Antibodies In Acute Lymphoblastic Leukemia Research Articles

Tumor-Suppressive Cross-Linking of Anti-T. cruzi Antibodies in Acute Lymphoblastic Leukemia.

Parasites have been associated with possible anticancer activity, including Trypanosoma cruzi, which has been linked to inhibiting the growth of solid tumors. To better understand this antitumor effect, we investigated the association of anti-T. cruzi antibodies with B cells of the acute lymphoblastic leukemia (ALL) SUPB15 cell line. The antibodies were generated in rabbits. IgGs were purified by affinity chromatography. Two procedures (flow cytometry (CF) and Western blot(WB)) were employed to recognize anti-T. cruzi antibodies on SUPB15 cells. We also used CF to determine whether the anti-T. cruzi antibodies could suppress SUPB15 cells. The anti-T. cruzi antibodies recognized 35.5% of the surface antigens of SUPB15. The complement-dependent cytotoxicity (CDC) results demonstrate the cross-suppression of anti-T. cruzi antibodies on up to 8.4% of SUPB15 cells. For the WB analysis, a band at 100 kDa with high intensity was sequenced using mass spectrometry, identifying the protein as nucleolin. This protein may play a role in the antitumor effect on T. cruzi. The anti-T. cruzi antibodies represent promising polyclonal antibodies that have the effect of tumor-suppressive cross-linking on cancer cells, which should be further investigated.

Anti-CD19 monoclonal antibodies in acute lymphoblastic leukemia in children

Creation of monoclonal antibodies for leukaemia treatment is rapidly developing area of science. A number of compounds were effective for treatment of acute lymphoblastic leukaemia (ALL) at children. While unconjugated humanized antibodies have good tolerability and can be combined with chemotherapy, immunoconjugates, delivering the toxic compounds directly to the target cells, have more serious adverse effects. Antigens with high and selective expression are the ideal targets for usage with antibodies and suitable for studies of phases I/II and III in case of pediatric ALL. Antigens with stable expression on the cells surfaces, suitable both for unconjugated antibodies, leading to antibodies-dependent cell and complement-dependent cytotoxicity, and for bispecific antibodies with participation of T-cells. Monoclonal antibodies have quite different mechanism of action in comparison with routine chemotherapy and, of course, can change the strategy of ALL treatment in future.

Effectivity Of Complement-Dependent Cytotoxicity Induced By Therapeutic Antibodies In Acute Lymphoblastic Leukemia Is Dictated By Antigen Expression Levels and Curtailed By Membrane-Bound Complement Regulatory Proteins

