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What is the immunogenicity of CAR-T cells in cancer treatment?
What is the immunogenicity of CAR-T cells in cancer treatment? Pre-existing and/or treatment-induced immunity to chimeric antigen receptor (CAR) constructs containing mouse-derived single-chain variable fragments is associated with treatment failure in some patients and may limit the success of re-dosing strategies .
Little is known about the possible impact of immunogenicity on the persistence and function of CAR T cells.
New technologies aimed at enhancing the performance of CAR T cells and/or the application of allogeneic CAR T cells may further amplify the possibility of anti-CAR immune responses. Therefore, strategies to overcome such risks need to be developed.
Various monitoring, mitigation, and management methods can be used to reduce the risk of anti-CAR immunity, although a validated analysis that can fully evaluate the anti-CAR immune response is still an unmet need.
We advocate the inclusion of CAR-related immunogenicity analysis in preclinical and clinical studies of CAR T cell therapy.
Chimeric antigen receptor (CAR) is a fusion protein that can specifically redirect T cells to surface molecules expressed on tumor cells, independent of the conventional T cell receptor (TCR)-major histocompatibility complex (MHC) interaction. CAR is introduced into T cells through gene transfer [1,2].
The antigen recognition domain is usually composed of a mouse-derived monoclonal antibody as a continuous peptide single-chain variable fragment (scFv) guided by an extracellular spacer domain that provides flexibility.
ScFv binds to its target epitope and transmits activation signals through modular intracellular signaling domains. Currently, CAR T cells are produced in vitro from T cells derived from peripheral blood. These T cells are usually transduced with replication-defective vectors that integrate CAR expression sequences into the T cell genome. These CAR T cells are subsequently expanded in large numbers in culture. After being injected into the patient, these cells can recognize and eliminate tumor cells that express the target antigen.
Autologous (patient-derived) and allogeneic (donor-derived) CAR-T cells have successfully developed from preclinical to clinical development, and are currently used to treat cancer patients [3,4,5]. Among the most successful CAR T cell products to date, three of which target the B cell lineage antigen CD19 have been approved by the FDA (tisagenlecleucel, axicabtagene ciloleucel and brexucabtagene autoleucel).
CAR T cells targeting CD19 have become an important treatment option for patients with acute B-cell lymphocytic leukemia (B-ALL) or some aggressive B-cell non-Hodgkin’s lymphoma (NHL), in patients who have undergone extensive pretreatment Induction of complete remission has a wide range of diseases. These CAR T cells are also associated with cytokine release syndrome, immune effector cell-related neurotoxicity syndrome and other immune-mediated adverse events .
So far, the success of CAR T cells in patients with hematological malignancies has not been replicated in patients with solid tumors due to various obstacles, and these obstacles have been reviewed in detail elsewhere [7,8]. Therefore, current efforts are aimed at increasing the potency of CAR T cells and/or combining them with other treatments aimed at overcoming these obstacles.
CAR T cells may induce humoral and cellular anti-CAR immune responses against non-self components of CAR constructs or residual proteins derived from gene transfer vectors. These proteins are inherently immunogenic (Figure 1). In turn, this response may limit the efficacy, thereby inhibiting the success of subsequent administration of CAR T cells [9,10,11]. In addition, many CAR scFvs currently in clinical development are derived from mouse or other non-human monoclonal antibodies (Supplementary Table 1).
A pre-existing antibody that widely recognizes mouse immunoglobulin scFv has been detected in a subgroup of patients, called human anti-mouse antibody (HAMA) [12,13]. Human or humanized scFv can also contain non-self sequences, because the variable binding fragments are produced through multiple gene recombination events and somatic hypermutation. Antibodies against specific antibody sequences (such as those contained in human scFv) are called anti-idiotypic antibodies [14,15]. In addition, the expression of proteins encoded by several human genes in a single peptide CAR chain will produce fusion sequences at junctions that do not normally exist in humans.
So far, there is no conclusive evidence that this type of anti-CAR immune response can lead to other reported adverse events, such as cytokine release syndrome and immune effector cell-related neurotoxicity syndrome; therefore, such toxicity is not the focus of this review.
Figure 1 | The mechanism of anti-CAR immune response. The acquired anti-chimeric antigen receptor (CAR) immune response can be humoral or cellular. In the context of the major histocompatibility complex (MHC), cellular immunity may originate from the processing and cross-presentation of foreign (mouse, virus, or non-self-human) sequences to CAR molecules. CAR peptides from apoptotic or necrotic CAR T cells can be displayed by antigen presenting cells and used to trigger T cell responses in secondary lymphoid organs (Figure a).
CAR-specific CD8+ T cells or cytotoxic CD4+ T cells  can eliminate CAR T cells that recognize and present CAR fragments through MHC-mediated. Humoral immunity can be triggered by CAR protein in apoptotic bodies presented by follicular dendritic cells to B cells. With the support of anti-CAR T cells, CAR-specific B cells can be expanded and then undergo class switching and plasma cell differentiation to produce different classes of immunoglobulins with different functions.
Anti-CAR antibodies may induce CAR T cell death through a variety of mechanisms, including antibody-dependent cytotoxicity, that is, the interaction between the CAR-binding antibody and the Fc receptor (FcR) domain of innate immune cells, such as natural killer (NK) Cells or macrophages cause cytotoxicity, or phagocytosis (Figure b) and complement-dependent cytotoxicity by releasing perforin and/or granzyme, which is due to CAR binding antibody to activate the complement cascade reaction, leading to the formation of a membrane attack complex And cell lysis (Figure c). After CAR is bound, anti-CAR IgE can also bind to the FcR of mast cells, thereby promoting degranulation and CAR T cell death (Figure d). Excessive release of multiple vasoactive mediators in mast cell granules may cause systemic allergic reactions, which can be fatal in one patient . KIR, killer immunoglobulin-like receptor.
