CD4 Protein Research Articles

Construction and identification of a stable CT26 cell line expressing CD19-FLUC-GFP

Solid tumors lack well-defined targets for chimeric antigen receptor T-cell (CAR-T) therapy. Therefore, introducing a known target molecule, CD19, into solid tumor cell lines via lentiviral transduction to investigate the cytotoxicity of CD19 CAR-T cells can potentially support CAR-T cell therapy against solid tumors. In this study, a stable colon cancer CT26 cell line, CT26-CD19-FLUC-GFP, expressing CD19, firefly luciferase (FLUC), and green fluorescent protein (GFP), was constructed using a triple-plasmid lentiviral system. The growth characteristics of this cell line were consistent with those of the CT26 cell line. Subsequent flow cytometry analysis confirmed stable expression of CD19 and GFP in CT26-CD19-FLUC-GFP cells after serial passaging up to the 5th, 10th, and 22nd generations. Further validation revealed significantly higher levels of CD19 mRNA and FLUC expression in CT26-CD19-FLUC-GFP cells continuously passaged up to the 22nd generation compared to the control CT26 cells. In comparison to T cells, CD19 CAR-T cells demonstrated substantial cytotoxicity against CT26-CD19-FLUC-GFP cells and MC38-CD19 cells. One week after intraperitoneal implantation of CT26-CD19-FLUC-GFP cells into mice, FLUC expression in the peritoneal region could be detected. These results indicate the successful establishment of a stable CT26 cell line expressing CD19-FLUC-GFP, which can be specifically targeted by CD19 CAR-T cells.

Discovery and preclinical development of a therapeutically active nanobody-based chimeric antigen receptor targeting human CD22

Chimeric antigen receptor (CAR-) T cell therapies targeting B-cell restricted antigens CD19, CD20, or CD22 can produce potent clinical responses for some B-cell malignancies, but relapse remains common. Camelid single domain antibodies (sdAbs or nanobodies) are smaller, simpler, and easier to recombine than single-chain variable fragments (scFvs) used in most CARs, but fewer sdAb-CARs have been reported. Thus, we sought to identify a therapeutically active sdAb-CAR targeting human CD22. Immunization of an adult Llama glama with CD22 protein, sdAb-cDNA library construction, and phage panning yielded >20 sdAbs with diverse epitope and binding properties. Expressing CD22-sdAb-CAR in Jurkat cells drove varying CD22-specific reactivity not correlated with antibody affinity. Changing CD28 to CD8-transmembrane design increased CAR persistence and expression in vitro. CD22-sdAb-CAR candidates showed similar CD22-dependent CAR-T expansion in vitro, though only membrane-proximal epitope targeting CD22-sdAb-CARs activated direct cytolytic killing and extended survival in a lymphoma xenograft model. Based on enhanced survival in blinded xenograft studies a lead CD22sdCAR-T was selected; achieving comparable complete responses to a benchmark short-linker m971-scFv CAR-T in high-dose experiments. Finally, immunohistochemistry and flow cytometry confirm tissue and cellular level specificity of the lead CD22-sdAb. This presents a complete report on pre-clinical development of a novel CD22sdCAR therapeutic.

Non-Contact Electric Field May Induced Higher CD4, CD8, Caspase-8, and Caspase-9 Protein Expression in Breast Tumor Tissue of Rats (Rattus norvegicus Berkenhout, 1769)

Non-contact electric field therapy has been studied as one of less invasive and safe alternative method in cancer therapy. Some studies reported that non-contact electric field therapy with low intensity (50 - 60 V/m) and medium frequency (150 kHz) could slower the rat’s breast tumor growth and did not cause significant damage on the rat's kidney and liver. However, further study needed to evaluate the potency of non-contact electric field to be developed into effective cancer therapy. Thus, we investigated the immune response of non-contact electric field therapy through the detection of CD4 and CD8 protein expression and the mechanism of apoptosis through the detection of caspase-8 and caspase-9 proteins on breast tumor tissue of rats. Twenty four rats were divided into 4 groups: 1. non-induction - non-therapy (NINT), 2. non-induction - therapy (NIT), 3. induction - non-therapy (INT), and 4. induction - therapy (IT). DMBA (7,12-dimethylbenz(α)anthracene) 20 mg/kg body weight was used to induced breast tumor formation. Rats with breast nodules that had reached 1 cm in size were exposed to an ECCT device with 150 kHz and 50 - 60 V/m electric fields. Further, the breast tissues were collected for routine histological and immunohistochemistry preparation for CD4, CD8, caspase 8, as well as caspase 9 detection. The data were statistically analyzed using Mann-Whitney U-Test method, with a significance level of p < 0.05 using the SPSS 16.0 version. The scoring results were compared between the INT and IT groups. Our results showed that non-contact electric field therapy could suppress breast tumor growth and improved its histological structure. Interestingly, the breast tissue of IT group qualitatively had slightly more necrotic and apoptotic cells than that of INT group. Moreover, the IT group showed by higher CD4 and CD8 as well as higher caspase 8 and caspase 9 expression. Collecting all the data together, we concluded that non-contact electric field therapy potent to improve histological structure of breast tumor.

3D Amplified Single-Cell RNA and Protein Imaging Identifies Oncogenic Transcript Subtypes in B-Cell Acute Lymphoblastic Leukemia.

