Fraction Of Antibodies Research Articles

B-293 Enzyme linked immunosorbent assay development and full validation for the quantification of the antibody conjugated fraction of an antiFAP ADC in human serum

Abstract Background Cancer remains a global public health issue. Despite recent progress in early diagnosis and treatment strategies, development of anti-tumor therapies with reduced systemic toxicity remains a challenge. Antibody-drug conjugates (ADCs), a new, highly-potent drug class are synthesized by linking a small molecule cytotoxic drug to a tumor specific antibody, are an emerging treatment option for many cancers. Despite advances in ADCs, study of the pharmacokinetics (PK) is challenging. A complete PK profile requires at least three analytical methods to determine: conjugated antibody, total antibody, and free drug by Ligand Binding Assays (LBA) and liquid chromatography/tandem mass spectrometry (LC-MS/MS) respectively. Objective To develop and validate an ELISA method to quantify the conjugated antibody fraction of an anti-fibroblast activation protein (FAP) ADC against solid tumors in human serum. Methods Conjugated antibody assay: This indirect sandwich ELISA utilizes human FAP as capture antigen (Abcam) with a proprietary rabbit polyclonal anti-drug IgG as detection antibody. The signal is amplified by the use of a second antibody conjugated to Horse Radish Peroxidase (Abcam). Absorbance is measured at 450nm. Immunoassay method validation was done in compliance with the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) M10 which establishes the limit for precision in CV% and accuracy in Relative Error (RE%) at ± 20% each (± 25% for LLOQ and ULOQ). Additionally, the total error (TE%) for accuracy and precision batches, should be ≤ 30% (≤ 40% at LLOQ and ULOQ). Results The weighted calibration curves were linear over a concentration range 0.5 to 100 µg/mL. A four-parameter regression model with a 1/Y2 weighting factor was adopted to best fit the OD450 values and concentrations. The intra- and inter-assay accuracy and precision for RE% and CV% was <12%, with a TE% < 23% for all concentrations tested. The quantification limit of 0.5 µg/mL complies with ICH criteria with a RE%, CV% and TE% of -10%, 8,5% and 15% respectively. Selectivity and specificity test fall within the criteria indicating that no individual matrix or co-medication affects the samples. The results from the dilution linearity obtained were an RE% of 11, -16 and -6 for 1:50, 1:100 and 1:200 respectively. Furthermore, not hook effect was observed as the dilution QC results gave above the quantification limit. Finally, the analyte was found to be stable at -20° and -80°for 148 days, at room temperature for 24hr and after 5 freeze/thaw cycles with a maximum RE and CV of 12 and 14% respectively in all the stability samples. Conclusions We have developed and optimized an ELISA method for measuring the antibody conjugated fraction of the ADC in serum. This method complies with the criteria of the ICH50 for selectivity, specificity, accuracy and precision, dilution linearity and hook effect in a concentration range between 0.5 - 100 µg/mL. Further, serum specimens are stable for 24h at room temperature, 5 freeze/thaw cycles and 148 days at -20 and -80 °C.

Machine learning evaluation for identification of M-proteins in human serum.

Serum electrophoresis (SPEP) is a method used to analyze the distribution of the most important proteins in the blood. The major clinical question is the presence of monoclonal fraction(s) of antibodies (M-protein/paraprotein), which is essential for the diagnosis and follow-up of hematological diseases, such as multiple myeloma. Recent studies have shown that machine learning can be used to assess protein electrophoresis by, for example, examining protein glycan patterns to follow up tumor surgery. In this study we compared 26 different decision tree algorithms to identify the presence of M-proteins in human serum by using numerical data from serum protein capillary electrophoresis. For the automated detection and clustering of data, we used an anonymized data set consisting of 67,073 samples. We found five methods with superior ability to detect M-proteins: Extra Trees (ET), Random Forest (RF), Histogram Grading Boosting Regressor (HGBR), Light Gradient Boosting Method (LGBM), and Extreme Gradient Boosting (XGB). Additionally, we implemented a game theoretic approach to disclose which features in the data set that were indicative of the resulting M-protein diagnosis. The results verified the gamma globulin fraction and part of the beta globulin fraction as the most important features of the electrophoresis analysis, thereby further strengthening the reliability of our approach. Finally, we tested the algorithms for classifying the M-protein isotypes, where ET and XGB showed the best performance out of the five algorithms tested. Our results show that serum capillary electrophoresis combined with decision tree algorithms have great potential in the application of rapid and accurate identification of M-proteins. Moreover, these methods would be applicable for a variety of blood analyses, such as hemoglobinopathies, indicating a wide-range diagnostic use. However, for M-protein isotype classification, combining machine learning solutions for numerical data from capillary electrophoresis with gel electrophoresis image data would be most advantageous.

Affinity Chromatography for Anti-Glucosylated Adhesin Antibody Purification: Depletion of Nonspecific Anti-Protein Antibodies and Antibody Recovery with Unconventional Elution Solutions.

