Evaluation of Bispecific T-Cell Engagers Targeting Murine Cytomegalovirus.

Human cytomegalovirus is a ubiquitous herpesvirus that, while latent in most individuals, poses a great risk to immunocompromised patients. In contrast to directly acting traditional antiviral drugs, such as ganciclovir, we aim to emulate a physiological infection control using T cells. For this, we constructed several bispecific T-cell engager (BiTE) constructs targeting different viral glycoproteins of the murine cytomegalovirus and evaluated them in vitro for their efficacy. To isolate the target specific effect without viral immune evasion, we established stable reporter cell lines expressing the viral target glycoprotein B, and the glycoprotein complexes gN-gM and gH-gL, as well as nano-luciferase (nLuc). First, we evaluated binding capacities using flow cytometry and established killing assays, measuring nLuc-release upon cell lysis. All BiTE constructs proved to be functional mediators for T-cell recruitment and will allow a proof of concept for this treatment option. This might pave the way for strikingly safer immunosuppression in vulnerable patient groups.

Discovery and preclinical evaluation of KYS202004A, a novel bispecific fusion protein targeting TNF-α and IL-17A, in autoimmune disease models.

The treatment of autoimmune and inflammatory diseases often requires targeting multiple pathogenic pathways. KYS202004A is a novel bispecific fusion protein designed to antagonize TNF-α and IL-17A, pivotal in the pathophysiology of autoimmune and inflammatory diseases. Our initial efforts focused on screening for optimal structure by analyzing expression levels, purity, and binding capabilities. The binding affinity of KYS202004A to TNF-α and IL-17A was evaluated using SPR. In vitro, we assessed the inhibitory capacity of KYS202004A on cytokine-induced CXCL1 expression in HT29 cells. In vivo, its efficacy was tested using a Collagen-Induced Arthritis (CIA) model in transgenic human-IL-17A mice and an imiquimod-induced psoriasis model in cynomolgus monkeys. KYS202004A demonstrated significant inhibition of IL-17A and TNF-α signaling pathways, outperforming the efficacy of monotherapeutic agents ixekizumab and etanercept in reducing CXCL1 expression in vitro and ameliorating disease markers in vivo. In the CIA model, KYS202004A significantly reduced clinical symptoms, joint destruction, and serum IL-6 concentrations. The psoriasis model revealed that KYS202004A, particularly at a 2  mg/kg dose, was as effective as the combination of ixekizumab and etanercept. This discovery represents a significant advancement in treating autoimmune and inflammatory diseases, offering a dual-targeted therapeutic approach with enhanced efficacy over current monotherapies.

Mechanistic computational modeling of monospecific and bispecific antibodies targeting interleukin-6/8 receptors.

The spread of cancer from organ to organ (metastasis) is responsible for the vast majority of cancer deaths; however, most current anti-cancer drugs are designed to arrest or reverse tumor growth without directly addressing disease spread. It was recently discovered that tumor cell-secreted interleukin-6 (IL-6) and interleukin-8 (IL-8) synergize to enhance cancer metastasis in a cell-density dependent manner, and blockade of the IL-6 and IL-8 receptors (IL-6R and IL-8R) with a novel bispecific antibody, BS1, significantly reduced metastatic burden in multiple preclinical mouse models of cancer. Bispecific antibodies (BsAbs), which combine two different antigen-binding sites into one molecule, are a promising modality for drug development due to their enhanced avidity and dual targeting effects. However, while BsAbs have tremendous therapeutic potential, elucidating the mechanisms underlying their binding and inhibition will be critical for maximizing the efficacy of new BsAb treatments. Here, we describe a quantitative, computational model of the BS1 BsAb, exhibiting how modeling multivalent binding provides key insights into antibody affinity and avidity effects and can guide therapeutic design. We present detailed simulations of the monovalent and bivalent binding interactions between different antibody constructs and the IL-6 and IL-8 receptors to establish how antibody properties and system conditions impact the formation of binary (antibody-receptor) and ternary (receptor-antibody-receptor) complexes. Model results demonstrate how the balance of these complex types drives receptor inhibition, providing important and generalizable predictions for effective therapeutic design.