The cellular immune response to transgenic cytotoxic T cells has been recorded in cancer patients and HIV patients, sometimes related to the immune rejection of adoptively transferred T cells [9,10,11]. In particular, T cells specific for CAR transgene or residual protein derived from gene transfer vectors have been shown to consume and inactivate injected CAR T cells . Similarly, the presence of humoral immunity and the development of antibodies against CAR constructs may interfere with CAR T cell activity by neutralizing antigen-binding fragments and/or promoting early CAR T cell apoptosis. After the application of CAR T cells, IgE-mediated HAMA triggers degranulation of mast cells and there is also a small risk of severe systemic allergic reactions. These patients use mouse-derived scFv and/or mouse-derived monoclonal antibodies to carry out CAR T cells. Presensitization [17,18] (Figure 1). However, the effect of CAR-specific immunogenicity on the clinical outcome after CAR T cell infusion is still poorly understood and there are few studies. Therefore, with the development of new CAR structures to improve efficacy and durability, understanding the origin and mechanism of CAR T cell immunogenicity remains critical, especially in patients with solid tumors. Here, we reviewed the published clinical data on the immune response of patients with hematological diseases or solid malignancies to CAR T cells, and discussed strategies for investigating, reducing and managing immunogenicity risks at different stages of CAR T cell product development.
Clinical evidence of anti-CAR immunity
Antibodies against CD19-directed CAR do not seem to impair the initial clinical response
Both tisagenlecleucel and axicabtagene ciloleucel [19,20,21,22] have observed pre-existing humoral immunity to CD19-specific CAR T cells. The vast majority of B-ALL patients (84.6%; n = 88) treated with tisagenlecleucel  have pre-existing anti-CAR antibodies, and the proportion reported in patients with refractory diffuse large B-cell lymphoma is similarly high (91.4 %).
The immunogenicity during treatment is defined as the increase in anti-CAR antibodies after infusion, and it has been reported that 5% and 36.7% of patients with diffuse large B-cell lymphoma and B-ALL, respectively [19,21]. According to reports, much fewer patients who received axicabtagene ciloleucel had pre-existing anti-CAR antibodies (3%; n=94) , while in patients who received brexucabtagene autoleucel, the combination of the two drugs did not Different tests to confirm the pre-existing anti-CAR antibody . All three products contain the same mouse-derived scFv (FMC63); therefore, the difference in the percentage of patients with pre-existing antibodies reported is notable and can reflect the different assays used for testing. Obviously, the presence of pre-existing antibodies or their increase after infusion of these products is not associated with a worse clinical response. This lack of clinical consequences has also been observed in other studies involving CAR T cells with mouse scFv targeting CD19 or CD20 [24,25].
Cell response after infusion of CD19-specific CAR may lead to treatment failure
Compared with pre-existing antibodies, the presence of CAR-specific cytolytic T cells after infusion is associated with treatment failures in some but not all clinical trials [9,10,26,27,28]. The first report on cellular immunity that may negate the therapeutic effect involved two patients with follicular lymphoma receiving multiple dose escalations of first-generation autologous CD19-directed CAR T cell infusions. These CAR T cells are produced through an inefficient manufacturing process, using T cell clones and integrated plasmid vectors, requiring extensive in vitro culture . 24 hours after the first infusion (108 cells/m²), cells were detectable in both patients, but they could not even last for 1 week. Despite the reinfusion at a higher dose and the use of IL-2 support, CAR T cells did not survive in the body. This failure may partly reflect the manufacturing method, but it may be exacerbated by the T cell-mediated anti-CAR response that exists before the first infusion and then expands after the administration. This reaction is directed against the hygromycin phosphotransferase-HSV1-thymidine kinase fusion (HyTK) suicide gene delivered by the CAR itself or co-delivered .
Similarly, T cell-mediated anti-CAR responses were also detected in subsequent experiments. These experiments involve the use of mouse-based scFv-based second-generation CD19-directed CAR T cells and, to a lesser extent, the use of all-human CAR construction Body [27,29]. It is worth noting that the enhanced lymphatic depletion of cyclophosphamide and fludarabine has been identified as a factor that may reduce the degree of anti-CAR cellular immunity [27,28]. Before the initial administration of CD19 targeting CAR T cells, a conditioning regimen containing these two drugs is currently considered the standard of care [30-32].
The success of reinfusion of CD19-specific CAR T cells may be limited by cellular immunity
Although patients with several hematological malignancies have a high rate of complete remission after the first infusion of CD19-directed CAR T cells after lymphocyte clearance, disease recurrence is still a problem. About 30-50% of patients develop disease within 12 months Relapse . According to experiments, antigen escape due to lack of CD19 expression is observed in 7-25% of patients . For those with recurrence of CD19-positive disease, repeated infusions of the initial CD19-targeted CAR T cell product seems attractive. However, the clinical response to a second or subsequent infusion is usually not ideal, and complete remission is usually seen in <25% of patients .
