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The Cause Dolution strategy and determination of CAR-T tonic signal

Cause Dolution strategy and determination of CAR-T tonic signal.   Adoptive cell transfer (ACT) using autologous T cells engineered to express chimeric antigen receptors (CAR) has been shown to be an efficient strategy for the treatment of relapsed or refractory B-cell malignancies (1-3).

CAR T cell therapy has now become a standard method of cancer treatment and is no longer regarded as a purely experimental treatment. However, the field is still in its infancy, and these great advances have not yet been reproduced in patients with advanced solid tumors (7-9).

In order to fully understand how the CAR structure determines the function and describe the complexity of CAR cell signaling and the network of interactions between CAR-T cells and other cells in the tumor microenvironment (TME) in vivo, much work remains to be done. Continue to devote a lot of energy to optimizing the CAR structure itself to enhance anti-tumor efficacy, metabolism, proliferation capacity and durability (10, 11).

It is becoming more and more obvious that the nuances of CAR design can produce amplification effects in vitro, especially in vivo, and the extracellular targeting part of CAR, hinge, spacer, transmembrane domain (TMD) and intracellular co-stimulation The best choice of domain (ICD) is crucial.

CAR tonic signaling can be defined as the uncoordinated and continuous activation of T cells in a ligand-independent or independent manner. In the absence of spatial and/or temporal control of CAR cell surface expression, this constitutive or chronic cell signal transduction may have a major deleterious effect on CAR T cell effector function and survival, and may lead to in vitro cytolytic ability. The huge difference between and the anti-tumor efficacy in vivo (18-21). This article focuses on the current ongoing research aimed at identifying and solving various forms of CAR conditioning signals and the introduction of detection schemes.

The reason for the tonic signal

1. CAR structure

In the past two decades, CAR design has undergone many iterative developments with the goal of optimizing the function and durability of CAR T cell effectors (27). The anti-tumor efficacy depends on the optimal CAR binding to the target epitope and the formation of cytolytic immune synapses between CAR T cells and target cells.

The interval length not only affects the flexibility of the CAR (45), but also affects the distance between the target cell and the CAR T cell membrane (46). To ensure the best immune synapse formation, especially in the membrane, the interval length is becoming more and more important. Proximal epitope (44).

2. Endogenous TCR tonic signal

MHC class II interactions are related to the maintenance of T cell reactivity and proliferation ability after activating the associated antigen (50,57). This may occur through a two-step process in lymphoid tissue, in which natural T cells with TCR exhibit low to moderate affinity for self-peptides presented on steady-state DCs, which may induce TCR signaling.

Cross-presentation of foreign peptides by activated DCs can further enhance this function, thereby enhancing the function of T cell effectors (12). The “pseudo-dimer” effect (presumed for CD4+ T cell/MHC class II interaction) can take advantage of this further, so that the recognition of MHC-loaded self-peptides and foreign peptides enhances the T cell’s response to the latter Sex (58).

At the same time, the high-affinity combination of TCR with its own MHC can maintain peripheral tolerance, leading to unrestricted tonic signaling, T cell tolerance and failure, anergy, apoptosis and/or enhanced regulatory T cells (Treg) function.

This model is illustrated in Figure 2 and demonstrates how TCR tonic signaling may promote a dynamic balance between T cell efficacy and positive and negative regulators of autoimmunity. This paradigm is similar to the selection of thymic T cells, which actively selects intermediate TCR and MHC affinities, and induces TCR tonic signaling required for subsequent responses to surrounding foreign peptides (12).

