Bactericidal human monoclonal antibody 1B1 shows specificity for meningococcal factor H binding protein variant 2 and displaces human factor H.

Thousands of disease cases and hundreds of deaths occur globally each year due to invasive meningococcal disease. Neisseria meningitidis serogroup B (MenB) is the leading cause of such disease in developed countries. Two vaccines, 4CMenB and MenB-fHbp, that protect against MenB are available and include one or two forms respectively of factor H binding protein (fHbp), a key protective antigen. Studies of circulating meningococci have identified over 1380 different fHbp amino acid sequences, which form three immunologically distinct clusters, termed variants 1, 2, and 3. Neither of the current vaccines contains a variant 2 antigen, which is less well characterized than fHbp variants 1 and 3. We characterized the interaction of fHbp variant 2 with humAb 1B1 using biochemical methods and live meningococcal assays. Further, we determined the crystal structure of the complex at 2.4 Å resolution, clearly revealing the epitope and providing the first detailed report of an antibody with distinct specificity for fHbp variant 2. Extensive mutagenesis and binding studies elucidated key hotspots in the interface. This combination of structural and functional studies provides a molecular explanation for the bactericidal potency and specificity of humAb 1B1 for fHbp variant 2. Our studies, focused on fHbp variant 2, expand the understanding of this previously under characterized group of the vast family of variants of fHbp, a virulence factor present on all meningococci. Moreover, the definition of a protective conformational epitope on fHbp variant 2 may support the design and development of novel variant 2-containing MenB vaccines affording greater breadth of protection.

Interleukin-17A (IL-17A) is involved in antibody specificity to conformational epitopes.

The specificity of antibodies (Ab) is essential for the precise recognition of foreign or dangerous molecules. We have shown that mice infected with non-pathogenic Lactate Dehydrogenase Elevating Virus (LDV) inoculated with human growth hormone (hGH) or Ovalbumin (OVA), exhibit modified specificity of anti-hGH or anti-OVA Ab associated with the secretion of IFN-γ, IL-13, and IL-17. Cytokines are directly or indirectly involved in the isotypes, specificity, and affinity of Ab. Accordingly, here we investigated the effect of IL-17 neutralization on Ab specificities to OVA and Diphtheria Toxoid (DTx) in a mouse model of viral infection. Thereby, we employed an anti-cytokine "auto-vaccination" with an OVA/IL-17A complex or a Monoclonal Ab (MAb) anti-IL-17A (MM17/F3). Competitive ELISA assays were used to estimate the quality of the humoral immune response and the amount of Abs to conformational versus linear antigenic determinants. Results indicated that the OVA/IL-17A complex increased Abs levels to conformational epitopes of OVA, while LDV prolonged antibodies for a longer period. Mice treated with MM17F3 MAb showed an increase in Abs to conformational epitopes of OVA. A similar effect, confirmed by a competitive Western-blot assay, was produced by LDV. Moreover, an increased level of IgM, IgG1, and IgG2a was found in infected animals. Similarly, MAb anti-IL-17A treatment increased the proportion of Ab to conformational epitopes of DTx in uninfected mice, while LDV decreased this parameter. In conclusion, our findings demonstrate a correlation between IL-17A neutralization and a change in Ab specificity to OVA or DTx, presenting a novel strategy for obtaining Abs with higher specificity.

BTN1A1 is a novel immune checkpoint mutually exclusive to PD-L1.