Design of Crosslinking Antibodies For T-Cell Activation: Experimental and Computational Analysis of PD-1/CD137 Bispecific Agents.

Bispecific and multispecific agents have become increasingly utilized in cancer treatment and immunotherapy, yet their complex design parameters present a challenge in developing successful therapeutics. Bispecifics that crosslink receptors on two opposing cells can provide specific activation of a receptor only when these cells are in close spatial proximity, such as an immune cell and cancer cell in a tumor. These agents, including T cell activating bispecifics, can avoid off-tumor toxicity through activation only in the tumor microenvironment by utilizing a tumor target to cluster T-cell receptors for a selective costimulatory signal. Here, we investigate a panel of PD-1/CD137 targeted Humabody VH domains to determine the key factors for T cell activation, such as affinity, valency, expression level, domain orientation, and epitope location. Target expression is a dominant factor determining both specificity and potency of T cell activation. Given an intrinsic expression level, the affinity can be tuned to modulate the level of activation and IC50 and achieve specificity between low and high expression levels. Changing the epitope location and linker length showed minor improvements to activation at low expression levels, but increasing the valency for the target decreased activation at all expression levels. By combining non-overlapping epitopes for the target, we achieved higher receptor activation at low expression levels. A kinetic model was able to capture these trends, offering support for the mechanistic interpretation. This work provides a framework to quantify factors for T cell activation by cell-crosslinking bispecific agents and guiding principles for the design of new agents.

Blinatumomab as postremission therapy replaces consolidation and substantial parts of maintenance chemotherapy and results in stable MRD negativity in children with newly diagnosed B-lineage ALL.

The bispecific T cell-binding antibody blinatumomab (CD19/CD3) is widely and successfully used for the treatment of children with relapsed or refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Here, we report the efficacy of a single course of blinatumomab instead of consolidation chemotherapy to eliminate minimal residual disease (MRD) and maintain stable MRD-negativity in children with primary BCP-ALL.Between February 2020 and November 2022, 177 children with non-high-risk BCP-ALL were enrolled in the ALL-MB 2019 pilot study (NCT04723342). Patients received the usual risk-adapted induction therapy according to the ALL-MB 2015 protocol. Those who achieved a complete remission at the end of induction (EOI) received treatment with blinatumomab immediately after induction at a dose of 5 µg/m2/day for 7 days and 21 days at a dose of 15 µg/m2/day, followed by 12 months of maintenance therapy. MRD was measured using multicolor flow cytometry (MFC) at the EOI, then immediately after blinatumomab treatment, and then four times during maintenance therapy at 3-month intervals.All 177 patients successfully completed induction therapy and achieved a complete hematological remission. In 174 of these, MFC-MRD was measured at the EOI. 143 patients (82.2%) were MFC-MRD negative and the remaining 31 patients had varying degrees of MFC-MRD positivity.MFC-MRD was assessed in all 176 patients who completed the blinatumomab course. With one exception, all patients achieved MFC-MRD negativity after blinatumomab, regardless of the MFC-MRD score at EOI. One adolescent girl with high MFC-MRD positivity at EOI remained MFC-MRD positive. Of 175 patients who had completed 6 months of maintenance therapy, MFC-MRD data were available for 156 children. Of these, 155 (99.4%) were MFC-MRD negative. Only one boy with t(12;21) (p13;q22)/ETV6::RUNX1 became MFC-MRD positive again. The remaining 174 children had completed the entire therapy. MFC-MRD was examined in 154 of them, and 153 were MFC-MRD negative. A girl with hypodiploid BCP-ALL showed a reappearance of MFC-MRD with subsequent relapse.In summary, a single 28-day course of blinatumomab immediately after induction, followed by 12 months of maintenance therapy, is highly effective in achieving MRD-negativity in children with newly diagnosed non-high risk BCP-ALL and maintaining MRD-negative remission at least during the treatment period.

Long-term survival follow-up for tebentafusp in previously treated metastatic uveal melanoma.

