WO2024030583A2

WO2024030583A2 - Novel constructs for chimeric antigen receptors and uses thereof

Info

Publication number: WO2024030583A2
Application number: PCT/US2023/029440
Authority: WO
Inventors: Nicholas G. Minutolo; Michael KLICHINSKY; Lauren Candice SHAW
Original assignee: Carisma Therapeutics Inc.
Priority date: 2022-08-03
Filing date: 2023-08-03
Publication date: 2024-02-08
Also published as: WO2024030583A3

Abstract

The present disclosure pertains to modified immune cells comprising one or more chimeric antigen receptors (CARs) and methods of using and making immune cells comprising one or more CARs.

Description

NOVEL CONSTRUCTS FOR CHIMERIC ANTIGEN RECEPTORS AND

USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S Provisional Application 63/394,828, filed .August 3, 2022, the contents of which are hereby incorporated by reference in their entirety.

BACKGROUND

[0002] Although immunotherapies have been investigated for many diseases and disorders, including cancer, Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), systemic amyloidosis, prion disease, cardiovascular disease, atherosclerosis, fibrosis, functional limitations that have been encountered still need to be addressed.

[0003] Therefore, a need exists for the development of new therapeutic modalities optimized to target specific antigens.

SUMMARY OF THE INVENTION

[0004] The present disclosure encompasses, among other things, compositions comprising modified immune cells (e.g., stem cells, macrophages, monocytes, and/or dendritic cells) comprising chimeric antigen receptors (CARs) and methods of producing the same. The present disclosure also encompasses, among other things, nucleic acid construct comprising one or more nucleic acid sequences encoding CARs described herein and methods of producing the same. The present disclosure provides, inter alia, CARs comprising specific intracellular domains (e g., one or more of aMyd88 intracellular domain or a portion thereof, CD40 intracellular domain or a portion thereof, or FcRy intracellular domain).

[0005] In some embodiments, modified immune cells described herein comprising or expressing CARs described herein exhibit, one or more of the following: (i) increased tumor killing (e.g., one or more of phagocytosis, lysis, apoptosis, or production of tumor killing cytokines (e.g., TNFa)), (ii) increased viability, (iii) increased expression of a CAR, (iv) increased expression of M/1 markers (e.g., one or both of CD80 or ( 1)86). (v) decreased expression of M2 markers (e.g., one or both of CD 163 or CD206), or (vi) reduced background cytokine release (e.g., one or more of TNFa, IL-6, or IL-8) upon stimulation, e.g., each relative to modified immune cells of the same type comprising a similar CAR (e.g., a CAR described herein, e.g., without one or more of a Myd88 intracellular domain or a portion thereof, CD40 intracellular domain or a portion thereof, or FcRy intracellular domain but with the other components of the comparator CAR, e.g., without any of aMyd88 intracellular domain or a portion thereof, CD40 intracellular domain or a portion thereof, or FcRy intracellular domain, but with the other components of the comparator CAR).

[0006] In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, and/or dendritic cells) comprising or expressing CARs described herein (e.g., comprising one or more of a Myd88 intracellular domain or a portion thereof, CD40 intracellular domain or a portion thereof, or FcRy intracellular domain) do not exhibit killing (e.g,, by phagocytosis, lysis, apoptosis, or production of tumor killing cytokines (e.g., TNFa)) of tumor cells that do not express a target antigen relative to modified immune cells of the same type comprising a similar CAR (e.g., a CAR described herein, e.g., without one or more of a Myd88 intracellular domain or a portion thereof CD40 intracellular domain or a portion thereof, or FcRy intracellular domain but with the other components of the comparator CAR, e.g., without any of a Myd88 intracellular domain or a portion thereof, CD40 intracellular domain or a portion thereof, or FcRy intracellular domain, but with the other components of the comparator CAR).

[0007] In one aspect, the disclosure provi des modified immune cell s comprising a chimeri c antigen receptor (CAR), wherein the CAR comprises: (a) one or more extracellular domains; (b) a transmembrane domain; and (c) one or more intracellular domains comprising one or both of: (i) a MyD88 intracellular domain or a portion thereof, or (ii) a CD40 intracellular domain or a portion thereof and wherein the modified immune cell is or comprises a macrophage, monocyte, dendritic cell, or stem cell.

[0008] In some embodiments, one or more extracellular domains comprise a scFv, VHH antibody, centyrin, or darpin. In some embodiments, a transmembrane domain comprises CD28, CD8a, CD40, MyD88, CD64, CD32a, CD32c, CD16a, CD3zeta, ICOS, Dectin-1, DNGR1, SLAMF7, TRI J, TLR2, TLR3, TRL4, TLR5, TLR6, TLR7, TLR8, or TLR9 transmembrane domain In some embodiments, one or more intracellular domains further comprise one or more of a: CD3-zeta, FcRy, CD40, CD64, CD32a, CD32c, CD 16a, CD89, TLR1, TLR2, TLR3, TLR4, TLR5, TLR.6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROSE RYK, TIE2, TRK, VEGFR, CD 19, CD20, 41BB, CD28, 0X40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, V/I/LxYxxL/V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPb, CD22, PIR-B, LILRB1, GCSFR (CD 114), RAGE, CD30, CD 160, DR3, Fnl4, HVEM, CD160, NGFR, RANK, TNFR2, TROY, XEDAR, TRIF, 41 BBL (TNFSF9), ( 1)27. OX40L, CD32b, CD l ib, ITGAM, SLAMF7, CD206, CD163, CD209, Dectin-2, IL1R, IL2R, IL3R, IL4R, IL5R, H.6R, IL7R, IL8R, IL9R, ILIOR, ILHR, II.J 2R, IL13R, ll. MR, IL15R, ILI7R, IFNaR, IFNgR, TNFR, CSF1R, CSF2R, DAP 10, CD36, Dectin-1 , ICOSL, or Syk intracellular domain or a portion of any of the foregoing. In some embodiments, one or more intracellular domains further comprise a CD3-zeta intracellular domain.

[0009] In some embodiments, a CAR further comprises one or more extracellular leader domains. In some embodiments, one or more extracellular leader domains comprise a CD8 extracellular leader domain. In some embodiments, a CAR further comprises one or more extracellular hinge domains. In some embodiments, one or more extracellular hinge domains comprise a CD28 extracellular hinge domain, a CD8a extracellular hinge domain, a DAP10 extracellular hinge domain, a DNGR-1 extracellular hinge domain, a Dectin-1 extracellular hinge domain, or an IgG4 extracellular hinge domain.

[0010] In some embodiments, a CAR comprises, from the one or more extracellular hinge domains to the one or more intracellular domains: (i) a CD28 extracellular hinge domain, CD28 transmembrane domain, CD40 intracellular domain, and CD3-zeta intracellular domain; (ii) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, and CD3-zeta intracellular domain; (iii) a CD28 extracellular hinge domain, CD28 transmembrane domain, CD40 intracellular domain, truncated Myd88 domain, and CD3-zeta intracellular domain; (iv) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, a CD40 intracellular domain, and CD3-zeta intracellular domain; (v) a CD28 extracellular hinge domain, CD28 transmembrane domain, and CD40 intracellular domain; (vi) a CD28 extracellular hinge domain, CD28 transmembrane domain, and truncated Myd88 domain; (vii) a CD28 extracellular hinge domain, CD28 transmembrane domain, CD40 intracellular domain, and truncated Myd88 domain; (viii) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, and CD40 intracellular domain; or (ix) a CD28 extracellular hinge domain, CD28 transmembrane domain, CD40 intracellular domain, CD3-zeta intracellular domain, and a truncated Myd88 domain.

[0011] In some embodiments, a CAR comprises or has: (a) an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs: 1-9, 45, 47, 49, 51, 53, 55, 57, 59, or 61; (b) an amino acid sequence that differs from any one of the amino acid sequences of SEQ ID NOs: 1-9, 45, 47, 49, 51 , 53, 55, 57, 59, or 61 by no more than five substitutions, additions, or deletions; or (c) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the amino acid sequences of SEQ ID NOs: 1 -9, 45, 47, 49, 51, 53, 55, 57, 59, or 61.

[0012 ] In some embodiments, a CAR comprises, from the one or more extracellular hinge domains to the one or more intracellular domains: (i) a CDS extracellular hinge domain, CD8 transmembrane domain, CD40 intracellular domain, and CD3-zeta intracellular domain, (ii) a CDS extracellular hinge domain, CDS transmembrane domain, truncated Myd88 domain, and CD3-zeta intracellular domain; (iii) a CD8 extracellular hinge domain, CD 8 transmembrane domain, CD40 intracellular domain, truncated Myd88 domain, and CD3-zeta intracellular domain; (iv) a CD8 extracellular hinge domain, CD8 transmembrane domain, truncated Myd88 domain, CD40 intracellular domain, and CD3-zeta intracellular domain; (v) a CD8 extracellular hinge domain, CD8 transmembrane domain, and CD40 intracellular domain; (vi) a CD8 extracellular hinge domain, CD8 transmembrane domain, and truncated Myd88 domain; (vii) a CD8 extracellular hinge domain, CD8 transmembrane domain, CD40 intracellular domain and truncated Myd88 domain; (viii) a CD8 extracellular hinge domain, CD8 transmembrane domain, truncated Myd88 domain and CD40 intracellular domain; or (ix) a CD8 extracellular hinge domain, CD8 transmembrane domain, CD40 intracellular domain, CD3-zeta intracellular domain, and truncated Myd88 domain.

[0013] In some embodiments, a CAR comprises or has (a) an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs: 10-18; (b) an amino acid sequence that differs from any one of the amino acid sequences of SEQ ID NOs: 10-18 by no more than five substitutions, additions, or deletions; or (c) an amino acid sequence that is at least 80%, 85%, 90%. 95%, 98% or 99% identical to any one of the amino acid sequences of SEQ ID NOs: 10-18,

[0014] In another aspect, the disclosure provides pharmaceutical compositions comprising a modified immune cell of any aspect or embodiment described herein. In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0015] In another aspect, the disclosure provides nucleic acid constructs comprising one or more nucleic acid sequences encoding: (a) one or more extracellular domains; (b) a transmembrane domain; and (c) one or more intracellular domains comprising one or both of: (i) a MyD88 intracellular domain or a portion thereof, or (ii) a CD40 intracellular domain or a portion thereof, and wherein the nucleic acid construct encodes a chimeric antigen receptor (CAR) comprising (a) through (c) In some embodiments, a nucleic acid construct further comprises one or more nucleic acid sequences encoding: (d) one or more extracellular leader domains, (e) one or more extracellular hinge domains, or (f) one or more cleavage peptides. In some embodiments, a cleavage peptide is a P2A, F2A, E2A or T2A peptide.

[0016] In some embodiments, a nucleic acid construct encodes, from the one or more extracellular hinge domains to the one or more intracellular domains: (i) a CD28 extracellular hinge domain, CD28 transmembrane domain, CD40 intracellular domain, and CDS -zeta intracellular domain; (ii) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, and CD3-zeta intracellular domain; (iii) a CD28 extracellular hinge domain, CD28 transmembrane domain, CD40 intracellular domain, truncated Myd88 domain, and CD3-zeta intracellular domain; (iv) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, a CD40 intracellular domain, and CD3-zeta intracellular domain; (v) a CD28 extracellular hinge domain, CD28 transmembrane domain, and CD40 intracellular domain, (vi) a CD28 extracellular hinge domain, CD28 transmembrane domain, and truncated Myd88 domain; (vii) a CD28 extracellular hinge domain, CD28 transmembrane domain, CD40 intracellular domain, and truncated Myd88 domain; (viii) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, and CD40 intracellular domain, or (ix) a CD28 extracellular hinge domain, CD28 transmembrane domain, CD40 intracellular domain, CD3-zeta intracellular domain, and a truncated Myd88 domain. [0017] In some embodiments, a nucleic acid construct comprises or has: (a) a nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs: 19-27, 63, 65, 67, 69, 71, 73, 75, 77, or 79; (b) a nucleotide sequence that differs from any one of the nucleotide sequences of SEQ ID NOs: 19-27, 63, 65, 67, 69, 71, 73, 75, 77, or 79 by no more than five substitutions, additions, or deletions; or (c) a nucleotide acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the nucleotide sequences of SEQ ID NOs: 19-27, 63, 65, 67, 69, 71, 73, 75, 77, or 79.

[0018] In some embodiments, a nucleic acid construct encodes, from the one or more extracellular hinge domains to the one or more intracellular domains: (i) a CD8 extracellular hinge domain, CD8 transmembrane domain, CD40 intracellular domain, and CD3-zeta intracellular domain; (ii) a CD8 extracellular hinge domain, CDS transmembrane domain, truncated Myd88 domain, and CD3-zeta intracellular domain, (iii) a CD8 extracellular hinge domain, CD8 transmembrane domain, CD40 intracellular domain, truncated Myd88 domain, and CD3-zeta intracellular domain; (iv) a CD8 extracellular hinge domain, CD8 transmembrane domain, truncated Myd88 domain, CD40 intracellular domain, and CD3-zeta intracellular domain; (v) a CDS extracellular hinge domain, CD8 transmembrane domain, and CD40 intracellular domain; (vi) a CD8 extracellular hinge domain, CD8 transmembrane domain, and truncated Myd88 domain; (vii) a CD8 extracellular hinge domain, CD8 transmembrane domain, CD40 intracellular domain and truncated Myd88 domain; (viii) a CDS extracellular hinge domain, CD8 transmembrane domain, truncated Myd88 domain and CD40 intracellular domain; or (ix) a CD8 extracellular hinge domain, CD8 transmembrane domain, CD40 intracellular domain, CD3-zeta intracellular domain, and truncated Myd88 domain.

[0019] In some embodiments, a nucleic acid construct comprises or has: (a) a nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs: 28-36; or (b) a nucleotide sequence that differs from any one of the nucleotide sequences of SEQ ID NOs: 28-36 by no more than five substitutions, additions, or deletions; or (c) a nucleotide acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the nucleotide sequences of SEQ ID NOs: 28-36. [0020] In another aspect, the disclosure provides pharmaceutical compositions comprising a nucleic acid construct of any aspect or embodiment described herein. In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0021] In another aspect, the disclosure provides methods of treating a disease or disorder in a subject, comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition of any aspect or embodiment described herein, wherein at least one sign or symptom of the disease or disorder is improved in the subject after administration.

[0022] In another aspect, the disclosure provides methods of modifying an immune cell, the method comprising: delivering to the immune cell a nucleic acid of any aspect or embodiment described herein, thereby producing a modified immune cell, wherein the modified immune cell comprises or is a macrophage, monocyte, dendritic cell, or stem cell.

[0023] In another aspect, the disclosure provides modified immune cells comprising a chimeric antigen receptor (CAR), wherein the CAR comprises: (a) one or more extracellular domains; (b) a CD8 or CD28 extracellular hinge domain, (c) a CD8 or CD28 transmembrane domain, and (d) one or more intracellular domains comprising an FcRy intracellular domain; and wherein the modified immune cell is or comprises a macrophage, monocyte, dendritic cell, or stem cell. In some embodiments, one or more intracellular domains further comprise one or both of: (i) a MyD88 intracellular domain or a portion thereof, or (ii) a CD40 intracellular domain or a portion thereof. In some embodiments, the one or more intracellular domains further comprise one or more of a: CD3-zeta, FcRy, CD40 CD64, CD32a, CD32c, CD 16a, CD89, TLR1 , TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROSE RYK, TIE2, TRK, VEGFR, CD 19, CD20, 4 IBB, CD28, GCSFR (CD114), RAGE, CD30, CD160, DR3, Fnl4, HVEM, CD160, NGFR, RANK, TNFR2, TROY, XEDAR, TRIP, 0X40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, V/L-'LxYxxL/V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPb, CD22, PIR-B, LILRB1, 41BBL (TNFSF9), CD27, OX40L, CD32b, CDl lb, ITGAM, SLAMF7, CD206, CD163, CD209, Dectin-2, IL1R, IL2R, IL3R, IL4R, IL5R, IL6R, IL7R, IL8R, IL9R, IL10R, IL HR, IL12R, IL13R, IL14R, IL15R, IL17R, IFNaR, IFNgR, TNFR, CSF1R, CSF2R, DAP10, CD36, Dectin-1, ICOSL, or Syk intracellular domain or a portion of any of the foregoing. [0024] In some embodiments, a CAR comprises, from the CD8 or CD28 extracellular hinge domain to the one or more intracellular domains: (i) a CD28 extracellular hinge domain, CD28 transmembrane domain, and FcRy intracellular domain; (ii) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, a CD40 intracellular domain, and FcRy intracellular domain; (iii) a CD28 extracellular hinge domain, ('1)28 transmembrane domain, truncated Myd88 domain, FcRy intracellular domain, and a CD40 intracellular domain; or (iv) a CD8 extracellular hinge domain, CDS transmembrane domain, truncated Myd88 domain, CD40 intracellular domain, and FcRy intracellular domain.

[0025 ] In some embodiments, a CAR comprises or has: (a) an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs: 37-40, 46, 48, 50, 52, 54, 56, 58, 60, 62, or 126- 143; or (b) an amino acid sequence that differs from any one of the amino acid sequences of SEQ ID NOs: 37-40, 46, 48, 50, 52, 54, 56, 58, 60, 62, or 126-143 by no more than five substitutions, additions, or deletions; or (c) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the amino acid sequences of SEQ ID NOs: 37-40, 46, 48, 50, 52, 54, 56, 58, 60, 62, or 126-143.

[0026] In another aspect, the disclosure provides nucleic acid constructs comprising one or more nucleic acid sequences encoding: (a) one or more extracellular domains; (b) a CD8 or CD28 extracellular hinge domain, (c) a CD8 or CD28 transmembrane domain, and (d) one or more intracellular domains comprising an FcRy intracellular domain: and wherein the nucleic acid construct encodes a chimeric antigen receptor (CAR) comprising (a) through (d).

[0027] In some embodiments, a nucleic acid construct further comprises one or more nucleic acid sequences encoding: (i) a MyD88 intracellular domain or a portion thereof, or (ii) a CD40 intracellular domain or a portion thereof.

[0028] In some embodiments, a nucleic acid construct encodes, from the CD8 or CD28 extracellular hinge domain to the one or more intracellular domains: (i) a CD28 extracellular hinge domain, CD28 transmembrane domain, and FcRy intracellular domain; (ii) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, a CD40 intracellular domain, and FcRy intracellular domain; (iii) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, FcRy intracellular domain, and a CD40 intracellular domain; or (iv) a CDS extracellular hinge domain, CDS transmembrane domain, truncated Myd88 domain, CD40 intracellular domain, and FcRy intracellular domain.

[0029] In some embodiments, a nucleic acid construct comprises or has: (a) a nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs: 41-44, 64, 66, 68, 70, 72, 74, 76, 78, 80, or 149-166; (b) a nucleotide sequence that differs from any one of the nucleotide sequences of SEQ ID NOs: 41-44, 64, 66, 68, 70, 72, 74, 76, 78, 80, or 149-166 by no more than five substitutions, additions, or deletions, or (c) a nucleotide acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the nucleotide sequences of SEQ ID NOs: 41-44, 64, 66, 68, 70, 72, 74, 76, 78, 80, or 149-166.

[0030] In another aspect, the disclosure provides modified immune cells comprising a chimeric antigen receptor (CAR), wherein a CAR comprises: (a) one or more extracellular domains, (b) a transmembrane domain, and (c) a DAP10 intracellular domain or a MyD88 intracellular domain or a portion thereof; and wherein the modified immune cell is or comprises a macrophage, monocyte, dendritic cell, or stem cell. In some embodiments, one or more intracellular domains comprise a CD40 intracellular domain or a portion thereof. In some embodiments, a transmembrane domain comprises or is a DAP 10 transmembrane domain. In some embodiments, a CAR further comprises a DAP 10 extracellular hinge domain.

[0031] In some embodiments, a CAR comprises, from a DAP10 extracellular hinge domain to one or more intracellular domains: (i) a DAP 10 extracellular hinge domain, DAP 10 transmembrane domain, DAP10 intracellular domain, truncated Myd88 domain, and CD40 intracellular domain, (ii) a DAP10 extracellular hinge domain, DAP10 transmembrane domain, DAP10 intracellular domain, CD40 intracellular domain, and truncated Myd88 domain, or (iii) a DAP10 extracellular hinge domain, DAP10 transmembrane domain, DAP10 intracellular domain, and truncated Myd88 domain.

[0032] In some embodiments, a CAR comprises or has: (a) an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs: 81-108; or (b) an amino acid sequence that differs from any one of the amino acid sequences of SEQ ID NOs: 81 -108 by no more than five substitutions, additions, or deletions; or (c) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the amino acid sequences of SEQ ID NOs: 81-108. [0033] In some embodiments, a nucleic acid construct comprises one or more nucleic acid sequences encoding: (a) one or more extracellular domains, (b) a transmembrane domain, and (c) one or more intracellular domains comprising one or both of: (i) a DAP10 intracellular domain or a portion thereof, or (ii) a MyD88 intracellular domain or a portion there of; and wherein the nucleic acid construct encodes a CAR comprising (a) through (c). In some embodiments, a nucleic acid construct encodes one or more intracellular domains further comprising a CD40 intracellular domain or a portion thereof. In some embodiments, a nucleic acid construct encodes a transmembrane domain that comprises or is a DAP 10 transmembrane domain. In some embodiments, a nucleic acid construct encodes one or more nucleic acid sequences encoding a DAP 10 extracellular hinge domain.

[0034] In some embodiments, a nucleic acid construct encodes, from a DAP 10 extracellular hinge domain to one or more intracellular domains: (i) a DAP10 extracellular hinge domain, DAP10 transmembrane domain, DAP10 intracellular domain, truncated Myd88 domain, and CD40 intracellular domain, (ii) a DAP10 extracellular hinge domain, DAP 10 transmembrane domain, DAP 10 intracellular domain, CD40 intracellular domain, and truncated Myd88 domain, or (iii) a DAP 10 extracellular hinge domain, DAP10 transmembrane domain, DAP10 intracellular domain, and truncated Myd88 domain.

[0035] In some embodiments, a nucleic acid construct comprises or has: (a) a nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs: 109-121, 147, 148, or 172-185: (b) a nucleotide sequence that differs from any one of the nucleotide sequences of SEQ ID NOs: 109-121, 147, 148, , or 172-185 by no more than five substitutions, additions, or deletions; or (c) a nucleotide acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the nucleotide sequences of SEQ ID NOs: 109-121, 147, 148, or 172-185.

[0036] In another aspect, the disclosure provides modified immune cells comprising a chimeric antigen receptor (CAR), wherein a CAR comprises: (a) one or more extracellular domains, (b) a transmembrane domain, and (c) one or more intracellular domains comprising one or both of: a CD64 intracellular domain or a portion thereof or a MyD88 intracellular domain or a portion thereof; and wherein the modified immune cell is or comprises a macrophage, monocyte, dendritic cell, or stem cell. In some embodiments, a transmembrane domain comprises a CD64 transmembrane domain or portion thereof. In some embodiments, a CAR further comprises a CD8 extracellular hinge domain. In some embodiments, a CAR comprises, from a CD8 extracellular hinge domain to one or more intracellular domains, a CDS extracellular hinge domain, CD64 transmembrane domain, CD64 intracellular domain, and truncated Myd88 domain.

[0037] In some embodiments, a CAR comprises or has: (a) an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs: 123-125; or (b) an amino acid sequence that differs from any one of the amino acid sequences of SEQ ID NOs: 123-125 by no more than five substitutions, additions, or deletions; or (c) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the amino acid sequences of SEQ ID NOs: 123-125.

[0038] In some embodiments, a nucleic acid construct comprises one or more nucleic acid sequences encoding: (a) one or more extracellular domains, (b) a transmembrane domain, (c) one or more intracellular domains comprising one or both of: (i) a CD64 intracellular domain or a portion thereof, or (ii) a MyD88 intracellular domain or a portion there of; and wherein the nucleic acid construct encodes a CAR comprising (a) through (c). In some embodiments, a nucleic acid construct encoding a transmembrane domain comprising a CD64 transmembrane domain or portion thereof. In some embodiments, a nucleic acid construct encodes one or more nucleic acid sequences encoding a CD8 extracellular hinge domain. In some embodiments, a nucleic acid construct encodes from a CD8 extracellular hinge domain to one or more intracellular domains, a CD8 extracellular hinge domain, CD64 transmembrane domain, CD64 intracellular domain, and truncated Myd88 domain

[0039] in some embodiments, a nucleic acid construct comprises or has: (a) a nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs: 144-146; (b) a nucleotide sequence that differs from any one of the nucleotide sequences of SEQ ID NOs: 144-146 by no more than five substitutions, additions, or deletions; or (c) a nucleotide acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the nucleotide sequences of SEQ ID NOs: 144-146.

[0040] In some embodiments, a MyD88 intracellular domain or a portion thereof comprises one or more amino acid substitutions chosen from E52A, R32A, R32K, Y58A, E52A/R32A/Y58A, L93P, S34Y, and R98C. In some embodiments, one or more amino acid substitutions comprises or is R32K. In some embodiments, a CAR described herein exhibits decreased tonic signaling relative to a similar CAR comprising a MyD88 intracellular domain or a portion thereof without the one or more amino acid substitutions. In some embodiments, an immune cell described herein exhibits increased tumor kilting ability relative to an immune cell of the same type comprising a similar CAR comprising a MyD88 intracellular domain or a portion thereof without the one or more amino acid substitutions.

[0041] In another aspect, the disclosure provides pharmaceutical compositions comprising a nucleic acid construct of any aspect or embodiment described herein. In some embodiments, a pharmaceutical composition comprises a pharmaceutically acceptable carrier.

[0042] In another aspect, the disclosure provides methods of treating a disease or disorder in a subject, the method comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition of any aspect or embodiment described herein, wherein at least one sign or symptom of the disease or disorder is improved in the subject after administration.

[0043] In another aspect, the disclosure provides methods of modifying an immune cell, the method comprising: delivering to the immune cell a nucleic acid of any aspect or embodiment described herein, thereby producing a modified immune cell, wherein the modified immune cell comprises or is a macrophage, monocyte, dendritic cell, or stem cell.

BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The drawings are for illustration purposes only, not for limitation.

[0045] Figure l is a schematic for generation of chimeric antigen receptor (CAR) expressing macrophages used for experiment and an exemplary CAR construct.

[0046] Figures 2A-C are graphs demonstrating the viability (FIG. 2A), CAR expression percentage (FIG. 2B), and CAR mean fluorescence intensity (MFI) (FIG. 2C) of macrophages transduced with constructs described herein (see Table 1 in Example 2).

[0047] Figures 3A-D are graphs showing tumor killing ability of CAR macrophages with constructs described herein (see Table 1 in Example 2).

[0048] Figures 4A-B are graphs showing cytokine release of CAR expressing macrophages with constructs described herein (see Table I in Example 2). [0049] Figures 5A-C are graphs demonstrating the viability (FIG. 5A), CAR expression percentage (FIG. 5B), and CAR MFI (FIG. 5C) of macrophages transduced with constructs described herein (see Table 2 in Example 4).

[0050] Figures 6A~D are graphs showing the expression of Ml (FIGS. 6A-B) and M2 markers (FIGS, 6C-D) of CAR macrophages transduced with constructs described herein (see Table 2 in Example 4).

[0051] Figures 7A-D are graphs showing cytokine release of macrophages expressing (TAR constructs comprising either of a CD28 hinge (H) and CD28 transmembrane (I'M) (FIGS.7A-B) or CD8 H and CD8 TM (FIGS.7C-D) (see Table 1 in Example 2 and Table 2 in Example 4).

[0052] Figures 8A-B are graphs showing null tumor killing ability of CAR macrophages comprising truncated Myd88 domain (ICD), CD40 ICD, and CD3-zeta for antigen negative tumor cells

[0053] Figures 9A-C are graphs showing cytokine release of CAR macrophages comprising truncated Myd88 ICD, CD40 ICD, and CD3-zeta.

[0054] Figures 10A-C are graphs demonstrating viability (FIG. 10 A), CAR expression percentage (FIG. 10B), and MFI (FIG. 10C) for CAR macrophages comprising FcRy ICD described herein (see Table 3 in Example 8).

[0055] Figures 11A-D are graphs demonstrating the expression of Ml (FIGS. 11 A-B) and M2 markers (FIGS. 11C-D) of CAR macrophages described herein (see Table 3 in Example 8).

[0056] Figures 12A-D are graphs showing tumor killing ability of CAR macrophages described herein (see Table 3 in Example 8).

[0057] Figures 13A-C are graphs showing cytokine release of CAR macrophages described herein (see Table 3 in Example 8).

[0058] Figure 14 is a schematic of exemplary' CAR constructs with Myd88 ICDs disclosed herein. [0059] Figure 15 is a graph showing exemplary NFKB activation in HEK cells expressing CAR constructs disclosed herein (see Tables 4-7 in Example 10).

[0060] Figures 16A-C are graphs showing cytokine release (TNFa (FIG. 16A), IL-6 (FIG. 16B), and IL-8 (FIG. 16C)) of CAR macrophages disclosed herein (see Tables 4-7 in Example 10)

[0061] Figures 17A-C are graphs showing cytokine release (TNFa (FIG. 17A), IL-6 (FIG. 17B), and IL-8 (FIG. 17C)) of CAR macrophages disclosed herein (see Table 4-7 in Example 10).

[0062] Figures 18A-C are graphs showing cytokine release (TNFa (FIG. 21 A), IL-6 (FIG. 2 IB), and IL-8 (FIG. 20C)) of CAR macrophages disclosed herein (see Tables 4-7 in Example 10)

[0063] Figures 19A-B are graphs demonstrating viability (FIG. 19A) and percentage of cells recovered (FIG. 19B) of macrophages transduced with constructs disclosed herein (see Table 4-7 in Example 10).

[0064] Figures 20A-B are graphs demonstrating CAR MFI and CAR percentage (FIG.

20A) and CAR expression (FIG. 20B) of macrophages transduced with constructs described here in (see Tables 4-7 in Example 10).

[0065] Figures 21A-B are graphs demonstrating expression of Ml (FIG. 21A) and M2 markers (FIG, 21B) of CAR macrophages described herein (see Tables 4-7 in Example 10),

[0066] Figures 22A-D are graphs showing tumor killing ability of CAR macrophages described herein (see Table 4-7 in Example 10).

[0067] Figures 23A-C are graphs showing tumor killing ability of CAR macrophages described herein (see Table 4-7 in Example 10).

[0068] Figures 24A-D are graphs showing tumor killing ability of CAR macrophages described herein (see Table 4-7 in Example 10)

[0069] Figures 25A-C are graphs showing tumor killing ability of CAR macrophages described herein (see Table 4-7 in Example 10). [0070] Figure 26 shows domains of exemplary DAP10 CAR constructs with Myd88 ICDs.

[0071] Figure 27 is a series of graphs showing viability and recovery percentages of macrophages expressing anti-HER2 CAR constructs comprising a DAP 10 domain and Myd88 ICD relative to control constructs and UTD macrophages, as measured by flow cytometry.

[0072] Figure 28 is a series of graphs showing CAR expression via histogram plot and quantification by percent expression and mean fluorescence intensity in macrophages expressing anti-HER2 CAR constructs comprising a D API 0 domain and Myd88 ICD relative to control constructs and UTD macrophages, as measured by flow cytometry.

[0073] Figure 29 is a series of graphs showing expression of Ml markers (CD80, CD86) and M2 markers (CD163, CD206) via quantification by mean fluorescence intensity in macrophages expressing anti-HER2 CAR constructs comprising a DAP 10 domain and Myd88 ICD relative to control constructs and UTD macrophages, as measured by flow cytometry,

[0074] Figure 30 is a series of graphs showing exemplary tumor cell death at different time points of AU565 cells when co-cultured at different ratios with macrophages expressing anti-HER2 CAR constructs comprising a DAP10 domain and Myd88 ICD relative to control constructs and UTD macrophages, as measured by GFP intensity from tumor cell expression.

[0075] Figure 31 is a graph showing exemplary’ tumor cell death of AU565 cells when co-cultured at different ratios with macrophages expressing anti-HER2 CAR constructs comprising a DAP 10 domain and Myd88 ICD relative to control constructs and UTD macrophages, as measured by GFP intensity from tumor cell expression.

[0076] Figure 32 is a series of graphs showing exemplary tumor cell death at different time points of Panel and MDA468 cells when co-cultured at 3: 1 E:T (effectortarget) with macrophages expressing anti-HER2 CAR constructs comprising a DAP 10 domain and Myd88 ICD relative to control constructs and UTD macrophages, as measured by GFP intensity from tumor cell expression.

[0077] Figure 33A-B is a series of graphs showing exemplary cytokine secretion in macrophages expressing anti-HER2 CAR constructs comprising a DAP 10 domain and Myd88 ICD relative to control constructs and UTD macrophages when incubated with PBS, HER2, or Mesothelia TNFa, IL-8, IL6, ILlb, and IL12p70 cytokines were assessed.

[0078] Figure 34 shows domains of exemplary DAP 10 CAR constructs comprising an anti-HER2 binder, and several mutations investigated in certain CAR constructs.

[0079] Figure 35 is a schematic showing an exemplary/ experimental timeline for screening mutant Myd88 CAR sequences in HEK Nulll cells.

[0080] Figure 36 is a graph showing exemplary viability percentages of HEK cells expressing anti-HER2 CAR constructs comprising a DAP 10 domain and Myd88 ICD (labeled as Myd88) described herein (see Table 8-10 in Example 14) relative to control constructs and UTD cells.

