Nanobody-based trispecific T cell engager (Nb-TriTE) enhances therapeutic efficacy by overcoming tumor-mediated immunosuppression.

BACKGROUND: T cell engagers (TCEs) have been established as an emerging modality for hematologic malignancies, but solid tumors remain refractory. However, the upregulation of programmed cell death 1 (PD-1) is correlated with T cell dysfunction that confer tumor-mediated immunosuppression. Developing a novel nanobody-based trispecific T cell engager (Nb-TriTE) would be a potential strategy to improve therapeutic efficacy. METHODS: Given the therapeutic potential of nanobodies (Nbs), we first screened Nb targeting fibroblast activation protein (FAP) and successfully generated a Nb-based bispecific T cell engager (Nb-BiTE) targeting FAP. Then, we developed a Nb-TriTE by fusing an anti-PD-1 Nb to the Nb-BiTE. The biological activity and antitumor efficacy of the Nb-TriTE were evaluated in vitro and in both cell line-derived and patient-derived xenograft mouse models. RESULTS: We had for the first time successfully selected a FAP Nb for the generation of novel Nb-BiTE and Nb-TriTE, which showed good binding ability to their targets. Nb-TriTE not only induced robust tumor antigen-specific killing, potent T cell activation and enhanced T cell function in vitro, but also suppressed tumor growth, improved survival and mediated more T cell infiltration than Nb-BiTE in mouse models of different solid tumors without toxicity. CONCLUSIONS: This novel Nb-TriTE provides a promising and universal platform to overcome tumor-mediated immunosuppression and improve patient outcomes in the future.

Ex-vivo CS1-OKT3 dual specific bivalent antibody-armed effector T cells mediate cellular immunity against multiple myeloma.

Bispecific T cell engaging antibodies (bsAbs) have emerged as novel and powerful therapeutic agents for redirecting T cells towards antigen-specific tumor killing. The cell surface glycoprotein and SLAM family member, CS1, exhibits stable and high-level expression on malignant plasma cells including multiple myeloma, which is indicative of an ideal target for bsAb therapy. Here, we developed a CS1 bsAb (CS1-dbBiTE) using Click chemistry to conjugate intact anti-CS1 antibody (Elotuzumab) and anti-huOKT3 antibody at their respective hinge regions. Using a cellular therapy approach, human T cells were armed ex-vivo with CS1-dbBiTE prior to examining effector activity. Our data indicates that arming T cells with CS1-dbBiTE induced T cell activation and expansion and subsequent cytotoxic activity against CS1-bearing MM tumors, demonstrated by significant CD107a expression as well as inflammatory cytokine secretion. As expected, CS1-dbBiTE armed T cells showed significantly reduced effector activity in the absence of CS1 expression. Similarly, in MM mouse xenograft studies, armed T cells exhibited effective anti-tumor efficacy highlighted by reduced tumor burden in MM.1S tumor-bearing mice compared to controls. On the basis of these findings, the rationale for CS1 targeting by human T cells armed with CS1-dbBiTE presents a potentially effective therapeutic approach for targeting MM.

Novel NKG2D-directed bispecific antibodies enhance antibody-mediated killing of malignant B cells by NK cells and T cells.

