Monospecific Antibodies Research Articles

Asymmetric Fc Engineering for Bispecific Antibodies with Reduced Effector Function.

Asymmetric bispecific antibodies are a rapidly expanding therapeutic antibody class, designed to recognize two different target epitopes concurrently to achieve novel functions not available with normal antibodies. Many therapeutic designs require antibodies with reduced or silenced effector function. Although many solutions have been described in the literature to knockout effector function, to date all of them have involved the use of a specific antibody subtype (e.g., IgG2 or IgG4), or symmetric mutations in the lower hinge or CH2 domain of traditional homodimeric monospecific antibodies. In the context of a heterodimeric Fc, we describe novel asymmetric Fc mutations with reduced or silenced effector function in this article. These heteromultimeric designs contain asymmetric charged mutations in the lower hinge and the CH2 domain of the Fc. Surface plasmon resonance showed that the designed mutations display much reduced binding to all of the Fc gamma receptors and C1q. Ex vivo ADCC and CDC assays showed a consistent reduction in activity. Differential scanning calorimetry showed increased thermal stability for some of the designs. Finally, the asymmetric nature of the introduced charged mutations allowed for separation of homodimeric impurities by ion exchange chromatography, providing, as an added benefit, a purification strategy for the production of bispecific antibodies with reduced or silenced effector function.

Detection of LGI1 and CASPR2 antibodies with a commercial cell-based assay in patients with very high VGKC-complex antibody levels

BackgroundThe presence of VGKC-complex antibodies, without LGI1/CASPR2 antibodies, as a standalone marker for neurological autoimmunity remains controversial. Additionally, the lack of an unequivocal VGKC-complex antibody cut-off level defining neurological autoimmunity makes it important to test for monospecific antibodies. We aim to determine the performance characteristics of a commercial assay (Euroimmun, Lübeck, Germany) for LGI1/CASPR2 antibody detection in patients with very high VGKC-complex antibody levels and report their clinico-serological associations. MethodsWe identified 8 patients in our cohort with the highest VGKC-complex antibody levels (median 2663.5pM, range 933–6730pM) with VGKC-complex antibody related syndromes (Group A). Two other groups were identified; 1 group with suspected neuronal surface antibody syndromes and negative for VGKC-complex antibodies (Group B, n=8), and another group with cerebellar ataxia and negative for onconeuronal antibodies (Group C, n=8). ResultsSeven out of 8 patients (87.5%) in Group A had LGI1 and/or CASPR2 antibodies. One Group B patient had LGI1 antibodies but was negative on re-testing with a live cell assay. No Group C patients had monospecific antibodies. Inter-rater reliability was high; combining Groups A and B patients, the kappa statistic was 0.87 and 1.0 for LGI1 and CASPR2 antibodies respectively. ConclusionWe demonstrated that a high proportion of patients with very high VGKC-complex antibody levels and relevant clinical syndromes have LGI1 and/or CASPR2 antibodies detected by the commercial assay. Our findings lend support to the use of the assay for rapid and reliable detection of LGI1 and CASPR2 antibodies.

Clinical Pharmacology and Translational Aspects of Bispecific Antibodies.

Clinical Pharmacology and Translational Aspects of Bispecific Antibodies.

Generation of Monospecific Polyclonal Antibodies to Recombinant Filarial Antigen rWbL2 and Evaluation of Its Immunodiagnostic Utility in Filariasis

ABSTRACTIntroductionLymphatic filariasis is a mosquito-borne disease affecting nearly 120 million people across the world. Filarial antigen detection is a good indicator for mapping new filarial cases and for evaluation of filarial elimination programs as compared with the low sensitivity associated with the direct evidence of microfilaria (Mf) in blood samples. To overcome low sensitivity and night-time blood collection method for parasite detection in filariasis cases, the sandwich enzyme-linked immunosorbent assay (ELISA) was standardized for detection of circulating filarial antigen using monospecific polyclonal antibodies raised against recombinant filarial antigen rWbL2. In the present study, the specific antibodies raised against novel recombinant antigens rWbL2 were explored to develop suitable filarial antigen assays. It was possible to come out with a filarial antigen assay that could detect WbL2 or its equivalent antigen with 40% sensitivity (by using mouse anti-WbL2 antibody as capturing antibody), 60% sensitivity (using FSIgG human filarial serum immunoglobulin G as capturing antibody), and 100% specificity. These assays show promise to detect and monitor active filarial infection and thus prove to have potential as a useful diagnostic and monitoring tool in the elimination program.How to cite this articleGandhe MB, Gandhe SM. Generation of Monospecific Polyclonal Antibodies to Recombinant Filarial Antigen rWbL2 and Evaluation of Its Immunodiagnostic Utility in Filariasis. Indian J Med Biochem 2017;21(2):117-123.

