Treatment of secondary CNS lymphoma using CD19-targeted chimeric antigen receptor (CAR) T cells.

BACKGROUND: Aggressive B cell lymphoma with secondary central nervous system (CNS) involvement (SCNSL) carries a dismal prognosis. Chimeric antigen receptor (CAR) T cells (CAR-T) targeting CD19 have revolutionized the treatment for B cell lymphomas; however, only single cases with CNS manifestations successfully treated with CD19 CAR-T have been reported. METHODS: We prospectively enrolled 4 patients with SCNSL into our study to assess clinical responses and monitor T cell immunity. RESULTS: Two of four SNCSL patients responded to the CD19-targeted CAR-T. Only one patient showed a substantial expansion of peripheral (PB) CAR-T cells with an almost 100-fold increase within the first week after CAR-T. The same patient also showed marked neurotoxicity and progression of the SNCSL despite continuous surface expression of CD19 on the lymphoma cells and an accumulation of CD4+ central memory-type CAR-T cells in the CNS. Our studies indicate that the local production of chemokine IP-10, possibly through its receptor CXCR3 expressed on our patient's CAR-T, could potentially have mediated the local accumulation of functionally suboptimal anti-tumor T cells. CONCLUSIONS: Our results demonstrate expansion and homing of CAR-T cells into the CNS in SNCSL patients. Local production of chemokines such as IP-10 may support CNS infiltration by CAR-T cells but also carry the potential of amplifying local toxicity. Future studies investigating numbers, phenotype, and function of CAR-T in the different body compartments of SNSCL patients receiving CAR-T will help to improve local delivery of "fit" and highly tumor-reactive CAR-T with low off-target reactivity into the CNS.

A novel multicolor fluorescent spot assay for the functional assessment of chimeric antigen receptor (CAR) T-cell products.

BACKGROUND AIMS: Chimeric antigen receptor (CAR) T-cell (CAR-T) therapies have revolutionized the treatment of B-cell lymphomas. Unfortunately, relapses after CD19-targeted CAR-T are relatively common and, therefore, there is a critical need for assays able to assess the function and potency of CAR-T products pre-infusion, which will hopefully help to optimize CAR-T therapies. We developed a novel multicolor fluorescent spot assay (MFSA) for the functional assessment of CAR-T products on a single-cell level, combining the numerical assessment of CAR-T products with their functional characterization. METHODS: We first used a standard single-cell interferon (IFN)-γ enzyme-linked immune absorbent spot assay to measure CD19-targeted CAR-T responses to CD19-coated beads. We then developed, optimized and validated an MFSA that simultaneously measures the secretion of combinations of different cytokines on a single CAR-T level. RESULTS: We identified IFN-γ/tumor necrosis factor-α/granzyme B as the most relevant cytokine combination, and we used our novel MFSA to functionally and numerically characterize two clinical-grade CAR-T products. CONCLUSIONS: In conclusion, we have developed a novel assay for the quantitative and functional potency assessment of CAR-T products. Our optimized MFSA is cost-effective, easy to perform, reliable, can be performed overnight, allowing for a fast delivery of the product to the patient, and requires relatively minimal maintenance and training. The clinical value of our novel assay will be assessed in studies correlating the pre-infusion assessment of CAR-T products with the patients' outcome in a prospective fashion.

Mouse Paneth Cell-Enriched Proteome Enabled by Laser Capture Microdissection.

Paneth cells are antimicrobial peptide-secreting cells located at the base of the crypts of the small intestine. The proteome of Paneth cells is not well defined because of their coexistence with stem cells, making it difficult to culture Paneth cells alone in vitro. Using a simplified toluidine blue O method for staining mouse intestinal tissue, laser capture microdissection (LCM) to isolate cells from the crypt region, and surfactant-assisted one-pot protein digestion, we identified more than 1300 proteins from crypts equivalent to 18,000 cells. Compared with the proteomes of villi and smooth muscle regions, the crypt proteome is highly enriched in defensins, lysozymes, and other antimicrobial peptides that are characteristic of Paneth cells. The sensitivity of the LCM-based proteomics approach was also assessed using a smaller number of cell equivalent tissues: a comparable proteomic coverage can be achieved with 3600 cells. This work is the first proteomics study of intestinal tissue enriched with Paneth cells. The simplified workflow enables profiling of Paneth cell-associated pathological changes at the proteome level directly from frozen intestinal tissue. It may also be useful for proteomics studies of other spatially resolved cell types from other tissues.

