Characterization of novel CD19-specific VHHs isolated from a camelid immune library by phage display.

BACKGROUND: Monoclonal antibody (mAb)-based immunotherapies have achieved promising outcomes in the treatment of immunological and oncological indications. CD19 is considered one of the most qualified antigens in the treatment of B-cell neoplasms. VHHs (nanobodies) are known for their physicochemical advantages over conventional mAbs rendering them suitable therapeutics and diagnostic tools. Herein, we aimed to isolate CD19-specific VHHs from a novel immune library using phage display. METHODS: An immune VHH gene library was constructed. Using phage display and after five biopanning rounds, two monoclonal CD19-specific VHHs were isolated. The selected VHHs were expressed, purified, and characterized in terms of their affinity, specificity, sensitivity, and ability to target CD19-positive cell lines. Moreover, in silico analyses were employed for further characterization. RESULTS: A VHH library was developed, and because the outputs of the 4th biopanning round exhibited the most favorable characteristics, a panel of random VHHs was selected from them. Ultimately, two of the most favorable VHHs were selected and DNA sequenced (designated as GR37 and GR41). Precise experiments indicated that GR37 and GR41 exhibited considerable specificity, sensitivity, and affinity (1.15 × 107 M-1 and 2.08 × 107 M-1, respectively) to CD19. Flow cytometric analyses revealed that GR37 and GR41 could bind CD19 on the surface of cell lines expressing the antigen. Moreover, in silico experiments predicted that both VHHs target epitopes that are distinct from that targeted by the CD19-specific single-chain variable fragment (scFv) FMC63. CONCLUSION: The selected VHHs can be used as potential targeting tools for the development of CD19-based immunotherapeutics.

Addressing the obstacles of CAR T cell migration in solid tumors: wishing a heavy traffic.

Chimeric antigen receptor T cell (CAR-T) therapy has been recognized as one of the most prosperous treatment options against certain blood-based malignancies. However, the same clinical and commercial success have been out of range in the case of solid tumors. The main contributing factor in this regard is the hostile environment the tumor cells impose that results in the exhaustion of immune effector cells alongside the abrogation of their infiltration capacity. The discovery of the underlying mechanisms and the development of reliable counterstrategies to overcome the inaccessibility of CAR-Ts to their target cells might correlate with encouraging clinical outcomes in advanced solid tumors. Here, we highlight the successive physical and metabolic barriers that systemically administered CAR-Ts face on their journey toward their target cells. Moreover, we propose meticulously-devised countertactics and combination therapies that can be applied to maximize the therapeutic benefits of CAR-T therapies against solid tumors.

An integrative transcriptome analysis reveals potential predictive, prognostic biomarkers and therapeutic targets in colorectal cancer.

BACKGROUND: A deep understanding of potential molecular biomarkers and therapeutic targets related to the progression of colorectal cancer (CRC) from early stages to metastasis remain mostly undone. Moreover, the regulation and crosstalk among different cancer-driving molecules including messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs) and micro-RNAs (miRNAs) in the transition from stage I to stage IV remain to be clarified, which is the aim of this study. METHODS: We carried out two separate differential expression analyses for two different sets of samples (stage-specific samples and tumor/normal samples). Then, by the means of robust dataset analysis we identified distinct lists of differently expressed genes (DEGs) for Robust Rank Aggregation (RRA) and weighted gene co-expression network analysis (WGCNA). Then, comprehensive computational systems biology analyses including mRNA-miRNA-lncRNA regulatory network, survival analysis and machine learning algorithms were also employed to achieve the aim of this study. Finally, we used clinical samples to carry out validation of a potential and novel target in CRC. RESULTS: We have identified the most significant stage-specific DEGs by combining distinct results from RRA and WGCNA. After finding stage-specific DEGs, a total number of 37 DEGs were identified to be conserved across all stages of CRC (conserved DEGs). We also found DE-miRNAs and DE-lncRNAs highly associated to these conserved DEGs. Our systems biology approach led to the identification of several potential therapeutic targets, predictive and prognostic biomarkers, of which lncRNA LINC00974 shown as an important and novel biomarker. CONCLUSIONS: Findings of the present study provide new insight into CRC pathogenesis across all stages, and suggests future assessment of the functional role of lncRNA LINC00974 in the development of CRC.

