September 25, 2022

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What hurdles need to be overcome to overcome solid tumor CAR-T therapy?

What hurdles need to be overcome to overcome solid tumor CAR-T therapy?



 

What hurdles need to be overcome to overcome solid tumor CAR-T therapy?


Chimeric antigen receptor T cell (CAR-T) therapy has achieved certain breakthroughs in the treatment of hematological malignancies, but in the same period of PD-1 products, Keytruda alone will achieve more than $17 billion in revenue in 2021. However, compared with this, the total revenue of all listed CAR-T products in 2021 is also far behind.

There are many reasons (high treatment costs, insufficient production capacity, etc.), one of the important factors is that the indication is limited to hematological tumors, and the large Some of them are end-line treatments.

All CAR-T targeting solid tumors are still in the exploratory stage, and CAR-T therapy currently faces many challenges in solid tumors, including immunosuppressive tumor microenvironment (TME), tumor antigen heterogeneity , stromal barriers and tumor accessibility, as well as on-target/off-target dilemmas. In the first two articles, we introduced the marketed products and pipeline layout of CAR-T therapy respectively  and the preparation process and cost structure

Finally, let’s review the current obstacles in CAR-T treatment and some strategies for overcoming these obstacles. (What hurdles need to be overcome to overcome solid tumor CAR-T therapy?)

 

 

Suppressive tumor microenvironment

Unlike hematological malignancies, the accessibility of cancer cells in solid tumors is limited, which also includes several physical barriers, such as tumor vasculature and extracellular matrix, that hinder the penetration of infused CAR-T cells into tumor tissue.

A solid tumor is an organ-like, disorganized structure composed of proliferating tumor cells surrounded by supporting stromal cells and blood vessels that nourish the neovascular system of the tumor, and tumor growth can be controlled by innate and adaptive components.

The main innate immune cells in solid tumors are: neutrophils, macrophages, dendritic cells (DC), mast cells, natural killer cells (NK cells) and myeloid-derived suppressor cells (MDSC).

Adaptive immune cells: T and B cells and regulatory T cells (Tregs). All of these immune cells are associated with the non-tumor stromal cells that make up the TME: endothelial cells, fibroblasts, pericytes, and mesenchymal cells. These cells and the factors and molecules they secrete make up the TME.

The main obstacle encountered by CAR-T cells in the treatment of solid malignant tumors is that TME prevents T lymphocytes from being transported and infiltrated to tumors by inhibiting soluble factors and overexpressing negative immune checkpoints to establish an immunosuppressive environment, and T cells effectively infiltrate into the tumor.

Tumor stroma is a critical step in maintaining the antitumor activity of infused T cells and the success of cancer immunotherapy.

 

What hurdles need to be overcome to overcome solid tumor CAR-T therapy?

Figure 1 Tumor immunosuppressive microenvironment

 

 

 


 Tumor microenvironment hypoxia

 

(1) Hypoxia is a hallmark of the tumor microenvironment that affects tumor progression and alters treatment outcomes in cancer patients.

Stabilization and nuclear translocation of hypoxia-inducible factor (HIF), which further regulates gene transcription by binding to hypoxia-responsive element (HRE) regions in hypoxia-inducible gene promoters, leading to cellular adaptation to environmental changes. On the other hand, T cell receptor (TCR) activation signals or cytokines generated during infection and inflammation can regulate the synthesis and stability of HIF, which in turn affects the activation and differentiation of T lymphocytes.

TCR-mediated T cell stimulation and hypoxic conditions increase HIF abundance and effector function of cytotoxic T lymphocytes (CTLs) under persistent infection or tumor microenvironment. At this point, the Von Hippel-Lindau (VHL) factor partially suppresses the T-cell response to protect the body from the damaging effects of an overreaction.

Stabilization of HIF1α in T lymphocytes is paralleled by enhanced expression of glycolytic enzymes such as GLUT-1 as a glucose transporter and decreased oxidative phosphorylation rates, thus increasing HIF levels through both hypoxia-dependent and hypoxia-independent pathways and activity, as modulators of metabolic pathways, and involved in T cell proliferation, differentiation, and effector activity.

 

(2) Hypoxia promotes the breakdown of adenosine triphosphate (ATP) and inhibits adenosine kinase (which phosphorylates adenosine to AMP), resulting in enhanced adenosine.

The interaction of adenosine accumulated from hypoxic sources in the tumor microenvironment with specific receptors expressed by T cells (i.e., A2AR and A2BR) interferes with TCR signaling, thereby inhibiting the antitumor activity of T cells.

Importantly, the interaction of adenosine and G protein-coupled receptors (GPCRs) activates protease A (PKA), which in turn regulates TCR downstream signaling.

