Fc Region Of Human IgG1 Research Articles

Molecular characterization and expression of porcine Siglec-5

In this study we describe the characterization of the porcine orthologue of Siglec-5. A cDNa clone was obtained from a porcine cDNa library derived from swine small intestine which encodes a 555 a-a type 1 transmembrane protein with sequence homology to human Siglec-5. This protein consists of four Ig-like domains, a transmembrane region, and a cytoplasmic tail with two tyrosine-based signalling motifs. When expressed as a recombinant protein fused to the Fc region of human IgG1, porcine Siglec-5 was able to bind porcine red blood cells in a sialic acid-dependent manner. Monoclonal antibodies (mAb) were developed against porcine Siglec-5 and used to analyse its expression in bone marrow and blood cells, and lymphoid tissues. Porcine Siglec-5 expression was mainly restricted to myelomonocytic cells and their precursors, being detected also, although at low levels, on plasmacytoid dendritic cells and B lymphocytes. In lymphoid tissues, ellipsoids of the spleen and subcapsular and medullar sinuses of lymph nodes were positive for Siglec-5. These mAbs were able to precipitate, from granulocyte lysates, a protein of approximately 85kDa under non-reducing conditions, indicating that porcine Siglec-5 is expressed as a monomer in the plasma membrane.

PCSK9 Prosegment Chimera as Novel Inhibitors of LDLR Degradation

The proprotein convertase PCSK9, a target for the treatment of hypercholesterolemia, is a negative regulator of the LDL receptor (LDLR) leading to its degradation in endosomes/lysosomes and up-regulation of plasma LDL-cholesterol levels. The proprotein convertases, a family of nine secretory serine proteases, are first synthesized as inactive zymogens. Except for PCSK9, all other convertases are activated following the autocatalytic excision of their inhibitory N-terminal prosegment. PCSK9 is unique since the mature enzyme exhibits a cleaved prosegment complexed with the catalytic subunit and has no protease activity towards other substrates. Similar to other convertases, we hypothesized that the in trans presence of the PCSK9 prosegment would interfere with PCSK9's activity on the LDLR. Since the prosegment cannot be secreted alone, we engineered a chimeric protein using the Fc-region of human IgG1 fused to the PCSK9 prosegment. The expression of such Fcpro-fusion protein in HEK293 and HepG2 cells resulted in a secreted protein that binds PCSK9 and markedly inhibits its activity on the LDLR. This was observed by either intracellular co-expression of PCSK9 and Fcpro or by an extracellular in vitro co-incubation of Fcpro with PCSK9. Structure-function studies revealed that the inhibitory function of Fcpro does not require the acidic N-terminal stretch (residues 31–58) nor the C-terminal Gln152 of the prosegment. Fcpro likely interacts with the prosegment and/or catalytic subunit of the prosegment≡PCSK9 complex thereby allosterically modulating its function. Our data suggest a novel strategic approach for the design and isolation of PCSK9 inhibitors.

Cleavage of the interchain disulfide bonds in rituximab increases its affinity for FcγRIIIA

The Fc region of human IgG1 mediates effector function via binding to Fcγ receptors and complement activation. The H and L chains of IgG1 antibodies are joined by four interchain disulfide bonds. In this study, these bonds within the therapeutic IgG1 rituximab (RTX) were cleaved either by mild reduction followed by alkylation or by mild S-sulfonation; consequently, two modified RTXs – A-RTX (alkylated) and S-RTX (S-sulfonated) – were formed, and both were almost as potent as unmodified RTX when binding CD20 antigen. Unexpectedly, each modified RTX had a higher binding affinity for FcγRIIIA (CD16A) than did unmodified RTX. However, S-RTX and A-RTX were each less potent than RTX in an assay of antibody-dependent cellular cytotoxicity (ADCC). In this ADCC assay, each modified RTX showed decreased secretion of granzyme B, but no change in perforin secretion, from effector cells. These results provide significant information on the structures within IgG1 that are involved in binding FcγRIIIA, and they may be useful in the development of therapeutic antagonists for FcγRIIIA.

Comparison of pharmacokinetics and biological activity of two IL-15 heterodimeric forms in mice and macaques (P6356)

Abstract IL-15 is a member of the γ-chain family of cytokine important for the proliferation and function of NK and T cells. IL-15 acts on the surface of the cell in complex with IL-15Rα to engage IL-15Rβγ complex in nearby cells. We recently showed that in both murine and human sera IL-15 is in complex with soluble IL-15Rα (sIL-15Rα), suggesting that IL-15/IL-15Rα heterodimer is responsible for IL-15 bioactivity in vivo. We have generated stable cell lines secreting high levels of fully processed human IL-15/sIL-15Rα cytokine. We also prepared an additional form of human glycosylated IL-15 heterodimer in which the C-terminus of sIL-15Rα is fused to the Fc region of human IgG1. The biochemical properties and bioactivity of these IL-15 heterodimers were tested in mice and macaques. IL-15/IL-15RαFc was characterized by an increased bioactivity that reflected its superior plasma half-life in mice, in comparison to IL-15/sIL-15Rα. However, delivery of these two IL-15 heterodimers in macaques failed to reveal any major differences in their pharmacokinetic and pharmacodynamic profile. In macaques, both IL-15 heterodimeric forms resulted in similar levels of plasma IL-15 and induced similar proliferation of NK, γδ and CD8+ T cells. Also, both IL-15 heterodimers were effective in reducing tumor burden in a colon carcinoma mouse model. These results provide important insights for the choice of IL-15 for clinical use, and suggest caution when interpreting data using IL-15/IL-15RαFc in mice.

