Binding Of Monoclonal Antibodies Research Articles

Characterization of 7A5: A Human CD137 (4-1BB) Receptor Binding Monoclonal Antibody with Differential Agonist Properties That Promotes Antitumor Immunity.

The CD137 receptor plays a key role in mediating immune response by promoting T cell proliferation, survival, and memory. Effective agonism of CD137 has the potential to reinvigorate potent antitumor immunity either alone or in combination with other immune-checkpoint therapies. In this study, we describe the discovery and characterization of a unique CD137 agonist, 7A5, a fully human IgG1 Fc effector-null monoclonal antibody. The biological properties of 7A5 were investigated through in vitro and in vivo studies. 7A5 binds CD137, and the binding epitope overlaps with the CD137L binding site based on structure. 7A5 engages CD137 receptor and activates NF-κB cell signaling independent of cross-linking or Fc effector function. In addition, T cell activation measured by cytokine IFNγ production is induced by 7A5 in peripheral blood mononuclear cell costimulation assay. Human tumor xenograft mouse models reconstituted with human immune cells were used to determine antitumor activity in vivo. Monotherapy with 7A5 inhibits tumor growth, and this activity is enhanced in combination with a PD-L1 antagonist antibody. Furthermore, the intratumoral immune gene expression signature in response to 7A5 is highly suggestive of enhanced T cell infiltration and activation. Taken together, these results demonstrate 7A5 is a differentiated CD137 agonist antibody with biological properties that warrant its further development as a cancer immunotherapy. GRAPHICAL ABSTRACT: http://mct.aacrjournals.org/content/molcanther/19/4/988/F1.large.jpg.

Antibody Binding to the O-Specific Antigen of Pseudomonas aeruginosa O6 Inhibits Cell Growth.

Pseudomonas aeruginosa is an opportunistic pathogen that is inherently resistant to many antibiotics and represents an increasing threat due to the emergence of drug-resistant strains. There is a pressing need to develop innovative antimicrobials against this pathogen. In this study, we identified the O-specific antigen (OSA) of P. aeruginosa serotype O6 as a novel target for therapeutic intervention. Binding of monoclonal antibodies and antigen-binding fragments therefrom to O6 OSA leads to rapid outer membrane destabilization and inhibition of cell growth. The antimicrobial effect correlated directly with antibody affinity. Antibody binding to the O antigen of a second lipopolysaccharide (LPS) type present in P. aeruginosa or to the LPS core did not affect cell viability. Atomic force microscopy showed that antibody binding to OSA resulted in early flagellum loss, formation of membrane blebs, and eventually complete outer membrane loss. We hypothesize that antibody binding to OSA disrupts a key interaction in the P. aeruginosa outer membrane.

Vaccination with Consensus H7 Elicits Broadly Reactive and Protective Antibodies against Eurasian and North American Lineage H7 Viruses.

H7 subtype avian influenza viruses have caused outbreaks in poultry, and even human infection, for decades in both Eurasia and North America. Although effective vaccines offer the best protection against avian influenza viruses, antigenically distinct Eurasian and North American lineage subtype H7 viruses require the development of cross-protective vaccine candidates. In this study, a methodology called computationally optimized broadly reactive antigen (COBRA) was used to develop four consensus H7 antigens (CH7-22, CH7-24, CH7-26, and CH7-28). In vitro experiments confirmed the binding of monoclonal antibodies to the head and stem domains of cell surface-expressed consensus HAs, indicating display of their antigenicity. Immunization with DNA vaccines encoding the four antigens was evaluated in a mouse model. Broadly reactive antibodies against H7 viruses from Eurasian and North American lineages were elicited and detected by binding, inhibition, and neutralizing analyses. Further infection with Eurasian H7N9 and North American H7N3 virus strains confirmed that CH7-22 and CH7-24 conferred the most effective protection against hetero-lethal challenge. Our data showed that the consensus H7 vaccines elicit a broadly reactive, protective response against Eurasian and North American lineage H7 viruses, which are suitable for development against other zoonotic influenza viruses.

Comparisons of Antibody Populations in Different Pre-Fusion F VLP-Immunized Cotton Rat Dams and Their Offspring.

