Anti-idiotypic Antibodies Research Articles

Development of a specific anti-idiotypic nanobody for monitoring aflatoxin M1 in milk and dairy products

Anti-idiotypic antibodies (AIds) display stable protein conformation, offering an alternative to conventional antigens in immunoassays. In this work, an anti-idiotypic variable domain of heavy-chain antibody (VHH) or nanobody named VHH C4 binding to monoclonal antibody (mAb) 2C9 against aflatoxin M1 (AFM1) was screened out from an alpaca immunized phage-displayed library. VHH C4 shows outstanding features in specificity and sensitivity, the IC50 is 0.25 ng/mL and exhibit almost no cross-reactivity with other aflatoxin analogs. As our knowledge, it is the first report for developing an anti-idiotypic nanobody for AFM1. An environment-friendly competitive ELISA was developed using VHH C4 as a coating antigen. The results revealed a linear range from 0.10 ng/mL to 0.60 ng/mL, with a limit of detection (LOD) of 0.05 ng/mL. The recovery of spiked samples (pure milk, yogurt, and milk powder) ranged from 84.1% to 119.1%. The ELISA results of AFM1 content in naturally contaminated samples were in good agreement with those determined by high performance liquid chromatography (HPLC). The results demonstrated that the nanobody VHH C4 is a promising alternative antigen in developing immunological methods for the detection of AFM1 in dairy products.

Enhanced Non-Toxic Immunodetection of Alternaria Mycotoxin Tenuazonic Acid Based on Ferritin-Displayed Anti-Idiotypic Nanobody-Nanoluciferase Multimers.

The non-toxic immunoassay for mycotoxins is being paid more attention due to its advantages of higher safety and cost savings by using anti-idiotype antibodies to substitute toxins. In this study, with tenuazonic acid (TeA), a kind of highly toxic Alternaria mycotoxin as the target, an enhanced non-toxic immunoassay was developed based on the ferritin-displayed anti-idiotypic nanobody-nanoluciferase multimers. First, three specific β-type anti-idiotype nanobodies (AId-Nbs) bearing the internal image of TeA mycotoxin were selected from an immune phage display library. Then, the AId-Nb 2D with the best performance was exploited to generate a nanoluciferase (Nluc)-functionalized fusion monomer, by which a one-step non-toxic immunodetection format for TeA was established and proven to be effective. To further improve the affinity of the monomer, a ferritin display strategy was used to prepare 2D-Nluc fusion multimers. Finally, an enhanced bioluminescent enzyme immunoassay (BLEIA) was established in which the half maximal inhibitory concentration (IC50) for TeA was 6.5 ng/mL with a 10.5-fold improvement of the 2D-based enzyme-linked immunosorbent assay (ELISA). The proposed assay exhibited high selectivities and good recoveries of 80.0-95.2%. The generated AId-Nb and ferritin-displayed AId-Nb-Nluc multimers were successfully extended to the application of TeA in food samples. This study brings a new strategy for production of multivalent AId-Nbs and non-toxic immunoassays for trace toxic contaminants.

An Automated Multicycle Immunoaffinity Enrichment Approach Developed for Sensitive Mouse IgG1 Antibody Drug Analysis in Mouse Plasma Using LC/MS/MS.

In the immuno-oncology field, surrogate mouse monoclonal antibodies are often preferred in establishing proper PK/PD/efficacy correlations as well as supporting anticipated mouse to human translation. Thus, a highly sensitive and specific bioanalytical method is needed in quantifying those surrogate mouse antibodies after dosing in mice. Unfortunately, when specific reagents, such as recombinant target antigen and anti-idiotypic antibody, are not available, measuring mouse surrogate antibody drugs in mice is very challenging for ligand binding assay (LBA) due to the severe cross reactivity potential. Different from LBA, if at least one unique surrogate peptide can be identified from the surrogate antibody sequence, the immunoaffinity enrichment based LC/MS/MS assay may be able to differentiate the analyte response from the high endogenous immunoglobulin background and provide adequate sensitivity. Herein, a new automated multicycle immunoaffinity enrichment method was recently developed to extract a surrogate mouse IgG1 (mIgG1) antibody drug from mouse plasma using a commercially available antimouse IgG1 secondary antibody. In the assay, reuse of the capture antibody up to six times mostly resolved the binding capacity issue caused by the abundant endogenous mIgG1 and made the immunoaffinity enrichment step more cost-effective. Combined with a unique surrogate peptide identified from the antibody, the LC/MS/MS assay achieved a limit of quantitation of 5 ng/mL with satisfactory assay precision, accuracy, and dynamic range. The successful implementation of this novel approach in discovery pharmacokinetic (PK) studies eliminates the dependence on specially generated immunoaffinity capturing reagents.

