Fragment scFv Research Articles

Abstract 3238: Engineering chimeric antigen receptors for adoptive T cell therapy of cancers that express the Tn antigen

Abstract Chimeric antigen receptors (CARs) have shown remarkable promise in treating hematological malignancies, especially those expressing CD19 antigen. On the other hand, identification and efficacy of CARs against antigens in solid cancers has remained a significant challenge. Defects in glycosylation in solid cancers are common, and can result in formation of cell surface-expressed neoantigens that are aberrantly glycosylated. Here, we focused on antigens that result from defects in O-linked glycosylation, leading to formation of unique antigens that possess an N-acetyl-galactosamine linked to a serine or threonine residue on a surface-expressed protein (also called Tn antigen), instead of extended glycosylation patterns. 237 is a mouse monoclonal antibody that was generated by a mouse immunized with cells of a spontaneous murine cancer, and that binds to a Tn-antigen linked to the mouse protein, OTS8. Previously, we showed that a CAR generated from the 237 single chain variable fragment (scFv) recognized, with low affinity, the human cell line Jurkat. More recently, we demonstrated that the 237 CAR could eradicate established Jurkat leukemia in a mouse model. Here, we used structure-guided directed evolution to engineer the binding site of 237 to bind to multiple Tn linked antigens, including MUC1, with higher affinity. CARs containing these engineered scFv variants recognized a panel of glycosylation-defective mouse and human cancer cell lines more effectively than the wild-type 237 CAR, with no activity toward cell lines lacking these defects. Cancer cell lines lacking MUC1 were also effectively recognized by the engineered variants, indicating their broadened specificity toward multiple Tn-linked antigens. Consistent with this finding, the CAR variants also demonstrated greater sensitivity toward several Tn-linked human peptides. To extend these studies, we are currently developing additional mouse models to study the efficacy of the engineered 237 CARs in the control of solid tumors. In summary, we developed efficient chimeric antigen receptors that recognize multiple, cancer-associated Tn linked antigens, based on a single antibody scaffold. We believe that their specific recognition of Tn antigen, together with broadened peptide-backbone reactivity, holds promise for cancer-specific recognition yet minimal antigen escape against tumors with defects in O-linked glycosylation, because cancer-specific Tn-linked epitopes on several independent proteins can be simultaneously targeted. Citation Format: Preeti Sharma, Venkata VVR Marada, Monika Kizerwetter, Claire P. Schane, Yanran He, Steven P. Wolf, Karin Schreiber, Edward J. Roy, Henrik Clausen, Hans Schreiber, David M. Kranz. Engineering chimeric antigen receptors for adoptive T cell therapy of cancers that express the Tn antigen [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3238.

Abstract 5653: Discovery of Anti-NKG2A antibody from immunized phage display approach

Abstract The inhibitory receptor NKG2A-CD94 is mainly expressed on NK cells and some T cell and mediates an inhibitory signal through association with a non-classical MHC class I molecule HLA-E, which is commonly over-expressed in human cancers. Targeting NKG2A with monoclonal antibody holds potentials in immunotherapy of cancer. However, proteins with homologous sequences such as NKG2C/CD94 or NKG2E/CD94 complexes generally deliver an activating signal upon ligand binding. NKG2A-specific antagonizing antibodies would be needed to further explore therapeutic potentials of targeting NKG2A for immunotherapy of cancer. We have developed and characterized panels of monoclonal antibodies (mAbs) that specifically recognize NKG2A. Balb/c and SJL mice had been immunized with recombinant NKG2A/CD94 protein, recombinant plasmids encoding hNKG2A or stable cell line with ectopic expression of hNKG2A. Splenocytes from such immunized animals were collected for construction of phage-displayed antibody library in a single chain variable fragment (scFv) format. The NKG2A-specific antibodies, that had no detectable binding to NKG2C, NKG2E or CD94, were isolated through multiple rounds of phage panning and ELISA-based screening. Such antibodies also block HLA-E ligand binding and enhanced cytotoxicity of NK cell lines and primary NK cells on cancer cells. The final lead is a humanized antibody which behaves well in stability test. Lab scale processes are successfully developed and confirmed. The safety and efficacy will be further validated. Citation Format: Teddy Yang, Jing Gao, Jingyun Yao, Dongxu Wang, Ken Dai. Discovery of Anti-NKG2A antibody from immunized phage display approach [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5653.