BACKGROUND: Tebentafusp, a bispecific (gp100×CD3) ImmTAC, significantly improved overall survival (OS) outcomes for HLA-A*02:01+ adult patients with untreated metastatic uveal melanoma (mUM) and showed promising survival in previously treated mUM with 1-year OS of 62% in the primary analysis of study IMCgp100-102. Here we report long-term outcomes from this phase 1/2 study in pretreated mUM. PATIENTS AND METHODS: Patients with previously treated mUM received tebentafusp weekly intravenous at 20 µg dose 1, 30 µg dose 2 and either 54, 64, 68, or 73 µg (phase 1) or 68 µg (phase 2) dose 3+. The primary objective was overall response rate. Secondary objectives included OS and safety. OS was estimated by Kaplan-Meier methods. Association between OS and baseline covariates, on-treatment Response Evaluation Criteria in Solid Tumors (RECIST) response, baseline tumor biopsy and circulating-tumor DNA (ctDNA) changes were assessed. RESULTS: 146 patients were treated with tebentafusp: 19 in phase 1 and 127 in phase 2. With a median follow-up duration of 48.5 months, the median OS was 17.4 months (95% CI, 13.1 to 22.8), and the 1-year, 2-year, 3-year and 4-year OS rates were 62%, 40%, 23% and 14%, respectively. Improved survival was associated with lower ctDNA baseline levels and greater ctDNA reductions by week 9 on-treatment, with 100% 1-year, 73% 2-year and 45% 3-year OS rates for patients with ctDNA clearance. Baseline gp100 expression was not associated with survival, despite more RECIST responses among patients with higher expression. No new safety signals were reported with long-term dosing. CONCLUSIONS: This study represents the longest follow-up of a Tcell receptor bispecific to date and confirms the durable survival benefits achieved with tebentafusp in previously treated mUM with good tolerability long-term. A role for ctDNA reduction as an early indicator of clinical benefit was again suggested for patients treated with tebentafusp.

Format-tuning of in vivo-launched bispecific T cell engager enhances efficacy against renal cell carcinoma.

BACKGROUND: Advanced clear cell renal cell carcinoma (ccRCC) is a prevalent kidney cancer for which long-term survival rates are abysmal, though immunotherapies are showing potential. Not yet clinically vetted are bispecific T cell engagers (BTEs) that activate T cell-mediated cancer killing through intercellular synapsing. Multiple BTE formats exist, however, with limited cross-characterizations to help optimize new drug design. Here, we developed BTEs to treat ccRCC by targeting carbonic anhydrase 9 (CA9) while characterizing the persistent BTE (PBTE) format and comparing it to a new format, the persistent multivalent T cell engager (PMTE). These antibody therapies against ccRCC are developed as both recombinant and synthetic DNA (synDNA) medicines. METHODS: Antibody formatting effects on binding kinetics were assessed by flow cytometry and intercellular synaptic strength assays while potency was tested using T-cell activation and cytotoxicity assays. Mouse models were used to study antibody plasma and tumor pharmacokinetics, as well as antitumor efficacy as both recombinant and synDNA medicines. Specifically, three models using ccRCC cell line xenografts and human donor T cells in immunodeficient mice were used to support this study. RESULTS: Compared with a first-generation BTE, we show that the PBTE reduced avidity, intercellular synaptic strength, cytotoxic potency by as much as 33-fold, and ultimately efficacy against ccRCC tumors in vivo. However, compared with the PBTE, we demonstrate that the PMTE improved cell avidity, restored intercellular synapses, augmented cytotoxic potency by 40-fold, improved tumor distribution pharmacokinetics by 2-fold, and recovered synDNA efficacy in mouse tumor models by 20-fold. All the while, the PMTE displayed a desirable half-life of 4 days in mice compared with the conventional BTE's 2 hours. CONCLUSIONS: With impressive efficacy, the CA9-targeted PMTE is a promising new therapy for advanced ccRCC, which can be effectively delivered through synDNA. The highly potent PMTE format itself is a promising new tool for future applications in the multispecific antibody space.

