The thalamic reticular nucleus orchestrates social memory.

Social memory has been developed in humans and other animals to recognize familiar conspecifics and is essential for their survival and reproduction. Here, we demonstrated that parvalbumin-positive neurons in the sensory thalamic reticular nucleus (sTRNPvalb) are necessary and sufficient for mice to memorize conspecifics. sTRNPvalb neurons receiving glutamatergic projections from the posterior parietal cortex (PPC) transmit individual information by inhibiting the parafascicular thalamic nucleus (PF). Mice in which the PPCCaMKII→sTRNPvalb→PF circuit was inhibited exhibited a disrupted ability to discriminate familiar conspecifics from novel ones. More strikingly, a subset of sTRNPvalb neurons with high electrophysiological excitability and complex dendritic arborizations is involved in the above corticothalamic pathway and stores social memory. Single-cell RNA sequencing revealed the biochemical basis of these subset cells as a robust activation of protein synthesis. These findings elucidate that sTRNPvalb neurons modulate social memory by coordinating a hitherto unknown corticothalamic circuit and inhibitory memory engram.

Phase 1 Study of CK0801 in Treatment of Bone Marrow Failure Syndromes.

BACKGROUND: An inflammatory bone marrow microenvironment contributes to acquired bone marrow failure syndromes. CK0801, an allogeneic T regulatory (Treg) cell therapy product, can potentially interrupt this continuous loop of inflammation and restore hematopoiesis. METHODS: In this phase 1 dose-escalation study of CK0801 Treg cells, we enrolled patients with bone marrow failure syndromes with suboptimal response to their prior therapy to determine the safety and efficacy of this treatment for bone marrow failure syndromes. RESULTS: We enrolled nine patients with a median age of 57 years (range, 19 to 74) with an underlying diagnosis of aplastic anemia (n=4), myelofibrosis (n=4), or hypoplastic myelodysplasia (n=1). Patients had a median of three prior therapies for a bone marrow failure syndrome. Starting dose levels of CK0801 were 1 × 106 (n=3), 3 × 106 (n=3), and 10 × 106 (n=3) cells per kg of ideal body weight. No lymphodepletion was administered. CK0801 was administered in the outpatient setting with no infusion reactions, no grade 3 or 4 severe adverse reactions, and no dose-limiting toxicity. At 12 months, CK0801 induced objective responses in three of four patients with myelofibrosis (two had symptom response, one had anemia response, and one had stable disease) and three of four patients with aplastic anemia (three had partial response). Three of four transfusion-dependent patients at baseline achieved transfusion independence. Although the duration of observation was limited at 0.9 to 12 months, there were no observed increases in infections, no transformations to leukemia, and no deaths. CONCLUSIONS: In previously treated patients, CK0801 demonstrated no dose-limiting toxicity and showed evidence of efficacy, providing proof of concept for targeting inflammation as a therapy for bone marrow failure. (Funded by Cellenkos Inc.; Clinicaltrials.gov number, NCT03773393.).

Allogeneic cord blood regulatory T cells decrease dsDNA antibody and improve albuminuria in systemic lupus erythematosus.

Background: Lupus nephritis (LN) constitutes the most severe organ manifestations of systemic lupus erythematosus (SLE), where pathogenic T cells have been identified to play an essential role in 'helping' B cells to make autoantibodies and produce inflammatory cytokines that drive kidney injury in SLE. Regulatory T cells (Tregs), responsible for decreasing inflammation, are defective and decreased in SLE and have been associated with disease progression. We hypothesize that treatment with allogeneic, healthy Tregs derived from umbilical cord blood (UCB) may arrest such an inflammatory process and protect against kidney damage. Methods: UCB-Tregs function was examined by their ability to suppress CellTrace Violet-labeled SLE peripheral blood mononuclear cells (PBMCs) or healthy donor (HD) conventional T cells (Tcons); and by inhibiting secretion of inflammatory cytokines by SLE PBMCs. Humanized SLE model was established where female Rag2-/-γc-/- mice were transplanted with 3 × 106 human SLE-PBMCs by intravenous injection on day 0, followed by single or multiple injection of UCB-Tregs to understand their impact on disease development. Mice PB was assessed weekly by flow cytometry. Phenotypic analysis of isolated cells from mouse PB, lung, spleen, liver and kidney was performed by flow cytometry. Kidney damage was assessed by quantifying urinary albumin and creatinine secretion. Systemic disease was evaluated by anti-dsDNA IgG Ab analysis as well as immunohistochemistry analysis of organs. Systemic inflammation was determined by measuring cytokine levels. Results: In vitro, UCB-Tregs are able to suppress HD Tcons and pathogenic SLE-PBMCs to a similar extent. UCB-Tregs decrease secretion of several inflammatory cytokines including IFN-γ, IP-10, TNF-α, IL-6, IL-17A, and sCD40L by SLE PBMCs in a time-dependent manner, with a corresponding increase in secretion of suppressor cytokine, IL-10. In vivo, single or multiple doses of UCB-Tregs led to a decrease in CD8+ T effector cells in different organs and a decrease in circulating inflammatory cytokines. Improvement in skin inflammation and loss of hair; and resolution of CD3+, CD8+, CD20+ and Ki67+ SLE-PBMC infiltration was observed in UCB-Treg recipients with a corresponding decrease in plasma anti-double stranded DNA IgG antibody levels and improved albuminuria. Conclusions: UCB-Tregs can decrease inflammatory burden in SLE, reduce auto-antibody production and resolve end organ damage especially, improve kidney function. Adoptive therapy with UCB-Tregs should be explored for treatment of lupus nephritis in the clinical setting.