The activating receptor natural killer group 2, member D (NKG2D) represents an attractive target for immunotherapy as it exerts a crucial role in cancer immunosurveillance by regulating the activity of cytotoxic lymphocytes. In this study, a panel of novel NKG2D-specific single-chain fragments variable (scFv) were isolated from naïve human antibody gene libraries and fused to the fragment antigen binding (Fab) of rituximab to obtain [CD20×NKG2D] bibodies with the aim to recruit cytotoxic lymphocytes to lymphoma cells. All bispecific antibodies bound both antigens simultaneously. Two bibody constructs, [CD20×NKG2D#3] and [CD20×NKG2D#32], efficiently activated natural killer (NK) cells in co-cultures with CD20+ lymphoma cells. Both bibodies triggered NK cell-mediated lysis of lymphoma cells and especially enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) by CD38 or CD19 specific monoclonal antibodies suggesting a synergistic effect between NKG2D and FcγRIIIA signaling pathways in NK cell activation. The [CD20×NKG2D] bibodies were not effective in redirecting CD8+ T cells as single agents, but enhanced cytotoxicity when combined with a bispecific [CD19×CD3] T cell engager, indicating that NKG2D signaling also supports CD3-mediated T cell activation. In conclusion, engagement of NKG2D with bispecific antibodies is attractive to directly activate cytotoxic lymphocytes or to support their activation by monoclonal antibodies or bispecific T cell engagers. As a perspective, co-targeting of two tumor antigens may allow fine-tuning of antibody cancer therapies. Our proposed combinatorial approach is potentially applicable for many existing immunotherapies but further testing in different preclinical models is necessary to explore the full potential.

Bispecific Antibody Detection Using Antigen-Conjugated Synthetic Nucleic Acid Strands.

We report here the development of two different sensing strategies based on the use of antigen-conjugated nucleic acid strands for the detection of a bispecific antibody against the tumor-related proteins Mucin1 and epidermal growth factor receptor. Both approaches work well in serum samples (nanomolar sensitivity), show high specificity against the two monospecific antibodies, and are rapid. The results presented here demonstrate the versatility of DNA-based platforms for the detection of bispecific antibodies and could represent a versatile alternative to other more reagent-intensive and time-consuming analytical approaches.

Anti-PD-L1 × anti-CD3 bispecific T-cell engager-armed T cells can overcome immunosuppression and redirect T cells to kill breast cancer cells expressing PD-L1.

T cell-based immunotherapy has transformed cancer treatment. Nonetheless, T cell antitumor activity can be inhibited by an immune checkpoint molecule expressed on cancer cells, program death ligand 1 (PD-L1), which interacts with the PD-1 on T cells. We generated αPD-L1 × αCD3 bispecific T-cell engager-armed T cells (BATs) to prevent PD-L1/PD-1 interaction and hence to redirect T cells to kill cancer cells. αPD-L1 × αCD3 bispecific T-cell engagers (BTEs) were produced from Chinese hamster ovary (CHO) cells to arm human primary T cells. Flow cytometry was used to investigate BTE binding to BATs. The cytotoxicity of BATs against PD-L1-expressing breast cancer (BC) cell lines was assessed in 2-dimensional (2D) and 3-dimensional (3D) culture models. The binding stability of BTE on BATs and their efficacy after cryopreservation were also examined. The CHO cell BTE expression yield was 3.34 mg/ml. The binding ability on T cells reached 91.02 ± 4.2 %. BATs specifically lysed PD-L1-expressing BC cells, with 56.4 ± 15.3 % HCC70 cells and 70.67 ± 15.6 % MDA-MB-231 cells lysed at a 10:1 effector-to-target ratio. BATs showed slight, nonsignificant lysis of PD-L1-negative BC cells, MCF-7, and T47D. Moreover, BATs significantly disrupted MDA-MB-231 3D spheroids expressing PD-L1 after 48 and 72 h of coculture. Cryopreserved BATs maintained BTE binding stability, cell viability, and anticancer activity, comparable to fresh BATs. αPD-L1 × αCD3 BATs induced the cytolysis of PD-L1-expressing BC cells in 2D and 3D coculture assays. BATs can be prepared and preserved, facilitating their use and transportation. This study demonstrates the potential of αPD-L1 × αCD3 BATs in treating cancers with positive PD-L1 expression.

Target-independent Immune-cell Activation by Aggregates of T Cell-redirecting Bispecific Antibodies.

