Antigen-binding Capacity Research Articles

Glycan profile of CHO derived IgM purified by highly efficient single step affinity chromatography

Immunoglobulin M (IgM) antibodies are reckoned as promising tools for therapy and diagnostic approaches. Nevertheless, the commercial success of IgMs is hampered due to bottlenecks in recombinant production and downstream processing. IgMs are large, complex and highly glycosylated proteins that are only stable in a limited range of conditions. To investigate these sensitive IgM antibodies we optimized the elution conditions for a commercially available IgM affinity matrix (CaptureSelect™). Applying a small-scale screening system, we optimized our single step purification strategy for high purity, high yield and retained antigen binding capacity. Here we show that IgMs are sensitive to aggregation at very acidic conditions (pH ≤ 3.0) despite often being used for affinity chromatography. We combined pH 3.5 with a high salt concentration to prevent aggregation during elution. The elution strategy presented in this paper will improve IgM processes for further applications. The herein used IgMs were produced in Chinese hamster ovary (CHO) cells. We present the first detailed glycan analysis of IgM produced in CHO cells with predominantly complex type structures at Asn171, Asn332 and Asn395 and oligomannosidic structures at Asn402 and Asn563 similar to human serum-IgM.

Oriented antibody immobilization on self-assembled monolayers applied as impedance biosensors

Oriented immobilization of antibodies on a sensor chip is crucial for enhancing both the sensitivity and antigen-binding capacity of immunosensors. Here, we report a comparative study of the effect of oriented and random antibody immobilization on the binding efficiency by electrochemical impedance spectroscopy (EIS). Oriented immobilization of anti-myoglobin immunoglobulin G (anti-Myo IgG) was achieved by bonding to an Fc receptor of protein G (PrG) on a self-assembled monolayer (SAM), which results in the myoglobin (Myo) binding sites being exposed outside the sensing surface. Random immobilization of anti-Myo IgG was achieved by direct covalent attachment to the SAM surface. Both immobilizations were applied to interdigitated electrodes to enhance the electrochemical signal, and the Myo biosensor performance was then evaluated by a series of EIS measurements. We found that (i) the rate of the normalized charge transfer resistance for the oriented sample was 3 times higher than that for the random sample and (ii) the detection limit was 0.001 ng/mL, which is the lowest recorded detection limit among Myo immunosensors based on EIS. These findings indicate that oriented antibody immobilization is crucial for preparing highly sensitive EIS-based biosensors.

Surface engineering of poly(methylmethacrylate): Effects on fluorescence immunoassay.

The authors present surface engineering modifications through chemistry of poly(methylmethacrylate) (PMMA) that have dramatic effects on the result of surface-bound fluorescence immunoassays, both for specific and nonspecific signals. The authors deduce the most important effect to be clustering of antibodies on the surface leading to significant self-quenching. Secondary effects are attributable to the formation of sparse multilayers of antibody. The authors compare PMMA as an antibody support surface with ultraviolet-ozone oxidized PMMA and also to substrates that were, after the oxidation, surface modified by a four-unit poly(ethyleneglycol) carboxylic acid (PEG4), a branched tricarboxylic acid, and a series of carboxylic acid-terminated dendrimers, from generation 1.5 to 5.5. Fluorescence immunoassay and neutron reflectometry were used to compare the apparent antibody surface loading, antigen binding and nonspecific binding on these various surfaces using anti-human IgG as a model antibody, chemically coupled to the surface by amide formation. Simple physical adsorption of the antibody on PMMA resulted in a thick antibody multilayer with small antigen binding capacity. On the carboxylated surfaces, with chemical coupling, a simple monolayer was formed. The authors deduce that antibody clustering was driven by conformational inflexibility and high carboxylate density. The PEG4-modified surface was the most conformationally flexible. The dendrimer-modified interfaces showed a collapse and densification. In fluorescence immunoassay, the optimal combination of high specific and low nonspecific fluorescence signal was found for the G3.5 dendrimer.

