Chimeric Antigen Receptor T-cell Constructs Research Articles

Human 3D Ovarian Cancer Models Reveal Malignant Cell-Intrinsic and -Extrinsic Factors That Influence CAR T-cell Activity.

In vitro preclinical testing of chimeric antigen receptor (CAR) T cells is mostly carried out in monolayer cell cultures. However, alternative strategies are needed to take into account the complexity and the effects of the tumor microenvironment. Here, we describe the modulation of CAR T-cell activity by malignant cells and fibroblasts in human three-dimensional (3D) in vitro cell models of increasing complexity. In models combining mucin-1 (MUC1) and TnMUC1 CAR T cells with human high-grade serous ovarian cancer cell spheroids, malignant cell-intrinsic resistance to CAR T-cell killing was due to defective death receptor signaling involving TNFα. Adding primary human fibroblasts to spheroids unexpectedly increased the ability of CAR T cells to kill resistant malignant cells as CCL2 produced by fibroblasts activated CCR2/4+ CAR T cells. However, culturing malignant cells and fibroblasts in collagen gels engendered production of a dense extracellular matrix that impeded CAR T-cell activity in a TGFβ-dependent manner. A vascularized microfluidic device was developed that allowed CAR T cells to flow through the vessels and penetrate the gels in a more physiological way, killing malignant cells in a TNFα-dependent manner. Complex 3D human cell models may provide an efficient way of screening multiple cytotoxic human immune cell constructs while also enabling evaluation of mechanisms of resistance involving cell-cell and cell-matrix interactions, thus accelerating preclinical research on cytotoxic immune cell therapies in solid tumors. Significance: Three-dimensional in vitro models of increasing complexity uncover mechanisms of resistance to CAR T cells in solid tumors, which could help accelerate development of improved CAR T-cell constructs.

Transforming CLL management with immunotherapy: Investigating the potential of CAR T-cells and bispecific antibodies

Immunotherapies, such as chimeric antigen receptor (CAR) T-cell therapy and bispecific antibodies or T-cell engagers, have revolutionized the treatment landscape for various B-cell malignancies, including B-acute lymphoblastic leukemia and many non-Hodgkin lymphomas. Despite their significant impact on these malignancies, their application in chronic lymphocytic leukemia (CLL) management is still largely under investigation. Although the initial success of CD19-directed CAR T-cell therapy was observed in 3 multiply relapsed CLL patients, with 2 of them surviving over 10 years without relapse, recent CAR T-cell therapy trials in CLL have shown reduced response rates compared to their efficacy in other B-cell malignancies. One of the challenges with using immunotherapy in CLL is the compromised T-cell fitness from persistent CLL-related antigenic stimulation, and an immunosuppressive tumor microenvironment (TME). These challenges underscore a critical gap in therapeutic options for CLL patients intolerant or resistant to current therapies, emphasizing the imperative role of effective immunotherapy. Encouragingly, innovative strategies are emerging to overcome these challenges. These include integrating synergistic agents like ibrutinib to enhance CAR T-cell function and persistence and engineering newer CAR T-cell constructs targeting diverse antigens or employing dual-targeting approaches. Bispecific antibodies are an exciting "off-the-shelf" prospect for these patients, with their investigation in CLL currently entering the realm of clinical trials. Additionally, the development of allogeneic CAR T-cells and natural killer (NK) cells from healthy donors presents a promising solution to address the diminished T-cell fitness observed in CLL patients. This comprehensive review delves into the latest insights regarding the role of immunotherapy in CLL, the complex landscape of resistance mechanisms, and a spectrum of innovative approaches to surmount therapeutic challenges.

CD33 CAR T-Cells (CD33CART) for Children and Young Adults with Relapsed/Refractory AML: Dose-Escalation Results from a Phase I/II Multicenter Trial

