As the leading cause of cancer-related deaths,lung cancer remains a noteworthy threat to human health.Although immunotherapies,such as immune checkpoint inhibitors(ICIs),have significantly increased the efficacy of lu...As the leading cause of cancer-related deaths,lung cancer remains a noteworthy threat to human health.Although immunotherapies,such as immune checkpoint inhibitors(ICIs),have significantly increased the efficacy of lung cancer treatment,a significant percentage of patients are not sensitive to immunotherapies and patients who initially respond to treatment can quickly develop acquired drug resistance.Bispecific antibodies(bs Abs)bind two different antigens or epitopes simultaneously and have been shown to enhance antitumor efficacy with suitable safety profiles,thus attracting increasing attention as novel antitumor therapies.At present,in addition to the approved bs Ab,amivantamab,three novel bs Abs(KN046,AK112,and SHR-1701)are being evaluated in phase 3 clinical trials and many bs Abs are being evaluated in phase 1/2 clinical trials for patients with non-small cell lung cancer(NSCLC).Herein we present the structure,classification,and mechanism of action underlying bs Abs in NSCLC and introduce related clinical trials.Finally,we discuss challenges,potential solutions,and future prospects in the context of cancer treatment with bsAbs.展开更多
Advances in antibody engineering have led to the generation of more innovative antibody drugs,such as bispecific antibodies(bs Abs).Following the success associated with blinatumomab,bs Abs have attracted enormous int...Advances in antibody engineering have led to the generation of more innovative antibody drugs,such as bispecific antibodies(bs Abs).Following the success associated with blinatumomab,bs Abs have attracted enormous interest in the field of cancer immunotherapy.By specifically targeting two different antigens,bs Abs reduce the distance between tumor and immune cells,thereby enhancing tumor killing directly.There are several mechanisms of action upon which bs Abs have been exploited.Accumulating experience on checkpoint-based therapy has promoted the clinical transformation of bs Abs targeting immunomodulatory checkpoints.Cadonilimab(PD-1×CTLA-4)is the first approved bs Ab targeting dual inhibitory checkpoints,which confirms the feasibility of bs Abs in immunotherapy.In this review we analyzed the mechanisms by which bs Abs targeting immunomodulatory checkpoints and their emerging applications in cancer immunotherapy.展开更多
Objective This study aimed to explore the value of M701,targeting epithelial cell adhesion molecule(EpCAM)and CD3,in the immunotherapy of ovarian cancer ascites by the in vitro assay.Methods The expression of EpCAM in...Objective This study aimed to explore the value of M701,targeting epithelial cell adhesion molecule(EpCAM)and CD3,in the immunotherapy of ovarian cancer ascites by the in vitro assay.Methods The expression of EpCAM in ovarian cancer tissues was analyzed by databases.The EpCAM expression and immune cell infiltration in different foci of ovarian cancer were detected by 8-channel flow cytometry.The toxic effect of M701 on OVCAR3 was tested using the in vitro cytotoxicity assay.The 3D cell culture and drug intervention experiments were performed to evaluate the therapeutic effect of M701 in ovarian cancer specimens.Flow cytometry was used to examine the effect of M701 on the binding of immune cells to tumor cells and the activation capacity of T cells.Results The results of the bioinformatic analysis showed that the expression of EpCAM in ovarian cancer tissue was significantly higher than that in normal ovarian tissue.The 8-channel flow cytometry of clinical samples showed that the EpCAM expression and lymphocyte infiltration were significantly heterogeneous among ovarian cancer patients and lesions at different sites.The in vitro experiment results showed that M701 had a significant killing effect on OVCAR3 cells.M701 also obviously killed primary tumor cells derived from some patients with ovarian cancer ascites.M701 could mediate the binding of CD3^(+)T cells to EpCAM^(+)tumor cells and induce T cell activation in a dose-dependent manner.Conclusion M701 showed significant inhibitory activity on tumor cells derived from ovarian cancer ascites,which had a promising application in immunotherapy for patients with ovarian cancer ascites.展开更多
Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody(BsAb).To choose an ideal format of anti-CD3 x anti-transferrin receptor(TfR)bispecific antibodies fo...Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody(BsAb).To choose an ideal format of anti-CD3 x anti-transferrin receptor(TfR)bispecific antibodies for clinical application,we constructed TfR bispecific T-cell engager(BiTE)in two extensively applied formats,including single-chain tandem singlechain variable fragments(scFvs)and double-chain diabodies,and evaluated their functional characterizations in vitro.Results demonstrated that TfR-BiTE in both formats directed potent killing of TfR+HepG2 cells.However,compared to two・chain diabodies,scFvs were more efficient in antigen binding and TfR target killing.Furthermore,different domain orders in scFvs would also be evaluated because single-TfR-CD3-His was preferable to single-CD3-TfR-His in immunotherapeutic strategies.Thus,the single-chain tandem TfR-CD3 format was favored for further investigation in cancer therapy.展开更多
Bispecific antibodies (BsAbs) of anti CD3×anti idiotype (Id) to B cell lymphocytic leukemia (CLL) were prepared by chemical conjugation and direct hybridization technique of hybridoma and hybridoma without scr...Bispecific antibodies (BsAbs) of anti CD3×anti idiotype (Id) to B cell lymphocytic leukemia (CLL) were prepared by chemical conjugation and direct hybridization technique of hybridoma and hybridoma without screening markers. The specificity of BsAbs from culture supernatants or ascites was assayed by indirect ELISA and indirect immunoflurescence (IF). The results showed that BsAbs could specifically react with homologous serum IgM from patients with B CLL and cells carrying CD3 marker respectively. Cell combination test and LDH assay demonstrated that BsAb significantly increased the conjugate formation between lymphocyte activated kill (LAK) cells and Daudi cells, and enhanced the cytotoxic activity of LAK cells against Daudi cells.展开更多
