Pretargeted radioimmunotherapy using genetically engineered antibody-streptavidin fusion proteins for treatment of non-hodgkin lymphoma.

Purpose: Pretargeted radioimmunotherapy (PRIT) using streptavidin (SAv)-biotin technology can deliver higher therapeutic doses of radioactivity to tumors than conventional RIT. However, "endogenous" biotin can interfere with the effectiveness of this approach by blocking binding of radiolabeled biotin to SAv. We engineered a series of SAv FPs that downmodulate the affinity of SAv for biotin, while retaining high avidity for divalent DOTA-bis-biotin to circumvent this problem.Experimental Design: The single-chain variable region gene of the murine 1F5 anti-CD20 antibody was fused to the wild-type (WT) SAv gene and to mutant SAv genes, Y43A-SAv and S45A-SAv. FPs were expressed, purified, and compared in studies using athymic mice bearing Ramos lymphoma xenografts.Results: Biodistribution studies showed delivery of more radioactivity to tumors of mice pretargeted with mutant SAv FPs followed by (111)In-DOTA-bis-biotin [6.2 +/- 1.7% of the injected dose per gram (%ID/gm) of tumor 24 hours after Y43A-SAv FP and 5.6 +/- 2.2%ID/g with S45A-SAv FP] than in mice on normal diets pretargeted with WT-SAv FP (2.5 +/- 1.6%ID/g; P = 0.01). These superior biodistributions translated into superior antitumor efficacy in mice treated with mutant FPs and (90)Y-DOTA-bis-biotin [tumor volumes after 11 days: 237 +/- 66 mm(3) with Y43A-SAv, 543 +/- 320 mm(3) with S45A-SAv, 1129 +/- 322 mm(3) with WT-SAv, and 1435 +/- 212 mm(3) with control FP (P < 0.0001)].Conclusions: Genetically engineered mutant-SAv FPs and bis-biotin reagents provide an attractive alternative to current SAv-biotin PRIT methods in settings where endogenous biotin levels are high. Clin Cancer Res; 17(23); 7373-82. (C)2011 AACR.

Rescue of the mature B cell compartment in BAFF-deficient mice by treatment with recombinant Fc-BAFF.

BAFF deficiency in mice impairs B cell development beyond the transitional stage 1 in the spleen and thus severely reduces the size of follicular and marginal zone B cell compartments. Moreover, humoral immune responses in these mice are dramatically impaired. We now addressed the question whether the decrease in mature B cell numbers and the reduced humoral immune responses in BAFF-deficient mice could be overcome by the injection of recombinant BAFF. We therefore engineered a recombinant protein containing the human IgG1 Fc moiety fused to receptor-binding domain of human BAFF (Fc-BAFF). At 1 week after the second injection of this fusion protein a complete rescue of the marginal zone B cell compartment and a 50% rescue of the follicular B cell compartment was observed. Moreover these mice mounted a T cell-dependent humoral immune response indistinguishable from wild-type mice. By day 14 upon arrest of Fc-BAFF treatment mature B cell numbers in the blood dropped by 50%, indicating that the life span of mature B cells in the absence of BAFF is 14 days or less. Collectively these findings demonstrate that injection of Fc-BAFF in BAFF-deficient mice results in a temporary rescue of a functional mature B cell compartment.

Expression and presentation of endogenous mouse mammary tumor virus superantigens by thymic and splenic dendritic cells and B cells.

Tolerance against superantigens (SAgs) encoded by endogenous mouse mammary tumor virus (Mtv) loci involves the intrathymic deletion of SAg-reactive T cells expressing a particular TCR V beta-chain, presumably upon presentation of the SAg by specialized APC. However, although the role of dendritic cells (DC) in the induction of tolerance against conventional Ags has been demonstrated, little is known about the role played by DC in tolerance induction against Mtv SAgs. Moreover, there is conflicting evidence concerning the capacity of DC to express and present Mtv SAgs. In this report we have analyzed the expression of Mtv SAgs in highly purified thymic and splenic DC and B cells by reverse transcriptase-PCR, using primers amplifying Mtv SAg-specific spliced mRNAs. DC express Mtv SAgs at levels comparable to B cells, but display a differential expression pattern of the various Mtv loci compared with B cells. Furthermore, our results show that DC are able to induce the deletion of SAg-reactive thymocytes in an in vitro assay, indicating that Mtv SAgs are functionally expressed on the DC surface. Collectively, our data are consistent with the hypothesis that DC play a role in the induction of intrathymic tolerance to Mtv SAgs.

Selectively expanding subsets of T cells in mice by injection of interleukin-2/antibody complexes: implications for transplantation tolerance.

