Redirection of CD4+ and CD8+ T lymphocytes via an anti-CD3 × anti-CD19 bi-specific antibody combined with cytosine arabinoside and the efficient lysis of patient-derived B-ALL cells.

BACKGROUND: B-acute lymphoblastic leukemia (B-ALL) is derived from B cell progenitors. Recently, the development of appropriate combinations of chemotherapy and immunotherapy represents a promising approach for eliminating cancer. We previously constructed an anti-CD3 × anti-CD19 bi-specific antibody in a diabody configuration and its disulfide-stabilized format (ds-diabody). The combination of the diabody or ds-diabody and Ara-C was highly effective in enhancing the cytotoxicity of T cells against the CD19+ human leukemia cell-line, Nalm-6, both in vitro and in vivo. This study verified whether B-ALL patient-derived cells were sensitive to the diabody or ds-diabody and low-dosage Ara-C combination. METHODS: This study aimed to detect the B7 family members B7.1 (CD80) and B7.2 (CD86) that were expressed in B-ALL patient-derived cells pre-treated by Ara-C (0.25 µM) and to determine the targeted killing ability of T cell subtypes induced by the diabody or ds-diabody combination with Ara-C both in vitro and in vivo. We also determined the levels of the cytokines that were released by activated CD4+ or CD8+ T cells during therapy. RESULT: Low-dose Ara-C enhanced CD80 and CD86 expression in nearly 50 % of specimens of B-ALL patient-derived cells. A combination of diabody or ds-diabody and Ara-C enhanced T cell against B-ALL cells in vitro and in vivo. Both CD8+ and CD4+ T cells were potently activated. Expression of CD25 and CD69 was augmented equally by CD4+ or CD8+ T cells. However, CD8+ T cells made the major contribution by redirecting target cell lysis in a granzyme B and perforin-dependent mechanism. CD4+ T cells played an important immunomodulatory role by secreting IL2. Consequently, IL3, IL6, TNFα, and IFNγ were also released by CD4+ or CD8+ T cells following diabody-mediated T cell activation. CONCLUSION: T cell therapy induced by diabody or ds-diabody combined with low dose of Ara-C was effective against cancer cell-lines and in clinical trials. In vivo, the ds-diabody was more efficient than its parent diabody due to its enhanced stability.

The DNA damage response induces antigen presenting cell-like functions in fibroblasts.

The DNA damage response (DDR) alerts the immune system to the danger posed by DNA damage through the induction of damage-associated molecular pattern molecules, chemokines, and ligands for activating immune receptors such as lymphocyte function-associated antigen 1 (LFA-1), NKG2D, and DNAX accessory molecule 1 (DNAM-1). Here we provide evidence that OVA(257-264) -pulsed fibroblasts gain the ability to activate naïve OT-I CD8(+) T cells in response to DNA damage. The ability of fibroblasts to activate OT-I CD8(+) T cells depended on the upregulation of ICAM-1 on fibroblasts and DNAM-1 expression of CD8(+) T cells. OVA(257-264) -pulsed fibroblasts were able to induce a protective T-cell response against B16-OVA cells in a DDR-dependent manner. Hence, the DDR may alert the immune system to the presence of potentially dangerous cells by upregulating the expression of ligands that can induce the activation of innate and adaptive immune cells.

Proinflammatory cytokines mediate the systemic inflammatory response associated with high-dose cytarabine treatment in children.

BACKGROUND: Treatment with high-dose cytarabine (1-beta-D-arabinofuranosylcytosine) is often associated with an acute febrile reaction sometimes including abdominal pain, myalgia, and rash. The similarity of these symptoms to those caused by hypersecretion of cytokines in the systemic inflammatory response syndrome (SIRS) prompted us to investigate the plasma levels of proinflammatory cytokines during treatment of children with high-dose cytarabine. PROCEDURE: Sixteen children treated for hematological malignancies and in clinical remission were studied during treatment with six infusions of cytarabine given every 12 hr at a dose of 2 g/m(2). Blood samples for analysis of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), interleukin-1gamma (IL-1gamma), interleukin-6 (IL-6), interleukin-8 (IL-8), interleukin-10 (IL-10) and interleukin-1 receptor antagonist (IL-1ra) were obtained prior to treatment and subsequently at 12, 36 and 60 hr. Additional samples were collected as soon as fever occurred. RESULTS: Thirteen of 16 patients developed fever at a median time of 30 hr following start of treatment. At 12 hr levels of TNF-alpha were elevated followed by a rise in IL-6, IFN-alpha, and IL-1ra, peaking at the onset of fever. Thereafter these levels slowly declined whereas low IL-10 levels became detectable. CONCLUSIONS: We conclude that high-dose cytarabine treatment often induces release of TNF-alpha followed by the sequential release of other proinflammatory cytokines. Most likely these cytokines mediate the development of symptoms comprising the cytarabine syndrome.