Bio-Orthogonal Cross-Linking Chemistry Enables In Situ Protein Encapsulation and Provides Sustained Release from Hyaluronic Acid Based Hydrogels

Chemically cross-linked hydrogels are promising systems for protein delivery applications, but their utility may be limited due to the possibility of protein reaction with hydrogel precursors. Herein, a catalyst-free inverse-demand Diels-Alder reaction between tetrazine and norbornene groups was used to demonstrate the bio-orthogonal nature of cross-linking chemistry that is chemically inert to proteins. Tetrazine-modified hyaluronic acid and norbornene-modified polyethylene glycol were used as hydrogel precursors for in situ encapsulation of a model protein, Fab1. Measurement of gelation kinetics demonstrates that network formation and gel stiffness are temperature-dependent but independent of Fab1 concentration. In vitro release testing shows that Fab1 is completely released from the hydrogel matrix over a period of several weeks. Analytical characterization suggests that Fab1 is released without any physical or chemical modifications and retains its antigen binding capacity. Thus, the bio-orthogonal and catalyst-free aqueous phase chemistry enables efficient in situ protein encapsulation in a single step and provides sustained protein release.

Trastuzumab and Pertuzumab Plant Biosimilars: Modification of Asn297-linked Glycan of the mAbs Produced in a Plant with Fucosyltransferase and Xylosyltransferase Gene Knockouts.

Plant biosimilars of anticancer therapeutic antibodies are of interest not only because of the prospects of their practical use, but also as an instrument and object for study of plant protein glycosylation. In this work, we first designed a pertuzumab plant biosimilar (PPB) and investigated the composition of its Asn297-linked glycan in comparison with trastuzumab plant biosimilar (TPB). Both biosimilars were produced in wild-type (WT) Nicotiana benthamiana plant (PPB-WT and TPB-WT) and transgenic ΔXTFT N. benthamiana plant with XT and FT genes knockout (PPB-ΔXTFT and TPB-ΔXTFT). Western blot analysis with anti-α1,3-fucose and anti-xylose antibodies, as well as a test with peptide-N-glycosidase F, confirmed the absence of α1,3-fucose and xylose in the Asn297-linked glycan of PPB-ΔXTFT and TPB-ΔXTFT. Peptide analysis followed by the identification of glycomodified peptides using MALDI-TOF/TOF showed that PPB-WT and TPB-WT Asn297-linked glycans are mainly of complex type GnGnXF. The core of PPB-WT and TPB-WT Asn297-linked GnGn-type glycan contains α1,3-fucose and β1,2-xylose, which, along with the absence of terminal galactose and sialic acid, distinguishes these plant biosimilars from human IgG. Analysis of TPB-ΔXTFT total carbohydrate content indicates the possibility of changing the composition of the carbohydrate profile not only of the Fc, but also of the Fab portion of an antibody produced in transgenic ΔXTFT N. benthamiana plants. Nevertheless, study of the antigen-binding capacity of the biosimilars showed that absence of xylose and fucose residues in the Asn297-linked glycans does not affect the ability of the glycomodified antibodies to interact with HER2/neu positive cancer cells.

Assessing microenvironment immunogenicity using tumor specimen exomes: Co-detection of TcR-α/β V(D)J recombinations correlates with PD-1 expression.

