Bispecific antibodies targeting immunomodulatory checkpoints for cancer therapy

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.

Efficacy of Bispecific Antibody Targeting EpCAM and CD3 for Immunotherapy in Ovarian Cancer Ascites:An Experimental Study

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.

Preparation and Characterization for Bispecific Antibodies of Anti-CD3×Anti-Idiotype to BCellLym phocytic Leukem ia

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.

Construction,expression and binding specificity of bispecific CD3×VEGFR-2 and CD3×NCAM antibodies in the single chain and diabody format

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.

Bispecific antibody drug conjugates:Making 1+1>2

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.

Stepping forward:T-cell redirecting bispecific antibodies in cancer therapy

T cell-redirecting bispecific antibodies are specifically designed to bind to tumor-associated antigens,thereby engaging with CD3 on the T cell receptor.This linkage between tumor cells and T cells actively triggers T cell activation and initiates targeted killing of the identified tumor cells.These antibodies have emerged as one of the most promising avenues within tumor immunotherapy.However,despite success in treating hematological malignancies,significant advancements in solid tumors have yet to be explored.In this review,we aim to address the critical challenges associated with T cellredirecting bispecific antibodies and explore novel strategies to overcome these obstacles,with the ultimate goal of expanding the application of this therapy to include solid tumors.

Disulfide-stabilized single-chain antibody-targeted superantigen: Construction of a prokaryotic expression system and its functional analysis

AIM: To construct the expression vector of B3 (scdsFv)-SEA (D227A) and to identify its binding and cytotoxic ability to B3 antigen positive carcinoma cell lines.METHODS: This fusion protein was produced by a bacterial expression system in this study. It was expressed mainly in the inclusion body. The gene product was solubilized by guanidine hydrochloride, refolded by conventional dilution method, and purified using SP-sepharose cation chromatography.RESULTS: The expression vector B3 (scdsFv)-SEA-PETwas constructed, the expression product existed mainly in the inclusion body, the refolding product retained the binding ability of the single-chain antibody and had cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37 ℃.CONCLUSION: This genetically engineered B3 (scdsFv)-SEA fusion protein has bifunction of tumor targeting and tumor cell killing and shows its promises as an effective reagent for tumor-targeted immunotherapy.

Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen

BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable ( ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator. METHODS: mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANT-AB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg. the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay ( ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E. coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE. RESULTS: The variable heavy ( VH ) and variable light (VL) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa. CONCLUSION: The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody.

Bioinformatics-led design of single-chain antibody molecules targeting DNA sequences for retinoblastoma

The development of single-chain Fv antibody (scFv) by recombinant gene expression is an important milestone for cancer therapy. Single-chain antibodies are reconstructed for cancer-targeted therapy to provide good penetration into tumor tissue and to improve their pharmacokinetics in vivo, offering a clinically valuable application. The relationship needs to be analyzed that there may be some variations between the structure and function of the fusion proteins, and the relationship between the structure and function of protein molecules was obtained through analyzing relevant literature at home and abroad as well as modeling analysis. Through our analysis of the interaction region between antibody and antigen, and of the binding sites for molecular conformation, it is clear that existing antibodies need to be modified at the DNA sequence level, enhancing the biological activity of the antibodies. Based on the view that bio-molecular computer models are closely integrated with biological experiments, a bio-molecular structure-activity relationship model can be established in terms of molecular conformation, physical and chemical properties and the biological activity of single-chain antibodies. Two enlightenments are obtained from our analysis. On one hand, the structure-activity relationship is clear for new immune molecules at the gene expression level. On the other hand, a single-chain antibody molecule can be designed and optimized for the cancer-oriented treatment. In this article, we provided the theoretical and experimental basis for the development of single-chain antibodies appropriate for retinoblastoma therapy.

Selection for Anti-transferrin Receptor Bispecific T-cell Engager in Different Molecular Formats

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.

