Disclosed are methods of making a genetically modified immune cell for modifying a tumor microenvironment (TME) and methods of modifying a tumor microenvironment (TME). In some embodiments, the method can include delivering a first vector to an immune cell, wherein the first vector comprises a nucleic acid encoding a protein that induces T-cell proliferation, promotes persistence and activation of endogenous or adoptively transferred NK or T cells and/or induces production of an interleukin, an interferon, a PD-1 checkpoint binding protein, HMGB1, MyD88, a cytokine or a chemokine. Methods of modulating the suppression of the immune response in a tumor microenvironment, minimizing the proliferation of tumor and suppressive cells, and increasing the efficiency of an anti-cancer therapy, anti-infection therapy, antibacterial therapy, anti-viral therapy, or anti-tumoral therapy are also provided.

The disclosure features amphiphilic ligand conjugates comprising a chimeric antigen receptor (CAR)ligand and a lipid. The disclosure also features compositions and methods of using the same, for example, to stimulate proliferation of CAR expressing cells.

Bi-specific chimeric antigen receptors (CARs) capable of binding to both CD19 and CD22 and immune cells expressing such. Also provided herein are therapeutic uses of such immune cells (e.g., CAR-T cells) for eliminating disease cells such as cancer cells.

The present disclosure provides rAAV vectors and rAAV virions that specifically express exogenous nucleic acid sequences in CD14.sup.+ cells. The rAAV vectors or virions are useful for specifically expressing exogenous nucleic acid sequences encoding, for example, cancer antigens, viral antigens, and/or bacterial antigens in monocytes and dendritic cells. The rAAV transduced CD14.sup.+ cells can be used as antigen presenting cells that induce antigen-specific T cell responses. The present disclosure further provides methods producing rAAV virions and methods of immunotherapy.

The current invention provides monocytic cells transfected with chimeric antigen receptor (CAR) to selectively home to tumors and upon homing differentiate into dendritic cells capable of activating immunity which is inhibitory to said tumor. In one embodiment of the invention, monocytic cells are transfected with a construct encoding an antigen binding domain, a transcellular or structural domain, and an intracellular signaling domain. In one specific aspect of the invention, the antigen binding domain interacts with sufficient affinity to a tumor antigen, capable of triggering said intracellular domain to induce an activation signal to induce monocyte differentiation into DC.

The current invention provides monocytic cells transfected with chimeric antigen receptor (CAR) to selectively home to tumors and upon homing differentiate into dendritic cells capable of activating immunity which is inhibitory to said tumor. In one embodiment of the invention, monocytic cells are transfected with a construct encoding an antigen binding domain, a transcellular or structural domain, and an intracellular signaling domain. In one specific aspect of the invention, the antigen binding domain interacts with sufficient affinity to a tumor antigen, capable of triggering said intracellular domain to induce an activation signal to induce monocyte differentiation into DC.

Disclosed are combination therapies for cancer utilizing the leverage of the adaptive immune system through chimeric antigen receptor (CAR) T cells, combined with leveraging the innate immune system by using CAR-macrophages (CAR-M) and CAR-natural killer (NK) cells. In some embodiments, the invention teaches the initial modification of the tumor microenvironment by administration of CAR-M and CAR-NK. The alteration of the tumor microenvironment results in reduction of barriers for CAR-T cells to enter the tumor, which allows for efficacy of CAR-T in treatment of solid tumors. In some embodiments adjuvant immunotherapies are utilized to expand immunological attack such as addition of complement, immunotherapeutic antibodies, chemotherapy and radiotherapy approaches.

A method for producing CAR-M1 macrophages expressing a chimeric antigen receptor in vitro and in vivo includes using a conjugate of a non-viral gene delivery system and a chimeric antigen receptor gene. The CAR-M1 macrophages are produced in vivo by delivering genes encoding a chimeric antigen receptor and IFN-?, specifically to macrophages in the body, and thus does not require culturing and preparing an in-vitro cellular therapeutic agent, thus reducing the manufacturing costs of therapeutic agents. The CAR-M1 macrophages are a safer therapy since a non-viral vector is used, as compared to the production of CAR-M1 macrophages by gene delivery using a viral vector, and are a novel therapeutic candidate having the advantage of high anticancer efficiency for solid cancers, due to CAR-M1 macrophages in which intrinsic properties of macrophages infiltrating solid cancers and cancer cell phagocytosis are improved.

Therapeutic and diagnostic methods are provided, which methods relate to the induction of expression of calreticulin on phagocytic cells. Specifically, the methods relate to macrophage-mediated programmed cell removal (PrCR), the methods comprising increasing PrCR by contacting a phagocytic cell with a toll-like receptor (TLR) agonist; or down-regulating PrCR by contacting a phagocytic cell with an inhibitor of Bruton's tyrosine kinase (BTK). In some embodiments, an activator of TLR signaling or a BTK agonist is provided in combination with CD4 7 blockade.

The present invention relates to novel immunoregulatory macrophage cells which are useful in the treatment of different immunological and non-immunological diseases and conditions. The cells are characterized by a specific marker and activity pattern which distinguishes them from other cells. The novel immunoregulatory macrophage cells have a high phagocytosing capacity and are capable to suppress the proliferation of T cells. The invention also provides a novel process for preparing immunoregulatory macrophage cells in suspension culture from blood monocytes. The process is amenable to a high degree of automation. In a still further aspect, the invention relates to a pharmaceutical composition comprising the immunoregulatory macrophage cells of the invention.