Immunogenicity Testing Research Articles

Assessing immunogenicity barriers of the HIV-1 envelope trimer

Understanding the balance between epitope shielding and accessibility on HIV-1 envelope (Env) trimers is essential to guide immunogen selection for broadly neutralizing antibody (bnAb) based vaccines. To investigate the antigenic space of Env immunogens, we created a strategy based on synthetic, high diversity, Designed Ankyrin Repeat Protein (DARPin) libraries. We show that DARPin Antigenicity Analysis (DANA), a purely in vitro screening tool, has the capability to extrapolate relevant information of antigenic properties of Env immunogens. DANA screens of stabilized, soluble Env trimers revealed that stronger trimer stabilization led to the selection of highly mutated DARPins with length variations and framework mutations mirroring observations made for bnAbs. By mimicking heterotypic prime-boost immunization regimens, DANA may be used to select immunogen combinations that favor the selection of trimer-reactive binders. This positions DANA as a versatile strategy for distilling fundamental antigenic features of immunogens, complementary to preclinical immunogenicity testing.

Preclinical development and characterization of a human plasma-derived high-purity factor X concentrate for therapeutic use.

Hereditary factor X deficiency is a rare bleeding disorder, with limited treatment options. This paper describes the approach to pre-clinical development and characterization of a high-purity plasma-derived factor X concentrate, to achieve orphan drug marketing authorization for the treatment of hereditary factor X deficiency. A chromatographic process was developed, to purify factor X from human plasma for fractionation. The product was characterized using in vitro, in vivo and ex vivo tests for potency, purity, thrombogenicity, immunogenicity, toxicity and stability. The production process complied with good pharmaceutical manufacturing practice. It achieved 6000-fold purification and 100-fold concentration of the factor X protein compared to human plasma. The factor X protein was 94%-96% pure. Other residual plasma proteins were well below levels in plasma, minimizing potential interference in hemostasis after therapeutic administration. Effective virus-reduction during manufacture, and the absence of thrombogenicity, toxicity and immunogenic potential were confirmed, addressing the main safety concerns historically associated with plasma-derived therapeutics. The freeze-dried product remained stable between +2°C and +30°C for at least three years. After reconstitution with water for injections, the factor X activity was maintained for at least 48h at +18°C to +22°C. Targeted pre-clinical development of the first highly-purified concentrate to treat hereditary factor X deficiency is described. Following international guidelines for nonclinical safety testing, particular strategies were adopted for unmodified products derived from human blood plasma. This approach may also be relevant to the development of other ultra-orphan medicinal products.

Addressing Domain Specificity in the Development of a Cell-Based Binding Assay for the Detection of Neutralizing Antibodies Against a CD47xPD-L1 Bispecific Antibody

PF-07257876 is a bispecific antibody being developed for the treatment of certain advanced or metastatic solid tumors. To support clinical development of PF-07257876, neutralizing antibody (NAb) assays were developed as part of a tiered immunogenicity testing approach. Because PF-07257876 targets both CD47 and PD-L1, determination of domain specificity of a NAb response may provide additional insight relating to PK, efficacy, and safety. Due to limitations of functional cell systems, two cell-based binding assays were developed using electrochemiluminescence to detect domain-specific NAb. While both NAb assays utilized a cell-based binding approach and shared certain requirements, such as sensitivity and tolerance to potentially interfering substances, the development of each assay faced unique challenges. Among the hurdles encountered, achieving drug tolerance while preserving domain specificity for CD47 proved particularly challenging. Consequently, a sample pretreatment procedure to isolate NAb from potentially interfering substances was necessary. The sample pretreatment procedure developed was based on a bead-extraction and acid dissociation (BEAD) approach. However, the use of the standard BEAD approach with whole drug to capture NAb resulted in loss of NAb detection under certain circumstances. Specifically, mock samples containing a mixture of NAb positive controls against both binding domains of the bispecific antibody produced false-negative results in the cell-based binding assay. An adaptation made to the standard BEAD approach restored domain-specific NAb detection, while also contributing to an assay sensitivity of 1 µg/mL in the presence of a clinically relevant drug tolerance level of up to 400 µg/mL.Graphical

Validation of Luminex immunological and competitive Luminex immunological assays for clinical immunogenicity assessment of a 14-valent recombinant human papillomavirus vaccine.

