Julia F. Agee

Immunologically diagnosed malignancy in Sjögren's pseudolymphoma

Studies of lymphocyte markers in a patient with Sjögrens syndrome who exhibited histologically benign lymphoproliferation in the lung revealed a malignant cell clone. T and B cells were quantitated according to their ability to form spontaneous rosettes with sheep erythrocytes and to fluoresce with fluorescein-conjugated antiserums, respectively. Circulating lymphocytes were 66 percent T cells (N = 58 +/- 2 per cent) and 14 percent B cells (N = 22+/- 1 percent), the latter exhibiting normal polyclonal distribution of membrane immunoglobulins. However, lymphocyte suspensions obtained from fresh lymph node and from biopsy specimens from a lymphoid lung nodule revealed 95 percent and 88 percent B cells, with 1 percent and 2 percent T cells, respectively. Moreover, when cryostat-frozen sections from both tissues were reacted with each of the heavy and light chain-specific antiserums, most cells demonstrated the presence of intracytoplasmic mu kappa immunoglobulin exclusively. Twenty-two months later, a clinically and histologically classic lymphoma developed. Repeat marker studies performed on cells freshly isolated and on frozen sections from the histologically malignant lymph node revealed persistence of the monoclonal marker on most cells.

Clone Emergence and Evolution in Chronic Lymphocytic Leukemia: Characterization of Clinical, Laboratory and Immunophenotypic Profiles of 25 Patients

Chronic lymphocytic leukemia (CLL) is a lymphoproliferative disease usually involving B-cells. Characteristically, B-CLL cells express CD5, CD19, CD20, CD23, cCLLa, Fc and mouse RBC receptors, and low density monoclonal surface immunoglobulins (Moslgs). In order to shed light into the presentation and natural history of B-CLL, patients with a sustained, mild relative (>45%) or absolute lymphocytosis (<104/μL) and a non-diagnostic marrow (waived if lymphocyte count <5 × 103μL) were phenotyped in search of a B-cell clone. Immunophenotyping, initially done by fluorescence microscopy was confirmed by one and two color flow cytometry. Positive and negative controls included 95 patients with overt B-CLL and 45 healthy volunteers, respectively. While patients with overt B-CLL exhibited a cCLLa+ (100%), CD19+ (92%), MosIgs+ (92%) and CD5+ (80%) clone, healthy volunteers did not. Out of 39 patients with lymphocytosis of unknown significance (LUS), 15 had relatively normal immunophenotypes. However, 24 exhibited a ...

A simple technique for the rapid enrichment of class and subclass hybridoma switch variants: A 1000-fold enrichment in half the time, for half the cost

Switching parental hybrids in vitro to downstream switch variant clones producing more desirable monoclonal antibodies (MoAbs) requires either labor intensive and time consuming subcloning techniques, or fluorescence activated sorting of the desired clones. We tested the hypothesis that enrichment of downstream switch variant clones might be achieved by selective lysis of upstream hybridoma cells followed by expansion of the enriched downstream clone. Using a parental hybridoma with surface and secretory IgM, we attempted to enrich downstream switch variant clones producing class (IgG) and subclass (IgG1 or IgG2a) MoAbs. Enrichment of downstream IgG, IgG1 and IgG2a MoAb-producing switch variants was achieved by single or repeated antibody-dependent, complement-mediated lysis of the upstream IgM-bearing parental hybridoma cells followed by limited subcloning. Two exposures of parental hybridoma cells to lysis followed by plating at 100 cells/well enriched the frequency of switch variants up to 1235-fold, enabling the development of IgG1 or IgG2a-producing subclones exhibiting high yield antibody production. Using this protocol, production time and costs were reduced by > 50% when compared to the standard technique. This novel technique for the rapid isolation and expansion of switch variant clones should be ideal for most laboratories, particularly those without access to cell sorting capabilities.

Chronic lymphatic leukemia evolving into chronic myelocytic leukemia

A diagnosis of chronic lymphatic leukemia (CLL) was made in an 83‐year‐old man on the basis of marked lymphocytosis (131.1 × 109/L) and infiltration of the marrow (77%) by small lymphoid cells. The hemoglobin was 11.8 g/dL and the platelet count was 427.5 × 109/L. With minimal and unsustained treatment, abrupt resolution of the lymphocytosis occurred with reciprocal emergence of a classic picture of chronic myelogenous leukemia (CML). During the hematologic evolution, blood lymphoid cells were isolated for surface marker, cytogenetic, and functional studies. E‐rosette receptors, Fcreceptors, and membrane immunoglobulins were found on only 7.5%, 7.4%, and 10.0% of these cells. These cells also failed to express a CLL‐associated antigen we have detected on ≈︁ 95% of the cells of all CLL patients studied, regardless of cell phenotype. In addition, the patients lymphoid cells responded poorly to leucoagglutinin (LPHA) stimulation, showed increased spontaneous metabolic activity and exhibited the Philadelphia chromosome. These observations suggest that the patients transient initial lymphocytosis was not due to CLL, but perhaps represented myeloid precursors in circulation prior to terminal differentiation.