Abstract 1264: Mutated CD80 may facilitate T-cell activation by inhibiting PDL1-PD1 suppression and by costimulating.

Abstract CD80 functions as a costimulatory signal to activate T cells when it binds to CD28 or as an apoptotic signal when it binds to CTLA4. We recently identified another function for CD80 and showed that co-expression of CD80 by Program Death Ligand-1+ (PDL1+) human tumor cells prevents PDL1 binding to its receptor PD1 on activated T cells, and enhances activation of tumor-reactive T cells. To distinguish if CD80 is facilitating T cell activation by preventing PDL1-PD1 interactions and/or by functioning as a costimulatory molecule, we have generated a recombinant CD80 molecule that does not bind to CD28. Since we hypothesize that CD80 may be a therapeutic for inhibiting tumor-mediated immune suppression via PDL1-PD1 interactions, the recombinant CD80 is also designed to not interact with CTLA4. Using site directed mutagenesis we generated CD80 with the following mutations: H96A, L97A, and E99A (CD8096,97,99). CD28 and CTLA4 bind to 35.3% and 37.4%, respectively, of tumor cells expressing wild type CD80. In contrast, only 1.2% and 1.4% of tumor cells expressing CD8096,97,99 bind CD28 and CTLA4, respectively, demonstrating that the mutant CD80 does not bind to CD28 or CTLA4. Previous studies demonstrated that CD80 prevents PDL1-PD1 interactions by binding to PDL1 and obscuring its detection. Only 7.05% of CD8096,97,99 mutants have detectable PDL1 (vs. 98% of untransfected parental human tumor cells), demonstrating that the CD8096,97,99 mutant does not bind CD28 or CTLA4, but retains the ability to inhibit PDL1-PD1 interactions. We are also generating a soluble form of CD8096,97,99 (sCD8096,97,99) that could be administered to cancer patients to prevent tumor cell-mediated PDL1 immune suppression. The construct for the soluble form was generated by ligating the two extracellular domains of CD80 containing the 96,97,99 mutations to the Fc region of human IgG1, followed by incorporation into the Pet21a+ plasmid containing the T7 promoter. Recombinant protein will be isolated from transformed E. coli using a protein G affinity column, and tested in functional experiments with PDL1+ tumor cells to determine if CD80 increases T-cell activation by inhibiting PDL1-PD-1 immune suppression and/or by facilitating costimulation. This work was funded, in part, by grants from the HHMI Precollege and Undergraduate Science Education Program (52006949), CBI (T32 GM066706), and NIH (R01CA84232 and RO1CA115880). Citation Format: Sonia P. Dalal, Samuel Haile, Preethi Somasundaram, Suzanne Ostrand-Rosenberg. Mutated CD80 may facilitate T-cell activation by inhibiting PDL1-PD1 suppression and by costimulating. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1264. doi:10.1158/1538-7445.AM2013-1264

Intracellular Trafficking of an Antibody Bipolar Bridged Complex of HSV‐1 gE‐gI, IgG, and a Viral Antigen

Herpes Simplex Virus 1 (HSV‐1) glycoproteins, gE and gI, mimic a human Fc‐receptor to hinder host immune responses. gE‐gI binds the Fc region of human IgG (hIgG), which can be internalized and released into endocytic organelles. gE‐gI also mediates antibody bipolar bridging (ABB), a process whereby IgG Fabs bind an antigen while its Fc region binds to gE‐gI. The cellular fate of the ABB complex is unknown. Here, we engineered an in vitro system to determine whether ABB complexes are internalized and degraded in host cells. Human cell lines transiently expressing gE‐gI and an HSV‐1 antigen, gD, normally localized at the cell surface of HSV‐1 virions and infected cells, were incubated with two forms of a monoclonal IgG anti‐gD antibody: one with human Fc to allow ABB and one with mouse Fc, a control which binds gD but not gE‐gI and cannot enable ABB. A hIgG against an irrelevant antigen was used as another non ABB control. 4D time‐lapse confocal fluorescence data indicate that gD is internalized in an ABB specific fashion and internalization is dependent upon gE‐gI. Internalized hIgGs bound to a surface viral antigen were targeted to lysosomes and biochemical analysis of enriched lysosomal compartments revealed degraded gD. These results suggest that gE‐gI plays an active role in clearing the infected cell surface of both host IgG and viral antigens, providing HSV‐1 with a mechanism to evade immune responses.

