Bispecific immune cell engager enhances the anticancer activity of CD16+ NK cells and macrophages in vitro, and eliminates cancer metastasis in NK humanized NOG mice.

BACKGROUND: In a prior report, we detailed the isolation and engineering of a bispecific killer cell engager, referred to as BiKE:E5C1. The BiKE:E5C1 exhibits high affinity/specificity for the CD16a activating receptor on natural killer (NK) cells and human epidermal growth factor receptor 2 (HER2) on cancer cells. In vitro studies have demonstrated that BiKE:E5C1 can activate the NK cells and induce the killing of HER2+ ovarian and breast cancer cells, surpassing the performance of the best-in-class monoclonal antibody, Trazimera (trastuzumab). To advance this BiKE technology toward clinical application, the objective of this research was to demonstrate the ability of BiKE:E5C1 to activate CD16+ immune cells such as NK cells and macrophages to kill cancer cells, and eradicate metastatic HER2+ tumors in NK humanized NOG mice. METHODS: We assessed BiKE:E5C1's potential to activate CD16-expressing peripheral blood (PB)-NK cells, laNK92 cells, and THP-1-CD16A monocyte-macrophages through flowcytometry and antibody-dependent cell-mediated cytotoxicity/phagocytosis (ADCC) assays. Subsequently, laNK92 cells were selected as effector cells and genetically modified to express the nanoluciferase gene, enabling the monitoring of their viability in NK humanized NOG mice using quantitative bioluminescent imaging (qBLI). To evaluate the functionality of BiKE:E5C1 in vivo, we introduced firefly luciferase-expressing ovarian cancer cells via intraperitoneal injection into hIL-15 and hIL-2 NOG mice, creating a model of ovarian cancer metastasis. Once tumor establishment was confirmed, we treated the mice with laNK92 cells plus BiKE:E5C1 and the response to therapy was assessed using qBLI. RESULTS: Our data demonstrate that BiKE:E5C1 activates not only laNK92 cells but also PB-NK cells and macrophages, significantly enhancing their anticancer activities. ADCC assay demonstrated that IgG1 Fc region had no impact on BiKE:E5C1's anticancer activity. In vivo results reveal that both hIL-15 and hIL-2 NOG mouse models support the viability and proliferation of laNK92 cells. Furthermore, it was observed that BiKE:E5C1 activates laNK92 cells in mice, leading to eradication of cancer metastasis in both NK humanized hIL-15 and hIL-2 NOG mouse models. CONCLUSIONS: Collectively, our in vivo findings underscore BiKE:E5C1's potential as an immune cell engager capable of activating immune cells for cancer cell elimination, thereby expanding the arsenal of available BiKEs for cancer immunotherapy.

Isolation and characterization of human anti-CD20 single-chain variable fragment (scFv) from a Naive human scFv library.

CD20 is a receptor expressed on B cells with anonymous functions. The receptor is the target of some food and drug administration (FDA) approved monoclonal antibodies (mAb), such as Rituximab and Obinutuzumab. Blocking CD20 using the aforementioned mAbs has improved Non-Hodgkin Lymphoma (NHL) therapy. All commercial mAbs on the market were raised in non-human animal models. Antibody humanization is inevitable to mitigate immune response. In order to keep the affinity of antibody intact, humanizations are only applied to frameworks which do not eliminate immune response to foreign CDRs sequences. To address this issue, human monoclonal antibody deemed imperative. Herein, we report the isolation and characterization of a fully human single-chain variable fragment (scFv) against the large loop of CD20 from naïve human antibody library. After three rounds of phage display, a library of enriched anti-CD20 scFv was obtained. The polyclonal phage ELISA demonstrated that after each round of phage display, the population of anti-CD20 scFv became dominant. The scFv, G7, with the most robust interaction with CD20 was selected for further characterization. The specificity of G7 scFv was evaluated by ELISA, western blot, and flow cytometry. Detecting CD20 in western blot showed that G7 binds to a linear epitope on CD20 large loop. Next, G7 scFv was also bound to Raji cell (CD20+) while no interaction was recorded with K562 cell line (CD20-). This data attested that the epitope recognized by G7 scFv is accessible on the cell membrane. The affinity of G7 scFv was estimated to be 63.41 ± 3.9 nM. Next, the sensitivity was evaluated to be 2 ng/ml. Finally, G7 scFv tertiary structure was modeled using Graylab software. The 3D structure illustrated two domains of variable heavy (VH) and variable light (VL) connected through a linker. Afterward, G7 scFv and CD20 were applied to in-silico docking using ClusPro to illustrate the interaction of G7 with the large loop of CD20. As the selected scFv from the human antibody library is devoid of interspecies immunogenic amino acids sequences, no humanization or any other modifications are required prior to clinical applications.