CRISPR/Cas9-mediated knockout of clinically relevant alloantigenes in human primary T cells.

BACKGROUND: The ability of CRISPR/Cas9 to mutate any desired genomic locus is being increasingly explored in the emerging area of cancer immunotherapy. In this respect, current efforts are mostly focused on the use of autologous (i.e. patient-derived) T cells. The autologous approach, however, has drawbacks in terms of manufacturing time, cost, feasibility and scalability that can affect therapeutic outcome or wider clinical application. The use of allogeneic T cells from healthy donors may overcome these limitations. For this strategy to work, the endogenous T cell receptor (TCR) needs to be knocked out in order to reduce off-tumor, graft-versus-host-disease (GvHD). Furthermore, CD52 may be knocked out in the donor T cells, since this leaves them resistant to the commonly used anti-CD52 monoclonal antibody lymphodepletion regimen aiming to suppress rejection of the infused T cells by the recipient. Despite the great prospect, genetic manipulation of human T cells remains challenging, in particular how to deliver the engineering reagents: virus-mediated delivery entails the inherent risk of altering cancer gene expression by the genomically integrated CRISPR/Cas9. This is avoided by delivery of CRISPR/Cas9 as ribonucleoproteins, which, however, are fragile and technically demanding to produce. Electroporation of CRISPR/Cas9 expression plasmids would bypass the above issues, as this approach is simple, the reagents are robust and easily produced and delivery is transient. RESULTS: Here, we tested knockout of either TCR or CD52 in human primary T cells, using electroporation of CRISPR/Cas9 plasmids. After validating the CRISPR/Cas9 constructs in human 293 T cells by Tracking of Indels by Decomposition (TIDE) and Indel Detection by Amplicon Analysis (IDAA) on-target genomic analysis, we evaluated their efficacy in primary T cells. Four days after electroporation with the constructs, genomic analysis revealed a knockout rate of 12-14% for the two genes, which translated into 7-8% of cells showing complete loss of surface expression of TCR and CD52 proteins, as determined by flow cytometry analysis. CONCLUSION: Our results demonstrate that genomic knockout by electroporation of plasmids encoding CRISPR/Cas9 is technically feasible in human primary T cells, albeit at low efficiency.

Effective suppression of tumour cells by oligoclonal HER2-targeted delivery of liposomal doxorubicin.

Synergistic effect of combined antibodies targeting distinct epitopes of a particular tumour antigen has encouraged some clinical trial studies and is now considered as an effective platform for cancer therapy. Providing several advantages over conventional antibodies, variable domain of heavy chain of heavy chain antibodies (VHH) is now major tools in diagnostic and therapeutic applications. Active targeting of liposomal drugs is a promising strategy, resulting in enhanced binding and improved cytotoxicity of tumour cells. In the present study, we produced four anti-HER2 recombinant VHHs and purified them via native and refolding method. ELISA and flow cytometry analysis confirmed almost identical function of VHHs in refolded and native states. Using a mixture of four purified VHHs, PEGylated liposomal doxorubicin was targeted against HER2-overexpressing cells. The drug release was analyzed at pH 7.4, 6.4 and 5.5 and dynamic light-scattering detector and TEM micrograph was applied to characterize the produced nanoparticles. The binding efficiency of these nanoparticles to BT474 and SKBR3 as HER2-positive and MCF10A as HER2-negative cell line was examined by flow cytometry. Our results indicated effective encapsulation of about 94% of the total drug in immunoliposomes. Flow cytometry results verified receptor-specific binding of targeted liposomes to SKBR3 and BT474 cell lines and more efficient binding was observed for liposomes conjugated with oligoclonal VHHs mixture compared with monoclonal VHH-targeted liposomes. Oligoclonal nanoparticles also showed more cytotoxicity compared with non-targeted liposomes against HER2-positive tumour cells. Oligoclonal targeting of liposomes was represented as a promising strategy for the treatment of HER2-overexpressing breast cancers.

