Antibody 2F5 Research Articles

Antibody-Mediated Delivery of VEGFC Ameliorates Experimental Chronic Colitis.

Crohn's disease (CD) and ulcerative colitis (UC) are two distinct forms of inflammatory bowel disease (IBD) characterized by an expanded lymphatic network with impaired functionality both in mouse models and in human patients. In this study, we investigated whether targeted delivery of the pro-lymphangiogenic vascular endothelial growth factor C (VEGFC) to the site of inflammation may represent a new, clinically feasible strategy for treating IBD. To achieve targeting of inflamed tissue, we developed a fusion protein consisting of human VEGFC fused to the F8 antibody (F8-VEGFC), which specifically binds to the extradomain A (EDA) of fibronectin, a spliced isoform almost exclusively expressed in inflamed tissues. The therapeutic activity of intravenously administered F8-VEGFC, compared to a targeted construct lacking VEGFC (F8-SIP), was investigated in a mouse model of dextran sodium sulfate (DSS)-induced colitis. The presence of EDA fibronectin was detected in both human and mouse inflamed colon tissue. Biodistribution studies of radiolabeled F8-VEGFC revealed a specific accumulation of the antibody in the colon of DSS-administered mice, as compared to an untargeted VEGFC fusion protein (KSF-VEGFC) (binding the irrelevant hen egg lysozyme antigen). Systemic treatment with F8-VEGFC significantly reduced the clinical and histological signs of inflammation, expanded the lymphatic vascular network, reduced the density of immune cells, and also decreased the expression of inflammatory cytokines in the inflamed colon. Overall, these results reveal that administration of F8-VEGFC represents a novel and promising approach for the treatment of IBD.

Enhanced Binding Efficiency of Microcantilever Biosensor for the Detection of Yersinia

A novel microcantilever sensor was batch fabricated for Yersinia detection. The microcantilever surface modification method was optimized by introducing a secondary antibody to increase the number of binding sites. A novel microfluidic platform was designed and fabricated successfully. A 30 μL solution could fully react with the microcantilever surface. Those routines enhanced the binding efficiency between the target and receptor on the microcantilever. With this novel designed microfluidic platform, the specific adsorption of 107 Yersinia on the beam surface with modified F1 antibody was significantly enhanced.

Design and characterisation of a novel interleukin-15 receptor alpha fusion protein and analysis of interleukin-15 complexation.

Interleukin-15 (IL15) is one of the most important cytokines currently being considered for cancer therapy applications. It is thought that by administering IL15 in complex with its cognate receptor alpha chain (IL15Rα) its biological activity could be increased manifold. We produced a fusion protein of mouse IL15Rα and the F8 antibody, that targets the alternatively-spliced extra-domain A (EDA) of fibronectin, which is overexpressed in many types of cancer. The fusion protein F8IL15Rα was cloned, expressed and characterized in vitro and its ability to bind to mouse IL15 was assessed with both size exclusion chromatography (SEC) and surface plasmon resonance (SPR) experiments. Furthermore, mouse and human IL15 and their corresponding Fc fused IL15Rα subunits were purchased, characterized and used to compare the capacity of F8IL15Rα to generate complexes. Surprisingly, none of the IL15Rα fusion proteins showed IL15 complexation on SEC. However, on SPR, F8IL15Rα displayed the ability to bind IL15. In a cell-based activity assay none of the IL15Rα fusions were able to increase cellular proliferation in combination with IL15 compared to IL15 alone. A better understanding of the molecular requirements for effective IL15 signalling are likely to be important for the development of IL15-based biopharmaceuticals.

Characteristics of Artificial Immunogens Containing Peptide Mimotopes of HIV-1 Epitopes Recognized by Monoclonal Antibodies 2F5 and 2G12

The paper describes construction of TBI-based recombinant proteins TBI-2F5 and TBI-2G12 that contain peptide mimotopes of HIV-1 epitopes recognized by broadly neutralizing antibodies 2F5 and 2G12, respectively. The capacity of the immunogens to induce neutralizing antibodies was evaluated. The sera of BALB/c mice immunized with recombinant proteins TBI, TBI-2F5, and TBI-2G12 neutralized HIV-1 env-pseudoviruses. Moreover, pooled serum from mice immunized with TBI-2F5 and TBI-2G12 neutralized env-pseudoviruses of HIV-1 subtype B more effectively than individual sera.

