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Hepatocellular carcinoma (HCC) More information about our demonstration works on HCC...

SwordS
Antigen density heterogeneity and limited treatment, two obstacles in improving therapeutic effect and applicability of immunotherapy

Target antigen density heterogeneity

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)

Limited treatment cannot suit all cases

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)

Read more: Current methods to improve recognition and their imperfections

Current methods to improve recognition are not perfect
Emerging methods to improve recognizing precision, like dual recognition ^(6,7)and tunable sensitivity ⁽⁵⁾, have been proved capableof eliminating specific tumor cells. However,these methods cannot completely solve the problem.

Specific tumor antigens for dual recognition is hard to find
The first step for immunotherapy is to select a highly specific tumor antigen as the target. However, qualified candidates for tumor antigens are rare in most cases. Taking HCC (hepatocellular carcinoma) as an example, although HCC-associated antigens, like EpCAM⁽⁸⁾, NY-ESO-1⁽⁹⁾, and GPC3⁽¹⁰⁾, are potential targets of cellular immunotherapies for advanced HCC. However, only GPC3, is wildly accepted as the tumor antigen of HCC owing to its high specificity.^(6,11,12). Only one tumor antigen for HCC prevents the use of dual recognition.

Tunable sensitivity requires optimized scFv– it is very get
The affinity of single-chain variable fragment (scFv) is essentialfor recognition. Even though recently highthroughput methods have been developed to screen forscFvs with different affinity to the same antigen⁽¹³⁾, specialized knowledge and high expense render it impossible to perform inall laboratories. Only one or two laboratories around the world have the capability to develop scFv with tunable sensitivity.

One important theory of traditional Chinese medicine:
suit the remedy to the case

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. Here, we report the “SwordS”, SynNotch-Stripe system, which can spontaneously generate non-monotonic responses by targeting different surface tumor antigen density of tumor cells at different states.(Figure 2)

SwordS enables antigen density dependent
tri-response for therapeutic applications

SwordS consists of two main modules, SynNotch and Stripe, and one supportive module, SynTF-SynPro(Figure 3).

Module 1: SynNotch
SynNotch,an engineered transmembrane receptor, bridges intra and extracellularinformation.

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 its targeting surface antigen, induced cleavages take 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 is an ideal platform for customized antigen sensing behavior.

SynNotch provides us an exciting platform for sensing and treating tumor because 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.

Module 1: Module 2: Stripe
Stripe consists of 3 interactional circuits.

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)

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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 achieves combined immunotherapy through an adjustable signal sorting module.

Stripe can spontaneously generate tri-response depending on the intensity of its input: a low intensity plateau, a hump-like signal peak for medium intensity, 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 medium, 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 cells with low surface antigen signal could be ignored, reducing the off-target effect. Meanwhile, the expression of therapeutic factor I or II can be adjusted via the signal sorting module to suit different tumor conditions.