41BB - YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

CD3zeia - linker TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG

(GGSGG) - hDHFR GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

(Amino acid 2-187 of YGVSWLRQPPRKGLEWLGVIWGSETTYY SALKS

WT; Y122I) -stop) RLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY

YYGGSY AMD YWGQGTSVTVSSTTTP APRPPTP AP

TI A SQPL SLRPE ACRP A AGGA VHTRGLDF ACDI YI

W APL AGTC G VLLL SL VITL YCKRGRKKLL Y IFKQP

FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRS

AD AP A YKQGQNQL YNELNL GRREE YD VLDKRRG

RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE

1G MKGERRRG K GHD GL Y QGL ST ATKDT YD ALHM

QALPPRGGSGGVGSLNCIVAVSQNMGIGKNGDLP

WPPLRNEFRYFQRMTTTSSVEGKQNLVIMGKKT

WFSIPEKNRPLKGRINLVLSRELKEPPQGAHFLSRS

LDDALKLTEQPELANKVDMVWIVGGSSVIKEAM

NHPGHLKLFVTRIMQDFESDTFFPEIDLEKYKLLP

EYPGVLSDVQEEKGIKYKFEVYEKND*

OT-CD19-057 (CD8a MALP VT ALLLPL ALLLHAARPDIQMTQTTSSLSAS L006 1023 leader -CD L9 scFV - LGDRVTISCRASQDTSKYLNWYQQKPDGTVKLLI

CD8a-Tm - 41BB - YHTSRLFISGVPSRFSGSGSGTDYSLTLSNLEQEDIA

CD3zeia - BamHi (GS)- TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG

stop) GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

YGVSWIRQPPRKGLEWLGVTWGSE'i ^'L YYNSALKS RLTUKDNSKSQVFLKMNSLQTDDTA1YYCAKH '

YYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAP

TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI

WAPLAGTCGVLLLSLVrTLYCKRGRKKLLYIFKQP

FMRP VQTTOEEDGCS CRFPF F FFGGCELR VKFSRS

ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG

RDPEMGGKPRRKNPQEG LYNELQKDKMAE A YSE

IGMKGERRRGKGHDGLYQGLSTATKDTYDALHM

QALPPRGS*

OT-CD19-058 (CD8a MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS 1007 1033 leader -CD 19 scFV - LGDR\01SCRASQDISKYLNWYOQKPDGTVKLLI

CD8a-Tm - 41BB - YHTSRLHS G VP SRF S G S G S GTD YSLTI SNLEQEDI A

CD3zeia - p2A - BamHI TYFCOQG TLPYTFGGGT LEiTGGGGSGGGGSG

(GS)- stop) GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

YGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKS

RLTIIKDNSkSQVFLKMNSLQTDDTAIYYCAKHY

YYGGSYAMDYWGQGTSVTVSSTTTPAPRPPTPAP

TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI

WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQP

FMRPVQTTQEEDGCSCRFPFFF.F.GGCELR VKFSRS

ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG

RDPEMGGKPRRKNPQEGLYNELQ DKMAEAYSE

IGMKGERRRGKGHDGLYQGLSTATKDTYDALHM

QALPPRGATNFSLLKQAGDVEENPGPGS*

OT-CDl.9-059 (CD8a MALPVTAT T J PLALLLHAARPYPYDVPDYADIQM 1008 1034 leader - HA Tag - CD 19 TQTTSSLSASLGDRVTISCRASQDISKYLNWYQQK

scFV - CD8a-Tm - 41BB PDG T VKLLIYHT SRLHS G VP S RF S G S G S GTD Y SLTI

- CDSzeta - BamHI (GS)- SNLEQEDIATYFCQOGNTLPYTFGGGTKLEITGGG

stop) GSGGGGSGGGGSEVKLQESGPGLVAPSQSLSVTC

TVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSET

TYYNS.ALKSRLTIIKD SKSOVFLKTv NSLQTDDTA

IYYCAKHYYYGGSYAMDYWGQGTSVTV'SSTTTP

APRPPTP APTIA SQPL SLRPE ACRP A AGG A VHTRG

LDFACDIYIWAPLAGTCGVLLLSLVTTLYCKRGRK

KLL YIFKQPFMRP VQTTQEED GC S CRFPEEEEGGC

ELR VKF SRS AD AP A YKQGQN QL YNELNL GRREE

YDVLDKRRGRDPEMGGKPRR PQEGLYNELQK

DKMAEAYSEIGMKGERRRGKGHDGLYQGLSTAT

KDTYDALHMQALPPRGS*

OT-CD 19-060 (CD8a MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS 1009 1035 leader - CD19 scFV- LGDRVTCSCRASQDISKYLNWYQQKPDGTVKLLI

CD8a-Tm- 41BB - YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

CD3zeia - Linker (SG)- TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG

Furin - BamHI (GSt- GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

stop) YGVSWIRQPPRKGLEWLGVTWGSh'i ^'iYYNSALKS

RLTin DNSKSQVFLKMNSLQTDDTAIYYCAKHY

YYGGSYAMDYWGOGTSVTVSSTTTPAPRPPTPAP

TLASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI

WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQP

FMRPVQTTQEEDGCSCRFPFFFFGGCELR VKFSRS

ADAPAYKQGQNQLYTNIELNLGRREEYDVLDKRRG

RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE

IGMKGERRRGKGHDGLYQGLSTATKDTYDALHM

QALPPRSGESRRVRRNKRSKGS*

OT-CD19-063 (CD8a MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS 1010 1036 leader - CD 19 scFV - LGDRVnSCRASQDISKYLNWYQQKPDGTVKLLI CD8a-Tm - 41BB - YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

CD3zeta - stop) TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG

GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

YGVSWlRQPPRKGLEWLGVIWGSE'l iYYNSALKS

RLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY

YYGGSYAMDYWGQGTSVTVSS ! Ί ^" 1 PAPRPPTP AP

TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI

WAPLAGTCGVLLLSLVTTLYCKRGRKKLLYIFKQP

FMRPVOTTQEEDGCSCRFPEEEEGGCELRVKFSRS

ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG

RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE

IGMKGERRRGKGHDGLYQGLSTATKDTYDALHM

QALPPR*

OT-CD 19-064 (CD8a MALPVT T J J PLALLLHAARPDiQMTQTTSSLSAS 1215-1231 1037 leader - CD 19 scFV - LGDRVnSCRASQDISKYLNWYOQKPDGTV T T J

CD8a-Tm - 4 IBB - YHTSRLHS G VP SRF S G S G S GTD YSLTI SNLEQEDi A

CD3zeta - Linker (TR)- TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG

HA Tag - FKBP (E31G, GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

F36V, R71G, K105E) - YGVSWIRQPPRKGLEWLGVIWGSETIYYNSALKS

stop-IRES- mCherry) RLTUKDNSKSQVFLKMNSLQTDDTAIYYCAKH '

YYGGSYAMDYWGQGTSVTVSSTi PAPRPPTPAP

TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI

WAPLAGTCGVLLLSLVITLYCKRGRKKLLY1FKQP

FMRPVQTTOEEDGCSCRFPFFF.EGGCELRVKFSRS

ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG

RDPEMGGKPRRKNPQEG L YNELQKDKMAE A YSE

IGMKGERRRGKGHDGLYQGLSTATKDTYDALHM

QALPPRTR YPYDVPDY AG VO VETISPGDGR TFPK

RGQTCVVHYTG^GDGKKVDSSRDRNKPFKFML

GKQEVIRU VEEGVAQMSVGQGAKLTISPDYAYG

ATGHPGIIPPHATL D ⁷FLLELE*MHRSAAAAT*I

PPPPPLSLPPP*RYWPKPLGIRPVCVCLYVIFHHIAV

FWQCEGPETWP CLLDEH S * G SFP SRQRN ARS VEC

REGS S S SGSFLKTN VC SDPLQA AEPPTWROVPLR

PKATCIRYTCKGGTTPVPRCELDSCGKSQMALLK

RIQOGAEGCPEGTPLYGI*SGASVHMLYMCLVEV

ΚΚΊ^■bRPPEPRGRGFPLK TMIIWPQP* * ARARRIT

WPSSRSSCASRCTWRAP*TATSSRSRARARAAPTR

APRPPS*R*PRVAPCPSPGTSCPLSSCTAPRPT*STP

PTSPTT*SCPSPRASSGSA**TSRTAAW*P*PRTPPC

RTASSSTR*SCAAPTSPPTAP*CRRRPWAGRPPPSG

CTPRTAP*RARSSRG*S*RTAATTTLRSRPPTRPRS

PCSCPAPTTSTSSWTSPPTTRTTPSWNSTNAPRAA

TPPAAWTSCTS*

OT-CD 19-066 (CD8a MALPVTAT 1 1 PLALLLHAARPDIQMTQTTSSLSAS 1015 1039 leader - CD19 scFV - LGDRVnSCRASQDISKYLNWYQQKPDGTVKLLI

CD8a-Tm - 41BB - YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA

CD3zeia - Linker (GSt- TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG

P2A peptide -mCherry GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

(MIL) - stop) YGVSWlRQPPRKGLEWLGVIWGSE'l iYYNSALKS

RLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHY

YYGGS YAMDYWGQGTS VTVS S'i'i'i Ϋ APRPPTP AP

TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI

WAPL AGTC GVLLL SLWLYCKRGRKKLLYIFKQP

FMRPVOTTQEEDGCSCRFPEEEEGGCELRVKFSRS

ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG

RDPEMGGKPRRKNPOEGLYNELQKDKMAEAYSE

IGMKGERRRGKGHDGLYQGLSTATKDTYDALHM QALPPRGSGATNFSLLKQAGDVEENPGPLSKGEE

DNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGR

PYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSK

AYVKOTADIPDYLKLSFPEGFKWERVMNFEDGG

VVTVTQDSSLQDGEFIYKVKLRGT FPSDGPVMQ

KKTMGWEASSERMYPEDGALKGEI QRL LKDG

GHYDAEVKTTYKAKKPVQLPGAYNVNIKLDSTSH

NEDYTIVEQYERAEGRHSTGGMDELYK*

OT-CAR 19-IL15-001 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS 1016 1040

(CD8a leader - CD 19 LGDRVnSCRASQDISKYLNWYQOKPDGTVKLLI

scFV -CD8a-Tm - 41BB - H1 SRLHSG SRFSGSGSGTDYSLTISNLEQEDIA

CD3zeia ~ T .inker fGS)~ TYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSG

P2A - IL15 -Linker (SG3- GGGSEVKLQESGPGLVAPSOSLSVTCTVSGVSLPD

(SG4)3-SG3-SLQ) - YGVSWIRQPPRKGLEWLGVIWGbE'l'lYY SALKS

iL15Ra - stop) RLTIKDNSKSOVFLKMNSLQTDDTATfYCAKHY

YYGGS YAMDY WGQGTS VTVS S I "1 "1 PAPRPPTP AP

TI A SQPL SLRPE ACRP A AGGA VHTRGLDF ACDI YI

W APL AGTC G VLLL SL V1TL YCKRGRKKLL Y IFKQP

FMRPVQTTQEEDGCSCRFPEEEEGGCELRVKt SRS

ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG

RDPEMGGKPRRK PQEGLYNELQKDKMAEAYSE

1G MKGERRRG GHD GL Y QGL ST ATKDT YD ALHM

QALPPRGSGATNFSLLKQAGDVEENPGPNWVNVI

SDLKK IEDLIQ SMH1D A TL YTE SD VHP S C K VT AM

CFLLELQVISLESGDASIHDTVENLIILAN SLSSNG

NVTESGCKECEELEEKNIKEFLOSFVHIVQMFI TS

SGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMS

VEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLT

EC^'L KATNVAHWTTPSLKCTRDPALWORP.APP

STVTTAGVTPQPESLSPSG EPAASSPSSNNTAATT

AAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTT

AKNWELTASASHOPPGVYPQGHSDTTVAISTSTV

LLCGLSAVSLLACYLKSRQTPPLASVE-MEAMEAL

PVTWGTSSRDEDLENCSHHL*

OT-CAR19-IL15-002 MALPVTAT J ,T PLALLLHAARPDIQMTQTTSSLSAS 1017 1041 (CD8a leader -CD 19 LGDRVnSCRASQDISKYLNWYOQKPDGTV T T J

scFV -CD8a-Tm -41BB - YHTSRLHS G VP SRF S G S G S GTD YSLTI SNLEQEDI A

CD3zeia - Linker (GS)- TYFCOQGNTLPYTFGGGT LEiTGGGGSGGGGSG

P2A - Linker GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

(MLLLVTST T T CELPHP YGVSWlRQPPRKGLEWLGViWGSETlYYNSALKS

AFLLIP) (SEQ ID NO: RLTUKDNSKSQVFLKMNSLQTDDTAIYYCA HY'

1031) - ILi 5 - Linker YYGGSY AMD YWGQGTSVWSS^'lTi 'PAPRPPTP AP

(SG3-(SG4)3-SG3-SLQ) TIASQPLSLRPEACRPAAGGA VHTRGLDF ACDIYI

- IL15Ra - stop) WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQP

FMRPVQTTQEEDGCSCRFPPFF.F.GGCELRVKFSRS

ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRG

RDPEMGGKPRRKNPQEGLYNELQ DKMAEAYSE

IGM GERRRGKGHDGLYQGLSTATKDTYDALHM

QALPPRGSGATNFSLLKQAGDVEENPGPMLLLVT

SLLLCELPHPAFLLIPNWVNVISDLKKIEDLIQSMH

IDATLYTESDVHPSCKVTAMKCFI J FT .QVISLESG

DA SIHDTVENLIIL ANNSL S SNGNVTE S GCKECEEL

EEKNIKEFLOSFVHrVQ FINTSSGGGSGGGGSGG

GGSGGGGSGGGSLQITCPPPMSVEHADiWVKSYS

LYSRERYICNSGFKRKAGTSSLTECVLNKATNVA

HWTTPSLKCIRDPALVHORPAPPSTVTTAGVTPQP

ESLSPSU EPAASSPSSNNTAATTAAIVPGSOLMPS

KSPSTGTTEISSHESSHGTPSQTTAKNWELTASAS HQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLA

CYL SRQTPPLASVEMEAMEALPVTWGTSSRDED LENCSHHL*

OT-CD19-IL 1 5-006 MALPVTALLLPLALLLHAARPDIQMTQTTSSLSAS 1018 1042 (CD8a leader -CD 19 LGDRVTISCRASQDTSKYLNWYQQKPDGTVKLLI

scFV - CD8a-Tm - 41BB YH'i^'bRLHSGWSRFSGSGSGTDYSLTIS LEQEDIA

- CD3zeta - Linker (GS)- TYFCOQGNTLPYTFGGGTKLEITGGGGSGGGGSG

P2A - IgE Leader - IL.15 - GGGSEVKLQESGPGLVAPSQSLSVTCTVSGVSLPD

Linker (SG3-(SG4)3- YGVSWIRQPPRKGLEWLGVrWGSE¹1 ^"1¹ YYNSALKS

SG3-SLQ) - IL15Ra - PxLlll DNSkSQVFL-KMNSLQTDDTAIYYCAKHY'

stop) YYGGSYAMDYWGOGTSVTVSS^'ri'TPAPRPPTPAP

TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYI

WAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKOP

FMRPVQTTQEEDGCSCRFPFFF.F.GGCELRV FSRS

ADAPAYKQGONQLYNELNLGRREEYDVLD RRG

RDPEMGU PRRKNPQEGLYNELQ DKMAEAYSE

IGMKGERRRGKGHDGLYQGLSTATKDTYDAL

QALPPRGSGATNFSLLKQAGDVEENPGPMDWTW

ILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHID

ATLYTESDVHPSC VTAM CFLLELQVISLESGDA

SIHDTVE NLIIL ANN SLS SNGNVTES GCKECEELEE NIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGG

SGGGGSGGGSLQTTCPPPMSVEHADIWVKSYSLYS

RERYICNSGFKRKAGTSSLTECVLNKAT VAHWT

TPSLKCIRDP ALVHQRPAPPSTVTTA GVTPQPE SLS

P S GKEP A A S SP S SNNT A A TT A AIVPGS QL3V1P SK SP S

TGTTEI S SHE S S H G TP S QTT AKNWELT A S A SHQPP

GVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLK

SRQTPPLASVEMEAMEALPVTWGTSSRDEDLENC

SHHL*

[00229] Constructs disclosed in Table 10 which are transcriptionally controlled by a CMV promoter, in some instances may be placed under the transcriptional control of a different promoter to test the role of promoters in CD 19 CAR expression. In one embodiment, the CMV promoter may be replaced by an EFla promoter. In one embodiment, the CMV promoter of the, OT-CD 19-001 construct, may be replaced to generate OT-CD19N-017 constract, with a EFla promoter. In another embodiment, the CMV promoter of the CD 19 CAR, OT-CD 19 CAR-002 constract, may be replaced to generate OT-CD 19N-018 construct, with a EFla promoter. In another embodiment, the CMV promoter of the CD 19 CAR, OT-CD 19 CAR-003 constract, may be replaced to generate OT-CD 19N-019 constract, with a EF 1 a promoter. In another embodiment, the CMV promoter of the CD 19 CAR, OT-CD 19 CAR-004 construct, may be replaced to generate OT-CD 19 -020 constract, with a EFla promoter. In another embodiment, the CMV promoter of the CD19 CAR, OT-CD 19 CAR-005 constract, may be replaced to generate OT-CD 19N-021 constract, with a EFla promoter. In another embodiment, the CMV promoter of the CD 19 CAR, OT-CD 19 CAR-006 constract, may be replaced to generate OT- CD 19N-022 construct, with a EFla promoter. In another embodiment, the CMV promoter of the CD 19 CAR, OT-CD 19 CAR-007 construct, may be replaced to generate OT-CD19N-023 construct, with a EFla promoter. In another embodiment, the CMV promoter of the CD19 CAR, OT-CD19 CAR-008 construct, may be replaced to generate OT-CD19N-024 construct, with a EFla promoter. In another embodiment, the CMV promoter of the CD 19 CAR, OT-CD19 CAR- 009 construct, may be replaced to generate OT-CD19N-025 construct, with a EFla promoter.

[00230] In one embodiment, the CAR construct comprises a CD 19 scFV (e.g., CAT13.1 E10 or FMC63), a CD8a spacer or transmembrane domain, and a 4- IBB and€ϋ3ζ endodomam. These constructs with CAT13.1E10 may have increased proliferation after stimulation in vitro, increased cytotoxicity against the CD 19+ targets, and increased effector and target interactions as compared to constructs with FMC63.

[00231] In some embodiments, the payloads of the present invention may be tuned using the catalytic domains of the E3 ubiquitin ligases. The catalytic domains of E3 ligases may be fused to an antibody or a fragment of the antibody. The payload is fused to the antigen recognized by the antibody or a fragment of the antibody that is fused to the E3 ligases catalytic domain. The E3 ligases useful in the present invention include, but are not limited to Ring E3 ligase, HECT E3 ligases and RBR E3 ligases. Any of the methods taught by Kanner SA et al. (2017) eLife: 6: e29744 may be useful in the present invention (the contents of which are incorporated by reference in their entirety).

[00232] In some embodiments, the payloads described herein, may be regulated by E3 ubiquitin ligases constructs. The E3 ligases constructs may comprise the catalytic domain of E3 ligases fused to an SRE and an antibody or a fragment of an antibody. The payloads are fused to the antigen recognized by the antibody or a fragment of an antibody, that is appended to the catalytic domain of E3 ligases. In the absence of the stimulus corresponding to the SRE, the E3 ubiquitin ligases constructs are destabilized, which in turn, allows the expression of the payloads fused to the antigen. In the presence of ligand corresponding to the SRE, the E3 ubiquitin ligases constructs are stabilized and available to bind to the antigen fused to the payloads. Binding of the E3 Isgases constructs to the antigens, targets the protein for degradation. The E3 ubiquitin ligases constructs may be used to regulate any payload described herein, provided the payload is fused to an antigen recognized by the antibody or the fragment of the antibody in the E3 ubiquitin ligases construct. In some embodiments, the payload is a chimeric antigen receptor. The E3 ubiquitin ligases constructs may be used to design logic gates. In one embodiment, the E3 ubiquitin ligases constructs may be used to generate a NOT gate, wherein one ligand induces the expression of the payload, while another inhibits the expression of the payload. In some embodiments, the NOT gate may be generated using the E3 ubiquitin ligases constructs and by fusing the pay loads-antigen fusion protein to a second a SRE that is distinct from the SRE in the E3 ubiquitin ligase construct.

[00233] 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.

Tandem CAR (TanCAR)

[00234] 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 niulti specific 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, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30 amino acids.

Split CAR

|0Θ235] In some embodiments, the components including the targeting moiety, transmembrane domain and intracellular signaling domains of the present in v ention 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 iigand 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 CO3C intracellular domain.

[00236] 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.

[00237] In some aspects, the two parts of the split C AR system contain heterodimerization domains that conditionally interact upon binding of a heierodimerizing 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 a! ,, J Am Chem Soc , 2008, 130(40): 13186-13187) and ABA (abscisic acid) induced dimerization of PP2C and PYL domains (Cutler ei al., Annu Rev Plant Biol. 2010, 61 : 651-679). The dual regulation using inducible assembly (e.g., iigand 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

[00238] 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

[00239] 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 ceils, thereby reducing CAR mediated tissue toxicity.

Activation-conditional CAR

[00240] 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. IL2 promoter or NFAT binding sites. In some embodiments, activation of the immune cell may be achieved by a constitutively expressed CAR (International Publication No: WQ2016126608; the contents of which are incorporated herein by reference in their entirety).

Cytokines, chemokines and other soluble factors

[00241] In accordance with the present invention, CARs of the present invention may be utilized along with other 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 ceil growth, differentiation, migration and survival, to a number of effector activities. For example, activated T cells produce a variety of cytokines for cytotoxic function to eliminate tumor cells.

[00242] 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 (TNFs), interferons (IFNs), TGF beta and chemokines. 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.

[00243] In some embodiments, payloads of the present invention may be cytokine receptors, recombinant receptors, variants, analogs and derivatives thereof; or signal components of cytokines.

[00244] In some embodiments, cytokines of the present invention may be utilized to improve expansion, survival, persistence, and potency of immune cells such as CD8+TEM, natural killer cells and tumor infiltrating lymphocytes (TIL) 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) Pleiotropisrn, 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 cytokine 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.

[00245] In some embodiments, the payload of the present invention may be an interleukin (IL) cytokine. Interleukins (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 ILL IL1 alpha (also called hematopoietin-l), ILlbeta (catabolin), IL ! delta, ILIepsilon, ILleta, IL ! zeta, interleukin- 1 family member 1 to 11 (IL1F1 to IL1F11), interleukin- 1 hornoiog 1 to 4 (IL1H1 to IL1H4), IL1 related protein 1 to 3 (ILIRPI to ILIRP3), IL2, 1L3, 1L4, IL5, IL6, IL7, U .K. IL9, IL10, IL10C, IL10D, ILl 1 , ILl la, H i l b. IL12, IL13, IL14, IL15, IL16, !! . ! ?. IL17A, ΪΠ 7Β, IL17C, IL17E, IL17F, ILl 8, ILl 9, IL20, IL20 like (IL20L), 1121 , 11,22, 11,23, IL23A, IL23-pl9, IL23-p40, IL24, 1125, IL26, IL27, IL28A, IL28B, IL29, IL30, IL31, IL32, IL33, IL34, IL35, IL36 alpha, IL36 beta, IL36 gamma, IL36R , IL37, IL37a, IL37b, IL37c, IL37d, IL37e and TL38. In other aspects, the payload of the present invention may be an interleukin receptor selected from CD121a, CDwl21b, IL2Ra/CD25, IL2Rp7CD122, IL2Ry/CD132, CDwl31, CDI24, CD 13 I, CDw l25, CD126, CD130, CD I27, CDw210, IL8RA, ILl lRa, CD212, CD213al , CD213a2, IL14R, ILlSRa, CDw217, IL18R , IL18Rp\ IL20R , and IL20Rp.

[00246] In one embodiment, the payload of the invention may comprise ILl 2. ILl 2 is a heterodimeric protein of two subunits (p35, p40) that is secreted by antigen presenting cells, such as macrophages and dendritic cells. ILl 2 is type 1 cytokine that acts on natural killer (NK) cells, macrophages, CD8⁺ Cytotoxic T cells, and CD4⁺ T helper cells through STAT4 pathway to induce IFN-γ production in these effector immune cells (reviewed by Trinchieri G, Nat Rev Immunol. 2003; 3(2): 133-146). IL12 can promote the cytotoxic activity of NK cells and CD8⁺ T cells, therefore has anti-tumor function. Intravenous injection of recombinant ILl 2 exhibited modest clinical efficacy in a handful of patients with advanced melanoma and renal cell carcinoma (Goliob et al., Clin. Cancer Res . 2000; 6(5): 1678— 1692) . IL12 has been used as an adjuvant to enhance cytotoxic immunity using a melanoma antigen vaccine, or using peptide pulsed peripheral blood mononuclear cells; and to promote NK cell activity in breast cancer with trastuzumab treatment. Local delivery of IL 12 to the tumor microenvironment promotes tumor regression in several tumor models. These studies ail indicate that locally increased IL12 level can promote anti-tumor immunity. One major obstacle of systemic or local administration of recombinant 1L12 protein, or through oncolytic viral vectors is the severe side effects when IL12 is presented at high level. Developing a system, that tightly controls IL.12 level may provide a safe use of IL12 in cancer treatment.

[00247] 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 interleukm. Likewise, TNFalpha, T Fot, TNF-aipha, TNF-a, TNF alpha and TNF a all refer to the same protein.

[00248] In one aspect, the effector module of the invention may be a DD-TL12 fusion polypeptide. This regulatable DD-IL12 fusion polypeptide may be directly used as an

immunotherapeutic agent or be transduced into an immune effector cell (T cells and TIL cells) to generate modified T cells with greater in vivo expansion and survival capabilities for adoptive cell transfer. The need for harsh preconditioning regimens in current adoptive cell therapies may be minimized using regulated IL12 DD-IL12 may be utilized to modify tumor microenvironment and increase persistence in solid tumors that are currently refractory to tumor antigen targeted therapy. In some embodiments, CAR expressing T cells may be armored with DD regulated IL12 to relieve immunosuppression without systemic toxicity.

[00249] In some embodiments, the 1L12 may be a Flexi IL12, wherein both p35 and p40 subunits, are encoded by a single cDNA that produces a single chain polypeptide. The single chain polypeptide may be generated by placing p35 subunit at the N terminus or the c terminus of the single chain polypeptide. Similarly, the p40 subunit may be at the N terminus or C terminus of the single chain polypeptide. In some embodiments, the IL12 constructs of the invention may be placed under the transcriptional control of the CMV promoter (SEQ ID NO. 716), an EF la promoter (SEQ ID NO. 717, SEQ ID NO. 908) or a PGK promoter (SEQ ID NO. 718). Any portion of IL12 that retains one or more functions of full length or mature IL12 may be useful in the present invention. In some aspects, the DD-IL12 comprises the amino acid sequences listed in Table 1 1 . The amino acid sequences in Table 1 1 may comprise a stop codon which is denoted in the table with a "*" at the end of the amino acid sequence.

Table 11 : DD-IL12 constructs

Linker - GGSGG 629 679-680

Linker - GGGGSGGGGSGGGGS 720 910-915

Linker GS GGATCC

Spacer ATNF SLLKQAGD VEENPGP 745 746

Furin cleavage - SARNRQKRS 721 750 site

Furin cleavage - ARNRQKRS 722 751 site

Modified Furin - ESRR VRRNKR SK 630 681-683

P2A Cieavable - GAT FSLLKQAGD VEENPGP 925 926 Peptide

p40 rWELKKD VVELDWYPDAPGEMVVLTCD 723 752-761,

TPEEDGITWTLDQSSEVXGSGKTLTIQVKEF 632-634

GDAGQYTCHKGGEVLSHSLLLLHKKEDGI

WSTDILKDQKEPKNKTFLRCEAK YSGRFT

CWWLTTISTDLTFSVKSSRGSSDPQGVTCG

AATLSAERVRGDNKEYEYSVECQEDSACP

AAEESLPIEVMVDAVHKLKYENYTSSFFIRD

IIKPDPPKNLQLKPLK SRQ VEVSWEYPDT

WSTPHSYFSLTFCVQVQGKSKREKKDRVFT

DKTSATVICRKNASTSVRAQDRYYSSSWSE

WASVPCS

p40 (K217N) rWELKKDVYWELDWYPDAPGEMWLTCD 747 748

TPEED GITWTLDQ S SE VL. GS GKTLTiQ VKEF

GDAGQYTCHKGGEVLSHSLLLLHKKEDGI

WSTDILKDQKEPKNKTFLRCEAKNYSGRFT

CWWLTnSTDLTFSVKSSRGSSDPQGVTCG

AATLSAERVRGDNKEYEYSVECQEDSACP

AAEESLPIEVMVDAVHKLKYENYTSSFFIRD

I IKPDPPNNLQLKPLKN SRQ VE V S WE YPDT

WSTPHSYFSLTFCVQVQGKSKREKKDRVFT

DKTSATVICRKNASISVRAQDRYYSSSWSE

WASVPCS

p35 RNLPVATPDPGMFPCLHHSQNLLRAVSNM 724 762-771,

LQKARQTLEFYPCTSEEIDHEDITKDKTSTV 1012

EACLPLELTKNESCLNSRETSFITNGSCLASR

KTSFMMALCLSSIYEDLKMYQVEFKTMNA

KLLMDPKRQIFLDQNMLAVIDELMQALNF

N SET VPQK S SLEEPDF YKT IKLCILL H AFRI

RAVTIDRVMSYLNAS

ecDHFR ISLIAALAVDYVIGMENAMPWNLPADLAW 9 692, 772, (Amino acid 2- FKRNTLNKPVIMGRHTWESIGRPLPGRKNII 814, 687, 159 of WT) LSSQPGTDDRVTWVKSVDEAIAACGDVPEI 988, 991 (R12Y, Y100I) MVIGGGRVIEQFLPKAQKLYLTHiDAEVEG

DTHFPDYEPDDWESVFSEFHD AD AQN SHS Y

CFEILERR

FKBP (F36V, GVQVETISPGDGRTFPKRGQTCVVHYTGML 11 684-686, L 106P) ED GKK VD S SRDRNKPFKFML GKQE VIRGW 987, 989

EEGVAQMSVGQRAKLTISPDYAYGATGHP

GnPPHATLVFDVELLKPE

FKBP (F36V, GVQVETISPGDGRTFPKRGQTCVVHYTGML 12 688-691, E31G, R71G, GDGKKVDSSRDRNKPFKFMLGKQEVIRGW 994, 1013, K105E) F ^ GVAQMS VGQGAKLTISPDYAYGATGHP 3028

GnPPHATLVFDVELLELE

hDHFR VGSLNCIVAVSQNMGIGKNGDLPWPPLRNE 46 773

(Amino acid 2- FR YFFRM i'l'l'S S VEGKQNL VTMGKKTWF SI

187 of WT: PEKNRPLKGRiNLVLSRELKEPPQGAHFLSR

Q36F, Y122I, SLDDALKLTEQPELANKVDMVWIVGGSSVI

A125F KEFMNHPGHLKLFVTRIMQDFESDTFFPEID LE YKLLPEYPGVLSDVQEEKGIKYKFEVY

EKND

hDHFR VGSLNCrVAVSQ MGVGKNGDLPWPPLR 894 979

(Amino acid 2- EFRWQRMTTTSSWGKQNLVIMGKKTWFS

187 of WT) IPEKNRPLKGRL LVLSRELKEPPQGAHFLS

i7v> R SLDD ALKL TEQPEL ANK VDMVWI VGG S S

WKEAMNOTGHLKLEVTRIMQDFESDTFFP EIDLEKYKLLPEYPGVLSDVOEEKGIKYKFE

\ Y! k\D

hDHFR VGSLNCIVAVSQNMGIGKNGDLPWPPLRNE 895 694, 995 (Amino acid 2- FRWQRMTTTSSVEGKQNLVTMGKKTWFSI

187 of WT) PEK RPLKGRINLVLSRELKEPPQGAHFLSR

(Y122I) SLDDALKLTEQPELANKVDMVWIVGGSSVI

KEAMNHPGHLKLFVTRIMQDFESDTFFPEID LEKYKLLPEYPGVLSDVQEEKGIKYKFEVY EKND

OT-IL12-001 CMV MCHOQLVTSWFSLVFLASPLVAGVQVETTSP 727 774 (p40 signal GDGRTFPKR GQTCWHYTGMLEDGKKVD S

sequence - SRDRNKPFKFMLGKQEVLRGWEEGVAQMS

FKBP (F36V, VGQRAKLTISPDYAYGATGHPGIIPPHATLV

L106P) - linker FDVELLKPEGGSGGIWELKKDVYVYELDW

(GGSGG) - YPDAPGEMWLTCDTPEEDGITWTLDOSSE

p40 - linker2 VLGSGKTLTIOVKEFGDAGQYTCHKGGEV ,

(G4S)3 - p35- SHSLLLLHKKEDGIWSTDILKDOKEPKNKTF

stop) LRCEAK YSGRFTCWWLTTISTDLTFSVKS

SRGS SDPQGVTCGAATLS AERVRGDNKEYE

YSVECQEDSACPAAEESLPIEVMVDAVHKL

KYENYTSSFFIRDIIKPDPPKNLQLKPLKNSR

QVEVSWEYPDTWSTPHSYFSLIFCVQVQG

KSKREKKDRVFTDKTSATV1CRKNASISVR

AQDR Y Y S S S WSEW A S VPC S GG GG S G GG GS

GGGGSRNLPVATPDPGMFPCLHHSQNLLRA

VSNMLQKARQTLEFYPCTSEEIDHEDITKDK

TSTVEACLPLS.TKNESCLNSRETSFITNGSC

LASRKTSFMMALCLSSIYEDLKMYQVEFKT NAKLLMDPKRQIFLDQNMLAVIDELMQA

LNFNSETVPQKSSLEEPDFYKTKIKLCILLI-I

AFRIRA VTIDRVM S YLNA S *

OT-IL 12-002 CMV MGVQVETISPGDGRTFPKRGOTCWHYTG 728 775 (Met - FKBP MLEDGKK VD S SRDRNKPFKFML GKQEVTR

(F36V, L106P) G WEEG VAQM S VGQR AKLTI SPD Y AY G ATG

- linker HPGI1P PH A TL VFD VEL L KPE GG S G GMCHQ

(GGSGG) - p40 QLVISWFSLVFLASPLVAIWELKKDVYVVE

signal sequence LDWYPDAPGEMWLTCDTPEEDGITWTLD

- p40 - linker QSSEVLGSGKTLT1QVKEFGDAGQYTCHKG

((G4S)3) - p35 GEVLSHSLUXHKKEDG!WSTD!LKDQKEP

- stop) KNKTFLRCEAKNYSGRFTCWWLTTISTDLT

FSVKSSRGSSDPQGVTCGAATLSAERVRGD

NKEYEYSVECQEDSACPAAEESLPIEVMVD

AVHKLKYENYTSSFFIRDIIKPDPPKNLQLK

PLKNSRQVEVSWEYPDTWSTPHSYFSLTFC

VQVQGK SKREKKDR VFTDKTS AT VTCRKN

ASISWAQDRYYSSSWSEWASVPCSGGGGS

GGGGSGGGGSRNLPVATPDPGMFPCLHHS

QNLLR A VSNMLQK ARQTLEF YPCT SEEIDH

EDITKDKTSTVEACLPLELTKNESCLNSRET

SFITNGSCLASRKTSFMMALCLSSIYEDLKM

YQV FKTN NAKLLMDPKRQIFLDONMLAV

IDELMQALNFNSETVPOKSSLEEPDFYKTKI

KLCILLHAFRIRA VTIDRVMS YLN AS * OT-IL 12-003 CMV MCHQQLVISWFSLVFLASPLVAGVQVETISP 729 776

(p40 signal GDG RTFPK RGQTC WH YTGMLEDG KK VD S

sequence - SRDRN PFKFMLGKQEVIRGWEEGVAOMS

FKBP (F36V, VGQRAKLTiSPDYAYGATGHPGITPPHATLV

L 106P)- furin FDVELLKPESARNRQKRSIWFI KKDVYWE

(SARNRQKRS LDWYPDAPGEMWLTCDTPEEDGITWTLD

) - p40- linker QSSEVT JSGKTLTTQVKEFGDAGQYTCFIKG

((G4S)3)~ p35 - GE VL S H S L LLLHK KED G IW STD ILKDQKEP

stop) KNKTFLRCEAK YSGRFTCWWLTTISTDLT

FSVKSSRGSSDPOGVTCGAATLSAERVRGD

NKEYEY^'SVECQEDSACPAAEESLPIEVMVD

AVHKLKYE YTSSFFIRDIIKPDPPK LQLK

PLK SRQVEVSWEYPDT STPHSYFSLTFC

VQVQGKSKREKKDRVFTDKTSATVICRKN

ASISVRAQDRYYSSSWSEWASVPCSGGGGS

GGGGSGGGGSRNLPVATPDPGMFPCLHHS

ONLLRAVSNMLO ARQTLEFYPCTSEEIDH

EDITKDKT STVE ACLPLELTKNE S CLN SRET SFITNGSCL ASRKTSFMMALCL S STYEDLKM YQVEFKTMNAKLLMDPKRQIFLDQNMLAV IDELMQALNFNSEWPQKSSLEEPDFYKTKI KLCILLHAFRIRAVTIDRVMSYL AS*

