WO2018161017A1 - Cd19 compositions and methods for immunotherapy - Google Patents

Cd19 compositions and methods for immunotherapy Download PDF

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Publication number
WO2018161017A1
WO2018161017A1 PCT/US2018/020741 US2018020741W WO2018161017A1 WO 2018161017 A1 WO2018161017 A1 WO 2018161017A1 US 2018020741 W US2018020741 W US 2018020741W WO 2018161017 A1 WO2018161017 A1 WO 2018161017A1
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WO
WIPO (PCT)
Prior art keywords
hdhfr
seq
domain
car
amino acid
Prior art date
Application number
PCT/US2018/020741
Other languages
English (en)
French (fr)
Inventor
Vipin Suri
Michael Joseph BRISKIN
Brian DOLINSKI
Kutlu Goksu ELPEK
Dan Jun LI
Scott Francis HELLER
Michelle Lynn OLS
Dexue Sun
Nicole KOSMIDER
Abhishek KULKARNI
Vijaya BALAKRISHNAN
Tucker EZELL
Original Assignee
Obsidian Therapeutics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to CN201880025103.XA priority Critical patent/CN110831961B/zh
Priority to EP18760420.2A priority patent/EP3589646A4/en
Priority to JP2019568594A priority patent/JP7341900B2/ja
Priority to AU2018227583A priority patent/AU2018227583B2/en
Priority to SG11201907922PA priority patent/SG11201907922PA/en
Priority to EA201991827A priority patent/EA201991827A1/ru
Priority to CA3055202A priority patent/CA3055202A1/en
Priority to KR1020197029002A priority patent/KR102746901B1/ko
Application filed by Obsidian Therapeutics, Inc. filed Critical Obsidian Therapeutics, Inc.
Publication of WO2018161017A1 publication Critical patent/WO2018161017A1/en
Priority to US16/558,224 priority patent/US11629340B2/en
Priority to US17/646,212 priority patent/US12104178B2/en
Priority to AU2023214349A priority patent/AU2023214349A1/en
Priority to JP2023139792A priority patent/JP2023164900A/ja
Priority to US18/787,024 priority patent/US20240401004A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4211—CD19 or B4
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
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    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0026—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5)
    • C12N9/0028—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on CH-NH groups of donors (1.5) with NAD or NADP as acceptor (1.5.1)
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    • C07K2317/00—Immunoglobulins specific features
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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Definitions

  • the present invention relates to compositions and methods for immunotherapy.
  • compositions comprise destabilizing domains (DDs) which tune protein stability.
  • DDs destabilizing domains
  • 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.
  • ACT adoptive cell transfer
  • TME tumor microenvironment
  • immunotherapeutic agent expression is needed in case of adverse events.
  • 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.
  • 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.
  • SRE stimulus response element
  • 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.
  • 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.
  • DD destabilizing domain
  • the composition for inducing an immune response may comprise an effector module.
  • the effector module may comprise a stimulus response element (SRE) operably linked to at least one payload.
  • the payload may be an immunotherapeutic agent.
  • the immunotherapeutic agent may be selected from, but is not limited to a chimeric antigen receptor (CAR) and an antibody.
  • CAR chimeric antigen receptor
  • the SRE of the composition may be responsive to or interact with at least one stimulus.
  • 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.
  • 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.
  • 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).
  • 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, ⁇ 72 ⁇ ), 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, I
  • 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.
  • hDHFR Amino acid 2-187 of WT; Y 122L HI31R, E144G
  • hDHFR Ammo acid 2-187 of WT; E31D, F32M, V I 161
  • hDHFR Amino acid 2-187 of WT; Q36F, N65F, Y122I
  • 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 ( ⁇ 7 ⁇ , 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 (R138
  • 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, ⁇ 72 ⁇ , R78G, E82G, F89L, D96G, N108D, Ml 12V, W114R, Y122D, K123E, 1139V, Q141R, D142G, F148L, E151G, E155G, Y157R, Q171R, Y183C, E184G, K185de3, D187N).
  • the stimulus of the SRE may be Trimethoprim or Methotrexate.
  • 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.
  • 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.
  • 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.
  • 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.
  • the extracellular target moiety may be an scFv derived from an antibody.
  • the scFv may specifically bind to a CD 19 antigen
  • the scFv of the CAR may be a CD 19 scFv
  • 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.
  • the CD19 scFv may comprise an amino acid sequence selected from the group consisting of any of SEQ ID NOs: 123-267 and 624.
  • the intracellular signaling domain of the CAR may be a signaling domain derived from T cell receptor CDSzeta.
  • 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.
  • the CAR may include a co-stimulatory domain.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the transmembrane domain may be derived from the CD3 epsilon chain of a T-cell receptor.
