Efficient and economic protein labeling for NMR in mammalian expression systems: Application to a preT-cell and T-cell receptor protein.

Protein nuclear magnetic resonance (NMR) spectroscopy relies on the ability to isotopically label polypeptides, which is achieved through heterologous expression in various host organisms. Most commonly, Escherichia coli is employed by leveraging isotopically substituted ammonium and glucose to uniformly label proteins with 15N and 13C, respectively. Moreover, E. coli can grow and express proteins in uniformly deuterium-substituted water (D2O), a strategy useful for experiments targeting high molecular weight proteins. Unfortunately, many proteins, particularly those requiring specific posttranslational modifications like disulfide bonding or glycosylation for proper folding and/or function, cannot be readily expressed in their functional forms using E. coli-based expression systems. One such class of proteins includes T-cell receptors and their related preT-cell receptors. In this study, we present an expression system for isotopic labeling of proteins using a nonadherent human embryonic kidney cell line, Expi293F, and a specially designed media. We demonstrate the application of this platform to the ß subunit common to both receptors. In addition, we show that this expression system and media can be used to specifically label amino acids Phe, Ile, Val, and Leu in this system, utilizing an amino acid-specific labeling protocol that allows targeted incorporation at high efficiency without significant isotopic scrambling. We demonstrate that this system can also be used to express proteins with fluorinated amino acids. We were routinely able to obtain an NMR sample with a concentration of 200 µM from 30 mL of culture media, utilizing less than 20 mg of the labeled amino acids.

Histological Assessment of Synovial Sarcoma Before and After TCR-T Cell Therapy and Cryoablation: A Case Report.

BACKGROUND/AIM: Cancer/testis antigens (CTAs) are well-known molecular targets with expression restricted to testicular germ cells and malignant tumors. T-cell receptor (TCR)-engineered T-cell (TCR-T) therapy against CTAs in patients with sarcoma has shown substantial progress, but resistance to TCR-T therapy remains a critical problem. In this report, we present a case of synovial sarcoma treated with TCR-T therapy targeting the New York-esophageal squamous cell carcinoma (NY-ESO)-1 protein. Histological findings were compared before and after TCR-T therapy and before and immediately after cryoablation. CASE REPORT: A 68-year-old man received additional wide resection for synovial sarcoma in the left leg. Due to multiple metastases, he was enrolled in a clinical trial of TCR-T therapy for NY-ESO-1. The tumor demonstrated a 34.9% reduction in diameter. However, disease progression occurred by day 84 after TCR-T therapy. Six months after disease progression, cryoablation was performed for right posterior rib lesion and tumor specimens were obtained by needle biopsy both before and immediately after cryoablation. Ten months after the diagnosis of disease progression, the patient died. Expression levels of NY-ESO-1, human leukocyte antigen, and immune checkpoint proteins remained unchanged before and after TCR-T therapy. Beta catenin was up-regulated in recurrent tumor tissues after TCR-T therapy compared to levels observed before TCR-T therapy. Immediately after cryoablation, immunoreactivity for NY-ESO-1 showed a slightly reduction. CONCLUSION: Up-regulation of beta-catenin in synovial sarcoma with recurrence after TCR-T therapy may be involved in T-cell exclusion and resistance to TCR-T therapy. Needle biopsy after cryoablation can be performed with sufficient pathological diagnostic accuracy including immunostaining.

Lymphocyte disturbance and functional assessment of the [Asp521Asn] ZAP70 mutation.

Activated zeta-chain-associated protein kinase 70 (ZAP70) phosphorylates the TCRαß:CD3:zeta complex to diversify and amplify TCR signaling. Patients with ZAP70 mutations can present with phenotypes of immune dysregulation as well as infection. We identified the first Taiwanese boy with the [Asp521Asn] ZAP70 mutation who presented with recurrent pneumonia, inflammatory bowel disease-like diarrhea, transient hematuria and autoimmune hepatitis. He had isolated CD8 lymphopenia, eosinophilia, hypogammaglobulinemia, and impaired lymphocyte proliferation. Downstream CD3/CD28 signaling, phosphorylation of AKT, ZAP70 and Ca2+ influx were decreased in [Asp521Asn] ZAP70 lymphocytes. Immunophenotyping analysis revealed expansion of transitional B and CD21-low B cells, Th2-skewing T follicular helper cells, but lower Treg cells. The Asp521Asn-ZAP70 hindered TCR-CD3 downstream phosphorylation and disturbed lymphocyte subgroup "profiles" leading to autoimmunity/autoinflammation. Further large-scale studies are warranted to clarify this lymphocyte disturbance. The prognosis significantly depends on hematopoietic stem cell transplantation, but not the genotype, the presence of opportunistic infections or immune dysregulation.

