Treatment Of B-cell Malignancies Research Articles

Ocular Surface Squamous Neoplasia In Situ Secondary to Bruton Tyrosine Kinase Inhibitor Therapy.

Bruton tyrosine kinase (BTK) inhibitors have become an important therapeutic option in treating B-cell malignancies including non-Hodgkin lymphoma and chronic lymphocytic leukemia. However, a noted morbidity associated with their use is the potential for the development of second primary malignancy related to immunomodulation. In this case, we report a second primary malignancy of ocular surface squamous neoplasia (OSSN) owing to BTK therapy. The OSSN was successfully treated and has been monitored without recurrence. To our knowledge, this is the first case of OSSN secondary to BTK therapy.

Augmenting CAR T-cell Functions with LIGHT.

Chimeric antigen receptor (CAR) T-cell therapy has resulted in remarkable clinical success in the treatment of B-cell malignancies. However, its clinical efficacy in solid tumors is limited, primarily by target antigen heterogeneity. To overcome antigen heterogeneity, we developed CAR T cells that overexpress LIGHT, a ligand of both lymphotoxin-β receptor on cancer cells and herpes virus entry mediator on immune cells. LIGHT-expressing CAR T cells displayed both antigen-directed cytotoxicity mediated by the CAR and antigen-independent killing mediated through the interaction of LIGHT with lymphotoxin-β receptor on cancer cells. Moreover, CAR T cells expressing LIGHT had immunostimulatory properties that improved the cells' proliferation and cytolytic profile. These data indicate that LIGHT-expressing CAR T cells may provide a way to eliminate antigen-negative tumor cells to prevent antigen-negative disease relapse.

In vivo CAR T-cell generation in nonhuman primates using lentiviral vectors displaying a multidomain fusion ligand

AbstractChimeric antigen receptor (CAR) T-cell therapies have demonstrated transformative efficacy in treating B-cell malignancies. However, high costs and manufacturing complexities hinder their widespread use. To overcome these hurdles, we have developed the VivoVec platform, a lentiviral vector capable of generating CAR T cells in vivo. Here, we describe the incorporation of T-cell activation and costimulatory signals onto the surface of VivoVec particles (VVPs) in the form of a multidomain fusion protein and show enhanced in vivo transduction and improved CAR T-cell antitumor functionality. Furthermore, in the absence of lymphodepleting chemotherapy, administration of VVPs into nonhuman primates resulted in the robust generation of anti-CD20 CAR T cells and the complete depletion of B cells for >10 weeks. These data validate the VivoVec platform in a translationally relevant model and support its transition into human clinical testing, offering a paradigm shift in the field of CAR T-cell therapies.

Progress and pitfalls of gene editing technology in CAR-T cell therapy: a state-of-the-art review.

CAR-T cell therapy has shown remarkable promise in treating B-cell malignancies, which has sparked optimism about its potential to treat other types of cancer as well. Nevertheless, the Expectations of CAR-T cell therapy in solid tumors and non-B cell hematologic malignancies have not been met. Furthermore, safety concerns regarding the use of viral vectors and the current personalized production process are other bottlenecks that limit its widespread use. In recent years the use of gene editing technology in CAR-T cell therapy has opened a new way to unleash the latent potentials of CAR-T cell therapy and lessen its associated challenges. Moreover, gene editing tools have paved the way to manufacturing CAR-T cells in a fully non-viral approach as well as providing a universal, off-the-shelf product. Despite all the advantages of gene editing strategies, the off-target activity of classical gene editing tools (ZFNs, TALENs, and CRISPR/Cas9) remains a major concern. Accordingly, several efforts have been made in recent years to reduce their off-target activity and genotoxicity, leading to the introduction of advanced gene editing tools with an improved safety profile. In this review, we begin by examining advanced gene editing tools, providing an overview of how these technologies are currently being applied in clinical trials of CAR-T cell therapies. Following this, we explore various gene editing strategies aimed at enhancing the safety and efficacy of CAR-T cell therapy.

In silico and in vivo analysis reveal impact of c-Myc tag in FMC63 scFv-CD19 protein interface and CAR-T cell efficacy

Anti-CD19 CAR-T cell therapy represents a breakthrough in the treatment of B-cell malignancies, and it is expected that this therapy modality will soon cover a range of solid tumors as well. Therefore, a universal cheap and sensitive method to detect CAR expression is of foremost importance. One possibility is the use of epitope tags such as c-Myc, HA or FLAG tags attached to the CAR extracellular domain, however, it is important to determine whether these tags can influence binding of the CAR with its target molecule. Here, we conducted in-silico structural modelling of an FMC63-based anti-CD19 single-chain variable fragment (scFv) with and without a c-Myc peptide tag added to the N-terminus portion and performed molecular dynamics simulation of the scFv with the CD19 target. We show that the c-Myc tag presence in the N-terminus portion does not affect the scFv’s structural equilibrium and grants more stability to the scFv. However, intermolecular interaction potential (IIP) analysis reveals that the tag can approximate the complementarity-determining regions (CDRs) present in the scFv and cause steric impediment, potentially disturbing interaction with the CD19 protein. We then tested this possibility with CAR-T cells generated from human donors in a Nalm-6 leukemia model, showing that CAR-T cells with the c-Myc tag have overall worse antitumor activity, which was also observed when the tag was added to the C-terminus position. Ultimately, our results suggest that tag addition is an important aspect of CAR design and can influence CAR-T cell function, therefore its use should be carefully considered.