Abstract 525: A novel anti-PD1-IL15 immunocytokine potentiates anti-tumor T cell activity of PD-1 checkpoint inhibition and IL-2R-beta-gamma agonism

Abstract Therapeutic antibodies targeting immune checkpoint inhibitors (ICI) such as PD-1/PD-L1 effectively expand and reactivate T cells in patients, leading to long-lasting response in multiple tumor types. However, only a fraction of patients responded to approved ICIs, majority are either resistant or quickly become refractory. Other immunotherapy modalities including immunostimulatory cytokines IL-2, IL-12 and IL-15 demonstrated clinical benefits as monotherapy or in combination with ICIs. Cytokines exhibit in general a short half-life and systemic toxicities that limit their use as the optimal doses in treating cancer. An antibody-based immunocytokine fusion such as Kadmon lead candidate KD033 (anti-PDL1/IL15) and Roche anti-PD1/CEA/FAP-IL2v can extend the half-life of cytokine and reduce systemic toxicities. In this study, a fully human single-chain variable fragment (scFv) antibody against mouse PD1 (mPD1) have been isolated from the phage display library. The variable domains of these antibodies were fused to either human IgG1a or mouse IgG2a constant domain first, and then fused to either human IL15 receptor alpha sushi domain-hIL15 complex (hSD15) or mouse IL15 receptor alpha sushi domain-mIL15 complex (mSD15). These variations of fusion have been tested in in vitro binding and functional assays, and in vivo in PD1/PDL1 resistant mouse syngeneic model LL2 (Lewis Lung Carcinoma). We demonstrated that hSD15 fusion antibody can stimulate both IL2 dependent human and murine cells growth, surprisingly, mSD15 fusion antibody can only stimulate murine cell growth. Although mSD15 fusion antibody EC50 was 10 folds better than hSD15 fusion antibody in stimulating mouse spleen cell growth in vitro, it did not show a better efficacy than hSD15 fusion antibody in LL2 model. In vivo, mSD15 fusion antibody had higher maximum tolerate dose than hSD15 fusion antibody (up to 12mg/kg). In addition, anti-mPD1/mSD15 showed better efficacy than either the mIL15 and anti-mPD1 alone or the combination of anti-mPD1 plus mIL15 in vitro and in vivo. Together these preclinical data have confirmed that PD1-IL15 could be a promising next generation IL-15 fusion for cancer immunotherapy. Citation Format: Dan Lu, Zhanna Polonskaya, Tzi-Pei Chang, Stella Martomo, Xenia Luna, Zhikai Zhang, Faical Miyara, Jeedar Patel. A novel anti-PD1-IL15 immunocytokine potentiates anti-tumor T cell activity of PD-1 checkpoint inhibition and IL-2R-beta-gamma agonism [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 525.

Abstract 6606: Develop dual-targeted CAR-T cells to achieve RCC cures

Abstract Clear cell renal cell carcinoma (ccRCC) is the major type of RCC and is among the 10 most common cancers in both men and women. ccRCC is characterized by the loss of chromosome 3p, where the von-Hippel-Lindau (VHL) gene is found. Its gene product, pVHL suppresses the hypoxia inducible factor (HIF) transcription factors and the deletion of VHL leads to HIF hyperactivation, upregulating the expression of many downstream genes involved in angiogenesis, metabolism, and cell-cycle regulation. Two gene products, carbonic anhydrase IX (CAIX) and CD70 which we are pursuing as therapeutic targets are downstream genes of the HIF-1α pathway and thus commonly overexpressed in ccRCC. Despite the development of checkpoint blockade inhibitors (CBIs) for the treatment of this disease, curative therapies are rare. Chimeric Antigen Receptor (CAR)-T cells are a new type of “living drug”, in which T cells are engineered to express a single chain variable antibody fragment (scFv) linked to an intracellular signaling motif that includes CD3ζ (z) activation domain (first generation), with CD28 or 41BB (second generation), or both (third generation) costimulatory domains. CAR-T cells have proven to be a powerful, clinically translatable immunotherapy for hematologic malignancies. However, these results have not been translatable to solid tumors due to inefficient homing of CAR-T cells, the suppressive tumor microenvironment (TME), and on-target off-tumor toxicities resulting from the sharing of CAR-T target epitopes on healthy tissues. To translate CAR-T cell therapy to ccRCC, we developed dual-targeted CAR-T cells against both CAIX and CD70, in which dual CAR can target double positive (CAIX+ CD70+) and single positive (CAIX+ CD70-, CAIX- CD70+) tumor cell populations to address this heterogeneity. By IHC staining, we demonstrated that these two target antigens are overexpressed in ccRCC and represent circa 90% of the tumor cell population regardless of disease stages. Here we build on our previous findings and demonstrate that 41BB CAR and CD4/CD8 CAR-T cell mixtures exhibit the superior efficacy and persistence in our ccRCC orthotopic NSG-SGM3 mouse model compared to other CAR constructs and CD8 alone. A panel of dual-targeted CARs have been encoded in our 41BB CAR construct and assessed in vitro and in vivo by using Celigo imaging cytometry assay and humanized ccRCC orthotopic NSG-SGM3 mouse model respectively. Dual-targeted anti-CAIX/CD70 CD4/8 CAR-T cells outperformed singly-targeted CAR-T cells with elevated efficacy. In summary, anti-CAIX/CD70 CAR-T cells hold a great promise to achieve ccRCC cures. Citation Format: Yufei Wang, Marion Grimaud, Alicia Buck, Atef Fayed, Matthew Chang, Rebecca Jennings, Maura Sticco-Ivins, Miriam Ficial, Leo L. Chan, Sabina Signoretti, Quan Zhu, Wayne A. Marasco. Develop dual-targeted CAR-T cells to achieve RCC cures [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6606.