Introduction: Current therapies for patients with acute myeloid leukemia (AML) push the limits of chemotherapy intensity but cure only 30% of adults and 70% of children. Recently developed antibody-based therapies and molecularly-targeted agents only modestly improve outcomes for select patient subsets. Thus, new approaches are needed. Given the success of chimeric antigen receptor (CAR) T-cell therapies for acute lymphoblastic leukemia (ALL), we developed and optimized a novel CD33 CAR T-cell (CD33CART) construct for clinical testing in a multicenter Phase I/II clinical trial in children, adolescents, and young adults (AYA) with relapsed/refractory (r/r) AML. ( Qin H, et al. JITC 2021) We report interim results following completion of the dose-escalation phase. Methods: This multicenter phase I/II clinical trial (NCT03971799) was conducted as a 3+3 dose escalation study through the Pediatric Transplantation and Cell Therapy Consortium with National Marrow Donor Program sponsorship and managed by CIBMTR CRO. Autologous CD33CART product were centrally manufactured on a ClinicMACS Prodigy® by the Biopharmaceutical Development Program at the Frederick National Laboratory for Cancer Research (NCI). Eligibility criteria were r/r AML in subjects < 35 years old with adequate organ/performance status and an identified allogeneic stem cell transplant (SCT) donor. Bone marrow assessment was used for standard morphologic evaluation and central flow cytometric minimal residual disease (MRD) quantification. All subjects received pre-CD33CART lymphodepleting chemotherapy (LD) with fludarabine (75-120 mg/m 2) and cyclophosphamide (900-1000 mg/m 2). CD33CART doses levels (DLs) were: DL1: 3 x 10 5 CD33 CAR+ T-cells/kg; DL2: 1 x 10 6/kg; DL3: 3 x 10 6/kg and DL4: 1 x 10 7/kg. Adverse event grading used CTCAE v5 with incorporation of ASTCT consensus definitions for cytokine release syndrome (CRS) and immune effector cell associated neurotoxicity syndrome (ICANS). Dose-limiting toxicities (DLTs) included > grade 3 CRS, > grade 3 ICANS, and persistent > grade 3 non-hematologic toxicity that did not resolve < grade 2 in 72 hours. CD33CART persistence was assessed via flow cytometric evaluation of the blood and bone marrow. Data cut-off was June 1, 2023. Results: A total of 24 subjects (median age of 16 years, range 1-34 years), were enrolled, 12 (50%) of whom underwent a prior SCT. CD33CART products were successfully manufactured for 23 subjects and infused into 19. In 4 non-infused subjects with manufactured products, 1 died from progressive disease (PD) prior to LD, 2 transitioned to palliative care and 1 withdrew consent to seek alternative therapy. Manufacturing was not performed in one subject due to death from PD after enrollment. One morbidly obese subject (BMI=49.3) enrolled at DL4 but received treatment at DL3 due to weight-based manufacturing limitations. The median time from enrollment to infusion was 47 days (range, 24-242). (Table) Three subjects each were infused at DL1 and DL2 without DLTs. At DL3, 1 subject experienced DLT (grade 4 CRS) prompting expansion at this dose level, and no subsequent DLTs were observed. At DL4, 1 subject experienced a prolonged grade 3 CRS (> 72 hours) and grade 3 ICANS, both constituting DLT and necessitating expansion of the cohort without additional DLTs seen. Across all dose-levels, CRS was seen in 13 (68%) patients and was > grade 3 in 4 (21%) with onset typically within the first 24 hours post-infusion. Complete remission (CR) was only seen at DL4 and achieved in 2 subjects, both achieving an MRD negative CR alongside myeloid aplasia. One SCT naïve subject developed candidemia during aplasia but was able to proceed to an allogenic SCT, achieved engraftment, and was in remission at Day +100 post SCT. The second patient (post-two prior SCTs) declined third SCT, had spontaneous count recovery and remained in remission until day +119 (MRD+ relapse). Transient CD33CART expansion was detected in 9 (47.4%) subjects overall and in all 6 (100%) subjects at DL4. Conclusions: CD33CART manufacturing is feasible in children and AYAs with r/r AML with acceptable toxicity experienced in treated subjects. CD33CART expansion was best in subjects treated at DL4 (1 x 10 7/kg) with MRD negative CRs and transient myeloid aplasia occurring in 2 of 5 (40%) subjects evaluable for response (Table). Based on early clinical efficacy at DL4, enrollment continues in the phase 2 portion.

Anti-GnRH Receptor Monoclonal Antibody, GHR106 is First-inClass GnRH Antagonist

During the last decade, a monoclonal antibody, GHR106 was generated and characterized extensively biologically and immunologically. This antibody targets specific human pan cancer marker and is being evaluated for potential therapeutic applications in cancer immunotherapy and fertility regulations. GHR106 was generated against N1-29 oligopeptide located in the extracellular domains of human GnRH receptor found either in the anterior pituitary or in most of cancer cells. In vitro culture of cancer cells revealed that this antibody can induce apoptosis of cancer cells following 24-48 hours incubations. Anti-tumor activities of GHR106 were evaluated by typical nude mouse experiments in which the effective volume reduction of implanted tumor was observed. Humanized forms of GHR106 were made available in CAR (chimeric antigen receptor) T-cell constructs. GHR106 was shown separately to induce cytotoxic killings of cancer cells in vitro by releasing cytokines following incubations of tumor cells with CAR-T cell constructs. In addition, GHR106 also acts as GnRH antagonist by a specific targeting to pituitary GnRH receptor for reversible suppressions of reproductive hormones. These were demonstrated in “Proof of Concept” rabbit experiments. A single subcutaneous injection with 1-3mg/kg of GHR106 to rabbits of either sex could result in 60 to 90% reductions of gonadotropins, estrogen and/or testosterone over a period of one to two weeks. Based on these preclinical assessments, it can be concluded that GHR106 is restricted in tissue expressions and suitable for cancer immunotherapy. It can also act as long-acting GnRH antagonist to target specifically on GnRH receptor in anterior pituitary for numerous gynecological diseases including ovulation inhibition in IVF/ART, endometriosis-premenstrual syndrome, precocious puberty, uterine fibroids and/or polycystic ovarian syndrome.

Bispecific PSMA antibodies and CAR-T in metastatic castration-resistant prostate cancer.

Prostate cancer is the most common cancer among men and the second leading cause of cancer-related deaths in men in the United States. The treatment paradigm for prostate cancer has evolved with the emergence of a variety of novel therapies which have improved survival; however, treatment-related toxicities are abundant and durable responses remain rare. Immune checkpoint inhibitors have shown modest activity in a small subset of patients with prostate cancer and have not had an impact on most men with advanced disease. The discovery of prostate-specific membrane antigen (PSMA) and the understanding of its specificity to prostate cancer has identified it as an ideal tumor-associated antigen and has revived the enthusiasm for immunotherapeutics in prostate cancer. T-cell immunotherapy in the form of bispecific T-cell engagers (BiTEs) and chimeric antigen receptor (CAR) T-cell therapy have shown exceptional success in treating various hematologic malignancies, and are now being tested in patients with prostate cancer with drug design centered on various target ligands including not just PSMA, but others as well including six-transmembrane epithelial antigen of the prostate 1 (STEAP1) and prostate stem cell antigen (PSCA). This summative review will focus on the data surrounding PSMA-targeting T-cell therapies. Early clinical studies with both classes of T-cell redirecting therapies have demonstrated antitumor activity; however, there are multiple challenges with this class of agents, including dose-limiting toxicity, 'on-target, off-tumor' immune-related toxicity, and difficulty in maintaining sustained immune responses within a complex and overtly immunosuppressive tumor microenvironment. Reflecting on experiences from recent trials has been key toward understanding mechanisms of immune escape and limitations in developing these drugs in prostate cancer. Newer generation BiTE and CAR T-cell constructs, either alone or as part of combination therapy, are currently under investigation with modifications in drug design to overcome these barriers. Ongoing innovation in drug development will likely foster successful implementation of T-cell immunotherapy bringing transformational change to the treatment of prostate cancer.