BACKGROUND: While Programmed cell death protein 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) blockade is a potent antitumor treatment strategy, it is effective in only limited subsets of patients with cancer, emphasizing the need for the identification of additional immune checkpoints. Butyrophilin 1A1 (BTN1A1) has been reported to exhibit potential immunoregulatory activity, but its ability to function as an immune checkpoint remains to be systematically assessed, and the mechanisms underlying such activity have yet to be characterized. METHODS: BTN1A1 expression was evaluated in primary tumor tissue samples, and its ability to suppress T-cell activation and T cell-dependent tumor clearance was examined. The relationship between BTN1A1 and PD-L1 expression was further characterized, followed by the development of a BTN1A1-specific antibody that was administered to tumor-bearing mice to test the amenability of this target to immune checkpoint inhibition. RESULTS: BTN1A1 was confirmed to suppress T-cell activation in vitro and in vivo. Robust BTN1A1 expression was detected in a range of solid tumor tissue samples, and BTN1A1 expression was mutually exclusive with that of PD-L1 as a consequence of its inhibition of Janus-activated kinase/signal transducer and activator of transcription signaling-induced PD-L1 upregulation. Antibody-mediated BTN1A1 blockade suppressed tumor growth and enhanced immune cell infiltration in syngeneic tumor-bearing mice. CONCLUSION: Together, these results confirm that the potential of BTN1A1 is a bona fide immune checkpoint and a viable immunotherapeutic target for the treatment of individuals with anti-PD-1/PD-L1 refractory or resistant disease, opening new avenues to improving survival outcomes for patients with a range of cancers.

Western Blot of Tau Protein from Mouse Brains Extracts: How to Avoid Signal Artifacts.

Tau is a microtubule-associated protein enriched in the axonal compartment. Its most well-known function is to bind and stabilize microtubules. In Alzheimer's disease and other neurodegenerative diseases known as tauopathies, tau undergoes several abnormal post-translational modifications including hyperphosphorylation, conformational changes, oligomerization, and aggregation. Numerous mouse models of tauopathies have been developed, and Western blotting remains an invaluable tool in studying tau protein physiological and pathological changes in these models. However, many of the antibodies that have been developed to analyze tau post-translational modifications are mouse monoclonal, which are at risk of producing artifactual signals in Western blotting procedures. This risk does not arise due to their lack of specificity, but rather because the secondary antibodies used to detect them will also react with the heavy chain of endogenous mouse immunoglobulins (Igs), leading to a non-specific signal at the same molecular weight as tau protein (around 50 kDa). Here, we present the use of anti-light-chain secondary antibodies as a simple and efficient technique to prevent non-specific Ig signals around 50 kDa. We demonstrate the efficacy of this method by either eliminating or identifying artifactual signals when using monoclonal antibodies directed at non-phosphorylated epitopes (T49, Tau3R, Tau4R), phosphorylated epitopes (MC6, AT180, CP13), or an abnormal tau conformation (MC1), in wild-type (WT) mice with tau hyperphosphorylation (hypothermic), transgenic mice overexpressing human tau (hTau mice), and tau knockout (TKO) mice.

Haemolytic transfusion reactions caused by non-ABO red cell antibodies reported to the Norwegian Haemovigilance System 2004-2020.

BACKGROUND AND OBJECTIVES: The aim of this study was to analyse the reports received in the Norwegian Haemovigilance System from 2004 to 2020 on acute and delayed haemolytic transfusion reactions caused by non-ABO red cell antibodies. MATERIALS AND METHODS: Antibody specificity, clinical symptoms and outcomes were included when available. RESULTS: After transfusion of 3.7 million red cell concentrates, reports on 78 cases of haemolytic transfusion reactions caused by non-ABO red cell antibodies were received, corresponding to an incidence of 1 in 47,000 transfused red cell concentrates. There were 30 acute and 48 delayed haemolytic transfusion reactions. A total of 113 red cell antibodies were found: 82 alloantibodies, 6 autoantibodies and 25 cases where the antibody specificity could not be determined. Two fatalities occurred: one caused by anti-Wra and one caused by an unidentified red cell antibody. The most frequently reported antibody specificities were those in the Rh and Kidd blood group systems, representing 24% and 14%, respectively, of all the antibodies identified. In six cases, errors occurred, leading to the issuing of blood units without the required phenotype match. CONCLUSIONS: Despite the possible underreporting, the low number of serious haemolytic transfusion reactions reflects an adequate pre-transfusion practice by the Norwegian blood banks.

Enhanced CD1d phosphatidylserine presentation using a single-domain antibody promotes immunomodulatory CD1d-TIM-3 interactions.