Using multiple platforms for critical reagents selection process to support pharmacokinetic ligand-binding assay development.

We have evaluated the utility of epitope binning on biolayer interferometry (BLI) as a strategy to funnel the selection of candidate pairs suitable for pharmacokinetic assay development. Totally, 8 anti-Idiotypic monoclonal antibodies in 64 possible combinations were tested by BLI, ELISA and Gyrolab®. Two epitope binning approaches were utilized, in-tandem and classic sandwich. Both formats identified four mutually exclusive bins providing 31 and 25 possible antibody pair combinations, respectively. In contrast, the ELISA and Gyrolab yielded 18 and 9 positive pairs, respectively, with only a partial correlation to the BLI results. Several positive pairs by ELISA and Gyrolab, screened negative by BLI. Just over half of the pairs predicted by BLI were positive on ELISA and less than a quarter were positive on Gyrolab. This evaluation showed, in our case, that BLI was limited in its ability to predict candidate pairs that would be successful in pharmacokinetic method development.

In Vitro and In Vivo Anti-Candida Activity and Structural Analysis of Killer Peptide (KP)-Derivatives.

The previously described decapeptide AKVTMTCSAS (killer peptide, KP), derived from the variable region of a recombinant yeast killer toxin-like anti-idiotypic antibody, proved to exert a variety of antimicrobial, antiviral, and immunomodulatory activities. It also showed a peculiar self-assembly ability, likely responsible for the therapeutic effect in animal models of systemic and mucosal candidiasis. The present study analyzed the biological and structural properties of peptides derived from KP by substitution or deletion of the first residue, leaving unchanged the remaining amino acids. The investigated peptides proved to exert differential in vitro and/or in vivo anti-Candida activity without showing toxic effects on mammalian cells. The change of the first residue in KP amino acidic sequence affected the conformation of the resulting peptides in solution, as assessed by circular dichroism spectroscopy. KP-derivatives, except one, were able to induce apoptosis in yeast cells, like KP itself. ROS production and changes in mitochondrial transmembrane potential were also observed. Confocal and transmission electron microscopy studies allowed to establish that selected peptides could penetrate within C. albicans cells and cause gross morphological alterations. Overall, the physical and chemical properties of the first residue were found to be important for peptide conformation, candidacidal activity and possible mechanism of action. Small antimicrobial peptides could be exploited for the development of a new generation of antifungal drugs, given their relative low cost and ease of production as well as the possibility of devising novel delivery systems.

The Impact of the Hybridoma Technology on the R&D of Idiotypic Antibodies.