Screening for scFv-fragments that are stable and active in the cytosol.

Intrabodies are antibodies that are not secreted but bind to their antigens inside the cell producing them. Intrabodies targeting antigens in the endoplasmatic reticulum were successfully used in vitro and in vivo. However, many target antigens interesting for research or therapy are located in the reducing environment of the cytosol, where correct folding and formation of disulfide bonds cannot be ensured. The majority of different scFv fragments, when expressed in the cytosol of the cell, do not fold correctly, are not stable or cannot bind their antigen. Such scFv antibodies are therefore not suited as intrabodies.In this study, we evaluated fast and simple screening methods to identify scFv fragments that are stable and functional in the cytosol. We analyzed various phage display derived human scFv antibodies recognizing extracellular signal-regulated kinase 2 (Erk2) for stability and antigen binding under reducing and non-reducing conditions. Further, we developed an assay allowing to measure the interaction of the scFv intrabodies with their antigen in the cytosol of in living cells, by using a Split-Luciferase (Split-Luc) assay. ScFv fragments showing antigen binding in the cytosol could successfully be identified.

Rapid Evaluation of Antibody Fragment Endocytosis for Antibody Fragment-Drug Conjugates.

Antibody–drug conjugates (ADCs) have emerged as the most promising strategy in targeted cancer treatment. Recent strategies for the optimization ADCs include the development of antibody fragment–drug conjugates (FDCs). The critical factor in the successful development of ADCs and FDCs is the identification of tumor antigen-specific and internalizing antibodies (Abs). However, systematic comparison or correlation studies of internalization rates with different antibody formats have not been reported previously. In this study, we generated a panel of scFv-phage Abs using phage display technology and their corresponding scFv and scFv-Fc fragments and evaluated their relative internalization kinetics in relation to their antibody forms. We found that the relative rates and levels of internalization of scFv-phage antibodies positively correlate with their scFv and scFv-Fc forms. Our systematic study demonstrates that endocytosis of scFv-phage can serve as a predictive indicator for the assessment of Ab fragment internalization. Additionally, the present study demonstrates that endocytic antibodies can be rapidly screened and selected from phage antibody libraries prior to the conversion of phage antibodies for the generation of the conventional antibody format. Our strategic approach for the identification and evaluation of endocytic antibodies would expedite the selection for optimal antibodies and antibody fragments and be broadly applicable to ADC and FDC development.