Antibodies from sera of a multiple sclerosis (MS) patient subpopulation preferentially recognize the hyperglucosylated adhesin protein HMW1ct(Glc) of the pathogen Haemophilus influenzae. This protein is the first example of an N-glucosylated native antigen candidate, potentially triggering pathogenic antibodies in MS. Specific antibodies in patients' sera can be isolated exploiting their biospecific interaction with antigens by affinity chromatography. Herein, the proteins HMW1ct and HMW1ct(Glc) were first immobilized on appropriately functionalized supports and further used to purify antibodies directly from MS patients sera. We describe a protocol to obtain an antibody fraction specifically recognizing the glusosylated residues on the HMW1ct(Glc) adhesin protein depleting antibodies to the unglucosylated HMW1ct sequence. Different elution solutions have been tested to recover the purified antibody fraction, strongly bound to the immobilized HMW1ct(Glc) adhesin protein.

Hemolysis and Free Heme Impact Therapeutic Activity of Anti-CD20 Antibody

Hemolysis is a process of destruction of erythrocytes that can occur in different hereditary or acquired pathologies. It is accompanied by release of hemoglobin and heme, which both manifest potent pro-oxidative and pro-inflammatory activities. If released in large quantities hemoglobin and heme can directly contribute to systemic inflammation and tissue injury in hemolytic diseases. Moreover, it was demonstrated that heme binds to a significant fraction (ca. 10 %) of human antibodies (Abs) and this binding can result in considerable functional changes. Thus, heme-bound human IgG acquire reactivity towards a large set of unrelated proteins, including autoantigens. In a previous study we observed that 14 % of a repertoire of 117 clinical-stage therapeutic mAbs are able to bind heme and as a result they acquired autoreactivity and polyreactivity. Among these Abs some are specific for CD20 and used in lymphoma therapy (e.g. Rituximab). Importantly, therapeutic mAbs can be administered in patients with ongoing hemolysis. Thus, it is of critical importance to decipher molecular mechanism and functional consequences of interaction of heme with therapeutic mAbs and understand how the therapy of lymphoma can be affected by underling hemolysis and heme release. The present study has the following objective: to elucidate the effect of heme on the therapeutic activity of anti-CD20 therapy in in vitro and in vivo models of B cell lymphoma. Effect of heme on anti-CD20 therapeutic Abs was assessed by i) binding assays - ELISA, western blot; protein microarrays; absorbance spectroscopy; surface plasmon resonance, and ii) molecular modeling. The impact of heme on functional and therapeutic activity of Rituximab was analyzed by i) site-directed mutagenesis; ii) flow cytometry; iv) in vitro and in vivo models of human B cell lymphoma. Our data reveled molecular and functional details about the interaction of heme with Rituximab and other anti-CD20 Abs. Thus, we characterized the interaction of Abs with heme, and identified in case of Rituximab the putative binding site for heme that overlapped with antigen binding site. By using a protein array with >9800 human proteins, we identified the target proteins of heme-bound Rituximab. A mouse model of human B cell lymphoma revealed that the therapeutic activity of Rituximab was compromised following exposure to cell free heme under condition of acute intravascular hemolysis. Our study delineates that a fraction of currently used therapeutic mAbs show high sensitivity to heme and their therapeutic activity can be compromised under hemolytic conditions. This finding suggests that therapeutic interventions that reduce free heme and /or intravascular hemolysis should precede therapy with these Abs. Moreover, we identified sequence features of Abs that determine the sensitivity to heme. This finding can help to reduce sensitivity of heme of therapeutic Abs by protein engineering.

Deciphering the antigen specificities of antibodies by clustering their complementarity determining region sequences.

Determining antigen and epitope specificity is an essential step in the discovery of therapeutic antibodies as well as in the analysis adaptive immune responses to disease or vaccination. Despite extensive efforts, deciphering antigen specificity solely from BCR amino acid sequence remains a challenging task, requiring a combination of experimental and computational approaches. Here, we describe and experimentally validate a simple and straightforward approach for grouping antibodies that share antigen and epitope specificities based on their CDR sequence similarity. This approach allows us to identify the specificities of a large number of antibodies whose antigen targets are unknown, using a small fraction of antibodies with well-annotated binding specificities.

Natural Antibodies Produced in Vaccinated Patients and COVID-19 Convalescents Recognize and Hydrolyze Oligopeptides Corresponding to the S-Protein of SARS-CoV-2.