A novel virus-like particle (VLP)-based multivalent recombinant human papillomavirus (HPV) vaccine was developed and evaluated in human, including 14 HPV-type specific VLP antigens (HPV6, 11, 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59). The pseudovirus-based neutralizing assay (PBNA) method is widely used for immunogenicity assessment of HPV vaccine in clinical trials. However, as many as 14 antigen-specific antibody levels need be determined, PBNA is, for many reasons, challenging and time-consuming. In this study, we developed a Luminex immunological assay (LIA) and a competitive Luminex immunological assay (cLIA). These methods increase the throughput, reproducibility and precision, as well as reduce the complexity. All assay parameters showed good characteristics in the validation of both methods, benefiting from highly purified and structurally correct VLPs, high specific antibodies, standard VLP-microspheres and PE-mAbs conjugating process, adequate assay development and stable system. Validation data support the use of both methods for immunogenicity assessment in clinical trials. LIA showed higher sensitivity than cLIA, and due to limited epitopes of mAb, cLIA detected lower antibody responses, and therefore, fewer antibodies. This work not only supports clinical trials of 14-valent HPV vaccines more efficiently and reliably, but also provides a set of validation strategies and usable standards for general vaccine immunogenicity testing.

High ionic strength dissociation assay reduces dimeric target interference in immunogenicity testing.

Aim: The presence of di-/multi-meric forms of soluble target in biological samples can interfere in anti-drug antibody (ADA) assays, leading to increased background values and potentially false positivity. The authors investigated the use of the high ionic strength dissociation assay (HISDA) to reduce target interference in two different ADA assays. Results: Interference caused by homodimeric FAP was successfully eliminated to enable cut point determination after applying HISDA. Biochemical experiments confirmed the dissociation of homodimeric FAPafter treatment with high ionic strength conditions. Conclusion: HISDA is a promising approach to simultaneously achieve high drug tolerance and reduced interference by noncovalently bound dimeric target molecules in ADA assays without extensive optimization, which is particularly advantageous in routine use.

Salmonella sheep abortion: Distribution, diagnosis, and control measures

ABSTRACT The purpose of the study is to identify and isolate the causative agent of Salmonella sheep abortion in the sheep breeding industry of the Republic of Kazakhstan. The study aims to provide a basis for the development and testing of vaccines against salmonella sheep abortion using the isolated epizootic strains of Salmonella abortus-ovis AN 9/2 and Salmonella abortus-ovis 372 as control strains for immunogenicity testing. Biomaterials and pathologic materials were investigated of 114 abortions, dead ewes, and newborn lambs using the bacteriological method with the diagnostic purpose from 2009 to 2019. As a result of the bacteriological studies, the causative agent of salmonella sheep abortion was isolated and identified – Salmonella abortus-ovis. The study concludes that salmonella sheep abortion is a significant infectious disease that can cause massive economic losses and high mortality rates in sheep breeding. Prevention and control measures, such as regular cleaning, disinfection of premises, clinical examination, and thermometry of lambs, bacteriological studies, and vaccination against salmonella sheep abortion, are essential in reducing the incidence of the disease and increasing animal productivity.

Assay development considerations to improve drug tolerance in direct competitive ligand binding neutralizing antibody assays, pretreatment strategies

Neutralizing anti-drug antibodies (ADAs) may affect safety, efficacy, and pharmacokinetic profile of a biotherapeutic drug and thus their assessment is of particular importance during immunogenicity testing. Neutralizing antibody (NAb) assays typically rely on NAbs ability to block the drug-target interaction. Higher NAb concentration and/or higher binding affinity of NAb to the drug, lowers the drug-target binding interaction. However, in the presence of high concentrations of residual circulating drug, as often seen for drugs with longer half-lives or in repeat-dose studies, NAbs may exist as drug bound complexes. In direct NAb assay formats, the NAb-drug complexes present in the sample could result in the NAb being unable to block the drug-target interaction eventually leading to a false negative response. The residual free circulating drug present in the sample may bind to the target in the NAb assay thereby competing with the drug used in the assay and inhibiting the assay signal, leading to a false positive response. For traditional ADA assays, multiple approaches involving acid treatment have been described to mitigate circulating drug interference issue. Here, we report two acid-treatment approaches that utilize the Dynabeads extraction with acid dissociation and Affinity Capture Elution (ACE) principle to improve drug tolerance in NAb assays.