Analysis of the Glycosylation Profile of Disease-Associated Water-Soluble Prion Protein Using Lectins.

The disease-associated water-soluble form of hamster prion protein (ws-PrPSc) has recently been found to be less stable than classical PrPSc. Since the stability of PrP to degradation correlates with its glycosylation level, the aim of this study was to investigate whether there are differences between the glycosylation of ws-PrPSc and classical PrPSc of hamster which might account for the ws-PrPSc minor stability compared with that of the classical PrPSc. Thus, ws-PrP and classical PrP were captured from noninfected or scrapie-infected hamster brain homogenate [high-speed supernatant (SHS) and high-speed pellet (PHS)] and blood plasma by anti-PrP antibodies (3F4 and 6H4) and subjected to screening for glycans by lectins under denaturing or nondenaturing procedures in a sandwich lectin-ELISA. Glycans have been found in minor quantities and differently exposed on ws-PrPSc from SHS and plasma compared with classical PrPSc from PHS. These differences have been shown to be potentially responsible for the instability of ws-PrPSc. Treatment of infected blood with GdnHCl significantly (P<0.01) increased the detection of ws-PrPSc in ELISA, reflecting an increase in its stability, and showed efficacy in removing high-abundance proteins in silver-stained gels. This increase in ws-PrPSc stability is due to an interaction of GdnHCl not only with high-abundance proteins but also with the ws-PrPSc glycosylation with particular regard to the mannose sugar. Analysis of lectins immunoreactivity toward total proteins from plasma collected before and at different time points after infection revealed that mannose might exert a stabilizing effect toward all of hamster blood glycoproteins, regardless of scrapie infection. Since low levels of ws-PrPSc/soluble-infectivity have been estimated both in blood and brain of hamster, this glycosylation-related instability may have negatively influenced the propensity of ws-PrPC to convert to ws-PrPSc both in blood and the brain. Therefore, PrPC glycosylation characteristics may provide a tool for the determination risk of prion transmissibility.

Immunotherapy of triple-negative breast cancer with cathepsin D-targeting antibodies

BackgroundTriple-negative breast cancer (TNBC) treatment is currently restricted to chemotherapy. Hence, tumor-specific molecular targets and/or alternative therapeutic strategies for TNBC are urgently needed. Immunotherapy is emerging as an exciting treatment option for TNBC patients. The aspartic protease cathepsin D (cath-D), a marker of poor prognosis in breast cancer (BC), is overproduced and hypersecreted by human BC cells. This study explores whether cath-D is a tumor cell-associated extracellular biomarker and a potent target for antibody-based therapy in TNBC.MethodsCath-D prognostic value and localization was evaluated by transcriptomics, proteomics and immunohistochemistry in TNBC. First-in-class anti-cath-D human scFv fragments binding to both human and mouse cath-D were generated using phage display and cloned in the human IgG1 λ format (F1 and E2). Anti-cath-D antibody biodistribution, antitumor efficacy and in vivo underlying mechanisms were investigated in TNBC MDA-MB-231 tumor xenografts in nude mice. Antitumor effect was further assessed in TNBC patient-derived xenografts (PDXs).ResultsHigh CTSD mRNA levels correlated with shorter recurrence-free survival in TNBC, and extracellular cath-D was detected in the tumor microenvironment, but not in matched normal breast stroma. Anti-cath-D F1 and E2 antibodies accumulated in TNBC MDA-MB-231 tumor xenografts, inhibited tumor growth and improved mice survival without apparent toxicity. The Fc function of F1, the best antibody candidate, was essential for maximal tumor inhibition in the MDA-MB-231 model. Mechanistically, F1 antitumor response was triggered through natural killer cell activation via IL-15 upregulation, associated with granzyme B and perforin production, and the release of antitumor IFNγ cytokine. The F1 antibody also prevented the tumor recruitment of immunosuppressive tumor-associated macrophages M2 and myeloid-derived suppressor cells, a specific effect associated with a less immunosuppressive tumor microenvironment highlighted by TGFβ decrease. Finally, the antibody F1 inhibited tumor growth of two TNBC PDXs, isolated from patients resistant or not to neo-adjuvant chemotherapy.ConclusionCath-D is a tumor-specific extracellular target in TNBC suitable for antibody-based therapy. Immunomodulatory antibody-based strategy against cath-D is a promising immunotherapy to treat patients with TNBC.

