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Generation Of Fviii-Specific Chimeric Antigen Receptor (Car) Tregs To Suppress Inhibitor Development In Hemophilia A Mice

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Title:
Generation Of Fviii-Specific Chimeric Antigen Receptor (Car) Tregs To Suppress Inhibitor Development In Hemophilia A Mice
Series Title:
19th Annual Undergraduate Research Symposium
Creator:
Saboungi, Rania
Language:
English
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Undetermined

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Center for Undergraduate Research
Center for Undergraduate Research
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Conference papers and proceedings
Poster

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Abstract:
Hemophilia A is an X-linked clotting factor disorder in which patients have a deficiency in coagulation factor VIII (FVIII). The development of inhibitory antibodies in hemophilia A patients who receive FVIII replacement therapy demand new therapies for tolerance. Chimeric antigen receptors (CAR) are recombinant molecules that combine antigen binding and T-cell signaling functions by redirecting immune reactivity toward a chosen antigen. We combined the specificity of CAR technology with regulatory T cell (Treg) therapy to produce FVIII specific Tregs. We did this by complexing the single-chain antigen recognizing variable fragments of a human FVIII antibody to primary and co-stimulatory T cell signaling domains (CD3ζ and CD28), then inserting the CAR molecule into Tregs to generate FVIII specific CAR-Tregs. We tested for FVIII specific activation and proliferation of FVIII CAR-Tregs in vitro. Antigen recognition and signaling by CAR-Tregs may also result in activation-induced cell death (AICD) of the transduced cells. To help find a balance between activation and AICD of the CAR-Treg, we made a series of mutations in the Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) of the CD3ζ domain, by site-directed mutagenesis. This has provided us a superior cell-therapy for immune tolerance to hemophilia. We will test for prevention of inhibitory antibody formation by adoptively transferred CAR Tregs in mice. Inhibitors will be generated by once weekly injections of FVIII and inhibitory titers in treated and untreated mice will be tested by ELISA and the Bethesda assay. ( en )
General Note:
Research authors: Rania Saboungi, Moanaro Biswas, Roland Herzog - University of Florida
General Note:
Faculty Mentor: Hemophilia A is an X-linked clotting factor disorder in which patients have a deficiency in coagulation factor VIII (FVIII). The development of inhibitory antibodies in hemophilia A patients who receive FVIII replacement therapy demand new therapies for tolerance. Chimeric antigen receptors (CAR) are recombinant molecules that combine antigen binding and T-cell signaling functions by redirecting immune reactivity toward a chosen antigen. We combined the specificity of CAR technology with regulatory T cell (Treg) therapy to produce FVIII specific Tregs. We did this by complexing the single-chain antigen recognizing variable fragments of a human FVIII antibody to primary and co-stimulatory T cell signaling domains (CD3ζ and CD28), then inserting the CAR molecule into Tregs to generate FVIII specific CAR-Tregs. We tested for FVIII specific activation and proliferation of FVIII CAR-Tregs in vitro. Antigen recognition and signaling by CAR-Tregs may also result in activation-induced cell death (AICD) of the transduced cells. To help find a balance between activation and AICD of the CAR-Treg, we made a series of mutations in the Immunoreceptor Tyrosine-based Activation Motifs (ITAMs) of the CD3ζ domain, by site-directed mutagenesis. This has provided us a superior cell-therapy for immune tolerance to hemophilia. We will test for prevention of inhibitory antibody formation by adoptively transferred CAR Tregs in mice. Inhibitors will be generated by once weekly injections of FVIII and inhibitory titers in treated and untreated mice will be tested by ELISA and the Bethesda assay. - Center for Undergraduate Research,

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University of Florida
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Copyright Rania Saboungi. Permission granted to University of Florida to digitize and display this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.

