Development of 177Lu‐nanobodies for radioimmunotherapy of HER2‐positive breast cancer: evaluation of different bifunctional chelators

Nanobodies show favourable pharmacokinetic characteristics for tumor targeting, including high tumor‐to‐background‐ratios. Labelled with a therapeutic radionuclide, nanobodies could be used as an adjuvant treatment option for HER2‐overexpressing minimal residual disease. The therapeutic radionuclide Lutetium‐177 is linked to the nanobody using a bifunctional chelator. The choice of the bifunctional chelator could affect the in vivo behaviour of the radiolabeled nanobody. Consequently, we compared four different bifunctional chelators ‐ p‐SCN‐Bn‐DOTA, DOTA‐NHS‐ester, CHX‐A”‐DTPA or 1B4M‐DTPA ‐ in order to select the optimal chemical link between Lutetium‐177 and a HER2 targeting nanobody. MS results revealed different degrees of chelator‐conjugation. High stability in time was observed, together with nanomolar affinities on HER2‐expressing tumor cells. Ex vivo biodistributions as well as SPECT/micro‐CT analyses showed high activities in tumors expressing medium HER2 levels with low background activity except for the kidneys. The 1B4M‐DTPA‐coupled conjugate was further evaluated in a high HER2‐expressing tumor model. Here, tumor uptake values of 5.99 ± 0.63, 5.12 ± 0.17, 2.83 ± 0.36 and 2.47 ± 0.38 %IA/g were obtained at 1, 3, 24 and 48h p.i., which coincided with exceptionally low background values, except for the kidneys, and unprecedented tumor‐to‐background ratios. No specific binding was observed in a HER2‐negative model. In conclusion, the in‐house developed anti‐HER2 nanobody 2Rs15dHIS can be successfully labeled with ¹⁷⁷Lu using different bifunctional chelators. Both macrocyclic and acyclic chelators show high stability in time. High specific tumor uptake combined with the lowest background uptake was measured using the 1B4M‐DTPA‐based conjugate. Copyright © 2012 John Wiley & Sons, Ltd.     

基于纳米抗体CDR3区抗原表位展示的β-HCG 抗体制备及鉴定

摘要：目的 通过纳米抗体CDR3区展示β-HCG C端表位的方式,验证纳米抗体在抗原表位展示中的作用。方法 采用基因合成的方法,将β-HCG C端表位（氨基序列151~165）插入到纳米抗体CDR3区,酶切、连接原核表达载体pET24a,IPTG诱导表达。将His标签填料纯化得到的高纯度目的蛋白免疫新西兰大耳白兔,经5次免疫后,间接ELISA检测效价,抗原偶联纯化介质进行免疫多抗纯化,Western blot进行多抗特异性检测。结果 成功构建原核表达重组载体,并获得高表达菌株,IPTG诱导后,目的蛋白主要以包涵体的形式存在,亲和层析获得纯度大于98%的目的蛋白,包涵体经复性后免疫,效价可达1∶512 000,Western blot特异性检测显示免疫多抗能够特异性结合β-HCG。结论 纳米抗体CDR3区β-HCG抗原C端表位展示的方法,可用于抗β-HCG抗体的制备,并为今后纳米抗体表位展示相关研究奠定基础。     

Temporal and spatial organization of ESCRT protein recruitment during HIV-1 budding