Towards an HIV Vaccine Based on Immune Network Theory II

We have designed immunization protocols based on the co-section model to induce a broadening antigen antibody response against HIV1. As antigen were various antigen-antibody complexes used, including the monoclonal antibody 1F7 that is expressed on anti-HIV1 antibodies with different antigen specificities. Rabbits were used to test this concept using. Antibodies that neutralized HIV-1 of the same strain as that of the gp120 used were detected, but they did not include broadly neutralizing antibodies (BnAbs). Immunization with gp120 in adjuvant induced antibodies binding to gp120, gp41, Nef and p24, a finding predicted by the immune network theory. Furthermore a complex of 1F7 and B12 induced antibodies against gp120 and gp41, demonstrating an antigen broadening response towards achieving the goal of inducing BnAbs through immunization.

Targeted delivery of calreticulin to ED-A fibronectin leads to tumor-growth retardation

We report the design and characterization of novel fusion proteins, consisting of the F8 antibody and of murine calreticulin (Calr). The F8 antibody recognizes the alternatively-spliced ED-A domain of fibronectin, an extracellular matrix component found in most tumor types, while calreticulin has previously been described as an “eat-me” signal for dendritic cells and phagocytes. Four fusion proteins, differing in antibody formats and peptide linkers, were produced in mammalian cells, purified to homogeneity and tested in vitro and in vivo. A quantitative biodistribution in F9 tumor-bearing mice revealed that the homobivalent F8-F8-Calr format, featuring a tandem diabody structure, had the best tumor-homing properties and, for this reason, this protein was studied in therapy experiments in CT26 tumor-bearing mice. Intravenous administration of F8-F8-Calr led to a tumor growth retardation, which could be further improved by combination with anti-PD1 antibody treatment. Immunohistochemical analysis revealed an increased density of CD8+ T cells, CD11c+ dendritic cells and F4/80+ macrophages in tumor tissue. Even though F8-F8-Calr did not lead to cancer cures at the doses tested, the excellent tolerability profile and the ability to favor a leukocyte infiltration into the neoplastic mass suggests that the targeted delivery of calreticulin may be considered for combination therapy approaches.

Guiding the humoral response against HIV-1 toward a MPER adjacent region by immunization with a VLP-formulated antibody-selected envelope variant.

Preventive HIV-1 vaccine strategies rely on the elicitation of broadly neutralizing antibody (bNAb) responses, but their induction in vivo by vaccination remains challenging. Considering that the ability of an epitope to elicit effective humoral immunity depends on its exposure on the virion, we have used a reverse genetics approach to select variants from an HIV-1 AC10_29 randomly mutated envelope library that showed increased affinity for a selected bNAb (4E10 bNAb targeting the HIV-1 MPER region). Isolated envelope sequences were analyzed by deep-sequencing showing a small number of dominant changes, including the loss of four potential N-linked glycosylation sites and disruption of the V1/V2 loop. Accordingly, the dominant variant (LR1-C1), showed not only increased affinity for MPER bNAbs 4E10 and 2F5, but also higher affinity for an additional antibody targeting the V3 loop (447-52D) that could be a consequence of an open conformation tier 1-like Env. Furthermore, the amino acids specific for the selected variant are associated with an increased sensitivity for 4E10 and 2F5 antibodies. In vivo studies showed that sera from mice immunized with LR1-C1 viruses possessed an improved neutralizing activity compared to the wild-type AC10_29 env. While Virus Like Particles (VLPs) carrying this envelope were unable to induce detectable neutralizing activity in immunized rabbits, one animal showed antibody response to the 4E10-proximal region. Our data establish a novel approach that has the potential to yield HIV envelope immunogen sequences that direct antibody responses to specific envelope regions.

