CAR-T therapy against hematological and solid cancer

Adoptive cell therapy is a promising alternative option for treating both solid and hematological cancers, with CAR-T therapy standing out as a prominent approach. In this method, T cells undergo genetic modification with chimeric antigen receptors (CARs) to improve their targeting abilities.1-2

The effectiveness of CAR-T cell treatment relies on the intricate interplay of phenotype, activation, and functional profiling of these modified cells. Immunophenotypic analysis of CAR-T cells plays a crucial role in guaranteeing treatment quality and enabling continual monitoring of treatment response.1

During the immunophenotyping process, engineered T cells are segregated according to their markers to characterize the composition of the cell population in the specimen. The deliberate identification and separation of specific subsets of CAR-T cells is vital for enhancing treatment responses.2

This article describes the features and role of CAR-T therapy in the immunophenotyping and treatment of solid cancers.

Immunophenotyping is an important method that couples specific antibodies with fluorescent compounds to uncover protein expression in cell populations to detect and categorize the tagged cells.

This technique exploits variations in surface markers among T cells, reflecting differences in their activation, differentiation, and memory status.2

These markers offer insights into immune cell development, function, proliferation potential, and long-term viability. The specific surface marker profiles correlate with the effectiveness of CAR-T cell therapy.3

Table 1 presents key markers for immunophenotypic analysis, including CD3, CD4, CD8, CD45RA, CD34R0, CCR7, CD27, and CD95.

Table 1. A comprehensive phenotype of markers of T cell subsets (DOI: 10.1007/978-1-0716-0146-4). Source: Sino Biological Inc. 

Flow cytometry (FCM) is the preferred approach for assessing the immunophenotype and functionality of CAR-T cell subsets.4 The success of experiments is directly influenced by the quality of Flow cytometry (FACS) antibodies.

FCM also allows for the monitoring of CAR-T cells in a patient's blood, which is crucial for predicting the probability and severity of cytokine release syndrome (CRS) or neurotoxicity.5

To aid research on immunophenotyping, Sino Biological has established a comprehensive collection of recombinant T cell biomarkers and T cell-related FACS exhibiting high affinity and specificity, including CD3, CD4, CD8, and CD45.

Figure 1. Quality products for T cell markers offered by Sino Biological. Image Credit: Sino Biological Inc.

Enhancing the effectiveness of CAR-T therapy requires the pre-selection of T cell populations before CAR engineering. T lymphocytes can be immunophenotyped based on CD3 expression.

This can be further categorized into CD4+ and CD8+ T cells, which are distinguished by their subsets and their effector and memory functions. They demonstrate various characteristics regarding internal and external markets, epigenetic factors, genetic programs, and metabolic pathways.6  

These subsets are intrinsically tied to CD3 molecule interactions in the T cell receptor (TCR) signaling pathway. They significantly impact CAR-T cell phenotype and therapeutic efficacy.5-7

Certain studies suggest that CD4+ CAR-T cells may trigger more powerful antitumor responses against specific solid tumors when compared to CD8+ cells.8

Figure 2. Schematic overview of the T cell subsets https://doi.org/10.1111/all.15104. Image Credit: Sino Biological Inc.

From the standpoint of the activation phase, Naive and memory T cells have prolonged persistence and heightened antitumor activity, and thus surpass effector T cells in therapeutic potential.9

CAR-T cells that display a memory-like phenotype, identified by surface markers like CD62L, CD45RA, CCR7, and CD45RO, exhibit superior efficacy.3

Exhaustion-like phenotypes, on the other hand, are characterized by the expression of inhibitory receptors (PD-1, LAG3, TIM-3, CTLA4, and TIGIT) and exhibit limited therapeutic potency.3,10

CAR-T cell products derived from preselected naive/stem memory T cells (TN/SCM) show greater expansion potential and a smaller risk of cytokine release syndrome (CRS).11

T cell activation plays a crucial role in CAR-T cell production, influencing transduction efficiency, cell expansion rate, and differentiation. The expression of surface receptors, including CD69, CD25, CD71, and MHC-II, assists in assessing T cell activation phases.12

Co-stimulatory molecules like CD26, CD27, CD28, CD30, CD154, or CD40L may additionally be expressed upon activation.13

Activated T cells can induce toxicities through the release of inflammatory cytokines such as TNF-α and IFN-γ.14-15 Monitoring these cytokine concentrations ensures the effectiveness of the T cell population (Figure 3). Tailoring the dosage of T cell stimulation affects CAR-T cell functionality, resulting in a more consistent and potent therapeutic product.16-17

Figure 3. Different T cell phenotypes are profiled for the expression of 3 activation markers: CD69 (early), CD25 (late), and HLA-DR (even later). The 2 effector cytokines (IFNγ and TNFα) are also quantified using sandwich ELISA. Image Credit: Sino Biological Inc.

