Figure 1. Manufacturing process of CAR-T cell therapy.

CAR Structure

CAR is a specially designed synthetic protein used to enhance the ability of cells to specifically recognize and eliminate tumor cells. It consists of three main parts: the extracellular domain, the transmembrane domain, and the intracellular domain. The extracellular domain mainly includes a single-chain variable fragment (scFv) and a connecting hinge region. scFv is an engineered protein structure that helps CAR-T cells recognize and bind to specific markers on the surface of tumor cells, also known as tumor-associated antigens (TAA). This recognition mechanism is independent of the major histocompatibility complex (MHC) system, thereby helping immune cells bypass the MHC-mediated immune escape strategies that tumor cells may adopt. The connecting hinge region is a key connection site, usually derived from molecules such as IgG or CD8α/CD28. The length and structure of this region can be adjusted to regulate the CAR signal strength and immune cell function. The transmembrane domain has an important influence on the expression and stability of CAR on the cell surface, and is also a key factor in signal transmission efficiency and CAR-T cell function. Commonly used transmembrane domains are derived from proteins such as CD4, CD8α, and CD28. The intracellular domain contains a co-stimulatory domain and an activation domain. Among them, the key role of the co-stimulatory domain is to enhance the activity and persistence of CAR-modified immune cells, such as CD28 promoting T cell proliferation and initial killing function, and 4-1BB prolonging the survival time of modified cells and enhancing their memory function. The activation domain usually uses the CD3ζ chain, which contains a key signaling element, the immunoreceptor tyrosine activation motif (ITAM). When CAR binds to the target antigen, these elements can transmit activation signals to CAR-T cells, thereby prompting them to perform immune functions, such as secreting cytokines and directly killing tumor cells.

Application of T Cells in CAR

T cells are an important component of circulating lymphocytes, play a central role in the human immune system and have a variety of surface molecular characteristics. These characteristics allow T cells to be classified into several subtypes according to their function, including, but not limited to, initial T cells, cytotoxic T cells, helper T cells, regulatory T cells and memory T cells. According to the different compositions of T cell surface receptors (TCRs), T cells can be further classified into αβ T cells (expressing TCRs composed of α chains and β chains) and γδ T cells (expressing TCRs composed of γ chains and δ chains). Among these, αβ T-cells are the most widely studied T-cell subtypes, especially the CD8+ cytotoxic T-cell subtype within them, which can directly kill tumor cells and is also the most commonly used subtype of CAR-T cells in anti-tumor therapy. CAR-T therapy targeting tumor cells is the earliest and most widely used form of CAR technology. Rosenberg originally proposed the concept of using a patient’s own immune cells to attack tumor cells and demonstrated this idea in experiments using tumor-infiltrating lymphocytes (TILs) from melanoma patients.

To overcome the problem of low immunogenicity of certain tumors, Israeli scientist Professor Zelig Eshhar developed CAR technology and created the first generation of CAR-T cells that can bypass MHC restrictions. He fused scFv corresponding to specific TAA with FcεRI receptor (γ chain) or CD3 complex (ζ chain) to construct CARs, and successfully expressed these CARs on the surface of T cells using genetic engineering technology, so that T cells can directly and specifically recognise and combine with TAA to kill tumor cells. However, these cells are unable to maintain long-term viability. To overcome this limitation, Carl H. June’s team developed second-generation CAR-T cells, which significantly increased their efficacy by introducing a costimulatory domain (such as CD137). The first patient with acute lymphoblastic leukaemia to be treated with second-generation CAR-T cells has survived more than 10 years since treatment in 2012, becoming a miracle in the history of medicine. The third generation of CAR-T cells attempts to increase activity by adding a co-stimulatory domain to the second generation, but studies have shown that their anti-tumor efficacy is not significantly better than the second generation. The fourth generation of CAR-T cells introduces specific cytokines or suicide genes to improve therapeutic efficacy while controlling potential toxicity. The fifth generation aims to achieve large-scale production of universal CAR-T cells to improve the availability and cost-effectiveness of treatment, but it must overcome higher technical barriers and meet more stringent safety requirements.

Although CAR technology continues to innovate and evolve, second-generation CAR technology remains the preferred technology for cell transformation. To date, ten CAR-T cell therapy products have been approved by the FDA for worldwide marketing, but the CAR-T cell therapies currently on the market are all for blood cancers. In the treatment of solid tumours, the clinical effect of CAR-T therapy has not yet met expectations, indicating that CAR technology and CAR-T therapy still face major development challenges.

Cat. No.	Product Name	Application
CABT-L0438Y	Magic Rabbit Anti-G4S linker monoclonal antibody, clone H2	FC	Inquiry
CABT-L0438YP	Magic Rabbit Anti-G4S linker monoclonal antibody, clone H2 [PE]	FC	Inquiry
CABT-L0438YF	Magic Rabbit Anti-G4S linker monoclonal antibody, clone H2 [FITC]	FC	Inquiry
CABT-L0438YA	Magic Rabbit Anti-G4S linker monoclonal antibody, clone H2 [APC]	FC	Inquiry
CABT-L0101YU	Mouse Anti-G4S linker monoclonal Antibody, clone 746	IA	Inquiry
CABT-L0101Y	Mouse Anti-G4S linker monoclonal Antibody, clone 746 [PE]	FC	Inquiry
CABT-L0102Y	Mouse Anti-G4S linker monoclonal Antibody, clone 746 [APC]	FC	Inquiry
DMABB-JX390	Magic Rabbit Anti-Whitlow 218 linker monoclonal antibody, clone F4	WB, FC	Inquiry
DMABB-JX391	Magic Rabbit Anti-Whitlow 218 linker monoclonal antibody, clone F4 [PE]	WB, FC	Inquiry
DMABB-JX392	Magic Rabbit Anti-Whitlow 218 linker monoclonal antibody, clone F4 [APC]	WB, FC	Inquiry
DMABB-JX393	Magic Rabbit Anti-Whitlow 218 linker monoclonal antibody, clone F4 [AF488]	WB, FC	Inquiry
DMABB-JX394	Magic Rabbit Anti-Whitlow 218 linker monoclonal antibody, clone F4 [AF647]	WB, FC	Inquiry
DMABB-JX395	Magic Rabbit Anti-Whitlow 218 linker monoclonal antibody, clone F4 [AF594]	WB, FC	Inquiry
DMABB-JX396	Magic Rabbit Anti-Whitlow 218 linker monoclonal antibody, clone F4 [Biotin]	WB, FC	Inquiry