Oral administration of Lactobacillus delbrueckii subspecies bulgaricus OLL1073R-1 suppresses inflammation by decreasing interleukin-6 responses in a murine model of atopic dermatitis

The oral intake of Lactobacillus spp. can provide beneficial effects to the host by modulating the immune response. Atopic dermatitis (AD) is an allergic inflammatory disease mediated by various immune responses. In this study, we examined the effect of a Lactobacillus strain, Lactobacillus delbrueckii ssp. bulgaricus OLL1073R-1 (OLL1073R-1), on AD development in a murine model of AD that was developed by the topical application of mite antigen in NC/Nga mice. The oral intake of heat-killed OLL1073R-1 cells inhibited both the development of dermatitis and the elevation of an acute inflammation marker, serum amyloid A. Another bacterial strain, Lactobacillus rhamnosus OLL2984, exerted no inhibitory effects on dermatitis. The oral intake of heat-killed OLL1073R-1 cells also attenuated secretion of IL-6 from lymph node cells in response to mite antigen and reduced IL-6 levels in inflamed tissues, such as auricles. Production of IFN-γ or IL-4 was not influenced by OLL1073R-1 intake. We also found that inhibition of IL-6 signaling by gp130-Fc (a fusion protein consisting of the extracellular portion of glycoprotein 130 fused to the Fc region of human IgG1) markedly decreased the severity of dermatitis in NC/Nga mice. Moreover, secretion of IL-6 by lymph node cells was augmented in NC/Nga mice compared with that in BALB/c mice. These results indicate that IL-6 plays an essential role in the development of dermatitis in the NC/Nga mouse model of AD, and that OLL1073R-1 inhibits dermatitis, at least in part, by suppressing the IL-6 response.

Expression of osteoprotegerin from a replicating adenovirus inhibits the progression of prostate cancer bone metastases in a murine model

Metastatic involvement of the skeleton is a frequent consequence of advanced prostate cancer. These skeletal metastases cause a number of debilitating complications and are refractory to current treatments. New therapeutic options are being explored, including conditionally replicating adenoviruses (CRAds). CRAds are engineered to selectively replicate in and destroy tumor cells and can be ‘armed’ with exogenous transgenes for enhanced potency. We hypothesized that a CRAd armed with osteoprotegerin (OPG), an inhibitor of osteoclastogenesis, would inhibit the progression of prostate cancer bone metastases by directly lysing tumor cells and by reducing osteoclast activity. Although prostate cancer bone metastases are predominantly osteoblastic in nature, increased osteoclast activity is critical for the growth of these lesions. Ad5-Δ24-sOPG-Fc-RGD is a CRAd that carries a fusion of the ligand-binding domains of OPG and the Fc region of human IgG1 in place of the viral E3B genes. To circumvent low tumor cell expression of the native adenoviral receptor, an arginine–glycine–aspartic acid (RGD) peptide insertion within the viral fiber knob allows infection of cells expressing αv integrins. A 24-base pair deletion (Δ24) within viral E1A limits replication to cells with aberrant retinoblastoma cell cycle regulator/tumor suppressor expression. We have confirmed that Ad5-Δ24-sOPG-Fc-RGD replicates within and destroys prostate cancer cells and, in both murine and human coculture models, that infection of prostate cancer cells inhibits osteoclastogenesis in vitro. In a murine model, progression of advanced prostate cancer bone metastases was inhibited by treatment with Ad5-Δ24-sOPG-Fc-RGD but not by an unarmed control CRAd.

A CD19/Fc fusion protein for detection of anti-CD19 chimeric antigen receptors

BackgroundChimeric Antigen Receptors (CARs) consist of the antigen-recognition portion of a monoclonal antibody fused to an intracellular signaling domain capable of activating T-cells. CARs displayed on the surface of transduced cells perform non-MHC-restricted antigen recognition and activating intracellular signaling pathways for induction of target cytolysis, cytokine secretion and proliferation. Clinical trials are in progress assessing the use of mature T-lymphocytes transduced with CARs targeting CD19 antigen to treat B-lineage malignancies. CD19 is an attractive target for immunotherapy because of its consistent and specific expression in most of the stages of maturation and malignancies of B-lymphocyte origin, but not on hematopoietic stem cells. Antibodies against the extracellular domain of the CAR molecule (anti-Fab, Fc or idiotype) have been used for detection of CAR expression in research and clinical samples by flow cytometry, but may need development for each construct and present significant background in samples from xenograft models.MethodsA specific reagent for the detection of anti-CD19 CAR expression was developed, a fusion protein consisting of human CD19 extracellular domains and the Fc region of human IgG1 (CD19sIg). Genes encoding CD19sIg fusion proteins were constructed by fusing either exons 1 to 3 (CD19sIg1-3) or exons 1 to 4 (CD19sIg1-4) of the human CD19 cDNA to a human IgG1Fc fragment. These fusion proteins are intended to work in similar fashion as the MHC Tetramers used for identification of antigen-specific T-cells, and may also have other applications in studies of activation of anti-CD19 CAR bearing cells. The CD19sIg proteins were produced from 293 T cells by stable lentiviral vector transduction and purification from culture medium.ResultsELISA assays using several different monoclonal antibodies to CD19 demonstrated dose-related specific binding by the fusion molecule CD19sIg1-4, but no binding by CD19sIg1-3. Conjugation of the CD19sIg1-4 fusion protein to Alexa Fluor 488 allowed specific and sensitive staining of anti-CD19 CAR-bearing cells for flow cytometry assays, detecting as low as 0.5% of CAR-modified primary cells with minimal background staining.ConclusionsThis fusion molecule is a sensitive reagent for detection of anti-CD19 CAR derived from any monoclonal antibody present in CAR-modified T-cells.