Adjunct Therapy with T Regulatory Cells Decreases Inflammation and Preserves the Anti-Tumor Activity of CAR T Cells.

With greater accessibility and an increased number of patients being treated with CAR T cell therapy, real-world toxicity continues to remain a significant challenge to its widespread adoption. We have previously shown that allogeneic umbilical cord blood-derived (UCB) regulatory T cells (Tregs) can resolve inflammation and treat acute and immune-mediated lung injuries. Allogeneic, cryopreserved UCB Tregs have shown a clinical benefit in patients suffering from COVID-19 acute respiratory distress syndrome. The unique properties of UCB Treg cells include a lack of plasticity under inflammatory micro-environments, no requirement for HLA matching, a long shelf life of cryopreserved cells, and immediate product availability, which makes them attractive for treating acute inflammatory syndromes. Therefore, we hypothesized that adjunct therapy with UCB Tregs may resolve the undesirable inflammation responsible for CAR T cell therapy-associated toxicity. In in vitro analysis, no interference from the addition of UCB Tregs was observed on CD19 CAR T cells' ability to kill CD19 Raji cells at different CAR T: Raji cell ratios of 8:1 (80.4% vs. 81.5%); 4:1 (62.0% vs. 66.2%); 2:1 (50.1% vs. 54.7%); and 1:1 (35.4% vs. 44.1%). In the xenogeneic B-cell lymphoma model, multiple injections of UCB Tregs were administered 3 days after CD19 CAR T cell injection, and no detrimental effect of add-on Tregs was noted on the circulating CD8+ T effector cells. The distribution of CAR T cells in multiple organs remained unaffected by the addition of the UCB Tregs. Specifically, no difference in the overall tumor burden was detected between the UCB Treg + CAR T vs. CAR T alone recipients. No tumor was detected in the liver or bone marrow in CAR T cells + UCB Tregs recipients, with a notable corresponding decrease in multiple circulating inflammatory cytokines when compared to CART alone recipients. Here we show the proof of concept for adjunct therapy with UCB Tregs to mitigate the hyper-inflammatory state induced by CAR T cells without any interference in their on-target anti-tumor activity. Administration of UCB Tregs after CAR T cells allows sufficient time for their synapse formation with tumor cells and exerts cytotoxicity, such that the UCB Tregs are diverted to interact with the antigen-presenting cells at the site of inflammation. Such a differential distribution of cells would allow for a two-pronged strategy of a UCB Treg "cooling blanket" effect and lay the groundwork for clinical study.

Allogeneic cord blood regulatory T cells can resolve lung inflammation.

