While CD19-directed chimeric antigen receptor (CAR) T cells can induce remission in patients with B cell acute lymphoblastic leukemia (ALL), a large subset relapse with CD19
disease. Like CD19, CD22 ...is broadly expressed by B-lineage cells and thus serves as an alternative immunotherapy target in ALL. Here we present the composite outcomes of two pilot clinical trials ( NCT02588456 and NCT02650414 ) of T cells bearing a 4-1BB-based, CD22-targeting CAR in patients with relapsed or refractory ALL. The primary end point of these studies was to assess safety, and the secondary end point was antileukemic efficacy. We observed unexpectedly low response rates, prompting us to perform detailed interrogation of the responsible CAR biology. We found that shortening of the amino acid linker connecting the variable heavy and light chains of the CAR antigen-binding domain drove receptor homodimerization and antigen-independent signaling. In contrast to CD28-based CARs, autonomously signaling 4-1BB-based CARs demonstrated enhanced immune synapse formation, activation of pro-inflammatory genes and superior effector function. We validated this association between autonomous signaling and enhanced function in several CAR constructs and, on the basis of these observations, designed a new short-linker CD22 single-chain variable fragment for clinical evaluation. Our findings both suggest that tonic 4-1BB-based signaling is beneficial to CAR function and demonstrate the utility of bedside-to-bench-to-bedside translation in the design and implementation of CAR T cell therapies.
Commercial anti-CD19 chimeric antigen receptor T-cell therapies (CART19) are efficacious against advanced B-cell non-Hodgkin lymphoma (NHL); however, most patients ultimately relapse. Several ...mechanisms contribute to this failure, including CD19-negative escape and CAR T dysfunction. All four commercial CART19 products utilize the FMC63 single-chain variable fragment (scFv) specific to a CD19 membrane-distal epitope and characterized by slow association (on) and dissociation (off) rates. We hypothesized that a novel anti-CD19 scFv that engages an alternative CD19 membrane-proximal epitope independent of FMC63 and that is characterized by faster on- and off-rates could mitigate CART19 failure and improve clinical efficacy.
We developed an autologous CART19 product with 4-1BB co-stimulation using a novel humanized chicken antibody (h1218). This antibody is specific to a membrane-proximal CD19 epitope and harbors faster on/off rates compared to FMC63. We tested h1218-CART19 in vitro and in vivo using FMC63-CART19-resistant models. We conducted a first-in-human multi-center phase I clinical trial to test AT101 (clinical-grade h1218-CART19) in patients with relapsed or refractory (r/r) NHL.
Preclinically, h1218- but not FMC63-CART19 were able to effectively eradicate lymphomas expressing CD19 point mutations (L174V and R163L) or co-expressing FMC63-CAR19 as found in patients relapsing after FMC63-CART19. Furthermore, h1218-CART19 exhibited enhanced killing of B-cell malignancies in vitro and in vivo compared with FMC63-CART19. Mechanistically, we found that h1218-CART19 had reduced activation-induced cell death (AICD) and enhanced expansion compared to FMC63-CART19 owing to faster on- and off-rates. Based on these preclinical results, we performed a phase I dose-escalation trial, testing three dose levels (DL) of AT101 (the GMP version of h1218) using a 3 + 3 design. In 12 treated patients (7 DLBCL, 3 FL, 1 MCL, and 1 MZL), AT101 showed a promising safety profile with 8.3% grade 3 CRS (n = 1) and 8.3% grade 4 ICANS (n = 1). In the whole cohort, the overall response rate was 91.7%, with a complete response rate of 75.0%, which improved to 100% in DL-2 and -3. AT101 expansion correlates with CR and B-cell aplasia.
We developed a novel, safe, and potent CART19 product that recognizes a membrane-proximal domain of CD19 with fast on- and off-rates and showed significant efficacy and promising safety in patients with relapsed B-cell NHL.