In addition, despite increased lymphatic depletion, the cytotoxic T cell population specific for CAR has been shown to expand in a subset of patients after the initial infusion. The detection of such cells is related to poor expansion of CAR T cell products after re-administration. To date, little is known about which specific epitopes in CAR constructs are recognized by anti-CAR cytotoxic T cells. The few existing studies have mostly found that T cells are specific to mouse scFv FMC63 used in several CD19-specific CARs [27,29], and to a lesser extent, to other parts of the transgene (such as signal peptide linker or hinge Domain) has specificity. 3 out of 19 patients who received CD19-specific CAR T cells)  (Supplementary Table 1).
Immunogenicity for hematological indications other than CD19 CAR T cells
Another promising CAR T cell candidate currently in clinical development targets plasma cell malignancies. Specifically, CAR T cells redirected to recognize B-cell maturation antigen (BCMA) induced effective anti-tumor responses in seven large clinical cohorts (as of July 2020) in patients with advanced multiple myeloma, albeit completely The remission rate is much lower than in B-ALL or NHL[36-38] patients, the response is not as durable as CD19 targeting CAR T cells. All trials used lymphatic removal (cyclophosphamide or fludarabine and cyclophosphamide) in most patients, and only one study provided data on CAR immunogenicity [36-42].
In a method recently described by Xu et al. , a bispecific CAR targets two different BCMA epitopes, using two camel-derived antigen binding domains combined in a single CAR construct to induce The highest complete remission rate reached 76% (13/17) of patients with multiple myeloma. Anti-CAR antibodies were detected in all patients whose disease relapsed after initial complete remission and were associated with a decrease in the number of circulating CAR T cells. Therefore, in patients receiving products that do not consume endogenous B cells, humoral immunity may be related to CAR T cell inactivation and treatment failure. In fact, the plasma cells consumed by BCMA-directed CAR T cells may not be enough to attenuate the early B cell responses that drive humoral immunity to CAR (Figure 2).
Several new CAR T cell methods are being developed for other indications, such as T cell malignancies (NCT03590574) . So far, the introduction of CAR constructs specific to pan-T cell lineage antigens has proven to be challenging because CAR T cells can kill each other, a phenomenon involving self-killing between CAR T cells [44,45 ]. This process will not only limit the effective manufacture of products, but also reduce the persistence of cells in the body.
An exception to these limitations is the development of anti-CD5 CAR T cells , in which a subset of CD5 specific CAR T cells down-regulates endogenous CD5 expression, thereby avoiding cannibalism and expanding clinical applications. Early reports from the ongoing phase I trial show that this CD5-targeted CAR T cell is effective for patients with T-cell acute lymphoblastic leukemia or T-cell lymphoma, without T-cell hypoplasia. The continuous depletion of endogenous T cells may theoretically hinder the adaptive immune response to tumors and pathogens, but it may also limit the degree of anti-CAR immunity, thereby increasing the persistence of CAR T cells.
Early trials of CAR T cell therapy in solid tumors reveal anti-CAR immunity
The use of CAR T cells to induce clinical responses in patients with advanced solid tumors has also proven to be challenging, in part due to poor T cell infiltration of the tumor and/or limited CAR T cell persistence7,8. In some cases, this Related to this, the anti-CAR immune response [48,49] (Figure 2). For example, Kershaw et al.  conducted a phase I trial to evaluate the safety of adoptive immunotherapy in patients with metastatic ovarian cancer using autologous first-generation CAR T cells targeting the α-folate receptor (FRα) Sex. These researchers observed that the antibody-mediated immune response to CAR T cells inhibited IFNγ release and cytotoxic activity against FRα-positive tumor cells. As observed in the experiment, this response greatly reduces the efficacy of CAR T cells and may lead to their rapid clearance.
Hege et al.  reported the results of the first two clinical trials (conducted in the 1990s) that evaluated CAR T cells in patients with solid tumors, with a focus on persistence and immunogenicity. These first-generation CAR T cell products contain humanized antibodies (huCC49) against tumor-associated glycoprotein (TAG-72), and the anti-CAR antibody against mouse-derived TAG-induced anti-idiotypes by forming the binding domain of huCC49 Immune response . Although most patients were repeatedly infused with high doses of CAR T cells (1×1010), this immune response resulted in the elimination of the infused TAG-72 CAR T cells within <14 weeks. A previous study using a fully mouse-derived CC49 variant showed that the antibody itself may have an antigen-binding epitope with particularly high immunogenic potential, leading to a HAMA response in most patients (54%) . Subsequent research led to the next generation of humanized huCC49 constructs with only the specificity determining residues of the mouse TAG-72 binding epitope transplanted to human antibodies. These early trials demonstrated the importance of minimizing mouse components when designing CAR T cell products for solid tumors.
Analyze the immunogenicity of next-generation CAR in solid tumors
Early studies involving first-generation CAR constructs helped to clarify the possibility of immunogenic response in solid tumor patients [16,48,49]. However, after the improvement in anti-tumor response was observed in preclinical models and patients with hematological malignancies, the limited efficacy of these CAR T cells prompted the switch to second-generation CAR. For example, Ahmed et al. [56,57] used autologous T cells to express a second-generation CAR based on the mouse HER2-specific monoclonal antibody FRP5. In one trial, FRP5-scFv HER2 extradomain-specific CAR T cells were used in 19 patients with advanced HER2-positive sarcoma. These researchers observed low but detectable levels of HER2-specific CAR transgene in peripheral blood for up to 2 years after repeated infusions. Similar CAR T cell dynamics were observed in another clinical study conducted in the same group involving patients with progressive glioblastoma. Although these and other trials have shown unsatisfactory clinical responses [56-59], the effects of cellular and humoral anti-CAR responses have not been studied in depth as a factor that may lead to treatment failure.
