Monovalent Antigen Research Articles

The CH1 domain influences the expression and antigen sensing of the HIV-specific CH31 IgM-BCR and IgG-BCR

How different classes of the B cell antigen receptor (BCR) sense viral antigens used in vaccination protocols is poorly understood. Here, we study antigen binding and sensing of human Ramos B cells expressing a BCR of either the IgM or IgG1 class with specificity for the CD4-binding-site of the envelope (Env) protein of the HIV-1. Both BCRs carry an identical antigen binding site derived from the broad neutralizing antibody (bnAb) CH31. We find a five times higher expression of the IgG1-BCR in comparison to the IgM-BCR on the surface of transfected Ramos B cells. The two BCR classes also differ from each other in their interaction with cognate HIV Env antigens in that the IgG1-BCR and IgM-BCR bind preferentially to polyvalent and monovalent antigens, respectively. By generating an IgM/IgG1 chimeric BCR, we found that the class-specific BCR expression and antigen-sensing behavior can be transferred with the CH1γ domain from the IgG1-BCR to the IgM-BCR. Thus, the class of CH1 domain has an impact on BCR assembly and expression as well as on antigen sensing.

Abstract 3190: Twin Fc-immune cell engager (ICE): A novel antibody platform for cancer immunotherapy with enhanced avidity for Fcγ receptors and complement

Abstract Immunoglobulin G (IgG)-based mAbs have become a dominant class of cancer immunotherapy due to their excellent drug-like properties, long half-life, and generally safe profiles. Typical IgG-based therapeutic mAbs have strong binding to target antigens owing to the valency effect of two antigen-binding fragments (Fabs). However, the single crystallizable fragment (Fc) lacks the valency effect to engage immune effector elements. As Fcγ receptors (FcγR) such as FcγRIIa and FcγRIIIa and C1q complement component have low affinity for Fc, IgG-based mAbs require high antibody opsonization density to exhibit avidity for these low affinity Fcγ receptors and C1q. This may also account for the suboptimal efficacy of IgG-based mAbs against tumors with less abundant antigen. To enhance effector functions of IgG1 by overcoming such limitation, we devised a novel platform, Twin Fc-ICE, in which a single Fab region is attached by two Fc regions which allow for high avidity binding to Fcγ receptors and C1q. The two Fc regions of Twin Fc-ICE are arranged in parallel, not in tandem, to adopt natural topologies of multiple Fc regions presented by IgG1-opsonized targets. Supporting the avidity effect of Twin Fc-ICE for FcγR binding, we showed that CTN001, a Twin Fc-ICE with trastuzumab Fab, have markedly increased binding to FcγRs including FcγRI, FcγRIIa and FcγRIIIa compared with trastuzumab. The avid FcγR binding was further demonstrated by flow cytometry analysis of human blood which revealed significantly increased binding of CTN001 relative to trastuzumab for immune cells expressing activating FcγRs such as natural killer (NK) cells, monocytes, and dendritic cells (DCs). The monovalent antigen binding of Twin Fc-ICE requires Fab with high affinity. However, provided that high affinity is achieved, the monovalent Fab adds to the two Fc regions to maximize the Fc load on cancer cells expressing low levels of antigen. Indeed, flow cytometry analysis revealed that treatment of CTN001 alone results in higher Fc load on HER2-expressing cancer cells than combination of two anti-HER2 IgG1 mAbs, trastuzumab and pertuzumab. In consistent with increased Fc load on target cells and avid FcγR binding, CTN001 induces enhanced effector functions such as CDC, ADCC and ADCP against HER2-expressing cancer cells compared with trastuzumab, which will be presented in a separate poster. CTN001 exhibits comparable thermal stability with trastuzumab and can be readily purified to >95% purity with typical purification platforms. The serum half-life of CTN001 at 10 mg/kg intravenous dose in rats is found non-inferior to that of trastuzumab (~12 days versus ~7 days). Taken together, Twin Fc-ICE provides an optimized platform with drug-like properties, which leverages avidity effects of Fc to potentiate therapeutic efficacy of antibody. Citation Format: Eun Shik Choi, Hyunkyu Park, Jiseon Bae, Ki-June Lee, Eunmi Kim, Sujin Yoon, Deokjae Lee, Gi-Hyeok Yang. Twin Fc-immune cell engager (ICE): A novel antibody platform for cancer immunotherapy with enhanced avidity for Fcγ receptors and complement [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3190.

