CAR-T Cell Products Research Articles

New Targets of CAR-T Therapy for B-cell lymphoma

B-cell lymphoma is a kind of hematologic malignancy with high morbidity and mortality, which is usually treated by chemoimmunotherapy followed by an autologous transplant. But the traditional therapy usually leads to relapse and refractory. Now FDA has approved four CAR-T cell products that all target CD19 to give relapsed or refractory patients another chance to control cancer. These achievements may thank the CD19, but the CD19 also leads to some problems including the off-tumor effect and antigen escape that result in off-tumor effects and relapses. These problems prevent CAR-T therapy from being widely used in treating B-cell lymphoma. To solve these problems, new targets should be discovered. Scientists have already done a lot of exploration in this area. To give the scientists some directions, this paper will briefly introduce the two problems, the characteristics that are required for the ideal targets, and some ideal future targets which are understudy now.

CAR T cell-induced systemic cytokine toxicity: current understanding and innovative designs

CARs engineering has emerged as a promising treatment for tumor. However, CAR T cell-induced systemic cytokine toxicity is one of the roadblocks limiting the widespread application of the therapy. Current study contributes to some toxicity-related monitoring and management guidelines. Researchers have further developed novel engineering strategies to produce inducible CARs and passively or autonomously control CAR T cells. This review described the recent bench and clinical outcomes of novel engineering CAR T-cell products and discuss their function, advantages, and limitations.

CAR-T manufactured from frozen PBMC yield efficient function with prolonged in vitro production.

Chimeric antigen receptor (CAR)-T cells are engineered to identify and eliminate cells expressing a target antigen. Current manufacturing protocols vary between commercial CAR-T cell products warranting an assessment of these methods to determine which approach optimally balances successful manufacturing capacity and product efficacy. One difference between commercial product manufacturing methods is whether T cell engineering begins with fresh (unfrozen) patient cells or cells that have been cryopreserved prior to manufacture. Starting with frozen PBMC material allows for greater manufacturing flexibility, and the possibility of collecting and storing blood from patients prior to multiple lines of therapy. We prospectively analyzed if second generation anti-CD19 CAR-T cells with either CD28 or 4-1BB co-stimulatory domains have different phenotype or function when prepared side-by-side using fresh or cryopreserved PBMCs. We found that cryopreserved PBMC starting material is associated with slower CAR-T cell expansion during manufacture but does not affect phenotype. We also demonstrate that CAR-T cell activation, cytokine production and in vitro anti-tumor cytotoxicity were not different when CAR-T cells were manufactured from fresh or cryopreserved PBMC. As CAR-T cell therapy expands globally, the need for greater flexibility around the timing of manufacture will continue to grow. This study helps support the concept that cryopreservation of PBMCs could be the solution to these issues without compromising the quality of the final CAR-T product.

621MO Preclinical study of DASH CAR-T cells manufactured in 48 hours

Patients with acute lymphoblastic leukemia (ALL) or relapsed/refractory (r/r) large B-cell lymphoma (LBCL) often progress rapidly, and current manufacture process of chimeric antigen receptor (CAR) T cells takes too long and resulting products are less optimal for in vivo expansion and function. The “DASH CAR-T” manufacturing process we developed enabled production of CAR-T cells of naïve phenotypes within 48 hours. We examined the efficacy of DASH CAR-T cells in a murine model and laid the foundation for novel clinical trials for patients with ALL or r/r B-ALL.

Key Points from the FDA's Guidance for CAR T-Cell Products

Key Points from the FDA's Guidance for CAR T-Cell Products

Identification of genomic determinants contributing to cytokine release in immunotherapies and human diseases

