November 28, 2021

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Interaction of next-generation CAR cell therapy with tumors and TME

Interaction of next-generation CAR cell therapy with tumors and TME

Interaction of next-generation CAR cell therapy with tumors and TME



 

Interaction of next-generation CAR cell therapy with tumors and TME


introduce

In this review, we studied current and future CAR design strategies, T cells expressing CAR or tumor-specific T cell receptor (TCR), and the interaction between engineered T cells and the tumor microenvironment (TME) , Pay special attention to improving the effectiveness and safety of adoptive T cell therapy in the treatment of solid tumors (Figure 1).

 

Interaction of next-generation CAR cell therapy with tumors and TME
Figure 1. CAR-T cell engineering method. Strategies for designing CAR-T cells to improve the function of solid tumors include focusing on CAR engineering, T cell engineering, and TME interaction optimization.

 

The immune evasion and immunosuppressive properties of TME lead to the poor efficacy of CAR-T cell therapy observed in solid tumors. The signs of TME have been extensively reviewed elsewhere, including

(1) physical barriers for immune cells to penetrate tumors,

(2) up-regulated checkpoint ligands,

(3) tumor stroma niche, 

(4) abundant immunosuppression And metastasis-promoting soluble factors, 

(5) regulating the expression of chemokines to preferentially recruit leukocytes with immunosuppressive phenotypes.

 

These factors in turn promote the design of CAR-T cells, which respond to TME elements to enhance the efficacy of CAR-T cells (Figure 5).

 

Interaction of next-generation CAR cell therapy with tumors and TME

Figure 5. Strategies to optimize the interaction between CAR-T cells and tumors CAR-T cells have been designed to utilize, reverse or circumvent tumor-driven immunosuppressive factors and axes through various mechanisms.

 

 

 


Tumor homing and penetration

 

The efficacy of CAR-T cell therapy in solid tumors is significantly hindered by poor immune cell infiltration. T cell migration is regulated by the chemokine axis. Tumor cells can up-regulate or down-regulate chemokines and regulate the expression of chemokines in tumor-related cells, leading to poor recruitment of CAR-T cells. Designing CAR-T cells to overexpress chemokine receptors that are overexpressed in TME can make the tumor’s defense mechanism target itself. For example, GD2 mesothelin-targeted CAR-T cells have been designed to co-express CCR2b, the main isotype of CCL2 chemokine receptors, causing T cells to homing to CCL2-expressing neuroblastoma and malignant pleural space, respectively. Dermatoma xenografts.

 

Similarly, CAR-T cells co-expressing CCR4 showed improved migration to tumors expressing CCL17 and CCL22 in vivo, while those cells expressing CXCR1 or CXCR2 showed enhanced homing of tumor-derived IL-8.

 

Once CAR-T cells reach the tumor site, their infiltration will be hindered by the high-density structure of extracellular matrix (ECM) associated with solid tumor nodules. Therefore, CAR-T cells modified to express heparinase, an enzyme that degrades ECM, have been shown to improve tumor invasion and overall survival in a variety of xenograft models.

 

CAR-T cells that successfully reach solid tumors are then faced with a variety of inhibition and escape characteristics, which can lead to CAR-T cell dysfunction. In order to improve the therapeutic effect in this immunosuppressive environment, CAR-T cells have been engineered to produce proteins:

(1) Improve CAR-T cell function in an autocrine manner;

(2) Destroy immunosuppressive components;

and/or

( 3) Induce TME remodeling to enhance the endogenous anti-tumor immune response (Figure 5).

 

 

Each of these strategies will be discussed in detail below.

 

 

 

 


Autocrine stimulation of CAR-T cells in TME

 

Cytokines are signaling proteins that can greatly enhance or eliminate CAR-T cell functions. Co-expression of CAR and immunostimulatory cytokines can significantly enhance the proliferation, survival and effector functions of CAR-T cells in immunosuppressive TME.

For example, T cells that constitutively co-express CD19 targeting CAR plus IL-2, IL-7, IL-15, or IL-21 have been shown to achieve better tumor control in vivo compared to CAR-only T cells .

 

Interestingly, although the receptor complexes of these four cytokines contain a common γ chain (γc), each cytokine has different effects on the proliferation, subtype differentiation and function of engineered T cells, which emphasizes T cells The complexity of biology and various potential results can be achieved through different engineering strategies.

T cells co-expressing IL-12, IL-15, IL-18 and/or IL-21 and CARs targeting multiple antigens are also described, thereby improving efficacy, proliferation and/or persistence in vivo.

However, constitutive overexpression of immunostimulatory cytokines can also increase toxicity. The regulatory strategies discussed earlier in this review, such as inducible promoters, can be used to regulate cytokine production and related toxicity.

 

 

 


Destruction of immunosuppressive components

 

The expression of immune checkpoint receptors and ligands (such as PD-1 and PD-L1) is common in TME, and they can effectively inhibit the cytotoxicity of CAR-T cells and induce anergy.

