Modulation of immunogenicity by engineered antigen-specific regulatory T cells: Fighting fire with fireman or police CARs

Transcription

1 Modulation of immunogenicity by engineered antigen-specific regulatory T cells: Fighting fire with fireman or police CARs David W. Scott Department of Medicine Uniformed Services University of the Health Sciences Bethesda, MD, USA 10th Open Scientific EIP Symposium Immunogenicity of Biopharmaceuticals Lisbon, Portugal, February 26, 2019

2 Our lab has focused on developing novel approaches to induce specific unresponsiveness ( immune tolerance ) and applying these to modulate human diseases and prevent/reverse adverse immune responses. Dealing with immunogenicity and adverse responses One such approach is regulatory T-cell therapy, which has been proposed to treat autoimmune diseases, allergy and transplant rejection, as well as to suppress undesirable antibody responses to bio-therapeutics, such as FVIII.

3 Rationale and issues Clinical studies with expanded human regulatory T-cell therapy are already in progress. However, these are polyclonal T cells that include a diverse repertoire of relativities. Caveats: The frequency of relevant regulatory T cells (Tregs) may be quite low. Expanded polyclonal Tregs (multiple specificities) may be non-specifically immunosuppressive.

4 Approaches Enrich and expand Tregs with antigen/tetramer, etc. Possible solution based on chimeric antigen receptor (CAR) therapy for cancer: Engineer specificity into polyclonal Tregs via retroviral transduction of specific receptors, e.g.: T-cell receptor (TCR) CAR (scfv) or Antigen (B-cell antibody receptor=bar)

5 Approach to transduce expanded polyclonal Tregs

6 Transduced antigen-specific polyclonal Tregs

7 Engineering antigen-specificity into polyclonal T cells: Four flavors TCR Treg CAR Treg *BAR Treg *BAR CD8 CD4 CD3 APC Treg TCR Signaling domains Teff ScFv Signaling domains? Treg CAR Signaling domains Antigenspecific B cell Treg BAR Signaling domains Antigenspecific B cell CD8 BAR *B-cell antibody receptor=bar

8 Engineering antigen-specificity into polyclonal T cells: TCR V-regions APC Teff CD4 CD3 Treg TCR Signaling domains TCR Treg effects FVIII in hemophilia A MBP in multiple sclerosis Aihong (Allan) Zhang Yong Chan Kim

9 Systems and targets Multiple systems: Hemophilia inhibitors (FVIII) Autoimmunity, e.g., MS (MBP) or Type 1 diabetes Allergy (OVA) Future targets (ADA s)

10 Hemophilia Tenase complex X-linked blood clotting disorder FVIII mutations cause Hemophilia A* FIX mutations cause Hemophilia B *Deletions, inversions, missense, stop codons

11 Hemophilia Tenase complex What is standard treatment for bleeds? X-linked blood clotting disorder FVIII mutations cause Hemophilia A* FIX mutations cause Hemophilia B *Deletions, inversions, missense, stop codons

12 The unwelcome response to a human protein, FVIII Hemophilia A patients can mount an immune response to FVIII depending, in part, on the nature of their mutation Because they lack FVIII, they did not develop immune tolerance to therapeutic FVIII NH 2 A1 A2 B A3 C1 C2 COOH Specific antibodies against FVIII inhibit clotting by binding to domains required its bio-activity ( inhibitors )

13 Application in Hemophilia A

14 Immunology 101: The immune Response to FVIII FVIII protein FVIII peptide Antigen presenting cells FVIII-specific helper T cell Activated FVIII-specific T cells FVIII protein Cytokines FVIII-specific regulatory T cell aka Police CARs FVIII peptide FVIII-specific TCR FVIII-neutralizing inhibitor antibody Activated FVIII-specific B cells FVIII-specific plasma cells

15 CD25 Foxp3 CD25 CD45RA Foxp3 SSC CD25 CD25 CD45RA Isolation of naive T cells and regulatory T cells from normal donor PBMC Treg (CD4 + CD25 hi CD127 l ) T naive (helper) (CD4 + CD25 - CD45RA + ) FSC CD4 CD127 CD4 T naive CD127 CD4 Helios T reg CD127 Helios

16 Foxp3 GFP GFP + Mock Antigen-specific upregulation of Treg markers in Tregs Induction of Foxp3 and GARP OVA peptide FVIII peptide Anti-CD3e Induction of LAP and GITR LAP GITR Mock GARP pc2 ( ) No antigen

17 FVIII-C2-specific immunosuppression by Treg17195 Teff : Treg ratio No Treg Teff/Treg ratio YC Kim et al. Blood 125: 1107, 2015.

