PD-1单抗联合顺铂或吉西他滨化疗对KRAS突变非小细胞肺癌A549细胞移植瘤小鼠模型的治疗作用

目的:探讨程序性死亡受体-1(PD-1)单抗联合顺铂或吉西他滨在KRAS基因突变非小细胞肺癌(NSCLC)A549细胞移植瘤小鼠模型治疗中的作用。方法:构建免疫系统-肿瘤双人源化A549细胞小鼠移植瘤模型,将60只小鼠按随机数字表法分成6组(10只/组),分别为对照组(200μL/kg PBS)、PD-1单抗组(20 mg/kg PD-1单抗)、顺铂组(3 mg/kg顺铂)、PD-1单抗+顺铂组(20 mg/kg PD-1单抗+3 mg/kg顺铂)、吉西他滨组(30 mg/kg吉西他滨)和PD-1单抗+吉西他滨组(20 mg/kg PD-1单抗+30 mg/kg吉西他滨)。TUNEL和DAPI双染色法检测移植瘤组织中细胞凋亡水平,测量移植瘤体积和质量并计算肿瘤生长抑制率,免疫组化法检测移植瘤微血管密度(MVD)。结果:成功构建免疫系统-肿瘤双人源化NSCLC A549细胞小鼠移植瘤模型,PD-1单抗+顺铂组移植瘤的细胞凋亡率、肿瘤生长抑制率均最高,移植瘤体积、质量和MVD均最小,与其他5组小鼠比较差异均有统计学意义(均P<0.05)。结论:顺铂与PD-1单抗具有协同活性,而吉西他滨拮抗PD-1单抗的治疗作用。提示PD-1单抗联合顺铂对KRAS突变NSCLC A549细胞移植瘤小鼠的疗效更好。

From targeting the tumor to targeting the immune system: Transversal challenges in oncology with the inhibition of the PD-1/PD-L1 axis

After that the era of chemotherapy in the treatment of solid tumors have been overcome by the "translational era", with the innovation introduced by targeted therapies, medical oncology is currently looking at the dawn of a new "immunotherapy era" with the advent of immune checkpoint inhibitors(CKI) antibodies.The onset of PD-1/PD-L1 targeted therapy has demonstrated the importance of this axis in the immune escape across almost all human cancers.The new CKI allowed to significantly prolong survival and to generate durable response, demonstrating remarkable efficacy in a wide range of cancer types.The aim of this article is to review the most up to date literature about the clinical effectiveness of CKI antibodies targeting PD-1/PD-L1 axis for the treatment of advanced solid tumors and to explore transversal challenges in the immune checkpoint blockade.

DNA-delivered monoclonal antibodies targeting the p53 R175H mutant epitope inhibit tumor development in mice

tThe tumor suppressor p53 is the most common mutated gene in cancer,with the R175H as the most frequent p53 missense mutant.However,there are currently no approved targeted therapies or immunotherapies against mutant p53.Here,we characterized and inves-tigated a monoclonal antibody(mAb)that recognizes the mutant p53-R175H for its affinity,specificity,and activity against tumor cells in vitro.We then delivered DNA plasmids expres-sing the anti-R175H mAb or a bispecific antibody(BsAb)into mice to evaluate their therapeutic effects.Our results showed that the anti-R175H mAb specifically bound to the p53-R175H an-tigen with a high affinity and recognized the human mutant p53-R175H antigen expressed on HEK293T or MC38 cells,with no cross-reactivity with wild-type p53.In cultured cells,the anti-R175H mAb showed higher cytotoxicity than the control but did not induce antibody-dependent cellular cytotoxicity.We made a recombinant MC38 mouse cell line(MC38-p53-R175H)that overexpressed the human p53-R175H after knocking out the endogenous mutant p53 alleles.In vivo,administration of the anti-R175H mAb plasmid elicited a robust anti-tumor effect against MC38-p53-R175H in mice.The administration of the anti-R175H BsAb plasmid showed no therapeutic effects,yet potent anti-tumor activity was observed in combination with the anti-PD-1 antibody.These results indicate that targeting specific mutant epitopes using DNA-delivered mAbs or BsAbs presents a form of improved natural immunity derived from tumor-infiltrating B cells and plasma cells against intracellular tumor antigens.