3. CAR tonic signal independent of ligand

In a study by Frigault and colleagues, ligand-independent tonic signaling that leads to continuous T cell expansion in vitro was shown to depend on the integration of the CD28 transmembrane and cytoplasmic domains in the CAR structure (21). Other members of the CD28 immunoglobulin superfamily (such as ICOS) seem to be unable to induce constitutive expansion when replacing CD28 in other similar CAR structures, while costimulation of ICD with 4-1BB seems to enhance ligand-independent proliferation ( 62), but it has not yet proven its sustained expansion and constitutive cytokine release (11);

Frigault and colleagues evaluated a set of 12 CARs targeting c-Met, mesothelin, and CD19 (21). They contain immunoglobulin G4 (IgG4) hinges or CD8a stems, and carry CD28, ICOS or CD8a transmembrane domains. The intracellular signaling domain contains CD28, 4-1BB, or ICOS that binds in cis to CD3z. The lentiviral vector is used with the elongation factor 1α (EF-1a) promoter.

As expected, after in vitro activation with anti-CD3/anti-CD28 magnetic beads and subsequent viral transduction, most CAR T cells showed a predictable and rapid initial proliferation pattern, and subsequently in the absence of exogenous presence Revert to resting state under IL-2. Interestingly, some CAR constructs show sustained expansion for up to 60 to 90 days in the absence of IL2 or target ligands.

Long and colleagues elaborated on the data of Frigault and colleagues (21). They were able to prove that costimulation of CD28 enhances CAR T cell failure mediated by tonic signaling, while costimulation of 4-1BB can limit it (18) .

This discovery and data highlighting the differences between CD28 and 4-1BB ICD in CAR T cells’ response to hypoxia, oxidative metabolism and negative regulation of apoptosis, as well as data generated by many research groups that emphasize 4-1BB-mediated mitochondria Biogenesis, persistence and central memory differentiation (10, 66, 67) are directly related to the best CAR design in the future.

ndeed, in clinical trials evaluating CD19-directed CARs containing CD28 and 4-1BB, similar differences in CAR persistence have been demonstrated (68-70).

The latest data from Klein Geltink and colleagues emphasize the complexity of CD28-mediated costimulation. At least in the initial stage of T cell activation, it has shown mitochondria with potential metabolic capacity, which is important for future T-cell responses (71 ). Therefore, the timing and duration of CD28 and 4-1BB signaling may be crucial for optimizing the metabolism and differentiation of CAR T cells.

Correlation with the later described studies related to assessing the contribution of hinges and spacers to the tonic signal, the modified GD2 BBz CARs lacked the IgG1 hinge-CH2-CH3 spacer and used CD8a TMD and scFv peptides derived from CD19 FMC63 scFv Connector. The relative contribution of these changes to improving CAR depletion seems to be limited. This is based on subsequent experiments using GD2 28z CAR, which incorporates a CD19 FMC63-derived peptide linker and lacks the IgG1 hinge-CH2-CH3 spacer. child.

This modified CAR did not show exhaustive improvement in the body, nor did it have anti-tumor effects. The CAR tonic signaling phenotype, which is predominantly failure, is shown in Figure 3B.

Hudecek and colleagues showed that the use of a full-length IgG4 Fc motif (including hinge, CH2, and CH3 modules) in CD19 and receptor tyrosine kinase-like orphan receptor 1 (ROR1)-directed CARs is associated with obvious tumor-related and banding Compared with CARs with truncated IgG4 spacers of CH2 and CH3, the non-dependent capture of CAR T cells in the lungs of NSG mice has reduced anti-tumor efficacy and durability (44). The authors speculate that CARs with full-length IgG4 Fc spacers are isolated by FcgR-expressing lung resident Ly6Cþ monocytes, which highlights the finding that a small number of CAR-T cells that can escape to the surrounding have a highly activated phenotype. Has significant significance.

Sufficient data (18, 20) indicate that different tonic signaling patterns may occur in different hinge/spacer domains, and this is likely to occur at the level of scFv oligomerization, which can be achieved by the flexibility and length of these domains To promote.

Mediated by the down-regulation of related chemokine receptor expression, the unrestricted activation caused by tonic signaling may lead to impaired CAR T cell transport to TME.

