WO2024124164A2

WO2024124164A2 - Compositions and methods for delivery of immunostimulatory cytokines to chimeric antigen receptor immune cells

Info

Publication number: WO2024124164A2
Application number: PCT/US2023/083172
Authority: WO
Inventors: Avery D. Posey; John T. Keane; Oula DAGHER
Original assignee: University of Pennsylvania Penn; US Department of Veterans Affairs
Current assignee: University of Pennsylvania Penn; US Department of Veterans Affairs
Priority date: 2022-12-09
Filing date: 2023-12-08
Publication date: 2024-06-13
Anticipated expiration: 2025-06-09
Also published as: WO2024124164A3

Abstract

The invention includes anti-single-chain variable fragment (scFv) antibodies or antigen-binding fragments thereof, wherein the scFv is an scFv of a CAR, and uses thereof (e.g., for targeting one or more immunostimulatory cytokines to modified immune cells which express the CAR and/or for activating said CAR-expressing immune cells without affecting cytotoxic potential or inducing exhaustion). The invention includes an antibody-linked cytokine, wherein the antibody is the anti-scFv antibody or antigen-binding fragment, and use thereof in a method of treating cancer. The invention further includes a lipid nanoparticle (LNP) harboring nucleoside-modified RNA(s) encoding at least one immunostimulatory cytokine, wherein the LNP comprises the anti-scFv antibody or antigen-binding fragment linked to the surface of the LNP, and use thereof.

Description

COMPOSITIONS AND METHODS FOR DELIVERY OF IMMUNOSTIMULATORY CYTOKINES TO CHIMERIC ANTIGEN RECEPTOR IMMUNE CELLS

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/386,826, filed Dec. 9, 2022, which application is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

The durable responses of chimeric antigen receptor (CAR) T cells in hematological tumors have inspired development of CAR T cell therapies for the treatment of solid tumors. While CAR T cells against solid tumors show promise, they have not been as successful as those targeting hematological malignancies due to a number of factors, including an immunosuppressive tumor microenvironment (TME). CAR T cells directed against solid tumor targets do not typically encounter their cognate target in the blood, but instead undergo limited homeostatic CAR T cell expansion in the blood and traffic to tumor sites. The number of CAR T cells that reach the tumor are insufficient to eradicate disease. Furthermore, increasing the infusion dose of CAR T cells has revealed on-target off-tumor toxicity.

There is an urgent need in the art for tools and methods to augment anti-tumor efficacy of CAR adoptive cell therapies in order to improve patient clinical outcomes. The present invention addresses this need.

SUMMARY OF THE INVENTION

In one aspect, the invention provides an anti-single-chain variable fragment (scFv) antibody or antigen-binding fragment, wherein the anti-scFv antibody or antigen-binding fragment is capable of specifically binding to a linker peptide of an scFv, wherein the linker peptide links a variable light chain (VL) of the scFv to a variable heavy chain (VH) of the scFv, and further wherein the scFv is an scFv of a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain comprising the scFv, a transmembrane domain, and an intracellular domain.

In some embodiments, the anti-scFv antibody or antigen-binding fragment further comprises a heavy chain variable region (VH) comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and a light chain variable region (VL) comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein:

(i) the HCDR1 comprises or consists of the amino acid sequence GFTFSDYY (SEQ ID NO: 59), the HCDR2 comprises or consists of the amino acid sequence ISDGGSYT (SEQ ID NO: 60), the HCDR3 comprises of consists of the amino acid sequence AREGDLGSFWFAFW (SEQ ID NO: 61), the LCDR1 comprises or consists of the amino acid sequence QDISNY (SEQ ID NO: 62), the LCDR2 comprises or consists of the amino acid sequence YTS (SEQ ID NO: 63), and the LCDR3 comprises or consists of the amino acid sequence QQGNTLP (SEQ ID NO: 64);

(ii) the HCDR1 comprises or consists of the amino acid sequence GFSLTDYG (SEQ ID NO: 69), the HCDR2 comprises or consists of the amino acid sequence VWGGGST (SEQ ID NO: 70), the HCDR3 comprises of consists of the amino acid sequence AKLYGHYYIMDY (SEQ ID NO: 71), the LCDR1 comprises or consists of the amino acid sequence ESVDSYGNSF (SEQ ID NO: 72), the LCDR2 comprises or consists of the amino acid sequence LAS (SEQ ID NO: 73), and the LCDR3 comprises or consists of the amino acid sequence QQNNEDPFT (SEQ ID NO: 74); or

(iii) the HCDR1 comprises or consists of the amino acid sequence GFTFSSYG (SEQ ID NO: 79), the HCDR2 comprises or consists of the amino acid sequence ISSGGSYT (SEQ ID NO: 80), the HCDR3 comprises of consists of the amino acid sequence ARQDYGSPFAY (SEQ ID NO: 81), the LCDR1 comprises or consists of the amino acid sequence QDINKY (SEQ ID NO: 82), the LCDR2 comprises or consists of the amino acid sequence YTS (SEQ ID NO: 83), and the LCDR3 comprises or consists of the amino acid sequence LQYDNLLWT (SEQ ID NO: 84).

In some embodiments:

(i) the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 67;

(ii) the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 75 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 77; or

(iii) the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 86 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 88.

In some embodiments, the anti-scFv antibody or antigen-binding fragment further comprises a heavy chain and a light chain, wherein the heavy chain comprises the VH and the light chain comprises the VL.

In some embodiments, the scFv of the CAR targets a tumor antigen, preferably wherein the tumor antigen is selected from the group consisting of alpha feto-protein (AFP)/HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD13, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, claudin 6, c-Met, DLL3, DR5, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa)/folate binding protein (FBP), folate receptor beta (FRb), follicle-stimulating hormone receptor (FSHR), GD-2, Glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, interleukin 13 receptor subunit alpha (IL3Ra), interleukin 13 receptor subunit alpha 2 (IL13Ra2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE- A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4/FAP, NKG2D- Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), Mullerian inhibiting substance type 2 receptor (MISIIR), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), P16, PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), R0R1, R0R2, TAG72, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUCl), TROP2, Glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRa4; GFRalpha4), VEGFR2, and any combination thereof.

In some embodiments, the scFv of the CAR targets TnMUCl.

In some embodiments, the intracellular domain of the CAR comprises a costimulatory domain and an intracellular signaling domain.

In some embodiments, one or more of the following applies: (i) the intracellular domain of the CAR comprises a costimulatory domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), 0X40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lek, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR);

(ii) the intracellular domain of the CAR comprises an intracellular signaling domain of a protein selected from the group consisting of a human CD3 zeta chain (CD3Q, FcyRIII, FcsRI, DAP10, DAP12, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof;

(iii) the intracellular signaling domain of the CAR comprises an intracellular signaling domain of CD3 zeta or a variant thereof;

(iv) the intracellular domain of the CAR comprises a costimulatory domain of a protein selected from CD28 and 4-1BB (CD137), or both CD28 and 4-1BB (CD 137);

(v) the intracellular domain of the CAR comprises a costimulatory domain of CD28 and an intracellular signaling domain of CD3 zeta; and/or

(vi) the intracellular domain of the CAR comprises a costimulatory domain of 4-1BB (CD 137) and an intracellular signaling domain of CD3 zeta.

In one aspect, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the anti-scFv antibody or antigen-binding fragment described herein, optionally wherein the nucleotide sequence further encodes a ribosome slip sequence, optionally wherein the ribosome slip sequence is selected from the group consisting of P2A, T2A, E2A, F2A, and variants thereof.

In one aspect, the invention provides a vector comprising the nucleic acid molecule described herein.

In one aspect, the invention provides a cell comprising the nucleic acid molecule described herein or the vector described herein.

In one aspect, the invention provides an antibody-linked cytokine comprising an immunostimulatory cytokine linked to an anti-scFv antibody or antigen-binding fragment, wherein the anti-scFv antibody or antigen-binding fragment is capable of specifically binding to a linker peptide of an scFv, wherein the linker peptide links a variable light chain (VL) of the scFv to a variable heavy chain (VH) of the scFv, and further wherein the scFv is an scFv of a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain comprising the scFv, a transmembrane domain, and an intracellular domain.

In some embodiments, the anti-scFv antibody or antigen-binding fragment comprises a heavy chain variable region (VH) comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and a light chain variable region (VL) comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein:

(ii) the HCDR1 comprises or consists of the amino acid sequence GFSLTDYG (SEQ ID NO: 69), the HCDR2 comprises or consists of the amino acid sequence VWGGGST (SEQ ID NO: 70), the HCDR3 comprises of consists of the amino acid sequence AKLYGHYYIMDY (SEQ ID NO: 71), the LCDR1 comprises or consists of the amino acid sequence ESVDSYGNSF (SEQ ID NO: 72), the LCDR2 comprises or consists of the amino acid sequence LAS (SEQ ID NO: 73), and the LCDR3 comprises or consists of the amino acid sequence QQNNEDPFT (SEQ ID NO: 74);

(iii) the HCDR1 comprises or consists of the amino acid sequence GFTFSSYG (SEQ ID NO: 79), the HCDR2 comprises or consists of the amino acid sequence ISSGGSYT (SEQ ID NO: 80), the HCDR3 comprises of consists of the amino acid sequence ARQDYGSPFAY (SEQ ID NO: 81), the LCDR1 comprises or consists of the amino acid sequence QDINKY (SEQ ID NO: 82), the LCDR2 comprises or consists of the amino acid sequence YTS (SEQ ID NO: 83), and the LCDR3 comprises or consists of the amino acid sequence LQYDNLLWT (SEQ ID NO: 84); or.

(iv) the HCDR1 comprises or consists of the amino acid sequence KFSFNKKYYMC (SEQ ID NO: 1), the HCDR2 comprises or consists of the amino acid sequence WIGCVDTGDAFIGY (SEQ ID NO: 2), the HCDR3 comprises of consists of the amino acid sequence RGVYPINTGYYYFDL (SEQ ID NO: 3), the LCDR1 comprises or consists of the amino acid sequence EDITNSLA (SEQ ID NO: 4), the LCDR2 comprises or consists of the amino acid sequence NLLIYRASTLAS (SEQ ID NO: 5), and the LCDR3 comprises or consists of the amino acid sequence QQGYSSTNVDNI (SEQ ID NO: 6).

In some embodiments:

(ii) the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 75 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 77;

(iii) the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 86 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 88; or

(iv) the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9.

In some embodiments, the immunostimulatory cytokine comprises a single polypeptide chain or is engineered to be expressed as a single polypeptide chain.

In some embodiments, the immunostimulatory cytokine is selected from IL-12, IL-18, and IL-23. In some embodiments, the anti-scFv antibody or antigen-binding fragment comprises a first polypeptide comprising the heavy chain and a second polypeptide comprising the light chain, and further wherein the N-terminus of the immunostimulatory cytokine is linked to the C- terminus of the first polypeptide comprising the heavy chain.

In some embodiments, the immunostimulatory cytokine is linked to the first polypeptide comprising the heavy chain via (i) a (G4S)I linker or (ii) a poly-alanine linker comprising two or more consecutive alanine residues.

In some embodiments, the poly-alanine linker consists of two alanine residues.

In some embodiments, the immunostimulatory cytokine is IL-12.

In some embodiments, the IL-12 is engineered to be expressed as a single polypeptide chain.

In some embodiments, the IL-12 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19.

In some embodiments, the first polypeptide comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11, a polyalanine linker, and single chain IL-12 comprising the amino acid sequence set forth in SEQ ID NO: 19, and the second polypeptide comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 13.

In some embodiments, the immunostimulatory cytokine is IL-18.

In some embodiments, the IL-18 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21.

In some embodiments, the first polypeptide comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11, a polyalanine linker, and IL-18 comprising the amino acid sequence set forth in SEQ ID NO: 21, and the second polypeptide comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 13.

In some embodiments, the immunostimulatory cytokine is IL-23.

In some embodiments, the IL-23 is engineered to be expressed as a single polypeptide chain. In some embodiments, the IL-23 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23.

In some embodiments, the first polypeptide comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11, a polyalanine linker, and single chain IL-23 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the second polypeptide comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 13.

In some embodiments, the anti-scFv antibody or antigen-binding fragment further comprises a heavy chain signal peptide and/or a light chain signal peptide, optionally wherein the heavy chain signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15 and/or the light chain signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17.

In some embodiments, the scFv of the CAR targets a tumor antigen, preferably wherein the tumor antigen is selected from the group consisting of alpha feto-protein (AFP)/HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD13, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, claudin 6, c-Met, DLL3, DR5, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa)/folate binding protein (FBP), folate receptor beta (FRb), follicle-stimulating hormone receptor (FSHR), GD-2, Glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, interleukin 13 receptor subunit alpha (IL3Ra), interleukin 13 receptor subunit alpha 2 (IL13Ra2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE- A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4/FAP, NKG2D- Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), Mullerian inhibiting substance type 2 receptor (MISIIR), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), P16, PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), ROR1, ROR2, TAG72, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUCl), TROP2, Glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRa4; GFRalpha4), VEGFR2, and any combination thereof.

In some embodiments, the scFv of the CAR targets TnMUCl. In some embodiments, the intracellular domain of the CAR comprises a costimulatory domain and an intracellular signaling domain.

In some embodiments, one or more of the following applies:

(i) the intracellular domain of the CAR comprises a costimulatory domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), 0X40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lek, TNFR-I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR);

In one aspect, the invention provides the antibody-linked cytokine described herein, for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the antibody linked cytokine to the subject, and further wherein the administering is performed before, simultaneously with, or after infusion of the subject with a population of modified immune cells which express the CAR.

In one aspect, the invention provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody-linked cytokine described herein, optionally wherein the nucleotide sequence comprises the amino acid sequence set forth in SEQ ID NO: 26, the amino acid sequence set forth in SEQ ID NO: 28, or the amino acid sequence set forth in SEQ ID NO: 30, or the nucleic acid molecule comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30.

In some embodiments, the nucleotide sequence encoding the antibody-linked cytokine further encodes a ribosome slip sequence, optionally wherein the ribosome slip sequence is selected from the group consisting of P2A, T2A, E2A, F2A, and variants thereof.

In one aspect, the invention provides a lipid nanoparticle (LNP), wherein the LNP comprises:

(a) at least one nucleoside-modified ribonucleic acid (RNA) encoding at least one immunostimulatory cytokine, wherein each immunostimulatory cytokine is encoded by a distinct nucleoside-modified RNA;

(b) at least one ionizable lipid; and

(c) an anti-scFv antibody or antigen-binding fragment, wherein the anti-scFv antibody or antigen-binding fragment is capable of specifically binding to a linker peptide of an scFv, wherein the linker peptide links a variable light chain (VL) of the scFv to a variable heavy chain (VH) of the scFv, and further wherein the scFv is an scFv of a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain comprising the scFv, a transmembrane domain, and an intracellular domain; wherein the at least one ionizable lipid at least partially encapsulates the at least one nucleoside-modified RNA; and further wherein the anti-scFv antibody or antigen-binding fragment is linked to the surface of the LNP.

In some embodiments, the at least one immunostimulatory cytokine comprises any one or more of IL- 12, IL- 18, and IL-23. In some embodiments, the at least one immunostimulatory cytokine comprises a single polypeptide chain or is engineered to be expressed as a single polypeptide chain.

In some embodiments, the at least one immunostimulatory cytokine comprises or consists of IL-12.

In some embodiments, the IL- 12 is engineered to be expressed as a single polypeptide chain.

In some embodiments, the at least one immunostimulatory cytokine comprises or consists of IL-18.

In some embodiments, the at least one immunostimulatory cytokine comprises or consists of IL-23.

In some embodiments, the IL-23 is engineered to be expressed as a single polypeptide chain.

In some embodiments, the IL-23 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23.

In some embodiments, the at least one nucleoside-modified RNA is messenger RNA (mRNA).

In some embodiments, the at least one nucleoside-modified RNA comprises pseudouridine and/or 1-methyl-pseudouridine.

In some embodiments, the at least one nucleoside-modified RNA is in vitro transcribed (IVT) RNA.

In some embodiments, the at least one ionizable lipid is a cationic lipid. In some embodiments, the anti-scFv antibody or antigen-binding fragment comprises a heavy chain variable region (VH) comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and a light chain variable region (VL) comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein:

(iv) the HCDR1 comprises or consists of the amino acid sequence KFSFNKKYYMC (SEQ ID NO: 1), the HCDR2 comprises or consists of the amino acid sequence WIGCVDTGDAFIGY (SEQ ID NO: 2), the HCDR3 comprises of consists of the amino acid sequence RGVYPTNTGYYYFDL (SEQ ID NO: 3), the LCDR1 comprises or consists of the amino acid sequence EDITNSLA (SEQ ID NO: 4), the LCDR2 comprises or consists of the amino acid sequence NLLIYRASTLAS (SEQ ID NO: 5), and the LCDR3 comprises or consists of the amino acid sequence QQGYSSTNVDNI (SEQ ID NO: 6).

In some embodiments:

(i) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11; and/or

(ii) the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13.

In some embodiments, the scFv of the CAR targets a tumor antigen, preferably wherein the tumor antigen is selected from the group consisting of alpha feto-protein (AFP)/HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD13, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, claudin 6, c-Met, DLL3, DR5, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa)/folate binding protein (FBP), folate receptor beta (FRb), follicle-stimulating hormone receptor (FSHR), GD-2, Glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, interleukin 13 receptor subunit alpha (IL3Ra), interleukin 13 receptor subunit alpha 2 (IL13Ra2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE- A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4/FAP, NKG2D- Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), Mullerian inhibiting substance type 2 receptor (MISIIR), New York esophageal squamous cell carcinoma- 1 (NY-ESO-1), Pl 6, PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), R0R1, R0R2, TAG72, TIM-3, TM4SF1, Tn-glycoform ofMUCl (TnMUCl), TR0P2, Glycosyl -phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRa4; GFRalpha4), VEGFR2, and any combination thereof.

In some embodiments, the scFv of the CAR targets TnMUCl.

In some embodiments, one or more of the following applies:

In one aspect, the invention provides the LNP described herein, for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the LNP to the subject, and further wherein the administering is performed before, simultaneously with, or after infusion of the subject with a population of modified immune cells which express the CAR.

In one aspect, the invention provides a pharmaceutical composition comprising the antibody-linked cytokine described herein, or the LNP described herein, and at least one pharmaceutically acceptable carrier, diluent, and/or excipient.

In one aspect, the invention provides the pharmaceutical composition described herein, for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the pharmaceutical composition to the subject, and further wherein the administering is performed before, simultaneously with, or after infusion of the subject with a population of modified immune cells which express the CAR.

In one aspect, the invention provides a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody-linked cytokine described herein, or a therapeutically effective amount of the LNP described herein, wherein the administering is performed before, simultaneously with, or after infusion of the subject with a population of modified immune cells which express the CAR.

In some embodiments, each cell of the population of modified immune cells is a T cell, an autologous cell, a human cell, or any combination thereof.

In some embodiments, the administering comprises subcutaneous injection, intraperitoneal injection, intradermal injection, intravenous injection, intramuscular injection, intrasternal injection, or infusion techniques.

In some embodiments, the administering comprises administering a first dose.

In some embodiments, the administering further comprises administering one or more subsequent doses.

In some embodiments, the cancer is selected from breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and thyroid cancer.

In some embodiments, the subject is a human.

In one aspect, the invention provides a recombinant IL-23, wherein the recombinant IL- 23 is engineered to be expressed as a single polypeptide chain.

In some embodiments, the recombinant IL-23 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23, or wherein the recombinant IL-23 comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description of preferred embodiments of the invention will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, non-limiting illustrative embodiments are shown in the drawings. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

FIG. 1A is a schematic of CAR construct including the single chain cytokine, P2A self- cleaving linker, and the 5E5 CAR with a CD2 costimulatory domain for constitutive expression of cytokine in CAR T cells.

FIG. IB shows validation of CAR expression on the surface of primary human T cells by flow cytometry.

FIG. 1C shows increased secretion of immunostimulatory cytokines measured by ELISA for each specific CAR-cytokine group. T cells were either non-stimulated and allowed to rest overnight, or stimulated overnight by tumor cells presenting antigen or antigen-independently by PMA/Ionomycin.

FIG. ID shows that secretion of cytokines does not increase population doublings of T cells in multiple donors.

FIG. 2A shows the percentage of T cells in each memory phenotype, ns is nonsignificant.

FIG. 2B shows the percentage of cells expressing 1, 2, or 3 exhaustion markers PD-1, Lag-3, and Tim-3. *=p< 05, **=p< 01, ***=p< 001, ****=p< 0001. ns is non-significant.

FIG. 3 shows cell lysis of MCF7 breast cancer cells by primary T cells from 2 donors. ND567 is presented at a 1: 1 effector to target (E:T) ratio and ND224 at a 3 : 1 E:T ratio. Time of co-culture is in hours (h). The top graphs show cytotoxicity measured by loss of impedance and the bottom graphs show cytotoxicity measured by loss of fluorescent target cell count. *=p< 05, **=p< 01, ***=p< 001, ****=p< 0001. ns is non-significant.

FIG. 4A shows serial caliper measurements of MCF7 tumor growth in NSG mice from the day of T cell treatment to the first sacrifice. *=p< 05, **=p< 01, ***=p< 001, ****=p< 0001. ns is non-significant.

FIG. 4B shows Kaplan-Meyer survival curve of tumor-bearing mice. *=p<.05, **=p< 01, ***=p< .001, ****=p< 0001. ns is non-significant.

FIG. 4C shows T cell counts on D33 and D63 post T cell treatment as well as postmortem immunohistochemical (IHC) analysis of tumors using anti-hCD3 staining. Interferon gamma and tumor necrosis factor alpha serum concentration levels extracted from 10-plex Luminex data are also shown. *=p< 05, **=p< 01, ***=p< 001, ****=p< 0001. ns is nonsignificant.

FIG. 4D shows weight change as a percentage of original weight of xenograft mice. *=p< 05, **=p< 01, ***=p< 001, ****=p< 0001. ns is non-significant. FIG. 5 shows in vitro killing of the MCF7 cell line by CAR-T cells either constitutively secreting cytokines or CAR-T cells dosed with antibody-linked cytokines (i.e., KIP4- 163 -linked cytokines). Time of co-culture is in hours (h). Loss of impedance infers killing of tumor cells. *=p< 05, **=p< 01, ***=p< 001, ****=p< 0001. ns is non-significant.

FIG. 6 is a chart which shows that the KIP4-163 anti-(G4S) antibody does not bind to a “short” (G₄S)I linker but does bind to a “long” (G4S)4 linker.

FIGs. 7A - 7B show that the KIP4-163 anti-(G4S) antibody and its cytokine conjugates activate CAR-T cells. All anti-(G4S) antibody cytokine conjugates significantly increased activation of CAR-T cells above that of CAR-T cells without anti-(G4S) antibody as well as that of non-transduced T cells with anti-(G4S) antibody plated. FIG. 7A is chart showing the expression levels of CD69+ cells by flow cytometry staining of FMC63BBz CD19-targeting CAR-T cells. FIG. 7B is a chart showing expression levels of CD69+ 5E5CD2z Tn-MUCl- targeting CAR-T cells.

FIGs. 8A - 8C show that activation of CAR-T cells with KIP4-163 anti-(G4S) antibody - IL12 conjugate drives a higher expression level of CD25, a moderate late activation marker important for T cell responses, than that seen with CAR-T cells activated with KIP4-163 alone.. FIG. 8A is a chart showing the flow cytometry data for FDC6BBz oncofetal fibronectin targeting CAR-T cells. FIG. 8B is chart showing the flow cytometry data for FMC63BBz CD 19- targeting CAR-T cells. FIG. 8C is chart showing the flow cytometry data for mAblO9BBz CEACAM6-targeting CAR-T cells.

FIG. 9 is a graph showing that, at an E:T ratio of 3: 1, KIP4-163 antibody-cytokine conjugates increase antitumor efficacy of CAR-T cells in vitro as compared to CAR-T cells that are kept alone or incubated with KIP4-163. 5 ug/mL of KIP4-163 anti-(G4S) antibody or a normalized concentration of antibody-cytokine conjugates was added in soluble form to wells of culture plates containing anti-Tn-MUCl CAR-T cells cocultured with MCF7 breast cancer cells at suboptimal E:T ratios. The control conditions (CAR alone and CAR with KIP4-163) did not control the growth of the tumor cells, whereas CAR-T cells with any of the antibody-cytokine conjugates totally eradicated tumor cells. *=p< 05, **=p< 01, ***=p<.001, ****=p<.0001. ns is non-significant.

FIGs. 10A - 10D show that antibody-cytokine conjugates increase the cytotoxic potential of CAR-T cells in a dose-dependent manner. FIG. 10A is a graph showing varying the concentration of unconjugated KIP4-163 anti-(G4S) antibody does not enhance CAR-T cell cytotoxicity. FIG. 10B is a graph showing that increasing the concentration of anti-(G4S) antibody-IL18 conjugate is directly proportional to its enhancing of CAR-T cell anti -tumor efficacy. FIG. IOC is a graph showing that anti-(G4S) antibody-IL12 conjugate is highly effective in potentiating CAR-T cell cytotoxicity at even the lowest concentrations tested. FIG. 10D is a graph showing that increasing the concentration of anti-(G4S) antibody-IL23 conjugate is directly proportional to its enhancing of CAR-T cell anti-tumor efficacy FIG. 11 is a chart showing that CAR-T cells uptake KIP4-163 anti-(G4S) antibody-cytokine conjugates whereas KIP4-163 anti-(G4S) antibody alone stays on the cell surface for at least 48 hours.

FIGs. 12A - 12D show that the KIP4-163 anti-(G4S) antibody-IL12 conjugate reduces tumor burden and increases survival in xenograft mice. FIG. 12A is a series of graphs showing total flux versus days for the condition indicated on each graph. NSG mice were injected on day -5 with 1E6 Nalm6 CBG luciferase+ cells. On day -1, mice were imaged and normalized by their total flux. On day 0, 1E6 CAR+ T cells were injected. On day 1 and weekly afterwards, 10 ug of KIP4-163 anti-(G4S) antibody or a normalized amount of antibody-cytokine conjugate were injected IV. FIG. 12B is a graph showing total average flux per group versus days as described for FIG. 12A. CAR-T cell-treated mice injected with KIP4-163 anti-(G4S) antibody- IL12 conjugate had lower average flux than mice injected with PBS. FIG. 12C is a chart showing the T cell count on day 14. Supplementing CAR-T cell-treated mice with anti-(G4S) antibody-IL12 resulted in an increase in peripheral blood T cell counts two weeks after T cell injection compared with mice injected with PBS alone. FIG. 12D is a graph showing survival of the mice versus days. Mice treated with CAR-T cells plus anti-(G4S) antibody-IL12 had significantly longer survival than mice treated with CAR-T cells plus PBS.

FIGs. 13A - 13B show ELISA screening data for specific antibody producing B-cells from mice immunized against either (G4S)s or whitlow linker peptides, after the third round of immunization, and prior to fusion with mouse myeloma cell lines for hybridoma generation. The x-axis indicates the dilutions applied on retroorbital blood serum. FIG. 13A is graph of the anti- (G4S)3 antibody ELISA screening data. FIG. 13B is graph of the anti -Whitlow antibody ELISA screening data.

FIGs. 14A - 14B show data from ELISA screening for hybridomas post fusion. FIG. 14A is chart of the anti-(G4S).3 hybridoma post fusion ELISA screening data. FIG. 14B is chart of the anti-Whitlow hybridoma post fusion ELISA screening data.

FIGs. 15A - 15D show data from ELISA screening for top scoring single cell clone (SCC) hybridomas subcloned from 43-B4 anti(G4S)3 or 44-A2 anti -whitlow bulk parent hybridomas, respectively. FIG. 15A is chart of the top scoring anti-(G4S)3 SCC hybridoma round

1 ELISA screening data. FIG. 15B is chart of the top scoring anti-(G4S)3 SCC hybridoma round

2 ELISA screening data. FIG. 15C is chart of the top scoring anti-Whitlow SCC hybridoma round 1 ELISA screening data. FIG. 15DC is chart of the top scoring anti-Whitlow SCC hybridoma round 2 ELISA screening data.

FIG. 16 is a chart of the luciferase assay data showing the % specific lysis (killing) of 293T-CBG+ cells expressing transmembrane-bound OD002, OD007, or OD008 scFv(s) against (G4S)3 or whitlow linkers by CAR-T cells that contain (G4S)3 or whitlow linkers in the scFv domain. 293T-CBG+ cells expressing KIP4-163 or anti-CD3 (0KT3) scFv are used as positive controls. Data are from 3 replicates; 2-way ANOVA; post hoc Tukey. *: p<0.05; **: p<0.01; ***: p<o ooi; ****• p<Q Q001; ns: non-significant. The CD19BBz CAR contains a (G4S)3 linker and the 4D5.5BBz CAR contains a whitlow linker.

FIGs. 17A - 17D show data from real-time xCelligence impedance analysis of specific killing of 293T cells expressing transmembrane-bound OD002 scFv by CAR-T cells that contain (G4S)3 or whitlow linkers in the scFv domain. The CD19BBz CAR contains a (G4S)3 linker and the 4D5.5BBz anti-ErbB2 CAR contains a whitlow linker. The E:T is 3: 1, n = 3, and ND580 donor. Loss of impedance implies target cell killing. FIG. 17A is a graph of the eSight real-time xCelligence impedance analysis for the conditions indicated. FIG. 17B is a graph of the eSight real-time xCelligence impedance analysis for the conditions indicated. FIG. 17C is a graph of the eSight real-time xCelligence impedance analysis for the conditions indicated. FIG. 17D is a graph of the eSight real-time xCelligence impedance analysis for the conditions indicated.

FIGs. 18A - 18D show data from real-time xCelligence impedance analysis of specific killing of 293T cells expressing transmembrane-bound OD007 scFv by CAR-T cells that contain (G4S)3 or whitlow linkers in the scFv domain. The CD19BBz CAR contains a (G4S)3 linker and the 4D5.5BBz CAR contains a whitlow linker. The E:T is 3: 1, n = 3, and ND580 donor. Loss of impedance implies target cell killing. FIG. 18A is a graph of the eSight real-time xCelligence impedance analysis for the conditions indicated. FIG. 18B is a graph of the eSight real-time xCelligence impedance analysis for the conditions indicated. FIG. 18C is a graph of the eSight real-time xCelligence impedance analysis for the conditions indicated. FIG. 18D is a graph of the eSight real-time xCelligence impedance analysis for the conditions indicated.

FIGs. 19A - 19D show data from real-time xCelligence impedance analysis of specific killing of 293T cells expressing transmembrane-bound OD008 scFv by CAR-T cells that contain (G4S)3 or whitlow linkers in the scFv domain. The CD19BBz CAR contains a (G4S)3 linker and the 4D5.5BBz CAR contains a whitlow linker. The E:T is 3: 1, n = 3, and ND580 donor. Loss of impedance implies target cell killing. FIG. 19A is a graph of the eSight real-time xCelligence impedance analysis for the conditions indicated. FIG. 19B is a graph of the eSight real-time xCelligence impedance analysis for the conditions indicated. FIG. 19C is a graph of the eSight real-time xCelligence impedance analysis for the conditions indicated. FIG. 19D is a graph of the eSight real-time xCelligence impedance analysis for the conditions indicated.

FIGs. 20A - 20B show the effects of incubation of CAR-T cells with KIP4-163 (anti- (G4S)3 antibody)-conjugated beads. The data are from flow staining analysis where n = 3 donors. 2 -way ANOVA, post hoc Tukey. FIG. 20A is chart showing that incubation with KIP- 163 beads enhances lentivirus (LV) transduction efficiency in pre-stimulated CAR-T cells requiring second round of stimulation. FIG. 20B is a chart showing that incubation of CAR-T cells with KIP4-163 beads enriches for CAR-expressing T cells. NTD: non-transduced; TD: CD19BBz transduced T cells; B2MK0: beta-2-microglobulin knockout cells. *=p<.05, **=p<.01. ns is non-significant.

FIGs. 21A - 21D show results from real-time xCelligence impedance analysis and ELISA for CAR-T cells incubated overnight with KIP4- 163 -conjugated beads, which leads to anti-CD19BBz CAR-T cell activation and secretion of IL-2 and IFNy, while preserving targetspecific cytotoxicity and safety against normal cells. FIG. 21A is a graph of the eSight real-time xCelligence impedance analysis for the conditions indicated (n = 3 replicates, same donor). FIG. 21B is a graph of the eSight real-time xCelligence impedance analysis for the conditions indicated (n = 3 replicates, same donor). FIG. 21C is a graph of the eSight real-time xCelligence impedance analysis for the conditions indicated (n = 3 replicates, same donor). FIG. 21D shows charts of ELISA assay results for the conditions indicated (n = 2 replicates). *=p<.05, **=p<.01, ***=p< 001, ****=p< 0001. ns is non-significant.

FIGs. 22A - 22E show that long-term (7 days) incubation of KIP4- 163 -conjugated beads with CAR-T cells significantly increases early and late T cell activation markers. FIG. 22A is a heatmap showing activation marker expression levels for the conditions indicated. FIG. 22B is a chart of flow staining analysis (n = 3 donors, 2-way ANOVA, post hoc Tukey) showing % CD4 versus CD8 T cell subset expression for the conditions indicated. FIG. 22C is a chart of flow staining analysis (n = 3 donors, 2-way ANOVA, post hoc Tukey) showing % CD25 expression for the conditions indicated. FIG. 22D is a chart of flow staining analysis (n = 3 donors, 2-way ANOVA, post hoc Tukey) showing % CD69 expression for the conditions indicated. FIG. 22EC is a chart of flow staining analysis (n = 3 donors, 2-way ANOVA, post hoc Tukey) showing % HLA-DR expression for the conditions indicated. *=p<.05, **=p<.01, ***=p< 001, ****=p< 0001. ns is non-significant.

FIGs. 23A - 23E show that long-term (7 days) incubation of KIP4- 163 -conjugated beads with CAR-T cells does not impact T cell memory profile. FIG. 23A is a heatmap showing relative phenotypes for the conditions indicated. FIG. 23B is a chart of flow staining analysis (n = 3 donors, 2-way ANOVA, post hoc Tukey) showing % effector T cells for the conditions indicated. FIG. 23C is a chart of flow staining analysis (n = 3 donors, 2-way ANOVA, post hoc Tukey) showing % naive T cells for the conditions indicated. FIG. 23D is a chart of flow staining analysis (n = 3 donors, 2-way ANOVA, post hoc Tukey) showing % TCM -TSCM T cells for the conditions indicated. FIG. 23E is a chart of flow staining analysis (n = 3 donors, 2-way ANOVA, post hoc Tukey) showing % TEM T cells for the conditions indicated. *=p< 05, **=p< 01, ***=p< 001, ****=p< 0001. ns is non-significant.

FIGs. 24A - 24F show that long-term (7 days) incubation of KIP4- 163 -conjugated beads with CAR-T cells induces mild-to-moderate exhaustion of CAR-T cells as compared to unstimulated or 5 days CD3/CD28 bead activated and exhausted CAR-T cells. FIG. 24A is a heatmap showing exhaustion markers expression for the conditions indicated at day 8 post stim. FIG. 24B is a chart of flow staining analysis (n = 3 donors, 2-way ANOVA, post hoc Tukey) showing % CD39⁺_KLRGT T cells for the conditions indicated. FIG. 24C is a chart of flow staining analysis (n = 3 donors, 2-way ANOVA, post hoc Tukey) showing % EOMES _CD39⁺ T cells for the conditions indicated. FIG. 24D is a chart of flow staining analysis (n = 3 donors, 2- way ANOVA, post hoc Tukey) showing % PD-1 _KLRG1 ⁺ T cells for the conditions indicated. FIG. 24E is a chart of flow staining analysis (n = 3 donors, 2-way ANOVA, post hoc Tukey) showing % PD-1⁺_TIM3⁺ T cells for the conditions indicated. FIG. 24F is a chart of flow staining analysis (n = 3 donors, 2-way ANOVA, post hoc Tukey) showing % EOMES _Tbet⁺ T cells for the conditions indicated. *=p< 05, **=p< 01, ***=p< 001, ****=p< 0001. ns is non- significant.

FIGs. 25A - 25D show data from tSNE clustering and x-shift clustering of exhaustion marker panels of T cells derived from 3 donors and stimulated with CD3/CD28 beads for 5 days, KIP4-163 beads for 7 days, or none. FIG. 25A is the cluster map. FIG. 25B is a chart plotting the % events by cluster. FIG. 25C is a heatmap that details the color-coded cluster expression profiles. FIG. 25D shows cluster maps for the tSNE-clustering based on CD4 or CD8 expression, as indicated.

FIGs. 26A - 26L show representative tSNE clustering of exhaustion markers taken from T cells derived from the same donor. FIG. 26A: Representative x-shift clusters of exhaustion panel from one donor plotted as EOMES versus CD39 expression. FIG. 26B: Representative x- shift clusters of exhaustion panel from one donor plotted as EOMES versus CD39 expression. FIG. 26C: Representative x-shift clusters of exhaustion panel from one donor plotted as tSNE plots based on KLRG-1 exhaustion marker single expression profile. FIG. 26D: Representative x-shift clusters of exhaustion panel from one donor plotted as tSNE plots based on KLRG-1 exhaustion marker single expression profile. FIG. 26E: Representative x-shift clusters of exhaustion panel from one donor plotted as tSNE plots based on PD-1 exhaustion marker single expression profile. FIG. 26F: Representative x-shift clusters of exhaustion panel from one donor plotted as tSNE plots based on PD-1 exhaustion marker single expression profile. FIG. 26G: Representative x-shift clusters of exhaustion panel from one donor plotted as tSNE plots based on TIM-3 exhaustion marker single expression profile. FIG. 26H: Representative x-shift clusters of exhaustion panel from one donor plotted as tSNE plots based on TIM-3 exhaustion marker single expression profile. FIG. 261: Representative x-shift clusters of exhaustion panel from one donor plotted as tSNE plots based on CD27 exhaustion marker single expression profile. FIG. 26 J: Representative x-shift clusters of exhaustion panel from one donor plotted as tSNE plots based on CD27 exhaustion marker single expression profile. FIG. 26K: Representative x-shift clusters of exhaustion panel from one donor plotted as tSNE plots based on Tbet exhaustion marker single expression profile. FIG. 26L: Representative x-shift clusters of exhaustion panel from one donor plotted as tSNE plots based on Tbet exhaustion marker single expression profile.

DETAILED DESCRIPTION

The present invention relates generally to compositions, methods and kits for augmenting anti-tumor efficacy of CAR adoptive cell therapies. The invention includes an anti-single-chain variable fragment (scFv) antibody or antigen-binding fragment, wherein the scFv is an scFv of a CAR, and uses thereof for targeting one or more immunostimulatory cytokines to modified immune cells which express the CAR. The invention includes an antibody-linked cytokine, wherein the antibody is the anti-scFv antibody or antigen-binding fragment, and use thereof in a method of treating cancer in a subject in need thereof. The invention further includes a lipid nanoparticle (LNP) harboring nucleoside-modified RNA(s) encoding at least one immunostimulatory cytokine, wherein the LNP comprises the anti-scFv antibody or antigenbinding fragment linked to the surface of the LNP, and use thereof in a method of treating cancer in a subject in need thereof. The invention further includes pharmaceutical compositions and kits comprising the antibody-linked cytokine of the invention or the LNP of the invention.

In one aspect, the invention includes an anti-single-chain variable fragment (scFv) antibody or antigen-binding fragment, wherein the anti-scFv antibody or antigen-binding fragment is capable of specifically binding to a linker peptide of an scFv, wherein the linker peptide links a variable light chain (VL) of the scFv to a variable heavy chain (VH) of the scFv, and further wherein the scFv is an scFv of a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain comprising the scFv, a transmembrane domain, and an intracellular domain.

In one aspect, the invention includes an antibody-linked cytokine, comprising an immunostimulatory cytokine linked to an anti-scFv antibody or anti gen -binding fragment, wherein the anti-scFv antibody or antigen-binding fragment is capable of specifically binding to a linker peptide of an scFv, wherein the linker peptide links a variable light chain (VL) of the scFv to a variable heavy chain (VH) of the scFv, and further wherein the scFv is an scFv of a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain comprising the scFv, a transmembrane domain, and an intracellular domain. The invention further includes use of the antibody -linked cytokine of the invention for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the antibody linked cytokine to the subject, and further wherein the administering is performed before, simultaneously with, or after infusion of the subject with a population of modified immune cells which express the CAR.