Production of single-chain Fv–Fc fusion protein in stably transformed insect cells

We describe the secretory production of a mouse anti-bovine ribonuclease A single-chain variable fragment (scFv) fused with the Fc region of human IgG1 in stably transformed lepidopteran insect cells. Use of the insect-derived Bip and melittin signal peptides resulted in higher yields of the secreted scFv–Fc fusion protein than use of the baculovirus gp64 signal peptide. After cotransfection with an expression vector that contains the Bip signal sequence upstream of the DNA encoding the scFv–Fc and a selection vector that carries a neomycin resistance gene, Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were cultured in the presence of G418. Colonies of resistant cells were obtained around 2 weeks after adding G418, and clonal cells were screened by enzyme-linked immunosorbent assay (ELISA) of the culture supernatant. The yield of the scFv–Fc protein secreted from the most productive clone was around 60mg/l in a shake-flask culture. To improve the productivity, we investigated the effect of medium supplements of sodium butyrate (NaBu), dimethyl sulfoxide (DMSO), and sericin. Supplementing culture medium with sericin increased the scFv–Fc protein yield to 82mg/l, but productivity was not increased by either NaBu or DMSO. These results indicate that the stably transformed insect cells may allow for the efficient production of scFv–Fc and other Fc fusion proteins.

The deficient cleavage of M protein-bound IgG by IdeS: Insight into the escape of Streptococcus pyogenes from antibody-mediated immunity

IdeS (IgG-degrading enzyme of Streptococcus pyogenes) is a virulence factor for S. pyogenes, group A Streptococcus (GAS). IdeS is believed to allow GAS to evade antibody-mediated phagocytosis by cleaving IgG at the lower hinge region. Human immunoglobulins bind to the GAS surface by two mechanisms: Specific antibodies attach at the Fab region to their specific antigens on the bacterial surface. Immunoglobulins can also attach nonspecifically at the Fc region to streptococcal M and M-like proteins. This phenomenon is believed to form the host-like coat and to block the recognition of Fc region by Fc receptor on phagocytes and antibody-dependent cell-mediated cytotoxicity. It is not known whether IdeS preferentially cleaves IgG attached at the Fab or Fc regions. To explore this issue, we used Sepharose beads coated with protein A or L or M protein as surrogate markers for specific (Fab) and nonspecific (Fc) binding sites. We found that IdeS cleaved Fab-bound IgG as rapidly as soluble IgG. In contrast, Fc-bound IgG was cleaved about 4 fold less than soluble IgG. In a competitive binding assay, we found that M protein had a greater affinity than IdeS to attach to the Fc region of human IgG. Thus, IdeS exhibited preferential IgG endopeptidase activity for Fab-bound IgG while allowing the non-specific binding of IgG to remain attached to M protein. We propose that this preferential enzymatic activity accounts for the ability of GAS to resist immunoglobulin-mediated phagocytosis and cytotoxicity.

Gγ recruitment system incorporating a novel signal amplification circuit to screen transient protein‐protein interactions

Weak and transient protein-protein interactions are associated with biological processes, but many are still undefined because of the difficulties in their identification. Here, we describe a redesigned method to screen transient protein-protein interactions by using a novel signal amplification circuit, which is incorporated into yeast to artificially magnify the signal responding to the interactions. This refined method is based on the previously established Gγ recruitment system, which utilizes yeast G-protein signaling and mating growth selection to screen interacting protein pairs. In the current study, to test the capability of our method, we chose mutants of the Z-domain derived from Staphylococcus aureus protein A as candidate proteins, and the Fc region of human IgG as the counterpart. By introduction of an artificial signal amplifier into the previous Gγ recruitment system, the signal transduction responding to transient interactions between Z-domain mutants and the Fc region with significantly low affinity (8.0 × 10(3) M(-1)) was successfully amplified in recombinant haploid yeast cells. As a result of zygosis with the opposite mating type of wild-type haploid cells, diploid colonies were vigorously and selectively generated on the screening plates, whereas our previous system rarely produced positive colonies. This new approach will be useful for exploring the numerous transient interactions that remain undefined because of the lack of powerful screening tools for their identification.