Successful administration of cytarabine after a previous anaphylactic reaction.

Cytarabine (Cyt) is an antimetabolite used primarily in the treatment of leukemia, and both immediate and delayed hypersensitivity reactions have been reported. We studied a 9-year-old girl with lymphoblastic leukemia, who developed three anaphylactoid reactions during Cyt treatment courses over a 1-year period. Three years later, Cyt was required again. Although a skin test was negative to Cyt at the concentration of 4 micrograms/ml, we decided on placebo-controlled administration of the drug. The Cyt was well tolerated, and urine values of N-methylhistamine showed no important variations throughout this period compared to those during the placebo administration. Skin tests carried out 14 days after the study were positive at the concentration of 4 micrograms/ml. The history of different episodes of allergic reactions to Cyt, the last one being the most severe, indicated the possible participation of an immediate hypersensitivity phenomenon, but because no studies had been carried out initially, we could not establish the presence of IgE antibodies. These results indicate that good tolerance existed after the control administration procedure. The long interval, 3 years, between the allergic episode and our protocol and the appearance of a positive skin test 14 days after the protocol indicated that the subject had lost sensitivity and become resensitized after the controlled administration procedure.

Anaphylactic shock due to cytarabine in a leukemic child.

In a young child with acute promyelocytic leukemia, treatment with a variety of chemotherapeutic agents produced an acute anaphylactic reaction. When cytarabine was removed from the chemotherapeutic regimen, no further anaphylaxis occurred. Employing an enzyme-linked immunosorbent assay with cytarabine coated onto the wells in bovine serum albumin, specific IgE antibodies to this drug could be demonstrated. Similar antibodies could not be demonstrated in the serum of normal controls or of two other patients receiving cytarabine. We therefore document anaphylactic shock mediated by specific IgE antibodies to cytarabine.

Effect of uracil arabinoside on metabolism and cytotoxicity of cytosine arabinoside in L5178Y murine leukemia.

Pretreatment of L5178Y murine leukemia cells with uracil arabinoside (ara-U) enhances the cytotoxicity of cytosine arabinoside (ara-C). This effect is mediated by the cytostatic effect of ara-U, which causes a delay of cell progression through S-phase. Consequently, the specific activity of enzymes that peak during S-phase increases, and deoxycytidine kinase increases 3.6-fold over untreated controls. This allows enhanced anabolism of ara-C to nucleotides, as well as increased incorporation into DNA with ultimate synergistic cytotoxicity. It is postulated that the systemic metabolism of high-dose ara-C to sustained high levels of ara-U in patients with acute leukemia may enhance the activity of subsequent doses of ara-C, and thus contribute to a means for pharmacologic self-potentiation, contributing to the unique therapeutic activity of high-dose ara-C.

Sensitive radioimmunoassay for cytarabine and uracil arabinoside in plasma.

A sensitive and specific radioimmunoassay for cytarabine (ara-C) or uracil arabinoside (ara-U) was established by using [3H]ara-C and anti-ara-C antiserum or [3H]ara-U and anti-ara-U antiserum. The antiserum was prepared by immunizing rabbits with ara-C or ara-U hapten conjugated to human albumin. In the present assay, as low as 2.5 ng/ml (0.01 microM) of ara-C or 5 ng/ml (0.02 microM) of ara-U in blood plasma samples could be detected. The cross-reaction of ara-C or ara-U structurally related compounds with each antiserum was so small that the nucleosides could be determined without any purification procedure. The plasma concentration of ara-C and ara-U in mice following oral administration of an antileukemic agent, cytosine arabinoside-5'-stearylphosphate (C18PCA) (100 mg/kg), or ara-C (48 mg/kg) was determined by using this method. In the case of C18PCA administration, ara-C was detectable in blood for at least 24 hours longer than in the case of ara-C administration. This method is suitable for the analysis of ara-C and ara-U, the metabolites of depot drugs of ara-C such as C18PCA.

Visceral herpesvirus infections in leukemic patients receiving cytarabine.