The S-protein is the major antigen of the SARS-CoV-2 virus, against which protective antibodies are generated. The S-protein gene was used in adenoviral vectors and mRNA vaccines against COVID-19. While the primary function of antibodies is to bind to antigens, catalytic antibodies can hydrolyze various substrates, including nucleic acids, proteins, oligopeptides, polysaccharides, and some other molecules. In this study, antibody fractions with affinity for RBD and S-protein (RBD-IgG and S-IgG) were isolated from the blood of COVID-19 patients vaccinated with Sputnik V. The fractions were analyzed for their potential to hydrolyze 18-mer oligopeptides corresponding to linear fragments of the SARS-CoV-2 S-protein. Here, we show that the IgG antibodies hydrolyze six out of nine oligopeptides efficiently, with the antibodies of COVID-19-exposed donors demonstrating the most significant activity. The IgGs of control donors not exposed to SARS-CoV-2 were found to be inactive in oligopeptide hydrolysis. The antibodies of convalescents and vaccinated patients were found to hydrolyze oligopeptides in a wide pH range, with the optimal pH range between 6.5 and 7.5. The hydrolysis of most oligopeptides by RBD-IgG antibodies is inhibited by thiol protease inhibitors, whereas S-IgG active centers generally combine several types of proteolytic activities. Ca2+ ions increase the catalytic activity of IgG preparations containing metalloprotease-like active centers. Thus, the proteolytic activity of natural antibodies against the SARS-CoV-2 protein is believed to be due to the similarity of catalytic antibodies' active centers to canonical proteases. This work raises the question of the possible physiological role of proteolytic natural RBD-IgG and S-IgG resulting from vaccination and exposure to COVID-19.

Host immune signatures as predictors of response to immunotherapy-based regimens in patients with metastatic renal cell carcinoma (mRCC)

Abstract Background Treatment options for mRCC have evolved to include VEGF targeted therapies (VEGF-TT), immune checkpoint inhibitors (ICIs), or combinations of both. However, clinical responses to systemic therapies in mRCC remain largely unpredictable and robust biomarkers are still lacking. The interaction between the tumor and its immune microenvironment has been shown to influence clinical outcomes in patients treated with ICI-based regimens. The aim of this study was to characterize the T-cell and B-cell immune repertoires in patients with mRCC treated with VEGF-TT, ICI or a combination of both, and evaluate their associations with clinical outcomes. Methods We identified patients with mRCC at Dana-Farber Cancer Institute treated with VEGF-TT, ICI or both, and for whom tumor and/or blood samples were available. T-cell receptor sequencing (TCR-seq) was performed on peripheral blood mononuclear cells (PBMCs), collected before and during therapy. Bulk RNA-sequencing (RNA-seq) was performed on available pre-treatment PBMCs and primary tumor samples. Immunoglobulin heavy chain (IgH) isotypes were inferred from bulk RNA-seq data using TRUST4. Parameters of the T-cell and B-cell repertoires were evaluated in responders vs. non-responders to systemic therapies, and between pre- and on-treatment samples within patient subgroups. Results In total, blood (PBMC) samples from 386 patients were available across all treatment cohorts (186 VEGF-TT, 126 ICI and 74 ICI+VEGF-TT). Following quality-control, TCR-seq data were available for 367 patients (228 pre-treatment and 139 on-treatment), while RNA-seq data were available for 105 PBMC and 17 tumor-derived pre-treatment samples. In the TCR-seq analysis, responders to ICI-based regimens (ICI or VEGF-TT+ICI) presented a trend towards an increased baseline (pre-treatment) TCR clonality as compared to non-responders (p=0.06) (Fig. 1A), corresponding to a less polyclonal T cell repertoire in responders at baseline. No significant changes in clonality were seen between pre- and on-treatment samples among responders to ICI regimens (p=0.14), as opposed to non-responders where a significant increase was identified (p=0.001). Therefore, responders to ICI-based regimens seem to have a more oligoclonal TCR repertoire (increased clonality) at baseline with no treatment-induced changes, whereas non-responders to ICI-based regimens seem to evolve from a more polyclonal to a more oligoclonal TCR repertoire in response to treatment. The analysis of IgH isotypes in baseline blood samples showed higher fraction of IgG1 in responders (vs. non-responders) to ICI regimens (p=0.01) (Fig. 1B). This was further confirmed in the analysis of IgH isotypes inferred from tumor samples (p=0.04). No significant differences in IgH isotype fractions were identified between responders and non-responders to VEGF-TT. Furthermore, while shared clonotypes were detected between blood and tumor samples, there were no differences in the Jaccard similarity index between responders and non-responders to ICI-based or VEGF-TT regimens. Figure 1: (A) Evaluation of pre-treatment and post-treatment TCR clonality in responders vs. non-responders across treatment cohorts. (B) Differences in the fraction of IGH isotypes between responders and non-responders across treatment cohorts. Conclusions We were successfully able to characterize T-cell receptor and B-cell IgH repertoires in a large cohort of patients with mRCC, and evaluate their associations with ICI response. Our results show that baseline TCR clonality and IgG1 antibody fraction are associated with the response to ICI regimens, suggesting a potential role for immune biomarker development. CDMRP DOD Funding: yes

Application of Magnetic Particles for Fast Determination of Immunoreactive Fraction of 68Ga-Labelled VHH Antibodies to PD-L1.