NCT05338931; Date: 2022-04-01.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Immunotherapy has revolutionized the treatment of cancer. In particular, immune checkpoint blockade, bispecific antibodies, and adoptive T-cell transfer have yielded unprecedented clinical results in ...hematological malignancies and solid cancers. While T cell-based immunotherapies have multiple mechanisms of action, their ultimate goal is achieving apoptosis of cancer cells. Unsurprisingly, apoptosis evasion is a key feature of cancer biology. Therefore, enhancing cancer cells’ sensitivity to apoptosis represents a key strategy to improve clinical outcomes in cancer immunotherapy. Indeed, cancer cells are characterized by several intrinsic mechanisms to resist apoptosis, in addition to features to promote apoptosis in T cells and evade therapy. However, apoptosis is double-faced: when it occurs in T cells, it represents a critical mechanism of failure for immunotherapies. This review will summarize the recent efforts to enhance T cell-based immunotherapies by increasing apoptosis susceptibility in cancer cells and discuss the role of apoptosis in modulating the survival of cytotoxic T lymphocytes in the tumor microenvironment and potential strategies to overcome this issue.
Bendamustine has been retrospectively shown to be an effective and safe lymphodepletion regimen prior to the anti-CD19 chimeric antigen receptor T cell (CART) products tisagenlecleucel and ...axicabtagene ciloleucel, as well as the anti-BCMA CART products idecabtagene vicleucel and ciltacabtagene autoleucel. However, bendamustine as lymphodepletion prior to lisocabtagene maraleucel (liso-cel), a 4-1BB co-stimulated, fixed CD4:CD8 ratio anti-CD19 CART product, has not been described yet. Thus, we studied a cohort of sequentially-treated patients with large B-cell lymphomas who received bendamustine lymphodepletion before liso-cel at the University of Pennsylvania between 5/2021 and 12/2023 (n = 31). Patients were evaluated for toxicities and responses. Of note, 7 patients (22.6%) would have dnot met the inclusion criteria for the registrational liso-cel clinical trials, mostly due to older age. Overall and complete response rates were 76.9% and 73.1%, respectively. At a median follow-up of 6.3 months, the 6-month progression-free and overall survival were 59.9% and 91.1%, respectively. Rates of cytokine-release syndrome (CRS) and neurotoxicity (ICANS) of any grade were 9.7% and 9.7%, respectively, with no grade ≥ 3 events. No infections were reported during the first 30 days following liso-cel infusion. Neutropenia ≥ grade 3 was observed in 29.0% of patients; thrombocytopenia ≥ grade 3 occurred in 9.7%. In conclusion, bendamustine lymphodepletion before liso-cel appears to be a strategy that can drive tumor responses while ensuring a mild toxicity profile.
Primary resistance to CD19-directed chimeric antigen receptor T-cell therapy (CART19) occurs in 10% to 20% of patients with acute lymphoblastic leukemia (ALL); however, the mechanisms of this ...resistance remain elusive. Using a genome-wide loss-of-function screen, we identified that impaired death receptor signaling in ALL led to rapidly progressive disease despite CART19 treatment. This was mediated by an inherent resistance to T-cell cytotoxicity that permitted antigen persistence and was subsequently magnified by the induction of CAR T-cell functional impairment. These findings were validated using samples from two CAR T-cell clinical trials in ALL, where we found that reduced expression of death receptor genes was associated with worse overall survival and reduced T-cell fitness. Our findings suggest that inherent dysregulation of death receptor signaling in ALL directly leads to CAR T-cell failure by impairing T-cell cytotoxicity and promoting progressive CAR T-cell dysfunction. SIGNIFICANCE: Resistance to CART19 is a significant barrier to efficacy in the treatment of B-cell malignancies. This work demonstrates that impaired death receptor signaling in tumor cells causes failed CART19 cytotoxicity and drives CART19 dysfunction, identifying a novel mechanism of antigen-independent resistance to CAR therapy.