OT-IL 12-004 CMV MCHQQLVISWFSLVFLASPLVArWELKKDV 730

(p40 signal YWE-LDWYPDAPGEMWLTCDTPEEDGIT

sequence - p40 WTLDQS SEVLG SGKTLTIOVKEFGD AGQYT

- linker CHKGGEVLSHSLLLLHKKEDGIWSTDILKD

((G4S)3) - p35 O EPKNKTTLRCEAK YSGRFTCWWLTTTS

- furin TDLTF S VK S SRG S SDPQG VTC G A ATL S AER

(ARNRQKRS) VRGDNKEYEYSVECQEDSACPAAEESLPIE

- FKBP (E31G, VMVDAVHKLKYENYTSSFFIRDIIKPDPPKN

F36V, R71G, LQLKPLKNSRQVEVSWEYPDTWSTPHSYFS

K105E) -stop) LTFCVQVQGKSKREKKDRVFTDKTSATVIC

RKNASISVRAQDRYYSSSWSEWASVPCSGG

GGSGGG GS GG GG SRNLP V ATPDPGMFPCL

HHSQNLLRAVSNMLQKARQTLEFYPCTSEE

lDHEDrrKDKTST\¾ACLPLELTKNESCLNS

RETSF3TNGSCLASRKTSFMMALCLSSIYED

LKMYQVEFKTMNAKLLMDPKRQIFLDQN LAVTDELMQALNFNSETVPQKSSLEEPDF

YKTKIKLCII i HAFRIRAVTTDRVMSYL AS

ARNRQKR S G VQ \'ΈΤί SP GD GR TFPKR GQTC

WHYTGMLGDGKKVD S SRDRNKPFKFNiL

GKQEVIRGWEEGVAQMSVGQGAKL^SPD

YAYGATGHPGnPPHATLVFDVELLELE*

OT-IL12-005 CMV MCHQQLViSWFSLVFLASPLVAIWELKKDV 731 778 (p40 signal Y V VELDWYPD APGEM V VLTCDTPEEDGIT

sequence- p40 - WTLDQ S SEVLG SGKTLTIQVKEFG D AGQYT

linker- CHKGGEVLSHSLLLLHKKEDGIWSTDILKD

((G4S)3) - p35 QKEPKNKTFLRCEAKNYSGRFTCWWLTnS

- linker TDLTF S VK S SRG S SDPQG VTC G A A TL S AER

(GGSG) - VRGDNKEYEY SVECQEDSACPAAEESLPIE

FKBP (E3 1.G, VMVDAVHKLKYENYTSSFFIRDIIKPDPPKN

F36V, R71G, LQLKPLKNSRQVEVSWEYPDTWSTPHSYFS

K105E) - stop) LTFCVQVQGKSKREKKDRVFTDKTSATVIC

RKNASISVRAQDRYYSSSWSEWASVPCSGG

GGSGGGGSGGGGSRNLPV ATPDPGMFPCL

HHSQNLLRAVSNMLQKARQTLEFYPCTSEE

IDHEDriKDKTSTVEACLPLELTKNESCLNS

RETSFITNGS CL A SRKTSFMMALCLS SIYED

LKMYQVEFKTMNAKLLMDPKRQIFLDQN MLAVrDELMQALNFNSETVPQKSSLEEPDF

YKmiKLClLLHAFRlPvAVTIDRVMSYLNAS

GGSGGVQVETISPG DGRTFP RGQTC VVHY TG MLGDGKKVD S SRDRNKPFKFMLGK QE V

rRG EEGVAQMSVGQGAKLTiSPDYAYGA

TGHPGIIPPHATLVFDVELLELE*

OT-IL 12-006 CMV MCHQOLVISWFSLVFLASPLVAIWELKKDV 732 779 (p40 signal YWELDWYPDAPGEMVVLTCDTPEEDGIT

sequence- p40 - WTLDQ S SE VL G SGKTLTIQ VKEFGD A GQ YT

linker CHKGGEVLSHSLLLLHKKEDGIWSTDILKD

((G4S)3)- p35 QKEPKNKTFLRCEAK YSGRFTCWWLTTIS

- stop) TDLTF SVKSSRGS SDPQGVTCGAATL S AER

VRGDNKEYEYSVECQEDSACPAAEESLP1E

VMVDAVFlKLKYENYTSSFFIRDriKPDPPKN

LQLKPL NSRQVEVS EYPDTWSTPHSYFS

LTFCVQVQGKSKREKKDRVFTDKTSATVTC

RKNASISVRA QDRYY S S SWSEWA SVPCSGG

GGSGGGGSGGGGSRNLPVATPDPGMFPCL

HHSQNLLRAVSNMLQKARQTLEFYPCTSEE

IDHEDITKDKTSTVEA CLPLELTKNES CLNS

RETSFITNGSCLASRKTSFMMALCLSSIYED

LKMYQ FKTMNAKLLMDPKRQIFLDQN

ML A VIDELMQ ALNFN SET VPQK S SLEEPDF

YKTKIKLCTLLHAFRIRAVTIDRVMSYLNAS

*

OT-1L12-007 CMV MCHQQLVTSWFSLVFLASPLVAISLIAALAV 733 780

(p40 signal DYVIGMENAMPWNLPADLAWFKRNTLNK

sequence, PVTMGRHTWESIGRPLPGRKNIILSSQPGTD

ecDHFR DRVTWVKSVDEAIAACGDVPEIMVIGGGR

(Amino acid 2- VEQFLPKAQKLYLTHIDAEVEGDTHFPDYE

159 of WT) PDDWESWSEFHDADAQNSHSYCFEILERR

(R12Y, 1001) - ESRRVRRN KSKIWELKKDVYVVELDWYP

furin site DAPGEMVVLTCDTPEEDGITWTLDQSSEVL

(ESRRVRRNK G S GKTLTIQ VKEFGD AGQYTCHK GGEVL SH

RSK) - p40 - SLLLLHKKEDGIWSTDILKDQKEPKNKTFLR

linker ((G4S)3) CEAKNYSGRFTCWWLTTISTDLTFSVKSSR

~ p35) GSSDPQGVTCGAATLSAERVRGDNKEYEY

SVECQEDSACPAAEESLPIEVMVDAVHKLK

YENYTSSFFIRDIIKPDPPKNLQLKPLKNSRO

^'^TSWEYPD^'TV\'^TSTPHSYFSLTFCVQVQGKS

KREKKDRVFTDKTSATVICRKNASISVRAQ

DRYYSSSWSEWASVPCSGGGGSGGGGSGG

GGSRNLPVATPDPGMFPCLHHSQNLLRAVS

NMLQKARQTLEFYPCTSEEIDHEDITKDKTS

TVE A CLPL ELTKNE SCLN SRETS FIT GS C L

ASRKTSFMMALCLSSIYEDLKMYQVEFKT

M AKLLMDPKRQIFLDQNMLAVIDELMQA

LNFNSETVPQKSSLEEPDFYKTKIKLCILLH

AFRIRAVTIDRVMSYLNAS

OT-IL 12-008 CMV MCHQQLVISWFSLVFLASPLVAVGSLNCIV 734 781 (p40 signal AVSQ MGIGKNGDLPWPPLRNEFRYFFRM

sequence; TTTSSVEGKQNLVIMGKKTWFSIPEKNRPL

hDHFR KGRI LVL SRELKEPPQGAHFL SR SL DD ALK

(Amino acid 2- LTEQPELANKVDMVWIVGGSSVIKEFMNHP

187 ofWT) GHLKLFVTRIMQDFESDTFFPEIDLEKYKLL

(Q36K, Y122I, PEYPG\1. SD VQEEKGIKY KF E VYEK DE SR

A125F) - funn R VRRNKR SKI WELKKD VY V VELD WYPD AP

site GEMVVLTCDTPEEDGITWTLDQSSEVLGSG

(ESRRVRRNK KTL;nQV EFGDAGQYTCHKGGEVLSHSLL

RSK)- p40 - LLHKKEDGIWSTDILKDQKEPKNKTFLRCE Iinkerf(G4S)3) AKNYSGRFTCWWLTnSTDLTFSVKSSRGS

- p35) SDPQGVTCGAATLSAERVRGDN EYEYSV

ECQEDSACPAAEESLPIEVMVDAVHKLKYE

NYTSSFFIRDIIKPDPPKNLQLKPLKNSRQVE

VSWEYPDTWSTPHS YFSLTFC VQVQGKSKR

EKKDRVFTDKTSATVICRK ASISVRAODR

YYSSSWSEWASVPCSGGGGSGGGGSGGGG

SRNLPVATPDPGMFPCLHHSO LLRAVS M

LQKARQTLEFYPCTSEEIDHEDITKDKTSTV

EACLPLELTK ESCLNSRETSFITNGSCLASR

KTSFmiALCLSSIYEDLKMYQVEFKTMNA

KLLMDPKRQIFLDQNMLAVLDELMQALNF

NSET QKSSLEEPDFYKTKLKLCILLHAFRI

R A VTIDR WIS YLNA S

OT-IL12-009 CMV MCHQQLVIS WFSLVFLA SPLVATWELK D V 735 782 (p40 signal YWELDWYPDAPGEMVVLTCDTPEEDGIT

sequence- p40 WTLDQSSEVLGSGKTLTIQVKEFGDAGQYT

- linker CHKGGEVLSHSLLLLHKKEDGIWSTDILKD

((G4S)3) - p35 OKEPK KTFLRCEAK YSGRFTCWWLTnS

- furin TDLTF S VK S SR G S SDPQG VTC G A ATL S AER

(ESRRVRRNK VRGDNKEYEYSVECQEDSACPAAEESLPIE

RSK) - FKBP VIVTVDAVHKLKYENYTSSFFIRDIIKPDPPKN

(E31G, F36V, LQLKPLK SRQ VE- VS WEYPDTWS TPH S YF S

R71G, K 105E)- LTFCVQVQGKSKREKKDRVFTDKTSATVIC

stop) RK ASISVRAQDRYYSSSWSEWASVPCSGG

GGSGGGGS GGGG SRNLP VATPDPGMFPCL

HHSQ LLRAVSNMLQKAROTLEFYPCTSEE

IDHEDITKDKTSTVEACLPLELTK ES CLN S

RETSFITNGS CL ASRKTSFMMALCL S SIYED

LKNWQVEFKTMNAKLLMDPKRQIFLDQN

ML AVIDELMQ ALNFN SET VPQ S SLEEPDF

YKTKI LCILLHAFRIRAVTIDRVMSYLNAS

ESR VRR KR S G VQVET1SPGDGRTFPKR

GQTC HYTGMLGDGKKVDSSRDRNKPFK

FMLGKQEVIRGWEEGVAQMSVGQGAKLTI

SPDYAYGATGHPGnPPHATLVFDVFl LELE

OT-IL12-019 PGK MCHQQLVISWFSLVFLASPLVATWELKKDV 732 779 (p40 signal YWELDWYPDAPGEMVVLTCDTPEEDGIT

sequence- p40~ WTLDQSSEVLGSGKTLTIQVKEFGDAGQYT

linker((G4S)3)- CHKGGEVLSHSLLLLHKKEDGIWSTDILKD

p35-stop) QKEPKNKTFLRCEAKNYSGRFTCWWLTTIS

TDLTF SVKSSRGS SDPQGVTCGAATL S AER

VRGDNKEYEYSVECQEDSACPAAEESLPIE

VMVDAVHKLKYENYTSSFFIRDIIKPDPPKN

LQLK LKN SRQ VE V SWEYPDTW S TPH S YF S

LTFCVQVQGKSKREKKDRVFTDKTSATVIC

RKNASISVRA QDRYY S S S WSEWA SVPCSGG

GGSGGGGSGGGGSRNLPVATPDPGMFPCL

HHSQNLLRAVSNMLQKARQTLEFYPCTSEE

IDHEDITKDKTSTVEACLPLFI TKNESCLNS

RETSFITNGSCLASRKTSFMMALCLSSIYED

LKMYQVEFKTMNAKLLMDPKRQIFLDQN

ML AVIDELMQ ALNFN SETVPQK S SLEEPDF

YKTKIKLCILLHAFRIRA VTIDR VMSYLNAS

OT-IL12-020 EFla MCHQQLVIS WFSLVFLASPLVATWELKKDV 732 779 (p4() signal YWELDWYPDAPGEMVVLTCDTPEEDGIT

sequence- p40- WTLDQSSEVLGSGKTLTIQVKEFGDAGQYT

CHKGGEVLSHSLLLLHKKEDGIWSTDILKD Iinker((G4S)3)- QKEPKNKTFLRCEAK YSGRFTCWWLTTIS

p35-stop) TDLTF SVKSSRGS SDPQGVTCGAATL SAER

VRGDNKEYEYSVECQEDSACPAAEESLP1E

VMVDAVHKLKYENYTSSFFIRDllKPDPPKN

LQLKPL NSRQVEVS EYPDTWSTPHSYFS

LTFCVQVQGKSKREKKDRVFTDKTSATVIC

RKN ASIS VR A QDRYY S S SWSEWA SVPCSGG

GGSGGGGSGGGGSRNLPVATPDPGMFPCL

HHSQNLLRAVSNMLQKARQTLEFYPCTSEE

IDHEDITKDKTSTVEA CLPLELTKNES CLNS

RETSFITNGSCLASRKTSFMMALCLSSIYED

LKMYQ FKTMNAKLLMDPKRQIFLDQN

ML A VIDELMQ ALNFN SETVPQK S SLEEPDF

YKTKIKLCTLLHAFRIRAVTIDRVlVlSYLNAS

*

OT-IL1.2-021 No promoter MCHQQLVISWFSLVFLASPLVA WELKKDV 732 779

(p40 signal YWELDWYPDAPGEMVVLTCDTPEEDGIT

sequence- p40- WTLDQ S SEVLG SGKTLTIQ VKEFG DAGQYT

linker((G4S)3)- CHKGGEVLSHSLLLLHKKEDGIWSTDILKD

p 35 -stop) QKEPKNKTFLRCEAKNYSGRFTCWWLTTIS

TDLTF S VK S SRG S SDPQG VTC G A A TL S AER

VRGDNKEYEYSVECQEDSACPAAEESLPIE

VMVD A VHKLKYE YTS S.F F1RDIIKPDPPKN

LQLKPLKN SRQVE VS WEYPDTWS TPH S YF S

LTFCVQVOGKSKREKKDRVFTDKTSATVIC

RKNASISVRAQDRWSSSWSEWASWCSGG

GGSGGGGSGGGGSRNLPVATPDPGMFPCL

HHSONLLRAVSNMLOKARQTLEFYPCTSEE

IDHEDITKDKTSTVEACLPLELTKNESCLNS

RETSFITNGSCLASRKTSFMM4LCLSSIYED

LKMYQVEFKTM AKLL 1DPKRQIFLDQN

MLA VIDELMQ ALNFN SETVPQKSSLEEPDF

YKmiKLClLLHAFRlRAVTIDRVMSYLNAS

*

OT-IL 12-022 PGK MCHQQLVISWFSLVFLASPLVArWELKKDV 731 778 (p40 signal YWELDWYPDAPGEMVVLTCDTPEEDGIT

sequence- p40 - WTLDQS SEVLG SGKTLTIQVKEFGD AGQYT

linker- CHKGGEVLSHSLLLLHKKEDGIWSTDILKD

((G4S)3) - p35 QKEPKNKTFLRCEAKNYSGRFTCWWLTnS

- linker TDLTF S VK S SRG S SDPQG VTC G A ATL SAER

(GGSG) - VRGDNKEYEYSVECQEDSACPAAEESLPIE

FKBP (E31G, VMVDAVHKLKYENYTSSFFIRDIIKPDPPKN

F36 R71G, LQLKPLKNSRQVEVSWEYPDTWSTPHSYFS

K105E) - stop) LTFCVQVQGKSKREKKDRVFTDKTSATVIC

RKNASISVRAQDRYYSSSWSEWASVPCSGG

GGSGGG GS GG GG SRNLP V ATPDPGMFPCL

HHSQNLLRAVSNMLQKARQTLEFYPCTSEE

roHED3TKDKTSTVEACLPLELTKNESCLNS

RETSF3TNGSCLASR 1 t- MMALCLSSIYED

LKMYQ VE FKTMNAKLLMDPKRQIFL DQN

MLAVTDELMQALNFNSETVPQKSSLEEPDF

YKTKIKLCILLHAFRIRAVTTDRVMSYL AS

GGSGGVQVETISPGDGRTFPKRGQTCVVHY

TGMLGDGKK VD S SRDRNKPFKFMLGKQEV

IRGWEEGVAQMSVGQGAKLTISPDYAYGA

TGHPGIIPPHATLVFDVELLELE*

OT-IL12-023 EFla MCHQQLVISWFSLVFLASPLVArWELKKDV 731 778 (p4() signal YWELDWYPDAPGEMVVLTCDTPEEDGIT

sequence- p40 - WTLDQS SEVLG SGKTLTIQ VKEFG DAGQYT

linker- CHKGGEVLSHSLLLLHKKEDGIWSTDILKD ((G4S)3) - p35 QKEPKNKTFLRCEAKNYSGRFTCWWLTTIS

- linker TDLTF SVKSSRGS SDPQGVTCGAATL SAER

(GGSG) - VRGDNKEYEYSVECQEDSACPAAEESLP1E

FKBP (E31G, VMVDAVHKLKYENYTSSFFIRDllKHUPPKN

F 6V, R73 G, LQLKPL NSRQVEVS EYPDTWSTPHSYFS

K 105E) - stop) LTFCVQVQGKSKREKKDRVFTDKTSATVIC

RKNASISVRA QDRYY S S SWSEWA SVPCSGG

GGSGGGGSGGGGSRNLPVATPDPGMFPCL

HHSQNLLRAVSNMLQKARQTLEFYPCTSEE

IDHEDITKDKTSTVEA CLPLELTKNES CLNS

RETSFITNGSCLASRKTSFMMALCLSSFYED

LKMYTJVEFKTMNAKLLMDPKRQIFLDQN

ML A VIDELMQ ALNFN SETVPQK S SLEEPDF

YKTKIKLCTLLHAFRIRAVTIDRVlVlSY'LNAS

GGSGGVQVTiTISPGDGRTFPKRGQTC HY

TGMLGDGKKVD S SRDRNKPFKFMLGKQEV

IRGWEEGVAQMSVGQGAKLTISPDYAYGA

TGHPGIIPPHATLVFDVELLELE*

OT-IL 12-024 No promoter MCHQQLVISWFSLVFLASPLVAIWELKKDV 731 778 (p40 signal YWELDWYPDAPGEMWLTCDTPEEDGIT

sequence- p40 - WTLDQSSEVLGSGKTLTIQVKEFGDAGQY

linker- CHKGGEVTSHSLLLLHKKEDGIWSTDILKD

((G4S)3) - p35 OKEPK KTFLRCEAKNYSGRFTCWWLTnS

- linker TDLTF S VK S SR G S SDPQG VTC G AATL SAER

(GGSG) - VRGDNKEYEYSVECQEDSACPAAEESLPIE

FKBP (E31G, VMVOAVHKLKYENYTSSF IRDIIKPDPPKN

F36V, R71G, LQLKPLKN SRQVE VS WEYPDTWS TPH S YF S

K105E) - stop) LTFCVQVQGKSKREKKDRVFTDKTSATVIC

RKNASISVRAQDRYYSSSWSEWASVPCSGG

GGSGGGGS GGGG SRNLP VATPDPGMFPCL

HHSQNLLRAVSNMLQKARQTLEFYPCTSEE

roHEDlTKDKTSTVEACLPLELTKNESCLNS

RETSF1TNGSCLASRKTSFMMALCLSSIYED

LKMY'QVEFKTMNAKLLMDPKRQIFLDQN

ML A VIDELMQ ALNFN SETVPQK S SLEEPDF

YKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

GGSGGVQVETISPGDGRTFPKRGQTCVVHY

TGMLGDGKKVDSSRDRNKPFKFMLGKQEV

IRGWEEGVAQMSVGQGAKLTISPDYAYGA

TGHPGIIPPHATLVFDVELLELE*

OT-IL 12-025 PGK MCHQQLVISWFSLVFLASPLVAIWELKKDV 731 778 (p40 signal YWELDWYPDAPGEMWLTCDTPEEDGIT

sequence- p40^■-- WTLDQ S SEVLG SGKTLTIQVKEFG DAGQYT

linker- CHKGGEVLSHSLLLLHKKEDGIWSTDILKD

((G4S)3) - p35 QKEPKNKTFLRCEAKNYSGRFTCWWLTnS

- linker TDLTF SV SSRGS SDPQG VTC G A A ^'TL S AER

(GGSG) - VRGDNKEYEYSVECQEDSACPAAEESLPIE

FKBP (E31.G, VMVDAVHKLKYENYTSSF FIRDIIKPDPPKN

F36V, R71G, LQLKPLKNSRQVEVSWEYPDTWSTPHSYFS

K105E) - stop) LTFCVQVQG SKREKKDRVFTDKTSATVIC

RKNASISVRAQDRYYSSSWSEWASVPCSGG

GGSGGG GS GG GG SRNLP VATPDPGMFPCL

HHSQNLLRAVSNMLQKARQTLEFYPCTSEE

IDHEDITKDKTSTVEACLPLELTKNESCLNS

RETSFITNGS CL A SRKTSFMMALCLS SIYED

LKMYQVE-FKTMNAKLLMDPKRQIFLDQN

ML A VIDELMQ ALNFN SETWQK S SLEEPDF

Y'KTKiKLCILLHAFRIRAVTIDRVMSYLNAS

GGSGGVQWTISPGDGRTFPKRGQTCWHY

TGMLGDGKKVD S SRDRNKPFKFMLGKQEV IRGWEEGVAQMSVGQGAKLTISPDYAYGA

TGHPGITPPHATLVFDVELLELE*

OT-IL 12-026 EFla MCHQQLVTSWFSLWLASPLVAIWELKKDV 731 778 (p40 signal YWELDWYPDAPGE WLTCDTPEEDGIT

sequence- p40 - WTLDQSSEVLGSGKTLTIQVKEFGDAGQYT

linker- CHKGGEVLSHSLLLLHKKEDGIWSTDILKD

((G4S)3) - p35 QKEPK KTFLRCEAKNYSGRFTCWWLTTIS

- linker TDLTF S VKS SRG S SDPQGVTCGAATL SAER

(GGSG) - VRGDNKEYEY S VECQED S ACP AAEESLP1E

FKBP (E31G, VMVDAVHKLKYENYTSSFF1RDIIKPDPPKN

F36V, R 1G, LQLKPLKNSRQVEVSWEYPDTWSTPHSYFS

K105E) - stop) LTFCVQVQGKSKREKKDRVFTDKTSATVIC

RKNASISVRAQDRYYSSSWSEWASVPCSGG

GGSGGGGSGGGGSRNLPVATPDPGMFPCL

HHSQNLLRAVSNMLQKARQTLEFYPCTSEE

IDHEDITKDKTSTVEACLPLELTKNESCLNS

RETSFITNGSCLASRKTSFMMALCLSSIYED

LKMYQVEFKTMNAKLLMDPKRQIFLDQN

ML A VIDELMQ ALNFN SETVPQK S SLEEPDF

YKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

GGSGGVQVETISPGDGRTFPKRGQTCW¾Y

TGMLGDGKK VD S SRDRNKPFKFMLGKQEV

IRGWEEGVAQMSVGQGAKLTISPDYAYGA

TGHPUllPPHATLVFDVELLELE*

OT-IL12-027 No promoter MCHQQLVISWFSLVFLASPLVATWELKKDV 731 778 (p40 signal YWELDWYPDAPGEMWLTCDTPEEDGIT

sequence- p40 -- WTLDQSSEVLGSGKTLTIQVKEFGDAGQYT

linker-

(CG4S)3) - p35 QKE PKNKTFLR CEAKNY S GRFTCWVVLTTIS

- linker TDLTF S VK S SR G S SDPQG VTC G A ATL S AER

(GGSG) - VRGDNKEYEYSVECQEDSACPAAEESLPIE

FKBP (E31G, VMVDAVUKLKYENYTSSFFIRDilKPDPPKN

F36V, R71G, LQLKPLKN SRQ VE- VS WEYPDTWS TPH S YF S

K105E) - stop) LTFCVQVQGKSKREKKDRVFTDKTSATVIC

RKNASISVRAQDRYYSSSWSEWASVPCSGG

GGSGGGGS GGGG SRNLP VATPDPGMFPCL

HHSQNLLRAVSNMLQKARQTLEFYPCTSEE

IDHEDITKDKTSTVEACLPLELTKNES CLNS

RETSFITNGS CL ASRKTSFMMALCL S SIYED

LKMYQVEFKTMNAKLLMDPKRQIFLDQN

ML A VIDELMQ ALNFN SETVPQK S SLEEPDF

YKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

GGSGGVQVETISPGDGRTFPKRGQTCWHY

TGMLGDG KKVD S SRDRNKPFKFMLGKQEV

IRGWEEGVAQMSVGQGAKLTISPDYAYGA

TGHPGIIPPHATLVFDVELLELE*

OT-IL 12-028 PGK MCHQQLVISWFSLVFLASPLVATWELKKDV 730 _*7T (p40 signal YWELDWYPDAPGEMWLTCDTPEEDGIT

sequence - p40 WTLDQS SEVLG SGKTLTIQVKEFGD AGQYT

- linker CHKGGEVLSHSLLLLHKKEDGIWSTDTLKD

((G4S)3) - p35 QKEPKNKTFLRCEAKNYSGRFTCWWLTTIS

- furin TDLTF S VK S SRG S SDPQG VTC G A A TL S AER

(ARNRQKRS) VRGDNKEYEYSVECQEDSACPAAEESLPIE

- FKBP (E3 ί G, VMVDAVHKLKYENYTSSFFIRDIIKPDPPKN

F36 R71G, LQLKPLKNSRQVEVSWEYPDTWSTPHSYFS

K105E) -stop) LTFCVQVQGKSKREKKDRVFTDKTSATVIC

RKNASISVRAQDRYYSSSWSEWASVPCSGG

GGSGGG GS GG GG SRNLP VATPDPGMFPCL

HHSQNLLRAVSNMLQKARQTLEFYPCTSEE

IDHEDTTKDKTSTVEACLPLELTKNESCLNS RETSFITNGSCLASRKTSFMMALCLSSIYED

LKMYQVEFKT NAKLLMDPKRQIFLDQN

MLAVIDELMQALNFNSETVPQKSSLEEPDF

YKTKIKLCILLHAFRIRAVTTDRVMSYLNAS

ARNRQKRSGVQVETISPGDGRTFPKRGQTC

WHYTGMLGDGKKVD S SRDRNKPFKFML

GKQEVIRGWEEGVAQMSVGQGAKLTISPD

YAYGATGHPGIIPPHATLVFDVELLELE*

OT-IL 12-029 EFla MCHQQLVISWFSLVFLASPLVAIWELKKDV 735 782

(p40 signal YWELDWYPDAPGEMVVLTCDTPEEDGIT

sequence- p40 WTLDQ S SE VL G SGKTLTIQ V EFGD A GQ YT

- linker CHKGGEVLSHSLLLLHKKEDGIWSTDILKD

((G4S)3) - p35 QKEPK KTFLR CEAKNY S GRFTCWWLTTl S

- furin ^'ID LTF S VK S SR G S SDPQG VTC G A ATL S AER

(ESRRVRRNK VRGDNKEYE Y S VECQED S A CP A AEE SLP1E

RS ) - FKBP VMVDAVHKLKYENTYTSSFFIRDIIKPDPPKN

(E31G, F36V, LQLKPLKNSRQVEVS EYPDTWSTPHSYFS

R71G, K 105E)- LTFCVQVQGKSKREKKDRVFTDKTSATVTC

stop) RKNASISVRAQDRYYSSSWSEWASVPCSGG

GGSGGGGSGGGGSRNLPVATPDPGMFPCL

HHSQNLLRAVSNMLQKARQTLEFYPCTSEE

IDHEDITKDKTSTVEA CLPLELTKNES CLNS

RETSFITNGSCLASRKTSFMMALCLSSIYED

LKMYQVEFKTMNAKLLMDPKRQIFLDQN

ML A VIDELMQ ALNFN SET VPQK S SLEEPDF

YKTKiKLCILLHAFRIRAVTIDRVMSYLNAS

ESRRVRRNKRSKGVQVETISPGDGRTFPKR

GQTCWHYTGMLGDGKKVDSSRDRNKPFK

FMLGKQEVIRGWEEGVAQMSVGQGAKLTI

SPD YAYGATGHPGIIPPHATLVFDVELLELE

*

OT-IL12-030 No promoter MCHQQLVISWFSLVFLASPLVAIWELKKDV 735 782 (p40 signal YWELDWYPDAPGEMVVLTCDTPEEDGIT

sequence- p40 WTLDQSSE 'LGSGKTLTIQ 'KEFGDAGQYT

- linker CHKGGEVLSHSLLLLHKKEDGIWSTDILKD

((G4S)3) - p35 QKEPKNKTFLRCEAKNYSGRFTCWWLTnS

- furin TDLTF S VKS SRG S SDPQGVTCGAATL S AER

(ESRRVRRNK VRGDNKEYEYSVECQEDSACPAAEESLPIE

RSK) - FKBP VMVDAVHKLKYENYTSSFFIRDIIKPDPPKN

(E31G, F36V, LQLKPLKN SRQ VE VS WEYPDTW S TPH S YF S

R71G, K105E)- LTFCVQVQGKSKREKKDRVFTDKTSATVTC

stop) RKNASISVRAQDRYYSSSWSEWASVPCSGG

GGSGGGGSGGGGSRNLPVATPDPGMFPCL

HHSQNLLRAVSNMLQKARQTLEFYPCTSEE

IDHEDITKDKTSTVEA CLPLELTKNESCLNS

RETSFIT GSCLASRKTSFMMALCLSSIYED

LKMYQVEFKTMNAKLL 1DPKRQIFLDQN

MLAVIDELMQALNFNSETVPQKSSLEbPDF

YKTKIKLCILLHAFRIRAVTIDRVMSYLNAS

ESRR\¾RNKRSKGVQVETISPGDGRTFPKR

GQTCWHYTGMLGDGKKVDSSRDRNKPFK

FMLGKQEVIRGWEEGVAQMSVGQGAKLTI

SPDYAYGATGHPGIIPPHATLVFDVELLELE

*

OT-IL 12-046 EFla MCHQQLATSWFSLVFLASPLVAGVQW/nSP 727 774 (p40 signal GDGRTFPKRGQTCWHYTGMLEDGKKVDS

sequence^■-- SRDRNKPFKFML GKQEV1RGWEEGVAQMS

FKBP (F36V, VGQRAKLTTSPDYAYGATGHPGIIPPHATLV

L106P) - FDVELLKPEGGSGGIWELKKDVYWELDW

Linker YPDAPG EMWLTCDTPE EDGITWTLDQS SE (GGSGG) - VI. G S GKTLT1Q VKEF GD AGQYTCH KGG E VL

p40 - Linker SHSLLLLH EDGIWSTDILKDQKEPKNKTF

((G4S)3) - p35 L RCEAKN YS GRFTC W WLT ISTDLTF SVKS S

- stop) RGSSDPQGVTCGAATLSAERVRGDNKEYE

Y S VECQE D S A CP A AEE SLP1EVM VD AVHKL

KYENYT S SFFIRD I I PDPPKNLQL KPL KN SR QVEVSWEYPDTWSTPHSYFSLTFCVQVQGK SKREK DRVFTDKTS AT VICRKNA SIS VRAO DRYYSSSWSEWASVPCSGGGGSGGGGSGG GGSRNLPVATPDPGMFPCLHHSQNLLRAVS NMLQKARQTLEFYPCTSEEIDHEDITKDKTS TVE ACLPLELTKNES CLN SRETSFITNG S CL A SRKTSF^IMALCLSSIYEDLKMYQ^FKTMN

AKLLMDPKRQIFLDQ MLAVIDELMQALNF NSETVPQK S SLEEPDF YKTKIKL CILLH AFRI RA VTIDRVMS YLN AS *

[00250] In one embodiment, the payload of the invention may comprise 1LI5. Interieukin 15 is a potent immune stimulator}⁷ cytokine and an essential survival factor for T cells, and Natural Killer cells. Preclinical studies comparing IL2 and 11,15, have shown than IL15 is associated with less toxicity than IL2. In some embodiments, the effector module of the invention may be a DD-IL15 fusion polypeptide. IL15 polypeptide may also be modified to increase its binding affinity for the IL15 receptor. For example, the asparagine may be replaced by aspartic acid at position 72 of IL15 (SEQ ID NO. 2 of US patent publication US20140134128A1; the contents of which are incorporated by reference in their entirety). In some embodiments, the IL15 constracts of the invention may be placed under the transcriptional control of the CMV promoter (SEQ ID ). 716), an EF 1 a promoter (SEQ ID NO. 717, SEQ ID NO. 908) or a PGK promoter (SEQ ID 718). In some aspects, the DD-IL15 comprises the amino acid sequences listed in Table 12. The ammo acid sequences in Table 12 may comprise a stop codon which is denoted in the table with a "*" at the end of the amino acid sequence.

Table 12: DD IL15 constructs

TMGWEASSERMYPEDGALKGEIKQRLKL

KDGGHYDAEVKTTYKAKKPVQLPGAYN YNIKLD1TSHNEDYT1VEQYERAEGRHSTG

G DELYK

IL15 NWVNVISDLKKiEDLIQSMHIDATLYTESD 785 794-797,

VHP S CK VT AMKCFLLELQ VI SLES GD A SIH 1001 DTVENLIILANNSLSSNGNVTESGCKECEE LEE NIKEFLQSFVHIVQMFINTS*

ecDHFR (Amino acid ISLIAALAVDYVIGMENAMPWNLPADLA 9 692, 772, 2-159 of WT) (R I2Y, WTKRNTLNKPVIMGRHTWESIGRPLPGRK 814, 687, Y100I) NHL S SQPGTDDR VTWVKS VDE AI AACGD 988, 991

VPEIMVIGGGR\TEOFLPKAQKLYLTHIDA

EVEGDTHFPDYEPDDWESVFSEFHDADA QN SH S Y CFEILERR*

hDHFR (Amino acid VGSLNCIVAVSQNMGIGKNGDLPWPPLR 895 694, 995 2-187 of WT) NEFRYFQRMTTTSSVEGKQNLVI GKKT

(Y 1221) WF S IPEK NRPLK GR INL VL SRELKEPPQ G A

HFLSRSLDDALKLTEQPELANKVDMVWI

VGGSSVr EAMNHPGHLKLFVTRIMQDFE-

SDTFFPEIDLEKYKLLPEYPG VL SD VQEEK

GIKYKFEVYE-KND

OT-IL15-001 (IL2 CMV M YRMQLL S C I AL SL AL VTN SN W V VI SDL 786 799 signal sequence- KKIEDLlOSMHiDATLYTESDVI-IPSCKVTA

1L 15 -stop) MKCFLLELQVTSLESGDASIHDTVENLIILA

NNSLSSNGNVTESGCKECEELEEKMKEFL QSFVHIVQMFINTS*

OT-iL 15-002 (IL2 CMV MYRMQLLSCIALSLALVTNSEFSTEFISLIA 787 800 signal sequence- ALAVDYVIGMENAMPWNLPADLAWFKR

iinker[EFSTEF]- NTLNKPVIMGRHTWESIGRPLPGRKNIILS

ecDHFR (amino acid SQPGTDDR VTWVKSVDEAIAACGDVPE1

2-159 of WT. R12Y, MVIGGGRVIEQFLPKAQKLYLTHIDAEVE

1001)- linker GDTHFPDYEPDDWESVFSEFHDADAQNS

[GGSGG]- IL15- HS YCFEILERRGGSGGNWVN V! SDLKKIE

stop) DL IQ SMH ID ATL YTE SD VHP S CK VT AMK C

FLLELQVISLESGDASIHDTVENLIILANNS LS S NGN VTESGCKECEELE EKN IKEFLQSF VHI VQMFINTS *

OT-IL 1 -062 (IgE EFla MDWTWILFLVAAATRVHSYPYDVPDYA 631 749 leader - HA Tag - NWVNVISDLKKIEDL!QSMHIDATLYTESD

JL15 - BamHI (GS) - VHPSCKVT AMKCFLLELQ VISLESGDAS1H

stop) DTVENLIILANNSLSSNGNVTESGCKECEE

LEEKNiKEFLQSFVHIVQMFINTSGS*

OT-IL15-132 (IgE EFla NfDWTWILFLVAAATRVHSNWV VISDLK 725 1055 leader -IL 15 - KIEDLIQ SMHID ATL YTE SD VHP S CK VT A

BamHI (GS) - P2A MKCFLLELQVISLESGDASIHDTVENLIILA

cleavabie peptide - NN SL S SNGNVTES GCKECEELEEKNIKEFL

mCheny (MIL) - QSFVHIVQMFINTS G S G ATNF SLLKQ AGD

stop) VEENPGPL SKGEEDNMAILKEFMRFKVmi

EGSVNGHEFEIEGEGEGRPYEGTQTAKLK VTKGGPLPFAWDILSPQFMYGSKAYVKH PADIPDYLKLSFPEGF WERVMNFEDGGV VT VTQD S SLQD GEFI YK VKLRGTN FP SD G P VMQ KTMGWE A S SERMYPED G ALKGE I KQRLKL DGGHYDAEVKTTYKAKKPVQ LPGAYNVNIKLDITSHNIiDYTIVT-iQYERAE

GRHSTGG DELYK*

OT-IL15-134 (IgE EFla MDWTWILFLVAAATRVHSNWVNVISDLK 726 1056 leader -IL 15 - Linker KIEDLIQ SMHID ATL- YTE SD VHP S CK VT A

(GS) - hDHFR (WT MKCFLLELQVISLESGDASIHDTVENLIILA

2-187, Y122I) - NN SL S SNGN VTES GCKECEELEEKNIKEFL BamHI (GS) - P2A QSFVHIVQMFINTSGSVGSLNCIVAVSQN

cleavable peptide -- MG I G KNGDLPWPPL RNEFR YFQ MTTTS S

mChen (MIL) - VEGKQNLVrMGKKTWFSlPEKNRPLKGRI

stop) NLVLSRELKEPPQGAHFLSRSLDDALKLT

EQPELANKVDMVWrVGGSSVIKEAMNHP

GHLKLFVTRIMQDFESDTFFPEIDLEKYKL

LPEYPGV SDVQPPKGTKY FEVYEKNDG

SGATNFSLLKQAGDVEENPGPLSKGEEDN

MAIIKEFMRFKVHMEGSVNGHE-FEIE-GEG

EGRPYEGTQTAKLKVTKGGPLPFAWDILS

PQFMYGSKAYVKHPADIPDYLKLSFPEGF

KWER VMNFED GG WTVTQD S SLQD GEFI

YK LRGTNFPSDGPV QKKTMGWEAS

SERMYPEDGALKGEIKORLKLKDGGHYD

AE.VKTTYKAKKPVQLPGAYNVNIKLDITS

HNEDYTrVEQYERAEGRHSTGGMDELYK

*

Description/ Promoter Amino Acid Sequence Amino Nucleic

Construct ID Acid SEQ Acid SEQ

ID NO ID NO

IL2 signal, sequence - MYRMQLL SCI ALSLAL VTN S 783 788-791

IgE Leader - MDWTWILFLVAAATRVHS 801 810, 930,

931

Linker - EFSTEF 784 792-793

Linker - GGSGG 629 676-680

HA Tag - YPYDVPDYA 1024 1025-1027

BamHI - GS - GGATCC

P2A Cleavable - GATNFSLLKQAGDVEENPGP 925 926 Peptide

rriCherry (MIL) L SKGEED M AUKEFMRFK VHMEG S VN G 1029 1030

HEFEIEGEGEGRPYEGTQTAKLKVTKGGP

LPFAWDILSPQFMYGSKAYVKHPADIPDY

LKLSFPEGFKWERVMNFEDGGVVTVTQD

S S LQD GER YK VKLR GTNFP S DGP VMQK

TM G WE AS S ERMYPED G A LKGEIK QRLKL

KDGGHYDAEVKTTYKAKKPVQLPGAYN

VNKLDITSHNEDYTIVEQYERAEGRHSTG

GMDELYK

IL15 NWVNVISDL KIEDL1QSMHIDATLYTESD 785 794-797,

VHPSCKVTAMKCFLLELQVISLESGDAS1H 1001 DTVENLI!LANNSLS SNGN VTESGCKECEE

LEEKNIKEFLQSFVHIVQMFTNTS*

ecDHFR (Amino acid ISLIAALAVDYVIGMENAMPWNLPADLA 9 692, 772, 2-159 of WT) (R12Y, WFKRNTLNKPVIMGRHTWESIGRPLPGRK 814, 687, Y100I) Nim S SQPGTDDR VTWVKS VDE AI AACGD 988, 991

VPEIMVIGGGRVIEQFLPKAQKLYLTHIDA

EVEGDTHFPDYEPDDWESVFSEFHDADA

QNSHSYCFEILERR*

hDHFR (Amino acid VGSLNCIVAVSQN GIGKNGDLPWPPLR 895 694, 995 2-187 of WT) NEFR YFQRMTTTS S VEGKQNL V MGKKT

(Y122I) WFSIPEKNRPLKGRINLVLSRELKEPPQGA

HFL SR SLDD ALKLTEQPEL ANK VDMVWI

VGGSSVIKEAM HPGHLKLFVTRIMODFE SDTFFPEIDLEKYKLLPEYPG VL SD VQEEK GIKY FEWEKND

OT-IL15-001 (1L2 CMV MYRMQLLSCIALSLALVTNSNWV VISDL 786 799 signal sequence- KKIEDLIQSMHIDATLYTESDVHPSCKVTA

IL15-stop) MKCFLLELOVISLESGDASIHDTVENLIILA

NNSLS SNGNVTE SGCKECEELEEKNIKEFL OSFWIVQMFINTS* OT-IL 15-002 (IL2 CMV MYRMQLL SCI ALSLALVTN SEFSTEFi SL1A 787 800 signal sequence- ALAVDYVIGMENAMPWNLPADLAWFKR

iinkerjEFSTEFj- NTL NKP V1MGRHTWE SI GRPLPGRKN1IL S

ecDHFR (amino acid SQPGTDDRVTWVKSVDEAIAACGDVPEI

2-159 of WT, R 12Y, MVIGGGRVIEQFLP AQKLYLTHIDAEVE

1001)- linker GDTHFPDYEPDDWESVFSEFHDADAQNS

[GGSGG]- IL15- HSYCFE1LERRGGSGGNWVNVISDLKKIE

stop) DLIQSMHIDATLYTESDVHPSCKVTAMKC

FLLELQ TSLESGDASIHDTVENLIILANNS

LSSNGNVTESGCKECEELEEKNIKEFLQSF

VHI VQMFINTS *

OT-IL 15-062 (IgE EFla MDWTWILFLVAAATRVHSYPYDVPDYA 631 749 leader - HA Tag -- NWVWISDLKKIEDLIQSMHTOATLYTESD

IL15 - BamHI (GS) - VHPSCKVTAMKCFT T FT QVISLESGDASIH

stop) DTVENLIILANNSLSSNGNVTESGCKECEE

LEE KN IKEFLQSF VHI VQMFINT S G S *

OT-IL15-132 (IgE EFla MDWTWILFLVAAATRVHSNWVNVISDLK 725 1055 leader -1L 15 - KIEDLIQSMH1DATLYTESDVHPSCKVTA

BamHI (GS) - P2A MKCFT 1 Fi .QVISLESGDASIHDTVENLIILA

cleavable peptide - NN SL S S N GN VTES GCKECEEL EEKNIKEFL

mCherry (MIL) - QSFVHIVQMFINTSGSGATNFSLLKQAGD

stop) VEENPG PL SKGEE DNMA IKEFMRFK VHM

EGSVNGHEFEIEGEGEGRPYEGTQTAKLK

V KGGPLPFAWDILSPQFMYGSKAYVKH

PADIPDYLKLSFPEGFKWERVMNFEDGGV

VT VTQD S SLQD GEF I YK VKLR GT FP S D G

PVMQKKTMGWEASSERMYPEDGALKGEI

KQRLKLKDGGHYDAEVKTTYKAKKPVQ

LPGAY V IKLDITSHNEDYTIVEQYERAE

GRHSTGGMDELYK*

OT-IL15-134 (IgE EFla MDWTWILFLVAAATRVHSNWVNVISDLK 726 1056 leader -IL 15 - Linker KIEDLIQSMHIDATLYTESDVHPSCKVTA

(GS) - hDHFR (WT MKCFLLELQVISLESGDASIHDTVENLIILA

2-187, Y122I) - NN SL S S N GN VTES GCKECEEL EEKNIKEFL

BamHI (GS) - P2A QSFVHIVQMFINTSGSVGSLNCIVAVSQN

cleavable peptide - S

mCherry (MIL) -

stop) NLVLSRELKEPPQGAHFLSRSLDDALKLT

EQPELANKVDMVWrVGGSSVIKEAMNHP

GHLKLFVTRIMODFESDTFFPEIDLEKYKL

LPEYPGVLSDVQEEKGIKYKFEVYE-KNDG

SGATNFSLLKQAGDVEENPGPLSKGEEDN

MAIIKEFMRFKVHTVIEGSVNGHE-FEIE-GEG

EGRPYEGTQTAKLKVTKGGPLPFAWDILS

PQFMYGSKAYVKHPADIPDYLKLSFPEGF

KWER VMNFE-D GG V VT VTQD S SLQD GEFI

YKVKLRGTNFPSDGPVMQKKTMGWEAS

SERM ^rPEDGALKGEIKQRLKLKDGGHYD

AE.VKTTYKAKKPVQLPGAYNVNIKLDITS

HNEDYTIVEQYERAEGRHSTGGMDELYK

*

[00251] A unique feature of 1L15 mediated activation is the mechanism of trans-presentation in which IL15 is presented as a complex with the alpha subunit of TL15 receptor (ILlSRa) that binds to and activates membrane bound 11. 15 beta/gamma receptor, either on the same cell or a different ceil. The IL15/IL15Ra complex is more effective in activating 1L15 signaling, than 1L15 by itself. Thus, in some embodiments, the effector module of the invention may include a DD-IL15/IL15Ra fusion polypeptide. In one embodiment, the payload may be IL15/IL15Ra fusion polypeptide described in US Patent Publication NO.: US20160158285AI (the contents of which are incorporated herein by reference in their entirety). The IL15 receptor alpha comprises an extracellular domain called the sushi domain which contains most of the structural elements necessary for binding to IL15. Thus, in some embodiments, payload may be the IL15/'IL15Ra sushi domain fusion polypeptide described in US Patent Publication NO.: US20090238791A1 (the contents of which are incorporated herein by reference in their entirety).

[00252] Regulated IL15/IL15Ra may be used to promote expansion, survival and potency of CD8TEM cell populations without impacting regulatory T cells, NK cells and TIL cells. In one embodiment, DD-IL15 IL15Ra may be utilized to enhance CD 19 directed T cell therapies in B cell leukemia and lymphomas. In one aspect, IL15/IL15Ra may be used as payload of the invention to reduce the need for pre-conditioning regimens in current CAR-T treatment paradigms.

[00253] The effector modules containing DD-IL15, DD-IL15/IL15Ra and/or DD-IL15/lLI5Ra sushi domain may be designed to be secreted (using e.g. IL2 signal sequence) or membrane bound (using e.g. IgE or CD8a signal sequence).

[00254] In some aspects, the DD-IL115/TLl 5Ra comprises the amino acid sequences provided in Table 13a, 13b, and 13c. The amino acid sequences in Tables 13a, 13b and 13c may comprise a stop codon which is denoted in the table with a "*" at the end of the amino acid sequence.

