Interleukin-27 inhibits pediatric B-acute lymphoblastic leukemia cell spreading in a preclinical model.

B-acute lymphoblastic leukemia (B-ALL) represents the most common pediatric hematological tumor that derives from the aberrant proliferation of early B lymphocytes in the bone marrow. Although most of the B-ALL children take advantage from current therapeutic protocols, some patients relapse and need alternative therapies. With this background, we investigated whether interleukin (IL)-27, an immunomodulatory cytokine with antitumor properties, may function as an antitumor agent against pediatric B-ALL cells. Here we show for the first time that pediatric B-ALL cells functional IL-27R and that IL-27 dampens directly tumor growth in vivo and in vitro through mechanisms elucidated in this study. The novelty of these results deals with the first demonstration that (1) B-ALL cells from pediatric patients injected intravenously (i.v.) into NOD/SCID/Il2rg(-/-) (NSG) mice gave rise to leukemic spreading that was severely hampered by IL-27; (2) IL-27-treated mice, compared with controls, showed significant reduction of putative B-ALL-initiating cells and blasts in the peripheral blood (PB), bone marrow (BM) and spleen; and that (3) IL-27 reduced in vitro B-ALL cell proliferation and angiogenesis, induced apoptosis and downregulated miR-155. Our results strongly encourage the development of future clinical trials to evaluate the toxicity and efficacy of IL-27 in childhood B-ALL patients.

Antitumor effect of donor marrow graft rejection induced by recipient leukocyte infusions in mixed chimeras prepared with nonmyeloablative conditioning: critical role for recipient-derived IFN-gamma.

Some patients lose chimerism following nonmyeloablative hematopoietic cell transplantation (HCT), yet, surprisingly, enjoy sustained tumor remissions. We hypothesized that host-versus-graft (HVG) alloresponses might induce antitumor effects against recipient tumors. We explored this question in mice by administering recipient leukocyte infusions (RLIs) to mixed chimeras established with nonmyeloablative conditioning. Mixed chimeras were prepared in the B10.A (H2a)-->B6 (H2b) strain combination using depleting anti-T-cell monoclonal antibodies (mAbs), cyclophosphamide, and thymic irradiation. B6 myeloid leukemia cells (MMB3.19) were administered 7 days following donor lymphocyte infusion (DLI) or RLI on day 35. Conversion to full donor chimerism occurred without graft-versus-host disease (GVHD) following DLI, whereas RLI led to loss of chimerism. Both RLI and DLI significantly delayed tumor mortality. In another strain combination (B10.BR [H2k]-->BALB/c [H2d]), RLI-induced or spontaneous loss of chimerism was associated with antitumor effects against the host-type B-cell lymphoma A20. HCT was essential for the antitumor effect of RLI. RLI induced elevated serum interferon-gamma (IFN-gamma) levels, and recipient-derived IFN-gamma was critical for their antitumor effects. Thus, HVG reactions (spontaneous or induced by RLI) mediate antitumor effects against hematologic malignancies via a recipient-derived IFN-gamma-mediated mechanism. A novel approach to achieving anti-tumor effects without the risk of GVHD is suggested.

T cell costimulus-independent and very efficacious inhibition of tumor growth in mice bearing subcutaneous or leukemic human B cell lymphoma xenografts by a CD19-/CD3- bispecific single-chain antibody construct.

We have recently demonstrated that a recombinant single-chain bispecific Ab construct, bscCD19xCD3, in vitro induces rapid B lymphoma-directed cytotoxicity at picomolar concentrations with unstimulated peripheral T cells. In this study, we show that treatment of nonobese diabetic SCID mice with submicrogram doses of bscCD19xCD3 could prevent growth of s.c. human B lymphoma xenografts and essentially cured animals when given at an early tumor stage. The effect was dose dependent, dependent on E:T ratio and the time between tumor inoculation and administration of bscCD19xCD3. No therapeutic effect was seen in the presence of human lymphocytes alone, a vehicle control, or with a bispecific single-chain construct of identical T cell-binding activity but different target specificity. In a leukemic nonobese diabetic SCID mouse model, treatment with bscCD19xCD3 prolonged survival of mice in a dose-dependent fashion. The human lymphocytes used as effector cells in both animal models did not express detectable T cell activation markers at the time of coinoculation with tumor cells. The bispecific Ab therefore showed an in vivo activity comparable to that observed in cell culture with respect to high potency and T cell costimulus independence. These properties make bscCD19xCD3 superior to previously investigated CD19 bispecific Ab-based therapies.

Linomide administration following bone marrow transplantation in mice.

The effect of linomide, an immunomodulatory drug, on natural killer (NK) cells and T cell-dependent immune responses following syngeneic or allogeneic bone marrow transplantation (BMT) was investigated in BALB/c mice inoculated with B-cell leukemia (BCL1). Linomide given in the drinking water had no impact on graft survival or graft versus leukemia (GVL) effects. Although linomide regulates anti-self reactivity in mice with experimental and spontaneous autoimmune disorders, the anti-tumor effects induced by allogeneic donor lymphocytes were not affected. This indicates that different mechanisms regulate anti-self and anti-leukemia effects. Alternatively, linomide might affect the homing of self-reactive lymphocytes to specific target organs in autoimmune disorders, although the homing process may not be relevant to the control of leukemia by alloreactive lymphocytes.

Tumor-derived multiple chaperone enrichment by free-solution isoelectric focusing yields potent antitumor vaccines.