Respiratory syncytial virus (RSV) infection poses a significant risk for infants. Since the direct vaccination of infants is problematic, maternal vaccination may provide a safer, more effective approach to their protection. In the cotton rat (CR) model, we have compared the immunization of pregnant CR dams with virus-like particles assembled with the prototype mutation stabilized pre-fusion F protein, DS-Cav1, as well two alternative mutation stabilized pre-fusion proteins (UC-2 F, UC-3 F) and showed that the alternative pre-fusion F VLPs protected the offspring of immunized dams significantly better than DS-Cav1 F VLPs (Blanco, et al. J. Virol. 93: e00914). Here, we have addressed the reasons for this increased protection by characterizing the specificities of antibodies in the sera of both immunized dams and their offspring. The approach was to measure the levels of total anti-pre-F IgG serum antibodies that would block the binding of representative pre-fusion specific monoclonal antibodies to soluble pre-fusion F protein targets. Strikingly, we found that the sera in most offspring of DS-Cav1 F VLP-immunized dams had no mAb D25-blocking antibodies, although their dams had robust levels. In contrast, all offspring of UC-3 F VLP-immunized dams had robust levels of these D25-blocking antibodies. Both sets of pup sera had significant levels of mAb AM14-blocking antibodies, indicating that all pups received maternal antibodies. A lack of mAb D25-blocking antibodies in the offspring of DS-Cav1 F VLP-immunized dams may account for the lower protection of their pups from challenge compared to the offspring of UC-3 F VLP-immunized dams.

Structure and mechanism of monoclonal antibody binding to thejunctional epitope of Plasmodium falciparumcircumsporozoite protein.

Lasting protection has long been a goal for malaria vaccines. The major surface antigen on Plasmodium falciparum sporozoites, the circumsporozoite protein (PfCSP), has been an attractive target for vaccine development and most protective antibodies studied to date interact with the central NANP repeat region of PfCSP. However, it remains unclear what structural and functional characteristics correlate with better protection by one antibody over another. Binding to the junctional region between the N-terminal domain and central NANP repeats has been proposed to result in superior protection: this region initiates with the only NPDP sequence followed immediately by NANP. Here, we isolated antibodies in Kymab mice immunized with full-length recombinant PfCSP and two protective antibodies were selected for further study with reactivity against the junctional region. X-ray and EM structures of two monoclonal antibodies, mAb667 and mAb668, shed light on their differential affinity and specificity for the junctional region. Importantly, these antibodies also bind to the NANP repeat region with equal or better affinity. A comparison with an NANP-only binding antibody (mAb317) revealed roughly similar but statistically distinct levels of protection against sporozoite challenge in mouse liver burden models, suggesting that junctional antibody protection might relate to the ability to also cross-react with the NANP repeat region. Our findings indicate that additional efforts are necessary to isolate a true junctional antibody with no or much reduced affinity to the NANP region to elucidate the role of the junctional epitope in protection.

Unfolding of monomeric lipoprotein lipase by ANGPTL4: Insight into the regulation of plasma triglyceride metabolism

The binding of lipoprotein lipase (LPL) to GPIHBP1 focuses the intravascular hydrolysis of triglyceride-rich lipoproteins on the surface of capillary endothelial cells. This process provides essential lipid nutrients for vital tissues (e.g., heart, skeletal muscle, and adipose tissue). Deficiencies in either LPL or GPIHBP1 impair triglyceride hydrolysis, resulting in severe hypertriglyceridemia. The activity of LPL in tissues is regulated by angiopoietin-like proteins 3, 4, and 8 (ANGPTL). Dogma has held that these ANGPTLs inactivate LPL by converting LPL homodimers into monomers, rendering them highly susceptible to spontaneous unfolding and loss of enzymatic activity. Here, we show that binding of an LPL-specific monoclonal antibody (5D2) to the tryptophan-rich lipid-binding loop in the carboxyl terminus of LPL prevents homodimer formation and forces LPL into a monomeric state. Of note, 5D2-bound LPL monomers are as stable as LPL homodimers (i.e., they are not more prone to unfolding), but they remain highly susceptible to ANGPTL4-catalyzed unfolding and inactivation. Binding of GPIHBP1 to LPL alone or to 5D2-bound LPL counteracts ANGPTL4-mediated unfolding of LPL. In conclusion, ANGPTL4-mediated inactivation of LPL, accomplished by catalyzing the unfolding of LPL, does not require the conversion of LPL homodimers into monomers. Thus, our findings necessitate changes to long-standing dogma on mechanisms for LPL inactivation by ANGPTL proteins. At the same time, our findings align well with insights into LPL function from the recent crystal structure of the LPL•GPIHBP1 complex.

Immunogenicity of HIV-1 Vaccines Expressing Chimeric Envelope Glycoproteins on the Surface of Pr55 Gag Virus-Like Particles.