18 Can this approach work to prevent or reverse inhibitor responses in hemophilia A mice?

19 Protocol of FVIII-specific suppression of secondary antibody formation by engineered FVIII-specific human Tregs Immunization in vivo Reactivation in vitro HLA.DR1XE16 Immunized mice rfviii n wks spleen FVIII 7 days ELIspot assay to detect anti-fviii antibody No FVIII + FVIII

20 Tregs 0 Tregs 1/2 SHOW FIGURE QUANTITATION No FVIII + FVIII 17195* Mock 1/4 1/8 1/16 Kim YC et al. Blood 125: 1107, *TCR vs. C2 domain peptide

21 Engineering antigen-specificity into polyclonal T cells: Single chain (scfv) CARs TCR Treg CAR Treg APC? Teff CD4 CD3 TCR ScFv Treg Signaling domains Signaling domains Treg CAR

22 What about CAR (chimeric single chain Fv) Tregs? Antigen on B cell or APC or endothelial cell surface? ScFv vs. antigen CAR Signaling domains Treg CAR Treg

23 Both TCR- or scfv engineered human Tregs suppress the secondary anti-fviii response in vitro scfv (A2) TCR (C2) Yoon et al., Blood, 129: , Anja N. Schmidt Jeongheon Yoon

24 TCR- or scfv engineered human Tregs suppress the anti-fviii response in vivo Mock TCR vs. C2 scfv vs. A2

25 Scheme for bystander suppression of multiple T-cell clones by a single Treg C2 A2 C1 xxx? TCR T effector APC? Treg?

26 Question: Can antigen-expressing BAR T-cell therapy modulate antibody responses by directly engaging antigen-specific B cells? BAR = B-cell antibody receptor

27 BAR (B-cell Antibody Receptor) Treg FVIII-specific B cell FVIII domain Structure of BAR (B-cell Antibody Receptor) Treg or CD8 cell FVIII-specific BAR Treg/CD8 Kalpana Parvathaneni

28 Engineering antigen-specificity into polyclonal T cells: Targeting the B cell TCR Treg CAR Treg BAR Treg BAR CD8 APC Teff? FVIIIspecific B cell FVIIIspecific B cell CD4 CD3 TCR ScFv Treg Signaling domains Signaling domains Treg CAR Signaling domains Treg BAR Signaling domains BAR CD8

29 A2/C2 BAR mcd8-mediated elimination of anti-fviii B cells from E16-mouse spleen cells stimulated with LPS A. αfviii IgM+ ELISPOT assay by LPSstimulated E16 B cells No LPS LPS (1 μg/ml) E/T ratio: 5:1 No CD8 OVA BAR A2/ C2 BAR B. Quantification of number of spots BAR-CD8 mediated Elimination of a FVIII IgM + B C 2500 # of Spots per 1xE6 Cells no CD8s A2 /C2 BAR OVA BAR no LPS Naive B cells + LPS 1mg/mL FVIII-coated wells

30 Survival of 2JLO-injected NSG mice with BAR CD8 T-cell therapy A2 BAR Control OVA BAR Control Day 10 Day 18 Day 25

31 Can BAR engineered CD4 Tregs target and suppress FVIIIspecific B cells FVIIIspecific B cell FVIIIspecific B cell FVIII domain CD8 Treg BAR CD8 BAR Treg

32 A n ti-f V III a n tib o d y (µ g /m l) Prevention of anti-fviii antibody development in naïve E16 mice by BAR human Tregs in vivo O b 2 F 3 h T re g s F V III B A R h T re g s

33 Systems and targets Multiple systems: Hemophilia inhibitors (FVIII) Autoimmunity, e.g., MS (MBP) or Type 1 diabetes Allergy (OVA) Future targets (ADA s)

34 BAR (B-cell Antibody Receptor) Treg Can BAR Tregs be used to modulate allergy? OVA-specific B cell Mast cell with FceR

35 Allergic response

36 Future: CAR or BAR cell therapy not only for hemophilia, but also for allergy, transplantation, autoimmunity or other monogenic diseases (and ADA?) Suppression of specific T and B cells Isolated T cells Generic donor Engineered T cells Viral systems Virally Transduced T cells

37 Summary Antigen-specific TCRs, single chain Fvs and antigen domains (BARs) have now been engineered for retroviral transduction into human T effectors and human (mouse) Tregs. These Tregs specifically suppressed both proliferation and cytokine production by antigen-specific T effectors, and antibody formation in vitro and in vivo in multiple model systems. Recent data with BAR CD8 s and Tregs (expressing antigen domains) may allow multiple approaches to regulate adverse immune responses. e.g., Ovalbumin-BAR itregs are able to suppress both active and passive anaphylaxis. Expansion of these studies to Tregs in a larger species (hemophilic dogs) is in progress, with human clinical studies on the horizon.

38 Acknowledgements Yongchan Kim Allan Zhang Jeongheon Yoon Kalpana Parvathaneni Maha Abdeladhim Kai Wucherpfennig Harvard Ethan Shevach NIH Kate Pratt USUHS Anja Schmidt Frankfurt Chris Königs Frankfurt Edward Mitre Laura Kropp USUHS USUHS Supported by NIH/NHLBI grants RO1 HL and R21 HL and NMSS RG

39 Obrigado Gracias