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Later, the researchers proved that M-type phospholipase A2 receptor 1 (PLA2R1) is the main human target antigen of IMN. Anti-PLA2R1 antibodies are found in 70% of IMN patients. In 2014, scientists discovered THSD7A antibodies in the sera of patients with PLA2R1-negative IMN. The discovery of PLA2R1 and THSD7A autoantibodies has made scientists more convinced that the immune system plays an important role in the pathogenesis of MN. At present, MN is considered to be an autoimmune disease, mainly caused by autoantibodies that recognize the target antigen of glomerular podocytes. The dense electronic deposits and epithelium under the foot processes can activate the complement system to form membrane attack complexes and cause podocyte damage. Systematic detection of target antigens is the key to revealing the pathogenesis of MN. The discovery and research of potential target antigens in the pathogenesis of MN may help the specific and targeted diagnosis and treatment of MN patients.

PLA2R Gene

M-type phospholipase A2 receptor 1 (PLA2R1) is a type I transmembrane receptor that belongs to the mammalian mannose receptor family and is mainly expressed in podocytes. In situ immune complexes are formed by PLA2R autoantibodies expressed by IMN patients bound to the surface of podocytes. Subsequently, the complement system will be activated through the bypass and mannose-binding lectin pathway, resulting in the formation of C5b9 attack membrane complexes, thereby damaging the podocytes.

Clinical studies have found that serum samples from 26 (70%) of 37 patients with idiopathic but not secondary membranous nephropathy specifically identified the presence of non-reduced glomerular extracts 185-kD glycoprotein. Mass spectrometry of the reactive protein band identified the M-type phospholipase A2 receptor. The reactive serum specimen recognizes recombinant PLA2R and binds to the same 185-kD glomerulin as the monospecific antibody. Anti-PLA2R autoantibodies in serum samples of patients with membranous nephropathy are mainly IgG4, which is the main immunoglobulin subclass in glomerular deposits. Further research found that PLA2R is normally expressed in human glomerular podocytes and co-localizes with IgG4 in the glomerular immune deposits of patients with membranous nephropathy. Studies have found that IgG can be eluted from these deposits in patients with idiopathic membranous nephropathy, but not membranous lupus or IgA nephropathy. The results of the study indicate that most patients with idiopathic membranous nephropathy have antibodies against conformation-dependent epitopes in PLA2R.

THSD7A Gene

Thrombospondin type 1 domain 7A (THSD7A) is expressed in placental vascular endothelial cells and plays a role in endothelial cell migration and angiogenesis. A previous study evaluated the expression of THSD7A by immunofluorescence staining of kidney biopsy samples and concluded that THSD7A is expressed in podocyte foot processes.
The THSD7A antibody was detected by Tomas et al. In 2014, anti-PLA2R1 was negative in the serum of European and Boston IMN patients. At the same time, immunohistochemistry of kidney biopsy samples showed that THSD7A is a 250 kDa glomerulin that is localized in podocytes. Studies have shown that THSD7A is the second autoantigen involved in the pathogenesis of adult IMN. In this study, 15 of the 154 patients with idiopathic membranous nephropathy without anti-PLA2R1 antibodies had serum samples that responded to THSD7A. IgG4 is the main anti-THSD7A autoantibody identified in these patients, although other subtypes are weakly present in most serum samples. Immunofluorescence staining of kidney biopsy samples from healthy controls showed linear glomerular expression of THSD7A and co-localized with podocyte foot processes, but not with glomerular basement membrane or endothelial cells.

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In the process of apoptosis, caspases (cysteine-aspartic proteases) promote cell death through the hydrolysis of more than 400 proteins. Studies have found that caspase is mainly activated through internal and external cell death pathways.

The intrinsic cell death pathway is a pathway that is mainly regulated by Bcl-2 family proteins and completes cell death through mitochondria. The key step in the intrinsic cell death pathway is the permeabilization of the outer mitochondrial membrane. After the outer mitochondrial membrane is permeabilized, the release of proteins from the space between the mitochondrial membranes promotes the activation of caspase and cell apoptosis. The released cytochrome C binds to APAF-1 and induces caspase-9 activation. Caspase 9 then activates caspase 3 and 7, leading to apoptosis.

Early Markers of Intrinsic Apoptotic Pathway

Bcl-2 Family
The Bcl-2 family regulates cell apoptosis through the effects of pro-apoptotic and anti-apoptotic members. This family consists of more than 20 cytoplasmic proteins containing Bcl-2 homology (BH) domains, which are essential for the function of apoptosis. Each member of the Bcl-2 family contains at least one BH motif to promote its function. The members of the Bcl-2 family are divided into three functional categories, namely anti-apoptotic, pro-apoptotic multi-domain effector and only Bcl-2 homolog 3 (BH3) activator.
Among them, the main family members are:

Bcl-2

Bcl-2 is the initial member of the Bcl-2 family and plays a role in inhibiting cell apoptosis. Bcl-2 is a ubiquitously expressed mitochondrial membrane protein, which can inhibit apoptotic cell death by isolating BAX and BAK.