  • 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.
  • the transmembrane domain may be derived from an immunoglobulin selected from IgGl, IgD, IgG4, and an IgG4 Fc region.
  • 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.
  • the CAR of the effector module may further comprise a hinge region near the transmembrane domain.
  • the hinge region may comprise an amino acid sequence selected from the group consisting of any of SEQ ID NOs: 426-504.
  • 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.
  • TSA tumor specific antigen
  • TAA tumor associated antigen
  • an antigenic epitope selected from a tumor specific antigen (TSA), a tumor associated antigen (TAA), or an antigenic epitope.
  • the antigen may be an antigenic epitope.
  • the antigenic epitope may be CD 19.
  • 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.
  • the antibody may comprise an amino acid sequence selected from the group consisting of any of SEQ ID NOs: 123-267 and 624.
  • 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.
  • 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.
  • 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.
  • the present invention also provides polynucleotides comprising the compositions of the invention.
  • the polynucleotides may be a DNA or RNA molecule.
  • the polynucleotides may comprise spatiotemporally selected codons.
  • the amino acids may be selected from the group consisting of the amino acids listed above.
  • polynucleotides of the invention may be a DNA molecule.
  • the polynucleotides may be an RNA molecule.
  • the RNA molecule may be a messenger molecule.
  • the RNA molecule may be chemically modified.
  • 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.
  • the present invention also provides vectors comprising polynucleotides described herein.
  • the vector may be a viral vector.
  • 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.
  • the present invention also provides immune cells for adoptive cell transfer (ACT) which may express the compositions of the invention, the polynucleotides described herein.
  • 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.
  • the immune cells may be autologous, allogeneic, syngeneic, or xenogeneic in relation to a particular individual subject.
  • 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.
  • the second immunotherapeutic agent may be selected from a cytokine, and a cytokine- cytokine receptor fusion.
  • the second immunothe apeutic agent may be a cytokine.
  • the cytokine may be IL12 or IL15.
  • the second immunotherapeutic agent may be a cytokine- cytokine receptor fusion polypeptide.
  • 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.
  • 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.
  • 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.
  • compositions of the invention are administered to the subject.
  • the polynucleotides of the invention are administered to the subject.
  • the immune cells of the invention are administered to the subject.
  • 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.
  • the binding domains of the library may be generated using phage display techniques with the CD 19 antigen as the seed sequence.
  • 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.
  • 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.
  • the present invention also provides chimeric antigen receptors that may comprise the
  • SRE stimulus response element
  • the effector module comprises a stimulus response element
  • SRE protein of interest
  • POI protein of interest
  • the SRE may be a destabilizing domain (DD).
  • 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.
  • FKBP FK506 binding protem
  • E. coli DHFR Dihydrofolate reductase
  • hDHFR human DHFR
  • the biocircuit system is a DD biocircuit system
  • 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.
  • CAR chimeric agent receptor
  • the SRE and payload may be operably linked through one or more linkers and the positions of components may vary within the effector module.
  • 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.
  • linker sequences with specific sequences and lengths
  • 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.
  • the DD may stabilize the immunotherapeutic agent with a stabilization ratio of at least one in the presence of the stimulus.
  • the DD may destabilize the immunotherapeutic agent in the absence of ligand with a destabilization ratio between 0, and 0.99.
  • 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 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.
  • 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.
  • the vector of the invention may be a non- viral vector, such as a nanoparticles and liposomes.
  • 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).
  • 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).
  • TILs tumor infiltrating lymphocytes
  • the engineered cell may be used for adoptive cell transfer for treating a disease (e.g., a cancer).
  • the present invention also provides methods for induc
  • the FMC63 binding domain may be included in the payloads and effector modules of the invention.
  • FIG. 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.
  • SRE stimulus response element
  • 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.
  • 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.
  • Figure 4 shows representative effector modules carrying two pay loads with a cleavage site.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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 .
  • 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.
  • a and B bound stimuli from two different presenters
  • a single receiver e.g., another single cell
  • 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.
  • Figure 10 depicts a single-stimulus-bridged receiver biocircuit system.
  • 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).
  • 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.
  • Figure 12 depicts a biocircuit system which requires a dual activation.
  • 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).
  • 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.
  • CAR chimeric antigen receptor
  • 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
  • Figure 15 shows schematic representation of CAR systems having one (A) or two (B and C) SREs incorporated into the effector module.
  • 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,
  • 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.
  • 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).
  • an antigen e.g. two antigens
  • 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.
  • 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.
  • 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.
  • Figure 22 denotes the frequency of IFNgamma positive T cells.
  • 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.