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Anti-CD19 chimeric antigen receptor (CAR) T cell therapy has led to unprecedented responses in patients with high-risk hematologic malignancies. However, up to 60% of patients still experience ...disease relapse and up to 80% of patients experience CAR-mediated toxicities, such as cytokine release syndrome or immune effector cell-associated neurotoxicity syndrome. We investigated the role of the intestinal microbiome on these outcomes in a multicenter study of patients with B cell lymphoma and leukemia. We found in a retrospective cohort (n = 228) that exposure to antibiotics, in particular piperacillin/tazobactam, meropenem and imipenem/cilastatin (P-I-M), in the 4 weeks before therapy was associated with worse survival and increased neurotoxicity. In stool samples from a prospective cohort of CAR T cell recipients (n = 48), the fecal microbiome was altered at baseline compared to healthy controls. Stool sample profiling by 16S ribosomal RNA and metagenomic shotgun sequencing revealed that clinical outcomes were associated with differences in specific bacterial taxa and metabolic pathways. Through both untargeted and hypothesis-driven analysis of 16S sequencing data, we identified species within the class Clostridia that were associated with day 100 complete response. We concluded that changes in the intestinal microbiome are associated with clinical outcomes after anti-CD19 CAR T cell therapy in patients with B cell malignancies.
We report a T cell lymphoma (TCL) occurring 3 months after anti-CD19 chimeric antigen receptor (CAR) T cell immunotherapy for non-Hodgkin B cell lymphoma. The TCL was diagnosed from a thoracic lymph ...node upon surgery for lung cancer. The TCL exhibited CD8
cytotoxic phenotype and a JAK3 variant, while the CAR transgene was very low. The T cell clone was identified at low levels in the blood before CAR T infusion and in lung cancer. To assess the overall risk of secondary primary malignancy after commercial CAR T (CD19, BCMA), we analyzed 449 patients treated at the University of Pennsylvania. At a median follow-up of 10.3 months, 16 patients (3.6%) had a secondary primary malignancy. The median onset time was 26.4 and 9.7 months for solid and hematological malignancies, respectively. The projected 5-year cumulative incidence is 15.2% for solid and 2.3% for hematological malignancies. Overall, one case of TCL was observed, suggesting a low risk of TCL after CAR T.
Introduction: Adoptive cell therapies (ACT) are currently revolutionizing cancer treatment. In particular, chimeric antigen receptor (CAR) T cells have demonstrated unprecedented responses against ...aggressive B cell malignancies with six FDA-approved products either targeting CD19 or BCMA. Other strategies for solid cancers such as T cell receptor (TCR)-redirected or tumor infiltrating T cells will soon become commercially available for melanoma and other cancers. However, despite this exciting new wave of ACT, the majority of patients will ultimately fail treatment, even in the most potent products such as CART19. One key mechanism of failure is the inability of adoptively transferred T cells to fully activate and function in the tumor bed. Thus, there is a dire need to develop effective strategies to enhance T cell activation to improve long-term responses in patients. Most of the current strategies are focused on reducing T cells' exhaustion, but another avenue is to study early T cell activation as a key step for long-term efficacy. We sought to define a broadly applicable strategy to disinhibit T cell activation by focusing on CD5, a transmembrane protein shown to negatively regulate T cell activation and proliferation through mediators such as SHP-1, CBL, and CBL-B ( Fig. 1A). We previously presented