PUMA
PUMA, also known as Bcl-2 binding component 3 or JFY-1, is a member of the BH3-only protein family. Studies have found that PUMA expression is regulated by p53 tumor suppressor protein. When cells respond to stress and DNA damage, the up-regulated p53 binds to the PUMA promoter, resulting in PUMA transcription. PUMA acts as a triggering factor for apoptosis through mitochondrial signal transduction and interaction with Bcl-xL, Bcl-2 and other anti-apoptotic proteins.

NOXA

NOXA is the pro-apoptotic member of BH3-only in the Bcl-2 protein family. Studies have found that NOXA is located in mitochondria and is the transcription target of p73 and p53. NOXA replaces the pro-apoptotic proteins BAK and BIM from members of the anti-apoptotic protein Bcl-2 family, thereby promoting apoptosis during cell survival. In addition, NOXA can also be upregulated independently of p53.

BAD

BAD is the only pro-apoptotic member of BH3 in the Bcl-2 protein family. BAD has a pro-apoptotic effect, and its exact role in apoptotic signal transduction depends on the post-translational modification status of BAD. Non-phosphorylated BAD forms heterodimers with two anti-apoptotic Bcl-2 protein family members, Bcl-xL and Bcl-2. The combination of BAD and Bcl-xL promotes cell apoptosis by inhibiting the anti-apoptotic function of Bcl-xL. After phosphorylation of serine, BAD cannot heterodimerize with Bcl-xL. This combination retains BAD in the cytoplasm, allowing Bcl-xL to inhibit cell apoptosis.

BID

BID is a pro-apoptotic molecule that can form heterodimers with other members of the Bcl-2 family (including the agonist BAX or the antagonist Bcl-2). BID contains a BH3 domain, which is required for its interaction with Bcl-2 family proteins and its pro-apoptotic activity. BID is cleaved to produce a shorter active form called truncated BID (tBID). tBID translocates to the mitochondria, leading to the activation of the intrinsic pathway.

BAX

BAX, also known as Bcl-2-like protein 4 or Bcl-2-related X protein, is a member of the pro-apoptotic multi-domain effector family. After inducing apoptosis, BAX translocates to mitochondria and induces MOMP.

BAK

BAK is a member of the pro-apoptotic multi-domain effector family. Due to its similar structure to BAX, it is considered to have significant homology with BAX. The study found that Mcl-1 and Bcl-xL isolate BAK in non-apoptotic cells. After the onset of apoptosis, NOXA and BAD release BAK, which causes the outer mitochondrial membrane to permeate and release pro-apoptotic factors.

Extrinsic Cell Death Pathway

After the death receptor binds to the ligand, the receptor oligomerizes and recruits adaptor proteins, such as serine/threonine protein kinase 1 (RIPK1), RIPK3, and Fas-related proteins ( FADD), eventually formed as a death-inducing signaling complex (DISC). As part of the DISC assembly, the former caspase 8 can be cleaved into its active form caspase-8 by oligomerization, which in turn cleaves and activates the effector caspase (caspase 3 and caspase 7). One of the early effects of this activation is the inhibition of phosphatidylserine (PS) flippase, resulting in exposure of PS on the outer plasma membrane.

Early Markers of Extrinsic Apoptotic Pathway

Phospholipid Asymmetry

Phospholipid asymmetry is the controlled distribution of different lipid species in the lipid bilayer. One of the early apoptotic events is the rearrangement of lipids in the plasma membrane. This change causes PS to be exposed on the outer surface of the cell. This exposure depends on the inhibition of flippase and the signal from the phagocytic cell.

Annexin V

Fluorophore-conjugated annexin V is commonly used to assess changes in plasma membrane asymmetry. However, PS exposure may be short-lived, and this phenomenon is called “PS flip”. In the early detection of apoptosis, Annexin V and pSIVA can be used together with propidium iodide to distinguish dead cells from cells in the early stages of apoptosis.

Initiator Caspase

Caspase-8 is the promoter caspase, a key signal molecule in the apoptosis mediated by CD95 and tumor necrosis factor receptor 1 (TNFR1). After activation, the cell death receptor Fas is connected to the adaptor molecule FADD through the corresponding death domain (DD). Then, FADD binds to procaspase 8 through the death effector domain (DED), and then undergoes oligomerization and autocatalytic activation. Activated caspase-8 cleaves and activates effector caspase and Bcl-2 family member BID. The activity of the starting protein caspase (caspase-8 and caspase-10) can be determined by Western blot or flow cytometry. The determination of caspase-8 activity by Western blotting depends on the detection of caspase-8 active fragments with a suitable caspase antibody.

Effector Caspases

Caspase-3 and caspase-7 are called “effector” caspases. The promoter caspase is automatically proteolyzed, and the effector caspase is cleaved by the promoter caspase. This hierarchical structure allows amplification of chain reactions. Effector caspases control many of the phenotypic changes observed during apoptosis, such as membrane blistering and DNA fragmentation.

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Caspase Type

It has been determined that there are at least 11 caspases:

Among these caspases, caspase 1 and caspase 11, and possibly caspase 4, are considered not to directly participate in the transduction of apoptosis signals. They are mainly involved in the activation of interleukin precursors. Caspase 2, caspase 8, caspase 9, and caspase 10 are involved in the initiation of cell apoptosis. Caspase 3, caspase 6, and caspase 7 are involved in the execution of apoptosis. Among them, caspase 3 and 7 have similar substrate and inhibitor specificities. They degrade PARP, DFF-45 (DNA fragmentation factor-45), which leads to inhibition of DNA repair and initiates DNA degradation. The substrates of caspase-6 are lamin A and keratin 18. Their degradation leads to the disintegration of the nuclear lamina and cytoskeleton.

