Allogeneic CAR-T Cells Research Articles

Allogeneic chimeric antigen receptor-T cells with CRISPR-disrupted programmed death-1 checkpoint exhibit enhanced functional fitness

Background aimsTherapeutic disruption of immune checkpoints has significantly advanced the armamentarium of approaches for treating cancer. The prominent role of the programmed death-1 (PD-1)/programmed death ligand-1 axis for downregulating T cell function offers a tractable strategy for enhancing the disease-modifying impact of CAR-T cell therapy. MethodsTo address checkpoint interference, primary human T cells were genome edited with a next-generation CRISPR-based platform (Cas9 chRDNA) by knockout of the PDCD1 gene encoding the PD-1 receptor. Site-specific insertion of a chimeric antigen receptor specific for CD19 into the T cell receptor alpha constant locus was implemented to drive cytotoxic activity. ResultsThese allogeneic CAR-T cells (CB-010) promoted longer survival of mice in a well-established orthotopic tumor xenograft model of a B cell malignancy compared with identically engineered CAR-T cells without a PDCD1 knockout. The persistence kinetics of CB-010 cells in hematologic tissues versus CAR-T cells without PDCD1 disruption were similar, suggesting the robust initial debulking of established tumor xenografts was due to enhanced functional fitness. By single-cell RNA-Seq analyses, CB-010 cells, when compared with identically engineered CAR-T cells without a PDCD1 knockout, exhibited fewer Treg cells, lower exhaustion phenotypes and reduced dysfunction signatures and had higher activation, glycolytic and oxidative phosphorylation signatures. Further, an enhancement of mitochondrial metabolic fitness was observed, including increased respiratory capacity, a hallmark of less differentiated T cells. ConclusionsGenomic PD-1 checkpoint disruption in the context of allogeneic CAR-T cell therapy may provide a compelling option for treating B lymphoid malignancies.

Abstract 899: Deciphering the benefits of variable delivery routes and molecular armoring to enhance efficacy of MUC1-CAR T-cells in targeting triple-negative breast cancer

Abstract As immune-therapies continue to develop for solid tumors, the tumor microenviroment (TME) poses many challenges that CAR T-cells need to overcome for efficient tumor cell clearance. Tumor immune evasion mechanisms, presence of immunosuppressive factors and cellular resistance to tumor infiltration are key mechanisms that the TME uses to reduce efficacy of T-cells against the tumor. Therapeutic options for triple negative breast cancer (TNBC) remain limited to date despite it being the most aggressive subtype of breast cancers and carrying the poorest prognosis. Tumor-associated MUC1 antigen is overexpressed in a large number of TNBC patients offering an effective discriminatory target for CAR T-cell therapy. Here, we present innovative strategies exploring alternative delivery routes and purposeful armoring of CAR T-cells to boost efficiency while preserving safety. To maintain anti-tumor CAR T-cell cytotoxic and proliferation in the hostile TME of solid tumors, we developed state-of-the-art multiplexed gene editing using high precision TALEN® technology. With that aim, we armored allogeneic CAR T-cells with specific attributes to release immune inflammatory cytokines and protect from inhibitory effects of the TGFB1 and PD1 pathways. A combination of strategies using allogeneic CAR T-cells with diverse attributes were evaluated against TNBC tumors in various in vivo pre-clinical models. Local CAR T-delivery resulted in effective control of tumor growth and robust antigen specific recognition. Furthermore, we assessed how the delivery route contributes to CAR-T cell distribution. CAR T-cell activity could also be increased systemically upon tumor-specific release of immunostimulatory cytokines without compromising safety. Our results demonstrate not only superior activity of armored MUC1-CAR T-cells resulting in tumor clearance but also in the recovery of normal glands. Thus, innovative strategies can be used to allow CAR T-cell efficiency in the hostile tumor microenvironment while preserving safety. Overall, our pre-clinical results underline the capability of armored allogenic MUC1-CAR T-cells to excel in the immune suppressive tumor micro-environment, suggesting that allogenic MUC1-CAR T-cell therapy could be an effective option in treating relapsed/refractory TNBC patients with limited therapeutic options. Citation Format: Piril Erler, Hana Cho, Jordan Skinner, Laurent Poirot, Beatriz Aranda-Orgilles. Deciphering the benefits of variable delivery routes and molecular armoring to enhance efficacy of MUC1-CAR T-cells in targeting triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 899.

Abstract 1775: Expansion of human T cells using xenogeneic free and gamma irradiated human platelet lysate