T-cell receptor (TcR) recombinations can be recovered from tumor specimen, whole exome sequences (WXS) files. However, it is not yet clear how these recombinations represent lymphocytes or an anti-tumor immune response. Here we report the identification of productive TcR-β recombinations in WXS files representing primary and metastatic melanoma. The recombinations are identifiable in about 20% of the cancer genome atlas melanoma samples. This frequency of detection is lower than the frequency of TcR-α VJ recombinations, consistent with the occurrence of biallelic TcR-α recombinations and possibly consistent with the fact that only one junctional recombination is required for TcR-α whereas two recombinations are required to form a TcR-β gene. Nevertheless, the ratio of productive TcR-β to unproductive TcR-β samples, in comparison to the ratio of productive to unproductive TcR-α or TcR-γ positive-samples, is very high. This result indicates that detection of a productive TcR-β VDJ recombination represents a comparatively high standard for potential antigen binding capacity, when employing a tumor specimen exome file for the assessment. Additionally, PD-1 expression and antigen presentation functions correlated with the co-detection of TcR-α and -β recombinations (e.g., p < 0.0004), suggesting that co-detection of TcR-α and -β recombinations represents an anti-melanoma response that has been blunted by the advent of PD-1 expression. We further show that the algorithm for detecting the TcR-β VDJ recombinations is applicable to exome files generated from mouse tissue, thus providing for opportunities to develop empirical paradigms for interpreting the identification of TcR V(D)J recombinations in tissue resident lymphocytes.

Development of biohybrid immuno-selective membranes for target antigen recognition

Membranes are gaining increasing interest in solid-phase analytical assay and biosensors applications, in particular as functional surface for bioreceptors immobilization and stabilization as well as for the concentration of target molecules in microsystems.In this work, regenerated cellulose immuno-affinity membranes were developed and they were used for the selective capture of interleukin-6 (IL-6) as targeted antigen. Protein G was covalently linked on the membrane surface and it was successfully used for the oriented site-specific antibody immobilization. The antibody binding capacity of the protein G-coupled membrane was evaluated. The specific anti IL-6 antibody was immobilized and a quantitative analysis of the amount of IL-6 captured by the immuno-affinity membrane was performed. The immobilization procedure was optimized to eliminate the non-specific binding of antigen on the membrane surface. Additionally, the interaction between anti IL-6 antibody and protein G was stabilized by chemical cross-linking with glutaraldehyde and the capture ability of immuno-affinity membranes, with and without the cross-linker, was compared. The maximum binding capacity of the protein G-coupled membrane was 43.8µg/cm2 and the binding efficiency was 88%. The immuno-affinity membranes showed a high IL-6 capture efficiency at very low antigen concentration, up to a maximum of 91%, the amount of captured IL-6 increased linearly as increasing the initial concentration. The cross-linked surface retained the antigen binding capacity demonstrating its robustness in being reused, without antibody leakage or reduction in antibody binding capacity. The overall results demonstrated the possibility of a reliable application of the immuno-affinity membrane developed for biosensors and bioassays also in multiple use.

A comparative analysis of the efficiency of bird and mammalian antibodies in HBsAg sandwich assay

The antigen-binding capacities of chicken and goat polyclonal antibodies and mouse monoclonal antibodies specific for the hepatitis B virus surface antigen were compared via enzyme immunoassay. It was shown that the use of both adsorbed and detecting chicken antibodies reduces significantly the analytical sensitivity of the sandwich method for the detection of this antigen. It was assumed that the reduced flexibility of IgY molecules hinders bivalent binding to antigen, which leads to the formation of more stable immune complexes.

Molecular Characterization of Two Monoclonal Antibodies against the Same Epitope on B-Cell Receptor Associated Protein 31.