Local sustained release of PD-1 monoclonal antibody and lenvatinib by thermo-sensitive hydrogel for improving tumor immunotherapy

In clinic,the combination of intravenous pembrolizumab(PD-1 monoclonal antibody)with oral Lenvatinib(LEN)exhibited an enhanced synergistic benefit for cancer therapy.However,the clinical outcomes were always limited by the problems of inconsistent pharmacokinetic profiles of two drugs,lower drug accumulation in tumor and obvious side effects during the combination therapy.Here,in situ-forming thermosensitive hydrogels based on PLGA-PEG-PLGA triblock copolymers were prepared for local administration of anti-PD1 and LEN(P&L@Gel)to improve therapeutic efficacy and safety.After peritumoral or surgical resection site injection,the significant increased concentrations of both drugs in tumor were observed with the local sustained release of P&L@Gel.In comparison with the group of intraperitoneal anti-PD1 plus oral LEN(P-ip&L-po),significantly higher tumor inhibition efficiency on CT26 tumor models could be obtained in P&L@Gel group,even at the dose of one-eighth of the former,same tumorinhibition effects could be achieved.The enhanced antitumor efficacy of P&L@Gel group was probably associated with the 2.2 folds of increased level of CD8+T cells and the polarization of tumor associated macrophage from M2 to M1 along with the increased drug accumulation.Moreover,compared with the obvious side effects of P-ip&L-po group,no significant changes of PLT,ALT and UA in blood,as well as IL-1αand IL-1βin mice paws were observed between P&L@Gel group and untreated group.These results suggested that local administration of anti-PD1 and LEN with thermosensitive hydrogel could offer a potential strategy for tumors or tumor postoperative adjuvant treatment.

Crystal clear： visualizing the intervention mechanism of the PD-1/PD-L1 interaction by two cancer therapeutic monoclonal antibodies

Antibody-based PD-IIPD-L1 blockade therapies have taken center stage in immunotherapies for cancer, with multiple clinical successes. PD-1 signaling plays pivotal roles in tumor-driven T-cell dysfunction. In contrast to prior approaches to generate or boost tumor-specific T-cell responses, antibody-based PD-1/PD-L1 blockade targets tumor-induced T-cell defects and restores pre- existing T-cell function to modulate antitumor immunity. in this review, the fundamental knowledge on the expression regulations and inhibitory functions of PD-1 and the present understanding of antibody-based PD-1/ PD-L1 blockade therapies are briefly summarized. We then focus on the recent breakthrough work concerning the structural basis of the PD-IIPD-Ls interaction and how therapeutic antibodies, pembrolizumab targeting PD-1 and avelumab targeting PD-L1, compete with the binding of PD-1/PD-L1 to interrupt the PD-1/PD-L1 interaction. We believe that this structural informationwill benefit the design and improvement of therapeutic antibodies targeting PD-1 signaling.

β淀粉样肽1～42单克隆抗体的制备

目的 制备稳定分泌β -淀粉样肽 1～ 4 2 (Aβ1-42 )单克隆抗体的杂交瘤细胞 ,产生高效价抗体。方法 通过基因工程技术 ,将Aβ1-42 基因融合于乙肝核心抗原 (HBcAg)的主要免疫优势区 (MIR) ,制备Aβ和HBcAg的融合蛋白 ,免疫BALB/c小鼠 ,取脾细胞与SP2 / 0细胞融合 ,筛选能稳定分泌Aβ1-42 单克隆抗体的杂交瘤细胞。结果 得到两株能稳定分泌Aβ1-42 单克隆抗体的杂交瘤细胞 ,Aβ1-42 单克隆抗体的Ig亚类为IgG3 。结论 制备得到的Aβ1-42 单克隆抗体特异性强 。

^(35)S 标记抗人肝癌单克隆抗体Hepama-1的制备

采用Ｈ２３５ＳＯ４与苯胺合成３５Ｓ－苯胺硫酸盐，经高温转换成３５Ｓ－对氨基苯磺酸，然后再用碳二亚胺交联到抗人肝癌单克隆抗体Ｈｅｐａｍａ－１上。所得３５Ｓ－ＭＡｂＨｅｐａｍａ－１用Ｅｌｉｓａ方法检测仍保持良好的免疫活性。产品最高比活度可达５１．８ＧＢｑ／ｇ，放化纯度＞９５％，放化产率以Ｈ２３５ＳＯ４投料计算最高可达２７％，以３５Ｓ－对氨基苯磺酸投料计算最高可达４５％，适用于做荷瘤裸鼠动物模型放免治疗研究实验。

Elevated nuclear PIGL disrupts the cMyc/BRD4 axis and improves PD-1 blockade therapy by dampening tumor immune evasion