BackgroundCytokine release syndrome (CRS) is a strong immune system response that can occur as a result of the reaction of a cellular immunotherapy with malignant cells. While the frequency and management of CRS in CAR T-cell therapy has been well documented, there is emerging interest in pre-emptive treatment to reduce CRS severity and improve overall outcomes. Accordingly, identification of genomic determinants that contribute to cytokine release may lead to the development of targeted therapies to prevent or abrogate the severity of CRS.MethodsForty three clinical CD22 CAR T-cell products were collected for RNA extraction. 100 ng of mRNA was used for Nanostring assay analysis which is based on the nCounter platform. Several public datasets were used for validation purposes.ResultsWe found the expression of the PFKFB4 gene and glycolytic pathway activity were upregulated in CD22 CAR T-cells given to patients who developed CRS compared to those who did not experience CRS. Moreover, these results were further validated in cohorts with COVID-19, influenza infections and autoimmune diseases, and in tumor tissues. The findings were similar, except that glycolytic pathway activity was not increased in patients with influenza infections and systemic lupus erythematosus (SLE).ConclusionOur data strongly suggests that PFKFB4 acts as a driving factor in mediating cytokine release in vivo by regulating glycolytic activity. Our results suggest that it would beneficial to develop drugs targeting PFKFB4 and the glycolytic pathway for the treatment of CRS.

Zamtocabtagene Autoleucel (MB-CART2019.1): An Investigational CAR-T Cell Product with Tandem Targeting of CD19 and CD20 as a Potential Treatment Option for Patients with Relapsed/ Refractory B Cell Non-Hodgkin Lymphoma

Presenting at the European Hematology Association (EHA) 2022 Congress, Peter Borchmann from the University of Cologne, Germany, discussed a novel tandem cluster of differentiation (CD) 19 and CD20 chimeric antigen receptor (CAR)-T therapy for the treatment of patients with relapsed or refractory B cell non-Hodgkin lymphoma. Borchmann presented the 2-year follow up data from DALY 1 trial, which showed that beyond a favourable safety profile, zamtocabtagene autoleucel (MB-CART2019.1) led to durable complete remissions.

Importance of CAR-T cell therapy monitoring using high-throughput assays

Advances in the development of chimeric antigen receptor (CAR)-T cells have undermined the paramount importance of this technology for the success of adoptive T-cell immunotherapy. The bespoke production of autologous CAR-T cells is a lengthy and costly process. Thus, the development of more cost-effective allogeneic 'off-the-shelf' CAR-T cells provides a more readily available treatment option. The exploration of methods to reduce costs and to determine which CAR-T cells are the most effective is key for providing this breakthrough treatment to most patients. The process from the design and development of CAR-T cells, through pre-clinical and clinical testing and manufacturing, to patient monitoring involves a variety of high-throughput tools that enable the monitoring of all processes to ensure the safety and efficacy of the treatment.

RWD67 Experiences with and Perceptions of CAR T-Cell Therapy for Multiple Myeloma Among Community-Based Hematologists/Oncologists

While the first CAR T-cell (CAR-T) therapy was approved in 2017, idecabtagene vicleucel, a BCMA-directed agent, is the first CAR-T product approved to treat relapsed/refractory multiple myeloma (MM), in March 2021. The experiences with and perceptions of this CAR-T therapy regimen among community-based hematologists/oncologists (cH/O) are of value to patients, pharma manufacturers, and payers.

S103: EFFICACY AND SAFETY OF ARI0002H, AN ACADEMIC BCMA-DIRECTED CAR-T CELL THERAPY WITH FRACTIONATED INITIAL THERAPY AND BOOSTER DOSE IN PATIENTS WITH RELAPSED/REFRACTORY MULTIPLE MYELOMA