[0081] Figure 37 is a series of graphs showing exemplary CAR expression via quantification by percent expression and mean fluorescence intensity in HEK cells expressing anti-HER2 CAR constructs comprising a DAP10 domain and Myd88 ICD (labelled as M88) described herein (see Table 8-10 in Example 14) relative to control constructs and UTD cells

[0082] Figure 38 is a graph showing exemplary NFKB activation in HEK cells expressing anti-HER2 CAR constructs comprising a DAP10 domain and Myd88 ICD (labeled as Myd88) described herein (see Table 8-10 in Example 14) relative to control constructs and UTD cells when incubated with PBS, HER2, or Mesothelia

[0083] Figure 39A-B is a series of graphs showing exemplary/ viability percentages and cells recovered of HEK cells expressing anti-HER2 CAR constructs comprising a DAP 10 domain and Myd88 ICD (labeled as Myd88) described herein (see Table 8-10 in Example 14) relative to control constructs and UTD cells.

[0084] Figure 40 is a series of graphs showing exemplary CAR expression via quantification by percent expression and mean fluorescence intensity in HEK cells expressing anti-HER2 CAR constructs comprising a DAP 10 domain and Myd88 ICD (labelled as M88) described herein (see Table 8-10 in Example 14) relative to control constructs and UTD cells.

[0085] Figures 41A-B are graphs of Ml (FIG. 41 A) and M2 markers (FIG. 41B) of CAR macrophages transduced with constructs described herein (see Table 8-10 in Example 14). [0086] Figure 42 is a graph showing tumor killing ability of CAR macrophages described herein (see Table 8-10 in Example 14),

[0087] Figures 43A-C are graphs showing tumor killing ability of CAR macrophages described herein (see Table 8-10 in Example 14) of HER2 expressing (AU562) and HER2 nonexpressing control cells (PANCI).

[0088] Figures 44A-C is a series of graphs showing exemplary cytokine secretion in macrophages expressing anti-HER2 CAR constructs comprising a DAP 10 domain and Myd88 ICD relative to control constructs and UTD macrophages when incubated with PBS, HER2, or Mesothelin. TNFa (FIG. 44A), IL-6 (FIG. 44B), and IL-8 (FIG. 44C) cv-okines were assessed.

[0089] Figure 45 shows domains of exemplar}- CAR constructs disclosed herein comprising a Myd88 and/or DAP10 co- stimulators' domain.

[0090] Figure 46 is a graph showing exemplary NFKB activation in HEK cells expressing anti-HER2 CAR constructs comprising a DAP 10 domain, Myd88 ICD (labeled as M88), and FcRy domain (labeled as y) disclosed herein relative to control constructs and LTD cells.

[0091] Figure 47 are graphs showing exemplary viability percentages and cells recovered of HEK cells expressing anti-HER2 CAR constructs comprising a DAP 10 domain, Myd88 ICD (labeled as M88), and FcRy domain (labeled as y) described herein relative to control constructs and UTT) cells

[0092] Figure 48 is a series of graphs showing exemplar}' CAR expression via quantification by percent expression and mean fluorescence intensity in HEK cells expressing anti-HER2 CAR constructs comprising a DAP10 domain, Myd88 ICD (labeled as M88), and FcRy domain (labeled as y) described herein relative to control constructs and UTD cells.

[0093] Figures 49A-B are graphs of Ml (FIG. 49A) and M2 markers (FIG. 49B) of CAR macrophages transduced with constructs described herein (see Tables 11-13 in example 17).

[0094] Figure 50 is a graph showing tumor killing ability of CAR macrophages described herein (see Tables 11-13 in example 17). [0095] Figures 51A-B is a series of graphs showing exemplary' cytokine secretion in macrophages expressing anti-HER2 CAR constructs comprising a DAPIO domain, Myd88 ICD, and FcRy domain relative to control constructs and UTD macrophages when incubated with PBS, HF.R2, or Mesothelin. TNFa (FIG. 50A) and IL-6 (FIG. 50B) cytokines were assessed.

DEFINITIONS

[0096] In order for the present invention to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the specification. The publications and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference.

[0097] 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.

[ 0098 ] Approximately or about: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1 %, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0099] Activation: As used herein, the term “activation” refers to the state of a cell, for example a monocyte, macrophage, dendritic cell, or stem cell that has been sufficiently stimulated to induce detectable cellular proliferation or has been stimulated to exert its effector function. Activation can also be associated wrth induced cytokine production, phagocytosis, cell signaling, target cell killing, and/or antigen processing and presentation.

[0100] Activated monocytes/macrophages/dendritic cells’. As used herein, the term “activated monocytes/macrophages/dendritic cells” refers to, among other things, monocyte/macrophage/dendritic cells/stem cells that are undergoing cell division or exerting effector function. The term “activated monocytes/macrophages/dendritic cells/stem cells” refers to, among others thing, cells that are performing an effector function or exerting any activity not seen in the resting state, including phagocytosis, cytokine secretion, proliferation, gene expression changes, metabolic changes, and other functions.

[0101] Agent As used herein, the term “agent” (or “biological agent” or “therapeutic agent”), refers to a molecule that may be expressed, released, secreted or delivered to a target by a modified cell described herein. An agent includes, but is not limited to, a nucleic acid, an antibiotic, an anti-inflammatory agent, an antibody or fragments thereof, an antibody agent or fragments thereof, a growth factor, a cytokine, an enzyme, a protein (e.g., an RNAse inhibitor), a peptide, a fusion protein, a synthetic molecule, an organic molecule (e.g., a small molecule), a carbohydrate, a lipid, a hormone, a microsome, a derivative or a variation thereof, and any combinations thereof. An agent may bind any cell moiety, such as a receptor, an antigenic determinant, or other binding site present on a target or target cell. An agent may diffuse or be transported into a ceil, where it may act intracellularly.

[0102] Antibody. As used herein, the term “antibody” refers to a polypeptide that includes canonical immunoglobulin sequence elements sufficient to confer specific binding to a particular target antigen. As is known in the art, intact antibodies as produced in nature are approximately 150 kD tetrameric agents comprising two identical heavy chain polypeptides (about 50 kD each) and two identical light chain polypeptides (about 25 kD each) that associate with each other into what is commonly referred to as a “Y-shaped” structure. Each heavy chain comprises at least four domains (each about 110 amino acids long) - an amino-terminal variable (VH) domain (located at the tips of the Y structure), followed by three constant domains: CHI, CH2, and the carboxy -terminal CH3 (located at the base of the Y’s stem). A short region, known as the “switch”, connects the heavy chain variable and constant regions. The “hinge” connects CH2 and CH3 domains to the rest of the antibody. Two disulfide bonds in this hinge region connect the two heavy chain polypeptides to one another in an intact antibody. Each light chain comprises two domains - an amino-terminal variable (VL) domain, followed by a carboxyterminal constant (CL.) domain, separated from one another by another “switch”. Intact antibody tetramers comprise two heavy chain-light chain dimers in which the heavy and light chains are linked to one another by a single disulfide bond, two other disulfide bonds connect the heavy chain hinge regions to one another, so that the dimers are connected to one another and a tetramer is formed. Naturally-produced antibodies are also glycosylated, typically on the CH2 domain. Each domain in a natural antibody has a structure characterized by an “immunoglobulin fold” formed from two beta sheets (e.g., 3-, 4-, or 5-stranded sheets) packed against each other in a compressed antiparallel beta barrel. Each variable domain contains three hypervariable loops known as “complementarity determining regions” (CDR1, CDR2, and CDR3) and four somewhat invariant “framework” regions (FR1, FR2, FR3, and FR4). When natural antibodies fold, the FR regions form the beta sheets that provide the structural framework for the domains, and the CDR loop regions from both the heavy and light chains are brought together in three- dimensional space so that they create a single hypervariable antigen binding site located at the tip of the Y structure. The Fc region of naturally-occurring antibodies binds to elements of the complement system, and also to receptors on effector cells, including, for example, effector cells that mediate cytotoxicity. Affinity and/or other binding attributes of Fc regions for Fc receptors can be modulated through glycosylation or other modification. In some embodiments, antibodies produced and/or utilized in accordance with the present invention (e.g., a CAR) include glycosylated Fc domains, including Fc domains with modified or engineered glycosylation. In some embodiments, any polypeptide or complex of polypeptides that includes sufficient immunoglobulin domain sequences as found in natural antibodies can be referred to and/or used as an “antibody”, whether such polypeptide is naturally produced (e.g., generated by an organism reacting to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial system or methodology. In some embodiments, an antibody is polyclonal. In some embodiments, an antibody is monoclonal, hi some embodiments, an antibody has constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, antibody sequence elements are humanized, primatized, chimeric, etc, as is known in the art. Moreover, the term “antibody”, as used herein, can refer in appropriate embodiments (unless otherwise stated or clear from context) to any of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation.

For example, in some embodiments, an antibody utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments. Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions; single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); camel oid antibodies; masked antibodies (e.g., Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPs™”); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®; Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc.].

[0103] Antibody agent: As used herein, the term “antibody agent” refers to an agent that specifically binds to a particular antigen. In some embodiments, the term encompasses any polypeptide or polypeptide complex that includes immunoglobulin structural elements sufficient to confer specific binding. Exemplary antibody agents include, but are not limited to monoclonal antibodies or polyclonal antibodies. In some embodiments, an antibody agent may include one or more constant region sequences that are characteristic of mouse, rabbit, primate, or human antibodies. In some embodiments, an antibody agent may include one or more sequence elements are humanized, primatized, chimeric, etc., as is known in the art. In many embodiments, the term “antibody agent” is used to refer to one or more of the art-known or developed constructs or formats for utilizing antibody structural and functional features in alternative presentation. For example, in some embodiments, an antibody agent utilized in accordance with the present invention is in a format selected from, but not limited to, intact IgA, IgG, IgE or IgM antibodies; bi- or multi- specific antibodies (e.g., Zybodies®, etc); antibody fragments such as Fab fragments, Fab’ fragments, F(ab’)2 fragments, Fd’ fragments, Fd fragments, and isolated CDRs or sets thereof; single chain Fvs; polypeptide-Fc fusions, single domain antibodies (e.g., shark single domain antibodies such as IgNAR or fragments thereof); cameloid antibodies; masked antibodies (e.g.. Probodies®); Small Modular ImmunoPharmaceuticals (“SMIPsTM”); single chain or Tandem diabodies (TandAb®); VHHs; Anticalins®; Nanobodies® minibodies; BiTE®s; ankyrin repeat proteins or DARPINs®;

.Avimers®; DARTs; TCR-like antibodies;, Adnectins®; Affilins®; Trans-bodies®; Affibodies®; TrimerX®; MicroProteins; Fynomers®, Centyrins®; and KALBITOR®s. In some embodiments, an antibody agent may lack a covalent modification (e.g., attachment of a glycan) that it would have if produced naturally. In some embodiments, an antibody agent may contain a covalent modification (e.g., attachment of a glycan, a payload [e.g., a detectable moiety, a therapeutic moiety, a catalytic moiety, etc], or other pendant group [e.g., poly-ethylene glycol, etc.]. In many embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes one or more structural elements recognized by those skilled in the art as a complementarity determining region (CDR); in some embodiments an antibody agent is or comprises a polypeptide whose amino acid sequence includes at least one CDR (e.g., at least one heavy chain CDR and/or at least one light chain CDR) that is substantially identical to one found in a reference antibody. In some embodiments an included CDR is substantially identical to a reference CDR in that it is either identical in sequence or contains between 1 -5 amino acid substitutions as compared with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that it shows at least 96%, 96%, 97%, 98%, 99%, or 100% sequence identity with the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that, is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that at least one amino acid within the included CDR is substituted as compared with the reference CDR but the included CDR has an amino acid sequence that, is otherwise identical with that of the reference CDR. In some embodiments an included CDR is substantially identical to a reference CDR in that 1-5 amino acids within the included CDR are deleted, added, or substituted as compared with the reference CDR but the included CDR has an amino acid sequence that is otherwise identical to the reference CDR. In some embodiments, an antibody agent is or comprises a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent is a polypeptide protein having a binding domain which is homologous or largely homologous to an immunoglobulin-binding domain. In some embodiments, an antibody agent is not and/or does not comprise a polypeptide whose amino acid sequence includes structural elements recognized by those skilled in the art as an immunoglobulin variable domain. In some embodiments, an antibody agent may be or comprise a molecule or composition which does not include immunoglobulin structural elements (e.g., a receptor or other naturally occurring molecule which includes at least one antigen binding domain).

[0104] Antibody fragment. As used herein, the term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, scFv antibodies, and multi specific antibodies formed from antibody fragments and human and humanized versions thereof.

[0105] Antibody heavy chain: As used herein, the term “antibody heavy chain” refers to the larger of the two types of polypeptide chains present in all antibody molecules in their naturally occurring conformations.

[0106] Antibody light chain: As used herein, the term “antibody light chain” refers to the smaller of the two types of polypeptide chains present in all antibody molecules in their naturally occur ri n g conf orm ati on s .

[0107] Synthetic antibody: As used herein, the term “synthetic antibody” refers to an antibody that is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage described herein. The term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which 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.

[010§] Antigen: As used herein, the term “antigen” or “Ag” refers to a molecule that is capable of provoking an immune response. This immune response may involve either antibody production, the activation of specific immunologically-competent cells, or both, A 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.

.A skilled artisan will understand that any DNA that comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an immune response 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 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

[0109] Anti-himor effect: As used herein, the term “anti-tumor effect” refers to a biological effect which can be manifested by a decrease in tumor volume, a decrease in the number of tumor cells, a decrease in the number of metastases, an increase in life expectancy, or amelioration of various physiological symptoms associated with the cancerous condition. An “anti-tumor effect” can also be manifested by the ability of the peptides, polynucleotides, cells and antibodies of the invention in prevention of the occurrence of a tumor in the first place.

[0110] Autologous: As used herein, the term “autologous” refers to any material derived from an individual to which it is later to be re-introduced into the same individual.

[Oil 1] Allogeneic: As used herein, the term “allogeneic” refers to any material (e.g., a population of cells) derived from a different animal of the same species.

[0112] Xenogenic: As used herein, the term “xenogeneic” refers to any material (e.g., a population of cells) derived from an animal of a different species.

[0113] Cancer: As used herein, the term “cancer” refers to a disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body Examples of various cancers include but are not limited to, 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 the like. In certain embodiments, the cancer is medullary thyroid carcinoma.

[0114] Conservative sequence modifications: As used herein, the term “conservative sequence modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions.

Modifications can be introduced into an antibody compatible with various embodiments by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for the ability to bind antigens using the functional assays described herein.

[0115] Co-stimulatory ligand: As used herein, the term “co-stimulatory ligand” refers to a molecule on an antigen presenting cell (e.g., an APC, dendritic cell, B cell, and the like) that specifically binds a cognate co-stimulatory molecule on a monocyte/macrophage/dendritic cell, thereby providing a signal which mediates a monocyte/macrophage/dendritic cell response, including, but not limited to, proliferation, activation, differentiation, and the like. A co- stimulatory ligand can include, but is not limited to, CD7, B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1 BBL, OX40L, inducible costimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor and a ligand that specifically binds with B7-H3. A co-stimulatory ligand also encompasses, inter alia, an antibody that specifically binds with a co-stimulatory molecule present on a monocyte/macrophage/dendritic cell, such as, but not limited to, CD27, CD28, 4-1BB, 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen- 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83.

[0116] Cytotoxic: As used herein, the term “cytotoxic” or “cytotoxicity” refers to killing or damaging cells. In one embodiment, cytotoxicity of the metabolically enhanced cells is improved, e.g. increased cytolytic activity of macrophages.

[0117] Effective amount As used herein, “effective amount” and “therapeutically effective amount” are interchangeable, and refer to an amount of a compound, formulation, material, or composition, described herein effective to achieve a particular biological result or provides a manufacturing, therapeutic or prophylactic benefit. Such results may include, but are not limited to, anti-tumor activity as determined by any means suitable in the art.

[0118] Effector function: As used herein, “effector function” or “effector activity” refers to a specific activity carried out by an immune cell in response to stimulation of the immune cell. For example, an effector function of macrophages to engulf and digest cellular debris, foreign substances, microbes, cancer cells and other unhealthy cells by phagocytosis.

[0119] Encoding: As used herein, “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or 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.

[0120] Endogenous: As used herein “endogenous” refers to any material from or produced inside a particular organism, cell, tissue or system

[0121] Exogenous: As used herein, the term “exogenous” refers to any material introduced from or produced outside a particular organism, cell, tissue or system. [0122] Expand: As used herein, the term “expand” refers to increasing in number, as in an increase in the number of cells, for example, monocytes, macrophages, and/or dendritic cells. In one embodiment, monocytes, macrophages, or dendritic cells that are expanded ex vivo increase in number relative to the number originally present in a culture. In another embodiment, monocytes, macrophages, or dendritic cells that are expanded ex vivo increase in number relative to other cell types in a culture. In some embodiments, expansion may occur in vivo. 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).

[0123] Expression: As used herein, the term “expression” of a nucleic acid sequence refers to generation of any gene product from a nucleic acid sequence. In some embodiments, a gene product can be a transcript. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g, by splicing, editing, 5’ cap formation, and/or 3’ end formation); (3) translation of an RNA into a polypeptide or protein, and/or (4) post-translational modification of a polypeptide or protein

[0124] Expression vector: As used herein, the term “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 cisacting 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, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0125] Fragment: As used herein, the terms “fragment” or “portion,” as used interchangeably herein, refers to a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole structure. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a nucleotide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, or more monomeric units (e.g., nucleic acids) as found in the whole nucleotide. In some embodiments, a nucleotide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the monomeric units (e.g., residues) found in the whole nucleotide. The whole material or entity may in some embodiments be referred to as the “parent” of the whole.

[0126] Homology: ks> used herein, the term “homology” refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical. In some embodiments, polymeric molecules are considered to be “homologous” to one another if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% similar (e.g., containing residues with related chemical properties at corresponding positions). As will be understood by those skilled in the art, a variety of algorithms are available that permit comparison of sequences in order to determine their degree of homology, including by permitting gaps of designated length in one sequence relative to another when considering which residues “correspond” to one another in different sequences. Calculation of the percent homology between two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-corresponding sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position; when a position in the first sequence is occupied by a similar nucleotide as the corresponding position in the second sequence, then the molecules are similar at that position. The percent homology between the two sequences is a function of the number of identical and similar positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.

[0127] Identity: As used herein, the term “identity'’ 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.

[0128] Siibstantial identity: As used herein, the term “substantial identity” refers to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary' skill in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues in corresponding positions. As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. In some embodiments, two sequences are considered to be substantially identical if at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over a relevant stretch of residues. In some embodiments, the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues. In the context of a CDR, reference to “substantial identity” typically refers to a CDR having an amino acid sequence at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to that of a reference CDR.

[0129] Immune cell: As used herein, the term “immune cell,” refers to a cell that is involved in an immune response, e.g., promotion of an immune response. Examples of immune cells include, but are not limited to, macrophages, monocytes, dendritic cells, neutrophils, eosinophils, mast cells, platelets, large granular lymphocytes, Langerhans' cells, natural killer (NK) cells, T-lymphocytes, or B -lymphocytes. A source of immune cells (e.g., macrophages, monocytes, or dendritic cells) can be obtained from a subject.

[0130] Immune response: As used herein the term “immune response” refers to a cellular and/or systemic 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.

[0131] Immunoglobulin: As used herein, the term “immunoglobulin” or “Ig,” refers to a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as a BCR (B cell receptor) or antigen receptor. The five members included in this class of proteins are IgA, IgG, IgM, IgD, and IgE. IgA is the primary antibody that is present in body secretions, such as saliva, tears, breast milk, gastrointestinal secretions and mucus secretions of the respiratory and genitourinary tracts. IgG is the most common circulating antibody. IgM is the main immunoglobulin produced in the primary' immune response in most subjects It is the most efficient immunoglobulin in agglutination, complement fixation, and other antibody responses, and is important in defense against bacteria and viruses. IgD is an immunoglobulin that has no known antibody function, but may serve as an antigen receptor. IgE is an immunoglobulin that mediates immediate hypersensitivity by causing release of mediators from mast cells and basophils upon exposure to allergen.

[0132] Isolated: As used herein, the term “isolated” refers to something 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. [0133] Lentivinis: .As used herein, the term “lentivirus” refers to a genus of the Retroviridae family. Lend viruses are unique among the retroviruses in being able to infect nondividing cells; they can deliver a significant amount of genetic information into the DNA of a host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. Vectors derived from lentiviruses offer the means to achieve significant levels of gene transfer in vivo.

[0134] Modified: As used herein, the term ‘"modified” refers to 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.

[ 0135] Modulating: As used herein the term “modulating,” refers to mediating a detectable increase or decrease in the level of a response and/or a change in the nature of a response in a subject compared with the level and/or nature of a response in the subject in the absence of a treatment or compound, and/or compared with the level and/or nature 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, preferably, a human.

[0136] Nucleic acid. As used herein, the terra “nucleic acid” refers to a polymer of at least three nucleotides. In some embodiments, a nucleic acid comprises DNA. In some embodiments, a nucleic acid comprises RNA. in some embodiments, a nucleic acid is single stranded. In some embodiments, a nucleic acid is double stranded. In some embodiments, a nucleic acid comprises both single and double stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non- phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and/or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises one or more, or all, non-natural residues. In some embodiments, a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5 -methyl cytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-broniouridine, C5-fluorouridine, C 5 -iodouridine, C5-propynyl -uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2- ami noadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 0(6)- methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'- fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g , by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000 or more residues long.

[0137] Operably linked: As used herein, the term “operably linked” refers to functional linkage between, for example, 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 sequences are contiguous and, where necessary' to join two protein coding regions, in the same reading frame

[0138] Overexpressed tnwior antigen: i\s> used herein, the term “overexpressed” tumor antigen or “overexpression” of a tumor antigen refers to an abnormal level of expression of a tumor antigen in a cell from a disease area like a solid tumor within a specific tissue or organ of the patient relative to the level of expression in a normal cell from that tissue or organ. Patients having solid tumors or a hematological malignancy characterized by overexpression of the tumor antigen can be determined by standard assays known in the art.

[0139] Polynucleotide: As used herein, the term “polynucleotide” refers to 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 li brary or a cell genome, using ordinary cloning technology and PCR™, and the like, and by synthetic means.

[0140] Polypeptide: As used herein, the term “polypeptide” refers to any polymeric chain of residues (e.g., amino acids) that are typically linked by peptide bonds. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-naturai amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only nonnatural amino acids. In some embodiments, a polypeptide may comprise D-amino acids, L- amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide’s N-terminus, at the polypeptide’s C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications may be selected from the group consisting of acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and/or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and/or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. Tn some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term “ polypeptide” may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and/or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary' polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class) For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very⁷ high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 20 or more amino acids, in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or more contiguous amino acids. In some embodiments, a useful polypeptide may comprise or consist of a fragment of a parent polypeptide. In some embodiments, a useful polypeptide as may comprise or consist of a plurality of fragments, each of which is found in the same parent polypeptide in a different spatial arrangement relative to one another than is found in the polypeptide of interest (e.g., fragments that, are directly linked in the parent may be spatially separated in the polypeptide of interest or vice versa, and/or fragments may be present in a different order in the polypeptide of interest than in the parent), so that the polypeptide of interest is a derivative of its parent polypeptide.

[0141J Protein: As used herein, the term “protein” refers to a polypeptide (i e., a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. Tn some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.

[0142] Signal transduction pathway: As used herein, the term “signal transduction pathway” refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of a cell. The phrase “cell surface receptor” includes molecules and complexes of molecules capable of receiving a signal and transmitting signal across the plasma membrane of a cell.

[0143] Single chain antibodies: As used herein, the term “single chain antibodies” refers to antibodies formed by recombinant DNA techniques in which immunoglobulin heavy and light chain fragments are linked to the Fv region via an engineered span of amino acids. Various methods of generating single chain antibodies are known, including those described in U.S. Pat. No. 4,694,778; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad Sei. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242: 1038-1041.

[0144] Specifically binds: As used herein, the term “specifically binds,” with respect to an antigen binding domain, such as an antibody agent, refers to an antigen binding domain or antibody agent which recognizes a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, an antigen binding domain or antibody agent that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antigen binding domain or antibody agent as specific. In another example, an antigen binding domain or antibody agent 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 antigen binding domain or antibody agent as specific. In some instances, the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antigen binding domain or antibody agent, 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 antigen binding domain or antibody agent recognizes and binds to a specific protein structure rather than to proteins generally If an antigen binding domain or antibody agent 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 antigen binding domain or antibody agent, will reduce the amount of labeled A bound to the antibody.

[0145] Stimtilalion: As used herein, the term “stimulation,” refers to a primary response induced by binding of a stimulatory molecule (e.g., an FcR complex, a TLR complex, or a TCR/CD3 complex), for example, with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via Fc receptor machinery, via a synthetic CAR. Stimulation can mediate altered expression of certain molecules, such as downregulation of TGF-beta, and/or reorganization of cytoskeletal structures, and the like. As used herein, the term “stimulatory' molecule,” refers to a molecule of a monocyte, macrophage, or dendritic cell that specifically binds with a cognate stimulatory ligand present on an antigen presenting cell. In some embodiments, a stimulatory molecule comprises an FcR extracellular domain comprising a CD64 (FcyRI), CD32a (FcyRIIa), CD32b (FcyRIIb), CD32c, CD 16a (FcyRIIIa), CD fob (FcyRIIIb), FcsRI, FcsRII, FcaRI (CD89) or CD40 domain. In some embodiments, a stimulatory' molecule comprises a TLR extracellular domain comprising a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain. As used herein, the term “stimulatory⁷ ligand,” refers to a ligand that when present on an antigen presenting cell (e.g , an aAPC, a macrophage, a dendritic cell, a B-cell, and the like) or tumor cell can specifically bind with a cognate binding partner (referred to herein as a “stimulatory molecule”) on a monocyte, macrophage, or dendritic cell thereby mediating a response by the immune cell, including, but not limited to, activation, initiation of an immune response, proliferation, and the like. Stimulaton^? ligands are well-known in the an and encompass, inter cilia. Toll-like receptor (TL,R) ligand, an anti -toll -like receptor antibody, an agonist, and an antibody for a monocyte/macrophage receptor. In addition, cytokines, such as interferon-gamma, are potent stimulants of macrophages.

[0146] Subject: As used herein, the term “subject” refers to an organism, for example, a mammal (e.g., a human, a non-human mammal, a non-human primate, a primate, a laboratory animal, a mouse, a rat, a hamster, a gerbil, a cat, or a dog). In some embodiments a human subject is an adult, adolescent, or pediatric subject. In some embodiments, a subject is suffering from a disease, disorder or condition, e.g., a disease, disorder, or condition that can be treated as provided herein, e.g., a cancer or a tumor listed herein. In some embodiments, a subject is susceptible to a disease, disorder, or condition; in some embodiments, a susceptible subject is predisposed to and/or shows an increased risk (as compared to the average risk observed in a reference subject or population) of developing the disease, disorder, or condition. In some embodiments, a subject displays one or more symptoms of a disease, disorder, or condition. In some embodiments, a subject does not display a particular symptom (e.g., clinical manifestation of disease) or characteristic of a disease, disorder, or condition. In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered

[0147] Substantially purified: J\s, used herein, the term “substantially purified”, for example as applied to a cell, refers to a cell that is essentially free of other cell types. A substantially purified cell also refers to a cell which has been separated from other cell types with which it is normally associated in its naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, this term refers simply to cell that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro. [0148] Target: As used herein, the term “target” refers to a ceil, tissue, organ, or site within the body that is the subject of provided methods, systems, and/or compositions, for example, a cell, tissue, organ or site within a body that is in need of treatment or is preferentially bound by, for example, a CAR.

[0149] Target site: As used herein, the term “target site” or “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.

[0150] T cell receptor: 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, A TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. A TCR comprises a heterodimer of an alpha (a) and beta (P) chain, although in some cells the TCR comprises gamma and delta (y/S) chains. TCRs may exist in alpha/beta and gamma/ delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain comprises two extracellular domains, a variable and constant domain. In some embodiments, a 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 .

[0151] Therapeutic: As used herein, the term “therapeutic” refers to a treatment and/or prophylaxis. A therapeutic effect is obtained by suppression, remission, or eradication of a disease state.

[0152] Transfected: As used herein, the term “transfected” or “transformed” or “transduced” 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.

[0153] Treat: As used herein, the term “treat,” “treatment,” or “treating” refers to partial or complete alleviation, amelioration, delay of onset of, inhibition, prevention, relief, and/or reduction in incidence and/or severity of one or more symptoms or features of a disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who does not exhibit signs or features of a disease, disorder, and/or condition (e.g., may be prophylactic). In some embodiments, treatment may be administered to a subject who exhibits only early or mild signs or features of the disease, disorder, and/or condition, for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject who exhibits established, severe, and/or late-stage signs of the disease, disorder, or condition. In some embodiments, treating may comprise administering to an immune cell (e.g., a monocyte, macrophage, or dendritic cell) or contacting an immune cell with a modulator of a pathway activated by m vitro transcribed mRNA.

[0154] Tumor: As used herein, the term “tumor” refers to an abnormal growth of cells or tissue In some embodiments, a tumor may comprise cells that are precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and/or non -metastatic. In some embodiments, a tumor is associated with, or is a manifestation of, a cancer. In some embodiments, a tumor may be a disperse tumor or a liquid tumor. In some embodiments, a tumor may be a solid tumor.

[0155] Vector: As used herein, the term “vector” refers to a composition of matter that 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 constmed 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 vims vectors, retroviral vectors, lenti viral vectors, and the like.

[0156] 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.

DETAILED DESCRIPTION

[0157] The present disclosure encompasses, among other things, compositions comprising modified immune cells (e.g., stem cells, macrophages, monocytes, and/or dendritic cells) comprising novel chimeric antigen receptors (CARs) described herein and methods of using and producing the same. The present disclosure also encompasses, among other things, compositions comprising modified immune cells (e.g., stem cells, macrophages, monocytes, and/or dendritic cells) comprising novel nucleic acid constructs encoding CARs described herein and methods of using and producing the same. In some embodiments, CARs of the present disclosure comprise one or more of: (i) a Myd88 intracellular domain or a portion thereof, (ii) CD40 intracellular domain or a portion thereof, or (iii) FcRy intracellular domain (also known as FCER1G). In some embodiments, CARs of the present disclosure comprise one or both of (i) a CD8 or CD28 extracellular hinge domain, or (ii) a CD8 or CD28 transmembrane domain.

[0158] In some embodiments, modified immune cells described herein comprising or expressing CARs described herein exhibit increased tumor killing, e.g., relative to a modified immune cell of the same type comprising a similar CAR (e.g., a CAR described herein, e g., without one or more of a Myd88 intracellular domain or a portion thereof, CD40 intracellular domain or a portion thereof, or FcRy intracellular domain but with the other components of the comparator CAR, e.g., without any of a Myd88 intracellular domain or a portion thereof, CD40 intracellular domain or a portion thereof, or FcRy intracellular domain, but with the other components of the comparator CAR). In some embodiments, modified immune cells comprising CARs described herein do not exhibit killing of tumor cells that do not express a target antigen, e.g., relative to modified immune cells of the same type comprising a similar CAR (e.g., a CAR described herein, e.g., without one or more of a Myd88 intracellular domain or a portion thereof, CD40 intracellular domain or a portion thereof, or FcRy intracellular domain but with the other components of the comparator CAR, e.g., without any of a \1vd88 intracellular domain or a portion thereof CD-40 intracellular domain or a portion thereof, or FcRy intracellular domain, but with the other components of the comparator CAR). In some embodiments, tumor killing comprises or is one or more of phagocytosis, lysis, apoptosis, or production of tumor killing cytokines (e.g., TNFa).

[0159] In some embodiments, modified immune cells described herein comprising or expressing CARs described herein exhibit increased viability, e.g., relative to a modified immune cell of the same type comprising a similar CAR (e.g., a CAR described herein, e.g , without one or more of a Myd88 intracellular domain or a portion thereof, CD40 intracellular domain or a portion thereof, or FcRy intracellular domain but with the other components of the comparator CAR, e.g., without any of a Myd88 intracellular domain or a portion thereof, CD40 intracellular domain or a portion thereof, or FcRy intracellular domain, but with the other components of the comparator CAR). In some embodiments, modified immune cells described herein comprising or expressing CARs described herein exhibit increased expression of a CAR, e.g., relative to a modified immune cell of the same type comprising a similar CAR (e.g., a CAR described herein, e.g., without one or more of a Myd88 intracellular domain or a portion thereof, CD40 intracellular domain or a portion thereof, or FcRy intracellular domain but with the other components of the comparator CAR, e.g., without any of aMyd88 intracellular domain or a portion thereof, CD40 intracellular domain or a portion thereof, or FcRy intracellular domain, but with the other components of the comparator CAR).

[0160] In some embodiments, modified immune cells described herein comprising or expressing C ARs described herein exhibit increased expression of Ml markers (e.g., one or both of CD80 or CD86), e.g., relative to a modified immune cell of the same type comprising a similar CAR (e.g., a CAR described herein, e.g., without one or more of a Myd88 intracellular domain or a portion thereof, CD40 intracellular domain or a portion thereof, or FcRy intracellular domain but with the other components of the comparator CAR, e.g., without any of a Myd88 intracellular domain or a portion thereof, CD40 intracellular domain or a portion thereof, or FcRy intracellular domain, but with the other components of the comparator CAR). In some embodiments, modified immune cells described herein comprising or expressing CARs described herein exhibit decreased expression of M2 markers ( e.g., one or both of CD 163 or CD206), e.g., relative to a modified immune cell of the same type comprising a similar CAR (e.g., a CAR described herein, e.g., without one or more of a Myd88 intracellular domain or a portion thereof, CD40 intracellular domain or a portion thereof, or FcRy intracellular domain but with the other components of the comparator CAR, e.g., without any of a Myd88 intracellular domain or a portion thereof CD40 intracellular domain or a portion thereof or FcRy intracellular domain, but with the other components of the comparator CAR). In some embodiments, modified immune cells described herein comprising or expressing CARs described herein exhibit reduced background cytokine release (e.g., one or more of TNFa, IL-6, or IL-8) prior to stimulation, each relative to modified immune cells of the same type comprising a similar CAR (e.g., a CAR described herein, e.g., without one or more of aMyd88 intracellular domain or a portion thereof CD40 intracellular domain or a portion thereof, or FcRy intracellular domain but with the other components of the comparator CAR, e.g., without any of a Myd88 intracellular domain or a portion thereof, CD40 intracellular domain or a portion thereof, or FcRy intracellular domain, but with the other components of the comparator CAR). As used herein, the term “background cytokine release” refers to the level of cytokine released by a modified cell as described herein, prior to stimulation by a target antigen.