Abstract Commercially available human platelet lysate (hPL) is produced using expired human platelets obtained from accredited blood banks in the United States. These platelets were originally intended for use in patient transfusion. The safety of platelets used in transfusion is managed by the U.S. Food & Drug Administration (FDA), as well as the American Association of Blood Banks (AABB). These organizations set standards, including testing for transmissible diseases. The United States record for blood safety is well established, with extremely low rates of disease transmission, making the platelet units used for hPL manufacture low risk. The Covid-19 pandemic has increased awareness of emerging infectious diseases, even though transmission of Covid-19 via blood transfusion has not been documented. For that reason, gamma irradiated hPL offers an additional safety measure in the clinic. Chimeric Antigen Receptor (CAR) expressing T-cells have demonstrated potent clinical efficacy in patients with hematological malignancies. In addition, there are several phase I clinical trials evaluating the use of CAR-T-cells for targeting of solid tumor-associated antigens. Some of the challenging issues found during production of CAR-T cells are the efficiency of T cell transduction to generate CAR-T cells, the expansion of T cells to clinically relevant numbers and the long-term survival in vivo of the therapeutic cells. The use of human platelet lysate has been demonstrated to improve these issues. Our data from experiments performed using human CD3+ from donors demonstrates that human platelet lysates offer an improved performance on T cell expansion versus serum derived products. hPL efficiently promotes T cell expansion, with higher cell yields and lower cell exhaustion rate. Additionally, we efficiently developed a protocol for suspension culture of T cells, which could facilitate the large-scale expansion of allogeneic CAR-T cells. Citation Format: Vanesa Alonso-Camino. Expansion of human T cells using xenogeneic free and gamma irradiated human platelet lysate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1775.

524 - CB-011, a BCMA-Specific Allogeneic CAR-T Cell Therapy, Engineered with Next-Generation CRISPR Technology to Express an HLA-E Fusion Transgene to Prevent Immune Cell-Mediated Rejection, for r/r Multiple Myeloma

524 - CB-011, a BCMA-Specific Allogeneic CAR-T Cell Therapy, Engineered with Next-Generation CRISPR Technology to Express an HLA-E Fusion Transgene to Prevent Immune Cell-Mediated Rejection, for r/r Multiple Myeloma

Allogeneic gene-edited HIV-specific CAR-T cells secreting PD-1 blocking scFv enhance specific cytotoxic activity against HIV Env+ cells invivo

HIV-specific chimeric antigen receptor (CAR) T-cells have been developed to target HIV-1 infected CD4+ T-cells that express HIV Env proteins. However, T cell exhaustion and the patient-specific autologous paradigm of CAR-T cell hurdled clinical applications. Here, we created HIV-specific CAR-T cells using human peripheral blood mononuclear cells and a 3BNC117-E27 (3BE) CAR construct that enabled the expression of programmed cell death protein (PD-1) -blocking scFv E27 and the single-chain variable fragment of the HIV-1-specific broadly neutralizing antibody 3BNC117 to target native HIV Env. Compared with T cells expressing 3BNC117-CAR alone, 3BE CAR-T cells showed greater cytotoxic activity against HIV Env+ cells with stronger proliferation capability, higher killing efficiency, and enhanced cytokine secretion in the presence of HIV Env-expressing cells. Furthermore, we manufactured TCR-deficient 3BE CAR-T cells through gene editing and demonstrated that these CAR-T cells could effectively kill HIV Env ​+ ​cells in vivo without the occurrence of severe graft-versus-host disease (GvHD) in NSG mice. These data suggest that we have provided a feasible approach to the generation of “off-the-shelf” anti-HIV CAR-T cells in combination with PD-1 checkpoint blockade immunotherapy, which can be a powerful therapeutic candidate for the functional cure of HIV.

Clinical Pharmacology and Determinants of Response to UCART19, an Allogeneic Anti-CD19 CAR-T Cell Product, in Adult B-cell Acute Lymphoblastic Leukemia.

UCART19 is an "off-the-shelf" genome-edited anti-CD19 chimeric antigen receptor (CAR)-T cell product, manufactured from unrelated healthy donor cells. UCART19 was administered to 25 adult patients with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL) in the CALM trial. All patients underwent lymphodepletion with fludarabine and cyclophosphamide ± alemtuzumab and received one of three ascending doses of UCART19. Given the allogeneic nature of UCART19, we analyzed the impact of lymphodepletion, HLA disparities, and host immune system reconstitution on its kinetics, along with other factors known to affect autologous CAR-T cell clinical pharmacology. Responder patients (12/25) had higher UCART19 expansion (C max) and exposure (AUCTlast) than nonresponders (13/25), as measured by transgene levels in peripheral blood. The persistence of CAR+ T cells did not exceed 28 days in 10/25 patients and lasted beyond 42 days in 4/25. No significant correlation was found between UCART19 kinetics and administered cell dose, patient and product characteristics or HLA disparities. However, the number of prior lines of therapy and absence of alemtuzumab negatively impacted UCART19 expansion and persistence. Alemtuzumab exposure positively affected IL7 and UCART19 kinetics, while negatively correlating with host T lymphocyte AUC0-28. UCART19 expansion is a driver of response in adult patients with R/R B-ALL. These results shed light on the factors associated with UCART19 kinetics, which remain highly affected by the impact of alemtuzumab on IL7 and host-versus-graft rejection. First description of the clinical pharmacology of a genome-edited allogeneic anti-CD19 CAR-T cell product showing the crucial role of an alemtuzumab-based regimen in sustaining UCART19 expansion and persistence through increased IL7 availability and decreased host T lymphocyte population.

Mechanistic Modeling of the Interplay Between Host Immune System, IL-7 and UCART19 Allogeneic CAR-T Cells in Adult B-cell Acute Lymphoblastic Leukemia.

