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Despite impressive progress, more than 50% of patients treated with CD19-targeting chimeric antigen receptor T cells (CAR19) experience progressive disease. Ten of 16 patients with large B cell lymphoma (LBCL) with progressive disease after CAR19 treatment had absent or low CD19. Lower surface CD19 density pretreatment was associated with progressive disease. To prevent relapse with CD19 − or CD19 lo disease, we tested a bispecific CAR targeting CD19 and/or CD22 (CD19-22.BB.z-CAR) in a phase I clinical trial ( NCT03233854 ) of adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL) and LBCL. The primary end points were manufacturing feasibility and safety with a secondary efficacy end point. Primary end points were met; 97% of products met protocol-specified dose and no dose-limiting toxicities occurred during dose escalation. In B-ALL ( n  = 17), 100% of patients responded with 88% minimal residual disease-negative complete remission (CR); in LBCL ( n  = 21), 62% of patients responded with 29% CR. Relapses were CD19 −/lo in 50% (5 out of 10) of patients with B-ALL and 29% (4 out of 14) of patients with LBCL but were not associated with CD22 −/lo disease. CD19/22-CAR products demonstrated reduced cytokine production when stimulated with CD22 versus CD19. Our results further implicate antigen loss as a major cause of CAR T cell resistance, highlight the challenge of engineering multi-specific CAR T cells with equivalent potency across targets and identify cytokine production as an important quality indicator for CAR T cell potency. Bispecific CAR T cells targeting CD19 and CD22 exhibit clinical activity and low toxicity in patients with large B cell lymphoma and B cell acute lymphoblastic leukemia, with relapses associated with loss of CD19 but not CD22.

Approximately 60% of patients with large B cell lymphoma treated with chimeric antigen receptor (CAR) T cell therapies targeting CD19 experience disease progression, and neurotoxicity remains a challenge. Biomarkers associated with resistance and toxicity are limited. In this study, single-cell proteomic profiling of circulating CAR T cells in 32 patients treated with CD19-CAR identified that CD4 + Helios + CAR T cells on day 7 after infusion are associated with progressive disease and less severe neurotoxicity. Deep profiling demonstrated that this population is non-clonal and manifests hallmark features of T regulatory (T Reg ) cells. Validation cohort analysis upheld the link between higher CAR T Reg cells with clinical progression and less severe neurotoxicity. A model combining expansion of this subset with lactate dehydrogenase levels, as a surrogate for tumor burden, was superior for predicting durable clinical response compared to models relying on each feature alone. These data credential CAR T Reg cell expansion as a novel biomarker of response and toxicity after CAR T cell therapy and raise the prospect that this subset may regulate CAR T cell responses in humans. Single-cell proteomic profiling of circulating CAR T cells in patients treated with CD19-CAR shows that CD4 + Helios + CAR T cells on day 7 after infusion are associated with progressive disease and less severe neurotoxicity.

Background Chimeric Antigen Receptor (CAR) T cells are now standard of care (SOC) for some patients with B cell and plasma cell malignancies and could disrupt the therapeutic landscape of solid tumors. However, access to CAR-T cells is not adequate to meet clinical needs, in part due to high cost and long lead times for manufacturing clinical grade virus. Non-viral site directed CAR integration can be accomplished using CRISPR/Cas9 and double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) via homology-directed repair (HDR), however yields with this approach have been limiting for clinical application (dsDNA) or access to large yields sufficient to meet the manufacturing demands outside early phase clinical trials is limited (ssDNA). Methods We applied homology-independent targeted insertion (HITI) or HDR using CRISPR/Cas9 and nanoplasmid DNA to insert an anti-GD2 CAR into the T cell receptor alpha constant (TRAC) locus and compared both targeted insertion strategies in our system. Next, we optimized post-HITI CRISPR EnrichMENT (CEMENT) to seamlessly integrate it into a 14-day process and compared our knock-in with viral transduced anti-GD2 CAR-T cells. Finally, we explored the off-target genomic toxicity of our genomic engineering approach. Results Here, we show that site directed CAR integration utilizing nanoplasmid DNA delivered via HITI provides high cell yields and highly functional cells. CEMENT enriched CAR T cells to approximately 80% purity, resulting in therapeutically relevant dose ranges of 5.5 × 10 8 –3.6 × 10 9 CAR + T cells. CRISPR knock-in CAR-T cells were functionally comparable with viral transduced anti-GD2 CAR-T cells and did not show any evidence of off-target genomic toxicity. Conclusions Our work provides a novel platform to perform guided CAR insertion into primary human T-cells using nanoplasmid DNA and holds the potential to increase access to CAR-T cell therapies.