Evaluating the Patient with Neurotoxicity after Chimeric Antigen Receptor T-cell Therapy

Chimeric antigen receptor (CAR) T-cells are now a well-established treatment for hematologic malignancies. Their use in clinical practice has expanded quite rapidly and hospitals have developed CAR T-cell protocols to evaluate patients for associated toxicities, and particularly for neurotoxicity. There are many variables that influence the risk for developing this complication, many of which are not fully understood. The severity can be related to a particular product. Clinical vigilance is critical to facilitate early recognition of neurotoxicity, hence the importance of pre-CAR T-cell neurological evaluation of each patient. While details of such an evaluation may slightly differ between institutions, generally a comprehensive neurological evaluation including assessment of cognitive abilities along with magnetic resonance imaging (MRI) of the brain is a gold standard. Management of neurotoxicity requires a well-orchestrated team approach with specialists from oncology, neurology, oftentimes neurosurgery and neuro-intensive care. Diagnostic work-up frequently includes detailed neurologic evaluation with comparison to the baseline assessment, imaging of the brain, electroencephalogram, and lumbar puncture. While steroids are uniformly used for treatment, many patients also receive tocilizumab for an underlying and frequently concomitant cytokine release syndrome (CRS) in addition to symptom-driven supportive care. Novel CAR T-cell constructs and other agents allowing for potentially lower risk of toxicity are being explored. While neurotoxicity is predominantly an early, and reversible, event, a growing body of literature suggests that late neurotoxicity with variable clinical presentation can also occur.

Proteomic Profiles of Cytokine Release Syndromes Following Lisocabtagene Maraleucel and Idecabtagene Vicleucel

Introduction: Adoptive cell therapy with chimeric antigen receptor (CAR) T-cells has improved responses in refractory diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (MM). Most CAR-T recipients experience cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) - similar yet clinically distinct inflammatory syndromes related to T-cell activation and expansion of multiple cytokines. Differences in tumor biology between MM and DLBCL and how they contribute to differences in cytokine expression is not well understood. Given different CAR T-cell constructs have varying levels of activation and toxicity, cytokine profiles between products and between different disease entities may inform management. In this study, we used multiplex proteomic profiling to evaluate differences in plasma cytokine signatures between two CAR T-cell products and diseases. Methods: Between September 2021 and March 2022, 20 patients received CAR T-cell therapy and consented to the Yale School of Medicine hematologic disease tissue bank (HIC#1401013259). Two longitudinal cohorts were prospectively enrolled: (1) patients who received lisocabtagene maraleucel (liso-cel) for DLBCL refractory after two lines of therapy, and (2) those who received idecabtagene vicleucel (ide-cel) for MM refractory after 4 lines of therapy. For each patient, the first sample was collected prior to lymphodepleting chemotherapy on day -5 with subsequent samples collected on days 0 (prior to CAR T-cell infusion) and 1, 2, 3, and 7 (after CAR T-cell infusion). Plasma samples were processed and frozen within 1 hour of collection. Proteomic profiling was performed at Eve Technologies (Calgary, Alberta, Canada). The Human 71-Plex Discovery Assay measuring 71 total cytokines and chemokines was used to interrogate each sample time point. Statistical analysis was performed in GraphPad Prism (GraphPad Software, San Diego, CA) and R (R Core Team). Levels of individual proteins were compared using Wilcoxon tests. P-values <0.05 were considered statistically significant. Results: Among 13 patients who received liso-cel for DLBCL (including 2 with transformed follicular lymphoma and 3 with double/triple hit pathology), the median age was 66 years (44-82), 7 (54%) were males, 8 (62%) had KPS ≥ 90, 8 (62%) had bulky disease, median lines of therapy were 4 (2-10), and 11 (85%) received systemic bridging therapy. Seven patients (54%) had any grade CRS, with one patient having grade 3 CRS; 2 (15%) had grade 1 and 2 ICANS. Four patients (31%) received an IL-6 inhibitor. At 3 months of follow-up, the objective response rate was 77% (10/13), of whom two patients had a complete response and 60% (6/10) had any grade CRS. Among 7 patients who received ide-cel for MM, with R-ISS score of 1, 2, and 3 in 4 (57%), 1 (14%), and 2 (29%) patients, respectively, the median age was 61 years (54-79), 5 (71%) were males, 4 (57%) had KPS ≥ 90, 3 (43%) had extramedullary disease, median lines of therapy were 7 (4-13), and 5 patients (71%) received systemic bridging therapy. Six patients (86%) had any grade CRS with no patients having grade ≥ 3 CRS; 3 (43%) had grade 1 ICANS. Four patients (57%) received an IL-6 inhibitor. Five patients (63%) received growth factor support on day 7. At 3 months of follow-up, the objective response rate was 86% (6/7) of whom two patients had a complete response and 83% (5/6) had any grade CRS. In a longitudinal analysis, significantly higher levels of IL-3, IL-5, IL-6, IL-10, IL-15, IL-309, and TNFα were noted during CRS (panel 1). In a comparison between CAR constructs, ide-cel was characterized by significantly higher levels of Eotaxin-2, FLT-3L, IL-5, IL-6, IL-15, I-309, MCP-1, and MCP-4 compared to liso-cel (panel 2). Many of these elevated cytokine proteins are implicated as chemoattractants for other leukocyte subtypes including but not limited to neutrophils, monocytes, eosinophils which may contribute to inflammation and may modulate response, toxicity, or relapse. Conclusions: Ide-cel is associated with a distinct cytokine and chemokine signature compared to liso-cel. This study provides pre-clinical evidence for the role of IL-5 inhibitors to treat or prevent CRS following MM-directed CAR T-cell therapy. Further studies with larger cohorts of patients are needed to validate these findings. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Targeting of low ALK antigen density neuroblastoma using AND logic-gate engineered CAR-T cells

