chimeric-antigen-receptor-t-cell-manufacturing-on-an-automated-cell

Chimeric Antigen Receptor T Cell Manufacturing on an Automated Cell Processor

Chimeric antigen receptor (CAR)-T cells represent a promising immunotherapeutic approach for the treatment of various malignant and non-malignant ...

Children’s Hospital of Philadelphia

The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms.

Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy.

Engineered T cell therapies have begun to demonstrate impressive clinical responses in patients with B cell malignancies. Despite this efficacy, many patients are unable to receive T cell therapy because of failure of in vitro expansion, a necessary component of cell manufacture and a predictor of in vivo activity. To evaluate the biology underlying these functional differences, we investigated T cell expansion potential and memory phenotype during chemotherapy in pediatric patients with acute lymphoblastic leukemia (ALL) and non-Hodgkin lymphoma (NHL). We found that patients with T cell populations enriched for early lineage cells expanded better in vitro and that patients with ALL had higher numbers of these cells with a corresponding enhancement in expansion as compared to cells from patients with NHL. We further demonstrated that early lineage cells were selectively depleted by cyclophosphamide and cytarabine chemotherapy and that culture with interleukin-7 (IL-7) and IL-15 enriched select early lineage cells and rescued T cell expansion capability. Thus, early lineage cells are essential to T cell fitness for expansion, and enrichment of this population either by timing of T cell collection or culture method can increase the number of patients eligible to receive highly active engineered cellular therapies.

Early memory phenotypes drive T cell proliferation in patients with pediatric malignancies.

Potent CD19-directed immunotherapies, such as chimeric antigen receptor T cells (CART) and blinatumomab, have drastically changed the outcome of patients with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). However, CD19-negative relapses have emerged as a major problem that is observed in approximately 30% of treated patients. Developing approaches to preventing and treating antigen-loss escapes would therefore represent a vertical advance in the field. Here, we found that in primary patient samples, the IL-3 receptor α chain CD123 was highly expressed on leukemia-initiating cells and CD19-negative blasts in bulk B-ALL at baseline and at relapse after CART19 administration. Using intravital imaging in an antigen-loss CD19-negative relapse xenograft model, we determined that CART123, but not CART19, recognized leukemic blasts, established protracted synapses, and eradicated CD19-negative leukemia, leading to prolonged survival. Furthermore, combining CART19 and CART123 prevented antigen-loss relapses in xenograft models. Finally, we devised a dual CAR-expressing construct that combined CD19- and CD123-mediated T cell activation and demonstrated that it provides superior in vivo activity against B-ALL compared with single-expressing CART or pooled combination CART. In conclusion, these findings indicate that targeting CD19 and CD123 on leukemic blasts represents an effective strategy for treating and preventing antigen-loss relapses occurring after CD19-directed therapies.

Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies.

T cells made with messenger RNA (mRNA) encoding chimeric antigen receptor (CAR) offer a safe alternative to those transduced with viral CARs by mitigating the side effects of constitutively active T cells. Previous studies have shown that mRNA CAR T cells are transiently effective but lack persistence and potency across tumor types. It was hypothesized that the efficacy of mRNA CARs could be improved by utilizing recent advancements in RNA technology, such as incorporating a modified nucleoside, 1-methylpseudouridine, into the mRNA and applying a novel purification method using RNase III to eliminate dsRNA contaminants. T cells electroporated with nucleoside-modified and purified mRNA encoding CD19 CAR showed an initial twofold increase in CAR surface expression, as well as a twofold improvement in cytotoxic killing of leukemia cells that persisted up to 5 days. T cells generated with nucleoside-modified and purified CAR mRNA also showed reduced expression of checkpoint regulators and a differential pattern of genetic activation compared to those made with conventional mRNA. In vivo studies using a leukemia mouse model revealed that the most robust 100-fold suppression of leukemic burden was achieved using T cells electroporated with purified mRNAs, regardless of their nucleoside modification. The results provide a novel approach to generate mRNA for clinical trials, and poise mRNA CAR T cells for increased efficacy during testing as new CAR targets emerge.

Purification of mRNA Encoding Chimeric Antigen Receptor Is Critical for Generation of a Robust T-Cell Response.

We previously described a mechanism of acquired resistance of B-cell acute lymphoblastic leukemia to CD19-directed chimeric antigen receptor T-cell (CART) immunotherapy. It was based on in-frame insertions in or skipping of CD19 exon 2. To distinguish between epitope loss and defects in surface localization, we used retroviral transduction and genome editing to generate cell lines expressing CD19 exon 2 variants (CD19ex2vs) bearing vesicular stomatitis virus G protein (VSVg) tags. These lines were negative by live-cell flow cytometry with an anti-VSVg antibody and resistant to killing by VSVg-directed antibody-drug conjugates (ADCs), suggestive of a defect in surface localization. Indeed, pulse-chase and α-mannosidase inhibitor assays showed that all CD19ex2vs acquired endoplasmic reticulum (ER)-specific high-mannose-type sugars but not complex-type glycans synthesized in the Golgi apparatus. When fused with green fluorescent protein (GFP), CD19ex2vs (including a mutant lacking the relevant disulfide bond) showed colocalization with ER markers, implying protein misfolding. Mass spectrometric profiling of CD19-interacting proteins demonstrated that CD19ex2vs fail to bind to the key tetraspanin CD81 and instead interact with ER-resident chaperones, such as calnexin, and ER transporters involved in antigen presentation. Thus, even the intact domains of CD19ex2vs cannot be easily targeted with ADCs or current CD19 CARTs but could serve as sources of peptides for major histocompatibility complex (MHC)-restricted presentation and T-cell receptor (TCR)-mediated killing.