Association of VLA4, 5, 6 and PSGL1 expression levels with engraftment in autologous HPSC transplantation in multiple myeloma patients.

The most promising therapy for leukemia is hematopoietic stem cell transplantation. Engraftment of HPSCs mainly depends on some factors such as adhesion molecules, including VLAs. This study tried to delineate the relationship between HPSCs engraftment and expression level of PSGL1 and VLA4, 5, and 6 genes in candidate MM patients for autologous bone marrow transplantation. Firstly, the CD 34+ HPSCs were collected from multiple myeloma (MM) patients after five days of G-CSF therapy through apheresis processes. Then, the patients were categorized into two groups of good and bad prognosis depending on engraftment time (Less or more than 18 days). Followingly, the expression of PSGL1 and VLA4, VLA5, and VLA6 genes were assessed by the qRT-PCR technique in each patient. Finally, the correlation between the genes and engraftment time was investigated to determine the prognostic role of each gene on HPSCs transplantation. Our findings demonstrated that there is a significant correlation between VLA4 (P=< 0.0001) and 5 (P = 0.005) levels and HPSCs engraftment time. As the higher levels of VLA4 and 5, the shorter time HPSCs engraftment occurs. In contrast, there was no significant correlation between VLA6 (P = 0.2) and PSGL1 (P = 0.3) genes levels and engraftment time. So that, the patients with a good prognosis had a higher level of VLA4 and VLA5, but no relation was found between VLA6 and PSGL1. It is concluded that VLA4 and VLA5 expression could be considered a significant prognostic factor for HPSC transplantation.

Liposome-based nanocarriers loaded with anthrax lethal factor and armed with anti-CD19 VHH for effectively inhibiting MAPK pathway in B cells.

OBJECTIVE: One of the vital signaling pathways in cancer development and metastasis is mitogen-activated protein kinases (MAPKs). Bacillus anthracis Lethal Toxin (LT) is a potent MAPK signaling inhibitor. This toxin is comprised of two distinct domains, Lethal Factor (LF), MAPK inhibitor, and Protective Antigen (PA). To enter various cell lines, LF must be associated with the protective antigen (PA), which facilitates LF delivery. In the current study, to block MAPK signaling, LF was loaded into anti-CD19 immunoliposomes nanoparticle to deliver the cargo to Raji B cells. METHODS: The liposome nanoparticle was prepared using classical lipid film formation, then conjugated to anti-CD19 VHH. The binding efficiency was measured through flow cytometry. The targeted cytotoxicity of LF immunoliposome was confirmed by BrdU lymphoproliferation assay. This was followed by Real-Time PCR to assess the effect of formulation on pro-apoptotic genes. The inhibitory effect of LF on MAPK signaling was confirmed by western blot. RESULTS: Liposome nano-formulation was optimized to reach the maximum LF encapsulation and targeted delivery. Next, phosphorylation of MAPK pathway mediators like MEK1/2, P38 and JNK were inhibited following the treatment of Raji cells with LF-immunoliposome. The treatment also upregulated caspase genes, clearly illustrating cell death induced by LF through pyroptosis and caspase-dependent apoptosis. CONCLUSIONS: In conclusion, anti-CD19 VHH immunoliposome was loaded with LF, a potent MAPK inhibitor targeting B cells, which curbs proliferation and ushers B cells toward apoptosis. Thus, immunoliposome presents as a versatile nanoparticle for delivery of LF to block aberrant MAPK activation. To use LF as a therapy, it would be necessary to materialize LF without PA. In the current study, PA was substituted with anti-CD19 immunoliposome to make it targeted to CD19+ while keeping the normal cells intact.