Background aimsThe targeting of solid cancers with chimeric antigen receptor (CAR) T cells faces many technological hurdles, including selection of optimal target antigens. Promising pre-clinical and clinical data of CAR T-cell activity have emerged from targeting surface antigens such as GD2 and B7H3 in childhood cancer neuroblastoma. Anaplastic lymphoma kinase (ALK) is expressed in a majority of neuroblastomas at low antigen density but is largely absent from healthy tissues. MethodsTo explore an alternate target antigen for neuroblastoma CAR T-cell therapy, the authors generated and screened a single-chain variable fragment library targeting ALK extracellular domain to make a panel of new anti-ALK CAR T-cell constructs. ResultsA lead novel CAR T-cell construct was capable of specific cytotoxicity against neuroblastoma cells expressing low levels of ALK, but with only weak cytokine and proliferative T-cell responses. To explore strategies for amplifying ALK CAR T cells, the authors generated a co-CAR approach in which T cells received signal 1 from a first-generation ALK construct and signal 2 from anti-B7H3 or GD2 chimeric co-stimulatory receptors. The co-CAR approach successfully demonstrated the ability to avoid targeting single-antigen-positive targets as a strategy for mitigating on-target off-tumor toxicity. ConclusionsThese data provide further proof of concept for ALK as a neuroblastoma CAR T-cell target.

EXTH-84. A NOVEL DUAL-SPECIFIC CHIMERIC ANTIGEN RECEPTOR T CELL WITH HIGH SPECIFICITY FOR EGFR AND EGFRVIII IMPROVES SURVIVAL IN EGFR EXPRESSING MEDULLOBLASTOMA

Abstract BACKGROUND Medulloblastoma is the most common malignant brain tumor in children 0-19 years of age and current treatment requires surgical resection, followed by high dose chemotherapy and radiotherapy. Therapy carries high morbidity, with late effects including neurocognitive decline, endocrine dysfunction, and subsequent malignancies. Chimeric antigen receptor (CAR) T cell therapy presents the potential for less toxic and more effective treatment for medulloblastoma. Epidermal growth factor (EGFR) is expressed in medulloblastoma derived cell lines and appears to serve an important role in the metastatic potential of this tumor type. D2C7 is a recombinant monoclonal antibody short chain variable fragment (scFv) with dual specificity, binding to wild type EGFR (EGFRwt) and its mutant EGFR variant III (EGFRvIII). We previously developed a novel, third-generation chimeric antigen receptor T-cell construct utilizing the D2C7 scFv and performed analysis on both glioblastoma and medulloblastoma tumor models. METHODS U87 and U87vIII glioblastoma and DAOY medulloblastoma cell lines were characterized by flow cytometry to evaluate for EGFRwt and EGFRvIII expression. Cytotoxicity assays were performed utilizing flow cytometry on serially diluted effector: target ratios of U87, U87vII, and DAOY mixed with D2C7 CAR. DAOY was orthotopically implanted into the frontal lobe of NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. After tumor implantation, D2C7 CAR was introduced within the tumor bed. RESULTS EGFR was detected in U87, U87vIII, and DAOY on flow analysis. EGFRvIII was expressed only on U87vIII. D2C7 CAR demonstrated effective in vitro cytotoxicity against all three cell lines with &amp;gt;90% cytotoxicity achieved at an effector: target ratio of 2.5:1. In vivo, D2C7 CAR treatment significantly prolonged survival compared to treatment with mock CAR. CONCLUSION D2C7 CAR demonstrates efficacy in vitro and in mouse models of medulloblastoma and glioblastoma. Future steps in our work will set out to assess the D2C7 CAR in NSG mice with DAOY in the posterior fossa.

Digital polymerase chain reaction strategies for accurate and precise detection of vector copy number in chimeric antigen receptor T-cell products

Background aimsVector copy number (VCN), an average quantification of transgene copies unique to a chimeric antigen receptor (CAR) T-cell product, is a characteristic that must be reported prior to patient administration, as high VCN increases the risk of insertional mutagenesis. Historically, VCN assessment in CAR T-cell products has been performed via quantitative polymerase chain reaction (qPCR). qPCR is reliable along a broad range of concentrations, but quantification requires use of a standard curve and precision is limited. Digital PCR (dPCR) methods were developed for absolute quantification of target sequences by counting nucleic acid molecules encapsulated in discrete, volumetrically defined partitions. Advantages of dPCR compared with qPCR include simplicity, reproducibility, sensitivity and lack of dependency on a standard curve for definitive quantification. In the present study, the authors describe a dPCR assay developed for analysis of the novel bicistronic CD19 × CD22 CAR T-cell construct. MethodsThe authors compared the performance of the dPCR assay with qPCR on both the QX200 droplet dPCR (ddPCR) system (Bio-Rad Laboratories, Inc, Hercules, CA, USA) and the QIAcuity nanoplate-based dPCR (ndPCR) system (QIAGEN Sciences, Inc, Germantown, MD, USA). The primer–probe assay was validated with qPCR, ndPCR and ddPCR using patient samples from pre-clinical CAR T-cell manufacturing production runs as well as Jurkat cell subclones, which stably express this bicistronic CAR construct. ResultsddPCR confirmed the specificity of this assay to detect only the bicistronic CAR product. Additionally, the authors’ assay gave accurate, precise and reproducible CAR T-cell VCN measurements across qPCR, ndPCR and ddPCR modalities. ConclusionsThe authors demonstrate that dPCR strategies can be utilized for absolute quantification of CAR transgenes and VCN measurements, with improved test–retest reliability, and that specific assays can be developed for detection of unique constructs.

Infectious Complications and Preventative Strategies following Chimeric Antigen Receptor T-cells (CAR-T cells) Therapy for B-Cell Malignancies.