The Forgotten YY2 in Reported YY1 Expression Levels in Human Cancers.

The transcription factor Yin Yang 1 (YY1) has been reported to be overexpressed in the majority of human cancers and that overexpression has prognostic significance. YY1 regulates several properties associated with cancer cells, including cell survival, cell proliferation, endothelial-mesenchymal transition, metastases, and resistance to both chemotherapeutics and immunotherapeutics. Although the majority of published reports focus on YY1 levels, little has been reported on the expression and activity of YY1 family member Yin Yang 2 (YY2). YY1 and YY2 share more than 50% homologies in DNA and amino acid sequences and share the same C-terminal zinc finger domains involved in DNA binding. This survey of the reported literature revealed that the antibodies used in published immunohistochemistry analyses were not uniquely specific for YY1. Most were likely cross-reactive with YY2. Furthermore, data from the Human Protein Atlas regarding YY1 and YY2 expression in various cancers were generated using antibodies that did not discriminate between YY1 and YY2. This review analyzed the commercially available antibodies listed against YY1 and YY2 and determined their cross-reactivities. A summary is of the published studies on the expression levels of YY1 in human cancers and their potential cross-reactivities with YY2 is also provided. Well-documented monospecific antibodies to both YY1 and YY2 have to be developed and used when examining the expression levels of YY1 and YY2 in human cancers to elucidate the accurate relationship between them and clinical significance of each.

The role of astroglial proteins in the brains of rats under the influence of doxorubicin and Humilid

Doxorubicin causes toxic side effects which result in profound changes in various organs including the heart muscle, which leads to the development of cardiomyopathy, with further complications, as our earlier studies have shown. But there are complications in treatment by anthracycline chemotherapy. The reduction of toxicity of cytotoxic drugs without reduction of their antitumor action remains an important medical challenge. The S-100b and glial fibrillar acid protein (GFAP) are the key astroglial proteins which are widely used as biomarkers for neurotraumas and strokes. Thus the purpose of our work was to study the GFAP and S-100b levels under the experimental model of doxorubicin-induced cardiomyopathy and the effect of the cytostatic separately or combined with Humilid. The level of studied proteins was measured by ELISA using monospecific antibodies against GFAP and S-100b (Sigma, USA), secondary anti-IgG labeled with horseradish peroxidase (Sigma, USA) and GFAP and S-100b standards (Sigma, USA). The experiment was conducted on white Wistar male rats weighing 210 ±50 g, which were divided into 3 groups with 8 animals in each: 1 – control, rats received saline i/p, 2 – doxorubicin 1 mg/kg i/p once weekly during 4 weeks, 3 – doxorubicin by the same scheme plus 0.01% solution of Humilid during 4 weeks. Under impact of doxorubicin an increase of fibrillar GFAP content in the rats’ hippocampus was shown, which may have occurred as a result of the proliferation of astrocytes. An increase of concentration of S-100b to 20–26% compared with the control animals was also detected in the hippocampus, thalamus and the cerebral cortex of the studied animals. Concomitant administration of doxorubicin with Humilid partially prevented the increase in concentration of GFAP and S-100b in the rats’ hippocampus. The reciprocal change in the level of S-100b and GFAP in the peripheral nervous system of the heart tissue was defined with a high degree of correlation under impact of doxorubicin. Accordingly, we have proposed the hypothesis that the increase in of the filament GFAP in the hippocampus may occur due to the development of the ischemic state under the impact of doxorubicin-induced cardiomyopathy. Humilid with doxorubicin prevented changes in the researched astroglial proteins of the heart muscle and brain parts of rats.

