Introduction: Classical Hodgkin Lymphoma (cHL) is frequently accompanied by the 9p24.1 amplicon, which contains the PD-L1 and PD-L2 immune checkpoint genes and results in their overexpression. Blockade of PD-1/PD-L1 and PD-1/PD-L2 interactions with anti–PD-1 antibodies is clinically effective; however, it has not been established if blockade of the PD-1/PD-L1 interaction alone is sufficient for therapeutic effect. Avelumab is a fully human IgG1 monoclonal antibody that selectively binds to PD-L1, leaving the PD-1/PD-L2 interaction intact, thus enabling assessment of the contribution of PD-L2 in the clinical response to PD-1 checkpoint blockade. Methods: In the phase 1 JAVELIN Hodgkin study (NCT02603419), eligible patients (pts) with histologically confirmed cHL were required to have disease progression following either autologous (auto) or allogeneic (allo) stem cell transplant (SCT), or to be SCT-ineligible. Pts were randomised in equal proportions across 5 avelumab dosing regimens: 70 mg, 350 mg, 500 mg Q2W, 500 mg Q3W, or 10 mg/kg Q2W. Endpoints included safety (NCI CTCAE v4.03) and the objective response rate (ORR) by Response Criteria for Malignant Lymphoma. Results: As of Feb 9, 2017, 31 pts were randomised and had a median age of 38 years (range 22–81). Five and 8 pts had disease progression following auto-SCT and allo-SCT, respectively; the remaining pts were SCT-ineligible. Pts received a median of 6 cycles (range 1–23) of avelumab to date. In 30 pts analyzed for safety, the most common treatment-related adverse events (TRAEs) of any grade were infusion-related reaction (IRR; 26.7%), nausea (20.0%), rash (20.0%), and fatigue (13. 3%).Two pts (6.7%) discontinued treatment due to IRR. Grade ≥ 3 TRAEs occurred in 11 pts (36.7%); there were no treatment-related deaths. Two pts who had received prior allo-SCT developed grade 3 liver graft vs host disease (GVHD), which completely resolved after immunosuppressive therapy and discontinuation of avelumab. ORR across all 31 pts was 54.8% (95% CI 36.0–72.7) with 2 complete responses (CRs; 6.5%) and 15 partial responses (PRs; 48.4%). Responses were observed in all dosing groups (ORR range 14.3%–83.3%). ORR in the 5 post-auto SCT pts was 20.0% (95% CI 0.5–71.6) with 1 PR. ORR in the 8 post-allo SCT pts was 75.0% (95% CI 34.9–96.8) with 1 CR (12.5%) and 5 PRs (62.5%). Conclusions: Avelumab appears to have clinical activity with an acceptable tolerability profile in pts with heavily pretreated cHL. The ORR was similar to that observed with PD-1 inhibitors, indicating that targeting PD-L2 may not be necessary or sufficient for the therapeutic effect observed following PD-1 checkpoint blockade in cHL. The high ORR observed in the post-allo SCT setting suggests that checkpoint inhibitors may enhance the graft vs lymphoma response; however, more mature data are required to assess the benefit–risk of PD-1 checkpoint blockade regarding GVHD in this setting. Keywords: classical Hodgkin lymphoma (cHL); Reed-Sternberg cells.