Simultaneous in situ detection of transcript and protein markers at the single-cell level is essential for gaining a better understanding of tumor heterogeneity and for predicting and monitoring treatment responses. However, the limited accessibility to advanced 3D imaging techniques has hindered their rapid implementation. Here, we present a 3D single-cell imaging technique, termed 3D digital rolling circle amplification (4DRCA), capable of the multiplexed and amplified simultaneous digital quantification of single-cell RNAs and proteins using standard fluorescence microscopy and off-the-shelf reagents. We generated spectrally distinguishable DNA amplicons from molecular markers through an integrative protocol combining single-cell RNA and protein assays and directly enumerated the amplicons by leveraging an open-source algorithm for 3D deconvolution with a custom-built automatic gating algorithm. With 4DRCA, we were able to simultaneously quantify surface protein markers and cytokine transcripts in T-lymphocytes. We also show that 4DRCA can distinguish BCR-ABL1 fusion transcript positive B-cell acute lymphoblastic leukemia cells with or without CD19 protein expression. The accessibility and extensibility of 4DRCA render it broadly applicable to other cell-based diagnostic workflows, enabling sensitive and accurate single-cell RNA and protein profiling.

Immunoinformatics Approach to Design a Chimeric CD70-Peptide Vaccine against Renal Cell Carcinoma.

Renal cell carcinoma (RCC) accounts for the majority of cancer-related deaths worldwide. Overexpression of CD70 has been linked to advanced stages of RCC. Therefore, this study aims to develop a multiepitope vaccine targeting the overexpressed CD70 using immunoinformatics techniques. In this investigation, in silico multiepitope vaccines were constructed by linking specific CD70 protein epitopes for helper T lymphocytes and CD8+ T lymphocytes. To enhance immunogenicity, sequences of cell-penetrating peptide (CPP), penetratin (pAntp), along with the entire sequence of tumor necrosis factor-α (TNF-α), were attached to the N-terminal and C-terminal of the CD70 epitopes. Computational assessments were performed on these chimeric vaccines for antigenicity, allergenicity, peptide toxicity, population coverage, and physicochemical properties. Furthermore, refined 3D constructs were subjected to a range of analyses, encompassing structural B-cell epitope prediction and molecular docking. The chosen vaccine construct underwent diverse assessments such as molecular dynamics simulation, immune response simulation, and in silico cloning. All vaccines comprised antigenic, nontoxic, and nonallergenic epitopes, ensuring extensive global population coverage. The vaccine constructs demonstrated favorable physicochemical characteristics. The binding affinity of chimeric vaccines to the TNF receptor remained relatively stable, influenced by the alignment of vaccine components. Molecular docking and dynamics analyses predicted stable interactions between CD70-CPP-TNF and the TNF receptor, indicating potential efficacy. In silico codon optimization and cloning of the vaccine nucleic acid sequence were accomplished using the pET28a plasmid. Furthermore, this vaccine displayed the capacity to modulate humoral and cellular immune responses. Overall, the results suggest therapeutic potential for the chimeric CD70-CPP-TNF vaccine against RCC. However, validation through in vitro and in vivo experiments is necessary. This trial is registered with NCT04696731 and NCT04046445.

A transcriptomic approach to explore the immune landscape of patient with pancreatic ductal adenocarcinoma with prognostic impact.

681 Background: Pancreatic ductal adenocarcinoma (PDAC) is characterized by its immunologically cold tumor microenvironment (TME) with scarce T cell infiltration and few molecular signatures of immune activation. To date, the immunotherapies including the immune checkpoint blockade of PD-1, and CTLA-4 either single or in combination have shown modest results in PDAC tumors. Recently, the Lymphocyte Activation Gene (LAG3) has emerged as a promising checkpoint target as indicated by preclinical and clinical data. The aim of this study is to explore the immune profile of a series of PDAC and to evaluate its prognostic impact. Methods: A 5-mm thick section of Formalin-Fixed Paraffin-Embedded (FFPE) tissue from a retrospective cohort of 28 PDAC cases were analyzed using the Precision Immunooncology panel (PIP, HTG Molecular Diagnostics) on a NextSeq 550 sequencer (Illumina). The RNA expression of 1392 immune-related genes were analyzed. Principal Components (PC) and Manhattan distance were studied for data visualization and clustering. Maxstat algorithm (maxstat v0.7-25) was used to establish optimal cut-offs for variable categorization. Log-rank and Cox regression were used for both univariate and multivariate for survival. All tests were two-tailed. The statistical analysis was performed using R studio (R version 4.0.3). Lastly, immunohistochemistry (IHC) for CD4, CD8, CD20, PD1, PD-L1, and LAG-3 proteins was performed on tissue microarrays (TMAs) to assess their correlation with RNA expression. Results: Unsupervised analysis of the gene expression identified two clusters of patients with differentially prognostic information. These two groups were defined with a logistic regression model of 14 genes ( BTLA, CXCR5, DLX6, KLHDC9, KRT13, LAG3, LGSN, MICB, SIGLEC5, SPINK5, SPN, TBX21, UPK2, ZIC5) showing for cluster1 and cluster 2 a median survival of 13 (0-97.583) and 57 (0.67-256.08) months, respectively (p-value = 0.0088). This predictive model was independently validated in the PDAC dataset of the Cancer Genome Atlas (TCGA) (p<0.0001). Interestingly, cluster 1, was characterized by the significant overexpression of LAG3 which has recently been proposed as the next immune checkpoint (IC) receptor target. Moreover, Cluster 1, exhibited the overexpression of other ICs such as PD1, PD-L1, IDO1, LIF and CTLA4, defining cluster 1 as an immunologically hot tumor. IHC for LAG3 and other ICs showed a moderate correlation with HTG results. Conclusions: In this study, a prognostic transcriptomic-based signature of 14 genes has been defined and validated for PDAC. This signature clearly identifies two prognostic groups that could constitute the basis for tailored immunotherapy with specific IC inhibitors. LAG-3 is a promising target for immunotherapy in PDAC patients.