Background: ARI0002h is a lentiviral autologous CAR T-cell product with a 4-1BB co-stimulatory domain and a humanized single chain variable fragment targeting BCMA. In pre-clinical studies, this academic CAR-T has demonstrated potent in vitro and in vivo activity. Aims: We report the safety and efficacy results of the CARTBCMA-HCB-01 multicenter clinical trial for patients with relapsed/refractory multiple myeloma (RRMM) (NCT04309981) who received ARI0002h in 5 Spanish centers. Methods: Patients (pts) aged 18-75 years old with RRMM were eligible for this study if they had measurable disease, received ≥2 prior regimens, including a proteasome inhibitor, an immunomodulatory drug and an anti-CD38 antibody, and were refractory to the last line of treatment. Bridging therapy was allowed after apheresis. Cyclophosphamide (300 mg/m2) and fludarabine (30 mg/m2) were used as lymphodepletion regimen. The targeted dose was 3x106/kg CAR+ cells and was administered in a fractionated manner (10%/30%/60%), with at least 24h between infusions. A second dose of 3x106 CAR+ cells/kg was planned at least 4 months after the first dose in pts who achieved any grade of response any response and had not or serious complications after the first administration. Primary objectives were overall response rate (ORR; at least partial response -PR-) within 3 months of the first infusion and rate of cytokine release syndrome (CRS) and/or neurological toxicity in the first 30 days. Response was assessed as per IMWG criteria and bone marrow minimal residual disease (MRD) was analyzed by next-generation flow (NGF). Results: As of February 9th 2022, 35 pts (median age 61 years) with RRMM were included in the trial. Four pts could not receive ARI-0002h due to MM progression and one died of infection. Therefore, 30 pts received ARI0002h cells (modified intention-to-treat population), of which 47% received bridging therapy. Median CAR-T cell production time was 11 days (range 9-14) with a 100% manufacture success. Median follow-up after ARI0002h administration for surviving pts was 16 months. The ORR of 30 evaluable pts was 100%, with a stringent complete remission (sCR) plus very good partial response (VGPR) rate of 90%. Median time to first response was one month. Of 26 MRD-evaluable pts at day +100, 92% were MRD-negative in bone marrow by NGF. 53% of patients were alive and without progression at 16 months. Median overall survival (OS) was not reached and the 16-month OS rate was 80% (Figure 1). AEs reported in >70% of pts were CRS (87%; grade [gr] 3/4 0%; gr 1 73%), neutropenia (97%; gr 3/4 100%), anemia (85%; gr 3/4 43%), and thrombocytopenia (79%; gr 3/4 70%). Median duration of CRS was 4 days (range 1-12). No CAR-T cell-related neurotoxicity cases were reported. Tocilizumab and corticosteroids were administered in 76% (mainly for persistent grade 1 CRS) and 12% of pts, respectively. ARI0002h cells demonstrated peak expansion on day 14 (range 7 days-6 months). 24 out of 28 eligible pts (86%) received the second dose (range 1.2-3x106 CAR+ cells/kg). Median time after first infusion was 4 months and 38% received a second lymphodepletion regimen. No relevant toxicities after second infusions were reported. 7 pts (29%) improved their response after reinfusion. Image:Summary/Conclusion: ARI0002h is the first European academic CART for RRMM that has demonstrated deep and durable responses and a favorable safety profile, including the absence of neurotoxicity and the feasibility of a second booster dose.