Caspase Activation

Due to the correlation between caspase and cell apoptosis, it exists in the cell in an inactive zymogen state, and can perform its function after activation. When the general protease is activated, only the N-terminal peptide is removed, while the activation of caspase requires cleavage at the aspartic acid site in the junction region of the two subunits, resulting in a two-subunit composition heterodimer, this is the enzyme with activity. Usually the N-terminal peptide is also removed during activation, but whether or not the N-terminal peptide is removed for caspase 7 has no effect on the activity. At present, it is believed that there is an upstream and downstream relationship between the initiator of apoptosis (caspase 2, 8, 9 and 10) and the performer (caspase 3, 6 and 7), that is, the initiator activates the performer. Apoptosis initiators (caspase 2, and 10) is activated by its multimeric form. Caspase 8 and 10 contain tandemly repeated “death effector” domains (DED), while caspase 2 and 9 contain different but similar caspase recruitment domains (CARD). These two domains can recruiting caspase 2, 8 And 10. In addition, caspase-8 terminates apoptosis, can cleave RIP1 and rip3, and effectively terminate necrosis.

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Significance of Apoptosis Markers
Clinical studies have shown that the inhibition of apoptosis is a hallmark of human cancer, and the ideal strategy for many targeted therapies is to specifically induce tumor cell death in vivo. Therefore, mechanism-based therapy in oncology can directly induce tumor cell apoptosis by targeting the molecular components of the apoptosis regulatory pathway, or indirectly induce apoptosis by regulating drug targets. Either way, there is an urgent need to apply proven and effective apoptosis biomarkers in clinical trials of apoptosis anticancer therapies. In addition, in basic apoptosis-related molecular mechanism research or tumor scientific research, it is also necessary to use accurate biomarkers to determine the relationship between research objects and apoptosis.
Molecularly, apoptosis is activated through death receptor-mediated external pathways or mitochondrial-directed internal pathways. The ligand binds to the death receptor on the plasma membrane, which triggers the activation of the initiator caspases 8 in an external pathway. In some cell types, this directly activates effector caspases, such as caspase 3. In other cells (and most cancer cells), caspase 8 can also amplify death signals by participating in internal pathways. The latter is controlled by pro-apoptotic and anti-apoptotic Bcl-2 family proteins. Under the stimulation of apoptosis, changes in the interaction of proteins within the family of mitochondria determine the release of cytochrome c. Cytoplasmic cytochrome c activates the apoptotic body complex, the promoter caspase 9 and the effector caspase. Afterwards, Caspase cleaves cytokeratins (CKs) and activates the activities of poly(ADP-ribose) polymerase and endonuclease (to generate nucleosomal DNA (nDNA)). These processes constitute a series of irreversible events and lead to the formation of apoptotic bodies. In addition, these processes promote the exposure of phosphatidylserine on the outer surface of the plasma membrane, allowing phagocytes to recognize dying cells.

Participants in many of the above molecular events are potential biomarkers of apoptosis (Fig 1). Usually in in vitro studies, most of these molecules can be used as markers to be detected by chemical methods. However, the detection of apoptosis in vivo is challenging. It is usually asynchronous, and the half-life of apoptotic cells in the tissue is very short. Therefore, biomarker analysis time is critical to the level of apoptosis detected in patient samples.

Ideal Characteristics of Biomarkers

Compared with in vitro studies, biomarkers used in clinical research have more stringent requirements. This marker must not only meet the requirements of scalable hypothesis testing in early clinical trials, but also confirm whether the drug has reached the tumor target (Proof of Mechanism, POM) and achieved the expected tumor results (Proof of Concept) in subsequent treatments , POC). PD biomarkers compared before and after drug treatment may reflect changes in the drug target (for example, protein phosphorylation, direct DNA damage, and enzyme activity), or those changes at the end of the target (for example, downstream signaling events, gene expression Variety). The cell fate after target regulation should be obvious (for example, changes in proliferation, apoptosis, or angiogenesis). The biomarkers measured in tumors and/or replacement body fluids cover a wide range of molecules (proteins, nucleic acids, lipids and sugars) as well as circulating intact cells. The ideal biomarker should provide a minimally invasive/non-invasive indirect continuous readout of disease/drug activity.

Therefore, ideal biomarkers should pursue the following standards:

• Specificity of biological process/target
• It can be accurately quantified in clinical samples and has sufficient dynamic range to detect changes after drug treatment
• Provide fast, reliable and powerful measurement
• It can be verified as an internationally recognized standard
• There is almost no overlap in levels between untreated patients and treated patients
• The baseline level does not vary from patient to patient
• Have a level related to the total burden of disease and not affected by unrelated conditions
• Have a level closely related to the proximal or distal effect of the treatment, thereby contributing to POM or POC
• Can be measured in easily available clinical samples

Common Apoptotic Markers

Table 1：Biomarkers of apoptosis.(Singhal et al.; 2005)

The exogenous apoptotic pathway and the intrinsic apoptotic pathway are the two main pathways leading to the execution of apoptosis, as well as the TNF pathway. The external apoptotic pathway that initiates apoptosis is triggered by death ligands that bind to death receptors present in the cell membrane. The intrinsic apoptotic pathway initiates apoptosis by activating Caspase-3 or cleaving the BH3 interaction domain death agonist (Bid) through the Puma and Noxa protein subgroups. The TNF pathway is mainly caused by the binding of TNF-α and TNFR1 through the intermediate membrane protein TNF receptor-associated death domain (TRADD) and Fas-associated death domain protein (FADD) to initiate the pathway leading to caspase activation. Therefore, apoptosis involves members of the Bcl-2 family (Bcl-2, Bcl-xL, Bcl-w, Mcl-1), Bax protein, caspase 8, death receptor, p53/p73 gene/P21 WAFI, and Changes in the level of NF-κB and other related genes can be detected by immunohistochemistry. In addition, most of them can also be detected by ELISA and flow cytometry.