Clinically Applicable Assessment of Tisagenlecleucel CAR T Cell Treatment by Digital Droplet PCR for Copy Number Variant Assessment.

Chimeric antigen receptor (CAR) T cell therapy is an innovative immunotherapy for treating cancers in both children and adults with proven utility in numerous clinical trials. Significantly, some CAR T cell therapies have now been approved by relevant national regulatory bodies across numerous countries for clinical therapeutic use outside of clinical trials. One such recently licensed product is tisagenlecleucel, a CAR T therapy approved for the treatment of B-cell acute lymphoblastic leukemia (B-ALL) using autologous T cells from the patient. The genetically engineered T cells target a protein called CD19, common to B cells, through a CAR incorporating a 4-1BB costimulatory domain to improve response. Since tisagenlecleucel is now a standard of care treatment for B-ALL, it is clinically essential to be able to accurately monitor these CAR T cells in patients. Assessment of the copy number variant (CNV) of the CAR T cell products allows this within a clinically acceptable timeframe for optimal patient benefit. However, no standardized method with high reproducibility and efficiency has been described within a routine clinical laboratory setting. Here, we demonstrated a novel digital droplet PCR (ddPCR)-based methodology for the study of CNV (ddPCR-CNV) in 4-1BB CD19-specific CAR T cells with universal applicability across clinical diagnostic laboratories.

Structural assessment of HLA-A2-restricted SARS-CoV-2 spike epitopes recognized by public and private T-cell receptors.

T cells play a vital role in combatting SARS-CoV-2 and forming long-term memory responses. Whereas extensive structural information is available on neutralizing antibodies against SARS-CoV-2, such information on SARS-CoV-2-specific T-cell receptors (TCRs) bound to their peptide-MHC targets is lacking. Here we determine the structures of a public and a private TCR from COVID-19 convalescent patients in complex with HLA-A2 and two SARS-CoV-2 spike protein epitopes (YLQ and RLQ). The structures reveal the basis for selection of particular TRAV and TRBV germline genes by the public but not the private TCR, and for the ability of the TCRs to recognize natural variants of RLQ but not YLQ. Neither TCR recognizes homologous epitopes from human seasonal coronaviruses. By elucidating the mechanism for TCR recognition of an immunodominant yet variable epitope (YLQ) and a conserved but less commonly targeted epitope (RLQ), this study can inform prospective efforts to design vaccines to elicit pan-coronavirus immunity.

Quality Assessment of Pre-Clinical Studies of Chimeric Antigen Receptor T-Cell Therapy Products: A Point of Focus on Safety.

BACKGROUND: Serious adverse reactions have been reported with the use of Chimeric Antigen Receptor (CAR) T-cell therapy in a clinical setting despite the success of these products in pre- clinical stages of development. OBJECTIVE: We evaluated the quality of available pre-clinical safety data of CAR T-cell therapy products. METHODS: A 21 items safety checklist was designed specifically for CAR T-cell. Literature was searched using search/MeSH terms in PubMed (October 2019 - February 2020). Studies were screened from title and abstract. Original pre-clinical researches related to CAR T-cell anti-cancer therapy were included. RESULTS: Of the search results, 152 studies (3 in vivo, 39 in vitro, and 110 combined) were included. Only 7.9% of studies were specifically designed to evaluate/ improve product safety. Eleven studies included target antigen(s), and no study included co-stimulatory molecule(s) expressed exclusively by the tumor tissue and/or CAR T-cells. One study used CRISPR-Cas9 for CAR gene insertion. The use of switch-off mechanism and purity assessment of CAR T-cell products were reported in 13.2% and 8.6% studies, respectively. Of the 113 studies with in vivo components, immuno- competent animal models were used in 24.8%. Measurements of blood pressure, temperature, body weight, and serum cytokines were reported in 0, 2.7, 29.2, and 27.4% studies, respectively. The tissue distribution and CAR T-cells persistence were reported in 26.5% of studies. The surface expression level of CAR, functional characterization of the product, and use of control were reported in >90% of studies. CONCLUSION: The majority of the checklist parameters were not reported in the pre-clinical publications to be adequately predictive of the safety of CAR T-cells in a clinical setting.