Previously, we showed that B-cell receptor associated protein 31 (BAP31), an endoplasmic reticulum (ER) membrane chaperone, is also expressed on the cell surface by two monoclonal antibodies (MAbs) 297-D4 and 144-A8. Both MAbs recognize the same linear epitope on the C-terminal domain of BAP31, although they were independently established. Here, flow cytometric analysis showed that 144-A8 had additional binding properties to some cells, as compared to 297-D4. Quantitative antigen binding assays also showed that 144-A8 had higher antigen binding capacity than 297-D4. Affinity measurement revealed that 144-A8 had 1.54-fold higher binding affinity than 297-D4. Analysis of the heavy- and light-chain variable region sequences of two MAbs revealed that both MAbs belonged to the same heavy chain (Igh-V3660 VH3) and light chain subgroup (IGKV21) with just two amino acid differences in each framework region, indicating that both MAbs arise from the same germline origin. Seven amino acid differences were found between the complementarity determining regions (CDRs) of the two MAbs. Molecular modeling of the epitope-paratope complexes revealed that the epitope appeared to reside in closer proximity to the CDRs of 144-A8 than to those of 297-D4 with the stronger hydrogen bond interactions with the former than the latter. More interestingly, an additional hydrophobic interaction appeared to be established between the leucine residue of epitope and the paratope of 144-A8, due to the substitution of H-Tyr101 for H-Phe101 in 144-A8. Thus, the different binding specificity and affinity of 144-A8 appeared to be due to the different hydrogen bonds and hydrophobic interaction induced by the alterations of amino acids in CDRs of 144-A8. The results provide molecular insights into how the binding specificities and affinities of antibodies evolve with the same epitope in different microenvironments.

Intramolecular trimerization, a novel strategy for making multispecific antibodies with controlled orientation of the antigen binding domains.

Here, we describe a new strategy that allows the rapid and efficient engineering of mono and multispecific trivalent antibodies. By fusing single-domain antibodies from camelid heavy-chain-only immunoglobulins (VHHs) to the N-terminus of a human collagen XVIII trimerization domain (TIEXVIII) we produced monospecific trimerbodies that were efficiently secreted as soluble functional proteins by mammalian cells. The purified VHH-TIEXVIII trimerbodies were trimeric in solution and exhibited excellent antigen binding capacity. Furthermore, by connecting with two additional glycine-serine-based linkers three VHH-TIEXVIII modules on a single polypeptide chain, we present an approach for the rational design of multispecific tandem trimerbodies with defined stoichiometry and controlled orientation. Using this technology we report here the construction and characterization of a tandem VHH-based trimerbody capable of simultaneously binding to three different antigens: carcinoembryonic antigen (CEA), epidermal growth factor receptor (EGFR) and green fluorescence protein (GFP). Multispecific tandem VHH-based trimerbodies were well expressed in mammalian cells, had good biophysical properties and were capable of simultaneously binding their targeted antigens. Importantly, these antibodies were very effective in inhibiting the proliferation of human epidermoid carcinoma A431 cells. Multispecific VHH-based trimerbodies are therefore ideal candidates for future applications in various therapeutic areas.

Clinical Activity and Persistence of Anti-CD22 Chimeric Antigen Receptor in Children and Young Adults with Relapsed/Refractory Acute Lymphoblastic Leukemia (ALL)