CD1a and skin T cells: a pathway for therapeutic intervention.

The CD1 and MR1 protein families present lipid antigens and small molecules to T cells, complementing well-studied major histocompatibility complex-peptide mechanisms. The CD1a subtype is highly and continuously expressed within the skin, most notably on Langerhans cells, and has been demonstrated to present self and foreign lipids to T cells, highlighting its cutaneous sentinel role. Alteration of CD1a-dependent T-cell responses has recently been discovered to contribute to the pathogenesis of several inflammatory skin diseases. In this review, we overview the structure and role of CD1a and outline the current evidence implicating CD1a in the development of psoriasis, atopic dermatitis and allergic contact dermatitis.

Exploring the Expression and Function of T Cell Surface Markers Identified through Cellular Indexing of Transcriptomes and Epitopes by Sequencing.

By utilizing both protein and mRNA expression patterns, we can identify more detailed and diverse immune cells, providing insights into understanding the complex immune landscape in cancer ecosystems. This study was performed by obtaining publicly available Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE-seq) data of peripheral blood mononuclear cells (PBMCs) from the Gene Expression Omnibus database. A total of 94674 total cells were analyzed, of which 32412 were T cells. There were 228 protein features and 16262 mRNA features in the data. The Seurat package was used for quality control and preprocessing, principal component analysis was performed, and Uniform Manifold Approximation and Projection was used to visualize the clusters. Protein and mRNA levels in the CITE-seq were analyzed. We observed that a subset of T cells in the clusters generated at the protein level divided better. By identifying mRNA markers that were highly correlated with the CD4 and CD8 proteins and cross-validating CD26 and CD99 markers using flow cytometry, we found that CD4+ and CD8+ T cells were better discriminated in PBMCs. Weighted Nearest Neighbor clustering results identified a previously unobserved T cell subset. In this study, we used CITE-seq data to confirm that protein expression patterns could be used to identify cells more precisely. These findings will improve our understanding of the heterogeneity of immune cells in the future and provide valuable insights into the complexity of the immune response in health and disease.

The mutation and low expression of ARID1A are predictive of a poor prognosis and high immune infiltration in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) has the poorest prognosis among all breast cancer subtypes. While several tumor types are excepted to have a curative response to immunotherapy through the AT-rich interaction domain 1A (ARID1A) gene, its role in TNBC remains unclear. The expression of the ARID1A gene and immune infiltration in TNBC were analyzed by way and function enrichment analysis. Additionally, 27 gene mutations, including ARID1A mutation, were detected in paraffin-embedded TNBC specimens and normal breast specimens using Next Generation Sequencing (NGS). Immunohistochemical staining was employed to detect the expression of AIRD1A, TP53, Ki67, CD4, CD8, and PD-L1 proteins in TNBC and the adjacent normal tissue samples. The bioinformatics analysis revealed that ARID1A was mutated in TNBC and significantly associated with tumor immune infiltration. NGS analysis showed a high mutation rate of ARID1A (35%) in TNBC, but the mutation status of ARID1A was not associated with age at onset, lymph node metastasis, pathological grade, or Ki67 index. Low expression or loss of AIRD1A was more commonly observed in TNBC tissues (36/108) as compared to normal tissues (3/25). Positive expression of CD8 and PD-L1 was observed in TNBC tissues with low ARID1A expression. ARID1A mutation was associated with low protein expression, and patients with ARID1A mutation or low protein expression had shorter progression-free survival. The ARID1A mutation and low expression are associated with poor prognosis and high immune infiltration in TNBC, and might be biomarkers for TNBC prognosis and immunotherapy efficacy.

Hippocampal and gut AMPK activation attenuates enterocolitis-like symptoms and co-occurring depressive-like behavior in ulcerative colitis model mice: Involvement of brain-gut autophagy

Patients with inflammatory bowel disease, including ulcerative colitis (UC) and Crohn's disease, have a high incidence of psychiatric disorders, including depression and anxiety. However, the underlying pathogenic mechanism remains unknown. Dextran sulfate sodium (DSS)-treated mice, a model of UC, exhibit depressive-like behavior and reduced adenosine monophosphate-activated protein kinase (AMPK) activity, which regulates various physiological functions in the brain and gut. However, comprehensive studies on UC pathophysiology with co-occurring depression focused on brain-gut AMPK activity are lacking. Therefore, we aimed to investigate whether resveratrol (RES), an AMPK activator, prevented DSS-induced UC-like symptoms and depressive-like behavior. DSS treatment induced UC-like pathology and depressive-like behavior, as assessed via the tail suspension test. Moreover, western blotting and immunohistochemical studies revealed that DSS increased p-p70S6 kinase (Thr389), p62, tumor necrosis factor-α, interleukin (IL)-1β, IL-18, NLR family pyrin domain containing 3 (NLRP3), cleaved caspase-1, cleaved Gasdermin-D (GSDMD), and cleaved caspase-3 expression levels in the rectum and hippocampus, and increased CD40, iNOS, and Kelch-like ECH-associated protein 1 expression levels, and the number of Iba1-positive cells in the hippocampus, and decreased p-AMPK and LC3II/I expression levels, and the number of NF-E2-related factor 2 (Nrf2)-positive cells, and reduced neurogenesis in the hippocampus. These changes were reversed by the RES administration. RES also enhanced PGC1α and SOD1 expression in the hippocampus of DSS-treated male mice. Moreover, NLRP3 staining was observed in the neurons and microglia, and cleaved GSDMD staining in neurons in the hippocampus of DSS-treated mice. Notably, RES prevented UC-like pathology and depressive-like behavior and enhancement of autophagy, decreased rectal and hippocampal inflammatory cytokines and inflammasome, and induced the Nrf2-PGC1α-SOD1 pathway in the hippocampus, resulting in neurogenesis in the hippocampal dentate gyrus. Our findings suggest that brain-gut AMPK activation may be an important therapeutic strategy in patients with UC and depression.