BACKGROUND AIMS: CD4+CD25+CD127lo regulatory T cells (Tregs) are responsible for maintaining immune homeostasis. Tregs can be rendered defective and deficient as a result of the immune imbalance seen in lung injury, and such dysfunction can play a major role in continued tissue inflammation. The authors hypothesized that adoptive therapy with healthy allogeneic umbilical cord blood (UCB)-derived Tregs may be able to resolve inflammation. RESULTS: Ex vivo-expanded UCB Tregs exhibited a unique phenotype with co-expression of CD45RA+CD45RO+ >80% and lung homing markers, including CD49d. UCB Tregs did not turn pathogenic when exposed to IL-6. Co-culture with increasing doses of dexamethasone led to a synergistic increase in UCB Treg-induced apoptosis of conventional T cells (Tcons), which translated into significantly higher suppression of proliferating Tcons, especially at a lower Treg:Tcon ratio. Multiple injections of UCB Tregs led to their preferential accumulation in lung tissue in an immune injury xenogenic model. A significant decrease in lung resident cytotoxic CD8+ T cells (P = 0.0218) correlated with a sustained decrease in their systemic distribution compared with controls (P < 0.0001) (n = 7 per arm) as well as a decrease in circulating human soluble CD40 ligand level (P = 0.031). Tissue architecture was preserved in the treatment arm, and a significant decrease in CD3+ and CD8+ burden was evident in immunohistochemistry analysis. CONCLUSIONS: UCB Treg adoptive therapy is a promising therapeutic strategy for treatment of lung injury.

Mesenchymal stem cells internalize Mycobacterium tuberculosis through scavenger receptors and restrict bacterial growth through autophagy.

Human mesenchymal stem cells (MSCs) express scavenger receptors that internalize lipids, including oxidized low-density lipoprotein (oxLDL). We report that MSCs phagocytose Mycobacterium tuberculosis (Mtb) through two types of scavenger receptors (SRs; MARCO and SR-B1), as blockade of the receptors with antibodies or siRNA knockdown decreased the uptake of Mtb. MSCs also expressed mannose receptor (MR) that was found to endocytose rhodamine-labeled mannosylated BSA (rMBSA), though the receptor was not involved in the uptake of Mtb. Dil-oxLDL and rMBSA taken up into MSC endosomes colocalized with Mtb phagosomes, thus suggesting that the latter were fusion competent. Phagocytosed Mtb did not replicate within MSCs, thus suggesting an intrinsic control of bacterial growth. Indeed, MSCs exhibited intrinsic autophagy, which was up-regulated after activation with rapamycin. SiRNA knockdown of autophagy initiator beclin-1 enhanced Mtb survival, whereas rapamycin-induced autophagy increased intracellular killing of Mtb. In addition, MSCs secreted nitric oxide after Mtb infection, and inhibition of NO by N(G)-monomethyl-L-arginine enhanced intracellular survival of Mtb. MSCs can be grown in large numbers in vitro, and autologous MSCs transfused into tuberculosis patients have been found to be safe and improve lung immunity. Thus, MSCs are novel phagocytic cells with a potential for immunotherapy in treating multidrug-resistant tuberculosis.

The therapeutic effects of rGel/BLyS fusion toxin in in vitro and in vivo models of mantle cell lymphoma.

Mantle cell lymphoma (MCL) is an incurable, aggressive histo-type of B-cell non-Hodgkin lymphoma associated with both high relapsed rates and relatively short survival. Because MCL over-expresses receptors for B lymphocyte stimulator (BLyS) and displays constitutively active NF-κB, agents targeting these pathways may be of therapeutic relevance in this disease. To investigate the potential clinical use of the rGel/BLyS fusion toxin in combination with bortezomib, we evaluated this fusion toxin for its ability to inhibit MCL growth in severe combined immunodeficiency (SCID) xenograft model. Compared with PBS-treated mice, mice treated with this fusion toxin prolonged both median (84 days vs. 125 days) and overall survival (0% vs. 40%) (p=0.0027). Compared with bortezomib alone-treated mice, mice treated with rGel/BLyS plus bortezomib showed significantly increased median (91 days vs. 158 days) and overall survival (0% vs. 20%) (p=0.0127). Histopathologic analysis of peritoneal intestinal mesentery from MCL-SCID mice showed no demonstrable microscopic lymphomatous involvement at 225 days after treatment with rGel/BLyS. Combination treatment resulted in a synergistic growth inhibition, down-regulation of NF-κB DNA-binding activity, inhibition of cyclin D1, Bcl-x(L), p-Akt, Akt, p-mTOR, and p-Bad, up-regulation of Bax, and induction of cellular apoptosis. Our findings demonstrate that rGel/BLyS is an effective therapeutic agent for both primary and salvage treatment of aggressive MCL expressing constitutively active NF-κB and BLyS receptors and may be an excellent candidate for clinical development.

Bax345/BLyS: a novel, completely human fusion protein targeting malignant B cells and delivering a unique mitochondrial toxin.