The biological activity of interleukin (IL)-2 and other cytokines in vivo can be augmented by binding to certain anti-cytokine monoclonal antibodies (mAb). Here, we review evidence on how IL-2/anti-IL-2 mAb complexes can be used to cause selective stimulation and expansion of certain T-cell subsets. With some anti-IL-2 mAbs, injection of IL-2/mAb complexes leads to expansion of CD8 T effector cells but not CD4 T regulatory cells (Tregs); these complexes exert less adverse side effects than soluble IL-2 and display powerful antitumor activity. Other IL-2/mAb complexes have minimal effects on CD8 T cells but cause marked expansion of Tregs. Preconditioning mice with these complexes leads to permanent acceptance of MHC-disparate pancreatic islets in the absence of immunosuppression.

Radiolabeled fragments of monoclonal antibodies against carcinoembryonic antigen for localization of human colon carcinoma grafted into nude mice.

Four monoclonal antibodies against carcinoembryonic antigen (CEA) have been selected from 32 hybrids that produce antibodies against this antigen, by the criteria of high affinity for CEA and low cross-reactivity with granulocyte glycoprotein(s). The specificity of tumor localization in vivo of the four MAb, and their F(ab')2 and Fab fragments was compared in nude mice bearing grafts of a serially transplanted, CEA-producing, human colon carcinoma. The distribution of radiolabeled MAb and their fragments after intravenous injection was analyzed by direct measurement of radioactivity in tumor and normal organs, as well as by whole-body scanning and by autoradiography of tumor sections. Paired labeling experiments, in which 131I-labeled antibody or fragments and 125I-labeled control IgG are injected simultaneously, were undertaken to determine the relative tumor uptakes of each labeled protein. The tumor antibody uptake divided by that of control IgG defines the specificity index of localization. Tumor antibody uptakes (as compared with the whole mouse), ranging between 7 and 15, and specificity indices ranging between 3.4 and 6.8, were obtained with the four intact MAb at day 4-5 after injection. With F(ab')2 fragments of the four MAb, at day 3, the tumor antibody uptakes ranged between 12 and 24 and the specificity indices between 5.3 and 8.2. With the Fab fragments prepared from the two most promising MAb, the antibody uptakes reached values of 34 and 82 at day 2-3 and the specificity indices were as high as 12 and 19. The scanning results paralleled those obtained by direct measurement of radioactivity. With intact MAb, tumor grafts of 0.5-1 g gave very contrasted positive scans 3 d after injection. Using MAb fragments, tumors of smaller size were detectable earlier. The best results were obtained with Fab fragments of MAb 35, which gave clear detections of tumors weighing only 0.1 g as early as 48 h after injection. Autoradiographs of tumor sections from mice injected with 125I-labeled MAb demonstrated that the radioactivity was localized in the tumor tissues and not in the stromal connective tissue of mouse origin. The highest radioactivity concentration was localized in areas known to contain CEA such as the pseudolumen of glands and the apical side of carcinoma cells. The penetration of radioactivity in the central part of tumor nodules and the pseudolumen appeared to be increased with the use of MAb fragments.

Transcript profiling suggests that differential metabolic adaptation of mice to a high fat diet is associated with changes in liver to muscle lipid fluxes.

Genetically homogenous C57Bl/6 mice display differential metabolic adaptation when fed a high fat diet for 9 months. Most become obese and diabetic, but a significant fraction remains lean and diabetic or lean and non-diabetic. Here, we performed microarray analysis of "metabolic" transcripts expressed in liver and hindlimb muscles to evaluate: (i) whether expressed transcript patterns could indicate changes in metabolic pathways associated with the different phenotypes, (ii) how these changes differed from the early metabolic adaptation to short term high fat feeding, and (iii) whether gene classifiers could be established that were characteristic of each metabolic phenotype. Our data indicate that obesity/diabetes was associated with preserved hepatic lipogenic gene expression and increased plasma levels of very low density lipoprotein and, in muscle, with an increase in lipoprotein lipase gene expression. This suggests increased muscle fatty acid uptake, which may favor insulin resistance. In contrast, the lean mice showed a strong reduction in the expression of hepatic lipogenic genes, in particular of Scd-1, a gene linked to sensitivity to diet-induced obesity; the lean and non-diabetic mice presented an additional increased expression of eNos in liver. After 1 week of high fat feeding the liver gene expression pattern was distinct from that seen at 9 months in any of the three mouse groups, thus indicating progressive establishment of the different phenotypes. Strikingly, development of the obese phenotype involved re-expression of Scd-1 and other lipogenic genes. Finally, gene classifiers could be established that were characteristic of each metabolic phenotype. Together, these data suggest that epigenetic mechanisms influence gene expression patterns and metabolic fates.