Antitumor efficacy and safety of unedited autologous CD5.CAR T cells in relapsed/refractory mature T-cell lymphomas

AbstractDespite newer targeted therapies, patients with primary refractory or relapsed (r/r) T-cell lymphoma have a poor prognosis. The development of chimeric antigen receptor (CAR) T-cell platforms to treat T-cell malignancies often requires additional gene modifications to overcome fratricide because of shared T-cell antigens on normal and malignant T cells. We developed a CD5-directed CAR that produces minimal fratricide by downmodulating CD5 protein levels in transduced T cells while retaining strong cytotoxicity against CD5+ malignant cells. In our first-in-human phase 1 study (NCT0308190), second-generation autologous CD5.CAR T cells were manufactured from patients with r/r T-cell malignancies. Here, we report safety and efficacy data from a cohort of patients with mature T-cell lymphoma (TCL). Among the 17 patients with TCL enrolled, CD5 CAR T cells were successfully manufactured for 13 out of 14 attempted lines (93%) and administered to 9 (69%) patients. The overall response rate (complete remission or partial response) was 44%, with complete responses observed in 2 patients. The most common grade 3 or higher adverse events were cytopenias. No grade 3 or higher cytokine release syndrome or neurologic events occurred. Two patients died during the immediate toxicity evaluation period due to rapidly progressive disease. These results demonstrated that CD5.CAR T cells are safe and can induce clinical responses in patients with r/r CD5-expressing TCLs without eliminating endogenous T cells or increasing infectious complications. More patients and longer follow-up are needed for validation. This trial was registered at www.clinicaltrials.gov as #NCT0308190.

Soluble CD83 modulates human-monocyte-derived macrophages toward alternative phenotype, function, and metabolism.

Alterations in macrophage (Mφ) polarization, function, and metabolic signature can foster development of chronic diseases, such as autoimmunity or fibrotic tissue remodeling. Thus, identification of novel therapeutic agents that modulate human Mφ biology is crucial for treatment of such conditions. Herein, we demonstrate that the soluble CD83 (sCD83) protein induces pro-resolving features in human monocyte-derived Mφ biology. We show that sCD83 strikingly increases the expression of inhibitory molecules including ILT-2 (immunoglobulin-like transcript 2), ILT-4, ILT-5, and CD163, whereas activation markers, such as MHC-II and MSR-1, were significantly downregulated. This goes along with a decreased capacity to stimulate alloreactive T cells in mixed lymphocyte reaction (MLR) assays. Bulk RNA sequencing and pathway analyses revealed that sCD83 downregulates pathways associated with pro-inflammatory, classically activated Mφ (CAM) differentiation including HIF-1A, IL-6, and cytokine storm, whereas pathways related to alternative Mφ activation and liver X receptor were significantly induced. By using the LXR pathway antagonist GSK2033, we show that transcription of specific genes (e.g., PPARG, ABCA1, ABCG1, CD36) induced by sCD83 is dependent on LXR activation. In summary, we herein reveal for the first time mechanistic insights into the modulation of human Mφ biology by sCD83, which is a further crucial preclinical study for the establishment of sCD83 as a new therapeutical agent to treat inflammatory conditions.

Innate TCRβ-chain engagement drives human T cells toward distinct memory-like effector phenotypes with immunotherapeutic potentials.

Clonotypic αβ T cell responses to cargoes presented by major histocompatibility complex (MHC), MR1, or CD1 proteins underpin adaptive immunity. Those responses are mostly mediated by complementarity-determining region 3 motifs created by quasi-random T cell receptor (TCR) gene rearrangements, with diversity being highest for TCRγδ. Nonetheless, TCRγδ also displays nonclonotypic innate responsiveness following engagement of germline-encoded Vγ-specific residues by butyrophilin (BTN) or BTN-like (BTNL) proteins that uniquely mediate γδ T cell subset selection. We now report that nonclonotypic TCR engagement likewise induces distinct phenotypes in TCRαβ+ cells. Specifically, antibodies to germline-encoded human TCRVβ motifs consistently activated naïve or memory T cells toward core states distinct from those induced by anti-CD3 or superantigens and from others commonly reported. Those states combined selective proliferation and effector function with activation-induced inhibitory receptors and memory differentiation. Thus, nonclonotypic TCRVβ targeting broadens our perspectives on human T cell response modes and might offer ways to induce clinically beneficial phenotypes in defined T cell subsets.