The PubMed data set was scanned with the title and abstract term "Idiotype" followed by secondary searches with "Vaccine" and "Clinical trial." The retrieved references were analyzed from the period before and after hybridoma technology (1975). In 1963, Oudin and Kunkel discovered that antibodies against antibodies can be raised to identify determinants unique to an antibody termed idiotype or individual antigenic determinant. Two laboratories reported that anti-idiotypic antibodies can suppress specific antibody responses in mice. In 1974, Jerne proposed a network of idiotypes and anti-idiotypes and the functionality of the idiotype network was confirmed. This prompted the proposal of a symmetrical regulatory immune response. By 1989, the concept and the functional parameters of the immune idiotype network were established in the prehybridoma period. It was not until 1981 that monoclonal anti-idiotypic antibodies were used as tools to study the expression of idiotypic determinants on antibodies and to categorize functional properties in the immune network as network antigens in 1989. Hybridoma-generated monoclonal anti-idiotypic antibodies provided the tools to precisely identify different idiotypic regions on antibodies and test these as targets to induce network cascades. The initial distinction of Ab2s as alpha and beta were expanded to include gamma and delta. The initial concept of Ab2beta being an antigen internal image, used as vaccine, was challenged showing that targeting all idiotopes on B cell receptors can induce specific antibodies. After the discovery of the hybridoma technology a wave of idiotype topic publications occurred, that declined by 2015. In 1985, in this wave of reports on anti-idiotypes, their importance to vaccines dominated. These vaccines targeted in animal models parasite, bacterial, and viral diseases, and cancer. The reported data indicated a therapeutic response in inbred mice. The issue of idiotype matching between mouse haplotypes of vaccine origin and treated mice were raised. In 1995, the human clinical trials in different cancers using anti-Id vaccines were reported. Only one such vaccine received conditional approval in Argentina and Cuba, whereas the other trials failed in phase II and III. The reasons for this failure were subsequently discussed. Although the use of the Milstein and Kohler hybridoma technology and subsequently alternative methods to produce monoclonal animal and human antibodies created a new class of drugs, commonly referred as "Biological," it failed on the promise therapeutic of anti-Id vaccines.

New immunological markers for the identification of lung cancer risk groups among workers in hazardous industries in Kuzbass region

Polycyclic aromatic hydrocarbons are widely known as risk factor in relation to lung cancer development. In this connection, we suggested to use idiotypic and anti-idiotypic antibodies IgG and IgA classes against polycyclic aromatic hydrocarbons in human blood serum as new biomarkers for lung cancer risk. The blood serums of 202 healthy men and 275 men with lung cancer were analyzed by ELISA based on idiotypic and anti-idiotypic antibodies IgG and IgA classes against polycyclic aromatic hydrocarbons. Obtained data were analyzed. It was suggested to use ratio of idiotypic and anti-idiotypic antibodies rather than distinguish level each of them separately. The neural networks for idiotypic and anti-idiotypic antibodies of IgG class (AUC = 0.95), idiotypic and anti-idiotypic antibodies of IgA class (AUC = 0.86), and idiotypic and anti-idiotypic antibodies of IgA and IgG classes (AUC = 0.93) were built as models for lung cancer predictions. Finally, the ELISA data of 52 Kuzbass region coal miners were identified as a group of lung cancer risk using obtained models. So, suggested markers antibodies in human blood serum were not only identified lung cancer patients also elicited group of lung cancer risk among healthy people.

Recombinant Anti-idiotypic Antibodies in Ligand Binding Assays for Antibody Drug Development.

Sensitive and reproducible pharmacokinetic (PK) assays and immunogenicity assessment are required as part of the complex and lengthy development process for biotherapeutic proteins. Ligand binding assays (LBAs) are included in a range of approaches applied to understand the nature and properties of the drug as well as the induction of anti-drug antibodies (ADA) against the therapeutic, which can cause adverse events and loss of efficacy. Currently, most biotherapeutics are monoclonal human or humanized antibodies. Anti-idiotypic antibodies, targeting the idiotopic determinants of individual antibody drugs are recognized as perfect reagents for such LBAs. Here we describe the typical setups for these assays and how different types of anti-biotherapeutic antibodies can be used to establish selective and sensitive assays.

Development and Therapeutic Potential of Foot-and-Mouth Disease Virus Specific Anti-Idiotype Antibodies

Development and Therapeutic Potential of Foot-and-Mouth Disease Virus Specific Anti-Idiotype Antibodies

Phage Display Selection of an Anti-Idiotype-Antibody with Broad-Specificity to Deoxynivalenol Mycotoxins.

The use of synthetic antibody libraries and phage displays provides an efficient and robust method for the generation of antibodies against a wide range of targets with highly specific binding properties. As the in vitro selection conditions can be easily controlled, these methods enable the rapid generation of binders against difficult targets such as toxins and haptens. In this study, we used deoxynivalenol mycotoxin as a target to generate anti-idiotype-antibodies with unique binding properties from synthetic antibody libraries. The binding of the selected anti-idiotype antibodies can be efficiently inhibited with the addition of free isoforms of deoxynivalenol. The antibody was consecutively used to develop deoxynivalenol-specific ELISA and TRF-immunoassays, which can detect deoxynivalenol and two of the most common metabolic isoforms in the range of 78–115 ng/mL.