To improve the efficacy of lenvatinib in combination with programmed death-1(PD-1)blockade therapy for hepatocellular carcinoma(HCC),we screened the suppressive metabolic enzymes that sensitize HCC to lenvatinib and PD-1 blockade,thus impeding HCC progression.After analysis of the CRISPR‒Cas9 screen,phosphatidylinositol-glycan biosynthesis class L(PIGL)ranked first in the positive selection list.PIGL depletion had no effect on tumor cell growth in vitro but reprogrammed the tumor microenvironment(TME)in vivo to support tumor cell survival.Specifically,nuclear PIGL disrupted the interaction between cMyc/BRD4 on the distant promoter of target genes and thus decreased the expression of CCL2 and CCL20,which are involved in shaping the immunosuppressive TME by recruiting macrophages and regulatory T cells.PIGL phosphorylation at Y81 by FGFR2 abolished the interaction of PIGL with importinα/β1,thus retaining PIGL in the cytosol and facilitating tumor evasion by releasing CCL2 and CCL20.Clinically,elevated nuclear PIGL predicts a better prognosis for HCC patients and presents a positive correlation with CD8+T-cell enrichment in tumors.Clinically,our findings highlight that the nuclear PIGL intensity or the change in PIGL-Y81 phosphorylation should be used as a biomarker to guide lenvatinib with PD-1 blockade therapy.

Selection and characterization of the novel anti-human PD-1 FV78 antibody from a targeted epitope mammalian cell-displayed antibody library

Currently,display-based methods are well established and widely used in antibody engineering for affinity maturation and structural stability improvement.We obtained a novel anti-human programmed death 1(PD-1)antibody using computer-aided design and a mammalian cell display technology platform.We used computer-aided modeling and distance geometry methods to predict and assign the key residues that contributed to the binding of human PD-L1 to PD-1.Then,we analyzed the sequence of nivolumab(an anti-human PD-1 antibody,referred to as MIL75 in the article)to determine the template for antibody design and library construction.We identified a series of potential substitutions on the obtained template and constructed a virtual epitope-targeted antibody library based on the physicochemical properties and each possible location of the assigned key residues.The virtual antibody libraries were displayed on the surface of mammalian cells as the antigen-binding fragments of full-length immunoglobulin G.Then,we used flow cytometry and sequencing approaches to sort and screen the candidates.Finally,we obtained a novel anti-human PD-1 antibody named FV78.FV78 competitively recognized the PD-1 epitopes that interacted with MIL75 and possessed an affinity comparable to MIL75.Our results implied that FV78 possessed equivalent bioactivity in vitro and in vivo compared with MIL75,which highlighted the probability and prospect of FV78 becoming a new potential antibody therapy.

Predefined GPGRAFY-Epitope-Specific Monoclonal Antibodies with Different Activities for Recognizing Native HIV-1 gp120

A seven-amino acid epitope GPGRAFY at the tip of the V3 loop in HIV-1 gp120 is the principal neutralizing epitope, and a subset of anti-V3 antibodies specific for this epitope shows a broad range of neu-tralizing activity. GPGRAFY-epitope-specific neutralizing antibodies were produced using predefined GPGRAFY-epitope-specific peptides instead of a natural or recombinant gp120 bearing this epitope. All six monoclonal antibodies (mAbs) could recognize the GPGRAFY-epitope on peptides and two of the antibod-ies, 9D8 and 2D7, could recognize recombinant gp120 in enzymelinked immunosorkentassy (ELISA) as-says. In the flow cytometry analysis, the mAbs 9D8 and 2D7 could bind to HIV-Env+ CHO-WT cells and the specific bindings could be inhibited by the GPGRAFY-epitope peptide, which suggests that these two mAbs could recognize the native envelope protein gp120 expressed on the cell membrane. However, in syncytium assays, none of the mAbs was capable of inhibiting HIV-Env-mediated cell membrane fusion. The different activities for recognizing native HIV-1 gp120 might be associated with different antibody affinities against the epitopes. The development of conformational mimics of the neutralization epitope in the gp120 V3 loop could elicit neutralizing mAbs with high affinity.

PD-1/PD-L1 blockade in cervical cancer: current studies and perspectives

Cervical cancer (CC) is the fourth most commonly diagnosed female malignancy and a leading cause of cancer-related mortality worldwide, especially in developing countries. Despite the use of advanced screening and preventive vaccines, more than half of all CC cases are diagnosed at advanced stages, when therapeutic options are extremely limited and side effects are severe. Given these circumstances, new and effective treatments are needed. In recent years, exciting progress has been made in immunotherapies, including the rapid development of immune checkpoint inhibitors. Checkpoint blockades targeting the PD-1/PD-L1 axis have achieved effective clinical responses with acceptable toxicity by suppressing tumor progression and improving survival in several tumor types. In this review, we summarize recent advances in our understanding of the PD-1/PD-L1 signaling pathway, including the expression patterns of PD-1/PD-L1 and potential PD-l/PD-Ll-related therapeutic strategies for CC.