Determining the immune environment of cutaneous T-cell lymphoma lesions through the assessment of lesional blood drops.

Detailed analysis of the cells that infiltrate lesional skin cannot be performed in skin biopsy specimens using immunohistochemistry or cell separation techniques because enzyme treatments applied during the isolation step can destroy small amounts of protein and minor cell populations in the biopsy specimen. Here, we describe a method for isolating T cells from drops of whole blood obtained from lesions during skin biopsy in patients with cutaneous T-cell lymphoma. Lesional blood is assumed to contain lesional resident cells, cells from capillary vessels, and blood overflowing from capillary vessels into the lesion area. The lesional blood showed substantial increases in distinct cell populations, chemokines, and the expression of various genes. The proportion of CD8+CD45RO+ T cells in the lesional blood negatively correlated with the modified severity-weighted assessment tool scores. CD4+CD45RO+ T cells in the lesional blood expressed genes associated with the development of cancer and progression of cutaneous T-cell lymphoma. In addition, CD8+CD45RO+ T cells in lesional blood had unique T-cell receptor repertoires in lesions of each stage. Assessment of lesional blood drops might provide new insight into the pathogenesis of mycosis fungoides and facilitate evaluation of the treatment efficacy for mycosis fungoides as well as other skin inflammatory diseases.

Signaling through polymerization and degradation: Analysis and simulations of T cell activation mediated by Bcl10.

The adaptive immune system serves as a potent and highly specific defense mechanism against pathogen infection. One component of this system, the effector T cell, facilitates pathogen clearance upon detection of specific antigens by the T cell receptor (TCR). A critical process in effector T cell activation is transmission of signals from the TCR to a key transcriptional regulator, NF-κB. The transmission of this signal involves a highly dynamic process in which helical filaments of Bcl10, a key protein constituent of the TCR signaling cascade, undergo competing processes of polymeric assembly and macroautophagy-dependent degradation. Through computational analysis of three-dimensional, super-resolution optical micrographs, we quantitatively characterize TCR-stimulated Bcl10 filament assembly and length dynamics, and demonstrate that filaments become shorter over time. Additionally, we develop an image-based, bootstrap-like resampling method that demonstrates the preferred association between autophagosomes and both Bcl10-filament ends and punctate-Bcl10 structures, implying that autophagosome-driven macroautophagy is directly responsible for Bcl10 filament shortening. We probe Bcl10 polymerization-depolymerization dynamics with a stochastic Monte-Carlo simulation of nucleation-limited filament assembly and degradation, and we show that high probabilities of filament nucleation in response to TCR engagement could provide the observed robust, homogeneous, and tunable response dynamic. Furthermore, we demonstrate that the speed of filament disassembly preferentially at filament ends provides effective regulatory control. Taken together, these data suggest that Bcl10 filament growth and degradation act as an excitable system that provides a digital response mechanism and the reliable timing critical for T cell activation and regulatory processes.

Preclinical assessment of the efficacy and specificity of GD2-B7H3 SynNotch CAR-T in metastatic neuroblastoma.

The ability to utilize preclinical models to predict the clinical toxicity of chimeric antigen receptor (CAR) T cells in solid tumors is tenuous, thereby necessitating the development and evaluation of gated systems. Here we found that murine GD2 CAR-T cells, specific for the tumor-associated antigen GD2, induce fatal neurotoxicity in a costimulatory domain-dependent manner. Meanwhile, human B7H3 CAR-T cells exhibit efficacy in preclinical models of neuroblastoma. Seeking a better CAR, we generated a SynNotch gated CAR-T, GD2-B7H3, recognizing GD2 as the gate and B7H3 as the target. GD2-B7H3 CAR-T cells control the growth of neuroblastoma in vitro and in metastatic xenograft mouse models, with high specificity and efficacy. These improvements come partly from the better metabolic fitness of GD2-B7H3 CAR-T cells, as evidenced by their naïve T-like post-cytotoxicity oxidative metabolism and lower exhaustion profile.