P1427: MONITORING CAR-T CELL IN B-CELL LYMPHOMA PATIENTS THROUGH SINGLE CHAIN VARIABLE FRAGMENT BY DIGITAL PCR

Background: CD19-targeted chimeric antigen receptor (CAR)-T cell is a treatment for B-cell lymphoma patients. CAR-T cell monitoring is important to ensure a correct follow-up, being multiparametric flow cytometry (MFC) the actual gold standard technique. However, other molecular techniques such as digital PCR (dPCR) could add complementary information that could be valuable to predict CAR-T response. Both available commercial antiCD19 CAR-T cell therapies, Tisagenlecleucel (tisa-cel) (Kymriah®) and Axicabtagen Ciloleucel (axi-cel) (Yescarta®) share the single variable fragment domain (FMC63), which makes possible the measurement by dPCR. Aims: To evaluate the usefulness of digital PCR for monitoring CAR-T cell levels in comparison to multiparametric flow cytometer. Methods: Forty-five patients diagnosed with diffuse large B-cell lymphoma (36), transformed-follicular lymphoma (7) and mediastinal primary large B-cell lymphoma (2), treated consecutively with anti-CD19 CAR-T cell therapy between June 2019 and May 2021 were included in this study. One hundred and forty-two peripheral blood (PB) samples were collected at days +7, +14, +30, +90 (40, 39, 40 and 23 samples, respectively) after infusion. DNA was purified using the Maxwell® RSC Whole Blood DNA Kit (Promega, USA). dPCR assays were performed on the QIAcuity One platform (QIAgen, Germany). MFC analysis was performed on a DxFLEX cytometer (Beckman Coulter), using CD19 (20-291) protein-FITC (ACRO Biosystems). To assess the sensitivity of molecular methodologies, serial dilutions from 100% to 0.001% of CAR-T were analyzed by dPCR. Correlation of CAR-T cell detection between MFC and dPCR was calculated using Pearson’s test. Results: A high correlation between dPCR and MFC was found (r = 0.90), demonstrating the usefulness of dPCR to quantify absolute number of CAR copies (Figure 1A and 1B). dPCR improved the detection capacity of MFC, detecting CAR-T in 15 samples that were negative by MFC because of low CAR-T cell presence in PB (Figure 1C), as MFC measures CAR-T events in total PB the low content of CAR-T could reduce MFC sensitivity. Median number of CAR-T cells detected by dPCR was greater than that quantified by MFC in each days of the follow-up (Figure 1D). Different CAR-T cell products (Yescarta and Kymriah) differ in its expansion rates, therefore tisa-cel expand before axi-cel (near day 7th and day 14th, respectively) (Figure 1C). Image:Summary/Conclusion:FMC63 quantification through dPCR was effective for CAR-T measurement, even improving MFC sensitivity, especially when CAR-T counting in PB is low in late monitoring days. dPCR monitoring could complement data given by MFC and being a very sensible gold standard technique in late monitoring as its sensitivity is very superior to MFC in those follow-up days.

P1446: PHASE I STUDY DATA UPDATE OF PHE885, A FULLY HUMAN BCMA-DIRECTED CAR-T CELL THERAPY MANUFACTURED USING THE T-CHARGETM PLATFORM FOR PATIENTS WITH RELAPSED/REFRACTORY (R/R) MULTIPLE MYELOMA (MM)

Background: Chimeric antigen receptor (CAR)-T cell therapy (tx) is approved for treatment of r/r MM. However, prolonged product manufacturing times and a need for longer duration of response are a major obstacle in this population, necessitating fast manufacture of a reliable and durable CAR-T cell product. Aims: Herein we report updated clinical data from a Phase I trial assessing the fully human B-cell maturation antigen (BCMA) CAR-T cell product PHE885 in adult patients (pts) with r/r MM. Methods: This multicenter, open-label study (NCT04318327) characterizes the safety and efficacy of PHE885 manufactured using the novel T-ChargeTM platform, which takes <2 d to manufacture the final product and relies on in vivo expansion after CAR-T cell infusion. Pts with MM r/r to ≥2 prior lines of tx, including an immunomodulatory drug, proteasome inhibitor, and anti-CD38 monoclonal antibody, were eligible. Pts received fludarabine and cyclophosphamide for lymphodepletion prior to a single PHE885 intravenous injection. Primary objectives were safety, including dose-limiting toxicities (DLTs) and adverse events (AEs). Secondary objectives included clinical response, T-ChargeTM process evaluation, and cellular kinetics. Results: At abstract submission, 22 pts were infused with PHE885 at the following fixed doses: 2.5×106 (n=4), 5×106 (n=12), 10×106 (n=5), and 14.3×106 (n=1) CAR-T cells; 15 pts (4 at 2.5×106, 10 at 5×106, and 1 at 14.3×106) were evaluable for safety and efficacy at the data cutoff for the abstract (October 13, 2021). Longer follow up of these 15 pts and data from additional pts treated at doses of 5×106 and 10×106 will be presented at the annual meeting. Of the 15 pts evaluable at data cutoff, DLTs were reported in 3 pts and included asymptomatic grade (gr) 3 transaminitis (n=1, 14.3×106), asymptomatic gr ≥3 elevated pancreatic enzymes (n=1, 5×106), and gr 4 neutropenia (n=1, 2.5×106). Most frequent tx-related gr ≥3 AEs included neutropenia (n=15, 100%), anemia (n=13, 87%), and thrombocytopenia (n=11, 73%). All pts experienced cytokine release syndrome (CRS) per Lee 2014 criteria; 2 had gr ≥3 CRS (n=1, 5×106; n=1, 14.3×106). Median times to CRS onset and resolution were 7 d (range, 4-12 d) and 11 d (range, 9-22 d), respectively. Tocilizumab (n=13, 87%), steroids (n=11, 73%), anakinra (n=4, 27%), and pressors (n=3, 20%) were used to manage CRS. Gr 2 neurotoxicity occurred in 4 pts (27%). Pts had an overall response rate of 93%, stringent complete response in 33%, very good partial response (VGPR) in 20%, and PR in 40%. Of evaluable pts at 3 mo post infusion, 43% (3/7) had minimal residual disease (MRD) at <10–5 sensitivity[JC(1], while 33% (2/6) were MRD negative at <10–6 by next-generation sequencing. Robust expansion of BCMA CAR-T cells was observed in all pts via qPCR and flow cytometry; maximum expansion (geometric mean Cmax) was 316,000 copies/μg by qPCR and 76% of circulating T cells by flow cytometry. Maximum expansion was reached at a median of 14.2 d by qPCR (13.9 d by flow cytometry). Naive-like T cells present in the leukapheresis were preserved during manufacturing of most PHE885 products as measured by flow cytometry, and an increase in naive/Tscm populations is observed in ≥VGPR pts post-infusion. Summary/Conclusion: Low doses of PHE885 manufactured by T-ChargeTM in <2 d have a manageable safety profile and encouraging clinical activity in pts with r/r MM. PHE885 expands rapidly in vivo, persists for prolonged periods, and demonstrates a relatively naive T-cell phenotype.