Adjuvanted nanoliposomes displaying six hemagglutinins and neuraminidases as an influenza virus vaccine

Inclusion of defined quantities of the two major surface proteins of influenza virus, hemagglutinin (HA) and neuraminidase (NA), could benefit seasonal influenza vaccines. Recombinant HA and NA multimeric proteins derived from three influenza serotypes, H1N1, H3N2, and type B, are surface displayed on nanoliposomes co-loaded with immunostimulatory adjuvants, generating "hexaplex" particles that are used to immunize mice. Protective immune responses to hexaplex liposomes involve functional antibody elicitation against each included antigen, comparable to vaccination with monovalent antigen particles. When compared to contemporary recombinant or adjuvanted influenza virus vaccines, hexaplex liposomes perform favorably in many areas, including antibody production, Tcell activation, protection from lethal virus challenge, and protection following passive sera transfer. Based on these results, hexaplex liposomes warrant further investigation as an adjuvanted recombinant influenza vaccine formulation.

Interaction of b-cell receptors and antigens with different spatial arrangement

B-cell receptors can interact with antigen epitopes on various objects: macromolecules, microorganisms or on the surface of other cells, e.g., follicular dendritic cells. Accordingly, B cells, on the one hand, have the ability to evaluate the location of pathogen surface epitopes, and, on the other hand, they must adapt their receptor apparatus to different epitope locations and antigen-bearing surface properties. Indeed, B-cell receptors and antibodies better bind objects with regular and dense epitope arrangement characteristic of many pathogens. As a result, such epitope arrangement can be recognized as a pathogen-associated geometric pattern, but the conditions for such recognition depend on the isotype of membrane immunoglobulin and the degree of B cell maturity. Young B cells express membrane IgM, which is involved in B cell development and the selection of their repertoire. Receptors with IgM do not impose strict requirements on epitope location and can activate B cells even upon binding a monovalent antigen. Receptors with membrane IgD are expressed later and predominate on naive B cells before entering the immune response. These receptors are optimized for two-point antigen binding and strictly require this type of interaction to induce an activation signal. Before contact with antigen, B-cell receptors are grouped in discrete membrane zones — nanoclusters, due to close interactions with the actin cytoskeleton. Contact with the antigen leads to the detachment of receptors from the cytoskeleton, rise in their mobility and the combining nanoclusters into microclusters — large clusters enriched with signaling molecules. The most dynamic changes are observed upon contact with an antigen fixed on the membrane of adjacent cell. In this case, free actin moves to the periphery of the intercellular contact zone, where it forms the cytoskeleton of the processes carrying receptor clusters. The processes spread across the surface of the partner cell and then contract, moving the antigen-binding microclusters to the center of the contact zone. Finally, the microclusters combine into a central cluster of the immune synapse, the intensity of the activation signal drops, and the cell prepares for endocytosis of antigens grouped at the local site. Thus, the structure of B-cell receptors can contribute to the response of the B-lymphocyte to antigens with a characteristic spatial location, while the dynamic interaction between B-cell receptor apparatus and the cytoskeleton allows optimizing the binding of antigens presented on various carriers. Knowledge on spatial aspects of antigen recognition may be useful for the construction of vaccines based on virus-like particles or antigens on other artificial carriers.

Development and characterization of narsoplimab, a selective MASP-2 inhibitor, for the treatment of lectin-pathway-mediated disorders.

