Anti-CD30 CAR T cells as consolidation after autologous haematopoietic stem-cell transplantation in patients with high-risk CD30+ lymphoma: a phase 1 study.

BACKGROUND: Chimeric antigen receptor (CAR) T cells targeting CD30 are safe and have promising activity when preceded by lymphodepleting chemotherapy. We aimed to determine the safety of anti-CD30 CAR T cells as consolidation after autologous haematopoietic stem-cell transplantation (HSCT) in patients with CD30+ lymphoma at high risk of relapse. METHODS: This phase 1 dose-escalation study was performed at two sites in the USA. Patients aged 3 years and older, with classical Hodgkin lymphoma or non-Hodgkin lymphoma with CD30+ disease documented by immunohistochemistry, and a Karnofsky performance score of more than 60% planned for autologous HSCT were eligible if they were considered high risk for relapse as defined by primary refractory disease or relapse within 12 months of initial therapy or extranodal involvement at the start of pre-transplantation salvage therapy. Patients received a single infusion of CAR T cells (2 × 107 CAR T cells per m2, 1 × 108 CAR T cells per m2, or 2 × 108 CAR T cells per m2) as consolidation after trilineage haematopoietic engraftment (defined as absolute neutrophil count ≥500 cells per µL for 3 days, platelet count ≥25 × 109 platelets per L without transfusion for 5 days, and haemoglobin ≥8 g/dL without transfusion for 5 days) following carmustine, etoposide, cytarabine, and melphalan (BEAM) and HSCT. The primary endpoint was the determination of the maximum tolerated dose, which was based on the rate of dose-limiting toxicity in patients who received CAR T-cell infusion. This study is registered with ClinicalTrials.gov (NCT02663297) and enrolment is complete. FINDINGS: Between June 7, 2016, and Nov 30, 2020, 21 patients were enrolled and 18 patients (11 with Hodgkin lymphoma, six with T-cell lymphoma, one with grey zone lymphoma) were infused with anti-CD30 CAR T cells at a median of 22 days (range 16-44) after autologous HSCT. There were no dose-limiting toxicities observed, so the highest dose tested, 2 × 108 CAR T cells per m2, was determined to be the maximum tolerated dose. One patient had grade 1 cytokine release syndrome. The most common grade 3-4 adverse events were lymphopenia (two [11%] of 18) and leukopenia (two [11%] of 18). There were no treatment-related deaths. Two patients developed secondary malignancies approximately 2 years and 2·5 years following treatment (one stage 4 non-small cell lung cancer and one testicular cancer), but these were judged unrelated to treatment. At a median follow-up of 48·2 months (IQR 27·5-60·7) post-infusion, the median progression-free survival for all treated patients (n=18) was 32·3 months (95% CI 4·6 months to not estimable) and the median progression-free survival for treated patients with Hodgkin lymphoma (n=11) has not been reached. The median overall survival for all treated patients has not been reached. INTERPRETATION: Anti-CD30 CAR T-cell infusion as consolidation after BEAM and autologous HSCT is safe, with low rates of toxicity and encouraging preliminary activity in patients with Hodgkin lymphoma at high risk of relapse, highlighting the need for larger studies to confirm these findings. FUNDING: National Heart Lung and Blood Institute, University Cancer Research Fund at the Lineberger Comprehensive Cancer Center.

IL-12 reprograms CAR-expressing natural killer T cells to long-lived Th1-polarized cells with potent antitumor activity.

Human natural killer T cells (NKTs) are innate-like T lymphocytes increasingly used for cancer immunotherapy. Here we show that human NKTs expressing the pro-inflammatory cytokine interleukin-12 (IL-12) undergo extensive and sustained molecular and functional reprogramming. Specifically, IL-12 instructs and maintains a Th1-polarization program in NKTs in vivo without causing their functional exhaustion. Furthermore, using CD62L as a marker of memory cells in human NKTs, we observe that IL-12 maintains long-term CD62L-expressing memory NKTs in vivo. Notably, IL-12 initiates a de novo programming of memory NKTs in CD62L-negative NKTs indicating that human NKTs circulating in the peripheral blood possess an intrinsic differentiation hierarchy, and that IL-12 plays a role in promoting their differentiation to long-lived Th1-polarized memory cells. Human NKTs engineered to co-express a Chimeric Antigen Receptor (CAR) coupled with the expression of IL-12 show enhanced antitumor activity in leukemia and neuroblastoma tumor models, persist long-term in vivo and conserve the molecular signature driven by the IL-12 expression. Thus IL-12 reveals an intrinsic plasticity of peripheral human NKTs that may play a crucial role in the development of cell therapeutics.

