Cells comprising a suppressor of gene expression and/or a syntheticpathway activator and/or an inducible payload

A system comprising:

• a. a first chimeric polypeptide comprising a priming receptor;
• b. a second chimeric polypeptide comprising a chimeric antigen receptor (CAR);
• c. a third chimeric polypeptide comprising a synthetic pathway activator (SPA);
• and
• d. a cytokine.

A system comprising:

• a. a first chimeric polypeptide comprises a priming receptor;
• b. a second chimeric polypeptide comprises a chimeric antigen receptor (CAR);
• and
• c. a cytokine.

A system comprising:

• a. a first chimeric polypeptide comprising a priming receptor;
• b. a second chimeric polypeptide comprising a chimeric antigen receptor (CAR);
• c. a suppressor of gene expression, and
• d. one or both of:
• i. a third chimeric polypeptide comprising a synthetic pathway activator (SPA);
• and/or
• ii. a cytokine.

One or more recombinant nucleic acids comprising at least one nucleic acid fragment comprising:

• a. a nucleotide sequence encoding a priming receptor comprising a first extracellular antigen-binding domain;
• b. a nucleotide sequence encoding a chimeric antigen receptor comprising a second extracellular antigen-binding domain;
• and
• c. a nucleotide sequence encoding a cytokine.

One or more recombinant nucleic acids comprising at least one nucleic acid fragment comprising:

• a. a nucleotide sequence encoding a priming receptor comprising a first extracellular antigen-binding domain;
• b. a nucleotide sequence encoding a chimeric antigen receptor comprising an second extracellular antigen-binding domain;
• and
• c. a nucleotide sequence encoding a synthetic pathway activator.

One or more recombinant nucleic acids comprising at least one nucleic acid fragment comprising:

• a. a nucleotide sequence encoding a priming receptor comprising a first extracellular antigen-binding domain;
• b. a nucleotide sequence encoding a chimeric antigen receptor comprising an second extracellular antigen-binding domain;
• c. a nucleotide sequence encoding a synthetic pathway activator;
• and
• d. a nucleotide sequence encoding a cytokine.

One or more recombinant nucleic acids comprising at least one nucleic acid fragment comprising:

• a. a nucleotide sequence encoding a priming receptor comprising a first extracellular antigen-binding domain;
• b. a nucleotide sequence encoding a chimeric antigen receptor comprising an second extracellular antigen-binding domain;
• and
• c. a nucleotide sequence of a suppressor of gene expression;
• and
• d. one or both of:
• i. a nucleotide sequence encoding a synthetic pathway activator;
• and/or
• ii. a nucleotide sequence encoding a cytokine.

A primary immune cell comprising at least one recombinant nucleic acid comprising:

• a. a nucleic acid sequence encoding a priming receptor comprising a first extracellular antigen-binding domain;
• b. a nucleic acid sequence encoding a chimeric antigen receptor comprising a second extracellular antigen-binding domain;
• and
• c. a nucleic acid sequence encoding a synthetic pathway activator and/or a nucleic acid sequence encoding a cytokine;
• wherein the recombinant nucleic acid is inserted into a target region of the genome of the primary immune cell, wherein the primary immune cell does not comprise a viral vector for introducing the recombinant nucleic acid into the primary immune cell.

A viable, virus-free, primary cell comprising a ribonucleoprotein complex (RNP)-recombinant nucleic acid complex, wherein the RNP comprises a nuclease domain and a guide RNA, wherein recombinant nucleic acid comprises:

• a. a nucleic acid sequence encoding a priming receptor comprising a first extracellular antigen-binding domain;
• b. a nucleic acid sequence encoding a chimeric antigen receptor comprising a second extracellular antigen-binding domain;
• and
• c. a nucleic acid sequence encoding a synthetic pathway activator that constitutively activates cytokine signaling and/or a nucleic acid sequence encoding a cytokine;
• wherein the 5′ and 3′ ends of the recombinant nucleic acid comprise nucleotide sequences that are homologous to genomic sequences flanking an insertion site in the genome of the primary cell.

A viable, virus-free, primary cell comprising a ribonucleoprotein complex (RNP)-recombinant nucleic acid complex, wherein the RNP comprises a nuclease domain and a guide RNA, wherein recombinant nucleic acid comprises:

• a. a nucleic acid sequence encoding a priming receptor comprising a first extracellular antigen-binding domain that specifically binds to ALPG/P;
• b. a nucleic acid sequence encoding a chimeric antigen receptor comprising a second extracellular antigen-binding domain that specifically binds to MSLN;
• and
• c. a nucleic acid sequence encoding a synthetic pathway activator that constitutively activates cytokine signaling and/or a nucleic acid sequence encoding a cytokine;
• wherein the 5′ and 3′ ends of the recombinant nucleic acid comprise nucleotide sequences that are homologous to genomic sequences flanking an insertion site in the genome of the primary cell.