Structure-guided engineering of the affinity and specificity of CARs against Tn-glycopeptides

The potency of adoptive T cell therapies targeting the cell surface antigen CD19 has been demonstrated in hematopoietic cancers. It has been difficult to identify appropriate targets in nonhematopoietic tumors, but one class of antigens that have shown promise is aberrant O-glycoprotein epitopes. It has long been known that dysregulated synthesis of O-linked (threonine or serine) sugars occurs in many cancers, and that this can lead to the expression of cell surface proteins containing O-glycans comprised of a single N-acetylgalactosamine (GalNAc, known as Tn antigen) rather than the normally extended carbohydrate. Previously, we used the scFv fragment of antibody 237 as a chimeric antigen receptor (CAR) to mediate recognition of mouse tumor cells that bear its cognate Tn-glycopeptide epitope in podoplanin, also called OTS8. Guided by the structure of the 237 Fab:Tn-OTS8-glycopeptide complex, here we conducted a deep mutational scan showing that residues flanking the Tn-glycan contributed significant binding energy to the interaction. Design of 237-scFv libraries in the yeast display system allowed us to isolate scFv variants with higher affinity for Tn-OTS8. Selection with a noncognate human antigen, Tn-MUC1, yielded scFv variants that were broadly reactive with multiple Tn-glycoproteins. When configured as CARs, engineered T cells expressing these scFv variants showed improved activity against mouse and human cancer cell lines defective in O-linked glycosylation. This strategy provides CARs with Tn-peptide specificities, all based on a single scFv scaffold, that allows the same CAR to be tested for toxicity in mice and efficacy against mouse and human tumors.

Comparative analysis of rapid agglutination latex test using single-chain antibody fragments (scFv) versus the gold standard Vero cell assay for Shiga toxin (Stx) detection

The latex agglutination test using single-chain antibody fragments (scFvStx1 and scFvStx2) coupled to latex particles, was compared with the gold standard Vero cell assay for Shiga toxin (Stx) detection, aiming to estimate the diagnosis potential of these scFv fragments in a rapid and straightforward test. The latex complexes identified the presence of the toxins up to a 1:8 dilution in the majority of the evaluated strains. Moreover, the Stx concentration was indirectly determined in Stx-producing Escherichia coli (STEC) strains, allowing detection limit inference. A Stx dilution curve was constructed, and the data was analyzed in a non-linear model by second-order polynomial regression for prediction (p-value of 0.001 and a R2 above 0.98 were considered for correlations). The detection limit was 30 ng/mL for Stx1 and 10 ng/mL for Stx2. The scFvStx1 and scFvStx2 coupled to latex nanoparticles provide a toxin assay with a competitive Stx detection limit, which has a low cost and short execution time. The diagnostic method proposed here, using, for the first time, recombinant antibody fragments, raises the possibility of developing a more affordable test to be used in the routine detection and surveillance of STEC infections.

Construction and evaluation of an antibody phage display library targeting heparan sulfate

Heparan sulfate (HS) is a linear polysaccharide with high structural diversity. Different HS epitopes have been detected and localized using single chain variable fragment (scFv) antibodies from a ‘single pot’ phage display library containing a randomized complementarity determining region of the heavy chain (CDR3). In this study, we created a new library containing anti-HS scFvs that all harbor a dp-38 heavy chain segment where the CDR3 region was engineered to contain the XBBXBX heparin binding consensus site (X = any amino acid, B = R, K or H). The library contained ~1.73 × 106 unique antibodies and was biopanned against HS from several sources. The selected antibodies were sequenced and chemically/immunohistologically characterized. A number of 67 anti-HS scFv antibodies were selected, of which 31 contained a XBBXBX CDR3 sequence. There was a clear preference for glycine at the first and proline at the fourth position of the CDR3. The sequence GZZP(R/K)X (Z = R, K or H, but may also contain N, S, or Q) was unusually overrepresented. Selected antibodies reacted with HS/heparin, but not with other glycosaminoglycans. Antibodies reacted differentially with respect to N-, 2-O, or 6-O-desulfated heparin preparations, and showed distinct topologies of HS epitopes in rat kidney sections. The library may be instrumental in the selection of a large pool of HS epitope-specific antibodies, and - since all antibodies differ only in their 6 amino acid CDR region - may be a tool for a rational design of antibodies recognizing specific HS sulfation patterns.

Construction, Expression, and Identification of Double Light Chain (VL-VL) Antibody from a Unique Bt Cry1-Specific Monoclonal Antibody