A mathematical mechanistic pharmacokinetic/pharmacodynamic model supports and captures quantitatively the beneficial impact of lymphodepleting patients before the infusion of an allogeneic CAR-T cell product. Mediation through IL-7 increase and host T lymphocytes decrease is underlined, and the model can be further used to optimize CAR-T cell therapies lymphodepletion regimen.

Development of an Off-the-Shelf Chimeric Antigen Receptor (CAR)-T Cell Therapy for T-Cell Acute Lymphoblastic Leukemia (T-ALL) without Gene Editing

CAR-T cell therapy has revolutionized the treatment of B-lineage ALL and other B-cell malignancies, but the development of cell therapies for T-cell malignancies has lagged behind. For T-ALL, CD7 is an excellent CAR target because it is consistently and highly expressed in leukemic cells and is retained during chemotherapy and at relapse. However, CD7 is also expressed in normal T-cells and the expression of anti-CD7 CARs triggers T-cell fratricide, resulting in a marked decrease in viable and/or effective CAR-T cells. We developed an anti-CD7 CAR-T cell product (PCART7) by utilizing a CD7 protein expression blocker (PEBL) to downregulate CD7 surface expression and prevent fratricide. PEBLs consist of a target binding domain fused to an intracellular retention domain that enables relocalization of surface proteins intracellularly (Png et al. Blood Adv 2017; Kamiya et al. Blood Adv 2018; Kamiya et al., JCI 2019). Autologous PCART7 cells lacking surface CD7 and expressing anti-CD7 CARs can be effectively produced under cGMP conditions and the technology is being tested in clinical trials for patients with relapsed/refractory T-ALL or T-cell lymphoblastic lymphoma. Generation of autologous CAR-T cells, however, may be hampered by the presence of a high percentage of leukemic cells in peripheral blood and/or low T cell numbers. An allogeneic anti-CD7 CAR-T cell product available "off-the shelf" would represent a useful tool to reduce leukemic burden and provide a bridge to hematopoietic cell transplant or to autologous CAR-T cell therapy. Because allogeneic T-cells can be activated upon recognition of host tissue antigens by their T-cell receptor (TCR)/CD3 complex, their infusion carries a risk of causing graft-versus-host disease (GvHD). We optimized a CD3 PEBL that can effectively downregulate surface TCR/CD3. We then developed a lentiviral-based process that allows simultaneous expression of CD3 and CD7 PEBLs together with anti-CD7 CAR. The PEBLs retained surface CD7 and TCR/CD3 intracellularly, thereby minimizing fratricide and abrogating TCR-mediated antigen recognition. PCART7 cells lacking TCR/CD3 were manufactured from healthy donor T-cells by using a double transduction with a bicistronic lentiviral vector delivering CD7 PEBL and anti-CD7 CAR (PCART7 LVV), and a second lentiviral vector delivering CD3 PEBL. Robust expansion of PCART7-CD3 PEBL cells was observed and more than 90% of the cells were of the desired CAR+CD7-CD3- phenotype. Following depletion of residual CD3/TCRαβ+ cells, CD3- cell purity was >99% (Fig. 1A). These results were reproduced across multiple donors. The efficient downregulation of surface TCR/CD3 by CD3 PEBL was similar to that achieved by CRISPR knockout of the TCR alpha chain locus (TRAC) and completely abrogated activation in response to TCR stimulation. In vitro short- and long-term assays confirmed strong cytotoxic potency against CD7+ leukemic cells but not CD7- target cells. Importantly, PCART7 cells were equally cytotoxic regardless of whether they were co-transduced with CD3 PEBL. In a xenograft model using the CCRF-CEM T-ALL cell line, PCART7-CD3 PEBL cells, like PCART7 cells, effectively suppressed tumor growth and improved survival (Fig. 1B). This study demonstrates the feasibility of using two PEBLs simultaneously for intracellular protein retention without perturbing critical CAR-T cell functions. The use of PEBL technology to generate allogeneic anti-CD7 CAR-T cells fits seamlessly in cGMP manufacturing protocols and avoids the risk of genotoxicity arising from the use of gene-editing techniques. Because the resulting allogeneic anti-CD7 CAR-T cells had high potency and were devoid of alloreactivity potential, they represent an attractive new tool for off-the-shelf CAR-T cell therapy of T-ALL and other CD7+ malignancies. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Allogeneic CAR-T Therapy Enabled By Base Editing of Lck to Resist Dasatinib Used to Prevent Rejection Mediated through Both T and NK in Host