The HIV-1 envelope glycoprotein (Env) is present on the surface of the virion at a very low density compared to most other enveloped viruses. Substitution of various parts of the stalk domain of Env (gp41) with the corresponding elements from other viral glycoproteins has been shown to increase Env spike density on the cell membrane and surface of virus-like particles (VLPs). In this study, chimeric Env antigens were generated by replacing the transmembrane and cytoplasmic domains of HIV-1 Env with the corresponding regions from the influenza H5 hemagglutinin (HA) (gp140HA2tr) and by replacing the entire gp41 region of Env with the HA2 subunit of HA (gp120HA2). Recombinant DNA and modified vaccinia Ankara (MVA) vaccines expressing HIV-1 subtype C mosaic Gag and gp150 Env or either of the chimeras were generated. Surprisingly, no significant differences were found in the levels of expression of gp150 Env or either of the chimeras on the surface of cells or on Gag VLPs. Differences were, however, observed in the binding of different monoclonal antibodies to the HIV-1 Env. Monoclonal antibodies, which recognized a V1 / V2 quaternary epitope at the tip of the native Env trimer, bound gp150 and gp140HA2tr chimera but failed to bind to the gp120HA2 chimera. Autologous Tier 2 neutralizing antibodies (NAbs) were produced by rabbits inoculated with DNA and MVA vaccines expressing the gp140HA2tr chimera or gp150 Env, but not those immunized with the gp120HA2 Env. These results showed that the addition of an HA2 stalk to HIV-1 gp120 did not improve immunogenicity, but rather that the full-length gp150 was required for optimal presentation of epitopes for the elicitation of a neutralizing antibody response to HIV-1.

Impact of Peri-Transplant Rituximab and Host/Donor Fc Receptor Polymorphisms for Patients with Relapsed or Refractory CD20+ B-Cell Malignancies

We previously reported results of allogeneic hematopoietic cell transplantation (HCT) in 93 older or medically unfit patients with non-Hodgkin's lymphoma (NHL) or chronic lymphocytic lymphoma (CLL) after conditioning with fludarabine (FLU) and 2-3 Gy total body irradiation (TBI). Three-year survival ranged from 45% to 52% depending on the underlying histology and disease stage. The major complication in those studies was disease relapse (24% at 1 year). The current prospective study in 63 NHL patients tested the hypothesis that disease relapse could be reduced, and overall survival (OS) improved, by peri-transplant rituximab (RTX). Treatment (Figure 1) consisted of RTX (375 mg/m²/day) on days -3, +10, +24 and +38 in addition to 2-3 Gy TBI ± FLU (30 mg/m² × 3 days). As pharmacokinetics (PK) and optimal dosing of RTX in allogeneic HCT are undefined, we collected PK data to optimize RTX dosing. RTX levels were assayed with murine anti-idiotype monoclonal antibody (18C9) binding to RTX. Median therapeutic RTX levels of >25 μg/mL were achieved through day 84 after HCT, with peak median level of 200.5 μg/mL at day 64. RTX levels were not correlated with relapse or graft-vs-host disease (GVHD) rates. NHL patients had significantly higher median RTX levels than CLL patients (day 84, p=0.003, Figure 2). We collected data on donor and recipient FCγRIIIa receptor polymorphisms expressed predominantly on natural killer cells, thought to affect responsiveness to RTX. This was done to identify patients or donor/recipient pairs most likely to benefit from peri-transplant RTX. FCγRIIIa polymorphisms were determined by PCR and single-strand confirmation polymorphism (SSCP) from DNA isolated from patient cells. Recipients with F/F and V/F FCγRIIIa polymorphisms experienced higher relapse rates compared to recipients with V/V polymorphism (p=0.15). Overall, almost a third of pre-HCT F/F patients relapsed. Probably due to small numbers, no difference in outcome was found with V/V donor pairing for these patients. Results are shown in Figure 3. Relapse rates at 5 years were similar among RTX-treated patients and historical controls (32% vs, 28%; P=0.94). RTX-treated patients experienced higher 5-year OS and progression-free survival; (47% vs, 38%; P=0.13) (41 vs, 32%; P=0.12), respectively, but none of the differences was statistically significant. Non-relapse mortality was lower among RTX-treated patients versus controls (27% vs, 40; P=0.05), respectively; incidences of grade 2-4 acute GVHD and grade 3-4 acute GVHD were similar (60% vs, 57%, 13% vs. 17%); and 5-year chronic GVHD was higher among RTX-treated patients (62% vs, 47%). These findings suggest that for patients with relapsed or refractory NHL, peri-transplant RTX neither reduced relapse nor improved OS or other post-HCT parameters. The role of donor-recipient pairing by FCγRIIIa polymorphisms in outcome remains to be determined.

