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.

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.

Emicizumab prophylaxis in people with hemophilia A and inhibitors: a systematic review and meta-analysis.

BACKGROUND: Until recently, the treatment of people with hemophilia A and inhibitors (PwHAi) was based on the use of bypassing agents (BPA). However, the advent of emicizumab as prophylaxis has demonstrated promising results. OBJECTIVES: We aimed to compare the bleeding endpoints between PwHAi on BPA and those on emicizumab prophylaxis. DESIGN AND SETTING: Systematic review of interventions and meta-analysis conducted at the Universidade Federal de Goiás, Goiânia, Goiás, Brazil. METHODS: The CENTRAL, MEDLINE, Scopus, and LILACS databases were searched on February 21, 2023. Two authors conducted the literature search, publication selection, and data extraction. The selected publications evaluated the bleeding endpoints between PwHAi on emicizumab prophylaxis and those on BPA prophylaxis. The risk of bias was evaluated according to the Joanna Briggs Institute criteria. A meta-analysis was performed to determine the annualized bleeding rate (ABR) for treated bleeds. RESULTS: Five publications (56 PwHAi) were selected from the 543 retrieved records. Overall, bleeding endpoints were lower during emicizumab prophylaxis than during BPA prophylaxis. All the publications had at least one risk of bias. The only common parameter for the meta-analysis was the ABR for treated bleeds. During emicizumab prophylaxis, the ABR for treated bleeds was lower than during BPA prophylaxis (standard mean difference: -1.58; 95% confidence interval -2.50, -0.66, P = 0.0008; I2 = 68.4%, P = 0.0031). CONCLUSION: Emicizumab was superior to BPA in bleeding prophylaxis in PwHAi. However, both the small population size and potential risk of bias should be considered when evaluating these results. SYSTEMATIC REVIEW REGISTRATION: CRD42021278726, https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=278726.

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.

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.

The bispecific B7H3xCD3 antibody CC-3 induces T cell immunity against bone and soft tissue sarcomas.

Sarcomas are rare and heterogeneous malignancies that are difficult to treat. Approximately 50% of patients diagnosed with sarcoma develop metastatic disease with so far very limited treatment options. The transmembrane protein B7-H3 reportedly is expressed in various malignancies, including different sarcoma subtypes. In several cancer entities B7-H3 expression is associated with poor prognosis. In turn, B7-H3 is considered a promising target for immunotherapeutic approaches. We here report on the preclinical characterization of a B7-H3xCD3 bispecific antibody in an IgG-based format, termed CC-3, for treatment of different sarcoma subtypes. We found B7-H3 to be expressed on all sarcoma cells tested and expression on sarcoma patients correlated with decreased progression-free and overall survival. CC-3 was found to elicit robust T cell responses against multiple sarcoma subtypes, resulting in significant activation, release of cytokines and effector molecules. In addition, CC-3 promoted T cell proliferation and differentiation, resulting in the generation of memory T cell subsets. Finally, CC-3 induced potent target cell lysis in a target cell restricted manner. Based on these results, a clinical trial evaluating CC-3 in soft tissue sarcoma is currently in preparation.

[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.

Application of blinatumomab, a bispecific anti-CD3/CD19 T-cell engager, in treating severe systemic sclerosis: A case study.

Systemic sclerosis, a severe inflammatory autoimmune disease, shares a common thread with cancer through the underlying mechanism of inflammation. This inflammatory milieu not only drives the immune dysregulation characteristic of autoimmune diseases but also plays a pivotal role in the pathogenesis of cancer. Among the cellular components involved, B cells have emerged as key players in hematologic tumor and autoimmune disease, contributing to immune dysregulation and persistent tissue fibrosis in systemic sclerosis, as well as tumor progression and immune evasion in cancer. Consequently, novel therapeutic strategies targeting B cells hold promise in both conditions. Recent exploration of CD19 CAR T cells in severe systemic sclerosis patients has shown great potential, but also introduced possible risks and drawbacks associated with viral vectors, prolonged CAR T cell persistence, lengthy production timelines, high costs, and the necessity of conditioning patients with organotoxic and fertility-damaging chemotherapy. Given these challenges, alternative CD19-depleting approaches are of high interest for managing severe systemic autoimmune diseases. Here, we present the pioneering use of blinatumomab, a bispecific anti-CD3/anti-CD19 T cell engager in a patient with progressive, severe systemic sclerosis, offering a promising alternative for such challenging cases.