Antibody-mediated delivery of VEGF-C potently reduces chronic skin inflammation.

VEGF-C is an important mediator of lymphangiogenesis and has been shown to alleviate chronic inflammation in a variety of disease models. In this study, we investigated whether targeted delivery of VEGF-C to sites of inflammation and site-specific activation of lymphatic vessels would represent a clinically feasible strategy for treating chronic skin inflammation. To this end, we generated a fusion protein consisting of human VEGF-C fused to the F8 antibody (F8-VEGF-C), which is specific for the alternatively spliced, angiogenesis-marking extradomain A (EDA) of fibronectin. In two mouse models of psoriasis-like skin inflammation, mediated by transgenic VEGF-A overexpression or repeated application of imiquimod, intravenous treatment with F8-VEGF-C but not with untargeted VEGF-C significantly reduced ear skin edema and was as effective as the clinically used TNF-α receptor-Fc fusion protein (TNFR-Fc). Treatment with F8-VEGF-C led to a marked expansion of lymphatic vessels in the inflamed skin and significantly improved lymphatic drainage function. At the same time, treatment with F8-VEGF-C significantly reduced leukocyte numbers, including CD4+ and γδ T cells. In sum, our results reveal that targeted delivery of VEGF-C and site-specific induction of lymphatic vessels represent a potentially new and promising approach for the treatment of chronic inflammatory diseases.

Personalizing Amyloidosis Therapy with Real Time PET Imaging of Fibril-Reactive Monoclonal Antibody Cael-101

Background AL amyloidosis is the most common type of systemic amyloidosis in western countries and has a poor prognosis, with a median survival of 12 to 18 months. Despite the improved prognosis gained by eliminating the offending plasma cell clone, mortality remains high due to multi-organ dysfunction caused by persistent, insoluble amyloid fibril deposits. The amyloid fibril-reactive murine monoclonal antibody 11-1F4 was designed to target amyloid deposits by directly binding to a conformational epitope present on human light-chain amyloid fibrils. The murine form of this antibody has demonstrated potential to bind amyloid in mice and humans (Blood. 2010 116: 2241) and to clear insoluble fibrils in mice with induced human AL amyloidomas, demonstrating the feasibility of using immunotherapy to elicit rapid destruction of amyloid fibrils. Of great translational importance, a chimeric form of 11-1F4 was produced (CAEL-101) and recently demonstrated therapeutic potential in an open-label, dose-escalation phase 1a/b study where 67% of patients with cardiac or renal amyloidosis demonstrated organ response. There was a statistically significant change in Global Longitudinal Strain with 9/10 patients showing improvement (p=0.004). Since we have shown that CAEL-101 successfully improved organ function, the overall goal of this work is for the first time to explore the diagnostic potential of CAEL-101 radiolabeled with a positron emitting radioisotope for systemic amyloidosis as well as to explore its use as a companion biomarker to stratify patients for CAEL-101 immunotherapy.Methods We obtained human amyloid extracts from the heart (κ1), liver (κ1), spleen (λ1) and kidney (λ6). Lyophilized human amyloid extracts were suspended in 25ml of sterile PBS and homogenized for 3 minutes and centrifuged at 12,000g for 30 minutes. 100mg of the resulting pellet was resuspended in sterile saline. Balb/c mice were then injected subcutaneously with amyloid extract. For imaging experiments, cGMP grade CAEL-101 was radiolabeled with 124I, a positron emitting radioisotope used for PET imaging, with the standard iodegen reaction. Approximately 5 days after human amyloid extract was implanted to form subcutaneous amyloidomas, animals were injected with 200μCi of [124I]CAEL-101 and imaged 1 and 4 days post injection using an Inveon microPET scanner. SUVmax for amyloidomas and contralateral background were obtained by drawing regions of interest in the PMOD software package and calculating tumor-to-background (T:B) ratios at 1 and 4 days post tracer infusion.Results We found that [124I]CAEL-101 successfully imaged 100% of mice bearing human amyloid extracts (κ1, λ1 and λ6 subtypes derived from heart, liver, spleen, and kidney). Human amyloidomas were visualized at both at 1 and 4 days post tracer infusion, with significantly increasing T:B radio by day 4, as expected when imaging large molecular weight antibodies. T:B ratios ranged from 2.1 to 4.2 at 4 days. Mice implanted with κ subtypes demonstrated significantly better in vivo T:B ratios (4.1 +/- 0.20), compared to λ subtypes (2.8 +/- 0.46), although all amyloidomas exhibited T:B uptake > 2.1, which would be clinically significant.Conclusions We have demonstrated for the first time the potential of using radiolabeled CAEL-101 as a companion diagnostic to image real-time targeting of human amyloidosis in vivo. This is highly translatable due to the fact that CAEL-101 has shown great promise in early stage clinical trials to clear insoluble amyloid plaques. Importantly, we successfully used PET imaging to visualize cardiac derived amyloid fibrils from AL amyloidosis patients. Therefore, we anticipate that dedicated gated cardiac PET/CT imaging of radiolabeled CAEL-101 will be successful at visualizing cardiac amyloid deposits in patients, especially with the rich blood flow in cardiac tissue and newer generation highly sensitive, high resolution digital PET scanners, in contrast to the non-cardiac optimized whole body scans used in prior studies with antibody-based PET. Given that we were able to image 100% of implanted human amyloidomas derived from heart, spleen, liver and kidney consisting of both κ and λ subtypes, we envision using CAEL-101 PET imaging to (1) diagnose systemic amyloidosis, (2) stratify patients for CAEL-101 immunotherapy, and (3) quantify peripheral organ amyloid fibril deposition pre and post anti-amyloid therapy. [Display omitted] DisclosuresSolomon:Caelum Biosciences: Consultancy, Equity Ownership. Lentzsch:Bayer: Consultancy; BMS: Consultancy; Janssen: Consultancy; Caelum Biosciences: Consultancy, Other: Dr. Lentzsch recused herself as an investigator from the Phase 1a/b trial testing CAEL-101 in 11/2017., Patents & Royalties: Shareholder for Caelum Biosiences. Mintz:Caelum Biosciences: Research Funding.