[0161] In some embodiments, a CAR described herein comprises: (a) one or more extracellular domains; (b) a transmembrane domain (e.g., a CD28 transmembrane domain or CDS transmembrane domain); and (c) one or more intracellular domains comprising one or both of: (i) a MyD88 intracellular domain or a portion thereof, or (ii) a CD40 intracellular domain or a portion thereof In some embodiments, one or more intracellular domains further comprise a CD3-zeta intracellular domain.

[0162] In some embodiments, a CAR further comprises one or more extracellular hinge domains. In some embodiments, one or more extracellular hinge domains comprise a CD28 extracellular hinge domain or a CD8a extracellular hinge domain. In some embodiments, a CAR described herein comprises: (a) one or more extracellular domains, (b) a CD28 extracellular hinge domain; (c) a CD28 transmembrane domain; and (d ) one or more intracellular domains comprising one or both of: (i) a MyD88 intracellular domain or a portion thereof or (ii) a CD40 intracellular domain or a portion thereof, and optionally further comprising a CD3-zeta domain. In some embodiments, a CAR described herein comprises: (a) one or more extracellular domains; (b) a CD8 extracellular hinge domain; (c) a CDS transmembrane domain; and (d) one or more intracellular domains comprising one or both of (i) a MyD88 intracellular domain or a portion thereof or (ii) a CD40 intracellular domain or a portion thereof, and optionally further comprising a CD3-zeta domain. In some embodiments, a CAR described herein comprises: (a) one or more extracellular domains; (b) a CD8 extracellular hinge domain; (c) a CD28 transmembrane domain, and (d) one or more intracellular domains comprising one or both of: (i) a MyD88 intracellular domain or a portion thereof, or (ii) a CD40 intracellular domain or a portion thereof and optionally further comprising a CD3 -zeta domain. In some embodiments, a CAR described herein compri ses:

(a) one or more extracellular domains; (b) a CD28 extracellular hinge domain; (c) a CD8 transmembrane domain; and (d) one or more intracellular domains comprising one or both of: (i) a MyD88 intracellular domain or a portion thereof, or (ii) a CD40 intracellular domain or a portion thereof, and optionally further comprising a CD3-zeta domain

[0163] In some embodiments, a CAR described herein comprises: (a) one or more extracellular domains, (b) a transmembrane domain, and (d) one or more intracellular domains comprising an FcRy intracellular domain. In some embodiments, a CAR described herein comprises: (a) one or more extracellular domains; (b) a CDS extracellular hinge domain or CD28 extracellular hinge domain, (c) a CDS extracellular hinge domain or CD28 transmembrane domain, and (d) one or more intracellular domains comprising an FcRy intracellular domain. In some embodiments, one or more intracellular domains further comprise one or both of: (i) a MyD88 intracellular domain or a portion thereof, or (ii) a CD40 intracellular domain or a portion thereof.

In some embodiments, a CAR described herein comprises: (a) one or more extracellular domains;

(b) a CD28 extracellular hinge domain, (c) CD28 transmembrane domain, and (cl) one or more intracellular domains comprising an FcRy intracellular domain, and optionally one or both of: (i) a MyD88 intracellular domain or a portion thereof, or (ii) a CD40 intracellular domain or a portion thereof. In some embodiments, a CAR described herein comprises: (a) one or more extracellular domains; (b) a CD8 extracellular hinge domain, (c) a CD8 transmembrane domain, and (d) one or more intracellular domains comprising an FcRy intracellular domain, and optionally one or both of: (i) a My D88 intracellular domain or a portion thereof, or (ii) a CD40 intracellular domain or a portion thereof. In some embodiments, a CAR described herein comprises: (a) one or more extracellular domains; (b) a CD28 extracellular hinge domain, (c) a CD8 transmembrane domain, and (d) one or more intracellular domains comprising an FcRy intracellular domain, and optionally one or both of: (i) a MyD88 intracellular domain or a portion thereof, or (ii) a CD40 intracellular domain or a portion thereof. In some embodiments, a CAR described herein comprises: (a) one or more extracellular domains; (b) a CD8 extracellular hinge domain, (c) CD28 transmembrane domain, and (d) one or more intracellular domains comprising an FcRy intracellular domain, and optionally one or both of: (i) a MyD88 intracellular domain or a portion thereof, or (ii) a CD40 intracellular domain or a portion thereof.

Immune Cells

[0164] The present disclosure provides, among other things, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising at ieast one chimeric antigen receptor (CAR) described herein. In some embodiments, a population of immune cells described herein comprises stem cells, monocytes, macrophages, dendritic cells, and/or precursors thereof In some embodiments, a population of immune cells comprises a substantially purified population of stern cells, monocytes, macrophages, or dendritic cells, or a cell line

[0165] In some embodiments, an immune cell is activated, e.g., an immune cell exhibits increased cytokine production, chemokine production, phagocytosis, cell signaling, target cell killing, and/or antigen presentation, e.g., relative to an inactive cell. In some embodiments, an activated immune cell exhibits changes in gene expression, e.g., an induction of pro- inflammatory gene expression, e.g., relative to an inactive cell In some embodiments, an activated immune cell exhibits changes in gene expression, e.g., an induction of antiinflammatory gene expression, e.g., relative to an inactive cell In certain embodiments, activated immune cells are undergoing cell division. In some embodiments, targeted effector activity of an immune cell is enhanced by inhibition of CD47 and/or SIRPa activity. CD47 and/or SIRPa activity may be inhibited by treating an immune cell with an ant.i-CD47 or anti- SIRPa antibody or by any method known to those skilled in the art.

[0166] In some embodiments, immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) are obtained (e.g., isolated) from a subject Immune cells may be autologous or sourced from allogeneic or universal donors. Cells can be obtained from a number of sources including peripheral blood mononuclear cells, bone marrow; lymph node tissue, spleen tissue, umbilical cord, tumors, and/or induced pluripotent stem cells, such as embryonic stem cells (ESCs). In certain embodiments, cells can be obtained from a unit of blood collected from a subject using any number of separation techniques known to a skilled artisan, such as Ficoll separation. In some embodiments, cells from circulating blood of a subject are obtained by apheresis or leukapheresis. Cells collected by apheresis may be washed to remove a plasma fraction and resuspended in a variety of buffers (e.g., phosphate buffered saline (PBS)) or culture media). In some embodiments, enrichment of immune cells (e.g. monocytes) comprises plastic adherence. In some embodiments, following enrichment, differentiation of immune cells (e.g. monocytes) comprises stimulation with GM-CSF. In some embodiments, a composition comprising blood cells (e.g., monocytes, lymphocytes, platelets, plasma, and/or red blood cells), such as a leukapheresis composition (e.g., a leukopak) is used for enrichment. In some embodiments, a leukapheresis composition (e.g., a leukopak) comprises a sample from a healthy human donor. In certain embodiments, apheresis of immune cells (e.g. monocytes) is followed by mobilization with GM-CSF. Tn certain embodiments, selection of immune cells (e g., monocytes) comprises CD 14 positive selection using microbeads (e.g., MACS® MicroBeads on a CliniMACS Prodigy device). In some embodiments, an immune cell precursor (e.g., precursors to macrophages, moriocytes, or dendritic cells including, but not limited to induced pluripotent stem cells, or iPSCs) is used in compositions and methods described herein. Immune cell precursors may be differentiated in vivo or ex vivo into immune cells. Non-limiting examples of precursor immune cells include hematopoietic stem cells, common myeloid progenitors, myeloblasts, monoblasts, promonocytes, or intermediates thereof. For example, induced pluripotent stem cells may be used to generate monocytes, macrophages, and/or dendritic cells. Induced pluripotent stem cells (iPSCs) may be derived from normal human tissue, such as peripheral blood, fibroblasts, skin, keratinocytes, or renal epithelial cells. Autologous, allogeneic, or universal donor iPSCs could be differentiated toward a myeloid lineage (e.g., a monocyte, macrophage, dendritic cell, or precursor thereof).

[0167] Immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein can be isolated from peripheral blood, for example, by lysing red blood cells and depleting lymphocytes and red blood cells, such as by centrifugation through a PERCOLL™ gradient. Alternatively, immune cells can be isolated from umbilical cord tissue. A specific subpopulation of immune cells can be further isolated by positive or negative selection techniques. In some embodiments, immune cells can be depleted of cells expressing certain antigens, including, but not limited to, CD34, CD3, CD4, CD8, CD56, CD66b, CD19, or CD20. In some embodiments, enrichment of an immune cell population, for example, by negative selection can be accomplished using a combination of antibodies directed to surface markers unique to the negatively selected cells. By way of non-limiting example, cell selection can also comprise negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on negatively selected cells.

[0168] During isolation of a desired population of immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein by positive or negative selection, immune cell concentration and surface (e.g., particles, such as beads) can be varied. It may be desirable to significantly decrease volume in which beads and cells are mixed together to ensure maximum contact area of cells and beads.

[0169] In some embodiments, prior to administration, modified immune cells (e g , stem cells, macrophages, monocytes, or dendritic cells) described herein (e.g,, comprising at least one CAR described herein) are treated with a pro-inflammatory' agent. In some embodiments, treatment with a pro-inflammatory' agent increases anti-tumor activity of modified immune cells described herein. In some embodiments, treatment with at least one pro-inflammatory agent promotes Ml phenotype (e.g., a switch from M2 to Ml phenotype) in modified immune cells described herein. In some embodiments, at least one pro-inflammatory agent comprises or is a CD40 agonist (e.g., CD40L). In some embodiments, at least one pro-inflammatory' agent comprises or is a 41BB-ligand agonist (e g., 4-IBB) In some embodiments, at least one pro- inflammatory' agent comprises or is a CD40 agonist (e.g., CD40L) and a 41BB-ligand agonist (e.g., 4-IBB).

[0170] In some embodiments, modified immune cells (e g , stem cells, macrophages, monocytes, or dendritic cells) described herein (e.g., comprising at least one CAR described herein) have been treated with one or more pro-inflammatory agents. In some embodiments, a modified immune cell described herein exhibits increased anti -tumor activity relative to an unmodified cell of the same type. In some embodiments, one or more pro-inflammatory agents comprises or is a CD40 agonist (e.g., CD40L). In some embodiments, one or more pro- inflammatory' agents comprises or is a 41BB-ligand agonist (e.g., 4-IBB). In some embodiments, one or more pro-inflammatory agents comprises or is a CD40 agonist (e.g., CD40L) and a 41BB-ligand agonist (e.g., 4-IBB). The disclosure provides methods of treating a disease or disorder in a subject, comprising: delivering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a modified macrophage, monocyte, or dendritic cell described herein.

[0171] The disclosure also provides methods of modifying immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein comprising a CAR described herein, wherein the method comprises treating an immune cell described herein with one or more pro-inflammatory agents, thereby producing a modified immune cell described herein that, exhibits increased anti-tumor activity relative to an cell of the same type comprising the CAR or a similar CAR that has not been treated with one or more pro-inflammatory agents. In some embodiments, one or more pro-inflammatory' agents comprises or is a CD40 agonist (e.g., CD40L). In some embodiments, one or more pro-inflammatory agents comprises or is a 41BB- ligand agonist (e g., 4-1BB). In some embodiments, one or more pro-inflammatory agents comprises or is a CD40 agonist (e.g., CD40L) and a 41BB-ligand agonist (e.g., 4-1BB). The disclosure provides methods of treating a disease or disorder in a subject, comprising: delivering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a immune cells described herein modified by methods described herein

[0172] In some embodiments, modified immune cells (e g , stem cells, macrophages, monocytes, or dendritic cells) described herein (e.g., comprising a CAR described herein) are administered to a subject in combination with a pro-inflammatory agent. In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) described herein (e.g., comprising a CAR described herein) are administered to a subject substantially simultaneously, before, or after a pro-inflammatory agent. In some embodiments, administration with a pro-inflammatory agent increases anti-tumor activity of modified immune cells described herein. In some embodiments, administration with a pro-inflammatory agent promotes Ml phenotype (e.g., a switch from M2 to Ml phenotype) in immune cells described herein. In some embodiments, a pro-inflammatory agent comprises or is a CD40 agonist (e.g., CD40L). In some embodiments, a pro-inflammatory agent comprises or is a 41BB-ligand agonists (e.g., 4- IBB).

Macrophages [0173] Macrophages are immune cells specialized for detection, phagocytosis, and destruction of target cells, such as pathogens or tumor cells. Macrophages are potent effectors of the innate immune system and are capable of at least three distinct anti-tumor functions: 1) phagocytosis of dead and dying cells, microorganisms, cancer cells, cellular debris, or other foreign substances; 2) cytotoxicity against tumor cells; and 3) presentation of tumor antigens to orchestrate an adaptive anti-tumor immune response.

[0174] Macrophages are abundant in the tumor microenvironment of numerous cancers and can adopt a number of phenotypes that are collectively referred to as tumor-associated macrophages (TAMs). The immunosuppressive nature of the tumor microenvironment typically results in more M2-like TAMs, which further contribute to the general suppression of anti -tumor immune responses. Recent studies, however, have identified that TAMs are able to be “reprogrammed” via pro-inflammatory signals, and that the switch from a M2 phenotype to a more Ml phenotype is associated with productive anti -tumor immune responses. Inducing endogenous TAMs to switch to MI -type cells and engineering macrophages that cannot be subverted into M2 would greatly enhance anti-tumor immunotherapy and represent a significant advance in the field.

[0175] In some embodiments, a macrophage comprises or is an undifferentiated or Mt) macrophage. In certain embodiments, a macrophage comprises or expresses one, two, three, four, five, or six of CD14, CD16, CD64, CD68, CD71 , or CCR.5. Exposure to various stimuli can induce MO macrophages to polarize into several distinct populations, which may be identified by macrophage phenotype markers, cytokine production, and/or chemokine secretion.

[0176] In some embodiments, a macrophage comprises or is a polarized macrophage. Under classical conditions of activation, MO macrophages can be exposed to pro-inflammatory signals, such as EPS, IFNy, and GM-CSF, and polarize into pro-inflammatory (i.e., Ml) macrophages. Generally, pro-inflammatory (Ml) macrophages are associated with pro- inflammatory immune responses, such as Th! and Thl7 T cell responses. Exposure to other stimuli can polarize macrophages into a diverse group of “alternatively activated” or antiinflammatory' (i.e., M2) macrophages.

[0177] In some embodiments, a macrophage comprises or is a pro-inflammatory (Ml) macrophage. In some embodiments, a macrophage expresses one or more markers of pro- inflammatory (Ml) macrophages (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of CD86, CD80, MHC II, IL-1R, TLR2, TLR4, iNOS, SOCS3, CD83, PD-L1, CD69, MHC I, CD64, CD32, CD 16, IL1R, a IFIT family member, or an ISG family member).

[0178] In some embodiments, a macrophage comprising or expressing at least one CAR described herein secretes relatively high levels of one or more inflammatory cytokines (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of IL-1, TNF, IL-12, IL- 18, IL-23, IFNa, IFNp, IFNy, IL-2, IL-6, IL-8, or IL33) or chemokines (e.g., one or both of CC or CXC chemokines) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, or 16 of the CXC chemokines; e.g., I, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 of the CC chemokines; eg., one of the CX3C chemokines, e.g., one or both of the C chemokines), e.g., relative to a macrophage comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a macrophage comprising or expressing at least one CAR described herein stimulates an immune response and/or inflammation, e.g., relative to a macrophage comprising a similar CAR (e.g., a CAR described herein).

[0179] In some embodiments, a macrophage comprises or is an anti-inflammatory (M2) macrophage (e.g., an M2a, M2b, M2c, and M2d macrophage). An M2a macrophage can be induced by IL-4, IL-13, and/or fungal infection. An M2b macrophage can be induced by IL-1R ligands, an immune complex, and/or LPS. An M2c macrophage can be induced by IL-10 and/or TGFp An M2d macrophage can be induced by IL-6 and/or adenosine. In some embodiments, a macrophage comprising or expressing at least one CAR described herein decreases an immune response in a subject, e.g., relative to a macrophage comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a macrophage expresses one or more markers of antiinflammatory' (M2) macrophages (e.g., one, two, or three of CD206, CD163, or CD209). In some embodiments, a macrophage comprising or expressing at least one CAR described herein exhibits increased secretion of one or more anti-inflammatory cytokines (e.g., one or both of IL- 10 or TGFp), e.g., relative to a macrophage comprising a similar CAR (e.g., a CAR described herein).

[0180] In some embodiments, a macrophage comprises at least one upregulated pro- inflammatory (Ml) marker and/or at least one downregulated anti-inflammatory (M2) marker as compared to a control macrophage that does not comprise at least one CAR as provided herein and/or the same macrophage before delivery of at least one CAR described herein. In some embodiments, at least one pro-inflammatory (MI) marker (e.g., HLA DR, CD86, CD80, PD-L1, CD83, CD69, MHC I, CD64, CD32, CD 16, ILIR, an IFIT family member, and/or an ISG family member) is upregulated in a macrophage. In some embodiments, at least one anti-inflammatory (M2) marker (e.g., CD206, CD163, and/or CD209) is downregulated in a macrophage.

[0181 J In some embodiments, a macrophage comprising or expressing at least one CAR described herein exhibits increased phagocytosis, e.g., relative to a macrophage comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a macrophage comprising or expressing at least one CAR) described herein exhibits increased cytotoxicity against a tumor cell, e.g., relative to a macrophage comprising a similar CAR (e.g , a CAR described herein). In some embodiments, a macrophage comprising or expressing at least one CAR described herein exhibits increased tumor antigen presentation (e.g., post-phagocytosis presentation) and/or increased antigen processing, e.g., relative to a macrophage comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a macrophage comprising or expressing at. least, one CAR exhibits increased tumor killing (e.g., by phagocytosis, lysis, apoptosis, or production of tumor killing cytokines (e.g., TNFa)), e.g,, relative to a macrophage comprising a similar CAR (e.g., a CAR described herein).

[0182] In some embodiments, a macrophage comprising or expressing at least one CAR described herein exhibits one or both of increased expression of one or more genes typically associated with increased effector function (e.g., phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion) (e.g., CD80, CD86, MHC -I, MHC-II, CD40, 41BBL, TNF, IFN-a, IFN-p, IFN-y, IL2, IL 12, IL6, II..8, ILlb, and/or CXCL12) or decreased expression of one or more genes typically associated with decreased effector function (e.g., phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion) (e.g., CD163, CD206, TGFp, IL-10, and/or IL4), e.g., relative to a macrophage comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a macrophage comprising or expressing at. least one CAR described herein exhibits increased production of ROS, e.g., relative to a macrophage comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a macrophage comprising or expressing at. least, one CAR described herein exhibits metabolic reprogramming (e g , of an interferon signaling pathway, TH1 pathway, PTEN signaling, PI3K signaling, MTOR signaling, TLR signaling, CD40 signaling, 4 IBB signaling, 41BBL signaling, macrophage maturation signaling, dendritic cell maturation signaling, CD3-zeta signaling, FcRy signaling, CD64 signaling, CD32a signaling, CD32c signaling, CD 16a signaling, TLR1 signaling, TLR2 signaling, TLR3 signaling, TLR.4 signaling, TLR5 signaling, TLR6 signaling, TLR7 signaling, TLR8 signaling, TLR9 signaling, ALK signaling, AXL signaling, DDR2 signaling, EGFR signaling, EphAl signaling, INSR signaling, cMET signaling, MUSK signaling, PDGFR signaling, PTK7 signaling, RET signaling, R0R1 signaling, ROS1 signaling, RYK signaling, TIE2 signaling, TRK signaling, VEGFR signaling, CD40 signaling, CD 19 signaling, CD20 signaling, 4 IBB signaling, CD28 signaling, 0X40 signaling, GITR signaling, TREM-1 signaling, TREM-2 signaling, DAP 12 signaling, MR signaling, ICOS signaling, MyD88 signaling, V/I/LxYxxL/V signaling, SIRPa signaling, CD45 signaling, Siglec-10 signaling, PD1 signaling, SHP-l signaling, SHP-2 signaling, KIR-2DL signaling, KIR-3DL signaling, NKG2A signaling, CD 170 signaling, CD33 signaling, BTLA signaling, CD32b signaling, SIRPp signaling, CD22 signaling, PIR-B signaling, and/or LILRB1 signaling), e.g., relative to a macrophage comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a macrophage comprising or expressing at least one CAR described herein exhibits induction of cell survival mechanisms, e.g., relative to a macrophage comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a macrophage comprising or expressing at least one CAR described herein exhibits induction of cell death mechanisms, e.g., relative to a macrophage comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a macrophage comprising or expressing at least one CAR described herein exhibits one, two, three, four, or five of increased resistance to phagocytic checkpoints, increased expression of chemokine receptors to aid in trafficking, increased expression of chemokines to recruit other immune cells, increased expression of ECM degrading enzymes (e.g., MMPs to degrade tumor ECM and/or exhibit anti fibrotic activity), and/or increased proliferation, e.g., relative to a macrophage comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a macrophage comprising or expressing at least one CAR described herein exhibits one, two, three, or four of improved duration of CAR expression, improved stability of the CAR on the cell surface, increased level of CAR expression, and/or decreased background activity of the CAR, e.g., relative to a macrophage comprising a similar CAR (e.g., a CAR described herein). [0183] In some embodiments, a macrophage comprising or expressing at least one CAR described herein decreases one or more signs and/or symptoms of an infection (e.g., of an infectious agent) in a subject, e.g., relative to a macrophage comprising a similar CAR (e.g., a CAR described herein). In some embodiments, an infectious agent comprises or is a virus, a protozoa (e.g., trypanosome, malaria, or toxoplasma), a bacteria (e.g., mycobacterium, salmonella, or listeria), a fungi (e.g., Candida), or a combination thereof. In some embodiments, a virus comprises hepatitis vims (e.g., hepatitis A, hepatitis B, hepatitis C, or hepatitis E), retrovirus, human immunodeficiency virus (e.g., HIV1 or HIV2), T cell leukemia virus, a Lymphotropic virus (e.g., HTL.V1 or HTL.V2), heqtes simplex virus (e.g., herpes simplex virus type 1 or type 2), Epstein-Barr virus, cytomegalovirus, varicella-zoster virus, poliovirus, measles virus, Rubella virus, Japanese encephalitis vims, mumps virus, influenza virus, adenovirus, enterovirus, rhinovirus, coronavirus (e.g , severe acute respiratory syndrome (SARS) virus, Middle East respiratory' syndrome (MERS) virus, or severe acute respiratory syndrome coronavirus 2 (SARS-CoV2)), Ebola virus. West Nile virus, or a variant or combination thereof.

[0184] In some embodiments, a macrophage comprising or expressing a at least one CAR described herein decreases formation and/or degrades existing aggregates via phagocytosis of at least one protein aggregate in a subject (e.g., a subject, having a neurodegen erative disease, an inflammatory disease, a cardiovascular disease, a fibrotic disease, amyloidosis, or a combination thereof), e.g., relative to a macrophage comprising a. similar CAR (e.g., a CAR described herein). In some embodiments, a neurodegenerative disease is selected from the group consisting of tauopathy, a-synucleopathy, presenile dementia, senile dementia, Alzheimer's disease, progressive supranuclear palsy (PSP), Pick's disease, primary' progressive aphasia, frontotemporal dementia, corti cobasal dementia, Parkinson's disease, dementia with Lewy bodies, Down's syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS ), Hallervorden-Spatz syndrome, polyglutamine disease, trinucleotide repeat disease, and prion disease. In some embodiments, an inflammatory disease is selected from the group consisting of systemic lupus erythematosus, vasculitis, rheumatoid arthritis, periodontitis, ulcerative colitis, sinusitis, asthma, tuberculosis, Crohn’s disease, chronic infection, hereditary periodic fever, a malignancy, systemic vasculitides, cystic fibrosis, bronchiectasis, epidermolysis bullosa, cyclic neutropenia, an immunodeficiency, Muckle- Wells (MWS) disease, and Familiar Mediterranean Fever (FMF). In some embodiments, amyloidosis is selected from the group consisting of Primary Amyloidosis (AL), Secondary Amyloidosis (AA), Familial Amyloidosis (ATTR) , Beta- 2 Microglobulin Amyloidosis, Localized Amyloidosis, Heavy Chain Amyloidosis (AH), Light Chain Amyloidosis (AL), Primary Systemic Amyloidosis, ApoAI Amyloidosis, ApoAII Amyloidosis, ApoAIV Amyloidosis, Apolipoprotein C2 Amyloidosis, Apolipoprotein C3 Amyloidosis, Corneal lactoferrin amyloidosis, Transthyretin-Related Amyloidosis, Dialysis amyloidosis, Fibrinogen amyloidosis, Lect2 amyloidosis (ALECT2), and Lysozyme amyloidosis. In some embodiments, a cardiovascular disease is selected from the group consisting of atherosclerosis, coronary artery disease, peripheral artery disease, hypertensive heart disease, metabolic syndrome, hypertension, cerebrovascular disease, and heart failure. In some embodiments, a fibrotic disease is selected from the group consisting of pulmonary' fibrosis, idiopathic pulmonary fibrosis, cirrhosis, cystic fibrosis, scleroderma, cardiac fibrosis, radiation- induced lung injury, steatohepatitis, glomerulosclerosis, interstitial lung disease, liver fibrosis, mediastinal fibrosis, retroperitoneal cavity fibrosis, bone marrow fibrosis, and skin fibrosis.

Monocytes

[0185] Monocytes are multipotent cells that circulate in the blood, bone marrow, and spleen, and generally do not proliferate when in a steady state. Monocytes can vary in size significantly in the range of about 10-30 pm in diameter. A ratio of nucleus to cytoplasm for a monocyte can range from about 2: 1 to about 1: 1. Typically, monocytes comprise chemokine receptors and pathogen recognition receptors that mediate migration from blood to tissues, such as during an infection. Monocytes can produce inflammatory cytokines, take up cells and/or toxic molecules, and differentiate into dendritic cells or macrophages.

[0186] In some embodiments, a monocyte comprises or expresses one or more phenotypic markers. Exemplarily phenotypic markers for human monocyte cells include, but are not limited to, CD9, GDI lb, GDI 1c, CDwl2, CD13, GDI 5, CDwl7, CD31, CD32, CD33, CD35, CD36, CD38, CD43, CD49b, CD49e, CD49f, CD63, CD64, CD65s, CD68, CD84, CD85, CD86, CD87, CD89, CD91, CDw92, CD93, CD98, CD101, CD102, CD111, CD112, GDI 15, CD116, CD119, CDwl21b, CDwl23, CD127, CDwl28, CDwl31, CD147, CD155, CD156a, CD 157, CD 162 CD 163, CD 164, CD 168, CD 171, CD 172a, CD 180, CD206, CD 13 lai, CD213 2, CDw210, CD226, CD281, CD282, CD284, and CD286. Exemplarily phenotypic markers for mouse monocyte cells include, but are not limited to, GDI la, CD 1 lb, CD16, CD18, CD29, CD31, CD32, CD44, CD45, CD49d, GDI 15, CD1 16, Cdwl31, CD281, CD282, CD284, CD286, F4/80, and CD49b. In certain embodiments, monocytes comprise one, two, or three of GDI lb, CD 14, or CD 16. In certain embodiments, monocytes comprise CD 14+ CD 16- monocytes, CD 14 CD i b monocytes, or CD 14- CD 16+ monocytes.

[0187] In some embodiments, a monocyte differentiates into a macrophage. In some embodiments, a monocyte differentiates into a dendritic cell (DC). Monocytes can be differentiated into macrophages or DCs by any technique known in the art. For example, differentiation of monocytes into macrophages can be induced by macrophage colony stimulating factor (M-CSF). Differentiation of monocytes into DCs can be induced by granulocyte-macrophage colony stimulating factor (GM-CSF) in combination with IL-4.

[0188] In some embodiments, a monocyte comprising or expressing at least one CAR described herein exhibits increased secretion of one or more cytokines (e.g., one, two, three, four, five, six, or seven of INF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, or IL-1), e.g., relative to a monocyte comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a monocyte comprising or expressing at least one CAR described herein exhibits increased phagocytosis, e.g., relative to a monocyte comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a monocyte comprising or expressing at least one CAR described herein exhibits enhanced survival, e g., relative to a monocyte comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a monocyte comprising or expressing at least one CAR) described herein exhibits enhanced differentiation into macrophages (e.g., Ml or M2 macrophages), e.g., relative to a monocyte comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a monocyte comprising or expressing at least one CAR) described herein exhibits enhanced differentiation into DCs (e.g., resident or migrating DCs and/or in lymphoid and non-lymphoid tissue), e.g., relative to a monocyte comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a monocyte comprising or expressing at least one CAR) described herein exhibits increased cytotoxicity against a tumor cell, e.g., relative to a monocyte comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a monocyte comprising or expressing at least one CAR) described herein exhibits increased tumor antigen presentation (e g., post- phagocytosis presentation) and/or increased antigen processing, e g., relative to a monocyte comprising a similar CAR (e.g,, a CAR described herein). In some embodiments, a monocyte comprising or expressing at least one CAR) described herein exhibits increased tumor killing (e.g., by phagocytosis, lysis, apoptosis, or production of tumor killing cytokines (e.g., TNFa), e.g., relative to a monocyte comprising a similar CAR (e.g , a CAR described herein).

|0189| In some embodiments, a monocyte comprising or expressing at least one CAR described herein exhibits one or both of increased expression of one or more genes typically associated with increased effector function (e.g., phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion) or decreased expression of one or more genes typically associated with decreased effector function (e.g., phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion), e.g., relative to a monocyte comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a monocyte comprising or expressing at least one CAR described herein exhibits increased production of ROS, e.g., relative to a monocyte without a CAR described herein. In some embodiments, a monocyte comprising or expressing at least one CAR described herein exhibits metabolic reprogramming, e.g., relative to a monocyte comprising a similar CAR (e.g,, a CAR described herein). In some embodiments, a monocyte comprising or expressing at. least one CAR described herein exhibits induction of cell survival mechanisms, e.g., relative to a monocyte comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a monocyte comprising or expressing at least one CAR described herein exhibits induction of cell death mechanisms, e.g., relative to a monocyte comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a monocyte comprising or expressing at least one CAR described herein exhibits one, two, three, four, or five of increased resistance to phagocytic checkpoints, increased expression of chemokine receptors to aid in trafficking, increased expression of chemokines to recruit other immune cells, increased expression of ECM degrading enzymes (e.g., MMPs to degrade tumor ECM and/or exhibit anti fibrotic activity), or increased proliferation, e.g., relative to a monocyte without a CAR described herein. In some embodiments, a monocyte comprising or expressing at least one CAR described herein and at. least, one CAR described herein exhibits one, two, three, or four of improved duration of CAR expression, improved stability of the CAR on the cell surface, increased level of CAR expression, and/or decreased background activity of the CAR, e.g., relative to a monocyte without a CAR described herein. Dendritic Cells

[0190] Dendritic cells (DCs) are bone marrow-derived, specialized antigen presenting cells that are involved in initiating immune responses and maintaining tolerance of the immune system to self-antigens. Dendritic cells may be found in both lymphoid and non-lymphoid organs and are generally thought to arise from lymphoid or myeloid lineages.

[0191] In some embodiments, a DC comprises or expresses one or more phenotypic markers. Exemplarily phenotypic markers for DCs include, but are not limited to, CD11c, CD83, CD la, CDlc, CD141, CD207, CLEC9a, CD123, CD85, CD 180, CD 187, CD205, CD281, CD282, CD284, CD286 and partially CD206, CD207, CD208 and CD209.

[0192] Immature DCs can be characterized by a high capacity for antigen capture, but relatively low T cell stimulatory capability. Inflammatory' mediators promote DC maturation. Once DCs reach the mature stage, there is a dramatic change in properties relative to immature DCs, such as a decrease in antigen capture ability and/or an increased ability to stimulate T ceils. In some embodiments, a DC comprises or is an immature DC. In other embodiments, a DC comprises or is a mature DC.

[0193] Without wishing to be bound by theory, it is believed that modification of a DC cell to comprise or express at least one CAR described herein can allow mature DCs to simultaneously exhibit increased antigen capture ability and T cell stimulation, e.g., relative to a DC comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a DC comprising or expressing at least one CAR described herein mediates tumor antigen presentation, e.g., increased tumor antigen presentation relative to a DC comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a DC comprising or expressing at least one CAR described herein mediates tumor T cell stimulation, e.g., increased T cell stimulation relative to a DC comprising a similar CAR (e.g., a CAR described herein).

[0194] In some embodiments, a DC comprising or expressing at least one CAR described herein exhibits increased secretion of one or more cytokines (e.g., one, two, three, four, five, six, or seven of TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, or 11,-1), e.g., relative to a DC comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a DC comprising or expressing at least one CAR described herein exhibits increased phagocytosis, e.g., relative to a DC comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a DC comprising or expressing at least one CAR described herein exhibits increased tumor antigen presentation (e.g., post-phagocytosis presentation), increased antigen processing, increased antigen cross presentation, increased T cell priming, and/or stimulation of T cells, e.g., relative to a DC comprising a similar CAR (e.g., a CAR described herein).