T cell-redirecting bispecific antibodies (bsAbs) have been under development as a new class of biotherapeutics for cancer immunotherapy. T cell-redirecting bsAbs simultaneously bind tumor-associated antigens on tumor cells and CD3 on T cells, resulting in T cell-mediated cytotoxicity against tumor cells. In this study, we prepared a tandem scFv-typed bsAb targeting HER2 and CD3 (HER2-CD3), and evaluated the impact of aggregation of HER2-CD3 on the in vitro immunotoxicity. A cell-based assay using CD3-expressing reporter cells revealed that the aggregates of HER2-CD3 directly activated CD3-expressing immune cells in the absence of target antigen (HER2)-expressing cells. Comparison of the aggregates generated under various stress conditions indicated the possibility that insoluble protein particles, which were detected by qLD analysis and contained non-denatured functional domains, contributed to the activation of CD3-expressing immune cells. In addition, HER2-CD3 aggregates stimulated hPBMCs and strongly induced the secretion of inflammatory cytokines and chemokines. The cytokine/chemokine-release profiles suggested that the aggregates could induce inflammatory responses not only by CD3-mediated T cell activation but also by other immune cell activations. These results indicated the potential risk of aggregation of T cell-redirecting bsAbs, which could induce unwanted immune cell activation and inflammation and thereby immune-mediated adverse reactions.

Adeno-Associated Virus-Mediated Immunotherapy Based on Bispecific Tandem scFv for Alzheimer's Disease.

BACKGROUND: Patients with Alzheimer's disease (AD) have considerably increased globally as a result of population aging, placing a significant burden on the global economy and the medical system. The outcome of clinical trials for AD immunotherapy that solely targeted amyloid-ß (Aß) or phosphorylated tau protein (p-Tau) was unsatisfactory. Therefore, blocking both Aß and p-Tau's pathological processes simultaneously while also preventing their interaction may be the key to developing an effective AD therapy. OBJECTIVE: To develop a novel immunotherapy based on bispecific tandem scFv (TaFv) against AD. METHODS: Bispecific single-chain antibody that targets both Aß and p-Tau were obtained using E. coli expression system. Biological ability of TaFvs were determined by ELISA, SDS-PAGE, and immunohistochemical assay. Recombinant adeno-associated virus 9 (rAAV9) were packaged to create TaFv. The in vivo activity of rAAV9 were detected in mouse, using biophotonic imaging and frozen section methods. RESULTS: The outcomes demonstrated that both Aß and p-Tau had a high affinity for the bispecific TaFv. Additionally, it can bind to the amyloid plaques and neuronal tangles in the brain slices of an AD mouse model. Moreover, the rAAV9 could infect neuronal cells, transverse the blood-brain barrier, and express TaFv in the mouse brain. CONCLUSION: This novel immunotherapy offers a fresh concept for the immunotherapy of AD and successfully delivers the double target antibody into the brain, acting on both pathogenic substances Aß and p-Tau.

CD3 Target Affinity Chromatography Mass Spectrometry as a New Tool for Function-Structure Characterization of T-Cell Engaging Bispecific Antibody Proteoforms and Product-Related Variants.

T-cell engaging bispecific antibodies (TCBs) targeting CD3 and tumor-specific antigens are very promising therapeutic modalities. Since CD3 binding is crucial for the potency of TCBs, understanding the functional impact of CD3 antigen-binding fragment modifications is of utmost importance for defining critical quality attributes (CQA). The current CQA assessment strategy requires the integration of structure-based physicochemical separation and functional cell-based potency assays. However, this strategy is tedious, and coexisting proteoforms with potentially different functionalities may not be individually assessed. This increases the degree of ambiguities for defining meaningful CQAs, particularly for complex bispecific antibody formats such as TCBs. Here, we report for the first time a proof-of-concept study to separate and identify critically modified proteoforms of TCBs using functional CD3 target affinity chromatography (AC) coupled with online mass spectrometry (MS). Our method enabled functional distinction of relevant deamidated and glycosylated proteoforms and the simultaneous assessment of product-related variants such as TCB mispairings. For example, CD3 AC-MS allowed us to separate TCB mispairings with increased CD3 binding (i.e., knob-knob homodimers) within the bound fraction. The functional separation of proteoforms was validated using an established workflow for CQA identification based on thoroughly characterized ion-exchange fractions of a 2+1 TCB. In addition, the new method facilitated the criticality assessment of post-translational modifications in stress studies and structural variants in early stage clone selection. CD3 AC-MS has high impact for streamlining the integration of functional and structural characterizations of the large landscape of therapeutic CD3 targeting TCBs from early stage research to late stage characterization.