Introduction: 4-1BB (CD137, TNFRSF9) receptor agonists enhance cytotoxic T-cell and NK cell responses, including antibody (Ab)-dependent cellular cytotoxicity, and have shown antitumor activity in preclinical models. Utomilumab (Uto), a fully human IgG2 monoclonal Ab, binds to human 4-1BB with high affinity and specificity and activates 4-1BB while blocking binding to endogenous 4-1BB ligand. Initial findings from the dose-finding cohorts of this study were presented previously. We report here updated results and data from the expansion cohort in patients (pts) with rituximab (R)-refractory follicular lymphoma (FL). Methods: The dose-finding component of this phase I study evaluated Uto at doses ranging from 0.03 to 10 mg/kg in combination with R at the standard 375 mg/m2 dose in pts with relapsed or refractory CD20+ non-Hodgkin's lymphoma (NHL), using a time-to-event continuous-reassessment-method design. In the expansion cohort, pts with R-refractory FL received Uto at the 1.2 mg/kg dose level. Pts received Uto on day 1, every 4 weeks up to 24 months and R from day 7, weekly for 4 weeks. The primary endpoint was dose-limiting toxicity (DLT) in the first 2 cycles, with safety, pharmacokinetics, and antitumor activity as secondary endpoints, and pharmacodynamics as an exploratory endpoint. Results: As of Nov 2016, 48 pts with CD20+ NHL including FL (n = 33), mantle cell (n = 6), diffuse large B cell (n = 3), marginal zone (n = 2), small lymphocytic (n = 2), and other (n = 2) lymphoma were treated with Uto combined with R. Approximately 50% of pts had received ≥3 prior anticancer therapies; 24/33 pts with FL had R-refractory disease. No DLTs were observed, and no pt discontinued treatment due to treatment-related adverse events (AEs). Of the 48 treated pts, 27% had grade ≥ 3 treatment-emergent AEs. The most common treatment-related AEs were fatigue (25%), infusion-related reaction (23%), and diarrhea (10%). Uto exposure appeared to increase with increasing doses. One (2%) of 43 anti-drug Ab (ADA) evaluable pts had treatment-induced ADA/neutralizing Ab against Uto. The objective response rate (ORR) across all Uto dose levels tested was 23% (11/48). In all FL pts, the ORR was 27% (9/33) and 33% (8/24) in FL pts with R-refractory disease with 4 complete and 4 partial responses. In the expansion cohort of pts with R-refractory FL, the ORR was 44% (4/9). Pharmacodynamic effects of Uto, including increases in circulating CD8+ T cells and soluble 4-1BB, were observed at dose levels between 0.06 and 10 mg/kg. Enrollment into the expansion is ongoing. Conclusions: The combination of Uto with R showed a highly favorable tolerability profile with no DLTs observed and no substantial hematologic, hepatic, or immune-related toxicity reported. The preliminary evidence of clinical activity observed in R-refractory FL pts supports further evaluation of Uto plus R, especially in pts requiring treatment regimens with reduced toxicity. Keywords: B-cell lymphoma; non-Hodgkin lymphoma (NHL); rituximab.

PurposeThis multicenter, dose-escalation study evaluates the safety, pharmacokinetics, and efficacy of galiximab (anti-CD80 monoclonal antibody) in patients with relapsed or refractory follicular lymphoma.Patients and MethodsPatients had follicular lymphoma that had relapsed or failed to respond to primary therapy; the majority (90%) presented with stage III or IV disease. Four weekly intravenous infusions of galiximab were administered at doses of 125, 250, 375, or 500 mg/m2.ResultsThirty-seven patients received galiximab treatment and were evaluated for safety; 35 were assessable for response. Antibody infusions were safe and well tolerated with no dose-limiting toxicities. A total of 22 (60%) of 37 patients experienced adverse events related to galiximab. All but one of the events were grade 1 or 2; the most common were fatigue, nausea, and headache. Cytopenias were rare; only one patient experienced anemia and febrile neutropenia, which were unrelated to galiximab and resolved after treatment. No patient developed antigaliximab antibody formation. The mean serum half-life ranged from 13 to 24 days. The overall response rate was 11% (two complete responses and two partial responses). Time to best response was delayed (months 3, 6, 9, and 12). Twelve patients (34%) maintained stable disease. Nearly half of all patients (49%) had a decrease in indicator lesions. Two responders remain on study without progression (22 and 24.4 months).ConclusionThe favorable safety profile of galiximab and evidence of single-agent biologic activity and dose-dependent pharmacokinetics support further evaluation of galiximab as a treatment for follicular lymphoma, possibly in combination with other lymphoma therapies.