the preliminary preclinical results of CD5 knocked out (KO) anti-CD19 CAR T cells (CART19) ( Blood (2020) 136 (Supplement 1): 51-52.) and demonstrated how CD5 KO dramatically increased T cell proliferation and enhanced tumor control. Here, we translate this effect to enhance ACT in multiple models including CAR T and TCR-redirected cells. We also compared early disinhibition of T cell activation (CD5 KO) compares with the highly studied PD1 KO to assess the translational potential of this strategy. Methods and Results: We initially tested CD5 KO CAR T cells in a model of pancreatic ductal cancer adenocarcinoma (PDAC; AsPC1 cell line). We deleted CD5 in anti-mesothelin CART cells (CARTmeso) using a clinically relevant CAR construct (M5 clone) (NCT03054298). CD5 KO CARTmeso cells demonstrated enhanced tumor control (by both tumor volume and bioluminescence) and prolonged overall survival ( Fig. 1B). As observed in the CART19 model, CD5 KO T cells showed increased expansion and persistence. Furthermore, CD5 KO CARTmeso cells established prolonged immune memory, as long-term surviving mice cleared rechallenged tumors (day 74). To study the effects of CD5 KO in TCR-redirected cells, we generated TCR α constant (TRAC) KO T cells further transduced with TCR-GP100 lentivirus and compared these to cells with a double KO of both TRAC and CD5 using the GP100+ melanoma cell line DM6. TRAC CD5 KO TCR-GP100 cells showed significantly reduced GP100+ DM6 tumor growth in vitro when compared to TRAC KO TCR-GP100 cells using a live imaging system, suggesting that CD5 KO could enhance the efficacy of adoptive T cell therapies as a whole. Immune checkpoint inhibitors targeting the PD1:PDL1 axis are routinely used in the clinic. More recently, deletion of PD1 in adoptively transferred T cells has shown feasibility in the clinic and there are currently over 30 clinical trials testing PD1 KO or inhibition in CART therapy for cancer. We, therefore, aimed to compare this established strategy to reduce T cell exhaustion with the disinhibition of early CART activation with CD5 deletion. To this goal, we used a clinically relevant sgRNA to knock out PD1 from CART19 cells and compared this to Mock and CD5 KO CART19 cells against CD19+ Nalm6 B-ALL cells in vivo. In a challenge dose model, PD1 KO CART19 were unable to enhance CART efficacy, while CD5 KO CART19 cells maintained strong tumor control that correlated with increased overall survival. We further compared CD5 versus PD1 KO CARTmeso in an in vivo solid tumor model of mesothelin+ PDAC. CD5 KO CARTmeso cells demonstrated enhanced tumor control and stronger response rates as determined by tumor volume and bioluminescence imaging. This increased tumor control allowed for enhanced survival that is likely explained by the significantly increased T cell count in CD5 KO CARTmeso-treated mice. Conclusion: In conclusion, this study demonstrates that CD5 is a negative regulator and possible novel immune checkpoint for adoptive T cell immunotherapies. We show that CD5 deletion leads to the enhancement of ACT anti-tumor function in several clinically relevant models of liquid and solid tumors.
T cell neoplasms, such as peripheral T cell lymphomas (PTCL) and acute T cell lymphoblastic leukemia (T-ALL), have some of the worst prognoses of all hematological malignancies. Poor outcomes are ...driven by a limited number of effective treatments, particularly in relapsed and refractory settings. While chimeric antigen receptor T cell (CAR-T) immunotherapy has been successful in other hematological diseases, its clinical use against T cell neoplasms is limited due to a lack of unique tumor-specific antigens. Additionally, CAR specificity against pan-T cell antigens causes CAR-T fratricide and insufficient manufacturability for therapeutic