Bispecific antibodies(bsAbs)hold promises for enhanced therapeutic potential surpassing that of their parental monoclonal antibodies.However,bsAbs pose great challenges in their manufacturing,and one of the common rea...Bispecific antibodies(bsAbs)hold promises for enhanced therapeutic potential surpassing that of their parental monoclonal antibodies.However,bsAbs pose great challenges in their manufacturing,and one of the common reasons is their susceptibility to aggregation.Building on previous studies demonstrating the functionality and potential manufacturability of Fab-scFv format bsAb,this investigation delved into the impact of environmental factors-such as pH,buffer types,ionic strength,protein concentrations,and temperatures-on its stability and the reversal of its self-associated aggregates.Mildly acidic,low-salt conditions were found optimal,ensuring bsAb stability for 30 days even at elevated temperature of 40°C.Furthermore,these conditions facilitated the reversal of its self-associated aggregates to monomers during the initial 7-day incubation period.Our findings underscore the robustness and resilience of Fab-scFv format bsAb,further confirming its potential manufacturability despite its current absence as commercial products.展开更多
Bispecific antibodies hold significant potential as next-generation biotherapeutics owing to their ability to simultaneously bind to two targets.However,the development of bispecific antibodies as biotherapeutics has ...Bispecific antibodies hold significant potential as next-generation biotherapeutics owing to their ability to simultaneously bind to two targets.However,the development of bispecific antibodies as biotherapeutics has been hindered by the high levels of byproducts produced,including both high molecular weight and low molecular weight variants.In addition,the inevitable expression of homodimers in host cells presents further obstacles to the commercial development of bispecific antibodies as therapeutics.These byproducts,which share similar physicochemical properties with the target,pose several challenges for downstream purification processes.In this study,we present a non-protein A purification platform that employ a one-step polishing chromatography to purify bispecific antibodies.Mixed-mode Capto adhere resin was used to capture the target protein at pH 7.90±0.10,followed by anion exchange chromatography as a polishing step.Overall,the results of this two-step chromatography purification method demonstrated at final product purity of 98%as assessed by size-exclusion high-performance liquid chromatography(SEC-HPLC)and 98%by reversed-phase-high-performance liquid chromatography(RP-HPLC),with residual host cell proteins controlled at 10 ppm and an excellent recovery rate of approximately 60%.This study presents a non-protein A capture platform,offering a simplified,streamlined,and competitive alternative to conventional affinity chromatography.展开更多
Bispecific antibodies are recombinant proteins with novel immunological properties and therapeutic potential. Recombinant protein quality and activity of several bispecific antibodies comprising different variable dom...Bispecific antibodies are recombinant proteins with novel immunological properties and therapeutic potential. Recombinant protein quality and activity of several bispecific antibodies comprising different variable domain combinations with respect to the parental monospecific single chain fragments (scFv) were evaluated after expression in bacteria or mammalian cells. The parental scFv proteins humanized anti-NCAM scFv, murine anti-VEGFR-2 scFv, murine and humanized anti-CD3 scFv, respectively, could successfully be expressed in E. coli, whereas the murine anti-NCAM scFv version could not be reliably detected. Bispecific CD3 × VEGFR-2 and CD3 × NCAM anti-bodies were expressed in the bispecific single chain and the single chain diabody format. However, the diabody derived from the murine anti-NCAM scFv could not efficiently be expressed in E. coli or in mammalian cells. Significant binding of the CD3 × NCAM single chain diabody comprising the humanized version of anti-CD3 and humanized version of anti-NCAM was efficient to both antigens. Nevertheless, binding of the bispecific single chain version to the NCAM antigen was inefficient in comparison to CD3 binding. In conclusion, the data could indicate that the result of scFv expression in bacteria may be predictive for the chances of success for functional expression of more complex bispecific derivatives.展开更多
Bispecific killer cell engagers(BiKEs)are a powerful tool to incite the killing power of natural killer(NK)cells.Here,we posited that the BiKE technology could be utilized to deplete activated immune cells expressing ...Bispecific killer cell engagers(BiKEs)are a powerful tool to incite the killing power of natural killer(NK)cells.Here,we posited that the BiKE technology could be utilized to deplete activated immune cells expressing programmed death-1(PD-1^(+)cells),and hence treat autoimmune diseases since these cells drive the disorders.We designed and generated PD-1 BiKE that targets an activating NK cell receptor,CD16,and PD-1.PD-1 BiKE showed specific binding to PD-1^(+)cells and engaged CD16 simultaneously.PD-1 BiKE enhanced NK cell-mediated apoptosis and depletion of PD-1^(+)Raji cells,but not PD-1-Raji cells.Further,PD-1 BiKE induced apoptosis of primary PD-1^(+)T lymphocytes that are highly relevant to autoimmune disease progression.The BiKE depleted 42%of primary T cells that were stimulated in vitro.Importantly,those ablated primary T cells were activated cells.Meanwhile,naive T cells were spared by the BiKE treatment,supporting the crucial selectivity of PD-1 BiKE-directed cell depletion.Lastly,PD-1 BiKE is more effective than a conventional depleting antibody in the depletion of PD-1^(+)cells.The current work supports PD-1 BiKE is a selective,potent,and safe tool to deplete PD-1^(+)cells.展开更多