CD80 is an immune costimulatory molecule expressed on the cell surface of follicular and other lymphomas. Galiximab, a macaque-human chimeric anti-CD80 monoclonal antibody, has been shown to have antitumor activity in patients with relapsed or refractory, follicular lymphoma. Preclinical work suggested antibody-dependent cellular cytotoxicity (ADCC) as a mechanism for galiximab-induced cell lysis. Single-nucleotide polymorphisms (SNPs) for high-affinity Fc gamma receptor IIIa and high interferon-gamma production have been associated with increased ADCC activity. For this report, genomic DNA from peripheral blood of clinical study patients was analyzed for Fc gamma receptor IIIa and interferon-gamma gene SNPs after PCR amplification. In addition, the frequency of interferon-gamma secreting cells in peripheral blood samples was evaluated by ELISPOT. SNP and ELISPOT results were then evaluated for correlation with clinical response (CR or PR) to galiximab and reduction in tumor burden (SPD of indicator lesions). Of 35 follicular lymphoma patients evaluable for response to galiximab, 2 CRs and 1 PR were observed; time to best response was delayed (Months 3, 9, and 12). Of 34 patients with Fc gamma receptor IIIa SNP results, 6 were homozygous for the high-affinity receptor. Of 33 pts with interferon-gamma SNP results, 4 were homozygous for the high-producing allele. None of the 3 responders were homozygous for high-affinity Fc gamma receptor IIIa or high interferon-gamma production, and the degree of tumor burden reduction was smaller in these homozygotes than in the others. In addition, pretreatment interferon-gamma ELISPOT data did not predict clinical response to galiximab or tumor burden reduction. In summary, high-affinity Fc gamma receptor IIIa and high interferon-gamma production do not predict clinical responsiveness of follicular NHL to galiximab, suggesting that ADCC may not be a primary mechanism of action for this antibody. Ongoing studies are evaluating other potential mechanisms including the immunomodulatory effects of blocking CD80.

Background. Islet xenotransplantation will most likely be performed in diabetic patients treated with immunosuppressive drugs. The importance of the galactosyl alpha(1–3) galactose (Gal&agr;1–3Gal) antigen in immunosuppressed islet xenograft recipients has not been studied. Methods. Fetal porcine islet-like cell clusters (ICCs) were transplanted into the renal subcapsular space of both Gal-knockout mice and ordinary mice. Transplantations were performed in untreated mice and mice immunosuppressed with cyclosporine A (CsA) plus 15-deoxyspergualin (DSG). Studies were also performed in immunosuppressed Gal-knockout mice that had been actively immunized against Gal&agr;1–3Gal. Evaluation was performed 12 days after transplantation using morphologic techniques. The levels of serum immunoglobulin (Ig)G and IgM to the Gal&agr;1–3Gal antigen or to the ICCs were determined. Results. No difference in the morphologic appearance could be seen between ordinary mice and Gal-knockout mice. No deposits of IgG, IgM, or C3 could be detected. Almost no difference could be seen between immunosuppressed Gal-knockout mice and immunosuppressed ordinary mice. In immunosuppressed, immunized Gal-knockout mice, the results were similar. In ordinary mice treated with CsA+DSG, the levels of anti-Gal IgM were lower than they were in untreated mice, whereas the levels of anti-Gal IgG were similar. In Gal-knockout mice (including immunized animals) treated with CsA+DSG, the levels of anti-Gal IgG and IgM were lower than they were in untreated Gal-knockout mice. Conclusions. After renal subcapsular transplantation, antibodies against Gal&agr;1–3Gal have no major influence on islet xenograft rejection in the pig-to-mouse model. Immunosuppression, which inhibits rejection in the pig-to-mouse model, is equally effective when transplantation is performed across the Gal&agr;1–3Gal barrier.

