Exploratory Prognostic Biomarkers of Complement-Mediated Thrombotic Microangiopathy (CM-TMA) in Adults with Atypical Hemolytic Uremic Syndrome (aHUS): Analysis of a Phase III Study of Ravulizumab.

BACKGROUND: Clinically validated biomarkers for monitoring of patients with complement-mediated thrombotic microangiopathy (CM-TMA) including atypical hemolytic uremic syndrome (aHUS) are unavailable. Improved characterization of biomarkers in patients with aHUS may inform treatment and monitoring for patients with CM-TMA. METHODS: This analysis used data collected from 55/56 (98.2 %) adult patients with aHUS enrolled in the global Phase III study of ravulizumab (NCT02949128). Baseline (pre-treatment) patient serum, plasma and urine biomarker levels were compared with the maximum observed levels in normal donors and evaluated for associations with pre-treatment plasma exchange/infusion and dialysis status. Biomarkers were also assessed for associations with key clinical measures at baseline and with changes at 26 and 52 weeks from treatment initiation via linear regression analyses. RESULTS: Complement-specific urine levels (factor Ba and sC5b-9) were elevated in >85 % of patients and are significantly associated with pre-treatment kidney dysfunction. Baseline levels of other evaluated biomarkers were elevated in >70 % of patients with aHUS, except for plasma sC5b-9 and serum sVCAM-1. Lower levels of urine complement markers at baseline are significantly associated with improvements in total urine protein and estimated glomerular filtration rate at 26 and 52 weeks of treatment. Clinical assessment of complement activation by a receiver operating characteristic analysis of Ba and sC5b-9 was more sensitive and specific in urine matrix than plasma. CONCLUSION: This analysis identified a set of biomarkers that may show utility in the prognosis of CM-TMA, including their potential for measuring and predicting response to anti-C5 therapy. Further studies are required to enhance patient risk stratification and improve management of these vulnerable patients. CLINICAL TRIALS REGISTRATION: NCT02949128, ClinicalTrials.gov.

Phase I study of samalizumab in chronic lymphocytic leukemia and multiple myeloma: blockade of the immune checkpoint CD200.

PURPOSE: Samalizumab is a novel recombinant humanized monoclonal antibody that targets CD200, an immunoregulatory cell surface member of the immunoglobulin superfamily that dampens excessive immune responses and maintains self-tolerance. This first-in-human study investigated the therapeutic use of samalizumab as a CD200 immune checkpoint inhibitor in chronic lymphocytic leukemia (CLL) and multiple myeloma (MM). EXPERIMENTAL DESIGN: Twenty-three patients with advanced CLL and 3 patients with MM were enrolled in an open-label phase 1 study (NCT00648739). Patients were assigned sequentially to one of 7 dose level cohorts (50 to 600 mg/m2) in a 3 + 3 study design, receiving a single dose of samalizumab intravenously once every 28 days. Primary endpoints were safety, identification of the maximum tolerated dose (MTD), and pharmacokinetics. Secondary endpoints were samalizumab binding to CD200, pharmacodynamic effects on circulating tumor cells and leukocyte subsets, and clinical responses. RESULTS: Twenty-one patients received > 1 treatment cycle. Adverse events (AEs) were generally mild to moderate in severity. Samalizumab produced dose-dependent decreases in CD200 expression on CLL cells and decreased frequencies of circulating CD200 + CD4+ T cells that were sustained at higher doses. The MTD was not reached. Decreased tumor burden was observed in 14 CLL patients. One CLL patient achieved a durable partial response and 16 patients had stable disease. All MM patients had disease progression. CONCLUSIONS: Samalizumab had a good safety profile and treatment was associated with reduced tumor burden in a majority of patients with advanced CLL. These preliminary positive results support further development of samalizumab as an immune checkpoint inhibitor. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00648739 registered April 1, 2008.

Eculizumab reduces complement activation, inflammation, endothelial damage, thrombosis, and renal injury markers in aHUS.

Atypical hemolytic uremic syndrome (aHUS) is a genetic, life-threatening disease characterized by uncontrolled complement activation, systemic thrombotic microangiopathy (TMA), and vital organ damage. We evaluated the effect of terminal complement blockade with the anti-C5 monoclonal antibody eculizumab on biomarkers of cellular processes involved in TMA in patients with aHUS longitudinally, during up to 1 year of treatment, compared with in healthy volunteers. Biomarker levels were elevated at baseline in most patients, regardless of mutational status, plasma exchange/infusion use, platelet count, or lactate dehydrogenase or haptoglobin levels. Eculizumab reduced terminal complement activation (C5a and sC5b-9) and renal injury markers (clusterin, cystatin-C, ß2-microglobulin, and liver fatty acid binding protein-1) to healthy volunteer levels and reduced inflammation (soluble tumor necrosis factor receptor-1), coagulation (prothrombin fragment F1+2 and d-dimer), and endothelial damage (thrombomodulin) markers to near-normal levels. Alternative pathway activation (Ba) and endothelial activation markers (soluble vascular cell adhesion molecule-1) decreased but remained elevated, reflecting ongoing complement activation in aHUS despite complete terminal complement blockade. These results highlight links between terminal complement activation and inflammation, endothelial damage, thrombosis, and renal injury and underscore ongoing risk for systemic TMA and progression to organ damage. Further research regarding underlying complement dysregulation is warranted. This trial was registered at www.clinicaltrials.gov as #NCT01194973.

Blockade of CD200 in the presence or absence of antibody effector function: implications for anti-CD200 therapy.