Th2 lymphoproliferative disorder of LatY136F mutant mice unfolds independently of TCR-MHC engagement and is insensitive to the action of Foxp3+ regulatory T cells.

Mutant mice where tyrosine 136 of linker for activation of T cells (LAT) was replaced with a phenylalanine (Lat(Y136F) mice) develop a fast-onset lymphoproliferative disorder involving polyclonal CD4 T cells that produce massive amounts of Th2 cytokines and trigger severe inflammation and autoantibodies. We analyzed whether the Lat(Y136F) pathology constitutes a bona fide autoimmune disorder dependent on TCR specificity. Using adoptive transfer experiments, we demonstrated that the expansion and uncontrolled Th2-effector function of Lat(Y136F) CD4 cells are not triggered by an MHC class II-driven, autoreactive process. Using Foxp3EGFP reporter mice, we further showed that nonfunctional Foxp3(+) regulatory T cells are present in Lat(Y136F) mice and that pathogenic Lat(Y136F) CD4 T cells were capable of escaping the control of infused wild-type Foxp3(+) regulatory T cells. These results argue against a scenario where the Lat(Y136F) pathology is primarily due to a lack of functional Foxp3(+) regulatory T cells and suggest that a defect intrinsic to Lat(Y136F) CD4 T cells leads to a state of TCR-independent hyperactivity. This abnormal status confers Lat(Y136F) CD4 T cells with the ability to trigger the production of Abs and of autoantibodies in a TCR-independent, quasi-mitogenic fashion. Therefore, despite the presence of autoantibodies causative of severe systemic disease, the pathological conditions observed in Lat(Y136F) mice unfold in an Ag-independent manner and thus do not qualify as a genuine autoimmune disorder.

Deletion of delta-opioid receptor in mice alters skin differentiation and delays wound healing.

In addition to their well-known antinociceptive action, opioids can modulate non-neuronal functions, such as immune activity and physiology of different cell types. Several findings suggest that the delta-opioid receptor (DOR) and its endogenous ligands (enkephalins) are important players in cell differentiation and proliferation. Here we show the expression of DOR in mouse skin and human skin cultured fibroblasts and keratinocytes using RT-PCR. In DOR knock-out (KO) mice, a phenotype of thinner epidermis and higher expression of cell differentiation marker cytokeratin 10 (CK 10) were observed compared with wild type (WT). Using a burn wound model, significant wound healing delay (about 2 days) and severe epidermal hypertrophy were shown at the wound margin of DOR KO mice. This wound healing delay was further investigated by immunohistochemistry using markers for proliferation, differentiation, re-epithelialization, and dermal repair (CK 6, CK 10, and collagen IV). The levels of all these markers were increased in wounds of KO mice compared with WT. During the wound healing, the epidermal thickness in KO mice augments faster and exceeds that of the WT by day 3. These results suggest an essential role of DOR in skin differentiation, proliferation, and migration, factors that are important for wound healing.

A new infectious mammary tumor virus in the milk of mice implanted with C4 hyperplastic alveolar nodules.

We have previously characterized an infectious mouse mammary tumor virus [(MMTV(SW)] which induces a strong superantigen response in vivo. Here we describe the isolation and characterization of MMTV(C4) which was derived from milk of mice implanted with hyperplastic alveolar nodules. MMTV(C4) stimulates V beta 2 expressing T cells after local injection in vivo. Comparison with known open reading frame (orf) sequences revealed high homology to Mtv-6, an endogenous virus interacting with V beta 3-expressing T cells. The carboxyl-terminal amino acids were, however, altered. High homology including the carboxyl-terminal orf amino acids were found with MMTV(C3H-K). We show here that MMTV(C3H-K) has lost its superantigen function. Sequence comparisons permitted the characterization of few key amino acids which could be important for T cell receptor interaction and superantigen processing.

Radio-immunothérapie du lymphome folliculaire: un pas vers la guérison [Radioimmunotherapy of follicular lymphoma: a step towards cure?]

Advanced stage follicular lymphoma is incurable by conventional treatment. Important progress has been observed with the development of new therapies based on monoclonal antibodies and on the use of radioimmunotherapy (RIT) in the treatment of non-Hodgkin lymphomas (NHL). Rituximab in combination with chemotherapy in the upfront setting significantly improved treatment outcome as compared with chemotherapy alone. Different studies also indicate that RIT has an important role in the management of NHL and could be beneficial in combination with chemotherapy. These two new treatment options have clearly distinctive mechanisms of action, rituximab being an exclusively biological treatment and RIT adding targeted systemic radiation therapy. Both RIT and the unlabeled antibody treatments might be further improved by different strategies including repetition of RIT or combination of different antibodies. We present here our experience with RIT using 131I-tositumomab (Bexxar) and discuss different topics regarding RIT, like the use of different antibodies, the best choice of the radioisotope or the place of radio-imaging. From the therapeutic point of view, we argue that the debate should not be as to which one among antibody immunotherapy or RIT should be best added to chemotherapy, but that all three treatments might be optimally combined with the aim to get the highest chance of cure for advanced stage follicular lymphoma.

