[Development of novel bispecific antibody therapy for multiple myeloma].

Over the past decade, new therapeutic modalities have markedly improved clinical outcomes for patients with multiple myeloma. Recently, immunotherapy using both bispecific antibodies (BsAb) and chimeric antigen receptor T cells (CAR-T cells) has induced further anti-myeloma responses. Different agents must be combined to overcome the heterogeneity of myeloma cell clones, and new modalities for the treatment of refractory myeloma must also be developed to strengthen therapeutic effects. We have developed a novel BiTE (bispecific T-cell engager)-based modality, referred to as bridging-BiTE (B-BiTE). B-BiTE is able to bind to both an Fc domain of a human immunoglobulin G monoclonal antibody (mAb) and the human CD3 molecule. This enables rapid generation of a mAb/B-BiTE complex and safely induces dual-lymphoid activation of both human T cells and NK cells against myeloma cells. Importantly, sequential immunotherapy using two different mAb/B-BiTE complexes can produce deep and durable anti-myeloma responses. To further advance treatment of multiple myeloma, it is important to determine how to combine and sequence immunotherapy with other agents while considering management of unique adverse events caused by activated immune cells.

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.

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.

Multinational proficiency tests for EGFR exon 20 insertions reveal that the assay design matters.

Insertion mutations in exon 20 of the epidermal growth factor receptor gene (EGFR exon20ins) are rare, heterogeneous alterations observed in non-small cell lung cancer (NSCLC). With a few exceptions, they are associated with primary resistance to established EGFR tyrosine kinase inhibitors (TKIs). As patients carrying EGFR exon20ins may be eligible for treatment with novel therapeutics-the bispecific antibody amivantamab, the TKI mobocertinib, or potential future innovations-they need to be identified reliably in clinical practice for which quality-based routine genetic testing is crucial. Spearheaded by the German Quality Assurance Initiative Pathology two international proficiency tests were run, assessing the performance of 104 participating institutes detecting EGFR exon20ins in tissue and/or plasma samples. EGFR exon20ins were most reliably identified using next-generation sequencing (NGS). Interestingly, success rates of institutes using commercially available mutation-/allele-specific quantitative (q)PCR were below 30% for tissue samples and 0% for plasma samples. Most of these mutation-/allele-specific (q)PCR assays are not designed to detect the whole spectrum of EGFR exon20ins mutations leading to false negative results. These data suggest that NGS is a suitable method to detect EGFR exon20ins in various types of patient samples and is superior to the detection spectrum of commercially available assays.

Bispecific T-cell engagers in childhood B-acute lymphoblastic leukemia.

Immunotherapy has revolutionized treatment for a wide variety of cancers yet its use has been relatively limited in childhood malignancies. With the introduction of bispecific T-cell engagers (BiTE®) and chimeric antigen T-cell receptor technologies, previously refractory patients have attained remission, including molecularly negative states of disease, thus providing the possibility of long-term cure. Blinatumomab is a widely available CD3-CD19 BiTE that has dramatically changed the landscape of therapy for some children with precursor-B acute lymphoblastic leukemias (B-ALL) and lymphoblastic lymphomas. Challenges remain with using BiTE in a broader population although the appeal of now-confirmed reduced toxicity and deeper molecular remissions suggests that this approach will be an essential part of future treatment of childhood B-ALL. Herein, we review some of the pertinent literature covering clinical trials with blinatumomab and address future approaches and combination trials including BiTE.

Naked antibodies and antibody-drug conjugates: targeted therapy for childhood acute lymphoblastic leukemia.

The treatment of childhood acute lymphoblastic leukemia (ALL) has reached overall survival rates exceeding 90%. The present and future challenges are to cure the remainder of patients still dying from disease, and to reduce morbidity and mortality in those who can be cured with standard-of-care chemotherapy by replacing toxic chemotherapy elements while retaining cure rates. With the novel therapeutic options introduced in the last years, including immunotherapies and targeted antibodies, the treatment of ALL is undergoing major changes. For B-cell precursor ALL, blinatumomab, an anti-CD19 bispecific antibody, has established its role in the consolidation treatment for both high- and standard-risk first relapse of ALL, in the presence of bone marrow involvement, and may also have an impact on the outcome of high-risk subsets such as infant ALL and Philadelphia chromosome-positive ALL. Inotuzumab ozogamicin, an anti-CD22 drug conjugated antibody, has demonstrated high efficacy in inducing complete remission in relapsed ALL, even in the presence of high tumor burden, but randomized phase III trials are still ongoing. For T-ALL the role of CD38-directed treatment, such as daratumumab, is gaining interest, but randomized data are needed to assess its specific benefit. These antibodies are currently being tested in patients with newly diagnosed ALL and may lead to major changes in the present paradigm of treatment of pediatric ALL. Unlike the past, lessons may be learned from innovations in adult ALL, in which more drastic changes are piloted that may need to be translated to pediatrics.