Overactivation of the lectin pathway of complement plays a pathogenic role in a broad range of immune-mediated and inflammatory disorders; mannan-binding lectin-associated serine protease-2 (MASP-2) is the key effector enzyme of the lectin pathway. We developed a fully human monoclonal antibody, narsoplimab, to bind to MASP-2 and specifically inhibit lectin pathway activation. Herein, we describe the preclinical characterization of narsoplimab that supports its evaluation in clinical trials. ELISA binding studies demonstrated that narsoplimab interacted with both zymogen and enzymatically active forms of human MASP-2 with high affinity (KD 0.062 and 0.089 nM, respectively) and a selectivity ratio of >5,000-fold relative to closely related serine proteases C1r, C1s, MASP-1, and MASP-3. Interaction studies using surface plasmon resonance and ELISA demonstrated approximately 100-fold greater binding affinity for intact narsoplimab compared to a monovalent antigen binding fragment, suggesting an important contribution of functional bivalency to high-affinity binding. In functional assays conducted in dilute serum under pathway-specific assay conditions, narsoplimab selectively inhibited lectin pathway-dependent activation of C5b-9 with high potency (IC50 ~ 1 nM) but had no observable effect on classical pathway or alternative pathway activity at concentrations up to 500 nM. In functional assays conducted in 90% serum, narsoplimab inhibited lectin pathway activation in human serum with high potency (IC50 ~ 3.4 nM) whereas its potency in cynomolgus monkey serum was approximately 10-fold lower (IC50 ~ 33 nM). Following single dose intravenous administration to cynomolgus monkeys, narsoplimab exposure increased in an approximately dose-proportional manner. Clear dose-dependent pharmacodynamic responses were observed at doses >1.5 mg/kg, as evidenced by a reduction in lectin pathway activity assessed ex vivo that increased in magnitude and duration with increasing dose. Analysis of pharmacokinetic and pharmacodynamic data revealed a well-defined concentration-effect relationship with an ex vivo EC50 value of approximately 6.1 μg/mL, which was comparable to the in vitro functional potency (IC50 33 nM; ~ 5 μg/mL). Based on these results, narsoplimab has been evaluated in clinical trials for the treatment of conditions associated with inappropriate lectin pathway activation, such as hematopoietic stem cell transplantation-associated thrombotic microangiopathy.

Antigen footprint governs activation of the B cell receptor

Antigen binding by B cell receptors (BCR) on cognate B cells elicits a response that eventually leads to production of antibodies. However, it is unclear what the distribution of BCRs is on the naïve B cell and how antigen binding triggers the first step in BCR signaling. Using DNA-PAINT super-resolution microscopy, we find that most BCRs are present as monomers, dimers, or loosely associated clusters on resting B cells, with a nearest-neighbor inter-Fab distance of 20–30 nm. We leverage a Holliday junction nanoscaffold to engineer monodisperse model antigens with precision-controlled affinity and valency, and find that the antigen exerts agonistic effects on the BCR as a function of increasing affinity and avidity. Monovalent macromolecular antigens can activate the BCR at high concentrations, whereas micromolecular antigens cannot, demonstrating that antigen binding does not directly drive activation. Based on this, we propose a BCR activation model determined by the antigen footprint.

Features of the immune response of rainbow trout to vaccination of antigens Yersinia ruckeri, Aeromonas salmonicida, Listonella anguillarum and Renibacterium salmoninarum

The antigens of Yersinia ruckeri, Aeromonas salmonicida sp.salmonicida, Listonella anguillarum and Renibacterium salmoninarum were immunized to rainbow trout of different ages immersion and intraperitaneal, then waited 300 degree days, after which they were infected with homologous and heterologous strains of the studied microorganisms. Prepared antigens and their various combinations were used in the experiments. After the control infection, the level of protective immunity was compared. The level of antibacterial protection obtained by the introduction of polyvalent variants was comparable to that acquired from the introduction of monovalent antigens. A number of cross–reactions were detected - so vaccination with pure A. salmonicida antigen provided some protection against infection with L. anguillarum and Y. ruckeri. In addition, the addition of R. salmoninarum antigen in combination enhanced the protective effect against other diseases to a greater extent than from homologous infection.

Lymph node-targeting adjuvant/neoantigen-codelivering vaccines for combination glioblastoma radioimmunotherapy.