Background: Arsenic trioxide (As-2 O-3 ) is an anticancer drug that has been reported to induce apoptosis and inhibit differentiation in human plasmacytoma and normal plasma/B cells without significant myelosuppression. We assessed the ability of As-2 O-3 as single therapy or in combination with an anti-CD20 monoclonal antibody (mAb) and whole body irradiation (WBI) to deplete B and plasma cells, both in vitro and in vivo, and to reduce the level of anti-alphaGal1-3Gal antibody (anti-Gal Ab) in baboons. Methods: In vitro the effect of As-2 O-3 on antibody secretion (anti-Gal IgM, total IgG and IgM) was measured by enzyme-linked immunospot assay (ELISPOT). Its inhibition of proliferation of baboon splenocytes and the NCI-H929 human plasmacytoma cell line was measured by tritiated thymidine uptake. In vivo: all baboons (n=7) had undergone splenectomy. The effects of As-2 O-3 (0.18 to 0.36 mg/kg) on B/plasma cell depletion and anti-Gal Ab production were assessed in three baboons. For comparison, three baboons received either WBI (2 x 150 cGy) or anti-CD20 mAb (20 mg/kg x 4 doses), or both WBI and anti-CD20 mAb. A final baboon received As-2 O-3 + WBI (150 cGy) + anti-CD20 mAb. Anti-Gal Ab levels were measured daily by ELISA. Depletion of B cells from blood and bone marrow (BM) was monitored by flow cytometry and by histology of lymph nodes (LN). Autopsy was performed in three baboons. Results: In vitro: As-2 O-3 (at 5 x 10(-6) mol/l) reduced anti-Gal IgM and total IgM secretors by 76% (P =0.53) and 95% (P < 0.001), respectively, but did not reduce total IgG secretors. As-2 O-3 inhibited in a dose-dependent manner the proliferation of activated splenocytes and of the NCI-H929 plasmacytoma cell line; complete inhibition was achieved at a dose of 1 x 10(-5) mol/l. In vivo: As-2 O-3 was found to be toxic at the doses given and was associated with the deaths of two of the four baboons that received it. Daily intravenous therapy with As-2 O-3 alone reduced B cells (CD20+) in the blood (by 50 to 90%), BM (40%) and LN (20 to 30%), but anti-Gal Ab levels were not significantly decreased. Anti-CD20 mAb therapy alone or WBI alone depleted B cells by 100% in the blood and BM, and 80 to 100% in the LN. The combination of anti-CD20 mAb + WBI led to depletion of B cells in blood, BM and LN for 3 months, but reduction of anti-Gal Ab remained marginal. The combination of As-2 O-3 + anti-CD20 mAb + WBI did not reduce anti-Gal Ab levels further. At autopsy in the latter baboon, B cells remained present in Peyer's patches and tonsils. Conclusions: In vitro: As-2 O-3 reduced B/plasma cell numbers and suppressed IgM secretors, but not IgG secretors. In vivo: As-2 O-3 was not as effective as either anti-CD20 mAb or WBI in depleting B/plasma cells, and was largely ineffective in reducing anti-Gal Ab levels. Its administration was associated with considerable toxicity. Autopsy in one baboon suggested that B cells in Peyer's patches and tonsils may be resistant to therapy and remain a source of continuing production of anti-Gal Ab.

The treatment of primary central nervous system lymphoma (PCNSL) with chemo- and radiotherapy is efficient in terms of tumor response. However, time to tumor progression often is of short duration and leptomeningeal relapse is common [1]. We present a 66-year-old man in third relapse of a CD20-positive PCNSL. After treatment with intravenous and intraventricular administration of the chimeric anti-CD20 monoclonal antibody rituximab, a total clearing of lymphoma cells in the cerebrospinal fluid (CSF) was achieved. There was no change in the size of the parenchymal tumor mass but there was slight improvement of clinical symptoms after therapy. Rituximab infusions (375 mg/m(2)) were first given systemically on days 1 and 8. Intraventricular injections of rituximab via Ommaya reservoir were given on days 16 (10 mg), 17 (40 mg), 24 (25 mg) and 25 (25 mg). Reversible side effects such as nausea, chills and hypotension were observed only immediately after intraventricular administration of 40 mg rituximab. Antibody levels in CSF were measured at 7 timepoints during and after the treatment period. These data suggest that intraventicular treatment with rituximab is safe and feasible with a potential activity on leptomeningeal tumor manifestation. Efficacy and pharmacokinetics of rituximab in PCNSL should be investigated in future trials.