In one aspect, the invention includes a lipid nanoparticle (LNP) comprising: (a) at least one nucleoside-modified ribonucleic acid (RNA) encoding at least one immunostimulatory cytokine, wherein each immunostimulatory cytokine is encoded by a distinct nucleoside- modified RNA; (b) at least one ionizable lipid; and (c) an anti-scFv antibody or antigen-binding fragment, wherein the anti-scFv antibody or antigen-binding fragment is capable of specifically binding to a linker peptide of an scFv, wherein the linker peptide links a variable light chain (VL) of the scFv to a variable heavy chain (VH) of the scFv, and further wherein the scFv is an scFv of a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain comprising the scFv, a transmembrane domain, and an intracellular domain; wherein the at least one ionizable lipid at least partially encapsulates the at least one nucleoside-modified RNA; and further wherein the anti-scFv antibody or antigen-binding fragment is linked to the surface of the LNP. The invention further includes use of the LNP of the invention for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the LNP to the subject, and further wherein the administering is performed before, simultaneously with, or after infusion of the subject with a population of modified immune cells which express the CAR.

In one aspect, the invention includes a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody -linked cytokine of the invention or a therapeutically effective amount of the LNP of the invention, wherein the administering is performed before, simultaneously with, or after infusion of the subject with a population of modified immune cells which express a CAR.

The invention further includes a recombinant IL -23, wherein the recombinant IL-23 is engineered to be expressed as a single polypeptide chain.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice of and/or for the testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used according to how it is defined, where a definition is provided. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

It is also to be understood that the methods described in this disclosure are not limited to particular methods and experimental conditions disclosed herein as such methods and conditions may vary.

Furthermore, the experiments described herein, unless otherwise indicated, use conventional molecular and cellular biological and immunological techniques within the skill of the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, N.Y. (1987-2008), including all supplements, Molecular Cloning: A Laboratory Manual (Fourth Edition) by MR Green and J. Sambrook, and Harlow et al., Antibodies: A Laboratory Manual, Chapter 14, Cold Spring Harbor Laboratory, Cold Spring Harbor (2013, 2nd edition).

Unless otherwise defined, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. In the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The use of “or” means “and/or” unless stated otherwise. The use of the term “including,” as well as other forms, such as “includes” and “included,” is not limiting.

Generally, nomenclature used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein is well-known and commonly used in the art. The methods and techniques provided herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. Enzymatic reactions and purification techniques are performed according to manufacturer’s specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well- known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

Compounds of the present disclosure may contain one or more asymmetric centers and may thus give rise to enantiomers, diastereomers, and other stereoisomeric forms. The present disclosure is meant to encompass all such possible forms, as well as their racemic and resolved forms and mixtures thereof.

So that the disclosure may be more readily understood, select terms are defined below. The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

“About,” as used herein, when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20% or ±10%, in some instances ±5%, in some instances ±1%, and in some instance ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

“Activation,” as used herein, refers to the state of a T cell that has been sufficiently stimulated to induce detectable cellular proliferation. Activation can also be associated with induced cytokine production, and detectable effector functions. The term “activated T cells” refers to, among other things, T cells that are undergoing cell division.

As used herein, to “alleviate” a disease means reducing the severity of one or more symptoms of the disease.

The term “antibody” or “Ab” or “immunoglobulin” are terms of art and can be used interchangeably and refer to a protein, or polypeptide sequence which is or is derived from an immunoglobulin molecule having at least one antigen binding site which specifically binds to a specific epitope on an antigen (See, e.g., Harlow et al., 1998, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins. The antibodies useful in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chainantibody heavy chain pair, intrabodies, hetero-conjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single chain Fv (scFv), nanobodies, intracellular antibodies, intrabodies, camelized antibodies, camelid antibodies, IgNAR antibodies, affybodies, Fab fragments, F(ab') fragments, F(ab)2, disulfide-linked Fvs (sdFv), anti- idiotypic (anti-Id) antibodies (including, e.g., anti-anti-Id antibodies), and antigen-binding fragments of any of the above. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA or IgY), any class, (e.g., IgGl, IgG2, IgG3, IgG4, IgAl or IgA2), or any subclass (e.g., IgG2a or IgG2b) of immunoglobulin molecule. In certain embodiments, antibodies described herein are IgG antibodies, or a class (e.g., human IgGl or IgG4) or subclass thereof. Full-length antibodies are typically tetramers comprising two heavy chain and two light chain immunoglobulin molecules.

As used herein, the term “monoclonal antibody” refers to an antibody obtained from a population of homogenous or substantially homogeneous antibodies. The term “monoclonal” is not limited to any particular method for making the antibody. Generally, a population of monoclonal antibodies can be generated by cells, a population of cells, or a cell line. In specific embodiments, a “monoclonal antibody,” as used herein, is an antibody produced by a single cell (e.g., a hybridoma or host cell producing a recombinant antibody), wherein the antibody binds to a coronavirus spike protein epitope (e.g., an epitope of a SARS-CoV-2 spike protein receptor binding domain (RBD)) as determined, e.g., by ELISA or other antigen-binding or competitive binding assay known in the art or in the Examples provided herein. In particular embodiments, a monoclonal antibody can be a chimeric antibody, a human antibody, or a humanized antibody. Methods for generating a humanized antibody are known in the art. In certain embodiments, a monoclonal antibody is a monovalent antibody or multivalent (e.g., bivalent) antibody. In particular embodiments, a monoclonal antibody is a monospecific or multi-specific antibody (e.g., bispecific antibody). Monoclonal antibodies described herein can, for example, can be made by the hybridoma method as described in Kohler et al.; Nature, 256:495 (1975) or can be isolated from phage libraries, for example. Other methods for the preparation of clonal cell lines and of monoclonal antibodies expressed thereby are well known in the art (see, for example, Chapter 11 in: Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel et al., eds., John Wiley and Sons, New York). Monoclonal antibodies may be identified by high-throughput direct sequencing of fully recombined VDJ sequences of B cell receptor (BCR) repertoires from single cells of animals immunized with an antigen for which the desired monoclonal antibody will specifically bind as described herein. See, e.g. Goldstein et al., Communications Biology (2019)2:304; Homs et al., Cell Reports (2020) 30:905-913). The identified monoclonal antibodies are then produced recomb inantly.

The terms “antibody fragment,” “antigen-binding fragment,” and “antigen-binding domain” are used interchangeably and refer to at least one portion of an intact antibody, or recombinant variants thereof, and comprising or consisting of the antigen-binding domain, e.g., an antigenic determining variable region of an intact antibody, that is sufficient to confer recognition and specific binding of the antibody fragment to a target, such as an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, scFv, linear antibodies, single domain antibodies such as sdAb (either VL or VH), VHH domains, and multi-specific (e.g., bi specific) antibodies formed from antibody fragments. The term “scFv” refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain (variable light chain, VL) and at least one antibody fragment comprising a variable region of a heavy chain (variable heavy chain, VH), wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N- terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL.

An “antibody heavy chain,” as used herein, refers to the larger of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.

An “antibody light chain,” as used herein, refers to the smaller of the two types of polypeptide chains present in antibody molecules in their naturally occurring conformations. Antibodies and antibody fragments may be generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage or by a synthetic DNA or RNA molecule encoding the antibody. Said DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art. The RNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the RNA has been obtained by transcribing DNA (synthetic or cloned) or other technology, which is available and well known in the art.

“Humanized” forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies can comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications are made to further refine and optimize antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321 : 522-525, 1986; Reichmann et al., Nature, 332: 323-329, 1988; Presta, Curr. Op. Struct. Biol., 2: 593-596, 1992.

The term “antigen” or “Ag” as used herein is defined as a molecule that provokes an adaptive immune response. This immune response may involve either antibody production, or the activation of specific immunogenically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA or RNA. A skilled artisan will understand that any DNA or RNA, which comprises a nucleotide sequence or a partial nucleotide sequence encoding a protein that elicits an adaptive immune response therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated or synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell or a biological fluid.

As used herein, the term “autologous” refers to any material derived from the same individual to which it is later to be re-introduced into the individual.

A “co- stimulatory molecule” refers to the cognate binding partner on a T cell that specifically binds with a co-stimulatory ligand, thereby mediating a co-stimulatory response by the T cell, such as, but not limited to, proliferation. Co-stimulatory molecules include, but are not limited to an MHC class I molecule, BTLA and a Toll ligand receptor.

A “co-stimulatory signal”, as used herein, refers to a signal, which in combination with a primary signal, such as TCR/CD3 ligation, leads to T cell proliferation and/or upregulation or downregulation of key molecules.

A “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate. In contrast, a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.

An “effective amount” as used herein, means an amount which provides a therapeutic or prophylactic benefit.

“Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an RNA (such as an mRNA), to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

“Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) RNA, and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

The term “epitope” as used herein is defined as a small chemical molecule on an antigen that can elicit an immune response, inducing B and/or T cell responses. An antigen can have one or more epitopes. Most antigens have many epitopes; i.e., they are multivalent. In general, an epitope is roughly about 10 amino acids and/or sugars in size. Preferably, the epitope is about 4- 18 amino acids, more preferably about 5-16 amino acids, and even more most preferably 6-14 amino acids, more preferably about 7-12, and most preferably about 8-10 amino acids. One skilled in the art understands that generally the overall three-dimensional structure, rather than the specific linear sequence of the molecule, is the main criterion of antigenic specificity and therefore distinguishes one epitope from another. Based on the present disclosure, a peptide used in the present invention can be an epitope.

The term “expand” as used herein refers to increasing in number, as in an increase in the number of T cells. In one embodiment, the T cells that are expanded ex vivo increase in number relative to the number originally present in the culture. In another embodiment, the T cells that are expanded ex vivo increase in number relative to other cell types in the culture. The term “ex vivo," as used herein, refers to cells that have been removed from a living organism, (e.g., a human) and propagated outside the organism (e.g., in a culture dish, test tube, or bioreactor).

The term “expression” as used herein is defined as the transcription and/or translation of a particular nucleotide sequence driven by its promoter. “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector comprises sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., Sendai viruses, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

“Identity” as used herein refers to the subunit sequence identity between two polymeric molecules particularly between two amino acid molecules, such as, between two polypeptide molecules. When two amino acid sequences have the same residues at the same positions; e.g., if a position in each of two polypeptide molecules is occupied by an arginine, then they are identical at that position. The identity or extent to which two amino acid sequences have the same residues at the same positions in an alignment is often expressed as a percentage. The identity between two amino acid sequences is a direct function of the number of matching or identical positions; e.g., if half (e.g., five positions in a polymer ten amino acids in length) of the positions in two sequences are identical, the two sequences are 50% identical; if 90% of the positions (e.g., 9 of 10), are matched or identical, the two amino acids sequences are 90% identical.

“Homologous” refers to the sequence similarity or sequence identity between two polypeptides or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The percent of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions compared X 100. For example, if 6 of 10 of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, a comparison is made when two sequences are aligned to give maximum homology.

The term “immune response” as used herein is defined as a cellular response to an antigen that occurs when lymphocytes identify antigenic molecules as foreign and induce the formation of antibodies and/or activate lymphocytes to remove the antigen. “Instructional material(s)” as used herein includes a publication, a recording, a diagram, or any other medium of expression that can be used to communicate the usefulness of a composition and/or compound of the invention in a kit. The instructional material may describe a method of using the composition and/or compound of the invention in a method of the invention. The instructional material of the kit may, for example, be affixed to a container that contains the compound and/or composition of the invention or be shipped together with a container that contains the compound and/or composition. Alternatively, the instructional material may be shipped separately from the container with the intention that the recipient uses the instructional material and the compound cooperatively. Delivery of the instructional material may be, for example, by physical delivery of the publication or other medium of expression communicating the usefulness of the kit, or may alternatively be achieved by electronic transmission, for example by means of a computer, such as by electronic mail, or download from a website.

“Isolated” means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.

By the term “modified” as used herein, is meant a changed state or structure of a molecule or cell of the invention. Molecules may be modified in many ways, including chemically, structurally, and functionally. Cells may be modified through the introduction of nucleic acids.

By the term “modulating,” as used herein, is meant mediating a detectable increase or decrease in the level of a response in a subject compared with the level of a response in the subject in the absence of a treatment or compound, and/or compared with the level of a response in an otherwise identical but untreated subject. The term encompasses perturbing and/or affecting a native signal or response thereby mediating a beneficial therapeutic response in a subject, such as, a human.

In the context of the present invention, the following abbreviations for the commonly occurring nucleosides (nucleobase bound to ribose or deoxyribose sugar via N-glycosidic linkage) are used. “A” refers to adenosine, “C” refers to cytidine, “G” refers to guanosine, “T” refers to thymidine, and “U” refers to uridine.

The term “oligonucleotide” typically refers to short polynucleotides. It will be understood that when a nucleotide sequence is represented by a DNA sequence (i.e., A, T, C, G), this also includes an RNA sequence (i.e., A, U, C, G) in which “U” replaces “T ”

Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns. In addition, the nucleotide sequence may contain modified nucleosides that are capable of being translated by translational machinery in a cell. For example, an mRNA where all of the uridines have been replaced with pseudouridine, 1 -methyl psuedouridine, or another modified nucleoside.

“Parenteral” administration of an immunogenic composition includes, e g., subcutaneous (s.c.), intraperitoneal, intradermal, intravenous (i.v.), intramuscular (i.m.), or intrastemal injection, or infusion techniques.

The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, “nucleic acid” and “polynucleotide” as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides” and which comprise one or more “nucleotide sequence(s)”. The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences (i.e., “nucleotide sequences”) which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence. Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types. “Polypeptides” include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others. The polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.

The term “operably linked” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the latter. For example, a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA or RNA sequences are contiguous and, where necessary to join two protein coding regions, in the same reading frame.

The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in situ, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

In certain instances, the polynucleotide or nucleic acid of the invention is a “nucleoside- modified nucleic acid” or “nucleoside modified RNA” which refers to a nucleic acid or RNA molecule comprising at least one modified nucleoside. A “modified nucleoside” refers to a nucleoside with a modification compared to a reference nucleoside. For example, over one hundred different nucleoside modifications have been identified in RNA (Rozenski, et al., 1999, The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197).

The term “pseudouridine” refers to the natural product which is a C-glycosyl pyrimidine that consists of uracil having a beta-D-ribofuranosyl residue attached at position 5 (i.e., 5-(beta- D-Ribofuranosyl)uracil). In some embodiments, the term refers to m'acp³!]/ (l-methyl-3-(3- amino-3 -carboxypropyl) pseudouridine. In another embodiment, the term refers to m¹⁽P (1- methylpseudouridine). In another embodiment, the term refers to \|/m (2'-O-methylpseudouridine. In another embodiment, the term refers to m⁵D (5-methyldihydrouridine). In another embodiment, the term refers to m³\|/ (3-methylpseudouridine). In another embodiment, the term refers to a pseudouridine moiety that is not further modified. In another embodiment, the term refers to a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the term refers to any other pseudouridine known in the art. Each possibility represents a separate embodiment of the present invention.

The term “promoter” as used herein is defined as a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a polynucleotide sequence. For example, the promoter that is recognized by bacteriophage RNA polymerase and is used to generate the mRNA by in vitro transcription.

By the terms “specifically binds” or “targets,” as used herein with respect to an antibody, including an scFv, e.g., an scFv of a CAR, is meant an antibody which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more other species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific. In another example, an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally. If an antibody is specific for epitope “A”, the presence of a molecule containing epitope A (or free, unlabeled A), in a reaction containing labeled “A” and the antibody, will reduce the amount of labeled A bound to the antibody.

By the term “stimulation,” is meant a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-beta, and/or reorganization of cytoskeletal structures, and the like.

A “stimulatory molecule,” as the term is used herein, means a molecule on a T cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell.

A “stimulatory ligand,” as used herein, means a ligand that when present on an antigen presenting cell (e.g., an aAPC, a dendritic cell, a B-cell, and the like) can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a T cell, thereby mediating a primary response by the T cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulatory ligands are well-known in the art and encompass, inter alia, an MHC Class I molecule loaded with a peptide, an anti-CD3 antibody, a superagonist anti-CD28 antibody, and a superagonist anti-CD2 antibody.

A “target site” or “target sequence” refers to a nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur. In some embodiments, a target sequence refers to a genomic nucleic acid sequence that defines a portion of a nucleic acid to which a binding molecule may specifically bind under conditions sufficient for binding to occur.

As used herein, the term “T cell receptor” or “TCR” refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR is composed of a heterodimer of an alpha (a) and beta (0) chain, although in some cells the TCR consists of gamma and delta (y/8) chains. TCRs may exist in alpha/beta and gamma/delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable and constant domain. In some embodiments, the TCR may be modified on any cell comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T cell, regulatory T cell, natural killer T cell, and gamma delta T cell.

The term “therapeutic” as used herein means a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, diminution, remission, or eradication of at least one sign or symptom of a disease or disorder.

The term “therapeutically effective amount” refers to the amount of the subject compound that will elicit the biological or medical response of a tissue, system, or subject that is being sought by the researcher, veterinarian, medical doctor or other clinician. The term “therapeutically effective amount” includes that amount of a compound that, when administered, is sufficient to prevent development of, or alleviate to some extent, one or more of the signs or symptoms of the disorder or disease being treated. The therapeutically effective amount will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.

To “treat” a disease or disorder as the term is used herein, means to reduce the frequency or severity of at least one sign or symptom of a disease or disorder experienced by a subject.

The term “transfected” or “transformed” or “transduced” as used herein refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

The phrase “under transcriptional control” or “operatively linked” as used herein means that the promoter is in the correct location and orientation in relation to a polynucleotide to control the initiation of transcription by RNA polymerase and expression of the polynucleotide.

A “vector” is a composition of matter which comprises an isolated nucleic acid and which can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term “vector” includes an autonomously replicating plasmid or a virus. The term should also be construed to include non-plasmid and non-viral compounds which facilitate transfer of nucleic acid into cells, such as, for example, polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated virus vectors, retroviral vectors, and the like.

Ranges: throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present invention that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present invention that consist essentially of, or consist of, the recited processing steps.

Anti-scFv antibodies

The present invention relates to compositions and methods for treating cancer in a subject in need thereof, wherein the methods and compositions comprise an anti-scFv antibody or antigen-binding fragment thereof. In certain embodiments, the anti-scFv antibody or antigen- binding fragment thereof is used to deliver one or more immunostimulatory cytokines (as an antibody-linked cytokine or as a lipid nanoparticle (LNP) comprising nucleoside-modified RNA encoding the immunostimulatory cytokine) to CAR-expressing immune cells (e.g., CAR T cells) in the subject, thereby enhancing the anti -tumor efficacy of the CAR adoptive therapy. Those skilled in the art will appreciate that the anti-scFv antibody or antigen-binding fragment will bind to any CAR having an scFv comprising the linker peptide epitope, and that binding of the anti- scFv antibody or antigen-binding fragment to the scFv of the CAR is not dependent upon the target antigen of the CAR or on the other domains of the CAR (e.g., the transmembrane or intracellular domain). In certain embodiments, the linker peptide epitope comprises, e.g., a (G₄S)₃ linker (GGGGSGGGGSGGGGS) (SEQ ID NO: 32), a (G₄S)₄ linker (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 57), or a Whitlow linker (GSTSGSGKPGSGEGSTKG) (SEQ ID NO: 58)). Further, skilled artisans will understand that only routine experimentation is needed to generate an scFv directed to a given tumor antigen, wherein the scFv comprises the linker peptide epitope (i.e., SEQ ID NO: 32, SEQ ID NO: 57, or SEQ ID NO: 58) to which the anti-scFv antibody or antigen-binding fragment of the invention binds.

In one aspect, the invention provides an anti-single-chain variable fragment (scFv) antibody or antigen-binding fragment thereof, wherein the anti-scFv antibody or antigen-binding fragment is capable of specifically binding to a linker peptide of an scFv, wherein the linker peptide links a variable light chain (VL) of the scFv to a variable heavy chain (VH) of the scFv, and further wherein the scFv is an scFv of a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain comprising the scFv, a transmembrane domain, and an intracellular domain.

In some embodiments, the anti-scFv antibody or antigen-binding fragment is a monoclonal antibody or antigen-binding fragment derived therefrom.

The anti-scFv antibody or antigen-binding fragment of the invention comprises a heavy chain variable region (VH) comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and a light chain variable region (VL) comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3).

In some embodiments, the HCDR1 comprises or consists of the amino acid sequence GFTFSDYY (SEQ ID NO: 59), the HCDR2 comprises or consists of the amino acid sequence ISDGGSYT (SEQ ID NO: 60), the HCDR3 comprises of consists of the amino acid sequence AREGDLGSFWFAFW (SEQ ID NO: 61), the LCDR1 comprises or consists of the amino acid sequence QDISNY (SEQ ID NO: 62), the LCDR2 comprises or consists of the amino acid sequence YTS (SEQ ID NO: 63), and the LCDR3 comprises or consists of the amino acid sequence QQGNTLP (SEQ ID NO: 64).

In some embodiments, the HCDR1 comprises or consists of the amino acid sequence GFSLTDYG (SEQ ID NO: 69), the HCDR2 comprises or consists of the amino acid sequence VWGGGST (SEQ ID NO: 70), the HCDR3 comprises of consists of the amino acid sequence AKLYGHYYIMDY (SEQ ID NO: 71), the LCDR1 comprises or consists of the amino acid sequence ESVDSYGNSF (SEQ ID NO: 72), the LCDR2 comprises or consists of the amino acid sequence LAS (SEQ ID NO: 73), and the LCDR3 comprises or consists of the amino acid sequence QQNNEDPFT (SEQ ID NO: 74).

In some embodiments, the HCDR1 comprises or consists of the amino acid sequence GFTFSSYG (SEQ ID NO: 79), the HCDR2 comprises or consists of the amino acid sequence ISSGGSYT (SEQ ID NO: 80), the HCDR3 comprises of consists of the amino acid sequence ARQDYGSPFAY (SEQ ID NO: 81), the LCDR1 comprises or consists of the amino acid sequence QDINKY (SEQ ID NO: 82), the LCDR2 comprises or consists of the amino acid sequence YTS (SEQ ID NO: 83), and the LCDR3 comprises or consists of the amino acid sequence LQYDNLLWT (SEQ ID NO: 84).

In some embodiments, the anti-scFv antibody or antibody fragment targets SEQ ID NO: 32 and SEQ ID NO: 58, wherein HCDR1 comprises or consists of the amino acid sequence GFTFSDYY (SEQ ID NO: 59), the HCDR2 comprises or consists of the amino acid sequence ISDGGSYT (SEQ ID NO: 60), the HCDR3 comprises of consists of the amino acid sequence AREGDLGSFWFAFW (SEQ ID NO: 61), the LCDR1 comprises or consists of the amino acid sequence QDISNY (SEQ ID NO: 62), the LCDR2 comprises or consists of the amino acid sequence YTS (SEQ ID NO: 63), and the LCDR3 comprises or consists of the amino acid sequence QQGNTLP (SEQ ID NO: 64).

In some embodiments, the anti-scFv antibody or antibody fragment targets SEQ ID NO: 32, wherein the HCDR1 comprises or consists of the amino acid sequence GFSLTDYG (SEQ ID NO: 69), the HCDR2 comprises or consists of the amino acid sequence VWGGGST (SEQ ID NO: 70), the HCDR3 comprises of consists of the amino acid sequence AKLYGHYYIMDY (SEQ ID NO: 71 ), the LCDR1 comprises or consists of the amino acid sequence ESVDSYGNSF (SEQ ID NO: 72), the LCDR2 comprises or consists of the amino acid sequence LAS (SEQ ID NO: 73), and the LCDR3 comprises or consists of the amino acid sequence QQNNEDPFT (SEQ ID NO: 74).

In some embodiments, the anti-scFv antibody or antibody fragment targets SEQ ID NO: 32 and SEQ ID NO: 58, wherein the HCDR1 comprises or consists of the amino acid sequence GFTFSSYG (SEQ ID NO: 79), the HCDR2 comprises or consists of the amino acid sequence ISSGGSYT (SEQ ID NO: 80), the HCDR3 comprises of consists of the amino acid sequence ARQDYGSPFAY (SEQ ID NO: 81), the LCDR1 comprises or consists of the amino acid sequence QDINKY (SEQ ID NO: 82), the LCDR2 comprises or consists of the amino acid sequence YTS (SEQ ID NO: 83), and the LCDR3 comprises or consists of the amino acid sequence LQYDNLLWT (SEQ ID NO: 84).

In certain embodiments, the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 67.

In some embodiments, the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 75 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 77.

In some embodiments, the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 86 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 88.

In certain embodiments, the anti-scFv antibody or antigen-binding fragment further comprises a heavy chain and a light chain, wherein the heavy chain comprises the VH and the light chain comprises the VL.

In certain embodiments, the anti-scFv antibody or antibody fragment targets SEQ ID NO: 32 and SEQ ID NO: 58, wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 67.

In some embodiments, the anti-scFv antibody or antibody fragment targets SEQ ID NO: 32, wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 75 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 77. In some embodiments, the anti-scFv antibody or antibody fragment targets SEQ ID NO: 32 and SEQ ID NO: 58, wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 86 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 88.

The anti-scFv antibody or antigen-binding fragment of the invention is capable of specifically binding to a linker peptide of an scFv (e.g., SEQ ID NO: 32, SEQ ID NO: 57, or SEQ ID NO: 58), wherein the scFv is the extracellular scFv of a CAR. In some embodiments, the scFv of the CAR targets (z.e., specifically binds) a tumor antigen. In certain embodiments, the tumor antigen is selected from the group consisting of alpha feto-protein (AFP)/HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD 13, CD 19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, claudin 6, c-Met, DLL3, DR5, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa)/folate binding protein (FBP), folate receptor beta (FRb), follicle-stimulating hormone receptor (FSHR), GD-2, Glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), HERZ, HLA-A2, ICAM1, interleukin 13 receptor subunit alpha (IL3Ra), interleukin 13 receptor subunit alpha 2 (IL13Ra2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE- A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4/FAP, NKG2D- Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), Mullerian inhibiting substance type 2 receptor (MISIIR), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), P16, PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), R0R1, R0R2, TAG72, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUCl), TROP2, Glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRa4; GFRalpha4), VEGFR2, and any combination thereof. In some embodiments, the scFv of the CAR targets TnMUCl.

The anti-scFv antibody or antigen-binding fragment of the invention is capable of specifically binding to a linker peptide of an scFv (e.g., SEQ ID NO: 32, SEQ ID NO: 57, or SEQ ID NO: 58), wherein the scFv is the extracellular scFv of a CAR. In some embodiments, the intracellular domain of the CAR comprises a costimulatory domain and an intracellular signaling domain.

In some embodiments of the anti-scFv antibody or antigen-binding fragment, the intracellular domain of the CAR comprises a costimulatory domain of a protein selected from the group consisting of proteins in the TNFR superfamily, CD28, 4-1BB (CD137), 0X40 (CD134), PD-1, CD7, LIGHT, CD83L, DAP10, DAP12, CD27, CD2, CD5, ICAM-1, LFA-1, Lek, TNFR- I, TNFR-II, Fas, CD30, CD40, ICOS, NKG2C, and B7-H3 (CD276), or a variant thereof, or an intracellular domain derived from a killer immunoglobulin-like receptor (KIR).

In some embodiments of the anti-scFv antibody or antigen-binding fragment, the intracellular domain of the CAR comprises an intracellular signaling domain of a protein selected from the group consisting of a human CD3 zeta chain (CD3Q, FcyRIII, FcsRI, DAP 10, DAP 12, a cytoplasmic tail of an Fc receptor, an immunoreceptor tyrosine-based activation motif (ITAM) bearing cytoplasmic receptor, TCR zeta, FcR gamma, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d, or a variant thereof.

In some embodiments of the anti-scFv antibody or antigen-binding fragment, the intracellular domain of the CAR comprises an intracellular signaling domain of CD3 zeta or a variant thereof.

In some embodiments of the anti-scFv antibody or antigen-binding fragment, the intracellular domain of the CAR comprises a costimulatory domain of a protein selected from CD28 and 4-1BB (CD137), or both CD28 and 4-1BB (CD137).

In some embodiments of the anti-scFv antibody or antigen-binding fragment, the intracellular domain of the CAR comprises a costimulatory domain of CD28 and an intracellular signaling domain of CD3 zeta.

In some embodiments of the anti-scFv antibody or antigen-binding fragment, the intracellular domain of the CAR comprises a costimulatory domain of 4-1BB (CD137) and an intracellular signaling domain of CD3 zeta.

The invention further provides a nucleic acid molecule comprising a nucleotide sequence encoding the anti-scFv antibody or antigen-binding fragment of the invention. In some embodiments, the nucleotide sequence encoding the anti-scFv antibody or antigen-binding fragment further encodes a ribosome slip sequence. In some embodiments, the ribosome slip sequence is selected from the group consisting of P2A, T2A, E2A, F2A, and variants thereof.

In various embodiments, the anti-scFv antibodies and antigen-binding fragments thereof of the invention are useful for activating CAR-expressing immune cells without affecting the cytotoxic potential or inducing exhaustion of said immune cells.

In various embodiments, the anti-scFv antibodies and antigen-binding fragments thereof of the invention are useful for enhancing viral transduction of CAR-expressing cells.

In various embodiments, the anti-scFv antibodies and antigen-binding fragments thereof of the invention are useful for enriching for CAR-expressing cells.

In various embodiments, the anti-scFv antibodies and antigen-binding fragments thereof of the invention (including bead conjugates thereof) are useful in kits and tools for development, generation, or purification of CAR-containing cells.

In various embodiments, the anti-scFv antibodies and antigen-binding fragments thereof of the invention are useful for delivering a payload (such as, but not limited to, a cytokine) to CAR-expressing cells, either by linking said payload directly to the anti-scFv antibody or antigen-binding fragment, or via a lipid nanoparticle (LNP), wherein the LNP comprises said payload and the LNP further comprises the anti-scFv antibody or antigen-binding fragment attached to the surface of the LNP.

Antibody-linked cytokines

In some aspects, the invention provides an antibody-linked cytokine, pharmaceutical compositions comprising the antibody-linked cytokine, and uses thereof in methods and kits for treating cancer in a subject in need thereof. The antibody -linked cytokines of the invention deliver an immunostimulatory cytokine to CAR-expressing immune cells (e.g., CAR T cells) via the linked anti-scFv antibody or antigen-binding fragment of the invention. The anti-scFv antibody binds to the extracellular scFv domain of the CAR, delivering the linked immunostimulatory cytokine to the CAR-expressing immune cells (e.g., CAR T cells) in the subject, thereby enhancing the anti-tumor efficacy of the CAR adoptive therapy. It is contemplated herein that, by enhancing the anti-tumor cytotoxicity of CAR-T cells, the antibody- linked cytokines of the invention permit the injection of lower doses of CAR-T cells, possibly lowering both off-tumor toxicity and cost associated with CAR-T cell therapy.

In one aspect, the invention provides an antibody-linked cytokine comprising an immunostimulatory cytokine linked to an anti-scFv antibody or antigen-binding fragment (e.g., the anti-scFv antibody or antigen-binding fragment of the invention, or e.g., KIP4-163), wherein the anti-scFv antibody is linked to the immunostimulatory cytokine via a linker peptide that is not recognized or bound by the anti-scFv antibody. In some embodiments, the anti-scFv antibody is linked to the immunostimulatory cytokine via a (G₄S)i linker (i.e., SEQ ID NO: 56). That is, the invention provides an antibody-linked cytokine, comprising an immunostimulatory cytokine linked to an anti-scFv antibody or antigen-binding fragment, wherein the anti-scFv antibody or antigen-binding fragment is capable of specifically binding to a linker peptide of an scFv (e.g., a (G₄S)3 linker (GGGGSGGGGSGGGGS) (SEQ ID NO: 32) and a (G₄S)₄ linker (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 57), or a Whitlow linker (GSTSGSGKPGSGEGSTKG) (SEQ ID NO: 58)), wherein the linker peptide links a variable light chain (VL) of the scFv to a variable heavy chain (VH) of the scFv, and further wherein the scFv is an scFv of a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain comprising the scFv, a transmembrane domain, and an intracellular domain. In certain embodiments, the anti-scFv antibody of the anti-scFv antibody-cytokine conjugate is KIP4-163 (see, e.g., W02019060713A1).

The anti-scFv antibody of the antibody-cytokine conjugate of the invention comprises a heavy chain variable region (VH) comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and a light chain variable region (VL) comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3). In some embodiments, the HCDR1 comprises or consists of the amino acid sequence GFTFSDYY (SEQ ID NO: 59), the HCDR2 comprises or consists of the amino acid sequence ISDGGSYT (SEQ ID NO: 60), the HCDR3 comprises of consists of the amino acid sequence AREGDLGSFWFAFW (SEQ ID NO: 61), the LCDR1 comprises or consists of the amino acid sequence QDISNY (SEQ ID NO: 62), the LCDR2 comprises or consists of the amino acid sequence YTS (SEQ ID NO:

63), and the LCDR3 comprises or consists of the amino acid sequence QQGNTLP (SEQ ID NO:

64). In some embodiments, the HCDR1 comprises or consists of the amino acid sequence GFSLTDYG (SEQ ID NO: 69), the HCDR2 comprises or consists of the amino acid sequence VWGGGST (SEQ ID NO: 70), the HCDR3 comprises of consists of the amino acid sequence AKLYGHYYIMDY (SEQ ID NO: 71), the LCDR1 comprises or consists of the amino acid sequence ESVDSYGNSF (SEQ ID NO: 72), the LCDR2 comprises or consists of the amino acid sequence LAS (SEQ ID NO: 73), and the LCDR3 comprises or consists of the amino acid sequence QQNNEDPFT (SEQ ID NO: 74). In some embodiments, the HCDR1 comprises or consists of the amino acid sequence GFTFSSYG (SEQ ID NO: 79), the HCDR2 comprises or consists of the amino acid sequence ISSGGSYT (SEQ ID NO: 80), the HCDR3 comprises of consists of the amino acid sequence ARQDYGSPFAY (SEQ ID NO: 81), the LCDR1 comprises or consists of the amino acid sequence QDINKY (SEQ ID NO: 82), the LCDR2 comprises or consists of the amino acid sequence YTS (SEQ ID NO: 83), and the LCDR3 comprises or consists of the amino acid sequence LQYDNLLWT (SEQ ID NO: 84). In some embodiments, the HCDR1 comprises or consists of the amino acid sequence KFSFNKKYYMC (SEQ ID NO: 1), the HCDR2 comprises or consists of the amino acid sequence WIGCVDTGDAFIGY (SEQ ID NO: 2), the HCDR3 comprises of consists of the amino acid sequence RGVYPINTGYYYFDL (SEQ ID NO: 3), the LCDR1 comprises or consists of the amino acid sequence EDITNSLA (SEQ ID NO: 4), the LCDR2 comprises or consists of the amino acid sequence NLLIYRASTLAS (SEQ ID NO: 5), and the LCDR3 comprises or consists of the amino acid sequence QQGYSSTNVDNI (SEQ ID NO: 6).

In certain embodiments, the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 65 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 67. In some embodiments, the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 75 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 77. In some embodiments, the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 86 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 88. In some embodiments, the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9. In certain embodiments, the anti- scFv antibody or antigen-binding fragment further comprises a heavy chain and a light chain, wherein the heavy chain comprises the VH and the light chain comprises the VL

In some embodiments, the immunostimulatory cytokine comprises any one IL-12, IL-18, and IL-23. In some embodiments, the immunostimulatory cytokine comprises IL-12. In some embodiments, the immunostimulatory cytokine comprises IL-18. In some embodiments, the immunostimulatory cytokine comprises IL-23.

In some embodiments of the antibody-linked cytokine, the immunostimulatory cytokine comprises a single polypeptide chain. In some embodiments, the at least one immunostimulatory cytokine is engineered to be expressed as a single polypeptide chain.

In certain embodiments of the antibody-linked cytokine, the immunostimulatory cytokine comprises a single chain version of TL-12, wherein a single linked polypeptide chain comprises both the IL-12A and IL-12B subunits of IL-12. In some embodiments, the IL-12 comprises or consists of SEQ ID NO: 19. In some embodiments, the IL-12 comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19. The invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding a single chain version of IL- 12, wherein a single linked polypeptide chain comprises both the IL-12A and IL-12B subunits of IL-12. In certain embodiments, the nucleic acid molecule comprises SEQ ID NO: 20. In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 20.

In certain embodiments of the antibody-linked cytokine, the immunostimulatory cytokine comprises IL-18. In some embodiments, the IL-18 comprises or consists of SEQ ID NO: 21. In some embodiments, the IL- 18 comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21. The invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding IL- 18. In certain embodiments, the nucleic acid molecule comprises SEQ ID NO: 22. In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 22.

In certain embodiments of the antibody-linked cytokine, the immunostimulatory cytokine comprises a single chain version of IL-23, wherein a single linked polypeptide chain comprises both the IL-23 A and IL-12B subunits of IL-23. In some embodiments, the IL-23 comprises or consists of SEQ ID NO: 23. In some embodiments, the IL-23 comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23. The invention also provides a nucleic acid molecule comprising a nucleotide sequence encoding a single chain version of IL-23, wherein a single linked polypeptide chain comprises both the IL-23A and IL-12B subunits of IL-23. In certain embodiments, the nucleic acid molecule comprises SEQ ID NO: 24. In certain embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 24.

In some embodiments of the antibody-linked cytokine, the anti-scFv antibody or antigenbinding fragment comprises a first polypeptide comprising the heavy chain and a second polypeptide comprising the light chain, wherein the N-terminus of the immunostimulatory cytokine is linked to the C-terminus of the first polypeptide comprising the heavy chain. In some embodiments, the immunostimulatory cytokine is linked to the first polypeptide comprising the heavy chain via a poly-alanine linker comprising two or more consecutive alanine residues. In some embodiments, the poly-alanine linker consists of two alanine residues. In some embodiments, the immunostimulatory cytokine is linked to the first polypeptide comprising the heavy chain via a glycine-serine linker.

In certain embodiments of the antibody-linked cytokine, the first polypeptide comprises a heavy chain comprising SEQ ID NO: 11, a polyalanine linker, and single chain IL-12 comprising SEQ ID NO: 19, and the second polypeptide comprises a light chain comprising SEQ ID NO: 13.

In certain embodiments of the antibody-linked cytokine, the first polypeptide comprises a heavy chain comprising SEQ ID NO: 11, a polyalanine linker, and IL-18 comprising SEQ ID NO: 21, and the second polypeptide comprises a light chain comprising SEQ ID NO: 13.

In certain embodiments of the antibody-linked cytokine, the first polypeptide comprises a heavy chain comprising SEQ ID NO: 11, a polyalanine linker, and single chain IL -23 comprising SEQ ID NO: 23, and the second polypeptide comprises a light chain comprising SEQ ID NO: 13.

In certain embodiments of the antibody-linked cytokine, the anti-scFv antibody or antigen-binding fragment further comprises a heavy chain signal peptide and/or a light chain signal peptide. In some embodiments, the heavy chain signal peptide comprises or consists of SEQ ID NO: 15 and/or the light chain signal peptide comprises or consists of SEQ ID NO: 17.

The invention further provides a nucleic acid molecule comprising a nucleotide sequence encoding the antibody-linked cytokine of the invention. In some embodiments, the nucleotide sequence encoding the antibody-linked cytokine comprises a nucleotide sequence encoding a ribosome slip sequence. In some embodiments, the ribosome slip sequence is selected from the group consisting of P2A, T2A, E2A, F2A, and variants thereof. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 26. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 28. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 26. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 28. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least

99% sequence identity to SEQ ID NO: 30.

Those of skill in the art will understand that various isoforms and splice variants of the immunostimulatory cytokines exist, many of which have similar and/or identical biological functions. The invention includes all such isoforms and splice variants of the immunostimulatory cytokines.

Table 1 provides exemplary nucleotide and amino acid sequences of the invention.

Table 1: Exemplary nucleotide and amino acid sequences

In certain embodiments, the at least one immunostimulatory cytokine comprises or consists of IL-12. In some embodiments, the IL-12 comprises or consists of SEQ ID NO: 19. In certain embodiments the IL- 12 comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least

88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least

95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:

19.

In certain embodiments, the at least one immunostimulatory cytokine comprises or consists of IL-18. In some embodiments, the IL-18 comprises or consists of SEQ ID NO: 21. In certain embodiments the IL-18 comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least

21.

In certain embodiments, the at least one immunostimulatory cytokine comprises or consists of IL-23. In some embodiments, the IL-23 comprises or consists of SEQ ID NO: 23. In certain embodiments the IL-23 comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23.

In some embodiments, the immunostimulatory cytokine comprises more than one domain or subunit (e.g., two or more subunits) of the immunostimulatory cytokine. In certain embodiments, the two or more subunits of the immunostimulatory cytokine can be expressed as a fusion protein. In some embodiments, the nucleoside-modified RNA encodes a first subunit of the immunostimulatory cytokine and a second subunit of the immunostimulatory cytokine, wherein the first subunit and the second subunit are linked to each other via a flexible linker, such as a glycine serine linker or polyalanine linker.