Lung cancer remains the leading cause of cancer-related deaths worldwide.Immune checkpoint inhibitors(ICIs)and bispecific antibodies(bsAbs)are transforming the treatment landscape,particularly in non-small cell lung c...Lung cancer remains the leading cause of cancer-related deaths worldwide.Immune checkpoint inhibitors(ICIs)and bispecific antibodies(bsAbs)are transforming the treatment landscape,particularly in non-small cell lung cancer(NSCLC).Perioperative immunotherapy trials,such as RATIONALE-315,have demonstrated significant improvements in pathological response rates and event-free survival for resectable NSCLC.Ivonescimab,a bsAb targeting PD-1 and VEGF,has shown promising efficacy in patients with EGFR mutations or PD-L1-positive tumors.The HARMONi-A trial highlighted improved progression-free survival(PFS)with ivonescimab compared to standard regimens,while HARMONi-2 revealed significant benefits in first-line PD-L1-positive NSCLC,including those with squamous histology.These advancements underscore the potential of bsAbs to overcome treatment resistance and improve outcomes.However,challenges remain,including optimizing treatment cycles,managing toxicity,and identifying predictive biomarkers.Future research should focus on refining therapeutic strategies,exploring combination therapies,and addressing unresolved questions regarding long-term efficacy and safety.Integrating these approaches has the potential to transform NSCLC management and improve patient survival globally.展开更多
To evaluate the efficacy and safety of KN026,a novel bispecific HER2(ECD2 and ECD4)antibody,plus KN046,a PD-L1,and CTLA4 bispecific antibody,in patients with advanced HER2-positive solid tumors.We conducted two sequen...To evaluate the efficacy and safety of KN026,a novel bispecific HER2(ECD2 and ECD4)antibody,plus KN046,a PD-L1,and CTLA4 bispecific antibody,in patients with advanced HER2-positive solid tumors.We conducted two sequentially designed phase Ib and Il studies with similar target populations and evaluation schedules.The primary endpoints included safety,maximum tolerated dose(MTD),the recommended phase Il dose(RP2D)for the phase Ib study,and the objective response rate(ORR)and duration of response(DoR)for the phase llstudy.Hereby,we solely report the results from 113 nonbreast cancer patients.In phase Ib,MTD was not reached.Dose 3 was confirmed to be acceptable for the phase lIl study.An objective response has been exclusively observed in HER2-positive patients.Any grade treatment-related adverse events(TRAEs)were reported in 108(95.6%)patients.The most common TRAEs were infusion reactions(38.9%),anemia(37.2%),elevated AST(31.0%),and diarrhea(30.1%).Among the 108 patients evaluated for efficacy,the overall ORR was 55.6%(95%Cl,45.7%,65.1%).In the HER2-positive GC subgroup,38 patients received this regimen as the 1st-line treatment and 30 patients achieved an objective response,with an ORR of 78.9%(95%Cl,62.7%,90.4%).Among 27 pretreated patients,the ORR was 44.4%(95%Cl,25.5%,64.7%).In the other HER2-positive solid tumor subgroup(n=34),the ORR was 52.9%(95%CI 35.1%,70.2%).Thus,KN026 plus KN04 exhibits promising efficacy and acceptable safety profiles in HER2-positive nonbreast cancer,as does the 1st-line treatment for GC.展开更多
Adoptive immune cell-based therapies have shown promise in cancer treatment,yet their efficacy against solid tumors is often limited by the immunosuppressive tumor microenvironment(TME).To overcome these barriers,we d...Adoptive immune cell-based therapies have shown promise in cancer treatment,yet their efficacy against solid tumors is often limited by the immunosuppressive tumor microenvironment(TME).To overcome these barriers,we design an innovative immune cell cocktail as a combinatorial biomaterial platform,which harnessing the complementary functions of neutrophils and natural killer(NK)cells derived from human pluripotent stem cells(hPSCs).Using CRISPR/Cas9,we introduce an anti-fluorescein isothiocyanate(FITC)chimeric antigen receptor(CAR)construct into the AAVS1 safe harbor locus of hPSCs,allowing for the differentiation of CAR-modified neutrophils and NK cells.These CAR neutrophils exhibit robust anti-tumor activity,forming immune synapses with tumor cells tagged via a bispecific adapter(FITC-folate),even in hypoxic TMEs,while CAR NK cells demonstrate antigen-specific cytotoxicity.Together,the cocktail biomaterial composed of CAR neutrophils and CAR NK cells creates a synergistic anti-tumor effect:having neutrophils enhance TME modulation,and NK cells provide targeted cytotoxicity.This biomaterial offers a scalable and off-the-shelf solution for producing CAR neutrophils and CAR NK cells,potentially reducing needs for high-dose exogenous cytokines and minimizing immune-related toxicities.Our findings suggest that hPSC-derived CAR neutrophils and CAR NK cells may form an effective immuno-cocktail biomaterial,offering a feasible strategy for advancing solid tumor immunotherapy through cellular synergy and TME adaptation.展开更多
Dear Editor,Chimeric antigen receptor T(CAR-T)cells have achieved substantial advances in the treatment of B-cell malignancies.Despite high initial response rates,some patients relapse,characterized by targeted antige...Dear Editor,Chimeric antigen receptor T(CAR-T)cells have achieved substantial advances in the treatment of B-cell malignancies.Despite high initial response rates,some patients relapse,characterized by targeted antigen loss(Shalabi et al.,2018).展开更多
To the Editor:Immunotherapy has become an important approach to combat hematological malignancies such as lymphoma.Bispecific antibodies can redirect immune cells to fight against tumors,utilizing either adaptive or i...To the Editor:Immunotherapy has become an important approach to combat hematological malignancies such as lymphoma.Bispecific antibodies can redirect immune cells to fight against tumors,utilizing either adaptive or innate immune systems.AFM13,a tetravalent bispecific antibody against CD30/CD16a,is dependent on natural killer(NK)cells to treat CD30+lymphoma1.展开更多
Bispecific antibody‒drug conjugates(BsADCs)represent an innovative therapeutic category amalgamating the merits of antibody‒drug conjugates(ADCs)and bispecific antibodies(BsAbs).Positioned as the next-generation ADC a...Bispecific antibody‒drug conjugates(BsADCs)represent an innovative therapeutic category amalgamating the merits of antibody‒drug conjugates(ADCs)and bispecific antibodies(BsAbs).Positioned as the next-generation ADC approach,BsADCs hold promise for ameliorating extant clinical challenges associated with ADCs,particularly pertaining to issues such as poor internalization,off-target toxicity,and drug resistance.Presently,ten BsADCs are undergoing clinical trials,and initial findings underscore the imperative for ongoing refinement.This review initially delves into specific design considerations for BsADCs,encompassing target selection,antibody formats,and the linker–payload complex.Subsequent sections delineate the extant progress and challenges encountered by BsADCs,illustrated through pertinent case studies.The amalgamation of BsAbs with ADCs offers a prospective solution to prevailing clinical limitations of ADCs.Nevertheless,the symbiotic interplay among BsAb,linker,and payload necessitates further optimizations and coordination beyond a simplistic“1+1”to effectively surmount the extant challenges facing the BsADC domain.展开更多
