Cancers: CAR-T and CAR-NK solid tumor targeting strategies

The emergence of engineered adoptive cell therapy (ACT) has changed the treatment landscape of hematological tumors and brought hope
for the treatment of solid tumors.
However, the application of cell therapy in solid tumors is affected
by poor tumor migration, penetration of the physical barrier, and active tumor inhibition.
Therefore, precise guidance of endogenous or adoptively metastatic lymphocytes into solid tumor masses is necessary to achieve the best anti-tumor results and will also improve patient safety
.

Traditional therapies, including chemotherapy and radiation, have been shown to stimulate T cells to migrate into tumors to enhance the effectiveness of treatment, while radiation therapy can have a distal effect
on distant metastases.
Similarly, inflammatory events within the tumor are directly induced by injection of pathogen-associated molecular patterns or oncolytic viruses into the tumor, stimulating systemic and local events at the tumor site to encourage lymphocyte migration and antigen-specific anti-tumor responses
.
In addition, antigen recognition itself is a major stimulus for
migration.

To date, many explorations and attempts have been made, and many strategies have emerged that can be used to enhance the migration and penetration
of adoptive metastatic cells, including CAR-NK and CAR-T cells, in solid tumor immunotherapy.

Chemokines and their receptors

A chemokine is a cytokine that regulates the migration and transport
of immune cells.
Chemotaxis gradients are essential
for recruiting effector cells to sites of inflammation, including the tumor microenvironment (TME).
Chemokine expression in solid tumors is secreted by stromal cells, tumor cells, and tumor-associated immune cells to determine which immune cells are recruited to TME, thereby aiding or hindering tumor growth
.

Multiple chemokine/chemokine receptor strategies have been used in preclinical studies of immunotherapeutic T cells to promote CAR-T cell targeting of tumors, including utilizing the CXCR3, CXCR2, CCR5, CCR2, and CCR3 axes
.
There are currently no approved cancer chemokine immunotherapy strategies, but multiple studies have shown good clinical potential
.

CXCR3

The CXCR3 axis is a key pathway for solid tumor immune cell recruitment, and the ligands of CXCR3 include CXCL9, CXCL10, and CXCL11
.
After activation, CXCR3 was induced and highly expressed on CD4+ T cells, CD8+ T cells, NK cells, and NKT cells polarized by effector TH1, and the expression of CXCR3 ligands in tumors, the increase in serum CXCR3 ligand levels, and CXCR3 on T cells enhanced the recruitment of T cells and achieved positive preclinical results
in a range of cancers.

The tumor local expression of CXCL11 by oncolytic vaccinia virus in a mouse model of mesothelioma successfully increased the transport of endogenous cytotoxic T lymphocytes to tumors and induced systemic anti-tumor immunity, highlighting the importance of
CXCR3 axis for lymphocyte migration.
In addition, the use of epigenetic regulators (DNA methylation and H3K27 trimethylation inhibitors) restored tumor expression of CXCL9 and CXCL10, improved CAR-T cell efficiency in mouse models of ovarian cancer, and improved T cell infiltration and tumor growth control
.
In a mouse myeloma model, a combination of two oncolytic adenoviruses encoding CXCL10 and IL-18 into established tumors resulted in smaller tumor growth and complete regression
of 80% of mouse tumors.

CXCR2

In a range of tumor models, regulation of CXCR2 expression on T cells has been shown to improve therapeutic outcomes
.
CXCR2 is specific for a variety of ligands overexpressed on tumors, including CXCL1, CXCL2, CXCL5, and CXCL8
.
These ligands are also expressed by various invasive immune cells and are often considered to be pro-tumor chemokines
.
For example, CXCL8 directly promotes invasion and metastasis and can promote metastatic niches
by recruiting granulocyte-derived suppressor cells and neutrophils.
One strategy for utilizing pro-tumor cytokine expression is to express their receptors, such as CXCR2, extopic on ideal effector cells to target them to tumors
.

The expression of CXCR2 in primary T cells enhanced the accumulation of T cells in tumor in a mouse model of melanoma, improving tumor regression and survival
.
An ongoing phase I/II trial aimed at treating patients with metastatic melanoma with CXCR2-transduced tumor-infiltrating lymphocytes followed by high-dose IL-2 has not yet been reported (NCT01740557).

CXCR2 expression has also shown prospect
in CAR-T cell therapy.
In hepatocellular carcinoma tumor models, the forced expression of CXCR2 on CAR-T cells increased migration and accumulation within tumors, improving treatment outcomes
.
Similarly, ectopic expression of CXCR1 or CXCR2 enhanced metastasis to tumors and induced tumor regression and improved survival in glioblastoma, pancreatic, and ovarian cancer models
.