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Generation of FVIII-Specific Chimeric Antigen Receptor (CAR) Tregs to Suppress Inhibitor Development in Hemophilia A Mice Introduction Hemophilia A is an X-linked clotting factor disorder in which patients have a deciency in coagulation factor VIII (FVIII). Current treatment is based on FVIII replacement therapy; however, 20-30% of patients develop inhibitory anti-FVIII antibodies (inhibitors), preventing the FVIII treatment from working. Regulatory T cells (Tregs) are immunosuppressants expressing the biomarkers CD4, FoxP3, and CD25. FoxP3 is the transcription factor regulating the development and function of Tregs. Consequently, the role of Tregs of the CD4 + CD25 + FoxP3 + phenotype in tolerance to coagulation factors has emerged, as they can suppress both the B and T cells involved in inhibitor formation. Antigen specic Tregs were generated by engineering and retrovirally transducing a chimeric antigen receptor (CAR) molecule with specicity to human FVIII. CARs are recombinant receptors that provide both antigen-binding and T-cell activating functions by redirecting immune reactivity toward a chosen antigen. Antigen recognition via the single chain antibody variable regions triggers signaling via the CD28 and CD3 co-stimulatory domains, which induces CAR-Treg activation and proliferation, without the need for MHC restriction. Antigen recognition and signaling by CAR-Tregs may also result in activation-induced cell death (AICD) of the transduced cells. Thus, the goal of this investigation is to not only test if FVIII specic CARTregs reverse inhibitor formation in a mouse model of hemophilia A, but to also nd a balance between antigen specic activation and AICD of the CAR-Treg Methods Results Conclusion Acknowledgements A single chain variable fragment (ScFv) from a human immunoglobulin directed against human FVIII was generated and fused to the CD3 and CD28 signaling domains of a 2nd generation CAR, then packaged into a retroviral system, pMys-IRES-GFP (which allows the delivery of genes to mammalian cells). CD3/CD28 bead activated CD4 + T cells were then retrovirally transduced to generate FVIII CAR-Tregs (s timulation of CD3 and CD28 is required for Treg expansion). Binding of FVIII-CAR Tregs to Fc-FVIII was tested using an A647 conjugated Fc antibody. To test for activation of the FVIII-CAR Tregs, upregulation of the CD69 activation marker was assessed 24 hours after stimulation with Fc-FVIII To nd a balance between antigen specic activation and AICD of the CAR-Treg, we made a series of mutations in the Immunoreceptor Tyrosine-based Activation Motifs of the CD3! domain, ITAM-1 and ITAM-3, by site-directed mutagenesis. We tested for prevention of inhibitory antibody formation by adoptively transferred CAR Tregs in mice. Inhibitors were generated by once weekly injections of FVIII, and inhibitory antibody titers in treated and untreated mice will be tested by ELISA and the Bethesda assay. Special thanks to Moanaro Biswas for her mentorship and the Herzog lab for providing all resources. GFP percentage indicated 12.37% transduced cells. FVIII CAR-Tregs bound Fc-FVIII (Eloctate) in a specic manner, with no binding observed to control Fc-FIX. Stimulation for 24 hours with Fc-FVIII resulted in upregulation of the CD69 activation marker for the GFP+ transduced cells only, indicating activation of FVIII-CAR Tregs. No non-specic activation with the unrelated Fc-FIX antigen was observed. The native CAR, with all ITAMs of the CD3 chain domain left intact, lead to over-activation and some induced cell death. A crippled CAR, with both ITAM-1 and ITAM-3 mutated, did not provide sufcient activation signals. A CAR with either ITAM-1 or ITAM-3 mutated provided an optimized signaling that did not cause cell death. Percent upregulation of the CD69 activation marker used to asses activation of the cells. Percent GFP loss used to assess percent cell death. We are currently testing for the suppression of inhibitor development in hemophilia A mice following adoptive immunotherapy with FVIII CAR-Tregs and continued weekly administrations of FVIII. We demonstrate that CAR-Tregs directed to human FVIII show antigen-specic binding, activation and proliferation. The suppressive activity of FVIII CARTreg on inhibitory antibody formation is now being evaluated in hemophilia A mice that are given factor replacement therapy with FVIII. Our results from testing mutations in the signaling domain of the FVIII-CAR should provide us with a superior cell-therapy protocol for immune tolerance to hemophilia. At the end of the study, we hope to establish that FVIII specic suppression by redirection of Tregs would reduce inhibitor titers to manageable levels. If successful, the approach for expression in human cells can be developed, with the goal of translation to the clinic. Rania Saboungi, Moanaro Biswas and Roland Herzog Pediatrics Dept, University of Florida, Gainesville, FL, United States, 32610