Enhanced anti-angiogenetic effect of transferrin receptor-mediated delivery of VEGF-trap in a glioblastoma mouse model.

Glioblastoma (GBM) is a common and aggressive brain cancer that accounts for 60% of adult brain tumors. Anti-angiogenesis therapy is an attractive option due to the high vasculature density of GBM. However, the best-known anti-angiogenic therapeutics, bevacizumab, and aflibercept, have failed to show significant benefits in GBM patients. One of the reasons is the limited brain penetration of antibody-based therapies due to existence of the blood-brain barrier (BBB), which is further strengthened by the blood vessel normalization effects induced by anti-angiogenic therapies. To investigate if increased drug concentration in the brain by transferrin receptor (TfR)-mediated delivery across the BBB can enhance efficacy of anti-angiogenic antibody therapies, we first identified an antibody that binds to the apical domain of the mouse TfR and does not compete with the natural ligand transferrin (Tf) binding to TfR. Then, we engineered two bispecific antibodies fusing a vascular endothelial growth factor (VEGF)-Trap with the TfR-targeting antibody. Characterization of the two bispecific formats using multiple in vitro assays, which include endocytosis, cell surface and whole-cell TfR levels, human umbilical vein endothelial cell growth inhibition, and binding affinity, demonstrated that the VEGF-Trap fused with a monovalent αTfR (VEGF-Trap/moAb4) has desirable endocytosis without the induction of TfR degradation. Peripherally administered VEGF-Trap/moAb4 improved the brain concentration of VEGF-Trap by more than 10-fold in mice. The distribution of VEGF-Trap/moAb4 was validated to be in the brain parenchyma, indicating the molecule was not trapped inside the vasculature. Moreover, improved VEGF-Trap brain distribution significantly inhibited the angiogenesis of U-87 MG GBM tumors in a mouse model.

Bispecific antibodies for immune cell retargeting against cancer.

INTRODUCTION: Following the approval of the T cell engaging bispecific antibody blinatumomab, immune cell retargeting with bispecific or multispecific antibodies has emerged as a promising cancer immunotherapy strategy, offering alternative mechanisms compared to immune checkpoint blockade. As we gain more understanding of the complex tumor microenvironment, rules and design principles have started to take shape on how to best harness the immune system to achieve optimal anti-tumor activities. AREAS COVERED: In the present review, we aim to summarize the most recent advances and challenges in using bispecific antibodies for immune cell retargeting and to provide insights into various aspects of antibody engineering. Discussed herein are studies that highlight the importance of considering antibody engineering parameters, such as binding epitope, affinity, valency, and geometry to maximize the potency and mitigate the toxicity of T cell engagers. Beyond T cell engaging bispecifics, other bispecifics designed to recruit the innate immune system are also covered. EXPERT OPINION: Diverse and innovative molecular designs of bispecific/multispecific antibodies have the potential to enhance the efficacy and safety of immune cell retargeting for the treatment of cancer. Whether or not clinical data support these different hypotheses, especially in solid tumor settings, remains to be seen.