CD200 is an immunosuppressive molecule overexpressed in multiple hematologic malignancies such as B cell chronic lymphocytic leukemia, multiple myeloma, and acute myeloid leukemia. We previously demonstrated that up-regulation of CD200 on tumor cells suppresses antitumor immune responses and that antagonistic anti-human CD200 mAbs enabled human PBMC-mediated tumor growth inhibition in xenograft NOD/SCID human (hu)-mouse models. Ab variants with effector function (IgG1 constant region (G1)) or without effector function (IgG2/G4 fusion constant region (G2G4)) exhibited high antitumor activity in a human tumor xenograft model in which CD200 was expressed. In this report, we seek to select the best candidate to move forward into the clinic and begin to decipher the mechanisms of tumor cell killing by comparing anti-CD200-G1 vs anti-CD200-G2G4 in two related animal models. In a CD200-expressing xenograft NOD/SCID hu-mouse model where CD200 ligand/receptor interactions are already established before initiating treatment, we find that anti-CD200-G1 is a less effective Ab compared with anti-CD200-G2G4. Separately, in a model that evaluates the effect of the Abs on the immune cell component of the xenograft NOD/SCID hu-mouse model distinctly from the effects of binding to CD200 on tumor cells, we find that the administration of anti-CD200-G1 Abs completely abolished human PBMC-mediated tumor growth inhibition. Along with supporting in vitro studies, our data indicate that anti-CD200-G1 Abs efficiently mediate Ab-dependent cellular cytotoxicity of activated T cells, critical cells involved in immune-mediated killing. These studies suggest important implications regarding the selection of the constant region in anti-CD200 immunotherapy of cancer patients.

Inhibition of terminal complement components in presensitized transplant recipients prevents antibody-mediated rejection leading to long-term graft survival and accommodation.

Ab-mediated rejection (AMR) remains the primary obstacle in presensitized patients following organ transplantation, as it is refractory to anti-T cell therapy and can lead to early graft loss. Complement plays an important role in the process of AMR. In the present study, a murine model was designed to mimic AMR in presensitized patients. This model was used to evaluate the effect of blocking the fifth complement component (C5) with an anti-C5 mAb on prevention of graft rejection. BALB/c recipients were presensitized with C3H donor skin grafts 7 days before heart transplantation from the same donor strain. Heart grafts, transplanted when circulating anti-donor IgG Abs were at peak levels, were rejected in 3 days. Graft rejection was characterized by microvascular thrombosis and extensive deposition of Ab and complement in the grafts, consistent with AMR. Anti-C5 administration completely blocked terminal complement activity and local C5 deposition, and in combination with cyclosporine and short-term cyclophosphamide treatment, it effectively prevented heart graft rejection. These recipients achieved permanent graft survival for >100 days with normal histology despite the presence of systemic and intragraft anti-donor Abs and complement, suggesting ongoing accommodation. Furthermore, double-transplant experiments demonstrated that immunological alterations in both the graft and the recipient were required for successful graft accommodation to occur. These data suggest that terminal complement blockade with a functionally blocking Ab represents a promising therapeutic approach to prevent AMR in presensitized recipients.

In vivo targeting of DC-SIGN-positive antigen-presenting cells in a nonhuman primate model.

In vivo targeting of antigen-presenting cells (APCs) with antigens coupled to antibodies directed against APC-specific endocytic receptors is a simple and a promising approach to induce or modulate immune responses against those antigens. In a recent in vitro study, we have shown that targeting of APCs with an antigen coupled to an antibody directed against the endocytic receptor DC-SIGN effectively induces a specific immune response against that antigen. The aim of the present study was to determine the ability of the murine antihuman DC-SIGN antibody AZN-D1 to target APCs in a cynomolgus macaque model after its administration in vivo. Immunohistochemical analysis demonstrated that macaques injected intravenously with AZN-D1 have AZN-D1-targeted APCs in all lymph nodes (LNs) tested and in the liver. DC-SIGN-positive cells were mainly located in the medullary sinuses of the LNs and in the hepatic sinusoids in the liver. No unlabeled DC-SIGN molecules were found in the LN of AZN-D1-injected macaques. Morphologic criteria and staining of sequential LN sections with a panel of antibodies indicated that the DC-SIGN-targeted cells belong to the myeloid lineage of APCs. In conclusion, this is the first study that shows specific targeting of APCs in vivo by using antibodies directed against DC-SIGN.

Effective induction of naive and recall T-cell responses by targeting antigen to human dendritic cells via a humanized anti-DC-SIGN antibody.

Current dendritic cell (DC)-based vaccines are based on ex vivo-generated autologous DCs loaded with antigen prior to readministration into patients. A more direct and less laborious strategy is to target antigens to DCs in vivo via specific surface receptors. Therefore, we developed a humanized antibody, hD1V1G2/G4 (hD1), directed against the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) to explore its capacity to serve as a target receptor for vaccination purposes. hD1 was cross-linked to a model antigen, keyhole limpet hemocyanin (KLH). We observed that the chimeric antibody-protein complex (hD1-KLH) bound specifically to DC-SIGN and was rapidly internalized and translocated to the lysosomal compartment. To determine the targeting efficiency of hD1-KLH, monocyte-derived DCs and peripheral blood lymphocytes (PBLs) were obtained from patients who had previously been vaccinated with KLH-pulsed DCs. Autologous DCs pulsed with hD1-KLH induced proliferation of patient PBLs at a 100-fold lower concentration than KLH-pulsed DCs. In addition, hD1-KLH-targeted DCs induced proliferation of naive T cells recognizing KLH epitopes in the context of major histocompatibility complex (MHC) classes I and II. We conclude that antibody-mediated targeting of antigen to DCs via DC-SIGN effectively induces antigen-specific naive as well as recall T-cell responses. This identifies DC-SIGN as a promising target molecule for DC-based vaccination strategies.