The nucleotide sequence(s) encoding the immunostimulatory cytokine may be derived from any animal which expresses the immunostimulatory cytokine. Non-limiting examples include a mouse, a rat, a pig, a simian, and a human. In certain embodiments, the immunostimulatory cytokine is an engineered and/or variant version of a naturally-occurring immunostimulatory cytokine. Such engineered immunostimulatory cytokines include recombinant, edited, tagged, and/or fusion immunostimulatory cytokines.

In certain embodiments, the immunostimulatory cytokine is a variant of a naturally occurring immunostimulatory cytokine. Tolerable variations of the nucleotide and amino acid sequences of the immunostimulatory cytokines will be known to those of skill in the art. For example, in certain embodiments the immunostimulatory cytokine or a subunit thereof comprises an amino acid sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least

84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least

91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

98%, or at least 99% sequence identity to any naturally-occurring or known reference amino acid sequence of the immunostimulatory cytokine or subunit thereof.

In other embodiments, the immunostimulatory cytokine or a subunit thereof is encoded by a nucleotide sequence that has at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any naturally-occurring or known reference nucleotide sequence encoding the immunostimulatory cytokine or subunit thereof. Lipid Nanoparticles

The present invention relates to compositions and methods for treating cancer in a subject in need thereof. In some aspects, the invention provides a lipid nanoparticle (LNP), pharmaceutical compositions comprising the LNP and uses thereof in methods and kits for treating cancer in a subject in need thereof. The LNPs of the invention are directed to deliver immunostimulatory cytokines to CAR-expressing immune cells (e.g., CAR T cells) via a surface-linked anti-scFv antibody or antigen-binding fragment of the invention. The anti-scFv antibody binds to the extracellular scFv domain of the CAR, delivering the LNP comprising nucleoside-modified RNA(s) encoding immunostimulatory cytokine(s) to the CAR-expressing immune cells (e.g., CAR T cells) in the subject, thereby enhancing the anti -tumor efficacy of the CAR adoptive therapy.

In one aspect, the invention provides an LNP comprising: (a) at least one nucleoside- modified ribonucleic acid (RNA) encoding at least one immunostimulatory cytokine, wherein each immunostimulatory cytokine is encoded by a distinct nucleoside-modified RNA; (b) at least one ionizable lipid; and (c) an anti-scFv antibody or antigen-binding fragment, wherein the anti- scFv antibody or antigen-binding fragment is capable of specifically binding to a linker peptide of an scFv, wherein the linker peptide links a variable light chain (VL) of the scFv to a variable heavy chain (VH) of the scFv, and further wherein the scFv is an scFv of a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain comprising the scFv, a transmembrane domain, and an intracellular domain; wherein the at least one ionizable lipid at least partially encapsulates the at least one nucleoside-modified RNA; and further wherein the anti-scFv antibody or antigen-binding fragment is linked to the surface of the LNP.

In some embodiments, the at least one immunostimulatory cytokine comprises any one or more of IL-12, IL-18, and IL-23. In some embodiments, the at least one immunostimulatory cytokine comprises IL- 12. In some embodiments, the at least one immunostimulatory cytokine comprises IL-18. In some embodiments, the at least one immunostimulatory cytokine comprises IL-23. In some embodiments, the at least one immunostimulatory cytokine comprises IL-12 and IL-18. In some embodiments, the at least one immunostimulatory cytokine comprises IL-12 and IL-23. In some embodiments, the at least one immunostimulatory cytokine comprises IL-18 and IL-23. In some embodiments, the at least one immunostimulatory cytokine comprises IL-12, IL- 18, and IL-23. In some embodiments, the at least one immunostimulatory cytokine comprises a single polypeptide chain. In some embodiments, the at least one immunostimulatory cytokine comprises two polypeptide chains. In some embodiments, the at least one immunostimulatory cytokine is engineered to be expressed as a single polypeptide chain.

In certain embodiments, the LNP comprises a first nucleoside-modified RNA encoding an IL-12A (p35) subunit of IL-12 and a second nucleoside-modified RNA encoding an IL-12B (p40) subunit of IL-12. In some embodiments, the LNP comprises a nucleoside-modified RNA encoding a single chain version of IL-12, wherein a single linked polypeptide chain comprises both the IL-12A and IL-12B subunits of IL-12. In certain embodiments, the LNP comprises a nucleoside-modified RNA comprising SEQ ID NO: 20. In certain embodiments, the LNP comprises a nucleoside-modified RNA comprising a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 20. In some embodiments, the IL-12 comprises or consists of SEQ ID NO: 19. In some embodiments, the IL- 12 comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19.

In certain embodiments, the LNP comprises a nucleoside-modified RNA encoding IL-18. In certain embodiments, the LNP comprises a nucleoside-modified RNA comprising SEQ ID NO: 22. In certain embodiments, the LNP comprises a nucleoside-modified RNA comprising a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 22. In some embodiments, the IL-18 comprises or consists of SEQ ID NO: 21. In some embodiments, the IL- 18 comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21.

In certain embodiments, the LNP comprises a first nucleoside-modified RNA encoding an IL-23A (pl9) subunit of IL-23 and a second nucleoside-modified RNA encoding an IL-12B (p40) subunit of IL-12/IL-23. In some embodiments, the LNP comprises a nucleoside-modified RNA encoding a single chain version of IL-23, wherein a single linked polypeptide chain comprises both the IL-23A and IL-12B subunits of IL-23. In certain embodiments, the LNP comprises a nucleoside-modified RNA comprising SEQ ID NO: 24. In certain embodiments, the LNP comprises a nucleoside-modified RNA comprising a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 24. In some embodiments, the IL-23 comprises or consists of SEQ ID NO: 23. In some embodiments, the IL-23 comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23.

In some embodiments, the at least one nucleoside-modified RNA is messenger RNA (mRNA). In some embodiments, the at least one nucleoside-modified RNA comprises pseudouridine and/or 1-methyl-pseudouridine. In some embodiments, the at least one nucleoside- modified RNA is in vitro transcribed (IVT) RNA. In certain embodiments, the at least one nucleoside-modified RNA is IVT mRNA comprising pseudouridine and/or 1-methyl- pseudouridine.

In some embodiments, the at least one ionizable lipid encapsulates the at least one nucleoside-modified RNA. In certain embodiments, the at least one ionizable lipid is a cationic lipid. Non-limiting examples of cationic lipids are described herein.

High efficiency and improved specificity for the in vivo delivery of the RNA cargo can be achieved with the use of optimally formulated lipid nanoparticles and incorporation of cellspecific antibodies to minimize potential off-target effects, as recently demonstrated [Rurik JG, et al. Science. 2022 Jan 7;375(6576):91-96], The present invention utilizes an anti-scFv antibody or antigen-binding fragment to target the LNP and the immunostimulatory cytokines expressed therefrom to the modified immune cells (e.g., T cells) expressing a CAR.

The term “lipid nanoparticle” refers to a particle having at least one dimension on the order of nanometers (e.g., 1-1,000 nm), which includes one or more lipids, for example a lipid of Formula (I), (II) or (III). In some embodiments, lipid nanoparticles are included in a formulation comprising a nucleoside-modified RNA as described herein. In some embodiments, such lipid nanoparticles comprise a cationic lipid (e.g., a lipid of Formula (I), (II) or (III)) and one or more excipient selected from neutral lipids, charged lipids, steroids and polymer conjugated lipids (e.g., a pegylated lipid such as a pegylated lipid of structure (IV), such as compound iVa). In some embodiments, the nucleoside-modified RNA is encapsulated in the lipid portion of the lipid nanoparticle or an aqueous space enveloped by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other undesirable effects induced by the mechanisms of the host organism or cells, e.g., an adverse immune response.

In various embodiments, the lipid nanoparticles have a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, and are substantially non-toxic. In certain embodiments, the nucleoside- modified RNA, when present in the lipid nanoparticles, is resistant in aqueous solution to degradation with a nuclease.

The LNP may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated. The term “lipid” refers to a group of organic compounds that are derivatives of fatty acids (e.g., esters) and are generally characterized by being insoluble in water but soluble in many organic solvents. Lipids are usually divided in at least three classes: (1) “simple lipids” which include fats and oils as well as waxes; (2) “compound lipids” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids.

In some embodiments, the LNP comprises at least one ionizable lipid. In some embodiments, the ionizable lipid is a cationic lipid.

In some embodiments, the LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.

In some embodiments, the LNP comprises a cationic lipid. As used herein, the term “cationic lipid” refers to a lipid that is cationic or becomes cationic (protonated) as the pH is lowered below the pK of the ionizable group of the lipid, but is progressively more neutral at higher pH values. At pH values below the pK, the lipid is then able to associate with negatively charged nucleic acids. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that assumes a positive charge on pH decrease.

In certain embodiments, the cationic lipid comprises any of a number of lipid species which carry a net positive charge at a selective pH, such as physiological pH. Such lipids include, but are not limited to, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N- (2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N- dimethylammonium bromide (DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N — (N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N- (l-(2,3-dioleoyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoracetate (DOSPA), dioctadecylamidoglycyl carboxy spermine (DOGS), l,2-dioleoyl-3- dimethylammonium propane (DODAP), N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), and N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE). Additionally, a number of commercial preparations of cationic lipids are available which can be used in the present invention. These include, for example, LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1,2- dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO/BRL, Grand Island, N.Y.); LIPOFECT AMINE® (commercially available cationic liposomes comprising N-(l-(2,3- dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium tri fluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.). The following lipids are cationic and have a positive charge at below physiological pH: DODAP, DODMA, DMDMA, l,2-dilinoleyloxy-N,N- dimethylaminopropane (DLinDMA), 1 ,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

In some embodiments, the cationic lipid is an amino lipid. Suitable amino lipids useful in the invention include those described in WO 2012/016184, incorporated herein by reference in its entirety. Representative amino lipids include, but are not limited to, 1, 2-dilinoley oxy-3 - (dimethylamino)acetoxypropane (dLin-DAC), l,2-dilinoleyoxy-3-morpholinopropane (dLin- MA), l,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3- dimethylaminopropane (dLin-S-DMA), l-linoleoyl-2-linoleyloxy-3 -dimethylaminopropane (dLin-2-DMAP), l,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (dLin-TMA.Cl), 1,2- dilinoleoyl-3-trimethylaminopropane chloride salt (dLin-TAP.Cl), l,2-dilinoleyloxy-3-(N- methylpiperazino)propane (dLin-MPZ), 3-(N,N-dilinoleylamino)- 1,2-propanediol (dLinAP), 3 - (N,N-dioleylamino)- 1,2-propanediol (DOAP), l,2-dilinoleyloxo-3-(2-N,N- di methyl ami no)ethoxypropane (dLin-EG-DMA), and 2,2-dilinoleyl-4-dimethylaminomethyl- [1,3] -di oxolane (dLin-K-DMA).

Suitable amino lipids include those having the formula:

wherein Ri and R2 are either the same or different and independently optionally substituted C10-C24 alkyl, optionally substituted C10-C24 alkenyl, optionally substituted C10-C24 alkynyl, or optionally substituted C10-C24 acyl;

R3 and R4 are either the same or different and independently optionally substituted Ci-Ce alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl or R? and R4 may join to form an optionally substituted heterocyclic ring of 4 to 6 carbon atoms and 1 or 2 heteroatoms chosen from nitrogen and oxygen;

R5 is either absent or present and when present is hydrogen or Ci-Ce alkyl; m, n, and p are either the same or different and independently either 0 or 1 with the proviso that m, n, and p are not simultaneously 0; q is 0, 1, 2, 3, or 4; and

Y and Z are either the same or different and independently O, S, or NH.

In some embodiments, Ri and R2 are each linoleyl, and the amino lipid is a dilinoleyl amino lipid. In some embodiments, the amino lipid is a dilinoleyl amino lipid.

A representative useful dilinoleyl amino lipid has the formula:

wherein n is 0, 1, 2, 3, or 4. In some embodiments, the cationic lipid is a dLin-K-DMA. In some embodiments, the cationic lipid is dLin-KC2-DMA (dLin-K-DMA above, wherein n is 2)

In some embodiments, the cationic lipid component of the LNPs has the structure of Formula (I):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

X is selected from the group consisting of -N(R^A)- and -N(R^A)2-;

L¹ and L² are each independently -O(C= carbon-carbon double bond; each occurrence of R^A is independently

R^la and R^lb are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R^la is H or C1-C12 alkyl, and R^lh together with the carbon atom to which it is bound is taken together with an adjacent R^lb and the carbon atom to which it is bound to form a carbon-carbon double bond;

R^2a and R^2b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R^2a is H or C1-C12 alkyl, and R^2b together with the carbon atom to which it is bound is taken together with an adjacent R^2b and the carbon atom to which it is bound to form a carbon-carbon double bond;

R^3a and R^3b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R^3a is H or C1-C12 alkyl, and R^3b together with the carbon atom to which it is bound is taken together with an adjacent R^3b and the carbon atom to which it is bound to form a carbon-carbon double bond;

R^4a and R^4b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R^4a is H or C1-C12 alkyl, and R^4b together with the carbon atom to which it is bound is taken together with an adjacent R^4b and the carbon atom to which it is bound to form a carbon-carbon double bond; R⁵ and R⁶ are each independently methyl or cycloalkyl;

R⁷ is, at each occurrence, independently H or C1-C12 alkyl;

R⁸ and R⁹ are each independently C1-C12 alkyl; or R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom; a and d are each independently an integer from 0 to 24; b and c are each independently an integer from 1 to 24; and e is 1 or 2.

In certain embodiments of Formula (I), at least one of R^la, R^2a, R^3a or R^4a is C1-C12 alkyl, or at least one of L¹ or L² is -O(C=O)- or -(C=O)O-. In other embodiments, R^la and R^lb are not isopropyl when a is 6 or n-butyl when a is 8.

In still further embodiments of Formula (I), at least one of R^la, R^2a, R^3a or R^4a is C1-C12 alkyl, or at least one of L¹ or L² is -O(C=O)- or -(C=O)O-; and

R^la and R^lb are not isopropyl when a is 6 or n-butyl when a is 8.

In other embodiments of Formula (I), R⁸ and R⁹ are each independently unsubstituted Ci- C12 alkyl; or R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring comprising one nitrogen atom;

In certain embodiments of Formula (I), any one of L¹ or L² may be -O(C=O)- or a carbon-carbon double bond. L¹ and L² may each be -O(C=O)- or may each be a carbon-carbon double bond.

In some embodiments of Formula (I), one of L¹ or L² is -O(C=O)-. In other embodiments, both L¹ and L² are -O(C=O)-.

In some embodiments of Formula (I), one of L¹ or L² is -O(C=O)-. In other embodiments, both L¹ and L² are -O(C=O)-. In some other embodiments of Formula (I), one of L¹ or L² is a carbon-carbon double bond. In other embodiments, both L¹ and L² are a carboncarbon double bond.

In still other embodiments of Formula (I), one of L¹ or L² is -O(C=O)- and the other of L¹ or L² is -(C=O)O-. In more embodiments, one of L¹ or L² is -O(C=O)- and the other of L¹ or L² is a carbon-carbon double bond. In yet more embodiments, one of L¹ or L² is -(C=O)O- and the other of L¹ or L² is a carbon-carbon double bond.

It is understood that “carbon-carbon” double bond, as used throughout the specification, refers to one of the following structures:

wherein R^a and R^b are, at each occurrence, independently H or a substituent. For example, in some embodiments R^aand R^b are, at each occurrence, independently H, C1-C12 alkyl or cycloalkyl, for example H or C1-C12 alkyl.

In other embodiments, the lipid compounds of Formula (I) have the following structure (la):

In other embodiments, the lipid compounds of Formula (I) have the following structure (lb):

In yet other embodiments, the lipid compounds of Formula (I) have the following structure (Ic):

In certain embodiments of the lipid compound of Formula (I), a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.

In some other embodiments of Formula (I), b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.

In some more embodiments of Formula (I), c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.

In some certain other embodiments of Formula (I), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6.

In more embodiments, d is 7. In yet other embodiments, d is 8. In some embodiments, d is 9. In other embodiments, d is 10. In more embodiments, d is 11. In yet other embodiments, d is 12. In some embodiments, d is 13. In other embodiments, d is 14. In more embodiments, d is 15. In yet other embodiments, d is 16.

In some other various embodiments of Formula (I), a and d are the same.

In some other embodiments, b and c are the same. In some other specific embodiments, a and d are the same and b and c are the same.

The sum of a and b and the sum of c and d in Formula (I) are factors which may be varied to obtain a lipid of Formula (I) having the desired properties. In some embodiments, a and b are chosen such that their sum is an integer ranging from 14 to 24.

In other embodiments, c and d are chosen such that their sum is an integer ranging from 14 to 24. In further embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24. In still more embodiments, a. b, c and d are selected such the sum of a and b and the sum of c and d is 12 or greater.

In some embodiments of Formula (I), e is 1. In other embodiments, e is 2.

The substituents at R^la, R^2a, R^3a and R^4a of Formula (I) are not particularly limited. In certain embodiments R^la, R^2a, R^3a and R^4a are H at each occurrence. In certain other embodiments, at least one of R^la, R^2a, R^3a and R^4a is C1-C12 alkyl. In certain other embodiments, at least one of R^la, R^2a, R^3a and R^4a is Ci-Cs alkyl. In certain other embodiments, at least one of R^la, R^2a, R^3a and R^4a is Ci-Ce alkyl. In some of the foregoing embodiments, the Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

In certain embodiments of Formula (I), R^la, R^lb, R^4a and R^4b are C1-C12 alkyl at each occurrence.

In further embodiments of Formula (I), at least one of R^la, R^lb, R^4a and R^4b is H or R^la, R^lb, R^4a and R^4b are H at each occurrence.

In certain embodiments of Formula (I), R^lb together with the carbon atom to which it is bound is taken together with an adjacent R^lb and the carbon atom to which it is bound to form a carbon-carbon double bond. In other embodiments of the foregoing R^4b together with the carbon atom to which it is bound is taken together with an adjacent R^4b and the carbon atom to which it is bound to form a carbon-carbon double bond.

The substituents at R⁵ and R⁶ of Formula (I) are not particularly limited in the foregoing embodiments. In certain embodiments one or both of R⁵ or R⁶ is methyl.

In certain other embodiments one or both of R⁵ or R⁶ is cycloalkyl for example cyclohexyl. In these embodiments, the cycloalkyl may be substituted or not substituted. In certain other embodiments, the cycloalkyl is substituted with C1-C12 alkyl, for example tertbutyl.

The substituents at R⁷ are not particularly limited in the foregoing embodiments of Formula (I). In certain embodiments, at least one R⁷ is H. In some other embodiments, R⁷ is H at each occurrence. In certain other embodiments R⁷ is C1-C12 alkyl.

In certain other of the foregoing embodiments of Formula (I), one of R⁸ or R⁹ is methyl. In other embodiments, both R⁸ and R⁹ are methyl.

In some different embodiments of Formula (I), R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring. In some embodiments of the foregoing, R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring.

In various different embodiments, the lipid of Formula (I) has one of the structures set forth in Table 2 below.

In some embodiments, the LNPs comprise a lipid of Formula (I), at least one nucleoside- modified ribonucleic acid (RNA) encoding at least one immunostimulatory cytokine, wherein each immunostimulatory cytokine is encoded by a distinct nucleoside-modified RNA, and one or more excipients selected from neutral lipids, steroids and pegylated lipids. In some embodiments the lipid of Formula (I) is compound 1-5. In some embodiments the lipid of Formula (I) is compound 1-6.

In some other embodiments, the cationic lipid component of the LNPs has the structure of Formula (II):

00 or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:

L¹ and L² are each independently -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)x-, -S-S-, - C(=O)S-, -SC(=O)-, -NR^aC(=O)-, -C(=O)NR^a-, -NR^aC(=O)NR^a, -OC(=O)NR^a-, -NR^aC(=O)O-, or a direct bond;

G¹ is C1-C2 alkylene, -(C=O)- , -O(C=O)-, -SC(=O)-, -NR^aC(=O)- or a direct bond;

G² is -C(=O)- , -(C=O)O-, -C(=O)S-, -C(=O)NR^a or a direct bond;

G³ is Ci-Ce alkylene;

R^a is H or C1-C12 alkyl;

R^la and R^lb are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R^la is H or C1-C12 alkyl, and R^lb together with the carbon atom to which it is bound is taken together with an adjacent R^lb and the carbon atom to which it is bound to form a carbon-carbon double bond;

R^2a and R^2b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R^2a is H or C1-C12 alkyl, and R^2b together with the carbon atom to which it is bound is taken together with an adjacent R^2b and the carbon atom to which it is bound to form a carbon-carbon double bond;

R^3a and R^3b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R^3a is H or C1-C12 alkyl, and R^3b together with the carbon atom to which it is bound is taken together with an adjacent R^3b and the carbon atom to which it is bound to form a carbon-carbon double bond;

R^4a and R^4b are, at each occurrence, independently either: (a) H or C1-C12 alkyl; or (b) R^4a is H or C1-C12 alkyl, and R^4b together with the carbon atom to which it is bound is taken together with an adjacent R^4b and the carbon atom to which it is bound to form a carbon-carbon double bond;

R⁵ and R⁶ are each independently H or methyl; R⁷ is C4-C20 alkyl;

R⁸ and R⁹ are each independently C1-C12 alkyl; or R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring; a, b, c and d are each independently an integer from 1 to 24; and x is 0, 1 or 2.

In some embodiments of Formula (II), L¹ and L² are each independently -O(C=O)-, -(C=O)O- or a direct bond. In other embodiments, G¹ and G² are each independently - (C=O)- or a direct bond. In some different embodiments, L¹ and L² are each independently - O(C=O)-, -(C=O)O- or a direct bond; and G¹ and G² are each independently -(C=O)- or a direct bond.

In some different embodiments of Formula (II), L¹ and L² are each independently - C(=O)-, -O-, -S(O)_X-, -S-S-, -C(=O)S-, -SC(=O)-, -NR^a-, -NR^O)-, -C(=O)NR^a-, -NR^aC(=O)NR^a, -OC(=O)NR^a-, -NR^aC(=O)O-, -NR^aS(O)_xNR^a-, -NR^aS(O)x- or -S(O)xNR^a-.

In other of the foregoing embodiments of Formula (II), the lipid compound has one of the following

(IIA) (IIB)

In some embodiments of Formula (II), the lipid compound has structure (IIA). In other embodiments, the lipid compound has structure (IIB).

In any of the foregoing embodiments of Formula (II), one of L¹ or L² is -O(C=O)-. For example, in some embodiments each of L¹ and L² are -O(C=O)-.

In some different embodiments of Formula (II), one of L¹ or L² is -(C=O)O-. For example, in some embodiments each of L¹ and L² is -(C=O)O-.

In different embodiments of Formula (II), one of L¹ or L² is a direct bond. As used herein, a “direct bond” means the group (e.g., L¹ or L²) is absent. For example, in some embodiments each of L¹ and L² is a direct bond.

In other different embodiments of Formula (II), for at least one occurrence of R^la and R^lb, R^la is H or C1-C12 alkyl, and R^lh together with the carbon atom to which it is bound is taken together with an adjacent R^lb and the carbon atom to which it is bound to form a carbon-carbon double bond.

In still other different embodiments of Formula (II), for at least one occurrence of R^4a and R^4b, R^4a is H or C1-C12 alkyl, and R^4b together with the carbon atom to which it is bound is taken together with an adjacent R^4b and the carbon atom to which it is bound to form a carbon-carbon double bond.

In more embodiments of Formula (II), for at least one occurrence of R^2a and R^2b, R^2a is H or C1-C12 alkyl, and R^2b together with the carbon atom to which it is bound is taken together with an adjacent R^2b and the carbon atom to which it is bound to form a carbon-carbon double bond.

In other different embodiments of Formula (II), for at least one occurrence of R^3a and R^3b, R^3a is H or C1-C12 alkyl, and R^3b together with the carbon atom to which it is bound is taken together with an adjacent R^3b and the carbon atom to which it is bound to form a carbon-carbon double bond.

In various other embodiments of Formula (II), the lipid compound has one of the following structures (IIC) or (IID):

wherein e, f, g and h are each independently an integer from 1 to 12.

In some embodiments of Formula (II), the lipid compound has structure (IIC). In other embodiments, the lipid compound has structure (IID). In various embodiments of structures (IIC) or (IID), e, f, g and h are each independently an integer from 4 to 10.

In certain embodiments of Formula (II), a, b, c and d are each independently an integer from 2 to 12 or an integer from 4 to 12. In other embodiments, a, b, c and d are each independently an integer from 8 to 12 or 5 to 9. In some certain embodiments, a is 0. In some embodiments, a is 1. In other embodiments, a is 2. In more embodiments, a is 3. In yet other embodiments, a is 4. In some embodiments, a is 5. In other embodiments, a is 6. In more embodiments, a is 7. In yet other embodiments, a is 8. In some embodiments, a is 9. In other embodiments, a is 10. In more embodiments, a is 11. In yet other embodiments, a is 12. In some embodiments, a is 13. In other embodiments, a is 14. In more embodiments, a is 15. In yet other embodiments, a is 16.

In some embodiments of Formula (II), b is 1. In other embodiments, b is 2. In more embodiments, b is 3. In yet other embodiments, b is 4. In some embodiments, b is 5. In other embodiments, b is 6. In more embodiments, b is 7. In yet other embodiments, b is 8. In some embodiments, b is 9. In other embodiments, b is 10. In more embodiments, b is 11. In yet other embodiments, b is 12. In some embodiments, b is 13. In other embodiments, b is 14. In more embodiments, b is 15. In yet other embodiments, b is 16.

In some embodiments of Formula (II), c is 1. In other embodiments, c is 2. In more embodiments, c is 3. In yet other embodiments, c is 4. In some embodiments, c is 5. In other embodiments, c is 6. In more embodiments, c is 7. In yet other embodiments, c is 8. In some embodiments, c is 9. In other embodiments, c is 10. In more embodiments, c is 11. In yet other embodiments, c is 12. In some embodiments, c is 13. In other embodiments, c is 14. In more embodiments, c is 15. In yet other embodiments, c is 16.

In some certain embodiments of Formula (II), d is 0. In some embodiments, d is 1. In other embodiments, d is 2. In more embodiments, d is 3. In yet other embodiments, d is 4. In some embodiments, d is 5. In other embodiments, d is 6.

In some embodiments of Formula (II), e is 1. In other embodiments, e is 2. In more embodiments, e is 3. In yet other embodiments, e is 4. In some embodiments, e is 5. In other embodiments, e is 6. In more embodiments, e is 7. In yet other embodiments, e is 8. In some embodiments, e is 9. In other embodiments, e is 10. In more embodiments, e is 11. In yet other embodiments, e is 12.

In some embodiments of Formula (II), f is 1. In other embodiments, f is 2. In more embodiments, f is 3. In yet other embodiments, f is 4. In some embodiments, f is 5. In other embodiments, f is 6. In more embodiments, f is 7. In yet other embodiments, f is 8. In some embodiments, f is 9. In other embodiments, f is 10. In more embodiments, f is 11. In yet other embodiments, f is 12.

In some embodiments of Formula (II), g is 1. In other embodiments, g is 2. In more embodiments, g is 3. In yet other embodiments, g is 4. In some embodiments, g is 5. In other embodiments, g is 6. In more embodiments, g is 7. In yet other embodiments, g is 8. In some embodiments, g is 9. In other embodiments, g is 10. In more embodiments, g is 11. In yet other embodiments, g is 12.

In some embodiments of Formula (II), h is 1. In other embodiments, e is 2. In more embodiments, h is 3. In yet other embodiments, h is 4. In some embodiments, e is 5. In other embodiments, h is 6. In more embodiments, h is 7. In yet other embodiments, h is 8. In some embodiments, h is 9. In other embodiments, h is 10. In more embodiments, h is 11. In yet other embodiments, h is 12.

In some other various embodiments of Formula (II), a and d are the same. In some other embodiments, b and c are the same. In some other specific embodiments and a and d are the same and b and c are the same.

The sum of a and b and the sum of c and d of Formula (II) are factors which may be varied to obtain a lipid having the desired properties. In some embodiments, a and b are chosen such that their sum is an integer ranging from 14 to 24. In other embodiments, c and d are chosen such that their sum is an integer ranging from 14 to 24. In further embodiment, the sum of a and b and the sum of c and d are the same. For example, in some embodiments the sum of a and b and the sum of c and d are both the same integer which may range from 14 to 24. In still more embodiments, a. b, c and d are selected such that the sum of a and b and the sum of c and d is 12 or greater.

The substituents at R^la, R^2a, R^3a and R^4a of Formula (II) are not particularly limited. In some embodiments, at least one of R^la, R^2a, R^3a and R^4a is H. In certain embodiments R^la, R^2a, R^3a and R^4a are H at each occurrence. In certain other embodiments, at least one of R^la, R^2a, R^3a and R^4a is C1-C12 alkyl. In certain other embodiments, at least one of R^la, R^2a, R^3a and R^4a is Ci-Cs alkyl. In certain other embodiments, at least one of R^la, R^2a, R^3a and R^4a is Ci-Ce alkyl. In some of the foregoing embodiments, the Ci-Cs alkyl is methyl, ethyl, n-propyl, iso-propyl, n- butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.

In certain embodiments of Formula (II), R^la, R^lb, R^4a and R^4b are C1-C12 alkyl at each occurrence.

In further embodiments of Formula (II), at least one of R^lb, R^2b, R^3b and R^4b is H or R^lb, R^2b, R^3b and R^4b are H at each occurrence.

In certain embodiments of Formula (II), R^lb together with the carbon atom to which it is bound is taken together with an adjacent R^lb and the carbon atom to which it is bound to form a carbon-carbon double bond. In other embodiments of the foregoing R^4b together with the carbon atom to which it is bound is taken together with an adjacent R^4b and the carbon atom to which it is bound to form a carbon-carbon double bond. The substituents at R⁵ and R⁶ of Formula (II) are not particularly limited in the foregoing embodiments. In certain embodiments one of R⁵ or R⁶ is methyl. In other embodiments, each of R⁵ or R⁶ is methyl.

The substituents at R⁷ of Formula (II) are not particularly limited in the foregoing embodiments. In certain embodiments R⁷ is Ce-Ci6 alkyl. In some other embodiments, R⁷ is Ce- C9 alkyl. In some of these embodiments, R⁷ is substituted with -(C=O)OR^b, -O(C=O)R^b, - C(=O)R^b, -OR^b, -S(O)_xR^b, -S-SR^b, -C(=O)SR^b, -SC(=O)R^b, -NR^aR^b, -NR^aC(=O)R^b, - C(=O)NR^aR^b, -NR^aC(=O)NR^aR^b, -OC(=O)NR^aR^b, -NR^aC(=O)OR^b, -NR^aS(O)_xNR^aR^b, - NR^aS(O)xR^b or -S(O)xNR^aR^b, wherein: R^a is H or C1-C12 alkyl; R^b is C1-C15 alkyl; and x is 0, 1 or 2. For example, in some embodiments R⁷ is substituted with -(C=O)OR^b or -O(C=O)R^b.

In various of the foregoing embodiments of Formula (II), R^b is branched C1-C15 alkyl. For example, in some embodiments R^b has one of the following structures:

In certain other of the foregoing embodiments of Formula (II), one of R⁸ or R⁹ is methyl. In other embodiments, both R⁸ and R⁹ are methyl.

In some different embodiments of Formula (II), R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5, 6 or 7-membered heterocyclic ring. In some embodiments of the foregoing, R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 5-membered heterocyclic ring, for example a pyrrolidinyl ring. In some different embodiments of the foregoing, R⁸ and R⁹, together with the nitrogen atom to which they are attached, form a 6-membered heterocyclic ring, for example a piperazinyl ring.

In still other embodiments of the foregoing lipids of Formula (II), G³ is C2-C4 alkylene, for example C3 alkylene. In various different embodiments, the lipid compound has one of the structures set forth in Table 2 below.

Table 2: Representative Lipids of Formula (II)

In some embodiments, the LNPs comprise a lipid of Formula (II), at least one nucleoside- modified ribonucleic acid (RNA) encoding at least one immunostimulatory cytokine, wherein each immunostimulatory cytokine is encoded by a distinct nucleoside-modified RNA, and one or more excipient selected from neutral lipids, steroids and pegylated lipids. In some embodiments, the lipid of Formula (II) is compound II-9. In some embodiments, the lipid of Formula (II) is compound II- 10. In some embodiments, the lipid of Formula (II) is compound II- 11. In some embodiments, the lipid of Formula (II) is compound 11-12. In some embodiments, the lipid of Formula (II) is compound 11-32. In some other embodiments, the cationic lipid component of the LNPs has the structure of Formula (III):

or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one ofL¹ or L² is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)_X-, -S-S-, -C(=O)S-, SC(=O)- , -NR^aC(=O)-, -C(=O)NR^a-, NR^aC(=O)NR^a-, -OC(=O)NR^a- or -NR^aC(=O)O-, and the other of L¹ or L² is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)_X-, -S-S-, -C(=O)S-, SC(=O)-, -NR^aC(=O)-, -C(=O)NR^a-, ,NR^aC(=O)NR^a-, -OC(=0)NR^a- or -NR^aC(=0)0- or a direct bond;

G¹ and G² are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;

R^a is H or C1-C12 alkyl;

R^B is selected from the group consisting

A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;

R¹ and R² are each independently C6-C24 alkyl or C6-C24 alkenyl;

R³ is H, OR⁵, CN, -C(=O)OR⁴, -OC(=O)R⁴ or -NR⁵C(=O)R⁴;

R⁴ is C1-C12 alkyl;

R⁵ is H or Ci-Ce alkyl;

R⁶ is, at each occurrence, independently H, OH or C1-C24 alkyl; nl and n2 are each independently an integer ranging from 0 to 15, wherein the sum of nl and n2 is an integer ranging from 1 to 15; and x is 0, 1 or 2.

In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIA) or (IIIB):

In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).

In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or (HID):

wherein y and z are each independently integers ranging from 1 to 12. In any of the foregoing embodiments of Formula (III), one of L¹ or L² is -O(C=O)-. For example, in some embodiments each of L¹ and L² are -O(C=O)-. In some different embodiments of any of the foregoing, L¹ and L² are each independently -(C=O)O- or -O(C=O)-. For example, in some embodiments each of L¹ and L² is -(C=O)O-.

In some different embodiments of Formula (III), the lipid has one of the following structures (HIE) or (IIIF) :

In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ):

In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.

In some other of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.

In some of the foregoing embodiments of Formula (III), R⁶ is H. In other of the foregoing embodiments, R⁶ is C1-C24 alkyl. In other embodiments, R⁶ is OH.

In some other foregoing embodiments of Formula (III), R¹ or R², or both, is C6-C24 alkenyl. For example, in some embodiments, R¹ and R² each, independently have the following structure:

wherein:

R^7a and R^7b are, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, wherein R^7a, R^7b and a are each selected such that R¹ and R² each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.

In some of the foregoing embodiments of Formula (III), at least one occurrence of R^7a is H. For example, in some embodiments, R^7a is H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R⁷¹¹ is C i-Cs alkyl. For example, in some embodiments, Ci-Cs alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, tertbutyl, n-hexyl or n-octyl.

In different embodiments of Formula (III), R¹ or R², or both, has one of the following

In some of the foregoing embodiments of Formula (III), R³ is OH, CN, -C(=O)OR⁴, - OC(=O)R⁴ or -NHC(=O)R⁴. In some embodiments, R⁴ is methyl or ethyl.

In various different embodiments, the cationic lipid of Formula (III) has one of the structures set forth in Table 3 below.

Table 3: Representative Compounds of Formula (III)

In some embodiments, the LNPs comprise a lipid of Formula (III), at least one nucleoside-modified ribonucleic acid (RNA) encoding at least one immunostimulatory cytokine, wherein each immunostimulatory cytokine is encoded by a distinct nucleoside-modified RNA, and one or more excipient selected from neutral lipids, steroids and pegylated lipids. In some embodiments, the lipid of Formula (III) is compound III-3. In some embodiments, the lipid of Formula (III) is compound III-7. In certain embodiments, the cationic lipid is present in the LNP in an amount from about 30 to about 95 mole percent. In some embodiments, the cationic lipid is present in the LNP in an amount from about 30 to about 70 mole percent. In some embodiments, the cationic lipid is present in the LNP in an amount from about 40 to about 60 mole percent. In some embodiments, the cationic lipid is present in the LNP in an amount of about 50 mole percent. In some embodiments, the LNP comprises only cationic lipids.

In certain embodiments, the LNP comprises one or more additional lipids which stabilize the formation of particles during their formation.

Suitable stabilizing lipids include neutral lipids and anionic lipids.

The term “neutral lipid” refers to any one of a number of lipid species that exist in either an uncharged or neutral zwitterionic form at physiological pH.

Representative neutral lipids include diacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides, sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

Exemplary neutral lipids include, for example, di stearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE) and dioleoyl-phosphatidylethanolamine 4- (N-maleimidomethyl)-cyclohexane-l-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1- stearioyl-2-oleoyl-phosphatidyethanol amine (SOPE), and l,2-dielaidoyl-sn-glycero-3- phophoethanolamine (transDOPE). In some embodiments, the neutral lipid is 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC).

In some embodiments, the LNPs comprise a neutral lipid selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various embodiments, the molar ratio of the cationic lipid (e.g., lipid of Formula (I)) to the neutral lipid ranges from about 2: 1 to about 8: 1.

In various embodiments, the LNPs further comprise a steroid or steroid analogue. A “steroid” is a compound comprising the following carbon skeleton:

In certain embodiments, the steroid or steroid analogue is cholesterol. In some of these embodiments, the molar ratio of the cationic lipid (e.g., lipid of Formula (I)) to cholesterol ranges from about 2: 1 to 1 : 1.

The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamines, N- succinylphosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

In certain embodiments, the LNP comprises glycolipids (e g., monosialoganglioside GMi). In certain embodiments, the LNP comprises a sterol, such as cholesterol.

In some embodiments, the LNPs comprise a polymer conjugated lipid. The term “polymer conjugated lipid” refers to a molecule comprising both a lipid portion and a polymer portion. An example of a polymer conjugated lipid is a pegylated lipid. The term “pegylated lipid” refers to a molecule comprising both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art and include l-(m onom ethoxy-poly ethyleneglycol)-2, 3- dimyristoylglycerol (PEG-s- DMG) and the like.

In certain embodiments, the LNP comprises an additional, stabilizing -lipid which is a polyethylene glycol-lipid (pegylated lipid). Suitable polyethylene glycol-lipids include PEG- modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramides (e g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines, PEG-modified di acylglycerols, PEG-modified dialkylglycerols. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In some embodiments, the polyethylene glycol-lipid is N-[(methoxy polyethylene glycol)2ooo)carbamyl]-l,2- dimyristyloxlpropyl-3-amine (PEG-c-DMA). In some embodiments, the polyethylene glycol- lipid is PEG-c-DOMG). Tn other embodiments, the LNPs comprise a pegylated diacylglycerol (PEG-DAG) such as l-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG- DMG), a pegylated phosphatidylethanoloamine (PEG-PE), a PEG succinate diacylglycerol (PEG-S-DAG) such as 4-O-(2’,3’-di(tetradecanoyloxy)propyl-l-O-(O- methoxy(polyethoxy)ethyl)butanedioate (PEG-S-DMG), a pegylated ceramide (PEG-cer), or a PEG dialkoxypropylcarbamate such as co-methoxy(polyethoxy)ethyl-N-(2,3- di(tetradecanoxy)propyl)carbamate or 2,3-di(tetradecanoxy)propyl-N-(co- methoxy(polyethoxy)ethyl)carbamate. In various embodiments, the molar ratio of the cationic lipid to the pegylated lipid ranges from about 100: 1 to about 25: 1.

In some embodiments, the LNPs comprise a pegylated lipid having the following structure (IV):

or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:

R¹⁰ and R^{1 1} are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and z has mean value ranging from 30 to 60.

In some of the foregoing embodiments of the pegylated lipid (IV), R¹⁰ and R¹¹ are not both n-octadecyl when z is 42. In some other embodiments, R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 18 carbon atoms. In some embodiments, R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 12 to 16 carbon atoms. In some embodiments, R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms. In some embodiments, R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms. In other embodiments, R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing 16 carbon atoms. In still more embodiments, R¹⁰ and R¹¹ are each independently a straight or branched, saturated or unsaturated alkyl chain containing 18 carbon atoms. In still other embodiments, R¹⁰ is a straight or branched, saturated or unsaturated alkyl chain containing 12 carbon atoms and R¹¹ is a straight or branched, saturated or unsaturated alkyl chain containing 14 carbon atoms.