Prolonged cytopenias after immune effector cell therapy and lymphodepletion in patients with leukemia, lymphoma and solid tumors

Background aimsThe success of chimeric antigen receptor (CAR) T-cell therapy in treating B-cell malignancies has led to the evaluation of CAR T-cells targeting a variety of other malignancies. Although the efficacy of CAR T-cells is enhanced when administered post-lymphodepleting chemotherapy, this can trigger bone marrow suppression and sustained cytopenia after CD19.CAR T-cell therapy. Additionally, systemic inflammation associated with CAR T-cell activity may contribute to myelosuppression. Cytopenias, such as neutropenia and thrombocytopenia, elevate the risk of severe infections and bleeding, respectively. However, data on the incidence of prolonged cytopenias after immune effector therapy in the solid tumor context remain limited. ObjectiveWe compared the incidence of prolonged cytopenias after immune effector therapy including genetically modified T-cells, virus-specific T-cells (VSTs) and NKT-cells, as well non-gene-modified VSTs for leukemia, lymphoma, and solid tumors (ST) to identify associated risk factors. MethodsA retrospective analysis was conducted of 112 pediatric and adult patients with relapsed and/or refractory cancers who received lymphodepleting chemotherapy followed by immune effector therapy. Patients treated with 13 distinct immune effector cell therapies through 11 single-center clinical trials and 2 commercial products over a 6-year period were categorized into 3 types of malignancies: leukemia, lymphoma and ST. We obtained baseline patient characteristics and adverse events data for each participant, and tracked neutrophil and platelet counts following lymphodepletion. ResultsOf 112 patients, 104 (92.9%) experienced cytopenias and 88 (79%) experienced severe cytopenias. Patients with leukemia experienced significantly longer durations of severe neutropenia (median duration of 14 days) compared with patients with lymphoma (7 days) or ST (11 days) (P = 0.002). Patients with leukemia also had a higher incidence of severe thrombocytopenia (74.1%), compared with lymphoma (46%, P = 0.03) and ST (14.3%, P < 0.0001). Prolonged cytopenias were significantly associated with disease type (63% of patients with leukemia, 44% of patients with lymphoma, and 22.9% of patients with ST, P = 0.006), prior hematopoietic stem cell transplant (HSCT) (66.7% with prior HSCT versus 38.3% without prior HSCT, P = 0.039), and development of immune effector cell-associated neurotoxicity syndrome (ICANS) (75% with ICANS versus 38% without ICANS, P = 0.027). There was no significant association between prolonged cytopenias and cytokine release syndrome. ConclusionsImmune effector recipients often experience significant cytopenias due to marrow suppression following lymphodepletion regardless of disease, but prolonged severe cytopenias are significantly less common after treatment of patients with lymphoma and solid tumors.

Abstract 1236: Characterization of CBX-250, a first-in-class TCR-mimetic-based T-cell engager targeting a cathepsin G peptide-HLA complex for the treatment of myeloid leukemia

Abstract While T-cell engaging therapies have demonstrated clear clinical benefit in the treatment of B-cell malignancies, their application to myeloid malignancies remains a challenge due to the limited repertoire of suitable tumor specific surface antigens. Targeting HLA-restricted peptides (pHLA) derived from intracellular cancer antigens provides an opportunity to explore the entire cancer proteome. CG1 (FLLPTGAEA) has been validated as an HLA-A*02:01 restricted leader peptide from Cathepsin G (CTSG) and is abundantly presented by leukemic versus normal myeloid cells. TCR-mimetic (TCRm) antibodies are ideally suited to target pHLA with high potency and specificity and can be engineered and manufactured using standard antibody technologies. Here we report the preclinical characterization of CBX-250, a novel CG1/HLA-A2 TCRm-CD3 bispecific T-Cell Engager (TCE). CBX-250 induced potent T-cell activation and T-cell mediated killing of leukemia cell lines with varying levels of target antigen density with sub-nM EC50 in vitro. In vivo, a closely related precursor of CBX-250 demonstrated potent tumor control in various AML and CML CDX models at doses as low as 0.01mpk. Although CTSG is a serine protease stored in neutrophil azurophilic granules, the CG1 peptide is preferentially presented by leukemia cells, as validated by mass spectrometry. Moreover, when co-culturing PBMCs with HLA-A2 neutrophils, we did not observe any CBX-250-mediated T-cell activation or IFNγ production. In silico predictions identified HLA-A2-restricted human peptides that share sequence or structural similarity to CG1. We determined the cross-reactivity risk of CBX-250 to be low, based on T-cell activation and T-cell mediated cytotoxicity assays against cells pulsed with supra-physiological concentrations of these peptides. Moreover, we observed no activity against target-negative cancer cells, including a CTSG-KO cell line. To further assess the specificity of CBX-250, we screened a panel of normal cells and found no meaningful T-cell activation or IFNγ production at CBX-250 concentrations exceeding its EC90. Finally, the CBX-250 precursor molecule demonstrated favorable safety in a double transgenic mouse model expressing human HLA-A2 and CG1. Following sequence optimization, CBX-250 demonstrated robust serum and pH stress stability and a favorable melting temperature, while retaining excellent potency and specificity. A full suite of analytical and biophysical assessments supports CBX-250’s favorable developability profile. In summary, these data provide strong preclinical evidence of the potency, specificity, safety, and developability of CBX-250, a novel, first-in-class, off-the-shelf, TCRm-based TCE for the treatment of myeloid malignancies. Citation Format: Geraldine Paulus, Benjamin Lee, Preethi Sankaran, Tanzila Rahman, Jessica Jimenez, Delainey O'Connor, Simon Yue, Yu Huang, Melissa Bikowitz, Sarah Jaffe, Shawn O'Malley, Bhupal Ban, Galina Gabriely, Tao Wang, Amanda Mak, Michael Princiotta, Chunhua Shi, Helen He, Ningping Feng, Jun Yan, Timothy Heffernan, Gheath Alatrash, Jeffrey Molldrem, Dmitri Wiederschain. Characterization of CBX-250, a first-in-class TCR-mimetic-based T-cell engager targeting a cathepsin G peptide-HLA complex for the treatment of myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1236.

