CAR race to cancer immunotherapy: from CAR T, CAR NK to CAR macrophage therapy

Abstract

Adoptive cell therapy with chimeric antigen receptor (CAR) immunotherapy has made tremendous progress with five CAR T therapies approved by the US Food and Drug Administration for hematological malignancies. However, CAR immunotherapy in solid tumors lags significantly behind. Some of the major hurdles for CAR immunotherapy in solid tumors include CAR T cell manufacturing, lack of tumor-specific antigens, inefficient CAR T cell trafficking and infiltration into tumor sites, immunosuppressive tumor microenvironment (TME), therapy-associated toxicity, and antigen escape. CAR Natural Killer (NK) cells have several advantages over CAR T cells as the NK cells can be manufactured from pre-existing cell lines or allogeneic NK cells with unmatched major histocompatibility complex (MHC); can kill cancer cells through both CAR-dependent and CAR-independent pathways; and have less toxicity, especially cytokine-release syndrome and neurotoxicity. At least one clinical trial showed the efficacy and tolerability of CAR NK cell therapy. Macrophages can efficiently infiltrate into tumors, are major immune regulators and abundantly present in TME. The immunosuppressive M2 macrophages are at least as efficient as the proinflammatory M1 macrophages in phagocytosis of target cells; and M2 macrophages can be induced to differentiate to the M1 phenotype. Consequently, there is significant interest in developing CAR macrophages for cancer immunotherapy to overcome some major hurdles associated with CAR T/NK therapy, especially in solid tumors. Nevertheless, both CAR NK and CAR macrophages have their own limitations. This comprehensive review article will discuss the current status and the major hurdles associated with CAR T and CAR NK therapy, followed by the structure and cutting-edge research of developing CAR macrophages as cancer-specific phagocytes, antigen presenters, immunostimulators, and TME modifiers.

Keywords: Adoptive cell transfer; CAR NK cells; CAR T therapy; CAR macrophage; Chimeric antigen receptor (CAR); Cytokine release syndrome; Immunotherapy; Tumor microenvironment.

Fig. 1

Timeline of CAR T therapy FDA approvals

Fig. 2

CAR structure for CAR T, CAR NK and CAR macrophage. CARs for CAR T, CAR NK and CAR macrophage have similar structures: the extracellular domain including the antigen binding domain and a spacer which is involved in engagement of target cells; transmembrane domain which docks CAR to immune cells and is also involved in other functions of CAR, such as stability and interaction with other membrane proteins; and the intracellular signaling domain which is involved in signaling transduction and activation of immune cells. For the target binding domain, in addition to scFv, native protein/peptide, cytokine and camelid nanobody have also been used. For the intracellular domain, in addition to the function to activate immune cells, other domains to regulate TME have also been used. Three generations of CAR structure are mainly determined by the difference of the intracellular domains. The first-generation CAR contains a single CD3ζ signaling domain. It has limited activities in CAR T cells as T cell activation requires a primary signal from T cell receptor complex with CD3 and a co-stimulatory signal from CD28. However, this generation of CAR has been used in CAR NK and CAR macrophage as a co-stimulatory signal is not required. The difference of the second- and third-generation CAR over the first-generation one is the addition of one and two co-stimulatory signaling domains. In the FDA-approved CAR T cells, these co-stimulatory domains are usually CD28 or 4-1BB. In CAR NK and CAR macrophage, their specific or other ITAM-containing domains are used for the intracellular signaling domain. (This figure was created at BioRender.com.)

Fig. 3

Harnessing NK cells for cancer immunotherapy. Several approaches are currently being actively pursued to exploit NK cells for cancer immunotherapy. A CAR NK cells. In CAR NK cells, artificial cell surface receptor on NK cells specifically recognizes tumor antigens on target/cancer cells and CAR NK cells destroy those cells. CAR can use the same CAR construct as used in CAR T cells with CD3ζ intracellular domain, or NK-specific activating domains, such as 2B4, DAP10 and DAP12. B Blockage of negative regulators on NK cells. The activity of NK cells is tightly regulated by both activating and inhibiting signaling pathways. The human killer cell immunoglobulin-like receptors (KIR; also known as CD158) are key negative regulators of NK cells. Engagement of KIR by MHC-I molecules on normal nucleated cells inhibits NK cell activity and induces “self” tolerance. Blockage of KIR activates NK cells to kill target cells. C Antibody-dependent cell-mediated cytotoxicity (ADCC). Antibody binds to its cognate antigen on target/cancer cells. Then the Fc region of the antibody is recognized by the Fc receptor, CD16, on NK cells which subsequently kills target/cancer cells coated with antibody. D Bi- and tri-specific killer engagers (BiKEs and TriKEs). Similar to ADCC, BiKEs and TriKEs bridge NK cells to target/cancer cells for cell killing. While the Fc portion of an antibody binds to the Fc receptor to mediate cell killing at ADCC, BiKEs and TriKEs contain a single variable portion (VH and VL) of antibody to engage the Fc receptor (CD16) on NK cells and another (for BiKE) or two other (for TriKE) variable portions of antibodies to bind to the antigen(s) on target/cancer cells. This figure was created at BioRender.com