Bifunctional amyloid-reactive peptide promotes binding of antibody 11-1F4 to diverse amyloid types and enhances therapeutic efficacy

Amyloidosis is a malignant pathology associated with the formation of proteinaceous amyloid fibrils that deposit in organs and tissues, leading to dysfunction and severe morbidity. More than 25 proteins have been identified as components of amyloid, but the most common form of systemic amyloidosis is associated with the deposition of amyloid composed of Ig light chains (AL). Clinical management of amyloidosis focuses on reducing synthesis of the amyloid precursor protein. However, recently, passive immunotherapy using amyloid fibril-reactive antibodies, such as 11-1F4, to remove amyloid from organs has been shown to be effective at restoring organ function in patients with AL amyloidosis. However, 11-1F4 does not bind amyloid in all AL patients, as evidenced by PET/CT imaging, nor does it efficiently bind the many other forms of amyloid. To enhance the reactivity and expand the utility of the 11-1F4 mAb as an amyloid immunotherapeutic, we have developed a pretargeting "peptope" comprising a multiamyloid-reactive peptide, p5+14, fused to a high-affinity peptide epitope recognized by 11-1F4. The peptope, known as p66, bound the 11-1F4 mAb in vitro with subnanomolar efficiency, exhibited multiamyloid reactivity in vitro and, using tissue biodistribution and SPECT imaging, colocalized with amyloid deposits in a mouse model of systemic serum amyloid A amyloidosis. Pretreatment with the peptope induced 11-1F4 mAb accumulation in serum amyloid A deposits in vivo and enhanced 11-1F4-mediated dissolution of a human AL amyloid extract implanted in mice.

Serological Epidemiological Investigation of Tibetan Sheep (Ovis aries) Plague in Qinghai, China.