Designing an ELP-intein system: toward a more realistic outlook.

Despite the ever-growing demand for proteins in pharmaceutical applications, downstream processing imposes many technical and economic limitations to recombinant technology. Elastin-like polypeptides tend to aggregate reversibly at a specific temperature. These biopolymers have been joined with self-cleaving inteins to develop a non-chromatographic platform for protein purification without the need for expensive enzymatic tag removal. Following the design and expression of an ELP-intein-tagged GFP, herein, we report certain complications and setbacks associated with this protein purification system, overlooked in previous studies. Based on our results, a recovery rate of 68% was achieved using inverse transition cycling. Fluorescence intensity analysis indicated a production yield of 11 mg GFP fusion protein per liter of bacterial culture. The low expression level is attributable to several factors, such as irreversible aggregation, slipped-strand mispairing or insufficiency of aminoacyl tRNAs during protein translation of the highly repetitive ELP tag. While the goals we set out to achieve were not entirely met, a number of useful tips could be gathered as a generic means for implementing ELP-intein protein purification. Overall, we believe that such reports help clarify the exact capacity of emerging techniques and build a fairly realistic prospect toward their application.

Oligo-clonal nanobodies as an innovative targeting agent for cancer therapy: New biology and novel targeting systems.

Variable heavy chain of HcAb (VHH), the smallest intact antibody fragment, possesses sub-nanomolar affinity to antigens. In spite of conventional antibodies, these fragments recognize concave and linear epitopes. VHHs are one the best weapon for targeted drug delivery in nanomedicine and biopharmaceutics. HER2 is overexpressed in 20-25% of breast and ovarian cancers. For many reasons, HER2 is a prominent target for drug delivery to breast tumor. In this study, we designed a robust prokaryotic expression system to express functional VHHs against HER2 receptor. This system showed high recombinant yields besides purified VHHs flow cytometry verified great capabilities of these molecules to pinpoint ecto-domain of HER2 receptor in MC4L2 HER2+ while insignificant non-specific binding to MC4L2 HER2-confirm nanobodies trivial cross-reaction. In the next step, we evaluated cooperative effect of four distinctive VHHs (oligoclonal VHHs) targeting different epitopes on HER2. As our result proved, using oligoclonal nanobodies as targeting moiety enhance targeting efficacy in comparison with monoclonal VHH.

Optimization of an anti-HER2 nanobody expression using the Taguchi method.

Despite being widely used in immunotherapy of cancer, whole antibodies are limited by several disadvantages. This has led to the advent of novel biomolecules such as nanobodies. Taguchi method is a statistical experimental design to study the effect of multiple variables in biological processes. In an effort to overexpress a recombinant anti-human epidermal growth factor receptor type 2 (HER2) nanobody, we performed a detailed study to find optimal condition of temperature, induction, culture media, vector, and host strain, using Taguchi methodology. A total of 16 various experiments were designed. Total protein of the formulated cultures were assessed by Bradford test and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by size exclusion high performance liquid chromatography to quantify the relative concentration of the nanobody in different expression settings. Western blotting was performed to confirm the expression of the anti-HER2 nanobody. When, individually, optimum parameters determined by Taguchi were applied, including SHuffle strain cultured in LB medium, induced with 0.4 mM isopropyl-ß-D-thio-galactoside for 18 h at 24°C, production yield further increased by about 9% (25.4 mg/L), compared to the highest expression setting. Flow cytometry and enzyme-linked immunosorbent assay result indicated improved protein binding in optimized conditions. Overall, our findings provide a basis for further investigations on economical production of recombinant nanobodies to improve production yield and activity.