4. Ligand-dependent CAR tonic signal

Theoretically, non-specific T cell adhesion can enhance ligand-independent and ligand-dependent signal transduction, for example, through the ICAM-1/LFA-1 interaction, thereby reducing TCR-mediated T cell threshold activation (56 ). This process is known to help mature DCs to initiate the initiation of naive T cells in lymphoid structures, and these DCs are prone to express and regulate cell surface adhesion molecules (105). These interactions may also promote TCR-mediated tonic signal transduction after combining with MHC that self-present MHC, and due to the influence on the downstream signal transduction pathway shared with CAR, similar phenomena may occur after CAR ACT in vivo.

Since ICAM-1, ICAM-2, VCAM-1 and other adhesion molecules are usually overexpressed on tumor cells and TME (106), when targeting solid tumors, these interactions may have an impact on CAR signal transduction. Especially useful. Interestingly, the tumor cell surface expression of adhesion molecules can be induced by exposure to activated CAR-T cells. For example, after A549 lung cancer cells with low mesothelin expression are exposed to the cytokines secreted by activated mesothelin-directed 28z CARs, A549 cells up-regulate ICAM-1 and are susceptible to CAR-T cells to enhance bystander cytotoxicity. This also proves the up-regulation of LFA-1 on the cell surface (107).

Potential strategies to solve CAR strong signal

1. Engineering or changing the targeting part.

Select the master gene sequence shared by VH and VL (111); by engineering disulfide bonds between the VH and VL domains, in the absence of peptide linkers or in combination to ensure maximum stability (112); by Introduce charged mutations in the VH and VL domains (113); graft by complementarity determining regions (CDR) (114); or use a combination of these strategies. Linkers of 15-20 residues are generally considered to be the most thermodynamically stable (109).

Due to the hydrophobic nature of the residues in the CDR, which mediate the binding to the target antigen, aggregation of single-chain variable fragments may also occur in the absence of domain exchange. Various techniques have been deployed to resist aggregation without reducing binding affinity. Examples include inserting two or more negatively charged residues at each edge of the third CDR (CDR3; Reference 115) of scFv, or introducing glycosylation sites in the second CDR to compensate for the third The presence of hydrophobic residues in each CDR (116).

The further improvement of scFv stability, especially at the VH:VL interface, can also be achieved using advanced calculation models. Ultimately, the biophysical properties of scFv depend on their germline sequence, but are affected by somatic hypermutation in the framework region. Computational modeling has been used to reduce these hypermutations to germ line sharing and optimize the stability of the VH:VL interface.

By simulating molecular dynamics in a computer, especially with regard to the poor stability of the two domains, scFv can be systematically designed to improve internal stability and minimize aggregation (118). These strategies are summarized in Figure 4B.

In general, the use of low-affinity scFv may not only provide a way to distinguish high-level antigen-expressing tumor cells from low-level normal cells (hence, safety; reference 120), but it can also make normal tissues more sensitive. The ligand-dependent tonic signal caused by the presence of widespread low-level antigen expression is minimized.

Alternative targeting moieties, such as camelid single-domain antibodies (VHH) called “Nanobodies”, have high homology with human VH sequences and are the smallest known single-chain antibodies (121). In essence, domain exchange cannot be performed, although their efficacy in CAR still needs to be fully clarified in experimental models. Interestingly, due to their small size and the length of the CDRs (which form elongated loops), they can access cryptic epitopes (such as catalytic sites in enzymes) or large structures that usually cannot escape immune surveillance (122). Like murine scFv, the potential immunogenicity of camel nanobodies is being solved using sequence humanization technology.

The problem of scFv aggregation can also be circumvented by using endogenous receptors or ligands as the extracellular targeting part of CAR. A variety of such constructs have been designed, some of which have been clinically evaluated (9, 124).

2. Adjust the hinge

Experiments conducted by Watanabe and colleagues have shown that a medium-length IgG2 hinge/spacer can eliminate the tonic signal of CAR without destroying the cytolytic ability (20). The 28z CAR transduced with the second-generation lentivirus was used to target multiple antigen targets (including CD19, mesothelin, PSCA, HER2 and MUC1) with or without the IgG4-CH3 hinge/spacer domain.