[0195] In some embodiments, a DC comprising or expressing at least one CAR described herein exhibits one or both of increased expression of favorable genes or decreased expression of unfavorable genes, e.g., relative to a DC comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a DC comprising or expressing at least one CAR described herein exhibits increased production of ROS, e.g., relative to a DC comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a DC comprising or expressing at least one CAR described herein exhibits metabolic reprogramming, e.g., relative to a DC comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a DC comprising or expressing at least one CAR described herein exhibits induction of cell survival mechanisms, e.g., relative to a DC comprising a similar CAR (e.g., a CAR described herein).

[0196] In some embodiments, a DC comprising or expressing at least one CAR described herein exhibits induction of cell death mechanisms, e.g., relative to a DC comprising a similar CAR (e.g., a CAR described herein). In some embodiments, a DC comprising or expressing at. least one CAR described herein exhibits one, two, three, four, or five of increased resistance to phagocytic checkpoints, increased expression of chemokine receptors to aid in trafficking, increased expression of chemokines to recruit other immune cells, increased expression of ECM degrading enzymes (e.g., MMPs to degrade tumor ECM and/or exhibit anti fibrotic activity), or increased proliferation, e.g., relative to a DC without a CAR described herein. In some embodiments, a DC comprising or expressing at least one CAR described herein and at least one CAR described herein exhibits one, two, three, or four of improved duration of CAR expression, improved stability of the CAR on the cell surface, increased level of CAR expression, and/or decreased background activity of the CAR, e.g., relative to a DC without a CAR described herein. Chimeric Antigen Receptors (CAR)

[0197] The term “chimeric antigen receptor” or “CAR,” as used herein, refers to an artificial cell surface receptor that is engineered to be expressed on an immune effector cell and specifically targets a cell and/or binds an antigen. CARs may be used, for example, as a therapy with adoptive cell transfer. For example, in some embodiments, immune cells (e.g,, stem cells, macrophages, monocytes, and/or dendritic cells) are removed from a patient (e.g., from blood, tumor or ascites fluid) and modified so that they express a receptor specific to a particular form of antigen. In some embodiments, such modified immune cells are then reintroduced to the same or a different subject as a therapeutics. In some embodiments, CARs have been expressed with specificity to an antigen, for example, a tumor associated antigen. In some embodiments, a CAR comprises an extracellular domain, a transmembrane domain and an intracellular domain.

[0198] In some embodiments, a modified immune cell, for example, a modified stern cell, macrophage, monocyte, or dendritic cell, is generated by expressing a CAR therein. In some embodiments, an immune cell comprises a CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the immune cell comprises a stem cell, macrophage, monocyte, or dendritic cell.

[0199] In some embodiments, a CAR may further comprise one or more of: one or more extracellular leader domains, one or more extracellular hinge domains and one or more intracellular co-stimulatory domains.

[0200] In some embodiments, a CAR comprises a spacer domain or hinge between an extracellular domain and a transmembrane domain (i.e., an extracellular hinge domain). In some embodiments, a CAR comprises a spacer domain or hinge between an intracellular domain and a transmembrane domain (i.e , an intracellular hinge domain). As used herein, the term “spacer domain” or “hinge” refers to any oligo- or polypeptide that functions to link a transmembrane domain to either an extracellular domain or to an intracellular domain in a polypeptide chain. In some embodiments, a spacer domain or hinge may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. In some embodiments, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length, may form a linkage between a transmembrane domain and an intracellular domain of a CAR An example of a linker includes a glycine-serine doublet. [0201 ] In some embodiments, an immune cel! (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR may comprise one or more control systems including, but not limited to: a safety switch (e.g., an on switch, and off switch, a suicide switch), a logic gate, for example an AND gate (e.g., two or more CARs, each of which lacks one or more signaling domains such that activation of both/all CARs is required for full immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) activation or function), an OR gate (e.g., two or more CARs, each with an intracellular domain such as CD3g and a co- stimulatory domain), and/or a NOT gate (e.g., two or more CARs, one of which includes an inhibitory' domain that antagonizes the function of the other CAR[s]).

[0202] The present disclosure also provides immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising a nucleic acid sequence (e.g., an isolated nucleic acid sequence) encoding a CAR, wherein the nucleic acid sequence comprises a nucleic acid sequence encoding an extracellular domain, a nucleic acid sequence encoding a transmembrane domain and a nucleic acid sequence encoding an intracellular domain, wherein the cell is a stem cell, macrophage, monocyte or dendritic cell that expresses the CAR

[0203] In some embodiments, a CAR comprises an extracellular domain that is operably linked to another domain of the CAR, such as a transmembrane domain or an intracellular domain, for expression in an immune cell. In some embodiments, a nucleic acid encoding an extracellular domain is operably linked to a nucleic acid encoding a transmembrane domain and the nucleic acid encoding the transmembrane domain is operably linked to a nucleic acid encoding an intracellular domain.

[0204] In some embodiments, an effector activity of an immune cell comprising a CAR is directed against a target cell comprising an antigen that specifically binds an antigen binding domain of the CAR. In some embodiments, a targeted effector activity directed against a target cell is or comprises phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion.

[0205] In some embodiments, a CAR described herein comprises at least one domain (e.g., an extracellular domain, a transmembrane domain, and/or an intracellular domain) that inhibits anti -phagocytic signaling in an immune cell described herein (e.g., a stem cell, macrophage, monocyte, or dendritic cell). In some embodiments, a CAR described herein improves effector activity of an immune cell described herein (e.g., a stem cell, macrophage, monocyte, or dendritic cell), e.g., by enhancing inhibition of CD47 and/or SIR Pc activity. In some embodiments, a CAR described herein binds CD47, e.g., and serves as a dominant negative receptor, inhibiting SIRPa activity (e.g., a CD47 sink). In some embodiments, a CAR described herein that binds SIRPa, e.g., comprises an activating receptor (e.g., comprises a CD3z intracellular domain). In some embodiments, a CAR described herein inhibits at least one interaction of CD47 and SIRPa. In some embodiments, a CAR is or comprises a phagocytic logic gate.

[0206] In some embodiments, an immune cell described herein (e.g., comprising at least one CAR described herein) comprises or expresses at least one variant or fragment of SIRPa (e.g., a dominant negative SIRPa or a high-affinity engineered variant of SIRPa (e.g., CV1)), 5F9 scFv, B6H12 scFv (e.g , a humanized B6H12 scFv), PD1 (e.g., a dominant negative PD1 or HAC-I), anti-PDl scFv (e.g., E27 or durvalumab), Siglec-10, Siglec-9, Siglec-11, and/or SHP-1. In some embodiments, a variant or fragment comprises a mutated intracellular domain. In some embodiments, a variant or fragment does not comprise or express at least one intracellular domain (e.g., an immune cell comprises or expresses an ant.i-CD47 scFv, CDS hinge domain, and CD8 transmembrane). In some embodiments, an immune cell described herein (e.g., comprising or expressing at least one CAR described herein) comprises a dominant negative receptor, e.g., blocking an inhibitory checkpoint.

[0207] In some embodiments, a CAR described herein further comprises a cleavage peptide (e.g., a P2A, F2A, E2A and/or T2A peptide) and at least one second CAR comprising at least one inhibitory domain of anti -phagocytic signaling. In some embodiments, at least one second CAR comprises a SIRPa (e.g., a high-affinity engineered variant of SIRPa (e.g., CV1)), 5F9 scFv, B6H12 scFv (e.g., a humanized B6H12 scFv), or a CD47 binding extracellular domain or a fragment thereof. In some embodiments, at least one second ('A R. comprises a SIRPa transmembrane domain or a fragment thereof. In certain embodiments, a second CAR further comprises a hinge domain (e.g., a CD8 hinge domain). In certain embodiments, at least one second CAR comprises: (i) a leader sequence (e.g., a CDS leader); ii) an extracellular domain (e.g., a SIRPa, CV1, 5F9 scFv, or B6H12 scFv (e.g., a humanized B6H12 scFv) extracellular domain); and ii) a transmembrane domain (e.g., a SIRPa transmembrane domain). Tn some embodiments, a CAR described herein further comprises a cleavage peptide (e.g., a P2A peptide) and at least one marker protein (e.g., CD20 or a fragment thereof, CD 19 or a fragment thereof, NGFR or a fragment thereof, a synthetic peptide, and/or a fluorescent protein).

[0208] In some embodiments, an immune cell described herein (e.g., comprising or expressing at least one CAR described herein) comprises or expresses one or more phosphatase dead domains (e.g. a phosphatase dead Shpl, phosphatase dead 72-5ptase (INPP5E), phosphatase dead Shp2, and/or phosphatase dead SHIP- 1 domain) and/or a constitutively active kinase domain (e.g., a constitutively active LYN domain). In some embodiments, a CAR described herein further comprises a cleavage peptide (e.g., a P2A, F2A, E2A and/or T2A peptide) and one or more phosphatase dead domains (e.g. a phosphatase dead Shpl , phosphatase dead 72-5ptase (INPP5E), phosphatase dead Shp2, and/or phosphatase dead SHIP-1 domain) and/or a constitutively active kinase domain (e.g., a. constitutively active LYN domain).

CAR Extracellular Domains

[0209] The present disclosure provides chimeric antigen receptors (CAR) comprising extracellular domains. In some embodiments, a CAR extracellular domain comprises an Fc receptor (FcR) extracellular domain. In some embodiments, a CAR extracellular domain comprises a toll-like receptor (TLR) extracellular domain. In some embodiments, a CAR extracellular domain comprises a leader domain. In some embodiments, a CAR extracellular domain comprises an antigen binding domain. In some embodiments, a CAR extracellular domain comprises a hinge domain. In some embodiments, a CAR extracellular domain comprises one or more of an FcR extracellular domain, a TLR extracellular domain, a leader domain, an antigen binding domain and a hinge domain. In some embodiments, a CAR extracellular domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a CAR extracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein),

FcR Extracellular Domains [0210] In some embodiments, an FcR extracellular domain comprises a full-length FcR extracellular domain. In some embodiments, an FcR extracellular domain comprises a portion of a full-length FcR extracellular domain. In some embodiments, an FcR extracellular domain (or portion thereof) is or comprises a human FcR extracellular domain. In some embodiments, an FcR extracellular domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an FcR extracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an FcR extracellular domain comprises a FcRy, CD64 (FcyRI), CD32a (FcyRIIa), CD32b (FcyRIIb), CD32c, CD 16a (FcvRIIIa), CD 16b (FcyRIIIb), FcsRI, FcsRII, or FcaRI (CD89) domain.

TLR Extracellular Domains

[0211] In some embodiments, a TLR extracellular domain comprises a full-length TLR extracellular domain. In some embodiments, a TLR extracellular domain comprises a portion of a full-length TLR extracellular domain. In some embodiments, a TLR extracellular domain (or portion thereof) is or comprises a human TLR extracellular domain. In some embodiments, a TLR extracellular domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein) In some embodiments, a TLR extracellular domain may be a domain that is not endogenous to a particular immune cel I type (e.g., a modified immune cell as provided herein) In some embodiments, a TLR extracellular domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.

Leader Domains

[0212] In some embodiments, a CAR comprises one or more extracellular leader domains. In some embodiments, a nucleic acid encoding a CAR comprises a nucleic acid sequence encoding an extracellular leader domain, but the extracellular leader domain is cleaved from the CAR before the CAR is expressed in an immune cell In some embodiments, an extracellular leader domain is or comprises a human extracellular leader domain. In some embodiments, an extracellular leader domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an extracellular leader domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an extracellular leader domain comprises a CD8 extracellular leader domain. In some embodiments, an extracellular leader domain comprises a leader domain from a stimulatory or co-stirnulatory domain (e.g., a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD19, CD20, 41 BB, CD28, 0X40, GITR, TREM-1, TREM-2, DAP 12, MR, ICOS, MyD88 domain).

Antigen Binding Domains

[0213] In some embodiments, a CAR comprises an antigen binding domain that binds to an antigen, for example, on a target cell. In some embodiments, a CAR comprises an antigen binding domain that binds to an antigen associated with viral infection, bacterial infection, parasitic infection, autoimmune disease, and/or cancer cells. In some embodiments, a CAR antigen binding domain recognizes an antigen that acts as a cell surface marker on a target cell associated with a particular disease state

[0214] In some embodiments, a CAR antigen binding domain binds to a tumor antigen, such as an antigen that is specific for a tumor or cancer of interest In some embodiments a tumor antigen comprises one or more antigenic cancer epitopes. In some embodiments, a tumor antigen comprises CD19; CD123; CD22; CD30; CD171; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24), C-type lectin-like molecule- 1 (CLL-l or CLECL1), CD33; epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-l)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca-Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-Like Tyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72);

CD38; CD44v6, Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (GDI 17); Interleukin- 13 receptor subunit alpha-2 (!L-13Ra2 or CD213A2); Mesothelin; Interleukin 11 receptor alpha (IL-l lRa); prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2), Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR-beta); Stagespecific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2 (Her2/neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100 (gplOO); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abel son murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type-A receptor 2 (EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2- 3)bDGalp(l-4)bDGlcp(l-l)Cer); transglutaminase 5 (TGS5), high molecular weight-melanoma- associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); Folate receptor beta; tumor endothelial marker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD 179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide portion of globoH glycoceramide (GloboH); mammary gland differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); Hepatitis A virus cellular receptor 1 (FIAVCR1); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein -coupled receptor 20 (GPR20); lymphocyte antigen 6 complex, locus K 9 (LY6K); Olfactory receptor 51E2 (OR51E2); TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer/testis antigen 1 (NY-ESO-1); Cancer/testis antigen 2 (LAGE- la); Melanoma- associated antigen 1 (MAGE-A1); ETS translocation- variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA17); X Antigen Family, Member 1 A (XAGEl ); angiopoi etin-binding cell surface receptor 2 (Tie 2); melanoma cancer testis antigen- 1 (MAD- CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1 , tumor protein p53 (p53); p53 mutant; prostein; surviving; telomerase; prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (MelanA or MARTI); Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N- Acetyl glucosaminyl-transferase V (NA 17); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin Bl; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 IB I (CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein )-Like (BORIS or Brother of the Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3): Paired box protein Pax-5 (PAX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPV E7), intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72, Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Fc fragment of IgA receptor (FC AR or CD89); Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like modulecontaining mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75); Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); or immunoglobulin lambda-like polypeptide 1 (IGLL1). In certain embodiments, a tumor antigen comprises ERBB2 (Her2/neu). In certain embodiments, a tumor antigen comprises PSMA. In certain embodiments, a tumor antigen comprises Mesothelin.

[0215] In some embodiments, a CAR antigen binding domain binds to a misfolded protein antigen or a protein of a protein aggregate, such as a protein that is specific for a disease/disorder of interest. In some embodiments, the disease/disorder is a neurodegenerative disease/disorder, an inflammatory disease/disorder, a cardiovascular disease/disorder, a fibrotic disease/disorder, or amyloidosis (e.g., mediated by protein aggregates of immunoglobulin light chains or of transthyretin). In some embodiments, the neurodegenerative disease/disorder is selected from the group consisting of tauopathy, asynucleopathy, presenile dementia, senile dementia, Alzheimer's disease (mediated by protein aggregates of beta-amyloid), Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy (PSP), Pick' s disease, primary progressive aphasia, frontotemporal dementia, corticobasal dementia, Parkinson's disease, Parkinson's disease with dementia, dementia with Lewy bodies, Down syndrome, multiple system atrophy, amyotrophic lateral sclerosis (ALS), Hallervorden-Spatz syndrome, polyglutamine disease, trinucleotide repeat disease, Familial British dementia, Fatal Familial Insomnia, Gerstmann-Straussler-Scheinker Syndrome, Hereditary cerebral hemorrhage with amyloidosis (Icelandic) (HCHW A-I), Sporadic Fatal Insomnia (sFI), Variably Protease- Sensitive Prionopathy (VPSPr), Familial Danish dementia, and prion disease (such as Creutzfeldt-Jakob disease, CJD and Variant Creutzfeldt-Jakob Disease (vCJD)).

[0216] In some embodiments, a CAR antigen binding domain comprises any domain that binds to an antigen. In some embodiments, a CAR antigen binding domain is or comprises a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, or any fragment thereof, for example an scFv. In some embodiments, a CAR antigen binding domain is or comprises an aptamer, a darpin, a centyrin, a naturally occurring or synthetic receptor, an affibody, or other engineered protein recognition molecule. In some embodiments, a CAR antigen binding domain is or comprises a mammalian antibody or a fragment thereof. In some embodiments, a CAR antigen binding domain is derived, in whole or in part, from the same species in which the CAR will ultimately be used. For example, for use in humans, an antigen binding domain of a CAR comprises a human antibody, a humanized antibody, or a fragment thereof (e.g. a scFv). In some embodiments, a CAR antigen binding domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a CAR antigen binding domain may be a domain that is not endogenous to a particular immune cell type (e g , a modified immune cell as provided herein)

[0217] In some embodiments, a CAR comprises one or more antigen binding domains. In some embodiments, a CAR comprises two or more antigen binding domains. In some embodiments, a CAR is a bispecific CAR. In some embodiments, an immune cell comprises two or more different CARs comprising one or more antigen binding domains. In some embodiments, an immune cell comprising a bispecific CAR and/or comprising two or more different CARs comprising one or more antigen binding domains can reduce off-target and/or on-target off-tissue effects by requiring that two antigens are present. In some embodiments, an immune cell comprises a bispecific CAR and/or comprises two or more different CARs comprising one or more antigen binding domains, wherein the CARs provide distinct signals that in isolation are insufficient to mediate activation of the modified cell, but are synergistic together, stimulating activation of the modified cell. In some embodiments, such a construct may be referred to as an ‘AND’ logic gate. [0218] In some embodiments, an immune cell comprising a bispecific CAR and/or comprising two or more different CARs comprising one or more antigen binding domains can reduce off-target and/or on-target off-tissue effects by requiring that one antigen is present and a second, normal protein antigen is absent before the cell’s activity is stimulated. In some embodiments, such a construct may be referred to as a ‘NOT’ logic gate. In contrast to AND gates, NOT gated CAR-modified cells are activated by binding to a single antigen. However, the binding of a second receptor to the second antigen functions to override the activating signal being perpetuated through the CAR. Typically, such an inhibitory receptor would be targeted against an antigen that is abundantly expressed in a normal tissue but is absent in tumor tissue.

Hinge Domains

[0219] In some embodiments, a CAR comprises one or more extracellular hinge domains. In some embodiments, a CAR extracellular hinge domain is or comprises a human extracellular hinge domain. In some embodiments, a CAR extracellular hinge domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a CAR extracellular hinge domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, one or more CAR extracellular hinge domains comprise a CD8a extracellular hinge domain, a DNGR-1 extracellular hinge domain, a Dectin- 1 extracellular hinge domain, a DAP10 extracellular hinge domain, or an IgG4 or a CD28 extracellular hinge domain. In some embodiments, a CAR comprises or is a CD28 extracellular hinge domain. In some embodiments, a CAR comprises or is a CD8a extracellular hinge domain. In some embodiments, a CAR comprises or is a DAP 10 extracellular hinge domain. In some embodiments, a CAR extracellular hinge domain optimizes the physicochemical parameters of a CAR, e.g., optimal size relative to tumor antigen (e.g., allowing for exclusion of inhibitory' molecules), optimal flexibility, optimal protein folding, optimal protein stability, optimal binding, optimal homodimerization, and/or lack of homodimerization.

CAR Transmembrane Domains

[0220] In some embodiments, a CAR comprises a transmembrane domain, for example, that connects an extracellular domain to an intracellular domain. In some embodiments, a CAR transmembrane domain is naturally associated with one or more other domain(s) of a CAR In some embodiments, a CAR transmembrane domain can be modified to avoid binding to transmembrane domains of other surface membrane proteins, in order to minimize interactions with other members of a receptor complex. In some embodiments, a CAR transmembrane domain may be derived either from a naturally-occurring or from a synthetic source. In some embodiments a CAR transmembrane domain is derived from a naturally-occurring membranebound or transmembrane protein. In some embodiments, a CAR transmembrane domain is or comprises a human transmembrane domain. In some embodiments, a CAR transmembrane domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a CAR transmembrane domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a CAR transmembrane domain comprises a CD8a, CD64, DAP10, CD32a, CD32c, CD16a, TRL1, TLR2, TLR3, TRIA TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, IN SR, cMET, MUSK, PDGFR, PTK7, RET, R0R1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD19, CD2.0, 41BB, CD28, 0X40, GITR, TREM-1, TREMA, DAP12, MR, ICOS, MyD88, CD3-zeta, Dectin-1, DNGR1, SLAMF7, FcR v, V/I/LxYxxL/V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRPp, CD22, PIR-B, LILRB1, CD36, TRIF, or Syk transmembrane domain. In some embodiments, a CAR comprises or includes a CD28 transmembrane domain. In some embodiments, a CAR comprises or is a CD8 transmembrane domain In some embodiments, a CAR comprises or is aDAPIO transmembrane domain. In some embodiments, a CAR comprises or is a CD64 transmembrane domain.

FcR Transmembrane Domains

[0221 ] In some embodiments, an FcR transmembrane domain comprises a full-length

FcR transmembrane domain. In some embodiments, an FcR transmembrane domain comprises a portion of a full-length FcR transmembrane domain. In some embodiments, an FcR transmembrane domain is or comprises a human FcR transmembrane domain, or portion thereof. In some embodiments, an FcR transmembrane domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an FcR transmembrane domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an FcR transmembrane domain comprises a FcRy, CD64 (FcyRI), CD32a (FcyRIIa), CD32b (FcyRIIb), CD32c, CD 16a (FcyRIIIa), CD 16b (FcyRIIIb), FCERI, FcsRII, or FcaRI (CD89) domain. In some embodiments, an FcR transmembrane domain comprises or is FceRI. In some embodiments, an FcR transmembrane domain comprises or is FcRy (also known as FCER1G). In some embodiments, an FcR transmembrane domain comprises or is CD64 (FcyRI).

TLR Transmembrane Domains

In some embodiments, a TLR transmembrane domain comprises a full-length TLR transmembrane domain. In some embodiments, a TLR transmembrane domain comprises a portion of a full-length TLR transmembrane domain. In some embodiments, a TLR transmembrane domain is or comprises a human TLR transmembrane domain, or portion thereof. In some embodiments, a TLR transmembrane domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a TLR transmembrane domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a TLR transmembrane domain comprises a TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.

CAR IntraceUiilar Domains

[0222] In some embodiments, a CAR comprises one or more intracellular domains. In some embodiments, a CAR intracellular domain is or comprises a human intracellular domain, or portion thereof. In some embodiments, a C AR intracellular domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a CAR intracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a CAR intracellular domain and/or other cytoplasmic domain of a CAR is responsible for activation of the cell in which the CAR is expressed (e.g., an immune cell). In some embodiments, a CAR intracellular domain of a CAR is responsible for signal activation and/or transduction in an immune cell comprising said CAR. [0223] In some embodiments, a CAR intracellular domain of a CAR includes at least one domain responsible for signal activation and/or transduction. In some embodiments, a CAR intracellular domain is or comprises at least one of a co-stimulatory molecule and a signaling domain. In some embodiments, a CAR intracellular domain of a CAR comprises dual signaling domains. In some embodiments, a CAR intracellular domain of a CAR comprises more than two signaling domains.

[0224] In some embodiments, a CAR intracellular domain comprises a cytoplasmic portion of a surface receptor. In some embodiments, a CAR intracellular domain comprises a co-stimulatory molecule. In some embodiments, a CAR intracellular domain comprises a molecule that acts to initiate signal transduction in an immune cell.

[0225] In some embodiments, an intracellular domain of a CAR includes any portion of one or more co-stimulatory molecules, such as at least one signaling domain from CD3, Fc epsilon RI gamma chain, MyD88, any derivative or variant thereof, any synthetic sequence thereof that has the same functional capability, and any combination thereof.

FciR hsiri ti:eFsi:ar Domains

[0226] In some embodiments, an FcR intracellular domain comprises a full-length FcR intracellular domain. In some embodiments, an FcR intracellular domain comprises a portion of a full-length FcR intracellular domain. In some embodiments, an FcR intracellular domain is or comprises a human FcR intracellular domain, or portion thereof. In some embodiments, an FcR intracellular domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an FcR intracellular domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, an FcR intracellular domain comprises a FcRy, CD64 (FcyRI), CD32a (FcyRIIa), CD32b (FcyRIIb), CD32c, CD 16a (FcvRIIIa), CD 16b (FcyRIIIb), FcsRI, FceRII, or FcaRI (CD89) domain.

TLR Iriiracellulaf Domains

[0227] In some embodiments, a TLR intracellular domain comprises a full-length TLR intracellular domain. In some embodiments, a TLR intracellular domain comprises a portion of a full-length TLR intracellular domain. In some embodiments, a TLR intracellular domain is or comprises a human TLR intracellular domain, or portion thereof. In some embodiments, a TLR intracellular domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a TLR intracellular domain may be a domain that is not endogenous to a particular immune cell type (e g., a modified immune ceil as provided herein). In some embodiments, a TLR intracellular domain comprises a TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9 domain.

Signaling Domains

[0228] In some embodiments, a CAR comprises one or more intracellular signaling domains. In some embodiments, a CAR intracellular signaling domain is or comprises a human intracellular signaling domain, or portion thereof. In some embodiments, a CAR signaling domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a CAR signaling domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).

[0229] In some embodiments, one or more CAR intracellular signaling domains comprise a CD3 -zeta, FcR y, CD64, CD32a, CD32c, CD16a, CD40, CD89, TLRl, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD40, CD 19, CD20, 4 IBB, CD28, 0X40, GITR, TREM-1, TREM-2, DAP12, AIR, ICOS, MyD88, V/ELxYxxL/V, SIRPa, CD45, Siglec-10, PD1 , SHP-1, SHP-2, K1R-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SIRP0, CD22, PIR-B, LILRB1, Syk, 41BB ligand (41BBL; TNFSF9), CD27, OX40L, CD32b, CD 11b, ITGAM, SLA.MF7, CD206, CD 163, CD209, GCSFR (CD 114), RAGE, CD30, CD 160, DR3, Fill 4, HVEM, CD 160, NGFR, RANK, TNFR2, TROY, XEDAR, TRIF, Dectin-2, or one or more cytokine receptor signaling domains (e.g., an IL1R, an IL2R, an IL3R, an IL4R, an 1L5R, an LL6R, an IL7R, an IL8R, an IL9R, an 1L10R, an LL11R, an IL12R, an IL13R, an IL14R, an IL15R, an IL17R, an IFNaR, an IFNgR, an TNFR, an CSF1R, an CSF2R, DAP10, CD36, Dectin-1, or ICOSL intracellular signaling domain).

[0230] In some embodiments, a CAR intracellular domain is or comprises a MyD88 intracellular domain or a portion thereof. In some embodiments, a MyD88 intracellular domain or a portion thereof is a derivative or variant of a naturally occurring MyD88 domain or a portion thereof In some embodiments, a variant of a MyD88 intracellular domain or a portion thereof comprises at least one mutation relative to a naturally occurring MyD88 intracellular domain or a portion thereof. In some embodiments, a MyD88 intracellular domain or a portion thereof comprises one or more mutations selected from E52A, R32A, R32K, Y58A, E52A/R32A/Y58A, L93P, S34Y, and R98C as compared to a naturally occurring MyD88 intracellular domain or a portion thereof. In some embodiments, a MyD88 intracellular domain or a portion thereof is or comprises an amino acid sequence of:

MAAGGPGAGSAAPVSSTSSLPLAAENMRYRRRLSLFENVRTQVAADWTALAEEMDFE YLEIRQLETQADPTGRLLDAWQGRPGASVGRIJ.dff.J..TKI..GRDDVl.J..ELGPSIEEDCQKYI LKQQQEEAEKPLQ\EAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDI (SEQ ID NO: 122), or an amino acid sequence that differs by no more than three, by no more than two, or by no more than one substitutions, additions, or deletions. In some embodiments, a MyD88 intracellular domain is at least 80% identical (e.g., at least 85%, 90%, 95%, 99% identical) to SEQ ID NO: 122.

[0231] In some embodiments, a MyD88 intracellular domain or a portion thereof is or comprises an amino acid sequence of

MAAGGPGAGSAAPVSSTSSLPLAALNMRVRRRLSLFLNVRTQVAADWTALAEEMDFE YLEIRQLETQADPTGRLLDAWQGRPGASVGRLLELLTKLGRDDATLELGPSIEEDCQKYI LKQQQEEAEKPLQVAAVDSSVPRTAELAGITTLDDPLGHMPERFDAFICYCPSDIQFVQE N41RQLEQTNYREKLCVSDRDVLPGTCVWSIASEEIEKRCRRMVVVVSDDYLQSKECDFQ TKFALSLSPGAIIQKRLIPIKYKAMKKEFPSILRFITVCDYTNPCTKSWFWTRLAKALSLP (SEQ ID NO: 167), or an amino acid sequence that differs by no more than three, by no more than two, or by no more than one substitutions, additions, or deletions. In some embodiments, a MyD88 intracellular domain is at least 80% identical (e.g., at least 85%, 90%, 95%, 99% identical) to SEQ ID NO: 167

[0232] In some embodiments, an intracellular domain of a CAR comprises dual signaling domains, such as 41BB, CD28, ICOS, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, CD116 receptor beta chain, CSF1 -R, LRP1/CD91, SR- Al, SR-A2, MARCO, SR-CL1, SR-CL2, SR-C, SR-E, CR1, CR3, CR4, Dectin 1 , DEC-205, DC-SIGN, CD 14, CD36, LOX-1, CDllb, together with arty of the signaling domains listed in the above paragraph in any combination.

Co-stimidatory Domains

[0233] As used herein, a “co- stimulatory molecule” or “co-stimulatory domain” refers to a molecule in an immune cell that is used to heighten or dampen an initial stimulus. For example, pathogen-associated pattern recognition receptors, such as TLR or the CD47/SIRPa axis, are molecules on immune cells that, respectively, heighten or dampen an initial stimulus. In some embodiments, a CAR co-stimulatory domain comprises TCR, CD3-zeta, CD3 gamma, CD3 delta, CD3 epsilon, CD86, common FcR gamma, FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fcgamma Rlla, DAP 10, DAP 12, T cell receptor (TCR), CD27, CD28, 4-1BB (CD 137), 0X40, CD30, CD40, PD-1, ICOS, 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, NKpSO (KLRF1), CD 127, CD 160, CD 19, CD4, CD8alpha, CDSbeta, 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 I I a, LFA-1, ITGAM, CDl lb, 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), CD 160 (BY55), PSGL1, CD 100 (SEMA4D), CD69, SLAMF6 (NTB-A, LyI08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD 162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, NKp44, NKp30, NKp46, NKG2D, TRIF, other co-stimulatory 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 combinations thereof.

[0234] In some embodiments, a C AR co-stimulatory domain may be a domain that is endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein). In some embodiments, a CAR co-stimulatory domain may be a domain that is not endogenous to a particular immune cell type (e.g., a modified immune cell as provided herein).

[0235] As used herein, a “co-stimulatory signal” refers to a signal, which in combination with a primary signal, such as activation of a CAR on an immune cell, leads to activation of the immune ceil. Cleavage Peptides

[0236] As used herein, a cleavage peptide refers to a peptide that can induce the cleaving of a recombinant protein in a cell. In some embodiments, a cleavage peptide is a 2A peptide. In some embodiments, a cleavage peptide is or comprises a P2A, F2A, E2A or T2A peptide. In some embodiments, a nucleic acid described herein comprises one or more nucleic acid sequences encoding one or more cleavage peptides. In some embodiments, a nucleic acid comprising a nucleic acid sequence encoding a cleavage peptide also comprises one or more nucleic acid sequences encoding one or more intracellular domains and one or more nucleic acid sequences comprising one or more peptide agents, wherein translation of the nucleic acid results in a protein comprising one or more intracellular domains separated from one or more peptide agents by a cleavage peptide. In some embodiments, a first promoter is operably linked to one or more nucleic acids encoding a CAR and a second promoter is operably linked to one or more nucleic acids encoding a peptide agent. In some embodiments, a nucleic acid sequence comprising a CAR, and optionally one or more peptide agents, further comprises an internal ribosome entry site (IRES) sequence. An IRES sequence may be any viral, chromosomal or artificially designed sequence that initiates cap-independent ribosome binding to mRNA facilitates the initiation of translation.

CAR Peptide Agents

[0237] As used herein, a CAR peptide agent refers to a peptide co-expressed with a CAR in an immune ceil. In some embodiments, a CAR peptide agent is co-expressed with a CAR to ensure stoichiometric balance and optimal signaling of a CAR. In some embodiments, a CAR peptide agent forms a homodimer with an identical peptide agent. In some embodiments, a CAR peptide agent forms a heterodimer with a different peptide agent. In some embodiments, a nucleic acid described herein comprises one or more nucleic acid sequences encoding one or more CAR peptide agents. In some embodiments, a CAR peptide agent is or comprises an FcR gamma chain.

[023§] In some embodiments, a CAR peptide agent comprises any peptide, protein, receptor, secreted antibody or a fragment thereof (e.g., an scFv, Fab, Fab', F(ab')2, Fc, or nanobody). In some embodiments, a CAR peptide agent comprises one or more cytokines (e.g , one or more of IL-1, IL-2, IL-6, IL-8, TNF-a, IFNa, IFNb, IFN-y, GMCSF, or MCSF), CD40-L, dominant negative SIRPa, dominant negative PD1, dominant negative CD45, dominant negative SIGLEC 10, or dominant negative LILRB.

