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WO2024168276A2 - Compositions et méthodes pour immunothérapies - Google Patents

Compositions et méthodes pour immunothérapies Download PDF

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Publication number
WO2024168276A2
WO2024168276A2 PCT/US2024/015218 US2024015218W WO2024168276A2 WO 2024168276 A2 WO2024168276 A2 WO 2024168276A2 US 2024015218 W US2024015218 W US 2024015218W WO 2024168276 A2 WO2024168276 A2 WO 2024168276A2
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composition
domain
cell
endogenous
sequence
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PCT/US2024/015218
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English (en)
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WO2024168276A3 (fr
Inventor
Michael Yi
Nhung Nguyen
Michael Bethune
Trevor Bentley
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Cargo Therapeutics, Inc.
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Publication of WO2024168276A2 publication Critical patent/WO2024168276A2/fr
Publication of WO2024168276A3 publication Critical patent/WO2024168276A3/fr

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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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/93—Ligases (6)

Definitions

  • Immunotherapeutic cell compositions may be generated for the treatment of diseases and/or conditions such as, for example, certain cancers and autoimmune conditions. Many existing immunotherapies are autologous, made by isolating a subject’s own T-cells, engineering and expanding those cells ex vivo, and re-infusing the engineered cells to the original subject.
  • GVHD occurs when an engrafted immune cell (i.e., an allogeneic CAR T cell) recognizes the host as foreign and directs an immune response against the host.
  • a variety of approaches to minimize or eliminate graft rejection and GVHD in patients treated with allogeneic cell therapies including, for example, expression of certain recombinant proteins in grafted cells to improve the efficacy of grafted cell implantation while concomitantly decreasing the activation of host immune responses and/or the activation of graft cell immune responses against the host, disruption of graft cell genes that encode T-cell receptor (TCR) subunits (including CD3 subunits), and/or major histocompatibility complex (MHC) proteins.
  • TCR T-cell receptor
  • MHC major histocompatibility complex
  • Some current strategies for targeting the TCR do not allow direct selection of engineered cells that don’t express functional TCRs and may be limited to one specific TCR. Additionally, current manufacturing strategies for making allogeneic cell therapies do not sufficiently reduce the amounts of TCRs being expressed by WSGR Docket No.61078-729.601 the engineered cells. Thus, currently available methods are not well-suited for widespread use and there remains an urgent medical need for methods of making allogeneic cells that do not activate the host or graft immune response.
  • SUMMARY [4] Provided herein are improved methods and compositions for reducing endogenous or functional TCR complex expression and/or function within a cell. The methods and compositions are useful to reduce or eliminate expression and assembly of functional T cell receptor complexes.
  • the methods and compositions provided herein can be used to reduce or eliminate expression and assembly of functional T cell receptor complexes using transient expression and do not require genomic editing.
  • the methods and compositions provided herein can allow for direct selection of the cells with reduced expression of the endogenous functional TCR complex. Additionally, the methods and compositions provided herein can target multiple subunits of the TCR complex.
  • Various methods and compositions described herein can be combined with each other or with other methods and compositions.
  • the methods and compositions provided herein provide alternative means to generate allogeneic cell therapies than existing methods and compositions to meet the urgent medical need for strategies to engineer cells that do not activate the host or graft immune response, thereby facilitating widespread uses of off-the-shelf or universal allogeneic cell immunotherapy products.
  • compositions comprising recombinant polynucleic acids comprising a sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises a transmembrane domain derived from a subunit of the TCR, wherein the transmembrane domain is from TCR alpha, TCR beta, TCR gamma, TCR delta, CD3 delta, CD3 gamma or CD3 epsilon; and an intracellular domain comprising a functional ubiquitin ligase domain from a ubiquitin ligase; wherein the fusion polypeptide ubiquitinates an endogenous subunit of the TCR complex in a T cell when expressed in the T cell.
  • the ubiquitin ligase is a RING (really interesting new gene) finger protein (RNF) family ubiquitin ligase, a U-box E3 ligase family ubiquitin ligase, a membrane associated ring-CH-type finger (MARCH) family ubiquitin ligase, a gene related to anergy in lymphocytes (GRAIL) E3 ubiquitin ligase or a carboxy-terminus of HSC70 interacting protein (CHIP) ubiquitin ligase.
  • RNF RING (really interesting new gene) finger protein
  • MARCH membrane associated ring-CH-type finger
  • GRAIL gene related to anergy in lymphocytes
  • CHIP carboxy-terminus of HSC70 interacting protein
  • the ubiquitin ligase is a RING (really interesting new gene) WSGR Docket No.61078-729.601 finger protein (RNF) family ubiquitin ligase.
  • the RING (really interesting new gene) finger protein (RNF) family ubiquitin ligase is selected from the group consisting of RNF122, RNF133, RNF152, RNF130, RNF148, RNF149 and RNF150.
  • the ubiquitin ligase is a membrane associated ring-CH-type finger (MARCH) family ubiquitin ligase selected from the group consisting of MARCH1, MARCH2, MARCH3, MARCH4, MARCH6, MARCH8, and MARCH9.
  • MARCH membrane associated ring-CH-type finger
  • compositions comprising recombinant polynucleic acids comprising a sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises a transmembrane domain derived from a subunit of the TCR complex, wherein the subunit is selected from the group consisting of TCR alpha, TCR beta, TCR gamma, TCR delta, CD3 delta, CD3 gamma, CD3 epsilon or CD3 zeta; and an intracellular domain comprising a functional ubiquitin ligase domain from a ubiquitin ligase, wherein the ubiquitin ligase does not comprise a gene related to anergy in lymphocytes (GRAIL) E3 ubiquitin ligase, a carboxy-terminus of Hsc70 interacting protein (CHIP) ubiquitin ligase, a RING (really interesting new gene) finger protein(RNF) 122, RNF133, or
  • the ubiquitin ligase is an E3 ubiquitin ligase.
  • the ubiquitin ligase is a membrane associated ring-CH-type finger (MARCH) family ubiquitin ligase.
  • the membrane associated ring-CH-type finger (MARCH) family ubiquitin ligase is selected from the group consisting of MARCH1, MARCH2, MARCH3, MARCH4, MARCH6, MARCH8 and MARCH9.
  • the ubiquitin ligase is a RING (really interesting new gene) finger protein (RNF) family ubiquitin ligase.
  • the RNF family ubiquitin ligase is selected from the group consisting of RNF130, RNF148, RNF149 and RNF150.
  • the transmembrane domain is a CD3 zeta transmembrane domain.
  • the transmembrane domain is not a CD3 zeta transmembrane domain.
  • the transmembrane domain is a CD3 delta transmembrane domain.
  • the transmembrane domain is not a CD3 delta transmembrane domain.
  • the transmembrane domain is a CD3 gamma transmembrane domain. [18] In some embodiments, the transmembrane domain is not a CD3 gamma transmembrane domain. [19] In some embodiments, the transmembrane domain is a CD3 epsilon transmembrane domain. WSGR Docket No.61078-729.601 [20] In some embodiments, the transmembrane domain is not a CD3 epsilon transmembrane domain. [21] In some embodiments, the intracellular domain further comprises an intracellular domain derived from a subunit of the TCR complex.
  • the intracellular domain derived from a subunit of the TCR complex is disposed between the transmembrane domain and the ubiquitin ligase domain.
  • the intracellular domain derived from a subunit of the TCR complex is a CD3 zeta intracellular domain.
  • the intracellular domain derived from a subunit of the TCR complex is not a CD3 zeta intracellular domain.
  • the intracellular domain derived from a subunit of the TCR complex comprises a CD3 delta intracellular domain, a CD3 gamma intracellular domain or a CD3 epsilon intracellular domain.
  • the transmembrane domain is a CD3 zeta transmembrane domain; and the intracellular domain derived from a subunit of the TCR complex is a CD3 zeta intracellular domain.
  • the transmembrane domain is a CD3 delta transmembrane domain; and the intracellular domain derived from a subunit of the TCR complex is a CD3 delta intracellular domain.
  • the transmembrane domain is a CD3 gamma transmembrane domain; and the intracellular domain derived from a subunit of the TCR complex is a CD3 gamma intracellular domain.
  • the transmembrane domain is a CD3 epsilon transmembrane domain; and the intracellular domain derived from a subunit of the TCR complex is a CD3 epsilon intracellular domain.
  • the fusion protein lacks an extracellular domain.
  • the fusion protein comprises an extracellular domain derived from a subunit of the TCR complex.
  • the extracellular domain derived from a subunit of the TCR complex is derived from TCR alpha, TCR beta, TCR gamma, TCR delta, CD3 delta, CD3 gamma or CD3 epsilon or CD3 zeta.
  • the fusion protein comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 44-59 and 61.
  • compositions comprising recombinant polynucleic acids comprising a sequence encoding a mutant CD3 zeta polypeptide, wherein the mutant CD3 zeta polypeptide WSGR Docket No.61078-729.601 comprises a mutant CD3 zeta transmembrane domain, wherein the mutant CD3 zeta transmembrane domain comprises the sequence LCYLLXGILFIYGVILTALFL (SEQ ID NO: 999), wherein X is an amino acid selected from the group consisting of R, K and S.
  • compositions comprising recombinant polynucleic acids comprising a sequence encoding a mutant CD3 zeta polypeptide, wherein the mutant CD3 zeta polypeptide comprises a mutant CD3 zeta transmembrane domain, wherein the mutant CD3 zeta transmembrane domain comprises the sequence LCYLLXGILFIYGVILTALFL (SEQ ID NO:999), wherein X is an amino acid selected from the group consisting of E, N, A R, K and S; and wherein the mutant CD3 zeta polypeptide comprises a CD3 zeta extracellular domain that is at least 4 amino acids in length.
  • compositions comprising cells, wherein the cells express endogenous CD3 zeta and comprise recombinant polynucleic acids comprising a sequence encoding a mutant CD3 zeta polypeptide, wherein the mutant CD3 zeta polypeptide is expressed in the cell and forms a CD3 zeta dimer with the endogenous CD3 zeta.
  • compositions comprising cells, wherein the cells express endogenous CD3 zeta and comprise recombinant polynucleic acids comprising a sequence encoding a mutant CD3 zeta polypeptide, wherein the mutant CD3 zeta polypeptide is expressed in the cells and forms an exogenous CD3 zeta dimer with the endogenous CD3 zeta polypeptide; and wherein formation of a exogenous TCR complex comprising the exogenous CD3 zeta dimer, endogenous TCR alpha, endogenous TCR beta, endogenous CD3 gamma, endogenous CD3 delta and endogenous CD3 epsilon is reduced or inhibited compared to formation of a TCR complex comprising a endogenous CD3 zeta dimer, TCR alpha, TCR beta, CD3 gamma, CD3 delta and CD3 epsilon.
  • compositions comprising cells, wherein the cells express CD3 zeta and comprises a recombinant polynucleic acid comprising a sequence encoding a mutant CD3 zeta polypeptide, wherein the mutant CD3 zeta polypeptide is expressed in the cells and forms an exogenous CD3 zeta dimer with the endogenous CD3 zeta polypeptide; and wherein the KD of the exogenous CD3 zeta dimer for an endogenous TCR complex is higher than a K D of an endogenous CD3 zeta dimer to the endogenous TCR complex.
  • compositions comprising cells, wherein the cells expresses endogenous CD3 zeta, and comprise a recombinant polynucleic acid comprising a sequence encoding a mutant CD3 zeta polypeptide, wherein the mutant CD3 zeta polypeptide is expressed in the cells and forms an exogenous CD3 zeta dimer with the endogenous CD3 zeta; and wherein a K D of the exogenous CD3 zeta dimer for endogenous TCR alpha is higher than a KD of an endogenous CD3 zeta dimer to the endogenous TCR alpha.
  • compositions comprising cells, wherein the cells expresses endogenous CD3 zeta, and comprises a recombinant polynucleic acid comprising a sequences encoding a mutant CD3 zeta polypeptide, wherein the mutant CD3 zeta polypeptide is expressed in the cells and forms an exogenous CD3 zeta dimer with the endogenous CD3 zeta; and wherein the cells exhibit a reduced amount of endogenous TCR complexes relative to cells that do not express the mutant CD3 zeta polypeptide.
  • compositions comprising cells, wherein the cells express endogenous CD3 zeta and comprise recombinant polynucleic acids comprising sequences encoding a mutant CD3 zeta polypeptide, wherein the mutant CD3 zeta polypeptide is expressed in the cells and forms a CD3 zeta dimer with the endogenous CD3 zeta; and wherein the CD3 zeta dimer does not substantially interact with an endogenous TCR complex.
  • the endogenous TCR complex comprises endogenous TCR alpha, endogenous TCR beta, endogenous CD3 gamma, endogenous CD3 delta and endogenous CD3 epsilon.
  • the mutant CD3 zeta polypeptide lacks a CD3 zeta intracellular signaling domain.
  • the mutant CD3 zeta polypeptide comprises a mutant CD3 zeta transmembrane domain.
  • the mutant CD3 zeta transmembrane domain comprises the sequence LCYLLXGILFIYGVILTALFL (SEQ ID NO: 999), wherein X is an amino acid selected from the group consisting of R, K and S.
  • the mutant CD3 zeta transmembrane domain comprises the sequence LCYLLXGILFIYGVILTALFL (SEQ ID NO: 999), wherein X is an amino acid selected from the group consisting of E, N, A, R, K and S.
  • the mutant CD3 zeta polypeptide comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 19-22, 39-41, 62 and 64.
  • the mutant CD3 zeta polypeptide comprises an extracellular domain.
  • the extracellular domain comprises a CD3 zeta extracellular domain or fragment thereof.
  • the extracellular domain comprises a full length CD3 zeta extracellular domain.
  • the extracellular domain comprises from 4 to 9 amino acids.
  • the cells are mammalian cells or human cells.
  • composition comprising a recombinant polynucleic acid comprising a sequence encoding a mutant CD3 epsilon polypeptide, wherein the mutant CD3 epsilon polypeptide WSGR Docket No.61078-729.601 comprises a mutant CD3 epsilon transmembrane domain comprising a sequence of MSVATIVIVXICITGGLLLLVYYWS (SEQ ID NO: 1000), wherein X is an amino acid selected from the group consisting of R and K.
  • composition comprising a recombinant polynucleic acid comprising a sequence encoding a mutant CD3 epsilon polypeptide, wherein the mutant CD3 epsilon polypeptide comprises (a) a CD3 gamma transmembrane domain or a CD3 delta transmembrane domain; and (b) a CD3 epsilon intracellular domain.
  • the mutant CD3 epsilon polypeptide comprises a CD3 gamma extracellular domain or a CD3 delta extracellular domain.
  • the mutant CD3 epsilon polypeptide comprises (a) a CD3 gamma extracellular domain, a CD3 gamma transmembrane domain and a CD3 epsilon intracellular domain, or (b) a CD3 delta extracellular domain, a CD3 delta transmembrane domain and a CD3 epsilon intracellular domain.
  • the mutant CD3 epsilon polypeptide comprises an endogenous CD3 epsilon extracellular domain and a mutant CD3 gamma transmembrane domain or a mutant CD3 delta transmembrane domain.
  • the mutant CD3 epsilon polypeptide comprises a truncated CD3 epsilon intracellular domain.
  • the truncated CD3 epsilon intracellular domain comprises a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more amino acids of the intracellular domain.
  • the truncated CD3 epsilon intracellular domain comprises a deletion of 45 amino acids of the intracellular domain.
  • the truncated CD3 epsilon intracellular domain comprises the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQH NDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENC MEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPV (SEQ ID NO: ###).
  • the truncated CD3 epsilon intracellular domain comprises a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more amino acids from the N-terminus of the intracellular domain.
  • the truncated CD3 epsilon intracellular domain comprises a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more amino WSGR Docket No.61078-729.601 acids from the C-terminus of the intracellular domain.
  • the truncated CD3 epsilon intracellular domain comprises a deletion of 45 amino acids from the C-terminus of the intracellular domain.
  • the truncated CD3 epsilon intracellular domain comprises the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQH NDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENC MEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPV (SEQ ID NO: ###).
  • composition comprising a recombinant polynucleic acid comprising a sequence encoding a mutant CD3 epsilon polypeptide, wherein the mutant CD3 epsilon polypeptide comprises a mutant CD3 epsilon transmembrane domain and a truncated CD3 epsilon intracellular domain.
  • the truncated CD3 epsilon intracellular domain comprises a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more amino acids of the intracellular domain.
  • the truncated CD3 epsilon intracellular domain comprises a deletion of 45 amino acids of the intracellular domain.
  • the truncated CD3 epsilon intracellular domain comprises the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQH NDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENC MEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPV (SEQ ID NO: ###).
  • the truncated CD3 epsilon intracellular domain comprises a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more amino acids from the N-terminus of the intracellular domain.
  • the truncated CD3 epsilon intracellular domain comprises a deletion of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more amino acids from the C-terminus of the intracellular domain.
  • the truncated CD3 epsilon intracellular domain comprises a deletion of 45 amino acids from the C-terminus of the intracellular domain.
  • the truncated CD3 epsilon intracellular domain comprises the amino acid sequence MQSGTHWRVLGLCLLSVGVWGQDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILWQH WSGR Docket No.61078-729.601 NDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYVCYPRGSKPEDANFYLYLRARVCENC MEMDVMSVATIVIVDICITGGLLLLVYYWSKNRKAKAKPV (SEQ ID NO: ###).
  • the truncated CD3 epsilon intracellular domain lacks an ER retention signal.
  • the mutant CD3 epsilon transmembrane domain comprising a sequence of MSVATIVIVXICITGGLLLLVYYWS (SEQ ID NO: 1000), wherein X is an amino acid selected from the group consisting of A, N, R and K.
  • a composition comprising a cell, wherein the cell comprises the recombinant polynucleic acid of any of the compositions described herein.
  • the cell expresses endogenous CD3 epsilon and/or endogenous CD3 zeta.
  • the cell expresses endogenous CD3 gamma and/or endogenous CD3 delta.
  • the mutant CD3 epsilon polypeptide is expressed in the cell and forms a dimer comprising exogenous CD3 epsilon and endogenous CD3 gamma and/or forms a dimer comprising exogenous CD3 epsilon and endogenous CD3 delta.
  • the KD of the exogenous CD3 epsilon-CD3 gamma dimer for an endogenous TCR complex or a subunit thereof is higher than the K D of a dimer comprising an endogenous CD3 epsilon and an endogenous CD3 gamma for the endogenous TCR complex or the subunit thereof; and/or wherein the KD of the exogenous CD3 epsilon-CD3 delta dimer for an endogenous TCR complex is higher than the KD of an endogenous CD3 epsilon-CD3 delta dimer to the endogenous TCR complex or the subunit thereof.
  • the K D of the exogenous CD3 epsilon-CD3 gamma dimer for an endogenous TCR beta is higher than the KD of an endogenous CD3 epsilon-CD3 gamma dimer to the endogenous TCR beta.
  • formation of an exogenous TCR complex comprising the exogenous CD3 epsilon-CD3 gamma dimer and endogenous TCR alpha, endogenous TCR beta, and (i) an endogenous CD3 delta-CD3 epsilon dimer or (ii) the exogenous CD3 epsilon-CD3 delta dimer is inhibited or lower compared to formation of a TCR complex comprising an endogenous CD3 epsilon- CD3 gamma dimer, endogenous TCR alpha, endogenous TCR beta, and (i) an endogenous CD3 delta- CD3 epsilon or (ii) the exogenous CD3 epsilon-CD3 delta dimer.
  • the exogenous CD3 epsilon-CD3 gamma dimer does not substantially interact with an endogenous TCR complex and/or does not substantially interact with TCR beta.
  • the KD of the exogenous CD3 epsilon-CD3 delta dimer for an endogenous TCR alpha is higher than the K D of an endogenous CD3 epsilon-CD3 delta dimer to the endogenous TCR alpha.
  • formation of an exogenous TCR complex comprising the exogenous CD3 epsilon-CD3 delta dimer plus an endogenous TCR alpha and an endogenous TCR beta, and either (i) an endogenous CD3 gamma-CD3 epsilon dimer or (ii) the exogenous CD3 epsilon-CD3 gamma dimer is inhibited or lower compared to formation of a TCR complex comprising an endogenous CD3 epsilon-CD3 delta dimer, endogenous TCR alpha, endogenous TCR beta, and (i) an endogenous CD3 gamma-CD3 epsilon dimer or (ii) the exogenous CD3 epsilon-CD3 gamma dimer.
  • the exogenous CD3 epsilon-CD3 delta dimer does not substantially interact with an endogenous TCR complex and/or does not substantially interact with TCR alpha.
  • the cell expresses a reduced amount of functional endogenous TCR complexes relative to a cell that does not express the mutant CD3 epsilon polypeptide.
  • the mutant CD3 epsilon polypeptide lacks a CD3 epsilon intracellular signaling domain.
  • the mutant CD3 epsilon polypeptide comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 23, 24, 35-37, 60 and 61.
  • the mutant CD3 epsilon polypeptide comprises an extracellular domain.
  • the extracellular domain comprises a CD3 epsilon extracellular domain or fragment thereof.
  • the extracellular domain comprises a full length CD3 epsilon extracellular domain.
  • the recombinant polynucleic acid further comprises a sequence encoding an ER retention tag.
  • the ER retention tag is a E319K tag.
  • the ER retention tag comprises an amino acid sequence of KKKKRD. In some embodiments, the ER retention tag comprises a sequence at least 95% sequence identity to any of the sequences in Table 5. [80] In some embodiments, the recombinant polynucleic acid further comprises a sequence encoding an antigen binding domain. In some embodiments, the antigen binding domain binds to a subunit is CD3. In some embodiments, the antigen binding domain binds to CD58. In some embodiments, the antigen binding domain binds to MHCI. In some embodiments, the antigen binding domain is an scFv. In some embodiments, the antigen binding domain is a humanized scFv.
  • the antigen binding domain is a VHH. In some embodiments, the antigen binding domain comprises a variable heavy chain domain (VH) and a variable light chain domain (VL). In some embodiments, the heavy chain domain is linked to the light chain domain via a linker. In some embodiments, the linker is a Whitlow linker. In some embodiments, the antigen binding domain from WSGR Docket No.61078-729.601 N-terminal to C-terminal comprises VH-Whitlow linker – VL. In some embodiments, the antigen binding domain from N-terminal to C-terminal comprises VL-Whitlow linker – VH.
  • the recombinant polynucleic acid comprises at least 80% sequence identity to any of the sequence in Table 7.
  • the cell is a mammalian cell.
  • a composition comprising of a cell, wherein the cell comprises the recombinant polynucleic acid of the compositions described herein.
  • a composition comprising a recombinant polynucleic acid comprising a sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises (1) an antigen binding domain, wherein the antigen binding domain binds to a TCR subunit; (2) an ER-retention tag.
  • subunit is CD3.
  • composition comprising a recombinant polynucleic acid comprising a sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises (1) an antigen binding domain, wherein the antigen binding domain binds to CD58; (2) an ER-retention tag.
  • expression of the fusion polypeptide in a cell reduces cell surface expression of an endogenous TCR subunit or reduces functional TCR complex formation in the cell compared to the cell surface expression of the endogenous TCR subunit or the functional TCR complex formation in a cell that does not express the fusion polypeptide.
  • expression of the fusion polypeptide in a cell reduces cell surface expression of an endogenous TCR subunit or reduces functional TCR complex formation in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the cell surface expression of the endogenous TCR subunit or the functional TCR complex formation in a cell that does not express the fusion polypeptide.
  • expression of the fusion polypeptide in a cell reduces cell surface expression of an endogenous TCR subunit or reduces functional TCR complex formation in the cell by at least 90% compared to the cell surface expression of the endogenous TCR subunit or the functional TCR complex formation in a cell that does not express the fusion polypeptide.
  • the fusion polypeptide further comprises a KDEL sequence.
  • the KDEL sequence is positioned between the antigen binding domain and the transmembrane domain.
  • the antigen binding domain is an scFv. In some embodiments, the antigen binding domain is a humanized scFv.
  • the antigen binding domain is a fully humanized scFv. In some embodiments, the antigen binding domain is a WSGR Docket No.61078-729.601 VHH. In some embodiments, the antigen binding domain comprises a variable heavy chain domain (VH) and a variable light chain domain (VL). [87] In some embodiments, the heavy chain domain is linked to the light chain domain via a linker. In some embodiments, the linker is a Whitlow linker. In some embodiments, the antigen binding domain from N-terminal to C-terminal comprises VH-Whitlow linker – VL.
  • the antigen binding domain from N-terminal to C-terminal comprises VL-Whitlow linker – VH.
  • the fusion polypeptide further comprises a transmembrane domain.
  • the transmembrane domain comprises a CD8alpha transmembrane domain, a UGT2B17 transmembrane domain, or a Trem1 transmembrane domain.
  • the domain is a UGT2B17 transmembrane domain.
  • the transmembrane domain is a Trem1 transmembrane domain.
  • the ER retention tag is positioned at the C- terminus of the fusion polypeptide.
  • the ER retention tag is a E319K tag. In some embodiments, the ER retention tag comprises an amino acid sequence of KKKKRD. In some embodiments, the ER retention tag comprises a sequence at least 95% sequence identity to any of the sequences in Table 5. In some embodiments, the fusion polypeptide comprises at least 80% sequence identity to any of the sequence in Table 6. [89] Provided herein is a composition comprising a cell, wherein the cell comprises the fusion polypeptide of the composition described herein.
  • composition comprising a recombinant polynucleic acid comprising or encoding an shRNA against CD3 zeta, and a sequence encoding a dominant negative CD3 epsilon polypeptide.
  • expression of the recombinant polynucleic acid in a cell reduces cell surface expression of an endogenous TCR subunit or reduces functional TCR complex formation in the cell compared to the cell surface expression of the endogenous TCR subunit or the functional TCR complex formation in a cell that does not express the fusion polypeptide.
  • expression of the recombinant polynucleic acid in a cell reduces cell surface expression of an endogenous TCR subunit or reduces functional TCR complex formation in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the cell surface expression of the endogenous TCR subunit or the functional TCR complex formation in a cell that does not express the fusion polypeptide.
  • expression of the recombinant polynucleic acid in a cell reduces cell surface expression of an endogenous TCR subunit or reduces functional TCR complex formation in the cell by at least 90% compared to the cell surface expression of the endogenous TCR subunit or the functional TCR complex formation in a cell that does not express the fusion polypeptide.
  • the recombinant polynucleic acid further comprises a promoter.
  • the shRNA is in the intron region of the promoter.
  • the promoter with the shRNA in the intron comprises a sequence at least 90% sequence identity to SEQ ID NO: 1117 or 1119.
  • compositions comprising recombinant polynucleic acids comprising at least two of the following: a sequence encoding the fusion polypeptide encoded by the recombinant polynucleic acid of any of the compositions described herein; a sequence encoding the mutant CD3 zeta polypeptide encoded by any of the recombinant polynucleic acids of a compositions described herein; a sequence encoding the mutant CD3 epsilon polypeptide encoded by the recombinant polynucleic acid of a composition described herein; a sequence encoding the fusion polypeptide encoded by the recombinant polynucleic acid of the composition of described herein; and sequence comprises the shRNA against CD3 zeta encoded by the recombinant polynucleic acid of
  • the composition comprising a recombinant polynucleic acid comprising a sequence encoding the mutant CD3 epsilon polypeptide encoded by the recombinant polynucleic acid of the composition described herein; and a sequence encoding the fusion polypeptide encoded by the recombinant polynucleic acid of the composition described herein.
  • the composition comprising a recombinant polynucleic acid comprising a sequence encoding the mutant CD3 epsilon polypeptide encoded by the recombinant polynucleic acid of the composition described herein; and sequence comprises the shRNA against CD3 zeta encoded by the recombinant polynucleic acid of the composition described herein.
  • the dominant negative CD3 epsilon polypeptide comprises a sequence at least 80% sequence identity to any of the SEQ ID NOs: 23, 24, 35-37, 60 and 61.
  • the recombinant polynucleic acid further comprises a sequence encoding a P2A self- cleaving peptide, a T2A self-cleaving peptide, an E2A self-cleaving peptide, or an F2A self-cleaving peptide.
  • a composition comprising a cell, wherein the cell comprises the recombinant polynucleic acid of the composition described herein.
  • the composition further comprises a small non-coding ribonucleic acid sequence (RNA) or a sequence encoding the small non-coding RNA.
  • the small non-coding RNA is configured to reduce an expression of a subunit of an endogenous T-cell receptor (TCR) complex in a cell relative to a cell that does not comprise the small non-coding RNA.
  • TCR T-cell receptor
  • the small non-coding RNA comprises a micro ribonucleic acid (miRNA), a short hairpin RNA (shRNA), a silencing ribonucleic acid (siRNA), a self-amplifying RNA, or a combination thereof.
  • the small non-coding RNA targets a TCR alpha mRNA.
  • the small non-coding RNA targets a CD3 epsilon mRNA.
  • the small non-coding RNA comprises a sequence with at least 80% sequence identity any one of SEQ ID NOs: 71-76.
  • the composition further comprises a sequence encoding a chimeric antigen receptor (CAR).
  • the CAR comprises an extracellular domain comprising an antigen binding domain; a transmembrane domain; and an intracellular domain comprising an intracellular signaling domain.
  • the antigen binding domain is an anti-CD19 binding domain.
  • the anti-CD19 scFv comprises a sequence with at least about 80% sequence identity to a sequence selected from the sequences in Table 13.
  • the antigen binding domain is an anti-CD20 binding domain.
  • the scFv comprises a sequence with at least about 80% sequence identity to a sequence selected from the sequences in Table 14.
  • the antigen binding domain is an anti-CD22 binding domain.
  • the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1) of QTIWSY (SEQ ID NO: ), LCDR2 of AAS (SEQ ID NO: ), and LCDR3 of QQSYSIPQT (SEQ ID NO: ), respectively; and a heavy chain CDR1 (HCDR1) of GDSVSSNSAA (SEQ ID NO: ), HCDR2 of TYYRSKWYN (SEQ ID NO: ), and HCDR3 of AREVTGDLEDAFDI (SEQ ID NO: ).
  • VL variable light chain domain having a light chain CDR1 (LCDR1) of QTIWSY (SEQ ID NO: ), LCDR2 of AAS (SEQ ID NO: ), and LCDR3 of QQSYSIPQT (SEQ ID NO: ), respectively; and a heavy chain CDR1 (HCDR1) of GDSVSSNSAA (SEQ ID NO: ), HCDR2 of
  • the antigen binding domain comprises an scFv with at least about 80% sequence identity to a sequence selected from the sequences in Table 15.
  • the antigen binding domain binds to an antigen that is selected from the group consisting of: CD19, CD20, CD22, glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY-ESO- 1, LAGE-la, p53, protein, PSMA, HER2, survivin and telomerase, prostate-carcinoma tumor antigen- 1 (PCTA-1), MAGE, ELF
  • the antigen-binding domain binds to an antigen selected from the group consisting of CD19, CD20, and CD22. In some embodiments, the antigen-binding domain binds to CD19. In some embodiments, the antigen-binding domain binds to CD20. In some embodiments, the antigen-binding domain binds to CD22. In some embodiments, the antigen-binding domain binds to CD19 and CD20. In some embodiments, the antigen-binding domain binds to CD19 and CD22. In some embodiments, the antigen-binding domain binds to CD20 and CD22. In some embodiments, the antigen-binding domain binds to CD19 and CD22.
  • the antigen-binding domain binds to CD19, CD20 and CD22.
  • the intracellular domain of the CAR comprises an intracellular costimulatory domain derived from an intracellular costimulatory domain of CD28, 4-1BB/CD137, or CD2.
  • the intracellular domain of the CAR comprises an intracellular signaling CD22, CD79a, CD79b, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, or ZAP70.
  • the transmembrane domain of the CAR comprises a transmembrane domain from CD8 or CD28.
  • the extracellular domain of the CAR comprises a hinge domain from CD8 or CD28.
  • the extracellular domain of the CAR comprises a hinge domain from CD8 and the transmembrane domain of the CAR comprises a transmembrane domain from CD8.
  • the extracellular domain of the CAR comprises a hinge domain from CD28 and the transmembrane domain of the CAR comprises a transmembrane domain from CD28.
  • WSGR Docket No.61078-729.601 intracellular signaling domain from CD3zeta.
  • the CAR comprises an anti-CD20 scFv, a hinge domain from CD28, a transmembrane domain from CD28, and a cytoplasmic domain comprising a costimulatory domain from CD2 and an intracellular signaling domain from CD3zeta. from 4-1BB and an intracellular signaling domain from CD3zeta.
  • the CAR comprises a sequence with at least 80% sequence identity to any one of sequences in Table 16. [116] In some embodiments, the CAR comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 68-70.
  • the cell is a mammalian cell. [118] In some embodiments, the cell is a lymphocyte.
  • the cell is a T cell.
  • the cell is a population of cells.
  • the T cell is a cytotoxic T cell, an effector T cell, a helper T cell, a natural killer T cell, a regulatory T cell, a memory T cell, a stem cell memory T cell, a central memory T cell, an effector memory T cell, a na ⁇ ve T cell, or a suppressor T cell.