The above studies involved patients with solid tumors and did not include lymphatic removal before CAR T cell infusion. In contrast, in a case report published in July 2020, three cycles of lymphatic removal were followed by infusion of second-generation CAR T cells targeting HER2, allowing peripheral expansion and bioavailability of the infused cells. Degree can be improved . This method produced a complete response in children with metastatic rhabdomyosarcoma, which was then consolidated by repeated CAR T cell infusions without further lymphatic depletion, but with anti-PD-1 antibodies. The child’s longitudinal immune surveillance showed that the T cell pool was remodeled, with immunodominant clones and serum autoantibodies that were reactive to oncogenic pathway proteins. Despite the obvious strong endogenous immune response, the patient remained HAMA negative even after receiving a total of 15 infusions of CAR T cell products containing mouse scFv. It is difficult to draw a clear conclusion based on the observation of a single patient. Therefore, further research is needed to better clarify the anti-CAR immune response and its potential interaction with lymphatic depletion, especially if re-administration is required. Ultimately, the use of enhanced lymphatic removal to reduce immunogenicity may help improve CAR T cell engraftment in patients with solid tumors [61,62,63].
Reduce inherent CAR immunogenicity
Several components of the CAR construct may trigger an anti-CAR immune response in the patient. Various methods can be used to reduce the immunogenicity of these components.
Humanized tumor reactive scFv
Some researchers are developing and testing humanized scFv to circumvent the anti-CAR response associated with mouse-derived scFv. The humanized construct may be less immunogenic, and may be more suitable for the rescue treatment of patients with disease relapse after initial mouse-derived CAR T cell infusion (Figure 3) or as an option for patients not using CART cells [29,50,64,65,66] Compared with a similar cohort that received CAR T cells containing mouse-derived scFv29, the first treatment of B-cell NHL patients with a fully human CD19-directed CAR induced CAR specificity T cell response is less. Another strategy is to replace the traditional scFv with an immunoglobulin only heavy chain recognition domain that lacks light chains and potentially immunogenic linker sequences (such as heavy and light chains or other related connection points) . These heavy chain CAR constructs have shown strong target affinity and efficacy in preclinical models [68,69,70].
In November 2019, the first clinical efficacy demonstration of a BCMA-specific fully human heavy chain construct was reported . Theoretically, constructs derived only from human protein components can still initiate an immune response involving anti-idiotypic antibodies in patients [48,72]. Therefore, a head-to-head clinical trial that directly compares the immunogenicity of mouse-derived and humanized scFv is necessary.
Exchange scFv with alternative tumor-specific domains
In addition to scFv-based CARs, tumor cells can also be targeted through endogenous tumor-specific receptor-ligand interactions. The scFv portion of CAR can be replaced by the extracellular domain of the receptor or a ligand that binds to the receptor overexpressed by the tumor. The part currently under preclinical and early clinical evaluation includes the chimeric NKG2D receptor [73-75] membrane mooring IL-13 targeting tumor cells expressing IL-13Rα2, cytokine CARs76, 77, 78, and integrin αvβ6 binding peptide 79, 80 and regulatory protein-ζ Chimeras 81. The immunogenicity of this tumor-specific receptor-ligand interaction is expected to be lower than that of traditional scFv because they have human peptide sequences and therefore are likely to be recognized as self-proteins. In addition, a universal CAR design with an extracellular domain that can be loaded with tumor-targeting antibodies in vivo may be able to redirect CAR T cell specificity in the event of antigen escape 82,83. However, to date, there are no immunogenicity data on the use of this universal receptor strategy.
Variant CAR interval
Optimal binding of scFv to target epitopes is essential for effective CAR T cell therapy. In particular, proximal epitopes near the tumor cell membrane may require CAR T cells with longer and more flexible hinge domains. Many groups use the constant regions of various IgG heavy chains as hinges [84,85]. In their original form, the CH2 and CH3 domains in these IgG-derived spacers can be targeted by Fc receptors to innate immune cells, such as macrophages, granulocytes, and natural killer (NK) cells, and may cause immunity Original risk (Figure 1). In fact, the presence of the Fc binding spacer containing the CH2 domain reported by Jensen et al. may lead to poor persistence of CD19 targeting CAR T cells. And other clinical studies [9,25,86]. This phenomenon can be avoided by Fc receptor binding epitopes within the mutation interval, thereby preventing innate immune cell activation [87,88].
Immunogenicity of new CAR technology
Cytokine support technology aimed at enhancing the anti-tumor efficacy of CAR T cells, especially to overcome cytokine starvation in tumors with immunosuppressive microenvironment, is a broad field of preclinical and clinical research [7,89]. Systemic administration of cytokines (such as IL-15) increases the risk of immunogenic reactions in patients and may lead to toxic reactions, such as fever, chills, hypotension, thrombocytopenia, and lymphopenia . Therefore, other methods of using engineered CAR T cells to locally release soluble cytokines have been used, demonstrating effective activity in preclinical models [91,92,93]. Bystander T cell activation is a theoretical risk inherent in this paracrine cytokine support strategy [94,95]. The early clinical results of this approach are encouraging , although whether paracrine cytokine support may amplify any anti-CAR response remains to be elucidated. Alternatively, the constitutively active cytokine receptor or cytokine receptor signal domain in the CAR molecule can only provide immunomodulatory cytokine signals to CAR T cells without affecting bystander lymphocytes [97,98]. This strategy is studied in clinical trials (NCT03635632 and NCT04099797).