Antibodies to the Envelope Glycoprotein of Human T Cell Leukemia Virus Type 1 Robustly Activate Cell-Mediated Cytotoxic Responses and Directly Neutralize Viral Infectivity at Multiple Steps of the Entry Process

Infection of human cells by human T cell leukemia virus type 1 (HTLV-1) is mediated by the viral envelope glycoproteins. The gp46 surface glycoprotein binds to cell surface receptors, including heparan sulfate proteoglycans, neuropilin 1, and glucose transporter 1, allowing the transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. The envelope glycoproteins are recognized by neutralizing Abs and CTL following a protective immune response, and therefore, represent attractive components for a HTLV-1 vaccine. To begin to explore the immunological properties of potential envelope-based subunit vaccine candidates, we have used a soluble recombinant surface glycoprotein (gp46, SU) fused to the Fc region of human IgG (sRgp46-Fc) as an immunogen to vaccinate mice. The recombinant SU protein is highly immunogenic and induces high titer Ab responses, facilitating selection of hybridomas that secrete mAbs targeting SU. Many of these mAbs recognize envelope displayed on the surface of HTLV-1-infected cells and virions and several of the mAbs robustly antagonize envelope-mediated membrane fusion and neutralize pseudovirus infectivity. The most potently neutralizing mAbs recognize the N-terminal receptor-binding domain of SU, though there is considerable variation in neutralizing proficiency of the receptor-binding domain-targeted mAbs. By contrast, Abs targeting the C-terminal domain of SU tend to lack robust neutralizing activity. Importantly, we find that both neutralizing and poorly neutralizing Abs strongly stimulate neutrophil-mediated cytotoxic responses to HTLV-1-infected cells. Our data demonstrate that recombinant forms of SU possess immunological features that are of significant utility to subunit vaccine design.

Robust neutralising activity and activation of neutrophil cytotoxic responses mediated by antibodies targeting the HTLV-1 envelope glycoprotein

Infection of human cells by Human T-cell leukaemia virus (HTLV-1) is mediated by the viral envelope glycoproteins. The gp46 surface glycoprotein binds to a cell surface receptors, including heparan sulphate proteoglycans, neuropilin-1, and Glut-1, allowing the transmembrane glycoprotein to initiate fusion of the viral and cellular membranes. The envelope glycoproteins are recognised by neutralising antibodies and cytotoxic T lymphocytes following a protective immune response, and therefore represent attractive components for an HTLV-1 vaccine. Here we begin to explore the immunological properties of recombinant SU as a potential subunit vaccine candidate. We have used a soluble recombinant gp46 fused to the Fc-region of human IgG (sRgp46-Fc) as an immunogen to vaccinate mice and generate monoclonal antibodies (mAbs) targeting SU. The recombinant SU protein is highly immunogenic, inducing high titre antibody responses to the immunogen and facilitating selection of hybridomas that secrete anti-SU mAbs. Many of these mAbs recognise envelope expressed by HTLV-1 infected cells and these antibodies strongly stimulate neutrophil-mediated cytotoxic responses. Moreover, several of the mAbs directly and robustly antagonise envelope-mediated fusion and neutralize pseudovirus infectivity. While antibodies targeting either the receptor-binding domain or C-terminal domain of SU exhibit anti-viral activity, the most potently neutralizing mAbs tend to recognise epitopes that cluster in the N-terminal receptor-binding domain of SU. Thus, our data demonstrate that recombinant forms of SU possess immunological features that are of direct utility to subunit vaccine design.

TNF receptor II fusion protein with tandemly repeated Fc domains

The extracellular domain of tumour necrosis factor (TNF) receptor II fused with the human IgG1 Fc region (TNFRII-Fc), as well as antibodies against TNF, has been used to treat rheumatoid arthritis. However, TNFRII-Fc is less effective than these antibodies in terms of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) against cells bearing TNF on the cell surface. We hypothesized that these activities could be increased by fusing TNFRII with tandemly repeated Fc (TNFRII-Fc-Fc). The affinities of TNFRII-Fc-Fc for soluble TNF-α and transmembrane TNF-α and the TNF-α cytotoxicity-inhibitory activity were as potent as those of TNFRII-Fc. TNFRII-Fc-Fc showed much higher binding avidity for Fcγ receptors than TNFRII-Fc and was more potent in terms of both ADCC and CDC against cells expressing transmembrane TNF-α. TNFRII-Fc-Fc of 80 kDa, as well as TNFRII-Fc-Fc of 200 kDa, was detected. TNFRII-Fc-Fc (80 kDa) was as potent as TNFRII-Fc in terms of both ADCC and CDC. These results suggest that Fc multimerization of receptor-Fc fusion proteins can augment effector functions such as ADCC and CDC, and thereby have the potential to provide a superior therapeutic effect. This may be the case not only for TNFRII-Fc but also for other receptor-Fc fusion proteins.