The effect of CD154 blockade and macrophage depletion or inhibition on baboon humoral and cellular immune responses to pig antigens was studied in a pig-to-baboon peripheral blood mobilized progenitor cell (PBPC) transplantation model aimed at inducing tolerance. We infused pig PBPCs in baboons pretreated with a nonmyeloablative regimen along with murine anti-human CD154 monoclonal antibody (mAb) and macrophage-depleting or -inhibiting agents. Group 1 baboons (n=2) underwent a nonmyeloablative regimen and immunoadsorption of anti-Gal(alpha)1,3Gal (Gal) antibody (Ab) before intravenous infusion of high doses (1.3-4.6 x 10(10)cells/kg) of PBPCs. In group 2 (n=5), cyclosporine was replaced by 8 doses of anti-CD154 mAb over 14 days. Group 3 (n=3) received the group 2 regimen plus medronate liposomes (n=2) or commercially available human intravenous immunoglobulin G depleted of anti-Gal Ab (n=1) to deplete/inhibit recipient macrophages. Group 1 developed sensitization to Gal and also developed new Ab to non-Gal porcine antigens within 10 to 20 days. In group 2, no sensitization to Gal or non-Gal determinants was seen, but Gal-reactive antibodies did return to their preleukocyte transplantation levels. CD154 blockade, therefore, induced humoral unresponsiveness to pig cells. In group 3, sensitization to Gal was seen in all three baboons at 20 days, and Abs against new porcine determinants developed in one baboon. The depletion or inhibition of host macrophages, therefore, prevented the induction of humoral unresponsiveness by CD154 blockade. These results suggest that CD154 blockade induces humoral unresponsiveness by a mechanism that involves the indirect pathway of antigen presentation. In vitro investigation of baboon anti-pig mixed lymphocyte reaction confirmed that only the indirect pathway is efficiently blocked by anti-CD154 mAb. The mechanism in which blockade of the CD40-CD154 pathway induces its effect remains to be determined, but it could involve the generation of regulatory cells capable of suppressing the direct pathway.

E-, P-, and L-selectin counterreceptor activities, leukocyte trafficking, and lymphocyte homing are controlled prominently but incompletely by α(1,3)fucosyltransferase FucT-VII-dependent fucosylation. Molecular determinants for FucT-VII-independent leukocyte trafficking are not defined, and evidence for contributions by or requirements for other FucTs in leukocyte recruitment is contradictory and incomplete. We show here that inflammation-dependent leukocyte recruitment retained in FucT-VII deficiency is extinguished in FucT-IV−/−/FucT-VII−/− mice. Double deficiency yields an extreme leukocytosis characterized by decreased neutrophil turnover and increased neutrophil production. FucT-IV also contributes to HEV-born L-selectin ligands, since lymphocyte homing retained in FucT-VII−/− mice is revoked in FucT-IV−/−/FucT-VII−/− mice. These observations reveal essential FucT-IV-dependent contributions to E-, P-, and L-selectin ligand synthesis and to the control of leukocyte recruitment and lymphocyte homing.

