Cloning, Expression and Characterization of a Peptibody To Deplete Myeloid Derived Suppressor Cells in a Murine Mammary Carcinoma Model

Myeloid derived suppressor cells (MDSCs) are an immature heterogeneous population of myeloid lineage that attenuate the anti-tumor immune responses. Depletion of MDSCs has been shown to improve efficacy of cancer immunotherapeutic approaches. Here, we produced and characterized a recombinant peptibody capable of recognizing and depleting murine MDSCs. Using SOE-PCR, the coding sequence of the MDSC binding peptide and linker were synthesized and then ligated into a home-made expression plasmid containing mouse IgG2a Fc. The peptibody construct was transfected into CHO-K1 cells by lipofectamine 3000 reagent and the resulting fusion protein was purified with protein G column and subsequently characterized by ELISA, SDS-PAGE and immunoblotting. The binding profile of the peptibody to splenic MDSCs and its MDSC depletion ability were then tested by flow cytometry. The purified peptibody appeared as a 70 kDa band in Western blot. It could bind to 98.8% of splenic CD11b+/Gr-1+ MDSCs. In addition, the intratumoral MDSCs were significantly depleted after peptibody treatment compared to their PBS-treated negative control counterparts (P <0.05). In this study, a peptibody capable of depleting intratumoral MDSCs, was produced. Our results imply that it could be considered as a potential drug effective for immunotherapy of cancers.

Pre-existing antitherapeutic antibodies against the Fc region of the hu14.18K322A mAb are associated with outcome in patients with relapsed neuroblastoma

PurposePatients with cancer receiving tumor-reactive humanized monoclonal antibody (mAb) therapy can develop a human antihuman antibody (HAHA) response against the therapeutic mAb. We evaluated for HAHA in patients with neuroblastoma treated in a phase I study of humanized anti-GD2 mAb (immunoglobulin (Ig)G1 isotype), hu14.18K322A (NCT00743496). The pretreatment sera (collected prior to mAb treatment) from 9 of 38 patients contained antitherapeutic antibodies, even though they had no prior mAb exposure. We sought to characterize these pre-existing antitherapeutic antibodies (PATA).Experimental designThe PATA+ pretreatment samples were characterized via ELISA; clinical associations with PATA status were evaluated.ResultsPretreatment sera from eight of nine PATA+ patients also bound rituximab and demonstrated preferential ELISA reactivity against the Fc portions of hu14.18K322A and rituximab as compared with the Fab portions of these mAbs. These PATA+ sera also recognized dinutuximab (human IgG1 isotype) and mouse IgG2a isotype mAbs, but not a mouse IgG1 isotype or the fully human panitumumab (IgG2 isotype) mAb. Of the 38 treated patients, only 4 patients (all in the PATA+ cohort) demonstrated no disease progression for>2.5 years without receiving further therapy (p=0.002).ConclusionsThis study demonstrates an association between clinical outcome and the presence of PATA against determinant(s) on the Fc component of the therapeutic mAb, suggesting that the PATA may be playing a role in augmenting mAb-based antitumor effects. Further analyses for the presence of PATA in a larger cohort of patients with relapsed neuroblastoma, analyses of their clinical correlates, identification of their immunological targets, and potential antitumor mechanisms are warranted.

Mouse IgG2a Antibodies Specific for the Commensal Streptococcus mitis Show Stronger Cross-Reactivity with Streptococcus pneumoniae than IgG1 Antibodies

Here we show that mouse IgG2a and IgG1 antibodies specific for the commensal Streptococcus mitis cross-react with pathogen Streptococcus pneumoniae serotypes 2 and 4, although the cross-reactivity conferred by IgG2a is stronger than that by IgG1 antibodies. These findings may be important for understanding the S. mitis-induced IgG isotype responses and have consequences for the development of an effective pneumococcal vaccine.