Rapid identification of anti-idiotypic mAbs with high affinity and diverse epitopes by rabbit single B-cell sorting-culture and cloning technology.

The proactive generation of anti-idiotypic antibodies (anti-IDs) against therapeutic antibodies with desirable properties is an important step in pre-clinical and clinical assay development supporting their bioanalytical programs. Here, we describe a robust platform to generate anti-IDs using rabbit single B cell sorting-culture and cloning technology by immunizing rabbits with therapeutic drug Fab fragment and sorting complementarity determining regions (CDRs) specific B cells using designed framework control as a negative gate to exclude non-CDRs-specific B cells. The supernatants of cultured B cells were subsequently screened for binding to drug-molecule by enzyme-linked immunosorbent assay and the positive hits of B cell lysates were selected for cloning of their immunoglobulin G (IgG) variable regions. The recombinant monoclonal anti-IDs generated with this method have high affinity and specificity with broad epitope coverage and different types. The recombinant anti-IDs were available for assay development to support pharmacokinetic (PK) and immunogenicity studies within 12 weeks from the start of rabbit immunization. Using this novel rapid and efficient in-house approach we have generated a large panel of anti-IDs against a series of 11 therapeutic antibody drugs and successfully applied them to the clinical assay development.

Preparation of a Growth Hormone Receptor/Prolactin Receptor Bispecific Antibody Antagonist Which Exhibited Anti-Cancer Activity.

Prolactin receptor (PRLR) and growth hormone receptor (GHR) are closely related to the occurrence and development of breast cancer, and breast cancer cell endogenously express GHR, PRLR and GHR-PRLR heterodimer. In this case, the combined use of PRLR or GHR inhibitors may produce better anti-breast cancer potential than PRLR or GHR inhibitors alone. In this case, it is necessary to develop the dual-function GHR/PRLR antagonists with anti-breast cancer potential. For this, we used hybridoma technology to generate an anti-idiotypic antibody (termed H53). We then used various techniques, including competitive ELISA, competitive receptor binding analysis, and indirect immunofluorescence assay to identify H53, and the results show that H53 behaves as a typical internal image anti-idiotypic antibody (Ab2β). Further experiments indicate that H53 is a dual-function inhibitor, which not only inhibited PRLR-mediated intracellular signaling, but also blocked GHR-mediated intracellular signaling in a dose-dependent manner. Furthermore, H53 could inhibit PRL/GH-driven cancer cell proliferation in vivo and in vitro. This study indicates that H53 exhibits potential biological activity against breast tumors, which implies that internal image anti-idiotypic antibodies may be a useful strategy for the development of PRLR/GHR dual-function antagonists for breast cancer therapy.

Mitochondria Autoimmunity and MNRR1 in Breast Carcinogenesis: A Review.