Several chimeric antigen receptor T-cell constructs (CAR-T cells) are currently approved for the treatment of B-cell malignancies, including non-Hodgkin lymphoma and acute lymphoblastic leukemia. Additionally, multiple other products are being investigated and developed for other hematological malignancies and solid cancers. Patients receiving CAR-T cells are at increased risk of infectious complications that lead to increased morbidity and inferior mortality in these patients. In this review, we discuss the literature on the incidence and types of infection in patients in the early and late-phase after CAR-T cells infusion. Additionally, we summarize the current literature on prophylaxis against viral, bacterial, and fungal infections after CAR-T cells infusion and the utility of preventative and supportive measures including intravenous immunoglobulins and myeloid growth factors.

Abstract PO-111: The impact of race, ethnicity and obesity on CAR T-cell outcomes

Abstract Introduction: Chimeric antigen receptor (CAR) T-cells have revolutionized therapies for B-cell malignancies. However, while ethnic and racial minorities or those with obesity represent medically underserved populations for whom cancer outcomes are worse, it is unknown whether these factors also negatively impact outcomes of CAR T-cells. Methods: We conducted a retrospective review of 5 unique phase I CAR T-cell trials for children and adults with B-cell malignancies (including CD19, CD22 and BCMA targeted CAR T-cells for B-cell acute lymphoblastic leukemia (B-ALL), B-cell non-Hodgkin lymphoma (NHL) and multiple myeloma (MM)). In addition to evaluating basic demographics, complete remission (CR) rates and cytokine release syndrome (CRS) severity, graded by ASTCT, were stratified by race (white vs non-white), ethnicity and obesity, defined as BMI &amp;gt; 30. All individual protocols were IRB approved, and the retrospective study for this analysis is registered at: Clinicaltrials.gov NCT03827343. Statistics were done in GraphPad Prism, using non-parametric tests with p&amp;lt;0.05 considered significant. Results: 185 patients were treated with 1 of 5 unique CAR T-cell constructs. The median age was 22.7 years (range, 4.2-69 years). 138 had B-ALL; 23 had NHL and 24 had MM. The racial distribution was 154 (83.2%) white patients, and 31 (16.8%) non-white patients. The ethnic distribution (Hispanic versus non-Hispanic) was: 45 (24.3%) Hispanic, 139 (75.1%) non-Hispanic, and 1 unknown. Twenty-eight patients (15.1%) were obese. CR rates in those with B-ALL was 68.1% (94/138), in MM was 8.3% (2/24), and in NHL was 43.5% (10/23). CR rates did not vary by race or ethnicity. White CR rate 88/154 (57.1%) vs non-white CR rate was 18/31 (58.1%); p=not-significant (NS)) Hispanic CR rate was 26/44 (59.1%) vs Non-Hispanic CR rate of 79/136 (58.1%); p=NS. The overall CR rate in obese patients was 50% (14/28) versus 58.6% (92/157) in non-obese patients, p=NS. Grade &amp;gt;/= 3 CRS occurred in 35/185 (18.9%) patients and did not vary by race. Grade &amp;gt;/= 3 CRS in white patients was 30/154 (19.5%) vs non-White patients was 5/31 (16.1%); p=NS. Rates of Grade &amp;gt;/= 3 CRS were higher in Hispanic patients, (13/45, 28.9%) versus non-Hispanic patients (21/139, 15.1%), p=0.05 and in obese patients, (9/28, 32.1%) vs non-obese 26/157 (16.6%); p=NS). Conclusion: While CR rates did not vary by race (white versus non-white), ethnicity or obesity, a closer analysis by racial sub-populations and at active dose levels is a critical next step to evaluate for subtle disparities. Hispanic patients and those with obesity had a trend towards higher rates of Grade &amp;gt;/= 3 CRS, which needs further study. Given the tremendous potential of CAR T-cells across diverse populations and ability to overcome chemotherapeutic resistance seen in minority populations, CAR T-cells may represent an opportunity to improve outcomes for underserved populations without substantially increasing toxicity. Citation Format: Paul Borgman, John Ligon, Bonnie Yates, Haneen Shalabi, Toni Foley, Lauren Little, Jennifer Brudno, Lekha Mikkilineni, James Kochenderfer, Nirali N. Shah. The impact of race, ethnicity and obesity on CAR T-cell outcomes [abstract]. In: Proceedings of the AACR Virtual Conference: 14th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2021 Oct 6-8. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr PO-111.

Relapsed or Refractory Diffuse Large B-Cell Lymphoma: "Dazed and Confused".

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. Approximately 30% to 40% of patients will develop relapsed/refractory (R/R) DLBCL, leading to significant morbidity and mortality. Salvage chemoimmunotherapy followed by high-dose chemotherapy and autologous stem cell rescue (HDT-ASCR) is the standard of care for chemosensitive and transplant-eligible R/R DLBCL. In patients who are ineligible for HDT-ASCR or who fail HDT-ASCR, treatment is mostly with palliative intent. However, the recent advances with chimeric antigen receptor T-cell (CAR-T) therapy and several FDA-approved targeted agents are changing the current landscape of R/R DLBCL management. There is no one-size-fits-all approach, and guidance regarding optimal sequencing of subsequent therapies is an unmet need. This review highlights the approved CAR-T constructs, including their efficacy, adverse effects, and real-world data; bridging therapy to CAR-T; the role of emerging targeted agents, including bispecific antibodies; and the timing of these targeted agents in relation to CAR-T therapy. Providing individualized treatment with thoughtful sequencing of available agents is essential until future prospective randomized clinical trials provide more insights.

Efficacy and Safety of Innovative Experimental Chimeric Antigen Receptor (CAR) T-cells versus Axicabtagene ciloleucel (Yescarta) for the Treatment of Relapsed/Refractory Large B-Cell Lymphoma (LBCL): Matching Adjusted Indirect Comparisons (MAICs) and Systematic Review.