  • the cell is a B cell.
  • the B cell is a na ⁇ ve mature B cell, a plasmablast, a plasma cell, or a memory B cell.
  • the T cells in the population of cells are memory T cells.
  • the population of cells comprises at least 1 ⁇ 10 5 cells.
  • pharmaceutical compositions comprising any of the compositions described herein, and a pharmaceutically acceptable excipient or carrier.
  • the cancer is lymphoma or leukemia.
  • the cancer comprises a hematological cancer.
  • the hematological cancer comprises lymphoma.
  • the hematological cancer comprises leukemia.
  • the hematologic cancer is a lymphoma, e.g., a relapsed and/or refractory lymphoma.
  • the cancer is selected from the group consisting of one or more acute leukemias including but not limited to B-cell acute lymphoid leukemia (B-ALL), T-cell acute lymphoid leukemia (TALL), small lymphocytic leukemia (SLL), acute lymphoid leukemia (ALL) (e.g., relapsing and refractory ALL); one or more chronic leukemias including but not limited to chronic myelogenous leukemia (CML), and chronic lymphocytic leukemia (CLL).
  • B-ALL B-cell acute lymphoid leukemia
  • TALL T-cell acute lymphoid leukemia
  • SLL small lymphocytic leukemia
  • ALL acute lymphoid leukemia
  • CML chronic myelogenous
  • Additional hematologic cancers or conditions include, but are not limited to mantle cell lymphoma (MCL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B cell lymphoma (DLBCL), follicular lymphoma, hairy cell leukemia, small cell- or a large cell-follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, Marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, and “preleukemia.”
  • MCL mantle cell lymphoma
  • B cell prolymphocytic leukemia blastic plasmacytoid den
  • a disease as used herein includes, but is not limited to atypical and/or non-classical cancers, malignancies, precancerous conditions; and any combination thereof.
  • the cancer is Large B-Cell Lymphoma (LBCL).
  • the subject has been diagnosed with LBCL through histological confirmation by a pathologist or pathology lab.
  • histological confirmation of LBCL may involve one of the following: diffuse LBCL (DLBCL) not otherwise specified (NOS), including germinal center B-cell (GCB) type or active B-cell (ABC) type, high-grade B-cell lymphoma with or without MYC and BCL2 and/or BCL6 rearrangements (double-hit or triple-hit lymphoma), DLBCL associated with chronic DLBCL arising from follicular lymphoma (FL; transformed follicular lymphoma), DLBCL arising from marginal zone lymphoma (MZL; transformed marginal zone lymphoma), primary mediastinal (thymic) large B-cell lymphoma, or FL, Grade 3B.
  • the human subject with cancer is 18 years of age or older. In some embodiments, the human subject with cancer is younger than 18 years old.
  • the cancer is lung cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, kidney cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, skin cancer, esophageal cancer, and/or the cancer includes a sarcoma cell, a rhabdoid cancer cell, a neuroblastoma cell, retinoblastoma cell, or a medulloblastoma cell, and/or the cancer is uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM),
  • UCS uterine carcinosarcoma
  • FIG.1 shows an exemplary fusion polypeptide that is configured to mediate ubiquitination/degradation of endogenous TCR and/or functional TCR complex within a cell.
  • FIG.2 shows that cells expressing an exemplary fusion polypeptide had low levels of surface expression of functional TCR complex relative to various controls.
  • FIG.3 shows that cells expressing various exemplary fusion polypeptides or shRNA targeted to TCR/CD3 complex components had decreased levels of surface expression of functional TCR complex relative to various controls.
  • FIG.135 shows that FIG.
  • FIG. 4 shows an exemplary mutant polypeptide that is configured to inhibit endogenous functional TCR complex formation within a cell.
  • FIGs. 5A-B show that cells expressing various exemplary mutant polypeptides had significantly decreased levels of surface expression of endogenous functional TCR complex relative to various controls.
  • FIGs. 6A-B show that T cells lacking surface TCR/CD3 due to TRAC KO or expression of mutant CD3-epsilon polypeptide were not activatable through engagement of TCR, whereas control T cells are.
  • FIG.7 shows that T cells lacking surface TCR/CD3 due to TRAC KO or expression of mutant CD3-epsilon polypeptide do not mediate graft-vs-host killing in a Mixed Lymphocyte Reaction (MLR) assay, whereas control T cells do.
  • FIG.8A shows that T cells expressing ER-retained scFvs against TCR exhibit reduced amount of surface TCR complex relative to the control cells that do not express the scFvs.
  • FIG.8B shows the percentages of CD137+CD69+ cells in the population after T cell activation.
  • FIG. 8C shows the percentages of CD25 + T cells in the population after T cell activation.
  • FIG.9A shows the hinge, the transmembrane domain, and the C-terminal ER tag of the ER- retained anti-TCR scFv constructs.
  • FIG.9B shows that the level of leakiness of ER-retained scFv was low in most of the constructs.
  • FIG.10A depicts two polynucleic acid constructs. One construct comprises an EF1a promoter sequence encoding a BFP and a dominant negative CD3 epsilon. Fig.10B shows that BFP expression was not affected by inclusion of the CD3z shRNA in the intron.
  • FIG.10C shows that expressing the construct with the shRNA and the sequence encoding the DN CD3 epsilon further reduced TCR surface levels compared to expressing the construct without the shRNA.
  • FIG. 11A demonstrates different constructs, each comprises a humanized anti-TCR scFv linked to an ER-retention tag.
  • Fig.11B shows that the leakiness of ER-retained scFvs was low in all constructs.
  • FIG. 12A shows that expressing anti-TCR or anti-CD3 binders linked to either ER-retained CD3 or dominant negative CD3 reduces surface expression of the TCR complex.
  • FIG.12B shows that expressing anti-CD58 scFv linked to either ER-retained CD3 or dominant negative CD3 reduces surface expression of CD58 and the TCR/CD3 complex.
  • FIG.12C shows that expressing anti-MHCI scFv linked to either ER-retained CD3 or dominant negative CD3 reduces surface expression of MHCI and the TCR/CD3 complex.
  • DETAILED DESCRIPTION [144] Disclosed herein are compositions and methods comprising recombinant polypeptides and/or recombinant nucleic acids encoding the recombinant polypeptides.
  • the recombinant polypeptides comprise fusion polypeptides or a mutant polypeptide.
  • the fusion polypeptides comprise all or a portion of a sequence of a T-cell receptor (TCR) subunit.
  • TCR T-cell receptor
  • the fusion WSGR Docket No.61078-729.601 polypeptides comprise at least two domains comprising: (a) a first domain that comprises all or a portion of a sequence of a subunit of a TCR and (b) a second domain that mediates a post-translational embodiments, the first domain of a fusion polypeptide comprises a sequence of a transmembrane embodiments, the second domain of a fusion polypeptide comprises a sequence of a ubiquitin ligase.
  • the sequence of a ubiquitin ligase comprises the sequence of a functional ubiquitin ligase domain derived from ubiquitin ligase.
  • the fusion polypeptide comprises a portion of a mutant polypeptide.
  • the mutant polypeptide comprises Definitions [145] The singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.
  • the term “a cell” includes one or more cells, including mixtures thereof.
  • any listed range can be recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, and so forth.
  • each range discussed herein can be readily broken down into a lower third, middle third and upper third, and the like.
  • all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above.
  • a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles.
  • a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.
  • Percent (%) sequence identity” or “homology” with respect to the nucleic acid or amino acid sequences identified herein is defined as the percentage of nucleic acid or amino acid residues in a candidate sequence that are identical with the amino acid residues in the polypeptide being compared, after aligning the sequences considering any conservative substitutions as part of the sequence identity.
  • all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the recombinant polypeptides, methods and other aspects belong.
  • polynucleotides As used herein, the terms “polynucleotides,” “nucleic acids,” and “oligonucleotide,” are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides, or ribonucleotides, or analogs thereof, either in single-, double-, or multi- stranded form.
  • Polynucleotides may have any three-dimensional structure and may perform any known function or unknown function.
  • Various non-limiting examples of polynucleotides include: non-coding or coding regions of a gene fragment, or a gene, locus (loci) that is defined from linkage analysis, exons or introns, messenger RNA, transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA or RNA of any sequences, nucleic acid probes or primers.
  • loci locus that is defined from linkage analysis, exons or introns, messenger RNA, transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA or RNA of any sequences, nucle
  • a polynucleotide may be exogenous or endogenous to a cell.
  • a polynucleotide may exist in a cell-free environment.
  • a polynucleotide may be a gene or WSGR Docket No.61078-729.601 fragment thereof.
  • a polynucleotide may be DNA.
  • a polynucleotide may be RNA.
  • a polynucleotide may have any three-dimensional structure, and may perform any function, known or unknown.
  • a polynucleotide may comprise one or more analogs (e.g., altered backbone, sugar, or nucleobase). If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • analogs include: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, fluorophores (e.g., rhodamine or fluorescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudouridine, dihydrouridine, queuosine, and wyosine.
  • fluorophores e.g., rhodamine or fluorescein linked to the sugar
  • thiol containing nucleotides biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-
  • Non-limiting examples of polynucleotides include coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, eDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, cell-free polynucleotides including cell-free DNA (cfDNA) and cell-free RNA (cfRNA), nucleic acid probes, and primers.
  • loci locus
  • locus defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), short interfering
  • the sequence of nucleotides may be interrupted by non- nucleotide components.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • the terms also encompass an amino acid polymer that has been modified, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • polypeptides may denote an order of amino acids in a polypeptide in an amino to carboxyl terminus direction in which residues that neighbor each other in the sequence are contiguous in the primary structure of the polypeptide. Variants of the amino acid sequences described herein may be included in various embodiments.
  • variant refers to a protein or polypeptide in which one or more amino acid substitutions, deletions, and/or insertions are present as compared to the amino acid sequence of a protein or polypeptide, and the term includes naturally occurring allelic variants and alternative splice variants of a protein or polypeptide.
  • variant encompasses the replacement of one or more amino acids in an amino acid sequence with a similar or WSGR Docket No.61078-729.601 homologous amino acid(s) or a dissimilar amino acid(s). Some variants include alanine substitutions at one or more amino acid positions in an amino acid sequence.
  • a “variant” further refers to a protein with sequence homology to the native biologically active protein that, in some embodiments, retains at least a portion of the therapeutic and/or biological activity of the biologically active protein.
  • a variant protein may share at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity with the reference biologically active protein.
  • a “partial sequence” is a linear sequence of part of a polypeptide that is known to comprise additional residues in one or both directions.
  • a “fragment” is a truncated form of a native biologically active protein that, in some embodiments, retains at least a portion of the therapeutic and/or biological activity.
  • biologically active protein moiety includes proteins modified, as for example, by site directed mutagenesis, insertions, or accidentally through mutations.
  • amino acid refers to either natural and/or unnatural or synthetic amino acids, including but not limited to glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics. Standard single or three letter codes are used to designate amino acids.
  • the term “natural L-amino acid” means the L optical isomer forms of glycine (G), proline (P), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), cysteine (C), phenylalanine (F), tyrosine (Y), tryptophan (W), histidine (H), lysine (K), arginine (R), glutamine (Q), asparagine (N), glutamic acid (E), aspartic acid (D), serine (S), and threonine (T), wherein the redundancy for the genetic code is included in its entirety for all amino acid codons.
  • the term “conservative amino acid substitution” refers to a type of amino acid mutation where an amino acid of one chemical type (e.g., basic, acidic, polar, etc.) is substituted by an amino acid of the same chemical type.
  • the twenty amino standard amino acids commonly used to make human proteins are grouped into several categories based on the chemical properties of their side chains. Amino acids with aliphatic side chains are alanine (“ala”, or “A”), isoleucine (“ile”, or “I”), leucine (“Leu”, or “L”), methionine (“met”, or “M”) and valine (“val”, or “V”).
  • Amino acids with aromatic side chains are phenylalanine (“phe”), or “F”), tryptophan (“trp”, or “W”), and tyrosine (“tyr”, or “Y”).
  • Amino acids with polar neutral side chains are asparagine (“asn”, “N”), cysteine (“cys”, or “C”), glutamine (“gln”, or “Q”), serine (“ser”, or “S”), and threonine (“thr”, or “T”).
  • Amino acids with acidic side chains are aspartic acid (“asp”, “D”) and glutamic acid (“glu”, or “E”).
  • Non-conservative amino acid substitution refers to a type of amino acid mutation where an amino acid of one chemical type (e.g., basic, acidic, polar, etc.) is substituted by an amino acid of a different chemical type.
  • non-naturally occurring means polypeptide or polynucleotide sequences that do not have a counterpart to, are not complementary to, or do not have a high degree of homology with a wild-type or naturally occurring sequence found in a mammal.
  • a non-naturally occurring polypeptide may share no more than 99%, 98%, 95%, 90%, 80%, 70%, 60%, 50% or even less amino acid sequence identity as compared to a natural sequence when suitably aligned.
  • the terms “gene” or “gene fragment” are used interchangeably herein.
  • a gene or gene fragment may be genomic or cDNA, as long as the polynucleotide contains at least one open reading frame, which may cover the entire coding region or a segment thereof.
  • a “fusion gene” is a gene comprising at least two heterologous polynucleotides that are linked together in frame. [161] As used herein, the term “exogenous” refers to a molecule, nucleic acid, protein, or structure that is introduced into the cell by genetic or biochemical means.
  • an exogenous molecule, nucleic acid, protein, or structure can be the same type as an endogenous molecule, nucleic acid, protein, or structure found within the cell, or may be a type of molecule, nucleic acid, protein, or structure that is not normally found in the cell.
  • an “endogenous” molecule, nucleic acid, protein, or structure is one that is present in the particular cell and/or in the particular cell at its developmental stage.
  • an endogenous TCR comprises (1) an endogenous TCR alpha ( ) polypeptide and an endogenous TCR beta ( ) polypeptide or (2) an endogenous TCR gamma ( ) polypeptide and an endogenous TCR delta ( ) polypeptide.
  • an endogenous CD3 dimer comprises (1) an endogenous heterodimer comprising an endogenous CD3 epsilon ( ) polypeptide and an endogenous CD3 delta ( ) polypeptide, (2) an endogenous heterodimer comprising an endogenous CD3 epsilon ( ) polypeptide and an endogenous CD3 gamma ( ) polypeptide, and (3) an endogenous homodimer comprising two endogenous CD3 zeta ( ) polypeptides.
  • An exogenous gene may be expressed from a nucleic acid in the cytoplasm (e.g., a plasmid, mRNA, or other recombinant nucleic acid construct) of a cell or from the genome of a cell following transduction with an integrating vector (e.g., a lentiviral or other retroviral vector) or knock-in with CRISPR/Cas or any other gene-editing mechanism known to those skilled in the art.
  • a nucleic acid in the cytoplasm e.g., a plasmid, mRNA, or other recombinant nucleic acid construct
  • an integrating vector e.g., a lentiviral or other retroviral vector
  • knock-in with CRISPR/Cas e.g., a lentiviral or other retroviral vector
  • An exogenous dimer of protein subunits or polypeptides can comprise a heterodimer (i.e., a dimer having one endogenous polypeptide and one exogenous or WSGR Docket No.61078-729.601 mutant polypeptide) or a homodimer (i.e., a dimer having two exogenous or native polypeptides), depending on the protein.
  • a heterodimer i.e., a dimer having one endogenous polypeptide and one exogenous or WSGR Docket No.61078-729.601 mutant polypeptide
  • a homodimer i.e., a dimer having two exogenous or native polypeptides
  • an exogenous or mutant CD3 complex comprises (1) an exogenous or mutant heterodimer comprising an exogenous or mutant CD3 epsilon ( ) polypeptide and an endogenous CD3 delta ( ) polypeptide, (2) an endogenous heterodimer comprising an endogenous CD3 epsilon ( ) polypeptide and an endogenous CD3 gamma ( ) polypeptide, and (3) an endogenous homodimer comprising two endogenous CD3 zeta ( ) polypeptides.
  • an exogenous or mutant CD3 complex comprises (1) an endogenous heterodimer comprising an endogenous CD3 epsilon ( ) polypeptide and an endogenous CD3 delta ( ) polypeptide, (2) an exogenous or mutant heterodimer comprising an exogenous or mutant CD3 epsilon ( ) polypeptide and an endogenous CD3 gamma ( ) polypeptide, and (3) an endogenous homodimer comprising two endogenous CD3 zeta ( ) polypeptides.
  • an exogenous or mutant CD3 complex comprises (1) an endogenous heterodimer comprising an endogenous CD3 epsilon ( ) polypeptide and an endogenous CD3 delta ( ) polypeptide, (2) an endogenous heterodimer comprising an endogenous CD3 epsilon ( ) polypeptide and an endogenous CD3 gamma ( ) polypeptide, and (3) an exogenous or mutant homodimer comprising an exogenous or mutant CD3 zeta ( ) polypeptide and an endogenous CD3 zeta ( ) polypeptide or two exogenous or mutant CD3 zeta ( ) polypeptides.
  • an exogenous or mutant CD3 complex comprises (1) an exogenous or mutant heterodimer comprising an exogenous or mutant CD3 epsilon ( ) polypeptide and an endogenous CD3 delta ( ) polypeptide, (2) an exogenous or mutant heterodimer comprising an exogenous or mutant CD3 epsilon ( ) polypeptide and an endogenous CD3 gamma ( ) polypeptide, and (3) an endogenous homodimer comprising two endogenous CD3 zeta ( ) polypeptides.
  • an exogenous or mutant CD3 complex comprises (1) an exogenous or mutant heterodimer comprising a mutant or exogenous CD3 epsilon ( ) polypeptide and an endogenous CD3 delta ( ) polypeptide, (2) an endogenous heterodimer comprising an endogenous CD3 epsilon ( ) polypeptide and an endogenous CD3 gamma ( ) polypeptide, and (3) an exogenous or mutant homodimer comprising an exogenous or mutant CD3 zeta ( ) polypeptide and an endogenous CD3 zeta ( ) polypeptide or two exogenous or mutant CD3 zeta ( ) polypeptides.
  • an exogenous or mutant CD3 complex comprises (1) an endogenous heterodimer comprising an endogenous CD3 epsilon ( ) polypeptide and an endogenous CD3 delta ( ) polypeptide, (2) an exogenous or mutant heterodimer comprising an exogenous or mutant CD3 epsilon ( ) polypeptide WSGR Docket No.61078-729.601 and an endogenous CD3 gamma ( ) polypeptide, and (3) an exogenous or mutant homodimer comprising an exogenous or mutant CD3 zeta ( ) polypeptide and an endogenous CD3 zeta ( ) polypeptide or two exogenous or mutant CD3 zeta ( ) polypeptides.
  • Heterologous means derived from a genotypically distinct entity from the rest of the entity to which it is being compared. For example, a glycine rich sequence removed from its native coding sequence and operatively linked to a coding sequence other than the native sequence is a heterologous glycine rich sequence.
  • heterologous as applied to a polynucleotide, a polypeptide, means that the polynucleotide or polypeptide is derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared.
  • “Homology” or “homologous” refers to sequence similarity or interchangeability between two or more polynucleotide sequences or two or more polypeptide sequences.
  • Best Fit When using a program such as Best Fit to determine sequence identity, similarity or homology between two different amino acid sequences, the default settings may be used, or an appropriate scoring matrix, such as blosum45 or blosum80, may be selected to optimize identity, similarity or homology scores.
  • polynucleotides that are homologous are those which hybridize under stringent conditions as defined herein and have at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, more preferably 97%, more preferably 98%, and even more preferably 99% sequence identity to those sequences.
  • operably connected or “operably linked” refers to positioning of components such that they function in their intended manner.
  • the components can be operably connected by a fusion, a linker, and/or a spacer.
  • domain as used herein when referring to a polypeptide or protein, refers to a sequence that can form an independent functional and/or structural unit.
  • binding domain refers to a molecule, such as a protein, or polypeptide sequence, which specifically binds to a target.
  • binding domain refers to a molecule, such as a protein, or polypeptide sequence, which specifically binds to a target.
  • specifically binds means that the binding domain preferentially binds the corresponding target over other targets. In some embodiments, “specifically binds” means that the binding domains have a higher affinity for the target than for other targets.
  • a “therapeutically effective amount” of an agent is an amount or number sufficient to provide a therapeutic benefit in the treatment or management of a disease or disorder, or to delay or minimize one or more symptoms associated with the disease or disorder (e.g., a solid tumor, a hematologic malignancy, or other cancer).
  • the term “therapeutically effective amount” can encompass an amount that improves overall therapeutic efficacy, reduces or avoids symptoms of the disease or disorder, or enhances the therapeutic efficacy of another therapeutic agent.
  • an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.”
  • a “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s).
  • the exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment and will be ascertainable by one skilled in the art using known techniques.
  • the term “treat,” “treating” or “treatment” of any disease or disorder refers, in one instance, to ameliorating the disease or disorder (i.e., slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof).
  • “treat,” “treating” or “treatment” refers to alleviating or ameliorating at least one physical parameter including those which may not be discernible by the patient.
  • “treat,” “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both.
  • T cell receptor TCR
  • T cell receptor complex TCR complex
  • TCR/CD3 complex TCR/CD3 complex
  • CD3 subunits include CD3 delta ( ) subunits, CD3 epsilon ( ) subunits, CD3 gamma ( ) subunits, and CD3 zeta ( ) subunits.
  • TCR , TCR , TCR , and TCR subunits are members of the immunoglobulin protein family and are present as heterodimers comprising either an alpha ( ) and a beta ( ) polypeptide or a gamma ( ) polypeptide and a delta ( ) polypeptide.
  • T cells bearing an alpha/beta ( / ) TCR heterodimer are referred to as “ / T cells”.
  • T cells bearing a gamma/delta ( / ) TCR heterodimer are referred to as “ / T cells”.
  • Functional TCRs are capable of binding an antigenic peptide bound by an MHC/HLA molecule and transducing a signal to activate or inhibit the T cell.
  • the CD3 subunits include four different subunits: CD3 delta ( ), CD3 epsilon ( ), CD3 gamma ( ), and CD3 zeta ( ), typically arranged in an epsilon/delta ( / ) heterodimer (CD3 / ), an epsilon/gamma ( / ) heterodimer (CD3 / ) and a zeta/zeta ( / ) homodimer (CD3 / ).
  • / T cells comprise a TCR complex consisting of an / TCR and a CD3 consisting of a CD3 / heterodimer, CD3 / heterodimer and a CD3 / homodimer
  • / T cells comprise a TCR complex consisting of a / TCR and a CD3 consisting of a CD3 / heterodimer, CD3 / heterodimer and a CD3 / homodimer.
  • the signal transduction process mediated by functional TCR complexes is effectively split between the TCR /TCR or TCR /TCR subunits (which binds to an antigenic peptide presented in the context of an MHC/HLA molecule on an antigen-presenting cell (APC) and triggers a signal transduction cascade) and the CD3 subunits (which transduces the signal produced by the binding of a TCR to antigen presented in the context of an MHC/HLA molecule on an APC to activate or inhibit an antigen-specific adaptive immune response).
  • the CD3 subunits serve as scaffolding for the entire multi-protein complex and also propagate signals from the TCR via immune tyrosine-activating motifs, or “ITAMs”).
  • a functional TCR complex is present on the cell membrane on a T-cell.
  • the functional TCR complex binds antigenic peptides bound to major histocompatibility complex (MHC) molecules.
  • MHC major histocompatibility complex
  • the binding of the functional TCR complex and the antigenic peptide bound to an MHC molecule triggers signal transduction pathways that lead to activation or inhibition of immune responses against the antigenic peptide.
  • Cells or other molecular entities that comprise the antigenic peptide or derivative thereof e.g., a degenerate peptide relative to the antigenic peptide
  • binding of the functional TCR with non- peptide antigenic molecules can also trigger signal transduction pathways that lead to activation or inhibition of immune responses against the non-peptide antigenic molecules.
  • WSGR Docket No.61078-729.601 The stoichiometry of the functional TCR complex comprises: either (1) a heterodimer / TCR) or (2) a heterodimer comprising formation of a dimer between the subunits). Charged amino acid residues in the transmembrane domain of each subunit facilitate the electrostatic interaction/attraction for assembly of a functional TCR complex.
  • Mutation of a charged residue to one with a different charge or without a charge can impair the electrostatic interaction/attraction between the TCR complex subunits, thereby reducing or inhibiting the formation of TCR complexes.
  • different TCR complex subunits may also interact with one another via their extracellular domains. domains.
  • degrading or removing any one of the endogenous TCR polypeptides can result in the loss of an endogenous TCR complex or produce a dysfunctional TCR complex.
  • a functional TCR complex comprises a complete functional TCR complex or any intermediate TCR complexes comprising endogenous subunits of the TCR complex.
  • intermediate TCR complexes lack one or more TCR or CD3 subunits present in a functional TCR/CD3 complex.
  • the conserved aspartic th homodimer is mediated by a tyrosine residue at position 12 (Y12) and a threonine residue at position 17 (T17) (corresponding to the residues Y42 and T47 of SEQ ID NO: 1). Mutation of these residues see, e.g., Call et al., 2006). Other variations or mutations in the native sequences that disrupt the interactions between them are also described in Call et al., 2004.
  • TCR complex formation or assembly is initiated at the endoplasmic reticulum (ER; see, e.g., e.g., an ER-retention signal (see Delgado et al., J. Exp. Med.2005 Feb 21;201(4):555-66, which is over those of the other subunits and prevent the fully assembled TCR complex from being WSGR Docket No.61078-729.601 complex to the plasma membrane.
  • ER endoplasmic reticulum
  • Intracellular signaling domains regulate primary signaling of the functional TCR complex either stimulating signaling or inhibiting it.
  • Signaling domains that stimulate TCR activity comprise signaling motifs known as immunoreceptor tyrosine-based activation motifs (or “ITAMs”).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • the recombinant polypeptide(s) comprise one or more sequences of a TCR polypeptide, e.g., a TCR and a TCR , or a TCR and a TCR .
  • the recombinant polypeptide binds one or more TCR complex polypeptides.
  • a subset of the recombinant polypeptides does not bind all polypeptides in the TCR complex.
  • the recombinant polypeptide comprises a fusion polypeptide, a mutant polypeptide, a domain thereof, a combination of domains thereof, or any combinations thereof.
  • Fusion Polypeptides comprising two or more domains and/or recombinant polynucleic acids encoding the fusion polypeptides.
  • the fusion polypeptides comprise 2, 3, 4, 5, or more domains.
  • the fusion polypeptides comprise at least two domains.
  • the fusion polypeptide comprising at least two domains comprises at least a transmembrane domain and an intracellular domain.
  • the fusion polypeptide comprises two domains.
  • the fusion polypeptide comprising two domains comprises a transmembrane domain and an intracellular domain.
  • the fusion polypeptides disclosed herein mediate a post-translational modification(s) of an endogenous TCR, an intermediate TCR complex, a fully assembled TCR complex, or any combination thereof.
  • the post-translational modification(s) facilitated by the fusion polypeptides described herein is ubiquitination, and the post-translational modification(s) facilitates ubiquitin-mediated degradation of the endogenous TCR, the intermediate TCR complex, the fully assembled TCR complex, or any combination thereof.
  • the post-translational modification(s) may also facilitate a removal of the endogenous TCR, intermediate TCR complex, the fully assembled TCR complex, functional TCR complex, or any combinations thereof from the plasma membrane of a cell.
  • the post-translational modification(s) may facilitate endocytosis of the endogenous TCR, intermediate TCR complex, fully assembled TCR complex, functional TCR complex, or any combinations thereof from the plasma membrane of the cell.
  • Fusion Polypeptide Transmembrane Domain [183]
  • the transmembrane domain of the fusion polypeptide binds or interacts with an endogenous TCR or a polynucleotide sequence encoding the TCR.
  • the transmembrane domain of the fusion polypeptide binds or interact with any endogenous intermediate TCR complexes.
  • the transmembrane domain of the fusion polypeptide binds or interact with a fully assembled TCR complex.
  • the transmembrane domain of the fusion polypeptide binds or interact with a functional TCR complex. Binding or interaction of the transmembrane domain of the fusion polypeptide with an endogenous TCR subunit polypeptide (i.e., intermediate TCR complex, or a fully assembled and functional TCR complex (i.e.
  • the transmembrane domain of the fusion polypeptide comprises a sequence derived from a transmembrane domain of a TCR polypeptide. In some embodiments, the transmembrane domain of the fusion polypeptide comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acids of a transmembrane domain of a TCR polypeptide.
  • the transmembrane domain of the fusion polypeptide comprises about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 WSGR Docket No.61078-729.601 amino acids of a transmembrane domain of a TCR polypeptide. In some embodiments, the transmembrane domain of the fusion polypeptide comprises from about 10 to 25, 15 to 25, 10 to 20, or 10 to 15 amino acids of a transmembrane domain of a TCR polypeptide.
  • the transmembrane domain of the fusion polypeptide comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids of a transmembrane domain of a TCR polypeptide. In some embodiments, the transmembrane domain of the fusion polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids of a transmembrane domain of a TCR polypeptide.
  • the transmembrane domain of the fusion polypeptide comprises at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of a transmembrane domain of a TCR polypeptide. In some embodiments, the transmembrane domain of the fusion polypeptide comprises at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of a transmembrane domain of a TCR polypeptide.
  • the transmembrane domain of the fusion polypeptide comprises about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of a transmembrane domain of a TCR polypeptide. In some embodiments, the transmembrane domain of the fusion polypeptide comprises 100% sequence identity to a sequence of a transmembrane domain of a TCR polypeptide. [185] In some embodiments, the transmembrane domain of the fusion polypeptide comprises a polypeptide, or a combination thereof. In some embodiments, the transmembrane domain of the fusion polypeptide.
  • the transmembrane domain of the fusion polypeptide comprises a embodiments, the transmembrane domain of the fusion polypeptide comprises a sequence of a transmembrane domain of the fusion polypeptide does not comprise a sequence of a transmembrane [186] In some embodiments, the transmembrane domain of the fusion polypeptide comprises a transmembrane domain of the fusion polypeptide comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, WSGR Docket No.61078-729.601 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, embodiments, the transmembrane domain of the fusion polypeptide comprises at most about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, embodiments, the transmembrane domain of the fusion polypeptide comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
  • the transmembrane domain of the fusion polypeptide comprises at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of of the fusion polypeptide comprises about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% embodiments, the transmembrane domain of the fusion polypeptide comprises 100% sequence identity [187] In some embodiments, the transmembrane domain of the fusion polypeptide comprises a transmembrane domain of the fusion polypeptide comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, embodiments, the transmembrane domain of the fusion polypeptide comprises at most about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, embodiments, the transmembran
  • the transmembrane domain of the fusion polypeptide comprises at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of of the fusion polypeptide comprises about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% WSGR Docket No.61078-729.601 embodiments, the transmembrane domain of the fusion polypeptide comprises 100% sequence identity [188] In some embodiments, the transmembrane domain of the fusion polypeptide comprises a transmembrane domain of the fusion polypeptide comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, embodiments, the transmembrane domain of the fusion polypeptide comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32
  • the transmembrane domain of the fusion polypeptide comprises at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a transmembrane domain of the fusion polypeptide comprises at most about 60%, 65% 70%, 75%, 80%, polypeptide.
  • the transmembrane domain of the fusion polypeptide comprises about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of a the fusion polypeptide comprises 100% sequence identity to a sequence of a transmembrane domain [191] In some embodiments, the transmembrane domain of the fusion polypeptide comprises a transmembrane domain of the fusion polypeptide comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, polypeptide.
  • the transmembrane domain of the fusion polypeptide comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids of a WSGR Docket No.61078-729.601 the fusion polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or transmembrane domain of the fusion polypeptide comprises at least about 60%, 65% 70%, 75%, 80%, polypeptide.
  • the transmembrane domain of the fusion polypeptide comprises at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of of the fusion polypeptide comprises about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% embodiments, the transmembrane domain of the fusion polypeptide comprises 100% sequence identity [192] In some embodiments, the transmembrane domain of the fusion polypeptide comprises a transmembrane domain of the fusion polypeptide comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, polypeptide.
  • the transmembrane domain of the fusion polypeptide comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids of a the fusion polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or transmembrane domain of the fusion polypeptide comprises at least about 60%, 65% 70%, 75%, 80%, polypeptide.
  • the transmembrane domain of the fusion polypeptide comprises at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of of the fusion polypeptide comprises about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% embodiments, the transmembrane domain of the fusion polypeptide comprises 100% sequence identity [193] In some embodiments, the transmembrane domain of the fusion polypeptide comprises a transmembrane domain of the fusion polypeptide comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, WSGR Docket No.61078-729.601 polypeptide.
  • the transmembrane domain of the fusion polypeptide comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acids of a the fusion polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or transmembrane domain of the fusion polypeptide comprises at least about 60%, 65% 70%, 75%, 80%, polypeptide.
  • the transmembrane domain of the fusion polypeptide comprises at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of of the fusion polypeptide comprises about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% embodiments, the transmembrane domain of the fusion polypeptide comprises 100% sequence identity [194] In some embodiments, the transmembrane domain of the fusion polypeptide comprises at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of any one of SEQ ID NOs: 15-18.