CCR4

The receptor CCR4 is low expressed on CD8+ T cells with ligands CCL22 and CCL17
.
In a mouse model of Hodgkin lymphoma, the expression of CCR4 was enhanced by retroviral transduction of anti-CD30 CAR-T cells, the migration to lymphoma cells in vitro was enhanced, and the effect
of CAR-T cell therapy was improved.
Although this study used a blood cancer model, this approach can also be applied to solid tumors, as upregulation
of CCL22 and/or CCL17 expression was found in gastrointestinal, ovarian, and pancreatic cancers.
In a mouse model of pancreatic cancer, retroviral transduction with CCR4 antigen-specific cytotoxic T cells enhanced migration to tumor sites and eliminated tumors
in 40% of mice.
Interestingly, the study showed that CCR4 modifications enhanced interaction with DCs and enhanced the binding
of T cells LFA-1 to DC ICAM-1.
Therefore, this strategy has the potential not only to enhance tumor invasion, but also to enhance the activation and support
of immune cells inside the tumor.

CCL2

CCL2 is secreted by a range of tumors and tumor-supporting immune cells and induces migration
of pro-tumor immune cells such as macrophages, TH2, and regulatory T cells.
However, its homologous receptor CCR2b is only weakly expressed
on activated T cells.
Compared with CAR-T cells that do not express CCR2b, the transport to CCL2-secreting neuroblastoma on GDH-CAR-T cells expressing CCR2b is enhanced by 10-fold and the relative antitumor activity
is increased.
However, targeting the CCL2 pathway may have some unintended toxic consequences because CCL2 is expressed in a range of non-tumor cell types, including endothelial cells, smooth muscle, and fibroblasts, and has been implicated in a variety of diseases, such as rheumatoid arthritis, asthma, inflammatory bowel disease, and SARS-CoV-2 infection
.
Considering that the level of CCL2 produced by tumors is higher than that of normal tissue, CCR2b-transduced CAR-T cells need to be evaluated
experimentally.

CXCR4

The chemokine receptor CXCR4 is widely expressed in a variety of hematological and solid tumors
.
Its binding to its corresponding ligand CXCL12 (SDF1) promotes tumor cell proliferation, survival and metastasis
.
Inhibition of CXCR4 leads to increased T cell and NK cell invasion while enrollment
of cancer-associated fibroblasts (CAFs) decreases.
Therefore, inhibition of CXCR4 is a potential strategy
to enhance the infiltration of CAR-T and CAR-NK cells to tumor sites.

Recent studies have shown that the combination of anti-EGFRvIII CAR-T cells and the PARP inhibitor olaparib improves efficacy
.
olaparib can reduce CAF secretion of CXCL12, thereby reducing the migration and infiltration of MDSCs, and improving the effect
of CAR-T cell therapy in mouse models of breast cancer.

CCL5

CCL5 is recognized by a range of chemokine receptors expressed on activated T cells such as CCR1, CCR3, and CCR5, making CCL5 a good candidate for tumor-specific expression to facilitate adoptive lymphocyte migration
.
CCL5-armed oncolytic virus and CAR-T cell therapy have been successfully used in mouse models of
neuroblastoma.

In preclinical mouse models of NK cell therapy, the use of the CCL5 axis has also been shown to improve efficacy
.
The injected NK cells are designed to overexpress CCR5 and inject with CCL5-carrying vaccinia virus within the tumor, resulting in increased NK cell infiltration and tumor regression
.

Although chemokine regulation has shown promise in immunotherapy models, there are serious application problems that may limit its efficacy
.
An important consideration is that most chemokines may have nonspecific recruitment
.
For example, while CCL5 can enhance the anti-tumor immune response by recruiting CCR5+ cells such as T, NK, and DC cells, it can also recruit monocytes, macrophages, and regulatory T cells and promote cancer invasion
.
In summary, the role of the selected chemokine needs to be clarified to prevent accidental recruitment of inhibitory cells into the
tumor.

Oncolytic viruses

Oncolytic viruses (OV) have important immunomodulatory effects and have the potential to be used in combination strategies
alongside adoptive cell therapy.
The ability of OVs to regulate TME and affect host anti-tumor immune response provides a strong theoretical basis
for the use of OVs to target CAR-T cells to treat tumors.
Mechanically, OVs induce immunogenic cell death in tumor cells, release molecular patterns associated with injury and pathogens (DAMP and PAMP), tumor-associated antigens or neoantigens, and induce cytokine and chemokine expression
at tumor sites.
OVs can induce the expression of major histocompatibility complexes (MHCs) on tumor cells, thereby increasing antigen presentation
.
In addition, OV-mediated TME remodeling affects innate and adaptive immune cell function, enhances dendritic cell activation, lymphocyte infiltration and activation, and stimulates epitope diffusion
.

