NOVEL PLATFORMS FOR CO-STIMULATION, NOVEL CAR DESIGNS AND OTHER ENHANCEMENTS FOR ADOPTIVE CELLULAR THERAPY
The disclosure provides compositions and method that promote adoptive cellular therapy. The disclosure provides polynucleotides, vectors, systems and cells comprising chimeric antigen receptors (CARs), synthetic immune receptors (SIRs), and the like in combination the specific activators of NFkB activity, thus improving cellular proliferation, expression and reduced apoptosis, which improves cell persistence in adoptive cell therapy.
1 . At least one recombinant polynucleotide comprising:
a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP); and
b) a sequence encoding a TCR constant chain or a fragment thereof;
wherein the TFP incorporates into a TCR when expressed in a T cell or T cell population with impaired or abolished functional expression of an endogenously expressed TCR chain.
2 . The at least one recombinant polynucleotide of claim 1 , wherein the TFP is one or more of CD3ε-TFP (or TFPε), CD3γ-TFP (or TFPγ), and CD3δ-TFP (or TFPδ).
3 . The at least one recombinant polynucleotide of claim 1 , wherein the sequence encoding the TCR constant chain or a fragment thereof is
a) TCR alpha constant chain or fragment thereof, or
b) a TCR beta constant chain or a fragment thereof, or
c) a TCR gamma constant chain or fragment thereof, or
d) a TCR delta constant chain or a fragment thereof, or
e) a combination of i) and ii) or iii) and iv).
4 . The at least one recombinant polynucleotide of claim 1 , wherein the TFP comprises an antigen binding domain fused to a TCR chain, wherein the TCR chain is CD3ε, CD3γ, or CD3δ.
5 . The at least one recombinant polynucleotide of claim 4 , wherein the antigen binding domain of the TFP comprises an antibody, an antibody fragment, scFv, a Fv, a Fab, a (Fab′)2, a single domain antibody (SDAB), a diabody, a camelid vHH domain, a non-immunoglobulin scaffold, a ligand, or an antigen binding portion of a receptor.
6 . The at least one recombinant polynucleotide of claim 1 , wherein the TFP is expressed from an expression cassette placed in an endogenous TCR gene locus, optionally wherein the endogenous TCR gene locus is TCRα, TCRβ1, TCRβ2, TCRγ, or TCRδ gene locus.
7 . The at least one recombinant polynucleotide of claim 1 , wherein the expression of the TFP is under the control of a promoter and/or regulatory elements for an endogenous T cell gene, wherein optionally the endogenous T cell gene is TCRα, TCRβ1, TCRβ2, TCRγ, or TCRδ gene.
8 . A T cell or T cell population with impaired or abolished functional expression of an endogenously expressed TCR chain and expressing at least one recombinant polynucleotide comprising:
a) a sequence encoding a T cell receptor (TCR) fusion protein (TFP); and
b) a sequence encoding a TCR constant chain or a fragment thereof;
wherein the TFP incorporates into a TCR when expressed in a T cell or T cell population.
9 . The T cell or T cell population of claim 8 , wherein the TFP is one or more of CD3ε-TFP (or TFPε), CD3γ-TFP (or TFPγ) or CD3δ-TFP (or TFPδ).
10 . The T cell or T cell population of claim 8 , wherein the sequence encoding the TCR constant chain or a fragment thereof is
a) a TCR alpha constant chain or fragment thereof, or
b) a TCR beta constant chain or a fragment thereof, or
c) a TCR gamma constant chain or fragment thereof, or
d) a TCR delta constant chain or a fragment thereof, or
e) a combination of i) and ii) or iii) and iv).
11 . The T cell or T cell population of claim 8 , wherein the TFP comprises an antigen binding domain fused to a TCR chain, wherein the TCR chain is CD3ε, CD3γ, or CD3δ.
12 . The T cell or T cell population of claim 11 , wherein the antigen binding domain of the TFP comprises an antibody, an antibody fragment, scFv, a Fv, a Fab, a (Fab′)2, a single domain antibody (SDAB), a diabody, a camelid vHH domain, a non-immunoglobulin scaffold, a ligand, or an antigen binding portion of a receptor.
13 . The T cell or T cell population of claim 8 , wherein the TFP is expressed from an expression cassette placed in an endogenous TCR gene locus, optionally wherein the endogenous TCR gene locus is TCRα, TCRβ1, TCRβ2, TCRγ, or TCRδ gene locus.
14 . The T cell or T cell population of claim 8 , wherein the expression of the TFP is under the control of a promoter and/or regulatory elements for an endogenous T cell gene, wherein optionally the endogenous T cell gene is TCRα, TCRβ1, TCRβ2, TCRγ, or TCRδ gene.
15 . The T cell or a T cell population of claim 8 , wherein the T cell or a T cell population lacks the expression of a functional HLA.
16 . An induced pluripotent stem cell capable of giving rise to a T cell or a T cell population of claim 8 .
17 . A method of generating a T cell or a T cell population, comprising steps of introducing into a T cell:
(i) a recombinant polynucleotide encoding a T cell receptor (TCR) fusion protein (TFP);
(ii) a recombinant polynucleotide sequence encoding a TCR constant chain or a fragment thereof, optionally wherein the sequence encoding the TCR constant chain or a fragment thereof is
a) TCR alpha constant chain or fragment thereof, or
b) a TCR beta constant chain or a fragment thereof, or
c) a TCR gamma constant chain or fragment thereof, or
d) a TCR delta constant chain or a fragment thereof, or
e) a combination of i) and ii) or iii) and iv)
(iii) a homologous recombination system suitable for targeted integration of the recombinant polynucleotide encoding the TFP at an endogenous TCR gene locus, optionally wherein the endogenous TCR gene locus is TCRα, TCRβ1, TCRβ2, TCRγ or TCRδ gene locus.
18 . The method of claim 17 , further comprising a step of eliminating the expression of a functional HLA.
19 . The method of claim 17 , wherein:
i. the TFP is one or more of CD3ε-TFP (or TFPε), CD3γ-TFP (or TFPγ), or CD3δ-TFP (or TFPδ);
ii. the sequence encoding the TCR constant chain or a fragment thereof is:
a) a TCR alpha constant chain or fragment thereof, or
b) a TCR beta constant chain or a fragment thereof, or
c) a TCR gamma constant chain or fragment thereof, or
d) a TCR delta constant chain or a fragment thereof, or
e) a combination of i) and ii) or iii) and iv);
iii. the TFP comprises an antigen binding domain fused to a TCR chain, wherein the TCR chain is CD3ε, CD3γ, or CD3δ;
iv. the TFP is expressed from an expression cassette placed in an endogenous TCR gene locus, optionally wherein the endogenous TCR gene locus is TCRα, TCRβ1, TCRβ2, TCRγ, or TCRδ gene locus; and/or
v. the expression of the TFP is under the control of a promoter and/or regulatory elements for an endogenous T cell gene, wherein optionally the endogenous T cell gene is TCRα, TCRβ1, TCRβ2, TCRγ or TCRδ gene.
20 . A method of treating a subject with T cell therapy in need thereof, comprising administering to the subject a therapeutically effective amount of the T cell or T cell population of claim 8 .