Membrane-bound complement regulatory proteins (mCRPs) are known to protect cells from bystander lysis and spontaneous activation of complement. In the case of antibody therapy these mCRP's may down-modulate antibody-induced complement-dependent cytotoxicity (CDC). Two of these mCRPs, CD55 (DAF) and CD59 (MIRL), have been suggested as modulators of CDC. However, no strong correlation between their expression level and target cell lysis has been demonstrated thus far. Our goal was to elucidate the role of these mCRPs in the efficacy of antibody-induced CDC.We tested the effectivity of CDC induced by the therapeutic antibodies Alemtuzumab (Alt, anti-CD52), Rituximab (Rtx, anti-CD20), and Ofatumumab (Ofa, anti-CD20) in 16 cell lines showing differential expression of target antigens and mCRPs. These cell lines were generated in our laboratory from primary acute lymphoblastic leukemia (ALL) cells and their phenotype and karyotype greatly resembles the primary malignancy. CDC was induced by incubation of target cells with a serial dilution of the respective antibody in the absence or presence of human serum for a period of 18 hours. Subsequent target cell death was determined using flow cytometry by measuring the absolute number of surviving cells (propidium iodide negative, CD19 positive) per sample, using a fixed number of CytoCount™ beads as an internal reference, and calculating the magnitude of cell loss compared to untreated control samples.A strong correlation was found between antigen expression level and CDC induction by Alt, Rtx, and Ofa. Interestingly, considerable deviation from this correlation was observed especially in cell lines with intermediate antigen expression levels. To test whether this was due to differential expression of mCRPs, surface expression levels of CD55 and CD59 were measured by flowcytometry. The magnitude of CDC strongly correlated to the mCRP expression in the cell lines with intermediate antigen expression (MFI 1128±104 (CD52, n=5) 796±132 (CD20, n=3)), resulting in 51% (Alt) and 75% (Ofa) reduction of antibody-induced lysis in cell lines with high CD55/CD59 expression compared to cell lines with low CD55/CD59 expression. In the cell lines with high antigen expression (MFI 2802±67 (CD52) 2136±692 (CD20)), the protective effect of co-expression of mCRPs appeared to be overpowered by the high target antigen expression levels.To study the individual contribution of CD55 and CD59 to the protective effect against CDC, we induced the expression of CD55 and CD59 separately in the cohort of target cell lines with intermediate target antigen expression. Cell lines were transduced, using the bicistronic LZRS vector and the ϕ-NX-A retrovirus packaging cell line, with a construct coding for CD55 or CD59, respectively, coupled to the truncated nerve growth factor receptor (tNGFR) via an internal ribosome entry site (IRES)-sequence. Cells positive for membrane expression of the tNGFR marker and CD55 or CD59 were sorted using flowcytometric cell sorting. This resulted in cell lines with significantly increased expression levels of the respective mCRP compared to their MOCK transduced counterparts, yet never exceeding physiological levels as found in the original ALL cell lines. When compared to MOCK transduced cell lines, all CD55 transduced cell lines (n=4) showed a decreased sensitivity for Alt-induced CDC with a maximum decrease of 69%. Similarly, forced induction of CD59 expression significantly decreased the sensitivity to CDC compared to MOCK transduced cell lines when using Alt (maximum decrease 52%), Rtx (maximum decrease 56%), or Ofa (maximum decrease 30%) (n=5).In conclusion, we show that efficacy of CDC induction by therapeutic antibodies is dictated by the level of target antigen expression, but is dominantly influenced by expression of the mCRPs CD55 and CD59. This restraining effect on sensitivity to antibody-induced CDC was observed using Alemtuzumab as well as Rituximab and Ofatumumab, suggesting a general mechanism independent of target antigen or type of antibody. Therefore, mCRP expression on malignant cells is likely to be an important factor in the sensitivity to antibody treatment, especially when cells with intermediate antigen expression are targeted. Disclosures:No relevant conflicts of interest to declare.

Targeted therapy with monoclonal antibodies in acute lymphoblastic leukemia

To describe the current new targeted therapy with monoclonal and bispecific antibodies in adult acute lymphoblastic leukemia (ALL), to improve response rates and outcome. Blast cells in ALL express a variety of specific antigens, such as CD19, CD20, CD22, CD33 and CD52, and recently monoclonal antibodies (MoAbs) became available to target these antigens. The anti-CD20 MoAb rituximab has substantially improved the outcome in Burkitt lymphoma/leukemia, and is currently applied in de-novo B-precursor ALL. The MoAbs directed against CD22, linked to cytotoxic agents, either to calicheamicin (inotuzomab ozogamicin) or to plant or bacterial toxins (epratuzumab) are explored in refractory/relapsed childhood and adult ALL. Targeting CD19 is of great interest, as it is expressed in all B-lineage cells, including early precursors. The new bispecific antibody blinatumomab combines single chain antibodies to CD19 and CD3, and thereby T cells lyse the CD19 bearing B cells and is effective in patients with positive minimal residual disease (MRD) or refractory/relapsed ALL. Antibody therapy in ALL is very promising, with high rate of complete remission and MRD-negativity in advanced ALL. It is currently explored in de-novo ALL to establish the best setting in combination with chemotherapy or even as a monotherapy.