  • Figure 24A depicts T cell subpopulations expressing CD 19 CAR.
  • Figure 28B depicts cell death caused by CD 19 CAR expressing T ceils.
  • 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.
  • 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.
  • 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.
  • Figure 29 is a bar graph representing the effect of promoters on transgene expression.
  • 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.
  • Figure 31A shows IFNgamma concentration.
  • Figure 3 IB shows IL2 concentration.
  • 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.
  • 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.
  • Figure 34A shows that restimulation increased the expression of IL12.
  • Figure 34B and Figure 34C show that ligand increased production of IL12.
  • 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.
  • 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.
  • 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.
  • Figure 38A shows the regulation of IL12 with PGK and EFla promoters and FKBP domains.
  • Figure 38B shows the relative expression of IL12.
  • Figure 39 depicts the kinetics of ILlSRa surface expression on CD4 T cells after IMP treatment.
  • Figure 40 represents a western blot of IL15-IL15Ra protein in HCT116 tumors from mice treated with TMP for 17 days in xenograft assays,
  • Figure 41 is a graph of the results of the MSD assay of IL15 protein levels in HEK293 cells.
  • 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.
  • 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.
  • Figure 44 represents the regulation of membrane bound IL15 with PO or IP dosing of TMP
  • 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.
  • passive immunotherapy includes approaches where immune effector molecules such as tumor-antigen specific cytotoxic T cells or antibodies are
  • 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).
  • TAA tumor associated antigen
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • biocircuit systems 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).
  • SREs stimulus response elements
  • FIG. 1 The overall architecture of a biocircuit system of the invention is illustrated in Figure 1.
  • 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.
  • POI protein of interest
  • 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 particular stimulus e.g., a small molecule
  • WO2017/180587 the contents of each of which are herein incorporated by reference in their entirety.
  • biocircuit systems, effector modules, SREs and components that tune expression levels and activities of any agents used for immunotherapy are provided.
  • 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.
  • 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.
  • 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
  • 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.
  • TCR cancer specific T cell receptor
  • CAR anti-tumor specific chimeric antigen receptor
  • 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
  • the biocircuits of the invention include at least one effector module as a component of an effector module system.
  • 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).
  • POIs proteins of interest
  • SRE stimulation 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.
  • the "responsive" nature of an SRE to a stimulus may be characterized by a covending or non-covalent interaction, a direct or indirect association or a stractural or chemical reaction to the stimulus.
  • 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.
  • the present invention provides methods for modulating protein expression, function or level.
  • 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
  • the present invention provides methods for modulating protein, expression, function or level by measuring the stabilization ratio and destabilization ratio.
  • 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.
  • 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.
  • 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.
  • ' 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.
  • 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, or at
  • the stimulus of the present invention maybe ultrasound stimulation.
  • the SREs of the present in vention may derived from mechanosensitive proteins.
  • the SRE of the present invention may be the mechanically sensitive ion channel, Piezol .
  • 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.
  • 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.
  • 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.
  • the SRE is an element, when responding to a stimulus, can tune intracellular localization, intramolecular activation, and/or degradation of payloads.
  • 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.
  • 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.
  • an SRE may represent a destabilizing domain
  • 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.
  • 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.
  • effector modules of the present invention may include one or more degrons to tune expression.
  • 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.
  • 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.
  • the degron may be an N- degron, a phospho degron, a heat inducible degron, a photosensitive degron, an oxygen dependent degron.
  • the degron may be an Ornithine decarboxylase degron as described by Takeuchi et al. (Takeuchi J et al. (2008). Biochem J. 2008 Mar
  • 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.
  • each component 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.
  • 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 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.
  • 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.
  • 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.
  • proteins can contribute to protein immunogenicity, including, but not limited to protein sequence, route and frequency of administration and patient population.
  • protem engineering may be used to reduce the immunogenicity of the compositions of the invention.
  • modifications to reduce immunogenicity may include modifications that reduce binding of the processed peptides derived from the parent sequence to MHC proteins.
  • 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.
  • 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.
  • compositions of the invention 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).
  • 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.
  • 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.
  • LMP low molecular weight proteins
  • 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.
  • 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.
  • 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)
  • 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.
  • the SRE is a
  • DD stabilizing/destabilizing domain
  • 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 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.
  • 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.
  • FDD fluorescent destabilizing domain
  • 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.
  • 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.
  • 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 ⁇ -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.
  • 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.
  • Another non-limiting example is the stability control region (SCR, residues 97-1 18) of Tropomyosin (Tm), which controls protein stability.
  • SCR Stret Control region
  • 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.
  • 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.
  • 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.
  • 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.