Humanized mouse models with endogenously developed human natural killer cells for in vivo immunogenicity testing of HLA class I-edited iPSC-derived cells

Human induced pluripotent stem cells (hiPSCs) genetically depleted of human leucocyte antigen (HLA) class I expression can bypass T cell alloimmunity and thus serve as a one-for-all source for cell therapies. However, these same therapies may elicit rejection by natural killer (NK) cells, since HLA class I molecules serve as inhibitory ligands of NK cells. Here, we focused on testing the capacity of endogenously developed human NK cells in humanized mice (hu-mice) using MTSRG and NSG-SGM3 strains to assay the tolerance of HLA-edited iPSC-derived cells. High NK cell reconstitution was achieved with the engraftment of cord blood-derived human hematopoietic stem cells (hHSCs) followed by the administration of human interleukin-15 (hIL-15) and IL-15 receptor alpha (hIL-15Rα). Such “hu-NK mice” rejected HLA class I-null hiPSC-derived hematopoietic progenitor cells (HPCs), megakaryocytes and T cells, but not HLA-A/B-knockout, HLA-C expressing HPCs. To our knowledge, this study is the first to recapitulate the potent endogenous NK cell response to non-tumor HLA class I-downregulated cells in vivo. Our hu-NK mouse models are suitable for the non-clinical evaluation of HLA-edited cells and will contribute to the development of universal off-the-shelf regenerative medicine.

ESAT-6-Ag85C-polyHistag Antigen Fusion is Potential as Vaccine Candidate for Tuberculosis

BACKGROUND: BCG vaccine has been proven to be effective protection against tuberculosis (TB) meningitis and miliary TB. However, for protection against pulmonary TB, the results remains vary widely. Recombinant vaccine consisting of two immunodominant Mtb antigens ESAT-6-Ag85C-polyHistag (EAH) is currently being developed as a new TB vaccine candidate for booster. An immugonecity test for vaccine candidates is required in the initial phase to evaluate cellular immune response. This study was conducted to evaluate the cellular immune response by measuring interferon-gamma (IFN-γ) cytokines produced by T cells after ex vivo stimulation by TB EAH antigen fusion.METHODS: Peripheral blood mononuclear cells supernatant samples were collected from 16 new pulmonary TB subjects, 17 pulmonary TB in treatment subjects, and 10 healthy subjects. Samples were tested for IFN-γ level with enzyme-linked immunosorbent assay (ELISA). Kruskal-Wallis test was used to test the differences between IFN-γ levels among three groups, and followed by post-hoc analysis using Mann Whitney.RESULTS: The median of IFN-γ levels for new pulmonary TB, pulmonary TB in treatment, and healthy subjects were 17.09 (2.65-140.14) pg/mL, 4.36 (2.43-21.41) pg/mL, and 2.91 (2.39-3.85) pg/mL, respectively. There were significant differences of IFN-γ levels between new pulmonary TB group and pulmonary TB in treatment group (p=0.012), between new pulmonary TB group and healthy group (p=0.001), and also between pulmonary TB in treatment group and healthy group (p=0.035).CONCLUSION: Results show that TB EAH could stimulate cell-mediated immune responses in the three groups, with the highest IFN-γ levels are found in new pulmonary TB group, suggesting a potential immunodominant antigen fusion for vaccine candidate development.KEYWORDS: Ag85C, ESAT-6, immunogenicity test, IFN-γ, TB vaccine candidate, tuberculosis

Molecular Characterization and Selection of Indigenous SARS-CoV-2 Delta Variant for the Development of the First Inactivated SARS-CoV-2 Vaccine of Pakistan