BACKGROUND: CD1d is a monomorphic major histocompatibility complex class I-like molecule that presents lipid antigens to distinct T-cell subsets and can be expressed by various malignancies. Antibody-mediated targeting of CD1d on multiple myeloma cells was reported to induce apoptosis and could therefore constitute a novel therapeutic approach. METHODS: To determine how a CD1d-specific single-domain antibody (VHH) enhances binding of the early apoptosis marker annexin V to CD1d+ tumor cells we use in vitro cell-based assays and CRISPR-Cas9-mediated gene editing, and to determine the structure of the VHH1D17-CD1d(endogenous lipid) complex we use X-ray crystallography. RESULTS: Anti-CD1d VHH1D17 strongly enhances annexin V binding to CD1d+ tumor cells but this does not reflect induction of apoptosis. Instead, we show that VHH1D17 enhances presentation of phosphatidylserine (PS) in CD1d and that this is saposin dependent. The crystal structure of the VHH1D17-CD1d(endogenous lipid) complex demonstrates that VHH1D17 binds the A'-pocket of CD1d, leaving the lipid headgroup solvent exposed, and has an electro-negatively charged patch which could be involved in the enhanced PS presentation by CD1d. Presentation of PS in CD1d does not trigger phagocytosis but leads to greatly enhanced binding of T-cell immunoglobulin and mucin domain containing molecules (TIM)-1 to TIM-3, TIM-4 and induces TIM-3 signaling. CONCLUSION: Our findings reveal the existence of an immune modulatory CD1d(PS)-TIM axis with potentially unexpected implications for immune regulation in both physiological and pathological conditions.

A strong case for third-party testing.

A strategy to identify high-quality commercially available antibodies for research reveals extensive use of non-specific antibodies and offers solutions for future large-scale testing.

The expression of antibodies to Z-DNA in the blood of patients with systemic lupus erythematosus: Relationship to autoantibodies to B-DNA.

To explore the antibody response to Z-DNA, a DNA conformation with a zig-zag structure, blood of patients with systemic lupus erythematosus (SLE) and otherwise healthy individuals (NHS) were assayed by ELISA using brominated poly(dGdC), a synthetic Z-DNA antigen. These studies showed that SLE patients commonly express antibodies to Z-DNA; NHS also had binding in this assay. In SLE blood, levels of antibodies to Z-DNA were related to those to B-DNA using calf thymus DNA as a source of B-DNA; cross-reactivity was demonstrated by adsorption experiments using DNA cellulose. As shown by dissociation assays, antibody binding of SLE anti-Z-DNA is sensitive to the effects of ionic strength, suggesting electrostatic binding. Since Z-DNA structure can be found in bacterial DNA as well as bacterial biofilms, these findings suggest that, in SLE, anti-DNA antibody responses can result from stimulation by DNA of bacterial origin, with cross-reactivity leading to autoreactivity.

Validation and the Determination of Antibody Bioactivity Using MILKSHAKE and Sundae Protocols.

To develop reproducible results, it is critical that all reagents used in an experiment be validated in an alternative or independent method. We present two such independent methods for determining the specificity of antibodies: (1) "MILKSHAKE," which can be used to validate the liability and specificity of antibodies directed against post-translationally-modified epitopes, and (2) "Sundae," which is a more complete alanine-like scanning method that can be used to better understand the binding and bioactivity of specific residues of a protein. We apply both of these methods to the interaction between an antibody and its antigen.

Evaluation of tumor antigen-specific antibody responses in patients with metastatic triple negative breast cancer treated with cyclophosphamide and pembrolizumab.