Using the hybridoma cells 2D3 which secrete anti-Cry1 monoclonal antibody as DNA source, the variable region genes of heavy chain (VH) and light chain (VL) were amplified and assembled as single-chain variable fragment (scFv). The scFv-2D3 genes were cloned into pET-26b vectors for expression and scFv-2D3 expression was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot (WB). The activity of scFv-2D3 was determined by indirect enzyme-linked immune sorbent assay (ELISA). As a result, scFv-2D3 was successfully constructed and expressed, with specific recognition activities to the Cry1 toxins. Homologous modeling and molecular docking results showed that VL was the main combination domain of scFv-2D3 with Cry1 toxins. Therefore, VH and VL were separately expressed and then assayed by indirect ELISA, indicating that both VH and VL could recognize the Cry1 toxin, and the activity of VL was higher than that of VH, which confirming the molecular docking result. Afterward, double light chain antibodies (VL-VL) were designed and produced, and the binding activities of VL-VL against Cry1Aa, Cry1Ab, and Cry1Ac were significantly enhanced with OD450 values improved 1.18, 1.44, and 1.22 times, respectively, compared with scFv-2D3. For VL-VL-based double antibody sandwiched ELISA (DAS-ELISA), the limits of detection (LOD) and limits of quantification (LOQ) were 10.5–16.0 and 12.8–21.5 ng mL−1 for seven Cry1 toxins, respectively, which were lower than scFv-2D3 (14.8–29.2 and 39.0–50.3 ng mL−1). The results were beneficial to developing high-throughput and high-sensitive immune-detecting technology for Cry1 toxins.

Construction and expression of chimeric antigen receptors targeting epidermal growth factor receptor (EGFR) and programmed cell death ligand-1(PD-L1)

Objective To construct an expression system of lentivirus vector encoding epidermal growth factor receptor-specific chimeric antigen receptor (EGFR-CAR) and programmed cell death ligand-1 (PD-L1) antibody. Methods Human PD-L1-Fc protein was used to immunize BALB/c mice. Cell-fusion and subcloning were performed to screen stable hybridoma strains with high secretion of PD-L1-specific antibodies, which were identified by both ELISA and Western blot. The activity of the antibodies in blocking the binding of programmed cell death-1 (PD-1) to PD-L1 was determined by fluorescence-activated cell sorting (FACS). Antibody affinity was analyzed by Fortebio Octet96. A single-chain variable fragment (scFv) was further constructed after antibody full-length sequencing and humanization using CDR grafting method. Meanwhile, the genes encoding the light and heavy chain variable regions (VL and VH) were cloned from a hybridoma secreting antibody against human EGFR by 5′ RACE technology to construct scFv gene. The expression of scFv was confirmed using pcDNA3.1 vector. EGFR-CAR containing CD137 intracellular function domain and PD-L1-scFv was ligated using 2A gene. The synthetic single molecule was cloned into pLVX-EF1a-IRES-ZsGreen1 lentivirus expression vector, and then transfected into 293T cells using Lenti-X Packaging Single Shots (VSV-G) to prepare infectious virus. Expression of CAR on cell surface and the soluble form of PD-L1-scFv in the supernatant of transfected 293V cells were detected by FACS and ELISA. Results A PD-L1 antibody named 11E3 with high ligand-receptor blocking performance was obtained. The humanized antibody showed a stable affinity (2.67×10-10 mol/L) after directly grafting the mouse CDRs (CDR1, CDR2 and CDR3) to human frameworks. EGFR-scFv was effectively expressed in a form of Fc-fusion. Secretory CAR (CTZ0431-1) and membrane CAR (CTZ0431-2) expression plasmids were constructed using lentivirus vector containing EGFR-CAR and PD-L1-scFv. The infection efficiency in 293V cells was around 10%. EGFR-scFv on the cell membranes and PD-L1-scfv in the culture supernatants were detected after 293V cells were infected with CTZ0431-1. EGFR-scFv and PD-L1-scfv were expressed on the cell membranes of 293V cells infected with CTZ0431-2. The expression rate of CAR in LV-CART46407-1-transfected activated T cells was 39.3%. Conclusions The lentivirus vectors co-expressing EGFR-CAR with moderate binding affinity and PD-L1-scFv with high binding affinity were successful constructed, which provided an essential tool for investing EGFR- and PD-L1 double targeted CAR-T cell therapy against solid tumor. Key words: EGFR-CAR; PD-L1-scFv; Membrane expression; Secretory expression; Synthetic biotechnology

Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing

Recently, we reported PPARα-dependent DNA demethylation of the Fgf21 promoter in the postnatal mouse liver, where reduced DNA methylation is associated with enhanced gene expression after PPARα activation. However, there is no direct evidence for the effect of site-specific DNA methylation on gene expression. We employed the dCas9-SunTag and single-chain variable fragment (scFv)-TET1 catalytic domain (TET1CD) system to induce targeted DNA methylation of the Fgf21 promoter both in vitro and in vivo. We succeeded in targeted DNA demethylation of the Fgf 21 promoter both in Hepa1-6 cells and PPARα-deficient mice, with increased gene expression response to PPARα synthetic ligand administration and fasting, respectively. This study provides direct evidence that the DNA methylation status of a particular gene may determine the magnitude of the gene expression response to activation cues.