We review here the evidence for participation of mitochondrial autoimmunity in BC inception and progression and propose a new paradigm that may challenge the prevailing thinking in oncogenesis by suggesting that mitochondrial autoimmunity is a major contributor to breast carcinogenesis and probably to the inception and progression of other solid tumors. It has been shown that MNRR1 mediated mitochondrial-nuclear function promotes BC cell growth and migration and the development of metastasis and constitutes a proof of concept supporting the participation of mitochondrial autoimmunity in breast carcinogenesis. The resemblance of the autoantibody profile in BC detected by IFA with that in the rheumatic autoimmune diseases suggested that studies on the autoantibody response to tumor associated antigens and the characterization of the mtDNA- and nDNA-encoded antigens may provide functional data on breast carcinogenesis. We also review the studies supporting the view that a panel of autoreactive nDNA-encoded mitochondrial antigens in addition to MNRR1 may be involved in breast carcinogenesis. These include GAPDH, PKM2, GSTP1, SPATA5, MFF, ncRNA PINK1-AS/DDOST as probably contributing to BC progression and metastases and the evidence suggesting that DDX21 orchestrates a complex signaling network with participation of JUND and ATF3 driving chronic inflammation and breast tumorigenesis. We suggest that the widespread autoreactivity of mtDNA- and nDNA-encoded mitochondrial proteins found in BC sera may be the reflection of autoimmunity triggered by mitochondrial and non-mitochondrial tumor associated antigens involved in multiple tumorigenic pathways. Furthermore, we suggest that mitochondrial proteins may contribute to mitochondrial dysfunction in BC even if mitochondrial respiration is found to be within normal limits. However, although the studies show that mitochondrial autoimmunity is a major factor in breast cancer inception and progression, it is not the only factor since there is a multiplex autoantibody profile targeting centrosome and stem cell antigens as well as anti-idiotypic antibodies, revealing the complex signaling network involved in breast carcinogenesis. In summary, the studies reviewed here open new, unexpected therapeutic avenues for cancer prevention and treatment of patients with cancer derived from an entirely new perspective of breast carcinogenesis.

Specific Targeting of Lymphoma Cells Using Semisynthetic Anti-Idiotype Shark Antibodies.

The B-cell receptor (BCR) is a key player of the adaptive immune system. It is a unique part of immunoglobulin (Ig) molecules expressed on the surface of B cells. In case of many B-cell lymphomas, the tumor cells express a tumor-specific and functionally active BCR, also known as idiotype. Utilizing the idiotype as target for lymphoma therapy has emerged to be demanding since the idiotype differs from patient to patient. Previous studies have shown that shark-derived antibody domains (vNARs) isolated from a semi-synthetic CDR3-randomized library allow for the rapid generation of anti-idiotype binders. In this study, we evaluated the potential of generating patient-specific binders against the idiotype of lymphomas. To this end, the BCRs of three different lymphoma cell lines SUP-B8, Daudi, and IM-9 were identified, the variable domains were reformatted and the resulting monoclonal antibodies produced. The SUP-B8 BCR served as antigen in fluorescence-activated cell sorting (FACS)-based screening of the yeast-displayed vNAR libraries which resulted after three rounds of screening in the enrichment of antigen-binding vNARs. Five vNARs were expressed as Fc fusion proteins and consequently analyzed for their binding to soluble antigen using biolayer interferometry (BLI) revealing binding constants in the lower single-digit nanomolar range. These variants showed specific binding to the parental SUP-B8 cell line confirming a similar folding of the recombinantly expressed proteins compared with the native cell surface-presented BCR. First initial experiments to utilize the generated vNAR-Fc variants for BCR-clustering to induce apoptosis or ADCC/ADCP did not result in a significant decrease of cell viability. Here, we report an alternative approach for a personalized B-cell lymphoma therapy based on the construction of vNAR-Fc antibody-drug conjugates to enable specific killing of malignant B cells, which may widen the therapeutic window for B-cell lymphoma therapy.

CD58 Aberrations Limit Durable Responses to CD19 CAR in Large B Cell Lymphoma Patients Treated with Axicabtagene Ciloleucel but Can be Overcome through Novel CAR Engineering