  • the transmembrane domain of the fusion polypeptide comprises at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 15-18. In some embodiments, the transmembrane domain of the fusion polypeptide comprises about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 15-18. In some embodiments, the transmembrane domain of the fusion polypeptide comprises 100% sequence identity to a sequence of any one of SEQ ID NOs: 15-18.
  • the intracellular domain of the fusion polypeptide mediates or facilitates at least a post-translational modification of an endogenous TCR subunit polypeptide or a polynucleotide sequence encoding the TCR.
  • the intracellular domain of the fusion polypeptide mediates or facilitates at least a post-translational modification of an endogenous intermediate TCR complex polypeptide.
  • the intracellular domain of the fusion polypeptide mediates or facilitates at least a post-translational modification of a fully assembled TCR complex polypeptide.
  • the intracellular domain of the fusion polypeptide mediates or facilitates at least a post-translational modification of a subunit of a TCR complex.
  • the intracellular domain of the fusion polypeptide WSGR Docket No.61078-729.601 comprises an enzymatic domain that can generate a post-translational modification of a subunit of an endogenous TCR complex.
  • the fusion polypeptide mediates, facilitates, or generates a post-translational modification of a subunit of an endogenous TCR complex in vivo, in vitro, ex vivo, or a combination thereof.
  • the post-translational modification of the endogenous TCR polypeptide or endogenous TCR complex polypeptide facilitates degradation of an TCR subunit from a plasma membrane of the cell.
  • This post-translational modification facilitates removal of the endogenous TCR polypeptide TCR subunit from the cell’s plasma membrane.
  • the removal of the endogenous TCR or endogenous TCR complex from the plasma membrane of the cell generally occurs via endocytosis of the endogenous TCR or endogenous TCR complex from the plasma membrane of the cell.
  • the post- translational modification may also facilitate a removal of the endogenous TCR or endogenous TCR complex from an intracellular membrane of the cell (e.g., endoplasmic reticulum, Golgi apparatus, endosome, or lysosome).
  • the post-translational modification comprises protein conjugation.
  • the protein conjugation comprises ubiquitination, SUMOlyation, neddylation, or a combination thereof.
  • the post-translational modification comprises ubiquitination.
  • the ubiquitination comprises mono-ubiquitination (i.e., conjugation of one ubiquitin to a polypeptide or protein) or poly-ubiquitination (i.e., conjugation of more than one ubiquitin to a polypeptide or protein).
  • Poly-ubiquitination comprises conjugation of a first ubiquitin to a protein or polypeptide and further conjugation of one or more secondary ubiquitins to the first ubiquitin.
  • a secondary ubiquitin is conjugated to the previous ubiquitin.
  • poly-ubiquitination of a polypeptide or protein mediates degradation of the polypeptide or protein.
  • poly-ubiquitination of a polypeptide or protein prior to degradation comprises conjugation of a secondary ubiquitin to the lysine 29 (K29) or 48 (K48) of the first ubiquitin.
  • mono-ubiquitination of a polypeptide or protein may mediate endocytosis of the polypeptide or protein.
  • Poly-ubiquitination of a polypeptide or protein may also mediate endocytosis of the polypeptide or protein.
  • the intracellular domain of the fusion polypeptide provided herein comprises a functional ubiquitin ligase domain (a ubiquitin ligase) of a ubiquitin ligase.
  • a functional ubiquitin ligase domain catalyzes the ubiquitination of a protein or polypeptide in vivo, in vitro, ex vivo, or a combination thereof.
  • a functional ubiquitin ligase domain comprises an enzymatic domain capable of catalyzing the ubiquitination of a protein or polypeptide in vivo, in vitro, ex vivo, or a WSGR Docket No.61078-729.601 combination thereof.
  • a functional ubiquitin ligase domain also catalyzes secondary ubiquitination (i.e., the ubiquitination of a first ubiquitin conjugated to a protein or polypeptide) in vivo, in vitro, ex vivo, or a combination thereof.
  • the functional ubiquitin ligase domain comprises a functional ubiquitin ligase domain derived from ubiquitin ligase protein selected from the group consisting of a RING (really interesting new gene) finger protein (RNF) family ubiquitin ligase, a U-box E3 ligase family ubiquitin ligase, a membrane associated ring-CH-type finger (MARCH) family ubiquitin ligase, an anergy in lymphocytes (GRAIL) E3 ubiquitin ligase, a carboxyl-terminus of Hsc70 interacting protein (CHIP) ubiquitin ligase, and a combination thereof.
  • RMF RING (really interesting new gene) finger protein
  • MARCH membrane associated ring-CH-type finger
  • GRAIL anergy in lymphocytes
  • CHIP carboxyl-terminus of Hsc70 interacting protein
  • the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from an RNF family ubiquitin ligase. In some embodiments, the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from an U-box E3 ligase family ubiquitin ligase. In some embodiments, the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from a MARCH family ubiquitin ligase. In some embodiments, the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from a GRAIL E3 ubiquitin ligase.
  • the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from a CHIP ubiquitin ligase. In some embodiments, the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from an E3 ubiquitin ligase. In some embodiments, the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from a RING (really interesting new gene) finger protein (RNF) family ubiquitin ligase selected from the group consisting of RNF122, RNF133, RNF152, RNF130, RNF148, RNF149, RNF150 and a combination thereof.
  • RNF RING
  • the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from a membrane associated ring-CH-type finger (MARCH) family ubiquitin ligase selected from the group consisting of MARCH1, MARCH2, MARCH3, MARCH4, MARCH6, MARCH8, MARCH9 and a combination thereof.
  • MARCH membrane associated ring-CH-type finger
  • the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from the group consisting of RNF130, RNF148, RNF149, RNF150, and a combination. thereof.
  • the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from RNF122. In some embodiments, the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from RNF133. In some embodiments, the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from RNF152. In some embodiments, the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from RNF130. In some embodiments, the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from RNF148.
  • the functional ubiquitin ligase WSGR Docket No.61078-729.601 domain comprises a ubiquitin ligase domain derived from RNF149. In some embodiments, the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from RNF150. In some embodiments, the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from MARCH1. In some embodiments, the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from MARCH2. In some embodiments, the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from MARCH3.
  • the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from MARCH4. In some embodiments, the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from MARCH6. In some embodiments, the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from MARCH8. In some embodiments, the functional ubiquitin ligase domain comprises a ubiquitin ligase domain derived from MARCH9.
  • the intracellular domain of a fusion polypeptide does not comprise a ubiquitin ligase domain derived from the group consisting of GRAIL, CHIP, RNF122, RNF133, RNF152, and a combination thereof. In some embodiments, the intracellular domain of a fusion polypeptide does not comprise a ubiquitin ligase domain of GRAIL. In some embodiments, the intracellular domain of a fusion polypeptide does not comprise a ubiquitin ligase domain of CHIP. In some embodiments, the intracellular domain of a fusion polypeptide does not comprise a ubiquitin ligase domain of RNF122.
  • the intracellular domain of a fusion polypeptide does not comprise a ubiquitin ligase domain of RNF133. In some embodiments, the intracellular domain of a fusion polypeptide does not comprise a ubiquitin ligase domain of RNF152. [201] In some embodiments, the ubiquitin ligase domain of the fusion polypeptide comprises at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of any one of the sequences of Table 4.
  • the ubiquitin ligase domain of the fusion polypeptide comprises at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of any one of SEQ ID NOs: 115-127. In some embodiments, the ubiquitin ligase domain of the fusion polypeptide comprises at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 115-127.
  • the ubiquitin ligase domain of the fusion polypeptide comprises about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 115- 127. In some embodiments, the ubiquitin ligase domain of the fusion polypeptide comprises 100% sequence identity to a sequence of any one of SEQ ID NOs: 115-127. [202] In some embodiments, the intracellular domain of a fusion polypeptide does not comprise a functional ubiquitin ligase domain.
  • the intracellular domain without a functional ubiquitin ligase domain may facilitate ubiquitination of an endogenous TCR or endogenous TCR WSGR Docket No.61078-729.601 complex.
  • the intracellular domain of the fusion polypeptide without a functional ubiquitin ligase domain comprises a domain that recruits a ubiquitin ligase or a protein having a functional ubiquitin ligase domain.
  • the intracellular domain of the fusion polypeptide comprises a sequence of an intracellular domain of a TCR.
  • a dissociation constant (KD) of a fusion polypeptide comprising a TCR intracellular domain, or a fragment thereof to an endogenous TCR may be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower than that of a fusion polypeptide that does not comprise the TCR intracellular domain, the partial sequence thereof, or the fragment thereof.
  • the K D of a fusion polypeptide comprising a TCR intracellular domain, or a fragment thereof to an endogenous TCR may be at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower than that of a fusion polypeptide that does not comprise the TCR intracellular domain, the partial sequence thereof, or the fragment thereof.
  • the stability of a fusion polypeptide comprising a TCR intracellular domain, or a fragment thereof may be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% more than that of a fusion polypeptide that does not comprise the TCR intracellular domain, the partial sequence thereof, or the fragment thereof.
  • the stability of a fusion polypeptide comprising a TCR intracellular domain, or a fragment thereof may be at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% more than that of a fusion polypeptide that does not comprise the TCR intracellular domain, the partial sequence thereof, or the fragment thereof.
  • the intracellular domain of the fusion polypeptide comprises a sequence of an intracellular domain of a TCR.
  • the intracellular domain of the fusion polypeptide comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 or more amino acids of an intracellular domain of a TCR.
  • the intracellular domain of the fusion polypeptide comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, WSGR Docket No.61078-729.601 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids of an intracellular domain of a TCR.
  • the intracellular domain of the fusion polypeptide comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60 amino acids of an intracellular domain of a TCR.
  • the intracellular domain of the fusion polypeptide comprises at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of an intracellular domain of a TCR.
  • the intracellular domain of the fusion polypeptide comprises at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of an intracellular domain of a TCR. In some embodiments, the intracellular domain of the fusion polypeptide comprises about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of an intracellular domain of a TCR. In some embodiments, the intracellular domain of the fusion polypeptide comprises 100% sequence identity to a sequence of an intracellular domain of a TCR.
  • the TCR intracellular domain of the fusion polypeptide comprises the some embodiments, the TCR intracellular domain of the fusion polypeptide comprises the intracellular embodiments, the TCR intracellular domain of the fusion polypeptide does not comprise the polypeptide does not comprise a signaling domain capable of mediating a functional TCR complex- mediated signaling cascade. In some embodiments, the TCR intracellular domain of the fusion polypeptide does not comprise ITAM. In some embodiments, the TCR intracellular domain of the fusion polypeptide comprises a mutated ITAM, such as a mutated ITAM with reduced signaling activity.
  • the intracellular domain of the fusion polypeptide comprises a sequence that has at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of any one of SEQ ID NOs: 25, 26, 28, 30, 31, and 33. In some embodiments, the intracellular domain of the fusion polypeptide comprises a sequence that has at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 25, 26, 28, 30, 31, and 33.
  • the intracellular domain of the fusion polypeptide comprises a sequence that has about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 25, 26, 28, 30, 31, and 33. In some embodiments, the intracellular domain of the fusion polypeptide comprises a sequence that has 100% sequence WSGR Docket No.61078-729.601 identity to a sequence of any one of SEQ ID NOs: 25, 26, 28, 30, 31, and 33.
  • the intracellular domain of the fusion polypeptide comprises a sequence that has at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of any one of SEQ ID NOs: 27, 29, 32, and 34. In some embodiments, the intracellular domain of the fusion polypeptide comprises a sequence that has at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 27, 29, 32, and 34.
  • the intracellular domain of the fusion polypeptide comprises a sequence that has about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 27, 29, 32, and 34. In some embodiments, the intracellular domain of the fusion polypeptide comprises a sequence that has 100% sequence identity to a sequence of any one of SEQ ID NOs: 27, 29, 32, and 34. [208] In some embodiments, the intracellular domain of the fusion polypeptide comprises a sequence that has at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of any one of SEQ ID NOs: 25-34.
  • the intracellular domain of the fusion polypeptide comprises a sequence that has at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 25-34. In some embodiments, the intracellular domain of the fusion polypeptide comprises a sequence that has about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 25-34. In some embodiments, the intracellular domain of the fusion polypeptide comprises a sequence that has 100% sequence identity to a sequence of any one of SEQ ID NOs: 25- 34.
  • the fusion polypeptide comprises a functional ubiquitin ligase domain and an additional sequence or domain in the cytoplasmic domain, the additional sequence or domain is N-terminal to the functional ubiquitin ligase domain. In some embodiments, the fusion polypeptide comprises a functional ubiquitin ligase domain and an additional sequence or domain in the cytoplasmic domain, the additional sequence or domain is C-terminal to the functional ubiquitin ligase domain.
  • Extracellular Domain [210] In another aspect, the fusion polypeptides disclosed herein further comprise an extracellular domain.
  • the extracellular domain of the fusion polypeptide comprises a sequence or part of a sequence of an extracellular domain of an endogenous TCR polypeptide.
  • the addition of an endogenous TCR extracellular domain, or a fragment thereof WSGR Docket No.61078-729.601 to any of the fusion polypeptides disclosed herein increases the binding affinity of the fusion polypeptide for an endogenous TCR polypeptide TCR subunit.
  • the KD of a fusion polypeptide comprising a TCR extracellular domain, or a fragment thereof for an endogenous TCR polypeptide may be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower than the KD of a fusion polypeptide without a TCR extracellular domain, the partial sequence thereof, or the fragment thereof for the same endogenous TCR polypeptide.
  • the K D of a fusion polypeptide comprising a TCR extracellular domain, or a fragment thereof for an endogenous TCR polypeptide may be at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower than the K D of a fusion polypeptide lacking a TCR extracellular domain, the partial sequence thereof, or the fragment thereof for the same endogenous TCR polypeptide.
  • the addition of a TCR extracellular domain, or a fragment thereof to any of the fusion polypeptides disclosed herein increases the stability (e.g., the half life) of the fusion polypeptide.
  • the TCR extracellular domain, or a fragment thereof may be glycosylated by enzymes that mediate a similar glycosylation to an endogenous TCR.
  • the stability of a fusion polypeptide comprising a TCR extracellular domain, or a fragment thereof may be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% more than that of a fusion polypeptide lacking a TCR extracellular domain, the partial sequence thereof, or the fragment thereof.
  • the stability of a fusion polypeptide comprising a TCR extracellular domain, or a fragment thereof may be at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% more than that of a fusion polypeptide lacking a TCR extracellular domain, the partial sequence thereof, or the fragment thereof.
  • the extracellular domain of the fusion polypeptide comprises a sequence of an extracellular domain of a TCR.
  • the extracellular domain of the fusion polypeptide comprises at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 150, 200 or more amino acids of an extracellular domain of an endogenous TCR polypeptide.
  • the extracellular domain of the fusion polypeptide comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 150, or 200 amino acids of an extracellular domain of an endogenous TCR polypeptide.
  • the extracellular domain of the fusion polypeptide WSGR Docket No.61078-729.601 comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, 150, or 200 amino acids of an extracellular domain of an endogenous TCR polypeptide.
  • the extracellular domain of the fusion polypeptide comprises at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of an extracellular domain of an endogenous TCR polypeptide. In some embodiments, the extracellular domain of the fusion polypeptide comprises at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of an extracellular domain of an endogenous TCR polypeptide.
  • the extracellular domain of the fusion polypeptide comprises about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of an extracellular domain of an endogenous TCR polypeptide. In some embodiments, the extracellular domain of the fusion polypeptide comprises 100% sequence identity to a sequence of an extracellular domain of an endogenous TCR polypeptide. In some embodiments, the extracellular domain of an endogenous TCR polypeptide comprises an extracellular domain selected from the group consisting of the extracellular domain of a TCR polypeptide, the extracellular domain of a TCR polypeptide, the extracellular domain of a TCR polypeptide, and the extracellular domain of a TCR polypeptide.
  • the extracellular domain of the fusion polypeptide comprises the polypeptide. In some embodiments, the extracellular domain of the fusion polypeptide comprises the extracellular domain of the fusion polypeptide comprises the extracellular domain of an endogenous polypeptide. [214] In some embodiments, the extracellular domain of the fusion polypeptide comprises a sequence that has at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of any one of SEQ ID NOs: 11-14.
  • the extracellular domain of the WSGR Docket No.61078-729.601 fusion polypeptide comprises a sequence that has at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 11-14. In some embodiments, the extracellular domain of the fusion polypeptide comprises a sequence that has about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 11-14. In some embodiments, the extracellular domain of the fusion polypeptide comprises a sequence that has 100% sequence identity to a sequence of any one of SEQ ID NOs: 11- 14.
  • a protein domain e.g., the transmembrane domain, the intracellular domain, the extracellular domain, the TCR intracellular domain, or the ubiquitin ligase domain
  • a first domain is operatively linked to a second domain by a linker.
  • the first domain is operatively linked to a second domain by a first linker, and the first and second domains are operatively linked to a third domain by a second linker.
  • the first domain is operatively linked to a second domain, and/or a third domain, and/or a fourth domain by a third linker.
  • the first domain may be linked to three or more additional domains by additional linkers.
  • the linker may include one or more intervening amino acid residues that are positioned between the first domain and second domain, and/or are positioned between the first, second, or third domains and any additional domains. In principle, there are no particular limitations on the length and/or amino acid composition of the linker.
  • any arbitrary single- chain peptide comprising about one to about 300 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) can be used as a linker.
  • the linker includes at least about 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.
  • the linker includes no more than about 300, 250, 200, 150, 140, 130, 120, 110, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, or 30 amino acid residues.
  • compositions comprising of a cell, wherein the cell comprises the recombinant polynucleic acid of the compositions described herein.
  • a composition comprising a recombinant polynucleic acid comprising a sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises (1) an antigen binding domain, wherein the antigen binding domain binds to a TCR subunit; and (2) an ER-retention tag.
  • WSGR Docket No.61078-729.601 In one aspect, provided herein is a composition comprising a recombinant polynucleic acid comprising a sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises (1) an antigen binding domain, wherein the antigen binding domain binds to CD58; and (2) an ER- retention tag. [218] In one aspect, provided herein is a composition comprising a recombinant polynucleic acid comprising a sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises (1) an antigen binding domain, wherein the antigen binding domain binds to MHCI; and (2) an ER- retention tag.
  • expression of the fusion polypeptide in a cell reduces cell surface expression of an endogenous TCR subunit or reduces functional TCR complex formation in the cell compared to the cell surface expression of the endogenous TCR subunit or the functional TCR complex formation in a cell that does not express the fusion polypeptide.
  • expression of the fusion polypeptide in a cell reduces cell surface expression of an endogenous TCR subunit or reduces functional TCR complex formation in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the cell surface expression of the endogenous TCR subunit or the functional TCR complex formation in a cell that does not express the fusion polypeptide.
  • the fusion polypeptide with antigen binding domains further comprises a KDEL sequence.
  • the KDEL sequence is positioned between the antigen binding domain and the transmembrane domain.
  • the antigen binding domain is an scFv. In some embodiments, the antigen binding domain is a humanized scFv.
  • the antigen binding domain is a VHH. In some embodiments, the antigen binding domain comprises a variable heavy chain domain (VH) and a variable light chain domain (VL). [221] In some embodiments, the heavy chain domain is linked to the light chain domain via a linker. In some embodiments, the linker is a Whitlow linker. In some embodiments, the antigen binding domain from N-terminal to C-terminal comprises VH-Whitlow linker – VL. In some embodiments, the antigen binding domain from N-terminal to C-terminal comprises VL-Whitlow linker – VH. [222] In some embodiments, the fusion polypeptide with antigen binding domains further comprises a transmembrane domain.
  • the transmembrane domain comprises a CD8a transmembrane domain, a UGT2B17 transmembrane domain, or a Trem1 transmembrane domain.
  • the transmembrane domain is a UGT2B17 transmembrane domain.
  • the transmembrane domain is a Trem1 transmembrane domain.
  • the ER retention tag is positioned at the C-terminus of the fusion polypeptide.
  • the ER retention tag is a E319K tag.
  • the ER retention tag comprises an amino acid sequence of KKKKRD.
  • the ER retention tag comprises a sequence at least 95% sequence identity to any of the sequences in Table 5.
  • the fusion polypeptides with antigen binding domains comprise at least 80% sequence identity to any of the sequence in Table 6 and Table 7.
  • the fusion polypeptides with antigen binding domains comprise any one of SEQ ID NOs: 136-172, or a sequence that has about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 136-172.
  • a fusion polypeptide comprises any combinations described by formula (I).
  • a fusion polypeptide comprises any one of the following combination based on formula (I): a1b1, a1b2, a1b3, a1b4, a1b5, a1b6, a1b7, a1b8, a1b9, a1b10, a1b11, a1b12, a1b13, a1b14, a2b1, a2b2, a2b3, a2b4, a2b5, a2b6, a2b7, a2b8, a2b9, a2b10, a2b11, a2b12, a2b13, a2b14, a3b1, a3b2, a3b3, a3b4, a3b5, a3b6, a3b7, a3b8, a3b9, a3b
  • the fusion polypeptide comprises a sequence that has at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of any one of SEQ ID NOs: 44-59,61, and 136-172. In some embodiments, the fusion polypeptide comprises a sequence that has at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 44-59,61, and 136-172.
  • the fusion polypeptide comprises a sequence that has about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 44-59,61, and 136-172. In some embodiments, the fusion polypeptide comprises a sequence that has 100% sequence identity to a WSGR Docket No.61078-729.601 sequence of any one of SEQ ID NOs: 44-59,61, and 136-172.
  • the fusion polypeptide comprises a sequence that has at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of any one of SEQ ID NOs: 44-59,61, and 136-172. In some embodiments, the fusion polypeptide comprises a sequence that has at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 44-45 and 47-59.
  • the fusion polypeptide comprises a sequence that has about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 44-45 and 47-59. In some embodiments, the fusion polypeptide comprises a sequence that has 100% sequence identity to a sequence of any one of SEQ ID NOs: 44-45 and 47-59.
  • Mutant Polypeptides [228]
  • disclosed herein are recombinant polypeptides and/or recombinant polynucleic acids encoding the recombinant polypeptides comprising at least 1, 2, 3, 4, 5 or more domains.
  • the recombinant polypeptide comprises a transmembrane domain. In some embodiments, the recombinant polypeptide comprises an extracellular domain. In some embodiments, the transmembrane domain of the recombinant polypeptide comprises an amino acid residue that mediates its interaction with other endogenous polypeptides. In some embodiments, the recombinant polypeptide comprises an intracellular domain. In some embodiments, the recombinant polypeptide lacks an intracellular domain. In some embodiments, the intracellular domain of the recombinant polypeptide mediates the intracellular localization of the recombinant polypeptide. In some embodiments, the recombinant polypeptide does not directly or indirectly trigger a signaling cascade.
  • the recombinant polypeptides disclosed herein comprise a sequence of an endogenous polypeptide (a counterpart endogenous polypeptide).
  • the recombinant polypeptide is a mutant polypeptide relative to the counterpart endogenous polypeptide.
  • the mutant polypeptide has at least 1, 2, 3, 4, 5 or more mutations relative to the counterpart endogenous polypeptide.
  • the mutant polypeptide has altered properties relative to the counterpart endogenous polypeptide.
  • the mutant polypeptide has altered binding affinities to other endogenous polypeptides, altered intracellular localization, altered signaling capacity, or a combination thereof, relative to the counterpart endogenous polypeptide.
  • the mutant polypeptide is a dominant negative form of the counterpart endogenous polypeptide.
  • the mutant polypeptides disclosed herein comprise a sequence of a mutant TCR complex polypeptide.
  • the mutant polypeptide comprises a dominant negative form (dominant negative inhibitor) of an endogenous TCR complex subunit when expressed within a cell.
  • the mutant polypeptide inhibits the formation of functional TCR complex within the cell.
  • the mutant polypeptide binds at least one but not all endogenous TCR subunits.
  • the mutant polypeptide is capable of binding to an endogenous TCR complex (or a polypeptide sequence encoding the TCR).
  • the mutant polypeptide binds to an endogenous TCR complex subunit but not all endogenous TCR complex subunits.
  • the mutant polypeptide may prevent the bound endogenous TCR (or a polypeptide comprising a portion of the sequence of the endogenous TCR) from forming an endogenous or functional TCR complex.
  • the mutant polypeptide does not comprise a functional signaling domain or localization domain/signal.
  • the lack of a functional signaling domain prevents an exogenous TCR complex comprising the mutant polypeptide from triggering a signaling cascade started by an endogenous or functional TCR complex.
  • a lack of a localization domain prevents the assembly of a functional TCR complex or correct localization of the functional TCR complex.
  • the formation of the exogenous TCR complex comprising the mutant polypeptide results in a decreased amount of an endogenous or functional TCR complex.
  • the mutant polypeptide facilitates the formation of the intermediate TCR complexes within the cell.
  • the mutant polypeptide facilitates the transport of the intermediate TCR complexes to the plasma membrane within the cell.
  • exogenous as used herein when referring to TCR complex/oligomer may comprise a complex/oligomer that does not occur in a cell that has not contacted with an exogenous genetic material or a progeny derived thereof.
  • the exogenous genetic material may not be found in a native mammalian or human cell.
  • the exogenous genetic material may encode an exogenous polypeptide.
  • WSGR Docket No.61078-729.601 The exogenous polypeptide may comprise a sequence of a TCR or encode a polypeptide comprising a sequence of a TCR.
  • Mutant CD3 zeta polypeptide comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutated amino acid residues relative [235] its binding partner from participating in the endogenous TCR-activated signaling cascade within a cell. endogenous TCR complex subunits or participating in functional TCR complex formation within a formation of an exogenous TCR complex.
  • Transmembrane Domain configured to bind one but not all TCR complex subunits.
  • the transmembrane WSGR Docket No.61078-729.601 [237] [238] 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids of a transmembrane domain of polypeptide comprises at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of WSGR Docket No.61078-729.601 comprises about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a [239] polypeptide and other endogenous TCR complex subunits (e.g., combination thereof.
  • the mutation is at the position designated “X” in formula (II): LCYLLXGILFIYGVILTALFL (SEQ ID NO: 999).
  • the endogenous transmembrane domain has the sequence of SEQ ID NO: 15.
  • the mutant at the position designated “X” in SEQ ID NO: 999 does not have an acidic amino acid at the position designated “X” in SEQ ID NO: 999.
  • polypeptide has a non-acidic amino acid at the X amino acid of SEQ ID NO: 999. In some “X” in SEQ ID NO: 999.
  • the mutation at the X amino acid of SEQ ID NO: 999 comprises a deletion of the X amino acid of SEQ ID NO: 999 or a deletion of D36 (corresponding to the 36 th amino acid of SEQ ID NO: 999 or D36 of SEQ ID NO: 1 (corresponding to the 36 th amino acid of [240] N, A R, K, H, or S at the position polypeptide has K at the position designated “X” in SEQ ID NO: 999.
  • the WSGR Docket No.61078-729.601 polypeptide has G at the position designated “X” in SEQ ID NO: 999.
  • the polypeptide has W at the position designated “X” in SEQ ID NO: 999.
  • the [241] a sequence that has at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of any one of SEQ ID NOs: 19-22.
  • the transmembrane domain of the polypeptide comprises a sequence that has at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of SEQ ID NO: 15.
  • the 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of SEQ ID NO: sequence that has about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a WSGR Docket No.61078-729.601 polypeptide comprises a sequence that has 100% sequence identity to a sequence of SEQ ID NO: 15.
  • polypeptide comprises a sequence of an extracellular domain of a TCR.
  • the extracellular domain of the mutant CD3 comprises a TCR extracellular domain, or a fragment a polypeptide thereof or an endogenous TCR complex or a polypeptide thereof.
  • the dissociation constant (KD extracellular domain, or a fragment thereof for an endogenous TCR or a polypeptide thereof is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, the TCR extracellular domain, the partial sequence thereof, or the fragment thereof.
  • the K D fragment thereof for an endogenous TCR or polypeptide thereof may be at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or domain, the partial sequence thereof, or the fragment thereof.
  • the addition of a TCR extracellular domain, or a fragment thereof to a added TCR extracellular domain, or a fragment thereof is glycosylated by enzymes that mediate a similar glycosylation on the endogenous TCR.
  • the stability (e.g., half-life) of least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, extracellular domain, the partial sequence thereof, or the fragment thereof.
  • the thereof may be at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, comprise the TCR extracellular domain, the partial sequence thereof, or the fragment thereof.
  • WSGR Docket No.61078-729.601 may comprise at most about 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids of an extracellular domain of about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of an about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of polypeptide comprises at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence WSGR Docket No.61078-729.601 [245] least about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 60, 70, 80, 90, 100, polypeptide comprises at most about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
  • polypeptide comprises a sequence that has at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 11. In some embodiments, the extracellular domain of 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 11. In some embodiments, the extracellular 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 11. In some embodiments, the identity to SEQ ID NO: 11. Intracellular Domain [247] capable of triggering a signaling cascade induced by a functional TCR complex. In some embodiments, domain.
  • the CD3 intracellular domain has one or more mutations compared WSGR Docket No.61078-729.601 to the endogenous CD3 comprises one or more deletions, amino acid substitutions, insertions, or a combination thereof.
  • SEQ ID NO. 25 In more mutated ITAMs that are unable to trigger a signaling cascade induced by a functional TCR complex.
  • the intracellular domain of the one of SEQ ID NOs: 25-26 [251] sequence that has at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of any one of SEQ ID NOs: 27-34.
  • the intracellular domain of the one of SEQ ID NOs: 27-34 [252] sequence that has at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence WSGR Docket No.61078-729.601 identity to a sequence of any one of SEQ ID NOs: 25-34.
  • the cell expresses the endogenous TCR complex subunits.
  • the cell expresses at least one endogenous inhibits the formation of an endogenous TCR or endogenous TCR complex.
  • [254] i.e., in vitro or ex vivo [255] within the cell by at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more.
  • the dimer within the cell by at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.
  • the abundance of [256] when expressed within a cell (e.g., a mammalian or human cell), the mutant polypeptide.
  • the stoichiometry of the exogenous TCR complex is the same as expressed in a cell comprises at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% less than the amount of the functional TCR complex in the same cell.
  • the amount of the exogenous TCR complex expressed in a cell comprises at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% less than the amount of the functional TCR complex in the same cell.
  • interacting with endogenous CD3 as in endogenous CD3 or comprises an amino acid sequence for interacting with endogenous CD3 comprising one or more conservative amino acid substitutions that amino acid substitution, change, or mutation” refers to the substitution of one amino acid for another having similar chemical and physical characteristics (i.e., replacing serine with threonine, or leucine with isoleucine).
  • a conservative amino acid change generally has a less significant impact on the physical, structural, or chemical properties of a polypeptide relative to a non-conservative amino acid substitution.
  • the amino acids Y12 and T17 are not mutated or only mutated with a conservative mutation in 40%, or 50% of the binding affinity of an endogenous CD3 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% higher than the binding affinity of one endogenous CD3 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% higher than the binding affinity of one endogenous CD3 polypeptide for another readily determined using conventional techniques, e.g., by competitive ELISA (enzyme-linked immunosorbent assay), equilibrium dialysis, by using surface plasmon resonance (SPR) technology (e.g., the BIAcore 2000 instrument, using general procedures outlined by the manufacturer); by radioimmunoassay; or the
  • KD a KD of at most about 10 -6 molar (M), 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M, 10 -14 M, or 10 -15 polypeptide with a K D of at least about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10- 13 M, 10 -14 M, or 10 -15 A) of at most about 10 6 /M, 10 7 /M, 10 8 /M, 10 9 /M, 10 10 /M, 10 11 /M, 10 12 /M, 10 13 /M, 10 14 /M, or 10 15 A at least about 10 6 /M, 10 7 /M, 10 8 /M, 10 9 /M, 10 10 /M
  • [259] substantially interact with an endogenous TCR polypeptide or an endogenous TCR complex substantially interact with an subunitendogenous TCR subunit.
  • the binding affinity between them is about 10 -6 M or less.
  • the binding affinity between them is about 10 -6 M or measured by K D of 10 -6 M, 10 -5 M, 10 -4 M, 10 -3 M or more for an TCR subunitendogenous TCR subunit and substantially interacts with an TCR subunitendogenous TCR subunit.
  • K D at least about 10 M, 1 M, 10 -1 M, 10 -2 M, 10 -3 M, 10 -4 M, 10 -5 M, or 10 -6 constant (KA) at least about 10 1 /M, 10 2 /M, 10 3 /M, 10 4 /M, 10 5 /M, or 10 6 /M.
  • a K D at most about 10 M, 1 M, 10 -1 M, 10 -2 M, 10 -3 M, 10 -4 M, 10 -5 M, or 10 -6 M.
  • D at least about 10 M, 1 M, 10 -1 M, 10 -2 M, 10 -3 M, 10 -4 M, 10 -5 M, or 10 -6 binds an endogenous TCR subunit with a KA at least about 10 1 /M, 10 2 /M, 10 3 /M, 10 4 /M, 10 5 /M, or 10 6 a K A at most about 10 1 /M, 10 2 /M, 10 3 /M, 10 4 /M, 10 5 /M, or 10 6 /M.