P1459: SAFETY AND EFFICACY FINDINGS OF AUTO1, A FAST-OFF RATE CD19 CAR, IN RELAPSED/REFRACTORY B-CELL NON-HODGKIN’S LYMPHOMA (B-NHL), AND CHRONIC LYMPHOCYTIC LEUKEMIA (CLL) / SMALL LYMPHOCYTIC LYMPHOMA (SLL)

Background: We have previously described AUTO1, a CD19 CAR with a fast off-rate CD19 binding domain, designed to reduce CAR T-cell immune toxicity and improve engraftment. Its clinical activity has been tested in r/r paediatric and adult B-ALL (Ghorashian S et al., Nat Med 2019; Roddie C et al., JCO 2021). This data confirms the intended function of the receptor, with low levels of CRS/ICANS and long-term engraftment of CAR T-cells observed in both patient groups. Aims: We have initiated testing of AUTO1 in the setting of B-NHL and CLL/SLL (NCT02935257). Methods: Manufacturing: CAR T-cell products were generated using a semi-automated closed process from non-mobilised patient leukapheresate. Study design: Subjects ≥ 16y underwent lymphodepletion with fludarabine (30mg/m x3) and cyclophosphamide (60mg/kg x1) prior to AUTO1 infusion, with the exception of the DLBCL cohort who additionally received a single dose of pembrolizumab (200mg) on day -1 to potentiate CAR-T expansion. AUTO1 dose varies based on the indication. Split dosing of 230 x106 CD19 CAR T-cells at day 0 and day 9 is employed in the CLL cohort. A single dose of 200 x106 CD19 CAR T-cells is delivered to patients with B-NHL. Study endpoints include feasibility of manufacture, grade 3-5 toxicity and remission rates at 1 and 3 months. Results: As of 8th February 2022, we enrolled 23 patients: 11 low grade NHL (LG-NHL:7 with FL and 3 with MCL), 7 DLBCL and 5 CLL. Apheresis was successful in all 23 patients and product manufacture was successful in 22 (pending in the last). 19 patients were infused: 10 with LG-NHL, 6 with DLBCL and 3 with CLL. 1 CLL patient was pending infusion at time of data cut-off and 2 patients died pre-infusion: 1 MCL patient, from COVID-19 and 1 CLL patient, from intracerebral haemorrhage. Patients treated with AUTO1 had a median age of 60 years (range 39-79), had received a median of 3 prior lines of treatment (range 2-8). Grade 1 CRS was reported in 6/19 and Grade 2 CRS in 3/19. No ICANS was observed in the B-NHL and CLL cohorts. CAR engraftment was observed in 13/13 patients evaluated by qPCR with ongoing persistence in 12/13 patients at last follow-up. In the LG-NHL and DLBCL cohorts 10/10 and 4/5 evaluable patients respectively were in CMR by 18FDG PET-CT post-treatment. Responses were ongoing in 9/10 LG-NHL at 12 months and in 4/4 DLBCL at months 1, 3, 3 and 6. In the CLL cohort, 2/3 evaluable patients achieved MRD negative remission in the bone marrow with residual small volume lymph nodes by CT at 6 and 3 months of follow-up respectively. 1 CLL patient did not engraft and had SD at month 1. Summary/Conclusion: AUTO1 has a tolerable safety profile in patients with r/r B-NHL and CLL despite high disease burden. Early data shows excellent complete remission rates and excellent CAR engraftment/expansion. Additional patients, updated data and longer follow up will be presented.