Suicide gene and elimination of markers
Although CAR T cells are effective for patients with certain hematological malignancies, they also pose safety risks due to unpredictable adverse effects of targeting, non-tumor toxicity, and/or additional technologies. Suicide genes and elimination markers have been studied as strategies that can be introduced together with CAR transgenes to specifically eliminate genetically modified cell products when serious adverse events occur [11,99,100]. However, these methods are not without the risk of immune complications. For example, Berger et al.  found multiple immunogenic epitopes in T cells expressing herpes simplex virus thymidine kinase (HSV-TK) as a suicide-inducing gene, resulting in CD4+ T cells and CD8+ T-mediated epitopes. Rapid development of anti-CAR T cell response cells. Therefore, as mentioned earlier, the HSV-TK suicide gene may also cause the CD19-directed CAR T cell rejection observed in the experiment conducted by Jensen et al. .
Safety systems adapted from human-derived proteins (such as surface binding elimination markers, including truncated EGFR or iCaspase 9 suicide system) avoid the introduction of xenogeneic components that may increase the risk of immunogenicity [100,101,102]. In order to implement safe genes in CAR T cell products, many researchers choose to co-express CAR constructs and suicide switches on a single polycistronic construct. These constructs have internal ribosomal entry sites or viruses containing self-cleaving peptides. 2A sequence separation. The virus-derived 2A sequence has not been shown to be immunogenic in in vitro studies , although only prospective clinical trials can clearly solve the problem of its immunogenicity in patients. It is worth noting that studies published in 2000 showed that the insertion of internal ribosome entry sites may reduce the expression level of downstream transgenes (such as suicide switches). Recently, gene editing has been used to create transgene-free suicide switches to avoid the risk of transgene-related immunogenicity, although this method currently requires further research .
Gene editing using transiently delivered nucleases
Programmable nucleases provide a way to enhance CAR T cell function through gene editing. This method has been used to disrupt the expression of endogenous immune checkpoint proteins, thereby improving the performance of CAR T cells; 106-108], and it is possible to make them co-administered with therapeutic monoclonal antibodies . In addition, due to the reduced expression of cell surface antigens, gene editing may limit the extent of cannibalism, allowing clinical studies on CART cells to detect common T cell antigens other than CD5 (such as CD3 and CD7) [44,45]. Importantly, gene editing can also be used to evade T cell-mediated anti-CAR immunity by eliminating MHC expression on the cell surface, as demonstrated by the use of allogeneic CAR T cells in vitro and in vivo [106,110,111] (Figure 4).
Figure 4 Strategies to avoid immune elimination of allogeneic CAR T cells. a|The presence of incompatible major histocompatibility complex (MHC) can enhance the anti-chimeric antigen receptor (CAR) response to allogeneic CAR T cells. Resting T cells express MHC class I (MHC I), and T cells can also up-regulate MHC class II (MHC II) after activation. b| MHC acts as an effective inhibitory receptor through the killer immunoglobulin-like receptor (KIR) on natural killer (NK) cells.
Eliminating MHC I from the surface of resting CAR T cells can prevent the same immune response and also increase the risk of NK cell-mediated cytotoxicity. c | Multiple gene editing can be used to eliminate the expression of MHC on CAR T cells while retaining the less diverse human leukocyte antigen (HLA)-C subtype, which may minimize the risk of NK cell rejection. Careful MHC matching should also be performed on donor-derived products. d | Disrupting the functional expression of CIITA, the master transcription regulator encoding MHC II or the common constant chain CD74, can be used to remove MHC II from the cell surface without causing the same immune response to activated CAR T cells.
Electronics | The allogeneic immune defense receptor (ADR) targeting the activation marker 4-1BB on allogeneic specific T cells may improve the persistence of allogeneic CAR T cells. The introduction of ADR of B2M-negative and MHC I-deficient CAR T cells may actively eliminate the risk of NK cell lysis. f | Site-specific insertion of universal MHC I constructs (such as singlet HLA-E constructs loaded with bait peptides) into B2M genes can prevent NK cells from targeting allogeneic-induced pluripotent stem cells. G | Alternatively, overexpression of CD47 prevents effective alloreactive T cell/NK cell activation and antibody-dependent cytotoxicity (ADCC) by acting as a “don’t eat me” signal.
In addition to accidental off-target gene editing events, the induction of host immune response combined with pre-existing humoral and cellular immunity has been identified as a risk factor associated with the CRISPR-Cas9 genome editing system [112-117] may lead to T cell-mediated Guide to eliminate cells expressing Cas9 [117,118]. Nevertheless, Stadtmauer et al.  demonstrated that the temporary delivery of CRISPR-Cas9 components as ribonucleoprotein complexes did not affect the persistence of autologous TCR redirected T cells in three patients. The rapid degradation of immunogenic proteins and the dilution during ex vivo amplification may explain these results. Therefore, in contrast to constitutive overexpression, transient delivery may be the preferred method for CAR T cell gene editing in cells with intact antigen processing and is worthy of further study.
Immune and allogeneic CAR T cells
Ready-made allogeneic CAR T cell products created using cells from healthy donors are an attractive alternative to autologous methods and have the potential to expand the clinical applicability of these cells and ensure timely availability . However, the non-self MHC expressed on these CAR T cells can trigger a host-derived immune response, leading to the elimination of immune-mediated products. Allogeneic CAR T cells can be targeted by the patient’s alloreactive T cells and have specificity for the main foreign MHC antigens [120,121]. Even donors that match some human leukocyte antigens (HLA) may generate additional immunogenic epitopes due to minor antigenic differences caused by polymorphisms .