  • DDs destabilizing domain
  • 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 4/11/2017, or in US Provisional Application No. 62/466,596 filed
  • 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.
  • 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:
  • Candidate destabilizing domain sequence identified from protein domains of these proteins 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.
  • 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.
  • novel DDs derived from E.coli DHFR may comprise amino acids 2-159 of the wild type ecDHFR sequence. This may be referred to as an Mldei mutation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • polymerase chain reaction primers that amplify a different size product for each barcode may be used to identify each barcode on an agarose gel.
  • each barcode may have a unique quantitative polymerase chain reaction probe sequence that enables targeted amplification of each barcode.
  • 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).
  • DHF 7,8-dihydrofolate
  • THF 5,6,7,8, tetrahydrofolate
  • NADPH nicotinamide adenine dihydrogen phosphate
  • 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.
  • MTX methotrexate
  • DHF dihydrofolate reductase
  • the DDs of the invention may be hDHFR mutants including the single mutation hDHFR ( ⁇ 122 ⁇ ), hDHFR (K81R), hDHFR (F59S), hDHFR (II TV), hDHFR (N65D), hDHFR (A 107V), hDHFR (N127Y), hDHFR
  • 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, K
  • the stimulus is a small molecule that binds to a SRE to post- translationally regulate protein levels.
  • DHF ligands trimethoprim (TMP) and methotrexate (MTX) are used to stabilize hDHFR mutants.
  • TMP trimethoprim
  • MTX methotrexate
  • 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,
  • del means that the mutation is the deletion of the amino acid at that position relative to the wild type sequence.
  • hDHFR (Y122I, A125F) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 34
  • hDHFR (Y178H, E18IG) MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 37
  • hDHFR (I8V, K133E, Y163C) MVGSLNCVVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 40
  • hDHFR (K19E, F89L, E181G) MVGSLNCIVAVSQNMGIGENGDLPWPPLRNEFRYFQRM 42
  • hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFFRMT 46 Q36F, Y122I, A125F) TTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSR
  • 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
  • hDHFR V2A, R33G, Q36R, MAGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFGYFRR 872 L100P, K185R
  • hDHFR (G16S, 117 V, F89L, MVGSLNCIVAVSQNMSVGKNGDLPWPPLRNEFRYFQR 873 D96G, K123E, M140V, D I46G, MTTTSS ⁇ 3 ⁇ 4GKQNLVDv GKKTWFSIPEKNRPLKGRINL ⁇ 7 L K156R) SRELKEPPOGAHL-LSRSLDGALKLTEQPELANKVDMVW
  • 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
  • hDHFR ( ⁇ 7 ⁇ , L98S, K99R, MVGSLNCIVAVSQNMGNGKNGDLPWPPLRNEFRYFQR 875 M112T, E151G, E162G, E172G) MTTTSSWGKQNLVMGKKTWSIPEKNRPLKGRINLVL
  • hDHFR (R138G, D142G, F143S, MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 876 K156R, K158E, E162G, V166A, MTTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVL K177E, Y178C, K185E, N186S) SRELKEPPQGAHFLSRSLDD ALKLTEQPEL ANKVDMVW
  • hDHFR (K83 R, K99R, L i OOP, MVGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQR 877 E102G, N108D, K123R, H128R, MTTTSSVEGKQNLVIMGKKTWSIPEKNRPLKGRINL ⁇ D142G, F180L, K185E) SRELREPPQGAHFLSRSLDDALRPTGQPELADKVDMVW
  • ITDHFR V2A, I17V, N30D, MAGSLNCIVAVSQNMGVGKNGDLPWPPLRDGFRYFRR 879 E31G, Q36R, F59S, K69E, ⁇ 72 ⁇ , MTTTSSVEGKQNLVIMGKKTWSSIPEKNRPLEGRTNLV H88Y, F89L, N108D, 109E, LSRELKEPPQGAYLLSRSLDDALKLTEQPELADEAGMV V110A, 1115V, Y122D, L132P, WVVGGSSVDKEAM HPGHPKLSVTRrVQDFGSDAFFPE F135S, M140V, E144G, T147A, IDLEKCKLLPEYPGVLSDAQEERGIKYKFEVYEKSD
  • hDHFR Amino acid 2-187 of WT; VGSLNCIVAVSQNMGVGKNGDLPWPPLRNEFRYFQRM 882 117 V, Y122I) TTTSSVEGKQNLVTMGKKTWFSIPEKNRPLKGRINLVLS
  • hDHFR (Amino acid 2-187 of WT: VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 883 Y122L M140I) TTSSVEGKQNL-VIMGKKTWFSIPEKNRPLKGRINLVLSR
  • hDHFR (Amino acid 2-187 of WT; VGSLNCrVAVSQN GIGKNGDLPWPPLRNE-FRYFQRMT 884 N127Y, Y122I) TTSSVEGKQNLVIMGKKTWTSIPEKNRPLKGRINLVLSR
  • hDHFR (Amino acid 2-187 of WT; VGSLNCiVAVSQNMGiGKNGDLPWPPLRNEFRYFQRMT 885 Y122L H 131R, E 144G) TTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSR
  • 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
  • hDHFR Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNDMRYFQRM 887 E31D, F32M, VI 161) TTTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLS
  • hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 888 E162G, I176F) TTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSR
  • hDHFR (Amino acid 2-187 of WT; VGSLNCrVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 889 K185E) TTSSVEGKQNLV13 ⁇ 41GKKTWFS1PEKNRPLKGRINLVLSR
  • hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 890 Y122I, A125F) TTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSR
  • hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFFRMT 891 Q36F, N65F, Y122I) TTSSVEGKQNLVIMGKKTWTSIPEKFRPLKGRINL ⁇ SR