Expression of secreted human single-chain fragment variable antibody against human amyloid beta peptide in Pichia pastoris

BACKGROUND：Studies have shown that monoclonal or polyclonal antibody injections of amyloid β peptide are effective in removing amyloid β peptide overload in the brain. OBJECTIVE： Based on successful screening of a human single-chain fragment variable antibody specific to amyloidβpeptide, this paper aimed to express recombinant human single-chain variable antibody against amyloid β peptide. DESIGN, TIME AND SETTING： A single sample experiment was performed at the Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Hospital （Beijing, China） from January to July 2006. MATERIALS： Human single-chain fragment variable antibody gene against amyloid β peptide was screened from a human phage-display antibody library. METHODS： Human single-chain fragment variable antibody gene was mutated to eliminate a BamHI restriction site and cloned into a T easy plasmid for pT-scFvAβ construction, which was identified by PCR amplification and endonuclease digestion. Plasmid pT-scFvAβ was cut by EcoRI and NotI endonucleases, and the antibody gene was cloned into pPIC9K plasmid to construct pPIC9K-scFvAβ expression vector, which was confirmed by gene sequencing. Linearized pPIC9K-scFvAβ was used to transform a Pichia pastoris GS115 cell line, and the recombinant was induced by 0.5% methanol to express human single-chain fragment variable antibody specific to amyloid β peptide. MAIN OUTCOME MEASURES： Protein electrophoresis was used to identify PCR products, gene sequencing was used to verify the pPIC9K-scFvA sequence, and SDS-PAGE was used to detect recombinant expression of human single-chain fragment variable antibody specific to amyloid β peptide in Pichia pastoris. RESULTS： Gene sequencing confirmed pPIC9K-scFvAβ orientation. Recombinants were obtained by linearized pPIC9K-scFvAβ transformation. After induction with 0.5% methanol, the recombinant yeast cells secreted proteins of 33-ku size. CONCLUSION： The expression vector pPIC9K-scFvAβ was successfully constructed. Human single-chain fragment variable antibody specific to amyloid β peptide was recombinantly expressed in Pichia pastoris.

抗CD3/CD20双特异性抗体生物学活性测定方法的建立

目的建立基于报告基因的抗CD3/CD20双特异性抗体生物学活性检测方法。方法以WIL2-S细胞系作为靶细胞,以Jurkat-NF-κB-Luc转基因细胞系作为效应细胞,对抗体的量效范围、孵育时间、诱导时间、效靶比及细胞接种密度进行优化,构建可用于测定抗CD3/CD20双特异性抗体生物学活性的报告基因法。依据ICHQ2的指导原则对方法进行全面验证,包括专属性、准确性、精密度、线性、稳定性。结果成功建立了具有量效关系的抗CD3/CD20双特异性抗体生物学活性检测方法,该方法的抗体起始浓度为500 ng/mL,稀释倍数为1∶4,共计8个浓度点(500,125,31.25,7.81,1.95,0.49,0.12,0.031 ng/mL),效靶比为4∶1,诱导时间为4 h。本方法具有良好的专属性;5个不同稀释组回收率样品经测定,相对效价分别为59.63%±4.57%,75.54%±4.05%,99.98%±9.63%,123.90%±5.54%和142.51%±12.82%;对应的回收率分别为93.17%±7.15%,94.42%±5.06%,99.98%±9.63%,99.12%±4.43%以及91.21%±8.21%,CV分别为7.67%,5.35%,9.63%,4.47%和9.00%,上述结果的变异系数CV值均<10%;实测效价与理论效价线性回归分析相关系数R^(2)=0.9823,线性良好;本研究建立的方法可以敏感检测出抗体结构变化对生物活性的影响,对双特异性抗体的稳定性检测有一定的指导意义。结论利用转基因细胞法成功建立了抗CD3/CD20双特异性抗体生物学活性检测方法,该方法专属性强、准确性高、重复性好,可用于评价抗CD3/CD20双特异性抗体生物学活性。

双特异性抗体在多发性骨髓瘤治疗中的研究进展

多发性骨髓瘤(MM)是一种难以治愈的恶性浆细胞疾病,其发病率逐年增加,免疫调节剂、蛋白酶体抑制剂、抗CD38抗体、CAR-T、造血干细胞移植的应用明显改善了MM患者的预后,对于传统方案治疗后复发/难治的患者,需开发新的治疗手段改善其预后。双特异性抗体是一种新型免疫治疗方法,通过结合恶性浆细胞和细胞毒性免疫效应细胞(T细胞/NK细胞)上的靶标产生免疫突触,导致T/NK细胞活化和恶性浆细胞裂解。多项临床前及I期临床研究显示出良好的疗效,为复发/难治MM患者带来新的希望,未来与其他治疗方案联合可改善患者的预后。本文总结了近年来开发的双特异性抗体的分类、临床前及临床实验结果,为治疗MM提供参考。