Strategies for Dodging the Obstacles in CAR T Cell Therapy.

Chimeric antigen receptor (CAR) T cell therapy has offered cancer patients a new alternative therapeutic choice in recent years. This novel type of therapy holds tremendous promise for the treatment of various hematologic malignancies including B-cell acute lymphoblastic leukemia (B-ALL) and lymphoma. However, CAR T cell therapy has experienced its ups and downs in terms of toxicities and efficacy shortcomings. Adverse events such as cytokine release syndrome (CRS), neurotoxicity, graft rejection, on-target off-tumor toxicities, and tumor relapse have tied the rescuing hands of CAR T cell therapies. Moreover, in the case of solid tumor treatment, CAR T cell therapies have not yielded encouraging results mainly due to challenges such as the formidable network of the tumor microenvironments (TME) that operates in a suppressive fashion resulting in CAR T cell dysfunction. In this review, we tend to shine a light on emerging strategies and solutions for addressing the mentioned barriers. These solutions might dramatically help shorten the gap between a successful clinical outcome and the hope for it.

Synergistic effect of granzyme B-azurin fusion protein on breast cancer cells.

As one of the most prevalent malignancies, breast cancer still remains a significant risk for public health. Common therapeutic strategies include invasive surgery, chemotherapy and anti-herceptin antibodies. Adverse effects, drug resistance and low efficacy of current therapies necessitates the emergence of more effective platforms. Naturally released by the immune system, granzyme B activates multiple pro-apoptotic pathways by cleaving critical substrates. Bacterial cupredoxin, azurin, selectively targets cancer cells via a p53-dependent pathway. Fused by a linker, GrB-Azurin fusion protein was overexpressed in HEK293T cells, and purified by metal chromatography. SDS-PAGE, Western blotting and ELISA were performed to confirm successful expression, purification and analyze binding properties of the fusion protein. After treatment of various breast cancer cell lines with increasing concentrations of GrB-Azurin, quantitative real-time RT-PCR was used to measure relative expression of p21, Fas and DR5 pro-apoptotic genes. The results of DNA fragmentation and WST-1 cell viability assays indicated significant apoptosis induction in MDA-MB-231, MCF7 and SK-BR-3 cells, while insignificant cytotoxicity was detected on MCF 10A normal breast cells. Herein, we report the development of a novel biotherapeutic against breast cancer. Selective effectiveness of GrB-Azurin fusion protein on different breast cancer cells highlighted the potential of the designed construct as a candidate anti-cancer biodrug.

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.

Multivalent targeting and killing of HER2 overexpressing breast carcinoma cells with methotrexate-encapsulated tetra-specific non-overlapping variable domain heavy chain anti-HER2 antibody-PEG-liposomes: In vitro proof-of-concept.

The variable domain of the heavy chain antibodies (VHHs) is the smallest (15 kDa) intact single domain antigen-binding fragment. VHHs often exhibit sub-nanomolar affinity for their designated targets and therefore are receiving increasing attention in molecular targeting and nanomedicine engineering. We cloned and expressed four non-overlapping anti-HER2 VHHs in a prokaryotic expression system that yielded disulfide-bonded VHHs. Purified VHHs, before and after thiolation, were characterized by Western blot and their functionality against the ecto-domain of HER2 receptor was confirmed by ELISA and flow cytometry. Thiolated VHHs were conjugated to the reactive maleimide-PEG2000-distearoylphosphatidylethanolamine incorporated into the bilayer of small unilamellar vesicles. We show high target-binding avidity and efficient cytotoxicity of optimized tetra-specific multivalent methatoraxate-loaded VHH-PEG-liposomes (55-60 VHH/vesicle) in HER2 over-expressing breast carcinoma cell lines compared with the best performing monoclonal VHH conjugated vesicles of identical VHH surface density. The VHH expression and production methodology as well as the synergistic effect of the four non-overlapping VHHs in HER2 binding provides an efficient approach for design and engineering of anti-cancer nanomedicines and their future applications within the context of personalized and precision therapies and diagnostics are discussed.