CD19 CAR T cells have revolutionized the treatment of relapsed and refractory (R/R) large B cell lymphomas (LBCL), mediating durable complete responses in approximately 40-50% of patients. Besides a loss or decrease in CD19 expression, no studies have identified tumor specific factors driving inherent or acquired resistance to CAR T cells in LBCL. Mutations in and loss of expression of LFA-3 (CD58) have been described in approximately 20% of cases of LBCL. As the ligand for CD2 on T cells, CD58 provides costimulation to T cells and CD58 loss or mutation has been linked to immune resistance in LBCL. We evaluated CD58 status in fifty-one R/R LBCL patients treated at Stanford with commercial axicabtagene ciloleucel (axi-cel) through immunohistochemistry (IHC) on tumor biopsy samples and/or deep sequencing of circulating tumor DNA by CAPP-Seq. We identified 12/51 (24%) patients with a CD58 aberration (lack of expression by IHC or mutation by CAPP-Seq). Progression-free survival (PFS) was significantly decreased in patients with a CD58 aberration (median PFS for CD58 aberration 3 months vs. not reached for CD58 intact, p<0.0001). In fact, only 1/12 patients with a CD58 alteration achieved a durable, complete response to axi-cel, while the remaining 11 patients progressed, most commonly after a period of initial response. Partial responses were more common among patients with CD58 aberrations (58% for CD58 aberration vs 10% for CD58 intact, p<0.001), and complete responses were less common (25% for CD58 aberration vs 82% for CD58 intact, p<0.0001). To probe the biology of CAR T cell responses towards tumors lacking functional CD58, we generated a CD58 knockout Nalm6 model. CD19.CD28.ζ, CD19.4-1BB.ζ, and CD22.4-1BB.ζ CAR T cells demonstrated significantly reduced cytokine production and cytolytic activity in response to CD58 KO vs wildtype (WT) tumor cells. Additionally, while mice inoculated with WT Nalm6 and treated with any of the three CARs demonstrate complete responses and prolonged leukemia-free survival, mice inoculated with CD58KO Nalm6 demonstrated only partial responses, eventual tumor progression, and death from leukemia. CD2, the T cell ligand for CD58, plays both an adhesive role and a costimulatory role in T cells. CD2 knockout resulted in significantly reduced cytokine production after CAR stimulation. Re-expression of only the CD2 extracellular domain did not rescue CAR function, indicating that CD2 signaling is essential for full CAR activation. Additionally, when we stimulated CD19 CAR T cells with anti-idiotype antibody (CAR stimulation), soluble CD58 (CD2 stimulation), or both, we observed significantly enhanced phosphorylation of both CD3ζ and ERK by western blot in CAR T cells stimulated through both the CAR and CD2. Phosphorylation analysis by mass spectrometry revealed that CD2 stimulation enhances phosphorylation of proximal signaling molecules in the TCR pathway (LCK, LAT, CD3ε among others) and also mediators of actin-cytoskeletal rearrangement in CAR T cells, consistent with effects in natural T cell responses. To overcome CD58 loss in LBCL, we generated second- and third-generation CAR T cell constructs integrating CD2 costimulatory domains within the CAR molecule. While these cis constructs demonstrated increased potency against CD58KO cells in vitro, they were unable to ultimately overcome CD58 loss in vivo. However, when CARs were co-expressed with an additional CD2 receptor in trans, they mediated significant anti-tumor activity in vivo, overcoming CD58 knockout in tumor cells. In conclusion, we have identified that CD58 status is an important biomarker for durable response to CAR T cells in LBCL. We modeled the biologic basis for this finding and generated CAR T cells capable of overcoming CD58 loss in B cell malignancies. CD58 mutations have been reported in many cancers, including multiple myeloma and colon cancer, and are likely to play a role in immune evasion for CAR T cells as they are developed for additional histologies. These data provide rationale for investigating CD58 status for patients receiving CAR based therapeutics and devising next generation CARs capable of overcoming this newly discovered mechanism of resistance. Disclosures Majzner: Xyphos Biopharma: Consultancy; Zai Lab: Consultancy; Lyell Immunopharma: Consultancy; GammaDelta Therapeutics: Membership on an entity's Board of Directors or advisory committees; Aprotum Group: Consultancy; Illumina Radiopharmaceuticals: Consultancy. Kurtz:Roche: Consultancy; Genentech: Consultancy; Foresight Diagnostics: Other: Ownership. Sotillo:Lyell Immunopharma: Consultancy, Other: Consultancy. Alizadeh:Janssen: Consultancy; Genentech: Consultancy; Pharmacyclics: Consultancy; Chugai: Consultancy; Celgene: Consultancy; Gilead: Consultancy; Roche: Consultancy; Pfizer: Research Funding. Miklos:Miltenyi Biotec: Research Funding; Janssen: Consultancy, Other: Travel support; Pharmacyclics: Consultancy, Other: Travel support, Patents & Royalties, Research Funding; Novartis: Consultancy, Other: Travel support, Research Funding; Allogene Therapeutics Inc.: Research Funding; Juno-Celgene-Bristol-Myers Squibb: Consultancy, Other: Travel support, Research Funding; Kite-Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Adaptive Biotech: Consultancy, Other: Travel support, Research Funding. Mackall:BMS: Consultancy; Allogene: Current equity holder in publicly-traded company; Apricity Health: Consultancy, Current equity holder in private company; Nektar Therapeutics: Consultancy; NeoImmune Tech: Consultancy; Lyell Immunopharma: Consultancy, Current equity holder in private company.