The role of B cells in antitumor immunity is becoming increasingly appreciated, as B cell populations have been associated with response to immune checkpoint blockade (ICB) in patients with breast cancer and murine models of breast cancer. Deeper understanding of antibody responses to tumor antigens is needed to clarify the function of B cells in determining response to immunotherapy. We evaluated tumor antigen-specific antibody responses in patients with metastatic triple negative breast cancer treated with pembrolizumab following low-dose cyclophosphamide therapy using computational linear epitope prediction and custom peptide microarrays. We found that a minority of predicted linear epitopes were associated with antibody signal, and signal was associated with both neoepitopes and self-peptides. No association was observed between signal presence and subcellular localization or RNA expression of parent proteins. Patient-specific patterns of antibody signal boostability were observed that were independent of clinical response. Intriguingly, measures of cumulative antibody signal intensity relative to immunotherapy treatment showed that the one complete responder in the trial had the greatest increase in total antibody signal, which supports a potential association between ICB-dependent antibody boosting and clinical response. The antibody boost in the complete responder was largely driven by increased levels of IgG specific to a sequence of N-terminal residues in native Epidermal Growth Factor Receptor Pathway Substrate 8 (EPS8) protein, a known oncogene in several cancer types including breast cancer. Structural protein prediction showed that the targeted epitope of EPS8 was in a region of the protein with mixed linear/helical structure, and that this region was solvent-exposed and not predicted to bind to interacting macromolecules. This study highlights the potential importance of the humoral immune response targeting neoepitopes as well as self epitopes in shaping clinical response to immunotherapy.

Understanding the SARS-CoV-2 Virus Neutralizing Antibody Response: Lessons to Be Learned from HIV and Respiratory Syncytial Virus.

The SARS-CoV-2 pandemic commenced in 2019 and is still ongoing. Neither infection nor vaccination give long-lasting immunity and, here, in an attempt to understand why this might be, we have compared the neutralizing antibody responses to SARS-CoV-2 with those specific for human immunodeficiency virus type 1 (HIV-1) and respiratory syncytial virus (RSV). Currently, most of the antibodies specific for the SARS-CoV-2 S protein map to three broad antigenic sites, all at the distal end of the S trimer (receptor-binding site (RBD), sub-RBD and N-terminal domain), whereas the structurally similar HIV-1 and the RSV F envelope proteins have six antigenic sites. Thus, there may be several antigenic sites on the S trimer that have not yet been identified. The epitope mapping, quantitation and longevity of the SARS-CoV-2 S-protein-specific antibodies produced in response to infection and those elicited by vaccination are now being reported for specific groups of individuals, but much remains to be determined about these aspects of the host-virus interaction. Finally, there is a concern that the SARS-CoV-2 field may be reprising the HIV-1 experience, which, for many years, used a virus for neutralization studies that did not reflect the neutralizability of wild-type HIV-1. For example, the widely used VSV-SARS-CoV-2-S protein pseudotype has 10-fold more S trimers per virion and a different configuration of the trimers compared with the SARS-CoV-2 wild-type virus. Clarity in these areas would help in advancing understanding and aid countermeasures of the SARS-CoV-2 pandemic.

Cohort-Specific Serological Recognition of SARS-CoV-2 Variant RBD Antigens.

OBJECTIVE: Estimating the response of different population cohorts to new SARS-CoV-2 variants is important to customize measures of control. Our goal was to evaluate how antibodies from sera of infected and vaccinated people recognize antigens expressed by different SARS-CoV-2 variants. METHODS: We compared sera from vaccinated donors and four patient/donor cohorts: Sera from critically ill patients collected 2-7 days and more than 10 days after admission to an intensive care unit, a NIBSC/WHO reference panel of SARS-CoV-2 positive individuals, and ambulatory or hospitalized (but not critically ill) positive donors. Samples were tested with an anti-SARS-CoV-2 ELISA kit coated with SARS-CoV-2 RBD recombinant antigens including mutations present in eleven of the most widespread variants. RESULTS: Sera from vaccinated individuals exhibited higher antibody binding (P<0.001) than sera from infected (but not critically ill) individuals when tested against the wild type (WT) and each of 11 variants' receptor binding domain (RBD). Antibodies' binding to the SARS-CoV-2 antigens of at least 6 variants, including Variants of Concern (VOCs), was reduced in comparison to the WT in vaccinated and non-critically ill convalescence individuals. CONCLUSION: Understanding differences between population cohorts in the antibody titers against WT vs variant RBD antigens can help design variant-specific immunoassays for surveillance and evaluation of the epidemiology of new variants.