Vaccines are one of the efficient means available so far for preventing and controlling the infection rate of COVID-19. Several researchers have focused on the whole virus's (SARS-CoV-2) inactivated vaccines which are economically efficient to produce. In Pakistan, multiple variants of SARS-CoV-2 have been reported since the start of the pandemic in February 2020. Due to the continuous evolution of the virus and economic recessions, the present study was designed to develop an indigenous inactivated SARS-CoV-2 vaccine that might help not only to prevent the COVID-19 in Pakistan, it will also save the country's economic resources. The SARS-CoV-2 were isolated and characterized using the Vero-E6 cell culture system. The seed selection was carried out using cross-neutralization assay and phylogenetic analysis. The selected isolate of SARS-CoV-2 (hCoV-19/Pakistan/UHSPK3-UVAS268/2021) was inactivated using beta-propiolactone followed by vaccine formulation using Alum adjuvant, keeping the S protein concentration as 5 μg/dose. The vaccine efficacy was evaluated by in vivo immunogenicity testing in laboratory animals and in in vitro microneutralization test. The phylogenetic analysis revealed that all the SARS-CoV-2 isolates reported from Pakistan nested into different clades, representing multiple introductions of the virus into Pakistan. The antisera raised against various isolates from different waves in Pakistan showed a varied level of neutralization titers. However, the antisera produced against a variant (hCoV-19/Pakistan/UHSPK3-UVAS268/2021; fourth wave) efficiently neutralized (1:64-1:512) all the tested SARS-CoV-2 isolates. The inactivated whole virus vaccine of SARS-CoV-2 was safe and it also elicited a protective immune response in rabbits and rhesus macaques on the 35th-day post-vaccination. The activity of neutralizing antibodies of vaccinated animals was found at 1:256-1:1024 at 35 days post-vaccination, indicating the effectiveness of the double-dose regime of the indigenous SARS-CoV-2 vaccine.

The HLA ligandome of oropharyngeal squamous cell carcinomas reveals shared tumour-exclusive peptides for semi-personalised vaccination

BackgroundThe immune peptidome of OPSCC has not previously been studied. Cancer-antigen specific vaccination may improve clinical outcome and efficacy of immune checkpoint inhibitors such as PD1/PD-L1 antibodies.MethodsMapping of the OPSCC HLA ligandome was performed by mass spectrometry (MS) based analysis of naturally presented HLA ligands isolated from tumour tissue samples (n = 40) using immunoaffinity purification. The cohort included 22 HPV-positive (primarily HPV-16) and 18 HPV-negative samples. A benign reference dataset comprised of the HLA ligandomes of benign haematological and tissue datasets was used to identify tumour-associated antigens.ResultsMS analysis led to the identification of naturally HLA-presented peptides in OPSCC tumour tissue. In total, 22,769 peptides from 9485 source proteins were detected on HLA class I. For HLA class II, 15,203 peptides from 4634 source proteins were discovered. By comparative profiling against the benign HLA ligandomic datasets, 29 OPSCC-associated HLA class I ligands covering 11 different HLA allotypes and nine HLA class II ligands were selected to create a peptide warehouse.ConclusionTumour-associated peptides are HLA-presented on the cell surfaces of OPSCCs. The established warehouse of OPSCC-associated peptides can be used for downstream immunogenicity testing and peptide-based immunotherapy in (semi)personalised strategies.

A Novel Neutralization Antibody Assay Method to Overcome Drug Interference with Better Compatibility with Acid-Sensitive Neutralizing Antibodies

Immunogenicity testing to detect and characterize anti-drug antibody (ADA) is required for almost all biotherapeutics. Monoclonal antibody biotherapeutics usually have long half-lives and for high-dose indications such as oncology, high level of drug will be present in the testing samples and interfere with ADA and/or neutralization antibody (NAb) measurement. To overcome this drug interference, acid-dissociation-based sample pre-treatment such as Bead-Extraction and Acid Dissociation (BEAD) has been successfully applied. The main concern for these acid-dissociation-based methods, however, is that harsh acid treatment could denature positive control Abs as well as NAb species in testing samples. In addition, high amount of biotinylated drug is needed in order to have effective competition with high level of drug in the samples, which in turn requires expensive magnetic beads. And the whole process of magnetic beads handling is tedious if doing manually and often causes trouble during assay transfer. Here, we describe a novel method which we named as Precipitation, Acid Dissociation and Biotin-drug as Assay Drug (PABAD). This novel method will need only one step of acid dissociation, with much milder and shorter acid treatment to maximally preserve NAb activity. In addition, only a fraction of biotinylated-drug is needed and there is no need to use additional streptavidin (SA)-plate or SA-magnetic beads for extraction. Compared to a BEAD-based assay, PABAD demonstrates significantly improved recovery of acid-sensitive NAb positive controls (PCs) and similar recovery of acid-resistant NAb PCs.