In vivo antibody labeling route and fluorophore dictate labeling efficiency, sensitivity, and longevity.

Leukocytes migrate through the blood and extravasate into organs to surveil the host for infection or cancer. Recently, we demonstrated that intravenous (IV) anti-CD45.2 antibody labeling allowed for precise tracking of leukocyte migration. However, the narrow labeling window can make this approach challenging for tracking rare migration events. Here, we show that altering antibody administration route and fluorophore can significantly extend the antibody active labeling time. We found that while both IV and intraperitoneal (IP) anti-CD45.2 antibody labeled circulating leukocytes after injection, they had different kinetic properties that impacted labeling time and intensity. Quantification of circulating antibody revealed that while unbound IV anti-CD45.2 antibody rapidly decreased, unbound IP anti-CD45.2 antibody increased over one hour. Using in vitro and in vivo serial dilution assays, we found that Alexa Fluor 647 (AF647) and Brilliant Blue 700 (BB700) dyes had the greatest labeling sensitivity compared to other fluorophores. However, IP antibody injection with anti-CD45.2 BB700, but not AF647, resulted in continuous blood leukocyte labeling for over 6 hours. Finally, we leveraged IP anti-CD45.2 BB700 antibody to track slower migrating leukocytes into tumors. We found that IP anti-CD45.2 antibody injection allowed for the identification of ~seven times as many tumor-specific CD8+ T cells that had recently migrated from blood into tumors. Our results demonstrate how different injection routes and fluorophores affect anti-CD45.2 antibody leukocyte labeling and highlight the utility of this approach for defining leukocyte migration in the context of homeostasis and cancer.

Restricting CAR T Cell Trafficking Expands Targetable Antigen Space.

Chimeric antigen receptor (CAR) T cells are an effective treatment for some blood cancers. However, the lack of tumor-specific surface antigens limits their wider use. We identified a set of surface antigens that are limited in their expression to cancer and the central nervous system (CNS). We developed CAR T cells against one of these antigens, LINGO1, which is widely expressed in Ewing sarcoma (ES). To prevent CNS targeting, we engineered LINGO1 CAR T cells lacking integrin α4 (A4ko), an adhesion molecule essential for migration across the blood-brain barrier. A4ko LINGO1 CAR T cells were efficiently excluded from the CNS but retained efficacy against ES. We show that altering adhesion behavior expands the set of surface antigens targetable by CAR T cells.

Systematic single amino acid affinity tuning of CD229 CAR T cells retains efficacy against multiple myeloma and eliminates on-target off-tumor toxicity.

T cells expressing chimeric antigen receptors (CARs) have shown remarkable therapeutic activity against different types of cancer. However, the wider use of CAR T cells has been hindered by the potential for life-threatening toxicities due to on-target off-tumor killing of cells expressing low amounts of the target antigen. CD229, a signaling lymphocyte-activation molecule (SLAM) family member, has previously been identified as a target for CAR T cell-mediated treatment of multiple myeloma (MM) due to its high expression on the surfaces of MM cells. CD229 CAR T cells have shown effective clearance of MM cells in vitro and in vivo. However, healthy lymphocytes also express CD229, albeit at lower amounts than MM cells, causing their unintended targeting by CD229 CAR T cells. To increase the selectivity of CD229 CAR T cells for MM cells, we used a single amino acid substitution approach of the CAR binding domain to reduce CAR affinity. To identify CARs with increased selectivity, we screened variant binding domains using solid-phase binding assays and biolayer interferometry and determined the cytotoxic activity of variant CAR T cells against MM cells and healthy lymphocytes. We identified a CD229 CAR binding domain with micromolar affinity that, when combined with overexpression of c-Jun, confers antitumor activity comparable to parental CD229 CAR T cells but lacks the parental cells' cytotoxic activity toward healthy lymphocytes in vitro and in vivo. The results represent a promising strategy to improve the efficacy and safety of CAR T cell therapy that requires clinical validation.

Chimeric antigen receptor (CAR) T cells for the treatment of a kidney transplant patient with post-transplant lymphoproliferative disorder (PTLD).