Multiple myeloma masquerading as T3 toxicosis

We report a case of a 52-year-old man who was referred by his primary care physician to rule out thyroid illness. His thyroid function tests (TFTs) revealed isolated elevation of total T3 (TT3) with normal total T4 (TT4), free T3 (FT3), free T4 (FT4), and unsuppressed thyroid stimulating hormone (TSH) raising the suspicion of antibody interference or abnormal thyroid hormone-binding proteins. To identify antibody interference, we repeated the immunoassay using varied platforms and assay methods. The total T3 levels which were high in one-step immunoassay normalized in two-step immunoassay. On further evaluation, the patient was diagnosed with multiple myeloma and the antibody interference was secondary to IgG monoclonal antibody binding specifically to analog T3 leading to spurious results. Ours is the first report from India and the fourth case in literature to report a multiple myeloma antibody interfering with TFTs.

Quantified Cell Binding of Astatinated Immunoconjugates on Ovarian Cancer Cell Spheroids by Alpha Camera Imaging

Optimization of patient dosing for intraperitoneal α-radioimmunotherapy of microtumours can be performed with the help of a biokinetic model. The goal of this study was to investigate whether simulations of mathematical models can be confirmed by in vitro models. We used the α- camera micro-imaging technology to quantify cell binding of 211At-radiolabelled monoclonal antibodies to small ovarian cancer cell clusters. Various sized spheroids (NIH:OVCAR-3) were treated with 211At-MX35 and 211At-Farletuzumab (170kBq/mL) for different time periods. Cell clusters were stained and evaluated as a whole or by serial sectioning using the α-camera device and the γ-counter as reference method. Alpha camera images were frame-parsed to determine the activity uptake and distribution. Cell numbers were estimated by area evaluation of conventional α-camera images in case of whole spheroids or, in case of cross-sections, by manual counting of adjacent haematoxylin and eosin stained cryosections. Kinetic binding curves were derived using the total number of bound antibodies per cell and compared to model simulations. Quant imaging with the α-camera provides an accurate and precise method for activity quantification. In contrast to 211At-Farletuzumab, binding kinetics of 211At-MX35 were considerably different from the model simulation predictions. Experimental data showed equilibrium binding within 4 hours after treatment followed by a decline in activity due to cell death. The in vitro model did confirm that lower levels of activity uptake per cell were reached for larger spheroids, especially near the core. For this particular cell line, the antibody binding characteristics of MX35 led to higher activity levels. This study demonstrated that antibody binding characteristics play an important role in intraperitoneal α-radioimmunotherapy. Our observations indicate that radiation effects may occur already shortly after treatment initialization and thus elucidating a parameter that needs to be added in the models used for treatment planning. Key words: astatine-211, radioimmunotherapy, alpha-particle therapy, quantification, biokinetic model

Molecular evolution of the hemagglutinin-neuraminidase (HN) gene in human respirovirus 3

Human respirovirus 3 (HRV3) is a major causative agent of acute respiratory infections in humans. HRV3 can manifest as a recurrent infection, although exactly how is not known. In the present study, we conducted detailed molecular evolutionary analyses of the major antigen-coding hemagglutinin-neuraminidase (HN) gene of this virus detected/isolated in various countries. We performed analyses of time-scaled evolution, similarity, selective pressure, phylodynamics, and conformational epitope prediction by mapping to HN protein models. In this way, we estimated that a common ancestor of the HN gene of HRV3 and bovine respirovirus 3 diverged around 1815 and formed many lineages in the phylogenetic tree. The evolutionary rates of the HN gene were 1.1 × 10−3 substitutions/site/year, although the majority of these substitutions were synonymous. Some positive and many negative selection sites were predicted in the HN protein. Phylodynamic fluctuations of the gene were observed, and these were different in each lineage. Furthermore, most of the predicted B cell epitopes did not correspond to the neutralization-related mouse monoclonal antibody binding sites. The lack of a link between the conformational epitopes and neutralization sites may explain the naturally occurring HRV3 reinfection.