Background: Adoptive cellular therapy with genetically modified T-cells using viral-based vectors to express chimeric antigen receptors targeting the CD19 molecule have demonstrated dramatic clinical responses in patients ALL. However, not all patients respond and CD19-negative escape has been observed. Thus, additional targets are needed. CD22 is a B-lineage-restricted, transmembrane phosphoglycoprotein of the Ig superfamily that is widely expressed on B-precursor ALL. Therefore, CD22 represents a promising target. This phase I dose-escalation study represents the first in human testing of anti-CD22 CAR adoptive cell therapy. The primary objectives were to determine the feasibility of producing anti-CD22 CAR cells meeting established release criteria and to assess the safety of administering escalating doses of anti-CD22-CAR engineered T cells in children and young adults with relapsed or refractory CD22-expressing B cell malignancies following a cyclophosphamide/fludarabine preparative regimen. Secondary objectives include determination of anti-leukemia effects, measurement of persistence of adoptively transferred anti-CD22 CAR T cells, and evaluation of cytokine profiles.Design: Children and young adults with relapsed/refractory CD22-expressing hematologic malignancies were eligible. Study endpoints included toxicity, feasibility, and antigen-specific immune and clinical responses. All enrolled subjects underwent autologous leukopheresis for peripheral blood mononuclear cells. Cells were then CD3+ enriched and cultured in the presence of anti-CD3/-CD28 beads followed by lentiviral vector supernatant containing the anti-CD22 (M971BBz) CAR, with culture duration of 7-10 days. On Day-4 (cell infusion=Day 0), subjects began induction chemotherapy with fludarabine 25 mg/m2 on Days-4, -3 and -2 and cyclophosphamide 900 mg/m2 on day-2. The first dose level started at 3 x 105 transduced T-cells/recipient weight (kg).Results: 6 subjects, aged 7-22 years, with ALL have been treated to date. All enrolled subjects had previously undergone at least one prior allogeneic hematopoietic stem cell transplant and all had received treatment with CD19 directed CAR-T cell therapy. Five subjects had a CD19 negative antigen escape, and one subject was a non-responder to prior CD19 CAR therapy. All subjects had demonstration of CD22 expression on > 99% of their ALL, although the antigen binding capacity had variability from < 900 to > 13,000 sites/cells. All subjects underwent successful culture, expansion and infusion of anti-CD22 CAR T-cells at the first dose level. The second subject enrolled met criteria for dose-limiting toxicity by virtue of grade 3 diarrhea which led to dose expansion at the first dose-level to treat a total of 6 subjects. Two subjects had grade 1 cytokine release syndrome (CRS), one subject had grade 2 CRS and in two subjects CRS was not seen. Evidence for CAR-T cell expansion was seen in peripheral blood, bone marrow and cerebrospinal fluid (Table). Clinical responses were evaluated at day 28 (+/- 4 days) post-infusion and included two subjects who had disease progression, two with disease stabilization and one subject who attained a minimal residual disease (MRD) negative complete remission. Flow cytometric CAR persistence was detected out to 47 days post-infusion in the responding patient with remission maintained for 3 months post-infusion. One patient is actively undergoing treatment and is too early to evaluate.Conclusions: This first-in-human anti-CD22 CAR T-cell therapy is safe, feasible and clinically active in patients who have undergone previous CAR therapy. Understanding mechanisms which may determine clinical efficacy are being explored. Accrual to the next dose level at 1 x 106 transduced T cells/kg is planned. Table 1#Age/SexPrior HCTPrior anti-CD19 CARCD19 neg relapse?CD22 site densityPre-HCT disease burden (% leukemia in aspirate)Maximum CD22 CAR expansion (flow)CRSBest ResponsePBMarrowCSF122/MYYY208495-100%00n/aNonePD220/FY (2)YY134525%52.3%19.5%0%1MRD neg CR322/MYYY846>90%73%36%32%1SD422/MYYN258995%6%1%0%2SD57/FYYY283932%0%1.3%0%NonePD617/FYYY21851%n/an/an/an/an/aSD: stable disease; PD: progressive disease DisclosuresMackall:Juno: Patents & Royalties: CD22-CAR.

A New Class of T-Cell Bispecific Antibodies for the Treatment of Multiple Myeloma, Binding to B Cell Maturation Antigen and CD3 and Showing Potent, Specific Antitumor Activity in Myeloma Cells and Long Duration of Action in Cynomolgus Monkeys

A New Class of T-Cell Bispecific Antibodies for the Treatment of Multiple Myeloma, Binding to B Cell Maturation Antigen and CD3 and Showing Potent, Specific Antitumor Activity in Myeloma Cells and Long Duration of Action in Cynomolgus Monkeys

Augmented Binary Substitution: Single-pass CDR germ-lining and stabilization of therapeutic antibodies