Highlights of This Issue

The development of an ultra-sensitive laser capture microdissection (LCM)/RNA-seq approach for studying epithelial and stromal compartments of human aberrant crypt foci (ACF) is described that focuses on proximal colon lesions harboring somatic mutations to KRAS, BRAF or APC. Pro-inflammatory NF-kB target genes (TIMP1, RELA and RELB) were identified as specific to ACF epithelia. A subset of BRAF-mutant ACF displayed a senescence-associated transcriptome with increased expression of CDKN2A. LCM-captured ACF-associated stroma are transcriptionally distinct from normal, with up-regulation of genes related to immune cell infiltration and fibroblast activation. These findings underscore the dynamic, complex interplay occurring early in colorectal neoplasia, highlighting the role of activated stromal fibroblasts and potential therapeutic targeting of NF-kB signaling for cancer prevention.High-grade serous ovarian cancer (HGSC) is the most common and lethal ovarian cancer. Two tissues have been suggested as its origin: ovarian surface and fallopian tube epithelia (OSE and FTE). Klinkebiel and colleagues hypothesized that the HGSC methylome will more closely resemble the methylome of its tissue of origin. DNA methylome profiling of patient-matched primary normal OSE and FTE, as well as primary HGSC obtained from various sources was performed. The data support the FTE origin model and suggest that DNA methylome profiling is a valuable approach to examine cell lineage in cancer, due to its tissue-specificity and biochemical stability.The pathological role of the enzyme peptidyl arginine deiminase 2 (PADI2) has been studied in autoimmune diseases and, very recently, in breast and skin cancer. Here, down-regulation of PADI2 is an early event in the pathogenesis of colorectal cancer associated with poor prognosis. PADI2 mRNA and protein levels, in independent cohorts, were found to be low or absent in tumors as well as in ulcerative colitis and cell line samples. PADI2 up-regulation was detected in matched normal mucosa and in differentiated cells in vitro. Moreover, low levels of PADI2 in both tumors and adjacent mucosa correlate with decreased patient survival independently of inflammation. These data suggest that PADI2 is a potential biomarker or a point for therapeutic intervention.Low density lipoprotein (LDL) related protein 6 (LRP6), is an important receptor component of the WNT pathway, often dysregulated during tumorigenesis, and is therefore an attractive target for inhibitory antibodies. However, LRP6 is unusual in that it has two spatially distinct ligand binding sites. Monospecific antibodies are unable to block both sites, and often potentiate signaling instead of blocking it. Using domain antibody technology, Jackson and colleagues constructed a bispecific molecule that fully inhibits this complex receptor molecule. Importantly, this bi-specific antibody is active both in vitro using cancer cells and in vivo using patient-derived tumor models.

An Ethylene-Protected Achilles' Heel of Etiolated Seedlings for Arthropod Deterrence.

A small family of Kunitz protease inhibitors exists in Arabidopsis thaliana, a member of which (encoded by At1g72290) accomplishes highly specific roles during plant development. Arabidopsis Kunitz-protease inhibitor 1 (Kunitz-PI;1), as we dubbed this protein here, is operative as cysteine PI. Activity measurements revealed that despite the presence of the conserved Kunitz-motif the bacterially expressed Kunitz-PI;1 was unable to inhibit serine proteases such as trypsin and chymotrypsin, but very efficiently inhibited the cysteine protease RESPONSIVE TO DESICCATION 21. Western blotting and cytolocalization studies using mono-specific antibodies recalled Kunitz-PI;1 protein expression in flowers, young siliques and etiolated seedlings. In dark-grown seedlings, maximum Kunitz-PI;1 promoter activity was detected in the apical hook region and apical parts of the hypocotyls. Immunolocalization confirmed Kunitz-PI;1 expression in these organs and tissues. No transmitting tract (NTT) and HECATE 1 (HEC1), two transcription factors previously implicated in the formation of the female reproductive tract in flowers of Arabidopsis, were identified to regulate Kunitz-PI;1 expression in the dark and during greening, with NTT acting negatively and HEC1 acting positively. Laboratory feeding experiments with isopod crustaceans such as Porcellio scaber (woodlouse) and Armadillidium vulgare (pillbug) pinpointed the apical hook as ethylene-protected Achilles’ heel of etiolated seedlings. Because exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and mechanical stress (wounding) strongly up-regulated HEC1-dependent Kunitz-PI;1 gene expression, our results identify a new circuit controlling herbivore deterrence of etiolated plants in which Kunitz-PI;1 is involved.

Dual anti-idiotypic purification of a novel, native-format biparatopic anti-MET antibody with improved in vitro and in vivo efficacy.