  • hDHFR Amino acid 2-187 of WT, VGSLNCrVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 892 N127Y
  • hDHF Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFQRMT 893 H131R, E144G) TTSSVEGKQNLVTMGKKTWFSIPEKNRPLKGRINLVLSR
  • hDHFR (Amino acid 2-187 of WT; VGSLNCrVAVSQN GVGKNGDLPWPPLRNEFRYFQRM 894 117V) TTTSSVE-GKQNLVTMGKKTWFSIPEKNRPLKGRrNLVL-S
  • hDHFR (Amino acid 2-187 of WT; VGSLNCTVAVSQNMGTGKNGDLPWPPLRNEFRYFQRMT 895 ⁇ 122 ⁇ ) TTSSVEGKQNLVIMGKKTWFSIPEKNRPLKGRINLVLSR
  • hDHFR (Amino acid 2-187 of WT; VGSLNCIVAVSQNMGIGKNGDLPWPPLRNEFRYFKRMT 981 Q36K, Y122I) TTSSVEGKQNLVIMGKKTWSIPE RPLKGRINLVLSR
  • DD mutations that do not inhibit ligand binding may be preferentially selected.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • the immunotherapeutic agent induces an anti-cancer immune response in a cell, or in a subject.
  • antibodies, fragments and variants thereof are payloads of the present invention.
  • antibodies of the present invention include without limitation, any of those taught in Table 5 of copending commonly owned U.S. Provisional Patent
  • antibody fragments and variants may comprise antigen binding regions from intact antibodies.
  • 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.
  • an “antibody” may comprise a heavy and light variable domain as well as an Fc region.
  • 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.
  • 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.
  • 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.
  • CDR refers to a region of an antibody comprising a structure that is complimentary to its target antigen or epitope.
  • 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.
  • 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.
  • CDR-H3 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).
  • 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).
  • 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).
  • 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.
  • single chain Fv 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.
  • the Fv polypeptide linker enables the scFv to form the desired structure for antigen binding.
  • 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.
  • tascFv tandem scFv
  • 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 ⁇ filmed (Nelson, A.
  • maxibodies (bivalent scFv fused to the amino terminus of the Fc (CH2-CH3 domains) of IgG may also be included.
  • 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.
  • 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.
  • 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.
  • 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.
  • methods of the present invention may include intrabody-based therapies.
  • variable domain sequences and/or CDR sequences disclosed herein may be incorporated into one or more constructs for intrabody-based therapy .
  • 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.
  • eacli monoclonal antibody is directed against a single determinant on the antigen.
  • 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.
  • 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.
  • 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.
  • the antibody may be a humanized full-length antibody.
  • 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.
  • 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.
  • compositions, biocircuits, biocircuit components, effector modules including their SREs or payloads of the present invention may be antibody mimetics.
  • 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.
  • 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).
  • 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.
  • the antibody may comprise a modified Fc region.
  • 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.
  • payioads of the invention may encode multispecific antibodies that bind more than one epitope.
  • 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.
  • 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.
  • a multi-specific antibody is a "bispecific antibody" which recognizes two different epitopes on the same or different antigens.
  • bispecific antibodies are capable of binding two different antigens.
  • Such antibodies typically comprise antigen-binding regions from at least two different antibodies.
  • 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.
  • BsMAb 'Afunctional bispecific antibodies
  • 'Afunctional bispecific antibodies 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.
  • 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;
  • the antibody may be "miniaturized".
  • 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.
  • RA rheumatoid arthritis
  • 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).
  • miniaturized antibodies 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).
  • 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.
  • a sdAb may be a "Camel Ig or "camelid VHH".
  • 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).