Antibody–drug conjugates(ADCs)are biologically targeted drugs composed of antibodies and cytotoxic drugs connected by linkers.These innovative compounds enable precise drug delivery to tumor cells,minimizing harm to ...Antibody–drug conjugates(ADCs)are biologically targeted drugs composed of antibodies and cytotoxic drugs connected by linkers.These innovative compounds enable precise drug delivery to tumor cells,minimizing harm to normal tissues and offering excellent prospects for cancer treatment.However,monoclonal antibody-based ADCs still present challenges,especially in terms of balancing efficacy and safety.Bispecific antibodies are alternatives to monoclonal antibodies and exhibit superior internalization and selectivity,producing ADCs with increased safety and therapeutic efficacy.In this review,we present available evidence and future prospects regarding the use of bispecific ADCs for cancer treatment,including a comprehensive overview of bispecific ADCs that are currently in clinical trials.We offer insights into the future development of bispecific ADCs to provide novel strategies for cancer treatment.展开更多
Bispecific antibodies(bsAbs)refer to a large family of molecules that recognize two different epitopes or antigens.Although a series of challenges,especially immunogenicity and chain mispairing issues,once hindered th...Bispecific antibodies(bsAbs)refer to a large family of molecules that recognize two different epitopes or antigens.Although a series of challenges,especially immunogenicity and chain mispairing issues,once hindered the development of bsAbs,they have been gradually overcome with the help of rapidly developing technologies in the past 5 decades.In the meantime,an increasing number of bsAb platforms have been designed to satisfy different clinical demands.Currently,numerous preclinical and clinical trials are underway,portraying a promising future for bsAb-based cancer treatment.Nevertheless,bsAb drugs still face enormous challenges in their application as cancer therapeutics,including tumor heterogeneity and mutational burden,intractable tumor microenvironment(TME),insufficient costimulatory signals to activate T cells,the necessity for continuous injection,fatal systemic side effects,and off-target toxicities to adjacent normal cells.Therefore,we provide several strategies as solutions to these issues,which comprise generating multispecific bsAbs,discovering neoantigens,combining bsAbs with other anticancer therapies,exploiting natural killer(NK)-cell-based bsAbs and producing bsAbs in situ.In this review,we mainly discuss previous and current challenges in bsAb development and underscore corresponding strategies,with a brief introduction of several typical bsAb formats.展开更多
Bispecific antibody (BsAb) usually consists of two different antigen-binding arms, by which it is capable of simultaneously binding to target cells and effector cells, and can directly mediate the killing of target ce...Bispecific antibody (BsAb) usually consists of two different antigen-binding arms, by which it is capable of simultaneously binding to target cells and effector cells, and can directly mediate the killing of target cells by retargeting and activating effector cells. The development of BsAb research goes through three main stages: chemical cross-linking of murine-derived monoclonal antibody, hybrid hy-bridomas and engineered BsAb. Among them, engineered BsAb has more formats than the other two, such as diabody, ScdHLX, ScZip, ScCH3, ScFab and BsIgG, etc. Compared with former murine-derived BsAbs, engineered BsAb has lower immunogenicity and stronger penetrating capacity, and currently, some of them appear suitable for clinical application in yields and qualities. Up to now, several phase I and phase II clinical studies of BsAb, for instance, some (Fab’)2 and Diabodies, have been performed. Among those BsAbs, anti-CD3/anti-tumor BsAbs is most common, they not only can activate T cell and induce CD3AK展开更多
In recent years,the development of bispecific antibodies(bsAbs)has been rapid,with many new structures and target combinations being created.The boom in bsAbs has led to the successive issuance of industry guidance fo...In recent years,the development of bispecific antibodies(bsAbs)has been rapid,with many new structures and target combinations being created.The boom in bsAbs has led to the successive issuance of industry guidance for their development in the US and China.However,there is a high degree of similarity in target selection,which could affect the development of diversity in bsAbs.This review presents a classification of various bsAbs for cancer therapy based on structure and target selection and examines the advantages of bsAbs over monoclonal antibodies(mAbs).Through database research,we have identified the preferences of available bsAbs combinations,suggesting rational target selection options and warning of potential wastage of medical resources.We have also compared the US and Chinese guidelines for bsAbs in order to provide a reference for their development.展开更多
基金supported by the National Natural Science Foundation of China(Grant No.82272845)the Natural Science Foundation of Shandong(Grant No.ZR2023LZL001)。
文摘As the leading cause of cancer-related deaths,lung cancer remains a noteworthy threat to human health.Although immunotherapies,such as immune checkpoint inhibitors(ICIs),have significantly increased the efficacy of lung cancer treatment,a significant percentage of patients are not sensitive to immunotherapies and patients who initially respond to treatment can quickly develop acquired drug resistance.Bispecific antibodies(bs Abs)bind two different antigens or epitopes simultaneously and have been shown to enhance antitumor efficacy with suitable safety profiles,thus attracting increasing attention as novel antitumor therapies.At present,in addition to the approved bs Ab,amivantamab,three novel bs Abs(KN046,AK112,and SHR-1701)are being evaluated in phase 3 clinical trials and many bs Abs are being evaluated in phase 1/2 clinical trials for patients with non-small cell lung cancer(NSCLC).Herein we present the structure,classification,and mechanism of action underlying bs Abs in NSCLC and introduce related clinical trials.Finally,we discuss challenges,potential solutions,and future prospects in the context of cancer treatment with bsAbs.