We generated a fusion protein Bax(345)/BLyS containing the truncated form of Bax (Bax(345)) at the N-terminus followed by a 218 linker to the B lymphocyte stimulator (BLyS). Bax(345)/BLyS was cytotoxic to a panel of diffuse large B cell lymphoma and mantle cell lymphoma lines expressing the BLyS receptors. Specific delivery of Bax(345)/BLyS to malignant B cells drove cells into apoptosis by mitochondrial dysfunction and treatment of cells with Bax(345)/BLyS induced down-regulation of Mcl-1, X-IAP, and survivin. Bax(345)/BLyS represents a new class of targeted therapeutic agents with a unique mechanism of action and may have therapeutic potential for malignant B cells.

Cell-targeting fusion constructs containing recombinant gelonin.

Therapeutic agents capable of targeting tumor cells present as established tumors and micrometastases have already demonstrated their potential in clinical trials. Immunotoxins targeting hematological malignancies and solid tumors have additionally demonstrated excellent clinical activity. This review focuses on our design and characterization studies of constructs composed of recombinant gelonin toxin fused to either growth factors or single-chain antibodies targeting solid tumor cells, tumor vasculature or hematological malignancies. These agents demonstrate cytotoxicity at nanomolar or sub-nanomolar levels. All of these constructs display impressive selectivity and specificity for antigen-bearing target cells in vitro and in vivo and are excellent clinical trial candidates.

Homicide and bipolar I disorder: a 22-year study.

BACKGROUND: Non-serious offenses in manic phase have been mainly studied in patients with bipolar disorder. However, some authors reported that depressive phase is related with the violent and homicidal manifestations of bipolar disorder. AIMS: We investigated the characteristics of homicide by the polarity of mood episode in patients with bipolar I disorder. METHODS: Among the offenders who were sentenced to undergo treatment at the National Institute of Forensic Psychiatry from October 1987 to January 2008, a total 219 offenders whose final diagnoses were bipolar I disorder based on DSM-III-R and DSM-IV were selected. Retrospective medical chart review was performed for characteristics of mood episodes. Descriptions of offenders were supplemented by review of the written records of the police or prosecutors. RESULTS: The general rate of total offense was higher in the manic phase than in the depressive phase (86.8% vs. 13.2%). However, the rate of homicide was higher in the depressive phase than in the manic phase. The victims of homicide were more likely to be family members of the patients in depressive phase than in manic phases (96.2% vs. 63.9%, p=0.001). However, parricide was committed only in manic phases. Altruistic motivation of homicide was significantly higher in depressive phase (34.6% vs. 0%, p<0.001) whereas impulsivity was the most common one in manic phases. CONCLUSIONS: The risk of offenses, particularly homicide for family members, should not be overlooked in the depressive phases of bipolar I disorder.

Biodistribution, pharmacokinetics, and nuclear imaging studies of 111In-labeled rGel/BLyS fusion toxin in SCID mice bearing B cell lymphoma.

PURPOSE: We examined the biodistribution and pharmacokinetics of (111)In-labeled rGel/BLyS, a gelonin toxin (rGel)-B lymphocyte stimulator (BLyS) fusion protein. MATERIALS AND METHODS: rGel/BLyS was labeled with In-111 through DTPA with a labeling efficiency >95%. Biodistribution/imaging studies were obtained in severe-combined immunodeficiency mice bearing diffuse large B cell lymphoma OCI-Ly10. Pharmacokinetic studies were performed in BALB/c mice. RESULTS: In vitro, DTPA-conjugated rGel/BLyS displayed selective cytotoxicity against OCI-Ly10 cells and mantle cell lymphoma JeKo cells. In vivo, rGel/BLyS exhibited a tri-exponential disposition with a rapid initial mean distribution followed by an extensive mean distribution and a long terminal elimination phase. At 48 h after injection, uptake of the radiotracer in tumors was 1.25 %ID/g, with a tumor-to-blood ratio of 13. Tumors were clearly visualized at 24-72 h post-injection. Micro-SPECT-CT images and ex vivo analyses confirmed the accumulation of rGel/BLyS in OCI-Ly10 tumors. CONCLUSIONS: (111)In-DTPA-rGel/BLyS are distributed to B cell tumors and induce apoptosis in tumors. Preclinical antitumor studies using rGel/BLyS should use a twice-per-week treatment schedule.