HIV-1 virions assemble at the plasma membrane of mammalian cells and recruit the endosomal sorting complex required for transport (ESCRT) machinery to enable particle release. However, little is known about the temporal and spatial organization of ESCRT protein recruitment. Using multiple-color live-cell total internal reflection fluorescence microscopy, we observed that the ESCRT-I protein Tsg101 is recruited together with Gag to the sites of HIV-1 assembly, whereas later-acting ESCRT proteins (Chmp4b and Vps4A) are recruited sequentially, once Gag assembly is completed. Chmp4b, a protein that is required to mediate particle scission, is recruited to HIV-1 assembly sites ∼10 s before the ATPase Vps4A. Using two-color superresolution imaging, we observed that the ESCRT machinery (Tsg101, Alix, and Chmp4b/c proteins) is positioned at the periphery of the nascent virions, with the Tsg101 assemblages positioned closer to the Gag assemblages than Alix, Chmp4b, or Chmp4c. These results are consistent with the notion that the ESCRT machinery is recruited transiently to the neck of the assembling particle and is thus present at the appropriate time and place to mediate fission between the nascent virus and the plasma membrane.Live-cell fluorescence microscopy of assembling HIV-1 virions has established the temporal sequence in which various viral and host molecules are recruited to the assembly site (1–6). The HIV-1 genome is recruited first to the plasma membrane by a subdetectable number of molecules of the structural protein, Gag (3), and a steady accumulation of Gag ensues for 6–10 min (1, 2, 4). After Gag recruitment is completed, members of the endosomal sorting complex required for transport III (ESCRT-III) complex and the ATPase vacuolar protein sorting-associated protein 4A (Vps4A) are recruited transiently, for just a few minutes, to the site of assembly (4, 6). The ESCRT machinery functions during membrane fission in processes such as the formation of multivesicular bodies, the terminal stages of cytokinesis (7), and the budding of enveloped viruses such as HIV-1 (8, 9). These processes all have inverted topologies compared with the topology of endocytic events at the plasma membrane. HIV-1 hijacks the ESCRT machinery by recruiting its members through specific amino acid sequences, called late domains, in the major structural protein Gag. Specifically, the PTAP motif recruits tumor susceptibility gene 101 (Tsg101) and the LXXLF motif recruits ALG-2 interacting protein X (Alix), with PTAP being the functionally more important motif (10, 11). Biochemical and genetic assays have defined specific molecular interactions between ESCRT proteins that are recruited by Gag (8, 12–14), but a fine temporal and spatial mapping of the recruitment of viral and host components relative to each other is lacking. Here, using live-cell multiple-color total internal reflection fluorescence microscopy (TIR-FM), we demonstrate that the ESCRT protein Tsg101 is corecruited with Gag and accumulates progressively, whereas charged multivesicular body protein 4b (Chmp4b) and Vps4A are recruited sequentially and transiently to Gag assembly sites. Moreover, because diffraction-limited microscopy cannot resolve spatial differences the size of an HIV-1 virion (∼100 nm) (15–17), we determined the relative spatial positions of Gag and of several members of the ESCRT machinery in nascent virions using two-color superresolution imaging.     

抗卵泡刺激素受体纳米抗体的制备及鉴定

目的 获得抗卵泡刺激素受体(FSHR)的纳米抗体.方法 使用原核表达的重组蛋白His-FSHR234对新疆双峰驼单域抗体噬菌体展示文库进行亲和筛选,将筛选获得的重链抗体的可变区(vhh)基因亚克隆至pET30a表达载体,转化E.coli BL21 (DE3),IPTG诱导表达VHH重组蛋白,镍离子亲和层析柱纯化获得纳米抗体.ELISA检测纳米抗体的抗原结合活性.结果 His-FSHR234筛选富集后,随机挑选40个克隆进行鉴定,其中28个为阳性克隆,挑选4个vhh基因进行克隆,PCR和酶切鉴定目的基因大小与预计相符.SDS-PAGE显示,VHHFSHR-06、VHHFSHR-25、VHHFSHR-30、VHHFSHR-50分别在相对分子质量(Mr)31000、26000、25000、26000有目的条带.EHSA检测显示,4个纳米抗体对His-FSHR234重组蛋白均具有结合活性,其中VHHFSHR-06的抗原结合活性最高.结论 从新疆双峰驼单域抗体噬菌体展示文库中筛选获得了1个具有较高抗原结合活性的抗FSHR的纳米抗体.     

Intraoperative fluorescence delineation of head and neck cancer with a fluorescent Anti‐epidermal growth factor receptor nanobody

Intraoperative near‐infrared (NIR) fluorescence imaging is a technology with high potential to provide the surgeon with real‐time visualization of tumors during surgery. Our study explores the feasibility for clinical translation of an epidermal growth factor receptor (EGFR)‐targeting nanobody for intraoperative imaging and resection of orthotopic tongue tumors and cervical lymph node metastases. The anti‐EGFR nanobody 7D12 and the negative control nanobody R2 were conjugated to the NIR fluorophore IRDye800CW (7D12‐800CW and R2‐800CW). Orthotopic tongue tumors were induced in nude mice using the OSC‐19‐luc2‐cGFP cell line. Tumor‐bearing mice were injected with 25 µg 7D12‐800CW, R2–800CW or 11 µg 800CW. Subsequently, other mice were injected with 50 or 75 µg of 7D12‐800CW. The FLARE imaging system and the IVIS spectrum were used to identify, delineate and resect the primary tumor and cervical lymph node metastases. All tumors could be clearly identified using 7D12‐800CW. A significantly higher tumor‐to‐background ratio (TBR) was observed in mice injected with 7D12–800CW compared to mice injected with R2‐800CW and 800CW. The highest average TBR (2.00 ± 0.34 and 2.72 ± 0.17 for FLARE and IVIS spectrum, respectively) was observed 24 hr after administration of the EGFR‐specific nanobody. After injection of 75 µg 7D12‐800CW cervical lymph node metastases could be clearly detected. Orthotopic tongue tumors and cervical lymph node metastases in a mouse model were clearly identified intraoperatively using a recently developed fluorescent EGFR‐targeting nanobody. Translation of this approach to the clinic would potentially improve the rate of radical surgical resections.     