Primary malignant melanoma of the esophagus successfully treated with camrelizumab:A case report and literature review

An 83-year-old Chinese woman presented with a 3-month history of dysphagia.She also had a history of hypertension,type 2 diabetes,fundus hemorrhage,and cataract but no history of cutaneous,ocular,or other-site melanomas.Upper gastrointestinal tract angiography revealed gastritis and duodenal diverticulum;thus,an endoscopic review was recommended.Enhanced computed tomography of the chest and upper abdomen revealed the following:(1)Esophageal space-occupying lesions and mediastinal lymph node enlargement(considering the high possibility of esophageal cancer,further endoscopy was recommended)and(2)A small amount of right pleural effusion,with no significant lymph node infiltration or distant metastasis.Esophagoscopy identified a bulge mass blocking the esophagus from 23 to 30 cm from the incisors.The upper mass had a spherical clustering,while the lower mass significantly festered.Pathological biopsy samples were obtained from the esophagus 23 and 28 cm from the incisors.Tissue biopsy showed proliferation of large round tumor cells and melanocytes.Immunohistochemistry showed positive findings for HMB45 and MelanA;partially positive findings for S100,CK7,CK5/6,CAM5.2,LCA,P63,and TTF-1;and negative findings for Syn.The Ki-67 positivity index was approximately 60%.Based on these findings,the patient was diagnosed with malignant esophageal melanoma with enlarged mediastinal lymph nodes.She was then treated with five cycles of camrelizumab therapy combined with chemotherapy from October 18,2019,to May 5,2020.Gastroscopy review following two courses of combination therapy revealed that the esophagus was 23-25 cm away from the incisors,and there were two continuous uplifted and beaded masses that had a smooth and black surface,with each of them having a length and diameter of approximately 1 cm.Melanosis of the mucosa around the lumen was observed at 40 cm from the incisors to the cardia;the dentate margin was clear;and the cardia had no stenosis.The patient then received five courses of combination therapy and became consistently stable after partial remission.No severe adverse events related to the immunotherapy were recorded.Camrelizumab may be a viable treatment option for patients with PMME.Additional evidence from future clinical trials and research is necessary to fully validate our findings.

Successful Combination Therapy of Nivolumab and Radiation for Massive Mucosal Melanoma in Patient with Autoimmune-Myasthenia Gravis, Systemic Lupus Erythematosus and Rheumatoid Arthritis

A 58-year-old woman complicated with autoimmune diseases of myasthenia gravis (MG), rheumatoid arthritis, and systemic lupus erythematosus noticed an irregular black macule on her bilateral major labia, which was diagnosed as malignant melanoma. The melanoma lesion involving the vagina, uterus, and ventral side of rectum was not operable and was treated with nivolumab and concurrent radiotherapy with good control of the MG. This resulted in remarkable tumor shrinkage, possibly due to synergistic effects of both treatments. To our knowledge, few reports have described the effectiveness of combination therapy with nivolumab and radiation for malignant melanoma. The present case showed an enhanced anti-tumor effect with combination therapy.

B7-1 mAb对B系淋巴瘤Raji细胞的生长抑制和凋亡诱导作用

为了研究B7 1分子功能性mAb (6H2 )对表达B7 1分子的B系淋巴瘤Raji细胞的生物效应。分别运用免疫荧光标记和流式细胞仪技术 (FACS )、台盼蓝拒染试验、MTT试验和RT PCR半定量分析 ,分析 6H2对Raji细胞的生长抑制和凋亡诱导作用。结果显示 ,6H2能抑制天然表达B7 1分子的人B系淋巴瘤Raji细胞的生长 ,产生细胞周期G2 /M的阻滞 ,上调细胞表面CD18和CD95 (Fas )的表达 ,且能使Raji细胞中Bax的相对转录表达显著增加 ,从而诱导Raji细胞的凋亡。 6H2为功能性抗人B7 1分子特异性mAb ,具有抑制Raji细胞生长和凋亡诱导作用 ,在B系淋巴瘤研究和生物治疗中具有应用价值。

Current Status and Clinical Research Progress of Immunotherapy for Advanced Gastric Cancer

Advanced gastric cancer is a common digestive system tumor,and its treatment has always been a difficult problem.In recent years,with the rapid development of immunotherapy,the treatment effect of advanced gastric cancer has been significantly improved.This article introduces the current status and clinical research progress of immune checkpoint inhibitors in advanced gastric cancer.Commonly used immunotherapy methods include chemical drug therapy,biological therapy,and gene therapy,among which the immune checkpoint inhibitors are currently one of the most popular immunotherapy methods,including nivolumab,pembrolizumab,and atezolizumab,which target programmed death ligand 1(PD-L1)low expression(1%–49%)and PD-L1 high expression(≥50%).The results of clinical studies have shown that immunotherapy can significantly prolong the survival of patients with advanced gastric cancer while having lower toxic side effects and better tolerance.However,immunotherapy also has some problems,such as drug resistance and repeated infection.Future research directions include exploring new immunotherapy methods,combination therapy,and individualized therapy.