Antitumor activity of T cells secreting αCD133-αCD3 bispecific T-cell engager against cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a lethal cancer of bile duct epithelial cells with a high mortality rate and limited therapeutic options. An effective treatment is, therefore, urgently needed to improve treatment outcomes for these patients. To develop a new therapeutic option, we engineered T cells secreting αCD133-αCD3 bispecific T-cell engager and evaluated their antitumor effects against CD133-expressing CCA cells. The cDNA encoding αCD133-αCD3 bispecific T-cell engager (αCD133-αCD3-ENG) was cloned into pCDH lentiviral construct and its expression was tested in Lenti-X 293T cells. T cells from healthy donors were then transduced with engineered lentiviruses to create T cells secreting αCD133-αCD3 engager to evaluate their antitumor activities. The average transduction efficiency into T cells was approximately 60.03±21.65%. In the co-culture system containing T cells secreting αCD133-αCD3 engager (as effector cells) and mWasabi-luciferase-expressing CCA cells (KKU-100 and KKU-213A; as target cells), the effector T cells exhibited significantly higher cytolytic activities against the target CCA cells (49.0±9.76% and 64.10±13.18%, respectively) than those observed against the untransduced T cells (10.97±10.65%; p = 0.0103 and 9.80±11.05%; p = 0.0054) at an effector-to-target ratio of 5:1. In addition, the secreted αCD133-αCD3 engager significantly redirected both transduced T cells and bystander T cells to kill the target CCA cells (up to 73.20±1.68%; p<0.05). Moreover, the transduced and bystander T cells could kill the target CCA spheroids at a rate approximately 5-fold higher than that of the no treatment control condition (p = 0.0011). Our findings demonstrate proof-of-principle that T cells secreting αCD133-αCD3 engager can be an alternative approach to treating CD133-positive CCA, and they pave the way for future in vivo study and clinical trials.

Platelets subvert antitumor efficacy of T cell-recruiting bispecific antibodies.

T cell-based immunotherapy, for example, with T cell-recruiting bispecific antibody (bsAb), has revolutionized oncological treatment. However, many patients do not respond to treatment, and long-term remissions are still rare. Several tumor immune evasion mechanisms have been reported to counteract efficiency of T cell-engaging therapeutics. Platelets largely affect cancer pathophysiology by mediating tumor invasion, metastasis, and immune evasion. On treatment of patients in a clinical trial with a PSMA×CD3 bsAb (NCT04104607), we observed profound treatment-associated platelet activation, mirrored by a decrease of total platelet count. On modeling the treatment setting, we found that platelet activation significantly reduced bsAb-mediated CD4+ and CD8+ T-cell reactivity as revealed by impaired T-cell degranulation, secretion of perforin, and ultimately, inhibition of target cell lysis. This effect occurred in a transforming growth factor beta (TGF-ß)-dependent manner and was not restricted to PSMA×CD3 bsAb, but rather observed with various CD3-directed bispecific constructs, including the approved CD19×CD3 bsAb blinatumomab. BsAb-mediated T-cell reactivity could be restored by platelet inhibition and specifically by blocking the TGF-ß axis. Together, our findings demonstrate that platelets undermine the efficacy of T cell-recruiting bsAb and identify modulation of platelet function as a means to reinforce the effectiveness of bsAb treatment.

Cytofluorimetric assay to investigate variability in blinatumomab in vitro response.