In various embodiments, z spans a range that is selected such that the PEG portion of (II) has an average molecular weight of about 400 to about 6000 g/mol. In some embodiments, the average z is about 45.

In other embodiments, the pegylated lipid has one of the following structures:

wherein n is an integer selected such that the average molecular weight of the pegylated lipid is about 2500 g/mol.

In certain embodiments, the additional lipid is present in the LNP in an amount from about 1 to about 10 mole percent. In some embodiments, the additional lipid is present in the LNP in an amount from about 1 to about 5 mole percent. In some embodiments, the additional lipid is present in the LNP in about 1 mole percent or about 1.5 mole percent.

In some embodiments, the LNPs comprise a lipid of Formula (I), at least one nucleoside- modified ribonucleic acid (RNA) encoding at least one immunostimulatory cytokine, wherein each immunostimulatory cytokine is encoded by a distinct nucleoside-modified RNA, a neutral lipid, a steroid and a pegylated lipid. In some embodiments the lipid of Formula (I) is compound 1-6. In certain embodiments, the neutral lipid is DSPC. In some embodiments, the steroid is cholesterol. In certain embodiments, the pegylated lipid is compound iVa.

In certain embodiments, the LNP comprises one or more targeting moieties, which are capable of targeting the LNP to a cell or cell population. For example, in some embodiments, the targeting moiety is a ligand, which directs the LNP to a receptor found on a cell surface.

In certain embodiments, the LNP comprises one or more internalization domains. For example, in some embodiments, the LNP comprises one or more domains, which bind to a cell to induce the internalization of the LNP. For example, in some embodiments, the one or more internalization domains bind to a receptor found on a cell surface to induce receptor-mediated uptake of the LNP. In certain embodiments, the LNP is capable of binding a biomolecule in vivo, where the LNP-bound biomolecule can then be recognized by a cell-surface receptor to induce internalization. For example, in some embodiments, the LNP binds systemic ApoE, which leads to the uptake of the LNP and associated cargo.

Other exemplary LNPs and their manufacture are described in the art, for example in U.S. Patent Application Publication No. US20120276209, Semple et ak, 2010, Nat Biotechnok, 28(2): 172-176; Akinc et ak, 2010, Mol Then, 18(7): 1357-1364; Basha et ak, 2011, Mol Ther, 19(12): 2186-2200; Leung et ak, 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440- 18450; Lee et ak, 2012, Int J Cancer., 131(5): E781-90; Belliveau et ak, 2012, Mol Ther nucleic Acids, 1 : e37; Jayaraman et ak, 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Mui et ak, 2013, Mol Ther Nucleic Acids. 2, el39; Maier et ak, 2013, Mol Then, 21(8): 1570-1578; and Tam et ak, 2013, Nanomedicine, 9(5): 665-74, each of which is incorporated by reference in its entirety. Additionally, WO 2022/081752, incorporated herein by reference in its entirety, describes LNP manufacturing techniques for increasing the potency of nucleic acid loaded lipid nanoparticles.

The following Reaction Schemes illustrate methods to make lipids of Formula (I), (II) or (III).

GENERAL REACTION SCHEME 1

Embodiments of the lipid of Formula (I) (e.g., compound A-5) can be prepared according to General Reaction Scheme 1 (“Method A”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. Referring to General Reaction Scheme 1, compounds of structure A-l can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A mixture of A-l, A-2 and DMAP is treated with DCC to give the bromide A-3. A mixture of the bromide A-3, a base (e.g., N,N-diisopropylethylamine) and the N,N-dimethyldiamine A-4 is heated at a temperature and time sufficient to produce A- 5 after any necessary workup and or purification step.

GENERAL REACTION SCHEME 2

B-5

Other embodiments of the compound of Formula (I) (e.g., compound B-5) can be prepared according to General Reaction Scheme 2 (“Method B”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. As shown in General Reaction Scheme 2, compounds of structure B-l can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A solution of B-l (1 equivalent) is treated with acid chloride B-2 (1 equivalent) and a base (e.g., triethylamine). The crude product is treated with an oxidizing agent (e.g., pyridinum chlorochromate) and intermediate product B-3 is recovered. A solution of crude B-3, an acid (e.g., acetic acid), and N,N-dimethylaminoamine B-4 is then treated with a reducing agent (e.g., sodium triacetoxyborohydride) to obtain B-5 after any necessary work up and/or purification.

It should be noted that although starting materials A-l and B-l are depicted above as including only saturated methylene carbons, starting materials which include carbon-carbon double bonds may also be employed for preparation of compounds which include carbon-carbon double bonds.

GENERAL REACTION SCHEME 3

Different embodiments of the lipid of Formula (I) (e.g., compound C-7 or C9) can be prepared according to General Reaction Scheme 3 (“Method C”), wherein R is a saturated or unsaturated C1-C24 alkyl or saturated or unsaturated cycloalkyl, m is 0 or 1 and n is an integer from 1 to 24. Referring to General Reaction Scheme 3, compounds of structure C-l can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art.

GENERAL REACTION SCHEME 4

D-7

Embodiments of the compound of Formula (II) (e.g., compounds D-5 and D-7) can be prepared according to General Reaction Scheme 4 (“Method D”), wherein R^la, R^lb, R^2a, R^2b, R^3a, R^3b, R^4a, R^4b, R³, R⁶, R⁸, R⁹, L¹, L², G¹, G², G³, a, b, c and d are as defined herein, and R⁷ represents R⁷ or a C3-C19 alkyl. Referring to General Reaction Scheme 1, compounds of structure D-l and D-2 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A solution of D-l and D-2 is treated with a reducing agent (e.g., sodium triacetoxyborohydride) to obtain D-3 after any necessary work up. A solution of D-3 and a base (e.g. trimethylamine, DMAP) is treated with acyl chloride D-4 (or carboxylic acid and DCC) to obtain D-5 after any necessary work up and/or purification. D-5 can be reduced with LiAlH/i D-6 to give D-7 after any necessary work up and/or purification.

GENERAL REACTION SCHEME 5

Embodiments of the lipid of Formula (II) (e.g., compound E-5) can be prepared according to General Reaction Scheme 5 (“Method E”), wherein R^la, R^lb, R^2a, R^2b, R^3a, R^3b, R^4a, R^4b, R⁵, R⁶, R⁷, R⁸, R⁹, L¹, L², G³, a, b, c and d are as defined herein. Referring to General Reaction Scheme 5, compounds of structure E-l and E-2 can be purchased from commercial sources or prepared according to methods familiar to one of ordinary skill in the art. A mixture of E-l (in excess), E-2 and a base (e.g., potassium carbonate) is heated to obtain E-3 after any necessary work up. A solution of E-3 and a base (e.g. trimethylamine, DMAP) is treated with acyl chloride E-4 (or carboxylic acid and DCC) to obtain E-5 after any necessary work up and/or purification.

GENERAL REACTION SCHEME 6

General Reaction Scheme 6 provides an exemplary method (Method F) for preparation of Lipids of Formula (III). G¹, G³, R¹ and R³ in General Reaction Scheme 6 are as defined herein for Formula (III), and GL refers to a one-carbon shorter homologue of Gl. Compounds of structure F-l are purchased or prepared according to methods known in the art. Reaction of F-l with diol F-2 under appropriate condensation conditions (e.g., DCC) yields ester/alcohol F-3, which can then be oxidized (e.g., PCC) to aldehyde F-4. Reaction of F-4 with amine F-5 under reductive amination conditions yields a lipid of Formula (III).

It should be noted that various alternative strategies for preparation of lipids of Formula (III) are available to those of ordinary skill in the art. For example, other lipids of Formula (III) wherein L¹ and L² are other than ester can be prepared according to analogous methods using the appropriate starting material. Further, General Reaction Scheme 6 depicts preparation of a lipids of Formula (III), wherein G¹ and G² are the same; however, this is not a required aspect of the invention and modifications to the above reaction scheme are possible to yield compounds wherein G¹ and G² are different.

It will be appreciated by those skilled in the art that in the process described herein the functional groups of intermediate compounds may need to be protected by suitable protecting groups. Such functional groups include hydroxy, amino, mercapto and carboxylic acid. Suitable protecting groups for hydroxy include trialkylsilyl or diarylalkylsilyl (for example, t- butyldimethylsilyl , Lbutyldiphenylsilyl or trimethyl silyl), tetrahydropyranyl, benzyl, and the like. Suitable protecting groups for amino, amidino and guanidino include /-butoxycarbonyl, benzyloxycarbonyl, and the like. Suitable protecting groups for mercapto include -C(O)-R" (where R" is alkyl, aryl or arylalkyl), /;-methoxybenzyl, trityl and the like. Suitable protecting groups for carboxylic acid include alkyl, aryl or arylalkyl esters. Protecting groups may be added or removed in accordance with standard techniques, which are known to one skilled in the art and as described herein. The use of protecting groups is described in detail in Green, T.W. and P.G.M. Wutz, Protective Groups in Organic Synthesis (1999), 3rd Ed., Wiley. As one of skill in the art would appreciate, the protecting group may also be a polymer resin such as a Wang resin, Rink resin or a 2-chlorotrityl-chloride resin.

Chimeric Antigen Receptors (CARs)

The anti-single-chain variable fragment (scFv) antibody or antigen-binding fragment of the invention is capable of specifically binding to a linker peptide of an scFv of a CAR. The linker peptide links a variable light chain (VL) of the scFv to a variable heavy chain (VH) of the scFv. The CAR comprises an extracellular domain comprising the scFv (also referred to as an antigen binding domain), a transmembrane domain, and an intracellular domain.

Antigen Binding Domain of the CAR

As disclosed herein, the present invention relates to compositions and methods for treating cancer in a subject in need thereof, wherein the methods and compositions comprise an anti-scFv antibody or antigen-binding fragment which is used to deliver one or more immunostimulatory cytokines (as an antibody-linked cytokine or as a lipid nanoparticle (LNP) comprising nucleoside-modified RNA encoding the immunostimulatory cytokine) to CAR- expressing immune cells (e.g., CAR T cells) in the subject, thereby enhancing the anti-tumor efficacy of the CAR adoptive therapy. The CAR comprises an extracellular domain comprising the scFv (also referred to as an antigen binding domain), a transmembrane domain, and an intracellular domain.

Those skilled in the art will appreciate that the anti-scFv antibody or antigen-binding fragment will bind to any CAR having an scFv comprising the linker peptide epitope, e.g., a (G₄S)3 linker (GGGGSGGGGSGGGGS) (SEQ ID NO: 32), a (G₄S)₄ linker (GGGGSGGGGSGGGGSGGGGS) (SEQ ID NO: 57), or a Whitlow linker (GSTSGSGKPGSGEGSTKG) (SEQ ID NO: 58) and that binding of the anti-scFv antibody or antigen-binding fragment to the scFv of the CAR is not dependent upon the target antigen of the CAR or on the other domains of the CAR (e.g., the transmembrane or intracellular domain). Further, skilled artisans will understand that only routine experimentation is needed to generate an scFv (and a CAR comprising said scFv) directed to a given tumor antigen, wherein the scFv comprises the linker peptide epitope (i.e., SEQ ID NO: 32, SEQ ID NO: 57, or SEQ ID NO: 58) to which the anti-scFv antibody or antigen-binding fragment of the invention binds.

Despite removal of the constant regions and the introduction of a linker, scFv proteins retain the specificity of the original immunoglobulin. Single chain Fv polypeptide antibodies can be expressed from a nucleic acid comprising VH- and VL-encoding sequences as described by Huston, et al. (Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988). See, also, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent Publication Nos. 20050196754 and 20050196754. Antagonistic scFvs having inhibitory activity have been described (see, e.g., Zhao et al., Hybridoma (Larchmt) 2008 27(6):455-51; Peter et al., J Cachexia Sarcopenia Muscle 2012 August 12; Shieh et al., J Imunol 2009 183(4):2277-85; Giomarelli et al., Thromb Haemost 2007 97(6):955-63; Fife eta., J Clin Invst 2006 116(8):2252-61; Brocks et al., Immunotechnology 1997 3(3): 173-84; Moosmayer et al., Ther Immunol 1995 2(10:31-40). Agonistic scFvs having stimulatory activity have been described (see, e.g., Peter et al., J Bioi Chem 2003 25278(38):36740-7; Xie et al., Nat Biotech 1997 15(8):768-71; Ledbetter et al., Crit Rev Immunol 1997 17(5-6):427-55; Ho et al., BioChim Biophys Acta 2003 1638(3):257-66).

The scFv of the CAR targets (i.e., specifically binds to) a tumor antigen. In some embodiments, the scFv of the CAR targets a tumor antigen selected from the group consisting of alpha feto-protein (AFP)/HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD13, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, claudin 6, c-Met, DLL3, DR5, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa)Zfolate binding protein (FBP), folate receptor beta (FRb), follicle-stimulating hormone receptor (FSHR), GD-2, Glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, interleukin 13 receptor subunit alpha (IL3Ra), interleukin 13 receptor subunit alpha 2 (IL13Ra2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4/FAP, NKG2D-Ligands (MIC- A, MIC-B, and the ULBPs 1 to 6), Mullerian inhibiting substance type 2 receptor (MISIIR), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), P16, PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), ROR1, ROR2, TAG72, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUCl), TR0P2, Glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRa4; GFRalpha4), VEGFR2, and any combination thereof. In some embodiments, the scFv of the CAR targets TnMUC 1.

Non-limiting exemplary nucleotide and amino acid sequences of CARs and scFvs suitable for use in the invention are provided herein.

Anti-TnMucl scFv nucleotide sequence (SEQ ID NO: 40)

CAGGTGCAGCTGCAGCAGTCTGATGCCGAGCTCGTGAAGCCTGGCAGCAGCGTGAAGATCAGCT GCAAGGCCAGCGGCTACACCTTCACCGACCACGCCATCCACTGGGTCAAGCAGAAGCCTGAGCA GGGCCTGGAGTGGATCGGCCACTTCAGCCCCGGCAACACCGACATCAAGTACAACGACAAGTTC AAGGGCAAGGCCACCCTGACCGTGGACAGAAGCAGCAGCACCGCCTACATGCAGCTGAACAGCC TGACCAGCGAGGACAGCGCCGTGTACTTCTGCAAGACCAGCACCTTCTTTTTCGACTACTGGGG CCAGGGCACAACCCTGACAGTGTCTAGCGGAGGCGGAGGATCTGGCGGCGGAGGAAGTGGCGGA GGGGGATCTGAACTCGTGATGACCCAGAGCCCCAGCTCTCTGACAGTGACAGCCGGCGAGAAAG TGACCATGATCTGCAAGTCCTCCCAGAGCCTGCTGAACTCCGGCGACCAGAAGAACTACCTGAC CTGGTATCAGCAGAAACCCGGCCAGCCCCCCAAGCTGCTGATCTTTTGGGCCAGCACCCGGGAA AGCGGCGTGCCCGATAGATTCACAGGCAGCGGCTCCGGCACCGACTTTACCCTGACCATCAGCT CCGTGCAGGCCGAGGACCTGGCCGTGTATTACTGCCAGAACGACTACAGCTACCCCCTGACCTT CGGAGCCGGCACCAAGCTGGAACTGAAG

Anti-TnMucl scFv amino acid sequence (SEQ ID NO: 41)

QVQLQQSDAELVKPGSSVKI SCKASGYTFTDHAIHWVKQKPEQGLEWIGHFSPGNTDIKYNDKF KGKATLTVDRSSSTAYMQLNSLTSEDSAVYFCKTSTFFFDYWGQGTTLTVSSGGGGSGGGGSGG GGSELVMTQSPSSLTVTAGEKVTMICKSSQSLLNSGDQKNYLTWYQQKPGQPPKLLI FWASTRE SGVPDRFTGSGSGTDFTLT I SSVQAEDLAVYYCQNDYSYPLTFGAGTKLELK

Anti-EGFRvIII scFv nucleotide sequence (SEQ ID NO: 42)

ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCG

AGATTCAGCTCGTGCAATCGGGAGCGGAAGTCAAGAAGCCAGGAGAGTCCTTGCGGATCTCATG CAAGGGTAGCGGCTTTAACATCGAGGATTACTACATCCACTGGGTGAGGCAGATGCCGGGGAAG GGACTCGAATGGATGGGACGGATCGACCCAGAAAACGACGAAACTAAGTACGGTCCGATCTTCC AAGGCCATGTGACTATTAGCGCCGATACTTCAATCAATACCGTGTATCTGCAATGGTCCTCATT

GAAAGCCTCAGATACCGCGATGTACTACTGTGCTTTCAGAGGAGGGGTCTACTGGGGACAGGGA

ACTACCGTGACTGTCTCGTCCGGCGGAGGCGGGTCAGGAGGTGGCGGCAGCGGAGGAGGAGGGT

CCGGCGGAGGTGGGTCCGACGTCGTGATGACCCAGAGCCCTGACAGCCTGGCAGTGAGCCTGGG

CGAAAGAGCTACCATTAACTGCAAATCGTCGCAGAGCCTGCTGGACTCGGACGGAAAAACGTAC

CTCAATTGGCTGCAGCAAAAGCCTGGCCAGCCACCGAAGCGCCTTATCTCACTGGTGTCGAAGC

TGGATTCGGGAGTGCCCGATCGCTTCTCCGGCTCGGGATCGGGTACTGACTTCACCCTCACTAT

CTCCTCGCTTCAAGCAGAGGACGTGGCCGTCTACTACTGCTGGCAGGGAACCCACTTTCCGGGA

ACCTTCGGCGGAGGGACGAAAGTGGAGATCAAG

Anti-EGFRvIII scFv amino acid sequence (SEQ ID NO: 43)

MALPVTALLLPLALLLHAARPEIQLVQSGAEVKKPGESLRI SCKGSGFNIEDYYIHWVRQMPGK

GLEWMGRIDPENDETKYGPI FQGHVT ISADTS INTVYLQWSSLKASDTAMYYCAFRGGVYWGQG

TTVTVSSGGGGSGGGGSGGGGSGGGGSDWMTQSPDSLAVSLGERAT INCKSSQSLLDSDGKTY

LNWLQQKPGQPPKRLISLVSKLDSGVPDRFSGSGSGTDFTLT ISSLQAEDVAVYYCWQGTHFPG TFGGGTKVE IK

Human anti-CD19 CAR NT sequence (SEQ ID NO: 44)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGG

AAATTGTGATGACCCAGTCACCCGCCACTCTTAGCCTTTCACCCGGTGAGCGCGCAACCCTGTC

TTGCAGAGCCTCCCAAGACATCTCAAAATACCTTAATTGGTATCAACAGAAGCCCGGACAGGCT

CCTCGCCTTCTGATCTACCACACCAGCCGGCTCCATTCTGGAATCCCTGCCAGGTTCAGCGGTA

GCGGATCTGGGACCGACTACACCCTCACTATCAGCTCACTGCAGCCAGAGGACTTCGCTGTCTA

TTTCTGTCAGCAAGGGAACACCCTGCCCTACACCTTTGGACAGGGCACCAAGCTCGAGATTAAA

GGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGAAGCCAGGTCCAACTCCAAGAAA

GCGGACCGGGTCTTGTGAAGCC TC G AACTCTTTC CTGACTTGT CTGTG GCGGAGTGTC

TCTCCCCGATTACGGGGTGTCTTGGATCAGACAGCCACCGGGGAAGGGTCTGGAATGGATTGGA

GTGATTTGGGGCTCTGAGACTACTTACTACCAATCATCCCTCAAGTCACGCGTCACCATCTCAA

AGGACAACTCTAAGAATCAGGTGTCACTGAAACTGTCATCTGTGACCGCAGCCGACACCGCCGT

GTACTATTGCGCTAAGCATTACTATTATGGCGGGAGCTACGCAATGGATTACTGGGGACAGGGT

ACTCTGGTCACCGTGTCCAGCACCACGACGCCAGCGCCGCGACCACCAACACCGGCGCCCACCA

TCGCGTCGCAGCCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCA CACGAGGGGGCTGGACTTCGCCTGTGATTTTTGGGTGCTGGTGGTGGTTGGTGGAGTCCTGGCT

TGCTATAGCTTGCTAGTAACAGTGGCCTTTATTATTTTCTGGGTGAGGAGTAAGAGGAGCAGGC

TCCTGCACAGTGACTACATGAACATGACTCCCCGCCGCCCCGGGCCCACCCGCAAGCATTACCA

GCCCTATGCCCCACCACGCGACTTCGCAGCCTATCGCTCCAGAGTGAAGTTCAGCAGGAGCGCA

GACGCCCCCGCGTACCAGCAGGGCCAGAACCAGCTCTATAACGAGCTCAATCTAGGACGAAGAG

AGGAGTACGATGTTTTGGACAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAG

GAAGAACCCTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGT

GAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTCA

GTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC

Human anti-CD19 CAR AA sequence (SEQ ID NO: 45)

MALPVTALLLPLALLLHAARPEIVMTQSPATLSLSPGERATLSCRASQDISKYLNWYQQKPGQA

PRLLIYHTSRLHSGIPARFSGSGSGTDYTLT ISSLQPEDFAVYFCQQGNTLPYTFGQGTKLE IK

GGGGSGGGGSGGGGSQVQLQESGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIG

VIWGSETTYYQSSLKSRVT I SKDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQG

TLVTVSSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDFWVLVWGGVLA CYSLLVTVAFI I FWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSA DAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYS E I GMKGERRRGKGHDGL YQGLS TATKDT YDALHMQALPPR

Human anti-MSLN CAR AA sequence (SEQ ID NO: 46)

MALPVTALLLPLALLLHTWRPQVQLVQSGAEVEKPGASVKVSCKASGYTFTDYYMHWVRQAPGQ

GLEWMGWINPNSGGTNYAQKFQGRVTMTRDTS ISTAYMELSRLRSDDTAVYYCASGWDFDYWGQ

GTLVTVSSGGGGSGGGGSGGGGSGGGGSDIVMTQSPSSLSASVGDRVT ITCRASQS IRYYLSWY

QQKPGKAPKLLIYTAS ILQNGVPSRFSGSGSGTDFTLTI SSLQPEDFATYYCLQTYTTPDFGPG

TKVEIKTTTPAPRPPTPAPT IASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGV

LLLSLVI TLYCKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAP

AYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSE IG

MKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

Human anti-MSLN CAR NT sequence (SEQ ID NO: 47)

ATGGCCCTCCCTGTCACCGCCCTGCTGCTTCCGCTGGCTCTTCTGCTCCACGCCGCTCGGCCCC AAGTCCAACTCGTTCAATCAGGCGCAGAAGTCGAAAAGCCCGGAGCATCAGTCAAAGTCTCTTG

CAAGGCTTCCGGCTACACCTTCACGGACTACTACATGCACTGGGTGCGCCAGGCTCCAGGCCAG

GGACTGGAGTGGATGGGATGGATCAACCCGAATTCCGGGGGAACTAACTACGCCCAGAAGTTTC

AGGGCCGGGTGACTATGACTCGCGATACCTCGATCTCGACTGCGTACATGGAGCTCAGCCGCCT

CCGGTCGGACGATACCGCCGTGTACTATTGTGCGTCGGGATGGGACTTCGACTACTGGGGGCAG

GGCACTCTGGTCACTGTGTCAAGCGGAGGAGGTGGATCAGGTGGAGGTGGAAGCGGGGGAGGAG

GTTCCGGCGGCGGAGGATCAGATATCGTGATGACGCAATCGCCTTCCTCGTTGTCCGCATCCGT

GGGAGACAGGGTGACCATTACTTGCAGAGCGTCCCAGTCCATTCGGTACTACCTGTCGTGGTAC

CAGCAGAAGCCGGGGAAAGCCCCAAAACTGCTTATCTATACTGCCTCGATCCTCCAAAACGGCG

TGCCATCAAGATTCAGCGGTTCGGGCAGCGGGACCGACTTTACCCTGACTATCAGCAGCCTGCA

GCCGGAAGATTTCGCCACGTACTACTGCCTGCAAACCTACACCACCCCGGACTTCGGACCTGGA

ACCAAGGTGGAGATCAAGACCACTACCCCAGCACCGAGGCCACCCACCCCGGCTCCTACCATCG

CCTCCCAGCCTCTGTCCCTGCGTCCGGAGGCATGTAGACCCGCAGCTGGTGGGGCCGTGCATAC

CCGGGGTCTTGACTTCGCCTGCGATATCTACATTTGGGCCCCTCTGGCTGGTACTTGCGGGGTC

CTGCTGCTTTCACTCGTGATCACTCTTTACTGTAAGCGCGGTCGGAAGAAGCTGCTGTACATCT

TTAAGCAACCCTTCATGAGGCCTGTGCAGACTACTCAAGAGGAGGACGGCTGTTCATGCCGGTT

CCCAGAGGAGGAGGAAGGCGGCTGCGAACTGCGCGTGAAATTCAGCCGCAGCGCAGATGCTCCA

GCCTACAAGCAGGGGCAGAACCAGCTCTACAACGAACTCAATCTTGGTCGGAGAGAGGAGTACG

ACGTGCTGGACAAGCGGAGAGGACGGGACCCAGAAATGGGCGGGAAGCCGCGCAGAAAGAATCC

CCAAGAGGGCCTGTACAACGAGCTCCAAAAGGATAAGATGGCAGAAGCCTATAGCGAGATTGGT

ATGAAAGGGGAACGCAGAAGAGGCAAAGGCCACGACGGACTGTACCAGGGACTCAGCACCGCCA

CCAAGGACACCTATGACGCTCTTCACATGCAGGCCCTGCCGCCTCGG

M5 Human anti-MSLN scFv nucleotide sequence (SEQ ID NO: 48)

CAAGTCCAACTCGTTCAATCAGGCGCAGAAGTCGAAAAGCCCGGAGCATCAGTCAAAGTCTCTT

GCAAGGCTTCCGGCTACACCTTCACGGACTACTACATGCACTGGGTGCGCCAGGCTCCAGGCCA

GGGACTGGAGTGGATGGGATGGATCAACCCGAATTCCGGGGGAACTAACTACGCCCAGAAGTTT

CAGGGCCGGGTGACTATGACTCGCGATACCTCGATCTCGACTGCGTACATGGAGCTCAGCCGCC

TCCGGTCGGACGATACCGCCGTGTACTATTGTGCGTCGGGATGGGACTTCGACTACTGGGGGCA

GGGCACTCTGGTCACTGTGTCAAGCGGAGGAGGTGGATCAGGTGGAGGTGGAAGCGGGGGAGGA

GGTTCCGGCGGCGGAGGATCAGATATCGTGATGACGCAATCGCCTTCCTCGTTGTCCGCATCCG

TGGGAGACAGGGTGACCATTACTTGCAGAGCGTCCCAGTCCATTCGGTACTACCTGTCGTGGTA CCAGCAGAAGCCGGGGAAAGCCCCAAAACTGCTTATCTATACTGCCTCGATCCTCCAAAACGGC GTGCCATCAAGATTCAGCGGTTCGGGCAGCGGGACCGACTTTACCCTGACTATCAGCAGCCTGC AGCCGGAAGATTTCGCCACGTACTACTGCCTGCAAACCTACACCACCCCGGACTTCGGACCTGG AAC CAAGG T GGAGAT CAAG

M5 Human anti-MSLN scFv amino acid sequence (SEQ ID NO: 49)

QVQLVQSGAEVEKPGASVKVSCKASGYT FTDYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKF QGRVTMTRDTS I S TAYMELSRLRSDDTAVYYCASGWDFDYWGQGTLVTVSSGGGGSGGGGSGGG GSGGGGSDIVMTQS PSSLSASVGDRVT I TCRASQS IRYYLSWYQQKPGKAPKLL IYTAS ILQNG VPSRFSGSGSGTDFTLT I S SLQPEDFATYYCLQTYTTPDFGPGTKVE IK

Anti-GD2 scFv nucleotide sequence (SEQ ID NO: 50)

ATGGCCTTACCAGTGACCGCCTTGCTCCTGCCGCTGGCCTTGCTGCTCCACGCCGCCAGGCCGG GATCCGATGTTGTGATGACCCAAACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTC CATCTCTTG C AGAT C T AG T C AGAG T C T T G T ACAC C G T AAC G GAAAC AC C T AT T TAG AT T G G T AC CTGCAGAAGCCAGGCCAGTCTCCAAAGCTCCTGATTCACAAAGTTTCCAACCGATTTTCTGGGG TCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGA GGCTGAGGATCTGGGAGTTTATTTCTGTTCTCAAAGTACACACGTTCCTCCGCTCACGTTCGGT GCTGGGACCAAGCTGGAGCTGAAAGGAGGTGGCGGGTCAGGGGGTGGCGGAAGCGGAGGCGGCG GTTCAGGCGGAGGAGGCTCGGAGGTGCAGCTTCTGCAGTCTGGACCTGAGCTGGAGAAGCCTTC CGCTTCAGTGATGATATCCTGCAAGGCTTCTGGTTCCTCCTTCACTGGCTACAACATGAACTGG GTGAGGCAGAATATTGGAAAGAGCCTTGAATGGATTGGAGCTATTGATCCTTACTACGGTGGAA CTAGCTACAACCAGAAGTTCAAGGGCAGGGCCACATTGACTGTAGACAAATCGTCCAGCACAGC CTACATGCACCTCAAGAGCCTGACATCTGAGGACTCTGTCTATTACTGTGTAAGCGGAATGGAG TACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCATCCGGA

Anti-GD2 scFv amino acid sequence (SEQ ID NO: 51)

MALPVTALLLPLALLLHTWRPGSDWMTQTPLSLPVSLGDQAS I SCRS SQSLVHRNGNTYLHWY LQKPGQS PKLL IHKVSNRFSGVPDRFSGSGSGTDFTLKI SRVEAEDLGVYFCSQSTHVPPLT FG AGTKLELKGGGGSGGGGSGGGGSGGGGSEVQLLQSGPELEKPSASVMI SCKASGSS FTGYNMNW VRQNI GKSLEWI GAIDPYYGGTSYNQKFKGRATLTVDKS S S TAYMHLKSLTSEDSVYYCVSGME YWGQGTSVTVS S SG Anti-FAP scFv nucleotide sequence (SEQ ID NO: 52)

CAAATTGTTCTCACCCAGTCTCCAGCGCTCATGTCTGCTTCTCCAGGGGAGAAGGTCACCATGA

CCTGCACTGCCAGCTCAAGTGTTAGTTACATGTACTGGTACCAGCAGAAGCCACGATCCTCCCC

CAAACCCTGGATTTTTCTCACCTCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCCGT

GGGTCTGGGACCTCTTTCTCTCTCACAATCAGCAGCATGGAGGCTGAAGATGCTGCCACTTATT

ACTGCCAGCAGTGGAGTGGTTACCCACCCATCACATTCGGCTCGGGGACAAAGTTGGAAATAAA

AGGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGAAGCCAGGTCCAACTGCAGCAG

CCTGGGGCTGAACTGGTAAAGCCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCGTCTGGCTACA

CCATCACCAGCTACTCTCTGCACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATTGG

AGAGATTAATCCTGCCAATGGTGATCATAACTTCAGTGAGAAGTTCGAGATCAAGGCCACACTG

ACTGTAGACAGCTCCTCCAACACAGCATTCATGCAACTCAGCAGGCTGACATCTGAGGACTCTG

CGGTCTATTACTGTACAAGATTGGACGATAGTAGGTTCCACTGGTACTTCGATGTCTGGGGCGC AGGGACCACGGTCACCGTCTCCTCA

Anti-FAP scFv amino acid sequence (SEQ ID NO: 53)

QIVLTQSPALMSASPGEKVTMTCTASSSVSYMYWYQQKPRSSPKPWI FLTSNLASGVPARFSGR

GSGTS FSLT ISSMEAEDAATYYCQQWSGYPPITFGSGTKLE IKGGGGSGGGGSGGGGSQVQLQQ PGAELVKPGASVKLSCKASGYTI TSYSLHWVKQRPGQGLEWIGEINPANGDHNFSEKFE IKATL TVDSSSNTAFMQLSRLTSEDSAVYYCTRLDDSRFHWYFDVWGAGTTVTVSS

Human CD 19 scFv (SEQ ID NO: 54)

GAAATTGTGATGACCCAGTCACCCGCCACTCTTAGCCTTTCACCCGGTGAGCGCGCAACCCTGT

CTTGCAGAGCCTCCCAAGACATCTCAAAATACCTTAATTGGTATCAACAGAAGCCCGGACAGGC

TCCTCGCCTTCTGATCTACCACACCAGCCGGCTCCATTCTGGAATCCCTGCCAGGTTCAGCGGT

AGCGGATCTGGGACCGACTACACCCTCACTATCAGCTCACTGCAGCCAGAGGACTTCGCTGTCT

ATTTCTGTCAGCAAGGGAACACCCTGCCCTACACCTTTGGACAGGGCACCAAGCTCGAGATTAA

AGGTGGAGGTGGCAGCGGAGGAGGTGGGTCCGGCGGTGGAGGAAGCCAGGTCCAACTCCAAGAA

AGCGGACCGGGTCTTGTGAAGCCATCAGAAACTCTTTCACTGACTTGTACTGTGAGCGGAGTGT

CTCTCCCCGATTACGGGGTGTCTTGGATCAGACAGCCACCGGGGAAGGGTCTGGAATGGATTGG

AGTGATTTGGGGCTCTGAGACTACTTACTACCAATCATCCCTCAAGTCACGCGTCACCATCTCA

AAGGACAACTCTAAGAATCAGGTGTCACTGAAACTGTCATCTGTGACCGCAGCCGACACCGCCG TGTACTATTGCGCTAAGCATTACTATTATGGCGGGAGCTACGCAATGGATTACTGGGGACAGGG TACTCTGGTCACCGTGTCCAGC

Human CD 19 scFv (SEQ ID NO: 55)

E IVMTQSPATLSLSPGERATLSCRASQDI SKYLNWYQQKPGQAPRLLIYHTSRLHSGI PARFSG SGSGTDYTLTI SSLQPEDFAVYFCQQGNTLPYTFGQGTKLE IKGGGGSGGGGSGGGGSQVQLQE SGPGLVKPSETLSLTCTVSGVSLPDYGVSWIRQPPGKGLEWIGVIWGSETTYYQSSLKSRVT I S KDNSKNQVSLKLSSVTAADTAVYYCAKHYYYGGSYAMDYWGQGTLVTVSS

FDC6 (HL) fibronectin CAR (SEQ ID NO: 107)

MALPVTALLLPLALLLHAARPGSQVTLKESGPGILQPSQTLSLTCSFSGFSLSTSGMGVG WIRQPSGKGLEWLAHIWWDDTRRYNPALKSRLTISNDTSNNQVFLKIASVDTADTATY YCARMNGNYPAWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSDVLMTQTPLSLPVSL GDQASISCRSSQNIVHSNGNTYLEWYLLKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTD FTLKISRVEAEDLGIYYCFQGSHIPPTFGGGTKLEIKSGTTTPAPRPPTPAPTIASQPLSLRP EACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPF MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREE

YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR

FDC6 (LH) fibronectin CAR (SEQ ID NO: 108)

MALPVTALLLPLALLLHAARPGSDVLMTQTPLSLPVSLGDQASISCRSSQNIVHSNGNTY LEWYLLKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGIYYCFQGS HIPPTFGGGTKLEIKGGGGSGGGGSGGGGSQVTLKESGPGILQPSQTLSLTCSFSGFSLST SGMGVGWIRQPSGKGLEWLAHIWWDDTRRYNPALKSRLTISNDTSNNQVFLKIASVDT ADTATYYCARMNGNYPAWFAYWGQGTLVTVSSSGTTTPAPRPPTPAPTIASQPLSLRPE ACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPF MRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREE

YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHD GLYQGLSTATKDTYDALHMQALPPR mAblO9_BBz CEACAM6 CAR (SEQ ID NO: 109) MALPVTALLLPLALLLHAARPGSDIKMTQSPSSLSASVGDRVTITCGASENIYGALNWFQ RKPGKAPKLLIYGATNLADGMPSRFSGSGSGRDFTLTISSLQPEDFATYFCQNVLSIPYTF GQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKGSGYPFTDYTI HWVRQAPGQGLEWIGHISTYSGNTNNNQKFKGRATMTVDKSTSTAYMELRSLRSDDTT VYYCARGDYYGSFYKFEYWGQGTTVTVSSSGTTTPAPRPPTPAPTIASQPLSLRPEACRP AAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPV QTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQ GL ST ATKDTYD ALHMQ ALPPR

Transmembrane Domain

CARs of the present invention may comprise a transmembrane domain that connects the antigen binding domain of the CAR to the intracellular domain of the CAR. The transmembrane domain of the CAR is a region that is capable of spanning the plasma membrane of a cell (e.g., an immune cell or precursor thereof). The transmembrane domain is for insertion into a cell membrane, e.g., a eukaryotic cell membrane. In some embodiments, the transmembrane domain is interposed between the antigen binding domain and the intracellular domain of a CAR.

In some embodiments, the transmembrane domain is naturally associated with one or more of the domains in the CAR. In some embodiments, the transmembrane domain can be selected or modified by one or more amino acid substitutions to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, to minimize interactions with other members of the receptor complex.

The transmembrane domain may be derived either from a natural or a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein, e.g., a Type I transmembrane protein. Where the source is synthetic, the transmembrane domain may be any artificial sequence that facilitates insertion of the CAR into a cell membrane, e.g., an artificial hydrophobic sequence. Examples of the transmembrane domain of particular use in this invention include, without limitation, transmembrane domains derived from (i.e. comprise at least the transmembrane region(s) of) the alpha, beta or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD7, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134 (OX-40), CD137 (4-1BB), CD154 (CD40L), ICOS, CD278, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 or a transmembrane domain derived from a killer immunoglobulin-like receptor (KIR).

In certain embodiments, the transmembrane domain comprises a transmembrane domain of CD8. In certain embodiments, the transmembrane domain of CD8 is a transmembrane domain of CD8a.

In some embodiments, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.

The transmembrane domains described herein can be combined with any of the antigen binding domains described herein, any of the intracellular domains described herein, or any of the other domains described herein that may be included in the CAR.

In some embodiments, the transmembrane domain further comprises a hinge region. The CAR of the present invention may also include a hinge region. The hinge region of the CAR is a hydrophilic region which is located between the antigen binding domain and the transmembrane domain. In some embodiments, this domain facilitates proper protein folding for the CAR. The hinge region is an optional component for the CAR. The hinge region may include a domain selected from Fc fragments of antibodies, hinge regions of antibodies, CH2 regions of antibodies, CH3 regions of antibodies, artificial hinge sequences or combinations thereof. Examples of hinge regions include, without limitation, a CD8a hinge, artificial hinges made of polypeptides which may be as small as, three glycines (Gly), as well as CHI and CH3 domains of IgGs (such as human IgG4).

In some embodiments, a CAR includes a hinge region that connects the antigen binding domain with the transmembrane domain, which, in turn, connects to the intracellular domain. The hinge region is preferably capable of supporting the antigen binding domain to recognize and bind to the target antigen on the target cells (see, e.g., Hudecek et al., Cancer Immunol. Res. (2015) 3(2): 125-135). In some embodiments, the hinge region is a flexible domain, thus allowing the antigen binding domain to have a structure to optimally recognize the specific structure and density of the target antigens on a cell such as tumor cell (Hudecek et al., supra). The flexibility of the Hinge region permits the hinge region to adopt many different conformations.

In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. In some embodiments, the hinge region is a hinge region polypeptide derived from a receptor (e.g., a CD8-derived hinge region). In certain embodiments, the hinge region is a CD8a hinge.

The hinge region can have a length of from about 4 amino acids to about 50 amino acids, e.g., from about 4 aa to about 10 aa, from about 10 aa to about 15 aa, from about 15 aa to about 20 aa, from about 20 aa to about 25 aa, from about 25 aa to about 30 aa, from about 30 aa to about 40 aa, or from about 40 aa to about 50 aa. In some embodiments, the hinge region can have a length of greater than 5 aa, greater than 10 aa, greater than 15 aa, greater than 20 aa, greater than 25 aa, greater than 30 aa, greater than 35 aa, greater than 40 aa, greater than 45 aa, greater than 50 aa, greater than 55 aa, or more.

Suitable hinge regions can be readily selected and can be of any of a number of suitable lengths, such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 amino acids to 15 amino acids, from 3 amino acids to 12 amino acids, including 4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 amino acids to 8 amino acids, or 7 amino acids to 8 amino acids, and can be 1, 2, 3, 4, 5, 6, or 7 amino acids. Suitable hinge regions can have a length of greater than 20 amino acids (e.g., 30, 40, 50, 60 or more amino acids).