Bispecific antibodies are of great interest due to their ability to simultaneously bind and engage different antigens or epitopes. Nevertheless, it remains a challenge to assemble, produce and/or purify them. Here we present an innovative dual anti-idiotypic purification process, which provides pure bispecific antibodies with native immunoglobulin format. Using this approach, a biparatopic IgG1 antibody targeting two distinct, HGF-competing, non-overlapping epitopes on the extracellular region of the MET receptor, was purified with camelid single-domain antibody fragments that bind specifically to the correct heavy chain/light chain pairings of each arm. The purity and functionality of the anti-MET biparatopic antibody was then confirmed by mass spectrometry and binding experiments, demonstrating its ability to simultaneously target the two epitopes recognized by the parental monoclonal antibodies. The improved MET-inhibitory activity of the biparatopic antibody compared to the parental monoclonal antibodies, was finally corroborated in cell-based assays and more importantly in a tumor xenograft mouse model. In conclusion, this approach is fast and specific, broadly applicable and results in the isolation of a pure, novel and native-format anti-MET biparatopic antibody that shows superior biological activity over the parental monospecific antibodies both in vitro and in vivo.

Proteolytic fragments of fibronectin function as matrikines driving the chemotactic affinity of prostate cancer cells to human bone marrow mesenchymal stromal cells via the α5β1 integrin

ABSTRACTThe haematopoietic niche is contributed to by bone marrow-resident mesenchymal stromal cells (BM-MSCs) and subverted by prostate cancer cells. To study mechanisms by which BM-MSCs and prostate cancer cells may interact, we assessed the migration, invasion, adhesion and proliferation of bone-derived prostate cancer cells (PC-3) in co-culture with pluripotent human BM-MSCs. We observed a strong adhesive, migratory and invasive phenotype of PC-3 cells with BM- MSC-co-culture and set out to isolate and characterize the bioactive principle. Initial studies indicated that chemotaxis was secondary to a protein residing in the >100kDa fraction. Size-exclusion chromatography (SEC) recovered peak activity in a high-molecular weight fraction containing thrombospondin-1 (TSP1). While TSP1 immunodepletion decreased activity, put-back with purified TSP1 did not reproduce bioactivity. Further purification of the TSP1-containing high-molecular weight fraction of the BM-MSC secretome with heparin-affinity chromatography recovered bioactivity with highly restricted bands on polyacrylamide gel electrophoresis, determined by mass spectroscopy to be proteolytic fragments of fibronectin (FN). Put-back experiments with full-length FN permitted adhesion but failed to induce migration. Monospecific antibodies to FN blocked adhesion. Proteolytic cleavage of FN generated FN fragments which now induced migration. Neutralizing monoclonal antibodies to FN receptors α5 and β1 integrins, and α5 knockdown specifically blocked migration and adhesion. Conclusion: Fibronectin fragments (FNFr) function as matrikines driving the chemotactic affinity of prostate cancer cells via the α5β1 integrin. Taken together with the high-frequency of α5β1 expression in disseminated prostate cancer cells in bone marrow aspirates from patients, the FNFr/FN-α5β1 interaction warrants further study as a therapeutic target.

Measurement of anti-DFS70 antibodies in patients with ANA-associated autoimmune rheumatic diseases suspicion is cost-effective.

The presence of antinuclear antibodies (ANA) is associated with a wide range of ANA-associated autoimmune rheumatic diseases (AARD). The most commonly method used for the detection of ANA is indirect immunofluorescence (IIF) on HEp-2 cells. This method is very sensitive but unspecific. As a consequence, ANA testing on HEp-2 substrates outside a proper clinical specialist framework may lead to inappropriate referrals to tertiary care specialists and, worst case inappropriate and potentially toxic therapy for the patient. Among ANA, isolated anti-DFS70 antibodies represent a potentially important biomarker that can be clinically used to discriminate AARD from non-AARD patients in ANA IIF positive individuals. Therefore, their presence may avoid unnecessary follow-up testing and referrals. In our study, we investigated if the implementation of a new ANA workup algorithm allowing for the identification of anti-DFS70 antibodies is cost-effective through the reduction of both unnecessary follow-up testing and outpatient clinic visits generated by the clinical suspicion of a potential AARD. None of the 181 patients included with a positive monospecific anti-DFS70 antibody result developed SARD during the follow-up period of 10 years. The reduction in number of tests after ANA and anti-DFS70 positive results was significant for anti-ENA (230 vs. 114 tests; p < 0.001) and anti-dsDNA antibodies (448 vs. 114 tests; p < 0.001). In addition, the outpatient clinic visits decreased by 70 % (p < 0.001). In total, the adoption of the new algorithm including anti-DFS70 antibody testing resulted in a cost saving of 60869.53 € for this pilot study. In conclusion, the use of anti-DFS70 antibodies was clearly cost-efficient in our setting.