Commercially available magnetic particles coated with protein A have been used in our study. The antigen conjugated with the Fc fragment (PD-L1-Fc) was immobilized on the particles. The IRF value of 68Ga radionuclide-labeled nanobodies (VHH) against PD-L1 (68Ga-VHH-PD-L1) was determined using magnetic particles coated with antigen molecules and cells expressing the antigen on their surface. When VHH antibodies were conjugated to 68Ga radionuclide, protein molecules were modified using bifunctional chelating agents: tetraazacyclododecanetetraacetic acid (DOTA) or deferoxamine (DFO). The magnitude of IRF was defined as the ratio of radioactivity specifically bound to particles or cells to the total radioactivity added to the sample. The specificity of the 68Ga-VHH-PD-L1 radioimmunoconjugate binding to the antigen-coated magnetic particles has been proved. Some special aspects, which should be taken into consideration when using this method, have been established. The comparison of the IRF estimates using the antigen-expressing cells and magnetic particles has not revealed any significant differences in the results obtained in our study. Nevertheless, the presented method based on magnetic particles with immobilized antigen molecules requires only 15 min to determine the radioimmunoconjugate IRF, which is of fundamental importance for the routine assessment of the specificity of radiopharmaceuticals containing short-lived isotopes.

Association of host immune signatures with response to immunotherapy-based regimens in patients with metastatic renal cell carcinoma (mRCC).

4550 Background: Treatment options for mRCC have evolved to include VEGF targeted therapies (VEGF-TT), immune checkpoint inhibitors (ICIs), or combinations of both. The interaction between the tumor and its immune microenvironment has been shown to influence clinical outcomes in patients treated with immunotherapy-based regimens. The aim of this study was to characterize the T-cell and B-cell immune repertoires in patients with mRCC treated with VEGF-TT, ICI or a combination of both, and evaluate their associations with clinical outcomes. Methods: We identified patients with mRCC at Dana-Farber Cancer Institute treated with VEGF-TT, ICI or both, and for whom tumor and/or blood samples were available. Bulk RNA-sequencing (RNA-seq) and T-cell receptor sequencing (TCR-seq) were performed on peripheral blood mononuclear cells (PBMCs), collected before and during therapy. Bulk RNA-seq was additionally performed on available pre-treatment primary tumor samples. Immunoglobulin heavy chain (IgH) isotypes were inferred from bulk RNA-seq data using TRUST4. Parameters of the T-cell and B-cell repertoires were evaluated in responders vs. non-responders to systemic therapies, and between pre- and on-treatment samples within patient subgroups. Results: In total, blood (PBMC) samples from 386 patients were available across all treatment cohorts. Following quality-control, TCR-seq and RNA-seq data were available for 367 and 134 patient samples, respectively. Responders to ICI-based regimens (ICI or VEGF-TT+ICI) presented a trend towards an increased baseline (pre-treatment) TCR clonality as compared to non-responders (p=0.06), corresponding to a less polyclonal T cell repertoire in responders at baseline. No significant changes in clonality were seen between pre- and on-treatment samples among responders to ICI regimens (p=0.14), as opposed to non-responders where a significant increase was identified (p=0.001). The analysis of IgH isotypes in baseline blood samples showed higher fraction of IgG1 in responders (vs. non-responders) to ICI regimens (p=0.01). This was further confirmed in the analysis of IgH isotypes inferred from tumor samples (p=0.04). No significant differences in IgH isotype fractions were identified between responders and non-responders to VEGF-TT. Conclusions: We were successfully able to characterize T-cell receptor and IgH repertoires in a large cohort of patients with mRCC and evaluate their associations with ICI response. Our results show that baseline TCR clonality and IgG1 antibody fraction are associated with the response to ICI regimens, suggesting a potential role for immune biomarker development.

Clinical and Regulatory Challenges and Opportunities for Monoclonal Antibodies in Low- and Middle-Income Countries: Lessons from COVID-19 and Beyond.

Monoclonal antibodies are an effective and growing class of pharmaceuticals for the treatment and prevention of a broad range of non-communicable and infectious diseases; however, most low- and middle-income countries have limited access to these innovative products. Many factors contribute to the global inequity of access to these products; however, in this report, we focus on clinical and regulatory complexities as further highlighted by the coronavirus disease 2019 pandemic. Despite a higher prevalence of many diseases in low- and middle-income countries, only 12% of clinical trials for monoclonal antibodies are conducted in these countries. Additionally, only a fraction of the available monoclonal antibodies in the USA and European Union are authorized for use in low- and middle-income countries. Through learnings from desk research and global symposia with international partners, we present recommendations to harmonize processes and facilitate regional and international collaborations for more rapid approval of fit-for-purpose innovative monoclonal antibodies and biosimilars in low- and middle-income countries.