Structural and functional characterization of IgG- and non-IgG-based T-cell-engaging bispecific antibodies.

Bispecific T-cell-engaging antibodies are a growing class of therapeutics with numerous molecules being tested in clinical trials and, currently, seven of them have received market approval. They are structurally complex and function as adaptors to redirect the cytotoxicity of T cells to kill tumor cells. T-cell-engaging bispecific antibodies can be generally divided into two categories: IgG/IgG-like and non-IgG-like formats. Different formats may have different intrinsic potencies and physiochemical properties, and comprehensive studies are needed to gain a better understanding of how the differences in formats impact on structural and functional characteristics. In this study, we designed and generated bispecific T-cell-engaging antibodies with IgG-like (DVD-Ig) and non-IgG (BiTE) formats. Both target the same pair of antigens (EGFR and CD3) to minimize the possible influence of targets on functional characterization. We performed a side-by-side comparison to assess differences in the physiochemical and biological properties of these two bispecific T-cell-engaging antibodies using a variety of breast and ovarian cancer cell-based functional assays to delineate the structural-functional relationships and anti-tumor activities/potency. We found that the Fc portion of T-cell-engaging bispecific antibodies can significantly impact antigen binding activity, potency, and stability in addition to eliciting different mechanisms of action that contribute the killing of cancer cells.

A bispecific antibody exhibits broad neutralization against SARS-CoV-2 Omicron variants XBB.1.16, BQ.1.1 and sarbecoviruses.

The Omicron subvariants BQ.1.1, XBB.1.5, and XBB.1.16 of SARS-CoV-2 are known for their adeptness at evading immune responses. Here, we isolate a neutralizing antibody, 7F3, with the capacity to neutralize all tested SARS-CoV-2 variants, including BQ.1.1, XBB.1.5, and XBB.1.16. 7F3 targets the receptor-binding motif (RBM) region and exhibits broad binding to a panel of 37 RBD mutant proteins. We develop the IgG-like bispecific antibody G7-Fc using 7F3 and the cross-neutralizing antibody GW01. G7-Fc demonstrates robust neutralizing activity against all 28 tested SARS-CoV-2 variants and sarbecoviruses, providing potent prophylaxis and therapeutic efficacy against XBB.1 infection in both K18-ACE and BALB/c female mice. Cryo-EM structure analysis of the G7-Fc in complex with the Omicron XBB spike (S) trimer reveals a trimer-dimer conformation, with G7-Fc synergistically targeting two distinct RBD epitopes and blocking ACE2 binding. Comparative analysis of 7F3 and LY-CoV1404 epitopes highlights a distinct and highly conserved epitope in the RBM region bound by 7F3, facilitating neutralization of the immune-evasive Omicron variant XBB.1.16. G7-Fc holds promise as a potential prophylactic countermeasure against SARS-CoV-2, particularly against circulating and emerging variants.

Characterization of anti-drug antibody responses to the T-cell engaging bispecific antibody cibisatamab to understand the impact on exposure.

An appropriately designed pharmacokinetic (PK) assay that is sensitive for anti-drug antibody (ADA) impact on relevant exposure is an alternative strategy to understand the neutralizing potential of ADAs. However, guidance on how to develop such PK assays and how to confirm the functional ADA impact on exposure is missing. Here, the PK assay of a T-cell-engaging bispecific antibody, cibisatamab, was developed based on its mechanism of action (MoA). Using critical monoclonal anti-idiotypic (anti-ID) antibody positive controls as ADA surrogates, the impact on exposure was evaluated pre-clinically. In a phase I clinical trial (NCT02324257), initial data suggest that the combination of ADA and PK assays for correlation of the ADA response with cibisatamab exposure. To understand the neutralizing potential of patient-derived ADAs on drug activity, advanced ADA characterization has been performed. Structural binding analysis of ADAs to antibody domains of the drug and its impact on targeting were assessed. For this purpose, relevant patient ADA binding features were identified and compared with the specific monoclonal anti-ID antibody-positive controls. Comparable results of target binding inhibition and similar impacts on exposure suggest that the observed reduction of Cmax and Ctrough levels in patients is caused by the neutralizing potential of ADAs and allows a correlation between ADA response and loss of exposure. Therefore, the described study provides important functional aspects for the development of an appropriately designed PK assay for bispecific antibodies as an alternative option towards understanding the neutralizing ADA impact on exposure.