The rGel/BLyS fusion toxin inhibits STAT3 signaling via down-regulation of interleukin-6 receptor in diffuse large B-cell lymphoma.

Aberrant signal transducer and activator of transcription (STAT)3 signaling participates in the development and progress of human cancers. We previously generated a highly cytotoxic fusion toxin designated rGel/BLyS for receptor-mediated delivery of the rGel toxin to malignant B-cells. In this study, we examined this fusion toxin for its ability to impact STAT3 signaling in diffuse large B-cell lymphoma (DLBCL). The activated B cell-like DLBCL lines were found to express higher levels of interleukin-6 receptor (IL-6R) and STAT3 than did the germinal center B cell-like DLBCL lines. Treatment of DLBCL cells with rGel/BLyS resulted in down-regulation of IL-6R and inhibited STAT3 phosphorylation, STAT3-DNA binding activity, and IL-6-inducible STAT3 reporter gene activity. In agreement with these results, we additionally found that rGel/BLyS down-regulated levels of several STAT3 targets (c-Myc, p21, Mcl-1, and Bcl-x(L)) and p-SYK, a positive regulator of STAT3. Inhibition of IL-6R-mediated STAT3 signaling by rGel/BLyS led to growth inhibition, triggered accumulation of cells in the sub-G(1) phase of the cell cycle, and induced apoptosis. Our results indicate that rGel/BLyS is an excellent candidate for the treatment of aggressive DLBCL which is resistant to conventional chemotherapeutic regimens and STAT3 signaling pathway may be an attractive therapeutic target for non-Hodgkin's lymphoma.

The rGel/BLyS fusion toxin inhibits diffuse large B-cell lymphoma growth in vitro and in vivo.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive subtype of B-cell non-Hodgkin lymphoma (NHL) and accounts for 30%to 40%of NHL. Molecules targeting nuclear factor-kappaB (NF-kappaB) are expected to be of therapeutic value in those tumors where NF-kappaB seems to play a unique survival role such as activated B-cell (ABC)-subtype DLBCL. We previously generated a rGel/BLyS fusion toxin for receptor-mediated delivery of the rGel toxin specifically to malignant B cells. In this study, we examined this fusion toxin for its ability to suppress DLBCL growth in vitro and in vivo. rGel/BLyS was specifically cytotoxic to DLBCL lines expressing all three BLyS receptors and constitutively active NF-kappaB. Treatment with rGel/BLyS induced down-regulation of the phosphorylation of inhibitory subunit of NF-kappaB (IkappaB-alpha), inhibition of NF-kappaB DNA-binding activity, and accumulation of IkappaB-alpha. In agreement with these results, we additionally found that rGel/BLyS downregulated levels of several NF-kappaB targets including Bcl-xL, Mcl-1, survivin, and x-chromosome linked inhibitor-of-apoptosis. Treatment also induced up-regulation of Bax and apoptosis through caspase-3 activation and poly ADP-ribose polymerase cleavage. Importantly, rGel/BLyS significantly inhibited tumor growth (P < .05) in a DLBCL xenograft model. Thus, our results indicate that rGel/BLyS is an excellent candidate for the treatment of aggressive NHLs that are both dependent on NF-kappaB and are resistant to conventional chemotherapeutic regimens.

The immunocytokine scFv23/TNF targeting HER-2/neu induces synergistic cytotoxic effects with 5-fluorouracil in TNF-resistant pancreatic cancer cell lines.

Human pancreatic tumor cells are highly resistant to both tumor necrosis factor (TNF) and to chemotherapeutic agents. HER-2/neu expression has been proposed as a negative prognostic marker in pancreatic intraepithelial neoplasia. Our approach was to utilize HER-2/neu expression on the surface of tumor cells as a therapeutic target employing scFv23/TNF, immunocytokine composed of a single chain Fv antibody (scFv23) targeting the HER-2/neu and the cytokine TNF as the cytotoxic moiety, to deliver TNF directly to TNF-resistant pancreatic tumor cells. Using a panel of human pancreatic cell lines, which overexpress HER-2/neu, we evaluated the in vitro response of cells to TNF, scFv23/TNF, Herceptin, and a combination of scFv23/TNF with various chemotherapeutic agents. We found that all pancreatic cancer cell lines were highly resistant to the cytotoxic effects of TNF and that scFv23/TNF was highly cytotoxic to TNF-resistant HER-2/neu-expressing pancreatic cancer cell lines at levels rivaling that of conventional chemotherapeutic agents. Combination studies demonstrated a synergistic cytotoxic effect of scFv23/TNF with 5-fluorouracil (5-FU) in TNF-resistant pancreatic cancer cell lines. Mechanistic studies demonstrated that the 5-FU plus scFv23/TNF combination specifically resulted in a down-regulation of HER-2/neu, p-Akt and Bcl-2 and up-regulation of TNF-R1. In addition, the combination 5-FU plus scFv23/TNF induced apoptosis and this synergistic effect was dependent on activation of caspase-8 and caspase-3. Delivery of the cytokine TNF to HER-2/neu expressing pancreatic tumor cells, which are inherently resistant to TNF using scFv23/TNF may be an effective therapy for pancreatic cancer especially when utilized in combination with 5-FU.

