The Road to Personalized Myeloma Medicine: Patient-specific Single-domain Antibodies for Anti-idiotypic Radionuclide Therapy.

To this day, multiple myeloma remains an incurable cancer. For many patients, recurrence is unavoidably a result of lacking treatment options in the minimal residual disease stage. This is due to residual and treatment-resistant myeloma cells that can cause disease relapse. However, patient-specific membrane-expressed paraproteins could hold the key to target these residual cells responsible for disease recurrence. Here, we describe the therapeutic potential of radiolabeled, anti-idiotypic camelid single-domain antibody fragments (sdAbs) as tumor-restrictive vehicles against a membrane-bound paraprotein in the syngeneic mouse 5T33 myeloma model and analogously assess the feasibility of sdAb-based personalized medicine for patients with multiple myeloma. Llamas were immunized using extracts containing paraprotein from either murine or human sera, and selective sdAbs were retrieved using competitive phage display selections of immune libraries. An anti-5T33 idiotype sdAb was selected for targeted radionuclide therapy with the ß--particle emitter 177Lu and the α-particle emitter 225Ac. sdAb-based radionuclide therapy in syngeneic mice with a low 5T33 myeloma lesion load significantly delayed tumor progression. In five of seven patients with newly diagnosed myeloma, membrane expression of the paraprotein was confirmed. Starting from serum-isolated paraprotein, for two of three selected patients anti-idiotype sdAbs were successfully generated.

Association Between Immunosenescence Phenotypes and Pre-frailty in Older Subjects: Does Cytomegalovirus Play a Role?

Frailty is highly prevalent in old age and confers an important mortality risk. Although the causes of frailty are multiple, immunosenescence (IS)-predominantly driven by cytomegalovirus (CMV)-has been implicated in its pathophysiology. Thus far, research examining the association between IS and frailty states is sparse and equivocal. On the other hand, evidence is mounting in support of the view that frailty can be reversed, especially for those in the pre-frail stage. Therefore, we aimed to clarify the impact of CMV on IS and its relevance to pre-frailty. One hundred seventy-three persons aged 80 to 99 years were enrolled. Pre-frailty was defined according to Fried's criteria. Anti-CMV IgG and serum IL-6 were measured using Architect iSystem and Luminex, respectively. T-cell phenotypes were determined using flow cytometry. The prevalence of pre-frailty was 52.6%, increased with age (p = .001), and was greater in men than women (p = .044). No relationship was found between pre-frailty and positive CMV serology. Further, CMV-seropositivity was significantly associated with less naïve cells, more memory and senescence-prone phenotypes (all p < .001). After adjusting for potential confounders, only IL-6, age and sex were predictive of pre-frailty. We conclude that the presence of pre-frailty is independent from CMV infection in very old subjects.

Lymphoma-like monoclonal B cell lymphocytosis in a patient population: biology, natural evolution, and differences from CLL-like clones.

High-count monoclonal B cell lymphocytosis (MBL) with a chronic lymphocytic leukemia (CLL) phenotype is a well-known entity, featuring 1-4% annual risk of progression towards CLL requiring treatment. Lymphoma-like MBL (L-MBL), on the other hand, remains poorly defined and data regarding outcome are lacking. We retrospectively evaluated 33 L-MBL cases within our hospital population and compared them to 95 subjects with CLL-like MBL (C-MBL). Diagnoses of L-MBL were based on asymptomatic B cell clones with Matutes score < 3, B cells < 5.0 × 103/µl, and negative computerized tomography scans. We found that median B cell counts were considerably lower compared to C-MBL (0.6 vs 2.3 × 103/µl) and remained stable over time. Based on immunophenotyping and immunogenetic profiling, most L-MBL clones did not correspond to known lymphoma entities. A strikingly high occurrence of paraproteinemia (48%), hypogammaglobulinemia (45%), and biclonality (21%) was seen; these incidences being significantly higher than in C-MBL (17, 21, and 5%, respectively). Unrelated monoclonal gammopathy of undetermined significance was a frequent feature, as the light chain type of 5/12 paraproteins detected was different from the clonal surface immunoglobulin. After 46-month median follow-up, 2/24 patients (8%) had progressed towards indolent lymphoma requiring no treatment. In contrast, 41% of C-MBL cases evolved to CLL and 17% required treatment. We conclude that clinical L-MBL is characterized by pronounced immune dysregulation and very slow or absent progression, clearly separating it from its CLL-like counterpart.