Anti-Gal alpha1-3Gal antibodies (anti alpha Gal Ab) are a major barrier to clinical xenotransplantation as they are believed to initiate both hyperacute and acute humoral rejection. Extracorporeal immunoadsorption (EIA) with alpha Gal oligosaccharide columns temporarily depletes anti-alpha Gal Ab, but their return is ultimately associated with graft destruction. We therefore assessed the ability of two immunotoxins (IT) and two monoclonal antibodies (mAb) to deplete B and/or plasma cells both in vitro and in vivo in baboons, and to observe the rate of return of anti alpha Gal Ab following EIA.The effects of the mouse anti-human IT anti-CD22-ricin A (proportional to CD22-IT, directed against a B cell determinant) and anti-CD38-ricin A (proportional to CD38-IT, B and plasma cell determinant) and the mouse anti-human anti-CD38 mAb (proportional to CD38 mAb) and mouse/human chimeric anti-human anti-CD20 (alpha CD20 mAb, Rituximab, B cell determinant) on B and plasma cell depletion and anti alpha Gal Ab production were assessed both in vitro and in vivo in baboons (n=9) that had previously undergone splenectomy. For comparison, two baboons received nonmyeloablative whole body irradiation (WBI) (300 cGy), and one received myeloablative WBI (900 cGy). Depletion of B cells was monitored by flow cytometry of blood, bone marrow (BM) and lymph nodes (LNG, staining with anti-CD20 and/or anti-CD22 mAbs, and by histology of LN. EIA was carried out after the therapy and anti alpha Gal Ab levels were measured daily.In vitro proportional to CD22-IT inhibited protein synthesis in the human Daudi B cell line more effectively than proportional to CD38-IT. Upon differentiation of B cells into plasma cells, however, less inhibition of protein synthesis after proportional to CD22-IT treatment was observed. Depleting CD20-positive cells in vitro from a baboon spleen cell population already depleted of granulocytes, monocytes, and T cells led to a relative enrichment of CD20-negative cells, that is plasma cells, and consequently resulted in a significant increase in anti alpha Gal Ab production by the remaining cells, whereas depleting CD38-positive cells resulted in a significant decrease in anti alpha Gal Ab production. In vivo, WBI (300 or 900 cGy) resulted in 100% B cell depletion in blood and BM,> 80% depletion in LN, with substantial recovery of B cells after 21 days and only transient reduction in anti alpha Gal Ab after EIA. proportional to CD22-IT depleted B cells by > 97% in blood and BM, and by 60% in LN, but a rebound of B cells was observed after 14 and 62 days in LN and blood, respectively. At 7 days, serum anti alpha Gal IgG and IgM Ab levels were reduced by a maximum of 40-45% followed by a rebound to levels up to 12-fold that of baseline anti alpha Gal Ab by day 83 in one baboon. The results obtained with proportional to CD38-IT were inconclusive. This may have been, in part, due to inadequate conjugation of the toxin. Cell coating was 100% with proportional to CD38 mAb, but no changes in anti alpha Gal Ab production were observed. proportional to CD20 mAb resulted in 100% depletion of B cells in blood and BM and 80% in LN, with recovery of B cells starting at day 42. Adding 150cGy WBI at this time led to 100% depletion of B cells in the BM and LN. Although B cell depletion in blood and BM persisted for >3 months, the reduction of serum anti alpha Gal IgG or IgM Ab levels was not sustained beyond 2 days.proportional to CD20 mAb+WBI totally and efficiently depleted CD20- and CD22-positive B cells in blood, BM, and LN for >3 months in vivo, but there was no sustained clinically significant reduction in serum anti alpha Gal Ab. The majority of antibody secretors are CD38-positive cells, but targeting these cells in vitro or in vivo with alpha CD38-IT was not very effective. These observations suggest that CD20-and CD22-positive B cells are not the major source of anti alpha Gal Ab production. Future efforts will be directed towards suppression of plasma cell function.