Abstract 4950: Collagen, type I, alpha 1 (COL1A1): a potential urinary biomarker that can distinguish between benign prostate hyperplasia and localized prostate cancer

Abstract Benign prostate hyperplasia (BPH) is the most frequent benign disease among men worldwide and its incidence increases with age. Prostate cancer (PCa) and BPH share similar symptoms and an elevated serum prostate-specific antigen (PSA) can be observed with either benign or malignant growth of the prostate. It is the demographic overlap of BPH and PCa, and the lack of discrimination between these two prostate diseases by PSA, that defines the diagnostic dilemma faced by clinicians when treating prostate disease. The goal of the current study was to identify novel non-invasive, biomarkers that can distinguish between BPH and PCa. We utilized iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) mass spectrometry techniques to sensitively and accurately identify the urinary proteome of men with BPH vs. men with PCa. We then performed functional enrichment analysis and pathways enrichment analysis of these proteins using Ingenuity Pathway Analysis (IPA) tools to determine differentially expressed pathways and functions in PCa as compared to BPH. Network analysis identified differences in a number of signaling molecules including ERK1/2, TGFβ, PI3K, p38 MAPK and NFκB, with a high degree of interactivity. Protein expression was validated by immunoblot analyses using monospecific antibodies. Here, we focused on collagen, type I, alpha 1 (COL1A1), one of a number of extracellular matrix-associated proteins detected. We found that COL1A1 was significantly (P &amp;lt; 0.001) elevated in the urine of patients diagnosed with early or localized PCa but not in the urine of men with BPH. Complementary studies were conducted on human BPH and PCa cells. Our data suggest that COL1A1 may represent a novel non-invasive biomarker that can discriminate between BPH and early PCa. (Supported by The Ellison Foundation) Citation Format: Andrej Jedinak, Camille Vuichoud, Andrew El-Hayek, Kevin R. Loughlin, Marsha A. Moses. Collagen, type I, alpha 1 (COL1A1): a potential urinary biomarker that can distinguish between benign prostate hyperplasia and localized prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4950.

Epitope Mapping of Avian Influenza M2e Protein: Different Species Recognise Various Epitopes.

A common approach for developing diagnostic tests for influenza virus detection is the use of mouse or rabbit monoclonal and/or polyclonal antibodies against a target antigen of the virus. However, comparative mapping of the target antigen using antibodies from different animal sources has not been evaluated before. This is important because identification of antigenic determinants of the target antigen in different species plays a central role to ensure the efficiency of a diagnostic test, such as competitive ELISA or immunohistochemistry-based tests. Interest in the matrix 2 ectodomain (M2e) protein of avian influenza virus (AIV) as a candidate for a universal vaccine and also as a marker for detection of virus infection in vaccinated animals (DIVA) is the rationale for the selection of this protein for comparative mapping evaluation. This study aimed to map the epitopes of the M2e protein of avian influenza virus H5N1 using chicken, mouse and rabbit monoclonal or monospecific antibodies. Our findings revealed that rabbit antibodies (rAbs) recognized epitope 6EVETPTRN13 of the M2e, located at the N-terminal of the protein, while mouse (mAb) and chicken antibodies (cAbs) recognized epitope 10PTRNEWECK18, located at the centre region of the protein. The findings highlighted the difference between the M2e antigenic determinants recognized by different species that emphasized the importance of comparative mapping of antibody reactivity from different animals to the same antigen, especially in the case of multi-host infectious agents such as influenza. The findings are of importance for antigenic mapping, as well as diagnostic test and vaccine development.

VNAR single-domain antibodies specific for BAFF inhibit B cell development by molecular mimicry.