Orvacabtagene Autoleucel (orva-cel; JCARH125): A Fully Human BCMA-Targeted Second-Generation CAR T Cell Product Characterized By a Predominant Central Memory Phenotype with High in Vitro and In Vivo Proliferative Potential and Sustained In Vivo Persistence

Background: Orva-cel is an investigational B-cell maturation antigen (BCMA)-targeted chimeric antigen receptor (CAR) T cell product genetically modified with a lentiviral vector to express a CAR construct with a unique fully human single-chain variable fragment, optimized spacer, and 4-1BB costimulatory and CD3ζ activation domains. Orva-cel is currently being evaluated for efficacy and safety in the ongoing phase 1/2 EVOLVE study (NCT03430011) in heavily pretreated patients with relapsed/refractory multiple myeloma. We characterized orva-cel drug products, manufactured using the process in place for the phase 2 portion of the study and intended for commercial manufacturing, for CAR+ T cell purity, phenotype, and function. Methods: Immunophenotyping was performed by flow cytometry of both surface and intracellular markers, including CD3, CD4, CD8, CD45, CCR7, CD45RA, CD28, CD27, and active caspase 3. Cytokine production after challenge with BCMA+ target cells was assessed by intracellular cytokine staining and Luminex multiplex assay of secreted cytokines, including interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and granzyme B (GrB). CAR-mediated in vitro proliferative capacity was measured after anti-idiotypic antibody stimulation using the IncuCyte Live-Cell Analysis System (Sartorius, Göttingen, Germany). In vivo CAR+ T cell proliferation and persistence were assessed by quantitative polymerase chain reaction (qPCR). Results: The orva-cel manufacturing process was designed to enable consistent production of highly pure CD3+ cell products (median frequency of CD3+ T cells, 99.96%; quartiles 1-3 interquartile range, 99.9%-100.0%; n = 81). Orva-cel drug products were characterized by high frequencies of less-differentiated CAR+ T cells, leading to a dominant central memory-like population (CCR7+CD45RA- CAR+ T cells) and substantial frequencies of naïve-like cells (CCR7+CD45RA+ CAR+ T cells) (Figure). When assayed for in vitro functional activity, orva-cel drug products showed robust antigen-specific cytokine and effector molecule production (IFN-γ, TNF-α, IL-2, and GrB) upon challenge with BCMA+ tumor cells, as well as vigorous proliferation in response to CAR stimulation. Preliminary correlative analysis suggested that the early memory phenotype may be linked to increased CAR+ T cell proliferative capacity, as determined by in vitro experiments and in vivo PK parameters (ie, maximum CAR+ T cell concentration observed in the blood [Cmax], time to Cmax, area under the curve from Day 0 to 28 [AUC0-28], and CAR+ T cell persistence at Month 3 and Month 6). Consistent with the early CAR T cell memory phenotype, qPCR analysis showed robust in vivo proliferation of CAR+ T cells after infusion, with a median Cmax of 1.54 × 105 transgene copies/µg DNA and median AUC0-28 of 1.61 × 106 transgene copies/µg DNA*day, as well as long-term in vivo persistence, with CAR+ T cells detected in 69% of patients at 6 months postinfusion. Conclusions: The orva-cel manufacturing process results in drug products characterized by highly pure T cells, with high frequencies of early memory and polyfunctional CAR+ T cells. Orva-cel drug products showed robust antigen-specific degranulation, production of multiple cytokines, sustained in vitro and in vivo proliferation, and in vivo persistence. Disclosures Colonna: Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Navarro:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Devries:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Beckett:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment. Amsberry:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Radhakrishnan:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Piasecki:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Heipel:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Li:Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment; Bristol-Myers Squibb Company: Current equity holder in publicly-traded company. Kavita:Bristol Myers Squibb Company: Current Employment, Current equity holder in publicly-traded company. Works:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment. Mujacic:Bristol-Myers Squibb Company: Current equity holder in publicly-traded company; Juno Therapeutics, a Bristol-Myers Squibb Company: Current Employment.