For example, hinge regions include glycine polymers (G)n, glycine-serine polymers (including, for example, (GS)_n, (GSGGS)n and (GGGS)_n, where n is an integer of at least one), glycine-alanine polymers, alanine-serine polymers, and other flexible linkers known in the art. Glycine and glycine-serine polymers can be used; both Gly and Ser are relatively unstructured, and therefore can serve as a neutral tether between components. Glycine polymers can be used; glycine accesses significantly more phi-psi space than even alanine, and is much less restricted than residues with longer side chains (see, e.g., Scheraga, Rev. Computational. Chem. (1992) 2: 73-142). Exemplary hinge regions can comprise amino acid sequences including, but not limited to, (GGGGS)n, GGSG, GGSGG, GSGSG, GSGGG, GGGSG, GSSSG, GGGGS and the like.

In some embodiments, the hinge region is an immunoglobulin heavy chain hinge region. Immunoglobulin hinge region amino acid sequences are known in the art; see, e.g., Tan et al.. Proc. Natl. Acad. Sci. USA (1990) 87(1): 162-166; and Huck et al., Nucleic Acids Res. (1986) 14(4): 1779-1789. As non-limiting examples, an immunoglobulin hinge region can include one of the following amino acid sequences: DKTHT; CPPC; CPEPKSCDTPPPCPR (see, e.g., Glaser et al., J. Biol. Chem. (2005) 280:41494-41503); ELKTPLGDTTHT; KSCDKTHTCP;

KCCVDCP; KYGPPCP; EPKSCDKTHTCPPCP (human IgGl hinge); ERKCCVECPPCP (human IgG2 hinge); ELKTPLGDTTHTCPRCP (human IgG3 hinge); SPNMVPHAHHAQ (human IgG4 hinge); and the like.

The hinge region can comprise an amino acid sequence of a human IgGl, IgG2, IgG3, or IgG4, hinge region. In one embodiment, the hinge region can include one or more amino acid substitutions and/or insertions and/or deletions compared to a wild-type (naturally-occurring) hinge region. For example, His229 of human IgGl hinge can be substituted with Tyr, so that the hinge region comprises the sequence EPKSCDKTYTCPPCP; see, e.g., Yan et al., J. Biol. Chem. (2012) 287: 5891-5897. In one embodiment, the hinge region can comprise an amino acid sequence derived from human CD8, or a variant thereof.

Intracellular Domain

A CAR of the present invention also includes an intracellular signaling domain. The terms “intracellular signaling domain” and “intracellular domain” are used interchangeably herein. The intracellular signaling domain of the CAR is responsible for activation of at least one of the effector functions of the cell in which the CAR is expressed (e.g., immune cell). The intracellular signaling domain transduces the effector function signal and directs the cell (e.g., immune cell) to perform its specialized function, e.g., harming and/or destroying a target cell.

Examples of an intracellular domain for use in the invention include, but are not limited to, the cytoplasmic portion of a surface receptor, co-stimulatory molecule, and any molecule that acts in concert to initiate signal transduction in the T cell, as well as any derivative or variant of these elements and any synthetic sequence that has the same functional capability.

Examples of the intracellular signaling domain include, without limitation, the , chain of the T cell receptor complex or any of its homologs, e.g., q chain, FcsRIy and P chains, MB 1 (Iga) chain, B29 (Ig) chain, etc., human CD3 zeta chain, CD3 polypeptides (A, 8 and e), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lek, Fyn, Lyn, etc.), and other molecules involved in T cell transduction, such as CD2, CD5 and CD28. In one embodiment, the intracellular signaling domain may be human CD3 zeta chain, FcyRIII, FcsRI, cytoplasmic tails of Fc receptors, an immunoreceptor tyrosine-based activation motif (IT AM) bearing cytoplasmic receptors, and combinations thereof.

In one embodiment, the intracellular signaling domain of the CAR includes any portion of one or more co-stimulatory molecules, such as at least one signaling domain from CD2, CD3, CD8, CD27, CD28, ICOS, 4-1BB, PD-1, any derivative or variant thereof, any synthetic sequence thereof that has the same functional capability, and any combination thereof.

Other examples of the intracellular domain include a fragment or domain from one or more molecules or receptors including, but not limited to, TCR, CD3 zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon rib), CD79a, CD79b, Fcgamma Rlla, DAP10, DAP12, T cell receptor (TCR), CD8, CD27, CD28, 4-1BB (CD137), 0X9, 0X40, CD30, CD40, PD-1, ICOS, a KIR family protein, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, a ligand that specifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD127, CD 160, CD 19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD l id, ITGAE, CD 103, ITGAL, CD 11 a, LFA-1, ITGAM, cDlib, ITGAX, CD 11c, ITGB1, CD29, ITGB2, CD 18, LFA- 1, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD 150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, Toll-like receptor 1 (TLR1), TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, other costimulatory molecules described herein, any derivative, variant, or fragment thereof, any synthetic sequence of a co-stimulatory molecule that has the same functional capability, and any combination thereof.

Additional examples of intracellular domains include, without limitation, intracellular signaling domains of several types of various other immune signaling receptors, including, but not limited to, first, second, and third generation T cell signaling proteins including CD3, B7 family costimulatory, and Tumor Necrosis Factor Receptor (TNFR) superfamily receptors (see, e.g., Park and Brentjens, J. Clin. Oncol. (2015) 33(6): 651-653). Additionally, intracellular signaling domains may include signaling domains used by NK and NKT cells (see, e.g. Hermanson and Kaufman, Front. Immunol. (2015) 6: 195) such as signaling domains of NKp30 (B7-H6) (see, e.g., Zhang et al., J. Immunol. (2012) 189(5): 2290-2299), and DAP 12 (see, e.g., Topfer et al., J. Immunol. (2015) 194(7): 3201-3212), NKG2D, NKp44, NKp46, DAP10, and CD3z.

Intracellular signaling domains suitable for use in a CAR of the present invention include any desired signaling domain that provides a distinct and detectable signal (e.g., increased production of one or more cytokines by the cell; change in transcription of a target gene; change in activity of a protein; change in cell behavior, e.g., cell death; cellular proliferation; cellular differentiation; cell survival; modulation of cellular signaling responses; etc.) in response to activation of the CAR (i.e., activated by antigen and dimerizing agent). In some embodiments, the intracellular signaling domain includes at least one (e.g., one, two, three, four, five, six, etc.) ITAM motifs as described below. In some embodiments, the intracellular signaling domain includes DAP10/CD28 type signaling chains. In some embodiments, the intracellular signaling domain is not covalently attached to the membrane bound CAR, but is instead diffused in the cytoplasm.

Intracellular signaling domains suitable for use in a CAR of the present invention include immunoreceptor tyrosine-based activation motif (ITAM)-containing intracellular signaling polypeptides. In some embodiments, an ITAM motif is repeated twice in an intracellular signaling domain, where the first and second instances of the ITAM motif are separated from one another by 6 to 8 amino acids. In one embodiment, the intracellular signaling domain of the CAR comprises 3 ITAM motifs.

In some embodiments, intracellular signaling domains includes the signaling domains of human immunoglobulin receptors that contain immunoreceptor tyrosine based activation motifs (IT AMs) such as, but not limited to, FcgammaRI, FcgammaRIIA, FcgammaRIIC, FcgammaRIIIA, FcRL5 (see, e.g., Gillis et al., Front. Immunol. (2014) 5:254).

A suitable intracellular signaling domain can be an ITAM motif-containing portion that is derived from a polypeptide that contains an ITAM motif. For example, a suitable intracellular signaling domain can be an ITAM motif-containing domain from any ITAM motif-containing protein. Thus, a suitable intracellular signaling domain need not contain the entire sequence of the entire protein from which it is derived. Examples of suitable ITAM motif-containing polypeptides include, but are not limited to: DAP12, FCER1G (Fc epsilon receptor I gamma chain), CD3D (CD3 delta), CD3E (CD3 epsilon), CD3G (CD3 gamma), CD3Z (CD3 zeta), and CD79A (antigen receptor complex-associated protein alpha chain).

In one embodiment, the intracellular signaling domain is derived from DAP12 (also known as TYROBP; TYRO protein tyrosine kinase binding protein; KARAP; PLOSL; DNAX- activation protein 12; KAR-associated protein; TYRO protein tyrosine kinase-binding protein; killer activating receptor associated protein; killer-activating receptor-associated protein; etc.). In one embodiment, the intracellular signaling domain is derived from FCER1G (also known as FCRG; Fc epsilon receptor I gamma chain; Fc receptor gamma-chain; fc-epsilon Rl-gamma; fcRgamma; fceRl gamma; high affinity immunoglobulin epsilon receptor subunit gamma; immunoglobulin E receptor, high affinity, gamma chain; etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 delta chain (also known as CD3D; CD3 -DELTA; T3D; CD3 antigen, delta subunit; CD3 delta; CD3d antigen, delta polypeptide (TiT3 complex); OKT3, delta chain; T-cell receptor T3 delta chain; T-cell surface glycoprotein CD3 delta chain; etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 epsilon chain (also known as CD3e, T- cell surface antigen T3/Leu-4 epsilon chain, T-cell surface glycoprotein CD3 epsilon chain, AI504783, CD3, CD3epsilon, T3e, etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 gamma chain (also known as CD3G, T-cell receptor T3 gamma chain, CD3-GAMMA, T3G, gamma polypeptide (TiT3 complex), etc.). In one embodiment, the intracellular signaling domain is derived from T-cell surface glycoprotein CD3 zeta chain (also known as CD3Z, T-cell receptor T3 zeta chain, CD247, CD3-ZETA, CD3H, CD3Q, T3Z, TCRZ, etc.). In one embodiment, the intracellular signaling domain is derived from CD79A (also known as B-cell antigen receptor complex-associated protein alpha chain; CD79a antigen (immunoglobulin-associated alpha); MB-1 membrane glycoprotein; ig- alpha; membrane-bound immunoglobulin-associated protein; surface IgM-associated protein; etc.). In one embodiment, an intracellular signaling domain suitable for use in a CAR of the present disclosure includes a DAP10/CD28 type signaling chain. In one embodiment, an intracellular signaling domain suitable for use in a CAR of the present disclosure includes a ZAP70 polypeptide. In some embodiments, the intracellular signaling domain includes a cytoplasmic signaling domain of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, or CD66d. Tn one embodiment, the intracellular signaling domain in the CAR includes a cytoplasmic signaling domain of human CD3 zeta.

While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The intracellular signaling domain includes any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

The intracellular domains described herein can be combined with any of the antigen binding domains described herein, any of the transmembrane domains described herein, or any of the other domains described herein that may be included in a CAR.

In certain embodiments, the intracellular domain comprises a costimulatory domain of 4- 1BB. In certain embodiments, the intracellular domain comprises an intracellular domain of CD3(^ or a variant thereof. In certain embodiments, the intracellular domain comprises 4-1BB and CD3(^ domains.

Nucleic Acids

In one aspect, the invention includes a nucleic acid molecule comprising a nucleotide sequence encoding the anti-scFv antibody or antigen-binding fragment described herein. .

In one aspect, the invention includes a nucleic acid molecule comprising a nucleotide sequence encoding the antibody-linked cytokine of the invention. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 26. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 28. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 30. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30.

In some aspects, the invention includes a nucleoside-modified nucleic acid molecule (e.g., a nucleoside-modified RNA) encoding an immunostimulatory cytokine. In some embodiments, the immunostimulatory cytokine is selected from IL-12, IL-18, IL-23, and any combination thereof. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 20. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 22. In some embodiments, the nucleotide sequence comprises SEQ ID NO: 24. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 20, SEQ ID NO: 22, or SEQ ID NO: 24.

In certain embodiments, a nucleic acid of the present disclosure comprises a first polynucleotide sequence and a second polynucleotide sequence. The first and second polynucleotide sequence may be separated by a linker. A linker for use in the present disclosure allows for multiple proteins to be encoded by the same nucleic acid sequence (e.g., a multi ci str onic or bicistronic sequence), which are translated as a polyprotein that is dissociated into separate protein components. In certain embodiments, the nucleic acid comprises from 5’ to 3’ the first polynucleotide sequence, the linker, and the second polynucleotide sequence. In certain embodiments, the nucleic acid comprises from 5’ to 3’ the second polynucleotide sequence, the linker, and the first polynucleotide sequence.

In some embodiments, the linker comprises a nucleic acid sequence that encodes for an internal ribosome entry site (IRES). As used herein, “an internal ribosome entry site” or “IRES” refers to an element that promotes direct internal ribosome entry to the initiation codon, such as ATG, of a protein coding region, thereby leading to cap-independent translation of the gene. Various internal ribosome entry sites are known to those of skill in the art, including, without limitation, IRES obtainable from viral or cellular mRNA sources, e.g., immunoglobulin heavychain binding protein (BiP); vascular endothelial growth factor (VEGF); fibroblast growth factor 2; insulin-like growth factor; translational initiation factor eIF4G; yeast transcription factors TFIID and HAP4; and IRES obtainable from, e.g., cardiovirus, rhinovirus, aphthovirus, HCV, Friend murine leukemia virus (FrMLV), and Moloney murine leukemia virus (MoMLV). Those of skill in the art would be able to select the appropriate IRES for use in the present invention.

In some embodiments, the antibody or antibody-cytokine fusion of the invention is expressed from a single nucleic acid molecule comprising a nucleotide sequence encoding a ribosome slip sequence (also known as a self-processing site). In some embodiments, the nucleotide sequence encoding the antibody of the invention comprises the ribosome slip sequence positioned between the nucleotide sequence encoding the heavy chain and the nucleotide sequence encoding the light chain. In some embodiments, the nucleotide sequence encoding the antibody-linked cytokine encodes the heavy chain and the cytokine as a fusion polypeptide (linked via a linker such as a polyalanine linker) and the nucleotide sequence encoding the ribosome slip sequence is positioned between the nucleotide sequence encoding the cytokine and the nucleotide sequence encoding the light chain. Preferably, the “self-processing” site is a ribosomal skipping site, also known as a ribosome slip sequence. As used herein, the terms “self-cleavage” and “self-processing” relate to “cleavage” without proteases, for example by ribosomal skipping. Preferably, a nucleotide sequence encoding a self-processing site is a nucleotide sequence encoding the amino acid sequence Asp-Val/Ile-Glu-X-Asn-Pro-Gly-Pro, wherein X may be any amino acid (i.e., DX1EX2NPGP, wherein Xi is Vai or He and X2 may be any (naturally occurring) amino acid; SEQ ID NO: 33). Ribosomal skipping leads to the provision of separate entities in the course of mRNA translation. The underlying mechanism is based on non-formation of a covalent linkage between two amino acids, i.e. G (Gly) and P (Pro) during mRNA translation. Accordingly, the mRNA translation is not interrupted by the nonformation of a covalent bond between the Gly/Pro, but rather proceeds without stopping the ribosomal activity on the mRNA. In particular, the ribosomes do not form a peptide bond between these amino acids, if a sequence pattern Asp-Val/Ile-Glu-X-Asn-Pro-Gly-Pro occurs in a peptide sequence. Non formation of a covalent bond occurs between the C-terminal Gly-Pro position of the above amino acid stretch. Preferred self-processing sites are 2A-sites, such as T2A (EGRGSLLTCGDVEENPGP; SEQ ID NO: 34); F2A (KQTLNFDLLKLAGDVESNPGP; SEQ ID NO: 35 ); E2A (QCTNYALLKLAGDVESNPGP; SEQ ID NO: 36); or P2A (ATNFSLLKQAGDVEENPGP; SEQ ID NO: 37); or sequence variants thereof , e.g., the sequence variants according to SEQ ID NO: 38 (GSGATNFSLLKQAGDVEENPGP) or SEQ ID NO: 39 (RKRRGSGATNFSLLKQAGDVEENPGP).

In some embodiments, a linker further comprises a nucleic acid sequence that encodes a furin cleavage site. Furin is a ubiquitously expressed protease that resides in the trans-golgi and processes protein precursors before their secretion. Furin cleaves at the COOH- terminus of its consensus recognition sequence. Various furin consensus recognition sequences (or “furin cleavage sites”) are known to those of skill in the art, including, without limitation, Arg-Xl-Lys- Arg or Arg-Xl-Arg-Arg, X2-Arg-Xl-X3-Arg, and Arg-Xl-Xl-Arg, such as an Arg-Gln-Lys- Arg, where XI is any naturally occurring amino acid, X2 is Lys or Arg, and X3 is Lys or Arg. Those of skill in the art would be able to select the appropriate Furin cleavage site for use in the present invention.

In some embodiments, the linker comprises a nucleic acid sequence encoding a combination of a Furin cleavage site and a 2A peptide. Examples include, without limitation, a linker comprising a nucleic acid sequence encoding a Furin cleavage site and F2A, a linker comprising a nucleic acid sequence encoding a Furin cleavage site and E2A, a linker comprising a nucleic acid sequence encoding a Furin cleavage site and P2A, a linker comprising a nucleic acid sequence encoding a Furin cleavage site and T2A. Those of skill in the art would be able to select the appropriate combination for use in the present invention. In such embodiments, the linker may further comprise a spacer sequence between the Furin cleavage site and the 2A peptide. In some embodiments, the linker comprises a Furin cleavage site 5’ to a 2A peptide. In some embodiments, the linker comprises a 2A peptide 5’ to a Furin cleavage site. Various spacer sequences are known in the art, including, without limitation, glycine serine (GS) spacers (also known as GS linkers) such as (GS)n, (SG)n, (GSGGS)n and (GGGS)n, where n represents an integer of at least 1. Exemplary spacer sequences can comprise amino acid sequences including, without limitation, GGSG, GGSGG, GSGSG, GSGGG, GGGSG, GSSSG, and the like. Those of skill in the art would be able to select the appropriate spacer sequence for use in the present invention.

The nucleotide sequences encoding the anti-scFv antibody, antibody-linked cytokine, immunostimulatory cytokines, and CARs of the invention, as described herein, can alternatively comprise sequence variations with respect to the original nucleotide sequences, for example, substitutions, insertions and/or deletions of one or more nucleotides, with the condition that the resulting polynucleotide encodes a polypeptide according to the invention. Therefore, the scope of the present invention includes nucleotide sequences that are substantially homologous to the known and/or reference nucleotide sequences which encode an immunostimulatory cytokine of interest.

As used herein, a nucleotide sequence is “substantially homologous” to any of the nucleotide sequences described herein when its nucleotide sequence has a degree of identity with respect to the nucleotide sequence of at least 60%, of at least 65%, of at least 70%, of at least 65%, of at least 80%, of at least 85%, of at least 90%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, or of at least 99%. A nucleotide sequence that is substantially homologous to a nucleotide sequence encoding an immunostimulatory cytokine can typically be isolated from a producer organism of the an immunostimulatory cytokine based on the information contained in the nucleotide sequence by means of introducing conservative or non-conservative substitutions, for example. Other examples of possible modifications include the insertion of one or more nucleotides within the sequence, the addition of one or more nucleotides at the 3’ and/or 5’ end of the sequence, or the deletion of one or more nucleotides at the 3’ and/or 5’ end or from within the sequence. The degree of identity between two polynucleotides is determined using computer algorithms and methods that are widely known for the persons skilled in the art.

Further, the scope of the invention includes nucleotide sequences that encode amino acid sequences of the anti-scFv antibody, antibody-linked cytokine, immunostimulatory cytokines, and CARs of the invention that preserve the cellular function of the the anti-scFv antibody, antibody-linked cytokine, immunostimulatory cytokines, and CARs of the invention, respectively.

As used herein, an amino acid sequence is “substantially homologous” to a known or reference amino acid sequence when its amino acid sequence has a degree of identity with respect to the amino acid sequence of at least 60%, of at least 65%, of at least 70%, of at least 65%, of at least 80%, of at least 85%, of at least 90%, of at least 95%, of at least 96%, of at least 97%, of at least 98%, or of at least 99%. The identity between two amino acid sequences can be determined by using the BLASTP algorithm (BLAST Manual, Altschul, S., et ah, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et ah, J. Mol. Biol. 215: 403-410 (1990)).

In some embodiments, a nucleic acid of the present disclosure may be operably linked to a transcriptional control element, e.g., a promoter, and enhancer, etc. Suitable promoter and enhancer elements are known to those of skill in the art.

For expression in a bacterial cell, suitable promoters include, but are not limited to, lacl, lacZ, T3, T7, gpt, lambda P and trc. For expression in a eukaryotic cell, suitable promoters include, but are not limited to, light and/or heavy chain immunoglobulin gene promoter and enhancer elements; cytomegalovirus immediate early promoter; herpes simplex virus thymidine kinase promoter; early and late SV40 promoters; promoter present in long terminal repeats from a retrovirus; mouse metallothionein-I promoter; and various art-known tissue specific promoters. Suitable reversible promoters, including reversible inducible promoters are known in the art. Such reversible promoters may be isolated and derived from many organisms, e.g., eukaryotes and prokaryotes. Modification of reversible promoters derived from a first organism for use in a second organism, e.g., a first prokaryote and a second a eukaryote, a first eukaryote and a second a prokaryote, etc., is well known in the art. Such reversible promoters, and systems based on such reversible promoters but also comprising additional control proteins, include, but are not limited to, alcohol regulated promoters (e.g., alcohol dehydrogenase I (alcA) gene promoter, promoters responsive to alcohol transactivator proteins (AlcR), etc.), tetracycline regulated promoters, (e.g., promoter systems including Tet Activators, TetON, TetOFF, etc.), steroid regulated promoters (e.g., rat glucocorticoid receptor promoter systems, human estrogen receptor promoter systems, retinoid promoter systems, thyroid promoter systems, ecdysone promoter systems, mifepristone promoter systems, etc.), metal regulated promoters (e.g., metallothionein promoter systems, etc.), pathogenesis-related regulated promoters (e.g., salicylic acid regulated promoters, ethylene regulated promoters, benzothiadiazole regulated promoters, etc.), temperature regulated promoters (e.g., heat shock inducible promoters (e.g., HSP-70, HSP-90, soybean heat shock promoter, etc.), light regulated promoters, synthetic inducible promoters, and the like.

In some embodiments, the promoter is a CD8 cell-specific promoter, a CD4 cell-specific promoter, a neutrophil-specific promoter, or an NK-specific promoter. For example, a CD4 gene promoter can be used; see, e.g., Salmon et al. Proc. Natl. Acad. Sci. USA (1993) 90:7739; and Marodon et al. (2003) Blood 101:3416. As another example, a CD8 gene promoter can be used. NK cell-specific expression can be achieved by use of an Neri (p46) promoter; see, e.g., Eckelhart et al. Blood (2011) 117: 1565.

For expression in a yeast cell, a suitable promoter is a constitutive promoter such as an ADH1 promoter, a PGK1 promoter, an ENO promoter, a PYK1 promoter and the like; or a regulatable promoter such as a GALI promoter, a GAL 10 promoter, an ADH2 promoter, a PHOS promoter, a CUP1 promoter, a GALT promoter, a MET25 promoter, a MET3 promoter, a CYC1 promoter, a HIS3 promoter, an ADH1 promoter, a PGK promoter, a GAPDH promoter, an ADC1 promoter, a TRP1 promoter, a URA3 promoter, a LEU2 promoter, an ENO promoter, a TP1 promoter, and AOX1 (e.g., for use in Pichia). Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. Suitable promoters for use in prokaryotic host cells include, but are not limited to, a bacteriophage T7 RNA polymerase promoter; a trp promoter; a lac operon promoter; a hybrid promoter, e.g., a lac/tac hybrid promoter, a tac/trc hybrid promoter, a trp/lac promoter, a T7/lac promoter; a trc promoter; a tac promoter, and the like; an araBAD promoter; in vivo regulated promoters, such as an ssaG promoter or a related promoter (see, e.g., U.S. Patent Publication No. 20040131637), a pagC promoter (Pulkkinen and Miller, J. Bacteriol. (1991) 173(1): 86-93; Alpuche- Aranda et al., Proc. Natl. Acad. Sci. USA (1992) 89(21): 10079-83), a nirB promoter (Harborne et al. Mol. Micro. (1992) 6:2805-2813), and the like (see, e.g., Dunstan et al., Infect. Immun. (1999) 67:5133-5141; McKelvie et al., Vaccine (2004) 22:3243-3255; and Chatfield et al., Biotechnol. (1992) 10:888- 892); a sigma70 promoter, e.g., a consensus sigma70 promoter (see, e.g., GenBank Accession Nos. AX798980, AX798961, and AX798183); a stationary phase promoter, e.g., a dps promoter, an spv promoter, and the like; a promoter derived from the pathogenicity island SPL2 (see, e.g., WO96/17951); an actA promoter (see, e.g., Shetron-Rama et al., Infect. Immun. (2002) 70:1087- 1096); an rpsM promoter (see, e.g., Valdivia and Falkow Mol. Microbiol. (1996). 22:367); a tet promoter (see, e.g., Hillen, W. and Wissmann, A. (1989) In Saenger, W. and Heinemann, U. (eds), Topics in Molecular and Structural Biology, Protein— Nucleic Acid Interaction. Macmillan, London, UK, Vol. 10, pp. 143-162); an SP6 promoter (see, e.g., Melton et al., Nucl. Acids Res. (1984) 12:7035); and the like. Suitable strong promoters for use in prokaryotes such as Escherichia coli include, but are not limited to Trc, Tac, T5, T7, and pLambda. Non-limiting examples of operators for use in bacterial host cells include a lactose promoter operator (LacI repressor protein changes conformation when contacted with lactose, thereby preventing the Lad repressor protein from binding to the operator), a tryptophan promoter operator (when complexed with tryptophan, TrpR repressor protein has a conformation that binds the operator; in the absence of tryptophan, the TrpR repressor protein has a conformation that does not bind to the operator), and a tac promoter operator (see, e.g., deBoer et al., Proc. Natl. Acad. Sci. U.S.A. (1983) 80:21-25).

Other examples of suitable promoters include the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operatively linked thereto. Other constitutive promoter sequences may also be used, including, but not limited to a simian virus 40 (SV40) early promoter, a mouse mammary tumor virus (MMTV) or human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, a MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, the EF-1 alpha promoter, as well as human gene promoters such as, but not limited to, an actin promoter, a myosin promoter, a hemoglobin promoter, and a creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired, or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

In some embodiments, the locus or construct or transgene containing the suitable promoter is irreversibly switched through the induction of an inducible system. Suitable systems for induction of an irreversible switch are well known in the art, e.g., induction of an irreversible switch may make use of a Cre-l ox-mediated recombination (see, c.g., Fuhrmann-Benzakein, et al., Proc. Natl. Acad. Sci. USA (2000) 28:e99, the disclosure of which is incorporated herein by reference). Any suitable combination of recombinase, endonuclease, ligase, recombination sites, etc. known to the art may be used in generating an irreversibly switchable promoter. Methods, mechanisms, and requirements for performing site-specific recombination, described elsewhere herein, find use in generating irreversibly switched promoters and are well known in the art, see, c.g., Grindley et al. Annual Review of Biochemistry (2006) 567-605; and Tropp, Molecular Biology (2012) (Jones & Bartlett Publishers, Sudbury, Mass ), the disclosures of which are incorporated herein by reference.

In some embodiments, a nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a CAR inducible expression cassette. In one embodiment, the CAR inducible expression cassette is for the production of a transgenic polypeptide product that is released upon CAR signaling. See, e.g., Chmielewski and Ab ken, Expert Opin. Biol. Ther. (2015) 15(8): 1145-1154; and Abken, Immunotherapy (2015) 7(5): 535-544. In some embodiments, a nucleic acid of the present disclosure further comprises a nucleic acid sequence encoding a cytokine operably linked to a T-cell activation responsive promoter. In some embodiments, the cytokine operably linked to a T-cell activation responsive promoter is present on a separate nucleic acid sequence. In one embodiment, the cytokine is IL-12. A nucleic acid of the present disclosure may be present within an expression vector and/or a cloning vector. An expression vector can include a selectable marker, an origin of replication, and other features that provide for replication and/or maintenance of the vector. Suitable expression vectors include, e.g., plasmids, viral vectors, and the like. Large numbers of suitable vectors and promoters are known to those of skill in the art; many are commercially available for generating a subject recombinant construct. The following vectors are provided by way of example, and should not be construed in anyway as limiting: Bacterial: pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene, La Jolla, Calif., USA); pTrc99A, pKK223-3, pKK233-3, pDR540, and pRIT5 (Pharmacia, Uppsala, Sweden). Eukaryotic: pWLneo, pSV2cat, pOG44, PXR1, pSG (Stratagene) pSVK3, pBPV, pMSG and pSVL (Pharmacia).

Expression vectors generally have convenient restriction sites located near the promoter sequence to provide for the insertion of nucleic acid sequences encoding heterologous proteins. A selectable marker operative in the expression host may be present. Suitable expression vectors include, but are not limited to, viral vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest. Opthalmol. Vis. Sci. (1994) 35: 2543-2549; Borras et al., Gene Ther. (1999) 6: 515-524; Li and Davidson, Proc. Natl. Acad. Sci. USA (1995) 92: 7700-7704; Sakamoto et al., H. Gene Ther. (1999) 5: 1088-1097; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (see, e.g., Ali et al., Hum. Gene Ther. (1998) 9: 81-86, Flannery et al., Proc. Natl. Acad. Sci. USA (1997) 94: 6916-6921; Bennett et al., Invest. Opthalmol. Vis. Sci. (1997) 38: 2857- 2863; Jomary et al., Gene Ther. (1997) 4:683 690, Rolling et al., Hum. Gene Ther. (1999) 10: 641-648; Ali et al., Hum. Mol. Genet. (1996) 5: 591-594; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63: 3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., Proc. Natl. Acad. Sci. USA (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., Proc. Natl. Acad. Sci. USA (1997) 94: 10319- 23; Takahashi et al., J. Virol. (1999) 73: 7812-7816); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. Additional expression vectors suitable for use are, e.g., without limitation, a lentivirus vector, a gamma retrovirus vector, a foamy virus vector, an adeno-associated virus vector, an adenovirus vector, a pox virus vector, a herpes virus vector, an engineered hybrid virus vector, a transposon mediated vector, and the like. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, volumes 1-4, Cold Spring Harbor Press, NY), and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno- associated viruses, herpes viruses, and lentiviruses.

In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326, 193).

The invention further includes vectors, e.g., expression vectors or cloning vectors, harboring nucleotide sequences encoding various proteins and polypeptides of the invention (i.e., the anti-scFv antibody or antigen-binding fragment of the invention, the antibody-linked cytokines of the invention, the immunostimulatory cytokines of the invention, and CARs of the invention) as well as RNAs of the invention.

In some embodiments, an expression vector (e.g., a lentiviral vector) may be used to introduce a nucleic acid into an immune cell or precursor thereof (e.g., a T cell). Accordingly, an expression vector (e.g., a lentiviral vector) of the present invention may comprise a nucleic acid encoding for a CAR. In some embodiments, the expression vector (e.g., lentiviral vector) will comprise additional elements that will aid in the functional expression of the CAR encoded therein. In some embodiments, an expression vector comprising a nucleic acid encoding for a CAR further comprises a mammalian promoter. In one embodiment, the vector further comprises an elongation-factor- 1 -alpha promoter (EF-la promoter). Use of an EF-la promoter may increase the efficiency in expression of downstream transgenes (e.g., a CAR-en coding nucleic acid sequence). Physiologic promoters (e.g., an EF-la promoter) may be less likely to induce integration mediated genotoxicity, and may abrogate the ability of the retroviral vector to transform stem cells.

Other physiological promoters suitable for use in a vector (e.g., lentiviral vector) are known to those of skill in the art and may be incorporated into a vector of the present invention. In some embodiments, the vector (e.g., lentiviral vector) further comprises a non-requisite cis acting sequence that may improve titers and gene expression. One non-limiting example of a non-requisite cis acting sequence is the central polypurine tract and central termination sequence (cPPT/CTS) which is important for efficient reverse transcription and nuclear import. Other nonrequisite cis acting sequences are known to those of skill in the art and may be incorporated into a vector (e.g., lentiviral vector) of the present invention. In some embodiments, the vector further comprises a posttranscriptional regulatory element. Posttranscriptional regulatory elements may improve RNA translation, improve transgene expression and stabilize RNA transcripts. One example of a posttranscriptional regulatory element is the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Accordingly, in some embodiments a vector for the present invention further comprises a WPRE sequence. Various posttranscriptional regulator elements are known to those of skill in the art and may be incorporated into a vector (e.g., lentiviral vector) of the present invention. A vector of the present invention may further comprise additional elements such as a rev response element (RRE) for RNA transport, packaging sequences, and 5’ and 3’ long terminal repeats (LTRs). The term “long terminal repeat” or “LTR” refers to domains of base pairs located at the ends of retroviral DNAs which comprise U3, R and U5 regions. LTRs generally provide functions required for the expression of retroviral genes (e.g., promotion, initiation and polyadenylation of gene transcripts) and to viral replication. In one embodiment, a vector (e.g., lentiviral vector) of the present invention includes a 3’ U3 deleted LTR. Accordingly, a vector (e.g., lentiviral vector) of the present invention may comprise any combination of the elements described herein to enhance the efficiency of functional expression of transgenes. For example, a vector (e.g., lentiviral vector) of the present invention may comprise a WPRE sequence, cPPT sequence, RRE sequence, 5’LTR, 3’ U3 deleted LTR’ in addition to a nucleic acid encoding for a CAR.

Vectors of the present invention may be self-inactivating vectors. As used herein, the term “self-inactivating vector” refers to vectors in which the 3’ LTR enhancer promoter region (U3 region) has been modified (e.g., by deletion or substitution). A self-inactivating vector may prevent viral transcription beyond the first round of viral replication. Consequently, a selfinactivating vector may be capable of infecting and then integrating into a host genome (e.g., a mammalian genome) only once, and cannot be passed further. Accordingly, self-inactivating vectors may greatly reduce the risk of creating a replication-competent virus. In some embodiments, a nucleic acid of the present invention may be RNA, e.g., in vitro synthesized RNA. Methods for in vitro synthesis of RNA are known to those of skill in the art; any known method can be used to synthesize RNA comprising a sequence encoding an anti-scFv antibody or antigen-binding fragment, antibody-linked cytokine, immunostimulatory cytokine, and/or a CAR of the present disclosure. Methods for introducing RNA into a host cell are known in the art. See, e.g., Zhao et al. Cancer Res. (2010) 15: 9053. Introducing RNA comprising a nucleotide sequence encoding a polypeptide of the present disclosure into a host cell can be carried out in vitro, ex vivo or in vivo. For example, a host cell (e.g., an NK cell, a cytotoxic T lymphocyte, etc.) can be electroporated in vitro or ex vivo with RNA comprising a nucleotide sequence encoding an anti-scFv antibody or antigen-binding fragment, antibody-linked cytokine, immunostimulatory cytokine, and/or a CAR of the present disclosure.

In order to assess the expression of a polypeptide or portions thereof, the expression vector to be introduced into a cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In some embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, without limitation, antibiotic-resistance genes.

Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assessed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include, without limitation, genes encoding luciferase, betagalactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479: 79-82).

In some embodiments, a nucleic acid of the present disclosure is provided for the production of an anti-scFv antibody or antigen-binding fragment, antibody -linked cytokine, immunostimulatory cytokine, and/or a CAR as described herein, e.g., in a mammalian cell. In some embodiments, a nucleic acid of the present disclosure provides for amplification of the nucleic acid.

In vitro transcribed RNA

In some embodiments, the LNP and/or composition of the invention comprises in vitro transcribed (IVT) nucleoside-modified RNA encoding an immunostimulatory cytokine as described herein. In some embodiments, the LNP and/or composition of the invention comprises one or more IVT nucleoside-modified RNAs encoding one or more immunostimulatory cytokines, wherein each immunostimulatory cytokine is encoded by a distinct nucleoside- modified RNA.

In some embodiments, an IVT RNA can be introduced to a cell as a form of transient transfection. The RNA is produced by in vitro transcription using a plasmid DNA template generated synthetically. DNA of interest from any source can be directly converted by PCR into a template for in vitro mRNA synthesis using appropriate primers and RNA polymerase. The source of the DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence or any other appropriate source of DNA. In some embodiments, the desired template for in vitro transcription is an immunostimulatory cytokine, e.g., IL-12, IL-18, and/or IL-23.

In some embodiments, the DNA to be used for PCR contains an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In some embodiments, the DNA is a full length gene of interest of a portion of a gene. The gene can include some or all of the 5' and/or 3' untranslated regions (UTRs). The gene can include exons and introns. In some embodiments, the DNA to be used for PCR is a human gene. In another embodiment, the DNA to be used for PCR is a human gene including the 5' and 3' UTRs. In another embodiment, the DNA to be used for PCR is a gene from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi. In another embodiment, the DNA to be used for PCR is from a pathogenic or commensal organism, including bacteria, viruses, parasites, and fungi, including the 5' and 3' UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that contains portions of genes that are ligated together to form an open reading frame that encodes a fusion protein. The portions of DNA that are ligated together can be from a single organism or from more than one organism.

Genes that can be used as sources of DNA for PCR include genes that encode one or more immunostimulatory cytokines in an organism. In certain instances, the genes are useful for a short term treatment. In certain instances, the genes have limited safety concerns regarding dosage of the expressed gene.

In various embodiments, a plasmid is used to generate a template for in vitro transcription of mRNA, which is used for transfection.

Chemical structures with the ability to promote stability and/or translation efficiency may also be used. In certain embodiments, the RNA has 5' and 3' UTRs. In some embodiments, the 5' UTR is between zero and 3000 nucleotides in length. The length of 5' and 3' UTR sequences to be added to the coding region can be altered by different methods, including, but not limited to, designing primers for PCR that anneal to different regions of the UTRs. Using this approach, one of ordinary skill in the art can modify the 5' and 3' UTR lengths required to achieve optimal translation efficiency following transfection of the transcribed RNA.

The 5' and 3' UTRs can be the naturally occurring, endogenous 5' and 3' UTRs for the gene of interest. Alternatively, UTR sequences that are not endogenous to the gene of interest can be added by incorporating the UTR sequences into the forward and reverse primers or by any other modifications of the template. The use of UTR sequences that are not endogenous to the gene of interest can be useful for modifying the stability and/or translation efficiency of the RNA. For example, it is known that AU-rich elements in 3' UTR sequences can decrease the stability of mRNA. Therefore, 3' UTRs can be selected or designed to increase the stability of the transcribed RNA based on properties of UTRs that are well known in the art.

In some embodiments, the 5' UTR can contain the Kozak sequence of the endogenous gene. Alternatively, when a 5' UTR that is not endogenous to the gene of interest is being added by PCR as described above, a consensus Kozak sequence can be redesigned by adding the 5' UTR sequence. Kozak sequences can increase the efficiency of translation of some RNA transcripts, but does not appear to be required for all RNAs to enable efficient translation. The requirement for Kozak sequences for many mRNAs is known in the art. In other embodiments, the 5' UTR can be derived from an RNA virus whose RNA genome is stable in cells. In other embodiments, various nucleotide analogues can be used in the 3' or 5' UTR to impede exonuclease degradation of the mRNA. To enable synthesis of RNA from a DNA template without the need for gene cloning, a promoter of transcription should be attached to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for an RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter becomes incorporated into the PCR product upstream of the open reading frame that is to be transcribed. In some embodiments, the promoter is a T7 RNA polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleotide sequences for T7, T3 and SP6 promoters are known in the art.

In some embodiments, the mRNA has both a cap on the 5' end and a 3' poly(A) tail which determine ribosome binding, initiation of translation and stability mRNA in the cell. On a circular DNA template, for instance, plasmid DNA, RNA polymerase produces a long concatameric product, which is not suitable for expression in eukaryotic cells. The transcription of plasmid DNA linearized at the end of the 3' UTR results in normal sized mRNA, which is effective in eukaryotic transfection when it is polyadenylated after transcription.

On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenbom and Mierendorf, Nuc Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003).

The conventional method of integration of polyA/T stretches into a DNA template is molecular cloning. However, polyA/T sequence integrated into plasmid DNA can cause plasmid instability, which can be ameliorated through the use of recombination incompetent bacterial cells for plasmid propagation.

Poly(A) tails of RNAs can be further extended following in vitro transcription with the use of a poly(A) polymerase, such as E. coli polyA polymerase (E-PAP) or yeast polyA polymerase. In some embodiments, increasing the length of a poly(A) tail from 100 nucleotides to between 300 and 400 nucleotides results in about a two-fold increase in the translation efficiency of the RNA. Additionally, the attachment of different chemical groups to the 3' end can increase mRNA stability. Such attachment can contain modified/artificial nucleotides, aptamers and other compounds. For example, ATP analogs can be incorporated into the poly(A) tail using poly(A) polymerase. ATP analogs can further increase the stability of the RNA.