  • a sdAb may be a
  • immunoglobulin new antigen receptor (IgNAR).
  • VNAR variable new antigen receptor
  • CNAR constant new antigen receptor
  • immunoglobulin-based protein scaffolds 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.
  • CDR complementar - determining region
  • 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.
  • methods described herein include intrabody -based therapies.
  • variable domain sequences and/or CDR sequences disclosed herein are incorporated into one or more constructs for intrabody -based therapy.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • the polynucleotides have a modular design to encode at least one of the antibodies, fragments or variants thereof.
  • 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.
  • 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.
  • 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).
  • 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).
  • TSA tumor speci fic antigens
  • TAAs tumor associated antigens
  • 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.
  • TSA tumor specific antigen
  • a TSA may be a tumor neoantigen.
  • the tumor antigen specific antibody mediates complement-dependent cytotoxic response against tumor cells expressing the same antigen.
  • 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.
  • 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.
  • payloads of the present invention may be antibodies, fragments and variants thereof which are specific to CD 19 antigen.
  • 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).
  • 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.
  • 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.
  • a tumor specific antigen 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).
  • 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.
  • payloads of the invention may include FMC63-distinct antibodies, or fragments thereof.
  • FMC63-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.
  • antibodies of the invention may include CD19 antibodies, antibody fragments or variants that recognize CD 19 neoantigens including the CD 19 neoantigen lacking exon2.
  • the antibody or fragment thereof is immunologically speci fic to the CD 19 encoded by exon 1, 3 and/or 4.
  • 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.
  • 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.
  • CARs chimeric antigen receptors
  • chimeric antigen receptor refers to a synthetic receptor that mimics TCR on the surface of T cells.
  • a CAR is composed of an extracellular targeting domain, a transmembrane domain/region and an intracellular
  • 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 € ⁇ 3 ⁇ ), and/or one or more costimulator signaling domains, such as those from CD28, 4-1BB (CDI37) and OX-40 (CD134).
  • a "first-generation CAR" only has the 033 ⁇ signaling domain.
  • 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 0 ⁇ 3 ⁇ 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 .
  • 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.
  • 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.
  • 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).
  • 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).
  • this Tet-CAR may be the payioad of the effector module under the control of SREs (e.g., DDs) of the invention.
  • SREs e.g., DDs
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • selection of a CAR with a lower affinity may provide more T cell signaling and less toxicity.
  • 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.
  • TCR native T- cell receptor
  • 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).
  • TSA tumor specific antigen
  • 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.
  • 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
  • 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 ).
  • 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.
  • 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.
  • TSA tumor specific antigen
  • 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).
  • 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).
  • the targeting moiety of the CAR may be an FMC63 -distinct scFV.
  • 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.
  • targeting moiety may recognize a CD 19 antigen lacking exon2.
  • the targeting moiety recognizes a fragment of CD19 encoded by exon 1 , 3 and/or 4.
  • 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.
  • the targeting moieties of the present invention may be scFv comprising the amino acid sequences in Table 5.
  • CD 19 scFv 177 SEQ ID NO. 207 in US20160152723
  • CD 19 scFv 178 SEQ ID NO. 208 in US20160152723
  • 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)
  • ICR T cell receptor
  • the entire intracellular signaling domain can be employed.
  • 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.
  • the intracellular signaling domain of the present invention may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • 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.
  • the intracellular signaling domain is a CDS zeta (033 ⁇ ) signaling domain.
  • the intracellular region of the present invention further comprises one or more costimulatory signaling domains which provide additional signals to the immune effector cells.
  • 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).
  • CAR engineered immune cells e.g., CAR T cells.
  • 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.
  • the costimulatory signaling domain is derived from the cytoplasmic domain of CD28.
  • the costimulatory signaling domain is derived from the cytoplasmic domain of 4- IBB (CD 137).
  • 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.
  • 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), NK
  • SLAM signaling lympho
  • the intracellular signaling domain of the present invention may contain signaling domains derived from JAK-STAT.
  • 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.
  • ITAM Immunoreceptor Tyrosine-based Activation Motif
  • 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.