We are reporting a new strategy to overcome the rejection of the universal CAR-T cells by host through inhibiting host T and NK cells with dasatinib, while preserving the function of UCAR-T through base editing of LCK, a critical tyrosine kinase for T and NK cell activation. Autologous chimeric antigen receptor (CAR) T cells have changed the therapeutic landscape in hematological malignancies. The universal CAR-T (UCAR-T) therapies derived from healthy donor hold many promises, such as better function of CAR-T cells, off-shelf in availability, reduced variation in product quality and decreased cost. However, one challenge facing UCAR-T therapy is rapidly elimination of allogeneic cells by the host immune system. Although many strategies, such as clearance of host T/NK cells with alemtuzumab and knockout of B2M gene, have been developed and tested with some success in patients, novel ways to circumvent the surveillance of the host immune system against allogeneic UCAR-T cells are still under intensive investigation. The threonine (T316) in LCK, similar to T315 in Abl kinase, is a gatekeeping amino acid in ATP pocket. The T315I mutation of ABL kinase is the most prevalent mutation in imatinib resistant patients without affecting its kinase function. the LCK-T316M mutation has previously been shown to render T cells resistant to dasatinib. Therefore, we hypothesized that T316I mutation, achievable with cytosine base editing, can also make LCK resistant to dasatinib. In vitro experiments confirmed that in the presence of dasatinib, T cells with LCK-T316I point mutation could still be stimulated by anti-CD3/CD28 DynaBeads and expressed higher level of CD25, CD69, and 4-1BB while the wildtype T and NK cells were completely inhibited. CD19 targeting UCAR-T (Km UCAR-T) were generated by knocking out TRAC, B2M and base-editing of LCK simultaneously. Mixed lymphocyte reactions (MLRs) were performed by the incubation of Km UCAR-T and PBMC from HLA mismatched donors upon dasatinb or DMSO treatment. It was demonstrated that only the Km UCAR-T cells could survive and expand under the action of dasatinib. Besides, higher dasatinib concentrations could increase the proportion of Km UCAR-T cells even more. Dasatinib is an approved small molecule drug, with well understood safety profile in patients. Furthermore, dasatinib had no effect on the proliferation of T and NK cell, implying that the immune function can be quickly restored after withdrawing. Allogeneic CAR-T cells have the potential to overcome the limitations of autologous CAR-T cells and Km UCAR-T cells will be a new orthogonal approach that can be combined with other strategies to achieve the goal. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

CD82 Is Sufficient to Uniquely Identify Pluripotent Stem Cell-Derived Hemogenic Endothelium with the Hematopoietic Lineage Potency to Give Rise to Bona Fide Lymphocytes

Chimeric antigen receptor (CAR)-T and natural killer (NK) cells represent compelling opportunities for the treatment of hematological malignancies and solid tumors. However, autologous and donor-derived allogeneic CAR-T and NK cell therapies are limited based on the quantity and quality of the starting engineered cells. We previously described our off-the-shelf induced pluripotent stem cell (iPSC)-derived CAR-T (CAR-iT) and CAR-NK (CAR-iNK) cell platforms that eliminate some of the hurdles associated with patient-derived cells by creating a homogenous cell product that is derived from a renewable bank of iPSC master cell lines. A major challenge in the manufacturing of iPSC derived immune cells is the derivation of hemogenic endothelium (HE), which is the rare and transient direct precursor of hematopoietic cells, making its in vitro specification from iPSCs inefficient and challenging. Furthermore, there is not a reliable marker or set of markers for the identification of HE in culture. HE identification has traditionally relied on detection of the transcription factor RUNX1 and the establishment of reporter cell lines, neither of which are suitable for the manufacture of HE cells destined to become therapeutic immune cells. We have established a cytokine driven protocol for the differentiation of iPSCs that improves the efficiency of the derivation of RUNX1+ HE cells. To identify candidate HE surface markers, we compared iPSC-derived cells containing a high percentage of HE, based on RUNX1 expression, to control differentiated cells containing a low percentage of HE, via a cell surface antibody array. Through the array we identified several surface markers that were expressed by a higher percentage of CD34+ endothelial cells compared to control CD34+ cells. Several of the candidates identified, including CD44, CD61, CD226 and CD143 have been previously identified by other groups as candidate HE markers, thereby validating our screening method. We identified CD82 as a novel candidate HE marker, with expression uniquely restricted to the CD34+ endothelial population and positively correlating with RUNX1 expression (Fig. 1A). Furthermore, we demonstrated via limiting dilution assays that HE is enriched in the CD34+CD43-CD82+ population (HE frequency, 1/38) compared to the CD34+CD43-CD82- population(HE frequency, 1/736) (Fig. 1B). Even more strikingly, isolation of cells based on CD82 alone was sufficient to enrich for HE cells (HE frequency, 1/44) and outcompeted CD34 enrichment alone, suggestive of its unique ability to identify the HE population (Fig. 1B). To determine if CD82+ HE cells could give rise to lymphocytes we carried them forward into T cell differentiation and showed thatthey efficiently gave rise to CAR-iT cells (i.e. >95% CD7+ T lymphocytes). The identification of CD82 for the enrichment of iPSC derived HE is a critical step to the development of definitive hematopoiesis with ability to give rise to all lineages of the hematopoietic compartment and facilitates the scaled and consistent manufacture of high-quality T and NK cells for off-the-shelf therapeutic applications. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Allogeneic Donor-Derived Myeloid Antigen Directed CAR-T Cells - for Relapsed/Refractory Acute Myeloid Leukemia in Children after Allogeneic Hematopoietic Stem Cell Transplantation: Report of Three Cases