Clinical utility and implications of asparaginase antibodies in acute lymphoblastic leukemia

Hypersensitivity to asparaginase is common, but the differential diagnosis can be challenging and the diagnostic utility of antibody tests is unclear. We studied allergic reactions and serum antibodies to E. coli asparaginase (Elspar) in 410 children treated on St. Jude Total XV protocol for acute lymphoblastic leukemia. Of 169 patients (41.2%) with clinical allergy, 147 (87.0%) were positive for anti-Elspar antibody. Of 241 patients without allergy, 89 (36.9%) had detectable antibody. Allergies (P=0.0002) and antibodies (P=6.6 × 10(-6)) were higher among patients treated on the low-risk arm than among those treated on the standard/high-risk arm. Among those positive for antibody, the antibody titers were higher in those who developed allergy than in those who did not (P<1 × 10(-15)). Antibody measures at week 7 of continuation therapy had a sensitivity of 87-88% and a specificity of 68-69% for predicting or confirming clinical reactions. The level of antibodies was inversely associated with serum asparaginase activity (P=7.0 × 10(-6)). High antibody levels were associated with a lower risk of osteonecrosis (odds ratio=0.83; 95% confidence interval, 0.78-0.89; P=0.007). Antibodies were related to clinical allergy and to low systemic exposure to asparaginase, leading to lower risk of some adverse effects of therapy.

Clinical Utility and Implications of Asparaginase Antibodies in Acute Lymphoblastic Leukemia

Abstract 1481Clinical manifestations of allergy to asparaginase can be subtle, making the diagnosis challenging. The usefulness of antibody tests in differential diagnosis is unclear. We investigated clinical allergic reactions and serum antibodies to native E. coli asparaginase (Elspar) (at 4–5 time points per patient) in 410 children with newly diagnosed acute lymphoblastic leukemia (ALL) treated on St. Jude’s Total XV study. Of the 169 patients (41.2%) who exhibited clinical allergy to Elspar, 147 (87.0%) were positive for anti-Elspar antibodies as measured by ELISA. Of the 241 patients who never developed a clinical allergy, 89 (36.9%) had detectable antibodies. Antibody levels were estimated to be at their highest ∼ 50 days after an allergic reaction (Figure). Patients in the low-risk treatment arm were more likely than those in the standard/high-risk arm to have a clinical allergy (51% vs. 32%, P =.0002) and detectable antibodies (69% vs. 47%, P = 6.6 ×10−6) to Elspar. Of those patients positive for antibodies, the antibody exposure (area-under-the-level vs. time curve; AUC) over the entire course of asparaginase therapy (average ± standard deviation) was higher in those who developed allergy (30.5 ± 16.5 optical density × days) than in those who did not (10.8 ± 13.3; P < 1 × 10−15). Serum antibody measurements at week 7 of continuation therapy had a sensitivity of 87–88% and a specificity of 68–69% for predicting subsequent clinical reactions (occurring at weeks 7–9) or for confirming prior clinical reactions (occurring at weeks 1–6 of treatment). The median serum asparaginase activity was lower in patients positive for antibodies (0.05 IU/mL) than in those negative for antibodies (0.2 IU/mL; P = 7.0 × 10−6) 7 days after Elspar administration. After adjusting for age and treatment arm, children with higher antibody AUCs had a lower risk of symptomatic osteonecrosis (odds ratio 0.83; 95% confidence interval, 0.73–0.95; P =.007), a finding which may be caused by higher clearance of dexamethasone due to lower asparaginase activity (Yang et al, J Clin Oncol; 26: 1932–9, 2008). Antibodies to Elspar were significantly related to clinical allergy and were useful as a clinical test to predict or confirm clinical allergy to the drug. Antibodies also were associated with low plasma exposure to active asparaginase, consistent with a lower risk of other adverse effects of ALL therapy. [Display omitted] Anti-Elspar optical density (OD) level in serum samples (dots) relative to the number of days elapsed before (negative) or after (positive) clinical reaction to Elspar. The trend lines of median (solid) and quartiles (dashed) are shown. Disclosures:Evans:St. Jude Children’s Research Hospital: Employment, Patents & Royalties; Aldagen: Membership on an entity’s Board of Directors or advisory committees; NCI, NIH: Research Funding. Relling:St. Jude Children’s Research Hospital: Employment, Patents & Royalties; Sigma-Tau Pharmaceuticals, Inc:; NIH: Research Funding.