Next-generation CAR T cells to overcome current drawbacks.

As a rapidly emerging treatment in the oncology field, adoptive transfer of autologous, genetically modified chimeric antigen receptor (CAR) T cells has shown striking efficacy and is curative in certain relapsed/refractory patients with hematologic malignancy. This treatment modality of using a "living drug" offers many tantalizing and novel therapeutic strategies for cancer patients whose remaining treatment options may have otherwise been limited. Despite the early success of CAR T cells in hematologic malignancies, many barriers remain for widespread adoption. General barriers include cellular manufacturing limitations, baseline quality of the T cells, adverse events post-infusion such as cytokine release syndrome (CRS) and neurotoxicity, and host rejection of non-human CARs. Additionally, each hematologic disease presents unique mechanisms of relapse which have to be addressed in future clinical trials if we are to augment the efficacy of CAR T treatment. In this review, we will describe current barriers to hindering efficacy of CAR T-cell treatment for hematologic malignancies in a disease-specific manner and review recent innovations aimed at enhancing the potency and applicability of CAR T cells, with the overall goal of building a framework to begin incorporating this form of therapy into the standard medical management of blood cancers.

Template-based peptide modeling for celiac risk assessment of newly expressed proteins in GM crops.

Newly expressed proteins in genetically modified (GM) crops are subject to celiac disease risk assessment according to EFSA guidelines. Amino acid identity matches between short peptides (9aa) and known celiac restricted epitopes are required to be further evaluated through peptide modeling; however, validated methods and criteria are not yet available. In this investigation, several structures of HLA-DQ2.5/peptide/TCR (T-cell receptor) complexes were analyzed and two template-based peptide molding software packages were evaluated using various peptides including ones not associated with celiac disease. Structural characterization indicates that residues at P(position)1, P2, P5, P8, and P9 in the 9aa restricted epitopes also contribute to the binding of celiac peptides to the HLA-DQ2.5 antigen in addition to the presence of the motif Q/EX1PX2 starting at P4 or P6. The recognition of the HLA-DQ2.5/peptide complex by TCR is through specific interactions between the residues in the restricted epitopes and some loop structures in the TCR. The template-based software package GalaxyPepDock seems to be suitable for the application of peptide modeling when an estimated accuracy value of >0.95 combined with >160 interaction similarity score are used as a threshold for biologically meaningful in silico binding. Nevertheless, caution should be exercised when applying peptide modeling to celiac disease risk assessment until methods are rigorously validated and further evaluated to demonstrate its value in the risk assessment of newly expressed proteins in GM crops.

Summary of a workshop on preclinical and translational safety assessment of CD3 bispecifics.

Currently, there is a multitude of CD3 bispecifics with different molecular designs and binding properties in preclinical and clinical development for the treatment of liquid or solid tumors. The key safety concerns with CD3 bispecifics are excessive release of cytokines, which may translate to potentially life-threating cytokine release syndrome (CRS), target organ toxicity due to redirection of T-cells to normal tissues expressing the tumor-associated antigen (TAA) (off-tumor/on-target cytotoxicity), and, in some instances, neurotoxicity. Another key challenge is to arrive at a safe clinical starting dose and an efficient escalating strategy that allows patients in early dose cohorts the potential for clinical benefit in Phase 1 trials. To expand the therapeutic index and bring more treatment options to patients, there are intense efforts to overcome these challenges through improvements in molecular design, preclinical safety assessment strategies, and clinical management practices. A recent workshop at the U.S. Food and Drug Administration (FDA) with industry, academic, and regulatory agency representation was held to discuss the challenges and explore where such improvements to the development of CD3 bispecifics can be implemented. Here, the content of the presentations and the discussion that occurred during this workshop are summarized.

Costimulation Blockade in Transplantation.