In fact, data published in December 2020 (reference ) indicate that the short-term anti-tumor activity of allogeneic CD19-specific TCR requires chemotherapy and the anti-CD52 antibody alemtuzumab for pretreatment, alemtuzumab Consumption of B cells and T cells at the same time. Edit T cells in adults and children with refractory B-ALL. Importantly, alemtuzumab also reduces CD52-expressing CAR T cells after infusion; therefore, CD52 was deleted from the CAR T cells used in this experiment. Although alemtuzumab was added to lymphatic depletion, allogeneic CAR T cells showed limited persistence after reconstitution of endogenous T cells . These results emphasize the need to adopt strategies to overcome alloreactive immune disorders caused by even minor HLA mismatches.
Various methods have been used to generate CAR T cells with reduced MHC antigen expression so that patients can be treated regardless of HLA differences (Figure 4). Elimination of MHC class I (MHC I) by directly editing the HLA locus or disrupting the gene encoding β2-microglobulin (B2M) required for the expression of MHC I may prevent the allogeneic rejection of cells caused by CD8+ T[106,110] . However, due to the upregulation of MHCII due to T cell activation, CAR T cell rejection mediated by allogeneic specific CD4+ T cells may also occur. By deleting CIITA (the main transcription factor of MHC II gene) to eliminate MHC II expression, this rejection reaction can be alleviated .
These methods offer some hope for combating T cell-mediated rejection; however, paradoxically, the loss of HLA molecules makes allogeneic CAR T cells vulnerable to NK cell killing. Therefore, NK cell inhibitory transgenes have been developed to promote the persistence of transgenic cells in immunocompetent hosts (Figure 4). For example, Gornalusse et al.  developed non-polymorphic HLA-E molecules fused with B2M and bait antigens in human pluripotent stem cells to develop cells that are resistant to rejection by CD8+ T cells and NK cells. For example, other strategies involving multiple gene editing can be used to eliminate the highly polymorphic HLA-A and HLA-B alleles, while retaining the less diverse HLA-C, thereby preserving important endogenous NK cell inhibitory effects. Body . Alternatively, the overexpression of CD47 can inhibit the activation of alloreactive T cells and NK cells, and provide a “don’t eat me” signal to phagocytes, thereby preventing antibody-dependent cytotoxicity [126,127].
In July 2020, Mo et al.  published a report describing a “allogeneic immune defense receptor” that can realize CAR T by recognizing the activation marker 4-1BB in preclinical models Cell-mediated killing of alloreactive T cells and NK cells without the need for genetic modification of the HLA system. Interestingly, the allogeneic immune defense receptor expressed by engineered immune cells does not impair the expansion of allogeneic CAR T cells, nor does it impair the anti-tumor immune response in the mouse model .
Extensive lymphocyte clearance can also reduce the risk of immediate allogeneic rejection129,130. The disadvantage of this method is the possibility of graft-versus-host disease (GvHD) caused by the proliferation of persistent allogeneic CAR T cells or endogenous non-CAR donor T cells that retain the expression of alloreactive TCR. In order to reduce the risk of GvHD, researchers have transferred CAR transgenes into virus-specific T cells to produce safer products because the TCR of such cells is unlikely to be alloreactive [57,131,132,133,134].
Alternatively, CAR can be introduced into unconventional T cells with a restricted TCR pool (such as γδ T cells, mucosal-associated invariant T cells, NK T cells, and invariant NK T cells), because these have a smaller risk of GvHD [135,136,137 ]. As an alternative, gene editing can be used to destroy the TCR of allogeneic CAR T cells.
Anti-CAR immune response in clinical practice
Longitudinal studies of anti-CAR immune responses are particularly important to understand how immunogenicity can lead to treatment failure. The current professional association guidelines on the use of CD19-directed CAR T cells do not include recommendations for the evaluation or clinical management of suspected anti-CAR immunity [32,138,139,140]. In this section, we outline the need to develop validated detection methods to adequately monitor the immunogenicity of CAR T cell candidates and enable researchers to assess the clinical relevance of these responses, and where appropriate Implement appropriate evidence-based mitigation and management strategies (Figure 1).
Figure 5 | Monitoring, mitigation and management of anti-CAR immunity in the clinic. Prior to CAR T cell infusion, reducing anti-CAR immunity through a lymphatic clearance protocol can reduce the number of circulating lymphocytes and antigen-presenting cells. In addition, cytoreductive therapy may reduce the number of certain immunosuppressive cells in the tumor microenvironment, leading to tumor cell death and creating a pro-inflammatory environment, thereby promoting the anti-tumor efficacy of CAR T cells . Once patients are considered for treatment, monitoring of patients receiving CAR T cells should begin. Traditional cancer treatment options, including mouse-derived monoclonal antibodies 155, can induce human anti-mouse antibodies (HAMA) in patients [12,13].
Therefore, careful assessment of pre-existing immunity is important to avoid allergic reactions after CAR T cell infusion. Subsequently, monitoring in clinical trials should include monitoring of possible HAMA and specific anti-CAR immune responses, including antibody and T cell analysis. Before re-administration with the same CAR T cell product, repeated lymphocyte depletion can be used to reduce anti-CAR immunity, thereby realizing the management of low-level anti-CAR immunity. For patients who have progressed or relapsed after previous infusion of CAR T cells containing mouse single-chain variable fragments (scFvs), or who require subsequent infusions to prevent disease progression, humanized CAR T cell constructs can be considered . However, this method may incur additional costs and/or treatment delays.