Targeting the EGF receptor ectodomain in the context of cancer

With the discovery of the involvement of the ErbB family of transmembrane growth factor receptors in tumour malignancy, major efforts have been undertaken to develop agents able to specifically target these receptors. With varying success, these agents have been applied to either detect the presence of ErbB receptors on cancer cells or to neutralize receptor function in order to put a hold on the unbridled tumour growth. The two most exploited classes of ErbB-targeting agents are monoclonal antibodies binding the extracellular portion of the receptor and small molecules able to interfere with the intracellular tyrosine kinase activity. Here we focus on the various kinds of agents that have recently been developed to target the extracellular region of the EGFR, the best characterised member of the ErbB family. Because clinical successes are relatively poor, alternative but less developed approaches for receptor targeting are being evaluated. Much effort has been put into the development of smaller antibody fragments. In this context, EGFR-binding nanobodies and affibodies may prove to be a more efficient novel approach in targeting EGFR-positive tumours for therapeutic and diagnostic use.     

A class II MHC-targeted vaccine elicits immunity against SARS-CoV-2 and its variants

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (Spike_RBD) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHH_MHCII). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHH_MHCII-Spike_RBD elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHH_MHCII-Spike_RBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, has caused a global pandemic, infecting over 230 million people, and leading to millions of deaths (1). Rapid distribution of effective vaccines on a global scale is the most effective means of mitigating the political, social, and economic destabilization caused by the SARS-CoV-2 pandemic.The SARS-CoV-2 spike (S) protein is a trimeric transmembrane protein that binds to the cell surface receptor angiotensin-converting enzyme 2 (ACE2) via its receptor-binding domain (RBD) and mediates fusion with host membranes (2). SARS-CoV-2 S is the primary target for neutralizing antibodies and elicits both CD4 and CD8 T cell responses during infection (3–7). Most vaccines in current use or in development target S, or fragments of S, as the primary antigen (8). Because several variants of concern have emerged, many of which contain mutations in S that partially resist neutralization by vaccine-elicited and COVID-19–elicited antibodies, vaccines that offer protection against new variants are necessary (9–11).Leading vaccine candidates use an array of diverse vaccine platforms. These include inactivated virions, DNA-based vaccines, recombinant subunit preparations, lipid-encapsulated mRNA formulations, as well as live-attenuated, replication-incompetent viral vectored, and replication-competent viral vectored vaccines (8). None of them directly and specifically target antigen-presenting cells (APCs). We hypothesized that targeted delivery of antigen to professional class II MHC⁺ APCs would improve access to the processing and presentation pathways that generate CD4 and CD8 T cell responses, in addition to provoking a robust antibody response. Our earlier efforts to generate an anti-HPV16 CD8 T cell response relied on fusions of an anti-CD11b nanobody to the immunodominant epitope of the HPV16 E7 protein as a vaccine. Its success in eradicating even established tumors inspired us to pursue a similar effort to deliver the RBD of the SARS-CoV2 S protein as a fusion with a nanobody that targets APCs (12). Most vaccines in current use require specialized storage conditions (13–15). The development of vaccines with enhanced stability to allow storage at ambient temperature and rapidly adjustable to emerging variants of the virus therefore remains a priority. Moreover, vaccines that can be produced rapidly in a scalable manufacturing process would improve access.Here we report the development of a recombinant protein vaccine that consists of the SARS-CoV-2 Spike RBD (Spike_RBD) fused to an alpaca-derived nanobody that targets class II major histocompatibility (MHC II) complex antigens (VHH_MHCII-Spike_RBD). This vaccine delivers the antigen directly to class II MHC⁺ APCs. Immunization of both young and aged mice with two doses of VHH_MHCII-Spike_RBD resulted in robust binding and neutralizing antibody responses against SARS-CoV-2 and emerging variants. Immunization also induced prominent CD8 T cell responses against conserved Spike_RBD-derived epitopes. VHH_MHCII-Spike_RBD can be produced in high yield in mammalian cells and tolerates both storage at room temperature for at least 7 d and lyophilization without loss of efficacy.     