^'able 13a: DD-IL15/IL15Ra construct sequences

IL15 NWVNVISDL K IKDLIQSMHIDATLYTESDVHPSC VT 785 794-797, AMKCFLLELQ VI SLE S GD A S1HDT VENL1IL ANN SL S S N 1001 GNVTESGCKECEELEEKNIKEFLQSFVHIVOMFINTS*

iL15Ra rrCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAG 803 812-813,

TSSLTECVLNKATWAHWTTPSLKCIRDPALVHQRPA 1003

PPSTVTTAGVTPQPESLSPSGK AASSPSSNNTAATT

AAIVPGSOLMPSKSPSTGTTEISSHESSHGTPSOTTAK

WELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLS

AVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSS

RDEDLENC SHHL *

IL ISRa (31-205 of rrCPPPMSVEHADrWVKSYSLYSRERYICNSGFKRKAG 1.057 1058 Uniprot ID: TSSLTCCVLNKATNVAHWTTPSLKCIRDPALVHQRPA

Q13261.1) PPSTVTTAGVTPQPESLSPSGKEPAASSPSSN TAATTA

AI VPG SQLMP SK SP STGTTEI S SHE S SHGTP SQTT AKN WELTASASHQPPGVYPQGHSDTT

mCheny MSKGEEDNMAIIKEFMRFKVHMEGSV GHEFEIEGEG 1059 1060

EGRPYEGTOTAKLKVTKGGPLPFAWD3LSPQFMYGSK

AYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVT

VTQD S SLQDGEFI YK VKLR GTNFP SD GP QKKTMG

WEASSERMYPEDGALKGEIKQRL-KLKDGGHYDAEVK

TTYKAK PVQLPGAYNVNIKLDrrSHNEDYTlVEQYE

RAEGRHSTGGMDELYK *

tnCheny LSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEG 1029 1030

EGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSK

AYWHPADIPDYLKLSFPEGFKWERVMNFEDGGVVT

VTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMG

WEASSERMYPEDGALKGEIKQRLKL DGGHYDAEVK

TTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYE

RAEGRHSTGGMDELYK

HA Tag YPYDVPDYA 1024 1025-1027

Flag DYKDDDDK 1232 -

BamHI GS - GGATCC

P2A C!eavable GATNFSLLKQAGDVEENPGP 925 926 Peptide

ecDHFR (Amino ISLIAALAVDYVIGMENAMPWNLPADLAWFKRNTLN 9 692, 772, acid 2-159 of WT KPVmiGRHTWESlGRPLPGRKNIILSSQPGTDDRVTWV 814, 687, R12Y, Y100I) KSVDEAIAACGDVPEIMVIGGGRVIEQFLPKAQKLYLT 988, 9 1

HIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHS

YCFEILERR*

ecDHFR (Amino ISLIAALAVDHVIGMENAMPWNLPADLAWF^RNTLN 10 798, 815, 993 acid 2-159 of WT KPVrMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWV

R12H, E129K) KSVDEAIAACGDVPEMVIGGGRVYEOFLPKAQKLYL

THiDAEWGDTHFPDYKPDDWESVFSEFHDADAQNS

HSYCFEiLERR*

FKBP (E31G, GVQVETISPGDGRTFPKRGQTCWHYTGMLGDGKKV 12 688-691 , 994, F36V. R71G, DSSRDRNKPFKFMLGKQEVTRG EEGVAQMSVGQG 1013, 1028 K105E) A mSPDYAYGATGHPGIIPPHATLVFOVELLELE*

hDHFR (Amino VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 890 696, 973, acid 2-187 of WT; XlTTTSSVEGKQNLVDVlGK TWFSrPEKNRPLKGRINL 974, 996 Y1221, A125F) VLSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVD

MVWIVGGSSVI EFMNHPGHLKLFVTRIMQDFESDTF

FPEIDLEKYKLLPEYPGVLSDVQEEKGIKY .F EVYEKN

D*

hDHFR (Amino VGSLNCIVAVSONMGIGKNGDLPWPPLR EFRYFFRM 891 700, 975, acid 2-187 ofWT; TTTSSVEGKQNLVIMGKKTWFSIPEKFRPLKGREsrLVL 976, 998 Q36F, N65F, SRELKEPPQGAHFLSRSLDDALKLTEQPELANKVD V

Y122I) WIVGGSSViKEAMNTTPGHLKLFVTRIMQDFESD^'rf^'FPE

IDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEKND hDHFR (Amino VGSLNCIVAVSQNMGIGKNGDLPWPPLR EFRYFQR 889 972 acid 2-187 of WT; MTTTSSVEGKQNLVIMGK TWFSIPEKNRPLKGRINL

K185E) VLSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVD

MVWTVGGSS KEAMNHPGHLKLFVTRIMQDFESDT

FFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEE

ND*

hDHFR (Amino VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 888 970-972 acid 2-187 of WT; MTTTSSVTiGKQNLATMGK TWFSIPEXNRPLKGRINL

E162G, I176F) VLSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVD

MVWiVGGSSWKEAMNHPGHLKLFVTRIMQDFESDT

FFPEIDLEKY 1 I PGYPGVLSDVQEEKGFKYKFEVYE

KND*

hDHFR (Amino VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 892 977 acid 2-187 ofWT; MTTTSS\¾GKQNL\TMGKKTWFSIPEKNRPLKGRINL

N127Y) VLSREL EPPOGAHFLSRSLDDALKLTEQPELANKVD

VWIVGGSSVYKEA YHPGHLKLFVTRIMQDFESDT

FFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEK

ND

hDHFR (Amino VGSL CIVAVSQNMGVGKNGDLPWPPLRNEFRYFQR 894 979 acid 2-187 of WT; MTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINL

117V) VLSRELKEPPOGAHFLSRSLDDALKLTEQPELANKVD

MVWIVGGSS KEAM HPGHLKLFVTRIMODFESDT FFPEIDLEKYKLLPEYPGVLSDVOEEKGIKYKFE-VYEK

ND

hDHFR (Amino VGSLNCIVAVSQNMGVGKNGDLPWPPLRNEFRYFQR 882 969 acid 2-187 ofWT; MTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINL

117V, Y 1221) VLSRELKEPPQGAHFLSRSLDDALKLTEOPELA KVD

MVWIVGGSSVIKEA HPGHLKLFVTRIMQDFESDTF

FPEIDLEK YKLLPEYPG VL SD VQEEK G IKYKFEVYEKN

D

hDHFR (Amino VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 893 978 acid 2-187 ofWT; MTTTSSVEGKQNLVIMG KTWFSIPEKNRPLKGRINL

H131R, E144G) VLSRELKEPPQGAHFLSRSLDDALKLTEQPELANKVD

M IVGGSSVYKEA NHPGRLKLFVTRIMQDFGSDT

FFPEIDLEKYKLLPEYPGVLSDVQEEKGIKYKFEVYEK

ND

Table 13b: DD-IL15/IL15Ra constructs

VPEIMVIGGGRVYEQFLPKAQKLYLTHI

DAEVEGDTHFPDYKPDDWESVFSEFHD ADAQNSHSYCFEILERR*

OT-IL15-007 EFla MDWTWILFLVAAATRVHSNWVNVISDL 805 817 (igE signal KKIEDLIQSMHIDATLYTESDVHPSCKVT

sequence; 1L15; AMKCFLLELOVISLESGDASIHDTVENLII

linkerl (SG3- (SG4)5-SG3); EFLQSFVHIVQMFINTSSGGGSGGGGSGG

ILlSRa: linkerl GGSGGGGSGGGSLQ1TCPPPMSVEHAD1

(GGSGG); WV SY SLY SRER YI CNS GFKRKAGTS S L

F BP TECVLNKAT VAHWTTPSLKCIRDPALV

(E31G.F36V, HQRPAPPSTVTTAGVTPQPESLSPSGKEP

R71G, K105E)) AASSPSSNNTAATTAAIVPGSQLMPSKSP

STGTTE T S SHE S SHGTPSQTTAKN WELT A

SASHQPPGVYPQGHSDTTVAISTSTVLLC

GLSAVSLLACYLKSRQTPPLASVE EAM

EALPVTWGTSSRDEDLENCSHHLSGGVQ

VETISPGDGRTFPKRGQTCWHYTG LG

DGKKVDS SRDRNKPFKFMLGKQEVIRG

WEEGVAQMSVGQGAKLTISPDYAYGAT

GHPGIIPPHATLVFDVELLELE*

OT-IL15-008 EFla MDWTWILFLVAAATRVHSNWVNVISDL 806 818

(IgE signal KKIEDLIQSMHIDATLYTESDVHPSCKVT

sequence- 1L1.5- AMKCFLLELQVISLESGDASIHDTVENLII

linker (SG3- L ANNSLS SNGN VTES GCKECE E LEEKNIK

(SG4)3-SG3- EFLQSFVHIVQMFINTSSGGGSGGGGSGG

SLQ)- IL15Ra- GGSGGGGSGGGSLQITCPPPMSVEHADI

stop) WVKSYSLYSRERYICNSGFKRKAGTSSL

TECVLNKATNVAHWTTPSLKCIRDPALV

HQRPAPPSTVTTAGVTPQPESLSPSGKEP

AASSPSSNNTAATTAAIVPGSQLMPSKSP

S TGTTEI S SHE S SHGTP SQTT AKN WELT A

SASHQPPGVYPQGHSDTTVAISTSTVLLC

GLSAVSLLACYLKSRQTPPLASVEMEAM

E ALP VTWGTS SRDEDLENC SHHL *

OT-TL15-009 EFla MDWTWILFLVAAATRVHSNWVNVISDL 807 819

(IgE signal KKIEDLIQSMHIDATLYTESDVHPSCKVT

sequence- 1L1.5- AMKCFLLELQVISLESGDASIHDTVENLII

linker (SG3- L ANNSLS SNGN VTES GCKECE ELEEKNIK

(SG4)3-SG3- EFLQSFVHIVQMFINTSSGGGSGGGGSGG

SLQ)- IL15Ra- GGSGGGGSGGGSLQITCPPPMSVEHADI

linker (SG)- W VK SY SLY SRER YICNS GFKRKAGTS SL

ecDHFR (Amino TECM.NKATNVAHWTTPSLKCIRDPALV

acid 2-159 of HQRPAPPSTVTTAGVTPQPESLSPSGKEP

WT; R 12Y, AASSPSSNNTAATTAAIVPGSQLMPSKSP

Y100I)-stop) S TGTTEI S SHE S SHGTP SQTT AK WELT A

SASHQPPGVYPQGHSDTTVAISTSTVLLC

GLSAVSLLACYLKSRQTPPLASVEMEAM

E ALP VTWGTS SRDEDLENC SHHL S GI SLI

AALAVDYVIGMENAMPWNLPADLAWF

KRNTLNKPVIMGRHTWESIGRPLPGRKN

IILSSQPGTODRVTWVKSVDEAIAACGD

VPEIMVIGGGRVIEQFLPKAQKLYLTHID

AEVEGDTHFPDYEPDDWESVFSEFHDAD

A QN S H S YCFEILERR*

OT-IL15-010 EFla MDW ILFLVAAATR SNWWVISDL 808 820 (IgE signal KKIEDLIQSMHIDATLYTESDVHPSCKVT

sequence- IL15- AMKCFLLELQVISLESGDASIHDTVE-NLII

linker (SG3- LAN SLSSNGNVTESGCKECEELEEKNIK

(SG4)3-SG3- EFLQSFVHIVQMFINTSSGGGSGGGGSGG

- I l l - SLQ)- IL15Ra- GGSGGGGSGGGSLQITCPPPMSVEHAD1 iiriker (SG)- WV SY SLY SRER Yi CNS GFKRKAGTS SL

hDHFR (Y122I, TECVLNKAT VAHWTTPSLKCIRDPALV

A125F)-stop) HQRPAPPSTVTTAGVTPQPESLSPSGKEP

AASSPSSNNTAATTAAIVPGSQLMPSKSP

STGTTE I S SHE S SHGTPSQTTAKN WELT A

SASHQPPGVYPQGHSDTTVAISTSTVLLC

GLSAVSLLACYLKSRQTPPLASVE EAM

EALPVTWGTSSRDEDLENCSHHLSGVGS

LNCIVAVSQNMGIGKNGDLPWPPLRNEF

RYFQRMTTTS SVEGKQNL VMGKKTWF

SIPEKNRPLKGRINLVLSRELKEPPQGAH

FLSRSLDDALKL-TE-QPELA KVDMVWIV

GGSSVIKEFMNHPGHLKLFVTRIMQDFE

SDTFFPEIDLEKYKLLPEYPGVLSDVQEE

KGIKY KF E VYEKND *

OT-IL15-01 1 EFl a MDWT ILFLVAAATRVHSNWVNViSDL 809 821

(IgE signal K -EOLiQSMHIDATLYTESDVHPSCKVT

sequence- LL 15- AMKCFT I EI .QVISLESGDASIHDTVENLII

tinker (SG3- LAN SLSSNGNVTESGCKECEELEEK IK

(SG4)3-SG3- EFL-OSFVHIVQMFT TSSGGGSGGGGSGG

SLQ)- IL15Ra; GGSGGGGSGGGSLQITCPPPMSVEHADI

linker (SG)- WVKSYSLYSRERYICNSGFKRKAGTSSL

hDHFR (Amino TE-CVLNKATNVAHWTTPSLKCIRDPALV

acid 2-187 of HQRPAPPSTVTTAGVTPQPESLSPSGKEP

WT; Q36F, AASSPSSNNTAATTAAIVPGSQLMPSKSP

N65F, Y122I)- S TGT 1 El S SHE S SHGTP SQTT AKNWELT A

stop) SASHQPPGVYPQGHSDTTVAISTSTVLLC

GLSAVSLLACYLKSRQTPPLASVEMEAM

EALPVTWGTSSRDEDLENCSHHI .SGVGS

L NCI VA VSQNMGIGKN G DLP WPPLRNEF

RYFFRMTTTSSVEGKQNLVIMGKKTWFS

1PEKFRPLKGR1NL VL SREL KEPPQG AHFL

SRSLDDALKLTEQPELANKVDMVWIVG

GSSVIKEAMNHPGHLKLFVTRIMQDFES

DTFFPE IDLEKYKLLPEYPG VLS D VQEE K

GIK kF t. VYEKND*

OT-IL15-017 EFla MDWTWILFLVAAATRVHSNWVNVISDL 1061 1086 (IgE signal K iEDLIQSMHIDATLYTESDVHPSCKVT

sequence- IL15- AMKCFLLELQVTSI SGDASIHDTVENLII

linker (SG3- (SG4)3-SG3- EFLQSFVHIVQMFINTSSGGGSGGGGSGG

SLQ)- IL15Ra- GGSGGGGSGGGSLQITCPPPMSVEHADI

! ink r (SG)- WVKSYSLYSRERYICNSGFKRKAGTSSL

hDHFR (Amino TECVLNKATNVAHWTTPSLKCIRDPALV

acid 2-187 of HQRPAPPSTVTTAGVTPQPESLSPSGKEP

WT; K185E)- AASSPSSNNTAATTAAIVPGSQLMPSKSP

stop) ST 1 ! ^'EISSHESSHGTPSQTTAKNWELTA

SASHQPPGVYPQGHSDTTVAISTSTVLLC

GLSAVSLLACYLKSRQTPPLASVEMEAM

EALPVTWGTSSRDEDLENCSHHLSGVGS

LNCIVAVSQNMGIGKNGDLPWPPLRNEF

RYFQRMTTTSSVEGKQNLVIMGKKTWF

SIPEKNRPLKGRINLVLSRELKEPPQGAH

FLSRSLDDALKLTEQPELANKVDMVWIV

GGSSVYKE-AMNHPGHLKLFVTRIMQDF

ESDTFFPEIDLEKYKLLPEYPGVLSDVQE

EKGIK YKFE VYEEND *

OT-IL15-018 EFla MDWTWILFLVAAATRVHSNWVNVISDL 1062 1087

(IgE signal KKIEDLIQSMHIDATLYTESDVHPSCKVT sequence- IL.15- AMKCFT I ,ET .QVISLESGDASIHDTVENLII linker (SG3- L ANN SLSSN G NVTESGCKECEELEEKNIK

(SG4)3-SG3- EFLQSFVHIVQMFINTSSGGGSGGGGSGG

SLQ)- ]L15Ra- GGSGGGGSGGGSLQITCPPPMSVEHADI

linker (SG)- WVKSYSLYSRERYICNSGFKRKAGTSSL

hDHFR (Amino TECVLNKATNVAHWTTPSLKCIRDPALV

acici 2-187 of HQR P APP STVTT A G VTPQPESL SP S GKE P

WT; E162G, AASSPSSN TAATTAAIVPGSQL PSKSP

I176F)-stop) S TGTTEI S SHE S SHGTP SQTT AKN WELT A

SASHQPPGVYPQGHSDTTVAISTSTVLLC

GL S A VSLL A C YLK SRQTPPL A S VEME AM

EALPVTWGTSSRDEDLENCSHHLSGVGS

LNC^'IVAVSQNMGIGKNGDLPWPPLRNEF

RYFQRMTTTS S VEGKQNL VIMGKKTWF

SIPEKNRPLKGRINLVLSRELKEPPQGAH

FLSRSLDDALKLTEQPELANKVDMVWIV

GGSSVYKEAMNHPGHLKLFVTRIMQDF

ESDTFFPEIDLEKYKLLPGYPGVLSDVQE

EKGFKYKFE VYEK D *

OT-IL15-038 EFla MD WTWILFL V AAATR VH SNWVN VI SDL 1063 1088 (igE leader - KKIEDLIQSMHIDATLYTESDVHPSCKVT

IL 15 ~ Linker AMKCFLLELQVISLESGDASIHDTVE-NLII

(SG3-(SG4)3- LANNSLSSNGNVTESGCKECEELEEKNIK

SG3-SLQ) - EFLQSFVHIVQMFINTSSGGGSGGGGSGG

IL15Ra - Linker GGSGGGGSGGGSLQITCPPPMSVEHADI

(SG) - hDHFR WVKSYSLYSRERYICNSGFKRKAGTSSL

(Amino acid 2- TECVLNKATNVAHWTTPSLKCIRDPALV

187 of WT; HQRPAPPSTVTTAGVTPQPESLSPSGKEP

N127Y)-stop) AASSPSSNNTAATTAAIVPGSQLMPSKSP

S TGTTEI S SHE S SHGTP SQTT AKN WELT A

SASHQPPGVYPQGHSDTTVAISTSTVLLC

G L S A VS L L A C YLK SRQTPPL A S VEME AM

EALPVTWGTSSRDEDLENCSHHLSGVGS

LNC1VAVSQNMGIGKNGDLPWPPLRNEF

RYFQRMTTTS S VEGKQNL VTM GKKTWF

SIPEKNRPLKGRINLVLSRELKEPPQGAH

FLSRSLDDALKLTEQPELANKVDMVWIV

GGSSVYKEAMYHPGHLKLFVTRIMQDF

E SDTFFPE IDLEK YKLLPE YPG VL SD VQE

EKGIKYKFE VYEKND*

OT-IL15-051 EFla MD WTWILFL V AAATR VH SNWVN VI SDL 1064 1089 (IgE leader - KKIEDLIQSMHIDATLYTESDWPSCKVT

IL15 - Linker AMKCFi F ,FJ .QVISLESGDASIHDTVENLIi

(SG3-(SG4)3- LANNSLSSNGNVTESGCKECEELEEKNIK

SG3-SLQ) - HA EFLQSFVHIVQMFINTSSGGGSGGGGSGG

Tag - IL15Ra - GGSGGGGSGGGSLQYPYDVPDYAITCPP

stop) PMSVEHADIWVKSYSLYSRERYICNSGF

KRK AGTS SLTECVLNK ATN VAHVVTTPSL

K CIRDP AL VHQRP APPST VTT AGVTPQPE

SLSPSGKEPAASSPSSNNTAATTAAIVPG

SQLMPSKSPSTGTTEISSHESSHGTPSQTT

AKNWELTASASHQPPGVYPQGHSDTTV

AISTSTVLLCGLSAVSLLACYLKSRQTPP

LASVEMEAMEALPVTWGTSSRDEDLEN

CSHHL*

OT-IL15-053 EFla MD WTWILFL VAAATRVHSNWVNVISDL 1066 1091 (IgE leader - KKIFT)LIQSMHIDATLYTESDVHPSCKVT

IL15 - Linker AMKC FLLELQVISLES GD ASIHDTVENL II

(SG3(SG4)5SG3 LANNSLSSNGNVTESGCKECEELEEKNIK

EFLQSFVHIVQMFINTSSGGGSGGGGSGG S) - IL15Ra - GGSGGGGSGGGGSGGGGSGGGSITCPPP Stop) MSVEHADIWVKSYSLYSRERY1CNSGF

RKAGTSSLTECVLNKATNVAHWTTPSL

CIRDPALVHQRPAPPSTVTTAGVTPQPES

LSPSGKEPAASSPSSNNTAATTAAIVPGS

QLMPSKSPSTGTTEISSHESSHGTPSQTTA

K WELTASASHQPPGVYPQGHSDTTVAI

STSTVLLCGLSAVSLLACYL SRQTPPLA

S VEMEAMEALPVTWGTS SRDEDLENCS

HHL*

OT-IL15-054 EFla MD WTWILFL VAAATRVHSNWVNVISDL 1067 1092 (IgE leader - IF LIQSMHIDATLYTESDVHPSCKVT

IL15 - Linker AMKC FLLELQV1SLES GD ASIHDTVENL 11

(SG3(SG4)3S) - LANNSLSSNGNVTESGCKECEELEEKNIK

HA Tag -- Linker EFLQSFVHIVQMFINTSSGGGSGGGGSGG

(SG3S) - GGSGGGGSYPYDVPDYASGGGSITCPPP

IL15Ra - Stop) MSVEHADRWVKSYSLYSRERYICNSGFK

RKAGTSSI;TECVLNKATNVAI-IWTTPSLK

CffiDPALVHQRPAPPSTVTTAGVTPQPES

LSPSGKEPAASSPSSNNTAATTAAIVPGS

QLMPSKSPSTGTTEISSHESSHGTPSQTTA

KNWELTASASHQPPGVYPQGHSDTTVAI

STSTVLLCGLSAVSLLACYLKSRQTPPLA SVEMEAME ALP VTWGTS SRDEDLENCS HHL*

OT-TL15-055 EFla MD WTWILFL V A A A TR VH S N W VN VI SI) L 1068 1093 (IgE leader - KKIEDLIQSMHTDATLYTESDVHPSCKVT

IL 15 - Linker AMKCt LLELQVISLESGDASIHDTVENLII

(SG) - IL15Ra - LANNSLSSNGNVTESGCKECEELEEKNIK

Stop) EFLQSF VHI VQMFINTS S GITCPPPMS VEH

ADIWVK S Y SLY SRER YICNS GFKRKAGT

SSLTECVLNKATNVAHWTTPSLKCIRDP

ALVHQRPAPPSTVTTAG\TPQPESLSPSG

KEPAASSPSSN TAATTAAIVPGSQLMPS

KSPSTGTTEISSHESSHGTPSQTTAKNWE

LTASASHQPPGVYPQGHSDTTVAISTSTV

LLCGLSAVSLLACYLKSRQTPPLASVEM

EAMEALPVTWGTSSRDEDLENCSHHL*

OT-IL15-060 EFla MAPRRARGCRTLGLPALLLLLLLRPPAT 1069 1094 (IL15Ra signal RGNWVNVISDLKKIEDLIQSMHIDATLYT

peptide - IL 15 - E SD VHP S CK VT AMKCFLLELQ VI SLES G

Linker (SG3- DASIHDTVENLIILAN SLSSNGNVTESG

(SG4)3-SG3- CKECEELEEKNIKEFLQSFVHIVQMFINT

SLQ) - IL15Ra SSGGGSGGGGSGGGGSGGGGSGGGSLQI

- stop) TCPPPMSVEHADIWVKSYSLYSRERYIC

NSGFKRKAGTSSLTECVLNKATNVAHW

TTPSLKCIRDPALVHQRPAPPSTVTTAGV

TPQPESLSPSGKEPAASSPSSNNTAATTA

ArVPGSQLMPSKSPSTGTTEISSHESSHGT

PSQTTAKNWELTASASHQPPGVYPQGHS

DTTVAISTSTVLLCGLSAVSLLACYT .KSR

QTPPLASVEMEAMEALPVTWGTSSRDE

DLENCSHHL*

OT-IL15-063 EFla MD WTWILFL V A A ATR VH SNWVN VI SDL 1070 1095 (igE leader - KKIEDLIQSMHIDATLYTESDVHPSCKVT

IL 15 - Linker AMKCFLLELQ VISLESGDASIHDT NLII

(SG3-(SG4)3- LANNSLSSNGNVTESGCKECFFLEEKNIK

SG3-SLQ) - EFLQSFVHIVQMFINTSSGGGSGGGGSGG

IL15Ra - GGSGGGGSGGGSLQITCPPPMSVEHADI

WVKSYSLYSRERYICNSGFKRKAGTSSL BamHI (GS) - TECVLNKAT VAHWTTPSLKCIRDPALV stop) HQRPAPPSTVTTAGVTPQPESLSPSGKEP

AASSPSSNNTAATTAAIVPGSQLMPSKSP STGTTE T S SHE S SHGTPSQTTAKN WELT A SASHQPPGVYPQGHSDTTVAISTSTVLLC GLSAVSLLACYLKSRQTPPLASVE EAM EALPVTWGTSSRDEDLENCSHHLGS*

OT-IL15-064 EFla MD WTWILFL V A A ATR VH S N W VN VI SI) L 806 818 and OT-IL15- KKIEDLIQSMfflDATLYTESDVHPSCKVT

071 (IgE leader - AMKCFi F .FLQVISLESGDASIHDTVt! LII

IL15 - Linker LANNSLSSNGNVTESGCKECEELEEKNI

(SG3-(SG4)3- EFLOSFWIVQMFINTSSGGGSGGGGSGG

SG3-SLQ) - GGSGGGGSGGGSLQITCPPPMSVEHAD1

IL 15Ra - stop) WVKSYSLYSRERYICNSGFKRKAGTSSL

TECVLNKATNVAHWTTPSLKCIRDPALV

HQRPAPPSTVTTAGVTPQPESLSPSGKEP

AASSPSSNNTAATTAAIVPGSQLMPSKSP

STGTTEI S SHE S SHGTPSQTTAKN WELT A

SASHQPPGVYPQGHSDTTVAISTSTVLLC

GLSAVSLLACYLKSRQTPPLASVEMEAM

E ALP VTWGTS SRDEDLENC S HHL *

OT-1L15-066 EFla MD WTWILFL VAAATRVHSNWVNVISDL 807 819 (IgE leader - KKIEDLIQSMHIDATLYTESDVHPSCKVT

IL 15 - Linker AMKCFLLELQVISLESGDASIHDTVENLII

(SG3-(SG4)3- LANNSLSSNGNVTESGCKECEELEEKNIK

SG3-SLQ) - EFLQSFVHIVQMFINTSSGGGSGGGGSGG

IL15Ra - Linker GGSGGGGSGGGSLQITCPPPMSVEHADI

(SG) ecDHFR WVKSYSLYSRERYICNSGFKRKAGTSSL

(Amino acid 2- TECVLNKATNVAHWTTPSLKCIRDPALV

159 of WT, HQRPAPPSTVTTAGVTPQPESLSPSGKEP

R12Y, Y100I) - AASSPSSNNTAATTAAIVPGSQLMPSKSP

stop) S TGTTEI S SHE S SHGTP SQTT AKN WELT A

SASHQPPGVYPQGHSDTTVAISTSTVLLC

GLSAVSLLACYLKSRQTPPLASVEMEAM

E ALP VTWGTS SRDEDLENC SHHL S GI SLI

AALAVDYVIGMENAMPWNLPADLAWF

KRNTLNKPVIMGRHTWESIGRPLPGRKN

IILSSQPGTDDRVTWVKSVDEAIAACGD

VPEIMVIGGGRVIEQFLPKAQKLYLTHID

AEVEGDTHFPDYEPDDWESVFSEFHDAD

AQNSHSYCFEILERR*

OT-IL15-067 EFla MD WTWILFL VAAATR \Ή SNWYN VI SDL 1071. 1098

(IgE leader - KKIEDLIQSMHIDATLYTESDVHPSCKVT

IL 15 Linker AMKCFLLELQVISLESGDASIHDTVENLII

(SG3-(SG4)3- LANNSLSSNGNVTESGCKECEELEEKNIK

SG3-SLQ) - EFLQSFVHIVQMFINTSSGGGSGGGGSGG

IL15Ra - Linker GGSGGGGSGGGSLQITCPPPMSVEHADI

(SG) - ecDHFR WVKSYSLYSRERYICNSGFKRKAGTSSL

(Amino acid 2- TECVLNKATNVAHWTTPSLKCIRDPALV

159 of WT, HQRPAPPSTVTTAGVTPQPESLSPSGKEP

R12Y, Y100I) - AASSPSSNNTAATTAAIVPGSQLMPSKSP

stop) STGl'lEISSHESSHGTPSQTTAK WELTA

SASHQPPGVYPQGHSDTTVAISTSTVLLC

GLSAVSLLACYLKSRQTPPLASVEMEAM

EALPVTWGTSSRDEDLENCSHHLSGTST J

AALAVDYVIGMENAMPWNLPADLAWF

KRNTLNKPVIMGRHTWESIGRPLPGRKN

IILSSQPGTDDRVTWVKSVDEAIAACGD

VPEIMVIGGGRVIEQFLPKAQKLYLTHID AEVEGDTHFPDYEPDDWESVFSEFHDAD

A Q S H S YCFEILERR*

OT-IL15-070 EFla MAPRRARGCRTLGLPALLLLLLLRPPAT 1072 1101 (IL15Ra signal RGNWVNVISDLKKiEDLIQSMHIDATLYT

peptide - IL15 - E SD VHP S CK VT A KCFLLELQ VI SLES G

Linker (SG3- DASIHDTVENLimANNSLSSNGNVTESG

(SG4)3-SG3- CKECEELEEKNIKEFLQ SF\¾I VQMFLNT

SLQ) --- IL15Ra SSGGGSGGGGSGGGGSGGGGSGGGSLQl

- stop) TCPPPMSVEHADIWVKSYSLYSRERYIC

NSGFKRKAGTSSLTECVLN ATNVAHW

TTPSLKCIRDPALVHQRPAPPSTVTTAGV

TPQPESLSPSGKEPAASSPSSNNTAATTA

A1VPGSQLMPSKSP STGTTEIS S HES SHOT

P SQTTAKNWELTA S ASHQPPG VYPQGHS

DTTVAISTSTVLLCGLSAVSLLACYLKSR

QTPPLASVEMEAMEALPVTWGTSSRDE

DLENCSHHL*

OT-1L15-072 EFla MD WTWILFL V A A ATR VH S N W VN VI SD L 806 1096 (IgE leader■-- KKIEDLIQSMHIDATLYTESDVHPSCKVT

IL15 - Linker AMKCFLLELQ VISLESGDASIHDTVENLI!

(SG3-(SG4)3- LANNSLSSNGNVTESGCKECEELEEKNIK

SG3-SLQ) - EFLQSFWIVQMFINTSSGGGSGGGGSGG

IL15Ra -- stop) GGSGGGGSGGGSLQ1TCPPPMSVEHAD1

WVKSYSLYSRERYICNSGFKRKAGTSSL

TECVLNKATNVAHWTTPSLKCIRDPALV

HQRPAPPSTVTTAGVTPQPESLSPSGKEP

AASSPSSNNTAATTAAIVPGSQLMPSKSP

STGTTE T S SHE S SHGTPSQTTAKN WELT A

SASHQPPGVYPQGHSDTTVAISTSTVLLC

GL S A VSLL AC YLKSRQTPPL A S VEME AM

E ALP VTWGTS SRDEDLENC SHHL *

OT-1L15-089 EFla MD WTWILFL VAAATRVHSDYKDDDDK 1074 1102

(IgE leader■-- N WVN VI S DLKKIEDL IQ SMH ID ATL YTES

Π .ΛΟ Π ! ? D VHP S CK VT AMKC FLLELQ VI SLES GD A

Linker (SG3- SIHDTVENLIILANNSLSSNGNVTESGCK

(SG4)3-SG3- E CEE LEEKNIK EFLQSF VHTVQMFINT SSG

SLQ) - HA Tag GGSGGGGSGGGGSGGGGSGGGSLQYPY

- IL15Ra - DVPDYAITCPPPMSVEHADIWVKSYSLY

linker (GSG) - SRERYICNSGFKRKAGTSSLTECVLNKAT

ecDHFR (Amino N V AHWTTP SLKC 1RDP AL VHQR P APP ST

acid 2-159 of VTT AG VTPQPESL SP S GKEP A A S SP S SNN

WT, R12Y, TAATTAAI GSQLMPSKSPSTGTTEISSH

Y100I) - stop) ESSHGTPSQTTAKNWELTASASHQPPGV

YPQGHSDTTVAISTSTVLLCGLSAVSLLA

CYLKSRQTPPLASVEMEAMEALPVTWG

T S SRDEDLENC SHHLG S GI SLI A AL A VD Y

VIGMENAMPWNLPADLAWFKRNTLNKP

VIMGRHTWESIGRPLPGRKNIIL SSQPGT

DDRVTWVKSVDEAIAACGDVPEIMVIGG

GRVIEQFLPKAQKLYLTHIDAEVEGDTH

FPDYEPDDWESVFSEFHDADAQNSHSYC

FEILERR*

OT-IL15-109 EFla MD WTWILFL V A A ATR VH SN WVN VI SDL 1070 1095 (IgE leader - KKIEDLIQSMHIDATLYTESDVHPSCKVT

IL15 - Linker AMKCFLLELQVISLESGDASIHDTVENLII

(SG3-(SG4)3- LANNSLSSNGNVTESGCKECEELEEKNIK

SG3-SLQ) - EFLQSFWIVQMFINTSSGGGSGGGGSGG

IL ISRa - GGSGGGGSGGGSLQITCPPPMSVEHADI

BamHI (GS) - WVKSYSLYSRERYICNSGFKRKAGTSSL

stop) TECVLNKATNVAHWTTPSLKCIRDPALV HQRP APP STVTT AG VTPQPE SL SP S GKE P

AASSPSSNNTAATTAAiVPGSQLMPSKSP

STGTTEISSHESSHGTPSQTTAKNWELTA SASHQPPGVYPQGHSDTTVAISTSTVLLC G L S A VS L L A C YLK SRQTPPL A S VEME AM E ALP VTWGTS SRDE DLENC SHHLGS*

OT-IL15-110 EFla MDWTWILFLVAAATRVHSDYKDDDDK 1075 1103 (IgE ieader - NWVNVISDLKKIEDLIQSMHIDATLYTES

FLAG -- IL15 - D VHP S CK VT AM CF1 i F LQ VI SLE S GD A

Linker (SG3- SIHDT VENLIIL ANN SL S S N GN VTE S GCK

(SG4)3-SG3- ECEELEEKNKEFLQSFWIVQMFINTSSG

SLQ) - H A Tag GGSGGGGSGGGGSGGGGSGGGSLQYPY

- IL15Ra - DVPDYAITCPPPMSVEHADIWVKSYSLY

BamHI (GS) - SRERYICNSGFKRKAGTSSLTECVLNKAT

stop) NVAHWTTPSLKCIRDPALVHQRPAPPST

VTT AG VTPQPESL SP S GKEP A A S SP S SN

TAATTAAIVPGSQLMPSKSPSTGTTEISSH

E S SHGTP SOTTAKNWELTA S ASHQPPG V

YPQGHSDTTVAISTSTVLLCGLSAVSLLA

CYLKSRQTPPLASVEMEAMEALPVTWG

TSSRDEDLENCSHHLGS*

OT-IL15-114 EFla MDWTWILFLVAAATRVHSDYKDDDD 1076 1104

(IgE Ieader■-- NWVNVISDLKKIEDLIQSMHIDATLYTES

FLAG - SL 35 - D VHP S CK VT AMKC FLLELQ VI SLES GD A

Linker (SG3- SIHDTVENLIILANNSLSSNGNVTESGCK

(SG4)3-SG3- E CEE LEEKNIK EFLQSF VHIVQMFINT S S G

SLQ) -- HA Tag GGSGGGGSGGGGSGGGGSGGGSLQYPY

- IL15Ra - DVPDYAITCPPPMSVEHADIWVKSYSLY

Linker (GSG) - SRERYICNSGFKRKAGTSSLTECVLNKAT

hDHFR (Amino NVAHWTTPSLKCIRDPALVHQRPAPPST

acid 2-187 of VTT AG VTPQPESL SP S GKEP A A S SP S SNN

WT; K185E) - TAATTAAIVPGSQLMPSKSPSTGTTEISSH

stop) ESSHGTPSQTTAKNWELTASASHQPPGV

YPQGHSDTTVAISTSTVLLCGLSAVSLLA

CYLKSRQTPPLASVEMEAMEALPVTWG

T S SRDEDLENC SHHLG S G VG SLNCI V A V

SQNMGIGKNGDLPWPPLRNEFRYFQRM

TTTSSVEGKQNLVIMGKKTWFSiPEKNR

PLKGRINL VL SRELKEPPQG AHFL SRSLD

DALKLTEQPELANKVDMVWIVGGSSVY

KEAMNHPGHLKLFVTRIMQDFESDTFFP

EIDLEKYKLLPEYPGVLSDVQEEKGIKYK

FEVYEEND*

OT-IL15-115 EFla MDWTWILFLVAAATRVHSDYKDDDDK 1077 1.105

(IgE ieader - NWVNVISDLKKIEDLIQSMHIDATLYTES

FLAG - IL 15 - D VHP S CK VT AMKCFLLELQ VI SLES GD A

Linker (SG3- SIHDTVENLIIL ANNSL S SN GNVTESGCK

(SG4)3-SG3- ECEELEEKNrKEFLQSFVHIVQMFINTSSG

SLQ) - HA Tag GGSGGGGSGGGGSGGGGSGGGSLQYPY

- ILlSRa - DVPDYAITCPPPMSVEHADIWVKSYSLY

Linker (GSG) - SRERYICNSGFKRKAGTSSLTECVLNKAT

hDHFR (Amino NVAHWTTPSLKCIRDPALVHQRPAPPST

acid 2-187 of VTT AG VTPQPESL SP S GKEP A A S SP S SNN

WT; E162G, TAATTAAIVPGSQLMPSKSPSTGTTEISSH

I176F) - stop) E S SHGTP SQTTAKNWELTAS ASHQPPG V

YPQGHSDTTVAISTSTVLLCGLSAVSLLA

CYLKSRQTPPLASVEMEAMEALPVTWG

TSSRDEDLENC SHHLG SGVG SLNCTVAV

SQNMGIGKNGDLPWPPLRNEFRYFQRM

TTTSSVEGKQNLVIMGKKTWFSIPEKNR PLKGRINL VL S REL KEPPQG AHFL SR SLD

DALKLTEQPELA KVDMVWIVGGSSVY KEAMNHPGHLKLFVTRIMQDFESDTFFP EIDLEKYKLLPGYPGVLSDVQEEKGFKY KFEVYEKND*

OT-IL15-116 EFla MDWTWILFLVAAATRVHSDYKDDDDK 1078 1106 (IgE leader - NWVNVISDLKKIEDLIOSMHIDATLYTES

FLAG --- 1L 15 --- D VHP S CK VT AMKC FLLELQ VI SLES GD A

Linker (SG3- SIHDTVENLIILANNSLSSNGNVTESGCK

(SG4)3-SG3- E CEE LEEKN IKEFLQ SF VHi VQMF1NT S S G

SLQ) -- HA Tag GGSGGGGSGGGGSGGGGSGGGSLQYPY

- IL15Ra - DVPDYA!TCPPPMSVEHADIWVKSYSLY

Linker (GSG) - SRERYICNSGFKRKAGTSSLTECVLNKAT

hDHFR (Amino NVAHWTTPSLKC!RDPALVHQRPAPPST

acid 2-187 of VTT A G VTPQPESL SP S GKE PA A S SP S SNN

WT; H131R, TAATTAAIVPGSQLMPSKSPSTGTTETSSH

E144G) - stop) ESSHGTPSQTTAK WELTASASHQPPGV

YPQGHSDTTVAISTSTVLLCGLSAVSLLA

CYLKSRQTPPLASVEMEAMEALPVTWG

T S SRDEDLENC SHHLG S G VG SLNCI VA V

SQNMGIGKNGDLPWPPLRNEFRYFQRM

TTTSSVEGKQNLVIMGKKTWFSIPEKNR

PLKGRINL VLSRELKEPPQG AHFL SRSLD

DALKLTEQPELANKVDMVWIVGGSSVY

KEAMNHPGRLKLFVTRLMQDFGSDTFFP

EIDLEKYKLLPEYPGVLSDVQEEKGIKYK

FEVYEKND*

OT-TL15-117 EFla MDWTWILFLVAAATRVHSDYKDDDDK 1079 1 107 (IgE leader - NWVNViSDLKKIEDLIQSMHIDATLYTES

FLAG - IL15 - D VHP S CK VT AMKCFLLELQ VI SLES GD A

Linker (SG3- SIHDTVE-NLIILANNSLSSNGNVTESGCK

(SG4)3-SG3- F CEF LEEKNIKEFLQ SF VHI VQMFINT S S G

SLQ) - HA Tag GGSGGGGSGGGGSGGGGSGGGSLQYPY

- IL15Ra - D DYAITCPPPMSVEHADiWVKSYSLY

Linker (GSG) - SRERYICNSGFKRKAGTSSLTECVLNKAT

hDHFR( Amino NVAHWTTPSLKCIRDPALVHQRPAPPST

acid 2-187 of VTT AG VTPQPESL SP S GKEP A A S SP S SNN

WT; 117V) - TAATTAAIVPGSQLMPSKSPSTGTTEISSH

stop) ESSHGTPSQTTAKNWELTASASHQPPGV

YPQGHSDTTVAISTSTVLLCGLSAVSLLA

CYLKSRQTPPLASVEMEAMEALPVTWG

TSSRDEDLENCSHHLGSGVGSLNCIVAV

SQNMG VGKNGDLPWPPLRNEFR YFQRM

TTTSSVEGKQNLVIMGKKTWFS!PEKNR

PLKGRINL VL SRELKtPPQG AHFL SRSLD

DALKLTEQPELANKVDMVWIVGGSSVY

KEAMNHPG Hi ,KI .FVTRIMQDFESDTFFP

EIDLEKYKLLPEYPGVLSDVQEEKGIKYK

FEVYEKND*

OT-IL15-118 EFla MDWTWILFLVAAATRVHSDYKDDDDK 1080 1108 (IgE leader - NWVNVISDLKKIEDLIQSMHIDATLYTES

FLAG -- IL15 - D VHP S CK VT AMKCF1 i FLQVISLESGDA

Linker (SG3- SIHDTVENLIILANNSLSSNGNVTESGCK

(SG4)3-SG3- ECEELEEKNIKEFLQSFVHIVQMFINTSSG

SLQ) - H A Tag GGSGGGGSGGGGSGGGGSGGGSLQYPY

- ILl SRa - DWDYAITCPPPMSVEHADIWVKSYSLY

Linker (GSG) - SRERYICN SGFKRKAGTS SLTEC VLNKAT

hDHFR (Ammo NVAHWTTPSLKCIRDPALVHQRPAPPST

acid 2-187 of VTT AG VTPQPESL SP S GKEP A A S SP S SNN

TAATTAAIVPGSQLMPSKSPSTGTTEISSH WT, 127Y) - ESSHGTPSQTTAKNWELTASASHQPPGV stop) YPQGHSDTTVAISTSTVLLCGLSAVSLLA

CYLKSRQTPPLASVEMEAMEALPVTWG

T S SRDEDLEN C SHHLG S G VG SL N CI VA V

SQNMGIGKNGDLPWPPLRNEFRYFQRM

TTTSSVEGKQNLVIMGKKTWFSIPEK R

PLKGRINLVLSRELKEPPQGAHFLSRSLD

DAL LTEQPELANKVDMVWIVGGSSVY

KEAMYHPGHLKLFVTRIMQDFESDTFFP

EIDLEK YKLLPEYP G VL SD VQEEKGIKYK

FEVYEKND*

OT-IL15-119 EFla MD WTWILFL VAAATRVHSDYKDDDDK 1081 1109 (IgE leader - NWVN VI S DLKKIEDL IQ SMH ID ATL YTES

FLAG - IL 35 - D VHP S CK VT AMKC FLLELQ VI SLES GD A

Linker (SG3- SIHDTVENLIILANNSLSSNGNVTESGCK

(SG4)3-SG3- E CEE LEEKNIK EFLQSF VHIVQMFI T SSG

SLQ) - HA Tag GGSGGGGSGGGGSGGGGSGGGSLQYPY

- IL15Ra - DVPDYAITCPPPMSVEHADIWVKSYSLY

Linker (GSG) - SRER YICNSGFKRKAGTS SLTECVL KAT

hDHFR (Amino N VAHWTTP SLKC IRDP AL VHQRP APP ST

acid 2-187 of VTTAGVTPQPESLSPSG EPAASSPSSN

WT, I17V, TAATTAAIVPGSQLMPSKSPSTGTTEISSH

Y122I) - stop) ESSHGTPSQTTAKNWELTASASHQPPGV

YPQGHSDTTVAISTSTVLLCGLSAVSLLA

CYLKSRQTPPLASVEMEAMEALPVTWG

TSSRDEDLENCSHHI GSGVGSLNCIVAV

SQNMGVGKNGDLPWPPLRNEFRYFQRM

TTTSSVEGKQNLVIMGKKTWFSIPEKNR

PLKGRINL VL SRELkLPPQG AHFL SRSLD

DALKLTEQPELANKVDMVWIVGGSSVIK

EAMNHPGHLKLFVTRIMQDFESDTFFPEI

DLEKY 1 '· PEYPG VL S D VQEEKG IK YKF

EVYEKND*

OT-IL15-128 EFla MD WTWILFL V AAATR VH SNWVN VI SDL 1082 1 1 10 (IgE leader - KKIEDLIQSMHLOATLYTESDVHPSCKVT

IL15 - Linker AMKCFLLELQVISI SGDASIHDTVENLII

(SG3-(SG4)3- SG3-SLQ) - EFLQSFVHIVQMFINTSSGGGSGGGGSGG

IL15Ra- Spacer GGSGGGGSGGGSLQITCPPPMSVEHADI

- Flagx3 - WVKSYSLYSRERYICNSGFKRKAGTSSL

Spacer - BamHI TECVLNKATNVAHWTTPSLKCIRDPALV

(GS) - P2A HQRP APP STVTT AG VTPQPE SL SP S GKE P

cleavable peptide AASSPSSNNTAATTAAIVPGSQLMPSKSP

— tnCherry STG iEISSHESSHGTPSQTTAKNWELTA

(MIL) - stop) SASHQPPGVYPQGHSDTTVAISTSTVLLC

GLSAVSLLACYLKSRQTPPLASVEMEAM

EALPVTWGTSSRDEDLENCSHHLSRMD

YKD DDDKD YKDD DDKD YKDDDDK SR G

SGATNFSLLKQAGDVEENPGPLSKGEED

NMAIIKEFMRFKVHMEGSVNGHEFEIEG

EGEGRPYEGTQTAKLKVTKGGPLPFAW

DILSPQFMYGSKAYVKHPADIPDYLKLSF

PEGFKWERVMNFEDGGVVTVTQDSSLQ

D GEFIYK VKLR GTNFP SD GP VMQKKTM

GWEA S SERMYPED G ALKGEJKQRLKLK

DGGHYDAE TTYKAKKPVQLPGAYN

WIKLDITSHNEDYTIV QYERAEGRHST

GGMDELYK*

OT-TL15-129 EFla MD WTWILFL VAAATRVHSNWVN VISDL 1083 1 111 (IgE leader - KKIEDLIQSMHTDATLYTESDVHPSCKVT IL15 - Linker AMKCFT T ,KT .QVISLESGDASIHDTVENLII (SG3-(SG4)3- L ANN S LS SN G N VTES GC ECEELEEK IK SG3-SLQ) - EFLQSFVHIVQMFINTSSGGGSGGGGSGG IL 15Ra-BamHI GGSGGGGSGGGSLQITCPPPMSVEHADI (GS) - P2A WVKSYSLYSRERYICNSGFKRKAGTSSL cleavable peptide TECVLNKATNVAHWTTPSLKCIRDPALV - tnCherry HQR P APP STVTT A G VTPQPESL SP S GKE P (MlL)-stop) AASSPSSN TAATTAAIVPGSQL PSKSP