Adenovirus-mediated anti-AEG-1 ScFv expression driven by stathmin promoter inhibits tumor growth in cervical cancer

BackgroundAstrocyte-elevated gene-1 (AEG-1) is over-expressed in many cancer cells and has multiple key functions in tumor initiation and progression. Currently, targeted-AEG-1 siRNA is one of the most common techniques to down-regulate AEG-1 expression, but the lack of tumor specificity and available delivery system make it difficult to enter clinical trials.MethodsIn this study, we creatively developed an adenovirus-mediated anti-AEG-1 single-chain antibody fragment (ScFv) expression system driven by a tumor specific promoter, and experimented with it in human cervical carcinoma cells to investigate the effect on tumor’s proliferation and apoptosis.ResultsThe results showed that of HeLa and SiHa cells treated with this recombinant anti-AEG-1 ScFv adenovirus not only inhibited cell growth, but induced apoptosis both in vitro and in vivo. Furthermore, we also observed that the expressions of several apoptosis-related genes like Akt 1 and c-Myc decreased, while NF-κB (p65) and cleaved caspase 3 increased on protein levels in vivo.ConclusionWe concluded that stathmin promoter-driving anti-AEG-1 ScFv adenoviral system may be a breakthrough for its dual-specificity, and serve as an adjuvant tumor specific therapy method in the treatment for human cervical cancers.

PET imaging of PD-L1 expression in NSCLC: Pharmacokinetic and dosimetric comparison of full antibody and associated antibody fragments

The interaction of programmed death ligand 1 (PD-L1) with its receptor PD-1 located on T-cells promotes tumour cell immuno-escape. Non-small cell lung cancer (NSCLC) patients with tumours scored as PD-L1 positive tend to have more robust responses to PD-1/PD-L1 immunotherapy, making PD-L1 an attractive biomarker for patient selection. Immunohistochemistry (IHC) on tumour biopsies is currently the reference standard for assessing PD-L1 expression. However, this process faces several issues, including intra-tumoural PD-L1 heterogeneity, or primary vs. metastatic biopsies. PET imaging tools may provide a more holistic analysis of in vivo PD-L1 expression, thereby improving patient selection for PD-1/PD-L1 immunotherapy. PET tracers should ideally achieve high tumour-to-background ratios at early time points, while being rapidly cleared. For this purpose, we developed a cross-species (human/mouse) anti-PD-L1 antibody (named C4), as well as its Fab and scFv fragments. The best PD-L1 PET tracer will be selected by comparing pharmacokinetics, biodistribution, and dosimetry of the antibody/antibody fragments radiolabelled with 89Zr or 18F in nude mice bearing subcutaneous human NSCLC tumours. Murine 3D imaging-based dosimetry analysis will be performed using the RODES software developed by the IRSN Institute.

Assessment of an scFv Antibody Fragment Against ELTD1 in a G55 Glioblastoma Xenograft Model

Glioblastoma (GBM), the most common primary brain tumor found in adults, is extremely aggressive. These high-grade gliomas, which are very diffuse, highly vascular, and invasive, undergo unregulated vascular angiogenesis. Despite available treatments, the median survival for patients is dismal. ELTD1 (EGF, latrophilin, and 7 transmembrane domain containing protein 1) is an angiogenic biomarker highly expressed in human high-grade gliomas. Recent studies have demonstrated that the blood-brain barrier, as well as the blood-tumor barrier, is not equally disrupted in GBM patients. This study therefore aimed to optimize an antibody treatment against ELTD1 using a smaller scFv fragment of a monoclonal antibody that binds against the external region of ELTD1 in a G55 glioma xenograft glioma preclinical model. Morphological magnetic resonance imaging (MRI) was used to determine tumor volumes and quantify perfusion rates. We also assessed percent survival following tumor postdetection. Tumor tissue was also assessed to confirm and quantify the presence of the ELTD1 scFv molecular targeted MRI probe, as well as microvessel density and Notch1 levels. In addition, we used molecular-targeted MRI to localize our antibodies in vivo. This approach showed that our scFv antibody attached-molecular MRI probe was effective in targeting and localizing diffuse tumor regions. Through this analysis, we determined that our anti-ELTD1 scFv antibody treatments were successful in increasing survival, decreasing tumor volumes, and normalizing vascular perfusion and Notch1 levels within tumor regions. This study demonstrates that our scFv fragment antibody against ELTD1 may be useful and potential antiangiogenic treatments against GBM.