Glioblastoma multiforme (GBM) is the most common and lethal type of adult brain cancer. Current GBM standard of care, including radiotherapy, often ends up with cancer recurrence, resulting in limited long-term survival benefits for GBM patients. Immunotherapy, such as immune checkpoint blockade (ICB), has thus far shown limited clinical benefit for GBM patients. Therapeutic vaccines hold great potential to elicit anti-cancer adaptive immunity, which can be synergistically combined with ICB and radiotherapy. Peptide vaccines are attractive for their ease of manufacturing and stability, but their therapeutic efficacy has been limited due to poor vaccine co-delivery and the limited ability of monovalent antigen vaccines to prevent tumor immune evasion. To address these challenges, here, we report GBM radioimmunotherapy that combines radiotherapy, ICB, and multivalent lymph-node-targeting adjuvant/antigen-codelivering albumin-binding vaccines (AAco-AlbiVax). Specifically, to codeliver peptide neoantigens and adjuvant CpG to lymph nodes (LNs), we developed AAco-AlbiVax based on a Y-shaped DNA scaffold that was site-specifically conjugated with CpG, peptide neoantigens, and albumin-binding maleimide-modified Evans blue derivative (MEB). As a result, these vaccines elicited antitumor immunity including neoantigen-specific CD8+ T cell responses in mice. In orthotopic GBM mice, the combination of AAco-AlbiVax, ICB, and fractionated radiation enhanced GBM therapeutic efficacy. However, radioimmunotherapy only trended more efficacious over radiotherapy alone. Taken together, these studies underscore the great potential of radioimmunotherapy for GBM, and future optimization of treatment dosing and scheduling would improve the therapeutic efficacy.

Sensitivity and specificity for African horse sickness antibodies detection using monovalent and polyvalent vaccine antigen-based dot blotting.

The immune responses of animals infected with African horse sickness (AHS) virus are determined by enzyme-linked immunosorbent assay (ELISA), complement fixation, and virus neutralization test. During the outbreaks of AHS in Thailand, the immune response after vaccination has been monitored using commercial test kits such as blocking ELISA, which are expensive imported products unavailable commercially in Thailand. This study aimed to assess the sensitivity and specificity of anti-AHS virus antibodies using dot blotting based on monovalent and polyvalent strains of live attenuated AHS vaccine. A total of 186 horse sera, namely, 93 AHS-unvaccinated samples and 93 AHS-vaccinated samples, were used in this study. All sera underwent antibodies detection using commercial blocking ELISA and in-house dot blotting based on monovalent and polyvalent strains of live attenuated AHS vaccine. The numbers of true positive, false positive, true negative, and false negative results in the dot blotting were compared with those in blocking ELISA and the sensitivity and specificity of dot blotting were assessed. For the monovalent antigen, there were 78, 19, 74, and 15 true positive, false positive, true negative, and false negative results, respectively, while for the polyvalent antigen, the corresponding numbers were 84, 34, 58, and 9. Meanwhile, the diagnostic sensitivity and specificity for monovalent antigen were 83.87% and 79.57%, respectively, but 90.32% and 62.37% for polyvalent antigen. Dot blotting for AHS antibodies detection using vaccine antigen showed high sensitivity and rather a high specificity compared with the findings with the commercial ELISA test kit. In countries where commercial ELISA test kits are not available and when the size of a serum sample is small, dot blotting could become a good alternative test given its advantages, including its simplicity, rapidity, and convenience. To the best of our knowledge, these findings are the first report on the use of dot blotting for detecting AHS antibodies in horses. In conclusion, monovalent antigen-based dot blotting could be used as a reliable alternative serodiagnostic test for monitoring AHS humoral immune response, especially in vaccinated horses.

Vaccination with the Omicron spike RBD boosts broadly neutralizing antibody levels and confers sustained protection even after acquiring immunity to the original antigen.

The immune evasion of SARS-CoV-2 Omicron variants caused by multiple amino acid replacements in the receptor-binding domain (RBD) of the spike protein wanes the effectiveness of antibodies elicited by current SARS-CoV-2 booster vaccination. The vaccines that target Omicron strains have been recently developed, however, there has been a concern yet to be addressed regarding the negative aspect of the immune response known as original antigenic sin. Here, we demonstrate that the breadth of neutralizing antibodies against SARS-CoV-2 variants is barely elicited by immunizing monovalent viral antigens via vaccination or natural infection in mice and human subjects. However, vaccination of Omicron BA.1 RBD to pre-immunized mice with the original RBD conferred sustained neutralizing activity to BA.1 and BA.2 not only original pseudoviruses. The acquisition of neutralizing antibody breadth was further confirmed in vaccinated-then-Omicron convalescent human sera in which neutralizing activity against BA.1 and BA.2 pseudoviruses was highly induced. Thus, our data suggest that Omicron-specific vaccines or the infection with Omicron viruses can boost potent neutralizing antibodies to the Omicron variants even in the host pre-vaccinated with the original antigen.