  • K D at most about 10 M, 1 M, 10 -1 M, 10 -2 M, 10 -3 M, 10 -4 M, 10 -5 M, or 10 -6 M.
  • D at least about 10 M, 1 M, 10- WSGR Docket No.61078-729.601 1 M, 10 -2 M, 10 -3 M, 10 -4 M, 10 -5 M, or 10 -6 binds an endogenous TCR subunit with a KA at least about 10 1 /M, 10 ⁇ 2 /M, 10 3 M, 10 4 M, 10 5 M, or 10 6 K A at most about 10 1 /M, 10 2 /M, 10 ⁇ 3 /M, 10 4 /M, 10 5 /M, or 10 6 /M.
  • the KD complex polypeptide is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% higher than the K D for the same endogenous TCR complex polypeptide.
  • the KD polypeptide for an endogenous TCR complex polypeptide is at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% higher than the K D [264] In some embodiments, the KD polypeptide is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% higher than the K D endogenous TCR complex polypeptide.
  • the KD for an endogenous TCR complex polypeptide is at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% than the K D of the endogenous [265]
  • the KD polypeptide is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% than the K D D 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% than the K D D 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% than the K D D 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% than the K D D 40%, 45%, 50%, 100%, 2200%, 500%, 1000%, 10000%, or 100000% than the K D of the endogenous D of the 25%, 30%, 35%, 40%, 45%
  • the K D polypeptide is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% than the K D D 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% than the K D D of the mutant 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% than the KD of the the K D 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% than the K D polypeptide.
  • the K D polypeptide is at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% than the K D D 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% than the KD D of the mutant 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% than the KD of the the K D WSGR Docket No.61078-729.601 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% higher than the KD polypeptide.
  • the mutant acid position(s) that interact with an endogenous TCR polypeptide e.g., an endogenous TCR , an endogenous TCR , an endogenous TCR or an endogenous TCR .
  • the non- conservative mutation comprises mutating an acidic amino acid to a basic amino acid or a basic amino acid to an acidic amino acid.
  • TCR complex that is at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower than an amount of TCR complex that is at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 55%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower than an amount of TCR complex that is at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 5
  • transmembrane domain an extracellular domain that is at least about 4 amino acids in length
  • a transmembrane domain an extracellular domain that is at least about 4 amino acids in length
  • a [270] about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of comprises a sequence that has at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 38-41 and 62-64.
  • mutant polypeptide comprises a sequence of an endogenous WSGR Docket No.61078-729.601 transmembrane domain. In some embodiments, the mutant polypeptide comprises an endogenous intracellular domain.
  • [272] function of the endogenous TCR, such that it retains the ability to activate the TCR-initiated signaling (e.g., CD3 or CD3 ) from participating in the endogenous TCR activated signaling cascade within a e.g., CD3 or CD3 ) from binding to other endogenous TCR complex subunits or participating in functional TCR eliminates the formation of a functional TCR complex within a cell.
  • the mutant subunits within a cell Transmembrane Domain [273] domain capable of binding one but not all endogenous TCR subunits.
  • the mutant TCR polypeptide or endogenous TCR polypeptides comprise a transmembrane domain capable of binding to one but not all endogenous TCR WSGR Docket No.61078-729.601 [274] [275] thereof.
  • [276] comprises a sequence of a transmembrane domain of an endogenous TCR , an endogenous TCR polypeptide, an endogenous TCR polypeptide, or an endogenous TCR polypeptide.
  • the transmembrane 90%, 95%, or 99% sequence identity to a sequence of a transmembrane domain of an endogenous TCR , an endogenous TCR polypeptide, an endogenous TCR polypeptide, or an endogenous TCR polypeptide comprises about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of a transmembrane domain of an endogenous TCR , an endogenous TCR polypeptide, an endogenous TCR polypeptide, or an endogenous TCR polypeptide .
  • 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 amino acids 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 amino acids of a transmembrane comprises at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of a WSGR Docket No.61078-729.601 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of a transmembrane domain of an [278] the amino acid position designated “X” in the sequence MSVATIVIVXICITGGLLLLVYYWS transmembrane domain comprises an aspartic acid (D) residue at the amino acid designated X in comprises a glutamic acid (E) residue at the amino acid designated X in (SEQ ID NO: 1000 ).
  • the mutation at the amino acid position designated X in (SEQ ID NO: 1000 ) comprises a deletion of the amino acid designated X , corresponding to the deletion of polypeptide, or a combination thereof In some embodiments those further mutations comprise replacement of the amino acid designated X in (SEQ ID NO: 1000) (corresponding to position D137 of SEQ ID NO: 4) with at least 2, 3, 4, 5, 6, or more amino acids.
  • R, or K at the amino comprises an R, or K at the amino acid designated X in (SEQ ID NO: 1000).
  • the transmembrane domain of the one of SEQ ID NOs: 23-24 is
  • the transmembrane domain of the comprises at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of a transmembrane domain of an polypeptide, or a combination thereof.
  • Extracellular Domain [284] a sequence of an extracellular domain of a TCR.
  • the presence of an extracellular domain of an endogenous TCR polypeptide, or a fragment thereof increases the binding subunit.
  • the dissociation constant (K D comprising an extracellular domain of an endogenous TCR polypeptide, or a fragment thereof for an endogenous TCR polypeptide may be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower than that of a mutant the K D polypeptide, or a fragment thereof to an endogenous TCR may be at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% thereof.
  • the extracellular an endogenous TCR complex polypeptide comprises a domain configured to bind to an endogenous a domain configured to bind to an endogenous TCR subunit comprises a domain configured to bind to polypeptide comprising a domain configured to bind to an endogenous TCR subunit comprises a WSGR Docket No.61078-729.601 to an endogenous TCR subunit comprises a domain configured to bind to an endogenous CD3 polypeptide or a complex thereof.
  • the added extracellular domain of an endogenous TCR polypeptide, or a fragment thereof is glycosylated by enzymes that glycosylate the endogenous TCR extracellular domain of an endogenous TCR polypeptide, or a fragment thereof may be at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or endogenous TCR polypeptide, or a fragment thereof.
  • the stability of a mutant fragment thereof may be at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, that lacks an extracellular domain of an endogenous TCR polypeptide, the partial sequence thereof, or the fragment thereof.
  • the 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of an extracellular domain of an polypeptide comprises at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of an extracellular domain of an endogenous TCR polypeptide.
  • WSGR Docket No.61078-729.601 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of an extracellular domain of an endogenous TCR polypeptide. In some embodiments, the extracellular domain of the mutant endogenous TCR polypeptide.
  • Intracellular Domain [292] domain capable of transporting a non-functional intermediate TCR complex to the plasma membrane WSGR Docket No.61078-729.601 intracellular domain.
  • the sequence of a CD3 comprises one or more mutations. polypeptide comprise a deletion, an amino acid substation, an insertion, or a combination thereof. In e.g., SEQ ID NO. 33). In TCR complex to the plasma membrane of a cell.
  • [294] least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 amino 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 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, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 amino 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
  • the cell may facilitate the formation of an exogenous TCR or exogenous TCR complex.
  • the cell comprises one or more endogenous TCR complex subunits.
  • the cell polypeptides disclosed herein within a cell inhibits the formation of an endogenous TCR or endogenous TCR complex.
  • [297] i.e., in vitro or ex vivo
  • [298] i.e., in vitro or ex vivo [299] 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more.
  • WSGR Docket No.61078-729.601 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%. in vitro by at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, in vitro by at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%.
  • formation least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more.
  • the expression of the the cell by at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, polypeptide reduces the amount of dimers comprising polypeptides with the sequences of endogenous in vitro by at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more.
  • the mutant D of at least about 10 6 /M, 10 7 /M, 10 8 /M, 10 9 /M, 10 10 /M, 10 11 /M, 10 12 /M, 10 13 M, 10 14 /M, or 10 15 /M.
  • the mutant transmembrane domain comprising one or more conservative amino acid substitutions in the domain polypeptide comprises a native or endogenous sequence of SEQ ID NO: 14 that is not mutated.
  • the one or more non-conservative mutations comprise mutating an acidic amino acid to a basic amino acid or a polar amino acid.
  • the one or more non-conservative mutations comprises any other non-conservative mutation contemplated herein.
  • conservative substitutions of the amino acids present in the corresponding domain of the endogenous D137 mutation e.g., D137N, D137A, D137R, D137K, or D137S; corresponding to the D137 of SEQ ID NO: 4
  • an endogenous domain comprising one or more conservative amino acid substitutions e.g., SEQ ID NO: 14
  • endogenous TCR complex subunits e.g., WSGR Docket No.61078-729.601 inhibits the formation of the functional TCR complexes.
  • a cell expressing a at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower than that of a cell that does not express the disclosed herein comprises an amount of the functional TCR complex that is at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,
  • the amount of the functional TCR complex can be measured by any methods described herein.
  • the amount of the functional TCR complex can be measured by any of the methods described in EXAMPLE 1 or EXAMPLE 2. [304] / , endogenous TCR / , or a combination thereof with a KD of at most about 10 M, 1 M, 10 -1 M, 10 -2 M, 10 -3 M, 10 -4 M, 10 -5 M, or 10 -6 M.
  • K A association constant
  • / , or endogenous TCR / with a K D of at most about 10 M, 1 M, 10 -1 M, 10 -2 M, 10 -3 M, 10 -4 M, 10 -5 M, or 10 -6 M.
  • / , or endogenous TCR / with a KD at least about 10 M, 1 M, 10 -1 M, 10- 2 M, 10 -3 M, 10 -4 M, 10 -5 M, or 10 -6 WSGR Docket No.61078-729.601 TCR / , or endogenous TCR / , with a KA of at least about 10 1 /M, 10 2 /M, 10 3 /M, 10 4 /M, 10 5 /M, or 10 6 / , or endogenous TCR / , with a KA of at most about 10 1 /M, 10 2 /M, 10 3 /M, 10 4 M, 10 5 /M, or 10 6 /M.
  • [306] / , or endogenous TCR / with a KD at most about 10 M, 1 M, 10 -1 M, 10 -2 M, 10 -3 M, 10 -4 M, 10 -5 M, or 10 -6 M.
  • the / , or endogenous TCR / with a K D at least about 10 M, 1 M, 10 -1 M, 10- 2 M, 10 -3 M, 10 -4 M, 10 -5 M, or 10 -6 TCR / , or endogenous TCR / , with a KA at least about 10 1 /M, 10 ⁇ 2 /M, 10 3 /M, 10 4 /M, 10 5 /M, or 10 6 / , or endogenous TCR / , with a KA at most about 10 1 /M, 10 2 /M, 10 3 /M, 10 4 /M, 10 5 /M, or 10 6 /M.
  • [307] / , or endogenous TCR / with a KD at most about 10 M, 1 M, 10 -1 M, 10 -2 M, 10 -3 M, 10 -4 M, 10 -5 M, or 10 -6 M.
  • the / , or endogenous TCR / with a K D at least about 10 M, 1 M, 10 -1 M, 10- 2 M, 10 -3 M, 10 -4 M, 10 -5 M, or 10 -6 TCR / , or endogenous TCR / , with a KA at least about 10 1 /M, 10 2 /M, 10 3 /M, 10 4 /M, 10 5 /M, or 10 6 / , or endogenous TCR / , with a KA at most about 10 1 /M, 10 2 /M, 10 3 /M, 10 4 /M, 10 5 /M, or 10 6 /M.
  • the K D / , or endogenous TCR / comprises a K D at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% higher than the KD / , or endogenous TCR / .
  • the KD WSGR Docket No.61078-729.601 / , or endogenous TCR / comprises a K D at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% higher than the KD / , or endogenous TCR / .
  • the KD / , or endogenous TCR / comprises a K D at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% higher than the K D / , or endogenous TCR / .
  • the KD / , or endogenous TCR / comprises a KD at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% higher than the KD of the endogenous endogenous TCR / , or endogenous TCR / .
  • the K D of the exogenous endogenous TCR / , or endogenous TCR / comprises a KD at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% higher than the KD / , or endogenous TCR / .
  • the K D of the / , or endogenous TCR / comprises a K D at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% higher than the KD / , or endogenous TCR / .
  • the KD comprises a K D at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% higher than the K D D D at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% higher than the K D of the endogenous D of the exogenous D at least about 5%, 10%, 15%, 20%, 25%, WSGR Docket No.61078-729.601 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% higher than the K D D of the D at most about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 100%, 200%, 500%, 1000%, 10000%, or 100000% higher than the K D D of the D at most about 5%, 10%, 15%, 20%, 25%
  • the at least one TCR complex comprises one exogenous CD3 complex, wherein the exogenous CD3 complex comprises an exogenous CD3 /CD heterodimer, an endogenous CD3 /CD3 heterodimer, and an endogenous CD3 /CD3 homodimer. In some embodiments, the at least one TCR complex comprises one exogenous CD3 complex, wherein the exogenous CD3 complex comprises an endogenous CD3 /CD heterodimer, an exogenous CD3 /CD3 heterodimer, and an endogenous CD3 /CD3 homodimer.
  • the at least one TCR complex comprises one exogenous CD3 complex, wherein the exogenous CD3 complex comprises an exogenous CD3 /CD heterodimer, an exogenous CD3 /CD3 heterodimer, and an endogenous CD3 /CD3 homodimer.
  • the first respective exogenous TCR complex comprises an when comparing a first respective exogenous TCR complex and a second respective exogenous TCR wherein the first respective exogenous TCR complex and the second respective exogenous TCR first respective exogenous TCR complex and the second respective exogenous TCR complex exogenous TCR complex may be at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% less than the amount of the second respective exogenous TCR complex.
  • the first respective exogenous TCR complex may be at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% less than the amount of the second respective exogenous TCR complex.
  • WSGR Docket No.61078-729.601 [314]
  • the mutant when expressed within a cell (e.g., a mammalian or human cell), the mutant 1000%, 10000%, or 100000% higher than the amount of the functional TCR complex.
  • Domain combinations [315]
  • WSGR Docket No.61078-729.601 [316] about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of polypeptide comprises a sequence that has at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 23, 24, 35-37, 60, and 61. In some 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of any one of SEQ ID NOs: 23, has 100% sequence identity to a sequence of any one of SEQ ID NOs: 23, 24, 35-37, 60, and 61.
  • sequence identity to a sequence of any one of has at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence sequence that has about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a comprises a sequence that has 100% sequence identity to a sequence of any one of SEQ ID NOs: 35- 37.
  • WSGR Docket No.61078-729.601 [317] about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of SEQ ID NO: 18, and wherein D137 of SEQ ID NO:18 is replaced with K or R. In some embodiments, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 18, and wherein D137 of SEQ ID NO: 18 that has about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to a sequence of SEQ ID NO: 18, wherein D137 of SEQ ID NO: 18 is replaced with K or R.
  • polypeptide comprises a sequence that has at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to SEQ ID NO: 18, wherein D13 is replaced with K.
  • D137 of SEQ sequence that has about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to polypeptide comprises a sequence that has 100% sequence identity to SEQ ID NO: 18, wherein D137 a sequence that has at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence polypeptide comprises a sequence that has at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to SEQ ID NO: 18, wherein D137 is replaced with R.
  • the ER retention tag is a E319K tag. In some embodiments, the ER retention tag comprises an amino acid sequence of KKKKRD. In some embodiments, the ER retention tag comprises a sequence at least 95% sequence identity to any of the sequences in Table 5. In some comprises a sequence encoding an antigen binding domain. In some embodiments, the antigen binding WSGR Docket No.61078-729.601 embodiments, the TCR subunit is CD3.
  • the antigen binding domain binds to CD58. In some embodiments, the antigen binding domain binds to MHCI. In some embodiments, the antigen binding domain is an scFv. In some embodiments, the antigen binding domain is a humanized scFv. In some embodiments, the antigen binding domain is a VHH. In some embodiments, the antigen binding domain comprises a variable heavy chain domain (VH) and a variable light chain domain (VL). In some embodiments, the heavy chain domain is linked to the light chain domain via a linker. In some embodiments, the linker is a Whitlow linker.
  • the antigen binding domain from N-terminal to C-terminal comprises VH-Whitlow linker – VL. In some embodiments, the antigen binding domain from N-terminal to C-terminal comprises VL-Whitlow linker – VH. In some embodiments, the recombinant polynucleic acid comprises at least 80% sequence identity to any of the sequence in Table 7. RNA Interference [318] In another aspect, provided herein are compositions, systems, and methods for RNA interference to reduce or eliminate expression of an endogenous TCR or an endogenous TCR complex.
  • RNA interference and the term “RNAi” are synonymous and are used herein to refer to the process by which a polynucleotide or small interfering RNA (siRNA) comprising a ribonucleotide unit exerts an effect on a biological process.
  • the process includes, but is not limited to, gene silencing by degrading mRNA, tRNA, rRNA, and hnRNA.
  • a composition comprising a recombinant polynucleic acid comprising or encoding an shRNA against CD3 zeta, and a sequence encoding a dominant negative CD3 epsilon polypeptide.
  • expression of the recombinant polynucleic acid in a cell reduces cell surface expression of an endogenous TCR subunit or reduces functional TCR complex formation in the cell compared to the cell surface expression of the endogenous TCR subunit or the functional TCR complex formation in a cell that does not express the fusion polypeptide.
  • expression of the recombinant polynucleic acid in a cell reduces cell surface expression of an endogenous TCR subunit or reduces functional TCR complex formation in the cell by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the cell surface expression of the endogenous TCR subunit or the functional TCR complex formation in a cell that does not express the fusion polypeptide.
  • expression of the recombinant polynucleic acid in a cell reduces cell surface expression of an endogenous TCR subunit or reduces functional TCR complex formation in the cell by at least WSGR Docket No.61078-729.601 90% compared to the cell surface expression of the endogenous TCR subunit or the functional TCR complex formation in a cell that does not express the fusion polypeptide.
  • the recombinant polynucleic acid further comprises a promoter.
  • the shRNA is in the intron region of the promoter.
  • the sequence identity to SEQ ID NO: 117 the sequence identity to SEQ ID NO: 117.
  • the promoter with the shRNA in the intron comprises a sequence at least 90% sequence identity to SEQ ID NO: 115 or 116.
  • the dominant negative CD3 epsilon polypeptide comprises a sequence at least 80% sequence identity to any of the SEQ ID Nos: 23, 24, 35-37, 60 and 61.
  • the recombinant polynucleic acid further comprises a sequence encoding a P2A self-cleaving peptide, a T2A self-cleaving peptide, an E2A self-cleaving peptide, or an F2A self-cleaving peptide.
  • compositions comprising one or more RNAs that each comprise a small non-coding RNA or a sequence encoding thereof.
  • the small non-coding RNA comprises a micro ribonucleic acid (miRNA), a transfer ribonucleic acid (tRNA), a small interfering ribonucleic acid (siRNA), a self-amplifying RNA (saRNA), a short-hairpin RNA (shRNA), or a combination thereof.
  • the small non-coding RNA comprises an miRNA, an siRNA, an saRNA, an shRNA, or a combination thereof.
  • the small non-coding RNA comprises an miRNA, an siRNA, an shRNA, or a combination thereof. In some embodiments, the small non-coding RNA comprises an miRNA. In some embodiments, the small non-coding RNA comprises an siRNA. In some embodiments, the small non-coding RNA comprises an shRNA. [322] In some embodiments, the RNA compositions provided herein are administered to one or more cells and targeted to a protein of interest such that expression of one or more RNA compositions within the cell decreases or eliminates expression of the targeted protein by decreasing or eliminating transcription of the RNA encoding that protein.
  • an RNA composition composition comprising a small non-coding RNA reduces or eliminates expression of endogenous composition comprising a small non-coding RNA reduces or eliminates expression of endogenous WSGR Docket No.61078-729.601 composition comprising a small non-coding RNA reduces or eliminates expression of endogenous composition comprising a small non-coding RNA reduces or eliminates expression of endogenous [323] In some embodiments, an RNA composition comprising a small non-coding RNA decreases the expression of at least one endogenous TCR complex subunit.
  • an RNA composition comprising a small non-coding RNA decreases the expression of at least one of: embodiments, a cell comprising an RNA composition comprising a small non-coding RNA targeting an endogenous TCR subunit as described herein comprises at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% less of the endogenous TCR complex subunit relative to a cell that does not comprise an RNA composition comprising a small non-coding RNA targeting an endogenous TCR complex subunit.
  • a cell comprising an RNA composition comprising a small non-coding RNA targeting an endogenous TCR complex subunit as described herein comprises at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% less of the endogenous TCR complex subunit relative to a cell that does not comprise an RNA composition comprising a small non-coding RNA targeting an endogenous TCR complex subunit.
  • a cell comprising an RNA composition comprising a small non-coding RNA targeting an endogenous TCR complex subunit as described herein comprises at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% less of another endogenous TCR complex subunit relative to a cell that does not comprise an RNA composition comprising a small non-coding RNA targeting an endogenous TCR complex subunit.
  • a cell comprising an RNA composition comprising a small non-coding RNA targeting an endogenous TCR complex subunit as described herein comprises at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% less of another endogenous TCR complex subunit relative to a cell that does not comprise an RNA composition comprising a small non- coding RNA targeting an endogenous TCR complex subunit.
  • a cell comprising an RNA composition comprising a small non-coding RNA targeting an endogenous TCR complex WSGR Docket No.61078-729.601 subunit as described herein comprises at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% less of an endogenous TCR complex relative to a cell that does not comprise an RNA composition comprising a small non-coding RNA targeting an endogenous TCR complex subunit.
  • a cell comprising an RNA composition comprising a small non-coding RNA targeting an endogenous TCR complex subunit as described herein comprises at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% less of an endogenous TCR complex relative to a cell that does not comprise an RNA composition comprising a small non-coding RNA targeting an endogenous TCR complex subunit.
  • a cell comprising an RNA composition comprising a small non-coding RNA targeting an endogenous TCR complex subunit as described herein comprises at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% less of the functional TCR complex relative to a cell that does not comprise an RNA composition comprising a small non-coding RNA targeting an endogenous TCR complex subunit.
  • a cell comprising an RNA composition comprising a small non-coding RNA targeting an endogenous TCR complex subunit as described herein comprises at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% less of the functional TCR complex relative to a cell that does not comprise an RNA composition comprising a small non-coding RNA targeting an endogenous TCR complex subunit.
  • the RNA composition comprising a small non-coding RNA comprises an siRNA.
  • RNA and the phrase “small interfering RNA” both refer to unimolecular nucleic acids and to nucleic acids comprising two separate strands, in either case comprising a duplex domain comprising between 18 and 30 base pairs (bp), capable of reducing or eliminating expression of a specific gene by RNAi.
  • siRNA and short interfering RNA both encompass native (i.e., unmodified) nucleic acids as well as modified nucleic acids comprising nucleotides having moieties other than ribonucleotide moieties, including, but not limited to, modified nucleotides, modified internucleotide linkages, non-nucleotides, deoxynucleotides and analogs of the aforementioned nucleotides.
  • siRNAs can be duplexes, and can also comprise short hairpin RNAs, RNAs with loops as long as, for example, 4 to 23 or more nucleotides, RNAs with stem loop bulges, micro-RNAs, and short temporal RNAs.
  • RNAs having loops or hairpin loops can include structures where the loops are connected to the stem by linkers such as flexible linkers.
  • Flexible linkers can be comprised of a wide variety of chemical structures, as long as they are of sufficient length and materials to enable effective intramolecular hybridization of the stem elements. Typically, the length to be spanned is at least about WSGR Docket No.61078-729.601 10-24 atoms.
  • the siRNAs are hairpins, the sense strand and antisense strand are part of one longer molecule.
  • RNAi may include shRNA. [326]
  • RNAi may include microRNAs. MicroRNAs (miRNAs) belong to a class of small non-coding RNAs.
  • miRNAs regulate gene expression by interacting with mRNAs via base-pairing and reducing the production of proteins from these mRNAs by affecting their translation.
  • An miRNA binds to and regulates the translation of up to several hundred target messenger RNAs, which enables the coordinated expression of multiple related genes.
  • microRNAs comprising a conventional naturally occurring sequence, a chemically modified version or sequence, or a homologue thereof.
  • microRNAs presented herein are from 7 nucleotides to as long as 30 nucleotides in length, comprising a substantial portion that is double-stranded (i.e., base-paired), either having two separate strands or a hairpin structure.
  • an RNA composition comprising a microRNA comprises an RNA of 7, 8, 9, 10, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides in length.
  • an RNA composition comprising a microRNA comprises an RNA from 7 to 30 nucleotides, from 7 to 25 nucleotides, from 15 to 30 nucleotides, from 15 to 25 nucleotides, from 17 to 30 nucleotides, or from 17 to 25 nucleotides long.
  • a “miRNA mimic” is a double- stranded RNA molecule that retains at least a portion of the biological activity of the miRNA it is said to mimic, e.g.
  • a miRNA mimic has an enhanced biological activity as compared to the reference miRNA and as measured by a suitable assay, such as activity that is at least 10%, 25%, 50%, 75%, 90%, 100%, 200%, 300%, or more increased.
  • a gene editing system, and/or gene expression modulator of the present disclosure is provided to a cell.
  • the gene editing system, and/or gene expression modulator is provided to a recombinant cell of the disclosure.
  • the recombinant cell of the disclosure comprises a recombinant polypeptide, a CAR, and/or a gene editing system, and/or gene expression modulator.
  • the gene editing system, and/or gene expression modulator decreases and/or modulates expression of a gene that encodes an endogenous TCR polypeptide.
  • the gene editing system, and/or gene expression modulator decreases and/or modulates expression of a gene that encodes an endogenous MHC polypeptide.
  • the gene may be a gene that encodes only an extracellular domain of a T-cell receptor or MHC.
  • the gene may be a gene that encodes only an intracellular signaling domain of a T-cell receptor or MHC.
  • the endogenous gene may be selected from the group WSGR Docket No.61078-729.601 consisting of TCR , TCR RFXAP, a viral immunoevasin, ICAM1, CD80, CD58, OX40L, and any combination thereof.
  • a shRNA comprises a sequence that has at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of any one of SEQ ID NOs: 71-76.
  • a shRNA comprises a sequence that has at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of any one of SEQ ID NOs: 71-76.
  • T-cell receptor expression may be decreased using small-hairpin RNAs (shRNAs) that target nucleic acids encoding specific TCRs (e.g., (e.g., CD3 ) T cells.
  • shRNAs small-hairpin RNAs
  • targeting shRNAs have been designed for key components of the TCR complex.
  • an shRNA targeting CD3 epsilon comprises the sequence ctggaggcttgctgaaggctgtatgctgAACGCCAACTGATAAGAGGCAgttttggccactgactgacTGCCTCTTC AGTTGGCGTTcaggacacaaggcctgttactagcactcacatggaacaaatggccca (SEQ ID NO: ).
  • MHC expression may be decreased using small-hairpin RNAs (shRNAs) that target nucleic acids encoding MHCI and/or MHCII proteins in T cells (e.g., B2M, CIITA, NLRC5, RFX5, RFXANK, RFXAP, viral immunoevasins and the like).
  • shRNAs small-hairpin RNAs
  • targeting shRNAs have been designed for key components of the MHC complex.
  • an shRNA targeting B2M comprises the sequence: ctggaggcttgctgaaggctgtatgctgAATCTTTGGAGTACGCTGGATgttttggccactgactgacATCCAGCGCT CCAAAGATTcaggacacaaggcctgttactagcactcacatggaacaaatggccca (SEQ ID NO: ).
  • an RNAi construct comprises a sequence complementary to a coding sequence that encodes a polypeptide with at least about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of any one of SEQ ID NOs: 1-43 and 62-64.
  • an RNAi construct comprises a sequence complementary to a coding sequence that encodes a polypeptide with at most about 60%, 65% 70%, 75%, 80%, 85%, 90%, 95%, 99% or more sequence identity to a sequence of any one of SEQ ID NOs: 1-43 and 62-64.
  • compositions comprising recombinant polynucleic acids comprising at least two of the following: a sequence encoding the fusion polypeptide encoded by the recombinant polynucleic acid of any of the compositions described herein; a sequence encoding the mutant CD3 zeta polypeptide encoded by any of the recombinant polynucleic acids of a composition WSGR Docket No.61078-729.601 described herein; a sequence encoding the mutant CD3 epsilon polypeptide encoded by the recombinant polynucleic acid of a composition described herein; a sequence encoding the fusion polypeptide encoded by the recombinant polynucleic acid of the composition of described herein; and sequence comprises the shRNA against CD3 encoded by the recombinant polynucleic acid of the composition described herein.
  • the composition comprising a recombinant polynucleic acid comprising a sequence encoding the mutant CD3 epsilon polypeptide encoded by the recombinant polynucleic acid of the composition described herein; and a sequence encoding the fusion polypeptide encoded by the recombinant polynucleic acid of the composition described herein.
  • the composition comprising a recombinant polynucleic acid comprising a sequence encoding the mutant CD3 epsilon polypeptide encoded by the recombinant polynucleic acid of the composition described herein; and sequence comprises the shRNA against CD3 zeta encoded by the recombinant polynucleic acid of the composition described herein.
  • the dominant negative CD3 epsilon polypeptide comprises a sequence at least 80% sequence identity to any of the SEQ ID Nos: 23, 24, 35-37, 60 and 61.
  • the recombinant polynucleic acid further comprises a sequence encoding a P2A self- cleaving peptide, a T2A self-cleaving peptide, an E2A self-cleaving peptide, or an F2A self-cleaving peptide.
  • a composition comprising a cell, wherein the cell comprises the recombinant polynucleic acid of the composition described herein.
  • Chimeric Antigen Receptors (CARs) [337]
  • a recombinant polypeptide or recombinant nucleic acid encoding the recombinant polypeptide further comprises a sequence encoding a chimeric antigen receptor (CAR).
  • the CAR comprises an extracellular domain comprising an antigen binding domain, a transmembrane domain, and an intracellular domain comprising an intracellular signaling domain.
  • a cell expressing any of the recombinant or fusion polypeptides disclosed herein further comprises a CAR.
  • provided herein are recombinant nucleic acids encoding any of the recombinant fusion polypeptides described herein, and recombinant nucleic acids encoding a CAR. In some embodiments, both such recombinant nucleic acids are delivered to a cell.
  • a recombinant nucleic acid encoding a recombinant polypeptide may both be delivered to a cell and expressed by the cell.
  • the CAR comprises an extracellular domain comprising an antigen binding domain; a transmembrane domain; and an intracellular domain comprising an intracellular signaling domain.
  • the antigen binding domain is an anti-CD19 binding domain.
  • the anti-CD19 scFv comprises a sequence with at least about 80% sequence identity to a sequence selected from the sequences in Table 13.
  • the antigen binding domain is an anti-CD20 binding domain.
  • the scFv comprises a sequence with at least about 80% sequence identity to a sequence selected from the sequences in Table 14.
  • the antigen binding domain is an anti-CD22 binding domain.
  • the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1) of QTIWSY (SEQ ID NO: ), LCDR2 of AAS (SEQ ID NO: ), and LCDR3 of QQSYSIPQT (SEQ ID NO: ), respectively; and a heavy chain CDR1 (HCDR1) of GDSVSSNSAA (SEQ ID NO: ), HCDR2 of TYYRSKWYN (SEQ ID NO: ), and HCDR3 of AREVTGDLEDAFDI (SEQ ID NO: ).
  • VL variable light chain domain having a light chain CDR1 (LCDR1) of QTIWSY (SEQ ID NO: ), LCDR2 of AAS (SEQ ID NO: ), and LCDR3 of QQSYSIPQT (SEQ ID NO: ), respectively; and a heavy chain CDR1 (HCDR1) of GDSVSSNSAA (SEQ ID NO: ), HCDR2 of
  • the antigen binding domain comprises an scFv with at least about 80% sequence identity to a sequence selected from the sequences in Table 15. [345] In some embodiments, the antigen binding domain binds to an antigen that is selected from the group consisting of: CD19, CD20, CD22, glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY-ESO- 1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate-carcinoma tumor antigen- 1 (PCTA-1), MAGE
  • PCTA-1 prostate-car
  • the antigen-binding domain binds to an antigen selected from the group consisting of CD19, CD20, and CD22. In some embodiments, the antigen-binding domain binds to CD19. In some embodiments, the antigen-binding domain binds to CD20. In some embodiments, the antigen-binding domain binds to CD22. In some embodiments, the antigen-binding domain binds to CD19 and CD20. In some embodiments, the antigen-binding domain binds to CD19 and CD22. In some embodiments, the antigen-binding domain binds to CD20 and CD22.
  • the intracellular domain of the CAR comprises an intracellular costimulatory domain derived from an intracellular costimulatory domain of CD28, 4-1BB/CD137, or CD2.
  • the intracellular domain of the CAR comprises an intracellular signaling CD22, CD79a, CD79b, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, or ZAP70.
  • the transmembrane domain of the CAR comprises a transmembrane domain from CD8 or CD28.
  • the extracellular domain of the CAR comprises a hinge domain from CD8 or CD28.
  • the extracellular domain of the CAR comprises a hinge domain from CD8 and the transmembrane domain of the CAR comprises a transmembrane domain from CD8.