  • the intracellular domain of the present invention may comprise amino acid sequences of Table 6. Description Amino Acid Sequence
  • CD272 (BTLA1) co- RRH QGKQNEL SDTAGREINL VD AHLK SEQTE ASTRQN SQ stimulatory domain VLLSETGI YD NDPD LCFRMQEG SEVYS NPCLE E NKPG VY A
  • CD272 (BTLA1) co- CCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQ stimulatory domain NSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPG
  • CD30 co-stimulatoiy RRACRKRIRQKLHLCYPVQTSQPKLELVDSRPRRSSTQLR domain SGASVTEPVAEERGLMSQPLMETCHSVGAAYLESLPLQD
  • CD 148 intracellular RKKRKDAKNNEVSFSQIKPK S LIRVENFEAYF KQQAD 293 domain SNCGFAEEYEDLKLVGISOPKYAAELAENRGKNRYN VL
  • CD28 4- IBB, and/or RSKRSRLLHSDYMNMTPRRPGPTOKHYQPYAPPRDFAAY 300 CD3 signaling domain RSRFS ⁇ 7 VKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFP
  • CD28/CD3C AAAJEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSK 301
  • CD28-4-1BB MFWVLVWGGVLACYSLLVWAFIIFWVKRGRKKLLYIF 303 intracellular domain KQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL CD28-4-1BB lEVMYPPPYLDNEKSNGT!IHVKGKHLCPSPLFPGPSKPFW 304 intracellular domain VLVWGGVLACYSI VWAF1IFWVKRGRKKLLYIF QPF
  • CD3 delta chain ' MEHSTFLSGL ⁇ ATLLSQV ⁇ FKIPIEELE 307 intracellular signaling WVEGTVGTLLSDITRLDLG RILDPRG!YRCNGTDIYKDK domain ESTVQVHYRMCQSCVELDPATVAGTIVTDVIATLLLALGV
  • CD3 delta chain MEHSTTLSGLVLATLLSQVSPFKIPEELEDR VNCNTSIT 308 intracellular signaling WVEGWGTLLSDI RLDLGKRILDPRGIYRCNGTD!YKDK domain ESTVQVHYRTADTQALLR DQVYQPLRDRDDAOYSHLG
  • CD3 gamma MEQGKGLAVLILAIILLQGTLAOSIKGNHLVKVYDYOEDG 315 intracellular domain SVLLTCDAEAK ITWFKDGKMIGFLTEDKKKWNLGSNAK
  • CD3 gamma MEQGKGLA ⁇ TILAIILLQGTLAQSIKGNHLVKVYDYQEDG 318 intracellular domain SVLLTCDAEAKNITWFKDGKMIGFL-TE-DKKKWNLGSNAK
  • CDS zeta intracellular MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFI 319 domain YGVILTALFLRVKFSRSADAPAYQQGQNQLYNELNLGRR
  • CDS zeta intracellular MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFI 320 domain YGmTALFLRVKFSRSAD APAYQQGQNQL YNELNL GRR
  • CD3 zeta intracellular MKWKALFTAAILQAQLPITEAQSFGLLDPKLCYLLDGILFI 321 domain YGVILTALFLRM FSRSADAPAYQQGQNQLYNELNLGRR
  • CD3 zeta intracellular NQLYNELNLGRREEYDVLDKR 322 domain
  • CDS zeta intracellular DGLYQGLSTATKDTYDALHMQ 324 domain
  • CD3 zeta intracellular R VKF SRS AEPP A YQQGQNQL YNELNL GRREEYD VLDKRR 325 domain GRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMK
  • CD3 zeta intracellular MIP A WLLLLLL VEQ AA ALG EPQLC YILD AILFL VG I LTL 330 domain LVCRLKTOVRKAATTSYEKSRVKFSRSADAPAYOQGQNOL
  • CD3 zeta intracellular LRVKFSRSADAP A YQQGQNQL YNELNLGRREEYDVLDKR 331 domain RGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM
  • CDS zeta intracellular LR VKF S R S AD AP AYQQG QNQL .YNF i NLGRREEYD VLDKR 333 domain RGRDPEMGGKPQRRKNPQEGLYNELQKDKMAEAYSETG
  • CDS zeta intracellular NQLYNELNLGRREEYDVLDKR 335 domain
  • CD3 zeta intracellular EGLYNELQKDKMAEAYSE IG MK 336 domain
  • CDS zeta intracellular DGLYQGLSTATKDTYDALHMQ 337 domain
  • CDS zeta intracellular DPKLCYLLDGILFIYGVIL ALFLRVKFSRSADAPAYQOGO 341 domain NQLYNELNLGRREEYD ' V'LDKRRGRDPEMGGKPQRRKNP
  • CD3 zeta intracellular MKWKALFTAArLQAOLPITEAQSFGT .T .OPKLCYLLDGILFI 342 domain YGViLTALFLRV FSRSADAPAYQQGQNQLYNELNLGRR
  • CD7 A intracellular MPGGPGVLQALPATIFLLFLLSAVYLGPGCQALWMHKVP 344 domain ASLMVSLGEDAHFQCPH SSNNANVTWWRVLHGNYTWP
  • DAP 12 intracellular MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSP 354 domain GvLAG!vMGDLV ⁇ TVLIALAVYFLGRLVPRGRGAAEAAT
  • DAP 12 intracellular MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSP 355 domain GVLAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEATR
  • DAP 12 intracellular MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVNIGDL 356 domain VLTVLIALAVYFLGRLVPRGRGAAEAATRKQRITETESPY