5' caps on also provide stability to mRNA molecules. In some embodiments, RNAs produced by the methods to include a 5' cap structure. Such cap structure can be generated using Vaccinia capping enzyme and T -O-methyl transferase enzymes (Cell Script, Madison, WI). Alternatively , 5' cap is provided using techniques known in the art and described herein (Cougot, et ah, Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et ah, RNA, 7: 1468-95 (2001); Elango, et ah, Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

RNA can be introduced into target cells using any of a number of different methods, for instance, commercially available methods which include, but are not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830 (BTX) (Harvard Instruments, Boston, Mass.) or the Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg Germany), cationic liposome mediated transfection using lipofection, polymer encapsulation, peptide mediated transfection, or biolistic particle delivery systems such as “gene guns” (see, for example, Nishikawa, et al. Hum Gene Ther., 12(8):861-70 (2001). In certain embodiments RNA of the invention is introduced to a cell with a method comprising the use of TransIT®-mRNA transfection Kit (Mirus, Madison WI), which, in some instances, provides high efficiency, low toxicity, transfection. In certain embodiments RNA of the invention is introduced into a cell (e.g., a cell of a subject) via encapsulation within an LNP as described herein.

Nucleoside-modified RNA

In some embodiments, the LNP and/or composition of the present invention comprises a nucleoside-modified nucleic acid encoding an immunostimulatory cytokine as described herein.

In some embodiments, the LNP and/or composition of the present invention comprises one or more nucleoside-modified nucleic acid(s) encoding a plurality of immunostimulatory cytokines. In some embodiments, each immunostimulatory cytokine is encoded by a distinct nucleoside-modified RNA.

For example, in some embodiments, the LNP and/or composition comprises a nucleoside-modified RNA. In some embodiments, the LNP and/or composition comprises a nucleoside-modified mRNA. Nucleoside-modified mRNA have particular advantages over nonmodified mRNA, including for example, increased stability, low or absent innate immunogenicity, and enhanced translation. Nucleoside-modified mRNA useful in the present invention is further described in U.S. Patent Nos. 8,278,036, 8,691,966, and 8,835,108, each of which is incorporated by reference herein in its entirety. In certain embodiments, nucleoside-modified mRNA does not activate any pathophysiologic pathways, translates very efficiently and almost immediately following delivery, and serve as templates for continuous protein production in vivo lasting for several days (Kariko et al., 2008, Mol Ther 16: 1833-1840; Kariko et al., 2012, Mol Ther 20:948-953). The amount of mRNA required to exert a physiological effect is small and that makes it applicable for human therapy. For example, as described herein, nucleoside-modified mRNAs, each encoding an immunostimulatory cytokine (such as IL- 12, IL- 18, and IL-23), enhances the efficacy of CAR T cell therapy.

In certain instances, expressing a protein by delivering the encoding mRNA has many benefits over methods that use protein, plasmid DNA or viral vectors. During mRNA transfection, the coding sequence of the desired protein is the only substance delivered to cells, thus avoiding all the side effects associated with plasmid backbones, viral genes, and viral proteins. More importantly, unlike DNA- and viral-based vectors, the mRNA does not carry the risk of being incorporated into the genome and protein production starts immediately after mRNA delivery. For example, high levels of circulating proteins have been measured within 15 to 30 minutes of in vivo injection of the encoding mRNA. In certain embodiments, using mRNA rather than the protein also has many advantages. Half-lives of proteins in the circulation are often short, thus protein treatment would need frequent dosing, while mRNA provides a template for continuous protein production for several days. Moreover, purification of proteins is problematic and they can contain aggregates and other impurities that cause adverse effects (Kromminga and Schellekens, 2005, Ann NY Acad Sci 1050:257-265).

In certain embodiments, the nucleoside-modified RNA comprises the naturally occurring modified-nucleoside pseudouridine. In certain embodiments, inclusion of pseudouridine makes the mRNA more stable, non-immunogenic, and highly translatable (Kariko et ah, 2008, Mol Ther 16: 1833-1840; Anderson et ah, 2010, Nucleic Acids Res 38:5884-5892; Anderson et al., 2011, Nucleic Acids Research 39:9329-9338; Kariko et al., 2011, Nucleic Acids Research 39:el42; Kariko et al., 2012, Mol Ther 20:948-953; Kariko et al., 2005, Immunity 23: 165-175).

It has been demonstrated that the presence of modified nucleosides, including pseudouridines, in RNA suppress their innate immunogenicity (Kariko et al., 2005, Immunity 23: 165-175). Further, protein-encoding, in vitro-transcribed RNA containing pseudouridine can be translated more efficiently than RNA containing no or other modified nucleosides (Kariko et al., 2008, Mol Ther 16:1833-1840). Subsequently, it is shown that the presence of pseudouridine improves the stability of RNA (Anderson et al., 2011, Nucleic Acids Research 39:9329-9338) and abates both activation of PKR and inhibition of translation (Anderson et al., 2010, Nucleic Acids Res 38:5884-5892).

In certain embodiments, the nucleoside-modified nucleic acid molecule is a purified nucleoside-modified nucleic acid molecule. For example, in certain embodiments, the composition is purified to remove double-stranded contaminants. In certain instances, a preparative high performance liquid chromatography (HPLC) purification procedure is used to obtain pseudouridine-containing RNA that has superior translational potential and no innate immunogenicity (Kariko et al., 2011, Nucleic Acids Research 39:el42). Administering HPLC- purified, pseudourine-containing RNA coding for erythropoietin into mice and macaques resulted in a significant increase of serum EPO levels (Kariko et al., 2012, Mol Ther 20:948- 953), thus confirming that pseudouridine-containing mRNA is suitable for in vivo protein therapy. In certain embodiments, the nucleoside-modified nucleic acid molecule is purified using non-HPLC methods. In certain instances, the nucleoside-modified nucleic acid molecule is purified using chromatography methods, including but not limited to HPLC and fast protein liquid chromatography (FPLC). An exemplary FPLC -based purification procedure is described in Weissman et al., 2013, Methods Mol Biol, 969: 43-54. Exemplary purification procedures are also described in U.S. Patent Application Publication No. US2016/0032316, which is hereby incorporated by reference in its entirety.

The present invention encompasses RNA, oligoribonucleotide, and polyribonucleotide molecules comprising pseudouridine or a modified nucleoside. In certain embodiments, the composition comprises an isolated nucleic acid encoding an immunostimulatory cytokine, wherein the nucleic acid comprises a pseudouridine or a modified nucleoside. In certain embodiments, the composition comprises a vector, comprising an isolated nucleic acid encoding an immunostimulatory cytokine, wherein the nucleic acid comprises a pseudouridine or a modified nucleoside.

In some embodiments, the nucleoside-modified RNA of the invention is IVT RNA, as described elsewhere herein. For example, in certain embodiments, the nucleoside-modified RNA is synthesized by T7 phage RNA polymerase. In another embodiment, the nucleoside-modified mRNA is synthesized by SP6 phage RNA polymerase. In another embodiment, the nucleoside- modified RNA is synthesized by T3 phage RNA polymerase.

In some embodiments, the modified nucleoside is m'acp³'!' (l-methyl-3-(3-amino-3- carboxypropyl) pseudouridine. In another embodiment, the modified nucleoside is m¹⁽P (1- methylpseudouridine). In another embodiment, the modified nucleoside is m (2'-O- methylpseudouridine). In another embodiment, the modified nucleoside is m⁵D (5- methyldihydrouridine). In another embodiment, the modified nucleoside is m³T (3- methylpseudouridine). In another embodiment, the modified nucleoside is a pseudouridine moiety that is not further modified (T). In another embodiment, the modified nucleoside is a monophosphate, diphosphate, or triphosphate of any of the above pseudouridines. In another embodiment, the modified nucleoside is any other pseudouridine-like nucleoside known in the art.

In another embodiment, the nucleoside that is modified in the nucleoside-modified RNA the present invention is a modified uridine (U). In another embodiment, the modified nucleoside is a modified cytidine (C). In another embodiment, the modified nucleoside is a modified adenosine (A). In another embodiment, the modified nucleoside is a modified guanosine (G).

In another embodiment, the modified nucleoside of the present invention is m⁵C (5- methylcytidine). In another embodiment, the modified nucleoside is m⁵U (5-methyluridine). In another embodiment, the modified nucleoside is m⁶A (N⁶-methyladenosine). In another embodiment, the modified nucleoside is s²U (2 -thiouridine). In another embodiment, the modified nucleoside is T (pseudouridine). In another embodiment, the modified nucleoside is Um (2'-O-methyluridine).

In other embodiments, the modified nucleoside is m'A (1 -methyladenosine); m²A (2- methyladenosine); Am (2'-0-methyladenosine); ms²m⁶A (2-methylthio-N⁶-methyladenosine); i⁶A (N⁶-isopentenyladenosine); ms²i⁶A (2-methylthio-N⁶-isopentenyladenosine); io⁶A (N⁶-(cis- hydroxyisopentenyl)adenosine); ms²io⁶A (2-methylthio-N⁶-(cis-hydroxyisopentenyl) adenosine); g⁶A (N⁶-glycinylcarbamoyladenosine); t⁶A (N⁶ -threonylcarbamoyladenosine); ms²t⁶A (2- methylthio-N⁶ -threonyl carbamoyladenosine); m⁶t⁶A (N⁶-methyl-N⁶- threonylcarbamoyladenosine); hn⁶A(N⁶ -hydroxynorvalylcarbamoyladenosine); ms²hn⁶A (2- methylthio-N⁶ -hydroxynorvalyl carbamoyladenosine); Ar(p) (2'-O-ribosyladenosine (phosphate)); I (inosine); m¹! (1 -methylinosine); irflm (l,2'-O-dimethylinosine); m³C (3- methylcytidine); Cm (2'-O-methylcytidine); s²C (2 -thiocytidine); ac⁴C (N⁴-acetylcytidine); CC (5-formylcytidine); m⁵Cm (5,2'-O-dimethylcytidine); ac⁴Cm (N⁴-acetyl-2'-O-methylcytidine); k²C (lysidine); m'G (1 -methylguanosine); m²G (N²-methylguanosine); m⁷G (7- methylguanosine); Gm (2'-O-methylguanosine); m²2G (N²,N²-dimethylguanosine); m²Gm (N²,2'-O-dimethylguanosine); m²2Gm (N²,N²,2'-O-trimethylguanosine); Gr(p) (2'-0- ribosylguanosine (phosphate)); yW (wybutosine); ChyW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylwyosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galactosyl-queuosine); manQ (mannosyl-queuosine); preQo (7-cyano-7-deazaguanosine); preQi (7-aminomethyl-7- deazaguanosine); G⁺ (archaeosine); D (dihydrouridine); m’Um (5,2'-O-dimethyluridine); s⁴U (4- thiouridine); m⁵s²U (5-methyl-2-thiouridine); s²Um (2-thio-2'-O-methyluridine); acp³U (3-(3- amino-3-carboxypropyl)uridine); ho⁵U (5-hydroxyuridine); mo⁵U (5-methoxyuridine); cmo⁵U (uridine 5-oxyacetic acid); mcmo’U (uridine 5-oxyacetic acid methyl ester); chnrU (5- (carboxyhydroxymethyl)uridine)); mchm⁵U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm⁵U (5-methoxycarbonylmethyluridine); mcm⁵Um (5-methoxycarbonylmethyl-2'-O- m ethyluridine); mcm⁵s²U (5-methoxycarbonylmethyl-2-thiouridine); nm⁵s²U (5-aminomethyl-2- thiouridine); mnm⁵U (5-methylaminomethyluridine); mnmVU (5-methylaminomethyl-2- thiouridine); rnnmNe²!! (5-methylaminomethyl-2-selenouridine); ncm⁵U (5- carbamoylmethyluridine); ncm⁵Um (5-carbamoylmethyl-2'-O-methyluridine); cmnm⁵U (5- carboxymethylaminomethyluridine); cmnnfUm (5-carboxymethylaminomethyl-2'-O- methyluridine); cmnnfis²!! (5-carboxymethylaminomethyl-2-thiouridine); m A (N⁶,N⁶- dimethyladenosine); Im (2'-O-methylinosine); m⁴C (N⁴-methylcytidine); m⁴Cm (N⁴,2'-O- dimethylcytidine); hm⁵C (5-hydroxymethylcytidine); m³U (3 -methyluridine); cm⁵U (5- carboxymethyluridine); m⁶Am (N⁶,2'-O-dimethyladenosine); m Am (N⁶,N⁶,O-2'- trimethyladenosine); m²⁷G (N²,7-dimethylguanosine); m²’^{2 7}G (N²,N²,7-trimethylguanosine); m³Um (3,2'-O-dimethyluridine); m⁵D (5-methyldihydrouridine); fiCm (5-formyl-2'-O- methylcytidine); m'Gm (l,2'-O-dimethylguanosine); m'Am (l,2'-O-dimethyladenosine); rm⁵U (5-taurinomethyluridine); rm⁵s²U (5-taurinomethyl-2-thiouridine)); imG-14 (4- demethylwyosine); imG2 (isowyosine); or ac⁶A (N⁶-acetyladenosine).

In another embodiment, a nucleoside-modified RNA of the present invention comprises a combination of 2 or more of the above modifications. In another embodiment, the nucleoside- modified RNA comprises a combination of 3 or more of the above modifications. In another embodiment, the nucleoside-modified RNA comprises a combination of more than 3 of the above modifications.

In various embodiments, between 0.1% and 100% of the residues in the nucleoside- modified of the present invention are modified (e.g., either by the presence of pseudouridine or another modified nucleoside base). In some embodiments, the fraction of modified residues is 0.1%. In another embodiment, the fraction of modified residues is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%. In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.7%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 0.9%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 7%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 9%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 55%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 65%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 75%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 85%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 91%. In another embodiment, the fraction is 92%. In another embodiment, the fraction is 93%. In another embodiment, the fraction is 94%. In another embodiment, the fraction is 95%. In another embodiment, the fraction is 96%. In another embodiment, the fraction is 97%. In another embodiment, the fraction is 98%. In another embodiment, the fraction is 99%. In another embodiment, the fraction is 100%.

In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.

In another embodiment, 0.1% of the residues of a given nucleoside (i.e., uridine, cytidine, guanosine, or adenosine) are modified. In another embodiment, the fraction of modified residues is 0.2%. In another embodiment, the fraction is 0.3%. In another embodiment, the fraction is 0.4%. In another embodiment, the fraction is 0.5%.

In another embodiment, the fraction is 0.6%. In another embodiment, the fraction is 0.7%. In another embodiment, the fraction is 0.8%. In another embodiment, the fraction is 0.9%. In another embodiment, the fraction is 1%. In another embodiment, the fraction is 1.5%. In another embodiment, the fraction is 2%. In another embodiment, the fraction is 2.5%. In another embodiment, the fraction is 3%. In another embodiment, the fraction is 4%. In another embodiment, the fraction is 5%. In another embodiment, the fraction is 6%. In another embodiment, the fraction is 7%. In another embodiment, the fraction is 8%. In another embodiment, the fraction is 9%. In another embodiment, the fraction is 10%. In another embodiment, the fraction is 12%. In another embodiment, the fraction is 14%. In another embodiment, the fraction is 16%. In another embodiment, the fraction is 18%. In another embodiment, the fraction is 20%. In another embodiment, the fraction is 25%. In another embodiment, the fraction is 30%. In another embodiment, the fraction is 35%. In another embodiment, the fraction is 40%. In another embodiment, the fraction is 45%. In another embodiment, the fraction is 50%. In another embodiment, the fraction is 55%. In another embodiment, the fraction is 60%. In another embodiment, the fraction is 65%. In another embodiment, the fraction is 70%. In another embodiment, the fraction is 75%. In another embodiment, the fraction is 80%. In another embodiment, the fraction is 85%. In another embodiment, the fraction is 90%. In another embodiment, the fraction is 91%. In another embodiment, the fraction is 92%. Tn another embodiment, the fraction is 93%. In another embodiment, the fraction is 94%. In another embodiment, the fraction is 95%. In another embodiment, the fraction is 96%. In another embodiment, the fraction is 97%. In another embodiment, the fraction is 98%. In another embodiment, the fraction is 99%. In another embodiment, the fraction is 100%. In another embodiment, the fraction of the given nucleotide that is modified is less than 8%. In another embodiment, the fraction is less than 10%. In another embodiment, the fraction is less than 5%. In another embodiment, the fraction is less than 3%. In another embodiment, the fraction is less than 1%. In another embodiment, the fraction is less than 2%. In another embodiment, the fraction is less than 4%. In another embodiment, the fraction is less than 6%. In another embodiment, the fraction is less than 12%. In another embodiment, the fraction is less than 15%. In another embodiment, the fraction is less than 20%. In another embodiment, the fraction is less than 30%. In another embodiment, the fraction is less than 40%. In another embodiment, the fraction is less than 50%. In another embodiment, the fraction is less than 60%. In another embodiment, the fraction is less than 70%.

In another embodiment, a nucleoside-modified RNA of the present invention is translated in the cell more efficiently than an unmodified RNA molecule with the same sequence. In another embodiment, the nucleoside-modified RNA exhibits enhanced ability to be translated by a target cell. In another embodiment, translation is enhanced by a factor of 2-fold relative to its unmodified counterpart. In another embodiment, translation is enhanced by a 3-fold factor. In another embodiment, translation is enhanced by a 4-fold factor. In another embodiment, translation is enhanced by a 5-fold factor. In another embodiment, translation is enhanced by a 6- fold factor. In another embodiment, translation is enhanced by a 7-fold factor. In another embodiment, translation is enhanced by a 8-fold factor. In another embodiment, translation is enhanced by a 9-fold factor. In another embodiment, translation is enhanced by a 10-fold factor. In another embodiment, translation is enhanced by a 15-fold factor. In another embodiment, translation is enhanced by a 20-fold factor. In another embodiment, translation is enhanced by a 50-fold factor. In another embodiment, translation is enhanced by a 100-fold factor. In another embodiment, translation is enhanced by a 200-fold factor. In another embodiment, translation is enhanced by a 500-fold factor. In another embodiment, translation is enhanced by a 1000-fold factor. In another embodiment, translation is enhanced by a 2000-fold factor. In another embodiment, the factor is 10-1000-fold. In another embodiment, the factor is 10- 100-fold. In another embodiment, the factor is 10-200-fold. In another embodiment, the factor is 10-300-fold. In another embodiment, the factor is 10-500-fold. In another embodiment, the factor is 20-1000- fold. In another embodiment, the factor is 30-1000-fold. In another embodiment, the factor is 50- 1000-fold. In another embodiment, the factor is 100-1000-fold. In another embodiment, the factor is 200- 1000-fold. In another embodiment, translation is enhanced by any other significant amount or range of amounts.

In another embodiment, the nucleoside-modified RNA of the present invention exhibits significantly less innate immunogenicity than an unmodified in vitro-synthesized RNA molecule of the same sequence. In another embodiment, the modified RNA molecule exhibits an innate immune response that is 2-fold less than its unmodified counterpart. In another embodiment, innate immunogenicity is reduced by a 3-fold factor. In another embodiment, innate immunogenicity is reduced by a 4-fold factor. In another embodiment, innate immunogenicity is reduced by a 5-fold factor. In another embodiment, innate immunogenicity is reduced by a 6-fold factor. In another embodiment, innate immunogenicity is reduced by a 7-fold factor. In another embodiment, innate immunogenicity is reduced by a 8-fold factor. In another embodiment, innate immunogenicity is reduced by a 9-fold factor. In another embodiment, innate immunogenicity is reduced by a 10-fold factor. In another embodiment, innate immunogenicity is reduced by a 15-fold factor. In another embodiment, innate immunogenicity is reduced by a 20-fold factor. In another embodiment, innate immunogenicity is reduced by a 50-fold factor. In another embodiment, innate immunogenicity is reduced by a 100-fold factor. In another embodiment, innate immunogenicity is reduced by a 200-fold factor. In another embodiment, innate immunogenicity is reduced by a 500-fold factor. In another embodiment, innate immunogenicity is reduced by a 1000-fold factor. In another embodiment, innate immunogenicity is reduced by a 2000-fold factor. In another embodiment, innate immunogenicity is reduced by another fold difference.

In another embodiment, “exhibits significantly less innate immunogenicity” refers to a detectable decrease in innate immunogenicity. In another embodiment, the term refers to a fold decrease in innate immunogenicity (e.g., 1 of the fold decreases enumerated above). In another embodiment, the term refers to a decrease such that an effective amount of the nucleoside- modified RNA can be administered without triggering a detectable innate immune response. In another embodiment, the term refers to a decrease such that the nucleoside-modified RNA can be repeatedly administered without eliciting an innate immune response sufficient to detectably reduce production of the protein encoded by the modified RNA. In another embodiment, the decrease is such that the nucleoside-modified RNA can be repeatedly administered without eliciting an innate immune response sufficient to eliminate detectable production of the protein encoded by the modified RNA.

Pharmaceutical Compositions

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

Although the description of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts.

Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as nonhuman primates, cattle, pigs, horses, sheep, cats, and dogs.

Pharmaceutical compositions that are useful in the methods of the invention may be prepared, packaged, or sold in formulations suitable for intracameral, ophthalmic, oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, intravenous, intracerebroventricular, intradermal, intramuscular, or another route of administration. Other contemplated formulations include projected nanoparticles, liposomal preparations, resealed erythrocytes containing the active ingredient, and immunogenic-based formulations.

A pharmaceutical composition of the invention may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a “unit dose” is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient, which would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

In addition to the active ingredient, a pharmaceutical composition of the invention may further comprise one or more additional pharmaceutically active agents.

Controlled- or sustained-release formulations of a pharmaceutical composition of the invention may be made using conventional technology.

As used herein, “parenteral administration” of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, intracam eral injection, as well as intraocular, intravitreal, subcutaneous, intraperitoneal, intramuscular, intradermal, intrasternal injection, intratumoral, intravenous, intracerebroventricular and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e. powder or granular) form for reconstitution with a suitable vehicle (e.g. sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer systems.

Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parent erally-acceptable diluent or solvent, such as water or 1,3-butane diol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations that are useful include those that comprise the active ingredient in microcrystalline form, in a liposomal preparation, or as a component of a biodegradable polymer system.

As used herein, “additional ingredients” include, but are not limited to, one or more of the following: excipients; surface active agents; dispersing agents; inert diluents; granulating and disintegrating agents; binding agents; lubricating agents; sweetening agents; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents, demulcents; buffers; salts; thickening agents; fillers; emulsifying agents; antioxidants; antibiotics; antifungal agents; stabilizing agents; and pharmaceutically acceptable polymeric or hydrophobic materials. Other “additional ingredients” which may be included in the pharmaceutical compositions of the invention are known in the art and described, for example in Remington's Pharmaceutical Sciences (1985, Genaro, ed., Mack Publishing Co., Easton, PA), which is incorporated herein by reference.

Methods of Treating Cancer

The present invention provides a method of treating cancer in a subject in need thereof. In some aspects, the method comprises administering to the subject a therapeutically effective amount of the antibody-linked cytokine of the invention as described herein, wherein the administering is performed before, simultaneously with, or after infusion of the subject with a population of modified immune cells which express a CAR. In some aspects, the method comprises administering to the subject a therapeutically effective amount of the LNP of the invention as described herein, wherein the administering is performed before, simultaneously with, or after infusion of the subject with a population of modified immune cells which express a CAR

In some embodiments of the method, each cell of the population of modified immune cells is a T cell, an autologous cell, a human cell, or any combination thereof.

In some embodiments, the administering comprises subcutaneous injection, intraperitoneal injection, intradermal injection, intravenous injection, intramuscular injection, intrasternal injection, or infusion techniques. In some embodiments, the administering comprises administering a first dose. In some embodiments, the administering further comprises administering one or more subsequent doses.

In some embodiments, the cancer is selected from breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and thyroid cancer

Suitable subjects include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs. In some embodiments, the subject is selected from a human, a dog, and a cat. In some embodiments, the subject is a human.

In some embodiments, the administering comprises administering a first dose. In certain embodiments, the administering further comprises administering one or more subsequent doses.

It will be appreciated that the composition of the invention may be administered to a subject either alone, or in conjunction with another agent.

The therapeutic and prophylactic methods of the invention thus encompass the use of pharmaceutical compositions encoding at least one immunostimulatory cytokine described herein to practice the methods of the invention. The pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of from 1 ng/kg/day and 100 mg/kg/day. In some embodiments, the invention envisions administration of a dose, which results in a concentration of the compound of the present invention from 10 nM and 10 mM in a mammal. Typically, dosages which may be administered in a method of the invention to a mammal, such as a human, range in amount from 0.01 pg to about 50 mg per kilogram of body weight of the mammal, while the precise dosage administered will vary depending upon any number of factors, including but not limited to, the type of mammal and type of disease state being treated, the age of the mammal and the route of administration. In certain embodiments, the dosage of the compound will vary from about 0. 1 pg to about 10 mg per kilogram of body weight of the mammal. In certain embodiments, the dosage will vary from about 1 pg to about 1 mg per kilogram of body weight of the mammal.

The composition may be administered to a mammal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. The frequency of the dose will be readily apparent to the skilled artisan and will depend upon any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the mammal, etc.

Methods for administration of immune cells for adoptive cell therapy are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described, e.g., in US Patent Application Publication No. 2003/0170238 to Gruenberg et al; US Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8(10):577-85). See, e.g., Themeli et al. (2013) Nat Biotechnol. 31(10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438(1): 84-9; Davila et al. (2013) PLoS ONE 8(4): e61338. In some embodiments, the cell therapy, e.g., adoptive T cell therapy is carried out by autologous transfer, in which the cells are isolated and/or otherwise prepared from the subject who is to receive the cell therapy, or from a sample derived from such a subject. Thus, in some aspects, the cells are derived from a subject, e.g., patient, in need of a treatment and the cells, following isolation and processing are administered to the same subject.

In some embodiments, the cell therapy, e.g., adoptive T cell therapy, is carried out by allogeneic transfer, in which the cells are isolated and/or otherwise prepared from a subject other than a subject who is to receive or who ultimately receives the cell therapy, e.g., a first subject. In such embodiments, the cells then are administered to a different subject, e.g., a second subject, of the same species. In some embodiments, the first and second subjects are genetically identical. In some embodiments, the first and second subjects are genetically similar. In some embodiments, the second subject expresses the same HLA class or supertype as the first subject.

In some embodiments, the subject has been treated with a therapeutic agent targeting the disease or condition, e.g. the tumor, prior to administration of the cells or composition containing the cells. In some aspects, the subject is refractory or non-responsive to the other therapeutic agent. In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

In some embodiments, the subject is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden. In some aspects, the subject is initially responsive to the therapeutic agent, but exhibits a relapse of the disease or condition over time. In some embodiments, the subject has not relapsed. In some such embodiments, the subject is determined to be at risk for relapse, such as at a high risk of relapse, and thus the cells are administered prophylactically, e.g., to reduce the likelihood of or prevent relapse. In some aspects, the subject has not received prior treatment with another therapeutic agent.

In some embodiments, the subject has persistent or relapsed disease, e.g., following treatment with another therapeutic intervention, including chemotherapy, radiation, and/or hematopoietic stem cell transplantation (HSCT), e.g., allogenic HSCT. In some embodiments, the administration effectively treats the subject despite the subject having become resistant to another therapy.

The modified immune cells of the present invention can be administered to an animal, preferably a mammal, even more preferably a human, to treat a cancer. In addition, the cells of the present invention can be used for the treatment of any condition related to a cancer, especially a cell-mediated immune response against a tumor cell(s), where it is desirable to treat or alleviate the disease. The types of cancers to be treated with the modified cells or pharmaceutical compositions of the invention include, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Other exemplary cancers include but are not limited breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, thyroid cancer, and the like. The cancers may be non-solid tumors (such as hematological tumors) or solid tumors. Adult tumors/cancers and pediatric tumors/cancers are also included. In one embodiment, the cancer is a solid tumor or a hematological tumor. In one embodiment, the cancer is a carcinoma. In one embodiment, the cancer is a sarcoma. In one embodiment, the cancer is a leukemia. In one embodiment the cancer is a solid tumor.

Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases).

Carcinomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma.

Sarcomas that can be amenable to therapy by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas.

In certain exemplary embodiments, the modified immune cells of the invention are used to treat a myeloma, or a condition related to myeloma. Examples of myeloma or conditions related thereto include, without limitation, light chain myeloma, non-secretory myeloma, monoclonal gamopathy of undertermined significance (MGUS), plasmacytoma (e.g., solitary, multiple solitary, extramedullary plasmacytoma), amyloidosis, and multiple myeloma. In one embodiment, a method of the present disclosure is used to treat multiple myeloma. In one embodiment, a method of the present disclosure is used to treat refractory myeloma. In one embodiment, a method of the present disclosure is used to treat relapsed myeloma.

In certain exemplary embodiments, the modified immune cells of the invention are used to treat a melanoma, or a condition related to melanoma. Examples of melanoma or conditions related thereto include, without limitation, superficial spreading melanoma, nodular melanoma, lentigo maligna melanoma, acral lentiginous melanoma, amelanotic melanoma, or melanoma of the skin (e.g., cutaneous, eye, vulva, vagina, rectum melanoma). In one embodiment, a method of the present disclosure is used to treat cutaneous melanoma. In one embodiment, a method of the present disclosure is used to treat refractory melanoma. In one embodiment, a method of the present disclosure is used to treat relapsed melanoma.

In yet other exemplary embodiments, the modified immune cells of the invention are used to treat a sarcoma, or a condition related to sarcoma. Examples of sarcoma or conditions related thereto include, without limitation, angiosarcoma, chondrosarcoma, Ewing’s sarcoma, fibrosarcoma, gastrointestinal stromal tumor, leiomyosarcoma, liposarcoma, malignant peripheral nerve sheath tumor, osteosarcoma, pleomorphic sarcoma, rhabdomyosarcoma, and synovial sarcoma. In one embodiment, a method of the present disclosure is used to treat synovial sarcoma. Tn one embodiment, a method of the present disclosure is used to treat liposarcoma such as myxoid/round cell liposarcoma, differentiated/dedifferentiated liposarcoma, and pleomorphic liposarcoma. In one embodiment, a method of the present disclosure is used to treat myxoid/round cell liposarcoma. In one embodiment, a method of the present disclosure is used to treat a refractory sarcoma. In one embodiment, a method of the present disclosure is used to treat a relapsed sarcoma.

The cells of the invention to be administered may be autologous, with respect to the subject undergoing therapy.

The administration of the cells of the invention may be carried out in any convenient manner known to those of skill in the art. The cells of the present invention may be administered to a subject by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient transarterially, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In other instances, the cells of the invention are injected directly into a site of inflammation in the subject, a local disease site in the subject, alymph node, an organ, a tumor, and the like.

In some embodiments, the cells are administered at a desired dosage, which in some aspects includes a desired dose or number of cells or cell type(s) and/or a desired ratio of cell types. Thus, the dosage of cells in some embodiments is based on a total number of cells (or number per kg body weight) and a desired ratio of the individual populations or sub-types, such as the CD4+ to CD8+ ratio. In some embodiments, the dosage of cells is based on a desired total number (or number per kg of body weight) of cells in the individual populations or of individual cell types. In some embodiments, the dosage is based on a combination of such features, such as a desired number of total cells, desired ratio, and desired total number of cells in the individual populations.

In some embodiments, the populations or sub-types of cells, such as CD8⁺ and CD4⁺ T cells, are administered at or within a tolerated difference of a desired dose of total cells, such as a desired dose of T cells. In some aspects, the desired dose is a desired number of cells or a desired number of cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg. In some aspects, the desired dose is at or above a minimum number of cells or minimum number of cells per unit of body weight. In some aspects, among the total cells, administered at the desired dose, the individual populations or sub-types are present at or near a desired output ratio (such as CD4⁺ to CD8⁺ ratio), e.g., within a certain tolerated difference or error of such a ratio.

In some embodiments, the cells are administered at or within a tolerated difference of a desired dose of one or more of the individual populations or sub-types of cells, such as a desired dose of CD4+ cells and/or a desired dose of CD8+ cells. In some aspects, the desired dose is a desired number of cells of the sub-type or population, or a desired number of such cells per unit of body weight of the subject to whom the cells are administered, e.g., cells/kg. In some aspects, the desired dose is at or above a minimum number of cells of the population or subtype, or minimum number of cells of the population or sub-type per unit of body weight. Thus, in some embodiments, the dosage is based on a desired fixed dose of total cells and a desired ratio, and/or based on a desired fixed dose of one or more, e.g., each, of the individual sub-types or subpopulations. Thus, in some embodiments, the dosage is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4¹ to CD8¹ cells, and/or is based on a desired fixed or minimum dose of CD4⁺ and/or CD8⁺ cells.

In certain embodiments, the cells, or individual populations of sub-types of cells, are administered to the subject at a range of about one million to about 100 billion cells, such as, e.g., 1 million to about 50 billion cells (e.g., about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the foregoing values), such as about 10 million to about 100 billion cells (e.g., about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million cells, about 10 billion cells, about 25 billion cells, about 50 billion cells, about 75 billion cells, about 90 billion cells, or a range defined by any two of the foregoing values), and in some cases about 100 million cells to about 50 billion cells (e.g., about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value in between these ranges.

In some embodiments, the dose of total cells and/or dose of individual sub-populations of cells is within a range of between at or about IxlO⁵ cells/kg to about IxlO¹¹ cells/kg 10⁴ and at or about 10¹¹ cells/kilograms (kg) body weight, such as between 10^? and 10⁶ cells / kg body weight, for example, at or about 1 x 10⁵ cells/kg, 1.5 x IO³ cells/kg, 2 x 1 C cells/kg, or 1 x 10⁶ cells/kg body weight. For example, in some embodiments, the cells are administered at, or within a certain range of error of, between at or about 10⁴ and at or about 10⁹ T cells/kilograms (kg) body weight, such as between 10⁵ and 10⁶ T cells / kg body weight, for example, at or about 1 x 1 C T cells/kg, 1.5 x 10⁵ T cells/kg, 2 x 10⁵ T cells/kg, or 1 x 10⁶ T cells/kg body weight. In other exemplary embodiments, a suitable dosage range of modified cells for use in a method of the present disclosure includes, without limitation, from about IxlO⁵ cells/kg to about IxlO⁶ cells/kg, from about IxlO⁶ cells/kg to about IxlO⁷ cells/kg, from about IxlO⁷ cells/kg about IxlO⁸ cells/kg, from about IxlO⁸ cells/kg about IxlO⁹ cells/kg, from about IxlO⁹ cells/kg about IxlO¹⁰ cells/kg, from about IxlO¹⁰ cells/kg about IxlO¹¹ cells/kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO⁸ cells/kg. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about IxlO⁷ cells/kg. In other embodiments, a suitable dosage is from about IxlO⁷ total cells to about 5xl0⁷ total cells. In some embodiments, a suitable dosage is from about IxlO⁸ total cells to about 5xl0⁸ total cells. In some embodiments, a suitable dosage is from about 1.4xl0⁷ total cells to about l.lxlO⁹ total cells. In an exemplary embodiment, a suitable dosage for use in a method of the present disclosure is about 7xl0⁹ total cells.

In some embodiments, the cells are administered at or within a certain range of error of between at or about 10⁴ and at or about 10⁹ CD4⁺ and/or CD8⁺ cells/kilograms (kg) body weight, such as between 10⁵ and 10⁶ CD4⁺ and/or CD8⁺cells / kg body weight, for example, at or about 1 x 10⁵ CD4⁺ and/or CD8⁺ cells/kg, 1.5 x 10⁵ CD4⁺ and/or CD8⁺ cells/kg, 2 x 10⁵ CD4⁺ and/or CD8⁺ cells/kg, or 1 x 10⁶ CD4⁺ and/or CD8 cells/kg body weight. In some embodiments, the cells are administered at or within a certain range of error of, greater than, and/or at least about 1 x 10⁶, about 2.5 x 10⁶, about 5 x 10⁶, about 7.5 x 10⁶, or about 9 x 10⁶ CD4⁺ cells, and/or at least about l x 10⁶, about 2.5 x 10⁶, about 5 x 10⁶, about 7.5 x 10⁶, or about 9 x 10⁶ CD8+ cells, and/or at least about 1 x 10⁶, about 2.5 x 10⁶, about 5 x 10⁶, about 7.5 x 10⁶, or about 9 x 10⁶ T cells. In some embodiments, the cells are administered at or within a certain range of error of between about 10⁸ and 10¹² or between about IO¹⁰ and 10¹¹ T cells, between about 10⁸ and 10¹² or between about IO¹⁰ and 10¹¹ CD4⁺ cells, and/or between about 10⁸ and 10¹² or between about 10¹⁰ and 10¹¹ CD8⁺ cells. In some embodiments, the cells are administered at or within a tolerated range of a desired output ratio of multiple cell populations or sub-types, such as CD4+ and CD8+ cells or sub-types. In some aspects, the desired ratio can be a specific ratio or can be a range of ratios, for example, in some embodiments, the desired ratio (e.g., ratio of CD4⁺ to CD8⁺ cells) is between at or about 5: 1 and at or about 5: 1 (or greater than about 1:5 and less than about 5: 1), or between at or about 1 :3 and at or about 3 : 1 (or greater than about 1:3 and less than about 3: 1), such as between at or about 2: 1 and at or about 1 :5 (or greater than about 1 :5 and less than about 2: 1, such as at or about 5: 1, 4.5: 1, 4: 1, 3.5: 1, 3: 1, 2.5: 1, 2: 1, 1.9: 1, 1.8: 1, 1.7: 1, 1.6: 1, 1.5: 1, 1.4: 1, 1.3: 1, 1.2: 1, 1.1 : 1, 1 : 1, 1 : 1.1, 1 : 1.2, 1 : 1.3, 1 : 1.4, 1 : 1.5, 1 : 1.6, 1 : 1.7, 1 : 1.8, 1 : 1.9: 1 :2, 1 :2.5, 1 :3, 1 :3.5, 1 :4, 1 :4.5, or 1 :5. In some aspects, the tolerated difference is within about 1%, about 2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value in between these ranges.

In some embodiments, a dose of modified cells is administered to a subject in need thereof, in a single dose or multiple doses. In some embodiments, a dose of modified cells is administered in multiple doses, e g., once a week or every 7 days, once every 2 weeks or every 14 days, once every 3 weeks or every 21 days, once every 4 weeks or every 28 days. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof. In an exemplary embodiment, a single dose of modified cells is administered to a subject in need thereof by rapid intravenous infusion.

For the prevention or treatment of disease, the appropriate dosage may depend on the type of disease to be treated, the type of cells or recombinant receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes, previous therapy, the subject's clinical history and response to the cells, and the discretion of the attending physician. The compositions and cells are in some embodiments suitably administered to the subject at one time or over a series of treatments.

Following administration of the cells, the biological activity of the engineered cell populations in some embodiments is measured, e.g., by any of a number of known methods. Parameters to assess include specific binding of an engineered or natural T cell or other immune cell to antigen, in vivo, e.g., by imaging, or ex vivo, e.g., by ELISA or flow cytometry. In certain embodiments, the ability of the engineered cells to destroy target cells can be measured using any suitable method known in the art, such as cytotoxicity assays described in, for example, Kochenderfer et al., J. Immunotherapy, 32(7): 689-702 (2009), and Herman et al. J. Immunological Methods, 285(1): 25-40 (2004). In certain embodiments, the biological activity of the cells is measured by assaying expression and/or secretion of one or more cytokines, such as CD 107a, IFNy, IL-2, and TNF. In some aspects the biological activity is measured by assessing clinical outcome, such as reduction in tumor burden or load.

In certain embodiments, the subject is provided a secondary treatment. Secondary treatments include but are not limited to chemotherapy, radiation, surgery, and medications.

In some embodiments, the subject can be administered a conditioning therapy prior to adoptive cell therapy (e.g., CAR T cell therapy). In some embodiments, the conditioning therapy comprises administering an effective amount of cyclophosphamide to the subject. In some embodiments, the conditioning therapy comprises administering an effective amount of fludarabine to the subject. In preferred embodiments, the conditioning therapy comprises administering an effective amount of a combination of cyclophosphamide and fludarabine to the subject. Administration of a conditioning therapy prior to adoptive cell therapy (e.g., CAR T cell therapy) may increase the efficacy of the adoptive cell therapy. Methods of conditioning patients for T cell therapy are described in U.S. Patent No. 9,855,298, which is incorporated herein by reference in its entirety.

In some embodiments, a specific dosage regimen of the present disclosure includes a lymphodepletion step prior to the administration of the modified T cells. In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide and/or fludarabine.