Introduction The outcome of HSCT among children with chemorefractory acute myeloid leukemia (AML) is poor. The incidence of relapse exceeds 50%. We hypothesized that myeloid antigen-directed CAR-T cell therapy may induce remission in children with refractory AML. Patients and methods Three pediatric patients with R/R AML were enrolled in a compassionate-use program. All cases had AML, relapsing after multiple lines of treatment, including previous HSCT (n=3). One patient had CD123-positive leukemia cell population in the bone marrow, 2 pts had CD33-positive population. At the time of allogeneic CAR-T application disease burden was overt leukemia (n=3), all patients had full donor chimerism in the CD3 cells. Baseline characteristics of the pts are summarized in table 1. Allo-CAR-T were derived from previous HSCT donors (haplo n=2, MRD n=1). The FlA chemotherapy regimen was used to reduce tumor burden, followed by the fludarabine and cyclophosphamide for lymphodepletion before CAR-T cell infusion in all patients. One patient received CD123 directed allo-CAR-T cells, 2 pts - CD33 CAR-T cells. Both CD33 and CD123 constructs were 2-nd generation 4-1BBz CARs, modified based on the published Ukrainskaya et al. Clinical grade manufacturing was performed on Prodigy bioreactor platform with retroviral transduction. Results Cytokine release syndrome (CRS) occurred in 3 patients and was grade ≤3, all pts received tocilizumab with good effect. Two patients had neurologic events (ICANS grade 1-2). In one patient rash and liver toxicity coincided with CRS, potentially representing aGVHD, with full response to corticosteroids. All pts had grade III/IV hematological toxicities. The median time to CAR-T cell peak expansion was 14 days. At 4 weeks after CAR-T infusion two patients achieved complete MRD negative remission with incomplete count recovery and good CAR-T cell expansion. One pt. had MRD positive remission with CD33 loss on residual blast cells. Two patients (#1 and #2) on d +37 and +45 receivedTotal body irradiation (TBI) at 4 Gy and TCRαβ+ depleted graft from original donors. Abatacept on d 0, +7, +14 was used as GVHD prophylaxis. One pt. (#3) received second HSCT on d +30 from haploidentical donor with full intensity TBI-based regimen. All patients engrafted recovered hematopoietic function) and had MRD-negative remission with full donors chimerism on d+30. Two patients, #1 and #2, relapsed after 4 and 2 months after graft infusion, respectively. Patient #3 remains in MRD-negative remission on day +100. All patients alive with median follow up 164 days (102-196). Conclusion Our early experience suggests that allogeneic myeloid antigen-directed CAR-T cells are effective as cytoreduction and have manageable toxicity. Myeloid antigen-directed CAR-T therapy can be used as a bridge to allo HSCT in r/r AML. We have documented CAR-T expansion and persistence. Prospective testing of the approach is warranted. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A First-in-Human Phase 1, Multicenter, Open-Label Study of CB-010, a Next-Generation CRISPR-Edited Allogeneic Anti-CD19 CAR-T Cell Therapy with a PD-1 Knockout, in Patients with Relapsed/Refractory B Cell Non-Hodgkin Lymphoma (ANTLER Study)

Background: Autologous CAR-T cell therapies have shown significant benefit in the treatment of adults with relapsed/refractory B cell non-Hodgkin lymphoma (r/r B-NHL). However, these CAR-T cell therapies have inherent challenges including functional deficiencies in the patient's T cells that could yield an inconsistent or impaired product. In addition, the extended time and complexity in manufacturing the product can limit patient access and potentially require bridging therapy. CB-010 is an allogeneic, off-the-shelf anti-CD19 CAR-T cell therapy derived from healthy donor T cells; it is the lead program in Caribou's allogeneic CAR-T cell therapy platform. Patients (pts) receiving CB-010 do not require leukapheresis or bridging therapy. A next-generation CRISPR-Cas9 genome-editing technology developed at Caribou, which uses Cas9 and CRISPR hybrid RNA-DNA (chRDNA) guides to significantly reduce off-target editing, was implemented to generate 3 genome edits in the manufacture of CB-010: (i) knockout of the TRAC gene to eliminate TCR expression to reduce the risk of graft-versus-host disease (GvHD), (ii)site-specific insertion of a CD19-specific CAR into the TRAC locus, and (iii) knockout of the gene encoding PD-1, designed to limit premature CAR-T cell exhaustion and enhance antitumor activity. Statistically significant preclinical survival benefit across B-NHL subtypes and longer-duration efficacy in vivo compared to similar CAR-T cells without a PD-1 knockout support the clinical evaluation of CB-010. Methods and Study Design: CB-010 is being investigated in a multicenter, Phase 1 clinical trial in pts with r/r B-NHL. A 3+3 dose escalation design followed by expansion at the maximum tolerated dose (MTD) and/or the recommended phase 2 dose (RP2D) is being utilized. Primary objectives are to determine the safety and tolerability of CB-010 and the RP2D. Additional key objectives include preliminary antitumor activity and pharmacokinetics (PK). After serially receiving lymphodepletion therapy with cyclophosphamide (60mg/kg/day for 2 days) and fludarabine (25mg/m2/day for 5 days), pts receive a single dose infusion of CB-010 and are followed for safety and efficacy. Initial antitumor activity, as well as time to progression, overall survival, and disease response as measured by Cheson criteria will be evaluated. The study is actively enrolling patients. Cohort 1 at an initial single dose of 40 million CAR-T cells has been completed successfully with 6 patients. Dose escalation of CB-010 is ongoing, and patients are being enrolled in cohort 2 to receive a single dose of 80 million CAR-T cells. The ANTLER Phase 1 trial is registered on clinicaltrials.gov (NCT04637763).