We have utilized a free-solution/isoelectric focusing technique (FS-IEF) to obtain fractions rich in multiple chaperone proteins from clarified A20 tumor lysates. Vaccines prepared from chaperone-rich fractions are capable of providing protective immunity in mice subsequently challenged intravenously with the same A20 B cell leukemia cells. This protection is at least equal to that provided by purified, tumor-derived heat-shock protein 70, which was the best chaperone immunogen in our hands against this aggressive murine leukemia model. Dosage escalation studies, however, revealed that increasing vaccine dosages actually abrogated the protective effects. The physical nature of the enriched chaperones indicates that they are associated in complexes, which may have implications for their function. FS-IEF is relatively simple, rapid, and efficient, thus making combined multi-chaperone therapy feasible.

Immunoprotective activities of multiple chaperone proteins isolated from murine B-cell leukemia/lymphoma.

Although the use of tumor-derived heat shock/chaperone proteins (HSPs) as anticancer vaccines is gaining wider study and acceptance, there have thus far been no reports concerning chaperone antitumor activities against disseminated hematological malignancies. We have devised an efficient and effective method for purification of the chaperone proteins grp94/gp96, HSP90, HSP70, and calreticulin from harvested A20 murine leukemia/lymphoma tumor material. We have demonstrated that these purified proteins, when used as vaccines, can induce potent and specific immunity against a lethal tumor challenge. Individual chaperone proteins were differentially effective in their abilities to provide immune protection. The increase in survival generated by the most effective chaperone vaccine, HSP70, resulted from at least a 2-log reduction in tumor burden. Syngeneic granulocyte macrophage colony-stimulating factor producing fibroblasts were injected at the site of vaccination in an attempt to augment the immune response. Surprisingly, localized granulocyte macrophage colony-stimulating factor production inhibited the protective effects of chaperone vaccination. These studies provide evidence that chaperone proteins can be isolated from B-cell tumors and used effectively to immunize against disseminated lymphoid malignancies.

[Vaccination against cancer soon a therapeutic possibility. B-cell tumors, colonic cancer and melanoma may be suitable for this treatment]. / Vaccination mot cancer snart en behandlingsmöjlighet. B-cellstumörer, koloncancer och melanom kan lämpa sig för denna terapi.

The theoretical basis of cancer vaccination having been well established during the past two decades, the translation of this knowledge into clinically applicable immunisation procedures is now an urgent need. Numerous antigenic preparations are available that are capable of inducing specific anti-tumour immunity which can be augmented by appropriate cytokines. Promising tumour vaccination results have been obtained in B-cell malignancies, colorectal carcinoma, and melanoma; tumour regression has been noted in myeloma, non-Hodgkin lymphoma, colorectal carcinoma, and melanoma patients, and significantly prolonged disease-freed survival in non-Hodgkin lymphoma and colorectal carcinoma patients. The presence of only minimal residual disease would seem to be a clinical prerequisite for tumour vaccination.

[Characterization of tolerogen-producing cells and lymphoid tissues where self-tolerance is induced--a study on allogeneic bone marrow chimeras established with spontaneous leukemia prone mice].

SL/Kh strain of mice provides a spontaneous pre-B cell leukemia model and AKR/J mice provides a spontaneous thymoma model. Using these leukemia-prone mice, protective influence of bone marrow (BM) transplantation and tolerance induction to a host antigen, Mls-1a, after BM reconstitution were studied. When SL/Kh mice were reconstituted with BM cells from allogeneic C57BL/6 mice, these [B6-->SL] chimeric mice survived longer than non-treated SL or [SL-->SL] syngeneic chimeras. These findings are compatible to those obtained with [B6-->AKR] chimeras. In [D2-->SL] and [D2-->AKR] chimeras, V beta 6+ T cells reactive to Mls-1a had been clonally eliminated 5 weeks after BM reconstitution. On the other hand, minor graft versus host reaction (GVHR) abrogated the clonal elimination of V beta 6+ T cells in both [D2-->SL] and [D2-->AKR] chimeras. This abrogation appeared to be attributable to early disappearance of Mls-1a producing host T cells in the GVHR chimeras. The cells responsible for the Mls-1a production were revealed to be mainly CD8+CD44+ T cells by in vitro mixed lymphocyte reaction (MLR), although other T cell subsets also induced MLR under certain conditions. Administration of the CD8+CD44+ T cells from (AKR x BR)F1 mice induced clonal elimination of V beta 6+ T cells in the thymus and lymph nodes of [BR-->BR] syngeneic chimeras. By contrast, CD8+CD44- T cells induced partial reduction of V beta 6+ T cells only in lymph nodes. CD4+ T cells of Mls-1a type showed no tolerogenicity in this in vivo study, although these CD4+ T cells as well as CD8+ T cells evoked potentially MLR against Mls-1a. The present findings indicate that host CD8+CD44+ T cells are a major source of Mls-1a production in [Mls-1b-->Mls-1a] BM chimera system and suggest that CD44 expression on T cells is associated not only with high Mls-1a production but also with lymphoid tissues where the tolerogenic cells migrate.

Effect of a synthetic adjuvant for inducing anti-tumour immunity.

An acylated derivative of muramyl dipeptide (MDP), 6-O-(2-tetradecyl-hexadecanoyl)-muramyl-dipeptide (B30-MDP) is a strong adjuvant effective in inducing cell-mediated immunity. We used B30-MDP as an adjuvant for induction of anti-tumour immunity. Guinea-pigs which were injected repeatedly with a mixture of X-ray-treated leukaemic cells and B30-MDP dissolved in phosphate buffered saline resisted a challenge of leukaemia cells and showed no sign of leukocytosis. The immunity induced was tumour-specific and retained for more than 100 days. These results suggest that B30-MDP is useful as a simple but potent immunotherapeutic tool.