In some embodiments, the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg/m²/day and about 2000 mg/m²/day (e.g., 200 mg/m²/day, 300 mg/m²/day, or 500 mg/m²/day). In an exemplary embodiment, the dose of cyclophosphamide is about 300 mg/m²/day. In some embodiments, the lymphodepletion step includes administration of fludarabine at a dose of between about 20 mg/m²/day and about 900 mg/m²/day (e.g., 20 mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day). In an exemplary embodiment, the dose of fludarabine is about 30 mg/m²/day. In some embodiment, the lymphodepletion step includes administration of cyclophosphamide at a dose of between about 200 mg/m²/day and about 2000 mg/m²/day (e.g., 200 mg/m²/day, 300 mg/m²/day, or 500 mg/m²/day), and fludarabine at a dose of between about 20 mg/m²/day and about 900 mg/m²/day (e.g., 20 mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day). In an exemplary embodiment, the lymphodepletion step includes administration of cyclophosphamide at a dose of about 300 mg/m²/day, and fludarabine at a dose of about 30 mg/m²/day.

In an exemplary embodiment, the dosing of cyclophosphamide is 300 mg/m²/day over three days, and the dosing of fludarabine is 30 mg/m²/day over three days.

Dosing of lymphodepletion chemotherapy may be scheduled on Days -6 to -4 (with a -1 day window, i.e., dosing on Days -7 to -5) relative to T cell (e.g., CAR-T, TCR-T, a modified T cell, etc.) infusion on Day 0.

In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including 300 mg/m² of cyclophosphamide by intravenous infusion 3 days prior to administration of the modified T cells. In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including 300 mg/m² of cyclophosphamide by intravenous infusion for 3 days prior to administration of the modified T cells.

In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of between about 20 mg/m²/day and about 900 mg/m²/day (e.g., 20 mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day). In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including fludarabine at a dose of 30 mg/m² for 3 days.

In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at a dose of between about 200 mg/m²/day and about 2000 mg/m²/day (e.g., 200 mg/m²/day, 300 mg/m²/day, or 500 mg/m²/day), and fludarabine at a dose of between about 20 mg/m²/day and about 900 mg/m²/day (e.g., 20 mg/m²/day, 25 mg/m²/day, 30 mg/m²/day, or 60 mg/m²/day). In an exemplary embodiment, for a subject having cancer, the subject receives lymphodepleting chemotherapy including cyclophosphamide at a dose of about 300 mg/m²/day, and fludarabine at a dose of 30 mg/m² for 3 days. Cells of the invention can be administered in dosages and routes and at times to be determined in appropriate pre-clinical and clinical experimentation and trials. Cell compositions may be administered multiple times at dosages within these ranges. Administration of the cells of the invention may be combined with other methods useful to treat the desired disease or condition as determined by those of skill in the art.

It is known in the art that one of the adverse effects following infusion of CAR T cells is the onset of immune activation, known as cytokine release syndrome (CRS). CRS is immune activation resulting in elevated inflammatory cytokines. CRS is a known on-target toxicity, development of which likely correlates with efficacy. Clinical and laboratory measures range from mild CRS (constitutional symptoms and/or grade-2 organ toxicity) to severe CRS (sCRS; grade >3 organ toxicity, aggressive clinical intervention, and/or potentially life threatening). Clinical features include: high fever, malaise, fatigue, myalgia, nausea, anorexia, tachycardia/hypotension, capillary leak, cardiac dysfunction, renal impairment, hepatic failure, and disseminated intravascular coagulation. Dramatic elevations of cytokines including interferon-gamma, granulocyte macrophage colony-stimulating factor, IL- 10, and IL-6 have been shown following CAR T-cell infusion. One CRS signature is elevation of cytokines including IL-6 (severe elevation), IFN-gamma, TNF-alpha (moderate), and IL-2 (mild). Elevations in clinically available markers of inflammation including ferritin and C-reactive protein (CRP) have also been observed to correlate with the CRS syndrome. The presence of CRS generally correlates with expansion and progressive immune activation of adoptively transferred cells. It has been demonstrated that the degree of CRS severity is dictated by disease burden at the time of infusion as patients with high tumor burden experience a more sCRS.

Accordingly, the invention provides for, following the diagnosis of CRS, appropriate CRS management strategies to mitigate the physiological symptoms of uncontrolled inflammation without dampening the antitumor efficacy of the engineered cells (e.g., CAR T cells). CRS management strategies are known in the art. For example, systemic corticosteroids may be administered to rapidly reverse symptoms of sCRS (e.g., grade 3 CRS) without compromising initial antitumor response.

In some embodiments, an anti-IL-6R antibody may be administered. An example of an anti-IL-6R antibody is the Food and Drug Administration-approved monoclonal antibody tocilizumab, also known as atlizumab (marketed as Actemra, or RoActemra). Tocilizumab is a humanized monoclonal antibody against the interleukin-6 receptor (IL-6R). Administration of tocilizumab has demonstrated near-immediate reversal of CRS.

CRS is generally managed based on the severity of the observed syndrome and interventions are tailored as such. CRS management decisions may be based upon clinical signs and symptoms and response to interventions, not solely on laboratory values alone.

Mild to moderate cases generally are treated with symptom management with fluid therapy, non-steroidal anti-inflammatory drug (NSAID) and antihistamines as needed for adequate symptom relief. More severe cases include patients with any degree of hemodynamic instability; with any hemodynamic instability, the administration of tocilizumab is recommended. The first-line management of CRS may be tocilizumab, in some embodiments, at the labeled dose of 8 mg/kg IV over 60 minutes (not to exceed 800 mg/dose); tocilizumab can be repeated Q8 hours. If suboptimal response to the first dose of tocilizumab, additional doses of tocilizumab may be considered. Tocilizumab can be administered alone or in combination with corticosteroid therapy. Patients with continued or progressive CRS symptoms, inadequate clinical improvement in 12-18 hours or poor response to tocilizumab, may be treated with high- dose corticosteroid therapy, generally hydrocortisone 100 mg IV or methylprednisolone 1-2 mg/kg. In patients with more severe hemodynamic instability or more severe respiratory symptoms, patients may be administered high-dose corticosteroid therapy early in the course of the CRS. CRS management guidance may be based on published standards (Lee et al. (2019) Biol Blood Marrow Transplant, doi.org/10.1016/j.bbmt.2018.12.758; Neelapu et al. (2018) Nat Rev Clin Oncology, 15:47; Teachey et al. (2016) Cancer Discov, 6(6):664-679).

Features consistent with Macrophage Activation Syndrome (MAS) or Hemophagocytic lymphohistiocytosis (HLH) have been observed in patients treated with CAR-T therapy (Henter, 2007), coincident with clinical manifestations of the CRS. MAS appears to be a reaction to immune activation that occurs from the CRS, and should therefore be considered a manifestation of CRS. MAS is similar to HLH (also a reaction to immune stimulation). The clinical syndrome of MAS is characterized by high grade non-remitting fever, cytopenias affecting at least two of three lineages, and hepatosplenomegaly. It is associated with high serum ferritin, soluble interleukin-2 receptor, and triglycerides, and a decrease of circulating natural killer (NK) activity. Sources of Immune Cells

In certain embodiments, a source of immune cells (e.g. T cells) is obtained from a subject for ex vivo manipulation. Sources of target cells for ex vivo manipulation may also include, e.g., autologous or heterologous donor blood, cord blood, or bone marrow. For example the source of immune cells may be from the subject to be treated with the modified immune cells of the invention, e.g., the subject's blood, the subject's cord blood, or the subject's bone marrow. Nonlimiting examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. Preferably, the subject is a human.

Immune cells can be obtained from a number of sources, including blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, spleen tissue, umbilical cord, lymph, or lymphoid organs. Immune cells are cells of the immune system, such as cells of the innate or adaptive immunity, e.g., myeloid or lymphoid cells, including lymphocytes, typically T cells and/or NK cells. Other exemplary cells include stem cells, such as multipotent and pluripotent stem cells, including induced pluripotent stem cells (iPSCs). In some aspects, the cells are human cells. With reference to the subject to be treated, the cells may be allogeneic and/or autologous. The cells typically are primary cells, such as those isolated directly from a subject and/or isolated from a subject and frozen.

In certain embodiments, the immune cell is a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a natural killer T cell (NKT cells), a regulatory T cell (Treg), a stem cell memory T cell, a lymphoid progenitor cell a hematopoietic stem cell, a natural killer cell (NK cell) or a dendritic cell. In some embodiments, the cells are monocytes or granulocytes, e.g., myeloid cells, macrophages, neutrophils, dendritic cells, mast cells, eosinophils, and/or basophils. In an embodiment, the target cell is an induced pluripotent stem (iPS) cell or a cell derived from an iPS cell, e.g., an iPS cell generated from a subject, manipulated to alter (e.g., induce a mutation in) or manipulate the expression of one or more target genes, and differentiated into, e.g., a T cell, e.g., a CD8+ T cell (e.g., a CD8+ naive T cell, central memory T cell, or effector memory T cell), a CD4+ T cell, a stem cell memory T cell, a lymphoid progenitor cell or a hematopoietic stem cell.

In some embodiments, the cells include one or more subsets of T cells or other cell types, such as whole T cell populations, CD4+ cells, CD8+ cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, localization, and/or persistence capacities, antigen- specificity, type of antigen receptor, presence in a particular organ or compartment, marker or cytokine secretion profile, and/or degree of differentiation. Among the sub-types and subpopulations of T cells and/or of CD4+ and/or of CD8+ T cells are naive T (TN) cells, effector T cells (TEFF), memory T cells and sub-types thereof, such as stem cell memory T (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated effector memory T cells, tumor-infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa- associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T (Treg) cells, helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha/beta T cells, and delta/gamma T cells. In certain embodiments, any number of T cell lines available in the art, may be used.

In some embodiments, the methods include isolating immune cells from the subject, preparing, processing, culturing, and/or engineering them. In some embodiments, preparation of the engineered cells includes one or more culture and/or preparation steps. The cells for engineering as described may be isolated from a sample, such as a biological sample, e.g., one obtained from or derived from a subject. In some embodiments, the subject from which the cell is isolated is one having the disease or condition or in need of a cell therapy or to which cell therapy will be administered. The subject in some embodiments is a human in need of a particular therapeutic intervention, such as the adoptive cell therapy for which cells are being isolated, processed, and/or engineered. Accordingly, the cells in some embodiments are primary cells, e.g., primary human cells. The samples include tissue, fluid, and other samples taken directly from the subject, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic engineering (e.g. transduction with viral vector), washing, and/or incubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine and sweat, tissue and organ samples, including processed samples derived therefrom.

In some aspects, the sample from which the cells are derived or isolated is blood or a blood-derived sample, or is or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, gut associated lymphoid tissue, mucosa associated lymphoid tissue, spleen, other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testes, ovaries, tonsil, or other organ, and/or cells derived therefrom. Samples include, in the context of cell therapy, e.g., adoptive cell therapy, samples from autologous and allogeneic sources.

In some embodiments, the cells are derived from cell lines, e.g., T cell lines. The cells in some embodiments are obtained from a xenogeneic source, for example, from mouse, rat, nonhuman primate, and pig. In some embodiments, isolation of the cells includes one or more preparation and/or non-affinity based cell separation steps. In some examples, cells are washed, centrifuged, and/or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated based on one or more property, such as density, adherent properties, size, sensitivity and/or resistance to particular components.

In some examples, cells from the circulating blood of a subject are obtained, e.g., by apheresis or leukapheresis. The samples, in some aspects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and/or platelets, and in some aspects contains cells other than red blood cells and platelets. In some embodiments, the blood cells collected from the subject are washed, e.g., to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some aspects, a washing step is accomplished by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, the cells are resuspended in a variety of biocompatible buffers after washing. In certain embodiments, components of a blood cell sample are removed and the cells directly resuspended in culture media. In some embodiments, the methods include density-based cell separation methods, such as the preparation of white blood cells from peripheral blood by lysing the red blood cells and centrifugation through a Percoll or Ficoll gradient.

In one embodiment, immune are obtained cells from the circulating blood of an individual are obtained by apheresis or leukapheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. The cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media, such as phosphate buffered saline (PBS) or wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations, for subsequent processing steps. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca-free, Mg-free PBS. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

In some embodiments, the isolation methods include the separation of different cell types based on the expression or presence in the cell of one or more specific molecules, such as surface markers, c. ., surface proteins, intracellular markers, or nucleic acid. In some embodiments, any known method for separation based on such markers may be used. In some embodiments, the separation is affinity- or immunoaffinity-based separation. For example, the isolation in some aspects includes separation of cells and cell populations based on the cells' expression or expression level of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, followed generally by washing steps and separation of cells having bound the antibody or binding partner, from those cells having not bound to the antibody or binding partner.

Such separation steps can be based on positive selection, in which the cells having bound the reagents are retained for further use, and/or negative selection, in which the cells having not bound to the antibody or binding partner are retained. In some examples, both fractions are retained for further use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best carried out based on markers expressed by cells other than the desired population. The separation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but need not result in a complete absence of cells not expressing the marker. Likewise, negative selection, removal, or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but need not result in a complete removal of all such cells.

In some examples, multiple rounds of separation steps are carried out, where the positively or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells expressing multiple markers simultaneously, such as by incubating cells with a plurality of antibodies or binding partners, each specific for a marker targeted for negative selection. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed on the various cell types.

In some embodiments, one or more of the T cell populations is enriched for or depleted of cells that are positive for (marker+) or express high levels (marker¹¹*⁸¹¹) of one or more particular markers, such as surface markers, or that are negative for (marker -) or express relatively low levels (marker¹⁰") of one or more markers. For example, in some aspects, specific subpopulations of T cells, such as cells positive or expressing high levels of one or more surface markers, e.g., CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and/or CD45RO+ T cells, are isolated by positive or negative selection techniques. In some cases, such markers are those that are absent or expressed at relatively low levels on certain populations of T cells (such as non-memory cells) but are present or expressed at relatively higher levels on certain other populations of T cells (such as memory cells). In one embodiment, the cells (such as the CD8+ cells or the T cells, e.g., CD3+ cells) are enriched for (i.e., positively selected for) cells that are positive or expressing high surface levels of CD45RO, CCR7, CD28, CD27, CD44, CD 127, and/or CD62L and/or depleted of (e.g., negatively selected for) cells that are positive for or express high surface levels of CD45RA. In some embodiments, cells are enriched for or depleted of cells positive or expressing high surface levels of CD 122, CD95, CD25, CD27, and/or IL7-Ra (CD 127). In some examples, CD8+ T cells are enriched for cells positive for CD45RO (or negative for CD45RA) and for CD62L. For example, CD3+, CD28+ T cells can be positively selected using CD3/CD28 conjugated magnetic beads e.g., DYNABEADS® M-450 CD3/CD28 T Cell Expander).

In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD 14. In some aspects, a CD4+ or CD8+ selection step is used to separate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed to a relatively higher degree on one or more naive, memory, and/or effector T cell subpopulations. In some embodiments, CD8+ cells are further enriched for or depleted of naive, central memory, effector memory, and/or central memory stem cells, such as by positive or negative selection based on surface antigens associated with the respective subpopulation. In some embodiments, enrichment for central memory T (TCM) cells is carried out to increase efficacy, such as to improve longterm survival, expansion, and/or engraftment following administration, which in some aspects is particularly robust in such sub-populations. In some embodiments, combining TCM-enriched CD8+ T cells and CD4+ T cells further enhances efficacy.

In some embodiments, memory T cells are present in both CD62L+ and CD62L- subsets of CD8+ peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8+ and/or CD62L+CD8+ fractions, such as using anti-CD8 and anti-CD62L antibodies. In some embodiments, a CD4+ T cell population and a CD8+ T cell sub-population, e.g., a subpopulation enriched for central memory (TCM) cells. In some embodiments, the enrichment for central memory T (TCM) cells is based on positive or high surface expression of CD45RO, CD62L, CCR7, CD28, CD3, and/or CD 127; in some aspects, it is based on negative selection for cells expressing or highly expressing CD45RA and/or granzyme B. In some aspects, isolation of a CD8+ population enriched for TCM cells is carried out by depletion of cells expressing CD4, CD 14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment for central memory T (TCM) cells is carried out starting with a negative fraction of cells selected based on CD4 expression, which is subjected to a negative selection based on expression of CD 14 and CD45RA, and a positive selection based on CD62L. Such selections in some aspects are carried out simultaneously and in other aspects are carried out sequentially, in either order. In some aspects, the same CD4 expression-based selection step used in preparing the CD8+ cell population or subpopulation, also is used to generate the CD4+ cell population or sub-population, such that both the positive and negative fractions from the CD4- based separation are retained and used in subsequent steps of the methods, optionally following one or more further positive or negative selection steps.

CD4+ T helper cells are sorted into naive, central memory, and effector cells by identifying cell populations that have cell surface antigens. CD4+ lymphocytes can be obtained by standard methods. In some embodiments, naive CD4+ T lymphocytes are CD45RO-, CD45RA+, CD62L+, CD4+ T cells. In some embodiments, central memory CD4+ cells are CD62L+ and CD45RO+. In some embodiments, effector CD4+ cells are CD62L- and CD45RO. In one example, to enrich for CD4+ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD1 lb, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is bound to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow for separation of cells for positive and/or negative selection.

In some embodiments, the cells are incubated and/or cultured prior to or in connection with genetic engineering. The incubation steps can include culture, cultivation, stimulation, activation, and/or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulatory agent. Such conditions include those designed to induce proliferation, expansion, activation, and/or survival of cells in the population, to mimic antigen exposure, and/or to prime the cells for genetic engineering, such as for the introduction of a recombinant antigen receptor. The conditions can include one or more of particular media, temperature, oxygen content, carbon dioxide content, time, agents, e.g., nutrients, amino acids, antibiotics, ions, and/or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors, and any other agents designed to activate the cells. In some embodiments, the stimulating conditions or agents include one or more agent, e.g., ligand, which is capable of activating an intracellular signaling domain of a TCR complex. In some aspects, the agent turns on or initiates TCR/CD3 intracellular signaling cascade in a T cell. Such agents can include antibodies, such as those specific for a TCR component and/or costimulatory receptor, e.g., anti-CD3, anti-CD28, for example, bound to solid support such as a bead, and/or one or more cytokines. Optionally, the expansion method may further comprise the step of adding anti-CD3 and/or anti CD28 antibody to the culture medium (e.g., at a concentration of at least about 0.5 ng/ml). In some embodiments, the stimulating agents include IL-2 and/or IL- 15, for example, an IL-2 concentration of at least about 10 units/mL.

In another embodiment, T cells are isolated from peripheral blood by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient. Alternatively, T cells can be isolated from an umbilical cord. In any event, a specific subpopulation of T cells can be further isolated by positive or negative selection techniques.

The cord blood mononuclear cells so isolated can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD8, CD14, CD19, and CD56. Depletion of these cells can be accomplished using an isolated antibody, a biological sample comprising an antibody, such as ascites, an antibody bound to a physical support, and a cell bound antibody. Enrichment of a T cell population by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. A preferred method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4⁺ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8.

For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (z.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells/ml is used. In one embodiment, a concentration of 1 billion cells/ml is used. In a further embodiment, greater than 100 million cells/ml is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/ml Is used, n yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/ml is used. In further embodiments, concentrations of 125 or 150 million cells/ml can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion.

T cells can also be frozen after the washing step, which does not require the monocyteremoval step. While not wishing to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, in a non-limiting example, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or other suitable cell freezing media. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20°C or in liquid nitrogen.

In one embodiment, the population of T cells is comprised within cells such as peripheral blood mononuclear cells, cord blood cells, a purified population of T cells, and a T cell line. In another embodiment, peripheral blood mononuclear cells comprise the population of T cells. In yet another embodiment, purified T cells comprise the population of T cells.

In certain embodiments, T regulatory cells (Tregs) can be isolated from a sample. The sample can include, but is not limited to, umbilical cord blood or peripheral blood. In certain embodiments, the Tregs are isolated by flow-cytometry sorting. The sample can be enriched for Tregs prior to isolation by any means known in the art. The isolated Tregs can be cryopreserved, and/or expanded prior to use. Methods for isolating Tregs are described in U.S. Patent Numbers: 7,754,482, 8,722,400, and 9,555,105, and U.S. Patent Application No. 13/639,927, contents of which are incorporated herein in their entirety.

Expansion of Immune Cells

Whether prior to or after modification of cells to express a CAR, the cells can be activated and expanded in number using methods as described, for example, in U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681 ; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Publication No. 20060121005. For example, the T cells of the invention may be expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated by contact with an anti-CD3 antibody, or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore. For co-stimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, T cells can be contacted with an anti- CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) and these can be used in the invention, as can other methods and reagents known in the art (see, e.g., ten Berge et al., Transplant Proc. (1998) 30(8): 3975-3977; Haanen et al., J. Exp. Med. (1999) 190(9): 1319-1328; and Garland et al., J. Immunol. Methods (1999) 227(1-2): 53-63).

Expanding T cells by the methods disclosed herein can be multiplied by about 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater, and any and all whole or partial integers therebetween. In one embodiment, the T cells expand in the range of about 20 fold to about 50 fold.

Following culturing, the T cells can be incubated in cell medium in a culture apparatus for a period of time or until the cells reach confluency or high cell density for optimal passage before passing the cells to another culture apparatus. The culturing apparatus can be of any culture apparatus commonly used for culturing cells in vitro. Preferably, the level of confluence is 70% or greater before passing the cells to another culture apparatus. More preferably, the level of confluence is 90% or greater. A period of time can be any time suitable for the culture of cells in vitro. The T cell medium may be replaced during the culture of the T cells at any time. Preferably, the T cell medium is replaced about every 2 to 3 days. The T cells are then harvested from the culture apparatus whereupon the T cells can be used immediately or cryopreserved to be stored for use at a later time. In one embodiment, the invention includes cryopreserving the expanded T cells. The cryopreserved T cells are thawed prior to introducing nucleic acids into the T cell.

In another embodiment, the method comprises isolating T cells and expanding the T cells. In another embodiment, the invention further comprises cryopreserving the T cells prior to expansion. In yet another embodiment, the cryopreserved T cells are thawed for electroporation with the RNA encoding the chimeric membrane protein.

Another procedure for ex vivo expansion cells is described in U.S. Pat. No. 5,199,942 (incorporated herein by reference). Expansion, such as described in U.S. Pat. No. 5,199,942 can be an alternative or in addition to other methods of expansion described herein. Briefly, ex vivo culture and expansion of T cells comprises the addition to the cellular growth factors, such as those described in U.S. Pat. No. 5,199,942, or other factors, such as flt3-L, IL-1, IL-3 and c-kit ligand. In one embodiment, expanding the T cells comprises culturing the T cells with a factor selected from the group consisting of flt3-L, IL-1, IL-3 and c-kit ligand.

The culturing step as described herein (contact with agents as described herein or after electroporation) can be very short, for example less than 24 hours such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 hours. The culturing step as described further herein (contact with agents as described herein) can be longer, for example 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more days.

Various terms are used to describe cells in culture. Cell culture refers generally to cells taken from a living organism and grown under controlled condition. A primary cell culture is a culture of cells, tissues or organs taken directly from an organism and before the first subculture. Cells are expanded in culture when they are placed in a growth medium under conditions that facilitate cell growth and/or division, resulting in a larger population of the cells. When cells are expanded in culture, the rate of cell proliferation is typically measured by the amount of time required for the cells to double in number, otherwise known as the doubling time.

Each round of subculturing is referred to as a passage. When cells are subcultured, they are referred to as having been passaged. A specific population of cells, or a cell line, is sometimes referred to or characterized by the number of times it has been passaged. For example, a cultured cell population that has been passaged ten times may be referred to as a PIO culture. The primary culture, i.e., the first culture following the isolation of cells from tissue, is designated P0. Following the first subculture, the cells are described as a secondary culture (Pl or passage 1). After the second subculture, the cells become a tertiary culture (P2 or passage 2), and so on. It will be understood by those of skill in the art that there may be many population doublings during the period of passaging; therefore the number of population doublings of a culture is greater than the passage number. The expansion of cells (i.e., the number of population doublings) during the period between passaging depends on many factors, including but is not limited to the seeding density, substrate, medium, and time between passaging.

In one embodiment, the cells may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-gamma, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGF-beta, and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyLcysteine and 2-mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g. penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).

The medium used to culture the T cells may include an agent that can co-stimulate the T cells. For example, an agent that can stimulate CD3 is an antibody to CD3, and an agent that can stimulate CD28 is an antibody to CD28. A cell isolated by the methods disclosed herein can be expanded approximately 10 fold, 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80 fold, 90 fold, 100 fold, 200 fold, 300 fold, 400 fold, 500 fold, 600 fold, 700 fold, 800 fold, 900 fold, 1000 fold, 2000 fold, 3000 fold, 4000 fold, 5000 fold, 6000 fold, 7000 fold, 8000 fold, 9000 fold, 10,000 fold, 100,000 fold, 1,000,000 fold, 10,000,000 fold, or greater. In one embodiment, the T cells expand in the range of about 20 fold to about 50 fold, or more. In one embodiment, human T regulatory cells are expanded via anti-CD3 antibody coated KT64.86 artificial antigen presenting cells (aAPCs). Methods for expanding and activating T cells can be found in U.S. Patent Numbers: 7,754,482, 8,722,400, and 9,555, 105, contents of which are incorporated herein in their entirety.

In one embodiment, the method of expanding the T cells can further comprise isolating the expanded T cells for further applications. In another embodiment, the method of expanding can further comprise a subsequent electroporation of the expanded T cells followed by culturing. The subsequent electroporation may include introducing a nucleic acid encoding an agent, such as a transducing the expanded T cells, transfecting the expanded T cells, or electroporating the expanded T cells with a nucleic acid, into the expanded population of T cells, wherein the agent further stimulates the T cell. The agent may stimulate the T cells, such as by stimulating further expansion, effector function, or another T cell function.

Kits for Treating Cancer

In another aspect, the invention further provides a kit for treating cancer in a subject in need thereof, the kit comprising (i) a pharmaceutical composition comprising an antibody-linked cytokine of the invention as described herein, or an LNP of the invention as described herein, and at least one pharmaceutically acceptable carrier, diluent, and/or excipient; and (ii) instructional material(s) for administration of a therapeutically effective amount of the pharmaceutical composition to the subject. In some embodiments, the instructional material(s) included in the kit further comprises instructions for carrying out the method of the invention for treating cancer in a subject in need thereof.

The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other physical and electronic documents.

While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. It will be readily apparent to those skilled in the art that other suitable modifications and adaptations of the methods described herein may be made using suitable equivalents without departing from the scope of the embodiments disclosed herein. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto. Having now described certain embodiments in detail, the same will be more clearly understood by reference to the following examples, which are included for purposes of illustration only and are not intended to be limiting.

EXPERIMENTAL EXAMPLES

The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein. Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out the preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

Example 1

Materials and Methods

CAR T Cell Generation

All cytokine sequences were synthesized by Integrated DNA Technologies and developed using a pTRPE parental vector. Normal donor T cells were enriched from leukaphresed PBMCs and ex-vivo expanded by stimulating T cells with anti-CD3/CD28 Dynabeads (Cell Therapy Systems catalog number 40203D) for 16 hours followed by lentiviral transduction at MOI 5. T cells were de-beaded on day 5 and allowed to expand until day 9. RPMI medium supplemented with 10% FBS (Gibco catalog #16140-071), 1% HEPES (Gibco catalog #15630-080), 1% GlutaMAX (Gibco catalog #35050-061), and 1% Pen Strep (Gibco catalog #15140-122). Media changes were made every 2 days starting on day 5, and media was supplemented with 30IU/mL interleukin-2. Cells were counted and replated in the above media every 2 days starting on day 5, and maintained at .75E6 cells/mL. The MCF-7 cell line was maintained in log growth phase in Dulbecco’s Modified Essential Medium (Gibco catalog #10569-010) supplemented with 10% FBS and 1% Pen Strep.

Flow Cytometry

CAR-T cell identification was performed utilizing a biotin-SP-conjugated anti-mouse IgG, F(ab’)² antibody (lackson ImmunoResearch catalog #115-065-072) followed by a PE- conjugated streptavidin as a secondary reagent (BD Biosciences catalog #554061). The memory phenotype and exhaustion marker expression was analyzed by a 10-color flow cytometry panel. Flow Cytometry was analyzed using using a BD LSRFortessa Cell Analyzer and BD Bioscience’s Flowlo. All figures were generated and statistics were analyzed using GraphPad Prism. In vitro Cytokine Analysis

ELISAs for human IL-12p70, IL- 18, and IL-23 were performed utilizing Duoset ELISA technologies (Biotechne catalog #DY1270-05, #DY-318-05, and #DY-1290-05, respectively) following supplier-provided protocols. The in vitro Luminex was performed with a Milliplex Human 31 Plex Cytokine kit (Millipore catalog #SPRCUS707). Supernatant was collected from 100,000 T cells normalized to a normalized CAR+ percentage following overnight culture in media alone, a 1 :1 E:T co-culture of MCF-7 tumor cells, or 25ng/mL PMA and Ipg/mL lonomycin.

In vitro killing ofMCF7 cells

In vitro killing assays were performed on an Agilent xCELLigence RTCA eSight instrument. 1E4 MCF-7 tumor cells were cultured for 24 hours followed by addition of T cells in defined E:T ratios (1: 1 for ND567 and 3: 1 forND224 shown in FIG. 3). T cells were normalized for CAR positivity following thaw. In vitro killing of MCF7 cells by CAR-T cells either constitutively secreting cytokines or CAR-T cells dosed with the antibody-linked cytokine conjugate is shown by impedance. Tumor cells were plated as stated above. CAR-T cells were normalized for CAR positivity and plated 24 hours after tumor cells at a 3 : 1 E:T ratio. ,0122nmol of either antibody or antibody-linked cytokine conjugate was added into the culture at the same time as the CAR-T cells.

Mouse Xenograft Models

NODZscvc/Zgamma (NSG) mice were obtained from the Stem Cell and Xenograft Core. Female mice were inoculated with 1E6 MCF-7 cells in 50% PBS and 50% Matrigel (Corning catalog #354234) subcutaneously on the right rear flank. Palpable tumors were measured with calipers to randomize mice into treatment groups with normalized average tumor sizes. 1E6 CAR+ cells or an equivalent amount of non-transduced cells were injected via lateral tail vein in lOOpL PBS. Body weights were recorded daily for the first week and weekly thereafter. Caliper measurements of tumor volume were recorded weekly. Mice were sacrificed when tumor burden became greater than 2000 mm³, if body weight loss was more than 10%, or mice showed body condition deterioration. IHC was performed by the CHOP Pathology Core and analysis was done via Aperio ImageScope utilizing Positive Pixel Count v9. Luminex assay was performed a IQ- plex human panel (ThermoFisher catalog #LHC0001M) with serum collected following centrifugation of peripheral blood in a EDTA coated tube (BD catalog #365974) in a microcentrifuge tube for 10 minutes at 2000rpm.

Results

Creation and Validation of CAR-T Cells Constitutively Secreting Cytokines

CAR-T cells expressing the 5E5 CAR (VH and VL regions of anti-TnMUCl mAb 5E5 linked with a (G₄S)₃ (GGGGSGGGGSGGGGS) (SEQ ID NO: 32) synthetic linker to create the scFv binding region, a CD8a hinge region and transmembrane domain, a CD2 co- stimulatory domain, and a CD3z stimulatory domain was previously generated. Each of three cytokines (IL- 12, IL-18, and IL-23) were included in the CAR construct with the P2A self-cleaving peptide allowing expression of the CAR molecule and each cytokine in a single plasmid sequence (FIG. 1A). Because IL-12 and IL-23 are heterodimeric cytokines, a (G₄S)I (GGGGS) (SEQ ID NO: 56) linker between the two subunits was utilized to express each cytokine in a single chain format. Primary human T cells were transduced with lentiviral vectors to express the CAR and cytokines (FIG. IB) T cells were expanded for 9 days ex vivo and over the course of a 9-day expansion the secretion of cytokines did not lead to a significant increase in the fold expansion of T cells (FIG. ID). To test the cells for secretion of cytokines, the cells were cultured either alone, in a co-culture with a TnMUCl+ cell line (MCF7), or with PMA/ionomycin to stimulate the cells without antigen. All three cytokine-secreting T cells were able to selectively increase the amount of cytokine found in the culture media after 24 hours, and all secreted significantly less cytokines at rest compared to when activated antigen dependently or independently (FIG. 1C).

Secretion of IL- 12 Leads to a More Activated and More Effector -like Phenotype

T cell phenotype has become an important part of immunotherapy studies as early-stage phenotypes have been linked with better clinical outcomes. The main four T cell phenotypes are naive (TN: CD45RO-, CCR7+), terminal effector (TE: CD45RO-, CCR7-), effector memory (TEM: CD45RO+, CCR7-), and central memory (TCM: CD45RO+, CCR7+). Of these four subsets, TCM and TN are the least differentiated, which provides the capacity for durable persistence and are the desired memory phenotypes of CAR-T products. Analysis of the memory phenotype of the CAR-T cells after the 9-day CAR-T cell expansion showed that IL-12 secreting CAR-T cells had a small increase in the number of TEM cells, as compared to the phenotype of all other groups (FIG. 2A). The same cells were also analyzed for markers of T cell exhaustion: PD-1, LAG-3, and TIM-3. IL-12 secreting T cells showed a significant increase in the percentage of cells expressing multiple of these markers, as well as a significant decrease in cells not expressing any of the markers (FIG. 2B).

CAR-T Cells Constitutively Secreting Cytokines Increase Anti-Tumor Effects In Vitro

To test the efficacy of CAR-T cells in vitro, CAR-T cells were seeded with the breast cancer cell line MCF7 at defined effector to target ratios in an impedance based killing assay. A significant increase in tumor lysis was found for CAR-T cells secreting all three immunostimulatory cytokines as compared to both non-transduced T cells as well as CAR-T cells alone (FIG. 3). This effect was reproduced with multiple healthy donors. While a significant increase in tumor lysis was found, CAR-T cells secreting IL- 18 seemed to lose control of tumor growth after days of co-culture, in one donor creating a significant decrease in killing by the end of the assay as compared to CAR-T cells secreting IL- 12 and IL-23.

CAR-T Cells Constitutively Secreting IL- 12 and IL-23 Delay Tumor Growth at Low Doses in vivo

To further assess the ability of the cytokine-secreting CAR-T cells, NSG mice inoculated subcutaneously with MCF7 breast cancer cells were treated with 1E6 CAR+ T cells when the tumors became palpable. T cells secreting IL-12 and IL -23 significantly delayed tumor growth compared to non-transduced T cells, CAR-T cells alone, or CAR-T cells secreting IL-18. FIG. 4A shows average tumor size of the mice as measured by caliper until the first mouse had to be sacrificed due to tumor size exceeding acceptable volume. The decrease in tumor growth in IL- 12 and IL-23 secreting mice also lead to an increase in survival in both groups (FIG. 4B). As toxicity has been reported in past xenograft models in cytokine secreting T cells mouse weights were monitored throughout the experiment; no significant loss of weight was observed for any of the groups (FIG. 4D). Peripheral blood T cell count was measured at days 33 and 63 after T cell injection, and, at both time points, an increased number of T cells was found in the IL-12 and IL- 23 secreting groups. Postmortem, subcutaneous tumors were excised and analyzed utilizing an anti-human CD3 antibody. At time of death, the mean of the percent of pixels in the tumor section positive for CD3 was increased in the IL-12 and IL-23 secreting groups. Blood was also taken from these mice at time of death to analyze serum cytokine levels via a 10-plex Luminex panel. Serum levels of interferon gamma (IFNy) and tumor necrosis factor alpha (TNFoc) were increased in the IL- 12 and IL-23 secreting groups, indicative of an increase in antitumor activity (FIG. 4C)

Unexpected Superior Tumor Lysis for cells cultured with antibody-linked cytokines compared to CAR-T cells constitutively secreting the same cytokine

In vitro killing of the MCF7 cell line by CAR-T cells either constitutively secreting cytokines or CAR-T cells dosed with the antibody-linked cytokine of the invention is shown in FIG. 5. For these experiments with the anti-scFv antibody and antibody-cytokine conjugates, the anti-scFv antibody was KIP4-163. KIP4-163 was previously described see, e.g., W02019060713A1). Killing of tumor cells is shown by impedance. The cells constitutively secreting cytokines have a significant increase in tumor lysis as compared to CAR-T cells alone. CAR-T cells alone had a significant increase in tumor lysis when cultured with antibody-linked cytokine conjugates as compared to the antibody alone. CAR-T cells cultured with the antibody alone did not have an increase or decrease in killing efficacy as compared to CAR-T cells alone. Unexpectedly, cells cultured with antibody-linked cytokine conjugates showed superior tumor lysis as compared to CAR-T cells constitutively secreting the same cytokine.

Anti-(G4S) antibody does not bind to (G4S)I but does bind to (G4 )4

The typical linker used in the scFv of CARs is a (G4S)3 linker, and thus a “short” (G4S)I linker was used herein to link the anti-scFv antibody to the cytokines. To test whether the anti- scFv antibody will bind to the (G4S)I “short” linker, or to a longer linker, anti-CD22 CARs comprising an scFv having either a (G4S)I “short” linker or a (G4S)4 “long” linker were generated. CAR T cells expressing these anti-CD22 CARs were tested with plate bound KIP4- 163 anti-(G4S) antibody. The “long” linker CAR-T cells are highly activated by plate-bound anti- (G4S) antibody as evident by the increased surface expression of T cell activation marker CD69, whereas the “short” linker CAR-T cells are lowly activated or not activated by plate-bound anti- (G4S) antibody (FIG. 6). Anti-(G4S) antibody enhances lentivirus transduction efficiency in pre-stimulated CAR-T cells and enriches for CAR+ T cells

To assess the effect of anti-(G4S) antibody on T cell transduction, CAR-T cells were cultured alone, with KIP4-163 anti-(G4S) antibody beads, or with CD3/CD28 beads. NTD T cells were also tested with KIP4-163 anti-(G4S) antibody beads. Flow staining analysis was performed to assess for transduction efficiency and % CAR expression (FIGs. 20A - 20B). The results indicate that anti-(G4S) antibody enhances lentivirus transduction efficiency in pre-stimulated CAR-T cells and enriches for CAR+ T cells (FIGs. 20A - 20B).

Anti-(G4S) antibody activates CAR-T cells

CD19 targeting CAR T cells were incubated overnight with KIP4-163 beads and assessed by eSight real-time xCelligence impedance analysis and ELISA assays. The results indicate that anti-(G4S) antibody activates CAR-T cells by stimulating secretion of IL-2 and IFNy, while preserving target-specific cytotoxicity and safety against normal cells (FIGs. 21A - 21D).

Next, the effect of incubating the CD 19 targeting CAR T cells long term (for 7days) with KIP4-163 beads was assessed. The results indicate that long term activation of CAR-T cells using anti-(G4S) antibody significantly increases early and late activation markers (FIGs. 22A - 22E) but does not impact T cell memory profile (FIGs. 23A - 23E). Additionally, the long term activation of CAR-T cells using anti-(G4S) antibody induces mild-to-moderate exhaustion of CAR-T cells as compared to unstimulated or CD3/CD28 bead activated and exhausted CAR-T cells (FIGs. 24A - 24F). The exhaustion marker panels of CAR-T cells derived from 3 donors, and stimulated with CD3/CD28 beads, KIP4-163 beads, or none were assessed (FIGs. 25A - 25B). The heatmap details the color-coded cluster expression profiles (FIG. 25C). tSNE- clustering based on CD4 or CD8 expression is also plotted (FIG. 25D). Representative tSNE- clustering of exhaustion markers taken from T cells derived from the same donor are shown in FIGs. 26A -26L. Collectively, these data indicate that anti-(G4S) antibody could be utilized for mild CAR-T cell stimulation (such as for stimulation prior to LV transduction) without aggravating T cell exhaustion. Importantly, anti-(G4S) antibody-stimulated CAR-T cells maintain specific on-target cytotoxicity and off-target safety.

Anti-(G4S) antibody-cytokine conjugates selectively activates CAR-T cells The KTP4-163 anti-(G4S) antibody-cytokine conjugates were next tested for their ability to activate CAR-T cells. CAR-T cells (1E5) expressing either a CD19 targeting CAR (FMC63BBz) or a Tn-MUCl targeting CAR (5E5CD2z) were each co-cultured for 24 hours with plate-bound KIP4-163 anti-(G4S) antibody or anti-(G4S) antibody-cytokine conjugates and assessed for CD69 expression by flow cytometry. All anti-(G4S) antibody cytokine conjugates significantly increased activation of CAR-T cells above that of CAR-T cells without anti-(G4S) antibody as well as that of non-transduced T cells with anti-(G4S) antibody plated for both types of CAR-T cells (FIGs. 7A - 7B).