ALK inhibitors increase ALK expression and sensitize neuroblastoma cells to ALK.CAR-T cells.

Selection of the best tumor antigen is critical for the therapeutic success of chimeric antigen receptor (CAR) T cells in hematologic malignancies and solid tumors. The anaplastic lymphoma kinase (ALK) receptor is expressed by most neuroblastomas while virtually absent in most normal tissues. ALK is an oncogenic driver in neuroblastoma and ALK inhibitors show promising clinical activity. Here, we describe the development of ALK.CAR-T cells that show potent efficacy in monotherapy against neuroblastoma with high ALK expression without toxicity. For neuroblastoma with low ALK expression, combination with ALK inhibitors specifically potentiates ALK.CAR-T cells but not GD2.CAR-T cells. Mechanistically, ALK inhibitors impair tumor growth and upregulate the expression of ALK, thereby facilitating the activity of ALK.CAR-T cells against neuroblastoma. Thus, while neither ALK inhibitors nor ALK.CAR-T cells will likely be sufficient as monotherapy in neuroblastoma with low ALK density, their combination specifically enhances therapeutic efficacy.

Building safety into CAR-T therapy.

Chimeric antigen receptor T cell (CAR-T) therapy is an innovative immunotherapeutic approach that utilizes genetically modified T-cells to eliminate cancer cells using the specificity of a monoclonal antibody (mAb) coupled to the potent cytotoxicity of the T-lymphocyte. CAR-T therapy has yielded significant improvements in relapsed/refractory B-cell malignancies. Given these successes, CAR-T has quickly spread to other hematologic malignancies and is being increasingly explored in solid tumors. From early clinical applications to present day, CAR-T cell therapy has been accompanied by significant toxicities, namely cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), and on-target off-tumor (OTOT) effects. While medical management has improved for CRS and ICANS, the ongoing threat of refractory symptoms and unanticipated idiosyncratic toxicities highlights the need for more powerful safety measures. This is particularly poignant as CAR T-cell therapy continues to expand into the solid tumor space, where the risk of unpredictable toxicities remains high. We will review CAR-T as an immunotherapeutic approach including emergence of unique toxicities throughout development. We will discuss known and novel strategies to mitigate these toxicities; additional safety challenges in the treatment of solid tumors, and how the inducible Caspase 9 "safety switch" provides an ideal platform for continued exploration.

Patient-reported outcomes in CD30-directed CAR-T cells against relapsed/refractory CD30+ lymphomas.

Chimeric antigen receptor (CAR)-T cells targeting CD30 have demonstrated high response rates with durable remissions observed in a subset of patients with relapsed/refractory CD30+ hematologic malignancies, particularly classical Hodgkin lymphoma. This therapy has low rates of toxicity including cytokine release syndrome with no neurotoxicity observed in our phase 2 study. We collected patient-reported outcomes (PROs) on patients treated with CD30 directed CAR-T cells to evaluate the impact of this therapy on their symptom experience. We collected PROs including PROMIS (Patient-Reported Outcomes Measurement Information System) Global Health and Physical Function questionnaires and selected symptom questions from the NCI PRO-CTCAE in patients enrolled on our clinical trial of CD30-directed CAR-T cells at procurement, at time of CAR-T cell infusion, and at various time points post treatment. We compared PROMIS scores and overall symptom burden between pre-procurement, time of infusion, and at 4 weeks post infusion. At least one PRO measurement during the study period was found in 23 out of the 28 enrolled patients. Patient overall symptom burden, global health and mental health, and physical function were at or above baseline levels at 4 weeks post CAR-T cell infusion. In addition, PROMIS scores for patients who participated in the clinical trial were similar to the average healthy population. CD30 CAR-T cell therapy has a favorable toxicity profile with patient physical function and symptom burden recovering to at least their baseline pretreatment health by 1 month post infusion. Trial registration number: NCT02690545.

Co-transducing B7H3 CAR-NK cells with the DNR preserves their cytolytic function against GBM in the presence of exogenous TGF-ß.