Detection and Level Evaluation of Antibodies Specific to Environmental Bacteriophage I11mO19 and Related Coliphages in Non-Immunized Human Sera

Bacteriophages (phages) are viruses infecting bacteria. They are widely present in the environment, food, and normal microflora. The human microbiome is a mutually interdependent network of bacteria, bacteriophages, and human cells. The stability of these tri-kingdom interactions may be essential for maintaining immunologic and metabolic health. Phages, as with each other's antigens, may evoke an immune response during a human's lifetime and induce specific antibody generation. In this manuscript, we labeled these antibodies as naturally generated. Naturally generated antibodies may be one of the most important factors limiting the efficacy of phage therapy. Herein, we attempted to determine the physiological level of these antibodies specific to a population bacteriophage named I11mO19 in human sera, using an ELISA-based assay. First, we purified the phage particles and assessed the immunoreactivity of phage proteins. Then, affinity chromatography was performed on columns with immobilized phage proteins to obtain a fraction of human polyclonal anti-phage antibodies. These antibodies were used as a reference to elaborate an immunoenzymatic test that was used to determine the level of natural anti-phage antibodies. We estimated the average level of anti-I11mO19 phage antibodies at 190 µg per one milliliter of human serum. However, immunoblotting revealed that cross-reactivity occurs between some proteins of I11mO19 and two other coliphages: T4 and ΦK1E. The antigens probably share common epitopes, suggesting that the determined level of anti-I11mO19 phage might be overestimated and reflects a group of antibodies reactive to a broad range of other E. coli phages. Anti-I11mO19 antibodies did not react with Pseudomonas bacteriophage F8, confirming specificity to the coliphage group. In this work, we wanted to show whether it is possible to determine the presence and level of anti-phage antibodies in nontargeted-immunized sera, using an immunoenzymatic assay. The conclusion is that it is possible, and specific antibodies can be determined. However, the specificity refers to a broader coliphage group of phages, not only the single phage strain.

Method for direct measuring antibody affinity in biological sample.

We are reporting a new method to measure the affinity of specific antibodies in biological sample without the need of purification, or direct labeling. It was used to measure the affinity of anti-RBD (SARS-CoV2) IgG in plasma from Covid-19 recovered patient and vaccinated populations. Among the vaccinated population, we also measured the affinities of vaccine-induced antibodies against the RBD of the ancestral SARS-Cov-2 virus and the Omicron variants. The method has provided a quantitative way to evaluate antibodies produced by vaccination. The new method involves a classical Solution phase Equilibrium Titration step and an Imaging based Detection step (SET-ID). In the Solution phase Titration step, the antibody was kept at constant concentration (lower than the expected KD value) and mixed with various concentrations of antigen solution; In the Imaging Detection step, the amount of free antibody (not bound to antigen) at each antigen concentration was probed with small amount of microparticles coated with antigen and then the labeled secondary antibody. In this presentation, we will discuss the critical experimental condition, validation, and limitations. For verification, the binding curves of an antibody were compared using a well-established fluorescence spectroscopic-based method and the SET-ID method. The incubation time and the number of microparticles used in the second step were evaluated experimentally and theoretically to ensure minimal perturbation to the solution phase equilibrium, while the fraction of free antibody presented in the solution can still be measured by the signal on the microparticles.

Structure and neutralization mechanism of a human antibody targeting a complex Epitope on Zika virus.

We currently have an incomplete understanding of why only a fraction of human antibodies that bind to flaviviruses block infection of cells. Here we define the footprint of a strongly neutralizing human monoclonal antibody (mAb G9E) with Zika virus (ZIKV) by both X-ray crystallography and cryo-electron microscopy. Flavivirus envelope (E) glycoproteins are present as homodimers on the virion surface, and G9E bound to a quaternary structure epitope spanning both E protomers forming a homodimer. As G9E mainly neutralized ZIKV by blocking a step after viral attachment to cells, we tested if the neutralization mechanism of G9E was dependent on the mAb cross-linking E molecules and blocking low-pH triggered conformational changes required for viral membrane fusion. We introduced targeted mutations to the G9E paratope to create recombinant antibodies that bound to the ZIKV envelope without cross-linking E protomers. The G9E paratope mutants that bound to a restricted epitope on one protomer poorly neutralized ZIKV compared to the wild-type mAb, demonstrating that the neutralization mechanism depended on the ability of G9E to cross-link E proteins. In cell-free low pH triggered viral fusion assay, both wild-type G9E, and epitope restricted paratope mutant G9E bound to ZIKV but only the wild-type G9E blocked fusion. We propose that, beyond antibody binding strength, the ability of human antibodies to cross-link E-proteins is a critical determinant of flavivirus neutralization potency.

An Overview of ELISA-Based Initial Velocity Methods to Measure the Immunoreactive Fraction, Association Rate, And Equilibrium Constants of Monoclonal Antibodies

Three biochemical parameters—the on-rate (k+1), the dissociation rate (k-1), and the equilibrium (Ka) constants—describe the antibody interaction with a ligand. In addition, the antibody immune reactive fraction (IRF) is important because it affects k+1 and Ka constant values. The on-rate and dissociation constants are kinetic, but the equilibrium constant is a thermodynamic parameter, and the methods for their determination are different. Among the available methods to determine these parameters, only surface plasmon resonance (SPR)-based techniques allow calculation of the three antibody constants in a single experiment. However, conventional immunoassays are also suitable to determine antibody constants. Here, we describe some enzyme immunoassays (ELISA) methods developed in our laboratory to, i) quantitate mAb in culture supernatants from the initial velocity of the progress curve, ii) determine mAb IRF by a two-step ELISA method, iii) calculate mAb on-rate constant by kinetic ELISA based on time-course data of ligand binding to capture mAb, and, iv) describe a competitive-inhibition ELISA to measure the inhibition constant (Ki) of mAb binding to plate-bound ligand, in the presence of competing concentrations of mAb and soluble ligand.