S TGTTEI S SHE S SHGTP SQTT AKN WELT A

SASHQPPGVYPQGHSDTTVAISTSTVLLC

GL S A VSLL A C YLK SRQTPPL A S VEME AM

EALPVTWGTSSRDEDLENCSHHLGSGAT

NFSLLKQAGDVEENPGPLSKGEEDNMAI

IKEFMRFK MEGSVNGHEFEIEGEGEG

RPYEGTQTAKLKVTKGGPLPFAWDILSP

QFMYGSKAYVKHPADIPDYLKLSFPEGF

KWERVMNFEDGGWTVTQDSSLQDGEF

IYKVKLRGTNFPSDGPVMQKKTMGWEA

S SERMYPED G ALKGEIKQRLKLKDG GH

YDAEVKTTYKAKKPVQLPGAYNVNiKL

DITSHNED YTTVEQYERAEGRH STGG MD

ELYK*

Table 3c: IL15/IL15Ra constructs

STPPTSPTT*SCPSPRASSGSA*

*TSRTAAW*P*PRTPPCRTASS

STR* SC AAPTSPPTAP* CRRRP

WAGRPPPSGCTPRTAP*RARS

SRG* S * RTA ATTTLR SRPPTRP

RSPCSCPAPTTSTSSWTSPPTT RTTPSWNST APRAATPPAA WTSCTS

IgE leader - MDWTWILFLVAAATRVHSN 1115 1121 IL15 - WVNVISDL KIEDL1QSMHID

! ink r (GS) ATLYTESDVHPSCKVTAMKC

- hDI-IFR FT I Fl . Q VI S L E S GD A S IH DT VE

(Amino acid NLIIL AN SL S SNGNVTES GC

2-187 of KE CEE LEEKNIKEFLQSF VHT V

WT, Y122T) QMFINTSGSVGSLNCIVAVSQ

- stop NMGTGKNGDLPWPPLRNEFR

YFQRMTTTSSVEGKQNLVIM

GKKTWFSIPEKNRPLKGRINL

VL SRELKEPPQGAHFL SRSLD

DALKLTEQPELANKVDMVWI

VGGSSVKEAMNHPGHL-KLF

VTRIMQDFESDTFFPEIDLEKY

KLLPEYPGVL SDVQEEKGIKY

KFEVYEKND*

mCheny - MSKGH ί)Νλ ί Λ: ΙΚ ί iM K i Y 1116 1 122 stop HMEGS VNGHE FEIE GEGEGRP

YEGTQTAKLKVTKGGPLPFA WDILSPQFMYGSKAYVKHPA DIPDYLKLSFPEGFKWERVM NFEDGGVVT VTQD S SLQDGE FIYKVKLRGTNFPSDGP Q KK TMG WE A S SERMYPED G A LK GEIKQRLKLKDGGHYD AE VK TTYKAKKP VQLP G A YN V NIKLDITSHNEDYTIVEQYER AEGRHSTGGMDELYK*

OT-IL15- Full EFla MDWTWILFLVAAATRVHSN 1117, 1 123 123 and OT- constract WVNVISDLKKiEULTQSMHID 1 141 - IL15- ATLYTESDVHPSCKVTAMKC 1149

127(IgE FLL ELQ Vi S L E S GD AS IHDT VE

leader - NLIIL AN SL S SNGNVTES GC

IL 15 - KF CEELEEKNIKEFLQSF VHIV

BamHI (GS) OMFINTSGS*NLDNTTH*RSR

- stop - PSP SPPPN VTGRSRLE* GRCAF

spacer - VYMLFSTILPSFGNVRARKPG

IRES - PWLTSIPRGLSPLAKGMQGL

spacer - LN WKE A VPLE A S *RQTT S V A

mCheny - TLCRQRNPPPGDRCLCGQKP

stop) RV*DTPAKAAQPQCHWSWI

WERVKWLS S S VFNKGLKD A

OKVPHCMGSDLGPRCTCFTC

V* SRLKKRLGPPNHGDWFL*

KTR**YGHNHDEQGRGG*HG

HHQGVHALQGAHGGLRERP

RVRDRGRGRGPPLRGHPDRQ

AEGDQGWPPALRLGHPVPSV

H VRLQGLRE APRRHPRL L E A

VLPRGLQVGARDELRGRRRG

DRDPGLLPAGRRVHLQGEAA

RHQLPLRRPRNAEEDHGLGG LLRADVPRGRRPEGRDQAEA

EAEGRRPLRR* GQDHLQGQE

ARAAARRLQRQHQVGHHLP QRGLHHRGTVRTRRGPPLHR

RHGRAVQV

igE leader - MD WTWILFL V AA ATR VH SN 1118 1124 IL15 - WVNVISDLKKIEDLIQSMHID

BamHI (GS) ATLYTESDVHPSCKVTAMKC

- stop FLLELQ VI SEES GD A S 1HDT VE

NL 1IL ANN S L S SNGN VTE S G C

KECEELEEK IKEFLQSFVHrV

QMFINTSGS*

mCheny - MSKGEEDNMAIIKEFMRFKV 1119 1125 stop HMEGSVNGHEFEIEGEGEGRP

YEGTQTAKLKVTKGGPLPFA WDILSPQFMYGSKAYVKHPA DIPDYLKL SFPEGFKWER VM NFEDGG VVTVTQD S SLQDGE FIYKVKLRGTNFPSDGPVMQ KK TMG WE A S SERMYPED G A L GE1KQRLKLKDGGHYDAE

V TTYKAKKP VQLP G A Y N V NIKLDITSH EDYTTVEQYER AEGRHSTGGMDELYK*

[00255] In one embodiment, the payload of the present invention may comprise IL18. IL18 is a proinflammatory and immune regulatory cytokine that promotes IFN-y production by T and NK cells, IL18 belongs to the ILI family. Secreted ILI 8 binds to a heterodimer receptor complex, consisting of IL18Rct and /^-chains and initiates signal transduction. ILI 8 acts in concert with other cytokines to modulate immune system, functions, including induction of IFN-y production, Till responses, and NK cell activation in response to pathogen products. ILI 8 showed anticancer effects in several tumors. Administration of recombinant IL18 protein or IL18 transgene induces melanoma or sarcoma regression through the activation of CD4⁺ T and/or NK cell- mediated responses (reviewed by Srivastava et al., ( ^'HIT Med. Chem., 2010, 17: 3353-3357). The combination of TL18 with other cytokines, such as IL12 or co-stimulatory molecules (e.g., CD80) increases IL18 anti-tumor effects. For example, ILI 8 and IL12A/B or CD80 genes have been integrated successfully in the genome of oncolytic viruses, with the aim to trigger synergistically T cell -mediated anti-tumor immune responses (Choi et al.. Gene Tker., 2011, 18: 898-909). IL2/IL18 fusion proteins also display enhanced anti-tumor properties relative to either cytokine alone and low toxicity in preclinical models (Acres et al, Cancer Res., 2005, 65:9536- 9546),

[00256] ILI 8 alone, or in combination of ILI 2 and ILLS, activates NK cells. Preclinical studies have demonstrated that adoptively transferred IL12, IL15 and ILI 8 pre-activated NK cells display enhanced effector function against established tumors in vivo (Ni et al., J Exp Med. 2012, 209: 2351-2365; and Rom.ee et ai, Blood. 2012,120:4751-4760), Human IL12/IL15/iL18 activated NK cells also display memory -like features and secrete more IFN-y in response to cytokines (e.g., low concentration of IL2). In one embodiment, the effector module of the present invention may be a DD-IL18 fusion polypeptide.

[00257] In one embodiment, the pavload of the present invention may comprise IL21. IL21 is another pleiotropic type I cytokine that is produced mainly by T cells and natural killer T (NKT) cells. IL21 has diverse effects on a variety of cell types including but not limited to CD4^j" and CDS^"1" T cells, B cells, macrophages, monocytes, and dendritic cells (DCs). The functional receptor for IL21 is composed of IL21 receptor (IL21R) and the common cytokine receptor gamma chain, which is also a subunit of the receptors for IL2, IL4, IL7, IL9 and IL15. Studies provide compelling evidence that IL21 is a promising immunotherapeutic agent for cancer immunotherapy. IL21 promotes maturation, enhances cytotoxicity, and induces production of IFN-y and perforin by NK ceils. These effector functions inhibit the growth of B16 melanoma (Kasaian et al., Immunity, 2002, I6(4):559-569; and Brady et al, J Immunol.200 , 172(4):2048- 2058). IL21 together with IL15 expands antigen -specific CDS^'"^" T-cell numbers and their effector function, resulting in tumor regression (Zeng et al., J Exp Aied.2 05, 201(1): 139-148). 1L21 may also be used to rejuvenate multiple immune effector cells in the tumor microenvironment. IL21 may also directly induce apoptosis in certain types of lymphoma such as diffuse large B-cell lymphoma, mantle cell lymphoma, and chronic lymphocytic leukemia cells, via activation of STAT3 or STAT1 signal pathway. 1L21, alone or in combination with anti-CD20 mAb

(rituximab) can activate NK cell -dependent cytotoxic effects. Interestingly, discovery of the immunosuppressive actions of IL21 suggests that this cytokine is a '"double-edged sword"- IL21 stimulation may lead to either the induction or suppression of immune responses. Both stimulatory and suppressive effects of 1L21 must be considered when using IL21 -related immunotherapeutic agents. The level of IL21 needs to be tightly controlled by regulatory elements. In one aspect, the effector module of the present invention may be a DD-IL21 fusion polypeptide.

[00258] In som e em bodiments, payloads of the present invention may comprise type I interferons. Type I interferons (IFNs-I) are soluble proteins important for fighting viral infection in humans. IFNs-I include IFN-aipha subtypes (IFN- al, I FN- alb, IFN- ale), IFN-beta, IFN- delta subtypes (IFN-delta I, IFN-delta 2, IF -delta 8), IFN-gamma, IF -kappa, and IFN- epsilon, IFN-iambda, IFN-omega, IFN-tau and IFN-zeta. IFN-a and IFN-β are the main IFN-I subtypes in immune responses. All subtypes of IFN-I signal through a unique heterodimeric receptor, interferon alpha receptor (IFNAR), composed of 2 subunits, IFNAR1 and IFNAR2. IFNR activation regulates the host response to viral infections and in adaptive immunity. Several signaling cascades can be activated by 1FNR, including the Janus activated kinase-signai transducer and activation of transcription (JAK-STAT) pathway, the mitogen activated protein kinase (MAPK) pathway, the phosphoinositide 3-kinase (ΡΪ3Κ) pathway, the v-crk sarcoma virus CT10 oncogene homolog (avian)-like (CRKL) pathway, and NF-κΒ cascade. It has long been established that type 1 IFNs directly inhibit the proliferation of tumor cells and virus- infected cells, and increase MHC class I expression, enhancing antigen recognition, IFNs-I have also proven to be involved in immune system regulation. IFNs can either directly, through interferon receptor (IFNR), or indirectly by the induction of chemokines and cytokines, regulate the immune system . Type I IFNs enhance NK cell functions and promote survival of NK cells. Type I IFNs also affect monocytes, supporting the differentiation of monocytes into DC with high capacity for antigen presentation, and stimulate macrophage function and differentiation. Several studies also demonstrate that IFNs-I promote CD8⁺ T cell survival and functions. In some instances, it may be desirable to tune the expression of Type I IFNs using biocircuits of the present invention to avoid immunosuppression caused by long-term treatment with IFNs.

[ 00259 j New anticancer immunotherapies are being developed that use recombinant type I IFN proteins, type I IFN transgene, type I IFN-encoding vectors and type I IFN-expressing cells. For example, IFN-a has received approval for treatment of several neoplastic diseases, such as melanoma, RCC and multiple myeloma. Though type I IFNs are powerful tools to directly and indirectly modulate the functions of the immune system, side effects of systemic long-term treatments and lack of sufficiently high effi cacy have dampened the interest of IFN-a for clinical use in oncology. It is believed that if IFN levels are tightly regulated at the malignant tissues, type I IFNs are likely more efficacious. Approaches for intermittent delivery are proposed according to the observation that intermittency at an optimized pace may help to avoid signaling desensitizing mechanisms (negative feedback mechanisms) induced by IFNs-I (i.e., because of SOCS 1 induction) in the responding immune cells. In accordance with the present invention, the effector module may comprise a DD-IFN fusion polypeptide. The DD and its iigand control the expression of IFN to induce an antiviral and antitumor immune responses and in the meantime, to minimize the side effects caused by long-term exposure of IFN.

|00260] In some embodiments, payloads of the present invention may comprise members of tumor necrosis factor (TNF) superfamiiy. The term ' NF superfamily" as used herein refers to a group of cytokines that can induce apoptosis. Members of TNF family include TNF-alpha, TNF- beia (also known as lymphotoxin-alpha (LT-a)), lymphotoxin-beta (LT-β), CD40L(CD154), CD27L (CD70), CD30L(CD153), FASL(CD178), 4-1BBL (CD137L), QX40L, TRAIL (TNF- related apoptosis inducing ligand), APRIL (a oliferation -inducing ligand), TWEAK, TRANCE, TALL-1, G1TRL, LIGHT and TNFSF1 to TNFSF20 (TNF ligand superfamily member 1 to 20). In one embodiment, the payload of the invention may be TNF-alpha. TNF- alpha can cause cytolysis of tumor cells, and induce cell proliferation differentiation as well, in one aspect, the effector module of the present invention may comprise a DD-TNF alpha fusion polypeptide.

[00261] In some embodiments, payloads of the present invention may comprise inhibitory molecules that block inhibitory cytokines. The inhibitors may be blocking antibodies specific to an inhibitor}' cytokine, and antagonists against an inhibitory cytokine, or the like.

[00262] In some aspects, payloads of the present invention may comprise an inhibitor of a secondary cytokine IL35. IL35 belongs to the interleukin- 12 (11/12) cytokine family, and is a heterodimer composed of the IL27 β chain Ebi3 and the IL12 a chain p35. Secretion of bioactive IL35 has been described only in forkhead box protein 3 (Foxp3) ⁺ regulatory T cells (Tregs) (resting and activated Tregs). Unlike other membranes in the family, IL35 appears to function solely in an anti-inflammatory fashion by inhibiting effector T cell proliferation and perhaps other parameters (Collison et al, Nature, 2007, 450(7169): 566- 569).

[00263] In some embodiments, payloads of the present invention may comprise inhibitors that block the transforming growth factor beta, (TGF-β) subtypes (TGF-βΙ, ΤΟΡ-β2 and ΤΟΡ-β3), TGF-β is secreted by many cell types, including macrophages and is often complexed with two proteins LTBP and LAP. Serum proteinases such as plasmin catalyze the release of active TGF-β from, the complex from, the activated macrophages. It has been shown that an increase in expression of TGF-β correlates with the malignancy of many cancers. The immunosuppressive activity of TGF-β in the tumor microenvironment contributes to oncogenesis.

[00264] In some embodiments, payloads of the present invention may comprise inhibitors of IDO enzyme.

[00265] In some embodiments, payloads of the present invention may comprise chemokines and chemokine receptors. Chemokines are a family of secreted small cytokines, or signaling proteins that can induce directed chemotaxis in nearby responsive cells. The chemokine may be a SCY (small cytokine) selected from the group consisting of SCYA.1 -28 (CCL.1-28), SCYBl -16 (CXCLl-16), SCYCl-2 (XCLl-2), SCYD-1 and SCYE-1; or a C chemokine selected from XCL1 and XCL2; or a CC chemokine selected from CCLl, CCL2, CCL3, CCL4, CCL5, CCL6, CO .?. CCL8, CCL9, CCL10, (^"CI . 1 1. CCLl 2, CCLl 3, CCL14, CCL15, CCLl 6, CCLl 7, CCLl 8, CCLl 9, CCL20, CCL21 , CCL22, CCL23, CCL24, CCL25, CCL26, CCL27 and CCL28: or a CXC chemokine selected from CXCLl, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCLl 1, CXCLl 2, CXCLl 3, CXCLl 4, CXCL15, CXCL16 and CXCL17, ^' or a CX3C chemokine CX.1CLI . In some aspects, the chemokine receptor may be a receptor for the C chemokines including XCR1; or a receptor for the CC chemokmes including CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9 and CCR10; or a receptor for the CXC chemokines including CXCR1, CXCR2, CXCR3, CXCR4 and CXCR5; or a CX3C chemokine receptor CX3CR1.

[00266] In some embodiments, payloads of the present invention may comprise other immunomodulators that play a critical role in immunotherapy, such as GM-CSF (Granulocyte- macrophage colony stimulating factor), erythropoietin (EPO), MIP3a, monocyte chemotactic protein (MCP)-l, intracellular adhesion molecule (ICAM), macrophage colony stimulating factor (M-CSF), Interleukin-1 receptor activating kinase (iRAK-1), lactotransferrin, and granulocyte colony stimulating factor (G-CSF).

[00267] In some embodiments, the payload of the present invention may comprise

Amphiregulin. Amphiregulin (AREG) is an EGF-like growth factor which binds to the EGFR receptor and enhances CD4+ regulatory T cells (Tregs) function. AREG promotes immune suppression in the tumor environment. Thus, in some embodiment, the payloads of the present invention may comprise Amhiregulin to dampen immune response during immunotherapy.

[00268] In some embodiments, payloads of the present invention may comprise fusion proteins wherein a cytokine, chemokine and/or other soluble factor may be fused to other biological molecules such as antibodies and or ligands for a receptor. Such fusion molecules may increase the half-life of the cytokines, reduce systemic toxicity, and increase local concentration of the cytokines at the tumor site. Fusion proteins containing two or more cytokines, chemokines and or oilier soluble factors may be utilized to obtain synergistic therapeutic benefits. In one embodiment, payload may be a GM-CSF/1L2 fusion protein.

3. Additional effector module features

[00269] 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 w hich 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

[00270] 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.

[00271] 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 II signal peptide peptidases. Signal sequences derived from human 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).

[00272] 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" payioad, i.e., an immunotherapeutic agent as discussed herein.

[00273| 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 comprising amino acid of SEQ ID NO: 783, encoded by the nucleotide of SEQ ID NO: 788-791 and/or p40 signal sequence comprising the amino acid sequence of SEQ ID NO: 719, encoded by the nucleotide of SEQ ID NO: 736-744,

[00274] In some instances, signal sequences directing the payioad of interest to the surface membrane of the target cell may be used. Expression of the payioad on the surface of the target cell may be useful to limit the diffusion of the payioad to non-target in vivo environments, thereby potentially improving the safety profile of the payloads. Additionally, the membrane presentation of the payioad may allow for physiologically and qualitative signaling as well as stabilization and recycling of the payioad for a longer half-life. Membrane sequences may be the endogenous signal sequence of the N terminal component of the payioad 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 pay ioad is presented on the surface of the T cell. In some embodiments, the signal sequence may be IgE signal sequence comprising amino acid SEQ ID NO: 801 and nucleotide sequence of SEQ ID NO: 810, 930, or 931, CD8a signal sequence (also referred to as CD8a leader) comprising amino acid SEQ ID NO: 628 and nucleotide sequence of SEQ ID NO: 671-675, or lL15Ra signal sequence (also referred to as ILlSRa leader) comprising ammo acid SEQ ID NO: 932 and nucleotide sequence of SEQ ID NO: 933.

[00275] 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 NO.: 20070141666; and International patent application publication NO.: WOI 993018I 81; the contents of each of which are incorporated herein by reference in their entirety.

[00276] In other examples, a signal sequence may be a heterogeneous signal sequence from other organisms such as vims, 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. NO.: 8,093,016 to Cervm and Kim), bacterial lipoprotein signal sequences (e.g., PCX application publication NO.: WG199109952 to Lau and Hioux), E.coll enterotoxm Π signal peptides (e.g., U.S. Pat. NO.: 6,605,697 to Kwon et al.), E. coli secretion signal sequence (e.g., U.S. patent publication NO.: US2016090404 to Maiiey et al.), a lipase signal sequence from a methylotrophic yeast (e.g., U.S. Pat. NO.: 8,975,041), and signal peptides for DNases derived from Coryneform bacteria (e.g., U.S. Pat. NO.: 4,965, 197); the contents of each of which are incorporated herein by reference in their entirety.

[00277] 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. NO.: 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. NO.: 7,396,81 1; and Negi et al ., Database, 2015, 1-7; the contents of each of which are incorporated herein by reference in their entirety).

[00278] In some embodiments, signal sequences of the present invention, include without limitation, any of those taught in Table 6 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 VVO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.

Cleavage sites

[00279] 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.

[00280] The effector module may include one or more cleavage signai(s)/site(s) of any proteinases. The proteinases may be a serine proteinase, a cysteine proteinase, an endopeptidase, 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, caipain- , 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, eiastase, endoproteinase, enterokinase, factor Xa, formic acid, granzyme B, Matrix metallopeptidase-2, Matrix metallopeptidase-3, pepsin, proteinase K, SUMO protease, subtilisin, TEV protease, thermolysin, thrombin, trypsin and TAGZyme.

[00281] In one embodiment, the cleavage site is a furin cleavage site comprising the amino acid sequence SARNRQKRS (SEQ ID NO: 721), encoded by nucleotide sequence of SEQ ID NO: 750; or a revised furin cleavage site comprising the amino acid sequence ARNRQKRS (SEQ ID NO: 722), encoded by nucleotide sequence of SEQ ID NO: 751; or a modified furin site comprising the amino acid sequence ESRRVRRNKRSK (SEQ ID NO: 630), encoded by nucleotide sequence of SEQ ID NO: 681 -683.

[00282] 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 4/1 1 /2016, or in US Provisional Application No.

62/466,596 filed March 3, 2017 and the International Publication WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.

Protein tags

[00283] 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 numbe 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 halotag7), 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), biolumine scent tags (e.g. luciferase and its variants), affinity tags (e.g., maltose-binding protein (MBP) tag, glutathione-S-transferase (GST) tag), immunogenic affinity tags (e.g., protein A/G, IRS, AUI , AU5, glu-glu, KT3, S-tag, HSV, VSV-G, Xpress and V5), and other tags (e.g., biotin (small molecule), StrepTag (StrepII), SBP, biotin carboxyl carrier protein (BCCP), eXact, CBP, CYD, UPC, CBD mtein-chitm binding domain, Trx, ΝοφΑ, and NusA.

|0Θ284] 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.: US2013115635 and US2013012687; and International application publication NO.: WO2013091661 ; the contents of each of which are incorporated herein by- reference in their entirety.

[00285] 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.

[00286] In some embodiments, protein tags of the present invention, include w ithout limitation, any of those taught in Table 8 of copending commonly owned U.S. Provisional Patent

Application No. 62/320,864 filed on 4/1 1 /2016, or in US Provisional Application No.

62/466,596 filed March 3, 2017 and the International Publication WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.

Targeting peptides

[00287] 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 ceil 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.

[00288] 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.

[00289] 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 5 or fewer.

[00290] 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, 1 10,059; 8,706,219; and 8,772,449; and U.S. application publication NOs. : 2016089447; 2016060296; 2016060314; 2016060312; 201606031 1 ;

2016009772; 2016002613; 2015314011 and 2015166621; and international application publication NOs.; WO2015179691 and WO2015183044; the contents of each of which are incorporated herein by reference in their entirety.

[00291] 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, or in US Provisional Application No.

62/466,596 filed March 3, 2017 and the International Publication WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.

Linkers

[00292] 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.

[00293] 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 ammo acids in length. Linker length may also be optimized depending on the type of payioad utilized and based on the crystal structure of the payioad. 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), Isoleucme (1), Serine (S), Cysteine (C), Threonine (T), Methionine (M), Proline (P), Phenylalanine (F), Tyrosine (Y), Tryptophan (W), Histidine (H), Lysine (K), Arginine ( ), 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).

[00294] 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. Tire 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: 822: encoded by the nucleotide sequence SEQ ID NO: 823), GGSGG (SEQ ID NO: 629; encoded by any of the nucleotide sequences SEQ ID NO: 676-680), GGSGGG (SEQ ID NO: 824; encoded by any of the nucleotide sequences SEQ ID NO: 825-826), SGGGS (SEQ ID NO: 827; encoded by the nucleotide sequence SEQ ID NO: 828, 844, 909), GGSGGGSGG (SEQ ID NO: 829; encoded by the nucleotide sequence SEQ ID NO: 830), GGGGG (SEQ ID NO: 831), GGGGS (SEQ ID NO: 832) or (GGGGS)ii (n=l (SEQ ID NO: 832), 2 (SEQ ID NO: 833), 3 (SEQ ID NO: 720, encoded by the nucleotide sequence SEQ ID NO: 910-915), 4 (SEQ ID NO: 834), 5 (SEQ ID NO: 835), or 6 (SEQ ID NO: 836)), SSSSG (SEQ ID NO: 837) or (SSSSG)n (n=l (SEQ ID NO: 837), 2 (SEQ ID NO: 838), 3 (SEQ ID NO: 839), 4 (SEQ ID NO: 840), 5 (SEQ ID NO: 841), or 6 (SEQ ID NO: 842)),

SGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 802; encoded by the nucleotide sequence SEQ ID NO: 811, 916-920, 1002), EFSTEF (SEQ ID NO: 784; encoded by any of the nucleotide sequences SEQ ID NO: 792-793), GKSSGSGSESKS (SEQ ID NO: 845),

GGSTSGSGKS SEGKG (SEQ ID NO: 846), GSTSGSGKSSSEGSGSTKG (SEQ ID NO: 847), GSTSGSGKPGSGEGSTKG (SEQ ID NO: 848), VDYPYDVPDYALD (SEQ ID NO: 849; encoded by nucleotide sequence SEQ ID NO: 850), EGKSSGSGSESKEF (SEQ ID NO: 851), SGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGS (SEQ ID NO: 921 ; encoded by SEQ ID NO: 923 SGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 922; encoded by SEQ ID NO: 924), GS (encoded by GGTTCC), SG (encoded by AGCGGC), GSG (encoded by

GGATCCGGA or GGATCCGGT), or MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 1031; encoded by SEQ ID NO: 1032).

[00295 j 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 polyglycmes (such as (G)4 (SEQ ID NO: 1233), (G)5 (SEQ ID NO: 831), (G)8) (SEQ ID NO: 1234), poly(GA), and polyaianines. The linkers described herein are exemplary, and linkers that are much longer and which include other residues are contemplated by the present invention. |00296] 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 protem.

[00297] 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, chymotrypsin, ciostripain, enterokinase, Factor Xa, glutamyl endopeptidase, Granzyme B, Achromobacter proteinase I, pepsin, proline endopeptidase, proteinase K, Staphylococcal peptidase I, thermolysin, thrombin, trypsin, 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-nitrophenylsulfenyl)-3-methylindole), which cleaves tryptophan residues; dilute acids, which cleave at aspartyl-prolyl bonds; and e aspartic acid- proline add 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.

[00298] In other embodiments, a cleavable linker may be a "self-cleaving" Sinker 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 picomavirai 2A-like linker, CHYSEL sequences of porcine tescho virus (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. 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 viras that is recognized by a protease (2A peptidases) endogenous to the cell. The 2A peptide was identified among picornaviruses, a typical 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 Picornaviridae like equme rhinitis A viras, 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-transiationai cleavage of tins polyprotein at a single site that forms the junction between the viras capsid and replication polyprotein domains. The 2A sequences contain the consensus motif D-V/I-E-X-N-P-G-P (SEQ ID NO: 1235). 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 rem ain s fused to the C -terminus of th e 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 2 A peptides identified to date, four have been widely used namely FMDV 2 A (abbreviated herein as F2A); equine rhinitis A vims (ERAV) 2A (E2A); porcine teschovims-1 2A (P2A)

and Thoseaasigna vims 2A (T2A). In some embodiments, the 2 A peptide sequences useful in the present invention are selected from SEQ ID NO.8-1 1 of International Patent Publication WO2010042490, the contents of which are incorporated by reference in its entirety.

[00299] As a non-limiting example, the P2A cleavable peptide may be

GATNFSLLKQAGDVEENPGP (SEQ ID NO: 925; encoded by SEQ ID NO: 926).

[00300] The linkers of the present invention may also be non-peptide linkers. For example, alkyi linkers such as— H— (CKb) a-C(O)— , 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-Ce) lower acyl, halogen (e.g., CI, Br), CN, NH2, phenyl, etc.

[00301] 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.: WO2012/083424; the contents of each of which are incorporated herein by reference in their entirety.

[00302] 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 WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.

[00303] 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: 927), TATGGCCACAACCATG (SEQ ID NO: 928),

AATCTAGATAATACGACTCACTAGAGATCC (SEQ ID NO: 929),

GCTTGCCACAACCCACAAGGAGACGACCTTCC (SEQ ID NO: 1000), TCGCGAATG, or TCGCGA.

[00304] In one embodiment, the linker may be a BamHI site. As a non-limiting example, the BamHI site has the amino acid sequence GS and/or the DN A sequence GGATCC.

Embedded stimulus, signals and other regulatory- features |0030S] 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.

[00306] 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 WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.

[00307| 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. Exemplar}' 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.

Polynucleotides

[00308] 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. Exemplar ' 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 (PN As), locked nucleic acids (LN As, including L A having a β- D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-ammo-LNA having a 2'-amino functionalization, and 2'-amino- a-LNA having a 2'-amino functionalization) or hybrids thereof. |00309] 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 polypeptide 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.

|00310] 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 stracture, 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 stmctural 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 modifi cation, structural modifi cations are of a chemical nature and hence are chemical modifications. However, stmctural 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.

[00311 ] 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.

[00312] 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 stracture in the polynucleotide. An IRES 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.

[00313] In some embodiments, polynucleotides encoding biocircuits, effector modules, SREs and pay!oads of interest such as immunotherapeutic 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.

[00314] 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).

[00315] In som e em bodiments, 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 pavloads 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.

[00316] 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.)

[00317] In some embodiments, polynucleotides of the present invention may be modified. As used herem, 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: WO2013052523, the contents of which are incorporated herein by reference in its entirety.

[00318] 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 pyrimidme) or a derivative thereof (also referred to herein as "nucleobase"). As described herein, "nucleotide" is defined as a nucleoside including a phosphate group.

[00319] In some embodiments, the modification may be on the internucleoside 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, aikyl or aryl phosphonates, and phosphotriesters. Phosphorodithioat.es 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~phosphonat.es). Other modifications which may be used are taught in, for example, International Application NO: WO2013052523, the contents of which are incorporated herein by reference in their entirety.

[00320] Chemical modifications and/or substitution of the nucleotides or nucieobases 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)-l-(2- Hydroxypropyl)pseudouridine TP, l-(4-Methoxy-phenyl)pseudo-UTP,,2'-0-dimethyladenosine, l,2'-0-dimethylguanosine, l,2'-0-dimethylinosine, 1-Hexyl-pseiido-UTP, 1- Homoallylpseudouridine TP, 1-Hydroxymethylpseudouridine TP, 1-iso-propyl-pseudo-UTP, 1- Me-2-thio-pseudo-UTP, l-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-GTP, 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-1hio-cytidine, a-thio- guanosine, and/or a-thio-uridine.

[00321] 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 poly nucleotide 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 polynucleotide 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 l% to 70%, from l% to 80%, from l% to 90%, from l% 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%).

[00322] 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 tR A anticodon pools tend to vary among organisms, cell types, sub cellular locations and over time, the codon selection described herein is a spatiotemporal (ST) codon selection.

[00323] 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 ceil 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/codon/) and the CAI can be calculated by EMBOSS CAI program (http://emboss.sourceforge.net/). Codon optimization metliods 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 polynucleotide. Codon optimization tools, algorithms and services are known in the art, and non-limiting examples include sendees from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA), OptimumGene (GenScript,

Piscataway, NJ), algorithms such as but not limited to, DNAWorks v3.2.3 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. |00324] 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).

[00325] After optimization (if desired), the polynucleotide components are reconstituted and transformed into a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes.

[00326] 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, 1 1 11-1124; 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.

[00327] 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).

[00328] 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 read:!; rough, 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.

[00329] 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.

[00330] 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 AB AB AB 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.

[00331] 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, alky], 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 hormone receptors, non-peptidic species, such as lipids, lectins,

carbohydrates, vitamins, cofactors, or a drag. As non-limiting examples, they may be conjugates with other immune conjugates. [00332] In some embodiments, the compositions of the polynucleotides of the invention may 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 polymerase 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 are performed prior to Gibson assembly which 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 a method 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).

[00333] 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 NO: 684-686, 688-691 , 987-989, 994, 1013, and/or 1028, an ecDHFR mutant encoded by nucleotide sequence of SEQ ID NO: 687, 692, 772, 798, 814-815, 988, 991, and/or 993, hDHFR mutant encoded by nucleotide sequence of SEQ ID NO: 693-700, 773, 852-857 and/or 934-980, and/or 995-998.

[00334] In some embodiments, the polynucleotides of the invention may encode effector modules comprising the CD 19 CAR as the payload comprising the nucleotide sequence of SEQ ID NO: 701-715 and/or 1019-1042, or 1LI2 as the payload comprising the nucleotide sequence of SEQ ID NO. 774-782, or IL15 as the payload comprismg the nucleotide sequence of SEQ ID NO: 749, 799-800, and/or 1055-1056, or IL15/IL15Ra fusion polypeptide as the payload comprising the nucleotide sequence of SEQ ID NO: 816-821, 1086-1089, 1091-1095, 1098- 1111, 1120, and/or 1123.

Cells

[00335] 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 inv ention, 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 ceils (e.g., Thl, Th2, Thl7, Foxp3+ celis), 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) ceils, NK T ceils, tumor infiltrating lymphocytes (T^'iLs), 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 Ταβ cells and Τγδ 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 m their entirety).

[00336] In some embodiments, cells of the invention may be autologous, allogeneic, syngeneic, or xenogeneic in relation to a particular individual subject.

[00337] In some embodiments, cells of the invention may be mammalian cells, particularly- human cells. Ceils of the invention may be primary cells or immortalized cell lines.

[00338] In some embodiments, cells of the invention may include expansion factors as payload to trigger proliferation and expansion of the cells. Exemplary payloads include FAS such as KRAS, NRAS, RRAS, RRAS2, MRAS, ERAS, and HRAS, D1RAS such as DIRAS 1, DIRAS2, and DIRAS3, NKIRAS such as NKIRAS 1, and NKIRAS2, RAL such as RALA, and RALB, RAP such as RAP1A, RAP1B, RAP2A, RAP2B, and RAP2C, RASD such as RASD l, and RASD2, RASL such as RASL10A, RASL10B, RASL11 A, RASL11B, and RASL12, REM such as REM1, and REM2, GEM, RERG, RERGL, and RRAD.

[00339] 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., immunotherapeutic 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 inv ention 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 immunotherapeutic 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.

[00340] In some embodiments, immune cells of the invention may be T cells modified to express an antigen-specific T cell receptor (TCR), or an antigen specific chimeric antigen receptor (CAR) taught herein (known as CAR T cells). Accordingly, at least one polynucleotide encoding a CAR system (or a TCR) described herein, or a vector comprising the polynucleotide is introduced into a T cell. The T cell expressing the CAR or TCR binds to a specific antigen via the extracellular targeting moiety of the CAR or TCR, thereby a signal via the intracellular signaling domain (s) is transmitted into the T cell, and as a result, the T cell is activated. The activated CAR T cell changes its behavior including release of a cytotoxic cytokine (e.g., a tumor necrosis factor, and lymphotoxin, etc.), improvement of a cell proliferation rate, change in a cell surface molecule, or the like. Such changes cause destruction of a target cell expressing the antigen recognized by the CAR or TCR. In addition, release of a cytokine or change in a cell surface molecule stimulates other immune cells, for example, a B cell, a dendritic cell, a NK cell, and a macrophage.

[00341] The CAR introduced into a T cell may be a first-generation CAR including only the intracellular signaling domain from TCR CD3zeta, or a second-generation CAR including the intracellular signaling domain from TCR CDSzeta and a costimulatory signaling domain, or a third-generation CAR including the intracellular signaling domain from TCR CDSzeta and two or more costimulatory signaling domains, or a split CAR system, or an on/off switch CAR system. In one example, the expression of the CAR or TCR is controlled by a destabilizing domain (DD) such as a hDHFR. mutant, in the effector module of the invention. The presence or absence of hDHFR binding ligand such as TMP is used to tune the CAR or TCR expression in transduced T ceils or NK cells.

[00342] In some embodiments, CAR T cells of the invention may be further modified to express another one, two, three or more immunotherapeutic agents. The immunotherapeutic agents may be another CAR or TCR specific to a different target molecule; a cytokine such as IL2, 11. 12. IL15 and IL18, or a cytokine receptor such as IL15Ra; a chimeric switch receptor that converts an inhibitory signal to a stimulatory signal; a homing receptor that guides adoptively transferred cells to a target site such as the tumor tissue; an agent that optimizes the metabolism of the immune cell; or a safety switch gene (e.g., a suicide gene) that kills activated T ceils when a severe event is observed after adoptive cell transfer or when the transferred immune ceils are no- longer needed. These molecules may be included in the same effector module or in separate effector modules.

[00343] In one embodiment, the CAR T cell (including TCR T cell) of the invention may be an "armed" C AR T cell which is transformed with an effector module comprising a CAR and an effector module comprising a cytokine. The inducible or constitutive!}' secrete active cytokines furtlier armor CAR T ceils to improve efficacy and persistence. In this context, such CAR T cell is also referred to as "armored CAR T ceil". The "armor" molecule may be selected based on the tumor microenvironment and other elements of the innate and adaptive immune systems. In some embodiments, the molecule may be a stimulatory factor such as IL2, IL12, IL15, IL18, type I IFN, CD40L and 4-lBBL which have been shown to further enhance CAR T cell efficacy and persistence in the face of a hostile tumor microenvironment via different mechanisms (Y eku et al, Biochem Soc Trans., 2016, 44(2): 412-418).

[00344] In some aspects, the armed CAR T cell of the invention is modified to express a CD 1 CAR and IL12. Such T cells, after CAR mediated activation in the tumor, release inducible IL12 which augments T-ceil activation and attracts and activates innate immune ceils to eliminate CD 19-negative cancer cells.

[00345] In one embodiment, T cells of the invention may be modified to express an effector module comprising a CAR and an effector module comprising a suicide gene.

[00346] In one embodiment, the CAR T cell (including TCR T cell) of the invention may be transformed with effector modules comprising a cytokine and a safety switch gene (e.g., suicide gene). The suicide gene may be an inducible caspase such as caspase 9 which induces apoptosis, when activated by an extracellular stimulus of a biocircuit system. Such induced apoptosis eliminates transferred cell as required to decrease the risk of direct toxicity and uncontrolled cell proliferation.

[00347] In some embodiments, immune cells of the invention may be NK cells modified to express an antigen-specific T cell receptor (TCR), or an antigen specific chimeric antigen receptor (CAR) taught herein.

[00348] 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 CDS (T-cell co-receptor). NK cells are potent effector ceils 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.

[00349] Several pre-clinica3 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 (Seller et al., Immunotherapy, 2011, 3: 1445-1459). Adoptive transfer of NK cells expressing CAR such as DAP12-Based Activating CAR revealed improved eradication of tumor cells (Topfer et al., J Immunol, 2015; 194:3201-3212). NK cell engineered to express a CS-1 specific CAR also displayed enhanced cytolysis and interferon-γ (IFN-γ) production in multiple myeloma (Chu et al., Leukemia, 2014, 28(4): 917-927). [00350| NK cell activation is characterized by an array of receptors with acti vating 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 thai are generated from activating receptors and inhibitory receptors mainly determines the immediate cytotoxic activation.

[00351] NK cells may be isolated from peripheral blood mononuclear cells (PBMCs), or derived from human embryonic stem (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 ILLS, IL21, IL2, 41BBL, 11.12. IL18, MICA, 2B4, LFA-1, and BCM1/SLAMF2 (e.g., US patent publication NO: US20150190471 ).

[00352] Immune cells expressing effector modules comprising a CAR and/or other

immunotherapeutic agents can be used as cancer immunotherapy. The immunotherapy comprises the cells expressing a CAR and/or othe immunotherapeutic agents as an active ingredient, and may further comprise a suitable excipient. Examples of the excipient may include the aforementioned pharmaceutically acceptable excipients, including various cell culture media, and isotonic sodium chloride.

[00353] In some embodiments, cells of the present invention may be dendritic cells that are genetically modified to express the compositions of the invention. Such cells may be used as cancer vaccines.

Methods of CD 19 antibody development and characterization

[00354] In some embodiments, the present invention provides methods of producing CD 19 antibodies, antibody fragments or variants. Such methods may include the steps of: (1) preparing a composition with CD 19, (2) contacting a library of antibodies or antibody fragments or variable with the composition, and (3) identifying one or more CD19 antibodies. Also, provided herein are methods for identifying FMC63-distinct CD 19 antibodies, antibody fragments or variable. [ 00355 j In some embodiments, the present invention pro vides methods of identifying CD 19 scFvs. Such methods may involve screening phagemid libraries for CD 19 scFvs. Phagemid libraries expressing recombinant scFvs associated with the surface of bacteria or bacteriophages are useful in the present inventions. Phagemid libraries may be generated by PCR implication of the polynucleotides encoding the heavy chain and the kappa light chain of the immunoglobulin IgM and infecting Cre recombinase positive bacteria with the vectors containing the PCR products at a high multiplicity of infection (MOI). The high MOI results in bacteria containing multiple phagemids, each of which encodes a different VH and VL genes, which can be recombined by the Cre recombinase. The resulting library that may be generated by

recombination is approximately 10 ^s unique scFvs. In some instances, libraries of CD 19 scFvs formatted into chimeric antigen receptor constructs may be screened to identify CD19scFvs useful in the present invention.

[00356] In some embodiments, scFvs immunologically specific to CD 19 may be identified using cells that ectopically express full length, a fragment or a portion of CD19. Cell lines with low endogenous CD 19 expression may be selected for ectopic expression. In some

embodiments, the CD 19 may be a naturally occurring isoform of human CD 19.

[00357] In some embodiments, fusion proteins comprising the extracellular domains of CD 19 (i.e. exon 1- exon 4) fused to the Fc region of human IgGl (CD 19sIg) are utilized to identify CDI9 specific scFvs. Such fusion proteins have been described by Oliveira et ai (2013) Journal of Translational Medicine 11 :23; the contents of which are incorporated herein by reference in their entirety.