IL-12 drives a later activation state

The anti-(G4S) antibody-cytokine conjugates were next tested to determine the extent to which they activate CAR-T cells (i.e., whether they drive a higher state of activation). CAR-T cells expressing either a CD 19 targeting CAR (FMC63BBz), an oncofetal fibronectin targeting CAR (FDC6BBz), or a CEACAM6 targeting CAR (mAblO9BBZ) were each co-cultured for 24 hours with plate-bound KIP4-163 anti-(G4S) antibody or anti-(G4S) antibody-cytokine conjugates and assessed for CD25 expression by flow cytometry (FIGs. 8A - 8C). CAR-T cells activated by the anti-(G4S)-IL12 conjugate expressed higher levels of CD25, indicating a higher activation state.

Anti-jC S) antibody-cytokine conjugates increase anti-tumor efficacy in CAR-T cells

Next, T cells expressing a Tn-MUCl targeting CAR (5E5CD2z) were co-cultured in vitro with MCF7 breast cancer cells at suboptimal E:T ratio in the presence or absence of soluble anti- (G4S) antibody or anti-(G4S) antibody-cytokine conjugates. The anti-(G4S) antibody was added at a concentration of 5 ug/mL and a normalized amount of the anti-(G4S) antibody-cytokine conjugates was used (i.e., 1 ug/mL of antibody corresponds to 1.7 ug/mL antibody-IL12 conjugate, 1.22 ug/mL of antibody-IL18 conjugate, and 1.66 ug/mL of antibody-IL23 conjugate). The results show that KIP4-163 antibody-cytokine conjugates increase antitumor efficacy of CAR-T cells in vitro (FIG. 9). The control conditions (CAR alone and CAR with KIP4-163 anti- (G4S) antibody) did not control the growth of the tumor cells, whereas CAR-T cells with any of the antibody-cytokine conjugates killed the tumor cells completely (FIG. 9).

Titrations of the anti-(G4S) antibody-cytokine conjugates showed a dose response for each. The anti-(G4S) antibody -IL12 conjugate was effective at even the lowest dose tested whereas the anti-(G4S) antibody-IL18 and anti-(G4S) antibody -IL23 were effective at the higher doses tested but lost efficacy as the dose decreased (FIGs. 10A - 10D).

To determine whether the CAR-T cells uptake the anti-(G4S) antibody-cytokine conjugates, CAR-T cells were incubated for one hour with 5 ug/mL anti-(G4S) antibody or a normalized amount of anti-(G4S) antibody-cytokine conjugates. Presence of the anti-(G4S) antibody on the cell surface was assessed using an anti-mouse Fc antibody, whereas presence of the anti-(G4S) antibody -cytokine conjugates on the cell surface was assessed using an anti IL12p40 antibody, as IL12p40 is a monomer present in both IL12 and IL23. The results demonstrate that the anti-(G4S) antibody is present on the cell surface for at least 48 hours, whereas the anti-(G4S) antibody-cytokine conjugates were taken up and used by the T cells (FIG. 11)

Anti-(G4S) antibody-IL12 conjugate reduces tumor burden and increases survival in xenograft mice

NSG mice were injected on day -5 with 1E6 Nalm6 CBG luciferase+ cells. On day -1, mice were imaged and normalized by their total flux. 1E6 CAR+ T cells were injected on day 0. On day 1 and weekly afterwards, 10 ug of anti-(G4S) antibody or anti-(G4S) antibody-cytokine conjugates were injected IV. CAR-T cell treated mice given anti-(G4S) antibody -IL12 conjugate had significantly longer survival than mice given PBS which correlated with a lower average flux as well as an increase in peripheral blood T cells two weeks after T cell injection (FIGs. 12A - 12D)

Example 2

Next, additional anti-scFv monoclonal antibodies were generated which selectively target a linker found in the scFv domain of CARs (i.e., either a (648)3 linker (GGGSGGGSGGGS) (SEQ ID NO: 32) or a whitlow linker (GSTSGSGKPGSGEGSTKG) (SEQ ID NO: 58). The results from ELISA screening of murine serum samples for specific antibody producing B-cells from mice immunized against either (648)3 or whitlow linker peptides, after the third round of immunization, and prior to fusion with mouse myeloma cell lines for hybridoma generation are shown in FIGs. 13A - 13B. Results from ELISA screening for anti-(G4S)3 linker and anti- whitlow linker hybridoma post fusion are shown in FIGs. 14A - 14B. Results from ELISA screening for top scoring anti-(G4S)3 linker and anti-whitlow linker single cell clone hybridomas are shown in FIGs. 15A - 15D.

Next, scFvs were generated, which scFvs are termed OD002, OD007, and OD008. The three new scFv sequences are based on NGS sequencing of RNA samples extracted from top scoring bulk hybridoma of either (G4S)3 or whitlow antibodies (i.e., before SCC), followed by immune repertoire analysis and assembly. For OD002, VH was only seen in the G4S NGS data file, while VL was common between G4S and whitlow NGS data files. For OD007, VH was only seen in the whitlow NGS data file, while VL was common between G4S and whitlow NGS data files. For OD008, VH appeared in both G4S and whitlow NGS data files, while VL only appeared in the whitlow NGS data file.

A luciferase assay was performed to assess the specific lysis of 293T-CBG+ cells expressing test OD002, OD007, or OD008 scFv(s) against (648)3 or whitlow linkers by CAR-T cells that contain (648)3 or whitlow linkers in the scFv domain (i.e., CD19 targeting CD19BBz CAR-T cells or ErbB2 targeting 4D5.5BBz CAR-T cells) (FIG. 16). 293T-CBG+ cells expressing KIP4-163 or anti-CD3 (0KT3) scFv were used as positive controls. Amino acid and nucleotide sequences for the CDRs and VH and VL chains of OD002, OD007, and OD008 scFvs are provided in Table 1 (SEQ ID NOs: 59 - 106).

The OD002, OD007, and OD008 scFvs were next expressed in 293T cells and real-time xCelligence impedence analysis was performed to assess specific killing of the 293T cells by CAR-T cells expressing a CAR that contain either the (648)3 linker or whitlow linker (i.e., CD19 targeting CD19BBz CAR-T cells or ErbB2 targeting 4D5.5BBz CAR-T cells). Representative results for the OD002, OD007, and OD008 scFvs are shown in FIGs. 17A - 17D, FIGs. 18A - 18D, and FIGs. 19A - 19D, respectively. Upon repeating the experiments multiple times, the data indicated that OD002 and OD008 appear to recognize scFvs containing either (648)3 or whitlow linkers. Additionally, OD008 likely has higher binding affinity to both linkers as compared to that of OD002, reflected by the superior cytotoxicity of OD008-expressing 293 T cells by CD19BBz or 4D5.5BBz CAR-T cells. On the other hand, OD007 is selective for binding to the (648)3 linker in view of the finding that 4D5.5BBz CAR-T cells (comprising a whitlow linker in the CAR scFv) failed to kill OD007-expressing 293T cells, whereas the OD007-expressing 293T cells underwent selective and potent killing by CD19BBz CAR-T cells containing (G4S).i as a linker in its scFv domain.

Enumerated Embodiments

The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance:

Embodiment 1 : An anti-single-chain variable fragment (scFv) antibody or antigen-binding fragment, wherein the anti-scFv antibody or antigen-binding fragment is capable of specifically binding to a linker peptide of an scFv, wherein the linker peptide links a variable light chain (VL) of the scFv to a variable heavy chain (VH) of the scFv, and further wherein the scFv is an scFv of a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain comprising the scFv, a transmembrane domain, and an intracellular domain.

Embodiment 2: The anti-scFv antibody or antigen-binding fragment of embodiment 1, comprising a heavy chain variable region (VH) comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and a light chain variable region (VL) comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein:

Embodiment 3 : The anti-scFv antibody or antigen-binding fragment of embodiment 1 or embodiment 2, wherein:

Embodiment 4: The anti-scFv antibody or antigen-binding fragment of any one of embodiments 1-4, further comprising a heavy chain and a light chain, wherein the heavy chain comprises the VH and the light chain comprises the VL.

Embodiment 5: The anti-scFv antibody or antigen-binding fragment of any one of embodiments 1-4, wherein the scFv of the CAR targets a tumor antigen, preferably wherein the tumor antigen is selected from the group consisting of alpha feto-protein (AFP)/HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD13, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD1 17, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, claudin 6, c-Met, DLL3, DR5, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa)/folate binding protein (FBP), folate receptor beta (FRb), follicle-stimulating hormone receptor (FSHR), GD-2, Glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), HER2, HLA- A2, ICAM1, interleukin 13 receptor subunit alpha (IL3Ra), interleukin 13 receptor subunit alpha 2 (IL13Ra2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4/FAP, NKG2D-Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), Mullerian inhibiting substance type 2 receptor (MISIIR), New York esophageal squamous cell carcinoma- 1 (NY-ESO-1), Pl 6, PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), R0R1, R0R2, TAG72, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUCl), TR0P2, Glycosyl-phosphatidylinositol (GPI)- linked GDNF family a-receptor 4 (GFRa4; GFRalpha4), VEGFR2, and any combination thereof.

Embodiment 6: The anti-scFv antibody or antigen-binding fragment of any one of embodiments 1-5, wherein the scFv of the CAR targets TnMUCl.

Embodiment 7: The anti-scFv antibody or antigen-binding fragment of any one of embodiments 1-7, wherein the intracellular domain of the CAR comprises a costimulatory domain and an intracellular signaling domain.

Embodiment 8: The anti-scFv antibody or antigen-binding fragment of embodiment 7, wherein one or more of the following applies:

Embodiment 9: A nucleic acid molecule comprising a nucleotide sequence encoding the anti- scFv antibody or antigen-binding fragment of any one of embodiments 1-8, optionally wherein the nucleotide sequence further encodes a ribosome slip sequence, optionally wherein the ribosome slip sequence is selected from the group consisting of P2A, T2A, E2A, F2A, and variants thereof.

Embodiment 10: A vector comprising the nucleic acid molecule of embodiment 9.

Embodiment 11 : A cell comprising the nucleic acid molecule of embodiment 9 or the vector of embodiment 10.

Embodiment 12: An antibody-linked cytokine, comprising an immunostimulatory cytokine linked to an anti-scFv antibody or antigen-binding fragment, wherein the anti-scFv antibody or antigen-binding fragment is capable of specifically binding to a linker peptide of an scFv, wherein the linker peptide links a variable light chain (VL) of the scFv to a variable heavy chain (VH) of the scFv, and further wherein the scFv is an scFv of a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain comprising the scFv, a transmembrane domain, and an intracellular domain.

Embodiment 13: The antibody-linked cytokine of any embodiment 12, wherein the anti-scFv antibody or antigen-binding fragment comprises a heavy chain variable region (VH) comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and a light chain variable region (VL) comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein:

Embodiment 14: The antibody-linked cytokine of embodiment 13, wherein:

Embodiment 15: The antibody -linked cytokine of embodiment 14, wherein the anti-scFv antibody or antigen-binding fragment further comprises a heavy chain and a light chain, wherein the heavy chain comprises the VH and the light chain comprises the VL.

Embodiment 16: The antibody-linked cytokine of any one of embodiments 12-15, wherein the immunostimulatory cytokine comprises a single polypeptide chain or is engineered to be expressed as a single polypeptide chain. Embodiment 17: The antibody -linked cytokine of any one of embodiments 12-16, wherein the immunostimulatory cytokine is selected from IL-12, IL-18, and IL-23.

Embodiment 18: The antibody-linked cytokine of any one of embodiments 15-17, wherein the anti-scFv antibody or antigen-binding fragment comprises a first polypeptide comprising the heavy chain and a second polypeptide comprising the light chain, and further wherein the N- terminus of the immunostimulatory cytokine is linked to the C-terminus of the first polypeptide comprising the heavy chain.

Embodiment 19: The antibody-linked cytokine of embodiment 18, wherein the immunostimulatory cytokine is linked to the first polypeptide comprising the heavy chain via (i) a (G₄S)I linker or (ii) a poly-alanine linker comprising two or more consecutive alanine residues.

Embodiment 20: The antibody -linked cytokine of embodiment 19, wherein the poly-alanine linker consists of two alanine residues.

Embodiment 21 : The antibody-linked cytokine of any one of embodiments 12-20, wherein the immunostimulatory cytokine is IL-12.

Embodiment 22: The antibody-linked cytokine of embodiment 21, wherein the IL-12 is engineered to be expressed as a single polypeptide chain.

Embodiment 23: The antibody-linked cytokine of embodiment 22, wherein the IL-12 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19.

Embodiment 24: The antibody -linked cytokine of embodiment 18, wherein the first polypeptide comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11, a polyalanine linker, and single chain IL-12 comprising the amino acid sequence set forth in SEQ ID NO: 19, and the second polypeptide comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 13.

Embodiment 25: The antibody -linked cytokine of any one of embodiments 12-20, wherein the immunostimulatory cytokine is IL- 18.

Embodiment 26: The antibody-linked cytokine of embodiment 25, wherein the IL-18 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21.

Embodiment 27: The antibody -linked cytokine of embodiment 18, wherein the first polypeptide comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11, a polyalanine linker, and IL- 18 comprising the amino acid sequence set forth in SEQ ID NO: 21, and the second polypeptide comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 13.

Embodiment 28: The antibody -linked cytokine of any one of embodiments 12-20, wherein the immunostimulatory cytokine is IL-23.

Embodiment 29: The antibody-linked cytokine of embodiment 28, wherein the IL-23 is engineered to be expressed as a single polypeptide chain.

Embodiment 30: The antibody-linked cytokine of embodiment 29, wherein the IL-23 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23. Embodiment 31 : The antibody -linked cytokine of embodiment 18, wherein the first polypeptide comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11, a polyalanine linker, and single chain IL-23 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the second polypeptide comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 13.

Embodiment 32: The antibody-linked cytokine of any one of embodiments 15-31, wherein the anti-scFv antibody or antigen-binding fragment further comprises a heavy chain signal peptide and/or a light chain signal peptide, optionally wherein the heavy chain signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15 and/or the light chain signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17.

Embodiment 33: The antibody-linked cytokine of any one of embodiments 12-32, wherein the scFv of the CAR targets a tumor antigen, preferably wherein the tumor antigen is selected from the group consisting of alpha feto-protein (AFP)/HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD13, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, claudin 6, c-Met, DLL3, DR5, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa)/folate binding protein (FBP), folate receptor beta (FRb), follicle-stimulating hormone receptor (FSHR), GD-2, Glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, interleukin 13 receptor subunit alpha (IL3Ra), interleukin 13 receptor subunit alpha 2 (IL13Ra2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4/FAP, NKG2D-Ligands (MIC -A, MIC-B, and the ULBPs 1 to 6), Mullerian inhibiting substance type 2 receptor (MISIIR), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), P16, PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), R0R1, R0R2, TAG72, TIM-3, TM4SF1, Tn- glycoform of MUC1 (TnMUCl), TROP2, Glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRa4; GFRalpha4), VEGFR2, and any combination thereof.

Embodiment 34: The antibody-linked cytokine of any one of embodiments 12-33, wherein the scFv of the CAR targets TnMUCl .

Embodiment 35: The antibody-linked cytokine of any one of embodiments 12-34, wherein the intracellular domain of the CAR comprises a costimulatory domain and an intracellular signaling domain.

Embodiment 36: The antibody-linked cytokine of embodiment 35, wherein one or more of the following applies:

Embodiment 37: The antibody -linked cytokine of any one of embodiments 12-36, for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the antibody linked cytokine to the subject, and further wherein the administering is performed before, simultaneously with, or after infusion of the subject with a population of modified immune cells which express the CAR.

Embodiment 38: A nucleic acid molecule comprising a nucleotide sequence encoding the antibody-linked cytokine of any one of embodiments 12-36, optionally wherein the nucleotide sequence comprises the amino acid sequence set forth in SEQ ID NO: 26, the amino acid sequence set forth in SEQ ID NO: 28, or the amino acid sequence set forth in SEQ ID NO: 30, or the nucleic acid molecule comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30.

Embodiment 39: The nucleic acid molecule of embodiment 38, wherein the nucleotide sequence encoding the antibody -linked cytokine further encodes a ribosome slip sequence, optionally wherein the ribosome slip sequence is selected from the group consisting of P2A, T2A, E2A, F2A, and variants thereof.

Embodiment 40: A vector comprising the nucleic acid molecule of embodiment 38 or embodiment 39.

Embodiment 41 : A cell comprising the nucleic acid molecule of embodiment 38 or embodiment 39 or the vector of embodiment 40.

Embodiment 42: A lipid nanoparticle (LNP), wherein the LNP comprises:

(b) at least one ionizable lipid; and

Embodiment 43 : The LNP of embodiment 42, wherein the at least one immunostimulatory cytokine comprises any one or more of IL- 12, IL- 18, and IL-23.

Embodiment 44: The LNP of embodiment 42 or embodiment 43, wherein the at least one immunostimulatory cytokine comprises a single polypeptide chain or is engineered to be expressed as a single polypeptide chain.

Embodiment 45: The LNP of any one of embodiments 42-44, wherein the at least one immunostimulatory cytokine comprises or consists of IL-12.

Embodiment 46: The LNP of embodiment 45, wherein the IL-12 is engineered to be expressed as a single polypeptide chain.

Embodiment 47: The LNP of embodiment 46, wherein the IL-12 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19.

Embodiment 48: The LNP of any one of embodiments 42-44, wherein the at least one immunostimulatory cytokine comprises or consists of IL- 18. Embodiment 49: The LNP of embodiment 48, wherein the IL-18 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21.

Embodiment 50: The LNP of any one of embodiments 42-44, wherein the at least one immunostimulatory cytokine comprises or consists of IL-23.

Embodiment 51 : The LNP of embodiment 50, wherein the IL-23 is engineered to be expressed as a single polypeptide chain.

Embodiment 52: The LNP of embodiment 51, wherein the IL-23 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23.

Embodiment 53: The LNP of any one of embodiments 42-52, wherein the at least one nucleoside-modified RNA is messenger RNA (mRNA).

Embodiment 54: The LNP of any one of embodiments 42-53, wherein the at least one nucleoside-modified RNA comprises pseudouridine and/or 1-methyl-pseudouridine.

Embodiment 55: The LNP of any one of embodiments 42-54, wherein the at least one nucleoside-modified RNA is in vitro transcribed (IVT) RNA.

Embodiment 56: The LNP of any one of embodiments 42-55, wherein the at least one ionizable lipid is a cationic lipid. Embodiment 57: The LNP of any one of embodiments 42-56, wherein the anti-scFv antibody or antigen-binding fragment comprises a heavy chain variable region (VH) comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and a light chain variable region (VL) comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein:

Embodiment 58: The LNP of embodiment 57, wherein:

Embodiment 59: The LNP of embodiment 57 or embodiment 58, wherein the anti-scFv antibody or antigen-binding fragment further comprises a heavy chain and a light chain, wherein the heavy chain comprises the VH and the light chain comprises the VL.

Embodiment 60: The LNP of embodiment 59, wherein:

(i) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11; and/or (ii) the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13

Embodiment 61 : The LNP of any one of embodiments 57-60, wherein the anti-scFv antibody or antigen-binding fragment further comprises a heavy chain signal peptide and/or a light chain signal peptide, optionally wherein the heavy chain signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15 and/or the light chain signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17.

Embodiment 62: The LNP of any one of embodiments 42-61, wherein the scFv of the CAR targets a tumor antigen, preferably wherein the tumor antigen is selected from the group consisting of alpha feto-protein (AFP)/HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD13, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, claudin 6, c-Met, DLL3, DR5, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa)/folate binding protein (FBP), folate receptor beta (FRb), follicle-stimulating hormone receptor (FSHR), GD-2, Glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, interleukin 13 receptor subunit alpha (IL3Ra), interleukin 13 receptor subunit alpha 2 (IL13Ra2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4/FAP, NKG2D-Ligands (MIC -A, MIC-B, and the ULBPs 1 to 6), Mullerian inhibiting substance type 2 receptor (MISIIR), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), P16, PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), ROR1, ROR2, TAG72, TIM-3, TM4SF1, Tn- glycoform of MUC1 (TnMUCl), TROP2, Glycosyl-phosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRa4; GFRalpha4), VEGFR2, and any combination thereof.

Embodiment 63: The LNP of any one of embodiments 42-62, wherein the scFv of the CAR targets TnMUCl .

Embodiment 64: The LNP of any one of embodiments 42-63, wherein the intracellular domain of the CAR comprises a costimulatory domain and an intracellular signaling domain.

Embodiment 65: The LNP of embodiment 64, wherein one or more of the following applies:

Embodiment 66: The LNP of any one of embodiments 42-65, for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the LNP to the subject, and further wherein the administering is performed before, simultaneously with, or after infusion of the subject with a population of modified immune cells which express the CAR.

Embodiment 67: A pharmaceutical composition comprising the antibody -linked cytokine of any one of embodiments 12-36, or the LNP of any one of embodiments 42-65, and at least one pharmaceutically acceptable carrier, diluent, and/or excipient.

Embodiment 68: The pharmaceutical composition of embodiment 67, for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the pharmaceutical composition to the subject, and further wherein the administering is performed before, simultaneously with, or after infusion of the subject with a population of modified immune cells which express the CAR.

Embodiment 69: A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody-linked cytokine of any one of embodiments 12-36 or a therapeutically effective amount of the LNP of any one of embodiments 42-65, wherein the administering is performed before, simultaneously with, or after infusion of the subject with a population of modified immune cells which express the CAR.

Embodiment 70: The method of embodiment 69, wherein each cell of the population of modified immune cells is a T cell, an autologous cell, a human cell, or any combination thereof.

Embodiment 71 : The method of embodiment 69 or embodiment 70, wherein the administering comprises subcutaneous injection, intraperitoneal injection, intradermal injection, intravenous injection, intramuscular injection, intrastemal injection, or infusion techniques.

Embodiment 72: The method of any one of embodiments 69-71, wherein the administering comprises administering a first dose.

Embodiment 73: The method of embodiment 72, wherein the administering further comprises administering one or more subsequent doses. Embodiment 74: The method of any one of embodiments 69-73, wherein the cancer is selected from breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and thyroid cancer.

Embodiment 75: The method of any one of embodiments 69-74, wherein the subject is a human.

Embodiment 76: A recombinant IL-23, wherein the recombinant IL-23 is engineered to be expressed as a single polypeptide chain.

Embodiment 77: The recombinant IL-23 of embodiment 76, wherein the recombinant IL-23 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23, or wherein the recombinant IL-23 comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23.

The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated herein by reference in their entirety. While this invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of this invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims are intended to be construed to include all such embodiments and equivalent variations.

Claims

CLAIMS What is claimed:

1. An anti-single-chain variable fragment (scFv) antibody or antigen-binding fragment, wherein the anti-scFv antibody or antigen-binding fragment is capable of specifically binding to a linker peptide of an scFv, wherein the linker peptide links a variable light chain (VL) of the scFv to a variable heavy chain (VH) of the scFv, and further wherein the scFv is an scFv of a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain comprising the scFv, a transmembrane domain, and an intracellular domain.

2. The anti-scFv antibody or antigen-binding fragment of claim 1, comprising a heavy chain variable region (VH) comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and a light chain variable region (VL) comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein:

(iii) the HCDR1 comprises or consists of the amino acid sequence GFTFSSYG (SEQ ID NO: 79), the HCDR2 comprises or consists of the amino acid sequence ISSGGSYT (SEQ ID NO: 80), the HCDR3 comprises of consists of the amino acid sequence ARQDYGSPFAY (SEQ ID NO: 81), the LCDR1 comprises or consists of the amino acid sequence QDINKY (SEQ ID NO: 82), the LCDR2 comprises or consists of the amino acid sequence YTS (SEQ ID NO: 83), and the LCDR3 comprises or consists of the amino acid sequence LQYDNLLWT (SEQ ID NO: 84). The anti-scFv antibody or antigen-binding fragment of claim 1 or claim 2, wherein:

(iii) the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 86 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 88. The anti-scFv antibody or antigen-binding fragment of any one of claims 1-4, further comprising a heavy chain and a light chain, wherein the heavy chain comprises the VH and the light chain comprises the VL. The anti-scFv antibody or antigen-binding fragment of any one of claims 1-4, wherein the scFv of the CAR targets a tumor antigen, preferably wherein the tumor antigen is selected from the group consisting of alpha feto-protein (AFP)/HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD 13, CD 19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, claudin 6, c-Met, DLL3, DR5, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa)Zfolate binding protein (FBP), folate receptor beta (FRb), follicle-stimulating hormone receptor (FSHR), GD-2, Glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, interleukin 13 receptor subunit alpha (IL3Ra), interleukin 13 receptor subunit alpha 2 (IL13Ra2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE- A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4/FAP, NKG2D-Ligands (MIC -A, MIC-B, and the ULBPs 1 to 6), Mullerian inhibiting substance type 2 receptor (MISIIR), New York esophageal squamous cell carcinoma- 1 (NY-ESO-1), Pl 6, PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), R0R1, R0R2, TAG72, TIM-3, TM4SF1, Tn-glycoform ofMUCl (TnMUCl), TR0P2, Glycosylphosphatidylinositol (GPI)-linked GDNF family a-receptor 4 (GFRa4; GFRalpha4), VEGFR2, and any combination thereof. The anti-scFv antibody or antigen-binding fragment of any one of claims 1-5, wherein the scFv of the CAR targets TnMUC 1. The anti-scFv antibody or antigen-binding fragment of any one of claims 1-7, wherein the intracellular domain of the CAR comprises a costimulatory domain and an intracellular signaling domain. The anti-scFv antibody or antigen-binding fragment of claim 7, wherein one or more of the following applies:

(vi) the intracellular domain of the CAR comprises a costimulatory domain of 4-1BB (CD 137) and an intracellular signaling domain of CD3 zeta. A nucleic acid molecule comprising a nucleotide sequence encoding the anti-scFv antibody or antigen-binding fragment of any one of claims 1-8, optionally wherein the nucleotide sequence further encodes a ribosome slip sequence, optionally wherein the ribosome slip sequence is selected from the group consisting of P2A, T2A, E2A, F2A, and variants thereof. A vector comprising the nucleic acid molecule of claim 9. A cell comprising the nucleic acid molecule of claim 9 or the vector of claim 10. An antibody-linked cytokine, comprising an immunostimulatory cytokine linked to an anti-scFv antibody or antigen-binding fragment, wherein the anti-scFv antibody or antigen-binding fragment is capable of specifically binding to a linker peptide of an scFv, wherein the linker peptide links a variable light chain (VL) of the scFv to a variable heavy chain (VH) of the scFv, and further wherein the scFv is an scFv of a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain comprising the scFv, a transmembrane domain, and an intracellular domain. The antibody-linked cytokine of any claim 12, wherein the anti-scFv antibody or antigenbinding fragment comprises a heavy chain variable region (VH) comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and a light chain variable region (VL) comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein:

(iv) the HCDR1 comprises or consists of the amino acid sequence KFSFNKKYYMC (SEQ ID NO: 1), the HCDR2 comprises or consists of the amino acid sequence WIGCVDTGDAFIGY (SEQ ID NO: 2), the HCDR3 comprises of consists of the amino acid sequence RGVYPINTGYYYFDL (SEQ ID NO: 3), the LCDR1 comprises or consists of the amino acid sequence EDITNSLA (SEQ ID NO: 4), the LCDR2 comprises or consists of the amino acid sequence NLLIYRASTLAS (SEQ ID NO: 5), and the LCDR3 comprises or consists of the amino acid sequence QQGYSSTNVDNI (SEQ ID NO: 6). The antibody-linked cytokine of claim 13, wherein:

(iv) the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9. The antibody-linked cytokine of claim 14, wherein the anti-scFv antibody or antigenbinding fragment further comprises a heavy chain and a light chain, wherein the heavy chain comprises the VH and the light chain comprises the VL. The antibody-linked cytokine of any one of claims 12-15, wherein the immunostimulatory cytokine comprises a single polypeptide chain or is engineered to be expressed as a single polypeptide chain. The antibody-linked cytokine of any one of claims 12-16, wherein the immunostimulatory cytokine is selected from IL-12, IL-18, and IL-23. The antibody-linked cytokine of any one of claims 15-17, wherein the anti-scFv antibody or antigen-binding fragment comprises a first polypeptide comprising the heavy chain and a second polypeptide comprising the light chain, and further wherein the N-terminus of the immunostimulatory cytokine is linked to the C-terminus of the first polypeptide comprising the heavy chain. The antibody-linked cytokine of claim 18, wherein the immunostimulatory cytokine is linked to the first polypeptide comprising the heavy chain via (i) a (G4S)I linker or (ii) a poly-alanine linker comprising two or more consecutive alanine residues. The antibody-linked cytokine of claim 19, wherein the poly-alanine linker consists of two alanine residues. The antibody-linked cytokine of any one of claims 12-20, wherein the immunostimulatory cytokine is IL- 12. The antibody-linked cytokine of claim 21, wherein the IL-12 is engineered to be expressed as a single polypeptide chain. The antibody-linked cytokine of claim 22, wherein the IL-12 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19. The antibody-linked cytokine of claim 18, wherein the first polypeptide comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11, a polyalanine linker, and single chain IL-12 comprising the amino acid sequence set forth in SEQ ID NO: 19, and the second polypeptide comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 13. The antibody-linked cytokine of any one of claims 12-20, wherein the immunostimulatory cytokine is IL-18. The antibody-linked cytokine of claim 25, wherein the IL-18 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21. The antibody-linked cytokine of claim 18, wherein the first polypeptide comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11, a polyalanine linker, and IL- 18 comprising the amino acid sequence set forth in SEQ ID NO: 21, and the second polypeptide comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 13. The antibody-linked cytokine of any one of claims 12-20, wherein the immunostimulatory cytokine is IL-23. The antibody-linked cytokine of claim 28, wherein the IL-23 is engineered to be expressed as a single polypeptide chain. The antibody-linked cytokine of claim 29, wherein the IL-23 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23. The antibody-linked cytokine of claim 18, wherein the first polypeptide comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 11, a polyalanine linker, and single chain IL-23 comprising the amino acid sequence set forth in SEQ ID NO: 23, and the second polypeptide comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 13. The antibody-linked cytokine of any one of claims 15-31, wherein the anti-scFv antibody or antigen-binding fragment further comprises a heavy chain signal peptide and/or a light chain signal peptide, optionally wherein the heavy chain signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15 and/or the light chain signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17. The antibody-linked cytokine of any one of claims 12-32, wherein the scFv of the CAR targets a tumor antigen, preferably wherein the tumor antigen is selected from the group consisting of alpha feto-protein (AFP)/HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD13, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, claudin 6, c-Met, DLL3, DR5, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa)/folate binding protein (FBP), folate receptor beta (FRb), follicle-stimulating hormone receptor (FSHR), GD-2, Glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, interleukin 13 receptor subunit alpha (IL3Ra), interleukin 13 receptor subunit alpha 2 (IL13Ra2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4/FAP, NKG2D-Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), Mullerian inhibiting substance type 2 receptor (MISIIR), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), P16, PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), R0R1, R0R2, TAG72, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUCl), TROP2, Glycosyl-phosphatidylinositol (GPI)- linked GDNF family a-receptor 4 (GFRa4; GFRalpha4), VEGFR2, and any combination thereof. The antibody-linked cytokine of any one of claims 12-33, wherein the scFv of the CAR targets TnMUCl. The antibody-linked cytokine of any one of claims 12-34, wherein the intracellular domain of the CAR comprises a costimulatory domain and an intracellular signaling domain. The antibody-linked cytokine of claim 35, wherein one or more of the following applies:

(vi) the intracellular domain of the CAR comprises a costimulatory domain of 4-1BB (CD 137) and an intracellular signaling domain of CD3 zeta. The antibody-linked cytokine of any one of claims 12-36, for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the antibody linked cytokine to the subject, and further wherein the administering is performed before, simultaneously with, or after infusion of the subject with a population of modified immune cells which express the CAR. A nucleic acid molecule comprising a nucleotide sequence encoding the antibody-linked cytokine of any one of claims 12-36, optionally wherein the nucleotide sequence comprises the amino acid sequence set forth in SEQ ID NO: 26, the amino acid sequence set forth in SEQ ID NO: 28, or the amino acid sequence set forth in SEQ ID NO: 30, or the nucleic acid molecule comprises a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30. The nucleic acid molecule of claim 38, wherein the nucleotide sequence encoding the antibody-linked cytokine further encodes a ribosome slip sequence, optionally wherein the ribosome slip sequence is selected from the group consisting of P2A, T2A, E2A, F2A, and variants thereof. A vector comprising the nucleic acid molecule of claim 38 or claim 39. A cell comprising the nucleic acid molecule of claim 38 or claim 39 or the vector of claim 40. A lipid nanoparticle (LNP), wherein the LNP comprises:

(b) at least one ionizable lipid; and

(c) an anti-scFv antibody or antigen-binding fragment, wherein the anti-scFv antibody or antigen-binding fragment is capable of specifically binding to a linker peptide of an scFv, wherein the linker peptide links a variable light chain (VL) of the scFv to a variable heavy chain (VH) of the scFv, and further wherein the scFv is an scFv of a chimeric antigen receptor (CAR), wherein the CAR comprises an extracellular domain comprising the scFv, a transmembrane domain, and an intracellular domain; wherein the at least one ionizable lipid at least partially encapsulates the at least one nucleoside-modified RNA; and further wherein the anti-scFv antibody or antigen-binding fragment is linked to the surface of the LNP. The LNP of claim 42, wherein the at least one immunostimulatory cytokine comprises any one or more of IL- 12, IL- 18, and IL-23. The LNP of claim 42 or claim 43, wherein the at least one immunostimulatory cytokine comprises a single polypeptide chain or is engineered to be expressed as a single polypeptide chain. The LNP of any one of claims 42-44, wherein the at least one immunostimulatory cytokine comprises or consists of IL- 12. The LNP of claim 45, wherein the IL-12 is engineered to be expressed as a single polypeptide chain. The LNP of claim 46, wherein the IL-12 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 19 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 19. The LNP of any one of claims 42-44, wherein the at least one immunostimulatory cytokine comprises or consists of IL-18. The LNP of claim 48, wherein the IL-18 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 21 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 21. The LNP of any one of claims 42-44, wherein the at least one immunostimulatory cytokine comprises or consists of IL-23. The LNP of claim 50, wherein the IL-23 is engineered to be expressed as a single polypeptide chain. The LNP of claim 51, wherein the IL-23 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23. The LNP of any one of claims 42-52, wherein the at least one nucleoside-modified RNA is messenger RNA (mRNA). The LNP of any one of claims 42-53, wherein the at least one nucleoside-modified RNA comprises pseudouridine and/or 1-methyl-pseudouridine. The LNP of any one of claims 42-54, wherein the at least one nucleoside-modified RNA is in vitro transcribed (IVT) RNA. The LNP of any one of claims 42-55, wherein the at least one ionizable lipid is a cationic lipid. The LNP of any one of claims 42-56, wherein the anti-scFv antibody or antigen-binding fragment comprises a heavy chain variable region (VH) comprising three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) and a light chain variable region (VL) comprising three light chain complementarity determining regions (LCDR1, LCDR2, and LCDR3), wherein:

(iv) the HCDR1 comprises or consists of the amino acid sequence KFSFNKKYYMC (SEQ ID NO: 1), the HCDR2 comprises or consists of the amino acid sequence WIGCVDTGDAFIGY (SEQ ID NO: 2), the HCDR3 comprises of consists of the amino acid sequence RGVYPINTGYYYFDL (SEQ ID NO: 3), the LCDR1 comprises or consists of the amino acid sequence EDITNSLA (SEQ ID NO: 4), the LCDR2 comprises or consists of the amino acid sequence NLLIYRASTLAS (SEQ ID NO: 5), and the LCDR3 comprises or consists of the amino acid sequence QQGYSSTNVDNI (SEQ ID NO: 6). The LNP of claim 57, wherein:

(iv) the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 7 and/or the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 9. The LNP of claim 57 or claim 58, wherein the anti-scFv antibody or antigen-binding fragment further comprises a heavy chain and a light chain, wherein the heavy chain comprises the VH and the light chain comprises the VL. The LNP of claim 59, wherein:

(i) the heavy chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 11 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 11 ; and/or

(ii) the light chain comprises or consists of the amino acid sequence set forth in SEQ ID NO: 13 or comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 13. The LNP of any one of claims 57-60, wherein the anti-scFv antibody or antigen-binding fragment further comprises a heavy chain signal peptide and/or a light chain signal peptide, optionally wherein the heavy chain signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 15 and/or the light chain signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 17. The LNP of any one of claims 42-61, wherein the scFv of the CAR targets a tumor antigen, preferably wherein the tumor antigen is selected from the group consisting of alpha feto-protein (AFP)/HLA-A2, AXL, B7-H3, BCMA, CA-1X, CD2, CD3, CD4, CD5, CD7, CD8, CD13, CD19, CD20, CD22, CD30, CD33, CD38, CD44v6, CD70, CD79a, CD79b, CD80, CD86, CD117, CD123, CD133, CD147, CD171, CD276, CEA, claudin 18.2, claudin 6, c-Met, DLL3, DR5, epidermal growth factor receptor (EGFR), EGFRvIII, EpCAM, EphA2, fibroblast activation protein (FAP), folate receptor alpha (FRa)/folate binding protein (FBP), folate receptor beta (FRb), follicle-stimulating hormone receptor (FSHR), GD-2, Glycolipid F77, glypican 2 (GPC2), glypican-3 (GPC3), HER2, HLA-A2, ICAM1, interleukin 13 receptor subunit alpha (IL3Ra), interleukin 13 receptor subunit alpha 2 (IL13Ra2), LAGE-1, Lewis Y, LMP1 (EBV), MAGE-A1, MAGE-A3, MAGE-A4, Melan A, mesothelin, MG7 (glycosylated CEA), MMP, MUC1, Nectin4/FAP, NKG2D-Ligands (MIC-A, MIC-B, and the ULBPs 1 to 6), Mullerian inhibiting substance type 2 receptor (MISIIR), New York esophageal squamous cell carcinoma-1 (NY-ESO-1), P16, PD-L1, prostate stem cell antigen (PSCA), prostate specific membrane antigen (PSMA), R0R1, ROR2, TAG72, TIM-3, TM4SF1, Tn-glycoform of MUC1 (TnMUCl), TROP2, Glycosyl-phosphatidylinositol (GPI)- linked GDNF family a-receptor 4 (GFRa4; GFRalpha4), VEGFR2, and any combination thereof. The LNP of any one of claims 42-62, wherein the scFv of the CAR targets TnMUCl . The LNP of any one of claims 42-63, wherein the intracellular domain of the CAR comprises a costimulatory domain and an intracellular signaling domain. The LNP of claim 64, wherein one or more of the following applies:

(vi) the intracellular domain of the CAR comprises a costimulatory domain of 4-1BB (CD 137) and an intracellular signaling domain of CD3 zeta. The LNP of any one of claims 42-65, for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the LNP to the subject, and further wherein the administering is performed before, simultaneously with, or after infusion of the subject with a population of modified immune cells which express the CAR. A pharmaceutical composition comprising the antibody-linked cytokine of any one of claims 12-36, or the LNP of any one of claims 42-65, and at least one pharmaceutically acceptable carrier, diluent, and/or excipient. The pharmaceutical composition of claim 67, for use in a method of treating cancer in a subject in need thereof, wherein the method comprises administering a therapeutically effective amount of the pharmaceutical composition to the subject, and further wherein the administering is performed before, simultaneously with, or after infusion of the subject with a population of modified immune cells which express the CAR. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody-linked cytokine of any one of claims 12-36 or a therapeutically effective amount of the LNP of any one of claims 42-65, wherein the administering is performed before, simultaneously with, or after infusion of the subject with a population of modified immune cells which express the CAR. The method of claim 69, wherein each cell of the population of modified immune cells is a T cell, an autologous cell, a human cell, or any combination thereof. The method of claim 69 or claim 70, wherein the administering comprises subcutaneous injection, intraperitoneal injection, intradermal injection, intravenous injection, intramuscular injection, intrasternal injection, or infusion techniques. The method of any one of claims 69-71, wherein the administering comprises administering a first dose. The method of claim 72, wherein the administering further comprises administering one or more subsequent doses. The method of any one of claims 69-73, wherein the cancer is selected from breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, and thyroid cancer. The method of any one of claims 69-74, wherein the subject is a human. A recombinant IL-23, wherein the recombinant IL-23 is engineered to be expressed as a single polypeptide chain. The recombinant IL-23 of claim 76, wherein the recombinant IL-23 comprises or consists of the amino acid sequence set forth in SEQ ID NO: 23, or wherein the recombinant IL- 23 comprises a polypeptide comprising an amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 23.