Optimization of the Process for Preparing Bivalent Polysaccharide Conjugates to Develop Multivalent Conjugate Vaccines against Streptococcus pneumoniae or Neisseria meningitidis and Comparison with the Corresponding Licensed Vaccines in Animal Models.

This study aimed to describe, optimize and evaluate a method for preparing multivalent conjugate vaccines by simultaneous conjugation of two different bacterial capsular polysaccharides (CPs) with tetanus toxoid (TT) as bivalent conjugates. Different molecular weights (MWs) of polysaccharides, activating agents and capsular polysaccharide/protein (CP/Pro) ratio that may influence conjugation and immunogenicity were investigated and optimized to prepare the bivalent conjugate bulk. Using the described method and optimized parameters, a 20-valent pneumococcal conjugate vaccine and a bivalent meningococcal vaccine were developed and their effectiveness was compared to that of corresponding licensed vaccines in rabbit or mouse models. The immunogenicity test revealed that polysaccharides with lower MWs were better for Pn1-TT-Pn3 and MenA-TT-MenC, while higher MWs were superior for Pn4-TT-Pn14, Pn6A-TT-Pn6B, Pn7F-TT-Pn23F and Pn8-TT-Pn11A. For activating polysaccharides, 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) was superior to cyanogen bromide (CNBr), but for Pn1, Pn3 and MenC, N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDAC) was the most suitable option. For Pn6A-TT-Pn6B and Pn8-TT-Pn11A, rabbits immunized with bivalent conjugates with lower CP/Pro ratios showed significantly stronger CP-specific antibody responses, while for Pn4-TT-Pn14, higher CP/Pro ratio was better. Instead of interfering with the respective immunological activity, our bivalent conjugates usually induced higher IgG titers than their monovalent counterparts. The result indicated that the described conjugation technique was feasible and efficacious to prepare glycoconjugate vaccines, laying a solid foundation for developing extended-valent multivalent or combined conjugate vaccines without potentially decreased immune function.

111. pVACsplice: Predicting neoantigens from tumor-specific alternative splicing events derived from regulatory mutations

Neoantigens are tumor-specific peptides presented on the cell surface by MHC that can be recognized by the adaptive immune system. Personalized immunotherapies, such as cancer vaccines, rely on neoantigen prediction to identify sequences that can activate T cells to recognize and destroy the tumor. The majority of cancer vaccine trials have utilized neoantigens derived from missense mutations and small insertions and deletions. However, other mutation types contribute to the overall neoantigen landscape, including aberrantly spliced transcripts arising from cis-acting regulatory mutations. In this study, we explore the potential immunogenicity of alternative splicing events and present pVACsplice, a tool to expand the capability of pVACtools, a suite of tools for neoantigen prediction (http://www.pvactools.org). pVACsplice assembles alternative transcripts from tumor-specific splicing patterns, identifies sequence changes by comparison to a reference, and predicts neoantigens from the novel peptide sequences. Matched whole exome sequencing and RNA sequencing datasets from glioblastoma, melanoma, and colorectal cancer cohorts will be analyzed with pVACsplice to obtain binding affinity estimates. We will compare these results to neoantigen predictions from other mutation sources and across cancer types to discover the prevalence of immunogenic splicing events. Finally, we will perform immunogenicity testing with a set of high quality candidates to validate our predictions. We hope to increase the number of candidates for personalized vaccines by adding this functionality to our standard neoantigen prediction workflow. This tool could help generate a more accurate portrait of the neoantigen landscape in tumors, and in turn, enhance responses to personalized immunotherapies. Neoantigens are tumor-specific peptides presented on the cell surface by MHC that can be recognized by the adaptive immune system. Personalized immunotherapies, such as cancer vaccines, rely on neoantigen prediction to identify sequences that can activate T cells to recognize and destroy the tumor. The majority of cancer vaccine trials have utilized neoantigens derived from missense mutations and small insertions and deletions. However, other mutation types contribute to the overall neoantigen landscape, including aberrantly spliced transcripts arising from cis-acting regulatory mutations. In this study, we explore the potential immunogenicity of alternative splicing events and present pVACsplice, a tool to expand the capability of pVACtools, a suite of tools for neoantigen prediction (http://www.pvactools.org). pVACsplice assembles alternative transcripts from tumor-specific splicing patterns, identifies sequence changes by comparison to a reference, and predicts neoantigens from the novel peptide sequences. Matched whole exome sequencing and RNA sequencing datasets from glioblastoma, melanoma, and colorectal cancer cohorts will be analyzed with pVACsplice to obtain binding affinity estimates. We will compare these results to neoantigen predictions from other mutation sources and across cancer types to discover the prevalence of immunogenic splicing events. Finally, we will perform immunogenicity testing with a set of high quality candidates to validate our predictions. We hope to increase the number of candidates for personalized vaccines by adding this functionality to our standard neoantigen prediction workflow. This tool could help generate a more accurate portrait of the neoantigen landscape in tumors, and in turn, enhance responses to personalized immunotherapies.