Mouse IgG2a antibodies specific for the commensal Streptococcus mitis show stronger cross-reactivity with Streptococcus pneumoniae than IgG1 antibodies

Here we show that mouse IgG2a and IgG1 antibodies specific for the commensal Streptococcus mitis cross-react with the pathogen Streptococcus pneumoniae, although the cross-reactivity conferred by IgG2a is stronger than IgG1 antibodies. These findings may have implications for designing S. mitis-based vaccines against pneumococcal infections.

Treating B Cell Lymphomas with Anti CD81 Antibodies

The tetraspanin CD81 associates with CD19 on B cells; this molecular complex functions as co-receptor to lower the threshold of BCR-initiated B cell activation. Recently we have shown the importance of CD81 in tumor growth and metastasis of solid tumors (Vences-Catalan et al., 2015). However, the role of CD81 in lymphoid malignancies has not been explored. Previous studies demonstrated anti-proliferative effects of anti-CD81 antibodies on human B cell lymphomas using in vitro assays (Oren et al., 1990). Here we tested the therapeutic effect of an anti-human CD81 antibody in vivo against Raji and SUP-B8 B cell lymphomas using a xenograft model in SCID mice. Our studies demonstrated that our anti-human CD81 antibody (mouse IgG1) had therapeutic effect comparable to Rituximab (human IgG1) (Figure 1A). Yet, the two antibodies differ in their ability to mediate antibody-dependent cell cytotoxicity (ADCC), Rituximab is known to be highly effective, whereas the mouse IgG1 anti-CD81 antibody is not expected to mediate ADCC. To enhance the anti-CD81-mediated ADCC, we class switched the hybridoma to mouse IgG2a; we also engineered a chimeric antibody containing human IgG1ADCC-HIGH Fc constant region. Indeed, mouse IgG2a and the chimeric human IgG1 anti-CD81 mAb showed a remarkable increase in NK cell-mediated ADCC as well as complement-dependent cytotoxicity (CDC) when compared to Rituximab in vitro (data not shown) and in vivo (Figure 1B). These results suggest that CD81 can be a potential therapeutic target on B cell lymphomas by virtue of both its direct cytotoxic effect and as a mediator of ADCC and CDC. The humanized IgG1 version is being developed as a therapeutic candidate. Comparable efficacy of anti-CD81 to Rituximab. SCID mice were I.V.-injected with 1.5x106 Raji-GFP-Luc cells, tumors growth proceeded for 5 days before IP injection of 4 weekly doses of 100 ug of the indicated antibodies. (A) Survival of Raji-GFP-Luc bearing SCID mice given anti CD81 (n=30), Rituximab (n=20) or control MsIgG1 (n=30). (B) In vivo bioluminescence imaging of tumor growth in mice injected (left to right) with control mouse IgG1; anti-CD81 (MsIgG1); anti-CD81 MsIgG2a; chimeric anti-CD81 (HuIgG1) and Rituximab. Comparable efficacy of anti-CD81 to Rituximab. SCID mice were I.V.-injected with 1.5x106 Raji-GFP-Luc cells, tumors growth proceeded for 5 days before IP injection of 4 weekly doses of 100 ug of the indicated antibodies. (A) Survival of Raji-GFP-Luc bearing SCID mice given anti CD81 (n=30), Rituximab (n=20) or control MsIgG1 (n=30). / (B) In vivo bioluminescence imaging of tumor growth in mice injected (left to right) with control mouse IgG1; anti-CD81 (MsIgG1); anti-CD81 MsIgG2a; chimeric anti-CD81 (HuIgG1) and Rituximab. Disclosures Levy: Kite Pharma: Consultancy; Five Prime Therapeutics: Consultancy; Innate Pharma: Consultancy; Beigene: Consultancy; Corvus: Consultancy; Dynavax: Research Funding; Pharmacyclics: Research Funding.