T cells expressing VHH-directed oligoclonal chimeric HER2 antigen receptors: towards tumor-directed oligoclonal T cell therapy.

BACKGROUND: Adoptive cell therapy with engineered T cells expressing chimeric antigen receptors (CARs) originated from antibodies is a promising strategy in cancer immunotherapy. Several unsuccessful trials, however, highlight the need for alternative conventional binding domains and the better combination of costimulatory endodomains for CAR construction to improve the effector functions of the engineered T cells. Camelid single-domain antibodies (VHHs), which are the smallest single domain antibodies, can endow great targeting ability to CAR-engineered T cells. METHODS: We have developed a method to generate genetically engineered Jurkat T cells armed with a CAR comprising the anti-HER2 VHH as targeting moiety. From an immune camel library, five VHH clones were selected as a set of oligoclonal anti-HER2 VHHs that exhibited diverse binding abilities and joined them to CD28-CD3ζ and CD28-OX40-CD3ζ signaling endodomains. Jurkat T cells expression of VHH-CARs and cell functions were evaluated. RESULTS: The oligoclonal engineered T cells showed higher proliferation, cytokine secretion and cytotoxicity than each individual VHH-CAR-engineered Jurkat T cells. CONCLUSIONS: The combination of superior targeting ability of oligoclonal VHHs with the third generation CAR can substantially improve the function of engineered T cells. GENERAL SIGNIFICANCE: Antigen-specific directed oligoclonal T cells are alternatively promising, but safer systems, to combat tumor cells.

A nanobody directed to a functional epitope on VEGF, as a novel strategy for cancer treatment.

Compelling evidence suggests that vascular endothelial growth factor (VEGF), due to its essential role in angiogenesis, is a critical target for cancer treatment. Neutralizing monoclonal antibodies against VEGF are important class of drugs used in cancer therapy. However, the cost of production, large size, and immunogenicity are main drawbacks of conventional monoclonal therapy. Nanobodies are the smallest antigen-binding antibody fragments, which occur naturally in camelidae. Because of their remarkable features, we decided to use an immune library of nanobody to direct phage display to recognition of novel functional epitopes on VEGF. Four rounds of selection were performed and six phage-displayed nanobodies were obtained from an immune phage library. The most reactive clone in whole-cell ELISA experiments, was purified and assessed in proliferation inhibition assay. Purified ZFR-5 not only blocked interaction of VEGF with its receptor in cell ELISA experiments, but also was able to significantly inhibit proliferation response of human umbilical vein endothelial cells to VEGF in a dose-dependent manner. Taken together, our study demonstrates that by using whole-cell ELISA experiments, nanobodies against antigenic regions included in interaction of VEGF with its receptors can be directed. Because of unique and intrinsic properties of a nanobody and the ability of selected nanobody for blocking the epitope that is important for biological function of VEGF, it represents novel potential drug candidate.

The role of albumin and PPAR-α in differentiation-dependent change of fatty acid profile during differentiation of mesenchymal stem cells to hepatocyte-like cells.

Differentiation of mesenchymal stem cells (MSCs) to hepatocyte-like cells is associated with morphological and biological changes. In this study, the effect of hepatogenic differentiation on fatty acid profile and the expression of proliferator-activated receptors-α (PPAR-α) have been studied. For this purpose, MSCs isolated from human umbilical cord were differentiated into hepatocyte-like cells on selective culture media. The morphological and biochemical changes, PPAR-α expression and reactive oxygen species (ROS) levels were studied during the differentiation process. Besides, the cells were processed to determine changes in fatty acid profile using gas chromatography analysis. The results showed that hepatic differentiation of the MSCs is associated with a decrease in major polyunsaturated fatty acids in mature hepatocytes, whereas there was an increase in the saturated fatty acid (SFA) levels during hepatocyte maturation. The differentiation-dependent shift in the ratio of SFA/USFA was associated with changes in albumin and PPAR-α expression, whereas changes in fatty acid profile were independent of ROS production and lipid peroxidation in differentiating cells. In conclusion, these data may suggest that hepatocyte formation during the stem cell differentiation is associated with a shift in the fatty acid profile that is probably a normal phenomenon in hepatogenic differentiation of the MSCs.