Autopsy evidence of herpesvirus infection was found in visceral organs of four leukemic patients who had received large doses of cytarabine (cytosine arabinoside; Ara-C) shortly before their death. In three of these patients the infection was clinically unsuspected; in the fourth, cutaneous herpes zoster developed after administration of 300 mg of cytarabine daily for the preceding five days. Although cytarabine exhibits pronounced in vitro virucidal activity against herpes viruses and has been successfully used in clinical treatment of severe herpesvirus infections, the present findings and a review of the recent literature cast doubt on the antiviral effectiveness of this drug, particularly in already immunosuppressed patients, and suggest instead that such patients actually have an increased risk for development of disseminated herpesvirus infection owing to further depression of host defenses by the drug.

Cytarabine-induced anaphylaxis. Demonstration of antibody and successful desensitization.

An anaphylactic episode occurred in a patient receiving cytarabine (Ara-C) for acute myeloid leukemia. Specific allergy of the patient to cytarabine was demonstrated in vivo by intradermal testing and in vitro by four-hour passive cutaneous anaphylaxis in guinea pigs. Desensitization to cytarabine was successfully performed. This case represents the first known report of both cytarabine anaphylaxis and densensitization, and provides immunologic evidence implicating an allergic reaction to cytarabine.

A radioimmunoassay for cytosine arabinoside.

A radioimmunoassay (RIA) for cytosine arabinoside (AraC) has been developed using antiserum raised in a sheep to an AraC monophosphate-ovalbumin conjugate. The antibody shows only 0.008% cross-reactivity with uracil arabinoside (AraU) and low (0.023%) cross-reactivity with other commonly co-administered drugs such as cytotoxic and antibacterial agents, and also a number of naturally occurring nucleosides and nucleotides. It does however cross-react by 125% with AraC monophosphate and by 109% with AraC triphosphate. As little as 1 ng/ml of AraC can be detected in plasma, serum, urine and cerebrospinal fluid (CSF) with no need for prior extraction. This RIA has been used to follow the disappearance of AraC from the plasma of patients receiving the drug.

[Selective inhibition of the primary humoral immune response in mice with a combination of deoxycytidine with Cytosar]. / Izbiratel'noe ugnetenie pervichnogo gumoral'nogo immunnogo otveta u myshei kombinatsiei dezoksitsitidina s tsitozarom.

Cytosine arabinoside administration in lethal doses to C57BL/6j female mice immunized with red blood cells leads, under deoxycytidine protection, to reduction of serum hemagglutinin level on day 5 without toxicosis. Simultaneous injection of the metabolite and the antimetabolite proves to be optimum.

A radioimmunoassay for 1-beta-D-arabinofuranosyluracil with reference to cross-reactivity of 1-beta-D-arabinofuranosylcytosine with an antibody.

Antibodies directed against 1-beta-D-arabinofuranosyluracil have been produced in rabbits by immunization with a conjugate of 1-(5-O-succinyl-beta-D-arabinofuranosyl)uracil with human serum albumin. Two of four antibodies so obtained showed high specificity for 1-beta-D-arabinofuranosyluracil and allowed the development of a sensitive and reliable radioimmunoassay for this substrate. On the other hand, one antibody had a high affinity for 1-beta-D-arabinofuranosylcytosine. The binding of 1-beta-D-arabinofuranosylcytosine to this antibody was practically constant between pH 5.2 and 9.0, whereas 1-beta-D-arabinofuranosyluracil binding was affected drastically by pH. The pH-binding profile for 1-beta-D-arabinofuranosylcytosine and 1-beta-D-arabinofuranosyluracil was reminiscent of the specificity of ara-C-specific antibodies, which we previously obtained after immunization of rabbits with 1-(5-O-succinyl-beta-D-arabinofuranosyl)cytosine as a hapten.

A radioimmunoassay method for 1-beta-D-arabinofuranosyluracil using antibodies directed against 1-beta-D-arabinofuranosylcytosine.

Above pH 7.0 1-beta-D-arabinofuranosyluracil (ara-U) shows marked pH-dependent cross-reactivity with antibodies directed towards 1-beta-D-arabinofuranosylcytosine. Since this peculiar phenomenon has not been observed with other nucleosides and nucleotides thus far tested, it is probably the result of base-catalyzed tautomerism of ara-U to its enolic form which renders it more structurally similar to 1-beta-D-arabinofuranosylcytosine. By performing the radioimmunoassay at both pH 6.2 and 8.6 we could determine 1-beta-D-arabinofuranosylcytosine and ara-U simultaneously. This method for ara-U assay is simple, fairly reliable, and applicable to blood level studies.