The plague, which is caused by the Gram-negative coccobacillus bacterium Yersinia pestis, has been classified as a reemerging infectious disease by the World Health Organization. The Qinghai-Tibet Plateau natural plague focus is the largest plague focus in China, and Marmota himalayana is the primary host of the plague. Tibetan sheep (Ovis aries) were first identified as naturally infected hosts of Y. pestis based on etiological evidence in 1975, and activities such as slaughtering or skinning Tibetan sheep that have been infected by Y. pestis or died from Y. pestis infection had caused severe human plague in Qinghai. Tibetan sheep are important domestic livestock in the Qinghai-Tibet Plateau. Knowledge regarding the infection rate of Y. pestis in Tibetan sheep is important for understanding the range of infection and improving measures to control plague epidemics in this area. In this study, a serological survey involving 12,710 Tibetan sheep in all 44 counties in Qinghai Province was conducted. The total positive rate of indirect hemagglutination assay for Y. pestis in Tibetan sheep in Qinghai was 0.68% (86/12,710). Serological positivity to the Y. pestis F1 antibody was found in Tibetan sheep in all prefectures, except the Haidong and Haibei prefectures in Qinghai, with the seropositive rate in different counties ranging from 0.33% to 5.2% and the titers in the positive sera ranging from 1:20 to 1:5120. In addition, the seropositive rates in animal plague focus counties were higher than the rates in non-animal plague counties. Such results indicated a widespread infection of Tibetan sheep with Y. pestis in Qinghai, even though only sporadic epidemics of Tibetan sheep plague have been reported in Qinghai.

Degradation of anti-HIV antibodies 2F5 and PG9 by proteases of Nicotiana benthamiana

Degradation of anti-HIV antibodies 2F5 and PG9 by proteases of Nicotiana benthamiana

A safety study of newly generated anti-podoplanin-neutralizing antibody in cynomolgus monkey (Macaca fascicularis)

Hematogenous metastases are enhanced by platelet aggregation induced by tumor cell-platelet interaction. Podoplanin is a key molecule to enhance the platelet aggregation and interacts with C-type lectin-like receptor 2 (CLEC-2) on platelet via PLAG domains. Our previous reports have shown that blocking podoplanin binding to platelets by neutralizing antibody specific to PLAG4 domain strongly reduces hematogenous metastasis. However, podoplanin is expressed in a variety of normal tissues such as lymphatic vessels and the question remains whether treatment of tumors with anti-podoplanin neutralizing antibodies would be toxic. Monkeys are the most suitable species for that purpose. PLAG3 and PLAG4 domains had high homology among various monkey species and human. PLAG domain deleted mutants were indicated that monkey PLAG4 domain played a more crucial role in podoplanin-induced platelet aggregation than did the PLAG3 domain as in human. Moreover, newly established neutralizing antibodies (1F6, 2F7, and 3F4) targeting the monkey PLAG4 domain blocked interaction between monkey podoplanin and CLEC-2. Especially, the 2F7 neutralizing antibody strongly suppressed platelet aggregation and pulmonary metastasis. Furthermore, inhibiting podoplanin function with 2F7 neutralizing antibody exhibited no acute toxicity in cynomolgus monkeys. Our results suggested that targeting podoplanin with specific neutralizing antibodies may be an effective anticancer treatment.

A linear epitope coupled to DsRed provides an affinity ligand for the capture of monoclonal antibodies

Monoclonal antibodies (mAbs) dominate the market for biopharmaceutical proteins because they provide active and passive immunotherapies for many different diseases. However, for most mAbs, two expensive manufacturing platforms are required. These are mammalian cell cultures for upstream production and Protein A chromatography for product capture during downstream processing. Here we describe a novel affinity ligand based on the fluorescent protein DsRed as a carrier for the linear epitope ELDKWA, which can capture the HIV-neutralizing antibody 2F5. We produced the DsRed-2F5-Epitope (DFE) in transgenic tobacco (Nicotiana tabacum) plants and purified it using a combination of heat treatment and immobilized metal-ion affinity chromatography, resulting in a yield of 24 mg kg−1 at 90% purity. Using a design-of-experiments approach, we coupled up to 15 mg DFE per mL Sepharose. The resulting affinity resin was able to capture 2F5 from the clarified extract of N. benthamiana plants, achieving a purity of 97%, a recovery of >95% and an initial dynamic binding capacity at 10% product breakthrough of 4 mg mL−1 after a contact time of 2 min. The resin capacity declined to 15% of the starting value within 25 cycles when 1.25 M magnesium chloride was used for elution. We confirmed the binding activity of the 2F5 product by surface plasmon resonance spectroscopy. DFE is not yet optimized, and a cost analysis revealed that boosting DFE expression and increasing its capacity by fourfold will make the resin cost-competitive with some Protein A counterparts. The affinity resin can also be exploited to purify idiotype-specific mAbs.