[00358] Also, provided herein are methods to identify FMC63-distinct scFvs, which include scFvs that are immunologically specific to and bind to an epitope of the CD 19 antigen that is different or unlike the epitope of CD 19 antigen that is bound by FMC63. In some embodiments, FMC63-distinct scFvs are identified by screening the scFv library with a complex consisting of human CD 19 bound to FMC63. The CD 19 of Rhesus macaque (Macaca mulatto.) herein referred to as Rhesus CD 19, bears 88% homology to the human CD 19. Despite this high degree of homology, the Rhesus CD 19 is not recognized by FMC63, indicating that the FMC63 epitope is in the region of human CD 19 that is non-homologous to Rhesus CD19. Thus, in some embodiments, Rhesus CDI9 may be used to screen scFv libraries for FMC63-distinct scFvs. Mutations in the region of Rhesus CD 19 that is non-homologous to the human CD 19 have been previously utilized to identify residues of human CD 19 that confer binding to FMC63

(Sommermeyer et al. (2017) Leukemia Feb 16. doi: 10.1038/leu.2017.57). In some

embodiments, the mutational analysis described by Sommenneyer et ai. may be utilized to design human CD 19 mutants that are unable to bind to FMC63. Such mutants may include human CD19 (H218R, A237D, M243V, E244D, P250T) and human CD19 (H218R, A237D) and may be utilized to screen scFv libraries for FMC63-distinct scFvs. Sotillo et al have identified a splice variant of human CD 19 lacking exon 2 in cancer patients (Sotillo et al. (2015) Cancer Discov. 2015 Dec;5(12): 1282-95). The splice variant lacking exon 2 is not recognized by FMC63 and may also be used to screen scFv libraries for FMC63 -distinct scFvs.

|0Θ359] CD19 IgG fusion molecules generated by fusing the Fc region of human IgGl with the human CD19-compiete extracellular domains, i.e., exons 1-4 (CD19sIgGl-4) or extracellular domains lacking exon 2, i.e., exons 1 , 3 and 4 (CD19sIgGl,3,4) may also be utilized to screen scFv libraries for FMC63 -distinct scFvs.

[00360] CD19 proteins, variants and mutants useful in the invention are provided in Table 14.

Table 4: CD19 proteins, variants and mutants

DRDMWVVDTGLLLTRATAQDAGKYYCHRGNWTKSFYL

EITARPALWHWLLRIGGWKVPAVTLTYLIFCLCSLVGILQ

LORAI^TRR RKRM'roPTRRFF VTPPPGSGPQNQYGNV

LSLPTPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQVDGA

VGSR SPPGAGPEE EEGEGYE EPD SEEG SEF YEND SNFGQD

QL SQD GS G YE NPEDEPL GPEDED SF SN AE S YENEDE ELTQ

PVARTMDFLSPHGSAWDPSREATSLGSQSYEDMRGLLYA

APQLRTIRGQPGP HEEDADSYENMDNPDGPD AWGGGG

RMGTWSAR

Human CD19 (H218R, MPPPRLI^FLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLK 861 A237D, M.243V, E244D, GTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGIHMRPLAIW P250T) LFIFNVSQQMGGFYLCQPGPPSF AWQPGWTWVEGSGE

LFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVW

AKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLS

CGVPPDSVSRGPLSWTHVRP GP ST T .SLEL DDRPDRD

MWVVDTGLLLTRATAQDAGKYYCHRGNLT SFHLEITA

RPVLWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQR

ALVLRRKRKRM DPTRRFFKVTPPPGSGPQNQYGNVLSLP

TPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSR

SPPGVGPEEEEGEGYEEPDSEED SEFYEND SNL GQDQLSQ

DGSGYE-NPEDEPLGPEDEDSFSNAESYENEDEELTQPVAR

TMDFL SPHG S A WDP SRE ATSL AG SQ S YEDMR GIL Y A APQ

LRSIRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRM

GTWSTR

Human CD! 9 (H238R, PPPRLLFFLLFI_^TPMEVRPEEPLVVKVEEGDNAVLQCLK 862 A237D) GTSDGPTQQLTWSRESPLKPFLKLSLGLPGLG1HMRPLAIW

LFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGE

LFRWNVSDLGGLGCGL NRSSEGPSSPSGKLMSPKLYVW

AKDRPEIWEGEPPCLPPRD S LNQSL SQDLTM APGSTLWLS

CGVPPD SVSRGPLSWTH VRPKGPK SLL SLELKDDRPDRD

MWVMF GT T J -PRAT AQDAGKYYCHRGNLTMSFHLF.rr A

RPVTWHWLLRTGGWKVSAVTLAYLIFCLCSLVGILHLQR

ALVLRRKRKRMTDPTORFFKVTPPPGSGPQNQYGNVLSLP

TPTSGLGRAQRWAAGLGGTAPSYGNPSSDVQADGALGSR

SPPGVGPEEEEGEGYEEPDSEEDSEFYENDSNL GQDQLSQ

DG S GYENPEDEPL GPEDED SF SN AE S YENEDEELTQP VAR

TMDFL SPHG S AWDP SRE ATSL AG SQ S YEDMRGIL Y A APQ

LRSLRGQPGPNHEEDADSYENMDNPDGPDPAWGGGGRM

GTWSTR

Human C D 1 (Delta MPPPRLLFFLLFLTPMEVRPEEPLVVKVEGELFRWNVSDL 863 exon 2) GGLGCGLKNRSSEGPSSPSGKLMSPKLYVWAKDRPEIWE

GEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGVPPDSVS

RGPL S WTH VHPKGPK SLL SLELKDDRP ARDMW VMETGL

LLPRATAQDAGKYYCHRGNLTMSFHLEITARPVLWHWLL

RTGGWKVSAVTLAYLIFCLCSLVGILHLQRALVLRRKRKR

MTDPTRRFFKVTPPPGSGPQNQYGNVLSLPTPTSGLGRAQ

RWAAGLGGTAPSYGNPSSDVQADGALGSRSPPGVGPEEE

EGEGYEEPDSEEDSEFYENDSNLGQDQLSQDGSGYENPED

EPL GPEDED SF SN AE S YENEDEELTQP VARTMDFL SPHG S

AWDPSREATSLGSQSYEDMRGILYAAPQLRSIRGQPGPNH

EEDADSYENMDNPDGPDPAWGGGGRMGTWSTR

Human CD 19 (Exon I- MPPPRLLFFLLFLTPMEVRPEEPLVVKVEEGDNAVLQCLK 864 4) GTSDGPTQQLTWSRESPLKPFLKLSLGLPGLGEHMRPLAIW

LFIFNVSQQMGGFYLCQPGPPSEKAWQPGWTVNVEGSGE

LFRWNVSDLGGLGCGLKNRSSEGPSSPSGKLMSPKLYVW

AKDRPEIWEGEPPCLPPRDSLNQSLSQDLTMAPGSTLWLS

CGVPPDSVSRGPLSWTHVHPKGPKSLLSLELKDDRPARD

MWVMETGLLLPRATAQDAGKYYCHRGNLTMSFHLEITA

RP Human CD 19 (Exon PPRLLFFLLFLTP IEVRPEEPLVVKVEGELFRWNVSDL 865

13,4) GGLGCGLKN SSEGPSSPSGKLMSPKLYVWAKDRPEIWE

GEPPCLPPRDSLNQSLSQDLTMAPGSTLWLSCGYPPDSVS RGPLSWTHVHPKGPKSLLSLEL DDRPARDMWVMETGL LLPRATAQDAGKYYCHRGNLTMSFHLEITARP

III. PHARMACEU TICAL COMPOSITIONS AND FORMULATIONS

[00361] 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 oilier components of the invention, and optionally at least one pharmaceutically acceptable excipient or inert ingredient.

[00362] 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 contain s 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.

[00363] 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.

[00364] 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.

[00365] 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 acti ve 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.

[00366] 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), catiomc lipids and combinations thereof.

[00367] 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 PEGyiated lipids. In another aspect, the lipid may be a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC 3 -DMA, DLin-KC2-DMA and DODMA,

[00368] 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.

[00369] 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.

[00370] 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 admin stration 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.

[00371 j 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.

IV. APPLICATIONS

[00372] In one aspect of the present invention, methods for reducing a tumor 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 ceil modified to express the biocircuit, effector module, DD, and payload of interest (i.e., immunotherapeutic agent).

[00373] 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 phannaceuticai 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). [00374| 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.

1. Adoptive cell transfer (adoptive immunotherapy)

[00375J In some embodiments, cells winch 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 CD 19 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, Ciirr. Opin. Immunol. 2009, 21 : 233- 240).

[00376] 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 effect CAR therapies, in the manipulation or regulation of TILs, in allogeneic ceil therapy, in combination T cell therapy with other treatment lines (e.g. radiation, cytokines), to encode engineered TCRs, or modified TCRs, or to enhance T cells other than TCRs (e.g. by introducing cytokine genes, genes for the checkpoint inhibitors PD1, CTLA4).

[00377] 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 ceils within the subject.

[00378] 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 IL12 and IL15 as payload using SREs of the present invention to reduce the need for preconditioning (Pengram et al. (2012) Blood 119 (18): 4133- 41; the contents of which are incorporated by reference in their entirety).

[00379] 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, memory T cells, regulatory T cells (Tregs), helper T cells, cytokine -induced killer (CIK) cells, and any combination thereof. In other embodiments, immune stimulatory cells for ACT may be generated from embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC). In some embodiments, autologous or allogeneic immune cells are used for ACT.

[00380] In some embodiments, cells used for ACT may be T cells engineered to express CARs comprising an antigen-binding domain specific to an antigen on tumor cells of interest. In other embodiments, cells used for ACT may be NK cells engineered to express CARs comprising an antigen-binding domain specific to an antigen on tumor cells of interest. In addition to adoptive transfer of genetically modified T cells (e.g., CAR T cells) for immunotherapy, alternate types of CAR-expressing leukocytes, either alone, or in combination with CAR T cells may be used for adoptive immunotherapy. In one example, a mixture of T cells and NK cells may be used for ACT. The expression level of CARs in T cells and NK ceils, according to the present invention, is tuned and controlled by a small molecule that binds to the DD(s) operably linked to the CAR in the effector module.

[00381] In some embodiments, the CARs of the present invention may be placed under the transcriptional control of the T cell receptor alpha constant (TRAC) locus in the T cells to achieve uniform CAR expression while enhancing T cell potency. The TRAC locus may be disrupted using the CRISPR/Cas 9, zinc finger nucleases (ZFNs), TALENs followed by the insertion of the CAR construct. Methods of engineering CAR constructs directed to the TRAC locus are described in Eyquem J. et al (2017) Nature.543(7643): 1 13-117 (the contents of which are incorporated herein by reference in their entirety).

[00382] 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 lFN-γ, 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).

[00383] Other examples of genetic modification may include the introduction of chimeric antigen receptors (CARs) and the down-regulation of inhibitory NK cell receptors such as NKG2A.

[00384] 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.

[00385] 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 No.: US20160348072A1 and International Patent Publication NO: WO2016168595 Al ; 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 NO.: US20150152387A 1, U.S. Patent NO.: 7,435, 596; and Oy er, I.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 IL 15, IL21 , IL12 and 4-1BBL.

[00386] In some instances, sub populations of immune cells may be enriched for ACT. Methods for immune cell enrichment are taught in International Patent Publication NO.:

WO2015039100A1. 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. NO.: 9,512,401 (the content of each of which are incorporated herein by reference in their entirety).

[00387] In some embodiments, immune cells for ACT may be depleted of select sub populations to enhance T ceil expansion. For example, immune ceils may be depleted of Foxp3+ T lymphocytes to minimize the ant-tumor immune response using methods taught in US Patent Publication NO.: US 20160298081A1; the contents of which are incorporated by reference herein in their entirety. |00388] In some embodiments, activation and expansion of T ceils 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 NO.: WO2017015427, the content of which are incorporated herein by reference in their entirety.

[00389] 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.

[00390] 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 (ATOs) described by Sect CS et al. 2017. Nat 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 iigand (DLL1 ). 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.

[00391] 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 ceils 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.

[00392] In some embodiments, the multiple immunotherapeutic agents introduced into the immune ceils for ACT (e.g., T cells and NK ceils) may be controlled by the same biocircuit system. In one example, a cytokine such as IL12 and a CAR construct such as CD 19 CAR are linked to the same hDHFR destabilizing domain. The expression of IL12 and CD19 CAR is tuned using TMP simultaneously. In other embodiments, the multiple immunotherapeutic agents introduced into the immune cells for ACT (e.g., T ceils and NK ceils) may be controlled by different biocircuit systems. In one example, a cytokine such as IL12 and a CAR construct such as CD 19 CAR are linked to different DDs in two separate effector modules, thereby can be timed separately using different stimuli. In another example, a suicide gene and a CAR construct may be linked to two separate effector modules.

[00393] 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.

[00394] 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. The immunotherapeutic agents may be a second CAR or TCR specific to a different target molecule; a cytokine or a cytokine receptor; a chimeric switch receptor that converts an inhibitory signal to a stimulatory signal; a homing receptor that guides adoptively transferred cells to a target site such as the tumor tissue; an agent that optimizes the metabolism of the immune cell; 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.

[00395] 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 and IL15) into immune cells to promote immune cell proliferation and survival . Transduction of cytokine genes (e.g., gamma-cytokines IL2 and IL15) into cells will be able to propagate immune cells without addition of exogenous cytokines and cytokine expressing NK ceils have enhanced tumor cytotoxicity.

[00396] 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 ceil activation. T ceil 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.

[00397] In some embodiments, the biocircuits, and their components may be utilized to prevent T cell exhaustion in the context of Chimeric Antigen Receptor - T cell therapy (CAR-T). In this context, exhaustion in some instances, may be caused by the oligomerization of the scFvs of the CAR on the cell surface which leads to continuous activation of the intracellular domains of the CAR. As a non-limiting example, CARs of the present invention may include scFvs that are unable to oligomerize. As another non-limiting example, CARs that are rapidly internalized and re-expressed following antigen exposure may also be selected to prevent chronic scFv oligomerization on cell surface. In one embodiment, the framework region of the scFvs may be modified to prevent constitutive CAR signaling (Long et al. 2014. Cancer Research. 74(19) S I: the contents of which are incorporated by reference in their entirety). Tunable biocircuit systems of the present invention may also be used to regulate the surface expression of the CAR on the T cell surface to prevent chronic T cell activation. The CARs of the invention may also be engineered to minimize exhaustion. As a non-limiting example, the 41-BB signaling domain may be incorporated into CAR design to ameliorate T cell exhaustion. In some embodiments, any of the strategies disclosed by Long H A et al. may be utilized to prevent exhaustion (Long A H et al. (2015) Nature Medicine 21, 581-590; the contents of which are incorporated herein by reference in their entirety).

[00398] In some embodiments, the tunable nature of the biocircuits of the present invention may be utilized to reverse human T cell exhaustion observed with tonic CAR signaling. Reversibly silencing the biological activity of adoptively transferred cells using compositions of the present invention may be used to reverse tonic signaling which, in turn, may reinvigorate the T cells. Reversal of exhaustion may be measured by the downregulation of multiple inhibitory receptors associated with exhaustion.

[00399] In some embodiments, T cell metabolic pathways may be modified to diminish the susceptibility of T cells to exhaustion. Metabolic pathways may include, but are not limited to glycolysis, urea cycle, citric acid cycle, beta oxidation, fatty acid biosynthesis, pentose phosphate pathway, nucleotide biosynthesis, and glycogen metabolic pathways. As a non-limiting example, payloads that reduce the rate of glycolysis may be utilized to restrict or prevent T cell exhaustion (Long et al. Journal for Immunotherapy of Cancer 2013, l(Suppl 1 ): P21; the contents of which are incorporated by reference in their entirety). In one embodiment, T cells of the present invention may be used in combination with inhibitors of glycolysis such as 2-deoxyglucose, and rapamycin.

[00400] In some embodiments, effector modules of the present invention, useful for immunotherapy 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).

[00401] In some embodiments, payloads of the invention m ay 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, T1M3, VISTA, and CD96.

[00402] In one embodiment, the payload of the invention may be a CD276 CAR (with CD28, 4- IBB, and CD3 zeta intracellular domains), that does not show an upregulation of the markers associated with early T cell exhaustion (see International patent publication No.

WO2017044699; the contents of which are incorporated by reference in their entirety).

[00403] 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 CDS 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 CDS populations of TILs to enhance TIL mediated immune response.

2. Cancer vaccines

[00404] 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.

[00405] 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 (CTL) response. Peptides used for cancer vaccines may also 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(I2): el238539; the contents of which are incorporated herein by reference in their entirety). |00406] 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 1 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 NO.: US20160317633A 1, the contents of which are incorporated herein by reference in their entirety. 3. Combination treatments

[00407] 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 for enhancement of immunotherapy.

[00408] 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 immunostimulants 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 deliver}' carriers include, polymer microspheres, immune stimulating complexes, emulsions (oil-m-water or water-in-oil), aluminum, salts, liposomes or virosom.es.

[00409] 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. Dual regulation of CAR and cytokines and ligands to segregate the kinetic control of target-mediated activation from intrinsic cell T cell expansion. Such dual regulation also minimizes the need for pre-conditioning regimens in patients. As a non-limiting example, DD regulated CAR e.g. CD 19 CAR may be combined with cytokines e.g. IL 12 to enhance the anti-tumor efficacy of the CAR (Pegram H.J., et al. Tumor-targeted T cells modified to secrete 11,12 eradicate systemic tumors without need for prior conditioning. Blood.2012; ! 19:4133-41; the contents of each of which are incorporated herein by reference in their entirety). As another non-limiting example, Merchant et al. combined dendritic cell- based vaccinations with recombinant human IL7 to improve outcome in high-risk pediatric sarcomas patients (Merchant, M.S et. al. Adjuvant immunotherapy to Improve Outcome in High-Risk Pediatric Sarcomas. Clin Cancer Res. 2016. 22( 13):3182-91; the contents of each of which are incorporated herein by reference in their entirety). |00410] In some embodiments, immune effector ceils modified to express one or more antigen- specific TCRs or CARs may be combined with compositions of the invention comprising immunotherapeutic agents that convert the immunosuppressive tumor microenvironment.

[00411] In one aspect, effector immune cells modified to express CARs specific to different target molecules on the same cell may be combined. In another aspect, different immune ceils modified to express the same CAR construct such as NK cells and T cells may be used in combination for a tumor treatment, for instance, a T cell modified to express a CD 19 CAR may be combined with a NK cell modified to express the same CD 19 CAR to treat B cell malignancy.

[00412] In other embodiments, immune cells modified to express CARs may be combined with checkpoint blockade agents.

[00413] In some embodiments, immune effector ceils modified to expressed biocircuits, effector modules, DDs and payloads of the invention may be combined with cancer vaccines of the invention.

[004 4] 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 ceils, reducing the incidence or number of metastases, reducing tumor size, inhibiting tumor growth, reducing the blood supply to a tumor or cancer ceils, 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.

[00415] In some embodiments, anti-cancer agent or therapy may be a chemotherapeutic agent, or radiotherapy, immunotherapeutic agent, surger ', or any other therapeutic agent which, in combination with the present invention, improves the therapeutic efficacy of treatment.

[00416] In one embodiment, an effector module comprising a CD 19 CAR may be used in combination with amino pyrimidine derivatives such as the Burkit's tyrosine receptor kinase (BTK) inhibitor using methods taught in International Patent Application NO.: WO2016164580, the contents of which are incorporated herein by reference in their entirety.

[00417] 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.

[00418] Exemplary chemotherapies include, without limitation, Acivicm: Aciarubicm;

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 semusiine (methyl-CC U); thylenimines/methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa),

hexamethylmelamine (HMM, altretamine); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); 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'- difiuorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine, 2'-deoxycoformycin

(pentostatin), eiymrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2- chlorodeoxyadenosine (cladribine, 2- CdA); natural products including antimitotic dasgs such as paciitaxel, 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, idarabicin, bleomycins, plicamycin (mithramycin), 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 VGF/VEGF 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 (ο,ρ'-DDD) and aminoglutettiimide; 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 deacetyiase 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 Giivac) and erlotimb (an EGF receptor inhibitor) now marketed as Tarveca; anti-virals such as oseltamivir 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; Veran ine; Verdins; Verteporfin; Vinorelbine; Vinxaltine; Vitaxin; Vorozoie; Zanoierone; Zeniplatin; Ziiascorb; and Zinostatin stimalamer; ΡΒΚβ small -molecule inhibitor, GSK2636771 ; pan-PBK inhibitor (BKM120); BRAF inhibitors. Vemurafenib (Zelboraf) and dabrafenib (Tafinlar); or any analog or derivative and variant of the foregoing.

|00419] Radiotherapeutic agents and factors include radiation and waves that induce DNA damage for example, γ-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 thai all of these factors effect a broad 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.

[00420] In some embodiments, the chemotherapeutic agent may be an immunomodulatory agent such as lenalidomide (LEN). Recent studies have demonstrated that lenalidomide can enhance antitumor functions of CAR modified T cells (Otahal et al., Oncoimmiinology, 2015, 5(4): e l 115940). Some examples of anti-tumor antibodies include toeiiizumab, siltuximab.

[00421] Other agents may be used in combination with compositions of the invention may also include, but not limited to, agents that affect the upreguiation 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.

[00422] 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.

4. Diseases

[00423] 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. |00424] 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

[00425] 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, mediastinum (chest), mouth, ovaries, pancreas, penis, prostate, skin, small intestine, stomach, spinal marrow, tailbone, testicles, thyroid and uterus.

[00426] 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/ieukemia, squamous cell carcinoma, small cell carcinoma, large cell undifferentiated carcinomas, basal cell carcinoma and sinonasal undifferentiated carcinoma.

[00427] 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, iymphangiosarcoma, 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.

[00428] 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, Hypopharyngeal cancer, infiltrating ductal carcinoma, Infiltrating lobular carcinoma. Inflammatory breast cancer, Intestinal Cancer, Intrahepatic bile duct cancer, Invasive / infiltrating breast cancer, Islet cell cancer, Jaw cancer, Kaposi sarcoma, Kidney cancer,

Laryngeal cancer, Leiomyosarcoma, Lepto meningeal 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, Nasopharyngeal 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, Oropharyngeal cancer, Osteogenic sarcoma, Osteosarcoma, Ovarian cancer, Ovarian epithelial cancer, Ovarian germ cell tumor, Ovarian primar ' 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.

Infections diseases

[00429] In some embodiment, biocircuits 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-IL15Ra payioads of the invention may be used to increase immune cell proliferation and/or persistence of the immune cells useful in treating infectious diseases.

[00430] "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-ί, HSV-II, CMV, or VZV), a poxvirus (e-g~, an orthopoxvirus such as variola or vaccinia, or molluscum contagiosum), a picornavirus (e.g., rhinovirus or enterovirus), an orthomyxovirus (e.g., influenzavirus), a paramyxovirus (e.g., parainfluenza virus, mumps virus, measles vims, and respiratory syncytial 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 hepadnavirus (e.g., hepatitis B vims), a flavivirus (e.g., hepatitis C virus or Dengue virus), or a retrovirus (e.g., a lentivirus 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 Bordeteila; (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 trvpanosome infection and prions that cause human disease such as Creutzfeldt-Jakob Disease (CJD), variant Creutzfeldt- Jakob Disease (vCJD), Gerstmann-Straussler-Scheinker syndrome, Fatal Familial Insomnia and kuru. 5. Microbiome

[00431] 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 en vironmen sally 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, respiratory tract, gastrointestinal tract, and the reproductive tract.

[00432] 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. Commensal Bifidobacterium promotes antitumor immunity and facilitates anti-PD-Ll efficacy. Science 2015: 350: 1084-9; the contents of which are incorporated herein by reference in their entirety). In one embodiment, protein, RNA and/or other biomolecules derived from the microbiome may be used as a payload to influence the efficacy of the anti-cancer immunotherapies.

6. Tools and agents for making therapeutics

[00433] 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, type of cells, m ethod of gene transfers, method and time of ex vivo expansion, preconditioning and the amount and type of tumor burden in the subject. Such parameters may be optimized using tools and agents described herein.

Cell lines

[00434] The present disclosure provides a mammalian cell that has been genetically modified with tlie compositions of the invention. Suitable mammalian cells include primary cells and immortalized cell lines. Suitable mammalian cell lines include, but are not limited to Human embryonic kidney cell line 293, fibroblast cell line NIH 3T3, human colorectal carcinoma cell line HCT1 16, ovarian carcinoma cell line SKOV-3, immortalized T cell lines (e.g. Jurkat cells and SupTl cells), lymphoma cell line Raji cells, NALM-6 cells, K562 cells, HeLa cells, PC 12 cells, HL-60 cells, NK cell lines (e.g. NKL, NK92, NK962, and YTS), 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

[00435] 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 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, β-galactosidase, chloramphenicol acetyltransferase, β-glucuronidase, peroxidase, β-lactamase, catalytic antibodies, bioluminescent proteins e.g. luciferase, and fluorescent proteins such as Green fluorescent protein (GFP).

[00436] 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.

[00437] In some embodiments, the preferred reporter moiety may be luciferase proteins. In one embodiment, the reporter moiety is the Renilla luciferase (SEQ ID NO. 866, encoded by nucleic acid sequence of SEQ ID NO. 867), or a firefly luciferase (SEQ ID NO. 868, encoded by nucleic acid sequence of SEQ ID NO. 869).

Animal models

[00438] 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 w herein mouse ceils 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-Foxnl^nu/J, B6; 129S7-/¾gi^ft!!¾ion!/J, B6.129$7-Ragl^mlMom/J, B6. CBl7~Prkdc^SC!dlSzJ, NOD.129S7(B6)-i?ag/^te¾fom/J, -NOO.Cg-Ragl^^P fl^z/Sz, NOD.CB Yl-Prkd^lSzi, T (yD.Cg-Prkdc^sadB2m^tmlu"^c/J, ΝΟΌ-scid IL2Rgⁿⁱ⁾ⁱⁱ, Nude (nu) mice, SCID mice, NOD mice, RAG1/RAG2 mice, NOD-Scid mice, IL2rg««// mice, blmnull mice, ΉΟΏ-scid IL2rynull mice, ^~NQO~scid-B2mnull mice, beige mouse, and HLA transgenic mice. Cellular assays

[00439] In some embodiments, the effectiveness of the compositions of the inventions as immunotherapeutic 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. |00440] Provided herein are methods for functionally characterizing cells expressing S E, 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, CDS, CD4, CD 8, CD 14, CD20, CDl lb, CD 16, CD45 and HLA-DR, CD 69, CD28, CD44, IFNgamma. Markers for T cell exhaustion include PDI, TIMS, BTLA, CD 160, 2B4, CD39, and LAG3. Examples of cell surface markers for antigen presenting cells include, but are not limited to, MHC class I, MHC Class II, CD40, CD45, B7-1, B7-2, lFN-γ receptor and IL2 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 Π, B7-2, CD 18, 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, CD 14, CD 15, CD 16, 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, CXCR.4, CXCR3, NKG2D, CD71, CD69, CCR5, Phospho JAK/STAT, phospho ERK, phospho p38/ MAPK, phospho AKT, phospho STAT3, Granuiysin, Granzyme B, Granzyme K, IL10, IL22, IFNg, LAP, Perforin, and TNFa.

V. DELIVERY MODALITIES AND/OR VECTORS

Vectors

[00441] The present invention also provides vectors that package polynucleotid es 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 al. (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.

[00442] 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.

[00443] 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 Elongation Growth Factor- 1. Alpha (EF-1. alpha). Other constitutive promoters may also be used, including, but not limited to simian vims 40 (SV40), mouse mammary tumor virus (MMTV), human immunodeficiency vims (HIV), long terminal repeat (LTR), promoter, an avian leukemia vims promoter, an Epstein-Barr vims immediate early promoter, a Rous sarcoma vims promoter as well as human gene promoters including, but not limited to the phosphoglycerate kinase (PGK) promoter, 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 metallothionine promoter, glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter may be used. In some embodiments, the promoter may be selected from the SEQ ID NO.: 716-718.

[00444] 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.

[00445] 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.

[00446] In some embodiments, the recombinant expression vector may comprise regulatory sequences, such as transcription and translation initiation and termination codons, which are specific to the type of host cell into which the vector is to be introduced.

i . Lentiviral vectors

[00447] In some embodiments, lentiviral vectors/particles may be used as vehicles and deliver}' modalities. Lenti viruses 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 lenti vims include the Human Immunodeficiency Viruses: HIV-1 and HIV -2, the Simian Immunodeficiency Vims (SIV), feline immunodeficiency vims (FIV), bovine immunodeficiency vims (BIV), Jembrana Disease Vims (JDV), equine infectious anemia vims (EIAV), equine infectious anemia, virus, visna-maedi and caprine arthritis encephalitis vims (CAEV).

[00448] Typically, lenti viral particles making up the gene delivery vehicle are replication defective on their own (also referred to as "self-inactivating"). Lentivirases 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. Bioiecknol, 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.

[00449] Lentiviral particles may be generated by co-expressing the vims 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 vims, 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 ceil 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 eiectroporation, generally together with a dominant selectable marker, such as neo, DHFR, Gin synthetase or ADA , followed by selection in the presence of the appropriate drag and isolation of clones.

[00450] 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.

[00451] Ceils that can be used to produce high-titer lentiviral particles may include, but are not limited to, HEK293T cells, 293G cells, STAR cells (Relander et al., Mol ! hcr . 2005, 1 1 : 452- 459), FreeStyle™ 293 Expression System (ThermoFisher, Waltham, MA), and other HEK293T- based producer cell Sines (e.g., Stewart et al., Hum Gene Ther. _2011, 2,2.(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).

[00452] 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 (PIRYV), Vesicular stomatitis Aiagoas virus (VSAV), Vesicular stomatitis Indiana virus (VSTV) and Vesicular stomatitis New Jersey virus (VSNJV) and/or stains provisionally classified in the vesiculovims genus as Grass carp rhabdovirus, BeAn 157575 virus (BeAn 157575), Boteke virus (BTKV), Calchaqui virus (CQFV), Eel virus American (EVA), Gray Lodge virus (GLOV), Jurona virus (JURY), Klamath virus (KLAVj. Kwatta virus (KWAV), La Joya virus (LJV), Malpais Spring virus (MSPV), Mount Elgon bat virus (MEB V), Ferine t 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

nucleopolyhedroviras (AcMNPV), Anagrapha falcifera nuclear polyhedrosis virus, Bombyx mori nuclear polyhedrosis virus, Choristoneura fiimiferana nucleopolyhedroviras, Orgyia pseudotsugata single capsid nuclear polyhedrosis virus, Epiphyas postvittana

nucleopolyhedroviras, Hypharitria cunea nucleopolyhedroviras, Galleria mellonella nuclear polyhedrosis virus, Dhori virus, Thogoto virus, Antheraea pemyi nucleopolyhedroviras or Batken vims.

[00453] Additional elements provided in lentiviral particles may comprise retroviral LTR (long- terminal repeat) at either 5' or 3' terminus, 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) Posttranscriptionai Regulatory Element (WPRE) which enhances the expression of the transgene, and increases titer. The effector module is linked to the vector.

[00454] 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. [ 00455 j Lentivirus vectors used may be selected from, but are not limited to pLVX, pLenti, pLenti6, pLJMl, FUGW, pWPXL, pWPI, pLenti CMV puro DEST, pLJMl-EGFP, pULTRA, pInducer2Q, pHIV-EGFP, pCW57.1 , pTRPE, pELPS, pRRL, and plionll,

[00456] Lenti viral 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 NO.: WO2012079000; the contents of each of which are incorporated herein by reference in their entirety).

2. Retroviral vectors (γ-retroviral vectors)

[00457] 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 lenti viral 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 ceil types. Example species of Gamma retroviruses include the murine leukemia viruses (MLVs) and the feline leukemia viruses (FeLV).

[00458] In some embodiments, gam ma-retro viral vectors derived from a mammalian gamma- retrovirus such as murine leukemia viruses (MLVs), are recombinant. Hie MLV families of gamma retroviruses include the ecotropic, amphotropic, xenotropic and polytropic subfamilies. Ecotropic viruses are able to 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 vims 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.

[00459] 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- poi) 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.

[00460] 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 vims envelope protein (GALV) or vesicular stomatitis virus G protein (VSV-G), or Simian endogenous retrovirus envelop protein, or Measles Vims H and F protems, or Human immunodeficiency virus gp!20 envelope protein, or cocal vesiculovirus envelop protein (See, e.g., U.S. application publication NO.: 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 (Waehier 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 protems, can direct the attachment of viral vectors to target cells for transduction (Yang et aL, Biotechnol 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).

[00461] 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.

[00462] In some embodiments, polynucleotides encoding the biocircuit, biocircuit components, effector module, SRE are inserted within the recombinant viral genome. The other components of the viral mRNA of a recombinant gamma-retroviral vector may be modifi ed by insertion or removal of naturally occurring sequences (e.g., insertion of an IRES, insertion of a heterologous polynucleotide 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 US- region of the 3 -LTR. These modifications may increase the titers and the ability of infection. [00463] 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 NO.: 2007/048285; PCX application publication NOs.: WO2010/113037; WO2014/121005;

WO2015/056014; 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)

[00464] 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, AAV 8, AAV 9, AAV I O, AAV1 1, AAV 12 and AAVrhlO.

[00465] 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, AAV1 (SEQ ID NO: 6 and 64 of US20030138772), AAV 2 (SEQ ID NO: 7 and 70 of US20030138772), AAV3 (SEQ ID NO: 8 and 71 of US20030138772), AAV4 (SEQ ID NO: 63 of US20030138772), AAV5 (SEQ ID NO: 114 of US20030138772), AAV6 (SEQ ID NO: 65 of US20030138772), AAV7 (SEQ ID NO: 1-3 of US20030138772), AAV 8 (SEQ ID NO: 4 and 95 of US20030138772), AAV9 (SEQ ID NO: 5 and 100 of

US20030138772), AAVIO (SEQ ID NO: 117 of US20030138772), AAVi l (SEQ ID NO: 118 of US20030138772), AAV 12 (SEQ ID NO: 119 of US20030138772), AAVrhlO (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 -1 13 of US20030138772, the contents of which are herein incorporated by reference in their entirety.

[00466] 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.: WO1998/011244, WO2005/033321 and WO2014/14422; the contents of each of which are incorporated herein by reference in their entirety.

[00467] 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. |00468] 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.

[00469] 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.

[00470] Such vectors or viral genomes may also include, in addition to at least one or two ITRs (inverted terminal repeats), certain regulatory elements necessaiy 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.

[00471] In some embodiments, more than one effector module or SRE (e.g. DD) may be encoded in a viral genome.

4. Oncolytic viral vector

[00472] 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 thy midine 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, NO,: 9,226,977).

5. Messenger RNA (mRNA)

[00473] 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 polypeptide of interest in vitro, in vivo, in situ or ex vivo. Such mRNA molecules may have the stractural 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.

[00474] 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 WO2005005622, 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 WO2008140615, United States patent application national phase entry serial number 12/520,072 filed on July 2, 2,009 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

WO2007064952 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 WO2009077134, 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, 201 published as US201 1 065103, 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 WQ2007036366, national phase European patent EP1934345 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 US20120046346 and PCT application PCT/US2011/32679 filed April 15, 2011 published as WO20110130624; 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/US1998/019492 filed on September 18, 1998 published as WQ19990I4346; The Scrrpps Research Institute in PCT application number PCT/US2010/00567 filed on February 24, 2010 published as WO2010098861, and United States patent application national phase entry serial number 13/203,229 filed November 3, 2011 published as US20120053333; Ludwig- Maximillians University in PCT application number PCT/EP2010/004681 filed on July 30, 2010 published as WO201 1 123 16; 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, 201 1 published as US20120009649, and PCT applications PCT/US2010/59305 filed December 7, 2010 published as WO2011071931 and PCT US2010/59317 filed on December 7, 2010 published as WO2011071936; The Trustees of the University of Pennsylvania in PCT application number PCT/US2006/32372 filed on August 21, 2006 published as WO2007024708, and United States patent application national phase entry serial number 11/990,646 filed on March 27, 2009 published as US20090286852; Curevac GMBH in German patent application serial numbers DE10 2001 027 283.9 filed June 5, 2001, DEIO 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 WO2002098443, PCT/EP2002/14577 filed on December 19, 2002 published as WO2003051401 ,

PCT/EP2007/09469 filed on December 31 , 2007 published as WO2008052770,

PCT/EP2008/03033 filed on April 16, 2008 published as WO2009127230, PCT/EP2006/004784 filed on May 19, 2005 published as WO2006122828, PCT/EP2008/00081 filed on January 9, 2007 published as WO2008083949, and United States patent application serial numbers 10/729,830 filed on December 5, 2003 published as US20050032730, 10/870,110 filed on June 18, 2004 published as US20050059624, 1 1/914,945 filed on July 7, 2008 published as

US20080267873, 12/446,912 filed on October 27, 2009 published as US2010047261 now abandoned, 12/522,214 filed on January 4, 2010 published as US20100189729, 12/787,566 filed on May 26, 2010 published as US201 10077287, 12/787,755 filed on May 26, 2010 published as US20100239608, 13/185, 119 filed on July 18, 201 1 published as US201 10269950, and

13/106,548 filed on May 12, 2011 published as US20110311472 ail of which are herein incorporated by reference in their entirety,

[00475] 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. WO201 1 05799 (the contents of which are incorporated herein by reference in their entirety).

VI. DOSING. DELIVERY AND ADMINISTRATIONS

[00476] 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.

1. Delivery to cells

[00477] 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. [00478| 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 Sine 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 ceils.

[00479] Vectors may also be transferred to cells by non-viral methods by physical methods such as needles, electroporation, 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 cationie lipids, lipid nano emulsions, nanoparticles, peptide based vectors, or polymer based vectors.

[00480] 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 ait. Hie 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 peptide s taught in the International Patent Publication, WO2016161516 and

WO2017175072 may be useful in the present invention (the contents of each of which are herein incorporated by reference in their entirety),

2. Dosing

[00481] 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.

[00482] Compositions in accordance with the invention are typically formulated in dosage unit form for ease of administration and uniformity 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.

[00483] Compositions of the invention may be used in varying doses to avoid T cell energy, prevent cytokine release syndrome and minimize toxicity associated with immunotherapy. 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, IL6, IFN-γ, and TNF-a are elevated.

[00484] In some embodiments, the neurotoxicity may be associated with CAR or TIL therapy. Such neurotoxicity may be associated CD19-CARs. Toxicity may be due to excessive T cell infiltration into the brain. In some embodiments, neurotoxicity may be alleviated by preventing the passage of T cells through the blood brain barrier. This can be achieved by the targeted gene deletion of the endogenous aipha-4 integrin inhibitors such as tysabri/natalizumab may also be useful in the present invention.

3. Administration

[00485] 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 is described in U.S. Pat. NOs. : 7,435, 596; the contents of which are incorporated by reference herein in its entirety.

[00486] 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 WO2017/180587, the contents of each of which are incorporated herein by reference in their entirety.

[00487] 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.

[00488] In some embodiments, the ceils 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.

[00489] In some embodiments, the compositions for immunotherapy may be administered in vivo. In some embodiments, polypeptides of the present invention comprising biocircuits, effector molecules, S Es, payloads of interest (immunotherapeutic agents) and compositions of the invention may be delivered in vivo to the subject. In vi vo delivery of immunotherapeutic agents is well described in the art. For example, methods of delivery of cytokines are described in the E.P. Pat. No.: EP0930892 Al, the contents of which are incorporated herein by reference.

[00490] In one embodiment, the payloads of the present invention may be administered in conjunction with inhibitors of SUP- 1 and/or SHP-2. The tyrosine-protein phosphatase SHPl (also known as PTPN6) and SHP2 (also known as PTPN11) are involved in the Programmed Cell Death (PDl) inhibitor}' signaling pathway. The intracellular domain of PD1 contains an immunoreceptor tyrosine-based inhibitory- motif (ΪΤΙΜ) and an immunoreceptor tyrosine-based switch motif (ITSM). 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 l; 162(5):2717-24 ,; Berehtold S (1998) Mol Endocrinol.

Apr; 12(4):556-67 and Schram et al. (2012) Am J Physiol Heart Circ Physiol. l ;3Q2(l):H231 -43.; may be useful in the invention (the contents of each of which are incorporated by reference in their entirety).

Routes of delivery

[00491] 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.

|0Θ492] 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, mtra-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) intracav itary (into the base of the penis), intra v agmai administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), transvaginal, insufflation (snorting), sublingual, subiabial, 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, intraarticular, intrabiliary, intrabronchial, intrahursal, intracartilaginous (within a cartilage),

intracaudal (within the cauda equine), intracisteraal (within the cistema magna

cerebellomedularis), intracorneal (within the cornea), dental intracomal, intracoronary (within the coronaiy 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), intraepidermai (to the epidermis), intraesophageal (to the esophagus), intragastric (within the stomach),

intragingival (withm 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 (withm 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 (withm the pleura), intraprostatic (withm 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), mtratumor (within a tumor), intratympanic (within the aurus media), intravascular (within a vessel or vessels), intraventricular (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, respirator}' (within the respirator}- 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, submucosal, topical, transplacental (through or across the placenta), transtracheal (through the wall of the trachea), transtympanic (across or through the tympanic cavity), ureteral (to the ureter), urethral (to the urethra), vaginal, caudal block, diagnostic, nerve block, biliary perfusion, cardiac perfusion, photopheresis or spinal. VII. DEFINITIONS

[00493] At various places in the present specification, features or functions of the compositions of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual sub combination of the members of such groups and ranges. The following is a non-limiting list of term definitions.

[00494] Activity; As used herein, the term "activity" refers to the condition in which things are happening or being done. Compositions of the invention may have activity and this activity may involve one or more biological events. In some embodiments, biological events may include ceil signaling events. In some embodiments, biological events may include cell signaling events associated protein interactions with one or more corresponding proteins, receptors, small molecules or any of the biocircuit components described herein.

[00495] Adoptive ceil therapy (ACT): The terms "Adoptive cell therapy" or 'Adoptive celi transfer", as used herein, refer to a cell therapy involving in the transfer of cells into a patient, wherein cells may have originated from the patient, or from another individual, and are engineered (altered) before being transferred back into the patient. The therapeutic cells may be derived from, the immune system., such as Immune effector cells: CD4+ T cell: CD8+ T cell, Natural Killer cell (NK cell); and B cells and tumor infiltrating lymphocytes (TILs) derived from the resected tumors. Most commonly transferred cells are autologous anti-tumor T cells after ex vivo expansion or manipulation. For example, autologous peripheral blood lymphocytes can be genetically engineered to recognize specific tumor antigens by expressing T-cell receptors (TCR) or chimeric antigen receptor (CAR).

[00496] Agent: As used herein, the term "agent" refers to a biological, pharmaceutical, or chemical compound. Non-limiting examples include simple or complex organic or inorganic molecule, a peptide, a protein, an oligonucleotide, an antibody, an antibody derivative, antibody fragment, a receptor, and soluble factor.

[00497] Agonist: the term "agonist" as used herein, refers to a compound that, in combination with a receptor, can produce a cellular response. An agonist may be a ligand that directly binds to the receptor. Alternatively, an agonist may combine with a receptor indirectly by, for example, (a) forming a complex with another molecule that directly binds to the receptor, or (b) otherwise resulting in the modification of another compound so that the other compound directly binds to the receptor. An agonist may be referred to as an agonist of a particular receptor or family of receptors, e.g., agonist of a co-stimulatory receptor.

[00498] Antagonist: the term "antagonist" as used herein refers to any agent that inhibits or reduces the biological activity of the target(s) it binds. |00499] Antigen: the term "antigen^"' as used herein is defined as a molecule that provokes an immune response when it is introduced into a subject or produced by a subject such as tumor antigens which arise by the cancer development itsel This immune response may involve either antibody production, or the activation of specific immunologically-competent cells such as cytotoxic T lymphocytes and T helper cells, or both. An antigen can be derived from organisms, subunits of proteins/antigens, killed or inactivated whole cells or lysates. In the context of the invention, the terms "antigens of interest" or ''desired antigens" refers to those proteins and/or other biomolecules provided herein that are immunospecifically bound or interact with antibodies of the present inv ention and/or fragments, mutants, variants, and/or alterations thereof described herein. In some embodiments, antigens of interest may comprise any of the polypeptides or payloads or proteins described herein, or fragments or portions thereof.