BACKGROUND: The T-cell engager antibody blinatumomab (BlincytoT⁢M) represents a promising rescue therapy for relapsed/refractory CD19+ acute lymphoblastic leukemia (B-ALL), although ~20-30% of patients still do not respond to treatment. Blinatumomab creates a tight synapsis between CD3+ T-lymphocytes and leukemic CD19+ B-cells, resulting in a granzyme B (GzB)-mediated specific lysis of leukemic cells. METHODS: Aim of the study was to provide evidence that variability in blinatumomab response could have a genetic basis in PAX5, one of the most often mutated genes in B-ALL, affecting the CD19 surface expression on lymphoblasts, and could be explored in vitro by means of a cytofluorimetric assay, staining both surface antigens (CD45, CD19 and CD3) and intracytoplasmic markers (7AAD, Syto16). Two human immortalized B-ALL cell lines (NALM6 and REH) were chosen for their different PAX5 and CD19 protein levels, as verified by western blot and flow cytometry, respectively. RESULTS: In contrast to NALM6, REH cells do not express the full-length PAX5 protein and show less CD19 on the cell surface (fluorescence peak median intensity: 9155 versus 28895). Co-cultures of CD3+ T-lymphocytes from healthy donors and B-ALL cell lines were seeded at an effector-to-target cell ratio of 1:10 for simulating the condition existing in the bone marrow due to the malignant invasion of blast cells. Co-cultures were exposed in vitro to blinatumomab and the simultaneous increase in blast mortality and T-lymphocytes activation induced by the drug was observed at day +7 (both effects: p < 0.0001 versus untreated, two-way ANOVA, Bonferroni post-test), and was particularly pronounced in REH compared to NALM6 co-cultures (p < 0.05). Surprisingly, daily release of GzB in supernatants, measured by an ELISA assay, was significantly lower in drug-exposed REH co-cultures compared to NALM6 at early time-points (days +3 and +4, p-value < 0.0001, three-way ANOVA), reaching a comparable plateau only towards the end of the incubation period (at day +5). Only 2 out of 5 primary co-cultures of leukemic and mononuclear cells isolated from bone marrow aspirates of B-ALL patients (age: median 10.7 years, interquartile range (IQR) 3.4; males: 60%) responded to the drug in vitro (simultaneous blast mortality and T-lymphocyte activation: both effects: p < 0.0001 versus untreated) and at different drug concentrations. Results were unrelated to the percentages of immature CD19+ B-cells in the diagnostic samples. CONCLUSIONS: In conclusion, cytofluorimetric analysis can highlight the different response induced by blinatumomab among co-cultures. Whether and how this difference is affected by PAX5-regulated CD19 expression is unclear and whether it is predictive of in vivo response to therapy remains to be established. Further dedicated studies are required to investigate these issues in detail.

Human immunocompetent choroid-on-chip: a novel tool for studying ocular effects of biological drugs.

Disorders of the eye leading to visual impairment are a major issue that affects millions of people. On the other side ocular toxicities were described for e.g. molecularly targeted therapies in oncology and may hamper their development. Current ocular model systems feature a number of limitations affecting human-relevance and availability. To find new options for pharmacological treatment and assess mechanisms of toxicity, hence, novel complex model systems that are human-relevant and readily available are urgently required. Here, we report the development of a human immunocompetent Choroid-on-Chip (CoC), a human cell-based in vitro model of the choroid layer of the eye integrating melanocytes and microvascular endothelial cells, covered by a layer of retinal pigmented epithelial cells. Immunocompetence is achieved by perfusion of peripheral immune cells. We demonstrate controlled immune cell recruitment into the stromal compartments through a vascular monolayer and in vivo-like cytokine release profiles. To investigate applicability for both efficacy testing of immunosuppressive compounds as well as safety profiling of immunoactivating antibodies, we exposed the CoCs to cyclosporine and tested CD3 bispecific antibodies.

Immunomodulation of T- and NK-cell Responses by a Bispecific Antibody Targeting CD28 Homolog and PD-L1.

Checkpoint blockade therapies targeting PD-1/PD-L1 and CTLA-4 are clinically successful but also evoke adverse events due to systemic T-cell activation. We engineered a bispecific, mAb targeting CD28 homolog (CD28H), a newly identified B7 family receptor that is constitutively expressed on T and natural killer (NK) cells, with a PD-L1 antibody to potentiate tumor-specific immune responses. The bispecific antibody led to T-cell costimulation, induced NK-cell cytotoxicity of PD-L1-expressing tumor cells, and activated tissue-resident memory CD8+ T cells. Mechanistically, the CD28H agonistic arm of the bispecific antibody reduced PD-L1/PD-1-induced SHP2 phosphorylation while simultaneously augmenting T-cell receptor signaling by activating the MAPK and AKT pathways. This bispecific approach could be used to target multiple immune cells, including CD8+ T cells, tissue-resident memory T cells, and NK cells, in a tumor-specific manner that may lead to induction of durable, therapeutic antitumor responses.

Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells.