Abstract 3811: Targeted Interleukin 2 and synergy with immune check-point inhibitors

Abstract Systemic high-dose Interleukin 2 (IL2) is able to elicit complete responses in patients suffering from metastatic melanoma. However, the majority of patients cannot receive the whole treatment dose due to severe toxicities. Other approved drugs for the treatment of metastatic melanoma include chemotherapeutics (e.g. Dacarbazine), a protein kinase B-raf inhibitor (Vemurafenib) and an immune check-point inhibitor (Ipilimumab, an anti CTLA-4 antibody). These drugs provide a clear benefit to patients, but cancer cures are still rare. Targeting IL2 to the site of disease represents a promising strategy to circumvent the toxicity and maximize the efficacy of this immunomodulatory payload. Various formats of targeted IL2 fusion proteins are currently in clinical development. Furthermore, recent successful approaches in the field of immunotherapy focus on the inhibition of check-point pathways, aiming to release the immunosuppressive brakes of the immune system. To explore the possibility of improving therapeutic efficacy of the single agents, a targeted version of IL2 (namely F8-IL2, where the F8 antibody recognizes the alternatively-spliced extra-domain A of fibronectin) was investigated in combination with an anti PD-1, anti PD-L1, or anti CTLA-4 antibody in an immunocompetent mouse model of CT26 colon carcinoma. In a first study, the combination of F8-IL2 and CTLA-4 blockade led to significant tumor growth retardation (curing 3/ 5 mice). Increasing the dose of F8-IL2 from 30 to 45 µg led to a 100 % cure rate with no signs of toxicity (body weight loss &amp;lt;5 %). Moreover, all cured mice acquired protective long-term immunity, which prevented tumor growth upon subsequent rechallenge with CT26 colon carcinoma cells. Furthermore, high dose F8-IL2 combined with the anti PD-1 antibody showed better therapeutic results than the respective monotherapies. In contrast, combination of F8-IL2 with the anti PD-L1 antibody did not improve the therapeutic outcome. Taken together, these data provide a strong rationale for the clinical use of targeted IL2 in combination with Ipilimumab. Citation Format: Cornelia Deborah Hutmacher-Berndt, Dario Neri. Targeted Interleukin 2 and synergy with immune check-point inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3811.

Comparison of the genotypic and phenotypic properties of HIV-1 standard subtype B and subtype B/B′ env molecular clones derived from infections in China

Although a number of standardized human immunodeficiency virus 1 (HIV-1) pseudoviruses have been generated to assess neutralizing antibodies, subtype B/B′ has not been comprehensively characterized either genotypically or phenotypically. Full-length env genes were isolated from plasma samples derived from B/B′-infected individuals, including former plasma donors and men who had sex with men. The neutralization properties of these pseudoviruses were determined by testing against sCD4, 16 neutralizing monoclonal antibodies and 44 plasma samples, including samples from those infected with the three major prevalent clades in China. Twenty-eight pseudoviruses were successfully constructed, including 15 B′ and 13 B strains. Compared with subtype B strains from North America and Europe, the B′ strains from China showed greater variation in the V3 loop and higher susceptibility to the neutralizing antibody 2F5 and B/B′ plasma samples. The B′ strains from China showed significantly lower susceptibility to some trimer apex-binding neutralizing antibodies (PGT145, CH01, CH02, CH03, and CH04) than the B strains from Western countries. The 28 B-pseudotyped and B′-pseudotyped viruses were grouped into high, medium, and low clusters based on their overall neutralization sensitivity to plasma samples. The different genotypic and phenotypic properties of the standard subtype B from those of the Western viruses compared to the B and B′ strains from China suggest that clones from HIV-1-infected individuals in China are more suitable for the evaluation of candidate vaccines targeting the subtype B/B′ viruses circulating in China.