Abstract 882: Harnessing single cell multi-omics data to identify predictors of clinical outcome in CD19 CAR T cell therapy

Abstract Background: Chimeric antigen receptor (CAR) T cell therapy is effective in treating B-cell malignancies, but factors influencing the persistence of functional CAR+ T cells, such as product composition, patients’ lymphodepletion, and immune reconstitution, are not well understood. To shed light on this issue, we conducted single-cell multi-omics analysis of transcriptional, clonal, and phenotypic profiles from pre- to 1-month post-infusion of CAR+ and CAR− T cells from patients who received a donor-derived 4-1BB CAR product targeting CD19. Methods: Longitudinal blood samples and infusion products were obtained from 10 individuals with large B cell lymphoma or B-cell acute lymphoblastic leukemia (CARTELL cohort in Sydney Australia1). CD45+ cells and enriched CAR+ T cells were single cell sorted for simultaneous scRNA-seq, 41 barcoded proteins (N=41) and T cell receptors, n= 115,000 cells across 21 samples. Machine learning methods were used to identify subsets of immune cells associated with clinically relevant parameters. Results: Following infusion, CAR+ and CAR− T cells showed similar differentiation profiles with clonally expanded populations across heterogeneous phenotypes, demonstrating clonal lineages and phenotypic plasticity. These findings were validated by performing the same analysis on two publicly available scRNA-seq data sets from two cohorts 2, 3 of paediatric (N=18) and adult (N=32) individual with large B cell lymphoma who received CAR19 T cells. Analysis of mass-cytometry data from our cohort and from 28 patients with B cell lymphoma treated with commercial CAR19 T cells (axi-cel)4, identified an association of CAR− and also CAR+ T cells, both expressing an 2B4− CD11b+ CD8+ phenotype, with complete remission or progressive disease at 3 and 6 months post-infusion. Finally, we report additional analysis on the immunogenomics of circulating CAR T cells in two patients who developed CAR T cell induced lymphoma and compared it with ascites and CAR-Lymphoma cells. Discussion: These results suggest that CAR+ and CAR− CD8+ T cells share a differentiation trajectory terminating in an NK-like phenotype that is associated with clinical outcome, and that non-CAR-derived signals may influence patients’ immune recovery and outcomes.

Abstract 6703: Innovative mRNA strategies to encode CD19/CD3 T cell engagers in the treatment of B cell hematological malignancies