Cord blood (CB)-derived natural killer (NK) cells that are genetically engineered to express a chimeric antigen receptor (CAR) are an attractive off-the-shelf therapy for the treatment of cancer, demonstrating a robust safety profile in vivo. For poor prognosis brain tumors such as glioblastoma multiforme (GBM), novel therapies are urgently needed. Although CAR-T cells demonstrate efficacy in preclinical GBM models, an off-the-shelf product may exhibit unwanted side effects like graft-versus-host disease. Hence, we developed an off-the-shelf CAR-NK cell approach using a B7H3 CAR and showed that CAR-transduced NK cells have robust cytolytic activity against GBM cells in vitro. However, transforming growth factor (TGF)-ß within the tumor microenvironment has devastating effects on the cytolytic activity of both unmodified and CAR-transduced NK cells. To overcome this potent immune suppression, we demonstrated that co-transducing NK cells with a B7H3 CAR and a TGF-ß dominant negative receptor (DNR) preserves cytolytic function in the presence of exogenous TGF-ß. This study demonstrates that a novel DNR and CAR co-expression strategy may be a promising therapeutic for recalcitrant CNS tumors like GBM.

Inadvertent Transfer of Murine VL30 Retrotransposons to CAR-T Cells.

For more than a decade, genetically engineered autologous T-cells have been successfully employed as immunotherapy drugs for patients with incurable blood cancers. The active components in some of these game-changing medicines are autologous T-cells that express viral vector-delivered chimeric antigen receptors (CARs), which specifically target proteins that are preferentially expressed on cancer cells. Some of these therapeutic CAR expressing T-cells (CAR-Ts) are engineered via transduction with γ-retroviral vectors (γ-RVVs) produced in a stable producer cell line that was derived from murine PG13 packaging cells (ATCC CRL-10686). Earlier studies reported on the copackaging of murine virus-like 30S RNA (VL30) genomes with γ-retroviral vectors generated in murine stable packaging cells. In an earlier study, VL30 mRNA was found to enhance the metastatic potential of human melanoma cells. These findings raise biosafety concerns regarding the possibility that therapeutic CAR-Ts have been inadvertently contaminated with potentially oncogenic VL30 retrotransposons. In this study, we demonstrated the presence of infectious VL30 particles in PG13 cell-conditioned media and observed the ability of these particles to deliver transcriptionally active VL30 genomes to human cells. Notably, VL30 genomes packaged by HIV-1-based vector particles transduced naïve human cells in culture. Furthermore, we detected the transfer and expression of VL30 genomes in clinical-grade CAR-T cells generated by transduction with PG13 cell-derived γ-retroviral vectors. Our findings raise biosafety concerns regarding the use of murine packaging cell lines in ongoing clinical applications.

Targeting brain lesions of non-small cell lung cancer by enhancing CCL2-mediated CAR-T cell migration.

Metastatic non-small cell lung cancer (NSCLC) remains largely incurable and the prognosis is extremely poor once it spreads to the brain. In particular, in patients with brain metastases, the blood brain barrier (BBB) remains a significant obstacle for the biodistribution of antitumor drugs and immune cells. Here we report that chimeric antigen receptor (CAR) T cells targeting B7-H3 (B7-H3.CAR) exhibit antitumor activity in vitro against tumor cell lines and lung cancer organoids, and in vivo in xenotransplant models of orthotopic and metastatic NSCLC. The co-expression of the CCL2 receptor CCR2b in B7-H3.CAR-T cells, significantly improves their capability of passing the BBB, providing enhanced antitumor activity against brain tumor lesions. These findings indicate that leveraging T-cell chemotaxis through CCR2b co-expression represents a strategy to improve the efficacy of adoptive T-cell therapies in patients with solid tumors presenting with brain metastases.

The Emerging Role of CAR T Cell Therapy in Relapsed/Refractory Hodgkin Lymphoma.