Monitoring anti-CAR immunity in clinical trials and clinical practice
Observation of fatal allergic reactions caused by HAMA after infusion of mesothelin-specific CAR T cells17 may prompt regulatory agencies to recommend that immunogenicity risks be addressed in a phase I safety trial if immune reactions are suspected (Figure 5) . Various detection methods have been designed in various clinical trials to test the humoral or cellular immune response to CAR T cells (Box 1). However, with the exception of HAMA, these tests are rarely certified according to the clinical laboratory improvement amendments and are not currently in widespread use.
At the same time, by definition, HAMA is only valuable for detecting scFv antibodies derived from mice. In particular, HAMA assessment can be performed prior to subsequent infusion of mouse-derived CAR T cells, which may help to elucidate the possible mechanisms of primary drug resistance and/or limited response to a second or subsequent infusion . With the humanization of new constructs, HAMA assessment may become less relevant and new detection methods developed, whether for humanized constructs or potentially for constructs of non-mouse, non-human origin, Will become a growing demand. Before such accompanying tests are available, researchers may consider freezing serum samples and peripheral blood mononuclear cells collected at multiple time points for later analysis.
Although there are reports of effective immune responses to non-human CAR sequences and mutated oncoproteins, few clinical studies involving patients with solid tumors have investigated anti-CAR antibody responses other than HAMA49,60. Given that CAR T cell expansion and persistence are still not ideal in solid tumor trials, the role of the immune response to the CAR construct used has become increasingly important and should be re-examined. Therefore, it is important to establish validation tests that characterize the humoral and cellular immune responses to CAR constructs and related transgenes.
The test designed to detect anti-CAR antibodies can be based on cells or enzyme-linked immunosorbent assay (ELISA). Ideally, it should be able to measure the immunogenicity of all CAR components expressed on the surface of T cells and predict their effects on cellular products (such as cytotoxicity). Potential)  (Box 1). For cellular immune responses, analysis that can map the most immunogenic epitopes in CAR constructs is most likely to provide information for better CAR design and the development of new technologies to improve efficacy. Overall, these efforts can also benefit from the implementation of generic terminology and lessons learned from the immunogenicity assessment and reporting of biological agents (such as therapeutic proteins) [143,144,145,146].
Box 1 aims to monitor the detection of anti-CAR immunity
The cytotoxicity assay uses irradiated CAR-expressing targets to measure the effects of T cells expanded in vitro and/or monocytes in peripheral blood samples on target cells transduced by chimeric antigen receptor (CAR) (including T cells). Cells and autologous lymphocyte lines). This type of test usually uses 51Cr labeling to quantify target cell death after 4-6 hours of co-cultivation 10, 24, 27, 99.
Degranulation analysis measures the surface expression of CD107a (also known as Lamp1), which indicates that cytolytic vesicles are secreted on patient-derived T cells that have recently been expanded in vitro. These T cells are derived from peripheral blood samples, using flow cytometry and transradiation. A total of 10,156 CAR-expressing T cells were cultured.
In vitro proliferation test
In vitro proliferation assays are usually assessed by 3H-thymidine incorporation or flow cytometry (dilution of proliferation dyes, such as carboxyfluorescein diacetate succinimide), after stimulation with irradiated CAR-expressing target cells or CAR-peptide mixtures Then, the proliferation of patient-derived T cells in vitro was measured. Esters or similar) 26.
Enzyme-linked immunosorbent spot (ELISpot) detection
ELISpot analysis detects IFNγ released by CAR-specific T cells derived from patients expanded in vitro. These cells are usually derived from peripheral blood mononuclear cells and then expanded in the presence of irradiated CAR-expressing T cells and IL-2 for 2 weeks. After co-cultivation, these cells are exposed to irradiated CAR-expressing target cells or CAR-derived peptide libraries. The advantage of this method is that it can identify different protein fragments recognized by anti-CAR T cells28,29,103.
Intracellular cytokine staining and flow cytometry
Intracellular cytokine staining and flow cytometry involve the detection of IFNγ or other cytokine-producing T cells in peripheral blood mononuclear cells after stimulation with mouse-derived CAR sequence spanning peptides (or potentially non-mouse CAR peptides). This method uses fluorescent dye-labeled antibodies for intracellular staining, usually including but not limited to extracellular staining of IFNγ, TNF and/or IL-2 and/or activation markers (such as CD137 and CD40 ligands), and then use flow Cytometry for quantification19,21.
T cell receptor (TCR) profiling
TCR spectroscopy involves next-generation sequencing or TCR Vβ spectrotyping using flow cytometry. This method can identify expanded T cell clones in vivo based on individual TCR complementarity defined regions or TCR Vβ usage. If TCR library analysis is performed before infusion, this type of analysis can be used to detect infused clones.
Humoral anti-CAR immunity
Enzyme-linked immunosorbent assay (ELISA)
ELISA is a standardized test that can perform semi-quantitative analysis of human anti-mouse antibody (HAMA)17. To this end, patient serum is added to the wells coated with mouse immunoglobulin. After washing, the human immunoglobulin bound to mouse immunoglobulin is visualized by staining with a secondary antibody. Anti-human IgG is usually linked to a reporter enzyme (such as horseradish peroxidase). Subsequently, the enzyme-mediated luminescence or color change in the solution is monitored for quantification. Standards run in the same test can estimate HAMA levels.