In vivo fate mapping with SCL regulatory elements identifies progenitors for primitive and definitive hematopoiesis in mice.

One of the principal issues facing biomedical research is to elucidate developmental pathways and to establish the fate of stem and progenitor cells in vivo. Hematopoiesis, the process of blood cell formation, provides a powerful experimental system for investigating this process. Here, we employ transcriptional regulatory elements from the stem cell leukemia (SCL) gene to selectively label primitive and definitive hematopoiesis. We report that SCL-labelled cells arising in the mid to late streak embryo give rise to primitive red blood cells but fail to contribute to the vascular system of the developing embryo. Restricting SCL-marking to different stages of foetal development, we identify a second population of multilineage progenitors, proficient in contributing to adult erythroid, myeloid and lymphoid cells. The distinct lineage-restricted potential of SCL-labelled early progenitors demonstrates that primitive erythroid cell fate specification is initiated during mid gastrulation. Our data also suggest that the transition from a hemangioblastic precursors with endothelial and blood forming potential to a committed hematopoietic progenitor must have occurred prior to SCL-marking of definitive multilineage blood precursors.

Effects of an interleukin-15 antagonist on systemic and skeletal alterations in mice with DSS-induced colitis.

Inflammatory bowel diseases are commonly complicated by weight and bone loss. We hypothesized that IL-15, a pro-inflammatory cytokine expressed in colitis and an osteoclastogenic factor, could play a central role in systemic and skeletal complications of inflammatory bowel diseases. We evaluated the effects of an IL-15 antagonist, CRB-15, in mice with chronic colitis induced by oral 2% dextran sulfate sodium for 1 week, followed by another 1% for 2 weeks. During the last 2 weeks, mice were treated daily with CRB-15 or an IgG2a control antibody. Intestinal inflammation, disease severity, and bone parameters were evaluated at days 14 and 21. CRB-15 improved survival, early weight loss, and colitis clinical score, although colon damage and inflammation were prevented in only half the survivors. CRB-15 also delayed loss of femur bone mineral density and trabecular microarchitecture. Bone loss was characterized by decreased bone formation, but increased bone marrow osteoclast progenitors and osteoclast numbers on bone surfaces. CRB-15 prevented the suppression of osteoblastic markers of bone formation, and reduced osteoclast progenitors at day 14, but not later. However, by day 21, CRB-15 decreased tumor necrosis factor α and increased IL-10 expression in bone, paralleling a reduction of osteoclasts. These results delineate the role of IL-15 on the systemic and skeletal manifestations of chronic colitis and provide a proof-of-concept for future therapeutic developments.

Soybean response to starter nitrogen and Bradyrhizobium inoculation on a Cerrado oxisol under no-tillage and conventional tillage systems

In Brazil, Bradyrhizobium inoculation has successfully replaced the use of N fertilizer on soybean [Glycine max (L) Merr.] crops. However, with the expansion of no-tillage cropping systems in the Cerrados region, the idea that it is necessary to use small N rates at the sowing to overcome problems related with N immobilization has become widespread, mainly when soybean is cultivated after a non-legume crop. In this study we examined soybean response to small rates of N fertilizer under no-tillage (NT) and conventional tillage (CT) systems. Four experiments (a completely randomized block with five replicates) were carried out in a red yellow oxisol, during the periods of 1998/1999 and 1999/ 2000, under NT and CT. The treatments consisted of four urea rates (0, 20, 30 and 40 kg ha-1 N). All treatments were inoculated with Bradyrhizobium japonicum strains SEMIA 5080 and SEMIA 5079, in the proportion 1 kg of peat inoculant (1,5 x 10(9) cells g-1) per 50 kg of seeds. In both experiments, soybean was cultivated after corn and the N fertilizer was band applied at sowing. In all experiments, N rates promoted reductions of up to 50 % in the nodule number at 15 days after the emergence. Regardless of the management system, these reductions disappeared at the flowering stage and there was no effect of N rates on either the number and dry weight of nodules or on soybean yields. Therefore, in the Brazilian Cerrados, when an efficient symbiosis is established, it is not necessary to apply starter N rates on soybean, even when cultivated under notillage systems.