  • the extracellular domain of the CAR comprises a hinge domain from CD28 and the transmembrane domain of the CAR comprises a transmembrane domain from CD28.
  • intracellular signaling domain from CD3zeta In some intracellular signaling domain from CD3zeta.
  • the CAR comprises an anti-CD20 scFv, a hinge domain from CD28, a transmembrane domain from CD28, and a cytoplasmic domain comprising a costimulatory domain from CD2 and an intracellular signaling domain from CD3zeta. from 4-1BB and an intracellular signaling domain from CD3zeta.
  • the CAR comprises a sequence with at least 80% sequence identity to any one of sequences in Table 16.
  • CAR Extracellular Domain [352]
  • the extracellular domain of the CAR comprises an antigen binding domain.
  • the antigen binding domain can be any domain that specifically binds to an antigen.
  • the antigen is an antigen expressed by a tumor cell.
  • the antigen binding domain comprises a single chain variable fragment (scFv), a nanobody, a ligand, or a receptor.
  • the antigen binding domain can be any molecule that binds to the selected antigen with sufficient affinity and specificity, and is often an antibody or an antibody derivative, such as an scFv, single domain antibody (sdAb), Fab' fragment, (Fab')2 fragment, nanobody, diabody, or the like.
  • the antigen binding domain can be a receptor or a receptor fragment that binds specifically to the target antigen.
  • the antigen binding domain can be attached to the rest of the receptor directly (covalently) or indirectly (for example, through the noncovalent binding of two or more binding partners).
  • Antibody derivatives are molecules that resemble antibodies in their mechanism of ligand binding, and include, for example, nanobodies, duobodies, diabodies, triabodies, minibodies, F(ab')2 fragments, Fab fragments, single chain variable fragments (scFv), single domain antibodies (sdAb), and functional fragments thereof. See, e.g., D.L. Porter et al., N Engl J Med ( 2011) 365(8):725-33 (scFv); E.L.
  • Antibody derivatives can also be prepared from therapeutic antibodies, for example without limitation, by preparing a nanobody, duobody, diabody, triabody, minibody, F(ab')2 fragment, Fab fragment, single chain variable fragment (scFv), or single domain antibody (sdAb) based on a therapeutic antibody.
  • Antibody derivatives can also be designed using phage display techniques (see, e.g., E.
  • the antigen binding domain specifically binds to CD19.
  • the antigen binding domain is an anti-CD19 binding domain.
  • the antigen binding domain comprises an scFv with a variable light chain domain (VL) having a light chain CDR1 (LCDR1) comprising the amino acid sequence of RASQDISKYLN (SEQ ID NO: ), LCDR2 comprising the amino acid sequence of SRLHSGV (SEQ ID NO: ) and LCDR3 comprising the amino acid sequence of GNTLPYTFG (SEQ ID NO: ), respectively.
  • VL variable light chain domain having a light chain CDR1 (LCDR1) comprising the amino acid sequence of RASQDISKYLN (SEQ ID NO: ), LCDR2 comprising the amino acid sequence of SRLHSGV (SEQ ID NO: ) and LCDR3 comprising the amino acid sequence of GNTLPYTFG (SEQ ID NO: ), respectively.
  • the antigen binding domain comprises an scFv with a variable light chain domain (VL) having at least about 80% sequence identity to the amino acid sequence WSGR Docket No.61078-729.601 DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFS GSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: ).
  • VL variable light chain domain
  • the antigen binding domain comprises an scFv with a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1) comprising the amino acid sequence of DYGVS (SEQ ID NO: ), HCDR2 comprising the amino acid sequence of VIWGSETTYYNSALKS (SEQ ID NO: ), and HCDR3 comprising the amino acid sequence of YAMDYWG (SEQ ID NO: ), respectively.
  • VH variable heavy chain domain having a heavy chain CDR1 (HCDR1) comprising the amino acid sequence of DYGVS (SEQ ID NO: ), HCDR2 comprising the amino acid sequence of VIWGSETTYYNSALKS (SEQ ID NO: ), and HCDR3 comprising the amino acid sequence of YAMDYWG (SEQ ID NO: ), respectively.
  • the antigen binding domain comprises an scFv with a variable heavy chain domain (VH) having at least about 80% sequence identity to the amino acid sequence EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNS ALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: ).
  • VH variable heavy chain domain
  • the antigen binding domain comprises an scFv with at least about 80% sequence identity to the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFS GSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGSEVK LQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKS RLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: ).
  • the antigen binding domain comprises an scFv with at least about 80% sequence identity to the amino acid sequence EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNS ALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSG GGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLI YHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: ).
  • the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 65. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 85% identity of SEQ ID NO: 65. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 90% identity of SEQ ID NO: 65. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 65. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 65. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 65.
  • the antigen binding domain comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 65.
  • the antigen binding domain comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 65.
  • the antigen binding domain comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 65.
  • the antigen binding domain comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 65.
  • the antigen binding domain consists of an amino acid sequence of SEQ ID NO: 65.
  • the antigen binding domain consists of an amino acid sequence with at least 85% identity of SEQ ID NO: 65.
  • the antigen binding domain consists of an amino acid sequence with at least 90% identity of SEQ ID NO: 65. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 65. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 96% identity of SEQ ID NO: 65. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 65. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 65. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 65.
  • the antigen binding domain consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 65. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 65. [358] In some embodiments, the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 66. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 85% identity of SEQ ID NO: 66. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 90% identity of SEQ ID NO: 66. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 66.
  • the antigen binding domain comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 66. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 66. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 66. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 66. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 66. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 66.
  • the antigen binding domain consists of an amino acid sequence of SEQ ID NO: 66. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 85% identity of SEQ ID NO: 66. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 90% identity of SEQ ID NO: 66. In some embodiments, the antigen WSGR Docket No.61078-729.601 binding domain consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 66. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 96% identity of SEQ ID NO: 66.
  • the antigen binding domain consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 66. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 66. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 66. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 66. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 66. [359] In some embodiments, the antigen binding domain specifically binds to CD22.
  • the antigen binding domain is an anti-CD22 binding domain.
  • the antigen binding domain comprises an scFv with a variable light chain domain (VL) having a light chain CDR1 (LCDR1) comprising the amino acid sequence QTIWSY (SEQ ID NO: ), LCDR2 comprising the amino acid sequence AAS (SEQ ID NO: ) and LCDR3 comprising the amino acid sequence QQSYSIPQT (SEQ ID NO: ), respectively.
  • VL variable light chain domain having a light chain CDR1 (LCDR1) comprising the amino acid sequence QTIWSY (SEQ ID NO: ), LCDR2 comprising the amino acid sequence AAS (SEQ ID NO: ) and LCDR3 comprising the amino acid sequence QQSYSIPQT (SEQ ID NO: ), respectively.
  • the antigen binding domain comprises an scFv with a variable light chain domain (VL) having at least about 80% sequence identity to the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFS GRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEI (SEQ ID NO: ).
  • VL variable light chain domain
  • the antigen binding domain comprises an scFv with a variable heavy chain domain (VH) having a heavy chain CDR1 (HCDR1) comprising the amino acid sequence GDSVSSNSAA, HCDR2 comprising the amino acid sequence TYYRSKWYN (SEQ ID NO: ) and HCDR3 comprising the amino acid sequence AREVTGDLEDAFDI (SEQ ID NO: ), respectively.
  • VH variable heavy chain domain
  • the antigen binding domain comprises an scFv with a variable heavy chain domain (VH) having at least about 80% sequence identity to the amino acid sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYN DYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTV SS (SEQ ID NO: ).
  • VH variable heavy chain domain
  • the antigen binding domain comprises an scFv with at least about 80% sequence identity to the amino acid sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYN DYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTV SSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQ SGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK (SEQ ID NO: ).
  • the antigen binding domain comprises an scFv with at least about 80% sequence identity to the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFS GRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKGGGGSQVQLQQSGPGLVK PSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINP DTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSS (SEQ ID NO: ).
  • the antigen binding domain comprises an scFv with at least about 85, 90, 95, 97, 98, or 99% sequence identity to the amino acid sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYN DYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTV SSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQ SGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK (SEQ ID NO: ).
  • the antigen binding domain comprises an scFv with 100% sequence identity to the amino acid sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYN DYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTV SSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQ SGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK (SEQ ID NO: ).
  • the antigen binding domain comprises an amino acid sequence of SEQ ID NO: 67. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 85% identity of SEQ ID NO: 67. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 90% identity of SEQ ID NO: 67. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 95% identity of SEQ ID NO: 67. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 96% identity of SEQ ID NO: 67. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 97% identity of SEQ ID NO: 67.
  • the antigen binding domain comprises an amino acid sequence with at least 98% identity of SEQ ID NO: 67. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99% identity of SEQ ID NO: 67. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.5% identity of SEQ ID NO: 67. In some embodiments, the antigen binding domain comprises an amino acid sequence with at least 99.9% identity of SEQ ID NO: 67. In some embodiments, the antigen binding domain consists of an amino acid sequence of SEQ ID NO: 67. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 85% identity of SEQ ID NO: 67.
  • the antigen binding domain consists of an WSGR Docket No.61078-729.601 amino acid sequence with at least 90% identity of SEQ ID NO: 67. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 95% identity of SEQ ID NO: 67. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 96% identity of SEQ ID NO: 67. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 97% identity of SEQ ID NO: 67. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 98% identity of SEQ ID NO: 67.
  • the antigen binding domain consists of an amino acid sequence with at least 99% identity of SEQ ID NO: 67. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.5% identity of SEQ ID NO: 67. In some embodiments, the antigen binding domain consists of an amino acid sequence with at least 99.9% identity of SEQ ID NO: 67.
  • the antigen binding domain binds to an antigen that is selected from the group consisting of glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN- CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, GD3, B
  • an antigen that is selected
  • the antigen binding domain of a CAR provided herein is operatively linked to a transmembrane domain by a hinge domain.
  • the antigen binding domain of a CAR provided herein is directly linked to a transmembrane domain by a hinge domain.
  • the CARs provided herein comprise a CD28 hinge domain.
  • a CAR provided herein comprises a hinge domain having the amino acid sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: ).
  • the CARs provided herein comprise a CD8alpha hinge domain.
  • the CARs WSGR Docket No.61078-729.601 provided herein comprise the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY (SEQ ID NO: ).
  • a hinge or spacer domain is a portion of an immunoglobulin, including, but not limited to, one or more heavy chain constant regions, e.g., CH2 and CH3.
  • the spacer domain may include the amino acid sequence of a naturally occurring immunoglobulin hinge region or an altered immunoglobulin hinge region.
  • the spacer domain includes the CH2 and/or CH3 of IgG 1, lgG4, or IgD.
  • Illustrative spacer domains suitable for use in the CARs described herein include the hinge region derived from the extracellular regions of type 1 membrane proteins such as CD8a and CD28, which may be wild-type hinge regions from these molecules or variants thereof.
  • the hinge domain includes a CD8alpha or CD28 hinge region.
  • the hinge is a PD-1 hinge or CD152 hinge.
  • the CAR further includes an extracellular spacer domain, which may include a hinge domain.
  • the hinge domain is generally a flexible polypeptide connector region disposed between the targeting moiety and the transmembrane domain.
  • Exemplary hinge domain sequences include those from IgG subclasses (such as IgGl and IgG4), IgD, CD28, and CD8 domains.
  • the hinge domain provides structural flexibility to flanking polypeptide regions.
  • the hinge domain may consist of natural or synthetic polypeptides. It will be appreciated by those skilled in the art that hinge domains may improve the function of the CAR by promoting optimal positioning of the antigen binding domain in relationship to the portion of the antigen recognized by it. In some embodiments, a hinge domain may not be required for optimal CAR activity.
  • a hinge domain comprising a short sequence of amino acids promotes CAR activity by facilitating antigen-binding by, for example, relieving steric constraints that could otherwise alter antibody binding kinetics.
  • the hinge domain is linked downstream of the antigen-binding domain of a CAR and upstream of the transmembrane domain of a CAR.
  • suitable hinge domains include those derived from CD8a, CD28, CTLA4, CD4, PD1, IgGl, PGK, or IgG4.
  • the hinge domain can include regions derived from a human CD8a (also known as CD8a) molecule, a CD28 molecule, and any other receptors that provide a similar function in providing flexibility to flanking regions.
  • the CAR disclosed herein includes a hinge domain derived from a CD8a hinge domain.
  • the CAR disclosed herein includes a hinge domain derived from a CD28 or CD8 hinge domain.
  • the hinge domain has about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% sequence identity to a CD8alpha, CD28, CTLA4, CD4, PD1, IgGl, PGK, or IgG4 hinge domain.
  • the spacer domain further comprises a linker including one or more intervening amino acid residues that are positioned between the antigen binding domain and the extracellular hinge domain.
  • the linker is positioned downstream from the antigen binding domain and upstream from the hinge domain.
  • the length and/or amino acid composition of the linker there are no particular limitations to the length and/or amino acid composition of the linker.
  • any arbitrary single-chain peptide comprising about one to about 300 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more amino acid residues) can be used as a linker.
  • the linker includes at least about 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.
  • the linker includes no more than about 300, 250, 200, 150, 140, 130, 120, 110, 100, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, or 30 amino acid residues.
  • the length and amino acid composition of the extracellular spacer can be optimized to vary the orientation and/or proximity of the antigen binding domain and the extracellular hinge domain to one another to achieve a desired activity of the CAR.
  • the orientation and/or proximity of the antigen binding domain and the extracellular hinge domain to one another can be varied and/or optimized as a “tuning” tool or effect to enhance or reduce the efficacy of the CAR.
  • the orientation and/or proximity of the antigen binding domain and the hinge domain to one another can be varied and/or optimized to create a partially functional version of the CAR.
  • the extracellular spacer domain includes an amino acid sequence corresponding to an IgG4 hinge domain and an IgG4 CH2-CH3 domain.
  • the spacer domain can be a synthetic polypeptide spacer, such as a spacer having a random sequence, a (gly-gly-ser)n (“GGSn”) sequence, or a variation thereof such as (SGG)n, (GGGS) n , (SGGG) n , (GSGGG) n , and the like, where n can range from about 1 to about 15.
  • the synthetic polypeptide spacer domain can also include a naturally occurring sequence, such as a hinge domain derived from CD8alpha, IgG, and the like.
  • CAR Transmembrane Domain [374]
  • the extracellular domain of the CAR is operably connected to the transmembrane domain.
  • the extracellular domain is operably connected to the transmembrane domain by a spacer.
  • the transmembrane domain of the CAR serves to transduce the external signal received by the extracellular domain to the intracellular domain.
  • the transmembrane domain can be selected from a transmembrane region of a transmembrane protein such as, for example, Type I transmembrane proteins, an artificial hydrophobic sequence or a combination thereof.
  • transmembrane domains include the transmembrane regions of the alpha, beta, delta, or gamma polypeptides of the T cell receptor of the T cell receptor, CD28, CD3 delta, CD3 gamma, CD3 epsilon, or CD3 zeta polypeptides of CD3, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD28, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154.
  • Synthetic transmembrane domains comprise a triplet of phenylalanine, tryptophan and valine.
  • a short oligo- or polypeptide linker may form the linkage between the transmembrane domain and the intracellular signaling domain of the CAR.
  • a glycine-serine doublet provides a particularly suitable linker between the transmembrane domain and the intracellular signaling domain.
  • the CAR comprises a transmembrane domain from a polypeptide selected from the group consisting of: CD4, CD8alpha, CD28, CD154, and PD-1; and one or more intracellular costimulatory signaling domains from a polypeptide selected from the group consisting of: 4-1BB, CD28, CD134, and CD137; and an intracellular signaling domain from a polypeptide CD79a, CD79, and CD665.
  • a CAR may further include a spacer domain between the antigen- binding portion and the transmembrane domain, e.g., a CD8alpha hinge.
  • the CAR comprises a transmembrane domain from CD28.
  • the CAR comprises a transmembrane domain with the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: ). In some embodiments, the CAR comprises a transmembrane domain from CD8alpha. In some embodiments, the CAR comprises a transmembrane domain with the amino acid sequence IWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: ). [376] The transmembrane domain may be derived either from a natural, synthetic, semi-synthetic, or recombinant source.
  • the TM domain is derived from (e.g., includes at least the transmembrane region(s) or a functional portion thereof) of the alpha or beta chain of the T-cell , CD4, CD5, CD8alpha, CD9, CD16, CD22, CD27, CD28, CD33, CD35, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD152, CD154, and/or PD-1.
  • the transmembrane domain may include, for example without limitation, all or part of the transmembrane domain of the CD3zeta chain), CD28, CD2, CD4, OX40, 4-1BB (CD137), ICOS (CD278), ILRB (CD122), IL-2RG (CD132), CTLA-4, PD-1, or CD40, or a sequence derived from such a transmembrane domain.
  • the cytoplasmic signaling domain in general comprises a domain that transduces the event of ligand binding into an intracellular signal that activates the T cell.
  • the CD3 WSGR Docket No.61078-729.601 intracellular domain/activating domain is frequently used, although others such as MyD88 can be used.
  • the transmembrane domain is the transmembrane domain from CD3 , CD2, CD8, or CD28. In an embodiment, the transmembrane domain is derived from the transmembrane domain from CD2 or CD28. In some embodiments, the transmembrane domain has about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% sequence identity to a CD3 , CD28, CD2, CD4, OX40, 4-1BB (CD137), FcERIy, ICOS (CD278), ILRB (CD122), IL-2RG (CD132), or CD40 transmembrane domain.
  • a CAR includes a transmembrane domain derived from CD8a or CD28 and a short polypeptide linker, e.g., between 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids in length, that links the transmembrane domain and the intracellular signaling domain of the CAR or anti-CD2 fusion protein.
  • a glycine-serine linker may be employed as such a linker, for example.
  • CAR Intracellular Domain [379] The transmembrane domain of the CAR is operably connected to the intracellular domain.
  • the serves to transduce the received external signal to kick-start the downstream signaling cascade.
  • the comprises an intracellular signaling domain.
  • the intracellular domain comprises an intracellular signaling domain from CD2. In some embodiments, the intracellular domain comprises a truncated CD2 intracellular domain. In some embodiments, the intracellular signaling domain comprises an amino acid sequence of KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRP PPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN (SEQ ID NO:?).
  • the intracellular signaling domain consists of an amino acid sequence of KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRP PPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN (SEQ ID NO:?).
  • the intracellular domain comprises an intracellular signaling domain CD79a, CD79b, CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and/or ZAP70.
  • the CAR comprises an intracellular domain comprising an intracellular signaling domain from 4-1BB (CD137).
  • the CAR comprises an intracellular domain comprising an intracellular signaling domain with the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: ).
  • WSGR Docket No.61078-729.601 [381]
  • the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD3zeta.
  • the CAR comprises an intracellular domain comprising an intracellular signaling domain with the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: ).
  • the CAR comprises an intracellular domain comprising an intracellular signaling domain with the amino acid sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLY NELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: ).
  • the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD3zeta and an intracellular signaling domain from 4-1BB (CD137).
  • the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD2.
  • the CAR comprises an intracellular domain comprising an intracellular signaling domain with the amino acid sequence KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPAT (SEQ ID NO: ). In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence PATSQHPPPPPGHRSQAPSHRPPPPGHRVQH (SEQ ID NO: ). [384] In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain from CD3epsilon. In some embodiments, the CAR comprises an intracellular domain comprising an intracellular signaling domain with the sequence RPPPVPNPDYEPIRKGQRDLYSGLNQRRI (SEQ ID NO: ).
  • the CAR comprises an intracellular domain comprising a truncated CD3epsilon intracellular domain.
  • TCR T cell receptor
  • Signals generated through the T cell receptor (TCR) alone may be insufficient for full activation of the T cell and a secondary or costimulatory signal may also be required.
  • T cell activation can be mediated by two distinct classes of intracellular signaling domains: primary signaling domains that initiate antigen-dependent primary activation through the TCR (e.g., a TCR/CD3 complex) and costimulatory signaling domains that act in an antigen- independent manner to provide a secondary or costimulatory signal.
  • the CAR may include an intracellular signaling domain that includes one or more costimulatory signaling domains and a primary signaling domain.
  • ITAM-containing primary signaling domains suitable for use in a , CD35, CD22, CD79a, CD79b, and CD665.
  • a CAR includes a CD3 primary signaling domain and WSGR Docket No.61078-729.601 one or more costimulatory signaling domains.
  • a CAR includes a 4-1BB costimulatory signaling domain.
  • the intracellular primary signaling and costimulatory signaling domains are operably linked to the carboxyl terminus of the transmembrane domain.
  • a CAR lacks a CD2 intracellular signaling domain.
  • the CAR includes one or more costimulatory signaling domains to enhance the efficacy and expansion of T cells expressing the CAR.
  • Exemplary costimulatory domains suitable for use in CARs contemplated in particular embodiments include TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and/or ZAP70.
  • the costimulatory signaling domain has at least about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to a costimulatory signaling domain from TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and/or ZAP70 domain.
  • a CAR includes one or more costimulatory signaling domains selected from the group consisting of CD2, 4- 1BB, CD28, CD137, and CD134, and a CD3zeta primary signaling domain.
  • a costimulatory molecule can be represented in the following protein families: TNF receptor proteins, Immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocytic activation molecules (SLAM proteins), and activating NK cell receptors.
  • the CAR comprises two or more intracellular signaling domains.
  • the CAR comprises a first signaling domain and a second signaling domain or fragments thereof independently selected from the group consisting of a CD3zeta intracellular signaling domain, a CD28 intracellular signaling domain, a 4-1BB intracellular signaling domain, an OX-40 intracellular signaling domain, an inducible co-stimulator (ICOS) intracellular signaling domain, a CD27 intracellular signaling domain, and a MyD88/CD40 intracellular signaling domain.
  • a CAR may include a first intracellular signaling domain or fragment thereof that is a CD3zeta intracellular signaling domain and a second intracellular signaling domain or fragment thereof that is a CD28 intracellular signaling domain.
  • a CAR may include a first intracellular signaling domain or fragment thereof that is a CD3zeta intracellular signaling domain and a second intracellular signaling domain or fragment thereof that is a 4-1BB intracellular signaling domain.
  • a CAR may include a first intracellular signaling domain or fragment thereof that is a CD3zeta intracellular signaling domain, a second WSGR Docket No.61078-729.601 intracellular signaling domain or fragment thereof that is a 4-1BB intracellular signaling domain, and a third intracellular signaling domain or fragment thereof that is a CD3 epsilon intracellular signaling domain.
  • the cytoplasmic signaling domain has CD665, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD278 (ICOS), DAP10, LAT, KD2C, SLP76, TRIM, and/or ZAP70 cytoplasmic signaling domain.
  • CARs of the disclosure comprise a CD2 co-stimulatory domain, and one or more additional co-stimulatory domains to increase cytokine production or sensitivity, reduce or prevent anergy, and/or to increase proliferation and cytotoxic activity.
  • co-stimulatory domains can be derived from co-stimulatory proteins such as B7-1 (CD80), B7-2 (CD86), CTLA-4, PD-1, CD278, CD122, CD132, B7- H2, B7- H3, PD-L1, PD-L2, B7-H4, PDCD6, BTLA, 41BB (CD137), FcERTy, CD40L, 4- 1BBL, GITR, BAFF, GITR-L, BAFF-R, HVEM, CD27, LIGHT, CD27L, OX40, OX40L, CD30, CD30L, TAC1, CD40, CD244, CD84, BLAME, CD229, CRACC, CD2F-10, NTB-A, CD48, SLAM (CD150), CD58, ikaros, CD53, integrin a4, CD82, integrin a4b1, CD90, integrin a4b7, CD96, LAG-3, CD160, LMIR, CRTAM,
  • the cytoplasmic signaling domain has about 70, 75, 80, 85, 90, 92, 93, 94, 95, 96, 97, 98, 99 or about 100% sequence identity to an B7-1 (CD80), B7-2 (CD86), CTLA-4, PD-1, CD278, CD122, CD132, B7- H2, B7-H3, PD-L1, PD-L2, B7-H4, PDCD6, BTLA, 41BB (CD137), FcERTy, CD40L, 4- 1BBL, GITR, BAFF, GITR-L, BAFF-R, HVEM, CD27, LIGHT, CD27L, OX40, OX40L, CD30, CD30L, TAC1, CD40, CD244, CD84, BLAME, CD229, CRACC, CD2F-10, NTB-A, CD48, SLAM (CD150), CD58, ikaros, CD53, integrin a4, CD82, integrin a4b
  • the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD28 hinge domain, a CD28 transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain.
  • the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD8 hinge domain, a CD8 WSGR Docket No.61078-729.601 transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain.
  • the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD28 hinge domain, a CD28 transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD19 binding domain, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD28 hinge domain, a CD28 transmembrane domain, and an intracellular domain comprising a CD28 intracellular signaling domain.
  • the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD8h transmembrane domain, and an intracellular domain comprising a CD3 zeta intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD28 hinge domain, a CD28 transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain. In some embodiments, the CAR comprises an extracellular domain comprising an anti-CD22 binding domain, a CD8 hinge domain, a CD8 transmembrane domain, and an intracellular domain comprising a 4-1BB (CD137) intracellular signaling domain.
  • the CAR comprises an amino acid sequence of SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70. In some embodiments, the CAR comprises an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 68. In some embodiments, the CAR comprises an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 69.
  • the CAR comprises an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 70.
  • the CAR consists of an amino acid sequence of SEQ ID NO: 68, SEQ ID NO: 69, or SEQ ID NO: 70.
  • the CAR consists of an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 68.
  • the CAR consists of an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 69. In some embodiments, the CAR consists of an amino acid sequence with at least 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identity of SEQ ID NO: 70.
  • WSGR Docket No.61078-729.601 CAR Retention Tag [391]
  • the CAR further comprises a protein retention tag. In some embodiments, the protein retention tag is operably linked to the intracellular domain of the CAR.
  • the protein retention tag is operably linked to the extracellular domain of the CAR.
  • the protein retention tag is selected from the group consisting of an ER retention tag, a Golgi apparatus (Golgi) retention tag, a lysosome retention tag, a plasma membrane retention tag, a mitochondria retention tag, a peroxisome retention tag, a cytosolic retention tag, and a nuclear retention tag.
  • the protein retention tag is an ER retention tag.
  • the ER retention tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity to any one of the ER retention tags of Table 5.
  • the ER retention tag comprises an amino acid sequence LYKYKSRRSFIDEKKMP (SEQ ID NO: 400).
  • the ER retention tag comprises an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 400.
  • the ER retention tag consists of an amino acid sequence of SEQ ID NO: 400. In some embodiments, the ER retention tag consists of an amino acid sequence with at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% identity of SEQ ID NO: 400. In some embodiments, the ER retention tag comprises the amino acid sequence KKMP (SEQ ID NO: 410). In some embodiments, the ER retention tag consists of an amino acid sequence of SEQ ID NO: 410. [393] In some embodiments, the protein retention tag is a Golgi retention tag.
  • the Golgi retention tag comprises the amino acid sequence YQRL (SEQ ID NO: 380). In some embodiments, the Golgi retention tag consists of the amino acid sequence YQRL (SEQ ID NO: 380). In some embodiments, the protein retention tag is a lysosome retention tag. In some embodiments, the lysosome retention tag comprises the amino acid sequence KFERQ (SEQ ID NO: 390). In some embodiments, the lysosome retention tag consists of the amino acid sequence KFERQ (SEQ ID NO: 390). [394] In some embodiments, a protease cleavage site is disposed between the protein retention tag and the CAR.
  • the protease cleavage site is disposed between the protein retention tag and the intracellular domain of the CAR. In some embodiments, the protease cleavage site is disposed between the protein retention tag and the extracellular domain of the CAR.
  • Protease cleavage sites are to be understood as amino acid residues that are recognized by proteases and/or WSGR Docket No.61078-729.601 amino acid residues whose peptide bond is cleaved by proteases. In some embodiments, a protease cleavage site comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids.
  • protease cleavage site also can be a variant of a cleavage site of a known protease as long as it is recognized/cleaved by the protease.
  • protease cleavage sites include, but are not limited to protease cleavage sites for proteases from the serine protease family, or for metalloproteases, or for a protease from the cysteine protease family, and/or the aspartic acid protease family, and/or the glutamic acid protease family.
  • serine proteases cleavage sites include, but are not limited to, cleavage sites for chymotrypsin-like proteases, and/or subtilisin-like proteases, and/or alpha/beta hydrolases, and/or signal peptidases.
  • metalloprotease recognition sites include, but are not limited to, cleavage sites for metallocarboxypeptidases or metalloendopeptidases.
  • the protease cleavage site is TEV protease cleavage site.
  • compositions comprising any of the recombinant polypeptides, siRNAs, miRNAs, shRNAs, and/or chimeric antigen receptors disclosed herein.
  • present disclosure provides compositions comprising any of the recombinant polynucleic acids disclosed herein.
  • the composition comprises any combination of the recombinant polypeptides and recombinant polynucleic acids disclosed herein.
  • compositions described herein comprise at least polypeptides as described herein, at least 1, 2, 3, 4, or more of the small non-coding RNAs targeting an endogenous TCR polypeptide as described herein, at least 1, 2, 3, 4, or more of the small non- coding RNAs targeting an endogenous TCR subunit as described herein, or at least 1, 2, 3, 4, or more of the CARs as described herein.
  • compositions provided herein comprise WSGR Docket No.61078-729.601 mutant polypeptides as described herein, at most 1, 2, 3, 4, or more of the small non-coding RNAs targeting an endogenous TCR polypeptide as described herein, at most 1, 2, 3, 4, or more of the small non-coding RNAs targeting an endogenous TCR subunit as described herein, or at most 1, 2, 3, 4, or more of the CARs as described herein.
  • compositions provided herein mutant polypeptides as described herein comprise 1, 2, 3, 4, or more of the small non-coding RNAs targeting an endogenous TCR polypeptide as described herein, comprise 1, 2, 3, 4, or more of the small non-coding RNAs targeting an endogenous TCR subunit as described herein, or comprise 1, 2, 3, 4, or more of the CARs as described herein.
  • the compositions provided herein comprise any one or more of the targeting an endogenous TCR subunit described herein. In some embodiments, the compositions one or more of the CARs described herein. In some embodiments, the compositions provided herein WSGR Docket No.61078-729.601 one or more of the small non-coding RNAs targeting an endogenous TCR polypeptide described herein. In some embodiments, the compositions provided herein comprise any one or more of the targeting an endogenous TCR subunit described herein. In some embodiments, the compositions one or more of the CARs described herein.
  • compositions provided herein more of the small non-coding RNAs targeting an endogenous TCR polypeptide described herein.
  • the compositions provided herein the CARs described herein.
  • the compositions provided herein comprise any one or more of the small non-coding RNAs targeting an endogenous TCR polypeptide described herein and another one or more of the small non-coding RNAs targeting another endogenous TCR polypeptide described herein.
  • compositions provided herein comprise any one or more of the small non-coding RNAs targeting an endogenous TCR polypeptide described herein and any one or more of the CARs described herein. In some embodiments, the compositions provided herein comprise any one or more of the small non-coding RNAs targeting an endogenous TCR subunit described herein and any one or more of the CARs described herein. In some embodiments, the compositions provided herein comprise any one or more of the CARs described herein and another one or more of the CARs described herein.
  • the cell comprising the compositions described herein comprises any one or more of the polynucleic acids that encode any of the various recombinant polypeptides, siRNAs, miRNAs, shRNAs, and/or chimeric antigen receptors disclosed herein.
  • the cell comprises at least two of any recombinant polypeptides, siRNAs, miRNAs, shRNAs and/or chimeric antigen receptors
  • the cell comprises at least two of any recombinant polypeptides, siRNAs, miRNAs, shRNAs, and/or chimeric antigen receptors.
  • the cell comprises at least two of any recombinant polypeptides, siRNAs, miRNAs, shRNAs, and/or chimeric antigen receptors
  • the cell comprises the polynucleic acid that encodes the at least two of any recombinant polypeptides, siRNAs, miRNAs, shRNAs, and/or chimeric antigen receptors.
  • the polynucleic acid may be monocistronic. Monocistronic polynucleic acid may express only one of the at least two of any WSGR Docket No.61078-729.601 recombinant polypeptides, siRNAs, miRNAs, shRNAs, and/or chimeric antigen receptors.
  • the polynucleic acid may be polycistronic.
  • Polycistronic polynucleic acid may express at least two of the at least two of any recombinant polypeptides, siRNAs, miRNAs, shRNAs, and/or chimeric antigen receptors.
  • each monocistronic polynucleic acid may encodes only one of the at least two of any recombinant polypeptides, siRNAs, miRNAs, shRNAs, and/or chimeric antigen receptors.
  • TCR targeting agent the small non-coding RNAs targeting an endogenous TCR polypeptide described herein, or the small non-coding RNAs targeting an endogenous TCR subunit may be referred as a “TCR targeting agent”.
  • a cell comprising a composition that comprises two TCR targeting agents may have an amount of the functional TCR complex that is at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower, relative to a cell that does not comprise the composition.