Targeting high affinity and epitope-distinct oligoclonal nanobodies to HER2 over-expressing tumor cells.

Modern anti-HER2 antibody therapy tends to exploit a panel of different antibodies against different epitopes on the antigen. For this aim, nanobodies are very striking targeting agents and can be easily produced against any cell-specific membrane antigen. The oligoclonal nanobodies can be used to block more than one functional epitope on a target antigen and inhibit the generation of escape variants associated with cancer therapy. In this study, 12 nanobody clones selected from an immune camel library were examined for their ability to differ between tumor markers. These oligoclonal nanobodies targeted breast cancer cells better than each individual nanobody. In epitope mapping, several nanobodies overlapped in the epitope recognized by trastuzumab and some of the non-overlapping nanobodies could affect the binding of trastuzumab to HER2. This study demonstrates that the oligoclonal nanobodies are potential therapeutic tools that can be used instead of, or in combination with trastuzumab to assess tumor viability during treatment.

Occurrence of endocrine disruption chemicals (Bisphenol A, 4-nonylphenol, and Octylphenol) in muscle and liver of, Cyprinus Carpino Common, from Anzali wetland, Iran.

Phenolic endocrine disrupting chemicals are environmental pollutants with xenostrogen effects in wildlife and humans. The aim of this study was to determine 4-nonylphenol, Octylphenol, and Bisphenol A residues in various tissues of carp fish samples from Anzali wetland, Iran. 4-NP, OP, and BPA were detected with GC-MS in the muscle of fish from sampling location with maximal concentrations of 8.17, 9.67 and 5.87 µg/gdw, respectively. The highest concentrations of these compounds were found in the liver by HPLC. Since many endocrine disrupting substances were significantly lipophilic, distributing of these compounds into fish tissue has been correlated with lipid content.

New cell sources for CAR-based immunotherapy.

Chimeric antigen receptor (CAR) T cell therapy, in which a patient's own T lymphocytes are engineered to recognize and kill cancer cells, has achieved striking success in some hematological malignancies in preclinical and clinical trials, resulting in six FDA-approved CAR-T products currently available in the market. Despite impressive clinical outcomes, concerns about treatment failure associated with low efficacy or high cytotoxicity of CAR-T cells remain. While the main focus has been on improving CAR-T cells, exploring alternative cellular sources for CAR generation has garnered growing interest. In the current review, we comprehensively evaluated other cell sources rather than conventional T cells for CAR generation.

Effects of polybrene and retronectin as transduction enhancers on the development and phenotypic characteristics of VHH-based CD19-redirected CAR T cells: a comparative investigation.

Chimeric antigen receptor T cells (CAR T cells) have improved the prognosis of patients with certain hematologic malignancies. However, broader clinical application of this type of therapy is dependent on production protocols. We characterized VHH-based CD19-redirected CAR T cells generated using the transduction enhancers (TEs) polybrene or retronectin. The proliferation rate of activated T cells transduced using polybrene concentrations > 6 mg/mL decreased compared with untreated group. There was a direct relationship between polybrene concentration and transduction efficacy. Moreover, we demonstrated the proliferation of retronectin-transduced T cells increased in a dose-dependent manner (4-20 µg/mL). Whereas, different retronectin concentrations did not mediate a significant increase in T cell transduction rate. Moreover, lentiviral transduction rate was also dependent on the concentration of lentiviruses. At optimized TE concentrations, multiplicity of infection (MOI) of > 10 decreased living T cell transduction rate. Additionally, we demonstrated that CAR T cell phenotype is highly affected by TE type. Naïve T cell differentiation to central memory T cell was observed in the beginning of the expansion process and effector memory T cells became the predominant subset in the second week of expansion. Importantly, retronectin increased the proliferation of CAR T cells alongside medicating higher transduction rates, resulting in more naïve and central memory T cells. We demonstrated that a higher percentage of CAR T cells were generated using retronectin (with a less differentiated phenotype) making retronectin a more effective TE than polybrene for long-term CAR T cell processing in preclinical or clinical studies.