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.

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.

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.

T-Cell redirecting bispecific antibodies: a review of a novel class of immuno-oncology for advanced prostate cancer.

Novel T-cell immunotherapies such as bispecific T-cell engagers (BiTEs) are emerging as promising therapeutic strategies for prostate cancer. BiTEs are engineered bispecific antibodies containing two distinct binding domains that allow for concurrent binding to tumor-associated antigens (TAAs) as well as immune effector cells, thus promoting an immune response against cancer cells. Prostate cancer is rich in tumor associated antigens such as, but not limited to, PSMA, PSCA, hK2, and STEAP1 and there is strong biologic rationale for employment of T-cell redirecting BiTEs within the prostate cancer disease space. Early generation BiTE constructs employed in clinical study have demonstrated meaningful antitumor activity, but challenges related to drug delivery, immunogenicity, and treatment-associated adverse effects limited their success. The ongoing development of novel BiTE constructs continues to address these barriers and to yield promising results in terms of efficacy and safety. This review will highlight some of most recent developments of BiTE therapies for patients with advanced prostate cancer and the evolving data surrounding BiTE constructs undergoing clinical evaluation.

Striking the Balance with a PD-L1×4-1BB Bispecific Antibody.

Antibody-based immune checkpoint blockade therapy has revolutionized the field of cancer immunotherapy, yet its efficacy remains limited in immunologically cold tumors. Combining checkpoint inhibitors with costimulatory agonists improves tumoricidal activity of T cells but also can lead to off-target hepatotoxicity. Although bispecific antibodies confer tumor selectivity to alleviate undesirable adverse effects, toxicity concerns persist with increased dosing. In this issue of Cancer Research, Yuwen and colleagues introduce ATG-101, a tetravalent PD-L1×4-1BB bispecific antibody with high programmed death ligand 1 (PD-L1) affinity and low 4-1BB affinity, aiming to mitigate hepatotoxicity. ATG-101 demonstrates PD-L1-dependent 4-1BB activation, leading to selective T-cell activation within the tumor microenvironment. ATG-101 exhibits potent antitumor activity, even in large, immunologically cold, and monotherapy-resistant tumor models. Single-cell RNA sequencing reveals significant shifts of immune cell populations in the tumor microenvironment from protumor to antitumor phenotypes following ATG-101 treatment. In cynomolgus monkeys, no serious cytokine storm and hepatotoxicity are observed after ATG-101 treatment, indicating a broad therapeutic window for ATG-101 in cancer treatment. This study highlights the potential of tetravalent bispecific antibodies in cancer immunotherapy, with implications for various antibody-based treatment modalities across different fields. See related article by Yuwen et al., p. 1680.

[Pharmacoeconomic analysis of anti-angiogenic drugs for diabetic macular edema]. / Farmakoekonomicheskii analiz primeneniya antiangiogennykh preparatov pri diabeticheskom makulyarnom oteke.

Diabetic macular edema (DME) is a degenerative disease of the macular area in diabetes mellitus and can lead to vision loss, disability, and significantly reduced quality of life. Faricimab is the only bispecific antibody for DME therapy that targets two pathogenic pathways (Ang-2 and VEGF-A). PURPOSE: This study comparatively evaluates the clinical and economic feasibility of faricimab and other angiogenesis inhibitors in patients with DME. MATERIAL AND METHODS: This article analyzed literature on the efficacy and safety of intravitreal injections (IVI) of ranibizumab 0.5 mg, aflibercept 2 mg, and faricimab 6 mg. A model of medical care was developed for patients with DME receiving anti-angiogenic therapy. Pharmacoeconomic analysis was performed using cost minimization and budget impact analysis (BIA) methods. Modeling time horizon was 2 years. The research was performed from the perspective of the healthcare system of the Russian Federation. RESULTS: The efficacy and safety of faricimab in a personalized regimen (up to one IVI in 16 weeks) are comparable to those of aflibercept and ranibizumab, administered in various regimens. The use of faricimab is associated with the lowest number of IVIs. Over 2 years, the maximum costs of drug therapy were associated with the use of ranibizumab (about 914 thousand rubles), while the minimum costs were associated with the use of faricimab (614 thousand rubles). The reduction in inpatient care costs with faricimab therapy was 36% compared to aflibercept (216 and 201 thousand rubles in inpatient and day hospitals, respectively) and 82% compared to ranibizumab (486 and 451 thousand rubles in inpatient and day hospitals, respectively). BIA demonstrated that the use of faricimab will reduce the economic burden on the healthcare system by 11.3 billion rubles (9.8%) over 2 years. CONCLUSION: The use of faricimab is a cost-effective approach to treatment of adult patients with DME in Russia.