基金supported by the National Natural Science Foundation of China(Grant Nos.82130077 and 81961128025)。
文摘Advances in antibody engineering have led to the generation of more innovative antibody drugs,such as bispecific antibodies(bs Abs).Following the success associated with blinatumomab,bs Abs have attracted enormous interest in the field of cancer immunotherapy.By specifically targeting two different antigens,bs Abs reduce the distance between tumor and immune cells,thereby enhancing tumor killing directly.There are several mechanisms of action upon which bs Abs have been exploited.Accumulating experience on checkpoint-based therapy has promoted the clinical transformation of bs Abs targeting immunomodulatory checkpoints.Cadonilimab(PD-1×CTLA-4)is the first approved bs Ab targeting dual inhibitory checkpoints,which confirms the feasibility of bs Abs in immunotherapy.In this review we analyzed the mechanisms by which bs Abs targeting immunomodulatory checkpoints and their emerging applications in cancer immunotherapy.
基金This work was supported by the National Key Research&Development Program of China(No.2021YFC2701402).
文摘Objective This study aimed to explore the value of M701,targeting epithelial cell adhesion molecule(EpCAM)and CD3,in the immunotherapy of ovarian cancer ascites by the in vitro assay.Methods The expression of EpCAM in ovarian cancer tissues was analyzed by databases.The EpCAM expression and immune cell infiltration in different foci of ovarian cancer were detected by 8-channel flow cytometry.The toxic effect of M701 on OVCAR3 was tested using the in vitro cytotoxicity assay.The 3D cell culture and drug intervention experiments were performed to evaluate the therapeutic effect of M701 in ovarian cancer specimens.Flow cytometry was used to examine the effect of M701 on the binding of immune cells to tumor cells and the activation capacity of T cells.Results The results of the bioinformatic analysis showed that the expression of EpCAM in ovarian cancer tissue was significantly higher than that in normal ovarian tissue.The 8-channel flow cytometry of clinical samples showed that the EpCAM expression and lymphocyte infiltration were significantly heterogeneous among ovarian cancer patients and lesions at different sites.The in vitro experiment results showed that M701 had a significant killing effect on OVCAR3 cells.M701 also obviously killed primary tumor cells derived from some patients with ovarian cancer ascites.M701 could mediate the binding of CD3^(+)T cells to EpCAM^(+)tumor cells and induce T cell activation in a dose-dependent manner.Conclusion M701 showed significant inhibitory activity on tumor cells derived from ovarian cancer ascites,which had a promising application in immunotherapy for patients with ovarian cancer ascites.
基金National Natural Science Foundation of China(No.31570937 and No.81871391)Natural Science Foundation of Hubei Province of China(No.2017CFB707)+1 种基金the Fundamental Research Funds for the Central Universities of China(No.HUST:2018KFYYXJJ086)Graduates'Innovation Foundation of Huazhong University of Science and Technology(No.5003510001).
文摘Selecting an ideal molecular format from diverse structures is a major challenge in developing a bispecific antibody(BsAb).To choose an ideal format of anti-CD3 x anti-transferrin receptor(TfR)bispecific antibodies for clinical application,we constructed TfR bispecific T-cell engager(BiTE)in two extensively applied formats,including single-chain tandem singlechain variable fragments(scFvs)and double-chain diabodies,and evaluated their functional characterizations in vitro.Results demonstrated that TfR-BiTE in both formats directed potent killing of TfR+HepG2 cells.However,compared to two・chain diabodies,scFvs were more efficient in antigen binding and TfR target killing.Furthermore,different domain orders in scFvs would also be evaluated because single-TfR-CD3-His was preferable to single-CD3-TfR-His in immunotherapeutic strategies.Thus,the single-chain tandem TfR-CD3 format was favored for further investigation in cancer therapy.
文摘Bispecific antibodies (BsAbs) of anti CD3×anti idiotype (Id) to B cell lymphocytic leukemia (CLL) were prepared by chemical conjugation and direct hybridization technique of hybridoma and hybridoma without screening markers. The specificity of BsAbs from culture supernatants or ascites was assayed by indirect ELISA and indirect immunoflurescence (IF). The results showed that BsAbs could specifically react with homologous serum IgM from patients with B CLL and cells carrying CD3 marker respectively. Cell combination test and LDH assay demonstrated that BsAb significantly increased the conjugate formation between lymphocyte activated kill (LAK) cells and Daudi cells, and enhanced the cytotoxic activity of LAK cells against Daudi cells.