BiSpec Pairwise AI: guiding the selection of bispecific antibody target combinations with pairwise learning and GPT augmentation.

PURPOSE: Bispecific antibodies (BsAbs), capable of targeting two antigens simultaneously, represent a significant advancement by employing dual mechanisms of action for tumor suppression. However, how to pair targets to develop effective and safe bispecific drugs is a major challenge for pharmaceutical companies. METHODS: Using machine learning models, we refined the biological characteristics of currently approved or in clinical development BsAbs and analyzed hundreds of membrane proteins as bispecific targets to predict the likelihood of successful drug development for various target combinations. Moreover, to enhance the interpretability of prediction results in bispecific target combination, we combined machine learning models with Large Language Models (LLMs). Through a Retrieval-Augmented Generation (RAG) approach, we supplement each pair of bispecific targets' machine learning prediction with important features and rationales, generating interpretable analytical reports. RESULTS: In this study, the XGBoost model with pairwise learning was employed to predict the druggability of BsAbs. By analyzing extensive data on BsAbs and designing features from perspectives such as target activity, safety, cell type specificity, pathway mechanism, and gene embedding representation, our model is able to predict target combinations of BsAbs with high market potential. Specifically, we integrated XGBoost with the GPT model to discuss the efficacy of each bispecific target pair, thereby aiding the decision-making for drug developers. CONCLUSION: The novelty of this study lies in the integration of machine learning and GPT techniques to provide a novel framework for the design of BsAbs drugs. This holistic approach not only improves prediction accuracy, but also enhances the interpretability and innovativeness of drug design.

Targeted desialylation and cytolysis of tumour cells by fusing a sialidase to a bispecific T-cell engager.

Bispecific T-cell engagers (BiTEs) bring together tumour cells and cytotoxic T cells by binding to specific cell-surface tumour antigens and T-cell receptors, and have been clinically successful for the treatment of B-cell malignancies. Here we show that a BiTE-sialidase fusion protein enhances the susceptibility of solid tumours to BiTE-mediated cytolysis of tumour cells via targeted desialylation-that is, the removal of terminal sialic acid residues on glycans-at the BiTE-induced T-cell-tumour-cell interface. In xenograft and syngeneic mouse models of leukaemia and of melanoma and breast cancer, and compared with the parental BiTE molecules, targeted desialylation via the BiTE-sialidase fusion proteins enhanced the formation of immunological synapses, T-cell activation and T-cell-mediated tumour-cell cytolysis in the presence of the target antigen. The targeted desialylation of tumour cells may enhance the potency of therapies relying on T-cell engagers.

Targeting CEACAM5-positive solid tumors using NILK-2401, a novel CEACAM5xCD47 κλ bispecific antibody.