Preclinical characterization and clinical translation of pharmacodynamic markers for MK-5890: a human CD27 activating antibody for cancer immunotherapy.

BACKGROUND: Immune checkpoint inhibitors (ICI) have radically changed cancer therapy, but most patients with cancer are unresponsive or relapse after treatment. MK-5890 is a CD27 agonist antibody intended to complement ICI therapy. CD27 is a member of the tumor necrosis factor receptor superfamily that plays a critical role in promoting responses of T cells, B cells and NK cells. METHODS: Anti-CD27 antibodies were generated and selected for agonist activity using NF-кB luciferase reporter assays. Antibodies were humanized and characterized for agonism using in vitro T-cell proliferation assays. The epitope recognized on CD27 by MK-5890 was established by X-ray crystallography. Anti-tumor activity was evaluated in a human CD27 knock-in mouse. Preclinical safety was tested in rhesus monkeys. Pharmacodynamic properties were examined in mouse, rhesus monkeys and a phase 1 dose escalation clinical study in patients with cancer. RESULTS: Humanized anti-CD27 antibody MK-5890 (hIgG1) was shown to bind human CD27 on the cell surface with sub-nanomolar potency and to partially block binding to its ligand, CD70. Crystallization studies revealed that MK-5890 binds to a unique epitope in the cysteine-rich domain 1 (CRD1). MK-5890 activated CD27 expressed on 293T NF-κB luciferase reporter cells and, conditional on CD3 stimulation, in purified CD8+ T cells without the requirement of crosslinking. Functional Fc-receptor interaction was required to activate CD8+ T cells in an ex vivo tumor explant system and to induce antitumor efficacy in syngeneic murine subcutaneous tumor models. MK-5890 had monotherapy efficacy in these models and enhanced efficacy of PD-1 blockade. MK-5890 reduced in an isotype-dependent and dose-dependent manner circulating, but not tumor-infiltrating T-cell numbers in these mouse models. In rhesus monkey and human patients, reduction in circulating T cells was transient and less pronounced than in mouse. MK-5890 induced transient elevation of chemokines MCP-1, MIP-1α, and MIP-1ß in the serum of mice, rhesus monkeys and patients with cancer. MK-5890 was well tolerated in rhesus monkeys and systemic exposure to MK-5890 was associated with CD27 occupancy at all doses. CONCLUSIONS: MK-5890 is a novel CD27 agonistic antibody with the potential to complement the activity of PD-1 checkpoint inhibition in cancer immunotherapy and is currently undergoing clinical evaluation.

The Quest for Anti-α-Synuclein Antibody Specificity-Lessons Learnt From Flow Cytometry Analysis.

The accumulation of alpha-synuclein (aSyn) is the hallmark of a group of neurodegenerative conditions termed synucleopathies. Physiological functions of aSyn, including those outside of the CNS, remain elusive. However, a reliable and reproducible evaluation of aSyn protein expression in different cell types and especially in low-expressing cells is impeded by the existence of a huge variety of poorly characterized anti-aSyn antibodies and a lack of a routinely used sensitive detection methods. Here, we developed a robust flow cytometry-based workflow for aSyn detection and antibody validation. We test our workflow using three commercially available antibodies (MJFR1, LB509, and 2A7) in a variety of human cell types, including induced pluripotent stem cells, T lymphocytes, and fibroblasts, and provide a cell- and antibody-specific map for aSyn expression. Strikingly, we demonstrate a previously unobserved unspecificity of the LB509 antibody, while the MJFR1 clone revealed specific aSyn binding however with low sensitivity. On the other hand, we identified an aSyn-specific antibody clone 2A7 with an optimal sensitivity for detecting aSyn in a range of cell types, including those with low aSyn expression. We further utilize our workflow to demonstrate the ability of the 2A7 antibody to distinguish between physiological differences in aSyn expression in neuronal and non-neuronal cells from the cortical organoids, and in neural progenitors and midbrain dopaminergic neurons from healthy controls and in patients with Parkinson's disease who have aSyn gene locus duplication. Our results provide a proof of principle for the use of high-throughput flow cytometry-based analysis of aSyn and highlight the necessity of rigorous aSyn antibody validation to facilitate the research of aSyn physiology and pathology.