In addition to the natural immunomodulation of OVs, their genetic modifications allow tumors to locally express a wide range of peptides or non-coding RNAs to further enhance the inflammatory signature of tumors, supporting immune cell migration and activation
.
In a series of preclinical studies, OV, which carries a range of cytokines, chemokines and bispecific T cell junctions, has been used to remodel TME and improve the efficacy of
CAR-T cell therapy.
TILT-123 is an oncolytic adenovirus carrying TNF-α and IL-2 with the potential to
combine strategies with CAR-T cells.
Showing convincing synergies with PD-L1 inhibition in a range of preclinical models, TILT-123 is currently enrolling two clinical trials for clinical studies as a single agent or in combination with tumor-infiltrating lymphocytes for the treatment of metastatic melanoma (NCT04695327, NCT04217473).

Another use of OV and CAR-T cell therapy is to use transgenic OV to induce target antigen expression
in tumors.
This principle has been demonstrated in preclinical studies that CD19 antigens are displayed on the surface of solid tumor cells through OV vectors, so as to achieve effective targeting by anti-CD19 CAR-T cell therapy
.
There are currently two phase I clinical trials (NCT01953900, NCT03740256) that are testing the synergistic effect
of oncolytic viruses and CAR-T cells in the treatment of solid tumors.

Tumor-induced promoter

In addition to the constitutive expression of genes that have been used to enhance CAR-T cell therapy, targeted genes
that can be specifically expressed in tumors can be used by using a promoter system that induces them.
Triggering site-specific release of inflammatory mediators, including chemokines, by tumor antigens is a useful way
to encourage migration of other CAR-T cells and bystander cells to the tumor.

Inducible promoters activated by recognizing tumor antigens, either by CAR or TCR in T cells or TILs, have been used to overexpress multiple genes, especially antitumor cytokines, and have been shown to enhance anti-tumor effects
while improving safety.

IL-12 has been shown to enhance CAR-T cell activity in vivo, but the toxicity of IL-12 is also obvious
.
Preclinical tests for targeted expression mainly include IL-2-based NFAT promoters to target IL-12 expression to tumor sites
.
Using these antigen-triggered promoters to immobilize IL-12 expression at the tumor site can improve the killing effect of CAR-T cells on tumors in vivo and reduce the number of
CAR-T cells needed to effectively kill tumors.
This strategy minimizes the observed systemic toxicity of IL-12 expression
.
Local expression of IL-12 by CAR-T cells has also been shown to activate innate immune cells at tumor sites, such as macrophages, helping to eliminate antigen-negative tumor cells
.

NFAT-induced promoters have also shown advantages in non-CAR-ACT, such as TCR and TIL-ACT
.
Compared to non-engineered TILs, TILs that selectively express IL-12 at tumor sites produce an enhanced anti-tumor immune response and reduce the dose required 10 to 100-fold
in clinical trials of metastatic melanoma (NCT01236573).
Although some toxicity has been observed, the induction system it uses has shown potential
for use in human therapy.

The development of promoters that respond to non-TAA signals is another developing area of research that can take advantage of alterations
in tumor metabolite characteristics.
A hypoxic tumor microenvironment sensor
has been developed using hypoxia-inducing elements in CMV promoters.
The "HiTA system" limits CAR-T cell gene expression to a low-oxygen environment and ignores the normobic tissue
that expresses TAA.
The hypoxia-induced expression system can also be used to express other transgenes or the CAR itself to focus and enhance the immune response
.

Renovation of the CAR structure

The selection and design of CAR structures are key considerations for optimizing migration and antitumor activity
.
Systems such as Dual CARs and Universal CARs (UniCAR) are effective ways to
improve the targeting and safety of CAR-T cell tumors.

Dual CAR-T cells express CAR for different antigens, and can express more than one CAR on a single T cell or T cells transduced with two sets of different CAR structures to achieve targeting of multiple antigens
.
These methods have been successfully applied to the clinical treatment of a range of solid tumors in vivo, with good results
in clinical trials.
For example, by targeting CD38 and BCMA in vivo, combining two independent CAR-T cells has successfully treated multiple bone marrow cancer in clinical practice, the objective response rate has reached 87.
5%, and after nearly 1 year of follow-up, 76.
9% of patients have no recurrence and metastasis
.
In addition, the dual-target CAR-T for CD19/CD22 has also entered clinical trials (NCT03233854).