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Induced Protein

The processing protein used in the study is Yamanaka factors, which includes Oct3 / 4, Sox2, Klf4 and c-Myc. As we all know, Yamanaka factors are often used to induce the pluripotency of adult somatic cells in biological research. The induced iPS shows both pluripotency and youthful characteristics. Is it possible to simply rewind the aging clock without causing pluripotency? The researchers explored this hypothesis by strictly controlling the duration of exposure of these protein factors. Existing experimental results show that iPS cells can be induced by repeatedly exposing adult cells (such as the cells that make up the skin) to Yamanaka factors for two weeks. Researchers began to wonder whether exposing adult cells to Yamanaka protein for a few days instead of weeks could trigger this young reversal without causing omnipotence. Therefore, the researchers implemented strict control over the duration of protein factor exposure by introducing short-lived RNA information encoding Yamanaka factors into adult cells every day. Over time, these proteins will reverse the fate of the cells-pushing them back along the axis of development time until they resemble the young embryonic pluripotent cells they originated from.

Yamanaka Induces Cell Rejuvenation

Researchers have shown that after being induced to briefly express a set of proteins involved in embryonic development, old human cells will return to a younger and more vibrant state at the molecular level. Since the aging of muscle cells is the most outstanding performance in the aging process of the body, the researchers conducted research experiments on muscle stem cells. Although muscle stem cells have a natural ability to renew themselves, this ability will diminish with age. After protein treatment and repair of the existing muscle stem cells of old mice in vitro, they were transplanted back into the body, and it was found that the appearance of muscle stem cells was obviously rejuvenated. In fact, researchers at the Salk Institute for Biological Research discovered in 2016 that the brief expression of four Yamanaka factors in prematurely aging mice would extend the lifespan of the animals by about 20%. But it is not clear whether this method is applicable to humans. Researchers want to explore whether aging human cells will rejuvenate in a similar way, and whether this method can be extended to many organizations. To this end, the researchers devised a method that uses the instability of messenger RNA to temporarily express 4 kinds of Yamanaka factor and 2 other proteins in human skin and vascular cells. The researchers then compared the gene expression patterns of treated and control cells obtained from the elderly with untreated cells obtained from the young. It was found that cells from the elderly showed signs of reversal of aging only four days after exposure to reprogramming factors. Untreated elderly cells express high levels of genes related to aging, while treated elderly cells are more similar to young cells in gene expression patterns.

Further research found that during protein induction, these cells not only released any memory of their previous identities, but also recovered their youthful state. They completed this transformation by wiping the DNA to remove molecular tags, which not only distinguish the source of the cells but also indicate the age of the cells. The results of the experiment found that the treated cells were younger with untreated elderly cells. Next, they compared several signs of aging between the cells of young people, the untreated cells of the elderly, and the treated cells of the elderly, including how the cells perceive nutrition, metabolize compounds to produce energy, and dispose of cellular waste. Finally, it was found that the aging markers decreased and the cell metabolic capacity increased. Therefore, it is shown that Yamanaka induction can indeed achieve cell rejuvenation to a certain extent.

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Heart Regeneration
Although heart cells have a slight ability to regenerate. However, it is generally believed that the regenerative capacity of the human heart muscle is seriously insufficient, and it is not enough to make up for the severe loss of myocardium caused by catastrophic myocardial infarction or other heart disease. Studies have found that the heart of some vertebrates (such as zebrafish and salamanders) does undergo a regenerative reaction after injury; under normal conditions, mouse and human cardiomyocytes rarely divide. But after a serious injury, the remaining cardiomyocytes will start DNA synthesis and re-enter the cell cycle. Therefore, the division of existing cardiomyocytes seems to be the most important factor for heart regeneration in mice and humans. The dedifferentiation of cardiomyocytes near the damaged area occurs before their proliferation and is characterized by the loss of expression of myocardial contractile proteins (such as α-myosin heavy chain and troponin T). Studies find zebrafish heart regeneration may be mainly caused by undifferentiated stem cells or progenitor cells from the outer layer of the heart (epithelium). Further research on salamanders and zebrafish will more clearly define whether cardiac regeneration in these organisms requires dedifferentiation, proliferation and subsequent cardiomyocyte differentiation, or whether regeneration is driven by the recruitment of stem cells to the injured site. In contrast, in mammalian hearts, cardiomyocytes rarely divide after a myocardial infarction, although transgene overexpression of specific genes in mice increases the division of cardiomyocytes.

There is strong evidence that endothelial cells are renewed by bone marrow-derived progenitor cells, but the idea that cardiomyocytes are renewed by such cells has been heatedly debated. Less controversial is that adult mammalian heart muscle has a resident cardiac stem cell (CSC) population, which has the potential to differentiate into cardiomyocytes and other cell types (such as endothelial and vascular smooth muscle cells). The study found that CSCs can support the basic turnover of cardiomyocytes, but this turnover occurs at a very low rate without damage. CSCs have high proliferation and differentiation potential in vitro, and it may be a promising therapeutic direction to expand autologous CSCs in vitro or stimulate the regeneration of these cells in vivo.

The recognition that there is indeed a regeneration mechanism in the mammalian myocardium has aroused intense attention. Researchers have discovered that it may hinder the existence of aplastic disorders, including ischemia, inflammation and fibrosis at various stages of myocardial infarction. This unfavorable microenvironment may prevent the activation of resident CSCs, thereby reducing the success rate of exogenous cell therapy. Certain components of the inflammatory response may be essential to promote angiogenesis and progenitor cell recruitment, but excessive inflammation may also prevent the recruitment and survival of progenitor cells. Similarly, after myocardial infarction, a certain degree of fibrosis is required to prevent myocardial rupture, but dense fibrosis presents a strong physical barrier to regenerative cells.

Which Stem Cells Are Used In Heart Therapy?
Perhaps the most confusing aspect of current cardiac regeneration is the different cell types, which are considered to be candidates for cardiac therapy. Multiple cell candidates reflect that human research on cell regeneration is not deep enough, so further research and exploration are needed.