[00500] Approximately: As used herein, the term ^"'approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less in either direction (greater than or less than) of the stated reference value unless otheroise stated or otherwise evident from the context (except where such number would exceed 100 of a possible value).

[00501] Associated with: As used herein, the terms "associated with," "conj gated," "linked," "attached," and "tethered," when used with respect to two or more moieties, mean that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serve as linking agents, to form a structure that is sufficiently- stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions. An "association" need not be strictly through direct covalent chemical bonding. It may also suggest ionic or hydrogen bonding or a hybridization based connectivity sufficiently stable such that the "associated" entities remain physically associated.

[00502] Autologous: the term "autologous" as used herein is meant to refer to any material derived from the same individual to which it is later to be re-introduced into the individual.

[00503] Barcode: the term "barcode" as used herein refers to polynucleotide or amino acid sequence that distinguishes one polynucleotide or amino acid from another.

[00504] Cancer: the term "cancer" as used herein refers a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues ultimately metastasize to distant parts of the body through the lymphatic system or bloodstream. [00505] Co-stimuiatory molecule: As used herein, in accordance with its meaning in immune T cell activation, refers to a group of immune cell surface receptor/ligands which engage between T cells and APCs and generate a stimulatory signal in T cells which combines with the stimulatory signal in T cells that results from T cell receptor (TCR) recognition of antigen/MHC complex (pMHC) on APCs

[00506] Cytokines; the term '"cytokines", as used herein, refers to a family of small soluble factors with pleiotropic functions that are produced by many cell types that can influence and regulate the function of the immune system.

[00507] Delivery: the term "delivery" as used herein refers to the act or manner of delivering a compound, substance, entity, moiety, cargo or payload. A "delivery agent" refers to any agent which facilitates, at least in part, the in vivo delivery of one or more substances (including, but not limited to a compound and/or compositions of the present invention) to a ceil, subject or other biological system cells.

[00508] Destabilized: As used herein, the term "destable," "destabilize," "destabilizing region" or "destabilizing domain" means a region or molecule that is less stable than a starting, reference, wild-type or native form of the same region or molecule.

[00509] Engineered: As used herein, embodiments of the invention are "engineered" when they are designed to have a feature or property, whether structural or chemical, that varies from a starting point, wild type or native molecule.

[00510] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (i) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end processing); (3) translation of an RNA into a polypeptide or protein; (4) folding of a polypeptide or protein; and (5) post-translational modification of a polypeptide or protein.

[00511] Feature: As used herein, a "feature" refers to a characteristic, a property, or a distinctive element.

[00512] Formulation: As used herein, a "formulation" includes at least a compound and/or composition of the present invention and a deliver}' agent.

[00513] Fragment: A "fragment," as used herein, refers to a portion. For example, fragments of proteins may comprise polypeptides obtained by digesting full-length protein. In some embodiments, a fragment of a protein includes at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250 or more amino acids. In some embodiments, fragments of an antibody include portions of an antibody. [00514] Functional. As used herein, a '^"functional" biological molecule is a biological entity with a structure and in a form, in which it exhibits a property and/or activity by which it is characterized,

[00515] Immune cells: the term "an immune cell", as used herein, refers to any cell of the immune system that originates from a hematopoietic stem cell in the bone marrow, which gives rise to two major lineages, a myeloid progenitor cell (which give rise to myeloid cells such as monocytes, macrophages, dendritic cells, megakaryocytes and granulocytes) and a lymphoid progenitor cell (which give rise to lymphoid cells such as T cells, B cells and natural killer (NK) cells). Exemplary immune system cells include a CD4+ T cell, a CD8+ T cell, a CD4- CD 8- double negative T cell, a T γδ cell, a Τ β cell, a regulatory T cell, a natural killer cell, and a dendritic cell. Macrophages and dendritic cells may be referred to as "antigen presenting cells" or "APCs," which are specialized cells that can activate T cells when a major histocompatibility complex (MHC) receptor on the surface of the APC complexed with a peptide interacts with a TCR on the surface of a T cell,

[00516] Immunotherapy: the term "immunotherapy" as used herein, refers to a type of treatment of a disease by the induction or restoration of the reactivity of the immune system towards the disease.

[005 7] Tmmunotkerapeiitic agent: the term "immunotherapeutic agent" as used herein, refers to the treatment of disease by the induction or restoration of the reactivity of the immune system towards the disease with a biological, pharmaceutical, or chemical compound.

[00518] In vitro: As used herein, the term "in vitro" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).

[00519] In vivo: As used herein, the term "in vivo" refers to events that occur within an organism (e.g., animal, plant, or microbe or cell or tissue thereof).

[00520] Linker: As used herein, a linker refers to a moiety that connects two or more domains, moieties or entities. In one embodiment, a linker may comprise 10 or more atoms. In a further embodiment, a linker may comprise a group of atoms, e.g., 10-1 ,000 atoms, and can be comprised of the atoms or groups such as, but not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl, and imine. In some embodiments, a linker may comprise one or more nucleic acids comprising one or more nucleotides. In some embodiments, the linker may comprise an amino acid, peptide, polypeptide or protein. In some embodiments, a moiety bound by a linker may include, but is not limited to an atom, a chemical group, a nucleoside, a nucleotide, a nucleobase, a sugar, a nucleic acid, an amino acid, a peptide, a polypeptide, a protem, a protein complex, a payload (e.g., a therapeutic agent), or a marker (including, but not limited to a chemical, fluorescent, radioactive or bioiuminescent marker). The Sinker can be used for any useful purpose, such as to form multimers or conjugates, as well as to administer a payload, as described herein. Examples of chemical groups that can be incorporated into the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amido, amino, ether, thioether, ester, alkylene, heteroalkylene, aryl, or heterocyclyl, each of which can be optionally substituted, as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (e.g., ethylene or propylene glycol monomelic units, e.g., (Methylene glycol, dipropylene glycol, triethySene glycol, tripropylene glycol, tetraethylene glycol, or tetraethylene glycol), and dextran polymers. Other examples include, but are not limited to, cleavable moieties within the linker, such as, for example, a disulfide bond (-S-S-) or an azo bond (-N=N-), which can be cleaved using a reducing agent or photolysis. Non-limiting examples of a selectively cleavable bonds include an amido bond which may be cleaved for example by the use of tris(2- carboxyethyl) phosphine (TCEP), or other reducing agents, and/or photolysis, as well as an ester bond which may be cleaved for example by acidic or basic hydrolysis.

[00521] Checkpoint/factor. As used herein, a checkpoint factor is any moiety or molecule whose function acts at the junction of a process. For example, a checkpoint protein, ligand or receptor may function to stall or accelerate the cell cycle.

[00522] Metabolite: Metabolites are the intermediate products of metabolic reactions catalyzed by enzymes that naturally occur within cells. This term is usually used to describe small molecules, fragments of larger biomolecules or processed products.

[0Θ523] Modified: As used herein, the term '"modified" refers to a changed state or structure of a molecule or entity as compared with a parent or reference molecule or entity. Molecules may be modified in many ways including chemically, structurally, and functionally. In some

embodiments, compounds and/or compositions of the present invention are modified by the introduction of non-natural ammo acids.

[00524] Mutation: As used herein, the term, "mutation" refers to a change and/or alteration. In some embodiments, mutations may be changes and/or alterations to proteins (including peptides and polypeptides) and/or nucleic acids (including polynucleic acids). In some embodiments, mutations comprise changes and/or alterations to a protein and/or nucleic acid sequence. Such changes and/or alterations may comprise the addition, substitution and or deletion of one or more amino acids (in the case of proteins and/or peptides) and/or nucleotides (in the case of nucleic acids and or polynucleic acids e.g., polynucleotides). In some embodiments, wherein mutations compri se the addition and/or substitution of amino acids and/or nucleotides, such additions and/or substitutions may comprise 1 or more amino acid and/or nucleotide residues and may include modified amino acids and/or nucleotides. The resulting construct, molecule or sequence of a mutation, change or alteration may be referred to herein as a mutant.

[00525] Neoantigen the term "neoantigen", as used herein, refers to a tumor antigen that is present in tumor cells but not normal cells and do not induce deletion of their cognate antigen specific T cells in thymus (i.e., central tolerance). These tumor neoantigen s may provide a "foreign" signal, similar to pathogens, to induce an effective immune response needed for cancer immunotherapy. A neoantigen may be restricted to a specific tumor. A neoantigen be a peptide/protein with a missense mutation (missense neoantigen), or a new peptide with long, completely novel stretches of amino acids from novel open reading frames (neoORFs). The neoORFs can be generated in some tumors by out-of-frame insertions or deletions (due to defects in DNA mismatch repair causing microsatellite instability), gene-fusion, read-through mutations in stop codons, or translation of improperly spliced RNA (e.g., Saeterdal et al., Proc Natl Acad Sci USA , 2001 , 98: 13255-13260).

[00526] Off-target: As used herein, "off target" refers to any unintended effect on any one or more target, gene, cellular transcript, cell, and/or tissue.

[00527] Operahiy linked: As used herein, the phrase "operably linked" refers to a functional connection between two or more molecules, constructs, transcripts, entities, moieties or the like.

[00528] Pay load or pay load of interest (POI): the terms "pay load" and "payioad of interest (POI)", as used herein, are used interchangeable. A payioad of interest (POI) refers to any protein or compound whose function is to be altered. In the context of the present invention, the POI is a component in the immune system, including both innate and adaptive immune systems. Payloads of interest may be a protein, a fusion construct encoding a fusion protein, or non- coding gene, or variant and fragment thereof. Payioad of interest may, when ammo acid based, may be referred to as a protein of interest.

[00529] Pharmaceutically acceptable excipients: the term "pharmaceutically acceptable excipient," as used herein, refers to any ingredient other than active agents (e.g., as described herein) present in pharmaceutical compositions and having the properties of being substantially nontoxic and non-inflammatory in subjects. In some embodiments, pharmaceutically acceptable excipients are vehicles capable of suspending and/or dissolving active agents. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrates, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, giidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium, stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, rnaltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch giycolate, sorbitol, starch (com), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[00530] Pharmaceutically acceptable salts: Pharmaceutically acceptable salts of the compounds described herein are forms of the disclosed compounds wherein the add or base moiety is in its salt form (e.g., as generated by reacting a free base group with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines: alkali or organic salts of acidic residues such as carboxylic acids; and the like. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzene sulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsuifonate, citrate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maieate, malonate, methanesuifonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraetliylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts include the conventional non-toxic salts, for example, from non-toxic inorganic or organic acids. In some embodiments, a pharmaceutically acceptable salt is prepared from a parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington^'s Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Eastern, Pa,, 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P.H. Stahl and C.G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., j ournal of Pharmaceutical Science, 66, 1-19 (1977), each of which is incorporated herein by reference in its entirety. Pharmaceutically acceptable solvate: The term "pharmaceutically acceptable solvate/' as used herein, refers to a crystalline form of a compound wherein molecules of a suitable solvent are incorporated in the crystal lattice. For example, solvates may be prepared by crystallization, recrystallization, or precipitation from a solution that includes organic solvents, water, or a mixture thereof. Examples of suitable solvents are ethanol, water (for example, mono-, di-, and tri-hydrates), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), N, N^'-dimethylfonnamide (DMF), N, N'-dimemylacetamide (DM AC), 1,3-dimethyl- 2-imidazolidinone (DMEU), 1 ,3-dimethyl-3,4,5,6-tetrahydro-2-(lH)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone, benzyl benzoate, and the like. When water is the solvent, the solvate is referred to as a '^"hydrate.'¹ In some embodiments, the solvent incorporated into a solvate is of a type or at a level that is physiologically tolerable to an organism to which the solvate is administered (e.g., in a unit dosage form of a pharmaceutical composition).

[00531] Stable: As used herein "stable" refers to a compound or entity that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent.

[00532] Stabilized: As used herein, the term ^"'stabilize", ^"'stabilized," "stabilized region" means to make or become stable. In some embodiments, stability is measured relative to an absolute value. In some embodiments, stability is measured relative to a secondar ' status or state or to a reference compound or entity.

[00533] Standard CAR: As used herein, the term "standard CAR" refers to the standard design of a chimeric antigen receptor. The components of a CAR fusion protein including the extracellular scFv fragment, transmembrane domain and one or more intracellular domains are linearlv constructed single fusion protein.

[00534] Stimulus response element (SRE): the term, "stimulus response element (SRE), as used herein, 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 modulatoiy 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. One non-limiting example of an SRE is a destabilizing domain (DD).

[00535] Subject: As used herein, the term "subject" or "patient" refers to any organism to which a composition in accordance with the invention may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.

[00536] T cell: A T cell is an immune cell that produces T cell receptors (TCRs). T cells can be naive (not exposed to antigen; increased expression of CD62L, CCR7, CD28, CD3, CD127, and CD45RA, and decreased expression of CD45RO as compared to TCM), memory T cells (TM) (antigen-experienced and long-lived), and effector ceils (antigen-experienced, cytotoxic). TM can be further divided into subsets of central memory T cells (TCM, increased expression of CD62L, CCR7, CD28, CD 127, CD45RO, and CD95, and decreased expression of CD54RA as compared to naive T ceil and effector memor - T cells (TEM, decreased expression of CD62L, CCR7, CD28, CD45RA, and increased expression of CD127 as compared to naive T cells or TCM). Effector T cells (TE) refers to antigen-experienced CD8+ cytotoxic T lymphocytes that have decreased expression of CD62L, CCR7, CD28, and are positive for granzyrne and perforin as compared to TCM. Other exemplary T cells include regulatory T cells, such as CD4+ CD25+ (Foxp3+) regulator}' T cells and Tregl7 cells, as well as Trl, Th3, CD8+CD28-, and Qa-1 restricted T cells.

[00537] T cell receptor: T cell receptor (TCR) refers to an immunoglobulin superfamily member having a variable antigen binding domain, a constant domain, a transmembrane region, and a short cytoplasmic tail, which is capable of specifically binding to an antigen peptide bound to a MHC receptor. A TCR can be found on the surface of a cell or in soluble form and generally is comprised of a heterodimer having a and β chains (also known as TCRa and TCRp, respectively), or γ and δ chains (also known as TCRy and TCR5, respectively). The extracellular portion of TCR chains (e.g., a-chain, β-chain) contains two immunoglobulin domains, a variable domain (e.g., a-chain variable domain or V_A, β-chain variable domain or Vp) at the N-terminus, and one constant domain (e.g., a-chain constant domain or C_a and β-chain constant domain or Cp,) adjacent to the cell membrane. Similar to immunoglobulin, the variable domains contain complementary determining regions (CDRs) separated by framework regions (FRs). A TCR is usually associated with the CD3 complex to form a TCR complex. As used herein, the term "TCR complex" refers to a complex formed by the association of CD3 with TCR. For example, a TCR complex can be composed of a CD3y chain, a CD35 chain, two CD3e chains, a homodimer of€ϋ3ζ chains, a TCRa chain, and a TCRp chain. Alternatively, a TCR complex can be composed of a CD3y chain, a CD35 chain, two CD3s chains, a homodimer of Οϋ3ζ chains, a TCRy chain, and a TCR5 chain. A "component of a TCR complex," as used herein, refers to a TCR chain (i.e., TCRa, T(^"R|¾. TCRy or TCR5), a CD3 chain (i.e., CD3y, CD35, CD3e or €Τ)3ζ), or a complex formed by two or more TC chains or CDS chains (e.g., a complex of TCRa and TCR 3, a complex of TCRy and TCR5, a complex of CD3e and CD35, a complex of CD3y and CD3e, or a sub-TCR complex of TCRa, TCRp, CD3y, CD38, and two CD3e chains.

[00538] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to an infection, disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the infection, disease, disorder, and/or condition. In some embodiments, a therapeutically effective amount is provided in a single dose. In some embodiments, a therapeutically effective amount is administered in a dosage regimen comprising a plurality of doses. Those skilled in the art will appreciate that in some embodiments, a unit dosage form may be considered to comprise a therapeutically effective amount of a particular agent or entity if it comprises an amount that is effective when administered as part of such a dosage regimen.

[00539] Treatment or treating: As used herein, the tenns "treatment" or "treating" denote an approach for obtaining a beneficial or desired result including and preferably a beneficial or desired clinical result. Such beneficial or desired clinical results include, but are not limited to, one or more of the following: reducing the proliferation of (or destroying) cancerous cells or other diseased, reducing metastasis of cancerous cells found in cancers, shrinking the size of the tumor, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and/or prolonging survival of individuals.

[00540] Time: As used herein, the term "tune" means to adjust, balance or adapt one thing in response to a stimulus or toward a particular outcome. In one non-limiting example, the SREs and/or DDs of the present invention adjust, balance or adapt the function or structure of compositions to which they are appended, attached or associated with in response to particular stimuli and/or environments. [00541] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments in accordance with the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[00542] In the claims, articles such as "a," "an," and '"the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include '^"or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or the entire group members are present in, employed in or otherwise relevant to a given product or process.

[00543] It is also noted that the term ^"'comprising" is intended to be open and permits but does not require the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of is thus also encompassed and disclosed.

[00544] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise e vident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[00545] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinaiy skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any antibiotic, therapeutic or active ingredient; any method of production; any method of use; etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[00546] It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the purview of the appended claims without departing from the true scope and spirit of the invention in its broader aspects. EXAMPLES

Example I. Generation of novei !igand responsive SREs or DDs by mutagenesis screening Study design

[00547] To engineer constructs that display ligand dependent stability, a candidate ligand binding domain (LBD) is selected and a cell-based screen using yellow fluorescent protein (YFP) as a reporter for protein stability is designed to identify mutants of the candidate LBD possessing the desired characteristics of a destabilizing domain: low protein levels in the absence of a ligand of the LBD, (i.e., low basal stability), large dynamic range, robust and predictable dose-response behavior, and rapid kinetics of degradation (Banaszynski, et al., (2006) Cell; 126(5): 995-1004). The candidate LBD binds to a desired ligand but not endogenous signaling molecules.

[00548] The candidate LBD sequence (as a template) is first mutated using a combination of nucleotide analog mutagenesis and error-prone PCR, to generate libraries of mutants based on the template candidate domain sequence. The libraries generated are cloned in-frame at either the 5'- or 3 '-ends of the YFP gene, and a retroviral expression system is used to stably transduce the libraries of YFP fusions into NIH3T3 fibroblasts.

[00549] The transduced NIH3T3 cells are subjected to three to four rounds of sorting using fluorescence-activated cell sorting (FACS) to screen the libraries of candidate DDs. Transduced NIH3T3 cells are cultured in the absence of the high affinity ligand of the ligand binding domain (LBD), and cells that exhibit low levels of YFP expression are selected through FACS.

Screening Strategy I

[00550] The selected cell population is cultured in the presence of the high affinity ligand of the ligand binding domain for a period of time (e.g., 24 hours), at which point cells are sorted again by FACS. Cells that exhibit high levels of YFP expression are selected through FACS and the selected cell population is split into two groups and treated again with the high affinity ligand of the ligand binding domain at different concentrations; one group is treated with the lower concentration of the ligand and the other is treated with a high concentration of the ligand, for a period of time (e.g., 24 hours), at which point cells are sorted again by FACS. Cells expressing mutants that are responsive to lower concentrations of the ligand are isolated.

[00551] The isolated cells responsive to the lower concentration of the ligand are treated with the ligand again and cells exhibiting low fluorescence levels are collected 4 hours following removal of the ligand from the media. This fourth sorting is designed to enrich cells that exhibit fast kinetics of degradation (Iwamoto et al, Chem Biol. 2010 Sep 24; 17(9): 981-988). Screening Strategy II

[00552] The selected cell population is subject to additional one or more sorts by FACS in the absence of high affinit ⁷ ligand of LBD and cells that exhibit low levels of YFP expression are selected for further analysis. Cells are treated with high affinity ligand of the ligand binding domain, for a period of time (e.g. 24 hours), and sorted again by FACS. Cells expressing high levels of YFP are selected for through FACS. Cells with high expression of YFP are treated with ligand again and cells exhibiting low fluorescence levels are collected 4 hours following removal of the ligand from the media te enrich cells that exliibit fast kinetics of degradation. Any of the sorting steps may be repeated to identify DDs with ligand dependent stability.

[00553] The cells are recovered after sorting. The identified candidate cells are harvested and the genomic DNA is extracted. Hie candidate DDs are amplified by PCR and isolated. Hie candidate DDs are sequenced and compared to the LBD template to identify the mutations in candidate DDs.

Example 2. DD regulated recombinant IL12 expression

[00554] FKBP (DD)-1L12 and DHFR (DD)-1L12 constructs were packaged into pLVX IRES- Puro lentiviral vectors with CMV, EF la, or PGK promoters or without a promoter. The IL12 consists of two subunits, p40 and p35 which are separated by a linker. A p40 signal sequence was inserted next to the DD or IL12. In several constmcts, a furin protease cleavage site or a modified furin site was included.

[00555] HEK293T cells were transiently transfected with 200ng or 1 ^s of FKBP-IL.12 plasmids (OT-IL 12-001 to OT-ILI 2-005), and subsequently treated with 10μΜ Shield-1 or vehicle control for 6 hours. Culture media was collected from transfected cells and diluted 1 :50 to measure IL12 levels using p40 ELISA, The stabilization ratio was defined as fold change in IL12 expression with ligand treatment compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. Stabilization ratio greater than 1 is desired. The average IL12 ELISA readings and stabilization ratio are presented in Table 15.

Table 15: Ligand dependent IL12 induction

[ 00556 j OT-IL 12-002 and OT- IL12-004 showed low level of 1L12 expression in the absence of ligand when compared to IL12 levels in HEK 293T parental cells. Treatment with Shield- 1 resulted in an increase in TL12 levels in OT-IL12-002, OT-IL12-004, and OT-IL12-005 constructs and a stabilization ratio between 2 and 4. These data show that OT-IL 12-002 and OT- IL12-004 are destabilized in the absence of these constructs are stabilized by Shield- 1.

[00557] IL12 expression was measured in cells following stable transduction . 500,000 cells stably transduced with OT-IL 12-004 were plated in a 12 well plate and incubated overnight in growth media consisting of Dulbecco's Modified Eagle medium (DMEM) and 10% fetal bovine serum (FBS). The next day, cells were treated with ΙμΜ Shield- 1 or vehicle control for 6 or 24 hours. Following treatment with Shield- 1, growth media was collected from the cells and diluted 10, 40, 160 or 640 fold and IL12 levels were quantified using IL12-p40 ELISA. The stabilization ratio was defined as fold change in IL12 expression with ligand treatment compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. Stabilization ratio greater than 1 is desired. The average IL12 ELISA readings and stabilization ratio at 6 hours are presented in Table 16.

Table 16: Ligand dependent IL12 induction (6 hours)

[00558] IL12 stabilization ratio greater than 1 was observed at 10, 40 and 160-fold dilutions of media, indicating that IL12 is stabilized by Shield- 1 treatment at these dilutions at 6 hours.

[00559] The average IL12 ELISA readings and stabilization ratio at 24 hours are presented in Table 17.

Table 17: Ligand dependent ILL 2 induction (24 hours)

|00S60] IL12 stabilization ratio was greater than 1 at all media dilutions tested and the highest stabilization ratio was observed at 40-fold dilution of media at 24 hours, suggesting ligand dependent stabilization.

[00561] To evaluate Shield-l dependent FKBP-1L12 induction over time, 2 million cells were plated in growth medium, and incubated overnight in the presence of ΙμΜ Shield-l or vehicle control. Cells were then incubated for with the ligand for 2, 4,6, 8, 24, 48, or 72 hours and growth media was collected for the cells at all time points. Growth media was diluted 400-fold and TL 12 levels were measured using TL 12 p40 ELISA . The stabilization ratio was defined as fold change in 1L12 expression with ligand treatment compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. Stabilization ratio greater than 1 is desired. Average IL12 ELISA readings and stabilization ratio are presented in Table 18.

Table 18: IL12 induction over time

[00562] Stabilization ratio increased over time and peaked at 48 hours, suggesting that IL12 is stabilized by Shield-l with increasing duration of ligand treatment.

[00563] To evaluate the dependence of FKBP-IL12 production on Shield-l dose levels, OT- 1L12-004 transduced H.EK293T cells were plated at different densities (40,000 cells, 20,000 cells, 10,000 cells or 5,000 cells per well) onto a 96-well plate. Following overnight incubation, cells were treated with growth medium containing 0 to 10μΜ Shield-l for 24 hours. Media was then collected, diluted 400-fold and FKBP-IL12 levels were measured using IL12-p40 ELISA. Average IL12 ELISA readings are presented in Table 1 .

Table 19: Dose and cell number dependent IL12 induction

Shield-l 40000 20000 10000 5000

(μΜ) ceils/well ceils/well cells/well cells/well

10 623.77 656.70 214.11 193.62

3.333333 670.64 618.30 ' 273.74 207.55

1.111111 677.27 872.24 322.56 203.71

0,37037 368.17 582.71 250.49 172.50

0.123457 197.29 343.34 156.98 95.92

0.041152 171.50 205.68 63.79 48.89

0.013717 117.25 103.56 13.30 -2.35

0.004572 66.34 60.58 2.11 -8.53

0.001524 100.43 39.55 ' -13.58 -21.76 [00564] A dose dependent IL12 induction was observed at all cell numbers tested. 11,12 induction increased with Shield- 1 up to a dose of Ι μΜ; following which IL12 induction plateaued. Notably, greater IL12 induction was observed at 2000 and 4000 cells/well.

Example 3. DD regulated recombinant IL12 mediated functions in HEK293T cells

[00565] HEK -Blue sensor cells (InvivoGen, San Diego, CA) were utilized to evaluate whether DD regulated IL12 is capable of regulating signaling downstream of IL12. in these cells, the IL12 receptor, STAT4 and downstream transcriptional elements are linked to a reporter gene such that IL12 signaling can be monitored. One million HEK 293T were transfected with 200ng of OT-IL 12-003 plasmid using Lipofectamme 2000 (Thermo Fisher Scientific, Waltham, MA). 48 hours after transfection, cells were treated with growth media containing 10μΜ Shield- 1, incubated for another 24 hours, following which, media was collected. 50,000 HEK 293 Blue sensor cells were plated onto 96 well plates and incubated overnight with media (at different dilutions) from Shield- 1 treated OT-IL 12-003 expressing HEK293T ceils. After overnight incubation, 20 μΐ media was removed from each well and incubated with 180 μΐ Quanti-Blue reagent (InvivoGen, San Diego, CA) for 30 minutes at 37°C. Absorption was measured at 620 nm using a spectrophotometer. To generate a standard curve, 180 μΐ Quanti-Blue reagent was mixed with 20 μΐ of recombinant IL 12 at following concentrations 500, 250, 125, 62.5, 31.25, 15.62, 7.8 and 3.9 pg/ml. Functional IL12 concentrations were determined by comparing the optical density of each sample with IL12 standard curve. Measurable levels of functional IL12 were reached with 640-fold dilutions of 11,12 containing growth media and further plateaued at higher concentrations of the media (Figure 19A),

[0Θ566] The dependence of functional IL12 production on the dose of Shield- 1 used was measured. 10,000 HEK293T ceils stably transduced with OT-IL 12-004 were plated onto 96 well plates and treated with growth media containing 10, 3.33, 1.11, 0.37, 0.12, 0.04, 0.01, 0.005, 0.002 or 0 μΜ Shield- 1 for 24 hours. Following Shield- 1 treatment, media from cells was diluted 200-fold and 20μΕ of the diluted media was added to HEK Blue sensor cells. After overnight incubation, 20 μΐ of media w as removed from each well and incubated with 180 μΐ Quanti-Blue reagent (InvivoGen, San Diego, CA) for 30 minutes at 37°C. Absorption was measured at 620 nm using a spectrophotometer. To generate a standard curve, 180 μΐ Quanti-Blue reagent was mixed with 20 μΐ of recombinant IL12 at following concentrations 500, 250, 125, 62.5, 31.25, 15.62, 7.8 and 3.9 pg/ml. Functional IL12 concentrations were determined by comparing the optical density of each sample with IL12 standard curve. A dose dependent increase in the levels of functional IL12 levels was observed (Figure 19B).

Example 4. BP regulated recombinant IL12 expression in vivo

[00567] SKOV3 tumor cells expressing FBP regulated-IL12 (#OT-IL 12-009) or parental cells were implanted into SCID Beige mice (Day 0). Mice implanted with FKBP IL12 were dosed intraperitoneally with Shield- 1 (l Omg/kg) or vehicle control on Day 2 and Day 7, while the parental cells were left untreated. Blood samples were collected at 0, 2, 4, 6, 8 and 24 hours after Shield- 1 dosing and plasma human 1L12 levels were measured using ELISA. The average adjusted concentration of plasma IL12 is presented in Figure 19C. At Day 2, IL12 levels increased in Shield- 1 treatment and the levels were higher than vehicle control at 4, 6, 8, and 24 hours. Maximum IL12 levels were detected in Shield- 1 treated mice at 8 hours following treatment. In contrast, at day 7, 1L12 levels were very low and almost comparable to the IL12 levels in parental SKOV3 cells.

[00568] The experiment was repeated 28 days following implantation of SKOV3 tumor cells. Mice were split into three groups, with the groups receiving 1, 2 or 3 doses of ligand or vehicle control. Mice received multiple doses with a two-hour interval. Blood samples were collected right before the first dose (0 hours), and 6 hours and 24 hours after the first dosing. Plasma IL12 levels were measured and average IL12 concentrations are shown in Figure 19D. The two dose and three dosing scheme resulted in higher plasma IL12 levels when compared to vehicle treated samples. Peak plasma IL12 levels was detected at 6 hours following shield- 1 treatment with all dosing schemes, and the highest ILI 2 plasma levels were detected with the three-dose regimen . This demonstrates the ligand dependent stabilization of ILI 2 in vivo.

Example 5. DP regulated IL15

[00569] To test ligand dependent IL15 production, 1 million HEK-293T cells were plated in a 6-well plate in growth media containing DMEM and 10% FBS and incubated overnight at 37°C at 5% C02. Cells were then transfected with lOOng of OT-IL15-G0I(constitutive) or OT-IL15- 002 (ecDHFR-IL15) using Lipofectamine 2000 and incubated for 48 hrs. Following the incubation, media was exchanged for growth medium with 10μΜ Trimethoprim or vehicle control and further incubated for 24 hrs. Media was collected and the undiluted samples or samples diluted 4, 16, 256, 1024, 4096 or 16384-foid were tested using human IL15 ELISA. The stabilization ratio was defined as fold change in ILI 5 expression with ligand treatment compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. Stabilization ratio greater than 1 is desired. Average ILI 5 ELISA readings and stabilization ratio are presented in Table 20. Table 20: DD-IL15 induction

[00570| The 16-fold, 4-fold diluted, and undiluted media samples showed stabilization ratio greater than 1 ,5, suggesting a Trimethoprim dependent stabilization of IL15 at these dilutions. Example 6. BP regulated expression of IL15-IL15Ra fusion molecule

[00571] A fusion molecule is generated by fusing membrane bound IL15, IL15 Receptor alpha subimit (IL15R.a) and DDs such as ecDHFR (DD), FKBP (DD), or human DHFR (DD), These fusion molecules were cloned into pLVX-EFla-IRES-Puro vector.

[0Θ572| To test ligand dependent IL15-IL15Ra production, 1 million HEK-293T cells were plated in a 6-well plate in growth media containing DMEM and 10 FBS and incubated overnight at 37°C, 5% C02. Cells were then transfected with lOOng of constitutive IL15-IL15Ra (OT- IL15-008) or DD linked IL15-IL15Ra (OT-IL15-006, OT-IL15-007, OT-IL15-009, OT-IL15- 010, OT-IL 15-011) using Lipofectamine 2000 and incubated for 24 hrs. Following the incubation, media is exchanged for growth medium with or without 10μΜ Trimethoprim, or ΙμΜ Shield- 1 and further incubated for 24 hrs. Cells were harvested and IL15 levels are analyzed via western blotting using human IL15 antibody (Abeam., Cambridge, UK). OT-IL 15-009 showed the strong ligand (Trimethoprim) dependent stabilization of 11,15, while OT-IL 15-006 and OT- IL 15 -007 showed modest ligand dependent stabilization of IL15 (Figure 20A).

[00573] Surface expression of membrane bound IL15-IL15Ra constructs (OT-IL15-006, OT~ 11,15-007, OT-IL15-008, OT-IL15-009, OT-IL15-010, OT-IL15-011 ) was determined by FACS using anti-IL15 and anti-IL15Ra antibodies. HEK293T cells were transfected with IL15-IL15Ra constructs and then treated with suitable ligand (Shield- 1 or Trimethoprim). 48 hours after transfection, cells were analyzed using FACS. As expected, constitutive IL15~IL15Ra constract OT-TL15-008 showed high surface expression of IL15 and ILlSRa both in the presence and absence of ligand. Consistent with the results from the western blot, OT-IL 15-009 showed the strong ligand (Trimethoprim) dependent surface expression of IL15 and IL15Ra (Figure 20B, Figure 20C). 1005741 Membrane bound-IL15-IL15Ra constructs (OT-IL15-008 to OT IL15-011) were transduced into human colorectal carcinoma cell line, HCT-116 and stable integrants were selected with 2μg of puromycin. Stably integrated cells were then incubated for 24 hours in the presence or absence of ΙΟμΜ Trimethoprim or Ι Μ Methotrexate.

[00575] Surface expression of IL15 -IL15Ra fusion constructs was examined by staining with PE- conjugated IL15Ra antibody (Cat no. 330207, Biolegend, San Diego, CA). The median fluorescence intensity obtained with the different constracts in the presence or absence of the corresponding ligand is presented in Table 21 .

Table 21: Surface expression of IL15-IL15Ra fusion constracts

[00576| The stabilization ratio was calculated as the fold change in GFP intensity in ligand treated samples compared to treatment with DMSO (i.e. in the absence of ligand) with the same construct. The destabilization ratio was calculated as the fold change in GFP intensity in the DD regulated constructs compared to the constitutive construct (OT-IL15-008) in the absence of the ligand. Destabilization ratios less than 1 and stabilization ratios greater than 1 are desired in DDs. The ratios are presented in Table 22.

Table 22: IL15-IL15Ra destabilization and stabilization ratios

[00577] Destabilization ratios less than one was observed with OT-IL 15-006 (ecDHFR (R12H, E129K)) and OT-IL15-011 (hDHFR (Q36F, N65F, Y122I)) indicating a strong destabilization in the absence of ligand. Stabilization ratio greater than 1 was observed with all constructs with TMP treatment and with both OT-IL 15 -010 and 1 1 with MTX treatment. These data show that OT-IL 15-006 and OT-IL 15-01 1 are both strongly destabilized in the absence of ligand and strongly stabilized in the presence of ligand. [00578| The expression and ligand-dependent stabilization of IL15-lL15Ra constructs (OT- IL15-008 to OT-IL15-011) was measured in HCT-116. Cells were incubated with

ΙΟμΜ Trimethoprim or ΙμΜ Methotrexate or DMSO for 24 hours. Following incubation, cells were harvested and cell extracts were prepared. Cell extracts were run on SDS-PAGE and western blotted with anti-IL15 antibody (Catalog No. 7213, Abeam, Cambridge, UK). As shown in Figure 20D, the IL15/lL15Ra constitutive construct (OT-IL15-008) showed ligand independent IL15 expression while the DD regulated constnicts (OT-IL15-009 to OT-IL15-011) showed ligand dependent 11,15 expression . The identit ⁷ of the IL15 bands was also confirmed by immunoblotting with the anti-human DHFR antibody (Catalog No. 117705, Genetex, Irvine, CA), As shown in Figure 20D, both IL15-IL15Ra fusion constructs (QT-IL15-010 and 011) showed ligand dependent expression of DHFR expression.

[00579| To evaluate the dose dependence of ligand induced stabilization, IL15-IL15Ra fusion constructs namely, OT-IL15-009 (ecDHFR (R12Y, Y 1001)), OT-IL15-010 (hDHFR (Y122I, A125F)), and OT-IL15-011 (hDHFR (Q36F, N65F, Y 122I)) were stably transduced into HCT- 1 16 cells and incubated with increasing concentrations of Trimethoprim for 24 hours. Surface expression of IL15-IL15Ra fusion construct was quantified by FACS using IL15Ra- PE antibody. The median fluorescence intensity with increasing doses of TMP is represented in Table 23.

Table 23: Surface expression of IL15-IL15Ra

[00580] As shown in Table 23, all three constructs showed a dose dependent increase in median fluorescence intensity indicating a dose dependent increase in surface expression of 1L15- IL15Ra fusion upon addition of DD stabilizing ligand. 100581 ] The time course of ligand dependent stabilization of 1L 15 -1L 15 Ra fusion constructs was measured in HCT-1 16 cells. Cells were transduced with OT-IL15-009 (ecDHFR (R12Y, Y100I) construct and incubated with ΙΟμΜ Trimethoprim for 0, 12, 16, 24, 48 or 72 hours. Following incubations, surface expression of TL15-IL15Ra fusion construct was quantified by FACS using IL15Ra- PE antibody and compared to parental untransfected cells. The median fluorescence intensity (MFI) over time is represented in Table 24.

Table 24: Time course of IL15-IL15Ra surface expression

1005821 As shown in Table 24, OT-IL15-009 (ecDHFR (R12Y, Y100I) showed a time- dependent increase in median fluorescence intensity indicating that the surface expression of IL15-IL15Ra fusion increased with increased duration of treatment with DD stabilizing ligand. Example 7. DD regulated CD19 CAR expression

[00583] A CD 19 CAR fusion polypeptide was linked to either FKBP-DD, ecDHFR -DD or human DHFR- DD and the constructs were cloned into pLVX-IRES-Puro vector.

[00584] FKBP, ecDHFR and hDHFR DDs were positioned either between the CD 19 scFv and the CDSahinge (OT-CD19C-002, OT-CD 19C-003), between the CD8ahinge and the transmembrane domain (OT-CD19C-004, OT-CD19C-005) or at the C terminus of the construct (OT-CD19C-007, OT-CD 19C-008, OT-CD19C-009, OT-CD19C-010, OT-CD19C-01 1). In some instances, a furin cleavage site was added between the DD and the CD 19 scFv. A constitutive ly expressed CAR construct, OT-CD19C-001 was used as a positive control.

[00585] To test ligand dependent expression of DD-CD19 CAR constructs, 1 million HEK 293T cells were cultured in growth medium containing DMEM and 10% FBS and transfected with CAR constructs using Lipofectamine 2000. 48 hours after transfection, cells were treated with ΙμΜ θΓΐΟμΜ Shield- 1, 10μΜ Trimethoprim, ΙμΜ Methotrexate, or vehicle control and incubated for 24 hours. Cells were harvested, lysed and immunoblotted for CD3 Zeta, a component of the CAR, using anti-CD247 (BD Pharmingen, Franklin Lanes, NJ) and Alexa 555- conjugated-goat-anti mouse antibody (red) (Li -Cor, Lincoln, NE). Lysates were also

immunoblotted for Actin with Alexa 488-conjugated secondary antibody (green) to confirm uniform protein loading in all the samples. Compared to the untreated control, OT-CD19C-002 and OT-CD19C-003 showed increased levels of CDS Zeta in the presence of ligands Shield- 1 and TMP respectively indicating the stabilization of the CD19 CAR (Figure 21 A). As shown in Figure 21B, OT-CD19C-008 and OT-CD19C-010 constructs showed strong increase in CDS Zeta levels in the presence of Methotrexate and low levels in the absence of hgand . indicating a strong ligand-dependent stabilization of CD19 CAR. OT-CD19C-007 and OT-CD19C-009 showed modest increase in CDS Zeta levels in the presence of Shield- 1 and Methotrexate respectively, indicating a modest ligand-dependent stabilization of CD 19 CAR. As expected, the constructively expressed, OT-CD19C-001 showed strong expression of CD 19 C AR in the absence of ligand treatment.

[00586] Ly sates from cells expressing CD 19 CAR constructs were also immunoblotted for 4 1- BB, a component of the CAR. As shown in Figure 21C, OT-CD 19C-008, QT-CD19C-009, OT- CD19C-010 and OT-CD19C-011 showed low levels of 4-1 BB in the absence of ligand and high levels of 4-1 BB in the presence of the ligand, Methotrexate, indicating a strong ligand dependent stabiiization of CD19 CAR using these constructs. OT-CD19C-003, OT-CD19C-006 and OT-CD19C-007 showed modest increase in 4- IBB expression levels with treatment of corresponding ligands- TMP and Shield- 1, indicating a modest ligand dependent stabilization of CD19 CAR. Constructs OT-CD19N-014 and OT-CD 19N-015, which contain a form cleavage site, showed an additional, smaller 4 IBB sized protein product upon treatment with MTX. This smaller sized 4-1BB protein band was only seen with the add ition of the ligand and its molecular weight is consistent with the size of the CD19 CAR in OT-CD 19-001. These data indicate that the furin cleavage occurs only with ligand treatment.

[00587| Surface expression of DD-CD19 CAR constructs in HEK 293T cells was measured using Fluorescence activated cell sorting (FACS) with Protein L-Biotin-Strepavidin- Allophycocyanin which binds to the kappa light chain of the CAR (ThermoFisher Scientific, Waltham, MA). Cells were treated with ΙμΜ Shield- 1, ΙμΜ Methotrexate, 10μΜ Trimethoprim or vehicle control for 24 hours and subject to FACS analysis. As shown in Figure 2 ID, surface expression of OT-CD 19C -002 with FKBP-DD was detected only in the presence of Shield- 1, while OT-CD 19C-003 with ecDHFR-DD showed surface expression only in the presence of Trimethoprim. As expected, constitutively expressed construct OT-C19C-001 showed high expression both in ligand and control vehicle treated cells. Additional constructs were analyzed by FACS with Protein L-Biotin-Strepavidin-AUophycocyanin in a separate experiment. The percentage of GFP positive cells obtained with each construct in the presence or absence of ligand is presented in Table 25. In Table 25, N/A indicates not applicable. Construct Ligand Percentage GFP positive ceils

No Ligand Ligand

OT-CD19C-001 N/A 46.8 45.4

OT-CD19C-006 TMP 46.6 43.6

OT-CD19C-007 Shield- 1 28.9 34

OT-CD19C-008 MTX 15.8 31.3

OT-CD19C-009 MTX 16.5 34.2

OT-CD19C-010 MTX 14.8 33

OT-CD19C-011 MTX 14.5 32.9

OT-CD19C-012 TMP 19.1 18.7

OT-CD19C-013 Shield- 1 0. 1 0.4

OT-CD 19C-014 MTX 4.68 16.2

OT-CD19C-015 MTX 3.04 18.2

[00588] An increase in the percentage GFP positive cells was observed with OT-CD19C-007, OT-CD19C-008, OT-CD19C-009, OT-CD19C-010, OT-CD19C-011, OT-CD19C-014, and OT- CD19C-015. The highest increase in the percentage of GFP positive cells was observed with OT- CD19C-014, and OT-CD19C-015 constracts.

|0Θ589] The mean fluorescence intensities are presented in Table 26. In Table 26, MFI represents mean fluorescence intensity . The stabilization ratio was calculated as the fold change in GFP intensity in ligand treated samples compared to treatment with DMSO (i.e. in the absence of ligand) with the same constmct. The destabilization ratio was calculated as the fold change in GFP intensity in the DD regulated constructs compared to the constitutive construct (OT- CD19C-001) m the absence of the ligand. Destabilization ratios less than 1 and stabilization ratios greater than 1 are desired.

OT-CD19C-010 MTX 85.6 160

0.19 1.87

OT-CD 1 C-01 1 MTX 83,3 172

0.18 2.06

OT-CD19C-012 TMP 1 12 105

0.25 0,94

OT-CD19C-013 Shield- 1 61 49.5

0.13 0.81

OT-CD19C-014 MTX 78.6 124

0.17 1.58

OT-CD19C-015 MTX 73.3 143

0.16 1.95

[005901 A destabilization ration less than 1 was observed with all constructs indicating that all DD regulated constructs are destabilized in the absence of hgand. A stabilization ratio of greater than 1 was observed with OT-CD19C-008, OT-CD19C-009, OT-CD19C-010, OT-CD19C-011, OT-CD19C-014 and OT-CD19C-015. Notably, these constructs were also destabilized in the absence of ligand and therefore represent suitable CD19-DD constructs.