Clinical characteristics of struma ovarii.

OBJECTIVE: To evaluate the clinical characteristics of struma ovarii. METHODS: Twenty-five cases of struma ovarii were reviewed retrospectively from June 1994 to April 2007. The presenting clinical, radiologic, and pathologic features of the patients were reviewed. RESULTS: The mean age of the patients in this study was 45.3 years. The majority was of premenopausal status. Sixteen patients had clinical symptoms such as low abdominal pain, palpable abdominal mass and vaginal bleeding. Although one patient had an abnormal thyroid function test, the laboratory findings normalized after operative treatment. CA-125 levels were elevated in 6 cases. Diagnosis by preoperative imaging studies were 8 dermoid cysts, while only 3 cases were diagnosed as struma ovarii. There were 4 cases of malignant struma ovarii, and no patients with recurrent disease. CONCLUSION: Struma ovarii is a rare tumor. The presented clinical, laboratory and radiological findings of patients are very diverse. The diagnosis was confirmed by pathologic findings. The treatment of benign struma ovarii is surgical resection only. The cases of malignant struma ovarii may need adjuvant treatment, but recurrence is uncommon.

Significance of postoperative CA-125 decline after cytoreductive surgery in stage IIIC/IV ovarian cancer.

OBJECTIVE: The purpose of this study was to evaluate whether the decline in serum CA-125 levels following primary cytoreductive surgery prior to starting adjuvant chemotherapy has a prognostic value in patients with stage IIIC/IV ovarian carcinoma. METHODS: A retrospective review was conducted of all patients with stage IIIC/IV ovarian carcinoma who underwent primary cytoreductive surgery followed by platinum-based chemotherapy from 1994 to 2007. Demographic, pathologic, treatment, and survival data were collected. Patients were included if serum CA-125 levels were drawn preoperatively and within one week prior to their first chemotherapy cycle, and whose postoperative CA-125 level declined. Percentage decline was calculated, and was compared with standard statistical tests in groups by 25% declination intervals. RESULTS: Of the 112 stage IIIC/IV patients, 81 (72.3%) met the above inclusion criteria. The median time from surgery to postoperative CA-125 sampling was 16 days (range: 7-42). A >/=75% decline was associated with a median progression-free survival (PFS) of 25 months (95% CI=0-63). This was significantly longer when compared with each of the other 25% interval groups. After multivariate analysis, independent prognostic factors included a >/=75% decline in CA-125 levels after surgery and the presence of residual tumor. Age, grade, histology, and preoperative CA-125 levels were not statistically significant factors. CONCLUSION: A >/=75% decline in serum CA-125 serum levels from primary cytoreductive surgery to the start of adjuvant chemotherapy has independent prognostic value for PFS in patients with stage IIIC/IV ovarian carcinoma.

The rGel/BLyS fusion toxin specifically targets malignant B cells expressing the BLyS receptors BAFF-R, TACI, and BCMA.