Therefore, immune checkpoint blockade and CAR-T cell therapy have a strong synergistic potential. Several ongoing clinical trials are evaluating the combination therapy of CAR-T cells and exogenous checkpoint inhibitors. In addition, CAR-T cells have been designed to secrete immune checkpoint inhibitors, including anti-PD-1 scFv and anti-PD-L1 antibodies, or express PD-1 dominant negative receptor (DNR).

In addition to improving efficacy, this approach can also avoid the toxicity associated with systemic immune checkpoint blockade by restricting the distribution of checkpoint inhibitors to the direct environment of productive T cells.

For example, it has been shown that anti-PD-1 scFvs secreted by CAR-T cells injected intraperitoneally (IP) are still located at the injection site. However, when the same number of conventional CAR-T cells were administered IP together with exogenous anti-PD-1 antibody, the antibody was detected by the system within 3 hours.

 

The solid tumor environment also contains a variety of soluble factors, which can promote tumorigenesis and inhibit CAR-T cell function. For example, prostaglandin E2 (PGE2) is a biologically active lipid that is usually up-regulated in tumors and promotes tumor survival by regulating cell proliferation, migration, apoptosis and angiogenesis.

In the context of CAR-T cell therapy, PGE2 together with adenosine inhibits T cell signal transduction and activation through the activation of protein kinase A (PKA), thereby reducing T cell proliferation and effector function. In two solid tumor models that highly express PGE2, CAR-T cells are designed to express a peptide inhibitor.

The peptide inhibitor that translocates ezrin-mediated PKA to immune synapses shows improved tumor invasion and killing . Similarly, the increased concentration of reactive oxygen species (ROS) and other bioreactive chemicals in TME play an important role in tumorigenesis.

Catalase is an enzyme that promotes the decomposition of hydrogen peroxide (H2O2), which is a ROS that can damage the activity of T cells in TME. By co-expressing the catalase gene in HER2 and carcinoembryonic antigen (CEA) specific CAR-T cells to increase intracellular catalase levels, it has been shown to enable CAR-T cells to metabolize inhibitory H2O2, thereby increasing them The ability of tumor cell lysis.

 

The abnormal expression of cytokines in TME plays a key role in tumor progression and resistance to CAR-T cell therapy. In particular, TGF-β plays multiple roles in cancer progression by interacting with tumor cells, stroma and innate and adaptive immune cells to induce

(1) the secretion of immunosuppressive chemokines, cytokines and growth factors;

(2) ECM remodeling and matrix deposition;

(3) immunosuppressive reprogramming of macrophages, neutrophils and T cells;

(4) inhibition of the maturation or proliferation of T cells and NK cells.

 

In order to eliminate these powerful effects, CAR-T cells have been designed to express TGF-β DNR, which can effectively inhibit endogenous TGF-β signaling, so that T cells have enhanced proliferation in prostate cancer xenograft models And anti-tumor effect.

Based on these results, a phase 1 clinical trial has been initiated to evaluate T cells co-expressing PSMA CAR and DNR for the treatment of relapsed and refractory metastatic prostate cancer (NCT03089203).

DNR is different from the TGF-β targeting CAR and TGF-β switch receptors discussed in the previous section because DNR does not transduce any signals that can stimulate engineered T cells.

Compared with DNR, whether the stimulation of CAR and switch receptors will bring additional clinical benefits remains to be seen.

 

In TME, IL-6 is often overexpressed by tumor cells, tumor-associated macrophages (TAM) and other resident cells. IL-6 supports tumorigenesis through multiple mechanisms and plays a central role in inducing CRS after CAR-T cell infusion.

Tocilizumab is a monoclonal antibody that targets IL-6 receptor α (IL-6Rα) and has become the standard treatment for CRS after CAR-T cell therapy. Recently, CD19-targeting CAR-T cells co-expressing non-signaling, membrane-bound IL-6 receptor (mbaIL6) have been shown to isolate IL-6 while maintaining anti-tumor efficacy in vivo. However, whether CAR-T cells designed in this way can prevent CRS remains to be seen.

 

 

 


TME reshapes to promote endogenous immune response

 

Tumors are good at selectively attracting or evading subpopulations of white blood cells, including CAR-T cells, to promote immune regulation or suppression. In addition, tumors are usually able to induce immunosuppressive or metastatic phenotypes on the local matrix, as well as anti-inflammatory or dysfunctional phenotypes of resident white blood cells.

Another way to improve the efficacy of CAR-T cell therapy is to reverse this immunosuppressive cell niche by remodeling the composition and phenotype of tumor cells. To achieve this, CAR-T cells have been designed to secrete cytokines or other soluble factors to induce TME remodeling in a paracrine or endocrine manner.

 

In germinal center lymphoma, the loss of expression of herpes virus entry mediator (HVEM) induces the secretion of non-redundant stromal activating factors, leading to acute lymphostromal activation. The over-activated matrix recruits TFH cells, which support malignant B cells through CD40/CD40L interaction and cytokine stimulation.

As a countermeasure, CD19 CAR-T cells have been shown to secrete HVEM in a soluble form, thereby enhancing tumor control in vivo.