Skeletal Myoblast
One of the earliest cell-based cardiac regeneration strategies was to inject autologous skeletal muscle myoblasts into ischemic myocardium. Myoblasts are resistant to ischemia, and can be differentiated into myotubes (but not into cardiomyocytes) in the laboratory animal experiments and improve ventricular function. The myocardial tube will not integrate with the surviving cardiomyocytes, so it will not beat synchronously with the surrounding myocardium. However, related clinical trials were terminated due to lack of efficacy, so it is unlikely that skeletal myoblasts will actually regenerate the heart muscle. Interestingly, studies found that mouse skeletal muscle contains a large number of non-satellite cells, which can differentiate into spontaneous pulsatile cells with cardiomyocyte characteristics, but no one has found similar cells in human skeletal muscle.

Bone Marrow-Derived Cells

In stem cell cardiac therapy, it was first reported that adult stem cells or progenitor cells transplanted into the infarcted heart of mice that can differentiate into cardiac myocytes are a subset of hematopoietic cells derived from bone marrow. The first evidence that adult bone marrow-derived progenitor cells are involved in the formation of cardiomyocytes in the adult human heart is based on reports of Y chromosome-positive cardiomyocytes in male recipients of transplanted female donor hearts. Animal studies using labeled hematopoietic stem cells for bone marrow transplantation and subsequent myocardial infarction have shown that cardiomyocytes are derived from transplanted cells, but the proportion is extremely low. Moreover, other studies in animals have not demonstrated that hematopoietic progenitor cells can differentiate into cardiomyocytes or improve heart function. Therefore, there is currently no consensus on whether bone marrow-derived progenitor cells differentiate into cardiomyocytes in vivo.

Embryonic stem cell

Embryonic stem (ES) cells are prototype stem cells. They clearly meet all the requirements of stem cells: cloning, self-renewal and multi-potency. ES cells can differentiate into any type of cells present in an adult organism, so it has the potential to completely regenerate the heart muscle. The two obstacles facing the clinical application of ES therapy are immune rejection and the tendency of injecting ES cells to form teratomas. With the increase in knowledge of ES cell differentiation and cardiac embryonic development pathways, ES cell differentiation may become more controllable. Methods to limit teratoma formation include genetic selection of differentiated ES cells, or differentiation of ES cells into cardiomyocytes or endothelial cells in vitro before injection. For example, tumor necrosis factor promotes the differentiation of ES cells into cardiomyocytes. If the differentiated ES cells are delivered to the myocardium in a rich survival mixture, they can survive and improve myocardial function. The inherent difficulty in controlling the growth and differentiation of ES cells and other pluripotent stem cells is that the timing of activating specific signaling pathways may be crucial. For example, recent studies on mouse and zebrafish embryos have shown that the role of the Wnt-β-catenin pathway in heart development depends on the stage of development.

Endogenous cardiac stem cells

Because allogeneic cells face immunological challenges that may require immune rejection, the isolation of endogenous adult mammalian CSCs based on cell surface markers has attracted great interest. However, no clear CSC mark has been determined so far. Mammalian heart muscle includes a small percentage of stem cells expressing cell surface markers Kit or Scal. In addition, some side population cells also express Kit and / or Sca1, and like Kit ⁺ CSC and Sca1 ⁺ CSC, side population cells can produce cardiomyocytes in vitro and in vivo. In addition to Kit ⁺ CSC, Sca1 ⁺ CSC and side population cells, the fourth type of CSC also expresses the transcription factor Isl1. The tracer experiments showed that during embryonic heart development, cells expressing Isl1 can differentiate into endothelial cells, endocardial cells, smooth muscle cells, conduction system cells, right ventricular myogenic cells and atrial myogenic cells. There are also cells that express Isl1 in the heart of adult mammals, but they are limited to the right atrium, are found in fewer numbers than the embryonic heart, and have unknown physiological effects. Recently, epicardial-derived progenitor cells with angiogenic potential have been described.

Stem cell therapy for heart disease faces some challenges. The most important question to be answered in preclinical research is which stem or progenitor cells are the best choice for treatment. So far, under certain circumstances (acute myocardial infarction), bone marrow-derived progenitor cell therapy has proven to be safe and beneficial, but the regeneration potential of this cell is still controversial. CSC may have the potential to target patients, but isolation and cultivation procedures are still in the early stages of development. ES cells have the greatest differentiation potential, but face moral barriers and the greatest risk of teratoma formation. Whether ES cell derivatives will be rejected by the host’s immune response is still under debate. However, in principle, rejection can be avoided by using cells from a pool of only 150 donors with different HLA types. If new technologies for reprogramming human and mouse fibroblasts into ES-like cells can be used, the use of patient-reprogrammed cells can reduce or even eliminate immune rejection. When designing a more rational cell-based treatment for heart disease, a key issue is to understand the mechanism by which each stem or progenitor cell type can affect myocardial function. Similarly, different cardiology, such as acute myocardial infarction and chronic ischemic cardiomyopathy, may require different types of stem or progenitor cells.

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Cell Survival

From the previous introduction, we can know that UPR can promote cell survival, thereby reducing ER stress and restoring homeostasis. However, long-term UPR activation induces apoptosis via the PERK–eIF2α–ATF4–CHOP pathway. Studies have shown that the CHOP encoded by the gene DDIT3 can induce the expression of pro-apoptotic genes (such as DR5, TRB3, BIM and PUMA), and inhibit the expression of BCL2, thereby triggering apoptosis when the ER stress is triggered. In addition, ATF4-CHOP heterodimer also initiates the restoration of mRNA translation, resulting in increased protein synthesis, ATP depletion, oxidative stress and cell death. If the Ddit3 gene is deleted from the cell, ER stress causes less protein aggregation in the endoplasmic reticulum and reduces oxidative stress and apoptosis. If any other UPR gene is deleted, this will not happen instead. In addition, CHOP also activates ER oxidase 1α (ERO1α), an oxidoreductase that mediates the transfer of electrons to molecular oxygen during the formation of disulfide bonds to produce hydrogen peroxide. This reaction increases the ability to generate reactive oxygen species (ROS) from the ER and inositol-1,4,5-triphosphate receptor (IP3R) -mediated Ca^{2 +} efflux. Ca^{2 +} released from ER is absorbed by mitochondria through the ER membrane associated with mitochondria, thereby promoting mitochondrial ROS production through different mechanisms. The flux of Ca^{2 +} between the endoplasmic reticulum and mitochondria may combine the protein folding ATP requirement with the mitochondrial production of ATP. Therefore, endoplasmic reticulum stress causes oxidative stress and impairs mitochondrial function, resulting in cell death in a CHOP-dependent manner.