Introduction. Attempts to achieve immunological tolerance to porcine tissues in nonhuman primates through establishment of mixed hematopoietic chimerism are hindered by the rapid clearance of mobilized porcine leukocytes, containing progenitor cells (pPBPCs), from the circulation. Eighteen hours after infusing 1-2x10(10) pPBPC/kg into baboons that had been depleted of circulating anti-alpha Gal and complement, these cells are almost undetectable by flow cytometry. The aim of the present study was to identify mechanisms that contribute to rapid clearance of pPBPCs in the baboon. This was achieved by depleting, or blocking the Fc-receptors of, cells of the phagocytic reticuloendothelial system (RES) using medronate liposomes (MLs) or intravenous immunoglobulin (Mg), respectively.Methods. Baboons (preliminary studies, n=4) were used in a dose-finding and toxicity study to assess the effect of MLs on macrophage depletion in vivo. In another study, baboons (n=9) received a nonmyeloablative conditioning regimen (NMCR) aimed at inducing immunological tolerance, including splenectomy, whole body irradiation (300 cGy) or cyclophosphamide (80 mg/kg), thymic irradiation (700 cGy), T-cell depletion, complement depletion with cobra venom factor, mycophenolate mofetil, anti-CD154 monoclonal antibody, and multiple extracorporeal immunoadsorptions of anti-aGal antibodies. The baboons were divided into three groups: Group 1 (n=5) NMCR+pPBPC transplantation; Group 2 (n=2) NMCR+ML+pPBPC tranplantation; and Group 3 (n=2) NMCR+IVIg+pPBPC transplantation. Detection of pig cells in the blood was assessed by fluorescence-activated cell sorter and polymerase chain reaction (PCR).Results. Preliminary studies: ML effectively depleted macrophages from the circulation in a dose-dependent manner. Group 1: On average, 14% pig cells were detected 2 hr postinfusion of 1x10(10) pPBPC/kg. After 18 hr, there were generally less than 1.5% pig cells detectable. Group 2: Substantially higher levels of pig cell chimerism (55-78%) were detected 2 hr postinfusion, even when a smaller number (0.5-1 x 10(10)/ kg) of pPBPCs had been infused, and these levels were better sustained IS hr later (10-52%). Group 3: In one baboon, 4.4% pig cells were detected 2 hr after infusion of 1x10(10) pPBPC/kg. After 18 hr, however, 7.4% pig cells were detected. A second baboon died 2 hr after infusion of 4 x 10(10) pPBPC/kg, with a total white blood cell count of 90,000, of which 70% were pig cells. No differences in microchimerism could be detected between the groups as determined by PCR.Conclusions. This is the first study to report an efficient decrease of phagocytic function by depletion of macrophages with MLs in a large-animal model. Depletion of macrophages with MLs led to initial higher chimerism and prolonged the survival of circulating pig cells in baboons. Blockade of macrophage function with IVIg had a more modest effect. Cells of the RES, therefore, play a major role in clearing pPBPCs from the circulation in baboons. Depletion or blockade of the RES may contribute to achieving mixed hematopoietic chimerism and induction of tolerance to a discordant xenograft.

BACKGROUND:In pig-to-primate organ transplantation, hyperacute rejection can be prevented, but the organ is rejected within days by acute vascular rejection, in which induced high-affinity anti-Gal alpha1-3Gal (alphaGal) IgG and possibly antibodies directed against new porcine (non-alphaGal) antigenic determinants are considered to play a major role. We have explored the role of an anti-CD40L monoclonal antibody in modifying the humoral response to porcine hematopoietic cells in baboons pretreated with a nonmyeloablative regimen.METHODS:Porcine peripheral blood mobilized progenitor cells obtained by leukapheresis from both major histocompatibility complex-inbred miniature swine (n=7) and human decay-accelerating factor pigs (n=3) were transplanted into baboons. Group 1 baboons (n=3) underwent whole body (300 cGy) and thymic (700 cGy) irradiation, T cell depletion with ATG, complement depletion with cobra venom factor, short courses of cyclosporine, mycophenolate mofetil, porcine hematopoietic growth factors, and anti-alphaGal antibody depletion by immunoadsorption before transplantation of high doses (2-4 x 10(10)/cells/kg) of peripheral blood mobilized progenitor cells. In group 2 (n=5), cyclosporine was replaced by eight doses of anti-CD40L monoclonal antibodies over 14 days. The group 3 baboons (n=2) received the group 1 regimen plus 2 doses of anti-CD40L monoclonal antibodies (on days 0 and 2).RESULTS:In group 1, sensitization to alphaGal (with increases in IgM and IgG of 3- to 6-fold and 100-fold, respectively) and the development of antibodies to new non-alphaGal porcine antigens occurred within 20 days. In group 2, no sensitization to alphaGal or non-alphaGal determinants was seen, but alphaGal-reactive antibodies did return to their pre- peripheral blood mobilized progenitor cells transplant levels. In group 3, attenuated sensitization to alphaGal antigens was seen after cessation of cyclosporine and mycophenolate mofetil therapy at 30 days (IgM 4-fold, IgG 8-30-fold), but no antibodies developed against new porcine determinants. In no baboon did anti-CD40L monoclonal antibodies prevent sensitization to its own murine antigens.CONCLUSIONS:We believe these studies are the first to consistently demonstrate prevention of a secondary humoral response after cell or organ transplantation in a pig-to-primate model. The development of sensitization to the murine elements of the anti-CD40L monoclonal antibodies suggests that nonresponsiveness to cell membrane-bound antigen (e.g., alphaGal) is a specific phenomenon and not a general manifestation of immunological unresponsiveness. T cell costimulatory blockade may facilitate induction of mixed hematopoietic chimerism and, consequently, of tolerance to pig organs and tissues.