Fc Receptors (FcR)

[0239] In some embodiments, a CAR comprises one or more antigen binding domains and an FcR extracellular domain, and/or the transmembrane domain of the CAR comprises an FcR transmembrane domain, and/or the intracellular domain of the CAR comprises an FcR intracellular domain. In some embodiments, a CAR comprises, from N-terminus to C-terminus, one or more extracellular domains, an FcR extracellular domain, an FcR transmembrane domain, and an FcR intracellular domain. In some embodiments, one or more of the FcR extracellular domain, the FcR transmembrane domain and the FcR intracellular domain is or comprises a human FcR. domain. In some embodiments, an FcR extracellular domain, an FcR transmembrane domain and an FcR intracellular domain together comprise a full-length FcR. In some embodiments, an FcR. extracellular domain, an FcR transmembrane domain and an FcR intracellular domain together comprise a portion of a full-length FcR. In some embodiments, an FcR extracellular domain comprises a portion of a full-length FcR extracellular domain. In some embodiments, an FcR transmembrane domain comprises a portion of a full-length FcR transmembrane domain. In some embodiments, an FcR intracellular domain comprises a portion of a full-length FcR intracellular domain.

Toll-Like Antigen Receptors (TLR)

[0240] In some embodiments, a CAR comprises one or more antigen binding domains and a toll-like receptor (TLR) extracellular domain and/or the transmembrane domain of the CAR comprises a TLR transmembrane domain and/or the intracellular domain of the CAR comprises a TLR intracellular domain. In some embodiments, a CAR comprises, from N- terniinus to C-terminus, one or more extracellular domains, a TLR extracellular domain, a TLR transmembrane domain, and a TLR intracellular domain. In some embodiments, one or more of the TLR extracellular domain, the TLR transmembrane domain and the TLR intracellular domain is or comprises a human TLR domain. In some embodiments, a TLR extracellular domain, a TLR transmembrane domain and a TLR intracellular domain together comprise a full- length TLR. In some embodiments, a TLR extracellular domain, a TLR transmembrane domain and a TLR intracellular domain together comprise portion of a full-length TLR. In some embodiments, a TLR extracellular domain comprises a portion of a full-length TLR extracellular domain. In some embodiments, a TLR transmembrane domain comprises a portion of a full- length TLR transmembrane domain. In some embodiments, a TLR intracellular domain comprises a portion of a full-length TLR intracellular domain.

Methods of Immune Cell Modification

[0241] The present disclosure provides, among other things, methods for modifying an immune ceil (e.g., a stem cell, monocyte, macrophage, or dendritic cell) comprising delivering to the immune cell a nucleic acid construct comprising one or more nucleic acids encoding one or more CARs described herein. Methods can comprise delivering to an immune cell (e.g., a stem cell, monocyte, macrophage, or dendritic cell), a nucleic acid construct comprising one or more nucleic acids encoding: at least one extracellular domain described herein, at least one transmembrane domain described herein, and at least one intracellular domain described herein.

Delivery Methods

[0242] A nucleic acid construct comprising one or more nucleic acid sequences encoding at least one CAR described herein can be introduced into an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) by physical, chemical, or biological methods. In some embodiments, the present disclosure provides methods for modifying an immune cell comprising producing a modified immune cell (e.g., a stem cell, monocyte, macrophage, or dendritic cell) ex vivo. In some embodiments, the present disclosure provides methods for modifying an immune cell comprising producing a modified immune cell (e.g., a stem cell, monocyte, macrophage, or dendritic cell) in a subject (i.e., in vivo). For example, in some embodiments, certain provided methods of modifying immune cells (e.g., stem cells, macrophages, monocytes, and/or dendritic cells) in a subject include administering/delivering compositions comprising one or more nucleic acid molecules, wherein at least a portion of the one or more nucleic, acid molecules encodes the CAR, and a delivery vehicle to the subject.

[0243] Physical methods for introducing a nucleic acid construct described herein into an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) can comprise electroporation, calcium phosphate precipitation, lipofection, particle bombardment, microinjection, or a combination thereof. A nucleic acid construct can be introduced into immune cells using commercially available methods, including electroporation (Amaxa Nucleofector-II® (Amaxa Biosystems, Cologne, Germany), ECM 830 BTX (Harvard Instruments, Boston, Mass.) Gene Pulser II® (BioRad, Denver, Colo.), or Multiporator® (Eppendort, Hamburg Germany)). A nucleic acid construct can also be introduced into immune cells using mRNA transfection, e.g., cationic liposome "mediated transfection, lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic particle delivery systems, such as “gene guns” (See, e.g., Nishikawa, et al. Hum Gene Then, 12(8):861-70 (2001), which is hereby incorporated by reference in its entirety),

[0244] Biological methods for introducing a nucleic acid construct described herein into an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) include use of DNA and RNA vectors. In one embodiment, a vector comprises a plasmid vector, a viral vector, a transposon, a retrotransposon (e.g., piggyback, sleeping beauty), a site directed insertion vector (e.g., CRISPR, Zn finger nucleases, TALEN), suicide expression vector, or another vector known in the art. Viral vectors, and especially retroviral vectors, have become widely used for inserting genes into mammalian cells (e.g., human cells). Viral vectors can also be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses (e.g. Ad5f35), or adeno-associated viruses (See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362, which are hereby incorporated by reference in their entirety). Retroviral vectors, such as lentivirus, are suitable tools to achieve long-term gene transfer that allow for long-term, stable integration of a transgene and its propagation in daughter cells. In some embodiments, a lentiviral vector is packaged with a VPX protein (e.g., as described in International Publication No. WO 2017/044487, which is hereby incorporated by reference in its entirety). In some embodiments, VPX comprises a virion- associated protein (e.g., an accessory protein for viral replication). In some embodiments, a VPX protein is encoded by human immunodeficiency virus type 2 (HTV-2). Tn some embodiments, a VPX protein is encoded by simian immunodeficiency virus ( SIV ). In some embodiments, an immune cell described herein (e.g., a stern cell, macrophage, monocyte, or dendritic cell) is transfected with a lentiviral vector packaged with a VPX protein. In some embodiments, VPX inhibits at least one antiviral factor of an immune cell described herein (e.g., a stem cell, macrophage, monocyte, or dendritic cell). In some embodiments, a lentiviral vector packaged with a VPX protein exhibits increased transfection efficiency of an immune cell described herein (e.g., a stem cell, macrophage, monocyte, or dendritic cell), e.g., relative to a lentiviral vector not packaged with a VPX protein. Tn some embodiments, an immune cell described herein (e.g., a stem cell, macrophage, monocyte, or dendritic cell) is one or both of electroporated or transfected with at least one VPX mRNA prior to transfection with a viral vector (e.g., an adenoviral vector, e.g., an Ad2 vector or an Ad5 vector (e.g., Ad5f35 adenoviral vector, e g , a helper-dependent Ad5F35 adenoviral vector)).

[0245] Chemical means for introducing a nucleic acid construct described herein into an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) include colloidal dispersion systems, macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems (e.g., oil-in-water emulsions, micelles, mixed micelles, nanoparticles, liposomes, and lipofectamine-nucleic acid complexes).

[ 0246 ] An exemplary' system for delivery of a nucleic acid construct described herein is a lipid-based system. A nucleic acid construct described herein may be encapsulated in an aqueous interior of a liposome, interspersed within a lipid bilayer, attached to a liposome via a linking molecule, attached to a lipid nanoparticle (LNP) via a linking molecule, entrapped in a liposome, entrapped in an LNP, complexed with a liposome, complexed with an LNP, dispersed in a solution or suspension comprising a lipid, mixed with a lipid, complexed with a micelle, or otherwise associated with a lipid. Lipids for use in methods described herein may be naturally occurring or synthetic lipids. Lipids can also be obtained from commercial sources. For example, dimyristyl phosphatidylcholine can be obtained from Sigma (St. Louis, MO); dicetyl phosphate can be obtained from K & K Laboratories (Plainview, NY); cholesterol can be obtained from Calbiochem-Behring; and dimyristyl phosphati dyl glycerol can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL.). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20°C. Tn some embodiments, a lipid-based system may comprise one or more lipids that facilitate targeting of the composition to a desired cell type or cell types (e.g., stem cells, monocytes, macrophages, or dendritic cells). In some embodiments, a delivery vehicle allows a composition to be preferentially taken up (e.g. endocytosed, phagocytosed) by an immune cell (e g., stem cell, monocyte, macrophage, or dendritic cell) relative to a composition that does not comprise the delivery vehicle.

Targeting Moieties

[0247] In some embodiments, a delivery vehicle may comprise one or more targeting moieties. In some embodiments, a targeting moiety may facilitate passive targeting of a composition to a desired target. In some embodiments, a targeting moiety may facilitate active targeting of a composition to a desired target.

[0248] In some embodiments, a targeting moiety may be or comprise one of more of an antibody (e.g., a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody) or any fragment thereof, for example an scFv, an aptamer, a darpin, a centyrin, a naturally occurring or synthetic receptor, an affibody, or other engineered protein recognition molecule, for example, to bind to one or more of CD14, CD l ib, CD163, CD206, CD33, and/or CD209. In some embodiments, a targeting moiety may be or comprise a small molecule.

[0249] In some embodiments, a targeting moiety may be or comprise a particular lipid or combination of hydrophobic entities, for example, present in or forming an exterior surface of a liposome or lipid nanoparticle (e.g., for targeting to a particular cell type or cell types).

Nucleic Acid Molecules

[0250] In some embodiments of the present disclosure, one or more nucleic acid molecules are or comprise DNA. In some embodiments of the present disclosure, one or more nucleic acid molecules are or comprise messenger RNA (mRNA). In some embodiments, mRNA according to the present disclosure may be synthesized as unmodified or modified mRNA. Typically, mRNAs are modified to enhance stability. Modifications of mRNA can include, for example, modifications of the nucleotides of the RNA. A modified mRNA according to the present disclosure can thus include, for example, backbone modifications, sugar modifications or base modifications. In some embodiments, a step of modifying an mRNA comprises causing the mRNA to include a modified nucleotide, an alteration to the 5’ or 3’ untranslated region (UTR), a cap structure, and/or a poly(A) tail

[0251] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding one or more CARs described herein) may contain RNA backbone modifications. Typically, a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are modified chemically. Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g. cytidine 5'-O-(l -thiophosphate)), boranophosphates, positively charged guanidinium groups etc., which comprises replacing the phosphodi ester linkage by other anionic, cationic or neutral groups.

[0252] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding one or more CARs described herein) may contain sugar modifications. A typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 2'-deoxy-2'-fluoro- oligoribonucleotide (2'-fluoro-2'-deoxycytidine 5 '-triphosphate, 2'-fluoro-2 -deoxyuridine 5'- tri phosphate), 2'-deoxy-2'-deamine-oligoribonucleotide (2'-amino-2'-deoxycyt.idine 5'- triphosphate, 2'-amino-2’-deoxyuridine 5'-triphosphate), 2'-O-alkyloligoribonucleotide, 2'-deoxy- 2'-C-alkyloligoribonucleotide (2'-O-methylcytidine 5 '-triphosphate, 2 -methyluridine 5'- triphosphate), 2'-C-alkyloligoribonucleotide, and isomers thereof (2'-aracytidine 5 '-tri phosph ate, 2'-arauridine 5 '-triphosphate), or azidotriphosphates (2'-azido-2'-deoxycytidine 5 '-triphosphate, 2'-azido-2'-deoxyuridine 5'-triphosphate).

[0253] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding one or more CARs described herein) comprise modified nucleotide comprising pseudouridine (PsU), 5-methoxyuridine (5moU), 5-methylcytidine/pseudouridine (5meC PsU), Nl -methyl- pseudouridine (NlmPsU), or combinations thereof.

[0254] In some embodiments, mRNAs of the present disclosure (e g., mRNAs encoding one or more CARs described herein) may contain modifications of the bases of the nucleotides (base modifications). A modified nucleotide which contains a base modification is also called a base-modi fi ed nucl eotide.

[0255] Typically, mRNA synthesis includes the addition of a “cap” on the N-terminal (5’) end, and a “tail” on the C -terminal (3 ’) end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of a “tail” serves to protect the mRNA from exonuclease degradation.

[0256] Thus, in some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding one or more CARs described herein) include a 5’ cap structure. A 5’ cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5’ nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5’ triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase. Examples of cap structures include, but are not limited to, m7G(5’)ppp (5'(A,G(5')ppp(5^!)A and G(5')ppp(5')G. In some embodiments, a cap comprises a CapO structure. A capO structures lack a 2'-O-methyl residue of the ribose attached to bases 1 and 2, In some embodiments, a cap comprises an AGCapl structure. An AGCapl structures has a 2'-O-methyl residue at base 2. In some embodiments, a cap comprises a Cap2 structure. Cap2 structures have a 2'-O-methyl residue attached to both bases 2 and 3. In some embodiments, a cap structure comprises AGCapl, mbAGCap l, or Anti-Reverse Cap Analog (ARC A). In some embodiments, a modified mRNA of the present disclosure comprises an m6AGCapl and modified nucleotides comprising pseudouridine (PsU).

[0257] In some embodiments, mRNAs of the present disclosure (e.g,, mRNAs encoding one or more CARs described herein) include a 3’ poly(A) tail structure. A poly(A) tail on the 3' terminus of mRNA typically includes about 10 to 400 adenosine nucleotides (SEQ ID NO: 45) (e.g., about 100 to 400 adenosine nucleotides, about 10 to 200 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about 10 to 100 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides). In some embodiments, mRNAs include a 3’ poly(C) tail structure. A suitable poly(C) tail on the 3' terminus of mRNA typically include about 10 to 200 cytosine nucleotides (SEQ ID NO: 46) (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides). A poly(C) tail may be added to a poly(A) tail or may be a substitute for the poly(A) tail.

[0258] In some embodiments, mRNAs of the present disclosure (e.g., mRNAs encoding one or more CARs described herein) include a 5’ and/or 3’ untranslated region. In some embodiments, a 5’ untranslated region includes one or more elements that affect an rnRNA’s stability or translation, for example, an iron responsive element. In some embodiments, a 5’ untranslated region may be between about 50 and 500 nucleotides in length.

[0259] In some embodiments, a 3’ untranslated region includes one or more of a polyadenylation signal, a binding site for proteins that affect an mRNA’s stability of location in a cell, or one or more binding sites for miRNAs, In some embodiments, a 3’ untranslated region may be between 50 and 500 nucleotides in length or longer.

[0260] One of skill in the art would recognize that for disclosed amino acid sequences there are several nucleotide sequences capable of generating that amino acid sequence. While certain exemplary nucleotide sequences are disclosed, it is specifically contemplated that each nucleotide sequence giving rise to a particular amino acid sequence may be useful in certain embodiments.

Administration of Additional Payloads

[0261] In some embodiments, methods of the present disclosure comprise one or more steps of treating an immune cell (e.g., a stem ceil, macrophage, monocyte, or dendritic cell) during the process of modifying the immune cell. In some embodiments, methods of the present disclosure comprise one or more steps of administering to a subject an additional payload for modulating an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) during the process of modifying the immune cell. In some embodiments, a composition may comprise one or more additional payloads. In some embodiments, a composition may comprise one or more additional payloads in the same delivery vehicle as one or more nucleic acid molecules. In some embodiments, a composition may comprise one or more additional payloads in a different delivery vehicle than the one used with one or more nucleic acid molecules. [0262] In some embodiments, methods of the present disclosure comprise a step of treating an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with a modulator of a pathway activated by in vitro transcribed mRNA. In some embodiments, an additional payload may be or comprise a modulator of a pathway activated by in vitro transcribed mRNA. In vitro transcribed (IVT) mRNA is recognized by various endosomal innate immune receptors (Toll-like receptor 3 (TLR3), TLR7 and TLR8) and cytoplasmic innate immune receptors (protein kinase RNA-activated (PKR), retinoic acid-inducible gene I protein (RIG-I), melanoma differentiation- associated protein 5 (MDA5) and 2'-5'-oligoadenylate synthase (OAS)). Signaling through these different pathways results in inflammation associated with type 1 interferon (IFN), tumor necrosis factor (TNF), interleukin-6 (IL-6), IL- 12 and the activation of cascades of transcriptional programs. Overall, these create a pro-inflammatory microenvironment poised for inducing specific immune responses Moreover, downstream effects such as slow-down of translation by eukaryotic translation initiation factor 2a (eIF2a) phosphorylation, enhanced RNA degradation by ribonuclease L (RNaseL), and overexpression and inhibition of replication of self-amplifying mRNA are of relevance for the pharmacokinetics and pharmacodynamics of IVT mRNA.

[0263] In some embodiments, a modulator of a pathway activated by in vitro transcribed mRNA comprises an RNase inhibitor. In some embodiments, a modulator of a pathway activated by in vitro transcribed mRNA comprises an RNaseL, RNase 1'2 or RNase 1 inhibitor. In some embodiments, a modulator of a pathway activated by in vitro transcribed mRNA comprises an RNaseL inhibitor In some embodiments, an RNaseL inhibitor comprises sunitinib. In some embodiments, an RNaseL inhibitor comprises ABCE1.

[0264] In some embodiments, treating an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell ) with an RNaseL inhibitor increases mRNA stability in a modified immune cell relative to mRNA stability in a modified immune cell of the same type that was not treated with an RNaseL inhibitor. In some embodiments, treating an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with an RNaseL inhibitor increases CAR expression in a modified immune cell relative to CAR expression in a modified immune cell of the same type that was not treated with an RNaseL inhibitor. In some embodiments, treating an immune ceil (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with an RNaseL inhibitor increases effector activity in a modified immune cell relative to effector activity in a modified immune cell of the same type that was not treated with an RNaseL inhibitor.

[0265] In some embodiments, administering to a subject an RNaseL inhibitor increases mRNA stability in a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) relative to mRNA stability in a modified immune cell of the same type in a subject that that was not administered an RNaseL inhibitor. In some embodiments, administering to a subject an RNaseL inhibitor increases CAR expression in a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) relative to CAR expression in a modified immune cell of the same type in a subject that was not administered an RNaseL inhibitor. In some embodiments, administering to a subject an RNaseL inhibitor increases effector activity in a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) relative to effector activity in a modified immune cell of the same type in a subject that was not administered an RNaseL inhibitor.

[0266] In some embodiments of the present disclosure, a step of treating an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) occurs before a step of delivering an mRNA to the immune cell. In some embodiments of the present disclosure, a step of administering an additional payload to a subject occurs before a step of administering a composition comprising an mRNA to the subject.

[0267] In some embodiments, methods of the present disclosure comprise a step of culturing an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with a cytokine or immune stimulating recombinant protein. In some embodiments, methods of the present disclosure comprise a step of administering to a subject a cytokine or immune stimulating recombinant protein. In some embodiments, a cytokine comprises IFN-a, IFN-P, IFN-y, TNFa, IL-6, STNGL, LPS, a CD40 agonist, a 4- IBB ligand, recombinant 4-1BB, a CD19 agonist, a TLR agonist (e.g., TLR-1 , TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8 or TLR-9), TGF-P (e.g., TGF-pl, TGF- P2, or TGF-P3), a glucocorticoid, an immune complex, interleukin-1 alpha (IL-1 ex), IL-lp, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-20, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), Leukemia inhibitory' factor ( L If ). oncostatin M (OSM), TNF-p, CD154, lymphotoxin beta (LT-p), an A proliferation-inducing ligand (APRIL), CD70, GDI 53, glucocorticoid-induced TNF receptor ligand (GIT RL), tumor necrosis factor superfamily member 14 (TNFSF14), OX40L (CD252), TALL-1 (Tumor necrosis factor ligand superfamily member 13B - TNFSF13B), TNF-related apoptosis-inducing ligand (TRAIL), TNF-related weak inducer of apoptosis (TWEAK), TNF-related activation-induced cytokine (TRANCE), erythropoietin (Epo), thyroid peroxidase precursor (Tpo), FMS-related tyrosine kinase 3 ligand (FLT-3L), stem cell factor (SCF), macrophage colony-stimulating factor (M-CSF), merozoite surface protein (MSP), a Nucleotide-binding oligomerization domaincontaining protein (NOD) ligand (e.g., NODI, N0D2, or NOD 1/2 agonists), a RIG-I-like receptor (RLR) ligand (e.g., 5'ppp-dsRNA, 3p-hpRNA, Poly(LC), or Poly(dA:dT)), a C-type lectin receptor (CLR) ligand (e.g., curdlan, p-glucan, HKCA, laminarin, pustulan, scleroglucan, WGP dispersible, WGP soluble, zymosan, zymosan depleted, furfurman, b-GlcCer, GlcC14C 18, HKMT, TDB, TDB-HSl 5, or TDM), a cyclic dinucleotide sensor ligand (e.g., C-Gas agonist or stimulator of interferon gene (STING) ligand), an inflammasome inducer (e.g., alum, ATP, CPPD crystals, hemozoin, MSU crystals. Nano- Si 02, Nigericin, or TDB), an aryl hydrocarbon (AhR) ligand (e.g., FICZ, indirubin, ITE, or L-kynurenine), an alpha-protein kinase 1 (ALPK1) ligand, a multi-PRR ligand, an NFKB/NFAT activator (e.g., concavaiin A, ionomycin, PHA-P, or PAIA) or combinations thereof. In some embodiments, a cytokine comprises IFN-p.

[0268] In some embodiments of the present disclosure, a step of culturing an immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) occurs after a. step of delivering an mRNA to the immune cell. In some embodiments of the present disclosure, a step of administering to a subject a cytokine or immune stimulating recombinant protein occurs after a step of administering a composition comprising an mRNA to the subject.

[0269] In some embodiments, culturing a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with a cytokine or immune stimulating recombinant protein increases the viability of the modified immune cell relative to a modified immune cell of the same type that vvas not cultured with the cytokine or immune stimulating recombinant protein. In some embodiments, culturing a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with a cytokine or immune stimulating recombinant protein increases protein (e.g., at least one CAR described herein) expression in the modified immune cell relative to a modified immune cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein. In some embodiments, culturing a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic ceil) with a cytokine or immune stimulating recombinant protein increases longevity of protein (e.g., at least one CAR described herein) expression relative to a modified immune cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein. In some embodiments, culturing a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with a cytokine or immune stimulating recombinant protein increases effector activity of the modified immune cell relative to a modified immune cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein. In some embodiments, culturing a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) with a cytokine or immune stimulating recombinant protein increases pro-inflammatory (Ml) polarization of the modified immune cell relative to a modified immune cell of the same type that was not cultured with the cytokine or immune stimulating recombinant protein.

[0270 j In some embodiments, administering to a subject a cytokine or immune stimulating recombinant protein increases the viability of a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein. In some embodiments, administering to a subject a cytokine or immune stimulating recombinant protein increases protein (e.g., at least one CAR described herein) expression of a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein. In some embodiments, administering to a subject a cytokine or immune stimulating recombinant protein increases longevity of protein (e.g., at least one CAR described herein) expression in a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein. In some embodiments, administering to a subject a cytokine or immune stimulating recombinant protein increases effector activity of a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein. In some embodiments, administering to a subject a cytokine or immune stimulating recombinant protein increases pro-inflammatory (Ml) polarization of a modified immune cell (e.g,, a stem cell, macrophage, monocyte, or dendritic cell) in the subject relative to a modified immune cell of the same type in a subject that was not administered the cytokine or immune stimulating recombinant protein.

Modified Immune Cells

[0271 J In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) is made by methods of the present disclosure. In some embodiments, a modified immune cell comprises at least one CAR described herein. In some embodiments a modified immune cell comprises one or more nucleic acids encoding at least one CAR described herein. In some embodiments, at least one CAR described herein comprises at least one extracellular domain, at least one transmembrane domain and at least one intracellular domain

[0272] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) of the present disclosure comprises at least one CAR described herein comprising an extracellular domain described herein that binds a tumor antigen, such as an antigen that is specific for a tumor or cancer of interest. In some embodiments, a tumor antigen comprises one or more antigenic cancer epitopes In some embodiments, a tumor antigen comprises one or more of: CD19; CD123; CD22; CD30; CD171 ; CS-1 (also referred to as CD2 subset 1, CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLL-1 or CLECL1); CD33; epidermal growth factor receptor variant III ( EGFRv III ); ganglioside G2 (GD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2~3)bDGalp(l-4)bDGlcp(l-l)Cer); TNF receptor family member B cell maturation (BCMA); Tn antigen ((Tn Ag) or (GalNAca- Ser/Thr)); prostate-specific membrane antigen (PSMA); Receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-Like Tyrosine Kinase 3 (FLT3); Tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; Carcinoembryonic antigen (CEA); Epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); Interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); Mesothelia; Interleukin 11 receptor alpha (IL-l lRa); prostate stem cell antigen (PSCA); Protease Serine 21 (Testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis(Y) antigen; CD24; Platelet-derived growth factor receptor beta (PDGFR- beta); Stage-specific embryonic antigen-4 (SSEA-4); CD20; Folate receptor alpha; Receptor tyrosine-protein kinase ERBB2 (Her2/neu); Mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); Prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); Ephrin B2, fibroblast activation protein alpha (FAP); insulin-like growth factor 1 receptor (IGF-I receptor), carbonic anhydrase IX (CAIX); Proteasome (Prosome, Macropain) Subunit, Beta Type, 9 (LMP2); glycoprotein 100 (gplOO); oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl), tyrosinase; ephrin type-A receptor 2 (EphA2); Fucosyl GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2- 3)bDGalp(l-4)bDGlcp(l- l)Cer); transglutaminase 5 (TGS5); high molecular weight-melanoma- associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2), Folate receptor beta, tumor endothelial marker 1 (TEM1/CD248); tumor endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD 179a; anaplastic lymphoma kinase (ALK); Polysialic acid; placenta-specific 1 (PLACI); hexasaccharide portion of globoid glycoceramide (GloboH); mammary gland differentiation antigen (N Y-BR-1); uroplakin 2 (UPK2); Hepatitis A vims cellular receptor 1 (HAVCR1 ); adrenoceptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20), lymphocyte antigen 6 complex, locus K 9 (LY6K), Olfactory receptor 51E2 (OR51E2), TCR Gamma Alternate Reading Frame Protein (TARP); Wilms tumor protein (WT1); Cancer/testis antigen 1 (NY-ESO-l); Cancer/testis antigen 2 (LAGE- la); Melanoma- associated antigen 1 (MAGE-A1); ETS translocation-variant gene 6, located on chromosome 12p (ETV6-AML); sperm protein 17 (SPA 17); X Antigen Family, Member 1A (XAGE1); angiopoi etin-binding cell surface receptor 2 (Tie 2), melanoma cancer testis antigen- 1 (MAD- CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostem; surviving; telomerase; prostate carcinoma tumor antigen-1 (PCTA-1 or Galectin 8), melanoma antigen recognized by T cells 1 (Mel an A or MARTI); Rat sarcoma (Ras) mutant; human Telomerase reverse transcriptase (h TER I); sarcoma translocation breakpoints; melanoma inhibitor of apoptosis (ML-IAP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), N-Acetyl glucosaminyl -transferase V (NAI7); paired box protein Pax-3 (PAX3); Androgen receptor; Cyclin B l ; v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog (MYCN); Ras Homolog Family Member C (RhoC); Tyrosinase-related protein 2 (TRP-2); Cytochrome P450 1B1 (CYP1B1); CCCTC-Binding Factor (Zinc Finger Protein)-Like (BORIS or Brother of the Regulator of Imprinted Sites), Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3); Paired box protein Pax-5 (P.AX5); proacrosin binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A kinase anchor protein 4 (AKAP-4); synovial sarcoma, X breakpoint 2 (SSX2); Receptor for Advanced Glycation Endproducts (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2), legumain; human papilloma virus E6 (HPV E6); human papilloma virus E7 (HPVE7); intestinal carboxyl esterase; heat shock protein 70-2 mutated (mut hsp70-2); CD79a; CD79b; CD72; Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FC AR or CD89), Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like modulecontaining mucin-like hormone receptor-like 2 (EMR2); lymphocyte antigen 75 (LY75);

Glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); or immunoglobulin lambda-like polypeptide 1 (IGLL1). In certain embodiments, a tumor antigen comprises ERBB2 (Her2/neu). In certain embodiments, a tumor antigen comprises PSMA. In certain embodiments, a tumor antigen comprises Mesothelin, Delta-like protein 3 (DLL3), c-Met, or CD7.

[0273] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a modified mRNA encoding at least one CAR provided herein exhibits increased viability relative to a modified immune cell of the same type comprising a similar CAR as provided herein. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a modified mRNA encoding at least one CAR described herein exhibits increased expression of an mRNA encoding at least one CAR described herein relative to a modified immune cell of the same type comprising unmodified mRNA encoding a similar CAR as provided herein. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising at least one CAR as provided herein exhibits increased CAR expression relative to a modified immune cell of the same type comprising unmodified mRNA encoding a similar CAR. In some embodiments, a modified immune cell (e g , a stem cell, macrophage, monocyte, or dendritic ceil) comprising a modified mRNA encoding at least one CAR as provided herein exhibits increased longevity of a mRNA encoding at least one CAR relative to a modified immune cell of the same type comprising unmodified mRNA encoding a similar CAR. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a modified mRNA encoding a CAR as provided herein exhibits increased longevity of the CAR relative to a modified immune cell of the same type comprising a similar CAR as provided herein. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as provided herein exhibits increased effector activity relative to a modified immune cell of the same type comprising unmodified mRNA encoding a similar CAR as provided herein. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a modified mRNA encoding a CAR as provided herein exhibits increased pro-inflammatory (Ml) polarization relative to a modified immune cell of the same type comprising unmodified mRNA encoding a similar CAR as provided herein.

[0274] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as provided herein maintains a pro-inflammatory phenotype over time. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as provided herein maintains a pro- inflammatory phenotype at least 4 hours, 2 days, 4 days, 7 days, 14 days, and/or 28 days after an immune ceil is modified with a nucleic acid encoding the CAR.

[0275] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as provided herein maintains an antiinflammatory phenotype over time. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as provided herein maintains an anti-inflammatory phenotype at least 4 hours, 2 days, 4 days, 7 days, 14 days, and/or 28 days after an immune cell is modified with a nucleic acid encoding the CAR.

[0276] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as provided herein maintains a pro-inflammatory phenotype and/or otherwise resists subversion when challenged by anti-inflammatory' cytokines. In some embodiments, the sensitivity of a modified immune cell to environmental cytokines is measured by generating a dose-response curve of pro-inflammatory markers by treating modified immune ceils comprising a CAR as provided herein. In some embodiments, the sensitivity of a modified immune cell to environmental cytokines is measured by generating a dose-response curve of pro-inflammatory markers by treating modified immune cells comprising a CAR as provided herein.

[0277] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as provided herein maintains an antiinflammatory phenotype and/or otherwise resists subversion when challenged by pro- inflammatory cytokines. In some embodiments, the sensitivity of a modified immune cell to environmental cytokines is measured by generating a dose-response curve of anti-inflammatory markers by treating modified immune cells comprising a CAR as provided herein with increasing concentrations of pro-inflammatory cytokines. In some embodiments, the sensitivity of a modified immune cell to environmental cytokines is measured by generating a doseresponse curve of anti-inflammatory markers by treating modified immune cells comprising a CAR as provided herein with increasing concentrations of anti-inflammatory cytokines.

[0278] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as provided herein has minimal effects on neighboring cells. In some embodiments, a modified immune cell (e.g,, a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as provided herein has significant effects on neighboring cells In some embodiments, the effect of a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as provided herein can be tested by co-culturing modified immune cells with unmodified immune cells and using flow cytometry to analyze the expression of pro-inflammatory and anti-inflammatory markers in the unmodified cells. In some embodiments, modified immune cells and unmodified immune cells can be cocultured in a culture dish where the modified immune cells and unmodified immune cells contact each other. In some embodiments, modified immune cells and unmodified immune cells can be co-cultured in a culture dish where the modified immune cells and unmodified immune cells are separated by a transwell assay membrane.

[0279] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as provided herein has minimal cytotoxic effects on neighboring cells. In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as provided herein has significant cytotoxic effects on neighboring cells (e.g., cancer cells). In some embodiments, modifying an immune cell to comprise a CAR as provided herein is not cytotoxic to the modified immune cell. In some embodiments, RNAseq data from modified immune cells are examined to determine if upregulation of genes indicative of cytotoxic effects is present.

[0280] In some embodiments, expression of a CAR provided herein in a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) increases at least one targeted effector function (e.g., phagocytosis, targeted cellular cytotoxicity, antigen presentation, or cytokine secretion) of the modified immune cell relative to an unmodified immune cell or a modified cell comprising a similar CAR.

[0281] In some embodiments, a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell) comprising a CAR as provided herein comprises one or more control systems including, but not limited to: a safety switch (e.g., an on switch, an off switch, a suicide switch), transcriptional control (e.g. cell-specific promoters, cell-state specific promoters, promoters downstream of CAR activation, promoters downstream of endogenous signaling pathways, or drug-inducible transcription), post-transcriptional control of CAR mRNA (e.g.

RNA-based inhibition with endogenous or recombinant miRNA), or post-translational control of CAR structure or stability (e.g. a CAR whose intracellular domain conditionally associates with the full structure by dmg/light-inducible association (to allow signaling) or dissociation (to inhibit signaling), or whose stability is drug-regulated for inducible stabilization (to allow⁷ signaling) or degradation (to inhibit signaling)). These control systems can be combined to create logic gates, for example an AND gate (e.g. a CAR with a CAR-inducible promoter and cytosolic domain that associates in a drug-dependent manner, thus requiring CAR activation and the presence of a small molecule), an OR gate (e.g. a CAR under control of a promoter that is transcriptionally active following CAR activation or small molecule addition), and/or a NOT gate (e.g. a CAR whose mRNA is degraded by endogenous miRNA expressed in natural immune cell signaling states (such as miRNA upregulated by a particular cytokine signaling pathway, thus only expressing a C AR in the absence of this cytokine)).