RAP-536 Accelerates Recovery From Chemotherapy-Induced Anemia (CIA) without Promoting Tumor Growth In Murine Models of Cancer and CIA

AbstractAbstract 4230We have previously shown that administration of ACE-536, a soluble form of a modified activin type IIB receptor, is an effective therapy in both acute and chronic animal models of anemia. We have further demonstrated that ACE-536 induces red cell development by promoting late stage erythroblast maturation.Anemia is a debilitating complication associated with several chronic diseases, including cancer, and is exacerbated by the use of cancer chemotherapy. Recombinant human erythropoietin (EPO) is the most commonly prescribed treatment for chemotherapy induced anemia. However, there is increasing evidence to suggest that the administration of EPO may accelerate tumor growth by promoting angiogenesis. Therefore, non-EPO based strategies to correct chemotherapy induced anemia without effects on underlying tumor are needed. Several members of the TGF-β superfamily have been shown to be involved in red blood cell production. We have developed ACE-536 as a modified form of the extracellular region of the activin type IIB receptor fused to the Fc region of human IgG1, which binds to and inhibits several ligands in the TGF-β superfamily. Here we report on the effects of RAP-536, a murine analog of ACE-536 containing the modified extracellular region of the activin type IIB receptor fused to the Fc region of a murine IgG, in rodent models of chemotherapy induced anemia and tumor growth.To evaluate the efficacy of RAP-536 in a rodent model of chemotherapy induced anemia 300 g Sprague-Dawley rats were administered a single intravenous dose of carboplatin (75 mg/kg) or phosphate-buffered saline (PBS). Blood samples were taken twice weekly to assess the induction of carboplatin induced anemia. By Day 11 rats treated with carboplatin showed an average drop of 34%, 32% and 36% in red blood cells, hemoglobin and hematocrit, respectively, and were randomized to receive RAP-536 twice weekly at a dose of 10 mg/kg or an equal volume of tris-buffered (TBS). Blood samples were taken twice weekly to evaluate recovery from anemia. Rats treated with RAP-536 exhibited a more rapid rate of recovery (ANOVA; P < 0.001) than those treated with TBS. Moreover, at study termination (Day 35), red blood cells, hemoglobin and hematocrit levels of RAP-536 treated rats were 28%, 23% and 23% greater, respectively, than TBS-treated controls (P < 0.01).EPO administration has previously been shown to accelerate the rate of tumor growth in a mouse tumor bearing model; therefore, we sought to investigate the effect of RAP-536 in this context. Eight-week old mice were injected subcutaneously in the right hind flank with 3×105 Lewis lung carcinoma cells expressing a luciferase reporter. Mice were randomized to receive weekly subcutaneous injections of TBS, RAP-536 (10 mg/kg) or EPO (200 U/kg). Tumor growth was assessed twice weekly by manual external caliper measurements and once weekly by luciferase activity using an IVIS in vivo imaging system. Tumors in EPO treated mice grew at a moderately accelerated rate compared to TBS and RAP-536 treated mice; however, this was not statistically significant. At study termination tumors were excised and weighed. Mice treated with EPO showed statistically significant larger tumors compared to TBS treated animals (P = 0.0126). On the other hand, mice treated with RAP-536 did not show statistically significant increases in tumor size (P = 0.1689).In conclusion, targeting the TGF-β signaling pathway with RAP-536 is a viable therapeutic approach for the treatment of anemia induced by chemotherapy treatment and may represent a safer alternative to EPO-based therapies. Disclosures:Cadena:Acceleron Pharma: Employment. Suragani:Acceleron Pharma: Employment. Mitchell:Acceleron Pharma: Employment. Ucran:Acceleron Pharma: Employment. Liharska:Acceleron Pharma: Employment. Sako:Acceleron Pharma: Employment. Monnell:Acceleron Pharma: Employment. Underwood:Acceleron Pharma: Employment. Kumar:Acceleron Pharma: Employment. Pearsall:Acceleron Pharma: Employment.

CD19 Fc-Fusion Protein for Detection of Cells Expressing Anti-CD19 Chimeric Antigen Receptors.