If CAR-T cells express RIAD (regulatory subunit I anchor-disrupting peptide) that prevents the localization of PKA in the immune synapse, the inhibitory effect of adenosine on mesothelin-CAR-T cell activity can be attenuated; additionally the use of small molecules For example, BAY 60–6583 (adenosine A2B receptor agonist) can improve the therapeutic effect of CAR-T cells by affecting multiple targets.

 

(3) The immune checkpoint PDL1 molecule is up-regulated in a HIF-1α-dependent manner, and the hypoxic environment hinders the function of infiltrating T cells.

Elimination of PD1-PDL1 interaction by PD1 blockade enhanced A2AR expression on tumor-infiltrating T lymphocytes, resulting in enhanced sensitivity to accumulated adenosine immunosuppression.

Thus targeting both the PD1/PDL1 axis and adenosine A2A (either by genetic or pharmacological approaches) could improve T cell performance in CAR-T cell therapy.

 

 

 

 


Restricts entry into tumor cells

 

Extracellular matrix

The extracellular matrix (ECM), as part of the peritumoral stroma, is composed of fibrin, glycoproteins, polysaccharides, and proteoglycans. Increased expression and density of ECM components in malignant tissues, especially overproduction and deposition of hyaluronic acid and collagen, hinder the penetration of therapeutic agents.

In addition, elevated collagen density impairs T cell proliferation and cytotoxic activity and induces a regulatory phenotype, and the strategy of CAR-T cell manufacturing may also result in down-regulation of ECM-degrading enzymes.

Given these facts, application of ECM-degrading enzymes such as hyaluronidase and collagenase could reduce ECM stiffness and facilitate anticancer agent delivery.

Caruana and its concomitant induce heparanase (HPSE) expression in CAR-modified T cells, thereby enhancing the ECM degradation capacity and antitumor activity of co-expressing CAR T cells (ie, expressing CAR and HPSE).

 

Tumor blood vessels

Cancer cells divide uncontrollably and need to form new blood vessels for nutrients and oxygen.

The development of abnormal vasculature, and the downregulation of adhesion molecules involved in T cell extravasation (under the influence of angiogenic factors such as bFGF and VEGF), act as a physical barrier to T cell penetration into the tumor bed.

Furthermore, endothelial cells in the tumor microenvironment promote the expression of FasL and inhibitory molecules such as PD-L1, TIM3, IDO-1, PGE2, and IL-10, thereby suppressing effector T cell activity.

These characteristics of tumor blood vessels depend in part on VEGF production and the overexpression of its receptors.

Therefore, CARs designed for VEGFR1 and VEGFR2 have shown good effects in destroying tumor blood vessels and reducing tumor cell proliferation by limiting nutrients and oxygen.

Therefore, tumor-specific immunotherapy outcomes can be improved by increasing tumor-specific T cell infiltration, persistence, and antitumor activity by concurrent infusion of VEGFR2-specific CAR-T cells and antigen-specific TCR-transduced T cells.

 

 

What hurdles need to be overcome to overcome solid tumor CAR-T therapy?

Figure 2 Strategies to enhance tumor trafficking and penetration

 

 

 

Cancer-associated fibroblasts

 

Cancer-associated fibroblasts (CAFs) are one of the most abundant components in the tumor stroma and represent a reactive tumor-associated fibroblast population that secretes various active factors (VEGF and PDGF) to promote tumor development, Metastasis and treatment resistance.

CAF expression can be targeted by immunotherapy with various molecules, of which FAP is the most promising target. It is a cell surface serine protease that is highly expressed on associated stromal cells (CASCs) in various human cancers, such as lung, prostate, pancreatic, colorectal and ovarian cancers.

The strategy described in the figure below targets key signals and effectors of CAFs and is designed to inhibit CAF functions, such as cytokine (TGFβ) and growth factor pathways (VEGF, PDGF).

For example, CAF-derived extracellular matrix proteins (MMPs) and associated signaling can be targeted with monoclonal antibodies (MAbs) to induce matrix depletion and increase immune T cell infiltration.

FAP targeting aims to block the ability of CAFs to exert tumor-promoting effects in the TME, either by using MAb/antibody-drug conjugates, immunoconjugates or peptide-drug complexes, FAP-specific CAR-T cells or genes Knockout strategy to complete.

 

 

What hurdles need to be overcome to overcome solid tumor CAR-T therapy?

Figure 3 Strategies to counteract the tumorigenic effects of CAF

 

 

 

 


Presence of immunosuppressive cells

 

Regulatory T cells

Tregs are involved in the tumor’s immunosuppressive environment, with immunosuppressive features such as secretion of IL-10 and TGF-β, and IL-2 required for the proliferation of competitive effector T cells, inhibiting infused T cell function. Tregs are immunosuppressive cells that are highly dependent on IL-2.