Example 8. in vitro T eel! assay development

[00591] The goal of the study was to determine the T cell stimulation regimen and dose of TL12 needed to maximize T cell persistence and T cell differentiation in vitro. The study recapitulates the design of the adoptive cell therapy regimen wherein the T ceils were initially exposed to the antigen in vitro which results in activation followed by a resting phase and finally in vivo transfer where the T cells encounter the antigen again. T cells were stimulated CD3/CD28 beads or soluble CD3/CD28 on day 0 and the CD3/CD28 stimulus was washed off at the end of 48 hours. Ceils were treated with a dose of IL12 ranging from 0.01 - 1000 ng/mL. On day 9, the Thl phenotype of the cells was evaluated by examining the frequency of IFNgamma positive CD4+ cells and CD8+ cells. On day 14, cells were divided into two groups- one group received a second CD3/CD28 stimulation and a second group that was not stimulated. On day 16, the Thl phenotype was evaluated in both groups using FACS. The results for day 16 are presented in Figure 22. IFN gamma expression was higher in cells that received a CD3/CD28 restimulation on day 14 compared to cells that did not receive second stimulation. This indicates that both antigen restimulation and 1L12 exposure were required for the Thl phenotype. Further, as little as 0.1 ng/mL of IL12 was able to cause Th 1- skewing and IF gamma production from T cells in vitro, and higher doses of IL12 further improved this effect.

Example 9. Measuring human T cell responses in vitro and in vivo

[00592] IL12 promotes the differentiation of naive T cells into Th l cells which results in the secretion of IFN gamma from T cells. Human T cells were treated with IL12 or left untreated and analyzed by flow cytometry for the expression of IFN gamma and T cell marker CD3. Treatment with IL12 resulted in the differentiation of T cells as measured by an increase in the percentage of IFN gamma positive T cells from 0.21 to 22.3 (see inset of Figure 23A).

[00593] To test if membrane bound IL15/TL15Ra fusion protein (OT-IL15-008) can induce human T cell expansion, human T cells were transduced with the construct. T cell proliferation was measured by evaluating forward and side scatter of the T cell population using flow cytometry. Transduction with membrane bound IL15/IL15Ra fusion construct resulted in the expansion of human T cells (58.9) compared to control untransfected cells (37.8) (Figure 23B).

[ 00594 j Tracking T cells following their adoptive transfer is critical to determine their distribution at different sites in the host, their identity and persistence over time. Human T cells were stimulated with CD3/CD28 beads and incubated with SQU/rnl of IL2. Cells were expanded in vitro for 7 days with IL2 supplementation on day 3 and day 5. On day 5, the CD3/CD28 beads were removed and the ceiis were cultured for two days. On day 7, cells were washed to remove 11.2. and 5 million human T cells were injected intravenously into immune compromised, NOD.Cg-Prfefc*** mice. Blood samples were obtained 4, 24, 120 and 168 hours after cell transfer. Mice were euthanized 168 hours after cell transfer and the bone marrow and spleen were harvested . Immune cells were isolated from all samples and analyzed for the presence of human T cells using CD 3 and CD45 cell surface markers. As shown in Figure 23C, the percentage of CD3 positive, CD45 positive human T cells in the blood was higher in animals injected with human T cells, especially at 120 and 168 hours. CD3 positive, CD45 positive human T cells were also detected in the spleen and bone marrow of animals injected with human T cells. As expected no CD3 positive, CD45 positive human T cells were detected in control animals that were not injected with human T cells.

[ 00595 j To determine the identity of the T cells following adoptive transfer, blood samples were collected from mice 48 hours after injection, CD4 and CD8 T cells were analyzed for surface expression of CD45RA and CD62L. Both markers are highly expressed in naive T cells but are lost as the T ceils become antigen exposure. As shown in Figure 23D, human CD4 and CDS T cells showed high surface expression of both markers prior to injecting into mice, but was lost 48 hours after in vivo cell transfer indicating that the human T cells are exposed to the antigen in vivo.

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[00596] DD-IL12 function is characterized in vivo by evaluating the ability of tumor cells expressing these constructs to establish tumors and proliferate under the treatment of corresponding synthetic iigands e.g. Shield- 1, Trimethoprim or Methotrexate. 2-10 million HCT- 116 cells stably transduced with the constructs are subcutaneousiy xenografted with 50 matrigel into mice capable of producing functional B and NK cells. Approximately, two weeks after injection, when the tumors reach a size of approximately 300 cubic mm, mice are dosed with corresponding stabilizing ligands e.g. Shield-l, Trimethoprim or Methotrexate at varying concentrations every two days. Shield-l is injected with a carrier consisting of 10

Dimethylacetamide, 10 Solutol HS15, and 80 saline. Tumor volume and body weight are monitored twice a week and the experiment is terminated once the tumors reach 1 00 cubic mm in size. Plasma and tumor samples are collected 8 hours after the last dose of the ligand andIL12 as well as the ligand levels are measured.

[00597] To evaluate the ability of IL12 expressing cells to form, tumors, HCT-116 cells stably transduced with DD-IL12 constructs are pretreated with corresponding stabilizing ligands, Shield-l, Trimethoprim or Methotrexate and subsequently xenografted into mice. Reduction in tumor growth and a concomitant increase in IL12 levels in ligand treated mice compared to untreated controls is indicative conditional regulation of IL12 in vivo.

Example 11. DP regulated recombinant IL12 mediated functions in T cells

[00598] Functional responses to DD-1L12 is evaluated in primary human T cells and in human cell lines/transformed hematopoietic cell lines e.g. Raji cells. Human T cells are purified from peripheral blood mononuclear cells (PBMCs) by negative selection using CD4+ T- cell isolation kit (Miltenyi Biotec, Germany). T cells are treated with growth media from HEK 293T cells expressing DD-IL12 constructs for 5 days. Ceils are then activated with beads conjugated with- CD3/CD28 beads (Thermo Fisher Scientific, Waltham, MA) at the ratio of 3 beads per T cell and cultured for 3 days. Functional response to DD-IL12 is determined by measuring Interferon gamma in CD3 positive cells using flow cytometry following treatment with ligand or vehicle control. 1L12 promotes the differentiation of naive T cells into Thl ceils which results in the secretion of IFN gamma from T cells.

[00599] To evaluate IL12 induced phosphoiylation of STAT4 (Signal transducer and activator of transcription 4), human T-cells are isolated from PBMCs and activated with

phytohemagglutinin (PHA, 2^ig/ml) for 3 days followed by treatment with 50 RJ/ml of

Interleukm 2 (IL2) for 24hrs. Cells are then washed, resuspended in fresh media and rested for 4 hrs in the presence of ligand or vehicle control. Supernatant from DD-IL12 expressing

HEK293T cells is added to the primary cells, followed by incubation for 30 minutes. Cells are then harvested and STAT4 phosphoiy lation is analyzed using STAT4 antibody (Cell Signaling Technology, Dan vers, MA). )] Activation via IL15 can sustain T cell persistence by conferring a survival advantage. In addition, IL15/ILI 5Ra fusion molecule has been shown to confer a memory phenotype on T cells and increase proliferation of NK cells (Hurton (2016), PNAS, 113: E7788-7797; the contents of which are incorporated herein by reference in their entirety).

[00601] To evaluate signaling by DD regulated IL15~IL15Ra fusion constructs, NK-92 cells are incubated with HCT-116 cells expressing DD regulated IL15-IL15Ra fusion constructs. Trans signaling by IL15/iL15Ra is expected to increase STATS phosphorylation in NK92, which is measured by western blotting, and by FACS, Proliferation of NK92 cells is also measured.

[00602] To evaluate the effect of DD regulated IL15~IL15Ra fusion constructs on primary- T cells, cells are transduced with the fusion constructs. T cell proliferation in the absence of exogenous IL15 supplementation is measured. The T cell memory phenotype is measured by quantifying CD62L expression by FACS.

[00603] To assess if DD-IL15/IL15R expressing T cells maintain prolonged persistence in vivo, DD modified T cells are injected into mice. Constructs are tagged with luciferase reporter to allow in vivo tracking in mice. Mice are treated with vehicle control or corresponding ligand, Shield- 1, Trimethoprim or Methotrexate depending on the construct utilized and monitored over a period of 40-50 days using bioluminescent imaging (PerkinElmer, Massachusetts). Mice treated with ligand are expected to retain T cells expressing DD-lL15/!L15Ra while T cells in vehicle control treated animals are not expected to persist.

Example 13. DD regulated CD19 CAR expression and function in T cells

[00604] Ligand dependent expression of DD-CD19 CAR constructs is evaluated in primary human T cells and in immortalized/transformed hematopoietic cell lines e.g. Raji cells, Jurkat cells and K562 cells. Human T cells are purified from peripheral blood mononuclear cells (PBMCs) by negative selection using CD4+ T- cell isolation kit (Miltenyi Biotec, Germany). Primary T cells and hematopoietic ceil lines are stably transduced with DD-CD 19 CAR constmcts. Cells are treated with ΟμΜ Shield- 1 , 10μΜ Tnmethoprim, ΙμΜ Methotrexate or vehicle control and immunoblotted for CD3 Zeta using anti CD247 antibody.

[00605] The production of functional DD-CD19 CAR is analyzed in primar ' human T cells or human cell lines (NALM6, K562, Jurkat and Raji cells). Cells are incubated with CD 19 expressing antigen presenting cells or CD 19/Fc fusion protein in the presence of DD stabilizing iigands Shield- 1, TMP or MTX. After incubation, cells are stained with fluorescently labelled anti-CD69 antibodies and analyzed by flow cytometry. Cells with high CD69 expression are considered to have a functional DD-CD19 CAR. Functional response to DD-CD19 CAR is also determined by measuring interferon gamma levels using ELISA. DD-CD19 CAR expressing cells are expected to demonstrate higher interferon gamma levels in the presence of ligand than untreated cells.

[00606] Cytolytic potential of DD-CD19 CAR expressing cells is evaluated in primary human T cells or human cell lines (e.g. NALM6, K562 and Raji) using Chromium-51 Release Assay. Target cells are loaded with of Naa ⁵¹Cr04, washed twice and resuspended in phenol red-free growth medium. Untreated or ligand treated DD-CD19 CAR and mock transduced cells are coincubated with CD 19 expressing target ceils at various effector: target cell ratios, and chromium release into the supernatant is measured using a liquid scintillation counter. Cells with DD-CD19 C AR are expected to demonstrate specific cytolysis only in the presence of ligand. Cells with DD-CD 19 CAR in the absence of ligand or mock transfected cells are expected to show minimal cytolytic activity.

[00607] The in vivo antitumor efficacy of DD-CD 19 CAR is also evaluated. Immune compromised mice are injected with luciferase expressing human leukemic cell lines (NALM-6). Subsequently, mice are injected with DD-CD19 CAR T cells via tail vein injections. Mice are subdivided into treatment groups and are treated with a range of ligand doses. Two control groups are also included in the study: a control group that did not receive any ligand and another group that did not receive any T cells. Tumor burden as measured by luciferase activity is monitored over time using bioiuminescent imaging. Mice treated with DD-CD19 CAR T cells and ligand are expected to have a reduced tumor burden when compared to control animals. Example 14. Evaluation of antitumor response of DD regulated pavioads in syngeneic mouse models

[00608] The efficacy of cancer immunotherapy in organisms with intact immune cells is evaluated using syngeneic mouse models e.g. pMEL-1 and 4T1 mouse models, immune cells such as T cells and NK cells are isolated from syngeneic mice and transduced with DD regulated pavioads such as DD-IL12, DD-IL15, DDlL15-!L15Ra, and DD-CD19 CAR,. Cells are then injected into mice bearing subcutaneous syngeneic tumors and treated with varying

concentrations of ligand, Shield- 1 , Trimethoprim or Methotrexate, depending on the DD used. Mice treated with immune cells transduced with DD regulated payload are expected to have a reduced tumor burden when compared to control animals.

Example 15. Optimizing workflow for discovery of DD-regulated immunotherapeutic agents

[00609] To identify DD-CD19 CAR constructs suitable for immunotherapy , constructs are introduced into cell lines e.g. HEK293T cells and Jurkat cells. The expression of the construct in the presence or absence of the corresponding ligand is tested. Constracts which show low basal expression in the absence of ligand and robust, ligand-dose responsive expression are selected for further analysis. If no DO-CD 19 CAR constructs show ligand-dependent expression, then constructs are redesigned and the experiment is repeated till a regulatable construct is identified. Next, the ligand dependent regulation of the DD-CD19 CAR constructs is tested in vitro in primary T cells. If the constructs show low basal expression in the absence of the ligand and ligand dose responsive expression, they are subject to in vivo PK/PD proof of concept experiments. Otherwise, the constracts are redesigned and the new constructs are subject to similar analysis. The constitutively expressing CD 19 CAR constructs are transduced into T cells and CD 19 CAR expression is measured in parallel to the regulated construct. If no expression is detected in vitro, efforts are re focused on testing DD- CD 19 CAR constracts in vitro in T cells. In contrast, if the constitutive constructs show expression, then the expression of CD 19 CAR is measured in vivo.

[00610] To test in vivo PK/PD, mice are injected with T cells expressing DD-CD 19 CAR constructs and the test group is dosed with the ligand corresponding to the DD, while the control group is dosed with the appropriate vehicle control. Constructs that display ligand-dependent expression of CD 19 C AR are selected for in vivo functional proof of concept experiments. Parallel experiments are also conducted using the constitutive CD 19 CAR constructs. If constitutive CD 19 CAR expression is detected in vivo, then the constracts are selected for functional experiments. If no expression is detected in vivo, then constracts are redesigned.

[00611] Functional analysis in vivo is performed by testing if the constitutive and DD regulated CD 19 CAR expressing T cells display anti-tumor activity in a constitutive or ligand dependent manner respectively. If yes, then in vivo proof of concept is achieved and constructs suitable for immunotherapy are identified. If none of the DD regulated constructs show anti-tumor activity, then alternate dosing regimens are explored. If the constitutive CD19 CAR constructs do not sho anti-tumor activity, then efforts are focused on identifying DD-CD 19 CAR constructs that show in vivo expression in T cells.

Example 16. Co-expression of DD regulated payloads

[00612] Toxicity related to systemic administration of interleukins can be circumvented by- using CAR-T cells to deliver interleukins to the target tissue. This combinatorial approach also has greater anti-tumor activity than interleukin and CAR therapy alone. Cells are co-transfected with CD19 CAR (constitutive or DD regulated) and DD-Interleukin e.g. DD-IL12, DD-IL15 and DD-IL15/IL15Ra constracts. Transfected cells are treated with stabilizing ligands depending on the DD utilized. CD 19 CAR expression is evaluated by immnnoblotting for CDS zeta. DD-IL12, DD-IL15 and DD-IL15/BL15Ra expression in the media is measured by ELISA.

Example 17. CAR expression and functionality in T cells

[006 3] Primary T cells were transduced with CD 19 CAR constructs . Surface expression of CD 19 CAR construct was measured using Fluorescence activated cell sorting (FACS) with Protein L-Biotin-Strepavidin-Allophycocyanin which binds to the kappa light chain of the CAR (ThermoFisher Scientific, Waltham, MA) . To determine the percentage of the CD4 and CDS sub populations of CAR T cells, cells were analyzed by anti CD4, anti CDS antibodies and Protein L. As shown in Figure 24A, 67.3 % of CAR positive cells obtained were CD4 positive, while only 14.2% ceils were CDS positive.

[00614] To test the ability of CD 19 CAR cells to kill target cells, primary T cell populations transduced with OT-CD 19N-001 or OT-CD 19N-017 were cocultured with K562 cells expressing CD 19 (target cells) at a ratio of 5 : 1. Additional control combinations of T cells and target cells were also set up. These included CAR expressing T cells co cultured with K562 cells, T cells co cultured with K562 cells expressing CD 19 and K562 cells expressing CD 19 without T cell co culture. K562 cells were fluorescently labelled with NucLight Red and co cultured with T cells for 30 hours. Ceil death was monitored by labelling cells with Annexin V and K562 target cell death was measured by evaluating Annexin V staining in NucLight Red positive cells. The ratio of Annexin V staining per target cell area was calculated. As shown in Figure 24B, OT-CD 19N- 001 or OT-CD 19N-017 expressing T cells were effective in killing target K562 cells expressing CD 19. A low level of target cell killing was observed when iintransduced T cells were cocultured with GD I 9 expressing target cells. As expected, cell death was minimal in the co- culture of OT-CD 19N-001 expressing T cells and K562 cells (without CD 19 expression). These data sho that CD 19 CAR cells are effective in killing their corresponding target cells.

Example 18. Regulated expression of IL15-IL15Ra in T cells

[00615] DD regulated IL15-IL15Ra constructs such as OT-1L 15-009 or constitutively expressed constructs such as OT-IL15-008 were transduced into T cells such as primary T cells or SupTl cells. The transduction was carried out at two different lentivirus concentrations, 5 μΐ and 20 ill for the DD regulated construct using Lentiboost™(Sirion Biotech, Germany). 4 days after transduction, ceils were treated with 10μΜ TMP or DMSO control for 24 and 48 hours. Samples were analyzed with an anti IL1 Ra antibody using FACS. Additional controls samples such as cells treated with Lentiboost only, untransduced cells treated with DMSO or TMP, and Isotype controls were included in the FACS analysis. The FACS results are depicted in Figure 25A for 24 hours of TMP treatment and in Figure 25B for 48 hours of TMP treatment. In both figures, DMSO-A and TMP-A indicate cells treated with 5 μΐ of lenti virus and DMSO- B and TMP-B indicate cells treated with 20 μΐ of lentivirus. Treatment of T ceils expressing OT-IL15-009 with TMP for 24 hours resulted in an increase in the expression of ILlSRa in T cells with both doses of lentivirus used. Additionally, very low levels of ILlSRa were detected in the DMSO treated samples under the same conditions as well as in untransduced T cells. As expected, the constitutively expressed construct, OT-IL15-008 showed high expression of ILlSRa. TMP dependent expression of OT-IL15-0Q9 was not observed in SupTl cells (Figure 25A). Similar results were observed for both T cells and SupTl cells at 48 hours (Figure 25B). These results show that tight regulation of IL15-IL15Ra constructs can be achieved in primary T cells.

[00616] The surface expression of IL15 and ILlSRa was measured for OT-IL15-008 and OT- IL15-009. The percentage of cells expressing IL15, ILlSRa or both on the cell surface is presented in Table 27.

Table 27: Surface expression of IL15 and ILlSRa

[00617] As shown in Table 27, the percentage of cells with detectable surface expression of IL15 and ILlSRa was less than 5% with both constructs. Further, the percentage of cells with surface expression of LL 15Ra was much higher than the percentage of cells with detectable surface expression of ILLS.

[00618] The effect of increasing doses of TMP on IL15Ra expression in T cells was measured using the OT-IL 15-009 construct. T cells were treated with a range of doses of TMP starting from 0.156 μΜ to 160 μΜ for 24 hours. TL15Ra expression was measured using FACS. As shown in Figure 25C, the percentage of ILlSRa expressing T cells with OT-IL 15-009 cells was detected even at the lowest concentration of TMP and the percentage of ILlSRa positive cells at the lowest concentration of TMP was higher than the untreated control. The percentage of ILlSRa cells increased with increasing doses of TMP. [00619] ILl 5-Π,Ι 5Ra fusion constructs, OT-1L15-008, OT-1L15-009, and OT-IL15-010 were stably expressed in HCT116 cells treated with increasing doses of TMP ranging from 10μΜ, 33μΜ, and ΙΟΟμΜ TMP for 24 hours. Cell lysates were immunoblotted with anti ILiSRa antibody. As shown in Figure 26, IL15Ra expression of OT-IL15-009 was virtually undetectabie in the absence of TMP, and addition of increasing doses of TMP resulted in an increase in ILI 5Ra levels. Modest increase in ILiSRa expression was observed with OT-IL15-010 construct with the addition of TMP. As expected, the constitutive construct, OT-IL15-008 showed strong expression of lL15Ra both in the presence and absence of iigand.

Example 20. Effect of IL15-IL15Ra on T cell persistence and T cell memory phenotype

[00620] The effect of constitutive!}' expressed !L15-lL15Ra fusion construct, OT-1L15-008 on T cell persistence was measured in NSG mice. T cells were transduced with OT-DL15-008 and 4 million cells T cells were injected intravenously into NSG mice (number of mice =3), As a control, additional mice were injected with iintransduced T cells. Blood samples were obtained from mice at 2, 3, 4, 5 and 6 weeks after injection and analyzed by FACS for the presence of CD8 and/or CD4 positive human T cells expressing ILl 5 and ILiSRa, The percentage of human T cells in the blood was calculated as the percentage of total T cells i.e. human T cells (measured using anti-human CD45 antibody) and the mouse T ceils and endothelial cells (measured using the anti-mouse CD45 antibody). As shown in Figure 27 A, the percentage of T cells in the blood at 2 weeks was greater in mice injected with T cells transduced with OT-IL 15-008 compared to control mice that were injected with untransduced T cells. This observed increase in T cells decreased over 3,4, and 5 weeks, and the percentage of T cells was comparable between the two cohorts. At 6 weeks, one of the mice injected with OT-IL 15-008 transduced T cells showed a higher percen tage of human T cells in the blood. Tims, at 2 w eeks, the frequency of human T cells in the blood is increased in the blood of mice injected w ith OT-IL 15-008 transduced T cells.

[00621] The number of T cells in the blood was measured by comparing the number of human T cells in 50 uL of mouse blood using anti-human CD45 antibody as a marker for human T cells and anti-murine CD3 antibody as a marker for murine endothelial cells. As shown in Figure 27B, the number of human T cells in the blood increased at 2 weeks in mice injected with OT-IL 15- 008 transduced T cells, as compared to mice injected with untransduced T cells. The difference between the two cohorts was diminished at 3 weeks and 4 weeks. At 6 weeks, one of the mice injected with OT-IL15-008 transduced T cells showed a higher number of human T cells in the blood. Thus, at 2 weeks, the frequency and number of human T cells in the blood is increased in the blood of mice injected with OT-IL15-008 transduced T cells. These data support the role of IL15-IL15Ra fusion proteins in T cell persistence. The increased T cell frequency and number observed at 6 weeks in one of the mice may be due to graft versus host disease.

[00622] The effect of OT-IL 15-008 expression on the CD4 and CDS subset of T ceils was measured prior to injecting into mice (Week 0) and 2 weeks after injection. As shown in Figure 27C, the ratio of CD4 and CD8 cells was 1 : 1 prior to injecting into mice. However, at 2 weeks, the proportion of CD4 positive cells was much higher than the CDS positive cells in the transduced cells, indicating that OT-IL15-008 causes a preferential expansion of CD4 positive cells. The expression of the OT-TL15-008 construct within the CD4 and CD 8 subsets was measured using anti IL15Ra antibody. As shown in Figure 27D, prior to injections, 25 % of the OT-IL 15-008 transduced CD4 T cells and CD 8 T ceils expressed IL15Ra. At week 2, the percentage of IL15Ra positive CD4 and CD8 T cells increased to 80% indicating a preferential expansion of T cells transduced with OT-IL15-008. As expected, untransduced control T cells were negative for ILlSRa expression.

Example 21. IL12 dependent, re-stimulation independent Thl markers

[00623] T cells require T cell receptor restimulation in vivo or in vitro stimulation with CD3/CD28 to produce IFNgamma. To study the effect of IL12 activity on T cells in the absence of restimulation, several T cell markers were explored. T cells were expanded using one of the following 4 expansions strategies (i) Day 10 cytokine switch from IL2 to IL12, CD3/CD28 stimulation from day 0 to day 10 with no restimulation (ii) Day 10 cytokine switch from 1L2 to IL12, CD3/CD28 stimulation from day 0 to day 10 and restimulation at with CD3/CD28 from day 12 to day 14 (iii) Day 10 cytokine switch from II.2 to IL12, CD3/CD28 stimulation from day 0 to day3 with no restimulation (iv) Day 10 cytokine switch from 1L2 to 1L12, CD3/CD28 stimulation from day 0 to day 3 and restimulation at with CD3/CD28 from day 12 to day 14. Markers tested include CD69, IFNg, Perforin, CXCR3, Granzyme B, CCR5, CXCR6, Ki-67 and T-bet. IFNg appears to be the most robust and consistent marker for IL12 activity on human T cells, but requires re -stimulation of T cells to induce production. Thl markers which increase in response to IL12 in the absence of re-stimulation and IL2 (likely in vivo conditions) include Ki- 67, T-bet, Perform, CXCR3, and CCR5. [00624] Immune cells such as Natural Killer cells depend on cytokines such as IL15 for their proliferation and survival. This dependence on cytokines can be used to test the functionality of DD regulated or constitutively expressed cytokines and cytokine fusion proteins.

[00625] The dependency of the NK-92 cells on cytokines for activation was tested. Cells were initially cultured for 3 days with IL2, following which, cells were washed twice and cultured in media without IL2 for 7 hours. The cells were cultured for 18 hours in the presence of 1L12 (10 ng/ml) or varying concentrations of IL15 (100 ng/ml, 20 ng/ml, 4 ng/ml, 0.8 ng/ml, 0.16 ng/ml, 0.032 ng/ml, 0,0064 ng/ml and 0.00128 ng/ml), NK-92 cell activation in response to IL15 and IL12 treatment was evaluated by FACS analysis using a panel of markers whose increased expression is associated with NK cell activation. These markers include NKG2D, CD71, CD69; chemokine receptors such as CCR5, CXCR4, and CXCR3, Perforin, Granzynie B and interferon gamma (IFNg). Prior to FACS analysis for IFNg, cells were cultured for 4 hours with Brefeidin A. NK ceils respond to external stimuli such as cytokines in their environment through the phosphorylation of proteins JAK/STAT, ERK, and p38/MAPK pathways which are important for cell activation, signaling and differentiation pathways. The phosphorylation of AKT, STATS and STATS in response to cytokine addition was measured by FACS. Since phosphorylation events are transient NK-92 cells were treated with the cytokines for 15 or 60 minutes, prior to the analysis. The fold change in mean fluorescence intensities compared to untreated for IL15 treatment are presented in Table 28,

Table 28: IL15 induced markers

[00626] Treatment with ILI 5 resulted in an increase in the expression of CD69, CXCR4, Perforin, Granzynie B, and IFNg. The effect of ILI 5 on these markers was dose dependent with a higher dose of 1L15 resulting in a corresponding upregulation of markers. Phosphorylation of STATS was increased both at 15 and 60 minutes after the addition of 1L2 or 1L15. Taken together, these results show that cytokines can activate NK cells.

[00627] The fold change in activation markers observed with IL.12 treatment are shown in Table 29.

Table 29: IL1 2 induced markers

|0Θ628] Treatment with IL12 resulted in an increase in the expression of markers CD69, CCR5, Perforin, Granzyme B, and IFNgamma. Further, IFNg levels secreted by NK-92 ceils into the media higher than untreated controls upon treatment with IL12. Treatment with IL2 resulted in an increase in the expression of CXCR4, Perforin, Granzyme B, and IFNg. Further, IFNg levels secreted by NK-92 cells into the supernatant was higher than untreated controls upon treatment with 1L2.

Example 23. Effect of cytokines on T cell expansion and activation

[00629] To test the requirement of IL2 and IL12 for T cells expansion and activation, T ceils were stimulated with soluble CD3/CD28, CD3/CD28 Dynabeads or left, unstimulated for two days. Each of these groups was further split into two sub groups. One sub group was treated with IL2 and 100 ng/ml of IL12 while the second sub group was treated with IL2 only for the duration of the stimulation. For the soluble CD3/CD28 stimulated cells, a third subgroup that was only treated with 100 ng/ml was also included. T cell expansion over the course of 14 days was measured and the fold change in T cells expansion is shown in Figure 28A. CD3/CD28 dynabeads plus IL2 with or without IL12 had the most profound impact on T cell expansion followed by the T cells treated with soluble CD3/CD28 plus IL2 with or without IL12.

Unstimulated cells and cell treated with soluble CD3/CD28 cells that did receive IL2 treatment were unable to expand over the course of the experiment. These results show that IL2 is required for T cell expansion, but IL12 may be dispensable.

[00630] The effect of IL12 on T cell activation was measured by determining the frequency of IFNgamma positive CD4+ and CD8 + T cells. IFNg is produced by activated T cells. Three different stimulation protocols were used. In the first protocol, cells were stimulated with CD3/CD28 dvnabeads for 2 days, following which the beads were washed off and the cells were treated with varying concentrations of IL12 for 7 days (from day 2 to day 9). At day 9, cells were restimulated with soluble CD3/CD28 and the frequency of IFNgamma positive cells was determined by FACS. The results are presented in Figure 28B as the percentage of cells. In the second protocol, following 2 day CD3/CD28 dynabeads stimulation, T cells were maintained in culture for a longer duration of 14 days i.e. from day 2 to day 16. At day 16 cells, were restimulated with soluble CD3/CD28. At day 16, the frequency of IFNgamma positive cells was measured. The results are presented in Figure 28C as the percentage of cells. In the third protocol, T cells were initially stimulated for 2 days with CD3/CD28 dynabeads and IL2, followed by treatment with IL2 only for 9 days (i .e. from day 2 to day 11), followed by IL12 treatment for 2 to 5 days. In the last two days of the experiment, cells were also restimulated with soluble CD3/CD28. IFNgamma positive CD4 and CDS ceils were measured using FACS. The third protocol mimics the environment that is presented to T cells in adoptive cell therapy, both during in vitro transduction and T cells expansion as well as the in vivo. The results are presented in Figure 28D as the percentage of cells. In both 7-day treatment with 1LI2 as well as 14-day treatment with IL12, shown in Figure 28B and Figure 28C respectively, restimulation with CD3/CD28 cells at the end of the experiment increased the percentage of IFNgamma positive celis. A half maximum effective concentration (EC50) of IL12 observed with the first protocol for CDS cells was 50 pg/ml. The EC50 of IL12 observed with the second protocol was 12 pg/ml for CD4 cells and 65 pg/ml for CDS cells. Long-term culture with CD3/CD28 further increased the dependence on re-stimulation and IL12 for IFNg production.

|0Θ631 ] The results obtained with the third stimulation protocol are presented in Figure 28D. Immune cells treated with IL12 for the final 5 day s of the experiment combined with CD3/CD28 restimulation showed the highest percentage of IFN gamma positive cells (EC50 = 24 pg/ml for CD4 cells and 40 pg/ml for CDS cells), followed by cells that received IL12 for 2 days. Thus, T cells expanded in vitro can later differentiate in response to IL12, but restimulation may be required for IFNg production.

[00632] Taken together these results indicate that IL12 can stimulate IFN production in T cells when restimulated with CD3/CD28.

Example 24. Promoter selection for expression of SREs in T cells

[00633] The expression of SREs in a vector can be driven by either the retroviral long terminal repeat (LTR) or by cellular or viral promoters located upstream of the SRE . The activity of the promoter may vary with the cell type and thus promoter selection must be optimized for each cell type. To identify optimal promoters, AcGFP (SEQ ID NO. 870) was cloned into pLVX. IRES Puro construct with a CMV or an EFla promoter. Patient derived T cells and Sup T^'l cells were transduced with the constructs and GFP expression was measured at day 3 and day 5 after transduction using FACS. As shown in Figure 29, both the CMV promoter and the EFla can drive the expression of GFP in SupTl cells and T cells. The percentage of GFP positive T cells was higher when GFP expression was driven by CMV promoter compared to an EFla promoter, both at 3 days and 6 days after transduction. In contrast, the percentage of GFP positive cells was much higher when GFP expression was driven by the EFla promoter when compared to the CMV promoter. Thus, the optimum, promoter suitable for expression differs based on the cell type.

Example 25. Effect of ligand on T cell proliferation

[00634] The effect of ligands specific to the SREs of the invention on immune cell proliferation was measured to identify concentrations of the ligand that did not inhibit T ceil growth or survival. T cells derived from two different donors were stimulated with CD3/CD28 and treated with ligand TMP at doses ranging from 0.04 μΜ to 160 μΜ or DMSO. The percentage of divided cells within the CD4 and the CDS populations of T cells was measured using FACS. Concentrations of TMP ranging from 0.04 μΜ to 40 μΜ showed no effect on the percentage of divided cells within the CDS and CD4 populations, while 160 μΜ concentration of TMP resulted in an 70-90% reduction in the percentage of divided cells. Tims, the optimal concentration of TMP for T cell based experiments was determined to be less than 160 μΜ.

Example 26. Effect of DD regulated CD19 CAR on tonic signaling

[00635] Chronic antigen activation can result in T cell exhaustion. To test if DD regulated CD19 CAR constmcts induce tonic signaling, irradiated K562 ceils expressing CD 19 are plated into culture plates 12 hours before the addition of T cells expressing DD regulated CD19 CAR constmcts with Interleukin 2. Cells are counted every two days and media is replaced. For repeated stimulations, ceils are transferred to a new plate with K562-CD19 cells after 24 hours (for two stimulations) or every 12 hours (for four stimulations). For each condition, T cells are counted and analyzed by FACS for CAR, phenotypic and exhaustion markers every 12 hours. DD regulated constmcts were analyzed in the presence or absence of ligand. Markers analyzed include CD25 and CD69 for activation status: CD62 and CD45RA for memory status; and exhaustion markers PDl, ΊΊΜ3 and LAG3. DD regulated CD 19 CAR constmcts are expected to induce a lower percentage of cells that are positive for all three exhaustion markers- i.e.PDl, ΊΊΜ3 and LAGS and a higher percentage of cells that are CD45A+/CD62L+ indicating less differentiated T cells. Constitutively expressed CD19CAR constructs may induce the expression of all three exhaustion markers and may have a more differentiated phenotype with a higher proportion of CD45-/CD62L- and CD45+/CD62L- cells.

Example 27. Functional analysis of DP regulated CD19 CAR

|00636] To test the ability of DD regulated CD 19 CAR cells to kill target ceils, primary T cell populations transduced with DD regulated CD 19 CAR constructs are co cultured with K562 cells expressing CD 19 (target cells) at a ratio of 5: 1 in the presence or absence of the ligand specific to the DD e.g. Shield-1, TMP or MTX. Additional control combinations of T cells and target cells are also set up. These include DD regulated CAR expressing T ceils co cultured with K562 ceils (in the presence or absence of the ligand), T cells co cultured with K562 cells expressing CD 19 and K562 cells expressing CD 19 without T cell co culture. The K562 cells are fluorescently labelled with NucLight Red and co cultured with T cells for 30 hours. Cell death is monitored by labelling cells with Annexin V and the cell death in target K562 cells is monitored by evaluating cells that are positive for both Annexin V and NucLight Red. The ratio of Annexin V staining per target cell area is calculated. DD-CD19CAR expressing T cells are expected to be effective in killing target K562 cells expressing CD19 only in the presence of the ligand specific to the DD. Minimal target cell death is expected to occur when untransduced T cells are cocultured with CD19 expressing target cells; and with DD-CAR T cells (with or without ligand treatment) plus K562 cells (without CD19 expression).

Example 28. Generation of CD19 scFvs using the large phage antibody libraries

Construction of Primary Phagemid Library

|00637] Total RNA is prepared from 40 different samples of human peripheral blood lymphocytes and cDNA is synthesized using random primers. IgM variable regions are amplified using an IgM 3' primer and 5'VH primers. Pooled primers are also used to amplify the Vk and VL. An additional PGR step is added to include restriction sites as well as to introduce a region of overlap containing an scFv loxP linker. scFvs are obtained by mixing equimolar amounts of VH and VL genes and performing assembly. The scFvs are then cloned into pDAN5 vectors to obtain a primary library of approximately 10 ^s.

Recombination and secondary of the secondary library

[00638] To induce recombination, bacterial strain BS 1365 (which express Cre-recombinases constitutively) are infected with primary phagemid library at an MOI of 20: 1. This results in bacteria containing multiple phagemids, each of which encodes different VH and VL genes, which can be recombined by the Cre recombinase. Since the phagemid arise from bacteria containing many different scFv, the phenotype and genotype are not coupled , Phagemids derived from the bacteria are used to infect bacteria that do not express Cre (e.g. DH5a) at a low MOI of < 0.1 to couple genotype to phenotype.

CD9 expression constructs and cell lines

[00639] Human CD 19 isoforms described in Table 14 are cloned into appropriate vectors and transfected into cell lines with low endogenous CD19 expression such as K562 and 3T3 cell lines. The CD 19 expressing lines i.e. K562- CD19 or 3T3 -CD 19 cells, are used for positive selection of scFvs in the phage display library. The parental K562 and 3T3 are used for the negative selection.

Selection of antibodies recognizing CD 19 on ceil surface

[00640] The secondary phage display library is pre-cleared by screening with parental cells to remove non-specific binding phages. The precl eared phages are incubated with K562-CD19 or 3T3-CD19 cells, and bound phages are recovered and amplified for next round of selection. Three rounds of selection are performed to enrich for CD 19 binders. FMC63 -distinct scFvs i.e. scFvs that bind to epitopes distinct from FMC63, are selected in a parallel selection process by blocking the FMC63 epitope with an excess of FMC63 antibody.

[00641 ] The affinity of the scFv to CD 19 is a critical aspect that determines the performance of the antibody in pharmacokinetic and immune response assays. Affinity measurements for 96 scFv clones are made using techniques such as ELISA and surface plasma resonance which provide on-rate (Ka), off-rate (Kd), and affinity constant (KD).

[00642] scFv clones with desired off rates are subjected to Sanger sequencing to identify unique clones that bind to CD 19 expressing cells but not parental cells. Identified clones are then subject to epitope binning, using a competitive immunoassay that is used to characterize and then sort a library of scFvs against a target protein, e.g. CD19. scFvs against CD 19 are tested against all other CD 19 scFvs identified from the library, in a pairwise fashion to identify scFvs that prevent the binding of other scFvs to an epitope of CD 19 antigen. After a profile is created for each CD 19 scFv, a competitive blocking profile is created for each scFv relative to the others. Closely related binning profiles indicate that the antibodies have the same or a closely related epitope are binned together.

[00643] scFvs obtained at each of step of the selection process are subject to deep sequencing methods such as Ion Torrent/MiSeq. Heavy chain CDR3 sequences, including those that do not bind to FMC63 are identified using the Abmining ToolBox (D'Angelo S et al. (2014) MAbs. 6(1): 160-172) and top ranking HCDR3s are identified. HCDR3 specific primers designed from the DNA sequence of the top ranked sequences are then used to amplify scFv clones by inverse PGR and the PGR product is cloned into expression vectors.

Example 29. CD19 scFv affinity

[00644] The affinity of the scFv to the CD19 antigen is a critical aspect that determines the performance of the antibody in pharmacokinetic and immune response assays. Affinity measurements are made using techniques such as ELISA and surface plasma resonance which provide on-rate (Ka), off-rate (Kd), and affinity constant (KD).

[00645] Antibodies with varying affinities are identified using cells that have high or low ectopic expression of CD19. K562-CD19 cells and parental K562 cells with Sow CD19 expression are sorted by FAGS using CD 19 antibodies e.g. FMC63 to determine surface expression of CD 19. Cells are sorted into bottom 5% (i.e. low CD 19 expressing cells), top 5% (high CD 19 expressing cells) and the rest of the population of cells is categorized as median CD19 expressing cells.

CD19/Fc fus on proteins

[00646] FMC63 binds to human CD 19 in the region encoded by exon 2. To identify' FMC63- distinct CD19 scFvs, human CD 19 (Exon 1-4) or human CD19 (Exon 1,3,4) are fused with IgG to generate CD19-lgG fusion proteins, CD19slgGl-4 and CD 19sIgGl,3,4 respectively. CD 19- IgG fusion proteins are used for antibody screening. 96-well plates are coated with capture antibodies and incubated with CD19sIgGl -4 or CD 19sIgG l,3,4 fusion protein. The plates are washed and incubated with candidate CD 19 scFvs identified in Example 28. The plates are washed again and incubated again with reporter (e.g. Alkaline phosphatase) conjugated detection antibodies and detected using reporter compatible detection methods. Capture antibodies may be antihuman IgG Fc antibodies, or the FMC63 antibody (as a control). Tire detection antibody may^¬ be anti-human IgM antibody. FMC63 -distinct CD19 scFvs are expected to bind to

(CD 19sIgG 1,3,4) and (CD19sIgGl-4). In contrast, candidate CD19 scFvs that bind to epitopes that are identical or overlap with FMC63's epitope are expected to only bind to (CD19sIgGI -4).

Competition assay

[00647] CD 19 expressing K562 cells are incubated with nano molar concentrations of tagged candidate CD 19 scFvs e.g. identified in Example 28 and fixed concentration of tagged FMC63 scFv for competition binding assays. Cells are washed and stained with the secondary antibody corresponding to the tag used in the candidate CD 19 scFv. Mean fluorescence intensity is measured using flow cytometry. As a negative control, CD19 K562 expressing cells are incubated with varying concentrations of tagged candidate CD 19 scFv alone or FMC63 alone. For FMC63-distinct CD 19 scFvs, it is expected that there will be no competition for binding to CD 19 between the candidate CD 19 scFvs and FMC63. Thus, the mean fluorescence intensity of the tagged candidate CD 19 scFv is expected to increase with increasing concentrations of the candidate CD 19 scFv, while the mean fluorescence intensity of tagged FMC63 antibody is not expected to decrease with increasing concentrations of the candidate CD 19 scFv. This would indicate that the FMC63 is not displaced from its epitope by the addition of the candidate CD 19 scFv, suggesting distinct binding epitopes. For candidate CD 19 scFv that bind to the same epitope as FMC63, a decrease in the fluorescence intensity of FMC63 with increasing concentrations of the candidate CD19 scFv is expected.

[00648] FMC63 -distinct CD 19 scFvs engineered to generate FMC63-distmct CD19 CAR constructs with destabilizing domains, linkers, transmembrane and intracellular domains described in Table 1, and Tables 6, 7, 8A or 8B. The ability of FMC63 -distinct CD 19 CAR to induce cell activation, cytotoxicity and proliferation is compared to the FMC63-CD19 based CAR constructs in Jurkat cells. Constructs are also analyzed for their ability to induce the upregulation of exhaustion markers, PDl, TIM3 and LAG3, and constructs that are positive for multiple exhaustion markers are excluded from the analysis. Constructs that can induce Jurkat cell activation and cytotoxicity but not exhaustion markers are transduced into T cells and their efficacy is compared with the constitutive!}' expressed FMC63-based CD19 CAR construct. It is expected that DD regulated FMC63 -distinct CD 19 CAR constructs will demonstrate superior cytotoxic capabilities with minimal tonic signaling as compared to FMC63 CD 19 CAR constructs.

2] To test the antigen specificity of cell killing by T cells engineered to express constitutive or DD-containing CAR construct, CD 19 was ectopically expressed in the antigen negative K562 cell line. CD19 expression was measured using anti-CD 19 antibody conjugated to Phycoerythrin (PE). Figure 30A shows the expression of CD19 in parental K562 cells and K562- CD19 cells, wherein CD 19 is ectopically expressed. |006S0] To test the ability of DD regulated CD 19 CAR cells to kill target ceils, primary T cell populations were transduced with DD regulated CD 19 CAR constructs, OT-CD 19-024 with human DHFR DD and an EFla promoter. Transduced T cells were co cultured with K562 cells expressing CD19 (target cells) at a ratio of 5: 1 in the presence or absence of TMP (ΙΟΟμΜ). Additional control combinations of T cells and target cells were also set up. These included DD regulated CAR expressing T ceils co cuitured with antigen-negative K562 ceils (in the presence or absence of the ligand), imtransduced T cells co cultured with K562 cells expressing CD19 and K562 ceils expressing CD 19 without T cell co culture. The T cells utilized for this experiment were transduced with the OT-CD 19-024 construct (or imtransduced) and expanded for 11 days using protocols described in previous examples, frozen, thawed and co-cultured with target ceils. Target cells were treated with Mitomycin C to prevent their proliferation. The K562 or K562- CD19 target cells stably expressing the fluorescent protein NucLight Red were co cultured with T cells for 300 hours. Ceil death was monitored by labelling cells with Annexin V and the cell death in target K562 and K562-CD19 cells was monitored by evaluating cells that were positive for both Annexin V and NucLight Red using the IncuCyte® Live Ceil Analysis System (Essen Biosciences, Ann Arbor, MI). The results are presented in Figure SOB, where the killed target cells represented on the y axis are based on target ceils that are positive for both NucLight Red and Annexin V. Figure 30C, shows the killed target size as measured in (μΜ/well) at day 5. Target cell killing was observed with the OT-CD 19-024 construct only in TMP treated co- cultures of T cells and K562 target cells ectopically expressing CD19. No cell killing was observed in untreated controls of the same co-culture set up and when T ceils were co cultured with parental K562 ceils that do not express CD19 in the presence or absence of ligand. These data show that regulated CARs display ligand- and target-dependent ceil killing with minimal basal off-state.