Expression and immunogenicity analysis of the capsid proteins of porcine circovirus types 2 to 4

Porcine circovirus (PCV) comprises four types, PCV1, PCV2, PCV3, and PCV4, which belong to the Circovirus genus of the family Circoviridae. PCV1 is nonpathogenic, whereas PCV2, PCV3, and PCV4 can infect pigs and cause disease. However, due to a lack of experimental evidence, whether vaccines based on PCV capsid (Cap) can induce cross-reactivity against PCVs remains controversial. In this study, recombinant truncated capsids (rCaps) of PCV2, PCV3, and PCV4 were highly and efficiently expressed and purified, followed by the development and evaluation of antibodies against PCVs. The results showed that monovalent and trivalent antigens based on the recombinant Caps had adequate immunogenicity to stimulate specific antibodies against the corresponding protein and virus. Furthermore, antisera prepared from the recombinant Caps also cross-reacted with different PCVs. Therefore, recombinant proteins can be used as candidate antigens to develop vaccines and ELISA diagnostic kits. In addition, the antibodies prepared in this study are promising candidates for the simultaneous prevention and treatment of PCVs in the clinic.

Mass Spectrometry-Based Quantification of the Antigens in Aluminum Hydroxide-Adjuvanted Diphtheria-Tetanus-Acellular-Pertussis Combination Vaccines.

Vaccines undergo stringent batch-release testing, most often including in-vivo assays for potency. For combination vaccines, such as diphtheria-tetanus-pertussis (DTaP), chemical modification induced by formaldehyde inactivation, as well as adsorption to aluminum-based adjuvants, complicates antigen-specific in-vitro analysis. Here, a mass spectrometric method was developed that allows the identification and quantitation of DTaP antigens in a combination vaccine. Isotopically labeled, antigen-specific internal standard peptides were employed that permitted absolute quantitation of their antigen-derived peptide counterparts and, consequently, the individual antigens. We evaluated the applicability of the method on monovalent non-adjuvanted antigens, on final vaccine lots and on experimental vaccine batches, where certain antigens were omitted from the drug product. Apart from the applicability for final batch release, we demonstrated the suitability of the approach for in-process control monitoring. The peptide quantification method facilitates antigen-specific identification and quantification of combination vaccines in a single assay. This may contribute, as part of the consistency approach, to a reduction in the number of animal tests required for vaccine-batch release.

Recombinant Full-Length Hepatitis C Virus E1E2 Dimer Elicits Pangenotypic Neutralizing Antibodies.

An effective prophylactic vaccine would be beneficial for controlling and eradicating hepatitis C virus (HCV) infections. However, the high diversity across HCV genotypes is a major challenge for vaccine development. Selection of the appropriate immunogen is critical to elicit broad HCV neutralizing antibodies (NAbs). To increase the antigenic coverage of heterodimer glycoproteins, we designed and produced recombinant E1E2 antigens for genotypes 1a/1b/2a/3a/6a from an IgG Fc-tagged precursor protein in FreeStyle 293-F cells. The recombinant E1 and E2 antigens were localized and associated with the endoplasmic reticulum and co-purified from membrane extracts. By examining the interactions with HCV entry co-receptors and the blockade of HCV infection, we found that these purified Fc-E1E2 proteins displayed correct folding and function. Mouse immunization results showed that each recombinant E1E2 antigen could elicit a pangenotypic antibody response to itself and other genotypes. We also found that the pentavalent formula triggered a relatively higher and more uniform NAb titer and T cell response than monovalent antigens. Taken together, our findings may provide a useful strategy for the vaccine development of HCV and other viruses with highly heterogeneous surface glycoproteins.

The Cross-Protective Immunity Landscape Among Different SARS-CoV-2 Variant RBDs.