Chimeric Antigen Receptor T-Cells: An Overview of Concepts, Applications, Limitations, and Proposed Solutions.

Adaptive immunity, orchestrated by B-cells and T-cells, plays a crucial role in protecting the body from pathogenic invaders and can be used as tools to enhance the body’s defense mechanisms against cancer by genetically engineering these immune cells. Several strategies have been identified for cancer treatment and evaluated for their efficacy against other diseases such as autoimmune and infectious diseases. One of the most advanced technologies is chimeric antigen receptor (CAR) T-cell therapy, a pioneering therapy in the oncology field. Successful clinical trials have resulted in the approval of six CAR-T cell products by the Food and Drug Administration for the treatment of hematological malignancies. However, there have been various obstacles that limit the use of CAR T-cell therapy as the first line of defense mechanism against cancer. Various innovative CAR-T cell therapeutic designs have been evaluated in preclinical and clinical trial settings and have demonstrated much potential for development. Such trials testing the suitability of CARs against solid tumors and HIV are showing promising results. In addition, new solutions have been proposed to overcome the limitations of this therapy. This review provides an overview of the current knowledge regarding this novel technology, including CAR T-cell structure, different applications, limitations, and proposed solutions.

Review: Sustainable Clinical Development of CAR-T Cells - Switching From Viral Transduction Towards CRISPR-Cas Gene Editing.

T cells modified for expression of Chimeric Antigen Receptors (CARs) were the first gene-modified cell products approved for use in cancer immunotherapy. CAR-T cells engineered with gammaretroviral or lentiviral vectors (RVs/LVs) targeting B-cell lymphomas and leukemias have shown excellent clinical efficacy and no malignant transformation due to insertional mutagenesis to date. Large-scale production of RVs/LVs under good-manufacturing practices for CAR-T cell manufacturing has soared in recent years. However, manufacturing of RVs/LVs remains complex and costly, representing a logistical bottleneck for CAR-T cell production. Emerging gene-editing technologies are fostering a new paradigm in synthetic biology for the engineering and production of CAR-T cells. Firstly, the generation of the modular reagents utilized for gene editing with the CRISPR-Cas systems can be scaled-up with high precision under good manufacturing practices, are interchangeable and can be more sustainable in the long-run through the lower material costs. Secondly, gene editing exploits the precise insertion of CARs into defined genomic loci and allows combinatorial gene knock-ins and knock-outs with exciting and dynamic perspectives for T cell engineering to improve their therapeutic efficacy. Thirdly, allogeneic edited CAR-effector cells could eventually become available as “off-the-shelf” products. This review addresses important points to consider regarding the status quo, pending needs and perspectives for the forthright evolution from the viral towards gene editing developments for CAR-T cells.