Ameli-01: A Phase I Trial of UCART123v1.2, an Anti-CD123 Allogeneic CAR-T Cell Product, in Adult Patients with Relapsed or Refractory (R/R) CD123+ Acute Myeloid Leukemia (AML)

Introduction: UCART123v1.2 is a genetically modified allogeneic T-cell product manufactured from healthy donor cells. Donor-derived T-cells are transduced using a lentiviral vector to express the anti-CD123 chimeric antigen receptor and are further modified using Cellectis' TALEN® technology to disrupt the T-cell receptor alpha constant (TRAC) and CD52 genes to minimize risk of graft-vs-host disease (GvHD) and allow use of anti-CD52-directed therapy as part of lymphodepletion (LD). AMELI-01 (NCT04106076) is a phase 1, open-label, dose-escalation trial evaluating the safety, tolerability, expansion, and persistence of UCART123v1.2 given at escalating dose levels after LD with either fludarabine and cyclophosphamide (FC) or FC with alemtuzumab (FCA) in patients (pts) with R/R CD123+ AML. Alemtuzumab was added to the LD regimen to sustain host T- and NK-cell depletion and to promote UCART123v1.2-cell expansion and persistence. Methods: Key eligibility criteria include pts 18-65 yrs, adequate organ function, ECOG PS ≤ 1, and blasts positive for CD123 by flow cytometry. Pts must have received ≥2 cycles of chemotherapy (one with standard dose cytarabine), ≥ 1 cycle of a high/intermediate dose cytarabine containing regimen, ≥ 2 cycles of an HMA combination regimen, or prior allogeneic HSCT. After LD with FC or FCA, pts received UCART123v1.2 at one of the following dose levels: 2.5x105 cells/kg (DL1); 6.25x105 cells/kg (DL2); 1.5x106 cells/kg (DL2i); or 3.03x106 cells/kg (DL3). The primary endpoint is the safety, tolerability, and MTD/RP2D of UCART123v1.2. DLTs are assessed over a 28d observation period after UCART123v1.2 infusion. Additional endpoints include investigator assessed anti-leukemic activity per 2017 ELN criteria, and expansion, trafficking, and persistence of UCART123v1.2 (assessed in peripheral blood [PB] and bone marrow [BM] by flow cytometry and vector copy number [VCN]). Measurable residual disease (MRD) was analyzed by local multiparameter flow cytometry with a sensitivity of 0.02%. Results: As of July 1, 2022, 16 pts received LD and UCART123v1.2; 8 pts received UCART123v1.2 after FC (DL1 [n=2]; DL2 [n=3]; DL2i [n=2]; DL3 [n=1]), and 8 pts received UCART123v1.2 after FCA (DL2). Pts were high risk and heavily pretreated with disease characteristics outlined in Table 1. Median baseline BM and PB % blasts was 37% (0-88%) and 19% (0-79%), and median baseline CD123 positivity by central flow cytometry in the FC and FCA arms was 82% (17-98%) and 84% (25-97%), respectively. Four pts experienced DLTs: 3 pts in FC arm (G4 CRS [1pt in DL2i and 1pt in DL3] and G3 ICANS [1pt in DL2i]); and 1 pt in FCA arm (G5 CRS in DL2). Cytokine release syndrome (CRS) occurred in 15/16 pts (FC arm [n=7]; FCA arm [n=8]), of which 3 pts experienced ≥ G3 CRS which were classified as DLTs as noted above (FC arm G4 [n=2]; FCA arm G5 [n=1]). Responses were assessed beginning on D28. Evidence of UCART123v1.2 activity was observed in 4/16 pts with best overall responses as follows: FC arm (DL2: 1 SD; DL2i: 1 MLFS); FCA arm (DL2: 1 SD and 1 MRD-negative CR). The pt in the DL2 FCA arm with SD achieved greater than 90% BM blast reduction (60% to 5%) at D28. The pt with CR, who had failed 5 prior lines of therapy including HSCT and had baseline G4 cytopenias, had CRi at D28 with full count recovery at D56 and remains in an MRD-negative CR beyond the 8-month f/u visit. Adequate lymphodepletion was not achieved in the FC arm, as host lymphocyte recovery was observed in 7/8 pts prior to D28, and only 3/8 pts had UCART123v1.2 expansion (Cmax ranged from 3,757 to 27,828 copies/μg DNA). In contrast, FCA LD resulted in robust lymphodepletion for greater than 28 days in all pts, and 6/8 demonstrated UCART123v1.2 expansion, with Cmax ranging from 13,177 to 330,530 copies/μg DNA, an almost 9-fold increase compared with FC LD. An increase in inflammatory markers, notably serum ferritin, and serum cytokines (including IL-2, IL-6, IL-10, IL-15, IFN-γ, and TNF-α) in the FCA arm correlated with both UCART123v1.2 expansion and CRS. Conclusions: Adding alemtuzumab to the FC regimen was associated with improved LD and significantly higher UCART123v1.2 cell expansion and persistence, which correlated with improved activity and safety, including one pt in the DL2 FCA arm who achieved a durable MRD-negative CR. Overall, these data support the safety and activity of UCART123v1.2 after FCA LD in pts with CD123+ R/R AML. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

A Novel Protein-Based Approach to Generate Allogeneic CAR-T Cells with Simultaneous TCR and MHC Class 1 Downregulation

A Novel Protein-Based Approach to Generate Allogeneic CAR-T Cells with Simultaneous TCR and MHC Class 1 Downregulation

Efficacy and safety of universal (TCRKO) ARI-0001 CAR-T cells for the treatment of B-cell lymphoma.