Recombinant nanobody against MUC1 tandem repeats inhibits growth,invasion, metastasis,and vascularization of spontaneous mouse mammary tumors

Alteration in glycosylation pattern of MUC1 mucin tandem repeats during carcinomas has been shown to negatively affect adhesive properties of malignant cells and enhance tumor invasiveness and metastasis. In addition, MUC1 overexpression is closely interrelated with angiogenesis, making it a great target for immunotherapy. Alongside, easier interaction of nanobodies (single‐domain antibodies) with their antigens, compared to conventional antibodies, is usually associated with superior desirable results. Herein, we evaluated the preclinical efficacy of a recombinant nanobody against MUC1 tandem repeats in suppressing tumor growth, angiogenesis, invasion, and metastasis. Expressed nanobody demonstrated specificity only toward MUC1‐overexpressing cancer cells and could internalize in cancer cell lines. The IC50 values (the concentration at which the nanobody exerted half of its maximal inhibitory effect) of the anti‐MUC1 nanobody against MUC1‐positive human cancer cell lines ranged from 1.2 to 14.3 nm. Similar concentrations could also effectively induce apoptosis in MUC1‐positive cancer cells but not in normal cells or MUC1‐negative human cancer cells. Immunohistochemical staining of spontaneously developed mouse breast tumors prior to in vivo studies confirmed cross‐reactivity of nanobody with mouse MUC1 despite large structural dissimilarities between mouse and human MUC1 tandem repeats. In vivo, a dose of 3 µg nanobody per gram of body weight in tumor‐bearing mice could attenuate tumor progression and suppress excessive circulating levels of IL‐1a, IL‐2, IL‐10, IL‐12, and IL‐17A pro‐inflammatory cytokines. Also, a significant decline in expression of Ki‐67, MMP9, and VEGFR2 biomarkers, as well as vasculogenesis, was evident in immunohistochemically stained tumor sections of anti‐MUC1 nanobody‐treated mice. In conclusion, the anti‐MUC1 tandem repeat nanobody of the present study could effectively overcome tumor growth, invasion, and metastasis.     

Macrophage-mediated tumor-targeted delivery of engineered Salmonella typhimurium VNP20009 in anti-PD1 therapy against melanoma

Bacterial antitumor therapy has great application potential given its unique characteristics, including genetic manipulation, tumor targeting specificity and immune system modulation. However, the nonnegligible side effects and limited efficacy of clinical treatment limit their biomedical applications. Engineered bacteria for therapeutic applications ideally need to avoid their accumulation in normal organs and possess potent antitumor activity. Here, we show that macrophage-mediated tumor-targeted delivery of Salmonella typhimurium VNP20009 can effectively reduce the toxicity caused by administrating VNP20009 alone in a melanoma mouse model. This benefits from tumor-induced chemotaxis for macrophages combined with their slow release of loaded strains. Inspired by changes in the tumor microenvironment, including a decrease in intratumoral dysfunctional CD8⁺ T cells and an increase in PDL1 on the tumor cell surface, macrophages were loaded with the engineered strain VNP-PD1nb, which can express and secrete anti-PD1 nanoantibodies after they are released from macrophages. This novel triple-combined immunotherapy significantly inhibited melanoma tumors by reactivating the tumor microenvironment by increasing immune cell infiltration, inhibiting tumor cell proliferation, remodeling TAMs to an M1-like phenotype and prominently activating CD8⁺ T cells. These data suggest that novel combination immunotherapy is expected to be a breakthrough relative to single immunotherapy.     

Anti-PD-L1&CXCR4双特异性纳米抗体的纯化与活性

针对细胞程序性死亡-配体1（PD-L1）和CXC趋化因子受体4型（CXCR4）两个靶点，设计anti-PD-L1&CXCR4双特异性纳米抗体的基因序列，C末端连接组氨酸标签（6 × His标签），通过pET-22b（+）重组表达质粒转化大肠埃希菌E.coli BL21，经 IPTG 诱导表达，以可溶性形式存在于菌体裂解上清液。为了提高双特异性纳米抗体的产量和纯度，采用3种不同的方法进行样品制备和纯化。结果表明，通过机械裂菌并改进缓冲液的盐离子和咪唑浓度以及pH，经His Trap FF亲和色谱柱纯化后，对双特异性纳米抗体分离效果较好，目的蛋白产量超过1 mg/L，纯度可达到97%。同时，anti-PD-L1&CXCR4双特异性纳米抗体能够与细胞表面两个抗原特异性结合，增强IL-2活化的人外周血单个核细胞（PBMC）对胰腺癌细胞株 AsPC-1的杀伤能力，为其后续体内药效学评价奠定了基础。