B cell-activating factor (BAFF) plays a dominant role in the B cell homeostasis. However, excessive BAFF promotes the development of autoreactive B-cells and several antibodies have been developed to block its activity. Bispecific antibodies with added functionality represent the next wave of biologics that may be more effective in the treatment of complex autoimmune disease. The single variable domain from the immunoglobulin new antigen receptor (VNAR) is one of the smallest antibody recognition units that could be combined with monospecific antibodies to develop bispecific agents. We isolated a panel of BAFF-binding VNARs with low nM potency from a semi-synthetic phage display library and examined their functional activity. The anti-BAFF VNARs blocked the binding of BAFF to all three of its receptors (BR3, TACI and BCMA) and the presence of the conserved DXL receptor motif found in the CDR3 regions suggests molecular mimicry as the mechanism of antagonism. One clone was formatted as an Fc fusion for functional testing and it was found to inhibit both mouse and human BAFF with equal potency ex vivo in a splenocyte proliferation assay. In mice, subchronic administration reduced the number of immature and transitional intermediates B cells and mature B cell subsets. These results indicate that VNAR single domain antibodies function as selective B-cell inhibitors and offer an alternative molecular format for targeting B-cell disorders.

Functional differences exist in contribution of VH and VL from polyspecific IgM and monospecific IgG antibodies in antigen recognition and virus neutralization functions.

Abstract We analyzed role of individual variable heavy (FdVH) and variable light (FdVL) domains in comparison with VH+VL pair (scFv) originating from a polyspecific bovine IgM, with an exceptionally long CDR3H (61 amino acids), and a monospecific IgG1 antibody in antigen (Ag) recognition and virus neutralization functions. To this end, recombinant FdVH, FdVL and scFv were constructed and expressed in Pichia pastoris from bovine polyspecific IgM and IgG1 encoding cDNA. The scFv1H12 showed polyspecific antigen recognition similar to parent IgM antibody with minor differences. Unlike variable light domain FdVL1H12, variable heavy domain FdVH1H12 recognized multiple antigens that differed from scFv1H12 and the parent IgM antibody. Nevertheless, role of FdVL1H12 in providing structural support to FdVH in antigen recognition is noted. By contrast, the individual FdVH073 and FdVL074, originating from induced BoHV-1 neutralizing IgG1 antibody, recognized target epitope on BoHV-1 relatively weakly when compared to VH+VL pair as scFv3-18L. Both VH and VL domains of induced IgG antibody are required to achieve BoHV-1 neutralization function. To conclude, there exist subtle functional differences in relative contribution of VH and VL from polyspecific IgM and monospecific IgG antibodies in antigen recognition and virus neutralization functions. [Supportd by NSERC Canada]

Su1997 A Mono-Specific Antibody Against Short Isoform of DCLK1 (DCLK1-S) Is a Novel Biomarker of Colon Carcinogenesis in Patients

Su1997 A Mono-Specific Antibody Against Short Isoform of DCLK1 (DCLK1-S) Is a Novel Biomarker of Colon Carcinogenesis in Patients

Generation of BiKEs and TriKEs to Improve NK Cell-Mediated Targeting of Tumor Cells.

Cancer immunotherapies have gained significant momentum over the past decade, particularly with the advent of checkpoint inhibitors and CAR T-cells. While the latter personalized targeted immunotherapy has revolutionized the field, a need for off-the-shelf therapies remains. The ability of NK cells to quickly lyse antibody-coated tumors and potently secrete cytokines without prior priming has made NK cells ideal candidates for antigen-specific immunotherapy. NK cells have been targeted to tumors through two main strategies: mono-specific antibodies and bi/tri-specific antibodies. Mono-specific antibodies drive NK cell antibody-dependent cell-mediated cytotoxicity (ADCC) of tumor cells. Bi/tri-specific antibodies drive re-directed lysis of tumor cells through binding of a tumor antigen and direct binding and crosslinking of the CD16 receptor on NK cells, thus bypassing the need for binding of the Fc portion of mono-specific antibodies. This chapter focuses on the generation of bi- and tri-specific killer engagers (BiKEs and TriKEs) meant to target NK cells to tumors. BiKEs and TriKEs are smaller molecules composed of 2-3 variable portions of antibodies with different specificities, and represent a novel and more versatile strategy compared to traditional bi- and tri-specific antibody platforms.