Despite favorable results of CAR T-cell therapy for relapsed/refractory large B-cell lymphoma (R/R LBCL), several challenges remain, including incomplete response, immune-mediated toxicity, and antigen-loss relapse. We delineated the relative clinical benefit of the novel approaches compared to the currently approved CAR T-cell therapies. In the absence of head-to-head comparisons and randomized controlled trials, we performed Matching Adjusted Indirect Comparisons to quantify the relative efficacy and safety of experimental CARs against Axicabtagene ciloleucel (Yescarta), the first FDA-approved CAR. A total of 182R/R LBCL patients from 15 clinical trials with individual patient data (IPD) were pooled into eight populations by their CAR T-cell constructs and +/- ASCT status. The study endpoints were Progression-Free Survival (PFS), grade ≥ 3 cytokine release syndrome (CRS), and grade ≥ 3 neurotoxicity (NT). Tandem CD19.CD20.4-1BBζ CARs indicated favorable efficacy and safety, whereas the co-infusion of CD19 & CD20 with 4-1BBζ showed no clinical benefit compared to Yescarta. Third generation CD19. CD28. 4-1BBζ, and sequential administration of autologous stem cell transplantation (ASCT) and CD19. CARs presented statistically insignificant yet improved PFS and safety except for ASCT combined intervention which had suggestively higher NT risk than Yescarta. CARs with modified co-stimulatory domains to reduce toxicity (Hu19. CD8.28Zζ and CD19. BBz.86ζ) presented remarkable safety with no severe adverse events; however, both presented worse PFS than Yescarta. Third-generation CARs demonstrated statistically significantly lower NT than Yescarta. CD20. 4-1BBζ data suggested targeting CD20 antigen alone lacks clinical or safety benefit compared to Yescarta. Further comparisons with other FDA-approved CARs are needed.

Infectious Complications in Patients Treated with Idecabtagene Vicleucel for Relapsed and Refractory Multiple Myeloma

Introduction:Chimeric antigen receptor (CAR) T-cell therapy is a novel adoptive immunotherapy utilizing autologous T cells expressing synthetic fusion proteins that target specific antitumor antigens. Over recent years, novel CAR T-cell constructs have shown efficacy for the treatment of hematologic malignancies. The B-cell maturation antigen (BCMA)-directed CAR T-cell product idecabtagene vicleucel (ide-cel) is the first approved CAR T-cell therapy for the treatment of multiple myeloma (MM). While ide-cel represents an important advance in MM treatment, it is critical to better characterize the risk of infectious diseases following this novel therapy.Methods:We investigated infectious complications in 27 (CRB-401, n/62; KarMMa, n/128) adult patients who received ide-cel for relapsed and refractory MM at two institutions. Patients were enrolled in an open label, multi-site Phase 1 or 2 clinical trial (NCT02658929; NCT03361748) evaluating the safety and efficacy of ide-cel. All participants received a 3-day cycle of lymphodepleting chemotherapy with fludarabine and cyclophosphamide 5 days prior to infusion and ide-cel was administered at target doses of 150×10 6 to 450×10 6 CAR-positive T cells. All patients but one received antiviral prophylaxis with val/acyclovir or famciclovir. Seventeen patients received pneumocystis prophylaxis with atovaquone or trimethoprim-sulfamethoxazole. Only 2 patients received antibacterial prophylaxis with levofloxacin and no patients received antifungal prophylaxis. Infections were retrospectively identified from day of cell infusion (day 0) up to day 100 after infusion. Infections were reported if patients experienced symptoms with a microbiologic or histopathologic diagnosis, or for symptomatic site-specific infections in conjunction with radiographic or exam findings and treatment with systemic antimicrobials. Infection severity was determined using the Blood and Marrow Transplant Clinical Trials Network criteria. Cytokine release syndrome (CRS) events were graded according to the Lee criteria. Patients were censored on date of disease relapse, the last day of the study period, or death.Results:Median age was 59 (range 41 - 79), 56% were males. Patients had received a median of 6 previous antimyeloma regimens (range 3 - 10); and 74% had undergone prior autologous hematopoietic cell transplantation. Following infusion of cells, 24 patients (89%) developed CRS with 54% of those receiving ≥ 1 dose of tocilizumab and 17% receiving ≥ 1 dose of corticosteroid. Only two patients (7%) developed CAR T cell associated neurotoxicity (ICANS) and one of those patients received treatment with corticosteroids. Eight patients experienced 19 infection-related events over the first 100 days after ide-cel infusion. To determine infection density, we evaluated 27 patients contributing 667 days at risk between d0 and d30 and 1777 days at risk between d0 and d100. Median time to first infection was 22 days (range 0 - 85). The estimated infection density was 1.8 infections per 100 patient days over the first 30 days, and decreased to 1.1 infections per 100 patient days from day 30 to d100. Among the infection events, bacterial infections were the most common (74%) with 6 bloodstream infections (32%) observed. Viral infections were less frequent (21% of events) and only one fungal infection (5% of events) was observed during the at-risk period. Four infections were of moderate severity; 10 were severe; and 5 were life-threatening. Eleven of the 27 patients (41%) had persistent neutropenia (absolute neutrophil count <1000) after day 30.Conclusions:Our study in this cohort of patients provides clarity on specific infectious complications in a unique population, and is of particular relevance given the recent FDA approval of ide-cel. Of note, these results represent a cross study single institution subgroup analysis that may not reflect the complete trial data. The overall incidence of infection was similar to what has previously been reported in patients receiving CD-19 directed CAR T-cell therapy, even with persistent neutropenia after one month documented in 41% of patients. Bacterial infections were the most common, and there were 5 life-threatening bacterial infections within the first 30 days after infusion. Notably, patients in this group experienced only 1 fungal infection, despite no patients receiving antifungal prophylaxis. [Display omitted] DisclosuresSperling: Adaptive: Consultancy. Branagan: Adaptive Biotechnologies: Consultancy; BeiGene: Consultancy; CSL Behring: Consultancy; Karyopharm: Consultancy; Pharmacyclics: Consultancy; Sanofi Genzyme: Consultancy. Nadeem: Takeda: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; GSK: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees. Yee: GSK: Consultancy; Janssen: Consultancy; Takeda: Consultancy; Karyopharm: Consultancy; Oncopeptides: Consultancy; Adaptive: Consultancy; Bristol Myers Squibb: Consultancy; Sanofi: Consultancy; Amgen: Consultancy. Raje: Celgene, Amgen, Bluebird Bio, Janssen, Caribou, and BMS: Other. Munshi: Abbvie: Consultancy; Amgen: Consultancy; Karyopharm: Consultancy; Takeda: Consultancy; Adaptive Biotechnology: Consultancy; Legend: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Novartis: Consultancy; Oncopep: Consultancy, Current equity holder in publicly-traded company, Other: scientific founder, Patents & Royalties; Janssen: Consultancy; Bristol-Myers Squibb: Consultancy. Hammond: Merck: Research Funding; F2G: Research Funding; Synexis: Research Funding; Biointelect: Consultancy.