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.

Pre-Clinical Assessment of SAR442257, a CD38/CD3xCD28 Trispecific T Cell Engager in Treatment of Relapsed/Refractory Multiple Myeloma.

Current treatment strategies for multiple myeloma (MM) are highly effective, but most patients develop relapsed/refractory disease (RRMM). The anti-CD38/CD3xCD28 trispecific antibody SAR442257 targets CD38 and CD28 on MM cells and co-stimulates CD3 and CD28 on T cells (TCs). We evaluated different key aspects such as MM cells and T cells avidity interaction, tumor killing, and biomarkers for drug potency in three distinct cohorts of RRMM patients. We found that a significantly higher proportion of RRMM patients (86%) exhibited aberrant co-expression of CD28 compared to newly diagnosed MM (NDMM) patients (19%). Furthermore, SAR442257 mediated significantly higher TC activation, resulting in enhanced MM killing compared to bispecific functional knockout controls for all relapse cohorts (Pearson's r = 0.7). Finally, patients refractory to anti-CD38 therapy had higher levels of TGF-ß (up to 20-fold) compared to other cohorts. This can limit the activity of SAR442257. Vactoserib, a TGF-ß inhibitor, was able to mitigate this effect and restore sensitivity to SAR442257 in these experiments. In conclusion, SAR442257 has high potential for enhancing TC cytotoxicity by co-targeting CD38 and CD28 on MM and CD3/CD28 on T cells.

International Myeloma Working Group immunotherapy committee consensus guidelines and recommendations for optimal use of T-cell-engaging bispecific antibodies in multiple myeloma.

Multiple myeloma remains an incurable disease, despite the development of numerous drug classes and combinations that have contributed to improved overall survival. Immunotherapies directed against cancer cell-surface antigens, such as chimeric antigen receptor (CAR) T-cell therapy and T-cell-redirecting bispecific antibodies, have recently received regulatory approvals and shown unprecedented efficacy. However, these immunotherapies have unique mechanisms of action and toxicities that are different to previous treatments for myeloma, so experiences from clinical trials and early access programmes are essential for providing specific recommendations for management of patients, especially as these agents become available across many parts of the world. Here, we provide expert consensus clinical practice guidelines for the use of bispecific antibodies for the treatment of myeloma. The International Myeloma Working Group is also involved in the collection of prospective real-time data of patients treated with such immunotherapies, with the aim of learning continuously and adapting clinical practices to optimise the management of patients receiving immunotherapies.

[Preparation of a dual-specific antibody targeting human CD123 and exploration of its anti-acute myeloid leukemia effects].