Background: Blocking the CD47 "don't eat me"-signal on tumor cells with monoclonal antibodies or fusion proteins has shown limited clinical activity in hematologic malignancies and solid tumors thus far. Main side effects are associated with non-tumor targeted binding to CD47 particularly on blood cells. Methods: We present here the generation and preclinical development of NILK-2401, a CEACAM5×CD47 bispecific antibody (BsAb) composed of a common heavy chain and two different light chains, one kappa and one lambda, determining specificity (so-called κλ body format). Results: NILK-2401 is a fully human BsAb binding the CEACAM5 N-terminal domain on tumor cells by its lambda light chain arm with an affinity of ≈4 nM and CD47 with its kappa chain arm with an intendedly low affinity of ≈500 nM to enabling tumor-specific blockade of the CD47-SIRPα interaction. For increased activity, NILK-2401 features a functional IgG1 Fc-part. NILK-2401 eliminates CEACAM5-positive tumor cell lines (3/3 colorectal, 2/2 gastric, 2/2 lung) with EC50 for antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity ranging from 0.38 to 25.84 nM and 0.04 to 0.25 nM, respectively. NILK-2401 binds neither CD47-positive/CEACAM5-negative cell lines nor primary epithelial cells. No erythrophagocytosis or platelet activation is observed. Quantification of the pre-existing NILK-2401-reactive T-cell repertoire in the blood of 14 healthy donors with diverse HLA molecules shows a low immunogenic potential. In vivo, NILK-2401 significantly delayed tumor growth in a NOD-SCID colon cancer model and a syngeneic mouse model using human CD47/human SIRPα transgenic mice and prolonged survival. In cynomolgus monkeys, single doses of 0.5 and 20 mg/kg were well tolerated; PK linked to anti-CD47 and Fc-binding seemed to be more than dose-proportional for Cmax and AUC0-inf. Data were validated in human FcRn TG32 mice. Combination of a CEACAM5-targeting T-cell engager (NILK-2301) with NILK-2401 can either boost NILK-2301 activity (Emax) up to 2.5-fold or allows reaching equal NILK-2301 activity at >600-fold (LS174T) to >3,000-fold (MKN-45) lower doses. Conclusion: NILK-2401 combines promising preclinical activity with limited potential side effects due to the tumor-targeted blockade of CD47 and low immunogenicity and is planned to enter clinical testing.

A novel anti-LAG-3/TIGIT bispecific antibody exhibits potent anti-tumor efficacy in mouse models as monotherapy or in combination with PD-1 antibody.

We report the generation of a novel anti-LAG-3/TIGIT bispecific IgG4 antibody, ZGGS15, and evaluated its anti-tumor efficacy in mouse models as monotherapy or in combination with a PD-1 antibody. ZGGS15 exhibited strong affinities for human LAG-3 and TIGIT, with KDs of 3.05 nM and 2.65 nM, respectively. ZGGS15 has EC50s of 0.69 nM and 1.87 nM for binding to human LAG-3 and TIGIT on CHO-K1 cells, respectively. ZGGS15 competitively inhibited the binding of LAG-3 to MHC-II (IC50 = 0.77 nM) and the binding of TIGIT to CD155 (IC50 = 0.24 nM). ZGGS15 does not induce ADCC, CDC, or obvious cytokine production. In vivo results showed that ZGGS15 had better anti-tumor inhibition than single anti-LAG-3 or anti-TIGIT agents and demonstrated a synergistic effect when combined with nivolumab, with a significantly higher tumor growth inhibition of 95.80% (p = 0.001). The tumor volume inhibition rate for ZGGS15 at 2 mg/kg was 69.70%, and for ZGGS15 at 5 mg/kg plus nivolumab at 1 mg/kg, it was 94.03% (p < 0.001). Our data reveal that ZGGS15 exhibits potent anti-tumor efficacy without eliciting ADCC or CDC or causing cytokine production, therefore having a safe profile.

A bispecific antibody targeting HLA-DQ2.5-gluten peptides potently blocks gluten-specific T cells induced by gluten ingestion in patients with celiac disease.

