Table 2.

Genetic engineering strategies and combinational therapies potentiating CAR T-cell efficacy

(A) Genetic engineering strategies potentiating CAR T cells
	Transgene Effects		Antigen targeted	Tumor targeted	References
Improve infiltration/migration	CCR2	Promotes CAR T-cell trafficking to the tumor following systemic administration	MSLN GD2	Mesothelioma Neuroblastoma	(54, 80)
	CCR4	Promotes CAR T-cell trafficking to the tumor following systemic administration	CD30	Lymphoma	(92)
	Heparanase	Degrades the extracellular matrix, thereby improving CAR T-cell tumor infiltration and efficacy	CSPG4 GD2	Melanoma Neuroblastoma	(93)
Improve CAR T-cell effector function	Active akt	The constitutive Akt expression improves CAR T-cell survival, proliferation, cytokine secretion and renders them resistant to Treg suppression	GD2	Neuroblastoma	(82)
	IL-12	Increases effector cytokine secretion, renders CAR T cells resistant to Treg-mediated inhibition, and induces host innate immune response	CD19 CD30 MUC-16 CEA VEGFR	Leukemia Lymphoma Ovarian Colon melanoma, sarcoma, and colon cancer stroma	(94–97)
	IL-15	Improves T-cell expansion and reduces PD-1 expression	CD19	Leukemia	(98, 99)
	IL-7 or IL-7R	Increases proliferation, survival and effector function of CAR T cells even in the presence of Tregs	CD19 GD2	Leukemia Neuroblastoma	(99, 100)
	IL-21	Increases CAR T-cell proliferation and cytotoxic efficacy	CD19	Leukemia	(99, 101)
	CD80 or 4-1BBL	Trans-/autocostimulation between CAR T cells enhancing effector functions	PSMA	Prostate	(102)
	CD40L	Enhances tumor cell immunogenicity, stimulates moDC and increases CAR T-cell cytotoxic efficacy	CD19	Leukemia	(103)
	4αβ chimeric cytokine receptor	4αβ generated by the fusion of IL-4R ectodomain and IL-2R and IL-15R subunit enhances CAR T-cell long term proliferation and cytotoxicity	MUC1 PSMA ERBBR	Breast Prostate Head and Neck	(104)
Counteract immunosuppression	shRNA CTLA4	Decreased CTLA4 expression enhances CAR T-cell proliferation and antitumor activity	CD19	Leukemia	(105)
Improve specificity and safety	iCAR ‘safety switch’	iCAR with PD-1 or CTLA-4 inhibitory intracellular domain linked to secondary antigen constrains CAR T-cell specificity to cancer cells expressing the primary antigen	PSMA	Prostate	(106)

(B) Preclinical investigation of combinational therapies potentiating CAR T-cell efficacy
	Agents Effects		Antigen targeted	Tumor targeted	References
Preconditioning	Radiotherapy	Total body irradiation (TBI)-induced lymphodepletion in the host promotes CAR T-cell efficacy	EGFRvIII	Glioblastoma	(107)
	Flutamide	Fludamide-induced androgen ablation acts in additive with CAR T cells in vitro	MUC-1	Prostate	(108)
	Valproate	Sodium Valproate-induced upregulation of tumor cell-surface NKG2DL expression enhances the immune recognition of CAR T cells in vitro	NKG2DL	Ovarian	(109)
Monoclonal antibodies	PD-L1 or PD-1 immune checkpoint blockade	Blocking the PD-1 immunosuppressive signaling enhances CAR T-cell proliferation, cytotoxicity and cytokine secretion	CEA MSLN HER2	Liver metastases from colon cancer Mesothelioma Sarcoma	(48, 65, 110)
	Bispecific antibodies EGFR/cMet or EGFR/Epcam	Bispecific antibodies link EGFR-transduced CAR T cells to antigen-expressing tumor cells enhancing CAR T-cell recruitment/retention and cytotoxicity	CEA cMet Epcam	Colon	(111)
	Anti GM-CSF or Gr-1	Reduction of Myeloid-derived suppressor cells population (MDSC)	CEA	Liver metastases from colon cancer	(65)
Small specific inhibitory drug	ABT-737	Improves CAR T-cell killing by restoring apoptosis pathway in tumor cells	CD19	Leukemia	(112)
	Rapamycin	Inhibition of mTor kinase decreases the expression of anti-apoptotic molecules and others (VEGF, PD-L1, IL-10) leading to a superior antitumor effect of CAR T cells engineered with mTor resistance	CD19	Leukemia	(113)
	BRAFi/MEKi	Inhibition of MAPK pathway blocks tumor cell growth and enhances apoptotic killing by CAR T cells in vitro	GD2	Melanoma	(114)
Oncolytic virus	Adenovirus vector expressing Rantes and IL-15	Adenovirus vector-mediated Rantes and IL-15 expression in the tumor enhances CAR T-cell infiltration and persistence	GD2	Neuroblastoma	(115)
Whole cell vaccine	Irradiated K562 cells expressing CD40L and OX40L	Vaccination boosts antitumor efficacy of CAR T cells	GD2	Lung Neuroblastoma	(116)