基金supported by Agency for Science,Technology,and Research(A*STAR)BMRC Central Research Fund.
文摘Bispecific antibodies(bsAbs)hold promises for enhanced therapeutic potential surpassing that of their parental monoclonal antibodies.However,bsAbs pose great challenges in their manufacturing,and one of the common reasons is their susceptibility to aggregation.Building on previous studies demonstrating the functionality and potential manufacturability of Fab-scFv format bsAb,this investigation delved into the impact of environmental factors-such as pH,buffer types,ionic strength,protein concentrations,and temperatures-on its stability and the reversal of its self-associated aggregates.Mildly acidic,low-salt conditions were found optimal,ensuring bsAb stability for 30 days even at elevated temperature of 40°C.Furthermore,these conditions facilitated the reversal of its self-associated aggregates to monomers during the initial 7-day incubation period.Our findings underscore the robustness and resilience of Fab-scFv format bsAb,further confirming its potential manufacturability despite its current absence as commercial products.
文摘Bispecific antibodies hold significant potential as next-generation biotherapeutics owing to their ability to simultaneously bind to two targets.However,the development of bispecific antibodies as biotherapeutics has been hindered by the high levels of byproducts produced,including both high molecular weight and low molecular weight variants.In addition,the inevitable expression of homodimers in host cells presents further obstacles to the commercial development of bispecific antibodies as therapeutics.These byproducts,which share similar physicochemical properties with the target,pose several challenges for downstream purification processes.In this study,we present a non-protein A purification platform that employ a one-step polishing chromatography to purify bispecific antibodies.Mixed-mode Capto adhere resin was used to capture the target protein at pH 7.90±0.10,followed by anion exchange chromatography as a polishing step.Overall,the results of this two-step chromatography purification method demonstrated at final product purity of 98%as assessed by size-exclusion high-performance liquid chromatography(SEC-HPLC)and 98%by reversed-phase-high-performance liquid chromatography(RP-HPLC),with residual host cell proteins controlled at 10 ppm and an excellent recovery rate of approximately 60%.This study presents a non-protein A capture platform,offering a simplified,streamlined,and competitive alternative to conventional affinity chromatography.
基金financial support of AK by a grant of the Clotten-Stiftung,Freiburg,GermanyPPM was supported by the Deutsche Forschungsgemeinschaft DFG grant SFB599.
文摘Bispecific antibodies are recombinant proteins with novel immunological properties and therapeutic potential. Recombinant protein quality and activity of several bispecific antibodies comprising different variable domain combinations with respect to the parental monospecific single chain fragments (scFv) were evaluated after expression in bacteria or mammalian cells. The parental scFv proteins humanized anti-NCAM scFv, murine anti-VEGFR-2 scFv, murine and humanized anti-CD3 scFv, respectively, could successfully be expressed in E. coli, whereas the murine anti-NCAM scFv version could not be reliably detected. Bispecific CD3 × VEGFR-2 and CD3 × NCAM anti-bodies were expressed in the bispecific single chain and the single chain diabody format. However, the diabody derived from the murine anti-NCAM scFv could not efficiently be expressed in E. coli or in mammalian cells. Significant binding of the CD3 × NCAM single chain diabody comprising the humanized version of anti-CD3 and humanized version of anti-NCAM was efficient to both antigens. Nevertheless, binding of the bispecific single chain version to the NCAM antigen was inefficient in comparison to CD3 binding. In conclusion, the data could indicate that the result of scFv expression in bacteria may be predictive for the chances of success for functional expression of more complex bispecific derivatives.
基金supported by the HCI Melanoma Center Grant,the National Institutes of Health AI139535 grant,and the National Multiple Sclerosis Society GR-1807-31630 grant to Mingnan Chen(USA)supported by the HCI Melanoma Center Grant,the National Institutes of Health AI139535 grant,and the National Multiple Sclerosis Society GR-1807-31630 grant to Mingnan Chen(USA).
文摘Bispecific killer cell engagers(BiKEs)are a powerful tool to incite the killing power of natural killer(NK)cells.Here,we posited that the BiKE technology could be utilized to deplete activated immune cells expressing programmed death-1(PD-1^(+)cells),and hence treat autoimmune diseases since these cells drive the disorders.We designed and generated PD-1 BiKE that targets an activating NK cell receptor,CD16,and PD-1.PD-1 BiKE showed specific binding to PD-1^(+)cells and engaged CD16 simultaneously.PD-1 BiKE enhanced NK cell-mediated apoptosis and depletion of PD-1^(+)Raji cells,but not PD-1-Raji cells.Further,PD-1 BiKE induced apoptosis of primary PD-1^(+)T lymphocytes that are highly relevant to autoimmune disease progression.The BiKE depleted 42%of primary T cells that were stimulated in vitro.Importantly,those ablated primary T cells were activated cells.Meanwhile,naive T cells were spared by the BiKE treatment,supporting the crucial selectivity of PD-1 BiKE-directed cell depletion.Lastly,PD-1 BiKE is more effective than a conventional depleting antibody in the depletion of PD-1^(+)cells.The current work supports PD-1 BiKE is a selective,potent,and safe tool to deplete PD-1^(+)cells.