新型冠状病毒感染与CAR-T耐药双重挑战下的格菲妥单抗治疗:难治性弥漫大B细胞淋巴瘤1例报告

本文总结1例嵌合抗原受体T(chimeric antigen receptor T,CAR-T)细胞治疗失败的难治性弥漫大B细胞淋巴瘤患者,在合并新型冠状病毒(coronavirus disease 2019,COVID-19)情况下接受格菲妥单抗治疗后获得完全缓解,且未见COVID-19感染加重等其他严重不良反应的治疗经验,同时回顾文献中格菲妥单抗治疗难治性弥漫大B细胞淋巴瘤的疗效,以及双特异性抗体在合并COVID-19的淋巴瘤患者中的安全性。

Unlocking the potential of bispecific ADCs for targeted cancer therapy

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.

Current status and future perspectives of bispecific antibodies in the treatment of lung cancer

Monoclonal antibodies have been successfully incorporated into the current therapeutical landscape of lung cancer in the last decades.Recently,with technological advances,bispecific antibodies(bsAbs)have also shown robust efficacy in the treatment of malignant cancers,including lung cancer.These antibodies target two independent epitopes or antigens and have been extensively explored in translational and clinical studies in lung cancer.Here,we outline the mechanisms of action of bsAbs,related clinical data,ongoing clinical trials,and potent novel compounds of various types of bsAbs in clinical studies,especially in lung cancer.We also propose future directions for the clinical development of bsAbs,which might bring a new era of treatment for patients with lung cancer.

Bispecific antibodies in cancer therapy:Target selection and regulatory requirements

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.

双/多特异性抗体药物差异化研发策略

双特异性抗体药物相比于治疗性单克隆抗体药物有效性和安全性更强,逐渐成为新一代的治疗手段。随着基因工程技术的发展和产业的成熟,双/多特异性抗体药物研究数量不断增加,应用范围也在不断拓展,为满足临床需求和创造临床价值奠定了基础。本文综述了双/多特异性抗体药物的研发阶段和适应症、靶点组合、结构形式和作用机制,并探讨了双/多特异性抗体药物研发的关键点和差异化研发策略,有助于创新药物的快速发展,为推动这一类药物的临床转化应用提供了理论支持,并为临床实践提供了更精准和有效的治疗选择。

T细胞依赖的双特异性抗体结构设计的研究进展

随着抗体工程的不断发展,靶向CD3的T细胞依赖的双特异性抗体已经发展出多种多样的结构,从而满足更多临床治疗的需求。依据是否含有Fc段可将T细胞依赖的双特异性抗体分为IgG样和非IgG样两种结构。其中轻重链错配问题是双特异性抗体最常见的制备难点,为解决该问题,目前已有多种技术应用于其结构设计中,包括杵臼技术、结构域交换技术等。由于T细胞依赖的双特异性抗体的作用机制是将T细胞重定向至肿瘤细胞继而发挥杀伤作用,因此Fc段杀伤功能的沉默、靶点的亲和力设计,以及免疫突触形成的距离设计均是T细胞依赖的双特异性抗体结构设计的重点。本文将围绕以上针对T细胞依赖的双特异性抗体结构设计的研究进展进行总结。

Advanced cervix cancer patient with chemotherapy-induced grade IV myelosuppression achieved complete remission with cadonilimab:A case report

BACKGROUND The prognosis for patients with advanced metastatic cervix cancer(MCC)is poor,and this disease continues to pose a considerable therapeutic challenge.Despite the administration of first-line regimens consisting of cisplatin,paclitaxel,and bevacizumab,survival rates for patients with metastasis remain poor.The emergence of bispecific antibodies(BsAbs)offers a novel treatment option for patients diagnosed with MCC.CASE SUMMARY In this report,we present a patient with MCC who was treated with cadonilimab monotherapy at a dose of 6 mg/kg every two weeks after chemotherapy was proven to be intolerable.The patient exhibited a sustained complete response for a duration of 6 months,demonstrating an optimistic outlook.CONCLUSION This case illustrates the considerable efficacy of cadonilimab for treating advanced MCC.Therefore,BsAb therapy is a promising strategy for effectively treating patients with advanced MCC and should be considered as an option when patients are intolerant to standard chemotherapy.