  • a cell comprising a composition that comprises two TCR targeting agents may have an amount of the functional TCR complex that is at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower, relative to a cell that does not comprise the composition.
  • a cell comprising a composition that comprises two TCR targeting agents may have an amount of the functional TCR complex that is at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower, relative to a cell that comprises only one of the TCR targeting agents.
  • a cell comprising a composition that comprises two TCR targeting agents may have an amount of the functional TCR complex that is at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower, relative to a cell that comprises only one of the TCR targeting agents.
  • a cell comprising a composition that comprises at least two TCR targeting agents may have an amount of the functional TCR complex that is at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower, relative to a cell that does not comprise the composition.
  • a cell comprising a composition that comprises at least two TCR targeting agents may have an amount of the functional TCR complex that is at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, WSGR Docket No.61078-729.601 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower, relative to a cell that does not comprise the composition.
  • a cell comprising a composition that comprises at least two TCR targeting agents may have an amount of the functional TCR complex that is at least about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower, relative to a cell that comprises only one of the TCR targeting agents.
  • a cell comprising a composition that comprises at least two TCR targeting agents may have an amount of the functional TCR complex that is at most about 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower, relative to a cell that comprises only one of the TCR targeting agents.
  • Disruption of Genes [401]
  • the recombinant cells of the disclosure comprising recombinant polypeptides and/or recombinant nucleic acids encoding the recombinant polypeptides may further comprise a disruption of a gene.
  • the gene may be a gene that encodes a T-cell receptor. In some embodiments, the gene may be a gene that encodes an MHC. In some embodiments, the gene may be a gene that encodes an extracellular domain of a T-cell receptor or MHC. In some embodiments, the gene may be a gene that encodes an intracellular receptor of a T-cell receptor or MHC.
  • the gene may be selected from the group consisting of TCRalpha, immunoevasin, ICAM1, CD80, CD58, OX40L, SUGT1, TAP1, TAP2, TAPBP, HLA-A, HLA-C, HLA-DR, HLA-DP, HLA-DQ, CD74, US11, K3, ICP47, and any combination thereof.
  • the disruption of the gene may include administering a gene editing platform to a recombinant cell of the disclosure.
  • the disruption of the gene may include knock-down (KD), knock-out (KO), and/or knock-in and or overexpression of the gene.
  • disruption of the gene comprises a modification of the gene, such as, for example, insertion, deletion, substitution, inversion, duplication, translocation, and/or frameshift or the like.
  • the gene editing technology may include, for example, e/Cas9 systems, TALENS, and/or Zinc finger Nucleases (ZFNs), meganucleases, type IIS restriction endonucleases (Fokl and Fokl fusions) and the like.
  • gene editing technology may include RNA interference (RNAi), including small-interfering RNAs (siRNAs).
  • gene editing technologies may include microRNAs (miRNAs).
  • Gene editing enables the possibility of permanently modifying a genomic sequence of interest by enabling targeted disruption, insertion, excision, and WSGR Docket No.61078-729.601 correction in both ex vivo and in vivo settings.
  • Exemplary gene editing systems and methods for modulating gene expression are described in detailed herein.
  • the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 5%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 10%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 15%.
  • the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 20%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 25%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 30%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 35%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 40%.
  • the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 45%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 50%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 55%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 60%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 65%.
  • the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 70%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 75%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 80%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 85%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 90%.
  • the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 95%. In some embodiments, the gene editing system, WSGR Docket No.61078-729.601 and/or gene expression modulator may decrease and/or modulate expression of the gene by at least about 100%. [404] In another aspect, the administration of the gene editing system, and/or gene expression modulator to a first cell may inhibit expression of a T-cell receptor on the cell. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 5%.
  • the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 10%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 15%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 20%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 25%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 30%.
  • the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 35%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 40%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 45%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 50%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 55%.
  • the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 60%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 65%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 70%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 75%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 80%.
  • the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 85%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 90%. In some embodiments, the WSGR Docket No.61078-729.601 gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 95%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the T-cell receptor by at least about 100%.
  • the administration of the gene editing system, and/or gene expression modulator to a first cell may inhibit expression of an MHC on the surface of the cell.
  • the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 5%.
  • the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 10%.
  • the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 15%.
  • the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 20%.
  • the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 25%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 30%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 35%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 40%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 45%.
  • the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 50%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 55%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 60%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 65%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 70%.
  • the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 75%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 80%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 85%. In some embodiments, the gene editing system, and/or gene expression modulator WSGR Docket No.61078-729.601 may decrease and/or modulate expression of the MHC by at least about 90%.
  • the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 95%. In some embodiments, the gene editing system, and/or gene expression modulator may decrease and/or modulate expression of the MHC by at least about 100%.
  • CRISPR/cas Gene Editing Systems [406] “CRISPR” or “CRISPR/Cas” as used herein refers to a set of clustered regularly interspaced short palindromic repeats, or a system comprising such a set of repeats. “Cas,” as used herein, refers to a CRISPR-associated protein.
  • CRISPR/Cas system refers to a system derived from CRISPR and Cas which can be used to silence or modify a target gene.
  • Naturally occurring CRISPR/Cas systems are found in approximately 40% of sequenced eubacteria genomes and 90% of sequenced archaea. Grissa et al. (2007) BMC Bioinformatics 8: 172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages and provides a form of acquired immunity. Barrangou et al. (2007) Science 315: 1709-1712; Marragini et al. (2008) Science 322: 1843-1845.
  • the CRISPR sequence sometimes called a CRISPR locus, comprises alternating repeats and spacers.
  • the spacers usually comprise sequences foreign to the bacterium such as a plasmid or phage sequence; in gene editing applications in eukaryotic cells, the spacers are derived from the eukaryotic target gene sequence.
  • RNA from the CRISPR locus is constitutively expressed and processed by Cas proteins into small RNAs. These comprise a spacer flanked by a repeat sequence. The RNAs guide other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Horvath et al.
  • Cse proteins e.g., CasA
  • Cascade a functional complex, Cascade, that processes CRISPR RNA transcripts into spacer-repeat units that Cascade retains.
  • Cas6 processes the CRISPR transcript.
  • the CRISPR- based phage inactivation in E. coli requires Cascade and Cas3, but not Cas1 or Cas2.
  • a simpler CRISPR system relies on the protein Cas9, which is a nuclease with two active cutting sites, one for each strand of the double helix. Combining Cas9 and modified CRISPR locus RNA can be used in a system for gene editing. Pennisi (2013) Science 341: 833-836.
  • the Cas9 is derived from a S. pyogenes Cas9.
  • the CRISPR/Cas systems can thus be used to edit a target gene (adding, replacing or deleting one or more base pairs), or introducing a premature stop which thus decreases expression of a target gene.
  • the CRISPR/Cas system can alternatively be used like RNA interference, turning off a target gene in a reversible fashion.
  • the RNA can guide the Cas protein to a target promoter, sterically blocking RNA polymerases.
  • TALEN Gene Editing System [412] “TALEN” refers to a transcription activator-like effector nuclease, an artificial nuclease which can be used to edit a target gene.
  • TALENs are produced artificially by fusing a TAL effector (“TALE”) DNA binding domain, e.g., one or more TALEs, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 TALEs to a DNA-modifying domain, e.g., a FokI nuclease domain.
  • TALEs Transcription activator-like effects
  • Zhang (2011), Nature Biotech. 29: 149-153 By combining an engineered TALE with a DNA cleavage domain, a restriction enzyme can be produced which is specific to any desired DNA sequence. These can then be introduced into a cell, wherein they can be used for genome editing.
  • TALEs are proteins secreted by Xanthomonas bacteria.
  • the DNA binding domain contains a repeated, highly conserved 33-34 amino acid sequence, with the exception of the 12th and 13th amino acids. These two positions are highly variable, showing a strong correlation with specific nucleotide recognition. They can thus be engineered to bind to a desired DNA sequence.
  • TALEN a TALE protein is fused to a nuclease (N), e.g., a wild-type or mutated FokI endonuclease.
  • N nuclease
  • Several mutations to FokI have been made for its use in TALENs; these, for example, improve cleavage specificity or activity. Cermak et al. (2011) Nucl. Acids Res. 39: e82; Miller et al. (2011) Nature Biotech.29: 143-8; Hockemeyer et al. (2011) Nature Biotech.29: 731-734; Wood et al. (2011) Science 333: 307; Doyon et al.
  • the FokI domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALE DNA binding domain and the FokI cleavage domain and the number of bases between the two individual TALEN binding sites appear to be important parameters for achieving high levels of activity. Miller et al. (2011) Nature Biotech.29: 143-8.
  • TALEN can be used inside a cell to produce a double-stranded break (DSB) in a target nucleic acid, e.g., a site within a gene.
  • a mutation can be introduced at the break site if the repair mechanisms improperly repair the break via non-homologous end joining. Huertas, P., Nat. Struct. Mol. Biol. (2010) 17: 11-16. For example, improper repair may introduce a frame shift mutation.
  • foreign DNA can be introduced into the cell along with the TALEN; depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to modify a target gene, e.g., correct a defect in the target gene, thus causing expression of a repaired target gene, or e.g., introduce such a defect into a wt gene, thus decreasing expression of a target gene.
  • a target gene e.g., correct a defect in the target gene, thus causing expression of a repaired target gene, or e.g., introduce such a defect into a wt gene, thus decreasing expression of a target gene.
  • ZFN Zinc Finger Nuclease Gene Editing System
  • ZFN Zinc Finger Nuclease Gene Editing System
  • a ZFN comprises a DNA-modifying domain, e.g., a nuclease domain, e.g., a FokI nuclease domain (or derivative thereof) fused to a DNA-binding domain.
  • the DNA-binding domain comprises one or more zinc fingers, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 zinc fingers.
  • a zinc finger is a small protein structural motif stabilized by one or more zinc ions.
  • a zinc finger comprises, for example, Cys2His2, and can recognize an approximately 3-bp sequence.
  • Various zinc fingers of known specificity can be combined to produce multi-finger polypeptides which recognize about 6, 9, 12, 15 or 18-bp sequences.
  • selection and modular assembly techniques are available to generate zinc fingers (and combinations thereof) recognizing specific sequences, including phage display, yeast one-hybrid systems, bacterial one-hybrid and two-hybrid systems, and mammalian cells.
  • Zinc fingers can be engineered to bind a predetermined nucleic acid sequence. Criteria to engineer a zinc finger to bind to a predetermined nucleic acid sequence are known in the art. Sera (2002), Biochemistry, 41:7074-7081; Liu (2008) Bioinformatics, 24:1850-1857. WSGR Docket No.61078-729.601 [420] A ZFN using a FokI nuclease domain or other dimeric nuclease domain functions as a dimer. Thus, a pair of ZFNs are required to target non-palindromic DNA sites. The two individual ZFNs must bind opposite strands of the DNA with their nucleases properly spaced apart. Bitinaite et al.
  • a ZFN can create a double-stranded break in the DNA, which can create a frame-shift mutation if improperly repaired, e.g., via non-homologous end joining, leading to a decrease in the expression of a target gene in a cell.
  • foreign DNA can be introduced into the cell along with the ZFN; depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to modify a target gene, e.g., correct a defect in the target gene, thus causing expression of a repaired target gene, or e.g., introduce such a defect into a wt gene, thus decreasing expression of a target gene, e.g., as described in WO2013/169802. Meganuclease Gene Editing System [422]
  • the term “meganuclease” refers to an artificial nuclease which can be used to edit a target gene.
  • Meganucleases are derived from a group of nucleases which recognize 15-40 base-pair cleavage sites. Meganucleases are grouped into families based on their structural motifs which affect nuclease activity and/or DNA recognition. [424] Strategies for engineering a meganuclease with altered DNA-binding specificity, e.g., to bind to a predetermined nucleic acid sequence are known in the art. E.g., Chevalier et al. (2002), Mol. Cell., 10:895-905; Epinat et al. (2003) Nucleic Acids Res 31: 2952-62; Silva et al.
  • a meganuclease can create a double-stranded break in the DNA, which can create a frame- shift mutation if improperly repaired, e.g., via non-homologous end joining, leading to a decrease in the expression of a target gene in a cell.
  • foreign DNA can be introduced into the cell along with the Meganuclease; depending on the sequences of the foreign DNA and chromosomal sequence, this process can be used to modify a target gene, e.g., correct a defect in the target gene, thus causing expression of a repaired target gene, or e.g., introduce such a defect into a wt gene, thus decreasing expression of a target gene, e.g., as described in Silva et al. (2011) Current Gene Therapy 11:11-27. WSGR Docket No.61078-729.601 Targeting RNA [426]
  • the gene editing systems of the disclosure may include a targeting RNA.
  • the targeting RNA is any ribonucleotide sequence having sufficient complementarity with a target polynucleotide sequence, e.g., a target DNA sequence, to hybridize with the target sequence.
  • the targeting RNA is capable of directing sequence-specific cleavage of DNA at or adjacent to the target DNA sequence by a polypeptide comprising a cleavage domain.
  • the targeting RNA is typically about 20 nucleotides.
  • a targeting RNA is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.
  • a targeting RNA is fewer than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length.
  • the targeting RNA comprises a sequence of 10 nucleic acids.
  • the targeting RNA comprises a sequence of 11 nucleic acids.
  • the targeting RNA comprises a sequence of 12 nucleic acids.
  • the targeting RNA comprises a sequence of 13 nucleic acids.
  • the targeting RNA comprises a sequence of 14 nucleic acids.
  • the targeting RNA comprises a sequence of 15 nucleic acids.
  • the targeting RNA comprises a sequence of 16 nucleic acids.
  • the targeting RNA comprises a sequence of 17 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 18 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 19 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 20 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 21 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 22 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 23 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 24 nucleic acids. In some embodiments, the targeting RNA comprises a sequence of 25 nucleic acids.
  • the targeting RNA comprises a sequence of 26 nucleic acids.
  • the degree of complementarity between a targeting RNA and its corresponding target DNA sequence when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non- limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • any suitable algorithm for aligning sequences include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.c
  • a WSGR Docket No.61078-729.601 targeting RNA to direct by the polypeptide comprising a cleavage domain at or adjacent to the target sequence may be assessed by any suitable assay.
  • the components of a gene editing system as described herein, including the targeting RNA to be tested may be provided to a host cell having the complimentary target DNA sequence, such as by transfection with vectors encoding the components of gene editing system, followed by an assessment of preferential cleavage within the target DNA sequence, such as by Surveyor assay as described herein.
  • cleavage of a target DNA sequence may be evaluated in a test tube by providing the target DNA sequence, components of a gene editing system, including the targeting RNA to be tested, and a control targeting RNA different from the test targeting RNA, and comparing binding or rate of cleavage at the target DNA sequence between the test and control targeting RNA reactions.
  • a targeting RNA may be selected to target any target DNA sequence.
  • the target DNA sequence is a sequence within a genome of a cell. Exemplary target sequences include those that are unique in the target genome.
  • a unique target sequence in a genome may include a sequence NNNNNNNNNNNNNNN, where N is A, G, T, or C, and has a single occurrence in the genome.
  • a targeting RNA is selected to reduce the degree of secondary structure within the targeting RNA. Secondary structure may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148).
  • the targeting RNA hybridizes to a continuous stretch of nucleic acids within the target DNA. In other embodiments the targeting RNA hybridizes to a discontinuous stretch of nucleic acids within the target DNA. In embodiments, the targeting RNA may hybridize to a single- stranded target DNA sequence, for example, though base pairing.
  • the targeting RNA may hybridize to a double-stranded target DNA sequence, for example by hybridizing to the major- or minor-groove edges of the base pairs of the target DNA sequence.
  • the targeting RNA hybridizes to a target DNA sequence that is actively transcribed, e.g., actively transcribed in the cell type being studied.
  • the targeting RNA hybridizes to a target DNA sequence that does not comprise condensed chromatin, e.g., does not comprise condensed chromatin in the cell type being studied.
  • RNAs and/or target DNA sequences are described in, for example: Wang T, et al., Science (2013), vol.343, pp.80-84 and WO2015/048577, which are hereby incorporated by reference in their entirety. It will be understood by one of ordinary skill that the targeting RNAs of the genome editing systems of the present disclosure are not limited to those disclosed, for example, in Wang, et al. It will be appreciated that unlike other known gene editing systems, targeting RNAs to virtually any sequence of target DNA can be designed. Guide RNA [433] In another aspect, the gene editing systems of the disclosure comprises a guide RNA.
  • the Guide RNA refers to ribonucleic acid sequence that is capable of binding to a guide RNA- binding domain, e.g., a guide RNA-binding domain as described herein.
  • the Guide RNA refers to an RNA aptamer.
  • aptamers, and their corresponding polypeptide-based guide RNA- binding domains are known in the literature, any of which are suitable for use in the present disclosure.
  • Non-limiting examples of guide RNA/guide RNA-binding domain pairs are described in detail herein.
  • the guide RNA is between 1-100, 1-90, 1-80, 1-70, 1-60, 1-50, 1-40, 1- 30, 1-20 or 1-10 nucleotides.
  • the guide RNA is between about 20 and about 100 nucleotides, e.g., between about 30 and about 90, e.g., between about 40 and about 80, e.g., between about 50 and about 70 nucleotides.
  • the guide RNA is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 or more nucleotides.
  • Additional guide RNA molecules may be discovered using RNA-based combinatorial libraries, screened against any guide RNA-binding molecule of interest. Those of ordinary skill will appreciate how to design and identify guide RNAs to any guide RNA-binding domain of interest.
  • libraries e.g., phage display libraries, of RNA aptamers may be designed according to known methods, and those libraries may be screened for specific binding to a useful guide RNA-binding domains, also according to known methods.
  • RNA molecules that bind to specific targets of interest may be identified using SELEX (e.g., Fitzwater et al., Methods Enzymol., vol.267, pp.275-301 (1996), which is incorporated herein in its entirety).
  • SELEX e.g., Fitzwater et al., Methods Enzymol., vol.267, pp.275-301 (1996), which is incorporated herein in its entirety.
  • Guide RNAs capable of specifically binding to guide RNA-binding domains may also be generated by rational design based on computer modeling and or structural biology.
  • WSGR Docket No.61078-729.601 Dominant Negative Inhibition
  • provided herein are dominant-negative inhibitor proteins capable of interrupting, reducing, or eliminating TCR expression and/or function.
  • the dominant-negative inhibitor proteins comprise any of the recombinant polypeptides, fusion polypeptides, or mutant polypeptides described herein.
  • the dominant-negative provided herein are recombinant polynucleic acids comprising a polynucleotide encoding all or a portion of a TCR complex component (e.g., TCR-alpha, TCR-beta, TCR-delta, TCR-gamma, CD3- gamma, CD3-delta, CD3-epsilon, or CD3-zeta) modified so that it: (1) lacks key signaling motifs (e.g., an ITAM) required for protein function; (2) does not associate properly with the other endogenous TCR components; or (3) associates properly with the other endogenous TCR components but is otherwise not functional (e.g., it does not properly transduce a signal or bind its cognate ligand).
  • a TCR complex component e.g., TCR-alpha,
  • the recombinant polynucleic acid comprising a polynucleotide encoding all or a portion of a TCR complex component further comprises an inhibitory signaling motif (e.g., a cytoplasmic domain from a KIR protein) which alters cell signaling and promotes inhibitory signals through the recruitment of phosphatases (e.g., SHP1 and SHP2).
  • an inhibitory signaling motif e.g., a cytoplasmic domain from a KIR protein
  • the recombinant polynucleic acids comprising a polynucleotide encoding all or a portion of a TCR complex component or another gene of interest further comprises a portion of a protein or other gene product that can serve as a tag to measure expression of the recombinant polynucleic acids (i.e., a portion of a protein that serves as a means to identify the over- expressed minigene).
  • polynucleotides encoding a truncated CD19 protein comprising the binding site for one or more specific anti-CD19 antibodies can be operably linked to the minigene so that the resulting cell that expresses the minigene will express the encoded protein and can be identified with anti-CD19 mAbs.
  • the ability to measure the presence or absence of the encoded protein by measuring expression of the tag enables one to assess the extent of minigene expression and/or to isolate cells expressing the recombinant protein (and thus that lack a functional TCR).
  • a recombinant cell of the disclosure comprises expression of a dominant negative (DN) protein selected from the group consisting of DN-TCRalpha, DN-TCRbeta, DN- DN- WSGR Docket No.61078-729.601 DN-RFX5, DN-RFXANK, DN-RFXAP, a DN-viral immunoevasin, DN-ICAM1, DN-CD80, DN- CD58, DN-CD2, DN-OX40L, DN-SUGT1, DN-TAP1, DN-TAP2, DN-TAPBP, DN-HLA-A, DN- HLA-C, DN-HLA-DR, DN-HLA-
  • DN dominant negative
  • the present disclosure describes cells comprising the recombinant fusion proteins disclosed herein, cells comprising the chimeric antigen receptors disclosed herein, cells comprising the recombinant polynucleic acids disclosed herein, cells comprising the compositions comprising the recombinant polynucleic acids disclosed herein, and/or cells comprising the recombinant fusion proteins and chimeric antigen receptors disclosed herein.
  • the recombinant fusion protein may be a recombinant polypeptide.
  • the cell may express a recombinant polypeptide and/or chimeric antigen receptor disclosed herein.
  • the recombinant polypeptide and/or CAR are expressed on the surface of the cell.
  • when the recombinant polypeptide is expressed on the surface of the cell it may be bound to an MHC molecule.
  • the MHC molecule may be a class 1 MHC molecule.
  • the transmembrane portion passes through the cell membrane, and the one or more intracellular signaling domains are disposed adjacent to the intracellular side of the cell membrane.
  • cells may be genetically engineered (e.g., transduced, transformed, or transfected) with, for example, a vector construct of the present disclosure that may be, for example, a viral vector or a vector for homologous recombination that includes nucleic acid sequences homologous to a portion of the genome of the host cell, or may be an expression vector for the expression of the polypeptides of interest.
  • a vector construct of the present disclosure may be, for example, a viral vector or a vector for homologous recombination that includes nucleic acid sequences homologous to a portion of the genome of the host cell, or may be an expression vector for the expression of the polypeptides of interest.
  • Cells may be either untransformed cells or cells that have already been transfected with at least one nucleic acid molecule.
  • the cell is an immune cell, a stem cell, a mammalian cell, a primate cell, or a human cell. In some embodiments, the cell is autologous or allogeneic. In some embodiments, the cell is a T cell, a CD8-positive T cell, a CD4-positive T cell, a regulatory T cell, a cytotoxic T cell, or a tumor infiltrating lymphocyte.
  • WSGR Docket No.61078-729.601 Cells may be transduced/transfected with a polynucleic acid encoding the recombinant polypeptide and/or CAR.
  • a cell may be transduced with a bicistronic nucleic acid encoding a recombinant polypeptide and a CAR. In some, a cell may be transduced with a nucleic acid encoding a recombinant polypeptide and an additional nucleic acid encoding a CAR. In some embodiments, the cell is further transduced with an additional nucleic acid encoding one or more additional therapeutic agents such as, for example, but not limited to, an antibody, an antibody fragment thereof, or a protein therapeutic. [445] In another aspect, allogeneic T-cells may be isolated and cultured ex vivo.
  • the allogeneic T-cells comprise a CAR of the disclosure.
  • a recombinant polypeptide may be administered to the cultured CAR-T cell.
  • a recombinant polypeptide may be co-cultured with or pre-bound to a CAR-T cell prior to administration to a patient.
  • the cultured, recombinant polypeptide/CAR-T cell may be administered to a patient.
  • autologous T-cells may be isolated from a subject, and cultured ex vivo.
  • the autologous T-cells comprise a CAR of the disclosure.
  • a recombinant polypeptide may be administered to the cultured CAR-T cell.
  • a recombinant polypeptide may be co-cultured with or pre-bound to a CAR-T cell prior to administration to a patient.
  • the cultured, B2M/CAR-T cell may be administered to a patient.
  • cells of the disclosure comprises a CAR, and/or a recombinant polypeptide, and/or a disruption of a gene.
  • a cell may be modified comprising disruption of a gene, for example, gene knock-out, knock-down, mutation, knock-in, and/or overexpression, by administering a gene editing system and/or gene modulator of the disclosure.
  • the cell comprising disruption of a gene may be a T-cell.
  • the cell comprising disruption of a gene may further comprise a CAR.
  • the cell comprising the disruption of a gene and a CAR may further comprise a recombinant polypeptide or sequence encoding a recombinant polypeptide.
  • the recombinant cell is an animal cell.
  • the animal cell may be a mammalian cell. In some embodiments, the animal cell may be a mouse cell. In some embodiments, the animal cell may be a human cell.
  • the recombinant cell may be an immune system cell, e.g., a lymphocyte (for example without limitation, a T cell, natural killer cell or NK cell, natural killer T cell or NKT cell, a B cell, a plasma cell, tumor-infiltrating lymphocyte (TIL)), a monocyte or macrophage, or a dendritic cell.
  • the immune system cell may be selected from the group consisting of B cells, T cells, monocytes, dendritic cells, and epithelial cells.
  • the immune system cell may be a T lymphocyte.
  • the WSGR Docket No.61078-729.601 immune cell may also be a precursor cell, i.e., a cell that may be capable of differentiating into an immune cell.
  • Techniques for transforming a wide variety of the above-mentioned host cells and species are known in the art and described in the technical and scientific literature.
  • the nucleic acid molecule may be introduced into a host cell by a transduction procedure, transfection procedure, electroporation procedure, or a biolistic procedure. Accordingly, cell cultures including at least one recombinant cell as disclosed herein are also within the scope of this application.
  • Cells of the present disclosure may be autologous/autogeneic (“self”) or non-autologous (“non- self,” e.g., allogeneic, syngeneic or xenogeneic).
  • autologous refers to cells derived from the same individual to which they are subsequently administered.
  • allogeneic refers to cells of the same species derived from different individuals that are different enough genetically that they are unlikely to interact antigenically.
  • “Syngeneic,” as used herein refers to cells of a different individual that are genetically identical to the cell in comparison.
  • the cells are T cells obtained from a mammal.
  • the mammal is a primate.
  • the primate is a human.
  • T cells can be obtained from a number of sources including, but not limited to, peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells are obtained from a unit of blood collected from an individual using any number of known techniques such as sedimentation, e.g., FICOLL separation.
  • an isolated or purified population of T cells may be used.
  • TCTL and TH lymphocytes are purified from PBMCs.
  • the TCTL and TH lymphocytes are sorted into naive (TN), memory (TMEM), stem cell memory (TSCM), central memory (TCM), effector memory (TEM), and effector (TEFF) T cell subpopulations either before or after activation, expansion, and/or genetic modification.
  • Suitable approaches for such sorting include, e.g., magnetic-activated cell sorting (MACS), where TN are CD45RA+ CD62L+ CD95-; TSCM are CD45RA+ CD62L+ CD95+; TCM are CD45RO+ CD62L+ CD95+; and TEM are CD45RO+ CD62L- CD95+.
  • MCS magnetic-activated cell sorting
  • TN are CD45RA+ CD62L+ CD95-
  • TSCM are CD45RA+ CD62L+ CD95+
  • TCM are CD45RO+ CD62L+ CD95+
  • TEM are CD45RO+ CD62L- CD95+.
  • An exemplary approach for such sorting may be described in Wang et al. (2016) Blood 127(24):2980- 90.
  • a specific subpopulation of T cells expressing one or more of the following markers: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127, and HLA-DR can be further isolated by positive or
  • a specific subpopulation of T cells expressing one or more of the markers selected from the group consisting of CD62L, CCR7, CD28, WSGR Docket No.61078-729.601 CD27, CD122, CD127, CD197; or CD38 or CD62L, CD127, CD197, and CD38, may be further isolated by positive or negative selection techniques.
  • compositions comprising a cell comprising a recombinant polynucleic acid comprising a sequence encoding a recombinant polypeptide and/or a CAR of the disclosure, wherein the cell expresses the recombinant polypeptide and/or CAR of the disclosure, wherein the cell expressing the recombinant polypeptide and/or CAR of the disclosure is a grafted cell, and wherein the grafted cell is administered to a host, and wherein the grafted cell count when measured after administration to the host is at least about 5% greater than a grafted cell count of grafted cells that do not express the recombinant polypeptide and/or CAR of the disclosure.
  • the grafted cell count when measured after administration to the host is at least about 10% greater than a grafted cell count of grafted cells that do not express the recombinant polypeptide and/or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 20% greater than a grafted cell count of grafted cells that do not express the recombinant polypeptide and/or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 30% greater than a grafted cell count of grafted cells that do not express the recombinant polypeptide and/or CAR of the disclosure.
  • the grafted cell count when measured after administration to the host is at least about 40% greater than a grafted cell count of grafted cells that do not express the recombinant polypeptide and/or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 50% greater than a grafted cell count of grafted cells that do not express the recombinant polypeptide and/or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 60% greater than a grafted cell count of grafted cells that do not express the recombinant polypeptide and/or CAR of the disclosure.
  • the grafted cell count when measured after administration to the host is at least about 70% greater than a grafted cell count of grafted cells that do not express the recombinant polypeptide and/or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 80% greater than a grafted cell count of grafted cells that do not express the recombinant polypeptide and/or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 90% greater than a grafted cell count of grafted cells that do not express the recombinant polypeptide and/or CAR of the disclosure.
  • the grafted cell count when measured after administration to the host is at least about 100% greater than a grafted cell count of grafted cells that do not express the recombinant polypeptide and/or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about WSGR Docket No.61078-729.601 150% greater than a grafted cell count of grafted cells that do not express the recombinant polypeptide and/or CAR of the disclosure.
  • the grafted cell count when measured after administration to the host is at least about 200% greater than a grafted cell count of grafted cells that do not express the recombinant polypeptide and/or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is at least about 300% greater than a grafted cell count of grafted cells that do not express the recombinant polypeptide and/or CAR of the disclosure. In some embodiments, the grafted cell count when measured after administration to the host is greater than 300% greater than a grafted cell count of grafted cells that do not express the recombinant polypeptide and/or CAR of the disclosure.
  • the cell is a mammalian cell.
  • the cell can be a human cell.
  • the cell can be a blood cell.
  • the blood cell is a lymphocyte.
  • the lymphocyte is a T cell.
  • the cell is a population of cells.
  • the population of cells is a population of blood cells.
  • the blood cells can be lymphocytes.
  • the lymphocytes can be T cells.
  • the population of cells is a homogeneous mixture of cells of the same cell type.
  • the population of cells is a heterogeneous mixture of cells of different cell types.
  • the population of cells comprises at least about 1 10 5 cells.
  • the population of cells comprises at least about 1 10 6 cells. In some embodiments, the population of cells comprises at least about 1 10 7 cells. In some embodiments, the population of cells comprises at least about 1 10 8 cells. In some embodiments, the population of cells comprises at least about 1 10 9 cells. In some embodiments, the population of cells comprises from about 1 10 5 cells to about 1 10 9 cells. In some embodiments, the population of cells comprises from about 1 10 5 cells to about 1 10 8 cells. In some embodiments, the population of cells comprises from about 1 10 5 cells to about 1 10 7 cells. In some embodiments, the population of cells comprises from about 1 10 5 cells to about 1 10 6 cells.
  • compositions [456]
  • the present disclosure describes a pharmaceutical composition comprising the composition comprising the recombinant polypeptides and/or CARs disclosed herein, and a pharmaceutically acceptable excipient or carrier.
  • the present disclosure describes a pharmaceutical composition comprising the compositions comprising the recombinant polynucleic acids disclosed herein, and a pharmaceutically acceptable excipient or carrier.
  • the present disclosure describes a pharmaceutical composition comprising the composition comprising the cells disclosed herein, and a pharmaceutically acceptable excipient or carrier.
  • the pharmaceutical WSGR Docket No.61078-729.601 compositions generally include a therapeutically effective amount of the cells.
  • terapéuticaally effective amount is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease (e.g., cancer) or disorder associated, e.g., with the target cell or a population thereof (e.g., cancer cells), as compared to a control.
  • An effective amount can be administered in one or more administrations.
  • a “therapeutically effective amount” of the cells disclosed herein may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the cells to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects.
  • the term “therapeutically effective amount” includes an amount that is effective to “treat” an individual, e.g., a patient.
  • a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments, to be administered, can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (individual).
  • a pharmaceutical composition of the present disclosure includes from 1 10 5 to 5 10 10 of the cells of the present disclosure, or greater than 5 10 10 cells of the present disclosure.
  • the cells of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. More particularly, the cells of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable excipients or diluents. [458] Formulations of the cells suitable for administration to a patient (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.
  • the cells may be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration.
  • parenteral e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.
  • Pharmaceutical compositions that include the cells of the present disclosure may be prepared by mixing the cells having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and/or tonicity agents.
  • Acceptable carriers, excipients and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, WSGR Docket No.61078-729.601 phenol, m-cresol, p-chlor-m- cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than
  • An aqueous formulation of the recombinant polypeptides, proteases, nucleic acids, expression vectors, and/or cells may be prepared in a pH-buffered solution, e.g., at pH ranging from about 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5.
  • buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers.
  • the buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.
  • a tonicity agent may be included in the formulation to modulate the tonicity of the formulation.