dosing. Early studies of CAR-T against CD5- and CD7-expressing T cell neoplasms have shown responses, however, 1) most patients ultimately relapse, and 2) in some patients, antigen negative relapse has occurred. To circumvent these obstacles, we hypothesized that targeting CD2, a pan-T cell antigen, using anti-CD2 CAR-T cells that are engineered to lack CD2, would permit effective CAR-T manufacturing against T cell neoplasms. In a retrospective analysis of clinical flow cytometry diagnostic samples, we found CD2 expression in 25 of 33 (75.8%) and 37 of 51 (72.5%) pediatric PTCL and T-ALL cases, respectively. Similarly, we found 82 of 89 (92.1%) adult PTCL cases with CD2 immunohistochemical expression, which exceeded CD7 expression (38 of 90, 42.2%) that is currently being evaluated in early phase clinical trials. We optimized early CD2 knockout (KO) during CAR-T cell manufacturing from human donor T cells (KO efficiency: 81.2% ± 2.4, n=9 donors). We next engineered a best-in-class, 4-1BB-costimulated, second-generation CD2KO anti-CD2 CAR-T product (CART2) via lentiviral transduction ( PANEL A) using seven distinct single-chain variable fragments (transduction efficiency: 60.2% ± 5.5, n=7 donors) based on cellular expansion and stressed in vivo efficacy against CD2+ T-ALL cells (Jurkat). There were no significant differences in population doublings during CART2 expansion as compared to both CD2 wild-type (CD2 WT) untransduced T cells (Mock UTD) and CD2KO untransduced T cells (CD2KO UTD) (6.20 ± 0.42 vs. 6.12 ± 0.62 vs. 6.67 ± 0.68, respectively, Day 15, n=3 donors), while CD2 WT CART2 cells failed to expand due to fratricide. CART2 cells had no significant differences in T cell memory phenotypes or T cell exhaustion markers, including PD1 and LAG3 expression, at the end of the expansion. Expanded CART2 cells eradicated primary patient-derived CD2+ Sezary cells and CD2+ T-ALL leukemic blasts as measured by in vitro cytotoxicity assays. Cytotoxicity correlated with CD8+ CART2 cells expressing IL-2 (20.4% vs. 0.2% vs. 0.35%), TNFα (28.5% vs. 0.27% vs. 0.2%), and IFNγ (11.27% vs. 0.18% vs. 0.14%) relative to Mock UTD and CD2KO UTD controls stimulated with CD2+ Jurkat cells (n=2 donors). Importantly, we also found both CD2KO UTD and CART2 cells were capable of TNFα and IFNγ release upon interaction with cytomegalovirus and influenza peptides, suggesting that T cells with CD2 perturbations are still able to mediate effector T cell responses. We further demonstrated that CART2 cells were highly effective in controlling systemic tumor burden of T-ALL patient-derived xenograft mouse (PDX) models and significantly improved median overall survival as compared to Mock UTD and CD2KO UTD controls (138 days vs. 30 days vs. 30 days, p=0.0007, Mantel-Cox) ( PANEL B). Long-term tumor control was proportional to CART2 peripheral blood persistence at Day 28 (CD2KO UTD: 186.6 ± 83.7, n=5 vs. CART2: 936.3 ± 241.7, n=5) and Day 100 (CD2KO UTD: N/A vs. CART2: 514.0 ± 441, n=3). We also found that rescue infusion of CART2 could eliminate relapsed T-ALL in PDX mice previously in a remission after initial treatment with anti-CD5 CAR-T cells, with long-term persistence of CD2-negative T cells thereafter. In conclusion, our data demonstrate that CART2 are manufacturable and highly efficacious against T-cell neoplasms. To our knowledge, this is the first report of prolonged efficacy using a CD2-targeted cellular monotherapy in human leukemia preclinical models. This product could be delivered alone or in combination with additional CARs for dual targeting, such as anti-CD5 or anti-CD7 CARs, to reduce the potential of antigen-negative escape. Further research is ongoing to study the effect of CD2 deletion in anti-CD19 CAR T cells and the possible implication for clinical translation.
