Local production of cytokines by genetically engineered tumor cells decreases their tumorigenicity and elicits protective immune responses against the parental tumor cells. An alternative approach to elicit a therapeutic immune response is to use fusion proteins that can target tumor cells and simultaneously activate effector cells. Fusion proteins between human IL-2, murine or human GM-CSF, and sFv of antihuman carcinoma antibody L6 have been constructed, expressed in both COS and Chinese hamster ovary (CHO) cells, and purified by affinity chromatography. The biologic activity of L6 sFV-hIL-2, L6 sFv-mGM-CSF, and L6 sFv-hGM-CSF was tested on human T cell blasts, factor-dependent FDCP-1, and TF-1 cells, respectively. The ability of soluble L6 sFv-hIL-2, L6 sFv-mGM-CSF, and L6 sFv-hGM-CSF to stimulate the proliferation of the indicator cells was found to be comparable to that of recombinant hIL-2, mGM-CSF, or hGM-CSF. Tumor cells coated with L6 sFV-mGM-CSF or L6 sFv-hGM-CSF were also tested in this way and were found to be potent stimulators, indicating that the cytokines were functionally active when bound to the tumor cell surface. This work demonstrates the feasibility of targeting sFv-cytokine fusion proteins for the activation of effector cells as an alternative to cytokine gene therapy.
4-1BB is an activation-induced costimulatory TNF receptor family member which is expressed on a variety of cell types such as activated T cells, NK cells, DCs, B cells, monocytes and Neutrophils. 4-1BB/4-1BBL interaction leads to a series of activation effects including increased cytotoxic T lymphocytes (CTL) activity, cytokine induction, prevention of activation-induced cells death (AICD). Agonistic monoclonal antibodies targeting 4-1BB have shown robust anti-tumor activity but has also caused substantial liver toxicity which hampered their clinical development. To better assist the efficacy and toxicity evaluation of anti 4-1BB antibodies, Biocytogen developed a 4-1BB/4-1BBL double humanized knock in mice, which is the second generation of humanized 4-1BB mice. In the double humanized mouse, the exons of mouse 4-1BBL and 4-1BB genes that encode the transmembrane domain and extracellular domain were replaced by human counterpart exons. The expression of knocked in exons were confirmed by both RT-PCR and Flow cytometry. In vivo efficacy study showed that 4-1BB antibodies significantly inhibited tumor growth in mice bearing the colon cancer cell line MC38 and this effect was dose-dependent. The tumor growth inhibition (TGI) reached approximately 70% in the mice group dosed 1mg/kg. More importantly, Toxicity study showed that compared to the single humanized 4-1BB mice, double humanized 4-1BB/4-1BBL mice have higher sensitivity on liver toxicity testing, reflected by sera AST and ALT level as well as liver pathological analysis. These results indicate that the double humanized B-h4-1BB/h4-1BBL mouse model, by mimicking a more human like 4-1BB and 4-1BBL interaction, has been proved to be a more advanced option for preclinical toxicity and efficacy evaluation for 4-1BB therapeutic antibodies compared to single humanized 4-1BB mice. Citation Format: Lei Zhao, Linlin Wang, Dirui Li, Li Hui, Qingcong Lin. Double-humanized 4-1BB/4-1BBL mouse is an advanced model for liver toxicity and efficacy evaluation of 4-1BB therapeutic antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 501.