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.

[T-cell recruiting immunotherapies in B-cell lymphoma - the future backbone for all therapy lines?] / T-Zell-rekrutierende Immuntherapien des B-Zell-Lymphoms ­ bald in allen Therapielinien?

The introduction of immunologically targeted therapies has represented a significant advancement in the treatment of B-cell lymphomas, particularly aggressive B-cell lymphoma. CD19 CAR-T cells such as Axicabtagen-Ciloleucel (Axi-cel) and Lisocabtagen Maraleucel (Liso-cel) have been approved since 2022 and 2023, respectively, for second-line therapy of Diffuse Large B-Cell Lymphomas (DLBCL), when there is primary refractory disease or relapse within 12 months after the end of first-line therapy. These therapies result in a significant improvement in progression-free survival compared to the previous standard therapy (salvage chemotherapy followed by high-dose chemotherapy and autologous stem cell transplantation). Especially in elderly patients or patients with underlying medical conditions, CAR-T cell therapies like Axi-cel and Liso-cel demonstrate acceptable tolerability and high efficacy.Furthermore, bispecific T-cell-engaging antibodies ("bispecifics") such as Glofitamab, Epcoritamab, and Mosunetuzumab also represent promising treatment options for patients with relapsed disease after failure of second- or later line therapy and show efficacy even in a subset of patients relapsing after CD19 CAR-T cells. However, randomized study results for these substances are not yet available. They are expected to be used in earlier lines of therapy in the future, especially in combination with standard chemotherapy regimens. Common side effects of bispecific antibody therapies are cytokine release syndrome (CRS) and immune-mediated cytopenias, whereas immune-cell associated neurotoxicity syndrome (ICANS) is relatively rare compared to CD19 CAR T cells. In summary, bispecifics represent a novel, highly effective immunotherapy for the treatment of lymphomas with a very favourable toxicity profile.

CNS Antitumor Activity of Amivantamab With Osimertinib in Epidermal Growth Factor Receptor-Mutated Non-Small Cell Lung Cancer With Acquired Mesenchymal-Epithelial Transition Amplification Resistance Mechanism: A Case Report.

NSCLC w/EGFRex19del & MET amp: durable intracranial + systemic response to amivantamab/osimertinib.

Modular design of bi- and multi-specific knob domain fusions.

Introduction: The therapeutic potential of bispecific antibodies is becoming widely recognised, with over a hundred formats already described. For many applications, enhanced tissue penetration is sought, so bispecifics with low molecular weight may offer a route to enhanced potency. Here we report the design of bi- and tri-specific antibody-based constructs with molecular weights as low as 14.5 and 22 kDa respectively. Methods: Autonomous bovine ultra-long CDR H3 (knob domain peptide) modules have been engineered with artificial coiled-coil stalks derived from Sin Nombre orthohantavirus nucleocapsid protein and human Beclin-1, and joined in series to produce bi- and tri-specific antibody-based constructs with exceptionally low molecular weights. Results: Knob domain peptides with coiled-coil stalks retain high, independent antigen binding affinity, exhibit exceptional levels of thermal stability, and can be readily joined head-to-tail yielding the smallest described multi-specific antibody format. The resulting constructs are able to bind simultaneously to all their targets with no interference. Discussion: Compared to existing bispecific formats, the reduced molecular weight of the knob domain fusions may enable enhanced tissue penetration and facilitate binding to cryptic epitopes that are inaccessible to conventional antibodies. Furthermore, they can be easily produced at high yield as recombinant products and are free from the heavy-light chain mispairing issue. Taken together, our approach offers an efficient route to modular construction of minimalistic bi- and multi-specifics, thereby further broadening the therapeutic scope for knob domain peptides.