Construction of a new anti-CD123 chimeric antigen receptor T cells and effect of anti-acute myeloid leukemia

目的构建一种新的靶向CD123的嵌合抗原受体T细胞(CAR-T细胞)，为CD123阳性白血病的免疫治疗提供实验参考。方法通过单克隆筛选技术获得能稳定分泌CD123抗体的杂交瘤细胞株6E11，将杂交瘤细胞扩增后腹腔注射至Balb/c小鼠腹腔内，收集腹水并处理、纯化得到单克隆抗体，测定抗体效价并对其特异性进行验证；RT-PCR法获得轻链和重链可变区序列，并以此为基础利用分子克隆技术构建一种新的靶向CD123嵌合抗原受体，包装病毒后感染T细胞制备CD123CAR-T细胞，通过功能实验初步探讨6E11CAR-T细胞体外抗白血病的能力。结果①获得1株稳定分泌抗人CD123抗体的杂交瘤细胞株6E11并获得其可变区序列。②6E11单克隆抗体对CD123蛋白亲和性高，解离常数(Kd值)为2.10nmol/L，特异性识别CD123阳性细胞且与CD123阴性细胞无交叉反应。 ③成功构建了CD123CAR慢病毒载体，感染T细胞后获得了靶向CD123的CAR-T细胞(6E11CAR-T)，感染效率大于60％。 ④6E11CAR-T能明显杀伤CD123阳性靶细胞MV4-11，效靶比1∶1时6E11CAR-T细胞对MV4-11细胞的杀伤比例明显高于 Vecor-T细胞[(98.60±1.20)％对(20.28±6.74)％，P<0.001]，但对CD123阴性靶细胞K562没有明显杀伤作用。 ⑤MV4-11细胞可以显著激活6E11CAR-T，但对Vecor-T细胞无明显激活作用[(26.33±3.30)％对(1.17±0.06)％，P<0.001]。⑥6E11CAR-T与MV4-11细胞共培养上清中细胞因子水平均显著高于Vecor-T组[IL-2：(92.90±1.51)pg/ml对(6.05±3.41)pg/ml，P<0.001；TNF-α：(1 407.20±91.95)pg/ml对(7.86±0.85)pg/ml，P<0.001；IFN-γ：(5 614.60±170.17)pg/ml对(8.42±2.70)pg/ml，P<0.001]但与 K562细胞共培养后，两组各细胞因子水平差异无统计学意义。 ⑦6E11CAR-T在与CD123阳性髓系白血病(AML)原代细胞共培养过程中被显著激活，且能有效杀伤原代 AML细胞。结论杂交瘤细胞株6E11能稳定分泌高效特异的抗人CD123单克隆抗体，可用于检测表达人CD123的细胞，也能应用在靶向人CD123蛋白的肿瘤免疫治疗中，以 6E11Ig可变区序列为抗原识别区的CD123CAR-T细胞，具有明确的体外抗白血病活性，为进一步的临床研究奠定了基础。

Construction and heterologous overexpression of two chimeric proteins carrying outer hydrophilic loops of Vairimorpha ceranae and Nosema bombycis ATP/ADP carriers

Microsporidia Nosema bombycis and Vairimorpha ceranae cause destructive epizootics of honey bees and silkworms. Insufficient efficiency of the antibiotic fumagillin against V. ceranae, its toxicity and the absence of effective methods of N. bombycis treatment demand the discovery of novel strategies to suppress infections of domesticated insects. RNA interference is one such novel treatment strategy. Another one implies that the intracellular development of microsporidia may be suppressed by single-chain antibodies (scFv fragments) against functionally important parasite proteins. Important components of microsporidian metabolism are non-mitochondrial, plastidic-bacterial ATP/ADP carriers. These membrane transporters import host-derived ATP and provide the capacity to pathogens for energy parasitism. Here, we analyzed membrane topology of four V. ceranae and three N. bombycis ATP/ADP transporters to construct two fusion proteins carrying their outer hydrophilic loops contacting with infected host cell cytoplasm. Interestingly, full-size genes of N. bombycis transporters may be derived from the Asian swallowtail Papilio xuthus genome sequencing project. Synthesis of the artificial genes was followed by overexpression of recombinant proteins in E. coli as insoluble inclusion bodies. The gene fragments encoding the loops of individual transporters were also effectively expressed in bacteria. The chimeric antigens may be used to construct immune libraries or select microsporidia-suppressing scFv fragments from synthetic, semisynthetic, naïve and immune antibody libraries. A further expression of such antibodies in insect cells may increase their resistance to microsporidial infections.

