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Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the adoptive immunotherapy of which is worth studying. CD133, a kind of cancer stem cell (CSC) antigen, together with glypican-3 (GPC3) have been proved to be highly expressed in HCC cells and both of them are used as targets to generate chimeric antigen receptor (CAR) T cells. But there are limitations like “on-target, off-tumor” toxicity, low transfection efficacy and weak antitumor ability in CAR T cells treatment.
Methods: First we fused anti-CD133 and anti-GPC3 single chain Fragment variable (scFv) structures with intracellular domains, respectively. Using non-viral minicircle DNA (mcDNA) vectors to generate co-specific CAR T cells (CoG133-CAR T cells) against CD133 and GPC3 double-positive HCC cells. We exhibited the transduction efficiency of CoG133-CAR T cells and the antigen expression of tumor cell lines. Finally, the antitumor efficacy of CoG133-CAR T cells both in vitro and in vivo was detected.
Results: GPC3-CAR and CD133-CAR were successfully prepared using non-viral mcDNA vectors to generate effector cells. For the GPC3 and CD133 double-positive HCC (Huh7) xenograft mice, co-specific CAR T cells possessed stronger tumor growth suppression compared to single-targeted CAR (GPC3-CAR and CD133-CAR) T cells which induced only one antigen-mediated signal pathway. The same results also occurred on the in vitro experiments including cytokine secretion, cytotoxicity and proliferation ability of CAR T cells. Vital organs from CoG133-CAR T cells and normal T cells respectively treated Huh7 xenograft mice were stained by hematoxylin and eosin (H&E), the images showed no difference.
Conclusions: The mcDNA vectors loading CAR structures were transfected into T cells by electroporation without genetic mutation or mismatch. Huh7 is an HCC cell line with two antigens of GPC3 and CD133 highly expressed. The antitumor efficacy of co-specific CAR (CoG133-CAR) T cells against Huh7 cells is significantly enhanced. The joint design of two specific targets and non-viral vectors leads much more safety, also.
Keywords
Hepatocellular carcinoma, Cancer immunotherapy, Nonviral mcDNA vector, Cospecific CAR T cells, Cancer stem cells
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CURRENT STATUS: Under Review
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Received 30 Nov, 2021
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Invitations sent on 28 Nov, 2021
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This preprint is under consideration at Cancer Cell International. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Primary research
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Under Review
Version 1
Posted 10 Nov, 2021
No community comments so far
Reviews received
Received 30 Nov, 2021
Reviewers invited
Invitations sent on 28 Nov, 2021
Editor assigned
On 02 Nov, 2021
Submission checks complete
On 01 Nov, 2021
Editor invited
On 01 Nov, 2021
First submitted
On 01 Nov, 2021
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the adoptive immunotherapy of which is worth studying. CD133, a kind of cancer stem cell (CSC) antigen, together with glypican-3 (GPC3) have been proved to be highly expressed in HCC cells and both of them are used as targets to generate chimeric antigen receptor (CAR) T cells. But there are limitations like “on-target, off-tumor” toxicity, low transfection efficacy and weak antitumor ability in CAR T cells treatment.
Methods: First we fused anti-CD133 and anti-GPC3 single chain Fragment variable (scFv) structures with intracellular domains, respectively. Using non-viral minicircle DNA (mcDNA) vectors to generate co-specific CAR T cells (CoG133-CAR T cells) against CD133 and GPC3 double-positive HCC cells. We exhibited the transduction efficiency of CoG133-CAR T cells and the antigen expression of tumor cell lines. Finally, the antitumor efficacy of CoG133-CAR T cells both in vitro and in vivo was detected.
Results: GPC3-CAR and CD133-CAR were successfully prepared using non-viral mcDNA vectors to generate effector cells. For the GPC3 and CD133 double-positive HCC (Huh7) xenograft mice, co-specific CAR T cells possessed stronger tumor growth suppression compared to single-targeted CAR (GPC3-CAR and CD133-CAR) T cells which induced only one antigen-mediated signal pathway. The same results also occurred on the in vitro experiments including cytokine secretion, cytotoxicity and proliferation ability of CAR T cells. Vital organs from CoG133-CAR T cells and normal T cells respectively treated Huh7 xenograft mice were stained by hematoxylin and eosin (H&E), the images showed no difference.
Conclusions: The mcDNA vectors loading CAR structures were transfected into T cells by electroporation without genetic mutation or mismatch. Huh7 is an HCC cell line with two antigens of GPC3 and CD133 highly expressed. The antitumor efficacy of co-specific CAR (CoG133-CAR) T cells against Huh7 cells is significantly enhanced. The joint design of two specific targets and non-viral vectors leads much more safety, also.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
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