Early results showed that 88% of patients had a successful response, with all complete responses; Of the 21 LBCL patients, 62 percent responded and 29 percent had a complete response
.

UniCAR creates a safety "switch"
for clinically controlling the immune response by introducing a second component, called the Targeting Module (TM).
TMs are fusion protein molecules of UniCAR-T cell target antigens with TAA-binding domains (e.
g.
, single-chain antibodies), TMs link CAR to TAA, and removal of TM can rapidly eliminate UniCAR-T cell activity
due to the short half-life of TM (15–45 minutes).

Currently, several UniCAR systems have entered clinical trials, almost all of which focus on hematological malignancies targeting a range of targets, including CD19 (NCT02808442 and NCT02746952), CD22 (NCT04150497), and CD123 (NCT03190278).

While the CD19 trial was largely successful, with 67 percent of patients achieving a complete response, other trials, such as the CD123 UniCAR trial (NCT04106076), ran into safety concerns
.
For solid tumors, only a few UniCAR clinical trials are registered, such as one for PMSA in prostate cancer (NCT04633148).

Targets the tumor microenvironment

Carcino-associated fibroblasts are a phenotypically heterogeneous cell population that builds and remodels the TME extracellular matrix (ECM).

Depleting or altering the function of CAF is currently an area of in-depth research that has the potential to be an attractive option
for enhancing ACT and immunotherapy.

Since CAF contributes to the ECM/cell barrier, reducing its number and activity has the potential to have a synergistic effect with CAR-T/NK cell therapy to increase lymphocyte migration
to tumors.
Several CARs have been developed that exert anti-tumor effects
by directly depleting CAF by targeting fibroblast-activating protein α (FAP-α).
Anti-FAP-α CAR-T cell therapy has shown convincing efficacy
in a variety of preclinical models of solid tumors.
There are two clinical trials utilizing anti-FAP-α CAR-T cells, a completed Phase 1 trial of pleural mesothelioma (NCT01722149) and a Phase 1 clinical trial utilizing fourth-generation CAR (NCT03932565).

Another different strategy is to remove immunosuppressive cells such as MDSCs and TAM
.
In a mouse model of neuroblastoma, removal of tumor local MDSCs improves the efficacy of CAR-T cells and tumor infiltration
.
By clearing TAM using antifolate receptor β-CAR-T cells, increased infiltration of endogenous tumor-specific CD8+ T cells
was found.
TAM-targeted CAR-T cell pretreatment enhances the efficacy of tumor-specific anti-mesothelin CAR-T cells, enabling tumor regression and prolonging survival
.

In addition, solid tumors often exhibit metabolic disturbances leading to the release of immunosuppressive metabolites, including lactic acid, adenosine, and reactive oxygen species (ROS), which effectively inhibit lymphocyte migration
.
The combination of CAR-T/NK cell therapy with tumor metabolism inhibitors may enhance their migration to tumors and their killing effect
on tumor cells.

Enhance DC cell activity

Dendritic cells (DCs) contribute to the homeostasis, activation, and migration potential
of the T cell compartment.
While adoptive T cell transfer, the use of strategies to stimulate and enhance endogenous DC activity will maximize T cell participation and activation, encourage epitope diffusion, and alter TME
in a way that is more conducive to local and systemic immune activity.

Flt3L is a key growth factor for mobilizing and expanding DC cells, and the secretion of Flt3L by engineered CAR-T cells increases DC precursor cells in bone marrow and tumors, increases DC secretion of IL-12 and TNF in tumors, and inhibits tumor growth
.
Critically, CAR-T cells secrete Flt3L while promoting endogenous CD8+ T cell tumor infiltration and leading to epitope diffusion, indicating that the use of Flt3L activates host DC cells and enhances the migration
of lymphocytes to solid tumors.

brief summary

CAR-T and CAR-NK cell therapy is a very promising way to
improve the prognosis of cancer patients.
Although they are effective against hematologic malignancies, overcoming solid tumors remains difficult
.
Recently, drugs that stimulate migration, improve the tumor microenvironment, and remove tumor barriers have been combined with CAR-T/NK-based therapies to improve the disappointing performance
of CAR-T and NK cells in solid tumors.
These strategies will provide a key pathway
to advance engineered T cell and NK cell therapies targeting solid tumors.

References:

1.
Controlling Cell Trafficking: Addressing Failures in CAR T and NK Cell Therapy of Solid Tumours.
Cancers (Basel).
 2022 Feb; 14(4): 978.