Cell-based flow cytometry
Cell-based flow cytometry involves incubating CAR-expressing target cells (such as Jurkat T cells or Chinese Hamster Ovary (CHO) cells) with patient serum samples, followed by washing and secondary staining to obtain anti-human IgG. Additional steps are required to remove the HAMA signal to identify specific anti-CAR/idiotypic antibodies that are not related to HAMA9,40,86,142.
ELISA anti-drug antibody bridging test
The ELISA anti-drug antibody bridging test uses two soluble CAR proteins, one is bound to the surface of blood vessels, and the other is linked to reporter genes such as alkaline phosphatase or horseradish peroxidase. The complex is only fixed during the wash phase in the presence of anti-CAR antibodies. An additional step is required to remove the HAMA signal. This method cannot recognize CAR epitopes in a specific conformation, which requires anchoring the entire CAR to the cell membrane [10,17,31,157].
Reduce immunogenicity by enhancing lymphocyte clearance
The lymphatic depletion conditioning protocol performed before CAR T cell therapy is expected to affect not only the lymphatic compartments (including B cells and T cells), but also bone marrow cells. When CAR T cells expand in the circulation and lymphatic tissues, temporarily removing dedicated antigen-presenting cells can reduce the possibility of antigen cross-presentation. It is well known that cyclophosphamide-based chemotherapy will deplete regulatory T cells, otherwise it will limit the anti-tumor efficacy and durability of CAR T cells. Most researchers usually use fludarabine/cyclophosphamide-based lymphatic ablation to improve treatment outcomes and overcome the possibility of immune rejection due to initial CAR T cell infusion [27,28,32]. Enhancing the program is also being considered for subsequent infusions. Interestingly, Gauthier et al.  demonstrated that prior to the first CAR infusion, enhanced lymphatic depletion (using fludarabine plus cyclophosphamide compared with cyclophosphamide alone) compared with the second The next higher dose of CD19 is related to the improved response of CAR T cells.
Use humanized CAR and other structures to manage recurrent disease
In order to avoid the premature inactivation of CAR T cells due to the immune response to mouse-derived scFv, humanized scFv can be considered as an alternative to CAR T cell products. Early studies involving humanized CD19-targeted CAR T cell constructs reported varying clinical response rates, including patients who had progressed or relapsed after previous infusion of CAR T cells containing mouse scFv. For example, Maude et al. [64,148] infused another CD19-specific CAR T cell product (CTL119 , Including the humanized form of FMC63). In this phase I study, 9 of 16 patients (56%) had a complete remission at 1 month. However, in the same trial, 22/22 CAR T-cell initial disease patients received CTL119 CAR T-cell products in complete remission at this time point . Whether the relatively low rate of complete remission in patients receiving reinfusion reflects an immune response to an epitope shared between the mouse and the humanized CAR transgene, or is caused by other resistance mechanisms, remains unknown [29, 64,65,66,149].
Targeting replacement antigens is another approach that may circumvent the anti-CAR T cell response if it is attributed to a specific epitope within a given scFv or another specific part of the CAR construct. Among these strategies, CD22-directed CAR T cells are currently the most mature alternative to B-cell malignancies. So far, CAR T cells targeting CD22 containing fully human scFv have shown a high degree of efficacy in the treatment of patients with disease relapse using mouse scFv anti-CD19 CAR T cells and are equally effective regardless of CD19 expression [150,151,152].
CAR T cells are currently an important therapy for patients with certain relapsed and/or refractory hematological malignancies. Despite the strong level of efficacy reported, the anti-CAR immune response is associated with treatment failure, although direct causality has not been established in most studies. Nevertheless, depletion of endogenous B cells through direct CAR targeting or lymphocyte clearance can reduce the risk of immune response to CAR T cells. However, CAR T cells targeting non-B-cell malignancies (such as solid tumors) can induce T-cell and B-cell responses, thereby increasing the risk of immunogenicity (Figure 2). This problem increases the obstacles to designing effective CAR immunotherapy for solid tumors, and it is necessary to strike a balance between inducing effective endogenous immunity to cancer cells and the response to genetically modified CAR T cells.
The development of technologies to improve the safety and/or functionality of CAR T cells, such as suicide genes or cytokine self-support, may also bring new immunogenic risks. In addition, the emergence and development of CAR T cells derived from allogeneic donors has aroused people’s attention to the issue of immunogenicity. In fact, the potential success of “universal” off-the-shelf CAR T cells deserves to include strategies aimed at avoiding immune recognition, preventing allogeneic immunity, and/or reducing the risk of immunogenic reactions. CAR constructs with humanized scFv and/or other less immunogenic components may also reduce the risk of anti-CAR immunity. In the clinic, enhanced lymphatic depletion is still essential for reducing or regulating the anti-CAR immune response and promoting better CAR T cell persistence.
While working to reduce the incidence and/or severity of anti-CAR immune responses, the immunogenicity of patients receiving CAR T cells in clinical trials should be appropriately monitored. As we report, in less than one-third of CAR T cell trials, there are available data to study anti-CAR immunity (Supplementary Table 1). In addition, many of the tests used to date (Box 1) lack standardization and validation, thus limiting the strength of correlation and performance comparison. We strongly advocate the inclusion of CAR-related immunogenicity analysis in preclinical and clinical studies in order to study the immunogenic response and its impact in detail.
The wider use of CAR T cells supports the need to monitor anti-CAR immunity and requires vigilance against possible CAR-related immunogenicity to ensure the safety and effectiveness of CAR T cells in cancer patients. This knowledge will increase our understanding of the clinical relevance of immunogenicity and provide information for the development of CAR T cell strategies in the future.
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