Abstract The advent of T Cell Engager (TCE) immunotherapies marks a transformative shift in cancer treatment paradigms, introducing a novel action mechanism. Blinatumomab, a bispecific CD19/CD3 TCE, has shown remarkable efficacy in treating refractory or relapsed B-cell Acute Lymphoblastic Leukaemia (B-ALL), including cases resistant to conventional chemotherapy, leading to its global approval for B-ALL management. Despite its pronounced clinical success, Blinatumomab faces challenges with patient adherence, primarily due to the protein’s short half-life. This necessitates continuous administration requested 24 hours injection throughout the treatment course. Additionally, the drug’s association with the induction of cytokine release syndrome (CRS), presumably linked to the hyperactivation of the cytokine network, presents a significant concern. To address these limitations, we leverage mRNA modality to develop innovative therapeutic mRNAs encoding CD19/CD3 TCE. These mRNA drugs hold the potential to improve protein exposure in vivo with a steady production of encoded protein in-situ. On the other hand, with the gradual expression of protein, mRNA shifted the transient, “pulse”-like pharmacokinetic (PK) feature of the protein drugs to a more “plateau”PK curve, which allows a natural priming of T cells to reduce the risk of CRS induction. In this work, we developed a series of mRNAs encoding CD19/CD3 TCE through our mRNA technology platform and LNP delivery platform. Comparative analyses demonstrated that the TCEs produced in vivo by these mRNAs match the structure and function of recombinantly expressed proteins. PK studies in both mice and non-human primates confirmed a favorable PK profile with over 10-fold increase in TCE exposure compared to their protein counterparts. This increased exposure correlated with superior antitumor efficacy in both ALL and lymphoma models, evidenced by the potent effects of single mRNA doses compared to multiple doses of protein-based treatments. Meanwhile IL6 cytokines induced in mRNA treated mice were significantly alleviated than those in protein treated animals, indicating a reduced risk of induction of CRS. One candidate mRNA demonstrated an optimal "plateau" pharmacokinetic profile, achieving extensive target cell eradication over 20 days with a single low dose of 0.001 mpk in non-human primates. An investigator-initiated clinical study with mRNA encoding CD19/CD3 TCE is underway in relapsed and/or refractory acute lymphoblastic leukemia patients. The preliminary data from the on-going study showed ABO2015 with an acceptable safety profile, and activation of peripheral T cells was observed in one patient with low dose. These findings underscore the significant clinical potential of mRNA-encoded CD19/CD3 TCE in treating B-cell malignancies, heralding a paradigm shift in the clinical application of protein-based T cell engagers. Citation Format: Xia Zhong, Liang Du, Zhenxing Yang, Xiaoli Xu, Jijun Yuan, Bo Ying. Innovative mRNA strategies to encode CD19/CD3 T cell engagers in the treatment of B cell hematological malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6703.

Abstract 24: Utilizing T cell receptor-based therapy to treat anaplastic thyroid cancer

Abstract Anaplastic thyroid cancer (ATC) is one of the most aggressive solid tumor in thyroid tissue, with a 6-month medium overall survival. The effectiveness of conventional treatments in extending the survival of patients is limited. Therefore, there is an urgent need to develop novel systemic therapeutic interventions. Adoptive cell therapy has revolutionized cancer immunotherapy by successfully treating B-cell malignancies through chimeric antigen receptor-based approaches. For solid tumors, there is a need to identify a broader spectrum of tumor target antigens, and the field has now shifted towards leveraging T cell receptors (TCRs) which can recognize both intracellular and extracellular antigen-derived epitopes. Our team has established an immunogenic epitope discovery pipeline, followed by a specialized protocol for empiric validation of candidate epitopes. We characterized immunogenic epitopes by coupling tandem mass spectrometry of MHC-eluted peptides with tumor proteome and MHC-peptide binding prediction databases. By integrating multi-omics data (genomics, transcriptomics, and immunopeptidomics) from both in-house and publicly available ATC databases, we find that ATC exhibits significant upregulation of pathways associated with microtubule-based movement, chromosome segregation, and kinetochore complex components. We have identified several HLA-A2- and A24-restricted immunogenic peptides representing these pathway-associated proteins that covered more than 60% of the patient population. These immunogenic peptides were subsequently utilized to isolate antigen-specific T cells from peripheral blood and identify cognate TCR. These peptide-specific T lymphocytes elicit cytotoxicity toward multiple HLA-matched ATC cell lines and tumor samples in vitro and in vivo. In conclusion, our studies contribute to the development of a new treatment paradigm for treating ATC. Citation Format: Shao-Hsi Hung, Ying C. Henderson, Pan Ke, Yulun Chiu, Shailbala Singh, Yunyun Chen, Stephen Y. Lai, Cassian Yee. Utilizing T cell receptor-based therapy to treat anaplastic thyroid cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 24.

The role of chimeric antigen receptor T cells targeting more than one antigen in the treatment of B-cell malignancies.

Several products containing chimeric antigen receptor T cells targeting CD19 (CART19) have been approved for the treatment of patients with relapsed/refractory non-Hodgkin's lymphoma (NHL) and acute lymphoblastic leukaemia (ALL). Despite very impressive response rates, a significant percentage of patients experience disease relapse and die of progressive disease. A major cause of CART19 failure is loss or downregulation of CD19 expression in tumour cells, which has prompted a myriad of novel strategies aimed at targeting more than one antigen (e.g. CD19 and CD20 or CD22). Dual targeting can the accomplished through co-administration of two separate products, co-transduction with two different vectors, bicistronic cassettes or tandem receptors. In this manuscript, we review the pros and cons of each strategy and the clinical results obtained so far.

Pirtobrutinib: A New and Distinctive Treatment Option for B-Cell Malignancies.