 

CAR-T cells designed to secrete IL-12 have been shown to reshape TME by reprogramming TAM to the M1 phenotype and reducing the presence of MDSC and Treg in a syngeneic mouse model.

Similarly, CAR-T cells that constitutively secrete IL-18 can change the composition of TME by increasing the number of M1 macrophages, activated dendritic cells (DC) and activated NK cells in the tumor, while reducing the levels of M2 macrophages and Tregs. .

A direct comparison of CAR-T cells expressing IL-12- and IL-18 shows that IL-18 is more effective in remodeling immunosuppressive TME in a syngeneic murine pancreatic cancer model. In addition, CD19 CAR-T cells expressing IL-18 induce the expansion of endogenous CD8+ T cells, NK cells, NKT cells, and DCs in the bone marrow, which may help control the expression of heterogeneous CD19 in a syngeneic mouse model Tumor.

 

Among DCs, traditional type 1 DCs (cDC1s) are particularly good at inducing tumor immunity through their ability to cross-present cell antigens and trigger Th1 cells.

Recently, it has been shown that T cells are designed to secrete Fms-like tyrosine kinase 3 ligand (Flt3L), a hematopoietic growth factor that promotes the proliferation of cDC1 and DC precursors in tumors (0).

In addition, when T cells are co-transduced to express Flt3L and anti-HER2 CAR, the combination therapy of these CAR-T cells and adjuvants induces an enhanced anti-tumor response and the spread of endogenous T cell epitopes in vivo.

 

CAR-T cells are also designed to co-express multiple immunomodulatory proteins. In one example, CAR-T cells are programmed to co-express CCL19 and IL-7 to induce endogenous immune cell recruitment and stimulate the recruited cells, respectively. In a mouse model of syngeneic mast cell tumor expressing hCD20, these CAR-T cells induce a large number of endogenous T cells and DC recruitment, thereby enhancing and durable tumor clearance.

 

CAR-T cells are also designed to regulate TME by expressing surface-bound pro-inflammatory ligands. For example, CD40L is usually transiently expressed on T cells after TCR stimulation, and its interaction with CD40 receptors on different immune cell types can lead to APC activation, DC permission, and CD40+ tumor cell apoptosis.

Constitutive CD40L expression on CD19 CAR-T cells leads to increased surface expression of costimulatory molecules, adhesion molecules, HLA molecules, and Fas death receptors on CD40+ tumor cells, thereby increasing their immunogenicity. These T cells also induce monocyte-derived DC to secrete pro-inflammatory IL-12 in vitro and show enhanced anti-tumor efficacy in vitro and in vivo.

It was subsequently demonstrated that CAR-T cells expressing CD40L can permit APC in lymphoid tissues in a syngeneic immunocompetent mouse model, and found that this permitting depends on the CD40L/CD40 interaction.

 

In addition, increased recruitment of macrophages, DCs, and endogenous CD4+ and CD8+ T cells to lymphoid tissues, as well as the recruitment of DC, CD4+ and CD8+ T cells to tumors, was observed. A slight increase in the level of Treg in the tumor was also observed, but the ratio of D8+ T cells to Treg did not change.

Therefore, CAR-T cells expressing CD40L can reshape TME and lymphoid tissues, activate endogenous T cells to inhibit the re-attack of antigen-negative tumors, and strongly indicate the induction of epitope spread.

Similarly, the surface expression of 4-1BBL on CAR-T cells is proposed to reshape TME through autocrine-induced type I interferon secretion, which may improve DC cross-priming, Treg inhibition, and angiogenesis inhibition.

 

Finally, CAR-T cells can promote the junction of endogenous non-engineered T cells and tumor cells through the secretion of bispecific T cell junction (BiTE). BiTE is composed of two fused scFvs. Cui et al. designed BiTE, in which one scFv targets EGFR that is overexpressed in glioblastoma cells, and the other targets CD3 on T cells.

CAR-T cells targeting EGFRvIII that are designed to secrete EGFR/CD3 BiTE have been shown to eliminate orthotopic tumor xenografts with heterogeneous GFRvIII expression.

 

 

 


Conclusion

CAR-T cell therapy shows great prospects in the treatment of hematological malignancies. However, solid tumors pose unique challenges and require further modification and adjustment of technology to successfully treat these stubborn malignant tumors.

Recent protein and cell engineering strategies have made great progress in improving the inherent adaptability and anti-tumor function of T cells, improving tumor targeting specificity, preventing tumor escape and recurrence, and modifying TME to enhance the effect of immunotherapy.

Although most of the engineering strategies reported to date have focused on providing the characteristics required by the individual, advances in genome editing methods and gene circuit design have provided the possibility of multi-layered methods that can simultaneously meet the needs of many in the development of T cell therapies. Key requirements.

 

At the same time, when advancing clinical transformation, it is necessary to carefully balance the biological complexity and potential crosstalk between different engineering features in T cells and between engineered and endogenous immune cells, tumor cells, and other tumor-related factors. CAR-T cells treat.

 

 

 

Interaction of next-generation CAR cell therapy with tumors and TME

(source:internet, reference only)


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