细胞角蛋白19片段抗原21-1含量化学发光微粒子定量免疫检测方法的建立

目的建立细胞角蛋白19片段抗原21-1(cytokeratin 19 fragment 21-1,CYFRA21-1,简称CY21-1)含量的化学发光微粒子免疫检测方法(chemiluminescent microparticle immunoassay,CMIA)。方法原核表达制备重组CY21-1(r CY21-1),用其免疫BALB/c小鼠后,收集小鼠脾脏细胞,常规杂交瘤技术与Sp2/0进行融合,制备CY21-1单抗。间接ELISA法筛选可稳定分泌CY21-1抗体的杂交瘤细胞,分别标记微性磁珠(magnetic beads,MB)和吖啶酯(acridinium ester,AE),筛选可特异检测CY21-1的单抗配对,建立CY21-1 CMIA,同时验证方法的线性及灵敏度,并与同类试剂盒进行比较。结果经筛选获得了一组能特异检测CY21-1的单抗配对MB*26B5-3E4*AE,建立的CY21-1 CMIA对检测CY21-1校准品的线性范围为0.1~1 000 ng/m L,R^2=0.992 3,检测灵敏度为0.076 ng/m L。该方法与美国雅培公司生产的Abbott CY21-1 CMIA诊断试剂盒平行检测250份临床血清标本的结果具有良好的相关性(R^2=0.961 6)。结论成功建立了CY21-1 CMIA,且具有良好的线性及灵敏度,为我国肺癌的临床筛查和早期诊断奠定了基础。

Assays for predicting and monitoring responses to lung cancer immunotherapy

Immunotherapy has become a key strategy for cancer treatment, and two immune checkpoints, namely, programmed cell death 1 （PD-1） and its ligand （PD-L1）, have recently emerged as important targets. The interaction blockade of PD-1 and PD-L1 demonstrated promising activity and antitumor efficacy in early phase clinical trials for advanced solid tumors such as non-small cell lung cancer （NSCLC）. Many cell types in multiple tissues express PD-L1 as well as several tumor types, thereby suggesting that the ligand may play important roles in inhibiting immune responses throughout the body. Therefore, PD-L1 is a critical immunomodulating component within the lung microenvironment, but the correlation between PD-L1 expression and prognosis is controversial. More evidence is required to support the use of PD-L1 as a potential predictive biomarker. Clinical trials have measured PD-L1 in tumor tissues by immunohistochemistry （IHC） with different antibodies, but the assessment of PD-L1 is not yet standardized. Some commercial antibodies lack specificity and their reproducibility has not been fully evaluated. Further studies are required to clarify the optimal IHC assay as well as to predict and monitor the immune responses of the PD-I/PD-L1 pathway.

Characterization of a novel bispecific antibody targeting tissue factor-positive tumors with T cell engagement

T cell engaging bispecific antibody(TCB)is an effective immunotherapy for cancer treatment.Through co-targeting CD3 and tumor-associated antigen(TAA),TCB can redirect CD3+T cells to eliminate tumor cells regardless of the specificity of T cell receptor.Tissue factor(TF)is a TAA that involved in tumor progression.Here,we designed and characterized a novel TCB targeting TF(TF-TCB)for the treatment of TF-positive tumors.In vitro,robust T cell activation,tumor cell lysis and T cell proliferation were induced by TF-TCB.The tumor cell lysis activity was dependent upon both CD3 and TF binding moieties of the TF-TCB,and was related to TF expression level of tumor cells.In vivo,in both tumor cell/human peripheral blood mononuclear cells(PBMC)co-grafting model and established tumor models with poor T cell infiltration,tumor growth was strongly inhibited by TF-TCB.T cell infiltration into tumors was induced during the treatment.Furthermore,efficacy of TF-TCB was further improved by combination with immune checkpoint inhibitors.For the first time,our results validated the feasibility of using TF as a target for TCB and highlighted the potential for TF-TCB to demonstrate efficacy in solid tumor treatment.