The objective was to evaluate the efficacy/safety of pirtobrutinib in the treatment of B-cell malignancies and distinguish it from other available Bruton's tyrosine kinase (BTK) inhibitors. A literature search of PubMed (January 2021 through November 2023) and Clinicaltrials.gov was conducted using terms pirtobrutinib, Jaypirca, and LOXO 305. Licensing trials of available BTK inhibitors were also reviewed. Relevant English-language clinical trials were evaluated. Pirtobrutinib was approved by the US Food and Drug Administration for the treatment of relapsed/refractory mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) based largely on a phase 1/2 study in B-cell malignancies. Pirtobrutinib demonstrated a 73% overall response rate (ORR) in the CLL population and 58% in MCL. Pirtobrutinib has activity in patients resistant to earlier-generation, covalent BTK inhibitors. In fact, the ORRs were similar in BTK-pretreated and naïve patients. Adverse effects include fatigue, diarrhea, bleeding, and infection. Atrial fibrillation, a class effect of BTK inhibitors, may be less common with pirtobrutinib. Compared with earlier-generation BTK inhibitors, pirtobrutinib is more selective for BTK and binds noncovalently to the receptor. Ongoing studies are evaluating pirtobrutinib's use in multiple B-cell malignancies and comparing it with other BTK inhibitors. The characteristics of pirtobrutinib render it useful in the treatment of B-cell malignancies no longer responding to a previous BTK inhibitor, and results from ongoing clinical trials may support future expanded use.

A chimeric antigen receptor-based cellular safeguard mechanism for selective in vivo depletion of engineered T cells.

Adoptive immunotherapy based on chimeric antigen receptor (CAR)-engineered T cells has exhibited impressive clinical efficacy in treating B-cell malignancies. However, the potency of CAR-T cells carriethe potential for significant on-target/off-tumor toxicities when target antigens are shared with healthy cells, necessitating the development of complementary safety measures. In this context, there is a need to selectively eliminate therapeutically administered CAR-T cells, especially to revert long-term CAR-T cell-related side effects. To address this, we have developed an effective cellular-based safety mechanism to specifically target and eliminate the transferred CAR-T cells. As proof-of-principle, we have designed a secondary CAR (anti-CAR CAR) capable of recognizing a short peptide sequence (Strep-tag II) incorporated into the hinge domain of an anti-CD19 CAR. In in vitro experiments, these anti-CAR CAR-T cells have demonstrated antigen-specific cytokine release and cytotoxicity when co-cultured with anti-CD19 CAR-T cells. Moreover, in both immunocompromised and immunocompetent mice, we observed the successful depletion of anti-CD19 CAR-T cells when administered concurrently with anti-CAR CAR-T cells. We have also demonstrated the efficacy of this safeguard mechanism in a clinically relevant animal model of B-cell aplasia induced by CD19 CAR treatment, where this side effect was reversed upon anti-CAR CAR-T cells infusion. Notably, efficient B-cell recovery occurred even in the absence of any pre-conditioning regimens prior anti-CAR CAR-T cells transfer, thus enhancing its practical applicability. In summary, we developed a robust cellular safeguard system for selective in vivo elimination of engineered T cells, offering a promising solution to address CAR-T cell-related on-target/off-tumor toxicities.

A review of Bruton's tyrosine kinase inhibitors in multiple sclerosis.

Bruton's tyrosine kinase (BTK) inhibitors are an emerging class of therapeutics in multiple sclerosis (MS). BTK is expressed in B-cells and myeloid cells, key progenitors of which include dendritic cells, microglia and macrophages, integral effectors of MS pathogenesis, along with mast cells, establishing the relevance of BTK inhibitors to diverse autoimmune conditions. First-generation BTK inhibitors are currently utilized in the treatment of B-cell malignancies and show efficacy in B-cell modulation. B-cell depleting therapies have shown success as disease-modifying treatments (DMTs) in MS, highlighting the potential of BTK inhibitors for this indication; however, first-generation BTK inhibitors exhibit a challenging safety profile that is unsuitable for chronic use, as required for MS DMTs. A second generation of highly selective BTK inhibitors has shown efficacy in modulating MS-relevant mechanisms of pathogenesis in preclinical as well as clinical studies. Six of these BTK inhibitors are undergoing clinical development for MS, three of which are also under investigation for chronic spontaneous urticaria (CSU), rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Phase II trials of selected BTK inhibitors for MS showed reductions in new gadolinium-enhancing lesions on magnetic resonance imaging scans; however, the safety profile is yet to be ascertained in chronic use. Understanding of the safety profile is developing by combining safety insights from the ongoing phase II and III trials of second-generation BTK inhibitors for MS, CSU, RA and SLE. This narrative review investigates the potential of BTK inhibitors as an MS DMT, the improved selectivity of second-generation inhibitors, comparative safety insights established thus far through clinical development programmes and proposed implications in female reproductive health and in long-term administration.

A Comprehensive Review of Small-Molecule Inhibitors Targeting Bruton Tyrosine Kinase: Synthetic Approaches and Clinical Applications.