Cold Agglutinin Disease Shows Highly Recurrent Gains of Chromosome 3, 12 and 18

Cold agglutinin disease (CAD) is a rare autoimmune hemolytic anemia, caused by a distinct type of B-cell lymphoproliferative disease of the bone marrow. Anemia is mediated by binding of monoclonal cold agglutinin antibodies to the erythrocyte surface I antigen at temperatures below central body temperature, followed by agglutination and complement activation. These antibodies are almost exclusively encoded by IGHV4-34. We recently found recurrent KMT2D and CARD11 gene mutations in CAD (Malecka, et al 2017). We analyzed 12 CAD samples from the CAD-5 study (NCT02689986) (Berentsen, et al 2017) using Affymetrix OncoScan CNV Plus Assay and exome sequencing data in order to detect chromosomal aberrations. All samples were sorted using fluorescence activated cell sorting prior to analysis. Complete or partial gain of chromosome 3 (+3 or +3q) was detected in all samples (Table 1). Additionally, most cases showed either gain of chromosome 12 or 18 (9/12) (Table 1). Additional small regions of recurrent gain or loss were also detected in other chromosomes. The recurrent changes in least 4 samples are: +1p36.31-p36.13, -8p21.3-p21.2, +9q34.2-q34.3, +11q13.1-q13.2, +17q25.3, +21q22.3, +22q13.31-q13.33. Gains and losses of large parts of chromosomes are clearly visible in both Affymetrix OncoScan CNV Plus Assay and exome sequencing data (Figure 1). In contrast smaller changes are often not visible in exome sequencing data and therefore could not be fully validated. Further validation will be required for smaller chromosomal changes. Gain of chromosome 3/3q was characteristic for all CAD cases analyzed. Gain of chromosome 3 in CAD has previously been reported in 9 of 26 patients (Gordon, et al 1990, Michaux, et al 1995). Gain of chromosome 3 is not a highly recurrent finding in most B-cell lymphomas with the exception of marginal zone lymphoma (MZL). Of interest, gain of chromosome 12 and 18, demonstrated in our study, is also a feature of MZL. These findings, together with our previous findings (Malecka, et al 2017), suggest that the CAD-associated lymphoproliferative disease, although exclusively present in the bone marrow, might be related to MZL. The CAD-5 study assessed the efficacy of bendamustine plus rituximab in CAD patients. We explored whether a correlation existed between presence or absence of trisomy 12 and 18 with response to therapy. Although the series is small, we found a trend towards poorer response in patients with trisomy 18 compared to patients with trisomy 12. Furthermore, both patients without trisomy 12 or 18 had the best responses (Table 1). Despite the limited number of cases the Pearson correlation was statistically significant (p=0.02). However, more samples need to be analyzed to confirm these results. A correlation between response to therapy and presence of trisomy 12 or 18 has previously been demonstrated for other B-cell lymphomas. Trisomy 12 is frequent in patients with small lymphocytic lymphoma (28-36%) and in chronic lymphocytic leukemia (10-25%) and in the latter +12 is associated with intermediate prognosis (Autore, et al 2018). Trisomy 3 and 18 are found to be correlated with advanced stage extranodal marginal zone lymphoma (Krugmann, et al 2005). There is evidence from previous studies that trisomy 18 may be associated with upregulation of BCL2, an anti-apoptotic gene located on chromosome 18. Whether BCL2 is upregulated in CAD with trisomy 18 and whether it might be the cause of therapy resistance, needs to be investigated. Such a study is of importance since BCL2 inhibitors might be considered to improve treatment responses. We conclude that gain of chromosome 3 is a highly recurrent finding in CAD-associated lymphoproliferative disease. Further gain of chromosome 12 and 18 might be predictors of therapy outcome. These genetic findings are similar to what has previously been demonstrated in nodal and extranodal MZL, suggesting that CAD-associated lymphoproliferative disease, a disease of the bone marrow, might be related to MZL. Disclosures Berentsen: Mundipharma: Research Funding; Apellis, Bioverativ (a Sanofi company), Momenta Pharmaceuticals, and True North Therapeutics: Consultancy; Alexion, Apellis, and Janssen-Cilag: Honoraria. Tjønnfjord:Mundipharma: Research Funding.