Although humanized antibodies have been highly successful in the clinic, all current humanization techniques have potential limitations, such as: reliance on rodent hosts, immunogenicity due to high non-germ-line amino acid content, v-domain destabilization, expression and formulation issues. This study presents a technology that generates stable, soluble, ultrahumanized antibodies via single-step complementarity-determining region (CDR) germ-lining. For three antibodies from three separate key immune host species, binary substitution CDR cassettes were inserted into preferred human frameworks to form libraries in which only the parental or human germ-line destination residue was encoded at each position. The CDR-H3 in each case was also augmented with 1 ± 1 random substitution per clone. Each library was then screened for clones with restored antigen binding capacity. Lead ultrahumanized clones demonstrated high stability, with affinity and specificity equivalent to, or better than, the parental IgG. Critically, this was mainly achieved on germ-line frameworks by simultaneously subtracting up to 19 redundant non-germ-line residues in the CDRs. This process significantly lowered non-germ-line sequence content, minimized immunogenicity risk in the final molecules and provided a heat map for the essential non-germ-line CDR residue content of each antibody. The ABS technology therefore fully optimizes the clinical potential of antibodies from rodents and alternative immune hosts, rendering them indistinguishable from fully human in a simple, single-pass process.

Investigation of Fragment Antibody Stability and Its Release Mechanism from Poly(Lactide-co-Glycolide)-Triacetin Depots for Sustained-Release Applications

Achieving long-term drug release from polymer-based delivery systems continues to be a challenge particularly for the delivery of large hydrophilic molecules such as therapeutic antibodies and proteins. Here, we report on the utility of an in situ-forming and injectable polymer-solvent system for the long-term release of a model antibody fragment (Fab1). The delivery system was prepared by dispersing a spray-dried powder of Fab1 within poly(lactide-co-glycolide) (PLGA)-triacetin solution. The formulation viscosity was within the range 1.0 ± 0.3 Pa s but it was injectable through a 27G needle. The release profile of Fab1, measured in phosphate-buffered saline (PBS), showed a lag phase followed by sustained-release phase for close to 80 days. Antibody degradation during its residence within the depot was comparable to its degradation upon long-term incubation in PBS. On the basis of temporal changes in surface morphology, stiffness, and depot mass, a mechanism to account for the drug release profile has been proposed. The unprecedented release profile and retention of greater than 80% of antigen-binding capacity even after several weeks demonstrates that PLGA-triacetin solution could be a promising system for the long-term delivery of biologics.

Controlling Rotavirus-associated diarrhea: Could single-domain antibody fragments make the difference?

Group A Rotavirus (RVA) remains a leading cause of severe diarrhea and child mortality. The variable domain of camelid heavy chain antibodies (VHH) display potent antigen-binding capacity, have low production costs and are suitable for oral therapies. Two sets of anti-RVA VHHs have been developed: ARP1-ARP3; 2KD1-3B2. Here, we explore the potential of both sets as a prevention strategy complementary to vaccination and a treatment option against RVA-associated diarrhea in endangered populations. Both sets have been expressed in multiple production systems, showing extensive neutralizing capacity against strains of RVA in vitro. They were also tested in the neonatal mouse model with various degrees of success in preventing or treating RVA-induced diarrhea. Interestingly, mitigation of the symptoms was also achieved with freeze-dried ARP1, so that it could be applied in areas where cold chains are difficult to maintain. 3B2 was tested in a pre-clinical trial involving gnotobiotic piglets where it conferred complete protection against RVA-induced diarrhea. ARP1 was used in the first clinical trial for anti-RVA VHHs, successfully reducing stool output in infants with RVA diarrhea, with no detected side effects.

Effects of Protein A in Detection of Canine Distemper Virus Through Immunosensor Construction

We have developed a detection system to canine distemper virus (CDV) by employing surface plasmon resonance (SPR) and compared the effect of oriented and random immobilization. Several parameters, such as the surface concentration of immobilized antibodies, antigen binding capacity, and the maximum surface concentration of immobilized antibody were optimized. The maximal antigen binding capacity was obtained by means of a SPR-sensor immobilized via protein A, which leads to an end-on orientation. CDV at 0.1 ng/mL can be detected using protein A. Urine analysis showed that all samples contained detectable levels of CDV, but the results revealed an improvement in sensitivity of the assay when it is specifically oriented. The described method is simple, fast, selective, and sensitive to the biorecognition element. The results emphasize how important it is to attach proteins on surfaces in such a way that their binding sites are oriented toward the solution phase.