Abstract LB009: A BCMA-specific allogeneic CAR-T cell therapy (CB-011) genome-engineered to express an HLA-E fusion transgene to prevent immune cell rejection

Abstract The approval and commercial launch of multiple first-generation CD19- or BCMA-directed, autologous CAR-T cell products have laid the foundation and opened a path for the development of more advanced cellular therapeutics, including CAR-T cell products with next-generation capabilities. Among these newer designs, allogeneic cell therapies are positioned to unlock the broad potential of engineered immune cells as a leading therapeutic modality. However, expansion, persistence, armoring, and trafficking of allogeneic CAR-T cells are critical to achieving long-term efficacy. Caribou Biosciences is advancing a BCMA-specific allogeneic CAR-T cell product candidate, CB-011, with an immune cloaking approach that includes both the removal of the endogenous B2M protein and the insertion of a B2M-HLA-E-peptide (B2M-HLA-E) fusion protein transgene. This strategy is designed to blunt CAR-T cell rejection by both recipient T cells and NK cells, and CB-011 is in preclinical development for relapsed or refractory multiple myeloma (r/r MM). We used Cas12a chRDNA guides in the manufacture of CB-011 to make four edits, including site-specific insertion of a proprietary humanized anti-BCMA CAR into the TRAC locus with high specificity and efficiency, thus eliminating TCR expression to prevent graft-versus-host disease (GvHD). In addition, we inserted a gene encoding a B2M-HLA-E fusion protein into the native B2M gene locus. This method simultaneously prevents the expression of the native B2M protein and expresses a minor HLA class I antigen HLA-E to blunt both T- and NK-mediated rejection of the CAR-T cells by the recipient’s immune system. B2M is a protein that stabilizes all HLA class I antigens on the cell surface, therefore its knockout eliminates endogenous HLA class I presentation on the surface of the CAR-T cells. To demonstrate that the B2M-HLA-E fusion protein expression protects CB-011 from NK-mediated cell killing, we developed an in vitro competition assay in which CB-011 cells are co-incubated with NK cells. We observed that CAR-T cells expressing the B2M-HLA-E fusion have a survival advantage over cells that do not express the fusion in the presence of NK cells in vitro, indicating that they could resist killing by a recipient’s NK cells and potentially circulate longer. The BCMA-specific CAR leads to long-term survival in mice bearing established orthotopically-engrafted MM tumor cells. Furthermore, mice treated with high doses of CB-011 did not experience symptoms typical of GvHD. This strategy should enable CB-011 CAR-T cells to remain in circulation longer in recipients, providing for increased potential of antitumor activity. Citation Format: Elizabeth Garner, Emilie Degagne, Suparna Roy, Paul Donohoue, Tristan Fowler, Morena Stanaway, Vanina Vicena, Devin Mutha, Benjamin Schilling, McKay Shaw, Mara Bryan, Leslie Edwards, Stephen Smith, Bryan Kohrs, Lynda Banh, Kyle McSweeney, Justin Skoble, Steven Kanner. A BCMA-specific allogeneic CAR-T cell therapy (CB-011) genome-engineered to express an HLA-E fusion transgene to prevent immune cell rejection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB009.

Abstract 551: UCART20x22: First allogeneic dual CAR T-cell therapy for the treatment of B-cell malignancies