Autologous T cells expressing the Chimeric Antigen Receptor (CAR) have been approved as advanced therapy medicinal products (ATMPs) against several hematological malignancies. However, the generation of patient-specific CAR-T products delays treatment and precludes standardization. Allogeneic off-the-shelf CAR-T cells are an alternative to simplify this complex and time-consuming process. Here we investigated safety and efficacy of knocking out the TCR molecule in ARI-0001 CAR-T cells, a second generation αCD19 CAR approved by the Spanish Agency of Medicines and Medical Devices (AEMPS) under the Hospital Exemption for treatment of patients older than 25 years with Relapsed/Refractory acute B cell lymphoblastic leukemia (B-ALL). We first analyzed the efficacy and safety issues that arise during disruption of the TCR gene using CRISPR/Cas9. We have shown that edition of TRAC locus in T cells using CRISPR as ribonuleorproteins allows a highly efficient TCR disruption (over 80%) without significant alterations on T cells phenotype and with an increased percentage of energetic mitochondria. However, we also found that efficient TCRKO can lead to on-target large and medium size deletions, indicating a potential safety risk of this procedure that needs monitoring. Importantly, TCR edition of ARI-0001 efficiently prevented allogeneic responses and did not detectably alter their phenotype, while maintaining a similar anti-tumor activity ex vivo and in vivo compared to unedited ARI-0001 CAR-T cells. In summary, we showed here that, although there are still some risks of genotoxicity due to genome editing, disruption of the TCR is a feasible strategy for the generation of functional allogeneic ARI-0001 CAR-T cells. We propose to further validate this protocol for the treatment of patients that do not fit the requirements for standard autologous CAR-T cells administration.

OPTIMIZED PROTOCOLS OF EXPANSION OF PERIPHERAL BLOOD-DERIVED δT-CELLS FOR THE ESTABLISHMENT OF AN OFF-THE-SHELF ADVANCED CELL THERAPY AGAINST CANCER

The use of autologous T cells expressing chimeric antigen receptors (CAR) has revolutionized the treatment of hematological malignancies. However, the use of allogeneic CAR-T cells offers many advantages over autologous therapies, including the immediate availability of cryopreserved batches for infusion and the use of healthy donor cells since it is possible to obtain high amounts of fully functional cells. Moreover, this strategy also allows for the scaling up of the production of CAR-T cells resulting in cost reduction. Cells that do not express TCR αβ, such as gamma delta (γδ) T, are an alternative source to allow widespread allogeneic therapy possible. Their ability to recognize targets in an MHC-independent manner and the expression of innate immunity receptors increases the potential of using these cells in immunotherapies. Aiming to improve the effectiveness of γδ T-cell-based immunotherapies against tumors, we tested various optimization strategies. The most widely used protocol for selectively expanding Vδ2T cells relies on zoledronic acid (ZOL) stimulation of PBMCs in combination with IL-2 or/and IL-15 cytokines. Here, we tested different concentrations of IL-2 (100 and 1,000 U/mL) and IL-15 (12.5 and 100 ng/mL) with 5 μM zoledronic acid. In addition, we compared cell expansion in three defined cell culture media, OpTmizer T Cell Expansion SFM, X-vivo10, and TexMACS (supplemented with 2% AB serum). A total of twenty-four different combinations of IL-2, IL-15, and media were tested in the present study.After twelve days of culture, cells were counted and labelled with anti-CD3, anti-CD8, anti-CD4, anti-Vδ2, anti-PD1, anti-CD69, and anti-NKG2D antibodies for flow cytometry analysis. Our results demonstrated that although TexMACS medium is capable of supporting T-cell activation and expansion, it was not optimal for maximal expansion of Vδ2T cells (fold expansion 5.99 ± 1.61). However, the fold expansion was similar in OpTmizer and X-vivo10 media (fold expansion 21.38 ± 5.13 and 18.37 ± 5.42, respectively). Among the medium supplementation strategies, cultivation in OpTmizer combined with 100 U/mL IL-2 + 12.5 ng/mL IL-15 led to highest expansion index (32.4-fold). As for the activation markers of Vδ2T cells, the frequency of NKG2D+ cells was similar for all conditions tested (above 85%). However, the percentage of CD69+ cells was higher in cells cultured in TexMACS medium compared to OpTmizer and X-vivo10 media (81.6±5.3, 69±9.9, 60.2±7.2, respectively). It is known that the Programmed Cell Death-1 receptor (PD-1) plays an important role in regulating immune cell function. We examined the expression of PD-1 after twelve days of expansion. We observed lower levels of PD-1 in cells expanded in X-vivo10 medium supplemented with 1,000 U/mL IL-2 + 100 ng/mL IL-15, whereas levels of expression were up-regulated significantly when cells were cultivated in the same conditions without IL-15 (28.7% versus 70.6%). Cells expanded in either OpTimizer or TexMACS demonstrated a similar frequency to PD-1 positive cells (41±13% and 49.7%±9.5). In conclusion, we reported an optimized protocol to improve the expansion and the quality of Vδ2 T cells. These findings set the groundwork for the establishment of an off-the-shelf advanced cellular immunotherapy using this alternative population of T cells.