Objective: To construct a novel dual-specific antibody targeting human CD123 (CD123 DuAb) and study its effects in acute myeloid leukemia (AML) . Methods: Based on the variable region of the CD123 monoclonal antibody independently developed at our institution, the CD123 DuAb expression plasmid was constructed by molecular cloning and transfected into ExpiCHO-S cells to prepare the antibody protein. Through a series of in vitro experiments, its activation and proliferation effect on T cells, as well as the effect of promoting T-cell killing of AML cells, were verified. Results: ① A novel CD123 DuAb plasmid targeting CD123 was successfully constructed and expressed in the Expi-CHO eukaryotic system. ②The CD123 DuAb could bind both CD3 on T cells and CD123 on CD123(+) tumor cells. ③When T cells were co-cultured with MV4-11 cells with addition of the CD123 DuAb at a concentration of 1 nmol/L, the positive expression rates of CD69 and CD25 on T cells were 68.0% and 44.3%, respectively, which were significantly higher than those of the control group (P<0.05). ④Co-culture with CD123 DuAb at 1 nmol/L promoted T-cell proliferation, and the absolute T-cell count increased from 5×10(5)/ml to 3.2×10(6)/ml on day 9, and CFSE fluorescence intensity decreased significantly. ⑤ With the increase in CD123 DuAb concentration in the culture system, T-cell exhaustion and apoptosis increased. When the CD123 DuAb was added at a concentration of 1 nmol/L to the culture system, the proportion of CD8(+) PD-1(+) LAG-3(+) T cells was 10.90%, and the proportion of propidium iodide (PI) (-) Annexin Ⅴ(+) T cells and PI(+) Annexin Ⅴ(+) T cells was 18.27% and 11.43%, respectively, which were significantly higher than those in the control group (P<0.05). ⑥ The CD123 DuAb significantly activated T cells, and the activation intensity was positively correlated with its concentration. The expression rate of CD107a on T cells reached 16.05% with 1 nmol/L CD123 DuAb, which was significantly higher than that of the control group (P<0.05). ⑦The CD123 DuAb promoted cytokine secretion by T cells at a concentration of 1 nmol/L, and the concentration of IFN-γ and TNF-α in the supernatant of the co-culture system reached 193.8 pg/ml and 169.8 pg/ml, respectively, which was significantly higher than that of the control group (P<0.05). ⑧When CD123 DuAb was added at a concentration of 1 nmol/L to the co-culture system of T cells and CD123(+) tumor cells, the killing intensity of T cells significantly increased, and the residual rates of CD123(+) MV4-11 cells, CD123(+) Molm13 cells, and CD123(+) THP-1 cells were 7.4%, 6.7%, and 14.6% on day 3, respectively, which were significantly lower than those in the control group (P<0.05) . Conclusion: In this study, a novel CD123 DuAb was constructed and expressed. In vitro experiments verified that the DuAb binds to CD123(+) tumor cells and T cells simultaneously, promotes T-cell activation and proliferation, and facilitates their anti-leukemia effect, which provides a basis for further clinical research.

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.

Control of acute myeloid leukemia and generation of immune memory in vivo using AMV564, a bivalent bispecific CD33 x CD3 T cell engager.

Off-the-shelf immunotherapeutics that suppress tumor growth and provide durable protection against relapse could enhance cancer treatment. We report preclinical studies on a CD33 x CD3 bivalent bispecific diabody, AMV564, that not only suppresses tumor growth, but also facilitates memory responses in a mouse model of acute myelogenous leukemia (AML). Mechanistically, a single 5-day treatment with AMV564 seems to reduce tumor burden by redirection of T cells, providing a time window for allogeneic or other T cells that innately recognize tumor antigens to become activated and proliferate. When the concentration of bispecific becomes negligible, the effector: target ratio has also shifted, and these activated T cells mediate long-term tumor control. To test the efficacy of AMV564 in vivo, we generated a CD33+ MOLM13CG bioluminescent human cell line and optimized conditions needed to control these cells for 62 days in vivo in NSG mice. Of note, not only did MOLM13CG become undetectable by bioluminescence imaging in response to infusion of human T cells plus AMV564, but also NSG mice that had cleared the tumor also resisted rechallenge with MOLM13CG in spite of no additional AMV564 treatment. In these mice, we identified effector and effector memory human CD4+ and CD8+ T cells in the peripheral blood immediately prior to rechallenge that expanded significantly during the subsequent 18 days. In addition to the anti-tumor effects of AMV564 on the clearance of MOLM13CG cells in vivo, similar effects were seen when primary CD33+ human AML cells were engrafted in NSG mice even when the human T cells made up only 2% of the peripheral blood cells and AML cells made up 98%. These studies suggest that AMV564 is a novel and effective bispecific diabody for the targeting of CD33+ AML that may provide long-term survival advantages in the clinic.

Cancer therapy with antibodies.

The greatest challenge in cancer therapy is to eradicate cancer cells with minimal damage to normal cells. Targeted therapy has been developed to meet that challenge, showing a substantially increased therapeutic index compared with conventional cancer therapies. Antibodies are important members of the family of targeted therapeutic agents because of their extraordinarily high specificity to the target antigens. Therapeutic antibodies use a range of mechanisms that directly or indirectly kill the cancer cells. Early antibodies were developed to directly antagonize targets on cancer cells. This was followed by advancements in linker technologies that allowed the production of antibody-drug conjugates (ADCs) that guide cytotoxic payloads to the cancer cells. Improvement in our understanding of the biology of T cells led to the production of immune checkpoint-inhibiting antibodies that indirectly kill the cancer cells through activation of the T cells. Even more recently, bispecific antibodies were synthetically designed to redirect the T cells of a patient to kill the cancer cells. In this Review, we summarize the different approaches used by therapeutic antibodies to target cancer cells. We discuss their mechanisms of action, the structural basis for target specificity, clinical applications and the ongoing research to improve efficacy and reduce toxicity.