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.

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.

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.

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.

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.

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.

Generation of high affinity human single-hain antibody against PreSl of hepatitis B virus from immune phage-display antibody library

BACKGROUND: A single-chain antibody ( ScFv) phage display library was created by cloning antigen-binding re- gions of VH (variable domain) and VL gene repertoires as fusion proteins with a minor coat protein of filamentous phage, from which high affinity completely humanized ScFv against PreS1 of hepatitis B virus could be screened and characterized. METHODS: A combinatorial library of phage-display hu- man ScFv genes, which were derived from peripheral blood lymphocytes immunized by peptide PreS1 in vitro, was constructed. The library contained 7 × 108 clones. RESULTS: After 3 rounds panning, a high affinity (K = 10-7-10-8 mol/L) ScFv specific to PreS1 was obtained. Sequence analysis showed that the VH belonged to the VH4 family and Vλ to Vλ4. CONCLUSIONS: The described ScFv may provide a more satisfactory therapy. This application further illustrates that the method of in vitro antigen stimulation is expeditious for the source of human immune antibody library.

Frontier of therapeutic antibody discovery:The challenges and how to face them

Therapeutic monoclonal antibodies have become an important class of modern medicines.The established technologies for therapeutic antibody discovery such as humanization of mouse antibodies,phage display of human antibody libraries and transgenic animals harboring human IgG genes have been practiced successfully so far,and many incremental improvements are being made constantly.These methodologies are responsible for currently marketed therapeutic antibodies and for the biopharma industry pipeline which are concentrated on only a few dozen targets.A key challenge for wider application of biotherapeutic approaches is the paucity of truly validated targets for biotherapeutic intervention.The efforts to expand the target space include taking the pathway approach to study the disease correlation.Since many new targets are multi-spanning and multimeric membrane proteins there is a need to develop more effective methods to generate antibodies against these difficult targets.The pharmaceutical properties of therapeutic antibodies are an active area for study concentrating on biophysical characteristics such as thermal stability and aggregation propensity.The immunogenicity of biotherapeutics in humans is a very complex issue and there are no truly predictive animal models to rely on.The in silico and T-cell response approaches identify the potential for immunogenicity;however,one needs contingency plans for emergence of antiproduct antibody response for clinical trials.

Anti-amyloid beta single-chain Fv ameliorates behavioral impairment in Alzheimer's disease mice via adeno-associated virus delivery

Intracranial delivery of human Fc-deleted antibody specific to amyloid-β peptide （Aβ, anti-Aβ single-chain Fv, scFv） via adeno-associated virus （AAV） inhibits amyloid deposition in transgenic mice. However, the effects of AAV-mediated Fc-deleted antibody on animal behavior remain unclear. In this study, the anti-Aβ scFv antibody gone, isolated from phage display, was fused to the 5＇ end of the scFv antibody gone for antibody secretion by 2 rounds of polymerase chain reaction amplification. The fused antibody cDNA was cloned into a pSNAV2 plasmid under the control of the cytomegalovirus promoter. The sequence verified expression vector pSNAV2/scFv was transferred to BHK-21 ceils, and stable transfected BHK-21/scFv cells were established by G418 selection and infected with the recombinant herpes simplex virus rHSV/repcap for AAV production. Recombinant AAV was injected into the left quadriceps femoris of PDAPP transgenic mice. After 3 months, Morris water-maze results confirmed significantly improved cognitive function in a mouse model of Alzheimer＇s disease. Key Words： Alzheimer＇s disease; adeno-associated virus; amyloid-β peptide; single-chain antibody; neurodegenerative diseases; neural regeneration