HIV-specific T cells are necessary for control of HIV-1 replication but are largely insufficient for viral clearance. This is due in part to these cells’ recognition of immunodominant but variable regions of the virus, which facilitates viral escape via mutations that do not incur viral fitness costs. HIV-specific T cells targeting conserved viral elements are associated with viral control but are relatively infrequent in people living with HIV (PLWH). The goal of this study was to increase the number of these cells via an ex vivo cell manufacturing approach derived from our clinically-validated HIV-specific expanded T-cell (HXTC) process. Using a nonhuman primate (NHP) model of HIV infection, we sought to determine i) the feasibility of manufacturing ex vivo -expanded virus-specific T cells targeting viral conserved elements (CE, CE-XTCs), ii) the in vivo safety of these products, and iii) the impact of simian/human immunodeficiency virus (SHIV) challenge on their expansion, activity, and function. NHP CE-XTCs expanded up to 10-fold following co-culture with the combination of primary dendritic cells (DCs), PHA blasts pulsed with CE peptides, irradiated GM-K562 feeder cells, and autologous T cells from CE-vaccinated NHP. The resulting CE-XTC products contained high frequencies of CE-specific, polyfunctional T cells. However, consistent with prior studies with human HXTC and these cells’ predominant CD8 + effector phenotype, we did not observe significant differences in CE-XTC persistence or SHIV acquisition in two CE-XTC-infused NHP compared to two control NHP. These data support the safety and feasibility of our approach and underscore the need for continued development of CE-XTC and similar cell-based strategies to redirect and increase the potency of cellular virus-specific adaptive immune responses.

Umbilical cord blood (CB) has emerged as an effective alternative donor source for hematopoietic stem cell transplantation. Despite this success, the prolonged duration of immune suppression following CB transplantation and the naiveté of CB T cells leave patients susceptible to viral infections. Adoptive transfer of ex vivo-expanded virus-specific T cells from CB is both feasible and safe. However, the manufacturing process of these cells is complicated, lengthy, and labor-intensive. We have now developed a simplified method to manufacture a single culture of polyclonal multivirus-specific cytotoxic T cells in less than 30 days. It eliminates the need for a live virus or transduction with a viral vector, thus making this approach widely available and GMP-applicable to target multiple viruses. The use of overlapping PepMixes as a source of antigen stimulation enable expansion of the repertoire of the T cell product to any virus of interest and make it available as a third party \"off the shelf\" treatment for viral infections following transplantation.

Mucormycosis is responsible for an increasing proportion of deaths after allogeneic bone marrow transplantation. Because this disease is associated with severe immunodeficiency and has shown resistance to even the newest antifungal agents, we determined the feasibility of reactivating and expanding -specific T cells for use as adoptive immunotherapy in transplant recipients. extract pulsed monocytes were used to stimulate peripheral blood mononuclear cells from healthy donors, in the presence of different cytokine combinations. The generated -specific T cell products were phenotyped after the third stimulation and further characterized by the use of antibodies that block class I/II molecules, as well as pattern recognition receptors. Despite the very low frequency of -specific T cells of healthy donors, we found that stimulation with interleukin-2 (IL-2)/IL-7 cytokine combination could expand these rare cells. The expanded populations included 17%-83% CD4 T cells that were specific for antigens. Besides interferon-γ (IFN-γ), these cells secreted IL-5, IL-10, IL-13, and tumor necrosis factor alpha (TNF-α), and recognized fungal antigens presented by HLA-II molecules rather than through nonspecific signaling. The method described herein is robust and reproducible, and could be used to generate adequate quantities of activated -specific T cells for clinical testing of safety and antifungal efficacy in patients with mucormycosis.

Mycobacterial Infections can be severe in patients with T-cell deficiency or phagocyte disorders, and treatment is frequently complicated by antimicrobial resistance. Restoration of T-cell immunity via stem cell transplantation facilitates control of mycobacterial infections, but presence of active infections during transplantation is associated with a higher risk of mortality. Adoptive T cell immunotherapy has been successful in targeting viruses, but has not been attempted to treat mycobacterial infections. We sought to expand and characterize mycobacterial-specific T-cells derived from healthy donors in order to determine suitability for adoptive immunotherapy. Mycobacteria-specific T-cells (MSTs) were generated from 10 healthy donors using a rapid expansion protocol targeting five known mycobacterial target proteins (AG85B, PPE68, ESXA, ESXB, and ADK). MSTs were compared to T-cells expanded from the same donors using lysate from or purified protein derivative from (sensitin). MST expansion from seven patients with primary immunodeficiency disorders (PID) and two patients with IFN-γ autoantibodies and invasive infections. MSTs expanded from healthy donors recognized a median of 3 of 5 antigens, with production of IFN-γ, TNF, and GM-CSF in CD4 T cells. Comparison of donors who received BCG vaccine ( = 6) to those who did not ( = 4) showed differential responses to PPE68 ( = 0.028) and ADK ( = 0.015) by IFN-γ ELISpot. MSTs expanded from lysate or sensitin also recognized multiple mycobacterial antigens, with a statistically significant differences noted only in the response to PPE68 ( = 0.016). MSTs expanded from patients with primary immunodeficiency (PID) and invasive mycobacterial infections showed activity against mycobacterial antigens in only two of seven subjects, whereas both patients with IFN-γ autoantibodies recognized mycobacterial antigens. Thus, MSTs can be generated from donors using a rapid expansion protocol regardless of history of BCG immunization. Most tested PID patients had no detectable T-cell immunity to mycobacteria despite history of infection. MSTs may have clinical utility for adoptive immunotherapy in T-cell deficient patients with invasive mycobacterial infections.