Bruton tyrosine kinase (BTK) is an essential enzyme in the signaling pathway of the B-cell receptor (BCR) and is vital for the growth and activation of B-cells. Dysfunction of BTK has been linked to different types of B-cell cancers, autoimmune conditions, and inflammatory ailments. Therefore, focusing on BTK has become a hopeful approach in the field of therapeutics. Small-molecule inhibitors of BTK have been developed to selectively inhibit its activity and disrupt B-cell signaling pathways. These inhibitors bind to the active site of BTK and prevent its phosphorylation, leading to the inhibition of downstream signaling cascades. Regulatory authorities have granted approval to treat B-cell malignancies, such as chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL), with multiple small-molecule BTK inhibitors. This review offers a comprehensive analysis of the synthesis and clinical application of conventional small-molecule BTK inhibitors at various clinical stages, as well as presents promising prospects for the advancement of new small-molecule BTK inhibitors.

Advances of cell therapy in lung cancer: a narrative review.

Lung cancer is the second most prevalent malignancy and has the highest death rate. The main approaches for lung cancer treatment include surgery, chemotherapy, radiotherapy, targeted therapy, and immunotherapy. However, the treatments of the disease need to be further improved. An increasing number of scientific investigations indicated cell therapy to be a successful new treatment for lung cancer. Cell therapy can improve the host's immunity to disease and can compensate for the shortcomings in the therapeutic effects of traditional treatments, particularly in the case of cancer treatment. However, due to its recent development, its clinical efficacy still needs to be further examined. In order to provide an updated source on cell therapy for lung cancer, this paper summarizes the clinical use of chimeric antigen receptor T cells (CAR-Ts), stem cells, cytokine-induced killer cells (CIKs), and tumor-infiltrating lymphocytes (TILs) and discusses recent clinical advancements. We performed a search of the PubMed database on March 28, 2023, and again on June 10, 2023. A review of retrieved literature related to cell therapy and treatments for lung cancer was completed. Cell therapy has been applied in clinical studies on the treatment of disorders of the hematologic system, digestive system, respiratory system, and other systems. CAR-T therapy has been successfully used in the treatment of B-cell malignancies, which suggests that cell therapy has broad prospects in the treatment of malignant tumors. CAR-T, stem cells, CIKs, and TILs exert antitumor activity and can recognize and could be used to treat lung cancer. Cell therapy represents a novel solution in the treatment of lung cancer. Cell therapy, when combined with traditional therapies, can compensate for the shortcomings of these methods. Further research is needed to reduce the occurrence of adverse reactions and provide a more effective approach in treating lung cancer.

In Vitro and In Vivo Specificity and Biodistribution of a Novel CD8-Targeted Fusosome

Autologouschimeric antigen receptor (CAR) T cells represent a groundbreaking therapy for treating B-cell malignancies. However, the complexity, cost, time and toxicity associated with manufacturing and administering current CAR T cell therapies hinder their broader applications. To overcome CAR T limitations, Sana has developed a novel fusosome platform that can deliver a T cell targeted CAR gene therapy via systemic administration, without ablative pre-conditioning. The fusosome is a lentiviral vector pseudotyped with a targeted fusogen that specifically directs gene transfer into CD8+ cells. Our CD8-targeted CD19-directed CAR fusosome, SG299, transduces CD8+ cells in lymphoid tissues, showing preclinical safety and high selectivity, laying the groundwork for clinical evaluation of an in vivo CAR gene therapy. In vitro tissue cross reactivity (TCR) study was assessed to evaluate binding of the CD8 fusogen binder, a CD8-binding scFv, on tissues from Nemestrina macaques and compared these with tissues from human donors. SG299 specificity was also evaluated in vitro using panel of cell lines representing a breadth of receptor types. Cells were exposed to 8 IU/cell SG299 and vector integration was evaluated using ddPCR. Reverse transcriptase inhibitor nevirapine was used as control for the in vitro studies. To evaluate in vivo specificity and biodistribution, healthy nemestrina macaques were treated with a single dose of SG299 at 1 or 4 x 10 9 IU/kg (n=2 or 4) or saline (n=1 or 2) via intravenous infusion and evaluated for up to 35 days for vector biodistribution in CD8 enriched PBMCs and at terminal timepoint in panel of 17 tissues using ddPCR. In addition, we also demonstrated SG299 specificity with help of flow cytometry, in NALM6 tumor model in NSG mice. Briefly, NSG mice engrafted with human PBMCs were challenged with NALM6 tumors and treated with either SG299 or saline. The TCR study demonstrated that the CD8 fusogen binder is specific for the target tissues containing T cells and similar binding across multiple human and Nemestrina tissues. SG299 fusosome demonstrated CD8-specificity evaluated by transduction of cell types with CD8 receptor (SupT1 and PanT cells). There was no detectable off-target transduction in sets of primary as well as immortalized cell lines evaluated including pulmonary cells, renal epithelial cells, lymphoblast, endothelial cells and CD8KO cells. VCN analysis of nemestrina macaques PBMC and tissue samples following treatment with SG299 demonstrated dose dependent integrations per diploid genome through Day 35. All animals dosed 4 x 10 9 IU/kg showed quantifiable VCN signal through Day 34 in PBMC population enriched for CD8+ cells (0.0032 - 0.6544 vc/dg, vector copy per diploid genome). One out of 4 animals dosed at 1 x 10 9 IU/kg showed quantifiable VCN signal at Day 34 (0.0276 vc/dg). Tissues collected at necropsy on Day 35 demonstrated quantifiable VCN signal in spleen, lymph node, CD8+ splenocyte and lymphocyte, draining lymph node, heart ventricle, and injection site, for animals dosed at 4 x 10 9 IU/kg. One of 4 animals dosed 1 x 10 9 IU/kg showed quantifiable VCN in spleen, CD8+ splenocyte, and injection site. Flow cytometric analysis of peripheral blood of mice treated with SG299 showed a dose dependent increase in CAR T cells, and the CAR T cells were CD8+ T cells specific with no CAR T cell detection amongst the CD4+ T cells. Collectively, these studies demonstrate high on target specificity of SG299 fusosome to transduce CD8+ T cells in vitro and in vivo. Therefore, SG299 represents a novel therapeutic opportunity to generate CAR T cells in vivo and potentially overcome challenges associated with ex vivo CAR T therapies.