Identification of the Unique Properties of IgGs and their Heavy chains in Blood Serum of Multiple Sclerosis Patients

Monitoring of multiple sclerosis (MS) requires of molecular markers. Recently, using TCA-precipitation/ extraction and MALDI TOF/TOF mass-spectrometry, we identified in blood serum of multiple sclerosis (MS) patients earlier unknown 46 kDa form of human unconventional myosin IC isoform b (Myo1C) (Myronovskij et al., 2015). During SDS-electrophoresis of TCA-extracted proteins of blood serum of 28 MS patients, 3 additional polypeptides with 55, 50 and 25 kDa molecular masses were detected. Western-blot analysis using monospecific anti-human IgG rabbit HRP-conjugated antibodies showed that 55 and 25 kDa polypeptides belong to heavy and light chains of IgG molecules, while 50 kDa protein corresponds to free heavy chains of IgGs. None of these polypeptides was found in fractions of TCA extracted polypeptides isolated from blood serum of healthy human donors or patients with systemic lupus erythematosis and rheumatoid arthritis.

Novel Immunogenic Epitopes in the NaPi-IIb Protein: Production of Monospecific Antibodies Using Synthetic Peptides Outlined on Isoform Specific Regions of the Type IIb Sodium-Dependent Phosphate Transporter (NaPi-IIb)

NaPi-IIb is a multiple passage protein membrane which is primarily responsible for phosphate uptake in the kidney and in the small intestine. Beyond its physiological functions, their involvement with carcinogenesis was initially described in mid-2003, due to its distinct level of expression in normal and tumor cells of the ovary. Although less common than cervical cancer, epithelial ovarian cancer is considered the most lethal gynecologic malignancy, which is mainly due to diagnosis in the advanced stages as a result of the absence of symptoms during the onset of the disease and the lack of tools for early detection. Here, we produce antibodies that are anti-synthetic peptides that are derived from the regions of second extracellular loop of NaPi-IIb, which is a non-overlapping portion of MX35 epitope. These two 15 distinct amino acid residue peptides, designated as Let#1 and Let#2, are engineered in a very thorough way to detect specific sites only in this isoform, thus excluding cross-reactivity with other carriers of the same family. The lack of immunogenicity of small peptides is surpassed by the conjugation with carrier proteins. Using immunochemical methods, we demonstrate that polyclonal antibodies that are mono-specific for the Let#1 and Let#2 peptides recognize proteins that express similar amino acid sequences during blood circulation. Additionally, using flow cytometry, we identify NaPi-IIb in NIH:OVCAR-3 cells. The clear identification of two shorter peptides on the extracellular loop of NaPi-IIb, which are far from the monoclonal antibody MX35-recognizing epitopes, adds new promising tools for ovarian cancer follow-up and staging.

Dual Agonist Surrobody Simultaneously Activates Death Receptors DR4 and DR5 to Induce Cancer Cell Death.

Death receptors of the TNF family are found on the surface of most cancer cells and their activation typically kills cancer cells through the stimulation of the extrinsic apoptotic pathway. The endogenous ligand for death receptors 4 and 5 (DR4 and DR5) is TNF-related apoptosis-inducing ligand, TRAIL (Apo2L). As most untransformed cells are not susceptible to TRAIL-induced apoptosis, death receptor activators have emerged as promising cancer therapeutic agents. One strategy to stimulate death receptors in cancer patients is to use soluble human recombinant TRAIL protein, but this agent has limitations of a short half-life and decoy receptor sequestration. Another strategy that attempted to evade decoy receptor sequestration and to provide improved pharmacokinetic properties was to generate DR4 or DR5 agonist antibodies. The resulting monoclonal agonist antibodies overcame the limitations of short half-life and avoided decoy receptor sequestration, but are limited by activating only one of the two death receptors. Here, we describe a DR4 and DR5 dual agonist produced using Surrobody technology that activates both DR4 and DR5 to induce apoptotic death of cancer cells in vitro and in vivo and also avoids decoy receptor sequestration. This fully human anti-DR4/DR5 Surrobody displays superior potency to DR4- and DR5-specific antibodies, even when combined with TRAIL-sensitizing proapoptotic agents. Moreover, cancer cells were less likely to acquire resistance to Surrobody than either anti-DR4 or anti-DR5 monospecific antibodies. Taken together, Surrobody shows promising preclinical proapoptotic activity against cancer cells, meriting further exploration of its potential as a novel cancer therapeutic agent.