Cell Growth And Differentiation
Cells need a large amount of protein in the process of proliferation and differentiation, which will cause an increase in protein synthesis, which is the main cause of endoplasmic reticulum stress and UPR activation. During ER stress, the monitoring mechanism delays the process of ER and cell division. It depends on the mitogen-activated protein kinase (MAPK) SLT2, but not on PERK, IRE1α and ATF6α (UPR sensors).
The discovery of the role of UPR in cell differentiation is demonstrated by the need for the IRE1α–XBP1 pathway in plasma cell differentiation. Since cell differentiation is related to the six-fold expansion of ER, the IRE1α–XBP1 pathway is required for the expansion of the secretory pathway in cells with large amounts of protein secretion. Interestingly,  the increase of immunoglobulins load is not reason of the activation of the IRE1α–XBP1 pathway . Instead, it is caused by a differentiation-dependent signal from the B cell receptor that upregulates genes encoding components such as secretory pathways and plasma cell transcription factors, such as Mist1. Surprisingly, deleting PERK, eIF2α-P or ATF6α did not cause defects in plasma cell differentiation. In addition, XBP1 can induce a wide range of secretory pathway genes, as well as increased endoplasmic reticulum and lysosomal protein content, mitochondrial quality and function, ribosome number, and protein synthesis levels. Therefore, the IRE1α–XBP1 pathway significantly promotes the characteristic phenotypes of specialized secretory cells, such as gastric zymogen cells, β cells, and intestinal Paneth cells.

Cellular Metabolism

When the researchers discovered that PERK deletion and the mutation of the PERK phosphorylation site of eIF2α could cause defects in glucose metabolism, the link between ER stress signals and metabolism was confirmed. ER homeostasis and UPR activation are key to glucose and lipid metabolism. The steady state of blood glucose is strictly controlled by the levels of insulin and glucagon in the blood. ER homeostasis and UPR activation in insulin-secreting beta cells and hepatocytes that respond to insulin and glucagon play an important role in maintaining glucose homeostasis. And related studies have found that each UPR sub-pathway seems to maintain a liposomal homeostasis through different mechanisms.

Protein Secretion
ER is responsible for post-translational modification, folding and transportation of secreted proteins (such as cytokines and hormones). Endoplasmic reticulum stress inhibits the synthesis and secretion of secreted proteins through various mechanisms. A well-studied example is insulin. In beta cells, proinsulin interacts with many ER proteins to promote their folding and transport. For example, embryo knockout of XBP1 significantly impaired proinsulin processing. Because increased ER pressure and excessive activation of IRE1α induced RIDD, which degrades mRNAs20 encoding proinsulin processing enzymes. Therefore, during the transcription, translation, and secretion stages, endoplasmic reticulum stress and UPR activation affect insulin levels. Similarly, endoplasmic reticulum stress and the UPR pathway also affect other secreted proteins at the level of transcription or translation. Because these secreted proteins affect the function of distant organs, endoplasmic reticulum stress not only damages the survival and function of secreted cells, but also affects the entire organism.

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Immune checkpoints refer to some inhibitory signaling pathways that exist in the immune system, such as the PD-1/PD-L1 pathway and the CTLA-4 pathway. Under normal circumstances, in order to prevent activated T cells from destroying normal human cells, the immune system can control the activation of T cells by activating immune checkpoints such as PD-1 / PD-L1 to prevent T cells from erroneously attacking normal cells. However, cancer cells specifically recognize PD-1 on the surface of T cells by expressing the surface protein PD-L1, stealing this control mechanism, thereby activating immune checkpoints to suppress the immune activity of T cells, which will cause cancer cells to escape immune recognition and grow rapidly. This opens up a whole new line of immunotherapy ideas for cancer treatment-blocking immune checkpoints such as CTLA-4 or PD-1 through immune checkpoint inhibitors, preventing cancer cells from stealing this control mechanism, thereby releasing the immune system’s own capabilities to attack cancer.

Immune checkpoint therapy suppresses the activity of immune checkpoints, releases the immune brake in the tumor microenvironment, and reactivates the immune response of T cells to tumors, thereby achieving an anti-tumor effect. There are two natural ligands for PD-1, PD-L1 and PD-L2. One of the most widely studied and applied immune checkpoint inhibitors is the PD-1 / PD-L1 inhibitor. However, only about 20% to 40% of patients can actually benefit from PD-1 / PD-L1 inhibitor therapy. Two important factors are the lack of tumor-specific T cells and T Cells undergoing functional depletion.

As one of the deadliest cancers, pancreatic cancer is notoriously resistant to immune checkpoint therapies. Recently, scientists have found that the immune checkpoint VISTA is overexpressed in immune cells (especially macrophages) infiltrated into pancreatic tumors, so when PD-L1 inhibition is performed, the active VISTA pathway reduces this to a greater extent T cell immune response in this tumor. This indicates that the possible failure of using PD-1 / PD-L1 inhibitors to treat pancreatic cancer is that the active VISTA pathway still inhibits the T cell immune response, and blocking VISTA is expected to improve the efficacy of PD-L1 inhibitors on pancreatic cancer. This also means that it is possible to target other immune checkpoints to improve the efficacy of existing cancer immunotherapy.