The Anti-Glucocorticoid Receptor Antibody Clone 5E4: Raising Awareness of Unspecific Antibody Binding.

Unspecific antibody binding takes a significant toll on researchers in the form of both the economic burden and the disappointed hopes of promising new therapeutic targets. Despite recent initiatives promoting antibody validation, a uniform approach addressing this issue has not yet been developed. Here, we demonstrate that the anti-glucocorticoid receptor (GR) antibody clone 5E4 predominantly targets two different proteins of approximately the same size, namely AMP deaminase 2 (AMPD2) and transcription intermediary factor 1-beta (TRIM28). This paper is intended to generate awareness of unspecific binding of well-established reagents and advocate the use of more rigorous verification methods to improve antibody quality in the future.

Induced antigen-binding polyreactivity in human serum IgA.

Previous studies have shown that polyreactive antibodies play an important role in the frontline defense against the dissemination of pathogens in the pre-immune host. Interestingly, antigen-binding polyreactivity can not only be inherent, but also acquired post-translationally. The ability of individual monoclonal IgG and IgE antibodies to acquire polyreactivity following contact with various agents that destabilize protein structure (urea, low pH) or have a pro-oxidative potential (heme, ferrous ions) has been studied in detail. However, to the best of our knowledge this property of human IgA has previously been described only cursorily. In the present study pooled human serum IgA and two human monoclonal IgA antibodies were exposed to buffers with acidic pH, to free heme or to ferrous ions, and the antigen-binding behavior of the native and modified IgA to viral and bacterial antigens were compared using immunoblot and ELISA. We observed a dose-dependent increase in reactivity to several bacterial extracts and to pure viral antigens. This newly described property of IgA may have therapeutic potential as has already been shown for pooled IgG with induced polyreactivity.

Development of a TCR-like antibody and chimeric antigen receptor against NY-ESO-1/HLA-A2 for cancer immunotherapy.

BACKGROUND: The current therapeutic antibodies and chimeric antigen receptor (CAR) T cells are capable of recognizing surface antigens, but not of intracellular proteins, thus limiting the target coverage for drug development. To mimic the feature of T-cell receptor (TCR) that recognizes the complex of major histocompatibility class I and peptide on the cell surface derived from the processed intracellular antigen, we used NY-ESO-1, a cancer-testis antigen, to develop a TCR-like fully human IgG1 antibody and its derivative, CAR-T cells, for cancer immunotherapy. METHODS: Human single-chain variable antibody fragment (scFv) phage library (~10∧11) was screened against HLA-A2/NY-ESO-1 (peptide 157-165) complex to obtain target-specific antibodies. The specificity and affinity of those antibodies were characterized by flow cytometry, ELISA, biolayer interferometry, and confocal imaging. The biological functions of CAR-T cells were evaluated against target tumor cells in vitro. In vivo antitumor activity was investigated in a triple-negative breast cancer (TNBC) model and primary melanoma tumor model in immunocompromised mice. RESULTS: Monoclonal antibody 2D2 identified from phage-displayed library specifically bound to NY-ESO-1157-165 in the context of human leukocyte antigen HLA-A*02:01 but not to non-A2 or NY-ESO-1 negative cells. The second-generation CAR-T cells engineered from 2D2 specifically recognized and eliminated A2+/NY-ESO-1+tumor cells in vitro, inhibited tumor growth, and prolonged the overall survival of mice in TNBC and primary melanoma tumor model in vivo. CONCLUSIONS: This study showed the specificity of the antibody identified from human scFv phage library and demonstrated the potential antitumor activity by TCR-like CAR-T cells both in vitro and in vivo, warranting further preclinical and clinical evaluation of the TCR-like antibody in patients. The generation of TCR-like antibody and its CAR-T cells provides the state-of-the-art platform and proof-of-concept validation to broaden the scope of target antigen recognition and sheds light on the development of novel therapeutics for cancer immunotherapy.