Assessing CAR T-Cell Therapy Response Using Genome-Wide Sequencing of Cell-Free DNA in Patients With B-Cell Lymphomas

Methods that enable monitoring of therapeutic efficacy of autologous chimeric antigen receptor (CAR) T-cell therapy will be clinically useful. The aim of this study is to demonstrate the feasibility of blood-derived cell-free DNA (cfDNA) to predict CAR T-cell therapy response in patients with refractory B-cell lymphomas. Whole blood was collected before and throughout CAR T-cell therapy until day 154. Low-coverage (∼0.4×), genome-wide cfDNA sequencing, similar to that established for noninvasive prenatal testing, was performed. The genomic instability number (GIN) was used to quantify plasma copy number alteration level. Twelve patients were enrolled. Seven (58%) patients achieved a complete response (CR); 2 (25%), a partial response. Median progression-free survival was 99 days; median overall survival was not reached (median follow-up, 247 days). Altogether, 127 blood samples were analyzed (median, 10 samples/patient [range 8-13]). All 5 patients who remained in CR at the time of last measurement had GIN <170 (threshold). Two patients who attained CR, but later relapsed, and all but one patient who had best response other than CR had last GIN measurement of >170. In 5 of 6 patients with relapsed or progressive disease, increasing GIN was observed before the diagnosis by imaging. The abundance of CAR T-cell construct (absolute number of construct copies relative to the number of human genome equivalents) also showed a trend to correlate with outcome (day 10, P = .052). These data describe a proof-of-concept for the use of multiple liquid biopsy technologies to monitor therapeutic response in B-cell lymphoma patients receiving CAR T-cell therapy.

EFFICACY AND SAFETY OF EXPERIMENTAL CHIMERIC ANTIGEN RECEPTOR (CAR) T-CELLS VERSUS AXICABTAGENE CILOLEUCEL (YESCARTA) FOR THE TREATMENT OF RELAPSED/REFRACTORY LARGE B-CELL LYMPHOMA: MATCHING ADJUSTED INDIRECT COMPARISONS AND SYSTEMATIC REVIEW

Despite the high response rates seen with Chimeric Antigen Receptor (CAR) T-cell therapy for relapsed/refractory Large B-cell lymphoma (R/R LBCL), post-therapy relapse remains a key challenge. To date, no study has evaluated the comparative efficacy and safety of experimental CAR T-cell products versus currently approved CAR T-cell therapies. To indirectly compare the efficacy and safety of novel, experimental CAR T-cell products against the first FDA-approved CAR T-cell construct, Axicabtagene ciloleucel (Yescarta). In compliance with the PRISMA guidelines, we performed a systematic review, which identified 16 independent, early-phase clinical trials consisting of 193 LBCL patients with individual patient data (IPD). We categorized eight pooled populations based on the target antigens (CD19, CD20), co-stimulatory domains (CD28, 4-1BB), and CAR T-cells administered with or without concomitant autologous stem cell transplant (ASCT). The pooled populations were categorized as follows: (1) dual targeting strategies, such as tandem CD19.CD20, n = 28; (2) co-infusion of CD19 and CD20 CARs, n = 21; (3) third-generation CARs, n = 26; (4) CD19 CARs with modified constructs for reduced toxicity, including CD19.BBz.86, n = 21; and (5) Hu19.CD828Z, n = 14; (6) CD19. 4-1BB.S manufactured in China, n = 24; (7) concomitant ASCT and CD19.CD28, n = 45; and (8) CD20 CARs with a 4-1BB co-stimulatory domain, n = 14. A Matching Adjusted Indirect Comparison (MAIC) statistical technique was applied to account for heterogeneity in the study population across trials. Estimates for the experimental CAR T-cell trials were adjusted using patient-level data to match the ZUMA-1 (Yescarta, n = 108) study population based on mutually reported key baseline covariates, including age, disease stage, histology, refractoriness, number of prior lines of therapy, and extranodal disease. The study endpoints for this study included progression-free survival (PFS), cytokine release syndrome (CRS), and neurotoxicity (NT). In the dual-targeting strategy, only tandem CD19.CD20.4-1BB was associated with a statistically significantly improved PFS (HR = 0.46; 95% CI, 0.23-0.92) and a safer NT profile (OR = 0.15; 95% CI, 0.03-0.76) compared to Yescarta. As for safety, significantly reduced NT (OR = 0.19; 95% CI, 0.04-0.94) was also noted with third-generation CAR T-cells. None of the other comparisons were statistically significant. Of note, no statistically significant associations regarding PFS or safety were seen with the sequential administration of ASCT followed by CAR T-cells within 4-7 days. CD19. BBz.86 and Hu19. CD828Z CAR T-cell products containing modified co-stimulatory domains aimed at reduced toxicity demonstrated no grade ≥3 CRS nor NT occurrence. Anti-CD20.4-1BB did not show a remarkable difference in terms of the selected endpoints for this study. Safety data for the co-infusion of CD19 and CD20 were not available for this trial. Our MAIC suggests a dual targeting approach using tandem CD19.CD20.4-1BB might have both superior efficacy and safety compared to Yescarta. Future studies comparing experimental CAR T-cell constructs with other CD19. 4-1BB-based approved CAR T-cell products, such as Tisagenlecleucel (Kymriah) and Lisocabtagene maraleucel (Breyanzi), may further contribute to clarify these findings.