Preclinical evaluation of invariant natural killer T cells in the 5T33 multiple myeloma model.

Immunomodulators have been used in recent years to reactivate host anti-tumor immunity in several hematological malignancies. This report describes the effect of activating natural killer T (NKT) cells by α-Galactosylceramide (α-GalCer) in the 5T33MM model of multiple myeloma (MM). NKT cells are T lymphocytes, co-expressing T and NK receptors, while invariant NKT cells (iNKTs) also express a unique semi-invariant TCR α-chain. We followed iNKT numbers during the development of the disease in both 5T33MM mice and MM patients and found that their numbers dropped dramatically at the end stage of the disease, leading to a loss of total IFN-γ secretion. We furthermore observed that α-GalCer treatment significantly increased the survival of 5T33MM diseased mice. Taken together, our data demonstrate for the first time the possibility of using a preclinical murine MM model to study the effects of α-GalCer and show promising results of α-GalCer treatment in a low tumor burden setting.

Diagnostic potential of CD34+ cell antigen expression in myelodysplastic syndromes.

The World Health Organization introduced flow cytometry as an additional criterion for diagnosis of myelodysplastic syndromes (MDS). Aberrant antigen expression on bone marrow (BM) blasts may identify "low-grade MDS." This study aimed to examine differences in antigen expression on CD34+ BM cells between patients with MDS and those with secondary cytopenia. BM aspirates of 175 patients with cytopenia were classified as MDS or secondary cytopenia. Expression of stem cell antigens (CD34, CD133), myeloid antigens (CD13, CD33), B-cell antigens (CD19, CD10), growth factor receptors (CD117, CD123), and chemokine receptor (CD184) was examined. Thirty-two normal adults and 49 patients with CD34+ acute myeloid leukemia (AML) were also examined. High percentage of CD34+ cells, CD117 and CD123 overexpression, and abnormal CD45 expression on these cells are the best markers for MDS. These phenotypic aberrancies correlate with number of blasts and degree of dysplasia, and were similar to those in CD34+ AML, thus reflecting the relationship between these disorders.

Cellular aging and senescence characteristics of human T-lymphocytes.

CD28-, CD57+ and KLRG1+ are cell surface markers that have been used to describe senescent T-lymphocytes in humans. However, the relationship among these phenotypes during aging, and their relationship with the concept of in vitro cellular aging have not been well established. Using five-colour flow cytometry, we analyzed peripheral blood T-lymphocytes for their expression of CD28, CD57 and KLRG1 in 11 young (Y) and 11 old (O) apparently healthy human subjects. The proportions of CD28- and CD57+ cells were significantly higher among the T-cell populations of O compared to Y subjects; the proportion of KLRG1+ cells was significantly higher only among CD8+ cells. Populations that were more frequent in the elderly participants were characterised as CD28+ CD57+, CD28- CD57+ or CD28- CD57-. The expression of p16 and p21, considered as markers for in vitro senescence, was higher in CD28+ CD57+ cells than in other subpopulations in both age groups. The expression of p21 was age-related, which was not the case for p16. Thus, although both p16 and p21 are involved in T-cell senescence, they appear to behave differently. CMV infection and shifts in subpopulations are unlikely as explanations of the observed differences. Their higher levels of p16 and p21 expression, coupled with their higher prevalence in the elderly participants make CD28+ CD57+ cells the subpopulation of T-cells most closely corresponding to the concept of senescent cells.