Co-expression of PD-L1 Antibodies Enhances the Anti-Tumor Efficacy


Chimeric antigen receptor T cell (CAR-T) therapy has shownsignificant efficacy in treating patients with refractory B-cellleukemia and lymphoma [1,2]. However, CAR-T therapy hasyielded less favorable results in treating patients with solidtumors such as pancreatic, ovarian or mesothelioma cancers[3-5]. One of the major obstacles in treating solid tumors withCAR-T is the scarcity of antigen that is uniformly expressed intumor cells such as CD19 in B cell hematologic malignancies[6,7]. Therefore, designing a CAR against an antigen that ishighly expressed in cancer cells but either has negligible orreduced expression in normal tissues to avoid off-tumor toxicityis extremely critical. Several CAR-T clinical trials in treating solidtumors demonstrated poor efficacy and severe toxicitiesincluding death because of the low expression of antigen in thenormal cells [7-13]. Moreover, the immunosuppressive tumormicroenvironment of solid tumors inhibits the T cell infiltrationand proliferation limiting the potential of CAR-T cellimmunotherapy [14,15].Mesothelin (MSLN) has emerged as one of the promising targetsfor CAR-T cell therapy for solid tumors because of itsoverexpression in cancer cells including pancreatic, ovarian andmesotheliomas but with a basal expression in normal tissues[16-18]. Mesothelin is encoded as a 71-kDa cell surfaceglycoprotein that is cleaved by a furin protease into an N-terminal 31 kDa soluble fragment megakaryocyte potentiating.


factor  (MPF)  and  C-terminal  40  kDa  membrane-boundmesothelin  [16,18].  Although  mesothelin  is  expressed  in  lung,heart,  spleen,  liver,  kidney,  and  testis  of  adult  tissues,  thebiological  function  of  mesothelin  is  not  known  [19].  Miceharboring  a  null  mutation  in  the  mesothelin  gene  had  aphenotype  similar  to  the  wild-type  suggesting  mesothelin  wasnot  required  for  growth  and  reproduction  [19].  However,mesothelin  is  believed  to  involve  in  cell  adhesion  in  tumorprogression since it binds to the CA125/MuC16 facilitating themetastatic  proliferation  of  ovarian  cancer  onto  peritonealmesothelial cells [20,21].Programmed  death  1  (PD-1)  is  expressed  on  B  and  T  cells,macrophages  and  some  of  dendritic  cells  and  upon  activationregulates  the  tolerance  and  autoimmunity  [22].  PD-1-/-  micedeveloped lupus-like autoimmune disease confirming the role ofPD-1 in maintaining tolerance and autoimmunity [23,24]. PD-L1, a ligand of PD-1, is expressed constitutively on cancer cellsand  delivers  pro-survival  signals  that  favor  tumor  progression[25].  PD-1  is  capable  of  transmitting  inhibitory  signals  whenengaged  with  PD-L1  expressed  on  tumor  cells  and  thusinhibiting  tumor-specific  T-cell  proliferation  and  cytokineproduction  [26].  Therefore,  eliminating  either  PD-1  or  PD-L1and  blocking  their  interaction  can  result  in  the  breakdown  oftumor immune suppression.In  this  study,  we  generated  a  novel  vector  that  co-expresseshuman MSLN-targeted CAR with secretory human anti-PD-L1antibodies  to  overcome  tumor-mediated  immunosuppression.We  investigated  its  anti-tumor  potential  in  pancreatic,  ovarianand mesothelioma xenograft mouse models. The MSLN-targetedCAR-T cells secreting the anti-PD-L1 antibodies efficiently killedmesothelin-expressing cells in vitro as well as showed potent anti-tumor activity in a xenograft mouse model.


In conclusion, our results demonstrate that second-generationanti-mesothelin HN1CAR-T cells secreting anti-PD-L1 IgG-Fcantibodies enhanced anti-tumor efficacy and increased cytokine production. This CAR design may diminish CAR-T cellexhaustion and improve treatment for solid tumors. We proposethat CAR-T cells targeting tumor antigen as well as secretingantibody to block checkpoint signaling may circumvent theexhaustion of CAR-T cells. We foresee that a combination ofCAR and anti-PD1/PD-L1 into a single construct should beamong the next steps to explore to achieve higher anti-tumorefficacy in solid tumors.


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