Itacitinib (INCB039110), a JAK1 Inhibitor, Reduces Cytokines Associated with Cytokine Release Syndrome Induced by CAR T-cell Therapy.

T cells engineered to express a chimeric antigen receptor (CAR) are a promising cancer immunotherapy. Such targeted therapies have shown long-term relapse-free survival in patients with B-cell leukemia and lymphoma. However, cytokine release syndrome (CRS) represents a serious, potentially life-threatening side effect often associated with CAR T-cell therapy. CRS manifests as a rapid (hyper)immune reaction driven by excessive inflammatory cytokine release, including IFNγ and IL6. Many cytokines implicated in CRS are known to signal through the JAK-STAT pathway. Here we study the effect of blocking JAK pathway signaling on CAR T-cell proliferation, antitumor activity, and cytokine levels in in vitro and in vivo models. We report that itacitinib, a potent, selective JAK1 inhibitor, was able to significantly and dose-dependently reduce levels of multiple cytokines implicated in CRS in several in vitro and in vivo models. Importantly, we also report that at clinically relevant doses that mimic human JAK1 pharmacologic inhibition, itacitinib did not significantly inhibit proliferation or antitumor killing capacity of three different human CAR T-cell constructs (GD2, EGFR, and CD19). Finally, in an in vivo model, antitumor activity of CD19-CAR T cells adoptively transferred into CD19+ tumor-bearing immunodeficient animals was unabated by oral itacitinib treatment. Together, these data suggest that itacitinib has potential as a prophylactic agent for the prevention of CAR T cell-induced CRS, and a phase II clinical trial of itacitinib for prevention of CRS induced by CAR T-cell therapy has been initiated (NCT04071366).

Development of CAR-T Cell Constructs with Broad Anti-Tumor Tropism

Chimeric antigen receptor T (CAR-T) cell therapy is a transformative approach to cancer eradication. CAR-T is expensive in part due to the restricted use of each CAR construct for a specific set of tumors such as B cell lymphoma targeted with CD19 and multiple myeloma targeted with BCMA. A CAR construct with broad anti-tumor activity can be advantageous due to wide applicability and scalability of production. We show that CD126, the IL-6 receptor alpha, is an antigen that is expressed by many hematologic and solid malignancies including multiple myeloma, non-Hodgkin lymphoma, acute myeloid leukemia, pancreatic and prostate adenocarcinoma, non-small cell lung cancer and malignant melanoma amongst others. High CD126 expression is a negative prognostic marker in many malignancies. The two CD126 targeting CAR-T cell constructs contain the CD28 anchoring domain followed by 4-1BB and CD3 zeta signaling domain. Lentiviral vectors were generated with triple plasmid (CAR, psPAX2 and pMD2.G) transfection of 293T cells and the vector concentrated by ultracentrifugation and used to transduce human T cells. T cells were isolated from leuko-reduction cones using negative selection with magnetic beads. The transduction efficiency was around 60%. The T cells were activated with anti-CD3/CD28 beads and expanded for two weeks before using for downstream experiments. CD126 CAR-T cells are able to kill many tumor cells in an antigen specific manner and with an efficiency that is directly proportional to the cell surface expression of CD126 expression (rho = 0.6, p = 0.0019). The presence of soluble CD126 in the culture media did not interfere with CAR-T cell killing. The CAR-T constructs bind murine CD126. However, injection of CD126 targeting CAR-T cells in NSG mice did not lead to any evidence of hepatotoxicity and weight loss despite possible expression of this antigen on hepatocytes. In vivo studies in NSG mice with multiple myeloma (RPMI-8226) and prostate adenocarcinoma (DU-145) xenograft models (n=10 tumors per group) showed that the intravenously injected CD126 targeted CAR-T cells (107) infiltrated the tumors, expanded, produced human interferon gamma and killed the tumor cells (p&amp;lt;0.001). Bioluminescence imaging showed control of tumor growth in the actively treated tumors compared to the controls (p&amp;lt;0.05). At post mortem, mice injected with CD126 targeted CAR-T cells had smaller residual tumors compared to controls injected with non-engineered human T cells from the same donor. Binding of sIL-6R by CAR-T cells could mitigate cytokine release syndrome. In support of this, murine SAA-3 levels (the equivalent of human CRP) were lower in mice injected with CD126 CAR-T compared to controls (p&amp;lt;0.05), suggesting that binding of sIL-6R by CAR-T cells could mitigate cytokine release syndrome. CD126 provides a novel therapeutic for CAR-T cells in a broad variety of tumors with low risk of toxicity. Disclosures Dingli: Apellis: Consultancy; Millenium: Consultancy; Janssen: Consultancy; Bristol Myers Squibb: Research Funding; Sanofi-Genzyme: Consultancy; Alexion: Consultancy; Rigel: Consultancy; Karyopharm Therapeutics: Research Funding.

Response to CAR-T Therapy Can be Monitored Using Genome-Wide Sequencing of Cell-Free DNA in Patients with DLBCL

Response to CAR-T Therapy Can be Monitored Using Genome-Wide Sequencing of Cell-Free DNA in Patients with DLBCL