Despite the success of desensitization protocols, antibody-mediated rejection (AMR) remains a significant contributor to renal allograft failure in patients with donor-specific antibodies. Plasmapheresis and high-dose intravenous immunoglobulin have proved to be effective treatments to prevent and treat AMR, but irreversible injury in the form of transplant glomerulopathy can commonly manifest months to years later. There is an unmet need to improve the outcomes for patients at risk for AMR. Updated Banff criteria now take into account the increasing understanding of the complex and heterogeneous nature of AMR phenotypes, including the timing of rejection, subclinical and chronic AMR, C4d-negative AMR, and antibody-mediated vascular rejection. Treatment for AMR is not standardized, and there is little in the way of evidence-based treatment guidelines. Refining more precisely the mechanisms of injury responsible for different AMR phenotypes and establishing relevant surrogate endpoints to facilitate more informative studies will likely allow for more accurate determination of prognosis and efficacious intervention using new therapeutic approaches. In addition to plasma exchange and intravenous immunoglobulin, a number of other add-on therapies have been tried in small studies without consistent benefit, including anti-CD20, proteasome inhibitors, complement inhibitors, anti-interleukin-6 receptor blockers, and immunoglobulin G-degrading enzyme of Streptococcus pyogenes (called IdeS).

In this survey, the effectiveness of the carbon nitride in the form of graphitic structure (CN) as a drug delivery system for hydroxyurea (HU) and carmustine (BCN) anticancer drugs was evaluated through density functional theory (DFT) in both aqueous and vacuum phase. Moreover, a type of possible carrier based on functionalization graphitic-carbon nitride with chitosan is established with the goal of improving therapeutic efficacy and boosting designed deliverer for anticancer drugs. To explore the effectiveness of CN and functionalized CN, some important characteristics of HU and BCN drugs, CN carriers, and drug/CN complexes were calculated. Optimized geometries show that the best appropriate site for the adsorption of selected drugs on the nanosheets surface is the parallel orientation. The calculated adsorption energy in all studied complexes is negative, indicating that all of the analyzed structures are reliable and the interaction of the drug molecules with the examined nanosheets is a spontaneous process. Also, it turned out that the adsorption of BCN on the CN and f-CN is greater than HU. In accordance with the findings of the NBO, in the HU/CN and HU/f-CN complexes, the charges transfer positions on the direction of the HU molecule to the nanosheet, while in the BCN/CN and BCN/f-CN structures, charge transfer happens from the nanosheet to the BCN drug. The values of solvation energy exhibited a remarkable enhancement in the stability of f-CN after adsorption of the drug in the presence of water solvent. Functionalized nanosheets have a high sensitivity according to their electrical characteristics, implying their potential for drug release. Thus, HU/f-CN and BCN/f-CN complexes are favorable systems and functionalized nanosheet is a better carrier in comparison to pristine nanosheet for the HU and BCN drug delivery system. Generally, the results imply that g-C3N4 might be utilized as an effective drug-deliverer design for the HU and BCN drugs to treat diverse kinds of cancer.