Affinity Capture Elution (ACE) ELISA Method Development and Validation for Novel RPH-104 Drug Immunogenicity Evaluation.

As the number of therapeutic protein products is growing rapidly, there is a strong need for the development of bioanalytical methods that are easy to perform, specific, sensitive, robust, and affordable. Methods for immunogenicity evaluation of therapeutic proteins take an important place in this field of bioanalytics. The aim of the study was to develop a method for immunogenicity testing of the novel RPH-104 drug using the Affinity Capture Elution (ACE) ELISA technique. RPH-104 is a promising Interleukin-1 (IL-1) inhibitor that is currently undergoing a series of clinical studies, including those on socially significant and orphan diseases. The developed method was validated for assay cut-point, sensitivity, selectivity, drug tolerance, hook effect, specificity, precision, and stability. Method sensitivity was established at 114.9 ng/mL, while low and high positive controls were equal to anti-RPH-104 antibody concentrations of 155 ng/mL and 2500 ng/mL, respectively. Method specificity was confirmed in the presence of the interfering compounds, namely IL-1α, IL-1β, and IL1-Ra. The developed and validated ELISA method was successfully applied to subject samples.

Oncology biosimilars: New developments and future directions.

Biologicals have become an integral part of cancer treatment both as therapeutic agents and as supportive care agents. It is important to know that biologics are large, complex molecular entities requiring extensive immunogenicity testing and pharmacovigilance strategies to ensure no immune response is evoked in the body. Oncology's pharmacological market is dominated by biologics; however, their high development and manufacturing costs are burdensome to health care systems. Biologics being the most expensive prescription drugs on the market limit the accessibility for necessary treatment in the case of many patients. As biologics patents expire, the development of biosimilars is underway in an effort to lower costs and enable patients to access new cancer therapies. Regulatory guidelines for biosimilars have now been established and are constantly being revised to address any issues, facilitating their robust development. Moreover, many scientific societies offer guidance to help stakeholders better understand current regulations and biosimilar's safety. Despite the potential cost benefits, lack of knowledge about biosimilars, and the possibility of immunogenicity have created an uncertain environment for healthcare professionals and patients. In this review, we provide an overview of relevant legislation and regulations, pharmacoeconomics, and stakeholder perceptions regarding biosimilars. The article also describes biosimilars in development, as well as the ones currently available on the market.

Neutralization Activity of Anti-drug Antibodies Against a Biotherapeutic Can Be Predicted from a Comprehensive Pharmacokinetics, Pharmacodynamics, and Anti-drug Antibody Data Analysis.