The Chronic Lymphocytic Leukemia Antigen (cCLLa) as Immunotherapy Target: Pharmacokinetics and Biodistribution of Two Divalent, Ricin-Based Immunotoxins in Xenografted Athymic Mice

The chronic lymphocytic leukemia (CLL) antigen (cCLLa) is potentially suitable for targeted immunotherapy given its restriction to clonal CLL cells and lack of expression by normal lymphocytes. In order to assess the pharmacokinetics and biodistribution of two potent anti-cCLLa immunotoxins (ITs) were examined in the mouse model. The IgG fraction of anti-cCLLa monoclonal antibody CLL2m was conjugated with 125I-labeled intact (RTA) or deglycosylated (dgA) ricin chain A, injected intravenously into athymic mice engrafted with cCLLa-expressing human tumors, and monitored over 120 hours. Blood concentrations of CLL2m/125I-RTA and CLL2m/125I-dgA were best fit to biexponential equations but the latter exhibited a lower alphaT1/2 and betaT1/2 (4.1 and 102 min vs 5.9 and 126 min), a smaller volume of distribution (5.1 g vs 9.7 g), and a lower blood clearance (2.2 g/hr vs 4.6 g/hr). Both ITs exhibited preferential tumor uptake that followed distinct kinetics: rising tumor uptake for 2 hrs post-injection (while tissue uptake decreased), reaching tumor/non-tumoral tissue uptake ratios up to 16.9; and slower dissociation rates of tumor- vs tissue-bound ITs (>45% vs <20% remaining tissue-bound 6 hrs post-injection, respectively). Non-specific liver uptake was not prominent for either IT. In vivo IT deconjugation reached 50% approximately 12 hours pos-injection. The pharmacokinetics and biodistribution data in the mouse model suggest that ricin-based anti-cCLLa ITs are suitable for use in human trials.

The Chronic Lymphocytic Leukemia Antigen (cCLLa) as Immunotherapy Target: Assessment of LD50 and MTD of Four Ricin-Based Anti-cCLLa Immunotoxins (ITs) in Balb/c Mice

The chronic lymphocytic leukemia (CLL) antigen (cCLLa) is a promising immunotherapy target given its disease-restricted expression, its highest prevalence among CLL surface antigens, and its lack of expression by normal T- and B-lymphocytes. The objectives of this study were to assess the 50% lethal dose (LD50) and the maximum tolerated dose (MTD) in Balb/c mice of four anti-cCLLa immunotoxins (ITs) derived from the intact monoclonal antibody (MoAb) or its Fab fraction, each conjugated to either ricin chain-A (RTA) or its deglycosylated derivative (dgA). The IgG fraction of anti-cCLLa monoclonal antibody CLL2m and its Fab fraction were conjugated to RTA or dgA to generate four ITs: IgG/RTA, IgG/dgA, Fab/RTA and Fab/dgA. Progressive concentrations of each IT (ranging between 2.60 mg/kg and 100.00 mg/kg) were injected intravenously into groups of 5 mice each. After injection, mice were monitored daily for 10 days for survival. Observed mortality data in each group were matched to those in Weils tables for estimating LD50 (mg/kg) from the moving average interpolation method. Estimated LD50 (in mg/kg) were: IgG/RTA, 13.33; Fab/RTA, 25.53; IgG/dgA, 55.33; Fab/dgA, 55.33. Their respective MTD (mg/kg), defined as the highest dose level survived by all mice, were 8.78, 13.17, 29.63 and 29.63. Depending on the animal-to-human extrapolation method used, the calculated LD50 and MTD in humans ranged from 1.2 mg/kg and 0.8 mg/kg (IgG/RTA), to 55.6 mg/kg and 36.9 mg/kg (IgG/dgA and Fab/dgA), respectively. The following conclusions are drawn. 1. Antibody valence exerted little influence on either the LD50 or the MTD; 2. The LD50 to MTD ratios were approximately 2:1; 3. dgA-derived ITs were approximately one half as toxic as their RTA-derived counterparts; and 4. Extrapolation of LD50 and MTD mouse data to humans resulted in dose levels comparable to or exceeding those reported in most IT human trials. These data suggest the suitability of anti-cCLLa ITs for clinical immunotherapy trials.