The gluten-free diet for celiac disease (CeD) is restrictive and often fails to induce complete symptom and/or mucosal disease remission. Central to CeD pathogenesis is the gluten-specific CD4+ T cell that is restricted by HLA-DQ2.5 in over 85% of CeD patients, making HLA-DQ2.5 an attractive target for suppressing gluten-dependent immunity. Recently, a novel anti-HLA-DQ2.5 antibody that specifically recognizes the complexes of HLA-DQ2.5 and multiple gluten epitopes was developed (DONQ52). OBJECTIVE: To assess the ability of DONQ52 to inhibit CeD patient-derived T-cell responses to the most immunogenic gluten peptides that encompass immunodominant T cell epitopes. METHODS: We employed an in vivo gluten challenge model in patients with CeD that affords a quantitative readout of disease-relevant gluten-specific T-cell responses. HLA-DQ2.5+ CeD patients consumed food containing wheat, barley, or rye for 3 days with collection of blood before (D1) and 6 days after (D6) commencing the challenge. Peripheral blood mononuclear cells were isolated and assessed in an interferon (IFN)-γ enzyme-linked immunosorbent spot assay (ELISpot) testing responses to gluten peptides encompassing a series of immunodominant T cell epitopes. The inhibitory effect of DONQ52 (4 or 40 µg/mL) was assessed and compared to pan-HLA-DQ blockade (SPVL3 antibody). RESULTS: In HLA-DQ2.5+ CeD patients, DONQ52 reduced T cell responses to all wheat gluten peptides to an equivalent or more effective degree than pan-HLA-DQ antibody blockade. It reduced T cell responses to a cocktail of the most immunodominant wheat epitopes by a median of 87% (IQR 72-92). Notably, DONQ52 also substantially reduced T-cell responses to dominant barley hordein and rye secalin derived peptides. DONQ52 had no effect on T-cell responses to non-gluten antigens. CONCLUSION: DONQ52 can significantly block HLA-DQ2.5-restricted T cell responses to the most highly immunogenic gluten peptides in CeD. Our findings support in vitro data that DONQ52 displays selectivity and broad cross-reactivity against multiple gluten peptide:HLA-DQ2.5 complexes. This work provides proof-of-concept multi-specific antibody blockade has the potential to meaningfully inhibit pathogenic gluten-specific T-cell responses in CeD and supports ongoing therapeutic development.

The protective effect of anti-VEGF-A/Ang-2 bispecific antibody on retinal vein occlusion model mice.

(233/250) Retinal vein occlusion (RVO) causes macular edema and retinal ischemia resulting in visual field and vision loss. A bispecific antibody that blocks VEGF-A and angiopoietin-2 (Ang-2) has been recently launched and applied clinically to treat macular edema, but the role of Ang-2 in the pathogenesis of RVO is still unclear. In this study, we investigated the effects of the anti-VEGF-A/anti-Ang-2 bispecific antibody (BsAb) in a murine RVO model. By using RVO model mice, the expression of Ang-2 gene and protein was examined in the retina through real-time qPCR and Western blotting, respectively. A significant increase in Ang-2 was detected 1 day after occlusion. Immediately after occlusion, control IgG 400 µg/mL, anti-VEGF-A antibody 200 µg/mL, anti-Ang-2 antibody 200 µg/mL, and BsAb 400 µg/mL were intravitreally administered at 2 µL. Visual function was examined using electroretinograms, and apoptosis was examined using TUNEL staining. Interestingly, BsAb partially suppressed the decrease in amplitude of a and b waves compared to control IgG. Anti-Ang-2 antibody and BsAb reduced apoptosis-positive cells 1 day after occlusion. Comprehensive gene expression profiles were also examined using RNA sequencing analysis. RNA sequencing analysis of the retinal tissues showed that BsAb suppressed expression of gene groups associated with inflammatory response and vascular development compared to anti-VEGF-A antibody. Taken together, higher expression of Ang-2 contributes to the pathophysiology of RVO, providing a possible mechanism for the efficacy of BsAb in suppressing retinal dysfunction in RVO.

CEA-CD3 bispecific antibody cibisatamab with or without atezolizumab in patients with CEA-positive solid tumours: results of two multi-institutional Phase 1 trials.