Treatment for Hodgkin lymphoma (HL) has evolved considerably from the time it was originally described in the 19th century with many patients now being cured with frontline therapy. Despite these advances, upwards of 10% of patients experience progressive disease after initial therapy with an even higher percentage relapsing. Until recently there had been limited therapeutic options for relapsed and/or refractory HL outside of highly intensive chemotherapy with stem cell rescue. Improved understanding of the pathophysiology of HL, coupled with the emergence of more targeted therapeutics, has reshaped how we view the treatment of relapsed/refractory HL and its prognosis. With this, there has been an increased focus on immunotherapies that can reprogram the immune system to better overcome the immunosuppressive milieu found in HL for improved cancer cell killing. In particular, chimeric antigen receptor (CAR) T cells are emerging as a valuable therapeutic tool in this area. Building on the success of antibody-drug conjugates directed against CD30, CAR T cells engineered to recognize the same antigen are now reaching patients. Though still in its infancy, CAR T therapy for relapsed/refractory HL has shown exceptional promise in early-stage clinical trials with the potential for durable responses even in patients who had progressed through multiple lines of prior therapy. Here we will review currently available data on the use of CAR T cells in HL, strategies to optimize their effectiveness, and how this therapy may fit into the treatment paradigm of HL going forward.

Targeting disialoganglioside GD2 with chimeric antigen receptor-redirected T cells in lung cancer.

BACKGROUND: We explored whether the disialoganglioside GD2 (GD2) is expressed in small cell lung cancer (SCLC) and non-SCLC (NSCLC) and can be targeted by GD2-specific chimeric antigen receptor (CAR) T cells. METHODS: GD2 expression was evaluated in tumor cell lines and tumor biopsies by flow cytometry and immunohistochemistry. We used a GD2.CAR that coexpress the IL-15 to promote T-cell proliferation and persistence, and the inducible caspase 9 gene safety switch to ablate GD2.CAR-T cells in case of unforeseen toxicity. The antitumor activity of GD2.CAR-T cells was evaluated using in vitro cocultures and in xenograft models of orthotopic and metastatic tumors. The modulation of the GD2 expression in tumor cell lines in response to an epigenetic drug was also evaluated. RESULTS: GD2 was expressed on the cell surface of four of fifteen SCLC and NSCLC cell lines (26.7%) tested by flow cytometry, and in 39% of SCLC, 72% of lung adenocarcinoma and 56% of squamous cell carcinoma analyzed by immunohistochemistry. GD2 expression by flow cytometry was also found on the cell surface of tumor cells freshly isolated from tumor biopsies. GD2.CAR-T cells exhibited antigen-dependent cytotoxicity in vitro and in vivo in xenograft models of GD2-expressing lung tumors. Finally, to explore the applicability of this approach to antigen low expressing tumors, we showed that pretreatment of GD2low/neg lung cancer cell lines with the Enhancer of zeste homolog 2 inhibitor tazemetostat upregulated GD2 expression at sufficient levels to trigger GD2.CAR-T cell cytotoxic activity. CONCLUSIONS: GD2 is a promising target for CAR-T cell therapy in lung cancer. Tazemetostat treatment could be used to upregulate GD2 expression in tumor cells, enhancing their susceptibility to CAR-T cell targeting.

Pretherapy metabolic tumor volume is associated with response to CD30 CAR T cells in Hodgkin lymphoma.

Our group has recently demonstrated that chimeric antigen receptor T-cell therapy targeting the CD30 antigen (CD30.CAR-T) is highly effective in patients with relapsed and refractory (r/r) classical Hodgkin lymphoma (cHL). Despite high rates of clinical response, relapses and progression were observed in a subset of patients. The objective of this study was to characterize clinical and correlative factors associated with progression-free survival (PFS) after CD30.CAR-T cell therapy. We evaluated correlatives in 27 patients with r/r cHL treated with lymphodepletion and CD30.CAR-T cells. With a median follow-up of 9.5 months, 17 patients (63%) progressed, with a median PFS of 352 days (95% confidence interval: 116-not reached), and 2 patients died (7%) with a median overall survival of not reached. High metabolic tumor volume (MTV, >60 mL) immediately before lymphodepletion and CD30.CAR-T cell infusion was associated with inferior PFS (log rank, P = .02), which persisted after adjusting for lymphodepletion and CAR-T dose (log rank, P = .01 and P = .006, respectively). In contrast, receiving bridging therapy, response to bridging therapy, CD30.CAR-T expansion/persistence, and percentage of CD3+PD-1+ lymphocytes over the first 6 weeks of therapy were not associated with differences in PFS. In summary, this study reports an association between high baseline MTV immediately before lymphodepletion and CD30.CAR-T cell infusion and worse PFS in patients with r/r cHL. This trial was registered at www.clinicaltrials.gov as #NCT02690545.

Dual Targeting CAR-T Cells with Optimal Costimulation and Metabolic Fitness enhance Antitumor Activity and Prevent Escape in Solid Tumors.