In this case, after pre-exposure to anti-CD3/anti-CD2/anti-CD28 microbeads, during in vitro culture, the presence of hinges increases the amplification in a ligand-independent manner (especially after the 15th day later). Amplification) (136). The enhanced expansion of CAR T cells with hinges seems to depend on the proliferation of CD4β subparts, but if CD4β and CD8β T cells are cultured separately, this indicates that tonic signals may play different roles in CD4β and CD8β populations. Crosstalk can also occur between these two lineages.

3. The best choice for transmembrane domain

Although there are few reports on the role of the CAR transmembrane domain (TMD) in promoting tonic signal transduction, it is very clear that TMD is involved in the expression and stability of CAR cell surface and its ability to interact with other cell surface molecules. Plays a vital role, it helps signal transduction (137).

The use of unedited CD3z TMD can promote the heterodimerization of the endogenous CD3z chain, potentially lowering the antigen binding threshold required to initiate a cytotoxic response (138), and as an inevitable inference, can also enhance tonic signaling .

However, the surface expression of CD3z TMD containing CAR appears to be lower than that of CD28 or CD8a TMD (139).

4. The best choice for co-stimulating intracellular domains

In most cases involving scFv clustering, the CD28 domain appears to be harmful, while the 4-1BB domain is beneficial. However, there seems to be at least two different phenotypes: one is characterized by continuous expansion, terminal differentiation and aging (21,136,18).

In the latter case, it seems that the production of activated cytokines (such as IL2 and TNFa) is significantly reduced, and the use of the 4-1BB costimulatory domain can protect these cells from exhaustion and is associated with the memory T cell phenotype (18).

In the former case, the reduction of CAR surface expression or the shortening of the spacer can improve the negative effects of tonic signals in vivo and in vitro. In the experiments of Frigault and colleagues, the use of 4-1BB or ICOS costimulatory domains can also alleviate continuous expansion.

5. Control CAR expression

Regarding gene transduction using viral vectors, various strategies have been adopted to improve safety by minimizing the risk of producing replicating viruses and reducing the possibility of causing insertional mutagenesis.

Self-inactivation (SIN) vectors were developed using retrovirus and lentivirus by deleting/replacement of LTR elements. It is also possible to design a non-integrating lentivirus (NILV) by mutating the integrase gene or by modifying the attachment sequence of the LTR (144-146).

By limiting high-level CAR expression, these methods can reduce the possibility of tonic signals caused by CAR clustering. Frigault and colleagues demonstrated the use of lentiviral vectors to use the EF-1a promoter for continuous ligand-independent proliferation of CAR T cells, but not driven by CMV or the variable truncated PGK promoter (21). Recently, Gomes-Silva and colleagues demonstrated that the tonic signal mediated by 4-1BB is highly dependent on CAR surface expression, while the γ-retroviral LTR promoter is easy to amplify CAR expression in a CD-mediated positive feedback loop. 4-1BB-induced NFkB activation (73).

The use of IRES elements upstream of the LTR promoter may reduce CAR expression and thereby reduce tonic signal. A similar improvement was also observed after transduction with the SIN lentiviral vector.

6. Pharmacological strategy

Of course, people may think of using pharmacological agents to inhibit or reshape the negative effects of CAR tonic signals, such as fatigue and terminal differentiation. MAb inhibitors using up-regulated immune checkpoints (such as PD-1, LAG-3, or TIM-3) can potentially reverse this pattern (162). Various strategies have been tried to solve the latter.

These measures include activating the conventional Wnt/b-catenin pathway (which has been shown to promote the TSCM phenotype) by inhibiting glycogen synthase kinase 3 beta (GSK3b), which is a type of serine/threonine involved in the degradation of b-catenin Kinase, exist alone (163) or in the in vitro culture of tandem positive selection based on IL7, IL21 and CD8CD/CD62Lþ/CD45RAþ Streptomyces (164); use 2 deoxy sugar cose to inhibit glycolysis (165); It also reshapes mitochondrial function to replicate the memory T cell metabolic phenotype characterized by oxidative phosphorylation (OXPHOS) and fatty acid oxidation (FAO; reference 166). In addition, the phosphatidylinositol 3-kinase (PI3K)/Akt pathway is involved in the formation of T cell memory.