The Pfs230 N-terminal fragment, Pfs230D1+: expression and characterization of a potential malaria transmission-blocking vaccine candidate

BackgroundControl and elimination of malaria can be accelerated by transmission-blocking interventions such as vaccines. A surface antigen of Plasmodium falciparum gametocytes, Pfs230, is a leading vaccine target antigen, and has recently progressed to experimental clinical trials. To support vaccine product development, an N-terminal Pfs230 antigen was designed to increase yield, as well as to improve antigen quality, integrity, and homogeneity.MethodsA scalable baculovirus expression system was used to express the Pfs230D1+ construct (aa 552–731), which was subsequently purified and analysed. Pfs230D1+ was designed to avoid glycosylation and protease digestion, thereby potentially increasing homogeneity and stability. The resulting Pfs230D1+ protein was compared to a previous iteration of the Pfs230 N-terminal domain, Pfs230C1 (aa 443–731), through physiochemical characterization and in vivo analysis. The induction of functional antibody responses was confirmed via the standard membrane feeding assay (SMFA).ResultsPfs230D1+ was produced and purified to an overall yield of 23 mg/L culture supernatant, a twofold yield increase over Pfs230C1. The Pfs230D1+ protein migrated as a single band via SDS-PAGE and was detected by anti-Pfs230C1 monoclonal antibodies. Evaluation by SDS-PAGE, chromatography (size-exclusion and reversed phase) and capillary isoelectric focusing demonstrated the molecule had improved homogeneity in terms of size, conformation, and charge. Intact mass spectrometry confirmed its molecular weight and that it was free of glycosylation, a key difference to the prior Pfs230C1 protein. The correct formation of the two intramolecular disulfide bonds was initially inferred by binding of a conformation specific monoclonal antibody and directly confirmed by LC/MS and peptide mapping. When injected into mice the Pfs230D1+ protein elicited antibodies that demonstrated transmission-reducing activity, via SMFA, comparable to Pfs230C1.ConclusionBy elimination of an O-glycosylation site, a potential N-glycosylation site, and two proteolytic cleavage sites, an improved N-terminal Pfs230 fragment was produced, termed D1+, which is non-glycosylated, homogeneous, and biologically active. An intact protein at higher yield than that previously observed for the Pfs230C1 fragment was achieved. The results indicate that Pfs230D1+ protein produced in the baculovirus expression system is an attractive antigen for transmission-blocking vaccine development.

Research progress of adalimumab in pediatric Crohn ′s disease

Adalimumab is an anti-TNF-α recombinant IgG1, a fully humanized monoclonal antibody binding soluble human tumor necrosis factor alpha with high affinity and specificity.It has lower immunogenicity and can be used clinically for induction and maintenance of remission in Crohn′s disease.The effectiveness, sustainability and safety of adalimumab in the treatment of pediatric Crohn′s disease has been gradually confirmed, which may pave the way for new therapy to treat children with refractory Crohn′s disease. Key words: Adalimumab; Crohn′s disease; Children

Cetuximab, irinotecan and fluorouracile in fiRst-line treatment of immunologically-selected advanced colorectal cancer patients: the CIFRA study protocol

BackgroundCombination of chemotherapies (fluoropirimidines, oxaliplatin and irinotecan) with biologic drugs (bevacizumab, panitumumab, cetuximab) have improved clinical responses and survival of metastatic colorectal cancer (mCRC). However, patients’ selection thorough the identification of predictive factors still represent a challange. Cetuximab (Erbitux®), a chimeric monoclonal antibody binding to the Epidermal Growth Factor Receptor (EGFR), belongs to the Immunoglobulins (Ig) grade 1 subclass able to elicite both in vitro and in vivo the Antibody-Dependent Cell-mediated Cytotoxicity (ADCC). ADCC is the cytotoxic killing of antibody-coated target cells by immunologic effectors. The effector cells express a receptor for the Fc portion of these antibodies (FcγR); genetic polymorphisms of FcγR modify the binding affinity with the Fc of IgG1. Interestingly, the high-affinity FcγRIIIa V/V is associated with increased ADCC in vitro and in vivo. Thus, ADCC could partially account for cetuximab activity.Methods/designCIFRA is a single arm, open-label, phase II study assessing the activity of cetuximab in combination with irinotecan and fluorouracile in FcγRIIIa V/V patients with KRAS, NRAS, BRAF wild type mCRC. The study is designed with a two-stage Simon model based on a hypothetical higher response rate (+ 10%) of FcγRIIIa V/V patients as compared to previous trials (about 60%) assuming ADCC as one of the possible mechanisms of cetuximab action. The test power is 95%, the alpha value of the I-type error is 5%. With these assumptions the sample for passing the first stage is 14 patients with > 6 responses and the final sample is 34 patients with > 18 responses to draw positive conclusions. Secondary objectives include toxicity, responses’ duration, progression-free and overall survival. Furthermore, an associated translational study will assess the patients’ cetuximab-mediated ADCC and characterize the tumor microenvironment.DiscussionThe CIFRA study will determine whether ADCC contributes to cetuximab activity in mCRC patients selected on an innovative immunological screening. Data from the translational study will support results’ interpretation as well as provide new insights in host-tumor interactions and cetuximab activity.Trial registrationThe CIFRA trial (version 0.0, June 21, 2018) has been registered into the NIH-US National Library of Medicine, ClinicalTrials.gov database with the identifier number (NCT03874062).