Chimeric antigen receptor (CAR) T cells showed great activity in hematologic malignancies. However, heterogeneous antigen expression in tumor cells and suboptimal CAR-T cell persistence remain critical aspects to achieve clinical responses in patients with solid tumors. Here we show that CAR-T cells targeting simultaneously two tumor-associated antigens and providing transacting CD28 and 4-1BB costimulation, while sharing the sane CD3ζ-chain cause rapid antitumor effects in in vivo stress conditions, protection from tumor re-challenge and prevention of tumor escape due to low antigen density. Molecular and signaling studies indicate that T cells engineered with the proposed CAR design demonstrate sustained phosphorylation of T cell receptor-associated (TCR) signaling molecules and a molecular signature supporting CAR-T cell proliferation and long-term survival. Furthermore, metabolic profiling of CAR-T cells displayed induction of glycolysis that sustains rapid effector T cell function, but also preservation of oxidative functions, which are critical for T cell long-term persistence.

Fibrin gel enhances the antitumor effects of chimeric antigen receptor T cells in glioblastoma.

Regional delivery of chimeric antigen receptor (CAR) T cells in glioblastoma represents a rational therapeutic approach as an alternative to intravenous administration to avoid the blood-brain barrier impediment. Here, we developed a fibrin gel that accommodates CAR-T cell loading and promotes their gradual release. Using a model of subtotal glioblastoma resection, we demonstrated that the fibrin-based gel delivery of CAR-T cells within the surgical cavity enables superior antitumor activity compared to CAR-T cells directly inoculated into the tumor resection cavity.

Scalable Manufacturing of CAR T cells for Cancer Immunotherapy.

As of April 2021, there are five commercially available chimeric antigen receptor (CAR) T cell therapies for hematological malignancies. With the current transition of CAR T cell manufacturing from academia to industry, there is a shift toward Good Manufacturing Practice (GMP)-compliant closed and automated systems to ensure reproducibility and to meet the increased demand for cancer patients. In this review we describe current CAR T cells clinical manufacturing models and discuss emerging technological advances that embrace scaling and production optimization. We summarize measures being used to shorten CAR T-cell manufacturing times and highlight regulatory challenges to scaling production for clinical use. STATEMENT OF SIGNIFICANCE ∣: As the demand for CAR T cell cancer therapy increases, several closed and automated production platforms are being deployed, and others are in development.This review provides a critical appraisal of these technologies that can be leveraged to scale and optimize the production of next generation CAR T cells.

Bispecific binder redirected lentiviral vector enables in vivo engineering of CAR-T cells.

BACKGROUND: Chimeric antigen receptor (CAR) T cells have shown considerable promise as a personalized cellular immunotherapy against B cell malignancies. However, the complex and lengthy manufacturing processes involved in generating CAR T cell products ex vivo result in substantial production time delays and high costs. Furthermore, ex vivo expansion of T cells promotes cell differentiation that reduces their in vivo replicative capacity and longevity. METHODS: Here, to overcome these limitations, CAR-T cells are engineered directly in vivo by administering a lentivirus expressing a mutant Sindbis envelope, coupled with a bispecific antibody binder that redirects the virus to CD3+ human T cells. RESULTS: This redirected lentiviral system offers exceptional specificity and efficiency; a single dose of the virus delivered to immunodeficient mice engrafted with human peripheral blood mononuclear cells generates CD19-specific CAR-T cells that markedly control the growth of an aggressive pre-established xenograft B cell tumor. CONCLUSIONS: These findings underscore in vivo engineering of CAR-T cells as a promising approach for personalized cancer immunotherapy.

CAR T cells Targeting Human Immunoglobulin Light Chains Eradicate Mature B-cell Malignancies While Sparing a Subset of Normal B Cells.