Abstract Autologous CAR T-cell therapies have been transformative in the treatment of selected blood cancers. Despite this remarkable success, common mechanisms of resistance, such as tumor antigen escape, tumor heterogeneity and weaker CAR activity with low levels of antigen, emphasize the need to further optimize CAR T-cell therapies. In addition, there is a need to develop allogeneic “off-the-shelf” therapies that are readily available at the time of treatment decision. To address these challenges, we generated UCART20x22, the first allogeneic dual CAR T-cell targeting two well-validated antigens in B-cell malignancies, CD20 and CD22. Using in vitro cytotoxic and proliferation assays, we demonstrated that UCART20x22 displays strong activity against tumor cell lines with diverse CD20/CD22 antigen combinations, as well as increased activity against cells presenting both targets simultaneously. The specific activity of dual UCART20x22-cells persists overtime against tumor cells expressing both antigens (CD20, CD22) or only one. We also developed a pre-clinical model carrying subcutaneous lymphoma tumors expressing different antigen combinations in one single mouse. In this model, dual CAR T-cells provide efficient in vivo clearance of tumor cells expressing one or two antigens (CD20 and/or CD22) in a dose dependent manner, starting at a low dose of 1x106 CAR T-cells. Using a disseminated model of lymphoma with different combinations of antigen-expressing tumor cells, we demonstrated robust efficacy and persistence of UCART20x22 cells in the bone marrow. Furthermore, we used primary Non-Hodgkin Lymphoma patient samples expressing diverse CD22 and CD20 antigen levels to demonstrate that UCART20x22 displays robust and specific cytotoxic activity as well as IFNγ release in all tested combinations. Besides efficiently targeting two commonly expressed antigens in B-cell malignancies, UCART20x22 incorporates TALEN® mediated TRAC and CD52 specific gene editing to prevent Graft-vs-Host Disease and improve persistence in the presence of alemtuzumab (an anti-CD52 monoclonal antibody that can be used as part of a lymphodepleting regimen). These attributes allow the production of allogeneic CAR T-cells from healthy individuals that can be administered at the time of treatment decision. In summary, we show an efficient first in class allogeneic dual CAR T-cell product candidate with demonstrated in vitro and in vivo properties to overcome antigen escape in B-cell malignancies. Citation Format: Beatriz Aranda-Orgilles, Isabelle Chion-Sotinel, Steven Grudman, Ben Mumford, Chantel Dixon, Roman Galetto, Agnes Gouble, Laurent Poirot. UCART20x22: First allogeneic dual CAR T-cell therapy for the treatment of B-cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 551.

Engineering Next-Generation CAR-T Cells: Overcoming Tumor Hypoxia and Metabolism.

T cells engineered to express chimeric antigen receptors (CARs) have shown remarkable success in treating B-cell malignancies, reflected by multiple US Food and Drug Administration-approved CAR-T cell products currently on the market. However, various obstacles have thus far limited the use of approved products and constrained the efficacy of CAR-T cell therapy against solid tumors. Overcoming these obstacles will necessitate multidimensional CAR-T cell engineering approaches and better understanding of the intricate tumor microenvironment (TME). Key challenges include treatment-related toxicity, antigen escape and heterogeneity, and the highly immunosuppressive profile of the TME. Notably, the hypoxic and nutrient-deprived nature of the TME severely attenuates CAR-T cell fitness and efficacy, highlighting the need for more sophisticated engineering strategies. In this review, we examine recent advances in protein- and cell-engineering strategies to improve CAR-T cell safety and efficacy, with an emphasis on overcoming immunosuppression induced by tumor metabolism and hypoxia.

Toward Rapid, Widely Available Autologous CAR-T Cell Therapy – Artificial Intelligence and Automation Enabling the Smart Manufacturing Hospital

CAR-T cell therapy is a promising treatment for acute leukemia and lymphoma. CAR-T cell therapies take a pioneering role in autologous gene therapy with three EMA-approved products. However, the chance of clinical success remains relatively low as the applicability of CAR-T cell therapy suffers from long, labor-intensive manufacturing and a lack of comprehensive insight into the bioprocess. This leads to high manufacturing costs and limited clinical success, preventing the widespread use of CAR-T cell therapies. New manufacturing approaches are needed to lower costs to improve manufacturing capacity and shorten provision times. Semi-automated devices such as the Miltenyi Prodigy® were developed to reduce hands-on production time. However, these devices are not equipped with the process analytical technology necessary to fully characterize and control the process. An automated AI-driven CAR-T cell manufacturing platform in smart manufacturing hospitals (SMH) is being developed to address these challenges. Automation will increase the cost-effectiveness and robustness of manufacturing. Using Artificial Intelligence (AI) to interpret the data collected on the platform will provide valuable process insights and drive decisions for process optimization. The smart integration of automated CAR-T cell manufacturing platforms into hospitals enables the independent manufacture of autologous CAR-T cell products. In this perspective, we will be discussing current challenges and opportunities of the patient-specific but highly automated, AI-enabled CAR-T cell manufacturing. A first automation concept will be shown, including a system architecture based on current Industry 4.0 approaches for AI integration.

The EHA Research Roadmap: Malignant Lymphoid Diseases.

The EHA Research Roadmap: Malignant Lymphoid Diseases.