T cells play a pivotal role in orchestrating immune responses directed against a foreign (allogeneic) graft. For T cells to become fully activated, the T-cell receptor (TCR) must interact with the major histocompatibility complex (MHC) plus peptide complex on antigen-presenting cells (APCs), followed by a second "positive" costimulatory signal. In the absence of this second signal, T cells become anergic or undergo deletion. By blocking positive costimulatory signaling, T-cell allo-responses can be aborted, thus preventing graft rejection and promoting long-term allograft survival and possibly tolerance (Alegre ML, Najafian N, Curr Mol Med 6:843-857, 2006; Li XC, Rothstein DM, Sayegh MH, Immunol Rev 229:271-293, 2009). In addition, costimulatory molecules can provide negative "coinhibitory" signals that inhibit T-cell activation and terminate immune responses; strategies to promote these pathways can also lead to graft tolerance (Boenisch O, Sayegh MH, Najafian N, Curr Opin Organ Transplant 13:373-378, 2008). However, T-cell costimulation involves an incredibly complex array of interactions that may act simultaneously or at different times in the immune response and whose relative importance varies depending on the different T-cell subsets and activation status. In transplantation, the presence of foreign alloantigen incites not only destructive T effector cells but also protective regulatory T cells, the balance of which ultimately determines the fate of the allograft (Lechler RI, Garden OA, Turka LA, Nat Rev Immunol 3:147-158, 2003). Since the processes of alloantigen-specific rejection and regulation both require activation of T cells, costimulatory interactions may have opposing or synergistic roles depending on the cell being targeted. Such complexities present both challenges and opportunities in targeting T-cell costimulatory pathways for therapeutic purposes. In this chapter, we summarize our current knowledge of the various costimulatory pathways in transplantation and review the current state and challenges of harnessing these pathways to promote graft tolerance (summarized in Table 10.1).

Driving the CAR to the Bone Marrow Transplant Program.

PURPOSE OF REVIEW: The US Food and Drug Administration (FDA) approved two commercially available chimeric antigen receptor (CAR) T cell therapies for the treatment of relapsed B cell acute lymphoblastic leukemia (B-ALL) children and young adults less than 25 years of age and non-Hodgkin lymphoma in adults after promising results from early-phase single and multi-institutional clinical trials. In this review, we provide an overview of the practical aspects of a chimeric antigen T cell receptor (CAR-T) program development and the steps necessary for its successful implementation. RECENT FINDINGS: CAR-T therapy is a complex process and poses significant challenges as institutions prepare to deliver this therapy as a standard of care for the eligible patients. It requires a rigorous infrastructure with specific clinical, administrative, and regulatory demands. Institutions that led the clinical trials for CAR-T have adopted various approaches to integrate commercial CAR-T products into their program. Delivering commercial CAR-T cells outside the scope of clinical trials requires careful planning, allocation of resources, and utilization of existing infrastructure. Institutions may need to adapt the existing recommendations and guidelines and tailor them to meet the needs of their program and ensure appropriate financial reimbursement for this expensive but promising immunotherapy.

Recent Advances in CAR-T Cell Therapy for Non-Hodgkin Lymphoma.

Adoptive cellular immunotherapy with anti CD19 chimeric antigen receptor (CAR)-T cell has changed the treatment landscape in relapsed/refractory B cell lymphomas. They have emerged as effective therapy in patients with multiple relapsed/refractory disease, capable of sustaining durable remissions. Two CAR-T cell products (axicabtagene ciloleucel and tisagenlecleucel) are currently approved by the United States Food and Drug Administration. A third anti CD19 CAR-T cell, lisocabtagene ciloleucel is currently being evaluated in large clinical trials and may also be United States Food and Drug Administration-approved soon. CAR-T cell-related toxicities, including infections, cytokine release syndrome, and neurotoxicity are potential complications of therapy. With increasing use of CAR-T cells, the mechanism of toxicities and mitigation strategies needs to be developed. Additionally, reasons for CAR-T cell failure and progression following this therapy needs to be further studied. We describe the recent developments in this field, with emphasis on the complications of therapy and factors contributing to toxicities, efficacy, and resistance. We also describe the ongoing research in this field and the newer CAR-T cell constructs that are being developed to counter the challenges that have been identified in this field.

Assessment of TCR signal strength of antigen-specific memory CD8+ T cells in human blood.