MicroRNA miR-27a as a possible regulator of anti-inflammatory macrophage phenotype in preeclamptic placenta

IntroductionThe role of the TGFβ signaling pathway, an important cascade responsible for the anti-inflammatory polarization of macrophages, in the development of both early- and late-onset preeclampsia (eoPE and loPE), remains poorly understood. In this study, we examined the components of the TGFβ signaling cascade and macrophage markers within placental tissue in normal pregnancy and in PE. MethodsPatients with eoPE, loPE, and normal pregnancy were enrolled in the study (n = 10 in each group). Following techniques were used for the investigation: immunohistochemistry analysis, western blotting, qRT-PCR, isolation of monocytes by magnetic sorting, transfection, microRNA sequencing, and bioinformatic analysis. ResultsWe observed a significant decrease in the anti-inflammatory macrophage marker CD206 in the loPE group, alongside with a significant down-regulation of CD206 protein production in both eoPE and loPE groups. The level of CD68-positive cells and relative levels of CD163 and MARCO production were comparable across the groups. However, we identified a significant decrease in the TGFβ receptor 2 production and its gene expression in the PE group. Further analysis revealed a link between TGFBR2 and MRC1 (CD206) genes through a single miRNA, hsa-miR-27a-3p. Transfecting CD14-derived macrophages with the hsa-miR-27a-3p mimic significantly changed TGFBR2 production, indicating the potential role of this miRNA in regulating the TGFβ signaling pathway. We also revealed the up-regulation of hsa-miR-27a-5p and hsa-miR-27a-3p in the trophoblast BeWo b30 cell line under the severe hypoxia condition and the fact that TGFBR2 3′ UTR could serve as a potential target for these miRNAs. DiscussionOur findings uncover a novel potential therapeutic target for managing patients with PE, significantly contributing to a deeper comprehension of the underlying mechanisms involved in the development of this pathology.

Proteins as Vanguard: Biomedicine’s Revolutionary role in Cancer Treatment

The tides of cancer treatment are turning with proteins emerging as a stalwart component of biomedicine, poised to replace conventional chemotherapeutics. This editorial delves into the transformative potential of proteins in the field of biomedicine, highlighting their crucial role in personalized and targeted cancer therapies that hold the key to improved efficacy and reduced side effects. Biomedicine enables precision medicine by tailoring treatments based on the unique genetic makeup of individual patients. Targeted therapies, such as monoclonal antibodies and gene therapies, hold the promise of attacking cancer cells with unprecedented accuracy, minimizing damage to healthy tissues. Monoclonal antibodies, a key class of proteins, epitomize the precision targeting made possible by biomedicine. These engineered proteins seek out specific cancer cells, binding to surface markers with high affinity. This targeted approach minimizes collateral damage to healthy tissues, a notable advantage over the systemic impact of traditional chemotherapeutics [1]. 
 Proteins play a pivotal role in unleashing the power of the immune system through immunotherapy. Checkpoint inhibitors, designed as protein-based drugs, disrupt the signals that cancer cells use to evade immune detection. This reinvigorates the body's natural defense mechanisms, leading to sustained and specific anti-cancer responses [2]. At the forefront of biomedicine, CAR-T cell therapy involves engineering patients' own T cells to express chimeric antigen receptors (CARs), which are essentially protein structures. These receptors enable T cells to recognize and eliminate cancer cells with remarkable precision, showcasing the potential of protein-based therapies in reshaping cancer treatment strategies [3]. Proteins also serve as crucial biomarkers, offering insights into a patient's unique cancer profile. This information guides the development of personalized protein therapies tailored to target specific molecular vulnerabilities. Unlike the broad-spectrum nature of chemotherapeutics, proteins as biomedicine allows for more nuanced interventions. This individualized approach holds great promise for optimizing treatment outcomes and minimizing adverse effects. Several proteins are commercially available as biomedicines for cancer treatment. Trastuzumab is monoclonal antibody target the HER2 protein used in breast cancer. Rituximab target the CD20 protein on B cells. Pembrolizumab is an immune checkpoint inhibitor targeting PD-1 protein and used in various cancers, including melanoma, lung cancer, and head and neck cancers. Daratumumab target CD38 protein on myeloma cells and it is approved for the treatment of multiple myeloma. These examples represent a fraction of the protein-based biomedicines available for cancer treatment. 
 Despite the strides made in protein-based biomedicine, challenges such as cost, manufacturing complexities, and potential side effects persist. Ongoing research into refining protein therapies, optimizing delivery methods, and addressing these challenges is essential to realizing the full potential of proteins as a cornerstone of cancer treatment. Proteins, at the forefront of biomedicine's arsenal, are reshaping the landscape of cancer treatment. From precision targeting with monoclonal antibodies to the personalized potential of biomarker-driven therapies, the era of protein-based interventions heralds a new dawn in the fight against cancer. As research advances and challenges are met, proteins stand as vanguards in the journey towards replacing chemotherapeutics, offering renewed hope for improved patient outcomes and a paradigm shift in oncological care.