For example, Akt has been shown to phosphorylate and isolate forkhead box O (FOXO) transcription factors, thereby blocking molecules associated with less differentiated T cells (such as CD62L, CCR7, and IL7 receptor-a (IL7Ra or CD127)) transcription; ref.167].

7. Other T cell engineering strategies

In addition to inserting CARs, T cells can be further modified to optimize downstream signal transduction, express costimulatory molecules or secrete cytokines. All these methods can be used to mitigate the negative effects of tonic signaling. Possible strategies include overexpression of 4-1BBL when combined with 28z CAR (10); combined with BBz CAR, knocking out Cbl-b, an E3 ubiquitin protein ligase that can be promoted by regulating PI3K to enter CD28 (175, 176) No reaction. Overexpression of PGC-1a enhances the biogenesis of OXPHOS and mitochondria (171); or expression of the IL15/IL15R fusion protein tethered to the cell surface has been shown to promote the CD45RO-CCR7+CD95+TSCM phenotype (42).

The solution strategy summary is shown in the figure below:

All in all, chronic T cell activation has been observed in certain combinations of scFv, hinge, and costimulatory domains, and this may be increased due to high levels of CAR expression. In the case of CAR-T cells, chronic CAR signaling can also drive T cell failure, resulting in decreased T cell persistence and impaired anti-tumor activity. CAR showing tonic signal is related to accelerated T cell differentiation and exhaustion and impaired anti-tumor effect. Choosing a CAR whose configuration does not induce tonic signaling is important for enhancing antigen-specific T cell responses.

Introduce the tonic signal detection scheme

Because the phenotype and growth of CAR-T cells are different from those of control T cells, the tonic signal can be identified during the expansion of primary T cells.

1. Carry out CAR-T cell preparation according to the production process

Including T cell separation-T cell stimulation and transduction-demagnetization beads-CAR-T cell expansion; at the same time culturing untransduced T cells as a control.

2. Observe the following indicators:

(1) Cell expansion rate

Compare CAR-T with T cells to see if it will slow down significantly;

(2) Cell size

T cell size can also be used to anticipate tonic signaling. The volume of primitive T cells is about 160fl, but T cells will swell significantly when activated. The proliferating and metabolically active CAR-T cells grow to nearly 600 fl (or 10μm). A few days after stimulation, the size of T cells gradually returned to a resting state (about 300 fl on day 10-12). However, CAR-T cells with tonic signaling cannot return to a quiescent state and usually maintain a larger cell volume.

(3) Detect the secretion of cytokines

CAR-T cells with tonic signaling may secrete cytokines (IL-2 and IFN-γ), which continue to exceed the effect of the primary stimulus.

(4) Expression of activation, differentiation and inhibitory markers

Here, we will show how to use T cell activation, differentiation and inhibition (see Figure 2) to identify the tonic signal on CAR-T cells after the initial expansion. T cells can be analyzed at an earlier time point (ie, day 9 or day 10, when all T cells show similar activation marker levels), and at a later time point (ie, day 12 to 14) , The control T cells will be in a quiescent state, while the CAR-T cells with tonic signal will still be activated and show signs of differentiation and exhaustion.

(5) Analysis of Annexin V and 7-AAD staining on cell apoptosis

Excessive tonic CAR signal transduction usually leads to excessive activation of T cells, leading to increased apoptosis.

(6) Analyze tonic CAR signal by Western Blot

During in vitro expansion, the intensity of tonic CAR signal in T cells may change. Generally, 2 to 5 days after CAR transduction, the tonic signal in T cells is the highest. T cells with high spontaneous CAR signaling usually expand slowly, and are eventually eliminated by T cells with low tonic CAR signaling.

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