The performance of CAR-T cells is regulated by cell exhaustion, which reduces their persistence and killing activity, resulting in a diminished therapeutic potential against solid tumors.

Exhaustion markers such as PD-1, LAG3, TIM-3, CTLA4, and TIGIT provide insights into the state of CAR-T cell exhaustion, underscoring the importance of early T cell utilization to mitigate exhaustion-related limitations.18 The selection of different cytokine cocktails or suitable cytokines for CAR-T cell culture can significantly enhance the efficacy of CAR-T cells. 

Sino Biological has successfully developed an extensive range of high-quality GMP-grade cytokines, along with ELISA kits designed for the detection and measurement of residual cytokines. Additionally, Sino Biological offers a panel of recombinant exhaustion marker proteins with high purity and bioactivity.

Figure 4. Premium quality cytokines and ELISA kits. Image Credit: Sino Biological Inc.

Immunophenotyping shows significant potential in enhancing CAR-T cell therapy by improving therapeutic efficiency and predicting treatment responses.  

Sino Biological, a globally leading supplier of bioreagents and CRO services, provides comprehensive solutions for CAR-T cell therapy. These solutions encompass reagents and services that assist clients throughout each stage, from early target discovery to the preclinical phase of research and development.

Sino Biological offers a diverse range of reagents for the development of CAR-T cell therapy, featuring over 250 CAR-T target proteins, flow cytometry antibodies, GMP-grade cytokines, and GMP-grade SuperNuclease.

Stringent quality control measures are implemented to ensure the efficacy and safety of CAR-T cell products. These measures include exams of cell viability, CAR detection, population profiling, and cytotoxicity.

Figure 5. Comprehensive solutions for CAR-T therapies provided by Sino Biological. Image Credit: Sino Biological Inc.

Sino Biological is an international reagent supplier and service provider. The company specializes in recombinant protein production and antibody development. All of Sino Biological's products are independently developed and produced, including recombinant proteins, antibodies, and cDNA clones. Sino Biological is the researchers' one-stop technical services shop for the advanced technology platforms they need to make advancements. In addition, Sino Biological offers pharmaceutical companies and biotechnology firms pre-clinical production technology services for hundreds of monoclonal antibody drug candidates.

Sino Biological is committed to providing high-quality recombinant protein and antibody reagents and to being a one-stop technical services shop for life science researchers around the world. All of our products are independently developed and produced. In addition, we offer pharmaceutical companies and biotechnology firms pre-clinical production technology services for hundreds of monoclonal antibody drug candidates. Our product quality control indicators meet rigorous requirements for clinical use samples. It takes only a few weeks for us to produce 1 to 30 grams of purified monoclonal antibody from gene sequencing.

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Should Combination Therapy Be Standard for Benign Prostatic Hyperplasia?

The treatment of benign prostatic hyperplasia (BPH) that causes lower urinary tract symptoms has advanced enormously since the days when it was a simple choice between no treatment with reassurance, and transurethral resection of the prostate (TURP). Improvements in treatment arose from our recognition that one of the commonest urologic conditions affecting men required other therapeutic options that fitted somewhere between these two extremes. This, in turn, led to a huge amount of laboratory and clinical research that increased our understanding of BPH, and the number of pharmacologic and technological treatments available. As a result, there has been a dramatic decrease in the number of patients treated by TURP, and an equally dramatic increase in the number of patients treated by pharmacologic manipulation. Drug treatment of BPH was initially viewed with suspicion by urologists, but has gradually achieved widespread acceptance, with excellent consequences for patients.

Pharmacologic treatment of BPH is based on two concepts: first, that α-adrenergic blockade reduces smooth-muscle tone in the prostate and bladder neck; and second, that 5-α-reductase inhibition causes atrophy of prostate epithelium. Studies showed that, as monotherapy, both classes of drugs produced improvements in patients' symptoms and quality of life. Although their effects were not in any way comparable to the symptomatic improvements afforded by TURP, they tipped the balance irrevocably in favor of nonsurgical treatments for BPH.[1,2,3,4]

Urologists had further questions, however. Could these α-blockers or 5-α-reductase inhibitors prevent the long-term complications of BPH—such as acute urinary retention, recurrent urinary tract infection, or obstructive nephropathy and chronic renal failure—and could they prevent or delay the requirement for TURP? Also, would a combination of these drugs result in greater symptomatic relief and an improvement in peak urinary flow (Q max)?