Anti-CD25 Targeted Killing of Bicistronically Transduced Cells: A Novel Safety Mechanism Against Retroviral Genotoxicity

Gene therapy for Fabry disease, a deficiency in alpha-galactosidase A (alpha-gal A) activity, has the potential to provide a cure for the disorder with a single treatment. Despite modifications to existing vectors, concerns have arisen regarding the risk of genotoxicity associated with the use of retroviruses. To address safety concerns, we propose that expression of a cell surface protein, human CD25 (huCD25) in a bicistronic format, with any therapeutic gene such as alpha-gal A can provide a target that can be used to kill transduced cells selectively should transformative events occur. We show that an anti-CD25 antibody and immunotoxin can specifically target and eliminate transduced leukemia cells expressing CD25. In a murine leukemia model, antibody treatment reduced tumor burden 32-fold and increased survival compared with untreated mice. Furthermore, after a bone marrow transplant of therapeutically transduced cells into Fabry mice, antibody treatment reduced the number of retrovirally transduced huCD25-expressing cells in the peripheral blood. A systemic loss of transduced cells with functional consequences was also evident in the liver and spleen. This proof-of-principle study demonstrates that a targeted antibody can reduce tumor burden and selectively clear bicistronically transduced hematopoietic cells that express a target antigen, thus acting as a built-in safety mechanism.

Anti-CD25 Targeted Killing of Bicistronically Transduced Cells: A Novel Safety Mechanism Against Retroviral Genotoxicity

Anti-CD25 Targeted Killing of Bicistronically Transduced Cells: A Novel Safety Mechanism Against Retroviral Genotoxicity

Treatment with monoclonal antibodies in acute lymphoblastic leukemia: current knowledge and future prospects.

After rapid improvement of treatment results in adult acute lymphoblastic leukemia (ALL) from less than 10% to 30-40% in the past decades, more recently stagnation has been observed. In addition, a borderline for further intensification of chemotherapy appears to be reached in adult ALL patients. New, preferably non-chemotherapy, approaches are therefore urgently required. One of those is targeted therapy with monoclonal antibodies (MoAbs). ALL blast cells express a variety of specific antigens which may serve as targets, such as CD19, CD20, CD22, CD33, and CD52. Published results of MoAbs in ALL are reviewed. Most experience is available for anti-CD20 (rituximab) which led to a significant improvement of the outcome in B-cell non-Hodgkin's lymphoma (NHL). In ALL, rituximab is combined with chemotherapy mainly in mature B-ALL and Burkitt's lymphoma and preliminary results are promising. In the future, studies will also be done in B-precursor ALL. Another promising B-cell antibody is anti-CD22. Several CD19 MoAbs were also tested in phase I studies. However, results are not conclusive and these MoAbs are not generally available. Far less experience with MoAb therapy is available for T-ALL, but clinical studies are on the way with anti-CD52 and anti-CD25 in adult T-cell leukemia/lymphoma. Overall, it can be stated that MoAb therapy in ALL is a promising treatment approach. Monotherapy with MoAbs in relapsed ALL occasionally led to responses, but higher effectivity can be expected from a combination with chemotherapy and treatment in the state of minimal residual disease. Well-designed studies and joint efforts are required to explore optimal combinations, timing and dosage of MoAb therapy in ALL.

Clinical relevance of blast cell phenotype as determined with monoclonal antibodies in acute lymphoblastic leukemia of adults.

Over the past 10 years, leukemias and lymphomas have been analyzed extensively with antibodies and for enzyme activity [1, 2]. More recently, the introduction of monoclonal antibodies (MoAbs) has improved dramatically the precision, practicability, and standardization of immunophenotyping. Meanwhile, immunologic analysis of leukemia cells has become routine clinical practice, and evidence that cell typing with antibodies contributes to final diagnosis, differential diagnosis, and classification of leukemias is compelling [3].