AbstractAbstract 3756Chimeric Antigen Receptors (CARs) consist of the antigen-recognition portion of a monoclonal antibody fused to an intracellular signaling domain that is capable of activating T cells. CARs are displayed on the surface of transduced cells, performing non-MHC-restricted antigen recognition and activating intracellular signaling pathways for induction of target cytolysis, cytokine secretion and proliferation. Clinical trials are in progress assessing the use of modified mature T lymphocytes transduced with CARs targeting CD19 antigen to treat B-lineage malignancies. CD19 is an attractive target for immunotherapy because of its consistent and specific expression in most of the stages of maturation and malignancies of B lymphocyte origin, but not on hematopoietic stem cells. Antibodies against the extracellular domain of the CAR molecule (anti-Fab, Fc or idiotype) have been used for detection of CAR expression in research and clinical samples by flow cytometry, but may need development for each construct and present significant background in samples from xenograft models. We developed a specific reagent for the detection of anti-CD19 CAR expressed on cells, a soluble fusion protein consisting of human CD19 extracellular domains and the Fc region of human IgG1 (19sIg). Genes encoding 19sIg fusion proteins were constructed by fusing either Exons 1 to 3 (19sIg1-3) or Exons 1 to 4 (19sIg1-4) of the human CD19 cDNA to a human IgG1Fc fragment. These fusion proteins are intended to work in similar fashion as the MHC Tetramers used for identification of antigen-specific T cells, and may also have other applications in studies of activation of anti-CD19 CAR bearing cells. The 19sIg proteins were produced from 293T cells by stable lentiviral vector transduction and purification from culture medium. ELISA assays using several different monoclonal antibodies to CD19 demonstrated dose-related specific binding by the fusion molecule 19sIg1-4, but no binding to 19sIg1-3. Conjugation of the 19sIg1-4 fusion protein to Alexa Fluor 488 allowed specific and sensitive staining of anti-CD19 CAR-bearing cells for flow cytometry assays. Large-scale production and stable conjugation of this fusion molecule will provide a useful reagent to detect anti-CD19 CAR derived from any monoclonal antibody. Disclosures:No relevant conflicts of interest to declare.

Examination of the Relative Rates of Rituximab-Mediated Loss of CD20 From B Cells Via Trogocytosis Versus Internalization. Implications for Rituximab Therapy of CLL

AbstractAbstract 2404The use of the anti-CD20 monoclonal antibody (mAb) rituximab (RTX) in the immunotherapy of cancer has led to substantial advances in the treatment of B cell malignancies. However, many patients are refractory to RTX therapy and a substantial fraction of responding patients suffer relapses. In some patients the loss or lack of efficacy can be explained because levels of CD20 are substantially reduced on target B cells as a consequence of RTX therapy. This phenomenon may be due to growth and proliferation, over a period of at least several months, of a CD20-negative B cell clone. In contrast, we have reported that levels of CD20 expressed on circulating CLL cells can be reduced > 90% in less than one hour following RTX infusion. These clinical observations, as well as our results seen in vitro and in mouse models, suggest that after the body's effector mechanisms that promote mAb-mediated cell killing are exhausted or saturated, an alternative reaction predominates. RTX-CD20 complexes on targeted B cells are rapidly removed from the B cells by monocytes and macrophages through an Fc receptor-mediated endocytic process known as trogocytosis, or the shaving reaction. Beers et al. recently reported (Blood, 2010) that human CD20 transgenic mouse B cells opsonized with Rit m2a, a mouse IgG2a mAb with RTX activity, internalized both bound mAb and up to half of cell-surface CD20 after incubation for 2 hrs at 37C. They also reported that on average 40% of cell-surface CD20 was internalized when CLL cells opsonized with RTX were similarly treated. It would thus appear that as a consequence of RTX therapy, both trogocytosis by effector cells and direct internalization by B cells can promote loss of CD20 from B cells targeted by RTX. Both of these reactions will reduce the efficacy of RTX therapy, and a key question must focus on the relative rates of trogocytosis and internalization in promoting CD20 loss in vivo.To address these questions we examined human CD20 transgenic mouse 38C13 B cells (a kind gift from Dr. Josee Golay) for RTX-mediated CD20 internalization versus shaving. To test for internalization of cell-bound RTX, CD20+ 38C13 cells were reacted with Al488- or biotinylated RTX for 10 min to 2 hours at 37C, then washed and secondarily probed with the following reagents: 1, Al647 mAb HB43, specific for the Fc region of human IgG; 2, Al488-streptavidin to detect surface-bound biotinylated RTX; 3, Anti-Al488 antibodies that induce quenching of surface-bound Al488 RTX; or 4, The cells were subjected to an acid wash, re-equilibrated at neutral pH, and re-tested for Al488 RTX binding. Our flow cytometry results reveal rapid and stable binding of Al488-labeled or biotinylated RTX to the CD20+ 38C13 cells. After two hours at 37C, the amount of cell-bound RTX that had been internalized, defined by the 4 different assays, was much less than that reported by Beers, and ranged between 5 and 25%, suggesting that the degree of internalization may be model-dependent. Moreover, reaction of acceptor THP-1 cells (a monocytic line) with the RTX-opsonized cells leads to rapid shaving and removal of ~ 90% of bound RTX and CD20 from the 38C13 cells. After just 45 min at 37C in the presence of adhered THP-1 monocytes, there is a 10-fold reduction in fluorescence intensity signal (molecules of equivalent soluble fluorochrome) on the 38C13 cells attributable to either bound Al488 RTX or due to secondary development with Al647 anti-human IgG mAb HB43. Alternatively, Al488 RTX-opsonized CD20+ 38C13 cells were reacted in solution with THP-1 monocytes at varying THP-1/38C13 cell ratios. Within 45 min at 37C at a 3/1 ratio more than half of cell bound Al488 RTX and CD20 were removed from the 38C13 cells, and large amounts of Al488 RTX were found on the acceptor THP-1 cells. Our results indicate that the shaving reaction is considerably more rapid and indeed leads to the removal of much more bound anti-CD20 mAb RTX (and CD20) than the internalization reaction, suggesting that the shaving reaction is most likely primarily responsible for the rapid down-modulation of CD20 that is observed when CLL patients are treated with RTX. Disclosures:No relevant conflicts of interest to declare.