Glycosylation as regulator of human B-cell leukaemias in bone marrow

Leukemic B-cells are lodged in the bone marrow [BM], a complex organ composed of many cell types and extracellular matrix. Determining how the reciprocal interactions between these components are regulated is critical to our understanding of the factors that allow leukemia cells to survive, multiply and withstand treatment. All cells in the bone marrow are surrounded by a glycocalyx, a glycan-rich layer of high complexity, which regulates such cell-cell and cell-matrix interactions. However, the structure and function of the glycan components of the biomolecules that constitute this layer have not been explored in much detail. Gaps are difficult to fill due to technical limitations as well as the fact that the composition of the BM in health, disease and aging is not static. This also applies to B-lineage malignancies that develop or persist in BM such as B-cell precursor acute lymphoblastic leukemia and Multiple Myeloma, and the effects of their treatment. In contrast, the proteomes and transcriptomes of different human bone marrow cells have been studied more extensively. A combination of technologies now increasingly allows correlations to be made between the expression of glycosyltransferases and glycan structures in cell lines, which could be extrapolated to RNAseq data from primary cells. Glycopeptide analysis will also be invaluable in providing details of specific glycan occupancy on glycoproteins, even if only as a snapshot in time. Functional studies on CD19, CD138/SDC1 and BCMA/TNFRSF17 have already demonstrated the importance of their glycosylation. Additional studies using such approaches are likely to find many more other instances in which malignant B-cell homeostasis is regulated by glycosylation, and lead to the identification of new targets to treat B-cell malignancies.

Genomic Landscape of Ibrutinib- and/or Acalabrutinib-intolerant Patients with B-cell Malignancies Treated with Zanubrutinib in a Phase 2 Study

Introduction:Bruton tyrosine kinase inhibitors (BTKi) have shown remarkable utility in the treatment of B-cell malignancies; yet some patients have experienced toxicities to the BTKis, ibrutinib and acalabrutinib, which lead to dose reduction or treatment discontinuation. Zanubrutinib is a potent and selective next-generation BTKi. BGB-3111-215 (NCT04116437) is an ongoing, phase 2 study of the safety and efficacy of zanubrutinib monotherapy in patients with chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), Waldenström macroglobulinemia (WM), mantle cell lymphoma (MCL), or marginal zone lymphoma (MZL) who discontinued ibrutinib, acalabrutinib, or acalabrutinib and ibrutinib because of intolerance. Methods:Because the genomic profile of patients with B-cell malignancies who are intolerant to ibrutinib and/or acalabrutinib has not been extensively studied, mutations were assessed using a 106 gene-targeted next-generation sequencing panel (PredicineHEME TM, Predicine) on baseline and disease progression samples. Samples were sequenced to a median depth of &amp;gt;20,000 reads, with a validated sensitivity of 0.25% mutant allele frequency for all genomic regions, and 0.1% for mutational hotspots. Variant allele frequency (VAF) &amp;lt;0.1% for hotspot mutations and &amp;lt;0.25% for non-hotspot mutations were excluded from analysis; germline and CHIP mutations were also excluded from analysis. The association between gene mutations and progression-free survival (PFS, defined as time from the date of first zanubrutinib dose to date of first PD or death) was quantified by the log rank test and the hazard ratio and summarized by the Kaplan-Meier method. Results:As of June 6, 2022, baseline peripheral blood samples were collected from 71 (95.9%) of the 74 patients enrolled in the study. There were 46 patients with CLL, 11 with WM, eight with SLL, three with MCL, and three with MZL. A total of 56 patients were intolerant to ibrutinib only; 15 were intolerant to acalabrutinib or acalabrutinib and ibrutinib. At baseline, high mutation frequencies were observed in TP53, SF3B1, ATM, and NOTCH1 across disease indications ( Figure). The majority of TP53 mutations were missense mutations and located in the DNA binding domain. Nineteen different mutations were identified in ATM and were distributed across the whole gene with a majority clustered in the 3' regions encoding the FAT and PI3 kinase domains. All mutations (n=21) in SF3B1 were missense and 38% (n=8/21) of the mutations occurred at p.K700E/c.A2098G. In NOTCH1, the most common mutation (n=8/14, 57.1%) was a two base pair deletion (c.7541_7542delCT) in the PEST domain of exon 34. In patients with CLL/SLL with TP53, SF3B1, or ATM mutations at baseline, shorter progression-free survival was observed compared to patients without these mutations, suggesting that mutations in DNA damage response and RNA splicing/metabolism pathways are associated with less favorable prognosis. BTK mutations were detected in 4.2% (n=3/71) of baseline samples and in 57.1% (n=4/7) of samples available at disease progression (PD). All BTK mutations were at the C481 BTKi binding site. Two patients with baseline BTK mutations had PD and increased VAF of BTK mutants were detected for both patients at PD. A third patient died due to illness unrelated to treatment. Two patients acquired new BTKC481 mutations at PD. Conclusion: This is the first study to describe the genomic landscape of patients with B-cell malignancies who were intolerant to ibrutinib and/or acalabrutinib. Here we show that the gene mutational profile of these patients at baseline or at/after disease progression is comparable with patients with relapse/refractory disease who tolerate ibrutinib and, consistent with other studies, patients with mutations in TP53, SF3B1 or ATM genes had less favorable prognosis on BTKi. Further, intolerant patients who progressed on zanubrutinib acquired new BTK mutations and/or had an increase in the frequency of BTK mutations. These data contribute to the growing understanding of the mutational profiles of patients with B-cell malignancies, particularly those who were intolerant to BTKi, and further explores the association between mutations and response to subsequent zanubrutinib treatment. This study may provide supporting information to inform treatment guidelines for patients who become intolerant to BTKi.