Quantification of ADA of Humanized CD19 CAR-T Cell Therapy in R/R LBCL

Purpose: Humanized CD19 chimeric antigen receptor (CAR) T-cell therapy results in a high response rate in patients with relapsed/refractory (R/R) large B-cell lymphoma (LBCL). And many clinical trials have discovered the impact of anti-drug antibody (ADA) on CAR-T efficacy. But the ADA has yet been quantified. And its influence in patients with CAR-T infusion is still unknown. Patients and methods: We enrolled 30 patients with R/R LBCL treated with humanized CD19 CAR-T cells between April, 2022 and July, 2023, and collected their plasma at pre-FC, day7, 14, and follow up months for ADA testing using flow cytometry. Results: The detection of ADA within plasma of humanized CAR-T was performed using flow cytometry, and the relative quantification of ADA was achieved by setting up a calibration curve with CD19 protein as a standard. R 2 of the calibration curve can be 0.999 (Figure 1a). The physiologic threshold was determined as 0.1nM (95%CI) by using plasma from 10 normal subjects. 27/30 patients who experienced only one infusion and did not relapse had mostly low ADA (<3nM). Only one exception having a maximum value higher than 5nM with PFS maintained 1 year. All of the 27 patients had ADA presence before FC, but it did not affect CAR-T amplification. 3/30 patients who received second CAR-T cell infusion, 2 of which had 2 infusions of the same product, and 1 of which relapsed after relma-cel infusion and followed by the humanized product. 2 of the 3 patients (P08 and P09) who had a high ADA before the second infusion (of 3.52 and 3.27) had extremely limited CAR-T expansion after the second infusion (maximum CAR copies of 470 and 3950, respectively), and both relapsed 1 month after the second infusion (Figure 1b-1c). The other one patient (P18) had a low ADA both before and after the second infusion (maximum is before infusion, 0.65 nM), and had normal subsequent CAR copies amplification (maximum 60800 and minimum 10850), and did not relapse during the follow-up period (Figure 1d). We consider that ADA >3nM suggests poor CAR-T amplification and poor prognosis, but further validation is needed due to the small number of patients. Conclusions: The quantification of ADA is essential and credible, which contributes to the unification of standard. Pre-existing ADA does not affect CAR-T amplification for the first infusion but possibly suggests a negative impact on CAR-T efficacy and amplification of multiple-infusion. However, further research is needed.

Adoptively Infused Memory-like Natural Killer Cells Impact Adaptive Immune Responses in Patients with Acute Myeloid Leukemia

Natural killer (NK) therapies have been challenging owing to antigen escape, restricted trafficking, and limited tumor infiltration, translating into modest clinical benefit. We have shown that memory-like (ML)-NK cells acquire an in vivo differentiated phenotype distinct from conventional NK cells and are well-tolerated without cytokine release syndrome, GVHD, or neurotoxicity. WU-NK-101 (W-NK) is an allogeneic cytokine-reprogrammed, expanded, cryopreserved ML-NK cell product derived from healthy human peripheral blood mononuclear cells, with scalable manufacturability, that is currently in clinical development in relapsed/refractory (R/R) AML (NCT# 05470140). We conducted a multi-modal analysis of the immune tumor microenvironment (TME) in 13 patients with R/R AML receiving ML-NK cells (NCT#01898793) with the aim to determine whether TME modifications induced by ML-NK cells correlate with clinical benefit. NK cells were purified from leukapheresate, and activated with IL-12, IL-15, and IL-18 for 12-16 hours. Patients were lymphodepleted with fludarabine and cyclophosphamide. NK cells were infused in escalating doses: 0.5 × 10 6/kg, 1.0 × 10 6/kg, and all NK cells from a single leukapheresis, capped at 10 × 10 6/kg. Response was defined as <5% BM blasts on d+28. BM trephines for immune gene expression profiling (nCounter IO360®) and spatial proteomics (GeoMx™ DSP; n=53 immuno-oncology [IO] targets; NanoString Technologies, Seattle) were collected at baseline (BL) and on treatment (OT). Immune infiltration, NK and T cell number from BM samples was quantified manually on immunofluorescent imaging. Oncomine™ TCR Pan-Clonality assay (Thermo Fisher) was used to profile TCRb and g repertoires by NGS. The Shannon index was used to describe TCR repertoire diversity, and clonal evenness was used as a measure of the similarity of clone sizes. BM infiltration with CD8 + effectors, enhanced glycolysis and cell cycling was higher in BM samples from responders (CR) to ML-NK cells. By contrast, OXPHOS RNA scores and gene signatures of immune senescence and terminal T-cell exhaustion were higher in non-responders (NR). We employed machine learning to construct an 8-gene predictor of response which encompassed T/NK markers, such as CD7, and IFN regulatory factors and TLRs. This score was significantly higher at baseline in CR and positively correlated with response duration. Principal component analysis (PCA) of spatially resolved IO proteins separated CR from NR, particularly in OT specimens, where T-cell markers CD3/CD8 and inhibitory receptors CTLA4/LAG3 were among the top contributing features. CD34, type I IFN signaling molecule STING1 and PARP1 were significantly higher at baseline compared with OT, with OT showing higher expression of cytotoxicity markers and inhibitory receptors (e.g., Tim-3 and CD276). This observation strongly suggests modulation of the immune TME after the infusion of ML-NK cells. Response to ML-NK cells was associated with coordinated up-regulation of T cell, NK cell and B cell protein markers. To further substantiate the co-localization of T cells and NK cells in the BM FFPE sections (Fig. A), and its correlation with clinical efficacy we annotated the 93 ROIs in our spatial proteomics dataset based on a median split of CD3 and CD56 protein barcodes. ROIs from CR were largely CD3 highCD56 high, both at BL and OT. Conversely, ROIs from BM sections of NR to ML-NK cells had a predominantly CD3 lowCD56 low phenotype. Quantitative analysis of T-cell infiltration post ML-MK dosing showed a significant association with response, 0.9 (PD) vs. 5.3 (CR)-fold-increase ( P=0.01). Interestingly, data demonstrate selected intra-BM clonal expansion as evidenced by decreased TCR diversity (mean ± SEM, 4.6±0.3 vs. 3.3±0.4, pre vs. post; P=0.013)and increased clonality (Fig. B). Thus, pre-existing T-cell responses appear to be co-opted in the AML TME of CR, suggesting the potential for durable effectiveness of ML-NK cells. Multi-modal analyses of WU-NK-101 highlight unique transcriptional and functional states, which could underpin in vivo potency and persistence. WU-NK-101 are currently being evaluated in a phase 1 study of R/R AML (#NCT05470140).