BackgroundCAR T cells have been highly effective against refractory B cell malignancies but have not demonstrated sustained antitumor effects against solid tumors. Intense effort is underway to augment the potency of CAR T cells in order to overcome the suppressive tumor microenvironment, which is associated with T cell exhaustion. Adenosine is a major mediator of immune suppression. CD39 (ecto-ATP diphosphohydrolase-1) plays a central role in the generation of adenosine by catalyzing the metabolism of ATP into ADP/AMP. CD73 (5'-ectonucleotidase) subsequently metabolizes ADP/AMP into adenosine which mediates immune suppression through adenosine associated receptor signaling. CD39 is also expressed by exhausted CD8+ and tumor reactive T cells within the tumor microenvironment, where it is associated with tumor progression, but it remains unclear whether exhausted and/or tumor reactive CD39+CD8+ T cells mediate immune suppression via adenosine.MethodsWe developed a high affinity version of the disialoganglioside (GD2)-targeting CAR (HA-GD2) that spontaneously clusters on the surface of human T cells in the absence of antigen and mimics chronic antigen exposure leading to T cell exhaustion. Using this model, we demonstrate that exhausted CD39+CD8+ CAR T cells actively produce adenosine and mediate immune suppression through surface upregulation of CD39/73. In an attempt to generate adenosine resistance and enhance the function of exhausted CAR T cells, we knocked out CD39, CD73, or A2aR and overexpressed transmembrane-bound adenosine deaminase (ADA-TM).ResultsOnly overexpression of ADA-TM, which metabolizes adenosine to inosine, induced significant transcriptomic changes, higher frequency of stem- and central- like memory T cells, and a simultaneous decrease of exhausted T cell subpopulations. Direct exposure of HA-GD2 CAR T cells to high inosine concentration during cell manufacturing process, lead to a higher frequency of central-like memory cells and significant fitness enhancement associated with broad changes at the metabolic level. RNAseq and cyTOF analysis indicated decreased glycolytic flux, increased mitochondrial activity and glutamine and polyamine metabolism. Further, inosine altered the epigenetic state of HA-GD2 CAR T cells. We observed significant enrichment of IRF and NF-κB transcription factor motifs and motifs associated with memory differentiation in T cells grown in the presence of inosine. Finally, we showed that production of exhausted HA-GD2 and clinically-relevant version of GD2 CAR T cells in inosine-containing culture media enhances there in vivo efficacy, leading to improved survival of mice.ConclusionsWe propose introducing inosine during cell manufacturing process as a novel strategy for improving clinical outcomes of CAR T cell therapy.Ethics ApprovalImmunocompromised NOD-SCID-Il2rg−/− (NSG) mice were purchased from JAX and bred inhouse. All mice were bred, housed, and treated in ethical compliance with Stanford UniversityACUC (APLAC) approved protocolsProtocol ID 31287

BackgroundChimeric antigen receptor (CAR) therapy and hematopoietic stem cell transplantation (HSCT) are therapeutics for relapsed acute lymphocytic leukemia (ALL) that are increasingly being used in tandem. We identified a non-physiologic CD19+/CD3+ T-cell population in the leukapheresis product of a patient undergoing CAR T-cell manufacturing who previously received a haploidentical HSCT, followed by infusion of a genetically engineered T-cell addback product. We confirm and report the origin of these CD19+/CD3+ T cells that have not previously been described in context of CAR T-cell manufacturing. We additionally interrogate the fate of these CD19-expressing cells as they undergo transduction to express CD19-specific CARs.Main bodyWe describe the case of a preteen male with multiply relapsed B-ALL who was treated with sequential cellular therapies. He received an αβ T-cell depleted haploidentical HSCT followed by addback of donor-derived T cells genetically modified with a suicide gene for iCaspase9 and truncated CD19 for cell tracking (RivoCel). He relapsed 6 months following HSCT and underwent leukapheresis and CAR T-cell manufacturing. During manufacturing, we identified an aberrant T-cell population dually expressing CD19 and CD3. We hypothesized that these cells were RivoCel cells and confirmed using flow cytometry and PCR that the identified cells were in fact RivoCel cells and were eliminated with iCaspase9 activation. We additionally tracked these cells through CD19-specific CAR transduction and notably did not detect T cells dually positive for CD19 and CD19-directed CARs. The most likely rationale for this is in vitro fratricide of the CD19+ ‘artificial’ T-cell population by the CD19-specific CAR+ T cells in culture.ConclusionsWe report the identification of CD19+/CD3+ cells in an apheresis product undergoing CAR transduction derived from a patient previously treated with a haploidentical transplant followed by RivoCel addback. We aim to bring attention to this cell phenotype that may be recognized with greater frequency as CAR therapy and engineered αβhaplo-HSCT are increasingly coupled. We additionally suggest consideration towards using alternative markers to CD19 as a synthetic identifier for post-transplant addback products, as CD19-expression on effector T cells may complicate subsequent treatment using CD19-directed therapy.