Despite the fact that SARS-CoV-2 vaccines have been available in most parts of the world, the epidemic status remains grim with new variants emerging and escaping the immune protection of existing vaccines. Therefore, the development of more effective antigens and evaluation of their cross-protective immunity against different SARS-CoV-2 variants are particularly urgent. In this study, we expressed the wild type (WT), Alpha, Beta, Delta, and Lambda RBD proteins to immunize mice and evaluated their cross-neutralizing activity against different pseudoviruses (WT, Alpha, Beta, Delta, Lambda, and Omicron). All monovalent and pentavalent RBD antigens induced high titers of IgG antibodies against different variant RBD antigens. In contrast, WT RBD antigen-induced antibodies showed a lower neutralizing activity against Beta, Delta, Lambda, and Omicron pseudoviruses compared to neutralization against itself. Interestingly, Beta RBD antigen and multivalent antigen induced broader cross-neutralization antibodies than other variant RBD antigens. These data provide a reference for vaccine strain selection and universal COVID-19 vaccine design to fight the constant emergence of new SARS-CoV-2 variants.

Innate stimulation of B cells ex vivo enhances antibody secretion and identifies tumour-reactive antibodies from cancer patients.

Human B cells and their expressed antibodies are crucial in conferring immune protection. Identifying pathogen-specific antibodies following infection is possible due to enhanced humoral immunity against well-described molecules on the pathogen surface. However, screening for cancer-reactive antibodies remains challenging since target antigens are often not identified a priori and the frequency of circulating B cells recognizing cancer cells is likely very low. We investigated whether combined ex vivo culture of human B cells with three innate stimuli, interleukin-17 (IL-17), B-cell activation factor (BAFF), and the toll-like receptor 9 (TLR-9) agonist DNA motif CpG ODN 2006 (CpG), each known to activate B cells through different signalling pathways, promote cell activation, proliferation, and antibody production. Combined IL-17+BAFF+CpG prolonged B-cell survival and increased proliferation compared with single stimuli. IL-17+BAFF+CpG triggered higher IgG secretion, likely by activating differentiated, memory and class-switched CD19+CD20+CD27+IgD- B cells. Regardless of anti-FOLR antibody seropositive status, IL-17+BAFF+CpG combined with a monovalent tumour-associated antigen (folate receptor alpha [FOLR]) led to secreted antibodies recognizing the antigen and the antigen-expressing IGROV1 cancer cells. In a seropositive individual, FOLR stimulation favoured class-switched memory B-cell precursors (CD27-CD38-IgD-), class-switched memory B cells and anti-FOLR antibody production, while IL-17+BAFF+CpG combined with FOLR, promoted class-switched memory B-cell precursors and antibody-secreting (CD138+IgD-) plasma cells. Furthermore, IL-17+BAFF+CpG stimulation of peripheral blood B cells from patients with melanoma revealed tumour cell-reactive antibodies in culture supernatants. These findings suggest that innate signals stimulate B-cell survival and antibody production and may help identify low-frequency antigen-reactive humoral responses.

Signal Enhancement in Oriented Immunosorbent Assays: A Balance between Accessibility of Antigen Binding Sites and Avidity.

The sensitivity of immunoassays was reported to be increased by the orientation of antibodies. We investigated how the size and valence of antigens and orientation and valence of antibodies contribute to the analytical sensitivity of ELISA. Antigens differing in size and number of epitopes were compared using oriented and non-oriented ELISAs: the orientation of antibodies was obtained coating half-fragment antibodies on maleimide microplates, while, in non-oriented ELISA, whole antibodies were randomly physisorbed. The oriented assay performed better than the non-oriented one at each concentration (0.4–3.3 ng/mL) of a small monomeric antigen (cardiac Troponin I, 24 kDa, Rh 3 nm). No significant differences were observed with a large monovalent antigen (prostate-specific antigen-alpha(1) antichymotrypsin, 90 kDa, Rh > 3 nm), since its steric hindrance overcame the increased availability of antigen binding sites given by orientation. Large multivalent antigens (ferritin, 280 kDa, Rh 6 nm; α-fetoprotein, >70 kDa, Rh > 3.3 nm) performed better in non-oriented assays. In this case, the repeated epitopes on the surface of the antigens favored the engagement of both antigen binding sites of the whole IgG, thus suggesting that avidity represented the leading force in this experimental setting. In conclusion, the design of high-sensitivity ELISAs should consider the dimension and valency of antigens in addition to the affinity and avidity of antibodies.