Abstract 1324: A translational platform to design antibodies targeting triple negative breast cancer-specific antigens for cancer immunotherapy

Abstract Triple negative breast cancers (TNBCs) are characterised by the lack of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 (HER2) rendering them insensitive to current endocrine and anti-HER2 therapies, therefore novel treatment options are required. Breast cancers have been shown to be immunogenic and patient tumors feature infiltrating immune cells. Since monoclonal antibodies are an important therapeutic modality in the clinical management of many cancers, engineering antibodies against triple negative breast cancer specific antigens that may activate immune effector cells within tumors and in the circulation may lead to tumour clearance and may provide new treatment strategies for this patient group. Here we describe a platform for selection of TNBC-associated antigens, engineering of novel antibodies against these antigens and functional studies to elucidate antibody mechanisms of action and therapeutic potential in vitro and vivo. Our approach involves identification of tumour-associated antigens from patient tumor specimens using a transcriptomics approach and validation and evaluation of biological functions in breast cancer cells using cell culture models. Antibody heavy (VH) and light (VL) sequences are cloned in a dual expression vector system for recombinant expression in HEK293 cells to rapidly produce antibodies with reactivity to specific tumor antigens. The system is optimised for the seamless expression of V genes of any species including human sequences, along with human constant (C) genes of different antibody classes and subclasses. The latter permits the study of different class/subclass antibodies in order to identify those most effective in triggering immune cell activation and tumor clearance. Engineered monoclonal antibodies are subsequently tested for their antigen binding properties and capacity to activate immune effector cells against cancer cells in vitro. The therapeutic potential of selected candidates is evaluated in vivo using xenograft TNBC mouse models engrafted with components of the human immune system using adoptive transfer of healthy volunteer and patient derived peripheral blood cells. Xenograft models featuring human immune components serve to evaluate the therapeutic potential of monoclonal antibodies in the context of their capacity to redirect immune effector functions against cancer cells. Our findings demonstrate a translational pathway designed to facilitate the discovery and evaluation of novel antibody therapeutic options for triple negative breast cancer immunotherapy. Citation Format: Kristina M. Ilieva, Rebecca Marlow, Anthony Cheung, Erika Francesch, Panagiotis Karagiannis, Matthew Fittall, Silvia Crescioli, Andrew Tutt, Sophia Karagiannis. A translational platform to design antibodies targeting triple negative breast cancer-specific antigens for cancer immunotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1324. doi:10.1158/1538-7445.AM2015-1324

Hyperimmunised bovine milk and whey: influence of pH and enzymatic treatments on the antigen-binding capacity of immunoglobulin G.

Hyperimmunised bovine milk and whey (whole and defatted) were submitted at 37°C to different pH values (between 1 and 10) and enzymes (pepsin, trypsin and chymotrypsin) at their optimum pH and the IgG immunoactivity against Campylobacter jejuni was measured by means of ELISA assays. The kinetic antigen-binding capacity (ABC) losses follow a hyperbolic-type equation. The ABC of IgG is strongly reduced at low pH (1 and 2) and the effect is lower at alkaline pH (8 and 10). The presence of pepsin (at their optimum pH of 2) almost completely reduced the IgG ABC after 2 h of treatment. The ABCs are higher in whole products (milk and whey). The influence of trypsin and quimotrypsin on the ABCs is moderate (ABC losses lower that 25%). The ABC of IgG obtained from hyperimmunised bovine defatted milk and whey is largely reduced in conditions similar to those found in the human digestive tract. Only whole milk can maintain around 40% of their initial ABC. IgG encapsulation or other methods to protect the immunoglobulin activities could be an alternative to use these type of products in final foods formulae.