Example 33. In vitro CAR-T cell functional analysis

[00651] The efficacy of T ceils expressing DD regulated CD 19 CAR constructs in functionally interacting with target ceils is evaluated. To interact with the CD19CAR T cells, the chosen target cells express CD 19 naturally or ectopically. In this context, target cells which have high endogenous expression of CD19 such as Nalm6, Raji, Reh, Sent, Kopn8, and Daudi cells.

Alternatively, target cell lines may be engineered by ectopic expression of CD 19 in ceil lines that have low endogenous expression of CD19 such as K562. Multiple assays are used to measure functionality. Prior to co culture, the target ceils are optionally cuitured in the presence of presence of mitomycin C to prevent target ceil proliferation. This ensures that target cell growth does not out compete T cell growth. Cytotoxicity assays are used to measure the ability of T ceils induce target cell death. Target cells are engineered to express Renilla or Firefly luciferase and co cultured with T cells expressing DD regulated CD 19 CAR constructs for 18 to 24 hours in the presence of the ligand related to the DD or vehicle control. At the end of co culture, cells are lysed and luciferase activity is measured using appropriate substrate. Luciferase activity is expected to increase when DD regulated CD 19 CAR expressing T cells are co cultured with CD 19 expressing target cells in the presence of ligand. Cytotoxicity is not expected in vehicle control ceils or when the target cells do not express CD 19 are utilized.

[00652] Engagement of the CD 19 CAR with CD 19 antigen results in the activation of T cells which is measured 24 hours after co culture of CAR expressing T cells and target cells.

Activation of T cells is evaluated by measuring levels oflFNg, IL2, and CD69. T cell proliferation in response to antigen mediated T cell activation is measured by labelling T cells with Carboxyfluorescein succinimidyl ester, which is used to trace cells across multiple generations. Labelled T cells are cultured with Mitomycin treated target cells and cell proliferation is tracked over a period of 3 to 5 days. T cell proliferation and activation is expected to increase when DD regulated CD 19 CAR expressing T cells are co cultured with CD 19 expressing target cells in the presence of ligand. Both parameters are not expected in vehicle control cells or when the target cells do not express CD 19 are utilized.

[00653] Activation of T cells results in degranulation, an exocytic process by which cytotoxic T cells release molecules like perforin and granzymes which enable target cell killing.

Degranulation is measured by analysis of media for indications of exocytosis e.g. CD 107 by FACS and by markers of degranulation such as perforin and granzyme using immunoassays.

Example 34. Ligand dependent target cell death induced by DD regulated CD 9 CAR

[00654] To test the ability of DD regulated CD 19 CAR cells to kill target ceils, primary T cell populations are transduced with DD regulated CD 19 CAR constructs are co cultured with K562 cells expressing CD 19 (target cells) at a ratio of 5: 1 in the presence or absence of the ligand specific to the DD e.g. Shield- i (ΙμΜ), TMP (ΙΟΟμΜ) or MTX. Constmcts with FKBP, ecDHFR or human DHFR DDs may be utilized. Constructs with either CMV, EFla or PGK promoters may also be used. Multiple combinations of T ceils and target ceils are set up. These included DD regulated CAR expressing T cells co cultured with K562 cells (in the presence or absence of the ligand), T cells co cultured with K562 cells expressing CD 19 and K562 cells expressing CD19 without T cell co culture. Additional controls include target cells only;

untransduced T cells: T cells transduced with empty vector. The T cells utilized for this experiment are expanded for 11 days using protocols described in previous examples, frozen, thawed and transduced with the CD 19 CAR constructs. Target cells are treated with Mitomycin C to prevent their proliferation. The K562 cells are fluorescently labelled with NucLight Red and co cultured with T celis for 300 hours. Cell death is monitored by labelling cells with Amiexin V and the cell death in target K562 cells is monitored by evaluating cells that are positive for both Annexm V and NucLight Red using the IncuCyte® Live Cell Analysis System (Essen

Biosciences, Ann Arbor, MI). Target cell killing is expected with the DD regulated CAR constructs only in the presence of ligand and when K562 target cells ectopically expressing CD 19 are utilized. No cell killing is expected in untreated controls of the same co-culture set up and when T cells are co cultured with parental K562 cells that do not express CD19 in the presence or absence of ligand. Constitutive constructs are predicted to show cell killing both in the presence of ligand. Cell killing is also not expected in cocultures with untransduced T ceils, T cells transduced with empty vector; and cultures of target cells only.

[00655] To study the effect of ligand on the expression of cytokines in regulated CD 19 CAR constructs, T cell populations were transduced with empty vector, OT-CD 19-017, OT-CD 19- 023, OT-CD 19-024, or QT-CD19-G25. 5xl0⁴ transduced T celis were co cultured for 48 hours at an E:T (effector to target cell) ratio of 5: 1 in the presence or absence of TMP or Shield- 1. Target cells were treated with 50 ug/ml of Mitomycin C to prevent their proliferation. The cytokine concentration of IFNy and IL2 in the media supernatant were determined for each construct using MSD V-PLEX Proinflammatory Panel 1 Human Kit. The readout was obtained using a MESO QuickPlex SQ 120. As shown in Figure 31 A, a 6 fold increase in IFNy concentration was seen with the addition of ligand for OT-CD 19-024, and a 2 fold increase in IFNy concentration was seen with the addition of ligand for OT-CD 19-025. As shown in Figure 3 IB, a 6 fold increase in IL2 was seen for OT-CD 19-024 with the addition of ligand and a 9 fold increase was seen for OT-CD 19-025 with TMP.

[00656] HCTI 16 parental cells or cells transduced with IL12 constructs (OT-IL12-020, OT- IL12-026, or OT-IL12-029) were injected into immune compromised CD1 nude mice (n=4 per group) according to the study design in Table 30 below. HCTl 16 cells Day 15 Dose Concentration Route of Day 15 Dose

Vehicle n/a intraperitoneal

OT-IL 12-026 Shieid-i (lx) 10 mg/kg Intraperitoneal

Shield- 1 (3x, 2h apart) 10 mg kg Intraperitoneal

Vehicle n/a Intraperitoneal

OT-IL 12-029 Shield-1 (lx) 10 mg/kg Intraperitoneal

Shieid-i (3x, 2h apart) 10 mg/kg Intraperitoneal

Vehicle n/a Intraperitoneal

OT-IL 12-020

Shield-1 ( lx) 10 mg/kg intraperitoneal

Vehicle n/a Intraperitoneal

Parental

Shield-1 (lx) 10 mg kg Intraperitoneal

Tie mice were bled (blood harvested for plasma PK ane 1 11,12 MSD) at day 14 subcutaneous injection of 5x10⁶ cells (day 0), and 6, 10, and 24 hours post the day 15 dosing. At the end of the study, turnor and kidneys were minced with the razor in 500 ul PBS, spun down, and supernatant isolated for IL12 Meso Scale Diagnostic(MSD) assay.

[00658] As shown in Figure 32A, the basal plasma IL12 levels of the DD constructs were high, but the OT-IL12-026 and OT-IL12-029 constructs were still 100-fold lower than the constitutive (OT-IL 12-020) construct. When Figure 32A is shown as fold change from pre-dose plasma, OT- IL12-026 shows regulation at 6 and 10 hours. Figures 32B and 32C show that IL12 is detectable in kidney (Figure 32B) and tumor (Figure 32C) and the levels coordinate with plasma levels.

[00659] HCTl 16 parental cells or cells transduced with IL 12 constructs (OT-IL12-020, OT- IL12-026) were injected subcutaneously into Matrigel plus in female NSG mice (implant 200 ul matrigel plug with 1x10⁷ cells) (n=4) according to the study design in Table 31 below.

Table 31. Studv Design

|00660] Terminal collection of plasma (for IL12 MSD), plug supernatants and kidneys were collected. As shown in Figure 33 A, regulation of IL12 was achieved in vivo with high dose Aquashield. There was less regulation observed in the plasma (Figure 33B) and there was some flexi-IL12 detected in the kidneys (Figure 33C).

Example 38. Shield- 1 Can Induce ~40-5Gx Increases in IL12 Production by Primary

Human T Ceils Transduced with the IL12-026 Construct

[00661] On Day 0, primary human T cells were stimulated with Dynabeads (T-expander CD3/CD28) at a 3: 1 bead:cell ratio. The next day, lenti viruses (empty vector (pLVX-EFIa- IRES-Puro), OT-IL 12-020 (constitutive), or OT-IL 12-026 (regulated)) were added at a

multiplicity of infection (MOD of 10 in the presence of LentiBOOST and 5% FBS. On day 2, the cells were washed to remove the LentiBOOST and the bead:ceil ratio was reduced to 1 :3, and fresh 10% media and IL2 were added. On days 6, 9, and 13 the cells were counted for equal cell number plating, media replaced, iigand was added, and cells were either left unstimulated or restimuiated with soluble ImniunoCult™ Human CD3/CD28 T Cell Activator (StemCell Technologies). After overnight incubation (on days 7, 10, and 14), the supernatants were collected for IL12p40 and p7() MSD assay, and transduction efficiency was analyzed by FACS. OT-IL 12-026 T cells were found to be 7% transduced, and OT-IL 12-020 (constitutive) T ceils were 13% transduced on day 7. Restimulation was shown to increase the expression of IL12 (Figure 34A). Ligand increased production of IL12 by 10-day expanded OT-IL 12-026 expressing T cells by 40-50 fold (Figure 34B and Figure 34C).

Example 39. Dose Response of Shield- 1 on Transduced T Cells

[00662] Human T ceils were activated with CD3/CD28 Dynabeads (Life Technologies) for 1 day prior to transduction with lentiviruses (OT-IL12-026 or vector control), followed by 12-13 days of expansion in culture. T cells that had been transduced with different amounts of virus (4-40 MOI) were exposed to either a dose response of Shield- 1 for 24h (left panel). T cells that had been transduced at an MOI of 14 were treated with iuM Shield- 1 or vehicle control for increasing amounts of time (right panel). The levels of IL12 that had accumulated in the supernatants (from 100,000 cells per 200uL media) were measured using human IL12p40 MSD V-plex assay kits (Meso Scale Discovery).

[00663] From the analysis, it was shown that the increase in IL12 production by T cells expressing OT-IL12-026 is dose responsive to the ligand. Shield- 1 Figure 35A, and accumulates over time Figure 35 B. 'ινο Uose Kesponse, anc iice witn

Transferred T Cells Expressing OT-IL12-

[00664] Primary human T cells were stimulated with Dynabeads (T-expander CD3/CD28) at a 3: 1 beadiceil ratio. The next day, lentiviruses (OT-1L 12-020 (constitutive), OT-IL12-026 (regulated), or vector control) were added at a multiplicity of infection (MOI) of 10 in the presence of LentiBOOST and 5% FBS. The following day, T cells were washed to remove the LentiBOOST and the bead:cell ratio was reduced to 1 :3, and fresh 10% media and 1L2 were added. The T cells were expanded for a total of 10 days, and then 25xi0⁶ vector control or OT- IL12-026 transduced T cells or l OxlO⁶ constitutive OT-IL 12-020 transduced T cells were transferred into NSG mice (study day 0). Three days after cell transfer, the animals were dosed with either vehicle or AquaShieid (10, 50 or iOOmg/kg). Blood was sampled for plasma analysis of IL12p70 by MSD assay at 0, 4, 8, and 24h post dosing (Figure 36A). Clear dose responsive increases in plasma IL12 was observed.

[00665] On day 5 post T cell transfer, animals were dosed a second time with AquaShieid (Figure 36B). A second increase in plasma IL12 was observed upon repeat dosing with

AquaShieid.

Example 41. In Vivo Regulation of DD-12 Expressed in T Cells

[00666] To determine whether ligand can stabilize DD-IL12 in vivo upon sequential dosing of AquaShieid, T cells are transduced with DD-DL 12-expressing constructs (OT-IL 12-020 or OT- IL 12-026) and implanted into mice (n=4 per group) (day 0) as outlined in the study design below.

Table 32. Study Design

For each group, a pre-bleed sample is collected as well as samples at 4 hours and 24 hours after each dose. At the end of the study, tissue and organ samples are collected. FACS analysis is conducted to determine cell numbers and Thl markers. |00668] On Day 0, primary human T cells were stimulated with Dynabeads (T-expander CD3/CD28) at a 3: 1 bead:ceil ratio. The next day, lentiviruses (empty vector (pLVX-EFla- IRES-Puro), OT-IL 12-020 (constitutive), or OT-IL 12-026 (regulated)) were added at a multiplicity of infection (MOI) of 10 in the presence of LentiBOOST and 5% FBS. On day 2, the cells were washed to remove the LentiBOOST and the bead:ceil ratio was reduced to 1 :3, and fresh 10% media and IL2 were added.

|0Θ669] In vitro evaluation of these cells is shown under Figure 37A-37C.

|00670] After 10 days of expansion, T ceils were injected into NSG mice (12 x 10 ⁶ ceils injected, cells were 15% (constitutive) and 7.5% (regulated) IL12 positive by FACS). For each group, a pre-bleed sample was collected as well as plasma samples at 4 hours and 24 hours after each dose. At the end of the study, tissue and organ samples are collected. FACS analysis was conducted to determine T cell numbers in the blood and to assess Thl phenotypic markers.

[00671] As shown in Figure 37A, IL12 expression in response to sequential pulsed doses of ligand (50 mg/kg Aquashield administered orally on day 4 and 6 (50 mpk Aquashield q48hr)) was elevated in the plasma of mice with T cells expressing OT-IL 12-026 as compared to the vehicle treated controls. T cells expressing the empty vector control did not produce IL12. T cells transduced with OT-IL12-020 (IL12-020), the constitutive control, produced IL12 throughout the time course.

[00672] In Figure 37B, elevated plasma IL12 expression in response to sequential pulsed doses of ligand (50 mg/kg Aquashieid administered orally for 4 days (day 3-6) (50 mpk Aquashield QDx4)) was seen in mice bearing OT-IL12-026 expressing T cells as compared to the vehicle treated controls. Cells transduced with OT-IL 12-020 (IL12-020), the constitutive control, produced IL12 throughout the time course.

[00673] Figure 37C shows the IL12 expression over 1 1 days for the constitutive construct OT- IL 12-020 (IL 12-020). Ligand-regulated expression of IL12 from T cells expressing DD-IL12 from the construct OT-IL 12-026 was seen in mice treated with 50 mg/kg Aquashield administered orally on day 5 and 10 (50 mpk Aquashield d.5/10). T cells expressing the empty vector control did not produce IL12.

[00674] Figure 37D shows ligand-induced regulation of plasma IL12 expression from T cells expressing DD-IL12 from the construct OT-IL 12-026 when mice were treated orally with 50 mg/kg Aquashield on day 10 (50 mpk Aquashield dlO). The single ligand pulse increased plasma 11. 12 levels over those detected in vehicle-treated control mice harboring OT-IL12-026 expressing T cells. [00675| Regulation of 1L12 for all constructs shown in Figures 37A-37D did not impact IFNy levels, instead the levels of IFNy gradually rose over time. This is likely due to the exposure of the T cells to IL12 in culture during the in vitro expansion phase. However, ligand-induced regulation of IL12 increased granzyme B (GrB) (Figure 37E) and perforin expression (Figure 29F) by CD8+ T cells in vivo at day 7 post in vivo T cell transfer.

Example 42. Effect of PGK Promoter and N-terminal FKBP

[00676] HEK293T cells were transiently transfected with Lipofectamine 3000 and 2ug plasmid DNA each of: OT-IL12-019 (PGK promoter), OT-IL12-020 (EF1 alpha promoter), OT-1L12-025 (PGK promoter, C-terminal FKBP domain), OT-IL 12-026 (EFlalpha promoter, C-terminal FKBP domain), OT-IL12-046 (N-terminal FKBP). Ligand (luM Shield- 1) was added one day after transfection, and the cells were further cultured for 2 more days. IL12 secretion into the superaatants was quantitated by IL12p40 MSD assay. Genomic DNA (gDNA) and messenger RNA (mRNA) was purified from the cells. The levels of construct DNA integration into the cellular genome and levels of IL12 mRNA expression were quantitated by qPCR using primers specific to the WPRE element and IL12 within the respective constructs.

[00677] The gDNA qPCR analysis demonstrated that the FKBP DD-containing constructs had integrated to similar levels within the cellular genomes, and that the PGK promoter, as expected, generated less IL12 mRNA expression than the EFlalpha promoter (Figure 38A).

[00678] Due to the lower levels of mRNA transcription induced by the PGK promoter, the IL12p40 MSD assay also demonstrated that the PGK promoter reduced both basal and peak IL12 levels of secretion as compared to the construct using the EFlalpha promoter. The lower basal levels of IL12 production downstream of the PGK promoter resulted in ~2 fold improved ligand- induced IL12 regulation as compared with the construct with the EFlalpha promoter (Figure 38B). More specifically, the ligand-induced regulation of 1L12 expression increased from 6-fold to 13-fold with the change from the EFlalpha to the PGK promoter, respectively.

[00679] Constructs containing FKBP either at the N-terminus or at the C-tenminus of IL12 were integrated similarly into the cellular genome and generated similar levels of mRNA (Figure 38A). However, while C-terminal containing FKBP constructs regulate IL12 expression, the N- terminal-containing FKBP construct failed to regulate IL12 expression (Figure 38B).

Example 43. Kinetics of ligand-dependent stabilization of DD-IL15- ILlSRa

[00680] The on/off kinetics of ligand-dependent stabilization of DD-lL15-lL15Ra was measured in CD4 positive T cells. T cells were activated with CD3/CD28 beads at 3: 1 bead to T cell ratio in 24-well plates for 24 hrs. Lentivirus was added to wells in the presence of LentiBoost reagent, and cells were incubated for another 24 hrs and washed. Cells were resuspended in fresh media, and media was added every 2-3 days to expand and maintain cells at 0.5-lxl0⁶/ml. After 7 days of expansion, T cells transduced with the ecDHFR DD-IL15-IL15Ra fusion construct (OT-IL15-009) were treated with ΙΟΟμΜ ecDHFR iigand Trimethoprim (TMP) or vehicle control, DMSO. At multiple time points (i.e., 1, 2, 4, 6, 8, 15, 22 and 24 hrs) after TMP treatment, the transduced T cells were collected and analyzed for IL15Ra surface expression using anti-IL15Ra antibodies by flow cytometry. Untransduced T cells were used as a negative control. The T cells were sorted into CD4 positive and CDS positive populations and the percentage of IL15Ra positive CD4 positive T cells was analyzed. Figure 39 shows the kinetics of surface expression of IL15Ra on CD4 T cells after TMP treatment. Among the CD4 positive T cells transduced with the OT-IL 15-009 construct, the proportion of cells with surface expression of IL15Ra remained similar for both TMP treated and DMSO treated cells until 2 hrs after TMP treatment, and was comparable to that of untransduced ceils. However, from 4 hrs after TMP treatment, the cells transduced with the OT-IL 15-009 constract and treated with TMP exhibited an increased proportion of cells with surface expression of IL15Ra. This trend was observed until 22 hours after treatment with TMP. The CD4 positive T cells with surface- expressed IL15Ra cells constituted ~1% of untransduced cells, indicating that the proportion of cells that expressed endogenous ILiSRa is low.

Example 44. Ligand-dependent stabilization of DD-IL15-IL15Ra fusion molecules in vivo |00681] To examine whether iigand treatment induces stabilization of the DD-IL15-IL15Ra fusion molecules in vivo, HCT116 cells transduced with the OT-DL 15-009 constract were implanted subcutaneously in BALB/c nude mice and treated with TMP. TMP was orally administered to mice at a dose of 100 mg/kg, twice a day for 11 days after implantation, followed by administration of TMP at the dose of 300 mg/kg, twice a day for 6 days. As a negative control, separate mice implanted with HCT1 16 cells transduced with the OT-IL 15-009 construct were treated with the vehicle twice a day for 17 days. At 4 hrs after the last dosing of TMP or the vehicle control, tumors were harvested from the mice and analyzed for the levels of IL15-IL15Ra fusion molecules by western blotting. As shown in Figure 40, HCT116 tumors harvested from mice treated with TMP exhibited elevated levels of IL15-IL15Ra expression, compared to tumors treated with the vehicle. The GAPDH level was analyzed as a loading control. These data show that administration of iigand enabled stabilization of the DD-IL15- IL15Ra fusion molecule in vivo.

|0Θ682] Consistent with the efficacy of TMP -dependent IL15-IL15Ra stabilization in vivo, elevated levels of TMP (399.38 ng/g tumor) were observed in HCT116 tumors harvested from mice treated with TMP for 17 days. The levels of TMP associated with HCT116 tumors were considerably higher than those observed in mouse plasma at day 3 (15.67 ng/ml plasma) and at day 17 (99.5 ng/ml plasma), indicating that the orally administered TMP was successfully delivered to and accumulated in HCT1 16 tumors implanted in mice.

Example 45. Shedding resistant IL15-IL15Ra constructs

[00683] To maintain the efficiency of the trans-presentation of IL15 via the IL 15-IL 15 Ra fusion molecule, the IL15-IL15Ra shedding needs to be prevented. For this purpose, new DD-IL15- IL15Ra and constitutive IL15-IL15Ra constructs are designed through a variety of modifications on the IL15-IL15Ra fusion molecule. For example, the ILLS molecule or the IL15Ra molecule is truncated or mutated to remove presumable cleavage sites. ILlSRa has a cleavage site

(PQGHSDTT from the position 168 to 175 of SEQ ID NO. 803) in the extracellular domain immediately distal to the transmembrane domain of the receptor, as described by Bergamaschi C et al. (2008). J Biol Chem ;283(7):4189-99; Anthony SM et al. (2015). PLoS One. 10(3):

eO 120274), and International Patent Application Publication Nos. WO2014066527 and

WO2009002562 (the contents of each of which are incorporated herein by reference in their entirety). Tumor necrosis facto r-alpha-converting enzyme (TACE/ADAM17) has been implicated as a protease that cleaves between glycine (at the position 170 of SEQ ID NO. 803) and histidine (at the position 171 of SEQ ID NO. 803) and generates a naturally occurring soluble form of ILlSRa. The same mechanism can be responsible for the ILi5-ILi5Ra shedding. Hence, the cleavage site of ILlSRa is mutated such that cleavage by an endogenous protease is prevented. The mutation of the cleavage site is introduced by substitution, insertion or deletion of amino acid residues. The IL15-IL15Ra fusion molecule is also modified such that the full- length or truncated IL15-IL15Ra fusion molecule is fused to heterologous hinge domains and/or heterologous transmembrane domains. As non-limiting examples, variants of IL ! 5Ra can be utilized. Additionally, the length and sequence of the linkers that connect IL 15 and ILlSRa are modified.

[00684] To confirm that the modifications on the IL15~IL15Ra fusion molecule prevent shedding, the new DD-TL15-TL1 SRa or constitutive IL15-IL15Ra constructs are introduced into HCT-1 16 cells. Surface expression of IL15 and ILlSRa on the HCT-116 cells is examined by flow cytometry using anti-ILlS and ILlSRa antibodies to assess surface lL15-lL15Ra shedding. The presence or absence of ILLS in the cell culture supernatant is also analyzed by MSD assay. As a functional assay based on the sensitivity of NK cell activation by shed IL 15 in tumor supernatant, the transwell assay is conducted using HCT-116 ceils transduced with new DD- IL15-IL15Ra or constitutive IL15-IL15Ra expressing constructs and NK cells. The new DD-

— 233 IL15-IL15Ra-expressing constmcts that do not induce activation of NK cells in the presence of ligand and the new constitutive IL15-IL15Ra-expressing constructs that do not induce activation of NK cells are chosen for use in future experiments.

Example 46. Regulated expression of IL15-IL15Ra fusion molecule with C-terminal DD

$85] A fusion molecule is generated by fusing membrane bound IL15, IL15 Receptor alpha subunit (TL15Ra) and a human DHFR (DD). These fusion molecules were cloned into pLVX- EFla-IRES-Puro vector.

|00686] To test ligand dependent IL15-IL15Ra production, 1 million HEK-293T cells were plated in a 6-well plate in growth media containing DMEM and 10 FBS and incubated overnight at 37°C, 5% C02. Cells were then transfected with 1 OOng of constitutive IL15-IL15Ra (OT- IL15-008) or DD linked IL15-IL15Ra (OT-IL15-037 or OT-IL15-040) using Lipofectamine 2000 and incubated for 24 hrs. Following the incubation, media is exchanged for growth medium with or without 50μΜ Trimethoprim (TMP) and further incubated for 48 hrs. Cells were harvested and 11,15 levels are analyzed via western blotting using human IL15 antibody (Abeam, Cambridge, UK). The molecular weight of IL15Ra in OT-IL15-037 and OT-IL15-040 appeared to be the same as OT-IL15-008.

[00687] To test if IL15 is shed into the media, supernatant from HEK293 cells expressing IL15- IL15Ra fusion constructs was subject to immunoassays such as MSD (Rockville, Maryland). 48 hours after transfection, cells were analyzed and, as expected, constitutive IL15-IL15Ra construct OT-IL15-008 showed high surface expression of IL15 in the presence and absence of ligand. OT-IL15-037 and OT-IL15-040 showed the ligand (Trimethoprim) dependent surface expression of IL15 and IL15Ra (Figure 41). The detection of membrane bound IL15-IL15Ra fusion constmcts in the supernatant suggests that IL15 constmcts are likely shed from the cell surface.

Example 47. Effect TMP exposure to TMP in vitro on membrane bound IL15 expression

[00688] In order to determine if the dose and time of exposure to TMP in vitro influenced membrane bound IL15 expression, an in vitro dose response study was conducted with T cells expressing OT-IL 15-073.For this purpose, T cells were activated with CD3/CD28 beads at 3: 1 bead to T cell ratio in 24-well plates for 24 hrs. Lentivirus was added to wells. After 24 hrs, fresh media was added every 2-3 days to expand cells while maintaining cells at 0.5-lxl0⁶/ml . On day 11 of expansion. T cells treated with TMP starting at 100 uM, lOx dilutions and 9 points were analyzed after 2 hours in culture (washed 3x after TMP addition, fresh media added without TMP for 22 hours), 6 hours in culture, or 24 hours in culture and the results are shown in Figure 42A. As shown in Figure 42B and Table 33, this study showed that TMP iigand regulates membrane bound IL15 expression and the dose and time of exposure to TMP in vitro influences membrane bound IL15 expression.

^* lembrane Hound lLls Expression

Example 48. Regulated membrane bound IL15 expression in vivo

[ 00689 j To e valuate regulation of membrane bound IL15 in vivo, 2 constructs were selected for evaluation in vivo. Four group of T cells were used for this study and are outlined in Table 34. In Table 31, "N" represents the number of mice in each group.

[00690] The T ceils which were to be used as part of the in vivo study were evaluated 6 days post transduction, day of implant (day 9 post transduction) and 13 days post transduction and the cells in Groups 2-4 showed expression of the constructs.

|00691 ] T cells outlined in Table 31 were administered to mice by intravenous administration (3.9 x 10⁶ cells per mouse implanted). On day 3 the mice were dosed with 500 mg/kg of TMP 3 times (4 hours between doses) and bled 2 hours after each dose. The mice were again bled on day 4, 24 hours after the first TMP dose.

[00692] Figures 43A-43C show the expression of membrane bound IL15, 2, 6, 10, and 24 hours after the first TMP dose, using IL15 staining (Figure 43A), ILl SRa staining (Figure 43B), and IL15/'IL15Ra double ++ staimng (Figure 43C). Figure 43D are FACS plots for each mouse 10 hours after the first TMP dose. Figure 43E shows the expression of membrane bound IL15 in blood 2, 6, 10, and 24 hours after the first TMP dose and Figure 43F shows the plasma TMP levels 2, 6, 10, and 24 hours after the first TMP dose. [00693] In this study, T cells transduced with OT-IL15-071 or OT-IL15-073 (no lentiBoost) were administered intravenously to mice (15 x 10⁶ per mouse). 6 study groups were evaluated for this study: (1) untransduced, (2) OT-IL15-071 T cells, (3) OT-IL15-073 PO vehicle, (4) OT- IL15-073 PO TMP 500 mg/kg), (5) OT-IL15-073 IP vehicle, and (6) OT-IL15-073 IP TMP 300 mg/kg. The study design is shown in Table 35. PO dosing is 500 mg/kg TMP in 0.1M citrate and IP dosing is 300 mg kg TMP lactate in water.

Table 35, Study Design

[00694] The regulated expression in blood was analyzed 6 hours and 24 hours after the first dose, and 6 hours after the 5^th dose .

[00695] OT-IL 15-071 showed expression of membrane bound IL15 and the untransduced control did not show any expression.

[00696] Regulation of membrane bound IL15 was seen with repeat PO and IP dosing. As seen in Figure 44, regulated expression of membrane bound IL15 was detected 6 hours after the first dose on day 0, and 6 hours after dosing on day 5 (126 hrs) with both PO and IP dosing. There was no increase in expression in mice treated with vehicle.

[00697] While the present invention has been described at some length and with some particularity with respect to the several described embodiments, it is not intended that it should be limited to any such particulars or embodiments or any particular embodiment, but it is to be construed with references to the appended claims so as to provide the broadest possible interpretation of such claims in view of the prior art and, therefore, to effectively encompass the intended scope of the invention.

[00698] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, section headings, the materials, methods, and examples are illustrative only and not intended to be limiting.

Claims (51)

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.

2. The composition of claim. 1, wherein said at least one immunotherapeutic agent is selected from a chimeric antigen receptor (CAR) and an antibody.

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

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

5. The composition of claim 4, 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, and

(g) NQ02 comprising the amino acid sequence of SEQ ID NO. 7.

6. The composition of claim 5, wherein the parent protein is hDHFR and the DD comprises a mutant protein having:

(a) a single mutation selected from hDHFR (117V), hDHFR (F59S), hDHFR (N65D), hDHFR (K81R), hDHFR (A 107V), hDHFR (Y122I), hDHFR (N127Y), hDHFR

(M140I), hDHFR (K185E), hDHFR (N186D), hDHFR (M140I), hDHFR (Ammo acid 2- 187 of WT; 127Y), hDHFR (Amino acid 2-187 of WT; I17V), hDHFR (Amino acid 2- 187 of WT; Y 122I), and hDHFR (Amino acid 2-187 of WT: K185E);

(b) a double mutation selected from hDHFR (C7R, Y163C), hDHFR (A IOV, H88Y), hDHFR (Q36 , Y122I), hDHFR (M53T, R138I), hDHFR (T57A, I72A), hDHFR (E63G, 1176F), hDHFR (G21T, Y1221), hDHFR (L74N, Y1221), hDHFR (V75F, Y122I), hDHFR (L94A, T147A), DHFR (V121A, Y22I), hDHFR (Y122L A125F), hDHFR (H131R, E144G), hDHFR (T137R, F143L), hDHFR (Y178H, E18IG), hDHFR (Y183H, K185E), hDHFR (E162G, I176F) hDHFR (Amino acid 2-187 of WT; I17V, Y122I), hDHFR (Ammo acid 2-187 of WT; V ί 221. M140I), hDHFR (Ammo acid 2-187 of WT; N127Y, Y122I), hDHFR (Ammo acid 2-187 of WT; E162G, T 76F), and hDHFR (Ammo acid 2-187 of WT; H131R, E144G), and hDHFR (Ammo acid 2-187 of WT; Y1221, A125F);

(c) atriple mutation selected from hDHFR (V9A, S93R, P150L), hDHFR (18V, K133E, Y163C), hDHFR (L23S, VI 21 A, Y157C), hDHFR (K19E, F89L, E181G), hDHFR (Q36F, N65F, Y122I), hDHFR (G54R, M14QV, S168C), hDHFR (VI 10A, V136M,

K I77RK hDHFR (Q36F, Y1221, A125F), hDHFR (N49D, F59S, D153G), hDHFR (G21E, I72V, I176T), hDHFR (Amino acid 2-187 of WT; Q36F, Y122I, A125F), hDHFR (Ammo acid 2-187 of WT; Y122I, H131R, E144G), hDHFR (Amino acid 2-187 of WT; E31D, F32M, VI 161), and hDHFR (Amino acid 2-187 of WT; Q36F, N65F, Y122I); or

(d) a quadruple or higher mutation selected from hDHFR (V2A, R33G, Q36R, LI OOP, K185R), hDHFR (Ammo acid 2-187 of WT; D22S, F32M, R33S, Q36S, N65S), hDHFR (I17N, L98S, K99R, Ml 12T, E151G, E162G, E172G), hDHFR (G16S, I17V, F89L, D96G, K123E, M140V, D146G, K156R), hDHFR (K81R, K99R, LIOOP, E102G,

N108D, K123R, H128R, D142G, F180L, K185E), hDHFR (R138G, D142G, F143S, K156R, K158E, E162G, V166A, 177E, Y178C, K185E, N186S), hDHFR (N14S, P24S, F35L, M53T, K56E, R92G, S93G, N127S, H128Y, F135L, F143S, L159P, L160P, E173A, F180L), hDHFR (F35L, R37G, N65A, L68S, K69E, R71G, L80P, K99G, G117D, L132P, I139V, M140I, D142G, D146G, E173G, D187G), hDHFR (L28P,

N30H, M38V, V44A, L68S, N73G, R78G, A97T, K99R, A107T, K109R, D111N, L134P, F135V, T147A, I152V, K158R, E172G, V182A, E184R), hDHFR (V2A, I17V, N30D, E31G, Q36R, F59S, K69E, I72T, FI88Y, F89L, N108D, K109E, VI 1 OA, II 15V, Y122D, L132P, F135S, M140V, E144G, T147A, Y157C, V170A, K174R, N186S), hDHFR (L100P, E102G, Q103R, P104S, E105G, N108D, V113A, Wl 14R, Y122C, M126I, N127R, H128Y, L132P, F135P, I139T, F148S, F149L, I152V, D153A, D169G, V170A, I176A, K177R, V182A, K185R, N186S), and hDHFR (A10T, Q13R, NHS, N20D, P24S, N30S, M38T, T40A, K47R, N49S, K56R, I61T, K64R, K69R, Γ72Α, R78G, E82G, F89L, D96G, N108D, Ml 12V, W114R, Y 122D, K123E, 1139V, Q 141R, D142G, F148L, E151G, E155G, Y157R, Q171R, Y 183C, E184G, K185del, D187N).

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

8. The composition of claim 2, wherein the immunotherapeutic agent is a chimeric antigen receptor (CAR).

9. The composition of claim 8, wherein the chimeric antigen receptor (CAR) comprises

(a) an extracellular target moiety;

(b) a transmembrane domain;

(c) an intracellular signaling domain; and

(d) optionally, one or more co-stimulatory domains.

10. The composition of claim 9, wherein the CAR is 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.

11. The composition of claim 9, wherein the extracellular target moiety recognizes a target molecule on the surface of a cancer cell, wherein said target molecule on the surface of the cancer cell is selected from a cancer antigen, a plasma membrane lipid, a receptor and a membrane bound glycoprotein.

12. The composition of any of claims 9-11, 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

\ il l 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 protein of interest, a ligand, a receptor, a receptor fragment or a peptide aptamer.

13. The composition of claim 12, wherein the extracellular target moiety is a scFv derived from an antibody that specifically binds a CD 19 antigen.

14. The composition of claim 13, wherein the scFv is a CD 19 scFv is selected from one that comprises:

(a) a heavy chain variable region having an amino acid sequence independently selected from the group consisting of SEQ ID NO: 49-80, and a light chain variable region having an amino acid sequence independently selected from the group consisting of any of SEQ ID NOs: 81-122; or

(b) an amino acid sequence selected from the group consisting of any of SEQ ID NOs: 123-267 and 624.

15. The composition of claim 9, wherein

(a) the intracellular signaling domain of the CAR is the signaling domain derived from T ceil receptor CD3zeta or a ceil surface molecule selected from the group consisting of FcR gamma, FcR beta, CD 3 gamma, CD3 delta, CD3 epsiion, CDS, CD22, CD79a, CD79b, and CD66d; and

(b) the co-stimulatory domain is present and is selected from the group consisting of 2B4, HVEM, 1COS, LAG3, DAP10, DAP 12, CD27, CD28, 4-1BB (CD137), OX40 (CD134), CD30, CD40, ICOS (CD278), glueocortieoid-induced tumor necrosis factor receptor (GITR), lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT,

NKG2C, and B7-H3.

16. The composition of claim 15, wherein the intracellular signaling domain of the CAR is a T cell receptor CDSzeta signaling domain comprising the amino acid sequence of SEQ ID NO: 339.

17. The composition of claim 9, wherein the intracellular signaling domain of the CAR is a T cell receptor CD3zeta signaling domain comprising the amino acid sequence of SEQ ID NO: 626 and the co-stimulatory domain is present, said co-stimulatory domain being selected from amino acid sequence of any of SEQ ID NOs: 268-374.

1 8. The composition of claim 9, wherein the transmembrane domain is derived from any of the members of the group consisting of:

(a) a transmembrane region of an alpha, beta or zeta chain of a T-celi receptor;

(b) the CD3 epsilon chain of a T-cell receptor;

(c) a molecule selected from CD4, CDS, CD 8, CD8a, CD9, CD 16, CD22, CD33, CD28, CD37, CD45, CD64, CD80, CD86, CD 148, DAP 10, EpoRI, GITR, LAG3, ICOS, Her2, OX40 (CD 134), 4-1BB (CD 137), CD 152, CD 154, PD-1, or CTLA-4; and

(d) an immunoglobulin selected from IgGI , IgD, IgG4, and an IgCn Fc region.

19. The composition of claim 9, wherein the transmembrane domain comprises an amino acid sequence selected from the group consisting of any of SEQ ID NOs: 375-425 and 897-907.

20. The composition of claim 9, wherein the CAR further comprises

(e) a hinge region near the transmembrane domain, said hinge region comprising an amino ac d sequence selected from the group consisting of any of SEQ ID NOs: 426-504.

21. The composition of claim 2, wherein the immunotherapeutic agent is an antibody that is specifically immunoreactive to an antigen selected from a tumor specific antigen (TSA), a tumor associated antigen (TAA), or an antigenic epitope.

22. The composition of claim 21, wherein the antigen is an antigenic epitope and said antigenic epitope is CD 19.

23. The composition of claim 22, wherein the antibody is selected from one that comprises (a) a heavy chain variable region having an amino acid sequence independently selected from the group consisting of any of SEQ ID NOs: 49-80 and a light chain variable region having an amino acid sequence independently selected from the group consisting of any of SEQ ID NOs: 81-122; or

(b) an amino acid sequence selected from, the group consisting of any of SEQ ID NOs:

123-267.

24. The composition of claim 1 wherein said first effector module comprises the amino acid sequence of any of SEQ ID NO: 635-649, 1005-1010, 1015-1018, and 1215-1231.

25. The composition of claim 24, wherein said first SRE of the effector module stabilizes the immunotherapeutic agent by a stabilization ratio of 1 or more, wherein the stabilization ratio comprises the ratio of expression, function or level of the immunotherapeutic agent in the presence of the stimulus to the expression, function or level of the immunotherapeu tic agent in the absence of the stimulus.

26. The composition of any of claims 24-25, wherein the SRE destabilizes the

immunotherapeutic agent by a destabilizaiion ratio between 0, and 0.09, wherein the de stabilization ratio comprises the ratio of expression, function or level of the

immunotherapeutic agent in the absence of the stimulus specifi c to the SRE to the expression, function or level of the immunotherapeutic agent that is expressed constitutively, and in the absence of the stimulus specific to the SRE.

27. A polynucleotide encoding any of the compositions of claims 1-26.

28. The polynucleotide of claim 27, wherein the polynucleotide is a D A molecule, or a RNA molecule.

29. The polynucleotide of claim 28, wherein the polynucleotide is an RNA molecule and said RNA molecule is a messenger RNA.

30. The polynucleotide of claim 29, which is chemically modified.

31. The polynucleotide of claim. 28, which comprises spatiotemporally selected codons.

32. The polynucleotide of claim 29, further encoding a promoter, a linker, a signal peptide, a tag, a cleavage site and/or a targeting peptide,

33. A vector comprising a polynucleotide of any of claims 27-32.

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

35. The vector of claim 34, whic is a viral vector and wherein the viral vector is a retroviral vector, a lent! viral vector, a gamma retroviral vector, a recombinant AAV vector, an adeno viral vector, or an oncolytic viral vector.

36. An immune cell for adoptive cell transfer (ACT), which expresses any of the compositions of any of claims 1-26, the polynucleotides of any of claims 27-32, and/or is infected or transfected with the vector of any of claims 33-35.

37. Tire 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 (TIL), a memory T cell, a regulatory T (Treg) cell, a cytokine-induced killer (CDC) 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 immune ceil 49which further expresses a composition comprising a second effector module, said second effector module comprising a second SRE linked to a second immunotherapeutic agent wherein the second immunotherapeutic agent is selected from a cytokine, and a cytokine- cytokine receptor fusion.

39. The immune cell of claim 38, wherein the second immunotherapeutic agent is a cytokine.

40. The immune cell of claim 39, wherein the cytokine is IL12 or 1L15.

41. The immune cell of claim 38, wherein the second immunotherapeutic agent is a cytokine- cytokine receptor fusion polypeptide.

42. The immune cell of claim 41, wherein the cytokine-cytokine receptor fusion polypeptide is selected from a IL12-IL12 receptor fusion polypeptide, a IL15-IL15 receptor fusion polypeptide, and a IL15-IL15 receptor sushi domain fusion polypeptide.

43. The immune cell of claim 36 or 37, wherein the immune cell is autologous, allogeneic, syngeneic, or xenogeneic in relation to a particular individual subject.

44. A method of reducing a tumor volume or burden in a subject, comprising contacting said subject with a composition of any of claim s 1-2,6, the polynucleotides of any of claims 27-32, the vectors of any of claims 33-35 or the immune cells of any of claims 36-43, wherein the SRE responds to a stimulus and regulates the expression and function of the immunotherapeutic agent.

45. 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 1-26, the polynucleotides of any of claims 27-32, the vectors of any of claims 33-35 or the immune cells of any of claims 36- 43.

46. A method of identifying a domain of a CD 19 antigen which will not bind the FMC63 antibody (FMC63-distinct CD19 binding domain), said method comprising:

(a) preparing a composition comprising a CD19 antigen,

(b) contacting the composition in (a) with saturating levels of FMC63 antibody,

(c) contact the composition of step (b) with one or more selected members of a library of potential CD 19 binders; and

(d) identifying a binding domain on the CD 19 antigen based on the differential binding of the selected members of the library of CD19 binders compared to the binding of FMC63.

47. The method of claim 46, wherein said binding domains of the library are generated using phage display techniques with the CD19 antigen as the seed sequence.

48. The method of claim 47, wherein the binding domain is selected from 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 protem (dsFv), a unibody, a nanobody, or an antigen binding region of an antibody, and an antibody fragment.

49. The method of claim 48, wherein the CD19 antigen is selected from a whole or a portion of a human CD 19 antigen, and a whole or a portion of a Rhesus CD19 antigen.

50. A chimeric antigen receptor comprising the FMC63-distinct CD 19 binding domain obtained according to the method of any of claims 46-49.

51. An effector module comprising a stimulus response element (SRE) operably lined to the chimeric antigen receptor of claim 50.