  • Example tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof.
  • the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable.
  • the term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum.
  • Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.
  • a surfactant may also be added to the formulation to reduce aggregation and/or minimize the formation of particulates in the formulation and/or reduce adsorption.
  • Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene- polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS).
  • suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20 ) and polysorbate 80 (sold under the trademark Tween 80 ).
  • Suitable polyethylene- polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188 .
  • suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij .
  • Example concentrations of surfactant may range from about 0.001% to about 1% w/v.
  • the pharmaceutical composition includes cells of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof.
  • a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w/v).
  • kits for treating a disease, disorder, or condition in a subject in need thereof comprising administering a therapeutically effective amount of the pharmaceutical composition disclosed herein.
  • the pharmaceutical composition can be administered alone or in combination with other agents (e.g., an antibody or an antigen binding fragment thereof, or a molecule).
  • agents e.g., an antibody or an antigen binding fragment thereof, or a molecule.
  • a vaccine, an oncolytic virus, a checkpoint inhibitor, a T cell agonist antibody, chemotherapy, and/or a bispecific antibody can be combined with the pharmaceutical composition disclosed herein.
  • the pharmaceutical composition is administered with other cells (e.g., CAR T cells or other adoptively transferred T cells).
  • Administration “in combination with” one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
  • the one or more additional therapeutic agents, chemotherapeutics, anti-cancer agents, or anti-cancer therapies is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery.
  • chemotherapy and “anti-cancer agent” are used interchangeably herein.
  • Various classes of anti-cancer agents can be used.
  • Non-limiting examples include: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, podophyllotoxin, antibodies (e.g., monoclonal or polyclonal), checkpoint inhibitors, immunomodulators, cytokines, nanoparticles, radiation therapy, tyrosine kinase inhibitors (for example, imatinib mesylate), hormone treatments, soluble receptors and other antineoplastics.
  • the disease, disorder, or condition is a cancer, an inflammatory disease, a neuronal disorder, HIV/AIDS, diabetes (such as Type I diabetes), a cardiovascular disease, an infectious disease, or an autoimmune disease.
  • the disease, disorder, or condition is cancer.
  • the cancer is lymphoma or leukemia.
  • the disease, disorder, or condition is a hyperproliferative disorder.
  • Hyperproliferative disorders include cancers and hyperplasia characterized by the unregulated overgrowth of cells. Hyperproliferative disorders frequently display loss of genetic regulatory mechanisms and may express native proteins WSGR Docket No.61078-729.601 inappropriately (including expression of proteins from other cell types or developmental stages, expression of mutated proteins, and expression of proteins at levels higher or lower than normal).
  • B-cell hyperproliferative disorders include B-cell leukemias and lymphomas such as, but not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, precursor B lymphoblastic leukemia, hairy cell leukemia, diffuse large B- cell lymphoma (DLBCL), follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, Burkitt’s lymphoma, MALT lymphoma, Waldenstrom’s macroglobulinemia, and/or other disorders characterized by the overgrowth of B-lineage cells.
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • B-cell prolymphocytic leukemia precursor B lymphoblastic leukemia
  • hairy cell leukemia hairy cell leukemia
  • DLBCL diffuse large B- cell lymphoma
  • follicular lymphoma marginal
  • Hyperproliferative disorders include diseases such as, but not limited to, bladder cancer, including upper tract tumors and urothelial carcinoma of the prostate; bone cancer, including chondrosarcoma, Ewing's sarcoma, and osteosarcoma; breast cancer, including noninvasive, invasive, phyllodes tumor, Paget's disease, and breast cancer during pregnancy; central nervous system cancers, adult low-grade infiltrative supratentorial astrocytoma/oligodendroglioma, adult intracranial ependymoma, anaplastic astrocytoma/anaplastic oligodendroglioma/glioblastoma multiforme, limited (1-3) metastatic lesions, multiple (>3) metastatic lesions, carcinomatous lymphomatous meningitis, non-immunosuppressed primary CNS lymphoma, and metastatic spine tumors; cervical cancer; colon cancer, rectal cancer, anal carcinoma; esophageal cancer; gastric (
  • administering introducing” and “transplanting” are used interchangeably in the context of the placement of the recombinant polypeptides, nucleic acids, and/or gene editing molecules, and/or recombinant cells of the disclosure into a subject, by a method or route that results in at least partial localization of the introduced cells at a desired site, such as a site of injury or repair, such that a desired effect(s) is produced.
  • the recombinant polypeptides, nucleic acids, and/or gene editing molecules, or recombinant cells of the disclosure can be administered by any appropriate route that results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable.
  • the period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the lifetime of the patient, i.e., long-term engraftment.
  • an effective amount of the recombinant polypeptides, nucleic acids, and/or gene editing molecules, or recombinant cells is administered via a systemic route of administration, such as an intraperitoneal or intravenous route.
  • a systemic route of administration such as an intraperitoneal or intravenous route.
  • the terms “individual,” “subject,” “host” and “patient” are used interchangeably herein and refer to any subject for whom diagnosis, treatment or therapy is desired.
  • the subject is a mammal.
  • the subject is a human being.
  • the term “donor” is used to refer to an individual that is not the patient. In some embodiments, the donor is an individual who does not have or is not suspected of having the medical condition to be treated.
  • the recombinant polypeptides, nucleic WSGR Docket No.61078-729.601 acids, and/or gene editing molecules, or recombinant cells of the disclosure may be provided at (or after) the onset of a symptom or indication of a medical condition, e.g., upon the onset of disease.
  • the recombinant cells being administered according to the compositions and methods described herein comprises allogeneic T cells.
  • the recombinant cells being administered according to the compositions and methods described herein comprises allogeneic T cells. obtained from one or more donors.
  • the cell population being administered can be allogeneic blood cells, hematopoietic stem cells, hematopoietic progenitor cells, embryonic stem cells, or induced embryonic stem cells.
  • administered refers to the delivery of a recombinant cell composition of the disclosure into a subject by a method or route that results in at least partial localization of the cell composition at a desired site.
  • a cell composition can be administered by any appropriate route that results in effective treatment in the subject, i.e., administration results in delivery to a desired location in the subject where at least a portion of the composition delivered, i.e. at least 1 ⁇ 10 4 cells are delivered to the desired site for a period of time.
  • Modes of administration include injection, infusion, instillation, or ingestion.
  • injection refers to a method of administering a drug to a subject via a hypodermic syringe or other needle and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebrospinal, and intrasternal injection and infusion.
  • the route is intravenous.
  • administration by injection or infusion can be made. [475]
  • the cells are administered systemically.
  • systemic administration refers to the administration of a population of recombinant cells other than directly into a target site, tissue, or organ, such that it enters, instead, the subject's circulatory system and, thus, is subject to metabolism and other like processes.
  • the efficacy of a treatment comprising a composition for the treatment of a medical condition can be determined by the skilled clinician. However, a treatment is considered “effective treatment,” if any one or all of the signs or symptoms of, as but one example, tumor size is reduced (e.g., reduced by at least 10%), or other clinically accepted symptoms or markers of disease are improved or ameliorated.
  • Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or WSGR Docket No.61078-729.601 slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.
  • administration of a pharmaceutical composition comprises administering a recombinant polypeptide to a subject.
  • administration of a pharmaceutical composition comprises administering a recombinant polypeptide to a subject and administering a population of CAR-T cells to the subject.
  • the recombinant polypeptide and CAR-T cells may be administered at the same time.
  • the recombinant polypeptide may be administered before the CAR-T cells.
  • the recombinant polypeptide may be administered after the CAR-T cells.
  • the recombinant polypeptides, polynucleotides, and/or gene editing molecules of the disclosure may be introduced into a cell using biological methods.
  • biological methods may employ delivery methods such as vectors or synthetic liposomes, non-limiting examples described above; other examples may include non-viral biological agents, such as bacteria, bacteriophage, virus-like particles, erythrocyte ghosts, exosomes among others known in the art.
  • the polynucleotide may be introduced into a cell via any other delivery system.
  • the polynucleotide may be introduced into a cell using a cell transformation method.
  • the polynucleotide may be introduced into a cell using a cell transduction method. In some embodiments, the polynucleotide may be introduced into a cell by any transfection method wherein the transfection leads to uptake of any artificial introduction of foreign cargo e.g., nucleic acid into a cell. In some embodiments, the polynucleotide comprising the sequences encoding the fusion protein may be introduced into a cell using physical methods in which physical energy is applied for intracellular delivery. Such physical methods use application of force to generate transient pores in the cell membrane.
  • Some non-limiting examples of physical methods of delivering the polynucleotide or a cell bearing polynucleotide include, microfluidic electroporation, nanochannel electroporation, nanostraw electroporation, laser-induced photoporation, optical transfection, mechanoporation, ballistic gene delivery, cell squeezing, microinjection, nanofountain probe electroporation, particle bombardment, field-induced membrane disruption, sonoporation, optoporation, magnetoporation, constriction channel based intracellular delivery, thermoporation and any other electroporation-based cell delivery technique or device.
  • the polynucleotide may be introduced into a cell using chemical methods, such as chemical vector-based non-viral cargo delivery which may require modifying cell-penetrating peptides or proteins or endosomal escape to transfect cargo molecules into the cytoplasm directly.
  • chemical transfection methods are techniques that catalyze DNA cross-membrane transport.
  • chemical methods may use Ca2+phosphate, polycations or dendrimers including for example, without limitations, such methods as, use of cationic polymers e.g., diethylaminoethyl- dextran (DEAE-dextran).
  • Chemical methods of cell delivery may apply cell transfection with cationic lipids (non-viral vectors), also known as lipofection or lipid-mediated/liposome transfection, are used in cargo or gene transfection.
  • the recombinant polynucleotide comprising the sequences encoding the fusion protein maybe integrated to the genome of the cell.
  • the polynucleotide integrating into the cell may be single stranded.
  • the polynucleotide integrating may be double stranded DNA. In some embodiments, the polynucleotide may be short nucleotide sequences. In some embodiments, the polynucleotide may be long nucleotide sequences. In some embodiments, the integration into the genome of the cell may be transient integration in the cell. In some embodiments, the integration into the genome of the cell may be stable and integrate into the genome of the recipient cell. In some embodiments, the polynucleotide may integrate into the cell genome within a random locus. In some embodiments, the polynucleotide may integrate into the cell genome within a directed or targeted locus.
  • the polynucleotide may replicate when the cell genome replicates.
  • the cells bearing the polynucleotide comprising the sequences encoding the fusion protein having integrated or transduced or transformed into the cell may be characterized using various methods.
  • the recombinant polynucleotide comprising the sequences encoding the fusion protein may be encoded by a vector as described above.
  • the vector or cell comprising the recombinant polynucleotide is a recombinant vector or cell.
  • the recombinant cell or recombinant vector comprises a selectable biomarker.
  • the selectable marker that is expressed by the recombinant vector or a cell may be used to select and characterized the recombinant polynucleotide.
  • the selectable biomarker in the vector comprising the recombinant polynucleotide comprising the sequences encoding the fusion protein may be a fluorescent biomarker.
  • the selectable biomarker may be an antibiotic cassette.
  • the selectable marker may be a vector or molecule that produces a morphological change, wherein the WSGR Docket No.61078-729.601 morphological change denotes integration of the recombinant polynucleotide or cell or vector bearing the polynucleotide.
  • the selectable biomarker may be any selectable biomarker used in recombinant nucleic acid cloning technology or in the selection of recombinant molecules. Examples, of selectable markers without limitations, include, a transgene, a suicide gene, an activation biomarker, an antibiotic resistance cassette, a morphological change marker or a fluorescent marker.
  • Non-limiting examples of protein genes that may be used to encode fluorescent biomarker proteins include, green fluorescent protein (GFP) gene, enhanced green fluorescent protein (eGFP) gene, mScarlet fluorescent protein gene, red fluorescent protein (RFP) gene, infrared fluorescent protein (iRFP) gene, cyan fluorescent protein (CFP) gene, yellow fluorescent protein (YFP) gene, mCherry/texasRed gene, Cy5.5 fluorescent protein gene and many other fluorescent protein gene in the art.
  • GFP green fluorescent protein
  • eGFP enhanced green fluorescent protein
  • mScarlet fluorescent protein gene mScarlet fluorescent protein gene
  • red fluorescent protein (RFP) gene red fluorescent protein
  • iRFP infrared fluorescent protein
  • CFP cyan fluorescent protein
  • YFP yellow fluorescent protein
  • Cy5.5 fluorescent protein gene Cy5.5 fluorescent protein gene and many other fluorescent protein gene in the art.
  • Non-limiting examples of antibiotic selectable resistance marker genes include, for example, a kanamycin resistance gene, an ampicillin resistance gene, a streptomycin resistance gene, a neomycin resistance gene, a puromycin resistance gene, a gentamycin resistance gene, an erythromycin resistance gene, a Blasticidin S resistance gene, and a hygromycin B resistance gene among many others known in the art.
  • the polynucleotide integrating may be small interfering RNA or miRNA wherein the siRNA or miRNA may be short hairpin transcripts, or the short hairpins may be made from a selectable DNA vector.
  • Recombinant polypeptides and/or recombinant nucleic acids, gene editing molecules, guide RNA polynucleotides (RNA or DNA) and/or endonuclease polynucleotide(s) (RNA or DNA) of the present disclosure can be delivered by viral or non-viral delivery vehicles known in the art.
  • endonuclease polypeptide(s) may be delivered by viral or non-viral delivery vehicles known in the art, such as electroporation or lipid nanoparticles.
  • the DNA endonuclease may be delivered as one or more polypeptides, either alone or pre-complexed with one or more guide RNAs, or one or more crRNA together with a tracrRNA.
  • the recombinant polypeptides, polynucleotides, and/or gene editing molecules of the disclosure may be delivered by any known non-viral delivery vehicle.
  • the non- viral delivery vehicle comprises nanoparticles, liposomes, ribonucleoproteins, positively charged peptides, small molecule RNA-conjugates, aptamer-RNA chimeras, and RNA-fusion protein complexes.
  • non-viral delivery vehicles are described in Peer and Lieberman, Gene Therapy, 18: 1127-1133 (2011) (which focuses on non-viral delivery vehicles for siRNA that are also useful for delivery of other polynucleotides), which is incorporated herein by reference in its entirety.
  • WSGR Docket No.61078-729.601 Lipid Nanoparticles [485]
  • the recombinant polypeptides, polynucleotides, and/or gene editing molecules of the disclosure are delivered to a cell via a non-viral delivery vehicle.
  • the non-viral delivery vehicle comprises a lipid nanoparticle (LNP).
  • lipid nanoparticle or “LNP” are used interchangeably herein to refer to any particle having a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm.
  • a nanoparticle may range in size from 1-1000 nm, 1-500 nm, 1-250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25-60 nm.
  • LNPs may be made from cationic, anionic, or neutral lipids.
  • LNPs may be included in LNPs as ‘helper lipids’ to enhance transfection activity and nanoparticle stability. Limitations of cationic lipids include low efficacy owing to poor stability and rapid clearance, as well as the generation of inflammatory or anti-inflammatory responses. [488] LNPs may also be comprised of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids. [489] Any lipid or combination of lipids that are known in the art may be used to produce a LNP.
  • Exemplary lipids used to produce LNPs are: DOTMA, DOSPA, DOTAP, DMRIE, DC-cholesterol, DOTAP-cholesterol, GAP-DMORIE-DPyPE, and GL67A-DOPE-DMPE-polyethylene glycol (PEG).
  • Examples of cationic lipids are: 98N12-5, C12-200, DLin-KC2-DMA (KC2), DLin-MC3-DMA (MC3), XTC, MD1, and 7C1.
  • Exemplary neutral lipids are: DPSC, DPPC, POPC, DOPE, and SM.
  • Exemplary PEG-modified lipids are: PEG-DMG, PEG-CerC14, and PEG-CerC20.
  • the lipids may be combined in any number of molar ratios to produce an LNP.
  • the polynucleotide(s) may be combined with lipid(s) in a wide range of molar ratios to produce an LNP.
  • the recombinant polypeptides, polynucleotides, and/or gene editing molecules of the disclosure may each be administered separately to a cell or a patient.
  • the site- directed polypeptide such as a CRISPR/cas9 polypetide, may be pre-complexed with one or more guide RNAs, or one or more crRNA together with a tracrRNA.
  • the pre-complexed material may then be administered to a cell or a patient.
  • a ribonucleoprotein particle RNP
  • RNA forms specific interactions with RNA or DNA (i.e., base pairs). While this property is exploited in many biological processes, it also comes with the risk of promiscuous interactions in a nucleic acid-rich cellular environment.
  • RNPs WSGR Docket No.61078-729.601 ribonucleoprotein particles
  • Another benefit of the RNP is protection of the RNA from degradation.
  • the endonuclease in the RNP may be modified or unmodified.
  • the gRNA, crRNA, tracrRNA, or sgRNA may be modified or unmodified. Numerous modifications are known in the art and may be used.
  • the endonuclease and sgRNA may be generally combined in a 1:1 molar ratio.
  • the endonuclease, crRNA and tracrRNA may be generally combined in a 1:1:1 molar ratio.
  • a wide range of molar ratios may be used to produce a RNP.
  • the recombinant polypeptides, polynucleotides, and/or gene editing molecules of the disclosure are delivered by virus-derived expression vectors.
  • the virus- derived expression vector comprises a recombinant adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • rAAV Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions.
  • the AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived, and may be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV- 4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12, AAV-13 and AAV rh.74. Production of pseudotyped rAAV is disclosed in, for example, international patent application publication number WO 01/83692. See Table 1.
  • a method of generating a packaging cell involves creating a cell line that stably expresses all of the necessary components for AAV particle production.
  • a plasmid or multiple plasmids
  • a plasmid comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell.
  • AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6.
  • the packaging cell line is then infected with a helper virus, such as adenovirus.
  • a helper virus such as adenovirus.
  • the advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV.
  • Other examples of suitable methods employ adenovirus or baculovirus, rather than plasmids, to introduce rAAV genomes and/or rep and cap genes into packaging cells.
  • AAV vector serotypes can be matched to target cell types. For example, the following exemplary cell types may be transduced by the indicated AAV serotypes among others. See Table 2.
  • the virus-derived expression vector comprises a vector derived from lentivirus, alphavirus, enterovirus, pestivirus, baculovirus, herpesvirus, Epstein Barr virus, papovavirus, poxvirus, vaccinia virus, and herpes simplex virus.
  • the virus- derived expression vector comprises an expression vector derived from a lentiviral genome encoding any of the constructs described herein.
  • Cas9 mRNA, sgRNA targeting one or two loci in target gene, and donor DNA is each separately formulated into lipid nanoparticles, or are all co-formulated into one lipid nanoparticle, or co-formulated into two or more lipid nanoparticles.
  • Cas9 mRNA is formulated in a lipid nanoparticle, while sgRNA and donor DNA are delivered in an AAV vector.
  • Cas9 mRNA and sgRNA are co- formulated in a lipid nanoparticle, while donor DNA is delivered in an AAV vector.
  • RNA can be expressed from the same DNA, or can also be delivered as an RNA.
  • the RNA can be chemically modified to alter or improve its half-life, or decrease the likelihood or degree of immune response.
  • the endonuclease protein can be complexed with the gRNA prior to delivery.
  • Viral vectors allow efficient delivery; split versions of Cas9 and smaller orthologs of Cas9 can be packaged in AAV, as can donors for HDR.
  • a range of non-viral delivery methods also exist that can deliver each of these components, or non-viral and viral methods can be employed in tandem.
  • nano- particles can be used to deliver the protein and guide RNA, while AAV can be used to deliver a donor DNA.
  • WSGR Docket No.61078-729.601 Exosomes [503] The recombinant polypeptides, polynucleotides, and/or gene editing molecules of the disclosure, may be delivered via exosomes. Exosomes, a type of microvesicle bound by phospholipid bilayer, can be used to deliver nucleic acids to specific tissue. Many different types of cells within the body naturally secrete exosomes. Exosomes form within the cytoplasm when endosomes invaginate and form multivesicular-endosomes (MVE).
  • MVE multivesicular-endosomes
  • exosomes When the MVE fuses with the cellular membrane, the exosomes are secreted in the extracellular space. Ranging between 30-120 nm in diameter, exosomes can shuttle various molecules from one cell to another in a form of cell-to-cell communication. Cells that naturally produce exosomes, such as mast cells, can be genetically altered to produce exosomes with surface proteins that target specific tissues, alternatively exosomes can be isolated from the bloodstream. Specific nucleic acids can be placed within the engineered exosomes with electroporation. When introduced systemically, the exosomes can deliver the nucleic acids to the specific target tissue.
  • the recombinant polypeptides, polynucleotides, and/or gene editing molecules of the disclosure may be introduced into a cell using biological methods.
  • biological methods may employ delivery methods such as vectors or synthetic liposomes, non-limiting examples described above; other examples may include non-viral biological agents, such as bacteria, bacteriophage, virus-like particles, erythrocyte ghosts, exosomes among others known in the art.
  • the polynucleotide may be introduced into a cell via any other delivery system.
  • the polynucleotide may be introduced into a cell using a cell transformation method.
  • the polynucleotide may be introduced into a cell using a cell transduction method. In some embodiments, the polynucleotide may be introduced into a cell by any transfection method wherein the transfection leads to uptake of any artificial introduction of foreign cargo e.g., nucleic acid into a cell. In some embodiments, the polynucleotide comprising the sequences encoding the fusion protein may be introduced into a cell using physical methods in which physical energy is applied for intracellular delivery. Such physical methods use application of force to generate transient pores in the cell membrane.
  • Some non-limiting examples of physical methods of delivering the polynucleotide or a cell bearing polynucleotide include, microfluidic electroporation, nanochannel electroporation, nanostraw electroporation, laser-induced photoporation, optical transfection, mechanoporation, ballistic gene delivery, cell squeezing, microinjection, nanofountain probe electroporation, particle bombardment, field-induced membrane disruption, sonoporation, optoporation, magnetoporation, constriction channel based intracellular delivery, thermoporation and any other electroporation-based cell delivery technique or device.
  • Various physical delivery methods WSGR Docket No.61078-729.601 have long demonstrated the ability to deliver cargo molecules directly to the cell intracellular environment such as for example, the cytoplasm or nucleus of the cell.
  • the methods of delivery may depend on if the introduction may be for a single-cell intracellular delivery or not. art.
  • the polynucleotide may be introduced into a cell using chemical methods, such as chemical vector-based non-viral cargo delivery which may require modifying cell-penetrating peptides or proteins or endosomal escape to transfect cargo molecules into the cytoplasm directly.
  • the chemical transfection methods are techniques that catalyze DNA cross-membrane transport.
  • chemical methods may use Ca2+phosphate, polycations or dendrimers including for example, without limitations, such methods as, use of cationic polymers e.g., diethylaminoethyl- dextran (DEAE-dextran).
  • Chemical methods of cell delivery may apply cell transfection with cationic lipids (non-viral vectors), also known as lipofection or lipid-mediated/liposome transfection are used in cargo or gene transfection.
  • the recombinant polynucleotide comprising the sequences encoding the fusion protein maybe integrated to the genome of the cell.
  • the polynucleotide integrating into the cell may be single stranded.
  • the polynucleotide integrating may be double stranded DNA. In some embodiments, the polynucleotide may be short nucleotide sequences. In some embodiments, the polynucleotide may be long nucleotide sequences. In some embodiments, the integration into the genome of the cell may be transient integration in the cell. In some embodiments, the integration into the genome of the cell may be stable and integrate into the genome of the recipient cell. In some embodiments, the polynucleotide may integrate into the cell genome within a random locus. In some embodiments, the polynucleotide may integrate into the cell genome within a directed or targeted locus.
  • the polynucleotide may replicate when the cell genome replicates.
  • the cells bearing the polynucleotide comprising the sequences encoding the fusion protein having integrated or transduced or transformed into the cell may be characterized using various methods.
  • the recombinant polynucleotide comprising the sequences encoding the fusion protein may be encoded by a vector as described above.
  • the vector or cell comprising the recombinant polynucleotide is a recombinant vector or cell.
  • the recombinant cell or recombinant vector comprises a selectable biomarker.
  • the selectable marker that is expressed by the recombinant vector or a cell may be used to select and characterized the recombinant polynucleotide.
  • the selectable biomarker in the vector comprising the recombinant polynucleotide comprising the sequences encoding the fusion protein may be a fluorescent biomarker.
  • the selectable biomarker may be an antibiotic cassette.
  • the selectable marker may be a vector or molecule that produces a morphological change, wherein the morphological change denotes integration of the recombinant polynucleotide or cell or vector bearing the polynucleotide.
  • the selectable biomarker may be any selectable biomarker used in recombinant nucleic acid cloning technology or in the selection of recombinant molecules. Examples, of selectable markers without limitations, include, a transgene, a suicide gene, an activation biomarker, an antibiotic resistance cassette, a morphological change marker or a fluorescent marker.
  • Non-limiting examples of protein genes that may be used to encode fluorescent biomarker proteins include, green fluorescent protein (GFP) gene, enhanced green fluorescent protein (eGFP) gene, mScarlet fluorescent protein gene, red fluorescent protein (RFP) gene, infrared fluorescent protein (iRFP) gene, cyan fluorescent protein (CFP) gene, yellow fluorescent protein (YFP) gene, mCherry/texasRed gene, Cy5.5 fluorescent protein gene and many other fluorescent protein gene in the art.
  • Non-limiting examples of antibiotic selectable resistance marker gene include, kanamycin gene, ampicillin gene, streptomycin gene, neomycin gene, puromycin gene gentamycin gene, erythromycin gene, Blasticidin S gene, hygromycin B gene among many others known in the art.
  • kits [509] Also provided by the present disclosure are kits. In certain embodiments, provided are kits that include any of the recombinant nucleic acids, recombinant polypeptides, and/or expression vectors of the present disclosure, and instructions for introducing the recombinant nucleic acid, recombinant polypeptides, and/or expression vector into a cell.
  • the expression vector when the expression vector encodes a recombinant polypeptide that does not comprise the protease (trans configuration), the expression vector further encodes the protease.
  • the expression vector is configured to express the recombinant polypeptide and the protease from the same promoter.
  • the expression vector may be a bicistronic expression vector for expression of separate recombinant polypeptides and protease molecules under the same promoter in the cell.
  • kits of the present disclosure may further include any other reagents useful for regulatable signaling of the cell surface receptor, such as transfection/transduction reagents useful for introducing the nucleic acid or expression vector into cells of interest, e.g., immune cells (e.g., T cells) or other cells of interest.
  • transfection/transduction reagents useful for introducing the nucleic acid or expression vector into cells of interest, e.g., immune cells (e.g., T cells) or other cells of interest.
  • WSGR Docket No.61078-729.601 Components of the kits may be present in separate containers, or multiple components may be present in a single container.
  • a suitable container includes a single tube (e.g., vial), one or more wells of a plate (e.g., a 96-well plate, a 384-well plate, etc.), or the like.
  • the instructions of the kits may be recorded on a suitable recording medium.
  • the instructions may be printed on a substrate, such as paper or plastic, etc.
  • the instructions may be present in the kits as a package insert, in the labeling of the container of the kit or components thereof (i.e., associated with the packaging or sub packaging), etc.
  • the instructions are present as an electronic storage data file present on a suitable computer readable storage medium, e.g., portable flash drive, DVD, CD-ROM, diskette, etc.
  • the actual instructions are not present in the kit, but means for obtaining the instructions from a remote source, e.g., via the internet, are provided.
  • kits that includes a web address where the instructions can be viewed and/or from which the instructions can be downloaded. As with the instructions, the means for obtaining the instructions is recorded on a suitable substrate.
  • a composition comprising a recombinant polynucleic acid comprising a sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises (1) a transmembrane domain, wherein the transmembrane domain is from TCR alpha, TCR beta, TCR gamma, TCR delta, CD3 delta, CD3 gamma or CD3 epsilon; and (2) an intracellular domain comprising a functional ubiquitin ligase domain from a ubiquitin ligase; wherein the fusion polypeptide ubiquitinates an endogenous TCR subunit of a cell when expressed in the cell.
  • Embodiment 2 Embodiment 2.
  • the ubiquitin ligase is a RING (really interesting new gene) finger protein (RNF) family ubiquitin ligase, a U-box E3 ligase family ubiquitin ligase, a membrane associated ring-CH-type finger (MARCH) family ubiquitin ligase, an anergy in lymphocytes (GRAIL) E3 ubiquitin ligase or a carboxy-terminus of Hsc70 interacting protein (CHIP) ubiquitin ligase.
  • RNF RING
  • MARCH membrane associated ring-CH-type finger
  • GRAIL anergy in lymphocytes
  • CHIP carboxy-terminus of Hsc70 interacting protein
  • composition of embodiment 2, wherein the ubiquitin ligase is selected from the group consisting of RNF122, RNF133, RNF152, MARCH1, MARCH2, MARCH3, MARCH4, MARCH6, MARCH8, MARCH9, RNF130, RNF148, RNF149 and RNF150.
  • ubiquitin ligase is selected from the group consisting of RNF122, RNF133, RNF152, MARCH1, MARCH2, MARCH3, MARCH4, MARCH6, MARCH8, MARCH9, RNF130, RNF148, RNF149 and RNF150.
  • a composition comprising a recombinant polynucleic acid comprising a sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises (1) a transmembrane domain, wherein the transmembrane domain is from TCR alpha, TCR beta, TCR gamma, TCR delta, CD3 delta, CD3 gamma, CD3 epsilon or CD3 zeta; and (2) an intracellular domain comprising a functional ubiquitin ligase domain from a ubiquitin ligase, wherein the ubiquitin ligase does not comprise a gene related to anergy in lymphocytes (GRAIL) E3 ubiquitin ligase, a carboxy-terminus of Hsc70 interacting protein (CHIP) ubiquitin ligase, a RING (really interesting new gene) finger protein (RNF) 122, RNF133, or RNF152; and wherein the fusion polypeptide ubiquitinate
  • Embodiment 5 The composition of embodiment 1 or 4, wherein the ubiquitin ligase is an E3 ubiquitin ligase.
  • Embodiment 6. The composition of embodiment 4 or 5, wherein the ubiquitin ligase is a membrane associated ring-CH-type finger (MARCH) family ubiquitin ligase.
  • Embodiment 7. The composition of embodiment 6, wherein the ubiquitin ligase is selected from the group consisting of MARCH1, MARCH2, MARCH3, MARCH4, MARCH6, MARCH8 and MARCH9.
  • Embodiment 8 The composition of embodiment 4, wherein the ubiquitin ligase is selected from the group consisting of RNF130, RNF148, RNF149 and RNF150.
  • Embodiment 9 The composition of any one of embodiments 4-8, wherein the transmembrane domain is not a CD3 zeta transmembrane domain.
  • Embodiment 10. The composition of any one of embodiments 4-8, wherein the transmembrane domain is a CD3 zeta transmembrane domain.
  • Embodiment 11. The composition of any one of embodiments 1-9, wherein the transmembrane domain is a CD3 delta transmembrane domain.
  • Embodiment 12 The composition of any one of embodiments 1-9, wherein the transmembrane domain is a CD3 gamma transmembrane domain.
  • Embodiment 14 The composition of any one of embodiments 1-13, wherein the intracellular domain further comprises a TCR intracellular domain.
  • WSGR Docket No.61078-729.601 Embodiment 15. The composition of embodiment 14, wherein the TCR intracellular domain is between the transmembrane domain and the ubiquitin ligase.
  • Embodiment 16 The composition of embodiment 14 or 15, wherein the TCR intracellular domain is not a CD3 zeta intracellular domain.
  • Embodiment 17. The composition of embodiment 14 or 15, wherein the TCR intracellular domain is a CD3 zeta intracellular domain.
  • Embodiment 18 The composition of any one of embodiments 14-16, wherein the TCR intracellular domain comprises a CD3 delta intracellular domain, a CD3 gamma intracellular domain or a CD3 epsilon intracellular domain.
  • Embodiment 19 The composition of any one of embodiments 4-8, 14 or 15, wherein (a) the transmembrane domain is a CD3 zeta transmembrane domain; and (b) the TCR intracellular domain is a CD3 zeta intracellular domain.
  • Embodiment 20 The composition of any one of embodiments 14-16, wherein the TCR intracellular domain comprises a CD3 delta intracellular domain, a CD3 gamma intracellular domain or a CD3 epsilon intracellular domain.
  • the composition of any one of embodiments 1-22, wherein the fusion protein comprises a TCR extracellular domain.
  • composition of embodiment 24, wherein the TCR extracellular domain is from TCR alpha, TCR beta, TCR gamma, TCR delta, CD3 delta, CD3 gamma or CD3 epsilon or CD3 zeta.
  • Embodiment 26. The composition of any one of embodiments 1-25, wherein the fusion protein comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 44-59 and 61.
  • a composition comprising a recombinant polynucleic acid comprising a sequence encoding a mutant CD3 zeta polypeptide, wherein the mutant CD3 zeta polypeptide comprises a mutant CD3 zeta transmembrane domain, wherein the mutant CD3 zeta transmembrane WSGR Docket No.61078-729.601 domain comprises a sequence according to the formula LCYLLXGILFIYGVILTALFL (SEQ ID NO: 999), wherein X is an amino acid selected from the group consisting of R, K and S. Embodiment 28.