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication following kidney transplantation, and there is a critical and unmet need for PTLD treatments associated with more pronounced and durable responses. To date, reports on the use of CD19-targeted chimeric antigen receptor (CAR) T (CAR-T) cells in patients after solid organ transplant (SOT) have been anecdotal, clinical presentations and outcomes have been heterogenous, and a longitudinal analysis of CAR-T cell expansion and persistence in PTLD patients has not been reported. Our report describes a patient with a history of renal transplant who received CD19-directed CAR-T cell therapy for the treatment of refractory PTLD, diffuse large B cell lymphoma (DLBCL)-type. We show that even with the background of prolonged immunosuppression for SOT, it is possible to generate autologous CAR-T products capable of expansion and persistence in vivo, without evidence of excess T-cell exhaustion. Our data indicate that CAR-T cells generated from a SOT recipient with PTLD can yield deep remissions without increased toxicity or renal allograft dysfunction. Future clinical studies should build on these findings to investigate CAR-T therapy, including longitudinal monitoring of CAR-T phenotype and function, for PTLD in SOT recipients.

T cell responses against SARS-CoV-2 and its Omicron variant in a patient with B cell lymphoma after multiple doses of a COVID-19 mRNA vaccine.

Anti-SARS-CoV-2 antibodies are crucial for protection from future COVID-19 infections, limiting disease severity, and control of viral transmission. While patients with the most common type of hematologic malignancy, B cell lymphoma, often develop insufficient antibody responses to messenger RNA (mRNA) vaccines, vaccine-induced T cells would have the potential to 'rescue' protective immunity in patients with B cell lymphoma. Here we report the case of a patient with B cell lymphoma with profound B cell depletion after initial chemoimmunotherapy who received a total of six doses of a COVID-19 mRNA vaccine. The patient developed vaccine-induced anti-SARS-CoV-2 antibodies only after the fifth and sixth doses of the vaccine once his B cells had started to recover. Remarkably, even in the context of severe treatment-induced suppression of the humoral immune system, the patient was able to mount virus-specific CD4+ and CD8+ responses that were much stronger than what would be expected in healthy subjects after two to three doses of a COVID-19 mRNA vaccine and which were even able to target the Omicron 'immune escape' variant of the SARS-CoV-2 virus. These findings not only have important implications for anti-COVID-19 vaccination strategies but also for future antitumor vaccines in patients with cancer with profound treatment-induced immunosuppression.

Humoral immunity against SARS-CoV-2 variants including omicron in solid organ transplant recipients after three doses of a COVID-19 mRNA vaccine.

Objectives: Solid organ transplant recipients (SOTR) receiving post-transplant immunosuppression show increased COVID-19-related mortality. It is unclear whether an additional dose of COVID-19 vaccines can overcome the reduced immune responsiveness against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. Methods: We analysed humoral immune responses against SARS-CoV-2 and its variants in 53 SOTR receiving SARS-CoV-2 vaccination. Results: Following the initial vaccination series, 60.3% of SOTR showed no measurable neutralisation and only 18.9% demonstrated neutralising activity of > 90%. More intensive immunosuppression, antimetabolites in particular, negatively impacted antiviral immunity. While absolute IgG levels were lower in SOTR than controls, antibody titres against microbial recall antigens were higher. By contrast, SOTR showed reduced vaccine-induced IgG/IgA antibody titres against SARS-CoV-2 and its delta variants and fewer linear B-cell epitopes, indicating reduced B-cell diversity. Importantly, a third vaccine dose led to an increase in anti-SARS-CoV-2 antibody titres and neutralising activity across alpha, beta and delta variants and to the induction of anti-SARS-CoV-2 CD4+ T cells in a subgroup of patients analysed. By contrast, we observed significantly lower antibody titres after the third dose with the omicron variant compared to the ancestral SARS-CoV-2 and the improvement in neutralising activity was much less pronounced than for all the other variants. Conclusion: Only a small subgroup of solid organ transplant recipients is able to generate functional antibodies after an initial vaccine series; however, an additional vaccine dose resulted in dramatically improved antibody responses against all SARS-CoV-2 variants except omicron where antibody responses and neutralising activity remained suboptimal.