Assays [0282] A variety of assays may be performed to confirm the presence of a nucleic acid construct described herein and/or the presence of a protein (e.g., a CAR) in an immune cell (e.g,, a stem cell, macrophage, monocyte, or dendritic cell). For example, such assays include molecular biological assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR, and PCR; and biochemical assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots).

Other assay s of the present disclosure include, for example, fluorescence-activated cell sorting (FACS), immunofluorescent microscopy, MSD cytokine analysis, mass spectrometry (MS), RNA-Seq and functional assays.

[0283] A variety of assay s may be performed to determine various characteristics of a modified immune cell (e.g., a stem cell, macrophage, monocyte, or dendritic cell), such as, but not limited to, immune cell viability, nucleic acid expression, nucleic acid longevity, protein (e.g., CAR) expression, protein (e.g., CAR) longevity, effector activity, and pro-inflammatory' (Ml) polarization. For example, such assays include flow cytometry, quantitative PCR, and in vitro functional assays such as cytokine/chemokine secretion, phagocytosis, and specific lysis assays of target tumor cells.

Nucleic Acid Constructs

[0284] The present disclosure provides, among other things, nucleic acid molecules encoding at least one CAR described herein or a fragment thereof. An immune cell (e.g., stem cell, macrophage, monocyte, or dendritic cell) can comprise a nucleic acid molecule (e.g., an exogenous nucleic acid molecule) encoding at least one polypeptide (e.g., one or more CARs of the present disclosure) described herein.

[0285] 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. The phrase “nucleotide sequence that encodes a protein or an RNA” may also include introns to the extent that the nucleotide sequence encoding the protein may in some version contain an intron(s). The term “encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (e.g., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene, cDNA, or RNA, 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.

[0286] The term “operably linked” or “transcriptional control” refers to functional linkage between a regulatory sequence and a heterologous nucleic acid sequence resulting in expression of the heterologous nucleic acid sequence. 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. Operably linked DNA sequences can be contiguous with each other and, e.g., where necessary to join tw\o protein coding regions, are in the same reading frame.

[0287] Nucleic acid molecules encoding at least one protein (e.g., a CAR of the present disclosure) described herein or a fragment thereof can be a DNA molecule, an RNA molecule, or a combination thereof. In some embodiments, a nucleic acid molecule comprises or is a messenger RNA (mRNA) transcript encoding at least one protein (e.g., a CAR of the present disclosure) described herein or a fragment thereof. In some embodiments, a nucleic acid molecule comprises or is a DNA construct encoding at least one protein (e.g., a CAR of the present disclosure) described herein or a fragment thereof.

[0288] In some embodiments, all or a fragment of a protein (e.g., at least one CAR of the present disclosure) described herein is encoded by a codon optimized nucleic acid molecule, e.g., for expression in a cell (e.g., a mammalian cell). A variety of codon optimization methods are known in the art, e.g., as disclosed in US Patent Nos. 5,786,464 and 6,114,148, each of which is hereby incorporated by reference in its entirety.

[0289] Expression of nucleic acids described herein may be achieved by operably linking a nucleic acid encoding a protein (e.g., at least one CAR of the present disclosure) or fragment thereof to a promoter in an expression vector. Exemplary promoters (e.g., constitutive promoters) include, but are not limited to, an elongation factor-la promoter (EF-Ia) promoter, immediate early cytomegalovirus (CMV) promoter, ubiquitin C promoter, phosphoglycerokinase (PGK) promoter, simian vims 40 (SV40) early promoter, mouse mammary' tumor virus (MMTV) promoter, human immunodeficiency virus (HIV) long terminal repeat (LTR ) promoter, Moloney murine leukemia virus (MoMuLV) promoter, an avian leukemia vims promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, an actin promoter, a myosin promoter, a hemoglobin promoter, or a creatine kinase promoter. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter. A vector can also comprise additional promoter elements, e.g., enhancers, to regulate the frequency of transcriptional initiation.

[0290] In some embodiments, a vector comprising a nucleic acid molecule encoding a protein (e.g., at least one CAR of the present disclosure) or fragment thereof comprises or is a viral vector. Viral vector technology is well known in the art and is described (e.g., in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY). Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, or retroviral vectors (e.g., a lentiviral vector or a gamm ar etro viral vector). In some embodiments, a vector comprises a lentiviral vector (e.g., as described in US Patent No. 9,149,519 or International Publication No. WO 2017/044487, each of which is hereby incorporated by reference in its entirety).

[0291] In some embodiments, a viral vector comprises an adenoviral vector. Adenoviruses are a large family of viruses containing double stranded DNA. They replicate within the nucleus of a host cell, using the host’s cell machinery to synthesize viral RNA, DNA and proteins. Adenoviruses are known in the art to affect both replicating and non-replicating cells, to accommodate large transgenes, and to code for proteins without integrating into the host cell genome. In some embodiments, an adenoviral vector comprises an Ad2 vector or an Ad5 vector (e.g., Ad5G5 adenoviral vector, e.g., a helper-dependent Ad5F35 adenoviral vector).

[0292] In some embodiments, a viral vector is an adeno-associated virus (AAV) vector. AAV systems are generally well known in the art (see, e.g., Kelleher and Vos, Biotechniques, 17(6): 1 1 10-17 (1994); Cotten et al„ P.N.A.S. U.S.A., 89( 13 ): 6094-98 (1992); Curiel, Nat Immun, 13(2-3): 141-64 (1994); Muzyczka, Curr Top Microbiol Immunol, 158:97-129 (1992); and Asokan A, et al., Mol. Ther., 20(41:699-708 (2012)). Methods for generating and using recombinant AAV (rAAV) vectors are described, for example, in U.S. Pat. Nos. 5,139,941 and 4,797,368.

[0293] Several AAV serotypes have been characterized, including AAV1 , AAV2, AAV3 (e.g., AAV3B), AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, as well as variants thereof. Generally, any AAV serotype may be used to deliver a protein (e.g., at least one CAR of the present disclosure) or fragment thereof described herein. In some embodiments, an AAV serotype has a tropism for a particular tissue.

[0294] In some embodiments, CRISPR/Cas9 system has recently been shown to facilitate high levels of precise genome editing using adeno associated viral (AAV) vectors to serve as donor template DNA during homologous recombination (HR).

[0295] In some embodiments, a vector comprises a gammaretroviral vector (e.g., as described in Tobias Maetzig et al., “Gammaretroviral Vectors: Biology, Technology and Application"’ Viruses. 2011 Jun; 3(6): 677-713, which is hereby incorporated by reference in its entirety). Exemplary gammaretroviral vectors include Murine Leukemia Virus (MLV), Spleen- Focus Forming Virus (SFFV), and Myeloproliferative Sarcoma Vims (MPSV), and vectors derived therefrom .

[0296] In some embodiments, a vector comprises two or more nucleic acid sequences encoding a CAR, e.g., at least one CAR described herein, and a second CAR, e.g., a different CAR described herein. In some embodiments, two or more nucleic acid sequences encoding a CAR and a second CAR are encoded by a single nucleic molecule, e.g., in same frame and as a single polypeptide chain. In some embodiments, two or more CARs are separated by one or more cleavage peptide sites (e.g., an auto-cleavage site or a substrate for an intracellular protease). In certain embodiments, a cleavage peptide comprises a porcine teschovirus-1 (P2A) peptide, Thosea asigna virus (T2A) peptide, equine rhinitis A virus (E2A) peptide, foot-and- mouth disease virus (F2A) peptide, or a variant thereof.

[0297] In some embodiments, a vector comprises at least one nucleic acid sequence encoding a CAR, e.g., at least one CAR described herein, and at least one nucleic acid encoding at least one gene co-expressed with a CAR, e.g., a cytokine described herein (e.g., TNF, IL-12, IFN, GM-CSF, G-CSF, M-CSF, and/or IL-1) or a stimulatory ligand described herein (e.g., CD7, B7-1 (CD80), B7-2 (CD86), PD-L 1, PD-L2, 4-1 BBL, OX40L, ICOS-L, ICAM, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICE, HVEM, lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist or antibody that binds Toll ligand receptor, and/or a B7-H3 ligand.

Phannaceu tic al Compositions

[0298] The present disclosure, among other things, provides pharmaceutical compositions comprising modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprising one or more of CARs described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. The present disclosure, among other things, also provides pharmaceutical compositions comprising nucleic acids encoding one or more CARs described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

[0299] When “a therapeutically effective amount, “an immunologically effective amount,” “an anti-immune response effective amount,” or “an immune response-inhibiting effective amount” is indicated, a precise amount of a pharmaceutical composition described herein can be determined by a physician with consideration of individual differences in age, weight, immune response, and condition of the patient (subject).

[0300] Pharmaceutical compositions described herein may comprise buffers, such as neutral buffered saline or phosphate buffered saline (PBS); carbohydrates, such as glucose, mannose, sucrose, dextrans, or mannitol; proteins, polypeptides, or amino acids (e g., glycine); antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); serum and preservatives, such as cryoprotectant. In some embodiments, a pharmaceutical composition is substantially free of contaminants, e.g., there are no detectable levels of a contaminant (e.g., an endotoxin).

[0301 ] Pharmaceutical compositions described herein may be administered in a manner appropriate to the disease, disorder, or condition to be treated or prevented. Quantity and frequency of administration will be determined by such factors as condition of a patient, and type and severity of a patient’s disease, disorder, or condition, although appropriate dosages may be determined by clinical trials.

[0302] Pharmaceutical compositions described herein may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, liposomes, and suppositories. Preferred compositions may be injectable or infusible solutions. Pharmaceutical compositions described herein can be formulated for administration intravenously, subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, transarterially, or intraperitoneally.

[0303] In some embodiments, a pharmaceutical composition described herein is formulated for parenteral (e g., intravenous, subcutaneous, intraperitoneal, or intramuscular) administration. In some embodiments, a pharmaceutical composition described herein is formulated for intravenous infusion or injection. In some embodiments, a pharmaceutical composition described herein is formulated for intramuscular or subcutaneous injection. Pharmaceutical compositions described herein can be formulated for administered by using infusion techniques that are commonly known in immunotherapy (See, e.g., Rosenberg et al.. New Eng J. of Med. 319 :1676, 1988, which is hereby incorporated by reference in its entirety).

[0304] As used herein, the terms “parenteral administration” and “administered parenterally” refer to modes of administration other than enteral and topical administration, usually by injection or infusion, and include, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intratumoral, and intrasternal injection and infusion.

[0305] Pharmaceutical compositions comprising modified immune cells described herein may be administered at a dosage of about 10⁴ to about 10⁹ cells deg body weight (e g , about 10^s to about 10⁵ cells/kg body weight), including all integer values within those ranges. In some embodiments, a dose of immune cells described herein (e.g., stem cells, macrophages, monocytes, or dendritic cells) comprises at least about 1 x 10⁶, about 1.1 x 10⁶, about 2 x 10”, about 3.6 x 10⁶, about 5 x 10⁶, about 1 x 10', about 1.8 x 10', about 2 x 10⁷, about 5 x 10⁷, about 1 x 10^s, about 2 x 10⁸, about 5 x 10^s, about 1 x 10⁹, about 2 x 10⁹, or about 5 x 10⁹ cells. Pharmaceutical compositions described herein may also be administered multiple times at a certain dosage. An optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art by monitoring a patient for signs of a disease, disorder, or condition and adjusting treatment accordingly.

[0306] It may be desired to administer pharmaceutical compositions described herein to a subject and then subsequently redraw blood (or have apheresis performed), activate collected immune cells, and reinfuse a subject with activated immune cells. This process can be performed multiple times, e.g., every few weeks. Immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) can be activated from blood draws of from about 10 cc to about 400 cc. In some embodiments, immune cells (e.g., macrophages, monocytes, or dendritic cells) are activated from blood draws of about 20 cc, about 30 cc, about 40 cc, about 50 cc, about 60 cc, about 70 cc, about 80 cc, about 90 cc, or about 100 cc. Without being bound by theory, methods comprising multiple blood draw and reinfusions described herein may select for certain immune cell populations.

[0307] In some embodiments, pharmaceutical compositions described herein are administered in combination with (e.g., before, simultaneously, or following) a second therapy. For example, a second therapy can include, but is not limited to antiviral therapy (e.g., cidofovir, interleukin-2, Cytarabine (ARA-C), or natalizumab), chimeric antigen receptor- T cell (CAR-T) therapy, T-cell receptor (TCR)-T cell therapy, chemotherapy, radiation, an immunosuppressive agent (e.g., cyclosporin, azathioprine, methotrexate, mycophenolate, FK506 antibody, or glucocorticoids), an antagonist (e.g., one or more of a PD-1 antagonist, a PD-L1 antagonist, CTLA4 antagonist, CD47 antagonist, SIRPa antagonist, CD40 agonists, CSF1/CSF1R antagonist, or a STING agonist), or an immunoablative agent (e.g., an anti-CD52 antibody (e.g., alemtuzumab), an anti-CD3 antibody, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, a steroid, FR901228, or irradiation.

[0308] In some embodiments, pharmaceutical compositions described herein are administered in combination with (e.g., before, simultaneously, or following) bone marrow transplantation or lymphocyte ablative therapy or myeoablation therapy using a chemotherapy agent (e.g., fludarabine, external -beam radiation therapy (XRT), cyclophosphamide, or Rituxan). In certain embodiments, subjects undergo standard treatment with higli dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following transplant, subjects receive an infusion of a pharmaceutical composition comprising immune cells described herein. Pharmaceutical compositions described herein may be administered before or following surgery'.

[0309] A dosage of any aforementioned therapy to be administered to a subject will vary with a disease, disorder, or condition being treated and based on a specific subject. Scaling of dosages for human administration can be performed according to art-accepted practices. For example, a dose of alemtuzumab will generally be about 1 mg to about 100 mg for an adult, usually administered daily for a period of between about 1 day to about 30 days, e.g., a daily dose of about 1 mg to about 10 mg per day (e.g., as described in U.S. Patent No. 6,120,766, which is hereby incorporated by reference in its entirety).

[0310] In some embodiments, pharmaceutical compositions described herein are administered in combination with (e.g., before, simultaneously, or following) a checkpoint inhibitor. In some embodiments, a checkpoint inhibitor comprises or is pembrolizumab, ipilimumab, nivolumabm, azezolizumab, or a combination thereof. In some embodiments, one or more checkpoint inhibitors are administered concomitantly to a subject with a pharmaceutical composition disclosed herein. In some embodiments, one or more checkpoint inhibitors are administered to a subject 7, 14, 21, 28, 35, 42 or more days post-administration of a pharmaceutical composition disclosed herein. In some other embodiments, a pharmaceutical composition disclosed herein is administered to a subject 7, 14, 21, 28, 35, 42, or more days post-administration of a checkpoint inhibitor.

Methods of Treatment

[0311] The present disclosure, among other things, provides methods of treating a disease or disorder (e.g., a disease or a disorder described herein) in a subject comprising delivering a pharmaceutical composition described herein. In some embodiments, a therapeutically effective amount of a pharmaceutical composition described herein is administered to a subject having a disease or disorder. Pharmaceutical compositions described herein can be for use in the manufacture of a medicament for treating a disease or disorder in a subject or stimulating an immune response in a subject [0312] A subject to be treated with methods described herein can be a mammal, e g., a primate, e.g., a human (e.g,, a patient having, or at ri sk of having, a disease or disorder described herein). In some embodiments, modified immune cells (e.g., stem cells, macrophages, monocytes, or dendritic cells) may be autologous, allogeneic, or xenogeneic with respect to a subject. Pharmaceutical compositions described herein can be administered to a subject in accordance with a dosage regimen described herein, alone or in combination with one or more therapeutic agents, procedures, or modalities.

[0313] Pharmaceutical compositions described herein can be used to treat or prevent a disease associated with a tumor or cancer, a neurodegenerative disease or disorder, an inflammatory disease or disorder, a cardiovascular disease or disorder, a fibrotic disease or disorder, a disease associated with amyloidosis, and a combination of thereof.

[0314] A method of treating (e.g., one or more of reducing, inhibiting, or delaying progression of) a cancer or a tumor in a subject with a pharmaceutical composition described herein is provided. A subject can have an adult or pediatric form of cancer. A cancer may be at an early, intermediate, or late stage, or a metastatic cancer. A cancer can include, but is not limited to, a solid tumor, a hematological cancer (e.g., leukemia, lymphoma, or myeloma, e.g., multiple myeloma), or a metastatic lesion. Examples of solid tumors include malignancies, e g , sarcomas and carcinomas, e.g., adenocarcinomas of the various organ systems, such as those affecting the lung, breast, ovarian, lymphoid, gastrointestinal (e.g., colon), anal, genitals and genitourinary tract (e.g., renal, urothelial, bladder cells, prostate), pharynx, CNS (e.g., brain, neural or glial cells), head and neck, skin (e.g., melanoma, e.g., a cutaneous melanoma), pancreas, and bones (e.g., a chordoma).

[0315] In some embodiments, a cancer is selected from a lung cancer (e.g., a non-small cell lung cancer (NSCLC) (e.g., a non-small cell lung cancer (NSCLC) with squamous and/or non-squamous histology, or a NSCLC adenocarcinoma), or a small cell lung cancer (SCLC)), a skin cancer (e.g., a Merkel cell carcinoma or a melanoma (e.g., an advanced melanoma)), an ovarian cancer, a mesothelioma, a bladder cancer, a soft tissue sarcoma (e.g., a hemangiopericytoma (HPC)), a bone cancer (a bone sarcoma), a kidney cancer (e.g., a renal cancer (e.g., a renal cell carcinoma)), a liver cancer (e.g., a hepatocellular carcinoma), a cholangiocarcinoma, a sarcoma, a myelodysplastic syndrome (MBS), a prostate cancer, a breast cancer (e g., a breast cancer that does not express one, two or all of estrogen receptor, progesterone receptor, or Her2/neu, e.g., a triple negative breast cancer), a colorectal cancer (e.g., a relapsed colorectal cancer or a metastatic colorectal cancer, e.g., a microsatellite unstable colorectal cancer, a microsatellite stable colorectal cancer, a mismatch repair proficient colorectal cancer, or a mismatch repair deficient colorectal cancer), a nasopharyngeal cancer, a duodenal cancer, an endometrial cancer, a pancreatic cancer, a head and neck cancer (e.g., head and neck squamous cell carcinoma (HNSCC)), an anal cancer, a gastro-esophageal cancer, a thyroid cancer (e.g., anaplastic thyroid carcinoma), a cervical cancer (e.g., a squamous cell carcinoma of the cervix), a neuroendocrine tumor (NET) (e.g., an atypical pulmonary carcinoid tumor), a lymphoproliferative disease (e.g., a post-transplant lymphoproliferative disease), a lymphoma (e.g,, T-cell lymphoma, B-cell lymphoma, or a non-Hodgkin lymphoma), a myeloma (e.g , a multiple myeloma), or a leukemia (e g , a myeloid leukemia or a lymphoid leukemia)

[0316] In some embodiments, a cancer is a brain tumor, e.g., a glioblastoma, a gli osarcoma, or a recurrent brain tumor. In some embodiments, a cancer is a pancreatic cancer, e.g., an advanced pancreatic cancer. In some embodiments, a cancer is a skin cancer, e.g., a melanoma (e g., a stage II-IV melanoma, an HLA-A2 positive melanoma, an unresectable melanoma, or a metastatic melanoma), or a Merkel cell carcinoma. In some embodiments, a cancer is a renal cancer, e.g., a renal cell carcinoma (RCC) (e.g., a metastatic renal cell carcinoma). In some embodiments, a cancer is a breast cancer, e.g., a metastatic breast carcinoma or a stage IV breast carcinoma, e.g., a triple negative breast cancer (TNBC). In some embodiments, a cancer is a virus-associated cancer. In some embodiments, a cancer is an anal canal cancer (e.g., a squamous cell carcinoma of the anal canal). In some embodiments, a cancer is a cervical cancer (e.g., a squamous cell carcinoma of the cervix). In some embodiments, a cancer is a gastric cancer (e.g., an Epstein Barr Virus (EBV) positive gastric cancer, or a gastric or gastro-esophageal junction carcinoma). In some embodiments, a cancer is a head and neck cancer (e.g., an HPV positive and negative squamous cell cancer of the head and neck (SCCHN)). In some embodiments, a cancer is a nasopharyngeal cancer (NPC). In some embodiments, a cancer is a colorectal cancer, e.g., a relapsed colorectal cancer, a metastatic colorectal cancer, e.g., a microsatellite unstable colorectal cancer, a microsatellite stable colorectal cancer, a mismatch repair proficient colorectal cancer, or a mismatch repair deficient colorectal cancer. [0317] In some embodiments, a cancer is a hematological cancer. In some embodiments, a cancer is a leukemia, e.g., acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, chronic leukemia, or acute leukemia. In some embodiments, a cancer is a lymphoma, e.g., Hodgkin lymphoma (HL.), non-Hodgkin's lymphoma, lymphocytic lymphoma, or diffuse large B cell lymphoma (DLBCL) (e.g., a relapsed or refractory HL or DLBCL). In some embodiments, a cancer is a myeloma, e.g., multiple myeloma.

[031§] Pharmaceutical compositions described herein can be used to enhance or modulate an immune response in a subject. In one embodiment, a pharmaceutical composition described herein enhances, stimulates, or increases an immune response in a subject (e.g., a subject having, or at risk of, a disease or disorder described herein). In certain embodiments, a subject is, or is at risk of being, immunocompromised. For example, a subject is undergoing or has undergone a chemotherapeutic treatment and/or radiation therapy.

[0319] In some embodiments, a subject has, or is at risk of, developing an inflammatory disorder (e.g., a chronic or acute inflammatory disorder). In some embodiments, a subject has, or is at risk, of developing an autoimmune disease or disorder. Exemplary autoimmune diseases that can be treated with methods described herein include, but are not limited to, Alzheimer's disease, asthma (e.g., bronchial asthma), an allergy (e.g., an atopic allergy), Acquired Immunodeficiency Syndrome (AIDS), atherosclerosis, Behcet’s disease, celiac, cardiomyopathy, Crohn's disease, cirrhosis, diabetes, diabetic retinopathy, eczema, fibromyalgia, fibromyositis, glomerulonephritis, graft vs. host disease (GVHD), Guillain-Barre syndrome, hemolytic anemia, multiple sclerosis, myasthenia gravis, osteoarthritis, polychondritis, psoriasis, rheumatoid arthritis, sepsis, stroke, vasculitis, ventilator-induced lung injury, transplant rejection, Raynaud's phenomena, Reiter’s syndrome, rheumatic fever, sarcoidosis, scleroderma, Sjogren's syndrome, ulcerative colitis, uveitis, vitiligo, or Wegener's granulomatosis.

[0320] Administration of pharmaceutical compositions described herein may be carried out in any convenient manner (e.g., injection, ingestion, transfusion, inhalation, implantation, or transplantation). In some embodiments, a pharmaceutical compositions described herein is administered by injection or infusion. Pharmaceutical compositions described herein may be administered to a patient transarterially, subcutaneously, intravenously, intradermally, intratumorally, intranodally, intramedullar}', intramuscularly, or intraperitoneally. In some embodiments, a pharmaceutical composition described herein is administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or intramuscularly). In some embodiments, a pharmaceutical composition described herein is administered by intravenous infusion or injection. In some embodiments, a pharmaceutical composition described herein is administered by intramuscular or subcutaneous injection. Pharmaceutical compositions described herein may be injected directly into a site of inflammation, a local disease site, a lymph node, an organ, a tumor, or site of infection in a subject.

[0321] All publications, patent applications, patents, and other references mentioned herein, including GenBank Accession Numbers, are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein.

[0322] The disclosure is further illustrated by the following examples. An example is provided for illustrative purposes only. It is not to be construed as limiting the scope or content of the disclosure in any way.

EXAMPLES

[0323] The following examples are provided so as to describe to the skilled artisan how to make and use methods and compositions described herein, and are not intended to limit the scope of the present disclosure. Myd88 domains may be referred to as “M88” herein, including in the figures. CD3-zeta domains may be referred to as “Z” herein, FcRy domains may be referred to as “y’" herein, including in the figures.

Example 1: Lentiviral Transduction of Macrophages

[0324] In order to generate CAR-expressing macrophages, cells were thawed and resuspended in TexMACS culture medium with 10% FBS (TexlO) and 1% Penicillin/Streptomycin (Fig. 1). The thawed resuspended macrophages were subsequently spun into a pellet at 300 x g for a duration of 5 minutes. Next, supernatant was removed and the cell pellet was resuspended in TexlO with 10 ng/mL granulocyte-macrophage colony-stimulating factor (GMCSF) at a cell density of l.OxlO⁶ cells/mL. Macrophages were distributed into wells of a 6-well UpCell plate at a density of 1.5-2.0x l0⁶ ceils/weil (approximately 1.5-2.0 ml volume). VPX containing lentivirus at a desired MOI of 2-5 was added to each well. After 24 hours, plates were spun at 300xg for 5 minutes, supernatant removed, and cells were resuspended in 2 ml warm TexlO with 10 ng/mL GMCSF. After three or four days, fresh 2 mL Text with 10 ng/mL was added.

[0325] In order to harvest the macrophages for use in experiments, the UpCell plates were placed at 4°C for 30 minutes. Macrophages were then removed by gentle pipeting and transferred into a conical tube that was centrifuged at 300xg for 5 minutes. Next, the cell pellet was resuspended in TexlO, counted, and brought to a final concentration of l.OxlO⁶ macrophages per mL of Texl O.

Example 2: Viability, Receptor Expression, and Tumor Cell Killing of CAR Macrophages comprising a CD2§ Hinge and CD28 Transmembrane Domain

[0326] In this example, CAR-expressing macrophages were generated and evaluated for CAR expression and viability. First, human macrophages underwent culturing conditions and lentiviral transduction, as explained above. Macrophages were transduced with the CAR constructs shown in Table 1.

Table I. Exemplified CAR Constructs described herein comprising a CD28 hinge domain and a CD28 transmembrane domain.

[0327] Cells were plated at 50,000 cells per well and surface protein staining was performed using the following panel: CD80-FITC, CD86-PE, APC-anti-Trastuzumab, CD206- BV421, PerCP-Cy5.5-CD163, and LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen, Cat L34957). Detection of surface protein expression was completed using the .Attune flow cytometer (Thermo Fischer).

[0328] High viability was demonstrated across CAR constructs (Figs. 2A). All CAR macrophages had variable expression, with CTX 001 having highest percent expression (~90%) and CTX_589 having the lowest (~25%) (Fig. 2B). CAR MFI also showed some variability with

CTX 001 being the most abundant. Motably, the CAR constructs CTX 219, CTX 584, CTX_586, and CTX_588 were relatively comparable to each other. The lowest MFI values belonged to CAR constructs CTX 582, CTX 585, CTX 587, and CTX 589 (Fig. 2C).

[0329] For a killing assay of tumor cells, the desired number of macrophages plated in a volume of 100 pL Tex 10 in a 96 well flat-bottom dish. Next, these macrophages were incubated for a duration of 30-45 minutes at 37°C. This was then followed by addition of the desired number of tumor cells in a volume 100 pL TexlO, thus resulting in final volumes for all wells at 200 uL Tex lO. Prior to the experiment, the tumor cells, AU565, were engineered to express nuclear localized GFP.

[0330] Plates were incubated at room temperature for 15 minutes after tumor cell addition, followed by a 45 minute incubation period at 37°C. Incucyte® analysis of co-cultures was then monitored every' four hours The ratio between the effector macrophages (E) and target tumor cells (T) varied from 4: 1 E:T to 1 :2 E:T, along with a 0: 1 E:T target cell only control. The number of macrophages was kept constant at 10e3 macrophages per well. The change in fluorescence over time was measured to determine the amount of tumor cell killing occurring within the co-culture by reduction in GFP intensity relative to the initial time point (Time 0).

[0331] CAR construct containing truncated Myd88 domain (ICD) and CD3-zeta, CTX 582 and CTX 584, exhibited comparable tumor cell killing function with CTX 219 when the ratio of effector macrophages to tumor cells is 4: 1 (Figs. 3A-B). The superior tumor cell killing efficacy of CTX 584, which also comprises CD40 ICD in addition to Myd88 and CD3- zeta, becomes more evident when you have a 1 :2 E:T ratio (Figs. 3C-D).

Example 3: Cytokine Secretion of CAR Macrophages comprising a CD28 Hinge and CD28

Transmembrane Domain

[0332] In this example, a cytokine secretion assay was performed on macrophages transduced with CAR constructs noted in Table 1. For cytokine secretion assays, a HER2 or mesothelin antigen was resuspended in PBS to a concentration of 28 nM, Next, 100 pl, of either antigen solution was added to wells in a 96- well flat bottom plate and incubated at 37°C for 2 hours or 4°C overnight. PBS control wells that contain only PBS were also incubated. After incubation, wells were washed two times with 200 pL PBS, and were followed by the addition of 50,000 macrophages from each condition to a final volume of 200 iiL Tex 10. Next, plates were incubated for 24 hours and then centrifuged at 300 xg for 5 minutes. Subsequently, 125 pL of supernatant were removed and stored at -20°C. Cytokine analysis of TNFa and IL-12p70 was performed using the Proinflammatory Human Kit and Chemokine Human Kit (Meso Scale Discovery) per manufacturer instructions.

[0333] CAR constructs containing truncated Myd88 ICD and CD40 ICD (CTX_584, CTX 587, CTX 588, and CTX 589) were capable of potentiating TNFa secretion as well as IL- 12p70 secretion (Figs. 4A-B). The degree of cytokine secretion was greater than the Myd88 and Myd88 with CD3-zeta counterparts and CARs lacking an intracellular domain component.

Example 4: Viability and Receptor Expression of CAR Macrophages comprising a CD8 hinge domain and a CJD8 transmembrane domain

[0334] In this example, CAR expressing macrophages were generated and evaluated for CAR expression and viability. First, human macrophages underwent culturing conditions and lentiviral transduction, as explained above. Transduction of macrophages was performed with the CAR constructs shown in Table 2.

Table 2. Exemplified CAR Constructs described herein comprising a CD8 hinge domain and a CD8 transmembrane domain, except for CTX 219 (see Example 2).

[0335] Cells were plated at 50,000 cells per well in TexMACS media containing 10% FBS and incubated overnight at 37°C and 5% CO2. Surface protein staining was done using the following panel: CD80-FITC, CD86-PE, APC-anti-Trastuzumab, CD206-BV421, PerCP-Cy5.5- CD163, and LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen, Cat L34957).

Detection of surface protein expression using the Attune flow⁷ cytometer (Thermo Fischer).

[0336] High viability was demonstrated across CAR constructs (Fig. 5A). All CAR macrophages had variable expression, with CTX 219, CTX 595, and CTX 596 having highest percent expression (>80%), whereas CTX_592 and CTX_598 had the lowest percent expression in comparison (-20%) (Fig. SB). CAR MFI also showed some variability with CTX__219 being the most abundant. Further to this, the CAR constructs CTX_595,and CTX_597 were quite comparable to each other. The lowest MFI values belonging to CAR constructs CTX 590, CTX-591, CTX-592, CTX_593, CTX_594, CTX_596, and CTX_598 (Fig. 5C).

[0337] As shown in Figures 6A-B, expression of Ml markers, CD80 and CD86, was dramatically increased in macrophages expressing CAR constructs CTX 583 and CTX 584. Both of these CAR constructs’ ICD comprise a CD40, truncated My d88 domain, and CD3-zeta components. These two constructs demonstrated efficacious switching to a Ml phenotype when compared with M2 surface marker expression (Figs. 6C-D). This is surprising given these cells are not primed with CD40 ligand or co-cultured with tumor cells.

[0338] M2 surface marker expression (CD 163 and CD206) is comparably higher in a number of other CAR constructs, except the CAR construct CTX_594, which ICD only comprises CD40 ICD alone (Figs. 6C-D). These results are indicative of CAR expressing macrophages have plasticity to transition between M l and M2 phenotypes.

Example 5: Cytokine Secretion of CAR macrophages comprising: (i) a CD28 extracellular hinge domain and CD28 transmembrane member, or (ii) a CDS extracellular hinge domain and CD8 transmembrane member [0339] In this example, cytokine secretion assay was performed on all constructs described in Tables 1 and 2. First, a HER2 antigen was resuspended in PBS to a concentration of 28 nM. Next, 100 pL of either antigen solution was added to wells in a 96-well flat bottom plate and incubated at 37°C for 2 hours or 4°C overnight; PBS control wells that contain only PBS were also incubated. After incubation, wells were washed twice with 200 pL PBS, and were followed by the addition of 50,000 macrophages from each condition to a final volume of 200 pL TexlO. Next, plates were incubated for 24 hours and then centrifuged at 300 xg for 5 minutes. Subsequently, 125 pL of supernatant were removed and stored at -20°C. Cytokine analysis of TNFa and IL-8 was performed using the Proinflamrnatory Human Kit and Chemokine Human Kit (Meso Scale Discovery⁷) per manufacturer instructions.

[0340] CD28H/CD28TM CAR constructs that further comprise truncated My88 and CD40 ICD ■ ('TX 584 and CTX 588) were capable of potentiating both TNFa and IL-8 secretion upon HER2 introduction (Figs. 7A-B). The degree of cytokine secretion was notably⁷ greater for the CAR construct (CTX 584) comprising all three components of Myd88 truncated ICD, CD40 ICD, and CD3-zeta. In comparison with CD8H/CD8TM containing CAR constructs, CTX 598 (comprising CD40 ICD, CD.3-zeta, and truncated Myd88 domain) had dramatically higher IL-8 secretion than TNFa (Figs. 7C-D).