High-throughput isolation of cancer cells in spiral microchannel by changing the direction, magnitude and location of the maximum velocity.

Circulating tumor cells (CTCs) are scarce cancer cells that rarely spread from primary or metastatic tumors inside the patient's bloodstream. Determining the genetic characteristics of these paranormal cells provides significant data to guide cancer staging and treatment. Cell focusing using microfluidic chips has been implemented as an effective method for enriching CTCs. The distinct equilibrium positions of particles with different diameters across the microchannel width in the simulation showed that it was possible to isolate and concentrate breast cancer cells (BCCs) from WBCs at a moderate Reynolds number. Therefore we demonstrate high throughput isolation of BCCs using a passive, size-based, label-free microfluidic method based on hydrodynamic forces by an unconventional (combination of long loops and U-turn) spiral microfluidic device for isolating both CTCs and WBCs with high efficiency and purity (more than 90%) at a flow rate about 1.7 mL/min, which has a high throughput compared to similar ones. At this golden flow rate, up to 92% of CTCs were separated from the cell suspension. Its rapid processing time, simplicity, and potential ability to collect CTCs from large volumes of patient blood allow the practical use of this method in many applications.

T-cells engineered with a novel VHH-based chimeric antigen receptor against CD19 exhibit comparable tumoricidal efficacy to their FMC63-based counterparts.

Background: Chimeric antigen receptor (CAR)-T cell therapy has established itself as a potent therapeutic option for certain patients with relapsed/refractory (R/R) hematologic malignancies. To date, four CD19-redirected CAR-T cell products have been granted the United States Food and Drug Administration (FDA) approval for medical use. However, all of these products are equipped with a single-chain fragment variable (scFv) as their targeting domains. Camelid single-domain antibodies (VHH or nanobody) can also be used as alternatives to scFvs. In this study, we developed VHH-based CD19-redirected CAR-Ts, and compared them with their FMC63 scFv-based counterpart. Methods: Human primary T cells were transduced to express a second-generation 4-1BB-CD3ζ-based CAR construct whose targeting domain was based on a CD19-specific VHH. The expansion rate, cytotoxicity, and secretion of proinflammatory cytokines (IFN-γ, IL-2, and TNF-α) of the developed CAR-Ts were assessed and compared with their FMC63 scFv-based counterpart as they were co-cultured with CD19-positive (Raji and Ramos) and CD19-negative (K562) cell lines. Results: VHH-CAR-Ts showed an expansion rate comparable to that of the scFv-CAR-Ts. In terms of cytotoxicity, VHH-CAR-Ts mediated cytolytic reactions against CD19-positive cell lines, comparable to those of their scFv-based counterparts. Moreover, both VHH-CAR-Ts and scFv-CAR-Ts secreted remarkably higher and similar levels of IFN-γ, IL-2, and TNF-α upon co-cultivation with Ramos and Raji cell lines compared with while cultured alone or co-cultured with K562 cells. Conclusion: Our results demonstrated that our VHH-CAR-Ts could mediate CD19-dependent tumoricidal reactions as potently as their scFv-based counterparts. Moreover, VHHs could be applied as the targeting domains of CAR constructs to overcome the issues associated with the use of scFvs in CAR-T therapies.

Directed targeting of B-cell maturation antigen-specific CAR T cells by bioinformatic approaches: From in-silico to in-vitro.