CD19 Alterations Emerging after CD19-Directed Immunotherapy Cause Retention of the Misfolded Protein in the Endoplasmic Reticulum.

Unintentional transduction of B-cell acute lymphoblastic leukemia blasts during CART19 manufacturing can lead to CAR19+ leukemic cells (CARB19) that are resistant to CART19 killing. We developed an anti-CAR19 idiotype chimeric antigen receptor (αCAR19) to specifically recognize CAR19+ cells. αCAR19 CAR T cells efficiently lysed CARB19 cells in vitro and in a primary leukemia-derived xenograft model. We further showed that αCAR19-CART cells could be used as an "antidote" to deplete CART19 cells to reduce long-term side effects, such as B-cell aplasia.

A cellular antidote to specifically deplete anti-CD19 chimeric antigen receptor-positive cells.

While CD19-directed chimeric antigen receptor (CAR) T cells can induce remission in patients with B cell acute lymphoblastic leukemia (ALL), a large subset relapse with CD19 disease. Like CD19, CD22 is broadly expressed by B-lineage cells and thus serves as an alternative immunotherapy target in ALL. Here we present the composite outcomes of two pilot clinical trials ( NCT02588456 and NCT02650414 ) of T cells bearing a 4-1BB-based, CD22-targeting CAR in patients with relapsed or refractory ALL. The primary end point of these studies was to assess safety, and the secondary end point was antileukemic efficacy. We observed unexpectedly low response rates, prompting us to perform detailed interrogation of the responsible CAR biology. We found that shortening of the amino acid linker connecting the variable heavy and light chains of the CAR antigen-binding domain drove receptor homodimerization and antigen-independent signaling. In contrast to CD28-based CARs, autonomously signaling 4-1BB-based CARs demonstrated enhanced immune synapse formation, activation of pro-inflammatory genes and superior effector function. We validated this association between autonomous signaling and enhanced function in several CAR constructs and, on the basis of these observations, designed a new short-linker CD22 single-chain variable fragment for clinical evaluation. Our findings both suggest that tonic 4-1BB-based signaling is beneficial to CAR function and demonstrate the utility of bedside-to-bench-to-bedside translation in the design and implementation of CAR T cell therapies.

Antigen-independent activation enhances the efficacy of 4-1BB-costimulated CD22 CAR T cells.

To study the biology and identify markers of severe cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) in children after chimeric antigen receptor T-cell (CAR T) treatment.

Comprehensive Serum Proteome Profiling of Cytokine Release Syndrome and Immune Effector Cell-Associated Neurotoxicity Syndrome Patients with B-Cell ALL Receiving CAR T19.

Correction for Bagashev et al., "CD19 Alterations Emerging after CD19-Directed Immunotherapy Cause Retention of the Misfolded Protein in the Endoplasmic Reticulum".

Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 has been a clinical breakthrough for pediatric B-cell acute lymphoblastic leukemia (B-ALL), and loss of the CD19 target antigen on leukemic cells represents a major mechanism of relapse. Previous studies have observed CD19 mutations specific to CD19- relapses, and we sought to clarify and strengthen this relationship using deep whole-exome sequencing in leukemic cells expanded in a patient-derived xenograft. By assessing pre-treatment and relapse cells from 13 patients treated with CAR T-cell therapy, 8 of whom developed CD19- relapse and 5 of whom developed CD19+ relapse, we demonstrate that relapse-specific single-nucleotide variants and small indels with high allele frequency combined with deletions in the CD19 gene in a manner specific to those patients with CD19- relapse. Before CAR T-cell infusion, one patient was found to harbor a pre-existing CD19 deletion in the context of genomic instability, which likely represented the first hit leading to the patient's subsequent CD19- relapse. Across patients, preexisting mutations and genomic instability were not significant predictors of subsequent CD19- relapse across patients, with sample size as a potential limiting factor. Together, our results clarify and strengthen the relationship between genomic events and CD19- relapse, demonstrating this intriguing mechanism of resistance to a targeted cancer immunotherapy.

Article	Year
Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy. Cancer discovery\| PubMed ID: 26516065	2015
Early memory phenotypes drive T cell proliferation in patients with pediatric malignancies. Science translational medicine\| PubMed ID: 26738796	2016
Dual CD19 and CD123 targeting prevents antigen-loss relapses after CD19-directed immunotherapies. The Journal of clinical investigation\| PubMed ID: 27571406	2016
Purification of mRNA Encoding Chimeric Antigen Receptor Is Critical for Generation of a Robust T-Cell Response. Human gene therapy\| PubMed ID: 30024272	2019
CD19 Alterations Emerging after CD19-Directed Immunotherapy Cause Retention of the Misfolded Protein in the Endoplasmic Reticulum. Molecular and cellular biology\| PubMed ID: 30104252	2018
A cellular antidote to specifically deplete anti-CD19 chimeric antigen receptor-positive cells. Blood\| PubMed ID: 31703119	2020
Antigen-independent activation enhances the efficacy of 4-1BB-costimulated CD22 CAR T cells. Nature medicine\| PubMed ID: 33888899	2021
Comprehensive Serum Proteome Profiling of Cytokine Release Syndrome and Immune Effector Cell-Associated Neurotoxicity Syndrome Patients with B-Cell ALL Receiving CAR T19. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 35705524	2022
Correction for Bagashev et al., "CD19 Alterations Emerging after CD19-Directed Immunotherapy Cause Retention of the Misfolded Protein in the Endoplasmic Reticulum". Molecular and cellular biology\| PubMed ID: 35976030	2022
Characterization of Leukemic Resistance to CD19-Targeted CAR T-cell Therapy through Deep Genomic Sequencing. Cancer immunology research\| PubMed ID: 36255409	2023

Jessica Perazzelli

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