Assessment of the quality and the breadth of antigen (Ag)-specific memory T cells in human samples is of paramount importance to elucidate the pathogenesis and to develop new treatments in various diseases. T-cell receptor (TCR) signal strength, primarily controlled by TCR affinity, affects many fundamental aspects of T-cell biology; however, no current assays for detection of Ag-specific CD8+ T cells can assess their TCR signal strength in human samples. Here, we provide evidence that interferon regulatory factor 4 (IRF4), a transcription factor rapidly upregulated in correlation with TCR signal strength, permits the assessment of the TCR signal strength of Ag-specific CD8+ T cells in human peripheral blood mononuclear cells (PBMCs). Coexpression of IRF4 and CD137 sensitively detected peptide-specific CD8+ T cells with extremely low background in PBMCs stimulated for 18 hours with MHC class I peptides. Our assay revealed that human memory CD8+ T cells with high-affinity TCRs have an intrinsic ability to highly express CD25. Furthermore, HIV-specific CD8+ T cells in chronic HIV+ subjects were found to display primarily low-affinity TCRs with low CD25 expression capacity. Impairment in the functions of HIV-specific CD8+ T cells might be associated with their suboptimal TCR signals, as well as impaired responsiveness to interleukin-2.

The Other Side of CAR T-Cell Therapy: Cytokine Release Syndrome, Neurologic Toxicity, and Financial Burden.

Immune effector cells, including T cells and natural killer cells, which are genetically engineered to express a chimeric antigen receptor (CAR), constitute a powerful new class of therapeutic agents to treat patients with hematologic malignancies. Several CAR T-cell trials have shown impressive remission rates in patients with relapsed/refractory hematologic cancers. Although the clinical responses of these agents in hematologic malignancies have been very encouraging, they have also produced substantial morbidity and occasionally mortality resulting from toxicity. With more experience and collaboration, hopefully the toxicities and the costs will come down, increasing the availability of CAR T cells to patients in need.

Preclinical Assessment of Suitable Natural Killer Cell Sources for Chimeric Antigen Receptor Natural Killer-Based "Off-the-Shelf" Acute Myeloid Leukemia Immunotherapies.

The introduction of chimeric antigen receptors (CARs) to augment the anticancer activity of immune cells represents one of the major clinical advances in recent years. This work demonstrates that sorted CAR natural killer (NK) cells have improved antileukemia activity compared to control NK cells that lack a functional CAR. However, in terms of viability, effectiveness, risk of side effects, and clinical practicality and applicability, an important question is whether gene-modified NK cell lines represent better CAR effector cells than primary human donor CAR-NK (CAR-dNK) cells. Comparison of the functional activities of sorted CAR-NK cells generated using the NK-92 cell line with those generated from primary human dNK cells demonstrated that CAR-NK-92 cells had stronger cytotoxic activity against leukemia cells compared to CAR-dNK cells. CAR-NK-92 and CAR-dNK cells had similar CD107a surface expression upon co-incubation with leukemia cells. However, CAR-NK-92 cells secreted higher granzyme A and interleukin-17A levels, while CAR-dNK cells secreted more tumor necrosis factor alpha, interferon gamma, and granulysin. In addition, CAR-NK-92 cells revealed a significantly higher potential for adverse side effects against nonmalignant cells. In short, this work shows the feasibility for further development of CAR-NK strategies to treat leukemia.

An Economical, Quantitative, and Robust Protocol for High-Throughput T Cell Receptor Sequencing from Tumor or Blood.

The T cell receptor repertoire provides a window to the cellular adaptive immune response within a tumor, and has the potential to identify specific and personalized biomarkers for tracking host responses during cancer therapy, including immunotherapy. We describe a protocol for amplifying, sequencing, and analyzing T cell receptors which is economical, robust, sensitive, and versatile. The key experimental step is the ligation of a single-stranded oligonucleotide to the 3' end of the T cell receptor cDNA, which allows easy amplification of all possible rearrangements using only a single set of primers per locus, while simultaneously introducing a unique molecular identifier to label each starting cDNA molecule. After sequencing, this molecular identifier can be used to correct both sequence errors and the effects of differential PCR amplification efficiency, thus producing a more accurate measure of the true T cell receptor frequency within the sample. This method has been applied to the analysis of unfractionated human tumor lysates, subpopulations of tumor-infiltrating lymphocytes, and peripheral blood samples from patients with a variety of solid tumors.