Visualization of Multiple Myeloma Mouse Model with 89zr-Labeled Anti-CD38 Antibody By a New Imaging System; Whole-Gamma Imaging

Introduction Visualization and measurement of multiple myeloma (MM) cells in vivo by means of positron emission tomography (PET) is expected to determine biopsy sites, personalize treatments, and make an early diagnosis of recurrence. The positron radioisotope 89Zr is one of the promising radionuclides for PET image due to the long radioactive half-life of 3 days and stable labelling of antibodies. 89Zr also emits 909 keV single gamma-rays about 4 times more frequently than positrons, causing just noise to make PET images. To overcome this disadvantage of 89Zr, we propose a new imaging method that detects and uses the single gamma-rays as well as positrons for imaging, called whole gamma imaging (WGI). WGI is composed of PET and Compton imaging methods, equipped with two PET detector rings; both of the inner ring and the outer ring also acts as a scatterer and an absorber, respectively, for Compton imaging method (Fig. 1). In this study, we have firstly validated the affinity and specificity of antibodies recognizing CD38 and SLAMF7 in two types of human MM cell lines: RPMI8226 with CD38 expression and U226 with CD38 and SLAMF7. Then we carried out imaging in xenograft model mice with WGI using an 89Zr-antibody tracer and finally compared the quantitative values by WGI to those by ex-vivo biodistribution method. Methods Specific binding of antibodies was evaluated up to 10 × 106 cells of each MM cell type. An inhibition assay was performed using unlabeled IgG. 89Zr was produced in a cyclotron and neutralized 89Zr-oxalate was added to chelate-conjugated antibodies. Two severe combined immunodeficiency (SICD) mice after 3 Gy gamma-ray irradiation were implanted subcutaneously in the right flank with 3×107 MM cells in 100μL of PBS. On 12 days after the implantation, the tumor volume reached approximately 180 mm3 (mouse 1) and 110 mm3 (mouse 2). Based on the in-vitro study, 89Zr-anti-CD38 antibodies at the dose of 3.3 MBq were injected via a tail-vein. WGI imaging was performed on 4, 6, 8, and 12 days after the tracer injection. After WGI imaging on day 12, mice were euthanized for ex-vivo biodistribution analysis; blood was collected via cardiac puncture into counting tubes. Tumor and normal tissues: lung, liver, spleen, kidney, muscle, heart, bone (tibia), pancreas, stomach, intestine, brain, and lymph node were excised, and their weight and radioactivity were measured, and %ID/g values were calculated. Results High specific binding fractions of anti-CD38 antibodies were observed for RPMI8226 cells, reaching to approximately 50% at 10 × 106 cells; while only 5% for U226 cells. Those of anti-SLAMF7 antibodies were under 1% for both types of tumor cells. The inhibition assay using cold IgG blocked the binding of anti-CD 38 antibodies (Kd = 1.2 nM) and chelate-conjugated anti-CD38 antibodies (Kd = 1.4 nM) to RPMI8226. Then we decided to use 89Zr-anti-CD38 antibody and RPMI8226 cells for the WGI imaging study. WIG imaging showed that 89Zr-anti-CD38 antibody uptake in tumors increased over time, being up to standardized uptake value (SUV) 9.8 on 12 days after tracer injection. The uptake in the healthy tissues gradually decreased with time except for the bone (Fig. 2). Ex-vivo biodistribution revealed that the highest 89Zr-anti-CD38 antibody uptake in tumors was 92.4%ID/g (SUV, 18.5) and 97.5%ID/g (SUV, 19.5) in mice 1 and 2, respectively. That in healthy tissues was under 27.7%ID/g. Considering the specific activity of 89Zr-anti-CD38 antibodies of 4.2 MBq/nmol, 89Zr-anti-CD38 antibodies accumulated in RPMI8226 tumors at approximately 730 fmol/mg. Conclusion We proposed a new imaging method, called WGI, which clearly visualized MM cells and was able to estimate the uptake values of 89Zr-anti-CD38 antibody binding to specific antigen in MM tumors. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Moving the needle: Employing deep reinforcement learning to push the boundaries of coarse-grained vaccine models.