Increasing evidences suggest that the type I interferon (IFN) plays a critical role in the etiopathogenesis of systemic lupus erythematosus (SLE), which makes it a promising therapeutic target for the treatment of the disease. By screening a large size non-immune human antibody library, we have developed a human single-chain antibody (ScFv) AIFNα1bScFv01 and corresponding whole antibody AIFNα1bIgG01, that recognizes recombinant human interferon alpha1b (hIFNα1b) with high specificity and high affinity. The IgG antibody can downregulate the expression of ISG15 and IFIT-1 induced by either recombinant hIFNα1b or naive IFN-α presented in SLE patient's sera. The crystal structure of AIFNα1bScFv01-hIFNα1b complex solved to 2.8 Å resolution reveals that both Pro26-Gln40 region in loop AB and Glu147-Arg150 region in helice E of hIFNα1b contribute to binding with AIFNα1bScFv01. Four residues of above two regions (Leu30, Asp32, Asp35 and Arg150) are critical for the formation of antigen-antibody complexes. AIFNα1bScFv01 shares partial epitopes of IFNα1b with its receptor IFNAR2. AIFNα1bIgG01 has a much higher affinity for IFNα1b than IFNAR2 (K D = 0.747 nM versus 100 nM), making it unavailable for binding to IFNAR2 and preventing the activation of IFN-α-mediated signaling pathway. Thus, AIFNα1bIgG01 exhibits its neutralizing activity through competition with IFNRA2 to bind with IFN-α. Our results highlight the potential use of the human antibody for modulating the activity of IFN-α in SLE. Type-2 diabetes mellitus (T2DM) accounts for more than 90% of all diabetes worldwide. Over 100 million people worldwide have T2DM, and the prevalence is increasing dramatically in both the developed and developing countries. Amyloid deposits have been observed in a vast majority of the T2DM patients and these are primarily on account of misfolding and aggregation into fibrils of human islet amyloid polypeptide (hIAPP), a 37 residue endocrine hormone secreted by pancreatic β-cells. It has been suggested that intermediates produced in the process of fibrillization are cytotoxic to insulin producing β-cells. Hence, the inhibition of misfolding/fibrillization of hIAPP could be a possible strategy to mitigate T2DM. The misfolding of hIAPP involves structural transition from its native state (coil and/or helical and/or transient helical conformation) to β-sheet conformation. We have targeted hIAPP fibrillization by designing short peptides containing the helix inducing α,β-dehydrophenylalanine (∆Phe or ΔF) amino acid and the fibrillization inhibition was monitored by thioflavin-T assay and electron microscopy. We find that the short peptides inhibit fibrillization without any cytotoxic effect as tested on RIN4fm pancreatic cell line. Of these, the penta-peptide, FGA∆FL is the most effective inhibitor of hIAPP fibrillization. We successfully crystallized the penta-peptide and solved its 3D structure at atomic resolution using direct methods. Molecular conformation of the peptide reveals the occurrence of a nest-motif (Fig. A) involving the st...
Interleukin 17 (IL-17) is described as a pro-inflammatory cytokine produced by activated CD4 cells. Evidence shows that IL-17 is highly up-regulated at sites of inflammatory tissues of autoimmune diseases and amplifies the inflammation through synergy with other cytokines, such as TNF (tumor necrosis factor) α. Therefore, targeting IL-17/IL-17R, IL-17-producing pathways or IL-17-mediated signaling pathways can be considered for future therapy in autoimmune diseases. To evaluate the efficacy of IL17A and IL17F antibodies, we generated a double humanized B-hIL17A/hIL17F mouse in Biocytogen. In this model, the sequences encoding the extracellular domains of the mouse IL17A gene and the IL17F gene were replaced with the corresponding human sequences. IL17A and IL17F protein expression was detected in B-hIL17A/hIL17F mice by ELISA using a species-specific IL17A ELISA kit. In efficacy study of anti-human IL17A and IL17F antibody with psoriasis model induced in B-hIL17A/hIL17F mice, mice were treated with different dose of bimekizumab produced in house. Erythema and scaling score of the skin was significantly reduced compared with non-treatment group. The results suggest B-hIL17A/hIL17F mouse model is an effective tool for in vivo efficacy evaluation, which may represent a promising model for screening IL17 related drug candidates for the treatment of psoriasis disease. Citation Format: Chong Li, Lei Zhao, Tao Zhu, Yichao Chen, Leila Kokabee, Chaoshe Guo. Humanized IL17A/hIL17F mouse model provides a preclinical tool for the evaluation of therapeutic drugs targeting to IL-17 pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2956.
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