Opsonic monoclonal antibodies enhance phagocytic killing activity and clearance of Mycobacterium tuberculosis from blood in a quantitative qPCR mouse model

BackgroundPatients with impaired immunity often have rapid progression of tuberculosis (TB) which can lead to highly lethal Mycobacterium tuberculosis (MTB) sepsis. Opsonic monoclonal antibodies (MABs) directed against MTB that enhance phagocytic killing activity and clearance of MTB from blood may be useful to enhance TB immunity. MethodsBALB/c mice were immunized with ethanol-killed MTB (EK-MTB) and MABs were produced and screened by ELISA for binding to killed and live Mycobacterium smegmatis (SMEG) and MTB. MAB opsonophagocytic killing activity (OPKA) was examined using SMEG with HL60 and U-937 cells and MTB with U-937 cells. Clearance of MTB from blood was evaluated in Institute of Cancer Research (ICR) mice given opsonic anti-MTB MABs or saline (control) 24 h prior to intravenous infusion with 108 CFUs gamma-irradiated MTB (HN878). MTB levels in murine blood collected 0.25, 4 and 24 h post-challenge were assessed by qPCR. MAB binding to peptidoglycan (PGN) was examined by ELISA using PGN cell wall mixture and ultra-pure PGN. ResultsTwo MABs (GG9 and JG7) bound to killed and live SMEG and MTB (susceptible and resistant), and promoted OPKA with live MTB. MAB JG7 significantly enhanced OPKA of MTB. Both MABs significantly enhanced clearance of killed MTB from murine blood at 4 and 24 h as measured by qPCR. These opsonic MABs bound to PGN, a major cell wall constituent. ConclusionsAnti-MTB MABs that promote bactericidal phagocytic activity of MTB and enhance clearance of killed MTB from the blood, may offer an immunotherapeutic approach for treatment of MTB bacteremia or sepsis, and augment treatment of multi-drug resistant (MDR) or extensively drug resistant (XDR) TB.

Crack initiation and propagation in sweet cherry skin: A simple chain reaction causes the crack to 'run'.

Rain cracking severely affects the commercial production of many fleshy-fruit species, including of sweet cherries. The objectives were to investigate how the gaping macroscopic cracks (macrocracks) of a rain-cracked fruit can develop from microscopic cracks in the cuticle (microcracks). Incubating fruit in deionized water is well known to cause significant macrocracking. We found that after a lag phase of 2 h, the numbers and lengths of macrocracks increased. Macrocrack number approached an asymptote at 12 h, whereas macrocrack length continued to increase. The rate of macrocrack propagation (extension at the crack tip) was initially 10.8 mm h-1 but then decreased to a near-constant 0.5 mm h-1. Light microscopy revealed three characteristic zones along a developing macrocrack. In zone I (ahead of the crack), the cuticle was intact, the epidermal cells were unbroken and their cell walls were thin. In zone II, the cuticle was fractured, the first epidermal cells died and their cell walls began to thicken (swell). In zone III, most epidermal cells had died, their cell walls were swollen and cell:cell separation began along the middle lamellae. The thickness of the anticlinal epidermal cell walls and the percentage of intact living cells along a crack were closely and negatively related. Cracks were stained by calcofluor white, but there was no binding of monoclonal antibodies (mAbs) specific for hemicelluloses (LM11, LM21, LM25). Strong binding was obtained with the anti-homogalacturonan mAb (LM19), indicating the presence of unesterified homogalacturonans on the crack surface. We conclude that macrocrack propagation is related to cell death and to cell wall swelling. Cell wall swelling weakens the cell:cell adhesion between neighbouring epidermal cells, which separate along their middle lamellae. The skin macrocrack propagates like a ‘run’ in a fine, knitted fabric.

Efficient Innate Immune Killing of Cancer Cells Triggered by Cell-Surface Anchoring of Multivalent Antibody-Recruiting Polymers.