Introduction: CD19-directed Chimeric Antigen Receptor T cell (CART19) immunotherapy has revolutionized the treatment of B cell lymphoma. However, most CART19-treated patients either fail to respond ...or show disease progression after an initial response. In fact, up to 30% of lymphoma relapses following CART19 show loss of CD19 expression. In addition, several patients who respond well to CART19 display complete loss or severe reduction of their normal B cell repertoire, leaving them exposed to recurrent infections, limited response to vaccines, and requiring prophylactic measures such as intravenous immunoglobulins. A significant portion of mature B cell malignancies express B cell receptors (BCR) that use the same immunoglobulin heavy variable gene: IGHV4-34. In particular, ~30% of activated B cell (ABC) diffuse large B cell lymphomas (DLBCL), ~35% of primary central nervous system lymphomas, ~65% of vitreoretinal lymphomas, and ~35% of hairy cell leukemia variant express IGHV4-34. This suggests that the IGHV4-34 heavy chain is critical for driving the disease by delivering cell survival and proliferation signals, and multiple studies have shown that BCR signaling is required for ABC-DLBCL survival. However, while highly enriched in several types of B cell lymphomas, IGHV4-34 expressing B cells compose only ~5% of the normal B cell repertoire of healthy individuals. Therefore, we hypothesized that anti-IGHV4-34 CAR T cells would be highly effective and safe against B cell malignancies, as they would: (i) efficiently recognize IGHV4-34+ lymphoma cells while sparing normal B cells; and (ii) target a tumor driver that is essential for lymphoma cell survival ( Fig 1A). Methods and Results:We developed a novel CAR construct (CD8-41BB-CD3z) targeting the IGHV4-34+ BCR (CART4-34) using a single-chain variable fragment (scFv) derived from the 9G4 rat monoclonal antibody. We used tumor B cells that endogenously (HBL1) or exogenously (Jeko1, Bonna12, Mec1) express the IGHV4-34 BCR. Using cytotoxicity, cytokine secretion and proliferation assays in vitro we showed that CART4-34 specifically target all IGHV4-34+ tumor B cells tested while sparing IGHV4-34- tumor B cells and, most importantly, healthy B cells. However, while the initial CART4-34 showed strong cytotoxicity towards IGHV4-34+ cells in short-term in vitro assays, they were significantly inferior to CART19 in in vivo xenograft models. We hypothesized that the poor performance of CART4-34 compared to CART19 was due to the unique challenge of targeting the membrane-distal portion of the BCR, 18 nm from the plasma membrane. In contrast, CART19 targets an epitope of CD19 located just 5 nm from the membrane ( Fig 1B). Therefore, to improve immune synapse (IS) formation, we designed new CAR constructs with smaller extracellular domains by replacing the original CD8 hinge (44 amino acids (aa)) with either: (i) a short version of the IgG4 hinge (12aa); or (ii) a G4S linker (5aa). We found that both CART4-34 (IgG4) and CART4-34 (G4S) exhibited significantly improved cytotoxicity towards IGHV4-34+ tumor cells in short-term and long-term in vitro assays in all IGHV4-34+ cell lines tested, while maintaining specificity. Importantly, in in vivo human xenograft models of IGHV4-34+ lymphoma (HBL1), CART4-34 with short hinges showed significantly improved tumor control comparable to, or better than, CART19 ( Fig 1B). We also found that while CART19-resistant lymphoma cells had complete loss of CD19 surface expression, CART4-34-resistant tumor cells retained high expression of IgM, suggesting a reduced likelihood of antigen-negative escape owing to the critical role of the BCR for lymphoma B cell survival. Mechanistically, we found that while the original CART4-34 (CD8 hinge) formed IS with striking morphological differences compared to IS formed by CART19, the IS formed by CART4-34 with short hinges highly resembled those formed by CART19, providing a rationale for the improved anti-tumor potency of short-hinge CART4-34. Conclusion:We implemented a novel paradigm for the treatment of B cell malignancies: the specific targeting of the malignant clone, while sparing the remainder of the healthy B cell repertoire. We designed and optimized a best-in-class anti-IGHV4-34 CAR T cell product with potent anti-tumor effects and minimal toxicity towards IGHV4-34-negative B cells. The activity of CART4-34 will be investigated in a Phase I clinical trial.
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