The Evaluation and Comparison of Transcriptionally Targeted Noxa and Puma Killer Genes to Initiate Apoptosis Under Cancer-Specific Promoter CXCR1 in Hepatocarcinoma Gene Therapy.

BACKGROUND: Cancerous cells proliferate as fast as possible without a proper surveillance system. This rapid cell division leads to enormous mutation rates, which help a tumor establish. OBJECTIVES: This study evaluated the potential of inducing apoptosis using Noxa and Puma in a hepatocarcinoma cell line. METHODS: The current study generated two recombinant lentiviruses, pLEX-GCN and pLEX-GCP, bearing Noxa and Puma, respectively. Transduction of both genes to hepatocarcinoma (HepG2) was verified using fluorescent microscopic analysis, western blotting, and quantitative real-time polymerase chain reaction (PCR). To evaluate the potential of Noxa and Puma to initiate apoptosis, a caspase-9 real-time, MTT assay, and a 4', 6-diamidino-2-phenylindole (DAPI) reagent were performed to stain apoptotic cells. RESULTS: The data verified successful transduction to HepG2 and HEK293T. Higher relative expression of Noxa and Puma rather than the untransduced cell line showed these genes are expressed more in HepG2 in comparison to HEK293T. The results of the real-time PCR, MTT assay, and DAPI reagent illustrated that higher cells initiated apoptosis following Puma transduction rather than Noxa. CONCLUSIONS: In this approach, the suicide gene was transferred to transformed cells and ignited apoptosis to exterminate them. Puma is a more potent killer gene and has higher capabilities to start intrinsic apoptosis pathway.

Synergistic Effect of Expressed miR-128 and Puma Protein on Targeted Induction of Tumor Cell Apoptosis.

BACKGROUND: Puma is a highly robust pro-apoptotic protein. The protein becomes activated by p53 ensuing beyond-repair DNA damage. Downregulation of SIRT 1, by miR-128, elevates activated p53 that foment Puma indirectly. OBJECTIVES: In the present study, we used two-expression Adeno-Associated Virus (AAV) system for co-expression of miR-128 and Puma in order to evaluate apoptotic response; both in the tumor and normal cells, respectively. MATERIALS AND METHODS: Three recombinant AAVs constructs were generated. The First rAAV bearing Puma under the control of hTERT (p-AAV), the second construct designed such that to carry miR-128 downstream of CMV (mi-AAV), and the last construct comprises of the both CMV-miR-128 and hTERT- Puma. Real-Time PCR and western blotting were used to evaluate expression levels of the transduced genes. RESULTS: MTT assay and DAPI staining shown suicidal effect of each recombinant AAV vectors. p-AAV cytotoxicity was recorded for 62% of the tumor cells, while for normal cells it was only 20% cytotoxic. The second construct, mi-AAV, was not as potent and selective as p-AAV. This construct was shown to be 27% and 16% cytotoxic for BT-474 and HEK-293 cells, respectively. Co-expression of Puma and miR-128 (p-mi-AAV) was accomplished with a selective cytotoxicity toward BT-474. This construct was 85% toxic for tumor cells, although it was only 25% toxic for the normal cell line (HEK-293). CONCLUSIONS: In this study, we have shown that not only Puma is able to instigate apoptotic response but also its co-expression along with miR-128 could significantly enhance apoptosis in a synergistic manner.