B lymphocyte stimulator (BLyS) is crucial for B-cell survival, and the biological effects of BLyS are mediated by three cell surface receptors designated B cell-activating factor receptor (BAFF-R), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), and B-cell maturation antibody (BCMA). Increased expression of BLyS and its receptors has been identified in numerous B-cell malignancies. We generated a fusion toxin designated rGel/BLyS for receptor-mediated delivery of the recombinant gelonin (rGel) toxin to neoplastic B cells, and we characterized its activity against various B-cell tumor lines. Three mantle cell lymphoma (MCL) cell lines (JeKo-1, Mino, and SP53) and two diffuse large B-cell lymphoma (DLBCL) cell lines (SUDHL-6 and OCI-Ly3) expressing all three distinct BLyS receptors were found to be the most sensitive to the fusion toxin (IC(50) = 2-5 pmol/L and 0.001-5 nmol/L for MCL and DLBCL, respectively). The rGel/BLyS fusion toxin showed specific binding to cells expressing BLyS receptors and rapid internalization of the rGel component into target cells. The cytotoxic effects of rGel/BLyS were inhibited by pretreatment with free BLyS or with soluble BAFF-R, TACI, and BCMA decoy receptors. This suggests that the cytotoxic effects of the fusion toxin are mediated through BLyS receptors. The rGel/BLyS fusion toxin inhibited MCL cell growth through induction of apoptosis associated with caspase-3 activation and poly (ADP-ribose) polymerase cleavage. Our results suggest that BLyS has the potential to serve as an excellent targeting ligand for the specific delivery of cytotoxic molecules to neoplastic B cells expressing the BLyS receptors, and that the rGel/BLyS fusion toxin may be an excellent candidate for the treatment of B-cell malignancies especially MCL and DLBCL.

The growth factor fusion construct containing B-lymphocyte stimulator (BLyS) and the toxin rGel induces apoptosis specifically in BAFF-R-positive CLL cells.

The cytokine B lymphocyte stimulator (BLyS) mediates its effect through cell-surface receptors BAFF-R, TACI, and BCMA. BLyS receptors are expressed only on B cells and not present in other normal cells including normal T lymphocytes. Chronic lymphocytic leukemia (CLL) is a B-cell disease and CLL lymphocytes express BLyS receptors. Gelonin, a type 1 ribosome-inactivating toxin, lacks cell membrane binding domain and hence is nontoxic to intact cells. We generated a construct of recombinant gelonin (rGel) fused to BLyS to specifically target quiescent B-CLL lymphocytes. The construct rGel/BLyS specifically binds and internalizes through BAFF-R into CD19(+) B-CLL lymphocytes and induces apoptosis at nanomolar concentrations. In contrast, rGel alone was not able to internalize into these leukemic lymphocytes. Mechanistically, the rGel/BLyS construct inhibits protein synthesis with an IC(50) of less than 3 nM compared with more than 5000 nM for rGel toxin alone. This rGel/BLyS-mediated decrease in protein synthesis was associated with a decline in short-lived proteins such as MCL-1 and XIAP, the 2 survival proteins in B-CLL. There was a strong relationship between a decrease in these proteins and the cleavage of PARP, a hallmark feature of apoptosis. Taken together, these data suggest that the rGel/BLyS fusion toxin may have potential therapeutic efficacy for B-CLL patients.

The immunocytokine scFv23/TNF sensitizes HER-2/neu-overexpressing SKBR-3 cells to tumor necrosis factor (TNF) via up-regulation of TNF receptor-1.

Overexpression of HER-2/neu confers cellular resistance to tumor necrosis factor (TNF)-mediated cytotoxicity to SKBR-3 breast cancer cell lines. To understand the correlation between HER-2/neu expression and TNF resistance, we examined the unique signaling pathways associated with the cytotoxic effects of the immunocytokine scFv23/TNF, recombinant single-chain antibody fusion constructs containing TNF and targeting HER-2/neu, in TNF-resistant SKBR-3-LP cells. We found that treatment of HER-2/neu-overexpressing SKBR-3-LP cells with scFv23/TNF resulted in a 5- to 7-fold higher level of TNF receptor-1 expression 48 hours after exposure. In addition, treatment of SKBR-3-LP cells with scFv23/TNF resulted in down-regulation of Akt phosphorylation and induced apoptosis through cleavage of caspase-8, caspase-3, and poly(ADP-ribose) polymerase. ScFv23/TNF-induced cytotoxicity was inhibited by blocking of the binding of the TNF component of scFv23/TNF to TNF receptor-1 and was dependent on activation of caspase-8 and caspase-3. These results indicate that the immunocytokine scFv23/TNF sensitizes TNF-resistant HER-2/neu-overexpressing SKBR-3-LP cells to TNF-induced apoptosis via the overexpression of TNF receptor-1 and suggest that the overexpression of TNF receptor-1 plays a crucial role in TNF sensitivity in HER-2/neu-overexpressing cancer cells. ScFv23/TNF targeting the HER-2/neu may be an effective cytotoxic agent against HER-2/neu-overexpressing cancer cells, which are inherently resistant to TNF.