Bispecific antibodies (BsAbs) or fusion proteins (BsAbFPs) present a promising strategy for cancer immunotherapy. Numerous BsAbs targeting coinhibitory and costimulatory pathways have been developed for retargeting T cells and antigen presenting cells (APCs). It is challenging to assess the potency of BsAb that engages two different signaling pathways simultaneously in a single assay format, especially when the two antigen targets are expressed on different cells. To explore the potency of anti-PD-L1/CD40L BsAbFP, a fusion protein that binds to human CD40 and PD-L1, we engineered CHO cells as surrogate APCs that express T cell receptor activator and PD-L1, Jurkat cells with PD-1 and NFAT-luciferase reporter as effector T cells, and Raji cell with NFkB-luciferase that endogenously expresses CD40 as accessory B cells. A novel reporter gene bioassay was developed using these cell lines that allows anti-PD-L1/CD40L BsAbFP to engages both PD-1/PD-L1 and CD40/CD40L signaling pathways in one assay. As both reporters use firefly luciferase, the effects of activating both signaling pathways is observed as an increase in luminescence, either as a higher upper asymptote, a lower EC50, or both. This dual target reporter gene bioassay system reflects potential mechanism of action and demonstrated the ability of anti-PD-L1/CD40L BsAbFP to synergistically induce biological response compared to the combination of anti-PD-L1 monovalent monoclonal antibody and agonist CD40L fusion protein, or either treatment alone. The results also showed a strong correlation between the drug dose and biological response within the tested potency range with good linearity, accuracy, precision, specificity and stability indicating properties, suggesting that this "three-cell-in-one" dual target reporter gene bioassay is suitable for assessing potency, structure-function and critical quality attributes of anti-PD-L1/CD40L BsAbFP. This approach could be used for developing dual target bioassays for other BsAbs and antibodies used for combination therapy.

Discovery of a novel Igß and FcγRIIB cross-linking antibody, ASP2713, and its potential application in the treatment of systemic lupus erythematosus.

B cell-targeted therapies have evolved as established therapies for systemic lupus erythematosus (SLE); however, existing approaches still do not thoroughly satisfy clinical requirements due to limited efficacy against memory B cells, autoantibody-producing plasmablasts and disease heterogeneity. To provide a new treatment option for SLE, we created a novel anti-Igß antibody with enhanced affinity for Fc gamma receptor (FcγR) IIB called ASP2713. ASP2713 cross-reacted with both human and cynomolgus monkey Igß and showed increased binding affinity for human and monkey FcγRIIB compared to native human IgG1. This binding property allows dominant B cell binding and induction of intrinsic negative feedback signals. In human B cells, ASP2713 significantly and concentration-dependently induced FcγRIIB ITIM phosphorylation, while suppressing proliferation under B cell receptor stimulation. This pharmacological effect was also confirmed in in vitro B cell proliferation and antibody production assays using peripheral B cells isolated from patients with SLE. In a cynomolgus monkey tetanus toxoid-induced antibody production model, ASP2713 almost completely inhibited the increase in antigen-specific antibodies with superior efficacy to rituximab. Additionally, ASP2713 significantly suppressed recall antibody production in response to secondary tetanus toxoid immunization, indicating the memory B cell- and plasmablast-targeting potential of ASP2713. Our results suggest that ASP2713 may have therapeutic potential as a treatment for SLE, where B cells play a pathogenic role.

Discovery of a novel anti PD-L1 X TIGIT bispecific antibody for the treatment of solid tumors.