PURPOSE: CD19-redirected chimeric antigen receptor (CAR.CD19) T cells promote clinical responses in patients with relapsed/refractory B-cell non-Hodgkin lymphomas and chronic lymphocytic leukemia (CLL). However, patients showing sustained clinical responses after CAR.CD19-T treatment show increased infection risk due to compromised B-lymphocyte recovery. Mature B cell-derived malignancies express monoclonal immunoglobulins bearing either κ- or λ-light chains. We initially constructed CAR-T targeting the κ-light-chain (CAR.κ) and established a clinical study with it. After optimizing the CAR molecule, cells developed CAR-T targeting the λ-light chain (CAR.λ) and we explored their antitumor activity. EXPERIMENTAL DESIGN: Using Igλ+ lymphoma cell lines and patient-derived Igλ+ CLL cells, we evaluated the in vitro tumor cytotoxicity and cytokine profiles of CAR.λ. We also assessed the in vivo efficacy of CAR.λ in xenograft Igλ+ lymphoma models including a patient-derived xenograft (PDX) of mantle cell lymphoma, and the effects of λ- or κ-light chain-specific CAR-T on normal B lymphocytes in a humanized murine model. RESULTS: CAR.λ demonstrated antitumor effects against Igλ+ lymphoma cells and patient-derived CLL cells in vitro, and in vivo in xenograft and PDX Igλ+ lymphoma murine models. Antitumor activity of CAR.λ was superimposable to CAR.CD19. Furthermore, we demonstrated in the humanized murine model that λ- or κ-light chain-specific CAR-T cells only depleted the corresponding targeted light chain-expressing normal B cells, while sparing the reciprocal light chain carrying B cells. CONCLUSIONS: Adoptive transfer of CAR.λ and CAR.κ-T cells represents a useful and alternative modality to CAR.CD19-T cells in treating mature B-cell malignancies with minimal impact on humoral immunity.See related commentary by Jain and Locke, p. 5736.

Preclinical Evaluation of B7-H3-specific Chimeric Antigen Receptor T Cells for the Treatment of Acute Myeloid Leukemia.

PURPOSE: The development of safe and effective chimeric antigen receptor (CAR) T-cell therapy for acute myeloid leukemia (AML) has largely been limited by the concomitant expression of most AML-associated surface antigens on normal myeloid progenitors and by the potential prolonged disruption of normal hematopoiesis by the immunotargeting of these antigens. The purpose of this study was to evaluate B7-homolog 3 (B7-H3) as a potential target for AML-directed CAR T-cell therapy. B7-H3, a coreceptor belonging to the B7 family of immune checkpoint molecules, is overexpressed on the leukemic blasts of a significant subset of patients with AML and may overcome these limitations as a potential target antigen for AML-directed CAR-T therapy. EXPERIMENTAL DESIGN: B7-H3 expression was evaluated on AML cell lines, primary AML blasts, and normal bone marrow progenitor populations. The antileukemia efficacy of B7-H3-specific CAR-T cells (B7-H3.CAR-T) was evaluated using in vitro coculture models and xenograft models of disseminated AML, including patient-derived xenograft models. The potential hematopoietic toxicity of B7-H3.CAR-Ts was evaluated in vitro using colony formation assays and in vivo in a humanized mouse model. RESULTS: B7-H3 is expressed on monocytic AML cell lines and on primary AML blasts from patients with monocytic AML, but is not significantly expressed on normal bone marrow progenitor populations. B7-H3.CAR-Ts exhibit efficient antigen-dependent cytotoxicity in vitro and in xenograft models of AML, and are unlikely to cause unacceptable hematopoietic toxicity. CONCLUSIONS: B7-H3 is a promising target for AML-directed CAR-T therapy. B7-H3.CAR-Ts control AML and have a favorable safety profile in preclinical models.

Modifications to the Framework Regions Eliminate Chimeric Antigen Receptor Tonic Signaling.

Chimeric antigen receptor (CAR) tonic signaling, defined as spontaneous activation and release of proinflammatory cytokines by CAR-T cells, is considered a negative attribute because it leads to impaired antitumor effects. Here, we report that CAR tonic signaling is caused by the intrinsic instability of the mAb single-chain variable fragment (scFv) to promote self-aggregation and signaling via the CD3ζ chain incorporated into the CAR construct. This phenomenon was detected in a CAR encoding either CD28 or 4-1BB costimulatory endodomains. Instability of the scFv was caused by specific amino acids within the framework regions (FWR) that can be identified by computational modeling. Substitutions of the amino acids causing instability, or humanization of the FWRs, corrected tonic signaling of the CAR, without modifying antigen specificity, and enhanced the antitumor effects of CAR-T cells. Overall, we demonstrated that tonic signaling of CAR-T cells is determined by the molecular instability of the scFv and that computational analyses of the scFv can be implemented to correct the scFv instability in CAR-T cells with either CD28 or 4-1BB costimulation.