CRISPR-edited CAR-T cells: Using CRISPR-Cas9 to Improve CAR-T Therapy

One of the cornerstones of cancer immunotherapy, chimeric antigen receptor T cell (CAR-T) immunotherapy is a treatment comprising of T cells transfected with artificial receptors that target a specific tumor antigen, potentiating tumor destruction. Despite the effectiveness of this technique in treating hematopoietic malignancies, efficacy against other cancers leaves much to be desired. CAR-T therapy's anti-tumor effectiveness, safety, and accessibility are hampered by issues such T cell exhaustion, toxicity, and ineffective production techniques. With the advent of CRISPR-Cas9 technology, allowing ease of genome editing, it is now possible to address these challenges. By introducing a double-strand break at a particular genomic location, this gene editing technology can be utilized to target inhibitors of T lymphocyte function, directed to specific loci to produce a less toxic product, and engineer allogeneic CAR-T cells. However, CRISPR-Cas9 confers its own limitations, including off-target editing. This review introduces the applications of CRISPR technology to CAR-T therapy and evaluates how the technology can optimize the effectiveness, safety, and product availability of this cancer immunotherapy. This paper also addresses some of the potential drawbacks of CRISPR-edited CAR-T cells.

298P Stable, controllable, and scalable manufacturing of B7-H3 UCAR-T, an allogeneic CAR-T product for advanced glioma, from healthy donors

The CAR-T cell therapy has achieved great success in hematological malignancies and is gradually being used in solid tumors. Autologous CAR-T cell therapy targeting B7-H3 (CD276) has been studied and achieved preliminary results. However, autologous products are expensive and time-consuming to manufacture, and significant variability in patient T cells characteristics. We are developing the allogeneic universal CAR-T cells targeting B7-H3 (B7-H3 UCAR-T) for the treatment of advanced glioma, an off-the-shelf product candidate manufactured from serial healthy donor material and avoids many disadvantages of autologous CAR-T products.

300P The clinical results of an investigator initiated trial of allogeneic CAR-T cells targeting IL13Rα2 in the treatment of high-grade glioma

Glioblastoma is the most common intracranial malignant tumors, which is difficult to treat and has poor prognosis. T cell immunotherapy is becoming a powerful strategy for the treatment of cancer and may provide an opportunity to improve the prognosis of patients with advanced glioma.This trial has three purposes: 1) to evaluate the safety and feasibility of allogeneic CAR-T cells in human body; 2) Lumbar injection and intratumoral injection were compared; 3) Preliminarily evaluate the effectiveness of our product in patients with advanced glioma.

Serologic response and safety of COVID-19 vaccination in HSCT or CAR T-cell recipients: a systematic review and meta-analysis

BackgroundPatients receiving hematopoietic stem cell transplantation (HSCT) or chimeric antigen receptor T cell (CAR T-cell) therapy are immunocompromised and at high risk of viral infection, including SAR2-CoV-2 infection. However, the effectiveness and safety of COVID-19 vaccines in these recipients is not well characterized. The present meta-analysis evaluated the serologic response and safety of COVID-19 vaccines in these population.MethodsLiterature databases (MEDLINE, EMBASE, Web of Science, MedRvix and BioRvix) were searched for original studies with serologic response post COVID-19 vaccination in HSCT or CAR T-cell recipients published until July 14, 2022. The analysis included 27 observational studies with a total of 2899 patients receiving allogeneic HSCT (2506), autologous HSCT (286) or CAR T-cell therapy (107), and 683 healthy participants with serologic response data. Random effects models were used to pool the rate of serologic response to COVID-19 vaccination in HSCT or CAR T-cell recipients and odds ratio comparing with healthy controls.ResultsThe pooled seropositivity rates in HSCT and CAR T-cell recipients were 0.624 [0.506–0.729] for one dose, 0.745 [0.712–0.776] for two doses. The rates were significantly lower than those in healthy controls (nearly 100%). In subgroup analysis, CAR T-cell recipients exhibited an even lower seroconversion rate (one dose: 0.204 [0.094–0.386]; two doses: 0.277 [0.190–0.386]) than HSCT counterparts (one dose: 0.779 [0.666–0.862]; two doses: 0.793 [0.762–0.821]). The rates were comparable between autologous and allogeneic HSCT recipients. Other possible impact factors related to seropositivity were time interval between therapy and vaccination, use of immunosuppressive drugs and immune cell counts. Most vaccine-related adverse effects were mild and resolvable, comparable to general population.ConclusionsThis analysis revealed a diminished response to COVID-19 vaccines in HSCT or CAR T-cell recipients. Our findings may inform regular COVID-19 vaccination at appropriate intervals after HSCT or CAR T-cell therapy.