Novel CD20 Mutations As a Mechanism of Resistance to CD20-CD3 Targeted Therapies in Non-Hodgkin's Lymphoma

Non-Hodgkin's Lymphoma (NHL) is one of the most common blood cancer types in the world. CD20 is a B cell restricted lineage marker that is a well-established target for the treatment of NHL and anti-CD20 agents are included as in standard of care regimens. Given the success of these targeted therapies, strategies to further enhance therapeutic activity are being explored, including the CD20-CD3 bispecific molecule, mosunetuzumab, which is approved for treatment of relapsed and refractory Follicular Lymphoma in adults who have received >2 lines of treatment. Mosunetuzumab acts by redirecting CD3+ T cells to engage and eliminate CD20-expressing B cells. As with other targeted therapies, mutations and loss of target expression have been described following mosunetuzumab therapy [1]. Here we describe the impact of novel mutations identified in patient biopsy samples from the mosunetuzumab monotherapy trial, GO29781 that were located within the extracellular and transmembrane domains of CD20 on protein expression, localization and T-cell mediated killing. NHL cell lines, SU-DHL-16 and MAVER-1, were engineered using CRISPR-Cas9 system to create CD20 knock out (KO) cell lines. Wild-type (WT) or mutant CD20 were exogenously re-expressed in the CD20 KO cell lines and in the CD20 negative acute lymphoblastic leukemia cell line, REH, to create isogenic cell lines for further analysis. Complete CD20 loss was observed in cells expressing CD20 P160fs (frameshift) and Q187* (truncating) mutations. CD20 missense mutations located in the extracellular domain (C167G, K175E) expressed CD20 protein levels comparable to CD20 WT cells and membrane localization was confirmed by flow cytometry and immunofluorescence microscopy. To test the functional effect of CD20 mutations, in vitro co-culture cell killing assays were carried out using healthy donor CD8+ T cells, isogenic cell lines (3:1 effector to tumor cell ratio) and a proof-of-concept CD20-CD3 bispecific antibody. Differences in T-cell mediated killing were observed across mutation types. CD20 WT cell lines demonstrated 95-99% cell killing following 48 hour treatment while the CD20 KO, frameshift and truncating mutations all showed negligible T-cell mediated killing (~10-17% cell death). Cells expressing the missense mutations in the extracellular domain showed resistance to T-cell mediated killing (~30-45% cell death) compared to CD20 WT cells. T-cell activation was assessed by CD69 (early) and CD25 (late) surface markers. In all co-cultures, the level of CD69+ T cells was equivalent, while the CD69+CD25+ CD8 T cells were only significantly increased in the CD20 WT co-cultures, consistent with the high level of T-cell mediating killing. We have confirmed that these mutations can play a role in the development of resistance through both loss of target protein expression and interference within the anti-CD20 binding site. These studies help to characterize mechanisms of resistance to mosunetuzumab that could be extended to other anti-CD20 targeted agents. [1] Schuster et al. Journal of Clinical Oncology 2022, 40 (16), 7526.

SPI1 Is the Master Regulator of the Small Cell Variant of Anaplastic Large Cell Lymphoma Controlled By Methylation of SPI1 Gene Promoter Region

Introduction The small cell variant of anaplastic large cell lymphoma (SC-ALCL) is a subtype of anaplastic lymphoma kinase (ALK)-positive ALCL characterized by chemoresistance and poor prognosis, necessitating novel treatment strategies. Recent advances in targeted therapies have resulted in significant responses in various chemoresistant hematological malignancies. Therefore, understanding the underlying oncogenic mechanisms and identifying potential therapeutic targets are critical for overcoming SC-ALCL. Immunohistochemically, SC-ALCL comprises two distinct tumor cell populations: small tumor cells demonstrating negative to weak ALK and CD30 protein expression, and large tumor cells with robust expression of these proteins. We previously reported that small tumor cells display particular resistance to chemotherapy; however, the mechanisms underlying chemoresistance and the molecular characteristics of small tumor cells remain elusive. This study aimed to elucidate the molecular attributes of small tumor cells and pave the way for the development of innovative treatment approaches. Methods We investigated the characteristics of small tumor cells using patient blood samples diagnosed with SC-ALCL leukemic presentation and publicly available RNA-seq data from eight ALK-positive ALCL cell lines. Small and large tumor cells were separated from blood samples based on CD30 expression using CD30 - a phycoerythrin antibody and a magnetic bead separation kit. The presence of the NPM-ALK fusion gene was confirmed in both populations by PCR analysis. Bulk RNA-seq analysis was performed on four samples: small and large tumor cells at the onset and at the first (mainly occupied by small tumor cells) and second (mainly occupied by large tumor cells) relapses. Gene set enrichment analysis was used to identify gene expression patterns in small tumor cells. Signature genes of small tumor cells were identified by filtering with the following thresholds: false discovery rate-adjusted P-value <0.05, and fold-change >2 compared to large tumor cells. The transcription factors regulating the signature genes of small tumor cells were predicted using the g:Profiler software. Result Bulk RNA sequencing analysis of the four blood samples revealed that the SPI1 gene was significantly upregulated in small tumor cells compared to that in large tumor cells. SPI1 has emerged as the highest-scoring transcription factor, regulating the expression of 570 genes that characterize small tumor cells. Approximately 70% of these signature genes overlapped with the SPI1 target genes. Furthermore, gene set enrichment analysis revealed that small tumor cells display increased anti-apoptotic gene activity and decreased cell proliferation gene activity. These observations were validated by integrated gene expression analysis using publicly available RNA-seq data from eight ALK-positive ALCL cell lines (Figure 1). The study found high expression of SPI1 and SPI1 targeted genes in small tumor cells, accompanied by low expression of anti-apoptotic genes in ALK-positive ALCL cell lines, which were more analogous to large tumor cells than small tumor cells. Single-cell RNA-seq analysis validated the high expression of the SPI1 gene in small tumor cells. To explore the mechanism regulating SPI1 gene expression, we examined DNA methylation patterns in the SPI1 promoter region using conventional bisulfite sequencing. Conventional bisulfite sequencing revealed a hypomethylated SPI1 promoter region in small tumor cells and a hypermethylated region in large tumor cells, potentially accounting for the differential SPI1 expression. Conclusion This study highlights SPI1gene as the master regulator determining the unique characteristics of small tumor cells in SC-ALCL. Furthermore, the regulation of SPI1 gene expression appeared to be mediated by DNA methylation patterns within the SPI1 promoter region. These findings pave the way for innovative treatment strategies for SC-ALCL such as SPI1 targeting therapy and methylation-based therapies, offering promising avenues for treating SC-ALCL.