  • DAP 12 intracellular MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSP 358 domain GVXAGIVMGDLVLTVLIALAVYFLGRLVPRGRGAAEAAT
  • DAP 12 intracellular MGGLEPCSRLLLLPLLLAVSGLRPVQAQAQSDCSCSTVSP 359 domain GVLAGI GDLVLTVLIALAVYT ⁇ GRLVPRGRGAAEATR
  • DAP 12 intracellular MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAG1VMGDL 360 domain VLTVLIALAVYFLGRLVPRGRGAAEAATRKQRl ' lE'i'ESPY
  • DAP 12 intracellular MGGLEPCSRLLLLPLLLAVSDCSCSTVSPGVLAGIVMGDL 361 domain VLTVLIALAVYFLGRLVPRGRGAAEATRKQR1TETESPYQ
  • DAP 12 intracellular ESPYQELQGQRSDVYSDLNTQ 362 domain
  • DAP 12 intracellular ESPYQELQGQRSDVYSDLNTQ 363 domain
  • OX40-CD3 Zeta RRDQRLPPDAHKPPGGGSFRTPTQEEQADAHSTLAKIRVK 367 intracellular domain FSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGR
  • the CAR of the present invention may comprise a
  • transmembrane domain 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.
  • 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.
  • the transmembrane region may be derived from (i.e. comprise at least the
  • the transmembrane domain of the present invention may be synthetic.
  • the synthetic sequence may comprise predominantly hydrophobic residues such as leucine and valine.
  • 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.
  • a CD8a transmembrane domain a CD4 transmembrane domain
  • a CD 28 transmembrane domain a CD 28 transmembrane domain
  • CTLA-4 transmembrane domain a PD-1 transmembrane domain
  • PD-1 transmembrane domain a human IgG4 Fc region
  • 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.
  • 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.
  • 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.
  • CD28 Transmembrane FWVLWVGG ⁇ T.ACYSLLVTVAFIIFWVRSKRSRLLHSDYM 386 domain NMTPRRPGPTRKHYQP YAPPRDFAAYRS
  • CD28 Transmembrane FWVL VGGVLA ⁇ S ⁇ 392 domain and CD28 and NMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPA CD3 Zeta intracellular YQQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRR domain KNPQEGLYNELQKDKMAEAYSEI GMK GERRR GKGHDGLY
  • Ci)28 Transmembrane FWVLVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYM 393 domain and CD28, OX40, NMTPRRPGPTRKHYQPYAPPRDFAAYRSRDQRLPPDAHKP and CD3 Zeta intracellular PGGGSFRTPlQEEQADAHSTLA IRVKt SRSADAPAYQOGQ domain NQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE
  • CD28 Transmembrane FWVLV 'VGG ⁇ r LACYSLLVTVAFIIFW ⁇ r RRVKFSRSADAPA 394 domain and CDS Zeta YQQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRR intracellular domain KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY
  • CD28 transmembrane-CD3 AAAffiVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP 395 zeta signaling domain FW ⁇ WVGGVXACYSLLVTVAFIIFWVT SKRSRLLHSDYM ("28z") NMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPA
  • CDS 1 ransmembrane h MALPWALLLPLALLLHAARP 404 domain
  • FcERI a Transmembrane FFIPLLV ⁇ ILFAVDTGLFISTQQQVTFLL IKRTRKGFRLLNP 416 domain HPKPNPKN
  • Hinge region sequences useful in the present invention are provided in Table 8 A.
  • PKDTL ISRTTEWCVVVTDVSHEDPEVKFNWYVDGVEVH NAK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SN
  • ALPAPIE TISKAKGOPREPQVYTLPPSRDELTKNOVS LTCLVKGFYPSDIA ⁇ 3 ⁇ 4WESNGQPEN YKTTPPVLDSDGSF
  • Hinge (CHS) ESKYGPPCPPCPGQPREPOVYTLPPSQEEMTKNQVSLTCL 461

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JP7341900B2 (ja) 2023-09-11
AU2018227583B2 (en) 2023-06-01
JP2020511529A (ja) 2020-04-16
SG11201907922PA (en) 2019-09-27
EP3589646A1 (en) 2020-01-08
KR102746901B1 (ko) 2024-12-26
CN110831961A (zh) 2020-02-21
AU2018227583A1 (en) 2019-10-17
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CA3055202A1 (en) 2018-09-07
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