文摘Lung cancer remains the leading cause of cancer-related deaths worldwide.Immune checkpoint inhibitors(ICIs)and bispecific antibodies(bsAbs)are transforming the treatment landscape,particularly in non-small cell lung cancer(NSCLC).Perioperative immunotherapy trials,such as RATIONALE-315,have demonstrated significant improvements in pathological response rates and event-free survival for resectable NSCLC.Ivonescimab,a bsAb targeting PD-1 and VEGF,has shown promising efficacy in patients with EGFR mutations or PD-L1-positive tumors.The HARMONi-A trial highlighted improved progression-free survival(PFS)with ivonescimab compared to standard regimens,while HARMONi-2 revealed significant benefits in first-line PD-L1-positive NSCLC,including those with squamous histology.These advancements underscore the potential of bsAbs to overcome treatment resistance and improve outcomes.However,challenges remain,including optimizing treatment cycles,managing toxicity,and identifying predictive biomarkers.Future research should focus on refining therapeutic strategies,exploring combination therapies,and addressing unresolved questions regarding long-term efficacy and safety.Integrating these approaches has the potential to transform NSCLC management and improve patient survival globally.
基金supported by the National Natural Science Foundation of China(91959205 to L.S.)the Natural Science Foundation of Beijing Municipality(Z200015 to X.T.Z).
文摘To evaluate the efficacy and safety of KN026,a novel bispecific HER2(ECD2 and ECD4)antibody,plus KN046,a PD-L1,and CTLA4 bispecific antibody,in patients with advanced HER2-positive solid tumors.We conducted two sequentially designed phase Ib and Il studies with similar target populations and evaluation schedules.The primary endpoints included safety,maximum tolerated dose(MTD),the recommended phase Il dose(RP2D)for the phase Ib study,and the objective response rate(ORR)and duration of response(DoR)for the phase llstudy.Hereby,we solely report the results from 113 nonbreast cancer patients.In phase Ib,MTD was not reached.Dose 3 was confirmed to be acceptable for the phase lIl study.An objective response has been exclusively observed in HER2-positive patients.Any grade treatment-related adverse events(TRAEs)were reported in 108(95.6%)patients.The most common TRAEs were infusion reactions(38.9%),anemia(37.2%),elevated AST(31.0%),and diarrhea(30.1%).Among the 108 patients evaluated for efficacy,the overall ORR was 55.6%(95%Cl,45.7%,65.1%).In the HER2-positive GC subgroup,38 patients received this regimen as the 1st-line treatment and 30 patients achieved an objective response,with an ORR of 78.9%(95%Cl,62.7%,90.4%).Among 27 pretreated patients,the ORR was 44.4%(95%Cl,25.5%,64.7%).In the other HER2-positive solid tumor subgroup(n=34),the ORR was 52.9%(95%CI 35.1%,70.2%).Thus,KN026 plus KN04 exhibits promising efficacy and acceptable safety profiles in HER2-positive nonbreast cancer,as does the 1st-line treatment for GC.
基金supported by start-up package funding from The Hong Kong Polytechnic University(Y.C.)and the Shenzhen Science and Technology Program(No.JCYJ20241202130520027).
文摘Adoptive immune cell-based therapies have shown promise in cancer treatment,yet their efficacy against solid tumors is often limited by the immunosuppressive tumor microenvironment(TME).To overcome these barriers,we design an innovative immune cell cocktail as a combinatorial biomaterial platform,which harnessing the complementary functions of neutrophils and natural killer(NK)cells derived from human pluripotent stem cells(hPSCs).Using CRISPR/Cas9,we introduce an anti-fluorescein isothiocyanate(FITC)chimeric antigen receptor(CAR)construct into the AAVS1 safe harbor locus of hPSCs,allowing for the differentiation of CAR-modified neutrophils and NK cells.These CAR neutrophils exhibit robust anti-tumor activity,forming immune synapses with tumor cells tagged via a bispecific adapter(FITC-folate),even in hypoxic TMEs,while CAR NK cells demonstrate antigen-specific cytotoxicity.Together,the cocktail biomaterial composed of CAR neutrophils and CAR NK cells creates a synergistic anti-tumor effect:having neutrophils enhance TME modulation,and NK cells provide targeted cytotoxicity.This biomaterial offers a scalable and off-the-shelf solution for producing CAR neutrophils and CAR NK cells,potentially reducing needs for high-dose exogenous cytokines and minimizing immune-related toxicities.Our findings suggest that hPSC-derived CAR neutrophils and CAR NK cells may form an effective immuno-cocktail biomaterial,offering a feasible strategy for advancing solid tumor immunotherapy through cellular synergy and TME adaptation.
基金supported by the National Key R&D Program of China(2019YFA0906100 and 2019YFA0904200 to Y.J.C.)the Shenzhen Science and Technology Innovation Program(JCYJ20180504165501371 to Y.J.C.)+2 种基金the National Natural Science Foundation of China(32171464 and 81872783 to Y.J.C.),the Prevention and Control of COVID-2019 Research Program in University of Guangdong Province(2020KZDZX1176 to S.Z.)the Shenzhen Basic Research Program(JCYJ20180507182203049 to S.Z.)the Shenzhen Key Laboratory Foundation(ZDSYS20200811143757022 to S.Z.).
文摘Dear Editor,Chimeric antigen receptor T(CAR-T)cells have achieved substantial advances in the treatment of B-cell malignancies.Despite high initial response rates,some patients relapse,characterized by targeted antigen loss(Shalabi et al.,2018).
文摘To the Editor:Immunotherapy has become an important approach to combat hematological malignancies such as lymphoma.Bispecific antibodies can redirect immune cells to fight against tumors,utilizing either adaptive or innate immune systems.AFM13,a tetravalent bispecific antibody against CD30/CD16a,is dependent on natural killer(NK)cells to treat CD30+lymphoma1.