The first question has largely been answered by a 4-year trial with finasteride. This study showed that BPH is a progressive condition that can lead to a worsening of symptoms, acute urinary retention and a need for surgery. Treatment with finasteride alone produced a 50% reduction in the relative risk of patients with symptomatic BPH developing these problems.[5]

Two studies have attempted to answer the second question. The Veterans' Affairs Cooperative Studies Benign Prostatic Hyperplasia Study compared four groups of patients with symptomatic BPH, treated with terazosin, finasteride, a combination of both, or placebo.[6] The Prospective European Doxazosin and Combination Therapy Trial compared four groups of patients with symptomatic BPH treated with doxazosin, finasteride, a combination of both, or placebo.[7] Both trials had similar results: there was no additional benefit in taking a combination of finasteride and the α-blocker in question compared with taking the α-blocker alone. These trials were considered decisive by many, and the option of combination therapy was put aside. Both studies, however, were only of moderate duration (12 months in both cases), and have been criticized for their short duration, and the fact that only changes in symptom score and Q max were used to define success or failure of treatment. Some felt that the effect of combination therapy on the longer term consequences of the disease should be assessed.

This was the rationale for the Medical Therapy of Prostatic Symptoms (MTOPS) study. Rather than concentrate on the effect of combination therapy on longitudinal changes, the aim of this trial was to find out whether doxazosin or finasteride, either alone or in combination, could delay or prevent the clinical progression of BPH, its longer term complications, and the requirement for surgery, compared with placebo. The study enrolled 3,047 men, which provided 81% power to detect a 33% reduction in the incidence of disease progression in an active-therapy group, allowing for a 5% loss to follow-up per year.[8] Clinical disease progression was defined as the occurrence of any of the following: a ≥4-point increase from baseline in the American Urological Association symptom score, acute urinary retention, urinary tract infection, urosepsis, incontinence or a ≥1.5 mg/dl increase in serum creatinine level or to a value ≥50% above baseline. At 1 year there was little difference between the doxazosin and combination groups, but over the following 3 years combination therapy was significantly better than any other therapy at preventing progression. The number of patients that needed to be treated to prevent one instance of overall clinical progression was 8.4 for the combination group, 13.7 for the doxazosin group and 15.0 for finasteride group. In a preplanned subgroup analysis of patients with larger prostates, the number needed to treat was halved in the combination group. When individual progression events were looked at, an interesting observation could be made regarding the cumulative incidence of acute urinary retention. Combination therapy reduced the relative risk of developing retention by 81%. Finasteride delayed the time to acute urinary retention, and reduced the rate and relative risk of retention, whereas doxazosin only delayed its onset. The risk of invasive therapy was reduced by 64% in the finasteride group and by 67% in the combination group. Doxazosin alone did not reduce the cumulative risk. The number of patients that needed to be treated to prevent one patient from undergoing invasive therapy was 25.9 for the combination group, 60.1 for the doxazosin group and 29.0 for the finasteride group. Again, the number needed to treat was initially halved among patients with larger glands. Serum PSA was an accurate marker for prostate size.

Before reaching a conclusion on the use of combination therapy for BPH, one other piece of evidence should be presented. The Symptom Management After Reducing Therapy (SMART-1) trial examined the combination of dutasteride and tamsulosin, followed by withdrawal of tamsulosin in symptomatic men.[9] This trial enrolled a smaller group of patients and was not placebo-controlled. Patients were randomized to dutasteride and tamsulosin for 36 weeks, or to both for 24 weeks followed by dutasteride plus placebo for a further 12 weeks. Consistent with earlier trials, the combination produced a rapid improvement in symptoms. After tamsulosin withdrawal, the condition of patients with mild or moderate symptoms did not deteriorate, but the condition of patients with severe symptoms did.

Having reviewed the evidence, should we recommend a combination of an α-blocker and 5-α-reductase inhibitor as standard therapy for BPH? On the basis of a single large placebo-controlled trial[8] the answer has to be yes, but, in my opinion, it should not be recommended for every case. The trial referred specifically to a combination of doxazosin and finasteride; in order to accept that any combination of α-blocker and 5-α-reductase inhibitor can be used, proof of a class effect is required. One can draw one's own conclusions from the fact that many pharmaceutical companies are trying to disprove this class effect.[10] Candidates for combination treatment are patients with severe symptoms and larger prostates, for whom withdrawal of the α-blocker at 6 months is not an option. Serum PSA can be used as a surrogate marker for prostate size. If the patient is worried about the risk of invasive therapy, and does not have severe symptoms, either finasteride or a combination of finasteride plus an α-blocker can be used. A combination is significantly more effective than either agent alone in reducing the relative risk of disease progression, which was a more frequent event in the MTOPS trial than the development of acute urinary retention or the requirement for invasive therapy. In my opinion, combination therapy is here to stay, and has its own niche in the treatment of symptomatic BPH.

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Is ketamine therapy safe? Answers to questions after Matthew Perry’s death

Ketamine, once used primarily as a sedative or a psychedelic party drug, has become a popular fast-acting treatment for depression and other serious mental health conditions. But news that the actor Matthew Perry was undergoing ketamine therapy before his death has raised new questions about the drug’s safety.

The Los Angeles County Medical Examiner’s office revealed Friday that Perry died of the acute effects of ketamine. The actor was undergoing ketamine infusion therapy, according to the autopsy, and was last treated a week and a half before his death.