GlycoVariant Anti-CD37 Small Modular Immuno-Pharmaceutical Exhibits Superior Natural Killer Cell Mediated Cytotoxicity Against Chronic Lymphocytic Leukemia Cells at Low Concentrations and Low Antigen Density

AbstractAbstract 1847CD37 is a tetraspanin transmembrane family protein that is strongly expressed on the surface of mature human B cells and transformed mature B cell lymphoma and leukemia cells, including CLL cells. It is absent or minimally expressed on normal T cells, NK cells, monocytes, and granulocytes. Predominant expression of CD37 on CLL cells makes it an ideal candidate to target with potential agents for treatment of CLL. TRU-016, a small modular immunopharmaceutical protein (SMIP) that specifically binds to an extracellular region of CD37, is presently in clinical trials in CLL patients. TRU-016 includes humanized immunoglobulin variable regions (scFv) fused to a human IgG1 Fc region. We have previously reported that SMIP-016, the chimeric version of the humanized TRU-016, induced apoptosis in CLL B cells in the presence of goat anti-human Fc antibody cross-linker through a novel, caspase-independent pathway. Furthermore, SMIP-016 showed potent in vivo activity in a SCID xenograft mouse model. Aside from direct cytotoxicity, SMIP-016 mediates antibody-dependent cellular cytotoxicity (ADCC) by NK cells both in vitro and in vivo. In an attempt to enhance its ADCC function, a new variant of SMIP-016, SMIP-016GV, was designed with a modification of the glycosylation of the Fc portion of the molecule. SMIP-016GV exhibits enhanced binding to both low- and high-affinity molecular variants of human CD16 (FcγRIII) and augments ADCC potency when compared to SMIP-016. In this study, we compared SMIP-016GV and SMIP-016 in direct cytotoxicity and ADCC against CLL B cells. While SMIP-016 and SMIP-016GV mediated comparable direct cytotoxicity at 48 hrs in the presence of goat anti-human Fc crosslinker, the SMIP-016GV resulted in 2 to 4 fold increase in NK cell mediated ADCC function at all effector to target ratios tested. This increased ADCC with SMIP-016GV was observed using NK cell effectors derived from both normal as well as CLL-affected individuals. In addition, this enhanced cytotoxicity was sustained at concentrations of SMIP-016GV as low at 5E-6 μg/ml. These low concentrations of SMIP-016GV were also able to mediate superior ADCC in 697 cells expressing as few as 10,000 molecules of surface CD37 antigen. Furthermore, NK cells stimulated with the glycovariant were potently activated and released 3 to 4 fold more IFNγ compared to SMIP-016. Ongoing studies are aimed at defining other effector cells which may interact with SMIP-016GV via different Fcγ Receptors. Collectively, these results suggest potential use of the SMIP-016GV with enhanced ADCC function as an alternate for TRU-016 in B cell malignancies including CLL therapy. Disclosures:Siadak:Trubion Pharmaceuticals: Employment.

Cytoplasmic production of soluble and functional single-chain Fv-Fc fusion protein in Escherichia coli

We describe the soluble production of a mouse anti-bovine ribonuclease A single-chain variable fragment (scFv) fused with the Fc region of human IgG1 in Escherichia coli. Two production systems, secretory production using a pelB signal peptide and cytoplasmic production in a trxB/gor double mutant strain with an oxidizing cytoplasm, were investigated for efficient production of soluble and functional scFv-Fc fusion protein. Antigen-binding activity was observed in both systems but almost all of the scFv-Fc that was expressed formed insoluble aggregates. Hence, the co-expression of molecular chaperones was examined. Co-expression of GroEL/GroES showed a 4.6-fold increase in antigen-binding activity in the cytoplasmic production system but not in the secretory system. By contrast, the other two chaperones, DnaK/DnaJ/GrpE and trigger factor, had no effect in either production system. The protein solubility was also improved markedly by the co-expression of GroEL/GroES and approximately 70% of the 3A21 scFv-Fc protein was soluble. A practical productivity of more than 10mg/L was achieved with a simple batch shake-flask culture. These results indicate that the E. coli cytoplasmic production system with oxidizing cytoplasm and molecular chaperones might be one of the choices for the soluble production of scFv-Fcs and other Fc fusion proteins.