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
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
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 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(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 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 T-cell Engagers(BITEs)are a novel form of immunotherapy that overcome a deficiency of immune checkpoint inhibitors(ICI)by targeting a preidentified tumor associated antigen and redirecting a polyclonal popu...Bispecific T-cell Engagers(BITEs)are a novel form of immunotherapy that overcome a deficiency of immune checkpoint inhibitors(ICI)by targeting a preidentified tumor associated antigen and redirecting a polyclonal population of effector T-cells against the tumor.High grade serous ovarian cancer is a lethal disease in the recurrent setting and has not been amenable to ICI therapy.MUC16/CA125 is overexpressed in high grade serous ovarian cancer.BITEs targeting the tumor-retained portion of MUC16/CA125 have recently been described and are in early-phase clinical trials.To identify mechanisms of resistance to BITEs,we collected serum,peripheral blood mononuclear cells,and ascites samples from patients with disease progression on MUC16-directed bispecific antibodies.Analysis of these samples showed downregulation of MUC16/CA125,elevated secretion of VEGF,and epithelial-to-mesenchymal transition in tumor cells.Interestingly,hypoxia was determined to be a driver of these changes.These findings were prospectively validated in ovarian cancer cell lines with CRISPR/Cas9 knockout of MUC16/CA125 and VEGF.Peripheral blood mononuclear cells from patients with disease progression were capable of effective cytolysis ex vivo,suggesting that resistance to therapy was primarily tumor driven.Restoration of MUC16/CA125 expression did not restore cytotoxicity in the presence of increased VEGF secretion.Combination treatment with a VEGF inhibitor rescued cytotoxicity in hypoxia-conditioned ovarian cancer cell lines with preserved target antigen expression.Collectively,these data outline a link between hypoxia and the development of resistance to BITEs and posits inhibition of VEGF inhibition as a potentially important therapeutic intervention.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.
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 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.
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 Nile Albright Research Foundation(O.O.Y.)The Flatley Foundation(O.O.Y.)+2 种基金The Worden Family Foundation(O.O.Y.)The Barshad Family(O.O.Y.)the Vincent Memorial Hospital Foundation(S.K.B.).
文摘Bispecific T-cell Engagers(BITEs)are a novel form of immunotherapy that overcome a deficiency of immune checkpoint inhibitors(ICI)by targeting a preidentified tumor associated antigen and redirecting a polyclonal population of effector T-cells against the tumor.High grade serous ovarian cancer is a lethal disease in the recurrent setting and has not been amenable to ICI therapy.MUC16/CA125 is overexpressed in high grade serous ovarian cancer.BITEs targeting the tumor-retained portion of MUC16/CA125 have recently been described and are in early-phase clinical trials.To identify mechanisms of resistance to BITEs,we collected serum,peripheral blood mononuclear cells,and ascites samples from patients with disease progression on MUC16-directed bispecific antibodies.Analysis of these samples showed downregulation of MUC16/CA125,elevated secretion of VEGF,and epithelial-to-mesenchymal transition in tumor cells.Interestingly,hypoxia was determined to be a driver of these changes.These findings were prospectively validated in ovarian cancer cell lines with CRISPR/Cas9 knockout of MUC16/CA125 and VEGF.Peripheral blood mononuclear cells from patients with disease progression were capable of effective cytolysis ex vivo,suggesting that resistance to therapy was primarily tumor driven.Restoration of MUC16/CA125 expression did not restore cytotoxicity in the presence of increased VEGF secretion.Combination treatment with a VEGF inhibitor rescued cytotoxicity in hypoxia-conditioned ovarian cancer cell lines with preserved target antigen expression.Collectively,these data outline a link between hypoxia and the development of resistance to BITEs and posits inhibition of VEGF inhibition as a potentially important therapeutic intervention.
文摘小细胞肺癌(small-cell lung cancer,SCLC)约占肺癌的15%,恶性程度高,尽管对治疗敏感,但患者缓解持续时间短,预后极差。在化疗时代,广泛期SCLC(extensive stage SCLC,ES-SCLC)患者5年总生存率约为5%,到了免疫治疗时代,靶向程序性死亡受体1/程序性死亡配体1(programmed death 1/programmed death ligand 1,PD-1/PD-L1)的免疫检查点抑制剂联合化疗成为ES-SCLC一线治疗的新标准,疗效有所提升,但仍只有少数患者可以获得长期生存,5年生存率仅约10%左右。一个重要的原因是,ES-SCLC患者的后线治疗仍未取得实质性进展,需要开发ES-SCLC的新疗法。