C-Reactive Protein (CRP) Enhances Murine Antibody-Mediated Transfusion Related Acute Lung Injury (TRALI)

Transfusion-related acute lung injury (TRALI), a syndrome characterized by respiratory distress triggered by blood transfusions, is the leading cause of transfusion-related mortality. Mostly, TRALI has been attributed to passive infusion of human leucocyte antigen (HLA) and human neutrophil antigen (HNA) antibodies present in the transfused blood product. Several animal models have been developed to study the pathogenesis of antibody-mediated TRALI and various mechanisms for TRALI induction have been suggested, including involvement of endothelial cells, neutrophils and monocytes. In 2006, a murine of model of antibody-mediated TRALI was developed using a monoclonal MHC class I antibody (clone 34-1-2s). This antibody was shown to cause significant lung damage (excess lung water: pulmonary edema) within 2 hours of administration into BALB/c mice, which in follow-up studies was only reproducible after initial priming with the gram-negative endotoxin lipopolysaccharide (LPS). 34-1-2s was also shown to cause severe lung damage in severe combined immunodeficient (SCID) mice. We investigated 34-1-2s mediated TRALI in BALB/c mice, without LPS priming, and found no difference in TRALI severity when compared with injection with an control isotype antibody for 34-1-2s (Isotype Mouse IgG2a antibody), as examined by lung wet-to-dry ratios, a measure for pulmonary edema. Recently it was described that the acute phase protein C-reactive protein (CRP), heavily up-regulated during acute infections and also present at lower levels in healthy individuals, was able to enhance antibody-mediated platelet destruction both in vitro and in vivo via Fc-receptor mediated phagocytic responses. Considering the fact that TRALI has been shown to be mainly antibody-mediated, plus the fact that it has been suggested to be an Fc-dependent process as well, we investigated the effect of CRP in a murine antibody-mediated TRALI. We tested if CRP would be able to enhance antibody-mediated TRALI in the murine 34-1-2s based BALB/c TRALI model. For that purpose, we co-injected CRP together with 34-1-2s and compared that to co-injection of CRP together with control isotype mouse IgG2a or to injection with CRP alone. We found that CRP+34-1-2s injection resulted in significantly higher lung damage than CRP+isotype antibody, as well as than CRP alone, with at least 43% of the mice in the CRP+34-1-2s group having a lung wet-to-dry ratio of higher than 5, which is considered to represent severe lung damage. As the monocyte-derived neutrophil chemoattractant macrophage inflammatory protein 2 (MIP-2: murine equivalent of human IL-8) was recently shown to play a central role in murine (SCID) 34-1-2s-mediated TRALI induction, we measured MIP-2 values in our BALB/c TRALI model and found that CRP alone was capable of producing high levels of MIP-2, which were found to be even more increased when 34-1-2s was co-injected with CRP. We propose a mechanism in which CRP plays a synergistic role with 34-1-2s antibody to significantly increase the induction of antibody-mediated TRALI via enhanced stimulation of monocyte-derived MIP-2 secretion. Disclosures No relevant conflicts of interest to declare.

Type II (tositumomab) anti-CD20 monoclonal antibody out performs type I (rituximab-like) reagents in B-cell depletion regardless of complement activation

Anti-CD20 monoclonal antibodies (mAbs) are classified into type I (rituximab-like) or type II (tositumomab-like) based on their ability to redistribute CD20 molecules in the plasma membrane and activate various effector functions. To compare type I and II mAbs directly in vivo and maximize Fc effector function, we selected and engineered mAbs with the same mouse IgG2a isotype and assessed their B-cell depleting activity in human CD20 transgenic mice. Despite being the same isotype, having similar affinity, opsonizing activity for phagocytosis, and in vivo half-life, the type II mAb tositumomab (B1) provided substantially longer depletion of B cells from the peripheral blood compared with the type I mAb rituximab (Rit m2a), and 1F5. This difference was also evident within the secondary lymphoid organs, in particular, the spleen. Failure to engage complement did not explain the efficacy of the type II reagents because type I mAbs mutated in the Fc domain (K322A) to prevent C1q binding still did not display equivalent efficacy. These results give support for the use of type II CD20 mAbs in human B-cell diseases.