They bind to and deplete IL-2 in the surrounding environment, thereby reducing T cell availability by constitutively expressing the high-affinity IL-2 receptor (IL2R) subunit-alpha (CD25).

Treg cells also produce immunosuppressive cytokines (IL-10, IL-35, and TGFβ), which downregulate the activity of T cells and antigen-presenting cells (APCs).

In addition, Treg cells release large amounts of ATP, which is converted to adenosine (via CD39 and CD73), providing immunosuppressive signals to T cells and APCs.

 

Current strategies to eliminate Treg-mediated immunosuppressive mechanisms are mainly CAR-T cells designed to target Treg-expressed antigens for direct depletion. Other strategies are based on TME-based immunomodulation to enhance the performance of CAR-T cells:

(i) CAR-T cells express pro-inflammatory cytokines;

(ii) optimize costimulatory signaling structures to reduce IL-2 secretion and attenuate Treg expansion and tumorigenesis infiltration.

 

A final strategy is to confer intrinsic resistance to immunosuppression in CAR-T cells:

(i) a dominant negative receptor (DNR) designed to disrupt signaling, or (ii) a chimeric switch receptor (CSR or Switch) R) Convert a negative signal to a positive signal, or eliminate the expression of inhibitory receptors (such as PD1 for TGFβ receptors) by using genome editing tools (knockout).

 

What hurdles need to be overcome to overcome solid tumor CAR-T therapy?

Mechanisms by which Tregs exert immunosuppression in the TME (A),Strategies to overcome Treg-induced immunosuppression (B)

 

 

Tumor-Associated Macrophages (TAM)

Macrophages are one of the main effector cells of the immune system and play key roles in both innate and adaptive immune responses, constituting the first line of defense against foreign pathogens and helping to trigger adaptive antigen-specific responses.

Macrophages can be divided into two contrasting groups: classically activated macrophages or M1 macrophages (pro-inflammatory and anti-tumor) and alternately activated macrophages or M2 macrophages (anti-inflammatory and pro-tumor).

This difference is primarily due to polarization due to tissue exposure to soluble factors or pathogen-derived molecules.

M1 macrophages are pro-inflammatory cells that play a role in antitumor immunity by directing cellular immunity to TH1-type responses by secreting TNFα, IL-1β, and IL-12. Although M2 macrophages play a role in tissue homeostasis (stimulating Th2 responses to eliminate parasites, immune regulation, wound healing, and tissue repair), M2 macrophages can also promote tumor progression.

 

Tumor cells and stromal cells can secrete a variety of cytokines and chemokines that recruit macrophages around tumor cells and convert them into TAMs.

When macrophages are recruited to migrate into the tumor mesenchyme, they can adapt to their microenvironment by changing their phenotype.

TAMs are a specialized population of M2-like macrophages located in the TME and share some phenotypic features with M1 and M2 macrophages, but have specific transcriptional profiles distinct from these two types.

TAMs enhance tumor progression and metastasis by promoting genetic instability and enhancing angiogenesis, fibrosis, invasion, immunosuppression, and lymphocyte rejection.

On the one hand, macrophages produce inflammatory cytokines such as IL-17 and IL-23, which increase genetic instability, and on the other hand, they secrete inhibitory cytokines such as TGF-β and IL-10, through expression Immune checkpoint ligands such as PD-L1, PD-L2, B7-H4, or VISTA, or by generating reactive oxygen species (ROS), can hinder tumor immune surveillance and thus hinder T cell-mediated antitumor immunity.

 

Figure 5 Strategies to overcome TAM-induced inhibition in the TME

 

 

Various therapeutic strategies currently targeting macrophages aim to deplete or repolarize macrophages.

 

The first approach is to reduce or deplete TAMs by eliminating existing TAMs or by inhibiting further TAM recruitment, a strategy primarily by targeting (i) the colony stimulating factor 1 (CSF1)/CSF1 receptor (CSF1R) signaling pathway, (ii) chemokine/chemokine receptor axis, such as CCL2/CCR2, CCL5/CCR5, (iii) IL-8/CXCR2 or (iv) CXCL12/CXCR4 axis implementation.

 

The second approach is to repolarize TAMs to an M1-like phenotype by inhibiting PI3Kγ signaling, triggering inflammation through TLR agonists to activate Toll-like receptor (TLR: TLR3, TLR4, TLR7/8, and TLR9) signaling, or This is achieved by using the agonistic antibody CD40 antibody.

Antigen presentation and phagocytosis of TAMs can also be promoted by blocking anti-phagocytic surface proteins (such as SIRPα or Siglec-10) known as “don’t eat me” signals, while the antibodies block CD47 or phagocytosis expressed on cancer cells. CD24.