Primary Immune Responses and Affinity Maturation Are Controlled by IgD.

Mature B cells co-express IgM and IgD B cell antigen receptors (BCR) on their surface. While IgM BCR expression is already essential at early stages of development, the role of the IgD-class BCR remains unclear as most B cell functions appeared unchanged in IgD-deficient mice. Here, we show that IgD-deficient mice have an accelerated rate of B cell responsiveness as they activate antibody production within 24h after immunization, whereas wildtype (WT) animals required 3 days to activate primary antibody responses. Strikingly, soluble monovalent antigen suppresses IgG antibody production induced by multivalent antigen in WT mice. In contrast, IgD-deficient mice were not able to modulate IgG responses suggesting that IgD controls the activation rate of B cells and subsequent antibody production by sensing and distinguishing antigen-valences. Using an insulin-derived peptide we tested the role of IgD in autoimmunity. We show that primary autoreactive antibody responses are generated in WT and in IgD-deficient mice. However, insulin-specific autoantibodies were detected earlier and caused more severe symptoms of autoimmune diabetes in IgD-deficient mice as compared to WT mice. The rapid control of autoimmune diabetes in WT animals was associated with the generation of high-affinity IgM that protects insulin from autoimmune degradation. In IgD-deficient mice, however, the generation of high-affinity protective IgM is delayed resulting in prolonged autoimmune diabetes. Our data suggest that IgD is required for the transition from primary, highly autoreactive, to secondary antigen-specific antibody responses generated by affinity maturation.

Molecular requirements for B cell receptor engagement and activation

Abstract B cells are a central component of the adaptive branch of the immune system, responsible for humoral immunity through production of antibodies. Antigen engagement of the B cell receptor (BCR) is the first step in B cell activation; yet despite its recognized importance, the exact mechanism of BCR activation remains controversial. A central point of contention has been whether monovalent antigen is capable of activating the BCR or not. To address this question, we leveraged the B1-8hi knock-in BCR model, in which λ light-chain positive B cells display intermediate and high affinity binding to the haptens NP ((4-hydroxy-3-nitrophenyl)acetyl) and NIP ((4-hydroxy-3-iodo-5-nitrophenyl)acetyl), respectively, and combined this with a recently developed nucleic acid based scaffolding technology (Holliday junctions [HJs]) to prepare defined mono-, di- and trivalent displays of NP and NIP. Using confocal imaging, flow and imaging cytometry, as well as calcium-flux analyses, we find that HJs carrying 2 or 3 copies of either NP or NIP can bind and activate B1-8hi B cells across a wide range of concentrations, whereas HJs carrying a single NP require high concentrations for any measurable activity. Most interestingly, HJs carrying a single NIP are able to bind B1-8hi B cells at low concentrations, approaching equimolar stoichiometry, but despite measurable BCR engagement do not elicit activation. Our results may reconcile previous contradictory reports regarding monovalent antigen activation, by providing a framework for understanding complex interaction effects of antigen affinity, valency and concentration.

Abstract 554: Tumor-antigen 5T4-dependent activation of the CD137 costimulatory pathway by bispecific 5T4 x CD137 x CD137 TRIDENT™ molecules