Highly mutable infectious disease pathogens (hm-IDPs) such as HIV and influenza evolve faster than the human immune system can contain them, allowing them to circumvent traditional vaccination approaches and causing over one million deaths annually. Agent-based models can be used to simulate the complex interactions that occur between immune cells and hm-IDP-like proteins (antigens) during affinity maturation-the process by which antibodies evolve. Compared to existing experimental approaches, agent-based models offer a safe, low-cost, and rapid route to study the immune response to vaccines spanning a wide range of design variables. However, the highly stochastic nature of affinity maturation and vast sequence space of hm-IDPs render brute force searches intractable for exploring all pertinent vaccine design variables and the subset of immunization protocols encompassed therein. To address this challenge, we employed deep reinforcement learning to drive a recently developed agent-based model of affinity maturation to focus sampling on immunization protocols with greater potential to improve the chosen metrics of protection, namely the broadly neutralizing antibody (bnAb) titers or fraction of bnAbs produced. Using this approach, we were able to coarse-grain a wide range of vaccine design variables and explore the relevant design space. Our work offers new testable insights into how vaccines should be formulated to maximize protective immune responses to hm-IDPs and how they can be minimally tailored to account for major sources of heterogeneity in human immune responses and various socioeconomic factors. Our results indicate that the first 3 to 5 immunizations, depending on the metric of protection, should be specially tailored to achieve a robust protective immune response, but that beyond this point further immunizations require only subtle changes in formulation to sustain a durable bnAb response.

P120 Vaccine induced protection by secreted aspartyl proteinase 2 from Candida parapsilosis in Candida tropicalis mediated murine systemic candidiasis: a role of B-cells and antibodies

Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM ObjectivesNon-albicans Candida species are increasingly becoming more prevalent globally and emergence of drug resistance is another cause of concern. These challenges merit the need for developing novel immune therapies against Candida infections. We investigated the protective potential of secreted aspartyl proteinase (Sap2), a leading Candidate vaccine antigen identified from research based on C. albicans, in murine systemic candidiasis caused by non-albicans Candida species. Moreover, while the role of cellular immune responses in anti-Candida immunity is well established, less is known about the role of humoral immunity against systemic candidiasis. As such, we also investigated the role of humoral immunity in vaccine-mediated protection during murine systemic candidiasis.MethodsThe Sap2 genes were successfully cloned and expressed as recombinant proteins from C. albicans, C. tropicalis and C. parapsilosis strains prevalent in India. Groups of wild-type BALb/c mice were vaccinated with individual rSap2 protein along with alum as adjuvant, followed by systemic infection with a lethal dose of C. tropicalis. The protective potential of each rSap2 protein was evaluated using survival analysis and estimation of organ fungal burden. Histopathological assessment was performed using H&E and PAS staining. Serum cytokine levels and antigen-specific antibody titers were measured by ELISA. Cellular responses were analyzed in detail using flow cytometry. Functional evaluation of antibody role was performed using in vitro (biofilm inhibition and neutrophil-mediated killing) and in vivo (passive transfer) studies. B-cell epitope analysis was carried out using immunoinformatics approaches.ResultsMice vaccinated with rSap2 cloned from C. parapsilosis (Sap2-parapsilosis) showed highest increase in survival time (P = .02) and maximum reduction in organ fungal burden (spleen, kidney, lungs, brain) (P <.05); compared with sham immunized controls. Vaccination with rSap2 cloned from C. albicans did not improve survival in non-albicans C. tropicalis infection, despite the protein having ∼60% homology across species. Mice vaccinated with rSap2-parapsilosis also exhibited significantly higher levels of IFN gamma, IL-17, and IL-4 cytokine levels just before infection, which correlated with protection. In addition, rSap2-parapsilosis vaccination induced high titers of Sap2-specific antibodies, and a fraction of antibodies could bind whole fungus (which were predominantly of IgM isotype). Notably, sera from rSap2-parapsilosis vaccinated mice exhibited increased C. tropicalis biofilm inhibition ability and enhanced neutrophil-mediated fungal killing in vitro. Sap2-specificity was further confirmed by depleting antigen-specific antibodies. Passive transfer of Sap2-parapsilosis immune serum significantly reduced fungal burdens in naive mice, as compared to mice receiving sham-immune serum, upon infection. Higher numbers of total CD19 + B-cells, plasma cells, and Candida-binding B-cells in rSap2-parapsilosis vaccinated mice indicate a role of B-cells during early stages of Sap2-mediated immune response. Epitope analysis performed using identified B-cell epitopes provides insights about including important IgM and IgG epitopes, when designing multivalent or multi-epitope anti-Candida vaccine/s.ConclusionMice immunized with recombinant Sap2 cloned from C. parapsilosis exhibited increased survival during C. tropicalis mediated systemic candidiasis. Both Th1/Th17 mediated cellular immunity and humoral immunity contribute toward protection. Our results establish a role of B-cells and antibodies in generation of protective immune responses against Sap2 vaccine antigen during non-albicans Candida mediated murine systemic candidiasis.

Analysis of a SARS-CoV-2 convalescent cohort identified a common strategy for escape of vaccine-induced anti-RBD antibodies by Beta and Omicron variants.