Impact of enzymatic reduction on bivalent bispecific antibody fragmentation and loss of product purity upon reoxidation.

Antibody disulfide bond (DSB) reduction during manufacturing processes is a widely observed phenomenon attributed to host cell reductases present in harvest cell culture fluid. Enzyme-induced antibody reduction leads to product fragments and aggregates that increase the impurity burden on the purification process. The impact of reduction on bivalent bispecific antibodies (BisAbs), which are increasingly entering the clinic, has yet to be investigated. We focused on the reduction and reoxidation properties of a homologous library of bivalent BisAb formats that possess additional single-chain Fv (scFv) fragments with engineered DSBs. Despite all BisAbs having similar susceptibilities to enzymatic reduction, fragmentation pathways were dependent on the scFv-fusion site. Reduced molecules were allowed to reoxidize with and without low pH viral inactivation treatment. Both reoxidation studies demonstrated that multiple, complex BisAb species formed as a result of DSB mispairing. Furthermore, aggregate levels increased for all molecules when no low pH treatment was applied. Combined, our results show that complex DSB mispairing occurs during downstream processes while aggregate formation is dependent on sample treatment. These results are applicable to other novel monoclonal antibody-like formats containing engineered DSBs, thus highlighting the need to prevent reduction of novel protein therapeutics to avoid diminished product quality during manufacturing.

Selection of novel human scFvs against cancer antigen IL1RAP by phage and yeast surface display technology

The objective of this study was isolation of humanized single-chain variable fragment (scFv) antibodies against IL1RAP, a promising cancer marker in leukemia and other tumors. Here, we screened for antibody fragments that recognized IL1RAP using a phage-display library of humanized scFv through several rounds of bio-panning and further FACS selection of enriched yeast-display sub-library. Two specific scFv clones 02 and 61 were isolated with high affinity against IL1RAP. Selected IL1RAP scFv antibodies containing Fc segment were expressed and purified from 293F cells. scFv-02 expressed and purified from mammalian cells exhibited higher IL1RAP binding affinity on IL1RAP-positive 293F cell surface compared to scFv-61. In addition, scFv-02 was significantly specific to IL1RAP, as no binding signal of IL1RAP was detected on the surface of IL1RAP CRISPR-Cas9 edited cell line generated in this study. Two scFv antibodies targeting IL1RAP were isolated by phage and yeast display screening. scFv-02 could serve as a promising material for further development of IL1RAP targeted immunotherapy of leukemia and other diseases. Our work also modified the process for the selection of scFv against targeted antigen by stepwise using phage display, yeast display and mammalian cell display techniques.

The Use of a Shear Device to Monitor the Stability of a Single-Chain Variable Fragment (scFv) Fusion Protein MFECP1

Antibody based drugs are increasingly being used to treat a vast array of diseases because of their unique affinity to target specific antigen proteins on the surfaces of target cancer cells. Fusions of antibodies and conjugated biopharmaceuticals are progressively being used as this gives the opportunity to target other cytotoxic molecules to unwanted cells. It is critical to ensure these types of drug products are not fragile or uneconomical to produce at a large scale. A very small amount of precious protein solution can be characterised in an Ultra scale-down (USD) shear device to uncover if fusion proteins are prone to shear stress. This article presents how the purified and deglycosylated form of the MFECP1 fusion protein was quantified with an ELISA from 700-50 ng/ml, with a +/- 10% deviation in the standard curve. It also describes how the same MFECP1 fusion protein was analysed to establish the optimum experimental control conditions that were required to observe changes due to hydrodynamic-associated degradation in a shear device. Lastly, it looks at how a first order kinetic relationship can be used to model the rate of MFECP1 fusion protein degradation and how this was used to quantify the rate of protein loss during different shear environments with and without air/liquid interfaces.