Binding of monoclonal antibodies (mAbs) onto a cell surface triggers antibody-mediated effector killing by innate immune cells through complement activation. As an alternative to mAbs, synthetic systems that can recruit endogenous antibodies from the blood stream to a cancer cell surface could be of great relevance. Herein, we explore antibody-recruiting polymers (ARPs) as a novel class of immunotherapy. ARPs consist of a cell-binding motif linked to a polymer that contains multiple small molecule antibody-binding motifs along its backbone. As a proof of concept, we employ a lipid anchor that inserts into the phospholipid cell membrane and make use of a polymeric activated ester scaffold onto which we substitute dinitrophenol as an antibody-binding motif. We demonstrate that ARPs allow for high avidity antibody binding and drive antibody recruitment to treated cells for several days. Furthermore, we show that ARP-treated cancer cells are prone to antibody-mediated killing through phagocytosis by macrophages.

Elimination of erroneous results related to bovine mononuclear cell immunophenotyping by antibodies binding to Fc receptors

Blocking immunoglobulin G (IgG) binding receptors on leukocytes is an established and highly recommended preventive procedure for immunological assays. Failing to prevent such nonspecific binding can lead to erroneous results. Several studies testing different blocking reagents have been performed in murine or human cells, however, there are no specific studies on bovine cells. Our study aimed to investigate the efficiency of blocking reagents to inhibit the nonspecific binding of mouse monoclonal antibodies (mAbs) to bovine peripheral blood cells. We observed nonspecific interactions of IgG2a and IgG2b negative isotypes with bovine leukocytes, but not IgG1. We found that these nonspecific bindings could be eliminated by blocking with purified mouse IgG, whereas little or no blocking effect was observed when bovine serum or Mouse Seroblock FcR were applied. Moreover, in the absence of an efficient blocking reagent, the percentage of CD335 positive cells was significantly higher than in the group previously blocked with mouse IgG. Based on these results, and due to the lack of specific commercial blocking reagents for bovine cells, our recommendation is to use purified mouse IgG as a blocking reagent for immune assays targeting bovine leukocytes in order to enhance the accuracy of the results.

Galcanezumab in episodic migraine: subgroup analyses of efficacy by high versus low frequency of migraine headaches in phase 3 studies (EVOLVE-1 & EVOLVE-2)

BackgroundPatients with high-frequency episodic migraine (HFEM) have a greater disease burden than those with low-frequency episodic migraine (LFEM). Acute treatment overuse increases the risk of migraine chronification in patients with HFEM. Galcanezumab, a humanized monoclonal antibody binding calcitonin gene-related peptide (CGRP), is effective for migraine prevention with a favorable safety profile. Here, we investigate whether there are differences in galcanezumab efficacy in patients with LFEM or with HFEM.MethodsData were pooled from two double-blind, placebo-controlled phase 3 trials; EVOLVE-1 and EVOLVE-2. Patients were 18–65 years old, experienced 4–14 monthly migraine headache days (MHDs) for ≥1 year prior, with onset at < 50 years of age. Migraine headaches were tracked via electronic patient-reported outcome system and randomization was stratified by low (LFEM; 4–7 monthly MHDs) or high (HFEM; 8–14 monthly MHDs) frequency. Subgroup analysis compared the HFEM and LFEM subgroups with a linear or generalized linear mixed model repeated measures approach.ResultsThe intent-to-treat patients (N = 1773) had a mean age of 41.3 years, were mostly white (75%), female (85%), and 66% of patients had HFEM. In both the LFEM and HFEM subgroups, the overall (Months 1–6) and monthly changes from baseline in monthly MHDs and monthly MHDs with acute medication use compared with placebo were statistically significantly reduced for galcanezumab 120-mg and 240-mg. Galcanezumab (120-mg and 240-mg) significantly decreased the overall and monthly MHDs with nausea and/or vomiting, and with photophobia and phonophobia versus placebo in patients with LFEM or HFEM. In both subgroups, the mean overall (Months 1–6) and monthly percentages of patients with ≥50%, ≥75%, and 100% reduction in monthly MHDs from baseline were statistically significantly greater in patients receiving either dose of galcanezumab versus placebo. Galcanezumab (120-mg and 240-mg) significantly improved the Migraine-Specific Quality of Life Questionnaire role function-restrictive domain score as well as the Migraine Disability Assessment total score versus placebo for patients with LFEM or HFEM. There were no significant subgroup-by-treatment interactions.ConclusionsGalcanezumab was as effective in patients with HFEM as in those with LFEM. Associated symptoms, quality of life, and disability were similarly improved in patients with HFEM or LFEM.Trial registrationNCT02614183, NCT02614196.