AIMS: Chimeric Antigen Receptor (CAR) T-cell is a breakthrough in cancer immunotherapy. The primary step of successful CAR T cell therapy is designing a specific single-chain fragment variable (scFv). This study aims to verify the designed anti-BCMA (B cell maturation antigen) CAR using bioinformatic techniques with the following experimental evaluations. MAIN METHODS: Following the second generation of anti-BCMA CAR designing, the protein structure, function prediction, physicochemical complementarity at the ligand-receptor interface, and biding sites analysis of anti-BCMA CAR construct were confirmed using different modeling and docking server, including Expasy, I-TASSER, HDock, and PyMOL software. To generate CAR T-cells, isolated T cells were transduced. Then, anti-BCMA CAR mRNA and its surface expression were confirmed by real-time -PCR and flow cytometry methods, respectively. To evaluate the surface expression of anti-BCMA CAR, anti-(Fab')2 and anti-CD8 antibodies were employed. Finally, anti-BCMA CAR T cells were co-cultured with BCMA+/- cell lines to assess the expression of CD69 and CD107a as activation and cytotoxicity markers. KEY FINDINGS: In-silico results approved the suitable protein folding, perfect orientation, and correct locating of functional domains at the receptor-ligand binding site. The in-vitro results confirmed high expression of scFv (89 ± 1.15% (and CD8α (54 ± 2.88%). The expression of CD69 (91.97 ± 1.7%) and CD107a (92.05 ± 1.29%) were significantly increased, indicating appropriate activation and cytotoxicity. SIGNIFICANCE: In-silico studies before experimental assessments are crucial for state-of-art CAR designing. Highly activation and cytotoxicity of anti-BCMA CAR T-cell revealed that our CAR construct methodology would be applicable to define the road map of CAR T cell therapy.

Innovative Diagnostic Peptide-Based Technologies for Cancer Diagnosis: Focus on EGFR-Targeting Peptides.

Active targeting using biological ligands has emerged as a novel strategy for the targeted delivery of diagnostic agents to tumor cells. Conjugating functional targeting moieties with diagnostic probes can increase their accumulation in tumor cells and tissues, enhancing signal detection and, thus, the sensitivity of diagnosis. Due to their small size, ease of chemical synthesis and site-specific modification, high tissue penetration, low immunogenicity, rapid blood clearance, low cost, and biosafety, peptides offer several advantages over antibodies and proteins in diagnostic applications. Epidermal growth factor receptor (EGFR) is one of the most promising cancer biomarkers for actively targeting diagnostic and therapeutic agents to tumor cells due to its active involvement and overexpression in various cancers. Several peptides for EGFR-targeting have been identified in the last decades, which have been obtained by multiple means including derivation from natural proteins, phage display screening, positional scanning synthetic combinatorial library, and in silico screening. Many studies have used these peptides as a targeting moiety for diagnosing different cancers in vitro, in vivo, and in clinical trials. This review summarizes the progress of EGFR-targeting peptide-based assays in the molecular diagnosis of cancer.

Nanobody-based chimeric receptor gene integration in Jurkat cells mediated by φC31 integrase.

The crucial role of T lymphocytes in anti-tumor immunity has led to the development of novel strategies that can target and activate T cells against tumor cells. Recombinant DNA technology has been used to generate non-MHC-restricted chimeric antigen receptors (CARs). Here, we constructed a panel of recombinant CAR that harbors the anti-MUC1 nanobody and the signaling and co-signaling moieties (CD3ζ/CD28) with different spacer regions derived from human IgG3 with one or two repeats of the hinge sequence or the hinge region of FcγRII. The PhiC31 integrase system was employed to investigate if the recombination efficiency could be recruited for high and stable expression of T cell chimeric receptor genes. The effect of nuclear localization signal (NLS) and two different promoters (CMV and CAG) on efficacy of PhiC31 integrase in human T cell lines was evaluated. The presence of integrase in combination with NLS, mediated up to 7.6 and 8.5 fold increases in CAR expression in ZCHN-attB and ZCHHN-attB cassette integrated T cells, respectively. Our results showed that highly efficient and stable transduction of the Jurkat cell line by PhiC31 integrase is a feasible modality for generating anti-cancer chimeric T cells for use in cancer immunotherapy.