Adoptive immunotherapy with haploidentical natural killer cells and Anti-GD2 monoclonal antibody m3F8 for resistant neuroblastoma: Results of a phase I study

ABSTRACTNatural killer (NK) cell-mediated antibody-dependent toxicity is a potent mechanism of action of the anti-GD2 murine monoclonal antibody 3F8 (m3F8). Killer immunoglobulin-like receptor (KIR) and HLA genotypes modulate NK activity and are key prognostic markers in m3F8-treated patients with neuroblastoma. Endogenous NK-cells are suppressed in the setting of high tumor burden and chemotherapy. Allogeneic NK-cells however, demonstrate potent anti-neuroblastoma activity. We report on the results of a phase I clinical trial of haploidentical NK-cells plus m3F8 administered to patients with high-risk neuroblastoma after conditioning chemotherapy. The primary objective was to determine the maximum tolerated NK-cell dose (MTD). Secondary objectives included assessing anti-neuroblastoma activity and its relationship to donor-recipient KIR/HLA genotypes, NK function, and donor NK chimerism. Patients received a lymphodepleting regimen prior to infusion of haploidentical CD3-CD56+ NK-cells, followed by m3F8. Overall and progression free survival (PFS) were assessed from the time of first NK-cell dose. Univariate Cox regression assessed relationship between dose and outcomes. Thirty-five patients received NK-cells at one of five dose levels ranging from <1×106 to 50×106 CD3-CD56+cells/kg. One patient experienced grade 3 hypertension and grade 4 pneumonitis. MTD was not reached. Ten patients (29%) had complete or partial response; 17 (47%) had no response; and eight (23%) had progressive disease. No relationship was found between response and KIR/HLA genotype or between response and FcγRIII receptor polymorphisms. Patients receiving >10×106 CD56+cells/kg had improved PFS (HR: 0.36, 95%CI: 0.15–0.87, p = 0.022). Patient NK-cells displayed high NKG2A expression, leading to inhibition by HLA-E-expressing neuroblastoma cells. Adoptive NK-cell therapy in combination with m3F8 is safe and has anti-neuroblastoma activity at higher cell doses.

Novel antibody-cytokine fusion proteins featuring granulocyte-colony stimulating factor, interleukin-3 and interleukin-4 as payloads

Neutrophils can strongly influence disease activity in cancer and in chronic inflammation. Here, we report for the first time the construction and characterization of antibody-fusion proteins featuring granulocyte-colony stimulating factor and interleukin-3 as payloads capable of enhancing neutrophil activity and a novel antibody-interleukin-4 fusion protein with neutrophil inhibitory potential. We used the F8 antibody specific to the alternatively-spliced extra domain A (EDA) of fibronectin as a targeting agent, since the cognate antigen is strongly upregulated in diseases characterized by angiogenesis.The fusion proteins GCSF-F8, F8-IL3 and F8-IL4-F8, were cloned, expressed, and their targeting ability assessed, exhibiting preferential tumor uptake with tumor:blood ratios at 24 h after injection of 3.3, 18.2 and 27.3, respectively.In F9 tumor bearing-mice GCSF-F8 and F8-IL3 did not provide a therapeutic benefit, while F8-IL4-F8 showed a potent tumor growth retardation. In the collagen-induced model of arthritis, GCSF-F8 and F8-IL3 induced a worsening of the disease, while F8-IL4-F8 slowed arthritis progression but, surprisingly, exhibited substantial toxicity when used in combination with dexamethasone.Collectively, the results indicate that the novel fusion proteins could be expressed and efficiently delivered to the site of disease. However, they were not superior to other antibody-cytokine fusions previously described by our laboratory.