Example 6: Effect of CAR Macrophages comprising a truncated Myd88 ICD, CD40 ICD, and CD3-zeta on Antigen Negative Tumor Cells

[0341] In order to demonstrate that CAR constructs comprising truncated Myd88 ICD, CD40 ICD, and CD3-zeta and do not cause non-specific killing of antigen negative tumor cells, the desired number of macrophages were first plated in a volume of 100 pL Texl 0 in a 96 well flat-bottom dish. These macrophages were then incubated for a duration of 30-45 minutes at 37°C, followed by addition of the desired number of tumor cells in a volume of 100 pL TexlO, thus resulting in final volumes for all wells of 200 pL TexlO. The tumor cells that do not have the antigen of interest, MDA468, were engineered prior to the experiment to express nuclear localized GFP.

[0342] Plates were incubated at room temperature for 15 minutes after tumor cell addition, followed by a 45 minute incubation period at 37°C. Incucyte® analysis of co-cultures was then monitored. The ratio between the effector macrophages (E) and target tumor cells (T) was varied from 2:1 E:T to 1 :2 E:T, along with a 0:1 E:T target cell only control The number of tumor cells was kept constant at 10⁴ tumor cells per well. The change in fluorescence over time was measured every four hours to determine the amount of tumor cell killing that occurred within the co-culture by reduction in GFP intensity relative to the initial time point (Time 0).

[0343] CAR constructs CTX_584 and CTX_593, which each comprise truncated Myd88 ICD and CD40 ICD as well as CD3-zeta, exhibited null effects on tumor cells regardless of ratio (Figs. 8A-B). These constructs were comparable to the negative control of tumor cells alone, MDA468, and CTX 001 and CTX 219, which each lack truncated Myd88 ICD and CD40 ICD. These results demonstrate that inclusion of truncated Myd88 ICD and CD40 ICD in a CAR construct does not cause non-specific lysis of tumor cells that do not express the antigen of interest.

Example 7: Effect of truncated Myd88 ICD and CD40 IC'D on Cytokine Release

[0344] In this example, a cytokine secretion assay was performed to compare the effects of CAR constructs CTX_584 and CTX_594, which each comprise truncated Myd88 ICD and CD40 ICD as well as CD3-zeta. First, a HER2 or mesotheiin antigen was resuspended in PBS to a concentration of 28 nM. Next, 100 p.L of either antigen solution was added to wells in a 96- well flat bottom plate and incubated at 37°C for 2 hours or 4°C overnight. PBS control wells containing only PBS were also incubated. After incubation, wells were washed two times with 200 pL PBS, followed by addition of 50,000 macrophages from each condition to a final volume of 200 pL TexlO. Next, plates were incubated for 24 hours and then centrifuged at 300 xg for 5 minutes. Subsequently, 125 pL of supernatant were removed and stored at -20°C. Cytokine analysis of TNFa, IL-8, and IL-12p70 was performed using the Proinflammatory Human Kit and Chemokine Human Kit (Meso Scale Discovery) per manufacturer instructions.

[0345] Both CAR constructs CTX_584 and CTX_593, which each comprise truncated Myd88 ICD and CD40 ICD as well as CD3-zeta, increased TNFa, IL-12p70, and IL-8 cytokine release. This contrasts with CAR constructs that do not have a Myd88 and CD40 ICD, such as CTX 001 and CTX 219 (Figs. 9A-C). These results clearly point to the impact of the combination of truncated Myd88 ICD, CD40 ICD, and CD3-zeta on co- stimulation and activity of macrophages transduced with these exemplified CAR constructs.

Example 8: Viability, Receptor Expression, and M1/M2 Surface Expression of CAR Macrophages comprising a FcRy ICD

[0346] In this example, macrophages with CARs comprising a FcRy ICD (also known as FCERIG) were generated and evaluated for CAR expression, viability, and surface marker phenotype. First, human macrophages underwent culturing conditions and lentiviral transduction, as explained above. Transduction of macrophages was performed with the CAR constructs shown in Table 3.

Table 3. Exemplified CAR Constructs described herein comprising a FcRy ICD.

[0347] C ells were plated at 50,000 cells per well and surface protein staining was done using the following panel: CD80-FITC, CD86-PE, APC-anti-Trastuzumab, CD206-BV421, PerCP-Cy5.5-CD163, and LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen, Cat L34957). Detection of surface protein expression was completed using the Attune flow cytometer (Thermo Fischer).

[ 0348] High viability was demonstrated across all CAR constructs (Fig. IDA). All CAR macrophages had variable expression, with CTX 869 and CTX 872 having the highest percent expression (>50%), whereas CTX 870 and CTX 871 had the lowest percent expression in comparison (>20%) (Fig. 10B). CAR MFI also showed a similar trend in variability with both CTX 869 and CTX 872 being the most abundant in comparison to CTX 870 and CTX 871 (Fig. 10C). [0349] .As shown in Figures 11A-B, expression of Ml markers, CD80 and CD86, increased in macrophages comprising CAR constructs CTX 870, CTX 871, and CTX 872. All of these CAR constructs comprise ICDs with a Myd88 truncated ICD, CD40 ICD, and FcRy ICD, thus demonstrating the efficacious switching of CAR macrophages comprising Myd88 truncated ICD, CD40 ICD, and FcRy ICD to a Ml phenotype. In comparison, CAR constructs without all three components, CTX 313 and CTX 869, did not have as high of Ml surface protein expression. Instead, macrophages expressing these two CAR constructs had heightened expression of M2 markers, CD 163 and CD206 (Figs. 11C-D). These results demonstrate that CAR constructs comprising Myd88, CD40 ICD, and FcRy activate macrophages without priming or co-culturing with tumor cells.

Example 9: Tumor Cell Killing Function and Cytokine Release of FcRy CAR Macrophages

[0350] In this Example, CAR constructs comprising a FcRy domain were evaluated for their tumor cell killing function and cytokine release. For a killing assay, the desired number of macrophages were plated in a volume of 100 pL TexlO in a 96 well flat-bottom dish. These macrophages were then incubated for a duration of 30-45 minutes at 37°C, followed by addition of the desired number of tumor cells in a volume of 100 pL TexlO, resulting in a final volume for all wells of 200 gL TexlO. The tumor cells, AU565, were engineered prior to the experiment to express nuclear localized GFP.

[0351 ] Plates were incubated at room temperature for 15 minutes after tumor cell addition, followed by a 45 minute incubation period at 37°C. Incucyte® analysis of co-cultures was then monitored. The ratio between the effector macrophages (E) and target tumor cells (T) was varied from 1 :4 E:T to 2: 1 E:T. The number of macrophages was kept constant at 20,000 macrophages per well. The change in fluorescence over time was measured every four hours to determine the amount of tumor cell killing occurring within the co-culture by reduction in GFP intensity relative to the initial time point (Time 0).

[0352] All CAR constructs comprising a FcRy ICD exhibited comparable tumor cell killing function when the ratio of effector macrophages to tumor cells was 2:1 (Fig, 12A). The superior tumor cell killing efficacy of CAR constructs comprising a CD28 hinge and transmembrane domain in addition to a FcRy domain (CTX 869, CTX 870, CTX 871, and C TX 872) became more evident when you have a 1 :4 E:T ratio (Fig. 12B). Determination of tumor burden was then calculated by using the following equation: (Integrated intensity of CAR macrophages + AU565 tumor cells)/(Integrated intensity of AU565 cells alone). Notably, AU565 cells alone were run for each tumor cell count used in the assay to help in analysis. Remarkably, CAR constructs comprising a FcRy ICD (CTX_869, CTX_870, CTX_871, and CTX_872) showed diminished tumor burden whether the ratio was 2: 1 or 1 :4 E:T (Figs. 12C-D). These results demonstrate how CAR constructs comprising Myd88, CD40 ICD, and FcRy enhance macrophage killing of target tumor cells.

[0353] Next, a cytokine release assay was performed to compare the effects of these FcRy CAR construct. First, a HER2 or mesothelin antigen was resuspended in PBS to a concentration of 28 nM. Next, 100 pL of either antigen solution was added to wells in a 96-well flat bottom plate and incubated at 37°C for 2 hours or 4°C overnight. PBS control wells that contained only PBS 'were also incubated. After incubation, weds were washed two times with 200 pL PBS, followed by the addition of 50,000 macrophages from each condition to a final volume of 200 pL TexlO. Next, plates were incubated for 24 hours and then centrifuged at 300 xg for 5 minutes. Subsequently, 125 uL of supernatant was removed and stored at -20°C. Cytokine analysis of TNFa, IL-6, and IL-8 was performed using the Proinflammatory Human Kit and Chemokine Human Kit (Meso Scale Discovery) per manufacturer instructions.

[0354] CAR constructs comprising Myd88, CD40 ICD, and FcRy ICD all had notably high cytokine secretion of TNFa, IL-6, and IL-8 while maintaining reduced background cytokine release of the mesothelin antigen and PBS control (Figs. 13A-C). In comparison, CAR constructs comprising only FcRy ICD (CTX_313 and CTX_869) had minimal cytokine secretion. These results demonstrate the efficacy of CAR constructs comprising FcRy domain, Myd88, and CD40 ICD in maintaining high potentiation post-activation without the hindrance of background cytokine secretion.

Example 10: Reduced Tonic Signaling of CAR Macrophages comprising a Myd88 ICD mutant variant

[0355] In this example, CAR-expressing macrophages were generated (Fig. 14) and evaluated for tonic signaling (e.g., NFkB). First, a HEK Nulll cell line expressing the SEAP reporter gene under the control of the NFkB and API promoters was used Briefly, HEK Nulll reporter cells were plated in a six well plate and incubated overnight at 37°C and 5% CO2. Then, media was exchanged and a transfection mix was added containing 150 microliters Optimem, 9 microliters Lipofectamine 2000, and 2.5 micrograms of transfer plasmid. Transfer plasmids comprising the following CAR constructs shown in Tables 4-7 were used.

Table 4. Exemplified Control CAR Constructs described herein.

Table 5. Exemplified CAR Constructs described herein comprising a 0)28 hinge and transmembrane domain, CD40zeta ICD, and a mutated MydSS ICD.

Table 6. Exemplified CAR Constructs described herein comprising a CD28 hinge and transmembrane domain, CIMOgamma ICD, and a mutated Myd88 ICD.

Table 7. Exemplified CAR Constructs described herein comprising a CDS hinge domain, CD64 transmembrane domain, and a mutated Myd88 ICD.

[0356] Separately, 96-well plates were coated with either 28 nM HER2-his in PBS, 28 nM Mesothelin-his, or PBS alone and kept at 4°C. 24 hours after transfection, the HEK Null 1 reporter cells were collected and the pre-coated plated were washed twice with 200 microliters of PBS Then 5 x 10⁵ cells were plated as triplicates in the coated plates in a total volume of 200 microliters per well. As a positive control, 20 microliters of a 100 ng/mL stock of TNFa was added to untransduced cells. After 24 hours of stimulation, 20 microliters of supernatant from the culture was collected, and added to 180 microliter of QUANTI-Blue solution for an incubation period of 90 minutes. After incubation, the samples were measure at OD630. In order to analyze the data, the untransduced (UTD) triplicate values for each condition (PBS, Mesothelin, and HER2) were averaged and subtracted from the individual values of experimental wells of the same condition.

[0357] CAR constructs containing CD28 hinge and transmembrane domains with a CD3- zeta ICD and a truncated Myd88 ICD domain with either E52A, R32A, R32K, Y58A, Y58F, and R32A/E52A/Y58A mutations (CTXJ326, CTX 1328, CTX , 1330, CTX ,1332, CTXJ334, and CTX__1336) were selected for lentiviral production and further experimentation (Fig. 15). In addition, CAR constructs containing CD28 hinge and transmembrane domains with a Fc fragment of IgE, high affinity I, receptor for gamma (FcRy, also known to as FCER1 GIC) and a truncated Myd88 ICD domain with either R32A, R32K, Y58A, Y58F, and R32AZE52A/Y58A mutations (CTX__1329, CTX 1331, CTX 1333, CTX_1335, CTX_1337) were also selected for lentiviral production. Example 11: Cytokine Release, Receptor Expression, and M1/M2 Surface Expression of CAR Macrophages comprising a Myd88 ICD mutant variant

[0358] In this example, macrophages with CARs comprising a Myd88 ICD mutant variant from Example 10 were evaluated for CAR cytokine release, viability’, and surface marker phenotype.

[0359] A cytokine release assay’ was performed on macrophages as described in Example 9. Cytokine secretion was lost upon introduction of Myd88 mutations in some constructs (e.g., CTX-1327, CTX_1341, CTXJ344, CTX_1344, CTX J 346, CTXJ347, CTXJ342, and CTX 1345) when compared to their non-mutated counterparts (CTX 584, CTX 870, and CTX_879) (Figs. 16A-C and 17A-C). Minimum cytokine secretion was observed in macrophages transduced with CTX 1348, bearing a CD8 hinge and CD64 transmembrane domain with a Myd88 ICD comprising a R98C mutation. Additional cytokine release studies revealed that CAR constructs, CTX_1330, CTX_1331, and CTX_1348 maintained their HER2- specific cytokine release when compared to their non-mutated and control CAR counterparts (Figs. 18A-C).

[0360] Next, cells were plated at 50,000 cells per well and surface protein staining was done using the following panel: CD80-FITC, CD86-PE, APC-anti-Trastuzumab, CD206-BV421, PerCP-Cy5.5-CD163, and LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen, Cat L34957). Detection of surface protein expression was completed using the Attune flow cytometer (Thermo Fischer).

[0361] High viability was demonstrated across all CAR constructs (Fig. 19A). CAR macrophages bearing known tonic architectures that are non-Myd88 mutated constructs (CTX_584, CTX_870, and CTX_879) had slightly reduced recovery'’ (Fig. 19B). All CAR macrophages had variable CAR expression, with CTX 1331, CTX 1332, CTX 1333, and CTX_1348 having the highest percent expression (~>45%), whereas CTX_219 and CTX_1335 had the lowest percent expression (<20%) (Fig. 20A). CAR MFI also showed a similar trend in variability with CTX_1331, CTX_1332, CTX_1333, and CTX_1348 being the most abundant in comparison to their non-mutated counterparts (Figs. 20A-B). [0362] As shown in Fig. 21A, expression of the Ml marker, CD80, was reduced in macrophages including CAR constructs comprising Myd88 mutations when compared to their non-mutated counterparts. Furthermore, macrophages expressing these CAR Myd88 mutant variants had varying expression of the M2 markers, CD 163 and CD206 (Fig. 21B). These results demonstrate that CAR constructs comprising Myd88 truncated mutants were still capable of activing macrophages without priming or co-culturing with tumor cells.

Example 12: Tumor Cell Killing Function of CAR Macrophages Comprising a Myd88 ICD Mutant Variant

[0363] In this Example, CAR constructs comprising a mutated Myd88 ICD domain were evaluated for their tumor cell killing function in macrophages. For this assay, the desired number of macrophages were plated in a volume of 100 pL TexlO in a 96 well flat-bottom dish. These macrophages were then incubated for a duration of 30-45 minutes at 37°C, followed by addition of the desired number of tumor cells in a volume of 100 pL TexlO, resulting in a final volume for all wells of 200 pL Texl O. The tumor cells, AU565, were engineered prior to the experiment to express nuclear localized GFP.

[0364] Plates were incubated at room temperature for 15 minutes after tumor cell addition, followed by a 45 minute incubation period at 37°C. Incucyte® analysis of co-cultures was then monitored. The ratio between the effector macrophages (E) and target tumor cells (T) was varied from 1:1 E:T to 1:2 E:T. The change in fluorescence over time was measured every four hours to determine the amount of tumor cell killing occurring within the co-culture by reduction in GFP intensity relative to the initial time point (Time 0).

[0365] All CAR constructs comprising a Myd88 mutant variant ICD exhibited comparable tumor cell killing function to their non-mutated counterparts when the ratio of effector macrophages to tumor cells was 1:2 (Figs. 22A-D). The enhanced tumor cell killing efficacy of CAR constructs comprising a R32K Myd88 mutation with a CD28 hinge and transmembrane domain, CD40 ICD, and CD3z (CTX 1330); a R32K Myd88 mutation with a CD28 hinge and transmembrane domain, CD40 ICD, and FcRy (CTX_1331); and a R98C Myd88 mutation with a CD8 hinge domain and CD64 transmembrane domain (CTX 1348) became more evident at a 1: 1 E:T ratio (Figs. 23A-C, 24A-C, and 25A-C) [0366] Determination of tumor burden was then calculated using the following equation:

(Integrated intensity of CAR macrophages + AU565 tumor cells)/(Integrated intensity of AU565 cells alone). AU565 cells alone were run for each tumor cell count used in the assay to help in analysis. Remarkably, CAR constructs comprising a Myd88 ICD mutation (CTX 1330, CTX 1331, CTX 1348) showed diminished tumor burden relative to their non-mutated counterparts (Fig. 24D). These results demonstrate how CAR constructs comprising Myd88 mutated variants are capable of diminishing tumor burden and in some cases are likely superior to their non-mutated counterparts.

Example 13: Functional Effects of DAP10 CAR constructs with Myd88 ICD domain in macrophages

[0367] The present Example assesses cell viability’, CAR expression, M1/M2 phenotypic marker expression, tumor killing function, and cytokine secretion in macrophages expressing anti-HER2 CAR constructs comprising a DAP10 extracellular hinge domain and transmembrane domain with costimulation domains, such as a Myd88 ICD. Exemplary CAR constructs utilized in these experiments are described in Fig. 26.

[0368] Macrophages were thawed and plated at a density of 2 x IO⁶ cells per well in 6- well UpCell plates in 2 mL TexMacs with 10% FBS, 1% Pen/Strep, 10 ng/ml GM-CSF. After 2- 3 hours of rest, VPX lentivirus particles were diluted into TexMacs media and added to macrophages at specified multipli city of infection (MOIs). Media was fully exchanged 24 hours post-lentivirus addition. Cells were then tested 6-7 days post-transduction.

[0369] For testing of all experiments described herein, culture plates were placed at 4 ^CC for 30 minutes. Cells were removed from the plate, pelleted, and resuspended in TexMacs media. Next, cell counts were taken using an NC-200 or Vi-Cel Blue, and macrophages were subsequently used for experiments.

[0370] For assessment of viability, CAR expression, and M1/M2 markers, macrophages from each condition were plated at 50,000 cells per well, and surface protein staining was done using the following panel: CD80-FITC, CD86-PE, APC-anti-Trastuzumab, CD206-BV421 , PerCP-Cy5.5-CD163, and LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen, Cat L34957). Detection of surface protein expression was completed using the Attune flow cytometer (Thermo Fischer). Cell viability and recovery' are shown in Figure 27. Viability is defined as percentage of events captured via flow' cytometry that are negative for viability dye. Recovery is defined as number of live cells post-lifting on day 6 or 7 divided by total number of cells initially seeded for transduction with lentivirus at day 0. D.AP10 CAR constructs were not found to have major impact on macrophage recovery or viability. Expression of DAP 10 CAR constructs is quantified and shown in Figure 28.

[0371] Representative M1/M2 phenotypic marker expression in macrophages expressing DAP 10 CAR constructs is shown in Figure 29. CAR constructs comprising a Myd88 domain increased skewing of macrophages torvard a Ml phenotype as characterized by increased expression of M l markers and decreased expression of M2 markers.

[0372] For assessment of tumor killing function (e.g., cytotoxicity), the desired number of macrophages were plated in a volume of 100 pL TexlO in a 96 well flat-bottom dish. These macrophages were then incubated for a duration of 30-45 minutes at 37°C, followed by addition of the desired number of tumor cells in a volume of 100 pL TexlO, resulting in a final volume for all wells of 200 nL TexlO. The tumor cells (AU565, Panel, or MDA468 cells) were engineered prior to the present experiment to express nuclear localized GFP, AU565 cells express HER2 protein whereas Panel and MDA468 cell s do not express HER2 protein.

[0373] Plates were then incubated at room temperature for 15 minutes after addition of tumor cells, followed by 45 minutes incubation at 37 °C. GFP expression was then assessed in these co-cultures using an Incucyte. Plates were incubated at room temperature for 15 minutes after tumor cell addition, followed by a 45 minute incubation period at 37°C. Incucyte® analysis of co-cultures was then monitored. The ratio between the effector macrophages (E) and target tumor cells (T) was varied from 1:1 E:T to 1:16 E:T, The change in fluorescence over time was measured every four hours to determine the amount of tumor cell killing occurring within the coculture by reduction in GFP intensity relative to the initial time point (Time 0).

[0374] Change in AU565 tumor burden, as measured by GFP integrated intensity normalized to 4-hour or 0-hour time point, is shown in Figs. 30 and 31. DAP 10 CAR constructs displayed robust tumor cell killing function, but lost activity at lower E:T (effectortarget) ratios (e.g., 1 :16). Change in Panel and MD.A468 tumor burden, as measured by GFP integrated intensity normalized to 4-hour time point, is shown in Fig. 32. DAP10 CAR constructs did not exhibit tumor cell killing function against HER2' cell lines, except CTX 1364 which displayed some activity against Panel cells.

[0375] For a cytokine release assay, HER2 and Mesothelin antigens were diluted in PBS to a concentration of 28 nM, then 100 pL of antigen solution or control PBS was added to wells and incubated at 4 °C for 24 hours so that the antigens passively bound to the plate. Wells were then washed twice with PBS, and 50,000 macrophages were added to each well. Plates were incubated for 24 hours, followed by centrifugation at 300xg for 5 minutes. Supernatant was removed and stored at -20 °C. Cytokine levels were assessed using Proinfl amniatory Human Kit and Chemokine Human Kit (Meso Scale Discovery') following standard protocol instructions.

[0376] Supernatant cytokine levels of TNFa, IL-8, IL6, ILlb, and IL12p70 from macrophages expressing DAP10 CAR constructs are shown in Figs. 33A-B. CAR constructs comprising a Myd88 domain were observed to increase secretion of certain cytokines (e.g., TNFa and IL-8) when treated with HER2 antigen.

[0377] CTX 1364 (DAP10-M88-CD40) and CTX J 366 (DAP10-M88) expression led to Ml polarization. Robust tumor cell killing by macrophages expressing CTX_1364 was observed at low E:T ratios, but tapered off at very low E:T ratios (e.g., 1:16). CTX 1364 had some activity against Panel cells, but not MDA468 cells. CTX_1364 exhibited increased targetspecific cytokine secretion, but might also possess tonic activity. CTX 1366 also showed increased cytokine secretion, but to a lesser extent.

Example 14: Viability, Receptor Expression, and Tonic Signaling of DAP10 CAR constructs comprising a MydSS ICD mutant variant

[0378] In this example, CAR-expressing HEK cells were generated and evaluated for viability and tonic signaling. First, human macrophages underwent culturing conditions and lenti viral transduction, as described above in Example 1, with the CAR constructs described in . HEK Nulll reporter cells were transduced with CAR constructs shown in Tables 8-10 (Fig. 34). An exemplary experimental timeline for screening mutant Myd88 CAR sequences in Hek Null 1 cells is shown in Fig. 35. Table 8. Exemplified Control CAR Constructs described herein.

Table 9. Exemplified CAR Constructs described herein comprising a DAP10 hinge, transmembrane, and intracellular domain, CD40zeta ICD, and a mutated Myd88 ICD.

Table 10. Exemplified CAR Constructs described herein comprising a DAP10 hinge, transmembrane, and intracellular domain and a mutated Myd88 ICD.

[0379] Cells were plated at 50,000 cells per well and surface protein staining was done using the following panel: CD80-FI I C, CD86-PE, APC-anti-Trastuzumab, CD206-BV421, PerCP-Cy5.5-CD163, and LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogeri, Cat L34957). Detection of surface protein expression was completed using the Attune flow⁷ cytometer (Thermo Fischer).

[0380] High viability was demonstrated across all CAR constructs (Fig. 36). All CAR macrophages had variable expression, with CTX_1510, CTX_1511, CTX_1518, and CTX_1522 having the highest percent expression (->60%), whereas non-mutated constructs CTX ,1364, CTX_1366 and CTX_1525 had the lowest percent expression (~<45%). CAR MFI also showed a similar trend in variability with CTX 1510, CTX 1511, CTX 1518, and CTX 1522 being the most abundant in comparison to their non-mutated counterparts (Fig. 37).

[0381] The CAR constructs were then evaluated for tonic signaling using the methodology described in Example 10. DAP10 CAR constructs containing Myd88 ICD mutant variants had reduced tonic signaling compared to their non-mutated counterparts (Fig. 38). This present Example demonstrates the benefit of CAR constructs comprising a mutated Myd88 ICD in terms of reduced undesirable tonic signaling, while still having notable viability and expression. Example 15: Viability, Receptor Expression, and M1/M2 Surface Expression of DAP10 CAR Macrophages comprising a Myd88 ICD mutant variant

[0382] In this example, macrophages with DAP 10 CARs comprising a MyD88 mutant variant ICD were generated and evaluated for CAR expression, viability, and surface marker phenotype. First, human macrophages underwent culturing conditions and lenti viral transduction, as described in Example 1. Transduction of macrophages was performed with the CAR constructs described in Tables 8-10.

[0383] C ells per well were plated at 50,000 cells per well and surface protein staining was done using the following panel: CD80-FITC, CD86-PE, APC-anti-Trastuzumab, CD206- BV421, PerCP-Cy5.5-CD163, and LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen, Cat L34957). Detection of surface protein expression was completed using the Attune flow cytometer (Thermo Fischer).

[0384] High viability was demonstrated across all CAR constructs and variability for percent cells recovered (Figs. 39A-B). All CAR macrophages had variable expression, with CTX 1503 having relatively the highest percent expression (>60%), whereas CTX 1502, CTX_1518, CTX_1519 and CTX_1525 had the lowest percent expression in comparison (>20%) (Fig. 40). CAR MFI also showed a similar trend in variability with CTX 1510,

CTX_CTX_1511, and CTX_1522 being the most abundant in comparison to CTX_870, CTX 1503, CTX 1505, CTX 1513, and CTX 1525 (Fig. 40).

[0385] As shown in Figures 41A, expression of Ml markers, CD80 and CD86, increased in macrophages comprising CAR constructs CTX_1364 and CTX__1525. The macrophages expressing these two CAR constructs had reduced expression of M2 markers, CD 163 and CD206 (Figs. 41B). These results demonstrate that. CAR constructs comprising Myd88 and DAP 10 activate macrophages without priming or co-culturing with tumor cells.

Example 16: Tumor Cell Killing and Cytokine Release of DAP10 CAR constructs with Myd88 mutant variant ICD domain in macrophages

[0386] The present Example assesses tumor killing function and cytokine secretion in macrophages expressing anti-HER2 CAR constructs comprising a DAPIO extracellular hinge domain and transmembrane domain with costimulation domains, such as a Myd88 mutant variant ICD. Macrophages were transduced with CAR constructs as described in Tables 8-10.

[0387] Macrophages were thawed and plated at a density of 2 x 10⁶ cells per well in 6- well UpCell plates in 2 mL TexMacs with 10% FBS, 1% Pen/Strep, 10 ng/ml GM-CSF. After 2- 3 hours of rest, VPX lenti virus particles were diluted into TexMacs media and added to macrophages at specified multiplicity of infection (MOIs). Media was fully exchanged 24 hours post-lentivirus addition. Cells were then tested 6-7 days post-transduction.

[0388] For tumor kil ling assays, the desired number of macrophages were plated in a volume of 100 pL Tex 10 in a 96 well flat-bottom dish. These macrophages were then incubated for a duration of 30-45 minutes at 37°C, followed by addition of the desired number of tumor cells in a volume of 100 pL TexlO, resulting in a final volume for all wells of 200 pL TexlO. The tumor cells (AU565 and Panel) were engineered prior to the experiment to express nuclear localized GFP.

[0389] Plates were also incubated at room temperature for 15 minutes after tumor cell addition, followed by a 45 minute incubation period at 37°C. Incucyte® analysis of co-cultures was then monitored. The ratio between the effector macrophages (E) and target tumor cells (T) was varied from 4: 1 E:T to 1 : 16 E:T. The number of macrophages was kept constant at 20,000 macrophages per wel I .

[0390] Determination of tumor burden was then calculated using the following equation: (Integrated intensity of CAR macrophages + AU565 tumor ce11s)/(Integrated intensity of AU565 cells alone). AU565 cells alone were run for each tumor cell count used in the assay to help in analysis. Remarkably, DAP10 CAR constructs comprising a Myd88 ICD mutation (e.g., CTX-1504, CTXJ505, CTXJ 510, CTXJ 511, CTX_1519, CTX_1522, and CTX_1523) showed diminished tumor burden relative to their non-mutated counterparts (Fig. 42). These results demonstrate how DAP10 CAR constructs comprising Myd88 mutated valiants are capable of diminishing tumor burden.

[0391] All DAP10 CAR constructs comprising a Mydd88 mutant variant ICD exhibited notable tumor cell killing of HER2⁺ expressing AU565 cells, which gradually diminishes as the ratio from effector to target cells decreases from 4: 1 to 1 :16. This tumor cell killing is specific, as control HER2" Panel cells were relatively unaffected by the anti-HER2 tumor cell killing activity of the DAP 10 CAR macrophages, except CTX 1364 (Figs. 43A-C).

[0392] For a cytokine release assay, HER2 and Mesothelin antigens were diluted in PBS to a concentration of 28 nM, then 100 pL of antigen solution or control PBS was added to wells and incubated at 4 °C for 24 hours so that the antigens passively bound to the plate. Wells were then -washed twice with PBS, and 50,000 macrophages were added to each well. Plates were incubated for 24 hours, followed by centrifugation at 300xg for 5 minutes. Supernatant was removed and stored at -20 °C. Cytokine levels were assessed using Proinfl ammatory Human Kit and Chemokine Human Kit (Meso Scale Discovery) following standard protocol instructions.

[0393] Supernatant cytokine levels of TNFo, IL6, and IL-8 from macrophages expressing DAP 10 CAR constructs are shown in Figures 44A-C. DAP 10 CAR constructs with Myd88 mutants (CTX 1504, CTX 1570, CTX 1519, and CTX 1522) comprising a Myd88 domain were observed to increase secretion of certain cytokines (e.g , TNFa and IL-8) when treated with HER2 antigen.

Example 17: Tonic Signaling of CAR constructs comprising a Myd88 mutant variant ICD and FcRy ICD

[0394] In this example, CAR-expressing HEK cells were generated using the methods described in Example 10 for analysis of tonic signaling, except for the plates used were not coated with an antigen. HEK Null ! reporter cells were lipofected with CAR constructs described above and shown in Tables 11-13.

Table 11. Exemplified Control and Comparator CAR Constructs described herein.

Table 12. Exemplified CAR Constructs described herein comprising a CD28 hinge, CD28 transmembrane, FcRy intracellular domain, CD40 It’D. and a mutated Myd88 ICD.

Table 13. Exemplified CAR Constructs described herein comprising a CDS hinge, CDS transmembrane, FcRy intracellular domain, CD40 ICD, and a mutated Myd88 ICD.

[0395] CAR constructs containing either a CD8 or CD28 hinge and transmembrane domains with a truncated Myd88 ICD domain comprising R32A, Y58A, Y58F, S34Y, R98C, or R32AZE52A/Y58A mutations resulted in reduced tonic signaling compared to the DAPI0 CAR constructs (Fig. 46). The ability of mutations by themselves to lower tonic signaling may be variable CDS hinge and transmembrane containing CAR constructs with a truncated Myd88 ICD had further reduced tonic signaling when compared to their CD28 counterparts. This present Example demonstrates the benefit, of CAR constructs comprising a mutated Myd88 ICD in combination with a CDS or CD28 hinge and transmembrane domain in terms of reduced undesirable tonic signaling.

Example 18: Viability, Receptor Expression, and M1/M2 Surface Expression of CAR Macrophages comprising a Myd88 matant variant ICD and FcRy

[0396] In this example, macrophages with CARs comprising a CD8 or CD28 hinge and transmembrane with a Myd88 mutant variant ICD and FcRy ICD were evaluated for CAR expression, viability, and surface marker phenotype. First, human macrophages underwent culturing conditions and lentiviral transduction, as explained above. Transduction of macrophages was performed with the CAR constructs shown in Table 11-13.

[0397] Cells were plated at 50,000 cells per well and surface protein staining was done using the following panel: CD80-FITC, CD86-PE, APC-anti-Trastuzumab, CD206-B V421, PerCP~Cy5.5-CD163, and LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Invitrogen, Cat L34957). Detection of surface protein expression was completed using the Attune flow cytometer (Thermo Fischer).

[0398] High viability and cell recovery was demonstrated across all CAR constructs (Fig. 47). All CAR macrophages had variable expression, with CTX 1707, CTX CTX ...1711, CTX 1713, and CTX 1507 having the highest percent expression (>60%), whereas CTX 1701 and CTX_1706 had the lowest percent expression in comparison (>20%) (Fig. 48). CAR MFI also showed a similar trend in variability (Fig. 48).

[0399] As shown in Figures 49A-B, expression of Ml marker, CD86, increased in macrophages comprising CAR constructs comprising a Myd88 truncated mutant variant ICD with a CD8 or CD28 hinge and transmembrane domains (CTX 1702, CTX 1703, CTX 1705, C TX_ 1706, C i X 1707. CTX_1713, and CTXJ 714) relative to CAR constructs without Myd88 truncated mutant ICD. These same CAR constructs demonstrated decreased expression of the M2 marker, CD206, relative to CAR constructs with wild-type Myd88 truncated ICD. Thus, this present Example demonstrates that depending on the combination of mutations and CAR architecture, certain mutated Myd88 domains are effective at maintaining a less polarized Ml phenotype relative to control.

Example 19: Tumor Cell Killing Function and Cytokine Release of CAR Macrophages comprising a MydSS mutant variant ICD and FcRy ICD

[0400] In this Example, CAR constructs comprising a CD 8 or CD28 hinge and transmembrane domain with a Myd88 mutant variant ICD and FcRy ICD were evaluated for their tumor cell killing function and cytokine release. For a killing assay, the desired number of macrophages were plated in a volume of 100 pL TexlO in a 96 well flat-bottom dish. These macrophages were then incubated for a duration of 30-45 minutes at 37°C, followed by addition of the desired number of tumor cells in a volume of 100 pL TexlO, resulting in a final volume for all wells of 200 _suL Texl O. The tumor cells, AU565, were engineered prior to the experiment to express nuclear localized GFP.