 

A third TAM-targeting molecule is TGF-β, an anti-inflammatory cytokine normally expressed by macrophages during the resolution of injury.

Macrophages are both a source and target of TGF, by promoting the secretion of additional TGF-β, causing a positive feedback loop of TAM and maintaining the immunosuppressive TME.

Targeting TAM by TGF-β blockade has been used, also in combination with STING agonists or anti-PDL1 blockade, and has shown tumor regression in preclinical models.

 

The fourth approach is to enhance tumor cell phagocytosis. The latest research highlights the importance of multiple antigen targeting, which can both improve the effectiveness of CAR-T cell therapy and reduce off-target reactions, such as the generation of CD47-targeted CAR-T cells with two tandem CARs of TAG-72.

The dual targeting strategy enhances the ability of CAR-T cells to destroy tumor cells expressing low antigen levels, which is beneficial to increase the affinity of tandem CARs to tumor cells.

Some researchers have found that engineered NanoCAR-T cells that secrete anti-CD47 nanobodies can inhibit tumor growth while avoiding the toxicity encountered with systemic anti-CD47 therapy.

This TAM reprogramming strategy showed superior antitumor activity compared to standard CAR-T cells.

 

Myeloid-derived suppressor cells

MDSCs are immunosuppressive immature myeloid cells. There are two subtypes based on morphology and cell surface inhibitors: monocytic MDSC (M-MDSC) and polymorphonuclear/granulocyte MDSC (PMN-MDSC). They contribute to the tumor immunosuppressive environment by inducing Tregs and producing arginase (ARG1), inducible NOS (iNOS), and inhibitory cytokines such as IL-10 and TGF-β. Current strategies to increase the resistance of CAR-T cells to the immunosuppressive effects of MDSCs: (i) prevent MDSC differentiation and recruitment to the tumor bed; (2) deplete tumor-infiltrating MDSCs or (3) alleviate the immunosuppressive effects of MDSCs.

 

MDSCs play an immunosuppressive mechanism in the tumor microenvironment (A), Strategies to overcome MDSC-induced immunosuppression (B)

 

 

 

 


Expression of immune checkpoint molecules and immunosuppressive mediators

 

Immune checkpoints (ICs) ensure the maintenance of immune homeostasis by regulating the time course and intensity of immune responses.

However, receptor-based signaling cascades from ICs play a negative regulatory role in T cells, allowing tumors to evade immune surveillance by inducing immune tolerance.

The first major ICs identified as important receptors for T cell and CAR-T cell inhibition and apoptosis were CTLA-4 and PD-1.

Other immune receptors that have been extensively studied in cancer are LAG-3, TIGIT, T-cell immunoglobulin and mucin containing protein 3 (TIM3) and B and T lymphocyte attenuator (BTLA). Since PD1/PD-L1 inhibition is the most studied axis, let’s focus on PD-1/PD-L1 inhibition in CAR-T cell therapy.

 

PD-1, a member of the B7/CD28 family, plays a role in regulating T cell activity by interacting with two ligands, PD-L1 and PD-L2. PD-1/PD-L1 binding blocks the synthesis of IFN-γ and IL-2, thereby reducing T cell proliferation.

To prevent CAR-T cell exhaustion and immunosuppression in the TME, different strategies can be used, such as:

  1. The combination of CAR-T cells with immune checkpoint inhibitors (ICIs, such as anti-PD1 or PD-L1 antibodies);
  2. PD1-mediated inhibition can also be overcome by designing CAR-T cells that secrete PD-1-blocking or PD-L1-blocking single-chain variable fragments (scFv);
  3. Designing genetically modified CAR-T cells , the cells express the dominant negative PD-1 receptor (PD-1 DNR), which interferes with PD1 downstream signaling or PD-1 chimeric switch receptor (CSR), converting inhibitory signals into activating signals.
  4. The last strategy ablated PD1 expression by gene knockout or by shRNA (short hairpin RNA) inhibition.

 

 

Figure 7 The strategy of CAR-T cells to overcome the inhibition of negative immune checkpoint regulation (PD1/PD-L1 as an example)

 

 


In conclusion: What hurdles need to be overcome to overcome solid tumor CAR-T therapy?

 

The specificity of malignant tissues such as tumor vasculature and extracellular matrix hinders the infiltration of CAR-T cells, on the other hand, the presence of regulatory cells, overexpression of immune checkpoints and hypoxic conditions of the TME deplete adoptive transfer of T cells attenuate their antitumor activity.

At present, all CAR-T targeting solid tumors are still in the exploratory stage and face many challenges. Looking forward to overcoming these problems as soon as possible.

 

 

 

 

 

 

 

 

What hurdles need to be overcome to overcome solid tumor CAR-T therapy?

(source:internet, reference only)


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