基金This review was supported by the National Natural Science Foundation of China(82073318)Sichuan Science and Technology Program(2019YFS0003,China)the Support Program of Science&Technology Department of Sichuan Provincial(2023YFSY0046 and 2022NSFSC1365,China).
文摘Bispecific antibody‒drug conjugates(BsADCs)represent an innovative therapeutic category amalgamating the merits of antibody‒drug conjugates(ADCs)and bispecific antibodies(BsAbs).Positioned as the next-generation ADC approach,BsADCs hold promise for ameliorating extant clinical challenges associated with ADCs,particularly pertaining to issues such as poor internalization,off-target toxicity,and drug resistance.Presently,ten BsADCs are undergoing clinical trials,and initial findings underscore the imperative for ongoing refinement.This review initially delves into specific design considerations for BsADCs,encompassing target selection,antibody formats,and the linker–payload complex.Subsequent sections delineate the extant progress and challenges encountered by BsADCs,illustrated through pertinent case studies.The amalgamation of BsAbs with ADCs offers a prospective solution to prevailing clinical limitations of ADCs.Nevertheless,the symbiotic interplay among BsAb,linker,and payload necessitates further optimizations and coordination beyond a simplistic“1+1”to effectively surmount the extant challenges facing the BsADC domain.
基金supported by the National Natural Science Foundation of China(Nos.32070940 and 81991491)China Postdoctoral Science Foundation(No.2021M700115)+2 种基金Postdoctoral Innovation Talents Support Program(No.BX20220189)CAMS Innovation Fund for Medical Sciences(No.2019RU022)Fundamental Research Funds for the Central Universities(No.20720220006).
文摘Antibody–drug conjugates(ADCs)are biologically targeted drugs composed of antibodies and cytotoxic drugs connected by linkers.These innovative compounds enable precise drug delivery to tumor cells,minimizing harm to normal tissues and offering excellent prospects for cancer treatment.However,monoclonal antibody-based ADCs still present challenges,especially in terms of balancing efficacy and safety.Bispecific antibodies are alternatives to monoclonal antibodies and exhibit superior internalization and selectivity,producing ADCs with increased safety and therapeutic efficacy.In this review,we present available evidence and future prospects regarding the use of bispecific ADCs for cancer treatment,including a comprehensive overview of bispecific ADCs that are currently in clinical trials.We offer insights into the future development of bispecific ADCs to provide novel strategies for cancer treatment.
文摘Bispecific antibodies(bsAbs)refer to a large family of molecules that recognize two different epitopes or antigens.Although a series of challenges,especially immunogenicity and chain mispairing issues,once hindered the development of bsAbs,they have been gradually overcome with the help of rapidly developing technologies in the past 5 decades.In the meantime,an increasing number of bsAb platforms have been designed to satisfy different clinical demands.Currently,numerous preclinical and clinical trials are underway,portraying a promising future for bsAb-based cancer treatment.Nevertheless,bsAb drugs still face enormous challenges in their application as cancer therapeutics,including tumor heterogeneity and mutational burden,intractable tumor microenvironment(TME),insufficient costimulatory signals to activate T cells,the necessity for continuous injection,fatal systemic side effects,and off-target toxicities to adjacent normal cells.Therefore,we provide several strategies as solutions to these issues,which comprise generating multispecific bsAbs,discovering neoantigens,combining bsAbs with other anticancer therapies,exploiting natural killer(NK)-cell-based bsAbs and producing bsAbs in situ.In this review,we mainly discuss previous and current challenges in bsAb development and underscore corresponding strategies,with a brief introduction of several typical bsAb formats.
基金the State "863" High-Tech Program (Grant No. 102-09-03-03) by the Natural Science Foundation of Tianjin (GrantNo. 993803811).
文摘Bispecific antibody (BsAb) usually consists of two different antigen-binding arms, by which it is capable of simultaneously binding to target cells and effector cells, and can directly mediate the killing of target cells by retargeting and activating effector cells. The development of BsAb research goes through three main stages: chemical cross-linking of murine-derived monoclonal antibody, hybrid hy-bridomas and engineered BsAb. Among them, engineered BsAb has more formats than the other two, such as diabody, ScdHLX, ScZip, ScCH3, ScFab and BsIgG, etc. Compared with former murine-derived BsAbs, engineered BsAb has lower immunogenicity and stronger penetrating capacity, and currently, some of them appear suitable for clinical application in yields and qualities. Up to now, several phase I and phase II clinical studies of BsAb, for instance, some (Fab’)2 and Diabodies, have been performed. Among those BsAbs, anti-CD3/anti-tumor BsAbs is most common, they not only can activate T cell and induce CD3AK
基金supported by the National Key Research and Development Program of China(2022YFA1303803)National Natural Science Foundation of China(82073701)+1 种基金the Project Program of State Key Laboratory of Natural Medicines,China Pharmaceutical University(SKLNMZZ202209)supported by“Double First-Class”University Project(CPU2022PZQ07,China)。
文摘In recent years,the development of bispecific antibodies(bsAbs)has been rapid,with many new structures and target combinations being created.The boom in bsAbs has led to the successive issuance of industry guidance for their development in the US and China.However,there is a high degree of similarity in target selection,which could affect the development of diversity in bsAbs.This review presents a classification of various bsAbs for cancer therapy based on structure and target selection and examines the advantages of bsAbs over monoclonal antibodies(mAbs).Through database research,we have identified the preferences of available bsAbs combinations,suggesting rational target selection options and warning of potential wastage of medical resources.We have also compared the US and Chinese guidelines for bsAbs in order to provide a reference for their development.