In a new study, researchers from research institutions such as the French National Centre for Scientific Research found that as a new type of immune checkpoint inhibitor, NKG2A antibodies could potentially promote the anti-tumor effects of T cells and natural killer cells (NK cells) ability to better treat cancer patients when combined with existing cancer immunotherapy. The relevant research results were recently published in the journal Cell and the title of the paper was Anti-NKG2A mAb Is a Checkpoint Inhibitor that Promotes Anti-tumor Immunity by Unleashing Both T and NK Cells.

The key to this research is a receptor molecule called NKG2A. The researchers found that blocking this receptor enhances the immune activity of NK cells and T cells in mice, thereby increasing the antitumor immune response. They developed an NKG2A antibody called Monalizumab. It is a humanized monoclonal antibody.

Monalizumab mAb is also safe in clinical trials. As of October 2018, the number of patients participating in the treatment reached 40, with no additional safety issues. The most common adverse reactions were fatigue, fever and headache. These data show that the combination of Monalizumab mAb and cetuximab has a higher response rate and durable response for patients.

It can be seen that as a new type of immune checkpoint inhibitor, NKG2A antibody can promote the anti-tumor immune response by enhancing the activity of T cells and NK cells, so it can be used as a complement to the first generation of cancer immunotherapy.

References:

1. Pascale André et al. Anti-NKG2A mAb Is a Checkpoint Inhibitor that Promotes Anti-tumor Immunity by Unleashing Both T and NK Cells. Cell, 2019, doi:10.1016/j.cell.2018.10.014.

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Therefore, protein stability in the endoplasmic reticulum protein folding in cells is critical to cell function and survival. There are many processes in the endoplasmic reticulum that help cells maintain a steady state of protein folding. It is well known that the dynamic life cycle of a protein begins with the ribosome. Synthetic peptides usually carry an N-terminal signal sequence that is recognized by a signal recognition particle (SRP). This helps them enter the ER through the transporter complex. After entering the ER, the signal sequence is removed by a signal peptidase, and the nascent polypeptide is modified (eg, by N-glycosylation), assembled and folded to obtain its inherent conformation. At the same time, there are many general and substrate-specific protein disulfide isomerases and peptidyl propylene glycol isomerases in the ER, which can increase the rate of disulfide bond formation and cis-trans isomerization on proline residues and effectiveness. In addition, the endoplasmic reticulum environment of protein folding is very conducive to the folding of proteins and the formation of disulfide bonds due to the oxidative and kilomolar concentration of Ca2 +. The transport of proteins through the early secretion pathway is mainly determined by the composition of the ER companion protein, the protein folding state, and the structure of the N-chain glycan on the polypeptide. It is worth noting that protein folding is the most error-prone step in gene expression.

ER Pressure

Messenger RNA translation is a stage of gene expression that responds rapidly and reversibly to changes in cellular homeostasis and external stimuli through covalent modification of initiation factors. Because protein synthesis and secretion rates vary widely under different cell types and conditions, different levels of nutrition and energy are required to support protein folding needs. In addition, protein folding is also a process that requires energy, requiring coordinated action between organelles and different proteins, as well as resources from multiple metabolic pathways. Therefore, the correct folding efficiency of proteins in the endoplasmic reticulum is highly sensitive to changes in the intracellular environment or extracellular stimuli. Among them, changes in the endoplasmic reticulum homeostasis can lead to the accumulation of misfolded proteins and cause UPR. These include: increased levels of protein synthesis; impaired ubiquitination and proteasome degradation; insufficient autophagy; insufficient energy; excess or restricted nutrition; calcium level imbalance or redox homeostasis; the challenge of inflammation; and hypoxia.

Regulate Signal Path

Studies have found that in the endoplasmic reticulum, UPR helps cells adapt to endoplasmic reticulum stress. Existing data indicate that three UPR signaling pathways exist in multicellular animals: PRKR– eIF2α, IRE1α– XBP1, and activated transcription factor (ATF) 6α. Among them, PRKR and IRE1α are type I transmembrane proteins with ER cavity-like domain structure and cytoplasmic serine/threonine kinase domain. ATF6α is a type II transmembrane protein that contains a cytoplasmic loop AMP response element-binding protein (CREB) ATF basic leucine zipper domain. During acute endoplasmic reticulum stress, UPR is activated to restore endoplasmic reticulum protein folding in vivo. This equilibrium process involves transient decay of protein synthesis, increased protein folding and transport in the ER, and related protein degradation and autophagy. To reduce the load on protein folding, activated PERK causes phosphorylation to activate eIF2α, which temporarily suspends the translation of the original messenger RNA. Although total protein synthesis is reduced, eIF2α-P paradoxically up-regulates many mRNA-related genes, such as ATF4, increasing the protein’s ability to transport to the endoplasmic reticulum and other processes in the cell.
ATF6α and IRE1α activate the transcription pathway to increase the ability of cellular proteins to fold, transport and degradation. Under stress conditions, IRE1α  is activated. When activated IRE1α enters the nucleus, it not only improves protein folding and transport, but also promotes degradation pathways, and thus can resolve misfolded proteins. Activation of IRE1α also mediates mRNA decay and reduces the protein folding a load of the endoplasmic reticulum. When misfolded proteins accumulate in the ER, ATF6α is transported to the Golgi apparatus and processed to produce a cytoplasmic fragment that increases ER folding capacity mainly by inducing genes encoding protein chaperones. The results show that the three signal branches of UPR are not simultaneously activated by endoplasmic reticulum stress. Under stress conditions, ATF6α and IRE1α activation occurs immediately and then decays over time. PERK is immediately followed by activation and persists exist under chronic ER pressure regulation.

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