Modified Therapeutic Antibodies: Improving Efficacy

The prosperity of the biotherapeutics market reflects the feasibility and effectiveness of therapeutic antibodies for the treatment of cancers,inflammatory disorders,and refractory infections.As drawbacks emerge in clinical trials and practice,such as impeded binding,reduced effector functions,and frequent adverse reactions,modifications of therapeutic antibodies are unprecedently burgeoning in research and development(R&D).These modifications include:①modified glycosylation;②fragment of crystallizable domain(Fc)amino acid alterations;③cross-isotype or cross-subclass exchanges;④antibody–drug conjugates(ADCs);⑤single chain of variable region fragment(scFv)for chimeric antigen receptor T(CAR-T)cells;and⑥bispecific antibodies(bsAbs)in order to promote binding affinity,half-life in circulation,effectiveness toward target cells and,ultimately,to achieve overall improved efficacy.While many achievements have been made around the world in the past decades,China has been playing an active role in this realm,with its great demand for biotherapeutics with R&D potential.This review recapitulates the international progress that has been achieved with modified therapeutic antibodies,and then focuses on that of China in an independent section.

Fusion protein of single-chain variable domain fragments for treatment of myasthenia gravis

Single-chain variable domain fragment （scFv） 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pastoris. The afifnity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunolfuorescence staining. The ability of the fusion protein to block myas-thenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for speciifc immunosuppressive therapy of myasthenia gravis.

靶向BCMA/CD3的双特异性抗体药物的质量控制研究

目的 研究并建立靶向B细胞成熟抗原(BCMA)和白细胞分化抗原3(CD3)的双特异性抗体(双抗)药物的关键质量属性(CQA)质量控制方法。方法 从特异性鉴别、纯度、电荷异质性、产品相关杂质、翻译后修饰和生物学活性几个方面对靶向BCMA/CD3的双抗药物开展质量控制研究。采用基于赖氨酰肽链内切酶(Lys-C)酶切和反相高效液相色谱(RP-HPLC)技术的肽图法对靶向BCMA/CD3的双抗药物进行特异性鉴别确认;采用分子排阻色谱法(SEC)和还原/非还原十二烷基硫酸钠毛细管电泳法(CE-SDS)进行纯度控制;采用离子交换色谱法(IEC)测定电荷异质性和产品相关杂质;采用反相超高效液相色谱(RPUHPLC)和在线质谱联用的多属性监测方法(MAM)监测靶向BCMA/CD3双抗中的翻译后修饰;采用T细胞激活和报告基因法测定该双抗的生物学活性。结果 肽图法检测可有效识别双抗中的关键肽段,起到特异性鉴别确认的作用;SEC和CE-SDS法可有效区分双抗中的主成分、聚体和片段,对有效成分的纯度控制发挥关键作用;IEC法可有效区分酸碱变异体、目标双抗及亲代残留同源二聚体,有利于产品相关杂质的控制;MAM法可检测到天冬氨酸异构化和天冬酰胺脱酰胺化等翻译后修饰的存在,可作为CQA控制的有力补充;T细胞激活结合报告基因法可有效评估该双抗的生物学活性。结论 针对靶向BCMA/CD3的双抗药物的理化和生物学特性,研究并建立了针对其CQA的质量控制方法,从质量可控的角度确保该类双抗产品的临床安全性和有效性。

新型抗肿瘤药物在EGFR-TKI耐药非小细胞肺癌中的研究进展

表皮生长因子受体(EGFR)突变型非小细胞肺癌治疗引领了精准医学的发展,第三代EGFR酪氨酸激酶抑制剂(TKI)是目前晚期非小细胞肺癌的标准治疗手段,但耐药仍不可避免,EGFR-TKI耐药后治疗策略是临床研究探索的热点。近些年,随着双特异性抗体和抗体偶联药物等新型抗肿瘤药物的研发,改写了EGFR-TKI耐药人群的治疗格局。创新型抗肿瘤药物展现了非常令人鼓舞的疗效和极具潜力的应用前景,为EGFR-TKI耐药人群带来了全新的治疗选择。本文聚焦于双特异性抗体和抗体偶联药物在EGFR-TKI耐药人群中的最新研究进展,展望新型抗肿瘤药物的研发趋势。

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。