Historically, a neutralization antibody (NAb) assay is considered critical in immunogenicity assessment of biologic therapeutics, even with low anti-drug antibody (ADA) positive rates. In 2019, FDA new guidelines issued on immunogenicity testing acknowledged the possibility of using "a highly sensitive PD marker or an appropriately designed PK assay or both that generate data that inform clinical activity" to replace a NAb assay. In the current manuscript, we present data for PK, PD, and ADA assays which collectively succeed to replace the standalone NAb assay. The data include a total LC/MS-based PK assay, a serum neutralization antibody (SNA) assay that essentially measures pharmacodynamically functional PK and can detect NAb activity in the presence of 1:1 ratio of drug, and a highly drug-tolerant ADA assay. In addition, a model-based meta-analysis (MBMA) demonstrated that the ability of SNA assay to detect NAb at 1:1 ratio of drug is sensitive enough to monitor clinically meaningful efficacy change, which is 50% reduction of SNA titer. Our strategy of preparing a holistic data package discussed here may provide a roadmap to the community for alternatives in assaying neutralizing activity of ADA.

A humanized minipig model for the toxicological testing of therapeutic recombinant antibodies

The safety of most human recombinant proteins can be evaluated in transgenic mice tolerant to specific human proteins. However, owing to insufficient genetic diversity and to fundamental differences in immune mechanisms, small-animal models of human diseases are often unsuitable for immunogenicity testing and for predicting adverse outcomes in human patients. Most human therapeutic antibodies trigger xenogeneic responses in wild-type animals and thus rapid clearance of the drugs, which makes in vivo toxicological testing of human antibodies challenging. Here we report the generation of Göttingen minipigs carrying a mini-repertoire of human genes for the immunoglobulin heavy chains γ1 and γ4 and the immunoglobulin light chain κ. In line with observations in human patients, the genetically modified minipigs tolerated the clinically non-immunogenic IgG1κ-isotype monoclonal antibodies daratumumab and bevacizumab, and elicited antibodies against the checkpoint inhibitor atezolizumab and the engineered interleukin cergutuzumab amunaleukin. The humanized minipigs can facilitate the safety and efficacy testing of therapeutic antibodies.

Feasibility of using PALSAR technology as a signal amplifier for antibody bridging assay.

The immunogenicity testing of oligonucleotide drugs using an antibody bridging assay has been scarcely investigated. We developed a highly sensitive antibody bridging assay model and assessed it using probe alteration link self-assembly reactions (PALSAR) technology as a signal amplifier. Methods: The concentration of each probewas optimized, andthe bridging assay model was comparedwith and without signal amplification. Cut-point and analytical sensitivity were determined, and accuracy, precision and drug tolerance wereevaluated. Results: The PALSAR bridging assay achieved a net signal 21-36 times higher than that obtained with the conventional method. The analytical sensitivity achieved was 48.8ng/ml, with adequate accuracy, precisionand drug tolerance. Conclusion: PALSAR technology is feasible for developing an antibody bridging assay using oligonucleotides as capture and detection probes.

Safety and immunogenicity of the first Kazakh inactivated vaccine for COVID-19

ABSTRACT This article describes the results of a preclinical safety and immunogenicity study of QazCovid-in®, the first COVID-19 vaccine developed in Kazakhstan, on BALB/c mice, rats, ferrets, Syrian hamsters and rhesus macaques (Macaca mulatta). The study’s safety data suggests that this immunobiological preparation can be technically considered a Class 5 nontoxic vaccine. The series of injections that were made did not produce any adverse effect or any change in the general condition of the model animals’ health, while macroscopy and histology studies identified no changes in the internal organs of the BALB/c mice and rats. This study has demonstrated that a double immunization enhances the growth of antibody titers as assessed by the microneutralization assay (MNA) and the enzyme-linked immunosorbent assay (ELISA) in a pre-clinical immunogenicity test on animal models. The best GMT results were assessed in MNA and ELISA 7 days after re-vaccination; however, we noted that GMT antibody results in ELISA were lower than in MNA. A comparative GMT assessment after the first immunization and the re-immunization identified significant differences between model animal groups and a growth of GMT antibodies in all of them; also, differences between the gender groups were statistically significant. Moreover, the most marked MNA immune response to the QazCovid-in® vaccine was seen in the Syrian hamsters, while their SARS-CoV-2-specific antibody activity as assessed with ELISA was the lowest.