Cibisatamab is a bispecific antibody-based construct targeting carcinoembryonic antigen (CEA) on tumour cells and CD3 epsilon chain as a T-cell engager. Here we evaluated cibisatamab for advanced CEA-positive solid tumours in two open-label Phase 1 dose-escalation and -expansion studies: as a single agent with or without obinutuzumab in S1 (NCT02324257) and with atezolizumab in S2 (NCT02650713). Primary endpoints were safety, dose finding, and pharmacokinetics in S1; safety and dose finding in S2. Secondary endpoints were anti-tumour activity (including overall response rate, ORR) and pharmacodynamics in S1; anti-tumour activity, pharmacodynamics and pharmacokinetics in S2. S1 and S2 enrolled a total of 149 and 228 patients, respectively. Grade ≥3 cibisatamab-related adverse events occurred in 36% of S1 and 49% of S2 patients. The ORR was 4% in S1 and 7% in S2. In S2, patients with microsatellite stable colorectal carcinoma (MSS-CRC) given flat doses of cibisatamab and atezolizumab demonstrated an ORR of 14%. In S1 and S2, 40% and 52% of patients, respectively, developed persistent anti-drug antibodies (ADAs). ADA appearance could be mitigated by obinutuzumab-pretreatment, with 8% of patients having persistent ADAs. Overall, cibisatamab warrants further exploration in immunotherapy combination strategies for MSS-CRC.

Bispecific T cell engager-armed T cells targeting integrin ανß6 exhibit enhanced T cell redirection and antitumor activity in cholangiocarcinoma.

Advanced cholangiocarcinoma (CCA) presents a clinical challenge due to limited treatment options, necessitating exploration of innovative therapeutic approaches. Bispecific T cell engager (BTE)-armed T cell therapy shows promise in hematological and solid malignancies, offering potential advantages in safety over continuous BTE infusion. In this context, we developed a novel BTE, targeting CD3 on T cells and integrin αvß6, an antigen elevated in various epithelial malignancies, on cancer cells. The novel BTE was generated by fusing an integrin αvß6-binding peptide (A20) to an anti-CD3 (OKT3) single-chain variable fragment (scFv) through a G4S peptide linker (A20/αCD3 BTE). T cells were then armed with A20/αCD3 BTE (A20/αCD3-armed T cells) and assessed for antitumor activity. Our results highlight the specific binding of A20/αCD3 BTE to CD3 on T cells and integrin αvß6 on target cells, effectively redirecting T cells towards these targets. After co-culture, A20/αCD3-armed T cells exhibited significantly heightened cytotoxicity against integrin αvß6-expressing target cells compared to unarmed T cells in both KKU-213A cells and A375.ß6 cells. Moreover, in a five-day co-culture, A20/αCD3-armed T cells demonstrated superior cytotoxicity against KKU-213A spheroids compared to unarmed T cells. Importantly, A20/αCD3-armed T cells exhibited an increased proportion of the effector memory T cell (Tem) subset, upregulation of T cell activation markers, enhanced T cell proliferation, and increased cytolytic molecule/cytokine production, when compared to unarmed T cells in an integrin αvß6-dependent manner. These findings support the potential of A20/αCD3-armed T cells as a novel therapeutic approach for integrin αvß6-expressing cancers.

Antibody-drug conjugates, bispecific antibodies and CAR-T cells therapy in multiple myeloma.

INTRODUCTION: Modern immunotherapy approaches are revolutionizing the treatment scenario of relapsed/refractory (RR) multiple myeloma (MM) patients, providing an opportunity to reach deep level of responses and extend survival outcomes. AREAS COVERED: Antibody-drug conjugates (ADCs) and T-cell redirecting treatments, including bispecific antibodies (BsAbs) and chimeric antigen receptor (CAR) T cells therapy, have been recently introduced in the treatment of RRMM. Some agents have already received regulatory approval, while newer constructs, novel combinations, and applications in earlier lines of therapy are currently being explored. This review discusses the current landscape and possible development of ADCs, BsAbs and CAR-T cells immunotherapies. EXPERT OPINION: ADCs, BsAbs, and CAR-T therapy have demonstrated substantial activity in heavily pretreated, triple-class exposed (TCE) MM patients, and T-cell redirecting treatments represent new standards of care after third (European Medicines Agency, EMA), or fourth (Food and Drug Administration, FDA), line of therapy. All these three immunotherapies carry advantages and disadvantages, with different accessibility and new toxicities that require appropriate management and guidelines. Multiple on-going programs include combinations therapies and applications in earlier lines of treatment, as well as the development of novel agents or construct to enhance potency, reduce toxicity and facilitate administration. Sequencing is a challenge, with few data available and mechanisms of resistance still to be unraveled.