Genome-Wide CRISPR Screen Identifies MAD2L1BP, ANAPC15 and SLC39A7 As Intrinsic Regulators for Brentuximab Vedotin Sensitivity in Peripheral T-Cell Lymphoma Cells

Introduction: Brentuximab Vedotin (BV), an antibody-drug conjugate that specifically delivers the potent cytotoxic drug monomethyl auristatin E (MMAE) to CD30-positive cells, has been widely used as one of the key drugs for the treatment of peripheral T-cell lymphomas (PTCLs). BV shows relatively high response rate, however, the prognosis of PTCLs is not still satisfactory. The molecular mechanisms concerning sensitivity to BV remain to be clarified. Methods: To assess the cell-intrinsic genes affecting susceptibility to BV in PTCL cells, we performed a whole-genome loss-of-function CRISPR screen in DEL, an anaplastic large cell lymphoma (ALCL), ALK-positive cell line. DEL cells were transduced with the Brunello CRISPR knockout pooled library targeting 19,114 genes (4 sgRNAs/gene) followed by puromycin selection. Then, the transduced cells were treated with BV or DMSO control for 6 days. To rank the sgRNAs responsible for BV sensitivity, MAGeCK algorithm was used. Among the top 20 enriched genes, we further selected ones having at least 2 sgRNAs with a log2 fold change (BV/DMSO) < -0.5 or >0.5 as genes whose knock out confer BV sensitivity or resistance, respectively. Results: Through the whole-genome loss-of-function CRISPR screen, we identified 17 genes or 15 genes whose knock out confer BV sensitivity or resistance, respectively. TNF receptor superfamily member 8 (TNFRSF8, also known as CD30), a known BV target, was ranked as a top gene for BV resistance, confirming the reliability of our screening. Among the 17 enriched genes whose knock out confer BV sensitivity, endogenous mitotic checkpoint complex (MCC) inhibitors, namely MAD2L1BP and ANAPC15 were identified. As a confirmation, we knocked out MAD2L1BP or ANAPC15 in PTCL cell lines by CRISPR and found the enhanced susceptibility to BV of the cells. MAD2L1BP and ANAPC15, the endogenous MCC inhibitors, are known to facilitate anaphase-promoting complex/cyclosome (APC/C) activation. Therefore, we evaluated whether APC/C inhibition sensitize the PTCL cells to BV or not. Importantly, a combination use of proTAME, an APC/C inhibitor, and BV significantly suppressed cell proliferation of multiple PTCL lines, including ALCL, ALK-positive and adult T-cell leukemia/lymphoma (ATLL) (Fig. 1), compared to each single agent. In cell cycle analyses, PTCL lines treated with the combination demonstrated increased percentages of cells in G2/M phase, compared to each single agent. On the other hand, we found SLC39A7, a zinc channel in endoplasmic reticulum (ER), among 15 enriched genes whose knock out confer BV resistance. Since SLC39A7 has been reported to regulate TNFR1 cell surface expression (Fauster et al. Cell Death & Differentiation 2019), we hypothesized SLC39A7 could also affect the CD30 cell surface expression levels. In fact, flow cytometry analyses revealed downmodulation of CD30 cell surface expression in SLC39A7 knocked out PTCL lines (Fig. 2). We further evaluated the amount of CD30 protein expression by immunoblotting and found that SLC39A7 knocked out cells reduced the amount of mature form of CD30, which was consistent with the findings in flow cytometry. Intriguingly, we observed increasement of lower molecular weight band, which was considered as immature precursor CD30, in SLC39A7 knocked out cells, suggesting that impared glycobiology in ER might be a cause for surface CD30 downregulation in SLC39A7 knocked out PTCL cells. Finally, we treated PTCL lines with NVS-ZP7-4, an SLC39A7 inhibitor and the similar results were obtained regarding to CD30 expression by flow cytometry and immunoblotting. Conclusions: We performed an unbiased genome-wide CRISPR/Cas9 library screen and identified cell-intrinsic regulators for BV sensitivity in PTCLs. Our screening results suggested that the disruption of MAD2L1BP/ANAPC15 or APC/C inhibition would be a potential strategy to enhance therapeutic activity of BV in PTCLs. We also identified SLC39A7 downregulation could confer BV resistance by downregulating CD30 cell surface expression.