Abstract Introduction: Trophoblast glycoprotein 5T4 is expressed on the cell surface of multiple cancers yet sparingly on normal adult tissues. CD137 (4-1BB) is a co-stimulatory molecule expressed by activated T and NK cells that support cell activation, proliferation and survival. Previously we described bispecific tumor associated antigen x CD137 DART® molecules limiting CD137 mediated immunostimulation to the tumor microenvironment (AACR2017) as an approach to reduce the unwanted systemic CD137 effects associated with agonistic anti-CD137 mAbs. We subsequently identified a bispecific TRIDENT format bearing bivalent CD137 and monovalent tumor antigen engagement that provides maximal CD137 activation in a tumor antigen anchored manner. Here we apply that format to generate a 5T4 x CD137 x CD137 TRIDENT molecule that promotes CD137 activation in proximity to 5T4-expressing tumor cells leading to enhanced T-cell co-stimulation, proliferation and tumor cell killing activity when combined with CD3-engaging tumor-targeting bispecific DART molecules. Methods: TRIDENT molecules were constructed comprising bivalent CD137 binding and monovalent 5T4 binding. Binding properties were evaluated by surface plasmon resonance (SPR) and flow cytometry. Signaling was assessed using a NF-κB luciferase reporter cell line expressing CD137. Co-stimulatory activity was characterized with primary human or cynomolgus monkey T cells. T cells were incubated with or without antigen-expressing cells and submaximal concentrations of either anti-CD3 bead or CD3-engaging tumor-targeting bispecific DART molecules. Results: SPR and flow cytometry analyses demonstrate that 5T4 x CD137 x CD137 TRIDENT molecule binds human and monkey target antigens. The 5T4 x CD137 x CD137 TRIDENT molecule induces CD137 signaling in a reporter cell line and promotes cytokine release in primary human & cynomolgus monkey T-cell assays in the presence of 5T4+ tumor cells. In the absence of 5T4+ tumor cells, the TRIDENT molecule lacks agonistic activity. Furthermore, 5T4 x CD137 x CD137 TRIDENT molecule enhances T-cell proliferation and tumor cell cytolysis induced by CD3-targeted DART molecules. Consistent with preferential induction of CD137 by CD8 T-cells, 5T4 x CD137 x CD137 TRIDENT increases the fraction of CD8+ central memory and effector memory T cells in the presence 5T4+ tumor cells. Conclusions: 5T4 x CD137 x CD137 TRIDENT molecule promotes T-cell co-stimulation in a tumor antigen-dependent manner offering an opportunity to target CD137 immunostimulation, while potentially limiting non-specific systemic T-cell activation and related side effects. Citation Format: Liqin Liu, Ling Huang, Vatana Long, Yinhua Yang, Robert Burns, Jonathan Li, Jennifer DiChiara, Qihong Xu, Arin Whiddon, Chia-Ying K Lam, Jim Tamura, Valentina Ciccarone, Syd Johnson, Ezio Bonvini, Paul Moore. Tumor-antigen 5T4-dependent activation of the CD137 costimulatory pathway by bispecific 5T4 x CD137 x CD137 TRIDENT™ molecules [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 554.

Role of Multivalency and Antigenic Threshold in Generating Protective Antibody Responses.

Vaccines play a vital role in protecting our communities against infectious disease. Unfortunately, some vaccines provide only partial protection or in some cases vaccine-mediated immunity may wane rapidly, resulting in either increased susceptibility to that disease or a requirement for more booster vaccinations in order to maintain immunity above a protective level. The durability of antibody responses after infection or vaccination appears to be intrinsically determined by the structural biology of the antigen, with multivalent protein antigens often providing more long-lived immunity than monovalent antigens. This forms the basis for the Imprinted Lifespan model describing the differential survival of long-lived plasma cell populations. There are, however, exceptions to this rule with examples of highly attenuated live virus vaccines that are rapidly cleared and elicit only short-lived immunity despite the expression of multivalent surface epitopes. These exceptions have led to the concept that multivalency alone may not reliably determine the duration of protective humoral immune responses unless a minimum number of long-lived plasma cells are generated by reaching an appropriate antigenic threshold of B cell stimulation. Examples of long-term and in some cases, potentially lifelong antibody responses following immunization against human papilloma virus (HPV), Japanese encephalitis virus (JEV), Hepatitis B virus (HBV), and Hepatitis A virus (HAV) provide several lessons in understanding durable serological memory in human subjects. Moreover, studies involving influenza vaccination provide the unique opportunity to compare the durability of hemagglutinin (HA)-specific antibody titers mounted in response to antigenically repetitive whole virus (i.e., multivalent HA), or detergent-disrupted “split” virus, in comparison to the long-term immune responses induced by natural influenza infection. Here, we discuss the underlying mechanisms that may be associated with the induction of protective immunity by long-lived plasma cells and their importance in future vaccine design.