224. Development of Safer & Optimized CAR-T Cells Using Lentiviral Vectors

Chimeric antigen receptors (CAR) modified T cells have shown very promising clinical results in hematological malignancies. CAR are usually composed of a scFv antigen binding domain linked to a spacer and intracellular signaling domains. CAR introduction into T cells leads to cells with optimal effector functions and with a new antigen binding ability. This strategy is well suited to skirt immune escape mechanisms.Despite their efficacy, CAR-T cells still show toxicity due to a cytokine storm, even if it is manageable. Improving the safety while maintaining or even improving the anti-tumor efficacy of CAR-T cells is therefore a crucial therapeutic challenge.Based on our proprietary lentiviral vector technology, we are developing a CAR-T cell platform aiming at producing innovative CAR-T cells. The development conducted by Theravectys are: -Development of proprietary lentiviral vectors coding for CAR of the second (containing the CD3ζ and the 4-1BB cosignaling domains) and third generations (containing the CD3ζ, the CD28 and the 4-1BB domains) directed against CD19 (for CD19+ leukemias and lymphomas), LMP-1 and -2 (for EBV-induced leukemias). Our lentiviral vectors will allow optimal expression of CAR therefore leading to a better efficacy of CAR-T cells. Hematological malignancies will be used as a benchmark.-Development of an inducible RUSH (Retention Using Selective Hook) system which is based on the streptavidin anchored to the membrane of the endoplasmic reticulum (ER) through a hook, and on the SBP (streptavidin binding protein) introduced into the CAR structure. The interaction between the streptavidin-hook and the SBP-CAR allows the CAR retention inside the ER. The addition of biotin displaces the equilibrium of binding of streptavidin towards biotin instead of SBP, thus leading to the release of the CAR from the ER and its expression to the cytoplasmic membrane. View Large Image | Download PowerPoint SlideThe release of the CAR will stop with drug exhaustion (or antagonist) and remaining cells can be easily reactivated through reintroduction of biotin. This system will increase safety of CAR-T cells. A CD19-CAR RUSH will be used as a proof-of-concept of the RUSH system.Most of the scFv used to date are of murine origin. Neutralizing antibodies against these murine scFv can limit the efficacy of CAR. As an alternative, we are screening and engineering specific humanized camelid nanobodies to be used as binding domains since they are highly homologous to the human VH domain of antibodies and they display high antigen binding capacities. The proof-of-concept will be made with a second generation CAR containing a nanobody directed against Her2 as a binding domain.Theravectys’ technological platforms allows flexibility and reactivity in the CAR design, production and evaluation, thus leading to the fast generation of safe and optimized CAR-T cells.

The novel panel assay to define tumor-associated antigen-binding antibodies in patients with metastatic melanomas may have diagnostic value.

We aim to harness the natural humoral immune response by various technologies to get novel biomarkers. A complex antibody analysis in sera and in the tumor microenvironment leads to reveal tumor-specific antibodies. More strategies were introduced to select the most effective one to identify potential tumor antigen-binding capacity of the host. Epstein-Barr virus transformation and cloning with limiting dilution assay, magnetic cell sorting and antibody phage display with further methodological improvements were used in epithelial and neuroectodermal cancers. Column-purified sera of patient with melanoma were tested by immunofluorescence assay, while sera of further melanoma patients were processed for membrane-binding enzyme-linked immunosorbent assay. Some supernatants of selected B cell clones and purified antibodies showed considerable cancer cell binding capacity by immunofluorescence FACS analysis and confocal laser microscopy. Our native tumor cell membrane preparations helped to test soluble scFv and patients' sera for tumor binder antibodies. A complex tumor immunological study was introduced for patients with melanoma (ethical permission: ETT TUKEB 16462-02/2010); peripheral blood (n=57) and surgically removed primary or metastatic tumors (n=44) were gathered and processed at cellular immunological level. The technological developments proved to be important steps forward to the next antibody profile analyses at DNA sequence level. Cancer cell binding of patient-derived antibodies and natural immunoglobulin preparations of pooled plasma product intravenous immunoglobulins support the importance of natural human antibodies. Important cancer diagnostics and novel anticancer strategies are going to be built on these tools.