Alternative Hapten Design for Zearalenone Immunoreagent Generation.

Appropriate hapten design and synthesis have been identified as critical steps to generate high-performance immunoreagents and to develop sensitive and selective immunoanalytical methods. Antibodies and immunoassays for the major mycotoxin zearalenone have been reported and marketed. However, zearalenone haptens have mostly been prepared by the oxime active ester technique, and hapten characterization has generally been poor or non-existent. In the present study, novel haptens of zearalenone with longer linkers and with alternative tethering sites have been designed for immunizing and assay conjugate preparation. All of these molecules were purified and spectroscopically verified, and a structure-activity relationship evaluation was carried out. This approach revealed that the hapten with the linker at the carbonyl group generated antibodies with a higher affinity than the hapten functionalized at the phenyl moiety. Antibodies produced with the latter hapten, on the other hand, showed lower cross-reactivity values to the major zearalenone metabolites. Finally, similar immunoassay sensitivity was achieved with all of the antibodies when heterologous haptens were employed. Furthermore, by altering the structure of the competing antigen, the immunoassay selectivity was modified. These results demonstrate that immunochemical methods for zearalenone rapid analysis can still be improved in terms of sensitivity and selectivity.

Generalizable design parameters for soluble T cell receptor-based T cell engagers.

While most biological and cellular immunotherapies recognize extracellular targets, T cell receptor (TCR) therapeutics are unique in their ability to recognize the much larger pool of intracellular antigens found on virus-infected or cancerous cells. Recombinant T cell receptor (rTCR)-based therapeutics are gaining momentum both preclinically and clinically highlighted by recent positive phase III human clinical trial results for a TCR/CD3 bifunctional protein in uveal melanoma. Unlike antibody-based T cell engagers whose molecular formats have been widely and extensively evaluated, little data exist describing the putative activities of varied bifunctional formats using rTCRs. Here we generate rTCR/anti-CD3 bifunctionals directed toward NY-ESO-1 or MAGE-A3 with a variety of molecular formats. We show that inducing strong redirected lysis activity against tumors displaying either NY-ESO-1 or MAGE-A3 is highly restricted to small, tandem binding formats with an rTCR/antiCD3 Fab demonstrating the highest potency, rTCR/anti-CD3 single chain variable domain fragment showing similar but consistently weaker potency, and IgG-like or IgG-Fc-containing molecules demonstrating poor activity. We believe this is a universal trait of rTCR bifunctionals, given the canonical TCR/human leukocyte antigen structural paradigm.

Suitability of GRK Antibodies for Individual Detection and Quantification of GRK Isoforms in Western Blots.

G protein-coupled receptors (GPCRs) are regulated by GPCR kinases (GRKs) which phosphorylate intracellular domains of the active receptor. This results in the recruitment of arrestins, leading to desensitization and internalization of the GPCR. Aside from acting on GPCRs, GRKs regulate a variety of membrane, cytosolic, and nuclear proteins not only via phosphorylation but also by acting as scaffolding partners. GRKs' versatility is also reflected by their diverse roles in pathological conditions such as cancer, malaria, Parkinson's-, cardiovascular-, and metabolic disease. Reliable tools to study GRKs are the key to specify their role in complex cellular signaling networks. Thus, we examined the specificity of eight commercially available antibodies targeting the four ubiquitously expressed GRKs (GRK2, GRK3, GRK5, and GRK6) in Western blot analysis. We identified one antibody that did not recognize its antigen, as well as antibodies that showed unspecific signals or cross-reactivity. Hence, we strongly recommend testing any antibody with exogenously expressed proteins to clearly confirm identity of the obtained Western blot results. Utilizing the most-suitable antibodies, we established the Western blot-based, cost-effective simple tag-guided analysis of relative protein abundance (STARPA). This method allows comparison of protein levels obtained by immunoblotting with different antibodies. Furthermore, we applied STARPA to determine GRK protein levels in nine commonly used cell lines, revealing differential isoform expression.