The emergence of immune checkpoint inhibitors (ICIs), mainly based on PD-1/PD-L1 blockade has revolutionized the therapeutic landscape of cancer. Despite the huge clinical success ICIs have achieved, about 70% of patients still showed de novo and adaptive resistance. Exploring novel and complementary immune checkpoint molecules in addition to PD-1/PD-L1 is in great urgency. T cell immunoglobulin and ITIM domain (TIGIT) is a co-inhibitory molecule containing an immunoreceptor tyrosine-based inhibition motif (ITIM) within its cytoplasmic tail, and is highly expressed on regulatory T cells and activated CD4+ T, CD8+ T, and NK cells. We generated a novel single chain Fab heterodimeric bispecific IgG antibody format targeting PD-L1 and TIGIT with one binding site for each target antigen. The bispecifc antibody BiAb-1 is based on "knob-into-hole" technology for heavy chain heterodimerization with a glycine serine linker connecting the 3' end of Cκand the 5' end of VH to prevent wrong pairing of light chains. BiAb-1 was produced with high expression yields and show simultaneous binding to PD-L1 and TIGIT with high affinity. Importantly, cytokine production was enhanced by BiAb-1 from staphylococcal enterotoxin B (SEB) stimulated PBMCs. BiAb-1 also demonstrated potent anti-tumor efficacy in multiple tumor models and superior activity to PD-1/PD-L1 blockade molecules. In conclusion, we have applied rational antibody engineering technology to develop a monovalent heterodimeric bispecifc antibody, which combines the blockade of both PD-1/PD-L1 and TIGIT/CD155 pathways simultaneously and results in superior anti-tumor efficacy in multiple tumor models over existing anti PD-1/PD-L1 molecules.

Bispecific antibody-mediated redirection of NKG2D-CAR natural killer cells facilitates dual targeting and enhances antitumor activity.

BACKGROUND: Natural killer group 2D (NKG2D) is an activating receptor of natural killer (NK) cells and other lymphocytes that mediates lysis of malignant cells through recognition of stress-induced ligands such as MICA and MICB. Such ligands are broadly expressed by cancer cells of various origins and serve as targets for adoptive immunotherapy with effector cells endogenously expressing NKG2D or carrying an NKG2D-based chimeric antigen receptor (CAR). However, shedding or downregulation of NKG2D ligands (NKG2DL) can prevent NKG2D activation, resulting in escape of cancer cells from NKG2D-dependent immune surveillance. METHODS: To enable tumor-specific targeting of NKG2D-expressing effector cells independent of membrane-anchored NKG2DLs, we generated a homodimeric recombinant antibody which harbors an N-terminal single-chain fragment variable (scFv) antibody domain for binding to NKG2D, linked via a human IgG4 Fc region to a second C-terminal scFv antibody domain for recognition of the tumor-associated antigen ErbB2 (HER2). The ability of this molecule, termed NKAB-ErbB2, to redirect NKG2D-expressing effector cells to ErbB2-positive tumor cells of different origins was investigated using peripheral blood mononuclear cells, ex vivo expanded NK cells, and NK and T cells engineered with an NKG2D-based chimeric receptor. RESULTS: On its own, bispecific NKAB-ErbB2 increased lysis of ErbB2-positive breast carcinoma cells by peripheral blood-derived NK cells endogenously expressing NKG2D more effectively than an ErbB2-specific IgG1 mini-antibody able to induce antibody-dependent cell-mediated cytotoxicity via activation of CD16. Furthermore, NKAB-ErbB2 synergized with NK-92 cells or primary T cells engineered to express an NKG2D-CD3ζ chimeric antigen receptor (NKAR), leading to targeted cell killing and greatly enhanced antitumor activity, which remained unaffected by soluble MICA known as an inhibitor of NKG2D-mediated natural cytotoxicity. In an immunocompetent mouse glioblastoma model mimicking low or absent NKG2DL expression, the combination of NKAR-NK-92 cells and NKAB-ErbB2 effectively suppressed outgrowth of ErbB2-positive tumors, resulting in treatment-induced endogenous antitumor immunity and cures in the majority of animals. CONCLUSIONS: Our results demonstrate that combining an NKAB antibody with effector cells expressing an activating NKAR receptor represents a powerful and versatile approach to simultaneously enhance tumor antigen-specific as well as NKG2D-CAR and natural NKG2D-mediated cytotoxicity, which may be particularly useful to target tumors with heterogeneous target antigen expression.