Advancement in the development of single chain antibodies using phage display technology.

Phage display technology has become an important research tool in biological research, fundamentally changing the traditional monoclonal antibody preparation process, and has been widely used in the establishment of antigen-antibody libraries, drug design, vaccine research, pathogen detection, gene therapy, antigenic epitope research, and cellular signal transduction research.The phage display is a powerful platform for technology development. Using phage display technology, single chain fragment variable (scFv) can be screened, replacing the disadvantage of the large size of traditional antibodies. Phage display single chain antibody libraries have significant biological implications. Here we describe the types of antibodies, including chimeric antibodies, bispecific antibodies, and scFvs. In addition, we describe the phage display system, phage display single chain antibody libraries, screening of specific antibodies by phage libraries and the application of phage libraries.

Combination of T cell-redirecting strategies with a bispecific antibody blocking TGF-ß and PD-L1 enhances antitumor responses.

T cell-based immunotherapies for solid tumors have not achieved the clinical success observed in hematological malignancies, partially due to the immunosuppressive effect promoted by the tumor microenvironment, where PD-L1 and TGF-ß play a pivotal role. However, durable responses to immune checkpoint inhibitors remain limited to a minority of patients, while TGF-ß inhibitors have not reached the market yet. Here, we describe a bispecific antibody for dual blockade of PD-L1 and TFG-ß, termed AxF (scFv)2, under the premise that combination with T cell redirecting strategies would improve clinical benefit. The AxF (scFv)2 antibody was well expressed in mammalian and yeast cells, bound both targets and inhibited dose-dependently the corresponding signaling pathways in luminescence-based cellular reporter systems. Moreover, combined treatment with trispecific T-cell engagers (TriTE) or CAR-T cells significantly boosted T cell activation status and cytotoxic response in breast, lung and colorectal (CRC) cancer models. Importantly, the combination of an EpCAMxCD3×EGFR TriTE with the AxF (scFv)2 delayed CRC tumor growth in vivo and significantly enhanced survival compared to monotherapy with the trispecific antibody. In summary, we demonstrated the feasibility of concomitant blockade of PD-L1 and TGF-ß by a single molecule, as well as its therapeutic potential in combination with different T cell redirecting agents to overcome tumor microenvironment-mediated immunosuppression.

Combinatorially restricted computational design of protein-protein interfaces to produce IgG heterodimers.

Redesigning protein-protein interfaces is an important tool for developing therapeutic strategies. Interfaces can be redesigned by in silico screening, which allows for efficient sampling of a large protein space before experimental validation. However, computational costs limit the number of combinations that can be reasonably sampled. Here, we present combinatorial tyrosine (Y)/serine (S) selection (combYSelect), a computational approach combining in silico determination of the change in binding free energy (ΔΔG) of an interface with a highly restricted library composed of just two amino acids, tyrosine and serine. We used combYSelect to design two immunoglobulin G (IgG) heterodimers-combYSelect1 (L368S/D399Y-K409S/T411Y) and combYSelect2 (D399Y/K447S-K409S/T411Y)-that exhibit near-optimal heterodimerization, without affecting IgG stability or function. We solved the crystal structures of these heterodimers and found that dynamic π-stacking interactions and polar contacts drive preferential heterodimeric interactions. Finally, we demonstrated the utility of our combYSelect heterodimers by engineering both a bispecific antibody and a cytokine trap for two unique therapeutic applications.

[Methodological recommendations for surgical care in patients with hemophilia A receiving prophylactic therapy with emicizumab]. / Metodicheskie rekomendatsii po okazaniyu khirurgicheskoi pomoshchi u patsientov s gemofiliei A, poluchayushchikh profilakticheskuyu terapiyu emitsizumabom.

Methodological recommendations for surgical care in patients with hemophilia A receiving prophylactic therapy with emicizumab. Recommendations of the expert group. Moscow, 2024.