[0401] Plates were incubated at room temperature for 15 minutes after tumor cell addition, followed by a 45 minute incubation period at 37°C. Incucyte® analysis of co-cultures was then monitored. The ratio between the effector macrophages (E) and target tumor cells (T) was 1 : 1 E:T. The number of macrophages was kept constant at 20,000 macrophages per well. The change in fluorescence was measured at 72 hours to determine the amount of tumor ceil killing occurring within the co-culture by reduction in GFP intensity relative to the initial time point (Time 0). Determination of tumor burden was then calculated by using the following equation: (Integrated intensity of CAR macrophages + AU565 tumor cells)/(Integrated intensity of AU565 cells alone).

[0402] Tumor burden for CAR constructs CTX_1704 (R32K), CTX_1711 (R32A), and CTX 1713 (R32K) was greatly diminished, paralleling the DAP 10 CAR constructs with the Myd88 ICD (Fig. 50). The superior tumor cell killing efficacy of CAR constructs comprising either a CDS or CD28 hinge and transmembrane domain in addition to a Myd88 mutant variant domain was evident at a 1 : 1 E:T ratio. AU565 cells alone were run for each tumor cell count used in the assay to help in analysis. These results demonstrate that CAR constructs comprising Myd88 mutant variants, particularly R32A or R32K, with either a CD8 or CD28 hinge and transmembrane enhance macrophage killing of target tumor cells.

[0403] Next, a cytokine release assay was performed to compare the effects of these CAR constructs. First, a HER2 or mesothelin antigen was resuspended in PBS to a concentration of 28 iiM. Next, 100 uL of either antigen solution was added to wells in a 96-well flat bottom plate and incubated at 37°C for 2 hours or 4°C overnight. PBS control wells that contained only PBS w⁷ere also incubated. After incubation, w⁷ells were washed two times with 200 uL PBS, followed by the addition of 50,000 macrophages from each condition to a final volume of 200 uL TexlO. Next, plates were incubated for 24 hours and then centrifuged at 300 xg for 5 minutes. Subsequently, 125 pL of supernatant was removed and stored at -20°C. Cytokine analysis of TNFa, and IL-6 was performed using the Proinfl ammatory Human Kit and Chemokine Human Kit (Meso Scale Discovery⁷) per manufacturer instructions.

[0404] CAR constructs with a Myd88 truncated mutant variant of R32A or R32K (e.g., CTX_1704, CTX_1711 , and CTX_1713) all had notably high cytokine secretion of TNFa and IL-8 while maintaining reduced background cytokine release of the mesothelin antigen and PBS control (Figs. 51A-B). These results demonstrate the efficacy of CAR constructs comprising a CD8 or CD28 hinge and transmembrane domain with a FcRy and Myd88 R32A or R32K mutant variant domain in maintaining high potentiation post-activation without the hindrance of background cytokine secretion.

EXEMPLARY SEQUENCES

Table 14 - Exemplary Amino Acid Sequences for CARs described herein

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Table 15 - Exemplary Nucleotide Sequences for CARs described herein

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Table 16 - Exemplary Amino Acid Sequences for CARs described herein

Table 17 - Exemplary Nucleotide Sequences for CARs described herein

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Table 18 ■■■■ Exemplary Amino Acid Sequences for CARs described herein

Table 19 - Exemplary Nucleotide Sequences for CARs described herein

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I GAGGACTTCGCCACCTATTACTGTCAACAGCATTACAC CACTCCTCCCACCTTCGGCCAGGGCACCAAGGTGGAG ATCAAGAGAACCGGGTCAACCTCAGGTAGCGGCAAGC CGGGCTCCGGCGAGGGTTCCGAAGTGCAGCTGGTTGA GTC PGGCGGAGGCC fGGI CCAGCCCGGGGGT I CC 11 GC GCCTGTCTTGCGCCGCG AGCGG H'TT A AC ATC AAGGAT ACTTACATCCACTGGGTGAGGCAGGCACCAGGGAAGG GCCTGGAATGGGTGGCCAGGATCTATCCCACAAACGG ATACACACGCTACGCCGACTCAGTGAAGGGTCGCTTCA CCATCTCTGCGGATACCAGCAAGAACACCGCCTACTTG ( LAGATGAATTCTCTT AGAGCGGAGGACACTGC GG i GT ATTACTGTAGCCGGTGGGGTGGCGATGGATTCTATGCG AT GG A C GTG I’ GGGGC C AGGGC A C C C TGGT G A C C G IT TC CTCTAGCGGCATTGAAGTCATGTACCCTCCCCCTTATC TGGACAACGAGAAATCAAACGGCACCATTATCCACGT GAAGGGGAAGCACCTGTGCCCCTCCCCTCTTTTCCCCG GTCCATCTAAACCATTCTGGGTTTTGGTGGTCGTGGGT GGCGTCCTCGCTTGCTACTCTCTGCTTGTGACCGTGGCT TTCATTATCTTCTGGGTGATGGCCGCGGGTGGCCCAGG AGCGGGGAGTGCTGCCCCAGTCAGTTCCACCTCTTCCC TGCCGCTGGCTGCCCTCAACATGCGCGTCAGGAGACGT CTCAGCCTTTTCTTGAACGTTAGGACGCAAGTAGCCGC TGACTGGACCGCCCTGGCTGAAGAGATGGATTTCGAGT ACCTGGAGATCAGACAGCTGGAGACACAGGCGGACCC TACAGGCCGCTTGCTGGACGCCTGGCAGGGCCGCCCTG GCGCTAGCGTCGGTCGCCTCCTGGAGCTCCTGACTAAG 1^TGGGTTX}CGACX}ATGI<X:1TX:IX:GAAC/1XXIGGCCATC CATTGAAGAGGACTGTCAGAAGTACATCCTGAAGCAG C A AC A GGAGG A AGC T G A G A AGC C GC T GC A AGTC GC AG CGGTCGATTCAAGCGTCCCCCGTACTGCTGAGCTGGCA GGTATCACCACACTGGATGACCCTCTGGGACACATGCC CGAGCGCTTCGATGCTTTCATCTGCTACTGCCCCAGCG ACATCAAAAAGGTAGCTAAAAAGCCGACCAACAAGGC ACCGCATCCCAAGCAGGAGCCTCAGGAGATCAATTTC ccGGAiXjAcci'Cc:ccGGA-rcx:AA^rrAc^rrG(:cGCi'CCCG'r GCAAGAGACACTGCATGGCTGCCAGCCTGTCACCCAG GAAGACGGGAAGGAGTCCCGTATCAGTGTCCAAGAAA GACAGCGTCTTAAAATCCAGGTGCGCAAGGCGGCTAT TACAAGCTACGAAAAGAGCGACGGCGTGTACACTGGA CTCAGCACCAGAAACCAGGAAACTTACGAGACCCTGA

AACACGAGAAGCCCCCACAGTGA (SEQ ID NO: 80)

Table 20 - Exemplary Amino Acid Sequences for CARs described herein

CAR (N-terminns to C-terminus)

Amino Acid Sequence

Table 21 - Exemplary Nudeotide Sequences for CARs described herein

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Table 22 - Exemplary Amino Acid Sequences for CARs described herein

Table 23 ■■■• Exemplary Nucleotide Sequences for CARs described herein

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EQUIVALENTS

[0405] It is to be appreciated by those skilled in the art that various alterations, modifications, and improvements to the present disclosure will readily occur to those skilled in the art. Such alterations, modifications, and improvements are intended to be part of the present disclosure, and are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description and drawing are by way of example only and any invention described in the present disclosure if further described in detail by the claims that follow.

[0406] Those skilled in the art. will appreciate typical standards of deviation or error attributable to values obtained in assays or other processes described herein. The publications, websites and other reference materials referenced herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference in their entireties.

Claims

1. A modified immune cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises:

(a) one or more extracellular domains;

(b) a transmembrane domain; and

(c) one or more intracellular domains comprising one or both of:

(i) a MyD88 intracellular domain or a portion thereof, or

(ii) a CD40 intracellular domain or a portion thereof; and wherein the modified immune cell is or comprises a macrophage, monocyte, dendritic cell, or stem cell

2. The modified immune cell of claim I, wherein the one or more extracellular domains comprise a scFv, VHH antibody, centyrin, or darpin.

3. The modified immune cell of claim 1 or 2, wherein the transmembrane domain comprises one or more of a: CD28, CD8a, CD40, MyD88 CD64, CD32a, CD32c, CD 16a, CD3zeta, ICOS, Dectin- 1, DAP 10, DNGR1, SLAMF7, TRL1, TLR2, TLR3, TRIA, TLR5, TLR6, TLR7, TLR8, or TLR9 transmembrane domain.

4. The modified immune cell of any one of claims 1-3, wherein the one or more intracellular domains further comprise one or more of a: CD3-zeta, FcRy, CD40 CD64, CD 32a, CD32c, CD16a, CD89, TLR1, TLR2, TLR3, TLR4, TLR5, 1'1.116, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD19, CD20, 41BB, CD28, GCSFR (CD114), RAGE, CD30, CD160, DR3, Fnl4, HVEM, CD 160, NGFR, RANK, TNFR2, TROY, XEDAR, TRIP, 0X40, GITR, TREM-1, TREM-2, DAP 12, MR, ICOS, MyD88, V/I/LxYxxL/V, SIRPa, CD45, Siglec-10, PD1, SHP-1, SHP-2, KIR-2DL, K1R-3DL, NKG2A, CD 170, CD33, BTLA, CD32b, SIRPb, CD22, PIR-B, LILRB1, 41 BBL (TNFSF9), CD27, OX40L, CD32b, CDl lb, ITGAM, SLAMF7, CD206, CD163, CD209, Dectin-2, IL1R, IL2R, IL3R, IL4R, IL5R, IL6R, IL7R, IL8R, IL9R, TL10R, IL1 IR, TL12R, IL13R, IE14R, IL15R, TL17R, IFNaR, IFNgR, TNFR, CSFIR, CSF2R, DAP10, CD36, Dectin-1, ICOSL, or Syk intracellular domain or a portion of any of the foregoing.

5. The modified immune cell of any one of claims 1-4, wherein the one or more intracellular domains further comprise a CD3-zeta intracellular domain.

6. The modified immune cell of any one of claims 1-5, wherein the CAR further comprises one or more extracellular leader domains.

7. The modified immune cell of claim 6, wherein the one or more extracellular leader domains comprise a CD8 extracellular leader domain.

8. The modified immune cell of any one of claims 1-7, wherein the CAR further comprises one or more extracellular hinge domains.

9. The modified immune cell of claim 8, wherein the one or more extracellular hinge domains comprise one or more of a CD28 extracellular hinge domain, a CD8a extracellular hinge domain, a DAP10 extracellular hinge domain, a DNGR-1 extracellular hinge domain, a Dectin-1 extracellular hinge domain, or an IgG4 extracellular hinge domain.

10. The modified immune cell of claim 8 or 9, wherein the CAR comprises, from the one or more extracellular hinge domains to the one or more intracellular domains:

(i) a CD28 extracellular hinge domain, CD28 transmembrane domain, CD40 intracellular domain, and CD3-zeta intracellular domain;

(ii) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, and CD3-zeta intracellular domain;

(hi) a CD28 extracellular hinge domain, CD28 transmembrane domain, CD40 intracellular domain, truncated Myd88 domain, and CD3-zeta intracellular domain;

(iv) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, CD40 intracellular domain, and CD3-zeta intracellular domain; (v) a CD28 extracellular hinge domain, CD28 transmembrane domain, and CD40 i n t raced u I ar dom am ;

(vi) a CD28 extracellular hinge domain, CD28 transmembrane domain, and truncated Myd88 domain;

(vii) a CD28 extracellular hinge domain, CD28 transmembrane domain, CD40 intracellular domain, and truncated Myd88 domain;

(viii) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, and CD40 intracellular domain; or

(ix) a CD28 extracellular hinge domain, CD28 transmembrane domain, CD40 intracellular domain, CD3-zeta intracellular domain, and a truncated Myd88 domain.

1 1 . The modified immune cell of claim 10, wherein the CAR comprises or has:

(a) an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs: 1-9, 45, 47, 49, 51, 53, 55, 57, 59, or 61;

(b) an amino acid sequence that differs from any one of the amino acid sequences of SEQ ID NOs: 1-9, 45, 47, 49, 51, 53, 55, 57, 59, or 61 by no more than five substitutions, additions, or deletions; or

(c) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the amino acid sequences of SEQ ID NOs: 1-9, 45, 47, 49, 51, 53, 55, 57, 59, or 61 .

12. The modified immune cell of claim 8 or 9, wherein the CAR comprises, from the one or more extracellular hinge domains to the one or more intracellular domains:

(i) a CDS extracellular hinge domain, CD8 transmembrane domain, CD40 intracellular domain, and CD3-zeta intracellular domain;

(ii) a CD8 extracellular hinge domain, CD8 transmembrane domain, truncated Myd88 domain, and CD3-zeta intracellular domain;

(iii) a CD8 extracellular hinge domain, CD8 transmembrane domain, CD40 intracellular domain, truncated VivdSS domain, and CD3-zeta intracellular domain;

(iv) a CD8 extracellular hinge domain, CDS transmembrane domain, truncated Myd88 domain, CD40 intracellular domain, and CD3-zeta intracellular domain; (v) a CDS extracellular hinge domain, CDS transmembrane domain, and CD40 i ntracel lul ar dom a in ;

(vi) a CD8 extracellular hinge domain, CD8 transmembrane domain, and truncated Myd88 domain;

(vii) a CD8 extracellular hinge domain, CD8 transmembrane domain, CD40 intracellular domain and truncated Myd88 domain;

(viii) a CD8 extracellular hinge domain, CD8 transmembrane domain, truncated Myd88 domain and CD40 intracellular domain; or

(ix) a CD8 extracellular hinge domain, CD8 transmembrane domain, CD40 intracellular domain, CD3-zeta intracellular domain, and truncated Myd88 domain.

13. The modified immune cell of claims 12, wherein the CAR comprises or has:

(a) an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs: 10- 18;

(b) an amino acid sequence that differs from any one of the amino acid sequences of SEQ ID NOs: 10-18 by no more than five substitutions, additions, or deletions; or

(c) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the amino acid sequences of SEQ ID NOs: 10-18.

14. A pharmaceutical composition comprising a modified immune cell of any one of the previous claims.

15 The pharmaceutical composition of claim 14, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

16. A nucleic acid construct comprising one or more nucleic acid sequences encoding:

(a) one or more extracellular domains;

(b) a transmembrane domain; and

(c) one or more intracellular domains comprising one or both of:

(i) a MyD88 intracellular domain or a portion thereof, or

(ii) a CD40 intracellular domain or a portion thereof; and wherein the nucleic acid construct encodes a chimeric antigen receptor (CAR) comprising (a) through (c).

17. The nucleic acid construct of claim 16, further comprising one or more nucleic acid sequences encoding:

(d) one or more extracellular leader domains,

(e) one or more extracellular hinge domains, or

(f) one or more cleavage peptides.

18. The nucleic acid construct of claim 17, wherein the cleavage peptide is or comprises a P2A, F2A, E2A or T2A peptide.

19. The nucleic acid construct of claim 17 or 18, wherein the nucleic acid construct encodes, from the one or more extracellular hinge domains to the one or more intracellular domains:

(ii) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, and CD3-zeta intracellular domain,

(iii) a CD28 extracellular hinge domain, CD28 transmembrane domain, CD40 intracellular domain, truncated Myd88 domain, and CD3-zeta intracellular domain;

(iv) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, a CD40 intracellular domain, and CD3-zeta intracellular domain;

(v) a CD28 extracellular hinge domain, CD28 transmembrane domain, and CD40 i ntracellul ar dorrs ain ;

(viii ) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, and CD40 intracellular domain; or (ix) a CD28 extracellular hinge domain, CD28 transmembrane domain, CD40 intracellular domain, CD3~zeta intracellular domain, and a truncated Myd88 domain.

20. The nucleic acid construct of claim 19, wherein the nucleic acid construct comprises or has:

(a) a nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs: 19-27, 63, 65, 67, 69, 71, 73, 75, 77, or 79;

(b) a nucleotide sequence that differs from any one of the nucleotide sequences of SEQ ID NOs: 19-27, 63, 65, 67, 69, 71, 73, 75, 77, or 79 by no more than five substitutions, additions, or deletions; or

(c) a nucleotide acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the nucleotide sequences of SEQ ID NOs: 19-27, 63, 65, 67, 69, 71 , 73, 75, 77, or 79.

21. The nucleic acid construct of claim 17, wherein the nucleic acid construct encodes, from the one or more extracellular hinge domains to the one or more intracellular domains:

(i) a CD8 extracellular hinge domain, CD8 transmembrane domain, CD40 intracellular domain, and CD3-zeta intracellular domain;

(iii) a CDS extracellular hinge domain, CD8 transmembrane domain, CD40 intracellular domain, truncated Myd88 domain, and CD3-zeta intracellular domain;

(iv) a CDS extracellular hinge domain, CD8 transmembrane domain, truncated Myd88 domain, CD40 intracellular domain, and CD3-zeta intracellular domain;

(v) a CD8 extracellular hinge domain, CD8 transmembrane domain, and CD40 intracellular domain;

(vi) a CDS extracellular hinge domain, CD8 transmembrane domain, and truncated Myd88 domain;

(vii) a CD8 extracellular hinge domain, CD8 transmembrane domain, CD40 intracellular domain and truncated Myd88 domain; (viii) a CD8 extracellular hinge domain, CD8 transmembrane domain, truncated Myd88 domain and CD40 intracellular domain; or

22. The nucleic acid construct of claim 21, wherein the nucleic acid construct comprises or has:

(a) a nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs: 28-36, or

(b) a nucleotide sequence that differs from any one of the nucleotide sequences of SEQ ID NOs: 28-36 by no more than five substitutions, additions, or deletions; or

(c) a nucleotide acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the nucleotide sequences of SEQ ID NOs: 28-36.

23. A pharmaceutical composition comprising the nucleic acid construct of any one of claims 16-22

24. The pharmaceutical composition of claim 23, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

25. A method of treating a disease or disorder in a subject, the method comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition of any one of claim 14, 15, 23, or 24, wherein at least one sign or symptom of the disease or disorder is improved in the subject after adm ini strati on .

26. A method of modifying an immune cell, the method compri sing: delivering to the immune cell a nucleic acid of any one of claims 16-22, thereby producing a modified immune cell, wherein the modified immune cell comprises or is a macrophage, monocyte, dendritic cell, or stem cell

27. A modified immune cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises:

(a) one or more extracellular domains;

(b) a CD8 or CD28 extracellular hinge domain;

(c) a CD8 or CD28 transmembrane domain; and

(d) one or more intracellular domains comprising an FcRy intracellular domain; and wherein the modified immune cell is or comprises a macrophage, monocyte, dendritic cell, or stem cell

28. The modified immune cell of claim 27, wherein the one or more intracellular domains further comprise one or both of:

(i) a Myd88 intracellular domain or a portion thereof, or

(ii) a CD40 intracellular domain or a portion thereof.

29. The modified immune cell of any one of claims 27-28, wherein the one or more intracellular domains further comprise one or more of a: CD3-zeta, FcRy, CD40 CD64, CD32a, CD32c, CD16a, CD89, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TI..R7, TI..R8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, ROR1, ROS1, RYK, TIE2, TRK, VEGFR, CD19, CD20, 41BB, CD28, GCSFR (CD114), RAGE, CD30, CD160, DR3, Fnl4, HVEM, CD 160, NGFR, RANK, TNFR2, TROY, XEDAR, TRIF, 0X40, GITR, TREM-1, TREM-2, DAP12, MR, ICOS, MyD88, V/I/LxYxxL/V, SIRPa, CD45, Siglec-10, PD1, SI IP- 1. SHP-2, K1R-2DL, KIR-3DL, NKG2A, CD170, CD33, BTLA, CD32b, SlRPb, CD22, PIR-B, LILRB1, 41BBL (TNFSF9), CD27, OX40L, CD32b, CDllb, ITGAM, SLAMF7, CD206, CD 163, CD209, Dectin-2, IL1R, IL2R, IL3R, IL4R, IL5R, IL6R, IL7R, IL8R, IL9R, IL10R, IL11R, IL12R, IL13R, IL14R, IL15R, 1L17R, IFNaR, IFNgR, TNFR, CSF1R, CSF2R, DAP10, CD36, Dectin-1, ICOSL, or Syk intracellular domain or a portion of any of the foregoing.

30. The modified immune cell of claim 27 or 28, wherein the CAR comprises, from the CD8 or CD28 extracellular hinge domain to the one or more intracellular domains: (i) a CD28 extracellular hinge domain, CD28 transmembrane domain, and FcRy intracellular domain;

(ii) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, a CD40 intracellular domain, and FcRy intracellular domain; or

(iii) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, FcRy intracellular domain, and a CD40 intracellular domain; or

(iv) a CD8 extracellular hinge domain, CDS transmembrane domain, truncated Myd88 domain, CD40 intracellular domain, and FcRy intracellular domain.

31. The modified immune cell of any one of claims 27-30, wherein the CAR comprises or has:

(a) an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs: 37- 40, 46, 48, 50, 52, 54, 56, 58, 60, 62, or 126-143; or

(b) an amino acid sequence that differs from any one of the amino acid sequences of SEQ ID NOs: 37-40, 46, 48, 50, 52, 54, 56, 58, 60, 62, or 126-143 by no more than five substitutions, additions, or deletions; or

(c) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the amino acid sequences of SEQ ID NOs: 37-40, 46, 48, 50, 52, 54, 56, 58, 60, 62, or 126-143.

32. A nucleic acid construct comprising one or more nucleic acid sequences encoding:

(a) one or more extracellular domains;

(b) a CD8 or CD28 extracellular hinge domain,

(c) a CD8 or CD28 transmembrane domain, and

(d) one or more intracellular domains comprising an FcRy intracellular domain: and wherein the nucleic acid construct encodes a chimeric antigen receptor (CAR) comprising (a) through (d).

33. The nucleic acid construct of claim 31, further comprising one or more nucleic acid sequences encoding:

(i) a Myd88 intracellular domain or a portion thereof, or (ii) a CD40 intracellular domain or a portion thereof.

34. The nucleic acid construct of claim 32 or claim 33, wherein the one or more intracellular domains further comprise one or more of a: CD3-zeta, FcRy, CD40 CD64, CD32a, CD32c, CD 16a, CD89, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, ALK, AXL, DDR2, EGFR, EphAl, INSR, cMET, MUSK, PDGFR, PTK7, RET, R0R1, ROSE RYK, TTE2, TRK, VEGFR, GDIS), CD20, 41BB, CD28, GCSFR (CD114), RAGE, CD30, CD160, DR3, Fnl4, HVEM, CD 160, NGFR, RANK, TNFR2, TROY, XEDAR, TRIF, 0X40, GITR, TREM-1, TREM-2, DAP 12, MR, ICOS, MyD88, V/RLxYxxL/V, SlRPa, CD45, Siglec-10, PD1, SHP- 1, SI IP 2. KIR-2DL, KIR-3DL, NKG2A, CD 170, CD33, B TLA, CD32b, SIRPb, CD22, PIR-B, LILRB1, 41BBL (TNFSF9), CD27, OX40L, CD32b, CDllb, ITGAM, SLAMF7, CD206, CD 163, CD209, Dectin-2, IL1R, IL2R, IL3R, IL4R, TL5R, IL6R, IL7R, IL8R, FL OR , IL10R, IL11R, IL12R, IL13R, IL14R, IL15R, IL17R, IFNaR, IFNgR, TNFR, CSF1R, CSF2R, DAP 10, CD36, Dectin-1, ICOSL, or Syk intracellular domain or a portion of any of the foregoing

35. The nucleic acid construct of any one of claims 32-34, wherein the nucleic acid construct encodes, from the CDS or CD28 extracellular hinge domain to the one or more intracellular domains:

(i) a CD28 extracellular hinge domain, CD28 transmembrane domain, and FcRy intracellular domain;

(ii) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, a CD40 intracellular domain, and FcRy intracellular domain;

(iii) a CD28 extracellular hinge domain, CD28 transmembrane domain, truncated Myd88 domain, FcRy intracellular domain, and a CD40 intracellular domain, or

(iv) a CDS extracellular hinge domain, CD8 transmembrane domain, truncated MvdSS domain, CD40 intracellular domain, and FcRy intracellular domain.

36. The nucleic acid construct of any one of claims 32-35, wherein the nucleic acid construct comprises or has: (a) a nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs: 41-44, 64, 66, 68, 70, 72, 74, 76, 78, 80, or 149-166,

(b) a nucleotide sequence that differs from any one of the nucleotide sequences of SEQ ID NOs: 41-44, 64, 66, 68, 70, 72, 74, 76, 78, 80, or 149-166 by no more than five substitutions, additions, or deletions; or

(c) a nucleotide acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the nucleotide sequences of SEQ ID NOs: 41-44, 64, 66, 68, 70, 72, 74, 76, 78, 80, or 149-166.

37. A modified immune cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises:

(a) one or more extracellular domains;

(b) a transmembrane domain; and

(c) one or more intracellular domains comprising one or both of:

(i) a DAP 10 intracellular domain or a portion thereof, or

(ii) a MyD88 intracellular domain or a portion thereof; and wherein the modified immune cell is or comprises a macrophage, monocyte, dendritic cell, or stem cell

38. The modified immune cell of claim 37, wherein the one or more intracellular domains further comprise a CD40 intracellular domain or a portion thereof.

39 The modified immune cell of claim 37 or .38, wherein the transmembrane domain comprises or is a DAP10 transmembrane domain.

40. The modified immune cell of any one of claims 37-39, wherein the CAR further comprises a DAP 10 extracellular hinge domain.

41. The modified immune cell of claim 40, wherein the CAR comprises, from the DAP 10 extracellular hinge domain to the one or more intracellular domains: (i) a DAP10 extracellular hinge domain, DAP10 transmembrane domain, DAP 10 intracellular domain, truncated Myd88 domain, and CD40 intracellular domain;

(ii) a DAP10 extracellular hinge domain, DAP10 transmembrane domain, DAP10 intracellular domain, CD40 intracellular domain, and truncated Myd88 domain, or

(iii) a DAP 10 extracellular hinge domain, DAP 10 transmembrane domain, DAP 10 intracellular domain, and truncated Myd88 domain.

42. The modified immune cell of claims 41, wherein the CAR comprises or has:

(a) an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs: 81- 108;

(b) an amino acid sequence that differs from any one of the amino acid sequences of SEQ ID NOs: 81- 108by no more than five substitutions, additions, or deletions; or

(c) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the amino acid sequences of SEQ ID NOs: 81-108.

43. A nucleic acid construct comprising one or more nucleic acid sequences encoding:

(a) one or more extracellular domains;

(b) a transmembrane domain; and

(c) one or more intracellular domains comprising one or both of:

(i) a DAP 10 intracellular domain or a portion thereof, or

(ii) a MyD88 intracellular domain or a portion thereof; and wherein the nucleic acid construct encodes a chimeric antigen receptor (CAR) comprising (a) through (c)

44. The nucleic acid construct of claim 43, wherein the one or more intracellular domains further comprise a CD40 intracellular domain or a portion thereof.

45. T he nucleic acid construct of claim 43 or 44, wherein the transmembrane domain comprises or is a DAP10 transmembrane domain.

46. The nucleic acid construct of any one of claims 43-45, further comprising one or more nucleic acid sequences encoding a DAP 10 extracellular hinge domain.

47. The nucleic acid construct of claims 46, wherein the nucleic acid construct encodes, from the DAP10 extracellular hinge domain to the one or more intracellular domains:

(i) a DAP 10 extracellular hinge domain, DAP 10 transmembrane domain, DAP 10 intracellular domain, truncated Myd88 domain, and CD40 intracellular domain;

(ii) a DAP 10 extracellular hinge domain, DAP 10 transmembrane domain, DAP 10 intracellular domain, CD40 intracellular domain, and truncated Myd88 domain; or

(iii) a DAP10 extracellular hinge domain, DAP10 transmembrane domain, DAP10 intracellular domain, and truncated Myd88 domain.

48. The nucleic acid construct of claim 47, wherein the nucleic acid construct comprises or has:

(a) a nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs: 109- 121 , 147, 148, or 172-185, or

(b) a nucleotide sequence that differs from any one of the nucleotide sequences of SEQ ID NOs: 109-121, 147, 148, or 172-185 by no more than five substitutions, additions, or deletions, or

(c) a nucleotide acid sequence that, is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the nucleotide sequences of SEQ ID NOs: 109-121, 147, 148, or 172-185.

49 A modified immune cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises:

(a) one or more extracellular domains;

(b) a transmembrane domain; and

(c) one or more intracellular domains comprising one or both of:

(i) a CD64 intracellular domain or a portion thereof, or

50. The modified immune cell of claim 49, wherein the transmembrane domain comprises a CD64 transmembrane domain or portion thereof.

51. The modified immune cell of claim 49 or 50, wherein the CAR further comprises a CDS extracellular hinge domain.

52. The modified immune cell of claim 51, wherein the CAR comprises, from the CD8 extracellular hinge domain to the one or more intracellular domains, a CD8 extracellular hinge domain, CD64 transmembrane domain, CD64 intracellular domain, and truncated Myd88 domain

53. The modified immune cell of claims 52, wherein the CAR comprises or has:

(a) an amino acid sequence of any one of the amino acid sequences of SEQ ID NOs: 123- 125;

(b) an amino acid sequence that differs from any one of the amino acid sequences of SEQ ID NOs: 123-125 by no more than five substitutions, additions, or deletions; or

(c) an amino acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the amino acid sequences of SEQ ID NOs: 123- 125.

54. A nucleic acid construct comprising one or more nucleic acid sequences encoding:

(a) one or more extracellular domains;

(b) a transmembrane domain; and

(c) one or more intracellular domains comprising one or both of:

(i) a CD64 intracellular domain or a portion thereof, or

(ii) a MyD88 intracellular domain or a portion thereof; and wherein the nucleic acid construct encodes a chimeric antigen receptor (CAR) comprising (a) through (c).

55. The nucleic acid construct of claim 54, wherein the transmembrane domain comprises a CD64 transmembrane domain or portion thereof.

56. The nucleic acid construct of claim 49 or 50, further comprising one or more nucleic acid sequences encoding a CD8 extracellular hinge domain.

57. The nucleic acid construct of claim 56, wherein the nucleic acid construct encodes, from the CD8 extracellular hinge domain to the one or more intracellular domains, a CD8 extracellular hinge domain, CD64 transmembrane domain, CD64 intracellular domain, and truncated Myd88 domain.

58. The nucleic acid construct of claim 57, wherein the nucleic acid construct comprises or has:

(a) a nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs: 144- 146; or

(b) a nucleotide sequence that differs from any one of the nucleotide sequences of SEQ ID NOs: 144-146 by no more than five substitutions, additions, or deletions; or

(c) a nucleotide acid sequence that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to any one of the nucleotide sequences of SEQ ID NOs: 144-146.

59. The modified immune cell of any one of claims 1-13, 27-31, 37-42, or 49-52, wherein the MyD88 intracellular domain or a portion thereof comprises one or more amino acid substitutions chosen from E52A, R32A, R32K, Y58A, E52A/R32A/Y58 A, L93P, S34Y, and R98C.

60. The modified immune cell of claim 59, wherein the one or more amino acid substitutions comprises or is R32K.

61. The modified immune cell of claim 59 or 60, wherein the CAR exhibits decreased tonic signaling relative to a similar CAR comprising a MyD88 intracellular domain or a portion thereof without the one or more amino substitutions.

62. The modified immune cell of any one of claims 59-61, wherein the immune cell exhibits increased tumor killing ability relative to an immune cell of the same type comprising a similar CAR comprising a MyD88 intracellular domain or a portion thereof without the one or more amino acid substitutions.

63. The nucleic acid construct of any one of claims 16-22, 32-36, 43-48, or 54-58, wherein the MyD88 intracellular domain or a portion thereof comprises one or more amino acid substitutions chosen from E52A, R32A, R32K, Y58A, E52A/R32A/Y58A, L93P, S34Y, and R98C.

64. The nucleic acid construct of claim 63, wherein the one or more amino acid substitutions comprises or is R32K.

65. A pharmaceutical composition comprising a modified immune cell of any one of claims 27-31, 37-42, 49-53, or 59-62, or a nucleic acid construct of any one of claims 32-36, 43- 48, 54-58, 63, or 64.

66. The pharmaceutical composition of claim 65, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

67. A method of treating a disease or disorder in a subject, the method comprising: administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 65 or 66, wherein at least one sign or symptom of the disease or disorder is improved in the subject after administration.

68. A method of modifying an immune cell, the method comprising: delivering to the immune cell a nucleic acid of any one of claims 32-36, 43-48, 54-58, 63, or 64, thereby producing a modified immune cell, wherein the modified immune cell comprises or is a macrophage, monocyte, dendritic cell, or stem cell.

69. A method of producing a modified immune cell comprising a chimeric antigen receptor (CAR), the method comprising: administering to a subject a composition comprising:

(a) one or more nucleic acid molecules, wherein at least a portion of the one or more nucleic acid molecules encodes the CAR, and

(b) a delivery vehicle; wherein following administration of the composition one or more nucleic acid molecules are translated in an immune cell to produce a modified immune cell comprising the CAR, wherein the immune cell is a stem cell, monocyte, macrophage, or dendritic cell in the subject, and wherein the CAR comprises one or both of:

(i) a MyD88 intracellular domain or a portion thereof, or

(ii) a CD40 intracellular domain or a portion thereof.