An “Off-the-Shelf” CD2 Universal CAR-T Therapy Combined with a Long-Acting IL-7 for T-Cell Malignancies

INTRODUCTION: T-cell malignancies are associated with frequent relapse and high morbidity in children and adults, partly due to the lack of effective or targeted treatment options. Despite the use of multidrug chemotherapy, &amp;lt;50% of adults and &amp;lt;75% of children with T-ALL survive longer than five years. While CAR-T cell therapy is remarkable in treating B-cell malignancies, its use in T-cell malignancies is restricted due to fratricide, risk of immunodeficiency, and immune escape. METHODS: To broaden the use of CAR-T cells in pan T-cell malignancies, we developed an allogenic “universal” CD2-targeting CAR-T (UCART2), in which both CD2 and the T-cell receptor alpha subunit are both deleted by CRISPR/Cas9 editing to prevent both fratricide and GvHD. In vitro and in vivo efficacy of UCART2, alone or in combination with rhIL-7-hyFc (efineptakin alfa), a long-acting IL-7, was evaluated using CD2+ HH CBR-GFP T-ALL cell line and primary CD2+ T-ALL PDXs. To determine the impact of CD2 antigen loss on CAR-T cell function, CRISPR-mediated CD2 KO was performed in CD19 targeting CAR-T cells (UCART19ΔCD2). Single-cell secretome analyses on target-stimulated UCART19 and UCART19ΔCD2 were performed using the Isolight platform (IsoPlexis). The in vivo efficacy of UCART19 and UCART19ΔCD2 was evaluated in a CD19+ NALM6 GFP-CBR xenograft model. RESULTS: UCART2 demonstrated efficacy against T-ALL and CTCL in vitro and prolonged the survival of tumor-engrafted NSG mice in vivo. While CD2 deletion in UCART19 had little impact on in vitro killing efficacy, single-cell secretome analysis revealed that CD2 deletion in UCART19 reduced frequencies of the effector cytokines, including Granzyme-B and IFN-γ. We also observed that UCART19ΔCD2 (CD2-) had reduced anti-tumor efficacy compared to UCART19 (CD2+) in vivo in a CD19+ NALM6 GFP-CBR xenograft model. Of note, the reduced efficacy resulting from CD2 deletion was reversed when combined with rhIL-7-hyFc, a long-acting recombinant human interleukin-7. Treatment with rhIL-7-hyFc enhanced UCART2 expansion and persistence in vivo(Figure 1A) resulting in increased survival of NSG mice engrafted with both the primary patient T-ALL model (Figure 1B) and in a tumor re-challenge model in vivo. CONCLUSIONS: We have developed an allogenic “universal” CD2-targeting CAR-T cell UCART2, which is effective against T-ALL and CTCL/T-NHL. We found that CD2 deletion in CAR-T cells resulted in reduced secretion of effector cytokines and reduced anti-tumor activity in vivo, indicating that CD2 is critical for CAR-T function. However, when combined with rhIL-7-hyFc (efineptakin alfa), a long-acting recombinant IL-7, UCART2 induced durable complete responses in both primary and tumor rechallenge experiments in vivo. Our data suggest that UCART2, in combination with rhIL-7-hyFc, could be a suitable approach for treating T-cell malignancies.