Preparation and identification of an anti-idiotypic antibody antagonist (FG8) for EGFR that shows potential activity against liver cancer cells.

Currently, there are two categories of epidermal growth factor receptor (EGFR) antagonists, small molecule antagonists and anti-EGFR antibodies. In the current study, we developed a new EGFR antagonist employing the anti-idiotypic antibodies strategy. First, using EGF as an antigen, through a series of immunological protocols and hybridoma technology, we obtained an anti-idiotypic antibody against EGF receptor-binding epitopes. On this basis, we screened and characterized the anti-idiotype antibodies against EGFR through competitive ELISA, co-localization analysis, competitive receptor binding analysis, and immunofluorescence. Finally, an internal image anti-idiotype antibody called FG8 was successfully prepared. Experiment result shows that FG8 inhibits EGFR-mediated signaling pathways in vitro. Additionally, FG8 inhibits liver tumor cell proliferation as well as induces tumor cell apoptosis. The present study suggests that FG8 is a potential therapeutic agent for liver cancer. In addition, this study provides a novel method for the preparation of EGFR antagonists.

Immunomodulatory potential of anti-idiotypic antibodies for the treatment of autoimmune diseases.

The immune system is a complex network of specialized cells and organs that recognises and reacts against foreign pathogens while remaining unresponsive to host tissues. This ability to self-tolerate is known as immunological tolerance. Autoimmune disease occurs when the immune system fails to differentiate between self and non-self antigens and releases autoantibodies to attack our own cells. Anti-idiotypic (anti-ID) antibodies are important in maintaining a balanced idiotypic regulatory network by neutralising and inhibiting the secretion of autoantibodies. Recently, anti-ID antibodies have been advanced as an alternative form of immunotherapy as they can specifically target autoantibodies, cause less toxicity and side effects, and could provide long-lasting immunity. This review article discusses the immunomodulatory potential of anti-ID antibodies for the treatment of autoimmune diseases.

Activation and degranulation of CAR-T cells using engineered antigen-presenting cell surfaces.

Adoptive cell transfer of Chimeric Antigen Receptor (CAR)-T cells showed promising results in patients with B cell malignancies. However, the detailed mechanism of CAR-T cell interaction with the target tumor cells is still not well understood. This work provides a systematic method for analyzing the activation and degranulation of second-generation CAR-T cells utilizing antigen-presenting cell surfaces. Antigen-presenting cell surfaces composed of circular micropatterns of CAR-specific anti-idiotype antibodies have been developed to mimic the interaction of CAR-T cells with target tumor cells using micro-contact printing. The levels of activation and degranulation of fixed non-transduced T cells (NT), CD19.CAR-T cells, and GD2.CAR-T cells on the antigen-presenting cell surfaces were quantified and compared by measuring the intensity of the CD3ζ chain phosphorylation and the Lysosome-Associated Membrane Protein 1 (LAMP-1), respectively. The size and morphology of the cells were also measured. The intracellular Ca2+ flux of NT and CAR-T cells upon engagement with the antigen-presenting cell surface was reported. Results suggest that NT and CD19.CAR-T cells have comparable activation levels, while NT have higher degranulation levels than CD19.CAR-T cells and GD2.CAR-T cells. The findings show that antigen-presenting cell surfaces allow a quantitative analysis of the molecules involved in synapse formation in different CAR-T cells in a systematic, reproducible manner.

Purification and Characterization of Rabies Anti-Idiotype Antibodies Isolated from Chicken Egg Yolks

Purification and Characterization of Rabies Anti-Idiotype Antibodies Isolated from Chicken Egg Yolks