SummaryBackgroundEvolutionary pressure has led to the emergence of SARS-CoV-2 variants, with the most recent Omicron variant containing an unparalleled 30 mutations in the spike protein. Many of these mutations are expected to increase immune evasion, thus making breakthrough cases and re-infection more common.MethodsFrom June 2020 to December 2021 serial blood samples (initial post recovery, 6 months, 12 months) were collected from a COVID-19 convalescent cohort in Boston, MA. Plasma was isolated for use in Mesoscale Discovery based antibody binding assays. Unvaccinated donors or those vaccinated prior to the primary blood draw were excluded from this analysis, as were those who did not have at least two blood draws. Wilcoxon signed rank tests were used to compare pre- and post-vaccination titers and antibody response against different variants, while McNemar tests were used to compare the proportions of achieving ≥ 4 fold increases against different variants.FindingsForty-eight COVID convalescent donors with post-infection vaccination (hybrid immunity) were studied to evaluate the levels of cross-reactive antibodies pre- and post- vaccination against various SARS-CoV-2 Spike and RBD proteins. Vaccination with BNT162b2, mRNA-1273 or Ad26.COV2.S led to a 6.3 to 7.8 fold increase in anti-Spike antibody titers and a 7·0 to 7·4 fold increase in anti-WT, Alpha and Delta RBD antibody. However, a lower response was observed for Beta and Omicron RBDs with only 7/48 (15%) and 15/48 (31%) donors having a ≥4 fold increase in post-vaccination titers against Beta and Omicron RBDs. Structural analysis of the Beta and Omicron RBDs reveal a shared immune escape strategy involving residues K417-E484-N501 that is exploited by these variants of concern.InterpretationThrough mutations of the K417-E484-N501 triad, SARS-CoV-2 has evolved to evade neutralization by the class I/II anti-RBD antibody fraction of hybrid immunity plasma as the polyclonal antibody response post-vaccination shows limitations in the ability to solve the structural requirements to bind the mutant RBDs.FundingMassachusetts Consortium on Pathogen Readiness (280870.5116709.0016) and the National Institute of Allergy and Infectious Diseases (1R01AI161152-01A1).

Variations in antibody repertoires correlate with vaccine responses.

An important challenge in vaccine development is to figure out why a vaccine succeeds in some individuals and fails in others. Although antibody repertoires hold the key to answering this question, there have been very few personalized immunogenomics studies so far aimed at revealing how variations in immunoglobulin genes affect a vaccine response. We conducted an immunosequencing study of 204 calves vaccinated against bovine respiratory disease (BRD) with the goal to reveal variations in immunoglobulin genes and somatic hypermutations that impact the efficacy of vaccine response. Our study represents the largest longitudinal personalized immunogenomics study reported to date across all species, including humans. To analyze the generated data set, we developed an algorithm for identifying variations of the immunoglobulin genes (as well as frequent somatic hypermutations) that affect various features of the antibody repertoire and titers of neutralizing antibodies. In contrast to relatively short human antibodies, cattle have a large fraction of ultralong antibodies that have opened new therapeutic opportunities. Our study reveals that ultralong antibodies are a key component of the immune response against the costliest disease of beef cattle in North America. The detected variants of the cattle immunoglobulin genes, which are implicated in the success/failure of the BRD vaccine, have the potential to direct the selection of individual cattle for ongoing breeding programs.

Analysis of BNT162b2- and CVnCoV-elicited sera and of convalescent sera toward SARS-CoV-2 viruses.

The mRNA vaccine BNT162b2 (Comirnaty, BioNTech/Pfizer) and the vaccine candidate CVnCoV (Curevac) each encode a stabilized spike protein of SARS-CoV2 as antigen but differ with respect to the nature of the mRNA (modified versus unmodified nucleotides) and the mRNA amount (30μg versus 12μg RNA). This study characterizes antisera elicited by these two vaccines in comparison to convalescent sera. Sera from BNT162b2 vaccinated healthcare workers, and sera from participants of a phase I trial vaccinated with 2, 4, 6, 8, or 12μg CVnCoV and convalescent sera from hospitalized patients were analyzed by ELISA, neutralization tests, surface plasmon resonance (SPR), and peptide arrays. BNT162b2-elicited sera and convalescent sera have a higher titer of spike-RBD-specific antibodies and neutralizing antibodies as compared to the CVnCoV-elicited sera. For all analyzed sera a reduction in binding and neutralizing antibodies was found for the lineage B.1.351 variant of concern. SPR analyses revealed that the CVnCoV-elicited sera have a lower fraction of slow-dissociating antibodies. Accordingly, the CVnCoV sera almost fail to compete with the spike-ACE2 interaction. The significance of common VOC mutations K417N, E484K, or N501Y focused on linear epitopes was analyzed using a peptide array approach. The peptide arrays showed a strong difference between convalescent sera and vaccine-elicited sera. Specifically, the linear epitope at position N501 was affected by the mutation and elucidates the escape of viral variants to antibodies against this linear epitope. These data reveal differences in titer, neutralizing capacity, and affinity of the antibodies between BNT162b2- and CVnCoV-elicited sera, which could contribute to the apparent differences in vaccine efficacy.