The expression of the antigen on individual cells within a given tumor is different or heterogeneous. A solid tumor mass consists of numerous tumor cells. In these tumor cells, some may express relatively less tumor antigens, others may express relatively more tumor antigens. Meanwhile, a given tumor antigen is not only expressed on malignant cells, but may also be expressed on normal cells at a low level (Figure 1A). Thus, normal cells expressing low level of tumor antigens subsequently should not be targeted, otherwise would casuse complications. Carefully control the on-target/off-tumor effect is critical for the success of immunotherapy. (Figure 1B)
Carcinogenesis is a gradual progress driven by the accumulation of mutations. Tumor cells are highly heterogeneous in their surface tumor antigen expression(1), thus immune resistance(2), sensitivity to the treatment and so on. Meanwhile, efficient recognition by immunotherapy, as one of the fundamental challenges for solid tumors, is still in the way comparing with exciting results shown in treating hematological cancers(3).Currently, most existing immunotherapies exhaust in trying multiple methods to improve recognition(4-6), without considerating tumor heterogeneity. They focus narrowly on finding an ideal tumor antigen as the target and hope to generate a effective therapeutic response – monotonic response.We believe these conventional one-size-fits-all immunotherapies cannot adapt itself to all complex disease occasions in various types of tumors.(Figure 1B)
Tumor in different states need different treatments. To develop rational combined therapy, the key question is how to accurately manifest tumors’ condition. Fortunately, with improved understanding of oncogenesis and emerging therapies, clinical trials of rational combined therapy have become possible(14,15). Through investigation, we found that antigen density heterogeneity, tumor antigen level and expression pattern are associated with disease progression(16).Thus, Antigen density heterogeneity could be used to develop rational combined immunotherapy.
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SwordS consists of two main modules, SynNotch and Stripe, and one supportive module, SynTF-SynPro(Figure 3).
Synthetic Notch (SynNotch)(17)consists of three parts, the synthetic extracellular recognition domain (SynECD, e.g.scFv), the core transmembrane domain of wild Notch receptor(18), and the synthetic intracellular transcriptional domain (SynICD, e.g.SynTF). When the SynECD binds to itstargeting surface antigen, induced cleavagestake place on the core transmembrane domain of SynNotch, releasing the SynICD. The SynICDwould be transported into nucleus and activate the transcription of its corresponding promoter (Figure 4).
SynNotch provides us an exciting platform for sensing and treating tumorbecause its SynECD and SynICD are both customizable. SynECDcan be designed based on current available scFvs for different tumors such as α-GPC3 for HCC(11, 19),α-Her2 for glioma(4),α-CD19for acute lymphoid leukemia(20), etc.SynICD will trigger customized output after SynECD recognition.
SynPro S, an activating-form promoter triggered by the existence of initial signal (S), is the start point of the whole module. Meanwhile, activation strength of Promoter S (Pro S) is positively correlated with the concentration of S.SynTF X1/2, SynTF Y, are two orthogonal silencing-form synthetic transcription factor (SynTF) and their corresponding silencing-form synthetic promoter (SynPro) are SynPro X and SynPro Y. (SynTF X1 and SynTF X2 are the same transcription factor on two different open reading frams.)Importantly, the inhibition threshold of SynTF X must be much higher than SynTF Y. A, B, C are three response factors(Figure 5A)
.When S’s concentration is none or low. Expression level of circuit ①is low. Concentration of SynTFX1 and Y are not sufficient to inhibit SynPro X or SynPro Y. In this condition, the expression level of Circuit ②is normal.Thus,SynTF X2, the product of circuit ②, is in high concentration to sufficiently inhibit SynPro X and inhibit the expression of circuit ③ (Figure 5B). Only A (in green) is produced.
When S’s concentration is mediam.Expression level of ① circuit is mediam. Produced SynTF X1 is still not sufficient to inhibit high-threshold SynProX. However, SynTF Y produced by circuit ① can significantly inhibit low-threshold SynPro Y to express circuit ②. SynPro X cannot be inhibited by either SynTF X1 (not sufficient) or SynTF X2 (inhibited)(Figure 5C). Only B (in red) is produced.
When S’s concentration is high. Expression level of ① circuit is high. Both SynTF X1 and Y are in high concentration. Expression of circuit ② and ③ are inhibited(Figure 5D). C (in blue) is massively produced. C can have a slight couple-expression with X1/Y, when S’s concentration is low or mediam. However, these expressions can be neglected comparing to the dominantly expressed A (in low concentration of S) or B (in mediam concentration of S).
Stripe can spontaneously generate tri-response depending on the intensity of its input: a low intensity plateau, a hump-like signal peak formediamintensity, and a high intensity plateau (Figure 6).In a tumor therapy oriented project, we can separately designate the three outputs as no therapeutic factor expression, therapeutic factor I expression, and therapeutic factor II expression. That’s to say, if the input intensity is mediam, the output tends to be therapeutic factor I. If the input shifts towards high intensity, the output shifts to therapeutic factor II, and if the input shifts towards low intensity, output nothing. Thus, normal cellswith low surface antigen signal could be ignored, reducing the off-targeteffect.Meanwhile, the expression of therapeutic factor I orIIcanbe adjusted via the signal sorting module to suit different tumor conditions. |
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