  • a composition comprising a recombinant polynucleic acid comprising a sequence encoding a mutant CD3 zeta polypeptide, wherein the mutant CD3 zeta polypeptide comprises a mutant CD3 zeta transmembrane domain, wherein the mutant CD3 zeta transmembrane domain comprises a sequence according to the formula LCYLLXGILFIYGVILTALFL (SEQ ID NO: 999), wherein X is an amino acid selected from the group consisting of E, N, A R, K and S; and wherein the mutant CD3 zeta polypeptide comprises a CD3 zeta extracellular domain that is at least 4 amino acids in length.
  • Embodiment 29 Embodiment 29.
  • a composition comprising a cell, wherein the cell: (a) expresses endogenous CD3 zeta, and (b) comprises a recombinant polynucleic acid comprising a sequence encoding a mutant CD3 zeta polypeptide, wherein the mutant CD3 zeta polypeptide is expressed in the cell and forms an exogenous CD3 zeta dimer with the endogenous CD3 zeta.
  • Embodiment 30 Embodiment 30.
  • a composition comprising a cell, wherein the cell (a) expresses endogenous CD3 zeta, and (b) comprises a recombinant polynucleic acid comprising a sequence encoding a mutant CD3 zeta polypeptide, wherein the mutant CD3 zeta polypeptide is expressed in the cell and forms an exogenous CD3 zeta dimer with the endogenous CD3 zeta; and wherein formation of an exogenous TCR complex comprising: the exogenous CD3 zeta dimer, endogenous TCR alpha, endogenous TCR beta, endogenous CD3 gamma, endogenous CD3 delta and endogenous CD3 epsilon is inhibited or lower compared to formation of a TCR complex comprising: an endogenous CD3 zeta dimer, the endogenous TCR alpha, the endogenous TCR beta, the endogenous CD3 gamma, the endogenous CD3 delta and the endogenous
  • Embodiment 31 A composition comprising a cell, wherein the cell (a) expresses endogenous CD3 zeta, and (b) comprises a recombinant polynucleic acid comprising a sequence encoding a mutant CD3 zeta polypeptide, wherein the mutant CD3 zeta polypeptide is expressed in the cell and forms an exogenous CD3 zeta dimer with the endogenous CD3 zeta; and wherein a KD of the exogenous CD3 zeta dimer for an endogenous TCR complex is higher than a KD of an endogenous CD3 zeta dimer to the endogenous TCR complex.
  • Embodiment 32 A composition comprising a cell, wherein the cell (a) expresses endogenous CD3 zeta, and (b) comprises a recombinant polynucleic acid comprising a sequence encoding a mutant CD3 zeta polypeptide, wherein the mutant CD3
  • a composition comprising a cell, wherein the cell: WSGR Docket No.61078-729.601 (a) expresses endogenous CD3 zeta, and (b) comprises a recombinant polynucleic acid comprising a sequence encoding a mutant CD3 zeta polypeptide, wherein the mutant CD3 zeta polypeptide is expressed in the cell and forms an exogenous CD3 zeta dimer with the endogenous CD3 zeta; and wherein a KD of the exogenous CD3 zeta dimer for endogenous TCR alpha is higher than a KD of an endogenous CD3 zeta dimer to the endogenous TCR alpha.
  • Embodiment 33 Embodiment 33.
  • a composition comprising a cell, wherein the cell: (a) expresses endogenous CD3 zeta, and (b) comprises a recombinant polynucleic acid comprising a sequence encoding a mutant CD3 zeta polypeptide, wherein the mutant CD3 zeta polypeptide is expressed in the cell and forms an exogenous CD3 zeta dimer with the endogenous CD3 zeta; and wherein the cell exhibits a reduced amount of endogenous TCR complexes relative to a cell that does not express the mutant CD3 zeta polypeptide.
  • Embodiment 34 Embodiment 34.
  • a composition comprising a cell, wherein the cell: (a) expresses endogenous CD3 zeta, and (b) comprises a recombinant polynucleic acid comprising a sequence encoding a mutant CD3 zeta polypeptide, wherein the mutant CD3 zeta polypeptide is expressed in the cell and forms an exogenous CD3 zeta dimer with the endogenous CD3 zeta; and wherein the exogenous CD3 zeta dimer does not substantially interact with an endogenous TCR complex.
  • composition of any one of embodiments 31 or 33, wherein the endogenous TCR complex comprises endogenous TCR alpha, endogenous TCR beta, endogenous CD3 gamma, endogenous CD3 delta and endogenous CD3 epsilon.
  • Embodiment 36 The composition of any one of embodiments 27-35, wherein the mutant CD3 zeta polypeptide lacks a CD3 zeta signaling domain.
  • Embodiment 37. The composition of any one of embodiments 29-36, wherein the mutant CD3 zeta polypeptide comprises a mutant CD3 zeta transmembrane domain.
  • composition of embodiment 37 wherein the mutant CD3 zeta transmembrane domain comprises a sequence according to the formula LCYLLXGILFIYGVILTALFL (SEQ ID NO: 999), wherein X is an amino acid selected from the group consisting of E, N, A, R, K and S. WSGR Docket No.61078-729.601 Embodiment 39.
  • the composition of any one of embodiments 27-38, wherein the mutant CD3 zeta polypeptide comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 19-22, 39-41, 62 and 64.
  • Embodiment 40 Embodiment 40.
  • composition of any one of embodiments 27 and 29-39, wherein the mutant CD3 zeta polypeptide comprises an extracellular domain.
  • Embodiment 41 The composition of embodiment 40, wherein the extracellular domain is a CD3 zeta extracellular domain.
  • Embodiment 42 The composition of embodiment 40 or 41, wherein the extracellular domain is a full length CD3 zeta extracellular domain.
  • Embodiment 43 The composition of any one of embodiments 40-42, wherein the extracellular domain comprises at least 4 amino acids.
  • the composition of embodiment 28 or 43, wherein the extracellular domain comprises about 9 or 10 amino acids.
  • Embodiment 45 The composition of any one of embodiments 29-44, wherein the cell is a mammalian or human cell.
  • Embodiment 46 A composition comprising a recombinant polynucleic acid comprising a sequence encoding a mutant CD3 epsilon polypeptide, wherein the mutant CD3 epsilon polypeptide comprises a mutant CD3 epsilon transmembrane domain, wherein the mutant CD3 epsilon transmembrane domain comprises a sequence according to the formula MSVATIVIVXICITGGLLLLVYYWS (SEQ ID NO 1000), wherein X is an amino acid selected from the group consisting of R and K.
  • Embodiment 47 Embodiment 47.
  • a composition comprising a recombinant polynucleic acid comprising a sequence encoding a mutant CD3 epsilon polypeptide, wherein the mutant CD3 epsilon polypeptide comprises (a) a CD3 gamma transmembrane domain or a CD3 delta transmembrane domain; and (b) a CD3 epsilon intracellular domain.
  • Embodiment 48 The composition of embodiment 47, wherein the mutant CD3 epsilon polypeptide comprises a CD3 gamma extracellular domain or a CD3 gamma extracellular domain.
  • composition of embodiment 48 wherein the mutant CD3 epsilon polypeptide comprises (a) a CD3 gamma extracellular domain, a CD3 gamma transmembrane domain and a CD3 epsilon intracellular domain, or (b) a CD3 delta extracellular domain, a CD3 delta transmembrane domain and a CD3 epsilon intracellular domain.
  • Embodiment 50 The composition of any one of embodiments 47-49, wherein the mutant CD3 epsilon polypeptide comprises a mutant CD3 gamma transmembrane domain or a mutant CD3 gamma transmembrane domain.
  • WSGR Docket No.61078-729.601 Embodiment 51 Embodiment 51.
  • composition of any one of embodiments 46-50, wherein the mutant CD3 epsilon polypeptide comprises a truncated CD3 epsilon intracellular domain Embodiment 52.
  • a composition comprising a recombinant polynucleic acid comprising a sequence encoding a mutant CD3 epsilon polypeptide, wherein the mutant CD3 epsilon polypeptide comprises a mutant CD3 epsilon transmembrane domain; and wherein the mutant CD3 epsilon polypeptide comprises a truncated CD3 epsilon intracellular domain.
  • Embodiment 53 Embodiment 53.
  • composition of embodiment 51 or 52, wherein the truncated CD3 epsilon intracellular domain lacks an ER retention signal.
  • Embodiment 54. The composition of any one of embodiments 48-53, wherein the mutant CD3 epsilon transmembrane domain comprises a sequence according to the formula MSVATIVIVXICITGGLLLLVYYWS (SEQ ID NO: 1000), wherein X is an amino acid selected from the group consisting of A, N, R and K.
  • Embodiment 55 A composition comprising a cell, wherein the cell comprises the recombinant polynucleic acid of the composition of any one of embodiments 46-54.
  • composition of embodiment 55 wherein the cell expresses endogenous CD3 gamma and/or endogenous CD3 delta.
  • Embodiment 57 The composition of embodiment 56, wherein the mutant CD3 epsilon polypeptide is expressed in the cell and forms an exogenous CD3 epsilon-CD3 gamma dimer with the endogenous CD3 gamma and/or forms an exogenous CD3 epsilon-CD3 delta dimer with the endogenous CD3 delta.
  • Embodiment 58 Embodiment 58.
  • composition of embodiment 57 wherein the K D of the exogenous CD3 epsilon-CD3 gamma dimer for an endogenous TCR complex is higher than the KD of an endogenous CD3 epsilon-CD3 gamma dimer to the endogenous TCR complex; and/or wherein the K D of the exogenous CD3 epsilon-CD3 delta dimer for an endogenous TCR complex is higher than the K D of an endogenous CD3 epsilon-CD3 delta dimer to the endogenous TCR complex.
  • Embodiment 59 Embodiment 59.
  • Embodiment 60 The composition of embodiment 57 or 58, wherein the K D of the exogenous CD3 epsilon-CD3 gamma dimer for an endogenous TCR beta is higher than the K D of an endogenous CD3 epsilon-CD3 gamma dimer to the endogenous TCR beta.
  • composition of embodiment 59 wherein formation of an exogenous TCR complex comprising the exogenous CD3 epsilon-CD3 gamma dimer and endogenous TCR alpha, endogenous TCR beta, and (i) an endogenous CD3 delta-CD3 epsilon dimer or (ii) the exogenous CD3 epsilon-CD3 delta dimer is inhibited or lower compared to formation of a TCR complex comprising an endogenous CD3 epsilon-CD3 gamma dimer, endogenous TCR alpha, endogenous WSGR Docket No.61078-729.601 TCR beta, and (i) an endogenous CD3 delta-CD3 epsilon or (ii) the exogenous CD3 epsilon-CD3 delta dimer.
  • Embodiment 61 The composition of embodiment 59 or 60, wherein the exogenous CD3 epsilon-CD3 gamma dimer does not substantially interact with an endogenous TCR complex and/or does not substantially interact with TCR beta.
  • Embodiment 62 The composition of any one of embodiments 57-61, wherein the KD of the exogenous CD3 epsilon-CD3 delta dimer for an endogenous TCR alpha is higher than the K D of an endogenous CD3 epsilon-CD3 delta dimer to the endogenous TCR alpha.
  • Embodiment 63 Embodiment 63.
  • composition of embodiment 62 wherein formation of an exogenous TCR complex comprising the exogenous CD3 epsilon-CD3 delta dimer and endogenous TCR alpha, endogenous TCR beta, and (i) an endogenous CD3 gamma-CD3 epsilon dimer or (ii) the exogenous CD3 epsilon-CD3 gamma dimer is inhibited or lower compared to formation of a TCR complex comprising an endogenous CD3 epsilon-CD3 delta dimer, endogenous TCR alpha, endogenous TCR beta, and (i) an endogenous CD3 gamma-CD3 epsilon dimer or (ii) the exogenous CD3 epsilon-CD3 gamma dimer.
  • Embodiment 64 The composition of embodiment 62 or 63, wherein the exogenous CD3 epsilon-CD3 delta dimer does not substantially interact with an endogenous TCR complex and/or does not substantially interact with TCR alpha.
  • Embodiment 65 The composition of any one of embodiments 57-64, wherein the cell exhibits a reduced amount of functional endogenous TCR complexes relative to a cell that does not express the mutant CD3 epsilon polypeptide.
  • Embodiment 66 The composition of any one of embodiments 46-65, wherein the mutant CD3 epsilon polypeptide lacks a CD3 epsilon signaling domain cell.
  • Embodiment 67 The composition of embodiment 62 or 63, wherein the exogenous CD3 epsilon-CD3 delta dimer does not substantially interact with an endogenous TCR complex and/or does not substantially interact with TCR alpha.
  • Embodiment 65 The composition of any one of embodiments
  • composition of any one of embodiments 46-66, wherein the mutant CD3 epsilon polypeptide comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 23, 24, 35-37, 60 and 61.
  • Embodiment 68. The composition of any one of embodiments 46-67, wherein the mutant CD3 epsilon polypeptide comprises an extracellular domain.
  • Embodiment 69. The composition of embodiment 68, wherein the extracellular domain is a CD3 epsilon extracellular domain.
  • Embodiment 70 The composition of embodiment 68 or 69, wherein the extracellular domain is a full length CD3 epsilon extracellular domain.
  • a composition comprising a recombinant polynucleic acid comprising at least two of the following: (a) a sequence encoding the fusion polypeptide encoded by the recombinant polynucleic acid of the composition of any one of embodiments 1-26; (b) sequence encoding the mutant CD3 zeta polypeptide encoded by the recombinant polynucleic acid of the composition of any one of embodiments 27-45; and (c) a sequence encoding the mutant CD3 epsilon polypeptide encoded by the recombinant polynucleic acid of the composition of any one of embodiments 46-71.
  • Embodiment 73 The composition of any one of embodiments 1-72, wherein the composition further comprises a small non-coding ribonucleic acid sequence (RNA) or a sequence encoding the small non-coding RNA.
  • Embodiment 74 The composition of embodiment 73, wherein the small non-coding RNA is configured to reduce an expression of a subunit of an endogenous T-cell receptor (TCR) complex in a cell relative to a cell that does not comprise the small non-coding RNA.
  • TCR T-cell receptor
  • composition of embodiment 73 or 74, wherein the small non-coding RNA comprises a micro ribonucleic acid (miRNA), a short hairpin RNA, a silencing ribonucleic acid (siRNA), a self-amplifying RNA, or a combination thereof.
  • miRNA micro ribonucleic acid
  • siRNA silencing ribonucleic acid
  • the composition of embodiment 73 or 74, wherein the small non-coding RNA targets a TCR alpha RNA.
  • the composition of embodiment 73 or 74, wherein the small non-coding RNA targets a CD3 epsilon RNA Embodiment 78.
  • composition of embodiment 73 or 74, wherein the small non-coding RNA comprises a sequence with at least 80% sequence identity any one of SEQ ID NOs: 71-76.
  • Embodiment 79. The composition of any one of embodiments 1-78, wherein the composition further comprises a sequence encoding a chimeric antigen receptor (CAR).
  • Embodiment 80. The composition of embodiment 79, wherein the CAR comprises (a) an extracellular domain comprising an antigen binding domain; (b) a transmembrane domain; and (c) an intracellular domain comprising an intracellular signaling domain.
  • the composition of embodiment 80, wherein the antigen binding domain is an anti-CD19 binding domain.
  • VL variable light chain domain
  • LCDR1 light chain CDR1
  • VH variable heavy chain domain
  • Embodiment 83 Embodiment 83.
  • composition of embodiment 82, wherein the antigen binding domain comprises an scFv with at least about 80% sequence identity to any one of SEQ ID NOs: 65 and 66.
  • Embodiment 84. The composition of embodiment 80, wherein the antigen binding domain is an anti-CD22 binding domain.
  • composition of embodiment 84 wherein the antigen binding domain is an scFv comprising a variable light chain domain (VL) having a light chain CDR1 (LCDR1), LCDR2 and LCDR3 of QTIWSY, AAS and QQSYSIPQT, respectively; and a heavy chain CDR1 (HCDR1), HCDR2 and HCDR3 of GDSVSSNSAA, TYYRSKWYN and AREVTGDLEDAFDI, respectively.
  • VL variable light chain domain
  • HCDR1 heavy chain CDR1
  • HCDR2 and HCDR3 of GDSVSSNSAA
  • TYYRSKWYN TYYRSKWYN
  • AREVTGDLEDAFDI AREVTGDLEDAFDI
  • composition of embodiment 80 wherein the antigen binding domain binds to an antigen that is selected from the group consisting of: glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut HSP70-2, M-CSF, prostate- specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, HER2, survivin and telomerase, prostate-carcinoma tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor, GD2, GD3, B
  • Embodiment 88 The composition of any one of embodiments 80-87, wherein the intracellular domain of the CAR comprises an intracellular signaling domain from CD2.
  • WSGR Docket No.61078-729.601 Embodiment 89.
  • CD137 4-1BB
  • Embodiment 90 The composition of any one of embodiments 80-89, wherein the transmembrane domain of the CAR comprises a transmembrane domain from CD8 or CD28.
  • Embodiment 91 The composition of any one of embodiments 80-90, wherein the extracellular domain of the CAR comprises a hinge domain from CD8 or CD28.
  • Embodiment 92 The composition of any one of embodiments 80-91, wherein the CAR comprises a sequence with at least 80% sequence identity to any one of SEQ ID NOs: 68-70.
  • Embodiment 93 The composition of any one of embodiments 1-92, wherein the cell is a lymphocyte.
  • Embodiment 94 The composition of embodiment 93, wherein the cell is a T cell.
  • Embodiment 95 The composition of any one of embodiments 1-94, wherein the cell is a population of cells.
  • Embodiment 96. The composition of embodiment 95, wherein the population of cells comprises at least 1x10 ⁇ 5 cells.
  • Embodiment 97. A pharmaceutical composition comprising the composition of any one of embodiments 1-96, and a pharmaceutically acceptable excipient or carrier.
  • Embodiment 98. A method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of the pharmaceutical composition of embodiment 97.
  • Embodiment 99. The method of embodiment 98, wherein the cancer is lymphoma or leukemia.
  • the cancer is lung cancer, liver cancer, pancreatic cancer, stomach cancer, colon cancer, kidney cancer, brain cancer, head and neck cancer, breast cancer, skin cancer, rectal cancer, uterine cancer, cervical cancer, ovarian cancer, testicular cancer, skin cancer, esophageal cancer, and/or the cancer includes a sarcoma cell, a rhabdoid cancer cell, a neuroblastoma cell, retinoblastoma cell, or a medulloblastoma cell, and/or the cancer is uterine carcinosarcoma (UCS), brain lower grade glioma (LGG), thymoma (THYM), WSGR Docket No.61078-729.601 testicular germ cell tumors (TGCT), glioblastoma multiforme (GBM) and skin cutaneous melanoma (SKCM), liver hepatocellular carcinoma (LIHC), uveal melanoma (UVM
  • UCS uterine car
  • FIG. 1 depicts an exemplary fusion polypeptide comprising a ubiquitin ligase domain (a functional ubiquitin ligase domain).
  • the transmembrane domain of the fusion polypeptide contains a subunits (i.e any endogenous TCR complex subunits or endogenous TCR complex (for example, the functional TCR complex), facilitating their degradation.
  • the fusion polypeptide can contain the extracellular WSGR Docket No.61078-729.601 contain motifs/residues for post-translational modifications, such as glycosylation, that can promote the stability, expression level, or localization of the fusion polypeptide.
  • the inclusion of the extracellular domain may also increase the ability of the fusion polypeptide to interaction with other TCR complex subunits.
  • the inclusion of the additional intracellular domain, such as a sequence of intracellular domain may also contain a linker region to facilitate the folding of the ubiquitin ligase [518] FIG.
  • FIG. 2 shows that cells expressing a fusion polypeptide (CD3z_hTM_LG171; SEQ ID NO: had significantly lower amounts of TCR complexes on the plasma membrane, similar to the positive control (cells with a deletion of TRAC; labeled as “TRAC KO”), relative to the negative controls (NTD are un-transduced, unedited control cells; BFP are cells that expressed BFP only; Mock; cells that undergone electroporation but sgRNA was omitted and no editing occurs).
  • a fusion polypeptide CD3z_hTM_LG171; SEQ ID NO: had significantly lower amounts of TCR complexes on the plasma membrane, similar to the positive control (cells with a deletion of TRAC; labeled as “TRAC KO”), relative to the negative controls (NTD are un-transduced, unedited control cells; BFP are cells that expressed BFP only; Mock; cells that undergone electroporation but sgRNA was omitted and no editing occurs).
  • FIG. 3 shows that various fusion polypeptide constructs labeled on the x-axis (corresponding to SEQ ID NO: 44; 45; 47; 48; 49; 50; 51; 52; 53; 54; 55; 56; 57; 58; 59) with different combinations of CD3 subunits and ubiquitin ligase domains.
  • fusion polypeptides e.g., SEQ ID NOs: 46 or 61
  • Methods described herein can be used to test the effect of other fusion polypeptides (e.g., SEQ ID NOs: 46 or 61) on surface functional TCR expression.
  • Other variations of the fusion polypeptides are described herein and at least in Harris et al., ACS Synth Biol.2022 Jun 17;11(6):2029-2035, which is herein incorporated by reference in its entirety.
  • a fusion polypeptide with a TCR’s transmembrane domain does not comprise a ubiquitin ligase domain but a protein interacting domain that can interact and recruit a ubiquitin ligase.
  • the ubiquitin ligase recruited can then ubiquitinate an endogenous TCR or complex, thereby facilitating the degradation of the TCR or complex.
  • WSGR Docket No.61078-729.601 Example 2 – Strategies for reducing endogenous TCR expression or functional TCR complex formation using mutant polypeptides [521] Provided herein are strategies for reducing endogenous TCR expression of TCR complex formation using mutant polypeptides. [522] FIG.
  • FIG.5A shows that cells expressing various mutant and polypeptide polypeptide constructs labeled on the x-axis (corresponding to SEQ ID NOs: 44 (control); 35; 36; 37; 39; 40; 41 ) exhibited various levels of downregulations of the surface functional TCR complex expression.
  • Cells with TRAC knocked out (TRAC KO) or expressing a fusion polypeptide were used as positive controls.
  • FIG.5B shows that cells expressing mutant and polypeptides had significantly lower amounts of TCR complexes on the plasma membrane, similar to the positive control (cells with a deletion of TRAC gene; labeled as “TRAC KO”), relative to control, unmodified T cells.
  • Expression of a mutant complex expression by ⁇ 40% SEQ ID NO: 35.
  • Expression of a mutant mutation of transmembrane aspartic acid (and without its cytoplasmic domain) decreased the surface functional TCR complex expression even more to ⁇ 85% (SEQ ID NO: 36).
  • FIG.6A shows that control T cells with surface TCR/CD3 complex and T cells lacking surface TCR/CD3 complex due to TRAC KO or mutant expression exhibited similar (null) levels of activation markers CD25, CD69, and CD137 at baseline.
  • FIG. 6B shows that control T cells upregulated activation markers CD25, CD69, and CD137 following 72h activation with anti-CD3/anti- CD28 antibodies. By contrast, T cells lacking surface TCR/CD3 complex due to TRAC KO or mutant expression did not upregulate these activation markers.
  • FIG. 7 shows that control T cells with surface TCR/CD3 complex killed mock host T cells over the course of 9 days co-incubated in a mixed lymphocyte reaction. By contrast, T cells lacking surface TCR/CD3 complex due to TRAC KO or mutant expression did not kill mock host T cells in this assay.
  • Example 3 Strategies for reducing endogenous TCR expression or functional TCR complex formation using ER-retained scFvs against TCR
  • Constructs of scFv against TCR with an ER retention tag were expressed in primary T cells.
  • Fig. 8A shows significant reduction of surface TCR/CD3 complex expression on primary T cells engineered to express ER-retained scFvs against TCR. Extent of knockdown was dependent on ER- retention tag and addition of internal KDEL sequence.
  • Figs.8B and 8C show residual TCR activity after 28 days in culture. Prior to the 28 days in culture, T cells were purified with negative MACS selection (using anti-TCR antibody).
  • T cells were tested for capacity to activate pursuant to CD3/CD28 engagement via CD25, CD69, CD137 expression.
  • T cells are activated with TransACT (Miltenyi) for 72h in RPMI-1640, 10% fetal bovine serum, and non-essential amino acids.
  • TransACT Miltenyi
  • Fig.8B on day 28 post-purification, the percentage of CD137+CD69+ cells were significantly lower in the T cells with TRAC KO or expressing ER-retained scFvs against TCR than the control T cells with surface TCR/CD3 complex.
  • Fig.10A depicts two constructs, both with EF1a promoter driven expression of the BFP and CD3e DN transgenes.
  • the bottom construct included an additional CD3z targeting shRNA placed in the natural EF1a promoter intron.
  • Fig.10B shows that inclusion of the CD3z shRNA in the intron did not reduce the level of BFP expression compared to the version without the shRNA.
  • Fig. 10C shows that the combination of the CD3e DN and the shRNA further reduced TCR surface levels to the level of TRAC KO.
  • Example 5 Strategies for reducing endogenous TCR expression or functional TCR complex formation using a humanized anti-TCR scFv
  • Fig.11A Nine constructs with a humanized anti-TCR scFv fused to an ER retention domain were designed, as shown in Fig.11A. All constructs were co-transduced with BFP. Leakiness of the ER- retained anti-TCR scFv was assessed by the level of expression of the whitlow linker on non- permeabilized primary T cells.
  • Fig.11B shows that the leakiness of ER-retained scFvs was low in all constructs as the percentage of the whitlow positive cells were low.
  • TCR/CD3 reduction was dependent on the combination of variable humanized light and heavy chains as well as their orientation (i.e. V L- V H vs V H -V L ).
  • Constructs 4082, 4083, 4084, 4091, 4093 and 4094 reduced TCR/CD3 surface expression by more than 95%.
  • Example 6 Strategies for reducing endogenous TCR expression or functional TCR complex formation using a binder linked to either ER-retained CD3 or dominant negative CD3 [530] Constructs expressing an anti-TCR, an anti-CD3, an anti-CD58, or an anti-MHCl binder linked to either ER-retained CD3 or dominant negative CD3 were designed.
  • Fig. 12A shows significant reduction of surface TCR/CD3 complex expression on primary T cells engineered to express anti-TCR/CD3 binders linked to either ER-retained CD3 or dominant negative CD3 .
  • Fig. 12B shows significant reduction of surface CD58 and TCR/CD3 complex expression on primary T cells engineered to express anti-CD58 scFv linked to either ER-retained CD3 or dominant negative CD3 .
  • Fig.12C shows significant reduction of surface MHCI and TCR/CD3 complex expression on WSGR Docket No.61078-729.601 primary T cells engineered to express anti-MHCI scFv linked to either ER-retained CD3 or dominant negative CD3 .
  • the compositions described herein can prevent activation of T cells via the TCR/CD3 complex.
  • Activation of T cells [532] T cells were seeded into a well plate in RPMI-164 supplemented with 10% FBS with or without 2. Cells were then stained for CD25, CD69, and CD137 expression by flow cytometry. 2. T cell Mixed Lymphocyte Reaction [533] Cell Technologies). Purity is confirmed via flow cytometry.
  • Graft T cells are mixed at a 1:1 E:T ratio - host T cells in RPMI-1640 supplemented with 10% FBS + 20 IU/mL IL-2 and co- 2. Surviving host T cells counts are determined by flow cytometry.
  • the compositions described herein can inhibit alloreactive T cell killing.
  • Methods described herein can be used to test the effect of other fusion polypeptides (e.g., any one of SEQ ID NOs: 38, 42-43, 60, or 62-64) on surface functional TCR expression. Other variations of the fusion polypeptides are described herein and at least in Call et al., 2004, Call et al., 200, or Delgado et al., 2005.
  • Example 7 – Exemplary sequences [535] Exemplary CD3 fusion and mutant protein constructs are shown below in Table 3. Table 3: Exemplary CD3 fusion and mutant protein constructs WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 47 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601
  • Table 4 Exemplary functional ubiquitin ligase domain sequences WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 [537] Exemplary ER retention tag sequences are shown below in Table 5.
  • Exemplary ER retention tag sequences [538] Exemplary anti-TCR scFv constructs are shown below in Table 6 Table 6: Exemplary anti-TCR scFv constructs WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729
  • CD3 fusion or mutant protein constructs are human codon optimized, synthesized, and cloned into a MSGV1 retroviral vector by Gene Art (Life Technologies). Fusion or mutant proteins can be fused N-terminally or C-terminally with a second or third domain via a linker.
  • Retroviral vector production, T cell transduction and T cell gene editing [544] Retroviral supernatant is produced via transient transfection of 293 GP producer cells.293 GP cells are seeded onto poly-D-lysine (R&D Systems) coated plates the day prior to transfection.
  • plasmids encoding the genes of interest (CARs) and the fusion or mutant protein constructs (or small regulatory RNA constructs—siRNA, shRNA, and/or miRNA) are co-transfected with Lipofectamine 2000 (Life Technologies). Media is replaced 24h after transfection. Viral supernatant is harvested 24 to 48 h after media replacement and filtered through a for transduction.
  • Primary human T cells are isolated from fresh or frozen PBMCs obtained from healthy donor leukopaks from Stem Cell Technologies using negative MACS selection (Miltenyi). Isolated T cells are cryopreserved in Bambanker (GC Lymphotec Inc.).
  • T cells are activated with TransACT (Miltenyi) and 100 IU/mL IL-2 (Miltenyi) for 48 h and cultured in RPMI-1640 + 10% fetal bovine serum + non-essential amino acids (R10). T cells are transduced with retroviral vector on days 2 or 3 post activation.
  • TransACT TransACT
  • IL-2 Miltenyi
  • R10 fetal bovine serum + non-essential amino acids
  • Table 12 Exemplary CAR Proteins WSGR Docket No.61078-729.601 [548] Exemplary full length anti-CD19 scFv constructs are listed in Table 13 Table 13: Exemplary full length anti-CD19 scFv constructs WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 [549] Exemplary full length anti-CD20 scFv constructs are listed in Table 14.
  • Table 14 Exemplary full length anti-CD20 scFv constructs WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 [550] Exemplary anti-CD22 scFv construct, the heavy chain sequence, the light chain sequence, the variable regions, and the CDRs are listed in Table 15.
  • Table 15 Exemplary anti-CD22 scFv constructs WSGR Docket No.61078-729.601 [551] Exemplary anti-CD19, anti-CD20, and anti-CD22 CAR constructs are listed in Table 16. Table 16: Exemplary anti-CD19, anti-CD20, and anti-CD22 CAR constructs WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docke
  • T cells are activated with TransACT and 100 IU/mL IL-2 for 48-72 hr.
  • T cells are gene edited using 1:1 molar ratio of sgRNA:Cas9 on days 2 or 3 post activation via CRISPR/Cas9 using the Neon electroporator (Life Technologies).
  • T cells were gene edited with CRISPR/cas9 and assessed for KO by flow cytometry 5 days post-editing.
  • Exemplary sgRNA sequences to be used are shown in Table 17.
  • DN-RFX5or DN-RFXANK can be expressed.
  • Exemplary DN-RFX5 and DN-RFXANK constructs are show in Table 18.
  • Table 18 Exemplary DN-RFX5 and DN-RFXANK constructs WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601 WSGR Docket No.61078-729.601
  • Graft T cells weremixed at a 1:1 E:T ratio with primed, allogeneic host T cells in R10 + 20 IU/mL IL-2 and co-incubated for 48 h at 37 2. %Graft survival was determined by flow cytometry, gating on absolute event counts of graft T cells and normalizing in the absence of effector cells. Graft T cells transduced with B2M fusions were co-cultured with primed, alloreactive host T cells at a 1:1 ratio for 2 days. The compositions described herein can inhibit alloreactive T cell killing. 2. NK cell Mixed Lymphocyte Reaction [556] (Stem Cell Technologies). Purity was confirmed via flow cytometry.
  • Graft T cells were mixed at a 1:1 E:T ratio with activated, allogeneic host NK cells in R10 + 1000 IU/mL IL-2 and co-incubated for 48 WSGR Docket No.61078-729.601 2. %Graft survival was determined by flow cytometry, gating on absolute event counts of graft T cells and normalizing in the absence of effector cells. 3. PBMC Mixed Lymphocyte Reaction [557] (Stem Cell Technologies). Purity was confirmed via flow cytometry. Graft T cells were mixed at a 3:1 E:T ratio with allogeneic host PBMC cells in R10 + 20 IU/mL IL-2 and co-incubated for up to 12 days 2.

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Abstract

La présente demande concerne des polypeptides recombinants, des ARN non codants et/ou des récepteurs antigéniques chimériques modifiés. La demande concerne également des cellules modifiées pour exprimer un ou plusieurs polypeptides recombinants, des ARN non codants et/ou des récepteurs antigéniques chimériques modifiés. La demande concerne en outre leurs utilisations pour le traitement de maladies, d'états et/ou de troubles, et leurs utilisations pour la fabrication de populations de cellules.
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